JP2016020349A - Dual variable domain immunoglobulin and uses thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide engineered multivalent and multispecific binding proteins, production methods, and particularly methods of their uses in the prevention and/or treatment of acute and chronic inflammatory and other diseases.SOLUTION: The invention provides a binding protein comprising a polypeptide chain, the polypeptide chain comprising VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X1 is an amino acid or polypeptide, X2 is an Fc region, and n is 0 or 1.SELECTED DRAWING: Figure 1

Description

  The present invention relates to multivalent and multispecific binding proteins, methods for their production, and in particular to their use in the prevention and / or treatment of acute and chronic inflammation, cancer and other diseases.

  Processed proteins such as multispecific antibodies capable of binding to two or more antigens are known in the art. Such multispecific binding proteins can be made using cell fusion, chemical ligation or recombinant DNA techniques.

  Bispecific antibodies are based on somatic cell fusion of two different hybridoma cell lines that express mouse monoclonal antibodies with the desired specificity of the bispecific antibody (Milstein, C. and AC Cuello). , Nature, 1983.305 (5934): p.537-40). Due to the random pairing of two different Ig heavy and light chains in the resulting hybrid-hybridoma (or quadroma) cell line, up to 10 different immunoglobulin species are generated, only one of which functions Bispecific antibody. The presence of mispaired by-products and a greatly reduced production rate means that complex purification operations are required.

  Bispecific antibodies can be produced by chemical ligation of two different mAbs (see Staerz, UD, et al., Nature, 1985. 314 (6012): p. 628-31). . This approach does not give a uniform preparation. Other approaches have used chemical linkage of two different monoclonal antibodies or smaller antibody fragments (see Brennan, M., et al., Science, 1985.229 (4708): p. 81-3). Another method is to pair the two parent antibodies with a heterobifunctional crosslinker, but since the reaction of the crosslinker with the parent antibody is not site directed, preparation of the resulting bispecific antibody There is a problem that there is significant molecular non-uniformity in the object. In order to obtain a more homogeneous preparation of bispecific antibodies, two different Fab fragments have been chemically cross-linked at their hinge cysteine residues in a site-directed manner (Glennie, MJ. , Et al., J Immunol, 1987. 139 (7): p.2367-75). However, this method results in Fab'2 fragments and not complete IgG molecules.

  A wide variety of other recombinant bispecific antibody formats have recently been developed (see Kriangum, J., et al., Biomol Eng, 2001. 18 (2): p. 31-40). Among them, tandem single chain Fv molecules and diabodies and various derivatives thereof are the most widely used formats for the construction of recombinant bispecific antibodies. In general, the construction of these molecules begins with two single-chain Fv (scFv) fragments that recognize different antigens (Economides, AN, et al., Nat Med, 2003.9 (1): p. 47-52). A tandem scFv molecule (taFv) is a simple format that simply connects two scFv molecules with an additional peptide linker. The two scFv fragments present in these tandem scFv molecules form separate folding entities. A variety of linkers can be used to connect two scFv fragments and linkers having a length of up to 63 residues (Nakanishi, K., et al., Annu Rev Immunol, 2001. 19: p. 423-74). Parental scFv fragments can usually be expressed in soluble form in bacteria, but tandem scFv molecules are often observed to form insoluble aggregates in bacteria. Therefore, the use of refolding protocols or mammalian expression systems is generally applied to create soluble tandem scFv molecules. In a recent study, in vivo expression of tandem scFv induced against CD28 and anti-malignant melanoma-associated proteoglycans by transgenic rabbits and cattle has been reported (Gracie, IA, et al., J Clin. Invest, 1999.104 (10): p. 1393-401). In this construct, the two scFv molecules were connected by a CH1 linker and a serum concentration of up to 100 mg / L of bispecific antibody was found. A variety of strategies have been used to allow soluble expression in bacteria, including using a central linker with varying domain order or varying length or flexibility. Currently, several studies have reported the expression of soluble tandem scFv molecules in bacteria using very short Ala3 linkers or long linkers rich in glycine / serine (Leung, BP, et al. al., J Immunol, 2000.164 (12): p.6495-502; Ito, A., et al., J Immunol, 2003.170 (9): p.4802-9; al., J Neuroimmunol, 2002.125 (1-2): p.134-40). In a recent study, a tandem scFv repertoire containing a randomized central linker with a length of 3 or 6 residues to concentrate molecules produced in soluble and active form in bacteria. Of phage display was used. This approach resulted in the isolation of a preferred tandem scFv molecule with a 6 amino acid residue linker (see Arndt, M. and J. Krauss, Methods MoI Biol, 2003. 207: pp. 305-21). It is unclear whether this linker sequence is a general solution for soluble expression of tandem scFv molecules. Nevertheless, this study shows that phage display of tandem scFv molecules in combination with site-directed mutagenesis enriches these molecules (these molecules can be expressed in active form in bacteria). Showed that it is a powerful tool for.

  Bispecific diabody (db) uses a diabody format for expression. Diabodies are made from scFv fragments by reducing the length of the linker connecting the VH and VL domains to about 5 residues (Peipp, M. and T. Valerius, Biochem Soc Trans, 2002.30 ( 4): See p.507-11.) This reduction in linker size facilitates dimerization of the two polypeptide chains by cross-pairing of the VH and VL domains. A bispecific diabody can be used to link two polypeptide chains within the same cell, with either the structures VHA-VLB and VHB-VLA (VH-VL configuration) or VLA-VHB and VLB-VHA (VL-VH configuration). It is produced by expressing. A variety of different bispecific diabodies have been created in the past, many of which can be expressed in soluble form in bacteria. However, recent comparative studies have shown that the orientation of variable domains can affect the expression and formation of active binding sites (Mack, M., G. Riethmuller, and P. Kufer, Proc Natl. Acad Sci USA, 1995. 92 (15): p.7021-5). Nevertheless, soluble expression in bacteria represents an important advantage over tandem scFv molecules. However, because two different polypeptide chains are expressed in a single cell, an inactive homodimer can be produced along with an active heterodimer. This necessitates further purification steps to obtain a uniform preparation of the bispecific diabody. One approach to force the generation of a bispecific diabody is the creation of a knob-in-hole diabody (Holliger, P., T. Prospero, and G. Winter, Proc Natl Acad Sci USA, 1993). 90 (14): pp. 6444-8.18). This was shown for the bispecific diabody induced against HER2 and CD3. Replacing Val37 with Phe and Leu45 with Trp introduces a large knob in the VH domain that mutates Phe98 to Met and mutates Try87 to Ala in either the anti-HER2 or anti-CD3 variable domains To create a complementary hole in the VL domain. By using this approach, bispecific diabody production can be increased from 72% by the parent diabody to more than 90% by the knob-in-hole diabody. Importantly, the production rate was only slightly reduced as a result of these mutations. However, a decrease in antigen binding activity was observed for several constructs analyzed. Thus, this rather elaborate approach requires analysis of various constructs to identify mutations that produce heterodimeric molecules with unchanged binding activity. Furthermore, such an approach requires mutated modification of immunoglobulin sequences in the constant region, thus creating non-prototype and non-native forms of antibody sequences, which have increased immunogenicity, poor in vivo stability. And can result in undesirable pharmacokinetics.

  Single-stranded diabody (scDb) is another strategy to improve the formation of bispecific diabody-like molecules (Holliger, P. and G. Winter, Cancer Immunol Immunother, 1997. 45 (3-4). ): P.128-30; see Wu, AM, et al, Immunotechnology, 1996. 2 (1): p.21-36). A bispecific single chain diabody is made by connecting two dibody-forming polypeptide chains with an additional central linker having a length of about 15 amino acid residues. Thus, all molecules having a molecular weight corresponding to a monomeric single-stranded diabody (50-60 kDa) are bispecific. Several studies have shown that bispecific single-stranded diabodies are expressed in bacteria in a soluble and active form, and most of the purified molecules exist as monomers (Holliger). , P. and G. Winter, Cancer Immunol Immunoother, 1997. 45 (3-4): p.128-30; Wu, AM, 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, single chain diabodies combine the advantages of tandem scFv (all monomers are bispecific) and diabody (soluble expression in bacteria).

  More recently, diabodies have been fused to Fc to create more Ig-like molecules, termed di-diabodies (Lu, D., et al., J Biol Chem, 2004. 279). (4): p.2856-65). In addition, multivalent antibody constructs have been described that contain two Fab repeats in the heavy chain of IgG and are capable of binding to four antigen molecules (WO0177342A1, and Miller, K., et al., J Immunol, 2003. 170 (9): pp. 4854-61).

International Publication No. 0177342

Milstein, C.I. and A. C. Cuello, Nature, 1983.305 (5934): p. 537-40 Staerz, U .; D. , Et al. , Nature, 1985. 314 (6012): p. 628-31 Brennan, M.M. , Et al. , Science, 1985.229 (4708): p. 81-3 Glennie, M.M. J. et al. , Et al. , J Immunol, 1987. 139 (7): p. 2367-75 Kriankum, J. et al. , Et al. , Biomol Eng, 2001. 18 (2): p. 31-40 Economides, A.M. N. , Et al. Nat Med, 2003. 9 (1): p. 47-52 Nakanishi, K .; , Et al. , Annu Rev Immunol, 2001. 19: p. See 423-74 Gracie, I. et al. A. , Et al. , J Clin Invest, 1999.104 (10): p. 1393-401 Leung, B.M. P. , Et al. , J Immunol, 2000.164 (12): p. 6495-502 Ito, A.I. , Et al. J Immunol, 2003.170 (9): p. 4802-9 Karni, A .; , Et al. , J Neuroimmunol, 2002.125 (1-2): p. 134-40 Arndt, M.M. and J. et al. Krauss, Methods MoI Biol, 2003. 207: p. 305-21 Peipp, M.M. and T.A. Valerius, Biochem Soc Trans, 2002.30 (4): p. 507-11 Mack, M.M. , G. Riethmuller, and P.R. Kufer, Proc Natl Acad Sci USA, 1995. 92 (15): p. 7021-5 Holliger, P.M. , T. Prospero, and G.C. Winter, Proc Natl Acad Sci USA, 1993. 90 (14): p. 6444-8.18 Holliger, P.M. and G. Winter, Cancer Immunol Immunoother, 1997. 45 (3-4): p. 128-30 Wu, A .; M.M. , Et al, Immunotechnology, 1996. 2 (1): p. 21-36 Pluckthun, A.M. and P.M. Pack, Immunotechnology, 1997. 3 (2): p. 83-105 Ridgway, J.M. B. , Et al. , Protein Eng, 1996. 9 (7): p. 617-21 Lu, D .; , Et al. , J Biol Chem, 2004. 279 (4): p. 2856-65 Miller, K.M. , Et al. , J Immunol, 2003. 170 (9): p. 4854-61

  There is a need in the art for improved multivalent binding proteins capable of binding two or more antigens. The present invention provides a novel family of binding proteins that are capable of binding two or more antigens with high affinity.

SUMMARY OF THE INVENTION The present invention relates to a multivalent binding protein that can bind to two or more antigens. The present invention provides a novel family of binding proteins that are capable of binding two or more antigens with high affinity.

In one aspect, the invention includes a polypeptide chain, wherein said polypeptide chain is VD1- (X1) _n- VD2-C- (X2) _n (VD1 is the first variable domain and VD2 is the second A 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 in the binding protein are heavy chain variable domains. More preferably, the heavy chain variable domain is selected from the group consisting of a mouse heavy chain variable domain, a human heavy chain variable domain, a CDR grafted heavy chain variable domain, and a humanized heavy chain variable domain. In a preferred embodiment, VD1 and VD2 can bind to the same antigen. In another embodiment, VD1 and VD2 can bind to different antigens. Preferably C is a heavy chain constant domain. More preferably, X1 is a linker (provided that X1 is not CH1). Most preferably, X1 is, AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG ; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA (G 4 S) 4; SAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP QPKAAPSVVTLFPP; AKTTP; AKTTPPSVTPLAP; AKTTAP; AKTTAPPSVYPLAP; ASTKGP; ASTKGPSVFPLAP; GGGGSGGGGSGGGGGS; A linker selected from the group consisting of: ALMALS; GPAKELTPLKEAKVS; and GHEAAAVMQVQYPAS. Preferably X2 is an Fc region. More preferably, X2 is a variant Fc region.

In a preferred embodiment, the binding protein disclosed above comprises a polypeptide chain, said polypeptide chain being VD1- (X1) _n -VD2-C- (X2) _n (VD1 is the first heavy chain variable domain). VD2 is the second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker (but not CH1), and X2 is an Fc region.

In another embodiment, VD1 and VD2 in the binding protein are light chain variable domains. Preferably, the light chain variable domain is selected from the group consisting of a mouse light chain variable domain, a human light chain variable domain, a CDR grafted light chain variable domain and a humanized light chain variable domain. In one embodiment, VD1 and VD2 can bind to the same antigen. In another embodiment, VD1 and VD2 can bind to different antigens. Preferably C is a light chain constant domain. More preferably, X1 is a linker (provided that X1 is not CL1). Preferably, X1 is, AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG ; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA (G 4 S) 4; SAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVLFLPPP; AKTTP; AKTTPPSVTPLAP; AKTTAP; AKTTAPPSVYPLAP; a linker selected from the group consisting of ASTKGP and ASTKGPSVFPLAP. Preferably, the binding protein does not contain X2.

In a preferred embodiment, the binding protein disclosed above comprises a polypeptide chain, wherein the polypeptide chain is VD1- (X1) _n- VD2-C- (X2) _n (VD1 is the first light chain variable domain). VD2 is the second light chain variable domain, C is the light chain constant domain, X1 is a linker (but not CH1), and X2 does not contain the Fc region. .

In another preferred embodiment, the present invention comprises two polypeptide chains, wherein the first polypeptide chain is VD1- (X1) _n- VD2-C- (X2) _n (VD1 is the first heavy chain) A variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker (but not CH1), and X2 is an Fc region). And the second polypeptide chain is VD1- (X1) _n- VD2-C- (X2) _n (VD1 is the first light chain variable domain and VD2 is the second light chain variable domain) , C is a light chain constant domain, X1 is a linker (but not CH1), and X2 does not contain an Fc region. Most preferably, the dual variable domain (DVD) binding protein comprises four polypeptide chains, wherein the first two polypeptide chains are each VD1- (X1) _n -VD2-C- (X2) _n ( VD1 is the first heavy chain variable domain, VD2 is the second heavy chain variable domain, C is the heavy chain constant domain, X1 is a linker (but not CH1), and X2 is And the second two polypeptide chains are each VD1- (X1) _n -VD2-C- (X2) _n (VD1 is the first light chain variable domain); VD2 is the second light chain variable domain, C is the light chain constant domain, X1 is a linker (but not CH1), and X2 does not contain the Fc region. Such dual variable domain (DVD) proteins have four antigen binding sites.

  In another preferred embodiment, the binding proteins disclosed above are capable of binding to one or more targets. Preferably, the target is selected from the group consisting of cytokines, cell surface proteins, enzymes and receptors. Preferably, the binding protein is capable of modulating the biological function of one or more targets. More preferably, the binding protein is capable of neutralizing one or more targets. The binding protein of the present invention can bind to a cytokine selected from the group consisting of lymphokines, monokines and polypeptide hormones. In certain embodiments, the binding proteins are IL-1α and IL-1β; IL-12 and IL-18; TNFα and 1L-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 MEF; IL-13 and TGF-β; IL-13 and LHR agonists; IL-13 and CL25; IL-13 and SPRR2a; DL-13 and SPRR2b; IL-13 and ADAM8; and TNFα and PG 4, IL-13 and PED2, TNF and can be coupled to the pair of a cytokine selected from the group consisting of PEG2. In another embodiment, the binding proteins of the invention comprise 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; CSPGs 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 RGMA; NgR and RGMA; OMGp and RGMA; PDL-1 and CTLA-4; RGMA and RGMB; Te38 and TNFα; TNFα and Blys; TNFα and CD-22; TNFα and CTLA-4; TNFα and GP130; It is possible to bind to a target pair selected from the group consisting of IL-12p40; and TNFα and RANK ligands.

  In one embodiment, the binding proteins capable of binding to human IL-1α and human IL-1β are 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, a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 55, SEQ ID NO: 57 and SEQ ID NO: 59; and SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, It comprises a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 and SEQ ID NO: 60. In another embodiment, a binding protein capable of binding to mouse IL-1α and mouse IL-1β comprises the DVD heavy chain amino acid sequence of SEQ ID NO: 105 and the DVD light chain amino acid sequence of SEQ ID NO: 109.

  In one embodiment, the binding protein capable of binding to IL-12 and IL-18 is a DVD heavy 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. A chain amino acid sequence; and a DVD light chain amino acid sequence 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, a binding protein capable of binding to CD20 and CD3 comprises a DVD heavy chain amino acid sequence that is SEQ ID NO: 97 and a DVD light chain that is SEQ ID NO: 101.

  In another embodiment, the binding protein of the invention comprises BMP1, BMP2, BMP3B (GDF10), 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, EFNB1, IFNG, IFNW1, FIL1, FIL1 (ε), FLL1 (ζ), IL1A , IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL1I, 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 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL), NFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEG1), TNFSF18, GF1 (VEGF1), VEGF, R1 IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15R, IL21R1, IL21R1, IL18R1, IL18R1, IL18R1, IL1RAP, IL1RAPL1, IL1RAPL2 Binds to one, two or more cytokines, cytokine-related proteins and cytokine receptors selected from the group consisting of IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AEF1, HGF, LEP (leptin), PTN and THPO be able to.

  The binding proteins of the present invention include CCL1 (I-309), CCL2 (MCP-1 / MCAF), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8. (Mcp-2), CC11 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MDP-3b), CCL20 (MIP-3a), CCL21 (SLC / Exodas-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 (IP10), CXCL11 (I-TAC), CXCL12 (SDF1) , CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP (CXCL7), CX3CL1 (SCYD1), SCYE1, XCL1 (Lymphotactin), XCL2 (SCM-1b), BLR1 (MDR15), CCBP2 (D6 / CRA1 61) CKR1 / HM145), CCR2 (mcp-1RB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5 (CMKBR5 / ChemR13), CCR6 (CMKBR6 / CKR-L3 / ST) L22 / 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), HM748IL ), IL8RB (IL8Rb), LTB4R (GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, BD Selected from the group consisting of NF, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HDF1A, IL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL It can bind to one or more chemokines, chemokine receptors and chemokine related proteins. The binding protein of the present invention can bind to a cell surface protein selected from the group consisting of integrins. The binding protein of the invention can bind to an enzyme selected from the group consisting of a kinase and a protease. The binding protein of the present invention can bind to a receptor selected from the group consisting of a lymphokine receptor, a monokine receptor and a polypeptide hormone receptor.

  In preferred embodiments, the binding protein is multivalent. More preferably, the binding protein is multispecific. The multivalent and / or multispecific binding proteins have properties that are particularly desirable from a therapeutic standpoint. For example, multivalent and / or multispecific binding proteins are (1) taken up (and / or catabolized) faster than bivalent antibodies by cells expressing the antigen to which the antibody binds; (2) And / or (3) can induce cell death and / or apoptosis of cells expressing an antigen to which the multivalent antibody can bind. A “parent antibody” that provides at least one antigen binding specificity of a multivalent and / or multispecific binding protein is internalized (and / or catabolized) by cells expressing the antigen to which the antibody binds. ) Can be antibodies and / or agonists, cell death-inducing and / or apoptosis-inducing antibodies, and the multivalent and / or multispecific binding proteins described herein are one of these properties. One or more improvements may be indicated. Further, as described herein, when constructed as a multivalent binding protein, the parent antibody may lack any one or more of these properties, as described herein. These properties can be imparted when constructed in this way.

In another embodiment, the binding protein of the invention has at least 10 ² M ⁻¹ s ⁻¹ ; at least 10 ³ M ⁻¹ s ⁻¹ ; at least 10 ⁴ M ⁻¹ s as measured by surface plasmon resonance. ⁻¹ ; at least 10 ⁵ M ⁻¹ s ⁻¹ ; and at least 10 ⁶ M ⁻¹ s ⁻¹ , having a binding rate constant (K _on ) to one or more targets selected from the group consisting of Preferably, the binding proteins of the present invention have between 10 ² M ⁻¹ s ⁻¹ and 10 ³ M ⁻¹ s ⁻¹ as measured by surface plasmon resonance, 10 ³ M ⁻¹ s ⁻¹ and 10 ^{Between 4} M ⁻¹ s ⁻¹ , between 10 ⁴ M ⁻¹ s ⁻¹ and 10 ⁵ M ⁻¹ s ⁻¹ , or between 10 ⁵ M ⁻¹ s ⁻¹ and 10 ⁶ M ⁻¹ s ⁻¹ Having a binding rate constant (K _on ) for one or more targets.

In another embodiment, the binding protein has a maximum of about 10 ⁻³ s ⁻¹ ; a maximum of about 10 ⁻⁴ s ⁻¹ ; a maximum of about 10 ⁻⁵ s ⁻¹ ; and a maximum of about 10, as measured by surface plasmon resonance. Having a dissociation rate constant (K _off ) for one or more targets selected from the group consisting of selected from the group consisting of 10 ⁻⁶ s ⁻¹ . Preferably, the binding protein of the invention has a concentration of 10 ⁻³ s ⁻¹ to 10 ⁻⁴ s ⁻¹ , 10 ⁻⁴ s ⁻¹ to 10 ⁻⁵ s ⁻¹ , as measured by surface plasmon resonance. Or a dissociation rate constant (K _off ) for one or more targets between 10 ⁻⁵ s ⁻¹ and 10 ⁻⁶ s ⁻¹ .

In another embodiment, the binding protein is up to about 10 ⁻⁷ M; up to about 10 ⁻⁸ M; up to about 10 ⁻⁹ M; up to about 10 ⁻¹⁰ M; up to about 10 ⁻¹¹ M; up to about 10 ^−12. Having a dissociation constant (K _D ) for one or more targets selected from the group consisting of M and up to 10 ⁻¹³ M. Preferably, the binding protein of the invention is 10 ⁻⁷ M to 10 ⁻⁸ M; 10 ⁻⁸ M to 10 ⁻⁹ M; 10 ⁻⁹ M to 10 ⁻¹⁰ M; 10 ⁻¹⁰ M to 10 ^{− It has} a dissociation constant (K _D ) for IL-12 or IL-23 of ¹¹ M; 10 ⁻¹¹ M to 10 ⁻¹² M; or 10 ⁻¹² M to 10 ⁻¹³ M.

In another embodiment, the combined protein is a conjugate further comprising a factor selected from the group consisting of an immunoadhesion molecule, a contrast agent, a therapeutic agent and a cytotoxic agent. Preferably, the contrast agent is selected from the group consisting of a radioactive label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label and biotin. More preferably, the contrast agent is a radiolabel selected from the group consisting of ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho and ¹⁵³ Sm. is there. Preferably, the therapeutic or cytotoxic agent is selected from the group consisting of metabolic inhibitors, alkylating agents, antibiotics, growth factors, cytokines, anti-angiogenic agents, anti-mitotic agents, anthracyclines, toxins and apoptotic agents. Is done.

  In another embodiment, the binding protein is a crystallized binding protein and is present as a crystal. Preferably, the crystals are carrier-free pharmaceutical sustained release crystals. More preferably, the crystallized binding protein has a greater in vivo half-life than the soluble counterpart of said binding protein. Most preferably, the crystallized binding protein retains biological activity.

  In another embodiment, the binding protein is glycosylated. Preferably, the glycosylation is a human glycosylation pattern. One aspect of the present invention pertains to an isolated nucleic acid encoding any one of the binding proteins disclosed above. A further embodiment provides a vector comprising the isolated nucleic acid disclosed above, said vector comprising pcDNA; pTT (Durocher et al., Nucleic Acids Research 2002, VoI 30, No. 2); pTT3 (PTT with additional multiple cloning sites; pEFBOS (Mizushima, S. and Nagata, S., (1990) Nucleic acids Research Vol18, NO.17); pBV; pJV; pcDNA3.1 TOPO, pEF6TOPO and selected from pBJ Is done.

  In another embodiment, the host cell is transformed with the vector disclosed above. Preferably, the host cell is a prokaryotic cell. More preferably, the host cell is E. coli. In related embodiments, the host cell is a eukaryotic cell. Preferably, the eukaryotic cell is selected from the group consisting of protist cells, animal cells, plant cells and fungal cells. More preferably, the host cell is a mammalian cell including (but not limited to) CHO and COS; or a fungal cell such as Saccharomyces cerevisiae; or an insect cell such as Sf9.

  Another aspect of the present invention disclosed above comprises culturing any one of the host cells also disclosed above in a medium under conditions sufficient to produce a binding protein. A method for producing a binding protein is provided. Preferably 50% to 75% of the binding protein produced by this method is a bispecific tetravalent binding protein. More preferably, 75% to 90% of the binding protein produced by this method is a bispecific tetravalent binding protein. Most preferably, 90% to 95% of the produced binding protein is a bispecific tetravalent binding protein.

  Another embodiment provides a binding protein produced according to the methods disclosed above.

  One embodiment provides a composition for the release of binding protein, the composition comprising a formulation, the formulation comprising the crystallized binding protein and components disclosed above and at least one polymeric Including a carrier. Preferably, the polymer carrier is: poly (acrylic acid), poly (cyanoacrylate), poly (amino acid), poly (anhydride), poly (depsipeptide), poly (ester), poly (lactic acid), poly (lactic acid) Glycolic acid copolymer) or PLGA, poly (b-hydroxybutyrate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly [(organo) phosphazene] , Poly (orthoester), poly (vinyl alcohol), poly (vinyl pyrrolidone), maleic anhydride-alkyl vinyl ether copolymer, pluronic polyol, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharide, A polymer selected from one or more of the group consisting of licaminoglycans, sulfated polysaccharides, mixtures and copolymers thereof, preferably the ingredients are albumin, sucrose, trehalose, lactitol, gelatin , Hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol, and polyethylene glycol, another embodiment includes the step of administering to the mammal an effective amount of the composition disclosed above. Provide a method of treating.

  The present invention also provides a pharmaceutical composition comprising the binding protein disclosed above and a pharmaceutically acceptable carrier. In a further embodiment, the pharmaceutical composition comprises at least one additional therapeutic agent for treating the disease. Preferably, the additional agent is a therapeutic agent, contrast agent, cytotoxic agent, angiogenesis inhibitor (including but not limited to anti-VEGF antibody or VEGF-trap); kinase inhibitor (KDR and TIE- Co-stimulatory molecule blockers (including but not limited to anti-B7.1, anti-B7.2, CTLA4-Ig, anti-CD20); adhesion; Molecular blockers (including but not limited to anti-LFA-1 Abs, anti-E / L selectin Abs, small molecule inhibitors); anti-cytokine antibodies or functional fragments thereof (anti-IL-18, anti-TNF, anti-molecules) Including, but not limited to, IL-6 / cytokine receptor antibodies); methotrexate; cyclosporine; rapamycin; FK506; detectable label or repo TNF antagonists; anti-rheumatic drugs; muscle relaxants, anesthetics, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthesia, sedatives, local anesthesia, neuromuscular blocking agents, antimicrobial agents, anti-psoriatic agents, Corticosteroid, anabolic steroid, erythropoietin, immunization, immunoglobulin, immunosuppressant, growth hormone, hormone replacement agent, radiopharmaceutical, antidepressant, antipsychotic, stimulant, asthma, beta agonist, inhaled steroid, Selected from the group consisting of epinephrine or analogs, cytokines and cytokine antagonists.

  In another embodiment, the invention is a method of treating a human in a disease in which a target or multiple targets that can be bound by the binding proteins disclosed above are detrimental, in a human subject. The method is provided comprising administering to the human subject a binding protein as disclosed above such that the target or targets are inhibited and a treatment is achieved. Preferably, the disease is rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthritis, systemic lupus erythematosis, Crohn's disease, ulcerative colitis, Inflammatory bowel disease, insulin-dependent diabetes mellitus, thyroiditis, asthma, allergic disease, psoriasis, dermatitis, scleroderma, graft-versus-host disease, organ transplant rejection, acute or chronic immune disease related to organ transplantation, sarcoidosis Atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schlein purpura, microscopic vasculitis of the kidney, chronic active hepatitis, Uveitis, septic shock, toxic shock syndrome, septic syndrome, cachexia, infectious disease, parasitic disease, acquired immune deficiency syndrome, acute Acute myelitis, Huntington's disease, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignant tumor, heart failure, myocardial infarction, Addison's disease, sporadic, polyendocrine hypofunction syndrome I Type and polyendocrine hypofunction syndrome type II, Schmidt syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthritis, arthritis, Reiter's disease, psoriatic arthritis, ulcerative colitis Diseased synovitis, Chlamydia, Yersinia and Salmonella-related arthritis, spondyloarthropathy, atherosclerosis / atherosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, deciduous pemphigus, etc. Pemphigus, linear IgA disease, autoimmune hemolytic anemia, Coombs-positive hemolytic anemia, acquired pernicious anemia, juvenile pernicious anemia, myalgic encephalomyelitis Royal free disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosis hepatitis, idiopathic autoimmune hepatitis, acquired immune deficiency syndrome, acquired immune deficiency related diseases, hepatitis B, hepatitis C, Unclassifiable immunodeficiency (unclassifiable primary hypogammaglobulinemia), dilated cardiomyopathy, female infertility, ovarian dysfunction, early ovarian dysfunction, fibrotic lung disease, idiopathic interstitial pneumonia, inflammation Postinterstitial lung disease, interstitial pneumonia, connective tissue disease related interstitial lung disease, mixed connective tissue disease related lung disease, systemic sclerosis related interstitial lung disease, rheumatoid arthritis related interstitial lung disease Systemic lupus erythematosus-related lung disease, dermatomyositis / polymyositis-related lung disease, Sjogren's disease-related lung disease, ankylosing spondylitis-related lung disease, vasculitic diffuse lung disease, f Siderosis-related lung disease, drug-induced interstitial lung disease, fibrosis, radioactive fibrosis, obstructive bronchiolitis, chronic eosinophilic pneumonia, lymphocyte infiltrating lung disease, post-infectious interstitial lung disease , Ventilatory arthritis, autoimmune hepatitis, type 1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type 2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune-mediated hypoglycemia, melanoma Type B insulin resistance, hypoparathyroidism, acute immune disease related to organ transplantation, chronic immune disease related to organ transplantation, osteoarthritis, primary sclerosing cholangitis, type 1 psoriasis, type 2 Psoriasis, idiopathic leukopenia, autoimmune neutropenia, kidney disease NOS, glomerulonephritis, renal microvasculitis, Lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, Sperm autoimmunity, multiple sclerosis (all Subtype), sympathetic ophthalmitis, pulmonary hypertension secondary to connective tissue disease, Goodpasture syndrome, pulmonary symptoms of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjogren's syndrome, Takayasu / arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goiter autoimmune hypothyroidism (Hashimoto's disease), atrophic self Immune hypothyroidism, primary myxedema, lens-induced uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver disease, alcoholic cirrhosis, alcohol-induced liver injury, cholestasis (Choleocytosis), idiosyncratic liver disease, drug-induced hepatitis, non-alcoholic steatohepatitis, allergy and asthma, group B chain (GBS) infections, psychiatric disorders (eg depression and schizophrenia), diseases mediated by Th2 and Th1 types, acute and chronic pain (various forms of pain), and lung cancer, breast cancer, stomach cancer, bladder Cancers such as cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer and kidney cancer and hematopoietic malignancies (leukemia and lymphoma), abetalipoproteinemia, advanced cyanosis, 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, adenocarcinoma, aerial ectopic pulsation, AIDS dementia Complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, α-1-antitrypsin deficiency, amyotrophic lateral cord Disease, anemia, angina pectoris, anterior horn cell degeneration, anti-cd3 treatment, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aortic and aneurysms, aortic dissection, arterial Hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (persistent or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone transplant rejection, bone marrow transplant (BMT) rejection, leg block, Burkitt's lymphoma, burns, cardiac arrhythmia, cardiac dysfunction syndrome, cardiac tumor, cardiomyopathy, cardiopulmonary bypass inflammatory response, cartilage transplant rejection, cerebellar cortical degeneration, cerebellar disease, disordered or multifocal atrial tachycardia , Chemotherapy related diseases, chronic myelogenous leukemia (CML), chronic alcoholism, chronic inflammatory lesions, chronic Lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylic acid poisoning, colorectal cancer, congestive heart failure, conjunctivitis, contact dermatitis, pulmonary heart, coronary artery disease, Creutzfeldt-Jakob disease, culture Negative sepsis, cystic fibrosis, cytokine therapy-related disease, boxer brain, demyelinating disease, dengue hemorrhagic fever, dermatitis, dermatological symptoms, diabetes, diabetes mellitus, diabetic arteriosclerosis, diffuse Lewy body disease , Dilated congestive cardiomyopathy, basal ganglia disease, middle-aged Down syndrome, drug-induced motor disease induced by drugs that block central nerve dopamine receptors, drug sensitivity, eczema, encephalomyelitis, intracardiac Membrane inflammation, endocrine disease, epiglottitis, Epstein-Barr virus infection, erythema, extrapituitary tract and cerebellar disease, familial hemophagocytic lymphohistiocytosis autophagic lymphohistiocytosis), fatal thymus transplant rejection, Friedreich's ataxia, functional peripheral arterial disease, fungal sepsis, gas gangrene, gastric ulcer, glomerulonephritis, graft rejection of any organ or tissue, Gram-negative sepsis , Gram-positive sepsis, granulomas caused by intracellular organisms, hairy cell leukemia, Haller-Holden-Spatz disease, Hashimoto thyroiditis, hay fever, heart transplant rejection, hemochromatosis, hemodialysis, hemolytic uremic syndrome / thrombolytic platelets Reduced purpura, bleeding, hepatitis (type A), His bundle arrhythmia, HIV infection / HIV neuropathy, Hodgkin's disease, hyperkinetic disease, hypersensitivity reaction, hypersensitivity pneumonia, hypertension, hypokinetic disease, hypothalamus-pituitary- Adrenocortical system evaluation, idiopathic Addison disease, idiopathic pulmonary fibrosis, antibody-mediated cytotoxicity, asthenia, infant spinal cord Atrophy, aortic inflammation, influenza A, ionizing radiation exposure, iridocyclitis / uveitis / optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy , Kaposi's sarcoma, renal transplant rejection, Legionella, leishmaniasis, leprosy, corticospinal lesion, lipedema, liver transplant rejection, lymphedema, malaria, malignant lymphoma, malignant histiocytosis, Malignant melanoma, meningitis, meningococcusemia, metabolic / idiopathic, migraine, mitochondrial multi-system disorder, mixed connective tissue disease, monoclonal hypergammaglobulin blood Disease, multiple myeloma, multiple systemic degeneration (Mencel Degeline) Thomas Shy-Drager and Machado-Joseph), myasthenia gravis, Mycobacterium avium Intracellulare, Mycobacterium tuberculosis, myelodysplasia, myocardial infarction, fungal ischemic disease, nasopharyngeal cancer, newborn Chronic lung disease, nephritis, nephrosis, neurodegenerative disease, neurogenic I muscle atrophy, neutropenic fever, non-Hodgkin lymphoma, obstruction of abdominal aorta and its branches, obstructive arterial disease, okt3 therapy, testitis / Epididymis, testicularitis / vaginal reconstruction treatment, organ enlargement, osteoporosis, pancreas transplant rejection, pancreatic cancer, paraneoplastic transplantation, parathyroid transplant rejection, pelvic inflammatory disease, year-round Rhinitis, pericardial disease, peripheral atherosclerotic disease, peripheral vascular disease, peritonitis, pernicious anemia, Pneumocystis carini pneumonia, pneumonia, POEMS symptoms Groups (polyneuritis, organ hypertrophy, endocrine disease, monoclonal gamma globulinemia and skin change syndrome), post perfusion syndrome, post pump syndrome, myocardial infarction Post-open heart surgery syndrome, preeclampsia, progressive supranuclear palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon and disease, Raynaud's disease, Refsum's disease, regular narrow QRS tachycardia ), Renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcoma, scleroderma, senile chorea, senile dementia with Lewy bodies, seronegative arthritis, shock, sickle cell anemia, skin allograft Allograft rejection, skin change syndrome, small intestine transplant rejection, solid Tumor, specific arrhythmia, spinal ataxia, spinocerebellar degeneration, streptococcal myositis, cerebellar structural lesions, subacute sclerosing panencephalitis, eczema, cardiovascular syphilis, systemic anaphylaxis, systemic Inflammatory reaction syndrome, systemic onset juvenile rheumatoid arthritis, T cells or FABALL, telangiectasia, obstructive thromboangitis, thrombocytopenia, toxicity, transplantation, trauma / bleeding, type III hypersensitivity reaction, type IV hypersensitivity Unstable angina pectoris, uremia, urinary sepsis, hives, valvular heart disease, varicose veins, vasculitis, venous disease, venous thrombosis, ventricular fibrillation, viral and fungal infections, viral encephalitis / Aseptic meningitis, virus-related hemophagocytic syndrome, Wernicke-Korsakov syndrome, Wilson disease, any organ or tissue It is selected from the group comprising xenograft rejection.

