Fc-versions with altered binding with fcrn

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of immunology. Claimed is a version of Fc polypeptide of human IgG with substitutions 2591 and 308F, where numeration of positions is given in accordance with EU Kabat index. Described is a version of the said polypeptide, including one or several substitutions of the following: 428L, 434S, 307Q, 319L, 250I in addition to the said ones. Disclosed are: a nucleic acid, coding the said versions, a host cell for production of the said versions of polypeptide, which contains the coding nucleic acid, a method of obtaining the said versions of polypeptide, including application of the cell expressing the said polypeptide and containing the nucleic acid, which codes the said polypeptide.

EFFECT: application of the invention provides polypeptide, demonstrating higher affinity with human FcRn, which can be applied in therapy of different diseases.

11 cl, 32 dwg, 14 ex

 

The present application claims priority based on 35 U.S.. 119 (e) in respect of USSN 60/951536, filed July 24, 2007, and is a partial continuation of USSN 11/436266, filed may 17, 2006, which is a partial continuation of USSN 11/274065, filed November 14, 2005, both of which claimed priority based on 35 U.S.. 119 (e) in respect of USSN 60/627763, filed November 12, 2004; USSN 60/642886, filed January 11, 2005; USSN 60/649508, filed February 2, 2005; USSN 60/662468 filed March 15, 2005; USSN 60/669311 filed April 6, 2005; USSN 60/681607, filed may 16, 2005; USSN 60/690200, filed June 13, 2005; USSN 60/696609 filed July 5, 2005; USSN 60/703018, filed July 27, 2005; and USSN 60/726453, filed October 12, 2005, the full content of which is incorporated into this description by reference.

The technical field to which the invention relates.

The present invention relates to optimized variants of immunoglobulin IgG, engineering methods for their preparation and their application, particularly for therapeutic purposes.

The level of technology

Antibodies are immunologically proteins that bind with a specific antigen. Most mammals, including humans and mice, antibodies are formed of paired heavy and light polypeptide chains. Each circuit consists of individual domains of immunoglobulin (Ig) and hence, these proteins use the General term "immunoglobulin is". Each circuit consists of two separate areas, called "variable" and "constant" regions. Variable region light and heavy chains show a significant difference sequences of antibodies and are responsible for binding to the target antigen. Constant region show less difference sequences and are responsible for binding with a number of natural proteins to stimulate important biochemical reactions. In humans there are five different classes of antibodies, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (which includes subclasses IgG1, IgG2, IgG3 and IgG4 and IgM. The hallmark of these classes of antibodies are their constant region, despite the fact that subtle differences may exist in the V-region. Figure 1 shows the IgG1 antibody used in the present description as an example to describe the General structural features of immunoglobulins. IgG antibodies are protein tetramer, consisting of two heavy chains and two light chains. Heavy chain of IgG is composed of four immunoglobulin domains linked from N - to C-end in the order VH-CH1-CH2-CH3, which refers to the variable domain of the heavy chain constant domain 1 heavy chain constant domain 2 heavy chain and the constant domain 3 heavy chains, respectively (also oboznachaet the traveler as VH-Cγ1-γ2-γ3, that refers to the variable domain of the heavy chain constant domain gamma 1, constant gamma 2 domain and a constant domain gamma 3, respectively). Light chain IgG consists of two immunoglobulin domains linked from N - to C-end in the order VL-CL, which refers to the variable domain light chain and the constant domain of the light chain, respectively.

Variable region of the antibody contains antigennegative determinants of molecules and thus determines the specificity of the antibody against the target antigen. Variable region is so named because it differs most from its sequences from other antibodies within the same class. A large part of the variability of the sequences found in hypervariable sites (CDR). There are a total of 6 CDRs in the heavy and light chain has three domains, denoted by VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3. Variable area outside of the CDRs is called frame region (FR). Although it's not as diverse as the CDR, the variability of the sequence actually occurs in the field of FR between different antibodies. In General, this characteristic structure of antibodies provides a stable frame (area FR), on the basis of which a significant difference in binding antigens (CDR) may be detected by the immune system when the purchase specificity for a wide range of antigens. There are a number of high-resolution structures for a multitude of fragments of variable regions of various organisms, some of which are not bound to the antigen, and some of which are in complex with antigen. The sequence and structural characteristics of the variable regions of antibodies are well characterized (Morea et al., 1997, Biophys Chem 68:9-16; Morea et al., 2000, Methods 20:267-279, fully incorporated into the present description by reference), and conservative characteristics of antibodies has facilitated the development of many technologies generate antibodies (Maynard et al., 2000, Annu Rev Biomed Eng 2:339-376, fully incorporated into the present description by reference). For example, you can transplant the CDRs of one of the antibodies, e.g. antibodies mouse in the frame region of another antibody, e.g., human antibodies. This method is called in the art "humanization" and allows you to get a less immunogenic drugs based on antibodies from antibodies inhuman origin. Fragments containing the variable region can exist in the absence of other regions of antibodies, including, for example, antigennegative fragment (Fab), including VH-Cγ1 and VH-CL variable fragment (Fv)comprising VH and VL, single-chain variable fragment (scFv)comprising VH and VL connected in the same circuit, as well as many other parts of areabelow area (Little et al., 2000, Immunol Today 21:364-370, fully incorporated into the present description by reference).

The Fc region of the antibody interacts with a number of receptors and ligands Fc, passing a number of important functionality, called "effector functions". For IgG Fc region, as shown in figures 1 and 2, contain Ig domains γ2 and γ3 and the N-terminal hinge leading into γ2. An important family of Fc receptors for IgG class is a gamma Fc receptors (FcγR). These receptors mediate the interaction between antibodies and the cellular "link" of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290, both fully incorporated into the present description by reference). In humans this family of proteins includes FcγRI (CD64), including isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotype H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotype V158 and F158) and FcγRIIIb (including allotype FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, fully incorporated into the present description by reference). These receptors typically contain the extracellular domain, which mediates binding to Fc, a transmembrane domain, and intracellular domain, which may mediate any signal phenomenon inside the cell. These receptors are expressed in many immune cells, including onecity, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, b cells, large granular lymphocytes, Langerhans cells, natural killer (EC) cells and γγ T cells. In the formation of complex Fc/FcγR is recruiting these effector cells to sites of bound antigen, typically resulting in the phenomena of signal transmission within cells and important subsequent immune responses such as release of inflammatory mediators, activation of b-cells, endocytosis, phagocytosis, and cytotoxic attack. The ability to mediate cytotoxic and phagocytic effector function is a possible mechanism by which antibodies destroy the target cells. Mediated by the cell reaction in which nonspecific cytotoxic cells that Express FcγRs, recognize bound antibody on the target cell, and then cause the lysis of the specified target cells, called "antibody-dependent cretaceouspaleogene cytotoxicity (ADCC)" (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290, all fully incorporated into the present description by reference). Mediated by the cell reaction in which nonspecific cytotoxic cells that Express FcγRs, recognize bound antibody on the target cell, and then calls the t phagocytosis specified target cells, called "antibody-dependent cell-mediated phagocytosis (ADCP)". The number of structures was generated using the extracellular domains of human FcγRs, including FcγRIIa (access code in pdb 1H9V, fully incorporated into the present description by reference) (Sondermann et al., 2001, J Mol Biol 309:737-749, fully incorporated into the present description by reference) (the access code in pdb 1FCG, fully incorporated into the present description by reference) (Maxwell et al., 1999, Nat Struct Biol 6:437-442, fully incorporated into the present description by reference), FcγRIIb (access code in pdb 2FCB, fully incorporated into the present description by reference) (Sondermann et al., 1999, Embo J 18:1095-1103, fully incorporated into the present description by reference); and FcγRIIIb (access code in pdb 1E4J, fully incorporated into the present description by reference) (Sondermann et al., 2000, Nature 406:267-273, fully incorporated into the present description by reference). All FcγRs bind the same region on Fc, at the N-terminal region of domain γ2 and the preceding hinge, shown in figure 1. This interaction is structurally well characterized (Sondermann et al., 2001, J Mol Biol 309:737-749, fully incorporated into the present description by reference) and some patterns Fc person associated with the extracellular domain of human FcγRIIIb have been installed (access code in pdb 1E4K, fully incorporated in the present description the s by reference) (Sondermann et al., 2000, Nature 406:267-273, fully incorporated into the present description by reference) (access codes in pdb 1IIS and 1IIX, fully incorporated into the present description by reference) (Radaev et al., 2001, J Biol Chem 276:16469-16477, fully incorporated into the present description by reference), as well as the structure of the complex Fc/FcεRIα human IgE (access code in pdb 1F6A, fully incorporated into the present description by reference) (Garman et al., 2000, Nature 406:259-266, fully incorporated into the present description by reference). The reaction effector functions can be modified through the options in the field Fc (Lazar et al. 2006 Proc. Nat. Acad. Sci USA. 103(111):4005-4010, fully incorporated into the present description by reference).

The different IgG subclasses have different affinity values to FcγRs, with IgG1 and IgG3 typically significantly better contact with the receptor compared to IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65, fully incorporated into the present description by reference). All FcγRs bind the same region of the IgG Fc, but with different values of affinity: Kd with high affinity receptor FcγRI for IgG1 equal to 10-8M-1, whereas the low affinity receptors FcγRII and FcγRIII generally bound at the 10-6and 10-5respectively. The extracellular domains FcγRIIIa and FcγRIIIb are 96% identical; however, FcγRIIIb not contain what it intracellular signaling domain. In addition, while FcγRI, FcγRIIa/c, and FcγRIIIa are positive regulators of activation initiated by the immune complex, characterized by the presence of the intracellular domain, which contains immunoreceptor the tyrosine activating motif (ITAM), FcγRIIb contains immunoreceptor the tyrosine inhibitory motif (ITIM)and is therefore inhibitory. Thus, the first receptors are referred to as "activating receptor", and FcγRIIb is called "inhibiting receptor". These receptors also differ in the profiles and levels of expression in different immune cells. Another difficulty is the presence of a number of FcγR polymorphisms in the human proteome. Specific important polymorphism with clinical significance is a V158/F158 FcγRIIIa. IgG1 person associated with a higher affinity with the V158 allotype than F158 allotype. It was shown that this difference in affinity and probably its effect on ADCC and/or ADCP is an important factor in the effectiveness of the antibodies anti-SW, rituximab (Rituxan (Rituxan®), BiogenIdec). Patients with V158 allotype respond positively to treatment with rituximab; however, patients with F158 allotype, with lower affinity, less responsive (Cartron et al., 2002, Blood 99:754-758, fully incorporated into the present description by from ylki). Approximately 10-20% of individuals are homozygous V158/V158, 45% are heterozygous V158/F158 and 35-45% of individuals are homozygous F158/F158 (Lehrnbecher et al., 1999, Blood 94:4220-4232; Cartron et al., 2002, Blood 99:754-758, all fully incorporated into the present description by reference). Thus, 80-90% of people have low immune response, i.e. they have at least one allele F158 FcγRIIIa.

Overlapping but separate plot Fc, shown in figure 1, serves as a boundary for protein complement C1q. Like the binding of Fc/FcγR mediates ADCC, linking Fc/C1q mediates complementability cytotoxicity (CDC). C1q forms a complex with the serine proteases C1r and C1s to form complex C1. C1q can bind with six antibodies, despite the fact that linking the two IgG is sufficient to activate a cascade of reactions of complement. Like the Fc interaction with FcγR, different IgG subclasses have different affinity to C1q, with IgG1 and IgG3 typically significantly better contact with FcγRs than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65, fully incorporated into the present description by reference).

In IgG plot between Fc domains Cg2 and Cg3 (1) mediates interaction with the neonatal receptor FcRn, the binding of which returns have been subjected to endocytosis of antibody from endosome back to the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et a., 2000, Annu Rev Immunol 18:739-766, both fully incorporated into the present description by reference). This process is associated with impaired kidneys filter due to the large amount of full-length molecules, leads to a suitable time half-life of antibodies in serum in the range from one to three weeks. The binding of Fc to FcRn also plays a key role in the transport of antibodies. The binding site in the field of Fc to FcRn is also an area in which contact of the bacterial proteins a and G. the Strong binding of the protein data, typically used as a method of purification of antibodies by use of affinity chromatography with protein a or protein G during protein purification. Thus, the accuracy of this site in the Fc region is important both for clinical properties of antibodies and their treatment. The structure of the complex Fc/FcRn rats (Burmeister et al., 1994, Nature, 372:379-383; Martin et al., 2001, Mol Cell 7:867-877, both fully incorporated into the present description by reference) and Fc complexes with proteins a and G (Deisenhofer, 1981, Biochemistry 20:2361-2370; Sauer-Eriksson et al., 1995, Structure 3:265-278; Tashiro et al., 1995, Curr Opin Struct Biol 5:471-481, all fully incorporated into the present description by reference), give an idea of the Fc interaction with these proteins. The FcRn receptor is also responsible for the transfer of IgG in the gut of the newborn and in the lumen of the intestinal epithelium in adults (Ghetie nd Ward, Annu. Rev. Immunol., 2000, 18:739-766; Yoshida et al., Immunity, 2004, 20(6):769-783, both fully incorporated into the present description by reference).

Research domains Fc rats and humans have demonstrated the importance of certain residues of the Fc to bind FcRn. Sequences of rat and human have about 64% identity to the sequences in the Fc regions (residues 237-443 numbered according to Kabat et al.). Cm. 3, 4 and 5 on the subject of alignments Fc rat/man FcRn heavy chain and light chain of FcRn (beta-2-microglobulin). The model was created complex Fc/FcRn person on the basis of existing patterns of complex Fc/FcRn rats (Martin et al., 2001, Mol Cell 7:867-877, fully incorporated into the present description by reference). Sequences of rat and human contain some common residues critical for the binding of FcRn, for example N310 and N (Medesan et al., 1997 J. Immunol. 158(5):221-7; Shields et al., 2001, J. Biol. Chem. 276(9):6591-6604, both fully incorporated into the present description by reference). However, many provisions of the proteins of human and rat contain different amino acids, providing the specified residues in the sequence of a person excellent surroundings and possibly distinct identity than in the sequence of the rat. This variability limits the ability to transfer features from one homologue of another homologue.

In Fc mice random Muta the Oia and selection on the basis of phage display sites T, T and T leads to the triple mutant T252L/T254S/T256F with a 3.5-fold increase in the affinity to FcRn and 1.5-fold longer half-life in serum (Ghetie et al., 1997, Nat. Biotech. 15(7):637-640, fully incorporated into the present description by reference). The breach of an engagement Fc/FcRn by mutations at positions 253, 310 and 435 also leads to a reduction of the time half-life in vivo (Medesan et al J. Immunol. 1997 158(5):2211-7, fully incorporated into the present description by reference).

On the basis of the crystal structures of the complex Fc/FcRn rats were identified remains of Fc that are important for the binding of FcRn (Burmeister et al. Nature. 372:379-383 (1994); Martin et al. Molecular Cell. 7:867-877 (2001), both fully incorporated into the present description by reference). The initial structure of the complex Fc/FcRn was established in 1994 at a resolution of 6 Å (table 2A, Burmeister et al. Nature. 372:379-383 (1994), fully incorporated into the present description by reference). The structure of the higher resolution, established in 2001, Marin et al, presented a more detailed picture of the positions of the side chains (Martin et al. Molecular Cell. 7:867-877 (2001), fully incorporated into the present description by reference). This crystal structure of Fc rats associated with rat FcRn, was installed using the Fc dimer with one monomer containing mutations T252G/I253G/T254G/H310E/H433E/H435E violating wezwanie FcRn, and one monomer containing a monomer Fc wild-type.

Studies have been conducted mutations in human Fcγ some residues important for binding to FcRn, and these studies have demonstrated an increased half-life in serum. In human Fcγ1 Hinton et al. individually subjected to three mutations of the residues in the other 19 normal amino acids. Hinton et al. found that some mutants, the double mutant, increased affinity for binding to FcRn (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216. Hinton et al. Journal of Immunology 2006, 176:346-356, both fully incorporated into the present description by reference). Two mutations had increased half-life in monkeys. Shields et al. was subjected to mutation remains almost exclusively in A1A and investigated their binding to FcRn and FcγR (Shields et al., 2001, J. Biol. Chem., 276(9):6591-6604, fully incorporated into the present description by reference).

Dall'acqua et al. used phage display for selection of mutations Fc, which was associated with enhanced FcRn affinity. (Dall' Acqua et al. 2002, J. Immunol. 169:5171-5180, fully incorporated into the present description by reference). The selected DNA sequence mainly consisted of double and triple mutants. Dall'acqua et al. expressed proteins encoded by many of the selected sequences, and were found some who were associated with FcRn more firmly than if Fc wild-type.

For the introduction of antibodies and merged with Fc proteins as medicines required injection frequency associated with the clearance and time parameters half-life of the protein. Longer half-life in vivo reduces to carry out injections or to reduce the dose, which is obviously an advantage. Despite the fact that you have received in the past mutations in the Fc domain has led to some proteins having high affinity for binding to FcRn and increased half-life in vivo, these mutations have not determined the optimal mutation and increased time half-life in vivo.

One of the hallmarks of the Fc region is a conservative N-linked glycosylation occurring at N297, shown in figure 1. This carbohydrate or oligosaccharide, as it is sometimes called, plays a key structural and functional role for antibodies and is one of the main reasons for which antibodies should be obtained using expression systems mammalian (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Mimura et al., 2001, J Biol Chem 276:45539-45547.; Radaev et al., 2001, J Biol Chem 276:16478-16483; Shields et al., 2001, J Biol Chem 276:6591-6604; Shields et al., 2002, J Biol Chem 277:26733-26740; Simmons et al., 2002, J Immunol Methods 263:133-147; Radaev et al., 2001, J Biol Chem 276:16469-16477; and Krapp et al., 2003, J Mol Biol 325:979-989, all fully incorporated into the present description by reference).

Antibodies which were obtained for therapeutic use. Typical publications related to such therapies include Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200, Cragg et al., 1999, Curr Opin Immunol 11:541-547; Glennie et al., 2000, Immunol Today 21:403-410, McLaughlin et al., 1998, J Clin Oncol 16:2825-2833, and Cobleigh et al., 1999, J din Oncol 17:2639-2648, all of which are fully incorporated into this description by reference. Now for anticancer therapy any small reduction in mortality means success. Some variants of IgG specified in the present description, increase the ability of antibodies to limit further growth of or to destroy at least part of the target cancer cells.

Antitumor activity of antibodies is implemented by increasing their ability to mediate cytotoxic effector functions such as ADCC, ADCP, and CDC. Examples include Clynes et al., 1998, Proc Natl Acad Sci USA 95:652-656; Clynes et al., 2000, Nat Med 6:443-446 and Cartron et al., 2002, Blood 99:754-758, both of which are fully incorporated into the present description by reference.

IgG1 man is a most commonly used for therapeutic purposes antibody and therefore conducted the most research. However, different isotypes of IgG class, including IgG1, IgG2, IgG3 and IgG4, have unique physical, biological and clinical properties. In the art there is the need to create improved variations is tov IgG1, IgG2, IgG3 and IgG4. There is also the need to create these choices for improved binding to FcRn and/or longer half-life in vivo, no comparison with native IgG polypeptides. In addition, there is a need for a combination of variants with improved pharmacokinetic properties, with variants containing modifications to improve efficiency through the modified binding FR. This application meets these and other needs.

Disclosure of inventions

The present invention relates to Fc variants of the original polypeptide containing at least one modification in the Fc region of the specified polypeptide. In various embodiments, the implementation variant polypeptides exhibit altered binding to FcRn compared with the original polypeptide. In some embodiments, the modification may be selected from the group comprising: N, 246S, 247D, T, N, R, 248Q, 248R, 248Y, T, 249W, E, 250I, 250Q, 250V, 251D, 251E, 251H, 251I, 251K, 251M, 251N, 251T, 251V, 251Y, 252Q, 252Y, 253L, 253T, 253V, 254H, 254L, 254N, 254T, 254V, ^254N, 255E, 255F, 255H, 255K, 255S, 255V, 256E, 256V, 257A, 257C, 257D, 257E, 257F, 257G, 257H, 257I, 257K, 257L, 257M, 257N, 257Q, 257R, 257S, 257T, 257V, 257W, 257Y, 258R, 258V, 259I, 279A, 279D, 279C, 279F, 279G, 279H, 279I, 279K, 279M, 279N, 279P, 279Q, 279Q, 279R, 279S, 279T, 279W, 279Y, 280H, ^281A, ^281D, ^281S, ^281T, 282D, 282F, 282H, 282I, 282T, 283F, 283I, 283L, 283Y, 284H, 284K, 284P, 284Q, 284R, 284S, 284Y, 285S, 285V, 286D, 286#, 286L, 287H, 287S, 287V, 287Y, 288H, 288Q, 288S, 305H, 305T, 306F, 306H, 306I, 306N, 306T, 306V, 306Y, 307D, 307P, 307Q, 307S, 307, 307Y, 308C, 308D, 308E, 308F, 308G, 308H, 308I, 308K, 308L, 308M, 308N, 308Q, 308P, 308R, 308S, 308W, 308Y, 309F, 309H, 309N, 309Q, 309V, 309Y, 310K, 310N, 310T, 311A, 311L, 311P, 311T, 311V, 311W, 312H, 313Y, 315E, 315G, 315H, 315Q, 315S, 315T, 317H, 317S, 319F, 319F, 319L, 339P, 340P, 341S, 374H, 374S, 376H, 376L, 378H, 378N, 380A, 380T, 380Y, 382H, 383H, 383K, 383Q, 384E, 384G, 384H, 385A, 385C, 385F, 385H, 385I, 385K, 385L, 385M, 385N, 385P, 385Q, 385S, 385T, 385V, 385W, 385Y, 386E, 386K, 387#, 387A, 387H, 387K, 387Q, 389E, 389H, 426E, 426H, 426L, 426N, 426R, 426V, 426Y, 427I, 428F, 428L, 429D, 429F, 429K, 429N, 429Q, 429S, 429T, 429Y, 430D, 430H, 430K, 430L, 430Q, 430Y, 431G, 431H, 431I, 431P, 431P, 431S, 432F, 432H, 432N, 432S, 432V, 433E, 433P, 433S, 434A, 434F, 434H, 434L, 434M, 434Q, 434S, 434Y, 435N, 436E, 436F, 436L, 436V, 436W, 437E, 437V, 438H and 438K, where the numbering is given according to the EU Index in Kabat et al., and ^ means the insertion after the specified position, and # indicates a deletion of the specified position.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 250Q/252Y, 250Q/256E, 250Q/286D, 250Q/308F, 250Q/308Y, 250Q/311A, 250Q/311V, 250Q/380A, 250Q/428L, 250Q/428F, 250Q/434H, 250Q/434F, 250Q/434Y, 250Q/434A, 250Q/434M, and 250Q/434S.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 250E/252Y, E/E, 250E/286D, 250E/308F, 250E/308Y, E/A, 250E/311V, E/A, 250E/428L, 250E/428F, E/N, 250E/434F, 250E/434Y, E/A, E/434M, and 250E/434S.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 252Y/250Q, 252Y/250E, 252Y/256E, 252Y/286D, 252Y/308F, 252Y/308Y, 252Y/311A, 252Y/311V, 252Y/380A, 252Y/428L, 252Y/428F, 252Y/434H, 252Y/434F, 252Y/434Y, 252Y/434A, 252Y/434M, and 252Y/434S.

