CN105542011A - Bispecific or multispecific Ig JR binding protein - Google Patents

Bispecific or multispecific Ig JR binding protein Download PDF

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CN105542011A
CN105542011A CN201510796803.7A CN201510796803A CN105542011A CN 105542011 A CN105542011 A CN 105542011A CN 201510796803 A CN201510796803 A CN 201510796803A CN 105542011 A CN105542011 A CN 105542011A
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antibody
associated proteins
protein
disease
people
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贾儒
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Beijing Jiatai Xianke Pharmaceutical Co Ltd
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Beijing Jiatai Xianke Pharmaceutical Co Ltd
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Abstract

The invention relates to a multivalent and/or multispecific antigen binding protein capable of binding 2 or more species of antigens, and especially relates to a dual variable region immunoglobulin (Ig JR), a medicinal composition thereof, and a nucleic acid, a recombinant expression vector and a host cell used for preparing the Ig JR. The invention also relates to a use of the Ig JR in disease treatment.

Description

Dual specific or polyspecific Ig JR associated proteins
Invention field
Relate generally to dual specific of the present invention and/or polyspecific (antigen) associated proteins.In addition, the present invention relates to the polynucleotide of this kind of dual specific of coding and/or multi-specific binding protein, and comprise carrier and the host cell of this kind of polynucleotide.The invention still further relates to the method for generation of bispecific binding protein of the present invention and/or polyspecific, and these dual specifics and/or the purposes of multi-specific binding protein in the treatment of disease.
Background of invention
Antigen binding domain in antibody comprises two regions separated: variable region of heavy chain (V h) and variable region of light chain (V l: V κor V λ).Antigen binding site itself is made up of 6 polypeptide rings: 3 from VH district (H1, H2 and H3), 3 from VL district (L1, L2 and L3).The combination of constant gene segment C is reset and is caused coding V hdistrict and V lthe diversified one-level storehouse of the V gene in district.V hgene is by 3 constant gene segment Cs and V h, D and J hrestructuring.According to haplotype, the nearly 51 kinds of functional V of the mankind hsection (CookandTomlinson, ImmunolToday16:237 (1995)), 25 kinds of functional D sections (Corbett etc., J.Mol.Biol.268:69 (1997)) and 6 kinds of functional J hsection (Ravetch etc., Cell27:583 (1981)).V hthe region in VH district first and second antigen binding loops (H1 and H2) is formed in segment encodes polypeptide chain, and V h, D and J hsection forms VH district antigen iii coupling collar (H3) jointly.V lgene is only by 2 constant gene segment Cs and V land J lrestructuring forms.According to haplotype, the nearly 40 kinds of functional V of the mankind κsection ( andZachau, Biol.Chem.Hoppe-Seyler374:1001 (1993)), 31 kinds of functional V λsection (Williams etc., J.Mol.Biol.264:220 (1996) and Kawasaki etc., GenomeRes7:250 (1997)), 5 kinds of functional J κsection (Hieter etc., J.Mol.Biol.257:1516 (1982)) and 4 kinds of functional J λsection (VasicekandLeder, J.Exp.Med.172:609 (1990)).V lv is formed in segment encodes polypeptide chain lthe region in district first and second antigen binding loops (L1 and L2), and V land J lsection forms V jointly ldistrict's antigen iii coupling collar (L3).It is generally acknowledged, these initial storehouses have enough diversity, can select the antibody that at least can be combined with nearly all antigen with medium avidity." affinity maturation " through gene rearrangement can produce high-affinity antibody, and in the process, origination point suddenlys change, and is selected by immunity system according to the enhancing of binding ability.
Structure and the sequence of analyzing antibody show that 5 (H1, H2, L1, L2 and L3) in 6 antigen binding loops have number constrained backbone conformation and norm structure (ChothiaandLesk, J.Mol.Biol.196:901 (1987) and Chothia etc., Nature342:877 (1989)).Conformation of the main chain depends on concrete residue or the residue type of some key position in the length of (i) antigen binding loops and (ii) antigen binding loops and antibody framework.Length and the Key residues of analyzing antigen binding loops can be inferred by H1, H2, L1, L2 and L3 Conformation of the main chain of most people antibody-like sequence encoding (Chothia etc., J.Mol.Biol.227:799 (1992); Tomlinson etc., EMBOJ14:4628 (1995); Williams etc., J.Mol.Biol.264:220 (1996)).Although H3 district is in the diversity of sequence, length and configuration aspects much bigger (effect due to D section), but the existence of the special residue of key position or residue type determines that this district is also that becate length forms number constrained backbone conformation (Martin etc., J.Mol.Biol.263:800 (1996) in its length and antigen binding loops and antibody framework; Shirai etc., FEBSLetters399:1 (1996)).
Dual specific and multi-specificity antibody
Known in the artly comprise V hdistrict and V lthe dual specific of district's complementary pair and multi-specificity antibody.These dual specifics and multi-specificity antibody must comprise at least two couples of V hdistrict and V ldistrict, often couple of V h/ V lin conjunction with single antigen or epi-position.The method once described comprises hybrids knurl technology (MilsteinandCuelloAC, Nature305:537-40), microbody (Hu etc., CancerRes56:3055-3061 (1996)), double antibody (diabody) (Holliger etc., Proc.Natl.Acad.Sci.USA90:6444-6448 (1993); WO94/13804), chelating recombinant antibody (CRAb) (Neri etc., J.Mol.Biol.246:367-373 (1995)), two Fv antibody (biscFv) (Atwell etc., Mol.Immunol.33:1301-1312 (1996)), " convex-concave is coincide (knobsinholes) " stabilization antibody (Carter etc., ProteinSci.6:781-788 (1997)).In often kind of situation, often kind of antibody isotype comprises two or more antigen binding site, and the pattern of each antigen binding site is V hdistrict and V lthe complementary pair in district.Therefore, by a V hdistrict and its complementary V ldistrict, in conjunction with each antigen or epi-position, makes each antibody capable simultaneously in conjunction with two or more different antigen or epi-position.Each of these technology all has its distinctive shortcoming, such as: the V of non-activity in hybrids knurl technology h/ V lto greatly reducing mark shared by dual specific or polyspecific IgG, during coexpression, the same dimerization of heavy chain of antibody and/or the mispairing of not homospecific heavy chain of antibody and light chain reduce the productive rate of the construct of correct assembling, producing many non-functional by products, being separated the bi-specific antibody of wishing by being difficult to from it.In addition, most of dual specific or polyspecific method depend on different V h/ V lright combination or V hand V lthe combination of chain and regenerate two kinds of different V h/ V lbinding site.Therefore, the binding site ratio to often kind of antigen or epi-position can not be controlled in assembling molecule, so many assembling molecules are only in conjunction with a kind of antigen or epi-position, and not in conjunction with other antigen or epi-position.Can transform heavy chain or light chain (Carter etc. on sub-unit in some cases, 1997) to increase the number of the molecule in the site had in conjunction with two or more antigen or epi-position, but all molecules can not be made so all to have ability in conjunction with two or more antigens or epi-position.
The single variable region of heavy chain coming from natural antibody was also described, this variable region of heavy chain usually and light chain (from monoclonal antibody or storehouse, functional zone; See EP-A-0368684) combine.Show that these variable region of heavy chain energy specificitys and one or more related antigen interact, and be not combined into the part with two or more different antigen-specific from other heavy chain or variable region of light chain.But the shortcoming of this method is: the antibody variable region of separation may have one usually can hydrophobic interfaces interactional with light chain, it is exposed to solvent and can is viscosity, thus allows this single district to be attached on hydrophobic surface.In addition, lack two or more different heavy chains variable regions of light chain companion combination and can by the combination of hydrophobic interfaces, can stop they in conjunction with one instead of two when they are independent can in conjunction with part.And in this case, variable region of heavy chain is not combined with complementary light chain variable region, therefore stablize qualitative very poor, fold and easily open (WornandPluckthun, Biochemistry37:13120-7 (1998)).
A kind of multivalent antibody is disclosed in CN01810372, wherein two heavy chains link together, then different from two light chains matches thus produces multiple dual anti-mixture (such as, light chain 1 can match with heavy chain 1, also can match with heavy chain 2), but from multiple dual anti-mixture, be difficult to the bi-specific antibody that separation and purification goes out monomer.
Report in WO2007/024715 and transform dual variable region immunoglobulin as multivalence and multi-specific binding protein.But, the DVD-Ig associated proteins reported in WO2007/024715, two variable region of heavy chain connect together, two variable region of light chain connect together, middle with aminoacid sequence link, rotary freedom is restricted, causes losing most of degree of freedom, and likely reduce two special DVD-lg affinity of antibody (ClarissaG.Jakob 1, *rohintonEdalji, 2, Structurerevealsfunctionofthedualvariabledomainimmunoglo bulin (DVD-Ig tM) molecule, mAbs5:3,358 – 363; May/June2013) make antibody binding efficiency poor.
In view of the continuous increase of the number of dual specific and the possible application of multi-specificity antibody, and the difficulty relevant with multi-specificity antibody to dual specific available at present and deficiency, still there are the needs of the new improved form to this quasi-molecule.
Summary of the invention
The present invention relates to the multivalence in conjunction with 2 kinds or more antigens and/or multi-specific binding protein.The invention provides can with the associated proteins new family of high-affinity in conjunction with 2 kinds or more antigens.
In one embodiment, the invention provides a kind of associated proteins comprising the first polypeptide chain JRHC1, wherein said first polypeptide chain comprises VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3, and wherein VH1 is the first variable region of heavy chain, and CH1 is CH1, VL2 is the second variable region of light chain, CL is constant region of light chain, and X2 represents amino acid or the polypeptide of hinge area, CH2 and CH3 forms antibody Fc district, X1 is optional, and representative is used as amino acid or the polypeptide of joint.
In preferred embodiments, the invention provides a kind of associated proteins, wherein except the first above-mentioned polypeptide chain JRHC1, described associated proteins also comprises the second polypeptide chain JRLC1 and the 3rd polypeptide chain JRHC2, and wherein said second polypeptide chain comprises VL1-CL, and wherein VL1 is the first variable region of light chain, CL is constant region of light chain, and described 3rd polypeptide chain JRHC2 comprises VH2-CH1, wherein VH2 is the second variable region of heavy chain, and CH1 is CH1.
In preferred embodiments, the C-terminal of the VL2 of the first polypeptide chain and the C-terminal of the 3rd polypeptide chain are not be halfcystine simultaneously.In a preferred embodiment, the C-terminal of the VL2 of the first polypeptide chain JRHC1 is halfcystine, the C-terminal of the 3rd polypeptide chain JRHC2 is not halfcystine, and now the halfcystine C of HC1 chain can form intrachain disulfide bond with the halfcystine C in the EPKSCDKT of hinge area.In another preferred embodiment, the C-terminal of the VL2 of the first polypeptide chain JRHC1 is not halfcystine, and the C-terminal of the 3rd polypeptide chain JRHC2 is not halfcystine, and the halfcystine C in the hinge EPKSCDKT of now two HC1 chains forms interchain disulfide bond.In another preferred embodiment, the C-terminal of the VL2 of the first polypeptide chain JRHC1 is not halfcystine, the C-terminal of the 3rd polypeptide chain JRHC2 is halfcystine, now, the halfcystine of the hinge area EPKSCD of HC1 can form interchain disulfide bond with the halfcystine of the C-terminal of HC2, to improve protein-bonded stability.
In preferred embodiments, described variable region of heavy chain (VH1 or VH2) and variable region of light chain (VL1 or VL2) is selected from mouse heavy chain and light-chain variable domain, people's heavy chain and light-chain variable domain, the heavy chain of CDR grafting and variable region of light chain and humanized heavy chain and variable region of light chain.
Fc district can be the Fc district of native sequences, or variant Fc district.In a preferred embodiment, Fc district Shi Ren Fc district.In another preferred embodiment, Fc district comprises the Fc district from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.
In one embodiment, VH1 and VH2 can in conjunction with same antigen.In another embodiment, VH1 and VH2 can in conjunction with not synantigen.In one embodiment, VL1 and VL2 can in conjunction with same antigen.In another embodiment, VL1 and VL2 can in conjunction with not synantigen.
