Immunoglobulin with double variable domains and its applications

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed is binding protein for binding one or more targets, which contains four polypeptide chains forming four functional antigen-binding sites. Four polypeptide chains contain VD1-(X1)n-VD2-C-(X2)n. VD1 stands for first variable domain of heavy chain, VD2 stands for second variable domain of heavy chain, C stands for CH1 domain, X1 stands for polypeptide linker, on condition that it is not constant domain, and X2 stands for Fc-region, and n equals 0 or 1. Two polypeptide chains contain VD1-(X1)n-VD2-C. VD1 stands for first variable domain of light chain, VD2 stands for second variable domain of light chain, C stands for CL domain, X1 stands for linker, on condition that it is not constant domain; and n equals 0 or 1. Conjugate of binding protein with visualising detecting cytotoxic or therapeutic agent is described. Disclosed are: nucleic acids (NA), coding polypeptide chains, as well as expressing vectors, vectors for replication, host cells which contain them, and method of obtaining antibody applying cells. Described is pharmaceutical composition for treatment or preventing target-associated disease or disorder based on binding protein. Method of treatment by introduction of binding protein is described.

EFFECT: application of invention provides new format (DVD-Ig) of antigen-binding molecules, which in the same dosage possess higher activity with respect to target than respective full-size antibodies, which can be applied in medicine for prevention and treatment of various diseases.

45 cl, 27 tbl, 5 ex

 

Cross-reference to related application

In this application claims priority on provisional application U.S. No. 60/709911, filed August 19, 2005, and provisional application U.S. No. 60/732892, filed November 2, 2005.

The technical field to which the invention relates.

This invention relates to multivalent and multispecific binding proteins, methods of producing and, in particular, to their applications in the prevention and/or treatment of acute and chronic inflammation, cancer and other diseases.

The level of technology

Engineered proteins, such as multispecific antibodies, capable of binding two or more antigens are known in this field. Such multispecific binding proteins can be obtained by merging cells, chemical conjugation or by means of recombinant DNA.

Bespecifically antibody was obtained with the use of technology quadrom (see Milstein, C. and ..Cuello, Nature, 1983. 305(5934): p.537-40), based on somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies with the desired specificity especifismo antibodies. Due to the random mating of two different heavy and light chains of Ig in the resulting hybrid-hybridoma (or quadronno) cell lines were obtained up to ten different kinds of immunoglobu the ins, of which only one is functional bespecifically antibody. The presence of incorrectly paired by-products and significantly reduced outputs mean that required complex treatment methods.

Bespecifically antibodies can be obtained by chemical conjugation of two different mAb (see Staerz, U.D., et al., Nature, 1985. 314(6012): p. 628-31). This approach does not lead to homogeneous drug. Other approaches have used chemical conjugation of two different monoclonal antibodies or smaller fragments of antibodies (see Brennan, M., et al., Science, 1985. 229(4708): p. 81-3). Another way is to link the two primary antibodies with a hetero-bifunctional cross-linking agent, but received drugs bespecifically antibodies had the disadvantage of significant molecular heterogeneity, because the reaction of the crosslinking agent with the original antibody is not site-directed. For more homogeneous preparations bespecifically antibodies two different Fab-fragment chemically sewed together at their residues cysteine site-directed way (see Glennie, M.J., et al., J Immunol, 1987. 139(7): p. 2367-75). But this method leads to F(ab')2-fragments, not full IgG molecule.

Recently, we developed a wide variety of other formats recombinant bespecifically antibodies (see Kriangkum, J., et al., Biomol Eng, 2001. 18(2): p. 31-40). Among them the most Shir is used to formats for constructing recombinant bespecifically antibodies are tandem single-chain Fv molecules and diately and their various derivatives. Using the standard method of constructing these molecules begins with two single-chain Fv (scFv)fragments that recognize different antigens (see Economides, A.N., et al., Nat Med, 2003. 9(1): p. 47-52). Tandem scFv molecules(taFv) represent the direct format, directly connecting the two scFv molecules optional peptide linker. Two scFv-fragment present in these tandem scFv molecules, form the individual elements of the installation (folding). Can be used in different linkers to connect the two scFv fragments and linkers with up to 63 residues (see Nakanishi, K., et al., Annu Rev Immunol, 2001. 19: p. 423-74). Although the original scFv-fragments can usually be expressed in soluble form in bacteria, is often observed that tandem scFv molecules form insoluble aggregates in bacteria. Thus, the refolding protocols or systems mammalian expression used in the usual way to obtain a tandem scFv molecules. In a recent study reported expression in vivo transgenic rabbits and cattle tandem scFv directed against CD28 and associated with melanoma of proteoglycan (see Gracie, J.A., et al., J Clin Invest, 1999. 104(10): p. 1393-401). In this design two scFv molecules connected CH1-linker and found serum concentrations especifismo antibodies to 100 mg/L. To obtain the soluble expression in bacteria is x used a variety of strategies, including the variation of the order of the domains, or the use of secondary linkers of variable length or flexibility. Currently, several studies have reported the expression of soluble tandem scFv molecules in bacteria (see Leung, B.P., et al., J Immunol, 2000. 164(12): p. 6495-502; Ito, A., et al., J Immunol, 2003. 170(9): p. 4802-9; Karni, A., et al., J Neuroimmunol, 2002. 125(1-2): p. 134-40) using either a very short linker Ala3, or long rich in glycine/serine of linkers. In a recent study used a phage display tandem scFv-repertoire containing randomized average linkers in length from 3 or 6 residues, for enrichment against molecules that are produced in a soluble and active form in bacteria. This approach led to the selection of the preferred tandem scFv molecule with a linker of 6 amino acid residues (see Arndt, M. and J. Krauss, Methods Mol Biol, 2003. 207: p. 305-21). It is unclear whether this linker sequence General solution of the soluble expression of tandem scFv molecules. However, this study demonstrated that phage display tandem scFv molecules in combination with directed mutagenesis is a powerful tool for the enrichment of these molecules, which can be expressed in bacteria in the active form.

In bespecifically ditelo (Db) used for the expression of dimeric format. Diately the floor is up from scFv-fragments of a decrease in the length of the linker, connecting the VH - and VL-domain, up to about 5 residues (see Peipp, M. and T. Valerius, Biochem Soc Trans 2002. 30(4): p. 507-11). Reducing the size of the linker facilitates dimerization of two polypeptide chains crossover pairing of VH and VL domains. Bespecifically of diately get the expression of two polypeptide chains or with the structure of VHA-VLB and VHB-VLA (configuration VH-VL), or with the structure of the VLA-VHB and VLB-VHA (configuration VL-VH) in the same cell. In the past there were a large variety of different bespecifically dietel, and most of them can be expressed in soluble form in bacteria. However, a recent comparative study demonstrates that the orientation of the variable domains may influence the expression and formation of active binding sites (see Mack, M., G. Riethmuller, and P. Kufer, Proc Natl Acad Sci U S A, 1995. 92(15): p. 7021-5). However, soluble expression in bacteria is an important advantage compared with the tandem scFv molecules. However, because two different polypeptide chains are expressed in the same cell, together with the active heterodimers can be obtained inactive homodimer. This creates the need to introduce additional stages of purification to obtain homogeneous preparations bespecifically diatel. One approach to accelerate the generation bespecifically diatel is getting diatel type nob-into-hole (bulge-in-deepening) (see Holliger, P., T. Prospero, and G. Winter, Proc Natl Acad Sci U S A, 1993. 90(14): p. 6444-8 .18). It has been demonstrated to especifismo of diately directed against HER2 and CD3. A large "bulge" was introduced in the VH-domain replacement Val37 on Phe and Leu45 on Trp and complementary "deepening" received in VL-domain mutation Phe98 Met and Tyr87 in Ala in the variable domains of either anti-HER2 or anti-CD3. Using this approach, the production bespecifically diatel managed to increase from 72% source diately to more than 90% using the knob-into-hole diately. It is important that outputs only slightly decreased as a result of mutations. However, the reduction antigennegative activity was observed for several of the analyzed structures. Thus, this rather complex approach requires analysis of numerous structures for the identification of mutations that give heterodimeric molecule with unmodified binding activity. In addition, this approach requires mutational modification of immunoglobulin sequence in the constant region that creates negativnuu and unnatural form of a sequence antibodies that may result in increased immunogenicity, poor stability in vivo, as well as undesirable pharmacokinetics.

Stranded diately (scDb) represent an alternative strategy to improve education bespecifically dialaphone molecules (see Holliger, P and G. Winter, Cancer Immunol Immunother, 1997. 45(3-4): p. 128-30; Wu, A.M., et al., Immunotechnology, 1996. 2(1): p. 21-36). Bespecifically stranded diately receive a combination of the two datalibrary polypeptide chains with additional placed in the middle of the linker with a length of approximately 15 amino acid residues. Thus, all molecules with a molecular mass corresponding to the Monomeric single-chain Diethelm (50-60 kDa)are bespecifically. Several studies have demonstrated that bespecifically stranded diately expressed in bacteria in soluble and active form, and most of purified molecules are present as monomers (see Holliger, P. and G. Winter, Cancer Immunol Immunother, 1997. 45(3-4): p. 128-30; Wu, A.M., et al., Immunotechnology, 1996. 2(1): p. 21-36; Pluckthun, A. and P. Pack, Immunotechnology, 1997. 3(2): p. 83-105; Ridgway, J.B., et al., Protein Eng, 1996. 9(7): p. 617-21). Thus, stranded diately combine the advantages of tandem scFvs (all monomers are bespecifically) and dietel (soluble expression in bacteria).

Later ditelo was fused with the Fc to generate more Ig-like molecules, called di-diatesom (see Lu, D., et al., J Biol Chem, 2004. 279(4): p. 2856-65). In addition, it was described multivalent design antibodies containing two Fab-repeat in the heavy chain of IgG and can bind four molecules of antigen (see WO 0177342A1, and Miller, K., et al., J Immunol, 2003. 170(9): p. 4854-61).

In this area there is the duty to regulate the need for improved multivalent binding proteins, capable of binding two or more antigens. This invention provides a new family of binding proteins capable of binding two or more antigens with high affinity.

The invention

This invention relates to multivalent binding proteins capable of binding two or more antigens. The invention provides a new family of binding proteins capable of binding two or more antigens with high affinity.

In one of the embodiments, the invention provides a binding protein containing the polypeptide chain, and the specified polypeptide chain contains VD1-(X1)n-VD2-C-(X2)n, in which VD1 is the first variable domain, VD2 means of the second variable domain, C is a constant domain, X1 represents an amino acid or polypeptide, X2 represents an Fc region and n is 0 or 1. In a preferred embodiment, VD1 and VD2 of the binding protein means the variable domains of the heavy chain. More preferably variable domain of the heavy chain selected from the group consisting of mouse variable domain of the heavy chain, variable domain of the heavy chain human CDR-transplanted variable domain of the heavy chain and gumanitarnogo variable domain of the heavy chain. In a preferred embodiment, VD1 and VD2 are capable of ativate the same antigen. In another embodiment, VD1 and VD2 are capable of binding different antigens. Preferably means constant domain of the heavy chain. More preferably X1 means the linker with the proviso that X1 is not CH1. Most preferably, X1 denotes a linker selected from the group consisting of AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA(G4S)4;SAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVTLFPP; AKTTPP; AKTTPPSVTPLAP; AKTTAP; AKTTAPSVYPLAP; ASTKGP; ASTKGPSVFPLAP, GGGGSGGGGSGGGGS; GENKVEYAPALMALS; GPAKELTPLKEAKVS and GHEAAAVMQVQYPAS. Preferably X2 means Fc-region. More preferably, X2 is a variant Fc region.

In a preferred embodiment, the binding protein described above, contains the polypeptide chain, and the specified polypeptide chain contains VD1-(X1)n-VD2-C-(X2)n, in which VD1 is the first variable domain of the heavy chain, VD2 means of the second variable domain of the heavy chain, C is a constant domain of the heavy chain, X1 denotes a linker provided that it is not CH1, and X2 is an Fc-region.

In another embodiment, VD1 and VD2 of the binding protein are variable domain light chain. Preferably variable domain light chain selected from the group consisting of mouse variable domain of the light chain, variable domain of the light chain human CDR-transplanted in labeling domain light chain and gumanitarnogo variable domain of the light chain. In one embodiment, VD1 and VD2 are capable of binding the same antigen. In another embodiment, VD1 and VD2 are capable of binding different antigens. Preferably means constant domain of the light chain. More preferably X1 means the linker with the proviso that X1 is not CL1. Preferably X1 is a linker selected from the group consisting of AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA(G4S)4;SAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVTLFPP; AKTTPP; AKTTPPSVTPLAP; AKTTAP; AKTTAPSVYPLAP; ASTKGP and ASTKGPSVFPLAP. Preferably the binding protein does not contain X2.

In a preferred embodiment, the above-described binding protein contains a polypeptide chain, and the specified polypeptide chain contains VD1-(X1)n-VD2-C-(X2)n, in which VD1 is the first variable domain light chain, VD2 means of the second variable domain light chain, C is a constant domain of the light chain, X1 denotes a linker provided that it is not CH1, and X2 does not contain the Fc-region.

In another preferred embodiment, the invention provides a binding protein comprising two polypeptide chains, with the specified first polypeptide chain contains VD1-(X1)n-VD2-C-(X2)n, in which VD1 is the first variable domain of the heavy chain, VD2 means of the second variable domain of the heavy chain, means constant domain of the heavy chain, X1 means the linker provided that it is not CH1, and X2 is an Fc-region; and the specified second polypeptide chain contains VD1-(X1)n-VD2-C-(X2)n, in which VD1 is the first variable domain light chain, VD2 means of the second variable domain light chain, C is a constant domain of the light chain, X1 denotes a linker provided that it is not CH1, and X2 does not contain the Fc-region. Most preferably the binding protein with a dual variable domain (DVD) contains four polypeptide chains, the first two polypeptide chains contain VD1-(X1)n-VD2-C-(X2)n, respectively, where VD1 is the first variable domain of the heavy chain, VD2 means of the second variable domain of the heavy chain, C is a constant domain of the heavy chain, X1 denotes a linker provided that it is not CH1, and X2 is an Fc region; and the second two polypeptide chains contain VD1-(X1)n-VD2-C-(X2)n, respectively, where VD1 is the first variable domain light chain, VD2 means of the second variable domain light chain, C is a constant domain of the light chain, X1 denotes a linker provided that it is not CH1, and X2 does not contain the Fc-region. Such protein with dual variable domain (DVD) has four antigenspecific site.

In another preferred embodiment, the above-described binding proteins capable of binding one or more targets. Prefer the Ino target selected from the group consisting of cytokines, cell surface proteins, enzymes and receptors. Preferably the binding protein is capable of modulating a biological function of one or more targets. More preferably the binding protein is capable of neutralizing one or more targets. Binding protein according to the invention are capable of binding a cytokine selected from the group consisting of lymphokines, monokines and polypeptide hormones. In a specific embodiment, the binding protein capable of binding a pair of cytokines selected from the group consisting of IL-1α and IL-1β; IL-12 and IL-18, TNFα and IL-23, TNFα, and IL-13; TNF and IL-18; TNF and IL-12; TNF and IL-1β; TNF and MIF; TNF and IL-17; and TNF, and IL-15; TNF and VEGF; VEGFR and EGFR; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAM8; and TNFα and PGE4, IL-13 and PED2, TNF and PEG2. In another embodiment, the binding protein according to the invention are capable of binding a pair of targets selected from the group consisting of CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD38 and CD138; CD38 and CD20; CD38 and CD40; CD40 and CD20; CD-8 and IL-6; CSPG and RGM A; CTLA-4 and BTNO2; IGF1 and IGF2; IGF1/2 and Erb2B; IL-12 and TWEAK; IL-13 and IL-1β; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; PDL-1 and CTLA-4; RGM A and RGM B; Te38 and TNFα; TNFα and Blys; TNFα and CD-22; TNFα and CTLA-4; TNFα and GP130; TNFα and IL-12p40; and TNFα and RANK ligand.

In one embodiment, the binding protein capable of binding IL-1α of human rights and IL-1β person, contains linakis is now a sequence of the heavy chain DVD selected from the group consisting of SEQ ID NO. 33, SEQ ID NO. 37, SEQ ID NO. 41, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57 and SEQ ID NO. 59; and the amino acid sequence of the light chain DVD selected from the group consisting of SEQ ID NO. 35, SEQ ID NO. 39, SEQ ID NO. 43, SEQ ID NO. 46, SEQ ID NO. 49, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58 and SEQ ID NO. 60. In another embodiment, the binding protein capable of binding murine IL-1α and murine IL-1β, contains the amino acid sequence of the heavy chain DVD SEQ ID NO. 105 and amino acid sequence of the light chain DVD SEQ ID NO. 109.

In one embodiment, the binding protein capable of binding IL-12 and IL-18, contains the amino acid sequence of the heavy chain DVD selected from the group consisting of SEQ ID NO. 83, SEQ ID NO. 90, SEQ ID NO. 93, SEQ ID NO. 95 and SEQ ID NO. 114; and the amino acid sequence of the light chain DVD selected from the group consisting of SEQ ID NO. 86, SEQ ID NO. 91, SEQ ID NO. 94, SEQ ID NO. 46, SEQ ID NO. 96 and SEQ ID NO. 116.

In one embodiment, the binding protein capable of binding CD20 and CD3, contains the amino acid sequence of the heavy chain DVD SEQ ID NO. 97 and amino acid sequence of the light chain DVD SEQ ID NO. 101.

In another embodiment, the binding protein according to the invention are capable of binding one, two or more cytokines, cytokine-related proteins and receptors cytokines selected from the group consisting of BMP1, BMP2, BMP3B (GDF0), BMP4, BMP6, BMP8, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (aFGF), FGF2 (bFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1, IFNG, IFNW1, FILE1, FILE1 (EPSILON), FILE1 (ZETA), IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL17B, IL18, IL19, IL20, IL22, IL23, IL24, IL25,, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (TNF-a), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (ligand D27), TNFSF8 (CD30 ligand), TNFSF9 (ligand of 4-1BB), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, FIGF (VEGFD), VEGF, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN and THPO.

Binding protein according to the invention are capable of binding one or more chemokines, receptors chemokines and chemokine-related proteins selected from the group consisting of CCL1 (I-309), CCL2 (MCP-1/MCAF), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCL11 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL20 (MIP-3a), CCL21 (SLC/Exodus-2), CCL22 (MDC/STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK/ILC), CCL28, CXCL1 (GRO1), CXCL2 (GRO2), CXCL3 (GRO3), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL10 (IP-10), CXCL11 (I-TAC), CXCL12 (SDF1), CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP (CXCL7), CX3CL1 (SCYD1), SCYE1, XCL1 (lymphotactin), XCL2 (SCM-1b), BLR1 (MDR15), CCBP2 (D6/JAB61), CCR1 (CKR1/HM145), CCR2 (mcp-1RB/RA), CCR3 (CKR3/CMKBR3), CCR4, CCR5 (CMKBR5/ChemR13), CCR6 (CMKBR6/CKR-L3/STR22/DRY6), CCR7 (CKR7/EBI1), CCR8 (CMKBR8/TER1/CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), XCR1 (GPR5/CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28), CXCR4, GPR2 (CCR10), GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6 (TYMSTR/STRL33/Bonzo), HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R (GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HIF1A, IL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2 and VHL. Binding protein according to the invention are capable of binding protein cell surface selected from the group consisting of integrins. Binding protein according to the invention are capable of binding an enzyme selected from the group consisting of kinases and proteases. Binding protein according to the invention are capable of binding to a receptor selected from the group consisting of a receptor of lymphokine, receptor Monokini and receptor polypeptide hormone.

In a preferred embodiment, the binding protein is multivalent. More preferably the binding protein is multispecificity. Multivalent and/or multispecific binding proteins described above, have the desired properties, in particular, from a therapeutic point of view. For example, multivalent and/or multispecific binding protein can (1) be internality (and/or catabolization) faster than bivalent antibody, a cell expressing an antigen to which antibodies bind; (2) to be antibody-agonist; and/or (3) to induce death to EDI and/or apoptosis of cells, expressing the antigen with which the multivalent antibody can bind. "The original antibody, which provides at least one antigennegative specificity multivalent and/or multispecific binding protein may be an antibody that internalizes (and/or catabolized) a cell expressing the antigen with which the antibody binds); and/or may be an agonist, inducing cell death and/or inducing apoptosis, and is described here multivalent and/or multispecific binding protein can be improved (improvement) in one or more of these properties. In addition, the original antibody may be deprived of any one or more of these properties, but may be empowered by them in the construction as described here multivalent binding protein.

In another embodiment, the binding protein according to the invention has a rate constant of Association (Kon) to one or more targets selected from the group consisting of values of the specified constant comprising at least about 102M-1with-1; at least about 103M-1with-1; at least about 104M-1with-1; at least about 105M-1with-1; and is of at least approximately 10 6M-1with-1as measured by surface plasmon resonance. Preferably the binding protein according to the invention has a rate constant of Association (Kon) to one or more targets, comprising from 102M-1with-1up to 103M-1with1; 103M-1with1up to 104M-1with1; 104M-1with-1up to 105M-1with-1or from 105M-1with-1up to 106M-1with-1as measured by surface plasmon resonance.

In another embodiment, the binding protein has a rate constant of dissociation (Koff) to one or more targets selected from the group consisting of values of the specified constant, the components of at most approximately 10-3with-1; at most about 10-4with-1; at most about 10-5with-1; and at most about 10-6with-1as measured by surface plasmon resonance. Preferably the binding protein according to the invention has a rate constant of dissociation (Koff) to one or more targets, comprising from 10-3with-1up to 10-4with-1; 10-4with-1up to 10-5with-1; or from 10-5with-1on the 10 -6with-1as measured by surface plasmon resonance.

In another embodiment, the binding protein has a dissociation constant (KDin relation to one or more targets selected from the group consisting of values of the specified constant, the components of at most approximately 10-7M; at most about 10-8M; at most about 10-9M; at most about 10-10M; at most about 10-11M; at most about 10-12M; and at most 10-13M. Preferably the binding protein according to the invention has a dissociation constant (KDin relation to IL-12 or IL-23, comprising from 10-7M to 10-8M; 10-8M to 10-9M; 10-9M to 10-10M; 10-10up to 10-11M; 10-11M to 10-12M; or from 10-12up to 10 M-13M.

In another embodiment, the above-described binding protein is conjugated, optionally containing an agent selected from the group consisting of molecules immunoadhesin, agent imaging, therapeutic agent, and a cytotoxic agent. Preferably the agent visualization selected from the group consisting of a radioisotope label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magician who itoi label, and Biotin. More preferably the agent visualization is a radionuclide label selected from the group consisting of3H,14C,35S90Y99Tc111In125I131I177Lu,166Ho and153Sm. Preferably therapeutic or cytotoxic agent selected from the group consisting of an antimetabolite, an alkylating agent, an antibiotic, a growth factor, cytokine, antiangiogenic agent, antimitoticescoy agent, anthracycline, toxin and apoptotic agent.

In another embodiment, the above-described binding protein is a crystallized binding protein and exists in the form of a crystal. Preferably the crystal is not containing a pharmaceutical carrier crystal controlled release. More preferably crystallized binding protein has a greater half-life in vivo than the soluble counterpart of the specified binding protein. Most preferably crystallized binding protein retains biological activity.

In another embodiment, the above-described binding protein is glycosylated. Preferably glycosylation glycosylation is characteristic of the man.

One aspect of this invention relates to a selected nucleic acid, Cody is relevant for any of the above binding proteins. Additional option exercise provides a vector containing the above-described nucleic acid, and the specified vector selected from the group consisting of pcDNA; pTT (Durocher et al.,Nucleic Acids Research2002, Vol 30, No.2); pTT3 (pTT with additional multiple cloning site; pEFBOS (Mizushima, S. and Nagata, S., (1990)Nucleic acids ResearchVol 18, No. 17); pBV; pJV; pcDNA3.1 TOPO, pEF6 TOPO and a Shader with a.

In another aspect, the cell-master transform described above vector. Preferably the host-cell is a prokaryotic cell. More preferably the host-cell is E. coli. In a related embodiment, the host-cell is a eukaryotic cell. Preferably, the eukaryotic cell is selected from the group consisting of cell protist, animal cells, plant cells and fungal cells. More preferably the host-cell is a cell of a mammal, including, but not limited to, Cho and COS; or a fungal cell, such as Saccharomyces cerevisiae; or an insect cell such as Sf9.

In another aspect the present invention provides a method of obtaining the above-described binding protein, comprising culturing any of the host cells, as described above in a culture medium under conditions sufficient to produce the binding protein. Preferably 50%-75% binding protein obtained FPIC is BOM, are tetravalent binding protein with dual specificity. More preferably 75%-90% binding protein obtained in this way are tetravalent protein with dual specificity. Most preferably 90%-95% of the received binding protein are tetravalent binding protein with dual specificity.

Another variant implementation provides a binding protein, obtained as described above.

One of the embodiments provides a composition for the release of a binding protein, where the composition contains a ready-made form, which, in turn, contains the above-described crystallized binding protein and an ingredient; and at least one polymeric carrier. Preferably the polymeric carrier is a polymer selected from one or more groups consisting of poly(acrylic acid), poly(cyanoacrylates), poly(amino acids), poly(anhydrides), poly(depsipeptide), poly(esters), poly(lactic acid), copolymer of lactic and glycolic acids or PLGA, poly(β-hydroxybutyrate), poly(caprolactone), poly(dioxanone); poly(ethylene glycol), poly(hydroxypropyl)methacrylamide, poly[(organo)phosphazene], poly(complex arteparon), poly(vinyl alcohol), poly(vinylpyrrolidone), copolymers of maleic dioxide is IDA and alkylvinyl ether, polyols pluronics, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycosinolates, sulfated polysaccharides, their mixtures and copolymers. Preferably ingredient selected from the group consisting of albumin, sucrose, trehalose, lactate, gelatin, hydroxypropyl-β-cyclodextrin, methoxypolyethyleneglycol and polyethylene glycol. In another embodiment presents a method of treating a mammal, including a stage of introduction to the mammal an effective amount of the above composition.

This invention also provides a pharmaceutical composition comprising a binding protein described above and a pharmaceutically acceptable carrier. In the following embodiment, the pharmaceutical composition comprises at least one additional therapeutic agent for the treatment of disorders. Preferably the additional agent is selected from the group consisting of a therapeutic agent, agent visualization, a cytotoxic agent, angiogenesis inhibitors (including, but not limited to, anti-VEGF antibodies or VEGF-trap); inhibitors of kinases (including but not limited to, inhibitors of KDR and TIE-2); blockers molecules costimulation (including, but not limited to, anti-B7.1 and anti-B7.2, CTLA4-Ig, anti-CD20); bloccato the s adhesion molecules (including, but not limited to, anti-LFA-1-Ab, anti-E/L selectin-Ab inhibitors small molecule); anti-cytokine antibody or functional fragment (including, but not limited to, anti-IL-18, anti-TNF antibodies against IL-6/cytokine receptor); methotrexate; cyclosporin; rapamycin; FK506; detectable label or reporter; a TNF antagonist; an Antirheumatic agent, muscle relaxant, a narcotic agent, non-steroidal anti-inflammatory drug (NSAID), an analgesic agent, an anesthetic agent, a sedative, a local anesthetic means, a neuromuscular blocker, an antimicrobial agent, antipsoriatics agent, steroid, anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, immunosuppressant, growth hormone, hormone replacement drug, a radiopharmaceutical tools, antidepressant, antipsychotics, stimulant, anti-asthma drugs, beta-agonists, inhalation steroid, an epinephrine or analog, a cytokine, and a cytokine antagonist.

In another aspect, the invention provides a method of treatment of a subject-a person suffering from a disorder, in which the target or the target is able to communicate the above-described binding protein is (are) harmful involving the introduction the of this subject-the person described above binding protein so to inhibit the activity of the target or targets in the subject of human rights and to seek treatment. Preferably breach selected from the group consisting of arthritis, osteoarthritis, chronic juvenile arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin-dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis scleroderma, a disease graft-versus-host rejection of transplants of organs, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, grave's disease, nephrotic syndrome, syndrome of chronic fatigue, Wegener's granulomatosis, purpura's disease-Seleina, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, horii's disease, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemali the practical anemia, malignant diseases, cardiac insufficiency, myocardial infarction, Addison's disease, sporadic pluriglandular deficiency type I and pluriglandular deficiency type II syndrome Schmidt (acute) respiratory distress syndrome of adults, alopecia, alopecia (alopecia) areata, seronegative arthropathy, arthropathy, disease, Reiter, psoriatic arthropathy, arthropathy in ulcerative colitis, enteropathic synovitis associated with Chlamydia, Yersinia and Salmonella arthropathies, spondyloarthopathy, atheromatous disease/arteriosclerosis, atopic Allergy, autoimmune bullous diseases, ordinary water, leaf water, pemphigoid, linear IgA disease, autoimmune haemolytic anemia positive hemolytic anemia Coombs, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/British myalgic encephalitis, chronic mucocutaneous candidiasis, giant cell arteritis diagnostics, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, acquired immunodeficiency syndrome, a disease, related to the acquired immunodeficiency, hepatitis b, hepatitis C, common transient immunodeficiency (common transit hypogammaglobulinaemia), dilated cardio is ipatio, female infertility, ovarian function decline, premature ovarian function decline, fibroticheski lung disease, cryptogenic fibrosing alveolitis, polioptilidae interstitial lung disease, interstitial pneumonitis associated with connective tissue disease interstitial lung disease associated with disease mixed connective tissue of the lung disease associated with systemic sclerosis interstitial lung disease associated with rheumatoid arthritis interstitial lung disease associated with systemic lupus erythematosus disease of the lungs associated with dermatomyositis/polymyositis lung disease associated with disease Sjogren lung disease associated with ankylosing spondylitis disease of the lungs, vasculitis diffuse lung disease associated with hemosiderosis lung disease induced drug interstitial lung disease, fibrosis, radiation fibrosis, obliterating bronchiolitis, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, post-infectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, autoimmune hepatitis type 1 (classical autoimmune or lupoid is Apatite), autoimmune hepatitis type-2 (hepatitis C anti-LKM-antibodies), autoimmune mediated hypoglycemia, insulin resistance type with papillary pigment dystrophy of the skin (acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leukopenia, autoimmune neutropenia, renal disease NOS (BDU), glomerulonephritis, microscopic vasculitis of the kidneys, Lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS (BDU), autoimmunity sperm, multiple sclerosis (all subtypes), sympathetic OFTAL, pulmonary hypertension secondary to connective tissue disease, syndrome?, pulmonary manifestation nadeznogo of polyarteritis, acute rheumatic fever, rheumatoid spondylitis, of still's disease, systemic sclerosis, Sjogren syndrome, disease, Takayasu's disease/arteritis diagnostics, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease,), atrophic autoimmune hypothyroidism, primary myxedema, facog the frame uveitis, primary vasculitis, vitiligo, acute liver disease, chronic liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, cholestasis, idiosyncratic liver disease, drug-induced tool hepatitis, non-alcoholic steatohepatitis, Allergy and asthma, streptococcal infection group B (GBS), mental disorders (e.g., depression and schizophrenia), Th type 2 - and Th type 1-mediated diseases, acute and chronic pain (different forms of pain), and cancers such as lung cancer, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), abetalipoproteinemia, acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, atrial ectopic beats, AIDS dementia, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, deficiency of alpha-l-antitrypsin deficiency, and the side europejskiego sclerosis, anemia, angina, degeneration of anterior horn cells of the spinal cord, anti-cd3 therapy, antiphospholipid syndrome, allergic reactions to protivozachatochnye antibodies, aortic aneurysm and peripheral vascular, aortic dissection, arterial hypertension, arteriosclerosis, atrioventricular fistula, ataxia, atrial fibrillation (persistent or paroxysmal AF), atrial flutter, atrioventricular block, b-cell lymphoma, rejection, bone graft rejection, bone marrow transplant (BMT), interventricular blockade, Burkitt lymphoma, burns, cardiac arrhythmias, syndrome temporary cardiac arrest, heart tumors, cardiomyopathy, inflammatory reactions in artificial circulation of the blood, transplant rejection cartilage, cerebellar cortical degenerations, cerebellar violations, chaotic or multichrome atrial tachycardia associated with chemotherapy disorders, chronic malacitana leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colon cancer, congestive heart failure, conjunctivitis, contact dermatitis, pulmonary heart, coronary artery disease, is aesni of Creutzfeldt-Jakob, negative in the culture of sepsis, cystic fibrosis, associated with therapy with cytokines violations, Boxing dementia, demyelinating diseases, Dengue hemorrhagic fever, dermatitis, dermatologic conditions, diabetes, diabetes mellitus, diabetic arteriosclerotic diseases, cells with diffuse Lewy, dilated congestive cardiomyopathy, disorders of the basal (subcortical) nuclei of the brain, down's syndrome in middle age, induced by drugs disorders movement disorders induced by drugs which block CNS dopamine receptors, sensitivity to medications, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, infection with Epstein-Barr, erythromelalgia, extrapyramidal and cerebellar disorders, family hematopoietins lymphocytic histiocytosis, graft rejection, fetal thymus, Friedreich's ataxia, functional peripheral arterial disorders, fungal sepsis, gas (anaerobic) gangrene, gastric ulcers, glomerulonephritis, transplant rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas caused by intracellular organisms, leukemia pet is calendulis, disease Hallervorden-Spitze, Hashimoto thyroiditis, hay fever, transplant rejection hearts, hemochromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis (A), arrhythmia beam GIS, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, allergic reactions, allergic pneumonitis, hypertension, hypokinetic disorders of movement, diagnosis of the hypothalamic-pituitary-adrenal axis, idiopathic Addison's disease, idiopathic pulmonary fibrosis antibody-mediated cytotoxicity, asthenia, pediatric spinal muscular atrophy, inflammation of the aorta, influenza a, impact ionizing radiation, iridocyclitis/uveitis/optic neuritis, ischemic/reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, transplant rejection of kidney, infections with Mycobacterium Legionella, leishmaniasis, leprosy, lesions kortiko-spinal system, fat edema, transplant rejection liver, lymph edema, malaria, malignant lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic, cluster goal the main pain, mitochondrial Multisystem disorders, disease mixed connective tissue monoclonal gammopathy, multiple myeloma, degenerations, multiple systems (Mencel Dejerine-Thomas Shi-Drager and Machado-Joseph), heavy pseudoparallelism infants, ycobacterium avium intracellulare, tubercle bacilli, myelodysplasia syndrome, myocardial infarction, ischemic disorders of the myocardium, cancer of the nasopharynx, chronic lung disease of the newborn, nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscular atrophy, neurogenic fever, non-Jackinsky lymphoma, occlusion of the abdominal aorta and its branches, the occlusal arterial disorders, okt3 therapy, orchitis/epididymitis, procedures after treatment of orchitis/vasectomy, organomegaly, osteoporosis, transplant rejection, pancreas cancer, pancreatic cancer, paraneoplastic syndrome/hypercalcemia in malignant disease, transplant rejection parathyroid glands, inflammatory diseases of the renal pelvis, year-round rhinitis, diseases of the pericardium, peripheral atherosclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia, pneumonia, caused neumocystis carinii, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal Gampopa the AI syndrome and skin changes), postperfusion syndrome, potentializing syndrome, syndrome, post-MI-cardiotomy, pre-eclampsia, progressive supranuclear palsy, primary pulmonary hypertension, radiation therapy, phenomenon and disease, Raynaud's disease, disease, Raynaud's disease, diseases of Resume, tachycardia, regular narrow QRS, renovaskulyarnoy (renal-vascular) hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, scleroderma, senile horei, senile dementia associated with calves Levi, seronegative arthropathy, shock, sickle cell anemia, rejection, allograft skin syndrome skin changes, transplant rejection small intestine, solid tumors, specific arrhythmias, spinal ataxia, spinal-cerebellar degenerations, streptococcal myositis, structural lesions of the cerebellum, subacute sclerosing panencephalitis, syncope (syncope;), cardiovascular syphilis, systemic anaphylaxis, systemic inflammatory syndrome reactions, systemic juvenile rheumatoid arthritis, T-cell or FAB ALL (acute lymphoblastic leukemia), telangiectasia, obliterating thromboangiitis, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, allergic reactions type III Allergy type IV, unstable angina, uremia, urosepsis, urticaria valvular heart disease, varicose veins, vasculitis, venous diseases, venous thrombosis, atrial fibrillation (flicker) ventricular fibrillation, viral and fungal infections, acute encephalitis with a high risk of death/aseptic meningitis, hemophagocytosis syndrome associated with a high risk of death, syndrome Wernicke-Korsakov, Wilson disease, xenograft rejection of any organ or any fabric.

In another aspect, the invention provides a method of treatment of a patient suffering from disorders involving the stage of introduction of any of the above binding proteins before, concurrently or after administration of the second agent, as discussed above. In a preferred embodiment, the second agent is selected from the group consisting of budenoside, epidermal growth factor, corticosteroids, cyclosporin, sulfasalazin, aminosalicylates, 6-mercaptopurine, azathioprine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, inhibitors of thromboxane antagonists to receptor IL-1, monoclonal anti-IL-1β antibodies, monoclonal anti-IL-6 antibodies, growth factors, elastase inhibitors, pyridinedimethanol compounds, antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, IL-23, EMAP-II, GM-CSF, FGF, and PDGF, antibodies of CD2, CD3, CD4, CD8, CD 19, CD25, CD28, CD30, CD0, CD45, CD69, CD90 or their ligands, methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, Leflunomide, NSAIDs, ibuprofen, corticosteroids, prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, inhibitors of complement, adrenergic agents, IRAK, NIK, IKK, p38, inhibitors of MAP kinases, inhibitors of IL-1β-converting enzyme inhibitors, TNFα converting enzyme inhibitors transmission signal T cells, inhibitors of metalloproteinases, sulfasalazin, azathioprine, 6-mercaptopurine, inhibitors of angiotensin-converting enzyme, soluble receptors, cytokines, soluble TNF receptor P55 soluble TNF receptor P75, sIL-1RI, sIL-1RII, sIL-6R, anti-inflammatory cytokines, IL-4, IL-10, IL-11, IL-13 and TGFβ.

In a preferred embodiment, the above-described pharmaceutical composition is administered to a subject at least one method selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, vnutridomovogo, intracavitary, intracellular, intracerebellar, intracerebroventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardial, intraperitoneal, in triplicating, vnutripoliticheskoi, intra-lungs, intrarectal, internalname, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesicular, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal methods.

In one aspect the present invention provides at least one antiidiotypic antibody of at least one binding protein according to the invention. This antiidiotypic antibody includes any protein or peptide molecule that contains at least a portion of immunoglobulin molecules, such as, without limitation, at least one complementarity determining region (CDR) of a heavy or light chain or a ligand-binding part of the variable region of the heavy chain or light chain constant region of the heavy chain or light chain, frame area or any part thereof that may be included in the binding protein according to the invention.

In another embodiment, binding proteins according to the invention are capable of binding one or more targets selected from the group consisting of ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1; C19orf10 (IL27w); C3; C4; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6/JAB61); CCL1 (I-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a), CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL3 (MIP-1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2 (mcp-1RB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR13); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBI1); CCR8 (CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21Wap1/Cip1); CDKN1B (p27Kip1); CDKN1C; CDKN2A (p16INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; chitinases; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28); CXCL1 (GRO1); CXCL10 (IP-10); CXCL11 (I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2; human epidermal growth factor; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FILE1 (EPSILON); FILE1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10 (C10); fiberglass; GSN (gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HIP1; histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-α; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFN; IFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; IL1A; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2 IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25,; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); IL7; IL7R; IL8; IL8RA; IL8RB; IL8RB; IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b4-integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (keratin 19); KRT2A; KRTHB6 (specific for hair keratin type II); LAMA5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionein-III); MTSS1; MUC1 (mucin); MYC; MYD88; NCK2; narukana; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NR0B1; NR0B2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN;PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS1; RGS13; RGS3; RNF110 (ZNF144); robo2 on; S100A2; SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial monocyte-activating cytokine); SDF2; SERPINA1; SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-1); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Spr1); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB1I1; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TIMP3; tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAIP2 (B94); TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (ligand of 4-1BB); TOLLIP; Toll-like receptors; TOP2A (topoisomerase Iia); TP53 genes; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; version; VHL C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5/CCXCR1); YY1; and ZFPM2.

Brief description of drawings

Figa is a schematic representation of the structures of Dual Variable Domain (DVD)-Ig and shows the strategy of generating a DVD-Ig of the two primary antibodies; Figv is a schematic representation of the structures of DVD1-Ig, DVD2-Ig and two chimeric monospecific antibodies from two hybridoma clones 2D13.E3 (anti-IL-1α) and 13F5.G5 (anti-IL-1β).

Detailed description of the invention

This invention relates to multivalent and/or multispecific the Kim-binding proteins, capable of binding two or more antigens. Specifically, the invention relates to antibodies with dual variable domain (DVD-Ig) and their pharmaceutical compositions, as well as nucleic acids, recombinant expressing vectors and cells of the host for such DVD-Ig. This invention also includes methods of using the DVD-Ig according to the invention for the detection of specific antigens either in vitro or in vivo.

If there are no other specific instructions here, scientific and technical terms used in connection with the present invention, will have values that are commonly understood by experts in the field. Value and volume of these terms should be clear, however, in the case of any uncertainty, the definitions given here should be considered to prevail over any dictionary or other definition. In addition, unless the context explicitly requires another meaning, the terms in the singular number shall mean and the plural, and the terms in the plural will include the singular. In this application the use of "or" means "and/or"if no other instructions. In addition, the use of the term "comprising", as well as other forms, such as "includes" and "included" is not limiting. Terms such as "element"or "component" include both elements, and components containing one unit, and the elements and components that contain more than one subunit, if no specific other instructions.