In other options, the ante implementation of the Fc variant includes at least two modifications, selected from the group comprising: 256E/250Q, E/E, 256E/252Y, 256E/286D, 256E/308F, 256E/308Y, E/A, 256E/311V, E/A, 256E/428L, 256E/428F, E/N, 256E/434F, 256E/434Y, E/A, E/434M, and 256E/434S.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 286D/250Q, 286D/250E, 286D/252Y, 286D/256E, 286D/308F, 286D/308Y, 286D/311A, 286D/311V, 286D/380A, 286D/428L, 286D/428F, 286D/434H, 286D/434F, 286D/434Y, 286D/434A, 286D/434M, and 286D/434S.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 308F/250Q, 308F/250E, 308F/252Y, 308F/256E, 308F/286D, 308F/311A, 308F/311V, 308F/380A, 308F/428L, 308F/428F, 308F/434H, 308F/434F, 308F/434Y, 308F/434A, 308F/434M, and 308F/434S.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 308Y/250Q, 308Y/250E, 308Y/252Y, 308Y/256E, 308Y/286D, 308Y/311A, 308Y/311V, 308Y/380A, 308Y/428L, 308Y/428F, 308Y/434H, 308Y/434F, 308Y/434Y, 308Y/434A, 308Y/434M, and 308Y/434S.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 311A/250Q, A/E, 311A/252Y, A/E, 311A/286D, 311A/308F, 311A/308Y, A/A, 311A/428L, 311A/428F, A/N, 311A/434F, 311A/434Y, AA, A/434M, and 311A/434S.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 311V/250Q, 311V/250E, 311V/252Y, 311V/256E, 311V/286D, 311V/308F, 311V/308Y, 311V/380A, 311V/428L, 311V/428F, 311V/434H, 311V/434F, 311V/434Y, 311V/434A, 311V/434M, and 311V/434S.

In another embodiment, Fc variant contains m is Nisha least two modifications, selected from the group comprising: 380A/250Q, A/E, 380A/252Y, A/E, 380A/286D, 380A/308F, 380A/308Y, A/A, 380A/311V, 380A/428L, 380A/428F, A/N, 380A/434F, 380A/434Y, A/A, A/434M, and 380A/434S.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 428L/250Q, 428L/250E, 428L/252Y, 428L/256E, 428L/286D, 428L/308F, 428L/308Y, 428L/311A, 428L/311V, 428L/380A, 428L/434H, 428L/434F, 428L/434Y, 428L/434A, 428L/434M, and 428L/434S.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 434H/250Q, N/A, 434H/252Y, N/A, 434H/286D, 434H/308F, 434H/308Y, N/A, 434H/311V, N/A, 434H/428L and 434H/428F.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 434F/250Q, 434F/250E, 434F/252Y, 434F/256E, 434F/286D, 434F/308F, 434F/308Y, 434F/311A, 434F/311V, 434F/380A, 434F/428L and 434F/428F.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 434Y/250Q, 434Y/250E, 434Y/252Y, 434Y/256E, 434Y/286D, 434Y/308F, 434Y/308Y, 434Y/311A, 434Y/311V, 434Y/380A, 434Y/428L and 434Y/428F.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 434A/250Q, A/E, 434A/252Y, A/E, 434A/286D, 434A/308F, 434A/308Y, A/A, 434A/311V, A/A, 434A/428L and 434A/428F.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 434M/250Q, 434M/E, 434M/252Y, 434M/E, 434M/286D, 434M/308F, 44M/308Y, 434M/A, 434M/311V, 434M/A, 434M/428L and 434M/428F.

In another embodiment, the Fc variant includes at least two modifications selected from the group comprising: 434S/250Q, 434S/250E, 434S/252Y, 434S/256E, 434S/286D, 434S/308F, 434S/308Y, 434S/311A, 434S/311V, 434S/380A, 434S/428L and 434S/428F.

In another embodiment, the Fc variant includes at least one modification selected from the group comprising: Y319L, T307Q, V259I, M252Y, V259I/N434S, M428L/N434S, V308F/N434S, M252Y/S254T/T256E/N434S, M252Y/S254T/T256E/V308F, M252Y/S254T/T256E/M428L, V308F/M428L/N434S, V259I/V308F/N434S, T307Q/V308F/N434S, T250I/V308F/N434S, V308F/Y319L/N434S, V259I/V308F/M428L, V259I/T307Q/V308F, T250I/V259I/V308F, V259I/V308F/Y319L, T307Q/V308F/L309Y, T307Q/V308F/Y319L and T250Q/V308F/M428L.

In another embodiment, the Fc variant includes at least one modification selected from the group comprising: Y319L, T307Q, V259I, M252Y, V259I/N434S, M428L/N434S, V308F/N434S, V308F/M428L/N434S, V259I/V308F/N434S, T307Q/V308F/N434S, T250I/V308F/N434S, V308F/Y319L/N434S, V259I/V308F/M428L, V259I/T307Q/V308F, T250I/V259I/V308F, V259I/V308F/Y319L, T307Q/V308F/L309Y, T307Q/V308F/Y319L and T250Q/V308F/M428L.

In another embodiment, the Fc variant includes at least one modification selected from the group comprising: 250I, 250V, 252Q, 252Y, T, 256V, 259I, R, 307Q, 307S, 308F, 309N, 309Y, 311P, 319F, 319L, 428L and 434S.

In another embodiment, the Fc variant includes at least one modification selected from the group comprising: 250V/308F, 250I/308F, 254T/308F, 256V/308F, 259I/308F, 307P/208F, 307Q/308F, 307S/308F, 308F/309Y, 308F/309Y, V308F/311P, 308F/319L, 308F/319F, 308F/428L, 252Q/308F, M252Y/S254T/T256E, 259I/434S, 428L/434S, 308F/434S, 308F/428L/434S, 259I/308F/434S, 307Q/308F/434S, 250I/308F/434S, 308F/319L/434S, 259/308F/428L, 259I/307Q/308F, 250I/259I/308F, 259I/308F/319L, 307Q/308F/309Y, 307Q/308F/319L and 250Q/308F/428L.

In another embodiment, the present invention includes a method of treating a patient in need of such treatment, comprising introducing an effective amount of Fc-variant specified in the present description.

In another embodiment, the present invention includes a method of increasing the time half-life of the antibody or immunoadhesin by modifying the Fc according to the modifications specified in the present description.

Brief description of figures

Figure 1. Structure and function of antibodies. Model shown with full-length IgG1 antibodies person modeled using the humanized Fab patterns on the basis of an access code in pdb 1CE1 (James et al., 1999, J Mol Biol 289:293-301, fully incorporated into the present description by reference) and the structure of the Fc IgG1 person on the basis of an access code in pdb 1DN2 (DeLano et al., 2000, Science 287:1279-1283, fully incorporated into the present description by reference). Flexible hinge connecting areas Fab and Fc, not shown. IgG1 is a glycosilated of heterodimers, consisting of two light chains and two heavy chains. The Ig domains containing the antibody are labeled and contain VLand CLfor the light chain and VH, Shamma (γ1), Shama (γ2) and Shamma (γ3) for the heavy chain. The Fc region is labeled. The binding sites for choosing the corresponding proteins are labeled, including antigennegative center in variable regions and binding to FcγRs, FcRn, C1q, and proteins a and G in the Fc region.

Figure 2. Sequence human IgG used in accordance with the present invention with the EU numbering according to Kabat et al.

Figure 3. Example sequences of IgG human and rodents used in accordance with the present invention with the EU numbering according to Kabat.

Figure 4. An example of the sequences of the heavy chains of human FcRn and rodents used in accordance with the present invention.

Figure 5. Example sequences of beta-2-microglobulin human and rodents used in accordance with the present invention.

6. Model complex Fc/FcRn man, created on the basis of the structures of the rat (Burmeister et al., 1994, Nature, 372:379-383; Martin et al., 2001, Mol Cell 7:867-877, both fully incorporated into the present description by reference). Some residues of histidine is shown in the form of atoms spatial model in chains FcRn (light gray) and the Fc polypeptide (dark gray).

7. An illustration of some concepts used in the generation of variants that contain insertions or deletions.

Fig. Variants according to the present invention.

Fig.9. Variants according to the present invention.

Figure 10. Variants according to the present invention.

11. Chart vector pcDNA3.1 Zeo+, which can be is used in the creation of Fc variants.

Fig. Data competitive binding to FcRn wild-type Fc and Fc variant according to the present invention. On each chart Fc-variants according to the present invention shown as the left curve (red or dark gray), and the antibody anti - HER2 wild type is shown in the form of a right curve (blue or gray).

Fig. The conclusion regarding the abilities of Fc variants to bind FcRn. In columns from left to right shows a modification of the binding of FcRn used immunoglobulin, other modifications, the relative affinity to FcRn according to competitive analyses AlphaScreen™ compared with wild type (median value) and the number of analyses performed. Values of the relative affinity to FcRn greater than 1.0 show increased binding compared to wild type. Data were obtained at pH 6,0 (0,1M sodium phosphate, 25 mm sodium chloride).

Fig. Data binding to FcRn Fc-variants. Fc-variants included in alemtuzumab or antibody anti-HER2. Shows multiple increase in binding compared to the wild type, i.e. values greater than one indicate stronger binding to FcRn, while values less than one indicate decreased binding to FcRn.

Fig. Research results by means of surface plasmon resonance Fc variants with improved binding to FcRn. The histogram shows the fold is Velicina affinity for binding to FcRn each option compared to the Fc domain of the wild type.

Fig. Research by means of surface plasmon resonance antibody wild-type and variants according to the present invention. Shows the traces represent the Association and dissociation of Fc variant antibodies to FcRn at pH of 6.0.

Fig. Analyses of the binding of Fc variants according to the present invention with FcRn. Shows the data analysis of the direct binding measured using the AlphaScreen™ at pH 6,0 (a and b) and pH 7.0 (C).

Fig. Analyses of the binding of Fc variants according to the present invention with FcRn. Shows the unit of measurement of surface plasmon resonance, obtained by binding of Fc variants with surface-bound FcRn.

Fig. Measurement of surface plasmon resonance affinity for binding of Fc variants according to the present invention with human FcRn at pH of 6.0.

Fig. The measurement results of surface plasmon resonance (SPR) in the affinity for binding of Fc variants according to the present invention with FcRn human, macaque, and mouse. Values greater than one indicate an increased binding of Fc variants to FcRn, as determined by fitting curves Poland 1:1 model of binding Langmuir.

Fig. The conclusion regarding the abilities of Fc variants to bind FcRn. In columns from left to right shows a modification of the binding of FcRn used immunoglobulin, other modifications, the relative affinity to FcRn data competitive analysis AlphaScreen™ compared with wild type (average value) and the number of analyses performed. Values of the relative affinity to FcRn greater than 1.0 show increased binding compared to wild type. Data were obtained at pH 6,0 (0,1M sodium phosphate, 125 mm sodium chloride).

Fig. Amino acid sequence of the heavy and light chains of the antibody anti-HER2.

Fig. Amino acid sequences of the constant regions (SN-CH3) some of the heavy chain IgG1 used in the present description.

Fig. Amino acid sequences of the constant regions (SN-CH3) some heavy chain hybrid IgG1/2 used in the present description.

Fig. The binding of Fc variants with FRIIIA (V158 Allotype) according to competitive analyses AlphaScreen™.

Fig. The binding of the Fc variant protein according to competitive analyses AlphaScreen™.

Fig. Serum concentrations of wild-type and variants of the antibodies in mice with Nokin (knockin) human FcRn. Used antibodies anti-VEGF were wild-type (empty squares), V308F (filled squares), P257L (filled triangles) and P257N (crosses).

Fig. Examples of the types of FcRn binding according to the present invention. Antibodies anti-VEGF are listed with an indication of the amount of cultural environments and yield of purified protein.

Fig. The affinity of binding of the variants according to the present invention with human FcRn at pH of 6.0. Values are given as fold increase with asiausa ability of the specified alternative with respect to antibodies of the wild type. For example, option 434S binds to FcRn 4.4 times more durable than the antibody wild-type.

Fig. The binding of the antibody wild-type and variant antibodies to FcRn on the cell surface T.

Fig. Combined variants according to the present invention, containing numerous replacement.

Fig. Picture of the interactions of variant CH3 domain of man, containing 434S, labeled Ser434, and FcRn person.

The implementation of the invention

According to the present invention described getting new variants of the Fc domains, including options contained in the antibody, fused with the Fc proteins and immunoadhesin that have increased binding to FcRn receptor. As indicated in the present description, the binding to FcRn leads to a longer retention in serum in vivo.

In order to increase the retention Fc proteins in vivo increase in the affinity of binding should occur at pH values of about 6, while maintaining a lower affinity when pH of about 7.4. Despite ongoing research, believe that the Fc region have a longer half-life in vivo, as binding to FcRn at pH 6 in endosome sequestered Fc (Ghetie and Ward, 1997 Immunol Today. 18(12):592-598, fully incorporated into the present description by reference). Then endosomal compartment returns Fc to the cell surface. As tol is to compartment opens extracellular space, the higher the pH, ~7.4V, induces the release of Fc back into the blood. In mice Dall' Acqua et al. showed that the Fc mutants with increased binding to FcRn at pH 6 and pH of 7.4, in fact, reduced serum concentrations and half-life as Fc wild-type (Dall' Acqua et al. 2002, J. Immunol. 169:5171-5180, fully incorporated into the present description by reference). Consider that the increased affinity of Fc to FcRn at pH of 7.4 is not possible to release the Fc back into the blood. Therefore, mutations Fc, which will increase the half-life of Fc in vivo in the ideal case will increase the FcRn binding at lower pH values, at the same time allowing you to release Fc at higher pH values. The amino acid histidine changes its charged condition in the pH range of 6.0 to 7.4. Therefore, it is not surprising that the remains of His located in the complex Fc/FcRn (6) in terms of importance.

An additional aspect of the present invention represents an increase of binding FcRn compared to a wild-type, in particular at lower pH values, pH about 6.0 to facilitate binding of the Fc/FcRn in endosome. Also described Fc variants with altered binding to FcRn and changed the link to another class of Fc receptors, FcγR' (sometimes write FR), because it has been shown that differential binding to FcγR, in particular increased binding to FcγRIIb and a decreased binding to FcγRIIb, leads to an increased efficiency.

Definition

For a more complete understanding of this application the following are some definitions. It is implied that these definitions include grammatical equivalents.

In the present description, the term "ADCC" or "antibody-dependent cretaceouspaleogene cytotoxicity" means mediated cell reaction in which nonspecific cytotoxic cells expressing FcγR, recognize bound antibody on the target cell and then cause lysis of the target cells.

In the present description, the term "ADCP" or "antibody-dependent cell-mediated phagocytosis" means mediated cell reaction in which nonspecific cytotoxic cells expressing FcγR, recognize bound antibody on the target cell and then cause phagocytosis of the target cells.

In the present description the term "modification" means a replacement, insertions and/or deletions of amino acids in the polypeptide sequence, or a conversion to a movie, chemically associated with the protein. For example, the modification may be a modified structure of the carbohydrate or PEG attached to a protein. In the present description, the term "modified amino acid" means the replacement, insertion and/or deletion of amino acids in the polypeptide sequence.

At present the eat the description of the term "replacement of amino acids", or "substitution" means the replacement of amino acids in a specific position in the original polypeptide by another amino acid. For example, replacing E272Y refers to the variant polypeptide, in this case Fc-variant, in which the glutamic acid at position 272 is replaced by tyrosine.

In the present description, the term "insertion of amino acids" or "insertion" means adding amino acid sequence at a particular position in the original polypeptide sequence. For example-A or ^E means the insertion of glutamic acid after position 233 and before position 234. In addition, 233ADE or ^233ADE means the insertion AlaAspGlu after position 233 and before position 234.

In the present description, the term "deletion of amino acids" or "division" means the removal of the amino acid sequence in a specific position in the original polypeptide sequence. For example, He or A# indicates a deletion of glutamic acid at position 233. In addition, EDA233 or EDA233# indicates a deletion sequence GluAspAla starting at position 233.

In the present description, the term "variant protein" or "variant protein"or "option" means a protein that differs from the original protein by the presence of at least one modified amino acid. The term "variant protein" can refer to a protein, compositions containing the specified company code is OK or amino acid sequence that encodes it. Preferably the variant protein contains at least one modified amino acids compared to the original protein, for example, from about one to about ten modifications of amino acids, and preferably from about one to about five modifications of amino acids compared to the original protein. In the present description, the sequence variant protein preferably will have at least about 80% homology with the sequence of the original protein, and more preferably at least about 90% homology, most preferably at least about 95% homology. The term "variant protein" can refer to a variant protein, compositions containing the specified variant protein, or a DNA sequence that encodes it. Accordingly, in the present description, the term "variant antibody or variant antibody" means an antibody that differs from the original antibodies the presence of at least one modification of amino acids, the term "variant IgG" or "variant IgG" means an antibody that differs from the original IgG presence of at least one embodiment, amino acids, and the term "immunoglobulin" or "variant immunoglobulin" means an immunoglobulin sequence that differs from the source past the successive immunoglobulin by the presence of at least one modified amino acid. In the present description, the term "Fc variant" or "Fc variant" means a protein that contains a modification in the Fc domain. Fc-variants according to the present invention is determined in accordance with modifications of amino acids they contain. Thus, for example, I332E is an Fc variant containing replacement I332E relative to the source of the Fc polypeptide. Similarly, S239D/I332E/G236A determines Fc variant containing substitutions S239D, I332E and G236A relative to the source of the Fc polypeptide. The identity of the amino acid in the wild type could not be determined and in this case the above option is indicated 239D/332E/236A. Note that the order in which the proposed replacement is arbitrary and, for example, S239D/I332E/G236A represents the same Fc variant that and G236A/S239D/I332E, etc. To all of the provisions described according to the present invention, numbering is given according to the EU index or the EU numbering scheme (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, thereby fully incorporated into the present description by reference). The term "EU index" or "index EU according to Kabat", or "numbering scheme EU" refers to the numbering antibody EU (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, thereby fully incorporated into the present description by reference). The modification can be an addition, a deletion of the Il is the replacement. Replacement may include naturally occurring amino acids not naturally occurring amino acids. Options may contain non-natural amino acids. Examples include US 6586207; WO 98/48032; WO 03/073238; US 2004-0214988 A1; WO 05/35727A2; WO 05/74524A2; J.W. Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; J.W. Chin, & P.G. Schultz, (2002), ChemBioChem 11:1135-1137; J.W. Chin, et al., (2002), PICAS United States of America 99:11020-11024; and, L. Wang, & P.G. Schultz, (2002), Chem. 1-10, all of which are fully incorporated into this description by reference.

In the present description, the term "protein" is meant at least two covalently attached amino acids, thus the term includes proteins, polypeptides, oligopeptides and peptides. Piptadenia group may contain naturally occurring amino acids and peptide bonds, or synthetic coworkers peptide structure, "analogs", such as peptide (see Simon et al., PNAS USA 89(20):9367 (1992), which is fully incorporated into the present description by reference). Amino acids can be either naturally occurring or not occurring in nature that is obvious to experts in the field of technology. For example, homophenylalanine, citrulline and norleucine considered amino acids for the purposes of the present invention and can be used amino acids such as D-and L- (R or S) configuration. Variants according to the present invention may contain modify the promotion, including the use of unnatural amino acids using, for example, technologies developed by Schultz and colleagues, including, without limitation, the methods described Cropp & Shultz, 2004, Trends Genet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003, Science 301(5635):964-7, all of which are fully incorporated into this description by reference. In addition, the polypeptides can include synthetic derivatization of one or more side chains or terminal glycosylation, tahilramani (PEGylation), cyclic rearrangement, cyclization, the linkers to connect with other molecules, hybridization in proteins or domains of proteins and the addition of peptide tags or labels.

In the present description, the term "residue" refers to the position in the protein and associated amino acid identity. For example, Asparagine 297 (also referred to Asn297, also called N297) represents the remainder of the antibody is human IgG1.

In the present description, the term "Fab" or "Fab region" means a polypeptide containing domains VH, CH1, VL and CL immunoglobulin. The term "Fab" can refer to this region in isolation or in the context of full-length antibodies, fragments of antibodies or hybrid protein Fab.