In preferred embodiments, X1 is selected from the joint of lower group: EPKSCD; AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA (G 4s) 41sAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVTLFPP; AKTTPP; AKTTPPSVTPLAP; AKTTAP; AKTTAPSVYPLAP; ASTKGP; ASTKGPSVFPLAP, GGGGSGGGGSGGGGS; GENKVEYAPALMALS; GPAKELTPLKEAKVS and GHEAAAVMQVQYPAS.
Connect in a further preferred embodiment, above-disclosed associated proteins can in conjunction with one or more targets.Preferably target is selected from cytokine, cell surface protein, enzyme and acceptor.Preferably associated proteins can regulate the biological function of one or more targets.
In another embodiment, associated proteins of the present invention can in conjunction with being selected from following a kind, 2 kinds or more cytokines, cytokine associated protein and cytokine receptor: 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, IFNB1, IFNG, IFNW1, FIL1, FIL1 (EPSILON), FIL1 (ZETA), IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL17B, IL18, IL19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-β), LTB, TNF (TNF-α), TNFSF4 (OX40 part), TNFSF5 (CD40L), TNFSF6 (FasL), TNFSF7 (CD27 part), TNFSF8 (CD30 part), TNFSF9 (4-1BB part), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, FIGF (VEGFD), VEGF, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (Leptin), PTN and THPO.
Associated proteins of the present invention can in conjunction with being selected from one or more following chemokines, Chemokine Receptors and chemokine associated protein: CCL1 (I-309), CCL2 (MCP-1/MCAF), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCL11 (CCL11), CCL13 (MCP-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL20 (MIP-3a), CCL21 (SLC/exodus-2), CCL22 (MDC/STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2/ CCL11-2), CCL25 (TECK), CCL26 (CCL11-3), 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/JAB61), CCR1 (CKR1/HM145), CCR2 (mcp-1RB/RA), CCR3 (CKR3/CMKBR3), CCR4, CCR5 (CMKBR5/ChemR13), CCR6 (CMKBR6/CKR-L3/STRL22/DRY6), CCR7 (CKR7/EBI1), CCR8 (CMKBR8/TER1/CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), XCR1 (GPR5/CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28), CXCR4, GPR2 (CCR10), GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6 (TYMSTR/STRL33/Bonzo), HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R (GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HIF1A, IL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2 and VHL.Associated proteins of the present invention can in conjunction with the cell surface protein being selected from integrin.Associated proteins of the present invention can in conjunction with the enzyme being selected from kinases and proteolytic enzyme.Associated proteins of the present invention can in conjunction with the acceptor being selected from lymphokine receptor, monokine receptor and polypeptide hormone acceptor.
In a preferred embodiment, above-disclosed associated proteins can in conjunction with one or more targets be selected from VEGF-A, ANG-2, HER2, TNFalpha and anti-IL-17A.
In preferred embodiments, associated proteins is multivalence.More preferably associated proteins is polyspecific.Multivalence mentioned above and/or multi-specific binding protein have especially from the characteristic for the treatment of viewpoint needs.Such as, multivalence and/or multi-specific binding protein can (1) via cell internalization (and/or katabolism occur) quicker than bivalent antibody, the antigen that described cells expressing antibody combines with it; (2) be agonist antibody; And/or the necrocytosis of the cell of antigen that can combine with it of (3) abduction delivering multivalent antibody and/or apoptosis." parental antibody " of at least one antigen-binding specificity of multivalence and/or multi-specific binding protein is provided to be, via the antibody of cell internalization (and/or katabolism occurs) of expressing the antigen that antibody combines with it; And/or can be agonist, necrocytosis induction and/or apoptosis-inducing antibody, and as described herein multivalence and/or multi-specific binding protein can show in these characteristics in one or more improvement.In addition, parental antibody can lack in these characteristics one or more, but as described hereinly can give these characteristics when being configured to multivalent binding proteins.
In another embodiment, as measured by Octetlabelfreetechnology, the association rate constant (K that described associated proteins has for one or more targets described on) be selected from: at least about 10 2m -1s -1; At least about 10 3m -1s -1; At least about 10 4m -1s -1; At least about 10 5m -1s -1; With at least about 10 6m -1s -1.Preferably, as measured by Octetlabelfreetechnology, the association rate constant (K that associated proteins of the present invention has for one or more targets on) be selected from: 10 2m -1s -1to 10 3m -1s -1; 10 3m -1s -1to 10 4m -1s -1; 10 4m -1s -1to 10 5m -1s -1; Or 10 5m -1s -1to 10 6m -1s -1.
In another embodiment, as measured by Octetlabelfreetechnology, the dissociation rate constant (K that described associated proteins has for one or more targets described off) be selected from: at the most about 10 -3s -1; At the most about 10 -4s -1; At the most about 10 -5s -1; At the most about 10 -6s -1.Preferably, as measured by Octetlabelfreetechnology, the dissociation rate constant (K that described associated proteins has for one or more targets described off) be selected from: 10 -3s -1to 10 -4s -1; 10 -4s -1to 10 -5s -1; With 10 -5s -1to 10 -6s -1.
In another embodiment, associated proteins dissociation constant (K that one or more targets described are had d) be selected from: at the most about 10 -7m; At the most about 10 -8m; At the most about 10 -9m; At the most about 10 -10m; At the most about 10 -11m; At the most about 10 -12m; At the most about 10 -13m.Preferably, as measured by Octetlabelfreetechnology, the dissociation constant (K that associated proteins of the present invention has d) 10 -7m to 10 -8m, 10 -8m to 10 -9m, 10 -9m to 10 -10m, 10 -10m to 10 -11m; 10 -11m to 10 -12m; Or 10 -12m to 10 -13m.
In another embodiment, associated proteins mentioned above comprises the conjugate being selected from following reagent further: immunoadhesin molecule, developer, therapeutical agent and cytotoxic agent.Preferably developer is selected from radio-labeling, enzyme, fluorescent mark, luminescent marking, bioluminescence marker, magnetic mark and vitamin H.More preferably developer is selected from following radio-labeling: 3h, 14c, 35s, 90y, 99tc, 111in, 125i, 131i, 177lu, 166ho and 153sm.Preferably, treatment or cytotoxic agent are selected from metabolic antagonist, alkylating agent, microbiotic, somatomedin, cytokine, anti-angiogenic agent, antimitotic agent, anthracycline, toxin and apoptosis agent.
In another embodiment, associated proteins mentioned above is crystallized binding protein and exists as crystal.Preferably, crystal is DNAcarrier free Pharmaceutical controlled release crystal.More preferably, crystallized binding protein has the Half-life in vivo longer than described protein-bonded soluble counterpart.Most preferably, crystallized binding protein retains biological activity.
In another embodiment, associated proteins mentioned above is glycosylated.Preferably glycosylation pattern is people's glycosylation pattern.
One aspect of the present invention relates to the nucleic acid of the protein-bonded separation of the above-disclosed any one of coding.Further embodiment providing package is containing the carrier of the nucleic acid of above-disclosed separation, and wherein said carrier is selected from pcDNA; PTT (people such as Durocher, NucleicAcidsResearch2002, the 30th volume, No.2); PTT3 (has the pTT of other multiple clone site; PEFBOS (Mizushima, S. and Nagata, S., (1990) NucleicacidsResearch the 18th volume, No.17); PBV; PJV; PcDNA3.1, pcDNA3.1TOPO, pEF6TOPO and pBJ.
In yet another aspect, the above-disclosed vector of host cell.Preferably host cell is prokaryotic cell prokaryocyte.More preferably host cell is intestinal bacteria (E.coli).In the relevant embodiments, host cell is eukaryotic cell.Preferably eukaryotic cell is selected from protist cell, zooblast, vegetable cell and fungal cell.More preferably host cell is mammalian cell, includes but not limited to, CHO, COS or HEK293-F (HEK293-FreeStyle) cell; Or fungal cell's such as Saccharomyces cerevisiae (Saccharomycescerevisiae); Or insect cell such as Sf9.
Another aspect of the present invention provides produces above-disclosed protein-bonded method, under described method is included in and is enough to produce protein-bonded condition, cultivates same at above-disclosed any one host cell in the medium.
The invention still further relates to the protein produced according to aforesaid method.Further embodiment provides the protein produced according to above-disclosed method.
An embodiment provides for discharging protein-bonded composition, and wherein said composition comprises preparation, and described preparation comprises as above-disclosed crystallized binding protein and composition; And at least one polymeric carrier.Preferably polymeric carrier is selected from one or more following polymkeric substance: polyacrylic acid, polybutylcyanoacrylate, polyamino acid, polyanhydride, polyester peptide, polyester, poly(lactic acid), lactic acid-ethanol copolymer or PLGA, poly-b-butyric ester, polycaprolactone, poly-dioxanone (poly (dioxanone)), polyoxyethylene glycol, poly-(hydroxypropyl) Methacrylamide, poly-organophosphorus cyanogen, poe, polyvinyl alcohol, polyvinylpyrrolidone, maleic anhydride-alkyl vinyl ether co-polymer, pluronic polyvalent alcohol, white protein, alginate, Mierocrystalline cellulose and derivatived cellulose, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycosaminoglycan (glycaminoglycans), sulfated polysaccharides (polyeaccharides), adulterant (blends) and multipolymer thereof.Preferably, composition is selected from white protein, sucrose, trehalose, Saccharum lactis (lactitol), gelatin, hydroxypropyl-beta-cyclodextrin, methoxy poly (ethylene glycol) and polyoxyethylene glycol.Further embodiment provides and be used for the treatment of mammiferous method, described method comprises to the step of the above-disclosed composition of administration significant quantity.
Present invention also offers pharmaceutical composition, described pharmaceutical composition comprises as above-disclosed associated proteins and pharmaceutically acceptable carrier.In further embodiment, pharmaceutical composition comprises the other therapeutical agent of at least one that is used for the treatment of illness.Preferably other reagent is selected from: therapeutical agent, developer, cytotoxic agent, angiogenesis inhibitor (including but not limited to VEGF antibody or VEGF-trap); Kinase inhibitor (including but not limited to KDR and TIE-2 inhibitor); Costimulatory molecules blocker (including but not limited to anti-B7.1, anti-B7.2, CTLA4-Ig, anti-CD20); Adhesion molecule blockers (including but not limited to that anti-LFA-1Abs, anti-E/L select albumin A bs, micromolecular inhibitor); Anti-cytokine antibodies or its function fragment (including but not limited to anti-IL-18, anti-TNF, anti-IL-6/ cytokine receptor antibody); Methotrexate; S-Neoral; Rapamycin; FK506; Detectable label or reporter molecule; TNF antagonist; Rheumatism; Muscular flaccidity agent, narcotic, non-steroidal anti-inflammatory drug (NSAID), pain killer, narcotic, tranquilizer, neuromuscular blocking agents; Biocide, antipsoriatic, reflunomide, anabolic steroid, erythropoietin, immunization, immunoglobulin (Ig), immunosuppressor, tethelin, hormone replacement agent, radiopharmaceuticals, thymoleptic, antipsychotic drug, stimulant, asthma medications, beta-agonists, suck steroid, suprarenin or analogue, cytokine and cytokine antagonist.Preferably, described narcotic is local anesthetic.