Usually the item (and methods)used in connection with the culture of cells and tissues, molecular biology, immunology, Microbiology, genetics and chemistry of proteins and nucleic acids and hybridization described herein are items that are well known and commonly used in this field. Methods according to the invention are usually performed in accordance with conventional methods, well known in this field and described in various General and more specific references that are cited and discussed throughout the present description, unless otherwise specified. Enzymatic reactions and purification techniques performed in accordance with the descriptions of the manufacturer, as is usually done in the field or described here. The item (and laboratory procedures and methods)used in connection with analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry described herein are well known and commonly used in this field. Standard methods used for chemical syntheses, chemical analyses, pharmaceutical preparation, getting ready forms and delivery and for the treatment of patients.

For easier understanding of the present invention, the selected terms are defined below.

Used in this description, the term “polypeptide” refers to any polymer chain of amino acids. The terms “peptide” and “protein” are used interchangeably and refer to a polymer chain of amino acids. The term “polypeptide” includes native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. The polypeptide may be Monomeric or polymeric.

The term “isolated protein" or “isolated polypeptide" means a protein or polypeptide, which due to its origin or source not associated with natural and associated components that accompany it in its native state; essentially does not contain other proteins from the same species; is expressed by a cell from a different species or does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it occurs in nature, will be "isolated" from its natural and associated components. Protein can also be essentially exempt from natural and associated components selection using protein purification are well known in this field.

Used the in this description, the term “extract” refers to the manner essentially the release of chemical species, such as a polypeptide, from natural-associated components by isolation, for example, using methods of protein purification well known in this field.

“Biological activity” in this context refers to all biological properties inherent to the antigen. Biological properties include, but are not limited to, receptor binding, induction of cell proliferation, inhibition of cell growth, induction of other cytokines, induction of apoptosis and enzymatic activity.

The terms “specific binding” or “specifically binding” in this context with reference to the interaction of the antibody, protein or peptide with a second chemical particle means that this interaction depends on the presence of a particular structure (e.g., antigenic determinant or epitope) on the specified chemical particle; for example, the antibody recognizes a specific protein structure and associated with the specific structure of the protein, not protein as a whole. If the antibody is specific against epitope "A", the presence of molecules containing epitope A (or free, And unlabeled) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled And associated with the indicated antibody.

The term “antibody” in this context refers in a broad sense to any mole is OLE immunoglobulin (Ig), contains four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant or derivative, which preserves the main characteristics of the binding epitope of the Ig molecule. Such formats mutant, variant or derivative of the antibody known in the field. Their non-limiting variants are discussed below.

In the full-size antibody, each heavy chain comprises variable region heavy chain (abbreviated denoted here as HCVR or VH) and a constant region of the heavy chain. The constant region of the heavy chain consists of three domains, CH1, CH2 and CH3. Each light chain consists of a variable region light chain (abbreviated denoted here as LCVR or VL) and a constant region of the light chain. The constant region of the light chain consists of one domain, CL. Region VH and VL can be further subdivided into hypervariable regions, called complementarity determining regions (CDR), interspersed with regions that are more conservative, called frame regions (FR). Each VH and VL is composed of three CDRs and four FR located from amino end to the carboxy-end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The immunoglobulin molecules may be molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) and the and subclass.

The term “Fc region” is used to determine the C-terminal region of the heavy chain of immunoglobulin, which can be obtained by papain cleavage of intact antibodies. Fc-region may be native sequence Fc region or a variant Fc region. Fc region of immunoglobulin usually contains two constant domain, CH2 domain and CH3 domain, and optionally contains CH4 domain. In this area known replacement of amino acid residues in the Fc portion to change the effector functions of antibodies (Winter,et al.US PAT NOS 5,648,260; 5624821). Fc part of the antibody mediates several important effector functions, for example, induction of cytokines, ADCC, phagocytosis, complementability cytotoxicity (CDC) and the period of half-life/speed clearance of antibodies and complexes of antigen-antibody. In some cases, these effector functions are desirable for therapeutic antibodies, but in other cases may not be needed or even harmful, depending on therapeutic purpose. Some isotypes of human IgG, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγR and complement C1q, respectively. Neonatal Fc receptors (FcRn) are crucial determinants of the period half-life of antibodies in the bloodstream. In another embodiment, at least one amino acid residue is replaced in the constant region of the antibody, for example, Fc region of the antibody, so that the effector functions of antibodies are changed. Dimerization of two identical heavy chains of immunoglobulin mediated dimerization CH3 domains and is stabilized by disulfide bonds in the hinge region (Huber et al. Nature; 264: 415-20; Thies et al 1999 J Mol Biol; 293: 67-79.). Mutation of cysteine residues in the hinge regions to prevent the formation of disulfide bonds between the two heavy chains will destabilize the CH3 dimerization domains. The remains responsible for dimerization CH3, were identified (Dall'acqua 1998 Biochemistry 37: 9266-73.). Thus, it is possible to generate monovalent semi-Ig. Interestingly, these monovalent semi-Ig molecules have been found in nature for IgG subclasses and IgA (Seligman 1978 Ann Immunol 129: 855-70; Biewenga et al 1983 Clin Exp Immunol 51: 395-400). It was determined that the stoichiometry FcRn:Fc-region Ig was 2:1 (West et al. 2000 Biochemistry 39: 9698-708) and semi-Fc is sufficient for mediating the binding of FcRn (Kim et al 1994, Eur J Immunol; 24: 542-548). Mutations for the destruction of CH3 dimerization domain may not have a greater effect on its binding to FcRn as residues important for dimerization CH3, located on the inner surface of the partition β-folded structure CH3, while the region responsible for binding to FcRn, is located on the outer surface of the partition CH2-CH3 domains. However, the molecule semi-Ig can have a definite advantage in proniknoveniem fabric due to its small size compared to the size of conventional antibodies. In one embodiment, at least one amino acid residue is replaced in the constant region of the binding protein according to the invention, for example, Fc-region, so that the dimerization of the heavy chains is destroyed, which leads to molecules DVD-Ig.

The term “antigennegative part of the antibody (or simply “antibody”) refers in this context to one or more fragments of an antibody that retain the ability to specifically bind to the antigen. It was shown that antigennegative function of antibodies can be performed by fragments of a full-sized antibodies. Such antibodies can also be bespecifically, dual specific or multispecificity; specifically bind to two or more different antigens. Examples of binding fragments encompassed by the term “antigennegative part of the antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL domains, VH, CL and CH1; (ii) F(ab')2-fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH domains and CH1; (iv) an Fv fragment consisting of domains VL and VH of a single arm of an antibody, (v) a dAb fragment (Wardet al., (1989)Nature341:544-546, Winter et al., the PCT publication WO 90/05144 A1 included in this description by ssy is key), which contains a single variable domain; and (vi) selected complementarity determining region (CDR). In addition, although the two domains of Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables you to receive them in the form of a single protein chain in which the VL region and a VH mate with the formation of monovalent molecules (known as single-chain Fv (scFv); see, for example, Birdet al.(1988)Science242:423-426; and Hustonet al.(1988)Proc. Natl. Acad. Sci. USA85:5879-5883). It is assumed that such single-chain antibodies are also included in the term “antigennegative part of antibodies. This term also includes other forms of single-chain antibodies, such as diamela. Diately are bivalent, bespecifically antibodies, in which the domains VH and VL are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, forcing the domains to pair with complementary domains of another chain and creating two antigenspecific site (see, e.g., Holliger, P.,et al. (1993)Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R.J.,et al. (1994)Structure2:1121-1123). Such antigennegative part known in this field (Kontemann and Dubel eds., Antibody Engineering(2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5). In addition, single-chain antibodies also include “linear antibodies”, containing a pair of tandem Fv segments (VH-CH1-VH-CH1)which, together with complementary light chain polypeptide form a pair antigenspecific regions (Zapata et al. Protein Eng. 8(10):1057-1062 (1995); and U.S. patent No. 5641870).

The term “multivalent binding protein” is used throughout the description to refer binding protein containing two or more antigenspecific sites. Multivalent binding protein is preferably designed in such a way that it has three or more antigenspecific sites and is not normally found in nature antibody. The term “multispecificity binding protein” refers to a binding protein capable of binding two or more related or unrelated targets. Binding proteins with dual variable domain (DVD) according to the invention contain two or more antigenspecific sites and are tetravalent or multivalent binding proteins. DVD-binding proteins can be monospecific, i.e. able to bind one antigen, or multispecificity, i.e. capable of binding two or more antigens. DVD-binding proteins containing two DVD polypeptide heavy chains and two DVD polypeptide formed the second circuit, called DVD-Ig. Each half-DVD-Ig contains DVD polypeptide heavy chain DVD polypeptide light chains and two antigenspecific site. Each binding site contains a variable domain of the heavy chain variable domain and a light chain with 6 CDR in General, involved in the binding to the antigen, one antigennegative site.

The term “bespecifically antibody” refers in this context to the full-size antibody, which generate technology quadrom (see Milstein, C. and A.C. Cuello, Nature, 1983. 305(5934): p. 537-40), by chemical conjugation of two different mAb (see Staerz, U.D., et al., Nature, 1985. 314(6012): p. 628-31) or using "bump-in-the deepening", or similar approaches that introduce mutations in the Fc region (see Holliger, P., T. Prospero, and G. Winter, Proc Natl Acad Sci U S A, 1993.90(14): p. 6444-8 .18) with the formation of multiple different types of immunoglobulin, of which only one is functional bespecifically antibody. Molecular function bespecifically the antibody binds to one antigen (or epitope) on one of its two arms (one pair of HC/LC) and link it to a second antigen (or epitope) on his second shoulder (another pair of HC/LC). Under this definition bespecifically antibody has two different antigenspecific shoulder (both specificity and CDR sequences), and is monovalent for each antigen with which it is binding is raised.

The term “antibody with dual specificity” refers in this context to the full-size antibody that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC/LC) (see PCT publication WO 02/02773). Accordingly, the binding protein dual specificity has two identical antigenspecific shoulder with identical specificity and identical CDR sequences and is bivalent for each antigen to which it binds.

“Functional antigennegative site binding protein is a website that is able to bind the antigen target. Antigennegative affinity antigennegative site is not necessarily as strong as in the case of the original antibody derived from this antigennegative site, but this ability to bind the antigen should be measurable using any of various methods known to assess the binding of the antibody to the antigen. In addition, antigennegative the affinity of each of antigenspecific sites multivalent antibodies should not be here quantitatively the same.

The term” cytokine” is a generic term for proteins released by one cell population which act on another cell population as intercellular mediate the RC. Examples of such cytokines are lymphokines, Monokini and traditional polypeptide hormones. The cytokines also includes growth hormone, for example, human growth hormone, N-methionyl-human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); growth factor liver; fibroblast growth factor; prolactin, placental lactogenic; factor-alpha and factor-beta tumor necrosis; factor regression müller channel; associated with gonadotropin peptide mouse; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor such as NGF-alpha; platelet growth factor; transforming growth factors (TGF)such as TGF-α and TGF-β; insulin-like growth factor 1 and 11; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, -beta and-gamma, colony stimulating factors (CSFs)such as macrophage CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL)such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-18, IL-23; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit-ligand (KL). In the context, the term cytokine includes proteins from natural sources or from a culture of recombinant cells and biologically active equivalents of cytokines with native sequences.

The term “linker” is used to refer to polypeptides that contain two or more amino acid residues, connected by peptide bonds, and it is used to associate one or more antigenspecific parts. Such linker polypeptides are well known in the art (see, e.g., Holliger, P.,et al. (1993)Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R.J.,et al. (1994)Structure2:1121-1123). Preferred linkers include, but are not limited to, AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA(G4S)4; SAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVTLFPP; AKTTPP; AKTTPPSVTPLAP; AKTTAP; AKTTAPSVYPLAP; ASTKGP; ASTKGPSVFPLAP.

Constant domain of immunoglobulin means constant domain of the heavy or light chain. Amino acid sequence of the constant domain of the heavy chain and light chain of human IgG is known in this field.

The term “monoclonal antibody” refers in this context to an antibody obtained from a population essentially homogeneous antibodies, i.e, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. To the ome, in contrast to the preparations of polyclonal antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The definition of "monoclonal" should not be understood as the requirement for antibodies to any particular method.

The term “human antibody” in this context includes antibodies having variable and constant region derived from an immunoglobulin sequences of the germline of the person. Human antibodies according to the invention may include amino acid residues not encoded by the sequences of immunoglobulin germ line person (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody" in this context is not intended to include antibodies in which CDR sequences derived from the germline of another species of mammal, such as a mouse, have been grafted ("transplanted") on frame sequence of a human.

The term “recombinant human antibody” includes all human antibodies that receive, Express the shape, create or produce by recombinant means, such as antibodies expressed recombinant expressing vector, transfitsirovannykh cells of the host (additionally described in Section II C, below), antibodies isolated from a recombinant, combinatorial libraries of human antibodies (Hoogenboom H.R., (1997)TIB Tech.15:62-70; Azzazy H., and W.E. Highsmith, (2002)Clin. Biochem.35:425-445; Gavilondo J.V., and Larrick J.W. (2002)BioTechniques29:128-145; Hoogenboom H., and Chames P. (2000)Immunology Today21:371-378), antibodies isolated from an animal (e.g. a mouse)that is transgenic relative to the genes of the human immunoglobulin (see, e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green, L.L. (2002)Current Opinion in Biotechnology13:593-597; Little, M. et al (2000)Immunology Today21:364-370), or antibodies obtained, expressed, created or selected by any other means that involves splicing of immunoglobulin gene sequences to different DNA sequences. Such recombinant human antibodies have variable and constant region derived from an immunoglobulin sequences of the germline of the person. However, in some embodiments, the implementation of such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using an animal transgenic relatively Ig sequences of human somatic mutagenesis in ivo), and, therefore, amino acid sequence regions VH and VL of these recombinant antibodies are sequences that, although come from sequences of VH and VL germline man can not exist in nature in the repertoire of germline antibodies humans in vivo.

“Affinity matured” antibody is an antibody with one or more changes in one or more of its CDR, which lead to an improvement in the affinity of this antibody against the antigen compared to the original antibody, which does not have this changes (these changes). Preferred affinity Mature antibodies will have nanomolar or even picomolar affinity against the target antigen. Affine Mature antibodies produced by methods known in this field. Marks et al. BidlTechnology 10:779-783 (1992) describes affinity maturation by shuffling of domains VH and VL. Random mutagenesis of residues of CDR and/or framework described by Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995) and Hawkins et al, J. Mol. BioL 226:889-896 (1992).

The term “chimeric antibody” refers to antibodies that contain sequences of variable regions of heavy and light chains of one type and sequence of the constant regions from another species, such as antibodies, who have a mouse variable region heavy and light chain, associated with constant regions of a human.

The term “CDR-transplantirovannam antibody” refers to antibodies that contain sequences of variable regions of heavy and light chain from one species but in which the sequences of one or more CDR regions of VH and/or VL replaced with CDR sequences of another species, such as antibodies having murine variable regions of the heavy and light chain in which one or more murine CDRs (e.g., CDR3) are replaced with CDR sequences of a person.

The term "humanitariannet antibody" refers to antibodies that contain sequences of variable regions of heavy and light chains of the type of non-human (e.g., a mouse), but in which at least part of the sequence of the VH and/or VL was changed so that it is more "human-like", i.e. more similar to the variable sequences of the germline of the person. One of the types gumanitarnogo antibody is a CDR-transplantirovannam antibody in which the CDR sequences of a person entered in the sequence of VH and VL is not the man to replace the corresponding CDR of non-human.

The terms “Kabat numbering", “definitions of Kabat and tagging Kabat" are used here interchangeably. The terms, who are recognized in this area include the I to the system of numbering amino acid residues, which are more variable (i.e. hypervariable)than other amino acid residues in the variable regions of the heavy and light chain antibody, or antigennegative part (Kabatet al. (1971)Ann. NY Acad, Sci.190:382-391 and Kabat, E.A.,et al.(1991)Sequences of Proteins of Immunological Interest, Fifth EditionThe U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the variable regions of the heavy chain hypervariable region is in the range of positions of the amino acids 31-35 for CDR1, the provisions of amino acids 50-65 for CDR2, and the provisions of amino acids 95-102 for CDR3. For the variable region of the light chain hypervariable region is in the range of positions of the amino acids 24-34 for CDR1, the provisions of amino acids 50-65 for CDR2, and the provisions of amino acids 89-97 for CDR3.

In this context, the term “CDR" refers to the complementarity determining region in the variable sequences of the antibodies. In each of the variable regions of the heavy chain and light chain with three CDRs that are named CDR1, CDR2 and CDR3, for each of these variable regions. The term “set CDR” refers in this context to a group of three CDRs, which are found in a single variable region capable of binding an antigen. The exact boundaries of these CDR was defined differently in accordance with different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (197) and (1991)), not only provides unambiguous numbering system balances applicable to any variable regions of antibodies, but also provides precise boundaries of the residues that define the three CDR. These CDRs can be identified according to Kabat CDR. Cothia and co-authors (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that some Subpart in Kabat CDR accept almost identical conformation of the peptide skeleton, despite the great diversity at the level of amino acid sequence. These subparts were named L1, L2 and L3 and H1, H2 and H3, where "L" and "H" means the region of the light chain and heavy chain, respectively. These areas can be called the CDR Chotia, which have boundaries that overlap with the Kabat CDR. Other boundaries that define CDR, overlapping with the Kabat CDR, were described by Padlan (FASEB J. 9:pages 133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Other definitions of CDR boundaries may not conform strictly to any one of the aforementioned systems, but will, however, overlap with the Kabat CDR, although they may be shortened or lengthened in the light of predictions or experimental discovery that specific residues or groups of residues, or even full CDR essentially no effect on binding to the antigen. As used here the ways you can use CDR, defined in accordance with any of the specified system shall eat, although in the preferred embodiments implement the use of CDRs as defined by Kabat or Hatia.

In this context, the term "frame" or "frame sequence" refers to the remaining sequences of the variable regions minus CDR. Because the exact definition of a CDR sequence can be determined using different systems, the value of the frame sequence is considered according to different interpretations. Six CDRs (CDR-L1, -L2 and-L3 light chain and CDR-H1, -H2 and-H3 of the heavy chain) also share the frame region into four sub-region light chain and heavy chain (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without a description of specific subareas, such as FR1, FR2, FR3 or FR4, frame area cited by other authors, represents the combined FR in the variable regions of a single, naturally occurring chain of immunoglobulin. In this context, FR represents one of the four subareas and FR represents two or more of the four sub-fields constituting a frame area.

In this context, the term “gene antibody germ line” or “fragment of a gene" refers to a sequence of immunoglobulin encoded by non-lymphoid cells that had not undergone the maturation process, leading to genetic rearrangeable and is utali for the expression of specific immunoglobulin. (See, for example, Shapiro et al., Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484:13-30 (2001)). One of the benefits of various options for the implementation of the present invention stems from the recognition that genes antibody germline more likely than Mature antibody genes, retain the basic structure of the amino acid sequence characteristic of individuals in this form, and therefore less likely recognized as originating from an alien source when used therapeutically in this form.

In this context, the term “humanitariannet antibody” means an antibody or a variant, derivative, analog or fragment, which immunospecificity associated with interest antigen and which contain a frame region (FR), having essentially the amino acid sequence of human antibodies and complementarity determining region (CDR)having essentially the amino acid sequence of the antibody is non-human. Used in this description, the term "essentially" in the context of CDR refers to a CDR having the amino acid sequence at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of CDR of the antibody is non-human. Humanitariannet antibody contains essentially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv)in which all or substantially all of the CDR regions correspond to the CDR regions of an antibody of non-human (i.e., donor antibody) and all or essentially all of the framed areas are framed regions of the consensus sequence of human immunoglobulin. Preferably, humanitariannet antibody also comprises at least a portion of a constant region (Fc) of an immunoglobulin, usually that portion of human immunoglobulin. In some embodiments, the implementation humanitariannet antibody containing light chain and at least the variable domain of the heavy chain. The antibody may also include SN, hinge region, CH2, CH3 and CH4 region of the heavy chain. In some embodiments, the implementation humanitariannet antibody contains only humanitarian light chain. In some embodiments, the implementation humanitariannet antibody contains only humanitarian heavy chain. In some embodiments, the implementation humanitariannet antibody contains only humanitarianly variable domain light chain and/or humanitarian heavy chain.

In this context, the term “neutralizing” refers to neutralize the biological activity of the cytokine with specificity the immediate vicinity of the binding of the binding protein with a cytokine. Preferably the neutralizing binding protein binds to the cytokine and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85% or more.

The term “activity" includes such activities as the binding specificity/affinity of DVD-Ig in relation to two or more antigens.

The term “epitope" includes any protein determinant capable of specifically bind to immunoglobulin or T-cell receptor. In some embodiments, the implementation of the epitope determinants include chemically active surface groupings of molecules such as amino acids, the side chains of sugars, phosphoryl or sulfonyl, and, in some embodiments, the implementation may have specific three dimensional structural characteristics and/or specific characteristics of the charges. The epitope is a region of the antigen that binds the antibody. In some embodiments, the implementation is considered that the antibody specifically binds the antigen, mainly when it recognizes its antigen target in a complex mixture of proteins and/or macromolecules.

The term “surface plasmon resonance" in this context refers to an optical phenomenon that makes possible the analysis of real-time biospecific interactions by determining the changes in concentrations of Bel is in biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Jönsson, U.,et al.(1993)Ann. Biol. Clin.51:19-26; Jönsson, U.,et al.(1991)Biotechniques11:620-627; Johnsson, B.,et al.(1995)J. Mol. Recognit.8:125-131; and Johnnson, B.,et al.(1991)Anal. Biochem.198:268-277.

The term “Kon" in this context refers to the rate constant for the Association of the antibody with the antigen to form a complex of the antibody/antigen, as is well known in this field.

The term “Koff" in this context refers to the rate constant of dissociative antibodies from complex antibody/antigen, as is well known in this field.

The term “Kd" in this context refers to the dissociation constant of the specific interaction of antigen-antibody, as is well known in this field.

The term “labeled binding protein” in this context refers to a protein with the included label that provides an identification of the binding protein. Preferably, the label is detected marker, for example, the incorporation of radioactively labeled amino acids or accession to the polypeptide biotinyl groups that can be detected labeled Avidya (e.g., streptavidin containing a fluorescent marker or possessing enzymatic activity, which can be detected by optical or colorimetric method is MIS). Examples of labels for polypeptides include, but are not limited to, the following labels: radioisotopes or radionuclides (e.g.,3H,14C,35S90Y99Tc111In125I131I177Lu,166Ho or153Sm); fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinylate groups; predetermined polypeptide epitopes recognized by a secondary reporter (e.g., pairwise sequence latinboy lightning, binding sites for secondary antibodies, the binding domains of metals, epitope tags); and magnetic agents, such as gadolinium chelates.

The term “conjugate” refers to a binding protein, such as antibody chemically linked with the second chemical molecules, such as therapeutic or cytotoxic agent. The term “agent" is used herein to refer to chemical compounds, mixtures of chemical compounds, a biological macromolecule, or an extract derived from biological materials. Preferably therapeutic or cytotoxic agents include, but are not limited to, pertussis toxin, Taxol, cytochalasin B, gramicidin D, ethidiumbromid, emetine, mitomycin, etoposide, tenop the Zid, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogues or homologues.

The terms “crystal” and “crystallized” in this context refers to the antibody or antigennegative part which exists in the form of a crystal. Crystals are one of the forms considered solid state, which differs from other forms, such as amorphous solid state or liquid state. The crystals consist of regular, repeating, three-dimensional arrays of atoms, ions, molecules (e.g. proteins, such as antibodies or molecular ensembles (for example, complexes of antigen/antibody). These three-dimensional arrays are arranged in accordance with a specific mathematical relationships, which are well understood in this area. Fundamental elementary link, or element of a structure that is repeated in the crystal, called asymmetric structure element. The repetition of the asymmetric structure element in the structure, which corresponds to a specific, well-defined crystallographic symmetry, provides a unit cell of the crystal. The repetition of the unit cell by regular the movements in all three dimensions provides crystal. Cm. Giege, R. and Ducruix, A. Barrett, “Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, New York, (1999).”

The term “polynucleotide" in this context refers to a polymeric form of two or more nucleotides, either ribonucleotides or deoxynucleotides, or a modified form of any type of nucleotide. This term includes single-stranded and double-stranded forms of DNA, but preferably by means of double-stranded DNA.

The term "isolated polynucleotide" in this context would mean polynucleotide (e.g., genomic DNA, cDNA or polynucleotide synthetic origin, or some combination of the three), and, because of its origin, "selected polynucleotide", not associated with all polynucleotides or part of polynucleotide, with which the "isolated polynucleotide" is in nature; functionally associated with polynucleotide that is not associated with it in nature; or does not occur in nature in the form of a larger sequence.

The term “vector" in this context refers to a nucleic acid molecule capable of transporting another nucleic acid to which it was connected. One type of vector is a “plasmid", which refers to a circular double-stranded loop of DNA, which can be legirovanyh additional DNA segments. Another type of vector I which is a viral vector, in which additional DNA segments can be legirovanyh in the viral genome. Certain vectors are capable of Autonomous replication in a cell host, in which they are administered (e.g., bacterial vectors having a bacterial site of replication initiation, and epilimnia vectors mammals). Other vectors (e.g., non-opisanie vectors mammals) can be integrated into the genome of a host cell upon introduction into the cell of the host and thereby are replicated along with the genome of the host. Moreover, certain vectors are capable of driving the expression of genes with which they are functionally linked. Such vectors are called here "expressing recombinant vectors" (or simply "expressing vectors"). As a rule, expressing the vectors used in the methods of recombinant DNA, are often in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, this invention includes such other forms expressing vectors as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term “functionally linked" refers to the direct Soest is, in which the components described are in a relationship that allows them to function in the intended way. The regulatory sequence functionally linked" to the coding sequence are ligated in such a way that achieved the expression of the coding sequence under conditions compatible with the regulatory sequences. "Functionally related sequences include regulatory sequences of the expression, which are contiguous with the gene of interest and a regulatory sequence for expression, which areintransor at a distance to regulate gene of interest. The term “regulatory sequence of a gene” refers in this context to polynucleotide sequences which are necessary to complete the expression and processing of coding sequences to which they legirovanyh. Regulatory sequence for expression include appropriate sequence initiation, transcription termination, promoter and enhancer sequences; efficient signal processing RNA, such as splicing signals, polyadenylation; sequences that stabilize cytoplasmic mRNA; sequences that enhance the effect of what you want to make broadcast (i.e. the consensus Kozak sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such regulatory sequences differs depending on the host body; in prokaryotes, such regulatory sequences generally include promoter, the binding site of the ribosome and the sequence termination of transcription; in eukaryotes, generally, such regulatory sequences include promoters and the sequence termination of transcription. The term "regulatory sequence" includes components whose presence is important for the expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and sequences partners merge.

“Transformation" in this context refers to any process by which exogenous DNA enters the cell host. Transformation can occur under natural or artificial conditions using various methods, well known in this field. The transformation can be based on any known method for embedding alien sequences of nucleic acids in prokaryotic or eUK is ridicheskuyu cell host. The method chosen on the basis of the host cell, subject to transformation, and it may include, but are not limited to, viral infection, electroporation, lipofection and the bombarding particles. Such “transformed” cells include stably transformed cells in which the integrated DNA is capable of replication either as Autonomous plasmids can replicate, or as part of the chromosome of the host. They may include cells that transtorno Express integrated DNA or RNA for limited periods of time.

The term “recombinant a host cell” (or simply “a host cell”refers in this context to the cage, which was introduced exogenous DNA. It should be understood that such terms refer not only to the specific of the considered cell, but also to the progeny of such cells. Because certain modifications may occur in the following generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still covered used herein, the term "a host cell". Preferably the cell hosts include prokaryotic and eukaryotic cells, selected from any of the existing types. Preferred eukaryotic cells include cells about the earnest, fungi, plants and animals. Most preferably the cell hosts include, but are not limited to, line prokaryotic cells are E. coli; lines of mammalian cells SNO, SOME 293 and COS; the line of insect cells Sf9 and the fungal cell Saccharomyces cerevisiae.

Standard methods can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed in accordance with the descriptions of the manufacturer or as usually performed in the field, or as described in this document. The above methods and procedures can usually be performed in accordance with conventional methods, well known in this field, and as described in various General and more specific references that are cited and discussed throughout the present description. See, for example, the manual Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is included in this description by reference for any purpose.

“Transgenic organism”, as it is known in this field and as used herein, means an organism having cells that contain a transgene, where the transgene is introduced into the body (or an ancestor of the organism), expresses the polypeptide is not expressed in a natural way is in the body. “Transgene” is a DNA construct that is stable and functionally integrated into the host cell genome, from which it develops a transgenic organism, controlling the expression of the encoded gene product in one or more cell types or tissues of the transgenic organism.

The terms “regulate” and “modulate” are used interchangeably and refer in this context to change or modify the activity of interest molecules (e.g., biological activity of the cytokine). Modulation may be an increase or decrease in the magnitude of a certain activity or function of interest of the molecule. Typical examples of the activity and function of molecules include, but are not limited to, characteristics of the binding, enzymatic activity, cell receptor activation and signal transduction.

Accordingly, the term "modulator" means in this context, the connection is able to change or alter the activity or function of interest molecules (e.g., biological activity of the cytokine). For example, the modulator may cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator. In some embodiments, the implementation of the modulator is ing the inhibitor, which reduces the value of at least one activity or function of the molecule. Examples of inhibitors include, but are not limited to, proteins, peptides, antibodies, "Patiala", carbohydrate, or small organic molecules. "Patiala" described, for example, in WO01/83525.

The term “agonist" in this context refers to the modulator, which is in contact with interest the molecule causes an increase in the magnitude of a certain activity or function of this molecule compared to the magnitude of the activity or function observed in the absence of agonist. Specific interest agonists may include, but are not limited to, polypeptides, nucleic acids, carbohydrates, or any other molecules that bind to a given antigen.

The term “antagonist” or “inhibitor” in this context refers to the modulator, which is in contact with interest the molecule causes a decrease in the specific activity or function of this molecule compared to the magnitude of the activity or function observed in the absence of the antagonist. Specific interest antagonists include antagonists that block or modulate the biological or immunological activity of a specific antigen. Antagonists and inhibitors antigens may include the step in itself, but not limited to, proteins, nucleic acids, carbohydrates, or any other molecules that bind to a given antigen.

In this context the term “effective amount" refers to the amount of therapeutic agent that is sufficient to reduce or ameliorate the severity and/or duration of a violation or one or more symptoms, prevent progression of the violation, the initiation of regression disturbances, prevent the recurrence, development, occurrence or progression of one or more symptoms associated with the disorder, detecting violations, or enhance or improve the prophylactic or therapeutic effects of another therapeutic agent (e.g., prophylactic or therapeutic agent).

The term “sample" in this context is used in its broadest sense. “Biological sample” in this context includes, but is not limited to, any quantity of a substance from a living being or formerly living creature. Such beings include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood, serum, synovial fluid, cells, organs, tissue, bone marrow, lymph nodes and the village of the evaluation.

I. generating a DVD binding protein with a dual-variable-domain)

This invention relates to binding proteins with dual variable domain capable of binding one or more targets, and methods for their preparation. Preferably the binding protein contains a polypeptide chain, and the specified polypeptide chain contains VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X1 represents an amino acid or polypeptide, X2 represents an Fc region and n is 0 or 1. Binding protein according to the invention can be generated using different methods. This invention provides expressing vectors, cells are the owners of and the ways to generate binding protein.

A. generating a source of monoclonal antibodies

The variable domains of the DVD binding protein can be obtained from the source of antibodies, including polyclonal and monoclonal antibodies, capable of binding an antigen. These antibodies can be found in nature or can be produced using recombinant technology.

Monoclonal antibodies can be obtained using a wide variety of methods known in this field, including the use of what ibidem, recombinante technology and phage display technique, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma methods, including the methods known in this field and are described, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nded. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (these references are fully incorporated in this description by reference). The term “monoclonal antibody" in this context is used without limitation antibodies produced using hybridoma technology. The term “monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the way in which it is received. Hybridoma select, clone and subjected to additional screening for desirable characteristics, including a steady rise in hybridoma, high production of antibodies and the desired characteristics of the antibodies, as described in example 1 below. Hybridoma can be grown and propagated in vivo in syngeneic animals, animals that are deprived of the immune system, for example, Nude mice or in cell cultures in vitro. The methods of selection, cloning and propagation of hybridomas is well known to specialists in this field. In a preferred embodiment, domestic is hybridoma represent mouse hybridoma. In another preferred embodiment, hybridoma receive in the form of a non-human, non-mouse, such as rats, sheep, pigs, goats, cattle or horses. In another embodiment, hybridoma are hybridoma men with non-secretory myeloma cell fused with a human cell expressing an antibody able to bind a specific antigen.

Recombinant monoclonal antibodies are also separate, isolated lymphocytes using methods known in this field by way of breeding antibodies lymphocytes (SLAM), as described in U.S. patent No. 5627052, PCT publication WO 92/02551 and Babcock, J.S.et al.(1996)Proc. Natl. Acad. Sci. USA93:7843-7848. In this method, single cells secreting interest antibodies, for example, cells obtained from the immunized animal, identify and cDNA of the variable regions of the heavy and light chain release from the cells using back-transcriptase PCR, and then these variable regions Express, in the context of appropriate constant regions of immunoglobulin (e.g., constant regions of a human), in the cells of the host mammal, such as COS cells or Cho. Cell owners, transfetsirovannyh amplificatoare sequences to immunoglobu the ina, retrieved from the selected in vivo lymphocytes, can then be subjected to further analysis and selection in vitro, for example, penninga transfected cells with selection of cells expressing the antibody of interest to the antigen. Amplificatoare sequence of the immunoglobulin can then be manipulated in vitro, such as methods of affinity maturation in vitro, for example, the methods described in PCT publication WO 97/29131 and PCT publication WO 00/56772.

Monoclonal antibodies is also obtained by immunization of an animal-not human, containing some portion, or all, the locus of human immunoglobulin with interest antigen. In a preferred embodiment, the animal is a not-man is transgenic XENOMOUSE mouse, designed by genetic engineering of the mouse strain, which contains large fragments of the immunoglobulin loci of human rights and is defective in the production of mouse antibodies. See, for example, Green et al.Nature Genetics7:13-21 (1994) and U.S. patent 5916771, 5939598, 5985615, 5998209, 6075181, 6091001, 6114598 and 6130364. Cm. also WO 91/10741, published July 25, 1991, WO 94/02602, published February 3, 1994, WO 96/34096 and WO 96/33735, both published October 31, 1996, WO 98/16654, published April 23, 1998, WO 98/24893, published June 11, 1998, WO 98/50433 published November 12, 1998, WO 99/45031 published annoy September 10, 1999 WO 99/53049, published October 21, 1999, WO 00 09560, published on 24 February 2000 and WO 00/037504, published June 29, 2000. Transgenic XENOMOUSE mouse produces a repertoire of fully human antibodies, similar to the repertoire of antibodies adult, and generates antigen-specific mAb person. Transgenic XENOMOUSE mouse contains approximately 80% of the repertoire of human antibodies by introducing YAC fragments with the structure of the germ line and the size of mega-BP loci heavy chain of human and x light chain loci. Cm. Mendez et al.,Nature Genetics15:146-156 (1997), Green and JakobovitsJ. Exp. Med.188:483-495 (1998), the descriptions of which are included in this description by reference.

Howin vitrocan also be used to obtain a source of antibodies, in which a library of antibodies are screened to identify antibodies having the desired binding specificity. Methods such screening libraries of recombinant antibodies is well known in this area and include the methods described, for example, Ladneret al.U.S. patent No. 5223409; Kanget al.the PCT publication no WO 92/18619; Doweret al.the PCT publication no WO 91/17271; Winteret al.the PCT publication no WO 92/20791; Marklandet al.the PCT publication no WO 92/15679; Breitlinget al.the PCT publication no WO 93/01288; McCaffertyet al.the PCT publication no WO 92/01047; Garrardet al.the PCT publication no WO 92/09690; Fuchset al.(1991)Bio/Technology9:137-1372; Hayet al.(1992)Hum Antibod Hybridomas3:81-85; Huseet al.(1989)Science246:1275-1281; McCaffertyet al.,Nature(1990)348:552-554; Griffithset al.(1993)EMBO J12:725-734; Hawkinset al.(1992)J Mol Biol226:889-896; Clacksonet al.(1991)Nature352:624-628; Gramet al.(1992)PNAS89:3576-3580; Garradet al.(1991)Bio/Technology9:1373-1377; Hoogenboomet al.(1991)Nuc Acid Res19:4133-4137; and Barbaset al.(1991)PNAS88:7978-7982, in the publication of the patent application U.S. 20030186374 and PCT publication no WO 97/29131, the contents of each of which are included in this description by reference.

The source of the antibodies according to the invention can also be generated using various methods phage display technique, known in the field. In the methods of phage display of functional domains of antibodies are presented on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, such phage can be used to represent antigenspecific domains expressed from a repertoire or combinatorial libraries of antibodies (e.g., human or murine). Phage expressing antigennegative domain that binds an antigen, can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or bound to a solid surface or edge of the E. Phage used in these methods is typically filamentous phage including domains linking fd and M13, expressed from phage with Fab domains, Fv, or stabilized by disulfide Fv domains of antibodies recombinante merged with either protein gene III or protein VIII gene of phage. Examples of methods of phage display, which can be used to generate antibodies according to the invention include the methods described in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application no PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. patent№№ 5698426; 5223409; 5403484; 5580717; 5427908; 5750753; 5821047; 5571698; 5427908; 5516637; 5780225; 5658727; 5733743 and 5969108; each of which is fully incorporated into this description by reference.

As described in the above references, after selecting phage encoding region of the antibody from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigennegative fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in detail below. For example, the methods of recombinant obtain Fab fragments, Fab' and F(ab')2can b is to be used with application methods, known in this field, such as the methods described in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (these references are fully incorporated in this description by reference). Examples of methods that can be used to produce single-chain Fv antibodies, include the methods described in U.S. patent 4946778 and 5258498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).

Alternative screening libraries of recombinant antibodies using phage display to identify the source of the antibodies can be used other methodologies known in the field for screening large combinatorial libraries. One type of alternative expression systems is the expression system, in which a library of recombinant antibodies Express in the form of mergers RNA-protein as described in PCT publication no WO 98/31700 Szostak and Roberts and Roberts, R.W. and Szostak, J.W. (1997)Proc. Natl. Acad. Sci. USA94:12297-12302. In this system, create a covalent fusion between mRNA and peptide or protein that is encoded by in vitro translation of synthetic mRNAs that are puromycin, pipidinny acceptor antibiotic, at their 3'end. Thus, specific mRNAs may be enriched from a complex mixture of mRNAs (e.g., combinatorial libraries) on the Snov, properties of the encoded peptide or protein, for example, the antibodies or portion thereof, for example, binding of an antibody or part of the antigen with dual specificity. Nucleic acid sequences encoding antibodies or parts thereof, extracted from the screening of such libraries may be expressed by recombinant methods as described above (for example, in the cells of the host mammal), and, in addition, can be subjected to additional maturation affinity or additional rounds of screening mergers mRNA-peptide, which could be introduced mutations in the initially selected sequence, or other methods for affinity maturation in vitro of recombinant antibodies as described above.

In another approach the source of the antibodies can also be generated using the method, yeast display, known in this area. Methods yeast display use genetic methods to attach the domains of antibodies to the yeast cell wall and their expression on the surface of yeast. In particular, these yeasts can be used for expression antigenspecific domains expressed from a repertoire of combinatorial libraries of antibodies (e.g., human or mouse). Examples of methods yeast display, which can be used to obtain the source of the antibodies, include means, opisanie Wittrup, et al. in U.S. patent No. 6699658 included in this description by reference.

The above-described antibodies can be further modified to generate CDR-transplanted and humanized source of antibodies. CDR-transplanted original antibodies contain sequences of variable regions of heavy and light chains of human antibodies where one or more CDR regions of VHand/or VLreplaced with CDR sequences of mouse antibodies, capable of binding an antigen. Frame sequence from any human antibodies can serve as a template for transplantation CDR. However, a direct replacement chain for this frame often leads to some loss of binding affinity towards the antigen. The more homologous antibody is human relative to the original mouse antibody, the less likely is the possibility that the Association of the murine CDR-framed person will introduce disturbances in the CDR, which can reduce the affinity. Thus, it is preferable that the flexible frame of the person who is chosen to replace the murine variable framework, in addition to the CDR had at least 65% sequence identity with the framework of the variable region of mouse antibody. More preferably, the variable region of human the ka and mouse in addition to the CDR had at least 70% sequence identity. Even more preferably, the variable region of human and mouse, in addition to the CDR, had at least 75% sequence identity. Most preferably, the variable region of human and mouse, in addition to the CDR, had at least 80% sequence identity. Methods of obtaining such antibodies known in the art (see EP 239400; PCT publication WO 91/09967; U.S. patent No. 5225539; 5530101 and 5585089), for example, coating or surface change (EP 592106; EP 519596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS 91:969-973 (1994)) and shuffling circuit (U.S. patent No. 5565352).

