Stable and soluble antibodies

FIELD: medicine.

SUBSTANCE: invention relates to field of biochemistry, in particular to antibody or its antigen-binding fragment, which is specifically bind with human TNFα. Also disclosed are: separated molecule of nucleic acid, which codes said antibody, vector of expression, which contains said molecule of nucleic acid, and host cell, which contains said vector, for expression of said antibody. Disclosed are pharmaceutical composition for treatment of TNFα-mediated disease, which contains therapeutically effective quantity of said antibody, and method of treating TNFα-mediated disease with application of claimed composition.

EFFECT: invention makes it possible to effectively treat TNFα-mediated diseases.

16 cl, 9 dwg, 2 tbl, 2 ex

 

Cross-reference to related applications

According to §119 section 35 of the U. S. according to this application claims priority of the provisional application for U.S. patent No. 61/405798, filed October 22, 2010, and 61/484749, filed may 11, 2011, the full contents of which are herein by reference.

Area of technology

The present invention relates to methods for reducing the ability of antibodies to aggregation and to antibodies that are modified to reduce the ability to aggregate. The present invention also relates to antibodies that bind to the factor alpha tumor necrosis (TNFα). In particular, the present invention relates to a stable and soluble antibody that contains a modification that reduces the ability of aggregation, including to antibodies in the form of scFv and Fab fragments, which contain specific sequences of light chain and heavy chain, which is optimized in terms of stability, solubility and low immunogenicity. In addition, the present invention relates to methods of diagnosis and/or treatment of TNF-mediated disorders.

Background of the invention

Factor alpha tumor necrosis (TNFα, also known as cachectin) is a natural mammalian cytokine produced by numerous cell types, including Mont�the cítov and macrophages in response to endotoxin or other stimuli. TNFα is a key mediator of inflammation, immunological, and pathophysiological reactions (Grell, M., et al. (1995) Cell, 83: 793-802).

Soluble TNFα is formed by the cleavage of a transmembrane protein precursor (Kriegler, et al. (1988) Cell 53: 45-53), and synthesized polypeptides with a molecular mass of 17 kDa gather in soluble homotrimeric complexes (Smith, et al. (1987), J. Biol. Chem. 262: 6951-6954; for reviews TNFα see Butler, et al. (1986), Nature 320: 584; Old (1986), Science 230: 630). These complexes then bind to receptors on many cells. The binding causes a large number of proinflammatory effects, including (i) the release of other proinflammatory cytokines, such as interleukin(IL)-6, IL-8 and IL-1, (ii) the release of matrix metalloproteinases and (iii) increased expression of molecules involved in adhesion to endothelium, which additionally enhance the cascade of inflammatory and immune responses through the involvement of leukocytes in extravascular tissues.

A large number of disorders associated with elevated levels of TNFα, many of them are important in the field of medicine. It is established that the expression of TNFα are elevated in several human diseases, including chronic diseases such as rheumatoid arthritis (RA), inflammatory bowel disease, including Crohn's disease and ulcerative colitis, sepsis, congestive with�technol failure, asthma and multiple sclerosis. Transgenic mice carrying the gene for human TNFα, produce high levels of TNFα constitutive and developing wearing a spontaneous, destructive polyarthritis with similarities to RA (Keffer et al. 1991, EMBO J. 10, 4025-4031). Therefore, TNFα called proinflammatory cytokine.

Currently it is generally accepted that TNFα is a key factor in the pathogenesis of RA, which is a chronic, progressive and debilitating disease characterized by inflammation and destruction of multiple joints, systemic symptoms are fever and malaise and fatigue. RA also leads to chronic inflammation of the Bursa, usually with progression to joint cartilage and bone destruction. Elevated levels of TNFα detected in synovial fluid and peripheral blood of patients suffering from RA. When administered to patients suffering from RA, means that block TNFα, they reduce inflammation, relieve symptoms and slow joint damage (McKown et al. (1999), Arthritis Rheum. 42: 1204-1208).

Physiologically TNFα has also been associated with protection against specific infections (Cerami et al. (1988), Immunol. Today 9:28). TNFα is released by macrophages that have been activated by lipopolysaccharides of gram-negative bacteria. As such, TNFα, apparently, is meant having basic�e endogenous mediator involved in the development and pathogenesis endotoksicski shock associated with bacterial sepsis (Michie, et al. (1989), Br. J. Surg. 76: 670-671; Debets. et al. (1989), Second Vienna Shock Forum, p. 463-466; Simpson, et al. (1989) Crit. Care Clin. 5: 27-47; Waage et al. (1987). Lancet 1: 355-357; Hammerle. et al. (1989) Second Vienna Shock Forum p. 715-718; Debets. et al. (1989), Crit. Care Med. 17: 489-497; Calandra. et al. (1990), J. Infect. Dis. 161: 982-987; Revhaug et al. (1988), Arch. Surg. 123: 162-170).

It is found that, as in the case of other organ systems, TNFα also plays a key role in the Central nervous system, in particular in inflammatory and autoimmune disorders of the nervous system, including multiple sclerosis, Guillain-Barre, and myasthenia gravis, and degenerative disorders of the nervous system, including Alzheimer's disease, Parkinson's disease and Huntington's disease. TNFα is also implicated in disorders related systems of the retina and the muscles, including optic neuritis, macular degeneration, diabetic retinopathy, dermatomyositis, amyotrophic lateral sclerosis and muscular dystrophy, as well as in damage to the nervous system, including traumatic brain injury, acute spinal cord injury and stroke.

Hepatitis is another associated with TNFα inflammatory disease that can be caused, among other triggers, disease-causing viruses, including Epstein-Barr, cytomegalovirus and virus GE�of atit A-E. Hepatitis is a cause of acute inflammation of the liver in the region of the portal vein and the area of the slices, followed by fibrosis and tumor development. TNFα may also mediate cachexia in cancer, which is the most frequent cause of mortality in cancer (Tisdale, M. J. (2004), Langenbecks Arch surg. 389: 299-305).

The key role played by TNFα in inflammation, cellular immune response and pathology of many diseases has led to the search of the TNFα antagonists. One of the classes of TNFα antagonists intended for the treatment of TNFα-mediated diseases, are antibodies or fragments of antibodies that bind specifically to TNFα and thereby inhibit their action. Using antibodies against TNFα was found that blocking TNFα can eliminate the effects ascribed to TNFα, including, reduces IL-1, GM-CSF, IL-6, IL-8, adhesion molecules and tissue destruction (Feldmann et al. (1997), Adv. Immunol. 1997: 283-350). Among the specific inhibitors of TNFα, which recently went on sale, monoclonal, chimeric antibody is a mouse-human directed against TNFα (infliximab, RemicadeTM; Corporation Centocor/Johnson & Johnson) has demonstrated clinical efficacy in the treatment of RA and Crohn's disease. Despite these achievements, there remains a need for new and effective forms of antibodies or other antibodies for treatment associated with TNFα violations, such as RA. Frequent�spine, there is an urgent need for antibodies with optimal functional properties for an effective and durable treatment of arthritis and other TNFα-mediated disorders.

A brief summary of the invention

The present invention relates to antibodies comprising at least one reducing aggregation mutation, and to methods of producing such antibodies.

In one aspect of the present invention relates to methods for reducing the ability of antibodies to aggregation, comprising administering one or more reducing aggregation of modifications in the position of the residue involved in the interaction between the variable domain light chain and a variable domain heavy chain antibody, and substitution reduces the free energy between the variable domain light chain and the variable domain of the heavy chain of at least 0.5 kcal/mol, and therefore it decreases the aggregability of the modified antibodies as compared to that of the original antibody, in which no reducing aggregation modification(s).

In one aspect of the method according to the present invention includes the introduction of one or more amino acid substitutions in the region of contact of the variable domain of the light chain (VL) and the variable domain of the heavy chain (VH) of the antibody, and one or more substitutions are in positions of residues in�abusive to reduce free energy between the VL and VH by at least 10%, in a decrease in the ability to aggregation of the antibody compared to the original antibody. In a particular aspect, the sequence of the variable domain of the light chain of the antibody has at least 65% identical to the sequence SEQ ID NO:1. In other aspects, the sequence of the variable domain of the heavy chain at least 85% identical to the sequence SEQ ID NO:3 or the sequence SEQ ID NO:4.

In some aspects the method according to the present invention includes the modification of the residue at position 50 in accordance with the AHo numbering system and/or residue at the position 47 in accordance with the AHo numbering system variable domain of the light chain of the antibody, and therefore it decreases the ability to aggregation of the antibody compared to the original antibody. In other aspects the method according to the present invention, furthermore, includes the modification of residues in positions 12, 103 and 144 in accordance with the AHo numbering system variable domain of the heavy chain.

The present invention also relates to antibodies with reduced aggregability containing one or more reducing aggregation of modifications. In some aspects the antibody of the present invention is a Fab, Fab', F(ab)'2, single-chain Fv (scFv), Fv fragment, or a linear antibody. In other aspects the present invention relates to especif�cal or bivalent molecule, containing the antibody of the present invention.

In other aspects of reducing aggregation modification is at position 50 in accordance with the AHo numbering system variable domain of the light chain. In a specific aspect of reducing aggregation modification contains arginine (R) at position 50 in accordance with the AHo numbering system variable domain of the light chain. However, in another aspect, the reducing aggregation modification includes the replacement of lysine (K) to arginine (R) at position 50 in accordance with the AHo numbering system variable domain of the light chain.

However, in other aspects of reducing aggregation modification is in position 47 in accordance with the AHo numbering system variable domain of the light chain. In a specific aspect of reducing aggregation modification contains arginine (R) at position 47 in accordance with the AHo numbering system variable domain of the light chain. However, in another aspect, the reducing aggregation modification includes the replacement of lysine (K) to arginine (R) at position 47 in accordance with the AHo numbering system variable domain of the light chain.

The present invention also relates to a stable and soluble antibodies specific for TNFα, which contains an unusual sequence of light chain and heavy chain, which is optimized in terms of stability, �astronote, in vitro and in vivo binding to TNFα, and low immunogenicity. These antibodies are intended for diagnostic and/or treatment of TNFα-mediated disorders. The invention also relates to nucleic acids, vectors and cells-the hosts for expression of recombinant antibodies, the variable domains of light chains and variable domains of the heavy chains of the present invention, and to methods for their selection and use of these antibodies in medicine.

The present invention also relates to methods of treating TNFα-mediated disorders, comprising administering to the individual a pharmaceutical composition containing the antibody against TNFα according to the present invention. In some aspects TNF-alpha-mediated disorder is an ocular disease selected from the group consisting of uveitis, disease Behcet, retinitis, dry eye, glaucoma, Sjogren syndrome, diabetic neuropathy, scleritis, age-related macular degeneration and keratitis.

Specific preferred embodiments of the present invention will become apparent from the following more detailed description of some preferred embodiments and the claims.

Brief description of the drawings

Fig. 1 shows the titration curves for the provisions of residues VL47 (solid lines) and VL50 (dotted lines) in two different molecules�x scFv, 34rFW1.4 (black) and 578rFW1.4 (grey).

Fig. 2A demonstrates the stability 34rFW1.4 in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration of 60 mg/ml.

Fig. 2B demonstrates the stability 34rFW1.4_VLK50R_DHP in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration of 60 mg/ml.

Fig. 3A demonstrates the stability 34rFW1.4 in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration equal to 40 mg/ml.

Fig. 3B demonstrates the stability 34rFW1.4_VLK50R_DHP in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration equal to 40 mg/ml.

Fig. 4A demonstrates the stability 34rFW1.4 in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration equal to 20 mg/ml.

Fig. 4B demonstrates the stability 34rFW1.4_VLK50R_DHP in accelerated conditions, as determined by analysis with use�using size exclusion HPLC after incubation for 2 weeks at 40°C and using concentration, of 20 mg/ml.

Fig. 5A demonstrates the stability 34rFW1.4 in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration of 60 mg/ml.

Fig. 5B demonstrates the stability 34rFW1.4_VL_K50R in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration of 60 mg/ml.

Fig. 6A demonstrates the stability 34rFW1.4 in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration equal to 40 mg/ml.

Fig. 6B demonstrates the stability 34rFW1.4_VLK50R in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration equal to 40 mg/ml.

Fig. 7A shows the stability 34rFW1.4 in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration equal to 20 mg/ml.

Fig. 7B shows the stability 34rFW1.4_VLK50R in accelerated conditions, determined by analysis using size exclusion PLC after incubation for 2 weeks at 40°C and using concentration, of 20 mg/ml.

Fig. 8A demonstrates the stability 34rFW1.4 in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration of 60 mg/ml.

Fig. 8B demonstrates the stability 34rFW1.4_K47R in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration of 60 mg/ml.

Fig. 9A demonstrates the stability 34rFW1.4 in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration equal to 20 mg/ml.

Fig. 9B demonstrates the stability 34rFW1.4_K47R in accelerated conditions, determined by analysis using size exclusion HPLC after incubation for 2 weeks at 40°C and using a concentration equal to 20 mg/ml.

Detailed description of the present invention

The overall objective of the present invention is to provide a stable and soluble antibodies with a reduced ability to aggregation in solution. In a preferred embodiment implementation, the specified antibody is an antibody in the form of scFv or Fab fragment. The antibodies of the present invention, preferably, in�exclude light and heavy chains, disclosed in the present description.

Presented in the present description, a detailed description is an example and is intended only for illustrative discussion of preferred embodiments of the present invention, and is shown for the sake of providing what is believed to be the most rational and understandable description of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no attempt is made to show structural details of the present invention in more detail than is necessary for understanding principles of the present invention, on the basis of the description in conjunction with the drawings and/or examples to the person skilled in the art will understand how can be implemented in practice, some forms of the present invention.

For understanding of the present invention below defines some of the terms. Additional definitions are set forth throughout the detailed description. The following definitions and explaining how implied and can be assumed to be preferable to any further interpretation, unless they are not expressly modified in the following examples, or unless their use makes any interpretation meaningless or essentially meaningless. Vtech cases when the interpretation of the term would be meaningless or essentially meaningless, the definition should be taken from Webster dictionary, 3 rd edition or dictionary, known to specialists in this field, such as Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Used in the present description, the term "antibody" encompasses full-sized antibodies and any antigen-binding fragment (i.e., "antigen-binding portion "of an antigen-binding polypeptide" or "immune binder") or single chain. "Antibody" includes a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion. Each heavy chain comprises variable region heavy chain (in the present description abbreviated as VH) and the constant region of the heavy chain. The constant region of the heavy chain consists of three domains, CH1, CH2 and CH3. Each light chain comprises a variable region light chain (in the present description abbreviated as VL) and a constant region light chain. The constant region of the light chain consists of one domain, CL. VHand VL-region can be further subdivided into areas of hypervariability called complementarity determining sites (CDR), located interspersed with areas that �are more conservative called frame regions (FR). Each VHand VLis composed of three CDR and four FR located from amino end to the carboxyl end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant region of the antibodies may mediate the binding of the immunoglobulin with the tissues or host factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term "antigen-binding portion" of an antibody (or simply "part of the antibody") refers to one or more fragments of an antibody that retain the ability to specific binding with the antigen (e.g., TNF). It was found that the function of antibody related to antigen binding, can perform a full-sized fragments of the antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include (i) Fab fragment, a monovalent fragment consisting of the VL- VH-, CL and CH1 domains; (ii) F(ab')2-a fragment, a bivalent fragment comprising two Fab fragment linked by disulfide bridge at the hinge region; (iii) Fd fragment consisting of the VHand CH1 domains; (iv) Fv fragment consisting of the VLand VHdomains on�nogo antibodies shoulder, (v) a single domain or dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of VHdomain; and (vi) selected determines the complementarity plot (CDR) or (vii) a combination of two or more selected CDRs which may optionally be connected by a synthetic linker. Furthermore, although the two domains of Fv fragment, VLand VH, are encoded by separate genes, they can be combined using the methods of creating recombinant molecules via a synthetic linker that enables you to create them as a single protein chain in which the VLand VHregion coupled with the formation of monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). It is assumed that such single-chain antibodies are also included within the term "antigen-binding portion" of an antibody. These fragments of antibodies get, using conventional methods known to experts in this field, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding parts can be obtained by recombinant DNA methods or by using enzymatic or chemical cleavage of intact immunoglobulins. Antibodies may be antibodies of different isotypes, for example, an antibody of the IgG isotype (e.g., subtypes IgG1, IgG2, IgG3 and�and IgG4), IgA1, IgA2, IgD, IgE or IgM.