In yet another aspect, the invention provides the method that associated proteins disclosed herein is used for the treatment of the people experimenter suffering from illness, one or more targets that can be combined by above-disclosed associated proteins in described illness are harmful, described method comprises uses above-disclosed associated proteins to people experimenter, thus makes the activity inhibited of one or more targets in people experimenter and reach treatment.Preferably illness is selected from rheumatoid arthritis, osteoarthritis, JCA, septic arthritis, Lyme arthritis, arthritic psoriasis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, CrohnShi is sick, ulcerative colitis, inflammatory bowel, insulin-dependent diabetes, thyroiditis, asthma, allergic disease, psoriasis, dermatitis, scleroderma, graft versus host disease (GVH disease), organ-graft refection, the acute immune disease relevant to organ transplantation, the chronic immunological disorders relevant to organ transplantation, sarcoidosis, atherosclerosis, disseminated inravascular coagulation, KawasakiShi is sick, GraveShi is sick, nephrotic syndrome, Chronic Fatigue Syndrome, Wei Genashi granulomatosis, anaphylactoid purpura, kidney microvascular is scorching, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, emaciation, transmissible disease, parasitosis, acute transverse myelitis, huntington's chorea, Parkinson's disease, Alzheimer, apoplexy, primary biliary cirrhosis, hemolytic anemia, malignant tumour, in heart failure, myocardial infarction, AddisonShi is sick, sporadic pluriglandular I type lacks, pluriglandular II type lacks (Schmidt Cotard), adult's (acute) respiratory distress syndrome, bald head, alopecia areata, seronegative arthropathy, joint disease, ReiterShi is sick, arthropathia psoriatica, ulcerative colitis inflammatory joint disease, enteropathic synovitis, chlamydozoan, Yersinia and Salmonellas dependency joint disease, spondyloarthropathy, atheromatous disease/arteriosclerosis, atopic allergology, autoimmunity bullous diseases, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune hemolytic anemia, Coombs positive hemolytic anemias, acquired pernicious anemia, juvenile pernicious anemia, myalgia encephalitis/RoyalFree disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerotic hepatitis, hidden originality autoimmune hepatitis, acquired immune deficiency syndrome (AIDS), acquired immunodeficiency related diseases, hepatitis B, hepatitis C, common various immune deficiencies (common variable hypogammag lobulinemia), dilated cardiomyopathy, atocia, ovarian failure, premature ovarian failure, fibrotic lung disease, CFA, interstitial lung disease after inflammation, interstitial pneumonia, Connective Tissue Disease interstitial lung disease, mixed connective tissue disease associated pulmonary diseases, systemic scleroderma dependency interstitial lung disease, Arthritis and Rheumatoid Arthritis interstitial lung disease, systemic lupus erythematosus associated pulmonary diseases, dermatomyositis/polymyositis associated pulmonary diseases, the sick associated pulmonary diseases of family name, ankylosing spondylitis associated pulmonary diseases, symptomica dispersivity tuberculosis, haemosiderosis associated pulmonary diseases, drug-induced interstitial lung disease, fibrosis, radioactive fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrate tuberculosis, interstitial lung disease after infecting, urarthritis, autoimmune hepatitis, 1 type autoimmune hepatitis (traditional autoimmunity or lupoid hepatitis), 2 type autoimmune hepatitis (anti-LKM antibody hepatitis), the hypoglycemia of autoimmunization mediation, there is the Type B insulin resistance of acanthosis nigricans, hypoparathyroidism, osteoarthritis, primary sclerosing cholangitis, 1 type psoriasis, 2 type psoriasiss, idiopathic oligoleukocythemia, autoimmunity neutropenia, nephropathy NOS, glomerulonephritis, Lyme disease, discoid lupus erythematosus, idiopathic male infertility disease or NOS, Sperm autoimmunity, multiple sclerosis (all hypotypes), sympathetic ophthalmia, the pulmonary hypertension of connective tissue disease (CTD) secondary, Goodpasture Cotard, the lung performance of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, StillShi is sick, systemic scleroderma, Cotard, TakayasuShi disease/arteritis, AT, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, thyrocele property Autoimmune Thyroid hypofunction (HashimotoShi is sick), the hypofunction of atrophic Autoimmune Thyroid, primary myxedema, lens induced uveitis, primary angiitis, vitiligo, acute hepatopathy, chronic hepatopathy, alcoholic cirrhosis, the liver injury of alcohol induction, cholestasis, atopy hepatopathy, drug-induced hepatitis, nonalcoholic fatty liver disease, transformation reactions, B group streptococcus (GBS) infects, mental disorder (such as, depressed and schizophrenia), the disease of Th2 type and the mediation of Th1 type, acute and chronic pain (multi-form pain), cancer is lung such as, mammary gland, stomach, bladder, colon, pancreas, ovary, prostate gland and the rectum cancer, hematopoietic malignancies (leukemia and lymphoma), abetalipoproteinemia, acrocyanosis, acute and chronic parasitism or course of infection, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, gland cancer, atrial ectopic beat, AIDS dementia complex, the hepatitis of alcohol induction, allergic conjunctivitis, allergic contact dermatitis, rhinallergosis, allograft rejection, alpha-1-Antitrypsin deficiency, amyotrophic lateral sclerosis, anaemia, stenocardia, anterior horn cell sex change, AntiCD3 McAb is treated, antiphospholipid syndrome, anti-acceptor allergy, to advocate peace peripheral aneurysm, aortic dissection is formed, arterial hypertension, arteriosclerosis, arterio venous fistula, ataxia, atrial fibrillation (persistence or paroxysmal), auricular flutter, atrioventricular block, B cell lymphoma, bone graft repels, bone marrow transplantation (BMT) is repelled, bundle branch block, Burkitt lymphomas, burn, irregular pulse, the dizzy syndrome of heart shake, cardiac tumor, myocardosis, cardiopulmonary bypass inflammatory response, cartilage transplantation repels, cerebellar cortical degeneration, cerebellum illness, irregularity or multifocal atrial tachycardia, chemotherapy associated conditions, chronic granulocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathology, lymphocytic leukemia (CLL), chronic obstructive disease of lung (COPD), chronic poisoning by salicylic acid salt, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob is sick, culture negative sepsis, cystic fibrosis, cytokine therapy associated conditions, dementia pugilistica, demyelinating disease, dengue hemorrhagic fever, dermatological conditions, polyuria, diabetes, diabetic arteriosclerotic disease, diffusivity Lewy corpusculum is sick, DCMP, basal ganglion illness, middle age mongolism, by the drug-induced drug-induced dyskinesia blocking CNS Dopamine Receptors, drug susceptibility, eczema, encephalomyelitis, endocarditis, incretopathy, epiglottitis, ebv infection, erythromelalgia, extrapyramidal tract and cerebellum illness, familial Observation on Specificity of Blood-sucking lymphohistocysis disease, Fetal Thymus Transplant is repelled, FriedreichShi ataxia, functional peripheral disorder of artery, fungoid sepsis, gas gangrene, stomach ulcer, glomerulonephritis, the transplant rejection of any organ or tissue, Gram-negative sepsis, gram positive sepsis, due to the granuloma of intracellular biological, hairy cell, Hallervorden-Spatz is sick, HashimotoShi thyroiditis, spring fever, cardiac transplant rejection episode, hemochromatosis, hemodialysis, hemolytic uremic syndrome/thrombolysis thrombopenic purpura, hemorrhage, hepatitis A, Xinier reservoir irregular pulse, HIV/HIV neuropathy, Hokdkin disease, hyperkinetic dyskinesia, allergy, hypersensitivity pneumonitis, hypertension, hypokinetic dyskinesia, hypothalmus-pituitary-adrenal axis is evaluated, idiopathic AddisonShi is sick, idiopathic pulmonary fibrosis, antibody-mediated cytotoxicity, weak, infantile spinal muscular atrophy, aorta inflammation, influenza A, ionization radiation irradiation, iridocyclitis/uveitis/optic neuritis, ischemic damage and reperfusion damage, ishemic stroke, juvenile rheumatoid arthritis, JSMA, Kaposi sarcoma, renal transplant rejection, Legionnella, leishmaniasis, leprosy, cortex spinal cord system injury, lipedema, liver transplantation is repelled, lymphedema, malaria, malignant lymphoma, malignant histocytosis, malignant melanoma, meningitis, meningococcemia, metabolic migraine, idiopathic migraine, plastosome multisystem illness, MCTD, MG, multiple myeloma, multisystem sex change (Menzel, Dejerine-Thomas, Shy-Drager and Machado-Joseph), myasthenia gravis, mycobacterium in bird born of the same parents, Mycobacterium tuberculosis, myelodysplastic syndrome, myocardial ischemia illness, nasopharyngeal carcinoma, newborn infant's chronic lung disease, ephritis, nephrosis, neurodegenerative disease, neuropathic muscular atrophy, Neutropenic is had a fever, non Hodgkin lymphoma, aorta abdominalis and branch's obturation thereof, Occlusive arterial illness, OKT3 treats, testitis/epididymitis, testitis/vasotomy reverses operation, organomegaly, osteoporosis, pancreas transplant rejection, pancreas cancer, tumour related syndromes/the hypercalcemia of malignant tumour, parathyroid transplantation repels, inflammatory pelvic disease, perennial rhinitis, pericardial disease, periphery property atheromatosis, peripheral blood vessel illness, peritonitis, pernicious anemia, pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, incretopathy, MG and change of skin syndrome), postperfusion syndrome, syndrome after pump, syndrome after MI cardiotomy, preeclampsia, Progressive symmetric erythrokeratodermia core is benumbed, primary pulmonary hypertension, radiotherapy, RaynaudShi phenomenon, RaynaudShi is sick, RefsumShi is sick, conventional narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcoma, senile chorea, Lewy small body type senile dementia, seronegative arthropathy, apoplexy, sicklemia, skin allograft rejection, change of skin syndrome, small intestine transplantation repels, solid tumor, specific arrhythmia, spinal ataxia, spinocerebellar degeneration, suis myositis, cerebellum structural impairment, subacute sclerosing panencephalitis, faint, cardiovascular systems syphilis, systemic anaphylaxis, systemic inflammatory response syndrome, generalized seizure juvenile rheumatoid arthritis, T cell or FABALL, telangiectasis, thromboangiitis obliterans, thrombocytopenia, toxicity, graft, wound/hemorrhage, type III allergy, the allergy of IV type, unstable angina, uremia, urosepsis, urticaria, valvular heart disease, varix, vasculitis, venous disease, venous thrombosis, ventricular fibrillation, virus and fungi infestation, viral encephalitis/aseptic meningitis, virus associated erythrophage syndrome, Wernicke-Korsakoff syndrome, WilsonShi is sick, the xenograft rejection of any organ or tissue.
In preferred embodiments, above-disclosed pharmaceutical composition is used to experimenter via being selected from following at least one pattern: parenteral, subcutaneous, intramuscular, intravenously, intraarticular (intrarticular), in segmental bronchus, in abdomen, in capsule, in cartilage, in chamber, in body cavity, in cerebellum, Intraventricular, colonic, in neck, in stomach, in liver, in cardiac muscle, in bone, in pelvis, in pericardium, intraperitoneal, in pleura, in prostate gland, in lung, internal rectum, in kidney, in retina, in backbone, in synovial membrane, intrathoracic, intrauterine, intravesical, quick filling, vagina, rectum, buccal, sublingual, in nose and through skin.
In yet another aspect, the invention provides treatment and suffer from the method for the patient of illness, before described method is included in the second agent administration as discussed above, simultaneously or afterwards, use the protein-bonded step of above-disclosed any one.In preferred embodiments, the second reagent is selected from Budesonide, Urogastron, reflunomide, S-Neoral, sulfasalazine, aminosalicylate, Ismipur, azathioprine, metronidazole, lipoxygenase inhibitors, mesalazine, Olsalazine, Balsalazide, antioxidant, thromboxane inhibitors, IL-1 receptor antagonist, anti-il-i-beta monoclonal antibody, anti-IL-6 monoclonal antibody, somatomedin, elastase inhibitor, pyridyl-imidazolium compounds, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, IL-23, EMAP-II, GM-CSF, FGF and PDGF antibody or agonist, CD2, CD3, CD4, CD8, CD-19, CD25, CD28, CD30, CD40, CD45, CD69, the antibody of CD90 or its part, methotrexate, S-Neoral, FK506, rapamycin, mycophenlate mofetil, Leflunomide, NSAIDs, Ibuprofen BP/EP, reflunomide, Ultracortene-H, phosphodiesterase inhibitor, adenosine agonists, antithrombotics, complement inhibitor, adrenergic agent, IRAK, NIK, IKK, p38, map kinase inhibitor, IL-1 β converting enzyme inhibitor, TNF α converting enzyme inhibitor, T cell signal transduction inhibitor, inhibitors of metalloproteinase, sulfasalazine, azathioprine, Ismipur, angiotensin converting enzyme inhibitor, soluble cytokine receptor, solubility p55TNF acceptor, solubility p75TNF acceptor, sIL-1RI, sIL-1RII, sIL-6R, anti-inflammatory cytokines, IL-4, IL-10, IL-11, IL-13 and TGF β.