Humanized antibodies are antibody molecules from antibody species non-human, which binds the desired antigen having one or more complementarity determining regions (CDR) of the molecules of the immunoglobulin species of non-human and a frame region of the molecules of the immunoglobulin. Known Ig sequences person described in, for example,

www.ncbi.nlm.nih.gov/entrez- /query.fcgi; www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com/; www.antibodyresource.com/onlinecomp.html; www.public.iastate.edu/.about.pedro/research_tools.html; www.mgen.uni-heidelberg.de/SD/IT/IT.html; www.whfreeman.com/immunology/CH - 05/kuby05.htm; www.library.thinkquest.org/12429/Immune/Antibody.html; www.hhmi.org/grants/lectures/1996/vlab/; www.path.cam.ac.uk/.about.mrc7/m - ikeimages.html; www.antibodyresource.com/; mcb.harvard.edu/BioLinks/Immuno - logy.html.www.immunologylink.com/; athbox.wustl.edu/.about.hcenter/index.- html; www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913 .html- ; www.nal.usda.gov/awic/pubs/antibody/; www.m.ehime-u.acjp/.about.yasuhito- /Elisa.html; www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/lin - ks.html; www.biotech.ufl.edu/.about.fccl/protocol.html; www.isac-net.org/sites_geo.html; aximtl.imt.uni-marburg.de/.about.rek/AEP - Start.html; baserv.uci.kun.nl/.about.jraats/linksl.html; www.recab.uni-hd.de/immuno.bme.nwu.edu/; www.mrc-cpe.cam.ac.uk/imt-doc/pu - blic/INTRO.html; www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/; www.biochem.ucl.ac.uk/.about.martin/abs/index.html; antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html; www.unizh.ch/.about.honegger/AHOsem - inar/Slide01.html; www.cryst.bbk.ac.uk/.about.ubcg07s/; www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm; www.path.cam.ac.uk/.about.mrc7/h - umanisation/TAHHP.html; www.ibt.unam.mx/vir/structure/stat_aim.html; www.biosci.missouri.edu/smithgp/index.html; www.cryst.bioc.cam.ac.uk/.abo - ut.fmolina/Web-pages/Pept/spottech.html; www.jerini.de/fr roducts.htm; www.patents.ibm.com/ibm.html. Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health (1983),

each of which is incorporated in this description by reference. Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify the binding affinity, the velocity of the Association, the rate of dissociation, avidity, specificity, duration half-life or any other characteristics, as is well known in this field.

The frame remains in the frame areas of a person can be replaced by the corresponding residue from the CDR donor antibody to change, preferably improve, antigen binding. These frame replacement identify ways well known in this field, for example, TP is the study of interactions of residues of the CDR and framework residues to identify residues frame, important for binding of the antigen and of sequence comparison to identify unusual frame residues in specific positions (See, for example, Queen et al., U.S. patent No. 5585089; Riechmann et al., Nature 332:323 (1988), which are fully incorporated into this description by reference). Three-dimensional models of immunoglobulin are commonly available and known qualified specialists in this field. Available computer programs which illustrate and display probable three-dimensional conformational structures of selected candidate sequences of immunoglobulins. The study of these displays allows you to analyze the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., to analyze the remains, which affect the ability of the candidate immunoglobulin to bind its antigen. This way, FR residues can be selected and combined from the consensus and import sequences so that achieve the desired characteristics of the antibodies, such as increased affinity against the target antigen (antigen-targets). Typically, the CDR residues are directly and most substantially involved in the effect on binding to the antigen. Antibodies can be humaniterian using various methods known in this field, such as, but not ogranichivaya is camping them, the methods described in Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCT publication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592106; EP 519596, EP 239400, U.S. patents№№ 5565332, 5723323, 5976862, 5824514, 5817483, 5814476, 5763192, 5723323, 5766886, 5714352, 6204023, 6180370, 5693762, 5530101, 5585089, 5225539; 4816567, each of which is fully incorporated into this description by reference, including the quoted links.

The original monoclonal antibodies can be selected from various monoclonal antibodies, capable of binding specific target and is well known in this field. They include, but are not limited to, anti-TNF-antibody (U.S. patent No. 6258562), anti-IL-12 and/or anti-IL-12p40-antibody (U.S. patent No. 6914128); anti-IL-18 antibody (US 2005/0147610 A1), anti-C5, anti-CBL, an anti-CD147, anti-gp120, anti-VLA-4, anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L, anti-Id, anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-beta 2, anti-E-selectin, anti-Fact VII, anti-Her2/neu, anti-F gp, anti-CD11/18, anti-CD14, anti-ICAM-3, anti-CD80, anti-CD4, anti-CD3, anti-CD23, anti-beta2-integrin, anti-alfaretta, anti-CD52, anti-HLA-DR, anti-CD22, anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCR alpha beta, anti-CD2, anti-Hep B, anti-CA 125, anti-EpCAM, anti-gp120, anti-CMV, and the t-gpIIbIIIa, anti-IgE, anti-CD25, anti-CD33, anti-HLA, anti-VNR, anti-IL-1-alpha, anti-IL-beta, anti-IL-1 receptor, anti-IL-2 receptor, anti-IL-4, anti-IL-4 receptor, anti-IL5, anti-IL-5 receptor, anti-IL-6, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13 receptor anti-IL-17 and anti-IL-23 (see Presta LG. 2005 Selection, design, and engineering of therapeutic antibodies J Allergy Clin Immunol. 116:731-6 and

http://www.path.cam.ac.uk/~mrc7/humanisation/antibodies.html ).

The original monoclonal antibodies can also be selected from a variety of therapeutic antibodies approved for use in clinical trials or under development for clinical use. Such therapeutic antibodies include, but are not limited to, rituximab (Rituxan®, IDEC/Genentech/Roche) (see, for example, U.S. patent No. 5736137), a chimeric anti-CD20-antibody approved to treat non-Jackinsky lymphoma; HuMax-CD20, anti-CD20, currently developed by Genmab, anti-CD20-antibody described in U.S. patent No. 5500362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769 (PCT/US2003/040426, entitled "Immunoglobulin Variants and Uses Thereof"), trastuzumab (Herceptin®, Genentech) (see for example, U.S. patent No. 5677171), humanitariannet anti-Her2/neu-antibody approved to treat breast cancer; pertuzumab (rhuMab-2C4, Omnitarg®), currently developed by Genentech; an anti-Her2 antibody described in U.S. patent No. 4753894; cetuximab (Erbitux®, Imclone) (U.S. patent No. 4943533; PCT WO 96/40210), a chimeric anti-EGFR-antibody in the Kli is practical tests for various types of cancer; ABX-EGF (U.S. patent No. 6235883), currently developed by Abgenix-Immunex-Amgen; HuMax-EGFr (U.S. patent No. 10/172317), currently developed by Genmab; 425, EMD55900, EMD62000 and EMD72000 (Merck KGaA) (U.S. patent No. 5558864; Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al., 1987, J Cell Biochem. 35(4):315-20; Kettleborough et al., 1991, Protein Eng. 4(7):773-83); ICR62 (Institute of Cancer Research) (PCT WO 95/20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22(1-3):129-46; Modjtahedi et al., 1993, Br J Cancer. 1993, 67(2):247-53; Modjtahedi et al, 1996, Br J Cancer, 73(2):228-35; Modjtahedi et al, 2003, Int J Cancer, 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (U.S. patent No. 5891996; U.S. patent No. 6506883; Mateo et al, 1997, Immunotechnology, 3(1):71-81); mAb-806 (Ludwig Institue for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT WO 0162931A2) and SC100 (Scancell) (PCT WO 01/88138); alemtuzumab (Campath®, Millenium), humanitariannet monoclonal antibody currently approved for treatment of b-cell chronic leukocytic leukemia; muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed by Ortho Biotech/Johnson & Johnson, ibritumomab-tiuxetan (Zevalin®), anti-CD20-antibody developed by IDEC/Schering AG, gemtuzumab-ozogamicin (Mylotarg®), anti-CD33 (protein p67)-antibody developed by Celltech/Wyeth, alefacept (Amevive®), anti-LFA-3-Fc fusion developed by Biogen), abciximab (ReoPro®), developed by Centocor/Lilly, basiliximab (Simulect®), developed by Novartis, palivizumab (Synagis®), developed by Medimmune, infliximab (Remicade®), anti-TNF-antibody developed by Centocor, agave momab (Humira®), anti-TNF-antibody developed by Abbott, Humicade®, anti-TNF-antibody developed by Celltech, etanercept (Enbrel®), anti-TNF-Fc fusion developed by Immunex/Amgen, ABX-CBL, an anti-CD147-antibody developed by Abgenix, ABX-IL8, an anti-IL8-antibody developed by Abgenix, ABX-MA1, an anti-MUC18-antibody developed by Abgenix, pemtumomab (R1549, 90Y-muHMFG1), anti-MUC1 in the development of Antisoma, Therex (R1550), anti-MUC1-antibody developed by Antisoma, angioma (AS1405), developed by Antisoma, HuBC-1, developed by Antisoma, Triplatin (AS1407), developed by Antisoma, Antegren® (natalizumab), an anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibody, developed by Biogen, VLA-1-mAb, anti-VLA-1 integrin-antibody developed by Biogen, LTBR mAb, an anti-lymphotoxin beta receptor(LTBR)-antibody developed by Biogen, CAT-152, an anti-TGF-β2-antibody being developed by Cambridge Antibody Technology, J695, an anti-IL-12 antibody being developed by Cambridge Antibody Technology and Abbott, CAT-192, an anti-TGFβ1-antibody being developed by Cambridge Antibody Technology and Genzyme, CAT-213, an anti-eotaxin-antibody being developed by Cambridge Antibody Technology, LymphoStat-B® an anti-Blys-antibody being developed by Cambridge Antibody Technology and Human Genome Sciences, Inc., TRAIL-R1mAb, anti-TRAIL-R1 antibody being developed by Cambridge Antibody Technology and Human Genome Sciences, Inc., Avastin® bevacizumab, (rhuMAb-VEGF), an anti-VEGF antibody developed by Genentech, anti-HER family-receptor-antibody developed by Genentech, anti-tissue factor (ATF), an anti-tissue factor antibody being developed Gnentech, Xolair® (omalizumab), an anti-IgE antibody developed by Genentech, Raptiva® (efalizumab), anti-CD11a-antibody developed by Genentech and Xoma, MLN-02-antibody (formerly LDP-02), developed by Genentech and Millenium Pharmaceuticals, HuMax CD4, an anti-CD4 antibody, developed by Genmab, HuMax-IL15, an anti-IL15-antibody developed by Genmab and Amgen, HuMax-Inflam, being developed Genmab and Medarex, HuMax-Cancer, anti-heparanase I-antibody developed by Genmab and Medarex and Oxford GcoSciences, HuMax-lymphoma, developed by Genmab and Amgen, HuMax-TAC, developed by Genmab, IDEC-131, and anti-CD40L-antibody developed by IDEC Pharmaceuticals, IDEC-151 (clenoliximab), anti-CD4 antibody developed by IDEC Pharmaceuticals, IDEC-114, an anti-CD80-antibody developed by IDEC Pharmaceuticals, IDEC-152, anti-CD23 developed by IDEC Pharmaceuticals, anti-factor macrophage migration (MIF)antibodies developed by IDEC Pharmaceuticals, BEC2, antiidiotypic antibody developed Imclone, IMC-1C11, an anti-KDR-antibody developed by Imclone, DC101, an anti-flk-1 antibody developed by Imclone, anti-VE-cadherin antibodies developed by Imclone, CEA-Cide® (labetuzumab), anti-carcinoembryonic antigen (CEA)antibody developed by Immunomedics, LymphoCide® (epratuzumab), anti-CD22-antibody developed by Immunomedics, AFP-Cide, being developed Immunomedics, MyelomaCide developed by Immunomedics, LkoCide developed by Immunomedics, ProstaCide, being developed Immunomedics, MDX-010 anti-CTLA4-antibody developed by Medarex, MDX-060, an anti-CD30-antibody developed by Medarex, MDX-070, p is srabatyvanie Medarex, MDX-018 developed by Medarex, Osidem® (IDM-1) and anti-Her2 antibody, developed by Medarex and Measurement-Designed Molecules, HuMax®-CD4, an anti-CD4 antibody, developed by Medarex and Genmab, HuMax-IL15, an anti-IL15-antibody developed by Medarex and Genmab, CNTO 148, an anti-TNFα-antibody developed by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokine-antibody developed by Centocor/J&J, MOR101 and MOR102, anti-molecule-1 intercellular adhesion (ICAM-1)(CD54)antibodies developed by MorphoSys, MOR201, anti-receptor-3 fibroblast growth factor (FGFR-3)antibody developed by MorphoSys, Nuvion® (visilizumab), anti-CD3 antibody developed by Protein Design Labs, HuZAF®, an anti-gamma interferon-antibody developed by Protein Design Labs, Anti-α5β1-integrin-antibody developed by Protein Design Labs, anti-IL-12 antibody being developed Protein Design Labs, ING-1, an anti-Ep-CAM antibody developed by Xoma, Xolair® (malidoma), humanitariannet anti-IgE antibody developed by Genentech and Novartis, and MLN01, anti-beta2-integrin-antibody developed by Xoma, all cited above links in this paragraph are specifically included in this description by reference.

B. Designing molecules with dual variable domain (DVD-molecules)

The immunoglobulin molecule with dual variable domain (DVD-Ig) design so that two different variable domain of the light chain (VL) of two different original mAb linked in tandem directly or via a short linker way and recombinant DNA followed by a constant domain of the light chain. Similarly, the heavy chain contains two different variable domain of the heavy chain (VH)connected in tandem, followed by a constant domain SN and Fc-region (figa).

Variable domains can be obtained using methods of recombinant DNA from the original antibodies generated by any of the methods described above. In a preferred embodiment, the variable domain is a variable domain of a heavy or light chain. More preferably, the variable domain is a CDR-transplantirovannam or humanitarianly variable domain of a heavy or light chain. Most preferably variable domain is a variable domain of a heavy or light chain of human immunoglobulin.

In one embodiment, the first and second variable domains bind directly to each other using methods of recombinant DNA. In another embodiment, the variable domains linked through a linker sequence. Preferably bind two variable domain. Three or more variable domains may be linked directly or through a linker sequence. Variable domains can bind the same antigen, or may bind different antigens. DVD molecule is according to the invention can include one variable immunoglobulin domain and one variable domain of the non-immunoglobulin, for example, landspitali domain of the receptor, the active domain of the enzyme. DVD molecule may also contain 2 or more domains-Ig.

The linker sequence can be a single amino acid or polypeptide sequence. Preferably, the linker sequence is selected from the group consisting of AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA(G4S)4;SAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVTLFPP; AKTTPP; AKTTPPSVTPLAP; AKTTAP; AKTTAPSVYPLAP; ASTKGP; ASTKGPSVFPLAP; GGGGSGGGGSGGGGS; GENKVEYAPALMALS; GPAKELTPLKEAKVS and GHEAAAVMQVQYPAS. The choice of the linker sequence is based on the analysis of the structure of crystals of several Fab molecules. There is a natural flexible connection between the variable domain and the constant domain CH1/CL in the molecular structure of the Fab or antibody. This natural link contains approximately 10-12 amino acid residues, including 4-6 residues from the C-end of the V-domain and 4-6 residues from the N-terminal domain CL/SN. DVD-Ig according to the invention were generated using N-terminal 5-6 amino acid residues, or 11-12 amino acid residues, of CL or CH1 as the linker in the light chain and heavy chain DVD-Ig, respectively. N-terminal residues of CL or CH1 domains, in particular, the first 5-6 amino acid residues, adopt the conformation of a loop without strong secondary structures and, therefore, may day is painted as flexible linkers between the two variable domains. N-terminal residues of CL or CH1 domains are the natural lengthening of the variable domains, as they are part of Ig sequences and, therefore, minimize any greater degree of immunogenicity, potentially arising from the linkers and connections.

Other linker sequences may include any sequence of any length CL/CH1 domain but not all residues of the CL/CH1 domain; for example the first 5-12 amino acid residues of the CL/CH1 domains; the light chain linkers can be derived from Cκ or Cλ; and the heavy chain linkers can be derived from CH1 any isotypes, including Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, by cε and Cµ. Linker sequences can also be produced from other proteins such as Ig-like proteins (e.g., TCR, FcR, KIR); sequences on the basis of G/S (for example, G4S repeats); produced from the hinge region sequences; and other natural sequences from other proteins.

In a preferred embodiment, the constant region is associated with two related variable domains using recombinant DNA technologies. Preferably the sequence containing the associated variable domains of the heavy chain is linked to the constant domain of the heavy chain, and a sequence containing the associated variable domains of the light chain, connect is up with the constant domain of the light chain. Preferably constant domains are constant domain of the heavy chain of a human and the constant domain of the light chain of the human, respectively. Most preferably a heavy chain DVD connect the optional Fc-region. Fc-region can be Fc-area native sequence or variant Fc region. Most preferably the Fc region is an Fc-region of a person. In a preferred embodiment, the Fc-region comprises a Fc region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

In the most preferred embodiment, two polypeptide DVD heavy chain and two polypeptide DVD light chain combine to form a molecule of DVD-Ig. Detailed description of specific molecules DVD-Ig capable of binding specific targets, and methods for their preparation are presented in the Examples section below.

C. Receiving a DVD of proteins

Binding proteins according to the invention can be obtained by any of several methods known in this field. For example, expression of the host cells, where expressing vector (vectors)encoding the heavy chain DVD and a light chain DVD transferout in cell host by standard methods. It is assumed that various forms of the term "transfection" include a wide variety of ways, usually used for introducing exogenous DNA into a prokaryotic or eukaryotics the cell-master, for example, electroporation, precipitation of calcium phosphate, transfection using DEAE-dextran, and the like, Although the DVD proteins according to the invention can Express either prokaryotic or eukaryotic cells-the owners, the expression of a DVD of proteins in eukaryotic cells is the preferred and most preferred in the cells of the host mammal, because such eukaryotic cells (and in particular mammalian cells) should be more likely than prokaryotic cells to assemble and secrete a properly laid and immunologically active DVD-protein.

Preferred cells of mammalian hosts for expression of recombinant antibodies according to the invention include cells of Chinese hamster ovary cells Cho (including cells dhfr - Cho, described in Urlaub and Chasin, (1980)Proc. Natl. Acad. Sci. USA77:4216-4220, used with a DHFR-breeding marker, for example, described in R.J. Kaufman and P.A. Sharp (1982)Mol. Biol.159:601-621), NS0 myeloma cells, COS cells and SP2 cells. With the introduction of expressing recombinant vectors encoding DVD-proteins in the cells of the host mammal DVD-proteins produced by culturing the host cells for a period of time sufficient for expression of a DVD of proteins in the cells of the host or, more preferably, secretion of the DVD proteins into the culture medium, in to the grow th cells are the host. DVD-proteins can be extracted from the culture medium using standard cleaning methods.

In a preferred system for recombinant expression of a DVD of proteins according to the invention expressing recombinant vector encoding both the heavy chain DVD and light chain DVD, enter into the cells dhfr - CHO mediated by calcium phosphate transfection. This recombinant expressing the vector genes of the heavy and light chain DVD functionally linked to regulatory elements of the CMV enhancer/AdMLP promoter to run high levels of gene transcription. Recombinant expressing the vector also carries the gene for DHFR, which makes possible the selection of cells SNO, which were transliterowany this vector, using selection with methotrexate/amplification. Selected transformed cell hosts are cultivated for the expression of heavy and light chains DVD and intact DVD-protein extract of the culture medium. To obtain recombinant expressing vector using standard methods of molecular biology, transfection of host cells, selection of transformants, culturing host cells and extract DVD of the protein from the culture medium. Additionally, the invention provides a method of synthesis of DVD-protein according to the invention by culturing the host cell in a suitable culture with the food as long while not synthesized DVD-protein. The method further provides for allocation of a DVD of the protein from the culture medium.

An important feature of the DVD-Ig is that it can be obtained and purified in the same manner as a conventional antibody. Getting a DVD-Ig leads to a homogeneous, single main product with the desired dual specific activity without any modifications to the constant region or chemical modifications of any kind. Other previously described methods of generating "bespecifically", "multispecificity" and "multispecificity multivalent" full binding proteins does not lead to a single primary product, but lead instead to intracellular or Sekretareva the production of the mixture collected, inactive, monospecific, multispecificity, multivalent, full binding proteins and multivalent full binding proteins with a combination of various binding sites. For example, based on the scheme described by Miller and Presta (PCT publication WO 2001/077342(A1), 16 possible combinations of heavy and light chains. Thus, it seems only 6.25 per cent of the protein is desired active form. Department full-size forms of the protein from an inactive and partially active forms of the protein, using standard methods, chromatography, commonly used in onomastikon production, yet to be demonstrated.

Unexpectedly, the design multivalent full binding proteins with dual specificity” according to the invention leads to a light chain with dual variable domain and a heavy chain with dual variable domain that is collected primarily in the desired “having dual specificity multivalent full binding proteins”.

At least 50%, preferably 75% and more preferably 90% of the collected and expressed immunoglobulin molecules with dual variable domains are desired tetravalent protein with dual specificity. This aspect of the present invention is particularly increases the commercial application of the invention. Thus, the present invention includes a method of expression of the light chain with dual variable domain and a heavy chain with dual variable domain in a single cell leading to a single primary product having dual specificity tetravalent binding protein”.

The present invention provides a preferred method for the expression of light chain with dual variable domain and a heavy chain with dual variable domain in a single cell leading to a “primary product” “having dual specificity chetyrehvet the nogo binding protein”, where the “primary product” is more than 50% of all assembled protein, containing a light chain with dual variable domain and a heavy chain with dual variable domain.

This invention provides a preferred method for the expression of light chain with dual variable domain and a heavy chain with dual variable domain in a single cell leading to a “primary product” “having dual specificity tetravalent binding protein”, where the “primary product” is more than 75% of all assembled protein, containing a light chain with dual variable domain and a heavy chain with dual variable domain.

This invention provides a preferred method for the expression of light chain with dual variable domain and a heavy chain with dual variable domain in a single cell leading to a “primary product” “having dual specificity tetravalent binding protein”, where the “primary product” is more than 90% of all assembled protein, containing a light chain with dual variable domain and a heavy chain with dual variable domain.

II. Derivateservlet connecting DVD-proteins:

One of the embodiments provides a labeled binding protein, and this binding baie is OK according to the invention derivatization or linked to another functional molecule (e.g., another peptide or protein). For example, a labeled binding protein according to the invention can be formed functional binding of the binding protein according to the invention (chemical coupling, genetic fusion, noncovalent Association or otherwise) with one or more other molecular particles, such as another antibody (e.g., bespecifically antibody or diatel), the detected agent, cytotoxic agent, a pharmaceutical agent and/or a protein or peptide that can mediate Association of the binding protein with another molecule (such as a core part of streptavidin or polyhistidine label).

Applicable detected agents, which can be derivatization binding protein according to the invention include fluorescent compounds. Examples of fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonate, phycoerythrin etc. Binding protein can also be derivatization detectivesyme enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase, etc. When derivatization binding protein detektivami enzyme it will be detected by adding additional reagents that the enzyme uses to produce a detective shall be the product of the reaction. For example, when there is detected the agent is horseradish peroxidase, adding hydrogen peroxide and diaminobenzidine leads to a colored reaction product that can be detected, the binding protein can also be derivatization Biotin and detected indirectly by measuring the binding avidin or streptavidin.

Another variant of the invention provides for obtaining a crystallized binding protein, and ready-made forms and compositions containing such crystals. In one embodiment, the crystallized binding protein has a greater half-life in vivo than the soluble counterpart of this binding protein. In another embodiment, the binding protein retains biological activity after crystallization.

Crystallized binding protein according to the invention can be obtained in accordance with methods known in this field and are described in WO 02072636 included in this description by reference.

Another variant of the present invention provides glycosylated binding protein, in which the antibody or antigennegative part contains one or more carbohydrate residues. Getting protein in vivo may be subject to additional processing, known as post-translational modification. In particular, ostad the sugar (glucosamine residues) can be added enzyme, the process known as glycosylation. The obtained proteins bearing covalently linked oligosaccharide side chains, known as glycosylated proteins or glycoproteins. Antibodies are glycoproteins with one or more carbohydrate residues in the Fc domain and variable domain. Carbohydrate residues in the Fc domain having an important effect on the effector function of the Fc domain with minimal effect on the binding of the antigen or the period of the half-life of the antibody (R. Jefferis,Biotechnol. Prog.21(2005), pp. 11-16). In contrast, glycosylation of the variable domain may have an effect on antigennegative activity of antibodies. Glycosylation in the variable domain may have a negative effect on the binding affinity of antibodies, probably due to steric hindrances (Co, M.S., et al., Mol. Immunol. (1993) 30:1361-1367), or leads to increased affinity towards the antigen (Wallick, S.C., et al., Exp. Med. (1988) 168:1099-1109; Wright, A., et al., EMBO J.(1991) 10:2717 2723).

One aspect of this invention relates to the generation of mutants of the site of glycosylation, in which the O - or N-linked glycosylation site binding protein has been mutated. The person skilled in the art will be able to generate such mutants using standard well-known technologies. Mutants of the site of glycosylation, which is haunted retain biological activity, but have increased or decreased binding activity, are another object of the present invention.

In another embodiment, the glycosylation of the antibody or its antigennegative part according to the invention is modified. For example, can be obtained deglycosylation (glycosylamine) antibody (i.e. antibody devoid of glycosylation). Glycosylation can be altered, for example, to increase the affinity of antibodies against the antigen. Such carbohydrate modifications can be accomplished, for example, by changing one or more glycosylation sites in the sequence of the antibody. For example, can be produced by replacing one or more amino acids, which lead to the elimination of one or more sites of glycosylation variable regions with elimination by glycosylation at this site. This glycosylamine can increase the affinity of antibodies against the antigen. This approach is described in more detail in PCT publication WO20030164662 and U.S. patent No. 5714350 and 6350861, each of which is fully incorporated into this description by reference.

Additionally, or alternatively, may be created, modified binding protein according to the invention, which has an altered type of glycosylation, such as hypophyseal is consistent antibody having a smaller number fucosamine residues or an antibody having increased cut into two parts GlcNAc structures. Such altered pattern of glycosylation have been shown to increase ADCC-ability of antibodies. Such carbohydrate modifications can be accomplished, for example, expression of the antibody in the cell host with a modified apparatus glycosylation. Cells with the modified apparatus of glycosylation have been described in this area and can be used as host cells, which expressed recombinant antibodies according to the invention, to obtain thereby the antibody with altered glycosylation. See, for example, Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, but also European patent number EP 1176195; PCT publication WO 03/035835; WO 99/5434280, all of which are fully incorporated into this description by reference.

Glycosylation of the protein depends on the amino acid sequence of the protein of interest, as well as the host cell, in which this protein is expressed. Different organisms can produce different enzymes of glycosylation (e.g., glycosyltransferases and glycosidase) and have different available substrates (nucleotidase). Due to such factors glycosylation pattern of the protein and the composition helicoiling residues can vary dependent on the particular system of the host, in which is expressed a specific protein. Glucosamine residues used in this invention may include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid. Preferably glycosylated binding protein contains glycosylated residues, which create a picture of glycosylation of the protein of the person.

Qualified in this field specialists know that different glycosylation of proteins can lead to different characteristics of proteins. For example, the effectiveness of therapeutic protein produced in a microorganism host such as yeast, and glycosylation using endogenous pathway of yeast can be reduced as compared to the glycosylation of the same protein expressed in the cell of a mammal, such as a cell line Cho. These glycoproteins may also be immunogenic in humans and discover a reduced period of half-life in vivo after injection. Specific receptors in humans and other animals can learn specific glycosamine residues and to stimulate rapid clearance of the protein from the bloodstream. Other adverse effects may include changes in the packing of proteins, solubility, susceptibility to proteases, directed migration, transport, com is armenialization, secretion, the recognition of other proteins or factors of antigenicity or allergenicity. Thus, a practitioner can opt for a therapeutic protein with a specific composition and a specific pattern of glycosylation, for example, a composition and pattern of glycosylation is identical or at least similar to the composition and pattern of glycosylation produced in human cells or in species-specific cells of the intended subject is an animal.

The expression of glycosylated proteins that differ from the proteins of the host cell, can be achieved by genetic modification of the host cell for the expression of heterologous enzymes of glycosylation. Using methods known in this field practices can generate antibodies or their antigennegative part detecting glycosylation of proteins person. For example, strains of yeast can be genetically modified to Express not naturally occurring glycosylation enzymes, so glycosylated proteins (glycoproteins)produced in these yeast strains, detect glycosylation of proteins, identical glycosylation of animal cells, particularly human cells (patent application U.S. 20040018590 and 2002137134 and PCT publication WO2005100584 A2).

In addition to binding proteins, the invention also applies to antiidiotypic (an is-Id) antibody, specific in respect of such binding proteins according to the invention. Anti-Id antibody is an antibody which recognizes unique determinants generally associated with antigennegative region of another antibody. Anti-Id can be obtained by immunization of an animal binding protein or containing CDR region. Immunized animal will recognize and respond to idiotypical determinants of the immunizing antibody and produce anti-Id antibody. Anti-Id antibody can also be used as an immunogen for the induction of an immune response in another animal with obtaining the so-called anti-anti-Id antibodies.

In addition, a qualified specialist in this field will be clear that the protein of interest can be expressed using the library of host cells, genetically engineered for the expression of various enzymes of glycosylation, so that cells are the owners, members of this library, produce a protein of interest with variational distributions (paintings) glycosylation. Then the practitioner can select and highlight the protein of interest with a specific new paintings glycosylation. Preferably, a protein that has specifically chosen a new picture of glycosylation, shows improved and modified biological properties.

III. Use DVD-Ig

Given their ability to communicate with two or more antigen binding proteins according to the invention can be used for detecting antigens (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as the enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or immunohistochemistry tissue. DVD-Ig, directly or indirectly, mark detektivami substance to facilitate detection of the bound or unbound antibody. Suitable detected substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable complexes prosthetic groups include streptavidin/Biotin and avidin/Biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinyl-fluorescein, ancillary or phycoerythrin; examples of the fluorescent substance includes a luminal; and examples of suitable radioactive materials include3H,14C,35S90Y99Tc111 In125I131I177Lu,166Ho or153Sm.

Binding proteins according to the invention preferably are capable of neutralizing the activity of the antigens in vitro and in vivo. Thus, such a DVD-Ig can be used for inhibiting the activity of antigens, for example, in a cell culture containing these antigens, the subjects-people or other entities mammals that have antigens that cross-react binding protein according to the invention. In another embodiment, the invention provides a method for decreasing the activity of antigen in a subject suffering from diseases or disorders for which the activity of this antigen is harmful. Binding protein according to the invention can be introduced subject-to man for therapeutic purposes.

In this context, the term “a disorder in which the activity of the antigen is harmful” includes diseases and other disorders in which the presence of a specific antigen in a subject suffering from this disorder has also been shown to be responsible, or it is expected that it is either responsible for the pathophysiology of this disorder, or is a factor contributing to the deterioration of this violation. Thus, a disorder in which the activity of the antigen is harmful, submitted the violation, which, as expected, the decreased activity of the antigen will reduce the symptoms and/or progression of this violation. Such violations can be detected, for example, to increase the concentration of antigen in a biological fluid of a subject suffering from this disorder (e.g., to increase the concentration of antigen in serum, plasma, synovial fluid, etc. of the subject). Non-limiting examples of disorders that can be treated with the binding protein according to the invention, include examples of violations discussed below and in the section relating to pharmaceutical compositions of the antibodies according to the invention.

DVD-Ig according to the invention may bind to a single antigen or multiple antigens. Such antigens include, but are not limited to, the targets listed in the following databases are included in this description by reference. These databases target include the following lists:

Therapeutic target (http://xin.cz3.nus.edu.sg/group/cjttd/ttd.asp);

Cytokines and receptors cytokines (http://www.cytokinewebfacts.com/, http://www.copewithcytokines.de/cope.cgi and

http://cmbi.bjmu.edu.cn/cmbidata/cgf/CGF_Database/cytokine.medic.kumamoto-u.ac.jp/CFC/indexR.html);

Chemokines (http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.html);

Receptors chemokines and GPCR (http://csp.medic.kumamoto-u.ac.jp/CSP/Receptor.html, http://www.gpcr.org/7tm/);

Olfactory receptors (http://senselab.med.yale.edu/senselab/ORDB/default.asp);

R is the receptors (http://www.iuphar-db.org/iuphar-rd/list/index.htm);

Cancer target (http://cged.hgc.jp/cgi-bin/input.cgi);

Secreted proteins as potential antibody targets (http://spd.cbi.pku.edu.cn/);

Protein kinases (http://spd.cbi.pku.edu.cn/) and

Markers CD person (http://content.labvelocity.com/tools/6/1226/CD_table_final_locked.pdf) and (Zola H, 2005 CD molecules 2005: human cell differentiation molecules Blood, 106:3123-6).

DVD-Ig applicable as therapeutic agents for simultaneous blocking of two different targets to increase the efficiency/safety and/or increase coverage of patients. Such targets can include soluble targets (IL-13 and TNF) and the target-cell surface receptors (VEGFR and EGFR). You can also use the induction of changing the direction of cytotoxicity between tumor cells and T-cells (Her2 and CD3) for cancer treatment or between self-reactive cells and effector cells for autoimmunity/transplantation, or between any cell target and effector cells for elimination of inducing disease of the cells in any particular condition.

In addition, the DVD-Ig can be used to start the formation of clusters (clustering) and activation of the receptor in the target two different epitopes on the same receptor. This can be beneficial when receiving agonistic and antagonistic anti-GPCR-therapeutics. In this case, the DVD-Ig can be used for targeting d is a different epitope on the same cell for clustering/signal (two molecules on the cell surface) or signal (in one molecule). Similarly, the molecule DVD-Ig can be designed to run the ligation of CTLA-4 and a negative signal by targeting two different epitope (or 2 copies of the same epitope) of the extracellular domain of CTLA-4, which leads to negative regulation of the immune response. CTL-4 is a clinically validated target for therapeutic treatment of a number of immunological disorders. The interaction of CTLA-4/B7 negatively regulate the activation of T cells by attenuation of the progression of the cell cycle, production of IL-2 and proliferation of T cells after activation and the involvement of CTLA-4 (CD152) can negatively regulate the activation of T-cells and stimulate the induction of immunological tolerance. However, the strategy attenuate T-cell participation agonistic antibodies using TLA-4 was unsuccessful, since the activation of CTLA-4 requires ligation. Molecular interaction of CTLA-4/B7 takes place in the ranks of the “skewed lightning”, as shown by the structural analysis of crystals (Stamper 2001 Nature 410:608). However, none of the currently available agents, the binding of CTLA-4 does not possess the properties of ligation, including monoclonal anti-CTLA-4 antibodies. There have been several attempts to overcome this problem. In one case generated associated with the cellular membrane single-chain antibody, which is significantly inhibited and logainne rejection in mice (Hwang 2002 JI 169:633). In another case, the generated artificial associated with the surface of the APC single-chain antibody to CTLA-4 and showed that it attenuatum T-cell responses (Griffin 2000 JI 164:4433). In both cases, ligation of CTLA-4 was achieved close localized membrane-bound antibodies in artificial systems. Although these experiments provide the proven concept of immune down-regulation by running CTLA-4 negative signal transmission, the reagents used in these messages are not suitable for therapeutic use. For this purpose ligation of CTLA-4 can be achieved by using molecules DVD-Ig, which is aimed at two different epitope-target (or 2 copies of the same epitope) of the extracellular domain of CTLA-4. The reason for this is that the distance between the two binding site of IgG, approximately 150-170 angstroms, is too large for active ligation of CTLA-4 (30-50 E between 2 homodimers CTLA-4). However, the distance between the two binding sites on a DVD-Ig (on one shoulder) is much shorter, in the range of 30-50 Å, which makes it possible to correct ligation of CTLA-4.

Similarly, the DVD-Ig can be aimed at two different member of the set of cell surface receptors (e.g., IL-12R alpha and beta). In addition, the DVD-Ig can be aimed at and soluble CR1 protein/Pat the gene for faster clearance of soluble protein/pathogen target.

In addition, the DVD-Ig according to the invention can be used for tissue-specific delivery (target a tissue marker and a mediator of the disease for enhanced local PK and, therefore, higher efficiency and/or lower toxicity), including intracellular delivery (targeting internalize receptor and intracellular molecule), shipping in the inner part of the brain (targeting transferrin receptor and mediator of CNS diseases to cross the blood-brain barrier). DVD-Ig can also serve as a protein carrier for delivery of antigen specific location via binding of non-neutralizing epitope of this antigen, as well as to increase the period of the half-life of the antigen. In addition, the DVD-Ig can be designed either for a physical link with medical devices implanted patients, or to target these medical devices (see Burke, Sandra E.; Kuntz, Richard E.; Schwartz, Lewis B., Zotarolimus (ABT-578) eluting stents. Advanced Drug Delivery Reviews (2006), 58(3), 437-446; Surface coatings for biological activation andfunctionalization of medical devices, Hildebrand, H. F.; Blanchemain, N.; Mayer, G.; Chai, F.; Lefebvre, M.; Boschin, F., Surface and Coatings Technology (2006), 200(22-23), 6318-6324; Drug/device combinations for local drug therapies and infection prophylaxis, Wu, Peng; Grainger, David W., Biomaterials (2006), 27(11), 2450-2467; Mediation of the cytokine network in theimplantation of orthopedic devices., Marques, A. P.; Hunt, J. A.; Reis, Rui L., Biodegradabl Systems in Tissue Engineering and Regenerative Medicine (2005), 377-397). Briefly, the direction of the appropriate cell types to the site of the medical implant can stimulate the healing and restoration of normal tissue functions. Alternatively, there is also an inhibition of mediators (including, but not limited to, cytokines)released after implantation of the device, via DVD, associated with the device or aimed at the device. For example, for many years in invasive cardiology used stents to clear blocked arteries and improve blood flow to the heart muscle. However, it is known that the traditional stents from bare metal to cause restenosis (re-narrowing of the artery in the treated area) in some patients and can lead to blood clots. Was recently described coated with anti-CD34-antibody stent, which has reduced restenosis and preventing blood clots by capture of endothelial progenitor cells (EPC)circulating in the blood. Endothelial cells are the cells that line blood vessels, ensuring a smooth flow of blood. The EPC is attached to the solid surface of the stent, forming a smooth layer, which not only promotes healing but also prevents restenosis and thrombosis, complications previously associated with the use of stents (Aoji et al. 2005 J Am Coll Cardiol. 45(10):1574-9). In addition to improving outcomes for patients, which is m necessary stents, there are also complications for patients requiring surgery for cardiovascular bypass surgery. For example, prosthetic vascular channel (artificial artery), coated with anti-EPC-antibodies, could eliminate the need for the application of the arteries of the legs or arms of the patient for transplantation bypass grafting. This could reduce the time of surgery and anesthesia, which, in turn, will reduce the number of deaths during coronary artery bypass surgery. DVD-Ig construct such a way that it binds to a cell surface marker (such as CD34), and protein (or epitope of any kind, including, but not limited to lipids and polysaccharides), which was deposited on implantirovannomu device to facilitate recruitment of cells. Such approaches can also be used for other medical implants in General. Alternatively, the DVD-Ig can be applied to medical devices and after implantation and release all DVDs from this device (or any other need that may require additional fresh DVD-Ig, including aging and denaturation already inflicted DVD-Ig), this device could reboot the system by the introduction of a fresh DVD-Ig of the patient, and the DVD-Ig is designed for binding interest target (a cytokine, a cell surface marker (so the m as CD34), and so on) using a single set of binding sites and target marked on this unit (including proteins, epitope of any kind, including, but not limited to, lipids, polysaccharides, and polymers), using a different set of binding sites. This method has the advantage of prolonging the applicability of coated implants.

A. Application of DVD-Ig in various diseases

Molecules DVD-Ig according to the invention is applicable also as therapeutic molecules to treat various diseases. Such molecules DVD can associate one or more targets involved in a particular disease. Examples of such targets in various diseases described below.

1. Autoimmune and inflammatory reaction

It was believed that many proteins involved in General autoimmune and inflammatory responses, including C5, CCL1 (I-309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23 (MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11 (I-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1 (CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1 (endothelial activating monocytes cytokine), SPP1, TNF, TNFSF5, IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD, IRAK1, IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, RAF5, TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z, CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11, CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2, BMPR1A, BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1, IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11, IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA, LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF, TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21, TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2 and RNF110 (ZNF144). In one aspect is provided a DVD-Ig capable of binding one or more of the targets listed above.

2. Asthma

Allergic asthma is characterized by the presence of eosinophilia, metaplasia goblet (epithelial) cells, changes of epithelial cells, hyperresponsiveness of the Airways (AHR) and the expression of Th2 cytokines and Th1, as well as elevated levels of serum IgE. Currently, it is widely accepted that airway inflammation is a key factor underlying the pathogenesis of asthma involves a complex interaction of inflammatory cells such as T cells, b cells, eosinophils, mast cells and and macrophages, and their secreted mediators, including cytokines and chemokines. Currently, corticosteroids are the most important anti-inflammatory drugs in the treatment of asthma, but their mechanism of action is non-specific and there are security problems, especially in the youth population of patients. Therefore, to be justified is to develop more specific and targeted therapies. There is growing evidence that IL-13 in mice mimics many features of asthma, including AHR, mucus hypersecretion and fibrosis respiratory tract, regardless of eosinophilic inflammation (Finotto et al., International Immunology (2005), 17(8), 993-1007; Padilla et al., Journal of Immunology (2005), 174(12), 8097-8105).

It was assumed that IL-13 plays a Central role in the induction of pathological reactions associated with asthma. The development of therapies using monoclonal anti-IL-13 antibodies to reduce the effects of IL-13 in the lung is an exciting new approach, which provides an important perspective as a new treatment for asthma. However, other mediators differential immunological pathways are also involved in the pathogenesis of asthma, and blocking these mediators, along with IL-13, can bring additional therapeutic benefit. Such pairs of targets include, but are not limited to, IL-13 and Pro-inflammatory cytokines, such as FA is tor-α tumor necrosis (TNF-α). TNF-α may exacerbate the inflammatory response in asthma and may be associated with severity of disease (McDonnell, et al., Progress in Respiratory Research (2001), 31(New Drugs for Asthma, Allergy and COPD), 247-250). This suggests that blocking both IL-13 and TNF-α may have a favorable impact, particularly in severe airway disease. In a preferred embodiment, the DVD-Ig according to the invention is associated with the targets IL-13 and TNF-α, and is used to treat asthma.