The term "framework region" refers to adopted in the field of parts of the variable regions of the antibody are used, which is more divergent CDR-plots. Such a frame region is usually called the frame regions 1 through 4 inclusive (FR1, FR2, FR3 and FR4) and provide a frame for holding, in three-dimensional space, the three CDRs found in variable regions of the heavy or light chain of the antibody, so that the CDRs can form the antigen-binding surface. Such a frame region can also be called the skeletons, since they serve as a support for the presentation of more divergent CDR. As the antigen-binding molecules can be used by other CDR and framework region of the immunoglobulin superfamily, such as Ancienne repeats and fibronectin (see also, e.g., U.S. patent No. 6300064 and 6815540 and publication of the patent application U.S. No. 20040132028).

The term "epitope" or "antigenic determinant" refers to the place on the antigen to which an immunoglobulin or antibody specifically binds (e.g., TNF). An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

The terms "specific binding", "selective binding", "selectively binds�I" and "specifically binds" refers to binding of an antibody to an epitope of a given antigen. Typically, the antibody binds with an affinity (KD), approximately less than 10-7M, for example, approximately less than 10-8M, 10-9M or 10-10M or less, as determined using the technology using surface plasmon resonance (SPR) in a tool BIACORE.

The term "KD"refers to the equilibrium constant by the dissociation of the specific interaction of antibody with antigen. In some some embodiments, the antibodies of the present invention bind to TNF with the equilibrium constant by the dissociation (KD), constituting less than approximately 10-7M, e.g., less than approximately 10-8M, 10-9M or 10-10M or less, e.g., as defined using the technology using surface plasmon resonance (SPR) in a tool BIACORE.

As used in the present description, "identity" refers to the convergence of sequences of two polypeptides, molecules or of two nucleic acids. If both of the two compared sequences is the same Monomeric subunit in the form of bases or amino acids (for example, if a position in each of two DNA molecules is adenine, or the situation in each of two polypeptides is �ISIN), then the molecules are identical at that position. "Percent identity" of two sequences depends on the number of congruence, shared by the two sequences divided by the number of compared positions, x 100. For example, if the same 6 out of 10 positions in two sequences, the two sequences are identical at 60%. As an example, the DNA sequence CTGACT and CAGGTT identical 50% (match 3 of a total of 6 positions). Typically, the comparison is performed after aligning the two sequences to achieve the maximum identity. Such alignment can be provided using, for example, the method of Needleman and others ((1970) J. Mol. Biol. 48: 443-453), just implemented in computer programs such as the Align program (DNAstar, Inc.). The percent identity of two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which is incorporated into the ALIGN program (version 2.0), using a table of weight PAM120 residue, the penalty for the length of the skip = 12 and the penalty for pass = 4. In addition, the percent identity of two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)), which is incorporated into the GAP program in the GCG software package (available on the website http://www.gcg.com), using either a Blossum matrix 62, or �the connected PAM250, and the weighting factor for the passage of equal 16, 14, 12, 10, 8, 6 or 4, and the weight ratio of length equal to 1, 2, 3, 4, 5 or 6.

"Similar" sequences are sequences that, when aligned, have identical and similar amino acid residues and similar residues are conservative substitutions for the respective amino acid residues in an aligned reference sequence. In this regard, a "conservative substitution" of a residue in a reference sequence is replaced by a residue that is physically or functionally similar to the corresponding residue in the reference sequence, for example, which has similar size, shape, electric charge, chemical properties including the ability to form covalent or hydrogen bonds, etc. Thus, "modified as a result of the conservative(s) substitutions (substitutions)" sequence is a sequence that differs from the serial control or wild-type sequence that has one or more conservative substitutions. "Percent identity" of two sequences depends on the number of provisions that contain matching residues or conservative substitutions shared by the two sequences divided by the number of compared positions, x 100. On�reamer, if 6 out of 10 positions in two sequences are the same, and in 2 of the 10 provisions are conservative substitutions, the two sequences are similar by 80%.

Used in the present description, the term "conservative modifications" sequence, as expected, refers to modifications of amino acids which do not adversely affect the binding characteristics of the antibody containing the amino acid sequence, or do not change them. Such conservative modifications of the sequence includes substitutions, additions and deletions of nucleotides and amino acids. For example, modifications can be entered using standard methods known in this field, such as site-directed mutagenesis and mutagenesis using PCR. Conservative amino acid substitutions include substitutions, where amino acid residue substituted with amino acid residue having a similar side chain. In this area were installed family amino acid residues having similar side chains. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, sistei�, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a specific antibody is preferably replaced with another amino acid residue from the same family of side chains. Methods for determining the nucleotide and amino acid conservative substitutions which do not annul the binding with the antigen are well known in the art (see, e.g., Brummell et al, Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

As used in the present description, "amino acid consensus sequence" refers to amino acid sequences that can be obtained using a matrix of at least two, and preferably more, aligned amino acid sequences, and providing gaps in the alignment, so that you can determine the most frequently occurring amino acid residue at each position. A consensus sequence is a sequence that contains amino acids, which are represented with the highest frequency in each�position. In the case where two or more amino acids are represented equally in one position, the consensus sequence consists of two or all of these amino acids.

Amino acid sequence of the protein can be investigated at different levels. For example, persistence or variability can be manifested at the level of a single residue, the level of many residues, the set of residues with gaps, etc. Residues may be identical persistence of residue, or may be stored at the class level. Examples of classes of amino acids include polar, but uncharged R-groups (serine, threonine, asparagine and glutamine); positively charged R-groups (lysine, arginine and histidine); negatively charged R-groups (glutamic acid and aspartic acid); hydrophobic R groups (alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine) and specific amino acids (cysteine, glycine and Proline). The person skilled in the art known to other classes, and they can be defined using determinants of the structure or other data to determine substitutability. In a sense, the replaced amino acid may be any amino acid that can be substituted and which retains the functional conservatism in this position.

However, it will be understood that the biophysical properties of amino�slot one and the same class may differ in degree. For example, it will be understood that some of the hydrophobic R-groups (e.g., alanine, serine or threonine) are more hydrophilic (i.e., are more hydrophilic or less hydrophobic) than other hydrophobic R-groups (e.g., valine or leucine). The relative hydrophilicity or hydrophobicity can be determined using recognised in this area (see, e.g., Rose et al., Science, 229: 834-838 (1985) and Cornette et al., J. Mol. Biol, 195: 659-685 (1987)).

As used in the present description, in order to align one of the amino acid sequence (for example, the first sequence VHor VL) with one or more additional amino acid sequences (e.g., one or more sequences of the VHor VLin the database) the amino acid position in one sequence (e.g., the first sequence VHor VL) can be compared to a "corresponding position" in one or more additional amino acid sequences. As used in the present description, "the relevant provision" is an equivalent position in the compared sequence(s) after optimal alignment of sequences, i.e., after aligning the sequences to achieve the maximum percent identity or percent similarity.

The term "nucleic acid molecule" refers to DNA molecules or RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. Nucleic acid is "functionally linked" when it is set to its functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is functionally linked to the coding sequence if it affects the transcription of this sequence. In some embodiments, the present invention relates to a selected nucleic acid molecules that encode an antibody of the present invention, the variable domain light chain of the present invention and/or variable domain of the heavy chain of the antibody of the present invention. In some embodiments, the nucleic acid molecule of the present invention encodes a polypeptide that contains a variable region light chain that is at least 97% identical to SEQ ID NO:2 or SEQ ID NO:14; a polypeptide containing the variable region of the heavy chain that is at least 95% identical to SEQ ID NO:5; or an antibody that is at least 96% identical to SEQ ID NO:10 or SEQ ID NO:17.

The term "vector" refers to a nucleic acid molecule capable pyo�Enos another nucleic acid, with which it is connected. One type of vector is a "plasmid", which refers to the loop of circular, double-stranded DNA, which can be ligitamate additional segments of DNA. Another type of vector is a viral vector, where additional DNA segments can be ligitamate into the genome of the virus. Some vectors are capable of Autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial start replication, and epilimnia vectors mammals). Other vectors (e.g., napisanie vectors mammals) can be integrated into the genome of the host cell after introduction into a host cell and replicated along with the host genome.

The term "host" refers to a cell into which has been introduced an expression vector. The host cell may include bacterial, microbial cells, plant cells or animal. Bacteria that are amenable to transformation, include members of the Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. Suitable microbes include Saccharomyces cerevisiae and Pichia pastoris. Suitable cell lines of animal hosts include CHO cells (cell line of Chinese hamster ovary) cells and NS0.

The terms "treat" and "treatment" refer to therapeutic or preventative measures, description�referred to in the present description. In the methods of "treatment" is used the introduction of the antibodies of the present invention to the individual in need of such treatment, for example, an individual with a TNFα-mediated violation, or the individual in whose result may occur such violation, for the prevention, treatment, stitching the onset, reduce the severity or reduce the intensity of one or more symptoms of a violation, or repeated violations, or to increase the life span of the individual beyond their expected duration in the absence of such treatment.

The term "TNF-mediated breach" generally refers to painful conditions and/or symptoms associated with TNF, including any breach, for the occurrence, progression or the persistence of symptoms of which requires the involvement of TNF. Examples of TNF-mediated disorders include, but are not limited to, age-related macular degeneration, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, retrolental p., breast carcinoma, lung carcinoma, gastric carcinoma, esophageal carcinoma, colorectal carcinoma, liver carcinoma, carcinoma of the ovary, coma, arrhenoblastoma, cervical carcinoma, endometrial carcinoma, endometrial hyperplasia, endometriosis, fibrosarcoma, choriocarcinoma, head and neck cancer, carci�WMD nasopharynx, carcinoma of the larynx, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, carcinoma of the pancreas, retinoblastoma, astrocytoma, glioblastoma, Swannanoa, oligodendroglioma, medulloblastoma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, carcinoma of the urinary tract, thyroid carcinoma, Wilms tumor, renal cell carcinoma, carcinoma of the prostate, abnormal proliferation of cells of the vascular wall associated with phakomatoses, edema (such as that associated with brain tumors), Meigs syndrome, rheumatoid arthritis, psoriasis and atherosclerosis. TNF-mediated disorders also include dry eyes and associated with TNFα inflammatory processes, such as inflammation of the eye, allergic conjunctivitis, dermatitis, rhinitis and asthma, for example, and includes those cellular changes that are the result of the activity of TNFα, which leads directly or indirectly to associated with TNFα inflammatory process. In addition, TNF-mediated disorders also include angiogenesis in the eye, Behcet's disease, retinitis, glaucoma, Sjogren syndrome, diabetic neuropathy, scleritis, keratitis, and uveitis.

The term "effective dose" refers to the amount sufficient to achieve or at least� partially achieve the desired effect. The term "therapeutically effective dose" is defined as the amount sufficient to cure or at least partial suspension of the disease and its complications in patients already suffering from this disease. Quantity that is effective in the case of this application, will depend on the seriousness of the offence in respect of which the treatment is carried out, and the General condition of the own immune system of the patient.

The term "individual" refers to any human or non-human animal. For example, the methods and compositions of the present invention can be used for treating an individual with a TNF-mediated disorder.

The numbering system used in the present description for determining the positions of amino acid residues in the variable regions of heavy and light chains of the antibody correspond to the installed A. Honegger (J. Mol. Biol. 309 (2001) 657-670) (AHo numbering system). Table of correlations between the AHo numbering system and most commonly used system installed Kabat and others (Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) provided in A. Honegger, J. Mol. Biol. 309 (2001) 657-670.

Used in the present description, the term "aggregation" refers to the process of intermolecular interactions/associations between the monomer molecules in a liquid solution, resulting in images�of oligomeric state. Aggregation can be evaluated in degraded conditions using accelerated stability studies in a concentrated solution. Accelerated stability studies are designed to maximize degradation, aggregation, or chemical modifications of the compounds resulting from the use of extreme storage conditions. Accelerated stability studies, also known research in extreme conditions, usually carried out at 40°C and room temperature. These stability studies provide valuable information concerning the effect of exposure to the effects of environmental conditions outside of normal storage conditions indicated on the label, also known degraded conditions. Solutions with high concentrations of protein are widely used in the pharmaceutical industry. The behavior of proteins at high concentrations in solution may be very different from that predicted based on the analysis of dilute solution, due to thermodynamic ideality in these solutions. Non-ideality observed in these systems, related to protein-protein interactions (PPI). Different types of forces play a key role in determining the General nature and extent of these PPI, and their relative contributions influence the properties of the solute and solvent. �ol PPI due to these intermolecular forces determining the parameters of the solution including physical stability and self-Association and aggregation of proteins. Concentrated solutions are those solutions in which PPI has an effect on proteins in solution as a result of increasing degree of oligomerization. Concentrated solution can have, for example, concentration of protein, component of at least 10 mg/ml.

Soluble products of this process can be identified by analytical methods such as size exclusion HPLC. Used in the present description, the term "reducing aggregation modification refers to modification, such as amino acid replacement, which reduces the ability of the antibody to aggregation in a liquid solution compared to the original antibody, as described in the present description. "Source" antibody is an antibody that contains essentially the same sequence as that of the corresponding antibody, which comprises reducing the aggregation of the modification. For example, the original antibody may have the same CDR, and the modified antibody, and can have the same sequence as that of the modified antibody, except for the residue at position 47 and/or 50 in accordance with the AHo numbering system in the sequence of the variable domain of the light chain and may also be different according to the provisions of 12, 103 and 144 in accordance with the AHo numbering system in the last�coherence variable domain of the heavy chain. There also may be other differences, provided that the original antibody does not contain a reducing aggregation modifications that are present in the antibody, modified in accordance with the method of the present invention.

Used in the present description, the term "interaction" refers to the interaction between the two variable domain (variable domain of the light and heavy chains) antibody. The contact region includes amino acid residues that participate directly or indirectly in the interaction between variable domains. Such interaction includes, but is not limited to, all kinds of unrelated interactions, for example, forces van der Waals forces, hydrogen bond, electrostatic linkages and hydrophobic interactions between the two domains.