One aspect of the present invention provides for the protein-bonded at least one antiidiotypic antibody of at least one of the present invention.Antiidiotypic antibody comprises any protein or peptide that comprise molecule, described molecule comprises at least part of immunoglobulin molecules, such as but not limited to, at least one complementarity-determining region (CDR) of weight or light chain or its ligand binding moiety, heavy chain or variable region of light chain, heavy chain or constant region of light chain, framework region, maybe can mix any part in associated proteins of the present invention.
Particularly, the present invention relates to following aspect:
1. the associated proteins comprising the first polypeptide chain of a dual specific or polyspecific, wherein said first polypeptide chain comprises VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3, wherein VH1 is the first variable region of heavy chain, CH1 is CH1, and VL2 is the second variable region of light chain, and CL is constant region of light chain, X2 represents amino acid or the polypeptide of hinge area, CH2 and CH3 forms Fc district, and X1 is optional, and representative is used as amino acid or the polypeptide of joint.
2. according to the associated proteins of item 1, wherein said associated proteins also comprise the second polypeptide chain and or the 3rd polypeptide chain, wherein said second polypeptide chain comprises VL1-CL, wherein VL1 is the first variable region of light chain, CL is constant region of light chain, and described 3rd polypeptide chain comprises VH2-CH1, wherein VH2 is the second variable region of heavy chain, and CH1 is CH1.
3., according to the associated proteins of item 1 or 2, the C-terminal of the VL2 of wherein said first polypeptide chain and the C-terminal of described 3rd polypeptide chain are not be halfcystine simultaneously.
4., according to the associated proteins one of in aforementioned item, wherein said variable region of heavy chain (VH1 or VH2) and variable region of light chain (VL1 or VL2) is selected from mouse heavy chain and variable region of light chain, people's heavy chain and variable region of light chain, the heavy chain of CDR grafting and variable region of light chain and humanized heavy chain and variable region of light chain respectively.
5., according to the associated proteins one of in aforementioned item, wherein said Fc district is Fc district or the variant Fc district of native sequences.
6. according to the associated proteins one of in aforementioned item, Shi Ren Fc district of wherein said Fc district.
7., according to the associated proteins one of in aforementioned item, wherein said Fc district comprises the Fc district from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.
8., according to the associated proteins one of in aforementioned item, wherein said VH1 and described VH2 can in conjunction with same antigen.
9., according to the associated proteins one of in aforementioned item, wherein said VH1 and described VH2 can in conjunction with not synantigen.
10., according to the associated proteins one of in aforementioned item, wherein said VL1 and described VL2 can in conjunction with same antigen.
11. according to the associated proteins one of in aforementioned item, and wherein said VL1 and described VL2 can in conjunction with not synantigen.
12. according to the associated proteins one of in aforementioned item, and wherein said X1 is selected from: EPKSCD; AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA (G 4s) 41sAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVTLFPP; AKTTPP; AKTTPPSVTPLAP; AKTTAP; AKTTAPSVYPLAP; ASTKGP; ASTKGPSVFPLAP, GGGGSGGGGSGGGGS; GENKVEYAPALMALS; GPAKELTPLKEAKVS and GHEAAAVMQVQYPAS.
13. according to the associated proteins one of in aforementioned item, and wherein said associated proteins can in conjunction with one or more targets.
14. according to the associated proteins one of in aforementioned item, and wherein said target is selected from cytokine, cell surface protein, enzyme and acceptor.
15. according to the associated proteins one of in aforementioned item, and one or more targets wherein said are selected from VEGF-A, ANG-2, HER2, TNFalpha and anti-IL-17A.
16. according to the associated proteins one of in aforementioned item, and wherein said associated proteins can regulate the biological function of one or more targets.
17. according to the associated proteins one of in aforementioned item, and wherein said associated proteins is multivalent binding proteins.
18. according to the associated proteins one of in aforementioned item, and wherein said associated proteins is multi-specific binding protein.
19. according to the associated proteins one of in aforementioned item, and wherein said associated proteins has desired character, especially in treatment.
20. according to the associated proteins one of in aforementioned item, and wherein compared to bivalent antibody, described associated proteins is expressed the cell internalizing of the antigen of described antibodies and/or catabolic faster.
21. according to the associated proteins one of in aforementioned item, and wherein said associated proteins is agonist antibody.
22. according to the associated proteins one of in aforementioned item, and the necrocytosis of the cell of wherein said associated proteins abduction delivering antigen and/or apoptosis, wherein said associated proteins can in conjunction with described antigen.
23. according to the associated proteins one of in aforementioned item, wherein as measured by Octetlabelfreetechnology, and the association rate constant (K that described associated proteins has for one or more targets described on) be selected from: at least about 10 2m -1s -1; At least about 10 3m -1s -1; At least about 10 4m -1s -1; At least about 10 5m -1s -1at least 10 6m -1s -1.
24. according to the associated proteins one of in aforementioned item, wherein as measured by Octetlabelfreetechnology, and described association rate constant (K on) be selected from: 10 2m -1s -1to 10 3m -1s -1; 10 3m -1s -1to 10 4m -1s -1; 10 4m -1s -1to 10 5m -1s -1; With 10 5m -1s -1to 10 6m -1s -1.
25. according to the associated proteins one of in aforementioned item, wherein as measured by Octetlabelfreetechnology, and the dissociation rate constant (K that described associated proteins has for one or more targets described off) be selected from: at the most about 10 -3s -1; At the most about 10 -4s -1; At the most about 10 -5s -1; At the most about 10 -6s -1.
26. according to the associated proteins one of in aforementioned item, wherein as measured by Octetlabelfreetechnology, and described dissociation rate constant (K off) be selected from: 10 -3s -1to 10 -4s -1; 10 -4s -1to 10 -5s -1; With 10 -5s -1to 10 -6s -1.
27. according to the associated proteins one of in aforementioned item, wherein as measured by Octetlabelfreetechnology, and the dissociation constant (K that described associated proteins has for one or more targets described d) be selected from: at the most about 10 -7m; At the most about 10 -8m; At the most about 10 -9m; At the most about 10 -10m; At the most about 10 -11m; At the most about 10 -12m; At the most about 10 -13m.
28. according to the associated proteins of item 29, wherein as measured by Octetlabelfreetechnology, and described dissociation constant (K d) be selected from: 10 -7m to 10 -8m, 10 -8m to 10 -9m, 10 -9m to 10 -10m, 10 -10m to 10 -11m; 10 -11m to 10 -12m; Or 10 -12m to 10 -13m.
29. 1 kinds of associated proteins conjugates, it one of to comprise in aforementioned item described associated proteins, and preferably described associated proteins conjugate comprises further and is selected from following reagent: immunoadhesin molecule, developer, therapeutical agent and cytotoxic agent.
30. according to the associated proteins conjugate of item 31, and wherein said reagent is selected from following developer: radio-labeling, enzyme, fluorescent mark, luminescent marking, bioluminescence marker, magnetic mark and vitamin H.
31. according to the associated proteins conjugate of item 31 or 32, and wherein said developer is selected from following radio-labeling: 3h, 14c, 35s, 90y, 99tc, 111in, 125i, 131i, 177lu, 166ho and 153sm.
32. according to the associated proteins conjugate one of in aforementioned item, and wherein said reagent is selected from following treatment or cytotoxic agent: metabolic antagonist, alkylating agent, microbiotic, somatomedin, cytokine, anti-angiogenic agent, antimitotic agent, anthracycline, toxin and apoptosis agent.
33. according to the associated proteins one of in aforementioned item, and wherein said associated proteins is crystallized binding protein.
34. according to the crystallized binding protein of item 35, and wherein said crystal is DNAcarrier free Pharmaceutical controlled release crystal.
35. according to the crystallized binding protein of item 35, and wherein said associated proteins has the Half-life in vivo longer than described protein-bonded described soluble counterpart.
36. according to the crystallized binding protein of item 35, and wherein said associated proteins retains biologic activity.
37. according to the associated proteins one of in aforementioned item, and wherein said associated proteins is glycosylation associated proteins.
38. according to the associated proteins of item 39, and wherein said glycosylation associated proteins is people's glycosylation associated proteins.
39. 1 kinds of nucleic acid be separated, its protein-bonded aminoacid sequence of one of to encode in aforementioned item.
40. 1 kinds of carriers, it comprises the nucleic acid be separated according to item 41.
The carrier of 41. 41, wherein said carrier is selected from pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, pcDNA3.1, pcDNA3.1TOPO, pEF6TOPO and pBJ.
42. 1 kinds of host cells, it comprises the carrier according to item 42.
43. according to the host cell of item 44, and wherein said host cell is prokaryotic cell prokaryocyte.
44. according to the host cell of item 45, and wherein said prokaryotic cell prokaryocyte is intestinal bacteria.
45. according to the host cell of item 44, and wherein said host cell is eukaryotic cell.
46. according to the host cell of item 47, and wherein said eukaryotic cell is selected from protist cell, zooblast, vegetable cell and fungal cell.
47. according to the host cell of item 44, and wherein said host cell is mammalian cell or insect cell.
48. according to the host cell of item 49, and wherein said mammalian cell is CHO.
49. according to the host cell of item 49, and wherein said mammalian cell is COS.
50. according to the host cell of item 49, and wherein said mammalian cell is HEK293-F cell.
51. according to the host cell of item 48, and wherein said fungal cell is yeast cell.
52. according to the host cell of item 53, and wherein said yeast cell is Saccharomyces cerevisiae.
53. according to the host cell of item 49, and wherein said insect cell is insect Sf 9 cells.
Produce protein-bonded methods for 54. 1 kinds, it is included in is enough to produce under described protein-bonded condition, the host cell one of to cultivate in the medium in aforementioned item.
55. 1 kinds of protein, its method according to item 56 is produced.
56. 1 kinds of pharmaceutical compositions, its protein one of to comprise in aforementioned item and pharmaceutically acceptable carrier.
The pharmaceutical composition of 57. 58, it comprises the other reagent of at least one further.
The pharmaceutical composition of 58. 59, wherein said other reagent is selected from: therapeutical agent, developer, cytotoxic agent, angiogenesis inhibitor; Kinase inhibitor; Costimulatory molecules blocker; Adhesion molecule blockers; Anti-cytokine antibodies or its function fragment; Methotrexate; S-Neoral; Rapamycin; FK506; Detectable label or reporter molecule; TNF antagonist; Rheumatism; Muscular flaccidity agent, narcotic, non-steroidal anti-inflammatory drug (NSAID), pain killer, narcotic, tranquilizer, neuromuscular blocking agents; Biocide, antipsoriatic, reflunomide, anabolic steroid, erythropoietin, immunization, immunoglobulin (Ig), immunosuppressor, tethelin, hormone replacement agent, radiopharmaceuticals, thymoleptic, antipsychotic drug, stimulant, asthma medication, beta-agonists, suck steroid, suprarenin, cytokine and cytokine antagonist.
The pharmaceutical composition of 59. 60, wherein said narcotic is local anesthetic.
60. according to the protein one of in aforementioned item for the preparation of the purposes in the treatment disease of experimenter or the medicine of illness.