Animal models, such as the model of OVA-induced asthma mouse, which can be evaluated as inflammation and AHR, known in the field and can be used to determine the ability of different molecules DVD-Ig in the treatment of asthma. Animal models for the study of asthma described by Coffman, et al., The Journal of Experimental Medicine (2005), 201(12), 1875-1879; Lloyd, et al., Advances in Immunology (2001), 77, 263-295; Boyce et al., The Journal of Experimental Medicine (2005), 201(12), 1869-1873; and Snibson, et al., Journal of the British Society for Allergy and Clinical Immunology (2005), 35(2), 146-52. In addition to routine methods of evaluating the safety of these pairs of targets, specific tests on the degree of immunosuppression can be justified and useful in the selection of the best pairs of targets (see Luster et al., Toxicology (1994), 92(1-3), 229-43; Descotes, et al., Developments in biological standardization (1992), 77 99-102; Hart et al., Journal of Allergy and Clinical Immunology (2001), 108(2), 250-257).

Based on the above study and using the same model evaluation on the efficiency and the security can be defined other pairs of targets, which can bind molecules DVD-Ig and to be applicable for the treatment of asthma. Preferably such targets include, but are not limited to, IL-13 and IL-beta, because IL-beta is also involved in the inflammatory response in asthma; IL-13 cytokines and chemokines involved in inflammation, such as IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAM8. This invention also provides a DVD-Ig capable of binding one or more targets involved in asthma selected from the group consisting of CSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNA1, IFNB1, IFNG, histamine and histamine receptors, IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL18, IL19, KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL18R1, TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CCL24, CX3CL1, CXCL1, CXCL2, CXCL3, XCL1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STAT6, TBX21, TGFB1, TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR and chitinases.

3. Rheumatoid arthritis

Rheumatoid arthritis (RA), a systemic disease, characterized by chronic inflammatory reaction in Zinovii joints and is associated with degeneration of cartilage and erosion of periarticular bone. In diseased joints expressed many proinflammatory cytokines, including TNF, chemokines and growth factors. It has been shown that systemic injection of anti-TNF-antic the La or fused protein sTNFR in the model RA mouse is anti-inflammatory and protects the joints. Clinical studies in which the activity of TNF in patients with RA blocked introduced intravenous infliximab (Harriman G, Harper LK, Schaible TF. 1999 Summary of clinical trials in rheumatoid arthritis using infliximab, an anti-TNFalpha treatment. Ann Rheum Dis 58 Suppl 1:I61-4), a chimeric monoclonal anti-TNF antibody (mAB), provided evidence that TNF regulates the production of IL-6, IL-8, MCP-1 and VEGF, recruitment of immune and inflammatory cells into joints, angiogenesis, and reduction of blood levels of matrix metalloproteinases-1 and-3. A better understanding of the inflammatory pathway in rheumatoid arthritis has led to the identification of other therapeutic targets involved in rheumatoid arthritis. Hopeful treatment methods, such as methods of using antagonists of interleukin-6 (MRA), CTLA4Ig (abatacept, Genovese Mc et al. 2005 Abatacept for rheumatoid arthritis refractory to tumor necrosis factor alpha inhibition. N Engl J Med. 353:1114-23.), and anti-B cell therapy (rituximab, Okamoto H, Kamatani N. 2004 Rituximab for rheumatoid arthritis. N Engl J Med. 351:1909) has already been tested in randomized controlled trials over the past year. Identified other cytokines, and it was shown that they are useful in animal models, including interleukin-15, interleukin-17 and interleukin-18, and clinical trials of these agents are currently under way. Therapy using antibodies with dual specificity, uniting TNF-antibody and another mediator, has great potential to increase the clinical efficacy and/or coverage of patients. For example, blocking as TNF and VEGF could potentially eliminate inflammation and angiogenesis, both of which are involved in the pathophysiology of RA. We also blocking other pairs of targets involved in RA including, but not limited to, TNF and IL-18; TNF and IL-12; TNF and IL-23; TNF and IL-beta; TNF and MIF; TNF and IL-17; and TNF, and IL-15-specific DVD-Ig. In addition to routine methods of evaluating the safety of these pairs of targets, specific tests on the degree of immunosuppression can be justified and useful in the selection of the best pairs of targets (see Luster et al., Toxicology (1994), 92(1-3), 229-43; Descotes, et al., Developments in biological standardization (1992), 77 99-102; Hart et al., Journal of Allergy and Clinical Immunology (2001), 108(2), 250-257). Whether the molecule DVD-Ig applicable for the treatment of rheumatoid arthritis, may be assessed using animal models of RA animals such as the mouse model of collagen-induced arthritis. Other applicable models also well known in this area (see Brand DD., Comp Med. (2005) 55(2):114-22).

4. Systemic lupus erythematosus (SLE)

The hallmark of the immunopathogenesis of SLE is the activation of polyclonal B-cells, which leads to hyperglobulinemia, the production of antibodies and formation of immune complexes. Fundamental anomaly is, apparently, sposobnosti T cells to suppress the forbidden clones of b-cells due to generalized disturbed regulation of T cells. In addition, the interaction of B - and T-cells is facilitated by several cytokines, such as IL-10, and co-stimulating molecules such as CD40 and CD40L, B7 and CD28 and CTLA-4, which will trigger the second signal. These interactions, together with impaired phagocytic clearance of immune complexes and apoptotic material perpetuate this immune response obtained with tissue damage. These targets may be involved in SLE and can be potentially used for DVD-Ig for therapeutic interventions aimed at In-cell therapies: CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2, IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGS1, SLA2, CD81, IFNB1, IL10, TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3, MS4A1, ST6GAL1, CD1C, CHST10, HLA-A, HLA-DRA and NT5E.; co-stimulating signals: CTLA4 or B7.1/B7.2; inhibition of survival of b-cells: BlyS, BAFF; inactivation of complement C5; modulation of cytokines: a key principle is that the net biologic response in any tissue is the result of a balance between local levels of proinflammatory or anti-inflammatory cytokines (see Sfikakis PP et al 2005 Curr Opin Rheumatol 17:550-7). It is believed that SLE is run Th-2 disease with documented increases in serum IL-4, IL-6, IL-10. Absurd who are also DVD-Ig, capable of binding one or more targets selected from the group consisting of IL-4, IL-6, IL-10, IFN-α and TNF-α. The combination discussed above targets will increase therapeutic efficacy in SLE, which can be tested in several preclinical models of lupus (see Peng SL (2004) Methods Mol Med.; 102:227-72).

5. Multiple sclerosis

Multiple sclerosis (MS) is a complex autoimmune disease of the type of person mostly unknown etiology. Immunological destruction of the myelin basic protein (MBP) in the entire nervous system is the main pathology of multiple sclerosis. MS is a disease of complex pathologies, which includes infiltration of CD4+ and CD8+ T cells and the response in the Central nervous system. Expression in the CNS cytokines, reactive nitrogen molecule and co-stimulating molecules have been described in MS. The most important are the immunological mechanisms that contribute to the development of autoimmunity. In particular, antigen expression, interaction between cytokines and white blood cells and regulatory T cells, which contribute to the balance/modulation of other T cells such as Th1 cells and Th2, are important for identification of therapeutic targets.

IL-12 is a proinflammatory cytokine that is produced by ARS and stimulates trim is enservco Th1-effector cells. IL-12 is produced in the developing lesions of patients with MS, as well as in EAE-diseased animals. Previously it was shown that interference in IL-12-ways effectively prevents EAE in rodents and that neutralization in vivo of IL-12p40 by using anti-IL-12-mAb has a beneficial action induced by myelin model EAE normal monkeys.

TWEAK is a member of the TNF family, is constitutively expressed in the Central nervous system (CNS), proinflammatory, proliferative or apoptotic actions depending on cell types. Its receptor, Fn14, is expressed in the CNS endothelial cells, reactive astrocytes and neurons. The mRNA expression of TWEAK and Fn14 were increased in the spinal cord during experimental autoimmune encephalomyelitis (EAE). The treatment with anti-TWEAK-antibody when induced myelin-oligodendrocyte-glycoprotein (MOG) EAE in C57BL/6 mice led to a decrease in the severity of the disease and infiltration by leukocytes in the treatment of mice after priming phase.

One aspect of this invention refers to molecules DVD-Ig capable of binding one or more targets, preferably two targets selected from the group consisting of IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200, IFN-γ, GM-CSF, FGF, C5, CD52, and CCR2. The preferred implementation includes a dual specificity and the ti-IL-12/TWEAK-DVD-Ig as a therapeutic agent, useful for the treatment of MS. In this area there are several animal models for assessing the applicability of the molecules DVD for the treatment of MS (see Steinman L, et al., (2005) Trends Immunol. 26(11):565-71; Lublin FD., et al., (1985) Springer Semin Immunopathol.8(3):197-208; Genain CP, et al., (1997) J Mol Med. 75(3):187-97; Tuohy VK, et al., (1999) J Exp Med. 189(7):1033-42; Owens T, et al., (1995)Neurol Clin.13(1):51-73; and 't Hart BA, et al., (2005) J Immunol 175(7):4761-8. In addition to routine methods of evaluating the safety of these pairs of targets, specific tests on the degree of immunosuppression can be justified and useful in the selection of the best pairs of targets (see Luster et al., Toxicology (1994), 92(1-3), 229-43; Descotes, et al., Developments in biological standardization (1992), 77 99-102; Jones R. 2000 Rovelizumab (ICOS Corp). IDrugs.3(4):442-6).

6. Sepsis

The pathophysiology of sepsis is initiated by components of the outer membrane as gram-negative organisms (liposaccharide [LPS], lipid a endotoxin)and gram-positive organisms (lipoteichoic acid, peptidoglycan). These components of the outer membrane is able to communicate with the CD14 receptor on the surface of monocytes. Then, recently described by toll-like receptors signal is transferred into the cell, leading to the final production of proinflammatory cytokines factor-alpha tumor necrosis (TNF-α) and interleukin-1 (IL-I). Crushing inflammatory and immune responses are important signs of septic shock, and play a Central role in the pathogenesis in the facilities of the fabric, failure of multiple organs and death induced by sepsis. It has been shown that cytokines, in particular tumor necrosis factor (TNF) and interleukin (IL-1), are crucial mediators of septic shock. These cytokines have a direct toxic effect on the tissues; they also activate phospholipase A2. These and other actions lead to increased concentrations of platelet-activating factor, stimulating the activity of the synthase nitric oxide, stimulating tissue infiltration by neutrophils and stimulating the activity of neutrophils.

Treatment of sepsis and septic shock remains a clinical conundrum, and a recent prospective trials with biological response modifiers (i.e. anti-TNF, anti-MIF), focused on the inflammatory response, have shown only modest clinical benefits. Recently, interest has shifted towards methods of treatment aimed at the treatment of concomitant periods of immune suppression. Studies on experimental animals and critically ill patients showed that increased apoptosis of lymphoid organs and some parenchymal tissues contributes to immune suppression, anergy and dysfunction of organ systems. During sepsis syndromes apoptosis of lymphocytes could be triggered by the absence of IL-2 and release of glucocorticoids, Gerasimov or so-called cytokine is in the "death": factor alpha tumor necrosis or Fas-ligand. Apoptos flows through the autoactivation of cytosolic and mitochondrial caspases, which are influenced by Pro - and antiapoptotic members of the Bcl-2 family. In experimental animals the treatment of inhibitors of apoptosis can not only prevent apoptosis of lymphoid cells; it may also improve the outcome of the disease. Although clinical trials using anti-apoptotic agents remain distant, for the most part, due to technical difficulties associated with their introduction and targeting tissue, inhibition of lymphocyte apoptosis represents an attractive therapeutic target for septic patient. Similarly, having dual specificity agent, striking as an inflammatory mediator, and apoptotic mediator, may have an additional advantage. One aspect of this invention relates to a DVD-Ig capable of binding one or more targets involved in sepsis, preferably two targets selected from the group consisting of TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissue factor, MIP-2, ADORA2A, CASP1, CASP4, IL10, IL1B, NFKB1, PROC, TNFRSF1A, CSF3, IL10, IL1B, IL6, ADORA2A, CCR3, IL10, IL1B, IL1RN, MIF, NFKB1, PTAFR, TLR2, TLR4, GPR44, HMOX1, midkine, IRAK1, NFKB2, SERPINA1, SERPINE1, and TREM1. The effectiveness of such DVD-Ig in relation to sepsis can be assessed in preclinical animal models, known in this area (see Bura JA, et al.,(2005) Nat Rev Drug Discov. 4(10):854-65 and Calandra T, et al., (2000) Nat Med. 6(2):164-70).

7. Neurological disorders

7.1 Neurodegenerative diseases

Chronic neurodegenerative diseases typically associated with aging diseases characterized by progressive loss of neural functions (death of nerve cells, demyelination, loss of mobility and loss of memory. Emerging knowledge of the mechanisms underlying chronic neurodegenerative diseases (e.g. Alzheimer's disease) shows a complex etiology, and it was recognized that various factors contribute to their development and progression, for example, aging, glycemic status, production and multimerization amyloid, accumulation of advanced glycosylation end products (AGE), which bind to their receptor (RAGE) (receptor for AGE), increased oxidative stress brain, reduced cerebral blood flow, neurospine, including the release of inflammatory cytokines and chemokines, dysfunction of neurons and activation of microglia. Thus, these chronic neurodegenerative diseases are complex interactions between multiple cell types and mediators. Treatment strategies for these diseases is limited and mainly consists of either blocking vocal the positive processes of nonspecific anti-inflammatory agents (for example, corticosteroids, COX inhibitors) or agents for preventing loss of neuronal and/or synaptic function. These methods of treatment can stop the progression of the disease. Recent studies suggest that more targeted therapeutic agents, such as antibodies to soluble A-b-peptide (including A-b-oligomeric forms) can not only stop the progression of the disease, but may also support memory. These preliminary observations suggest that specific therapies, affecting more than one mediator (e.g., A-b and Pro-inflammatory cytokines, such as TNF)may provide even better therapeutic efficacy for chronic degenerative diseases than the efficiency observed with targeting mechanism is the only disease (for example, soluble A-balon) (see C.E. Shepherd, et al., Neurobiol Aging. 2005 Oct 24; Nelson RB., Curr Pharm Des. 2005; 11:3335; William L. Klein.; Neurochem Int. 2002; 41:345; Michelle C Janelsins et al., J Neuroinflammation. 2005; 2:23; Soloman B., Curr Alzheimer Res. 2004; 1:149; Igor Klyubin, et al., Nat Med. 2005;11:556-61; Arancio O, et al., EMBO Journal (2004) 1-10; Bornemann KD, et al., Am J Pathol. 2001; 158:63; Deane R, et al., Nat Med. 2003; 9:907-13; and Eliezer Masliah, et al., Neuron. 2005; 46:857).

Molecules DVD-Ig according to the invention may bind one or more targets involved in chronic neurodegenerative diseases such as Alzheimer's disease. Such mi the Yeni include, but not limited to, any mediator, soluble or cell surface, implicated in the pathogenesis of AD, for example, AGE (S100 A, amphoteric), proinflammatory cytokines (eg, IL-1), chemokines (such as MCP 1), molecules that inhibit nerve regeneration (e.g., Nogo, RGM A), molecules that enhance neurite outgrowth (neurotrophins). Efficiency molecules DVD-Ig can be validition on preclinical animal models such as transgenic mice, which sverkhekspressiya the precursor protein of amyloid or RAGE and develop disease symptoms similar to Alzheimer's disease. In addition, molecules DVD-Ig can be constructed and tested for efficacy in animal models, and the best therapeutic DVD-Ig can be selected for testing on patients, people. Molecules DVD-Ig can also be used to treat other neurodegenerative diseases such as Parkinson's disease. Alpha synuclein involved in the pathology of Parkinson's disease. DVD-Ig capable of target the alpha synuclein and inflammatory mediators, such as TNF, IL-1, MCP-1, can be an effective therapy for Parkinson's disease and also considered in this invention.

7.2 Regeneration of neurons and spinal cord injury

Despite the increasing knowledge of the pathological mechanisms, the damage is engaged in the spinal cord (SCI) still is a devastating condition and is a medical condition, characterized by a high demand for drugs. Most spinal cord injuries are a contusion or compression damage, and primary damage is typically followed by secondary mechanisms of damage (inflammatory mediators, such as cytokines and chemokines), which degrades the initial injury and lead to a significant increase in the area of damage, sometimes more than 10 times. These primary and secondary mechanisms in SCI is very similar to the mechanisms in the brain damage caused in any other way, for example, stroke. No satisfactory treatment, and bolus injection of high doses of methylprednisolone (MP) is the only therapy in the narrow window of time 8 h after injury. However, this processing is intended only to prevent secondary damage, without causing any significant functional recovery. She strongly criticized for the lack of unequivocal efficacy and severe harmful side effects, such as immunosuppression with subsequent infections and severe histopathological muscle changes. Not there are other approved drugs, biological agents or small molecules that stimulate endogenous regenerative potential, but in recent years persp the active principles of treatment and drug candidates discovered the efficacy in animal models of SCI. To a large extent the lack of functional recovery in SCI human caused factors, any abscopal neurite outgrowth in the areas of damage, scar tissue, myelin, and is also associated with damage to cells. These factors include myelin-associated proteins NogoA, OMgp, and MAG, RGM A, associated with scars CSPG (chondroitinsulfate) and inhibiting factors in reactive astrocytes (some semaphorin and Arini). However, in the area of damage is detected not only inhibiting growth of the molecules, but also stimulating neurite outgrowth factors, such as neurotrophins, laminin, L1, and others. This ensemble of inhibiting neurite outgrowth and growth stimulating molecules can explain the fact that blocking the only factors, such as NogoA or RGM A, led to the significant functional recovery in models of SCI rodents, because the reduction of inhibitory action could shift the balance from growth inhibition to stimulation of growth. However, the observed recovery by blocking molecules, inhibiting the growth of a single Narita were incomplete. To achieve a more rapid and more pronounced symptoms of recovery may be desirable or blocking of two molecules, inhibiting the growth of Narita, e.g., Nogo and RGM A or blocking molecules, inhibiting the growth of Narita and Wuxi is ivalsa function increases the growth of Narita molecules, for example, Nogo and neurotrophins, or blocking molecules, inhibiting the growth of Narita, e.g., Nogo and proinflammatory molecules, for example, TNF (see McGee AW, et al., Trends Neurosci. 2003; 26:193; Marco Domeniconi, et al., J Neurol Sci. 2005; 233:43; Milan Makwana1, et al., FEBS J. 2005; 272:2628; Barry J. Dickson, Science. 2002; 298:1959; Felicia Yu Hsuan Teng, et al., J Neurosci Res. 2005; 79:273; Tara Karnezis, et al., Nature Neuroscience 2004; 7, 736; Gang Xu, et al., J. Neurochem. 2004; 91; 1018).

In one aspect is provided a DVD-Ig capable of binding a pair of targets, such as NgR and RGM A; NogoA and RGM A; MAG and RGM A; OMGp and RGM A; RGM A and RGM B; CSPGs and RGM A; aggrecan, Malkin, negrocan, versican, phosphacan, Te38 and TNF-a; Aβ-globular-specific antibodies combined with antibodies that stimulate sprouting dendrites and axons. Pathology of dendrites is a very early sign of AD, and it is known that A NOGO limits the growth of dendrites. You can combine this type Ab with any of the SCI-candidate (myelin proteins) Ab. Other target DVD-Ig can include any combination of NgR-p75, NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo, Lingo-Troy, Lingo-p75, MAG or Omgp. In addition, the target may include any mediator, soluble or cell surface, implicated in the inhibition of neurite, for example, Nogo, Ompg, MAG, RGM A, semaphorin, afrina, soluble A-b, Pro-inflammatory cytokines (eg, IL-1), chemokines (such as MIP 1a), molecules that inhibit neuronal regeneration. The effectiveness of anti-nogo/anti-RGM A or similar mo is equl DVD-Ig can be validition on preclinical models of spinal cord injury in animals. In addition, molecules DVD-Ig can be constructed and tested for efficacy in animal models, and the best therapeutic DVD-Ig can be selected for testing on patients, people. In addition, can be designed molecules DVD-Ig, which are aimed at two different binding site of the ligand to one receptor, e.g., Nogo receptor, which binds three ligand Nogo, Ompg, and MAG and RAGE that binds A-b and S100 A. in Addition, inhibitors of the growth of neurites, e.g., nogo and nogo receptor, also play a role in preventing nerve regeneration in immunological diseases such as multiple sclerosis. It was shown that the interaction of nogo-a nogo receptor enhances recovery in models of multiple sclerosis in animals. Thus, the molecules of a DVD-Ig that can block the function of one's immune mediator, for example, cytokine, such as Ig-12, and molecule inhibition of the growth of Narita, for example, nogo or RGM, can provide faster and higher performance than blocking only or immune molecules or molecule inhibitor of the growth of Narita.

8. Oncological disorders

Therapy with monoclonal antibodies has emerged as an important therapeutic method for cancer (von Mehren M, et al, 2003Monoclonal antibody therapy for cancer. Annu Rev Med.; 54:343-69). Antibodies may be protivoopuxolevye actions by inducing apoptosis the change in the direction of cytotoxicity, interference in the interaction of the ligand-receptor or prevent the expression of proteins that are critical in relation to the neoplastic phenotype. In addition, antibodies can be targeted to components of the target tumor environment to distort vital structures, such as the formation of tumor-associated vasculature. Antibodies can also be targeted to receptors-target ligands are growth factors, such as the receptor for epidermal growth factor. Thus, this antibody inhibits the natural ligands that stimulate the growth of cells, preventing their binding to tumor target cells. Alternatively, antibodies can induce antiidiotypic network, complement-mediated cytotoxicity or antibody-dependent cellular cytotoxicity (ADCC). The use of antibodies with dual specificity, which is aimed at two separate tumor mediator will be, apparently, to create additional advantage compared to monospecific therapy. Discusses a DVD-Ig capable of binding the following pairs of targets to treat cancer: IGF1 and IGF2; IGF1/2 and Erb2B; VEGFR and EGFR; CD20 and CD3, CD138 and CD20, CD38 and CD20, CD38 and CD138, CD40 and CD20, CD138 and CD40, CD38 and CD40. Other combinations of targets include one or the number of members of the EGF family/erb-2/erb-3. Other targets (one or more)involved in oncological diseases that can connect a DVD-Ig, include, but are not limited to, the target selected from the group consisting of: CD52, CD20, CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FIBERGLASS, IGF1, IGF2, IL12A, IL1A, IL1B, IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2, EGF, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1, IGF1R, IL2, VEGF, BCL-2, CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1, IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3, IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL, TP53 GENES, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53 GENES, CHGB, GNRH1, IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3, NR2F6, NR4A3, ESR1, ESR2, NR0B1, NR0B2, NR1D2, NR1H2, NR1H4, NR1I2, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2, NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1, BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10, FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2, IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, INSL3, INSL4, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9, MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3, CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18, CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2 ON, CD44, ILK, ITGA1, APC, CD164, COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1, CDH12, CLDN3, CLN3, CYB5, CYC1, DAB2IP, DES, DNCL1, ELAC2, ENO2, ENO3, FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A, IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID, PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1, TPM2, TRPC6, ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR, LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB1, VEGF,VEGFC, ANGPTL3, BAI1, COL4A3, IL8, LAMA5, NRP1, NRP2, STAB1, ANGPTL4, PECAM1, PF4, PROK2, SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2, EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B (p27Kip1), CDKN2A (p16INK4a), COL6A1, CTNNB1 (b-catenin), CTSB (cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3, GSN (gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130), ITGA6 (a6-integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67), NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA), PTEN, SERPINB5 (maspin), SERPINE1 (PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A (topoisomerase Iia), TP53 genes, AZGP1 (zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cip1), CLDN7 (claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin), GABRP (GABAa), GNAS1, ID2, ITGA6 (a6-integrin), ITGB4 (b4-integrin), KLF5 (GC-block BP), KRT19 (keratin 19), KRTHB6 (keratin-specific hair type II), MACMARCKS, MT3 (metallothionein-III), MUC1 (mucin), PTGS2 (COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2, THBS4, and TNFAIP2 (B94).

IV. The pharmaceutical composition

This invention also provides pharmaceutical compositions containing the binding protein according to the invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing binding proteins according to the invention are compositions for use in, but not limited to, diagnosis, detective the AI or monitoring violations in prevention, therapy, treatment or weakening of violations or one or more symptoms and/or research. In a specific embodiment, the composition comprises one or more binding proteins according to the invention. In another embodiment, the pharmaceutical composition comprises one or more binding proteins according to the invention and one or more prophylactic or therapeutic agents other than binding proteins according to the invention, for the treatment of disorders. Preferred are prophylactic or therapeutic agents known that they are applicable or have been used or is currently used for the prevention, treatment, cure or ameliorate disorders or one or more of its symptoms. In accordance with these variants of the implementation of the composition may further contain a carrier, diluent or excipient.

Binding proteins according to the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises a binding protein according to the invention and a pharmaceutically acceptable carrier. In this context, a "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, Academy of Sciences of bacterially and antifungal agents, isotonic and delaying absorption agents and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of such carriers as water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In many cases it is preferable to include in the composition isotonic agents, for example, sugar, polyalcohol, such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as moisturizing or emulsifying agents, preservatives or buffers, which enhance the shelf life (expiration date) or the effectiveness of the antibodies or portions of antibodies.

Various delivery systems are known and can be used for introducing one or more antibodies according to the invention or a combination of one or more antibodies according to the invention and a prophylactic agent or therapeutic agent, applicable to the prevention, treatment, therapy or mitigate the violation or one or more symptoms of, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells that can Express the antibody or antibody fragment, receptor-mediated the initial endocytosis (see, for example, Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), the construction of a nucleic acid as part of a retroviral or other vector, etc. Ways of introducing preventive or therapeutic agent according to the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural introduction, intratumoral introduction and introduction through the mucous membrane (e.g., intranasal and oral methods). In addition, can be used pulmonary introduction, for example, using the inhaler or nebulizer, and finished form with aerosol forming agent. See, for example, U.S. patents№№ 6019968, 5985320, 5985309, 5934272, 5874064, 5855913, 5290540 and 4880078; and PCT publication nos WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346 and WO 99/66903, which are fully incorporated into this description by reference. In one embodiment, the binding protein according to the invention, a combined therapeutic agent or composition according to the invention is administered using pulmonary delivery technology pharmaceuticals Alkermes AIR® (Alkermes, Inc., Cambridge, Mass.). In a specific embodiment, prophylactic or therapeutic agents according to the invention is administered intramuscularly, intravenously, into the tumour, oral, intranasal, pulmonary, by whom do subcutaneously. Prophylactic or therapeutic agents can be entered in any convenient way, for example, by infusion or bolus injection, by absorption through epithelial or skin-mucous lining (for example, through the mucous membrane of the mouth, rectal and intestinal mucosa, and so on) and you can enter together with other biologically active agents. The administration can be systemic or local.

In a specific embodiment, it may be desirable introduction of prophylactic or therapeutic agents according to the invention locally to the area in need of treatment; this may be achieved, for example, but not limited to, local infusion, by injection, or by means of an implant, with the specified implant is porous or non-porous material, including membranes or matrices, such as membrane from Celestica, polymers, fibrous matrices (e.g., Tissuel®), or collagen matrices. In one embodiment, an effective amount of an antagonist of one or more antibodies according to the invention is administered locally to the affected area of a subject for the prevention, treatment, and/or mitigate the violation or its symptom. In another embodiment, an effective amount of one or more antibodies according to the invention is administered locally to the affected area in combination is with an effective amount of one or more therapeutic agents (e.g., one or more prophylactic or therapeutic agents), other than the binding protein according to the invention, to a subject for prevention, treatment, and/or mitigate the violation or its symptoms.

In another embodiment, the prophylactic or therapeutic agent can be delivered to the system with controlled release or delayed release. In one embodiment, can be used the pump to achieve controlled or sustained release (see, for example, Langer, above; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, can be used polymer materials to achieve controlled or sustained release of therapeutic agents according to the invention (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. patent No. 5679377; U.S. patent No. 5916597; U.S. patent No. 5912015; U.S. patent No. 5989463; U.S. patent No. 5128326; PCT publication no WO 99/15154; and PCT publication no WO 99/20253. Examples of polymers used in forms with delayed release include, but are not limited to, poly(2-Ki is roxithromycine), poly(methyl methacrylate), poly(acrylic acid), a copolymer of ethylene and vinyl acetate, poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), copolymers of lactide and glycolide (PLGA) and polyarteritis. In a preferred embodiment, the polymer used in the form of a sustained-release formulation is inert, does not contain leachable impurities, stable during storage, sterile, and biodegradiruemym. In another embodiment, the system with controlled or delayed release can be placed near the prophylactic or therapeutic target, which allows to use only part of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

System with adjustable release are discussed in the review Langer (1990, Science 249:1527-1533). Any method known to the skilled person skilled in the art, may be used to produce forms with delayed release, containing one or more therapeutic agents according to the invention. See, for example, U.S. patent No. 4526938, PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et al., 1996, "Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy &Oncology 39:179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of Long - Cirulating Emulsions," PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854 and Lam et al., 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery," Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, which are fully incorporated into this description by reference.

In a specific embodiment where the composition according to the invention is a nucleic acid encoding a prophylactic or therapeutic agent, the nucleic acid can be introduced in vivo to promote expression of the encoded it preventive or therapeutic agent, by constructing it as part of the expressing vector nucleic acid and the introduction of it so that it becomes intracellular, for example, using a retroviral vector (see, for example, U.S. patent No. 4980286), or by direct injection, or by use of microparticle bombardment (e.g., using a gene gun; Biolistic, Dupont), or coating with lipids or receptors cell surface, or transfairusa agents, or its introduction in connection with homeobox-like peptide which is known to be included in the core (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868). Alternatively, the nucleic acid can be introduced intracellularly and incorporated into the DNA of the host cell for the expression of homologous recombination.

The pharmacy is practical composition according to the invention is prepared in such a way that it is compatible with the intended method of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g. intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., local), transmucosal (crosslisted) and rectal administration. In a specific embodiment, the composition is in accordance with routine procedures as pharmaceutical compositions adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or local administration people. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include solubilizers agent and a local anesthetic such as lidocaine to ease pain at the injection site.

If the compositions according to the invention must be entered locally, they can be made in the form of ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known qualified specialist in this field. See, for example, Remington''s Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For not sprayed in a spray of local dosage forms usually the use of viscous-semi-solid or solid form, containing media or one or more excipients that are compatible with the local application and having a dynamic viscosity preferably greater than the viscosity of water. Suitable forms include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers or salts for effects on various properties, such as, for example, the osmotic pressure. Other suitable local dosage forms include spray aerosol preparations in which the active ingredient, preferably in combination with a solid or liquid inert carrier, Packed in a mixture with a pressurized volatile component (for example, gaseous propellant, such as freon) or in a bottle with a push valve. The moisturizer or hydrating agents may also be added to pharmaceutical compositions, and dosage forms, if desired. Examples of such optional ingredients are well known in this field.

If method according to the invention provides for intranasal introduction of the composition, the composition may be prepared in an aerosol form, spray fog or in the form of drops. In particular, prophylactic or therapeutic agents for use in accordance with this invention can be conveniently delivered in the form of an aerosol spray from pressurized packaging or nebulizer, with the use of a suitable propellant (e.g., DICHLORODIFLUOROMETHANE, trichloromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol dosage unit may be determined by providing a valve to deliver a measured quantity. Can be made capsules and cartridges (cartridges) (consisting, for example, from gelatin) for use in an inhaler or insufflator containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

If method according to the invention provides for oral administration, the compositions can be prepared in the form of tablets, capsules, starch wafers, gelatin capsules, solutions, suspensions, etc. Tablets or capsules can be prepared with conventional methods with pharmaceutically acceptable excipients such as binding agents (e.g., pre-gelatinizing corn starch, polyvinylpyrrolidone or hypromellose); fillers (e.g. lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrators (e.g., potato starch or sodium starch glycolate) or moisturizing agents (e.g. sodium lauryl sulphate). The tablets may be coated by methods well known in the field. Liquid preparations for oral administration can take the form of, without limitation, solutions, syrups or suspensions, or they can be presented as a dry product to rehydrate with water or other suitable filler before use. Such liquid preparations can be prepared in conventional ways with pharmaceutically acceptable additives such as suspendresume agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or Arabian gum); non-aqueous vehicles (e.g. almond oil, oily esters, ethyl alcohol or fractionated vegetable oils), preservatives (e.g. methyl or propyl-p-hydroxybenzoate or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents, if necessary. Preparations for oral administration can be suitably formulated for slow release, controlled wisweb the Denia or sustained release of a prophylactic or therapeutic agents.

The method according to the invention may include pulmonary introduction, for example, using the inhaler or nebulizer, the composition made with aerosol forming agent. See, for example, U.S. patents№№ 6019968, 5985320, 5985309, 5934272, 5874064, 5855913, 5290540 and 4880078; and PCT publication nos WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346 and WO 99/66903, each of which is fully incorporated into this description by reference. In a specific embodiment, the binding protein according to the invention, a combined therapeutic agent and/or composition according to the invention is administered using pulmonary delivery technology pharmaceuticals Alkermes AIR® (Alkermes, Inc., Cambridge, Mass.).

The method according to the invention may include the introduction of a composition formulated for parenteral administration by injection (for example, bolus injection or continuous infusion). Prepared for injection can be presented in a standard dosage form (e.g., in ampoules or mnogochasovykh containers with an added preservative. Songs can have such forms as suspensions, solutions or emulsions in oily or aqueous carriers, and may contain facilitate preparation agents, such as suspendida, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for Vasco is Denmark a suitable carrier (e.g., sterile does not contain pyrogens water) before use.

The methods according to the invention can additionally provide for the introduction of the compositions in the form of depot drugs. Such finished form prolonged action can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, these compositions can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil)or ion exchange resins, or in the form of poorly soluble derivatives (e.g., a poorly soluble salt).

The methods according to the invention encompass the introduction of the compositions as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as salts formed from hydrochloric, phosphoric, acetic, oxalic, tartaric acid, etc. and salts formed with cations such as salts formed from sodium, potassium, ammonium, calcium, ferric hydroxides, Isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Generally, the ingredients of the compositions are supplied either separately or in a mixture in a standard dosage form, for example, as a dry lyophilized powder or not soteriades the water concentrate in a hermetically sealed container, such as an ampoule or capsule (wafer)indicating the quantity of active agent. If the method of administration is by infusion, the composition may be placed in the infusion bottle containing sterile water or sterile saline pharmaceutical purity. If the method of administration is by injection, may be provided vial with sterile water for injection or saline solution, so that the ingredients can be mixed prior to introduction.

In particular, the invention also provides that one or more prophylactic or therapeutic agents, or pharmaceutical compositions according to the invention is Packed in a hermetically sealed container such as an ampoule or sachet indicating the quantity of this agent. In one embodiment, one or more prophylactic or therapeutic agents, or pharmaceutical compositions according to the invention are supplied as a dry sterilized lyophilized powder or not containing water concentrate in a hermetically sealed container and can be reconstituted (e.g., water or saline) to the appropriate concentration for administration to a subject. Preferably, one or more prophylactic or therapeutic agents, or pharmaceutical compositions according to izaberete the Oia are supplied as a dry sterile lyophilized powder in a hermetically sealed container in the standard dose of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg or at least 100 mg of Lyophilized prophylactic or therapeutic agents, or pharmaceutical compositions according to the invention should be stored at 2°C-8°C in the original container and these prophylactic or therapeutic agents or pharmaceutical compositions according to the invention should be administered within 1 week, preferably within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after reconstruction. In an alternative embodiment, one or more prophylactic or therapeutic agents, or pharmaceutical compositions according to the invention are supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of this agent. Preferably, the liquid form of the input composition is supplied in a hermetically sealed container with at least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 is g/ml, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml, or at least 100 mg/ml Liquid form should be stored at 2°C-8°C in its original container.

A pharmaceutical composition suitable for parenteral administration may be included binding proteins according to the invention. Preferably, the antibody or part of an antibody is prepared in the form of an injection solution containing 0.1-250 mg/ml binding protein. This injection solution may consist of either a liquid or from a freeze-dried dosage forms in a bottle of flint pebbles or amber vials or pre-filled syringe. The buffer may be L-histidine (1-50 mm), optimally 5-10 mm, at pH 5.0-7.0 (optimal pH 6.0). Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. The sodium chloride can be used to modify the toxicity of this solution at a concentration of 0-300 mm (preferably 150 mm for liquid dosage forms). The cryoprotectants can be included for a lyophilized dosage forms, mostly 0-10% sucrose (optimally 0.5 to 1.0%). Other suitable cryoprotectants include trehalose and lactose. Agents fillers can be included for a lyophilized dosage forms, mainly 1-10% mannitol (preferably 2-4%). As in the LM is coy, and in dried form can be used stabilizers, mainly 1-50 mm L-methionine (preferably 5-10 mm). Other suitable fillers include glycine, arginine, as a surfactant may be included Polysorbate-80 in the amount of 0-0,05% (optimally 0,005-0,01%). Additional surface-active agents include, but are not limited to, Polysorbate 20 and BRIJ. A pharmaceutical composition comprising a binding protein according to the invention, prepared in the form of an injectable solution for parenteral administration may additionally contain an agent that is used as the adjuvant, such as an agent used to increase the absorption or dispersion of a therapeutic protein (e.g. antibody). Especially used adjuvant is a hyaluronidase, such as Hylenex® (recombinant hyaluronidase human). The addition of hyaluronidase in the injection solution improves the bioavailability to humans after parenteral administration, especially subcutaneous injection. This addition makes it possible also large amounts at the injection site (i.e. more than 1 ml) with less pain and less discomfort, and minimal incidence of reactions injection site (see WO2004078140 and US2006104968 included in this description by reference).

Compositions according invented the Yu can be in various forms. They include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusium solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended route of administration and therapeutic application. Typical preferred compositions are in the form of injection or infusion solutions, such as compositions similar to the compositions used for passive immunization of humans with other antibodies. The preferred method of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under the conditions of preparation and storage. The composition may be prepared in the form of a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high concentration of the drug. Sterile injectable solutions can be prepared by incorporation of active compounds (i.e. antibodies which or part of the antibody) in the required amount in an appropriate solvent with one ingredient or combination of ingredients, listed above, if required, followed by sterilization by filtration. Generally, dispersions are prepared incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from the above ingredients. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and spray drying which yields a powder of the active ingredient plus any additional desired ingredient from its previously sterile-filtered solution. Should the fluidity of the solution can be maintained, for example, using a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be obtained by incorporating in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Binding proteins according to the invention can be administered in a number of ways known in this area, although for many types of therapeutic applications of the preferred route/method of administration is subcutaneous injection, intravenous injection or infusion. As will be Monday the IDT qualified in this field specialist the path and/or the method of administration will vary depending on the desired results. In some embodiments, the active compound can be prepared with a carrier that will protect this compound against rapid release, such as forms with controlled release, including implants, transdermal patches, microencapsulation delivery system. Can be used biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyarteritis and polylactic acid. Many methods of cooking such ready-made forms are patented or generally known qualified specialists in this field. See, for example,Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In some embodiments, the implementation of the binding protein according to the invention can be administered orally, for example, with an inert diluent or assimilated suitable for food carrier. This compound (and other ingredients, if desired) may also be enclosed in a capsule with a hard or soft shell, compressed into tablets, or incorporated directly into the diet of the subject. For personalnogo therapeutic introduction of these compounds may be incorporated with excipients and used in the Orme received inside tablets, buccal tablets, pastilles, capsules, elixirs, suspensions, syrups, wafers and the like For the introduction of compounds according to the invention otherwise than parenteral administration, it may be necessary to cover this compound material or joint injection of this compound with a material to prevent its inactivation.

Supplementary active compounds can also be included in these compositions. In some embodiments, the implementation of the binding protein according to the invention together or get together is injected with one or more additional therapeutic agents, which are applicable for the treatment of disorders in which the activity of IL-12 is harmful. For example, the binding protein according to the invention can be obtained together and/or put together with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules). In addition, one or more antibodies according to the invention can be used in combination with two or more previous therapeutic agents. In such combined methods of therapy can be advantageous to use a lower dose of an administered therapeutic agents, thus avoiding, thus, enabled the th toxicity or complications associated with different methods monotherapy.

In some embodiments, the implementation of the binding protein is associated with lengthening the half-life of the carrier, known in this area. Such media include, but are not limited to, the Fc-domain, polyethylene glycol and dextran. Such carriers are described, for example, in the patent application U.S. serial number 09/428082 and published application US patent # WO 99/25044, which are included in this description by reference for any purpose.

In a specific embodiment, nucleic acid sequences encoding the binding protein according to the invention or another prophylactic or therapeutic agent according to the invention, is administered for the treatment, prevention, cure or ameliorate disorders or one or more symptoms using gene therapy. Gene therapy is called therapy performed by introducing the subject expressed or expressed nucleic acid. In this embodiment of the invention, the nucleic acids produce coded antibody or preventive or therapeutic agent according to the invention, which mediates a prophylactic or therapeutic effect.

Any of the methods for gene therapy available in this field, can be used in soo is according to this invention. For the General overview of the methods of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215. Methods commonly known in the field of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). Detailed description of various methods of gene therapy are listed in US20050042664 A1, which is incorporated in this description by reference.