Unless otherwise stated, all technical and scientific terms used in this description have the meaning identical to that in which they usually refer to the average expert in the field of the present invention. Although methods and materials similar to those described in the present description, or their equivalent, can be used to implement or verify the present invention, the following describes the appropriate methods and materials. In case of conflict description �altoadige of the invention, including definitions, will prevail. In addition, the materials, methods and examples are only illustrative and are assumed not to be limiting.

Various aspects of the present invention are described with additional details in the following subsections. It is clear that the various implementation options, preferences and ranges can be combined arbitrarily. In addition, depending on the specific variant implementation, the chosen definition, implementation options or ranges may not be acceptable.

In one aspect of the present invention relates to antibodies that bind to TNFα and, thus, are suitable for blocking the functioning of TNFα in vivo.

In some embodiments, the antibodies of the present invention is optimized to reduce aggregation modification(s) relative to the original antibody so that the antibody of the present invention has a reduced ability to aggregate compared to the original/unmodified antibody. Such modifications(I) include specific replacement of amino acid residues involved in the interaction variable domain of the light chain (VL) and the variable domain of the heavy chain (VH). In some embodiments, reducing the aggregation modification contains at least one AMI�kislotno replacement, which reduces the free energy of interaction VL-VH compared to the free energy of interaction VL-VH source of antibody in the method of computer modeling, as described in the present description. Such modifications include amino acid substitutions of specific residues that contribute to the free energy of interaction VL-VH.

In some embodiments, reducing the aggregation modification of the present invention includes the substitution at position 50 in accordance with the AHo numbering system in VL. In one embodiment, the implementation of the replacement of an arginine (R) at position 50 in accordance with the AHo numbering system. In another embodiment of the arginine (R) at position 50 in accordance with the AHo numbering system replaces the lysine (K).

In other embodiments, reducing the aggregation modification of the present invention includes a substitution at position 47 in accordance with the AHo numbering system in VL. In one embodiment, the implementation of the replacement of an arginine (R) at position 47 in accordance with the AHo numbering system. In another embodiment of the arginine (R) at position 47 in accordance with the AHo numbering system replaces the lysine (K).

The AHo numbering system is described in detail in Honegger, A. and Pluckthun, A. (2001) J. Mol. Biol. 309: 657-670. Position 50 in accordance with the AHo numbering system in the variable domain light chain�and 42 corresponds to the position in accordance with the Kabat numbering system. Position 47 in accordance with the AHo numbering system in the variable domain light chain corresponds to the position 39 in accordance with the Kabat numbering system. The Kabat numbering system is described, moreover, in Kabat et al. (Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Table of correlations between the AHo numbering system and most often used by the numbering system established Kabat, etc., provided in A. Honegger, J. Mol. Biol. 309 (2001) 657-670.

The following table of ratios are given for two different numbering systems used for determining the positions of amino acid residues in the variable regions of heavy and light chains of the antibody. The Kabat numbering system is described, moreover, in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The AHo numbering system is described, in addition, Honegger, A. and Pluckthun, A. (2001) J Mol. Biol.309: 657-670).

Numbering in variable regions of the heavy chain

Table 1
Table of ratios to the provisions of residues in the variable domain of the heavy chain
KabatAHoKabatAHoKabatAHo
11445187101
22455288102
33465389103
44475490104
55485591105
66495692106
77505793107
*851 5894108
89525995109
91052a6096110
101152b6197111
111252c6298112
1213*6399113
13145364100114
14155465100a115
15165566100b116
16175667100c117
17185768100d118
18195869100e119
19205970100f120
20216071100g121
21226172100h122

22 236273100i123
23246374*124
24256475*125
25266576*126
26276677*127
*286778*128
27296879*129
28306980 *130
29317081*131
30327182*132
31337283*133
32347384*134
33357485*135
34367586*136
35377687101137
35a 387788102138
35b397889103139
*407990104140
*418091105141
*428192106142
36438293107143
374482a94108144
384582b95 109145
394682b96110146
40478397111147
41488498112148

42498599113149
435086100
Column 1: position of the residues in the Kabat numbering system. Column 2: relevant number in the AHo numbering system for the position specified in column 1. Column 3: position of the residues in the Kabat numbering system. Column 4: the number corresponding to the AHo numbering system for the position specified in column 3. Table�EC 5: the position of the residues in the Kabat numbering system. Column 6: the number corresponding to the AHo numbering system for the position specified in column 5.

Numbering in variable region light chain

55
Table 2
Table of ratios to the provisions of residues in the variable domain light chain
KabatAHoKabatAHoKabatAHo
11435183101
22445284102
33455385103
44465486104
554787105
66485688106
77495789107
88505890108
99*5991109
1010*6092110

1111*6193111
1212*62 112
1313*6395113
1414*6495a114
1515*6595b115
1616*6695c116
1717516795d117
1818526895e118
1919536995f119
20 205470*120
21215571*121
22225672*122
23235773*123
24245874*124
25255975*125
26266076*126
27276177 *127
*286278*128
27a296379*129
27b306480*130
27c316581*131
27d326682*132
27e336783*133
27f346884*134
* 35*85*135
2836*86*136

2937698796137
3038708897138
3139718998139
3240729099140
33417391100141
3442 7492101142
35437593102143
36447694103144
37457795104145
38467896105146
39477997106147
40488098107148
41498199108 149
425082100
Column 1: position of the residues in the Kabat numbering system. Column 2: relevant number in the AHo numbering system for the position specified in column 1. Column 3: position of the residues in the Kabat numbering system. Column 4: the number corresponding to the AHo numbering system for the position specified in column 3. Column 5: position of the residues in the Kabat numbering system. Column 6: the number corresponding to the AHo numbering system for the position specified in column 5.

The antibodies of the present invention may contain additional modifications as desired. For example, the antibody of the present invention may contain amino acid substitutions to reduce its immunogenicity in vivo according to methods described, for example, in the application for United States patent No. 12/973968, and/or replacement to increase the solubility of the antibody, as described in WO 09/155725. Thus, In one of the embodiments the antibody according to the present invention comprises a serine (S) at position 12 of the heavy chain (according to the AHo numbering system); serine (S) or threonine (T) at position 103 of the heavy chain (according to the AHo numbering system) and/�whether serine (S) or threonine (T) at position 144 the heavy chain (according to the AHo numbering system). In addition, the antibody can contain a serine (S) or threonine (T) at positions 97, 98 and/or 99 of the heavy chain (according to the AHo numbering system). Preferably, the antibody contains a serine (S) at position 12 of the heavy chain (according to the AHo numbering system), threonine (T) at position 103 of the heavy chain (according to the AHo numbering system) and threonine (T) at position 144 the heavy chain (according to the AHo numbering system).

In one of the embodiments the antibody according to the present invention contains a variable domain light chain containing SEQ ID NO:1:

EIVMTQSPSTLSASVGDRVIITC(X)n=1 to 50WYQQKPGRAPKLLIY(X)n=1 to 50GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)n=1 to 50FGQGTKLTVLG

In a preferred embodiment of the antibody of the present invention contains a variable domain light chain (CDRs underlined) containing SEQ ID NO:2:

EIVMTQSPSTLSASVGDRVIITCQSSQSVYGNIWMAWYQQKPGRAPKLLIYQASKLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQGNFNTGDRYAFGQGTKLTVLG

In another embodiment of the antibody of the present invention contains a variable domain of the heavy chain, containing SEQ ID NO:3 (the frame region of the variable domain of the heavy chain):

EVQLVESGGGLVQPGGSLRLSCTAS(X)n=1 to 50WVRQAPGKGLEWVG(X)n=1 to 50RFTISRDTSKNTVYLQMNSLRAEDTAVYYCAR(X)n=1 to 50WGQGTLVTVSS

However, in another embodiment of the antibody of the present invention contains a frame region of the variable domain of the heavy chain:

SEQ ID NO:4: frame�th scope variable domain of the heavy chain

EVQLVESGGGLVQPGGSLRLSCTVS(X)n=1 to 50WVRQAPGKGLEWVG(X)n=1 to 50RFTISKDTSKNTVYLQMNSLRAEDTAVYYCAR(X)n=1 to 50WGQGTLVTVSS

In a preferred embodiment of the antibody of the present invention contains a variable domain of the heavy chain (CDR are underlined) containing SEQ ID NO:5:

EVQLVESGGGSVQPGGSLRLSCTASGFTISRSYWICWVRQAPGKGLEWVGCIYGDNDITPLYANWAKGRFTISRDTSKNTVYLQMNSLRAEDTATYYCARLGYADYAYDLWGQGTTVTVSS

As used in the present description, the residues X are the places of insertion of CDR. X can be any natural amino acid; may be present at least three and up to 50 amino acids.

In one of the embodiments of the frame region of the variable domain of the light chain of the antibody according to the present invention comprises SEQ ID NO:1, and the frame region of the variable domain of the heavy chain comprises SEQ ID NO:3 or SEQ ID NO:4.

In another embodiment of the frame region of the variable domain of the light chain of the antibody of the present invention contains a sequence that is at least 65%, more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, more preferably, 99% identical SEQ ID NO:1. Most preferably, when the specified sequence contains an arginine (R) at position 50 in accordance with the AHo numbering system. In another embodiment, the implementation of the specified sequence contains an arginine (R) at position 47 in accordance with sist�my AHo numbering.

In another embodiment of the frame region of the variable domain of the heavy chain of the antibody of the present invention contains a sequence that is at least 80%, more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, more preferably, 99% identical SEQ ID NO:3. Preferably, when the specified antibody contains a serine (S) at position 12 of the heavy chain (according to the AHo numbering system), threonine (T) at position 103 of the heavy chain (according to the AHo numbering system) and threonine (T) at position 144 the heavy chain (according to the AHo numbering system).

In another embodiment of the frame region of the variable domain of the light chain of the antibody of the present invention contains a sequence that is at least 97%, 98%, preferably 99% identical to SEQ ID NO:2 or SEQ ID NO:14.

In another embodiment of the frame region of the variable domain of the heavy chain of the antibody of the present invention contains a sequence that is at least 95%, 96%, 97%, 98%, more preferably, 99% identical SEQ ID NO:5.

In another embodiment of the antibody of the present invention contains a sequence that is at least 96%, 97%, 98%, preferably 99% identical to SEQ ID NO:10 or SEQ ID NO:17.

In another embodiment of the carcass�tial region variable domain of the light chain of the antibody according to the present invention comprises SEQ ID NO:2 or SEQ ID NO:14, a frame region of the variable domain of the heavy chain contains SEQ ID NO:5.

In one of the embodiments of the antibodies or fragments of antibodies of the present invention are single-chain antibodies (scFv) or Fab fragments. In the case of scFv antibody VL domain may be linked to the VH-domain in one way or another orientation by a flexible linker. Appropriate structure of the linker prior art consists of a repetitive amino acid sequence GGGGS (SEQ ID NO:6) or their variants. In a preferred embodiment of the present invention is used as linker in the form of (GGGGS)4(SEQ ID NO:7) or its derivative, but also the possible variants of 1-3 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used for the present invention, described in Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol. Immunother. 50: 51-59. The location can be or VL-linker-VH or VH-linker-VL, wherein the first location is preferred. In the case of Fab-fragments selected the variable domains of the light chain VL are fused with a constant region light chain Ig Kappa man, while matching the variable domains of the heavy chain VH merged with the first (N-terminal) constant CH1 domain of human IgG. C-the end miaocheng disulf�crossing a bridge is formed between two constant domains.

Thus, in one embodiment of the antibody of the present invention contains the sequence:

SEQ ID NO:8

EIVMTQSPSTLSASVGDRVIITC(X)n=1 to 50WYQQKPGRAPKLLIY(X)n=1 to 50GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)n=1 to 50FGQGTKLTVLG GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTAS(X)n=1 to 50WVRQAPGKGLEWVG(X)n=1 to 50RFTISRDTSKNTVYLQMNS LRAEDTAVYYCAR(X)n=1 to 50WGQGTLVTVSS

In another embodiment of the antibody of the present invention contains the sequence:

SEQ ID NO:9

EIVMTQSPSTLSASVGDRVIITC(X)n=1 to 50WYQQRPGKAPKLLIY(X)n=1 to 50GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)n=1 to 50FGQGTKLTVLG GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVS(X)n=1 to 50WVRQAPGKGLEWVG(X)n=1 to 50RFTISKDTSKNTVYLQMNSLR AEDTAVYYCAR(X)n=1 to 50WGQGTLVTVSS

In a preferred embodiment of the antibody of the present invention contains the sequence:

SEQ ID NO:10

(34rFW1.4_VL_K50R_DHP):

EIVMTQSPSTLSASVGDRVIITCQSSQSVYGNIWMAWYQQKPGRAPKLLIYQASKLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQGNFNTGDRYAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTASGFTISRSYWICWVRQAPGKGLEWVGCIYGDNDITPLYANWAKGRFTISRDTSKNTVYLQMNSLRAEDTATYYCARLGYADYAYDLWGQGTTVTVSS

In one of the embodiments the antibody according to the present invention contains a variable domain light chain containing SEQ ID NO:11 (the frame region of the variable domain of the light chain of FW1.4 (KI27)):

EIVMTQSPSTLSASVGDRVIITC(X)n=1-50 WYQQKPGKAPKLLIY(X)n=1-50 GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)n=1-50 FGQGTKLTVLG

In another embodiment of the antibody of the present invention contains a variable domain light chain containing SEQ ID NO:12 (including replacement frame� the region of the variable domain of the light chain of FW1.4)):

EIVMTQSPSTLSASVGDRVIITC(X)n=1-50 WYQQKPGKAPKLLIY(X)n=1-50 GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)n=1-50 FGQGTKLTVLG

However, in another preferred embodiment of the antibody of the present invention contains the sequence:

SEQ ID NO:13

EIVMTQSPSTLSASVGDRVIITC(X)n=1 to 50WYQQRPGKAPKLLIY(X)n=1 to 50GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)n=1 to 50FGQGTKLTVLG

In another embodiment of the antibody of the present invention contains a variable domain light chain (CDRs underlined) containing SEQ ID NO:14:

EIVMTQSPSTLSASVGDRVIITCQSSQSVYGNIWMAWYQQRPGKAPKLLIYQASKLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQGNFNTGDRYAFGQGTKLTVL

Thus, in one embodiment of the antibody of the present invention contains the sequence:

SEQ ID NO:15:

EIVMTQSPSTLSASVGDRVIITC(X)n=1 to 50WYQQRPGKAPKLLIY(X)n=1 to 50GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)n=1 to 50FGQGTKLTVLG GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTAS(X)n=1 to 50WVRQAPGKGLEWVG(X)n=1 to 50RFTISRDTSKNTVYLQMNS LRAEDTAVYYCAR(X)n=1 to 50WGQGTLVTVSS

In another embodiment of the antibody of the present invention contains the sequence:

SEQ ID NO:16:

EIVMTQSPSTLSASVGDRVIITC(X)n=1 to 50WYQQRPGKAPKLLIY(X)n=1 to 50GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)n=1 to 50FGQGTKLTVLG GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVS(X)n=1 to 50WVRQAPGKGLEWVG(X)n=1 to 50RFTISKDTSKNTVYLQMNSLR AEDTAVYYCAR(X)n=1 to 50WGQGTLVTVSS

In one of the embodiments the antibody according to the present invention comprises the sequence:

SEQ ID NO: 17

(34rFW1.4_VL_K47R_DHP):

EIVMTQSPSTLSASVGDRVIITCQSSQSVYGNIWMAWYQQRPGKAPKLLIYQASKLASGVPSRFSGSGSGAEFTLTISLQPDDFATYYCQGNFNTGDRYAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTASGFTISRSYWICWVRQAPGKGLEWVGCIYGDNDITPLYANWAKGRFTISRDTSKNTVYLQMNSLRAEDTATYYCARLGYADYAYDLWGQGTTVTVSS

However, in another embodiment of the antibody of the present invention contains the sequence:

SEQ ID NO:18

(34rFW1.4)

EIVMTQSPSTLSASVGDRVIITCQSSQSVYGNIWMAWYQQKPGKAPKLLIYQASKLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQGNFNTGDRYAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTASGFTISRSYWICWVRQAPGKGLEWVGCIYGDNDITPLYANWAKGRFTISRDTSKNTVYLQMNSLRAEDTATYYCARLGYADYAYDLWGQGTTVTVSS

In one embodiment, the implementation of the VL of the original antibody constitutes or contains SEQ ID NO:11 or SEQ ID NO:12, or a sequence that is at least 65%, more preferably at 80%, 85%, 90%, 95%, 96%, 97%, 98%, more preferably, 99% identical SEQ ID NO:11 or SEQ ID NO:12. In another preferred embodiment, the implementation of the original VH of the antibody constitutes or contains SEQ ID NO:3 or SEQ ID NO:4, or a sequence that is at least 80%, preferably 85%, 90%, 95%, 96%, 97%, 98%, more preferably, 99% identical SEQ ID NO:3 or SEQ ID NO:4.

