Improved amino acid sequences against il-6r and polypeptides which contain thereof for treatment of il-6r associated diseases and disorders

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biochemistry, in particular to single variable domain, aimed against IL-6R, to polypeptide and construction, directed against IL-6R, containing said single variable domain, as well as to methods of obtaining them. Disclosed are nucleic acids, coding said single variable domain, polypeptide and construction, as well as genetic constructions, containing said nucleic acids. Described are host cells and host organisms, containing said nucleic acids. Invention also deals with composition for blocking interaction of IL-6/IL-6R, containing effective quantity of described single variable domain, polypeptide, construction, nucleic acid or genetic construction. Also disclosed is method of prevention and/or treatment of at least one of diseases or disorders, associated with IL-6, IL-6R, complex IL-6/IL-6R and/or signal pathways, in which IL-6, IL-6R or complex IL-6/IL-6R is involved and/or biological functions and reactions, win which IL-6, IL-6R or complex IL-6/IL-6R takes part with application of described single variable domain, polypeptide, construction or composition.

EFFECT: invention makes it possible to block interaction of IL-6/IL-6R effectively with increased affinity and biological activity.

25 cl, 70 dwg, 56 tbl, 61 ex

 

The technical field to which the invention relates.

The present invention relates to amino acid sequences directed against and/or can specifically bind (as defined here) with the receptor, interleukin-6 (IL-6R), as well as to compounds or constructs, and in particular proteins and polypeptides, containing or consisting primarily of one or more such amino acid sequences (also referred to as "amino acid sequences of the invention", "compounds of the invention", "a construction of the invention and polypeptides of the invention", respectively).

The invention also relates to nucleic acids encoding such amino acid sequences and polypeptides (also referred to as "nucleic acids of the invention" or "nucleotide sequence of the invention"); methods of producing such amino acid sequences and polypeptides; the cells of the host expressing or capable to Express such amino acid sequences or polypeptides; to compositions, in particular pharmaceutical compositions containing such amino acid sequences, polypeptides, nucleic acids and/or cells of the owners; and to the use of such amino acid sequences or polypeptides, Amu is inovah acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as prevention, therapeutic or diagnostic purposes as described in this invention.

Other aspects, embodiments, advantages and applications of the invention will become apparent from the further description.

The level of technology

The interaction of IL-6 protein, originally identified as a factor in the differentiation of b-cells (Harada et al., 1985, Proc. Natl. Acad. Sci. USA, 82: 5490-4; EP 0257406) receptor IL-6R (Yamasaki et al., 1988, Science, 241: 825-8; EP 0325474) leads to the formation of the complex IL-6/IL-6R. This complex binds to gp130 (Taga et al., 1989, Cell, 58; 573-81; EP 0411946), membrane protein on the target cell, which transmits various physiological actions of IL-6. It is now known that IL-6 is involved, among other things, the regulation of the immune response, hematopoiesis, and acute phase response, bone metabolism, angiogenesis and inflammation. Deregulation of IL-6 entails pathological processes in certain autoimmune and chronic inflammatory proliferative diseases (Ishihara and Harada, 2002, Biochim. Biophys. Acta, 1592: 281-96). As a consequence, inhibitors induced IL-6 signaling in the past has attracted a lot of attention (Harada et al., 1990, Immunol. Today, 11: 443-9). Polypeptides that specifically bind to IL-6 (Klein et al., 1991, Blood, 78: 1198-204; EP 0312996), IL-6R (EP 0409607) or gp130 (Saito et a., 1993, J. Immunol. Methods 163: 217-223; EP 0572118), as it turned out, show a significant inhibitory effect on the functioning of IL-6.

Super-production of IL-6 and alarms (in particular, the so-called transegalitarian) involved in various diseases and disorders, such as sepsis (Stames et al., 1999, J. Immunol., 148: 1968), various forms of cancer, such as multiple myeloma (MM), renal cell carcinoma (RCC), plasmocytoma leukemia (Klein et al., 1991), lymphoma, B-lymphoproliferative disorder (BLPD) and prostate cancer. Non-restrictive examples of other diseases caused by excessive production of IL-6 or alarms include resorption of bone (osteoporosis) (Roodman et al., 1992, J. Bone Miner. Res., 7: 475-8; Jilka et al 1992, Science, 257: 88-91), cachexia (Strassman et al., 1992, J. Clin. Invest. 89: 1681-1684), psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma (Emilie et al., 1994, Int. J. Immunopharmacol. 16: 391-6), inflammatory diseases and disorders, such as rheumatoid arthritis, systemic juvenile idiopathic arthritis, hypergammaglobulinemia (Grau et al., 1990, J. Exp. Med. 172: 1505-8); Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, a disease of Castellana, IgM-gammopathy, myxoma of the heart, asthma (particularly allergic asthma) and autoimmune insulin-dependent diabetes mellitus (Campbell et al., 1991, J. Clin. Invest. 87: 739-742). Others associated with IL-6 disease is Oia must be known to specialists in this field.

As you can see, for example, from the above works, the prior art described antibodies and fragments of antibodies against IL-6 person against IL-6R person and against gp130 person for the prevention and treatment associated with IL-6 related diseases. Their examples are Tocilizumab (see Woo et al., 2005, Arthritis Res. Ther. 7: 1281-8; Nishimoto et al., 2005, Blood 106: 2627-32; Ito et al., 2004, Gastroenterology, 126: 989-96; Choy et al., 2002, Arthritis Rheum. 46: 3143-50), BE8 (see Bataille et al., 1995, Blood 86: 685-91; Emilie et al., 1994, Blood 84: 2472-9; Beck et al., 1994, N. Engl. J. Med. 330: 602-5; Wendling et al., 1993, J. Rheumatol. 20: 259-62) and CNTO-328 company Centocor (see Journal of Clinical Oncology, 2004, 22/14S: 2560; Journal of Clinical Oncology, 2004, 22/14S: 2608; Int. J. Cancer, 2004, 111:592-5). Another active element known in this area for the prevention and treatment associated with IL-6 disease is the product of the merger of Fc and soluble gp130 (see Becker et al. 2004, Immunity, 21: 491-501; Doganci et al., 2005, J. Clin. Invest. 115: 313-25; Nowell et al., 2003, J. Immunol. 171: 3202-9; Atreya et al., 2000, Nat. Med. 6: 583-8). Amino acid sequence and nanotesla directed against IL-6R, and containing polypeptides described in WO 08/020079.

The invention

Specific, but not limiting purpose of the present invention is the provision of amino acid sequences, polypeptides and therapeutic compounds and compositions having improved therapeutic and/or pharmacological properties, along with other useful properties (such, for example, is to a large ease of access and/or reduced costs of goods), compared to amino acid sequences, antibodies and antelami prior art. These improved and useful properties will become clear from the further description. Without imposing any restrictions, amino acid sequences, polypeptides and therapeutic compounds and compositions provided by the invention can have the best binding and/or affinity, best avidity, better efficiency and/or activity, higher selectivity and/or they may be able to partially or preferably completely block the interaction between IL-6/IL-6R and/or to inhibit signaling through IL-6, IL-6R and/or IL-6/IL-6R.

In General, the purpose of the invention is to provide pharmacologically active agents, as well as compositions containing them, which can be used in the diagnosis, prevention and/or treatment of one or more associated with IL-6 diseases (as defined here); and the provision of methods of diagnosis, prevention and/or treatment of such diseases and/or disorders, which include the introduction and/or use of such funds and compositions.

The present invention relates to amino acid sequences (also referred to as "amino acid sequences of the invention"), directed against and/or can specifically bind (as op is Adelino here) with the receptor, interleukin-6 (IL-6R) with better affinity and/or avidity and/or with better efficiency and/or activity and is able to (partially or preferably fully) to block communication IL-6/IL-6R and/or to inhibit the conduction of a signal through IL-6, IL-6R and/or IL-6/IL-6R. More specifically, the present invention provides amino acid sequences that contain one or several segments of amino acid residues chosen from the following:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance;

and/or

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences with the affinity measures the I by means of surface plasmon resonance;

and/or

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance.

To associate with its epitope on IL-6R amino acid sequence should usually contain within its amino acid sequence one or more amino acid residues or one or more sections amino acid residues (as defined therein; each area contains one or more amino acid residues located near each other or in close proximity to each other, i.e. in the primary or tertiary structure of the amino acid sequence) via which the amino acid sequence of the invention may be contacted with the epitope on IL-6R. These amino acid residues or regions of amino acid residues thus form the "site" for binding to the epitope on IL-6R (which is also menue is camping here as "antigennegative website", as hereinafter defined).

The present invention provides a number of plots amino acid residues (as defined therein), which are particularly suited for binding to opredelennim epitope on IL-6R. These areas amino acid residues can be in and/or can be included in the amino acid sequence of the invention, in particular in such a way that they form a (included) antigennegative site amino acid sequences of the invention. Resulting amino acid sequence associated with a specific epitope on IL-6R that lies in, forms part of or coincides with (i.e. in the primary or tertiary structure) or is in close proximity to (i.e. in the primary or tertiary structure) the binding site of IL-6 to IL-6R (for example, competition with IL-6); and the obtained amino acid sequence capable of partially or preferably completely block the interaction between IL-6/IL-6R and/or to inhibit the passage of the signal through IL-6, IL-6R and/or IL-6/IL-6R. In this regard, amino acid sequences and polypeptides of the invention are preferably such that they can compete with IL-6 for binding to the receptor of IL-6. Amino acid sequences and polypeptides of the invention are preferably such that they can compete for binding with the recipe for the rum IL-6 with commercially available reconstructed chimeric monoclonal antibody human mouse against IL-6R Tocilizumab (MRA) (Chugai/Roche) or antigen-binding fragment (for example, see WO 92/19759 and the corresponding European patent EP 0628639 and Shinkura et al., 1998, Anticancer Research 18, 1217-1222), for example, in the method described in Example 11; and/or such that they can bind to the same epitope or binding site on IL-6R that Tocilizumab, or epitope, located next to the binding site and/or overlapping with the binding site.

Also amino acid sequences of the invention are preferably such that they can compete for binding to the receptor of IL-6 with control IgG provided in SEQ ID NO's:1 and 2, and/or control Fab, shown in SEQ ID NO's:3 and 4 (see Example 1); and/or such that they can bind to the same epitope or binding site on IL-6R that of control IgG or control Fab, or epitope, located next to the binding site and/or overlapping with the binding site. About receive and sequence control IgG or control Fab should refer to the following Example 1, and SEQ ID NO's:1-4.

It should be noted that the invention in its broadest sense is not limited to a specific structural role or function that these areas amino acid residues may have in an amino acid sequence of the invention, if only these areas of amino acid residues allow the amino acid sequence and is gaining contact with a specific epitope on IL-6R with an affinity and/or force (as hereinafter defined). Thus, in General, the invention in its broadest sense comprises any amino acid sequence that can bind to specific epitope on IL-6R and which contain one or several segments of amino acid residues described in it (in particular, and suitable combinations of two or more areas of amino acid residues), which are appropriately connected to each other through one or more additional amino acid sequences so that the entire amino acid sequence forms a binding domain and/or binding unit that is capable of contact with a specific epitope on IL-6R. However, it should also be noted that the presence of only one of such areas amino acid residues in the amino acid sequence of the invention itself may be sufficient to obtain amino acid sequences of the invention, the ability to communicate with a specific epitope on IL-6R (for example, again, you can refer to so-called "expedite-fragments" described in WO 03/050531).

Amino acid sequence containing one or more of these specific areas amino acid residues, exhibit improved properties, such for example as the best binding and/or affinity, the best avidity, best e is the efficiency and the power and/or greater selectivity in addition to their ability to partially or completely block the interaction between IL-6/IL-6R and/or to inhibit the passage of the signal through IL-6, IL-6R and/or IL-6/IL-6R.

More specifically, the amino acid sequence of the invention, containing one or more of these specific areas amino acid residues, can be contacted with IL-6R with an affinity (suitably measured and/or expressed as a value To the (real or perceived), the values of KD (real or perceived), the rate constant konand/or koffor as the value IC50as described hereinafter), preferably such that they:

- contact hIL-6 with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less;

and/or such that they:

- contact cynoIL-6R with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less;

and/or such that they:

- contact hIL-6 with a rate constant konfrom 104M-1s-1up to 107M-1s-1preferably from 105M-1s-1up to 107M-1s-1more preferably about 106M-1s-1 or more;

and/or such that they:

- contact the cynoIL-6R with a rate constant kon from 10 4M-1s-1up to 107M-1s-1preferably from 105M-1s-1up to 107M-1s-1more preferably about 106M-1s-1or more;

and/or such that they:

- contact hIL-6 with a rate constant kofffrom 10-3(t½ =0,69 s) up to 10-6s-1(giving almost irreversible complex with t½ in a few days), preferably between 10-4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less;

and/or such that they:

- contact cynoIL-6R with a rate constant kofffrom 10-3(t½=0,69 s) up to 10-6s-1(giving almost irreversible complex with a t½ in a few days), preferably between 10-4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less.

Some preferred values IC50for binding of the amino acid sequences of the invention to IL-6R will become apparent from the further description and the examples.

For example, when defining a method, TF-1, described by Kitamura et al. (1989, J. Cell PhysioL, 140: 323), the amino acid sequence of the invention may have IC50 values (at 100 ME IL-6/ml) between 10 nm and 50 PM, preferably m is wait 5 nm and 50 PM more preferably between 1 nm and 50 PM or less, such as about 750 or 500 PM or less. When defining this method TF-1 amino acid sequences of the invention may have IC50 values (5,000 IU of IL-6/ml) between 50 nm and 1 nm, preferably between 25 nm and 1 nm, more preferably between 10 nm and 1 nm or less, such as about 8 nm or less. When defining this method TF-1 amino acid sequences of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50received control IgG provided in SEQ ID NO:1 and 2, or control Fab, shown in SEQ ID NO:3 and 4 (see Example 1). When defining this method TF-1 amino acid sequences of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50obtained for the Tocilizumab (MRA).

In the determination method of the activity in the plasma when the values of Esso for IL-6 (e.g., in the presence of 27,29 ng IL-6/ml, as described the in Example 45) the amino acid sequence of the invention can have values IC 50between 500 PM and 50 PM, preferably between 250 PM and 50 PM, more preferably between 200 PM and 50 PM or less, such as 150 PM or less. In the determination method of the activity in the plasma when the values AS for IL-6 (e.g., in the presence 885 ng IL-6/ml as described in Example 45) the amino acid sequence of the invention can have values IC50between 1000 PM and 100 PM, preferably between 750 PM and Yopm, more preferably between 500 PM and 100 PM or less, 400 PM or less. When defining this method activity in plasma amino acid sequences of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50received control IgG provided in SEQ ID NO:1 and 2, or control Fab, shown in SEQ ID NO:3 and 4 (see Example 1). When defining this method activity in plasma amino acid sequences of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times sravnenie the value IC 50obtained for the Tocilizumab (MRA).

When defining a method binding with membrane IL-6R on the cell SNO amino acid sequences of the invention can have values IC50between 10 nm and 100 PM, preferably between 5 nm and 100 PM, more preferably between 2 nm and 10 PM or less, such as 2 nm or less.

In a preferred aspect, the amino acid sequence of the invention may contain two or more parcels of amino acid residues chosen from the following:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance; and/or

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, approximately the same or greater affinity compared to the amino acid sequence, contains the plot of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance; and/or

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance;

that (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to a) or b), the second stretch of amino acid residues corresponds to one of the amino acid sequence according to (C), (d), (e) or (f); (I) when the first stretch of amino acid residues corresponds to one of the amino acid sequence according to (C) or (d), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), e) or f); or (iii) when the first stretch of amino acid residues corresponds to one of the am is nekisnotnice sequences according to e) or f), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), C) or d);

Even more preferably the amino acid sequence of the invention contain three or more parcels amino acid residues, with the first stretch of amino acid residues selected from the group consisting of:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance;

the second stretch of amino acid residues selected from the group consisting of:

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to amino the PCI-e slot sequence, contains the plot of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance; and

the third stretch of amino acid residues selected from the group consisting of:

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance.

It should be noted that the invention is not limited by the origin of the amino acid sequence of the invention (or of the nucleotide sequence of the invention, used for its expression), nor how (was) created or obtained amino acid sequence or nucleotide sequence of the invention. Thus, the amino acid sequences of the invention can be a natural amino acid sequence (from any suitable species) or a synthetic or Polushin eticheskie amino acid sequence.

In one specific, but not restrictive aspect, the amino acid sequence of the invention may constitute such amino acid sequence, which contains immunoglobulin fold, or such amino acid sequence, which under certain conditions (such as physiological conditions) is able to form an immunoglobulin fold (e.g., by stacking). You can refer, among other things, to review Halaby et al. (1999, J. Protein Eng. 12: 563-71). Preferably the proper installation with the formation of immunoglobulin folds plots amino acid residues may properly form the antigen-binding site to bind a specific epitope on IL-6R or more preferably able to communicate with its epitope on IL-6R with an affinity (suitably measured and/or expressed as a value To the (real or perceived), the values of KA(real or perceived), the rate constant konand/or koffor as the value IC50as described hereinafter), which is defined in the present invention.

In another specific, but not restrictive aspect, the amino acid sequences of the invention are immunoglobulinemia sequence. In particular, but without limitation, amino acid n the coherence of the invention can be an amino acid sequence, which mainly consist of frame 4 plots (FR1 to FR4 respectively) and 3 complementarity determining areas (CDR1 to CDR3 respectively); or any suitable fragments of such amino acid sequences that still bind a specific epitope on IL-6R.

In such amino acid sequences of the invention the frame sequence can be any frame of the sequence, and examples of suitable frame sequences must be known in the art, for example, from standard references and further discussion and these works prior art.

Frame sequence preferably represent a frame sequence (suitable combinations thereof) of immunoglobulins or frame sequences obtained from the frame sequences of immunoglobulins (for example, by optimizing the sequence type of humanization or kanalizacji). For example, the frame sequence can be a frame sequence, originating from the variable domain of the light chain (e.g., sequence VLand/or variable domain of the heavy chain (e.g., sequence VH). In the case when the amino acid sequence of the invention t is aetsa the sequence of the variable domain of the heavy chain, it can represent the sequence of the variable domain of the heavy chain, originating from normal chetyrekhzvezdochnogo antibodies (type sequence VHoriginating from human antibodies), or the so-called sequence vHH(as hereinafter defined), originating from the so-called "antibody heavy chain" (as hereinafter defined). In one particularly preferred aspect, the frame sequence are either frame sequences that were obtained from the vhh sequence (in which the data frame of the sequence optionally can be partially or fully humanitarian) or normal sequence VHthat had been subjected to kanalizacji (as hereinafter defined).

For a General description of the antibody heavy chain and variable domains should address, inter alia, to refer here to the works of the prior art, and the work of the prior art set out on page 59 of WO 08/020079, and bibliography, see pages 41-43 of the International application WO 06/040153, and and references are prior art are included in the present invention by reference.

Amino acid sequence of the invention, in particular, may be a domain antibody (or an amino acid on sledovatelnot, suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb" (or an amino acid sequence suitable for use as a dAb) or nanotesla (as defined here, including, but not limited to the sequence VHH); Other single variable domains, or any suitable fragments of any of them.

In particular, the amino acid sequence of the invention can be nanotesla (Nanobody®) (as defined here), or any suitable fragment. [Note: Nanobody®(napothera), Nanobodies®(napothera) and Nanoclone®(nanoclay) are registered trademarks of Ablynx N.V.] Such nanotesla directed against IL-6R, also will be referred to here as "nanotesla of the invention".

In General, nanotesla can be defined as the amino acid sequence of the (General) structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4,

where FR1-FR4 mean frame sections 1 to 4, respectively, and CDR1-CDR3 mean areas of complementarity from 1 to 3, respectively, and one or more Hallmark residues defined in WO 08/020079 (table a-3 to a-8).

In General, nanotesla (in particular the sequence VHHand partially humanized Manotel is), in particular, can be characterized by the presence of one or more "Hallmark residues' in one or more frame sequences (e.g., as described in WO 08/020079, S. 61, line 24, to the S. 98, line 3).

In this respect, the amino acid sequences of the invention can basically be composed of 4 frame sections (FR1-FR4 respectively) and 3 phase determining complementarity (CDR1-CDR3, respectively), with:

- CDR1 selected from the group consisting of:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance; and/or

- CDR2 selected from the group consisting of:

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:84-91, provided that the amino acid sequence containing such site amino acid residues, with azeveda to IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance; and/or

- CDR3 selected from the group consisting of:

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance.

These preferred areas of complementarity (CDR1-CDR3, respectively), also referred to as "CDR(s) of the invention".

Preferably, the amino acid sequence of the invention essentially consist of 4 frame sections (FR1-FR4 respectively) and 3 complementarity determining plots (plots (CDR1-CDR3, respectively), with:

- CDR1 selected from the group consisting of:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, prefer the LNO no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance; and

- CDR2 selected from the group consisting of:

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance; and

- CDR3 selected from the group consisting of:

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the m is the same or greater affinity compared to the amino acid sequence, contains the plot of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance.

Such nanotesla can be obtained in any suitable manner and from any suitable source and may constitute, for example, natural sequence VHH(from any suitable species of Camelid) or synthetic or semi-synthetic amino acid sequence.

In one specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:80 or stretch of amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:80, provided that the amino acid sequence or nanotesla containing such site amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence or nanotesla containing the plot of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance.

In another specific aspect, the amino acid sequence or nanotesla of the invention contains at least a stretch of amino acid residues chosen from SEQ ID NO's:84, 89 or 91 or stretch of amino acid residues, having no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80, 89 or 91, provided that the amino acid sequence or nanotesla containing such site amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence or nanotesla containing the plot of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:84 or stretch of amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:84, provided that the amino acid sequence or nanotesla containing such site amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence or nanotesla containing the plot of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention contains at least Uch the drain amino acid residues, selected from SEQ ID NO's:93-94, or plot of amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-94, provided that the amino acid sequence or nanotesla containing such site amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence or nanotesla containing the plot of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:93 or stretch of amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:93, provided that the amino acid sequence or nanotesla containing such site amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence or nanotesla containing the plot of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance.

The following specific aspect, the amino acid sequentially the th or nanotesla of the invention comprises at least SEQ ID NO:80 and SEQ ID NO:84.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:80 and SEQ ID NO:93.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:84 and SEQ ID NO:93.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:80, SEQ ID NO:84 and SEQ ID NO:93.

Other preferred combinations of sequences of CDR1, CDR2 and CDR3 are also presented in table.A-1.

Preferred amino acid sequences of the invention can be selected from the group consisting of SEQ ID NO's:60-69; sequences that have no more than 2, preferably no more than 1 amino acid difference in one, two or all parts of the CDR of one of SEQ ID NO''s:60-69, provided that the amino acid sequence of no more than 2, preferably no more than 1 amino acid difference in one, two or all areas CDR binds to IL-6R with the same, about the same or greater affinity compared to the binding of one of SEQ ID NO''s:60-69, and affinity is measured by means of surface plasma resonance; and sequences that have no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:60-69, provided that aminomalononitrile not more than 2, preferably no more than 1 amino acid difference with one of SEQ ID NO's:60-69 binds to IL-6R with the same, about the same or greater affinity compared to the binding of one of SEQ ID NO''s:60-69, and affinity is measured by means of surface plasma resonance.

Such amino acid sequences of the invention preferably should be able to specifically bind with a specific epitope on IL-6R, and even more preferably capable of contact with a specific epitope on IL-6R with an affinity (suitably measured and/or expressed in the form of values of KD(real or perceived), the values of KA(real or perceived), the rate constant konand/or koffor as the value IC50as described hereinafter), which is defined in the present invention. Such amino acid sequences of the invention preferably should also have cellular activity and activity in the plasma, as defined in the present invention.

Amino acid sequence and nanotesla provided by the invention, preferably are substantially allocated (as defined here), or form part of a protein or polypeptide of the invention (also referred to as "polypeptides of the invention" or "proteins of the invention"), which may contain or generally composed of one or more amino acid sequences or of Manotel of the invention and optional further comprise one or more additional amino acid sequences or nantel (all of which are optionally linked via one or more suitable linkers).

Accordingly, in another aspect, the invention relates to compounds or constructs, and in particular proteins or polypeptides (also referred to as "compounds of the invention or polypeptides of the invention", respectively), which contain or consist mainly of one or more amino acid sequences or of Manotel of the invention (or suitable fragments), and optionally also contain one or more other groups, residues, molecules or units of binding. As will become clear from the further discussion, these additional groups, residues, molecules, binding unit or amino acid sequences can also provide additional functionality to the amino acid sequence of the invention (or compounds, constructs or polypeptide in which they are present), or may modify the properties of the amino acid sequence or nanotesla of the invention.

For example, such additional groups, residues, molecules or binding unit can be one or more additional amino acid sequences that connection, design or polypeptide is (drained) protein or (merged) the polyp is predom. In a preferred, but not restrictive aspect of such one or more other groups, residues, molecules or binding units are sequences of immunoglobulins. Even more preferably, these one or more other groups, residues, molecules or binding units are chosen from the group consisting of domain antibodies, amino acid sequences that are suitable for use as domain antibodies, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb", amino acid sequences that are suitable for use as a dAb, or nantel.

On the other hand, such groups, residues, molecules or binding unit can represent, for example, chemical groups, residues, molecules, which themselves may not be biologically and/or pharmacologically active. For example, and without limitation, such groups can be associated with one or more amino acid sequences or antelami invention therefore, to provide a "derived" amino acid sequence or polypeptide of the invention, as described below.

In the scope of the present invention also includes compounds, constructs or polypeptides containing or consisting mainly of single and multiple derivatives, as described herein, and optionally also containing one or more other groups, residues, molecules or units of the binding, not necessarily connected via one or more linkers. Preferably the data one or more other groups, residues, molecules or binding units are amino acid sequences.

In the compounds, constructs or polypeptides described above, one or more amino acid sequences or of Manotel of the invention and one or more groups, residues, molecules or binding units may be directly connected to each other and/or via one or more suitable linkers or spacers. For example, when one or more groups, residues, molecules or binding units are amino acid sequences, linkers can also be amino acid sequences, so that the resulting connection, design or polypeptide is fused protein or fused with polypeptide.

The process of designing/selecting and/or obtaining a compound or polypeptide of the invention on the basis of amino acid sequence or nanotesla of the invention, also referred to here as the "formatting" section of this amino acid sequence or nanotesla of the invention; and amino acid sequence or nanotesla izaberete the Oia, which form part of the compound or polypeptide of the invention, are considered to be '^rich" or sold "in the format of this compound or polypeptide of the invention. Examples of ways in which the amino acid sequence or nanotesla of the invention can be formatted, and examples of such formats will be clear to experts from the further discussion; and such formatted amino acid sequence or nanotesla constitute a further aspect of the invention.

For example, and without limitation, one or more amino acid sequences or of Manotel of the invention can be used as units of binding in such a protein or polypeptide, which may optionally contain one or more additional amino acid sequences that serve as binding units (i.e., against another epitope on IL-6R and/or against one or more other antigens, proteins or targets other than IL-6R) in order to obtain a monovalent, multivalent, multiperiodicity or multispecific polypeptide of the invention, respectively, which are all described here. Thus, the present invention also relates to compounds, constructs or polypeptides, which represents a monovalent constructs containing or consisting mainly of and inoculates sequence or nanotesla invention. The present invention thus also relates to compounds, constructs or polypeptides representing multivalent constructs, such, e.g., as the divalent or trivalent design. The present invention also relates to compounds, constructs or polypeptides representing multispecific constructs or polypeptides, such, e.g., as bispecific or trapezitinae design. The present invention also relates to compounds, constructs or polypeptides representing multipartitions design, such, e.g., as bipartites or tripartitions design.

In one specific aspect of the invention, the compounds or polypeptides of the invention may have a longer half-life compared with the corresponding amino acid sequence or nanotesla invention. Some preferred, but not restrictive examples of such compounds and polypeptides will become clear to the specialists from the further statement, and they are, for example, comprise amino acid sequences, nanotesla or polypeptides of the invention that have been chemically modified to increase their half-life (e.g., via Paglierani); amino acid sequences or napothera of the invention, containing at least Odie is an additional binding site protein serum (type serum albumin); or polypeptides of the invention containing at least one amino acid sequence or nanotesla of the invention, connected to at least one molecule (in particular, with at least one amino acid sequence) that increases the half-life of the amino acid sequence or nanotesla invention. Examples of polypeptides, amino acid sequences or of Manotel of the invention containing such increases the half-life of the molecule will be clear to experts from the further discussion; for example, they include, without limitation, polypeptides in which one or more amino acid sequences or of Manotel invention accordingly connected to one or more serum proteins or their fragments (type serum albumin (human) or suitable fragments) or with one or more binding units (such, for example, as domain antibodies, amino acid sequences that are suitable for use as domain antibodies, single domain antibodies, amino acid sequences suitable for use as a single domain antibody, "dAb", amino acid sequences that are suitable for use as a dAb, or nanotesla), are able to bind with serum proteins is, such as serum albumin (type serum human albumin), serum immunoglobulins IgG or transferrin; we shall refer to the further description and therein references); polypeptides that have amino acid sequence or nanotesla invention connects to the Fc-fragment (type Fc man) or a suitable part or fragment; or polypeptides in which one or more amino acid sequences or of Manotel invention suitably connected with one or more small proteins or peptides which are able to bind with serum proteins (including proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489).

In General, compounds or polypeptides of the invention with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times greater than the half-life of the corresponding amino acid sequence or nanotesla of the invention itself.

In a preferred, but not restrictive aspect of such compounds or polypeptides of the invention have a half-life in serum, which increased by more than 1 hours, preferably more than 2 hours, more predpochtitelnye than b hours as more than 12 hours or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence or nanotesla inventions themselves.

In another preferred, but not restrictive aspect of such compounds or polypeptides of the invention exhibit a half-life in human serum for at least 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half-life of at least 5 days (such as from 5 to 10 days), preferably at least 9 days (such as from 9 to 14 days), more preferably at least 10 days (such as about 10 to 15 days) or at least 11 days (such as from 11 to 16 days), more preferably at least 12 days (such as from 12 to 18 days or more) or more than 14 days (such as from 14 to 19 days).

Such proteins, polypeptides, compounds or constructs can also be significantly highlighted (as defined here).

Some preferred compounds, constructs or polypeptides of the invention include the following polypeptide sequence:

a) SEQ ID NO's:70-72;

b) a polypeptide sequence having no more than 2, preferably no more than 1 linakis is now the differences in one, two or all parts of the CDR of the invention from one of SEQ ID NO''s:70-72, provided that the polypeptide sequence of not more than 2, preferably no more than 1 amino acid difference in one, two or all parts of the CDR of the invention binds to IL-6R with the same, about the same or greater affinity compared to the binding of one of SEQ ID NO''s:70-72, and the affinity is measured by means of surface plasma resonance;

c) a polypeptide sequence having no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:70-72, provided that the polypeptide sequence of not more than 2, preferably no more than 1 amino acid difference with one of SEQ ID NO's:70-72 binds to IL-6R with the same, about the same or greater affinity compared to the binding of one of SEQ ID NO''s:70-72, and the affinity is measured by means of surface plasmon resonance.

Polypeptides with these sequences show a preferential property for use as pharmacologically active agents, such, e.g., as the good characteristics of the binding (high affinity and/or avidity), high efficiency and/or activity, along with the ability (partially or fully) to block the interaction between IL-6/IL-6R and/or to inhibit the conduction of a signal through IL-6, IL-6R and/or the comp is CEN IL-6/IL-6R.

More preferably, these polypeptides and compounds of the invention can bind to IL-6R with an affinity (suitably measured and/or expressed in the form of values of KD(real or perceived), the values of KA(real or perceived), the rate constant konand/or koffor as the value IC50as described hereinafter), preferably such that they:

- contact hIL-6R with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less;

and/or such that they:

- contact cynoIL-6R with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less;

and/or such that they:

- contact hIL-6R with a rate constant konfrom 104M-1s-1up to 107M-1s-1preferably from 105M-1s-1up to 107M-1s-1more preferably about 106M-1s-1or more;

and/or such that they:

- contact cynoIL-6R with a rate constant konfrom 104M-1s-1up to 107M-1s-1before occhialino from 10 5M-1s-1up to 107M-1s-1more preferably about 106M-1s-1or more;

and/or such that they:

- contact hIL-6R with a rate constant kofffrom 10-3(t½=0,69 s) up to 10-6s-1(giving almost irreversible complex with t½ in a few days), preferably between 10-4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less;

and/or such that they:

- contact cynoIL-6R with a rate constant kofffrom 10-3(t½=0,69 s) up to 10-6s-1(giving almost irreversible complex with t½ in a few days), preferably between 10-4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less.

Some preferred values IC50for binding of the polypeptides and compounds of the invention to IL-6R will become apparent from the further description and the examples.

For example, when defining a method, TF-1, described by Kitamura et al. (1989, J. Cell PhysioL, 140: 323), the polypeptides and compounds of the invention can have values IC50(ME at 100 IL-6/ml) between 10 nm and 50 PM, preferably between 5 nm and 50 PM, more preferably between 1 nm and 50 PM or less, such as about 750 or 500 PM Il is less. When defining this method TF-1 polypeptides and compounds of the invention may have IC50 values (5,000 IU of IL-6/ml) between 50 nm and 1 nm, preferably between 25 nm and 1 nm, more preferably between 10 nm and 1 nm or less, such as about 8 nm or less. When defining this method TF-1 polypeptides and compounds of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50received control IgG provided in SEQ ID NO:1 and 2, or control Fab, shown in SEQ ID NO:3 and 4 (see Example 1). When defining this method TF-1 amino acid sequences of the invention may have IC50 values that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50" received for Tocilizwnab (MRA).

In the determination method of the activity in the plasma when the values of the EU50for IL-6 (e.g., in the presence of 27,29 ng IL-6/ml as described in Example 45) polypeptides and compounds of the invention can have values IC50between 500 PM and 50 PM, before occhialino between 250 PM and 50 PM more preferably between 200 PM and 50 PM or less, such as 150 PM or less. In the determination method of the activity in the plasma when the values AS for IL-6 (e.g., in the presence 885 ng IL-6/ml as described in Example 45) polypeptides and compounds of the invention may have IC50 values between 1000 PM and 100 PM, preferably between 750 PM and 100 PM, more preferably between 500 PM and 100 PM or less, 400 PM or less. When defining this method activity in plasma polypeptides and compounds of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50received control IgG provided in SEQ ID NO:1 and 2, or control Fab, shown in SEQ ID NO:3 and 4 (see Example 1). When defining this method activity in plasma polypeptides and compounds of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50obtained for the Tocilizumab (MRA).

When defining a method of binding a meme is an early IL-6R on the cell SNO polypeptides and compounds of the invention can have values IC 50between 10 nm and 100 PM, preferably between 5 nm and 100 PM, more preferably between 2 nm and 10 PM or less, such as 2 nm or less.

In one specific aspect, polypeptides, compounds or constructs of the invention essentially consist of the amino acid sequence SEQ ID NO:70.

In another specific aspect, polypeptides, compounds or constructs of the invention essentially consist of the amino acid sequence SEQ ID NO:71.

The compounds or polypeptides of the invention can generally be obtained by a method that includes at least the stage of accession accordingly to one or more amino acid sequences, nanocell or monovalent constructs of the invention one or more other groups, residues, molecules or binding units, optionally via one or more suitable linkers, thus obtaining the compound or polypeptide of the invention. The polypeptides of the invention can also be obtained by the process, which typically includes at least the stage of obtaining a nucleic acid that encodes a polypeptide of the invention, ekspressirovali this nucleic acid in an appropriate manner and highlight the expressed polypeptide of the invention. Such methods can be known in itself by the way that should be clear specifications the sheets, for example, based on the methods described below.

Accordingly, the present invention also concerns the application of amino acid sequences of Manotel or monovalent constructs of the invention when receiving multivalent compounds, constructs or polypeptides. The way to obtain multivalent compounds, constructs or polypeptides includes adherence to the amino acid sequence, nanotesla or monovalent constructs of the invention at least one other group, residue, molecule or binding unit, optionally via one or more linkers.

In General, when the amino acid sequence or nanotesla of the invention (either containing compound, structure or polypeptide) is intended for administration to a subject (for example for therapeutic and/or diagnostic purposes as described herein), preferably either amino acid sequence or nanotesla does not occur naturally in a given subject; and, if they occur in a given subject in a natural way, in substantially the selected form (as defined here).

Amino acid sequence, nanotesla, polypeptides and compounds of the invention are directed against IL-6R person. However, they should preferably be cross what aktivnosti to IL-6R long-tailed macaques (cynomolgus monkey, Macaca fascicularis), which means that these amino acid sequences, nanotesla, polypeptides and compounds directed against (as defined here) and/or can specifically bind (as defined here) to IL-6R long-tailed macaques (Masasa fascicularis). Such cross-reactivity may have advantages from the point of view of drug development, as it gives the opportunity to test amino acid sequence, nanotesla, polypeptides and compounds against IL-6R man on the model of the disease in long-tailed macaques.

Amino acid sequence or nanotesla of the invention (as well as the containing compounds, constructs or polypeptides) has cross-reactivity to IL-6R person and long-tailed macaques means that the amino acid sequence or nanotesla of the invention (as well as the containing compounds, constructs or polypeptides) binds to IL-6R long-tailed macaques with an affinity (suitably measured and/or expressed in the form of values of KD(real or perceived), the values of KD (real or perceived), the rate constant konand/or koffor as the value IC50as described hereinafter), which is the same or will be at least 70% (preferably at least 80%, more predpochtitel is about not less than 90%, even more preferably at least 95%) of the affinity with which a given amino acid sequence or nanotesla of the invention (as well as the containing compounds, constructs or polypeptides) binds to IL-6R person. About the sequence of IL-6R and the corresponding sequence of cDNA long-tailed macaques can also refer to WO 09/010539 filed by Ablynx N. V. July 16, 2008, entitled "Receptor for interleukin-6 (IL-6) from Masasa fascicularis"; see SEQ ID NO:3 and Fig.1B about the cDNA sequence and SEQ ID NO:4 and Fig.3B about amino acid sequences.

Also in the scope of the present invention includes use of parts, fragments, analogs, mutants, variants, alleles and/or derivatives of the amino acid sequences or polypeptides according to the invention, and/or proteins or polypeptides containing or consisting essentially of one or more of such parts, fragments, analogs, mutants, variants, alleles and/or derivatives, as long as they can be applied as provided herein. Such parts, fragments, analogs, mutants, variants, alleles and/or derivatives will usually contain (at least part) functional antigennegative site for binding with a specific epitope on IL-6R, and more preferably they are able to specifically bind a specific epitope on L-6R, even more preferably they are able to bind a specific epitope on IL-6R with an affinity (suitably measured and/or expressed in the form of values of KD(real or perceived), the values of KA(real or perceived), the rate constant konand/or koffor as the value IC50as described hereinafter), which is defined in the present invention. Such parts, fragments, analogs, mutants, variants, alleles and/or derivatives preferably should also have cellular activity and activity in the plasma, as defined in the present invention. Some non-limiting examples of such parts, fragments, analogs, mutants, variants, alleles, derivatives of proteins or polypeptides will be clear to a person skilled from the further discussion. Additional fragments or polypeptides according to the invention can be obtained with the aid of a suitable combination (for example, by genetic fusion) one or more (smaller) parts or fragments described here.

In another aspect, the invention also relates to nucleic acids or nucleotide sequences coding for these amino acid sequences according to the invention, nanotesla according to the invention, the polypeptides of the invention (or fragments). Such nucleic acid is also referred to as "nucleic acids of the invention and can be in the form of genetic design as further disclosed herein. Accordingly, the present invention also relates to nucleic acids or nucleotide sequences are in the form of a genetic construct.

Nucleic acids of the invention can be of natural origin, synthetic or semi-synthetic, and may, for example, be sequences which are selected by PCR with a matrix of a suitable natural source (e.g., DNA or RNA isolated from cells), nucleotide sequences selected from the library (in particular from expression libraries), nucleotide sequences obtained by introducing mutations in the natural sequence (using any known suitable techniques, such as PCR errors), nucleotide sequences were obtained by PCR with overlapping primers, or sequences which were obtained using a known per se technologies of DNA synthesis.

In another aspect, the invention also applies to the owner or the cell master that Express (or capable of expressionate in suitable conditions) amino acid sequence according to the invention and/or nanotesla according to the invention and/or the polypeptide according to the invention; and/or which contain the nucleic acid according invented the Y. Some preferred, but non-limiting examples of such hosts or host cells will become apparent from the further description.

Further, the invention also relates to a product or composition containing or comprising at least one amino acid sequence according to the invention (or fragment), at least one nanotesla according to the invention, at least one polypeptide according to the invention, at least one compound or construct according to the invention, at least one monovalent construct according to the invention and/or at least one nucleic acid according to the invention and, optionally, one or more additional components of such compositions known per se, i.e. selected depending on the purpose of this composition. This product or the composition may be a pharmaceutical composition (as described here), the veterinary composition, product, or composition for diagnostic use (also described here). Some preferred versions of such products or compositions will become apparent from the further description.

The present invention also relates to a method of obtaining amino acid sequences of nantel, polypeptides, nucleic acids, host cells, products and compositions described here. The method of obtaining amino acid members is of telestai, nantel, polypeptides and/or monovalent constructs of the invention may include the following stages:

a) expressiona in suitable cells-the owners or the body of the host or in another suitable expression system of the nucleic acid according to the invention or the genetic constructs according to the invention, after which not necessarily must:

b) isolation and/or purification of resulting amino acid sequence, nanotesla, polypeptide or monovalent constructs according to the invention.

The method of obtaining amino acid sequences of nantel, polypeptides or monovalent constructs of the invention may include the following stages:

- cultivation and/or content host or host cell of the invention under such conditions that the host or a host cell of the invention expresses and/or produces at least one amino acid sequence, nanotesla, polypeptide, or monovalent construct according to the invention; and then optionally follows:

- isolation and purification of resulting amino acid sequence, nanotesla, polypeptide or monovalent constructs according to the invention.

Further, the invention relates to the application and use of amino acid sequences, polypeptides, compounds, constructs, nucleic acids, to etoc hosts products and compositions described herein, as well as to methods of preventing and/or treating disorders or diseases associated with IL-6R. Some preferred, but not limiting, examples of the application or use will become apparent from the further description.

Amino acid sequence, nanotesla, polypeptides, compounds, constructs and compositions of the present invention, in General, can be used to modulate, and in particular inhibit and/or prevent, binding of IL-6R to IL-6, and subsequent binding of the complex IL-6/IL-6R with gp130, thus modulate and in particular inhibit and/or prevent signalizovania induced IL-6, IL-6R, IL-6/IL-6R and/or gp130, that modulates the biological pathway involving IL-6, IL-6R, IL-6/IL-6R and/or gp130, and/or modulate the biological mechanisms, responses and/or effects of signalizovania in these ways.

In one aspect the present invention provides amino acid sequences, nanotesla, polypeptides, designs or compounds which are and/or which can be used as antagonists of IL-6R antagonists of signalizovania mediated IL-6R and/or mechanisms of biological pathways, reactions and/or effects involving IL-6R and/or signalizovania mediated IL-6R.

In this respect, the amino acid placentas the work, nanotesla, polypeptides, compounds of structure or composition of the present invention are such that they (a) specifically bind (as defined herein) with the receptor of IL-6; and (B) capable of downward regulation of the receptor for IL-6 and/or to the inhibition, reduction or downward regulation signalizovania receptor of IL-6 and/or pathways, mechanism(s), or signalizovania are involved in IL-6 or IL-6R. As it is clear to the specialist, such amino acid sequence, apatela, polypeptide, design or connection can, in General, to be used as antagonists of IL-6 receptor, IL-6 and/or biological pathways, mechanisms, and/or effects involving signalizovania-mediated IL-6, IL-6R and/or IL-6/IL-6R. Any such reduction or downward regulation (which may be a reduction in the relevant parameter for at least 1%, at least 5%, at least 10%, or more than 10%, or up to 50% or 100% of the value of the same parameter under the conditions, when the amino acid sequence, nanotesla, polypeptide, designs or the connection is not associated with the receptor IL-6), can be measured by any suitable method known per se, for example, this may be one of the methods described below and/or in the experimental part and/or mentioned here.

More konkretnou addition to the aforementioned properties (a) and (b), such antagonistic amino acid sequence, nanotesla, polypeptide, compound or construct associated with IL-6 in such a way that (C) is blocked, inhibited or reduced binding of IL-6 with IL-6R, compared with the way binds IL-6 with its receptor in the absence of amino acid sequence, nanotesla or polypeptide according to the invention.

Without limitation, such antagonistic amino acid sequences, nanotesla, polypeptides, designs or compounds can be contacted with a specific epitope on IL-6R in the vicinity of the binding site of IL-6 to IL-6R.

In addition to the above (a) (b) and (C) such antagonistic amino acid sequences or polypeptides of the invention can bind to IL-6R (for example, which is present in the complex IL-6/IL-6R) so that it (d) inhibits or prevents (e.g., fully or partially violates) the formation of the complex IL-6/IL-6R, so that decreases the binding of the complex, i.e., its affinity to, gp130 (or Vice versa, decreases the binding of gp130 to the complex, e.g., his affinity to it) that is modulated (e.g., decreases) signalizovania induced/caused by binding of the complex to gp130, compared with the formation of the complex and its binding to gp130 in the absence of amino acid members is Telenesti, nanotesla, polypeptide, compound or construct of the invention.

Amino acid sequence, nanotesla, polypeptides, designs, compounds or compositions of the invention also preferably (but without limitation) can be reduced (i.e., at least 1%, at least 10%, preferably at least 30%, more preferably at least 50%, even more preferably at least 75% or more) or completely inhibit the induction of C-reactive protein (CRP) in mammals (e.g., in humans or in a suitable animal model of inflammation, such as long-tailed macaques) when it is introduced this mammal in the appropriate therapeutic amount compared to mammals, which were not introduced amino acid sequence, nanotesla, polypeptide, compound, structure or composition of the invention.

Amino acid sequence, nanotesla, polypeptides, designs, compounds or compositions of the invention also preferably (but without limitation) such that they reduce (i.e., at least 1%, at least 10%, preferably at least 30%, more preferably at least 50%, even more preferably at least 75% or more) or completely inhibit the induction of the increase in the number of platelets in mammals (for example the person or in a suitable animal model of inflammation, such as long-tailed macaques) when it is introduced this mammal in the appropriate therapeutic amount compared to mammals, which were not introduced amino acid sequence, nanotesla, polypeptide, compound, structure or composition of the invention.

Amino acid sequence, nanotesla, polypeptides, compounds, constructs or compositions of the invention also preferably (but without limitation) such that they reduce (i.e., at least 1%, at least 10%, preferably at least 30%, more preferably at least 50%, even more preferably mo at least 75% or more) or completely inhibit the induction of fibrinogen in mammals (for example, in humans or in a suitable animal model of inflammation, such as long-tailed macaques) when it is introduced this mammal in the appropriate therapeutic amount compared to mammals, which were not introduced amino acid sequence, nanotesla, polypeptide, compound, structure or composition of the invention.

Accordingly, amino acid sequences, polypeptides, compounds, constructs or compositions of the invention can be used for the prevention and/or treatment of diseases and/or disorders associated with IL-6, IL-6R, IL-6/IL-6R (and n is necessarily with further complex with gp130), and/or from the signal path (paths), and/or biological function or reactions involving IL-6, IL-6R and/or IL-6/IL-6R (and optionally with further complex with gp130), in particular for the prophylaxis and/or treatment of diseases and/or disorders associated with IL-6, IL-6R, IL-6/IL-6R (and optionally with further complex with gp130), and/or from the signal path (paths), and/or their biological function or reactions involving IL-6, IL-6R and/or IL-6/IL-6R (and optionally with further complex with gp130), which are characterized by excessive or unwanted signalizovania mediated IL-6R or by (paths), in which he is involved. Examples of such diseases and/or disorders associated with IL-6, IL-6R, IL-6/IL-6R, and/or from the signal path (paths), and/or biological function and responses involving IL-6, IL-6R and/or IL-6/IL-6R will be clear to the person skilled in the art based on the information from the description above, and for example, include the following disorders or disorders: sepsis (Stames et al., 1999, J. ImmunoL, 148: 1968), various forms of cancer, such as multiple myeloma (MM), renal cell carcinoma (RCC), plasmocytoma leukemia (Klein et al., 1991), lymphoma, B-lymphoproliferative disorder (BLPD) and prostate cancer. Non-restrictive examples of other diseases caused by excessive production of IL-6 or C is alizirovannaya, include resorption of bone (osteoporosis) (Roodman et al., 1992, J. Bone Miner. Res., 7: 475-8; Jilka et al., 1992, Science, 257: 88-91), cachexia (Strassman et al., 1992, J. Clin. Invest. 89: 1681-1684), psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma (Emilie et al., 1994, Int. J. Immunopharmacol. 16: 391-6), inflammatory diseases and disorders, such as rheumatoid arthritis, systemic juvenile idiopathic arthritis, hypergammaglobulinemia (Grau et al., 1990, J. Exp. Med. 172: 1505-8); Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, a disease of Castellana, IgM-gammopathy, myxoma of the heart, asthma (particularly allergic asthma) and autoimmune insulin-dependent diabetes mellitus (Campbell et al., 1991, J. Clin. Invest. 87: 739-742). Others associated with IL-6 disease must be known specialist in this field. Such diseases and disorders are referred to in this description as "diseases and disorders associated with IL-6R".

Thus, and without limitation, amino acid sequence, nanotesla, polypeptides, designs, compounds or compositions of the invention can, for example, be used for the prevention and/or treatment of all diseases and disorders which are currently being prevented or treated with active active substances, which modulate signalizovania mediated IL-6R, for example, it can be metabolean, which are mentioned above. Also envisioned that amino acid sequence, nanotesla, polypeptides, designs, compounds or compositions of the invention can be used for the prevention and/or treatment of all diseases and disorders for which treatment with the use of such active substances currently being developed, proposed or will be proposed or developed in the future. Additionally it is envisaged that due to the exercise of their active properties, which are described later, amino acid sequence, nanotesla, polypeptides, designs, compounds or compositions of the invention can be used for the prevention and/or treatment of other diseases or disorders, in the treatment contain such active ingredients or their use is contemplated or is under development, and/or amino acid sequence, nanotesla, polypeptides, designs, compounds or compositions of the invention can provide new methods and treatment of the diseases or disorders described herein.

Accordingly, the present invention also relates to a method for preventing and/or treating at least one disease or disorder, the development of which can be prevented or which Leche is tsya introduction to the needy in the subject amino acid sequence, nanotesla, polypeptide, or monovalent constructs of the invention, the method includes an introduction to the needy in the subject of pharmaceutically active amount of at least one amino acid sequence, nanotesla, polypeptide, compound, (monovalent) the structure or composition of the invention.

The invention also relates to the use of amino acid sequence, nanotesla, polypeptide, compound or (monovalent) the construction of the invention in obtaining pharmaceutical compositions for preventing and/or treating at least one disease and disorders associated with IL-6, IL-6R, IL-6/IL-6R and/or signalling pathways, and/or biological function or reactions involving IL-6, IL-6R and/or IL-6/IL-6R, and/or for use in one or more of the methods described here.

The invention also relates to amino acid sequences, nanotesla, polypeptide, compound or (monovalent) structure according to the invention for use in the treatment and/or prophylaxis of at least one of the diseases or disorders associated with IL-6, IL-6R, IL-6/IL-6R and/or signalling pathways and/or the biological functions and responses involving IL-6, IL-6R and/or IL-6/IL-6R; and/or for use in one or more ways described ZV is camping.

In particular, the present invention provides amino acid sequences, nanotesla, proteins, polypeptides, compounds and/or designs which are suitable for prophylactic, therapeutic or diagnostic use in warm-blooded animals, particularly in mammals, preferably in humans.