In the present description the term "modification of IgG subclass" means the modification of amino acids, converting one amino acid of one of the IgG in the corresponding amino acid other isotype number of IgG. For example, as IgG1 contains tyrosine, a IgG2 contains phenylalanine in position EU 296, replacement F296Y in IgG2 consider a modification of the IgG subclass.

In the present description the term "non-naturally occurring modification" means a modification of amino acids, which is not izotopicheskoj. For example, since none of IgGs does not contain a glutamic acid at position 332, replacement I332E in IgG1, IgG2, IgG3 or IgG4 consider not found in nature modification.

In the present description, the terms "amino acid" and "amino acid identity" means one of the 20 naturally occurring amino acids, or any non-natural analogues that may be present in a specific position.

In the present description, the term "effector function" means biochemical reaction resulting from the interaction of the Fc region of the antibody to the receptor or ligand Fc. Effector functions include, without limitation ADCC, ADCP, and CDC.

In the present description, the term "effector cell" means a cell of the immune system expressing one or more Fc receptors and mediating one or more effector functions. Effector cells include, without limitation monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, b cells, large granular lymphocytes, Langerhans cells, natural the killer (EC) cells and γδ cells and can be from any organism, including, without limitation, humans, mice, rats, rabbits and monkeys.

In the present description, the term "ligand IgG Fc" means a molecule, preferably a polypeptide derived from any organism that binds to the Fc region of IgG antibodies to formation of the complex Fc/Fc ligand. The Fc ligands include, without limitation FcγRI, FcγRII, FcγRIII, and FcRn, C1q, C3, mannan binding lectin, mannosyl receptor, staphylococcal protein a, streptococcal protein G, and viral FcγR. The Fc ligands also include homologues of Fc receptor (FcRH), which are a family of Fc receptors that are homologous FcγR (Davis et al., 2002, Immunological Reviews 190:123-136, fully incorporated into the present description by reference). The Fc ligands may include unexplored molecules that bind Fc. Specific ligands IgG Fc are FcRn and gamma Fc receptors. In the present description, the term "Fc ligand" means a molecule, preferably a polypeptide derived from any organism that binds to the Fc region of the antibody with the formation of the complex Fc/Fc ligand.

In the present description, the term "Fc receptor gamma", "FcγR" or "FR" means any member of a family of proteins that binds to the Fc region of IgG antibodies and is encoded by gene FcγR. In humans this family includes, without limitation FcγRI (CD64), including isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including the allotype is N and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD 16), including isoforms FcγRIIIa (including allotype V158 and F158)and FcγRIIIb (including allotype FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, fully incorporated into the present description by reference), and any unexplored isoforms FcγRs or FcγR or allotype person. FcγR may be from any organism, including, without limitation, humans, mice, rats, rabbits and monkeys. FcγRs mouse include, without limitation FcγRI (CD64), FcγRII (CD32), FcγRIII (CD 16) and FcγRIII-2 (CD 16-2), as well as any unexplored mouse FcγR or an isoform or allotype FcγR.

In the present description, the term "FcRn" or "neonatal Fc receptor" means a protein that binds to the Fc region of the IgG antibody and at least partially encoded in the genome of FcRn. FcRn may be from any organism, including, without limitation, humans, mice, rats, rabbits and monkeys. As is known in the art, functional FcRn protein contains two polypeptide, often called "heavy chain and light chain". Light chain is a beta-2-microglobulin, and the heavy chain is encoded by gene FcRn. If this description is not specified otherwise, the term "FcRn" or "FcRn protein" refers to a complex FcRn heavy chain with beta-2-microglobulin. Sequence FcRn, in particular people of interest shown in the figures.

In the present about what Isani the term "original polypeptide" means unmodified peptide, then modify with obtaining options. The original polypeptide can be a naturally occurring polypeptide or variant, or version of a naturally occurring polypeptide. The term "original polypeptide" can refer to the polypeptide, compositions containing the specified source polypeptide or amino acid sequence that encodes it. Accordingly, in the present description, the term "source immunoglobulin" means unmodified peptide immunoglobulin, which modify with obtaining options, and the term "original antibody" means an unmodified antibody, which change with the receipt of variant antibodies. It should be noted that the term "original antibody" includes well-known commercially available, obtained by recombinant methods antibodies, as described below.

In the present description, the term "position" means the position in the protein sequence. Provisions can be numbered in the order or in accordance with the scheme, for example, by the EU index as in Kabat. For example, the position 297 is a position within the antibody is human IgG1.

In the present description, the term "antigen-target" means a molecule that is specifically bound variable region of the specified antibodies. Antigen-target can be a company code is OK carbohydrate, lipid or other molecule.

In the present description, the term "cell-target" means a cell expressing the antigen target.

In the present description, the term "variable region" means a region of the immunoglobulin, which contains one or more immunoglobulin domains, essentially encoded by any of the genes Vκ, Vλ and/or VH comprising the Kappa, lambda loci and genetic loci heavy chains of immunoglobulin, respectively.

In the present description, the term "wild type or WT" means amino acid sequence or nucleotide sequence that occurs in nature, including allelic variations. Protein wild type contains the amino acid sequence or nucleotide sequence, which purposefully not modified.

The present invention relates to antibodies, demonstrating modulated binding to FcRn (modulation, including increased and decreased binding). For example, in some cases, increased binding leads to cell recirculation antibodies, and, consequently, to increased time-life, such as therapeutic antibodies. Alternatively, reduced FcRn binding is necessary, for example, for diagnostic antibodies or therapeutic antibodies containing radioactive metal is I. In addition, antibodies that demonstrate increased binding to FcRn and altered binding to other Fc receptors, such as FcγR, are used according to the present invention. Accordingly, according to the present invention proposed antibodies.

Antibodies

The present invention relates to antibodies that contain amino acid modifications, modulating binding to FcRn. Of particular interest are antibodies, minimally containing the Fc region or a functional variant showing increased affinity for binding to FcRn at low pH and essentially not demonstrate altered binding at higher pH values.

Traditional structural units of antibodies, usually contain a tetramer. Each tetramer, as a rule, consists of two identical pairs of polypeptide chains, each pair contains one "light" (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). The light chain of man is divided into Kappa and lambda light chains. The heavy chain is divided into mu, Delta, gamma, alpha, or Epsilon, and they determine the isotype of antibodies IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, without limitation, IgG1, IgG2, IgG3 and IgG4. IgM has subclasses, including, without limitation, IgM1 and IgM2. So what Braz, in the present description, the term "isotype" refers to any of the subclasses of immunoglobulins defined chemical and antigenic properties of their constant regions. Known isotypes of immunoglobulin are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD and IgE.

Aminoterminal plot each chain contains a variable region consisting of approximately 100-110 or more amino acids primarily responsible for antigen recognition. In the variable regions of three loops connected to each of the V-domains of the heavy chain and light chain with the formation of antigennegative center. Each of the loops is called hypervariable area (hereinafter referred to as "CDR"), in which the variation of the amino acid sequence has the greatest value.

Carboxyterminal plot of each chain defines a constant region primarily responsible for effector function. Kabat et al. received numerous primary sequence of the variable regions of the heavy chains and light chains. On the basis of the degree of conservatism of these sequences, they classified the individual primary sequence of the CDR and framework region and made a list of them (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication No.91-3242, E.A. Kabat et al., which is entirely included in the present description means refers the key).

In the subclass of IgG immunoglobulins, there are several domains of the immunoglobulin heavy chain. In the present description, the term "immunoglobulin domain (Ig)" means the region of immunoglobulin having different tertiary structure. According to the present invention are domains of the heavy chains, including constant heavy (CH) domains and hinge domains. In the context of IgG antibodies of each isotype IgG contains three areas CH. Accordingly, the domains "CH" in the context of IgG are the following: "SN" refers to the provisions 118-220 according to the EU index as in Kabat. "CH2" refers to the provisions 237-340 according to the EU index as in Kabat and "CH3" refers to the provisions 341-447 according to the EU index as in Kabat.

Another type of domain Ig heavy chain represents the hinge area. In the present description, the term "hinge" or "hinge region"or "hinge region of the antibody"or "hinge region of immunoglobulin" means a flexible polypeptide containing amino acids between the first and second constant domain of the antibody. Structurally, the domain SN IgG ends at EU position 220, and the CH2 domain of IgG starts the position of the EU residue 237. Thus, for IgG hinge antibodies in the present description includes provisions 221 (D221 in IgG1) - 236 (G236 in IgG1), and the numbering is given according to the EU index as in Kabat. In some embodiments, implementation, such as Vkontakte Fc region, included the lower hinge, the term "lower hinge" in General refers to the provisions 226 or 230.

Of particular interest according to the present invention represent areas of Fc. In the present description, the term "Fc" or "Fc region" refers to the polypeptide containing the constant region of an antibody excluding the first immunoglobulin constant domain region, and in some cases part of the hinge. Thus, the term "Fc" refers to the last two immunoglobulin domains of IgA, IgD and IgG constant region, the last three immunoglobulin domains of IgE and IgM constant region and a flexible hinge N-terminal with respect to the specified domains. For IgA and IgM, Fc may contain j chain For IgG, as shown in figure 1, Fc contains the immunoglobulin domains Shamma and Shamma (Cg2 and Cg3) and the lower hinge region between Sgamma (Cg1) and Shamma (Cg2). Despite the fact that the boundaries of the Fc region may vary, the Fc region of the heavy chain of human IgG, as a rule, includes the remains of S or R relative to the carboxyl terminal, and the numbering is given according to the EU index as in Kabat. The term "Fc" may refer to this region in isolation or in the context of the Fc polypeptide, as described below. In the present description, the term "Fc polypeptide" means a polypeptide that contains all the Fc region or a part of it. The Fc polypeptides include antibodies, fused with the Fc proteins you Lennie area Fc and Fc fragments.

In some embodiments, the implementation of the antibodies are full-sized. In the present description, the term "full-size antibody" means a structure that forms the natural biological form of an antibody, including variable and constant region that contains one or more modifications as specified in the present description.

Alternatively, antibodies can be represented as a set of structures, including, without limitation, fragments of antibodies, monoclonal antibodies, bespecifically antibodies, Manantial, domain antibodies, synthetic antibodies (sometimes called in the present description "mimetics of antibodies), chimeric antibodies, humanized antibodies, hybrid antibodies (sometimes called in the present description, "conjugates of antibodies) and fragments for each of these, respectively.

Antibody fragments

In one of the embodiments the antibody is an antibody fragment. Of particular interest are antibodies that contain an Fc region, merged with Fc proteins and the constant region of the heavy chain (SN-hinge-CH2-CH3), also including hybrids constant region of the heavy chain.

Specific fragments of antibodies include, without limitation (i) the Fab fragment containing domains VL, VH, CL and SN, (ii) the Fd fragment containing domains VH and SN, (iii) the Fv fragment containing domains VL and VH one anti the ate; (iv) the dAb fragment (Ward et al., 1989, Nature 341:544-546, fully incorporated into the present description by reference), contains one variable region (v) selection of CDR, (vi) fragments, F(ab')2, a bivalent fragment comprising two linked Fab fragment (vii) single-chain Fv molecules (scFv), in which the VH domain and a VL domain are linked by peptide linker which allows the two mentioned domains to be associated with the formation of antigennegative center (Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883, fully incorporated into the present description by reference), (viii) bespecifically single-chain Fv (WO 03/11161, thereby fully incorporated into the present description by reference) and (ix) "diately" or "Triatel", polyvalent or polyspecific fragments obtained by gene fusion (Tomlinson et. al., 2000, Methods Enzymol. 326:461-479; WO 94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448, all fully incorporated into the present description by reference). Fragments of antibodies can be modified. For example, molecules can be stabilized by introduction of disulfide bridges linking domains VH and VL (Reiter et al., 1996, Nature Biotech. 14:1239-1245, fully incorporated into the present description by reference).

Chimeric and humanized antibodies

In some embodiments, the implement frame components can represent abuses different species. Therefore, if the protein is an antibody, the antibody can be a chimeric antibody and/or humanitariannet antibody. In General, the term "chimeric antibody"or "humanized antibody" refers to antibodies containing the combined area of more than one species. For example, "chimeric antibodies" traditionally contain variable area (s) of a mouse (or rat in some cases) and a constant area (s) of the person. The term "humanized antibody" generally refers to the antibodies of non-human origin containing frame region of the variable domain are replaced with sequences found in human antibodies. In General, humanitariannet antibody antibody completely, except CDR encoded by polynucleotides human origin or is identical to the antibody, with the exception of CDR. Region CDR, some or all of which are encoded by nucleic acids originating from nonhuman organism, transplanted in the framework of beta-folded layer of variable regions of human antibodies with obtaining antibodies, the specificity of which is determined by the embedded CDR. The receipt of such antibodies are described, for example, in WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536, all of which are fully included in the present described the e by reference. "Reverse mutation" selected acceptor residues frame in the corresponding donor residues are often required to restore affinity, lost in the original transplanted design (US 5530101; US 5585089; US 5693761; US 5693762; US 6180370; US 5859205; US 5821337; US 6054297; US 6407213, all fully incorporated into the present description by reference). Ideally, humanitariannet antibody will also contain at least a portion of constant region of immunoglobulin, usually immunoglobulin, and thus will contain the Fc region of a human. Humanized antibodies can also be obtained using mice with immune system, created by genetic engineering methods. Roque et al., 2004, Biotechnol. Prog. 20:639-654, fully incorporated into the present description by reference. Many technologies and methods of humanization and reverse engineering of antibodies of non-human origin are well known in the art (See. Tsurushita &Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of Cells, 533-545, Elsevier Science (USA) and in the references, all of which are fully incorporated into this description by reference). Methods of humanization include, without limitation, the methods described in Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al. 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'connor et al., 1998, Protein Eng 11:321-8, all of which are fully incorporated into this description by reference. Humanization or other ways to reduce the immunogenicity of the variable regions of the antibodies of non-human origin can include ways to restore the surface, for example, as described in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, which is fully incorporated into the present description by reference. In one embodiment, the initial antibody was subjected to affinity maturation, as is known in the art. Methods based on the structure, can be used for humanization and affinity maturation, for example, as described in USSN 11/004590. Methods based on the selection, can be used for humanization and/or affinity maturation variable regions of antibodies, including, without limitation, the methods described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37):22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, all of which are fully incorporated into this description by reference. Other methods of humanization may include transplantation of parts of CDRs, including, without limitation, the methods described in USSN 09/810510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 200, J. Immunol. 169:3076-3084, all of which are fully incorporated into this description by reference.

Bespecifically antibodies

In one of the embodiments of the antibodies according to the present invention include poly-specific antibody and in particular bespecifically antibody, also sometimes called "diatesom". These antibodies are antibodies that bind two (or more) different antigens. Diately can be obtained in a variety of ways known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449, fully incorporated into the present description by reference), for example, obtained chemically or interspecific hybrid.

Manantial

In one of the embodiments the antibody is Manantial. Manantial are minimized such antibodies proteins containing scFv connected to a CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061, fully incorporated into the present description by reference. In some cases, the scFv can be associated with the Fc region, and may include some or all of the hinge region.

Human antibodies

In one of the embodiments the antibody is a fully human antibody origin containing at least one modification as provided herein described is I. The term "antibody is fully human origin" refers to a human antibody containing the gene sequence of an antibody derived from a human chromosome with modifications specified in the present description.

Merged antibodies

In one of the embodiments of the antibodies according to the present invention are fused with the antibody proteins (sometimes called in the present description, "conjugates of antibodies"). One of the types of hybrids of antibody contains merged with Fc proteins, which bind the Fc region with a "partner" by conjugation. In the present description, the term "fused with Fc protein" means a protein in which one or more polypeptides functionally linked to the Fc region. In the present description, the term "fused with Fc protein" is synonymous with the terms "immunoadhesin", "hybrid Ig", "Ig Chimera", and "receptor globulin" (sometimes through the dash)used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200, both fully incorporated into the present description by reference). Merged with Fc protein combines the Fc region of immunoglobulin with a "partner" to merge, which in General can be any protein or small molecule. In fact, any protein or small molecule may be linked to the Fc with getting merged with Fc protein. Protein partner, the merger may include without limitation variable region of any of the antibodies, region of the receptor to bind to the target, the adhesive molecule, ligand, enzyme, cytokine, chemokine, or some other protein or domain of the protein. "Partners" merger, representing small molecules may include any therapeutic agent that directs merged with Fc protein to target for therapeutic intervention. Such targets can be any molecule, preferably the extracellular receptor, which is involved in the disease. Thus, the IgG variants can be combined with one or more "partners" to merge. In one of alternative embodiments a variant IgG kongugiruut or functionally linked with another therapeutic compound. therapeutic compound may be a cytotoxic agent, chemotherapeutic agent, toxin, radioisotope, cytokine or other therapeutically active agent. IgG can be associated with one of the many non-protein polymers, e.g. polyethylene glycol, polypropyleneglycol, polyoxyalkylene or copolymers of polyethylene glycol and polypropylenglycol.

In addition to merged with Fc proteins fused antibodies include a constant region of the heavy chain fused with one or more "partners" in the merger (including the variable region of any of the antibodies), while others merged antibodies represents the t is essentially or completely full-length antibodies with "partners" to merge. In one embodiment, the implementation of the role of "partner" merger lies in mediating binding to the target and thus it is functionally similar to the variable regions of the antibodies (and may actually be). In fact, any protein or small molecule may be connected to Fc with getting merged with Fc protein (or merged antibodies). Protein partners merger may include, without limitation, the scope of the receptor to bind to the target, the adhesive molecule, ligand, enzyme, cytokine, chemokine, or some other protein or domain of the protein. "Partners" merger, representing small molecules may include any therapeutic agent that directs merged with Fc protein to a target for therapeutic intervention. Such targets can be any molecule, preferably the extracellular receptor, which is involved in the disease.

"Partner" conjugation may be a protein or non-protein; the latter is usually obtained with the use of functional groups of antibodies and "partner" by conjugation. For example, linkers known in the art; for example, Homo - or heterobifunctional linkers are well known (see 1994 catalog of the Pierce Chemical Company, division of technology, devoted to the linkers, pages 155-200, incorporated into this description by reference).

Suitable conjugates include, without limitation label, as described below, medicines and cytotoxic agents, including, without limitation, a cytotoxic drug (e.g., chemotherapeutic agents or toxins, or active fragments of these toxins. Suitable toxins and their corresponding fragments include chain And diphtheria a chain of exotoxin a chain of ricin, a chain abrina, Curtin, krotin, vanomycin, inomycin and other Cytotoxic agents also include radiochemical substances obtained by conjugating radioisotopes to antibodies or by binding of a radionuclide to a chelating agent that has been covalently attached to the antibody. In additional embodiments, the implementation of the use calicheamicin, auristatin, geldanamycin, maytansine and duocarmycin and analogues; for the latest, see U.S. 2003/0050331 A1, which is fully incorporated into the present description by reference.

Covalent modifications of the antibody

Covalent modifications of the antibody included in the scope of the present invention and generally, but not always carry out their excision. For example, some types of covalent modifications of the antibody is introduced into the molecule by the interaction of specific amino acid residues of the antibody with the organic derivatizing agent (used to obtain derivatives), which can react is selected side chains or the N - or C-terminal residues.

Caseinline remains often subjected to interaction with α-halogenation (and corresponding amines), such as Chloroacetic acid or chloroacetamide obtaining carboxymethyl or carboxylatomethyl derivatives. Cysteinyl residues can also be derivatization by reaction with BROMOTRIFLUOROMETHANE, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetylation, N-alkylamide, 3-nitro-2-pirinelli sulphide, methyl-2-pyridyldithio, p-chloromercuribenzoate, 2-chloromercuri-4-NITROPHENOL, or chloro-7-nitrobenzo-2-oxa-1,3-diazoles etc.

Histidine remains derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent has a relative specificity in regard to the side chain of histidine. Also suitable para-brompheniramine; the above reaction is preferably carried out in 0.1 M cacodylate sodium at pH of 6.0.

Libinline and aminoterminal remains subjected to the interaction with the anhydrides of succinic acid or other carboxylic acids. The derivatization these agents leads to a change in charge disinilah residues. Other suitable reagents for derivatization residues containing alpha-amino group include imidiately, such as methylphenidate; pyridoxal phosphate; pyridoxal; harborhead; dinitrobenzoyl is about acid; O-methylisoleucine; 2,4-pentandiol and reaction with glyoxylate, being catalyzed by transaminases.

Argireline residues modified by reaction with one or more standard reagents, among which is phenylglyoxal, 2,3-butandiol, 1,2-cyclohexandione and ninhydrin. For derivatization residues arginine is necessary to carry out the reaction in an alkaline environment because of the high pKa values guanidino functional groups. Furthermore, these reagents can interact with the groups of lysine as well as the Epsilon-amino group of arginine.

Specific modification trailing residues can be with a particular interest made when introducing spectral labels into tyrosine residues by reaction with aromatic compounds, page or tetranitromethane. Most commonly used N-acetylimidazole, tetranitromethane with the formation of O-acetyltyrosine compounds and 3-nitro-derivatives, respectively. Tyrosine remains idiot using 125I or 131I with getting labeled proteins for use in radioimmunoassay, the above chloramines method is appropriate.

Side carboxyl groups (aspartyl or glutamyl) selectively modified by reaction with carbodiimides (R'-N=C=N--R', where R and R' may represent different alkyl groups, such as 1-C is clohessy-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. In addition, aspartamine and glutamine remains turned into asparaginyl and glutaminyl residues by reaction with ammonium ions.