The antibody of the present invention, which comprises reducing the aggregation modification, preferably, includes one or more CDRs from an antibody of rabbit. As is well known in this field, CDR rabbit differ from the CDR of a human or a rodent: they can contain cysteine residues that are linked by disulfide bridges with framed area or form of inter-CDR S-S-bridges. Moreover, rabbit CDR is often not related to any previously known canonical structure.

From�ikitelli a symptom of the present invention are bivalent and bespecifically molecules containing the antibody against TNFα or its fragment of the present invention. The antibody of the present invention, or antigen-binding portion may be subjected to derivatization or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate bespecifically molecule which binds to at least two different bindings sites or target molecules. The antibody of the present invention can be subjected to derivatization or associated with more than one other functional molecule to generate polyspecific molecules that bind to more than two different binding sites and/or target molecules; such polyspecific molecule, assumed to be also included in the term "bespecifically molecule", as used in the present description. Non-limiting examples bespecifically molecules include datelo, the single-stranded ditelo and tandem antibody, as is well known to specialists in this field.

To create bespecifically molecules of the present invention the antibody of the present invention can functionally bind (for example, via chemical bonding, genetic fusion, nequality communication or otherwise) with one or more binding mol�St, such as another antibody, antibody fragment, opukholespetsificheskaya or specific for pathogen antigens, peptidomimetic or mimetic binding, so the result is bespecifically molecule. Accordingly, the present invention includes bespecifically molecules containing at least one binding molecule having specificity against TNFα, and the second binding molecule having specificity against one or more epitopes of the target.

In one embodiment, the implementation bespecifically molecules of the present invention have binding specificity at least one antibody, or antibody fragment, including, for example, Fab, Fab', F(ab')2, Fv or single-chain Fv. The antibody may also be a light chain dimer or a heavy chain, or any minimal fragment, such as Fv or single-chain design, Ladner described. in U.S. patent No. 4946778, the content of which in incorporated by reference.

While preferred are monoclonal antibodies, other antibodies that can be used in bespecifically molecules of the present invention are murine, chimeric and humanized monoclonal antibodies.

Bespecifically molecules of the present invention �you can get by joining members of specificdate binding, using known in the field methods. For example, each binding specificity bespecifically molecules can be obtained separately, and then connect with each other. When specificnosti binding are proteins or peptides, a set of agents for coupling or crosslinking can be used for covalent compounds. Examples of crosslinking agents include protein A, carbodiimide, N-Succinimidyl-S-acetylthiourea (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), ortho-phenylenedimaleimide (oPDM), N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229: 81-83), and Glennie et al (1987) J. Immunol. 139: 2367-2375). The preferred agents are SATA and sulfo-SMCC, both of which can be purchased from Pierce Chemical Co. (Rockford, IL).

When specificnosti binding antibodies are, you can connect them by binding sulfhydryl residues, for example, via the C-terminal hinge region of the two heavy chains or other places, or natural, or artificially introduced. In a particularly preferred embodiment of the hinge region is subjected to modification with the inclusion of odd num� sulfhydryl residues, preferably one, prior to the connection.

Alternatively, both the specificity of binding can be encoded in the same vector and expressed and assembled in the same the host cell. This method is particularly applicable when bespecifically molecule is a protein Mat x Mat, the Mat x Fab, Fab x F(ab')2or ligand x Fab. Bespecifically molecule of the present invention may be a single-stranded molecule containing one single-chain antibody and the determinants of binding or single-stranded bespecifically molecule containing two binding determinants. Bespecifically molecule can contain at least two single-stranded molecules. In addition, bespecifically molecule can be a scFv that specifically binds to the first target, wherein the VH and VL of the indicated scFv connected by a flexible linker that contains a domain that provides specific binding with the second target. Suitable linkers are described, e.g., in international patent application WO 2010/006454. Methods of obtaining bespecifically molecules described, for example, in U.S. patent No. 5260203; U.S. patent No. 5455030; U.S. patent No. 4881175; U.S. patent No. 5132405; U.S. patent No. 5091513; U.S. patent No. 5476786; U.S. patent No. 5013653; U.S. patent No. 5258498 and U.S. patent No. 5482858.

Binding bespecifically molecules specific for Namistai can confirm, for example, - linked immunosorbent assay (ELISA), radioimmunoassay (RIA), the analysis using cell sorting device with excitation fluorescence (FACS), bioassay (e.g., growth inhibition), or by analysis using Western blot test. Through each of these reviews usually reveals the presence of special interest complexes protein-antibody through the use of labeled reagent (e.g., antibodies) specific to representing the interest of the complex. For example, the complex TNF-a antibody can be identified using, for example, associated with the enzyme antibody or antibody fragment which recognizes and specifically binds to the complexes of antibody-TNF. Alternatively, the complexes can be identified using any of a variety of other immunoassays. For example, the antibody can be marked with a radioactive isotope and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is included in the real description as references). The radioactive isotope can be identified in this way, as using a γ-counter or a scintillation counter, or by using autoradiography.

In another aspect, the present invention relates to the production of antibodies disclosed in the present description. Ways of getting ant�bodies having a reduced ability to aggregate in the solution provided in the present description, is based on the unexpected observation that due to modulation of the interaction between domains of the light chain and heavy chain of the antibody may reduce the ability of antibodies to aggregation, and that reduces aggregation mutations can likely be predicted by determining the free energy of interaction VL/VH, defined as the difference between the energy of the antibody (such as scFv) and energies of the individual variable domains. As demonstrated in the present description, reduces the aggregation of substitutions that modulate the interaction domains of the antibodies with the reduction in free energy between the variable domains can be made without affecting the stability or activity of binding of the antibody.

In one of the embodiments of the method according to the present invention comprises the steps:

(i) receipt of antibody containing the variable domain light chain (VL) variable domain and the heavy chain (VH);

(ii) determining one or more positions of the residues involved in the interactions between the variable domain light chain (VL) and the variable domain of the heavy chain (VH) of the antibody; and

(iii) modification of antibodies by introducing substitutions in a certain position(ies) OST�TKA from the condition, to make the change(s) reduced free energy between the VL and VH domains of at least 0.5 kcal/mol, preferably at least 1.0 kcal/mol and most preferably at least 2.0 kcal/mol (i.e., the free energy between the VL and VH domains with replacement is less than at least 0.5 kcal/mol in free energy between the respective VL and VH domains that do not contain amino acid substitution), thus reducing the aggregability of the modified antibodies as compared to that of the original antibody.

As noted above, the antibody can be, for example, Fab, Fab', F(ab)'2, single-chain Fv (scFv), Fv fragment, ditelo, the single-stranded datelo, tandem antibody or a linear antibody; in a preferred embodiment of the antibody is a single chain Fv (scFv).

In a preferred embodiment of the determination of one or more of the provisions of the residues involved in the interactions between the variable domain light chain and a variable domain heavy chain antibodies (i.e. stage (ii)) includes determining the free energy of interaction VL-VH. This can be accomplished through the use of well-known bioinformatics programs. One example of a suitable bioinformatics program is CHARMM (Chemistry at HARvard Macromolecular Mechanics).

To determine the free energy of the mutual�action VL-VH, usually provided detailed molecular view. The free energy of interaction represents the difference between the energy of the entire antibody, containing both the variable domains VL and VH, and the sum of the energy of the individual domains within the method using the implicitly given solvent. This includes the calculation of three separate energies: (1) G(a) antibodies; (2) G(b) VL and (3) G(c) VH. Accordingly, the free energy of interaction is equal to

G interaction = G(a) G(b)-G(c).

In one embodiment, the implementation of the method using the implicitly given solvent is GBMV or PBSA, known in this field.

The definition of free energy may also include the step of modeling the distribution of charges in the protein. Simulation of the specified charge distribution can be realized on the basis of electrostatic forces or van der Waals forces.

To determine the appropriate modifications, you can choose to replace one or more amino acid residues involved in the interactions. For example, create a molecular model of a protein containing one or more substitutions (e.g., replacing one or more residues to alanine) in the selected provisions, and determine the free energy of interaction in the view of the molecule with the replacement(s). If the free energy of interaction in a model of a molecule with replacement(s�) is less than the free energy of interaction in the original molecular models amino acid residue is chosen for replacement. Within CHARMm mutations can be constructed, for example, using the Protocol Build Mutants. One or more substitutions in the molecular model may be located in positions that are known or believed to be involved in the interaction VL/VH.

In one of the embodiments spend extra stage of energy minimization molecular models containing one or more substitutions in the selected position(s) in the region surrounding the mutation(s). The specified area can be set at the value equal to 10 angstroms.

In one embodiment, the implementation of a specific replacement for the position of balance is a charged amino acid.

The antibody obtained by the method according to the present invention may contain any suitable variable domain light chain or heavy chain, known in this field, and preferably contains at least one CDR of the antibody of the rabbit. In the present description describes some preferred the variable domains of the light and heavy chains. For example, the antibody of the present invention may contain a frame region VL of the antibody, which is at least 65%, more preferably at 80%, 85%, 90%, 95%, 96%, 97%, 98%, more preferably, 99% identical SEQ ID NO:11 or SEQ ID NO:12, containing, in addition, arginine (R) in position 47 in accordance with the system of numbering�AI AHo and/or at position 50 in accordance with the AHo numbering system variable domain of the light chain; and frame the VH region of the antibody that is at least 80%, preferably 85%, 90%, 95%, 96%, 97%, 98%, more preferably, 99% identical SEQ ID NO:3.

The modification of one or more of the provisions of the residues is carried out, preferably, in accordance with the methods set forth in PCT/CH2008/000285, which is incorporated into this description by reference in full. Briefly, in the case of specific subtype antibodies specific amino acids are present in specific positions of the residues frame region of the antibody. For example,

(a) in the case of variable regions of the heavy chain VH3 family of human preferred amino acids are:

(i) glutamine (Q) at amino acid position 1 using AHo numbering system or Kabat;

(ii) glutamine (Q) at position 6 the amino acids, using the numbering system AHo or Kabat;

(iii) threonine (T) or alanine (A) at amino acid position 7 using AHo numbering system or Kabat;

(iv) alanine (A), valine (V) or phenylalanine (F) at position 89 the amino acids, using AHo numbering system (position 78 amino acids, using the Kabat numbering system); and/or

(v) arginine (R), glutamine (Q), isoleucine (I) leucine (L), methionine (M) or phenylalanine (F) at position 103 the amino acids, using AHo numbering system (position 89 the amino acids, using the Kabat numbering system);

(b) in the case of variable region�STI heavy chain VH1a family man preferred amino acids are:

(i) glutamic acid (E) at amino acid position 1 using AHo numbering system or Kabat;

(ii) glutamic acid (E) at position 6 the amino acids, using the numbering system AHo or Kabat;

(iii) leucine (L) at position 12 the amino acids, using AHo numbering system (position 11 of the amino acids, using the Kabat numbering system);

(iv) methionine (M) at amino acid position 13 using AHo numbering system (position 12 amino acids, using the Kabat numbering system);

(v) glutamic acid (E) or glutamine (Q) at position 14 the amino acids, using AHo numbering system (amino acid position 13 using Kabat numbering system);

(vi) leucine (L) at position 19 the amino acids, using AHo numbering system (position 18 of the amino acids, using the Kabat numbering system);

(vii) isoleucine (I) at position 21 of the amino acid using AHo numbering system (amino acid position 20 using Kabat numbering system);

(viii) phenylalanine (F), serine (S), histidine (H) or aspartic acid (D) at position 90 the amino acids, using AHo numbering system (position 79 the amino acids, using the Kabat numbering system);

(ix) aspartic acid (D) or glutamine (Q) at position 92 the amino acids, using AHo numbering system (position 81 the amino acids, using the Kabat numbering system);

(x) glycine (G), asparagine (N) or Tr�onin (T) at position 95 the amino acids using AHo numbering system (position 82b of the amino acids, using the Kabat numbering system); and/or

(xi) threonine (T), alanine (A), Proline (P) or phenylalanine (F) at position 98 the amino acids, using AHo numbering system (position 84 the amino acids, using the Kabat numbering system);

(C) in the case of variable regions of the heavy chain family VH1b human preferred amino acids are:

(i) glutamic acid (E) at amino acid position 1 using AHo numbering system or Kabat;

(ii) threonine (T), Proline (P), valine (V) or aspartic acid (D) at position 10 the amino acids, using AHo numbering system (position 9 of the amino acids, using the Kabat numbering system);

(iii) leucine (L) at position 12 the amino acids, using AHo numbering system (position 11 of the amino acids, using the Kabat numbering system);

(iv) valine (V), arginine (R), glutamine (Q) or methionine (M) at amino acid position 13 using AHo numbering system (position 12 amino acids, using the Kabat numbering system);

(v) glutamic acid (E), arginine (R) or methionine (M) at position 14 the amino acids, using AHo numbering system (amino acid position 13 using Kabat numbering system);

(vi) arginine (R), threonine (T) or asparagine (N) at amino acid position 20 using AHo numbering system (position 19 the amino acids, using Syst�mu Kabat numbering);

(vii) isoleucine (I), phenylalanine (F) or leucine (L) at position 21 of the amino acid using AHo numbering system (amino acid position 20 using Kabat numbering system);

(viii) lysine (K) at position 45 the amino acids, using AHo numbering system (position 38 of the amino acids, using the Kabat numbering system);