More specifically, the present invention provides such amino acid sequences, nanotesla, proteins, polypeptides, compounds and/or designs that can be used to prevent, treat, reduce the symptoms and/or diagnosis of one or more disorders associated with IL-6 (as defined here) in a warm-blooded animal, particularly a mammal, preferably humans.

Other fields of application and use of amino acid sequences of nantel, polypeptides and compounds of the invention will become apparent to a person skilled from the further description.

Brief description of figures

Fig.1. Analysis of the immune response in Lam 81 and 82 by the ELISA method as described in Example 2.

Fig.2. Analysis of the immune response in Lam 81 and 82 method FACS as described in Example 2. Legend: L181 pre: preimmune serum from Lama 81; L181 PBL1: serum taken after 28 days from Lama 81; L182 pre: preimmune serum from Lama 82; L182 PBL2: serum taken after 43 days at Lama 82.

Fig.3. Schemes shall determine the method Alphascreen to identify nantel against IL-6 binding site on IL-6R.

Fig.4. Amino acid sequence of Manotel against IL-6R.

Fig.5. SDS-PAGE of purified nantel, obtained as described in Example 6.

Fig.6. Inhibition of the interaction between IL-6/IL-6R separate antelami when measuring method Alphascreen. As control was used MAb BR-6 and control Fab-fragment (described in Example 1).

Fig.7. The binding of Manotel against IL-6R with U266 cells during FACS analysis.

Fig.8. The binding of Manotel against IL-6R with U266 cells in the absence (top) and presence (bottom) of human plasma.

Fig.9. Individual profiles observed (icons) and predicted by the model (solid line) concentrations of IL6R304 in the plasma from the time the long-tailed monkeys after intravenous introduction 1 mg/kg (o), 5 mg/kg (Δ), 10 mg/kg (+), 25 mg/kg (×) and 100 mg/kg (0).

Fig.10. The binding of Manotel with IL-6R mice and humans. In each group of 3 columns left means IL-6R man, middle - IL-6R mouse, and right - idle test.

Fig.11. SDS-PAGE of purified bispecific of Manotel.

Fig.12. Inhibition of the interaction between IL-6/IL-6R bespecifically antelami when measuring method Alphascreen.

Fig.13. Analysis of the binding of divalent nantel with U266 cells by FACS method.

Fig.14. Analysis of the binding of trivalent nantel with U266 cells by FACS method.

Fig.15. Comparison of sequences IL6R03, IL6R04 and IL6R13 5 most homologous gametime serial what inotai person. For more explanation, see Example 23.

Fig.16A and B. Amino acid sequence optimized for sequence variants IL6R03, IL6R04 and IL6R13. For more explanation, see Example 23.

Fig.17. Inhibition of the interaction between IL-6/IL-6R-optimized sequence variants IL6R03.

Fig.18. Inhibition of the interaction between IL-6/IL-6R-optimized sequence variants IL6R04.

Fig.19. Inhibition of the interaction between IL-6/IL-6R-optimized sequence variants IL6R13.

Fig.20. Inhibition of the interaction between IL-6/IL-6R-optimized sequence variants IL6R13.

Fig.21. Inhibition of the interaction between IL-6/IL-6R by antelami wild-type and optimized sequence antelami against IL-6R.

Fig.22. Cellular activity is optimized by sequence of Manotel in comparison with antelami wild-type.

Fig.23. Activity in plasma using ELISA method optimized sequence of Manotel in plasma (a) and macaques ().

Fig.24. Activity in plasma using ELISA method optimized sequence of Manotel when the values of Esbo (left) and EC (right) for IL-6.

Fig.25. Mapping epitopes of Manotel to IL-6 as described in Example 29: analysis of competition covered on IL-6R (a-C) or with IL-6 matrix.

Fig.26. Amino acid placentas the activity ripened on the affinity variants IL6R65.

Fig.27. Curves binding nanotesla IL6R65 (referred to as source nanotesla) and ripened on the affinity of options.

Fig.28. The study IL6R65 and 5 ripe for affinity variants of the method for determining the activity in the plasma of humans and macaques.

Fig.29. Inhibition of IL-6-dependent cell proliferation, TF-1 ripe on the affinity of antelami. Cells were cultured in the presence of 2 ng/ml IL-6 people at various concentrations was Neotel. Proliferation was measured by incorporation of3H-thymidine.

Fig.30. Competition between IL-6 and two ripened on the affinity of antelami (b-C) in comparison with the competition between IL-6 and IL6R65 (a) when measured on the Biacore instrument.

Fig.31. The sequence of binding IL-6R of Manotel after the 2nd cycle of affinity maturation (combinatorial library CDR1/2+CDR3).

Fig.32. Inhibition of IL-6-dependent cell proliferation, TF-1. Cells were cultured in the presence of 2 ng/ml IL-6 people at various concentrations of Manotel. Proliferation was measured by incorporation of3H-thymidine.

Fig.33. The study IL6R65 and ripened on the affinity variants of the 2nd cycle method for determining the activity in the plasma of humans and macaques. Source = IL6R65.

Fig.34. Linking IL6R65 and RMRA with human PBMCs from whole blood.

Fig.35. The inhibition activity of membrane IL-6R formatted antelami and control IgG. Cells TF-1 braincube is ovali with serial dilutions IL6R304 (■), IL6R305 (▲), IL6R306 (▼) or control IgG (●), after which induced proliferation with ME 100 IL-6/ml After incubation for 72 h was assessed cell proliferation by the inclusion of3H-thymidine. Presents mean values ±S. E. of three measurements.

Fig.36. Inhibition of membrane IL-6R formatted antelami and control IgG at a high level of IL-6. Cells TF-1 plaincourault with serial dilutions A (♦), IL6R304 (■), IL6R305 (▲), IL6R306 (▼) or control IgG (●), after which induced proliferation with 5,000 IU of IL-6/ml After incubation for 72 h was assessed cell proliferation by the inclusion of3H-thymidine. Presents mean values ±S. E. of three measurements.

Fig.37. The effect of IL6R304 and IL6R305 on cell proliferation, TF-1. Cells TF-1 was inoculated at a density of 12,500 cells per well and incubated with or without 50 nm IL6R304 or IL6R305. Proliferation was induced with 100 ME IL-6/ml or cells incubated in the absence of growth factors. After incubation for 72 h was assessed cell proliferation by incorporation of H-thymidine. Each experimental point was measured 30 times. Presents mean values ±S. E.

Fig.38. Activity in plasma by the method of ELISA in the plasma of man. Inhibition of binding of IL-6 person with sIL-6R plasma control IgG (●), IL6R20A11 (♦), IL6R304 (■), IL6R305 (▲), IL6R306 (▼) or outsiders NB (×). Presents mean values ±S. E. of the two and the measurements. And, In competition with 25 ng/ml IL-6 (EC50). C,D: competition at 885 ng/ml IL-6 (ES).

Fig.39. Linking IL6R20A11 and rich variations with cells of SSC. Expressing IL-6R cells Cho (A) or negative cells Cho (C) were incubated with IL6R20A11 (♦), IL6R304 (■), IL6R305 (▲), IL6R306 (▼). The bound peroxidase nanotesla were detected using MAI C1.5.3.1 and antibodies against mouse with PE.

Fig.40. The binding of Manotel to IL-6R with human PBL whole blood. Left: lymphocytes (L, black), monocytes (M, dark gray) and granulocytes (G, light gray) was spotted on the characteristics of the FSC/SSC. In the middle: background fluorescence of the PE the three passed through the channel populations. Right: fluorescence RE after incubation with 1 μm IL6R305.

Fig.41. The binding of Manotel to IL-6R with human PBL. Processed EDTA blood from 2 donors were incubated with IL6R20A11 (♦), IL6R304 (■), IL6R305 (▲), IL6R306 (▼). The bound peroxidase nanotesla were detected using MAb c1.5.3.1 and antibodies against mouse with PE. A: lymphocytes; In: monocytes; S: granulocytes.

Fig.42. Curves linking rich ripe on the affinity of Manotel with albumin human and macaque.

Fig.43. Curves linking rich ripe on the affinity of Manotel with IL-6R humans and macaques.

Fig.44. Activity in plasma by the method of ELISA in the plasma of monkeys. Inhibition of binding of IL-6 person with sIL-6R plasma macaques control IgG (●), IL6R20A11 (♦), IL6R304 (■), IL6R305 (▲), IL6R306 (▼) or outsiders NB (x.

Fig.45. The cross-reactivity IL6R20A11 to sIL-6R from other species. Left:

linking IL6R20A11 with sIL-6R man on the tablet after inactivated with recombinant sIL-6R person (●), macaques (■) or mouse (▲). Right: binding IL6R20A11 with sIL-6R person after inactivated by plasma man (●), macaques (■), mouse (▲) or cavy (▼).

Fig.46. Competition for binding by ELISA method. IL6R20A11 (0,05 nm) plaincourault at various concentrations of IL-6R (●), LIF-R (■), CNTF-R (▲), OSM-R (▼) or IL-11R/Fc (♦). Available IL6R20A11 captured on sIL-6R and were detected through anti-His.

Fig.47. The scheme of experiments on the analysis of PK/PD IL6R304 and IL6R305 in vivo.

Fig.48. The effect of control IgG, IL6R304 and IL6R305 at increasing levels of CRP under the action of hIL-6 in individual long-tailed macaques. (A) Animals 27, 28 and 29 served as negative control and received only hIL-6. Control IgG (black icons), different doses of IL6R304 (blue icons) and different doses IL6R305 (D, red) was injected into/in, after which he entered the s/hIL-6 in a dose of 5 mcg/kg once a day for 7 days.

Fig.49. The average CRP levels for all groups obtained in the study of PK/PD IL6R304 and IL6R305 in vivo.

Fig.50. The effect of control IgG, IL6R304 and IL6R305 to increased levels of fibrinogen under the action of hIL-6 in individual long-tailed macaques. (A) Animals 27, 28 and 29 served as negative control and received only hIL-6. Control IgG (B, black C is acci), different doses of IL6R304 (blue icons) and different doses IL6R305 (D, red) was injected into/in, after which he entered the s/hIL-6 in a dose of 5 mcg/kg once a day for 7 days. The results are normalized by basal levels.

Fig.51. The mean fibrinogen level for all groups obtained in the study of PK/PD IL6R304 and IL6R305 in vivo.

Fig.52. The effect of control IgG, IL6R304 and IL6R305 to increase the number of platelets under the action of hIL-6 in individual long-tailed macaques. (A) Animals 27, 28 and 29 served as negative control and received only hIL-6. Control IgG (black icons), different doses of IL6R304 (blue icons) and different doses IL6R305 (D, red) was injected into/in, after which he entered the s/hIL-6 in a dose of 5 mcg/kg once a day for 7 days. The results are normalized by basal levels.

Fig.53. The average number of platelets for all groups obtained in the study of PK/PD IL6R304 and IL6R305 in vivo.

Fig.54. Individual graphs of the observed plasma concentrations with time after the on/in the introduction of a bolus IL6R304 (0,4-2-10 mg/kg) in long-tailed macaques. 11m, 12m and 13f mean three graphs on the left side, 17m, 18f and 14m - average graphics, and 15m and 16f - graphs on the right side.

Fig.55. Individual graphs of the observed plasma concentrations with time after the on/in the introduction of a bolus IL6R305 (0,4-2-10 mg/kg) in long-tailed macaques. 19m, 20f and 21f mean three graphs on the left side, 25m,26f and 22m - average graphics, and 23m and 24f - graphs on the right side.

Fig.56. Graphics mean observed plasma concentrations with time after the on/in the introduction of a bolus IL6R304 (0,4-2-10 mg/kg) and IL6R202 (2 mg/kg dose resulted in a 0.4-10 mg/kg) in long-tailed macaques.

Fig.57. Immunodetection antibodies against IL6R304 before the introduction and after different number of days after the on/in the introduction of IL6R304. Tablets for ELISA were covered IL6R304. Captions below each of the figures correspond to the groups on the histogram from left to right.

Fig.58. Immunodetection antibodies against IL6R304 before the introduction and after different number of days after the on/in the introduction of IL6R304. Tablets for ELISA were covered IL6R300. Captions below each of the figures correspond to the groups on the histogram from left to right.

Fig.59. Immunodetection antibodies against IL6R304 before the introduction and after different number of days after the on/in the introduction of IL6R304. Tablets for ELISA were covered ALB8. Captions below each of the figures correspond to the groups on the histogram from left to right.

Fig.60. Immunodetection antibodies against IL6R305 before the introduction and after different number of days after the on/in the introduction IL6R305. Tablets for ELISA were covered IL6R305. Captions below each of the figures correspond to the groups on the histogram from left to right.

Fig.61. Immunodetection antibodies against IL6R305 before the introduction and after different number of days after the on/in the introduction IL6R305. Tablets for ELISA were covered IL6R300. Under the ISI under each of the figures correspond to the groups on the histogram from left to right.

Fig.62. Immunodetection antibodies against IL6R305 before the introduction and after different number of days after the on/in the introduction IL6R305. Tablets for ELISA were covered ALB8. Captions below each of the figures correspond to the groups on the histogram from left to right.

Fig.63. The level of sIL-6R plasma long-tailed macaques after a single on/in the introduction of a bolus of Manotel to IL6R. A: 2 animals received counter. IgG in a dose of 5 mg/kg (■) or medium (▲); At: 3 animals received IL6R304 in a dose of 0.04 mg/kg; From: 3 animals received IL6R305 dose of 0.04 mg/kg

Fig.64. The overall level of sIL-6R plasma long-tailed macaques after a single on/in the introduction of a bolus of Manotel to IL6R. A: animals received IL6R304 (▲, ■, ●), contr. IgG (▼) or media (◊); In: animals received IL6R305 (▲, ■, ●), contr. IgG (▼) or media (◊). Presents mean values ±S. E. for each group.

Fig.65. The overall level of IL-6 in the plasma of long-tailed macaques after a single on/in the introduction of a bolus of Manotel to IL6R. The total concentration of IL-6 in plasma, endogenous IL-6 macaques and introduced IL-6 person, measured on the Gyrolab platform. Samples that were below the detection limit, given as a 9.6 PG/ml A: IL6R304;

In: IL6R305; P: positive (counter. IgG) and negative (buffer) control. Presents mean values ±S. E. for each treatment (n=3 for 0.4 and 2 mg/kg; n=2 to 10 mg/kg). D: concentration of endogenous IL-6 macaques in plasma of individual animals pic the e/in the introduction of a bolus of 10 mg/kg IL6R304 (■,●), IL6R305 (▲,▼) or third nantel (◊,●). Presents mean values ±S. E. of the 2 measurements.

Fig.66. General level (a) and the level of free (In) sIL6R plasma long-tailed macaques after a single intravenous introduction of different doses of IL6R304. Presents the average concentrations of each biomarker in plasma ±SD for each group. Media or IL6R304 these doses were administered at time 0. Cm. signature of Fig.W.

Fig.67. The overall level and the level of free sIL6R in plasma and the concentration of IL6R304 in long-tailed macaques after a single intravenous introduction 25 mg/kg IL6R304. Presents the average concentrations ±SD for a specific group. The chart shows the total sIL6R (solid line, squares), free sIL6R (solid line, circles) and the concentration of IL6R304 (dashed line, Delta) depending on time (days).

Fig.68. Individual charts observed (icons) and predicted by the model (solid line) to the total concentration of sIL6R of time after the on/in the 1 mg/kg (o), 5 mg/kg (Δ), 10 mg/kg (+), 25 mg/kg (×) and 100 mg/kg (◊) IL6R304.

Fig.69. The profiles of the average concentration of IL6R304 in the plasma from the time the long-tailed macaques after/in the introduction of a bolus 1,5,10,25 or 100 mg/kg IL6R304.

Fig.70. Open pharmacokinetic model with three compartments with linear and non-linear clearance from the Central compartment. CLNON-IL6Rlinear clearance, not mediated IL6R; Vc- volume Central compartment; Vd- volume deep peripheral compartment; CLdthe flow between the Central and deep compartment; Vs- small amount of peripheral compartment; CLdthe flow between the Central and the small compartment; a CLIL6Rnonlinear clearance mediated IL6R (Vmax- maximum metabolic rate and Kmthe concentration of IL6R304 corresponding to 50% of Vmax).

Disclosure of the invention

In the present description, the examples and the formula:

a) If not specified, or is not otherwise defined, all terms used have their usual in this field the value that must be known to specialists. For example, will refer to standard textbooks such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd ed.), vols.1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., eds., "Current Protocols in Molecular Biology", Green Publishing and Wiley Interscience, New York (1987); Lewin, "Genes II, John Wiley & Sons, New York, N. Y., (1985); Old et al., "Principles of Gene Manipulation: An Introduction to Genetic Engineering", 2nd ed University of California Press, Berkeley, CA (1981); Roitt et al., "Immunology" (6th ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., "Roitt's Essential Immunology", 10thed, Blackwell Publishing, UK (2001); and Janeway et al., "Immunobiology" (6th ed.). Garland Science Publishing/Churchill Livingstone, New York (2005), as well as the above work prior art.

(b) Unless otherwise specified, the term "immunoglobulin sequence" refers to the antibody heavy chains or to the Oba is the resultant 4-chain antibody - it is used as a General term covering and full of antibodies, and single chain, as well as all parts, domains or fragments (including antigennegative domains or fragments such as domains VHHor domains VH/VL, respectively). In addition, the term "sequence" in the present invention (for example, terms like "immunoglobulin sequence", "sequence antibodies, sequence of the variable domain sequence VHH" or "protein sequence") in General should be understood as including the corresponding amino acid sequence, and its encoding nucleic acid or nucleotide sequences, unless the context requires a more specific interpretation. Also, the term "nucleotide sequence" in the present invention encompasses a nucleic acid molecule with the nucleotide sequence, so that the terms "nucleotide sequence" and "nucleic acid" should be considered equivalent and are used interchangeably.

c) If not indicated otherwise, all methods, stages, techniques and procedures that are not specifically described in detail can be performed and carried out by a method known per se, as should be clear to the experts. Again, we refer, for example, at one hundred the standard textbooks, the above operation of the prior art and provides other links; as well as, for example, the following surveys: Presta, 2006, Adv. Drug Deliv. Rev., 58(5-6): 640-56; Levin and Weiss, 2006, Mol. Biosyst., 2(1): 49-57; Irving et al., 2001, J. Immunol. Methods 248(1-2): 31-45; Schmitz et al., 2000, Placenta, 21 Suppl. A, S106-12; Gonzales et al., 2005, Tumour BioL, 26(1); 31-43, which describes such methods engineering proteins as affinity maturation and other techniques to improve specificity and other desirable properties of proteins such as immunoglobulins.

d) Amino acid residues will be indicated according to the standard three-letter or one-letter encoding amino acids as listed in table.A-2.

Table A-2.
Single-letter and three-letter encoding amino acids
Nonpolar, uncharged (pH 6,0-7,0)(3)AlanineA1AA
ValineValV
LeucineLeuL
IsoleucineNoI
F is kilalanin PheF
Methionine(1)MetM
TryptophanTrpW
ProlineProP
Polar, uncharged (pH 6,0-7,0)Glycine(2)GlyG
SeriesSerS
ThreonineThrT
CysteineCys
AsparagineAsnN
GlutamineGlnQ
TyrosineTyrY
Polar, charged (at pH 6,0-7,0)LysineLysTo
ArginineArgR
Histidine(4)HisH
AspartateAspD
GlutamateGluE
Notes:
(1)Sometimes, too, is a polar uncharged amino acid.
(2)Sometimes also considered to be non-polar, uncharged amino acid.
(3)As should be known to the experts, the fact that the amino acid residue in this table as charged or uncharged at pH 6.0-7.0 and in no way reflects the charge of this amino acid residue at pH below 6.0 and/or at pH above 7.0 and are shown in table amino acid residues may be both charged and uncharged at higher or lower pH, which should be clear to the experts.
(4)As is known, the charge balance His strongly affected by small changes in pH, but the residue of His in General is almost uncharged at pH about 6.5.

(e) for the purposes of comparing two or more nucleotide sequences can be calculated degree of sequence identity is of lidosta" between a first nucleotide sequence and the second nucleotide sequence by dividing [the number of nucleotides in the first nucleotide sequence, which are identical with the nucleotides at corresponding positions in the second nucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by 100%, with each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence is compared with the first nucleotide sequence is a difference of one nucleotide (position).

On the other hand, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithm for mapping sequences of type NCBI Blast v2.0, using the default settings.

Some other methods, computational algorithms, and settings to determine the degree of sequence identity, for example, described in WO 04/037999, EP 0967284, EP 1085089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2357768-A.

Usually in order to determine the degree of "sequence identity" between two sequences of nucleotides in accordance with the method of calculation set forth above as the "first" nucleotide sequence is taken nucleotide sequence with the greatest number of nucleotides, and as the second nucleotide sequence is taken by another nucleotide sequence.

f) In the spruce comparing two or more amino acid sequences it is possible to calculate the degree of "sequence identity" between a first amino acid sequence and second amino acid sequence (also referred to as "amino acid identity") by dividing [the number of amino acid residues in the first amino acid sequence identical to amino acid residues in the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], with each deletion, insertion, substitution or addition of amino acid residue in the second amino acid sequence is compared with the first amino acid sequence is considered to be a difference of one amino acid residue (position), i.e., "amino acid difference" as defined here.

On the other hand, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithm such as those described above for determining the degree of identity of nucleotide sequences, again using the default settings. Usually, in order to determine the percentage of "sequence identity" between two amino acid residues in accordance with the method of calculation set forth above as the "first" amino acid sequence amino acid sequence is taken with the greatest number of amino acids, and as a "second" is aminokislotnoi sequence takes a different amino acid sequence.

Also, when determining the degree of sequence identity between two amino acid sequences professionals can consider the so-called "conservative" amino acid substitutions which in General can be described as amino acid substitutions in which one amino acid residue is replaced with another amino acid residue with a similar chemical structure and having no or almost no influence on the function, activity or other biological properties of the polypeptide. Such amino acid substitutions are well known in this field, for example, from WO 04/037999, GB 335 768-A, WO 98/49185, WO 00/46383 and WO 01/09300; a (preferred) types and/or combinations of such substitutions can be selected on the basis of the relevant provisions of the WO 04/037999 and WO 98/49185 and see them in other works.

Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a)-(e) is replaced with another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (C) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val, and Cys; and (e) aromatic mod and: Phe, Tyr and Trp.

Especially preferred are the following conservative substitutions: Ala to Gly or Ser; Arg to Lys; Asn at Gin or His; Asp for Glu; Cys to Ser; Gln for Asn; Glu for Asp; Gly to Ala or Pro; His to Asn or Gln; He to Leu or Val; Leu't or Val; Lys for Arg, Gln or Glu; Met to Leu, Tight or Ile; Phe for Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp for Tyr; Tyr to Trp; and/or Phe to Val, Not or Leu.

Any amino acid substitution in the Annex to the polypeptides described herein can also be based on the frequency analysis of the variability of amino acids between homologous proteins of different species developed Schuiz et al. (1978, "Principles of Protein Structure", Springer-Verlag), on the analysis of structure-forming potentials developed by Chou and Fasman (1974, Biochemistry 13: 211, 1978, Adv. Enzymol., 47: 45-149), and on the analysis of the hydrophobicity profiles of the proteins developed by Eisenberg et al. (1984, Proc. Natl. Acad. Sci. USA 81: 140-144), Kyte and Doolittle (1981, J. Mol. Biol. 157: 105-132) and Goldman et al. (1986, Ann. Rev. Biophys. Chem. 15: 321-353), which are all incorporated in its entirety by reference. Information about primary, secondary and tertiary structure of Manotel given in the present description and in the above work prior art. In addition, for this purpose, the crystal structure of the vhh domain Lama, for example in Desmyter et al. (1996, Nature Structural Biology, 3(9): 803), Spinelli et al. (1996, Natural Structural Biology, 3: 752-757) and Decanniere et al. (1999, Structure, 7(4): 361). For more information about some amino acid residues, which is in the normal domains V Hform a contact area VH/VLand possible camelicious substitutions in these positions can be found in the above previous works.

(g) Amino acid sequences and nucleic acid sequences are considered to be "exactly the same" if they have 100% sequence identity (as defined here) along its entire length.

h) When comparing two amino acid sequences, the term "amino acid difference" refers to an insertion, deletion or replacement of one amino acid residue in the same position of the first sequence, compared with the second sequence; it is envisaged that two amino acid sequences can contain one, two or more such amino acid differences.

i) When it is said that the nucleotide sequence or amino acid sequence "contains" another nucleotide sequence or amino acid sequence, respectively, or "mostly consists of" another nucleotide sequence or amino acid sequence, this may mean that the last nucleotide sequence or amino acid sequence was included in the said first nucleotide sequence or amino acid sequence, respectively,but more often it means what is called the first nucleotide sequence or amino acid sequence contains within its sequence a stretch of nucleotides or amino acid residues, respectively, which has the same nucleotide sequence or amino acid sequence, respectively, and the last sequence, regardless of how actually was made or received named the first sequence (which could be, for example, any suitable method described herein). As a non-restrictive example: when it is said that the amino acid sequence of the invention contains a stretch of amino acid residues, it may mean that this stretch of amino acid residues was introduced in the amino acid sequence of the invention, but more often it means that the amino acid sequence of the invention includes within its sequence a stretch of amino acid residues, regardless of how it was made or received this amino acid sequence of the invention. When it is said that nanotesla invention contains the CDR sequence, this may mean that the sequence of the CDR was introduced in nanotesla inventions, but most often it means that nanotesla the invention includes within its what sledovatelnot site amino acid residues of the same amino acid sequence, as this sequence of CDR, regardless of how it was made or received this nanotesla invention. It should also be noted that if the last amino acid sequence has a biological or structural function, it preferably has the same, similar or equivalent to the biological or structural function in the said first amino acid sequence (in other words, the mentioned first amino acid sequence preferably is such that the last sequence is able to perform almost the same, similar or equivalent to the biological or structural function). For example, when it is said that nanotesla invention contains the CDR sequence or frame sequence, respectively, then the sequence of CDR and frame sequence is preferably capable of functioning in this nanotesla as a CDR sequence or frame sequence, respectively. Also when it is said that the nucleotide sequence contains a different nucleotide sequence, called the first nucleotide sequence preferably is such that when it is ekspressirovannoj in the expression product (i.e., polypeptide) amino acid sequence encoded by the last nucleot the ne sequence, is part of the expression product (in other words, the last nucleotide sequence is in the same reading frame that called the first, large nucleotide sequence).

(j) the Nucleotide sequence or amino acid sequence is considered to be "almost" selected "state" - for example, compared to its natural source and/or the reaction medium or culture medium, from which it was received - if she was separated from at least one other component with which it is usually associated in this source or environment, such as other nucleic acids, other protein/polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component. In particular, the sequence of nucleic acid or amino acid sequence is considered to be "virtually allocated", if it was cleaned at least 2 times, preferably at least 10 times, more preferably at least 100 times and up to 1000 times or more. The sequence of the nucleic acid or amino acid sequence that is "almost" selected "state", preferably practically homogeneous, as determined matched with the existing method, once the appropriate chromatographic methods or by polyacrylamide gel electrophoresis.

k) the Term "domain" in the present invention generally refers to a globular plot amino acid sequence (such as chain antibodies, in particular, globular plot heavy chain of the antibody or polypeptide, consisting mainly of such globular plot. Usually this domain contains peptide loops (for example, 3 or 4 peptide loops), stabilized, for example, in the form of a sheet or disulfide bonds. The term "domain binding' refers to a domain that is directed against antigenic determinants (as defined here).

l) the Term "antigenic determinant" refers to the epitope on the antigen recognized antigennegative molecule (such as amino acid sequence, nanotesla or polypeptide of the invention), in particular, the antigen-binding site of this molecule. The terms "antigenic determinant" and "epitope" can be used interchangeably.

(m) Amino acid sequence (such as nanotesla, an antibody, a polypeptide of the invention or antigennegative protein or polypeptide in General or its fragment), which can (specifically) bind to, has an affinity and/or that has specificity for a specific antigenic determinant, epitope, antigen is or protein (or for at least one part thereof, fragment or epitope), is considered to be "against" or "against" this antigenic determinant, epitope, antigen or protein.

n) the Term "specificity" refers to the number of different types of antigens or antigenic determinants, which can communicate a certain antigennegative molecule or antigennegative protein (such as amino acid sequence, nanotesla or polypeptide of the invention). Specificity antigennegative protein can be determined based on affinity and/or avidity. Affinity, represented by the equilibrium constant for the dissociation of antigen from antigennegative protein (KD), is a measure of the binding strength between an antigenic determinant and antigennegative site on antigennegative protein: the smaller the value of KDthe greater the strength of binding between an antigenic determinant and antigennegative molecule (on the other hand, the affinity can be expressed in the form of affinity constant (KA), which is equal to 1/KD). As should be known to specialists (for example, from the further discussion), the affinity can be determined by means known per se, depending on the specific antigen. The avidity is a measure of the strength of bonding between antigennegative molecule (such as amino acid sequence, anotella or polypeptide of the invention) and the corresponding antigen. The avidity due to an affinity between the antigenic determinant and its antigennegative site on antigennegative molecule, and the number of corresponding binding sites located on antigennegative the molecule. As a rule, antigennegative proteins bind with their antigen with a dissociation constant (Ko) of 10-5up to 10-12liter/mol or less, preferably from 10-7up to 10-12liter/mol or less and more preferably from 10-8up to 10-12liter/mol (i.e., a constant of Association (KAfrom 105up to 1012liter/moles or more, preferably from 107up to 1012liter/moles or more and more preferably from 108up to 1012liter/mol). It is usually assumed that any value of KDmore than 104liter/mol (or any value of KAless than 104M-1indicates the nonspecific binding. Preferably monovalent immunoglobulin sequence of the invention will be contacted with the desired antigen with an affinity less than 500 nm, preferably less than 200 nm, more preferably less than 10 nm, such as less than 500 PM. Specific binding antigennegative protein antigen or antigenic determinant can be determined in any appropriate way known per se, including, for example, the analysis Scatchard and/or the methods of competitive binding, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and competitive methods sandwich and their different ways, known per se in the field; as well as other methods listed here.

The dissociation constant may be actual or apparent dissociation constant, as should be known to specialists. Methods for determining the dissociation constants must be known in the art, for example, they include the methods given here. In this regard, it should be clear that it is impossible to measure dissociation constants greater than 10-4liter/mol or 10-3liter/mol (e.g., 10-2liter/mol). Not necessarily that also must be known to specialists (actual or apparent), the dissociation constant can be calculated on the basis of (real or apparent) of the Association constants (KD) the ratio of [KD=1/KD]. Affinity refers to the strength or stability of the molecular interactions. The affinity is usually given in the form of Co or dissociation constants in units of liter/mole (or M-1). The affinity can also be expressed in terms of the Association constants, KA, which is equal to I/KDin units (liter/mol)-1(or M-1). In the present description, the stability of the interaction between two molecules (such as amino acid sequence, NAS the body or polypeptide of the invention and the desired target) mainly expressed in terms of the values of K Dfor their interaction; professionals should be clear that in light of the relations KA=i/KD determination of the strength of molecular interaction value of KDcan also be used to calculate the corresponding values of KD. The value of KDcharacterizes the strength of molecular interactions in thermodynamic sense, since it is associated with a free energy (DG) of binding of the well-known relation DG=RTln(KD) (which is equivalent to DG=RTln(KD), where R is the gas constant, T is the absolute temperature, and ln denotes the natural logarithm.

Values of KDfor biological interactions, which is considered to be significant (e.g., specific), are typically in the range of 10-10M (0.1 nm) to 10-5M (10000 nm). The stronger the interaction, the less KD.

KDcan also be expressed as the ratio of rate constants of dissociation of the complex, denoted as koffto speed his Association, denoted as kon(KD=koff/konand KA=kon/koff). The rate constant koffis expressed in units of s-1(where s is the designation of a second in the SI system). The rate constant konis expressed in units of M-1s-1. Constant koncan vary from 102M-1s-1up to about 107M1 s-1approaching limited by the diffusion rate constant of the Association for bimolecular interaction. Constant koffassociated with the half-life of this molecular interaction through the relation t½ =ln(2)/koff. Constant koffcan vary between 10-6s-1(almost irreversible complex with t½ in a few days) and 1 s-1(t½ =0,69 sec). The affinity of a molecular interaction between two molecules can be measured by various methods known per se, such as are well known biosensor method of surface plasmon resonance (SPR) (see, for example, Ober et al., Intern. Immunology, 13, 1551-1559, 2001), in which one molecule is fixed on a biosensor chip, and the other molecule is passed through the immobilized molecule in flow conditions, giving the measurement values of kon, koffand hence KD(or KA). This can be done, for example, using well known devices BIACORE. Professionals should also be clear that the measured value of KDmay correspond to an apparent KDif the measurement process in some way affects the inner affinity of binding of the expected molecule, for example, due to artifacts associated with the layering on the biosensor of one of the molecules. The apparent value of KDit can also be the intend, if a molecule contains more than one site of recognition for the other molecules. In this scenario, the measured affinity can affect the avidity of the interaction between two molecules.

Another approach that can be used to assess the affinity is a 2-step method ELISA (enzyme immunosorbent assay) by the method of Friguet et al. (1985, J. Immunol. Methods 77: 305-19). In this method, the measurement of equilibrium binding in solution and eliminate possible artifacts associated with adsorption of one of the molecules on the substrate such as plastic. However, the precise measurement of the KDcan be very time consuming, so often defined the apparent values of KDto assess the strength of binding of two molecules. It should be noted that if all measurements are performed in a consistent manner (i.e., the measurement conditions are maintained constant), measurements of the apparent constants KDcan be used as an approximation of the actual values of KDtherefore , in this paper the values of KDand the apparent KDgiven equal value.

Finally, it should be noted that in many situations, an experienced scientist can calculate the convenient determination of the affinity of the binding relative to some standard molecules. For example, to assess the strength of bonding between molecules a and b can be used, e.g., the mill is artnow molecule, for which it is known that it binds to and appropriately marked fluorophore or chromophore group or other chemical molecule such as Biotin, for ease of detection by the method of ELISA or FACS (fluorescence-activated sorting of cells) or in another format (fluorophore for fluorescence detection, the chromophore for detection of light absorption, Biotin for streptavidin-mediated detection using ELISA method). Typically, the molecule specification is contained in a fixed concentration, and the concentration And varies for a given concentration or amount of C. the result is the value of the IC50corresponding to the concentration And in which the signal measured for C in the absence And reduced by half. If you know KDref, i.e., KDmolecules are standard, and the total concentration of crefmolecules of the standard, the apparent KD for the interaction a-b can be obtained by the following formula: KD=IC50/(1+cref/KD ref). Note that if cref<<KDrefthen KD≈IC50. If the measurement IC50is carried out in a consistent way (e.g., supported by a fixed value of Cref) when compared communicating substances, the durability or stability of the molecular interactions can be estimated from IC50and this dimension is considered as the equivalent to K Dor seeming KDthroughout this text.

a) the half-life of the amino acid sequence, nanotesla, compound or polypeptide of the invention in General can be defined as the time required for the concentration of the amino acid sequence, nanotesla, compound or polypeptide in the serum decreased by 50% in vivo, for example due to degradation of the sequence or compound and/or clearance or Deposit of the sequence or compound by natural mechanisms. The half-life of the amino acid sequence, nanotesla, compound or polypeptide of the invention in vivo can be determined in any manner known per se, such as pharmacokinetic analysis. Appropriate methods should be known in the art and in General can include, for example, the stage corresponding to the introduction of warm-blooded jivotnim (i.e., person or other suitable mammals, such as mice, rabbits, rats, pigs, dogs or primates, for example, monkeys from the genus of Massa, in particular long-tailed macaques (cynomolgus monkeys. Macaco, fascicularis) and/or rhesus monkeys (Masasa mulatto), and baboon (Papio ursinus)~), the appropriate dose amino acid sequence, nanotesla, compound or polypeptide of the invention; the sampling of blood or other samples from this animal; determining the level or to the ncentratio amino acid sequence, nanotesla, compound or polypeptide of the invention in the blood sample; calculate from the graph obtained in this data the time when the level or concentration of the amino acid sequence, nanotesla, compound or polypeptide of the invention will be reduced by 50% as compared to the initial level after dosing. We will refer, for example, in "Experimental", which follows, and in standard textbooks, such as Kenneth A. et al. (Chemical Stability ofPharmaceuticals: A Handbook for Pharmacists) and Peters et al. (1996, Pharmacokinetic Analysis: A Practical Approach). We will refer also to Gibaldi M. and D. Perron (1982, "Pharmacokinetics", published by Marcel Dekker, 2nd rev. ed.).

Specialists must also be known (see, for example, pages 6 and 7 in WO 04/003019 and are there other references) that the half-life can be expressed using parameters such as t½ alpha, t½ -beta and area under the curve (AUC). In the present description "the longer half-life" means increasing any of these parameters, such as any two of these parameters, or almost all three of these parameters. In the present invention, "increasing time-life" or "increased half-life", in particular, means an increase in t½ -beta, as with the increase in t½ -alpha and/or the AUC or both, and without it.

p) In the context of the present invention, "modulating" or "modulate" in obselesence or the reduction or inhibition of activity, or Vice versa, increasing the activity of the target or antigen that is measured in an appropriate manner in vitro, in cells or in vivo. In particular, "modulating" or "to modulate" may mean either reducing or inhibiting the activity or Vice versa, increase (relevant or assumed) the biological activity of the target or antigen when measuring the appropriate method in vitro, in cells or in vivo (which usually depends on the target or antigen) at least 1%, preferably by at least 5%, such as 10% or 25%, for example at least 50%, 60%, 70%, 80% or 90% or more compared to the activity of the target or antigen when defining the same method under the same conditions but in the absence of amino acid sequence, nanotesla, polypeptide, compound or construct of the invention.

As should be known to specialists, "modulating" may also include the implementation of changes (as in the direction of increase and decrease) in affinity, avidity, specificity and/or selectivity of the target or antigen to one or more ligands, binding partners, partners of the Association in homomultimers or heteropolymers form or substrates; and/or implementation changes (as in the direction of increase and decrease) the sensitivity of the target or antigen to the resultant or more of the conditions in the environment, or the environment, in which the target or antigen (such as pH, ionic strength, presence of cofactors and other), compared with the same conditions but in the absence of amino acid sequence, nanotesla, polypeptide, compound or construct of the invention. As should be known to experts, this again can be defined in any suitable way and/or any suitable method known per se, depending on the specific target or antigen.

"Modulating" may also mean the implementation of changes (i.e., action as an agonist, antagonist or inverse agonist, respectively, depending on the target or antigen, and the desired biological or physiological effect) in respect of one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which participates the target or antigen (or member of their substrates, ligands, or path), such as signalling pathway or metabolic pathway and their associated biological or physiological effects). Again, as should be known to specialists, such action as an agonist or antagonist can be defined in any suitable way and/or any suitable method (in vitro, in cells or in vivo), known per se, depending on the specific target or antigen. In particular, the action in which the quality of the agonist or antagonist may be such, what is the desired biological or physiological activity increases or decreases, respectively, by at least 1%, preferably by at least 5%, such as 10% or 25%, for example at least 50%, 60%, 70%, 80% or 90% or more compared to the activity of the target or antigen when defining the same method under the same conditions but in the absence of amino acid sequence, nanotesla, polypeptide, compound or construct of the invention.

The modulation may, for example, to include allosteric modulation of the target or antigen; and/or the reduction or inhibition of binding of the target or antigen to one of its substrates or ligands and/or competing with a natural ligand, substrate for binding to the target or antigen.

The modulation may also include activation of the target or antigen, or the mechanism or pathway in which it participates. The modulation may, for example, also include the implementation of changes in relation to styling or conformation of the target or antigen, or about the ability of the target or antigen to styling, changing its conformation (for example, when binding with ligand), Association with other subunits or dissociation. The modulation may, for example, also include the implementation of changes in the ability of the target or antigen to transport other with the joining or to serve as a channel for other compounds (such as ions).

The modulation may be reversible or irreversible, but for the pharmaceutical and pharmacological purposes it is usually reversible.

In the context of the present invention, "modulating" or "to modulate" may mean the implementation of agonistic or antagonistic effect, respectively, in relation to IL-6, IL-6R and/or the biological pathways, reactions, signalizovania, mechanisms or effects involving IL-6 and/or IL-6R. In particular, "modulating" or "to modulate" may mean agonistic or antagonistic action (i.e., as a full or partial agonist or antagonist, respectively) when measured with a suitable method in vitro, in cells or in vivo (like those shown here), which leads to the change of the corresponding parameter by at least 1%, preferably by at least 5%, such as at least 10% or 25%, for example at least 50%, 60%, 70%, 80% or 90% or more compared with the same parameter when defining the same method under the same conditions but in the absence of amino acid sequence, nanotesla, polypeptide, compound or construct of the invention.

In the context of the present invention, "modulating, inhibiting and/or preventing binding of the complex IL-6/IL-6R with gp130" means that the amino acid sequence is, nanotesla, polypeptides, compounds or constructs of the present invention associated with a specific epitope on IL-6R (i.e. on him or IL-6R/IL-6R) in such a way that the formation of the complex IL-6/IL-6R reduced, inhibited and/or prevented (e.g., fully or partially broken) so that reduced, inhibited and/or prevented the binding of the complex to gp130, e.g., his affinity for the gp130 (or Vice versa, reduced, inhibited and/or prevented the binding of gp130 to the complex, e.g., his affinity to it), so that modulates (e.g., reduced, inhibited and/or prevented) signalizovania induced/caused by binding of the complex to gp130, compared with the formation of the complex and its binding to gp130 in the absence of amino acid sequence, nanotesla, polypeptide, compound or construct of the invention.

q) with respect to the target or antigen, the term "site of interaction on the target or antigen means a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is a site for binding to a ligand, receptor or other binding partner, a catalytic site, a cleavage site, a site allosteric interaction, a site involved in multimerization (such as gomme is Itachi or heterogenization) of the target or antigen; or any other site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is involved in a biological action or mechanism of the target or antigen. In a more General sense, "the site of interaction" can be any site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen, which can communicate amino acid sequence, nanotesla, polypeptide, compound or construct of the invention in such a way that is modulated (as defined here) of the target or antigen (and/or any ways of interaction, the holding signal, the biological mechanism or biological action, involving the target or antigen).

g) "part of the amino acid residues" means two or more amino acid residues adjacent to each other or are in close proximity to each other, i.e. in the primary or tertiary structure of the amino acid sequence. In the context of the present invention "part of the amino acid residues responsible (at least partially) for binding amino acid sequence, nanotesla, polypeptide, compound or construct of the invention with its specific epitope on IL-6R.

s) it is Considered that the amino acid sequence of nanotesla, polypeptide, compound or construct of the invention is "specific for" a first target or antigen compared to a second target or antigen when it binds to the first antigen with an affinity (as described above, and suitably expressed as values of KD, KA, velocity, Koffand/or speed Kon), which is at least 10 times, 100 times, and preferably at least 1000 times, and up to 10000 or more times better than the affinity with which a given amino acid sequence, nanotesla, polypeptide, compound or construct is associated with the second target or polypeptide. For example, the first amino acid sequence, nanotesla, polypeptide, compound or construct can contact the target or antigen with a value of KDthat is at least 10 times less, 100 times less, and preferably at least 1000 times less than and up to 10,000 or even more times less than the value of KDwith which this amino acid sequence, nanotesla, polypeptide, compound or construct is associated with the second target or polypeptide. Preferably, if the amino acid sequence, nanotesla, polypeptide, compound or construct "specific for" a first target or antigen compared to a second target or antigen, it is directed against (as defined here) this first target or antigen, but not directed against a second target or antigen.

t) If the amino acid sequence or nanotesla of the invention (as well as the containing compounds, constructs and polypeptides) "cross reacts with IL-6R from two different species (i.e., of the first kind and second kind), it means that the amino acid sequence or nanotesla of the invention (as well as the containing compounds, constructs and polypeptides) is associated with IL-6R from a second species with an affinity (suitably measured and/or expressed in the form of values of KD(real or perceived), the values of KD(real or perceived), speed Koffand/or speed Konvalue IC50as further described herein) that is the same or is at least 70% (preferably at least 80%, more preferably at least 90% and even more preferably at least 95%) of the affinity with which a given amino acid sequence or nanotesla of the invention (as well as the containing compounds, constructs and polypeptides) is associated with IL-6R from the first type.

u) As will be described further, the total number of amino acid residues from NSA calving may be of the order of 110-120, is preferably 112-115, and is most preferably 113. However, it should be noted that part, fra the cops, analogs or derivatives (as will be described in detail hereinafter) of Manotel have no special restrictions on the length and/or size, unless such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described here.

v) Amino acid residues of Manotel numbered according to the General numbering for vh domains given Kabat et al. ("Sequences of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), which was applied to vhh domains from Camelids in article Riechmann and Muyldermans (2000, J. Immunol. Methods 240 (1-2): 185-195; for example, see Fig.2 in this publication); it is given here. According to this numbering, FR1 of Manotel contains amino acid residues at positions 1-30, CDR1 of Manotel contains amino acid residues at positions 31-35, FR2 of Manotel contains amino acid residues at positions 36-49, CDR2 of Manotel contains amino acid residues at positions 50-65, FR3 of Manotel contains amino acid residues in positions 66-94, CDR3 of Manotel contains amino acid residues at positions 95-102, and FR4 of Manotel contains amino acid residues in positions 103-113. [In this regard it should be noted, as is well known about the domains VHand domains VHHthat the total number of amino acid residues in each of the CDR may vary and may not match the total number of amino acid the residue, described by Kabat numbering (i.e. one or more positions according to the Kabat numbering may not be occupied real sequence of any real sequence may contain more amino acid residues than the number permitted by Kabat numbering). This means that in General the numbering of Kabat can match or not match the actual numbering of amino acid residues in the real sequence. However, in General we can say that according to the Kabat numbering and regardless of the number of amino acid residues in areas CDR position 1 according to the Kabat numbering corresponds to the start of FR1 and Vice versa, position 36 according to the Kabat numbering corresponds to the start FR2 and Vice versa, position 66 according to the Kabat numbering corresponds to the start of FR3 and Vice versa, and position 103 according to the Kabat numbering corresponds to the start of FR4 and Vice versa].

Alternative methods of numbering amino acid residues of domain VHthat can also be applied similarly to domains VHHfrom Camelids and nanocell, is the method described by Chothia et al. (1989, Nature 342: 877-883), the so-called "AbM definition" and the so-called "contact definition". However, in the present description, claims and figures should be numbered according to Kabat as applied to domains VHHno Riechmann and Muyldermans, unless otherwise specified.

<> w) the Figures, sequence Listing and the Experimental part/Examples are given for further illustration of the invention and in no way should be interpreted or construed as limiting the scope of the invention and/or the accompanying claims, unless expressly stated otherwise.

The present invention provides lots amino acid residues (SEQ ID NO's:80-82, SEQ ID NO's:84-91 and SEQ ID NO's:93-95), which are particularly suited for binding to IL-6R. These areas amino acid residues may be present and/or can be inserted in the amino acid sequence of the invention, in particular, so that they were (part) antigennegative site amino acid sequences of the invention. These areas amino acid residues were generated as CDR sequence of an antibody heavy chain or sequence VHHraised against IL-6R and undergo further maturation of affinity (see Examples) to further enhance the affinity for binding to IL-6R, as well as other properties, such as efficiency and/or activity and/or selectivity, in addition to their ability to partially or completely block the interaction between IL-6/IL-6R and/or to inhibit signaling through IL-6, IL-6R and/or IL-6/IL-6R. These areas amino acid residues also imeout is here as a "sequence of CDR of the invention (i.e., "the sequence of CDR1 of the invention, the CDR2 sequence of the invention and the CDR3 sequence of the invention", respectively).

However, it should be noted that the invention in its broadest sense is not limited to the specific structural role or function that these areas amino acid residues may have in an amino acid sequence of the invention, if only these areas of amino acid residues allow the amino acid sequence of the invention to contact the IL-6R. Thus, in General, the invention in its broadest sense provides amino acid sequences which are able to bind to IL-6R with certain affinato, avidity, efficiency and/or durability, along with the ability to partially or completely block the interaction between IL-6/IL-6R and/or to inhibit signaling through IL-6, IL-6R and/or IL-6/IL-6R, and containing one or more CDR sequences as described herein, in particular a suitable combination of two or more such CDR sequences, which are appropriately connected to each other through one or more other amino acid sequences in such a way that this entire amino acid sequence forms a binding domain and/or binding unit that is capable of contact with IL-6. However, it should also be noted that the presence of only one such CDR sequence in an amino acid sequence of the invention itself may be sufficient to give the amino acid sequences of the invention ability to bind to IL-6R; again, you can refer to, for example, on the so-called "Expedite fragments" described in WO 03/050531.

So, in the specific, but not restrictive aspect, the amino acid sequence of the invention may contain at least one stretch of amino acid residues selected from the group consisting of:

sequences CDR1:

a) SEQ ID NO's:80-82;

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance;

and/or

- CDR2 sequences:

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference atomnoi of SEQ ID NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance;

and/or

- CDR3 sequences:

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance.

In particular, the amino acid sequence of the invention may be amino acid sequence containing at least one antigennegative site, and this antigennegative site contains at least one stretch of amino acid residues selected from the group consisting of sequences CDR1, the placenta is valnontey CDR2 and CDR3 sequences, as described above (or any suitable combinations thereof). In a preferred aspect, however, the amino acid sequence of the invention contains more than one, such as two or more sites of amino acid residues selected from the group consisting of the sequences of the invention CDR1, the sequence of the invention CDR2 and sequences of the invention CDR3.

Accordingly, the present invention also relates to amino acid sequences containing two or more sites of amino acid residues chosen from the following:

sequences CDR1:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance;

and/or

- CDR2 sequences:

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ D NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance;

and/or

- CDR3 sequences:

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance;

that: (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to a) or b), the second stretch of amino acid residues corresponds to one of the amino acid sequence according to (C), (d), (e) or (f); (ii) when the first stretch of amino acid residues corresponds to one of aminokislotnykh sequence according to (C) or (d), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), e) or f); or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequence according to e) or f), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), C) or d).

In a specific aspect, the present invention also relates to amino acid sequences containing three or more sections amino acid residues, with the first stretch of amino acid residues chosen from the following sequences CDR1:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, preferably no more than I amino acid differences from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance;

the second stretch of amino acid residues chosen from the following sequences, CDR2:

c) SEQ ID NO's:84-91; or

d) participation the Cove amino acid residues, having no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance; and

the third stretch of amino acid residues chosen from the following sequences of CDR3:

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance.

As described in the present invention, it also comprises the amino acid sequence containing one or several segments of amino acid residues that has no more than 2, predpochtitel is but no more than 1 amino acid difference from one of the sites of amino acid residues, shown in (a), (C) and/or (e), i.e., from one of the sequences CDR1 (i.e. one of SEQ ID NO's:80-82), from one of the CDR2 sequences (i.e. one of SEQ ID NO's:84-91) and/or from one of the CDR3 sequences (i.e. one of SEQ ID NO's:93-95).

The term "amino acid difference" refers to the inserts, divisions, or substitutions of one amino acid residue in one position of the site amino acid residues (or CDR sequence) shown in b), d) or f), compared with a plot of amino acid residues (or CDR sequence) from a), C) or e), respectively; means that the site amino acid residues (or CDR sequence) of b), d) and f), respectively, may contain one or at most two such amino acid differences compared with a plot of amino acid residues of a), C) or e), respectively.

"Amino acid difference" may be one, maximum two substitutions, divisions, or insert, or combinations thereof, that either improve the properties of the amino acid sequence of the invention, or at least not too much affect (reduce) the desirable properties or the balance or combination of desired properties of the amino acid sequence of the invention. In this regard, the derived amino acid sequence of the invention at least due the contact IL-6R with the same, about the same or a higher affinity compared to the amino acid sequence containing one or several segments of amino acid residues without one or two substitutions, deletions or insertions, and the affinity is measured by means of surface plasma resonance.