  In another embodiment, the present invention comprises the step of administering any one of the binding proteins disclosed above before, simultaneously with or after administration of the second agent as discussed above. A method of treating a patient suffering from a disease is provided. In a preferred embodiment, the second factor is budenoside, epidermal growth factor, corticosteroid, cyclosporine, sulfasalazine, aminosalicylate, 6-mercaptopurine, azathioprine, metronidazole, lipoxygenase inhibitor, mesalamine, olsalazine, balsalazide, anti Oxidizing agent, thromboxane inhibitor, IL-1 receptor antagonist, anti-IL-1β monoclonal antibody, anti-IL-6 monoclonal antibody, growth factor, elastase inhibitor, pyridinyl-imidazole compound, 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-E, GM-CSF, FGF and PDGF , CD2, CD3, CD4, CD8, CD 19, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or antibodies of these ligands, methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAID, ibuprofen, corticosteroid, prednisolone, phosphodiesterase inhibition Agent, adenosine agonist, antithrombotic agent, complement inhibitor, adrenergic agent, IRAK, NIK, IKK, p38, MAP kinase inhibitor, IL-1β converting enzyme inhibitor, TNFα converting enzyme inhibitor, T cell signaling inhibition Agent, metalloproteinase inhibitor, sulfasalazine, azathioprine, 6-mercaptopurine, angiotensin converting enzyme inhibitor, soluble cytokine receptor, soluble p55TNF receptor, soluble p75TNF receptor, sIL-1RI, sIL-1RII, sIL-6R, antiinflammatory cytokines, selected from IL-4, IL-10, IL-11, IL-13 and the group consisting of TGF [beta.

  In a preferred embodiment, the pharmaceutical composition disclosed above is parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavity, intracavity ( intracerebral), intracerebellum, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, pelvic, pericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intracolonic, kidney Administered to a subject by at least one mode selected from internal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vagina, rectum, intraoral, sublingual, intranasal and transdermal Is done.

  One aspect of the invention provides at least one anti-idiotype antibody to at least one binding protein of the invention. Anti-idiotype antibodies include at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy or light chain variable region that can be incorporated into a binding protein of the invention, Any protein or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule, including but not limited to a heavy or light chain constant region, a framework region or any portion thereof, is included.

  In another embodiment, the binding protein of the invention comprises ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGRDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPTL4; AMPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1; orf10 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6 / JAB61); CCL1 (I-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP) CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2) SCL; Exodus-2; CCL22 (MDC / STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2 / eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK / ILC); CCL28; 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); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD3 CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81: CD83: CD86: CDH1 (E-cadherin); CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; chitinase; CHST10; CKLFSF2; KLLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (Claudin-7); CLN3; CLU (clusterin); CMKLLR1; CMKOR1 (RDC1); (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-cadherin); CTSB (cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28); CXCL1 (GRO1); CXCL10 ( CXCL11 (I-TAC / IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GRO 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; EGF; EGFR; ELAC2; EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF 10; FGF11; FGF12; FGF12B; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FDL1 (ε); FIL1 (ζ); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; 2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10 (C10); GRP; GSN (gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HEP1; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; 1FNA6; IFNA7; IFNB1; IFNγ; IFNW1; IGF1; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19A; IL1B; IL1F10; IL1F1; IL1F9; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22RA2; IL27; 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; ITAK1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; HGB3; ITGB4 (b4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5P KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (keratin 19); KRT2 KRTHB6 (hair-specific type II keratin); LAMA5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARKKS; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; MUC1 (mucin) ); MYC; MYD88; NCK2; neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM2) NOXB; NROB1; NR0B2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F2; NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, ODZ1, OPRD1, P2RX7, PAP, PART1, PATE, PAWR, PCA3, PCNA, PDGFA, PDGFB, PECAM1, PF4 (CXCL4), PGF, PGR; PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PR PROL; PROC2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS1; RGS13; RGS3; RNF110 (ZNF144); ROBO2; S100D2; B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial monocyte activated 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; TIMP3; tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TNF9; TNF-a; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TR TNFSF11 (TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF6 (NF40) TNFSF5 (NF40F); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptor; TOP2A (topoisomerase Iia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; Versican; VHLC5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5 / CCXCR1); can bind to one or more targets selected from the group consisting of YY1 and ZFPM2 .

FIG. 1A is a schematic diagram of a dual variable domain (DVD) = Ig construct, showing a strategy for making DVD-Ig from two parent antibodies. FIG. 1B shows hybridoma clone 2D13. E3 (anti-IL-1α) and 13F5. FIG. 2 is a schematic diagram of constructs DVD1-Ig, DVD2-Ig and two chimeric monospecific antibodies from G5 (anti-IL-1β).

Detailed Description of the Invention The present invention relates to multivalent and / or multispecific binding proteins capable of binding to two or more antigens. Specifically, the present invention relates to dual variable domain immunoglobulins (DVD-Ig) and pharmaceutical compositions thereof and nucleic acids, recombinant expression vectors and host cells for making such DVD-Igs. Also encompassed by the present invention are methods of using the DVD-Ig of the present invention to detect specific antigens either in vitro or in vivo.

  Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. However, if there is any ambiguity, the meaning and scope of the term should be clear, and the definitions given in this specification are all dictionary or definition outside this specification. Dominate. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of “or” means “and / or” unless stated otherwise. Further, the use of the term “including” and other forms such as “includes” and “included” is not limiting. Also, terms such as “element” or “component” encompass elements and components comprising one unit and elements and components comprising more than one subunit unless specifically stated otherwise.

  Nomenclature generally used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein and these techniques Are well known and commonly used in the field. In general, the methods and techniques of the present invention are subject to conventional methods well known in the art, and various general and more specific references cited and discussed throughout this specification, unless otherwise specified. It can be carried out as described in. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly performed in the art or as described herein. Nomenclature used in connection with analytical chemistry, synthetic organic chemistry and medicinal chemistry and medicinal chemistry described herein, and analytical chemistry, synthetic organic chemistry and medicinal chemistry and medicinal chemistry experiments described herein Room operations and techniques are well known and commonly used in the field. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient treatment.

In order that the present invention may be more readily understood, certain terms are defined below.
The term “polypeptide” as used herein refers to any polymer chain of amino acids. The terms “peptide” and “protein” are used interchangeably with the term polypeptide and also denote a polymer chain of amino acids. The term “polypeptide” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. The polypeptide can be monomeric or multimeric.

  The term “isolated protein” or “isolated polypeptide” refers to a naturally associated component that is present with the protein or polypeptide in its native state for its origin or origin. A protein or polypeptide without which it is substantially free of other proteins from the same species, expressed by cells obtained from different species, or non-naturally occurring. Thus, a chemically synthesized polypeptide, or a polypeptide synthesized in a cell line different from the cell from which the polypeptide is originally derived, is “isolated” from the components that naturally accompany it. Proteins can also be made substantially free of inherent components by isolation using protein purification techniques well known in the art.

  As used herein, the term “recover” refers to a chemical species, such as a polypeptide, by substantially isolating components that naturally accompany it, eg, by isolation using protein purification techniques well known in the art. Represents a method to avoid inclusion.

  As used herein, “biological activity” refers to all inherent biological properties of an antigen. Biological properties include, but are not limited to, binding to a receptor; induction of cell proliferation, inhibition of cell proliferation, induction of other cytokines, induction of apoptosis and enzyme activity.

  With respect to the interaction of an antibody, protein or peptide with a second chemical species, as used herein, the term “specific binding” or “specifically binds” means that the interaction is a chemical species, eg, Depending on the presence of a particular structure (eg, an antigenic determinant or epitope) on the antibody, it means to recognize and bind to a specific protein structure rather than a protein in general. If the antibody is specific for epitope “A”, the presence of a molecule containing epitope A (ie, unlabeled free A) in a reaction containing labeled “A” and antibody Reduces the amount of labeled A bound to the antibody.

  The term “antibody” as used herein refers to any immunoglobulin (Ig) molecule or Ig molecule composed of four polypeptide chains (two heavy (H) chains and two light (L) chains). It is intended to broadly represent any functional molecule, variant, variant or derivative thereof that retains its essential epitope binding properties. Such mutant, variant or derivative antibody formats are known in the art. These non-limiting embodiments are discussed below.

  In full-length antibodies, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain CL. The VH and VL regions are further subdivided into hypervariable regions (referred to as complementarity determining regions (CDRs)) interspersed with more conserved regions (referred to as framework regions (FR)). Is possible. Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The immunoglobulin molecules can be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

  The term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain that can be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains (CH2 domain and CH3 domain) and optionally a CH4 domain. Replacing amino acid residues in the Fc portion to alter antibody effector function is well known in the art (Winter, et al US PAT NOS 5,648,260; 5624821). The Fc portion of an antibody mediates several important effector functions such as cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and antibody half-life / exclusion rate and antigen-antibody complexes . In some cases, these effector functions are desirable for therapeutic antibodies, but in other cases, depending on the therapeutic purpose, they may not be necessary or even harmful. Certain human IgG isotypes, in particular IgG1 and IgG3, mediate ADCC and CDC through binding to FcγR and complement C1q, respectively. The neonatal Fc receptor (FcRn) is an important component that determines the circulating half-life of antibodies. In yet another embodiment, at least one amino acid residue is substituted in the constant region of the antibody, eg, the Fc region of the antibody, such that the effector function of the antibody is altered. Dimerization of two identical heavy chains of an immunoglobulin is mediated by dimerization of the CH3 domain and stabilized by disulfide bonds within the hinge region (Huber et al. Nature; 264: 415-20). Thies et al 1999 J MoI Biol; 293: 67-79.). Mutation of cysteine residues in the hinge region to prevent heavy chain-heavy chain disulfide bonds destabilizes CH3 domain dimerization. The residues required for CH3 dimerization have been identified (Dall 'Acqua 1998 Biochemistry 37: 9266-73.). Therefore, it is possible to generate monovalent half-Ig. Interestingly, these monovalent half-Ig molecules have been found in nature for both IgG and IgA subclasses (Seligman 1978 Ann Immunol 129: 855-70 .; Biewenga et al 1983 Clin Exp Immunol 51 : 395-400). The stoichiometry of the FcRn: IgFc region has been determined to be 2: 1 (West et al. 2000 Biochemistry 39: 9698-708) and half-Fc is sufficient to mediate FcRn binding ( Kim et al 1994 Eur J Immunol; 24: 542-548.). Residues important for CH3 dimerization are located on the inner interface of the CH3b sheet structure, whereas the region required for FcRn binding is located on the outer interface of the CH2-CH3 domain Thus, mutations to disrupt CH3 domain dimerization may not have a greater adverse effect on its FcRN binding. However, half-Ig molecules may have certain advantages in tissue penetration because they are smaller in size than normal antibodies. In one embodiment, at least one amino acid residue is substituted in the constant region of the binding protein of the invention, eg, the Fc region, such that heavy chain dimerization is disrupted, resulting in a half-DVDIg molecule. Yes.

As used herein, the term “antigen-binding portion” (or simply “antibody portion”) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments can also be bispecific, bispecific or multispecific formats, and specifically bind to two or more different antigens. Examples of binding fragments encompassed by the term “antigen-binding portion” of an antibody include (i) Fab fragments (monovalent fragments consisting of VL, VH, CL and CH1 domains), (ii) F (ab ′) ₂ Fragment (bivalent fragment containing two Fab fragments linked by disulfide bridge in hinge region), (iii) Fd fragment consisting of VH and CH1 domains, (iv) consisting of VL and VH domains of single arm of antibody Fv fragment, (v) dAb fragment containing a single variable domain (Ward et al. (1989) Nature 341: 544-546, Winter et al., PCT publication WO 90/05144 A1 (incorporated herein by reference) And vi) an isolated complementarity determining region (CDR). In addition, the two domains of the Fv fragment, VL and VH, are encoded by distinct genes, but these are as a single protein chain in which the VL and VH regions combine to form a monovalent molecule. These can be ligated using recombinant methods (known as single chain Fv (scFv). For example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies such as diabodies are also encompassed. Diabodies are obtained by using linkers where the VH and VL domains are expressed on a single polypeptide chain but are too short to allow pairing between two domains on the same chain. , Bivalent bispecific antibodies that force both domains to pair with the complementary domains of another chain and create two antigen binding sites (see, eg, Holliger, P., et al. (1993)). Proc. Natl. Acad. Sd. USA 90: 6444-6448; Poljak et al (1994) Structure 2: 1121-1123). Such antibody binding moieties are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5). This chain antibody is also included in a “linear antibody” (VH-CH1-VH-CH1) comprising a pair of tandem Fv segments that form a pair of antigen-binding regions together with a complementary light chain polypeptide (Zapata et al. Protein Eng.8 (10): 1057-1062 (1995); and US Patent No. 5,641,870).

  Throughout this specification, the term “multivalent binding protein” is used to describe a binding protein comprising two or more antigen binding sites. A multivalent binding protein is preferably an antibody that is engineered to have three or more antigen binding sites and is generally non-naturally occurring. The term “multispecific binding protein” refers to a binding protein capable of binding to two or more related or unrelated targets. The dual variable domain (DVD) binding proteins of the present invention comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. DVDs can be monospecific (ie, capable of binding to one antigen) or multispecific (ie, capable of binding to two or more antigens). . A DVD binding protein comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides is designated DVDIg. Each half of the DVDIg contains a heavy chain light chain polypeptide and a light chain DVD polypeptide and two antigen binding sites. Each binding site includes a heavy chain variable domain and a light chain variable domain, with a total of 6 CDRs involved in antigen binding per antigen binding site.

  As used herein, the term “bispecific antibody” refers to quadroma technology (see Milstein, C. and A. C. Cuello, Nature, 1983.305 (5934): p. 537-40). Mutations in the Knob into hole or Fc region by chemical ligation of two different mAs (see Staerz, UD, et al., Nature, 1985. 314 (6012): p. 628-31) (See Holliger, P., T. Prospero, and G. Winter, Proc Natl Acad Sci USA, 1993. 90 (14): p. 6444-8.18). Several different antibodies, only one is a functional bispecific antibody Resulting in immunoglobulin species, representing a full-length antibody. Depending on the molecular function, a bispecific antibody binds to one antigen (or epitope) present on one of its two binding arms (one pair of HC / LC) and its second arm (HC / Bind to different antigens (or epitopes) present on different pairs of LCs. By this definition, a bispecific antibody has two different antigen binding arms (in both specificity and CDR sequences) and is monovalent for each antigen it binds.

  The term “bispecific antibody” as used herein refers to binding to two different antigens (or epitopes) present in each of its two binding arms (HC / LC pairs). Represents possible full-length antibodies (see PCT publication WO 02/02773). Thus, a bispecific binding protein has two identical antigen binding arms with the same specificity and the same CDR sequence, and is bivalent for each antigen to which it binds.

  A “functional antigen binding site” of a binding protein is a site capable of binding to a target antigen. The antigen-binding affinity of an antigen-binding site is not necessarily as strong as the parent antibody from which the antigen-binding site is derived, but its ability to bind to an antigen assesses antibody binding to the antigen. Must be measurable using any one of a variety of known methods. Furthermore, in the present specification, the antigen-binding affinity of each antigen-binding site of a multivalent antibody need not be quantitatively the same.

  The term “cytokine” is a generic term for proteins released by one cell population and acting against another cell population as intercellular mediators. Examples of such cytokines are lymphokines, monokines and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and glycoprotein hormones such as luteinizing hormone; hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; Muller tube inhibitor; mouse gonadotropin-related peptide Inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin TPO); neuronal growth factor such as NGF-α; platelet growth factor; transforming growth factor (TGF) such as TGF-α and TGF-β; Urin-like growth factors-1 and -11; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β and -γ; colony stimulating factors (CSF) such as macrophage-CSF (M-CSF); granules Sphere macrophages-CSF (GM-CSF); and granulocytes-CSF (G-CSF); 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 and other interleukins (IL); TNF-α or TNF-β, etc. Tumor necrosis factor; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins obtained from natural sources or from recombinant cell culture and biologically active equivalents of native sequence cytokines.

The term “linker” refers to a polypeptide comprising two or more amino acid residues linked by peptide bonds and is used to link one or more antigen binding moieties. Such linker polypeptides are well known in the art (see, eg, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ., Et al. (1994) Structure 2: 1121-1123). Preferred linkers, AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG ; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA (G 4 S) 4; SAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVTLFPP AKTTP; AKTTPPSVTPLAP; AKTTAP; AKTTAPSVYPLAP; ASTKGP; ASTKGPSVFPLAP.

  An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy and light chain constant domain amino acid sequences are known in the art.

  The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the antibodies making up the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigen. Furthermore, unlike polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” should not be construed as requiring production of the antibody by any particular method.

  As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention can be used, for example, in CDRs, particularly CDE3, amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, by random or site-directed mutagenesis in vitro or in vivo). Mutations introduced by somatic mutations). However, as used herein, the term “human antibody” shall not include antibodies in which CDR sequences derived from the germline of another mammalian species such as a mouse are grafted onto a human framework sequence. .

  As used herein, the term “recombinant human antibody” refers to an antibody (further described in Section II C below), recombinant, expressed using a recombinant expression vector transfected into a host cell. Antibodies isolated from combinatorial human antibody libraries (Hoogenboom HR, (1997) TIB Tech. 15: 62-70; Azzazzy H., and Highsmith WE, (2002) Clin. Biochem. 35: 425-445; Gavilondo JV, and Larryrick JW (2002) BioTechniques 29: 128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21: 371. 378), an antibody isolated from an animal (eg, a mouse) that has been transfected with a human immunoglobulin gene (eg, Taylor, LD, et al. (1992) Nucl. Acids Res. 20): 6287-6295; Kellermann SA, and Green LL (2002) Current Opinion in Biotechnology 13: 593-597; Little M. et al (2000) Immunology Today 21: 364-370) or human immunity. Prepared and expressed by recombinant means, such as antibodies prepared, expressed, produced or isolated by any other means including splicing of gene sequences to other DNA sequences It is, the produced, or is intended to include all human antibodies isolated. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are used for in vitro mutagenesis (or in vivo somatic mutagenesis if animals transgenic for human Ig sequences are used). Thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from the human germline VH and VL sequences and related to these sequences, but in vivo within the human antibody germline repertoire. Is a sequence that may not occur in nature.

  An “affinity matured” antibody is one or more changes in one or more CDRs of the antibody that result in improved affinity of the antibody for the antigen compared to the parent antibody without changes. An antibody having Preferred affinity matured antibodies have nM affinity or even pM affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art. “Marks et al. Bidl Technology 10: 779-783 (1992)” describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described by Barbas et al. ProcNat. Acad. Sci, USA 91: 3809-3813 (1994); Schier et al. Gene 169: 147-155 (1995); Yelton et al. J. et al. Immunol. 155: 1994-2004 (1995); Jackson et al. , J .; Immunol. 154 (7): 3310-9 (1995); and Hawkins et al, J. MoI. MoI. Biol. 226: 889-896 (1992).

  The term “chimeric antibody” refers to a heavy and light chain variable region sequence from one species, such as an antibody having a mouse heavy and light chain variable region linked to a human constant region, and a constant region from another species. Represents an antibody containing sequence.

  The term “CDR grafted antibody” is derived from certain species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (eg, CDR3) are replaced with human CDR sequences. Represents an antibody in which one or more sequences of the CDR regions of VH and / or VL are replaced with CDR sequences of another species.

  The term “humanized antibody” includes heavy and light chain variable region sequences from species other than human (eg, mouse), but at least some of the VH and / or VL sequences are more “human-like”. Ie, antibodies that have been modified to be more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences and the corresponding non-human CDR sequences are replaced.

  The terms “Kabat number”, “Kabat definition” and “Kabat label” are used interchangeably herein. These terms recognized in the art are amino acids that are more variable (ie, hypervariable) compared to other amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof. Represents a system for numbering residues (Kabat et al. (1971) Ann. NY Acad, Sci. 190: 382-391 and, Kabat, EA, et al. (1991) Sequences of Proteins of Immunologicals. Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24-34 for CDR1, amino acid positions 50-56 for CDR2, and amino acid positions 89-97 for CDR3.

  As used herein, the term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each variable region of the heavy and light chains, which are designated CDR1, CDR2 and CDR3 for each variable region. The term “CDR set” as used herein refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Intersect (National Institutes of Health, Bethesda, Md. (1987) and (1991)) can be applied to any region of antibodies. As well as providing precise residue boundaries that define three CDRs, which can be referred to as Kabat CDRs, Chothia and co-workers (Chothia & Lesk, J. Mol.Biol.196: 901-917 (1987) and Chothia et al., Nature 342: 877-883 (1989)) are within the Kabat CDR. We have found that certain subparts adopt nearly identical peptide backbone conformations despite having great diversity at the amino acid sequence level, these subparts being L1, L2 and L3 or H1, H2 and H3 (“L” and “H” denote light and heavy chain regions, respectively), which are sometimes referred to as Chothia CDRs, which are referred to as Kabat CDRs. Other boundaries that define CDRs that overlap with Kabat CDRs are Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol Biol 262 (5): 732-45 (1996). It is possible that yet another CDR boundary definition may not strictly follow one of the above systems, Nonetheless, it overlaps with Kabat CDRs, which are in light of the predicted or experimental findings that residues or, in some cases, specific residues or groups throughout the CDRs do not significantly affect antigen binding, Although the methods used herein can use CDRs defined according to any of these systems, preferred embodiments use CDRs defined by Kabat or Chothia. .

  As used herein, the term “framework” or “framework sequence” refers to the remaining sequence of the variable region minus the CDRs. Since the exact definition of CDR sequences can be determined by different systems, the significance of framework sequences is correspondingly subject to different interpretations. The six CDRs (light chain CDR-L1, -L2 and -L3 and heavy chain CDR-H1, -H2 and -H3) are the framework regions on the light and heavy chains, and the four on each chain. Dividing into subregions (FR1, FR2, FR3 and FR4), CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. As specified by others, without identifying specific subregions as FR1, FR2, FR3 or FR4, the framework regions are combined within the variable regions of a single naturally occurring immunoglobulin chain. Represents FR. As used herein, one FR represents one of the four subregions, and multiple FRs represent two or more of the four subregions that make up the framework region.

  As used herein, the term “germline antibody gene” or “gene fragment” is encoded by non-lymphoid cells that have not undergone a maturation process that results in genetic rearrangements and mutations for expression of a particular immunoglobulin. Represents the immunoglobulin sequence to be expressed. (See, eg, Shapiro et al., Crit. Rev. Immunol. 22 (3): 183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484: 13-30 (2001)). One advantage provided by the various embodiments of the present invention is that germline antibody genes are more prone to preserve the essential amino acid sequence structures characteristic of individuals within a species than mature antibody genes; This results from the recognition that when used therapeutically in that species, it is less likely to be recognized as originating from an exogenous source.

  As used herein, the term “humanized antibody” refers to a framework (FR) region that immunospecifically binds to an antigen of interest and substantially comprises the amino acid sequence of a human antibody and amino acids of a non-human antibody. An antibody or a variant, derivative, analog or fragment thereof comprising a complementary determining region (CDR) substantially having a sequence. With respect to CDR, the term “substantially” as used herein refers to at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98, relative to the amino acid sequence of the non-human antibody CDR. CDRs with amino acid sequences that are% or at least 99% identical. A humanized antibody comprises substantially all of at least one, typically two variable domains (Fab, Fab ′, F (ab ′) 2, FabC, Fv), with all or substantially all of the CDR regions. All correspond to those of non-human immunoglobulin (ie, donor antibody), and all or substantially all of the framework regions are of human immunoglobulin consensus sequence. Preferably, the humanized antibody comprises at least a portion of an immunoglobulin, typically a human immunoglobulin constant region (Fc). In some embodiments, a humanized antibody contains both the light chain and at least the variable domain of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, a humanized antibody contains only a humanized heavy chain. In certain embodiments, a humanized antibody contains only the humanized variable domain of the light chain and / or the humanized heavy chain.

  The term “neutralization” as used herein refers to the binding protein specifically binding to a cytokine and neutralizing the biological activity of the 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 activities such as the binding specificity / affinity of DVD-Ig for two or more antigens.

  The term “epitope” includes any polypeptide determinant capable of specific binding to an immunoglobulin or T cell receptor. In certain embodiments, the epitope determinant comprises a chemically active surface group of a molecule such as an amino acid, sugar side chain, phosphoryl or sulforyl, and in certain embodiments, specific three-dimensional structural features. And / or may have specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

  As used herein, the term “surface plasmon resonance” refers to detecting changes in protein concentration within a biosensor matrix using, for example, the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). Represents an optical phenomenon that enables analysis of real-time biospecific interactions. For further description, see Jonsson, US; , Et al. (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U .; , Et al. (1991) Biotechniques 11: 620-627; Johnsson, B .; , Et al. (1995) J. MoI. Mol. Recognit. 8: 125-131; and Johnson, B.M. , Et al. (1991) Anal. Biochem. 198: 268-277.

As used herein, the term “K _on ” is intended to represent a binding rate constant for the association of an antibody to an antigen to form an antibody / antigen complex, as is known in the art.

As used herein, the term “K _off ” is intended to represent the dissociation rate constant for the dissociation of an antibody from an antibody / antigen complex, as is known in the art.

As used herein, the term “K _d ” is intended to represent the dissociation constant of a particular antibody-antigen interaction, as is known in the art.

As used herein, the term “labeled binding protein” refers to a protein that has incorporated a label that provides identification of the binding protein. Preferably, the label is a detectable marker, eg, a radioactive marker or fluorescent marker that can be detected by incorporation of a labeled amino acid or marked avidin (eg, optical or colorimetric methods). The attachment of a biotin moiety to a polypeptide that can be detected by streptavidin containing enzymatic activity). Examples of labels for polypeptides include the following radioisotopes or radionuclides (eg, ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, Or ¹⁵³ Sm); fluorescent label (eg, FITC, rhodamine, lanthanide phosphor); enzymatic label (eg, horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent marker; biotinyl group, recognized by secondary reporter Certain polypeptide epitopes (eg, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic factors such as, but not limited to, gadolinium chelates.

  The term “conjugate” refers to a binding protein, such as an antibody, that is chemically linked to a second chemical moiety (such as a therapeutic or cytotoxic agent). In this specification, the term “factor” is used to denote an extract made from a chemical compound, a mixture of chemical compounds, a biological macromolecule or a biological material. Preferably, the therapeutic agent or cytotoxic factor includes pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione , Mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol and puromycin, and analogs or homologues thereof. is not.

  The terms “crystal” and “crystallized” as used herein refer to an antibody or antigen-binding portion thereof that exists in crystalline form. Crystals are a form of the solid state of matter, which is different from other forms such as an amorphous solid state or a liquid crystalline state. Crystals are composed of regularly repeating three-dimensional arrays of atoms, ions, molecules (eg, proteins such as antibodies) or molecular assemblies (eg, antigen / antibody complexes). These three-dimensional arrays are aligned according to specific mathematical relationships that are well understood in the art. The basic units or building blocks that are repeated in the crystal are called asymmetric units. Repeating asymmetric units in an arrangement that matches a given well-ordered crystalline symmetry gives a “unit cell” of the crystal. Repeating the unit cell with regular transformations in all three dimensions gives a crystal. Giege, R.A. 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).

  As used herein, the term “polynucleotide” refers to a polymeric form of two or more nucleotides (either ribonucleotides or deoxynucleotides or a modified form of any type of nucleotide). . The term includes single and double stranded forms of DNA, but preferably double stranded DNA.

  As used herein, the term “isolated polypeptide” shall mean a polynucleotide (eg, of genomic, cDNA or synthetic origin, or some combination thereof) for An “isolated polynucleotide” can act on a polynucleotide that is not originally linked, wherein the “isolated polynucleotide” is not associated with all or part of the polynucleotide that is originally found therein Or is not inherently present as part of a larger sequence.

  As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid linked to the nucleic acid molecule. One type of vector is a “plasmid”, which represents a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be introduced into the host cell and integrated into the genome of the host cell, thereby replicating with the host genome. In addition, certain vectors are capable of inducing the expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include other forms such as expression vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses) such as viral vectors that perform an equivalent function.

  The term “operably linked” refers to a juxtaposition where the components described are in a relationship permitting them to function in the intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. “Operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. As used herein, the term “expression control sequence” refers to a polynucleotide sequence that is necessary to affect the expression and processing of linked coding sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (ie, Kozak Consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such regulatory sequences varies depending on the host organism. In prokaryotes, such regulatory sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence. In eukaryotes, in general, such regulatory sequences can include a promoter and a transcription termination sequence. The term “regulatory sequence” is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, such as leader sequences and fusion pair sequences.

  As used herein, “transformation” refers to any process by which foreign DNA enters a host cell. Transformation can occur under natural or artificial conditions using a variety of methods well known in the art. Transformation can rely on any known method for inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. The method is selected based on the host cell being transformed and can include, but is not limited to, viral infection, electroporation, lipofection and particle irradiation. Such “transformed” cells include stably transformed cells in which the inserted DNA can replicate as autonomously replicating plasmids or as part of a host chromosome. included. These include cells that transiently express the inserted DNA or RNA over a limited time.

  The term “recombinant host cell” (or simply “host cell”) as used herein is intended to refer to a cell into which foreign DNA has been introduced. It should be understood that such terms represent not only the cells of interest, but also the progeny of such cells. Such progeny may not actually be identical to the parent cell, as certain modifications may occur in subsequent generations due to mutations or environmental effects, but as used herein. Still included within the scope of the term “host cell”. Preferably, host cells include prokaryotic and eukaryotic cells selected from any sphere of organisms. Preferred eukaryotic cells include protists, fungi, plant and animal cells. Most preferably, host cells include, but are not limited to, prokaryotic cell lines E. coli; mammalian cell lines CHO, HEK293 and COS; insect cell lines Sf9 and fungal cells Saccharomyces cerevisiae.

  Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly performed in the art or as described herein. In general, the techniques and procedures described above are described according to conventional methods well known in the art, and in various general and more specific references that are cited and discussed throughout this specification. Can be implemented. See, for example, “Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989) for all purposes, by reference, specification,”). See).

  A “transgenic organism” as is known in the art and used herein refers to an organism having cells that contain the transgene, and a transgene (or progeny of the organism) introduced into the organism. Expresses a polypeptide that is not naturally expressed in the organism. A “transgene” is a DNA construct that has been stably and operatively integrated into the genome of a cell from which a transgenic organism has developed and one or more cell types or Induce expression of the encoded gene product in the tissue.

  The terms “modulate” or “modulate” are used interchangeably and as used herein represent a change or alteration in the activity of a molecule of interest (eg, the biological activity of a cytokine). Modulation can be an increase or decrease in the magnitude of a certain activity or function of the molecule of interest. Typical activities and functions of a molecule include, but are not limited to, binding properties, enzyme activity, cell receptor activation and signal transduction.