(ix) threonine (T), Proline (P), valine (V) or arginine (R) at position 47 the amino acids, using AHo numbering system (position 40 the amino acids, using the Kabat numbering system);

(x) lysine (K), histidine (H) or glutamic acid (E) at position 50 of the amino acid using AHo numbering system (position 43 of the amino acids, using the Kabat numbering system);

(xi) isoleucine (I) at position 55 the amino acids, using AHo numbering system (amino acid position 48 using Kabat numbering system);

(xii) lysine (K) at position 77 the amino acids, using AHo numbering system (position 66 amino acids, using the Kabat numbering system);

(xiii) alanine (A), leucine (L) or isoleucine (I) at position 78 amino acids using AHo numbering system (position 67 the amino acids, using the Kabat numbering system);

(xiv) glutamic acid (E), threonine (T) or alanine (A) at position 82 the amino acids, using AHo numbering system (position 71 the amino acids, using the Kabat numbering system);

(xv) threonine (T), serine (S) or leucine (L) � position 86 the amino acids using AHo numbering system (position 75 the amino acids, using the Kabat numbering system);

(xvi) aspartic acid (D), asparagine (N) or glycine (G) at position 87 of the amino acid using AHo numbering system (position 76 amino acids, using the Kabat numbering system); and/or

(xvii) asparagine (N) or serine (S) at position 107 the amino acids, using AHo numbering system (position 93 the amino acids, using the Kabat numbering system);

(d) in the case of variable region light chain family V1 human preferred amino acids are:

(i) glutamic acid (E) or isoleucine (I) at amino acid position 1 using AHo numbering system or Kabat;

(ii) valine (V) or isoleucine (I) at amino acid position 3 using AHo numbering system or Kabat;

(iii) valine (V), leucine (L) or isoleucine (I) at amino acid position 4 using AHo numbering system or Kabat;

(iv) glutamine (Q) at position 24 the amino acids, using the numbering system AHo or Kabat;

(v) arginine (R) or isoleucine (I) at position 47 the amino acids, using AHo numbering system (position 39 the amino acids, using the Kabat numbering system);

(vi) arginine (R), glutamic acid (E), threonine (T), methionine (M) or glutamine (Q) at position 50 of the amino acid using AHo numbering system (position 42 of the amino acids, using the Kabat numbering system);/p>

(vii) histidine (H), serine (S) or phenylalanine (F) at position 57 the amino acids, using AHo numbering system (position 49 the amino acids, using the Kabat numbering system);

(viii) phenylalanine (F) at position 91 the amino acids, using AHo numbering system (position 73 amino acids, using the Kabat numbering system); and/or

(ix) valine (V), serine (S), glycine (G) or isoleucine (I) at position 103 the amino acids, using AHo numbering system (position 85 the amino acids, using the Kabat numbering system);

(e) in the case of variable region light chain family V3 human preferred amino acids are:

(i) threonine (T) at amino acid position 2 using AHo numbering system or Kabat;

(ii) threonine (T) at amino acid position 3 using AHo numbering system or Kabat;

(iii) isoleucine (I) at position 10 the amino acids, using the numbering system AHo or Kabat;

(iv) a tyrosine (Y) at position 12 the amino acids, using the numbering system AHo or Kabat;

(v) serine (S) at position 18 the amino acids, using the numbering system AHo or Kabat;

(vi) an alanine (A) at amino acid position 20 using AHo numbering system or Kabat;

(vii) methionine (M) at position 56 the amino acids, using AHo numbering system (amino acid position 48 using Kabat numbering system);

(viii) valine (V) or threonine (T) at position 74 in the amino acids, used�eswa the AHo numbering system (position 58 of the amino acid, using the Kabat numbering system);

(ix) asparagine (N) at position 94 of amino acids using AHo numbering system (position 76 amino acids, using the Kabat numbering system);

(x) a tyrosine (Y) or serine (S) at position 101 the amino acids, using AHo numbering system (position 83 the amino acids, using the Kabat numbering system); and/or

(xi) leucine (L) or alanine (A) at position 103 the amino acids, using AHo numbering system (position 85 the amino acids, using the Kabat numbering system);

f) in the case of variable region light chain family V1 human preferred amino acids are:

(i) leucine (L), serine (S) or glutamic acid (E) at amino acid position 1 using AHo numbering system or Kabat;

(ii) alanine (A), Proline (P), isoleucine (I) or tyrosine (Y) at amino acid position 2 using AHo numbering system or Kabat;

(iii) valine (V) or methionine (M) at amino acid position 4 using AHo numbering system or Kabat;

(iv) glutamic acid (E) at amino acid position 7 using AHo numbering system or Kabat;

(v) alanine (A) at position 11 the amino acids, using the numbering system AHo or Kabat;

(vi) threonine (T) or serine (S) at position 14 the amino acids, using the numbering system AHo or Kabat;

(vii) histidine (H) at position 46 of the amino acid using AHo numbering system (position 38 the amino�of islote, using the Kabat numbering system);

(viii) threonine (T), serine (S), asparagine (N), glutamine (Q) or Proline (P) at position 53 the amino acids, using AHo numbering system (position 45 the amino acids, using the Kabat numbering system);

(ix) arginine (R) or glutamine (Q) at position 82 the amino acids, using AHo numbering system (position 66 amino acids, using the Kabat numbering system);

(x) glycine (G), threonine (T) or aspartic acid (D) at position 92 the amino acids, using AHo numbering system (position 74 amino acids, using the Kabat numbering system); and/or

(xi) valine (V), threonine (T), histidine (H) or glutamic acid (E) at position 103 the amino acids, using AHo numbering system (position 85 the amino acids, using the Kabat numbering system). Accordingly, replacement, introduced in the method according to the present invention, subject to the ideas set out in PCT/CH2008/000285. The definition of the subtype known specialist in this field.

In one of the embodiments of the method according to the present invention includes the modification of antibodies at position 47 and/or 50 in accordance with the AHo numbering system variable domain of the light chain, in particular, the variable domain of the light chain V1. Preferably, the antibody is subjected to modification with the inclusion of arginine (R) at position 47 in accordance with the AHo numbering system and/or p�Appendix 50 in accordance with the AHo numbering system variable domain of the light chain. In some embodiments, the lysine (K) is replaced by arginine (R) at position 47 and/or position 50 in accordance with the AHo numbering system variable domain of the light chain. As the antibodies of the present invention may contain additional modifications if desired, the methods of the present invention may include the additional step of modifying antibodies, for example, with the inclusion of serine (S) at position 12 of the heavy chain (according to the AHo numbering system); serine (S) or threonine (T) at position 103 of the heavy chain (according to the AHo numbering system) and/or serine (S) or threonine (T) at position 144 the heavy chain (according to the AHo numbering system). In addition, the antibody can be modified with the inclusion of serine (S) or threonine (T) at positions 97, 98 and/or 99 of the heavy chain (according to the AHo numbering system). Preferably, when the method includes the step of modifying the antibody with the inclusion of serine (S) at position 12 of the heavy chain (according to the AHo numbering system), threonine (T) at position 103 of the heavy chain (according to the AHo numbering system) and threonine (T) at position 144 the heavy chain (according to the AHo numbering system).

The present invention furthermore relates to a method of creating a humanized antibody with low ability to aggregation in solution, including the selection� frame region of the variable domain of the light chain, which contains arginine (R) at position 47 and/or position 50 in accordance with the AHo numbering system. The method may also include the selection of a frame region of the variable domain of the heavy chain, which contains a serine (S) at position 12 of the heavy chain (according to the AHo numbering system); serine (S) or threonine (T) at position 103 of the heavy chain (according to the AHo numbering system) and/or serine (S) or threonine (T) at position 144 the heavy chain (according to the AHo numbering system). In one embodiment of the frame region, identified based on the selected one of the selection criteria, may be further modified by reducing the aggregation modification of the present invention. For example, in the case of identification of a frame region of the variable domain of the light chain, which contains an arginine (R) at position 50, the residue in the position according to the AHo numbering system can be replaced by a different amino acid such as arginine (R), or in the case of identification of a frame region of the variable domain of the heavy chain, which contains a serine (S) at the position 12 in accordance with the AHo numbering system, the residues at positions 103 and 144 in accordance with the AHo numbering system can be a replacement of threonine.

As used in the present description, a "humanized" antibody is an antibody which contains CDR, non-human and human or derived from the human sequence of frame regions of the variable domain of the heavy chain and/or variable domain of the light chain. Humanization of antibodies is well known in this field. In one embodiment, the implementation of a humanized antibody contains at least one, and preferably six CDR of the antibody produced in rabbit or selected from the CDR library.

Frame region of the variable domain of the antibody can be selected, for example, from a database (such as the Kabat database, Genbank (http://www.ncbi.nlm.nih.gov/genbank/), VBASE (http://vbase.mrc-cpe.cam.ac.uk/), VBASE2 (http://www.vbase2.org/), Kabat database related sequences representing the immunological proteins of interest (http://www.kabatdatabase.com/index.html), the universal protein resource (UniProt; http://pir.georgetown.edu and Abysis database (http://www.bioinf.org.uk/abs/), on the basis of identity and/or similarity to the sequence of frame regions of the variable domain of the antibody from which the CDR, or based in another preferred against sequences(and) the frame regions.

There are various computer programs to search the sequences of frame areas of a person who satisfies the selected requirement(s). For example, "KabatMan" is the version, allowing the possibility of searching in promosummary in the Kabat sequences of antibodies from the book of Sequences of Immunological Interest. Program KabatMan described in the article: Martin (1996) Accessing the Kabat Antibody Sequence Database by Computer PROTEINS: Structure, Function and Genetics, 25, 130-133, and is available on the website http://www.bioinf.org.uk/abs/simkab.html and http://www.bioinf.org.uk/abs/kabatman.html. In Abysis database, on the website http://www.bioinf.org.uk/abysis/ United data sequences from the Kabat, IMGT and PDB structural data from PDB. It provides full interface "point-and-click" which allows you to iterate over sequence data according to different criteria and displays the results in various formats. In the case of data from the PDB bust sequences can be combined with structural constraints.

In another aspect, the present invention relates to an antibody obtained using the method described in the present description. In a preferred embodiment of implementation, the specified antibody contains a frame region VL of the antibody, at least 80%, preferably 85%, 90%, 95%, 96%, 97%, 98%, more preferably, 99% identical SEQ ID NO:12 or SEQ ID NO:13; preferably, the antibody contains an arginine (R) at position 47 and/or at position 50 in accordance with the AHo numbering system variable domain of the light chain.

Additionally or alternatively, the frame region VH of the antibody constitutes or contains SEQ ID NO:3, or a sequence that is at least 80%, more preferably, n� 85%, 90%, 95%, 96%, 97%, 98%, more preferably, 99% identical SEQ ID NO:3.

In some embodiments, the present invention, furthermore, relates to:

(1) the Way of the ability to reduce aggregation of the antibody, representing heterodimeric complex that contains the variable domains of the heavy and light chains, which includes stages:

(a) obtaining detailed molecular representation antibodies;

(b) determine the free energy of interaction between the two domains;

(c) selecting one or more amino acid residues involved in the interactions, for replacement by providing a molecular model of the antibody containing one or more substitutions at the selected positions, and determining the free energy of interaction in the view of the molecule with the replacement(s);

(d) selecting an amino acid residue to be replaced if the free energy of interaction in a model of a molecule with replacement(s) is less than the free energy of interaction in the original molecular model;

(2) the Method according to (1), in which the free energy of interaction is determined by calculating the difference between the energy of the complex and the sum of energies of individual domains within the method using the implicitly given solvent;

(3) the Method according to (2), in which the solvent is GBMV or PBSA;

(4) the Method according to any �h preceding (1) to(3), further comprising a stage

(i) modeling the distribution of charges in the protein, and this stage is performed as part of stage a and b;

(5) the Method according to (4), in which the modeling of the distribution of charges is carried out on the basis of electrostatic forces or van der Waals forces;

(6) the Method according to any one of preceding (1) to(5), in which stage (c) includes the additional step of minimizing energy in the region around the mutation;

(7) the Method according to any one of preceding (1) to(7), wherein the antibody is a single chain Fv fragment (scFv).

The antibodies of the present invention can be obtained using conventional methods in the field of recombinant genetics. Knowing the sequence of the polypeptides can be obtained cDNA that encode them, using gene synthesis using known in the field methods. These cDNAs can be cloned into a suitable vector plasmid.

Standard methods for cloning and mutagenesis are well known to the person skilled in the art, may be used to attach linkers, shuffling of domains or create a merger to obtain Fab fragments. The basic methodologies that describe the General methods of this invention, are described in Molecular Cloning, A Laboratory Manual (Sambrook &Russell, 3rded. 2001) and in Current Protocols in Molecular Biology (Ausubel et al., 1999).

The DNA sequence containing the gene encoding scFv, or in the case of Fab-fragments containing either two separate gene, or bicistronic operon containing two genes for mergers VL-Cκ and VH-CH1, cloned into a suitable expression vector, preferably a vector with inducible promoter. Care must be taken in front of every gene was present corresponding to the binding site of ribosomes, which provides streaming. It should be understood that the antibodies of the present invention contain the described sequence, and do not consist of them. For example, the strategy of cloning may prescribe the creation of constructs with which to Express the antibody, in which there is one or a small number of additional residues at N-end. In particular, methionine, derived from the start-codon, may be present in the final protein in the cases when it is not cleaved posttranslation. Most designs for antibodies in the form of scFv leads to an additional alanine at the N-end. In a preferred embodiment of the present invention is chosen expression vector for periplasmic expression in E. coli (Krebber, 1997). The specified vector contains the promoter before splitting the signal sequence. The sequence encoding the peptide antibody, and then drained in reading frame with split signal sequence. This makes it possible EmOC�allenou delivery of the expressed polypeptide into the periplasm of the bacteria, where the signal sequence is cleaved. Then the antibody is minimized. In the case of Fab-fragments of both peptide mergers VL-Cκ and VH-CH1 must be related to the export signal. After reaching the peptides periplasmic forms a covalent S-S bond between C-terminal cysteines. If the preferred is the expression of the antibodies in the cytoplasm, these antibodies can usually be obtained with high yields from Taurus inclusion, which can be easily separated from other cell fragments and protein. In this case Taurus inclusion was dissolved in a denaturing agent, such as, for example, guanidine hydrochloride (GndHC1), and then subjected to refolding using renaturation procedures, well known to specialists in this field.

Plasmids expressing the polypeptides of scFv or Fab, is introduced into a suitable host, preferably a bacterial, a yeast cell or a cell of a mammal, most preferably a suitable strain of E. coli, for example, JM83 for periplasmic expression or BL21 for expression in the form of Taurus inclusion. The polypeptide can be obtained or from periplasm, or from Taurus inclusion and to clean using standard methods, such as ion-exchange chromatography, chromatography with reversed phase, affinity chromatography and/or gel filtration, which are well known to those skilled in Yes�sphere.

The antibodies of the present invention can be characterized in respect of yield, solubility and stability in vitro. For example, the ability to bind to TNF, preferably, human TNFα, can be investigated in vitro using ELISA or surface plasmon resonance (BIACore), using recombinant human TNF as described in WO9729131, with the last of these methods also allows to determine the rate constant (koffthat is, preferably, should be less than 10-3h-1. Preferred are values of Kd≤ 10 nm.