The derivatization with bifunctional agents suitable for crosslinking antibodies with water-insoluble auxiliary matrix or surface for use in many ways in addition to the methods described below. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetate)-2-Penilaian, glutaraldehyde, N-hydroxysuccinimide esters, for example esters of 4-azidoaniline acid, homobifunctional imidiately, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylester), and bifunctional maleimides, such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propionamide form a photoactivated intermediate that can form cross-links in the presence of light. Alternatively, reactive water-insoluble matrices such as brazian-activated carbohydrates and the reactive substrates described in U.S. patent 3969287; 3691016; 4195128; 4247642; 4229537 and 4330440, all of which are fully incorporated into the present description by reference, used for immobilization of proteins.

Glutaminyl and asparaginyl remnants repeatedly desamuduru with the image of the cation corresponding glutamyl and aspartyl residues, respectively. Alternatively, the data remains desamuduru in a moderately acidic environment. Any form of these residues included in the scope of the present invention. Other modifications include hydroxylation of Proline and lysine, phosphorylation of hydroxyl groups merilnyh or traveling residues, methylation of the α-amino groups of the side chains of lysine, arginine and histidine (I.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp.79-86 [1983], fully incorporated into the present description by reference), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Glycosylation

Another type of covalent modification is glycosylation. In another embodiment, variants of IgG specified in this description can be modified to contain one or more constructed glycoform. In the present description, the term "constructed glycoform" means a carbohydrate composition that is covalently attached to IgG, with specified carbohydrate composition differs chemically from the composition of the original IgG. Designed glycoform may be suitable for many tasks, including, without limitation, the strengthening or weakening of effector functions. Designed glycoform can be obtained in a variety of ways known in Dan the nd engineering (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; US 6602684; USSN 10/277370; USSN 10/113929; PCT WO 00/61739 A1; PCT WO 01/29246 A1; PCT WO 02/31140 A1; PCT WO 02/30954 A1, all fully incorporated into the present description by reference; (Potelligent® [Biowa, Inc., Princeton, NJ]; engineering technology glycosylation GlycoMAb® [Glycart Biotechnology AG, Zurich, Switzerland]). Many of these technologies are based on controlling the level fokusirovannyi and/or branched oligosaccharides covalently attached to the Fc region, for example, by IgG expression in various organisms or cell lines, created or otherwise obtained (e.g., cells Cho Lec-13 or cell YB2/0 hybridoma rats), by regulating enzymes in the path of glycosylation (e.g., FUT8 [α1,6-fucosyltransferase] and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifying carbohydrate (carbs) after the expression of IgG. The term "constructed glycoform" usually refers to the different carbohydrate or oligosaccharide; thus, the variant IgG, for example, the antibody or merged with Fc protein may contain constructed glycoform. Alternatively, the term "constructed glycoform" can refer to the variant IgG containing different carbohydrate or oligosaccharide. As is known in the art, the glycosylation profiles can depend ka is from the protein sequence (for example, the presence or absence of specific amino acid residues glycosylation below)and from the host cell or organism in which the protein is formed. Specific expression systems described below.

Glycosylation of peptides, as a rule, is either N-linked or O-linked. "N-linked" refers to the attachment of a carbohydrate fragment to the side chain of an asparagine residue. Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except Proline, are sequences of recognition enzymatic joining of carbohydrate fragment to the side chain of asparagine. Thus, the presence of any of these Tripeptide sequences in the polypeptide creates a potential centre of glycosylation. "O-linked glycosylation refers to the attachment of one of the sugars N-atsetilgalaktozamin, galactose, or xylose to hydroxyamino slot, most often serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

Adding centers glycosylation of the antibody convenient method is carried out by changing the amino acid sequence, so that it contains one or more of the above Tripeptide sequences (for N-with the centers provided glycosylation). This adjustment can also be made by adding or replacing one or more residues of serine or threonine to the original sequence (for O-linked centers glycosylation). For convenience, the amino acid sequence of the antibody preferably change through changes at the DNA level, particularly by mutations to DNA that encodes a target polypeptide at preselected bases such that formed the codons, which will be broadcast in essential amino acids.

Another way of increasing the number of carbohydrate fragments of antibodies is a chemical or enzymatic coupling of glycosides with protein. These procedures have the advantage that they do not require the formation of the protein in the cell host, which is capable of glycosylation in relation to N - and O-Svandovo glycosylation. Depending on the connection method, sugar (sugar) can be attached to (a) arginine and histidine, (b) free carboxyl groups, (C) free sulfhydryl groups such as groups of cysteine, (d) free hydroxyl groups such as groups of serine, threonine, or hydroxyproline, (e) aromatic residues such as residues phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. The above method is described in WO 87/05330 and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp.259-306, both of which are fully incorporated into the present description by reference.

Removal of the carbohydrate fragments that are present in the original antibody can be carried out chemically or enzymatically. For chemical deglycosylation should be protein effects connection triftormetilfullerenov acid or equivalent connection. This impact leads to the collapse of most or all Sugars except the linking sugar (N-acetylglucosamine or N-atsetilgalaktozamin), while the polypeptide remains intact. Chemical deglycosylation described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem. 118:131, both of which are fully incorporated into the present description by reference. Enzymatic cleavage of carbohydrate fragments of the polypeptides can be carried out through the use of a variety of endo - and ectoparasites, as described by Thotakura et al., 1987, Meth. Enzymol. 138:350, which is fully incorporated into the present description by reference. Glycosylation in the possible centers of glycosylation can be prevented by the use of compounds tunicamycin as described Duskin et al., 1982, J. Biol. Chem. 257:3105, which is fully incorporated into the present description by reference. Tunicamycin blocks the formation of protein-N-glycosidic linkages.

Labeled antibodies

In some embodiments, the implementation of covalent modification of the antibody according to the present invention includes adding one or more labels. In some cases they are treated as fused antibodies. The term "group for labeling" means any detektiruya tag. In some embodiments, the implementation group for labeling combined with the antibody by means of spacer elements groups of different lengths to reduce potential steric inconsistencies. Various methods of labeling proteins known in the art and they can be used when implementing the present invention.

In General, tags belong to many classes depend on the particular analysis, in which they are detected: a) isotopic labels, which may be a radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); (C) a redox-active fragments; (d) optical dyes, enzyme group (for example, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); (e) biotinylated group; and (f) predetermined polypeptide epitopes recognized by a secondary reporter (e.g., pairwise sequence latinboy "lightning", binding sites for secondary antibodies, the binding domains with metal, epitope tags and etc). In some embodiments, the implementation group for labeling combined with the antibody by means of spacer elements groups of different lengths to reduce potential steric inconsistencies. Various methods of labeling proteins known in the art and they can be used when implementing the present invention.

Specific labels include optical dyes, including, without limitation, chromophores, phosphors and fluorophores, the latter are specific in many cases. The fluorophores can be either fluorescent agents, representing "a small molecule or protein fluorescent agents.

The term "fluorescent label" refers to any molecule that may be about arozena through its inherent fluorescent properties. Suitable fluorescent labels include, without limitation, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarins, pyrene, malachite green, stilbene, Lucifer yellow, Cascade Blue, Texas red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5 to, LC Red 705, Oregon green dyes Alexa-Fluor (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, Rhodamine, and Texas red (Pierce, Rockford, IL), Cy 5, Cy Of 5.5, Cy 7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fluorophores described in the Molecular Probes Handbook by Richard P. Haugland, which is fully incorporated into the present description by reference.

Suitable protein fluorescent labels also include, without limitation, green fluorescent protein, including species such as green fluorescent protein Renilla, Ptilosarcus, or Aequorea (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., the access number in Genbank U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), "enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), the luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:2603-2607) and Renilla (WO 92/15673, WO 95/07463, WO 98/14605, WO 98/26277, WO 99/49019, U.S. Patents 5292658, 5418155, 5683888, 5741668, 5777079, 5804387, 5874304, 5876995, 5925558). All of the above is haunted sources in this paragraph is expressly incorporated into this description by reference.

Options IgG

In one of the embodiments of the present invention proposed a variant IgG proteins. Options IgG at least include an antibody fragment containing the region CH2-CH3 heavy chain. In addition, options include IgG Fc domains (e.g., including a lower hinge region), and IgG variants containing the constant region of the heavy chain (SN-hinge-CH2-CH3), which are suitable according to the present invention, all of which can be merged with "partners" to merge.

Variant IgG contains one or more modifications of amino acids compared to the starting polypeptide IgG, in some cases, compared to IgG wild-type. Variant IgG may have one or more optimized properties. Variant IgG differs in its amino acid sequence from the original IgG presence of at least one amino acid modifications. Thus, the IgG variants contain at least one modified amino acids compared to the original IgG. Alternatively, IgG variants may contain several modifications of amino acids compared to the original IgG, for example, from about one to fifty modifications of amino acids, preferably from about one to ten modifications of amino acids and most preferably from about one to about five meters is deficate amino acids compared to the original IgG.

Thus, sequence variants of IgG and sequence of the original polypeptide Fc are essentially homologous. For example, a variant sequence Fc-variant in the present description will have about 80% homology with a variant sequence of the original IgG, preferably at least about 90% homology and most preferably at least about 95% homology. Modifications can be genetically engineered using molecular biology or can be produced enzymatically or chemically.

The antigens of the target for antibodies

In fact, any antigen can be aiming to influence IgG variants, including without limitation proteins, subunits, domains, motifs and/or epitopes belonging to the following list of antigens target, which includes both soluble factors such as cytokines and membrane-bound factors, including transmembrane receptors: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-ISO-PCF2a, 8-oxo-dG, Adenosine receptor A1, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB / ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitripsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemia, anti-Id, ASPARTIC, predesign the th natriuretic factor, integrin av/b3, Ax1, b2M, B7-1, B7-2, B7-H, a stimulator of b-lymphocytes (BlyS), VASYA, VASYA-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a osteogenic BMP-3, BMP-4 BMP-2b, BMP-5, BMP-6.-1, BMP-7 (OP-1), BMP-8 BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, Bombezin, atrofichesky factor of bones, BPDE, BPDE-DNA, the PTS, the complement factor 3 (C3), Sa, C4, C5, CA, C10, CA, CAD-8, Calcitonin, Zamf, the carcinoembryonic antigen (CEA), carcinomas antigen, Cathepsin And, Cathepsin In, Cathepsin C/DPPI, Cathepsin D, Cathepsin E, Cathepsin N, Cathepsin L, Cathepsin About, Cathepsin S, Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (P67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, the toxin of Clostridium botulinum, the toxin of Clostridium perfringens, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, tumor-associated antigen cytokeratin, DAN, DCC, DcR3, DC-SIGN, the acceleration factor of disintegration, des(1-3)-IGF-I (IGF-1 brain), Dhh, digoxin, DNAM-1, Tnkase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EM, EMMPRIN, ENA, endotheliosis receptor, Enkephalinase, eNOS, Eot, eotaxin, Ersam, Ephrin B2/ EphB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, protein activator of fibroblasts (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (.-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha, GFR-A2, GFR-alpha, GITR, Glucagon, Glut 4, glycoprotein IIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, a factor that stimulates growth hormone, the Hapten NP-cap or NIP-cap), HB-EGF, NSS, glycoprotein gB shell HCMV, glycoprotein gH shell HCMV, HCMV UL, hematopoietic growth factor (HGF), Hep In gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3, (ErbB-3), Her4 (ErbB-4), glycoprotein gB of herpes simplex virus (HSV)glycoprotein gD of HSV, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120 V3 loop of gp 120 of HIV IIIB, HLA, HLA-DR, HM 1,24, HMFG PEM, HRG, Hrk, cardiac myosin man, the human cytomegalovirus (HCMV), the human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, Inhibin, iNOS, A-chain, insulin b-chain of insulin, insulin-like growth factor 1, integrin A2, integrin alpha, the integrin is life, integrin alpha/beta integrin alpha/beta, integrin alpha (V), integrin alpha/beta, integrin alpha/beta, integrin alpha, integrin beta, integrin beta2, interferon gamma, IP-10, I-TAC, JE, Kallickrein 2, Kallickrein 5, Kallickrein 6, Kallickrein 11, Kallickrein 12, Kallickrein 14, Kallickrein 15, Kallickrein L1, Kallickrein L2, Kallickrein L3, Kallickrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF, LECT2, Lefty, antigen Lewis Y related antigen Lewis-Y, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, lung surfactant, luteinizing hormone, receptor lymphotoxin-beta, Mac-1, MAdCAM, MAG, MAP2, MARC, SAM, SAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotease, receptor MGDF, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAS, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, inhibiting substance Muller, Mug, MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin, Neurotrophin-3, -4, or -6, Neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, REM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, Proinsulin, Prolactin, Protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, A-chain is alexina, In-circuit Relaxin, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3 the receptors of T cells (e.g., a receptor alpha/beta T cells), TdT, TECK, TEM, TEM, TEM, TEM, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, poly-specific TGF-beta, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-β RIII, TGF-beta, TGF-beta 2, TGF-beta, TCP beta, TCP beta, Thrombin, Timothy Ck-1, Thyrostimulin hormone, Tie, TIMP, TIQ, Tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF-R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2), TNFSF11 (TRANCE/RANK Ligand, ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A(TNF-a Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas Ligand, APO-1 Ligand, ART Ligand), TNFSF7 (CD27 Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137 Ligand), TP-1, t-PA, TPO, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen expressing cognate Lewis Y carbohydrate, TWEAK, THU, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4 integrin VNR, the factor a background of Villebranda, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, and receptors for hormones and growth factors.

To a person skilled in the art it is obvious that the above list of targets refers not only to specific proteins and biomolecules, but also to biochemical pathway or pathways that include them. For example, when referring to CTLA-4 as the antigen target suggest that ligands and receptors that are co-stimulating path of T cells, including CTLA-4, B7-1, B7-2, CD28, and any other unexplored ligands or receptors that communicate with the data of proteins are also targets. Thus, in the present description, the term "target" refers not only to the specific biomolecule, but also to the set of proteins that interact the specified target and participants of the biochemical pathway, applies the specified target. To a person skilled in the art it is also clear that any of the above antigens are targets, ligands or receptors that are associated with them, or other members of the relevant biochemical pathway may be functionally associated with an Fc variant according to the present invention with the formation merged with Fc protein. Thus, for example, merged with Fc protein, aiming affecting EGFR (the receptor for epidermal growth factor), can be created by the functional binding of Fc variants with EGF, TGF-b or any other ligand, explored or unexplored, which binds to EGFR. Accordingly, the Fc variant according to the present invention can be functionally linked to EGFR with education merged with Fc protein to bind to the EGF, TGF-b or any other ligand, explored or unexplored, which binds to EGFR. Thus, in reality, any polypeptide, as a ligand, receptor, or some other protein or domain of a protein, including, without limitation, the above-mentioned targets and proteins that comprise the relevant biochemical pathway may be functionally associated with an Fc variant according to the present invention with the formation merged with Fc protein.

The selection of the appropriate antigen depends on the application. For cancer is ecene the desired target, whose expression is restricted to cancer cells. Some of the targets, which proved especially amenable to therapy with antibodies represent a target with alarm functions. Other therapeutic antibodies exert their effects by blocking the transmission of the signal receptor by inhibiting the binding of the receptor with its sister ligand. Another mechanism of action of therapeutic antibodies is that they cause the lower (negative) regulation of the receptor. Other antibodies do not act by passing the signal through their target antigen. In some cases, the use of antibodies against infectious agents.

In one embodiment, the implementation of the Fc variants according to the present invention is included in the antibody to the cytokine. Alternatively, the Fc variants are merging or kongugiruut with the cytokine. In the present description, the term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. For example, as described in Penichet et al., 2001, J Immunol Methods 248:91-101, which is expressly incorporated into this description by reference, cytokines can be connected with the antibody to produce a number of necessary properties. The use of the AMI of such cytokines are lymphokines, Monokini and normal polypeptide hormones. Cytokines include growth hormone such as human growth hormone, N-methionyl the human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine, insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyrostimulin hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogenic; factor-alpha and-beta tumor necrosis; inhibitory substance Mueller; gonadotropin-associated peptide mouse; inhibin; activin; factor vascular endothelial growth; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet growth factor; transforming growth factors (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and-II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, -beta and-gamma; colony stimulating factors (CSFs)such as macrophage CSF (M-CSF); granulocyte-macrophage CSF (GM-CSF) and granulocyte-CSF (G-CSF); interleukins (IL)such as IL-1, IL-1alpha, IL-2, IL-g, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis factor such as TNF-alpha or TNF-beta; CA; and other polypeptide factors including LIF and kit ligand (KL). In the present description, the term cytokine includes proteins from natural East is nikov or from a culture of recombinant cells and biologically active equivalents of cytokines, containing the native sequence.

Cytokines and soluble targets, such as members of the TNF superfamily, constitute a preferred target for use in conjunction with variants according to the present invention. For example, antibodies anti-VEGF, anti-CTLA-4 and anti-TNF or fragments of these antibodies are antibodies that are particularly suitable for the use of Fc variants that increase binding to FcRn. Drugs against these targets is often used in the treatment of autoimmune diseases, with the required multiple injections over long periods of time. Therefore, a longer half-life in serum and less frequent courses of treatment, due to the variants according to the present invention, are preferred.

Fc-variants according to the present invention can be useful for a number of antibodies and merged with Fc proteins authorized for use in clinical trials or under development. In the present description the indicated antibodies and merged with Fc proteins are called "clinical products and candidates." Thus, in a preferred embodiment, the Fc polypeptides according to the present invention can find application in a range of clinical products and candidates. For example, the Fc polypeptides according infusion is he to the invention can be useful for a number of antibodies, sighting affecting CD20. For example, the Fc polypeptides according to the present invention can find application in the antibody, which is essentially similar to rituximab (Rituxan®, IDEC/Genentech/Roche) (see for example US 5736137), chimeric antibody, anti-CD20, approved for treating non-Hodgkin's lymphoma; HuMax-CD20, anti-CD20, currently under development Genmab, the antibody anti-CD20 described in US 5500362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769 (PCT/US2003/040426, entitled "Immunoglobulin Variants and Uses Thereof). The Fc polypeptides according to the present invention can be useful for a number of antibodies that attack affecting members of the family of receptors for epidermal growth factor, including EGFR (ErbB-1), Her2/neu (ErbB-2), Her3, (ErbB-3), Her4 (ErbB-4). For example, the Fc polypeptides according to the present invention can find application in the antibody, which is essentially the same as trastuzumab (trastuzumab) (Herceptin® (Herceptin®), Genentech) (see for example, US 5677171), humanitariannet antibody anti-Her2/neu approved for treatment of breast cancer; pertuzumab (pertuzumab) (rhuMab-2C4, Omnitarg™), currently under development Genmab; the antibody anti-Her2 described in US 4753894; cetuximab (cetuximab) (Erbitux® (Erbitux®), Imclone) (US 4943533; PCT WO 96/40210), the chimeric antibody anti-EGFR in clinical trials for a variety of cancers, ABX-EGF (US 6235883), currently under development Abgenix-Immunex-Amgen; HuMax-EGFr (USSN 10/172317), razrabatyvaemej currently Genmab; 425, EMD55900, EMD62000 and EMD72000 (Merck KGaA) (US 5558864; Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al., 1987, J Cell Biochem. 35(4):315-20; Kettleborough et al., 1991, Protein Eng. 4(7):773-83); ICR62 (Institute of Cancer Research) (PCT WO 95/20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22(1-3): 129-46; Modjtahedi et al., 1993, Br J Cancer. 1993, 67(2):247-53; Modjtahedi et al, 1996, Br J Cancer, 73(2):228-35; Modjtahedi et al, 2003, Int J Cancer, 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and the Centre de Immunologia Molecular, Cuba (US 5891996; US 6506883; Mateo et al, 1997, Immunotechnology, 3(1):71-81); mAb-806 (Ludwig Institue for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT WO 0162931A2); and SC100 (Scancell) (PCT WO 01/88138). In another preferred embodiment, the Fc polypeptides according to the present invention can find application in alemtuzumab (Grade® (Campath®), Millenium), humanitariannet monoclonal antibody currently approved for treatment of b-cell chronic lymphocytic leukemia. The Fc polypeptides according to the present invention can find application in a multitude of antibodies or merged with Fc protein, which is essentially the same as with other clinical products and candidates, including, without limitation, muromonab-CD3 (muromonab-CD3 (Orthoclone OKT3®), an antibody anti-CD3 developed by Ortho Biotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin®) (ibritumomab tiuxetan, Zevalin®), an antibody anti-CD20 developed by IDEC/Schering AG, gemtuzumab ozogamicin (Mylotarg®) (gemtuzumab ozogamicin, Mylotarg®), antibody anti-CD33 (protein P67), developed by Celltech/Wyeth, alefacept (AMEVIVE®) (lefacept, Amevive®), merged with Fc protein anti-LFA-3, developed by Biogen, abciximab (Reopro®) (abciximab, ReoPro®), developed by Centocor/Lilly, basiliximab (Simulect®) (basiliximab, Simulect®), developed by Novartis, palivizumab (Synagis®) (palivizumab, Synagis®), developed by MedImmune, infliximab (Remicade®) (infliximab, Remicade®), antibody anti-NF developed by Centocor, adalimumab (Humira®) (adalimumab, Humira®), antibody anti-NF developed by Abbott, Humicade™antibody anti-NF developed by Celltech, etanercept (Enbrel®) (etanercept, Enbrel®), merged with Fc protein anti-NF developed by Immunex/Amgen, ABX-CBL, an antibody anti-CD147 developed by Abgenix, ABX-IL8, an antibody anti-IL8 developed by Abgenix, ABX-MA1, an antibody anti-MUC18, developed by Abgenix, Pemtumomab (Pemtumomab) (R1549, 90Y-muHMFG1), anti-MUC1 developed by Antisoma, Therex (R1550), antibody anti-MUC1 developed by Antisoma, AngioMab (AS 1405), developed by Antisoma, HuBC-1, developed by Antisoma, Triplatin (Thioplatin) (AS1407), developed by Antisoma, Antegren® (natalizumab) (Antegren®, natalizumab), antibody anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7, developed by Biogen, VLA-1 mAb, an antibody anti-VLA-1 integrin, developed by Biogen, LTBR mAb, an antibody anti-lymphotoxin beta receptor (LTBR), developed by Biogen, CAT-152, an antibody anti-TGF-β2, developed by Cambridge Antibody Technology, J695, an antibody anti-IL-12, being developed by Cambridge Antibody Technology and Abbott, CAT-192, an antibody anti-TGFβ1, developed by Cambridge Antibody Technology and Genzyme, CAT-213, an antibody anti-Eotaxin1, RA is managed by Cambridge Antibody Technology, antibody anti-Blys LymphoStat-B™, developed by Cambridge Antibody Technology and Human Genome Sciences, Inc., TRAIL-R1mAb, antibody, anti-TRAIL-R1, developed by Cambridge Antibody Technology and Human Genome Sciences, Inc., Avastin™ (bevacizumab) (Avastin®, bevacizumab, rhuMAb-VEGF)antibody, anti-VEGF, developed by Genentech, an antibody of the family of anti-HER receptor, developed by Genentech, Anti-tissue factor (ATF), the antibody against tissue factor, developed by Genentech, Xolair™ (Omalizumab) (Xolair™, Omalizumab), antibody anti-IgE, developed by Genentech, Raptiva™ (Efalizumab) (Raptiva™, Efalizumab), antibody anti-CD11a, developed by Genentech and Xoma, antibody MLN-02 (formerly LDP-02), developed by Genentech and Millenium Pharmaceuticals, HuMax CD4 antibody, anti-CD4, developed by Genmab, HuMax-IL15, an antibody anti-IL15, developed by Genmab and Amgen, HuMax-Inflam, being developed Genmab and Medarex, HuMax-Cancer, an antibody anti-heparanase I, developed by Genmab and Medarex and Oxford GcoSciences, HuMax-Lymphoma, developed by Genmab and Amgen, HuMax-TAC, developed by Genmab, IDEC-131, and the antibody anti-CD40L developed by IDEC Pharmaceuticals, IDEC-151 (Clenoliximab (Clenoliximab)), antibody anti-CD4, developed by IDEC Pharmaceuticals, IDEC-114, an antibody anti-CD80, developed by IDEC Pharmaceuticals, IDEC-152, an anti-CD23, developed by IDEC Pharmaceuticals, antibodies to factor macrophage migration (MIF), developed by IDEC Pharmaceuticals, BEC2, antiidiotypic antibody developed by Imclone, IMC-1C11, an antibody anti-KDR developed by Imclone, DC101, an antibody anti-flk-1, developed by Imclone, and is Titel anti-VE cadherin, developed by Imclone, CEA-Cide™ (labetuzumab (labetuzumab)), antibody to carcinoembryonic antigen (CEA), developed by Immunomedics, LymphoCide™ (Epratuzumab (Epratuzumab)), antibody anti-CD22 developed by Immunomedics, AFP-Cide, being developed Immunomedics, MyelomaCide developed by Immunomedics, LkoCide developed by Immunomedics, ProstaCide, being developed Immunomedics, MDX-010, an antibody anti-CTLA4 developed by Medarex, MDX-060, an antibody anti-CD30 developed by Medarex, MDX-070, developed by Medarex, MDX-018 developed Medarex, Osidem™ (IDM-1) and the antibody anti-Her2 developed by Medarex and Measurement-Designed Molecules, HuMax™-CD4 antibody, anti-CD4, developed by Medarex and Genmab, HuMax-IL15, an antibody anti-IL15, developed by Medarex and Genmab, CNTO 148, an antibody anti-TNFα developed by Medarex and Centocor/J&J, CNTO 1275, an antibody anti-cytokine developed by Centocor/J&J, MOR101 and MOR102, antibodies (CD54) molecules intercellular adhesion-1 (ICAM-1), developed by MorphoSys, MOR201, the antibody to the receptor fibroblast growth factor 3 (FGFR-3), developed by MorphoSys, Nuvion® (visilizumab) (Nuvion®, visilizumab), antibody anti-CD3 developed by Protein Design Labs, HuZAF™antibody anti-gamma interferon developed by Protein Design Labs, Anti-α5β1 Integrin developed by Protein Design Labs, anti-IL-12 developed by Protein Design Labs, ING-1 antibody anti-EP-ITSELF developed by Xoma, and MLN01, the antibody is an anti-Beta2 integrin developed by Xoma; all of the above sources in this paragraph explicitly vklucheny the present description by reference.