The purposes of 61. 62, wherein said illness is selected from rheumatoid arthritis, osteoarthritis, JCA, septic arthritis, Lyme arthritis, arthritic psoriasis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, CrohnShi is sick, ulcerative colitis, inflammatory bowel, insulin-dependent diabetes, thyroiditis, asthma, allergic disease, psoriasis, dermatitis, scleroderma, graft versus host disease (GVH disease), organ-graft refection, the acute immune disease relevant to organ transplantation, the chronic immunological disorders relevant to organ transplantation, sarcoidosis, atherosclerosis, disseminated inravascular coagulation, KawasakiShi is sick, GraveShi is sick, nephrotic syndrome, Chronic Fatigue Syndrome, Wei Genashi granulomatosis, anaphylactoid purpura, kidney microvascular is scorching, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, emaciation, transmissible disease, parasitosis, acute transverse myelitis, huntington's chorea, Parkinson's disease, Alzheimer, apoplexy, primary biliary cirrhosis, hemolytic anemia, malignant tumour, in heart failure, myocardial infarction, AddisonShi is sick, sporadic pluriglandular I type lacks, pluriglandular II type lacks (Schmidt Cotard), adult's (acute) respiratory distress syndrome, bald head, alopecia areata, seronegative arthropathy, joint disease, ReiterShi is sick, arthropathia psoriatica, ulcerative colitis inflammatory joint disease, enteropathic synovitis, chlamydozoan, Yersinia and Salmonellas dependency joint disease, spondyloarthropathy, atheromatous disease/arteriosclerosis, atopic allergology, autoimmunity bullous diseases, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune hemolytic anemia, Coombs positive hemolytic anemias, acquired pernicious anemia, juvenile pernicious anemia, myalgia encephalitis/RoyalFree disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerotic hepatitis, hidden originality autoimmune hepatitis, acquired immune deficiency syndrome (AIDS), acquired immunodeficiency related diseases, hepatitis B, hepatitis C, common various immune deficiencies (common variable hypogammag lobulinemia), dilated cardiomyopathy, atocia, ovarian failure, premature ovarian failure, fibrotic lung disease, CFA, interstitial lung disease after inflammation, interstitial pneumonia, Connective Tissue Disease interstitial lung disease, mixed connective tissue disease associated pulmonary diseases, systemic scleroderma dependency interstitial lung disease, Arthritis and Rheumatoid Arthritis interstitial lung disease, systemic lupus erythematosus associated pulmonary diseases, dermatomyositis/polymyositis associated pulmonary diseases, the sick associated pulmonary diseases of family name, ankylosing spondylitis associated pulmonary diseases, symptomica dispersivity tuberculosis, haemosiderosis associated pulmonary diseases, drug-induced interstitial lung disease, fibrosis, radioactive fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrate tuberculosis, interstitial lung disease after infecting, urarthritis, autoimmune hepatitis, 1 type autoimmune hepatitis (traditional autoimmunity or lupoid hepatitis), 2 type autoimmune hepatitis (anti-LKM antibody hepatitis), the hypoglycemia of autoimmunization mediation, there is the Type B insulin resistance of acanthosis nigricans, hypoparathyroidism, osteoarthritis, primary sclerosing cholangitis, 1 type psoriasis, 2 type psoriasiss, idiopathic oligoleukocythemia, autoimmunity neutropenia, nephropathy NOS, glomerulonephritis, Lyme disease, discoid lupus erythematosus, idiopathic male infertility disease or NOS, Sperm autoimmunity, multiple sclerosis (all hypotypes), sympathetic ophthalmia, the pulmonary hypertension of connective tissue disease (CTD) secondary, Goodpasture Cotard, the lung performance of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, StillShi is sick, systemic scleroderma, Cotard, TakayasuShi disease/arteritis, AT, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, thyrocele property Autoimmune Thyroid hypofunction (HashimotoShi is sick), the hypofunction of atrophic Autoimmune Thyroid, primary myxedema, lens induced uveitis, primary angiitis, vitiligo, acute hepatopathy, chronic hepatopathy, alcoholic cirrhosis, the liver injury of alcohol induction, cholestasis, atopy hepatopathy, drug-induced hepatitis, nonalcoholic fatty liver disease, transformation reactions, B group streptococcus (GBS) infects, mental disorder (such as, depressed and schizophrenia), the disease of Th2 type and the mediation of Th1 type, acute and chronic pain (multi-form pain), cancer is lung such as, mammary gland, stomach, bladder, colon, pancreas, ovary, prostate gland and the rectum cancer, hematopoietic malignancies (leukemia and lymphoma), abetalipoproteinemia, acrocyanosis, acute and chronic parasitism or course of infection, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, gland cancer, atrial ectopic beat, AIDS dementia complex, the hepatitis of alcohol induction, allergic conjunctivitis, allergic contact dermatitis, rhinallergosis, allograft rejection, alpha-1-Antitrypsin deficiency, amyotrophic lateral sclerosis, anaemia, stenocardia, anterior horn cell sex change, AntiCD3 McAb is treated, antiphospholipid syndrome, anti-acceptor allergy, to advocate peace peripheral aneurysm, aortic dissection is formed, arterial hypertension, arteriosclerosis, arterio venous fistula, ataxia, atrial fibrillation (persistence or paroxysmal), auricular flutter, atrioventricular block, B cell lymphoma, bone graft repels, bone marrow transplantation (BMT) is repelled, bundle branch block, Burkitt lymphomas, burn, irregular pulse, the dizzy syndrome of heart shake, cardiac tumor, myocardosis, cardiopulmonary bypass inflammatory response, cartilage transplantation repels, cerebellar cortical degeneration, cerebellum illness, irregularity or multifocal atrial tachycardia, chemotherapy associated conditions, chronic granulocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathology, lymphocytic leukemia (CLL), chronic obstructive disease of lung (COPD), chronic poisoning by salicylic acid salt, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob is sick, culture negative sepsis, cystic fibrosis, cytokine therapy associated conditions, dementia pugilistica, demyelinating disease, dengue hemorrhagic fever, dermatological conditions, polyuria, diabetes, diabetic arteriosclerotic disease, diffusivity Lewy corpusculum is sick, DCMP, basal ganglion illness, middle age mongolism, by the drug-induced drug-induced dyskinesia blocking CNS Dopamine Receptors, drug susceptibility, eczema, encephalomyelitis, endocarditis, incretopathy, epiglottitis, ebv infection, erythromelalgia, extrapyramidal tract and cerebellum illness, familial Observation on Specificity of Blood-sucking lymphohistocysis disease, Fetal Thymus Transplant is repelled, FriedreichShi ataxia, functional peripheral disorder of artery, fungoid sepsis, gas gangrene, stomach ulcer, glomerulonephritis, the transplant rejection of any organ or tissue, Gram-negative sepsis, gram positive sepsis, due to the granuloma of intracellular biological, hairy cell, Hallervorden-Spatz is sick, HashimotoShi thyroiditis, spring fever, cardiac transplant rejection episode, hemochromatosis, hemodialysis, hemolytic uremic syndrome/thrombolysis thrombopenic purpura, hemorrhage, hepatitis A, Xinier reservoir irregular pulse, HIV/HIV neuropathy, Hokdkin disease, hyperkinetic dyskinesia, allergy, hypersensitivity pneumonitis, hypertension, hypokinetic dyskinesia, hypothalmus-pituitary-adrenal axis is evaluated, idiopathic AddisonShi is sick, idiopathic pulmonary fibrosis, antibody-mediated cytotoxicity, weak, infantile spinal muscular atrophy, aorta inflammation, influenza A, ionization radiation irradiation, iridocyclitis/uveitis/optic neuritis, ischemic damage and reperfusion damage, ishemic stroke, juvenile rheumatoid arthritis, JSMA, Kaposi sarcoma, renal transplant rejection, Legionnella, leishmaniasis, leprosy, cortex spinal cord system injury, lipedema, liver transplantation is repelled, lymphedema, malaria, malignant lymphoma, malignant histocytosis, malignant melanoma, meningitis, meningococcemia, metabolic migraine, idiopathic migraine, plastosome multisystem illness, MCTD, MG, multiple myeloma, multisystem sex change (Menzel, Dejerine-Thomas, Shy-Drager and Machado-Joseph), myasthenia gravis, mycobacterium in bird born of the same parents, Mycobacterium tuberculosis, myelodysplastic syndrome, myocardial ischemia illness, nasopharyngeal carcinoma, newborn infant's chronic lung disease, ephritis, nephrosis, neurodegenerative disease, neuropathic muscular atrophy, Neutropenic is had a fever, non Hodgkin lymphoma, aorta abdominalis and branch's obturation thereof, Occlusive arterial illness, OKT3 treats, testitis/epididymitis, testitis/vasotomy reverses operation, organomegaly, osteoporosis, pancreas transplant rejection, pancreas cancer, tumour related syndromes/the hypercalcemia of malignant tumour, parathyroid transplantation repels, inflammatory pelvic disease, perennial rhinitis, pericardial disease, periphery property atheromatosis, peripheral blood vessel illness, peritonitis, pernicious anemia, pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, incretopathy, MG and change of skin syndrome), postperfusion syndrome, syndrome after pump, syndrome after MI cardiotomy, preeclampsia, Progressive symmetric erythrokeratodermia core is benumbed, primary pulmonary hypertension, radiotherapy, RaynaudShi phenomenon, RaynaudShi is sick, RefsumShi is sick, conventional narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcoma, senile chorea, Lewy small body type senile dementia, seronegative arthropathy, apoplexy, sicklemia, skin allograft rejection, change of skin syndrome, small intestine transplantation repels, solid tumor, specific arrhythmia, spinal ataxia, spinocerebellar degeneration, suis myositis, cerebellum structural impairment, subacute sclerosing panencephalitis, faint, cardiovascular systems syphilis, systemic anaphylaxis, systemic inflammatory response syndrome, generalized seizure juvenile rheumatoid arthritis, T cell or FABALL, telangiectasis, thromboangiitis obliterans, thrombocytopenia, toxicity, graft, wound/hemorrhage, type III allergy, the allergy of IV type, unstable angina, uremia, urosepsis, urticaria, valvular heart disease, varix, vasculitis, venous disease, venous thrombosis, ventricular fibrillation, virus and fungi infestation, viral encephalitis/aseptic meningitis, virus associated erythrophage syndrome, Wernicke-Korsakoff syndrome, WilsonShi is sick, the xenograft rejection of any organ or tissue.
62. according to the purposes of item 62 or 63, wherein said Pharmaceutical formulations is for using to described experimenter via being selected from following at least one pattern: parenteral, subcutaneous, intramuscular, intravenously, intraarticular, in segmental bronchus, in abdomen, in capsule, in cartilage, in chamber, in body cavity, in cerebellum, Intraventricular, colonic, in neck, in stomach, in liver, in cardiac muscle, in bone, in pelvis, in pericardium, intraperitoneal, in pleura, in prostate gland, in lung, internal rectum, in kidney, in retina, in backbone, in synovial membrane, intrathoracic, intrauterine, intravesical, quick filling, vagina, rectum, buccal, sublingual, in nose and through skin.
63. 1 kinds of pharmaceutical compositions, its protein one of to comprise in aforementioned item and pharmaceutically acceptable carrier.
The pharmaceutical composition of 64. 65, it comprises the other reagent of at least one further.
The pharmaceutical composition of 65. 66, wherein said other reagent is selected from: therapeutical agent, developer, cytotoxic agent, angiogenesis inhibitor; Kinase inhibitor; Costimulatory molecules blocker; Adhesion molecule blockers; Anti-cytokine antibodies or its function fragment; Methotrexate; S-Neoral; Rapamycin; FK506; Detectable label or reporter molecule; TNF antagonist; Rheumatism; Muscular flaccidity agent, narcotic, non-steroidal anti-inflammatory drug (NSAID), pain killer, narcotic, tranquilizer, neuromuscular blocking agents; Biocide, antipsoriatic, reflunomide, anabolic steroid, erythropoietin, immunization, immunoglobulin (Ig), immunosuppressor, tethelin, hormone replacement agent, radiopharmaceuticals, thymoleptic, antipsychotic drug, stimulant, asthma medication, beta-agonists, suck steroid, suprarenin, cytokine and cytokine antagonist.
The pharmaceutical composition of 66. 67, wherein said narcotic is local anesthetic.
Protein-bonded fragment one of in 67. aforementioned items or part, it can specific binding dual specific antigen or polyspecific antigen, preferably described protein-bonded fragment or part are fragment or the part of antibody, are preferably Fab, Fab' and F (ab') 2, Fd, scFv (scFv), single-chain antibody, disulphide connect or the Fv (sdFv or dsFv) of disulphide stabilization.
Associated proteins one of in 68. aforementioned items, wherein said associated proteins is antibody.
69. 1 kinds of test kits, its protein one of to comprise in aforementioned item.
Accompanying drawing is sketched
Fig. 1. the structure of dual specific or polyspecific IgJR antibody.Figure 1A shows the strategy producing dual specific or polyspecific IgJR antibody from 2 kinds of parental antibodies; Figure 1B is the schematic diagram of three constructs of expressing dual specific or polyspecific IgJR antibody.