For example, depending on the host organism used for expression of amino acid sequences of the invention, such deletions and/or substitutions may be designed to remove one or more sites for post-translational modifications (such as one or more sites of glycosylation), which should be within the competence of specialists in this field.

In a preferred aspect of the invention, "amino acid difference" is an amino acid substitution. Amino acid substitutions may be one or a maximum of two substitutions that either improve the properties of the amino acid sequence of the invention, or at least not too much impair the desirable properties or the balance or combination of desired properties of the amino acid sequence of the invention. In this regard, the derived amino acid sequence of the invention should at least contact the IL-6R with the same, about the same or a higher affinity compared to the amino acid pic what ecovitality, containing one or several segments of amino acid residues without one or two substitutions, and the affinity is measured by means of surface plasma resonance.

Amino acid substitutions in one or more areas of amino acid residues can be conservative amino acid substitutions. "Conservative" amino acid substitutions in General is such amino acid substitutions in which one amino acid residue is replaced with another amino acid residue with a similar chemical structure and having no or almost no influence on the function, activity or other biological properties of the resulting amino acid sequence. Such conservative amino acid substitutions are well known in this field, for example, from WO 04/037999, GB 3357768-A, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or combinations of such substitutions can be selected on the basis of the relevant provisions of the WO 04/037999, and WO 98/49185 and given other references.

Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a)-(e) is replaced with another amino acid residue within the same group: (a) small aliphatic nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and the x (uncharged) amides: Asp, Asn, Glu and Gln; (C) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val, and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

Especially preferred are the following conservative substitutions: Ala to Gly or Ser; Arg to Lys; Asn for Gln or His; Asp for Glu; Cys to Ser; Gin for Asn; Glu for Asp; Gly to Ala or Pro; His to Asn or Gln; He to Leu or Val; Leu or Val; Lys for Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe for Met, Leu or Tyr; Ser for Thr; Thr to Ser; Trp for Tyr; Tyr to Trp; and/or Phe to Val, Ile or Leu.

In another aspect of the invention, amino acid substitutions at one or more sites of amino acid residues can give the amino acid sequences of high affinity for binding to IL-6R. This can be done by methods such as random or site-directed mutagenesis and/or other methods for affinity maturation known per se, such, e.g., as described in WO 09/004065, WO 2009/004066, WO 05/003345, WO 06/023144, EP 527809, EP 397834.

Without being restrictive, rules (partially or fully adhered) to replace amino acid residues in the CDR can be the following (i.e., substitution of amino acids with similar chemistry of the side chains):

- K is replaced by R;

- R is replaced by K;

- And is replaced by S or T;

- S is replaced with a or T;

- T is replaced by a or S;

- I is replaced by L or V;

- L is replaced by I or V;

- V is replaced by I or L;

p> - F is replaced by Y;

- Y is replaced by F;

- N is replaced with D;

- D is replaced with N;

- Q is replaced with E;

- E is replaced with Q;

- G is replaced with A;

- M is replaced by L;

- N, S, W and R remain constant.

In addition, and also without limitation, rules (partially or fully adhered) to replace amino acid residues in the CDR can be summarized in the following alternative way to replace the provisions 27-35 and provisions 50-58 (according to the numbering system of Kabat).

for provisions 27-35:

original amino acid residue in position 27 (Kabat numbering) is replaced with F; G; R; S; 2 out of F, G, R, S; 3 of F, G, R, S; or all of them, preferably all of them;

original amino acid residue in position 28 (Kabat numbering) is replaced with A; I; S; T; 2 out of A, I, S, T; 3 out of A, I, S, T; or all of them, preferably all of them;

original amino acid residue in position 29 (Kabat numbering) is replaced with F; G; L; S; 2 out of F, G, L, S; 3 of F, G, L, S; or all of them, preferably all of them;

original amino acid residue in position 30 (Kabat numbering) is replaced by D; G; S; T; 2 out of D, G, S, T; 3 out of D, G, S, T; or all of them, preferably all of them;

original amino acid residue in position 31 (Kabat numbering) is replaced with D; I; N; S; T; 2 out of D, I, N, S, T; 3 out of D, I, N, S, T; or all of them, preferably all of them;

- source the th amino acid residue in position 32 (Kabat numbering) is replaced with D; N;Y; 2 out of D, N, Y; or all of them, preferably all of them;

original amino acid residue in position 33 (Kabat numbering) is replaced with A; G; T; V; 2 A, G, T, V; 3 out of A, G, T, V; or all of them, preferably all of them;

original amino acid residue in position 34 (Kabat numbering) is replaced by I; M; or all of them, preferably all of them;

original amino acid residue in position 35 (Kabat numbering) is replaced with A; G; S; 2 out of A, G, S; or all of them, preferably all of them;

to the provisions of 50-58, if original amino acid sequence has the amino acid sequence in position 52a (Kabat numbering):

original amino acid residue in position 50 (Kabat numbering) is replaced with A; C; G; S; T; 2 out of a, C, G, S, T; 3 out of a, C, G, S, T; 4 out of a, C, G, S, T; or all of them, preferably all of them;

original amino acid residue in position 51 (Kabat numbering) is replaced by I;

original amino acid residue in position 52 (Kabat numbering) is replaced with N; R; S; T; 2 out of N, R, S, T; 3 out of N, R, S, T; or all of them, preferably all of them;

original amino acid residue in position 52a (Kabat numbering) is replaced by R; S; T; W; 2 from R, S, T, W; 3 from R, S, T, W; or all of them, preferably all of them;

original amino acid residue in position 53 (Kabat numbering)is replaced with D; G; N; S; T; 2 out of D, G, N, S, T; 3 out of D, G, N, S, T; 4 out of D, G, N, S, T; or all of them, preferably all of them;

original amino acid residue in position 54 (Kabat numbering) is replaced by D; G; or all of them, preferably all of them;

original amino acid residue in position 55 (Kabat numbering) is replaced by D; G; S; 2 out of D, G, S; or all of them, preferably all of them;

original amino acid residue in position 56 (Kabat numbering) is replaced by I; N; R; S; T; 2 I, N, R, S, T; 3 out of I, N, R, S, T; 4 out of I, N, R, S, T; or all of them, preferably all of them;

original amino acid residue in position 57 (Kabat numbering) is replaced by T;

original amino acid residue in position 58 (Kabat numbering) is replaced with D; H; N; S; Y; 2 out of D, H, N, S, Y; 3 out of D, H, N, S, Y; 4 of D, H, N, S, Y; or all of them, preferably all of them;

to the provisions of 50-58, if original amino acid sequence has amino acid sequence in position 52a (Kabat numbering),

original amino acid residue in position 50 (Kabat numbering) is replaced with A; G; R; S; T; 2 out of A, G, R, S, T; 3 out of A, G, R, S, T; 4 out of A, G, R, S, T; or all of them, preferably all of them;

original amino acid residue in position 51 (Kabat numbering) is replaced by I;

original amino acid residue in position 52 (Kabat numbering) replacement is conducted on N; S; T; 2 out of N, S, T; or all of them, preferably all of them;

original amino acid residue in position 53 (Kabat numbering) is replaced with N; R; S; T; Y; 2 out of N, R, S, T, Y; 3 N, R, S, T, Y; 4 N, R, S, T, Y; or all of them, preferably all of them;

original amino acid residue in position 54 (Kabat numbering) is replaced by D; G; R; S; 2 out of D, G, R, S; 3 out of D, G, R, S; or all of them, preferably all of them;

original amino acid residue in position 55 (Kabat numbering) is replaced by G;

original amino acid residue in position 56 (Kabat numbering) is replaced by G; N; R; S; T; 2 out of D, N, R, S, T; 3 out of D, N, R, S, T; 4 out of D, N, R, S, T; or all of them, preferably all of them;

original amino acid residue in position 57 (Kabat numbering) is replaced by T;

original amino acid residue in position 58 (Kabat numbering) is replaced with D; N; T; Y; 2 out of D, N, T, Y; 3 out of D, N, T, Y; or all of them, preferably all of them;

then in the CDR sequence (in any manner known per se, as described below), there may be one or more potentially useful substitutions (or combinations thereof) that are installed at the same time, and the resulting amino acid sequence can be tested for affinity for IL-6R and/or other desirable properties, such as the ability (partially or preferably fully) lock the th interaction of IL-6/IL-6R and/or to inhibit the passage of the signal through IL-6, IL-6R and/or IL-6/IL-6R. Thus, by a limited number of trial and error on the basis of this presentation specialist may find other suitable substitutions in the CDR (or their appropriate combinations).

Amino acid sequence of the invention can be any amino acid sequence that contains at least one stretch of amino acid residues, and this stretch of amino acid residues has an amino acid sequence corresponding to the sequence of at least one of the CDR sequences shown here. This amino acid sequence may not contain immunoglobulin fold. For example, and without limitation, this amino acid sequence may be suitable fragment of immunoglobulin sequence that contains at least one CDR sequence (as defined above), but not large enough for the formation of (full) immunoglobulin fold (again, you can refer to, for example, to "Expedite fragments" described in WO 03/050531). On the other hand, this amino acid sequence may be suitable "protein skeleton containing at least one stretch of amino acid residues corresponding CDR sequence, as defined here for amino acid serial is Inesta of the invention (i.e., as part antigennegative site). Appropriate frameworks for the presentation of amino acid sequences must be known in the art, for example, they contain, without limitation, linking frames based on or derived from immunoglobulins (i.e., other than immunoglobulin sequences already described here), protein skeletons originating from domains of protein a (such as Affibodies™), tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptor, composed anchirinah repetitions of Askerov and domain-bearing (Binz et al., 2005, Nat. Biotech., 23: 1257) and the binding molecules on the basis of DNA or RNA, including DNA or RNA aptamers (Ulrich et al., 2006, Comb. Chem. High Throughput Screen. 9(8): 619-32).

Again, any amino acid sequence of the invention contains one or more CDR sequences as defined here for the amino acid sequences of the invention (i.e., "CDR of the invention"), preferably such that it can specifically bind (as defined here) to IL-6R, more preferably is such that it can bind to IL-6R with the same affinity binding (suitably measured and/or expressed in the form of values of KD(actual or apparent), values of KA(actual or apparent), the rate constant konand/or speed koffeither the value of the IC50, as described later), as defined here. Any amino acid sequence of the invention contains one or more CDR sequences as defined here for the amino acid sequences of the invention preferably is such that it has activity on cells and activity in plasma, as defined here.

In addition, professionals must be known and what can be "transplanted" one or more sites CDR defined here for the amino acid sequences of the invention (i.e., "CDR of the invention"), on the other "skeletons", including the skeletons of a man or nimmanahaeminda frames. Appropriate frameworks and methodologies for such transplantation CDR should be clear to experts and well known in this field, for example, see US 7,180,370, WO 01/27160, EP 0605522, EP 0460167, US 7.054,297, Nicaise et al. (2004, Protein Science, 13: 1882-1891), Ewert et al. (2004, Methods, 34(2); 184-199), Kettleborough et al. (1991, Protein Eng. 4(7): 773-783), O'brien and Jones (2003, Methods Mol. Biol. 207: 81-100), Skerra (2000, J. Mol. Recognit. 13: 167-187), and Saerens et al. (2005, J. Mol. Biol. 352(3); 597-607), and is given in other references. For example, techniques known per se for transplantation CDR of a mouse or a rat skeleton plots and frameworks person, you can use the same way to obtain chimeric proteins containing one or more CDR sequences defined here for the amino acid sequences of the invention, the one or more frame sections or sequences of human rights.

Thus, in a specific aspect, the invention also encompasses chimeric amino acid sequence containing at least one CDR sequence selected from the group consisting of sequences CDR1 of the invention, the CDR2 sequences of the invention and CDR3 sequences of the invention (defined here for the amino acid sequences of the invention). Preferably, such a chimeric amino acid sequence containing at least one CDR sequence selected from the group consisting of sequences CDR1 of the invention (defined here for the amino acid sequences of the invention), and at least one CDR sequence selected from the group consisting of the CDR2 sequences of the invention (defined here for the amino acid sequences of the invention); or at least one CDR sequence selected from the group consisting of sequences CDR1 of the invention (defined here for the amino acid sequences of the invention), and at least one CDR sequence selected from the group consisting of the CDR3 sequences of the invention (defined here for the amino acid sequences of the invention); or such a chimeric polypeptide can include at least one follower is ity CDR, selected from the group consisting of the CDR2 sequences of the invention (defined here for the amino acid sequences of the invention), and at least one CDR sequence selected from the group consisting of the CDR3 sequences of the invention (defined here for the amino acid sequences of the invention). For example, such a chimeric polypeptide can contain one CDR sequence selected from the group consisting of the CDR3 sequences of the invention (defined here for the amino acid sequences of the invention), one CDR sequence selected from the group consisting of sequences CDR1 of the invention (defined here for the amino acid sequences of the invention), and one CDR sequence selected from the group consisting of the CDR2 sequences of the invention (defined here for the amino acid sequences of the invention). Combinations of CDR, shown here as a preferred amino acid sequences of the invention (see tab.A-1) should be preferred for these chimeric polypeptides.

In the data the chimeric polypeptides areas CDR can be linked to other amino acid sequences and/or with each other through amino acid sequence in which the amino-acid PEFC is the sequences preferably are frame sequences or act as a frame sequence or together form a frame for the presentation of the CDR.

According to one non-restrictive embodiment, the chimeric amino acid sequence contains at least two sequences of CDR (defined herein for the amino acid sequences of the invention) connected via at least one frame of the sequence in which preferably at least one of the two sequences of the CDR is CDR3 sequence, and a second CDR sequence is a sequence of CDR1 or CDR2. According to a preferred but not restrictive embodiment, the chimeric amino acid sequence contains at least three CDR sequence of the invention (defined herein for the amino acid sequences of the invention), United at least two frame sequences, in which preferably at least one of the three CDR sequences is a sequence of CDR3 and the other two CDR sequence are sequences of CDR1 or CDR2, preferably this one sequence CDR1 and a CDR2 sequence. According to one specifically preferred, but not restrictive embodiment, the chimeric amino acid sequence have the structure FR1'-CDR1-FR2'-CDR2-FR3'-CDR3-FR4'. in which CDR1, CDR2 and CDR3 are defined here for the amino acid sequence the invention, a FR1', FR2', FR3' FR4' is a frame sequence. In particular, FR1', FR2', FR3' FR4' may represent a sequence, respectively, of the frame section 1, frame 2, frame 3 phase and frame section 4 antibodies person (such as posledovatelnostyu) and/or parts or fragments of such frame sequences. You can also use parts or fragments of the chimeric polypeptide with the structure FR1'-CDR1-FR2'-CDR2-FR3'-CDR3-FR4'. Preferably, these parts or fragments such that they meet the criteria established for the amino acid sequences of the invention.

In such amino acid sequences of the invention the frame sequence can be any suitable frame sequence, and examples of suitable frame sequences must be known in the art, for example, from standard textbooks and further discussion and here works prior art.

Frame sequence preferably represent a frame sequence (suitable combinations thereof) of immunoglobulins or frame sequence, originating from the frame sequences of immunoglobulins (for example, when optimizing sequences of the type of humanization or kanalizacji). For example, a frame on which sledovatelnot can be a frame sequence, originating from the variable domain of the light chain (i.e., VLsequences) and/or variable domain of the heavy chain (i.e., VH-sequence). In one particularly preferred aspect, the frame sequence can be either a frame sequence derived from the sequences of the VHH(in which these frame sequences can optionally be partially or fully humanitarian) or conventional VHsequences that have been subjected kanalizacji (as defined here).

Frame sequence can preferably be such that the amino acid sequence of the invention is a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody); a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), antibody, "dAb" (or an amino acid sequence that is suitable for use as a dAb); or nanotesla (including the sequence VHH). Again, a suitable frame sequence must be known in the art, for example, from standard textbooks and further discussion and here works prior to level the equipment.

In particular, frame sequences present in the amino acid sequences of the invention may contain one or more Hallmark residues (as defined in WO 08/020079 (PL.from A-3 to a-8), so the amino acid sequence of the invention is nanotesla. Some preferred, but not restrictive examples of such frame sequences (or their appropriate combinations) will become clear from the further discussion (e.g., see tab.A-1). In General, nanotesla (in particular, the sequence VHHand (partially) humanized sequence VHH), in particular, can be characterized by the presence of one or more "Hallmark residues" in one or more frame sequences (e.g., as described in detail in WO 08/020079, page 61, line 24, to page 98, line 3).

In a preferred aspect, the amino acid sequence of the invention contains an immunoglobulin fold or capable, under suitable conditions to form an immunoglobulin fold. Preferably the amino acid sequence of the invention is an immunoglobulin sequence; and even more preferably amino acid sequence of the invention has the structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Accordingly, the present invention relates to amino acid placenta is valnontey, which mainly consist of frame 4 plots (FR1 to FR4 respectively) and 3 complementarity determining areas (CDR1 to CDR3, respectively), with:

- CDR1 selected from the group consisting of:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance;

and/or

- CDR2 selected from the group consisting of:

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface p is azmanova resonance;

and/or

- CDR3 selected from the group consisting of:

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance.

In this embodiment, the amino acid sequence contains at least one CDR sequence selected from the group consisting of sequences CDR1 of the invention (defined here for the amino acid sequences of the invention), the CDR2 sequences of the invention (defined here for the amino acid sequences of the invention) or the CDR3 sequences of the invention (defined here for the amino acid sequences of the invention). Preferably the amino acid sequence contains at least two CDR sequence selected from the group consisting of sequences CDR1 of the invention (defined here for amino acid sequences is Soberania), the CDR2 sequences of the invention (defined here for the amino acid sequences of the invention) or the CDR3 sequences of the invention (defined here for the amino acid sequences of the invention), so that at least one CDR sequence selected from the group consisting of sequences CDR1 of the invention (defined here for the amino acid sequences of the invention), and at least one CDR sequence selected from the group consisting of the CDR2 sequences of the invention (defined here for the amino acid sequences of the invention); or at least one CDR sequence selected from the group consisting of sequences CDR1 of the invention (defined here for the amino acid sequences of the invention), and at least one CDR sequence selected from the group consisting of the CDR3 sequences of the invention (defined here for the amino acid sequences of the invention); or at least one CDR sequence selected from the group consisting of the CDR2 sequences of the invention (defined here for the amino acid sequences of the invention), and at least one CDR sequence selected from the group consisting of the CDR3 sequences of the invention (defined who's here for the amino acid sequences of the invention); or in such a way that the amino acid sequence can contain three CDR sequence selected from the group consisting of sequences CDR1 of the invention (defined here for the amino acid sequences of the invention), from the CDR2 sequences of the invention (defined here for the amino acid sequences of the invention) and CDR3 sequences of the invention (defined here for the amino acid sequences of the invention). Thus, the present invention also relates to amino acid sequences that essentially consist of 4 frame sections (FR1 to FR4 respectively) and 3 complementarity determining areas (CDR1 to CDR3, respectively), with:

- CDR1 selected from the group consisting of:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance is;

and

- CDR2 selected from the group consisting of:

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance;

and

- CDR3 selected from the group consisting of:

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance.

Preferred combinations of CDR sequences for the amino acid sequences of the invention are presented in table.A-1.

Amino acid sequences of the invention can basically be composed of a sequence variable domain of the heavy chain, originating from normal chetyrekhzvezdochnogo antibodies, or can basically consist of a sequence variable domain of the heavy chain occurring antibodies of the heavy chain. Amino acid sequences of the invention can basically consists of a domain antibody (or an amino acid sequence suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), antibody, "dAb" (or an amino acid sequence suitable for use as a dAb) or nanotesla.

For a General description (single)domain antibodies, we refer to the above work prior art, as well as on the EP 0368684. Regarding the term "dAb" will refer, for example, Ward et al. (1989, Nature 341: 544-6), Holt et al. (2003, Trends Biotechnol. 21: 484-490); as well as, for example, in WO 06/030220, WO 06/003388 and other published patent applications of the company Domantis Ltd.

It should also be noted that although less preferred in the context of the present invention because derived from mammals, single domain antibodies or single variable domains can occur isoprenaline shark species (e.g., the so-called "IgNAR domains, for example, see WO 05/18629).

In particular, the amino acid sequence of the invention mainly consists of or can be nanotesla (Nanobody®) (as defined here) or a suitable fragment. [Note: Nanobody®, Nanobodies®and Nanoclone®are registered trademarks of the company Ablynx N. V.]. Such nanotesla directed against IL-6R, also will be referred to as "antelami of the invention".

For a General description of Manotel we will refer in the following description, as well as on the work of prior art, e.g., described in WO 08/020079 (p. 16).

In one specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:80 or stretch of amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:80, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasma resonance.

In another specific aspect, the amino acid posledovatelno the ü or nanotesla of the invention contains at least a stretch of amino acid residues, selected from SEQ ID NO's:84, 89 or 91; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:84, 89 or 91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasma resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:84; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:84, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasma resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention contains at least the area am is nekisnotnice residues, selected from SEQ ID nos:93-94; or the site amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from one of SEQ ID nos:

93-94, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasmon resonance.

The following specific aspect, the amino acid sequence or apatela of the invention comprises at least SEQ ID NO:93; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:93, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasma resonance.

The following specific aspect, the amino acid sequence or apatela of the invention comprises at least SEQ ID NO:80; or customerinitiated residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:80; and at least a stretch of amino acid residues chosen from SEQ ID NO:84, 89 and 91; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO:84, 89 and 91, provided that the amino acid sequence containing the data plots of the amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the data areas of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasma resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:0; any stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:80; and at least SEQ ID NO:84; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:84, provided that the amino acid sequence containing the data plots of the amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the data areas of amino acid residues without the 2 or 1 amino acid differences, moreover, the affinity is measured by the method of plasma resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:80; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:80; and at least a stretch of amino acid residues chosen from SEQ ID nos:93-94; or the site amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from one of SEQ ID nos:93-94, provided that the amino acid sequence containing the data plots amino acid residues, binds to IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the data areas of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasma resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention contains at least a stretch of amino acid residues chosen from SEQ ID NO:80; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:80; and at least SEQ ID NO:93; or a stretch of amino acid residues, containing not more than 2, preferably no more than the 1 amino acid difference from SEQ ID NO:93, provided that the amino acid sequence containing the data plots of the amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the data areas of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasma resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention contains at least a stretch of amino acid residues chosen from SEQ ID NO:84, 89 and 91; or a stretch of amino acid residues containing not more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO:84, 89 and 91; and at least a stretch of amino acid residues chosen from SEQ ID nos:93-94; or the site amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from one of SEQ ID nos:93-94, provided that the amino acid sequence containing the data plots of the amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the data areas of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasma resonance.

The following specific AC is the known amino acid sequence or nanotesla of the invention contains at least a stretch of amino acid residues, selected from SEQ ID NO:84, 89 and 91; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO:84, 89 and 91; and at least SEQ ID NO:93; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:93, provided that the amino acid sequence containing the data plots of the amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the data areas of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasmon resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:84; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:84, and at least a stretch of amino acid residues chosen from SEQ ID nos:93-94; or the site amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from one of SEQ ID nos:93-94, provided that the amino acid sequence containing the data plots amino acid residues, binds to IL-6R with the same, about the same or most of the affinity compared to the amino acid sequence, contains the data areas of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasmon resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:84; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:84; and at least SEQ ID NO:93; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:93, provided that the amino acid sequence containing the data plots of the amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the data areas of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasmon resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:80 and SEQ ID NO:84.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:80 and SEQ ID NO:93.

The following specific aspect, the amino acid sequence or nanotesla izobreteyonija at least SEQ ID NO:84 and SEQ ID NO:93.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:80; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:80; and at least a stretch of amino acid residues chosen from SEQ ID NO:84, 89 and 91; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO:84, 89 and 91; and at least a stretch of amino acid residues, selected from SEQ ID nos:93-94; or the site amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from one of SEQ ID nos:93-94, provided that the amino acid sequence containing the data plots of the amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the data areas of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasmon resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:80; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:80; and by at least a stretch of amino acid residues, selected from SEQ ID NO:84, 89 and 91; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO:84, 89 and 91; and at least SEQ ID NO:93; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:93, provided that the amino acid sequence containing the data plots of the amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the data areas of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasmon resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:80; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:80; and at least SEQ ID NO:84; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:84; and at least a stretch of amino acid residues chosen from SEQ ID nos:93-94; or the site amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from one of SEQ ID nos:93-94, provided h is of amino acid sequence, contains the data plots of the amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the data areas of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasma resonance.

The following specific aspect, the amino acid sequence or nanotesla of the invention comprises at least SEQ ID NO:80; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:80; and at least SEQ ID NO:84; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:84; and at least SEQ ID NO:93; or a stretch of amino acid residues, containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:93, provided that the amino acid sequence containing the data plots of the amino acid residues binds IL-6R with the same, about the same or greater affinity compared to the amino acid sequence containing the data areas of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of plasma resonance.

The following specific aspect, the amino acid placentas is required or nanotesla of the invention comprises at least SEQ ID NO:80, SEQ ID NO:84 and SEQ ID NO:93.

Preferred combinations of sequences of CDR1, CDR2 and CDR3, defined herein for the amino acid sequences of the invention, also shown in table.A-1.

In one preferred aspect, the amino acid sequences of the invention are selected from the group consisting of:

a) SEQ ID NO's:60-69;

b) amino acid sequences that have no more than 2, preferably no more than 1 amino acid difference in one, two or all of its CDR sequences of one of SEQ ID NO''s:60-69, provided that the amino acid sequence of no more than 2, preferably no more than 1 amino acid difference in one, two or all of its CDR sequences binds to IL-6R with the same, about the same or greater affinity compared to the binding of one of SEQ ID NO''s:60-69, and affinity is measured by means of surface plasma resonance; and

(C) sequences having no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:60-69, provided that the amino acid sequence of no more than 2, preferably no more than 1 amino acid difference with one of SEQ ID NO's:60-69 binds to IL-6R with the same, about the same or greater affinity compared to the binding of one of SEQ ID NO''s:60-69, and affinity is measured by the method of surface the spas plasma resonance.

In another preferred aspect, the amino acid sequences of the invention are selected from the group consisting of:

a) SEQ ID NO's:65-69;

b) amino acid sequences that have no more than 2, preferably no more than 1 amino acid difference in one, two or all of its CDR sequences of one of SEQ ID NO''s:65-69, provided that the amino acid sequence of no more than 2, preferably no more than 1 amino acid difference in one, two or all of its CDR sequences binds to IL-6R with the same, about the same or greater affinity compared to the binding of one of SEQ ID NO''s:65-69, and the affinity is measured by means of surface plasma resonance; and

c) sequences having no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:65-69, provided that the amino acid sequence of no more than 2, preferably no more than 1 amino acid difference with one of SEQ ID NO's:65-69 binds to IL-6R with the same, about the same or greater affinity compared to the binding of one of SEQ ID NO''s:65-69, and the affinity is measured by means of surface plasmon resonance.

In the following preferred aspect, the amino acid sequences of the invention are selected from the group consisting of:

a) SEQ ID NO:66;

b) the amino acid is the shaft sequences having no more than 2, preferably no more than 1 amino acid difference in one, two or all of its CDR sequences from SEQ ID NO:66, provided that the amino acid sequence of no more than 2, preferably no more than 1 amino acid difference in one, two or all of its CDR sequences binds to IL-6R with the same, about the same or greater affinity compared to the binding of SEQ ID NO:66, and the affinity is measured by means of surface plasma resonance; and

(C) sequences having no more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:66, provided that the amino acid sequence of no more than 2, preferably no more than 1 amino acid difference with SEQ ID NO; 66 binds to IL-6R with the same, about the same or greater affinity compared to the binding of SEQ ID NO:66, and the affinity is measured by means of surface plasmon resonance.

When comparing the two plots amino acid residues (or two CDR sequences), the term "amino acid difference in one, two or all of its CDR" refers to an insertion, deletion or replacement of one amino acid residue in one position of the site amino acid residues (or CDR sequence) contained in the amino acid sequence of the invention, as defined in paragraph (B) compared with a plot of amino acid residues (or CDR sequence), contained in the amino acid sequence of the invention, as defined in paragraph (a); assumes two site amino acid residues (or two sequences CDR) can contain one or at most two such amino acid differences.

By "amino acid difference in one, two or all of its CDRs" is meant that the amino acid sequence of the invention can have not more than 2, preferably no more than 1 amino acid difference in its CDR1 and/or not more than 2, preferably no more than 1 amino acid difference in its CDR2 and/or not more than 2, preferably no more than 1 amino acid difference in its CDR3 compared to the CDR1, CDR2 and/or CDR3 in one of the amino acid sequence of (a) (i.e. one of SEQ ID NO''s:60-69); once no more than 2, preferably no more than 1 amino acid difference in its CDR1 compared with in one of the CDR1 amino acid sequence of (a) (i.e. one of SEQ ID NO''s:60-69); or not more than 2, preferably no more than 1 amino acid difference in its CDR2 compared to the CDR2 in one of the amino acid sequence of (a) (i.e. one of SEQ ID NO''s:60-69); or not more than 2, preferably no more than 1 amino acid difference in its CDR3 compared to CDR3 in one of the amino acid sequence of (a) (i.e. one of SEQ ID NO''s:60-69); or not more than 2, preferably no more than 1 amino acid differences in SV who eat CDR1 compared with in one of the CDR1 amino acid sequence of (a) (i.e. one of SEQ ID NO''s:60-69) and not more than 2, preferably no more than 1 amino acid difference in its CDR2 compared to the CDR2 in one of the amino acid sequence of (a) (i.e. one of SEQ ID NO''s:60-69); or not more than 2, preferably no more than 1 amino acid difference in its CDR1 compared with in one of the CDR1 amino acid sequence of (a) (i.e. one of SEQ ID NO''s:60-69) and not more than 2, preferably no more than 1 amino acid difference in its CDR3 compared to CDR3 in one of the amino acid sequence of (a) (i.e. one of SEQ ID NO''s:60-69); or not more than 2, preferably no more than 1 amino acid difference in its CDR2 compared to the CDR2 in one of the amino acid sequence of (a) (i.e. one of SEQ ID NO''s:60-69) and not more than 2, preferably no more than 1 amino acid difference in its CDR3 compared to CDR3 in one of the amino acid sequence of (a) (i.e. one of SEQ ID NO''s:60-69); or not more than 2, preferably no more than 1 amino acid difference in its CDR1 compared with in one of the CDR1 amino acid sequence of (a) (i.e. one of SEQ ID NO''s:60-69), not more than 2, preferably no more than 1 amino acid difference in its CDR2 compared to the CDR2 in one of the amino acid sequence of (a) (i.e. one of SEQ ID NO''s:60-69) and not more than 2, preferably no more than 1 amino acid difference in its CDR3 compared to CDR3 in one of aminoxy the pilot sequences from (a) (i.e. one of SEQ ID NO''s:60-69).

"Amino acid difference in one, two or all of its CDRs" can represent one or a maximum of two substitutions, deletions or insertions in one or more areas CDR or any combination thereof, that either improve the properties of the amino acid sequence of the invention, or at least not deteriorate (decrease) too much of the desirable properties or the balance or combination of desired properties of the amino acid sequence of the invention. In this regard, the derived amino acid sequence of the invention should at least be contacted with IL-6R with the same, about the same or a higher affinity compared to the amino acid sequence containing one or several segments of amino acid residues without one or a maximum of two substitutions, deletions or insertions, and the affinity is measured by means of surface plasmon resonance. The obtained amino acid sequences are preferably such that they can communicate with a specific epitope on the receptor of IL-6 with an affinity (suitably measured and/or expressed in the form of values of KD(real or perceived), the values of KA(real or perceived), the rate constant konand/or rate constants koffor as the value IC50ka is described later), which is defined here. The obtained amino acid sequences preferably have activity on cells and activity in plasma, as defined here.

In one aspect of the invention, "amino acid difference in one, two or all of its CDRs" presents amino acid substitution. Amino acid substitutions may be one or a maximum of two substitutions in one or more areas CDR that either improve the properties of the amino acid sequence of the invention, or at least not too much impair the desirable properties or the balance or combination of desired properties of the amino acid sequence of the invention. In this regard, the derived amino acid sequence of the invention should at least be contacted with IL-6R with the same, about the same or a higher affinity compared to the amino acid sequence containing one or several segments of amino acid residues without one or a maximum of two substitutions, and the affinity is measured by means of surface plasma resonance. The obtained amino acid sequences are preferably such that they can communicate with a specific epitope on the receptor of IL-6 with an affinity (suitably measured and/or expressed in the form of values of kd(valid or is gudegast), values of KA(real or perceived), the rate constant konand/or rate constants koffor as the value IC50as described hereinafter), which is defined here. The obtained amino acid sequences preferably have activity on cells and activity in plasma, as defined here.

As discussed above, amino acid substitutions in the CDR can be any possible replacement, such as "conservative substitutions" (as defined above), and they can be done according to certain rules (as defined here) and/or may cause the improvement of the properties of the obtained amino acid sequences.

The invention also relates to amino acid sequences that have no more than 2, preferably no more than 1 amino acid differences from the one (full) sequences SEQ ID NO's:60-69.

When comparing two amino acid sequences, the term "amino acid difference" refers to an insertion, deletion or replacement of one amino acid residue in the same position of the first amino acid sequence compared with the second amino acid sequence, meaning that the two amino acid sequences can contain one or at most two such amino acid differences.

"Amino acid difference" can submit the best one or a maximum of two substitutions, deletions or insertions in the amino acid sequence, i.e., in one or more frame sections or in one or more areas CDR or any combination of them, that either improve the properties of the amino acid sequence of the invention, or at least not too much impair the desirable properties or the balance or combination of desired desired properties of the amino acid sequence of the invention. In this regard, the derived amino acid sequence of the invention should at least be contacted with IL-6R with the same, about the same or a higher affinity compared to the amino acid sequence containing one or several segments of amino acid residues without one or a maximum of two substitutions, and the affinity is measured by means of surface plasmon resonance. The obtained amino acid sequences are preferably such that they can communicate with a specific epitope on the receptor of IL-6 with an affinity (suitably measured and/or expressed in the form of values of KD(real or perceived), the values of KA(real or perceived), the rate constant konand/or rate constants koffor as the value IC50as described hereinafter), which is defined here. The resulting linakis is now the sequence is also preferably have activity on cells and activity in plasma, as defined here. The specialist will be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations thereof, and determine their influence on the properties of the resulting amino acid sequence.

In one aspect of the invention, "amino acid difference" is an amino acid substitution. Amino acid substitutions may be one or a maximum of two substitutions in one or more frame sections or in one or more areas CDR or any combination of them, that either improve the properties of the amino acid sequence of the invention, or at least not too much impair the desirable properties or the balance or combination of desired desired properties of the amino acid sequence of the invention. In this regard, the derived amino acid sequence of the invention should at least be contacted with IL-6R with the same, about the same or a higher affinity compared to the amino acid sequence containing one or several segments of amino acid residues without one or a maximum of two substitutions, and the affinity is measured by means of surface plasmon resonance. The obtained amino acid sequences are preferably such that they can communicate with a specific epitope on the receptor of IL-6 with DDPs is the your (suitably measured and/or expressed as a value To the (real or perceived), values of KD(real or perceived), the rate constant konand/or rate constants koffor as the value IC50as described hereinafter), which is defined here. The obtained amino acid sequences preferably have activity on cells and activity in plasma, as defined here. The specialist will be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations thereof, and determine their influence on the properties of the resulting amino acid sequence.

As described above, substitutions, insertions or deletions can be in one or more frame sections and/or in one or more sections of the CDR. As discussed above, the amino acid substitution in one or more CDRs may be any replacement, such as "conservative substitution" (as defined above), can be done according to certain rules (as defined here) and/or may cause the improvement of the properties of the obtained amino acid sequences.

When such substitutions, insertions or deletions are made at one or more frame parts, they can be produced by one or more Hallmark residues (e.g., as defined in WO 08/020079; PL.from A-3 to a-8) and/or one or more other provisions in the frame rests, although replacement, staccioli deletions balance Hallmark is usually less preferable (unless this is not appropriate human nature and humanizing substitutions as described here). As non-restrictive examples, the replacement may be, for example, a conservative substitution (as described herein) and/or amino acid residue may be replaced with another amino acid residue that naturally occurs in the same position in another domain VHH(see WO 08/020079, PL.from A-5 to a-8), although the invention is generally not limited to this.

Substitutions, insertions or deletions made (preferably) one or more frame sections, can be a replacement for the optimization of the sequence of frame sections, such, e.g., as human nature and humanizing substitutions. Some preferred, but not restrictive human nature and humanizing substitutions (and their appropriate combinations) will become known to the specialists of the presentation. Potentially useful human nature and humanizing substitutions can be ascertained by comparing the sequence of frame sections, one of the amino acid sequences of the invention, shown in a), with the corresponding frame sequence of one or more closely related sequences VHman, after which this amino acid sequence of the invention, shown in a), there may be one or more of the installed while potentially useful human nature and humanizing substitutions (or their combination is s) (in any way, known per se, as described later), and the resulting amino acid sequence can be tested for affinity for IL-6R, stability, ease and level of expression and/or other desirable properties defined here. Thus, by a limited number of trial and error, specialists of the presentation can be set to other suitable human nature and humanizing substitutions (or their appropriate combinations).

Depending on the host organism used for expression of amino acid sequences, nanotesla or polypeptide of the invention, such deletions and/or substitutions may be made and thus to remove one or more sites for post-translational modifications (such as one or more sites of glycosylation), which should be within the competence of specialists in this field. On the other hand, substitution or insertion can be designed to enter one or multiple sites for attachment of functional groups (as described here), for example, for site-specific tahilramani (again as described here).

As you can see from the data on the entropy of VHHand variability of VHHshown in the table.from A-5 to a-8 WO 08/020079, some amino acid residues in frame areas are more conservative than others. In General, although the invention in its broadest smiley not limited to, any substitutions, deletions or insertions, preferably produced in a less conservative positions. Also amino acid substitutions generally preferable amino acid deletions or insertions.

The obtained amino acid sequences of the invention or nanotesla invention should preferably be contacted with IL-6R with the same affinity (suitably measured and/or expressed in the form of values of KD(real or perceived), the values of KD (real or perceived), speed konand/or speed koffor as the value IC50as described hereinafter), preferably they are:

- contact hIL-6 with a dissociation constant (Ko) from 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less;

and/or such that they:

- contact cynoIL-6R with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less;

and/or such that they:

- contact hIL-6 with a rate constant konfrom 104M-1s-1up to 107M-1s-1preferably from 105M-1s-1up to 107M-1s -1more preferably about 106M-1s-1or more;

and/or such that they:

- contact cynoIL-6R with a rate constant konfrom 104M-1s-1up to 107M-1s-1preferably from 10 M-1s-1up to 107M-1s-1more preferably about 106M-1s-1or more;

and/or such that they:

- contact hIL-6 with a rate constant kofffrom 10-3(t½ =0,69 s) up to 10-1s-1(giving almost irreversible complex with t½ in a few days), preferably between 10-4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less;

and/or such that they:

- contact cynoIL-6R with a rate constant kofffrom 10-3s-1(t½ =0,69 s) up to 10-6s-1(giving almost irreversible complex with t½ in a few days), preferably between 10-4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less.

Some preferred values IC50for binding of the amino acid sequences of the invention to IL-6R will become clear from the further description and the examples.

Activity and/or the efficiency of amino acid sequences and nantel of the invention and compositions containing them can be tested by any suitable method in vitro, the cells, in vivo and/or animal model known per se, or any of their combinations, depending on specific diseases or disorders. Suitable methods and models in animals should be known to experts, they include, for example, analysis of cell proliferation in IL-6-dependent cell lines, including TF-1, XG1 and 7TD1, the model of collagen-induced arthritis, a model of transplantation of synovial tissue in SCID mice, a model of heterotransplantation various types of human cancer, including lymphoma, myeloma, prostate cancer and renal cell carcinoma, models of IBD, including the TNBS model in primates (e.g., such as described in Shinkura et al., 1998, Anticancer Research 18: 1217-1222), models of arthritic disease-human primates (e.g., as described in Vierboom et al., 2008, Drug Discov. Today: Dis Model doi:10.1016/j.ddmod. 2008.06.003), as well as methods and models for animals used in the following experimental part and there in the works prior art (Peake et al., 2006, Rheumatology 45: 1485-9; Wahid et al., 2000, Clin. Exp. ImmunoL, 122: 133-142; Matsuno et al., 1998, Arthritis and Rheumatism 41: 2014-2021; WO 08/020079).

For example, in the method TF-1, described by Kitamura et al. (1989, J. Cell Physiol. 140: 323), the amino acid sequence of the invention or nanotesla of the invention can have values IC50(ME at 100 IL-6/ml) between 10 nm and 50 PM, preferably between 5 nm and 50 PM, more preferably between 1 nm and 50 PM or less, such as note the RNO 750 or 500 PM or less. When defining this method TF-1 amino acid sequence of the invention or nanotesla of the invention can have values IC50(5,000 IU of IL-6/ml) between 50 nm and 10 nm, preferably between 25 nm and 1 nm, more preferably between 10 nm and 1 nm or less, such as 8 nm or less. When defining this method TF-1 amino acid sequences of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more times compared with the value of the IC50received control IgG provided in SEQ ID NO:1 and 2, or control Fab, shown in SEQ ID NO:3 and 4 (see Example 1). When defining this method TF-1 amino acid sequences of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more times compared with the value of the IC50obtained for the Tocilizumab (MRA).

In the determination method of the activity in the plasma when the values EC50for IL-6 (e.g., in the presence of 27,29 ng IL-6/ml as described in Example 45) the amino acid sequence of the invention is whether nanotesla of the invention can have values IC 50between 500 PM and 50 PM, preferably between 250 PM and 50 PM, more preferably between 200 PM and 50 PM or less, such as 150 PM or less. In the determination method of the activity in the plasma when the values AS for IL-6 (e.g., in the presence 885 ng IL-6/ml as described in Example 45) the amino acid sequence of the invention or nanotesla of the invention can have values IC50between 1000 PM and 100 PM, preferably between 750 PM and 100 PM, more preferably between 500 PM and 100 PM or less, 400 PM or less. When defining this method activity in plasma amino acid sequence of the invention or nanotesla of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more times compared with the value of the IC50received control IgG provided in SEQ ID NO:1 and 2, or control Fab, shown in SEQ ID NO:3 and 4 (see Example 1). When defining this method activity in plasma amino acid sequence of the invention or nanotesla of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, the school more preferably 5 times, 7 times or more times compared with the value of the IC50obtained for the Tocilizumab (MRA).

When defining a method binding with membrane IL-6R on the cell SNO amino acid sequence of the invention or nanotesla of the invention can have values IC50between 10 nm and 100 PM, preferably between 5 nm and 100 PM, more preferably between 2 nm and 10 PM or less, such as 2 nm or less.

As should be clear from the above presentation, the scope of the invention also includes use of parts or fragments, or combinations of two or more parts or fragments of amino acid sequences or of Manotel of the invention, as defined here, in particular parts or fragments of amino acid sequence SEQ ID NO's:60-69. Thus, according to one embodiments of the invention, the term "amino acid sequence of the invention" or "nanotesla of the invention in its broadest sense covers and such parts or fragments.

In General, such parts or fragments of amino acid sequences or of Manotel of the invention (including their analogs) have amino acid sequences in which, compared with the amino acid sequence of the corresponding full amino acid sequence or nanotesla of the invention, one or more amino acid residues at N-end, one or several who are amino acid residues at the C-end, one or more contiguous internal amino acid residues, or any combination were subjected to deletions and/or deleted.

The parts or fragments are preferably such that they can communicate with a specific epitope on the receptor of IL-6 with an affinity (suitably measured and/or expressed as a value To the (real or perceived), the values of KD(real or perceived), the rate constant konand/or koffor as the value IC50as described later), as defined here.

In particular, amino acid sequences, nanotesla and their parts or fragments are preferably such that they:

- contact hIL-6 with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less;

and/or such that they:

- contact cynoIL-6R with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less;

and/or such that they:

- contact hIL-6 with a rate constant konfrom 104M-1s-1up to 107M-1s-1preferably from 105M -1s-1up to 107M-1s-1more preferably about 106M-1s-1or more;

and/or such that they:

- contact cynoIL-6R with a rate constant konfrom 104M-1s-1up to 107M-1s-1preferably from 105M-1s-1up to 107M-1s-1more preferably about 106M-1s-1or more;

and/or such that they:

- contact hIL-6 with a rate constant kofffrom 10-3s-1(t½ =0,69 s) up to 10-6s-1(giving almost irreversible complex with t½ in a few days), preferably between 10-4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less;

and/or such that they:

- contact cynoIL-6R with a rate constant kofffrom 10-3s-1(t½ =0,69 s) up to 10-6s-1(giving almost irreversible complex with t½ in a few days), preferably between 10-4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less.

The affinity of the parts or fragments against the receptor of IL-6 may be determined by the method known per se, for example, described herein a method of measurement.

Luba is a part or fragment is preferably as follows, that they contain at least 10 contiguous amino acid residues, preferably at least 20 contiguous amino acid residues, more preferably at least 30 contiguous amino acid residues, such as at least 40 contiguous amino acid residues of the amino acid sequence of the corresponding full-sized amino acid sequence or nanotesla of the invention.

Also, any part or fragment is preferably such that they contain at least one CDR1, CDR2 and/or CDR3 or at least part of it (in particular, at least the CDR3 or at least part of it). More preferably, any part or fragment is such that they contain at least one CDR (preferably at least CDR3 or a part thereof) and at least one other CDR (i.e., CDR1 or CDR2) or at least part, preferably the United corresponding frame sequence or at least a part of it. More preferably, any part or fragment is such that they contain at least one CDR (preferably at least CDR3 or a part thereof) and at least part of the other two CDR, again preferably connected corresponding frame sequence or at least a part of it.

In accordance with another especially preferred, but not ogranichitel the NYM embodiment, such part or fragment contains at least CDR3, such as FR3, CDR3 and FR4 of the corresponding full-size nanotesla of the invention, for example as described in International application WO 03/050531 (Lasters et al.).

As mentioned above, it is also possible to combine two or more of such parts or fragments (of the same or different amino acid sequences or of Manotel of the invention), i.e. to get more parts or fragments (as defined here) amino acid sequences or nanotesla invention. Also, for example, you can combine one or more parts or fragments of amino acid sequence or nanotesla of the invention with one or more parts or fragments of domain VHperson.

In accordance with one preferred embodiment, the parts or fragments have a sequence that is at least 50%, preferably at least 60%, more preferably 70%, even more preferably at least 80%, such as 90%, 95% or 99% or more identical to one of the amino acid sequences or of Manotel for SEQ ID NO's:60-69.

Parts and fragments and encoding their nucleotide sequences can be obtained and need not be combined in any manner known per se. For example, such parts or fragments can be obtained by inserting a stop codona nucleic acid, encoding a full-sized amino acid sequence or nanotesla of the invention, and then expressive resulting nucleic acid by a method known per se (as described here). On the other hand, can be obtained nucleic acid encoding such parts or fragments, using the corresponding restriction nucleic acid that encodes a full-sized amino acid sequence or nanotesla invention or synthesizing such nucleic acid by a method known per se. Parts or fragments can also be obtained by the methods of peptide synthesis known per se.

The invention further relates to compounds or constructs, containing or consisting primarily of one or more amino acid sequences or of Manotel of the invention, and optionally also containing one or more other groups, residues, molecules or binding units, optionally connected via one or more linkers. In a preferred aspect of the data one or more other groups, residues, molecules or binding units are amino acid sequences. In another preferred aspect of the data is one or more linkers are amino acid sequences. Such compounds or constructs are also referred to as "polypeptides of the invention".

P is dipeptide of the invention may contain amino acid sequence or nanotesla of the invention, fused at its N end, With the end or at the N-end and C-end of at least one amino acid sequence, thus obtaining the fused protein containing the amino acid sequence or nanotesla of the invention and one or more amino acid sequences.

One or more additional amino acid sequences can be any suitable and/or desirable amino acid sequence. Additional amino acid sequence may change or not change, alter or otherwise influence the (biological) properties of the amino acid sequence or nanotesla of the invention and can give or not to give additional functionality to the amino acid sequence, nanotesla or polypeptide of the invention. Preferably, the additional amino acid sequence is such that it imparts one or more desired properties or functionalities of the amino acid sequence, nanotesla or polypeptide of the invention.

Examples of such amino acid sequences must be known in the art and in General can contain all of the amino acid sequences used in the fusion peptides on the basis of conventional antibodies and their fragments (including yield of scFv's and od is domenie antibodies). We will refer, for example, to review Holliger and Hudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

For example, such amino acid sequence may be an amino acid sequence that increases the half-life, solubility or absorption, reduces the immunogenicity or toxicity, eliminates or reduces undesirable side effects and/or imparts other desirable properties and/or reduce the undesired properties of polypeptides of the invention compared to the amino acid sequence or nanotesla the invention per se. Some non-restrictive examples of such amino acid sequences represented by serum proteins such as serum albumin (for example, see WO 00/27435) or molecule haptens (e.g., haptens, recognized by circulating antibodies, for example, see WO 98/22141).

Additional amino acid sequences can also provide a second binding site, which may be directed against any desired protein, polypeptide, antigen, antigenic determinant or epitope (including the same protein, polypeptide, antigen, antigenic determinant or epitope against which it is directed amino acid sequence or nanotesla of the invention, or another protein, polypeptide, antigen, antigenic determinant or epitope). For example, additional is minociclina sequence can provide a second binding site, directed against the protein (such, for example, as serum albumin human, or another protein such as IgG), which provides a longer half-life in serum. Such amino acid sequences, for example, include nanotesla, as well as small peptides and binding proteins described in WO 91/01743, WO 01/45746 and WO 02/076489, and dAb's described in WO 03/002609 and WO 04/003019. We will refer also to Harmsen et al. (2005, Vaccine, 23 (41): 4926-42), and EP 0368684, as WO 08/028977, WO 08/043821, WO 08/043822 and WO 08/068280 from Ablynx N. V.

Preferred amino acid sequences, which can give the amino acid sequences or nanocell inventions increased the half-life, can be selected from SEQ ID NO's:97-99.

Such amino acid sequences, in particular, can be directed against serum albumin (particularly serum albumin) and/or against IgG (particularly IgG person). For example, such amino acid sequences can be amino acid sequence directed against serum albumin (human), and amino acid sequences which are able to bind with the amino acid residues in serum albumin (human) that are not involved in binding of serum albumin to FcRn (for example, see WO 06/0122787), and/or amino acid is posledovatelnosti, the ability to communicate with those amino acid residues in serum albumin, which are not part of domain III of serum albumin (again, for example, see WO 06/0122787); amino acid sequences that provide or can provide longer half-life (for example, see WO 08/028977); amino acid sequences against serum albumin person that can cross-react with serum albumin from at least one of species of mammals, in particular, at least one species of Primate (such as, without limitation, monkeys from the genus of Masasa (in particular, long-tailed macaques (cynomolgus monkey, Masasa fascicularis) and/or rhesus monkeys (Masasa mulatto)) and baboon (Papio ursinus), again, we refer to WO 08/028977); amino acid sequences which are able to bind with serum albumin regardless of pH (for example, see WO 08/043821), and/or amino acid sequences that are conditional binding molecules (for example, see WO 08/043822).

In accordance with another embodiment, one or more additional amino acid sequences can contain one or more parts, fragments or domains of conventional 4-chain antibodies (in particular, antibodies) and/or heavy chain antibodies. For example, although this is usually less preferably, linakis is now a sequence or nanotesla of the invention can be connected with a conventional (preferably human) domain V Hor VLeither natural or synthetic analogue domain VHor VLagain optionally via a linker sequence (including other (single)domain antibodies type dAb's described by Ward et al.).