The Fc polypeptides according to the present invention can be included in the above clinical candidates and products or antibodies and merged with Fc protein essentially similar to them. The Fc polypeptides according to the present invention can be included in the options above clinical candidates and products, humanized, subjected to affinity maturation, created or modified in some other way.

In one of the embodiments, the Fc Polypeptides according to the present invention used for the treatment of autoimmune symptoms, symptoms of inflammation or transplant. The antigens of the target and clinical products and candidates that are suitable for the treatment of these disorders include without limitation antibodies anti-α4β7 integrin, such as LDP-02, antibodies anti-beta2 integrin, such as LDP-01, antibodies anti-complement (C5), such as 5G 1,1, antibodies anti-CD2, such as BTI-322, MEDI-507, antibodies anti-CD3, such as OKT3, SMART anti-CD3 antibodies, anti-CD4, such as IDEC-151, MDX-CD4, OKT4A, antibodies anti-CD11a antibodies, anti-CD14, such as IC14, antibodies anti-CD18 antibodies, anti-CD23, such as IDEC 152, antibodies anti-CD25, such as Zenapax, antibodies anti-CD40L, such as s, Antova, IDEC-131, antibodies, anti-CD64, such as MDX-33, antibodies, anti-CD80, such as IDEC-114, antibodies anti-CD147, such as ABX-CBL antibodies anti-E-selectin, such as CDP850, antibodies anti-gpIIb/IIIa, is e as Reopro/Abciximab, antibodies anti-ICAM-3, such as ICM3, antibodies anti-ICE, such as VX-740, antibodies anti-FcR1, such as MDX-33, antibodies anti-IgE, such as rhuMab-E25, antibodies anti-IL-4, such as SB-240683, antibodies anti-IL-5 such as SB-240563, SCH55700, antibodies anti-IL-S, such as AVH-IL8 antibodies anti-interferon gamma antibody, anti-TNF (TNF, TNFa, TNFa, TNF-alpha), such as CDP571, CDP870, D2E7, Infliximab, MAK-195F and antibodies anti-VLA-4, such as Antegren (Antegren).

Fc-variants according to the present invention, such as variants with increased binding to FcRn can be used in the molecules of the TNF inhibitors with improved properties. Suitable molecule inhibitors of TNF include any molecule, inhibitory action of TNF-alpha in a mammal. Suitable options include fused with Fc protein Enbrel® (etanercept) and antibodies Humira® (adalimumab) and Remicade® (infliximab). Monoclonal antibodies (such as Remicade and Humira)created using an Fc variant according to the present invention with increased binding FcFn, can lead to greater efficiency through increased time half-life.

In some embodiments, the implementation of the use of antibodies to infectious diseases. Antibodies to eukaryotic cells include antibodies that attack affecting yeast cells, including, without limitation, Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyce fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, plasmodium falciparium, and Yarrowia lipolytica.

Also suitable antibodies to the cells of fungi, including antigens of the target associated with Candida species, including, among others, Candida glabrata, Candida albicans, .krusei, .lusitaniae and .maltosa, as well as species of Aspergillus, Cryptococcus, Histoplasma, Coccidioides, Blastomyces, and Penicillium.

Antibodies directed against antigens of the target associated with protozoa, include without limitation antibodies associated with Trypanosoma, Leishmania species, including Leishmania donovanii;, Plasmodium spp., Pneumocystis carinii, Cryptosporidium parvum, Giardia lamblia, Entamoeba histolytica, and Cyclospora cayetanensis.

Also suitable antibodies to prokaryotic antigens, including antibodies to the appropriate bacteria, such as pathogenic and non-pathogenic prokaryotes, including, without limitation, Bacillus, including Bacillus anthracis; Vibrio, e.g., V. cholerae, E., e.g., Enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g. S. typhi; Microbacteria, for example, M. tuberculosis, M. leprae; Clostridium, e.g., C. botulinum, C. tetani, C. difficile, C. perfringens; Corynebacterium, for example, C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae; Staphylococcus for example, S. aureus; Haemophilus influenzae bacteria, for example, N. influenzae; Neisseria, for example, N. meningitidis, N. gonorrhoeae; Yersinia, for example, Y. lamblia, Y. pestis, Bacillus Pseudomonas, for example, P. aeruginosa, P. putida; Chlamydia, e.g., C. trachomatis; Bordetella, e.g., B. pertussis; Treponema, e.g., T. palladium; B. anthracis, Y. pesis, Brucella spp., F. tularensis, B. mallei, B. pseudomallei, B. mallei, B. pseudomallei, C. botulinum, Salmonella spp., SEB V. cholerae toxin B, E. coli O157:H7, Listeria spp., Trichosporon beigelii, Rhodotorula species, Hansenula anomala, Enterobacter sp., Klebsiella sp., Listeria sp., Mycoplasma sp. etc.

According to some aspects, antibodies directed against viral infections; these viruses include, without limitation, orthomyxovirus (e.g., influenza virus), paramyxoviruses (e.g respiratory syncytial virus, mumps virus, measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses, togaviruses (e.g., rubella virus), parvoviruses, poxviruses (e.g. variola virus, vaccinia virus), enteroviruses (e.g. poliovirus, Coxsackievirus), hepatitis viruses (including a, b and C), herpesviruses (e.g., herpes simplex virus, varicella-zoster pox, cytomegalovirus, Epstein-Barr), rotaviruses, noroviruses, Hantaviruses, arenaviruses, rhabdovirus (e.g., rabies virus), retroviruses (including HIV, HTLV-I and-II), papovaviruses (e.g. papillomavirus), polyomaviruses and picornaviruses, and the like,

Optimized properties of IgG variants

According to the present application is also suggested that IgG variants that are optimized for a variety of therapeutically important properties. In the present description variant IgG, which create or intend to demonstrate one or more optimized properties, called "optimizarea the major variant IgG". The most preferable properties that can be optimized include, without limitation, an increased or reduced affinity for FcRn and increased or decreased half-life in vivo. Appropriate options for implementation include antibodies, showing increased affinity for binding to FcRn at low pH, such as pH associated with endosomes, for example, a pH of 6.0 at the same time supporting a lower affinity at higher pH values, such as of 7.4, to allow increased absorption endosomes, but the normal speed of release. Similarly these antibodies with modulated binding to FcRn may have other desired properties, such as modulated binding to FcγR, such as specified in the application USSN 11/174287, 11/124640, 10/822231, 10/672280, 10/379392 and the patent application 11/256060 called "Variants of immunoglobulin IgG, with optimized effector function", filed October 21, 2005. I.e. optimized properties also include, without limitation increased or reduced affinity to FcγR. In one possible implementation options IgG optimize, as a result they have a high affinity to activating FcγR person, preferably FcγRIIIa, in addition to the characteristics of the binding of FcRn. In one possible alternative embodiment, the var is anti IgG optimize, as a result they have reduced affinity for the inhibitory receptor FcγRIIb person. I.e. specific options for implementation include the use of antibodies that demonstrate increased binding to FcRn and increased binding to FcγRIIIa. In other embodiments, implementation of the use of antibodies that demonstrate increased binding to FcRn and increased binding to FcγRIIIa. Assume that according to the specified options for the implementation of the proposed IgG polypeptides with enhanced therapeutic properties in humans, for example, enhanced effector function and higher anticancer activity. In an alternative embodiment, variants of IgG optimize, as a result they have increased or decreased affinity to FcRn and increased or decreased affinity to human FcγR, including but not limited to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb, including their allelic variants. Assume that according to the modalities for the implementation of the proposed IgG polypeptides with enhanced therapeutic properties in humans, for example, increased half-life and reduced effector function. In other embodiments, implementation options provide enhanced IgG affinity to FcRn and increased affinity to one or more FcγR, but reduced affinity to one linescale other FcγR. For example, the variant IgG may have increased binding to FcRn and FcγRIIIa, but reduced binding to FcγRIIb. Alternatively, the variant IgG may have reduced binding to FcRn and FcγR. In another embodiment, the variant IgG may have low affinity to FcRn and increased affinity to FcγRIIb, but reduced affinity to one or more FcγR. In yet another embodiment, the variant IgG may have low affinity to FcRn and increased affinity to FcγRIIb, but reduced affinity to one or more FcγR. In yet another embodiment, the variant IgG may have an increased half-life and reduced effector functions.

Preferred embodiments of include optimization of binding to FcRn and human FcγR, however, in alternative embodiments, the implementation of the IgG variants possess enhanced or reduced affinity for FcRn and FcγR non-human organisms, including, without limitation, rodents and non human primates. Options IgG that optimize for binding to FcRn is not of human origin, can find application in research. For example, the available models of mice for many diseases, allowing to test properties such as efficacy, toxicity and pharmacokinetic characteristics specified by antilego medicines. As is known in the art, the cancer cells can be transplanted or introduced by injection to mice to mimic human cancer, the method called "xenotransplantation". The testing options IgG, including IgG variants that are optimized in relation to FcRn, you can get useful information about characteristics of the clearance of the protein, its mechanism of clearance, etc. Options IgG can also be optimized in terms of increased functionality and/or properties of the solution in glycosylated form. The Fc ligands include, without limitation FcRn, FcγRs, C1q and proteins a and G, and can be from any source, including without limitation human, mouse, rat, rabbit, or monkey, preferably human. In an alternative preferred embodiment, variants of IgG optimize, with the result that they become more stable and/or more soluble compared to the glycosylated form of the original variant IgG.

Options IgG may contain modifications of modulating the interaction with ligands Fc, in addition to FcRn and FcγR, including, without limitation, complement proteins and homologues of the Fc receptor (FcRH). FcRH include, without limitation FcRH1, FcRH2, FcRH3, FcRH4, FcRH5 and FcRH6 (Davis et al, 2002, Immunol. Reviews 190:123-136, fully incorporated into the present description by reference).

Preferably specificity var is Anta IgG to the Fc ligand will determine its therapeutic usefulness. The usefulness of the specified variant IgG for therapeutic purposes will depend on the epitope and shape of the target antigen and the disease or symptom being treated. For most targets and symptoms of increased binding to FcRn may be preferred because of increased binding to FcRn may result in increased time half-life in serum. Longer half-life in serum allows less frequently or in a lower dose to enter a drug. It is preferable, when a therapeutic agent is injected in response to the symptom requiring re-introduction. For some targets and symptoms reduced binding to FcRn may be preferred. This may be the most preferred when it is necessary to Fc-variant with increased clearance and reduced half-life in serum, for example, the Fc polypeptides to be used as radiopharmaceuticals funds or radiotherapy funds.

Can be used variants of IgG, including IgG variants that have higher affinity to FcRn with an activating FcγR, and/or reduced affinity to inhibitory FcγRs. For some targets and symptoms may also be useful variants of IgG, providing differential selectivity for different Akti is arousih FcγR; for example, in some cases it may be necessary enhanced binding to FcγRIIa and FcγRIIIa but not FcγRI, whereas in other cases, increased binding only with FcγRIIa may be preferred. For some targets and symptoms may be preferable to use the IgG variants that alter FcRn binding and reinforcing as FcγR-mediated and complement-mediated effector functions, whereas in other cases it may be useful to use IgG variants that increase binding to FcRn or the half-life in serum and either FcγR-mediated or complement-mediated effector functions. For some targets or symptoms of cancer can be a useful weakening or destruction of one or more effector functions, for example by turning off the binding to C1q, one or more FcγR, or FcRn one or more other Fc ligands. For other targets, and symptoms may be preferable to use the IgG variants that provide enhanced binding to the inhibitory FcγRIIb, but the level of wild-type, reduced or remote linking activating FcγR. In particular, it may be appropriate, for example, in the case when a task variant IgG is the inhibition of inflammation or autoimmune disease or modulating the immune system in some way the m Since autoimmune diseases are generally long and the treatment is performed with repeated injections, treatment Fc variant has an increased half-life due to increased FcRn is the most preferred.

Can be modified to improve stability, solubility, function, or clinical application of IgG. In a preferred embodiment, IgG variants may contain modifications to reduce immunogenicity in humans. In the most preferred embodiment, the immunogenicity of the variant IgG reduced with the use of the method described in USSN 11/004590, which is fully incorporated into the present description by reference. In alternative embodiments, the exercise of options IgG humanitarium (Clark, 2000, Immunol Today 21:397-402, fully incorporated into the present description by reference.

Options IgG may contain modifications to reduce immunogenicity. Modifications to reduce immunogenicity may include modifications that reduce the binding of processed polypeptides derived from the original sequence, with MHC proteins. For example, the amino acid modifications can be made so that no or a minimum number of immune epitopes, which are assumed to bind with high affinity with Liu who passed the predominant MHC alleles. Some authentication methods to bind to the MHC epitopes in protein sequences are known in the art and can be used for the calculation of the epitopes in the variant IgG. See, for example, WO 98/52976; WO 02/079232; WO 00/3317; USSN 09/903,378; USSN 10/039,170; USSN 60/222,697; USSN 10/754,296; PCT WO 01/21823; and PCT WO 02/00165; Mallios, 1999, Bioinformatics 15:432-439; Mallios, 2001, Bioinformatics 17:942-948 (in Russian); Sturniolo et al., 1999, Nature Biotech. 17: 555-561; WO 98/59244; WO 02/069232; WO 02/77187; Marshall et al., 1995, J. Immunol. 154:5927-5933; Hammer et al., 1994, J. Exp. Med. 180:2353-2358, all of which are fully incorporated into this description by reference. Based on the sequence information can be used to determine the binding interaction of the indicated peptide-MHC (see, for example, Mallios, 1999, Bioinformatics 15:432-439; Mallios, 2001, Bioinformatics 17: p942-948; Sturniolo et. al., 1999, Nature Biotech. 17:555-561, all of which are fully incorporated into this description by reference).

Creating options IgG

Variants according to the present invention can be obtained in various ways. Options specified in this description can be insertions, deletions, substitutions, and other modifications or combinations of these and other changes. In particular, the new embodiment of the present invention is the creation of insertions and deletions, which either increase or reduce the binding of the Fc polypeptide with Fc ligand. As indicated in this is the description can be made insertions or deletions that increase or decrease the affinity of the polypeptide Fc to FcRn. Insertions and deletions can be obtained by rational approaches or approaches, including the use of random components, such as arbitrary and polypropolene creating libraries or screening. In an alternative embodiment, described replacement, increasing or decreasing the affinity of the Fc polypeptide for FcRn.

Modifications of the skeleton: insertions and deletions

Variant Fc polypeptides can be obtained by replacing the variant amino acid in place of the original amino acid in position in the Fc polypeptide. By replacing one or more amino acids in the variant amino acid in the Fc polypeptide modify the side chains at these positions. The most suitable replacement modify the properties of the Fc by changing the side chains of the Fc. Substituted side chains can interact directly or indirectly with a partner on the binding of the Fc, which is associated with the function or property of Fc. At least one replacement alters the covalent structure of one or more side chains of the original polypeptide Fc.

In the alternative, can be obtained with variant Fc polypeptides, changing the covalent structure of the skeleton of the original Fc polypeptide. In proteins, the backbone atoms represent the peptide nitrogen, al the FA-carbon, carbonyl or peptide carbonyl carbon and oxygen. The change in the covalent structure of the skeleton provides additional ways to modify the properties of the Fc polypeptides. Covalent structure of the skeleton of the Fc can be changed by adding atoms in the backbone, for example, by the insertion of one or more amino acids, or by removing atoms from the backbone, for example by deletion of one or more amino acids. Covalent structure of the skeleton may also be modified by replacement of individual backbone atoms to other atoms (Deechongkit et al., J Am Chem Soc. 2004. 126(51):16762-71, fully incorporated into the present description by reference). As is known in the art and illustrated in the present description, insertions or deletions of amino acids in the Fc polypeptides can be made by insertions or deletions of the corresponding nucleotides in the DNA encoding the specified Fc polypeptide. Alternatively, as is known in the art, insertions or deletions of amino acids may be carried out during the synthesis of polypeptides Fc.

The creation of insertions or deletions of amino acids that alter the interaction of the Fc polypeptide with one or more binding partners (for example, FR, FcRn, C1q), can be carried out by considering the structure of the complex of the polypeptide Fc and its partner swazilan the Y. In a less preferred embodiment, the creation can be done by looking at the structure of the polypeptide Fc and information about Fc region involved in the binding partner binding. This information can be obtained by conducting experiments on the mutagenesis experiments on phage display, by comparing homology, computer modeling or other methods.

Preferred positions in the amino acid sequence of insertions or deletions that affect the binding interaction of the Fc, but does not affect the overall structure, stability, expression or application of the Fc polypeptide, loops are involved in the interactions Fc/partner binding of Fc. To change the binding of FcRn polypeptide with Fc position 244-257, 279-284, 307-317, 383-390 and 428-435 represent the preferred location of the hinges for insertions or deletions (numbering according to EU index Kabat et al., Burmeister et al., 1994, Nature, 372:379-383; Martin et al., 2001, Mol Cell 7:867-877, all fully incorporated into the present description by reference). To change the binding of the Fc-gamma receptor with the Fc polypeptide provisions 229-239, 266-273, 294-299 and 324-331 represent the preferred location of the hinges for insertions or deletions (numbering according to EU index Kabat et al., code in PDB 1E4K. pdb Sondermann et al. Nature. 2000 406:267, all of which are fully VK is uceni in the present description by reference). Loops represent a region of the polypeptide that are not included in the alpha-helical structure or the structure of the beta-pleated layer. The position of the loops are provisions not included in the alpha-helical structure, or structure of the beta-pleated layer (van Holde, Johnson and Ho. Principles of Physical Biochemistry. Prentice Hall, New Jersey 1998, Chapter 1 pp 2-67, fully incorporated into the present description by reference). The position of the hinges are preferred, because the atoms of the skeleton, as a rule, are more flexible and less likely to be involved in the formation of hydrogen bonds compared to the atoms of the skeleton of the alpha-helices and beta-pleated layers. Therefore, the lengthening or shortening of the loop due to insertions or deletions of one or several amino acids is less likely to cause significant destructive changes of the Fc polypeptide, including stability, expression, or other difficulties.