Binding proteins according to the invention is applicable in the treatment of various diseases, where targets that are recognized by these binding proteins are harmful to health. Such diseases include, but are not limited to, rheumatoid arthritis, osteoarthritis, chronic juvenile arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin-dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis scleroderma, a disease graft-versus-host rejection of the transplant organs, acute or chronic immune disease associated with transplant is the situation of bodies, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, graves ' disease, nephrotic syndrome, disease, chronic fatigue, Wegener's granulomatosis, purple's disease-Seleina, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, horey chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignant disease, heart failure, myocardial infarction, Addison's disease, sporadic pluriglandular failure type I and pluriglandular deficiency type II syndrome Schmidt (acute) respiratory distress syndrome of adults, alopecia, alopecia (alopecia) alopecia, the seronegative arthropathy, arthropathy, disease, Reiter, the psoriatic arthropathy, arthropathy, ulcerative colitis, enteropathic synovitis associated with Chlamydia, Yersinia and Salmonella the arthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis, atopic Allergy, autoimmune bullous disease, utricularia vulgaris, the leaf bladderwort, pemphigoid, linear IgA disease, autoimmu the ing hemolytic anemia, positive hemolytic anemia of Coombs, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/British myalgic encephalitis, chronic mucocutaneous candidiasis, giant cell arteritis diagnostics, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, acquired immunodeficiency syndrome, a disease, related to the acquired immunodeficiency, hepatitis b, hepatitis C, common transient immunodeficiency (common transient hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, loss of ovarian function, premature loss of ovarian function, vibrationthe lung disease, cryptogenic fibrosing alveolitis, polioptilidae interstitial lung disease, interstitial pneumonitis associated with disease of the connective tissue interstitial disease the lungs, associated with a disease mixed connective tissue of the lung disease associated with systemic sclerosis interstitial lung disease associated with rheumatoid arthritis interstitial lung disease associated with systemic lupus erythematosus is a disease of the lungs that is associated with dermatomyositis/polymyositis lung disease, associated with a disease, Sjogren's disease of the lungs that is associated with ankiloziruyushchiy spondylitis disease of the lungs, masculine diffuse lung disease associated with hemosiderosis disease of the lungs induced drug interstitial lung disease, fibrosis, radiation fibrosis, obliterative bronchiolitis, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, post-infectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, autoimmune hepatitis type 1 (classical autoimmune or lupoid hepatitis), autoimmune hepatitis type-2 (hepatitis with anti-LKM-antibodies), autoimmune mediated hypoglycemia, insulin resistance type with papillary pigment dystrophy of the skin (acanthosis nigricans, hypoparathyroidism, acute immune disease associated with with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leukopenia, autoimmune neutropenia, renal disease NOS (BDU), glomerulonephritis, microscopic vasculitis of the kidneys, Lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS (BDU), autoimmunity sperm, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease syndrome?, pulmonary manifestation nadeznogo of polyarteritis, acute rheumatic fever, rheumatoid spondylitis, of still's disease, systemic sclerosis, Sjogren syndrome, Takayasu's disease/arteritis diagnostics, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo, acute liver disease, chronic liver disease, alcoholic cirrhosis, alcohol-induced liver injury, cholestasis, idiosyncratic liver disease, induced drug-induced hepatitis, non-alcoholic steatohepatitis, Allergy and asthma, streptococcal infection group (GBS), mental disorders (e.g., depression and schizophrenia), Th type 2 - and Th type 1-mediated diseases, acute and chronic pain (different forms of pain), and cancers such as lung cancer, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer and hematopoietic malignant diseases (leukemia and lymphoma), abetalipoproteinemia, acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, atrial ectopic beats, AIDS-associated complex of dementia, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, deficiency of alpha-l-antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina, degeneration of anterior horn cells of the spinal cord, anti-cd3 therapy, antiphospholipid syndrome, allergic reactions to protivozachatochnye antibodies, aortic aneurysm and peripheral vascular, aortic dissection, arterial hypertension, arteriosclerosis, atrioventricular fistula, ataxia, atrial fibrillation (persistent or paroxysmal AF), atrial flutter, atrioventricular blockade, b-cell lymphoma, rejection, bone graft rejection of bone marrow transplant (BMT), interventricular blockade, Burkitt's lymphoma, burns, cardiac arrhythmia syndrome temporary cardiac arrest, cancer of the heart, cardiomyopathy, inflammatory response in the artificial circulation, graft rejection cartilage, cerebellar cortical degeneration, cerebellar violations, chaotic or multichrome preset the th tachycardia, associated with chemotherapy disorders, chronic miliitary leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, cancer of the colon, congestive heart failure, conjunctivitis, contact dermatitis, pulmonary heart disease coronary artery disease Creutzfeldt-Jakob, negative in culture sepsis, cystic fibrosis, associated with therapy with cytokines violations, Boxing dementia, demyelinating diseases, Dengue haemorrhagic fever, dermatitis, dermatologic conditions, diabetes, diabetes mellitus, diabetic arteriosclerotic disease disease with diffuse calves Levi, dilated congestive cardiomyopathy, disorders of the basal (subcortical) brain nuclei, down's syndrome in middle age, induced by drugs of movement disorders, disorders induced by drugs which block CNS dopamine receptors, sensitivity to drugs, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, infection with Epstein-Barr, erythromelalgia, extrapyramidal and cerebellar violations, family hematopoietically lim is iitary histiocytosis, implant rejection, fetal thymus, ataxia, functional peripheral arterial disorders, fungal sepsis, gas (anaerobic) gangrene, gastric ulcer, glomerulonephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas caused by intracellular organisms, leukemia reticuloendothelial disease Hallervorden-Spitze, Hashimoto's thyroiditis, hay fever, graft rejection hearts, hemochromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purple, hemorrhage, hepatitis (A), arrhythmia beam GIS, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, allergic reactions, allergic pneumonitis, hypertension, hypokinetic movement disorders, evaluation of the hypothalamic-pituitary-adrenal axis, idiopathic Addison disease, idiopathic pulmonary fibrosis antibody-mediated cytotoxicity, weakness, childhood spinal muscular atrophy, inflammation of the aorta, influenza a, the effects of ionizing radiation, iridocyclitis/uveitis/optic neuritis, ischemic/reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, sarcomas is sarcoma, transplant rejection of kidney, infection with Mycobacterium Legionella, leishmaniasis, leprosy, damage kortiko-spinal system, fat edema, transplant rejection liver, lymph edema, malaria, malignant lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic, cluster headache, mitochondrial multi-system disturbance, disease mixed connective tissue, the monoclonal gammopathy, multiple myeloma, degeneration of multiple systems (Mencel Dejerine - Thomas Shi-Drager and Machado-Joseph), heavy pseudoparalysis myasthenia, ycobacterium avium intracellulare, the tubercle Bacillus, myelodysplasia syndrome, myocardial infarction, ischemic disorders of the myocardium, nasopharynx cancer, chronic lung disease of the newborn, nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscular atrophy, neurogenic fever, non-Hodgkins lymphoma, occlusion of the abdominal aorta and its branches, the occlusal arterial disorders, okt3 therapy, orchitis/epididymitis, procedure after treatment of orchitis/vasectomy, organomegaly, osteoporosis, transplant rejection, pancreas cancer, pancreatic cancer, paraneoplastic syndrome/hypercalcemia in malignant disease, graft rejection parathyroid same the drive, inflammatory disease of the renal pelvis, rhinitis, diseases of the pericardium, peripheral atherosclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia, pneumonia, caused neumocystis carinii, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy syndrome and skin changes), postperfusion syndrome, potentiality syndrome, post-MI-cardiotomy, pre-eclampsia, progressive supranuclear palsy, primary pulmonary hypertension, radiation therapy, phenomenon and Raynaud's disease, Raynaud's disease, a disease of Resume, tachycardia with regular narrow QRS, renovaskulyarnoy (renal-vascular) hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, scleroderma, senile horey, senile dementia associated with calves Levi, seronegative arthropathies, shock, sickle cell anemia, rejection of allograft skin syndrome skin changes, graft rejection of the small intestine, solid tumors, specific arrhythmias, spinal ataxia, spinal-cerebellar degenerations, streptococcal myositis, structural damage to the cerebellum, subacute sclerosing panencephalitis, syncope (syncope;), cardiovascular syphilis, systemic anaphylaxis syndrome systemn the th inflammatory response, systemic juvenile rheumatoid arthritis, T-cell or FAB ALL (acute lymphoblastic leukemia), telangiectasia, thromboangiitis obliterans, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, allergic reactions type III, type IV allergies, unstable angina, uremia, urosepsis, urticaria, valvular heart disease, varicose veins, vasculitis, venous diseases, venous thrombosis, atrial (shimmer) ventricular fibrillation, viral and fungal infections, acute encephalitis with a high risk of death/aseptic meningitis, hemophagocytosis syndrome associated with a high risk of death, syndrome Wernicke-Korsakov, Wilson disease, xenograft rejection of any organ or any fabric. (See Peritt et al. the PCT publication no WO A, Leonard et al., the PCT publication no WO 9524918 A1 and Salfeld et al., the PCT publication no WO 00/A).

Binding proteins according to the invention can be used to treat humans suffering from autoimmune diseases, in particular diseases associated with inflammation, including rheumatoid arthritis, spondylitis, Allergy, autoimmune diabetes, autoimmune uveitis.

Preferably, binding proteins according to the invention or their antigennegative part is used for the treatment of rheumatoid arthritis, Crohn's disease, multiple sclerosis, insulin-dependent diabetes mellitus and psoriasis.

Binding protein according to the invention can also enter one or more additional therapeutic agents applicable in the treatment of various diseases.

Binding protein according to the invention may be used separately or in combination for the treatment of such diseases. It should be clear that the binding proteins can be used alone or in combination with an additional agent, e.g., a therapeutic agent, and the specified additional agent is selected qualified for its intended purpose. For example, the additional agent may be a therapeutic agent, which is recognized in the field as an agent, applicable to the treatment of the disease or condition to be treated by the antibody according to the invention. The additional agent can be an agent that gives a useful feature of this therapeutic compositions, for example, an agent that affects the viscosity of the composition.

It should also be understood that combinations that must be included in the scope of this invention are combinations that are applicable for their intended purpose. The agents below are illustrative for these purposes and are not limiting. Combinations, which are part of the present invention, can the set of antibodies according to the invention and at least one additional agent, selected from the lists below. The combination can also include more than one additional agent, e.g., two or three additional agent, if this combination is such that the formed composition can perform its intended function.

Preferred combinations for the treatment of autoimmune and inflammatory diseases are non-steroidal anti-inflammatory drugs, also called NSAIDs, which include drugs like ibuprofen. Other preferred combinations are corticosteroids including prednisolone; the well known side effects of the use of steroids can be reduced or even eliminated by reducing the dose of steroid in the treatment of patients in combination with DVD-Ig according to the invention. Non-limiting examples of therapeutic agents for rheumatoid arthritis with which it is possible to combine the antibody or part of an antibody according to the invention, include the following: suppressive cytokines anti-inflammatory drug (CSAID); antibodies to other cytokines or growth factors, human or antagonists of other cytokines or growth factors of the person, for example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, IL-23, interferons, EMAP-II, GM-CSF, FGF, and PDGF. Binding proteins according invented the Yu or antihistamie part can be combined with antibodies to cell surface molecules, such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligands including CD154 (gp39 or CD40L).

Preferred combinations of therapeutic agents may interfere at different points in the autoimmune and subsequent inflammatory cascade; preferred examples include TNF antagonists like chimeric, humanized anti-TNF antibody or anti-TNF-human antibodies, D2E7 (PCT publication no WO 97/29131), CA2 (Remicade™), CDP 571, and soluble TNF receptors p55 or p75, their derivatives (p75TNFR1gG (Enbrel™) or p55TNFR1gG (Lenercept), and also inhibitors of TNFα converting enzyme (TACE); similarly, inhibitors of IL-1 (inhibitors of interleukin-1-converting enzyme, IL-1RA etc) can be effective for the same reason. Other preferred combinations include interleukin-11. Other preferred combinations include key members of the autoimmune response which may act parallel to the function of IL-12, depending on the function of IL-12 or with IL-12; particularly preferred are antagonists of IL-18, which includes antibodies, IL-18 or soluble receptors of IL-18 or IL-18 binding proteins. It was shown that IL-12 and IL-18 have overlapping but distinct functions, and the combination of antagonists to both of them may be the most effective. Another preferred combination is n is depleting anti-CD4 inhibitors. Other preferred combinations include antagonists of co-stimulating a pathway CD80 (B7.1) or CD86 (B7.2)including antibodies, soluble receptors or antagonistic ligands.

Binding proteins according to the invention can also be combined with such agents as methotrexate, 6-MP, azathioprine-sulfasalazin, mesalazine, olsalazine, chlorogenic/hydroxychloroquine, pencillamine, aurothiomalate (intramuscular and oral), azathioprine, cohesin, corticosteroids (oral, inhalation and introduce local injection), agonists beta-2-adrenergic receptors (salbutamol, terbutaline, salmeterol), xantina (theophylline, aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, cyclosporine, FK506, rapamycin, mycophenolate-mofetil, Leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, inhibitors of complement, adrenergic agents, agents which interfere with signaling by proinflammatory cytokines such as TNF-α or IL-1 (e.g., inhibitors IRAK, NIK, IKK, p38 or MAP kinase)inhibitors of IL-1β converting enzyme inhibitors, TNFα converting enzyme (TACE)inhibitors of the signal T-cells, such as kinase inhibitors, inhibitors of metalloproteinases, sulfasalazine, azathioprine, 6-Merck is peperina, inhibitors of angiotensin-converting enzyme, soluble receptors, cytokines and their derivatives (e.g., soluble TNF receptors p55 or p75 and their derivatives p75TNFRIgG (Enbrel™ and p55TNFRIgG (Lenercept)), sIL-1RI, sIL-1RII, sIL-6R), antiinflammatory cytokines (eg, IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib, folic acid, of hydroxychloroquine sulfate, rofecoksib, etanercept, infliximab, naproxen, valdecoxib, sulfasalazin, methylprednisolone, meloxicam, methylprednisolone acetate, gold-sodium-thiomalate, aspirin, triamcinolone acetonide, propoxyphene napsylat/Arar, folate, nabumetone, diclofenac, piroxicam, etodolac, diclofenac-sodium, oxaprozin, oxycodone hydrochloride, hydrocodone bitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra, recombinant human, tramadol hydrochloride, salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium, prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin, glucosamine of self/chondroitin, amitriptyline hydrochloride, sulfadiazine, oxycodone hydrochloride/acetaminophen, olopatadine hydrochloride misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740, roflumilast, IC-485, CDC-801 and mesopram. Preferred combinations include methotrexate or Leflunomide and, in the temperate is whether severe cases of rheumatoid arthritis, cyclosporine.

Non-limiting additional agents that may be used in combination with a binding protein for the treatment of rheumatoid arthritis include, but are not limited to, the following agents: non-steroidal anti-inflammatory drug (NSAID); suppressive cytokines anti-inflammatory drug (CSAID); CDP-571/BAY-10-3356 (humanitariannet anti-TNFα-antibody; Celltech/Bayer); cA2/infliximab (chimeric anti-TNFα-antibody; Centocor); 75 kdTNFR-IgG/etanercept (protein 75 KD TNF receptor-IgG; Immunex; see, for example,Arthritis & Rheumatism(1994) Vol. 37, S295;J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNF-IgG (protein of 55 KD TNF receptor-IgG; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (sustainable primaryservername anti-CD4 antibody; IDEC/SmithKline; see, for example,Arthritis & Rheumatism(1995) Vol.38, S185); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fused proteins; Seragen; see, for example,Arthritis & Rheumatism(1993) Vol.36, 1223); Anti-Tac (humanized anti-IL-2Rα; Protein Design Labs/Roche); IL-4 (anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10, an anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or agonists of IL-4 (e.g., agonist antibodies); IL-1RA (antagonist of IL-1-receptor; Synergen/Amgen); anakinra (Kineret®/Amgen); TNF-bp/s-TNF (soluble TNF binding protein; see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S284;Amer. J. Physiol. - Heart and Circulatory discrimination(1995) Vol.268pp. 37-42); R973401 (inhib the tor phosphodiesterase type IV; see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S282); MK-966 (COX-2 inhibitor; see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S81); iloprost (see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S82); methotrexate, thalidomide (see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S282) and thalidomide and related drugs (e.g., celgen); Leflunomide (anti-inflammatory agent and inhibitor of cytokines; see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S131;Inflammation Research(1996) Vol.45pp. 103-107); tranexamic acid (inhibitor of plasminogen activation; see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S284); T-614 (inhibitor of cytokines; see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S282); prostaglandin E1 (see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S282); tenidap (non-steroidal anti-inflammatory drug; see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S280); naproxen (non-steroidal anti-inflammatory drug; see, for example,Neuro Report(1996) Vol.7pp. 1209-1213); meloxicam (non-steroidal anti-inflammatory drug); ibuprofen (non-steroidal anti-inflammatory drug); piroxicam (non-steroidal anti-inflammatory drug); diclofenac (non-steroidal anti-inflammatory drug); indomethacin (non-steroidal protivovospalitel the th vehicle); sulfasalazin (see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S281); azathioprine (see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S281); ICE inhibitor (inhibitor of interleukin-1β converting enzyme); inhibitor of zap-70 and/or lck (inhibitor of the tyrosine kinase zap-70 or lck); VEGF inhibitor and/or an inhibitor of VEGF-R inhibitors of growth factor vascular endothelial cells or growth factor receptor vascular endothelial cells; inhibitors of angiogenesis); corticosteroid anti-inflammatory drugs (e.g., SB203580); TNF inhibitors-convertase; anti-IL-12 antibodies; anti-IL-18 antibodies; interleukin-11 (see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S296); interleukin-13 (see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S308); inhibitors of interleukin-17 (see, for example,Arthritis & Rheumatism(1996) Vol.39No. 9 (supplement), S120); gold; penicillamine; chloroquine; chlorambucil; hydroxychloroquine; cyclosporine; cyclophosphamide; total lymphoid irradiation; anti-timatic-globulin; anti-CD4 antibodies; CD5-toxins; orally administered peptides and collagen; lobenzarit-disodium; regulatory cytokines agents (CRA) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); antisense phosphorothioate oligodeoxynucleotide ICAM-1 (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone; orgotein; fractions floor is sulfate; minocycline; anti-IL2R antibodies; marine and Botanical lipids (fatty acids fish and plant seed; see, for example, DeLucaet al. (1995)Rheum. Dis. Clin. North Am.21:759-777); auranofin; phenylbutazone; meclofenamic acid; intravenous immunoglobulin; zileuton; azaribine; mycophenolate acid (RS-61443); tacrolimus (FK-506), sirolimus (rapamycin); amiprilose (terpentin); cladribine (2-chloromethoxypropyl); methotrexate; anti-virus agents and immunomodulatory agents.

In one embodiment, the binding protein or antihistamie part is administered in combination with one of the following agents for the treatment of rheumatoid arthritis: a KDR inhibitor small molecule (ABT-123), an inhibitor of Tie-2 small molecule; methotrexate; prednisone; celecoxib, folic acid; of hydroxychloroquine sulfate; rofecoksib; etanercept; infliximab; Leflunomide; naproxen; valdecoxib; sulfasalazin; methylprednisolone; ibuprofen; meloxicam; methylprednisolone acetate; gold sodium-thiomalate; aspirin; azathioprine; triamcinolone acetonide; propoxyphene napsylate/apap; folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium; oxaprozin; oxycodone hydrochloride, hydrocodone bitartrate/apap; diclofenac-sodium/misoprostol; fentanyl; anakinra, recombinant human; tramadol hydrochloride; salsalate; sulindac; cyanocobalamin/fa/pyridoxine; acetone is nopen; alendronate-sodium; prednisolone; morphine sulfate; lidocaine hydrochloride; indomethacin; glucosamine sulfate/chondroitin; cyclosporine; amitriptyline hydrochloride; sulfadiazine; oxycodone hydrochloride/acetaminophen; olopatadine hydrochloride; misoprostol; naproxen-sodium; omeprazole; mycophenolat-mofetil; cyclophosphamide; rituximab; IL-1 TRAP; MRA; CTLA4-IG, IL-18 BP; ABT-874; ABT-325; anti-IL-18; anti-IL-15; BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; roflumilast; IC-485; CDC-801 and mesopram.

Non-limiting examples of therapeutic agents for inflammatory bowel disease with which a binding protein according to the invention can be combined include the following agents: budenoside; epidermal growth factor; corticosteroids; cyclosporin, sulfasalazin; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; inhibitors of thromboxane; antagonists of IL-1-receptor; monoclonal anti-IL-1β antibodies; monoclonal anti-IL-6 antibodies; growth factors; elastase inhibitors; pyridinedimethanol compounds; antibodies to other cytokines or growth factors person or antagonists to other cytokines or growth factors of the person, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-17, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies according to the invention or their antigennegative parts can be combined is activated with antibodies to cell surface molecules, such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands. Antibodies according to the invention or their antigennegative parts can also be combined with such agents as methotrexate, cyclosporine, FK506, rapamycin, mycophenolate-mofetil, Leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, inhibitors of complement, adrenergic agents, agents which interfere with signaling by proinflammatory cytokines such as TNF-α or IL-1 (e.g., inhibitors of kinases IRAK, NIK, IKK, p38 or MAP), inhibitors of IL-1β converting enzyme inhibitors, TNFα converting enzyme inhibitors transmission signal T-cells, such as kinase inhibitors, inhibitors of metalloproteinases, sulfasalazin, azathioprine, 6-mercaptopurine, inhibitors of angiotensin-converting enzyme, soluble receptors, cytokines and their derivatives (e.g., soluble TNF receptors p55 or p75, sIL-1RI, sIL-1RII, sIL-6R) and antiinflammatory cytokines (eg, IL-4, IL-10, IL-11, IL-13 and TGFβ).

Preferred examples of therapeutic agents for Crohn's disease with which a binding protein can be combined include the following agents: TNF antagonists, for example anti-TNF antibodies, D2E7 (PCT publication No. WO 97/29131; HUMIRA), CA2 (REMICADE), CDP 571, design TNFR-Ig, inhibitors (p75TNFRIgG (ENBREL and p55TNFRIgG (LENERCEPT)), and PDE4 inhibitors. Antibodies according to the invention or their antigennegative part can be combined with corticosteroids, for example, budenoside and dexamethasone. Binding proteins according to the invention or their antigennegative parts can also be combined with such agents as sulfasalazin, 5-aminosalicylic acid and olsalazine, and agents which interfere with synthesis or action of proinflammatory cytokines such as IL-1, for example, inhibitors of IL-1β-converting enzyme and IL-1ra. Antibodies according to the invention or their antigennegative part can also be used with inhibitors of signal transmission T cells, for example, tyrosine kinase inhibitors 6-mercaptopurine. Binding proteins according to the invention or their antigennegative part can be combined with IL-11. Binding proteins according to the invention or their antigennegative part can be combined with the following tools: mesalamine, prednisone, azathioprine, mercaptopurine, infliximab, methylprednisolone sodium succinate, Diphenoxylate/Atropa sulfate, loperamide hydrochloride, methotrexate, aerosol, folate, ciprofloxacin/dextrose-water, hydrocodone bitartrate/Arar, tetracycline hydrochloride, fluocinonide, metronidazole, thimerosal/boric acid, cholestyramine/sucrose, ciprofloxacin hydrochloride, giostsiamina sulfate, meperidine hydrochlor is d, midazolam hydrochloride, oxcodone hydrochloride/acetaminophen, prometazina hydrochloride, sodium phosphate, sulfamethoxazole/trimethoprim, celecoxib, polycarbophil, propoxyphene napsylate, hydrocortisone, multivitamins, disodium-balsalazide, codeine phosphate/Arar, colesevelam hydrochloride, cyanocobalamin, folic acid, levofloxacin, methylprednisolone, natalizumab and interferon-gamma.

Non-limiting examples of therapeutic agents for multiple sclerosis with which binding proteins according to the invention can be combined include the following agents: corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-β1 (AVONEX; Biogen); interferon-B1b (BETASERON; Chiron/Berlex); interferon α-n3) (Interferon Sciences/Fujimoto), interferon-α (Alfa Wassermann/J&J), interferon-βA-IF (Serono/Inhale Therapeutics), Peg-interferon-α2b (Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; claribid; antibodies to other cytokines or growth factors or their receptors person or antagonists of other cytokines or growth factors or their receptors person, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Binding proteins according to the invention can be combined with antibodies to molecules of the cell surface is ness, such as CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their ligands. Binding of the antibodies according to the invention can also be combined with such agents as methotrexate, cyclosporine, FK506, rapamycin, mycophenolate-mofetil, Leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, inhibitors of complement, adrenergic agents, agents which interfere with signaling by proinflammatory cytokines such as TNF-α or IL-1 (e.g., inhibitors of kinases IRAK, NIK, IKK, p38 or MAP), inhibitors of IL-1β converting enzyme, TACE inhibitors, inhibitors of signal transmission T-cells, such as kinase inhibitors, inhibitors of metalloproteinases, sulfasalazin, azathioprine, 6-mercaptopurine, inhibitors of angiotensin-converting enzyme, soluble receptors, cytokines and their derivatives (e.g., soluble TNF receptors p55 or p75, sIL-1RI, sIL-1RII, sIL-6R) and antiinflammatory cytokines (eg, IL-4, IL-10, IL-11, IL-13 and TGFβ).

Preferred examples of therapeutic agents for multiple sclerosis with which binding proteins according to the invention can be combined include interferon-β, for example, IFNβ1a and IFNβ1b; Copaxone, corticosteroids, caspase inhibitors, for example inhibitors of caspase-1 inhibitors, IL-1, TNF inhibitors, and EN is the body to CD40-ligand and CD80.

Binding proteins according to the invention can be combined with such agents as alemtuzumab, dronabinol, Unimed, daclizumab, mitoxantrone, xaliproden hydrochloride, Fampridine, glatiramer acetate, natalizumab, cannabidiol, a-immunokine NNSO3, ABR-215062, AnergiX.MS antagonists of the receptor for chemokines, BBR-2778, calaguala, CPI-1189, LEM (encapsulated in the liposome mitoxantrone), THC.CBD (agonist cannabinoid) MBP-8298, mesopram (PDE4 inhibitor), MNA-715, anti-IL-6-receptor antibody, neurovax, pirfenidone-allotrope 1258 (RDP-1258), sTNF-R1, talampanel, teriflunomide, TGF-beta2, diplomatic, antagonists of VLA-4 (for example, TR-14035, VLA4 Ultrahaler, Antegran-ELAN/Biogen), antagonists of interferon-gamma agonists of IL-4.

Non-limiting examples of therapeutic agents for angina with which you can combine binding proteins according to the invention, include the following agents: aspirin, nitroglycerin, isosorbide Mononitrate, metoprolol succinate, atenolol, metoprolol tartrate, amlodipine besylate, diltiazem hydrochloride, the isosorbide dinitrate treatment, the clopidogrel bisulfate, nifedipine, atorvastatin calcium, potassium chloride, furosemide, simvastatin, verapamil hydrochloride, digoxin, propranolol hydrochloride, carvedilol, lisinopril, spironolactone, hydrochlorothiazide, enalapril maleate, nadolol, ramipril, enoxaparin sodium, heparin sodium, valsartan, sotalol, hydrochlori is, fenofibrate, ezetimibe, bumetanide, losartan-potassium, lisinopril/hydrochlorothiazide, felodipine, captopril, bisoprolol fumarate.

Non-limiting examples of therapeutic agents for ankylosing spondylitis with which you can combine binding proteins according to the invention, include the following agents: ibuprofen, diclofenac and misoprostol, naproxen, meloxicam, indomethacin, diclofenac, celecoxib, rofecoksib, sulfasalazin, methotrexate, azathioprine, minocycline, prednisone, etanercept, infliximab.

Non-limiting examples of therapeutic agents for asthma with which you can combine binding proteins according to the invention, include the following agents: albuterol, salmeterol/fluticasone, montelukast sodium, fluticasone propionate, budesonide, prednisone, salmeterola xinafoate, levalbuterol hydrochloride, albuterol sulfate/ipratropium, prednisolone sodium phosphate, triamcinolone acetonide, beclomethasone dipropionate, Pretoria bromide, azithromycin, pirbuterol acetate, prednisolone, theophylline anhydrous, methylprednisolone sodium succinate, clarithromycin, zafirlukast, formoterol fumarate, the vaccine of the influenza virus, methylprednisolone, amoxicillin trihydrate, flunisolide, injection against allergies, kromolin-sodium, Fexofenadine hydrochloride, flunisolide/menthol, amoxicillin/clavul is at, levofloxacin inhalation device, guaifenesin, dexamethasone sodium phosphate, moxifloxacin hydrochloride, doxycycline hyclate, guaifenesin/d-methorphan, p-pseudoephedrine/codeine/chlorphenyl, Gatifloxacin, cetirizine hydrochloride, mometasone furoate, salmeterola xinafoate, benzonatate, cephalexin, PE/hydrocodone/chlorphenyl, cetirizine hydrochloride/pseudoephedrine, phenylephrine/codeine/promethazine, codeine/promethazine, cefprozil, dexamethasone, guaifenesin/pseudoephedrine, chlorpheniramine/hydrocodone, nedocromil sodium, terbutalina sulfate, epinephrine, methylprednisolone, metaproterenol sulfate.

Non-limiting examples of therapeutic agents for COPD with which you can combine binding proteins according to the invention, include the following agents: albuterol sulfate/ipratropium, ipratropium bromide, salmeterol/fluticasone, albuterol, salmeterola xinafoate, fluticasone propionate, prednisone, theophylline anhydrous, methylprednisolone sodium succinate, montelukast sodium, budesonide, formoterol fumarate, triamcinolone acetonide, levofloxacin, guaifenesin, azithromycin, beclomethasone dipropionate, levalbuterol hydrochloride, flunisolide, Ceftriaxone sodium, amoxicillin trihydrate, Gatifloxacin, zafirlukast, amoxillin/clavulanate, flunisolide/menthol, chlorpheniramine/hydrocodone metaproterenol sulfate, methylp rednisolone, mometasone furoate, p-pseudoephedrine/codeine/chlorphenyl, pirbuterol acetate, p-pseudoephedrine/loratadine, terbutaline sulfate, Tiotropium bromide, (R,R)-formoterol, TgAAT, cilomilast, roflumilast.

Non-limiting examples of therapeutic agents for HCV with which you can combine binding proteins according to the invention, include the following agents: interferon-alpha-2a, interferon-alpha-2b, interferon-alpha con1, interferon-alpha-n1, targeted interferon-alpha-2a, targeted interferon-alpha-2b, ribavirin and PEG-interferon-Alfa-2b + ribavirin, ursodeoxycholic acid, glycyrrhizin acid, thymalfasin, Maximin, VX-497 and any compounds that are used to treat HCV through exposure to the following targets: polymerase HCV, HCV protease, HCV helicase, HCV IRES (internal binding site of the ribosome).

Non-limiting examples of therapeutic agents for idiopathic pneumovirus (interstitial pneumonia), which can be combined binding proteins according to the invention, include the following agents: prednisone, azathioprine, albuterol, colchicine, albuterol sulfate, digoxin, gamma interferon, methylprednisolone sodium succinate, lorazepam, furosemide, lisinopril, nitroglycerin, spironolactone, cyclophosphamide, ipratropium bromide, actinomycin d, alteplaza, fluticasone propionate, lewohl xacin, metaproterenol sulfate, morphine sulfate, oxycodone hydrochloride, potassium chloride, triamcinolone acetonide, tacrolimus anhydrous, calcium, interferon-alpha, methotrexate, mycophenolate-mofetil, interferon-gamma-1β.

Non-limiting examples of therapeutic agents for myocardial infarction with which you can combine binding proteins according to the invention, include the following agents: aspirin, nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin sodium, the clopidogrel bisulfate, carvedilol, atenolol, morphine sulfate, metoprolol succinate, warfarin sodium, lisinopril, isosorbide Mononitrate, digoxin, furosemide, simvastatin, ramipril, tenecteplase, enapril maleate, torsemide, retavase, losartan-potassium, chinaprice hydrochloride/MAG carb, bumetanide, alteplaza, enalaprilat, amiodarone hydrochloride, tirofibana hydrochloride monohydrate, diltiazem hydrochloride, captopril, irbesartan, valsartan, propanolol hydrochloride, fosinopril-sodium, lidocaine hydrochloride, eptifibatide, Cefazolin sodium, atropine sulfate, aminocaproic acid, spironolactone, interferon, sotalol hydrochloride, potassium chloride, docusate-sodium, dobutamine hydrochloride, alprazolam, pravastatin sodium, atorvastatin calcium, midazolam hydrochloride, meperidine hydrochloride, the isosorbide dinitrate treatment, epinephrine, dopamine hydro is lurid, bivalirudin, rosuvastatin, ezetimibe/simvastatin, avasimibe, cariporide.

Non-limiting examples of therapeutic agents for psoriasis with which you can combine binding proteins according to the invention, include the following agents: inhibitor small molecule KDR (ABT-123), inhibitor small molecule Tie-2, calcipotriene, clobetasol propionate, triamcinolone acetonide, halobetasol propionate, tazarotene, methotrexate, fluocinonide, betamethasone dipropionate stimulated, flucinolone acetonide, acitretin, shampoo-tar, betamethasone valerate, mometasone furoate, ketoconazole, pramoxine/fluotsinolon, hydrocortisone valerate, flurandrenolide, urea, betamethasone, clobetasol propionate/emoll, fluticasone propionate, azithromycin, hydrocortisone, moisturizing blend, folic acid, desonide, pimecrolimus, coal tar, diflorasone diacetate, etanercept folate, lactic acid, methoxsalen, hc/bismuth subgal/znox/resor, methylprednisolone acetate, prednisone, sunscreen, halcinonide, salicylic acid, anthralin, clocortolone pivalate, coal extracts, coal tar/salicylic acid, coal tar/salicylic acid/sulfur, desoximetasone, diazepam, emollient, fluocinonide/emollient mineral oil/castor oil/na lact, the miner is inoe oil/peanut oil, vaseline/isopropylmyristate, psoralen, salicylic acid, soap/tribromsalan, thimerosal/boric acid, celecoxib, infliximab, cyclosporine, alefacept, efalizumab, tacrolimus, pimecrolimus, PUVA, UVB, sulfasalazin.

Non-limiting examples of therapeutic agents for psoriatic arthritis with which you can combine binding proteins according to the invention, include the following agents: methotrexate, etanercept, rofecoksib, celecoxib, folic acid, sulfasalazin, naproxen, Leflunomide, methylprednisolone acetate, indomethacin, of hydroxychloroquine sulfate, prednisone, sulindac, betamethasone dipropionate stimulated, infliximab, methotrexate, folate, triamcinolone acetonide, diclofenac, dimethylsulfoxide, piroxicam, diclofenac sodium, Ketoprofen, meloxicam, methylprednisolone, nabumetone, tolmetin-sodium, calcipotriene, cyclosporine, diclofenac sodium/misoprostol, fluocinonide, glucosamine sulfate, thiomalate gold-sodium, hydrocodone bitartrate/apap, ibuprofen, risedronate sodium, sulfadiazine, tioguanin, valdecoxib, alefacept, efalizumab.

Non-limiting examples of therapeutic agents for restenosis, which can be combined binding proteins according to the invention, include the following agents: sirolimus, paclitaxel, everolimus, tacrolimus, ABT-578, acetaminophe is.

Non-limiting examples of therapeutic agents for ishialgii, which can be combined binding proteins according to the invention, include the following agents: hydrocodone bitartrate/apap, rofecoksib, cyclobenzaprine hydrochloride, methylprednisolone, naproxen, ibuprofen, oxycodone hydrochloride/acetaminophen, celecoxib, valdecoxib, methylprednisolone acetate, prednisone, codeine phosphate/apap, tramadol hydrochloride/acetaminophen, metaxalone, meloxicam, Methocarbamol, lidocaine hydrochloride, diclofenac sodium, gabapentin, dexamethasone, carisoprodol, Ketorolac-tromethamine, indomethacin, acetaminophen, diazepam, nabumetone, oxycodone hydrochloride) hydrochloride, diclofenac-sodium/misoprostol, propoxyphene napsylate/apap, Aza/oxycod/oxycodone Ter, ibuprofen/hydrocodone bit, tramadol hydrochloride, etodolac, propoxyphene hydrochloride, amitriptyline hydrochloride, carisoprodol/codeine FOS/Aza, morphine sulfate, mulitvitamin, naproxen sodium, orphenadrine citrate, temazepam.

Preferred examples of therapeutic agents for SLE (lupus), which can be combined proteins according to the invention, include the following agents: NSAIDs, such as diclofenac, naproxen, ibuprofen, piroxicam, indomethacin; COX2 inhibitors, for example, celecoxib, rofecoksib, valdecoxib; antimalarial with whom estva, for example, hydroxychloroquine; steroids, such as prednisone, prednisolone, budenoside, dexamethasone; cytotoxic agents such as azathioprine, cyclophosphamide, mycophenolate-mofetil, methotrexate; inhibitors of PDE4 or purine synthesis inhibitors, for example, CellCept. Binding proteins according to the invention can also be combined with such agents as sulfasalazin, 5-aminosalicylic acid, olsalazine, imuran, and agents which interfere with synthesis, production or action of proinflammatory cytokines such as IL-1, for example, caspase inhibitors, such as inhibitors of IL-1β-converting enzyme and IL-1ra. Binding proteins according to the invention can also be used with inhibitors of signal transmission T cells, for example, inhibitors tyrosinekinase; or molecules that target molecule activation of T cells, for example, CTLA-4-IgG or anti-B7 family antibodies, anti-PD-1 family antibodies. Binding proteins according to the invention can be combined with IL-11 or anti-cytokine-antibody, for example, fontolizumab (anti-IFNg-antibody), or protivoparazitarnymi antibodies, for example, anti-IL-6-receptor antibody and antibodies to surface molecules In cells. Antibodies according to the invention or their antigennegative part can also be used with LJP 394 (abetimus), agents that Deplete or inak is everyt b-cells, for example, rituximab (anti-CD20-antibody), lymphostat-B (anti-BlyS-specific antibody), TNF antagonists, for example anti-TNF antibodies, D2E7 (PCT publication no WO 97/29131; HUMIRA), CA2 (REMICADE), CDP 571, designs TNFR-Ig, (p75TNFRIgG (ENBREL) and p55TNFRIgG (LENERCEPT)).

The pharmaceutical compositions according to the invention may include a "therapeutically effective amount" or "prophylactically effective amount" binding protein according to the invention. "Therapeutically effective amount” means an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. A therapeutically effective amount of the binding protein can be determined by the person skilled in the art and may vary according to factors such as the disease state, age, sex and weight of the individual, and the binding ability of this protein to induce the desired response in the individual. Therapeutically effective amount is the amount at which any toxic or detrimental effects of the antibodies or parts of antibodies were outweighed by therapeutically beneficial effects. "Prophylactically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired prophylactic result. Usually, the since a prophylactic dose is used for the subjects prior to or at the early stage of the disease, it prophylactically effective amount will be less than therapeutically effective amount.

Dosing regimens can be tailored to provide the optimum desired response (e.g., therapeutic or prophylactic response). For example, there may be a single bolus may be entered several divided doses over time or the dose may be proportionally reduced or increased as dictated by the need for specific therapeutic situation. Especially, it is preferable to prepare parenteral compositions in a standard dosage form for ease of administration and uniformity of dosage. Standard dosage form is called here physically discrete units suitable as single doses to subjects of the mammal to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in Association with the required pharmaceutical carrier. Specification in respect of standard dosage forms is dictated by (a) the unique characteristics of the active compound and the particular achievable therapeutic or prophylactic effect, and (b) the limitations inherent in the type of compounding such an active compound for the influence of the sensitivity of individuals, and depends on the characteristics and limitations.

Exemplary, non-limiting range for a therapeutically or prophylactically effective amount of a binding protein according to the invention is 0.1-20 mg/kg, more preferably 1-10 mg/kg it Should be noted that the magnitude of the dose may vary depending on the type and severity of the condition to be relieved. In addition, it should be clear that for any particular subject, specific dosing regimens should be adjusted over time according to the individual need and the professional judgment of the person making the introduction of, or controlling the introduction of these compositions, and that these dose ranges are merely exemplary and are not intended to limit the scope or practice of the claimed composition.

Qualified in this field specialists will be obvious that other suitable modifications and adaptations of the methods of the invention described herein are obvious and may be made using suitable equivalents without deviating from the scope of this invention or described herein of embodiments. After a detailed description of the invention, the same will be clear with reference to the following examples, which are included only for C is lei illustration and are not intended to limit the present invention

Examples

Example 1: Generating a dual variable domain immunoglobulin (DVD-Ig)

With dual variable domains of the molecule of the immunoglobulin (DVD-Ig) design so that two different variable domain of the light chain (VL) of two different original mAb linked in tandem directly or via a short linker by means of recombinant DNA with subsequent constant domain of the light chain. Similarly, the heavy chain contains two distinct variable domain of the heavy chain (VH)connected in tandem, with subsequent constant domain CH1 and Fc-region (figa).

Example 1.1: Generation of mouse monoclonal antibodies to IL-1α and IL-1β

Monoclonal antibodies to IL-1α and IL-1β were generated as follows using hybridoma technology are well known in this field.

Example 1.1.A: Immunization of mice

Purified recombinant IL-1α of human rights and IL-1β mouse (R&D Systems) was used as immunogens, as well as coating antigens in the analysis of titer and ELISA-screening. Immunizing dose was in the range of 5.0 to 20.0 μg/mouse/injection for all antigens to both initial and re-immunization (booster immunizations). Adjuvant ImmunEasy was purchased from Qiagen (Waltham, MA) and used against adjuvant/antigen, comprising 20 ml of adjuvant ImmunEasy to 0.0 μg of antigen. Each group of animals subject to immunization, contained 5 mice with knockout of IL-1αβ, received from Dr. Yoichiro Iwakura (University of Tokyo, Minato-ku, Tokyo, Japan). Mice were immunized in accordance with the scheme of doses, described below. Cells MRC-5 were purchased from ATS (Manassas, VA) and used for bioanalysis IL-1. Kits ELISA IL-8 human and a control mouse anti-hIL-1α and-β antibodies (MAB200 and MAB201) was purchased from R&D Systems (Minneapolis, MN).