  Correspondingly, the term “modulator” as used herein is a compound capable of altering or altering the activity or function of a molecule of interest (eg, biological activity of a cytokine). . For example, a modulator may cause an increase or decrease in the magnitude of certain activities or functions of the molecule compared to the magnitude of activity or function observed in the absence of the modulator. In certain embodiments, the modulator is an inhibitor that reduces the magnitude of at least one activity or function of the molecule. Typical inhibitors include but are not limited to proteins, peptides, antibodies, peptibody, carbohydrates or small organic molecules. Pepti buddies are described, for example, in WO 01/83525.

  As used herein, the term “agonist” refers to the magnitude of a certain activity or function of a molecule as compared to the magnitude of activity or function observed in the absence of the agonist when contacted with the molecule of interest. Represents a modulator that causes an increase in Specific agonists of interest include, but are not limited to, polypeptides, nucleic acids, carbohydrates or any other molecule that binds to an antigen.

  As used herein, the term “antagonist” or “inhibitor” refers to a certain type of molecule compared to the magnitude of activity or function observed in the absence of the antagonist when contacted with the molecule of interest. Represents a modulator that causes a decrease in the magnitude of activity or function. Specific antagonists of interest include those that block or modulate the biological or immunological activity of the antigen. Antigen antagonists and inhibitors may include, but are not limited to, proteins, nucleic acids, carbohydrates, or any other molecule that binds to an antigen.

  As used herein, the term “effective amount” refers to reducing or reducing the severity and / or duration of a disease or one or more symptoms thereof, suppressing disease progression, and causing disease regression. Preventing the recurrence, development, onset or progression of one or more symptoms associated with the disease, detecting the disease, or the prophylactic or therapeutic effect of another therapy (eg, a prophylactic or therapeutic agent) Represents the amount of therapy that is sufficient to enhance or improve.

  As used herein, the term “sample” is used in the broadest sense. As used herein, a “biological sample” includes, but is not limited to, an organism or any amount of material obtained from what was an organism. Such organisms 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, urine, synovial fluid, cells, organs, tissues, bone marrow, lymph nodes and spleen.

I. The present invention relates to dual variable domain binding proteins capable of binding to one or more targets and methods of making the same. Preferably, the binding protein comprises a polypeptide chain, wherein said polypeptide chain is VD1- (X1) _n- VD2-C- (X2) _n (VD1 is the first variable domain and VD2 is the second variable domain) And C is a constant domain, X1 represents an amino acid or polypeptide, X2 represents an Fc region, and n is 0 or 1. The binding protein of the present invention can be prepared using various techniques. The present invention provides expression vectors, host cells and methods for making binding proteins.

A. Production of Parent Monoclonal Antibodies Variable domains of DVD binding proteins can be obtained from parent antibodies, such as polyclonal and monoclonal antibodies that can bind to the antigen of interest. These antibodies can exist naturally or can be made by recombinant techniques.

  Monoclonal antibodies can be prepared using a variety of techniques known in the art, such as the use of hybridoma, recombinant and phage display techniques, or combinations thereof. For example, monoclonal antibodies are known in the art, see, for example, “Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., N. and T-CeI Hybridomas 563-681 (Elsevier, NY, 1981), which is incorporated by reference in its entirety, such as those taught in hybridoma technology. Is possible. The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, such as any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Hybridomas are selected, cloned, and further screened for desired properties, such as robust hybridoma growth, high antibody production and desired antibody properties, as described below in Example 1. Hybridomas can be cultured and grown in vitro in syngeneic animals, in animals lacking the immune system (eg, nude mice), in vivo, or in cell culture. Methods for selecting, cloning and growing hybridomas are well known to those skilled in the art. In a preferred embodiment, the hybridoma is a murine hybridoma. In another preferred embodiment, the hybridoma can be produced in a non-human non-mouse species such as a rat, sheep, pig, goat, cow or horse. In another embodiment, the hybridoma is a human hybridoma in which a human nonsecretory myeloma is fused with a human cell that expresses an antibody capable of binding to a specific antigen.

  In addition, recombinant monoclonal antibodies are disclosed in U.S. Pat. S. Patent No. 5,627,052, PCT Publication WO92 / 02551 and Babcock, J. et al. S. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 7843-7848, from a single isolated lymphocyte using a technique referred to in the art as the selected lymphocyte antibody method (SLAM). Produced. In this method, single cells that secrete the antibody of interest are identified, for example, lymphocytes from an immunized animal, heavy and light chain variable region cDNAs are rescued from the cells by reverse transcriptase-PCR, These variable regions can then be expressed in a mammalian host cell such as a COS or CHO cell in association with an appropriate immunoglobulin constant region (eg, a human constant region). Host cells derived from lymphocytes that have been transfected with amplified immunoglobulin sequences and selected in vivo are then transfected cells, for example, to isolate cells that express antibodies to the antigen of interest. Can be further analyzed and selected in vitro. Amplified immunoglobulin sequences can be further manipulated in vitro, such as by in vitro affinity maturation methods, such as those described in PCT Publication WO 97/29131 and PCT Publication WO 00/56772.

  Monoclonal antibodies are also produced by immunizing non-human animals that contain some or all of the human immunoglobulin loci with the antigen of interest. In a preferred embodiment, the non-human animal is a XENOMOUSE transgenic mouse (a modified mouse strain that contains a large fragment of the human immunoglobulin locus and lacks mouse antibody production). For example, Green et al. Nature Genetics 7: 13-21 (1994) and United States Patents 5,916,771,5,939,598,5,985,615,5,998,209,6,075,181,6,091,001 See 6,114,598 and 6,130,364. WO 91/1074 published on July 25, 1991, WO 94/02602 published on February 3, 1994, both WO 96/34096 and WO 96/33735 published on October 31, 1996, 1998 WO 98/16654 published on April 23, WO 98/24893 published on June 11, 1998, WO 98/50433 published on November 12, 1998, published on September 10, 1999 See also WO 99/45031, WO 99/53049 published October 21, 1999, WO 0009560 published February 24, 2000, and WO 00/037504 published June 29, 2000. XENOMOUSE transgenic mice produce an adult-like human repertoire of fully human antibodies and produce antigen-specific human Mabs. XENOMOUSE transgenic mice contain about 80% of the human antibody repertoire through the introduction of megabase sized germline arranged YAC fragments at the human heavy and x light chain loci. Mendez et al. , Nature Genetics 15: 146-156 (1997), Green and Jakobovits J. et al. Exp. Med. 188: 483-495 (1998), the disclosures of which are incorporated herein by reference.

  In vitro methods can also be used to generate parent antibodies, and antibody libraries are screened to identify antibodies with the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art, see, eg, Ladner et al. U. S. Patent No. 5, 223, 409; Kang et al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al PCT Publication No. WO 92/01047; Garrard et al PCT Publication No. WO 92/09690; Fuchs et al (1991) Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum Antibody Hydromass 3: 81-85; Huse et al (1989) Science 246: 1275-1281; McCafferty et al, Nature (1990) 348: 552-554; -734; Hawkins et al. (1992) J Mol Biol 226: 889-896; Clackson et al. (1991) Nature 352: 624-628; Gram et al. (1992) PNAS 89: 3576-3580; Garrad et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19: 4133-4137; and Barbas et al (1991) PNAS 88: 7978-7882, US patent application publication 20030186374 and PCT Publication No. The methods described in WO 97/29131, the contents of each of which are incorporated herein by reference, are included.

  The parent antibody of the present invention can also be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. In particular, such phage can be used to display antigen binding domains expressed from repertoire or combinatorial antibody libraries (eg, human or mouse). Phage expressing an antigen binding domain that binds to the antigen of interest can be selected or identified using the antigen, for example, using a labeled antigen or an antigen bound or captured to a solid surface or bead. is there. Phages used in these methods are typically expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either phage gene III or gene VIII protein. Filamentous phage containing fd and M13 binding domains. Examples of phage display methods that can be used to generate the antibodies of the invention include “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); Advances in Immunology 57: 191-280 (1994); PCT application No. PCT / GB91 / 01134; PCT publications WO90 / 0280 WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US Pat. Nos. 5,698,426; 5,223,409; , 484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225 5,658,727; 5,733,743 and 5,969,108, each of which is incorporated herein by reference in its entirety.

  As described in the above references, after phage selection, the antibody coding region obtained from the phage is isolated and isolated to produce a complete antibody containing a human antibody or any other desired antigen-binding fragment. Can be used and expressed in any desired host, for example, mammalian cells, insect cells, plant cells, yeast and bacteria, as detailed below. For example, PCT publication WO92 / 22324; Mulinax et al. BioTechniques 12 (6): 864-869 (1992); and Sawai et al. AJRI 34: 26-34 (1995); and Better et al. Techniques for recombinantly producing Fab, Fab ′ and F (ab ′) 2 fragments using methods known in the art, such as the method disclosed in, Science 240: 1041-1043 (1988). (The above references are incorporated by reference in their entirety). Examples of techniques that can be used to generate single chain Fv and antibodies include US Pat. S. Pat. 4,946,778 and 5,258,498; 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).

  Instead of screening recombinant antibody libraries by phage display, other methods known in the art for screening large combinatorial libraries can be applied for parent antibody identification. One type of alternative expression system is PCT Publication NO. By Szostak and Roberts. WO 98/31700 and Roberts, R.W. W. and Szostak, J.A. W. (1997) Proc. Natl. Acad. Sci. A recombinant antibody library, such as that described in USA 94,12297-12302, is expressed as an RNA-protein fusion. In this system, a covalent fusion is created between the mRNA and the peptide or protein it encodes by in vitro translation of synthetic mRNA carrying puromycin, a peptidyl acceptor antibiotic at their 3 'end. Thus, a specific mRNA is based on the properties of the encoded peptide or protein, such as an antibody or part thereof (such as binding of the antibody or part thereof to a bispecific antigen). It is possible to concentrate from a mixture (eg a combinatorial library). Nucleic acid sequences encoding antibodies or portions thereof recovered from screening of such libraries can be expressed by recombinant means as described above (eg, in mammalian host cells), Furthermore, by further rounds of screening for mRNA-peptide fusions in which mutations have been introduced in the initially selected sequence, or as described above, by other methods for in vitro affinity maturation of recombinant antibodies. It can be subjected to further affinity maturation.

  In another approach, the parent antibody can be generated using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of the yeast. In particular, such yeast can be used to display antigen binding domains expressed from repertoire or combinatorial antibody libraries (eg, human or mouse). Examples of yeast display methods that can be used to generate parent antibodies include Wittrup, et al., Which is incorporated herein by reference. U. S. Patent NO. 6,699,658 are included.

The antibodies can be further modified to generate CDR-grafted and humanized parent antibodies. The CDR-grafted parent antibody contains heavy and light chain variable region sequences from a human antibody, and one or more of the V _H and / or _VL CDR regions can bind to the antigen of interest. It is replaced with the CDR sequence of a mouse antibody. Framework sequences obtained from any human antibody can serve as a template for CDR grafting. However, linear substitution on such frameworks often results in some loss of binding affinity for the antigen. The more homologous a human antibody is to the original mouse antibody, the less likely the combination of human framework and mouse CDR introduces distortions in the CDR that can reduce affinity. Accordingly, it is preferred that the human variable framework selected to replace the mouse variable framework other than the CDR has at least 65% sequence identity with the mouse antibody variable region framework. More preferably, the human and mouse variable regions, excluding the CDRs, have at least 70% sequence identity. More preferably, the human and mouse variable regions, excluding the CDRs, have at least 75% sequence identity. Most preferably, the human and mouse variable regions except the CDRs have at least 80% sequence identity. Methods for making such antibodies are known in the art (EP239,400; PCT publication WO91 / 09967; US Pat. Nos. 5,225,539; 5,530,101; and 5,585). 089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28 (4/5): 489-498 (1991); Studnikka et al., Protein. Engineering 7 (6): 805-814 (1994); Roguska et al., PNAS 91: 969-973 (1994)), and chain shuffling (US Pat. NO.5,565,352).

Humanized antibodies are obtained from non-human species antibodies that bind to the desired antigen having one or more complementarity determining regions (CDRs) derived from non-human species and framework regions derived from human immunoglobulin molecules. Antibody molecule. Known human Ig sequences have been disclosed. For example, www. ncbi. nlm. nih. gov / entrez- / query. fcgi; www. atcc. org / phage / hdb. html; www. scquest. com /; www. abcam. com /; www. antibodyresource. com / online comp. html; www. public. istate. edu /. about. pedro / research tools. html; www. mgen. uni-heidelberg. de / SD / IT / IT. html; www. whfreeman. com / immunology / CH-05 / kuby05. html; 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 / Immunology. html. www. immunologiclink. com /; pathbox. 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; aximl. imt. uni-marburg. de /. about. lek / AEP-Start. html; uci. kun. nl /. about. jraats / linksl. html; www. recab. uni-hd. de / immuno. bme. nwu. edu /; www. mrc-cpe. cam. ac. uk / imt-doc / public / 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 / wwwwagen. html; www. unith. ch /. about. honegger / AHOseminar / Slide01. html; www. cryst. bbk. ac. uk /. about. ubcg07s /; www. nimr. mrc. ac. uk / CC / ccaewg / ccaewg. html; www. path. cam. ac. uk /. about. mrc7 / humanization / TAHHP. html; www. ibt. unam. mx / vir / structure / stat aim. html; www. biosci. misouri. edu / smithgp / index. html; www. cryst. bioc. cam. ac. uk /. abo-ut. fmolina / Web-pages / Pept / spottech. html; www. jerini. de / fr products. html; www. patents. ibm. com / ibna. html. Kabat et al. , Sequences of Proteins of Immunological Interest, U.S.A. S. Dept. Health (1983), each incorporated herein by reference in its entirety. Such introduced sequences may be used to reduce immunogenicity, or for binding, affinity, binding rate, dissociation rate, binding force, specificity, half-life or any other suitable known in the art. Can be used to reduce, enhance or modify various properties.

  Framework residues in the human framework regions can be substituted with the corresponding residue from the CDR donor antibody to alter antigen binding, preferably to improve antigen binding. These framework substitutions can be performed by methods well known in the art, such as by modeling the interaction of CDRs with framework residues, to identify framework residues important for antigen binding, and for specific Identified by sequence comparisons to identify unusual framework residues at positions. (See, eg, Queen et al., US Pat. No. 5,585,089; Riechmann et al., Nature 332: 323 (1988), which are incorporated herein by reference in their entirety. See).). Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs that diagrammatically and display the three-dimensional structures that can be taken by the selected candidate immunoglobulin sequences are available. Examination of these displays allows analysis of the putative role of residues in the function of candidate immunoglobulin sequences, ie, analysis of residues that affect the ability of a candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen (s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Antibodies are prepared according to 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. MoI. 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); Studnikka 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. WO90 / 14443, WO90 / 14424, WO90 / 14430, EP229246, EP592,106; EP519,596, EP239,400, U.S. Pat. S. Pat. Nos. 5,565,332,5,723.323,5,976,862,5,824,514,5,817,483,5814476,5763192,5723323,5,76686,5,714,352,6,204, 023,6,180,370,5,693,762,5,530,101,5,585,089,5,225,539; 4,816,567 (each (references cited therein) Humanization using various techniques known in the art, such as, but not limited to, those described in, and incorporated herein by reference in their entirety. Is possible.

  The parent monoclonal antibody may be selected from a variety of monoclonal antibodies well known in the art that can bind to a specific target. These include anti-TNF antibody (US Patent No. 6,258,562), anti-IL-12 and / or anti-IL-12p40 antibody (US Patent No. 6,914,128); anti-IL-18 antibody (US2005) / 0147610A1), anti-C5, anti-CBL, 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-β2, anti-E-selectin, anti-factor VII, anti-Her2 / neu, anti-Fgp, anti-CD11 / 18, anti-CD14, anti-ICAM-3, anti-CD80, anti-CD4, anti-CD3, anti-CD23, anti-β2 -Integrin, anti-α4β7, anti-CD52, anti-HLADR, anti-CD22, anti-CD20, anti-MIF, anti-CD64 FcR), anti-TCRαβ, anti-CD2, anti-HepB, anti-CA125, anti-EpCAM, anti-gp120, anti-CMV, anti-gpIIbIIIa, anti-IgE, anti-CD25, anti-CD33, anti-HLA, anti-VNR integrin, anti-IL-1α, anti-IL -1β, anti-IL-1 receptor, anti-IL-2 receptor, anti-IL-4, anti-IL-4 receptor, anti-IL-5, 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 (Presta LG. 2005 Selection, design, and engineering of therapeutic antigens J Allergy Clin Immunol. 116: 6 and http://www.path.cam.ac. uk / ~ mrc7 / humanization / antibodies.html), but is not limited thereto.

The parent monoclonal antibody may also be selected from a variety of therapeutic antibodies approved for use, in clinical trials or in development for clinical use. Such therapeutic antibodies, rituximab ^{(Rituxan (R), IDEC /} Genentech / Roche) ( see, for example, U.S.Pat.NO.5,736,137), approved for the treatment of non-Hodgkin's lymphoma Chimeric anti-CD20 antibody; HuMax-CD20, an anti-CD20 currently under development by Genmab, U. S. Pat. NO. 5,500,362, anti-CD20 antibodies, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel) and PRO70769 ("Immunoglobulin Variants T and U PCs"). / US2003 / 040426), trastuzumab ^{(Herceptin (R), Genentech)} ( e.g., U.S.Pat.NO.5,677,171 reference), a humanized anti-Her2 / neu antibody approved to treat breast cancer ; pertuzumab ^{(rhuMab-2C4, Omnitarg (R} )), currently being developed by Genentech; U. S. Pat. No. Anti-Her2 antibody described in 4,753,894; cetuximab ^{(Erbitux (R), Imclone)} (U.S.Pat.NO.4,943,533; PCT WO96 / 40210), clinical trials for various cancers Chimeric anti-EGFR antibody; currently under development by ABX-EGF (US Pat. NO. 6,235,883), Abgenix-Immunex-Amgen; HuMax-EGFr (US Ser. NO. 10 / 172, 317), currently under development by Genmab; 425, EMD 55900, EMD 62000 and EMD 72000 (Merck KGaA) (US Pat. No. 5,558,864; Murthy et al. 1987, Arch Biochem Biophys. 252 (2). ): 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 WO95 / 20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22 (1-3): 129-46; Modjtahedi et al., 1993, Br J Cancer. 1993, 67 (2): Modjtahedi et al, 1996, Br J Cancer. 73 (2): 228-35; Modjtahedi et al, 2003, Int J Cancer, 10 5 (2): 273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (US Pat. No. 5,891,996; US Pat. NO. 6,506) Mateo et al, 1997, Immunotechnology, 3 (1): 71-81); mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering, Jungblue, et al., Jungblue, et al. (2): 639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National) Cancer Institute (PCT WO0162931A2); and SC10O (Scancell) (PCT WO01 / 88138); Alemtuzumab (Campath ^(R) , Millenium), a humanized monoclonal antibody currently approved for the treatment of B-cell chronic lymphocytic leukemia ; muromonab ^{-CD3 (OrthocloneOKT3 (R)),} Ortho Biotech / Johnson & anti-CD3 antibody developed by Johson, ibritumomab tiuxetan ^{(Zevalin (R)), IDEC} / anti-CD20 antibody developed by ScheringAG, gemtuzumab ozogamicin (Mylotarg ^{( R)} ), anti-CD33 (p67 tag) developed by Celltech / Wyeth Protein) antibodies, alefacept ^{(Amevive (R)),} anti-LFA-3Fc fusion developed by Biogen), abciximab developed by ^{Centocor / Lilly (ReoPro (R)} ), basiliximab developed by Novartis (Simulect ^{(R )),} Parijizumabu developed by Medimmune (Synagis ^(R)), infliximab ^(Remicade (R)), an anti-TNFα antibody developed by Centocor, adalimumab ^(Humira (R)), an anti-TNFα antibody developed by Abbott, Humicade ^(R), an anti-TNFα antibody developed by Celltech, etanercept ^{(Enbrel (R)), Immunex} / Amgen Anti-TNFαFc fusion developed by ABB-CBL, antibody developed by Abgenix CD147 antibody, ABX-IL8, anti-IL8 antibody developed by Abgenix, ABX-Mal, anti-MUC18 antibody developed by Abgenix, Pemutumomab (R1549,90Y-muHMFG1), anti-MUC1 being developed by Antisoma, Therex (R1550), anti-MUC1 antibody being developed by Antisoma, AngioMab being developed by Antisoma (AS1405), anc -1, Thioplatin (AS1407), Antegren ^(R) (natalizumab), B, developed by Antisoma Anti-α-4-β-1 (VLA-4) and α-4-β-7-antibodies developed by iogen, VLA-1 mAb, anti-VLA-1 integrin antibody developed by Biogen, LTBR mAb, Biogen Anti-lymphotoxin β receptor (LTBR) antibody developed by CAT-152, anti-TGF-β2 antibody developed by Cambridge Antibody Technology, J695, antibody developed by Cambridge Antibody Technology and AbbotIL12 antibody , CAT-192, Cambridge Antibody Technology and Genzyme, developed anti-TGFβ1 antibody, CAT-213, Cambridge Antibody Anti eotaxin 1 antibody being developed by dy Technology, Cambridge Antibody Technology and Human Genome Sciences Inc. LymphoStat-B ^(R) anti-Blys antibody, TRAIL-R1 mAb, Cambridge Antibody Technology and Human Genome Sciences Inc. Anti-TRAIL-R1 antibody, Avastin ^(R) bevacizumab, rhuMAb-VEGF), anti-VEGF antibody developed by Genentech, anti-HER receptor family antibody developed by Genentech, anti-tissue factor (ATF) ), Anti-tissue factor antibody developed by Genentech, Xolair ^(R) (omalizumab), anti-IgE antibody developed by Genentech, Raptiva ^(R) (Efelizumab), anti-CD11a antibody developed by Genentech and Xoma , MLN-02 antibody (formerly LDP-02), HuMax CD4, Ge, developed by Genentech and Millennium Pharmaceuticals Developed by anti-CD4 antibody developed by nmabu, anti-IL15 antibody developed by HuMax-IL15, Genmab and Amgen, developed by HuMax-Inflam, HuMax-Cancer, GenMax and Mdarex and Oxford GcoSciences developed by Genmab and Medarex Anti-heparanase I antibody, HuMax-Lymphoma developed by Genmab and Amgen, HuMax-TAC developed by Genmab, IDEC-131 and anti-CD40L antibody developed by IDEC Pharmaceuticals, IDEC-151 (Klenoliximab ), Developed by IDEC Pharmaceuticals Anti-CD4 antibody, IDEC-114, anti-CD80 antibody developed by IDEC Pharmaceuticals, IDEC-152, anti-CD23 developed by IDEC Pharmaceuticals, anti-macrophage migration factor (MIF) developed by IDEC Pharmaceuticals Anti-idiotype antibody developed by Imcone, anti-idrotype antibody developed by IMC-1C11, Imclone, anti-flk-1 antibody developed by ImClone, anti-flk-1 antibody developed by Imclone, anti-idiotype antibody developed by BEC2, Imclone VE-cadherin antibody, CEA-Cide ^{(R) (labetuzumab),} an anti-carcinoembryonic being developed by Immunomedics anti Original (CEA) antibody, LymphoCide ^(R) (epratuzumab), anti-CD22 antibody developed by Immunomedics, AFP-Cide developed by Immunomedics, MyelomaCide developed by Immunomedics, and developed by Immunomedics ProstaCide developed by Immunomedics, MDX-010, anti-CTLA4 antibody developed by Medarex, MDX-060, anti-CD30 antibody developed by Medarex, MDX-070 developed by Medarex, developed by Medarex MDX-018, Osidem ^(R) (IDM-I) And Medarex and Immuno-the Designed Molecules anti-Her2 antibody being developed ^by, HuMax (R) ^-CD4, an anti-CD4 antibody being developed by Medarex and Genmab, HuMax-IL15, Medarex and anti IL15 antibody being developed by Genmab Anti-TNFα antibody developed by CNTO148, Medarex and Centocor / J & J, anti-cytokine antibody developed by CNTO1275, CentocorJ & J, MOR101 and MOR102, anti-cell adhesion molecule-1 developed by MorphoSys (ICAM-I ) (CD54) antibody, MOR201, anti-fibroblast proliferation being developed by MorphoSys Child receptor receptor 3 (FGFR-3) antibody, Nuvion ^{(R) (visilizumab),} Protein Design anti-CD3 antibody being developed by ^Labs, HuZAF ^(R), anti-γ-interferon antibodies being developed by Protein Design Labs Anti-α5β1 integrin developed by Protein Design Labs, Protein Design
Anti being developed by Labs IL-12, ING-1 , anti-Ep-CAM antibody being developed by Xoma, Genentech and Xolair developed by Novartis ^(R) (omalizumab) humanized anti-IgE antibodies and MLN01, Xoma Including, but not limited to, anti-β2 integrin antibodies (all of the references cited above in this paragraph are expressly incorporated herein by reference). It is not a thing.

B. Construction of DVD molecule:
Dual variable domain immunoglobulin (DVD-Ig) molecules have two different light chain variable domains (VL) derived from two different parental mAbs linked in series via recombinant DNA technology, either directly or via a short linker, It is designed to be followed by a light chain constant domain. Similarly, the heavy chain comprises a constant domain CH1 and an Fc region (FIG. 1A) after two different heavy chain variable domains (VH) linked in series.

  Variable domains can be obtained using recombinant DNA technology obtained from a parent antibody produced by any one of the methods described above. In a preferred embodiment, the variable domain is a mouse heavy or light chain variable domain. More preferably, the variable domain is a CDR grafted or humanized variable heavy or light chain domain. Most preferably, the variable domain is a human heavy or light chain variable domain.

  In one embodiment, the first and second variable domains are directly linked to each other using recombinant DNA technology. In another embodiment, the variable domains are linked via a linker sequence. Preferably, two variable domains are linked. Three or more variable domains can also be linked directly or via a linker sequence. The variable domains can bind to the same antigen or can bind to different antigens. The DVD molecule of the present invention may comprise one immunoglobulin variable domain and one non-immunoglobulin variable domain, such as a receptor ligand binding domain, an enzyme active domain. A DVD molecule may also contain two or more non-Ig domains.

The linker sequence can be a single amino acid or polypeptide sequence. Preferably, the linker sequence, AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG ; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA (G 4 S) 4; SAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP QPKAAPSVVTLFPP; AKTTP; AKTTPPSVTPLAP; AKTTAP; AKTTAPPSVYPLAP; ASTKGP; ASTKGPSVFPLAP; GGGGSGGGGGSGGGGS; GENKVEY APALMALS; GPAKELTPLKEAKVS; and GHEAAAVMQVQYPAS. The choice of linker sequence is based on the crystal structure analysis of several Fab molecules. There is a natural flexible link between the variable domain in the Fab or antibody molecular structure and the CH1 / CL constant domain. This natural linkage comprises about 10 to 12 amino acid residues composed of 4-6 residues from the C-terminus of the V domain and 4-6 residues from the N-terminus of the CL / CH1 domain. DVDIg of the present invention was prepared using 5 to 6 amino acid residues at the N-terminus of CL or CH1, or 11 to 12 amino acid residues as linkers in the light and heavy chains of DVD-Ig, respectively. The N-terminal residue of the CL or CH1 domain, especially the first 5 to 6 amino acid residues, adopts a loop conformation without a strong secondary structure and therefore acts as a flexible linker between the two variable domains Is possible. Since the N-terminal residue of the CL or CH1 domain is part of the Ig sequence, it is a natural extension of the variable domain, thus minimizing to any extent any immunogenicity that may result from linkers and ligation. suppress.

  Other linker sequences can include any sequence of any length of the CL / CH1 domain, but not all residues of the CL / CH1 domain. For example, the first 5 to 12 amino acid residues of the CL / CH1 domain; the light chain linker can be derived from Cκ or Cλ; and the heavy chain linker can be Cγ1, Cγ2, Cγ3, Cγ4, Cα1. , Cα2, Cδ, Cε, and Cμ can be derived from any isotype of CH1. Linker sequences include Ig-like proteins (eg, TCR, FcR, KIR), G / S-based sequences (eg, G4S repeats); sequences derived from the hinge region and other natural sequences derived from other proteins, etc. It can also be derived from other proteins.

  In a preferred embodiment, the constant domain is linked to two linked variable domains using recombinant DNA technology. Preferably, the sequence comprising a linked heavy chain variable domain is linked to a heavy chain constant domain, and the sequence comprising a linked light chain variable domain is linked to a light chain constant domain. Preferably, the constant domains are a human heavy chain constant domain and a human light chain constant domain, respectively. Most preferably, the DVD heavy chain is further linked to the Fc region. The Fc region may be a native sequence Fc region or a variant Fc region. Most preferably, the Fc region is a human Fc region. In a preferred embodiment, the Fc region includes an Fc region obtained from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

  In the most preferred embodiment, two heavy chain DVD polypeptides and two light chain DVD polypeptides are combined to form a DVD-Ig molecule. A detailed description of specific DVD-Ig molecules that can bind to specific targets and methods of making them are described in the Examples section below.

C. Production of DVD Proteins The binding proteins of the present invention can be produced by any of a number known in the art. For example, in expression from a host cell, expression vectors encoding DVD heavy chain and DVD light chain are transfected into the host cell by standard techniques. Various forms of the term “transfection” refer to various techniques commonly used for introducing foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. It shall be included. While it is possible to express the DVD protein of the present invention in either prokaryotic or eukaryotic host cells, eukaryotic cells (particularly mammalian cells) are appropriately folded and immunized compared to prokaryotic cells. Expression of DVD proteins in eukaryotic cells is preferred, with expression in mammalian host cells being most preferred because of the greater tendency to assemble and secrete biologically active DVD proteins.

Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary (CHO cells) (Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220. And dhfr ^- CHO cells for use with DHFR selectable markers as described, for example, in RA Kaufman and PA Sharp (1982) Mol. Biol. 159: 601-621. ), NS0 myeloma cells, COS cells and SP2 cells. When a recombinant expression vector encoding a DVD protein is introduced into a mammalian host cell, or more preferably, the host cell is contained therein for a period of time sufficient to allow expression of the DVD protein in the host cell. DVD protein is produced by culturing host cells for a period of time sufficient to allow secretion of the DVD protein into the growing medium. DVD protein can be recovered from the culture medium using standard protein purification methods.

In a preferred system for recombinant expression of the DVD protein of the present invention, recombinant expression vectors encoding both DVD heavy chain and DVD light chain are introduced into dhfr ^- CHO cells by calcium phosphate-mediated transfection. The Within the recombinant expression vector, the DVD heavy and light chain genes are each operably linked to a CMV enhancer / AdMPL promoter control element to induce high levels of gene transcription. The recombinant expression vector also carries a DHFR gene that allows selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformant host cells are cultured to allow for expression of DVD heavy and light chains and intact DVD protein is recovered from the medium. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select for transformants, culture host cells, and recover DVD proteins from the medium. . Furthermore, the present invention provides a method for synthesizing the DVD protein of the present invention by culturing the host cell of the present invention in an appropriate medium until the DVD protein of the present invention is synthesized. The method can further comprise isolating the DVD protein from the medium.

  An important feature of DVD-Ig is that DVD-Ig can be produced and purified in a similar manner as a conventional antibody. The production of DVD-Ig results in a uniform single major product with the desired bispecific activity without any sequence modification of the constant region or any kind of chemical modification. Other previously described methods for making “bispecific”, “multispecific” and “multispecific multivalent” full-length binding proteins do not result in a single major product, instead Intracellular or secreted production of a mixture of assembled inactive monospecific, multispecific, multivalent full-length binding proteins and multivalent full-length binding proteins with different binding site combinations . By way of example, there are 16 possible combinations of heavy and light chains, based on the design described in Miller and Presta (PCT publication WO 2001/077342 (A1)). As a result, only 6.25% of the protein appears to be the desired active form. Typically, using standard chromatographic techniques used in large-scale manufacturing, it has not yet been possible to separate the fully active form of the protein from the inactive and partially active form of the protein. It has not been demonstrated.

  Surprisingly, the design of the “bispecific multivalent full-length binding protein” of the present invention is primarily a dual variable domain light chain that assembles into the desired “bispecific multivalent full-length binding protein”. And results in a double variable domain heavy chain.

  At least 50%, preferably 75% and more preferably 90% of the assembled and expressed dual variable domain immunoglobulin molecules are the desired bispecific tetravalent proteins. This aspect of the invention in particular enhances the commercial use of the invention. Thus, the present invention expresses a dual variable domain light chain and a double variable domain heavy chain in a single cell, resulting in a single major product of a “bispecific tetravalent full length binding protein” including.

  The present invention provides a preferred method for expressing a dual variable domain light chain and a double variable domain heavy chain in a single cell, resulting in a “major product” of a “bispecific tetravalent full-length binding protein” Here, however, the “major product” is over 50% of all assembled proteins including double variable domain light chains and double variable domain heavy chains.

  The present invention expresses dual variable domain light chains and double variable domain heavy chains in a single cell, resulting in a single “major product” of a “bispecific tetravalent full-length binding protein”. A preferred method is provided, wherein the “major product” is greater than 75% of all assembled proteins including double variable domain light chains and double variable domain heavy chains.

  The present invention expresses a dual variable domain light chain and a double variable domain heavy chain in a single cell, most often resulting in a single “major product” of a “bispecific tetravalent full-length binding protein”. A preferred method is provided, wherein the “major product” is greater than 90% of all assembled proteins including double variable domain light chains and double variable domain heavy chains.

II. Derivatized DVD binding protein:
One embodiment provides a labeled binding protein, wherein the binding protein of the invention is derivatized or linked to another functional molecule (eg, a peptide or protein). For example, the labeled binding protein of the present invention may comprise another antibody (eg, a bispecific antibody or diaboti), a detectable agent, a cytotoxic agent, a drug and / or another molecule (streptavidin core region or polyhistidine). The present invention (by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities such as proteins or peptides capable of mediating association of the binding protein Can be induced by functionally linking these binding proteins.