Insertions and deletions can be used to change the length of the polypeptide. For example, in the areas of the loops change the loop length leads to altered flexibility and conformational entropy of the specified loop. Insertions in the loop as a whole will increase the conformational entropy of the loop, which can be defined as the Boltzmann constant multiplied by the natural logarithm of the number of possible conformations (van Holde, Johnson and Ho. Principles of Physical Biochemistr. Prentice Hall, New Jersey 1998, pp 78, fully incorporated into the present description by reference). By the insertion of at least one amino acid in the polypeptide increases the total number of conformations of the polypeptide. These additional conformations can be useful for the formation of appropriate interactions Fc/partner Fc binding, because the binding of Fc-binding protein may be used one additional conformations of the polypeptide Fc. In this case, the insertion can lead to stronger interactions Fc/partner binding of Fc. If additional conformations are not used in the field of the binding specified by the insertion may lead to weaker interactions Fc/partner binding to Fc because of the additional conformations will compete with conformation capable of binding. Similarly, deletion polypeptide segment also can lead to either stronger or weaker interactions Fc/partner binding of Fc. If the deletion of a segment, reducing the possible number of backbone conformations, excludes conformation capable of binding, this deletion can lead to weaker interactions Fc/partner binding of Fc. If the deletion does not preclude a conformation capable of binding, deletion can lead to a stronger interaction is the textbooks Fc/partner binding Fc, since this deletion can eliminate conformations competing with conformation able to bind.

Insertions and deletions can be used to change the position and orientation of the amino acids in the Fc polypeptide. Since insertions and deletions cause a change in the covalent structure of the skeleton, they inevitably cause a change in the positions of the backbone atoms. 7 compared the position of the skeleton in some segments of the loop, labeled L1-L4, in three different cores. The reference structure of the skeleton contains four segments of the loop, whereas in frame with the deletion does not contain the segment L1, and the segment insertions contains an additional segment before (i.e. N-terminal), the segment L1. Deletions and insertions cause the greatest change in the structure of the skeleton near the site of insertions or deletions. By deletion of the segment located near the N-terminal region of the loop, such as a segment of the L1 loop is shortened, and the remaining segments move their positions closer to the N-end of loop. This causes a shift in the segment L2 to the former location of the segment L1 and N-end of loop. This change in the position of the segment L2 in the direction of the segment L1 can enhance the binding of the complex Fc/partner binding to Fc and is preferred when there is prior information that the amino acid or amino acids located in the L2, implementing tlaut appropriate interaction partner binding Fc, in L1. For example, if L2 contains alanine and tyrosine, and the replacement of two amino acids segment L1 to alanine and tyrosine leads to Fc variant has an increased binding, deletion of L1 can be obtained Fc-variant with enhanced affinity to the binding partner Fc.

Similarly the insertion of polypeptide segments in the polypeptide Fc at the N-terminal region of the loop causes a shift in the positions of the segments of the loop to the C-terminal site of the loop. 7 the insertion of one or several amino acids before (i.e., N-end) segment L1 changes the conformation of the skeleton, including the offset of the segment L1 to the C-terminal region of the loop. This type of insertion is preferred when it is known that located in the segment L1 amino acids provide the appropriate interaction in provisions L2, as indicated by the insertion can lead to stronger interactions Fc/partner binding of Fc. If necessary, the weaker interaction of the Fc/partner binding Fc, can be used for the insertion offset the wrong amino acid to another position. Built-in, remote and reference segments (L1-L4 7) can represent one or several amino acids in the Fc polypeptide.

Alternatively, insertions or deletions can be used in the limit the second area of the hinge means, similar insertion or deletions in the N-terminal region loops. Insertions in the C-end of the loop can result in offset of provisions N-terminal with respect to insertions into the N end of the loop. Deletions in the C-end of the loop can result in offset of provisions N-terminal with respect to deletions, to the end of the loop. The choice of using insertions or deletions in the N-terminal or C-terminal region of the loop carried out on the basis of amino acids arranged in a loop having high or low affinity Fc/partner binding of Fc and required offset provisions.

Insertions or deletions can be used in any area of the Fc polypeptide, including loops, alpha-helical region and the beta-pleated layers. Preferred sites for insertions and deletions include the area of the loops, which represent the areas that are not alpha-helical or beta areas-folded layers. Loops are preferred, as they generally accept the changes of the skeleton better than alpha-helix or beta-pleated layers. The most preferred sites for insertions or deletions leading to stronger interactions, protein/protein, located on the N-terminal or C-terminal parts of the loop. If the side chain loops involved in the interaction of the Fc/partner binding Fc, insertions, or deletions to these sites with less likely to lead to highly destructive changes in binding interactions. As a result of deletions within the exact center of the loop is more likely destruction of important residues on the border Fc/partner binding of Fc, but as a result of insertions within the exact center of the loop more likely inappropriate interactions on the border Fc/partner binding of Fc. The number of removed or embedded residues can be determined by the extent of the necessary changes of the skeleton, while preferred are insertions or deletions of 15 or less residues, more preferred are insertions or deletions of 10 or less residues, and most preferred are insertions or deletions of 5 or less residues.

Once constructed, the position and size of the deletion Fc-variant, define the entire polypeptide sequence, and the polypeptide can be obtained by methods known in the art.

However, insertional Fc-options include additional construction phase sequence containing at least one amino acid to be inserted. The insertion of polar residues, including Ser, Thr, Asn, Gln, Ala, Gly, His, are preferred in the provisions that are expected to be exposed to the Fc polypeptide. Small amino acids, including Ser, Thr, and Ala are the most preferred because of the small size less likely STERI the Eski prevents interactions Fc/partner binding of Fc. Ser and Thr can also form hydrogen bonds with atoms partner Fc binding.

Insertions also have additional flexibility, resulting in a built-in polypeptide can be obtained in such a way as to form a suitable interaction partner binding of the Fc, which is necessary when you want a stronger binding of the Fc/partner binding of Fc. The length of the insertions in the skeleton can be determined by modeling the variance of the skeleton with a built-in simple typical sequence. For example, insertions polyserena, polyglycine or polyalanine of different lengths can be constructed and simulated. Modeling can be implemented in a variety of ways, including homology modeling based on the known three-dimensional structures of homologues containing the specified insertion, and by means of computer simulation, including MODELLER (M.A. Marti-Renom et al. Annu. Rev. Biophys. Biomol. Struct. 29, 291-325, 2000) and ROSETTA (Kuhlman et al. (2003). Science 302, 1364-8), both fully incorporated into the present description by reference. As a rule, first get different conformation of the skeleton, and the final structure of the skeleton can be determined after establishing the identity of the side chain. Side chains can be created using algorithms PDA® (US 6188965; 6269312; 6403312; 6801861; 6804611; 6792356, 6950754 and USSN 09/782004; 09/927790; 10/101499; 10/666307; 10/666311; 10/21802, all fully incorporated into the present description by reference).

Can be made insertions or deletions to change the binding of the Fc polypeptides with FR similarly described method changes the properties of the binding of FcRn. Domains Fc contact FR in the position shown in figure 1. The structure of the complex Fc/FR, including codes in PDB 1T89 and 1IIS (Radaev S et al. J. Biol. Chem. v276, p.16469-16477, fully incorporated into the present description by reference)demonstrate the interacting residues and the loop between the two structures. All the results of mutagenesis, such as shown in US 11/124620 and US 6737056 (both of which are fully incorporated into the present description by reference) are useful in certain appropriate offsets in the frame.

Insertions or deletions may be made at any polypeptide, in addition to the Fc polypeptides, methods set forth in the present description. For example, insertions or deletions in the member of the TNF superfamily,, APRIL, can be produced using the three-dimensional structure (code PDB 1XU1.pdb, Hymowitz, et al. (2005) J. Biol. Chem. 280:7218, fully incorporated into the present description by reference). Insertions or deletions can be made to increase the binding of APRIL with its receptor TACI. The remains of the loops, the preferred sites of insertions or deletions, are the remains of Ser118-Val124, Asp164-Phe167, Pro192-Ala198 Pro221-Lys226. Data loops interact with TACI in complex APRIL/TACI and mediates the binding.

Polypeptides, including variants.

Options IgG can be based on sequences of human IgG and thus the sequence of human IgG is used as a "base" sequences with which to compare the other sequences, including, without limitation, the sequences of other organisms, for example, the sequence of rodents and primates. Options also IgG can contain a sequence of other classes of immunoglobulins, such as IgA, IgE, IgD, IgM, etc. it is Assumed that despite the fact that options create IgG in the context of a single source of IgG variants can be created in the context of another, the second source of IgG or "transferred" to him. This is carried out by determining the "equivalent" or "relevant" residues and substitutions between the first and second IgG, usually on the basis of the homology sequence or structural homology between the sequences of IgG. To establish the homology of amino acid sequence of the first specified IgG directly compared with the sequence of the second IgG. After alignment of the sequences using one or more equalization programs homology, known in the art (for example, using a conservative who's remnants as an intermediate species), providing for necessary insertions and deletions to maintain alignment (i.e. avoiding the removal of conservative residues through arbitrary deletion and insertion), determine the residues equivalent to specific amino acids in the primary sequence of the first variant IgG. Alignment conservative residues preferably should retain 100% of such residues. However, the alignment of more than 75% or 50% conservative residues are also suitable for the determination of equivalent residues. Equivalent residues can also be determined by establishing the structural homology between the first and second IgG, i.e. at the level of tertiary structure for IgGs, patterns which have been identified. In this case, the equivalent residues defined as residues, for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the ancestor or predecessor (N on N, CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. The alignment is carried out after the orientation and location of the best models with the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the proteins. Regardless of how you define equivalent or corresponding residues and regardless of the identity of the source of IgG, which create IgGs, unchanged is still there, what are investigated IgG variants can be created in the second source of IgG, which has significant homology sequence or structural homology with the variant IgG. Thus, for example, if you get a variant antibody in which the antibody is a human IgG1, by using the above methods or other methods for determining equivalent residues, the variant antibody may be created in a different source IgG1 antibody that binds to another antigen, the source IgG2 antibody man, the original antibody IgA man, the original antibody IgG2a or IgG2b mouse, etc. Again, as described above, the context of the original variant IgG does not affect the ability to transfer IgG variants in other source IgGs.

Methods for creating, receiving and screening options IgG. The described methods are not limited to any particular use or method of application. Preferred methods generally illustrate that one or more of the IgG variants can be created, obtained and experimentally subjected to screening to obtain the IgG variants with optimized effector function. Describes the many ways of creating, receiving, and testing of variants of the antibodies and protein in USSN 10/754296 and USSN 10/672280, both of which are fully incorporated into the present description by the your reference.

Many ways to create a protein can be used to create variants of IgG with optimized effector function. In one of the embodiments may be used based on the structure of the creation method, in which the available structural information is used to control substitutions, insertions or divisions. In the preferred embodiment, can be used in a computer a method of screening, in which replacement is made on the basis of their energy matching in computer calculations. See, for example, USSN 10/754296 and USSN 10/672280 and the sources, all of which are fully incorporated into this description by reference.

Sequence alignment can be used to control the substitutions identified in the regulations. To a person skilled in the art it is obvious that the use of sequence information may limit the introduction of substitutions that are potentially damaging to the structure of the protein. The source sequences can vary widely and can include one or more well-known databases, including without limitation the Kabat database (northwestern University (Northwestern University); Johnson & Wu, 2001, Nucleic Acids Res. 29:205-206; Johnson & Wu, 2000, Nucleic Acids Res. 28:214-218), database IMGT (IMGT, the international ImMunoGeneTics information sstem®; Lefranc et al., 1999, Nucleic Acids Res. 27:209-212; Ruiz et al., 2000 Nucleic Acids Res. 28:219-221; Lefranc et al., 2001, Nucleic Acids Res. 29:207-209; Lefranc et al., 2003, Nucleic Acids Res. 31:307-310), and VBASE, all of which are fully incorporated into this description by reference. Information about the sequences of the antibodies can be acquired, compiled, and/or the resulting alignments geneticheskih sequences or sequences of naturally occurring antibodies of any organism, including, without limitation, mammals. To a person skilled in the art it is obvious that the use of sequences of human or substantially human origin may also have the advantage that they are less immunogenic when administered to humans. Other databases that represent a more General database of nucleic acids or proteins, i.e. not specific antibodies include, without limitation SwissProt, GenBank Entrez database EMBL nucleotide sequences. Aligned sequences can include sequences of VH, VL, CH and/or CL. There are numerous based on the sequence program alignment and methods known in the art, and all of them are used in obtaining the sequence alignment.

Alternatively, how random or polyloc logo mutagenesis can be used to create amino acid modifications in the required positions. In these cases, the position is chosen randomly or amino acid changes carried out under the simplified rules. For example, all the remnants can be subjected to mutation with getting alanine, mutagenesis is called "alanine scanning". These methods can be combined with more complex engineering approaches, which use selection methods for screening higher levels of differences between sequences. As is well known in the art, there are many filtering technologies that can be used for these approaches, including, for example, display technologies such as phage display, ribosomal display, the display on the cell surface, etc. as described below.

Methods of obtaining and screening of IgG variants are well known in the art. General methods of molecular biology, antibodies, expression, purification, and screening are described in Antibody Engineering, edited by Duebel &Kontermann, Springer-Verlag, Heidelberg, 2001; and Hayhurst &Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard &Georgiou, 2000, Annu Rev Biomed Eng 2:339-76. See also the methods described in USSN 10/754296; USSN 10/672280; and USSN 10/822231; and 11/124620, all of which are fully incorporated into this description by reference.

Preferred variants according to the present invention include variants that are listed on Fig. Alternatively, predpochtitel the s variants according to the present invention include variants, shown in Fig.9. Additionally, in the alternative, the preferred options in accordance with the present invention include variants, shown in figure 10. These options showed increased binding to the Fc receptor, FcRn, as illustrated in the examples.

Creating options IgG

Options IgG can be created by any method known in the art. In one embodiment of the sequence variants of IgG is used to produce nucleic acids that encode the sequence of States and which can then be cloned in the cells of the host, expressed and analyzed if necessary. These technologies are carried out using well known procedures and the number of ways that you can find them in the application described in Molecular Cloning - A Laboratory Manual, 3rdEd. (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001), and Current Protocols in Molecular Biology (John Wiley & Sons), both of which are fully incorporated into the present description by reference. Nucleic acids encoding the IgG variants may be included in the expression vector for the expression of the protein. The expression vectors typically contain a protein functionally linked, i.e. in a functional dependency, and control or regulatory sequences, breeding markers, any partner who apostrophes merger and/or additional elements. Options IgG can be obtained by growing the host cell, transformed with nucleic acid, preferably an expression vector, containing nucleic acid encoding options IgG, in appropriate conditions to induce or call the expression of the protein. Can be used with a wide range of suitable host cells, including, without limitation, mammalian cells, bacteria, insect cells and yeast. For example, many cell lines, which may find applications described in the catalog of cell lines ATSS, available from the American type culture collection (American Type Culture Collection), fully incorporated into the present description by reference. Methods of introducing exogenous nucleic acid into cells-the hosts are well known in the art and will vary depending on the host cell.

In a preferred embodiment, variants of IgG purified or emit after expression. Antibodies may be isolated or purified in a variety of ways known to specialists in this field of technology. Standard purification methods include chromatography, electrophoresis methods, immunological methods, deposition methods, dialysis, filtration, concentration and methods chromatofocusing. As is well known in the art, many nature of the different proteins bind antibodies, for example, the bacterial proteins A, G and L, and these proteins may find use in cleaning. Often cleaning can contribute to a specific partner in the merger. For example, proteins can be purified using glutathione resin if used fusion with GST, Ni+2-affinity chromatography if using His-tag, or immobilized antibody anti-flag if the use flag-tag. For General guidance about appropriate cleaning methods, see Antibody Purification: Principles and Practice, 3rdEd., Scopes, Springer-Verlag, NY, 1994, which is fully incorporated into the present description by reference.

Screening options IgG

Fc variants may be subjected to screening using a variety of methods, including, without limitation, the ways in which using in vitro, in vivo analyses and analyses of cell-based and technology selection. The technology of automatic screening and screening of high performance can be used in screening procedures. In screening can use a partner to merge or label, for example, immune label, isotope label, or with a small molecule, a label such as a fluorescent or colorimetric dye.

In a preferred embodiment, functional and/or biophysical properties of Fc variants is subjected to screening analyze in vitro. In a preferred embodiment, the protein is subjected to screening on the subject of functionality, for example, its ability to catalyze the reaction or its affinity to bind against the target.

As is known in the art, subgroups methods of screening are those who select the appropriate members of the library. In the present description these methods are called "selection methods, and these methods find use in the present invention in the screening of Fc variants. When protein libraries are screened using the method of selection, only those library members that fit, i.e. that meet the selection criteria, miss, isolated and/or watching. Many selection methods known in the art, and they can be used in the present invention for screening protein libraries. Other selection methods that may find use in the present invention include methods that do not rely on the display, such as methods in vivo. Subgroup of selection methods, called "directed evolution", is a means including mating or "crossing" of candidate sequences in the selection process, sometimes with the inclusion of new mutations.

In a preferred embodiment, Fc variants is subjected is treningu using one or more cell-based assays or in vivo analyses. For these analyses treated and untreated proteins, as a rule add from the outside so that the cells are exposed to individual variants or pooling options related to the library. These analyses usually, but not always, based on the function of the Fc polypeptide; i.e., the ability of the Fc polypeptide to contact the target and mediate some biochemical reaction, for example, effector function, inhibition of binding of a ligand/receptor, apoptosis, etc. These assays often involve monitoring the response of cells to IgG, for example, survival, cell death, changes in cellular morphology, or transcriptional activation such as cellular expression of the natural gene or gene-reporter. For example, these assays may measure the ability of D-variants to induce ADCC, ADCP or CDC. For some tests it may be necessary to add more cells or components, i.e. in addition to the target cells, for example, serum complement or effector cells, such as monocytes peripheral blood (RVMS), macrophages, natural killer cells, etc. These extra cells can be from any organism, preferably humans, mice, rats, rabbits and monkeys. Antibodies can induce apoptosis in some cell lines expressing the target, or could the t to mediate the attack target cells by immune cells, added to the analysis. Monitoring of the death or viability of cells known in the art and include the use of dyes, immunochemical, cytochemical and radioactive reagents. Transcriptional activation can serve as a method of analysis functions in the analysis of cell-based. Alternatively, screening, cell-based exercise using cells transformed or transfected with nucleic acids encoding variants. Ie Fc-variants do not add external to the cells.

Biological properties of IgG variants can be characterized based on data from experiments involving cells, tissues and the whole organism. As is known in the art, drugs are often tested on animals, including, without limitation, mice, rats, rabbits, dogs, cats, pigs, and monkeys, in order to measure the effectiveness of the drug in the treatment of a disease or disease model or to measure the pharmacokinetic characteristics, toxicity and other properties of the drug. These animals can be considered as models of diseases. Drugs are often tested on mice, including, without limitation, the nude mice, SCID mice, mice with xenografts and transgenic mice (including what I nocini and knockouts). As a result of these studies it is possible to obtain important data for identifying potential protein that will be used as a drug. For testing can be used any organism, preferably mammals. For example, monkeys because of their genetic similarity to humans may represent a suitable model for treatment and, thus, can be used to test the efficacy, toxicity, pharmacokinetic characteristics or other properties of IgGs. Ultimately, it is necessary to conduct tests on humans to permit use as medicines and, therefore, these experiments necessarily assumed. Thus, IgGs can be tested on humans to determine their therapeutic efficacy, toxicity, immunogenicity, pharmacokinetic characteristics and/or other clinical properties.

How to use options IgG

Options IgG can find application in a wide range of products. In one embodiment, the implementation of the variant IgG is a therapeutic, diagnostic or experimental reagent, preferably therapeutic. Variant IgG can be used in the composition of an antibody which is a monoclonal or polyclonal. In preference the sustained fashion the embodiment variants of IgG is used to destroy target cells, which generate the antigen target, for example, cancer cells. In an alternative embodiment, variants of IgG is used for blocking, call antagonism or agonism to the target antigen, for example to call antagonism to cytokine or cytokine receptor. In an alternative preferred embodiment, variants of IgG is used for blocking, call antagonism or agonism to the target antigen and destruction of target cells producing antigen-target.

Options IgG can be used for various therapeutic purposes. In a preferred embodiment, the antibody-containing variant IgG is administered to a patient to treat disorders associated with the antibody. For present purposes, the term "patient" includes humans and other animals, preferably mammals and most preferably humans. In the present description, the term "disorder associated with an antibody or disorder susceptible to antibody"or "condition"or "disease" means a disorder that can be alleviated by introducing a pharmaceutical composition comprising a variant IgG. Disorders associated with the antibody include, without limitation, autoimmune diseases, immunological diseases, infectious diseases, inflammatory diseases, neurological diseases and oncologicas is e, and neoplastic diseases, including cancer. In the present description, the terms "cancer" and "cancer" to describe or refer to the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, without limitation, carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumor, mesothelioma, sandamu, meningioma, adenocarcinoma, melanoma, leukemia and lymphomatosa.