Accordingly, the compound or construct of the invention data is one or more groups, residues, molecules or binding units may be selected from the group consisting of domain antibodies, amino acid sequences that are suitable for use as domain antibodies, single domain antibodies, amino acid sequences that are suitable for use as single domain antibodies, antibody "dAb", amino acid sequences that are suitable for use as a dAb, or nantel.

In one aspect of the invention, connection, design or polypeptide of the invention containing at least one amino acid sequence or nanotesla of the invention may have an increased half-life compared with the corresponding amino acid sequence or nanotesla invention. Some preferred, but not restrictive examples of such compounds, constructs and polypeptides will become clear to experts from the following narrative, they can be, for example, compounds, constructs or polypeptides containing minocyclinee sequence, nanotesla or polypeptides of the invention, subjected to chemical modification to increase their half-life (for example, by tahilramani); or polypeptides of the invention containing at least one amino acid sequence or nanotesla of the invention, connected to at least one molecule (in particular, at least one amino acid sequence) that increases the half-life of Manotel invention. Examples of the compounds, constructs or polypeptides of the invention containing such increases the half-life of molecules or amino acid sequences will be known to experts from the following statement; for example, they include, without limitation, polypeptides in which one or more amino acid sequences or of Manotel invention accordingly connected to one or more serum proteins or fragments (such as serum albumin or suitable fragments) or with one or more binding units that can bind with proteins serum (such as, for example, nanotesla or (single)domain antibodies, who can contact these proteins serum as serum albumin, serum immunoglobulins IgG, or transferrin); polypeptides in which amino is sledovatelnot or nanotesla invention connects to the Fc-part (such as Fc man) or a suitable part or fragment; or polypeptides in which one or more amino acid sequences or of Manotel invention accordingly connected to one or more small proteins or peptides which are able to bind with serum proteins (such as proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489).

At least one amino acid sequence or nanotesla also can connect with one or more (preferably human) domains WithN1, CN2 and/or CH3, not necessarily through a linker sequence. For example, amino acid sequence or nanotesla, United with the appropriate domainN1, may be used, for example, together with suitable light chains, to obtain fragments of antibodies/structures analogous to conventional Fab fragments or F(ab')2-fragments, but in which one or (in the case of F(ab')2-fragment) both normal domain VHreplaced by the amino acid sequence or nanotesla invention. Amino acid sequences or nanotesla can also connect to the domain, CH3 (optionally through a linker) to obtain designs with increased half-life in vivo.

In accordance with one specific aspect of the polypeptide of the invention, one or more amino acid sequence is th or Manotel of the invention can be connected (optionally via a suitable linker or hinge section) with one or more constant domains (for example, constant domains 2 or 3, which can be used as part of or for the formation of Fc-part) with the Fc-part and/or one or more parts, fragments or domains of antibodies, giving one or more effector functions to the polypeptide of the invention and/or the ability to communicate with one or more Fc receptors. For example, for this purpose, and without limitation, one or more additional amino acid sequences can contain one or more domainsN2 and/or CN3 antibodies, such as antibody heavy chain (as described here), more preferably from conventional 4-chain antibodies; and/or can be (partially) section Fc, for example from IgG (e.g., IgG1, IgG2, IgG3 and IgG4), IgE, or from another human Ig, such as IgA, IgD or IgM. For example, in WO 94/04678 described antibodies of the heavy chains containing the domain VHHcamelid or humanitariannet derived (i.e. nanotesla), where the domain WithN2 and/or CN3 camelid replaced by domains WithN2, and CN3, receiving immunoglobulin that consists of 2 heavy chains, each of which contains nanotesla and domains WithN2 and CH3 person (but not domainN1), and that the immunoglobulin has an effector functions that are provided by the domainN2, and CN3, and this immunoglobulin can operate the work in the absence of light chains. Other amino acid sequences that can be appropriately associated with amino acid sequences or antelami of the invention to provide an effector function must be known to experts and should be selected based on the desired effector functions. We will refer, for example, in WO 04/058820, WO 99/42077, WO 02/056910 and WO 05/017148, and review Holliger and Hudson, supra; and WO 09/068628). Compound amino acid sequence or nanotesla of the invention with the Fc-part can also lead to longer half-life compared with the corresponding amino acid sequence or nanotesla invention. For some applications it may also be appropriate or even to be preferred using the Fc-part of and/or constant domains (i.e. domains WithN2 and/or CN3), which increased the half-life without any biologically significant effector function. Other suitable structures containing one or more amino acid sequences or of nantel and one or more constant domains with increased half-life in vivo, should be known in the art and can include, for example, two or more amino acid sequences or of Manotel associated with the domainN3, optionally, via a linker sequence. In the General case, the AE any protein or derivative with increased half-life preferably have a molecular weight more than 50 KD, i.e. limit cut-off for renal suction.

In another specific, but not restrictive aspect for the formation of a polypeptide of the invention it is possible to connect one or more amino acid sequences of the invention (optionally via a suitable linker or hinge section) with natural, synthetic or semi-synthetic constant domains (or their analogs, variants, mutants, parts or fragments) with a lower (or almost absent) tendency to coalesce into dimers (compared to the constant domains estestvennim way present in conventional 4-chain antibodies). Such Monomeric (i.e., not self-organizing) options Fc-chain or its fragments must be known to specialists. For example, Helm et al. (1996, J. Biol. Chem. 271: 7494) described Monomeric variants of Fc-circuit that can be used in the polypeptide chains of the invention.

Also Monomeric variants of Fc-circuit preferably such that they are still able to contact the complement or consistent with Fc-receptors (depending on the Fc-part of which they occur), and/or such that they still have some or all of the effector functions of the Fc-part from which they originated (or at a lower level, still suitable for the purpose). On the other hand, in this floor the peptide chains of the invention Monomeric Fc-circuit can be used to give increased time half-life of the polypeptide chain, while Monomeric Fc-circuit may not have or almost have affectingly functions.

In General, amino acid sequence or nanotesla of the invention (or the containing compounds, constructs or polypeptides with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times greater than the half-life of the corresponding amino acid sequence or nanotesla of the invention per se. For example, amino acid sequence, nanotesla, compounds, constructs or polypeptides of the invention with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably 6 hours, such as more than 12 hours or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.

In a preferred, but not restrictive aspect of the invention, such amino acid sequences, nanotesla, compounds, constructs and polypeptides of the invention exhibit a half-life in human serum for at least 12 hours, preferably at least 24 hours, more preferably at mere hours even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half-life of at least 5 days (such as from 5 to 10 days), preferably at least 9 days (such as from 9 to 14 days), more preferably at least 10 days (such as about 10 to 15 days) or at least 11 days (such as from 11 to 16 days), more preferably at least 12 days (such as from 12 to 18 days or more, than 14 days (such as from 14 to 19 days).

Additional amino acid sequence may also be a signal sequence or leader sequence that directs secretion of the amino acid sequence, nanotesla or polypeptide of the invention from a host cell after synthesis (for example, to provide pre-, Pro - or shall form the polypeptide of the invention, depending on the host cell used for expression of the polypeptide of the invention).

Additional amino acid sequence may be a sequence or signal that allows the amino acid sequence, nanotesla or polypeptide of the invention to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or allowing the amino acid sequence, nanotesla or polyp is ptib of the invention to penetrate or cross a biological barrier type cell membrane, cell layer type layer of epithelial cells, a tumor including solid tumors, or the blood-brain barrier. Suitable examples of such amino acid sequences must be known in the art, for example, they include, without limitation, vectors Peptrans" mentioned above, the sequence described Cardinale et al., and amino acid sequences and fragments of antibodies, known per se, which can be used to ekspressirovali or nantel and polypeptides of the invention in the form of so-called "Intertel" ("intrabodies"), for example as described in WO 94/02610, WO 95/22618, US 7,004,940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1512696; Cattaneo, A. & Biocca, S. (1997, Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag) and Kontermann (2004, Methods 34: 163-170), and provides additional references.

In accordance with one preferred, but not limiting embodiment, the amino acid sequence or nanotesla of the invention contains at least one additional amino acid sequence or nanotesla, so it's the polypeptide of the invention contains at least two, such as two, three, four, five or more amino acid sequences or of nantel, in which the amino-acid sequence or nanotesla can optionally be connected via one or more sequences Linke the offering (as defined here). The polypeptides of the invention containing two or more amino acid sequences or of Manotel invention, of which at least one amino acid sequence or nanotesla of the invention, also referred to hereinafter as "multivalent" polypeptides of the invention and the amino acid sequence or nanotesla present in such polypeptides, are also designated in the future as in a multivalent format". For example, bivalent' polypeptide of the invention contains two amino acid sequences and/or two nanotesla, not necessarily connected via a linker sequence, whereas a "trivalent" polypeptide of the invention contains three amino acid sequences and/or nanotesla, not necessarily connected across the two sequences of the linkers, and so on; and at least one of the amino acid sequences and/or nantel present in the polypeptide, and until all amino acid sequences and/or nantel present in the polypeptide, are amino acid sequences and/or antelami of the invention.

In the multivalent polypeptide of the invention two or more amino acid sequences or of Manotel may be the same or different and can be directed against the od of the CSOs and the same antigen or antigenic determinants (for example, against the same part or epitope or against different parts or epitopes) or, conversely, may be directed against different antigens or antigenic determinants or their combinations. For example, a bivalent polypeptide of the invention may include: (a) two identical amino acid sequences or nanotesla; (b) a first amino acid sequence or the first nanotesla directed against a first antigenic determinant of a protein or antigen and a second amino acid sequence or the second nanotesla directed against the same antigenic determinants of a given protein or antigen, and they differ from the first amino acid sequence, or the first nanotesla; (C) a first amino acid sequence or the first nanotesla directed against a first antigenic determinant of a protein or antigen and a second amino acid sequence or the second nanotesla, directed against different antigenic determinants of a given protein or antigen; or (d) a first amino acid sequence or the first nanotesla against the first protein or antigen and a second amino acid sequence or the second nanotesla against the second protein or antigen (i.e. different from the first antigen). Similarly trivalent polypep the ID of the invention may, for example and without limitation, include: (a) three identical amino acid sequences or nanotesla; (b) two identical amino acid sequences or two nanotesla against a first antigenic determinant of an antigen and a third amino acid sequence or a third nanotesla directed against different antigenic determinants on the same antigen; (C) two identical amino acid sequences or two napothera against a first antigenic determinant of an antigen and a third amino acid sequence or a third nanotesla directed against a second antigen different from the first antigen; (d) a first amino acid sequence or the first nanotesla, directed against a first antigenic determinant of a first antigen, a second amino acid sequence or the second nanotesla directed against a second antigenic determinant of this first antigen, and a third amino acid sequence or a third nanotesla directed against a second antigen different from the first antigen; or (e) a first amino acid sequence or the first nanotesla directed against a first antigen, a second amino acid sequence or the second nanotesla directed against a second antigen different from the first antigen, and a third amino acid is tnou sequence or third nanotesla, directed against a third antigen different from the first and second antigens.

The polypeptides of the invention containing at least two amino acid sequences and/or two nanotesla, in which at least one amino acid sequence or one nanotesla directed against a first antigen (i.e. against the receptor for IL-6) and at least one amino acid sequence or one nanotesla directed against a second antigen (i.e. different from the receptor IL-6), also referred to as the "multispecific" polypeptides of the invention and the amino acid sequence or nanotesla present in such polypeptides, also referred to as being in "multispecific format". So, for example, "bispecific" polypeptide of the invention is a polypeptide that contains at least one amino acid sequence or one nanotesla directed against a first antigen (i.e., receptor IL-6), and at least one amino acid sequence or another nanotesla directed against a second antigen (i.e. different from the receptor IL-6), whereas "trapezitinae" polypeptide of the invention is a polypeptide which contains at least one amino acid sequence or one nanotesla, directed against a first antigen (i.e., receptor IL-6), n is at least one amino acid sequence or another nanotesla, directed against a second antigen (i.e. different from the receptor IL-6), and at least one amino acid sequence or another nanotesla directed against a third antigen (i.e. different from the receptor, IL-6, and the second antigen), and so on

Accordingly, in its simplest form, bispecific polypeptide of the invention is a bivalent polypeptide of the invention (as defined here), containing the first amino acid sequence or the first nanotesla directed against the receptor for IL-6, and a second amino acid sequence or the second nanotesla directed against a second antigen, and the data of the first and second amino acid sequence or nanotesla may not necessarily be connected through a sequence of linkers (as defined here); while trapezitinae polypeptide of the invention in its simplest form is a trivalent polypeptide of the invention (as defined here), containing the first amino acid sequence or the first nanotesla, directed against the receptor for IL-6, second amino acid sequence or the second nanotesla directed against a second antigen and a third amino acid sequence or a third nanotesla directed against a third antigen, and the first, second and third amino acid after which outermost or nanotesla can optionally be connected via a sequence of one or more, preferably one or more, preferably two linkers.

In one specific aspect, the polypeptide of the invention is a trivalent bispecific polypeptide. Trivalent, bispecific polypeptide of the invention in its simplest form may be a trivalent polypeptide of the invention (as defined here), containing two identical amino acid sequences or two nanotesla against the receptor for IL-6, and a third amino acid sequence or a third nanotesla against another antigen, and the first, second and third amino acid sequence or nanotesla can optionally be connected via a sequence of one or more, preferably one or more, preferably two linkers.

In another specific aspect, the polypeptide of the invention is bispecific polypeptide. Bispecific polypeptide of the invention in its simplest form may be a bivalent polypeptide of the invention (as defined here), containing the first amino acid sequence or the first nanotesla against the receptor for IL-6, and a second amino acid sequence or the second nanotesla against another antigen, and the data of the first and second amino acid sequence or n is of the hotel can optionally be connected via a linker sequence.

In a preferred, but not restrictive example multispecific polypeptide of the invention contains at least one amino acid sequence or one nanotesla of the invention and at least one nanotesla, which provides a longer half-life. Some preferred, but not restrictive examples of such nantel include nanotesla directed against serum proteins such as serum albumin human, tyrosinase protein, transferrin (human), fibrinogen, the immunoglobulins such as IgG, IgE, or IgM, or one of the other whey proteins described in WO 04/003019.

For example, mice can be used nanotesla against serum albumin mouse (MSA), whereas for pharmaceutical applications can be used nanotesla against serum albumin human.

The following embodiment of the present invention is a polypeptide construct as described above, in which the at least one whey protein (human) is a protein from among serum albumin (human), serum immunoglobulin (human), tyrosinaemia protein (human) transferrin (human), fibrinogen (human), and so on

Accordingly, in a specific aspect, the polypeptide of the invention is trivalent, b is the specific polypeptide, containing two identical amino acid sequences or two nanotesla against the receptor for IL-6, and a third amino acid sequence or a third nanotesla directed against serum albumin (human), with these first, second and third amino acid sequence or nanotesla can optionally be connected via a sequence of one or more, preferably one or more, preferably two linkers.

In another specific aspect, the polypeptide of the invention is bispecific polypeptide containing the first amino acid sequence or the first nanotesla against the receptor for IL-6, and a second amino acid sequence or the second nanotesla directed against serum albumin (human), with these first, second and third amino acid sequence or nanotesla can optionally be connected via a linker sequence.

In accordance with one specific, but not limiting aspect of the invention, the polypeptides of the invention contain, in addition to one or more amino acid sequences or of Manotel of the invention, at least one nanotesla against serum albumin human. Although these nanotesla against serum albumin person and may be such as described in General privedennyh above applications company Ablynx N. V. (for example, see W04/062551), but in accordance with a particularly preferred, but not restrictive embodiment of the data nanotesla against serum albumin person essentially consist of the amino acid sequences selected from SEQ ID NO's:97-99.

Some preferred, but not limiting examples of polypeptides of the invention containing at least one amino acid sequence or one nanotesla against the receptor of IL-6 and at least one amino acid sequence or one nanotesla, providing a longer half-life, the following:

a) SEQ ID NO's:70-72;

b) a polypeptide sequence having no more than 2, preferably no more than 1 amino acid difference in one, two or all parts of the CDR of the invention from one of SEQ ID NO''s:70-72, provided that the polypeptide sequence of not more than 2, preferably no more than 1 amino acid difference in one, two or all parts of the CDR of the invention binds to IL-6R with the same, about the same or greater affinity compared to the binding of one of SEQ ID NO''s:70-72, and the affinity is measured by means of surface plasmon resonance; and

c) a polypeptide sequence having no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:70-72, provided that the polypeptide sequence of alnost not more than 2, preferably no more than 1 amino acid difference with one of SEQ ID NO's:70-72 binds to IL-6R with the same, about the same or greater affinity compared to the binding of one of SEQ ID NO''s:70-72, and the affinity is measured by means of surface plasma resonance.

Some preferred, but not restrictive examples of trivalent bispecific polypeptides of the invention are the following:

a) SEQ ID NO's:71-72;

b) a polypeptide sequence having no more than 2, preferably no more than 1 amino acid difference in one, two or all parts of the CDR of the invention from one of SEQ ID NO's:71-72, provided that the polypeptide sequence of not more than 2, preferably no more than 1 amino acid difference in one, two or all parts of the CDR of the invention binds to IL-6R with the same, about the same or greater affinity compared to the binding of one of SEQ ID NO''s:70-72, and the affinity is measured by means of surface plasma resonance; and

c) a polypeptide sequence having no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:71-72, provided that the polypeptide sequence of not more than 2, preferably no more than 1 amino acid difference with one of SEQ ID NO's:71-72 binds to IL-6R with the same, about the same or greater affinity p is compared with the binding of one of SEQ ID NO's:71-72, moreover, the affinity is measured by means of surface plasma resonance.

Some preferred, but not restrictive examples bispecific polypeptides of the invention containing one amino acid sequence or one nanotesla against the receptor for IL-6, and one amino acid sequence or one nanotesla, providing a longer half-life, the following:

a) SEQ ID NO:70;

b) a polypeptide sequence having no more than 2, preferably no more than 1 amino acid difference in one, two or all parts of the CDR of the invention from SEQ ID NO:70, provided that the polypeptide sequence of not more than 2, preferably no more than 1 amino acid difference in one, two or all parts of the CDR of the invention binds to IL-6R with the same, about the same or greater affinity compared to the binding of SEQ ID NO:70, and the affinity is measured by means of surface plasmon resonance, and

c) a polypeptide sequence having no more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:70, provided that the polypeptide sequence of not more than 2, preferably no more than 1 amino acid difference with SEQ ID NO:70 binds to IL-6R with the same, about the same or greater affinity compared to the binding of SEQ ID NO:70, and the affinity measures the I by means of surface plasmon resonance.

When comparing the two plots amino acid residues (or two CDR sequences), the term "amino acid difference in one, two or all parts of the CDR of the invention" refers to an insertion, deletion or replacement of one amino acid residue in one position of the site amino acid residues (or CDR sequence) of the invention contained in the polypeptide of the invention defined in paragraph (b), compared with a plot of amino acid residues (or CDR sequence) of the invention contained in the polypeptide of the invention defined in paragraph (a); assumes two site amino acid residues (or two sequences CDR) the invention can contain one or at most two such amino acid differences.

By "amino acid difference in one, two or all parts of the CDR of the invention" is meant that the amino acid sequence or nanotesla of the invention contained in the polypeptide of the invention may have no more than 2, preferably no more than 1 amino acid difference in its CDR1 and/or not more than 2, preferably no more than 1 amino acid difference in its CDR2 and/or not more than 2, preferably no more than 1 amino acid difference in its CDR3 (i.e. CDR1, CDR2 and/or CDR3, forming antigennegative site for binding of the compound or polypeptide of the invention with a specific epitope IL-6R) compared to the CDR1, CDR2 and/or CDR3 in amino acid sequence or nanotesla of the invention contained in one of the polypeptides of (a) (i.e. one of SEQ ID NO''s:60-69); as it is not more than 2, preferably no more than 1 amino acid difference in its CDR1 (i.e. CDR1, forming antigennegative site for binding of the compound or polypeptide of the invention with a specific epitope on IL-6R) than in CDR1 amino acid sequence or nanotesla of the invention contained in one of the polypeptides of (a) (i.e., CDR1 in one of SEQ ID NO''s:60-69); or not more than 2, preferably no more than 1 amino acid difference in its CDR2 (i.e., in CDR2, forming antigennegative site for binding of the compound or polypeptide of the invention with a specific epitope on IL-6R) in comparison with the CDR2 in amino acid sequence or nanotesla of the invention contained in one of the polypeptides of (a) (i.e., CDR2 in one of SEQ ID NO''s:60-69); or not more than 2, preferably no more than 1 amino acid difference in its CDR3 (i.e., at CDR3, forming antigennegative site for binding of the compound or polypeptide of the invention with a specific epitope on IL-6R) compared to CDR3 in amino acid sequence or nanotesla of the invention contained in one of the polypeptides of (a) (i.e., CDR3 in one of SEQ ID NO''s:60-69); or not more than 2, preferably no more than 1 amino acid difference in its CR1 (i.e., in CDR1, forming antigennegative site for binding of the compound or polypeptide of the invention with a specific epitope on IL-6R) than in CDR1 amino acid sequence or nanotesla of the invention contained in one of the polypeptides of (a) (i.e., CDR1 in one of SEQ ID NO''s:60-69), and not more than 2, preferably no more than 1 amino acid difference in its CDR2 (i.e., in CDR2, forming antigennegative site for binding of the compound or polypeptide of the invention with a specific epitope on IL-6R) compared to the CDR2 in amino acid sequence or nanotesla of the invention contained in one of the polypeptides of (a) (i.e., CDR2 in one of SEQ ID NO''s:60-69); or not more than 2, preferably no more than 1 amino acid difference in its CDR1 (i.e. CDR1, forming antigennegative site for binding of the compound or polypeptide of the invention with a specific epitope on IL-6R) than in CDR1 amino acid sequence or nanotesla of the invention contained in one of the polypeptides of (a) (i.e., CDR1 in one of SEQ ID NO''s:60-69), and not more than 2, preferably no more than 1 amino acid difference in its CDR3 (i.e., at CDR3, forming antigennegative site for binding of the compound or polypeptide of the invention with a specific epitope on IL-6R) compared to CDR3 in amino acid sequence or nanotesla of the invention, soteriades is in one of the polypeptides of (a) (i.e. CDR3 in one of SEQ ID NO''s:60-69); or not more than 2, preferably no more than 1 amino acid difference in its CDR2 (i.e., in CDR2, forming antigennegative site for binding of the compound or polypeptide of the invention with a specific epitope on IL-6R) in comparison with the CDR2 in amino acid sequence or nanotesla of the invention contained in one of the polypeptides of (a) (i.e., CDR2 in one of SEQ ID NO''s:60-69), and not more than 2, preferably no more than 1 amino acid difference in its CDR3 (i.e., at CDR3, forming antigennegative site for binding of the compound or polypeptide of the invention with a specific epitope on IL-6R) compared to CDR3 in amino acid sequence or nanotesla of the invention contained in one of the polypeptides of (a) (i.e., CDR3 in one of SEQ ID NO''s:60-69); or not more than 2, preferably no more than 1 amino acid difference in its CDR1 (i.e. CDR1, forming antigennegative site for binding of the compound or polypeptide of the invention with a specific epitope on IL-6R) than in CDR1 amino acid sequence or nanotesla of the invention contained in one of the polypeptides of (a) (i.e., CDR1 in one of SEQ ID NO''s:60-69), and not more than 2, preferably no more than 1 amino acid difference in its CDR2 (i.e., in CDR2, forming antigennegative site for binding of the compound or polypeptide of the invention with specific EPI the ops on IL-6R) compared to the CDR2 in amino acid sequence or nanotesla of the invention, contained in one of the polypeptides of (a) (i.e., CDR2 in one of SEQ ID NO''s:60-69), and not more than 2, preferably no more than 1 amino acid difference in its CDR3 (i.e., at CDR3, forming antigennegative site for binding of the compound or polypeptide of the invention with a specific epitope on IL-6R) compared to CDR3 in amino acid sequence or nanotesla of the invention contained in one of the polypeptides of (a) (i.e., CDR3 in one of SEQ ID NO''s:60-69).

"Amino acid difference in one, two or all of its CDRs" can represent one or a maximum of two substitutions, deletions or insertions in one or more sections of the CDR of the invention or combinations thereof, that either improve the properties of the compound or polypeptide of the invention or at least not too much affect (reduce) the desirable properties or the balance or combination of desired properties of the compound or polypeptide of the invention. In this respect, the obtained compound or polypeptide of the invention should at least be contacted with IL-6R with the same, about the same or a higher affinity compared with the compound or polypeptide that contains one or more plots CDR of the invention one or a maximum of two substitutions, divisions, or inserts, and the affinity is measured by means of surface plasma resonance. The compounds or polypep the IDA preferably are they can be contacted with a specific epitope on the receptor of IL-6 with an affinity (suitably measured and/or expressed in the form of values of KD(real or perceived), the values of KD (real or perceived), the rate constant konand/or rate constants koffor as the value IC50as described hereinafter), which is defined here. The compounds or polypeptides also preferably have activity on cells and activity in plasma, as defined here.

In one aspect of the invention, "amino acid difference in one, two or all of its CDRs" presents amino acid substitution. Amino acid substitutions may be one or a maximum of two substitutions in one or more areas CDR that either improve the properties of the compound or polypeptide of the invention or at least not too much affect (reduce) the desirable properties or the balance or combination of desired properties of the compounds or constructs of the invention. In this respect, the obtained compound or polypeptide of the invention should at least be contacted with IL-6R with the same, about the same or a higher affinity compared with the connection or structure that contains one or more plots CDR without one or a maximum of two substitutions, and affinity change aetsa by means of surface plasmon resonance. The compounds or polypeptides preferably are such that they can communicate with a specific epitope on the receptor of IL-6 with an affinity (suitably measured and/or expressed in the form of values of KA(real or perceived), the values of KD(real or perceived), the rate constant konand/or rate constants koffor as the value IC50as described hereinafter), which is defined here. The compounds or polypeptides also preferably have activity on cells and activity in plasma, as defined here. Specialist in General will be able to determine and select suitable substitutions based on the above statement, and optionally after a limited number of simple experiments that can, for example, include the introduction of a limited number of possible substitutions and determining their influence on the properties of the resulting compounds or polypeptides.

Amino acid substitutions in one or more CDRs of the invention can be any possible replacement, such as "conservative substitutions" (as defined above), and they can be done according to certain rules (as defined here) and/or may cause the improvement of the properties of the obtained compounds or polypeptides (as hereinafter defined).

The invention also relates to the of soedinenii or polypeptides, having no more than 2, preferably no more than 1 amino acid differences from the one (full) sequences SEQ ID NO's:70-72.

When comparing two compounds or polypeptides, the term "amino acid difference" refers to an insertion, deletion or replacement of one amino acid residue in the same position of the first connection, or a polypeptide as compared to the second compound or polypeptide; assumes that two connections or polypeptide can contain one or at most two such amino acid differences.

"Amino acid difference" can represent one or a maximum of two substitutions, deletions or insertions in connection or polypeptide, i.e., in one or more frame sections or in one or more areas CDR (which may be a CDR of the invention, i.e., in amino acid sequence or nanotesla of the invention, or other CDRs, i.e., in SEQ ID NO:98), in the sequence of the linker, or any combination thereof, that either improve the properties of the amino acid sequence of the invention, or at least not too much worse (less) desirable properties or the balance or combination of desired properties of the compound or polypeptide of the invention. In this respect, the obtained compound or polypeptide of the invention should at least contact with the receptor of IL-6 with t is Kim, about the same or greater affinity than compound or polypeptide without one or a maximum of two substitutions, deletions or insertions, and the affinity is measured by means of surface plasma resonance. The compounds or polypeptides preferably are such that they can communicate with a specific epitope on the receptor of IL-6 with an affinity (suitably measured and/or expressed in the form of values of KD(real or perceived), the values of KA(real or perceived), the rate constant konand/or rate constants koffor as the value IC50as described hereinafter), which is defined here. The compounds or polypeptides also preferably have activity on cells and activity in plasma, as defined here.

In one aspect of the invention, "amino acid difference" represents the amino acid replacement. Amino acid substitutions may be one or a maximum of two substitutions in the frame sections, in one or more areas CDR (which may be a CDR of the invention, i.e., in amino acid sequence or nanotesla of the invention, or other CDRs, i.e., in SEQ ID NO:98), in the sequence of the linker, or any combination thereof, that either improve the properties of consecutive amino acid the activity of the invention, or at least not too much affect (reduce) the desirable properties or the balance or combination of desired properties of the compound or polypeptide of the invention. In this respect, the obtained compound or polypeptide of the invention should at least contact with the receptor of IL-6 with the same, about the same or greater affinity than compound or polypeptide without one or a maximum of two substitutions, and the affinity is measured by means of surface plasmon resonance. The compounds or polypeptides preferably are such that they can communicate with a specific epitope on the receptor of IL-6 with an affinity (suitably measured and/or expressed in the form of values of KD(real or perceived), the values of KA(real or perceived), the rate constant konand/or rate constants koffor as the value IC50as described hereinafter), which is defined here. The compounds or polypeptides also preferably have activity on cells and activity in plasma, as defined here.

As described above, substitutions, insertions or deletions can be in one or more frame sections, in one or more areas of CDR and/or in one or more sequences of linkers. Replace, insert, or is elechi in CDR can be any possible replacement, insertions or deletions, such as "conservative substitutions" (as defined here), they can be done according to certain rules (as defined here) and/or may cause the improvement of the properties of the obtained compounds or polypeptides.

When such substitutions, insertions or deletions are made at one or more frame sections, they can represent any possible substitutions, insertions or deletions. They can be produced by one or more Hallmark residues (e.g., as defined in WO 08/020079; PL.from A-3 to a-8) and/or one or more other provisions in the frame rests, although substitutions, insertions or deletions balance Hallmark is usually less preferable (unless this is not appropriate human nature and humanizing substitution as described here). As non-restrictive examples, the replacement may be, for example, a conservative substitution (as described herein) and/or amino acid residue may be replaced with another amino acid residue that naturally occurs in the same position in another domain VHH(see WO 08/020079, PL.from A-5 to a-8), although the invention is generally not limited to this.

Substitutions, insertions or deletions made (preferably) one or more frame sections, can be a replacement for the optimization of the sequence, such, e.g., as human nature and humanizing substitutions. N what are preferred, but not limited to human nature and humanizing substitutions (and their appropriate combinations) will become clear to the specialists of the presentation. Potentially useful human nature and humanizing substitutions can be ascertained by comparing the sequence of frame sections, one of the amino acid sequences or of Manotel of the invention contained in one of the polypeptides of the invention, shown in a), with the corresponding frame sequence of one or more closely related sequences VHman, after which this amino acid sequence or nanotesla of the invention contained in one of the polypeptides of the invention, shown in a), there may be one or more of the installed while potentially useful human nature and humanizing substitutions (or combinations thereof) (any method known per se, as described later), and the resulting amino acid sequence can be tested for affinity for IL-6R, stability, ease and level of expression and/or other desirable properties defined here. Thus, by a limited number of trial and error, specialists of the presentation can be set to other suitable human nature and humanizing substitutions (or their appropriate combinations).

Depending on the host organism used to Express the connection is in or polypeptide of the invention, such deletions and/or substitutions may be made and thus to remove one or more sites for post-translational modifications (such as one or more sites of glycosylation), which should be within the competence of specialists in this field. On the other hand, substitution or insertion can be designed to enter one or multiple sites for attachment of functional groups (as described here), for example, for site-specific tahilramani (again as described here).

As you can see from the data on the entropy of VHHand variability of VHHshown in the table.from A-5 to a-8 WO 08/020079, some amino acid residues in frame areas are more conservative than others. In General, although the invention in its broadest sense and is not limited to, any substitutions, deletions or insertions, preferably produced in a less conservative positions. Also amino acid substitutions generally preferable amino acid or division of the insert.

The compounds of the invention or the polypeptide of the invention should preferably be contacted with IL-6R with the same affinity (suitably measured and/or expressed as a value To the (real or perceived), the values of KD(real or perceived), speed konand/or speed koffor in the de values IC 50as described hereinafter), preferably they are:

- contact hIL-b with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less;

and/or such that they:

- contact cynoIL-6R with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less;

and/or such that they:

- contact hIL-6 with a rate constant konfrom 104M-1s-1up to 107M-1s-1preferably from 105M-1s-1up to 107M-1s-1more preferably about 106M-1s-1or more;

and/or such that they:

- contact cynoIL-6R with a rate constant konfrom 104M-1s-1up to 107M-1s-1preferably from 105M-1s-1up to 107M-1s-1more preferably about 106M-1s-1or more;

and/or such that they:

- contact hIL-6 with a rate constant kofffrom 10-3s-1(t½ =0,69 s) up to 10-6s-1(giving almost irreversible complex with t½ in a few days), predpochtitelno from 10 -4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less;

and/or such that they:

- contact cynoIL-6R with a rate constant kofffrom 10-3s-1(t½ =0,69 s) up to 10-6s-1(giving almost irreversible complex with t½ in a few days), preferably between 10-4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less.

Some preferred values IC50for binding compounds and polypeptides of the invention to IL-6R will become clear from the further description and the examples.

Activity and/or efficacy of the polypeptides and compounds of the invention and compositions containing them can be tested by any suitable method in vitro, in cells, in vivo and/or animal model known per se, or any of their combinations, depending on specific diseases or disorders. Suitable methods and models in animals should be known to experts, they include, for example, analysis of cell proliferation in IL-6-dependent cell lines, including TF-1, XG1 and 7TD1, the model of collagen-induced arthritis, a model of transplantation of synovial tissue in SCID mice, a model of heterotransplantation different is the Idov cancer man including lymphoma, myeloma, prostate cancer and renal cell carcinoma, models of IBD, including the TNBS model in primates (e.g., such as described in Shinkura et al., 1998, Anticancer Research 18: 1217-1222), models of arthritic disease-human primates (e.g., as described in Vierboom et al., 2008, Drug Discov. Today: Dis Model doi:10.1016/j.ddmod. 2008.06.003), as well as methods and models for animals used in the following experimental part and there in the works prior art (Peake et al., 2006, Rheumatology 45: 1485-9; Wahid et al., 2000, Clin. Exp. Immunol., 122: 133-142; Matsuno et al., 1998, Arthritis and Rheumatism 41: 2014-2021; WO 08/020079).

For example, in the method TF-1, described by Kitamura et al. (1989, J. Cell Physiol. 140: 323), compounds of the invention or polypeptides of the invention can have values IC50(ME at 100 IL-6/ml) between 10 nm and 50 PM, preferably between 5 nm and 50 PM, more preferably between 1 nm and 50 PM or less, such as about 750 or 500 PM or less. When defining this method TF-1 amino acid sequence of the invention or nanotesla of the invention can have values IC50(5,000 IU of IL-6/ml) between 50 nm and 10 nm, preferably between 25 nm and 1 nm, more preferably between 10 nm and 1 nm or less, such as 8 nm or less. When defining this method TF-1 compounds of the invention or polypeptides of the invention can have values IC50that are at least as, or preferably better, at least the least two times, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more times compared with the value of the IC50received control IgG provided in SEQ ID NO:1 and 2, or control Fab, shown in SEQ ID NO:3 and 4 (see Example 1). When defining this method TF-1 compounds of the invention or polypeptides of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more times compared with the value of the IC50obtained for the Tocilizumab (MRA).

In the determination method of the activity in the plasma when the values of the EU50for IL-6 (e.g., in the presence of 27,29 ng IL-6/ml as described in Example 45) compounds of the invention or polypeptides of the invention can have values IC50between 500 PM and 50 PM, preferably between 250 PM and 50 PM, more preferably between 200 PM and 50 PM or less, such as 150 PM or less. In the determination method of the activity in the plasma when the values AS for IL-6 (e.g., in the presence 885 ng IL-6/ml as described in Example 45) compounds of the invention or polypeptides of the invention can have values IC50between 1000 PM and 100 PM, preferably between 750 PM and 100 PM, more preferably between 500 PM and 100 PM Il is less as of 400 PM or less. When defining this method activity in the plasma of the compounds of the invention or polypeptides of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more times compared with the value of the IC50received control IgG provided in SEQ ID NO:1 and 2, or control Fab, shown in SEQ ID NO:3 and 4 (see Example 1). When defining this method activity in the plasma of the compounds of the invention or polypeptides of the invention can have values IC50that are at least as, or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more times compared with the value of the IC50obtained for the Tocilizumab (MRA).

When defining a method binding with membrane IL-6R on the cell SNO compounds of the invention or polypeptides of the invention can have values IC50between 10 nm and 100 PM, preferably between 5 nm and 100 PM, more preferably between 2 nm and 10 PM or less, such as 2 nm or less.

In a preferred aspect, the compound or polypeptide of the invention contains or consists mainly of amino the PCI-e slot sequence SEQ ID NO:70. In another preferred aspect, the compound or polypeptide of the invention contains or consists mainly of the amino acid sequence of SEQ ID NO:71. Polypeptides with amino acid sequences exhibit improved properties, such, e.g., as the best binding and/or affinity, the best avidity, the best efficiency and the activity and/or greater selectivity, along with their ability to partially or completely block the interaction between IL-6/IL-6R and/or to inhibit signaling through IL-6, IL-6R and/or IL-6/IL-6R.

The invention also relates to monovalent constructs (also referred to as "monovalent constructs of the invention") containing, or consisting primarily of one amino acid sequence or nanotesla invention. Preferred monovalent constructs of the invention contain or essentially consist of SEQ ID NO''s:60-69, such as SEQ ID NO''s:65-69, for example, SEQ ID NO:66. Such monovalent constructs, as well as amino acid sequences and nanotesla of the invention can be used to obtain the compounds or polypeptides of the invention, such, e.g., as multivalent and/or multispecific compounds or polypeptides of the invention.

Accordingly, the present invention also concerns the application of amino acid sequences of Manotel or monovalent what's constructions of the invention to produce compounds constructs or polypeptides of the invention. The invention also concerns a method for obtaining compounds, constructs or polypeptides of the invention, including binding amino acid sequence, nanotesla or monovalent constructs of the invention with one or more other groups, residues, molecules and binding units. Such a method may include linking amino acid sequence, nanotesla or monovalent constructs of the invention with one or more other groups, residues, molecules and binding units via one or more linkers.

In a preferred aspect, one or more other groups, residues, molecules and the binding units are a unit of binding, such as amino acid sequences or nanotesla. Accordingly, the present invention also concerns the application of amino acid sequences of Manotel or monovalent constructs of the invention to obtain multivalent and/or multispecific compounds, constructs or polypeptides of the invention. The invention also relates to a method of obtaining a multivalent and/or multispecific compounds, constructs or polypeptides of the invention, including binding amino acid sequence, nanotesla or monovalent design and is gaining with one or more other binding units type amino acid sequence or nantel. Such a method may include linking amino acid sequence, nanotesla or monovalent constructs of the invention with one or more binding units via one or more linkers.

In a specific aspect, the present invention also concerns the use of a monovalent constructs containing or consisting primarily of one of SEQ ID NO's:60-69 (preferably SEQ ID NO''s:65-69, more preferably SEQ ID NO:66), to obtain multivalent and/or multispecific compounds, constructs or polypeptides of the invention. The invention also relates to a method of obtaining a multivalent and/or multispecific compounds, constructs or polypeptides of the invention, including binding of monovalent constructs containing or consisting primarily of one of SEQ ID NO's:60-69 (preferably SEQ ID NO''s:65-69, more preferably SEQ ID NO:66), with one or more binding units type amino acid sequence or nantel. Such a method may include the binding of monovalent constructs containing or mainly consisting of SEQ ID NO's:60-69 (preferably SEQ ID NO''s:65-69, more preferably SEQ ID NO:66), with one or more binding units via one or more linkers.

In another specific aspect, the present invention relates to the use of monovalent to the instructions, containing or consisting primarily of one of SEQ ID NO's:60-69 (preferably SEQ ID NO''s:65-69, more preferably SEQ ID NO:66), to obtain multivalent and/or multispecific compounds, constructs or polypeptides containing or mainly consisting of SEQ ID NO's:70-72 (preferably SEQ ID NO's:70-71, more preferably SEQ ID NO:70 or SEQ ID NO:71). The invention also relates to a method of obtaining a multivalent and/or multispecific compounds, constructs or polypeptides containing or mainly consisting of SEQ ID NO's:70-72 (preferably SEQ ID NO's:70-71, more preferably SEQ ID NO:70 or SEQ ID NO:71), including the binding of monovalent constructs containing or consisting primarily of one of SEQ ID NO's:60-69 (preferably SEQ ID NO''s:65-69, more preferably SEQ ID NO:66), with the amino acid sequence, containing or mainly consisting of SEQ ID NO's:98, through one or more linkers.

Suitable for multivalent and/or multispecific polypeptides of the spacers or linkers must be known in the art and in General can be any linkers or spacers used in this area for connection amino acid sequence. Preferably, such a linker or spacer suitable for use in the construction of proteins or polypeptides intended for pharmaceutical PR the changes.

Some particularly preferred spacers include spacers and linkers used in this area to connect the fragments of antibodies or domains of antibodies. They include the linkers mentioned in the above work prior art and, for example, the linkers used in this area to construct diatel or scFv fragments (in this respect, however, it should be noted that as used in ditelo and fragments of scFv sequences of the linkers must have the same length, the degree of mobility and other properties that allow appropriate domains VHand VLto connect with education full antigennegative site, there are no special restrictions for length or mobility used in the polypeptide of the invention linker, since each amino acid sequence or nanotesla themselves form a complete antigennegative site.

For example, the linkers can be any suitable amino acid sequence, in particular, amino acid sequences consisting of 1-50, preferably 1-30, such as 1-20 or 1-10 amino acid residues. Some preferred examples of such amino acid sequences include linkers Gly-Ser, for example, type (GlyxSery)z, such as (Gly4Ser)3or (Gly3Ser 2)3as described in WO 99/42077, servirovochnye parcels type hinge areas of natural antibody heavy chain or a similar sequence (of the type described in WO 94/04678).

Some other particularly preferred linkers are polyalanine (AAA), and the linkers are shown in table.In-8, of which especially preferred AAA, GS-7 and GS-9.

Other suitable linkers typically contain organic compounds or polymers, in particular those suitable for use in proteins for pharmaceutical applications. For example, the binding domains of antibodies was used molecules of polyethylene glycol, for example, see WO 04/081026.

In the framework of the invention provides that the length, degree of mobility and/or other properties used linkers (although they are not critical, as is the case with the linkers used in scFv fragments) may have some influence on the properties of the final polypeptide of the invention, including the affinity, specificity or avidity for the receptor of IL-6 or to one or more other antigens. From the above presentation specialist will be able to determine the optimal linkers for specific polypeptides of the invention, optionally after a certain number of simple experiments.

In the framework of the invention also provides that used linkers attach the polypeptide is m of the invention one or several favorable properties or functionalities and/or provide one or more sites for the formation of derivatives and/or for attachment of functional groups (e.g., as described here for the derived amino acid sequences of nantel, compounds and polypeptides of the invention). For example, linkers containing one or more charged amino acid residues, can provide improved hydrophilic properties, whereas linkers forming or containing a small epitopes or tags (labels) can be used for detection, identification and/or purification. Again, the presentation specialist will be able to determine the optimal linkers for specific polypeptides of the invention, optionally after a certain number of simple experiments.

Finally, if the polypeptides of the invention uses two or more linkers, they may be the same or different. Again, the presentation specialist will be able to determine the optimal linkers for specific polypeptides of the invention, optionally after a certain number of simple experiments.

Typically, for ease of expression and production of the polypeptide of the invention should be linear polypeptide. However, the invention in its broadest sense is not limited. For example, if the polypeptide of the invention contains three or more amino acid sequences or of nantel, it is possible to associate them with the linker with three or more "shoulders", and each "shoulder" connects the I amino acid sequence or nanotesla with the formation of the "star" design. It is also possible, although this is usually less preferred, to use the design of the ring.

The invention in its broadest sense also includes the derived amino acid sequences of nantel, compounds or polypeptides of the invention. Such derivatives can generally be obtained by modifying, in particular chemical and/or biological (e.g., enzymatic) modification of amino acid sequences of nantel, compounds or polypeptides of the invention and/or one or more amino acid residues that comprise amino acid sequences, nanotesla, compounds or polypeptides of the invention.

Examples of such modifications, as well as examples of amino acid residues within the amino acid sequences, sequences of nantel, compounds or polypeptides that can be modified in such a way (either on the protein backbone, or, preferably, on the side chain), methods and techniques that can be used to introduce such modifications and the possible applications and benefits of such modifications must be known to specialists.

For example, such modification may involve the introduction (e.g., through covalent bonds or any other suitable manner) of one or more functional groups, residues or molecules in the amino acid on sledovatelnot, nanotesla, compound or polypeptide of the invention, in particular, one or more functional groups, residues or molecules, which imparts one or more desired properties or functionalities of the amino acid sequence, nanotesla, compound or polypeptide of the invention. Examples of such functional groups must be known to specialists.

For example, such modification may involve the introduction (e.g., through covalent bonds or any other suitable manner) of one or more functional groups that increase the half-life, the solubility and/or absorption of amino acid sequence, nanotesla, compound or polypeptide of the invention that reduce the immunogenicity and/or toxicity of the amino acid sequence, nanotesla, compound or polypeptide of the invention, eliminating or weakening any unwanted side effects amino acid sequence, nanotesla, compound or polypeptide of the invention and/or giving other preferred properties and/or reduce the undesired properties of the amino acid sequence, nanotesla, compound or polypeptide of the invention; or any combination of two or more of the above. Examples of such functional groups and methods for their introduction should be a well-known expert and in General can include all functional groups and methods specified in the operations of the prior art described above, and also functional groups and methods known per se for modification of pharmaceutical proteins, in particular, to modify the antibodies or fragments of antibodies, including scFv and single-domain antibodies), for which we shall refer, for example, on Remington''s Pharmaceutical Sciences (1980, 16th ed., Mack Publishing Co., Easton, PA). Such functional groups may, for example, be connected directly (e.g., covalently) with the amino acid sequence, nanotesla, compound or polypeptide of the invention, or optionally via a suitable linker or spacer, as again, this should be known to specialists.

One of the most widely used methods to increase half-life and/or reduce immunogenicity of pharmaceutical proteins involves attaching a suitable pharmacologically acceptable polymer type poliatilenglikola (PEG) or its derivatives (such as methoxypolyethyleneglycol or mPEG). In General, can be any suitable shape tahilramani, such as tahilramani used to antibodies and fragments of antibodies (including single)domain antibodies and form scFv's); we will refer, for example, Chapman (2002, Nat. Biotechnol., 54: 531-545); Veronese and Harris (2003, Adv. Drug Deliv. Rev. 54: 453-456), Harris and Chess (2003, Nat. Rev. Drug. Discov., 2: 214-21) and WO 04/060965. Various reagents for tahilramani proteins is also commercially available, for example, from Nektar Therapeutics, USA.

Preferably applies sent tahilramani, in particular, through the cysteine residue (for example, see Yang et al. (2003, Protein Engineering, 16 (10): 761-770). For example, for this purpose it is possible to attach the PEG to the natural cysteine residue in the amino acid sequence, nanotime, compound or polypeptide of the invention can be modified amino acid sequence, nanotesla, compound or polypeptide of the invention so that it is convenient to introduce one or more cysteine residues for attachment of PEG or drain amino acid sequence containing one or more cysteine residues for attachment of PEG, with N - and/or C-end amino acid sequence, nanotesla, compound or polypeptide of the invention, all of the methods of protein engineering known per se specialists.

Preferably to the amino acid sequences of nantel, compounds or polypeptides of the invention uses a PEG with a molecular weight of more than 5000, such as more than 10,000 and less than 200000, such as less than 100,000; for example, in the range 20000-80000.

Another, usually less preferred modification comprises N-linked or 0-linked glycosylation, usually as part of cotranslational and/or post-translational modification, depending on the host cell used for expre the mechanisms of amino acid sequence, nanotesla, compound or polypeptide of the invention.

Another modification may involve the introduction of one or more detectable labels or other generating signals or groups of molecules, depending on the intended use of the labeled amino acid sequence, nanotesla, compound or polypeptide of the invention. Suitable labels, and methods of their acquisition, use and detection must be known in the art, for example, they include, without limitation, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine and fluorescent metals, such as152Eu or other metals of the lanthanide series), a fluorescence label, a chemiluminescent label, or a bioluminescent label (as luminal, isoluminol, aromaticheskie esters of acridine, imidazole, salt acridine, oxalate esters, dioxetane or GFP and its analogs), radioisotopes (such as3H,125I32P,35S14C,51Cr36Cl57Co.,58With,59Fe and15Se), metals, chelation of metals or metal cations (for example, such metal cations as99mTc123I111In131I97EN,67Cu67Ga and68Ga and other metals or metal cations, which are particularly suitable for use in diagnostics is the IR and imaging in vivo in vitro or in situ (such as157Gd55Mn162Dy,52Cr and56Fe), as well as chromophores and enzymes (such as malatdegidrogenaza, staphylococcal nuclease, Delta isomerase-5-steroid, yeast alcoholdehydrogenase, alpha glitserofosfatdegidrogenazy, triazolopyrimidine, bitinvertdata, horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase and acetylcholinesterase). Other suitable labels must be known in the art, for example, they include molecules that can be detected by NMR or ESR spectroscopy.

Such labeled amino acid sequence, nanotesla, compounds or polypeptides of the invention, for example, can be used in the analysis of in vitro, in vivo or in situ methods including immunoassays known per se, such as ELISA, RIA, EIA and other methods "sandwich principle" and so on), as well as for diagnostics and imaging in vivo, depending on the choice of the specific label.

As should be known in the art, other modifications may include the introduction of chelating groups, for example, for chelation of one or more metals or metal cations described above. Suitable chelating groups, for example, include, without limitation, Diethylenetriamine auxonne acid(DTPA) or ethylenediaminetetraacetic acid (EDTA).

Another modification may include the introduction of a functional group constituting one part of a specific binding pair, such as a pair of Biotin-(strept)avidin. This functional group can be used to link amino acid sequence, nanotesla, compound or polypeptide of the invention to another protein, polypeptide or chemical compound that is associated with the other half of the binding pair, i.e., through the formation of binding pairs. For example, amino acid sequence, nanotesla, compound or polypeptide of the invention can be anywhereman with Biotin and connected to another protein, polypeptide, compound or carrier conjugated with Avidya or streptavidin. For example, such a conjugated amino acid sequence, nanotesla, compound or polypeptide of the invention can be used as a reporter, for example, in a diagnostic system where generating the detected signal reagent anywhereman with Avidya or streptavidin. Such binding pairs, for example, can be used to link amino acid sequence, nanotesla, compound or polypeptide of the invention with the media, including suitable for pharmaceutical purposes. One non-restrictive examples are liposomal dosage forms, the description is by Cao and Suresh (2000, Journal of Drug Targetting, 8 (4); 257). Such binding pairs may be used to associate a pharmaceutically active agent with the amino acid sequence, nanotesla, compound or polypeptide of the invention.

Other possible chemical and enzymatic modifications must be known to specialists. Such modifications can be produced for research purposes (e.g., to study the relationship function-activity). We will refer, for example, Lundblad and Bradshaw (1997, Biotechnol. Appl. Biochem., 26: 143-151).

Derivatives are preferably such that they are associated with specific apropos on the receptor of IL-6 with an affinity (suitably measured and/or expressed in the form of values of KD(real or perceived), the values of KA(real or perceived), the rate constant konand/or koffor as the value IC50as described hereinafter), which is defined here.