In one of the embodiments of the present invention relates to antibodies that bind to TNFα and, accordingly, are suitable for blocking the functioning of TNFα in vivo. In a particular embodiment of the antibody against TNFα contains the sequence of SEQ ID NO:10 or SEQ ID NO:17.

With therapeutic use of antibodies against TNF of the present invention is administered to a mammal, preferably human, in a pharmaceutically acceptable dosage form such as those discussed above, including those that can be administered to the human intravenously in a bolus injection or by continuous infusion over a period of time, using intramuscular, intraperitoneal, intracerebroventricular, podlin�, intra-articular, intrasynovial, intrathecal, oral, local, intraocular, intranasal, aural, sublingual, percutaneous route or by inhalation, for example. Antibodies are also suitably administered orally tumors, about tumors, lesions inside or around lesions for manifestations of local and systemic therapeutic effects.

For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the type of disease in respect of which the treatment is carried out, as defined above, the severity and course of the disease, the antibody is a preventive or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the discretion of the attending physician. Antibodies respectively being administered to the patient once or over several treatments.

Antibodies against TNF of the present invention can be used for the treatment of TNF-mediated diseases. Depending on the type and severity of the disease from about 1 μg/kg to about 50 mg/kg (e.g., 0.1 to 20 mg/kg) of antibody is an initial possible dose for administration to a patient, for example, or using one or more separate administrations, or by continuous infusion. A typical daily or weekly dosage is probably in the range of�from about 1 μg/kg to about 20 mg/kg or more depending on the factors noted above. In the case of repeated administrations over several days or longer, depending on the condition, the treatment is repeated until the occurrence of a desired suppression of disease symptoms. However, there may be used other schemes of doses. The success of this therapy is easily traced using conventional techniques and assays, including, for example, x-rays of tumors.

In accordance with another variant implementation of the present invention, the efficiency of the antibody for preventing or treating disease may be improved by the introduction of antibodies periodically or in combination with another agent that is effective for those purposes, such as growth factor vascular endothelial (VEGF), an antibody capable of inhibiting or neutralizing the angiogenic activity of acidic and basic fibroblast growth factor (FGF), or growth factor hepatocyte (HGF), an antibody capable of inhibiting or neutralizing the coagulant activity of tissue factor, protein C, or protein S (see Esmon et al., PCT patent application no WO 91/01753, published 21 February 1991), an antibody capable of binding to HER2 receptor (see Hudziak et al., PCT patent application no WO 89/06692, published July 27, 1989), or with one or more conventional therapeutic agents such as, for example, alkylating agents, folic acid antagonists, an�metabolite metabolism of nucleic acids, antibiotics are analogues of pyrimidines, 5-fluorouracil, cisplatin, purine nucleosides, amines, amino acids, triazolinones or corticosteroids. Such other agents may be introduced in the composition or may be administered separately. Also antibody, respectively, to enter periodically or in combination with radiological treatments, whether or irradiation or introduction of radioactive substances.

The antibodies of the present invention can be used as a means for affinity purification. In this process, the antibodies are subjected to immobilization on a solid phase, such as Sephadex resin or filter paper, using the well-known in the field methods. Subjected to immobilization of the antibody are brought into contact with a sample containing protein-target (or its fragment) that binds an antibody, such as TNF, in the case of antibodies against TNFα, treated, and subsequently the substrate is washed with a suitable solvent that will remove essentially all of the material in the sample except the target protein that is bound to the immobilized antibody. Finally, the substrate is washed with another suitable solvent, such as glycine buffer, pH 5.0, that will release the protein target from the antibody.

Antibodies can also be used in diagnostic studies of protein-mi�Yeni, for example, for detecting its expression in specific cells, tissues, or serum. Such diagnostic methods can be used to diagnose cancer.

When applying for the diagnosis of antibody is usually labeled molecule visualized. There are numerous labels that can generally be divided into the following classes:

(a) Radioisotopes, such as111In99Tc,14C,131I,125I,3H,32P or35S. the Antibody can be tagged with a radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991), for example, and radioactivity can be measured using scintillation measurements.

(b) Fluorescent labels such as chelates of rare earth elements (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrine and Texas red are available. Fluorescent labels can be konjugierte with the antibody using the methods described in Current Protocols in Immunology, supra, for example. Fluorescence can be measured using a fluorimeter.

(c) Different labels in the form of combinations of enzyme-substrate available, and an overview of some of them is presented in U.S. patent No. 4275149. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate which can be measured with the use of� different ways. For example, an enzyme may catalyze a color change of the substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Methods of determining the change in fluorescence as described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using chemiluminometer, for example), and transfer energy to fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., Firefly luciferase and bacterial luciferase; U.S. patent No. 4737456), luciferin, 2,3-dihydropteridine, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, SharedAccess (e.g., glucose oxidase, galactosidase and glucose-6-phosphate-dehydrogenase), oxidase heterocyclic compounds (such as uricase and xanthine oxidase), lactoperoxidase, microbiocides, etc. Ways of kojouharova of enzymes to antibodies are described in O'sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone &H. Van Vunakis), Academic press, New York, 73: 147-166 (1981). Examples of combinations of enzyme-substrate include, for example:

(i) horseradish peroxidase (HRPO) with hydrogen peroxide as substr�the one moreover, hydrogenperoxide oxidizes dry precursor (e.g., orthophenylene (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-nitrophenylphosphate as chromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., para-nitrophenyl-β-D-galactosidase) or 4-methylumbelliferyl-β-D-galactosidase with a chromogenic substrate.

Numerous other combinations of enzyme-substrate are well known to specialists in this field. An overview is given in U.S. patent No. 4275149 and 4318980. Sometimes the label indirectly kongugiruut with the antibody. The person skilled in the art is aware of various ways to achieve this. For example, the antibody can be konjugierte with Biotin and any of the three broad classes of labels mentioned above can be konjugierte with Avidya, or Vice versa. Biotin selectively binds to Avidya, and therefore the label can be konjugierte with the antibody in this indirect way. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody kongugiruut with a small hapten (e.g., digoxin) and one of various types of labels mentioned above, kongugiruut with an antibody against hapten (for example, an antibody against digoxin). Thus, can be achieved indirect conjugation of the label with the antibody�.

In some embodiments, the implementation is not required to tag antibody, and its presence could be detected using a labeled antibody that binds to the antibody target.

The antibodies of the present invention can be used in any known method of research, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation reactions. Cm. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc. 1987).

The competitive binding assays rely on the ability of labeled standard to compete with the analyte in the sample for binding with a limited amount of antibody. For example, the amount of TNF protein in the sample is inversely proportional to the amount of standard that is associated with antibodies. To facilitate determining the amount of standard that is bound, the antibodies usually translated into an insoluble form prior to or after the competition, so that the standard and analyte that are bound with antibodies, can be easily separated from the standard and analyte that remain unbound.

Sandwich assays involve the use of two antibodies, each of which is capable of binding to a different immunogenic portions or epitopes detectable protein. In the case of sandwich-analysis of the analyte in the sample binds to the first antibody, which is immobilized�but on a solid support, and thereafter a second antibody binds to the analyte, forming, thus, insoluble three-part complex. See, for example, U.S. patent No. 4376110. The very second antibody may be labeled with the detectable component (direct sandwich assays), or it can be measured using the antibody against the immunoglobulin, which is marked with a detectable component (indirect sandwich assays). For example, one type of sandwich assay is an ELISA, in which case detected component is an enzyme.

In the case of immunohistochemistry, the tissue sample, such as a tumor sample may be fresh or frozen or may be placed in paraffin and fixed with the use of a latch, such as formalin, for example.

Antibodies can also be used for in vivo diagnostic assays of tumors. Typically, the antibody is labeled with a radionuclide (such as111In99Tc,14C,131I,125I,3H,32P or35S), so that the location of the tumor can be determined using immunoscintigraphy.

The antibody of the present invention can be in the set, packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic tests. In the case where the antibody labeled with the enzyme, the kit will include substrates and �factory, required by the enzyme (for example, the precursor of the substrate, which provides the detectable chromophore or fluorophore). In addition, may include other components, such as stabilizers, buffers (e.g., buffer to block or lysing buffer), etc. Relative amounts of the various reagents may vary in a wide range to achieve certain concentrations of reactants in solution, which substantially optimize the sensitivity of the analysis. In particular, the reagents can be in the form of dry powders, usually liofilizirovanny containing fillers that upon dissolution will create a reagent solution with a suitable concentration.

The present invention also relates to pharmaceutical preparations containing one or more antibodies of the present invention, for therapeutic purposes. In one of the embodiments of the present invention relates to antibodies against TNF for the treatment of TNF-mediated diseases.

The term "pharmaceutical preparation" refers to preparations which are in such form that it creates the opportunity for the biological activity of the antibody was steadily effective, and which contain no additional components which are toxic to individuals for whom the drug is to be injected. "Pharmaceutical industry�ski acceptable excipients (carriers, additives are fillers that can be motivated to enter the mammal to achieve an effective dose of active ingredient used.

"Stable" drug is a drug, wherein the antibody essentially maintains its physical stability and/or chemical stability and/or biological activity upon storage. In this area there are various analytical methods to determine the stability of the protein, and reviews are given in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90, for example. Stability can be determined by the selected temperature for a selected period of time. Preferably, when the drug is stable at room temperature (approximately 30°C) or at 40°C for at least 1 month and/or stable at about 2-8°C for at least 1 year, or during at least 2 years. In addition, the drug preferably is stable after freezing (for example, up to -70°C) and thawing of the drug.

An antibody "retains its physical stability" in a pharmaceutical drug if it shows signs of aggregation, precipitation and/or denaturation, as determined by visual examination of color and/or transparency, or Opredelenie through the scattering of UV light or gel filtration.

An antibody "retains its chemical stability" in a pharmaceutical drug, if the chemical stability at a particular time is such that the protein is considered to still retain its biological activity, as defined below. Chemical stability can be determined by detecting and quantifying chemically altered forms of the antibody. A chemical change can include a change in size (e.g., truncation), which can be estimated using gel filtration, electrophoresis in SDS-page and/or ionization laser desorption using a matrix/time-of-flight mass spectrometry (MALDI/TOF MS), for example. Other types of chemical changes include the change of the charge (for example, resulting from desametasone), evaluation can be performed using ion exchange chromatography, for example.

An antibody "retains its biological activity" in a pharmaceutical drug if the biological activity of the antibodies in a given time is within about 10% (within the errors of the study) biological activity demonstrated in that point in time when a pharmaceutical product has been received, as determined in the analysis of binding to the antigen, for example. Other tests "biological activity" konkretiziroval�s in the present description below.

"Isotonic" means that interest, the drug has essentially the same osmotic pressure as human blood. Isotonic preparations will generally have an osmotic pressure from about 250 to 350 mOsm. Izotonichnost can be determined using paranapanema or cryoscopic osmolarity system, for example.

"Polyol" is a substance with multiple hydroxyl groups, and includes sugars (reducing and nevosstanovlenie sugar), saharospirty and saharomiceta. Preferred in the present description, the polyols have a molecular weight which is less than about 600 kDa (e.g., is in the range from about 120 to about 400 kDa). "Reducing sugar" is a sugar that contains polyacetylene group IT, which can reduce the number of metal ions or react with the formation of covalent bonds with lysine and other amino groups in proteins, "nevosstanovlenie sugar" is a sugar that does not possess these properties of a reducing sugar. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Nevosstanovlenie sugars include sucrose, trehalose, sorbose, melezitose and raffinose�. Mannitol, xylitol, aritra, trait, sorbitol and glycerine are examples of charsperrow. As sharokina they include salt of L-gluconic acid and their metal salts. In the case where it is desirable that the drug was stable to freeze-thaw cycles, the polyol preferably is one that does not crystallize at freezing temperatures (e.g. -20°C) destabilization of antibody in the preparation. Nevosstanovlenie sugar include, but are not limited to, sucrose and trehalose.

As used in the present description, "buffer" refers to a buffered solution, which is resistant to pH changes due to the action of its acid-base the connected components. The buffer of the present invention has a pH ranging from about 4.5 to about 7; preferably, from about 4.8 to about 6.5. Examples of buffers that will control the pH within this range include acetate (e.g., materiality), succinates (e.g. matricectomy), gluconate, histidinate, citrate and incorporating other organic acid buffers. If desirable, is resistant to freeze-thaw cycles of the drug, the buffer preferably is a phosphate.

In the pharmacological importance, in the context of the present invention, "therapeutically effective�th number of" antibody refers to the amount, effective for the prevention or treatment of disorders, for the treatment of which the antibody is effective. "Disease/violation" is any condition, treatment with antibody which will have the effect. It includes chronic and acute disorders or diseases including those pathological conditions that cause a predisposition of a mammal to the offense in question.

"Preservative" is a compound that can be included in the drug to substantially reduce the activity of the bacteria, thereby obtaining a drug for multiple use, for example. Examples of potential preservatives include octadecyltrimethylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds) and benzene chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkylarene, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. The most preferred preservative is benzyl alcohol.

The present invention also relates to pharmaceutical compositions containing one or more antibodies together with at least one physiologists�Eski acceptable carrier or excipient. The pharmaceutical compositions may contain, for example, one or more of the following: water, buffers (e.g., neutral buffered saline or phosphate buffered saline), ethanol, mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates (e.g., glucose, mannose, sucrose or dekstrana), mannitol, proteins, adjuvants, polypeptides or amino acids such as glycine, antioxidants, complexing agents such as EDTA or glutathione and/or preservatives. As noted above, other active ingredients may (but need not) be included in the pharmaceutical compositions of the present invention.

A carrier is a substance which may be combined with the antibody before the introduction of the patient, often for the purpose of controlling the stability or bioavailability of the compound. Carriers for use within such formulations are typically biocompatible and may also be biodegradable. Carriers include, for example, monovalent or multivalent molecules such as serum albumin (e.g., human or bovine), ovalbumin, peptides, polylysine and polysaccharides, such as amylodextrin and polyamidoamine. Carriers also include materials solid substrates, such as granules and microparticles comprising, for example, polylactate, polyglycolic, copolymers�EP lactide with glycolide, polyacrylate, latex, starch, cellulose or dextran. The carrier can carry the connection in a number of ways, including covalent bond (either directly or via a linker group), non-covalent interaction or mixing.

Pharmaceutical compositions can be obtained for any suitable route of administration including, for example, ocular, intranasal, aural, sublingual, transdermal, local, oral, nasal, rectal or parenteral administration. In some embodiments, the preferred compositions are in a form suitable for oral administration. Such forms include, for example, pills, tablets, lozenges, pellets, suspensions in water or oil-based, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. However, in other embodiments, the compositions of the present invention can be obtained as a lyophilizate. Used in the present description, the term "parenteral(s)" includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal and intraperitoneal injection, as well as any similar way of injections or infusions.