  Useful detectable agents that can derivatize the binding proteins of the present invention include fluorescent compounds. Typical fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. The binding protein can also be derivatized with a detectable enzyme such as alkaline phosphatase, horseradish peroxidase, glucose oxidase. If the bound protein is derivatized with a detectable enzyme, the antibody is detected by adding additional reagents used by the enzyme to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a detectable colored reaction product. The bound protein can be derivatized with biotin and also detected through indirect measurement of avidin or streptavidin binding.

  Another embodiment of the invention provides crystallized binding proteins and formulations and compositions comprising such crystals. In one embodiment, the crystallized binding protein has an in vivo half-life that is greater than the soluble counterpart of the binding protein. In another embodiment, the binding protein retains biological activity after crystallization.

  The crystallized binding proteins of the present invention can be produced according to methods known in the art and as disclosed in WO02072636, incorporated herein by reference.

  Another embodiment of the invention provides a glycosylated binding protein wherein the antibody or antigen binding portion thereof comprises one or more carbohydrate residues. Emerging in vivo protein production can go through further processing known as post-translational modification. In particular, sugar (glycosyl) residues can be added enzymatically (a method known as glycosylation). The resulting protein with covalently linked oligosaccharide side chains is known as a glycosylated protein or glycoprotein. An antibody is a glycoprotein having one or more carbohydrate residues in the Fc domain and in the variable domain. Carbohydrate residues in the Fc domain have an important effect on the effector function of the Fc domain with minimal effects on the antigen binding or half-life of the antibody (R. Jefferis, Biotechnol. Prog. 21 (2005 ), Pp. 11-16). In contrast, glycosylation of variable domains can affect the antigen-binding activity of antibodies. Glycosylation in the variable domain can have a negative impact on antibody binding affinity, presumably due to steric hindrance (Co, MS, et al., MoI. Immunol. (1993). ) 30: 1361-1367) or may result in increased affinity for the antigen (Wallick, SC, et al., Exp. Med. (1988) 168: 1099-1109; Wright, A., et al. , EMBO J. (1991) 10: 2717 2723).

  One aspect of the invention relates to generating glycosylation site variants in which the O-linked or N-linked glycosylation sites of the binding protein are mutated. One skilled in the art can make such variants using standard well-known techniques. Glycosylation site variants that retain biological activity but have increased or decreased binding activity are another object of the present invention.

  In yet another embodiment, the glycosylation of the antibody or antigen binding portion of the invention is modified. For example, an aglycosylated antibody can be made (ie, the antibody lacks glycosylation). Glycosylation can be modified, for example, to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be achieved, for example, by changing one or more sites of glycosylation within the antibody sequence. For example, it is possible to make one or more amino acid substitutions that result in the removal of one or more variable region glycosylation sites to remove glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such an approach is described in PCT Publication WO2003016466A2 and U.S. Pat. S. Pat. Nos. 5,714,350 and 6,350,861, each of which is hereby incorporated by reference in its entirety.

  In addition or alternatively, modified linkages of the invention having altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased biantennary GlcNAc structure Proteins can be made. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modification can be achieved, for example, by expressing the antibody in a host cell having an altered glycosylation mechanism. Cells with altered glycosylation mechanisms have been described in the art, in which they are used as host cells to produce antibodies with altered glycosylation by expressing a recombinant antibody of the invention. Can do. See, for example, Shields, R .; L. et al. (2002) J. Org. Biol. Chem. 277: 26733-26740; Umana et al. (1999) Nat. Biotech. 17: 176-1 and European Patent No: EP 1,176,195; PCT Publications WO 03/035835; WO 99/54342 80, each of which is incorporated herein by reference in its entirety. .

  Protein glycosylation depends on the amino acid sequence of the protein of interest and the host cell in which the protein is expressed. Different organisms can produce different glycosylation enzymes (eg, glycosyltransferases and glycosidases) and have different substrates (nucleotide sugars) available. Due to such factors, the protein glycosylation pattern and the composition of glycosyl residues may vary depending on the host system in which the protein is expressed. Glycosyl residues useful in the present invention can include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid. Preferably, the glycosylated binding protein comprises a glycosyl residue such that the glycosylation pattern is human.

  It is known to those skilled in the art that different protein glycosylation can result in different protein properties. For example, the potency of a therapeutic protein produced in a microbial host such as yeast and glycosylated using the yeast endogenous pathway is reduced compared to the potency of the same protein expressed in a mammalian cell such as a CHO cell line. Can be done. Such glycoproteins can also be immunogenic in humans and exhibit a reduced in vivo half-life after administration. Specific receptors in humans and other animals can recognize specific glycosyl residues and facilitate rapid removal of proteins from the bloodstream. Other deleterious effects may include protein folding, solubility, protease sensitivity, transport, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity or allergenic changes. Accordingly, those skilled in the art will recognize specific glycosylation compositions and patterns, eg, glycosylation compositions and patterns that are the same or at least similar to those produced in human cells or in species-specific cells of the intended subject animal. You may like the therapeutic protein you have.

  Expressing a glycosylated protein different from that of the host cell can be accomplished by genetically modifying the host cell to express a heterologous glycosylation enzyme. Using techniques known in the art, one of skill in the art can generate antibodies or antigen-binding portions thereof that exhibit human protein glycosylation. For example, glycosylated proteins (glycoproteins) produced in yeast strains exhibit the same protein glycosylation as that of animal cells, particularly human cells (U. S patent applications 20040018590 and 200201337134 and PCT publication WO2005100584A2). ), Yeast strains have been genetically modified to express a non-naturally occurring glycosylation enzyme.

  In addition to binding proteins, the present invention also relates to anti-idiotype (anti-Id) antibodies specific for such binding proteins of the present invention. An anti-Id antibody is an antibody that recognizes unique determinants generally associated with the antigen-binding region of another antibody. Anti-Id can be prepared by immunizing an animal with a binding protein or CDR-containing region thereof. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce anti-Id antibodies. Anti-Id antibodies can also be used as “immunogens” to induce an immune response in yet another animal producing so-called anti-anti-Id antibodies.

  In addition, using a library of host cells that are genetically modified to express various glycosylation enzymes such that the member host cells of the library produce a protein of interest having a variant glycosylation pattern, It is understood by those skilled in the art that the protein of interest can be expressed. One skilled in the art can then select and isolate a protein of interest having a particular novel glycosylation pattern. Preferably, the protein with a particularly selected novel glycosylation pattern exhibits improved or modified biological properties.

III. Use of DVD-Ig Since the binding proteins of the invention can bind to two or more antigens, the binding proteins of the invention can be enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA) or tissues. Conventional immunoassays such as immunohistochemistry can be used to detect the antigen (eg, in a biological sample such as serum or plasma). DVD-Ig is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Examples of luminescent materials include luminol; and examples of suitable radioactive materials include ³ H ₁ , ¹⁴ C _, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho or ¹⁵³ Sm is included.

  The binding proteins of the invention are preferably capable of neutralizing antigen activity both in vitro and in vivo. Thus, such DVD-Ig inhibits antigenic activity, for example, in cell culture media containing the antigen, in human subjects, and in other mammalian subjects that have an antigen with which the binding protein of the invention cross-reacts. Can be used to In another embodiment, the present invention provides a method of reducing antigen activity in a subject suffering from a disease or disorder in which antigen activity is detrimental. The binding proteins of the invention can be administered to human subjects for therapeutic purposes.

  As used herein, the term “disease in which antigenic activity is detrimental” means that the presence of an antigen in a subject suffering from the disease is responsible for the pathophysiology of the disease or worsening of the disease Including illnesses and other illnesses that have been shown or suspected to be contributing factors. Thus, a disease in which antigenic activity is detrimental is a disease in which a decrease in antigenic activity is expected to alleviate the symptoms and / or progression of the disease. Such diseases are manifested, for example, by increased antigen concentrations in biological fluids of patients suffering from the disease (eg, increased concentrations of antigens in the subject's serum, plasma, synovial fluid, etc.). Can be done. Non-limiting examples of diseases that can be treated with the binding proteins of the present invention include those discussed below and in the section regarding pharmaceutical compositions of the antibodies of the present invention.

  The DVD-Ig of the present invention can bind to one antigen or multiple antigens. Such antigens include, but are not limited to, the targets listed in the following databases (the databases are incorporated herein by reference). These target databases include the following:

Target of therapy (http://xin.cz3.nus.edu.sg/group/cjttd/ttd.asp);
Cytokines and cytokine receptors (http://www.cytokinewebfacts.com/, http://www.copywithcytokines.de/cope.cgi and http://cmbi.bjmu.edu.cn/cmfidaCcn/cmbida Database / cytokine. medic. kumamoto-u. ac. jp / CFC / indexR. html);
Chemokines (http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.html);
Chemokine receptors and GPCRs (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);
Receptor (http://www.iufar-db.org/iufar-rd/list/index.html);
Cancer target (http://cged.hgc.jp/cgi-bin/input.cgi);
Proteins secreted as potential antibody targets (http://spd.cbi.pku.edu.cn/);
Protein kinases (http://spd.cbi.pku.edu.cn/) and human CD markers (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 is useful as a therapeutic agent to simultaneously block two different targets to enhance efficacy / safety and / or increase patient coverage. Such targets can include soluble targets (IL-13 and TNF) and cell surface receptor targets (VEGFR and EGFR). Such targets may be between tumor cells and T cells (Her2 and CD3) for cancer therapy, or between autoreactive and effector cells for autoimmunity / transplantation, or certain diseases It can also be used to induce re-induced cytotoxicity between any target cells and effector cells to eliminate disease-causing cells.

  In addition, DVD-Ig can be used to trigger receptor clustering and activation when designed to target two different epitopes on the same receptor. This can have utility in making anti-GPCR therapeutics with agonist and antagonist effects. In this case, DVD-Ig targets two different epitopes present on one cell for clustering / signaling (two cell surface molecules) or signaling (on one molecule). Can be used. Similarly, DVD-Ig molecules target CTLA-4 ligation and negative signals by targeting two different epitopes of the CTLA-4 extracellular domain (or two copies of the same epitope), resulting in downregulation of the immune response. It can be designed to provoke. CTLA-4 is a clinically validated target for therapeutic treatment of a number of immunological diseases. CTLA-4 / B7 interaction negatively regulates T cell activation by attenuating T cell proliferation following cell cycle progression, IL-2 production and activation, and CTLA-4 (CD152) Restraint down-regulates T cell activation and promotes induction of immune tolerance. However, since CTLA-4 activation requires ligation, the strategy of weakening T cell activation by constraining CTLA-4 by agonistic antibodies has not been successful. The molecular interaction of CTLA-4 / B7 is a “distorted zipper” sequence, as shown by crystal structure analysis (Stamper 2001 Nature 410: 608). However, none of the currently available CTLA-4 binding reagents, including anti-CTLA-4 monoclonal antibodies, have linking properties. Several attempts have been made to address this problem. In one case, a single chain antibody with a cellular component attached was made that significantly inhibited allogeneic rejection in mice (Hwang 2002 JI 169: 633). In another case, a single chain antibody against CLTA-4 linked to an artificial APC surface was made and shown to attenuate T cell responses (Griffin 2000 JI 164: 4433). In either case, CTLA-4 ligation was achieved with membrane-bound antibodies placed in close proximity in the artificial system. Although these experiments provide evidence for the concept of immune downregulation by causing negative signaling of CTLA-4, the reagents used in these reports are not suitable for therapeutic applications. To this end, CTLA-4 ligation can be achieved by using DVD-Ig molecules that target two different epitopes (or two copies of the same epitope) of the CTLA-4 extracellular domain. The rationale is that the distance through the two binding sites of IgG (approximately 150 to 170 mm) is too large for the active linkage of CTLA-4 (30 to 50 mm between two CTLA-4 homodimers). It is. However, the distance between the two binding sites on the DVD-Ig (one arm) is much shorter, in the range of 30 to 50 mm, allowing for proper coupling of CTLA-4.

  Similarly, DVD-Ig can target two different elements of the cell surface receptor complex (eg, IL-12Rα and β). In addition, DVD-Ig can target CR1 and soluble proteins / pathogens to induce rapid elimination of target soluble proteins / pathogens.

  In addition, the DVD-Ig of the present invention provides intracellular delivery (targeting internalization receptors and intracellular molecules), delivery into the brain (transferrin receptor and central nervous system to cross the blood brain barrier). Tissue-specific delivery (targeting tissue markers and disease mediators to obtain higher potency and / or lower toxicity with enhanced local PK), such as targeting disease mediators) Can be used for. DVD-Ig can also serve as a carrier protein to deliver the antigen to a specific location via binding of the antigen to a non-neutralizing epitope and can increase the half-life of the antigen It is. In addition, DVD-Ig can be physically linked to medical devices implanted in a patient or designed to target these medical devices (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 and functionalization of medical devices, Hildebrand, H Blanchemain, N .; Mayer, G .; Chai, F .; efbvre, M .; Boschin, F., Surface and Coatings Technology (2006), 200 (22-23), 6318-6324; , Biomaterials (2006), 27 (11), 2450-2467; Mediation of the cytokinetic network of the orthopedic devices, Marques, AP, Hunt, J. A. R .; dable Systems in Tissue Engineering and Regenerative Medicine (2005), see 377-397). In summary, inducing the appropriate type of cells to the site of the medical implant can promote healing and recovery of normal tissue function. Alternatively, inhibition of mediators (including but not limited to cytokines) released upon device implantation by a DVD coupled to the device or a DVD targeted to the device is also provided. For example, stents have been used for many years in interventional cardiology to clean blocked arteries and improve blood flow to the myocardium. However, traditional bullion stents are known to cause restenosis (reducing narrowing of arteries in the treated area) in some patients and can cause blood clotting. Recently, a stent coated with an anti-CD34 antibody has been described, which reduces the occurrence of restenosis and prevents the occurrence of blood clotting by capturing endothelial progenitor cells (EPC) circulating throughout the blood. To do. Endothelial cells are cells that are aligned with blood vessels and allow blood to flow smoothly. EPC adheres to the hard surface of the stent to form a smooth layer that not only promotes healing, but also prevents restenosis and blood clotting, complications that have traditionally been associated with the use of stents. (Aoji et al. 2005 J Am Coll Cardiol. 45 (10): 1574-9). In addition to improving results for patients in need of stents, improvements have been suggested for patients in need of cardiovascular bypass surgery. For example, an artificial blood vessel conduit (artificial artery) coated with an anti-EPC antibody eliminates the need to use an artery from the patient's foot or arm for bypass surgical implantation. This shortens the surgery and anesthesia time, thereby reducing coronary surgery death. DVD-Ig includes (but is not limited to) cell surface markers (such as CD34) and proteins (or lipids and polysaccharides) coated on implanted devices to facilitate cell mobilization. It is designed to bind to all kinds of epitopes). Such an approach is generally applicable to other medical implants. Alternatively, the DVD-Ig can be coated on a medical device and requires more fresh DVD-Ig when implanted (or such as aging and modification of a previously filled DVD-Ig). Release all DVDs from the device (according to all other demands obtained) and the device can be refilled by systemically administering a fresh DVD-Ig to the patient, in which case the DVD- Ig binds to a target of interest having one set of binding sites (cytokines, cell surface markers (such as CD34), etc.) and coated on a device having another set of binding sites (proteins, all types of Designed to bind to epitopes (including but not limited to lipids, polysaccharides and polymers) This technology is useful for coated implants It has the advantage that the extended.

A. Use of DVD-Ig in Various Diseases The DVD-Ig molecules of the present invention are also useful as therapeutic molecules for treating various diseases. Such DVD molecules can bind to one or more targets involved in a particular disease. Examples of such targets in various diseases are described below.

1. Human autoimmune and inflammatory responses C5, CCL1 (1-309), 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), CXCLII (I-TAC / IP- 9), CXCL12 (SDFl), CXCL13, CXCL1 4, 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 SCYE1 (endothelial monocyte activating 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, AFF4, TRAF4, TRAF4 ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z, CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44, HAVCR2, OPRD1, T2R7L4T TLR6, TLR7, TLR8, TLR9, TLR10, BLR1, CCL1, CCL2, C CL3, 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, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11, CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, RH, MPR1, MPH C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J01 8, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1, IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7ST, IL7ST IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11, IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, EL13RA1, IL13RA2, IL15, IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19 KITLG, LEP, LTA, LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, DGF1, 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 Many proteins, such as RNF110 (ZNF144), are presumed to be involved in general autoimmune and inflammatory responses. In one aspect, a DVD-Ig is provided that is capable of binding to one or more of the targets listed above.

2. Asthma Allergic asthma is characterized by the presence of eosinophilia, goblet cell dysplasia, epithelial cell changes, airway hypersensitivity (AHR) and Th2 and Th1 cytokine expression and elevated serum IgE levels. It is now widely accepted that airway inflammation is a central factor underlying the development of asthma, including inflammatory cells such as T cells, B cells, eosinophils, mast cells and macrophages, and cytokines. And complex interactions of these secreted mediators such as chemokines are involved. Corticosteroids are today the most important anti-inflammatory treatment for asthma, but their mechanism of action is nonspecific and there are safety concerns, especially in the young patient population. Thus, there is a need for the development of more specific and targeted therapies. There is increasing evidence that IL-13 in mice mimics many of the characteristics of asthma, such as AHR, mucosal hypersecretion and airway fibrosis, independent of eosinophilic inflammation. (Finoto et al., International Immunology (2005), 17 (8), 993-1007; Padilla et al., Journal of Immunology (2005), 174 (12), 8097-8105).

  IL-13 is presumed to have a central role in causing pathological responses with asthma. The development of anti-IL-13 monoclonal antibody therapy to suppress the effects of IL-13 in the lung is an exciting new approach that offers considerable promise as a novel treatment for asthma. However, other mediators of different immunological pathways are also involved in the development of asthma, and blocking these mediators in addition to IL-13 can provide additional therapeutic benefits. Such target pairs include, but are not limited to, pro-inflammatory cytokines such as IL-13 and tumor necrosis factor-α (TNF-α). TNF-α can amplify inflammatory responses in asthma and can be associated with disease severity (McDonnell, et al., Progress in Respiratory Research (2001), 31 (New Drugs for Asthma, Allergy and 7). -250). This blocks both IL-13 and TNF-α, suggesting that it may have beneficial effects, particularly in severe airway disease. In a preferred embodiment, the DVD-Ig of the present invention binds to the targets IL-13 and TNFα and is used to treat asthma.

  Animal models, such as the OVA-induced asthma mouse model, that can assess both inflammation and AHR are known in the art and used to measure the ability of various DVD-Ig molecules to treat asthma. obtain. Animal models for studying asthma are described by Coffman, et al. , Journal of Experimental Medicine (2005), 201 (12), 1875-1879; Lloyd, et al. , Advances in Immunology (2001), 77, 263-295; Boyce et al. , 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 the routine safety assessment of these target pairs, a specific test for the degree of immunosuppression is assured and can help in selecting the best target pair (Luster et al., Toxology (1994), 92 (1-3), 229-43; Descottes, et al., Developments in biologic standardization (1992), 77 99-102; -257).

  Based on the rationale disclosed above, and using the same evaluation model for efficacy and safety, other DVD-Ig molecules can bind and be useful for treating asthma A target pair can be determined. Preferably, such targets include IL-13 and IL-1β; IL-1β is also implicated in the inflammatory response in asthma; inflammation such as IL-13 and IL-9 IL-13 and cytokines and chemokines involved in IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL IL-13 and TGF-β; IL-13 and LHR agonists; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and IL-13 and ADAM8. It is not limited to. The present invention relates to 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, KTILG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL18R TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CL24, CX3CL1, CXCL1, CXCL2, CXCL3, XCL1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STAT1, TFX1, TBX1, TFX21 Also provided is a DVD-Ig capable of binding to one or more targets involved in asthma selected from the group consisting of CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR and chitinase.

3. Rheumatoid arthritis Rheumatoid arthritis (RA), a systemic disease, is characterized by a chronic inflammatory response in joint synovial fluid, with cartilage degeneration and adjacent articular bone. Many pro-inflammatory cytokines are expressed in affected joints, such as TNF, chemokines and growth factors. Systemic administration of anti-TNF antibodies or sTNFR fusion proteins to mouse models of RA has been shown to be anti-inflammatory and joint protective. A clinical study in which the activity of TNF in RA patients was blocked by intravenously administered infliximab (chimeric anti-TNF monoclonal antibody (mAB)) (Harriman G, Harper LK, Schable TF. 1999 Summary of clinical tristrips) arthritis using infliximab, an anti-TNF alpha treatment. Ann Rheum Dis 58 Suppl 1: 161-4) TNF is IL-6, IL-8, MCP-1 and VEGF production, immune and inflammatory cells into joints Evidence provided that it controls mobilization, angiogenesis, and reduction of blood levels of matrix metalloproteinases-1 and -3. A deeper understanding of the inflammatory pathway in rheumatoid arthritis has led to the identification of other therapeutic targets involved in rheumatoid arthritis. In the past, interleukin-6 antagonist (MRA), CTLA4Ig (adatacept, Genovese Mc et al 2005 Abatacept for rheumatoid arthritis refractory to tumor necrosis factor 35. Promising therapies such as (Rituximab, Okamoto H, Kamatani N. 2004 Rituximab for rheumatoid arthritis. N Engl J Med. 351: 1909) have already been tested in randomized controlled clinical trials. Other cytokines such as interleukin-15, interleukin-17, and interleukin-18 have been identified and shown to be beneficial in animal models, and clinical trials of these factors are currently underway. Bispecific antibody therapy in combination with anti-TNF and another mediator has great potential in increasing clinical efficacy and / or patient coverage. For example, blocking both TNF and VEGF, both involved in the pathophysiology of RA, has the potential to eradicate inflammation and angiogenesis. Including, but not limited to, TNF and IL-18, TNF and IL-12; TNF and IL-23; TNF and IL-1β; TNF and MDDF; TNF and IL-17; and TNF and IL-15. ), Blocking other pairs of targets involved in RA with specific DVD Igs. In addition to the routine safety assessment of these target pairs, a specific test for the degree of immunosuppression is assured and can help in selecting the best target pair (Luster et al., Toxology (1994), 92 (1-3), 229-43; Descottes, et al., Developments in biologic standardization (1992), 77 99-102; Hart et al., Journal of Allergy and Clinical Immunology (200) 1 (200) -257). The usefulness of DVDIg molecules for the treatment of rheumatoid arthritis can be assessed using preclinical animal RA models, such as the mouse model of arthritis induced by collagen. Other useful models are also well known in the art (see Brand DD., Comp Med. (2005) 55 (2): 114-22).

4). SLE
The immunopathogenic feature of SLE is polyclonal B cell activation, which results in hyperglobulinemia, autoantibody production and immune complex formation. The fundamental abnormality appears to be that T cells cannot suppress the forbidden B cell clones due to general T cell dysregulation. Furthermore, B and T cell interactions are facilitated by several cytokines, such as IL-10 and costimulatory molecules such as CD40 and CD40L and B7 and CD28 and CTLA-4 that initiate a second signal. These interactions, along with the elimination of damaged phagocytes of immune complexes and apoptotic material, perpetuate the immune response due to the resulting tissue injury. The following targets may be involved in SLE and have the potential to be used for the DVD-Ig approach for therapeutic intervention. B cell targeted therapy: 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, HDAC11, ILH KLF6, TNFRSF7, CD28, CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TN SF7, CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3, MS4A1, ST6GAL1, CD1C, CHST10, HLA-A, HLA-DRA and NT5E; costimulatory signals: CTLA4 or B7. 1 / B7.2; Inhibition of B cell survival "BlyS, BAFF; complement inactivation: C5; cytokine regulation. The central principle is that the overall biological response in any tissue is the result of a balance between local levels of pro-inflammatory or anti-inflammatory cytokines (Sfikkais PP et al 2005 Curr Opin). Rheumatol 17: 550-7). SLE is thought to be a Th2-induced disease with an elevation reported in the serum IL-4, IL-6, IL-10 literature. Also contemplated is a DVD Ig capable of binding to one or more targets selected from the group consisting of IL-4, IL-6, IL-10, INF-a and TNF-a. The target combinations discussed above enhance therapeutic efficacy against SLE that can be examined in a number of lupus preclinical models (Peng SL (2004) Methods Mol Med .; 102: 227). -72).

5. Multiple Sclerosis Multiple sclerosis (MS) is a complex human autoimmune disease whose etiology is largely unknown. Immunological destruction of myelin basic protein (MBP) through the nervous system is a major etiology of multiple sclerosis. MS is a disease of complex lesions with infiltration by CD ⁴⁺ and CD ⁸⁺ T cells and a response disease within the central nervous system. Expression in the central nervous system of cytokines, reactive nitrogen species and costimulatory molecules has all been described in MS. Of primary concern are immunological mechanisms that contribute to the development of autoimmunity. In particular, antigen expression, cytokine and leukemia interactions and regulatory T cells, which help balance / modulate other T cells such as Th1 and Th2 cells, are important areas for therapeutic target identification.

  IL-12 is a pro-inflammatory cytokine produced by APC and promotes differentiation of Th1 effector cells. IL-12 is produced in developing lesions in MS patients and animals affected by EAE. Previously, interference in the IL-12 pathway effectively suppresses EAE in rodents and in vivo IL-12p40 with anti-IL-12 mAb in a myelin-induced EAE model in common marmoset Neutralization has been shown to have a beneficial effect.

  TWEAK is a member of the TNF family that is constitutively expressed in the central nervous system (CNS) and has pro-inflammatory, proliferative or apoptotic effects depending on the cell type. Its receptor Fn14 is expressed in the central nervous system by endothelial cells, reactive astrocytes and neurons. TWEAK and FN14 mRNA expression increased in the spinal cord during experimental autoimmune encephalomyelitis (EAE). Anti-TWEAK antibody treatment in EAE induced in C57BL / 6 mice by myelin oligodendrocyte glycoprotein (MOG) reduced the severity of disease and reduced leukocyte infiltration when mice were treated after the prime phase. Brought.

  One embodiment of the present invention includes IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200, IFNγ, GM-CSF, FGF, C5, CD52. And a DVDIg molecule capable of binding to one or more, preferably two, targets selected from the group consisting of CCR2. Preferred embodiments include bispecific anti-IL-12 / TWEAKDVDIg as a beneficial therapeutic for the treatment of MS. Several animal models for evaluating the usefulness of DVD molecules for treating MS are known in the art (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; VK, et al., (1999) J Exp Med. 189 (7): 1033-42; Owens T, et al., (1995) Neurol Clin. L3 (1): 51-73; and 't Hart BA, et al., (2005) J Im unol 175 (7): see 4761-8). In addition to the routine safety assessment of these target pairs, a specific test for the degree of immunosuppression is assured and can help in selecting the best target pair (Luster et al., Toxology (1994), 92 (1-3), 229-43; Descottes, et al., Developments in biologic standardization (1992), 77 99-102; Jones R. 2000 Roverizumab (ICOS Corp)] Drugs. 3 (4): 442-6). reference).

6). Sepsis The pathophysiology of sepsis is initiated by the outer membrane components of both gram-negative organisms (lipopolysaccharide [LPS], lipid A, endotoxin) and gram-positive organisms (lipoteic acid, peptidoglycan). These outer membrane components can bind to the CD14 receptor on the surface of monocytes. With the recently described Toll-like receptor, the signal is then transmitted to the cells, and the final pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1) Bring about production. Overwhelming inflammatory and immune responses are an essential feature of septic shock and play a central role in the pathogenesis of tissue damage, multiple organ failure and death induced by sepsis. Cytokines, particularly tumor necrosis factor (TNF) and interleukin (IL) -1, have been shown to be critical mediators of septic shock. These cytokines have a direct toxic effect on tissues and also activate phospholipase A2. These and other effects result in increased concentrations of platelet activating factor, enhanced nitric oxide synthase activity, enhanced tissue infiltration by neutrophils and enhanced neutrophil activity.

  Treatment of sepsis and septic shock is still a clinical challenge, and some recent prospective clinical trials using biological response modifiers that target inflammatory responses (ie, anti-TNF, anti-MIF) Only showed clinical benefit. Recently, interest has shifted to treatments aimed at reversing the accompanying period of immunosuppression. Studies in laboratory animals and critically ill patients have shown that increased apoptosis of lymphoid organs and some parenchymal tissues contributes to this immunosuppression, anergy and organ system dysfunction. During sepsis syndrome, lymphocyte apoptosis can be triggered by the absence of IL-2 or by the release of glucocorticoids, granzymes or so-called “death” cytokines: tumor necrosis factor α or Fas ligand. Apoptosis proceeds via autoactivation of cytoplasmic and / or mitochondrial caspases that can be affected by pro-apoptotic and anti-apoptotic members of the Bcl-2 family. In experimental animals, treatment with inhibitors of apoptosis can not only suppress apoptosis of lymphoid cells, but also improve prognosis. Clinical trials with anti-apoptotic factors remain less feasible, mainly due to technical difficulties associated with their administration and tissue targeting, but inhibition of lymphocyte apoptosis is attractive for patients with sepsis A target for successful treatment. Similarly, bispecific factors that target both inflammatory mediators and apoptosis mediators may have additional benefits. One aspect of the present invention is TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptor, 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, GTAPR, LTR2 , HMOX1, Midkine, IRAK1, NFKB2, SERPINA1, SERPINE1 and TREM1 selected from the group consisting of one or more targets involved in sepsis, preferred DVDIg capable of binding to two targets. The efficacy of such DVDIg against sepsis can be assessed in preclinical animal models known in the art (Buras JA, et al., (2005) Nat Rev Drug Disc. 4 (10): 854- 65 and Calandra T, et al, (2000) Nat Med. 6 (2): 164-70)).

7). 7.1 Neurodegenerative diseases Chronic neurodegenerative diseases are usually age-dependent diseases characterized by progressive loss of neuronal function (neural cell death, demyelination), loss of motility and loss of memory. It is. Findings emerging as the underlying mechanism of chronic neurodegenerative diseases (eg, Alzheimer's disease) indicate complex etiology, and various factors such as age, glycemic status in their development and progression , Amyloid production and multimerization, accumulation of acquired glycation end products (AGE) binding to RAGE (receptor for AGE), increased brain oxidative stress, decreased brain blood flow, inflammatory cytokines and chemokines It has been observed that neuroinflammation, including release, neuronal dysfunction and microglia activation contribute. Thus, these chronic neurodegenerative diseases exhibit complex interactions between multiple cell types and mediators. Treatment strategies for such diseases are limited and include non-specific anti-inflammatory agents (eg, corticosteroids, COX inhibitors) or factors that suppress neuronal loss and / or synaptic function and the inflammatory process Occupying the majority. These therapies cannot stop disease progression. Recent studies have shown that more targeted therapies such as antibodies to soluble Ab peptides (Ab oligomeric forms) can not only help stop disease progression, but also maintain memory. It also suggests that it can help. These preliminary observations indicate that specific therapies that target more than one disease mediator (eg, Ab and pro-inflammatory cytokines (such as TNF)) target a single disease mechanism It has been suggested that it may provide a much better therapeutic effect on chronic neurodegenerative diseases than what has been observed in some cases (eg soluble A-baron) (CE. Shepherd, et al, Neurobiol Aging). 2005 Oct 24; Nelson RB., Curr Pharm Des. 2005; l 1: 3335; William L. Klein.; Neurochem Int.2002; 41: 345; Michel C Janelsins, et al., 2Ne. 23; Soloman B. et al. Irr Klyubin, et al., Nat Med. 2005; 11: 556-61; Arancio O, et al., EMBO Journal (2004) 1-10; Bornemann KD, et al., Curr Alzheimer Res. , 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).

  The DVD-Ig molecules of the present invention can bind to one or more targets involved in chronic neurodegenerative diseases such as Alzheimer's disease. Such targets include all mediators (soluble or cell surface) presumed to be involved in the pathogenesis of AD, such as AGE (S100A, amphoterin), pro-inflammatory cytokines (eg IL-1), chemokines (Eg, MCP1), molecules that inhibit nerve regeneration (eg, Nogo, RGM-A), and molecules that enhance neurite growth (neurotrophin), but are not limited thereto. The efficacy of DVD-Ig molecules can be validated in preclinical animal models such as transgenic mice that overexpress amyloid precursor protein or RAGE and develop Alzheimer's-like symptoms. In addition, DVD-Ig molecules can be constructed and tested for efficacy in animal models, and the best therapeutic DVD-Ig can be selected for testing in human patients. DVD-Ig molecules can also be used for the treatment of other neurodegenerative diseases such as Parkinson's disease. α-synuclein is involved in Parkinson's disease. DVD-Ig, which can target alpha-synuclein and inflammatory mediators (TNP, IL-1, MCP-1, etc.) can prove to be an effective treatment for Parkinson's disease, As envisioned in the present invention.

7.2 Regeneration of nerve cells and spinal cord injury Despite the growing knowledge of pathological mechanisms, spinal cord injury (SCI) is still a devastating symptom and is characterized by high medical demands. Indications. Many spinal cord injuries are contusions or compression injuries, usually secondary damage mechanisms that worsen the initial damage and lead to significant enlargement of the lesion site (especially more than 10 times) following the primary damage. (Inflammatory mediators such as cytokines and chemokines) occur. These primary and secondary mechanisms in SCI are very similar to those in brain damage caused by other means, such as stroke. There is no satisfactory treatment and high dose bolus injection of methylprednisolone (MP) is the only therapy used within a narrow time frame of 8 hours after injury. However, this treatment is only intended to prevent secondary damage without any significant functional recovery. Great criticism has been made for severe side effects such as lack of clear effects and subsequent immunosuppression with infections and severe histopathological muscle changes. While other drugs, biology or small molecules that stimulate endogenous regenerative potential have not been approved, promising therapeutic principles and drug candidates have recently shown efficacy in animal models of SCI. In many cases, the lack of functional recovery in human SCI is caused by factors that inhibit neurite outgrowth at the lesion site, in scar tissue, in myelin, and on injured cells. Such factors are myelin-associated proteins NogoA, OMgp and MAG, RGMA, scar-associated CSPG (chondroitin sulfate proteoglycan) and inhibitors for reactive astrocytes (some semaphorins and ephrins). However, at the lesion site not only growth inhibitory molecules are found, but also neurite growth stimulating factors such as neurotrophins, laminin L1 and others. This reduction in inhibitory effects can shift the balance from growth inhibition to growth promotion, so this coordination of neurite growth-inhibiting molecules and neurite growth-promoting molecules can lead to a single factor such as NogoA or RGMA. It can be explained that blocking resulted in significant functional recovery in the rodent SCI model. However, the recovery observed by blocking a single neurite growth inhibitory molecule was not complete. To achieve faster and more significant recovery, block two neurite growth inhibitory molecules (eg, Nogo and RGMA) or block neurite growth inhibitory molecules and It may be desirable to enhance function (eg, Nogo and neurotrophin) or block neurite outgrowth inhibiting molecules (eg, Nogo) and pro-inflammatory molecules (eg, TNF) (McGee AW , Et al., Trends Neurosci. 2003; 26: 193; Marco Domenonici, et al., J Neuro Sci.2005; 233: 43; Milan Makwanal, et al., FEBS J.2005; 272: 2628; Dickson, Science. 2 002; 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 J. ur. 2004; 91; 1018).