Fig. 2. the aminoacid sequence of anti-vegf and Anti-HER 2.
Fig. 3. the aminoacid sequence of anti-VEGF/HER2 dual specific IgJR antibody.In figure 3 a, be respectively from left to right in construct #B1JRHC1 chain: runic, signal protein; Light blue, VH1; Green, CH1; Redness, adaptor protein (X1); Black, VL2; Purple, CL; Redness, X2CH2CH3; In figure 3b, be respectively from left to right in construct #B2JRLC1 chain: runic, signal protein; Black, VL1; Purple, CL; In fig. 3 c, in construct #B3JRHC2 chain, be respectively from left to right: runic, signal protein; Light blue, VH2; Green, CH1.In figure 3 a, can lack or be substituted at the cysteine residues (C) (green overstriking) of the C-terminal of the VL2 of construct #B1JRHC1.In fig. 3 c, can add cysteine residues (C) (pink) at the C-end (purple) of construct #B3JRHC2.
Fig. 4. the expression vector collection of illustrative plates of dual specific IgJR antibody.The construct related in the present invention all uses pcDNA3.1 expression vector to express.
Fig. 5. the size exclusion chromatography of anti-VEGF/HER2 dual specific IgJR antibody and SDS-PAGE analyze, in fig. 5, only occur that the one corresponding to anti-VEGF/HER2 dual specific IgJR antibody is unimodal, molecular weight is about 248KD, in figure 5b, SDS-PAGE shows anti-VEGF/HER2 dual specific IgJR antibody and is made up of three kinds of protein, is respectively JRHC1, JRHC2 and JRLC1.
Fig. 6. the differential scanning calorimetric analysis of anti-VEGF/HER2 dual specific IgJR antibody, the Tm of display anti-VEGF/HER2 dual specific IgJR antibody is about 75 DEG C.
Fig. 7. the stability of anti-VEGF/HER2 dual specific IgJR antibody and preparation result, the optimum storage of display and preparation condition are pH5.8,10mM Histidine, 280mM sucrose, 0.02% Polysorbate 20.
Fig. 8 .Octet Real-time dynamics binding analysis display anti-VEGF/HER2 dual specific IgJR antibody is simultaneously in conjunction with VEGF and HER2.
Fig. 9. the anti-VEGF/effect of HER2 dual specific IgJR antibody in HUVEC cell proliferation.
Figure 10. the anti-VEGF/effect of HER2 dual specific IgJR antibody in SKBr-3 cell proliferation.
Figure 11. the aminoacid sequence of anti-VEGF antibodies and anti-ANG-2 antibody.
Figure 12. the aminoacid sequence of anti-VEGF/ANG-2 dual specific IgJR antibody.In fig. 12, be respectively from left to right in construct #A1JRHC1 chain: runic, signal protein; Light blue, VH1; Green, CH1; Redness, adaptor protein (X1); Black, VL2; Dark blue, CL; Redness, X2CH2CH3; In Figure 12 B, be respectively from left to right in construct #A2JRLC1 chain: runic, signal protein; Black, VL1; Purple, CL; In fig. 12 c, in construct #A3JRHC2 chain, be respectively from left to right: runic, signal protein; Light blue, VH2; Green, CH1.In fig. 12, can lack or be substituted at the cysteine residues (C) (green overstriking) of the C-terminal of the VL2 of construct #A1JRHC1.In fig. 12 c, can add cysteine residues (C) (pink) at the C-end of construct #A3JRHC2.
Figure 13. the real-time binding kinetics research of Octet of anti-VEGF/ANG-2 dual specific IgJR antibody.Octet Real-time dynamics binding analysis display anti-VEGF/ANG-2 dual specific IgJR antibody is simultaneously in conjunction with VEGF-A and ANG-2.
Figure 14. the aminoacid sequence of anti-TNFalpha and anti-IL-17A antibody.
Figure 15. the aminoacid sequence of anti-TNF alpha/IL-17A dual specific IgJR antibody.In Figure 15 A, be respectively from left to right in construct #C1JRHC1 chain: runic, signal protein; Light blue, VH1; Green, CH1; Redness, adaptor protein (X1); Black, VL2; Purple, CL; Redness, X2CH2CH3; In Figure 15 B, be respectively from left to right in construct #C2JRLC1 chain: runic, signal protein; Black, VL1; Purple, CL; In figure 15 c, in construct #C3JRHC2 chain, be respectively from left to right: runic, signal protein; Light blue, VH2; Green, CH1.In Figure 15 A, can lack or be substituted at the cysteine residues (C) (green overstriking) of the C-terminal of the VL2 of construct #C1JRHC1.In figure 15 c, can add cysteine residues (C) (pink) at the C-end of construct #C3JRHC2.
Figure 16 .Octet Real-time dynamics binding analysis display anti-TNF alpha/IL-17A dual specific IgJR antibody is simultaneously in conjunction with TNFalpha and IL-17A.
Detailed Description Of The Invention
The present invention relates to the multivalence in conjunction with 2 kinds or more antigens and/or multi-specific binding protein.Specifically, the present invention relates to dual variable region immunoglobulin (IgJR) and pharmaceutical composition thereof and for the preparation of the nucleic acid of this type of IgJR, recombinant expression vector and host cell.The present invention further comprises the purposes of IgJR of the present invention in treatment illness.
Unless otherwise defined herein, the Science and Technology term used together with the present invention should have the implication that those of ordinary skill in the art understand usually.The implication of term and scope should be clear, but when any potential indefinite property, definition provided herein has precedence over any dictionary or external definition.In addition, unless the context otherwise requires, singular references should comprise plural number, and plural term should comprise odd number.In addition, term " comprises " and other forms of use is nonrestrictive.Equally, unless otherwise expressly specified, term such as " element " or " component " contains the element and component that comprise a unit and the element comprised more than a subunit and component.
Usually, the nomenclature used together with cell and tissue culture described herein, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization and its technology be this area well-known and normally used those.Except as otherwise noted, Method and Technology of the present invention is generally well-known according to this area, and the ordinary method as various and more specifically described in reference is carried out, and described reference is quoted from start to finish at this specification sheets and discussed.Enzymatic reaction and purification technique according to the specification sheets of manufacturers, usually realize as this area or as described hereinly to carry out.The nomenclature used together with analytical chemistry described herein, synthetic organic chemistry and medical science and pharmaceutical chemistry and its laboratory procedure and technology be this area well-known and normally used those.Standard technique is used to be used for chemosynthesis, chemical analysis, medicine preparation, to prepare and send and patient treatment.
The present invention can more easily understand, and the term of selection is hereafter defining.
Definition
Term " immunoglobulin molecules " refers to the protein of the structure with naturally occurring antibody.Such as, the immunoglobulin (Ig) of IgG class is about 150, and 000 daltonian different tetramer glycoprotein, two light chains connected by disulfide linkage and two heavy chains form.Every bar heavy chain has variable region (territory) (VH), and also referred to as variable heavy chain domain or heavy chain variable domain, hinge area (HR) and three constant domain (CH1, CH2 and CH3), also referred to as CH.When IgE immunoglobulin like protein, heavy chain also has CH4 structural domain.Therefore, heavy chain immunoglobulin holds at N the polypeptide be made up of following structural domain to C extreme direction: VH-CH1-HR-CH2-CH3-(CH4).Similarly, from N end to C end, every bar light chain has variable region (VL), also referred to as variable light chain domain or light-chain variable domain, follows by constant light (CL) structural domain, also referred to as constant region of light chain.Therefore, light chain immunoglobulin holds at N the polypeptide be made up of following structural domain to C extreme direction: VL-CL.Immunoglobulin (Ig) is made up of two the Fab fragments connected by immunoglobulin hinge region and Fc structural domain substantially.
Term " antibody " herein, with implication use the most widely, contains the Multiple Antibodies structure including but not limited to monoclonal antibody, polyclonal antibody and antibody fragment, as long as the antigen-binding activity that their displays are wished.Antibody useful in the present invention also comprises the antigen-binding antibody fragment of antibody of the present invention, includes but not limited to Fab, Fab' and F (ab') 2, Fd, scFv (scFv), single-chain antibody, disulphide connect or the Fv (sdFv or dsFv) of disulphide stabilization.The present invention also comprises the single domain antibody comprising VL territory or VH territory.In addition, described antibody can produce by any method, such as phage display or produce in any organism, ovum or clone, comprise bacterium, insect, yeast (fungi), Mammals or other types generation there is cell or the clone of the antibody (as humanized antibody) of desired character.Can also pass through to combine the Fab part from different plant species and Fc district, or the framework region by retaining antigen determination region, framework region being modified to other species forms antibody.
What term " variable region " or " variable domain " referred to heavy chain of antibody or light chain relates to the structural domain that this antibody is combined with antigen.The heavy chain of natural antibody has similar structure usually with the variable domain (being respectively VH with VL) of light chain, and each structural domain comprises four conservative framework regions (FR) and three hypervariable regions (HVR).See such as Kindt etc., KubyImmunology, the 6th edition, W.H.FreemanandCo., 91 pages (2007).Single VH or VL structural domain can be enough to give antigen-binding specificity.
Term " Fc district " is used for defining the C end regions of heavy chain immunoglobulin in this article, and it comprises at least part of constant region.This term comprises native sequences Fc district and variant Fc district.Although the boundary in the Fc district of IgG heavy chain can slight variations, human IgG heavy chain Fc district is normally defined from Cys226 or the carboxyl terminal extending to heavy chain from Pro230.But C end Methionin (Lys447) in Fc district can presence or absence.Unless otherwise indicated herein, in Fc district or constant region, the numbering of amino-acid residue is according to Kabat etc., SequencesofProteinsofImmunologicalInterest, 5th edition PublicHealthService, NationalInstitutesofHealth, Bethesda, MD, EU numbering system described in 1991, also referred to as EU index.One of " subunit " finger-type two polypeptide becoming dimeric Fc structural domain of Fc structural domain used herein, namely comprise heavy chain immunoglobulin C hold constant region, can stably oneself combine polypeptide.Such as, the subunit of IgGFc structural domain comprises IgGCH2 and IgGCH3 constant region.
As use alpha nerein, term " hypervariable region " or " antigen-binding portion thereof of antibody " refer to the amino-acid residue of the antibody that responsible antigen combines.Hypervariable region comprises the amino-acid residue from " complementary determining region " or " CDR ".These regions, variable region of " framework " or " FR " district's right and wrong some hypervariable region residues as defined herein.Therefore, the light chain of antibody and heavy chain are from N-terminal to C-terminal inclusion region FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.CDR on each chain by this class amino framework acids separately.Especially, the CDR3 of heavy chain is the region contributing to antigen combination most.CDR and FR is according to Kabat etc., SequencesofProteinsofImmunologicalInterest, and the 5th edition, the standard definition of PublicHealthService, NationalInstitutesofHealth, Bethesda, MD (1991) is determined.
As used in this application, term " constant region " (constant domain) refers to the summation in the region of the non-variable region of antibody.Constant region does not participate in conjugated antigen directly, but demonstrates multiple effector function.Depend on the aminoacid sequence of the constant region of the heavy chain of antibody, antibody is divided into: IgA type, IgD type, IgE type, IgG type and IgM type, and the several of these can be divided into hypotype further, such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2.The CH corresponding with the different shaped of antibody is called α, δ, ε, γ and μ.The constant region of light chain that can find in whole five kinds of Antibody types is called κ (kappa) and λ (lambda).
Antibodies specific refers to the defined epitope of antibody to antigen or the Selective recognition of antigenic determinant.Such as natural antibody is monospecific.As used herein, term " polyspecific " refers to antibody or the associated proteins with two or more antigen binding site, the antigen that wherein at least two basic change is different or the different epi-positions of same antigen.Herein " specific antibody " and " " binding proteins specific " has identical implication, is used interchangeably.
According to the present invention, " bi-specific antibody " is antibody or the associated proteins with two different antigen-binding specificities.Antibody of the present invention is such as different at least two antigen (such as the VEGF of the first antigen and the ANG-2 as the second antigen) is polyspecific.In one embodiment of the invention, multi-specificity antibody according to the present invention is dual specific.