Briefly, a mixture of adjuvant-antigen was prepared first by careful mixing the adjuvant vial using a vortex. The desired number adjuvant was removed from the vial and placed in an autoclaved microcentrifuge tube 1.5 ml Antigen was prepared in PBS or saline with a concentration in the range 0.5-1.0 mg/ml. Then calculated the amount of antigen was added to the microcentrifuge tube containing the adjuvant and the solution gently mixed with a pipette up and down 5 times. The mixture adjuvant-antigen were incubated at room temperature for 15 minutes and then mixed again gently pipette up and down 5 times. The solution adjuvant-antigen extracted with a suitable syringe for injection of the animal. Only 5-20 μg of antigen were injected with the volume of 50-100 μl. Each animal was immunized and then were immunized 2-3 times depending on the title. Animals with good titles received final intravenous boost the-immunization before merging and generation hybrid.

Example 1.1.B: Screening hybridomas

Hybridoma generated as described above were subjected to screening and determined the titer of antibodies using ELISA:protein antigens were applied directly to the plates ELISA for detection of specific antibodies using standard ELISA techniques. Briefly, tablets ELISA were covered with 100 µl of either rhIL-1α, or rhIL-1β (1.0 microgram/ml in PBS) overnight at 4°C. the Tablets were washed 3 times with 250 μl PBS/0.5% tween20and blocked with 200 μl blocking buffer (2% BSA in PBS with 0.5% tween20). To each well was added to the diluted serum or hybrid supernatant (100 μl) and incubated at room temperature for 2 hours. Then the tablets were washed 3 times with PBS/0.5% tween20for detection used HRP-goat antibodies against mouse IgG and OD binding was observed at 450 nm. Hybridoma clones producing antibodies that detect specific binding activity in ELISA, was subclinically and purified, and the affinity (Biacore) and efficiency (MRC-5-bioanalysis) antibodies characterized as follows.

Example 1.1.C: Characterization of murine monoclonal antibodies to IL-1α and IL-1β

The following tests were used to characterize the antibodies produced by the hybridomas described in example 1.1.B.

Example 1.1.C.1: surface plasmon Resonance

Connect the surrounding interaction in real time between the captured antibody (mouse anti-rmIL1-antibody captured on the biosensor matrix by goat antibodies against mouse IgG) and rmIL-1 was measured using surface plasmon resonance (SPR)using the BIAcore system (Biacore AB, Uppsala, Sweden)according to the manufacturer's instructions and standard procedures. Briefly, rmIL-1 diluted in the working HBS buffer (Biacore AB) and aliquots of 50 µl were injected with through the matrices immobilized protein at the speed of a current of 5 ml/min concentration was rhIL1 62,5, 125, 187,5, 250, 375, 500, 750, 1000, 1500 and 2000 nm. To determine the dissociation constants (the speed of dissociation), Association constants (speed Association) used the kinetic BIAcore evaluation (version 3.1).

Example 1.1.C.2: Bioanalysis anti-IL-1 antibodies

Cell line MRC-5 line is fibroblastic human cells, which produce IL-8 in response to IL-1α and IL-1β person dependent dose-dependent manner (see Dinarello C. A., K. Muegge, and S.K. Durum. 2000. Current Protocols in Immunology 6:1). Cells MRC-5 were cultured in MEM supplemented with 10% FBS and grown at 37°C in an incubator with 5% CO2. To determine the neutralizing activities of mAb against recombinant IL-1α and IL-1β person with different concentrations (0-10 μg/ml) mAb (50 ml) was added to a 96-well plate and were preincubator with 50 μl of rhIL-1a or rhIL-1b (10-50 PG/ml) for 1 hour at 37°C. Supernatants were collected, diluted and the concentration of IL-8 was measured with the help of open the ELISA using a standard ELISA kit for IL-8 (R& D Systems). The activity of the antibodies was determined by its ability to inhibit the production of IL-8 cells MRC-5.

Based Biacore analysis and bioanalysis MRC-5 identified a number of mouse antibodies anti-hIL-1a and anti-hIL-1b with high affinity, as shown in table 1 below:

Table 1
Generation and characterization of mouse anti-hIL-1a/b mAb
No. clone mAbSpecificityKD(M)IC50(M)
3D12.E3hIL-1α1,11E-096,70E-10
18F4.2C8hIL-1α5,78E-108,90E-11
6H3.1A4.3E11hIL-1α3,54E-102,40E-10
13F5.G5hIL-1β2,91E-106,00E-10
1B12.4H4hIL-1β2,13E-105,30E-10
6B12.4F6hIL-1β3,20E-10

Example 1.1.D: Cloning and sequencing of mouse monoclonal antibodies to IL-1α and IL-1β

Cloning and sequencing of genes of variable domains of the heavy chain (VH) and light chain (VL) of all anti-IL-1a/b-mAb, described in table 1, and additional antibodies was performed after isolation and purification of total RNA from each hybridoma cell line using reagent Trizol (Invitrogen) according to the manufacturer's instructions. Amplification as gene VH and VL gene was performed using oligonucleotides IgGVH and IgκVL from a set of primers mouse Ig (Novagen, Madison, WI) with a set of One-tube RT-PCR (Qiagen) according to the manufacturer's recommendation. DNA fragments derived from productive amplification, cloned in the vector pCR-TOPO (Invitrogen) according to the manufacturer's instructions. Then multiple clones VH and VL sequenced by the method of dideoxy-termination circuit using an ABI sequencing machine 3000 (Applied Biosystems, Foster City, CA). Gene-sequences of VL and VH of all mAb is shown below in table 2.

Table 2
Murine monoclonal antibodies capable of binding IL-1α or IL-1β man
ProteinID sequence Sequence
12345678901234567890
VH 3D12.E3SEQ ID NO.:1QIQLVQSGPELKKPGETVKISCKASGYTFRNYGMNWVKQAPGKDLKRMAWINTYTGESTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGIYYYGSSYAMDYWGQGTSVTVSS
VL 3D12.E3SEQ ID NO.:2NIQMTQTTSSLSASLGDRVTISCRASQDISNCLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTLPYAFGGGTKLEINR
VH 18F4.2C8SEQ ID NO.:3EVQLQQSGAELVKPGASVKLSCTASGLNIKDTYMHWLKQRPEQGLEWIGRIDPANGNAKYDPRFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGDGNFHFDYWGQGTTLTVSS
VL 18F4.2C8SEQ ID NO.:4DIVMTQSQRFMSTSVGDRVSVTCKASQNVGTNIAWYQQKPGQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSVDLAEYFCQQYTRYPLTFGGGTKLEIKR
VH 6H3.1A4.3E11SEQ ID NO.:5QVQLQQPGAELVRPGASVKLSCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSETLYSQKFKDTAILTVDKSSSTAYMQLSSLTSEDSAVYYCARYGFDYWGQGTTLTVSS
VL 6H3.1A4.3E11SEQ ID NO.:6QIVLTQSPALMSASPGEKVTMTCSASSSVNYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPYTFGGGTKLEMKR
VH 13F5.G5SEQ ID NO.:7QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTSYMQLSGLTSEDSAMYFCVRFPTGNDYYAMDYWGQGTSVTVSS
VL 13F5.G5SEQ ID NO.:8NIVLTQSPASLAVSLGQRATISCRASESVDSYGNSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNDPFTFGSGTKLEIKR
VH 1B12.4H4SEQ ID NO.:9QVHLKESGPGLVAPSQSLSITCTVSGFSLTDYGVSWIRQPPGKGLEWLGLIWGGGDTYYNSPLKSRLSIRKDNSKSQVFLKMNSLQTDDTAVYYCAKQRTLWGYDLYGMDYWGQGTSVTVSS
VL 1B12.4H4SEQ ID NO.:10ETTVTQSPASLSMAIGEKVTIRCITSTDIDVDMNWYQQKPGEPPKLLISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLSEDVADYYCLQSDNLPLTFGAGTKLELKR
VH 6B12.4F6SEQ ID NO.:11EVQLQQSGPELVKTGTSVKISCKASGYSFTGYYMHWVRQSHGKSLEWIGYISCYNGFTSYNPKFKGKATFTVDTSSSTAYIQFSRLTSEDSAVYYCARSDYYGTNDYWGQGTTLTVSS
VL 6B12.4F6SEQ ID NO.:12QIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAEDAATYYCQQRSTYPYTFGGGTKLEIKR

Example 1.2: Generation and characterization of chimeric antibodies, the mouse-man

All of the above mAb was turned into a Chimera (constant region) and expressed, purified and characterized to confirm activity and used as controls for subsequent analysis of the DVD-Ig. To turn 3D12.E3 in chimeric form 3D12.E3-VL amplified using PCR using primers P1 and P2; meanwhile, Ck gene (pBOS vector generated in the laboratory of the authors when ABC) amplified using primers P3 and P4. Both PCR reactions were performed according to standard methods and PCR methods. Two of the PCR product was purified is from the gel and used together in the form of overlapping matrix for subsequent overlapping PCR reactions using primers P1 and P4 in a standard PCR conditions. The final PCR product, chimeric light chain 3D12.E3-VL-hCk, was subcloned into the expressing vector mammals pEF6 TOPO (Invitrogen) TOPO-cloning according to the manufacturer's instructions. Table 3 shows the sequences of PCR primers:

Table 3
P1: 5' ATG GTG TCC ACA GCT CAG TTC C 3'SEQ ID NO.13
P2: 5' GC AGC CAC CGT ACG CCG GTT TAT TTC CAG 3'SEQ ID NO.14
P3: 5' CGT ACG GTG GCT GCA CCA TCT GTC 3'SEQ ID NO.15
P4: 5' TCA ACA CTC TCC CCT GTT GAA GC-3'SEQ ID NO.16

For the conversion of the heavy chain 3D12.E3 in chimeric form 3D12.E3-VH amplified using PCR using primers P5 and P6; meanwhile, Cγ1 gene (pBOS vector generated in the laboratory of the authors when ABC) amplified using primers P7 and P8. Both PCR reactions were performed according to standard methods and PCR methods. Two PCR product was purified from the gel and used together in the form of overlapping matrix for subsequent overlapping PCR reactions using primers P5 and P8 in standard PCR conditions. The final PCR product, chimeric light chain 3D12.E3-VH-hCγ1, who was klonirovali in expressing vector mammals pDNA3.1 TOPO (Invitrogen) according to the manufacturer's instructions. Table 4 shows the sequences of PCR primers:

Table 4
P5: 5' ATG GCT TGG GTG TGG ACC TTG C 3'SEQ ID NO.17
P6: 5' GGG CCC TTG GTC GAC GCT GAG GAG ACG GTG ACT GAG G 3'SEQ ID NO.18
P7: 5' GCG TCG ACC AAG GGC CCA TCG GTC TTC C 3'SEQ ID NO.19
P8: 5' TC ATT TAC CCG GAG ACA GGG AGA GGC 3'SEQ ID NO.20

Similarly, chimeric form 13F5.G5-VH-Cγ1 was generated using primers P21/P22 (VH) and P7/P8 (hCγ1) and cloned into the vector pcDNA3.1 TOPO, and chimeric form 13F5.G5-VL-Cκ generated using primers P23/P24 (VL) and P3/P4 (hCk) and cloned in the vector pEF6 TOPO. Table 5 shows the sequences of PCR primers:

Table 5
P21: 5' ATA GAA TGG AGC TGG GTT TTC CTC 3'SEQ ID NO.21
P22: 5' GGG CCC TTG GTC GAC GC TGA GGA GAC GGT GAC TGA 3'SEQ ID NO.22
P23: 5' ATG GTC CTC ATG TCC TTG CTG TTC 3'SEQ ID NO.23
P24: 5' GC AGC CAC CGT ACG CCG TTT TAT TTC CAG CTT TG-3'/td> SEQ ID NO.24

For expression of a chimeric Ab 13F5.G5-VL-Cκ and 13F5.G5-VH-γ1 was coexpression in COS using lipofectamine (Invitrogen) for 72 hours, the medium was collected and IgG was purified Protein a-chromatography. Similarly, 13F5.G5-VL-Cκ and 13F5.G5-VH-γ1 was coexpression in COS using lipofectamine (Invitrogen) for 72 hours, the medium was collected and IgG was purified Protein a-chromatography. Both purified chimeric Ab characterized using Biacore and MRC-5-bioanalysis to confirm activity. The results showed that these chimeric Ab showed similar affinity and activity with affinity and activity of the original murine mAb.

Example 1.3: Construction, expression and purification with dual variable domains of IL-1α/β-immunoglobulin (DVD-Ig)

The design used to generate a DVD-Ig capable of binding hIL-1α and hIL-1β, illustrated in figv.Briefly, the original mAb, which includes two high-affinity murine Ab, anti-hIL-1α (clone 3D12.E3) and anti-hIL-1β (clone 13F5.G5), was obtained by immunization of Balb/c mice with recombinant protein of IL-1α (rhIL-1α) and recombinant protein IL-1β (rhIL-1β), respectively. Genes VL/VH these two hybridoma clones were isolated using RT-PCR using primer set for mouse Ig (Novagen, Madison, WI). Genes VL/VH first turned into chimeric antibodies (const areas immunodeficiency is globulina person) to confirm activity and efficiency. For generation DVD1-Ig VH and VL 13F5.G5 poured directly from the N end of the VH and VL 3D12.E3, respectively (as shown in figv). DVD2-Ig was designed in a similar way, except that he had the linker between the two variable domains in the light chain (linker sequence ADAAP)and heavy chain (linker sequence AKTTPP). These sequences were chosen from N-all mouse sequences Ck and CH1. These linker sequences selected from N-all mouse sequences Ck and CH1 are the natural lengthening of the variable domains and find flexible conformation without significant secondary structures based on the analysis of several crystal structures of Fab. Detailed methods PCR cloning are described below.

Example 1.3.A: Molecular cloning of hIL-1a/b-DVD1-Ig

13F5.G5-VH amplified using PCR using primers P21 and P25; meanwhile, 3D12.E3-VH-hγ1 amplified using primers P14 and P8. Both PCR reactions were performed according to standard methods and PCR methods. Two of the PCR product was purified from the gel and used together in the form of overlapping matrix for subsequent overlapping PCR reactions using primers P21 and P8 in standard PCR conditions. The final PCR product, a heavy chain DVD1-Ig hIL-1a/bDVD1-VH-hγ1, was subcloned into the former is reservasi vector pcDNA3.1 mammalian TOPO (Invitrogen) according to the manufacturer's instructions. Table 6 shows the sequences of PCR primers:

Table 6
P14: 5' CAG ATC CAG TTG GTG CAG TCT GG-3'SEQ ID NO.25
P25: 5' - CAC CAA CTG GAT CTG TGA GGA GAC GGT GAC TGA GG-3'SEQ ID NO.26

To generate light chain hIL-1a/bDVD1-Ig 13F5.G5-VL amplified using PCR using primers P23 and P26; meanwhile, 3D12.E3-VH-hκ amplified using primers P16 and P4. Both PCR reactions were performed according to standard methods and PCR methods. Two of the PCR product was purified from the gel and used together in the form of overlapping matrix for subsequent overlapping PCR reactions using primers P23 and P4 in a standard PCR conditions. The final PCR product, light chain hIL-1a/bDVD1-Ig hIL-1a/bDVD1-VL-hκ, was subcloned into the expressing vector mammals pEF6 TOPO (Invitrogen) according to the manufacturer's instructions. Table 7 shows the sequence of PCR primers:

Table 7
P16: 5' AAT ATC CAG ATG ACA CAG ACT ACA TCC 3'SEQ ID NO.27
P26: 5' GTGT CAT CTG GAT ATT CCG TTT TAT TTC CAG CTT TG-3' SEQ ID NO.28

Example 1.3.B: Molecular cloning of hIL-1a/b-DVD2-Ig

13F5.G5-VH amplified using PCR using primers P21 and P17; meanwhile, 3D12.E3-VH-hγ1 amplified using primers P18 and P8. Both PCR reactions were performed according to standard methods and PCR methods. Two of the PCR product was purified from the gel and used together in the form of overlapping matrix for subsequent overlapping PCR reactions using primers P21 and P8 in standard PCR conditions. The final PCR product, a heavy chain DVD2-Ig hIL-1a/bDVD2-VH-hγ1, was subcloned into the expressing vector pcDNA3.1 mammalian TOPO (Invitrogen) according to the manufacturer's instructions. Table 8 shows the sequence of PCR primers:

Table 8
P17: 5' tgg ggg tgt cgt ttt ggc tga gg-3'SEQ ID NO.29
P18: 5' GCC AAA ACG ACA CCC CCA CAG ATC CAG TTG GTG CAG 3'SEQ ID NO.30

To generate light chain hIL-1a/bDVD2-Ig 13F5.G5-VL amplified using PCR using primers P23 and P19; meanwhile, 3D12.E3-VH-hκ amplified using primers P20 and P4. Both PCR reactions were performed according to standard methods and PCR methods. Two cont the KTA PCR was purified from the gel and used together in the form of overlapping matrix for subsequent overlapping PCR reactions using primers P23 and P4 in a standard PCR conditions. The final PCR product, light chain hIL-1a/bDVD2-Ig hIL-1a/bDVD2-VL-hκ, was subcloned into the expressing vector mammals pEF6 TOPO (Invitrogen) according to the manufacturer's instructions. Table 9 shows the sequence of PCR primers:

Table 9
P19: 5' TGG TGC AGC ATC AGC CCG TTT TAT TTC 3'SEQ ID NO.31
P20: 5' GCT GAT GCT GCA CCA AAT ATC CAG ATG ACA CAG 3'SEQ ID NO.32

The final sequence hIL-1a/bDVD1-Ig and hIL-1a/bDVD2-Ig described in table 10:

Table 10
Amino acid sequence of hIL-1α/β-DVD1-Ig and hIL-1α/β-DVD2-Ig
ProteinID sequenceSequence
12345678901234567890
The region of the protein
DVD HEAVY VARIABLE
hIL-1a/bDVD1-Ig
SEQ ID NO.:33QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTSYMQLSGLTSEDSAMYFCVRFPTGNDYYAMDYWGQGTSVTVSSQIQLVQSGPELKKPGETVKISCKASGYTFRNYGMNWVKQAPGKDLKRMAWINTYTGESTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGIYYYGSSYAMDYWGQGTSVTVSS
VH 13F5.G5SEQ ID NO.:7 QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNW
VKQRPGQGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTSYMQLSGLTSEDSA
MYFCVRFPTGNDYYAMDYWGQGTSVTVSS
LinkerNo
3D12.E3 VHSEQ ID NO.:1QIQLVQSGPELKKPGETVKISCKASGYTFRNYGMNWVKQAPGKDLKRMAWINTYTGESTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGIYYYGSSYAMDYWGQGTSVTVSS
CHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
DVD LIGHT VARIABLE
hIL-1a/bDVD1-Ig
SEQ ID NO.:35NIVLTQSPASLAVSLGQRATISCRASESVDSYGNSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPFTFGSGTKLEIKRNIQMTQTTSSLSASLGDRVTISCRASQDISNCLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTLPYAFGGGTKLEINRR
13F5.G5 VLSEQ ID NO.:8NIVLTQSPASLAVSLGQRATISCRASESVDSYGNSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPFTFGSGTKLEIKR
LinkerNo
3D12.E3 VLSEQ ID NO.:2NIQMTQTTSSLSASLGDRVTISCRASQDISNCLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTLPYAFGGGTKLEINR
CL SEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
DVD HEAVY VARIABLE
hIL-1a/bDVD2-Ig
SEQ ID NO.:37QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTSYMQLSGLTSEDSAMYFCVRFPTGNDYYAMDYWGQGTSVTVSSAKTTPPQIQLVQSGPELKKPGETVKISCKASGYTFRNYGMNWVKQAPGKDLKRMAWINTYTGESTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGIYYYGSSYAMDYWGQGTSVTVSS
13F5.G5 VHSEQ ID NO.:7QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTSYMQLSGLTSEDSAMYFCVRFPTGNDYYAMDYWGQGTSVTVSS
LinkerSEQ ID NO.:38AKTTPP
3D12.E3 VHSEQ ID NO.:1QIQLVQSGPELKKPGETVKISCKASGYTFRNYGMNWVKQAPGKDLKRMAWINTYTGESTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGIYYYGSSYAMDYWGQGTSVTVSS
CHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
DVD LIGHT VARIABLE HIL-1a/bDVD2-IgSEQ ID NO.:39NIVLTQSPASLAVSLGQRATISCRASESVDSYGNSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPFTFGSGTKLEIKRADAAPNIQMTQTTSSLSASLGDRVTISCRASQDISNCLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTLPYAFGGGTKLEINR
SEQ ID NO.:8NIVLTQSPASLAVSLGQRATISCRASESVDSYGNSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPFTFGSGTKLEIKR
LinkerSEQ ID NO.:40ADAAP
3D12.E3 VLSEQ ID NO.:2NIQMTQTTSSLSASLGDRVTISCRASQDISNCLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTLPYAFGGGTKLEINR
CLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example S: Expression and purification of hIL-1a/bDVD1-Ig

Heavy and light chain each construct was subcloned into the vector pcDNA3.1 TOPO and pEF6 TOPO (Invitrogen Inc.), respectively, and sequenced to ensure accuracy. Plasmids encoding the heavy and light chains each design, transtorno expressed using reagents of lipofectamine 2000 and 293-pectin, respectively, in COS cells, and 293 cells embryonic human kidney (American Type Culture Collection, Manassas, VA). Environment culture cells were collected 72 hours after transient transfection, and antibodies were purified using Protein a-chromatography (Pierce, Rockford, IL) according to the manufacturer's instructions. Ab was analyzed using SDS-PAGE and quantified using A280 and BCA (Pierce, Rockford, IL). Table 1 shows the levels of expression of hIL-1a/bDVD1-Ig and hIL-1a/bDVD2-Ig comparable with the levels of expression of a chimeric Ab, indicating that the DVD-Ig can be efficiently expressed in mammalian cells.

td align="center"> 64,380 (64,371)
Table 11
Expression and analysis of molecular weight of hIL-1a/bDVD-Ig
The expression level (ng/ml)Molecular weight (daltons)
COSFreestyle 293Light chainHeavy chainFull length
Control00
3D12.E3-Ch2788388623,69649,914147,220
13F5.G5-Ch3260356224,08449,518147,204
DVD1-Ig2988330035,797 (35,790)200,346 (200,521)
DVD2-Ig2433348636,222 (36,220)64,976 (64,973)202,354 (202,573)
The molecular mass of the light chain, heavy chain and full length DVD1-Ig and DVD2-Ig determined experimentally mass spectrometry is shown in parentheses

Example 1.4:Mass spectrometry and SEC analysis of hIL-1a/b DVD-Ig

For measurement of molecular weight (MW) of light and heavy chains of the DVD-Ig 10 ál of DVD-Ig (0.8 µg/µl) was restored 1,0M DTT solution (5 μl). Column protein PLRP-S, 8 µ, 4000 A, and 1×150 mm (Michrom BioResource, Auburn, MA) was used to separate the heavy and light chains of the DVD-Ig. Agilent HP1100 Capillary HPLC (Agilent Technologies Inc., Pala Alto, CA) was used with a mass spectrometer QSTAR (Applied Biosystems, Foster City, CA). The valco valve was set for 10 minutes to stop the flow of waste to MS for desalting of the sample. Buffer And contained 0.02% TFUK, 0,08% FA, 0.1% of ACN and 99.8% of HPLC-H2O. The buffer contained 0.02% TFUK, 0,08% FA, 0.1% of HPLC-H2O and 99.8% of ACN. The flow rate HPLC was 50 μl/min, and injection volume of sample was 8.0 ml of the column oven Temperature was set at 60°C, and the gradient separation was a: 5% b for 5 minutes; 5% B To 65% B over 35 minutes; 65% B-95% B for another 5 minutes, 95% B To 5% B for 5 mine is. Scan TOFMS was equal to from 800 to 2500 Amu, and the cycles were equal to 3600. To determine the MW full-size DVD-Ig was used cartridge (cartridge) Protein MicroTrap (Michrom BioResource, Auburn, MA) for desalting of the sample. HPLC-gradient represented: 5% B for 5 minutes; 5% B To 95% B over 1 min, and 95% B To 5% B for the remaining 4 minutes. Scan QSTAR TOFMS was from 2000 to 3500'ail, and cycles were equal to 899. All the raw MS data were analyzed using software Analyst QS (Applied Biosystems). For SEC analysis of the DVD-Ig of purified DVD-Ig and chimeric Ab in PBS, were applied to a column of Superose 6 10/300 G2, 300×10 mm (Amersham Bioscience, Piscataway, NJ). For the SEC used the device HPLC, model 10A (Shimadzu, Columbia, MD). All proteins was determined using UV detection at 280 nm and 214 nm. The elution was isocratic at the speed of a current of 0.5 ml/min To study the stability of the samples in the concentration range of 0.2-0.4 mg/ml in PBS were subjected to 3 cycles of freezing-thawing between -80°C and 25°C or incubated at 4°C, 25°C or 40°C for 4 weeks and 8 weeks with subsequent SEC-analysis.

DVD-Ig and chimeric Ab was purified Protein a-chromatography. Exit cleaning (3-5 mg/l) corresponded to determine the amount of hIgG expression environment for each protein. The composition and purity of the purified DVD-Ig and chimeric Ab was analyzed using SDS-PAGE as in not reducing and reducing conditions. In non reducing conditions each of h is four proteins migrated as a single band. Proteins DVD-Ig was detected magnitude MW more than chimeric Ab, as expected. In non reducing conditions each of the four proteins gave two bands, one heavy chain and one light chain. Again, heavy and light chain DVD-Ig were larger than the heavy and light chains of the chimeric Ab. SDS-PAGE showed that each DVD-Ig is expressed in the form of single particles, and heavy and light chain effectively mate with the formation of IgG-like molecules. The sizes of the heavy and light chains, and also full-length protein of two molecules DVD-Ig match their calculated molecular mass based on amino acid sequences (see table 11).

To determine the exact molecular weight of a DVD-Ig was used mass spectrometry. As shown in table 1 was experimentally determined molecular mass of each DVD-Ig, including the light chain, heavy chain and a full-sized protein, is consistent with the predicted value. For additional studies of the physical properties of the DVD-Ig in the solution used the method of exclusion (gel permeation) chromatography size (SEC) for the analysis of each protein. As chimeric Ab and DVD2-Ig was detected only peak that indicates the physical homogeneity in the form of Monomeric proteins. Chimeric Ab 3D12.E3 found a smaller physical size than the chimeric Ab 13F5.G5 that has indicated that A chimeric 3D12.E3 took more compact, globular form. DVD1-Ig was detected main peak, and the peak in the form of a shoulder on the right, suggesting that part of DVD1-Ig, perhaps, is in aggregate form in these buffer conditions.

Example 1.5:Stability analysisin vitrohIL-1a/bDVD-Ig

Physical stability of the DVD-Ig was tested as follows. Purified antibodies in the concentration range of 0.2-0.4 mg/ml in PBS were subjected to 3 cycles of freezing-thawing between -80°C and 25°C or incubated at 4°C, 25°C or 40°C for 4 weeks and 8 weeks, followed by analysis by the method of exclusion chromatography size (SEC) (see table 12).

Table 12

Analysis of stability in vitro hIL-1a/bDVD-Ig using SEC

The degree of aggregation and fragmentation are shown in percent, while the percentage of Ab represents the intact molecule

Agg: aggregate

Ab: the intact antibody

Frgm: fragments

Both chimeric Ab showed a minor degree of aggregation and fragmentation, normal, normal IgG molecules. DVD1-Ig found some aggregation on S after cleaning. In the analysis of the stability of DVD1-Ig was also discovered aggregation in PBS in a variety of conditions; however, the percentage of aggregated forms of DVD1-Ig did not increase during prolonged storage or at higher temperatures. The percentage of fragmented forms of DVD1-Ig was normal to the ohms range, a similar range of chimeric Ab 3D12.E3. In contrast, DVD2-Ig showed exceptional stability. No aggregation or fragmentation is not defined for DVD2-Ig in all tested conditions, and 100% DVD2-Ig was preserved in the form of intact Monomeric molecule.

Example 1.6:Definition antigennegative affinity of hIL-1a/bDVD-Ig

The binding kinetics of the DVD-Ig with rhIL1-α and rhIL1-β was determined by measurements on the basis of resonance of the surface plasmon with the help of the device Biacore 3000 (Biacore AB, Uppsala, Sweden) using HBS-EP (10 mm HEPES, pH 7,4, 150 mm NaCl, 3 mm EDTA and 0.005% surfactant P20) at 25°C. All chemicals were obtained from Biacore AB (Uppsala, Sweden) or otherwise from a different source, as described here. Approximately 5000 RU of goat specific polyclonal antibodies against the Fcγ fragment human IgG (Pierce Biotechnology Inc, Rockford, IL)diluted in 10 mm sodium acetate (pH 4.5), was immobilized directly on a biosensor chip category "research" CM5 using a standard set for aminosilane, in accordance with the instructions and procedures of the manufacturer at 25 mg/ml of unreacted particles on the surface of the biosensor blocked by ethanolamine. The surface modified carboxymethylcysteine in flow cell 2 and 4 was used as the reaction surface. Not modified carboxymethyl extra without goat antibodies against human IgG in flow cell 1 and 3 was used as the reference (reference) surface. For the kinetic analysis of the equations of velocities derived from the model binding 1:1 Langmuir, customized for both phases of the Association and dissociation of all ten injection (using a global analysis fit) using software Bioevaluation 4.0.1. Purified samples of the DVD-Ig was diluted in HEPES-buffered saline for engagement through specific goat antibodies against IgG Fc man of the reaction surface and injectively on the reaction matrices at a speed of a current of 5 ml/min. rate Constants for Association and dissociation, kon (M-1s-1) and koff (s-1), was determined with the continuous speed of a current of 25 ml/min. rate Constants were obtained by performing kinetic measurements of binding at ten different concentrations of antigen in the range of 1.25-1000 nm. Then the equilibrium constant of dissociation (M) the reaction between DVD-Ig and rhIL1α/β was calculated from the kinetic constants of the velocity according to the following formula: KD=koff/kon. Aliquots of samples rhIL1α/β was injectively also simultaneously on the control reference and the reaction CM-surface for registration and subtraction of any background nonspecific binding with the exception of the greater part of the change of the refractive index and the injection of noise (background). The surface was regenerated by two subsequent injection of 25 ml of 10 mm glycine (pH of 1.5) at a speed of DC 5 ml/min Surface of immobilienbank the th anti-Fc antibody is fully regenerated and saved them an exciting full capacity for twelve cycles. The apparent stoichiometry of the captured complex DVD-Ig rhIL1α/β was calculated under conditions of saturating binding (equilibrium stationary state) using the following formula:

The Biacore analysis showed chimeric Ab, with the values of the kinetics and affinity of binding to IL-1, similar to the corresponding values of hybridoma mAb, indicating that you have selected the correct sequence VL/VH (table III). General parameters of the binding of these two DVD-Ig with hIL-1α were similar, with values of the affinity of DVD-Ig, which is only 2-3 times less than the value of the affinity of chimeric Ab 3D12.E3. The binding affinity of DVD2-Ig with hIL-1β was slightly less than the affinity of the chimeric Ab 13F5.G5, but 3 times higher than the affinity of DVD1-Ig. The binding affinity of these two DVD-Ig with hIL-1 compared with the affinity of chimeric Ab against hIL-1 was similar, as shown by equation stoichiometry with IL-1. Both chimeric Ab, being bivalent monospecific, was associated with IL-1α and IL-1β on Biocore with the stoichiometry of 1.6 and 1.7, respectively. It is normal for IgG due to intermolecular obstacles in close immobilization of the antibody on the sensor chip (Biacore, resulting in a stoichiometry in the range 1.5-2.0. The stoichiometry of both DVD-Ig against hIL-1α and hIL-1β was similar to the stoichiometry of these two chimeric Ab, the decree is Wai, both DVD-Ig had a bivalent ability of binding to each antigen.

Table 13
Functional characteristics of the molecule anti-IL-1-DVD-Ig
Antigenkon
(M-1 s-1)
koff
(s-1)
Kd
(M)
StoichiometryActivity
IC50(M)
3D13.E3hIL-1α6,43E+057,13E-041,11E-092,06,70E-10
3D12.E3-ChhIL-1α4,12E+055,52E-041,34E-091,67,00E-10
DVD1-IghIL-1α3,70E+041,05E-042,83E-091,82,30E-09
DVD2-IghIL-1α7,35E+04 2,52E-043,42E-092,02,90E-09
13F5.G5hIL-1β2,13E+066,21E-042,91E-101,86,00E-10
13F5.G5-ChhIL-1β1,41E+066,54E-044,62E-101,75,30E-10
DVD1-IghIL-1α6,09E+051,59E-032,60E-091,53,10E-09
DVD2-IghIL-1β1,19E+069,50E-047,98E-101,81,60E-09
The affinity and stoichiometry were measured using Biacore; Activity (IC50) was measured using MRC-5-bioanalysis.

In addition, tetravalent linking the DVD-Ig of two specific antigens were also analyzed using Biacore (table 14). DVD-Ig fun first is whether through goat antibody against the Fc of human touch on the Biacore chip and the first antigen was injectively and watched the signal link. Then, when the DVD-Ig is saturated with the first antigen was injectively second antigen and watched the second signal. This was done by first injection of IL-1β, and then IL-1α or the first injection of IL-1α, and then IL-1β for DVD2-Ig. In any sequence were determined by dual binding activity. Similar results were obtained for DVD1-Ig. Thus, each DVD-Ig was able to bind both antigens simultaneously in the form of tetravalent molecules with dual specificity. As shown in table IV, the stoichiometry of both DVD-Ig with the first antigen, or hIL-1α, or hIL-1β was greater than 1.5, similar to the stoichiometry of the monospecific bivalent binding. After the second injection of antigen, although DVD-Ig was already occupied by the first antigen, the stoichiometry of both DVD-Ig against a second antigen (i.e. hIL-1α or hIL-1β) was from 1.0 to 1.3. Thus, the DVD-Ig capable of binding two molecules of IL-1α and two molecules of IL-1β. DVD-Ig was first captured through goat antibody against the Fc of human touch on the Biacore chip and the first was injectively first antigen and watched the signal link with the subsequent injection of the second antigen.

Table 14
The analysis of the stoichiometry of hIL-1a/b DVD-Ig in tetravalent with dual binding specificity to IL-1α/β
The reaction single is CA Stoichiometry
Captured Ab1st antigen2nd antigenhIL-1α:DVD-IghIL-1β:DVD-Ig
DVD1-Ig: 932hIL-1α: 190hIL-1β: 752,31,0
DVD1-Ig: 1092hIL-1β: 141hIL-1α: 1071,11,5
DVD2-Ig: 1324hIL-1α: 209hIL-1β: 1371,81,3
DVD2-Ig: 1184hIL-1β: 159hIL-1α: 1311,21,6

Example 1.7:The definition of functional homogeneity DVD-Ig

Because DVD2-Ig was purified Protein a-chromatography instead of target-specific affinity chromatography, any potential wrongly placed and/or incorrectly paired domains VL/VH, if present, can be estimated by studies linking against these 2 different antigens. This analysis of binding was performed exclusion liquid is Oh chromatography (SEC). DVD2-Ig, one or after a 120 minute incubation period at 37°C with IL-1α, IL-1β or IL-1α and IL-1β, in equimolar ratio, was applied on the column. Each antigen was passed separately as controls. The SEC results showed that DVD2-Ig was able to bind IL-1α and IL-1β in solution, and this binding was led to the displacement signal SEC, indicating the increase of dynamic size DVD2-Ig, when he was in complex with any antigen. This offset signal DVD2-Ig was 100%, not partial, suggesting that all molecules DVD2-Ig were able to bind the antigen. In the presence of both IL-1α and IL-1β, was additional offset signal DVD2-Ig, indicating that all the molecules DVD2-Ig were able to bind both antigens in a uniform manner. This experiment demonstrated that the DVD-Ig expressively as functionally homogeneous protein. This is significant because it demonstrates that the DVD-Ig can be obtained in the form of a homogeneous, separate functional particles, which differs from all previously described bespecifically, multispecificity and multivalent immunoglobulin-like and made of immunoglobulin molecules.

Example 1.8:Determination of biological activity of DVD-Ig

The biological activity of DVD-Ig was measured using bian is Lisa MRC-5. Cell line MRC-5 line is fibroblastic human cells, which produce IL-8 in response to IL-1α and IL-1β person dependent on dose. Cells MRC-5 were obtained from ATS and cultured in medium MEM, supplemented with 10% FBS, at 37°C in an incubator with 5% CO2. To determine the neutralizing activity of DVD-Ig against IL-1α or IL-1β person of 50 µl Ab (A-1E-12 M) in MEM/10%FBS was added to the 96-well plate and were preincubator with 50 μl of hIL-1α or hIL-1β (200 PG/ml) for 1 hour at 37°C, 5% CO2. Then cells MRC-5 at a concentration of 1E5/ml was added (100 μl) to all wells and the plates were incubated over night at 37°C in an incubator with 5% CO2. Supernatant collected and the production of IL-8 human was measured using a standard ELISA (R&D Systems, Minneapolis, MN). Neutralizing activity of DVD-Ig was determined by its ability to inhibit the production of IL-8.

As shown in table 13, both DVD-Ig were able to neutralize hIL-1α and hIL-1β. In accordance with a binding affinity with hIL-1α neutralizing activity of DVD1-Ig and DVD2-Ig against hIL-1α were also similar, ie 3 times smaller than the neutralizing activity of chimeric Ab (see table 3). In accordance with a binding affinity against hIL-1β neutralizing activity DVD2-Ig against hIL-1β was slightly less than the neutralizing activity of chimeric Ab 13F5.G5, but 3 times higher than neutralizing the asset is ity DVD1-Ig. Overall there was no significant decrease of the biological activities of molecules DVD-Ig compared to the original mAb.

To determine whether the DVD-Ig to inhibit the production of IL-8 in the presence of both IL-1α and IL-1β, equal amounts of hIL-1α and hIL-1β was added in the same culture system analysis of MRC-5. As DVD1-Ig and DVD2-Ig were able to inhibit the synthesis of IL-8 cells MRC-5 in the presence of both IL-1α and IL-1β, with activities similar to the activities of nanoanalysis, attended by only one cytokine (table 13). In this analysis, which was attended by both IL-1α and IL-1β, dual inhibitory activity DVD2-Ig (1.2 nm) was higher than the inhibitory activity of DVD1-Ig (2,2 nm).

Example 2:Analysis of the size of the linker and orientation of the variable domain of the molecule in the DVD-Ig

Designed additional molecules DVD-Ig with different initial mAb pairs, as shown in table 15. For each pair mAb generated four different designs of DVD-Ig: 2 with a short linker and 2 long linker, each in two different orientations of domains: a-b-C (alpha-beta-const domain) and b-a-C (beta-alpha constant domain). The linker sequence was produced from N-terminal sequence SK or CN domain, as follows:

A short linker: light chain: TVAAP; heavy chain: ASTKGP;

Long linker: light chain: TVAAPSVFIFPP; heavy chain: ASTKGPSVFPLAP.

the CE construction heavy and light chains were subcloned into expressing the pBOS vector and expressed in COS cells or 293 cells wild-type.

For the design of new clones DVD the variable domains of the two mAb, as the light chain and heavy chain, the first Sociali in tandem using overlapping PCR as described for hIL-1abDVD1-Ig and hIL-1abDVD2-Ig. Then these articulated pieces was subcloned into the pBOS vector using homologous recombination. Briefly, the vector was linearizable restriction cleavage (2 μg of vector pBOS-hCk was digested with restrictase FspAI and BsiWI in O+ buffer and 2 μg of vector pBOS-hCγ z, a non uncoupled FspAI and SaII O+ buffer). Split samples were subjected to electrophoresis on 1% agarose gel and frame fragment was purified in 50 ál of water. For homologous recombination and transformation of competent DH5α cells were thawed on ice and mixed with 20-50 ng articulated PCR product and 20-50 ng of linearized vector (each 50 μl of DH5α cells). The mixture is gently mixed and incubated on ice for 45 minutes followed by a heat shock at 42°C for 1 minute. Then add 100 µl of the environment SOC and incubated at 37°C for 1 hour. Culture for transformation was inoculable on cups LB/agar containing ampicillin, and incubated at 37°C for 18-20 hours. Allocated bacterial clones from which the purified DNA was subjected her to sequencing analysis. The final clones with confirmed sequence was cotranslationally (mating HV and LC the same is ary Ab) in COS cells or 293 for the expression and purification of Ab, as described previously.

Characteristics of purified proteins DVD-Ig are summarized in table 16. The left part of table 16 shows the specificity and affinity of binding and neutralizing activity 2 pairs of mAb used for the design of new molecules hIL-1a/bDVD-Ig. Antibodies 18F4.2C8 and 1B12.4H4 (see example 1.1.D) used to construct hIL-1a/bDVD3a-Ig, hIL-1a/bDVD4a-Ig, hIL-1a/bDVD3b-Ig and hIL-1a/bDVD4b-Ig. hIL-1a/bDVD3a-Ig and hIL-1a/bDVD4a-Ig were in the orientation of a-b-C, with a short linker and a long linker, respectively. hIL-1a/bDVD3b-Ig and hIL-1a/bDVD4b-Ig were in orientation b-a-C, with short and long linker, respectively. Antibodies 6H3.1A4 and 6B12.4F6 used to construct hIL-1a/bDVD5a-Ig, hIL-1a/bDVD6a-Ig, hIL-1a/bDVD5b-Ig and hIL-1a/bDVD6b-Ig. hIL-1a/bDVD5a-Ig and hIL-1a/bDVD6a-Ig were in the orientation of a-b-C, with short and long linker, respectively. hIL-1a/bDVD5b-Ig and hIL-1a/bDVD6b-Ig were in orientation b-a-C, with short and long linker, respectively. Molecular cloning of these additional hIL-1a/bDVD-Ig was performed using methods previously described for hIL-1a/bDVD1-Ig (see example 1.3), using overlapping PCR techniques. Amino acid sequences of these additional hIL-1a/bDVD-Ig presented in table 15.