  In one embodiment, NgR and RGMA; NogoA and RGMA; MAG and RGMA; OMGp and RGMA; RGMA and RGMB; CSPG and RGMA; DVD-Ig capable of binding to a target pair, such as an Aβ globulomer specific antibody combined with an antibody that promotes axonal sprouting is provided. Dendritic lesions are a very early sign of AD and NOGOA is known to limit dendritic growth. Such kind of ab can be combined with any of the SCI candidate (myelin protein) Abs. Other DVD-Ig targets may include any combination of NgR-p75, NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo, Lingo-Troy, Lingo-p75, MAG or Omgp. Furthermore, the targets include all mediators (soluble or cell surface) that are presumed to be involved in neurite inhibition, such as Nogo, Ompg, MAG, RGMA, semaphorin, ephrin, soluble Ab, pro-inflammatory Sex cytokines (eg, IL-1), chemokines (eg, MIPIa), molecules that inhibit nerve regeneration may also be included. The efficacy of anti-nogo / anti-RGMA or similar DVD-Ig molecules can be validated in preclinical animal models of spinal cord injury. In addition, these DVD-Ig molecules can be constructed and tested for efficacy in animal models, and the best therapeutic DVD-Ig can be selected for testing in human patients. In addition, DVD-Ig molecules targeting two different ligand binding sites on RAGE that bind to a single receptor, eg, the three ligands Nogo, Ompg and MAG, and Rab that bind to Ab and S100A. It is possible to build. Furthermore, neurite growth inhibitors, such as nogo and nogo receptors, also play a role in suppressing nerve regeneration in immunological diseases such as multiple sclerosis. Inhibition of nogo-nogo receptor interaction has been shown to enhance recovery in animal models of multiple sclerosis. Thus, one immune mediator, eg, a cytokine such as IL-12, and a neurite growth inhibitory molecule, eg, a DVD-Ig molecule that can block the function of nogo or RGM, is immune or neurite growth inhibitory. It can be faster and have greater efficacy than blocking only the agent molecule.

8). Tumor Disease Monoclonal antibody therapy has emerged as an important treatment for cancer (von Mehren M, et al 2003 Monoclonal antibody therapy for cancer. Annu Rev Med .; 54: 343-69). Antibodies can exert anti-tumor effects by inducing apoptosis, re-induced cytotoxicity, interfering with ligand-receptor interactions, or suppressing expression of proteins critical for the neoplastic phenotype. Furthermore, antibodies can target components of the tumor microenvironment and disrupt essential structures such as the formation of vasculature associated with the tumor. The antibody can also target a receptor whose ligand is a growth factor (such as an epidermal growth factor receptor). Thus, the antibody inhibits the natural ligand that stimulates cell proliferation from binding to the targeted tumor cells. Alternatively, the antibody can induce an anti-idiotype network, complement-mediated cytotoxicity or antibody-dependent cytotoxicity (ADCC). The use of bispecific antibodies that target two separate tumor mediators may provide additional advantages over monospecific therapy. DVDIg that can bind to the following pairs of targets is also envisaged to treat tumor diseases. 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 target combinations include one or more members of the EGF / erb-2 / erb-3 family. Other targets (one or more) involved in tumor diseases to which DVDIg can bind include 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 FGF9, GRP, IGF1, IGF2, IL12A, IL1A, IL1B, IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2, EGF, FGF10, FGF18, F F2, FGF4, FGF7, IGF1, IGF1R, IL2, VEGF, BCL2, CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1, IGFBP6, IL1A, IL1PA BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3, IGFBP6, IL2, INSL4, MYC, N0X5, NR6A1, PAP, PR TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1, IG 1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3, NR2F6, NR4A3, ESR1, ESR2, NR0B1, NR0B2, NR1D2, NR1H2, NR1H2, NR1I2, C2 NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2, NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1, BRCA1, CHGA, CHGB1, CLU, CLU1, CLU1, CLU1 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, INSL3L, INSL4L KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9, MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, CDG, HCD1, TGF3, 44 CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18, CDH19, CDH20, CDH7, C H8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164, COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1, CDH12, CLDN3, CLN3, CYB5, CYC1, CYB5, CYC1, DNCL1, ELAC2, ENO2, ENO3, FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A, IL29, K6HF, KAI1, KRT2A, MIB1, PART3, PART3, PAPI, APG3 PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1, TPM , TRPC6, ANGPPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR, LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB1, VEGF, VEGFC, ANGPTL3, BAI1, CAI4 , NRP1, NRP2, STAB1, ANGPTL4, PECAM1, PECAM1, PF4, PROK2, SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, IFNA1, IFN1, IFNIL, IFN1 , EFNA3, EFNB2, EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PD GFA, TEK, TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, HCN1 Cadherin), CDKN1B (p27Kip1), CDKN2A (pl6INK4a), COL6A1, CTNNB1 (b-catenin), CTSB (cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-I) , GATA3, GSN (gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130), ITGA6 (a6 integrin), JUN, KLK5, RT19, 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, AZGP1 (zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1 / Cip1), CLDN7 (Claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin), GABRP (GABAa), GNAS1, ID2, I GA6 (a6 integrin), ITGB4 (b4 integrin), KLF5 (GC Box BP), KRT19 (keratin 19), KRTHB6 (hair-specific type II keratin), MACMARKCKS, MT3 (metallothionectin-III), MUC1 (mucin) ), PTGS2 (COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2, THBS4 and TNFA4 and TNFA4 ) Is selected from the group consisting of, but is not limited to.

IV. Pharmaceutical Composition The present invention also provides a pharmaceutical composition comprising the binding protein of the present invention and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a binding protein of the invention is for diagnosing, detecting or monitoring a disease, preventing, treating, managing or reducing the disease or one or more symptoms thereof. And / or used in research, but not limited to. In certain embodiments, the composition comprises one or more proteins of the invention. In another embodiment, the pharmaceutical composition comprises one or more binding proteins of the invention and one or more prophylactic or therapeutic agents other than the binding proteins of the invention for treating a disease. including. Preferably, the prophylactic or therapeutic agent is known to be useful for the prevention, treatment, management or alleviation of the disease or one or more symptoms thereof, or the disease or one or more symptoms thereof. Has been used or is currently used in the prevention, treatment, management or alleviation of According to these embodiments, the composition may further comprise a carrier, diluent or excipient.

  The binding proteins of the invention can be incorporated into a pharmaceutical composition suitable for administration to a subject. Typically, the pharmaceutical composition also provides a pharmaceutical composition comprising the binding protein of the invention and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Is included. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, polyalcohols such as sugar, mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers that enhance the shelf life or effectiveness of the antibody or antibody portion.

Various delivery systems are known, eg, liposomes, microparticles, microcapsules, recombinant cells capable of expressing antibodies or antibody fragments, receptor-mediated endocytosis (eg, Wu and Wu, J. Biol Chem. 262: 4429-4432 (1987)), encapsulated in a nucleic acid construct, etc. as part of a retrovirus or other vector, etc., and one or more antibodies of the invention or one of the invention. Can be used to administer prophylactic or therapeutic agents useful in preventing, managing, treating or alleviating or more antibodies and diseases or one or more symptoms thereof It is. Methods for administering the prophylactic or therapeutic agents of the present invention include parenteral administration (eg, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, intratumoral administration and mucosal administration (eg Including, but not limited to, intranasal and oral routes). In addition, pulmonary administration can be used, for example, by use of inhalation devices or nebulizers and formulations with added aerosolizing agents. For example, U.S. Pat. S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540 and 4,880,078; and PCT Publication Nos. See WO92 / 19244, WO97 / 32572, WO97 / 44013, WO98 / 31346 and WO99 / 66903, each of which is incorporated herein by reference in its entirety. In one embodiment, the composition of the binding protein, a combination therapy of the invention or may, AlkermesAIR ^(R) pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass .) Is administered with. In certain embodiments, the prophylactic or therapeutic agents of the invention are administered intramuscularly, intravenously, intratumorally, orally, intranasally, pulmonary or subcutaneously. The prophylactic or therapeutic agent can be administered by any convenient route, for example by infusion or bolus injection, by absorption through the epithelial or mucosal lining (eg, oral mucosa, rectum and intestinal mucosa, etc.) Can be administered with other active agents. Administration can be systemic or local.

In certain embodiments, it may be desirable to administer the prophylactic or therapeutic agent of the invention locally to the site in need of treatment. This can be achieved, for example, by local infusion, by injection or by means of implants, including but not limited to, an implant comprising a sialastic membrane, a polymer, a fibrous matrix (e.g. , Tissue ^(R) ) or a collagen matrix, such as a porous or non-porous material comprising a membrane and a matrix. In one embodiment, an effective amount of one or more antibody antagonists of the invention is locally administered to the affected site of a subject to prevent, treat, manage, and / or alleviate the disease or its symptoms. Administered. In another embodiment, an effective amount of one or more antibodies of the invention is used to prevent, treat, manage, and / or alleviate a disease or one or more symptoms thereof. In combination with an effective amount of one or more therapeutic agents (eg, one or more prophylactic or therapeutic agents) other than the binding proteins of the invention are administered locally to the affected site of the subject.

  In another embodiment, the prophylactic or therapeutic agent can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (Langer, supra; 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, polymeric materials can be used to achieve controlled release or sustained release of the therapeutic agents of the invention. (For example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, DrugDrug Bioavailability. See New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23: 61. Levy et al., 1985, Science 228: 190; 1989, Ann. eurol.25: 351; Howard et al., 1989, J. Neurosurg.7 1: 105); S. Pat. NO. 5,679,377; S. Pat. No. 5,916,597; S. Pat. NO. 5,912,015; S. Pat. NO. 5,989,463; S. Pat. NO. 5, 128, 326; PCT Publication No. WO99 / 15154; and PCT Publication No. See also WO 99/20253. Examples of polymers used in sustained release formulations include poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-covinyl acetate), poly (methacrylic). Acid), polyglycolide (PLG), polyanhydride, poly (N-vinylpyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactide (PLA), poly (lactide-co-glycolide) ( PLGA) and polyorthoesters are included, but are not limited to these. In a preferred embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, a controlled release or sustained release system can be placed near the prophylactic or therapeutic agent to require only a portion of the systemic dosage (e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

  Sustained release systems are discussed in the review by Langer (1990, Science 249: 1527-1533). Any technique known to those skilled in the art can be used to make sustained release formulations comprising one or more therapeutic agents of the present invention. For example, U.S. Pat. S. Pat. No. 4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et al. , 1996, “Intratumor Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology 39: 179-189, et al. , 1995, “Antibody Mediated Lung Targeting of Long-Circumulating Emulations,” PDA Journal of Pharmaceutical Science & Technology 50: 372-397. , 1997, “Biodegradable Polymeric Carriers for a FGF Antibody for Cardiovascular Application,” Pro. Lnt'l. Symp. Control. Rel. Bioact. Mater. 24: 853-854, and Lam et al. , 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibodies for Local Delivery," Proc. lnt'l. Symp. Control Rel. Bioact. Mater. 24: 759-760, each of which is incorporated herein by reference in its entirety.

  In certain embodiments where the composition of the invention is a nucleic acid encoding a prophylactic or therapeutic agent, the nucleic acid is constructed as part of a suitable nucleic acid expression vector, eg, a retroviral vector (US Pat. No. 4, 980, 286), or by direct injection or by the use of microparticle irradiation (eg, gene gun; Biolistic, Dupont) or administering a nucleic acid so that the nucleic acid is intracellular, or lipid Alternatively, by coating with a cell surface receptor or transfection agent, or by administering a nucleic acid linked to a homeobox-like peptide known to enter the nucleus (see, eg, Joliot et al., 1991, Proc. Natl.Acad.Sci.USA 88: 1864-1868), nucleic acid encoding. To facilitate expression of Bozai or therapeutic agents, it is possible to administer the nucleic acid in vivo. Alternatively, nucleic acids can be introduced into cells and incorporated into host cell DNA for expression by homologous recombination.

  A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral, intranasal (eg, inhalation), transdermal (eg, topical), transmucosal and rectal administration. It is not limited to. In a specific embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection (such as lignocamone).

  If the composition of the present invention is to be administered topically, the composition may be in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion or well known to those skilled in the art. It can be formulated in other forms. See, for example, “Remington's Pharmaceutical Sciences and Induction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995)”. In the case of a non-sprayable topical dosage form, a viscous to semi-solid or solid containing carrier or one or more excipients compatible with topical application and preferably having a kinematic viscosity greater than water A form is commonly used. Appropriate formulations are, if desired, sterilized or adjuvants (eg, preservatives, stabilizers, wetting agents, buffers or salts) to affect various properties such as, for example, osmotic pressure. Solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like mixed in with. For other suitable topical dosage forms, preferably the active ingredient in combination with a solid or liquid inert carrier is in a mixture with a pressurized volatile substance (eg a gaseous propellant such as freon) or Included are sprayable aerosol preparations packaged in squeeze bottles. If desired, wetting agents or wetting agents can be added to the pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art.

  Where the method of the invention involves intranasal administration of the composition, the composition can be formulated in aerosol form, spray, mist, or in the form of nasal drops. In particular, prophylactic or therapeutic agents for use in accordance with the present invention may be added using a suitable propellant (eg, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). It can be conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges (eg, composed of gelatin) may be formulated for use in inhalation or gas infusion devices containing a powder mixture of the compound and a suitable powder base such as lactose or starch. Where the method of the invention includes oral administration, the composition can be formulated orally in the form of tablets, capsules, cachets, gel caps, solutions, suspensions, and the like. Tablets or capsules may be combined by conventional means with a binder (eg, pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); a filler (eg, lactose, microcrystalline cellulose or calcium hydrogen phosphate); a lubricant. (Eg, magnesium stearate, talc or silica); prepared with pharmaceutically acceptable excipients such as disintegrants (eg, starch starch or sodium starch glycolate); or wetting agents (eg, sodium lauryl sulfate). Is possible. The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, but are not limited to, solutions, syrups or suspensions, or dry to make up with water or other suitable vehicle before use. It can be given as a product. Such liquid preparations are prepared by conventional means, such as suspensions (eg sorbitol syrup, cellulose derivatives or hardened edible fat); emulsifiers (eg lecithin or acacia); non-aqueous vehicles (eg almond oil, oily esters). , Ethyl alcohol or fractionated vegetable oils); and preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid. The preparation may also contain buffer salts, flavoring agents, coloring agents and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated for slow, controlled or sustained release of prophylactic or therapeutic agents.

The methods of the invention can include pulmonary administration, for example, by use of an inhalation device or nebulizer, use of a composition formulated with an aerosolizing agent. For example, U.S. Pat. S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540 and 4,880,078; and PCT Publication Nos. See WO92 / 19244, WO97 / 32572, WO97 / 44013, WO98 / 31346 and WO99 / 66903, each of which is incorporated herein by reference in its entirety. In certain embodiments, the composition of the binding protein, a combination therapy and / or the invention of the present invention, AlkermesAIR ^(R) pulmonary drug delivery technology (Alkermes, Inc.Cambridge, Mas.) Is administered with.

  The methods of the invention can include administration of a composition formulated for parenteral administration by injection (eg, by bolus injection or continuous infusion). Injectable formulations may be given in unit dosage form (eg, in ampoules or multiple dose containers) with added preservatives. The composition may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle (eg, sterile pyrogen-free water) before use.

  The methods of the invention can further comprise administration of a composition formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the composition can be formulated with a suitable polymeric or hydrophobic material (eg, as an acceptable emulsion in oil) or with an ion exchange resin, or as a poorly soluble derivative (eg, as a poorly soluble salt). .

  The methods of the present invention include the administration of compositions formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide And salts formed with cations such as those derived from ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

  In general, the components of the composition are lyophilized dry powder or anhydrous concentrate in sealed containers, such as syringes or sachets indicating the amount of active agent, separately or in unit dosage forms. Supplied as a mixture). Where the mode of administration is infusion, the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the mode of administration is by injection, a syringe of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

  In particular, the present invention is such that one or more of the prophylactic or therapeutic agents of the present invention or the pharmaceutical composition of the present invention is packaged in a sealed container such as a syringe or odor bag indicating the amount of the drug. Also provide. In one embodiment, one or more of the prophylactic or therapeutic agents of the invention or the pharmaceutical composition is supplied as a lyophilized dry sterile powder or anhydrous concentrate in a sealed container for administration to a subject. Can be reconstituted (e.g., with water or saline) to an appropriate concentration. Preferably, one or more of the prophylactic or therapeutic agents or pharmaceutical composition of the present invention is 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 a unit dosage of at least 100 mg supplied as a lyophilized dry sterile powder in a sealed container. The freeze-dried preventive or therapeutic agent or pharmaceutical composition of the present invention should be stored between 2 ° C. and 8 ° C. in its original container, and the preventive or therapeutic agent or pharmaceutical composition of the present invention. Items are reconstituted 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 should be administered within 1 hour. In another embodiment, one or more of the prophylactic or therapeutic agents of the present invention or the pharmaceutical composition of the present invention is provided in liquid form in a sealed container indicating the amount and concentration of the drug. Preferably, the liquid form of the administered composition is 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 mg / kg, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml or at least 100 mg / ml in a sealed container. The liquid form should be stored between 2 ° C and 8 ° C in its original container.

The binding proteins of the present invention can be incorporated into pharmaceutical compositions suitable for parenteral administration. Preferably, the antibody or antibody portion is prepared as an injectable solution containing 0.1 to 250 mg / mL binding protein. Injectable solutions may consist of liquid or lyophilized dosage forms in flint or amber containers, syringes or prefilled syringes. The buffer may be L-histidine (1 to 50 mM), optimally 5 to 10 mM, pH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to modify the toxicity of solutions at concentrations from 0 to 300 mM (optimally 150 mM for liquid dosage forms). For lyophilized dosage forms, it is possible to include a cryoprotectant, mainly 0 to 10% sucrose (optimally 0.5 to 1.0%). Other suitable cryoprotectants include trehalose and lactose. For lyophilized dosage forms it is possible to include fillers, mainly 1 to 10% mannitol (optimally 2 to 4%). In both liquid and lyophilized dosage forms, it is possible to use a stabilizer, mainly 1 to 50 mM L-methionine (optimally 5 to 10 mM). Other suitable fillers include glycine arginine and can be included as 0 to 0.05% polysorbate-80 (optimally 0.005 to 0.01%). Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants. A pharmaceutical composition comprising a binding protein of the invention prepared as an injectable solution for parenteral administration may be used as an adjuvant, such as those used to increase the absorption or dispersion of therapeutic proteins (eg, antibodies). Useful factors can further be included. Particularly useful adjuvant, Hylenex ^(R) (recombinant human hyaluronidase) is hyaluronidase, such as. The addition of hyaluronidase in an injectable solution improves human bioavailability after parenteral administration, particularly subcutaneous administration. Larger injection site volumes (ie, greater than 1 mL) are possible with less pain and discomfort and with minimal occurrence of injection site reactions (ie, WO2004078140 and US2006104968, incorporated herein by reference). See).

  The composition of the present invention may be in various forms. These include, for example, liquid, semi-solid and solid agents such as liquid solutions (eg, injectable and injectable solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories) Shape is included. The preferred form depends on the intended mode of administration and therapeutic application. A typical preferred composition is in the form of an injectable or injectable solution, such as a composition similar to that used for human passive immunization with other antibodies. The preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In preferred embodiments, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular injection or subcutaneous injection.

  The therapeutic composition typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can incorporate the active compound (ie, antibody, antibody portion) in the required amount in a suitable solvent with one or a combination of the ingredients listed above, and as needed It can be prepared by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion solvent and the required other ingredients from those listed above. In the case of a lyophilized sterile powder for the preparation of a sterile injectable solution, the preferred method of preparation is to obtain a powder of the active ingredient plus any additional desired ingredients from the previously sterile filtered solution. Give vacuum drying and powder drying. The proper fluidity of the solution can be maintained, for example, by using a coating material such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. . Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

  Although the binding proteins of the invention can be administered by various methods known in the art, for many therapeutic applications, the preferred route / mode of administration is subcutaneous injection, intravenous injection or infusion. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. In certain embodiments, the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, for example, “Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978”.

  In certain embodiments, the binding proteins of the invention can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compounds (and other ingredients, if desired) can also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the patient's diet. For oral therapeutic administration, the compound can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. In order to administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with a substance to prevent its inactivation or to co-administer the compound with a substance to prevent its inactivation. .

  Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, the binding proteins of the invention are co-formulated with one or more additional therapeutic agents useful for treating diseases in which IL-12 activity is detrimental, and / or Co-administered. For example, a binding protein of the invention is co-formulated with one or more additional antibodies that bind to other tablets (eg, antibodies that bind to other cytokines or antibodies that bind to cell surface molecules), and / or Or they can be co-administered. Furthermore, one or more antibodies of the present invention may be used in combination with two or more of the aforementioned therapeutic agents. Such combination therapies can advantageously use lower dosages of the therapeutic agent administered, thus avoiding the toxicity or complications that can occur with various monotherapy.

  In certain embodiments, the binding protein is linked to a half-life extending vehicle known in the art. Such vehicles include, but are not limited to, Fc domains, polyethylene glycol and dextran. Such vehicles are described in U.S. Pat. S. Application Serial NO. 09 / 428,082 and published PCT Application No. WO 99/25044 (incorporated herein by reference for all purposes).

  In certain embodiments, a nucleic acid sequence encoding a binding protein of the invention or another prophylactic or therapeutic agent of the invention treats and prevents a disease or one or more symptoms thereof by gene therapy; Administered to manage or alleviate. Gene therapy refers to a therapy performed by administering to a subject an expressed nucleic acid or an expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded antibody or prophylactic or therapeutic agent of the invention that mediates a prophylactic or therapeutic effect.

  In accordance with the present invention, any method for gene therapy available in the art can be used. For a general review of 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 generally known in the field of available recombinant DNA technology 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, 1990. A detailed description of various methods of gene therapy is disclosed in US200500042664 A1, which is incorporated herein by reference.

  The binding proteins of the invention are useful in treating various diseases where the target recognized by the binding protein is detrimental. Such diseases include rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, pyogenic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosis, Crohn's disease, ulcerative colitis Inflammatory bowel disease, insulin-dependent diabetes mellitus, thyroiditis, asthma, allergic disease, psoriasis, dermatitis, scleroderma, graft-versus-host disease, organ transplant rejection, acute or chronic immune disease related to organ transplantation, Sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schlein purpura, renal microvasculitis, chronic active hepatitis , Uveitis, septic shock, toxic shock syndrome, septic syndrome, cachexia, infectious disease, parasitic disease, acquired immune deficiency syndrome, acute Acute myelitis, Huntington's disease, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignant tumor, heart failure, myocardial infarction, Addison's disease, sporadic, polyendocrine hypofunction syndrome I Type and polyendocrine hypofunction syndrome type II, Schmidt syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthritis, arthritis, Reiter's disease, psoriatic arthritis, ulcerative colitis Diseased synovitis, Chlamydia, Yersinia and Salmonella-related arthritis, spondyloarthropathy, atherosclerosis / atherosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, deciduous pemphigus, etc. Pemphigus, linear IgA disease, autoimmune hemolytic anemia, Coombs-positive hemolytic anemia, acquired pernicious anemia, juvenile pernicious anemia, myalgic encephalomyelitis Royal free disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosis hepatitis, idiopathic autoimmune hepatitis, acquired immune deficiency syndrome, acquired immune deficiency related diseases, hepatitis B, hepatitis C, Unclassifiable immunodeficiency (unclassifiable primary hypogammaglobulinemia), dilated cardiomyopathy, female infertility, ovarian dysfunction, early ovarian dysfunction, fibrotic lung disease, idiopathic interstitial pneumonia, inflammation Postinterstitial lung disease, interstitial pneumonia, connective tissue disease related interstitial lung disease, mixed connective tissue disease related lung disease, systemic sclerosis related interstitial lung disease, rheumatoid arthritis related interstitial lung disease Systemic lupus erythematosus-related lung disease, dermatomyositis / polymyositis-related lung disease, Sjogren's disease-related lung disease, ankylosing spondylitis-related lung disease, vasculitic diffuse lung disease, f Siderosis-related lung disease, drug-induced interstitial lung disease, fibrosis, radioactive fibrosis, obstructive bronchiolitis, chronic eosinophilic pneumonia, lymphocyte infiltrating lung disease, post-infectious interstitial lung disease , Ventilatory arthritis, autoimmune hepatitis, type 1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type 2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune-mediated hypoglycemia, melanoma Type B insulin resistance, hypoparathyroidism, acute immune disease related to organ transplantation, chronic immune disease related to organ transplantation, osteoarthritis, primary sclerosing cholangitis, type 1 psoriasis, type 2 Psoriasis, idiopathic leukopenia, autoimmune neutropenia, kidney disease NOS, glomerulonephritis, renal microvasculitis, Lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, Sperm autoimmunity, multiple sclerosis (all Subtype), sympathetic ophthalmitis, pulmonary hypertension secondary to connective tissue disease, Goodpasture syndrome, pulmonary symptoms of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjogren's syndrome, Takayasu / arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goiter autoimmune hypothyroidism (Hashimoto's disease), atrophic self Immune hypothyroidism, primary myxedema, lens-induced uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver disease, alcoholic cirrhosis, alcohol-induced liver injury, cholestasis (Choleocytosis), idiosyncratic liver disease, drug-induced hepatitis, non-alcoholic steatohepatitis, allergy and asthma, group B chain (GBS) infections, psychiatric disorders (eg depression and schizophrenia), diseases mediated by Th2 and Th1 types, acute and chronic pain (various forms of pain), and lung cancer, breast cancer, stomach cancer, bladder Cancers such as cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer and kidney cancer and hematopoietic malignancies (leukemia and lymphoma), abetalipoproteinemia, advanced cyanosis, 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, adenocarcinoma, aerial ectopic pulsation, AIDS dementia Complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, α-1-antitrypsin deficiency, amyotrophic lateral cord Disease, anemia, angina pectoris, anterior horn cell degeneration, anti-cd3 treatment, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aortic and aneurysms, aortic dissection, arterial Hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (persistent or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone transplant rejection, bone marrow transplant (BMT) rejection, leg block, Burkitt's lymphoma, burns, cardiac arrhythmia, cardiac dysfunction syndrome, cardiac tumor, cardiomyopathy, cardiopulmonary bypass inflammatory response, cartilage transplant rejection, cerebellar cortical degeneration, cerebellar disease, disordered or multifocal atrial tachycardia , Chemotherapy related diseases, chronic myelogenous leukemia (CML), chronic alcoholism, chronic inflammatory lesions, chronic Lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylic acid poisoning, colorectal cancer, congestive heart failure, conjunctivitis, contact dermatitis, pulmonary heart, coronary artery disease, Creutzfeldt-Jakob disease, culture Negative sepsis, cystic fibrosis, cytokine therapy-related disease, boxer brain, demyelinating disease, dengue hemorrhagic fever, dermatitis, dermatological symptoms, diabetes, diabetes mellitus, diabetic arteriosclerosis, diffuse Lewy body disease , Dilated congestive cardiomyopathy, basal ganglia disease, middle-aged Down syndrome, drug-induced motor disease induced by drugs that block central nerve dopamine receptors, drug sensitivity, eczema, encephalomyelitis, intracardiac Membrane inflammation, endocrine disease, epiglottitis, Epstein-Barr virus infection, erythema, extrapituitary tract and cerebellar disease, familial hemophagocytic lymphohistiocytosis autophagic lymphohistiocytosis), fatal thymus transplant rejection, Friedreich's ataxia, functional peripheral arterial disease, fungal sepsis, gas gangrene, gastric ulcer, glomerulonephritis, graft rejection of any organ or tissue, Gram-negative sepsis , Gram-positive sepsis, granulomas caused by intracellular organisms, hairy cell leukemia, Haller-Holden-Spatz disease, Hashimoto thyroiditis, hay fever, heart transplant rejection, hemochromatosis, hemodialysis, hemolytic uremic syndrome / thrombolytic platelets Reduced purpura, bleeding, hepatitis (type A), His bundle arrhythmia, HIV infection / HIV neuropathy, Hodgkin's disease, hyperkinetic disease, hypersensitivity reaction, hypersensitivity pneumonia, hypertension, hypokinetic disease, hypothalamus-pituitary- Adrenocortical system evaluation, idiopathic Addison disease, idiopathic pulmonary fibrosis, antibody-mediated cytotoxicity, asthenia, infant spinal cord Atrophy, aortic inflammation, influenza A, ionizing radiation exposure, iridocyclitis / uveitis / optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy , Kaposi's sarcoma, renal transplant rejection, Legionella, leishmaniasis, leprosy, corticospinal lesion, lipedema, liver transplant rejection, lymphedema, malaria, malignant lymphoma, malignant histiocytosis, Malignant melanoma, meningitis, meningococcusemia, metabolic / idiopathic, migraine, mitochondrial multi-system disorder, mixed connective tissue disease, monoclonal hypergammaglobulin blood Disease, multiple myeloma, multiple systemic degeneration (Mencel Degeline) Thomas Shy-Drager and Machado-Joseph), myasthenia gravis, Mycobacterium avium Intracellulare, Mycobacterium tuberculosis, myelodysplasia, myocardial infarction, fungal ischemic disease, nasopharyngeal cancer, newborn Chronic lung disease, nephritis, nephrosis, neurodegenerative disease, neurogenic I muscle atrophy, neutropenic fever, non-Hodgkin lymphoma, obstruction of abdominal aorta and its branches, obstructive arterial disease, okt3 therapy, testitis / Epididymis, testicularitis / vaginal reconstruction treatment, organ enlargement, osteoporosis, pancreas transplant rejection, pancreatic cancer, paraneoplastic transplantation, parathyroid transplant rejection, pelvic inflammatory disease, year-round Rhinitis, pericardial disease, peripheral atherosclerotic disease, peripheral vascular disease, peritonitis, pernicious anemia, Pneumocystis carini pneumonia, pneumonia, POEMS symptoms Groups (polyneuritis, organ hypertrophy, endocrine disease, monoclonal gamma globulinemia and skin change syndrome), post perfusion syndrome, post pump syndrome, myocardial infarction Post-open heart surgery syndrome, preeclampsia, progressive supranuclear palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon and disease, Raynaud's disease, Refsum's disease, regular narrow QRS tachycardia ), Renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcoma, scleroderma, senile chorea, senile dementia with Lewy bodies, seronegative arthritis, shock, sickle cell anemia, skin allograft Allograft rejection, skin change syndrome, small intestine transplant rejection, solid Tumor, specific arrhythmia, spinal ataxia, spinocerebellar degeneration, streptococcal myositis, cerebellar structural lesions, subacute sclerosing panencephalitis, eczema, cardiovascular syphilis, systemic anaphylaxis, systemic Inflammatory reaction syndrome, systemic onset juvenile rheumatoid arthritis, T cells or FABALL, telangiectasia, obstructive thromboangitis, thrombocytopenia, toxicity, transplantation, trauma / bleeding, type III hypersensitivity reaction, type IV hypersensitivity Unstable angina pectoris, uremia, urinary sepsis, hives, valvular heart disease, varicose veins, vasculitis, venous disease, venous thrombosis, ventricular fibrillation, viral and fungal infections, viral encephalitis / Aseptic meningitis, virus-related hemophagocytic syndrome, Wernicke-Korsakov syndrome, Wilson disease, any organ or tissue Including but xenograft rejection, but are not limited to (Peritt et al. PCT publication No. WO2002097048A2, Leonard et al. , PCT publication NO. WO9524918A1, and Salfeld et al. , PCT publication NO. WO00 / 56772A1).

  The binding protein of the present invention is used to treat autoimmune diseases, particularly humans suffering from inflammation-related autoimmune diseases such as rheumatoid arthritis, spondylitis, allergy, autoimmune diabetes, and autoimmune uveitis. Can be used.

  Preferably, the binding protein or antigen binding portion thereof of the present invention is used to treat rheumatoid arthritis, Crohn's disease, multiple sclerosis, insulin dependent diabetes mellitus and psoriasis.

  The binding proteins of the invention can also be administered with one or more additional therapeutic agents useful in the treatment of various diseases.

  The binding proteins of the present invention can be used alone or in combination to treat such diseases. It is understood that the binding protein can be used alone or in combination with an additional agent, such as a therapeutic agent, said additional agent being selected by those skilled in the art for its intended purpose. Should. For example, the additional agent can be a therapeutic agent recognized in the art as useful for treating a disease or condition being treated by an antibody of the invention. The additional factor can also be a factor that imparts a beneficial attribute to the therapeutic composition, such as a factor that affects the viscosity of the composition.

  It should be further understood that the combinations to be included in the present invention are useful combinations for their intended purpose. The factors described below are for purposes of illustration and are not intended to be limiting. The combination that is part of the invention can be an antibody of the invention and at least one additional factor selected from the following list. In such combinations, the combination can also include two or more additional factors, eg, two or three additional factors, provided that the formed composition can perform its intended function. .