In some embodiments, the antibody of the present invention can be entered directly�military in the eye by injection into the tissues of the eye, such as periocular, conjunctival, subtenon injections, intracameral injection, injection into the vitreous inside the eye, subretinal, subconjunctival, retrobulbar, or intracanalicular injections; by direct injection into the eye using a catheter or other device for administration, such as a granule for retinal applications, insertion into the eye, suppository or an implant containing a porous, non-porous, or gelatinous material; by using eye drops or ointments for topical use; or using the device for slow release, located in the bag or implanted adjacent to the sclera (transscleral introduction) or in the sclera (intrascleral introduction), or inside the eye. Intracameral injection can be performed through the cornea into the anterior chamber to make possible the achievement means of the trabecular network. Intracanalicular injection can be performed in the venous collector channels, starting from the sclera sinus canal, or into the schlemm's canal.

For delivery to the eye, the antibody of the present invention may be combined with ophthalmologist acceptable preservatives, co-solvents, surface-active substances that increase the viscosity of the substances that increase the penetration of substances, buffers, chlorine�ohms sodium or water for water education, sterile suspension or solution for ophthalmic use. Products for local ophthalmic use may be packaged, for example, in multi-dose form. Therefore, you might need preservatives to prevent contamination by microbes during use. Suitable preservatives include chlorobutanol, methylparaben, propylparaben, generationy alcohol, Agudat denetria, sorbic acid, polyquaternium-1 or other agents known to specialists in this field. Such preservatives are typically used at a level of from 0.001 to 1.0% weight to volume. The compositions of the present invention in the form of a single dose will be sterile, but usually without preservatives. Such compositions, therefore, will not, as a rule, contain preservatives.

In some embodiments, compositions intended for local injection into the eye, in the form of eye drops or eye ointments, and the total number of antibodies would be approximately 0,001-1,0% (in weight ratio). Preferably, the amount of antibody against TNFα is from approximately 0.01 to approximately 1.0% (in weight ratio).

The compositions of the present invention in some cases will be introduced in the form of solutions for local administration. Aqueous solutions are, as a rule, predpochtitel�governmental, on the basis of the ease of obtaining, and also allowing the introduction of such compositions by the patient through the introduction of one or two drops of the solutions in the affected eye. However, the compositions may also be suspensions, viscous or poluvyazkie gels, or other types of solid or semi-solid compositions.

Compositions intended for oral use can be obtained in accordance with any method known in this field to obtain pharmaceutical compositions, and may contain one or more agents such as sweetening agents, corrigent, dyes and preservatives to produce attractive and tasty preparations. Tablets contain the active ingredient in admixture with physiologically acceptable excipients which are suitable for manufacture of tablets. Such fillers include, for example, inert diluents (e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate), granulating agents and improve tablet disintegration (for example, corn starch, or alginic acid), binders (e.g., starch, gelatin or acacia gum) and lubricants (e.g. magnesium stearate, stearic acid or talc). The tablets may be uncoated or they may be coated by known methods to delay disintegration and absorption in the gastro-�isicem tract and thereby provide a continuous action over a longer period of time. For example, a material with a time delay, such as glycerylmonostearate or glycerylmonostearate, can be used.

Preparations for oral administration can also be administered in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin or olive oil). Aqueous suspensions contain the antibody in admixture with excipients suitable for the preparation of suspensions, water-based. Such fillers include suspendresume means (for example, sodium carboxymethylcellulose, methylcellulose, gidropropilmetilzelluloza, sodium alginate, polyvinylpyrrolidone, tragacanth gum and acacia gum); and dispersants or wetting means (for example, natural phosphatides, such as lecithin, condensation products of accelerated with fatty acids, such as polyoxyethylenated, the condensation products of ethylene oxide with long chain aliphatic alcohols, such as heptadecafluorooctyl, the condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as polyoxyethylenesorbitan�, or condensation products of ethylene oxide with partial esters derived from fatty acids and lexicology anhydrides, such as polyethylenterephthalat). Aqueous suspensions can also contain one or more preservatives, for example ethyl or n-propyl-para-hydroxybenzoate, one or more coloring agents, one or more corrigentov, and one or more sweeteners, such as sucrose or saccharin. Syrups and elixirs can be derived sweeteners, such as glycerin, propylene glycol, sorbitol or sucrose. Such preparations can also contain one or more funds, reduce irritation, preservatives, corrigentov and/or dyes.

Suspension oil-based can be obtained by suspending the active ingredients in a vegetable oil (e.g., peanut oil, olive oil, sesame oil or coconut oil) or in mineral oil such as liquid paraffin. Suspensions can contain a thickening agent, such as yellow beeswax, hard paraffin or cetyl alcohol. Sweeteners, such as those described above, and/or corrigenda can be added to polucheniya oral medication with attractive taste. The preservation of such suspensions can be ensured by adding an antioxidant, such as ascorbic acid.

Dispersed �EROSKI and pellets, suitable for suspensions based on water by the addition of water provide the active ingredient in admixture with dispersing or wetting agent, suspenders agent and one or more preservatives. Examples of suitable dispersants and wetting agents are those mentioned above. There also may be additional excipients, for example, sweeteners, corrigenda and dyes.

Pharmaceutical compositions may also be in the form of emulsions of the type oil-in-water". The oil phase may be a vegetable oil (e.g., olive oil or peanut oil), mineral oil (e.g., liquid paraffin) or a mixture thereof. Suitable emulsifiers include natural gums (e.g., acacia gum or tragacanth gum), natural phosphatides (e.g., soy, lecithin and esters or partial esters derived from fatty acids and hexitol), anhydrides (e.g., orbitonasal) and condensation products of partial esters derived from fatty acids and hexitol, with ethylene oxide (e.g., polyoxyethylene). The emulsion may also contain one or more sweeteners, and/or corrigentov.

Pharmaceutical composition can be obtained in the form of a sterile injectable aqueous or oil-containing suspension, in which the modulator, depending on �ispolzuemogo media and concentration, or suspended, or dissolved in the carrier. Such a composition can be obtained in accordance with a known level using suitable dispersing agents, wetting agents and/or suspendresume agents, such as those noted above. Among the suitable carriers and solvents that may be used are water, 1,3-butanediol, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils may be used as solvent or suspendida environment. This purpose can be used any tasteless non-volatile oil, including synthetic mono - or diglycerides. In addition, fatty acids such as oleic acid, can be used when receiving injectable compositions, and tools such as local anesthetics, preservatives and/or buffering agents can be dissolved in the carrier.

Pharmaceutical composition can be obtained in the form of drugs with a slow release (i.e. drug, such as a capsule that mimics the slow release of modulator following the introduction). These drugs can usually be obtained using well-known technology, and input by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target location. Carriers for use in �such formulations are biocompatible, and may also be biodegradable; preferably, when the drug provides a relatively constant level of release modulator. The amount of antibodies contained in the product with a slow release, depends, for example, from the site of implantation, the rate and expected duration of release and the nature of diseases/disorders that are treated or warning.

Antibodies against TNFα, as described in the present description, can be administered in an amount which gives a concentration in the body fluids (e.g. blood, plasma, serum, cerebrospinal fluid, synovial fluid, lymph, intercellular interstitial fluid, tears or urine) that is sufficient for detectable binding to TNF and preventing or suppressing TNF-mediated diseases/disorders. It is believed that the dose is effective if it causes a positive effect in a patient as described above. Preferred dosages for systemic administration range from about 0.1 mg to about 140 mg per kilogram of body weight per day (from about 0.5 mg to about 7 g for each patient per day), the dose for oral administration, as a rule, exceed in approximately 5-20 times the dose for intravenous administration. The amount of antibody that can be combined with materials media policereport single dose, will vary depending on the owner in respect of which the treatment is carried out, and the particular route of administration. Form of dosage units will generally contain from about 1 mg to about 500 mg of active ingredient.

In some embodiments, pharmaceutical compositions may be packaged for treating conditions responsive to antibody directed against TNF. Packaged pharmaceutical compositions may include a container containing an effective amount of at least one of the antibodies described in the present description, and instructions (e.g., label) indicating that the contained composition is to be used for the treatment of diseases/disorders that are responsive to one of an antibody after administration to the patient.

The antibodies of the present invention can also be chemically modified. Preferred modifying groups are polymers, for example, optionally substituted, unbranched or branched polyalkene, polyalkylene and polyoxyalkylene polymer, or a branched or unbranched polysaccharide. Such effector group may increase the half-period of the existence of antibodies in vivo. Specific examples of synthetic polymers include optionally substituted, unbranched or isn�branched polyethylene glycol (PEG), the polypropylene glycol, polyvinyl alcohol or derivatives thereof. Specific natural polymers include lactose, amylose, dextran, glycogen or derivatives thereof. The size of the polymer may be varied if desired, but will generally be in the range for the average molecular weight of from 500 Da to 50,000 Da. In the case of local application, when the antibody is intended to penetrate into the fabric, the preferred molecular weight of the polymer is about 5000 Da. The polymer molecule can be attached to the antibody, for example, to the C-Terminus of the heavy chain Fab fragment via covalently linking the hinge peptide as described in WO0194585. As for attaching the PEG components, reference is made to "Poly(ethyleneglycol) Chemistry, Biotechnological and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York.

After receipt of interest antibodies, described above, receives its containing pharmaceutical composition. The antibody is introduced into the composition, not subjected to prior lyophilization, and representing in the present description, the interest of the product is an aqueous preparation. Preferably, the antibody in the medicament is an antibody fragment such as an scFv. A therapeutically effective amount of the antibody present in the drug, determine � into account the desired dose volumes and method(s) of administration, for example. Cited as an example the concentration of antibody in the medicament is from about 0.1 mg/ml to about 50 mg/ml, preferably from about 0.5 mg/ml to about 25 mg/ml and, most preferably, from about 2 mg/ml to about 10 mg/ml.

Get aqueous preparation containing the antibody in a pH-buffer solution, as described above. The concentration of the buffer can be from about 1 mm to about 50 mm, preferably, from about 5 mm to about 30 mm, depending, for example, from the buffer and the desired isotonicity of the preparation.

The composition is administered polyol, which performs the function of a means of supporting the correct tonicity of the solution, and can stabilize the antibody. In preferred embodiments, the product does not contain support the correct tonicity amounts of salt, such as sodium chloride, as this may cause precipitation of the antibody and/or may result in oxidation at low pH. In preferred embodiments, the polyol is nevosstanovlenie sugar, such as sucrose or trehalose. The polyol is added to the drug in quantities that can be changed to fit the desired isotonicity of the preparation. Preferably, the aqueous preparation is isotonic, in which case the appropriate concentration�and polyol in the product are in the range of from about 1% to about 15% weight to volume preferably, in the range from about 2% to about 10% weight to volume, for example. However, hypertonic or hypotonic drugs may also be suitable. Add the amount of polyol may also be adapted to the molecular weight of the polyol. For example, there may be added a smaller amount of a monosaccharide (e.g., mannitol) compared with the disaccharide (such as trehalose).

To containing the antibody to the drug may be added a surfactant. Cited as examples of the surfactant include nonionic surfactants, such as Polysorbate (e.g. Polysorbate 20, 80, etc.) or poloxamer (e.g., poloxamer 188). Usually the added amount of the surfactant is such that it reduces aggregation introduced in the preparation of the antibody/derivative antibodies and/or minimizes the formation of particles in the product, and/or reduces adsorption. For example, a surfactant may be present in the formulation at a level of from about 0,001% to about 0.5%, preferably from about 0.005% to about 0.2% and most preferably from approximately 0.01% to approximately 0.1%.

In one embodiment, the implementation of the drug contains certain�above Lenno agents (i.e. antibody, buffer, polyol and surfactant) and is essentially not contain one or more preservatives such as benzyl alcohol, phenol, m-cresol, chlorobutanol and benzene Cl. In another embodiment, the implementation of a preservative may be included in the drug, especially when the drug is a multi-dose drug. The concentration of preservative may be in the range of from about 0.1% to about 2%, most preferably from about 0.5% to about 1%. The drug may be administered one or more other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington's Pharmaceutical Sciences 21st edition, Osol, A. Ed. (2006), provided that they have no adverse effect on the desired characteristics of the drug. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the used doses and concentrations, and include additional buffering agents; co-solvents; antioxidants including ascorbic acid and methionine; complexing agents such as EDTA; metal complexes (e.g., complexes of Zn-protein); biodegradable polymers, such as polyesters; and/or salt-forming counterions such as sodium.

Used for in vivo introduction of drugs must be sterile. It's easy to implement�you by filtering through a sterile membrane filters, before or after receiving the drug.

The drug is administered to a mammal in need of treatment an antibody, preferably human, by known methods, such as intravenous administration in the form of a bolus injection or by continuous infusion over a period of time, using intramuscular, intraperitoneal, intracerebroventricular, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, local route or by inhalation, or through other means described in the present description. In some embodiments, the drug is administered to a mammal by intravenous injection. To this end, the drug can be administered using a syringe or dropper, for example.

Appropriate dosage ("therapeutically effective amount") of the antibody will depend, for example, from the disease in respect of which treated, the severity and course of the disease, the antibody is a preventive or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, the type of antibody, and the discretion of the attending physician. Antibody respectively administered to the patient once or over a number of treatments, and it can be administered to the patient at any time since diagnosis. The antibody can be entered � as the sole treatment or in combination with other drugs, or therapy, applicable to the treatment of the disease in question.

As a General assumption, administered a therapeutically effective amount of the antibody will be in the range from approximately 0.1 to approximately 100 mg/kg of body weight of the patient, or after a single, or repeated introduction, the typical usable range of the antibody will be from about 0.3 to about 20 mg/kg, more preferably, from about 0.3 to about 15 mg/kg, administered daily, for example. However, there may be used other schemes of doses. The efficiency of this therapy is easily traced using conventional methods.

In some embodiments, the pharmaceutical composition containing the antibody against TNFα according to the present invention, is administered to a patient with a TNF-mediated disorder.

In another embodiment of the present invention relates to a finished product containing the container that holds the aqueous pharmaceutical preparation of the present invention and which is provided with instructions regarding its use. Suitable containers include, for example, vials, ampoules and syringes. The container can be obtained from a variety of materials, such as glass or plastic. Cited as an example of a container is 3-20 cm3with�clanna ampoule is for single use. Alternatively, in the case of multi-dose medication container may be 3-100 cm3a glass bubble. The container will hold the drug and the label on the container, or connected with it, may indicate the area of application. The finished product may also include other materials desirable from a commercial point of view and the point of view of the consumer, including other buffers, diluents, filters, needles, syringes and inserts the instructions in the application.

The contents of any patents, patent applications and reference documents, which are listed throughout this description of the invention, included herewith by reference in their full amounts.

Unless the context dictates otherwise, as used in the present description, the terms in the singular shall include the plural, and the terms in the plural shall include the singular.

EXAMPLES

The present invention, furthermore, is illustrated by the following examples, which should not be considered as an additional constraint. The content of all shapes and all reference documents, patents and published patent applications, cited throughout this application, is intentionally incorporated into this description by reference in full.

Everywhere in the examples were used the following m�the materials and methods, except where otherwise indicated.

General materials and methods

Typically, the implementation in practice of the present invention are used, except where otherwise indicated, conventional methods of chemistry, molecular biology, recombinant DNA technology, immunology (especially, e.g., technologies of creation of antibodies) and standard methods for obtaining polypeptides. See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., C. S. H. L. Press, Pub. (1999); and Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons (1992).