In particular, these derivatives of the invention are preferably such that they:

- contact hIL-6 with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less;

and/or such that they:

- contact cynoIL-6R with a dissociation constant (KDfrom 1 nm to 1 PM is whether less preferably from 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less;

and/or such that they:

- contact hIL-6 with a rate constant konfrom 104M-1s-1up to 107M-1s-1preferably from 105M-1s-1up to 107M-1s-1more preferably about 106M-1s-1or more;

and/or such that they:

- contact cynoIL-6R with a rate constant konfrom 104M-1s-1up to 107M-1s-1preferably from 105M-1s-1up to 107M-1s-1more preferably about 106M-1s-1or more;

and/or such that they:

- contact hIL-6 with a rate constant kofffrom 10-3s-1(t½=0,69 s) up to 10-6s-1(giving almost irreversible complex with a t½ in a few days), preferably between 10-4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less;

and/or such that they:

- contact cynoIL-6R with a rate constant kofffrom 10-3s-1(t½ =0,69 s) up to 10-6s-1(giving almost irreversible complex with t½ in a few days), preferably the t 10 -4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less.

As indicated above, the invention also relates to proteins or polypeptides, which mainly consist of or contain at least one amino acid sequence, nanotesla, compound or polypeptide of the invention. By "mostly" means that the amino acid sequence of the protein or polypeptide of the invention or the same as the amino acid sequence, nanotesla, compound or polypeptide of the invention or corresponds to amino acid sequences, nanotesla, compound or polypeptide of the invention having a limited number of amino acid residues, such as 1-20 amino acid residues, for example, 1-10 amino acid residues, preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues inserted at the N-end-end or at the N-end and C-end amino acid sequence, nanotesla, compound or polypeptide.

The amino-acid residues may or may not change, alter or otherwise influence the (biological) properties of the amino acid sequence, nanotesla, compound or polypeptide of the invention and may or may not give up anicello the functionality of the amino acid sequence, nanotesla or polypeptide of the invention. For example, such amino acid residues:

(a) may include the N-terminal Met residue, for example, in the expression in heterologous cells-the owners or the body of the host;

(b) may be a signal sequence or leader sequence directing secretion of the amino acid sequence, nanotesla or polypeptide of the invention from a host cell after synthesis. Suitable secretory leader peptides must be known in the art and may be such as described below. Usually the leader sequence linked to the N-end amino acid sequence, nanotesla, compound or polypeptide, although the invention in its broadest sense is not limited to;

c) can form a sequence or signal that allows the amino acid sequence, nanotesla, compound or polypeptide to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or allowing the amino acid sequence, nanotesla or polypeptide of the invention to penetrate or cross a biological barrier type cell membrane, a cell layer type layer of epithelial cells, a tumor including solid tumors, or the blood-brain barrier. Suitable examples of the same, the amino acid sequence must be known to specialists. Some non-restrictive examples are small peptide vector ("vector Peptrans") described in WO 03/026700 and Temsamani et al., Expert Opin. Biol. Ther., 1, 773 (2001); Temsamani and Vidal, Drug Discov. Today, 9, 1012 (004) and Rousselle, J. Pharmacol. Exp. Ther., 296, 124-131 (2001), the sequence of membrane translocator described by Zhao et al., Apoptosis, 8, 631-637 (2003). C-terminal and N-terminal amino acid sequence for intracellular targeting fragments of antibodies, for example, described by Cardinale et al.. Methods, 34, 171 (2004). Other suitable methods for intracellular targeting include expressiona and/or the use of so-called "Intertel" containing the amino acid sequence, nanotesla, compounds or polypeptides of the invention, as described below;

d) can comprise a label ("tag"), for example, amino acid sequence or residue that allows or promotes the purification of amino acid sequence, nanotesla, compound or polypeptide, for example, affinity techniques directed against the sequence or residue. After this sequence, or the residue can be removed (e.g., by chemical or enzymatic cleavage) to obtain amino acid sequences, nanotesla, compound or polypeptide (for this purpose, the label may not necessarily be associated with the amino acid sequence, after what outlineto nanotesla, compound or polypeptide through tsepliaeva sequence of the linker or to contain ottsepleny motive). Some preferred, but not restrictive examples of such residues as multiple histidine residues, residues of glutathione tag and myc-tag type AAAEQKLISEEDLNGAA (SEQ ID NO:100);

e) can represent one or several amino acid residues that have been subjected to functionalization and/or can serve as a site for attachment of functional groups. Suitable amino acid residues and functional groups must be known in the art and include, without limitation, amino acid residues and functional groups specified here for the derived amino acid sequences of nantel, compounds and polypeptides of the invention.

Amino acid sequence, nanotesla, polypeptides and nucleic acids of the invention can be obtained by a method known per se, as should be clear to experts from here further description. For example, amino acid sequence, nanotesla and polypeptides of the invention can be obtained by any method known per se to generate antibodies, in particular, to obtain fragments of antibodies (including single-domain antibodies and scFv fragments). Some preferred, but not restrictive ways of obtaining and inability sequences nantel, polypeptides and nucleic acids include the methods and techniques described here.

As should be known to experts, one particularly useful method of obtaining amino acid sequences of nantel and/or polypeptides of the invention in General involves the following stages:

- expressiona in suitable cells-the owners or the body of the host (also referred to as the "host of the invention") or in another suitable expression system of the nucleic acid encoding this amino acid sequence, nanotesla or polypeptide of the invention (also referred to as "nucleic acid of the invention"), then necessarily follows:

- isolation and purification of resulting amino acid sequence, nanotesla or polypeptide of the invention.

In particular, such a method may include stages:

- cultivation and/or content owner according to the invention under such conditions that the host of the invention expresses and/or produces at least one amino acid sequence, nanotesla and/or polypeptide of the invention, after which not necessarily must:

- isolation and purification of resulting amino acid sequence, nanotesla or polypeptide of the invention.

Accordingly, the present invention also relates to nucleic acid and the sequence is of linesta nucleotides, coding for amino acid sequences, nanotesla, polypeptides or monovalent constructs of the invention (also referred to as "nucleic acid of the invention"). Nucleic acid of the invention can be single-stranded or double-stranded DNA or RNA, preferably in the form of double-stranded DNA. For example, the nucleotide sequence of the invention can be a genomic DNA, cDNA or synthetic DNA (such as DNA potrebitelskiy codons, which was subjected to a special adaptation for expression in specific cells-the owners or the body of the host).

In accordance with one embodiment of the invention, the nucleic acid of the invention is selected as defined here. Nucleic acid of the invention may also be located in and/or be part of a vector, such, for example, as a plasmid, cosmid or YAC, which again can be almost in the selected form.

Nucleic acids of the invention can be obtained or prepared by a method known per se, based on the information here about amino acid sequences, nanotech and/or polypeptides of the invention, and/or can be isolated from a suitable natural source. Besides, it should be clear to experts, to obtain a nucleic KIS is the notes of the invention can be connected in a suitable manner several nucleotide sequences, once at least one nucleotide sequence encoding amino acid sequence or nanotesla, together with nucleic acids, for example, encoding one or more linkers.

Methods for creating a nucleic acid of the invention should be known in the art, for example, they may include, without limitation, automated DNA synthesis; site-directed mutagenesis; the combination of two or more natural and/or synthetic sequences (or two or more parts thereof); the introduction of mutations, leading to the expression of the truncated expression product; introducing one or more restriction sites (e.g., to create cassettes and/or sites that are easy to split and/or merge using the appropriate restriction enzymes); and/or the introduction of mutations using PCR reaction using one or more "mismatched" primers. These and other methods should be known to experts, again, we refer to standard manuals such as Sambrook et al. and Ausubel et al., mentioned above, as well as in the following Examples.

Nucleic acid of the invention may also be located in and/or be part of the genetic constructs, as it should be known to experts. Such genetic constructs generally contain at least one NAA is eyouuu acid of the invention, which is not necessarily associated with one or more elements of genetic constructs known per se, such for example, as one or more suitable regulatory elements (as appropriate promoters, enhancers, terminators, and so on), and these additional elements of genetic constructs. Such genetic constructs containing at least one nucleic acid of the invention, also referred to as "the genetic constructs of the invention".

The genetic constructs of the invention can be a DNA or RNA, preferably double-stranded DNA. The genetic constructs of the invention can also be suitable for the transformation of the specified cells of the host or of the host body, or appearance, suitable for embedding in the genomic DNA of the specified host cells, or type, suitable for independent replication, maintenance and/or inheritance in a given organism, the host. For example, the genetic constructs of the invention can be in the form of a vector, such, for example, as a plasmid, cosmid, YAC, viral vector, or a transposon. In particular, the vector may be an expression vector, i.e. a vector that can provide expression in vitro and/or in vivo (e.g., in a suitable cell host, organism-the owner and/or the expression system).

In a preferred, but the e restrictive embodiment of the genetic structure of the invention includes;

a) at least one nucleic acid of the invention, functionally associated with

b) one or more regulatory elements, such as promoter and optionally a suitable terminator; and also optional

c) one or more additional elements of genetic constructs known per se;

while the terms "regulatory element", "promoter", "terminator" and "functionally linked" have their ordinary meaning in the art (as described below); and data "additional elements" present in the genetic constructs may for example be a sequence 3'- or 5'-UTR, leader sequences, selection markers, expression markers/genes reporters and/or elements that may contribute to or increase (efficiency) transformation or integration. These and other suitable elements for such genetic constructs must be known in the art and can, for example, depend on the type of construction, the anticipated cells of the host or of the host body; the way the data is the nucleotide sequence of the invention will be expressed (e.g., through constitutive, transient or inducible expression); and/or used by the transformation method. Thus, a regulatory sequence, promote the s and terminators known per se for the expression and production of antibodies and fragments of antibodies (including single-domain antibodies and scFv fragments), can be used essentially in the same way.

Preferably in the genetic structures of this invention at least one nucleic acid and data regulatory elements and optional data one or more additional elements "functionally related" to each other, this usually means that they are in a functional relationship with each other. For example, a promoter is considered to be "functionally related" to the coding sequence if the promoter is capable of initiating or otherwise to control/regulate the transcription and/or expression of the coding sequence (in this case, the coding sequence is understood as being "under the control of the promoter). In General, when two nucleotide sequences are functionally linked, they will be in the same orientation and usually in the same reading frame. They must also be practically related, even though it may not be required.

Preferably the regulatory and further elements of genetic constructs of the invention are such that they are able to provide intended them biological functions in the given tile is Ah-owners or the body of the host.

For example, a promoter, enhancer or the terminator must be "functional" in the specified cells-the owners or the body-master, meaning that (for example) the promoter must be able to initiate or otherwise control/regulate the transcription and/or expression of a nucleotide sequence - i.e., the coding sequences with which they are functionally related (as defined here).

Some particularly preferred promoters include, without limitation, promoters, known per se for the expression in these cells masters, in particular, the promoters for expression in bacterial cells, such as mentioned herein and/or those that were used in the Examples.

The selection marker should be such that he gave an opportunity with the relevant criteria to distinguish cells of the owners and/or organisms owners that have been successfully transformed with the nucleotide sequence of the invention from those of cells/organisms-owners that were not successfully transformed. Some preferred, but not restrictive examples of such markers are genes that provide resistance to antibiotics such as kanamycin or ampicillin), genes providing resistance to temperature or genes, to support the cells of host-Il is the body of the host in the absence of certain factors, compounds and/or (food) components in the environment that are necessary for the survival of the untransformed cells or organisms.

Leader sequence should be such that the specified cells-the owners or the body-master, she contributed desirable post-translational modifications and/or directed transcribed RNA in a desirable part or cell organelle. The leader sequence may also contribute to the secretion of the expression product from the cells. When this leader sequence can be any Pro-, pre -, or shall be a sequence running in the cells-the owners or the body of the host. The leader sequence may not be required for expression in bacterial cells. For example, a leader sequence, known per se for the expression and production of antibodies and fragments of antibodies (including single-domain antibodies and scFv fragments), can be used essentially in the same way.

Expression of the marker or gene-reporter should be such that in the cells-the owners or the body-master, they contributed to the detection of the expression level of the gene or the sequence of nucleotides located in a) genetic structure. Expression of the marker can optionally also to facilitate determination of the localization of the expressed is the product, e.g., in a certain part or organelle of a cell and/or in specific cells, tissues, organs or parts of a multicellular organism. Such genes reporters can be expressed in the form of protein, fused with the amino acid sequence, nanotesla or polypeptide of the invention. Some preferred, but not restrictive examples include fluorescent proteins such as GFP.

Some preferred, but not restrictive examples of suitable promoters, terminators and additional elements include those that can be used for expression in these cells of the host; in particular those that are suitable for expression in bacterial cells such as those that are listed here, and/or those that were used in the following Examples. For some (other) non-limiting examples of promoters, selection markers, leader sequences, expression markers and additional elements that may be present/be used in the genetic constructions of the invention, such as terminators, enhancers of transcription and/or translation and/or factors integration - shall refer to the General handbooks, such as Sambrook et al. and Ausubel et al., above, and the examples in WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, US 7,207,410, US 5,693,492 and EP 108589. Other examples should be known to specialists. We will refer also to the above operation of the prior art and described in other references.

The genetic constructs of the invention can generally be obtained by the appropriate connection of the nucleotide sequences of the invention with one or more additional elements described above, for example, using the techniques described in General reference books, such Sambrook et al. and Ausubel et al., above.

Often genetic constructions of the invention are obtained by introducing the nucleotide sequence of the invention into a suitable (expression) vector, known per se. Some preferred, but not restrictive examples of suitable expression vectors are shown in the following Examples, as well as specified previously.

Nucleic acids of the invention and/or the genetic constructs of the invention can be used to transform host cells or the host body, i.e., for expression and/or production of amino acid sequence, nanotesla or polypeptide of the invention. Suitable hosts or cells of the hosts must be known in the art and may be, for example, any suitable fungal, prokaryotic or eukaryotic cells or cell lines, or any eligible is fungal, prokaryotic or eukaryotic organisms, for example:

bacterial strains including gram-negative strains, such as strains of Escherichia coli; or Proteus, for example Proteus mirabilis; or Pseudomonas, for example, Pseudomonas fluorescens; and gram-positive strains, such as strains of Bacillus, for example. Bacillus subtilis or Bacillus brevis; or Streptomyces, for example, Streptomyces lividans; or Staphylococcus, for example, Staphylococcus carnosus; or Lactococcus, for example, Lactococcus lactis;

cells of fungi, including cells from species of Trichoderma, for example, Trichoderma reesei; or Neurospora, for example, Neurospora crassa; or Sordaria, for example, Sordaria macrospora; or Aspergillus, for example, Aspergillus niger or from Aspergillus sojae; or from other filamentous fungi;

- yeast cells, including cells from species of Saccharomyces, for example of Saccharomyces cerevisiae; or Schizosaccharomyces, for example, Schizosaccharomyces pombe; or Pichia, for example, Pichia pastoris or Pichia methanolica; or Hansenula, for example, Hansenula polymorpha', or Kluyveromyces, for example, Kluyveromyces lactis; or Arxula, for example, Arxula adeninivorans, or Yarrowia, for example, yarrow/a lipolytica,

cells or cell lines of amphibians such as Xenopus oocytes;

cells or cell lines from insects, such as cells/cell lines from Lepidoptera, including cells, Spodoptera SF9 and Sf21, or cells/cell lines from Drosophila, such as cell Schneider and KS;

- plants or plant cells, for example, in tobacco plants; and/or

cells or mammalian cell lines, for example, cells or cell lineis person, cells or cell lines of mammalian, including cells Cho cells KSS (for example, cells KSS-21) and cells or cell lines of human, such as HeLa cells, COS (for example COS-7) and PER.C6;

and other hosts or cells of owners, known per se for the expression and production of antibodies or fragments of antibodies (including single-domain antibodies and scFv fragments), which must be known to specialists. We will refer also to the above operation of the prior art and, for example, in WO 94/29457; WO 96/34103; WO 99/42077; Frenken et al. (1998, Res. Immunol. 149(6): 589-99), Riechmann and Muyldermans (1999, J. Immunol. Methods 231(1-2): 25-38), van der Linden (2000, J. Biotechnol. 80(3): 261-70), Joosten et al. (2003, Microb. Cell Fact. 2(1): 1), Joosten et al. (2005, Appl. Environ. Biotechnol. 66(4): 384-92.); and given other references.

Amino acid sequence, nanotesla and polypeptides of the invention it is also possible to introduce and Express in one or more cells, tissues or organs of a multicellular organism, for example, for prophylactic and/or therapeutic purposes (e.g., gene therapy). Of cells or tissue can enter the nucleotide sequence of the invention in any suitable way, for example, by themselves (e.g., using liposomes) or after embedding them into a suitable vector for gene therapy (for example, derived from retroviruses type adenovirus or parvovirus type adenoassociated the data viruses). As should be known to specialists, such gene therapy can be performed in vivo and/or in situ in the body of the patient by introducing a nucleic acid of the invention or a suitable vector for gene therapy encoding it, the patient or in specific cells or specific tissues or organs of a patient; or you can handle the appropriate cells (which are often taken from the body of the subject to treatment of a patient, such as explantion lymphocytes, bone marrow aspirates or biopsy tissue) in vitro nucleotide sequence of the invention, and then appropriately re-enter in the patient's body. All this can be done using vectors for gene therapy, methods and delivery systems, which are well known in the art and described, for example, in Culver, K. W. (1994, "Gene Therapy", p.xii, Mary Ann Liebert, Inc., Publishers, New York, N. Y.); Giordano (1996, Nature Medicine 2: 534-539); Schaper (1996, Circ. Res. 79:911-919); Anderson (1992, Science 256:808-813); Verma (1994, Nature 389: 239); Isner (1996, Lancet 348: 370-374); Muhlhauser (1995, Circ. Res. 77: 1077-1086); Onodera (1998, Blood 91:30-36); Verma (1998, Gene Ther. 5: 692-699); Nabel (1997, Ann. N. Y. Acad. Sci, 811: 289-292); Verzeletti (1998, Hum. Gene Ther. 9: 2243-51); Wang 1996, Nature Medicine 2: 714-716); WO 94/29469; WO 97/00957; US 5,580,859; or Schaper (1996, Current Opinion in Biotechnology 7: 635-640). For example, this area was described expression in situ fragments of scFv (Afanasieva et al. (2003, Gene Ther., 10:1850-1859) and dietel (Blanco et al., 2003, J. ImmunoL, 171:1070-1077).

For the expression of amino acid sequences, Anatel or polypeptides in cells can also Express in the form of so-called "Intertel", for example, as described in WO 94/02610, WO 95/22618, US 7,004,940, WO 03/014960, Cattaneo, A. & Biocca, S. (1997, Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag) and Kontermann (2004, Methods 34:163-170).

Amino acid sequence, nanotesla and polypeptides of the invention can be produced, for example, in the milk of transgenic mammals, for example, in the milk of rabbits, cows, goats or sheep (see for example US 5,741,957, US 5,304,489 and US 5,849,992 about common methods of introducing transgenes into mammals), in plants or plant parts, including leaves, flowers, fruits, seeds, roots or tubers (for example, in tobacco, corn, soybean or alfalfa), or, for example, in the pupae of the silkworm Bombix mori.

Moreover, amino acid sequence, nanotesla and polypeptides of the invention can also Express and/or to produce in a cell-free expression systems, and suitable examples of such systems must be known to specialists. Some preferred, but not restrictive examples include the expression in the system of wheat germ; in the lysate of rabbit reticulocytes; or in the system Zubay E. coli.

As indicated above, one advantage of the use of Manotel is that polypeptides based on them can be obtained by expression in a suitable bacterial system, and suitable bacterial expression systems, vectors, cells-owners, regulatory e the cops and other must be known to specialists, for example, from the above links. However, it should be noted that the invention in its broadest sense is not limited to expression in bacterial systems.

Preferably the invention uses the expression system (in vivo or in vitro) type bacterial expression system, which provides polypeptides of the invention in a form suitable for pharmaceutical use, and such expression systems again must be known to specialists. Professionals should also be aware that the polypeptides of the invention, suitable for pharmaceutical applications, can be obtained by the methods of synthesis of peptides.

For the production on an industrial scale, preferred heterologous hosts for the (industrial) receiving nantel or containing nanotesla protein drugs are strains of E. coli, Pichia pastoris, S. cerevisiae that are suitable for large scale expression/production/fermentation, in particular for large scale pharmaceutical expression/production/fermentation. Suitable examples of such strains should be known to specialists. Such strains and systems products/expression to the same are manufactured by companies such as Biovitrum (Uppsala, Sweden).

On the other hand, for large scale expression/production/fermentation, and in castnet is, for large-scale pharmaceutical expression/production/fermentation, you can use the lines of mammalian cells, in particular cells of the Chinese hamster ovary (Cho). Again, such systems the expression/production also produced some of the companies mentioned above.

The choice of a particular expression system, will depend in part on the need for some post-translational modifications, more specifically, in the glycosylation. The preparation of recombinant proteins containing nanotesla for which it is desirable or required glycosylation, entails the use of expressing owners from mammals that have the ability to glycosylate expressed protein. In this regard, professionals should be aware that the resulting profile of glycosylation (i.e., type, number and position of attached debris) will depend on the cell or cell line used for expression. Preferably used cell or cell line person (giving protein, which basically has the glycosylation profile of a person or line of cells of other mammals, which can provide a profile of glycosylation, which in fact and/or functionally is the same as the profile of glycosylation in humans or at least similar to it. In General, prokaryotic org the mechanisms of type E. coli not have the ability to glycosylation of proteins, and the use of lower eukaryotes type of yeast usually results in a glycosylation profile that differs from the glycosylation profile of a person. However, it should be understood that the invention can be used all the above cell-hosts and expression systems, depending on the intended amino acid sequence, nanotesla or polypeptide.

Thus, in accordance with one non-restrictive embodiment of the invention the amino acid sequence, nanotesla or polypeptide of the invention are glycosylated. In accordance with another non-restrictive embodiment of the invention, amino acid sequence, nanotesla or polypeptide of the invention are not glycosylated.

In accordance with one preferred, but not restrictive embodiment of the invention the amino acid sequence, nanotesla or polypeptide of the invention produced in bacterial cells, in particular, in bacterial cells, suitable for large scale pharmaceutical production, such as cells of the strains listed above.

In accordance with another preferred, but not restrictive embodiment of the invention the amino acid sequence, nanotesla or polypeptide of izobreteny is produced in yeast cells, in particular, in yeast cells, suitable for large scale pharmaceutical production, such as cells of the species listed above.

In accordance with another preferred, but not restrictive embodiment of the invention the amino acid sequence, nanotesla or polypeptide of the invention produced in mammalian cells, particularly human cells or cells from cell lines of human, particularly human cells or cells from cell lines of human, suitable for large scale pharmaceutical production, types of cell lines, above.

If to obtain amino acid sequences of nantel and polypeptides of the invention uses the expression in the cells of the host, the amino acid sequence, nanotesla or polypeptides of the invention can either be produced intracellularly (e.g., in the cytosol, in periplasm or inclusion bodies) and then released from the host cells and optionally further purified; or can produce extracellular (e.g., in the environment in which the cultured host cell), and then to stand out from the culture medium and optionally further purified. When using eukaryotic host cells usually preferable extracellular receive, because it greatly facilitates Yes inasea selection and subsequent processing of the obtained amino acid sequences, nantel, polypeptides and proteins. Bacterial cells of the type described above strains of E. coli normally do not secrete proteins from cells, with the exception of certain classes of proteins, such as toxins and hemolysin, and secretory products in E. coli indicates the translocation of proteins across the inner membrane in periplasmatic space. Periplasmatic products have several advantages over products in the cytosol. For example, the N-terminal amino acid sequence of the secreted product may be identical to the natural gene product after removal of the signal sequence for secretion of the specific signal peptidase. Besides proteasa activity in periplasm is much lower than in the cytoplasm. In addition, purification of proteins is simplified due to the smaller amount of ballast proteins in periplasm. Another advantage is that can form the correct disulfide bonds because periplasm provides a more oxidizing environment than the cytoplasm. Proteins subjected to superexpression in E. coli, are often insoluble aggregates called inclusion bodies. These calf inclusion can be in the cytosol or in periplasm; extraction of biologically active proteins from these Taurus inclusion requires denaturation process/re-styling. Many is their recombinant proteins, including therapeutic proteins, isolated from the Taurus inclusion. On the other hand, as it should be known to experts, it is possible to use recombinant strains of bacteria that have been genetically modified so that they secretively desired protein, in particular, amino acid sequence, nanotesla or polypeptide of the invention.

Thus, in accordance with one non-restrictive embodiment of the invention the amino acid sequence, nanotesla or polypeptide of the invention represents an amino acid sequence, nanotesla or polypeptide, which were obtained intracellular and were isolated from host cells, in particular bacterial cells or from Taurus inclusion in bacterial cells. In accordance with another non-restrictive embodiment of the invention the amino acid sequence, nanotesla or polypeptide of the invention were obtained extracellular and were isolated from the environment in which it was cultivated cells of the host.

Some preferred, but not restrictive examples of promoters for use in such cells-hosts include:

for expression in E. coli: lac-promoter (and its derivatives, such as the lacUV5 promoter); arabinosyl promoter; left (PL) and right (PR) promoter of phage lambda; the promoter of the trp operon; hybrid lac/trp promoters (tac, and trc); ramotar T7 (more specifically gene 10 of phage T7) and the other promoters of the T-phage; the promoter of the gene of resistance to tetracycline Tn10; engineering options above promoters comprising one or more copies of a sequence of external regulatory operator;

for expression in S. cerevisiae: constitutive: ADH1 (alcohol dehydrogenase 1), ENO (enolase), CYC1 (cytochrome C iso-1), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), PGK1 (phosphoglycerate), PYK1 (pyruvate kinase); adjustable: GAL1,10,7 (enzymes of galactose metabolism), ADH2 (alcoholdehydrogenase 2), PHO5 (acid phosphatase), CUP1 (copper metallothionein); heterologous: CaMV (35S promoter of cauliflower mosaic virus);

for expression in Pichia pastoris: the promoter AOH (alcoholiday I);

for expression in mammalian cells: enhancer/promoter immediate early genes of human cytomegalovirus (hCMV); variant promoter immediate early genes of human cytomegalovirus (hCMV), containing a sequence of two tetracycline operators, so that the promoter was regulated by the Tet repressor; the promoter timedancing (TK) of Herpes simplex virus; the enhancer/promoter of the long terminal repeat (RSV LTR) of the rous sarcoma virus; the promoter of the elongation factor 1α (hEF-1α) of human, chimpanzee, mouse or rat; the early SV40 promoter; the promoter of the long terminal repeat of HIV-1; β-actin promoter.

Some preferred, but not restrictive examples of vecto the s for use with these host cells include:

vector for expression in mammalian cells: pMAMneo (Clontech), pcDNA3 (Invitrogen), pMClneo (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110). pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and 1ZD35 (ATCC 37565), as well as systems for the expression of viral basis, such as those based on adenovirus;

vectors for expression in bacterial cells: vector pet (Novagen) and the vector pQE (Qiagen);

vectors for expression in yeast or other fungi: pYES2 (Invitrogen) and expression vector for Pichia (Invitrogen);

vector for expression in insect cells: pBlueBacII (Invitrogen) and other baculovirus vectors;

vector for expression in plants or plant cells: for example, vector-based virus cauliflower mosaic or mosaic virus tobacco, suitable strains of Agrobacterium or vector-based Ti-plasmid.

Some preferred, but not restrictive secretory sequences for use with these host cells include:

- for use in bacterial cells type E. coil: PelB, Bla, OmpA, OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LamB, and the like; TAT signal peptide, C-terminal secretion signal of hemolysin;

- for use in yeast cells: shall be a sequence of α-factor mating phosphatase (phol), invertase (Sue), and others;

- for use in mammalian cells: the property is built to an alarm event, if the protein target is of eukaryotic origin; signal peptide V-J2-C κ-chain Ig mouse; and others

Suitable methods for transformation of an organism or host cells according to the invention must be known in the art and may depend on the desired cells of the host/the host body and used a genetic design. Again, we refer to the above references and patent applications.

After the transformation can be performed phase identification and selection of those host cells or organisms hosts that have been successfully transformed with a nucleotide sequence/genetic construct of the invention. This can be, for example, qualifying on the basis of a selection marker that is present in the genetic constructs of the invention, or stage, including the detection of amino acid sequences of the invention, e.g., using specific antibodies.

Transformed cell hosts (which may be in the form of stable cell lines or organisms-owners (which may be in the form of a stable mutant lines or strains) are the following aspects of the present invention.

Preferably, these cells are the owners or organisms-the owners are such that they Express, or (at least) able to Express (e.g., in suitable conditions) amino acid posledovatelno the ü, nanotesla or polypeptide of the invention (and in the case of the host organism: at least one cell, part or organ). The invention also includes the next generation, the offspring and/or offspring cells of the host or of the host body according to the invention, which, for example, can be obtained by dividing cells either sexual or asexual reproduction.

To obtain/ekspressirovali amino acid sequences of the invention transformed cell hosts or transformed organism-owner in General can contain, support, and/or to cultivate in these conditions to (desirable) amino acid sequence, nanotesla or polypeptide of the invention expressively/produced. Suitable conditions should be known in the art and generally depend on the used cells/organisms-owners as well as from the regulatory elements controlling the expression of (relevant) nucleotide sequence of the invention. Again, we refer to the references and patent applications identified above in the section on genetic constructions of the invention.

In General, suitable conditions may include the use of a suitable environment, the presence of a suitable power source and/or the suitable nutrients, the use of a suitable temperature and optional presence podhodyashego the inducing factor or compounds (e.g., when the nucleotide sequence of the invention is under the control of the inducible promoter); and all of them can be selected by specialists. Again, under these conditions, amino acid sequences of the invention can be expressed constitutive manner, intermittent, or only when they are raised accordingly.

Professionals should also be aware that amino acid sequence, nanotesla or polypeptide of the invention may (first) be born in an immature form (as described above), and then subjected to post-translational modification, depending on the cells of the host/the host body. Moreover, amino acid sequence, nanotesla or polypeptide of the invention can be subjected to glycosylation, which again depends on the used cells masters/the host body.

Amino acid sequence, nanotesla or polypeptide of the invention can then be separated from the cells of the host/the host body and/or from the medium in which the cells of the host or master-organism was cultivated, using the methods of separation and/or purification of proteins, known per se, such as methods (preparative) chromatography and/or electrophoresis, methods of fractional planting, affine methods (e.g., using specific, tsepliaeva AMI is kislotno sequence, merged with the amino acid sequence, nanotesla or polypeptide of the invention) and/or preparative methods of immunology (e.g., using antibodies against the to-be-allocated amino acid sequence).

In General, for pharmaceutical use, the polypeptides of the invention can be prepared as a pharmaceutical preparation or compositions comprising at least one amino acid sequence, nanotesla or polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more additional pharmaceutically active polypeptides and/or compounds. As non-restrictive examples of such dosage forms can be in a form suitable for oral administration, for parenteral administration (such as intravenous, intramuscular or subcutaneous injection or intravenous infusion), for local use, for administration by inhalation, in the form of skin patches, implants, suppositories, and other Such suitable forms for administration - which can be solid, semi-solid or liquid, depending on the method of introduction - and the methods and media for use in their manufacture must be known in the art and described below.

So, following the ninth aspect of the invention concerns pharmaceutical compositions containing at least one amino acid sequence of the invention, at least one nanotesla of the invention or at least one polypeptide of the invention and at least one suitable carrier, diluent or excipient (i.e. suitable for pharmaceutical use), and optionally one or more additional active substances. In a preferred aspect of the invention concerns pharmaceutical compositions containing SEQ ID NO:70 and at least one suitable carrier, diluent or excipient (i.e. suitable for pharmaceutical use), and optionally one or more additional active substances. In another preferred aspect of the invention concerns pharmaceutical compositions containing SEQ ID NO:71 and at least one suitable carrier, diluent or excipient (i.e. suitable for pharmaceutical use), and optionally one or more additional active substances.

In General, amino acid sequence, nanotesla and polypeptides of the invention can be prepared and introduced by any suitable means, known per se, for which we shall refer, for example, in the above operation of the prior art (in particular to WO 04/041862, WO 04/041863, WO 04/041865 and WO 04/041867), as well as to the standard handbooks, such as Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, USA (1990) or Remington, the Science and Practice of Pharmacy, 21th ed., Lippincott, Williams and Wilkins (2005).

For example, amino acid sequence, nanotesla and polypeptides of the invention can be prepared and introduced by any method known per se for conventional antibodies and fragments of antibodies (including antibody scFv and diatel) and other pharmaceutically active proteins. Such dosage forms and methods for their production must be known in the art and include, for example, preparations suitable for parenteral administration (for example, intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecal injection) or local (percutaneous or intradermal) administration.

Preparations for parenteral administration may constitute, for example, sterile solutions, suspensions, dispersions or emulsions, suitable for infusion or injection. Suitable carriers or diluents for such drugs include, for example, without limitation, sterile water and aqueous buffer solutions, such as saline solution, phosphate buffer solutions, ringer's solution, dextrose and Hanks solution; water oil; glycerin; ethanol; glycols type propylene glycol, mineral oils, animal fats and vegetable oils, for example, peanut oil, soybean oil, as well as their p is Rhodesia mixture. Usually the preferred aqueous solutions or suspensions.

Amino acid sequence, nanotesla and polypeptides of the invention can also be administered by the methods of delivery for gene therapy. E.g., see U. S. 5,399,346, incorporated by reference in its entirety. When using the delivery method for gene therapy of primary cells, transfected with a gene encoding the amino acid sequence has apatela or polypeptide of the invention can additionally be transferout tissue-specific promoters for specific organs, tissue, grafts, tumors, or cells, as well as to transferout signal and a stabilizing sequence for subcellular expression.

Thus, amino acid sequences, nanotesla and polypeptides of the invention can be entered systematically, e.g., orally, in combination with such a pharmaceutically acceptable carrier, as an inert diluent or an assimilable suitable for food media. They can be enclosed in gelatin capsules with hard or soft shell, compressed into tablets, or directly included in the food in the diet of the patient. For oral therapeutic introduction of amino acid sequences, nanotesla and polypeptides of the invention can be combined with one or more excipients and used in the form of tablets taken orally, alkaline tablets cakes, capsules, elixirs, suspensions, syrups, wafers, etc., Such compositions and preparations should contain at least 0.1% of the amino acid sequence, nanotesla or polypeptide of the invention. Their content in the compositions and preparations can, of course, vary, and for convenience may be from 2% to 60% by weight of this standard dose forms. The content of amino acid sequences, nanotesla or polypeptide of the invention in such therapeutically useful compositions is such as to achieve the level of effective dose.

Tablets, pellets, pills, capsules, etc. can also contain the following: binders, such as tragacanth gum, gum Arabic, corn starch or gelatin; excipients such as calcium phosphate; dezintegriruetsja agents, such as corn starch, potato starch, alginic acid and others; emollients, such as magnesium stearate; sweeteners, such as sucrose, fructose, lactose or aspartame or you can add flavorings such as peppermint oil, oil of Grushenka or cherry flavoring. When the standard dose form is a capsule, it may contain, along with the materials of the above type, a liquid carrier, such as vegetable oil or polyethylene glycol. May contain various other materials in the form of Obolo the key or otherwise modifying the physical form of solid dose forms. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and other Syrup or elixir may contain the amino acid sequence, nanotesla or polypeptides of the invention, sucrose or fructose as sweeteners, methyl - and propylparaben as preservatives, a dye and flavoring type cherry or orange flavor. Of course, any material used in the preparation of a standard dose form should be pharmaceutically acceptable and practically non-toxic in the quantities used. In addition, amino acid sequences, nanotesla and polypeptides of the invention can be included in the drugs and devices for slow release.

Drugs and forms for oral administration can also be supplied intersolubility coating, which allows the construction of the invention to be stable in the gastric environment and pass into the intestine. In General, drugs and forms for oral administration can be properly prepared for delivery to any desired portion of the gastrointestinal tract. In addition, for delivery to the gastrointestinal tract may be a suitable candle.

Amino acid sequence, nanotesla and polypeptides of the invention can also be administered intravenously or NR is drybrushing by infusion or injection. Solutions of amino acid sequences of nantel and polypeptides of the invention or their salts can be prepared in water, optionally mixed with a nontoxic surfactant substance. It is also possible to prepare dispersions in glycerol, liquid polyethylene glycol, triacetine and their mixtures, as well as in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

Pharmaceutical dose forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which is adapted to ex tempore preparation of sterile solutions or dispersions for injection or infusion and need not be enclosed in liposomes. In any case, the final dose form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier can be a solvent or liquid dispersion medium containing, for example, water, ethanol and a polyol (e.g. glycerol, propylene glycol, liquid polyethylene glycol, etc.,), vegetable oils, non-toxic glycerin esters and their suitable mixtures. The proper fluidity can be maintained, for example, by the formation of liposomes, by the observance of the required particle size in the case of dispersions or using detergent is. To prevent the action of microorganisms using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the compositions for injection is achieved using the compositions of substances that slow down the absorption of, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by introduction of amino acid sequences of nantel and polypeptides of the invention in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by sterilization by filtration. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods for their preparation are methods of drying under vacuum and freeze drying, which give a powder of the active ingredient plus any additional desired ingredient present in the previously sterilized by filtration solutions.

For local application of amino acid sequence, nanotesla and polypeptides of the invention may be applied in pure form, if what they are liquid. However, in General it is desirable to apply them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be solid or liquid.

Applicable solid carriers include fine-grained solids such as talc, clay, microcrystalline cellulose, silica, alumina, etc. Applicable liquid carriers include water, hydroxyalkyl or glycols or a mixture of water-alcohol/glycol, which can dissolve or dispergirujutsja in effective quantities of amino acid sequence, nanotesla and polypeptides of the invention, optionally with non-toxic detergents. To optimize the properties for this application, you can add adjuvants, such as odorants and additional antimicrobial agents. The liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump or aerosol spray guns.

Together with liquid media for education spreading pastes, gels, ointments, Soaps, etc. for applying directly to the skin of the user, you can also use such thickening agents as synthetic polymers, fatty acids, salts and esters of fatty acids, fatty alcohols, modified cellulose or option is lirovannye mineral materials.

Examples of useful dermatological compositions which can be used for delivery of amino acid sequences of nantel and polypeptides of the invention on the skin, known in this area; for example, see Jacquet et al. (US 4,608,392), Geria (US 4,992,478), Smith et al. (US 4,559,157) and Wortzman (US 4,820,508).

The applicable dose amino acid sequences of nantel and polypeptides of the invention can be determined by comparison of their activity in vitro and activity in vivo in animal models. The extrapolation methods effective doses from mice and other animals to humans are known in the art; for example, see US 4,938,949.

Usually the concentration of the amino acid sequences of nantel and polypeptides of the invention in a liquid composition type of lotion is from 0.1 to 25 wt. -%, preferably from 0.1 to 10 wt%. Concentration in semi-solid and solid compositions of a type of gel or powder is 0.1-5%, preferably 0.5 to 2.5% of the mass.

The number of amino acid sequences of nantel and polypeptides of the invention required for treatment depends not only on the specific selected amino acid sequence, nanotesla or polypeptide of the invention, and the method of application, the nature of the subject to treatment of the disease and the age and condition of the patient, and ultimately is at the discretion of the attending physician or Clinician. Also the dosage of am is nekisnotnice sequences nantel and polypeptides of the invention varies depending on the cell, tumor, tissue, graft, or organ target.

The required dose for the convenience can be represented in a single dose or in the form of fractional doses, administered at appropriate intervals, for example, as two, three, four or more fractional doses per day. Itself fractional dose may be further divided, e.g., on a number of discrete loosely spaced injections, such as repeated inhalation of apparatus for injection (inhaler) or by depositing a few drops in the eye. The mode of administration may include prolonged daily treatment. By "continuous" is meant at least two weeks and preferably lasts several weeks, months, or years. The necessary modifications of this dose interval can be set as an ordinary specialist in this area only one simple selection, taking into account these provisions. Cm. Remington''s Pharmaceutical Sciences (Martin E. W., ed. 4), Mack Publishing Co., Easton, PA. In case of any complications, the dosage can be adjusted by the individual physician.

In another aspect, the invention concerns a method of prevention and/or treatment of at least one associated with IL-6R diseases and/or disorders, which includes an introduction to the needy in the subject of pharmaceutically active amount of amino acid pic is egovernance of the invention, nanotesla of the invention, the polypeptide of the invention, the compounds of the invention, the construction of the invention and/or containing pharmaceutical compositions.

In the context of the present invention, the term "prevention and/or treatment" includes not only the prevention and/or treatment of disease, but also generally includes the prevention of disease, slowing or reversing the disease, preventing or slowing the appearance of one or more symptoms associated with the disease, reduction and/or weakening of one or more symptoms associated with the disease, reduce the severity and/or duration of the disease and/or any related symptoms and/or prevent further enhance the severity of the disease and/or any related symptoms, prevention, the reduction or reversal of any physiological damage caused by the disease, and any pharmacological effect, which is beneficial for the patient under treatment.

Subject to treatment can be any warm-blooded animal, particularly a mammal and more preferably a human. As it should be known to experts, subject to treatment must be an individual suffering from or at risk of these diseases and violated the th.

The invention relates to a method of preventing and/or treating at least one disease and/or disorders associated with IL-6, IL-6R, IL-6/IL-6R, with its biological or pharmacological activity, and/or biological or signalling pathways involving IL-6, IL-6R and/or IL-6/IL-6R, which includes an introduction to the needy in the subject of pharmaceutically active amount of the amino acid sequence of the invention, nanotesla of the invention, the polypeptide of the invention, the compounds of the invention, the construction of the invention and/or containing pharmaceutical compositions. In particular, the invention relates to a method of preventing and/or treating at least one disease and/or disorders that can be prevented and/or treated by modulating IL-6, IL-6R, IL-6/IL-6R, their biological or pharmacological activity, and/or biological or signaling pathways involving IL-6, IL-6R and/or IL-6/IL-6R, which includes an introduction to the needy in the subject of pharmaceutically active amount of the amino acid sequence of the invention, nanotesla of the invention, the polypeptide of the invention, the compounds of the invention, the construction of the invention and/or containing pharmaceutical compositions. In particular, such pharmaceutically active amount may be the number, sufficient to modulate IL-6, IL-6R, IL-6/IL-6R, their biological or pharmacological activity, and/or biological or signaling pathways involving IL-6, IL-6R and/or IL-6/IL-6R.

The invention also relates to a method of preventing and/or treating at least one disease and/or disorders that can be prevented and/or treated by administration to the patient of amino acid sequences of the invention, nanotesla of the invention or of the polypeptide of the invention, which includes an introduction to the needy in the subject of pharmaceutically active amount of the amino acid sequence of the invention, nanotesla of the invention, the polypeptide of the invention, the compounds of the invention, the construction of the invention and/or containing pharmaceutical compositions.

More specifically the invention relates to a method of preventing and/or treating at least one disease and/or disorders selected from the group consisting of the listed diseases and disorders, which includes an introduction to the needy in the subject of pharmaceutically active amount of the amino acid sequence of the invention, nanotesla of the invention, the polypeptide of the invention, the compounds of the invention, the construction of the invention and/or containing pharmaceutical compositions.

In particular, the present izobreteny which relates to a method of prevention and/or treatment of sepsis, various forms of cancer, bone resorption, osteoporosis, cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, inflammatory diseases, which includes the introduction of a pharmacologically active amount of the amino acid sequence of the invention, nanotesla of the invention, the polypeptide of the invention and/or containing pharmaceutical compositions. Different types of cancer can be selected from the group consisting of multiple myeloma (MM), renal cell carcinoma (RCC), plasmacytomas leukemia, lymphoma, B-lymphoproliferative disorders (BLPD) and prostate cancer. Inflammatory diseases can be selected from the group consisting of rheumatoid arthritis, systemic juvenile idiopathic arthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, diseases of Castellana, IgM-gammopathy, Mikami heart, asthma, allergic asthma, and autoimmune insulin-dependent diabetes mellitus.

In one preferred aspect the present invention relates to a method of prevention and/or treatment of sepsis, various forms of cancer, bone resorption, osteoporosis, cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma and inflammatory ill the deposits, which includes the introduction of pharmaceutically active amount of SEQ ID NO:70 and/or its containing pharmaceutical compositions. In another preferred aspect the present invention relates to a method of prevention and/or treatment of sepsis, various forms of cancer, bone resorption, osteoporosis, cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, and inflammatory diseases, which includes the introduction of pharmaceutically active amount of SEQ ID NO:71 and/or its containing pharmaceutical compositions. Different types of cancer can be selected from the group consisting of multiple myeloma (MM), renal cell carcinoma (RCC), plasmacytomas leukemia, lymphoma, B-lymphoproliferative disorders (BLPD) and prostate cancer. Inflammatory diseases can be selected from the group consisting of rheumatoid arthritis, systemic juvenile idiopathic arthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, diseases of Castellana, IgM-gammopathy, Mikami heart, asthma, allergic asthma, and autoimmune insulin-dependent diabetes mellitus.

In another embodiment the invention relates to a method of immunotherapy, in particular for passive immunotherapy, which includes an introduction to a subject suffering from or is advergames risk of these diseases and disorders, pharmaceutically active amount of the amino acid sequence of the invention, nanotesla of the invention, the polypeptide of the invention, the compounds of the invention, the construction of the invention and/or containing pharmaceutical compositions.

In the above methods, the amino acid sequence, nanotesla, polypeptides, compounds and/or constructs of the invention and/or compositions containing them can be administered in any suitable way, depending on the specific pharmaceutical forms or compositions. Thus, amino acid sequences, nanotesla, polypeptides, compounds and/or constructs of the invention and/or compositions containing them can be administered, for example, oral, parenteral (e.g., intravenously, intraperitoneally, subcutaneously, intramuscularly, or by any other method of administration bypasses the gastrointestinal tract) through the nose, through the skin, topically or by suppository, by inhalation, again depends on the specific pharmaceutical forms or compositions. The Clinician will be able to choose a suitable route of administration, and a suitable pharmaceutical form or composition that should be used with this introduction, depending on the disease and/or disorders, which need to be prevented or treated, and other factors well known to clinicians.

Aminoisophthalate, nanotesla, polypeptides, compounds and/or constructs of the invention and/or compositions containing them are entered according to the mode of treatment that is appropriate for prevention and/or treatment of diseases and/or disorders, which need to be prevented or treated. The Clinician will generally be able to determine a suitable treatment regimen, the severity of the disease that should be treated, and/or the severity of its symptoms, the specific amino acid sequence, nanotesla, polypeptides, compounds or constructs of the invention, which should be used, the specific route of administration and the specific pharmaceutical form or composition that you want to use, age, gender, weight, diet, General condition of the patient, and like factors well known to clinicians.

In General, treatment should include the introduction of one or more amino acid sequences of nantel, polypeptides, compounds and/or constructs of the invention or one or more compositions containing, in one or more of pharmaceutically effective doses. The specific number of the injected dose can be set by the Clinician, again on the basis of the factors listed above.

In General, for the prevention and/or treatment of these diseases and disorders and depending on specific diseases or disorders that can be treated, the potential is social activity used by a specific amino acid sequence, nantel, polypeptides, compounds and constructions of the invention, the specific route of administration and the specific pharmaceutical form or composition, amino acid sequence, nanotesla, polypeptides, compounds and constructions of the invention should be administered in amounts of between 1 gram and 0.01 microgram per kg body weight per day, preferably between 0.1 g and 0.1 microgram per kg body weight per day, such as 1, 10, 100, 1000 μg per kg of body weight per day, either continuously (e.g., the infusion) as a single daily dose or as multiple fractional doses during the day. The Clinician will generally be able to determine the appropriate daily dose depending on the factors mentioned above. Also, it should be clear that in specific cases, the Clinician may depart from these quantities, for example, on the basis of the factors listed above, and its expert judgment. In General, some recommendations for input doses can be obtained from those doses that are usually entered for comparable conventional antibodies or fragments of antibodies against the same target and input in much the same way, but taking into account differences in the affinity/avidity, efficiency, biological distribution, time-life and similar factors well known to specialists.

Typically, in the above method is used, only one amino acid posledovatelno the ü, nanotesla, polypeptide, compound or construct of the invention. However, in the scope of this invention includes the use of two or more amino acid sequences of nantel, polypeptides, compounds and/or constructs of the invention in combination.

Amino acid sequence, nanotesla, polypeptides, compounds and constructions of the invention can also be used in combination with one or more additional pharmaceutically active compounds or principles, i.e. as a combined treatment regimen, which may give or not to give a synergistic effect. Again, the Clinician will be able to select such additional connections or beginning, as well as suitable combined treatment on the basis of the factors listed above, and its expert judgment.

In particular, amino acid sequences, nanotesla, polypeptides, compounds and constructions of the invention can be used in combination with other pharmaceutically active compounds or principles that are applied or could be applied for the prevention and/or treatment of diseases and disorders listed above, so you can get or not to get a synergistic effect. Examples of such compounds, and began, as well as the ways, methods and pharmaceutical forms or compositions for their introduction should be is swesty clinicians.

When using two or more substances or started as part of a combined treatment regimen you can enter them one and the same method of administration or different routes of administration, almost at the same time or at different times (e.g., almost simultaneously, sequentially or interleaved scheme). If the substance or the beginning entered at the same time in the same way of introduction, you can enter them as separate pharmaceutical forms or compositions or in combined form, or composition, as it should be known to experts.

Moreover, the use of two or more active substances or started as part of a combined treatment regimen, each of the substances or started can be entered in the same amount and in the same way as when using the connection or start in themselves, and such combined use may give or not to give a synergistic effect. However, if the combined use of two or more active substances or started results in a synergistic effect, it is possible to reduce the number of one, several or all of the input substances or started, and thus to obtain the desired therapeutic effect. For example, it may be useful to avoid, limit or reduce any undesirable side effects associated with the use of one or more substances or started, when they are used in their usual amounts, and yet to obtain the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment applied according to the invention, it is possible to determine and/or monitor in any way known per se for these diseases and/or disorders, as it should be clear to clinicians. The Clinician will also be able, when necessary and on an individual basis, amend, or modify specific treatment in order to obtain the desired therapeutic effect, avoid, limit, or reduce unwanted side effects and/or to achieve the appropriate balance between achieving the desired therapeutic effect, on the one hand, and the avoidance, limitation, or reduction of undesirable side effects.

In General, you should stick to the regimen to achieve the desired therapeutic effect and/or so long as to be saved to the desired therapeutic effect. Again, this can be determined by a Clinician.

In another aspect of the invention concerns the use of amino acid sequence, nanotesla, polypeptide, compound or (monovalent) the construction of the invention in the manufacture of pharmaceutical compositions for the prevention and/or treatment of at least one associated with IL-6R disease is.

The invention also concerns the use of amino acid sequence, nanotesla, polypeptide, compound or (monovalent) the construction of the invention in the manufacture of pharmaceutical compositions for the prevention and/or treatment of at least one of the diseases and disorders associated with IL-6, IL-6R, IL-6/IL-6R and/or signalling pathways and/or the biological functions and responses involving IL-6, IL-6R and/or IL-6/IL-6R; and/or for use in one or more ways described here.

The invention also concerns the use of amino acid sequence, nanotesla, polypeptide, compound or (monovalent) the construction of the invention in the manufacture of pharmaceutical compositions for preventing and/or treating at least one disease or disorders that can be prevented and/or treated by modulating IL-6, IL-6R, IL-6/IL-6R, their biological or pharmacological activity, and/or biological or signaling pathways involving IL-6, IL-6R and/or IL-6/IL-6R.

The invention also concerns the use of amino acid sequence, nanotesla, polypeptide, compound or (monovalent) the construction of the invention in the manufacture of pharmaceutical compositions for preventing and/or treating at least one disease or naru is to be placed, which can be prevented and/or treated by administration to the patient of amino acid sequence, nanotesla, polypeptide, compound or construct of the invention.