  A preferred combination for treating autoimmune and inflammatory diseases is a non-steroidal anti-inflammatory drug (including drugs such as ibuprofen), also referred to as NSAID. Another preferred combination is a corticosteroid such as prednisolone. Well-known side effects of steroid use can be reduced or even eliminated by gradually reducing the steroid dose required when treating patients in combination with the DVDIg of the present invention. . Non-limiting examples of therapeutic agents for rheumatoid arthritis that can be combined with the antibodies or antibody portions of the invention include the following: cytokine inhibitory anti-inflammatory drugs (CSAIDs); other human cytokines or growth factors (eg, , 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, interferon, EMAP-II, GM-CSF, FGF and PDGF) or antagonists thereof. The binding protein of the present invention or the antigen-binding portion thereof is CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or It is possible to combine with antibodies against cell surface molecules such as these ligands including CD154 (gp39 or CD40L).

Preferred combinations of therapeutic agents can interfere with autoimmunity and the subsequent inflammatory cascade in different ways. Preferred examples include chimeric, humanized or human TNF antibodies, D2E7 (PCT Publication WO 97/29131), CA2 (Remicade ^™ ), CDP571, and soluble p55 or p75 TNF receptors, derivatives thereof, (p75TNFRIgG (Enbrel ^™ ) Or TNF antagonists such as p55TNFR1gG (Lenecept) and TNFα converting enzyme (TACE) inhibitors, etc. Similarly, IL-1 inhibitors (interleukin-1 converting enzyme inhibitors, IL-1RA, etc.) have the same reason Other preferred combinations include interleukin 11. Still other preferred combinations are parallel to IL-12 function, dependent on IL-12 function, or IL-12. Self that can act in cooperation with -12 Central players of the epidemic response are included, particularly preferred are IL-18 antagonists such as IL-18 antibodies or soluble IL-18 receptors or IL-18 binding proteins, IL-12 and IL-18 are It has been shown that overlapping, but having different functions, antagonist combinations to both can be most effective, yet another preferred combination is a non-depleting anti-CD4 inhibitor. Yet another preferred combination includes antagonists of the costimulatory pathway CD80 (B7.1) or CD86 (B7.2), including antibodies, soluble receptors or antagonistic ligands.

The binding proteins of the present invention include methotrexate, 6-MP, azathioprine sulfasalazine, mesalazine, olsalazine, chloroquinine / hydroxychloroquine, penicillamine, gold thiomalate (intramuscular and oral), azathioprine, colchicine, corticosteroid (oral, inhalation and Local injection), β-2 adrenergic receptor agonists (salbutamol, terbutaline, salmeteral), xanthine (theophylline, aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, cyclosporine, FK506, rapamycin, mycofeno Lato mofetil, leflunomide, NSAID, eg corticosteroids such as ibuprofen, prednisolone, phosphodieste Factors that interfere with signal transduction by pro-inflammatory cytokines such as protease inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, TNF □ or IL-1 (eg, IRA, NIK, IKK, p38 Or MAP kinase inhibitor), IL-1β converting enzyme inhibitor, TNFα converting enzyme (TACE) inhibitor, T cell signaling inhibitor such as kinase inhibitor, metalloproteinase inhibitor, sulfasalazine, azathioprine, 6-mercaptopurine, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g., soluble p55 or p75TNF receptor and derivatives p75TNFRIgG (Enbrel ^TM and p55TNFRIgG (Lenercept)), sIL- IR1, sIL-1RII, IL-6R), anti-inflammatory cytokines (eg, IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab, naproxen, valdecoxib, sulfasalazine , Methylprednisolone, meloxicam, methylprednisolone acetate, sodium gold thiomalate, aspirin, trimcinolone acetonide, propoxyphen napsilate / apap, folato, nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium, oxaprodin, oxycodone hydrochloride, hydrocodone dihydrocodone Salt / apap, diclofenac sodium / misoprostol, fentanyl, anakinra, human recombinant, hydrochloric acid Tramadol, salsalate, sulindac, cyanocobalamin / fa / pyridoxine, acetaminophen, alendronate sodium, prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin, glucosamine sulfate / chondroitin, amitriptyline hydrochloride, sulfadiazine, oxycodone / acetaminophen hydrochloride, olopatadine hydrochloride , Misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab, IL-1TRAP, MRA, CTLA4-IG, IL-18BP, anti-IL-18, anti-IL15, BIRB-796, SCIO-469, VX-702 , AMG-548, VX740, Roflumilast, IC-485, CDC-801, and mesopram. Preferred combinations include methotrexate or leflunomide and in cases of moderate or severe rheumatoid arthritis include cyclosporine.

Non-limiting additional factors that can also be used in combination with binding proteins to treat rheumatoid arthritis include the following non-steroidal anti-inflammatory drugs (NSAIDs); cytokine-suppressing anti-inflammatory drugs (CSAIDs) CDP-571 / BAY-10-3356 (humanized anti-TNFα antibody; Celltech / Bayer); cA2 / infliximab (chimeric anti-TNFα antibody; Centocor); 75 kdTNFR-IgG / etanercept (75 kDTNF receptor-IgG fusion protein; See, for example, Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol.44, 235A; 55 kdTNF-IgG (55 kDTNF receptor-IgG Hoffmann-LaRoche); IDEC-CE9.1 / SB210396 (primatized non-depleting anti-CD4 antibody; IDEC / SmithKline; see, eg, Arthritis & Rheumatism (1995) Vol. 38, S185; DAB486-IL-2 and / or DAB389-IL-2 (IL-2 fusion protein; Seragen; see, eg, 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 (SCH52000; recombinant IL-10, anti-inflammatory support) Tokain; DNAX / Schering); IL- 4; IL-10 and / or IL-4 agonists (e.g., agonist antibodies); IL-1RA (IL- 1 receptor antagonist; Synergen / Amgen); anakinra (Kineret ^(R) / Amgen); TNF-bp / s-TNF (soluble TNF binding protein; see, eg, Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284; Amer. J. Physiol. (1995) Vol. 268, pp. 37-42); R97401 (phosphodiesterase type IV inhibitors; see, for example, Arthritis & Rheumatism (1996) Vol. 39, no. 9 (Appendix), S282; MK-966 (COX-2 inhibitor; see, eg, Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S81); Iloprost (eg, Arthritis & Rheumatism (1996 ) Vol.39, No. 9 (Addendum), S82); methotrexate; thalidomide (see, for example, Arthritis & Rheumatism (1996) Vol. ); Refnomide (anti-inflammatory and cytokine inhibitors; see, for example, Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S131; Inflammation Research. (1996) Vol. 45, pp. 103-107); tranexamic acid (inhibitors of plasminogen activation; see, for example, Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S284); T-614 (cytokine inhibitors; see, eg, Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); prostaglandin E1 (eg, Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S282); Tenidap (Nonsteroidal anti-inflammatory drugs; see, for example, Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Addendum), S280); Naproxen (Nonsteroidal anti-inflammatory) For example, Neuro Report (1996) Vol. 7, pp. 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 anti-inflammatory drug); Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S281); ICE inhibitors (inhibitors of the enzyme interleukin 1β converting enzyme); zap-70 and / or lck inhibitors (tyrosine kinase za) -70 or lck inhibitors); VEGF inhibitors and / or VEGF-R inhibitors (inhibitors of vascular endothelial growth factor or vascular endothelial growth factor receptor; inhibitors of angiogenesis); corticosteroid anti-inflammatory Drugs (eg, SB203580); TNF-convertase inhibitors; anti-IL-12 antibodies; anti-IL-18 antibodies; interleukin-11 (eg, Arthritis & Rheumatism (1996) Vol. 39, no. 9 (Supplement), see S296); interleukin-13 (see, eg, Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S308); interleukin-17 inhibitors (see, eg, Arthritis & Rheumatism (see 1996) Vol. 39, No. 9 (Supplement), S120); Gold; Penicillamine; Chloroquine; Chlorambucil; Hydroxychloroquine; Cyclosporine; Cyclophosphamide; Total lymphatic irradiation; Antithymocyte globulin; Toxins; Orally administered peptides and collagen; Robenzalito disodium; Cytokine regulators (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM I antisense phosphorothioate oligodeoxynucleotide (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulfate; minocycline Anti-IL2R antibodies; mice and plant lipids (fish and plant seed fatty acids; see, eg, DeLuca et al. (1995) Rheum. Dis. Clin. North Am. J ,: 759-777); auranofin; phenylbutazone; meclofenamine Flufenamic acid; intravenous immunoglobulin; zileuton; azaribidine; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapama) Isine); amiprilose (terrafectin); cladribine (2-chlorodeoxyadenosine); methotrexate; antiviral agents; and immunomodulators, but is not limited to these.

  In one embodiment, the binding protein or antigen binding portion thereof is administered in combination with one of the following factors for the treatment of rheumatoid arthritis. Small molecule inhibitor of KDR (ABT-123), small molecule inhibitor of Tie-2; methotrexate; prednisone; celecoxib; folic acid; hydroxychloroquine sulfate; rofecoxib; etanercept; infliximab; leflunomide; naproxen; Ibuprofen; Meloxicam; Methylprednisolone acetate, Gold sodium thiomalate; Aspirin; Azathioprine; Triamcinolone acetonide; Proxiphene napsilate / apap; Diclofenac sodium / misoprostol; Tanil; Anakinra, human recombinant; tramadol hydrochloride; salsalate; sulindac; cyanocobalamin / fa / pyridoxine; acetaminoen; sodium alendronate; prednisolone; morphine sulfate; lidocaine hydrochloride; indomethacin; Olocodazine / acetaminophen; olopatadine hydrochloride; misoprostol; naproxen sodium; omeprazole; mycophenolate mofetil; cyclophosphamide; rituximab, IL-1TRAP; MRA; CTLA4-IG; IL-18BP; -325 (anti-IL-18); anti-IL-15; BIRB-796; SCIO-469; VX-702; MG-548; VX740; roflumilast; IC-485; CDC-801 and mesopram.

  Non-limiting examples of therapeutic agents for inflammatory bowel disease that can be combined with the binding proteins of the invention include: budenoside; epidermal growth factor; corticosteroid; cyclosporine; sulfasalazine; amino salicylate; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitor; mesalamine; olsalazine; balsalazide; antioxidant; thromboxane inhibitor; IL-1 receptor antagonist; anti-IL-1β monoclonal antibody; Elastase inhibitors; pyridinyl-imidazole compounds; other human cytokines or growth factors (eg, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL) -16, IL-17, IL- 8, EMAP-II, GM-CSF, include antibodies or their antagonists to FGF and PDGF). The antibodies of the present invention or antigen-binding portions thereof can be combined with antibodies against cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 and their ligands. The antibody of the present invention or an antigen-binding portion thereof includes methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs such as corticosteroids such as ibuprofen and prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents , Complement inhibitors, adrenergic agents, factors that interfere with signal transduction by pro-inflammatory cytokines such as TNFα or IL-1 (eg, IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzyme Inhibitors, TNFα converting enzyme inhibitors, T cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurine, angiotensin converting enzyme inhibitors Soluble cytokine receptors and their derivatives (eg, soluble p55 or p75 TNF receptor, sIL-1RI, sIL-1RII, sIL-6R) and anti-inflammatory cytokines (eg, IL-4, IL-10, IL-11, It can also be combined with factors such as IL-13 and TGFβ).

  Preferred examples of therapeutic agents for Crohn's disease that can be combined with binding proteins include: TNF antagonists such as anti-TNF antibodies, D2E7 (PCT Publication No. 97/29131; HUMIRA), CA2 (REMICADE) , CDP571, TNFR-Ig constructs, (p75TNFRIgG (ENBREL) and p55TNFRIgG (LENERCEPT)) inhibitors and PDE4 inhibitors. The antibodies or antigen-binding portions thereof of the present invention can be combined with corticosteroids such as budenoside and dexamethasone. The binding proteins of the present invention or antigen binding portions thereof include factors such as sulfasalazine, 5-aminosalicylic acid and olsalazine as well as factors that interfere with the synthesis or action of pro-inflammatory cytokines such as IL-1. It can also be combined with a converting enzyme inhibitor and IL-1ra). The antibodies or antigen-binding portions thereof of the present invention can be used with T cell signaling inhibitors, such as the tyrosine kinase inhibitor 6-mercaptopurine. The binding protein of the present invention or the antigen-binding portion thereof can be combined with IL-11. The binding protein of the present invention or an antigen-binding portion thereof is mesalamine, prednisone, azathioprine, mercaptopurine, infliximab, methylprednisolone sodium succinate, diphenoxylate / atropine sulfate, loperamide hydrochloride, methotrexate, omeprazole, folato, ciprofloxacin / Dextrose-water, hydrocodone bitartrate / apap, tetracycline hydrochloride, fluocinonide, metronidazole, thimerosal / boric acid, cholestyramine / sucrose, ciprofloxacin hydrochloride, hyoscyamine sulfate, meperidine hydrochloride, midazolam hydrochloride, oxycodone hydrochloride / acetaminophen , Promethazine hydrochloride, sodium phosphate, sulfamethoxazole / trimethoprim, celecoxib, polycarbophil, na Sill acid propoxyphene, hydrocortisone, multivitamins, balsalazide disodium, codeine phosphate / acetaminophen (apap), can be combined colesevelam hydrochloride, cyanocobalamin, folic acid, levofloxacin, methylprednisolone, natalizumab and interferon gamma.

  Non-limiting examples of therapeutic agents for multiple sclerosis that can be combined with the binding proteins of the present invention include: corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide; Cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-β1a (AVONEX; Biogen); interferon-β1b (BETASERON; Chiron / Berlex); interferon α-n3) (Interferon Sciences / Fujimota) J & J), interferon-β1A-IF (Serono / Inhale Therapeutics), PEG interferon α2b (E zon / Schering-Plough), copolymer 1 (Cop-1; COPAXONE; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; clavribine; other human cytokines or growth factors (eg, 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) and their Antibodies to the receptor or antagonists thereof are included. The binding protein of the present invention is combined with an antibody against a cell surface molecule such as CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or a ligand thereof. Is possible. The binding proteins of the present invention include methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs such as corticosteroids such as ibuprofen and prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibition Agents, adrenergic agents, factors that interfere with signal transduction by pro-inflammatory cytokines such as TNFα or IL-1 (eg, IRAK, NIK, DCK, p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TACE Inhibitors, T cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurine, angiotensin converting enzyme inhibitors, soluble cytokines Receptors and derivatives thereof (eg soluble p55 or p75 TNF receptor, sIL-1RI, sIL-1RII, sIL-6R) and anti-inflammatory cytokines (eg IL-4, IL-10, IL-13 and TGFβ) ) And the like.

  Preferred examples of therapeutic agents for multiple sclerosis that can be combined with the binding proteins of the present invention include interferon-β, such as IFNβ1a and IFN1b; copaxone, corticosteroids, caspase inhibitors, Examples include caspase-1 inhibitors, IL-1 inhibitors, TNF inhibitors and antibodies to CD40 ligand and CD80.

  The binding proteins of the present invention include alemtuzumab, dronabinol, unimed, daclizumab, mitoxantrone, xaliproden hydrochloride, fampridine, glatiramer acetate, natalizumab, synnavidol, a-immunokine NNS03, ABR-215062, Anergix. MS, chemokine receptor antagonist, BBR-2778, caraguarin, CPI-1189, LEM (liposome encapsulated mitoxantrone), THC. CBD (cannabinoid agonist) MBP-8298, mesopram (PDE4 inhibitor), MNA-715, anti-IL-6 receptor antibody, neurobax, pirfenidone allotrap 1258 (RDP-1258), sTNF-R1, talam panel, teriflunomide, TGF It can also be combined with factors such as β2, tiprimotide, VLA-4 antagonists (eg TR-14035, VLA4Ultrahaler, Antegran-ELAN / Biogen), interferon gamma antagonists, IL-4 agonists and the like.

  Non-limiting examples of therapeutic agents for angina that can be combined with the binding proteins of the present invention include the following: aspirin, nitroglycerin, isosorbide mononitrate, metoprolol succinate, atenolol, tartaric acid Metoprolol, amlodipine besylate, diltiazem hydrochloride, isosorbide nitrate, clopidogrel bisulfate, nifedipine, atorvastatin calcium, potassium chloride, furosemide, simvastatin, verapamil hydrochloride, digoxin, propranolol hydrochloride, carvedilol, lisinopril, spironolactone, hydrochlorothiazide, enalapril maleate , Ramipril, enoxaparin sodium, heparin sodium, valsartan, sotalol hydrochloride, fenofibrate, ezetimibe, Tanido, potassium losartan, lisinopril / hydrochlorothiazide, felodipine, captopril, include bisoprolol fumarate.

  Non-limiting examples of therapeutic agents for ankylosing spondylitis that can be combined with the binding proteins of the present invention include: ibuprofen, diclofenac and misoprostol, naproxen, meloxicam, indomethacin, diclofenac, Celecoxib, rofecoxib, sulfasalazine, methotrexate, azathioprine, minocycline, prednisone, etanercept, infliximab.

  Non-limiting examples of therapeutic agents for asthma that can be combined with the binding proteins of the present invention include: albuterol, salmeterol / fluticasone, montelukast sodium, fluticasone propionate, budesonide, prednisone, xinafoic acid Salmeterol, levalbuterol hydrochloride, albuterol sulfate / ipratropium sulfate, prednisolone sodium phosphate, triamcinolone acetonide, beclomethasone dipropionate, ipratropium bromide, azithromycin, pyrbuterol acetate, prednisolone acetate, theophylline anhydride, methylprednisolone sodium succinate, methylprednisolone sodium succinate Zafirlukast, formoterol fumarate, influenza virus vaccine, methylprednisolone, aminoki Syrin trihydrate, flunisolide, allergy injection, cromolyn sodium, fexofenadine hydrochloride, flunisolide / menthol, amoxicillin / clavulanate, levofloxacin, inhalation aid, guaifenesin, dexamethasone sodium phosphate, maxifloxacin hydrochloride, doxycycline water Hycrate, guaifenesin / d-methorphan, p-ephedrine / cod / chlorphenyl, gatifloxacin, cetirizine hydrochloride, mometasone furoate, salmeterol xinafoate, benzonate, cephalexin, pe / hydrocodone / chlorphenyl hydrochloride, cetirizine hydrochloride / Pseudoephedrine, phenylephrine / cod / promethazine, codeine / promethazine, cefprozil, dexamethasone, guaifene On / pseudoephedrine, chlorpheniramine / hydrocodone, nedocromil sodium, terbutaline sulfate, epinephrine, methylprednisolone, include metaproterenol sulfate.

  Non-limiting examples of therapeutic agents for COPD that can be combined with the binding proteins of the present invention include: albuterol sulfate / ipratropium sulfate, ipratropium bromide, salmeterol / fluticasone, albuterol, salmeterol xinafoate, fluticasone propionate, Prednisone, theophylline anhydride, methylprednisolone sodium succinate, montelukast sodium, budesonide, formoterol fumarate, triamcinolone acetonide, levofloxacin, guaifenesin, azithromycin, beclomethasone dipropionate, levalbuterol hydrochloride, flunisolide sodium, ceftriaxone hydrochloride, Xylillin trihydrate, gatifloxacin, zafirlukast, amoxicillin / club Narate, flunisolide / menthol, chlorpheniramine / hydrocodone, metaproterenol sulfate, methylprednisolone, mometasone furoate, p-ephedrine / cod / chlorophenyl, pyrbuterol acetate, p-ephedrine / loratadine, terbutaline sulfate, tiotropium bromide, (R, R) -formoterol, TgAAT, silomilast, roflumilast are included.

  Non-limiting examples of therapeutic agents for HCV that can be combined with the binding proteins of the invention include: interferon-α-2a, interferon-α-2b, interferon-αcon1, interferon-α-nl, PEGylated interferon-α-2a, PEGylated interferon-α-2b, ribavirin, Peg interferon α-2b + ribavirin, ursodeoxycholic acid, glycyrrhizic acid, timalfacin, maxamine, VX-497 and the following targets: HCV Any compound used to treat HCV through interventions in polymerase, HCV protease, HCV helicase, HCVIRES (intrasequence ribosome entry site) is included.

  Non-limiting examples of therapeutic agents for idiopathic pulmonary fibrosis that can be combined with the binding proteins of the present invention include: prednisone, azathioprine, albuterol, colchicine, albuterol sulfate, digoxin, gamma interferon, Sodium methylprednisolone succinate, lorazepam, furosemide, lisinopril, nitroglycerin, spironolactone, cyclophosphamide, ipratropium bromide, actinomycin d, alteplase, fluticasone propionate, levofloxacin, metaproterenol sulfate, morphine sulfate, oxycodone chloride, potassium chloride , Triamcinolone acetonide, tacrolimus anhydride, calcium, interferon-α, methotrexate, mycophenolate mofetil, inter -Ferron-γ-1β is included.

  Non-limiting examples of therapeutic agents for myocardial infarction that can be combined with the binding proteins of the invention include: aspirin, nitroglycerin, metoprolol tartrate, sodium enoxaparin, sodium heparin, clopidogrel bisulfate, carvedilol , Atenolol, morphine sulfate, metoprolol succinate, warfarin sodium, lisinopril, isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril, tenecteplase, enalapril maleate, tolsemide, retabase, losartan potassium, quinapril hydrochloride, quinapril hydrochloride Enalaprilate, amiodarone hydrochloride, tirofiban hydrochloride m-hydrate, diltiazem hydrochloride, captopril, irbesartan, val Rutan, propranolol 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, isosorbide dinitrate, epinephrine, dopamine hydrochloride, bivalirudine, rosuvastatin, ezetimibe / simvastatin, abashimibu, cariporide.

  Non-limiting examples of therapeutic agents for psoriasis that can be combined with the binding proteins of the present invention include the following: small molecule inhibitor of KDR (ABT-123), small molecule inhibition of Tie-2 Agents, calcipotriene, clobetasol propionate, triamcinolone acetonide, halobetasol propionate, tazarotene, methotrexate, fluocinonide, augmented betamethasone dipropionate, fluocinolone acetonide, acitretin, tar shampoo, tar shampoo Mometasone, ketoconazole, paramoxine / fluocinolone, hydrocortisone valerate, flulandenolide, urea, betamethasone, clobetasol propionate / emollient, fluticasone propionate, a Thromycin, hydrocortisone, moisturizing formulation, folic acid, desonide, pimecrolimus, coal tar, diflorazone diacetate, etanercept folate, lactic acid, metoxalene, hc / bismuth subgal / zinc oxide / resor, methylprednisolone acetate, Prednisolone, sunscreen, halcinonide, salicylic acid, anthralin, crocortron pivalate, coal extract, coal tar / salicylic acid, coal tar / salicylic acid / sulfur, desoxymethasone, diazepam, emollient, fluocinonide / emollient, mineral oil / Castor oil / na lact, mineral oil / peanut oil, petroleum / isopropyl myristate, psoralen, salicylic acid, soap / tribromosaran, thimerosal / boric acid, celecoxib Infliximab include cyclosporine, alefacept, efalizumab, tacrolimus, pimecrolimus, PUVA, UVB, sulfasalazine.

  Non-limiting examples of therapeutic agents for psoriatic arthritis that can be combined with the binding proteins of the invention include the following: methotrexate, etanercept, rofecoxib, celecoxib, folic acid, sulfasalazine, naproxen, leflunomide, methyl acetate Prednisolone, Indomethacin, Hydroxychloroquine sulfate, Prednisone, Sulindac, Enhanced betamethasone dipropionate, Infliximab, Methotrexate, Folate, Triamcinolone acetonide, Diclofenac, Dimethylsulfoxide, Piroxicam, Diclofenac sodium, Ketoprofen methyl bromone , Tolmetin sodium, calcipotriene, cyclosporine, diclofenac sodium / misopro Tall, fluocinonide, glucosamine sulfate, gold sodium thiomalate, hydrocodone bitartrate / acetaminophen, include ibuprofen, risedronate sodium, sulfadiazine, thioguanine, valdecoxib, alefacept, it is efalizumab.

  Non-limiting examples of therapeutic agents for restenosis that can be combined with the binding proteins of the invention include the following: sirolimus, paclitaxel, everolimus, tacrolimus, ABT-578, acetaminophen. .

  Non-limiting examples of therapeutic agents for sciatica that can be combined with the binding proteins of the invention include the following: hydrocodone tartrate / acetaminophen, rofecoxib, cyclobenzaprine hydrochloride, methylprednisolone, Naproxen, ibuprofen, oxycodone / acetaminophen hydrochloride, celecoxib, valdecoxib, methylprednisolone acetate, prednisolone, codeine / acetaminophen phosphate, tramadol / acetaminophen hydrochloride, metaxalone, meloxicam, methcarbamol, lidocaine hydrochloride, diclofenac sodium, Gabapentin, dexamethasone, carisoprodol, ketorolac tromethamine, indomethacin, acetaminophen, diazepam, nabmethione, oxycodone hydrochloride Tizanidine hydrochloride, diclofenac sodium / misoprostol, propoxyphene / acetaminophen napsylate, asa / oxycod / coxicodone ter, ibuprofen / hydrocodone bitartrate, tramadol hydrochloride, etodolac, propoxyphene hydrochloride, amitriptyline hydrochloride, carisoprodol / phosphorus Acid codeine / asa, morphine sulfate, multivitamin, naproxen sodium, orphenadrine citrate, temazepam.

  Preferred examples of therapeutic agents for SLE (lupus) that can be combined with the binding proteins of the present invention include the following: NSAIDs such as diclofenac, naproxen, ibuprofen, piroxicam, indomethacin; COX2 inhibitors such as , Celecoxib, rofecoxib, valdecoxib; antimalarial agents such as hydroxychloroquine; steroids such as prednisone, prednisolone, budenoside, dexamethasone; cytotoxic agents such as azathioprine, cyclophosphamide, mycophenolate mofetil, methotrexate; PDE4 Or purine synthesis inhibitors such as Cellcept. The binding protein of the present invention includes factors such as sulfasalazine, 5-aminosalicylic acid, olsalazine, and imran, and factors that interfere with the synthesis, production, or action of proinflammatory cytokines such as IL-1, such as IL-1β converting enzyme. It can also be combined with inhibitors and caspase inhibitors such as IL-1ra. The binding proteins of the present invention also comprise a T cell signaling inhibitor, such as a tyrosine kinase inhibitor; or a molecule that targets a T cell activation molecule, such as a CTLA-4-IgG or anti-B7 antibody family antibody, Can be used with PD-1 family antibodies. The binding protein of the invention is combined with an IL-11 or anti-cytokine antibody, such as fonotizumab (anti-IFNg antibody), or an anti-receptor receptor antibody, such as an anti-IL-6 receptor antibody and an antibody against a B cell surface molecule It is possible. The antibodies of the present invention or antigen-binding portions thereof may also be LJP394 (abetimus) (a factor that depletes or inactivates B cells), such as rituximab (anti-CD20 antibody), lymphostat B (anti-BlyS antibody) , TNF antagonists such as anti-TNF antibody, D2E7 (PCT Publication NO. WO97 / 29131; HUMIRA), CA2 (REMICADE), CDP571, TNFR-Ig construct (can be used with p75TNFRIgG (ENBREL and p55TNFRIgG (LENERCEPT)).

  The pharmaceutical composition of the present invention may comprise a “therapeutically effective amount” or “prophylactically effective amount” of the binding protein of the present invention. “Therapeutically effective amount” refers to an amount effective to achieve the desired therapeutic result and over the period of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the binding protein can be determined by one of ordinary skill in the art and can vary according to factors such as the disease state, age, sex and individual weight and the ability of the binding protein to elicit the desired response within the individual. A therapeutically effective amount is also the amount by which any toxic or deleterious effect of an antibody or antibody portion can be surpassed by a therapeutically beneficial effect. A “prophylactically effective amount” refers to an amount that is effective at the dosage required to achieve the desired prophylactic result and for the period of time necessary to achieve the desired prophylactic result. Typically, since prophylactic medication is used in patients prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.

  Dosage regimens may be adjusted to provide the optimum desired response (eg, a therapeutic or prophylactic response). For example, a single bolus can be administered, multiple divided doses can be administered over time, or doses can be proportionally reduced or increased as indicated by the urgency of the treatment situation Can be. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. The dosage unit form used herein represents a physically separated unit suitable as a unitary dosage for the mammalian patient to be treated, each unit together with the required pharmaceutical carrier, Contains a predetermined amount of active compound calculated to produce the desired therapeutic effect. The specifications for the dosage unit forms of the present invention include (a) the specific characteristics of the active compound and the specific therapeutic or prophylactic effect to be achieved and (b) such active compound for the treatment of hypersensitivity in an individual. It is defined by restrictions inherent in the field of formulation and depends directly on these.

  A typical non-limiting range for a therapeutically or prophylactically effective amount of a binding protein of the invention is 0.1 to 20 mg / kg, more preferably 1 to 10 mg / kg. It should be noted that dosage values can vary depending on the type and severity of symptoms to be alleviated. The specific dosing regimen should be adjusted over time according to the individual judgment of the person administering or supervising the composition to any specific patient, according to individual requirements, and It is further understood that the dosage ranges set forth in the specification are exemplary only and are not intended to limit the scope or practice of the claimed compositions. Should.

  Other suitable modifications and adaptations of the methods of the invention described herein will be apparent, and appropriate equivalents may be made without departing from the scope of the invention or the embodiments disclosed herein. It will be apparent to those skilled in the art that these can be performed using. Although the invention has been described in detail herein, the invention will be more clearly understood by reference to the following examples, which are intended for purposes of illustration only and are not intended to limit the invention.

Example (Example 1)
Generation of Dual Variable Domain Immunoglobulins (DVD-Ig) Dual Variable Domain Immunoglobulin (DVD-Ig) molecules are composed of two different light chain variable domains (VL) derived from two different parental mAbs by recombinant DNA technology. It is designed to be linked in series, directly or via a short linker, followed by a light chain constant domain. Similarly, the heavy chain comprises a constant domain CH1 and an Fc region (FIG. 1A) after two different heavy chain variable domains (VH) linked in series.

Example 1.1
Production of mouse monoclonal antibodies against IL-1α and IL-1β Monoclonal antibodies against IL-1α and IL-1β were produced using hybridoma technology well known in the art as follows.
(Example 1.1.A)
Mouse Immunization Purified recombinant human IL-1α and mouse IL-1β (R & D Systems) were used as immunogens and coating antigens in titer assays and ELISA screens. For all antigens for both primary and boost immunizations, immunization doses ranged from 5.0 to 20.0 μg / mouse / injection. ImmunoEasy adjuvant was purchased from Qiagen (Waltham, Mass.) And used at an adjuvant / antigen ratio of 20 mL ImmunEasy adjuvant per 10.0 μg antigen. Each group of animals to be immunized is Dr. Five IL-1αβ KO mice obtained from Yoichiro Iwakura (University of Tokyo, Minato-ku, Tokyo, Japan) were included. Mice were immunized according to the dosing schedule described below. MRC-5 cells were purchased from ATCC (Manassas, VA) and used for IL-1 bioassay. Human IL-8 ELISA kit and control mouse anti-hIL-1α and β antibodies (MAB200 and MAB201) were purchased from R & D Systems (Minneapolis, Minn.).

  In summary, an adjuvant-antigen mixture was first prepared by gently mixing the adjuvant in the vial using vortexing. The desired amount of adjuvant was removed from the vial and placed in a heat sterilized 1.5 mL microcentrifuge tube. Antigen was prepared in PBS or saline at concentrations ranging from 0.5 to 1.0 mg / mL. The calculated amount of antigen was then added to the microcentrifuge tube with adjuvant and the solution mixed by gently pipetting up and down 5 times. The adjuvant-antigen mixture was incubated for 15 minutes at room temperature and then mixed again by gently pipetting up and down 5 times. For injection into animals, the adjuvant-antigen solution was aspirated into a suitable syringe. A total of 5 to 20 μg of antigen was injected in a volume of 50 to 100 μL. Each animal was immunized and then immunized 2-3 times depending on the titer. Animals with excellent titers received a final intravenous immunization prior to fusion and hybridoma generation.

(Example 1.1.B)
Hybridoma Screening Hybridomas prepared as described above were screened and antibody titers were measured using ELISA. Standard antigen techniques were used to coat protein antigens directly on ELISA plates to induce specific antibodies. In summary, ELISA plates were coated with 100 μL of either rhIL-1α or rhIL-1β (1.0 μg / mL in PBS) at 4 ° C. overnight. Plates were washed 3 times with 250 μL PBS / 0.5% Tween 20 and blocked with 200 μL blocking buffer (2% BSA in PBS plus 0.5% Tween 20). Diluted serum or hybridoma supernatant (100 μL) was added to each well and incubated for 2 hours at room temperature. The plates were then washed 3 times with PBS / 0.5% Tween 20, and the bound OD was observed at 450 nm using HRP goat anti-mouse IgG for detection. Hybridoma clones producing antibodies that showed high specific binding activity in ELISA were subcloned and purified, and antibody affinity (Biacore) and potency (MRC-5 bioassay) were characterized as follows.

(Example 1.1.C)
Characterization of mouse monoclonal antibodies against IL-1α and IL-1β Example 1.1. In order to characterize the antibody produced by the hybridoma described in B, the following assay was used.

Example 1.1.C.1
Surface plasmon resonance:
In accordance with the manufacturer's instructions and standard procedures, the captured antibody (via goat anti-mouse IgG, on the biosensor matrix by surface plasmon resonance (SPR) using the BIAcore system (Biacore AB, Uppsala, Sweden). The real-time binding interaction between rmIL-1 and mouse anti-rmIL1 antibody captured in In summary, rmIL-1 was diluted in HBS running buffer (Biacore AB) and 50 μL aliquots were injected through the immobilized protein matrix at a flow rate of 5 mL / min. The concentrations of rhIL1 used were 62.5, 125, 187.5, 250, 375, 500, 750, 1000, 1500 and 2000 nM. BIAcore kinetic evaluation software (version 3.1) was used to measure the dissociation constant (dissociation rate) and association constant (binding rate).