Table 15
Amino acid sequence of the heavy chain and light chain of six DVD-Ig capable of binding IL-1α and IL-1β
ProteinSequence
The region of the proteinID sequence12345678901234567890
DVD HEAVY VARIABLE
hIL-1a/b DVD3a-Ig
SEQ ID NO.:41EVQLQQSGAELVKPGASVKLSCTASGLNIKDTYMHWLKQRPEQGLEWIGRIDPANGNAKYDPRFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGDGNFHFDYWGQGTTLTVSSASTKGPQVHLKESGPGLVAPSQSLSITCTVSGFSLTDYGVSWIRQPPGKGLEWLGLIWGGGDTYYNSPLKSRLSIRKDNSKSQVFLKMNSLQTDDTAVYYCAKQRTLWGYDLYGMDYWGQGTSVTVSS
18F4.2C8 VHSEQ ID NO.:3EVQLQQSGAELVKPGASVKLSCTASGLNIKDTYMHWLKQRPEQGLEWIGRIDPANGNAKYDPRFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGDGNFHFDYWGQGTTLTVSS
LinkerSEQ ID NO.:42ASTKGP
1B12.4H4 VHSEQ ID NO.:9QVHLKESGPGLVAPSQSLSITCTVSGFSLTDYGVSWIRQPPGKGLEWLGLIWGGGDTYYNSPLKSRLSIRKDNSKSQVFLKMNSLQTDDTAVYYCAKQRTLWGYDLYGMDYWGQGTSVTVSS
CHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
DVD LIGHT VARIABLE HIL-1a/b DVD3a-IgSEQ ID NO.:43DIVMTQSQRFMSTSVGDRVSVTCASQNVGTNIAWYQQKPGQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSVDLAEYFCQQYTRYPLTFGGGTKLEIKR TVAAPETTVTQSPASLSMAIGEKVTIRCITSTDIDVDMNWYQQKPGEPPKLLISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLSEDVADYYCLQSDNLPLTFGAGTKLELKRR
18F4.2C8 VLSEQ ID NO.:4DIVMTQSQRFMSTSVGDRVSVTCKASQNVGTNIAWYQQKPGQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSVDLAEYFCQQYTRYPLTFGGGTKLEIKR
LinkerSEQ ID NO.:44TVAAP
1B12.4H4 VLSEQ ID NO.:10ETTVTQSPASLSMAIGEKVTIRCITSTDIDVDMNWYQQKPGEPPKLLISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLSEDVADYYCLQSDNLPLTFGAGTKLELKR
CLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
DVD HEAVY VARIABLE
hIL-1a/b DVD3b-Ig
SEQ ID NO.:45QVHLKESGPGLVAPSQSLSITCTVSGFSLTDYGVSWIRQPPGKGLEWLGLIWGGGDTYYNSPLKSRLSIRKDNSKSQVFLKMNSLQTDDTAVYYCAKQRTLWGYDLYGMDYWGQGTSVTVSSASTKGPEVQLQQSGAELVKPGASVKLSCTASGLNIKDTYMHWLKQRPEQGLEWIGRIDPANGNAKYDPRFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGDGNFHFDYWGQGTTLTVSS
1B12.4H4 VHSEQ ID NO.:9QVHLKESGPGLVAPSQSLSITCTVSGFSLTDYGVSWIRQPPGKGLEWLGLIWGGGDTYYNSPLKSRLSIRKDNSKSQVFLKMNSLQTDDTAVYYCAKQRTLWGYDLYGMDYWGQGTSVTVSS
LinkerSEQ ID NO.:42ASTKGP
18F4.2C8 VHSEQ ID NO.:3EVQLQQSGAELVKPGASVKLSCTASGLNIKDTYMHWLKQRPEQGLEWIGRIDPANGNAKYDPRFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCRGDGNFHFDYWGQGTTLTVSS
CHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
DVD LIGHT VARIABLE HIL-1a/b DVD3b-IgSEQ ID NO.:46ETTVTQSPASLSMAIGEKVTIRCITSTDIDVDMNWYQQKPGEPPKLLISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLSEDVADYYCLQSDNLPLTFGAGTKLELKRTVAAPDIVMTQSQRFMSTSVGDRVSVTCKASQNVGTNIAWYQQKPGQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSVDLAEYFCQQYTRYPLTFGGGTKLEIKR
1B12.4H4 VLSEQ ID NO.:10ETTVTQSPASLSMAIGEKVTIRCITSTDIDVDMNWYQQKPGEPPKLLISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLSEDVADYYCLQSDNLPLTFGAGTKLELKR
LinkerSEQ ID NO.:44TVAAP
18F4.2C8 VLSEQ ID NO.:4DIVMTQSQRFMSTSVGDRVSVTCKASQNVGTNIAWYQQKPGQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSVDLAEYFCQQYTRYPLTFGGGTKLEIKR
CLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
DVD HEAVY VARIABLE hIL-1a/b DVD4a-IgSEQ ID NO.:47EVQLQQSGAELVKPGASVKLSCTASGLNIKDTYMHWLKQRPEQGLEWIGRIDPANGNAKYDPRFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGDGNFHFDYWGQGTTLTVSSASTKGPSVFPLAPQVHLKESGPGLVAPSQSLSITCTVSGFSLTDYGVSWIRQPPGKGLEWLGLIWGGGDTYYNSPLKSRLSIRKDNSSQVFLKMNSLQTDDTAVYYCAKQRTLWGYDLYGMDYWGQGTSVTVSS
18F4.2C8 VHSEQ ID NO.:3EVQLQQSGAELVKPGASVKLSCTASGLNIKDTYMHWLKQRPEQGLEWIGRIDPANGNAKYDPRFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGDGNFHFDYWGQGTTLTVSS
LinkerSEQ ID NO.:48ASTKGPSVFPLAP
1B12.4H4 VHSEQ ID NO.:9QVHLKESGPGLVAPSQSLSITCTVSGFSLTDYGVSWIRQPPGKGLEWLGLIWGGGDTYYNSPLKSRLSIRKDNSKSQVFLKMNSLQTDDTAVYYCAKQRTLWGYDLYGMDYWGQGTSVTVSS
CHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
DVD LIGHT VARIABLE HIL-1a/bDVD4a-IgSEQ ID NO.:49DIVMTQSQRFMSTSVGDRVSVTCKASQNVGTNIAWYQQKPGQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSVDLAEYFCQQYTRYPLTFGGGTKLEIKRTVAAPSVFIFPPETTVTQSPASLSMAIGEKVTIRCITSTDIDVDMNWYQQKPGEPPKLLISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLSEDVADYYCLQSDNLPLTFGAGTKLELKR
18F4.2C8 VLSEQ ID NO.:4DIVMTQSQRFMSTSVGDRVSVTCKASQNVGTNIAWYQQKPGQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSVDLAEYFCQQYTRYPLTFGGGTKLEIKR
LinkerSEQ ID NO.:50TVAAPSVFIFPP
1B12.4H4 VLSEQ ID NO.:10 ETTVTQSPASLSMAIGEKVTIRCITSTDIDVDMNWYQQKPGEPPKLLISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLSEDVADYYCLQSDNLPLTFGAGTKLELKR
CLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
DVD HEAVY VARIABLE hIL-1a/b DVD4b-IgSEQ ID NO.:51QVHLKESGPGLVAPSQSLSITCTVSGFSLTDYGVSWIRQPPGKGLEWLGLIWGGGDTYYNSPLKSRLSIRKDNSKSQVFLKMNSLQTDDTAVYYCAKQRTLWGYDLYGMDYWGQGTSVTVSSASTKGPSVFPLAPEVQLQQSGAELVKPGASVKLSCTASGLNIKDTYMHWLKQRPEQGLEWIGRIDPANGNAKYDPRFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGDGNFHFDYWGQGTTLTVSS
1B12.4H4 VHSEQ ID NO.:9QVHLKESGPGLVAPSQSLSITCTVSGFSLTDYGVSWIRQPPGKGLEWLGLIWGGGDTYYNSPLKSRLSIRKDNSKSQVFLKMNSLQTDDTAVYYCAKQRTLWGYDLYGMDYWGQGTSVTVSS
LinkerSEQ ID NO.:48ASTKGPSVFPLAP
18F4.2C8 VHSEQ ID NO.:3EVQLQQSGAELVKPGASVKLSCTASGLNIKDTYMHWLKQRPEQGLEWIGRIDPANGNAKYDPRFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGDGNFHFDYWGQGTTLTVSS
CHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
DVD LIGHT VARIABLE HIL-1a/b DVD4b-IgSEQ ID NO.:52ETTVTQSPASLSMAIGEKVTIRCITSTDDVDMNWYQQKPGEPPKLLISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLSEDVADYYCLQSDNLPLTFGAGTKLELKR TVAAPSVFIFPPDIVMTQSQRFMSTSVGDRVSVTCKASQNVGTNIAWYQQKPGQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSVDLAEYFCQQYTRYPLTFGGGTKLEIKR
1B12.4H4 VLSEQ ID NO.:10ETTVTQSPASLSMAIGEKVTIRCITSTDIDVDMNWYQQKPGEPPKLLISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLSEDVADYYCLQSDNLPLTFGAGTKLELKR
LinkerSEQ ID NO.:50TVAAPSVFIFPP
18F4.2C8 VLSEQ ID NO.:4DIVMTQSQRFMSTSVGDRVSVTCKASQNVGTNIAWYQQKPGQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSVDLAEYFCQQYTRYPLTFGGGTKLEIKR
CLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
DVD HEAVY VARIABLE hIL-1a/b DVD5a-IgSEQ ID NO.:53QVQLQQPGAELVRPGASVKLSCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSETLYSQKFKDTAILTVDKSSSTAYMQLSSLTSEDSAVYYCARYGFDYWGQGTTLTVSSASTKGPEVQLQQSGPELVKTGTSVKISCKASGYSFTGYYMHWVRQSHGKSLEWIGYISCYNGFTSYNPKFKGKATFTVDTSSSTAYIQFSRLTSEDSAVYYCARSDYYGTNDYWGQGTTLTVSS
6H3.1A4.3E11 VHSEQ ID NO.:5QVQLQQPGAELVRPGASVKLSCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSETLYSQKFKDTAILTVDKSSSTAYMQLSSLTSEDSAVYYCARYGFDYWGQGTTLTVSS
LinkerSEQ ID NO.:42ASTKGP
6B12.4F6 VHSEQ ID NO.:11EVQLQQSGPELVKTGTSVKISCKASGYSFTGYYMHWVRQSHGKSLEWIGYISCYNGFTSYNPKFKGKATFTVDTSSSTAYIQFSRLTSESAVYYCARSDYYGTNDYWGQGTTLTVSS
CHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
DVD LIGHT VARIABLE HIL-1a/b DVD5a-IgSEQ ID NO.:54QIVLTQSPALMSASPGEKVTMTCSASSSVNYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPYTFGGGTKLEMKRTVAAPQIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAEDAATYYCQQRSTYPYTFGGGTKLEIKR
6H3.1A4.3E11 VLSEQ ID NO.:6QIVLTQSPALMSASPGEKVTMTCSASSSVNYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPYTFGGGTKLEMKR
LinkerSEQ ID NO.:44TVAAP
6B12.4F6 VLSEQ ID NO.:12QIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAEDAATYYCQQRSTYPYTFGGGTKLEIKR
CLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
DVD HEAVY VARIABLE hIL-1a/b DVD5b-IgSEQ ID NO.:55EVQLQQSGPELVKTGTSVKISCKASGYSFTGYYMHWVRQSHGKSLEWIGYISCYNGFTSYNPKFKGKATFTVDTSSSTAYIQFSRLTSEDSAVYYCARSDYYGTNDYWGQGTTLTVSSASTKGPQVQLQQPGAELVRPGASVKLSCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSETLYSQKFKDTAILTVKSSSTAYMQLSSLTSEDSAVYYCARYGFDYWGQGTTLTVSS
6B12.4F6 VHSEQ ID NO.:11EVQLQQSGPELVKTGTSVKISCKASGYSFTGYYMHWVRQSHGKSLEWIGYISCYNGFTSYNPKFKGKATFTVDTSSSTAYIQFSRLTSEDSAVYYCARSDYYGTNDYWGQGTTLTVSS
LinkerSEQ ID NO.:42ASTKGP
6H3.1A4.3E11 VHSEQ ID NO.:5QVQLQQPGAELVRPGASVKLSCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSETLYSQKFKDTAILTVDKSSSTAYMQLSSLTSEDSAVYYCARYGFDYWGQGTTLTVSS
CHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
DVD LIGHT VARIABLE HIL-1a/b DVD5b-IgSEQ ID NO.:56QIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAEDAATYYCQQRSTYPYTFGGGTKLEIKRTVAAPQIVLTQSPALMSASPGEKVTMTCSASSSVNYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPYTFGGGTKLEMKR
6B12.4F6 VLSEQ ID NO.:12QIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAEDAATYYCQQRSTYPYTFGGGTKLEIKR
LinkerSEQ ID NO.:44TVAAP
6H3.1A4.3E11 VLSEQ ID NO.:6
CLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
DVD HEAVY VARIABLE hIL-1a/b DVD6a-IgSEQ ID NO.:57QVQLQQPGAELVRPGASVKLSCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSETLYSQKFKDTAILTVDKSSSTAYMQLSSLTSEDSAVYYCARYGFDYWGQGTTLTVSSASTKGPSVFPLAPEVQLQQSGPELVKTGTSVKISCKASGYSFTGYYMHWVRQSHGKSLEWIGYISCYNGFTSYNPKFKGKATFTVDTSSSTAYIQFSRLTSEDSAVYYCARSDYYGTNDYWGQGTTLTVSS
6H3.1A4.3E11 VHSEQ ID NO.:5QVQLQQPGAELVRPGASVKLSCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSETLYSQKFKDTAILTVDKSSSTAYMQLSSLTSEDSAVYYCARYGFDYWGQGTTLTVSS
LinkerSEQ ID NO.:48ASTKGPSVFPLAP
6B12.4F6 VHSEQ ID NO.:11EVQLQQSGPELVKTGTSVKISCKASGYSFTGYYMHWVRQSHGKSLEWIGYISCYNGFTSYNPKFKGKATFTVDTSSSTAYIQFSRLTSEDSAVYYCARSDYYGTNDYWGQGTTLTVSS
CHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
DVD LIGHT VARIABLE HIL-1a/b DVD 6a-IgSEQ ID NO.:58QIVLTQSPALMSASPGEKVTMTCSASSSVNYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPYTFGGGTKLEMKRTVAAPSVFIFPPQIVLTQSPAIMSASPGEKVTITCSSSSVSYMHWFQQKPGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAEDAATYYCQQRSTYPYTFGGGTKLEIKRR
6H3.1A4.3E11 VLSEQ ID NO.:6QIVLTQSPALMSASPGEKVTMTCSASSSVNYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPYTFGGGTKLEMKR
LinkerSEQ ID NO.:50TVAAPSVFIFPP
6B12.4F6 VLSEQ ID NO.:12QIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAEDAATYYCQQRSTYPYTFGGGTKLEIKR
CLSEQ ID NO.:36RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
DVD HEAVY VARIABLE hIL-1a/b DVD6b-IgSEQ ID NO.:59EVQLQQSGPELVKTGTSVKISCKASGYSFTGYYMHWVRQSHGKSLEWIGYISCYNGFTSYNPKFKGKATFTVDTSSSTAYIQFSRLTSEDSAVYYCARSDYYGTNDYWGQGTTLTVSSASTKGPSVFPLAPQVQLQQPGAELVRPGASVKLSCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSETLYSQKFKDTAILTVDKSSSTAYMQLSSLTSEDSAVYYCARYGFDYWGQGTTLTVSS
6B12.4F6 VHSEQ ID NO.:11EVQLQQSGPELVKTGTSVKISCKASGYSFTGYYMHWVRQSHGKSLEWIGYISCYNGFTSYNPKFKGKATFTVDTSSSTAYIQFSRLTSEDSAVYYCARSDYYGTNDYWGQGTTLTVSS
LinkerSEQ ID NO.:48ASTKGPSVFPLAP
6H3.1A4.3E11 VHSEQ ID NO.:5QVQLQQPGAELVRPGASVKLSCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSETLYSQKFKDTAILTVDKSSSTAYMQLSSLTSEDSAVYYCARYGFDYWGQGTTLTVSS
CH SEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
DVD LIGHT VARIABLE HIL-1a/b DVD6b-IgSEQ ID NO.:60QIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAEDAATYYCQQRSTYPYTFGGGTKLEIKRTVAAPSVFIFPPQIVLTQSPALMSASPGEKVTMTCSASSSVNYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPYTFGGGTKLEMKRR
6B12.4F6 VLSEQ ID NO.:12QIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAEDAATYYCQQRSTYPYTFGGGTKLEIKR
LinkerSEQ ID NO.:50TVAAPSVFIFPP
6H3.1A4.3E11 VLSEQ ID NO.:6QIVLTQSPALMSASPGEKVTMTCSASSSVNYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPYTFGGGTKLEMKR
CLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Characteristics of new designs DVD summarized in table 16. The affinity (Kd) and biological activity (IC50) was determined using Biacore and MRC-5-bioanalysis, respectively. Analysis of SDS-PAGE of all new DVD-proteins showed a normal pattern of migration in Vosstania is counteracted, and in not reducing conditions, like normal antibody and DVD1/2-Ig.

Table 16

Characterization of new molecules DVD-Ig produced from new pairs mAb

NA: neutralizing activity was not determined.

Functional characterization of new molecules DVD revealed that with any orientation DVD with a long linker functioned better than DVD with a short linker, in relation to binding and neutralization of both antigens. As for the DVD with long linkers, DVD with orientation b-a-C showed good binding to both antigens and good neutralization of both antigens, while the DVD with the orientation of the a-b-C showed good binding and neutralization of IL-1α and reduced binding and neutralization of IL-1β (for example, DVD4b against DVD4). Molecule DVD-Ig, DVD4b, bound and neutralized as IL-1α and IL-1β with sub-nm and fully maintained the characteristics of binding and neutralization of the original mAb.

Example 3:The generation of a DVD-Ig capable of binding IL-12 and IL-18

Molecules DVD-Ig capable of binding IL-12 and IL-18, designed as described above, using two of the original mAb, one against IL-12p40 person (AVT) and another against IL-18 person (AVT). Generated four different designs of anti-IL-12/18-DVD-Ig: 2 with a short linker and 2 long linker, each in two different Orient the operations domain: 12-18-and 18-12-C (table VI). The linker sequence produced from N-terminal sequence domain of human Cλ/κ or CH1, were as follows:

For structures DVD1218 (ABT874 has a Vλ):

light chain (λ): short linker: QPKAAP; long linker: QPKAAPSVTLFPP

heavy chain (γ1): short linker: ASTKGP; long linker: ASTKGPSVFPLAP

For structures DVD1812 (ABT325 has a VK):

light chain (κ): short linker: TVAAP; long linker: TVAAPSVFIFPP

heavy chain (γ1): short linker: ASTKGP; long linker: ASTKGPSVFPLAP.

All designs heavy and light chains were subcloned into expressing the pBOS vector and expressed in COS cells or 293 cells wild type, followed by purification Protein A-chromatography. Purified material was subjected to SDS-PAGE, and their profiles were similar to profiles DVD2-Ig.

In table 17 below describes the structure of the heavy chains and light chains, used for the expression of each protein anti-IL12/IL18-DVD-Ig.

Table 17
Constructs for protein expression of anti-IL12/IL18-DVD-Ig
Protein a DVD-IgThe design of the heavy chainThe design of the light chain
DVD1218SLDVD1218HC-SLDVD1218LC-SL
DVD121LL DVD1218HC-LLDVD1218LC-LL
DVD1812SLDVD1812HC-SLDVD1812LC-SL
DVD1812LLDVD1812HC-LLDVD1812LC-LL

Example 3.1.1:Molecular cloning of DNA structures for DVD1218SL and DVD1218LL

To generate designs heavy chain DVD1218HC-LL and DVD1218HC-SL VH domain ABT-874 PCR-amplified using primers: Primer 1 and Primer 2L or Primer 2S, respectively; meanwhile VH domain ABT-325 amplified using primers: Primer 3L or Primer 3S and Primer 4, respectively. Both PCR reactions were performed according to standard methods and PCR methods. Two of the PCR product was purified from the gel and used together as overlapping matrix for subsequent overlapping PCR reactions using Primer 1 and Primer 4 in standard PCR conditions. Products overlapping PCR was subcloned into the double-split Srf I and Sal I expressing vector mammals pBOS-hCγ1,z, non-a (Abbott) using the standard approach homologous recombination.

To generate designs light chain DVD1218LC-LL and DVD1218LC-SL VL domain ABT-874 PCR-amplified using primers: Primer 5 and Primer 6L or Primer 6S, with therefore, its; meanwhile, the VL-domain ABT-325 amplified using primers: Primer 7L or Primer 7S and Primer 8, respectively. Both PCR reactions were performed according to standard methods and PCR methods. Two of the PCR product was purified from the gel and used together as overlapping matrix for subsequent overlapping PCR reactions using Primer 5 and Primer 8 in standard PCR conditions. These products are overlapping PCR was subcloned into the double-split Srf I and NotI expressing vector mammals pBOS-hCk (Abbott) using the standard approach of homologous recombination. The primers used for these constructs are shown in table 18 below:

Table 18

A similar approach was used to generate DVD1812SL and DVD1812LL, as described below.

Example 3.1.2:Molecular cloning of DNA structures for DVD1812SL and DVD1812LL

To generate designs heavy chain DVD1812HC-LL and DVD1812HC-SL VH domain ABT-325 PCR-amplified using primers: Primer 1 and Primer 9L or Primer 9S, respectively; meanwhile VH domain ABT-874 amplified using primers: Primer 10L or Primer 10S and Primer 4, respectively. Both PCR reactions were performed according to standard methods and PCR methods. Two of the PCR product was purified from the gel and used together as overlapping matrix for subsequent overlapping PCR reactions using Primer 1 and Primer 4 in standard PCR conditions. These products are overlapping PCR was subcloned into the double-split Srf I and Sal I expressing vector mammals pBOS-hCγ1,z, non-a (Abbott) using the standard approach of homologous recombination. Sequences of primers are listed below.

To generate designs light chain DVD1812LC-LL and DVD1812LC-SL VL domain ABT-325 PCR-amplified using primers: Primer 11 and Primer 12L or Primer 12S, respectively; meanwhile VL domain ABT-874 amplified using primers: Primer 13L or Primer 13S and Primer 14, respectively. Both PCR reactions were performed according to standard methods and PCR methods. Two of the PCR product was purified from the gel and used together as overlapping matrix for subsequent overlapping PCR reactions using Primers 11 and Primer 14 in a standard PCR conditions. These products are overlapping PCR was subcloned into the double-split Srf I and NotI expressing vector mammals pBOS-hCk (Abbott) using the standard approach of homologous recombination. The primers used for these structures are given in table 19 below:

Table 19

The final DNA sequence for the eight designs heavy and light chains of anti-IL12/IL18-DVD-Ig are shown in table 20:

Table 20
Amino acid sequence binding proteins DVD, capable of binding IL-12 and IL-18ProteinID sequenceSequenceThe region of the protein12345678901234567890DVD HEAVY VARIABLE
DVD1218HC-SLSEQ ID NO.:83QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSSASTKGPEVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSSABT-874 VHSEQ ID NO.:84QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSSLINKERSEQ ID NO.:42ASTKGPABT-325 VHSEQ ID NO.:85EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSSCHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK DVD LIGHT VARIABLE
DVD1218LC-SLSEQ ID NO.:86MTWTPLLFLTLLLHCTGSLSQSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPALLFGTGTKVTVLGQPKAAPEIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKRABT-874 VLSEQ ID NO.:87QSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPALLFGTGTKVTVLGLINKERSEQ ID NO.:88QPKAAPABT-325 VLSEQ ID NO.:89EIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKRCLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDVD HEAVY VARIABLE
DVD1218HC-LLSEQ ID NO.:90QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSSASTKGPSVFPLAPEVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSSABT-874 VHSEQ ID NO.:84QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSSLINKERASTKGPSVFPLAPABT-325 VHSEQ ID NO.:85EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSSCHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDVD LIGHT VARIABLE
DVD1218LC-LLSEQ ID NO.:91QSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPALLFGTGTKVTVLGQPKAAPSVTLFPPEIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKRABT-874 VLSEQ ID NO.:87QSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPALLFGTGTKVTVLGLINKERSEQ ID NO.:92QPKAAPSVTLFPPABT-325 VLSEQ ID NO.:89EIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKRCLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSNRGEC DVD HEAVY VARIABLE
DVD1812HC-SLSEQ ID NO.:93EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSSASTKGPQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSSABT-325 VHSEQ ID NO.:85EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSSLINKERSEQ ID NO.:42ASTKGPABT-874 VHSEQ ID NO.:84QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSSCHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDVD LIGHT VARIABLE
DVD1812LC-SLSEQ ID NO.:94EIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKRTVAAPQSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPALLFGTGTKVTVLGABT-325 VLSEQ ID NO.:89EIVMTQSPATLSVSPGERATLSCRASEISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKR LINKERSEQ ID NO.:44TVAAPABT-874 VLSEQ ID NO.:87QSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPALLFGTGTKVTVLGCLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDVD HEAVY VARIABLE
DVD1812HC-LLSEQ ID NO.:95EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSSASTKGPSVFPLAPQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSSABT-325 VHSEQ ID NO.:85EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSSLINKERSEQ ID NO.:48ASTKGPSVFPLAPABT-875 VHSEQ ID NO.:84QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSSCHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK DVD LIGHT VARIABLE
DVD1812LC-LLSEQ ID NO.:96EIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKRTVAAPSVFIFPPQSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPALLFGTGTKVTVLGABT-325 VLSEQ ID NO.:89EIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKRLINKERSEQ ID NO.:50TVAAPSVFIFPPABT-874 VLSEQ ID NO.:87QSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPALLFGTGTKVTVLGCLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example 3.2.:Definition antigennegative affinity of IL-12/IL-18 DVD-Ig

Binding affinity of anti-IL-12/18-DVD-Ig in respect of hIL-12 and hIL-18 were determined using Biacore (table 21). Neutralizing activity against IL-18 were determined using KG-1 analysis (Konishi, K., et al.). Briefly, samples of IL-18 (at a final concentration of 2 ng/ml) was preincubator with DVD-Ig (in final concentrations of 0-10 mg/ml) at 37°C for 1 hour and then added to the cells KG-1 (3×106/ml) in RPMI medium containing 10 ng/ml hTNF, with sleduyushei incubation at 37°C for 16-20 hours. Cultural supernatant collected and the production of IFN-γ person in each sample was determined using ELISA (R&D Systems). Activity of inhibiting molecules DVD against IL-18, represented by the values of the IC50shown in table VI. To determine the activity of inhibiting molecules anti-IL-12/18-DVD against IL-12 used the analysis of IL-12-induced production of IFN-γ from activated PHA blast cells (D'andrea, A et al.) For the production of IFN-γ in human PHA-blast cells were incubated for 18 hours with IL-12 people. Submaximal stimulation (55-75% of maximum) was obtained with the concentration of IL-12 person, 200 PG/ml. Supernatant analyzed for IFN-γ using specific IFN-γ human ELISA (Endogen, Cambridge, MA). Neutralizing IL-12 DVD inhibit induced IL-12 production of IFN-γ. Neutralizing activity DVD was determined by measuring the concentration DVD required for inhibition of 50% the production of IFN-γ RNA-blast human cells, as shown in table 21.

Table 21

Characterization of molecules with anti-IL-18/IL-12-DVD-Ig

The affinity (KD) was determined using Biacore and activity (IC50) was determined using KG-1-bioanalysis (IL-18) and PBMC analysis (IL-12).

Table 21 shows the specificity and affinity of binding and neutralizing activity 2 full-size mAb person used on the I design molecules with anti-IL-12/IL-18-DVD. As shown in table 4, these mAb have high affinity and high neutralizing activity. Summarizing the characteristics of the structures of anti-IL-18/IL-12-DVD shown in table VI. Analysis of SDS-PAGE of all new DVD-proteins showed a normal pattern of migration in reducing and not reducing conditions, similar to conventional antibody and DVD1/2-Ig. SEC analysis showed that all molecules were normal, detecting peaks in the range of 200 KD. These Biacore binding agreement with neutralizing activity of these biological tests.

Example 3.3:The biological activity of anti-IL-12/IL-18 DVD-Ig in vivo

As IL-12 and IL-18 are required for producing optimal IFN-γ in response to various stimuli. The biological activity of anti-IL-12/IL-18 DVD-Ig in vivo was determined using the model huPBMC-SCID mouse. For this model, anti-IL-12 antibody (ABT-874), anti-IL-18 antibody (ABT-325) or anti-IL-12/anti-IL-18 DVD-Ig were injected with I.P. Pavlova. or i.v. (250 mg/mouse each) with subsequent migration of svezheokrashennym human PBMC (huPBMC) I.P. Pavlova. the SCID mice. Fifteen minutes later mice stimulated dried cellsStaphylococcus aureus(SAC) for the induction of IFNγ production person. Animals (n=7-8/group) were killed after 18-20 hours, and serum levels huIFNγ was determined by ELISA method. ABT 874 and ABT-325 inhibited SAC-induced IFNγ by approximately 70%, which represents a Maxim which aspects of the inhibition of IFNγ each connection for this model. However, treatment of mice ABT-874 + ABT-325 and anti-IL-12/anti-IL-18 DVD-Ig inhibited IFNγ production by almost 100%. These results suggest that the molecule anti-IL-12/anti-IL-18 DVD-Ig is functionally active in vivo.

Example 3.4Pharmacokinetic and pharmacodynamic studies with anti-IL-12/IL-18 DVD-Ig

General pharmacokinetic and pharmacodynamic profile of anti-IL-12/IL-18 DVD-Ig was similar to the profile of the original mAb in mice, i.e. was 73% bioavailability comparable with normal IgG. Similar pharmacokinetics, i.e. rapid clearance after a day 6-8 observed for other mAb (e.g., humans, rats etc), possibly due to reaction with anti-human IgG.

Male SD rats were administered doses of anti-IL-12/IL-18 DVD-Ig, comprising 4 mg/kg, or i.v., or s.c. The early part of this PK-curves looked normal and very similar to this part of the Republic of Kazakhstan-the curve of the other antibodies person. The exact half-life in both groups could not be determined because of the rapid clearance of DVD-Ig, starting with day 6. The sudden drop in the concentration DVD-Ig after 6 could be caused by a reaction RAHA. However, a similar profile was also observed for one of the original antibody (ABT-874), used for the design of the DVD-Ig in this particular experiment, as well as other monospecific antibodies person, investigated previously. On the basis of the concentration of DVD-Ig to day 6 was determined bi is the availability of DVD-Ig in both groups s.c. and i.v. Two of the three rats found 80-95% bioavailability, and the average bioavailability of these three rats was 73%.

Example 3.5:Physical/chemical characterization of anti-IL-12/anti-IL-18 DVD-Ig

Results physical and chemical characteristics obtained from 293 cells, purified Protein a-chromatography of anti-IL-12/anti-IL-18 DVD-Ig are summarized in table 22.

Table 22
Physical/chemical characterization of anti-IL-12/anti-IL-18 DVD-Ig
Tested parametersAnalysis/methodologyResults/comments
The affinity (Kd)
IL-12
IL-18
Biacore
Biacore
38 PM (65 PM for ABT-874)
622 PM (137 PM for ABT-325)
Activity (IC50)
IL-12
IL-18
Analysis of PHA-blast cells
Analysis of cells KG-1
7 PM (5 PM for ABT-874)
180 PM (300 PM for ABT-325)
M.WMSHC: 64130 (tiora. 64127)
LC: 36072 (tiora. 36072)
Amino acid sequenceSequencing - MSAll the same the s
Disulfide bondPeptide mappingAll 20 disulfide bonds coincided
The glycosylation profileSimilar to other full-size antibodies person, available in the laboratory - NGA2F and NGA1F observed in the form of basic shapes
Heterogeneity chargeCautionable (WCX-10)Homogeneity
PIcIEF9,42 (ABT-874: 9,46)
Dynamic sizeDSL7,69 nm (5,34 nm for ABT-325)
Purity/unitsSDS-PAGE
SEC
AUC
Homogeneity as reducing (~64 KD HC - and ~36 KD LC-strips), and not regenerating (one page) gels
One peak (~100%)observed immediately after purification Protein A-chromatography with subsequent SEC
~16-17% of the aggregates observed after 2 cycles of freezing-thawing, according to AUC

Stability (freeze/Otti is the W) SEC~5% aggregates after 2 cycles of freezing-thawing, increased to ~13% after an additional 10 cycles of freezing and thawing. The reason for this is not decided (because of the way specific to this sequence or LC-hybrid lambda/Kappa)
PK profileRat i.v. and s.c.Similar to the profile (or limited profile) of the original mAb.
BioavailabilityRat i.v. against s.c.Average 73%; In General similar to the bioavailability of the original mAb

Example 3.6:Generation of additional anti-12/anti-18 DVD-Ig (1D4.1-ABT325)

Designed additional molecule anti-IL-12/IL-18 DVD-Ig with a different source of anti-IL-12-mAb (clone No. 1D4.1), as shown in table 23. Design 1D4.1-ABT325-DVD-Ig was generated with a short linker produced from N-terminal sequence of the domain SK and SN person, as follows:

A short linker: light chain: TVAAP; heavy chain: ASTKGP.

All designs heavy and light chain was subcloned into expressing vector pBOS, expressed in COS cells or 293 cells wild-type and characterized as described above. 1D4.1-ABT325-DVD-Ig fully preserves the activity of the two is similar mAb (table 24).

Table 23
Amino acid sequence 1D4.1-ABT325-DVD-Ig
ProteinID sequenceSequence
The region of the protein12345678901234567890
1D4.1-ABT325 DVD-Ig HEAVY VARIABLESEQ ID NO.:114EVTLRESGPALVKPTQTLTLTCTFSGFSLSKSVMGVSWIRQPPGKALEWLAHIYWDDDKYYNPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARRGIRSAMDYWGQGTTVTVSSASTKGPEVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSS
1D4.1 VHSEQ ID NO.:115EVTLRESGPALVKPTQTLTLTCTFSGFSLSKSVMGVSWIRQPPGKALEWLAHIYWDDDKYYNPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARRGIRSAMDYWGQGTTVTVSS
LINKERSEQ ID NO.:99ASTKGP
ABT-325 VHSEQ ID NO.:85EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSS
CHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPNNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
1D4.1-ABT325 DVD-Ig LIGHT VARIABLESEQ ID NO.:116DIVMTQSPDSLAVSLGERATINCKASQSVSNDVAWYQQKPGQPPKLLIYYASNRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQDYNSPWTFGGGTKVEIKRTVAAPEIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKR
1D4.1 VLSEQ ID NO.:117DIVMTQSPDSLAVSLGERATINCKASQSVSNDVAWYQQKPGQPPKLLIYYASNRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQDYNSPWTFGGGTKVEIKR
LINKERSEQ ID NO.:44TVAAP
ABT-325 VLSEQ ID NO.:89EIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKR
CLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Table 24
Characterization of molecules 1D4.1-ABT325-DVD-Ig
mAbThe affinity (KdM)Activity (IC50M)
IL-12IL-18IL-12IL-18
1D4.11,20E-10N/A4,18E-10N/A
ABT325N/A1,91E-10N/A6,87E-11
1D4.1-ABT325 DVD-Ig1,33E-101,59E-102,17E-101,20E-10
The affinity (Kd) was determined using Biacore and activity (IC50) was determined using KG-1-bioanalysis (IL-18) and PBMC analysis (IL-12)

Example 4:Generation of anti-CD20/anti-CD3-DVD-Ig

Anti-CD20/anti-CD3-DVD-Ig was generated using a murine anti-CD20 human (clone 5F1) and anti-human CD3 (clone OKT3) of the original antibody. The original design included 2 DVD-Ig with different orientations of the domains. Anti-CD3/anti-CD20-DVD-Ig was designed in the sequence VCD3-linker-VCD20-constant domain, and anti-CD20/anti-CD3-DVD-Ig was designed in the sequence VCD20-linker-VCD3-constant domain. However, in the preliminary study linking cell surface anti-CD20-binding activity was decreased in the molecule anti-CD3/anti-CD20-DVD-Ig, even though anti-CD3-activity was preserved in this structure of the E. In contrast, both anti-CD3 and anti-CD20-binding activity was fully retained in the molecule anti-CD20/anti-CD3-DVD-Ig, indicating that in this case was the optimal orientation of the domains for the combination of these two mAb/targets in the format of a DVD-Ig. Thus, the design of the anti-CD20/anti-CD3-DVD-Ig was selected for further studies.

Anti-CD20/anti-CD3-DVD-Ig was generated in the form of chimeric Ig, i.e. constant region was constant region of a human. To analyze the binding line Jurkat T-cells and Raji b cells was blocked by human IgG and then were stained with mouse anti-hCD3-mAb ACT, mouse anti-hCD20-mAb 1F5 and anti-CD20/anti-CD3-DVD-Ig. The cells are then washed and stained with FITC-labeled goat antibody against mouse IgG (without cross-reactivity with anti-hIgG). Anti-CD20/anti-CD3-DVD-Ig bound as T-cells and b-cells, whereas CD3 and CD20-mAb bound only T-cells or b-cells, respectively. Amino acid sequence of CD20/CD3-DVD-Ig are described in table 25.

Table 25
Amino acid sequence of CD20/CD3-DVD-Ig
ProteinID sequenceSequence
District Bel is and 12345678901234567890
DVD HEAVY VARIABLE
CD20CD3DVD-Ig
SEQ ID NO.:97QVQLRQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSHYGSNYVDYFDYWGQGTTLTVSSAKTTAPSVYPLAPQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS
5F1 VHSEQ ID NO.:98QVQLRQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSHYGSNYVDYFDYWGQGTTLTVSS
LINKERSEQ ID NO.:99AKTTAPSVYPLAP
OKT3 VHSEQ ID NO.:100ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
CHSEQ ID NO.:34ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
CD20CD3DVD-Ig VARIABLE LIGHTSEQ ID NO.:101QIVLSQSPAILSASPGEKVTMTCRASSSLSFMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYFCHQWSSNPLTFGAGTKLELKRADAAPTVSIFPPQIVLTQSPAIMSASPGEKVTMTCSSSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR
5F1 VLSEQ ID NO.:102QIVLSQSPAILSASPGEKVTMTCRASSSLSFMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYFCHQWSSNPLTFGAGTKLELKR
LINKERSEQ ID NO.:103ADAAPTVSIFPP
OKT3 VLSEQ ID NO.:104QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR
CLSEQ ID NO.:36TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example 5:The generation of mIL-1α/β DVD-Ig

To study the key issues relating to pharmacokinetics, efficacy in vivo penetration into the tissue and immunogenicity of molecules DVD-Ig, mouse molecules DVD-Ig against murine IL-1α/β was designed, as described below.

Example 5.1:The design of mIL-1α/β DVD-Ig

Murine molecules DVD-Ig against IL-1α/β mouse was designed using two murine mAb against IL-1α/β mouse (9H10 and 10G11)generated from mice with double KO (knockout) IL-1α/β. Mouse monoclonal antibody against murine IL-1α and against murine IL-1β, was generated by immunization of mice with double KO (knockout) IL-1α/β murine IL-1α or murine IL-1β, respectively. One mouse mAb against murine IL-1α (clone N) and one mouse on the Noah mAb against murine IL-1β (clone 10G11) were selected and used to generate molecules mIL-1α/β DVD-Ig. Experienced different sizes linker and different orientation domains. Final functional molecules mIL-1α/β DVD-Ig was designed in the orientation of V(anti-mIL-1β)-linker-V(anti-mIL-1β)-mouse constant domain (Cγ2 and Cκ). Methods cloning, expression and purification were similar to techniques for hIL-1α/β DVD-Ig. Cloning mIL-1α/β DVD-Ig was performed using a similar overlapping PCR and homologous recombination, as described for hIL-1α/β-DVD3-Ig. Sequence mIL-1α/β DVD-Ig are shown below in table 26:

Table 26
Amino acid sequence of mIL-1α/β DVD-Ig
ProteinID sequenceSequence
The region of the protein12345678901234567890
mIL-1 is/is the DVD-Ig HEAVY VARIABLESEQ ID NO.:105EVQLQQSGPELVKPGTSVKMSCKTSGYTFTSYVMHWVKQKPGQGLEWIGYIIPYNDNTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRNEYYGSSFFDYWGQGTTLTVSSAKTTAPSVYPLAPQVILKESGPGILQPSQTLSLTCSFSGFSLSTYGTAVNWIRQPSGKGLEWLAQIGSDDRKLYNPFLKSRITLSEDTSNSQVFLKITSVDTEDSATYYCANGVMEYWGLGTSVTVSS
10G11 VHSEQ ID NO.:106EVQLQQSGPELVKPGTSVKMSCKTSGYTFTSYVMHWVKQKPGQGLEWIGYIIPYNDNTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRNEYYGSSFFDYWGQGTTLTVSS
LINKERSEQ ID NO.:99AKTTAPSVYPLAP
9H10 VHSEQ ID NO.:107QVILKESGPGILQPSQTLSLTCSFSGFSLSTYGTAVNWIRQPSGKGLEWLAQIGSDDRKLYNPFLKSRITLSEDTSNSQVFLKITSVDTEDSATYYCANGVMEYWGLGTSVTVSS
CHSEQ ID NO.:108AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK
mIL-1 is/is the DVD-Ig LIGHT VARIABLESEQ ID NO.:109DIQMTQSPASLSASVGETVTITCRGSGILHNYLVWYQQKQGKSPQLLVYSAKILADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPFTFGSGTKLEIKRADAAPTVSIFPPSIVMTQTPKFLLVSAGDRVTITCKASQSVNHDVAWYQQMPGQSPKLLIYFASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPYTFGGGTKLEIKR
10G11 VLSEQ ID NO.:110DIQMTQSPASLSASVGETVTITCRGSGILHNYLVWYQQKQGKSPQLLVYSAKILADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPFTFGSGTKLEIKR
LINKERSEQ ID NO.:111ADAAPTVSIFPP
9H10 VLSEQ ID NO.:112SIVMTQTPKFLLVSAGDRVTITCKASQSVNHDVAWYQQMPGQSPKLLIYFASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPYTFGGGTKLEIKR
CLSEQ ID NO.:113ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

Mouse mIL-1α/β DVD-Ig retained the affinity/activity in vitro against both IL-1α and IL-1β. Table 27 shows the characteristics of mAb N (anti-mIL-1α), 10G11 (anti-mIL-1β) and mIL-1α/β DVD-Ig.