More specifically the invention concerns the use of amino acid sequence, nanotesla, polypeptide, compound or construct of the invention in the manufacture of pharmaceutical compositions for the prevention and/or treatment associated with IL-6R diseases, in particular for the prophylaxis and/or treatment of sepsis, various forms of cancer, bone resorption, osteoporosis, cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, inflammatory diseases, which includes the introduction of a pharmacologically active amount of the amino acid sequence of the invention, nanotesla of the invention, the polypeptide of the invention and/or containing pharmaceutical compositions. Different types of cancer can be selected from the group consisting of multiple myeloma (MM), renal cell carcinoma (RCC), plasmacytomas leukemia, lymphoma, B-lymphoproliferative disorders (BLPD) and prostate cancer. Inflammatory diseases can be selected from the group consisting of rheumatoid arthritis, systemic juvenile idiopathic arthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis, systemic lupus in which lanky (SLE), multiple sclerosis, a disease of Castellana, IgM-gammopathy, Mikami heart, asthma, allergic asthma, and autoimmune insulin-dependent diabetes mellitus.

The invention also relates to amino acid sequences, nantel, compounds or constructs, polypeptides, monovalent constructs of the invention or containing pharmaceutical compositions for use in preventing and/or treating at least one associated with IL-6R diseases and/or disorders.

The invention also relates to amino acid sequences, nantel, compounds or constructs, polypeptides, monovalent constructs of the invention or containing pharmaceutical compositions for use in preventing and/or treating at least one disease and/or disorders associated with IL-6, IL-6R, IL-6/IL-6R, their biological or pharmacological activity, and/or biological or signalling pathways involving IL-6, IL-6R and/or IL-6/IL-6R.

The invention also relates to amino acid sequences, nantel, compounds or constructs, polypeptides, monovalent constructs of the invention or containing pharmaceutical compositions for use in preventing and/or treating at least one disease and/or disorders that can be prevented and/or is Ekiti by modulating IL-6, IL-6R, IL-6/IL-6R, their biological or pharmacological activity, and/or biological or signaling pathways involving IL-6, IL-6R and/or IL-6/IL-6R.

The invention also relates to amino acid sequences, nantel, compounds or constructs, polypeptides, monovalent constructs of the invention or containing pharmaceutical compositions for use in preventing and/or treating at least one disease and/or disorders that can be prevented and/or treated by administration to the patient of amino acid sequences of the invention, nanotesla of the invention or of the polypeptide of the invention.

The invention also relates to amino acid sequences, nantel, compounds or constructs, polypeptides, monovalent constructs of the invention or containing pharmaceutical compositions for use in the prevention and/or treatment of sepsis, various forms of cancer, bone resorption, osteoporosis, cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, inflammatory diseases, which includes the introduction of a pharmacologically active amount of the amino acid sequence of the invention, nanotesla of the invention, the polypeptide of the invention and/or containing pharmaceutical compositions. According to CNAE types of cancer can be selected from the group consisting of multiple myeloma (MM), renal cell carcinoma (RCC), plasmacytomas leukemia, lymphoma, B-lymphoproliferative disorders (BLPD) and prostate cancer. Inflammatory diseases can be selected from the group consisting of rheumatoid arthritis, systemic juvenile idiopathic arthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, diseases of Castellana, IgM-gammopathy, Mikami heart, asthma, allergic asthma, and autoimmune insulin-dependent diabetes mellitus.

Subject to treatment can be any warm-blooded animal, particularly a mammal, more preferably human. As it should be known to experts, subject to treatment must be an individual suffering from or at risk of these diseases and disorders.

Again, in such pharmaceutical compositions, one or more amino acid sequences of nantel, polypeptides, compounds or constructs of the invention can also be combined appropriately with one or more other active principles such as those that are listed here.

The present invention is hereinafter disclosed in the following Examples, which in no way should be interpreted as restrictive. Full content of the W all references (including literature references, issued patents, published patent applications and pending patent applications) cited throughout the application are hereby expressly incorporated into it by reference, in particular, regarding the content of the above works.

ASPECTS

Aspect 1. Amino acid sequence directed against IL-6R, which contains one or several segments of amino acid residues chosen from the following:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance;

and/or

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid PEFC is a sequence, contains the plot of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance;

and/or

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance;

Aspect 2. Amino acid sequence according to aspect 1, containing two or more parcels of amino acid residues chosen from the following:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, etc is what the affinity is measured by means of surface plasma resonance;

and/or

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance;

and/or

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance.

that (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to a) or b), the second stretch of amino acid residues corresponds to one of the amino acid for which sledovatelnot according to C), d), e) or f); (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequence according to (C) or (d), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), e) or f); or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequence according to e) or f), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), C) or d).

Aspect 3. Amino acid sequence according to aspect 1 or 2, containing three or more parcels amino acid residues, with the first stretch of amino acid residues selected from the group consisting of:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance;

the second stretch of amino acid residues select the n group, consisting of:

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance; and

the third stretch of amino acid residues selected from the group consisting of:

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance.

Aspect 4. Amino acid sequence according to any one of aspects 1-3, which contains immunoglobulin fold or under appropriate conditions with osobne to form an immunoglobulin fold.

Aspect 5. Amino acid sequence according to any one of aspects 1-4, which is an immunoglobulin sequence.

Aspect 6. Amino acid sequence according to any one of aspects 1-5, which essentially consists of 4 frame sections (FR1 to FR4 respectively) and 3 complementarity determining areas (CDR1 to CDR4, respectively), with:

- CDR1 selected from the group consisting of:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance;

and/or

- CDR2 selected from the group consisting of:

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity for cf is the ranking, with the amino acid sequence, contains the plot of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance;

and/or

- CDR3 selected from the group consisting of:

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance.

Aspect 7. Amino acid sequence according to aspect 6, which essentially consists of 4 frame sections (FR1 to FR4 respectively) and 3 complementarity determining areas (CDR1 to CDR4, respectively), with:

- CDR1 selected from the group consisting of:

a) SEQ ID NO's:80-82; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:80-82, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or pain is their affinity compared to the amino acid sequence, contains the plot of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance;

and

- CDR2 selected from the group consisting of:

c) SEQ ID NO's:84-91; or

d) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:84-91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance;

and

- CDR3 selected from the group consisting of:

e) SEQ ID NO's:93-95; or

f) plots amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-95, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon rez is the mission.

Aspect 8. Amino acid sequence according to any one of aspects 1-7, which contains at least:

a) SEQ ID NO:80; or

b) plot of amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:80, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance.

Aspect 9. Amino acid sequence according to any one of aspects 1-8, which contains at least one stretch of amino acid residues chosen from the following:

a) SEQ ID NO's:84, 89 or 91; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:84, 89 or 91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by the method of surface the CSOs plasma resonance.

Aspect 10. Amino acid sequence according to any one of aspects 1-9, which contains at least:

a) SEQ ID NO:84; or

b) plot of amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:84, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance.

Aspect 11. Amino acid sequence according to any one of aspects 1 to 10, which contains at least one stretch of amino acid residues chosen from the following:

a) SEQ ID NO's:93-94; or

b) parts of the amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's:93-94, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmas the frame of the resonance.

Aspect 12. Amino acid sequence according to any one of aspects 1 to 11, which contains at least:

a) SEQ ID NO:93; or

b) plot of amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:93, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasma resonance.

Aspect 13. Amino acid sequence according to any one of aspects 1 to 12, which contains at least two segments of amino acid residues chosen from the following:

a. SEQ ID NO:80 and SEQ ID NO:84;

b. SEQ ID NO:80 and SEQ ID NO:93; or

C. SEQ ID NO:84 and SEQ ID NO:93.

Aspect 14. Amino acid sequence according to any one of aspects 1-13, which contains SEQ ID NO:80, SEQ ID NO:84 and SEQ ID NO:93.

Aspect 15. Amino acid sequence according to any one of aspects 1 to 14, which essentially consists of the sequence of the variable domain of the heavy chain, originating from normal chetyrekhzvezdochnogo antibodies, or essentially consists of the sequence of the variable domain of the heavy chain, originating from Antialias heavy chain.

Aspect 16. Amino acid sequence according to any one of aspects 1-15, which essentially consists of a domain antibody (or an amino acid sequence suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), antibody, "dAb" (or an amino acid sequence suitable for use as a dAb) or nanotesla.

Aspect 17. Amino acid sequence according to any one of aspects 1-16 selected from the group consisting of:

a) SEQ ID NO's:60-69;

(b) sequences having no more than 2, preferably no more than 1 amino acid difference in one, two or all of its CDRs from one of SEQ ID NO''s:60-69, provided that the amino acid sequence containing not more than 2, preferably no more than 1 amino acid difference in one, two or all of its CDRs, binds to IL-6R with about the same or greater affinity compared to one of SEQ ID NO''s:60-69, and affinity is measured by means of surface plasma resonance;

c) sequences having no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:60-69, provided that the amino acid sequence containing not more than 2, preferably no more than 1 amino acid Otley the Oia from one of SEQ ID NO''s:60-69, binds to IL-6R with about the same or greater affinity compared to the binding of one of SEQ ID NO''s:60-69, and affinity is measured by means of surface plasma resonance.

Aspect 18. Amino acid sequence according to aspect 17, selected from the group consisting of:

a) SEQ ID NO's:65-69;

(b) sequences having no more than 2, preferably no more than 1 amino acid difference in one, two or all of its CDRs from one of SEQ ID NO''s:65-69, provided that the amino acid sequence containing not more than 2, preferably no more than 1 amino acid difference in one, two or all of its CDRs, binds to IL-6R with about the same or greater affinity compared to one of SEQ ID NO''s:65-69, and the affinity is measured by means of surface plasma resonance;

c) sequences having no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:65-69, provided that the amino acid sequence containing not more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:65-69, binds to IL-6R with about the same or greater affinity compared to the binding of one of SEQ ID NO''s:65-69, and the affinity is measured by means of surface plasma resonance.

Aspect 19. Amino acid sequence according to aspect 18, selected from the group SOS is oasa from:

a) SEQ ID NO:66;

(b) sequences having no more than 2, preferably no more than 1 amino acid difference in one, two or all of its CDRs from SEQ ID NO:66, provided that the amino acid sequence containing not more than 2, preferably no more than 1 amino acid difference in one, two or all of its CDRs, binds to IL-6R with about the same or greater affinity compared to one of SEQ ID NO:66, and the affinity is measured by means of surface plasma resonance;

c) sequences having no more than 2, preferably no more than 1 amino acid difference from SEQ ID NO:66, provided that the amino acid sequence containing not more than 2, preferably no more than 1 amino acid difference from SEQ ID NO's:66, binds to IL-6R with about the same or greater affinity compared to the binding of SEQ ID NO:66, and the affinity is measured by means of surface plasma resonance.

Aspect 20. Amino acid sequence according to any one of aspects 1 to 19, which specifically binds to hIL-6R with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less.

Aspect 21. Amino acid sequence according to any one of aspects 1 to 20, which specifically concerns ivalsa with cynoIL-6R with a dissociation constant (K Dfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less.

Aspect 22. Amino acid sequence according to any one of aspects 1 to 21, which specifically binds to HIL-6R with a rate constant konfrom 104M-1s-1up to 107M-1s-1preferably from 105M-1s-1up to 107M-1s-1more preferably about 106M-1s-1or more.

Aspect 23. Amino acid sequence according to any one of aspects 1-22, which specifically binds to cynoIL-6R with a rate constant konfrom 104M-1s-1up to 107M-1s-1preferably from 107M-1s-1up to 107M-1s-1more preferably about 106M-ls-lor more.

Aspect 24. Amino acid sequence according to any one of aspects 1-23, which specifically binds to hIL-6R with a rate constant kofffrom 10-3(t½ =0,69 s) up to 10-6s-1(giving almost irreversible complex with t½ in a few days), preferably between 10-4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or men who that is

Aspect 25. Amino acid sequence according to any one of aspects 1 to 24, which specifically binds to cynoIL-6R with a rate constant kofffrom 10-3(t½ =0,69 s) up to 10-6s-1(giving almost irreversible complex with t½ in a few days), preferably between 10-4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less.

Aspect 26. Amino acid sequence according to any one of aspects 1-25, which in the determination method TF-1 is the value of the IC50(ME at 100 IL-6/ml) between 10 nm and 50 PM, preferably between 5 nm and 50 PM, more preferably between 1 nm and 50 PM or less, such as about 750 or 500 PM or less.

Aspect 27. Amino acid sequence according to any one of aspects 1-26, when determining which method TF-1 is the value of the IC50(5,000 IU of IL-6/ml) between 50 nm and 1 nm, preferably between 25 nm and 1 nm, more preferably between 10 nm and 1 nm or less, such as about 8 nm or less.

Aspect 28. Amino acid sequence according to any one of aspects 1-27, when determining which method TF-1 is the value of the IC50that is at least the same or preferably better, at least twice, preferably three times, more preferably four times, even more preferably is 5 times 7 times or more than 7 times in comparison with the value of the IC50received control IgG provided in SEQ ID NO:1 and 2, or control Fab, shown in SEQ ID NO:3 and 4.

Aspect 29. Amino acid sequence according to any of aspects 1-28, when determining which method TF-1 is the value of the IC50that is at least the same or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50obtained for the Tocilizumab (MRA).

Aspect 30. Amino acid sequence according to any one of aspects 1-29, which in the determination method of the activity in the plasma when the value EC50for IL-6 is set to the IC50between 500 PM and 50 PM, preferably between 250 PM and 50 PM, more preferably between 200 PM and 50 PM or less, such as 150 PM or less.

Aspect 31. Amino acid sequence according to any one of aspects 1-30, which in the determination method of the activity in the plasma when the value EC50for IL-6 is set to the IC50between 1000 PM and 100 PM, preferably between 750 PM and 100 PM, more preferably between 500 PM and 100 PM or less, 400 PM or less.

Aspect 32. Amino acid sequence according to any one of aspects 1 to 31, which in the determination method of the activity and in plasma according to aspect 30 or 31 is set to the IC 50that is at least the same or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50received control IgG provided in SEQ ID NO:1 and 2, or control Fab, shown in SEQ ID NO:3 and 4.

Aspect 33. Amino acid sequence according to any one of aspects 1-32, which in the determination method of the activity in the plasma according to aspect 30 or 31 is set to the IC50that is at least the same or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50obtained for the Tocilizumab (MRA).

Aspect 34. Amino acid sequence according to any one of aspects 1-33, which has the value of the IC50to associate with membrane IL-6R on the cell SNO between 10 nm and 100 PM, preferably between 5 nm and 100 PM, more preferably between 2 nm and 10 PM or less, such as 2 nm or less.

Aspect 35. Compound or construct that contains or essentially consists of one or more amino acid sequences according to any one of aspects 1-34 and optionally further comprises one or more others the other groups, residues, molecules or binding units, optionally connected via one or more linkers.

Aspect 36. Compound or construct according to aspect 35, in which data from one or more other groups, residues, molecules or units of bonding selected from the group consisting of domain antibodies, amino acid sequences suitable for use as domain antibodies, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb", the amino acid sequences suitable for use as a dAb, or nantel.

Aspect 37. Compound or construct according to any one of aspects 35 to 36, which is a multivalent construct, such, e.g., as the divalent or trivalent design.

Aspect 38. Compound or construct according to any one of aspects 35 to 37, which is multispecific design, such, e.g., as especifica or trapezitinae design.

Aspect 39. Compound or construct according to any one of aspects 35-39, which has an increased half-life compared with the corresponding amino acid sequence according to any one of aspects 1-34 per se.

Aspect 40. Compound or construct according to aspect 39, in which data from one or more other groups, residues, molecules and the and units-binding give the joint or structure increased half-life compared with the corresponding amino acid sequence according to any one of aspects 1 to 20.

Aspect 41. Compound or construct according to aspect 39, in which data from one or more other groups, residues, molecules or binding units, giving the joint or structure increased the half-life selected from the group consisting of serum proteins or their fragments, binding units that can bind with proteins serum, Fc portion, and small proteins or peptides are able to bind with serum proteins.

Aspect 42. Compound or construct according to aspect 39, in which data from one or more other groups, residues, molecules or binding units, giving the joint or structure increased the half-life selected from the group consisting of serum albumin person or its fragments.

Aspect 43. Compound or construct according to aspect 39, in which data from one or more other groups, residues, molecules or binding units, giving the joint or structure increased the half-life selected from the group consisting of binding units that can communicate with serum albumin (such as serum albumin) or a serum immunoglobulin (such as IgG).

Aspect 44. Compound or construct according to aspect 39, in which data from one or more other groups, residues, molecules or binding units, giving the connection or design the AI increased half-life, selected from the group consisting of domain antibodies, amino acid sequences suitable for use as domain antibodies, single domain antibodies, amino acid sequences that are suitable for use as single domain antibodies, antibody "dAb", the amino acid sequences suitable for use as a dAb, or nantel the ability to communicate with serum albumin (such as serum albumin) or a serum immunoglobulin (such as IgG).

Aspect 45. Compound or construct according to aspect 39, in which data from one or more other groups, residues, molecules or binding units, giving the joint or structure increased the half-life selected from SEQ ID NO's:97-99.

Aspect 46. Compound or construct according to any one of aspects 35 to 45, selected from the following polypeptide sequences:

a) SEQ ID NO's:70-72;

b) polypeptide sequences that have no more than 2, preferably no more than 1 amino acid difference in one, two or all parts of the CDR of the invention from one of SEQ ID NO''s:70-72, provided that the polypeptide sequence of not more than 2, preferably no more than 1 amino acid difference in one, two or all parts of the CDR of the invention binds to IL-6R with about the same or greater affinity for sravnenie is with linking one of SEQ ID NO''s:70-72, moreover, the affinity is measured by means of surface plasmon resonance.

c) a polypeptide sequence having no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s:70-72, provided that the polypeptide sequence of not more than 2, preferably no more than 1 amino acid difference with one of SEQ ID NO's:70-72 binds to IL-6R with about the same or greater affinity compared to the binding of one of SEQ ID NO''s:70-72, and the affinity is measured by means of surface plasmon resonance.

Aspect 47. Compound or construct according to any one of aspects 35-46, selected from the following polypeptide sequences:

a) SEQ ID NO's:70-71;

b) polypeptide sequences that have no more than 2, preferably no more than 1 amino acid difference in one, two or all parts of the CDR of the invention from one of SEQ ID NO's:70-71, provided that the polypeptide sequence of not more than 2, preferably no more than 1 amino acid difference in one, two or all parts of the CDR of the invention binds to IL-6R with about the same or greater affinity compared to the binding of one of SEQ ID NO's:70-71, and the affinity is measured by means of surface plasma resonance.

c) a polypeptide sequence having no more than 2, preferably no more than 1 amino the PCI-e slot differences from one of SEQ ID NO's:70-71, provided that the polypeptide sequence of not more than 2, preferably no more than 1 amino acid difference with one of SEQ ID NO's:70-71 binds to IL-6R with about the same or greater affinity compared to the binding of one of SEQ ID NO's:70-71, and the affinity is measured by means of surface plasma resonance.

Aspect 48. Compound or construct according to any one of aspects 35-47, which comprises or essentially consists of SEQ ID NO:70.

Aspect 49. Compound or construct according to any one of aspects 35-47, which contains or consists mainly of SEQ ID NO:71.

Aspect 50. Compound or construct according to any one of aspects 35-49, which specifically binds to hIL-6 with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less.

Aspect 51. Compound or construct according to any one of aspects 35-50, which specifically binds to cynoIL-6 with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less.

Aspect 52. Compound or construct according to any one of aspects 35-51, which specifically binds to hIL-6R with a constant ck is rosty k onfrom 104M-1s-1up to 107M-1s-1preferably from 105M-1s-1up to 107M-1s-1more preferably about 106M-1s-1or more.

Aspect of 53. Compound or construct according to any one of aspects 35 to 52, which specifically binds to cynoIL-6R with a rate constant konfrom 104M-1s-1up to 107M-1s-1preferably from 105M-1s-1up to 107M-1s-1more preferably about 106M-1s-1or more.

Aspect 54. Compound or construct according to any one of aspects 35-53, which specifically binds to hIL-6R with a rate constant kofffrom 10-3s-1(t½ =0,69 s) up to 10-6s-1(giving almost irreversible complex with t½ in a few days), preferably between 10-4s-1up to 10-6s-1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less.

Aspect 55. Compound or construct according to any one of aspects 35-54, which specifically binds to cynoIL-6R with a rate constant kofffrom 10-3s-1(t½ =0,69 s) up to 10-6s-1(giving almost irreversible complex with t½ in a few days), preferably between 10-4s-1up to 10-6 -1more preferably from 10-5s-1up to 10-6s-1how about 10-5s-1or less.

Aspect 56. Compound or construct according to any one of aspects 35-55, which in the determination method TF-1 is the value of the IC50(ME at 100 IL-6/ml) between 10 nm and 50 PM, preferably between 5 nm and 50 PM, more preferably between 1 nm and 50 PM or less, such as about 750 or 500 PM or less.

Aspect of 57. Compound or construct according to any one of aspects 35-56, which in the determination method TF-1 is the value of the IC50(5,000 IU of IL-6/ml) between 50 nm and 1 nm, preferably between 25 nm and 1 nm, more preferably between 10 nm and 1 nm or less, such as about 8 nm or less.

Aspect 58. Compound or construct according to any one of aspects 35-57, which in the determination method TF-1 is the value of the IC50that is at least the same or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50received control IgG provided in SEQ ID NO:1 and 2, or control Fab, shown in SEQ ID NO:3 and 4.

Aspect of 59. Compound or construct according to any one of aspects 35-58, which in the determination method TF-1 is the value of the IC50that is at least the same or pre is respectfully better at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50obtained for the Tocilizumab (MRA).

Aspect 60. Compound or construct according to any one of aspects 35-59, which in the determination method of the activity in the plasma when the value of the EU50for IL-6 is set to the IC50between 500 PM and 50 PM, preferably between 250 PM and 50 PM, more preferably between 200 PM and 50 PM or less, such as 150 PM or less.

Aspect 61. Compound or construct according to any one of aspects 35 to 60, which in the determination method of the activity in the plasma when the value ECos for IL-6 is set to the IC50between 1000 PM and 100 PM, preferably between 750 PM and 100 PM, more preferably between 500 PM and 100 PM or less, 400 PM or less.

Aspect 62. Compound or construct according to any one of aspects 35-61, which in the determination method of the activity in the plasma in 60 or 61 is set to the IC50that is at least the same or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50received control IgG provided in SEQ ID NO:1 and 2, or control Fab, shown in SEQ ID NO:3 and 4.

Aspect 3. Compound or construct according to any one of aspects 35-62, which in the determination method of the activity in the plasma in 60 or 61 is set to the IC50that is at least the same or preferably better, at least twice, preferably three times, more preferably four times, even more preferably 5 times, 7 times or more than 7 times in comparison with the value of the IC50obtained for the Tocilizumab (MRA).

Aspect 64. Compound or construct according to any one of aspects 35-63, which has the value of the IC50to associate with membrane IL-6R on the cell SNO between 10 nm and 100 PM, preferably between 5 nm and 100 PM, more preferably between 2 nm and 10 PM or less, such as 2 nm or less.

Aspect of 65. Monovalent construct that contains or essentially consisting of one amino acid sequence according to any one of aspects 1-34.

Aspect 66. The use of amino acid sequence according to any one of aspects 1-34 or a monovalent constructs according to aspect 65 when receiving a multivalent compound or construct according to any one of aspects 37-64.

Aspect of 67. The method of obtaining amino acid sequence according to any one of aspects 37-64, including linking amino acid sequence according to any one of aspects 1-34 or a monovalent constructs according to aspect 65 with one or more groups which, residues, molecules or units of the binding.

Aspect 68. The method according to the aspect of 67 to obtain multivalent compound or construct according to any one of aspects 37-64, including linking amino acid sequence according to any one of aspects 1-34 or a monovalent constructs according to aspect 65 with other groups, residues, molecules or binding units via one or more linkers.

Aspect 69. Nucleic acid or nucleotide sequence encoding the amino acid sequence according to any one of aspects 1-34 or a compound or construct according to any one of aspects 35-64, which can be obtained by expression of the coding its nucleic acid or nucleotide sequence or a monovalent constructs according to aspect 65.

Aspect 70. Nucleic acid or nucleotide sequence according to aspect 69, which is in the form of a genetic construct.

Aspect of 71. The host or a host cell that Express or under appropriate conditions can Express the amino acid sequence according to any one of aspects 1-34, compound or construct according to any one of aspects 35-64, which can be obtained by expression of the coding its nucleic acid or nucleotide sequence, or a monovalent construct according to aspect 65; and/or who will win nucleic acid or nucleotide sequence according to aspect 69, or a genetic construct according to aspect 70.

Aspect 72. The method of obtaining amino acid sequence according to any one of aspects 1-34, compound or construct according to any one of aspects 35-64, which can be obtained by expression of the coding its nucleic acid or nucleotide sequence, or a monovalent constructs according to aspect 65; and the method at least includes stages:

a) ekspressirovali in suitable cells-the owners or the body of the host or in another suitable expression system of nucleic acid or nucleotide sequence according to aspect 69, or genetic constructs according to aspect 70;

not necessarily followed:

b) isolating and/or cleaning resulting amino acid sequence according to any one of aspects 1-34, compound or construct according to any one of aspects 35-64, which can be obtained by expression of the coding its nucleic acid or nucleotide sequence, or a monovalent constructs according to aspect 65.

Aspect 73. The method of obtaining amino acid sequence according to any one of aspects 1-34, compound or construct according to any one of aspects 35-64, which can be obtained by expression of the coding its nucleic acid or nucleotide sequence, or a monovalent constructs according to aspect 65; and the method at least includes stages:

a) it is steverivonia and/or content host or host cell according to aspect 71 in such conditions, when this host or a host cell Express and/or secrete at least one amino acid sequence according to any one of aspects 1-34, compound or construct according to any one of aspects 35-64, which can be obtained by expression of the coding its nucleic acid or nucleotide sequence, or a monovalent construct according to aspect 65;

not necessarily followed:

b) isolating and/or cleaning resulting amino acid sequence according to any one of aspects 1-34, compound or construct according to any one of aspects 35-64, which can be obtained by expression of the coding its nucleic acid or nucleotide sequence, or a monovalent constructs according to aspect 65.

Aspect 74. A composition comprising at least one amino acid sequence according to any one of aspects 1-34, compound or construct according to any one of aspects 35-64, monovalent construct according to aspect 65 or nucleic acid or nucleotide sequence according to aspect 69 or 70.

Aspect of 75. The composition according to the aspect 74, which is a pharmaceutical composition.

Aspect of 76. The composition according to the aspect 74, which is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable carrier, diluent or excep the UNT and/or adjuvant, and optionally contains one or more additional pharmaceutically active polypeptides and/or compounds.

Aspect of 77. The method of prevention and/or treatment of at least one of the diseases or disorders associated with IL-6, IL-6R, IL-6/IL-6R and/or signalling pathways and/or the biological functions and responses involving IL-6, IL-6R or IL-6/IL-6R, which includes an introduction to the needy in the subject of pharmaceutically active amount of at least one amino acid sequence according to any one of aspects 1-34, compound or construct according to any one of aspects 35-64, monovalent constructs according to aspect 65 or composition according to any one of aspects 75-76.

Aspect of 78. The method according to aspect 77, wherein the diseases and disorders associated with IL-6, IL-6R, IL-6/IL-6R and/or signalling pathways and/or the biological functions and responses involving IL-6, IL-6R and/or IL-6/IL-6R, which are selected from the group comprising sepsis, various forms of cancer, bone resorption, osteoporosis, cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, and inflammatory diseases.

Aspect of 79. The method according to aspect 78, in which many forms of cancer is chosen from the group consisting of multiple myeloma (MM), renal cell carcinoma (RCC), plasmocytoma leukemia, lymphoma, B-lymphoproliferative disorder (BLPD) and prostate cancer.

Aspect 80. The method according to aspect 78, which in policeline disease selected from the group consisting of rheumatoid arthritis, systemic juvenile idiopathic arthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, diseases of Castellana, IgM-gammopathy, Mikami heart, asthma, allergic asthma, and autoimmune insulin-dependent diabetes mellitus.

Aspect of 81. The method of prevention and/or treatment of at least one of the diseases or disorders that can be prevented or treated by the introduction of the needy in the subject amino acid sequence according to any one of aspects 1-34, compound or construct according to any one of aspects 35-64 or monovalent constructs according to aspect 65, the method includes an introduction to the needy in the subject of pharmaceutically active amount of at least one amino acid sequence according to any one of aspects 1-34, compound or construct according to any one of aspects 35-64, monovalent constructs according to aspect 65 or composition according to any one of aspects 75-76.

Aspect 82. The use of amino acid sequence according to any one of aspects 1-34, compound or construct according to any one of aspects 35-64 or monovalent constructs according to aspect 65 upon receipt of a pharmaceutical composition for prevention and/or treatment of at least one of the diseases or disorders associated IL-6, IL-6R, IL-6/IL-6R and/or signalling pathways and/or the biological functions and responses involving IL-6, IL-6R and/or IL-6/IL-6R; and/or for use in one or more ways according to aspects 77-81.

Aspect of 83. Amino acid sequence according to any one of aspects 1-34, compound or construct according to any one of aspects 35-64 or monovalent construct according to aspect 65 for the prevention and/or treatment of at least one of the diseases or disorders associated with IL-6, IL-6R, IL-6/IL-6R and/or signalling pathways and/or the biological functions and responses involving IL-6, IL-6R and/or IL-6/IL-6R; and/or for use in one or more ways according to aspects 77-81.

Aspect of 84. The derived amino acid sequence according to any one of aspects 1-34, compound or construct according to any one of aspects 35-64 or monovalent constructs according to aspect 65.

Aspect of 85. The derivative according to aspect 84, can specifically bind to IL-6R.

Aspect of 86. Derivative according to any one of aspects 84-85, whose half-life in serum of at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times greater than the half-life of the corresponding amino acid sequence according to any one of aspects 1-34 per se, is connected to the I or construct according to any one of aspects 35-64 per se or a monovalent constructs according to aspect 65 per se.

Aspect of 87. Derivative according to any one of aspects 84-86, whose half-life in serum is increased more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence according to any one of aspects 1-34 per se, a compound or construct according to any one of aspects 35-64 per se or a monovalent construct according to aspect 65 per se.

Aspect 88. Derivative according to any one of aspects 84-87, whose half-life in serum is at least 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more; for example, at least 5 days (such as from 5 to 10 days), preferably at least 9 days (such as from 9 to 14 days), more preferably at least 10 days (such as about 10 to 15 days) or at least 11 days (such as from 11 to 16 days), more preferably at least 12 days (such as from 12 to 18 days or more) or more than 14 days (such as from 14 to 19 days).

Aspect of 89. Derivative according to any one of aspects 84-88, which is a pegylated derivative.

Aspect 90. Composition containing at least one derivative according to any one of aspects 84-89.

EXAMPLES

I. delanie binding IL-6R pantel

Example 1. Materials

The materials used for the selection of binding IL-6R of Manotel table.C-1.

You have created two representative of an antibody against IL-6R person described in EP 0628639 (Fab-fragment and the total IgG), which were used as control compounds. Fab-fragment and fully IgG designed on the basis of the L-chain, called "RVLa" (see EP 0628639 B1, PL.2, version (a)), and H-chain called "RVHf" (see EP 0628639 B1, PL.3, version (f)). This L-chain and the H chain were selected to construct control joints because according to EP 0628639 B1 (for example, see paragraph [0074]) reconstructed human antibody that contains the L chain and this N-chain, showed the ability to bind to IL-6R person on the same level as RM, monoclonal antibody mouse against IL-6R person (again, see EP 0628639 B1, paragraph [009] and see additional references).

Full control IgG consisted of amino acid sequence SEQ ID NO:1 (heavy chain) and SEQ ID NO:2 (light chain). Fab-fragment consisted of the amino acid sequences of SEQ ID NO:3 (sections VLb and VHf heavy chain fused with a plot of CH1 of IgG1 human) and SEQ ID NO:4 (variable region light chain human rebuilt RM-1, fused with a Ckappaperson.

Encoding DNA fragments were obtained by the method of collection is offered by the PCR, assembly PCR using overlapping oligonucleotides. The PCR products were cloned into a single dahlstrand vector, which allows to Express the functional, connected by a disulfide bond Fab-fragments in periplasmic E. coli. Full IgG was produced in cells SNO, 2 transfected expressing vectors containing the genes for the light and heavy chains. The gene encoding a heavy chain was obtained by merging VHf with a constant region of human IgG1. Light chain described in EP 0628639.

Example 2. Immunization

Were immunized two lamas (81 and 82) with IL-6R person (Peprotech) under the scheme of immunization are shown in table.-2.

Upon completion of the immunization schedule for each animal were analyzed immune responses by ELISA method. For this purpose, covered with neutravidin the microplate recorded biotinylated IL-6R (2 μg/ml). Added serial dilution of serum samples taken after 0, 28, 39, and 43 days (starting dilution 1:500), and were detected associated IgG Lama addition of HRP labeled goat antibodies against IgG Lama. As the substrate used TMB. The results are presented in Fig.1.

Immune responses were also analyzed by FACS method: serial dilutions (starting dilution 1:100) serum samples taken after 0, 28, and 43 days, incubated with cells U266 (human myeloma). Bound IgG Lama were detected using FITC labeled goat antibodies against IgG Lama. Results before the taulani in Fig.2.

Overall, these data suggest that both animals have developed a good immune response against IL-6R, and at least part of IgG Lama recognizes IL-6R on the cell surface U266.

Example 3. Designing libraries

For amplification of the coding nanotesla of gene fragments as starting material for RT-PCR used RNA isolated from PBLs and lymph nodes. These fragments were cloned in the expression vector derived from pUC119 which contained the LacZ promoter, the coding sequence of the protein pill coliphage, a resistance gene for ampicillin or carbenicillin, a multiple cloning site and a leader sequence gen3. In one frame with the coding sequence of nanotesla vector encodes C-terminal tag of C-myc and tag (His)6. The phage was obtained by the standard method and after sterilization by filtration kept at 4°C for further use. Features designed libraries are listed in the table.C-3.

Example 4. Selection

The selection of the above libraries was performed using different conditions are shown in table.C-4.

While all the conditions carried out only one cycle of selection. Each product selection analyzed for the degree of enrichment (the content of the phage in the eluate relative to control), diversity (profile HinfI) and the percentage of IL-6R-positive clones (ELISA).These parameters were selected the best sample for further analysis. The products from each selection again cloned as a pool in the expression vector derived from pUC119 which contained the LacZ promoter, a resistance gene for ampicillin or carbenicillin, a multiple cloning site and a leader sequence gen3. In one frame with the coding sequence of nanotesla vector encodes C-terminal tag of C-myc and tag (His)6. Selected colonies were cultured them in a deep 96-well plates (volume 1 ml), and then induced the expression of Manotel the addition of IPTG. Periplasmatic extracts were obtained according to standard procedures.

Example 5. Screening

Periplasmatic extracts were first analyzed for their ability to inhibit the interaction of IL-6/IL-6R. This was 2 independent samples to determine the method of Alphascreen, which is schematically shown in Fig.3. In the sample 1 Periplasmatic extracts were incubated with soluble receptor of IL-6 (1 nm), biotinylated IL-6 (3 nm), streptavidin coated donor beads and coated with MAb BN-12 acceptor beads (20 ág/ml). Nanotesla, positive under this definition, could inhibit either the interaction of the IL-6/IL-6R or the interaction of IL-6R/MAb BN-12. To distinguish between these 2 possibilities, was the second sample (sample 2). In this sample periplasmatic extracts were incubated with biotinylated IL-6R(0.3 nm), streptavidin coated donor beads and coated with MAb BN-12 acceptor beads (10 ág/ml). Nanotesla, which turned positive in the sample 1, but negative in the sample 2, was considered to be inhibitors of IL-6/IL-6R.

In both samples periplasmatic extracts were diluted 25 times, which roughly corresponds to a final concentration of 40 nm. Statistical summary of the screening programme are presented in the following table.C-5. Nanotesla showing the strongest inhibition, were selected for analysis of the rate constant koffon the Biacore instrument and DNA sequencing. In Fig.4 shows the protein sequence of the inhibitor of Manotel selected for further analysis on the cells. In table.C-6 lists the values of kofffor these inhibitor of Manotel.

Example 6. Expressiona and purification of Manotel

Selected nanotesla expressed in E. coli in the form of proteins tagged with C-myc and (His)6 in cultures of 50 or 250 ml. Expression was induced by adding 1 mm IPTG and continued for 4 h at 37°C. After centrifugation of cell cultures were prepared periplasmatic extracts by freezing and thawing of rainfall. These extracts were used as source material for affinity chromatography with immobilized metal (IMAC). Nanotesla was suirable from the column with 150 mm imidazole, and then subjected to di is Lisa against PBS. Total output and output per 1 liter of cell culture are listed in the table.C-7. SDS-PAGE of purified nantel (except RMSE) shown in Fig.5.

Example 7. Competition analysis mayeda proteins

14 purged of Manotel tested method Alphascreen on the inhibition of the interaction between IL-6/IL-6R. To IL-6R (0.3 nm) was added a serial dilution of purified proteins (concentration range: 500 nm - 10 PM) and incubated for 15 minutes then added 3 nm biotinylated IL-6 and coated with BN-12 acceptor beads and this mixture is incubated for 1 hour. Finally, added streptavidin donor beads and after 1 hour incubation, read the tablet on the reader Envision. As control included BR-6 and Fab-fragment described in Example 1. The results are presented in Fig.6.

For all 14 of nantel was observed curves dose-effect values IC50within 48 PM to 1.7 nm (table.C-8). The most powerful antelami in this method proved to be RMRS and RMRN. For RMRA was achieved only partial (-50%) inhibition of the interaction between IL-6/IL-6R.

Example 8. Determination of the affinity of the resulting nantel

The affinity constants (Kdsole nantel and control Fab-fragment described in Example 1 was determined by means of surface plasmon resonance (SPR) instrument Biacore 3000. Briefly, touch on the chip CM recorded IL-6R through the amino group PR is the density of 800-1000 Rel. units of Nanotesla was injected at 5 different concentrations between 1 and 50 PM. The flow rate was 45 μl/min in all experiments. Phase Association and dissociation were 3 and 10 min, respectively. The chip was regenerated with glycine/HCl pH to 1.5. Curves binding at various concentrations of Manotel used to calculate the kinetic parameters kon, koffand Kd(PL.C-9).

Example 9. The activity of Manotel on cells XG1

All cleared nanotesla tested method XG1. XG1 is IL-6-dependent cell line of human myeloma. Premaxilla proliferation is achieved at ~20 PG/ml IL-6. Definition mostly performed as described by Zhang et al. (1994, Blood 83: 3654-3663). As control included control Fab-fragment described in Example 1. The values of the IC50ranged from 90 PM to 50 nm (as shown in the table.C-10). A small subgroup of Manotel also tested by this method in the presence of 1 mg/ml HSA.

Example 10. The activity of Manotel on cells TF-1

Nanotesla also tested on their ability to inhibition of IL-6-dependent cell proliferation, TF-1 (ESAS no.93022307; 1989, J. Cell Physiol., 140: 323; 1993, Exp. Cell Res., 208: 35) by blocking the binding of IL-6 with IL-6R on the cell surface. For this, the serial cultivation of NanoTec has plaincourault with a fixed number of cells TF-1 for 2 hours at 37°C. then was added IL-6 to the end to the concentrations of 2 ng/ml IL-6-dependent proliferation of the cells was continued for 72 hours, and it was measured by the incorporation of tritium-labeled thymidine. The values of the IC50are given in table.C-11.

Example 11. Competition with reference Fab for binding to IL-6R

All 14 of Manotel analyzed for their ability to inhibition of binding of the control Fab described in Example 1, IL-6R, when determining based on the method of Alphascreen. In this method, 100 nm of purified nantel incubated with 0.4 nm biotinylated IL-6R. Added covered with control Fab acceptor beads and streptavidin coated donor beads and measured the concentration of complex control Fab/IL-6R. The values obtained in the presence of nantel, compared with control, which was not added nanotesla, getting the ratios between 2 values, expressed in %, which are listed in the table.S-12. All nanotesla except IL6R03, not shown or showed only partial inhibition of binding of the control Fab with IL-6R, which means that their epitopes do not overlap or only partially overlap with the epitope of the control Fab.

Example 12. The binding of Manotel with U266 cells

The binding of Manotel with membrane-bound IL-6R, expressed in U266 cells were analyzed by FACS method. The analysis was performed on the purified NAS calving of the selected clones (IL6R04, IL6R09, IL6R11, IL6R13 and IL6R14). The results are presented in Fig.7. All nanote is and contacted expressed on the cell surface IL-6R person.

Example 13. The binding of Manotel with IL-6R from human plasma

Soluble IL-6R is present in plasma at a concentration of 80-400 ng/ml to establish whether nanotesla IL6R03, IL6R04 and IL6R13 contact with IL-6R from plasma, investigated the effect of human plasma on the binding of Manotel with U266 cells. The human plasma inhibited the binding of U266 cells, indicating that all 3 nanotesla able to communicate with IL-6R from human plasma (see Fig.8).

Example 14. The cross-reactivity of Manotel to IL-6R mouse

The cross-reactivity of Manotel to IL-6R mice were analyzed by ELISA method. For this, 500 nm of Manotel inflicted on a microplate coated with IL-6R mice and humans at 1 µg/ml Detection was performed using antibodies against thuja and anti mouse HRP as the first and second antibodies, respectively. In Fig.10 shows the optical density. None of the investigated nantel was not observed binding to IL-6R mouse.

Example 15. The summary for selection of binding IL-6R of Manotel

Immunization 2 Lam recombinant IL-6R gave a set of the 14 unique nantel that can block the interaction between IL-6 and IL-6R. This set was subjected to detailed analysis and on the basis of all experimental data for further development were selected nanotesla IL6R03, IL6R04 and IL6R13. The most important characteristics of NanoTec are given in table.C-13.

II. Formatting nantel against IL-6R

Example 16. Getting multivalent structures

Nanotesla against IL-6R, described in the previous sections, is also expressed in the form bispecific structures consisting of C-terminal has napothera against SA (ALB1), the linker of the 9 amino acid Gly/Ser and N-terminal has napothera against IL-6R. In addition, designed a 4 trivalent, bispecific nanotesla consisting of C-terminal and N-terminal nanotesla against IL-6R and nanotesla against SA (ALB1) in the middle, all of which are connected via linkers of 9 amino acid Gly/Ser. The IDs of these nantel are given in table.C-14.

Example 17. Expressiona bispecific of Manotel against IL-6R

Design for bispecific of Manotel expressed in E. coli in the form of proteins labeled with status and (His)6and then was purified from culture medium by methods of affinity chromatography with immobilized metal ion (IMAC) and exclusive chromatography (SEC). Total output and output per 1 liter of cell culture are listed in the table.C-15. SDS-PAGE of purified nantel shown in Fig.11.

Example 18. Competition analysis meyado proteins

Peeled bispecific nanotesla tested method Alphascreen on the inhibition of the interaction between IL-6/IL-6R. To IL-6R (0.3 nm) was added a serial dilution of purified proteins (concentration range: 250 nm-5 PM), and incubated for 15 minutes then added 3 nm bioti lirovannomu IL-6 and coated with BN-12 acceptor beads and this mixture is incubated for 1 hour. Finally, added streptavidin donor beads and after 1 hour incubation, read the tablet on the reader Envision. As control included BR-6 and Fab-fragment described in Example 1. The results are presented in Fig.12.

For all nantel was observed curves dose-effect values IC50ranging from 123 PM to 1.67 nm (table.S-16).

Example 19. The activity of Manotel on cells XG1

Bispecific nanotesla tested method of proliferation XG1. The values of the IC50ranged from 60 PM to 65 nm. Nanotesla also tested by this method in the presence of 1 mg/ml serum albumin human. The values of the IC50For bispecific of Manotel ranged from 190 gr to 90 nm. As control included control IgG as described in Example 1. The values of the IC50are given in table.C-17.

When testing rich of Manotel method XG1 in the presence of albumin were observed decrease in activity. Activity formatted nantel IL6R24, IL6R44 and IL6R49 exceeded or were within the same range as the control IgG in the presence of serum albumin.

Example 20. Determination of affinity for IL-6R

Analyzed the binding bispecific of Manotel to IL-6R by means of surface plasma resonance. Determined kinetic parameters, which are listed in the table.C-18. Manotel in a bivalent format (IL6R23, IL6R24, IL6R33) is E. there was a significant drop affinity for IL-6R.

Example 21. Determination of affinity for serum albumin were Analyzed binding of rich nantel with serum albumin by the method of surface plasmon resonance. Defined constants affinity (Kd), which are listed in the table.C-19. For comparison included albumin binding nanotesla Alb-1 (SEQ ID NO:97). Affinity was within the previously formatted nantel containing the same building block against serum albumin, although overall they were less active. This was particularly evident in relation to the affinity to serum albumin mouse.

Example 22. Linking formatted nantel with U266 cells Binding formatted Manotel of the selected clones (IL6R23, IL6R24, IL6R29, IL6R33, IL6R44 and IL6R53) U266 cells were analyzed by FACS method. The results are presented in Fig.13 and 14. The divalent nanotesla IL6R23, IL6R24, IL6R29 and IL6R33 showed lower binding compared to the monovalent building blocks, whereas the trivalent of Manotel IL6R44 and IL6R53 observed the same binding.

III. Optimization of sequences of Manotel against IL-6R

Example 23. Strategy optimization sequences

The protein sequence of each of Manotel IL6R03, IL6R04 and IL6R13 were compared with the 5 closest sequences of the germline of the person with the greatest degree homola the GII (Fig.15). Amino acid differences in frame sections from the consensus sequences of the germline of the person selected colors. Amino acid differences, highlighted in light gray, selected for conversion to human, whereas the amino acids highlighted in dark grey color, was left unchanged. You have created a set of 4 optimized sequence variants for each of the 3 nantel (stage 1). These options were analyzed for several parameters, and the results used to create a second set of nantel (stage 2). The protein sequences of all optimized sequence variants shown in Fig.16.

Example 24. Optimization of sequences in stage 1 In the process of optimizing sequences in stage 1 were created and analyzed the following 12 options.

IL6R03: IL6R61, IL6R62, IL6R63 and IL6R64

IL6R04: IL6R71, IL6R72, IL6R73 and IL6R74

IL6R13: IL6R81, IL6R82, IL6R83 and IL6R84

Amino acid sequences of all of these various options is shown in Fig.16.

Study of optimized nantel method of competition for IL-6/IL-6R

Optimized sequence clones IL6R03, IL6R04 and IL6R13 tested method Alphascreen on the inhibition of the interaction between IL-6/IL-6R. To IL-6R (0.3 nm) was added a serial dilution of purified nantel and incubated for 15 minutes then added 3 nm biotinylated IL-6 and coated with BN-12 acceptor beads and this mixture is incubated for 1 hour. Finally, added streptavidin donor beads and after 1 hour incubation, read the tablet on the reader Envision. The control consisted of the original clones. The results are presented in Fig.17, Fig.18 and Fig.19.

All optimized sequence variants IL6R03 (IL6R61, 62 and 64) value IC50were within 2-fold of the value of the IC50IL6R03, while IL6R63 showed ~4 times smaller value IC50.

For optimized sequence variants IL6R04 there were no significant differences in the values of the IC50between IL6R04 and 4 optimized variants.

For optimized sequence variants IL6R13 values IC50The IL6R81 and IL6R83 were almost identical to the one in IL6R13, whereas in 2 variants carrying mutations RAT→KGL frame section 2 (IL6R82 and IL6R84), showed a decline in activity.

Determination of affinity

Optimized sequence variants IL6R03, IL6R04 and IL6R13 also were analyzed for binding to IL-6R on the Biacore instrument. The kinetic parameters are given in table.C-20.

For optimized sequence variants IL6R03 values of Kdthe IL6R61, 62 and 63, all were within 2-fold of the values of KdIL6R03. The value of Kdthe IL6R64 not been defined.

For optimized sequence variants IL6R04 there were no significant the differences in the values of K dbetween IL6R04 and 4 optimized variants.

For optimized sequence variants IL6R13 values of Kdthe IL6R81 and IL6R83 were very close to the value of Kdthe IL6R13, whereas in 2 variants carrying mutations RAT→KGL frame section 2 (IL6R82 and IL6R84), showed a strong decrease of the affinity.

In General, these observations are fully consistent with the results obtained in the analysis of competition method Alphascreen.

Example 25. Optimization of sequences in stage 2 Based on the affinity and activity at stage 1, it was decided to create the following set of options:

for IL6R03 United all mutations available options IL6R61, 62, 63 and 64, receiving ILR65;

for IL6R04 United all mutations available options IL6R71, 72, 73 and 74, receiving ILR75;

for IL6R13 was created a set of 6 new variants (IL6R85-90) carrying different mutations (combination) sequence RAT in FR2. These mutations were introduced based IL6R83, as this optimized sequence variant did not differ from IL6R13 either by affinity or activity.

Analysis of the rate constants koffthe optimized variants IL6R13

Optimized sequence variants IL6R85-90 in the form of periplasmatic extracts were analyzed on a Biacore instrument. To calculate the values of koffused dissociation curves (table.S-21). The nano is eating IL6R87-89 values of k offwere close to those of the IL6R13, while nantel IL6R85, 86 and 90, they were 10 times higher.

Study of optimized nantel method of competition for IL-6/IL-6R

Optimized sequence variants IL6R85-90 in the form of periplasmatic extracts tested method Alphascreen on their ability to inhibit the interaction of IL-6/IL-6R. The results are presented in Fig.20.

Nanotesla IL6R87, 88 and 89 were blocking the interaction of IL-6/IL-6R stronger than options IL6R13 - 85, 86 and 90. These results are fully consistent with the observations, sdelannym on the Biacore instrument. Comparison of the amino acid sequence optimized variants IL6R13 showed that the decrease in koffand activity of the responsible mutation T45L. Therefore, further study was selected as the most close person option without mutations T45L, i.e. IL6R88.

This option is expressed, purified and analyzed for inhibition of the interaction between IL-6/IL-6R with purified clones IL6R65 and IL6R75. Significant differences between the different optimized sequence clones (IL6R65, IL6R75 and IL6R88) and corresponding non-optimized variants (IL6R03, IL6R04 and IL6R13, respectively) was observed (Fig.21).

Determination of affinity

The affinity constants (IQ) - optimized sequences of clones IL6R65, 75 and 88 to IL-6R person was determined by the method of surface PLA the time of resonance of the instrument Biacore 3000. Briefly, touch on the chip CM recorded IL-6R through the amino group at a density of 800-1000 Rel.ed. The remaining reactive group iactiveaware. The binding of nantel was evaluated at various concentrations from 0.5 to 50 nm. Each sample was injected for 4 min at a flow rate of 45 μl/min, which contributes to the binding with the associated chip antigen. Then through the chip missed a buffer for binding without nantel with the same speed, which contributes to the dissociation of bound peroxidase nantel. Kinetic parameters were optimized for sequence variants IL6R03, 04 and 13 are given in table.C-22.

Significant differences in the affinity constants between the original and optimized sequence antelami was not observed.

Determination of the activity on cells

Optimized sequence nanotesla were analyzed by the method of XG-1. The results are presented in Fig.22. The values of the IC50are given in table.C-23. Optimization of the sequence had no significant effect on the activity suppression induced IL-6 proliferation when determining on the cells.

IV. Additional features optimized nantel

Example 26. Determination of affinity for IL-6R long-tailed macaques (supo)

Affinity IL6R03-IL6R65, IL6R04-IL6R75 and IL6R13-IL-6R88 to cynoIL-6R (macaques) were identified by means of SPR instrument Biacore 3000. Briefly, the and touch the chip CM recorded cynoIL-6R through the amino group at a density of 760 Rel.ed. The remaining reactive group iactiveaware. Linking NanoTec was evaluated at various concentrations from 1.25 to 100 nm. Each sample was injected for 4 min at a flow rate of 45 μl/min, which contributes to the binding with the associated chip antigen. Then through the chip missed a buffer for binding without nantel with the same speed, which contributes to the dissociation of bound peroxidase nantel. The kinetic parameters are given in table.C-24.