In one embodiment, the implementation of the variant IgG is the only therapeutically active agent, administered to the patient. Alternatively, the variant IgG is administered in combination with one or more therapeutic agents, including but not limited to cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitors, antihormonal agents, kinase inhibitors, antiangiogenic agents, cardioprotector or other therapeutic agents. Options IgG can be introduced simultaneously using one or more treatment regimens. For example, the variant IgG can be administered to the patient along with chemotherapy, radiation therapy, or both chemotherapy and radiation therapy. In one embodiment, the implementation of the variant IgG can be put together with one or more antibodies, which may or may not be a variant IG. According to another variant implementation, the variant IgG and one or more other types of anticancer therapy used to treat cancer cells ex vivo. It is assumed that the specified treatment ex vivo may be appropriate in bone marrow transplantation and in particular autologous bone marrow transplantation. Necessarily assume that the variants of IgG can be used in combination with still other therapeutic techniques such as surgery.

Many other therapeutic agents may be injected with IgG variants. In one embodiment, the implementation of IgG injected together with the antiangiogenic agent. In the present description, the term "angiogenic agent" means a compound that blocks or to some extent prevents the growth of blood vessels. Antiangiogenic factor may, for example, be a small molecule or protein, e.g. antibody, fused with the Fc protein or cytokine binding to the growth factor or growth factor receptor involved in the stimulation of angiogenesis. In the present description of the preferred angiogenic factor is an antibody, to bind to the growth factor vascular endothelial (VEGF). In an alternate embodiment, the IgG is administered in conjunction with a therapeutic agent, inducing or enhancing the adaptive immune response, n is the sample, an antibody that attack affects CTLA-4. In an alternate embodiment, the IgG is administered together with a tyrosine kinase inhibitor. In the present description, the term "tyrosine kinase inhibitor" means a molecule, to some extent inhibitory tyrosinekinase tyrosine kinase activity. In an alternative embodiment, variants of IgG injected together with the cytokine.

Pharmaceutical compositions are assumed in the case when you get a variant IgG and one or more therapeutically active agents. The compositions of the IgG variants receive for storage by mixing the necessary IgG purity possible with pharmaceutically acceptable carriers, excipients or stabilizers (Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980, fully incorporated into the present description by reference) in the form of lyophilized formulations or aqueous solutions. These compositions for application for introduction in vivo, preferably are sterile. This is easily carried out by filtration through a membrane for sterile filtration or other means. Variants of IgG and other therapeutically active agents specified in this description can also be obtained in the form of immunoliposome and/or enclosed in microcapsules.

The concentration of therapeutically active variant IgG in the composition may vary about is about 0.1 to 100 wt%. In a preferred embodiment, the IgG concentration is in the range of 0.003 to 1.0 mol. For the treatment of a patient can be entered therapeutically effective dose of a variant IgG. In the present description, the term "therapeutically effective dose" means the dose that provides the necessary effects. The exact dose will depend on the goals of treatment will be determined by the specialist in the art using known methods. The dose may be in the range from 0.01 to 100 mg/kg body weight or higher, such as 0.01, of 0.1, 1.0, 10, or 50 mg/kg of body weight, the dose of 1-10 mg/kg is preferred. As is known in the art, may be necessary amendments to the destruction of proteins, systemic or localized delivery, and rate of synthesis of new proteases, as well as the age, body weight, General health, sex, diet, duration of administration, drug interaction and the severity of the condition, which can be determined by the specialist in the art using standard research.

Introduction pharmaceutical composition comprising a variant IgG, preferably in the form of a sterile aqueous solution may be carried out in a variety of ways, for example, without limitation, orally, subcutaneously, intravenously, parenterally, intranasally, in the ear, inside the eye, rectal, who Aguinaldo, transdermal, topical (e.g., gels, salves, lotions, creams, etc.), administered intraperitoneally, intramuscularly, intra-lungs (for example, inhalation AERx® manufactured by Aradigm, or delivery system in light Inhance® produced by Nektar Therapeutics, etc). The drug is indicated in the present description, may be introduced simultaneously with other drugs, i.e. drugs that are specified in the present description, can be assigned in conjunction with other therapies or medicines, including for example, small molecules, and other biological drugs, radiation therapy, surgery, etc.

Examples

Below are examples to illustrate the present invention. These examples do not limit the present invention to any particular use or application method. For all positions, described according to the present invention, numbering is given according to the EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, fully incorporated into the present description by reference). For specialists in the field of technology relating to antibodies, it is clear that this agreement consists of the inconsistent numbering in specific areas of immunoglobulin sequence, providing a standardized reference to con ervative provisions in the collections of the immunoglobulin. Accordingly, the provisions of any specific immunoglobulin defined by the EU index will not necessarily be consistent with its ordinal sequence.

Example 1. The creation of DNA, expression and purification of Fc-options

Fc variants were created using the Fc domain of IgG1 man and variable domain trastuzumab (Herceptin®, Genentech). The Fc polypeptides were part of alemtuzumab, antibodies anti-HER2 or AS. Alemtuzumab (Grade®registered trade mark of the Millenium) is humanitariannet monoclonal antibody currently approved for treatment of b-cell chronic lymphocytic leukemia (Hale et al., 1990, Tissue Antigens 35:118-127, fully incorporated into the present description by reference). Trastuzumab (Herceptin® is a registered trademark of Genentech) is an antibody anti-HER2/neu for the treatment of metastatic breast cancer. Sequences of the heavy and light chains of the antibody anti-HER2 shown in Fig. AS is a monoclonal antibody anti-CD30. Variable region of Herceptin were collected using recursive PCR. Then this variable region was cloned from human IgG1 in the vector pcDNA3.1/Zeo(+) (Invitrogen), shown in figure 11. Plasmids were propagated in cells of E. coli One Shot TOP10 (Invitrogen) and purified using a set of Hi-Speed Plasmid Maxi Kit (Qiagen). Defined p is the sequence of the plasmids and the result confirmed the presence of the cloned inserts.

Carried out site-directed mutagenesis using the method Quikchange™ (Stratagene). Plasmids containing the necessary substitutions, insertions and deletions, reproduce in the cells of E. coli One Shot TOP10 (Invitrogen) and purified using a set of Hi-Speed Plasmid Maxi Kit (Qiagen). Sequenced DNA and confirmed the sequence.

Plasmids containing heavy chain gene (VH-Cγ1-Cγ2-Cγ3) (wild type or variants), was co-transfusional with plasmid containing light chain gene (VL-κ), in cells C. Medium was harvested 5 days after transfection and antibodies were purified from the supernatant using affinity chromatography with protein A (Pierce). Characteristics of the binding protein And some modified Fc shown in Fig. The antibody concentration was determined by analysis using bicinchoninic acid (ICA) (Pierce).

Example 2. Measure the affinity of binding

The binding polypeptides Fc with Fc ligands were analyzed by measurements of surface plasma resonance. Measurement of surface plasma resonance (SPR) was performed using the instrument BIAcore 3000 (BIAcore AB). Antibody wild-type or variant antibody recorded using immobilized protein L (Pierce Biotechnology, Rockford, IL) and measured the binding of the receptor with the analyte. Protein L is covalently linked with a touch h the SIP CM at a concentration of 1 μm in 10 mm sodium acetate, pH 4.5 on the sensor chip CM using N-hydroxysuccinimide/N-ethyl-N'-(-3-dimethylaminopropyl) carbodiimide (NHS/EDC) at a flow rate of 5 ál/min Flow cell 1 touch each chip was treated with NHS/EDC as a negative control binding. The movable buffer was a 01 M HEPES pH of 7.4, 0.15 M NaCl, 3 mm EDTA, 0.005% and about./about. surfactant P20 (HBS-EP, Biacore, Uppsala, Sweden), and the buffer regeneration of the chip was a 10 mm glycine-HCl pH to 1.5. 125 nm antibody anti-HER2 wild-type or variant antibody anti-HER2 was associated with protein L chip SM in HBS-EP at a speed of 1 μl/min for 5 minutes. Analyte FcRn-His-GST, FcRn, coupled with His-tag, glutathione-S-transferase in serial razvedennyh 1 of 250 nm was injected for 20 minutes of Association, 10 minute dissociation in HBS-EP at a speed of 10 ál/min Reaction, measured in units of the resonance (RU), received through 1200 seconds after the introduction of the receptor, reflecting colocaciones binding. The cycle only with the antibody and buffer provided the baseline analysis. Built graphics according to EN 1/log concentrations and customized to a sigmoidal dose-response relationships using curvilinear regression using GraphPad Prism.

The binding polypeptides Fc with Fc ligands also carried out using the AlphaScreen technology™ (Amplified Luminescent Proximity Homogeneous Assay). AlphaScreen™ is a non-radioactive Lumine the interest proximity analysis on the basis of beads. The excitation laser donor beads leads to the excitation of oxygen, which in the case reasonably close to the acceptor bead, will cause a cascade of chemiluminescent events, ultimately leading to fluorescence emission at 520-620 nm. The main advantage of AlphaScreen™ is its sensitivity. As one donor bead emits up to 60,000 of the excited oxygen molecules per second, the gain of the signal is extremely large, which can detect up to attomolar (10-18) levels. Antibody wild type was biotinilated standard methods for attachment to streptavidin coated donor bead and labeled ligand Fc, for example, FcRn, Rshaman or Protein a, was associated with glutathione chelate acceptor beads. AlphaScreen™ was used as a analysis direct linking, in which the interaction of the Fc/Fc ligand bring the donor and acceptor beads with obtaining the measured signal. In addition, AlphaScreen™ was used as a competitive analysis for screening the received Fc polypeptides. In the absence of competing polypeptides Fc antibody wild-type and FcRn interact and give a signal at 520-620 nm. Unlabeled Fc domains compete with the interaction of the wild-type Fc/FcRn, quantitatively reducing the fluorescence, which facilitates the determination of the values of the relative affine the STI to bind.

Example 3. The ability to bind FcRn Fc-options.

The affinity for binding to IgG1 Fc to FcRn was measured using a variant antibodies using AlphaScreen technology™. The Fc polypeptides were part of Alemtuzumab or Trastuzumab. Alemtuzumab (Grade®, Ilex) is humanitariannet monoclonal antibody currently approved for treatment of b-cell chronic lymphocytic leukemia (Hale et al., 1990, Tissue Antigens 35:118-127, fully incorporated into the present description by reference). Trastuzumab (Herceptin®, Genentech) is an antibody anti-HER2/neu for the treatment of metastatic breast cancer.

Received data contention AlphaScreen™ and the measured relative binding of Fc variants compared with the antibody wild type in 0.1 M sodium phosphate pH 6.0 m with 25 mm sodium chloride. Examples of signal AlphaScreen™ as a function of competing antibodies shown in Fig. Curves 12 for options, curves P257L, P257N, V279E, V279Q, V279Y, ^281S, E283F, V284E, L306Y, T307V, V308F and Q311V demonstrate higher affinity, because the curve of each variant is shifted in the left direction relative to the curve of the wild type. Data competition AlphaScreen™ Fc variant according to the present invention are summarized in Fig and 14. Additional data competition AlphaScreen™ in 0,1M phosphate sodium pH of 6.0 with 125 mm sodium chloride, summero the Ana on Fig. The data relative binding to FcRn variants compared to wild type. Values greater than one show improved binding of Fc variants to FcRn compared to a wild type. For example, options E283L and V284E have 9.5 and 26-fold stronger binding than wild type, respectively. Measurement of surface plasmon resonance of many variants also show increased binding to FcRn as shown in Fig and 16.

At position 257 all the options that are missing amino acid, Proline, and a substituted amino acid without the N skeleton of the covalent bond of the side chain, give the hull more flexibility, providing more space for the Fc domain to the best of FcRn binding. In particular, variations in the position 257 to L and N have a strong binding to FcRn at pH 6, demonstrating that chetyrekhtomnym side chain and the pattern of gamma branching side chain helps the Fc domain to carry out productive, i.e. strong interaction with FcRn. Position 308 interacts with the position 257. Both of these provisions in turn communicate with the N310, which is directly included in the interaction of the Fc/FcRn (table 2, Burmeister et al (1994) Nature 372:379-383, fully incorporated into the present description by reference). Fc-variants V308F and V08Y demonstrate a 2.9-fold and 4.3-fold increase in the affinity to FcRn compared to a wild-type (is high). Position 279 and 385 interact with FcRn as options V279E, V279Q and V279Y, and G385H, and G385N all have a stronger interaction with FcRn. These options are all presented relative to amino acids that can form hydrogen bonds. Area series Fc human IgG1 containing various modifications according to the present invention, shown in Fig.

Fc-variant N434Y has the strongest binding to FcRn at pH of 6.0, as shown in Fig. One option N434Y has a 16-fold increased binding. The combination of this option with other modifications led to stronger binding. For example, P257L/N434Y, ^281S/N434Y and V308F/N434Y demonstrate 830-fold, 180-fold and 350-fold increase in binding to FcRn.

Example 4. Variants containing insertions and deletions.

Carried insertions and deletions that alter the strength of interactions Fc/FcRn, and measured their ability to bind various ligands Fc. Created Fc variant containing an embedded Ser residue between residues 281 and 282 using EU numbering Kabat et al, with increasing ability to bind to FcRn Fc domain. This variant is denoted as ^281S, the "^" means the insertion after the specified position. Data AlphaScreen™, showing improved binding ^281S shown in fig.12b and 21A. Built-in sequence that can transform the conduct of a few residues, indicated after the number position. Fc-variant was created in Kappa, the antibody anti-HER2 IgG1-trastuzumab (Herceptin®, Genentech) using the methods described in the present description. The insertion at the site between residues 281 and 282 leads to the displacement of the residues of the loop Fc, C-terminal to residue 281, With the end of the loop and changing the position of the side chain. Fc-variants containing substitutions at positions 282, 283 and 284 showed that C-terminal offset this loop was appropriate (see Fig). Also created another variant, a deletion N286, sometimes referred to N286#, resulting in the shifted position of this loop, bind FcRn. This option shows increased binding to FcRn at pH 6,0 (fig.14b).

Data AlphaScreen™ demonstrate the binding of the variant ^281S and other variants to FcRn. These data AlphaScreen™ received in the result of the analysis of direct linking. Higher levels of chemiluminescent signals exhibit a stronger binding. Because concentrations of options increase in this analysis, receive stronger signals. The data at pH of 6.0, figa and 17b show an increased affinity ^281S, P257L, P257L/^281S (combined version with the replacement/insertion) and other variants compared to wild-type Fc. Also presents a double substitution T250Q/M428L, previously shown as having increased time Pology is neither in monkeys (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216, fully incorporated into the present description by reference). One insertion ^281S leads to increased binding of the Fc/FcRn. In addition, ^281S also leads to increased binding P257L when these two modifications combine option P257L/^281S, as shown on the data of ~40 nm. Data on Fig to show that these variants do not show increased binding to FcRn at pH 7.0. Reduced affinity at pH 7.0 is required for increased time half-life in vivo, as it helps to release the Fc polypeptides of the FcRn in the extracellular space, an important step in recycling Fc.

Experiments by the method of surface plasmon resonance also exhibit improved binding ^281S with FcRn. On Fig shows the units of the response resulting from the binding of various Fc variants to FcRn on the surface of the chip. After complete bonding option with the chip unit response register and marked on the ordinate. The insertion of ^281S demonstrates binding capacity comparable with other options that are specified in the present description as having high affinity to FcRn compared to a wild-type (as examples, see Fig, 14 and 15).

Option deletions containing a deletion N286, N286#, also shows a high affinity to FcRn compared to a wild type. This variant features a 2.0-fold increase is receiving affinity to FcRn, as shown in Fig. These data also represent data AlphaScreen™, the resulting competitive experiment at pH of 6.0. These options are used for inhibiting the binding of Fc wild-type, connected with the donor bead, FcRn, is connected to the acceptor beads. It was necessary in two times fewer free N286#, compared with wild-type Fc, for the inhibition of binding of donor/acceptor beads through complex Fc/FcRn. This demonstrates two times more durable binding N286# compared to wild type.

Other Fc variants that contain insertions or deletions, have low affinity to FcRn. Option insertions ^254N has significantly reduced binding to FcRn relative to expected, based on the nature and provisions of this option. In this embodiment, the insertion, Asn, located in the middle of the loop, FcRn binding. This insertion has only 1.1% of the affinity from the affinity of binding of wild-type (Fig).

Example 5. Combined variants with altered characteristics in relation to FcRn and FR

As shown in fig.13b for antibodies anti-HER2 Fc-variant P257L has a high affinity to FcRn compared with WT. P257L gave an average value of 2.6-fold increase in the affinity to FcRn in relation to human FcRn, pH 6.0 phosphate buffer and added with 25 mm NaCl. In is the result of adding I332E or S239D/I332E to option P257L received double and triple options, P257L/I332E and S239D/P257L/I332E, retaining high affinity to FcRn. Variant S239D/I332E has essentially unaltered binding to FcRn compared to a wild-type, as shown in the analysis of AlphaScreen™ fig.14b. Data double and triple variants had a 5 - and 4-fold higher affinity. Options I332E and S239D/I332E have improved binding FR, in particular with FRIII (Cm. US 11/124620, which is fully incorporated into the present description by reference). Binding capacity FR some variants according to the present invention shown in Fig. Binding capacity of protein And some variants according to the present invention shown in Fig. The protein binding And are often used in purification of proteins containing the Fc. Replacement V308F also enhances the binding of FcRn at pH 6,0 (fige). V308F has a 3-fold increased affinity as a replacement in the antibody anti-HER2, trastuzumab (Herceptin™, Genentech), and also has a high affinity for combining with substitutions that increase the binding FR, such as I332E, S239D/I332E and S298A/E333A/K334A (Lazar et al. 2006 Proc. Nat. Acad. Sci USA. 103(111):4005-4010, Shields et al. 2001 J. Biol. Chem. 276:6591-6604, both fully incorporated into the present description by reference). Increased binding G385H with FcRn is also saved when combined with improves the binding FR substitutions, especially in the variant slim replacement S239D/I332E/G385H.

Variants with increased binding to FcRn can be combined with options for reducing or eliminating binding FR and protein complement C1q. Improved binding to FcRn increases the effect of protecting the receptor, leading to increased time-life. Proteins containing the Fc can also be introduced into cells and metabolized through their interaction with FR and protein C1q. If the interaction of the Fc/FcR and F/protein C1q are not necessary for the effectiveness of antibodies, can be made deletions of these interactions. Deletions of these interactions can also reduce the destructive effect of the receptor, thereby also leading to increased time-life. In particular, options 234G, 235G, 236R, 237K, 267R, 269R, A, 325L and 328R (US 11/396495, fully incorporated into the present description by reference) can be combined with options that improve the binding of FcRn to obtain variants with improved affinity for FcRn and low affinity to FR or C1q. These include 235G/257C, A/N, 325A/257L, 234G/308F, 234G/434Y and 269R/308F/311V. These options can be obtained in the domains of the IgG1 Fc, despite the fact that reduced interaction with FR or C1q can also be obtained by introducing these mutations into proteins containing the Fc domains IgG2, IgG4 or IgG3. Making modify the Nations FcRn, such as 257N, 257L, M, 308F, 311V, IgG2 decreases the binding FR and increased interaction with FcRn.

Variants with reduced binding to FcRn may be combined with the variants with increased binding to FR or C1q. Reduced binding to FcRn, combined with increased binding FR, it may be useful to increase the number containing Fc protein available for call of effector functions. The decrease in binding to FcRn may lead to the reduction of the number containing Fc protein sequestered FcRn, and thus influence the bioavailability. Modifications such as I253V, S254N, S254# (deletion 254), TN and H435N, reduce binding to Fc/FcRn (Fig) and can be combined with the variants with improved binding FR, such as S239D, I332E, NE, G236A. The resulting Fc domains, such as domains, containing I253V/S239D/I332E, I332E/H435N or S254N/H268E have reduced binding to FcRn and increased binding FR.

Variants with reduced binding to FcRn may be combined with the variants with reduced binding FR. This combination reduced binding to FcRn and FR is useful in applications such as videografia, which contains Fc protein is subjected to tracking of radioactive or toxic aptly is. In the ideal case, the half-life of the protein containing radioactive label, similar to the half-life of the radionuclide itself. This ensures the removal of the label from the body at the same time as the decay of a radionuclide. Reduced interaction FR also provide optimal accessibility containing Fc protein target. For example, if containing Fc protein is an antibody, the decrease in binding FcR allows a greater number of antibodies to be available for antigen. Combination of options that affect FcRn and FR, such as 235G/254N, 236R/435N, 269R/I253V suitable for this application.

Example 6. Fc-variants in the binding of an antibody OST577 with human FcRn.

OST577 is an antibody to surface antigen of hepatitis b virus (Ehrlich et al. (1992) Hum. Antibodies Hybridomas 3:2-7, fully incorporated into the present description by reference). Sequences of the heavy and light chains were taken from the Kabat database with KADBID 000653 (heavy) and KADBID 007557 (light) (Martin AC, Proteins. 1996 Mau; 25(1):130-3, fully incorporated into the present description by reference). DNA encoding these heavy and light chains, were synthesized by Blue Heron Biolotechnology, Bothell, WA. Antibodies OST577 wild-type and variant antibodies OST577 expressed and purified as in the case of anti-HER2 (trastuzumab) in EXAMPLE 1. Analyses linking Biacore™ is sushestvovali, as described in EXAMPLE 2, with the hybrid protein FcRn person/glutathione-D-transferase (GST), attached to the chip surface. As shown in Fig, Fc-variants according to the present invention have altered binding to FcRn person. Variants with increased binding, it is easier to join FcRn on the surface and cause a greater increase in units response (RU's). All options shown with modifications in binding to FcRn, have a high affinity to FcRn compared to a wild-type protein. These include P257L, P257N, V308F, N434Y, P257L/N434Y and P257L/V308F. It was shown that the variant with the third largest EN at 975 seconds, T250Q/M428L, increases the half-life of antibodies OST577 in macaques (Hinton et al. 2004 Journal of Biological Chemistry 279(8):6213-6216, Hinton et al. 2006 Jounal of Immunology 176:346-356, both fully incorporated into the present description by reference). In the specified data set included the antibody contains a constant region of the heavy chain hybrid IgG1/IgG2, containing substitutions S239D/I332E. As described in the example. 5, the data replacement increases the affinity of the specified antibodies to FR. As shown in Fig, these substitutions do not alter the ability to bind FcRn, as Biacore™ traces hybrid S239D/I332E overlap with traces of wild-type, containing Kappa and lambda domains CL1.