Determining thermal stability

The IR spectra of attenuated total internal reflection Fourier-transform (FTIR-ATR) were obtained for various single chain molecules and subsequent, using cell FT-IR Bio-ATR Bruker Tensor. Molecules concentrated up to 3 mg/ml and dialyzed overnight at 4°C against PBS, pH 6.5, and running buffer was collected as a control. The denaturation profiles were obtained by exposure of the molecules heat using a wide range of temperatures from 5°C irregular (cables 25-95°C). Processing of all spectra was performed using OPUS software. Background as a primary buffer and non-stationary atmospheric background (CO2and H2O) you�Italy from the reflection spectrum of the protein. Then in the resulting reflection spectrum of the protein was amended on the baseline, and the spectra corresponding hamidou I protein was determined based on the width of the widest resolvable peak in the intended. Second derivative spectra were obtained for spectra with a bandwidth corresponding to hamidou I, using a function based on polynomials of the third degree, together with the smoothed function. Changes in the structure of the protein was assessed by analyzing the second derivatives of the spectra corresponding to hamidou I, using a linear calibration curve for the initial calculation based on the selection on a curve, considering the denaturation = 0% for measurements at 3 low temperatures, and the denaturation = 100% for measurements at 3 higher temperatures. The denaturation profiles were used to approximate mid points of conformational transitions under the action of heat (TM) for each option, using a sigmoidal model Boltzmann.

Solubility determination

The relative solubility of different scFv molecules was determined after increasing the aggregation and precipitation of the protein in the presence of ammonium sulfate. Ammonium sulfate was added to the protein in aqueous solutions with the creation of steps increases the saturation by 5% in the final mixture of salt-protein. Precipitation in the dynamic range was determined empirically, and the steps of saturation was reduced in this range �about 2.5% in the final mix. After adding ammonium sulfate to the sample was gently mixed and centrifuged for 30 minutes at 6000 rpm. Protein remaining in the supernatants were obtained for each cent saturation of ammonium sulphate. Solubility curves were established by determining the protein concentration in the supernatant using a spectrophotometer NanoDropTM1000. Measurement of the remaining soluble protein in the supernatants were normalized and used to estimate the average points of the relative solubility for each case, using a sigmoidal model Boltzmann.

The study of short-term stability

Protein was investigated after incubation for two weeks at 40°C against soluble aggregates and degradation products. Proteins with a concentration of 10 mg/ml, dialyzed overnight at 4°C against PBS with a wide range of pH values(3,5, 4,5, 5,5, 6,5, 7,0, 7,5 and 8.5). Control protein with the concentration in a standard PBS buffer (pH 6.5) was stored at -80°C for a 2-week period. The definition of degradation bands by electrophoresis in SDS-page was performed at time points t=0 and t=14 days, and soluble aggregates were determined using size exclusion HPLC. Determination of residual activity after 2 weeks at 40°C was carried out using Biacore.

Example 1

Optimization of antibodies against TNFα � the form of scFv, 34rFW1.4, to reduce aggregation

Antibody 34rFW1.4 demonstrates the ability to aggregation depending on the pH in solution. Antibody 578rFW1.4 (see international application WO2009155724), which has the same frame structure as that 34rFW1.4, demonstrates the ability to aggregation. To determine residues in 34rFW1.4, which can be modified to reduce its ability to aggregation, were created by homologous models, as described next.

The sequence of the variable domains used for creating homologous models of antibody 34rFW1.4 and antibodies 578rFW1.4. To create models used the search based on the BLAST algorithm to identify structures of samples for sequences of variable domains of the light chain (VL) and heavy chain (VH) for each antibody separately. BLOSUM80 matrix for the less divergent alignments) was used as matrix for alignments due to the high conservatism of frame regions in antibodies. Were the samples for each chain (VL/VH), which are identical in more than 70% of the query sequences.

The program MODELER software Discovery Studio 2.5.5 (DS 2.5.5) (Accelrys, Inc., San Diego, CA) was used to create 100 models scFv identified on the basis of samples of variable domains. The alignment control sequences, subjected to m�melirovanie, with the structures of the samples were used as input data. It was created 100 models containing all non-hydrogen atoms. The best model structure was selected based on the indicator of the PDF (probability density distribution) of physical energy. The relative orientation of the VH - and VL-domain was set to the corresponding one in the structure of the reference variable domains with the greatest homology with both sequences (VL and VH), subject to modeling. Duplicate conformations in the simulation excluded, added the ends, and added the missing atoms of the side chains. The force field CHARMM made to models scFv and were subjected to 2000 cycles of energy minimization using RMS fall = 0.01 and a generalized born model using molecular volume (GBMV) as the model implicitly using the specified solvent.

For each of the antibodies were made calculations of ionization protein residues and pK. The calculations were done using the implementation provided Protocol in Discovery Studio 2.5.5 (DS 2.5.5), based on theory developed by Bashford and Karplus, 1991. The results of calculations of ionization protein residues and pK for the two molecules were compared to the index pK1/2side chains and titration curves of each taraudage residue, including Asp, Glu, Arg, Lys, His, Tyr, Cys, N-terminal and C-terminal residues of antibodies.

Power floor� CHARMM made to models scFv, and carried out the pyrolysis at a pH of 7.4, titration curves and pK of individual residues was calculated based on the pH range from pH 2 to pH 14 using a pH jumps=0,2. Two conservative Lys residue at positions 47 and 50 in the light chain of the antibody 578rFW1.4 compared with Lys at the same position in 34rFW1.4 (see Fig. 1).

The introduction of preferred substitutions

Software Discovery Studio was used to perform simulations of molecular dynamics for predicting mutations that increase the interaction between VL and VH, thus preventing the collapse of domains and the formation of oligomers and/or aggregates of higher order. The stability of the interaction between the VL/VH of the antibody 34rFW1.4 defined energy as the free energy G. the Adaptation of the Protocol CHARMM (also included in DS 2.5.5) used to calculate the difference between the energies of all heterodimeric complex scFv and the sum of energies of each of the individual variable domains. The calculations were done within the method using the implicitly given solvent using the generalized born model using molecular volume (GBMV). Were calculated the energies of both domains and complex, and the result of the calculation was set as the difference between the energy of the complex and the sum of the energy of the individual domains according to the following calculation: G interaction = G(a) G(b) - G(c), where G(a) is a fun� energy antibodies, G(b) is the energy of the VL and G(c) is the energy of the VH.

Arginine (R) was chosen as a possible replacement residue is lysine (K) at positions 47 and 50, because the value of pKa for the side chain R (12,5) more pKa for K (10,5). Mutants K47R and K50R were created by replacing K by R separately in the relevant provisions. 2000 cycles of energy minimization was performed on the residues located at a distance of 10 angstroms or closer to the region surrounding the mutation, to ensure the adaptation of the new model of the molecules to change. The model is implicitly specified solvent for these cycles of energy minimization was GBMV. Predicted average ΔG for mutants was -94 kcal/mol. Therefore, in accordance with the assessment of the contribution of mutations is 4 kcal/mol compared to the original antibody against TNF in the form of scFv.

Example 2

Stability analysis of optimized antibodies 34rFW1.4

K50R mutant and mutant K47R were created in the antibody 34rFW1.4 (which is described in located on the simultaneous consideration of the patent office, international application no PCT/CH2009/000219, filed June 25, 2009, the content of which is incorporated into this description by reference in full). Another mutant 34rFW1.4 containing additional substitutions to reduce its immunogenicity in vivo, was obtained in accordance with methods described � the application for U.S. patent No. 12/973968. In particular, the third mutant, named 34rFW1.4_VLK50R_DHP, contained a serine (S) at position 12 of the heavy chain (according to the AHo numbering system), threonine (T) at position 103 of the heavy chain (according to the AHo numbering system) and threonine (T) at position 144 the heavy chain (according to the AHo numbering system). Stability studies of the original and mutant antibodies 34rFW1.4 was carried out at accelerated conditions, as described below.

Antibody 34rFW1.4, mutant 34rFW1.4_VL_K50R and 34rFW1.4_VLK50R_DHP concentrated up to 20, 40 and 60 mg/ml in the composition of phosphate-buffered saline (50 mm Na2HPO4, 150 mm NaCl, pH 6.5) and incubated for 2 weeks at 40°C. Antibodies 34rFW1.4 and 34rFW1.4_K47R concentrated up to 20 and 60 mg/ml in the composition of phosphate-buffered saline (50 mm Na2HPO4, 150 mm NaCl, pH 6.5) and incubated for 2 weeks at 40°C. the Samples were investigated before and after incubation for 14 days in relation to degradation, using electrophoresis in a 12.5% polyacrylamide gel with sodium dodecyl sulfate (SDS-page) in regenerating and Sevostyanova conditions. Size exclusion liquid chromatography high resolution (size exclusion HPLC) was used to determine the content of monomers and soluble aggregates in the samples before and after incubation period. The monomers were separated from demonomania States per column (TSKgel Super SW2000 (TOSOH Biosience), and calculated the percentage of Monomeric protein as the area corresponding to the monomer peak, divided by the total area of all peaks obtained. The research results for 34rFW1.4 and 34rFW1.4_VLK50R_DHP shown in Fig. 2A-B, 3A-B and 4A-B, respectively. Results for 34rFW1.4_VL_K50R shown in Fig. 5B, 6B and 7B. These experiments showed that 34rFW1.4_VLK50R_DHP has a reduced ability to aggregate compared to 34rFW1.4. Mutant 34rFW1.4_K47R also demonstrated such a reduction, as shown in Fig. 8B and 9B.

In addition, compared thermal stability and affinity of antibody binding 34rFW1.4 and 34rFW1.4_VLK50R_DHP. The results showed that the mutations introduced to create antibodies 34rFW1.4_VLK50R_DHP not affect the stability or activity of binding compared to the original antibody 34rFW1.4.

EQUIVALENTS

Numerous modifications and alternative embodiments of the present invention will be obvious to specialists in this field in view of the preceding description. Accordingly, this description should be considered only as an illustration and is intended for communication specialists in this field the best embodiment of the present invention. The details of the structure may vary significantly without departing from the scope of the essence of the present invention, and exclusive use of all modifications that are included in the volume�m the annexed claims, saved. It is assumed that the present invention is limited only to the extent that provides the attached claims and the applicable rules of law.

All literature and similar materials cited in this application, including patents, patent applications, articles, books, treatises, dissertations, Web pages, figures and/or application, regardless of the format of such literature and similar materials included in a verbal form by reference in their entirety. In the case where one or more of the incorporated literature and similar materials differs from that application or contradict it, including the specific terms, the use of terms that describe how or etc., dominates this proposal.

Used in the present description section headings are used only for organizational purposes and should not be construed as limiting the described object in any way.

Although the present invention is described in connection with various embodiments of the implementation and examples, it is assumed that the ideas of the present invention are limited to these options implementation or examples. On the contrary, the present invention includes various alternatives, modifications and equivalents, as will be clear to experts in this field.

The formula of the invention should not sciaticabrite described order or elements, unless indicated to this effect. It should be understood that various changes in form and details may be made without departing from the scope of the appended claims. Therefore, the claimed rights for all implementation options that fall within the scope and essence of the following claims and their equivalents.

1. The antibody, or antigen-binding fragment that specifically binds to human TNFα, comprising:
a) variable domain light chain containing the sequence of SEQ ID NO: 2 or SEQ ID NO: 14, and
(b) the variable domain of the heavy chain containing the sequence of SEQ ID NO: 5.

2. The antibody according to claim 1, wherein the variable domain light chain contains the sequence of SEQ ID NO: 2.

3. The antibody according to claim 1, wherein the variable domain light chain contains the sequence of SEQ ID NO: 14.

4. The antibody according to claim 1, optionally with a linker containing the sequence of SEQ ID NO: 7.

5. The antibody according to claim 1, which contains the sequence of SEQ ID NO: 10.

6. The antibody according to claim 1, which contains the sequence of SEQ ID NO: 17.

7. Pharmaceutical composition for the treatment of TNFα-mediated diseases, containing a therapeutically effective amount of the antibody according to claim 1 and a pharmaceutically acceptable carrier.

8. A method of treating TNFα-mediated disease, comprising administering to the individual the pharmaceutical�certification of the composition according to claim 7.

9. A method according to claim 8, in which TNFα-mediated disease is an ocular disease selected from the group consisting of uveitis, disease Behcet, retinitis, dry eye, glaucoma, Sjogren syndrome, diabetic neuropathy, scleritis, age-related macular degeneration and keratitis.

10. A method according to claim 8, in which the pharmaceutical composition is administered using an ocular, intranasal, aural, sublingual, transdermal, local, oral, nasal, rectal or parenteral administration.

11. A method according to claim 10, wherein the pharmaceutical composition is administered once or divided into treatment in the total dose, of from 0.1 to 100 mg of the antibody.

12. A method according to claim 8, in which TNFα-mediated disease is uveitis, a pharmaceutical composition is applied topically in the eye of the individual.

13. The selected nucleic acid molecule that encodes the antibody according to claim 1.

14. The expression vector containing the nucleic acid molecule according to claim 13.

15. A host for expression of the antibody containing the vector according to claim 14.

16. The antibody, or antigen-binding fragment of claim 1, wherein the antigen-binding fragment is an Fab, Fab', F(ab)'2, single-chain Fv (scFv), Fv fragment, or a linear antibody.



 

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EFFECT: present invention can find further application in diagnosing and therapy of beta-amyloid related diseases, such as Alzheimer disease.

9 cl, 4 dwg, 5 tbl, 17 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a completely human monoclonal antibody, which binds insulin-like growth factor-II (IGF-II) and has a cross responsiveness to IGF-I, as well as its antigen-binding fragment. There are disclosed a nucleic acid molecule coding an antibody according to the invention, a vector and a host cell for the expression of the antibody according the invention. There are described a pharmaceutical composition, as well as conjugates for treating and diagnosing malignant tumour, using the antibody according to the invention in preparing the therapeutic agent and a method for determining IGF-II and IGF-I levels in a patient's sample.

EFFECT: present invention can find further application in cancer therapy.

16 cl, 27 ex, 18 tbl

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and immunology. There are presented optimised genes of light and heavy chains of Infliximab, an anti-tumour necrosis factor alpha (TNF-alpha) antibody, as well as a cell line VKPM-N-131, and a method for antibody biosynthesis. Nucleotide sequences of the genes coding the light and heavy chains of Infliximab are optimised in order to provide the content of codones most specific for mammals; the G/C content is expected to make 50-60% of the total composition; the absence of expanded tracts of a degenerate composition and the absence of RNA secondary structures.

EFFECT: Chinese hamster ovary cell line (CHO) produced by transfection by expression structures containing the genetic sequences according to the invention, enables producing at least 50 mg/l of the monoclonal antibody Infliximab.

4 cl, 3 dwg, 4 ex

FIELD: biotechnologies.

SUBSTANCE: strain of cells of the Chinese hamster ovaries CHO-Inflix 20/5 is produced, transfected by vectors, expressing light and heavy chains of chimeric antibody against tumour necrosis factor alpha (TNF-α) of human being is produced. The strain is deposited into the Russian collection of cellular cultures under No. RKKK(P)760D.

EFFECT: invention allows to produce chimeric antibody with the specific efficiency no less than 33,5 picograms per cell a day.

4 dwg, 2 tbl, 4 ex

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