Although there were some differences in the affinity constants between IL6R04 and IL6R75, but from this experiment it is clear that both molecules have an affinity for IL-6R macaques was significantly lower than IL-6R person. On the contrary, IL6R03 and IL6R65 constant affinity for IL-6R macaques were in the same range as for IL-6R person. Beyond expectations, the crystal structure of the complex IL-6/IL-6R/gp130 (Boulanger et al.) shows that IL-6 binding site on IL-6R fully conservative between man and makokou, testifying that IL6R04 binds to another epitope.

Example 27. Testing-optimized sequence of Manotel method activity in plasma using a plasma of human and macaque

In order to evaluate the cross-reactivity IL6R65 and IL6R75 to IL-6R long-tailed macaques, were used to define activity in plasma using ELISA method, using as a source of sIL-6R plasma of humans or macaques. With the series of breeding nantel was preincubated plasma IL-6 person (50 ng/ml). Then spent the capture of SIL-6R plasma covered with BN-12 tablet and were detected associated IL-6 with biotinylated polyclonal antibodies and conjugate streptavidin-HRP.

As can be seen from Fig.23A, IL6R201 and IL6R65 able to completely block the binding of IL-6 with SIL-6R person, but IL6R65 was less strong than IL6R201 and the control IgG and control Fab described in Example 1. As can be seen from Fig.23C, IL6R65 able to completely block the binding of IL-6 with SIL-6R macaques with efficacy comparable with control Fab described in Example 1.

Example 28. Testing-optimized sequence of Manotel method activity in plasma at high concentrations of IL-6

The determination of the activity of plasma in plasma were also used to test the ability of Manotel to block IL-6 at high concentrations. IL6R04, IL6R65 and control Fab described in Example 1 was tested at values EU50for IL-6 (50 ng/ml) and AS for IL-6 (885 ng/ml). The results are presented in Fig.24. It is seen that IL6R04 proved to be the most active when ECso for IL-6. And when AS for IL-6 control Fab and IL6R65 still able to completely block SIL-6R plasma, although at higher concentrations, indicating that these 2 molecules bind epitope that overlaps with the IL-6-binding site. The value of the IC50the control Fab was increased from 0.55 nm is about of 8.47 nm, and the IC50The IL6R65 increased with 2,61 nm to 66,25 nm.

Example 29. A study of competition in the Biacore instrument

Experiments on Biacore was performed in order to investigate whether IL-6 and IL6R65 contact with IL-6R at the same time. Control Fab (Fig.25A), IL6R201/75 (Fig.25V) or IL6R65 (Fig.25C) planted on IL-6R, and then spent the binding of IL-6 complex. Almost no binding of IL-6 was not observed with control Fab and IL6R65. When IL-6 was planted on IL-6R and injected nanotesla, all 3 nanotesla was able to contact the complex. But in the case of control Fab and IL6R65, this was probably caused by the displacement of IL-6 due to lower affinity of IL-6 to IL-6R.

Finally, spent the binding of IL-6R covered with IL-6 chip in the presence or in the absence of nantel (Fig.25D). This confirmed previous results, namely that IL-6R is not captured in the presence of a control Fab or IL6R65.

In conclusion, we note that experiments on mapping of epitopes on the Biacore instrument showed that the control Fab, and IL6R65 sit down to the same epitope as IL-6. At that time, as IL6R65 and control Fab is able to completely block the binding of IL-6 with IL-6R, nanotesla IL6R201 not able to prevent the binding of IL-6 with IL-6R, when it is associated with the receptor.

V. affinity Maturation in IL6R65

Example 30. Diversification strategy for affinity maturation

Lots CDR nanotesla IL6R65, for example, the Ali randomization with the use of 2 of the following strategies.

1. Each residue in CDR replaced with other residues with similar chemical composition of the side chain:

K↔R

A↔S↔T

I↔L↔V

F↔Y

N↔D

Q↔E

G→A

M→L

H, C, W, P is left unchanged.

2. Each residue in CDR is replaced with the number of amino acids occurring in nature in this position. Introduced only those amino acids that are most frequently encountered in each position, to limit the diversity in this position. This approach was used only for randomization CDR1 and CDR2.

A randomized CDR1 and CDR2 were performed in parallel using the 2 strategies above, a CDR3 were subjected to randomization separately for strategy 1, the receiving end 3 of the library. All 3 libraries have created their own forces by PCR with overlapping and capacity using degenerate oligonucleotides. Theoretical diversity for each of the libraries was approximately 1×106. Fragments encoding options nantel, cloned into a vector for phage display. The actual size of all 3 libraries was approximately 1×108(a 100-fold coverage of theoretical diversity). Spent one cycle of selection using different concentrations of biotinylated IL-6R (0, 1, 10 and 100 PM) in solution. Under such conditions was not observed enrichment for the library CDR3, whereas for both libraries CDR1/2 was observed in a dose-zavisimosti. Products from libraries CDR1/2 were subjected to analysis periplasmatic extracts using ELISA method, and the clones with the highest signals were subsequently tested on Biacore instrument. The best 30 clones in the analysis using ELISA method showed values of koffbetween 2,1×10-3and 2.6×10-4s-1. All 5 nantel with the lowest values of koffsequenced, expressed and purified (Fig.26).

Example 31. Expressiona and purification of Manotel

Ripened on the affinity of nanotesla expressed in E. coli in the form of proteins labeled with status and (His)6in cultures with a volume of 250 ml. Expression was induced by adding 1 mm IPTG and continued for 4 h at 37°C. After centrifugation of cell cultures were prepared periplasmatic extracts by freezing and thawing of rainfall. These extracts were used as source material for affinity chromatography with immobilized metal ion (IMAC). Nanotesla was suirable from the column with 150 mm imidazole, and then absoluely on column Hiprep26/10.

Example 32. Determination of the affinity matured by affinity variants on the Biacore instrument

Nanotesla IL6R65 (optimized sequence) and 5 ripe for affinity variants were analyzed on a Biacore instrument. Registered binding curves for different concentrations of purified nantel and used them for races is the ETA of the affinity constants. Values of Kdthese 5 clones ranged from 0.34 to 0.95 nm, which corresponds to 13-fold improvement relative to IL6R65 (original nanotesla) for the best variant (Fig.27).

Example 33. Determination of the affinity matured by affinity variants of the method activity in plasma

All 5 ripened on the affinity of nantel and optimized sequence nanotesla also tested method activity in plasma. This nanotesla in various concentrations was mixed with containing soluble IL-6R plasma of human or long-tailed macaques at a fixed concentration of IL-6 person (2.4 or 42 nm). After 1 hour incubation mixture was transferred to the tablet Maxisorp coated with MAb BN-12 versus IL-6R (Diaclone). The amount of bound IL-6 was determined by the subsequent addition of the biotinylated polyclonal antibody against IL-6 (R&D Systems) and conjugate streptavidin-HRP. As the substrate used TMB. Measured the conversion of the substrate at 450 nm (Fig.28).

Example 34. The study ripened on the affinity options method TF-1

Ripened on the affinity of nanotesla also tested on their ability to inhibition of IL-6-dependent cell proliferation, TF-1 (ESAS.93022307; 1989, J. Cell Physiol., 140: 323; 1993, Exp. Cell Res., 208: 35) by blocking the binding of IL-6 with IL-6R on the cell surface. For this, the serial cultivation of NanoTec has plaincourault with pixaround the m number of cells TF-1 for 2 hours at 37°C. After that we added IL-6 to a final concentration of 2 ng/ml IL-6-dependent proliferation of the cells was continued for 72 hours, and it was measured by the incorporation of tritium-labeled thymidine (Fig.29).

The values of the IC50The ripened on the affinity of Manotel were up to 17 times better than IL6R65, but all the options were less active than the control IgG.

Example 35. A study of competition in the Biacore instrument

Experiments on Biacore was performed in order to investigate whether IL-6 contact IL-6R simultaneously with IL6R65 and 2 ripened on the affinity variants (7D6 and S). In these experiments, IL-6R planted covered with BN-12 chip and fed his nanotesla IL6R65 (Fig.30A), 7D6 (Fig.30V) or S (Fig.30C). Then evaluated the binding of IL-6 complex 1E-6R-nanotesla injection of the cytokine at a concentration of 100 nm. In addition, it also determined the binding of all 3 of Manotel with IL-6R complex with IL-6.

Results for IL6R65 (Fig.30A) are comparable with the results obtained previously, and confirm that IL6R65 and IL-6 recognize the same epitope on IL-6R. As expected, ripened on the affinity options IL6R65 and IL-6 also recognize the same epitope on IL-6R (Fig.30B-C).

Example 36. Further affinity maturation

Then created a set of 47 nantel containing various combinations of beneficial mutations in CDR1/2 and CDR3. These mutations were installed a thorough review of the om protein sequences and koff values for all clones of nantel, selected from libraries of randomization CDR. The koff values of these clones 2nd generation ranged from 4.2 E-04 to 4.5 E-05 s-1. For further analysis were selected 5 clones exhibiting the lowest values of koff(20F6, A, E, A, 21D11). Their sequence is shown in Fig.31.

Example 37. Expressiona of nantel and purification of variants from the second cycle

Ripened on the affinity of nanotesla expressed in E. coli in the form of proteins tagged with C-myc and (His)6in cultures with a volume of 250 ml. Expression was induced by adding 1 mm IPTG and continued for 4 h at 37°C. After centrifugation of cell cultures were prepared periplasmatic extracts by freezing and thawing of rainfall. These extracts were used as source material for affinity chromatography with immobilized metal ion (IMAC). Nanotesla was suirable from the column with 150 mm imidazole, and then absoluely on column Hiprep26/10.

Example 38. The melting point

The temperature resistance of Manotel analyzed by the method of thermal shift. Values of TP were similar in all ripened on the affinity of nantel and slightly higher in comparison with IL6R65. The values of Tmare given in table.25.

Example 39. Determination of the affinity variants of the 2nd cycle on the Biacore instrument

Kinetic parameters for nanotesla IL6R65 (optimized after which outermost) and 5 ripe for affinity variants was determined in the Biacore instrument KZT100. The rate constants of Association (ka) was determined from the binding curves at 2 different concentrations cleared nanotesla and fixed concentration of IL-6R, which is recorded on the chip through mAb BN-12. Values of kddetermined at one concentration of Manotel using IL-6R, covalently fixed on the chip. Values of ka, kdand KDare given in table.C-26.

Example 40. Study options from the 2nd cycle method TF-1

The biological activity of variants of the 2nd cycle was investigated by the method of TF-1, and the data shown in Fig.32. Ripened on the affinity clones from the 2nd cycle had 5-8 times higher activity than the clone S from the 1st cycle, and were 2.5-4 times stronger than standard control IgG as described in Example 1. The best clone was A with the value of the IC500.4 nm (4 times stronger than standard).

Example 41. Study options from the 2nd cycle method activity in plasma

Inhibition of soluble IL-6R were analyzed by the method of activity in the plasma at high and low concentrations of IL-6. Variants of the 2nd cycle were at least as strong as the control IgG in the determination method of the activity in the plasma (0.1 to 0.2 nm), which was at the limit of sensitivity of this method. At high concentrations of IL-6 (ES) ripened on the affinity of the mutants were still capable of blocking connect is the use of IL-6 with sIL-6R and turned out to be 3-4 times stronger than control IgG. In this method, there were no differences between the variants of the 1st and 2nd cycle. As expected, all investigated clones had cross-reactivity with Makaay (Fig.33).

Example 42. Binding to PBMCs human whole blood

Cleaned nanotesla IL6R65 and RMRA tested method FACS for binding to human PBMCs. Binding to cells was detected using biotinylated mAb against His and labeled with PE streptavidin (Fig.34). As IL6R65 and ripened on the affinity of the variant RMRA was associated with neutrophils, monocytes and a subpopulation of lymphocytes. This is consistent with the expression profile of IL-6R.

VI. Formatting ripened on the affinity of Manotel

Example 43. Getting multivalent structures

RMRA formatted in the form of divalent and trivalent of Manotel with binding albumin by nanoceram ALB8. A summary of the different nanocell presented in table.C-27.

Example 44. Neutralization of membrane IL-6R in the method TF-1

In the first experiment tested whether the formatted nanotesla to inhibit signaling through the IL-6R, using as a model system for cell line TF-1. Nanotesla A, IL6R304, IL6R305 and IL6R306 dose-dependently and completely blocked induced IL-6 cell proliferation, TF-1, which is mediated membrane IL-6R (Fig.35, PL.S-28).

These results show the Ute, all formatted nanotesla are stronger than the control IgG as described in Example 1, when the inhibition of the activity of membrane IL-6R. Compared to its monovalent equivalent of IL6R304, IL6R305 inhibits mediated IL-6 responses in ~7 times stronger, indicating a strong interaction IL6R305 with membrane IL-6R. IL6R306 less active than IL6R305, and shows only a 2-fold greater efficiency that IL6R304. This suggests that the format IL6R306 is less favourable and that IL6R306 impossible in a strong binding.

It was further examined whether the formatted nanotesla still completely inhibit signaling through the IL-6R at pathological concentrations of IL-6. Indeed, A, IL6R304, IL6R305 and IL6R306 dose-dependently and completely blocked cell proliferation, TF-1 induced IL-6 at 5000 IU/ml (Fig.36, PL.C-29). As expected, the values of IC50 were shifted compared with experiments conducted at 100 IU/ml In agreement with previous results, IL6R305 stronger than all blocked induced IL-6 signaling through membrane IL-6R.

Because nanotesla really interact with IL-6R, it was examined whether the binding of Manotel with this receptor to cause the activation of the cells, leading to their proliferation. Cells TF-1 were incubated with an excess of Manotel in the presence or absence of 100 is u/ml IL-6 (Fig.37). As expected, IL6R304 and IL6R305 completely prevented mediated IL-6 proliferation. In fact, IL6R305 and IL6R306 inhibited induced IL-6 proliferation to the level of the background (on3H-thymidine was measured in the absence of growth factors), suggesting that IL6R304 and IL6R305 completely blocked the effect of IL-6.

IL6R304 and IL6R305 did not cause cell proliferation, TF-1 in the absence of growth factors, suggesting that these compounds do have an antagonistic effect on the cells TF-1.

Example 45. Neutralization of sIL-6R rich ripe on the affinity of antelami method ELISA

Ability building block IL6R20A11 and its rich options to prevent binding of IL-6 person with sIL-6R plasma were analysed by activity in plasma using ELISA method. Since the concentration of sIL-6R plasma varies, it is necessary to use the same plasma in various experiments to compare the activity of Manotel. Also first performed titration of IL-6 during incubation in plasma, to determine at what concentration will be used IL-6 together with antelami. Values EU50and AS for IL-6 in plasma was 27,29 ng/ml and 885 ng/ml, respectively. These concentrations were used later for testing the activity of Manotel at normal and high concentrations of IL-6.

IL6R20A11 and its format is consistent variants compared with control IgG, described in Example 1. The obtained values IC50for different nantel are given in table.C-30. When the value of the EU50for IL-6 (Fig.38A,) and monovalent and divalent nanotesla fall in the same interval, and control IgG. Although IL6R304 has only one binding site, he showed the same value IC50as a control IgG (0,229 nm against 0,258 nm). IL6R305 was two times stronger than IL6R304 (IC50=0,137 nm), which is consistent with the presence of 2 binding sites. But IL6R306 was less strong than IL6R304 and control IgG, and showed IC50in 0,412 nm.

Most likely, they had reached the limit of detection sensitivity. In fact, the concentration of sIL-6R plasma was ~30 ng/ml or 0.6 nm. Therefore it was necessary to block antelami only 0.3 nm sIL-6R (50% in plasma), which means that the minimum value of the IC50that can be obtained is 0.15 nm. This corresponds to the obtained values of IC50. However, if the concentration of IL-6 increased to 885 ng/ml, nanocell will be harder to compete with IL-6 and you might find a greater difference in activity. Indeed, at high concentrations of IL-6 nanotesla IL6R20A11, IL6R304 and IL6R305 was stronger than the control IgG, a IL6R306 - no (Fig.38C, D). The ratio IC50at high and low concentrations of IL-6 are given in table.C-30. Obviously, the increase in L-6 more influenced control IgG and IL6R306, than other nanotesla. This was also observed when determining the method of TF-1 (see Example 44).

Example 46. Linking rich ripe on the affinity of Manotel with membrane IL-6R

In order to lock signalizovania from IL-6, you need to neutralizirati antelami and soluble and membrane IL-6R. Therefore, we analyzed the binding of different formatted nantel with expressing IL-6R cells by flow cytometry.

Linking expressing IL-6R cells SNO

To analyze the binding of Manotel against IL-6R with membrane IL-6R used stably transfected cells SNO expressing IL-6R person (Fig.39A). As a negative control was used IL-6R-negative cells Cho (Fig.39B). All 4 nanotesla showed saturable binding expressing IL-6R cells, whereas IL-6R-negative cells were detected only very weak signals at high concentration of Manotel.

Average values of fluorescence RE taken out in GraphPad graphics and built 4PL to determine the values of the EU50. They are listed in the table.C-31. Unlike method TF-1, IL6R305 as if did not influence the effect of avidity in these terms: he was only 2 times stronger IL6R304 (0,8984 against 1,939 nm). As in the methods TF-1 and ELISA in plasma, IL6R306 contacted worse with IL-6R-positive cells.

The binding of white blood cells is peripheral blood

To demonstrate the binding of Manotel with membrane IL-6R in the physiological conditions used PBL person. This matrix is very similar to the situation in vivo, as it contains HSA (~50 mg/ml), sIL-6R (~30 ng/ml), cells expressing membrane IL-6R (T-cells CD4+, monocytes, granulocytes), and IL-6R-negative cells (the majority of circulating b-cells, T-cells CD8+). Nanotesla incubated in EDTA treated blood from 2 donors, and the bound peroxidase nanotesla were detected by flow cytometry. Lymphocytes, monocytes and granulocytes were spotted on the characteristics of the FSC/SSC (Fig.40), and the bound peroxidase nanotesla were detected on the channel RE.

As can be seen from Fig.40 (right), the granulocytes and the monocytes were uniformly colored antelami. On the contrary, was painted only part of the lymphocytes. It is observed in the form of a double peak in the histogram RE. The average fluorescence 3 noise population after incubation with different concentrations of Manotel shown in Fig.41, and the obtained values of the EU50are given in table.C-32.

Example 47. Affinity rich ripe on the affinity of Manotel to serum albumin human and macaque (cyno)

Performed kinetic analysis bispecific, divalent and trivalent of Manotel IL6R304, IL6R305 and IL6R306 on human serum albumin and macaques method SPR instrument Biacore 3000. The results depict what aulani in Fig.42 and shown in table.C-33. Nanotesla IL6R304, 305 and 306 showed similar kinetic rate constants and affinity (17-23 nm) for the SA human and macaque. Affinity to SA from formatted nantel against IL-6R was 6.5 times lower than that of monovalent nanotesla ALB11 against SA by reducing 2.5 times the speed of the Association and increase 2.5 times the speed of dissociation.

Example 48. The affinity for IL-6R humans and macaques (cyno)

Conducted a kinetic analysis of IL-6R human and macaque method SPR on a Biacore instrument T 100. Due to the indications for a change in the conformation of IL-6R with its immobilization directly on the chip rate of the Association was measured on IL-6R captured BN-12. The dissociation rate was measured on immobilizovannoi directly IL-6R due to the absence of the gripper with a lower dissociation rate than the interaction HaHOTeno-IL-6R. The results are presented in table.C-34, and Fig.43.

Speed Association A (IL6R300; SEQ ID NO:66) were down less than 2 times when formatting with the building block versus SA (IL6R304). The dissociation rate for IL6R304 on IL-6R person was at or below the detection limit of the instrument Biacore. The dissociation rate on IL-6R macaques was ≥2-fold higher than IL-6R person, but still near the detection limit. The calculated affinity at IL6R304 was ≤14 PM on IL-6R man and 25 PM on IL-6R macaques.

Example 49. Interspecies cross-reactivity IL6R20A11

Cross-react is you want to make IL6R20A11 and its rich options to sIL-6R long-tailed macaques (cyno) were analysed by activity in plasma using ELISA method, using the plasma macaques. Also used competition ELISA method for determination of cross-reactivity IL6R20A11 to sIL-6R macaque and mouse.

Activity in plasma using ELISA method

First performed titration of IL-6 person during incubation in plasma macaques and found that the value of the EU50for IL-6 is 50,11 ng/ml. It is this concentration was subsequently used for testing cross-reactivity of Manotel to sIL-6R plasma macaques. As can be seen from Fig.44, IL6R20A11 and its rich options clearly showed cross reactivity to sIL-6R macaques. Observed the same rank order as in the plasma, and the ratio of the values of the IC50in the plasma of human and macaque plasma was similar for all compounds (table.C-35).

Competition ELISA method

Activity in plasma using ELISA method can be used only if BN-12 is capable of capturing sIL-6R plasma of this type and if the binding of IL-6 with sIL-6R can be detected. So I developed a more General method of competition in the ELISA method. This method is based on the binding IL6R20A11 with captured neutravidin complex IL-6R-Biotin. Briefly, 0.4 nm IL6R20A11 was preincubated with serial dilutions of plasma from various species containing endogenous sIL-6R, and then spent the capture of free IL6R20A11 on biotinylated sIL-6R person, immobilized on a covered what neutravidin tablet, and were detected using FITC-mAb against VHH and conjugate antibodies against FITC with HRP. Concentration IL6R20A11 0.4 nm corresponds to a concentration giving 50% of maximum signal (EC50=0,35±0,021 nm; n=4).

As can be seen from Fig.45, IL6R20A11 clearly showed cross reactivity to sIL-6R macaques, which confirms the results on Biacore. On the contrary, was not observed binding to sIL-6R mouse. Plasma of humans and macaques also competed for binding IL6R20A11 with recombinant sIL-6R, and plasma mouse - no. Actually there was an increase of signals at high plasma concentrations mouse that was probably due to the detection of mouse IgG in this method.

Example 50. Specificity IL6R20A11 to IL-6R

IL-6R belongs to the family of receptors of cytokines of the first type. Since the binding site of these cytokines receptors conservative, specificity IL6R20A11 to IL-6R were analyzed by analysis of binding with the receptor, related to IL-6R. Linking IL6R20A11 with LIF-R, CNTF-R, OSM-R and IL-11R/Fc was analyzed by competition for binding by ELISA method. As can be seen from Fig.46, sIL-6R from the positive control inhibited binding IL6R20A11 on the tablet. The value of the IC50for sIL-6R (0,03 nm) corresponds to the expected IC50based on the quantities used IL6R20A11 (0,025 nm and 0.05 nm). None of the cognate IL-6R protein does not compete for binding IL6R20A11 with SIL-6R, even when 00-fold molar excess (5 nm). Similar conditions were that CLF-1/CLC, IL12-p40, IL-27B and gp130/Fc also do not interact with 0.05 nm IL6R20A11, even at such high concentrations as 100 nm (results not shown).

VII. Analysis of PK/PD IL6R304 and IL6R305 in vivo

The aim of this study was to analyze the pharmacokinetics (PK), pharmacodynamics (PD) and the immunogenicity of 2-optimized sequence, ripened on the affinity of Manotel against the receptor of interleukin-6 (IL-6R), namely IL6R304 and IL6R305, long-tailed macaques after a single intravenous administration of a bolus. With the introduction of Manotel followed 7 daily subcutaneous injections of recombinant IL-6 person (h), starting from 24 hours after administration of Manotel. The ultimate goal of this study efficacy in vivo was to evaluate the ability of these nantel against IL-6R to inhibition induced hIL-6 parameters and compare their performance with each other and with the standard control from Example 1.

In humans and other primates, it was reported that recombinant hIL-6 induces the synthesis of acute phase proteins. Proteins of the acute phase is defined as the class of such proteins as C-reactive protein (CRP), serum amyloid a, haptoglobin, fibrinogen, albumin and transferrin concentrations in plasma increases or decreases by at least 25% in response to inflammation, mainly due to changes in their products hepato what it. Profiles production of cytokines and acute phase reaction differ in various inflammatory conditions. Therefore, changes of acute phase proteins reflect the presence and intensity of inflammation, which makes them important for the diagnosis. The main stimulators of the production of acute phase proteins are associated with inflammation cytokines that are produced during inflammation: IL-6, IL-1β, α-tumor necrosis factor (TNF-α), γ-interferon (INF-γ), transforming growth factor β (TGF-β), and possibly IL-8.

Example 51. Study design

In this study, 6 groups (groups 6-11, table.C-36) from 2-3 animals received a single on/injection IL6R304 or IL6R305. Tested 3 different doses of both nantel, namely 0,4,2 or 10 mg/kg in Addition, animals in group 12 (n=3) received the media and served as a negative control, whereas animals of group 13 (n=3) were administered 5 mg/kg control IgG (table.C-36).

Since TD1, i.e. 24 hours after administration of the test substances, all animals were injected daily for 7 days hIL-6 (5 μg/kg; Fig.47).

Before and after the introduction of hIL-6, at given points in time, took samples of blood from the head of the great saphenous vein or veins of the legs (see Fig.47). Additional blood samples were taken at the day of the TDO for the analysis of FC (see tab.S-37).

Example 52. The effect has Neotel caused by hIL-6 response of the acute phase (phase 2)

Studied the effect of Manotel floor and the positive control IgG on the induction of acute phase reactions in the daily introduction 7 times in a row hIL-6. Study parameters: levels of CRP, fibrinogen level and platelet count.

In the negative control group (group 12) levels of CRP were increased immediately after the first injection of hIL-6, and the maximum level was reached already on the 2nd day. The maximum level was 0.2-0.8 mg/ml, and this plateau was maintained until the 8th day, i.e. the day after the last injection of hIL-6 (Fig.48A).

These changes were completely suppressed by pre-treatment with 5 mg/kg control IgG (Fig.48V). The single treatment comparable dose (2 mg/kg) or 5-fold higher dose (10 mg/kg) of Manotel IL6R304 and IL6R305 also gave almost complete inhibition of the induction of CRP throughout the experiment (Fig.48S and D). Only animal No. 18 in the group with the highest dose of IL6R304 showed some induction, reaching a maximum level of CRP in the serum of about 0.1 mg/ml In the group with the lowest dose (0.4 mg/kg) both nanotesla gave complete inhibition within 7 days. Levels of CRP were increased on day 8 to a level comparable with the level in the negative control group (Fig.48). Mean levels of CRP in all groups is shown in Fig.49.

The fibrinogen level was slowly increased in the group of negative control to an average maximum of 5 times the basal level (Fig.50A). This maximum was achieved only on day 6 and continued for the next 2 days. On day 14, the level of fibrin is gene returned to the basal level. Control IgG completely inhibited the induction of fibrinogen (Fig.30V). Both nanotesla showed dose-dependent inhibition of induction of fibrinogen (Fig.50C and D). The groups with the highest dose inhibition was almost complete in both animals pretreated IL6R304 and 1 in 2 animals treated IL6R305. There was a small increase in the level of fibrinogen in animal No. 25 (10 mg/kg IL6R305) and all animals in the groups with medium and smallest doses of both nantel. But in these animals the fibrinogen level never exceeded 2-3 times the basal level. Mean levels of fibrinogen in all groups is shown in Fig.51.

All animals in the negative control group the number of platelets was slowly increased, starting from 5th day. The maximum level is achieved in 8-14 days and was 160-190% of the basal level. The effect of hIL-6 on the number of platelets is completely blocked for a single pretreatment with 5 mg/kg control IgG or ≥2 mg/kg of Manotel. Induction in the number of platelets was observed only in the groups with the lowest dose of nantel, starting in all animals on day 8. Maximum induction for IL6R304 was about 120-150% of the basal level, and for IL6R305 the maximum number of platelets was 160-180% of the basal level (Fig.52 and 53).

In conclusion, IL6R304 and IL6R305 showed similar dose-dependent and complete Engibarov is of all three parameters of the reactions of the acute phase.

Example 53. Plasma concentrations after intravenous introduction IL6R304 or IL6R305 (0,4-2-10 mg/kg) in long-tailed macaques

They took blood samples for pharmacokinetic analysis (FC) in plasma using ELISA method before administration and at the following time points after the introduction of IL6R304 or IL6R305: 5 and 30 minutes, 3 and 8 hours, 1, 2, 3, 4, 5, 6, 7, 8, 14, 21 and 29 days.

Individual graphs of the observed plasma concentrations with time after the on/in the introduction of IL6R304 (0,4-2-10 mg/kg) and IL6R305 (0,4-2-10 mg/kg) long-tailed makaka shown in Fig.54 and Fig.55, respectively.

Example 54. Accompanimental pharmacokinetic analysis IL6R304 and IL6R305 (0,4-2-10 mg/kg) in long-tailed macaques

Summaries of key parameters FC, obtained by accompanimental analysis of pharmacokinetic (PK) IL6R304 (0,4-2-10 mg/kg) and IL6R305 (0,4-2-10 mg/kg) in long-tailed macaques, are given in table.C-38, PL.C-39, PL.C-40, PL.C-41, PL.C-42 and table.C-43. These parameters FC was obtained by the method of accompanimental analysis (NCA) using WinNonlin Professional Software Version 5.1 (Pharsight Corp.). The final parameters for some of the animals counted only two points (R2=1 by default).

Concentrations of both nantel plasma long-tailed macaques intravenously decreased three-phase manner. In the first two days after the introduction it was noted the initial distribution and subsequent slower main (dominant) phase. Gradual the TES reduction at low concentrations resulted in the terminal phase, characterized by a short half-life.

Because antibodies against the drugs were found in plasma samples the majority of long-tailed macaques, changes the terminal half-life at low concentrations could be associated with an immunological mechanism of clearance. However, this is unlikely when considering the profiles of FC: at the lowest dose of the shortest half-life was observed in those points where it is not detected immunogenicity. Moreover, despite the presence of significant titer at high doses, still tends to reduce the time half-life (e.g., at 10 mg/kg IL6R304 in/in).

Based on the observed profiles of FC it is assumed that both nanotesla eliminated from the bloodstream at least two mechanisms. In this situation, the linear mechanism insatiable clearance must be a non-specific degradation of the connection. And the second mechanism is saturable clearance must be mediated by the target (e.g., internalization of the drug associated with membrane IL-6R, and subsequent clearance. At high concentrations of Manotel last mechanism of clearance must become rich and insignificant compared to the insatiable line clearance line clearance will be predominant (giving the main half-life). However, at low concentrations the rate of meth is bolism for a given concentration of Manotel increases, causing the terminal slope.

Due to the indirect target clearance parameters FC, obtained by analysis of the NCA, such as clearance and half-life are dependent on dose and time. The total clearance is maximum at the lowest dose: 24,8 and 35 ml/day/kg for IL6R304 and IL6R305 after 0.4 mg/kg compared with 10.4-9,00 and 5,93-7,76 ml/day/kg for IL6R304 and IL6R305 at higher doses. Accordingly, normalized to the dose of exposure is smaller at the lowest dose (dose=CL×AUC).

The dominant (main) the half-life of IL6R304 decreased from 6,61 days to 5.00 days and 1.73 days after the on/in the 10, 2 and 0.4 mg/kg of the Main half-life of IL6R305 decreased from 7.37 days to 4.29 days and 1.64 days after the on/in the 10, 2 and 0.4 mg/kg Because there is more data in the earlier points, the terminal phase is best characterized at the lowest dose: after the on/in the 0.4 mg/kg IL6R304 and IL6R305 there was a short terminal half-life in 0,530 days and 0,470 days respectively.

Based on these data PK, pharmacokinetic properties IL6R304 and IL6R305 can be considered close.

On the 29th day of testing monkeys 14T and monkeys 15f (2 mg/kg) was still found to have low levels of concentration of IL6R304. Based on the profiles of FC from other apes, such results were unexpected. It is possible that they indicate the second type of the saturable binding the deposits with the target which becomes appreciable only at very low concentrations. However, these results could be an artifact in the analysis of samples for FC using ELISA method.

Given the volume of distribution calculated by the method of the NCA, was low, ranging from 1 to 2 volumes of plasma approximately 40 ml/kg for both nantel, indicating limited distribution outside the vascular space. However, the true values of VSSmight be underestimated due to methodological errors associated with the NCA (e.g., distribution and subsequent degradation of Manotel in the peripheral space can not be attributed to the distribution and the total systemic clearance). The Vss values were fairly constant at different dose levels.

To illustrate the possible effects of binding to a target on the profile of FC in Fig.56 shows a comparison of the average profiles of FC in IL6R304 (0,4-2-10 mg/kg) and IL6R202 (2 mg/kg, SEQ ID NO:73). For clarity, the profile of FC IL6R202 (2 mg/kg) also resulted in a dose of 0.4 mg/kg and 10 mg/kg Shows that IL6R304 binds to IL-6R in long-tailed macaques, whereas for IL6R202 no binding to target the long-tailed macaques (WO 09/010539). Accordingly, at high concentrations, where the predominant linear clearance, profiles (and the corresponding half-life times) both nantel similar. And at low concentrations for IL6R304 there is postepenno change in the slope, most likely due to the indirect target clearance, whereas the IL6R202 there is no change of the terminal slope. However, low concentrations of IL6R304 and IL6R305 could be underestimated due to interference from IL-6R at the FC using ELISA method, especially at high levels of IL-6R.

Example 55. Detection of antibodies against IL6R304 and IL6R305

A number of plasma samples taken before administration and after different number of days after the introduction of IL6R304 or IL6R305 were subjected to screening for the presence of antibodies monkeys (isotype IgG), is able to connect with antelami or one or more of their building blocks. Samples from animals treated IL6R304 analyzed on tablets covered with IL6R304 (Fig.57), IL6R300 (Fig.58) or ALB8 (Fig.59). Samples from animals treated IL6R305 analyzed on tablets covered with IL6R305 (Fig.60), IL6R300 (Fig.61) or ALB8 (Fig.62).

Summary the appearance of antibodies against drugs (ADA) to complete nanotesla (IL6R304 and IL6R305) are given in table.C-44. Observed weak answers or no answer IL6R300 and A1b8. And after the on/in the introduction of IL6R304 all monkeys were found ADA (except animal No. 16, which it was impossible to determine ADA because of the high values before the introduction). Antibodies appeared after 1 week after injection in monkeys treated with 0.4 mg/kg, and 2 weeks after injection in monkeys treated with 2 mg/kg and 10 mg/kg, the highest titer of the ADA were obtained in animals No. 11 and 13 (at a dose of 0.4 mg/kg). After/in the introduction IL6R305 all monkeys were found ADA (except animal No. 23, in which the ADA was not found). Antibodies appeared after 1 week after injection in monkeys treated with 0.4 mg/kg, and 2 weeks after injection in monkeys treated with 2 mg/kg and 10 mg/kg of the highest titles of the ADA were obtained in animals No. 22 and 24 (at the dosage of 2 mg/kg) and animals No. 25 and 26 (at the dosage of 10 mg/kg).

Example 56. The effect of Manotel on the level of sIL-6R

As expected on the basis of published data (Nishimoto et al., 2008, Blood 112(10): 3959-64), the processing control IgG led to the increase in sIL-6R plasma, whereas the processing of the media - not (Fig.63A). Similarly, a small dose of IL6R304 caused a rapid increase of sIL-6R in all three monkeys (Fig.V). The treated IL6R305 animals sIL-6R plasma also increased, but not as noticeable as in other experimental groups (Fig.S).

VIII. The effectiveness of IL6R304

Example 57. The effect of Manotel on the level of sIL-6R in the study of effectiveness

We measured the overall level of sIL-6R plasma (free associated with antelami and is associated with IL-6) using ELISA method. As expected on the basis of published data (Nishimoto et al., 2008, Blood 112(10): 3959-64), the processing control IgG led to the increase in sIL-6R plasma, whereas the processing of the media - not (Fig.64A). Similarly, all treated antelami animals showed a rapid increase of sIL-6R. This mod is but to explain a slower clearance of the complex control IgG - or NB-sIL-6R in comparison with the free sIL-6R.

The maximum level of sIL-6R and the duration of effect was clearly dose dependent. In addition, the effect of nantel was more pronounced than that of control IgG (cf. dose of Manotel 2 mg/kg with control IgG (Fig.64). This can be explained by a more rapid clearance of immune complexes of antibodies via Fc. Beyond expectations, however, the increase in sIL-6R and duration of effect was more pronounced for IL6R304 (Fig.64A) than for IL6R305 (Fig.V).

Example 58. The effect of Manotel on the level of IL-6 in the study of effectiveness

The overall level of IL-6 in plasma (free associated with sIL-6R) was measured on the Gyrolab platform. When this definition to capture IL-6 used biotinylated mAb rat against IL-6 person and for detection of labeled Alexa mouse mAb against IL-6 person. In this method, measured and endogenous IL-6 long-tailed macaques, and recombinant IL-6 man, who had introduced daily from day 1 till day 8. Therefore, it is necessary to distinguish between the IL-6 measured up to 1 day (= only endogenous IL-6 macaques), and from 2 to 29 day (= input IL-6 man + of endogenous IL-6 macaques).

As can be seen from Fig.65, the introduction of IL6R304, IL6R305, control IgG and to a lesser extent placebo resulted in a transient increase in IL-6, which was the peak after 8 h after injection. However, this increase was significantly higher in treated antelami groups. The effect on IL-6 as if h is was specific for anti-IL-6R of nantel, since the introduction of a foreign nanotesla also caused a transient increase of IL-6 (Fig.65D). Early increase of IL-6 is most likely due to the stress resulting from the processing of animals, but also further increase the production of IL-6 under the action of a small amount of endotoxin in the preparations of Manotel.

During the phase of injection of IL-6 blood samples were taken before each daily administration of IL-6. In the placebo group, which received IL-6, but not received napothera or control IgG, almost no detectable IL-6 (Fig.65S). This is consistent with the short half-life of IL-6 person after s/C injection (Tsigos et al., 1997, J. Clin. Endocrinol. Metab. 82: 4167-70). However, in all animals treated with 2-10 mg/kg control IgG, IL6R304 or IL6R305, IL-6 was detected from 2 to 8 day. This can be explained by blocking receptor clearance of IL-6, which increases its half-life (Nishimoto et al., 2008, Blood 112(10); 3959-64). Thus, circulating IL-6 may serve as a pharmacodynamic biomarker for neutralization of IL-6R.

IX. Pharmacodynamics IL6R304

Example 59. Pharmacodynamic effects after a single dose of the long-tailed macaques

Also measured changes in the concentration of sIL6R in plasma after a single intravenous introduction IL6R304 in healthy (i.e. not subjected to stimulation) long-tailed macaques. In this study PK/PD single dose was 1-100 mg/kg, Took blood samples for analysis of the pharmacokinetics, immunogenicity and pharmacodynamics. To measure the General level of sIL6R used a method based on ELISA, and for measuring free sIL6R used method of binding ligands on the Gyrolab platform™. When determining the overall level of sIL6R to capture sIL6R (free and complex) used neeutralize monoclonal antibody against IL6R, and to detect - biotinylated polyclonal antibody against IL6R in combination with conjugate streptavidin-HRP. When determining free sIL6R to capture free sIL6R used biotinylated building block A, and for detection of the labeled Alexa neeutralize monoclonal antibody against IL6R.

The results of the study PK/PD in a single dose confirmed the dose-dependent effect of IL6R304 on: (i) maximum combined concentration of sIL6R (Fig.BA), (ii) the duration of raising the General level of sIL6R (Fig.66A) and (iii) the duration of suppression of free sIL6R (Fig.V). In General, concentrations of total and free sIL6R returned to the original level after some time (depending on the dose, the longest enhancement and suppression observed at the highest dose). The concentration of free sIL6R after administration of low dose of 1 mg/kg IL6R304 decreased approximately 8 days, and then increased to a level higher than the concentration of the treated carrier animals.

<> There was good inverse correlation between the General level of sIL6R, the level of free sIL6R (PD) and the concentration of IL6R304 confirming that sIL6R can be used as a biomarker for the presence of active drug. In Fig.67 presents one example that will clarify the interdependence between these 3 parameters (group average dose was chosen because this group shows the return to initial level): introduction IL6R304 led to increase the total concentration of sIL6R and to the formation of stable complex drug-sIL6R. At least reduce the concentration of IL6R304 in plasma decreased in parallel and the total concentration of sIL6R, since most of sIL6R consisted of complexional receptor. This is confirmed by the low concentration of free sIL6R, which is returned to the original level when Troubleshooting IL6R304 from the bloodstream. Low concentration of free sIL6R confirms that the measured total sIL6R really inactive and complexion with IL6R304 (Fig.67).

Example 60. Description of pharmacodynamic effects through modeling of PK/PD

The impact of the introduction of IL6R304 on the overall level sIL6R may be due to direct binding of IL6R304 with the receptor complex remains in the bloodstream due to increasing the half-life of the molecule IL6R304 (bound to albumin). As measurable changes in the total concentration of sIL6R follow the kinetics in the temporal delay, the relationship of PK/PD best describes the indirect response model, which was used to describe the effect of introduced/IL6R304 on the accumulation of sIL6R complex-IL6R304. The model describes the response to a drug that occurs due to inhibition of elimination of sIL6R when linking it with IL6R304. In this model, the indirect response of the rate of change of the overall complex of sIL6R-IL6R304 (response R) is described as:

dRdt=Kin-Kout*[1-Imax*CnIC50n+Cn]*R

where Kin- the rate of synthesis of zero order; R - the overall level of sIL6R; Imax- the maximum degree of inhibition; C - concentration of IL6R304 in plasma; n is the coefficient of the curve of dose-response; and Koutthe rate constant of elimination of the first order for sIL6R.

All available data on the overall parameters of sIL6R studies PK/PD in a single dose were exposed simultaneously in the model (WinNonlin Professional Software Version 5.1, Pharsight Corporation, Mountain View, California, USA), using pharmacokinetic function, described in Example 61, as the input function for m is Delhi PK/PD indirect response.

In Fig.68 presents individual observed and predicted model data on the total concentration of sIL6R time in long-tailed macaques after/in the introduction of 1, 5, 10, 25, or 100 mg/kg Evaluation of the pharmacodynamic parameters IL6R304 in long-tailed macaques are shown in table.C-45. All parameters were estimated with a sufficient degree of accuracy, as evidenced by the values of CV% of less than 50%.

All data after the on/in the introduction of the study PK/PD in one dose at the same time was introduced in the indirect response model describing the behavior of sIL6R, IL6R304 and sIL6R complex-IL6R304.

The average half-life of sIL6R was equal to approximately 5.8 h (=ln2/Koutwhen Kout=Kin/R0), and the rate of production was 2,49 ng/ml/h IL6R304 able to almost completely block the elimination of sIL6R through the primary path (Imax=97%). At this rate of elimination has reached a new high with low constant kout, which correlates with that of serum albumin long-tailed macaques. After this was established, a new basal level of total sIL6R. When evaluating IC50125 ng/ml or 4.48 nm IL6R304 was a strong inhibitor elimination complexioned sIL6R in long-tailed macaques.

X. Pharmacokinetics IL6R304

Example 61. The pharmacokinetics of the long-tailed macaques

This section provides data characterizing the pharmacokinetics the behavior/entered IL6R304 in one species cross-reactivity (long-tailed macaques).

The concentration of IL6R304 in healthy (not induced) long-tailed macaques were measured using a special method kits are used (immunoassay with increased fluorescence lanthanide when dissociation). The total concentration of active IL6R304 measured IL6R-dependent method.

The study PK/PD in a single dose of IL6R304 was administered to healthy male long-tailed macaques in the/in a single bolus at 0, 1, 5, 10, 25 and 100 mg/kg all animals took blood samples for analysis on FC, ADA (antibodies against the drug) and FD before the introduction and at the specified time points after administration of the dose. The samples were analyzed for FC, FD and ADA (also see Example 62).

For detection of antibodies against IL6R304 used tested electrochemiluminescent (ECL) method joint screening and confirmation. Briefly, used IL6R304 for capture and detection antibodies against the drug (ADA) in the format of a uniform definition for the device MSD Sector Imager 2400.

For the analysis of FC IL6R304 recorded on streptavidin coated tablets via biotinylated tool against nantel (3E8biv-bio). After the formation of the complex with the target IL6R to generate a fluorescent signal in the amplifying solution used labeled with europium mAb against IL6R.

The profiles of the average concentration of IL6R304 in plasma with time is shown in Fig.69. A summary of key pharmacokinetic parameters of IL6R304 in after/in the times is m a bolus in 1, 5,10,25 or 100 mg/kg are shown in table.C-46.

The pharmacokinetic profile after/in the introduction showed a three-phase reduction. In the first two days after injection was observed phase distribution with subsequent slower main (dominant) phase and a faster terminal phase. The phase distribution can be divided into fast (small compartment) and slow (deep compartment). Based on the phases of elimination, IL6R304 presumably subject to clearance by two mechanisms: linear insatiable (nonspecific elimination or CLNON-IL6Rand nonlinear saturable (indirect target or specific elimination, CLIL6R) mechanism of clearance. The latter may be due to the internalization of IL6R304 associated with membrane IL6R, and subsequent clearance of the complex IL6R304-mIL6R.

Since the clearance of IL6R304 is a combination of saturated and insatiable the way, the kinetics of plasma long-tailed macaques showed a nonlinear behavior with a half-life, which depends on the dose, and this dose level will depend on the time.

At saturation CLIL6Rwhen the CL is mainly determined CLNON-IL6Rgiven the half-life of IL6R304 in long-tailed macaques ranged from 5.8 to 8.9 days and was close to famous for serum albumin long-tailed macaques (Nguyen et al., 2006, Protein Eng. Des Sel. 19: 291). This is consistent with the expected and known cross-reactivity albumin binding part of the molecule IL6R304 to albumin long-tailed macaques.

The average exposure after administration of a single dose was increased slightly more than proportionally to the dose of between 1 and 5 mg/kg and between 10 and 25 mg/kg and in proportion to the dose of between 5 and 10 mg/kg Result in the group with a dose of 100 mg/kg should be treated with caution, as this group was composed of only one animal. In General, due to the limited number of monkeys at dose groups, assessment of dose proportionality was rough.

Linking IL6R304 with sIL6R led to an increase in the measured total concentration of sIL6R, which included sIL6R and complex s!L6R-IL6R304; it is assumed that this increase was due to slower clearance of the complex as compared to sIL6R.

On the basis of available data on immunogenicity, PK and PD concluded that the appearance of antibodies ADA could affect the profile PK/PD IL6R304 two animals from the group with the highest dose (animals 15 and 17). Both animals showed an unexpected decrease in the level of IL6R304 in the plasma simultaneously with the appearance of measurable ADA and with the reduction of the pharmacodynamic effect. Therefore, these animals were not considered in the analysis of PK/PD. The appearance of antibodies to ADA in other animals didn't seem to have an obvious effect on the profiles of PK/PD, so these data who were included in the analysis.

In one animal (animal 3) in the group with a dose of 1 mg/kg were observed indirect target clearance, although this is expected with such a low dose. In one animal (animal 6) in the group with a dose of 5 mg/kg mediated target clearance was still observed despite the higher the dose and the expected saturation of a given path. Because the variability in the endogenous concentrations of IL6R between animals can be high-mediated target clearance may be strong evidence of variability. In addition, when the concentration of IL6R304 close to the expected value of KM(here: 0,718 µg/ml), small changes in the concentration of IL6R304 leads to a large change in the nonlinear clearance. The combination of both can lead to significant confirmation mediated or not mediated only by the target clearance in groups with low doses, which is reflected in the large variation of the terminal values of time-life. The parameters FC in animals 3 and 6 were excluded from descriptive statistics, as biological variability precludes a meaningful assessment of the accuracy of the applied methods (table.C-46).

None of the animals in the placebo group were not subjected to systematic effects of IL6R304. All predosavia samples of the treated IL6R304 animals were outside the lower boundary of the quantitative op is edeline (LLOQ).

Animals from receiving treatment groups showed increased concentrations of IL6R304 in the plasma with increasing dose level. The highest average total exposure (AUCinf) was observed in the group with the highest dose (100 mg/kg) and was 540612 µg·h/ml

Average normalized to the dose values AUCinfincreased in proportion to dose in the range of 5 to 10 mg/kg and slightly more than proportionally to the dose of between 1 and 5 mg/kg and 10 and 25 mg/kg (of 1.3 and 1.4, respectively). A more than proportional increase between 25 and 100 mg/kg should be treated with caution, as the group with the highest dose was composed of only one animal. In General, due to the limited number of monkeys at dose groups, assessment of dose proportionality was rough.

Note that these accompanimental analysis indicated the difference of time half-life at a dose of 1 mg/kg, compared with higher doses. This is explained by the large contribution of saturated mediated target mechanisms of clearance compared with higher doses, dominated by insatiable mechanisms.

Based on the phases of elimination, IL6R304 presumably subject to clearance by two mechanisms: linear and nonlinear mechanism of clearance. Linear clearance mechanism, probably due to the insatiable and not mediated IL6R removing IL6R304 and corresponds to the slow and carried ecifically proteolytic degradation of IL6R304. Nonlinear and indirect IL6R the process of clearance is a saturable mechanism of clearance, which, in all probability, represents the binding of IL6R304 membrane IL6R with subsequent internalization and clearance.

Nonlinear pharmacokinetic behavior of IL6R304 in long-tailed macaques were fixed by substituting the data in the open pharmacokinetic model with three compartments, with linear and non-linear clearance from the Central compartment. The structural model shown in Fig.70.

All available data on individual/in concentrations in plasma from the study PK/PD in a single dose were exposed simultaneously in the model (WinNonlin Professional Software Version 5.1, Pharsight Corporation, Mountain View, California, USA), using the iterative weighted (1/ŷ*ŷ), where ŷ is the predicted concentration in the plasma. In Fig.71 presents the individual graphs of the observed and predicted in the model the concentration of IL6R304 in plasma long-tailed macaques from time to time after the on/in the introduction of 1, 5, 10, 25, or 100 mg/kg evaluation of pharmaco kinetic parameters of IL6R304 are given in table.C-47.

All data from the study PK/PD in one dose at the same time substituted in the open model with three compartments, with linear (CLNON-IL6R) and nonlinear (CLIL6R) clearance from the Central compartment. At low concentrations of IL6R304 (C<<<Km) transformed ladyship contribution is mediated IL6R clearance, which is equal to Vmax/Km. At high concentrations IL6R304 (WITH>>>Kmpath mediated IL6R ground is saturated and must proceed with maximum mass transfer (i.e., Vmax). Consequently, the total clearance (CL) is dominated by linear, not mediated IL6R path (CLNON-IL6R).

Nonlinear mediated IL6R component clearance explains the dependence of the half-life of IL6R304 time-and dose-dependent in long-tailed macaques.