Table 27
Description mDVD4-Ig
AntigenKD(M)IC50(M)
9H10mIL-1α1,73E-102,00E-10
10G11mIL-1β2,30E-103,70E-10
mIL-1α/βDVD-IgmIL-1α7,66E-102,00E-09
mIL-1β6,94E-108,00E-10

Example 5.2:In vivo activity of mIL-1α/β DVD-Ig on the model of arthritis

Therapeutic effects of anti-IL-1-alpha, anti-IL-beta, combined anti-IL-1-alpha/anti-IL-beta and mouse anti-IL-1alpha/beta-DVD4-Ig was evaluated in a murine model of collagen-induced arthritis, well known in this field. Briefly, male mice DBA-1 immunosera and bovine collagen type II in CFA introduction to the base of the tail. Mice were immunized again Simhasanam intraperitoneally (I.P. Pavlova) at day 21. After the occurrence of the disease in day 24-27 mice were selected and divided into separate groups of 10 mice each. Average rating arthritis control group and groups with antibodies against cytokines were compared at the beginning of processing. To neutralize IL-1 mice were injected with every second day 1-3 mg/kg anti-IL-1alpha-mAb, anti-IL-beta-mAb, a combination of anti-IL-1-alpha/anti-IL-beta-mAb or mouse anti-IL-1alpha/beta-DVD4-Ig intraperitoneally. Of the mice were examined three times per week visual external signs of arthritis in peripheral joints and defined assessment of disease activity.

Blockade of IL-1 in therapeutic mode effectively reduced the severity of arthritis, and anti-IL-beta-antibody was found higher efficiency (24% reduction in the average assessment of arthritis compared with the control group)than anti-IL-1-alpha antibody (10% reduction). An additional effect was observed between anti-IL-1-alpha and anti-IL-beta, with a 40% reduction in the average assessment of arthritis in mice treated with both anti-IL-1-alpha and anti-IL-beta-mAb. Unexpectedly, when the same dose level processing mDVD-Ig provided a 47% reduction in the average assessment of arthritis, demonstrating therapeutic efficacy in vivo mDVD-Ig. This efficiency, the inventors also observed in measurements puhani the joints on this model of the animal.

This invention incorporates by reference in their entirety ways well known in the field of molecular biology and drug delivery. These methods include, but are not limited to, the methods described in the following publications:

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Although we have described above a number of options and features, qualified specialists in this field will be clear that modifications and variations of the described embodiments and features may be made without deviating from this disclosure or the invention defined in the accompanying claims. Each of the mentioned in this description of the publications incorporated by reference.

1. Binding protein for binding one or more targets, containing the four polypeptide chains, where two polypeptide chains contain VD1-(X1)n-VD2-C-(x2)n (formula 1)where VD1 is the first variable domain of the heavy chain, VD2 means of the second variable domain of the heavy chain, With means CN domain, X1 means a polypeptide linker, provided that it does not t aetsa constant domain, and x2 means Fc region, and n is 0 or 1; and two polypeptide chains contain VD1-(X1) n-VD2-(formula 2)where VD1 is the first variable domain light chain, VD2 means of the second variable domain light chain, With mean CL domain, X1 means the linker, provided that it is not constant domain; and n is 0 or 1;
where the four polypeptide chains of the specified binding protein form four functional antigenspecific site.

2. Binding protein according to claim 1, where said target is selected from the group consisting of ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AIF1; AIG1; AKAR; AKAR; AMS; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; ARS; AROS; AR; AZGP1 (zinc-and-glycoprotein); V; V; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; WMR; WMR; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1; C19orf10 (IL27w); C3; C4; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6/JAB61); CCL1 (I-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a), CCL21 (MIP-2); SLC; Exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL3 (MIP-1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2 (mcp-1RB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR13); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBI1); CCR8 (CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38, CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20;CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21Wap1/Cip1); CDKN1B (p27Kip1); CDKN1C; CDKN2A (p16INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; chitinases; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28); CXCL1 (GRO1); CXCL10 (IP-10); CXCL11 (I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2; human epidermal growth factor; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FILE1 (EPSILON); FILE1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10 (C10); fiberglass; GSN (gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HIP1; histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-α; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFN; IFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; IL1A; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2 IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25,; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); IL7; IL7R; IL8; IL8R; IL8RB; IL8RB; IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC block BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (keratin 19); KRT2A; KRTHB6 (specific to hair keratin type II); LAMA5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionein-III); MTSS1; MUC1 (mucin); MYC; MYD88; NCK2; narukana; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NR0B1; NR0B2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS1; RGS13; RGS3; RNF110 (ZNF144); robo2 on; S100A2; SCGB1D2 (lipophilin); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial activating monocytes cytokine); SDF2; SERPINA1; SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-1); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Spr1); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TB4R2; TH; TSR; TDGF1; TECH; TGFA; TGFB1; TGFB1I1; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1); THBS2; THBS4; TRO; TIE (Tie-1); TIMP3; tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAIP2 (V); TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OH-ligand); TNFSF5 (CD40-l is English); TNFSF6 (FasL); TNFSF7 (D27-ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-W-ligand); TOLLIP; Toll-like receptors; TORA (topoisomerase Iia); TR; TRM1; TRM; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; version; VHL C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5/CCXCR1); YY1 and ZFPM2.

3. Binding protein according to claim 1, where the specified binding protein capable of binding two targets, where these two targets selected from the group consisting of CD138 and CD20; CD138 and CD40; CD20 and CD3; CD38 and CD138; CD38 and CD20; CD38 and CD40; CD40 and CD20; CD19 and CD20, CD-8 and IL-6; PDL-1 and CTLA-4; CTLA-4 and BTNO2; CSPGs and RGM A; IGF1 and IGF2; IGF1/2 and Erb2B; IL-12 and IL-18; IL-12 and TWEAK; IL-13 and ADAM8; IL-13 and CL25; IL-13 and 1b-beta; IL-13 and IL-25; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-9; IL-13 and LHR agonist; IL-13 and MDC; IL-13 and MIF; IL-13 and PED2; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and TARC; IL-13 and TGF-β; IL-1α and IL-1β; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; RGM a and RGM B; A and TNFα; TNFα and IL-12; TNFα and IL-12p40; TNFα and IL-13; TNFα and IL-15; TNFα and IL-17; TNFα and IL-18; TNFα and IL-beta; TNFα and IL-23; TNFα and MIF; TNFα and PEG2; TNFα and PGE4; TNFα and VEGF; and VEGFR and EGFR; TNFα and RANK-ligand; TNFα and Blys; TNFα and GP130; TNFα and CD-22; and TNFα and CTLA-4.

4. Binding protein according to any one of claims 1 to 3, where the specified binding protein capable of modulating a biological function of these one or more targets.

5. Binding protein according to any one of claims 1 to 3, where the specified binding protein able to neutralize the specified one or more targets.

6. Binding protein according to any one of claims 1 to 3, where the specified one or more targets selected from the group consisting of a cytokine,chemokine, protein is a cell surface enzyme and receptor.

7. Binding protein according to claim 6, where the indicated cytokine selected from the group consisting of lymphokines, monokines and polypeptide hormones.

8. Binding protein according to claim 7, where these cytokines are IL-1α and IL-1β.

9. Binding protein of claim 8, where the specified binding protein contains the amino acid sequence of the heavy chain DVD selected from the group consisting of SEQ ID NO. 33, SEQ ID NO. 37, SEQ ID NO. 41, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57 and SEQ ID NO. 59; and the amino acid sequence of the light chain DVD selected from the group consisting of SEQ ID NO. 35, SEQ ID NO. 39, SEQ ID NO. 43, SEQ ID NO. 46, SEQ ID NO. 49, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58 and SEQ ID NO. 60.

10. Binding protein according to claim 7, where these cytokines are TNF-α and IL-13.

11. Binding protein according to claim 7, where these cytokines are IL-12 and IL-18.

12. Binding protein according to claim 11, where the specified binding protein contains the amino acid sequence of the heavy chain DVD selected from the group consisting of SEQ ID NO. 83, SEQ ID NO. 90, SEQ ID NO. 93, SEQ ID NO. 95 and SEQ ID NO. 114; and the amino acid sequence of the light chain DVD selected from the group consisting of SEQ ID NO. 86, SEQ ID NO. 91, SEQ ID NO. 94, SEQ ID NO. 96 and SEQ ID NO. 116.

13. Binding protein according to claim 6, where the indicated chemokine selected from the group consisting of CCR2, CCR5 and CXCL-13.

14. Binding protein according to claim 6, where the protein of the cell surface is ti is the integrin.

15. Binding protein according to claim 6, where the proteins on the cell surface are CD-20 and CD3.

16. Binding protein according to clause 15, where the specified binding protein contains the amino acid sequence of the heavy chain DVD SEQ ID NO. 97 and amino acid sequence of the light chain DVD SEQ ID NO. 101.

17. Binding protein according to claim 6, where the specified enzyme selected from the group consisting of kinases and proteases.

18. Binding protein according to claim 6, where the specified receptor selected from the group consisting of a receptor of lymphokine, receptor Monokini and receptor polypeptide hormone.

19. Binding protein according to claim 6, where the specified binding protein has a rate constant of Association (Kon) to the said one or more targets selected from the group consisting of values of the specified constant comprising at least about 102M-1c-1; at least about 103M-1with-1; at least about 104M-1with-1; at least about 105M-1with-1and at least approximately 106M-1with-1as measured by the method of resonance of the surface plasmon.

20. Binding protein according to claim 6, where the specified binding protein has a rate constant of dissociation (Koff) in respect of the specified one or more targets selected from the group which, consisting of values of the specified constant, the components of at most approximately 10-3s-1; at most about 10-4s-1; at most about 10-5s-1and at most approximately 10-6s-1as measured by the method of resonance of the surface plasmon.

21. Binding protein according to claim 6, where the specified binding protein has a dissociation constant (KDin the said one or more targets selected from the group consisting of values of the specified constant, the components of at most approximately 10-7M; at most about 10-8M; at most about 10-9M; at most about 10-10M; at most about 10-11M; at most about 10-12M and at most approximately 10-13M

22. Conjugate binding protein for binding one or more targets containing binding protein described in any one of claims 1 to 21, and the agent selected from the group consisting of molecules immunoadhesin, agent imaging, therapeutic agent, and a cytotoxic agent.

23. Conjugate binding protein according to item 22, where the specified agent is the agent visualization selected from the group consisting of a radioactive label, an enzyme, a fluorescent label, luminescent the Noah marks, bioluminescent label, a magnetic label, and Biotin.

24. Conjugate binding protein according to item 23, where the specified agent visualization is a radioactive label selected from the group consisting of3H,14C,35S90Y99Tc111In125I131I177Lu,166But153Sm.

25. Conjugate binding protein according to item 22, where the specified agent is a therapeutic or cytotoxic agent selected from the group consisting of an antimetabolite, an alkylating agent, an antibiotic, a growth factor, cytokine, agent against angiogenesis, antimitoticescoy agent, anthracycline, toxin and apoptotic agent.

26. Binding protein according to claim 1, where the specified binding protein is a crystallized binding protein.

27. Crystallized binding protein on p, where the specified crystal is not containing a pharmaceutical carrier crystal controlled release.

28. Crystallized binding protein on p where the specified binding protein has a greater half-life in vivo than the soluble counterpart of the specified binding protein.

29. Crystallized binding protein on p where the specified binding protein retains biological activity.

30. The selected nucleic acid encoding a polypeptide chain, which is used for the floor is to be placed binding protein according to claim 1, where the encoded polypeptide chain contains a structure VD1-(X1)n-VD2-C-(X2)n, in which VD1 is the first variable domain of the heavy chain, VD2 means of the second variable domain of the heavy chain, With means CN domain, X1 means a polypeptide linker, provided that it is not a constant domain, and x2 means Fc region, and n is 0 or 1.

31. The selected nucleic acid encoding a polypeptide chain, which is used to obtain the binding protein according to claim 1, where the encoded polypeptide chain contains a structure VD1-(X1)n-VD2-C, where VD1 is the first variable domain light chain, VD2 means of the second variable domain light chain, With mean CL domain, X1 means a polypeptide linker, provided that it is not a constant domain, and n is 0 or 1.

32. A vector containing a selected nucleic acid according to item 30, the selected nucleic acid according p or a combination of the selected nucleic acid according to item 30 and the selected nucleic acid p, where specified, the vector is suitable for replication of these nucleic acids.

33. The expression vector containing the selected nucleic acid according to item 30, the selected nucleic acid according p or a combination of the selected nucleic acid according to item 30 and the selected nucleic acid p, where specified, the vector is suitable for ekspressirovali oksanacherkasova acids.

34. A vector containing
the first selected nucleic acid encoding a first polypeptide chain according to claim 1, containing VD1-(X1)n-VD2-C-(x2)n, in which VD1 is the first variable domain of the heavy chain, VD2 means of the second variable domain of the heavy chain, With means CN domain, X1 means the linker, provided that it is not a constant domain, x2 means Fc region, and n is 0 or 1; and
the second selected nucleic acid encoding a second polypeptide chain according to claim 1, containing VD1-(X1)n-VD2-C-(x2)n, in which VD1 is the first variable domain light chain, VD2 means of the second variable domain light chain, With mean CL domain, X1 means the linker, provided that it is not constant domain; and n is 0 or 1;
where specified, the vector is suitable for replication of these nucleic acids.

35. The expression vector containing
the first selected nucleic acid encoding a first polypeptide chain according to claim 1, containing VD1-(X1)n-VD2-C-(x2)n, in which VD1 is the first variable domain of the heavy chain, VD2 means of the second variable domain of the heavy chain, With means CN domain, X1 means the linker, provided that it is not a constant domain, x2 means Fc region, and n is 0 or 1; and
the second selected nucleic acid encoding a second polypeptide chain according to claim 1, containing VD1-(X1)n-VD2-C-(x2)n, in which VD1 first means is areally domain of the light chain, VD2 means of the second variable domain light chain, With mean CL domain, X1 means the linker, provided that it is not constant domain; and n is 0 or 1;
where specified, the vector is suitable for ekspressirovali these nucleic acids.

36. Vector on p or 35, where the specified vector selected from the group consisting of pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, pcDNA3.1 TOPO, pEF6 TOPO and a Shader with a.

37. A host cell containing a vector according p or 34, for replication of the specified vector.

38. A host cell containing the expression vector according p, 35 or 36, for ekspressirovali specified binding protein or part thereof, encoded by the specified vector.

39. A host cell according to clause 37 or § 38, which is a prokaryotic cell.

40. A host cell according to § 38, which is an E.coli cell.

41. A host cell according to § 38, which is a eukaryotic cell.

42. A host cell according to paragraph 41, where the specified eukaryotic cell is selected from the group consisting of cell protist, animal cells, plant cells and fungal cells.

43. A host cell according to paragraph 41, where the specified eukaryotic cell is an animal cell selected from the group consisting of mammalian cells, cells of birds and insect cells.

44. A host cell according to paragraph 41, which is the cell SNO.

45. A host cell according to paragraph 41, which is a COS cell.

46. A host cell according to § 42 where the specified gr is bcova a host cell is a yeast cell.

47. A host cell according to item 46, where this yeast cell is Saccharorayces cerevisiae.

48. A host cell according to item 43, where this insect cell is an Sf9 cell of an insect.

49. A method of obtaining a binding protein, involving the cultivation of the host cells described in any of PP-48, in a culture medium under conditions sufficient to produce the binding protein.

50. The method according to § 49, where 50-75% of the binding protein are tetravalent binding protein with dual specificity.

51. The method according to § 49, where 75-90% of the binding protein are tetravalent binding protein with dual specificity.

52. The method according to § 49, where 90-95% of the binding protein are tetravalent binding protein with dual specificity.

53. Binding protein according to claim 1, obtained by the method comprising culturing the host cell according to any one of p-48 in culture medium under conditions sufficient to produce the binding protein.

54. Pharmaceutical composition for treating or preventing diseases or disorders in an individual, containing an effective amount of the binding protein according to any one of claims 1 to 29 and 53, and a pharmaceutically acceptable carrier, where the specified binding protein binds to one or more targets that are associated with the decree of nami disease or disorder and which is harmful to the health of a specified individual.

55. The pharmaceutical composition according to item 54, optionally containing at least one additional agent.

56. The pharmaceutical composition according to § 55, where the specified additional agent selected from the group consisting of a therapeutic agent, agent visualization, a cytotoxic agent, angiogenesis inhibitors, kinase inhibitors, blockers molecules costimulation, blocking of adhesion molecules, anticytokine antibody or functional fragment, methotrexate, cyclosporine, rapamycin, FK506, detectable label or reporter, a TNF antagonist, an Antirheumatic agent, muscle relaxant, a narcotic agent, non-steroidal anti-inflammatory drug (NSAID), an analgesic agent, an anesthetic agent, a sedative, a local anesthetic means, a neuromuscular blocker, an antimicrobial agent, agent antipsoriatics, steroid, anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, immunosuppressant, growth hormone, hormone replacement drug, a radiopharmaceutical tools, antidepressant, antipsychotics, stimulant, anti-asthma drugs, beta-agonists, inhalation steroid, an epinephrine or analog, a cytokine, and a cytokine antagonist.

57. A method of treating sub is known for illness or violation by introducing to the subject a binding protein according to any one of claims 1 to 29 and 53 for binding one or more targets, associated with the specified disease or disorder.

58. The method according to § 57, where the specified disease or disorder selected from the group comprising rheumatoid arthritis, osteoarthritis, chronic juvenile arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin-dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis scleroderma, a disease graft-versus-host rejection of the transplant organs, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, graves ' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, purple's disease-Seleina, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acute transverse myelitis, horey chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignant disease, heart failure, myocardial infarction, Addison's disease, sporadic pluriglandular failure type is I, pluriglandular deficiency type II syndrome (Schmidt), (acute) respiratory distress syndrome of adults, alopecia, alopecia (alopecia) alopecia, the seronegative arthropathy, arthropathy, disease, Reiter, the psoriatic arthropathy, arthropathy, ulcerative colitis, enteropathic synovitis associated with Chlamydia, Yersinia and Salmonella the arthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis, atopic Allergy, autoimmune bullous disease, utricularia vulgaris, the leaf bladderwort, pemphigoid, linear IgA disease, autoimmune hemolytic anemia, positive hemolytic anemia of Coombs, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/British myalgic encephalitis, chronic mucocutaneous candidiasis, giant cell arteritis diagnostics, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, acquired immunodeficiency syndrome, a disease, related to the acquired immunodeficiency, hepatitis b, hepatitis C, common transient immunodeficiency (common transient hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, loss of ovarian function, premature loss of ovarian function, vibrationthe lung disease, cryptogenic fibrosing alveolitis, polyvocality is inoe interstitial lung disease, interstitial pneumonitis associated with disease of the connective tissue interstitial lung disease associated with disease mixed connective tissue of the lung disease associated with systemic sclerosis interstitial lung disease associated with rheumatoid arthritis interstitial lung disease associated with systemic lupus erythematosus is a disease of the lungs that is associated with dermatomyositis/polymyositis lung disease, associated with a disease, Sjogren's disease of the lungs that is associated with ankylosing spondylitis disease of the lungs, masculine diffuse lung disease associated with hemosiderosis disease of the lungs induced drug interstitial lung disease, fibrosis, radiation fibrosis, obliterative bronchiolitis, chronic eosinophilic pneumonia, lymphocytic infiltrative disease lung, post-infectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, autoimmune hepatitis type 1 (classical autoimmune or lupoid hepatitis), autoimmune hepatitis type-2 (hepatitis with anti-LKM-antibodies), autoimmune mediated hypoglycemia, insulin resistance type with papillary pigment dystrophy of the skin (acanthosis nigricans, hypoparathyroidism, acute immune disease, and solirovanie with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leukopenia, autoimmune neutropenia, renal disease NOS (BDU), glomerulonephritis, Lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS (BDU), autoimmunity sperm, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease syndrome?, pulmonary manifestation nadeznogo of polyarteritis, acute rheumatic fever, rheumatoid spondylitis, of still's disease, systemic sclerosis, Sjogren syndrome, Takayasu's disease/arteritis diagnostics, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary will Sekulic, vitiligo, acute liver disease, chronic liver disease, alcoholic cirrhosis, alcohol-induced liver injury, cholestasis, idiosyncratic liver disease, induced drug-induced hepatitis, non-alcoholic steatohepatitis, Allergy, streptococcal infection group B (GBS), the mental is such violations (e.g., depression and schizophrenia), Th type 2 - and Th type 1-mediated diseases, acute and chronic pain (different forms of pain), and cancers such as lung cancer, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer and hematopoietic malignant diseases (leukemia and lymphoma), abetalipoproteinemia, acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, atrial ectopic beats, complex, AIDS-dementia, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, deficiency of alpha-l-antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina, degeneration of anterior horn cells of the spinal cord, anti-CD3 therapy, antiphospholipid syndrome, allergic reactions to protivozachatochnye antibodies, aortic aneurysm and peripheral vascular, aortic dissection, arterial hypertension, arteriosclerosis, atrioventricular fistula, ataxia, atrial fibrillation (persistent or paroxysmal)the chatter is the W fibrillation, atrioventricular blockade, b-cell lymphoma, rejection, bone graft rejection of bone marrow transplant (BMT), interventricular blockade, Burkitt's lymphoma, burns, cardiac arrhythmia syndrome temporary cardiac arrest, cancer of the heart, cardiomyopathy, inflammatory response in the artificial circulation, graft rejection cartilage, cerebellar cortical degeneration, cerebellar violations, chaotic or multichrome atrial tachycardia associated with chemotherapy disorders, chronic miliitary leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic intoxication salicylate, cancer of the colon, congestive heart failure, conjunctivitis, contact dermatitis, pulmonary heart disease coronary artery disease Creutzfeldt-Jakob, negative in culture sepsis, cystic fibrosis, associated with therapy with cytokines violations, Boxing dementia, demyelinating diseases, Dengue haemorrhagic fever, dermatologic conditions, diabetes, diabetes mellitus, diabetic arteriosclerotic disease, a disease of the cells with diffuse Lewy, dilated congestive cardiomyopathy, narushenia.posle (subcortical) brain nuclei, down's syndrome in middle age, induced by drugs of movement disorders, disorders induced by drugs which block CNS dopamine receptors, sensitivity to drugs, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, infection with Epstein-Barr, erythromelalgia, extrapyramidal and cerebellar violations, family hematopoietically lymphocytic histiocytosis, implant rejection, fetal thymus, ataxia, functional peripheral arterial disorders, fungal sepsis, gas (anaerobic) gangrene, gastric ulcer, glomerulonephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas caused by intracellular organisms, leukemia reticuloendothelial disease Hallervorden-Spitze, Hashimoto's thyroiditis, hay fever, graft rejection hearts, hemochromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purple, bleeding, hepatitis a, arrhythmia beam GIS, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, allergic reactions, allergic pneumonitis, hypertension, hypokinetic movement disorders, di is gnostico of the hypothalamic-pituitary-adrenal axis, idiopathic Addison disease, idiopathic pulmonary fibrosis antibody-mediated cytotoxicity, weakness, childhood spinal muscular atrophy, inflammation of the aorta, influenza a, the effects of ionizing radiation, iridocyclitis/uveitis/optic neuritis, ischemic/reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, transplant rejection of kidney, infection with Mycobacterium Legionella, leishmaniasis, leprosy, damage kortiko-spinal system, fat edema, transplant rejection liver, lymph edema, malaria, malignant lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, migraine headache metabolic syndrome, idiopathic migraine headache, mitochondrial multi-system disturbance, disease mixed connective tissue, the monoclonal gammopathy, multiple myeloma, degeneration of multiple systems (Menzel, Dejerine-Thomas, Shi-Drager and Machado-Joseph), heavy pseudoparalysis the gravis, Mycobacterium avium intracellulare, the tubercle Bacillus, myelodysplasia syndrome, ischemic disorders of the myocardium, nasopharynx cancer, chronic lung disease of the newborn, nephritis, nephrosis, neurodegenerative diseases, neurogenic muscle and is Rafii, neurogenic fever, non-Hodgkins lymphoma, occlusion of the abdominal aorta and its branches, the occlusal blood disorders, OCC therapy, orchitis/epididymitis, procedure after treatment of orchitis/vasectomy, organomegaly, osteoporosis, transplant rejection, pancreas cancer, pancreatic cancer, paraneoplastic syndrome/hypercalcemia in malignant disease, graft rejection parathyroid glands, inflammatory disease of the renal pelvis, rhinitis, diseases of the pericardium, peripheral atherosclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia, pneumonia caused by Pneumocystis carinii, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, the monoclonal gammopathy syndrome and skin changes), postperfusion syndrome, potentiality syndrome, post-MI-cardiotomy, preeclampsia, progressive supranuclear palsy, primary pulmonary hypertension, radiation therapy, phenomenon, Raynaud's disease, Raynaud's disease, a disease of Resume, tachycardia with regular narrow QRS, renovaskulyarnoy (renal-vascular) hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, senile horey, senile dementia associated with calves Levi, seronegative arthropathies, shock, sickle the bottom-cell anemia, rejection of allograft skin syndrome skin changes, graft rejection of the small intestine, solid tumors, specific arrhythmias, spinal ataxia, spinal-cerebellar degenerations, streptococcal myositis, structural damage to the cerebellum, subacute sclerosing panencephalitis, syncope (syncope;), cardiovascular syphilis, systemic anaphylaxis, systemic inflammatory syndrome reactions, systemic juvenile rheumatoid arthritis, T-cell or FAB ALL (acute lymphoblastic leukemia), telangiectasia, obliterating thromboangiitis, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, allergic reactions type III, type IV allergies, unstable angina, uremia, urosepsis, urticaria, valvular heart disease, varicose veins, vasculitis, venous diseases, venous thrombosis, atrial (shimmer) ventricular fibrillation, viral and fungal infections, viral encephalitis/aseptic meningitis, viral hemophagocytosis syndrome, Wernicke-Korsakov, Wilson disease and xenograft rejection of any organ or any fabric.

59. The method according to § 57, where the aforementioned introduction of the subject to perform at least one method selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, NutriBar the registration number, intracapsular, vnutridomovogo, intracavitary, intracellular, intracerebellar, intracerebroventricular, the introduction of the inside of the colon/colon, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, vnutripoliticheskoi, intra-lungs, intrarectal, internalname, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesicular, bolus, vaginal, rectal, buccal, sublingual, intranasal and transdermal routes of administration.

60. Binding protein according to any one of claims 1 to 5, where the linker X1 is selected from the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO. 118); AKTTPKLEEGEFSEARV (SEQ ID NO. 119); AKTTPKLGG (SEQ ID NO. 120); SAKTTPKLGG (SEQ ID NO. 121); SAKTTP (SEQ ID NO. 122); RADAAP (SEQ ID NO. 123); RADAAPTVS (SEQ ID NO. 124); RADAAAAGGPGS (SEQ ID NO. 125); RADAAAA (G4S)4(SEQ ID NO. 126); SAKTTPKLEEGEFSEARV (SEQ ID NO. 127); ADAAP (SEQ ID NO. 40); ADAAPTVSIFPP (SEQ ID NO. 103); TVAAP (SEQ ID NO. 44); TVAAPSVFIFPP (SEQ ID NO. 50); QPKAAP (SEQ ID NO. 88); QPKAAPSVTLFPP (SEQ ID NO. 92); AKTTPP (SEQ ID NO. 38); AKTTPPSVTPLAP (SEQ ID NO. 128); AKTTAP (SEQ ID NO. 129); AKTTAPSVYPLAP (SEQ ID NO. 99); ASTKGP (SEQ ID NO. 42); ASTKGPSVFPLAP (SEQ ID NO. 48); GGGGSGGGGSGGGGS (SEQ ID NO. 130); GENKVEYAPALMALS (SEQ ID NO. 131); GPAKELTPLKEAKVS (SEQ ID NO. 132); and GHEAAAVMQVQYPAS (SEQ ID NO. 133).

61. Binding protein according to claim 3, where the two targets are IL-13 and IL-4.

62. Binding protein according to claim 3, where the decree is installed two targets are IL-1α and IL-1β.

63. Binding protein according to claim 3, where the two targets are TNFα and IL-17.

64. Binding protein according to claim 3, where the two targets are TNFα and VEGF.

65. Binding protein according to claim 3, where the two targets are VEGFR and EGFR.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to a recombinant cell of Ralstonia eutropha, designed to obtain 2-hydroxyisobutyric acid. The cell is transformed by a plasmid with the sequence SEQ ID NO: 2.

EFFECT: cell bearing said plasmid produces 2-hydroxyisobutyric acid in concentration of up to 0,72 mmol/kg.

4 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to immunology. Described are antibodies against VEGF, one on which contains complementary regions with amino acid sequences SEQ ID NO:1, 2, 3, 4, 6 and 7, another contains complementary regions with amino acid sequences SEQ ID NO:1, 2, 3, 5, 6 and 7, disclosed in description. Also described are polynucleotides, coding said antibodies; espression vectors, containing said polynucleotides, and host cells, intended for obtaining antibodies in accordance with the claimed invention. Claimed is method of obtaining antibodies against VEGF, which includes expression of vector in host cell and separation of antibody. Disclosed is method of obtaining immunocongugate of antibody against VEGF, which includes conjugation of antibody with drug or cytotoxic agent. Described is method of VEGF identification, which includes identification of complex VEGF-antibody against VEGF in biological sample. In addition, described are compositions for treatment of VEGF-associated disease, one of which contains efficient quantity of antibody against VEGF, and another - efficient quantity of polynucleotide, coding said antibody. Also disclosed are methods of: 1) treating tumour, cancer or VEGF-associated cell proliferative disease; 2) inhibition if angiogenesis in subject and 3) inhibition of vascular permeability; consisting in introduction to subject of efficient quantity of antibody against VEGF in accordance with claimed invention.

EFFECT: invention makes it possible to obtain antibodies against VEGF and apply them for treatment of VEGF-associated diseases.

41 cl, 16 dwg, 2 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly a method for specific collection of DNA molecules (DNA aptamers) with high affinity for a recombinant protein target. Said method involves synthesis of a single polypeptide chain of a recombinant protein containing a fragment of glutathione S-transferase, a protein target, a peptide sequence split by the B. Anthracis lethal factor, a peptide which is biotinylated in vitro under the action of an E.coli biotin-ligase enzyme, binding the obtained recombinant polypeptide with an oligonucleotide library and immobilising the protein on paramagnetic particles bearing glutathione, washing the paramagnetic particles with the immobilised polypeptide from unbound oligonucleotides in a liquid stream, splitting the protein target with the bound DNA aptamers from the surface of paramagnetic particles with the B. anthracis lethal factor, separating and amplifying the DNA sequence with affinity to the recombinant protein target in a polymerase chain reaction and obtaining a set of single-chain DNA aptamers that are specific to the protein target.

EFFECT: invention provides efficient production of DNA aptamers with high affinity for recombinant protein targets.

4 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers immunology and medicine. What is presented is an antibody for the recovery of the central nervous system, comprising an antigen-binding site that specifically binds to human Nogo A polypeptide or human NiG described by (SEQ ID NO: 2 and 3, respectively, presented in the description), wherein the antigen-binding site comprises: CDR-H1-6A3 (SEQ ID NO:8), CDR-H2-6A3 (SEQ ID NO:9) and CDR-H3-6A3 (SEQ ID NO:10); and CDR-L1-6A3 (SEQ ID NO:11), CDR-L2-6A3 (SEQ ID NO:12) and CDR-L3-6A3 (SEQ ID NO:13). There are also described a polynucleotide coding the above antibody; an expression vector comprising the above polynucleotide; and a host cell specified in bacterium, yeast or mammalian cell line comprising myeloma, hybridoma, or immortalised B-cell for producing the antibody according to the present invention. A pharmaceutical composition for the CNS recovery comprising an effective amount of the above antibody mixed with at least one acceptable carrier or solvent is also described. Using the polynucleotide, the expression vector or the host cell for the above pharmaceutical composition is also described. The invention enables producing the human Nogo A or NiG antibody effective in treating CNS injuries.

EFFECT: what is presented is a method for producing the above antibody involving the polynucleotide or vector expression in the host cell.

16 cl, 11 dwg, 9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry and discloses a polypeptide having antimicrobial activity, which includes an amino acid sequence having at least 70% identity with an amino acid sequence corresponding to positions 1-21 of SEQ ID NO:2. The invention also relates to structures of nucleic acids, vectors and host cells which include a polynucleotide which encodes the polypeptide according to the invention, as well as a method of producing such a polypeptide and use of the polypeptide to destroy microbe cells.

EFFECT: invention widens the range of antimicrobial polypeptides.

18 cl, 2 tbl, 2 ex

FIELD: chemistry.

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

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

17 cl, 2 tbl

FIELD: medicine.

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

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

86 cl, 20 tbl, 9 ex, 51 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and represents a method for preparing virus-like particles containing hepatitis C virus core, E1 and E2 structural antigens, and a method for particle purification. The method for preparing the virus-like particles involves Hansenula polymorpha yeast cell transformation with a recombinant plasmid containing one open reading frame carrying hepatitis C virus core-E1-E2 genes a direct expression of which leads to the formation of core, E1 and E2 antigens. That is followed by culturing and recovering the prepared particles. A method for virus-like particle purification involves bleaching of a damaged cell homogenate, diafiltration, concentration, precipitation with low pH, ion-exchange chromatography, sedimentation centrifugation in a density gradient, hydrophobic chromatography and gel chromatography.

EFFECT: invention enables preparing the high-yield virus-like particles applicable as a vaccine for preventing and/or treating the diseases caused by hepatitis C virus.

2 cl, 5 dwg, 5 ex

Anti-mif antibodies // 2509777

FIELD: chemistry.

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

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

22 cl, 14 dwg, 16 ex

FIELD: biotechnologies.

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

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

2 cl, 1 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers immunology and medicine. What is presented is an antibody for the recovery of the central nervous system, comprising an antigen-binding site that specifically binds to human Nogo A polypeptide or human NiG described by (SEQ ID NO: 2 and 3, respectively, presented in the description), wherein the antigen-binding site comprises: CDR-H1-6A3 (SEQ ID NO:8), CDR-H2-6A3 (SEQ ID NO:9) and CDR-H3-6A3 (SEQ ID NO:10); and CDR-L1-6A3 (SEQ ID NO:11), CDR-L2-6A3 (SEQ ID NO:12) and CDR-L3-6A3 (SEQ ID NO:13). There are also described a polynucleotide coding the above antibody; an expression vector comprising the above polynucleotide; and a host cell specified in bacterium, yeast or mammalian cell line comprising myeloma, hybridoma, or immortalised B-cell for producing the antibody according to the present invention. A pharmaceutical composition for the CNS recovery comprising an effective amount of the above antibody mixed with at least one acceptable carrier or solvent is also described. Using the polynucleotide, the expression vector or the host cell for the above pharmaceutical composition is also described. The invention enables producing the human Nogo A or NiG antibody effective in treating CNS injuries.

EFFECT: what is presented is a method for producing the above antibody involving the polynucleotide or vector expression in the host cell.

16 cl, 11 dwg, 9 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology, gene and protein engineering and specifically to recombinant plasmid DNA pG1-Rm7, which facilitates synthesis of hybrid protein G1-Rm7 in Escherichia coli cells, which is capable of biding the tumour necrosis factor and has bioluminescence of luciferase Renilla muelleri, where said plasmid DNA includes the nucleotide sequence SEQ ID NO: 1 and can be in medicine. The invention also relates to the protein pG1-Rm7 having molecular weight of 65.4 kDa, consisting of a single-strand anti tumour necrosis factor antibody, a GGSGGS peptide and modified luciferase Renalla muelleri and characterised by SEQ ID NO: 2.

EFFECT: invention enables to obtain a highly sensitive reporter for detecting a tumour necrosis factor via bioluminescent analysis.

2 cl, 4 dwg, 3 ex

FIELD: medicine.

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

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

86 cl, 20 tbl, 9 ex, 51 dwg

FIELD: biotechnologies.

SUBSTANCE: antibody against angiopoietin-2 (Ang-2) is presented; with that, the above antibody includes a variable area of heavy chain SEQ ID No: 7 (MEDI5) and a variable area of light chain, which has been chosen from SEQ ID No:3 (MEDI1); SEQ ID No:4 (MEDI2); SEQ ID No:5 (MEDI3); and SEQ ID No:6 (MEDI4). Nucleic acid coding the above antibody has been developed. The invention describes a composition for being used in inhibition of angiogenesis of cancer tumour of a mammal, which requires it; the above composition contains the extracted antibody in therapeutically effective amount and an excipient.

EFFECT: proposed variant antibodies have increased stability at storage, out of which they have been obtained, and are capable of competing with Tie-2 for binding with Ang-2.

12 cl, 17 dwg, 15 tbl, 13 ex

Anti-axl antibodies // 2506276

FIELD: chemistry.

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

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

23 cl, 20 dwg, 24 ex, 3 tbl

FIELD: biotechnologies.

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

EFFECT: improved activity and thermal stability.

14 cl, 6 dwg, 6 tbl, 11 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes a nanoantibody (a single-domain antibody) specifically bound to hemagglutinin of A-type flu virus H5N2 and suppressing infection of this virus, which is characterised with aminoacid sequence. Besides, a viral, an adenoviral, an adeno-associated and a lentiviral vector for expression of a nanoantibody and a composition for suppression of progress of infection of A-type flu virus H5N2 containing a nanoantibody and a viral vector are described.

EFFECT: invention can be further used in therapy of infection of A-type flu virus H5N2.

6 cl, 12 dwg, 18 ex, 1 tbl

FIELD: biotechnologies.

SUBSTANCE: invention proposes humanised anti-NKG2A antibody obtained from murine antibody Z270, which is characterised through amino-acid sequences of variable domains, and method of its obtainment. Besides, a pharmaceutical composition is described, which contains an antibody according to the invention, a treatment method and application of the antibody in production of a medicine to be injected into a patient who is a human being suffering the disorder chosen from cancer, virus disease, inflammatory disorder and autoimmune disorder.

EFFECT: invention can be further used in therapy.

14 cl, 20 dwg, 2 tbl, 16 ex

FIELD: biotechnologies.

SUBSTANCE: versions of an antibody or its fragment, which are specific in relation to β-amyloid protein, are proposed. Each version is characterised by the fact that it includes H- and L-chains, or areas VH and VL, each of which contains three corresponding CDR. The following is described: polypeptide VL, polypeptide VH, as well as coding nucleic acid, expression vector containing it and a cell carrying the vector, which are used for obtaining an antibody or its functional fragment. The following is proposed; a test kit, versions of pharmaceutical composition, a mixture to be used as a medicine based on the antibody or its functional fragment. Versions of the method used for production of an antibody are described: using a cell, nucleic acid or a vector. A composite preparation method, as well as an in vitro amyloid disease diagnostics method, a method for determination of a degree of loading with in vitro amyloidogenic patches, a method for curing or relief of actions of amyloid disease, which use an antibody or its functional fragment, are described. Inventions can be used in therapy and diagnostics of Alzheimer disease and other enlisted amyloid diseases.

EFFECT: proposed inventions provide new antibodies that bind the epitope contained in the area of 12-23 protein αβ1-42; with that, residues 15-20 have a fundamental importance.

44 cl, 18 dwg, 9 tbl, 16 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a humanized human monoclonal CD19 antibody prepared of an HB12B antibody, or a fragment thereof characterised by amino acid sequences of variable domains. Also, there are presented nucleic acids coding polypeptides having the sequences of the variable domains, and a cell expressing the antibody under the invention, and a pharmaceutical composition, and a method for treating a B-cell diseases or disorders in a human.

EFFECT: invention can find further application in treating various CD19-associated diseases, including autoimmune diseases, and preventing or treating the graft-versus-host disease (GVHD), and the humoral rejection and post-transplantation lymphoproliferative disorder in a human graft recipient.

21 cl, 45 dwg, 40 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to a method for obtaining an antibody, the pharmacokinetic properties of which have been changed at maintaining antigen-binding activity of a variable area, which provides for the following stages: (a) obtaining antibodies in which there has been modified a charge of amino-acid residues chosen from amino-acid residues in positions 31, 61, 62, 64 and 65 of the variable area of a heavy chain and in positions 24, 27, 53, 54 and 55 of the variable area of a light chain in compliance with numbering as per Kabat system, where modification of the charge of amino-acid residues leads to the change of 1.0 or more at a theoretical isoelectric point of the variable area of the antibody, and (b) extracting an antibody with stored antigen-binding activity from antibodies obtained at stage (a).

EFFECT: invention allows effective change in pharmacokinetic properties of an antibody, thus maintaining its antigen-binding activity.

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