As used herein, term " monospecific " antibody refers to the antibody with one or more binding site, and wherein each binding site is in conjunction with the identical epi-position of same antigen.
As used in this application, term " valency " refers to the concrete number of the binding site existed in antibody molecule.Such as natural antibody or full length antibody according to the present invention have two basic change site, and are divalence.Therefore, term " divalence ", " trivalent ", " tetravalence ", " pentavalent " and " sexavalence " refer in antibody molecule, there are two basic change site, three binding sites, four combinations site, five binding sites and six binding sites respectively.Multi-specificity antibody according to the present invention is at least " divalence ".
Antibody of the present invention can have three or more binding site, and be polyspecific, preferably dual specific or tri-specific.Even when exist more than three binding sites (namely antibody be tetravalence, pentavalent or sexavalence or multivalence), also can be dual specific according to multi-specificity antibody of the present invention.For the antibody had more than two antigen binding sites, some binding sites can be identical, as long as this protein has binding site to two kinds of different antigens.
" specific binding " means this combination and has selectivity to antigen, and can with not want or non-specific interaction distinguishes.The other technologies can be familiar with by enzyme-linked immunosorbent assay (ELISA) or those skilled in the art measure the ability of antibody binding specificity antigenic determinant, these other technologies are surperficial plasmon resonance (SPR) technology (analyzing on BIAcore instrument) (Liljeblad etc. such as, GlycoJ17, 323-329 (2000)), tradition combines and measures (Heeley, EndocrRes28, 217-229 (2002)) and Octettechnology (Do, T., Deng people, ProteinExpressionandPurification, 60 (2), 147-150, 2008).
As used herein, term " k off" (s _ 1) refer to the dissociation rate constant of specific antibodies-AI.This value is also called k dvalue.
As used herein, term " K on" (M _ 1xs -1) refer to the association rate constant of specific antibodies-AI.
" avidity " refers to the intensity of the summation of noncovalent interaction between the single combining site of molecule (such as acceptor) and its binding partners (such as part).Except as otherwise noted, " binding affinity " used herein refers to the interactional inherent binding affinity of 1:1 between the member that reflection combines (such as antigen-binding portion thereof and antigen, or acceptor and its part).The avidity of molecule X to its mating partner Y can be expressed as dissociation constant (K usually d), its be dissociate and association rate constant (be respectively k offand K on) ratio.Therefore, equivalent avidity can comprise different rate constants, as long as the ratio of rate constant keeps identical.Avidity can be measured by improving method known in the art, comprise described herein those.For measuring k off, K onand K dconcrete grammar be Real-time dynamics binding analysis (OctetReal-TimeKenitic).
" disulfide formation " refers to the process forming covalent linkage between two halfcystines existing in one or two polypeptide, and it can be illustrated as "-S--S-".
The protein-bonded generation of 1.IgJR
The present invention relates to the dual variable region associated proteins and preparation method thereof in conjunction with one or more targets.Described associated proteins comprises the first polypeptide chain JRHC1, wherein said first polypeptide chain comprises VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3, wherein VH1 is the first variable region of heavy chain, CH1 is CH1, and VL2 is the second variable region of light chain, and CL is constant region of light chain, X2 represents amino acid or the polypeptide of hinge area, CH2 and CH3 forms antibody Fc district, and X1 is optional, and representative is used as amino acid or the polypeptide of joint.Except the first above-mentioned polypeptide chain JRHC1, associated proteins also comprises the second polypeptide chain JRLC1 and the 3rd polypeptide chain JRHC2, wherein said second polypeptide chain comprises VL1-CL, wherein VL1 is the first variable region of light chain, CL is constant region of light chain, and described 3rd polypeptide chain JRHC2 comprises VH2-CH1, wherein VH2 is the second variable region of heavy chain, and CH1 is CH1.Associated proteins of the present invention can use various technology to produce.The invention provides and produce protein-bonded expression vector, host cell and method.
The cysteine residues (C) held at the C of the VL2 of the first polypeptide chain JRHC1 does not participate in maintaining the protein-bonded correct conformation of dual specific IgJR, therefore can lack or be substituted, usually Serine is replaced to, to improve the protein-bonded oxidation-resistance of dual specific IgJR and to prevent abnormal crosslinked.Another kind of situation, can join this antibody by one or more halfcystine key, to improve its stability.An optional method is: add halfcystine at the C-end of the 3rd polypeptide chain JRHC2, forms disulfide linkage with the halfcystine of the hinge area EPKSCD with the first polypeptide chain JRHC1.We find, are not simultaneously for improving IgJR protein-bonded stability when halfcystine at the C-terminal of the VL2 of the first polypeptide chain and the C-terminal of the 3rd polypeptide chain.Specifically, be halfcystine at the C-terminal of the VL2 of the first polypeptide chain JRHC1, when the C-terminal of the 3rd polypeptide chain JRHC2 is not halfcystine, now the halfcystine C of HC1 chain can form intrachain disulfide bond with the halfcystine C in the EPKSCDKT of hinge area.When the C-terminal of the VL2 of the first polypeptide chain JRHC1 is not halfcystine, the C-terminal of the 3rd polypeptide chain JRHC2 is not halfcystine, and the halfcystine C in the hinge EPKSCDKT of now two HC1 chains forms interchain disulfide bond.Be not halfcystine at the C-terminal of the VL2 of the first polypeptide chain JRHC1, when the C-terminal of the 3rd polypeptide chain JRHC2 is halfcystine, the halfcystine of the hinge area EPKSCD of HC1 can form interchain disulfide bond with the halfcystine of the C-terminal of HC2.These three kinds of situations all can improve the protein-bonded stability of IgJR.
A. the generation of monoclonal antibody.
Dual specific or the protein-bonded variable region of polyspecific IgJR can obtain from parental antibody, and comprising can the polyclone of binding purposes antigen and monoclonal antibody.These antibody can be naturally occurringly maybe can be produced by recombinant technology.
Monoclonal antibody can use extensively various technology known in the art to prepare, and comprises and uses hybridoma, recombinant chou and display technique of bacteriophage, or its combination.Such as, monoclonal antibody can use hybridoma technology to prepare, and comprises the people such as known in the art and such as Harlow, Antibodies:ALaboratoryManual, (ColdSpringHarborLaboratoryPress, the 2nd edition 1988); Hammerling, waits people: those of instruction in MonoclonalAntibodiesandT-CellHybridomas563-681 (Elsevier, N.Y., 1981) (described reference entirety is incorporated herein by reference).As used herein, term " monoclonal antibody " is not limited by the antibody that hybridoma technology produces.Term " monoclonal antibody " refers to derive from single clone, comprises any eucaryon, protokaryon or phage clone, instead of produces its antibody of method.Hybridoma is selected, clone and screen desired characteristic further, comprises strong hybridomas grew, high antibody produces and required antibody characteristic.Hybridoma can in syngeneic animal body, the immune animal of shortage such as nude mice or cultivate in cell cultures in vitro and expansion.The method selecting, clone and expand hybridoma is that those of ordinary skill in the art are well-known.In preferred embodiments, hybridoma is Mouse Hybridoma Cells.In a further preferred embodiment, hybridoma is at inhuman, non-mouse species, and such as rat, sheep, pig, goat, ox or Malaysia and China produce.In another embodiment, hybridoma is people's hybridoma, and wherein people's non-secretory myeloma can in conjunction with people's cytogamy of the antibody of specific antigen with expression.
Recombinant monoclonal antibodies also uses in this area and is called that the program of selection lymphocyte antibody method (SLAM) produces from lymphocyte that is single, that be separated, described SLAM is as U.S. Patent number 5,627,052, open WO92/02551 and Babcock of PCT, J.S. people is waited, described in (1996) Proc.Natl.Acad.Sci.USA93:7843-7848.In this approach, the unicellular of object antibody is secreted in qualification, such as derive from the lymphocyte of immune animal, and from cell, save heavy and variable region of light chain cDNAs by reverse transcriptase PCR, subsequently can at suitable constant region for immunoglobulin (such as, human constant region) in background, in mammalian host cell such as COS or Chinese hamster ovary celI, express these variable regions.With the immunoglobulin sequences transfection of amplification, derive from the lymphocytic host cell selected in body, further analyzed in vitro and selection can be experienced subsequently, such as by the cell of elutriation transfection to be separated the cell of expression for the antibody of object antigen.The immunoglobulin sequences of amplification can carry out extracorporeal treatment further, and such as, by external affinity maturation method, open WO97/29131 and PCT of such as PCT discloses those that describe in WO00/56772.
Monoclonal antibody is also by producing with object antigen immune non-human animal, and described non-human animal comprises some or all human immunoglobulin gene's seats.In preferred embodiments, non-human animal is XENOMOUSE transgenic mice, comprise human immunoglobulin gene's seat large fragment and in mouse antibodies generation defective engineered mouse species.See, such as, the people such as Green, NatureGenetics7:13-21 (1994) and United States Patent (USP) 5,916,771,5,939,598,5,985,615,5,998,209,6,075,181,6,091,001,6,114,598 and 6,130,364.Also be illustrated in WO91/10741 disclosed in 25 days July in 1991, WO94/02602 disclosed in the 3 days February in 1994, all WO96/34096 and WO96/33735 disclosed in the 31 days October in 1996, WO98/16654 disclosed in the 23 days April in 1998, WO98/24893 disclosed in the 11 days June in 1998, disclosed in the 12 days November in 1998 98/50433, WO99/45031 disclosed in 10 days September in 1999, WO99/53049 disclosed in the 21 days October in 1999, disclosed in the 24 days February in 2000 WO0009560 and disclosed in the 29 days June in 2000 WO00/037504.XENOMOUSE transgenic mice produces the adult sample people spectrum of human antibody, and produces antigen-specific people Mabs.By introduce megabasse size, the germline configuration YAC fragment of people's heavy chain gene seat and x light chain gene seat, XENOMOUSE transgenic mice comprises about 80% people's antibody repertoire.See people such as Mendez, NatureGenetics15:146-156 (1997), Green and JakobovitsJ.Exp.Med.188:483-495 (1998), the disclosure of described reference is hereby incorporated by.
In vitro method also may be used for preparing parental antibody, and wherein screening antibodies library has the antibody of required binding specificity with qualification.This kind of screening method about recombinant antibodies library is known in the art, and is included in the method described in following reference: such as, the people such as Ladner, U.S. Patent number 5,223,409; The people such as Kang, PCT publication number WO92/18619; The people such as Dower, PCT publication number WO91/17271; The people such as Winter, PCT publication number WO92/20791; The people such as Markland, PCT publication number WO92/15679; The people such as Breitling, PCT publication number WO93/01288; The people such as McCafferty, PCT publication number WO92/01047; The people such as Garrard, PCT publication number WO92/09690; The people such as Fuchs, (1991) Bio/Technology9:1370-1372; The people such as Hay, (1992) HumAntibodHybridomas3:81-85; The people such as Huse, (1989) Science246:1275-1281; The people such as McCafferty, Nature (1990) 348:552-554; The people such as Griffiths, (1993) EMBOJ12:725-734; The people such as Hawkins, (1992) JMolBiol226:889-896; The people such as Clackson, (1991) Nature352:624-628; The people such as Gram, (1992) PNAS89:3576-3580; The people such as Garrad, (1991) Bio/Technology9:1373-1377; The people such as Hoogenboom, (1991) NucAcidRes19:4133-4137; With people such as Barbas, (1991) PNAS88:7978-7982, US patent application discloses 20030186374 and PCT publication number WO97/29131, and the content of described each reference is incorporated herein by reference.