Example 7. The affinity of Fc variants to human FcRn, monkey and mouse.

Fc variants of the antibody anti-HER2, the trastuzumab was obtained as described in example 1. Traces of surface plasma resonance (SPR) was obtained as described in example 2, except that FcRn human, macaque or mouse attached to the chip surface. Were the two curves CPD for each Fc-variants with different amounts of GST-FcRn attached to the surface. Each curve was fitted to the model binding Langmuir and two Kd values obtained were averaged to obtain a characteristic value for each pair variant receptor. The results are presented on Fig as fold increase in Kd compared with wild-type trastuzumab. For example, option V308F/Q311V has 3.4 times stronger binding to human FcRn compared to a wild type. V308F/Q311V also has a 3.7 and 5.1 times more durable binding to FcRn monkey and mouse, respectively. It was shown that M428L variant increases the half-life of antibodies (Hinton et al. 2004 Journal of Biological Chemistry 279(8):6213-6216, fully incorporated into the present description by reference) and has a 2.4-, and 2.0 and 2.1-fold increased binding to FcRn human, monkey and mouse, respectively. Other options including P257L, P257N, N434Y, Q311V, V308F, V308F/N434Y, P257L/V308F and P257L/N434Y, also exhibit increased binding at pH of 6.0.

Example 8. Fc-Rn options in different domains Speakers.

Variants according to the present invention can be included in l is a constant battle domain using technologies of molecular biology and cleaning, mentioned in the present description, including the one described in example 1. Amino acid sequence of the constant domains of IgG1, IgG2, IgG3 and IgG4 can be used, as shown in figure 2. Additionally, there may be used a combination of two or more different constant domains. For example, on Fig are some of the modifications included according to the present invention in a hybrid IgG1 and IgG2. This hybrid contains the domain SN IgG2 and domains CH2 and CH3 IgG1. IgG3 has a shorter half-life in humans, compared to IgG1, IgG2 and IgG4 (7 days, compared to ~21 day, Janeway, Travers, Walport, Shiomchik. Immunology, 5thed. Garland Publishing, c2001, 4-16, fully incorporated into the present description by reference) and therefore is required in some applications.

Example 9. Obtaining variants in antibody anti-VEGF.

Antibodies anti-VEGF with altered binding was obtained using the methods described in the present description, including the example 1. Heavy chain of anti-VEGF wild-type contains the following amino acid sequence:

Light chain of anti-VEGF wild-type contains the following amino acid sequence:

Separate and combined variants with altered binding include the options shown in Fig that the demon is Trichet received options the amount used of cultural environments and the resulting output options antibodies. The numbering of these options are given according to the EU index as in Kabat et al. Options listed on Fig received either IgG1 or hybrid VH containing sequences of both IgG1 and IgG2. These options contain a variable region that binds the VEGF antigen. Assessment of all proteins by exclusion chromatography and electrophoresis gel in the presence of sodium dodecyl sulfate showed a purity of >90%.

Example 10. The half-life variants of antibodies in vivo

Investigated in mice pharmacokinetic characteristics of the antibodies of the wild type and variant antibodies. Used mouse not expressed FcRn mouse (mouse B6-FcgrtTm1Dcr) and were heterozygous with respect to "Nokia human FcRn (hFcRn Tg - transgene), as described in Petkova et al. International Immunology 2006 Dec; 18(12):1759-69. Petkova et al showed that N434A variant has an increased half-life in mice with "Nokin human FcRn, which is consistent with earlier results showing that N434A variant has an increased half-life in monkeys (application USA No.11/208422, publication number US 26067930 A1). 9-12 week old female mice were injected intravenously by injection of 2 mg/kg of the antibodies in groups of 6 mice per one antibody. Blood samples were collected after one hour and 1, 4, 8, 11, 15, 18, 21, 25 and 28 days of orbiting the bend. The concentration of each antibody in the serum was measured using enzyme-linked immunosorbent assay (ELISA), "sandwich"-type using anti-human antibody Fc and detection using europium.

On Fig shows the results of studies that are representative data from two separate studies. Shown are average and standard deviation of the mean values for the four samples. Obviously, option V308F has a longer half-life, remaining at measurable concentrations of up to 25 days. Wild type and variants P257L and P257N appear faster, with measurable concentrations of 15, 8 and 4 days, respectively. Serum concentration as a function of time customized to compartmental model using the software package WinNonLin (Pharsight Inc). The final half-life of options V308F was 4.9 days, while the final half-life of wild-type and variants P257L and P257N was 3.0, 1.9 and 0.9 days, respectively. The area under the curves (CPD) options V308F were 129 day*μg/ml, while the area under the curves of wild type and variants P257L and P257N was 70, 38 and 38 day*μg/ml, respectively.

Example 11. Experiments on binding to FcRn at pH of 6.0.

Options anti-VEGF according to the present invention were tested for their ability to bind to human FcRn with, and is Alisov Biacore, as described in example 2, with some modifications. FcRn man was covalently attached to the chip CM in 10 mm sodium acetate, pH 4.5, using N-hydroxysuccinimide/N-ethyl-N'-(-3-dimethylaminopropyl)carbodiimide (NHS/EDC) at a flow rate of 5 μl/min Used human FcRn contained GST and HIS-tagged variant for easier cleaning and other analyses. Approximately 3300 EN FcRn was attached to the chip. Flow cell 1 was treated with NHS/EDC as a negative control binding. The movable buffer consisted of 25 mm phosphate buffer pH of 6.0, 150 mm NaCl, 3 mm EDTA and 0.005% vol./about. surfactant P20. Antibodies were washed away with the chip FcRn with the same buffer at a pH of 7.4, which was quickly deleted all the tested variants. Traces of the Association and dissociation biacore customized to conformational exchange model to calculate the apparent equilibrium constants for the binding, Kd.

These results demonstrate that the variant V308F and many other variants have enhanced binding to FcRn. For antibodies anti-VEGF wild-type Kd was 18 nm, which differs significantly from the values according to Dall' Acqua et al (Dall' Acqua et al Journal of Immunology 2002, 169:5171-5180) due to differences in the analysis and reconciliation of data. Presents the format of the analysis led to reproducible results if the chip FcRn used soon after receipt. However, the chip FcRn was split PR is using, possibly due to the dissociation of one of the two circuits FcRn from the surface. The results show altered binding of these variants compared to anti-VEGF wild-type. On Fig shows fold increase of the strength of binding compared with control wild-type. Values greater than one indicate that the variant antibody has a higher affinity for FcRn than the wild-type protein. Option V308F, for example, binds FcRn 4.5 times more durable than the antibody wild type. Option V308F/M428L binds FcRn 12.3 times more durable, and option T307P/V308F binds FcRn at 3.16 times more strongly than the wild-type protein. The options shown in Fig not possess reduced affinity to FcRn compared to a wild-type (values were less than 1.0). Option N434S has an affinity to bind to FcRn 4.4 times more powerful than the wild type, comparable with V308F.

Example 12. Experiments binding transmembrane FcRn.

The alpha chain of FcRn and cDNA beta-2-microglobulin was ordered from OriGene Technologies Inc (Rockville, MD) and were transfusional cells T and this resulted in the expression of a functional FcRn on the cell surface. 20 µg Fcgrt and 40 µg DNA beta-2-microglobulin was transfusional using lipofectamine (Invitrogen Inc.) and the cells were left to grow for three days in DMEM media with 10% serum ultra low IgG content. Also grew the control of the s cells, not transfetsirovannyh two chains FcRn. Different amounts of antibodies anti-VEGF (wild type and variants) was associated with the cells for 30 minutes in 25 mm phosphate buffer pH of 6.0, 150 mm NaCl, 0,5% BSA, and then 6-9 times washed in 25 mm phosphate buffer pH of 6.0, 150 mm NaCl, 0,5% BSA plus 0,003% igepal. After washing, antibodies were fixed on the surface by treatment with linking 1% PFA. Then, the bound antibodies were detected using Fab'2labeled D, the Fab domains of human and measured the mean fluorescence intensity (MFI) using a BD FACS Canto II. The average values of two samples for each antibody presented on Fig. The curves match the data on Fig not provide interpretable values IS, as many curves do not form the upper base line when the saturation of the cells. However, antibodies can be ranked in order of their affinity to bind by submitting log[option]where MFI equal to 3000, EC(MFI=3000). Using this indicator antibodies can be listed in order from strongest affinity to FcRn to the weak affinity to FcRn as follows: V308F/M428L, V259I/V308F, T250I/V308F, T250Q/M428L, N434S, T307Q/V308F, P257L, T307S/V308F, V308F, T256V/V308F, V308F/L309Y and wild type.

Example 13. Features options, 434S.

Antibodies containing the modification 434S, have the most suitable properties, which make them the preferred option and in accordance with the present invention. In IgG1 human remnant wild type represents asparagine, Asn in position 434 so that this option can be specified N434S in the context of IgG1 or other Fc domains containing Asn, N at position 434. In General, this option may be denoted simply 434S. In the present description option 434S was successfully obtained in the antibody anti-HER2, trastuzumab, and the antibody anti-VEGF.

Ser at position 434 has the ability to form hydrogen bonds with FcRn, either directly or indirectly, i.e. mediated by water or molecules of the dissolved substance. Gamma-oxygen Ser at position 434 is located close to the carbonyl oxygen atoms Gly131 and Pro134 FcRn molecules, as shown in Fig. On Fig shows a model of the Fc domain of a human in complex with human FcRn. The domain Fc in the specified model contains replacement 434S, hence the residue 434 in the Ser on Fig. The model created using technologies such as PDA® (Dahiyat and Mayo, Protein Sci. 1996 May; 5(5):895-903), the crystal structure of the Fc domain of the rat associated with FcRn rats (Martin et al. Mol Cell. 2001 Apr; 7(4):867-77), and Pymol (Delano Scientific). The small size of the Ser fits easily between the two proteins.

Variant antibodies N434S has a 4.4-fold increased affinity for binding with respect to FcRn compared with the antibody wild type, as shown by measurements of the system Biacore™ (Fig). This option also played the duty to regulate increased binding to FcRn, associated with the surface of the cells, as shown by dimension counting cells (Fig).

Based on the results, shown in Fig and 30, assume that the preferred options containing 434S and other modifications include V308F/434S, 428L/434S, 252Y/434S, 259I/308F/434S, 250I/308F/434S, and 307Q/308F/434S.

Example 14. Additional options

Additional options can be based on data contained in the present description and literature (Dall' Acqua et al Journal of Biological Chemistry 2006 Aug 18; 281(33):23514-24; Petkova et al. International Immunity 2006 Dec; 18(12):1759-69; Dall' Acqua et al Journal of Immunology 2002, 169:5171-5180; Hinton et al, Journal of Biological Chemistry 2004 279(8):6213-6216; Shields et al. Journal of Biological Chemistry 2001 276(9):6591-6604; Hinton et al. Journal of Immunology 2006, 176:346-356, all fully incorporated into the present description by reference). These options include options that are listed on Fig.

Based on the results, shown in Fig and 30, and results Dall' Acqua et al (Journal of Biological Chemistry 2006 Aug 18; 281(33):23514-24 included in the present description by reference), the preferred options include Y319L, T307Q, V259I, M252Y, V259I/N434S, M428L/N434S, V308F/N434S, M252Y/S254T/T256E/N434S, M252Y/S254T/T256E/V308F, M252Y/S254T/T256E/M428L, V308F/M428L/N434S, V259I/V308F/N434S, T307Q/V308F/N434S, T250I/V308F/N434S, V308F/Y319L/N434S, V259I/V308F/M428L, V259I/T307Q/V308F, T250I/V259I/V308F, V259I/V308F/Y319L, T307Q/V308F/L309Y, T307Q/V308F/Y319L and T250Q/V308F/M428L.

Based on the results, shown in Fig and 30, the preferred options include Y319L, T307Q, V259I, M252Y, V259I/N434S, M428L/N434S, V308F/N434S, V308F/M428L/N434, V259I/V308F/N434S, T307Q/V308F/N434S. T250I/V308F/N434S, V308F/Y319L/N434S, V259I/V308F/M428L, V259I/T307Q/V308F, T250I/V259I/V308F, V259I/V308F/Y319L, T307Q/V308F/L309Y, T307Q/V308F/Y319L and T250Q/V308F/M428L.

While specific embodiments of the present invention have been described above to illustrate, for specialists in the art it is obvious that can be done numerous variations of the details in the scope of the present invention, which is defined by the attached claims. The contents of all of these sources are fully included in the present description.

1. Polypeptide that demonstrates increased binding to FcRn person specified polypeptide contains a variant Fc polypeptide Fc of human IgG, where the specified variant Fc contains an isoleucine at position 259, phenyalanine in position 308, where the specified variant Fc exhibits increased binding to FcRn compared with the specified Fc polypeptide, where the numbering is given according to the EU index in Kabatet al.

2. Polypeptide that demonstrates increased binding to FcRn person specified polypeptide contains a variant Fc polypeptide Fc of human IgG, where the specified variant Fc contains an isoleucine at position 259, phenyalanine in position 308, where the specified polypeptide further comprises one or more of the following modifications: 428L, 434S, 307Q, 319L, 250I, where the specified variant Fc shows increased hydrogen bonds is the W with human FcRn compared with the specified polypeptide Fc man the numbers are given according to the EU index in Kabat et al.

3. The polypeptide containing the variant Fc polypeptide according to any one of claims 1 and 2, where the specified polypeptide is an antibody, fused with the Fc protein or immunoadhesin.

4. The polypeptide containing the variant Fc polypeptide, according to claim 3, where this option contains the Fc region of IgG1 or Fc region of IgG2, or Fc region IgG3, or Fc region of IgG4.

5. The polypeptide containing the variant Fc polypeptide according to any one of p-4, where the specified polypeptide has a longer half-life in serum of the mammal compared with the protein containing the Fc region of the wild type.

6. The polypeptide containing the variant Fc polypeptide, according to claim 5, where the specified mammal is a human.

7. The polypeptide containing the variant Fc polypeptide according to any one of p-6, which is an antibody having specificity to an antigen selected from the group consisting of a growth factor, vascular endothelial (VEGF), tumor necrosis factor alpha (TNF-α), CD25, a receptor of the epidermal growth factor (EGFR) and IgE.

8. The polypeptide containing the variant Fc polypeptide according to any one of p-7, designed for use as a medicine.

9. The host cell for production of the polypeptide containing the variant Fc, according to any one of claims 1 to 8, the host cell contains a nucleic acid encoding the polypeptide, the soda is containing the specified variant Fc, according to any one of claims 1 to 8.

10. A method of obtaining a polypeptide containing the variant Fc, according to any one of claims 1 to 8, the method involves providing cells containing a nucleic acid encoding a specified polypeptide, where the specified cell is cultivated in suitable for expression of the indicated polypeptide conditions.

11. Nucleic acid encoding a polypeptide according to any one of claims 1 to 8.



 

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Fused rage proteins // 2513695

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biochemistry. Claimed is fused protein for treating diseases, mediated by advanced glycation end products (AGE), consisting of a fragment of a version of human receptor of advanced glycation end products (RAGE), which has two point mutations H217R and R221H, and a fragment of constant domain of human immunoglobulin IgG4, joined with linker if necessary. In addition, considered are: nucleic acid and recombinant host cell for obtaining fused protein, as well as pharmaceutical composition for treatment of AGE-mediated diseases, which contain fused protein.

EFFECT: invention ensures lower aggregation of fused protein.

13 cl, 19 dwg, 3 ex, 9 tbl

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biotechnology, in particular to novel peptide analogue of insulin-like growth factor-1 (IGF-1), which contains amino acid substitution of methionine in position 59 on Asn, Leu, Nle, Ile, Arg, A6c, Glu, Trp or Tyr, as well as other additional substitutions, inserts and deletions. Said peptide or its pharmaceutically acceptable salt is used in composition of pharmaceutical composition for treatment of IGF-1-mediated diseases, as well as in method of treating dwarfism.

EFFECT: invention makes it possible to obtain IGF-1 analogue-agonist, possessing higher biological activity with respect to native IGF-1.

17 cl, 2 tbl

FIELD: medicine.

SUBSTANCE: present group of inventions relates to biotechnology. What is presented is a humanised anti-CD79b antibody and its antigen-binding fragment produced of murine antibody MA79b and CD79b having a substantially analogous binding affinity thereto. A polynucleotide, a vector, a host cell and a method for producing the anti-CD79b antibody according to the invention; immunoconjugates, compositions and methods for cell growth inhibition, a method of treating an individual suffering cancer, a method of treating a proliferative disease and tumour in a mammal, a method for B-cell proliferation inhibition; a method for detecting the presence of CD79b in a sample and method for binding the antibody to the CD79b expressing cell are also disclosed.

EFFECT: given invention can find further application in therapy of the CD79b associated diseases.

86 cl, 20 tbl, 9 ex, 51 dwg

Anti-mif antibodies // 2509777

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and immunology. Invention discloses a monoclonal antibody and its antigen-binding parts which specifically bind the C-end or central part of the macrophage migration inhibitory factor (MIF). The anti-MIF antibody and its antigen-binding part further inhibit biological function of the human MIF. The invention also describes an isolated heavy and light chain of immunoglobulins obtained from anti-MIF antibodies, and molecules of nucleic acids which encode such immunoglobulins.

EFFECT: disclosed is a method of identifying anti-MIF antibodies, pharmaceutical compositions containing said antibodies and a method of using said antibodies and compositions for treating diseases associated with MIF.

22 cl, 14 dwg, 16 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to creation of recombinant plasmids providing expression of poly-epitopic tumour-associated antigens in dendritic cells capable of stimulation of specific cytocidal cells, and it may be used in medicine. Recombinant plasmid DNA pCI-UB-POLYEPI contains 11 epitopes of tumour-associated antigens of colorectal cancer, its size is 6 355 n. p. and it expresses the following amino acid sequence: DYKDDDDK-LLGVGTFVV-ADRIW-GLKAGVIAV-AAYARY-VLAFGLLLA-ADRIW-YQLDPKFITSI-AAYARY-IMIGVLVGV-ADRIW-YLSGADLNL-AAYARY-CGIQNSVSA-AAYARY-LLLLTVLTV-ADRIW-QYIKANSKFIGlTEL-ANIY-SIINFEKL-ARY-SASFDGWATVSVIAL-ARY-SERVRTYWIIIELKHKARE-ARY-IQNDTGFYTLHVIKSDLVNEE. Mature dendritic cells obtained by adding to immature dendritic cells of pro-inflammatory TNF-α (tumour necrosis factor) cytokine are transfected by constructed plasmid DNA pCl-UB-POLYEPI thus activating them. Then activated dendritic cells are cultured together with peripheral mononuclear blood cells of people sick with colorectal cancer for generation of antigen-specific antitumour cytocidal cells.

EFFECT: invention allows efficient generation of antigen-specific cytocidal cell with antitumour activity in vitro, required for immune response by the 1-st type T-helper to colorectal cancer antigens.

2 cl, 1 dwg, 4 ex

Anti-axl antibodies // 2506276

FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed are monoclonal antibodies which bind to the extracellular domain of receptor tyrosine kinase AXL and which at least partially inhibit AXL activity, as well as antigen-binding fragments. Also provided is an isolated nucleic acid molecule, a host cell and a method of producing a monoclonal antibody and an antigen-binding fragment thereof, as well as use of the monoclonal antibody or antigen-binding fragment thereof to produce a drug, pharmaceutical compositions, a method of diagnosing and a method of preventing or treating a condition associated with expression, overexpression and/or hyperactivity of AXL.

EFFECT: invention can be used in therapy and diagnosis of diseases associated with AXL.

23 cl, 20 dwg, 24 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically binds heparin-binding EGF-like growth factor (HB-EGF) and its antigen-binding fragment. Invention describes a nucleic acid molecule, an expressing vector, a host cell and a method for obtaining an antibody or its antigen-binding fragment, as well as use of antibody or its antigen-binding fragment for obtaining pharmaceutical composition for diagnostics, prevention or treatment of hyperproliferation disease, methods and sets for diagnostics and prevention or treatment of the state associated with HB-EGF expression. This invention can be further found in therapy of diseases determined with or related to HB-EGF expression.

EFFECT: improving efficiency of composition and treatment method.

34 cl, 43 dwg, 28 ex, 12 tbl

FIELD: biotechnologies.

SUBSTANCE: invention describes polynucleotide, expression vector, host cell and production method of humanised antibody together with their use, as well as medical preparation against rheumatoid arthritis, prophylaxis or treatment method of rheumatoid arthritis and use of humanised antibody at production of pharmaceutical preparation for prophylaxis or treatment of rheumatoid arthritis. This invention can be used in therapy of human diseases associated with α9 integrin.

EFFECT: improved activity and thermal stability.

14 cl, 6 dwg, 6 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed are versions of an isolated monoclonal antibody, specific to hGM-CSF, where each version is characterised by a heavy and light chain. Each of the versions is characterised by the fact that it contains six appropriate CDR. Described are: a pharmaceutical composition, and a set, representing medication, based on the antibody application. Disclosed are: a coding isolated nucleic acid, an expression vector, containing it, and a vector-carrying host cell, used for the antibody obtaining. Described is a method of obtaining the antibody with the cell application.

EFFECT: claimed inventions can be applied for treating disease or disorder, associated with superexpression of hGM-CSF.

25 cl, 9 dwg, 14 tbl, 15 ex

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