(Example 1.1.C.2)
Anti-IL-1 bioassay:
The MRC-5 cell line is a human lung fibroblast cell line that produces IL-8 in response to human IL-1α and IL-1β in a dose-dependent manner (Dinarello, CA, K. Muegge). , And S. K. Durum. 2000. Current Protocols in Immunology 6: 1). MRC-5 cells were cultured in 10% FBS complete MEM and grown at 37 ° C. in a 5% CO ₂ incubator. To measure the neutralizing potency of mAb against recombinant human IL-1α and IL-1β, different concentrations of mAb (50 μL) (0 to 10 μg / mL) were added to a 96 well plate and rhIL at 37 ° C. for 1 hour. Pre-incubated with 50 μL (10 to 50 pg / mL) of −1a or rhIL-1b. Supernatants were collected, diluted, and IL-8 concentration was measured by ELISA using a standard IL-8 ELISA kit (R & D Systems). Antibody potency was measured by the ability of the antibody to inhibit IL-8 production by MRC-5 cells.

  Based on the Biacore and MRC-5 bioassays, a number of mouse anti-hIL-1a and anti-hIL-1b antibodies with high affinity and potency were identified as shown in Table 1 below.

(Example 1.1.D)
Cloning and sequencing of mouse monoclonal antibodies against IL-1α and IL-1β Following isolation and purification of total RNA from each hybridoma cell line using Trizol reagent (Invitrogen) according to the manufacturer's instructions, Table 1 Cloning and sequencing of the variable heavy (VH) and light (VL) genes of all described anti-IL-1a / b mAbs and additional antibodies were performed. Using the One-tube RT-PCR kit (Qiagen) as directed by the manufacturer and using IgGVH and IgκVL oligonucleotides obtained from Mouse Ig-Primer Set (Novagen, Madison, WI), both VH and VL genes Amplification was performed. The DNA fragment resulting from productive amplification was cloned into the pCR-TOPO vector (Invitrogen) according to the manufacturer's instructions. The sequences of multiple VH and VL clones were then determined by the dideoxy chain termination method using an ABI 3000 sequencer (Applied Biosystems, Foster City, CA). The sequences of all mAb VL and VH genes are shown in Table 2 below.

(Example 1.2)
Generation and characterization of mouse-human chimeric antibodies All of the above mAbs were converted to chimeras (with human constant regions), expressed, purified, characterized to confirm activity, and then subjected to subsequent DVD-Ig analysis. Use as a control for. 3D12. To convert E3 to a chimeric form, primers P1 and P2 were used to E3-VL was PCR amplified and the human Ck gene (in a pBOS vector made in-house at ABC) was amplified using primers P3 and P4. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were gel purified and used together as a duplicate template for subsequent duplicate PCR reactions using standard PCR conditions and using primers P1 and P4. The final PCR product chimeric light chain 3D12. E3-VL-hCk was subcloned into the pEF6TOPO mammalian expression vector (Invitrogen). Table 3 shows the PCR primer sequences.

  3D12. In order to convert the E3 heavy chain to a chimeric form, primers P5 and P6 were used to generate 3D12. E3-VH was PCR amplified and the human Cγ1 gene (in the pBOS vector produced in-house at ABC) was amplified using primers P7 and P8. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were gel purified and used together as a duplicate template for subsequent duplicate PCR reactions using standard PCR conditions and using primers P5 and P8. According to the manufacturer's instructions, the final PCR product chimeric light chain 3D12. E3-VH-hCγ1 was subcloned into pcDNA3.1 TOPO mammalian expression vector (Invitrogen). Table 4 shows the PCR primer sequences.

  Similarly, chimera 13F5.. Using primers P21 / P22 (for VH) and P7 / P8 (for hCγ1). G5-VH-Cγ1 was generated and cloned into pcDNA3.1 TOPO vector and chimera 13F5. Using primers P23 / P24 (for VL) and P3 / P4 (for hCk). G5-VL-Ck was made and cloned into the pEF6TOPO vector. Table 5 shows the PCR primer sequences.

  In order to express the chimeric Ab, 13 hours in Lipofectamine (Invitrogen) for 13 hours. G5-VL-Cκ and 13F5. G5-VH-Cγ1 was co-expressed in COS, the medium was collected and IgG purified by protein A chromatography. Similarly, using Lipofectamine (Invitrogen) for 72 hours, 13F5. G5-VL-Cκ and 13F5. G5-VH-Cγ1 was co-expressed in COS, the medium was collected and IgG was purified by protein A chromatography. To confirm the activity, both purified chimeric Abs were characterized by Biacore and MRC-5 bioassays. The results showed that these chimeric Abs showed similar affinity and potency to that of the original mouse mAb.

(Example 1.3)
Construction, expression and purification of IL-1α / β dual variable domain immunoglobulin (DVD-Ig) The construct used to make DVD-Ig capable of binding to hIL-1α and hIL-1β is It is shown in FIG. 1B. In summary, by immunizing Balb / c mice with recombinant IL-1α protein (rhIL-1α) and recombinant IL-1β protein (rhIL-1β), respectively, two high affinity mouse Abs, anti-hIL- A parental mAb containing 1α (clone 3D12.E3) and anti-bIL-1β (clone 13F5.G5) was obtained. The VL / VH genes of these two hybridoma clones were isolated by RT-PCR using mouse IgPrimerKit (Novagen, Madison, WI). To confirm activity and potency, the VL / VH gene was first converted to a chimeric antibody (with human constant regions). To make DVD1-Ig, 13F5. G5 VH and VL are respectively 3D12. Fused directly to the N-terminus of E3 VH and VL (as shown in FIG. 1B). DVD2-Ig was similarly constructed except that it had a linker between the two variable domains in both the light chain (linker sequence ADAAP) and the heavy chain (linker sequence AKTTPP). These sequences were selected from the N-terminus of the mouse Ck and CH1 sequences. These linker sequences, selected from the N-terminus of mouse Ck and CH1, are natural extensions of the variable domain and, based on the analysis of several Fab crystal structures, provide a flexible conformation without significant secondary structure. Show. Detailed procedures for PCR cloning are described below.

(Example 1.3.A)
Molecular cloning of hIL-1a / bDVD1-Ig:
Using primers P21 and P25, 13F5. G5-VH was PCR amplified and using primers P14 and P8, 3D12. E3-VH-hCγ1 was amplified. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were gel purified and used together as a duplicate template for subsequent duplicate PCR reactions using standard PCR conditions and using primers P21 and P8. The final PCR product DVDIg heavy chain hIL-1a / bDVD1-VH-hCγ1 was subcloned into pcDNA3.1 TOPO mammalian expression vector (Invitrogen) according to the manufacturer's instructions. Table 6 shows the PCR primer sequences.

  In order to make hIL-1a / bDVD1-Ig light chain, primers P23 and P26 were used to generate 13F5. G5-VL was PCR amplified and using primers P16 and P4, 3D12. E3-VL-hCκ was amplified. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were gel purified and used together as a duplicate template for subsequent duplicate PCR reactions using standard PCR conditions and using primers P23 and P4. The final PCR product hIL-1a / bDVD1-Ig light chain hIL-1a / bDVD1-VL-hCκ was subcloned into the pEF6TOPO mammalian expression vector (Invitrogen) according to the manufacturer's instructions. Table 7 shows the sequences of the PCR primers.

(Example 1.3.B)
Molecular cloning of hIL-1a / bDVD2-Ig:
Using primers P21 and P17, 13F5. G5-VH was PCR amplified and using primers P18 and P8, 3D12. E3-VH-hCγ1 was amplified. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were gel purified and used together as a duplicate template for subsequent duplicate PCR reactions using standard PCR conditions and using primers P21 and P8. The final PCR product DVD2-Ig heavy chain hIL-1a / bDVD2-VH-hCγ1 was subcloned into the pcDNA3.1 TOPO mammalian expression vector (Invitrogen) according to the manufacturer's instructions. Table 8 shows the PCR primer sequences.

  In order to generate hIL-1a / bDVD2-Ig light chain, primers P23 and P19 were used to generate 13F5. G5-VL was PCR amplified and using primers P20 and P4, 3D12. E3-VL-hCκ was amplified. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were gel purified and used together as a duplicate template for subsequent duplicate PCR reactions using standard PCR conditions and using primers P23 and P4. The final PCR product hIL-1a / bDVD2-Ig light chain hIL-1a / bDVD2-VL-hCκ was subcloned into the pEF6TOPO mammalian expression vector (Invitrogen) according to the manufacturer's instructions. Table 9 shows the PCR primer sequences.

  The final sequences of hIL-1a / bDVD1-Ig and hIL-1a / bDVD2-Ig are listed in Table 10.

(Example 1.3.C)
Expression and Purification of hIL-1a / bDVD1-Ig The heavy and light chains of each construct were subcloned into pcDNA3.1 TOPO and pEF6TOPO vectors (Invitrogen Inc.), respectively, and sequenced to verify accuracy. Using Lipofectamine 2000 and 293fectin reagent, transiently express the plasmids encoding the heavy and light chains of each construct in COS cells and human fetal kidney 293 cells (American Type Culture Collection, Manassas, VA), respectively. I let you. 72 hours after transient transfection, cell culture medium was collected and antibodies were purified using protein A chromatography (Pierce, Rockford, IL) according to the manufacturer's instructions. Ab was analyzed by SDS-PAGE and quantified by A280 and BCA (Pierce, Rockford, IL). Table 11 shows that the expression levels of hIL-1a / bDVD1-Ig and hIL-1a / bDVD2-Ig are equivalent to those of the chimeric Ab, and that DVD-Ig is efficient in mammalian cells. It suggests that it can be expressed.

(Example 1.4)
hIL-1a / b DVD-Ig Mass Spectrometry and SEC Analysis To determine the molecular weight (MW) of the light and heavy chains of DVD-Ig, 10 μL of DVD-Ig (0. 8 μg / μL) was reduced. PLRP-S, 8μ, 4000 Å and 1 × 150 mm protein columns (Michrom BioResource, Auburn, Mass.) Were used to separate the heavy and light chains of DVD-Ig. A mass spectrometer QSTAR (Applied Biosystems, Foster City, Calif.) And an Agilent HPI10O Capillary HPLC (Agilent Technologies Inc., Pala Alto, Calif.) Were used. The Barco valve was set at 10 minutes to switch the flow from waste to MS to desalinate the sample. Buffer A was 0.02% TFA, 0.08% FA, 0.1% ACN and 99.8% HPLC-H2O. Buffer B contained 0.02% TFA, 0.08% FA, 0.1% CAN, 0.1% HPLC-H2O and 99.8% ACN. The HPLC flow rate was 50 μL / min and the sample injection volume was 8.0 mL. The column oven temperature was set to 60 ° C. and the separation gradient was 5% B for 5 minutes, 5% B to 65% B for 35 minutes, 65% B to 95% B for 5 minutes, and 95% B for 5 minutes. To 5% B. The TOFMS scan was 800 to 2500 amu and the cycle was 3600. To measure the MW of full-length DVD-Ig, a Protein MicroTrap cartridge (Michrome BioResource, Auburn, Mass.) Was used to desalinate the sample. The HPLC gradient was 5% B for 5 minutes, 5% to 95% B in 1 minute, and 95% B to 5% B in 4 minutes. The QSTARTOFMS scan was 2000 to 3500 amu and the cycle was 899. All MS raw data were analyzed using Analyst QS software (Applied Biosystems). For SEC analysis of DVD-Ig, purified DVD-Ig and chimeric Ab in PBS were applied to a Superose 6 10/300 G2, 300 × 10 mm column (Amersham Bioscience, Piscataway, NJ). An HPLC instrument model 10A (Shimadzu, Columbia, MD) was used for SEC. All proteins were measured using UV detection at 280 nm and 214 nm. The elution was homogeneous at a flow rate of 0.5 mL / min. For stability studies, samples in the concentration range of 0.2 to 0.4 mg / mL in PBS were subjected to 3 freeze-thaw cycles between -80 ° C and 25 ° C, or 4 weeks and 8 SEC analysis was performed after incubation at 4 ° C, 25 ° C or 40 ° C for a week.

  DVD-Ig and chimeric Ab were purified by protein A chromatography. The purification yield (3-5 mg / L) was consistent with the hIgG quantification of the expression solvent for each protein. The composition and purity of the purified DVD-Ig and chimeric Ab were analyzed by SDS-PAGE under both reducing and non-reducing conditions. In non-reducing conditions, each of the four proteins migrated as a single bond. The DVD-Ig protein showed a higher molecular weight than the chimeric Ab, as expected. In non-reducing conditions, each of the four proteins gave two bands (one heavy chain and one light chain). Similarly, the heavy chain and light chain of DVD-Ig are larger in size than that of chimeric Ab. In SDS-PAGE, each DVD-Ig is expressed as a single species, and the heavy and light chains are efficiently paired. Have been shown to form IgG-like molecules. The sizes of the heavy and light chains and the full-length protein of the two DVD-Ig molecules are consistent with their molecular weight calculated based on the amino acid sequence (see Table 11).

  Mass spectrometry was used to determine the exact molecular weight of DVD-Ig. As shown in Table 1, the molecular weight of each DVD-Ig (including light chain, heavy chain and full length protein) measured experimentally is in good agreement with the expected value. To further study the physical properties of DVD-Ig in solution, size exclusion chromatography (SEC) was used to analyze each protein. Both chimeric Ab and DVD2-Ig showed a single peak and physical homogeneity as a monomeric protein. 3D12. E3 chimera Ab is 13F5. Shows a smaller physical size than the G5 chimeric Ab, 3D12. This suggests that the E3 chimeric Ab takes a more compact spherical shape. DVD1-Ig shows a main peak and a right shoulder peak, suggesting that some of DVD1-Ig is probably in an aggregated form under current buffer conditions.

(Example 1.5)
Analysis of in vitro stability of hIL-1a / bDVD-Ig The physical stability of DVD-Ig was examined as follows. Purified antibodies in the concentration range of 0.2 to 0.4 mg / mL in PBS are subjected to 3 freeze / thaw cycles between −80 ° C. and 25 ° C., or 4 and 8 weeks, 4 After incubation at 25 ° C or 40 ° C, analysis using size exclusion chromatography (SEC) analysis was performed (see Table 12).

  Both chimeric Abs showed a slight degree of aggregation and fragmentation, normal for normal IgG molecules. DVD1-Ig showed some aggregation on SCE after purification. In stability analysis, DVD1-Ig also showed aggregation in PBS under different conditions, but the percentage of aggregated form of DVD1-Ig did not increase during prolonged storage or at higher temperatures. . The percentage of fragmented form of DVD1-Ig is in the normal range and chimera 3D12. E3. Similar to that of Ab. In contrast, DVD2-Ig showed significant stability. Under all conditions examined, no aggregation or fragmentation was detected for DVD2-Ig and 100% of DVD2-Ig maintained intact monomer molecules.

(Example 1.6)
Measurement of Antigen Binding Affinity of hIL-1a / bPVD-Ig The kinetics of DVD-Ig binding to rhIL1-α and rhIL1-β was determined by HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0 ° C. at 25 ° C. .005% surfactant P20) was measured by measurement based on surface plasmon resonance using a Biacore 3000 instrument (Biacore AB, Uppsala, Sweden). All chemicals were obtained from Biacore AB (Uppsala, Sweden) or from another source described herein. A goat anti-human IgG Fcγ fragment-specific polyclonal antibody (Pierce) diluted in 10 mM sodium acetate (pH 4.5) approximately using a standard amine coupling kit according to the manufacturer's instructions and operating at 25 mg / mL. 5000 RU from Biotechnology Inc, Rockford, IL) was immobilized directly across the CM5 research grade biosensor chip. The unreacted part on the biosensor surface was blocked with ethanolamine. The modified carboxymethyl dextran surface in flow cells 2 and 4 was used as the reaction surface. Unmodified carboxymethyldextran without goat anti-human IgG in flow cells 1 and 3 was used as the reference surface. For kinetic analysis, kinetic equations derived from the 1: 1 Langmuir binding model were simultaneously fitted to all 10 injection association and dissociation phases using Bioevaluation 4.0. 1 software (global fit). Analysis). To capture across the goat anti-human IgG Fc specific reaction surface, the purified DVD-Ig sample was diluted in HEPES buffered saline and injected onto the reaction matrix at a flow rate of 5 mL / min. Association and dissociation rate constants k _on (M ⁻¹ s ⁻¹ ) and k _off (s ⁻¹ ) were measured under a continuous flow rate of 25 mL / min. Rate constants were derived by performing kinetic binding measurements at 10 different antigen concentrations ranging from 1.25 to 1000 nM. The equilibrium dissociation constant (M) of the reaction between DVD-Ig and rhIL1α / β was then calculated from the kinetic rate constant by the following formula: K _D = k _off / k _on . In order to record and subtract all non-specific binding background to remove most of the refractive index change and injection noise, a preparative sample of rhIL1α / β sample was also simultaneously applied to the blank reference and reaction CM surface. Injected. Thereafter, 25 mL of 10 mM glycine (pH 1.5) was injected twice at a flow rate of 5 mL / min to regenerate the surface. The surface on which the anti-Fc antibody was immobilized was completely regenerated, and the complete capture ability was maintained over 12 cycles. The following formula:

を用いて、飽和結合条件（定常状態平衡）下で、捕捉されたＤＶＤ−Ｉｇ−ｒｈＩＬ１αβ複合体の見かけの化学量論を計算した。

Was used to calculate the apparent stoichiometry of the captured DVD-Ig-rhIL1αβ complex under saturated binding conditions (steady state equilibrium).

  Biacore analysis shows that the chimeric Ab has similar binding kinetics and affinity for IL-1 similar to the original hybridoma mAb, suggesting that the correct VL / VH sequence was isolated. (Table III). The overall binding parameters of the two DVD-Igs to hIL-1α are similar and the affinity of DVD-Ig is similar to that of chimeric 3D12. It was only 2-3 times lower than the affinity of E3Ab. The binding affinity of DVD2-Ig to hIL-1β was determined using the chimeric Ab13F5. Although slightly lower than G5, it was 3 times higher than the affinity of DVD1-Ig. As shown by stoichiometric evaluation for IL-1, the affinity of the two DVD-Igs for hIL-1 was similar compared to the affinity of the chimeric Ab for hIL-1. Both chimeric Abs that are bivalent monospecific bound to IL-1α and IL-1β on the Biocore with 1.6 and 1.7 stoichiometry, respectively. This is common for IgG due to intermolecular interference when the antibody is tightly immobilized on a Biacore sense chip, and the stoichiometry ranges from 1.5 to 2.0. The stoichiometry of both DVD-Igs to hIL-1α and hIL-1β is similar to the stoichiometry of the two chimeric Abs, and both DVD-Igs have a bivalent binding capacity for each antigen. It suggests.

  In addition, the tetravalent bispecific antigen binding of DVD-Ig was also analyzed by Biacore (Table 14). First, DVD-Ig was captured via the goat anti-human Fc antibody on the Biacore sensor chip, the first antigen was injected, and the binding signal was observed. As DVD-Ig was saturated by the first antigen, the second antigen was then injected and a second signal was observed. For DVD2-Ig, this was done by first injecting IL-1β followed by IL-1α, or first injecting IL-1α followed by IL-1β. Double binding activity was detected in any sequence. Similar results were obtained for DVD1-Ig. Thus, each DVD-Ig was able to bind both antigens simultaneously as a bispecific tetravalent molecule. As shown in Table IV, the stoichiometry of both DVD-Igs for the first antigen (either hIL-1α or hIL-1β) is greater than 1.5, and the monospecific bivalent binding Similar to stoichiometry. When the second antigen was injected, the DVD-Ig was already occupied by the first antigen, but the stoichiometry of both DVD-Igs relative to the second antigen (ie, either hIL-1α or hIL-1β). The argument was between 1.0 and 1.3. Thus, DVD-Ig can bind to two IL-1α and two IL-β molecules. First, DVD-Ig was captured via a goat anti-human Fc antibody on a Biacore sensor chip, a first antigen was injected, a binding signal was observed, and then a second antigen was injected.

(Example 1.7)
Measurement of functional homogeneity of DVD-Ig DVD2-Ig was purified by protein A chromatography instead of target specific affinity chromatography, so it was either misfolded and / or made a wrong pair If any VL / VH domain is present, it can be assessed by binding studies against two different antigens. Such binding analysis was performed by size exclusion liquid chromatography (SEC). DVD2-Ig was applied to the column alone or after an incubation period of 120 minutes at 37 ° C. with IL-1α, IL-1β or an equimolar ratio of both IL-1α and IL-1β. Each of the antigens was run alone as a control. The SEC results show that DVD2-Ig can bind IL-1α and IL-1β in solution, and such binding is a shift to the SEC signal (when complexed with either antigen, DVD2 -Indicates an increase in the dynamic size of Ig). The shift of the DVD2-Ig signal was not partial and was 100%, suggesting that all DVD-2Ig molecules can bind the antigen. In the presence of both IL-1α and IL-1β, there is an additional complete shift of the DVD2-Ig signal, indicating that all DVD2-Ig molecules can bind both antigens in a uniform manner. This experiment showed that DVD-Ig is expressed as a functionally homogeneous protein. This indicates that DVD-Ig can be produced as a uniform, single functional species distinct from all previously described bispecific, multispecific and multivalent immunoglobulin-like and immunoglobulin-derived molecules. So it has important suggestions.

Example 1.8
Measurement of biological activity of DVD-Ig The biological activity of DVD-Ig was measured using the MRC-5 bioassay. The MRC-5 cell line is a human lung fibroblast cell line that produces IL-8 in response to human IL-1α and IL-1β in a dose-dependent manner. MRC-5 cells were obtained from ATCC and cultured at 37 ° C. in 5% CO ₂ incubator in 10% FBS complete MEM. To measure the neutralizing activity of DVD-Ig against human IL-1α and IL-1β, 50 μL of Ab in MEM / 10% FBS (1E-7 to 11E-12M) was added to a 96-well plate, 37 ° C., 1 hour at 5% CO _2, were preincubated with hIL-l [alpha] or hIL-l [beta] of 50μL (200pg / mL). MRC-5 cells at a concentration of 1E5 / mL were then added to all wells (100 μL) and the plates were incubated overnight at 37 ° C. in a 5% CO ₂ incubator. Supernatants were collected and human IL-8 production was measured by standard ELISA (R & D Systems, Minneapolis, Minn.). The neutralizing activity of DVD-Ig was measured by the ability of DVD-Ig to inhibit IL-8 production.

  As shown in Table 13, both DVD-Igs were able to neutralize hIL-1α and hIL-1β. Consistent with the binding affinity to hIL-1α, the neutralization activity of DVD1-Ig and DVD2-Ig against hIL-1α was similar (ie, 1/3 of the neutralization activity of chimeric Ab (see Table III)). )). Consistent with its binding affinity for hIL-1β, the neutralizing activity of DVD2-Ig against hIL-1β is similar to that of chimeric Ab13F5. Although slightly lower than the neutralizing activity of G5, it was 3 times higher than the affinity of DVD1-Ig. Overall, there was no significant decrease in the biological activity of the DVD-Ig molecule compared to the original mAb.

  To determine whether DVD-Ig can inhibit IL-8 production in the presence of both IL-1α and IL-1β, hIL-1α and hIL- in the same culture system of the MRC-5 assay. An equal amount of 1β was added. Both DVD1-Ig and DVD2-Ig are similar in activity to the monoassay where only one cytokine is present, and IL-8 by MRC-5 cells in the presence of both IL-1α and IL-1β. Synthesis could be inhibited (Table 13). In this assay where both IL-1α and IL-1β are present, the dual inhibitory activity of DVD2-Ig (1.2 nM) was higher than the dual inhibitory activity of DVD1-IG (2.2 nM).

(Example 2)
Analysis of Linker Size and Variable Domain Orientation in DVD-Ig Molecules Additional DVD-Ig molecules with different parental mAb pairs were constructed as shown in Table 15. For each pair of mAbs, four different DVD-Ig constructs (short 2 with a linker and 2 with a long linker). The linker sequence was derived from the N-terminal sequence of the human Ck or CH1 domain, as shown below.

Short linker: light chain: TVAAP; heavy chain: ASTKGP
Long linker: light chain: TVAAPSVIFPPP; heavy chain ASTKGPSVFPLAP
All heavy and light chain constructs were subcloned into the pBOS expression vector and expressed in COS cells or Freestyle 293 cells.

  To construct a new DVD clone, two mAb variable domains (both light and heavy chains) were used using overlapping PCR as described for hIL-1abDVD1-Ig and hIL-1abDVD2-Ig. ) Were first connected in series. The ligated pieces were then subcloned into the pBOS vector using homologous recombination. In summary, the vector was linearized by restriction digestion (2 μg of pBOS-hCk vector was digested with FapAI and BsiWI in O + buffer, and 2 μg of pBSO-hCγz, non a, vector in O + buffer. Digested with FspAI and SaII). The digested sample was run on a 1% agarose gel and the backbone fragment was purified in 50 μL of water. For homologous recombination and transformation, DH5α transformed recipient cells were thawed on ice and mixed with 20-50 ng ligated PCR product and linearized vector 20-50 ng (in all 50 μLDH5α cells). ). The mixture was gently mixed and incubated on ice for 45 minutes, followed by heat shock at 42 ° C. for 1 minute. Then 100 μL of SOC medium was added and incubated at 37 ° C. for 1 hour. Transformation cultures were seeded on LB / agar plates containing ampicillin and incubated for 18-20 hours at 37 ° C. Bacterial clones were isolated from which DNA was purified and subjected to sequencing analysis. As previously described, clones with final sequence confirmation were co-transfected into COS or 293 cells (matching HV and LC of the same Ab pair) for Ab expression and purification.

  Properties of the purified DVD-Ig protein are summarized in Table 16. The left part of Table 16 shows the specificity, binding affinity and neutralizing capacity of the two pairs of mAbs used for the construction of the new hIL-1a / bDVD-1g molecule. To construct hIL-1a / bDVD3a-Ig, hIL-1a / bDVD4a-Ig, hIL-1a / bDVD3b-Ig and hIL-1a / bDVD4b-Ig, antibodies 18F4.2C8 and 1B12.4H4 (Example 1. 1.D) was used. hIL-1a / bDVD3a-Ig and hIL-1a / bDVD4a-Ig are in the ab-C orientation and have a short linker and a long linker, respectively. hIL-1a / bDVD3b-Ig and hIL-1a / bDVD4b-Ig are in the baC orientation and have a short linker and a long linker, respectively. Antibodies 6H3.1A4 and 6B12.4F6 were 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 are in the ab-C orientation and have a short linker and a long linker, respectively. hIL-1a / bDVD5b-Ig and hIL-1a / bDVD6b-Ig are in ba-C orientation and have a short linker and a long linker, respectively. Using the procedure described above for hIL-1a / bDVD1-Ig (see Example 1.3), molecular cloning of these additional hIL-1a / bDVD-Ig was performed using overlapping PCR operations. The amino acid sequences of these additional hIL-1a / bDVD-Ig are disclosed in Table 15.

The characteristics of the new DVD construct are summarized in Table 16. Affinity (K _d ) and biological activity (IC ₅₀ ) were measured by Biacore and MRC-5 bioassays, respectively. SDS-PAGE analysis of all new DVD proteins showed normal migration patterns similar to normal antibodies and DVD1 / 2-Ig under both reducing and non-reducing conditions.

  The functional characterization of the new DVD molecule showed that the DVD with the long linker outperformed the DVD with the short linker in terms of binding and neutralization of both antigens in any orientation. For DVDs with long linkers, DVDs with ba-C orientation showed excellent binding to both antigens and good neutralization of both antigens, whereas DVDs with a-b-C orientation , Showed excellent binding to IL-1α and excellent neutralization of IL-α, but binding to IL-1β and neutralization of IL-β was reduced (eg, DVD4b vs. DVD4a). The DVD-Ig molecule, DVD4b, bound and neutralized both IL-1α and IL-1β at less than nM and fully retained the binding and neutralizing properties of the parental mAb.

(Example 3)
Production of DVD-Ig capable of binding IL-12 and IL-18 As described above, one DVD-Ig molecule capable of binding IL-12 and EL-18, one for human IL-12p40 (ABT874) and one for It was constructed using two parental monoclonal antibodies that are against human EL-18 (ABT325). Four different anti-IL-12 / 18 DVD-Ig constructs were made, with two short linkers and two long linkers, each with two different domain orientations: 12-18-C and 18-12-C (Table VI). Linker sequence from the N-terminal sequence of human C [lambda] / C _k or CH1 domain were as follows.

DVD1218 constructs (. ABT874 is having a V _λ) with respect to:
Light chain (λ): short linker: QPKAAP, long linker: QPKAAPSVLFLFPP,
Heavy chain (vl): short linker: ASTKGP, long linker: ASTKGPSVFPLAP,
For the DVD 1812 construct (ABT 325 has V _k ):
Light chain (k): short linker: TVAAP, long linker: TVAAPSVIFFPP,
Heavy chain (vl): short linker: ASTKGP, long linker: ASTKGPSVFPLAP.

  All heavy and light chain productions were subcloned into the pBOS expression vector, expressed in COS cells or Freestyle 293 cells, and then purified by protein A chromatography. The purified material was subjected to SDS-PAGE and SEC and their properties were similar to those of DVD2-Ig.

  Table 17 below describes the production of heavy and light chains used to express each anti-IL12 / L18 DVD-Ig protein.

Example 3.1.1: Molecular Cloning of DNA Production for DVD1218SL and DVD1218LL ABT was used to create heavy chain constructs DVD1218HC-LL and DVD1218HC-SL using Primer 1 and Primer 2L or Primer 2S, respectively. While the -874 VH domain was PCR amplified, the VBT domain of ABT-325 was amplified using primer 3L or primer 3S and primer 4, respectively. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were gel purified and used together as a duplicate template for subsequent duplicate PCR reactions using primer 1 and primer 4 using standard PCR conditions. By using a standard homologous recombination approach to pBOS-hCvl (Abbott), a non-mammalian expression vector, double digested with Srf1 and SalI, overlapping PCR products are obtained. Subcloned.

  To make light chain constructs DVD1218LC-LL and DVD1218LC-SL, the VL domain of ABT-874 was PCR amplified using primer 5 and primer 6L or primer 6S, respectively, whereas primer 7L Alternatively, Primer 7S and Primer 8 were used to amplify the BT domain of ABT-325. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were gel purified and used together as a duplicate template for subsequent duplicate PCR reactions using primer 5 and primer 8, using standard PCR conditions. Overlapping PCR products were subcloned by using a standard homologous recombination approach to the pBOS-hCkl mammalian expression vector (Abbott) double digested with Srf1 and NotI. The primers used for these constructions are listed in Table 18 below.

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

Example 3.1.2: Molecular cloning of DNA production for DVD1812SL and DVD1812LL To create heavy chain constructs DVD1812HC-LL and DVD1812HC-SL, using primer 1 and primer 9L or primer 9S, respectively, The VH domain of ABT-325 was PCR purified. The VH domain of ABT-874 was amplified using primer 10L or primer 10S and primer 4, respectively. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were gel purified and used together as a duplicate template for subsequent duplicate PCR reactions using primer 1 and primer 4 using standard PCR conditions. By using a standard homologous recombination approach to pBOS-hCvl (Abbott), a non-mammalian expression vector, double digested with Srf1 and SalI, overlapping PCR products are obtained. Subcloned. The following is the primer sequence.

  To make the light chain constructs DVD1812LC-LL and DVD1812LC-SL, the VL domain of ABT-325 was PCR amplified using primer 11 and primer 12L or primer 12S, respectively, whereas primer 13L, respectively. Alternatively, primer 13S and primer 14 were used to amplify the BT domain of ABT-874. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were gel purified and used together as a duplicate template for subsequent duplicate PCR reactions using primer 11 and primer 14, using standard PCR conditions. Overlapping PCR products were subcloned by using a standard homologous recombination approach to the pBOS-hCkl mammalian expression vector (Abbott) double digested with Srf1 and NotI. The primers used for these constructions are listed in Table 19 below.

  The final DNA sequences for the production of the 8 heavy and light chains of anti-IL12 / L-18 DVD-Ig are as shown in Table 20.

(Example 3.2)
Measurement of Antigen Binding Affinity of IL-12 / L-18DVDIg The binding affinity of anti-IL-12 / 18DVD-Ig to hIL-12 and hIL-18 was measured by Biacore (Table 21). Neutralizing activity against EL-18 was measured by KG-1 assay (Konishi, K, et al.). Briefly, after pre-incubating an IL-18 sample (with a final concentration of 2 ng / mL) with DVD-Ig (with a final concentration between 0 and 10 mg / mL) for 1 hour at 37 ° C. After addition to KG-1 cells (3 × 10 ⁶ cells / mL) in RPMI medium containing 10 ng / mL hTNF, the cells were incubated at 37 ° C. for 16-20 hours. Culture supernatants were collected and human IFN-γ production in each sample was measured by ELISA (R & D Systems). Table 6 shows the inhibitory activity of DVD molecules against IL-18 expressed as IC50 values. To measure the inhibitory activity of anti-IL-12 / 18 DVD molecules against IL-12, an IFN-γ production assay induced by IL-12 from activated PHA blasts was utilized (D'Andrea, A et al.). To produce human IFN-γ, PHA blasts were incubated with human IL-12 for 18 hours. Sub-maximal stimulation (maximum 55-75%) was obtained at a human IL-12 concentration of 200 pg / mL. The supernatant was assayed for IFN-γ using a specific human IFN-γ ELISA (Endogen, Cambridge, Mass.). By neutralizing IL-12 DVD, IFN-γ production induced by IL-12 is prevented. As shown in Table 21, the neutralizing activity of the DVD was measured by measuring the concentration of DVD required to inhibit 50% of IFN-γ production by human PHA blasts.

  Affinity (Kd) was measured by Biacore, and the potency was measured by KG-1 bioassay (IL-18) and PBMC assay (IL-12).

  Table 21 shows the specificity, binding affinity and neutralizing activity of the two fully human monoclonal antibodies used to construct the anti-DL-12 / IL-18DVD molecule. As shown in Table VI, these monoclonal antibodies have high affinity and neutralizing activity. A summary of the features of making anti-IL-18 / IL-12 DVD is shown in Table VI. SDS-PAGE analysis of all new DVD proteins showed normal migration patterns under both reducing and non-reducing conditions, as with normal antibodies and DVD1 / 2-Ig. SEC analysis showed that all molecules were normal and exhibited a peak in the 200 kD region. Biacore binding data is consistent with neutralizing activity in biological assays.