Table B-1.
Amino acid sequences included in the composition control connections
Heavy chain control IgG, SEQ ID NO:1
QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRV TMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain of control IgG, SEQ ID NO:2
DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC
Heavy chain control Fab, SEQ ID NO:3
QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRV TMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSC
Light chain control Fab, SEQ ID NO:4
DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC
Table B-2.
The protein sequence of Manotel against IL-6R
RMRN, SEQ ID NO:5
EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSSGTTSTYYS DSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWRGWNDYWG QGTQVTVSS
PMP35E11, SEQ ID NO:6
EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEHEGVSCISSSDGSTYYADS VKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAERDVPARSLCGSYYWYDYRGQGTQVTVSS
PMP32C9/IL6R04, SEQ ID NO:7
EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQPGKEREGVSGISSSDGNTYYADS VKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSS
PMP35H4/IL6R13, SEQ ID NO:8
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTES MKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVTVSS
PMP32E10, SEQ ID NO:9
EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADS VKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSS
PMP30C11, SEQ ID NO:10
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAVISRSGSSTYYADS VKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCKAEWAGDYDYWGQGTQVTVSS
PMP35C10, SEQ ID NO:11
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAVIHWSSGSTYYADP VKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAFLPGPEGFHDYWGQGTQVTVSS
PMP34G9, SEQ ID NO:12
EVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYADS VKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSS
PMP31A4/IL6R03, SEQ ID NO:13
EVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADS VKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSS

RMRE, SEQ ID NO:14
EVQLVESGGGLVQAGGSLRLSCAASGNIFDDNTMGWTWNRQPPGKQRELVAIIATDGSTNYA DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLFSLRLGRDYWGQGTQVTVSS
PMP33A3, SEQ ID NO:15
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYGAIGWFRQAPGKEREGVSCISSSTGSTYYAD SVKGRFTISRDNGKNTVYLQMNSLKPEDTAVYYCAADKMWSPCLVAANEEALFEYDYWGQGTQVTVSS
PMP34A12, SEQ ID NO:16
EVQLVESGGGLVQPGGSLRLSCVASGFSLDYYVIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTIS RDNAKNTVYLQMNSLKPE DTAVYYCAADLLRTPEFCVDSAPYDYWGQGTQVTVSS
PMP28E11, SEQ ID NO:17
EVQLVESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYAD SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCALVHTTAQATGVPQREYEYEWWGQGTQVTVSS
PMP35F4, SEQ ID NO:18
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAIITWNSSTYYADS VKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAQYGLGYAEDYWGQGTQVTVSS
Table B-3. Protein sequences of multivalent nantel against IL6R
IL6R22, SEQ ID NO:19
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAVISRSGSSTYYAD SVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCKAEWAGDYDYWGQGTQVTVSSGGGGSG GGSAVQLVESGGGLVQPGNSLRLCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
IL6R23, SEQ ID NO:20
EVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADS VKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSSGGG GSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGS DTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
IL6R24, SEQ ID NO:21
EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYAD SVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVT VSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSS ISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
IL6R25, SEQ ID NO:22
EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYAD SVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTV SSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSI SGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
IL6R26, SEQ ID NO:23
EVQLVESGGGLVQAGGSLRLSCAASGNIFDDNTMGWTWNRQPPGKQRELVAIIATDGSTNYA DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLFSLRLGRDYWGQGTQVTVSSGGGGSGGGSAVQLVES GGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDT LYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
IL6R28, SEQ ID NO:24
EVQLVESGGGLVQPGGSLRLSCVASGFSLDYYVIGWFRQAPGKEREGVSCISSSDGSTYYAD SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLLRTPEFCVDSAPYDYWGQGTQVTV SSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVQAPGKEPEWVSSI SGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
IL6R29, SEQ ID NO:25
EVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYAD SVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSSGGGGSGG GSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS

IL6R30, SEQ ID NO:26
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAVIHWSSGSTYYAD PVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAFLPGPEGFHDYWGQGTQVTVSSGGGG SGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSD TLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
IL6R31, SEQ ID NO:27
EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEHEGVSCISSSDGSTYYAD SVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAERDVPARSLCGSYYWYDYRGQGTQVT VSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSS ISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
IL6R32, SEQ ID NO:28
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAIITWNSSTYYADS VKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAQYGLGYAEDYWGQGTQVTVSSGGGGSG GGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
IL6R33, SEQ ID NO:29
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTE SMKGRFTISRDNAKNTVYLQMNSLKPEDAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVT VSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSS ISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
IL6R34, SEQ ID NO:30
EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSSGTTSTYY SDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWRGWNDY WGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPG KEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
IL6R43, SEQ ID NO:31
EVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADS VKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSSGGG GSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGS DTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSEVQ LVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADSVKG RLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSS
IL6R44, SEQ ID NO:32
EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYAD SVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVT VSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSS ISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVT VSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSG ISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPE FFKYWGQGTQVTVSS
IL6R49, SEQ ID NO:33
EVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYAD SVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSSGGGGSGG GSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGG GSEVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYY ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSS
IL6R53, SEQ ID NO:34
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTE SMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVT VSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSS ISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVT VSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSA ISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPD EYDYWGQGTQVTVSS

Table B-4.
Protein sequence optimized p sequence of Manotel
IL6R61, SEQ ID NO:40
EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADS VKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSS
IL6R62, SEQ ID NO:41
EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADS VKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSS
IL6R63, SEQ ID NO; 42
EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADS VKGRLTISRDNAKNTLYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSS
IL6R64, SEQ ID NO:43
EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADS VKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSS
IL6R65, SEQ ID NO:44
EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADS VKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSS
IL6R71, SEQ ID NO:45
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYAD SVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS
IL6R72, SEQ ID NO:46
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYAD SVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS
IL6R73, SEQ ID NO; 47
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYAD SVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS
IL6R74, SEQ ID NO:48
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYAD SVKGRFTIS S DNAKNTLYLQMNSLRPEDTAVYYCAAE P PDS SWYL DGS PE FFKYWGQGTLVTVSS
IL6R75, SEQ ID NO:49 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYAD SVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS
IL6R81, SEQ ID NO:50
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTE SMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS
IL6R82, SEQ ID NO:51
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTE SMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS
IL6R83, SEQ ID NO:52
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTE SMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS
IL6R84, SEQ ID NO:53
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTE SMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS
IL6R85, SEQ ID NO:54
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGLEWVSAISWNGNNTYYTE SMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS
IL6R86, SEQ ID NO:55
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRALEWVSAISWNGNNTYYTE SMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS
IL6R87, SEQ ID NO:56
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKATEWVSAISWNGNNTYYTE SMKGRFTISRDNAKNTLYLQMNSLRPEDAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS
IL6R88, SEQ ID NO:57
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTE SMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS

IL6R89, SEQ ID NO:58
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGTEWVSAISWNGNNTYYTE SMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS
IL6R90, SEQ ID NO:59
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKALEWVSAISWNGNNTYYTE SMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS
Table B-5.
Protein sequence ripened on the affinity of Manotel
PMP7F4, SEQ ID NO:60
EVQLVESGGGLVQPGGSLRLSCAASGTTFKVNVMAWYRQAPGKGRELVAGIINGGSTTYADS VKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSS
PMP7C4, SEQ ID NO:61
EVQLVESGGGLVQPGGSLRLSCAASGTTFRINVMAWYRQAPGKGRELVAGIITNGSTSYADS VKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSS
PMP7D6, SEQ ID NO:62
EVQLVESGGGLVQPGGSLRLSCAASGSIFRVNVMAWYRQAPGKGRELVAAVINGGTTTYADS VKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSS
PMP7G7, SEQ ID NO:63
EVQLVESGGGLVQPGGSLRLSCAASGTTFKINIMAWYRQAPGKGRELVAGVITGGNTTYADS VKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSS
PMP7G8, SEQ ID NO:64
EVQLVESGGGLVQPGGSLRLSCAASGSTFRINVMAWYRQAPGKGRELVAGVINDGSTTYADS VKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSS
PMP20F6, SEQ ID NO; 65
EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIVSGGSTSYADS VKGRFTIS RDNAKNTLYLQMNSLRPEDTAVYYCAFITTNS DYDLGRRYWGQGTLVTVS S
PMP20A11, IL6R300, SEQ ID NO:66
EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADS VKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSS
PMP20E10, SEQ ID NO:67
EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIVSGGSTSYADS VKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSS
PMP21A10, SEQ ID NO:68
EVQLVESGGGLVQPGGSLRLSCAASGSIFKINVMAWYRQPGKGRELVAGIVTGGSTSYADS VKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSS
PMP21D11, SEQ ID NO:69
EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIVTGGSTSYADS VKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSS
Table B-6.
Protein sequence rich ripe for affinity/optimized sequence of Manotel
IL6R304, SEQ ID NO:70
EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYAD SVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISG SGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS
IL6R305, SEQ ID NO:71
EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYAD SVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIIS GGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQG TLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSS QGTLVTVSS

IL6R306, SEQ ID NO:72
EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYAD SVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRWGQGTLVTVSSG GGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISG SGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVS SGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGI ISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWG QGTLVTVSS
IL6R202, SEQ ID NO:73
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYA DSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTL VTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEW VSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQG TLVTVSS
Table B-7.
Preferred, but not restrictive examples svyazyvayus albumin of Manotel
ALB-1, SEQ ID NO:97
AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
ALB-8(gumanitarnoe ALB-1), SEQ ID NO:98
EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS
ALB-2, SEQ ID NO:99
AVQLVESGGGLVQGGGSLRLACAASERIFDLNLMGWYRQGPGNERELVATCITVGDSTNYADSVK GRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKIRRTWHSELWGQGTQVTVSS
Table B-8.
List of sequences of linkers
GS30, SEQ ID NO:101
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
GS15, SEQ ID NO:102
GGGGSGGGGSGGGGS
GS9, SEQ ID NO:103
GGGGSGGGS
GS7, SEQ ID NO:104
SGGSGGS
Top long hinge section Lama, SEQ ID NO:105
EPKTPKPQPAAA
Table C-1.
The materials used in the allocation of binding IL-6R of Manotel
ProviderDescription
IL-6 personDiacloneRecombinant protein obtained in E. coli
Bio-IL-6 personDiaclone/PEIL-6 person firm Diaclone, biotinylated on RE (6 betinov molecule)
Soluble IL-6R manPeprotechRecombinant protein obtained in cells NC (cat. No. 200-06R)
Soluble IL-6R manR&D SystemsRecombinant protein obtained in the cell is x Sf21 (cat. No. 227-SR)
MAb BR-6DiacloneNeutralizing MAb against IL-6R
MAbBN-12DiacloneNeitralizatsii MAb against IL-6R
MAb Ml 82BD BiosciencesBiotinylated MAb against IL-6R

Conjugate antibodies to IgG Lama (h&l) HRPBethyl LabsGoat RAG against IgG Lama
The cell line TF-1ESAS no.93022307J Cell Physiol 1989, 140:323; Exp Cell Res 1993, 208:35
Table C-2.
Scheme immunization
DayLama No. 081Lama No. 082Tissue samples
0100 mcg100 mcg 10 ml immune blood
7100 mcg100 mcg-
1450 mcg50 mcg-
2150 mcg50 mcg-
2850 mcg50 mcg10 ml immune blood
3550 mcg50 mcg-
39150 ml immune blood + biopsy of lymph node MOATS
43150 ml immune blood + PBL2
5250 mcg50 mcg-
59150 ml immune blood + NC1
Table C-3.
Characteristics libraries nantel obtained from immunized llamas
The size of the libraryThe content insertion (%)
Lama 816×10787
Lama 825×10778
Table C-4.
The conditions used for the selection of Manotel
MethodImmobilization/captureAntigen Concentration/amountWash
Magnetic beadsstreptavidinbio-IL-6R0,1,10, 100 ngtrypsin
Solutionstreptavidine ballsbio-IL-6R0,0,01, 0,1, 1Nmtrypsin
TabletBN-12IL-6R (Peprotech)of 0.1, 10, 100 nmtrypsin
TabletBN-12IL-6R (R&D)0,1,10, 100 nmtrypsin
Table C-5.
Statistics screening
Method The number of clones when skerningeQty inhibitors(%)Qty prosecutionand clonesThe number of unique sequences
IL-6-IL-6R153672 (4,7%)4614

Table C-6.
Values of koffthe inhibiting of Manotel
NanoteslaIDkoff(s-1)NanoteslaIDkoff(s-1)
RMRNIL6R141,14 E-04PMP34G9IL6R091,39 E-03
RMRE IL6R114,17 E-04PMP31A4IL6R031,60 E-03
RMRSIL6R04a 1.50 E-04PMP32E2IL6R068,86 E-04
RMRNIL6R131,78 E-04PMP33A3IL6R072,42 E-04
RMREIL6R051,27 E-03PMP34A12IL6R08n/o
RMRSOLIL6R022,94 E-03PMP28E11IL6R01n/o
RMRS IL6R105,09 E-04PMP35F4IL6R128,96 E-04
Table C-7.
The output of Manotel in cell cultures
NanoteslaOutput (mg)Output (mg/l)NanoteslaOutput (mg)Output (mg/l)
RMRN0,140,6PMP34G90,091,8
RMRE0,652,6PMP31A41,064,2
RMRS0,336,5PMP32E2 of 1.576,3
RMRN0,499,8PMP33A30,331,3
RMRE0,783,1PMP34A120,572,3
RMRS0,632,5PMP28E110,081,6
RMRS0,532.1PMP35F40,241.0
Table C-8.
The values of the IC50The selected nantel
ID NanoteslaIC50(M)
IL6R01PMP28E117.26 E-10
IL6R02RMRS1.69 E-09
IL6R03PMP31A47,16 E-10
IL6R04RMRS9.30 E-11
IL6R05RMRE6,26 E-10
IL6R06PMP32E21,21 E-09
IL6R07PMP33A31.44 E-09
IL6R08PMP34A121,18 E-09
IL6R09PMP34G9 2,38 E-10
IL6R10RMRS5,96 E-10
IL6R11RMRE1,58 E-10
IL6R12PMP35F45,17 E-10
IL6R13RMRN4.77 E-11
IL6R14RMRN2.22 E-10
Table C-9.
Kinetic parameters of the selected subset of 14 of inhibiting nantel against IL-6R
IDNanoteslakoff(s-1)kon(1/Ms)KD(nm)
IL6R01PMP28E11 2,62 E+050,4
IL6R02RMRS2,94 E-038,40 E+055,9
4,95 E-03
IL6R03PMP31A41,47 E-034,84 E+053,0
1.60 E-03
IL6R04RMRS9,42 E-05the 3.65 E+050,3
1.50 E-04
IL6R05RMREof 1.41 E-03the 1.44 E+059,8
1.27 E-03

td align="center" namest="c6" nameend="c7"> 0,8
IL6R06PMP32E28,86 E-04
EUR 7.57 E-031.07 E+067,1
IL6R07RMRA2,42 E-04n/an/a
IL6R08RMRA1.97 E-031,94 E+0510,2
IL6R09PMP34G91,29 E-036,41 E+052,0
1,30 E-03
1,11 E+061,2
1.39 E-03
IL6R10RMRS5,26 E-044,14 E+051,3
5,09 E-04
IL6R11RMRE3,40 E-043,91 E+050,9
3,96 E-04
2,15 E+051,9
4,17 E-04
IL6R12PMP35F41,16 E-03
8,96 E-046,78 E+051,7
IL6R13RMRN1,21 E-042,31 E+050,5
1,09 is -04
of 1.37 E+050,8
1,78 E-04
IL6R14RMRNof 1.00 E-04as 4.02 E+050,3
1,14 E-04
Table C-10.
The values of the IC50for inhibition of antelami cell proliferation XG-1
NanoteslaIC50(nm)IC50(nm) + HSA
IL6R01n/a
IL6R0231,0
IL6R0316,2of 17.5
IL6R040,1 0,1
IL6R057,3
IL6R0642,1
IL6R07a 50.5
IL6R0836,6
IL6R092,73,0
IL6R102,5
IL6R11of 5.4
IL6R122,8
IL6R131,41,3
IL6R140,6
Control Fab6,0
Table C-11.
The values of the IC50for inhibition of antelami proliferation of TF1 cells
NanoteslaIC50(nm)
IL6R01n/a
IL6R0294,7
IL6R0362,1
IL6R040,4
IL6R0538,0
IL6R06137,9
IL6R07374,9
IL6R0824,3
IL6R098,7
IL6R109,9

IL6R119,9
IL6R126,8
IL6R135,2
IL6R141.5
Control Fab9,2
Table C-12.
Competition with reference Fab for binding to IL-6R in the analysis method alphascreen
NanoteslaResidual binding of the control Fab with IL-6R (%)
IL6R0149
IL6R0286
IL6R035
IL6R0450
IL605 64
IL6R0636
IL6R0780
IL6R0899
IL6R0962
IL6R10102
IL6R1140
IL6R12103
IL6R1325
IL6R1496
Table C-13.
Summary characteristics of Manotel
NanoteslaKD(nm)IC50(nm) (IL-6/IL-6R)IC50(nm) on XG-1IC50(nm) TF-1Competition with counter. Fab (%)
IL6R010,40,73n/an/a49
IL6R025,91,6931,094,786
IL6R033,00,7216,262,15
IL6R040,30,090,10,450
IL6R059,80,637,338,064
IL6R067,11,2142,1137,936
IL6R07 N/a 0the 1.44a 50.5374,980
IL6R0810,21,1836,624,399
IL6R092,00,242,78,762
IL6R101,30,602,59,9102
IL6R110,90,16of 5.49,940
IL6R121,70,522,86,8103
IL6R130,50,05 1,45,225
IL6R140,30,220,61,596
Table C-14.
Item ID formatted nantel
IDFormatSEQ ID NO:
IL6R22PMP30C11-9GS-ALB119
IL6R23PMP31A4-9GS-ALB120
IL6R24PMP32C9-9GS-ALB121
IL6R25PMP32E10-9GS-ALB122
IL6R26PMP32E2-9GS-ALB123
IL6R28PMP34A12-9GS-ALB124

/tr>
IL6R29PMP34G9-9GS-ALB125
IL6R30PMP35C10-9GS-ALB126
IL6R31PMP35E11-9GS-ALB127
IL6R32PMP35F4-9GS-ALB128
IL6R33PMP35H4-9GS-ALB129
IL6R34PMP40H5-9GS-ALB130
IL6R43PMP31A4-9GS-ALB1-9GS-A31
IL6R44PMP32C9-9GS-ALB1-9GS-32C932
IL6R49PMP34G9-9GS-ALB1-9GS-34G933
IL6R53PMP35H4-9GS-ALB1-9GS-35H434
Table C-15.
The output of the if expression bispecific of Manotel against IL-6R
NanoteslaIDOutput (mg)Output(mg/l)NanoteslaIDOutput(mg)Output(mg/l)
RMRSIL6R221,14,2PMP34G9IL6R293,313,2
RMRAIL6R230,30,6RMRSIL6R30 0,93,7
RMRSIL6R240,52,0RMREIL6R311,87,3
RMREIL6R251,35,0PMP35F4IL6R320,51,1
RMREIL6R261,14,2RMRNIL6R331,97,5
RMRAIL6R282,18,4RMRNIL6R340,40,9
Table C-16.
The values of the IC50bivalent of Manotel when competition analysis method alphascreen
NanoteslaIC50(M)
IL6R225,59 F-10
IL6R241,45 E-10
IL6R25to 6.43 E-10
IL6R261,67 E-09
IL6R283,26 E-10
IL6R291.23 E-10
IL6R303,43 E-10
IL6R311.31 E-10
IL6R322,68 E-10
IL6R331.39 E-10
IL6R341,46 E-10
Control Fabof 5.92 E-10
Table C-17.
The values of the IC50formatted Manotel in the analysis method of the proliferation of XG-1
NanoteslaIC50(nm)IC50(nm) + HSA
IL6R2250,2
IL6R2316,990,4
IL6R240,20,5
IL6R258,4
IL6R2665,3
IL6R284,4
IL6R29 3,6the 13.4
IL6R3027,2
IL6R314,6
IL6R321,6

Values of Kdformatted Manotel for binding to serum albumin from different species
IL6R332,615,4
IL6R340,82,5
IL6R440,070,17
IL6R490,060,19
IL6R530,130,61
Control IgG6,0 0,47
Table C-18.
Kinetic parameters of binding to IL-6R
Nanoteslakd(s-1)ka(1/Ms)KD(nm)
IL6R225.7 E-033.3 E+0516,9
IL6R231.5 E-033.2 E+054,6
IL6R241.1 E-043,7 E+050,3
IL6R251.2 E-031,2 E+0510,3
IL6R266.9 E-034.5 E+0515,5
IL6R285.3 E-042.4 E+052,2
IL6R291.5 E-037,1 E+052,1
IL6R301.2 E-03the 1.6 E+057,5
IL6R313.8 E-04the 1.6 E+052,3
IL6R321.3 E-031.0 E+061,3
IL6R331,25 E-041.1 E+051,1
IL6R341.1 E-042.6 E+050,4
Table C-19.
IDPeopleMouseMacaque, supoRHBaboon
Kd(nm)Kd(nm)Kd(nm)Kd(nm)Kd(nm)
IL6R2211,1108
IL6R231627527,623,823,2
IL6R241512228,328,3 of 40.3
IL6R2513,9122
IL6R269,473
IL6R2810,6180
IL6R2910,8831920,626,8
IL6R3012,1113
IL6R31the 13.4 86,8
IL6R3210179
IL6R3327,398,624,524,632,3
IL6R349,21111514,718,9
IL6R4451,499343
IL6R5335497
ALB-10,66,5
Table C-20.
Kinetic parameters were optimized for sequence variants IL6R03,04 and 13
IL6R03 (3,0 nm)IL6R61ToIn(nm)2
ka(1/Ms)8,50 E+05
kd(1/s)1,70 E-03
IL6R62KD(nm)2,2
ka(1/Ms)9,29 E+05
kd(1/s)2,07 E-03
IL6R63KD(nm)
ka(1/Ms)9,90 E+05
kd(1/s)the 3.65 E-03
IL6R64KD(nm)n/o
ka(1/Ms)n/a 0
kd(1/s)of 1.00 E-03
IL6R04 (0.3 nm)IL6R71KD(nm)0,2
ka(1/Ms)7,03 E+05
kd(1/s)1,53 E-04
IL6R72KD(nm)0,3
ka(1/Ms) 5,43 E+05
kd(1/s)1,80 E-04
IL6R73KD(nm)0,3
ka(1/Ms)6,98 E+05
kd(1/s)2,33 E-04
IL6R74KD(nm)0,2
ka(1/Ms)to 7.67 E+05
kd(1/s)1,22 E-04
IL6R13 (0.7 nm)IL6R81KD(nm)0,4
ka(1/Ms)3,20 E+05
kd(1/s)1,28 E-04
IL6R82KD(nm) 5,1
ka(1/Ms)to 6.19 E+05
kd(1/s)3,14 E-03
IL6R83KD(nm)03
ka(1/Ms)3,50 E+05
kd(1/s)1,20 E-04
IL6R84KD(nm)of 5.4
ka(1/Ms)a 7.62 E+05
kd(1/s)4.09 to E-03
Table C-21.
Values of koffthe optimized sequence variants IL6R13
IDkoff(s-1)
IL6R132.1 E-04
IL6R852.1 E-03
IL6R861.7 E-03
IL6R871.1 E-04
IL6R882.6 E-04
IL6R891.9 E-04
IL6R901.9 E-03
Table C-22.
Kinetic parameters were optimized for sequence variants IL6R03, 04 and 13
IL6R03 (3,0 nm)IL6R65KD(nm)4
ka(1/Ms)6,00 E+05
kd(1/s)2.35 E-03
IL6R04 (0.3 nm)IL6R75 KD(nm)0,1
ka(1/Ms)1,00 E+06
kd(1/s)1E-04
IL6R13 (0.7 nm)IL6R88KD(nm)0,9
ka(1/Ms)2.30 E+05
kd(1/s)2,13 E-04

Table C-23.
The IC50 values of The optimized sequence and nantel wild type when parsing method XG-1
The values of the IC50(nm)
The original nanoteslaOptimized nanotesla
IL6R0317IL6R6526
IL6R04 0,1IL6R750,04
IL6R131,4IL6R883,3
Table C-24.
Kinetic parameters of the binding of Manotel with IL-6R macaques
IDka(1/Ms)kd(1/s)kd(nm)
IL6R033,70 E+051,64 E-034,4
IL6R651.65 E+051.97 E-0312
IL6R045,86 E+04of 1.00 E-02171
IL6R201*2,18 E+05 6,11 E-0328,1
* IL6R201 is a version IL6R75 without labels
Table C-25.
The values of Tmthe IL6R65 and ripened on the affinity of Manotel to IL-6R
Tm(the first time)Tm(the second time)The average value. Tm
IL6R6570,4470,6470,5471,0770,67
PMP7F476,6876,5576,5876,4476,56
A74,5674,2274,3974,3974,39
E75,295,22 75,3175,3875,30
A74,4274,0374,1274,1974,19
21D1174,0374,2974,45sampled at 74.2574,26
20F674,3674,2974,4574,3274,36
Table C-26.
Kinetic parameters of affinity at IL6R65 and ripened on the affinity of options
He apatelaka(M-1·s-1)kd(s-1)KD(PM)
IL6R653.8 E-033800
E1.0 E+06*3.3 E-0533
21D111.0 E+06*3.5 E-0535
A1.0 E+06*1.2 E-0512
20F61.0 E+06*3.3 E-0533
A1.0 E+061.9 E-0519
* Assessment

Table C-27.
Formats ripened on the affinity of Manotel against IL6R
FormatOptimized nanoteslaName
20A11-9GS-ALB8IL6R304
A-9GS-A-9GS-ALB8IL6R305
A-9GS-ALB8-9GS-AIL6R306
Table C-28.
The activity of rich nantel and controls in the analysis method of the proliferation of TF-1 at 100 ME IL-6/ml
ConnectionIC50(nm)SD(nm)Repeat
A0,2830,2563
IL6R3040,7150,3902
IL6R3050,0980,0464
IL6R3060,341rate £ 0.1623
Control Fab6,2620,7062
Control IgG0,9210,2754
Table C-29.
The activity of rich Manotel in the analysis method of the proliferation of TF-1 at 5,000 IU of IL-6/ml
ConnectionIC50(nm)
A15,22
IL6R30415,48
IL6R3055,49
IL6R30623,19
the completed IgG 144,5
Table C-30.
The values of the IC50(nm) formatted nantel to neutralize the binding of IL-6 with sIL-6R plasma of man
ConnectionIC50the normal level IL6IC50at a high level of IL6Ratio (high/ low)
Control IgG0,2581,696,54
IL6R20A110,1980,3561,80
IL6R3040,2290,6342,77
IL6R3050,1370,3352,44
IL6R3060,4122,395,80

Table C-31.
Values EU50(nm) for binding formatted ripened on the affinity of Manotel cells SNO 4D5 (4PL)
ConnectionEC50(nm)
IL6R20A111,396
IL6R3041,939
IL6R3050,8984
IL6R3066,154
Table C-32.
The values of EC50(nm) for binding formatted ripened on the affinity of Manotel with PBL from 2 donors
ConnectionL1L2M1M2G1G2
IL6R20A113,5 2,9243,6214,7772,4155,614
IL6R3049,27320,688,24114,175,98517,32
IL6R3054,28225,792,9064,2622,4344,927
IL6R3066049,5949,3859,3453,9977,68
L: lymphocytes: monocytes, G: granulocytes
Table C-33.
Kinetic is a mini-options to associate the formatted ripened on the affinity of Manotel with serum albumin human and macaque
NanoteslaSerum albumin manSerum albumin macaques
ka(M-1s-1)kd(s-1)KD(nm)ka(M-1s-1)kd(s-1)KD(PM)
ALB11the 5.45 E+051,68 E-03is 3.085,11 E+051,53 E-062,99
IL6R3042,15 E+054,75 E-0322,11,95 E+054,56 E-0323,4
IL6R305a 2.01 E+05 4,07 E-0320,32,03 E+05a 3.87 E-0319,1
IL6R3062.25 E+053,83 E-0317,12,12 E+053,70 E-0317,4
Table C-34.
Kinetic parameters for the binding of rich ripe on the affinity of Manotel with IL-6R human and macaque
NanoteslaIL-6R manIL-6R macaques
ka(M-1s-1)kd(s-1)KD(PM)ka(M-1s-1)kd(s-1)KD(PM)
IL6R3001E+06 H/OH/O1E+06n/an/a
IL6R3047E+05≤1E-05*≤148E+052E-0525
* Detection limit
Table C-35.
Comparison of IC50 values (nm) for neutralizing the binding of hIL-6 with sIL-6R plasma macaques and humans
ConnectionIC50in the plasma of manIC50plasma macaquesAttitude (human/macaque)
Control IgG0,2580,1661,55
IL6R20A110,198 0,1171,69
IL6R3040,2290,1371,67
IL6R3050,1370,07911,73
IL6R3060,4120,3211,29

Table C-36.
Dose groups for analysis of in vivo PK/PD IL6R304 and IL6R305
GroupThe subject of tester depositsDose of nantel (mg/kg century. so, in/in)The dose of IL-6 (m kg/kg C. T., p/K)Number and sex of animalsNo. of animals
6IL6R304 0,45 mg/kg, once a day on protag. 7 days starting 24 hours after administration of Manotel2 m, 1 W11, 12, 13
721 m, 2 W14, 15, 16
8101 m, 1 W17, 18
9IL6R3050,41 m, 2 W19, 20, 21
1022 m, 1 W22, 23, 24
11101 m, 1 W25, 26
12A negative. control02 m, 1 W 27, 28, 29
13Position. control5 mg/kg control IgG1 m, 2 W30, 31, 32
Table C-37.
The time of sampling for analysis FC
GroupThe time of sampling in animalsQty sample
Group 6-13day 0: before the introduction of nantel and after 5 min, 30 min, 3 h and 8 h after injection of Manotel352
days 1, 2, 3, 4, 5, 6, 7; before the introduction of IL-6
days 8, 14, 21 and 29
Table C-38.
The main parameters of the FC IL6R304 after a single on/in the introduction of a bolus dose of 0.4 mg/kg in long-tailed macaques
IL6R304: 0.4 mg/kg
Unit11m12m13fAverageCV %
Vssml/kg45,144,638,842,88
CLml/day/kg24,828,2a 21.524,814
MRTday1,811,581,801,738
t1/2λz1day1,921,541,72 1,7311
λz1 bottomday1111,000
λz1 topday4454,3313
R2t1/2λz1is 0.9980,9970,999is 0.9980
t1/2λz2day0,5660,5040,5210,5306
λz2 bottomday 5454,6712
λz2 topday767to 6.679
R2t1/2λz20,9671,000,9380,9683
AUClastday×µg/ml16,014,118,616,214
AUCextrap%0,3720,3650,3250,354 7
AUCinfday×µg/ml16,114,218,616,314
AUCinf/Dday×kg/ml0,040being 0.0360,0470,04114

Table C-39.
The main parameters of the FC IL6R304 after a single on/in the introduction of a bolus dose of 2 mg/kg in long-tailed macaques. The final parameters for some of the animals counted only two points (R2=1 by default)
IL6R304: 2 mg/kg
Unit14m15f16fAverage CV %
Vssml/kg56,055,9to 49.353,77
CLml/day/kg9,9911,0the 10.110,46
MRTdaythe ceiling of 5.60of 5.064,865,177
t1/2λz1day5,794,344,875,0015
λz1 bottomday2 2220
λz1 topday141414140
R2t1/2λz10,9940,9790,9910,9881
t1/2λz12day1,511,521,30the 1.449
λz2 bottomday141414140
λz2 in which RNI day212121210
R2t1/2λz21,001,001,001,000
t1/2λz3day5,615,95-5,784
λz3 bottomday2121-210
λz3 topday2929 -290
R2t1/2λz31,001,001,001,000
AUClastday×µg/ml2001811971925
AUCextrap%0,2950,2470,0900,21151
AUCinfday×µg/ml2001811971935
AUCinf/D day×kg/ml0,1000,0910,0990,0965
Table C-40.
The main parameters of the FC IL6R304 after a single on/in the introduction of a bolus dose of 10 mg/kg in long-tailed macaques
IL6R304: 0.4 mg/kg
Unit17m18fAverageCV%
Vssml/kg76,5and 88.882,710
CLml/day/kg7,6610,359,0021
MRTday10,08,579,2911
t1/2λz1day7,15between 6.086,6111
λz1 bottomday1110
λz1 topday2929290
R2t1/2λz10,9900,990 0,9900
AUClastDeng×kg/ml1230932108119
AUCextrap%5,793,50with 4.6435
AUCinfday×µg/ml1306966113621
AUCinf/Dday×kg/ml0,1310,0970,11421

Table C-41.
The main parameters of the FC IL6R305 after a single on/in the introduction of a bolus dose of 0.4 mg/kg in long-tailed macaques. The final parameters for some of the animals counted only two points (R2=1 by default)
IL6R305: 0.4 mg/kg
Unit19m20f21fAverageCV %
Vssml/kg59,272,563,965,210
CLml/day/kg33,538,036,035,86
MRT day1,771,911,771,824
t1/2λz1day1,791,251,891,6421
λz1 bottomday11110
λz1 topday5544,6712
R2t1/2λz1 0,9970,9810,9970,9921
t1/2λz2day0,446-0,4950,4707
λz2 bottomday5-550
λz2 topday6-660
R2t1/2λz21,00 -1,001,000
AUClastdengming/ml11,849,8411,0210,909
AUCextrap%0,765to 6.580,8552,73122
AUCinfdengming/ml11,910,511,111,26
AUCinf/Ddanggg/ml0,0300,026 0,0280,0286
Table C-42.
The main parameters of the FC IL6R305 after a single on/in the introduction of a bolus dose of 2 mg/kg in long-tailed macaques. The final parameters for some of the animals counted only two points (R=1 by default)
IL6R305: 2 mg/kg
Unit22m23m24fAverageCV%
Vssml/kg27,528,030,428,65
CLml/day/kgof 5.81and 5.30of 6.68 to 5.9312
MRTday4,735,284,554,858
t1/2λz1day4.26 deaths4,564,044,296
λz1 bottomday22220
λz1 topday141414140
R2t1/2λz1 0,9850,9540,9860,9752
t1/2λz2day1,332,341,161,6140
λz2 bottomday141414140
λz2 topday212121210
R2t1/2λz211 110
AUClastday×µg/ml34437429933911
AUCextrap%0,0890,7910,0410,307137
AUCinfday×µg/ml34437729934011
AUCinf/Dday×kg/ml0,1720,1890,1500,170
Table C-43.
The main parameters of the FC IL6R305 after a single on/in the introduction of a bolus dose of 10 mg/kg in long-tailed macaques. The final parameters for some of the animals counted only two points (R2=1 by default)
IL6R305: 10 mg/kg
Unit25m26fAverageCV %
Vssml/kgto 38.359,248,730
CLml/day/kg6,918,607,7615
MRT day5,54to 6.886,2115
t1/2λz1day5,639,107,3733
λz1 bottomday2220
λz1 topday1414140
R2t1/2λz10,9410,9680,9552
t1/2λz2day1,261,161,216
λz2 bottomday2121210
λz2 topday2929290
R2t1/2λz21,001,001,000
AUClastday×µg/ml144711621305 15
AUCextrap%0,0080,0090,00811
AUCinfday×µg/ml14471162130515
AUCinf/Dday×kg/ml0,1450,1160,13015
Table C-44.
Summary the appearance of antibodies against IL6R304 and IL6R305 to full nanocell
IL6R304 Macaque ADA-put.*IL6R305 Macaque ADA-put.*
0.4 mg/kg11m >7 days0.4 mg/kg19m>7 days
12m>7 days20f>7 days
13f>7 days21f>7 days
2 mg/kg14m>7 days2 mg/kg22m>14 days
15f>14 days23mnot found
16funknown due to the high level before the introduction of24f>14 days
10 mg/kg17m>14 days 10 mg/kg25m> 14 days
18f>14 days26f> 14 days
* >7 days: TD 7 negative., ≥TD 14 will believe.; >14 days: TD 14 the negative., ≥TD 21 will believe.
Table C-45.
Pharmacodynamic parameters of IL6R304 in long-tailed macaques
Score% CV
Kin(ng/ml/h)2,407,37
R0(ng/ml)21,45,23
Imax(%)0,9700,34
IC50(ág/ml)0,14615,8
IC50(nm)5,23
t1,4315,3

Table C-47.
Pharmacokinetic parameters of IL6R304 in long-tailed macaques
Score%CV
Vc(ml/kg)45,65,23
Vin(ml/kg)14,815,2
Vs(ml/kg)24,916,1
Vdss(ml/kg)85,3
CLNON-IL6R(ml/h/kg)0,2373,39
CLd(ml/h/kg)0,047529,9
CLs(ml/h/kg) 2,8640,4
Vmax(µg/h/kg)is 1.97111,2
Km(ág/ml)0,71430,4
CLIL6R(ml/h/kg)was 2.76

1. Single variable domain that is directed against IL-6R, which essentially consists of 4 frame sections, from FR1 to FR4, respectively, and 3 complementarity determining plots from CDR1 to CDR3, respectively, in which:
-CDR1 is chosen from:
a) SEQ ID NO: 80;
and
-CDR2 selected from the group consisting of:
b) SEQ ID NO's: 84, 89 and 91; or
(C) plot of amino acid residues that has no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO's: 84, 89 and 91, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without the 2 or 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance;
and
-CDR3 selected from the group consisting of:
d) SEQ ID NO's: 93-94; or
e) plot of amino acid residues, Meuse is about no more than 1 amino acid difference from one of SEQ ID NO's: 93-94, provided that the amino acid sequence containing the site of amino acid residues binds IL-6R with about the same or greater affinity compared to the amino acid sequence containing the site of amino acid residues without 1 amino acid differences, and the affinity is measured by means of surface plasmon resonance.

2. Single variable domain under item 1, which contains SEQ ID NO: 80, SEQ ID NO: 84 and SEQ ID NO: 93.

3. Single variable domain under item 1 or 2, which is selected from the group consisting of:
a) SEQ ID NO's: 65-69;
b) sequences having no more than 2, preferably no more than 1 amino acid differences in the CDR2 and/or CDR3 from SEQ ID NO''s: 65-69, provided that the amino acid sequence of no more than 2, preferably no more than 1 amino acid difference in the CDR2 and/or CDR3, binds to IL-6R with about the same or greater affinity compared to the binding of one of SEQ ID NO''s: 65-69, and the affinity is measured by means of surface plasmon resonance;
c) sequences having no more than 2, preferably no more than 1 amino acid difference from one of SEQ ID NO''s: 65-69, provided that the amino acid sequence of no more than 2, preferably no more than 1 amino acid difference with one of SEQ ID NO's: 65-69 binds to IL-6R with about the same or is more affinity compared to the binding of one of SEQ ID NO''s: 65-69, moreover, the affinity is measured by means of surface plasmon resonance.

4. The polypeptide that is directed against IL-6R, which essentially consists of two or more single variable domains according to any one of paragraphs.1-3.

5. The polypeptide under item 4, in which a single variable domains are connected through one or more linker sequences.

6. Design directed against IL-6R, containing one or more single variable domains according to any one of paragraphs.1-3, and optionally containing one or more other groups or units of binding, which gives the polypeptide an increased half-life and in which one or more other groups or units of bonding selected from the group consisting of groups to tahilramani, serum albumin, Fc fragments, or a domain antibody, single domain antibody, “dAb”, or nantel the ability to communicate with serum albumin or serum immunoglobulin.

7. Design by p. 6, which contains one or more other binding units that can communicate with serum albumin human.

8. Design by p. 6, which contains one or more other binding units that can communicate with IgG.

9. Design by p. 6, which is selected from the following polypeptides:
SEQ ID NO's: 70-72.

10. Design by p. 6, which is contains or essentially consists of SEQ ID NO: 70 or SEQ ID NO: 71.

11. The polypeptide according to any one of paragraphs.4 and 5, which specifically binds to hIL-6R with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less.

12. Structure according to any one of paragraphs.6-10, which specifically binds to hIL-6R with a dissociation constant (KDfrom 1 nm to 1 PM or less, preferably 500 PM to 1 PM or less, more preferably 100 PM to 1 PM or less and even more preferably from 50 PM to 1 PM or less.

13. A method of obtaining a structure according to any one of paragraphs.6-10, which involves attaching one or more single variable domains according to any one of paragraphs.1-3 to one or more groups, or units of bonding selected from the group consisting of groups to tahilramani, serum albumin, Fc fragments, or a domain antibody, single domain antibody, “dAb”, or nantel the ability to communicate with serum albumin or serum immunoglobulin.

14. The method according to p. 13, where serum albumin is a serum albumin of a person.

15. The method according to p. 13, where serum immunoglobulin is an IgG.

16. Nucleic acid encoding a single variable domain according to any one of paragraphs.1-3.

17. Genetic design is required for the expression and/or production of single variable domain according to any one of paragraphs.1-3, containing at least one nucleic acid according to p. 16, functionally connected to one or more regulatory elements and, optionally, one or more further elements of genetic constructs, such as selection marker gene, a reporter, a leader sequence or factors integration.

18. Nucleic acid encoding a polypeptide according to any one of paragraphs.4 and 5, or design on PP.6-10 or 12, which can be obtained by expression of the coding its nucleic acids.

19. Genetic construction for the expression and/or production of the polypeptide according to any one of paragraphs.4 and 5, or constructs according to any one of paragraphs.6-10 or 12, which can be obtained by expression of the coding its nucleic acid containing at least one nucleic acid on p. 18, functionally connected to one or more regulatory elements and, optionally, one or more further elements of genetic constructs, such as selection marker gene, a reporter, a leader sequence or factors integration.

20. A host cell that expresses, or in appropriate circumstances, able to Express the single variable domain according to any one of paragraphs.1-3, the polypeptide according to any one of paragraphs.4 and 5 or construct according to any one of paragraphs.6-10 or 12, which can be perceived by whom and when the expression of the coding its nucleic acid, and which contains a nucleic acid or a genetic construct according to any one of paragraphs.16-19.

21. The body is the master, which is a rabbit, a cow, a goat, or a sheep, which expresses or in appropriate circumstances, able to Express the single variable domain according to any one of paragraphs.1-3, the polypeptide according to any one of paragraphs.4 and 5 or construct according to any one of paragraphs.6-10 or 12, which can be obtained by expression of the coding its nucleic acid, and which contains a nucleic acid or a genetic construct according to any one of paragraphs.16-19.

22. The body is the master, which is a plant that expresses or in appropriate circumstances, able to Express the single variable domain according to any one of paragraphs.1-3, the polypeptide according to any one of paragraphs.4 and 5 or construct according to any one of paragraphs. 6-10 or 12, which can be obtained by expression of the coding its nucleic acid, and which contains a nucleic acid or a genetic construct according to any one of paragraphs.16-19.

23. The method of obtaining single variable domain according to any one of paragraphs.1-3, the polypeptide according to any one of paragraphs.4 and 5 or constructs according to any one of paragraphs.6-10 or 12, which can be obtained by expression of the coding its nucleic acid, the method at least includes the steps:
a) ekspressirovali in a suitable cell host or organism-the owner or back emochnoy system of expression of nucleic acids or genetic constructs according to any one of paragraphs.16-19;
or
cultivation and/or content of the host organism according to any one of paragraphs.21 and 22 or the host cell under item 20 in such conditions, in which the host organism or a host cell Express and/or produce at least one single variable domain according to any one of paragraphs.1-3, the polypeptide according to any one of paragraphs.4 and 5 or construct according to any one of paragraphs.6-10 or 12, which can be obtained by expression of the coding its nucleic acid;
optional with the subsequent:
b) isolating and, optionally, additional cleaning obtained with this single variable domain according to any one of paragraphs.1-3, the polypeptide according to any one of paragraphs.4 and 5 or constructs according to any one of paragraphs.6-10 or 12, which can be obtained by expression of the coding its nucleic acids.

24. Composition type of pharmaceutical composition for blocking the interaction of IL-6/IL-6R containing a pharmaceutically effective amount of a single variable domain according to any one of paragraphs.1-3, the polypeptide according to any one of paragraphs.4 and 5, the structure according to any one of paragraphs.6-10 or 12, or a nucleic acid or genetic constructs according to any one of paragraphs.16-19.

25. The method of prevention and/or treatment of at least one of the diseases or disorders associated with IL-6, IL-6R, IL-6/IL-6R and/or signalling pathways are involved in IL-6, IL-6R or IL-6/IL-6R and/or biological what functions and reactions, involving IL-6, IL-6R or IL-6/IL-6R, and the method includes the introduction to the needy in the subject of pharmaceutically active amount of at least one single variable domain according to any one of paragraphs.1-3, the polypeptide according to any one of paragraphs.4 and 5, the structure according to any one of paragraphs.6-10 or 12 or compositions by p. 24.



 

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16 cl, 8 dwg, 6 tbl, 11 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents a combined recombinant protein of the formula: S-L-R, including SR10, SR13, SR15, SdR10, SdR13 or SdR15, which specifically recognises melanoma cells, where S - streptavidin monomer, L - linker having amino-acid sequence Ser-Arg-Asp-Asp-Asp-Asp-Lys containing a restriction site with enteropeptidase and marked as "d", or amino-acid sequence Ser-Arg-Ala-Gly-Ala,R - melanoma-addressing oligopeptide representing R10 having amino-acid sequence Asp-Gly-Ala-Arg-Tyr-Cys-Arg-Gly-Asp-Cys-Phe-Asp-Gly, or R13 having amino-acid sequence Leu-Ser-Gly-Cys-Arg-Gly-Asp-Cys-Phe-Glu-Glu, or R15 having amino-acid sequence Asp-Gly-Phe-Pro-Gly-Cys-Arg-Gly-Asp-Cys-Ser-Gln-Glu. This invention also describes recombinant plasmid DNAs pSR and pSdR for expression of the specified combined proteins, bacterial strains Escherichia coli MG1655/pSR and MG1655/pSdR, producers of the specified combined proteins and a producing method of melanoma-addressing oligopeptide R from combined recombinant proteins SdR10, SdR13 or SdR15.

EFFECT: invention allows producing combined proteins that provide selective and effective binding to receptors on the surface of melanoma cells and can be used in diagnostics and therapy of cancer of a human being.

9 cl, 7 dwg, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented group of inventions refers to biotechnology, and concerns a DLK1-Fc fused protein and using it for the metastases inhibition, a polynucleotide coding such a protein, an expression vector containing the polynucleotide, a host cell producing the above fused protein, a method for producing the fused protein by culturing the above host cell, a composition containing the above fused protein, and a method for the metastases inhibition. The characterised fused protein contains a DLK1 extracellular soluble domain consisting of the amino acid sequence SEQ ID NO:4 and Fc domain of a human antibody.

EFFECT: group of inventions can be used for preparing a therapeutic agent for reduction of cancer cell migration and the metastases inhibition.

11 cl, 36 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and medicine, and concerns an vaccine against influenza caused by known viral strains of influenza A and B, as well as potential reassortants. The presented polyvalent influenza vaccine is based on a hybrid protein containing N1, N3 and N5 protein fragments of influenza A virus, a haemagglutinin fragment of influenza B virus and FliC1 and FliC2 flagellin components (SEQ ID NO:1) bridged flexibly. The protein-coding nucleotide sequence (SEQ ID NO:2) is optimised for the high expression in Escherichia coli cells.

EFFECT: using the characterised vaccine enables providing the general protection against influenza.

3 cl, 4 dwg, 1 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biochemistry, in particular to a polypeptide, which is capable, when fused with a biologically active polypeptide, of increasing its half-life time in serum, as well as to its application for increasing the half-life time in the circulation of erythropoietin, the stimulation factor of granulocyte colony, p40 and a receptor of the tumour necrosis factor. Disclosed are a molecule of nucleic acid, coding the said polypeptide, containing it expression vector, as well as a method of obtaining the said polypeptide by an introduction of a nucleic acid molecule, which codes it, into a mammalian host cell, its growth, as well as collection of the expressed polypeptide.

EFFECT: invention makes it possible to increase the time of half-life of the biologically active polypeptide in serum in an efficient way.

15 cl, 9 dwg, 1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, more specifically to modified von Willebrand factor (VWF), and can be used in medicine. A recombinant method is used to preparing modified VWF fused in C-terminal of its primary translation product with N-terminal of albumin by the linker SSGGSGGSGGSGGSGGSGGSGGSGGSGGSGS. The prepared modified VWF is used as a part of the pharmaceutical composition for treating or preventing coagulation failure.

EFFECT: invention enables preparing the modified VWF which maintains its ability to N-terminal dimerisation and C-terminal multimerisation with a prolonged half-period of functional blood plasma occurrence as compared to the half-period of functional VWF occurrence.

17 cl, 5 dwg, 4 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: inventions refer to biotechnology and concern a fused protein for the specific inhibition of blood coagulation, an expression plasmid DNA coding this fused protein, a bacterium of the genus Escherichia transformed by this DNA, and to a method for preparing the fused protein. The presented fused protein contains thioredoxin I E.coli and infestine-4 and is characterised by the sequence SEQ ID NO:2. The plasmid DNA contains the sequence SEQ ID NO:1 coding the presented fused protein and controlled by a promoter functioning in a bacterial cell. The method for preparing the above fused protein involves culturing the above bacteria in a nutrient medium, breaking the bacterial cells and purifying the above fused protein with using a metal chelate chromatography and an anion-exchange chromatography.

EFFECT: characterised solutions enables preparing the protein providing the above specificity to be used in blocking the contact activation of blood coagulation by inhibition of the XIIa factor and the absence of inhibition of the Xa factor.

4 cl, 10 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering and can be used for methane-producing cell permeability control. What is prepared is a polypeptide able to permeate into a methane-producing cell and to increase its permeability, characterised by an amino acid sequence SEQ ID NO:117, 118 or 119 or being at least 90% identical to the above sequence, or at least 15 sequential amino acids of the above sequence. What is also prepared is a polynucleotide coding the above polypeptide cloning and expressing vectors used for producing host cells producing the polypeptide or used for the vector replication. The polypeptide can contain a fluorescent tag on an N-terminal amino acid residue.

EFFECT: invention enables providing higher methane-producing cell permeability.

18 cl, 35 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to biotechnology and represents a polypeptide construction for treatment, prevention and relief of disorders, associated with an adhesion of platelets and platelet-mediated aggregation or its dysfunction, which includes one or more single-domain antibodies, aimed against the von Willebrand factor (vWF), and one or more single-domain antibodies aimed against serum albumen (SA). The invention also relates to nucleic acid, coding such polypeptide construction, to compositions, containing the said construction, and to its application for obtaining medications for prevention, treatment and relief of the said disorders.

EFFECT: claimed invention makes it possible to extend an assortment of medications for treatment, prevention and relief of disorders, associated with the platelet adhesion and platelet-associated aggregation or its dysfunction.

15 cl, 30 dwg, 32 tbl, 69 ex

FIELD: medicine.

SUBSTANCE: invention refers to biochemistry, particularly to artificial immunogenic proteins having the properties of melanoma antigens. What is declared is an artificial gene coding MEL-TCI-A0201 polyepitope immunogenic protein containing multiple cytotoxic restricted HLA-A*0201 and T-helper epitopes of NY-ESO-1, MART1, MAGE-A1, MAGE-A3, MAGE-A11, MAGE-C1 melanoma antigens, having a sequence of 1,535 base pairs presented in Fig. 3. There are also declared a recombinant plasmid DNA containing the above artificial gene, and MEL-TCI-A0201 immunogenic protein with the properties of the melanoma antigens.

EFFECT: invention enables providing higher immunogenicity of the artificial polyepitope T-cell immunogen inducing a higher level of cytotoxic T-lymphocyte response.

3 cl, 11 dwg, 3 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is presented is an antibody representing a neutralising VEGFR-2/KDR antibody with its hypervariable regions being identical to the hypervariable regions of TTAC 0001 of VEGFR-2/KDR antibody fused with a binding domain of angiopoietin 2 which is Tie-2 ligand for treating cancer by angiogenesis inhibition. A DNA coding the above antibody, an expression vector containing the above DNA, and a CHO host cell transformed by the above vector for preparing the antibody are also described. What is also presented is a method for preparing the antibody involving: host cell incubation, and the antibody recovery from a culture fluid of CHO cell. What is described is a pharmaceutical composition for treating an angiogenesis-related disease, containing an effective amount of the above antibody and at least one pharmaceutically acceptable carrier.

EFFECT: invention enables preparing the VEGFR-2/KDR antibody fused with the binding domain of angiopoietin 2 which may be used for effective treatment of a disease related to excessive angiogenesis.

13 cl, 10 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is described is a recovered human antibody or its antigen-binding fragment. The antibody binds to human interleukin-4 alpha-receptor (hlL-4R). There are also described a nucleic acid molecule coding this antibody, an expression vector, a host cell, a method for producing such antibody and a therapeutic composition containing this antibody.

EFFECT: presented group of inventions can be used in medicine for treating asthma and atopic dermatitis.

15 cl, 3 tbl, 3 ex

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