(Example 3.3)
In vivo bioactivity of anti-IL-12 / IL-18DVD-Ig The huPBMC-SCID mouse model was used to measure the bioactivity of anti-DL-12 / IL-18DVD-Ig in vivo. In this model, anti-IL-12 antibody (ABT-874), anti-EL-18 antibody (ABT-325) or anti-IL-12 / anti-IL-18DVD-Ig were injected intraperitoneally or intravenously (250 mg / each mouse). ) Subsequently, freshly purified human PBMC (huPBMC) was injected intraperitoneally into SCID mice. After 15 minutes, dry S. aureus cells (SAC) were administered to mice to induce human IFNγ production. Animals (n = 7-8 / group) were sacrificed 18-20 hours later and serum huIFNγ levels were measured by ELISA. ABT874 and ABT-325 inhibit IFNγ induced by SAC by 70%, corresponding to maximal IFNγ inhibition with each compound in this model. However, treatment of mice with ABT-874 + ABT-325 and anti-IL-12 / anti-IL-18DVD-Ig inhibited IFNγ production almost 100%. These results suggest that anti-IL-12 / anti-IL-18DVD-Ig molecules are functionally active in vivo.

(Example 3.4)
Pharmacokinetic and pharmacodynamic studies of anti-IL-12 / IL-18DVD-Ig The general pharmacokinetic and pharmacodynamic properties of anti-EL-12 / IL-18DVD-Ig are shown in parental monoclonal antibodies in mice. It is the same. That is 73% bioavailability, comparable to normal IgG. A similar pharmacokinetic feature, ie rapid clearance after 6-8 days, was also observed for other monoclonal antibodies (eg, human, rat, etc.), presumably due to the anti-human IgG response.

  Male SD rats were administered anti-IL-12 / IL-18DVD-Ig at either 4 mg / kg, either intravenously or subcutaneously. The early part of the PK curve looked normal and was very similar to that of other human antibodies. Due to the rapid clearance of DVD-Ig starting on day 6, the exact half-life in both groups was not obtained. The sharp drop in DVD-Ig concentration after day 6 can be due to the RAHA reaction. However, not only for other monospecific human antibodies already studied, but also for one of the parent antibodies (ABT-874) used for the construction of this DVD-Ig in this experiment. Characteristics were observed. For both subcutaneous and intravenous injections, the bioavailability of DVD-Ig was estimated based on DVD-Ig concentrations up to day 6. Two of the three rats showed a bioavailability of 80-95%, with an average bioavailability between the three being 73%.

(Example 3.5)
Physical / chemical characteristics of anti-IL-12 / anti-IL-18DVD-Ig The results of the physical and chemical characteristics of anti-IL-12 / anti-IL-18DVD-Ig purified from protein 293 from 293 cells Are summarized in Table 22.

(Example 3.6)
Generation of additional anti-12 / anti-18 DVD-Ig (1D4.1-ABT325) As shown in Table 23, additional anti-EL- of different parent anti-IL-12 monoclonal antibodies (clone number 1D4.1) 12 / IL-18DVD-Ig molecules were constructed. A 1D4.1-ABT325DVD-Ig construct was made using a short linker derived from the N-terminal sequence of the human Ck and CH1 domains as follows.

Short linker: light chain: TVAAP, heavy chain: ASTKGP
All heavy and light chain productions were subcloned into the pBOS expression vector and expressed in COS cells or Freestyle 293 cells and then characterized as described above. 1D4.1-ABT325 DVD-Og fully retains the activity of the two original monoclonal antibodies (Table 24).

Example 4
Generation of anti-CD20 / anti-CD3 DVD-Ig Mouse anti-human CD20 (clone 5F1) and anti-human CD3 (clone OKT3) parent antibodies were used to generate anti-CD20 / anti-CD3 DVD-Ig. The first construct included two DVD-Igs with different domain orientations. V _CD3 - a steady order to build the anti-CD3 / anti _{CD20DVD-Ig, V CD20} - - linker _{V CD20} linker _{-V CD3} - in steady order to construct anti-CD20 / anti-CD3 DVD-Ig. However, in preliminary cell surface binding studies, anti-CD20 binding activity was reduced in anti-CD3 / anti-CD20 DVD-Ig molecules, but anti-CD3 activity was preserved in this design. In contrast, both anti-CD3 binding activity and anti-CD20 binding activity are fully preserved in anti-CD20 / anti-CD3 DVD-Ig, which is related to the combination of these two monoclonal antibodies / targets in the DVD-Ig format. , Showed the optimal domain orientation. Therefore, the anti-CD20 / anti-CD3 DVD-Ig construct was selected for subsequent studies.

  Anti-CD20 / anti-CD3 DVD-Ig was produced as a chimeric Ig. That is, the constant region was a human constant region. For binding analysis, human T cell line Jurkat and B cell line Raji were blocked with human IgG, followed by mouse anti-hCD3 monoclonal antibody OKT3, mouse anti-hCD20 monoclonal antibody 1F5, and anti-CD20 / anti-CD3 DVD-Ig. Stained. Cells were then washed and stained with FITC-labeled goat anti-mouse IgG (no cross-reactivity with anti-hIgG). Anti-CD20 / CD3DVD-Ig bound to both T cells and B cells, whereas CD3 and CD20 monoclonal antibodies bound to only T cells or only B cells, respectively. The amino acid sequence of CD20 / CD3DVD-Ig is disclosed in Table 25.

(Example 5)
Production of mIL-1α / βDVD-Ig In order to study important issues regarding pharmacokinetics, in vivo efficacy, tissue penetration and immunogenicity of DVD-Ig molecules, as described below Mouse EL1α / βDVD-Ig molecules were constructed.

(Example 5.1)
Construction of mIL-1α / βDVD-Ig Using two mouse anti-mouse IL-1α / β monoclonal antibodies (9H10 and 10G11) made from DL1αβ double knockout mice, anti-mouse IL-1α / βDVD-Ig molecules Built. Mouse anti-mouse DL-1a and mouse anti-mouse IL-1β monoclonal antibodies were generated by immunizing IL-1α / β double knockout mice with mouse IL-1α or mouse IL-1β, respectively. One mouse anti-mouse IL-1α (clone 9H10) and one mouse anti-mouse IL-1β monoclonal antibody (clone 10G11) were selected and used to generate mIL-1α / βDVD-Ig molecules. Various linker sizes and different domain orientations were examined. The final functional mIL-1α / βDVD-Ig molecule was constructed with the orientation of V (anti-mIL-1β) -linker-V (anti-mIL-1β) -mouse constant region (Cγ2a and CK). Cloning, expression and purification procedures were similar to that of hIL-1α / βDVD-Ig. Cloning of mIL-1α / βDVD-Ig was performed using overlapping PCR and homologous recombination similar to that described for hIL-1α / βDVD3-Ig. The sequence of mIL-1α / βDVD-Ig is shown in Table 26 below.

  Mouse mIL-1α / βDVD-Ig retained affinity / in vitro efficacy for both IL-1α and IL-1β. Table 27 shows characterization of monoclonal antibodies 9H10 (anti-m1L-1α), 10G11 (anti-m1L-1β) and mIL-1α / βDVD-Ig.

(Example 5.2)
In vivo activity of mIL-lot / βDVD-Ig in an arthritis model Treatment of anti-EL-1α, anti-IL-1β, combined anti-IL-1α / anti-IL-1β, and mouse anti-IL-1α / βDVD4-Ig The effect was evaluated in a collagen-induced arthritis mouse model well known in the art. Briefly, male DBA-1 mice were immunized at the base of the tail using bovine type II collagen in CFA. On day 21, mice were boosted intraperitoneally (intraperitoneally) with zymosan. After disease onset on days 24-27, mice were selected and the mice were divided into 10 individual groups. Mean arthritis scores for the control and anti-cytokine groups were comparable to the beginning of treatment. To neutralize IL-1, anti-IL-1α monoclonal antibody, anti-IL-1β monoclonal antibody, anti-IL-1α / anti-IL-β monoclonal antibody combination, or mouse anti-IL-1α / β DVD4-Ig Mice were injected intraperitoneally every other day with 1-3 mg / kg. Mice were carefully examined three times a week for visual aspects of arthritis in the peripheral joints and a score for disease activity was determined.

  IL-1 blockade in the treatment mode effectively relieves the severity of arthritis and anti-IL-1β is more effective than anti-IL-1α (10% decrease in mean arthritis score compared to the subject group) (24% reduction). Further effects were observed between anti-IL-1α and anti-IL-1β, and the mean arthritis score was reduced by 40% in mice treated with both anti-IL-1α and anti-IL-1β monoclonal antibodies. Surprisingly, at the same dose level, mDVD-Ig treatment showed a 47% reduction in mean arthritis score, indicating the therapeutic efficacy of mDVD-Ig in vivo. Similar efficacy was also observed in the results of joint swelling measurements in this animal model.

  The present invention incorporates by reference the entire techniques well known in the fields of molecular biology and drug delivery. These techniques include, but are not limited to, techniques described in the following publications.

  Although a number of embodiments and features have been described above, modifications to the described embodiments and features may be made without departing from the invention as defined in the disclosure of the specification or the appended claims. And those skilled in the art will appreciate that variations can be made. Each of the publications listed herein is incorporated by reference.

Claims (76)

A binding protein comprising a polypeptide chain, wherein said polypeptide chain is VD1- (X1) _n- VD2-C- (X2) _n (VD1 is the first variable domain and VD2 is 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.).   The binding protein according to claim 1, wherein the VD1 and VD2 are heavy chain variable domains.   The binding protein of claim 2, wherein the heavy chain variable domain is selected from the group consisting of a mouse heavy chain variable domain, a human heavy chain variable domain, a CDR grafted heavy chain variable domain, and a humanized heavy chain variable domain. .   The binding protein according to claim 2, wherein VD1 and VD2 are capable of binding to the same antigen.   The binding protein according to claim 2, wherein VD1 and VD2 are capable of binding to different antigens.   The binding protein according to claim 2, wherein C is a heavy chain constant domain.   The binding protein according to claim 6, wherein X1 is a linker (provided that X1 is not CH1). Linker AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA (G 4 S) 4; SAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVTLFPP; AKTTPP; AKTTPPSVTPLAP; AKTTAP; AKTTAPPSYPLAP; ASTKGP; ASTKGPSVFPLAP; GGGGSGGGGGGGGS; GENKVEYAPALMALS; 8. The binding protein of claim 7, selected from the group consisting of GPAKELTPLKEAKVS; and GHEAAAVMQVQYPAS.   The binding protein according to claim 7, wherein X2 is an Fc region.   The binding protein of claim 9, wherein the Fc region is a variant Fc region. A binding protein comprising a polypeptide chain, wherein said polypeptide chain is VD1- (X1) _n- VD2-C- (X2) _n (VD1 is the first heavy chain variable domain and VD2 is the second heavy chain) The binding protein comprising a variable domain, C is a heavy chain constant domain, X1 is a linker (but not CH1), and X2 is an Fc region.   The binding protein according to claim 1, wherein the VD1 and VD2 are light chain variable domains.   13. The binding protein of claim 12, wherein the light chain variable domain is selected from the group consisting of a mouse light chain variable domain, a human light chain variable domain, a CDR grafted light chain variable domain, and a humanized light chain variable domain. .   13. A binding protein according to claim 12, wherein VD1 and VD2 are capable of binding to the same antigen.   13. A binding protein according to claim 12, wherein VD1 and VD2 are capable of binding to different antigens.   13. A binding protein according to claim 12, wherein C is a light chain constant domain.   The binding protein according to claim 16, wherein X1 is a linker (provided that X1 is not CL1). Linker AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA (G 4 S) 4; SAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVTLFPP; AKTTPP; AKTTPPSVTPLAP; AKTTAP; AKTTAPPSYPLAP; ASTKGP; ASTKGPSVFPLAP; GGGGSGGGGGGGGS; GENKVEYAPALMALS; 18. A binding protein according to claim 17 selected from the group consisting of GPAKELTPLKEAKVS; and GHEAAAVMQVQYPAS.   18. A binding protein according to claim 17, wherein the binding protein does not comprise X2. A binding protein comprising a polypeptide chain, wherein said polypeptide chain is VD1- (X1) _n- VD2-C- (X2) _n (VD1 is the first light chain variable domain and VD2 is the second light chain) The binding protein comprising a variable domain, C is a light chain constant domain, X1 is a linker (but not CH1), and X2 does not contain an Fc region. A binding protein comprising first and second polypeptide chains, wherein the first polypeptide chain is VD1- (X1) _n- VD2-C- (X2) _n (VD1 is the first heavy chain variable domain) VD2 is the second heavy chain variable domain, C is the heavy chain constant domain, X1 is a linker (where X1 is not CH1), and X2 is an Fc region). And the second polypeptide chain is VD1- (X1) _n- VD2-C- (X2) _n (VD1 is the first light chain variable domain and VD2 is the second light chain variable domain) , C is a light chain constant domain, X1 is a linker, where X1 is not CH1, and X2 does not contain an Fc region. A binding protein comprising four polypeptide chains, the two polypeptide chains being VD1- (X1) _n -VD2-C- (X2) _n (VD1 is the first heavy chain variable domain and VD2 is the second A heavy chain variable domain, wherein C is a heavy chain constant domain, X1 is a linker (where X1 is not CH1), and X2 is an Fc region); and two poly The peptide chain is VD1- (X1) _n- VD2-C- (X2) _n (VD1 is the first light chain variable domain, VD2 is the second light chain variable domain, and C is the light chain constant domain) And wherein X1 is a linker (where X1 is not CH1) and X2 does not contain an Fc region).   2. The binding protein of claim 1, wherein the binding protein is capable of binding to one or more targets.   The target is: ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AIF1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; 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- 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 (M 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-L1PR); -9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDH1 (E-cadherin); CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21Wap1 / Cip1); CDKN1B (p27Kip1); CEBPB; CER1; CHGA; CHGB; chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKL CLFSF6; CKLSF7; CKLSFSF8; CLDN3; CLDN7 (Claudin-7); CLN3; CLU (clusterin); CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL4A3; COL4A3; ); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-cadherin); 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); CXCL (ENA-78 / LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9 / CKR-L2); CXCR4; CXCR6 (TYMSTR / STRL33 / Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHB4; EPO; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; 2B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF8; FGF9; FGFR3; FIGF (VEGFD); FDL1 (EPSILON); FIL1 (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); PR31; GPR44; GPR81 (FKSG80); GRCC10 (C10); GRP; GSN (gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HEP1; histamine and histamine receptor; HLA-A; HLA -DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICESL; ID2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; 1FNA6; IFNA7; IFNB1; IFNγ; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18RAP1; IL19; IL1A; IL1B; IL1F5; IL1F6; IL1F6; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2IL1RN; IL2; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL24; IL27; IL2RB; IL2RG; IL3; IL30; IL3 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; HGB3; ITGB4 (b4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GCBxBP); KL; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (keratin 19); KRT2A; KRTBB6 ( LAMA5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARKKS; MAG or Omgp; MAP2K7 (C-Jun); MDK; MIB1; Midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; NCK2; neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NR1B1; NR1D2; NR1H2; NR1H3; NR1H2; NR1I2; NR1I3; NR2C1; NR2E1; NR2E3; NR2F1; NR2F6; NR3C1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; Phoscan; PIAS2; PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; P OC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS1; RGS13; RGS3; RNF110 (ZNF144); ROBO2; S100A2; SCGB1D2 (SCB1A2); SCMB2A2 (mammaglobin 1); SCYE1 (endothelial monocyte-activating cytokine); SDF2; SERPINA1; SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-1); SERPINF1; SHBG; SLA2; SLC33A2; SLC33A2 SLC43A1; SLIT2; SPP1; SPRR1B (Spr1); ST6GAL1; STAB1; STAT6; STEAP; ST TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB1I1; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1); THBS2; THBS4; TIMP3; tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TNF-a; TNFAIP2 (B94); TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF 11 (TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (FS40 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptor; TOP2A (topoisomerase Iia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF4; TRAF5; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; Versican; VHL 5; VLA-4; XCL1 (Lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5 / CCXCR1); YY1 and is selected from the group consisting of ZFPM2, binding protein of claim 23.   The binding protein can bind to two targets, which are 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 RGMA; 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 IL-1β; 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-1 And MDC; IL-13 and MIF; IL-13 and PED2; IL-13 and SPRR2a; 1L-13 and SPRR2b; IL-13 and TARC; IL-13 and TGF-β; IL-1α and IL-1β; Ngo and RGMA; NogoA and RGMA; OMGp and RGMA; RGMA and RGMB; Te38 and TNFα; TNFα and IL-12; TNFα and IL-12p40; TNFα and IL-13; TNFα and IL-15; TNFα and IL-18; TNFα and IL-1; TNFα and IL-23; TNFα and MIF; TNFα and PEG2; TNFα and PGE4; TNFα and VEGF; and VEGFR and EGFR; TNFα and RANK ligand; ys; TNF [alpha] and GP130; TNF [alpha] and CD-22; and is selected from the group consisting of TNF [alpha] and CTLA-4, binding protein of claim 1.   24. The binding protein of claim 23, wherein the binding protein is capable of modulating the biological function of one or more targets.   24. The binding protein of claim 23, wherein the binding protein is capable of neutralizing one or more targets.   26. A binding protein according to any one of claims 23 to 25, wherein the target is selected from the group consisting of cytokines, chemokines, cell surface proteins, enzymes and receptors.   30. The binding protein of claim 28, wherein the cytokine is selected from the group consisting of lymphokines, monokines and polypeptide hormones.   30. The binding protein of claim 29, wherein the cytokine is IL-1α or IL-1β.   The binding protein is 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. DVD heavy chain amino acid sequence; and 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 32. The binding protein of claim 30, comprising a DVD light chain amino acid sequence selected from:   30. The binding protein of claim 29, wherein the cytokine is TNF- [alpha] and IL-13.   30. The binding protein of claim 29, wherein the cytokine is IL-12 and IL-18.   A DVD heavy chain amino acid sequence wherein the binding protein is 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 SEQ ID NO: 86, SEQ ID NO: 91, SEQ ID NO: 94, sequence 34. The binding protein of claim 33, comprising a DVD light chain amino acid sequence selected from the group consisting of a DVD light chain amino acid sequence selected from the group consisting of No. 46, SEQ ID NO: 96, and SEQ ID NO: 116.   29. The binding protein of claim 28, wherein the chemokine is selected from the group consisting of CCR2, CCR5 and CXCL-13.   29. A binding protein according to claim 28, wherein the cell surface protein is an integrin.   29. A binding protein according to claim 28, wherein the cell surface proteins are CD-20 and CD3.   38. The binding protein of claim 37, wherein the binding protein comprises a DVD heavy chain amino acid sequence that is SEQ ID NO: 97 and a DVD light chain that is SEQ ID NO: 101.   29. A binding protein according to claim 28, wherein the enzyme is selected from the group consisting of kinases and proteases.   29. The binding protein of claim 28, wherein the receptor is selected from the group consisting of a lymphokine receptor, a monokine receptor and a polypeptide hormone receptor. The binding protein has at least about 10 ² M ⁻¹ S ⁻¹ ; at least about 10 ³ M ⁻¹ S ⁻¹ ; at least about 10 ⁴ M ⁻¹ S ⁻¹ ; 10. A binding rate constant (K _on ) to said one or more targets selected from the group consisting of 10 ⁵ M ⁻¹ S ⁻¹ ; and at least about 10 ⁶ M ⁻¹ S ^−1. 28. A binding protein according to 28. The binding protein has a maximum of about 10 ⁻³ s ⁻¹ ; a maximum of about 10 ⁻⁴ s ⁻¹ ; a maximum of about 10 ⁻⁵ s ⁻¹ ; and a maximum of about 10 ⁻⁶ s ⁻ as measured by surface plasmon resonance. 29. A binding protein according to claim 28 having a dissociation rate constant ( _Koff ) to said one or more targets selected from the group consisting of ¹ . Up to about 10 ⁻⁷ M; up to about 10 ⁻⁸ M; up to about 10 ⁻⁹ M; up to about 10 ⁻¹⁰ M; up to about 10 ⁻¹¹ M; up to about 10 ⁻¹² M and up to 10 ⁻ 30. The binding protein of claim 28, having a dissociation constant (K _D ) for the one or more targets selected from the group consisting of ¹³ M.   44. A binding protein conjugate comprising the binding protein according to any one of claims 1 to 43, further comprising a factor selected from the group consisting of an immunoadhesion molecule, a contrast agent, a therapeutic agent and a cytotoxic agent.   45. The binding protein conjugate according to claim 44, wherein the agent is a contrast agent selected from the group consisting of a radioactive label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. The contrast agent is a radioactive label selected from the group consisting of ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, and ¹⁵³ Sm. 46. A binding protein conjugate according to Item 45.   The agent is a therapeutic agent or cytotoxicity selected from the group consisting of metabolic inhibitors, alkylating agents, antibiotics, growth factors, cytokines, anti-angiogenic agents, anti-mitotic agents, anthracyclines, toxins and apoptotic agents. The binding protein conjugate according to claim 45, which is an agent.   The binding protein according to claim 1, wherein the binding protein is a crystallized binding protein.   49. The crystallized binding protein of claim 48, wherein the crystal is a pharmaceutical sustained release crystal without a carrier.   49. The crystallized binding protein of claim 48, wherein the binding protein has a longer half-life in vivo than the soluble counterpart of the binding protein.   49. The crystallized binding protein of claim 48, wherein the binding protein retains biological activity.   23. An isolated nucleic acid encoding the binding protein amino acid sequence of any one of claims 1 to 22.   53. A vector comprising the isolated nucleic acid of claim 52.   54. The vector of claim 53, wherein the vector is selected from the group consisting of pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, pcDNA3.1 TOPO, pEF6TOPO and pBJ.   54. A host cell comprising the vector of claim 53.   56. The host cell of claim 55, wherein the host cell is a prokaryotic cell.   The host cell is E. coli. 57. The host cell according to claim 56, which is E. coli.   56. The host cell of claim 55, wherein the host cell is a eukaryotic cell.   59. The host cell according to claim 58, wherein the eukaryotic cell is selected from the group consisting of protist cells, animal cells, plant cells and fungal cells.   59. The host cell according to claim 58, wherein the eukaryotic cell is an animal cell selected from the group consisting of mammalian cells, avian cells and insect cells.   59. A host cell according to claim 58, wherein the host cell is a CHO cell.   59. A host cell according to claim 58, wherein the host cell is COS.   59. A host cell according to claim 58, wherein the host cell is a yeast cell.   64. The host cell of claim 63, wherein the yeast cell is Saccharomyces cerevisiae.   59. A host cell according to claim 58, wherein the host cell is an insect Sf9 cell.   66. A method of producing a binding protein comprising culturing the host cell of any one of claims 55 to 65 in a medium under conditions sufficient to produce the binding protein.   68. The method of claim 66, wherein 50% to 75% of the produced binding protein is a bispecific tetravalent binding protein.   68. The method of claim 66, wherein 75% to 90% of the produced binding protein is a bispecific tetravalent binding protein.   68. The method of claim 66, wherein 90% to 95% of the produced binding protein is a bispecific tetravalent binding protein.   68. A protein produced according to the method of claim 66.   71. A pharmaceutical composition comprising the binding protein of any one of claims 1 to 51 and 70 and a pharmaceutically acceptable carrier.   72. The pharmaceutical composition of claim 71, further comprising at least one additional therapeutic agent.   Said additional agents are therapeutic agents, contrast agents, cytotoxic agents, angiogenesis inhibitors; kinase inhibitors; costimulatory molecule blockers; adhesion molecule blockers; anti-cytokine antibodies or functional fragments thereof; methotrexate; cyclosporine; FK506; detectable label or reporter; TNF antagonist; anti-rheumatic drug; muscle relaxant, anesthetic, non-steroidal anti-inflammatory drug (NSAID), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocker, Antimicrobial agent, anti-psoriatic agent, corticosteroid, anabolic steroid, erythropoietin, immunization, immunoglobulin, immunosuppressant, growth hormone, hormone replacement agent, radiopharmaceutical, antidepressant, antipsychotic, stimulant, asthma drug , Beta agonists, steroids for inhalation, epinephrine or analogs, cytokines and cytokines It is selected from the group consisting of down antagonists, pharmaceutical composition according to claim 72.   71. A method of treating a disease or disorder in a subject by administering to the subject the binding protein of any one of claims 1 to 51 and 70 so that treatment is achieved.   Rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthritis, systemic lupus erythematosis, Crohn's disease, ulcerative colitis, inflammatory Bowel disease, insulin-dependent diabetes mellitus, thyroiditis, asthma, allergic disease, psoriasis, dermatitis, scleroderma, graft-versus-host disease, organ transplant rejection, organ transplant-related acute or chronic immune disease, sarcoidosis, atheroma Atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schlein purpura, microscopic vasculitis of the kidney, chronic active hepatitis, uvea Inflammation, septic shock, toxic shock syndrome, septic syndrome, cachexia, infectious disease, parasitic disease, acquired immune deficiency syndrome, acute transverse spine Inflammation, Huntington's disease, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignant tumor, heart failure, myocardial infarction, Addison's disease, sporadic hypoendocrine syndrome type I and multiple Endocrine hypofunction syndrome type II, Schmidt syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthritis, arthritis, Reiter's disease, psoriatic arthritis, ulcerative colitis, enteric disease Meningitis, Chlamydia, Yersinia and Salmonella-related arthritis, spondyloarthropathy, atherosclerotic / atherosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, deciduous pemphigus, pemphigoid, Linear IgA disease, autoimmune hemolytic anemia, Coombs-positive hemolytic anemia, acquired pernicious anemia, juvenile pernicious anemia, myalgic encephalomyelitis / Roya Free disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosis hepatitis, idiopathic autoimmune hepatitis, acquired immune deficiency syndrome, acquired immune deficiency related disease, hepatitis B, hepatitis C, classification Impaired immunodeficiency (unclassifiable primary hypogammaglobulinemia), dilated cardiomyopathy, female infertility, ovarian dysfunction, early ovarian dysfunction, fibrotic lung disease, idiopathic interstitial pneumonia, post-inflammation Interstitial lung disease, interstitial pneumonia, connective tissue disease related interstitial lung disease, mixed connective tissue disease related lung disease, systemic sclerosis related interstitial lung disease, rheumatoid arthritis related interstitial lung disease, Systemic lupus erythematosus-related lung disease, dermatomyositis / polymyositis-related lung disease, Sjogren's disease-related lung disease, ankylosing spondylitis-related lung disease, vasculitic diffuse lung disease, hemosidero Cis-related lung disease, drug-induced interstitial lung disease, fibrosis, radiofibrosis, obstructive bronchiolitis, chronic eosinophilic pneumonia, lymphocyte infiltrating lung disease, post-infection interstitial lung disease , Ventilatory arthritis, autoimmune hepatitis, type 1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type 2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune-mediated hypoglycemia, melanoma Type B insulin resistance, hypoparathyroidism, acute immune disease related to organ transplantation, chronic immune disease related to organ transplantation, osteoarthritis, primary sclerosing cholangitis, type 1 psoriasis, type 2 Psoriasis, idiopathic leukopenia, autoimmune neutropenia, kidney disease NOS, glomerulonephritis, renal microvasculitis, Lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, Sperm autoimmunity, multiple sclerosis (all sub Ip), sympathetic ophthalmitis, pulmonary hypertension secondary to connective tissue disease, Goodpascher syndrome, pulmonary symptoms of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjogren's syndrome , Takayasu / arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goiter autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmunity Hypothyroidism, primary myxedema, lens-induced uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver disease, alcoholic cirrhosis, alcohol-induced liver injury, cholestasis ), Idiosyncratic liver disease, drug-induced hepatitis, non-alcoholic steatohepatitis, allergy and asthma, group B streptococci (GB) ) Infections, mental disorders (eg depression and schizophrenia), diseases mediated by Th2 and Th1 types, acute and chronic pain (various forms of pain), and lung cancer, breast cancer, stomach cancer, bladder cancer, Cancers such as colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer and kidney cancer and hematopoietic malignancies (leukemia and lymphoma), abetalipoproteinemia, advanced cyanosis, 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, adenocarcinoma, aerial ectopic pulsation, AIDS dementia complex, Alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, α-1-antitrypsin deficiency, amyotrophic lateral sclerosis, poor Angina pectoris, anterior horn cell degeneration, anti-cd3 therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aortic and aneurysms, aortic dissection, arterial hypertension, arteries Sclerosis, arteriovenous fistula, ataxia, atrial fibrillation (persistent or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone transplant rejection, bone marrow transplant (BMT) rejection, leg block, Burkitt lymphoma , Burn, cardiac arrhythmia, cardiac dysfunction syndrome, cardiac tumor, cardiomyopathy, cardiopulmonary bypass inflammatory response, cartilage transplant rejection, cerebellar cortical degeneration, cerebellar disease, disordered or multifocal atrial tachycardia, chemotherapy Related diseases, chronic myelogenous leukemia (CML), chronic alcoholism, chronic inflammatory lesions, chronic lymph Leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylic acid poisoning, colorectal cancer, congestive heart failure, conjunctivitis, contact dermatitis, pulmonary heart, coronary artery disease, Creutzfeldt-Jakob disease, culture negative sepsis Cystic fibrosis, cytokine therapy-related disease, boxer brain, demyelinating disease, dengue hemorrhagic fever, dermatitis, dermatological symptoms, diabetes, diabetes mellitus, diabetic arteriosclerotic disease, diffuse Lewy body disease, dilated Congestive cardiomyopathy, basal ganglia disease, middle-aged Down syndrome, drug-induced motor disease induced by drugs that block central nerve dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis Endocrine disease, epiglottis, Epstein-Barr virus infection, erythema, extrapituitary tract and cerebellar disease, familial hemophagocytic lymphohistiocytosis phagocytic lymphohistiocytosis), fatal thymus transplant rejection, Friedreich ataxia, functional peripheral arterial disease, fungal sepsis, gas gangrene, gastric ulcer, glomerulonephritis, graft rejection of any organ or tissue, gram-negative sepsis , Gram-positive sepsis, granulomas caused by intracellular organisms, hairy cell leukemia, Haller-Holden-Spatz disease, Hashimoto thyroiditis, hay fever, heart transplant rejection, hemochromatosis, hemodialysis, hemolytic uremic syndrome / thrombolytic platelets Reduced purpura, bleeding, hepatitis (type A), His bundle arrhythmia, HIV infection / HIV neuropathy, Hodgkin's disease, hyperkinetic disease, hypersensitivity reaction, hypersensitivity pneumonia, hypertension, hypokinetic disease, hypothalamus-pituitary- Adrenocortical system evaluation, idiopathic Addison disease, idiopathic pulmonary fibrosis, antibody-mediated cytotoxicity, asthenia, infant spinal muscular atrophy Aortic inflammation, influenza A, ionizing radiation exposure, iridocyclitis / uveitis / optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma , Kidney transplant rejection, legionella, leishmaniasis, leprosy, corticospinal lesions, lipemia, liver transplant rejection, lymphedema, malaria, malignant lymphoma, malignant histiocytosis, malignant melanoma Meningitis, meningococcusemia, metabolic / idiopathic, migraine, mitochondrial multi-system disorder, mixed connective tissue disease, monoclonal hypergammaglobulinemia, multiple Myeloma, multiple systemic degeneration (Mencel Degeline-Thoma Shy-Drager and Machado-Joseph), myasthenia gravis, Mycobacterium avium Intracellulare, Mycobacterium tuberculosis, myelodysplasia, myocardial infarction, fungal ischemic disease, nasopharyngeal cancer, newborn chronic Lung disease, nephritis, nephrosis, neurodegenerative disease, neurogenic I muscle atrophy, neutropenic fever, non-Hodgkin lymphoma, obstruction of abdominal aorta and its branches, obstructive arterial disease, okt3 therapy, testitis / testis Epidermitis, orchitis / vaginal reconstruction, organ enlargement, osteoporosis, pancreatic transplant rejection, pancreatic cancer, paraneoplastic syndrome / hypercalcemia of malignant tumor, parathyroid transplant rejection, pelvic inflammatory disease, perennial Rhinitis, pericardial disease, peripheral atherosclerotic disease, peripheral vascular disease, peritonitis, pernicious anemia, Pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (multiple Neuritis, organ hypertrophy, endocrine disease, monoclonal gamma globulinemia and skin change syndrome, post perfusion syndrome, post pump syndrome, post-myocardial infarction open heart surgery Syndrome, pre-eclampsia, progressive supranuclear palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon and disease, Raynaud's disease, Refsum's disease, regular narrow QRS tachycardia, kidney Vascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcoma, scleroderma, senile chorea, senile dementia with Lewy bodies, seronegative arthritis, shock, sickle cell anemia, skin allograft Rejection, skin change syndrome, small intestine transplant rejection, solid tumor, Specific arrhythmia, spinal ataxia, spinocerebellar degeneration, streptococcal myositis, cerebellar structural lesions, subacute sclerosing panencephalitis, eczema, cardiovascular syphilis, systemic anaphylaxis, systemic inflammatory response Syndrome, systemic onset juvenile rheumatoid arthritis, T cells or FABALL, telangiectasia, obstructive thromboangiitis, thrombocytopenia, toxicity, transplantation, trauma / bleeding, type III hypersensitivity reaction, type IV hypersensitivity, no Stable angina, uremia, urinary sepsis, hives, valvular heart disease, varicose veins, vasculitis, venous disease, venous thrombosis, ventricular fibrillation, viral and fungal infections, viral encephalitis / sterile Meningitis, virus-related hemophagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson disease, xenotranslocation of any organ or tissue It is selected from the group comprising rejection method according to claim 74.   The administration to a subject can be parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavity, intracerebral, intracerebellar, intraventricular, Intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, pelvic, pericardial, intraperitoneal, intraperitoneal, intraprostatic, intrapulmonary, intracolonic, intrarenal, intraretinal, intraspinal, smooth 75. The method of claim 74, according to at least one manner selected from intramembrane, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, oral, sublingual, intranasal and transdermal.

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