Parental antibody of the present invention also can use various phage display method known in the art to produce.In phage display method, functional antibodies structural domain is at phage particle displaying on surface, and described phage particle carries its polynucleotide sequence of coding.Especially, this kind of phage may be used for showing by the antigen-binding domains composed or combinatorial antibody library (such as, people or mouse) is expressed.The phage of expressing the antigen-binding domains of binding purposes antigen can carry out selecting or identifying with antigen, the antigen of such as applying marking or the antigen being combined by solid surface or pearl or catch.The phage used in these methods is generally the filobactivirus comprising fd and M13 binding domains, described binding domains is by the phage expression with Fab, Fv or the stable Fv antibody domain of disulphide, and described antibody domain and phage gene III or gene VIII protein matter are recombinated and merged.The example that may be used for the phage display method preparing antibody of the present invention to comprise disclosed in following reference those: the people such as Brinkman, J.Immunol.Methods182:41-50 (1995); The people such as Ames, J.Immunol.Methods184:177-186 (1995); The people such as Kettleborough, Eur.J.Immunol.24:952-958 (1994); The people such as Persic, Gene1879-18 (1997); The people such as Burton, AdvancesinImmunology57:191-280 (1994); PCT application PCT/GB91/01134; The open WO90/02809 of PCT; WO91/10737; WO92/01047; WO92/18619; WO93/11236; WO95/15982; WO95/20401; And U.S. Patent number 5,698,426; 5,223,409; 5,403,484; 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; Described reference separately entirety is incorporated herein by reference.
As described in reference above, after phage is selected, antibody coding region from phage can carry out being separated and for generation of complete antibody, and express in any required host, described complete antibody comprises people's antibody or Fab needed for any other, described host comprises mammalian cell, insect cell, vegetable cell, yeast and bacterium, such as, As described in detail below.Such as, also can adopt the technology of recombinant production Fab, Fab ' and F (ab ') 2 fragment, wherein use methods known in the art, such as disclosed in following reference those: the open WO92/22324 of PCT; The people such as Mullinax, BioTechniques12 (6): 864-869 (1992); With people such as Sawai, AJRI34:26-34 (1995); And the people such as Better, Science240:1041-1043 (1988) (described reference entirety is incorporated herein by reference).The example of technology that may be used for producing strand Fvs and antibody comprises those that describe in following reference: United States Patent (USP) 4,946,778 and 5,258,498; The people such as Huston, MethodsinEnzymology203:46-88 (1991); The people such as Shu, PNAS90:7995-7999 (1993); And the people such as Skerra, Science240:1038-1040 (1988).
As substituting by phage display screening recombinant antibodies library, the additive method for screening large combinatorial library known in the art can be applied to the qualification of parental antibody.As the PCT publication number WO98/31700 of Szostak and Roberts, and Roberts, and Szostak R.W., J.W. describe in (1997) Proc.Natl.Acad.Sci.USA94:12297-12302, the alternative expression system of a class is the wherein recombinant antibodies library expression system of expressing as RNA-protein fusions.In such systems, by carrying the In Vitro Translation of the synthesis mRNA of tetracycline-peptidyl receptor antibiotic on its 3 ' end, between the peptide or protein of mRNA and coding thereof, producing covalency merge.Therefore, based on the peptide of coding or the character of protein such as antibody or its part, the such as combination of antibody or its part and dual specific antigen, specific mRNA can from enrichment in the complex mixture of mRNAs (such as, combinatorial library).The encoding antibody reclaimed by this class library screening or the nucleotide sequence of its part, can by recombination method as above (such as, in mammalian host cell) express, and in addition can by the other screening circulation of mRNA-peptide fusions, or by implementing further affinity maturation for the additive method of the external affinity maturation of recombinant antibodies as mentioned above, sudden change is introduced in the initial sequence selected in described mRNA-peptide fusions.
In another approach, parental antibody also can use yeast display method known in the art to produce.In yeast display method, use genetic method to be bound by yeast cells wall to make antibody domain, and they are presented on yeast surface.Especially, this primary yeast may be used for showing by the antigen-binding domains composed or combinatorial antibody library (such as, people or mouse) is expressed.The example that may be used for the yeast display method preparing parental antibody is included in the people such as Wittrup, U.S. Patent number 6,699, disclosed in 658 those, described patent is incorporated herein by reference.
Above-mentioned antibody can carry out modifying producing CDR grafting with humanized parental antibody further.The parental antibody of CDR grafting comprises weight from people's antibody and light-chain variable sequence, and wherein replace with can the CDR sequence of murine antibody of binding purposes antigen in one or more CDR districts of VH and/or VL.Frame sequence from anyone antibody can serve as the template for CDR grafting.But the direct chain on this kind of framework is replaced and is usually caused losing with some of the binding affinity of antigen.People's antibody and initial murine antibody get over homology, make mouse CDR and people's framework combinations by less for the possibility introducing the distortion that can reduce avidity in CDR.Therefore, it is preferred for selecting to replace the variable framework of mouse with the variable framework of people with murine antibody variable region framework except CDR with the sequence iden of at least 65%.The sequence iden that people except CDR and mouse variable region have at least 70% is preferred.The sequence iden that people except CDR and mouse variable region have at least 75% is more preferably.The sequence iden that people except CDR and mouse variable region have at least 80% is most preferred.Method for the production of this kind of antibody be known in the art (see EP239,400; The open WO91/09967 of PCT; U.S. Patent number 5,225,539; 5,530,101; With 5,585,089), veneer or resurfacing (EP592,106; EP519,596; Padlan, MolecularImmunology28 (4/5): 489-498 (1991); The people such as Studnicka, ProteinEngineering7 (6): 805-814 (1994); The people such as Roguska, PNAS91:969-973 (1994)) and chain reorganization (U.S. Patent number 5,565,352).
Humanized antibody is the antibody molecule from the non-human species antibody combining required antigen, has from one or more complementarity-determining regions (CDR) of non-human species and the framework region from human normal immunoglobulin molecule.Known people Ig sequence is open on following website, such as,
Its separately entirety be incorporated herein by reference.As known in the art, the sequence of this kind of input may be used for reducing immunogenicity, or minimizing, enhancing or modification combination, avidity, association rate, dissociation rate, avidity, specificity, transformation period or any other suitable feature.
Framework residues in people framework region can replace with the corresponding residue from CDR donor antibody, to change, preferably improves antigen and combines.These frameworks replace to be identified by method well-known in the art, such as by the interaction modeling of CDR and Framework residues with qualification to antigen in conjunction with important Framework residues and gene comparision to identify rare Framework residues on location.(see, such as, the people such as Queen, U.S. Patent number 5,585,089; The people such as Riechmann, Nature332:323 (1988), described reference entirety is incorporated herein by reference).Three dimensional immunoglobulin model is normally obtainable and be that those skilled in the art are familiar with.To illustrate and the computer program showing the possible three-dimensional conformation structure of selected candidate immunoglobulin sequences sequence is obtainable.These inspections shown allow to analyze residue may act in candidate immunoglobulin sequences functional nucleotide sequence plays, and namely analyzing influence candidate immunoglobulin sequences is in conjunction with the residue of the ability of its antigen.By this way, FR residue can be selected and from total and list entries combination, thus make to reach required antibody characteristic and such as the avidity of one or more target antigens is increased.Generally speaking, CDR residue directly and most significantly relate to antigen combine impact.Antibody can use multiple technologies known in the art to carry out humanization, such as but not limited to describe in following reference those: the people such as Jones, Nature321:522 (1986); The people such as Verhoeyen, Science239:1534 (1988)), the people such as Sims, J.Immunol.151:2296 (1993); Chothia and Lesk, J.Mol.Biol.196:901 (1987), the people such as Carter, Proc.Natl.Acad.Sci.U.S.A.89:4285 (1992); The people such as Presta, J.Immunol.151:2623 (1993), Padlan, MolecularImmunology28 (4/5): 489-498 (1991); The people such as Studnicka, ProteinEngineering7 (6): 805-814 (1994); Roguska. people is waited, PNAS91:969-973 (1994); PCT open WO91/09967, PCT/:US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP229246, EP592,106; EP519,596, EP239,400, U.S. Patent number 5,565,332,5,723,323,5,976,862,5,824,514,5,817,483,5814476,5763192,5723323,5,766886,5,714,352,6,204,023,6,180,370,5,693,762,5,530,101,5,585,089,5,225,539; 4,816,567, described reference separately entirety is incorporated herein by reference, and comprises the reference wherein quoted.
Parental monoclonal antibody can be selected from can in conjunction with particular target and various monoclonal antibody well-known in the art.These include but not limited to, anti-TNF antibodies (U.S. Patent number 6,258,562), anti-IL-12 and/or anti-IL-12p40 antibody (U.S. Patent number 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-CD 40 L, anti-Id, anti-ICAM-1, anti-cxcl 13, anti-CD2, anti-EGFR, anti-TGF-beta 2, anti-CD62L, anti-FactVII, anti-Her2/neu, anti-Fgp, anti-CD11/18, anti-CD14, anti-ICAM-3, anti-CD80, anti-CD4, AntiCD3 McAb, anti-CD23, anti-β 2 integrin, anti alpha 4 β 7, anti-CD52, anti-HLADR, anti-CD22, anti-CD20, anti-MIF, anti-CD 64 (FcR), anti-tcr α β, anti-CD2, anti-HepB, anti-CA 125, anti-EpCAM, anti-gp120, anti-CMV, anti-gpIIbIIIa, anti-IgE, anti-CD25, anti-CD 33, anti-HLA, anti-VNR integrin, anti-IL-1 Alpha anti-il-i-beta, anti-IL-1 acceptor, anti-IL-2 acceptor, anti-IL-4, anti-IL-4 acceptor, anti-IL5, anti-IL-5 acceptor, anti-IL-6, anti-IL-8, anti-IL-9, anti-il-13, anti-il-13 acceptor, anti-IL-17 and anti-il-23 are (see PrestaLG.2005Selection, design, andengineeringoftherapeuticantibodiesJAllergyClinImmunol .116:731-6.
Parental monoclonal antibody can also be selected from approval in clinical trial, or the various therapeutic antibodies used in clinical application exploitation.This type of therapeutic antibodies includes but not limited to, the appropriate uncommon agate (RituxanIDEC/Genentech/Roche) of profit (see such as U.S. Patent number 5,736,137), the chimeric anti-CD20 antibodies of approval treatment non_hodgkin lymphoma, HuMax-CD20, anti-CD20 in being developed by Genmab at present, at U.S. Patent number 5, 500, the anti-C20 antibody described in 362, AME-133 (AppliedMolecularEvolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel) and PRO70769 (PCT/US2003/040426, name is called " ImmunoglobulinVariantsandUsesThereof "), Herceptin (trastuzumab) (Herceptin, Genentech) (see such as U.S. Patent number 5, 677, 171), the anti-Her2/neu antibody of humanization of approval treatment mammary cancer, handkerchief trastuzumab (pertuzumab) (rhuMab-2C4, Omnitarg), in being developed by Genentech at present, at U.S. Patent number 4,753, the anti-Her2 antibody described in 894, Cetuximab (cetuximab) (Erbitux, Imclone) (U.S. Patent number 4,943,533, PCTWO96/40210, for the chimeric anti-egfr antibodies in the clinical trial of kinds cancer, ABX-EGF (U.S. Patent number 6,235,883), in being developed by Abgenix-Immunex-Amgen at present, HuMax-EGFr (U.S.Ser.No.10/172,317), in being developed by Genmab at present, 425, EMD55900, EMD62000 and EMD72000 (MerckKGaA) (U.S. Patent number 5,558,864, the people such as Murthy, 1987, ArchBiochemBiophys.252 (2): 549-60, the people such as Rodeck, 1987, JCellBiochem.35 (4): 315-20, the people such as Kettleborough, 1991, ProteinEng.4 (7): 773-83), ICR62 (InstituteofCancerResearch) (PCTWO95/20045, the people such as Modjtahedi, 1993, J.CellBiophys.1993,22 (1-3): 129-46, the people such as Modjtahedi, 1993, BrJCancer.1993,67 (2): 247-53, the people such as Modjtahedi, 1996, BrJCancer, 73 (2): 228-35, the people such as Modjtahedi, 2003, IntJCancer, 105 (2): 273-80), TheraCIMhR3 (YMBiosciences, CanadaandCentrodeImmunologiaMolecular, Cuba (U.S. Patent number 5,891,996, U.S. Patent number 6,506,883, the people such as Mateo, 1997, Immunotechnology, 3 (1): 71-81), mAb-806 (LudwigInstitueforCancerResearch, MemorialSloan-Kettering) (people such as Jungbluth, 2003, ProcNatlAcadSciUSA.100 (2): 639-44), KSB-102 (KSBiomedix), MR1-1 (IVAX, NationalCancerInstitute) (PCTWO0162931A2), with SC100 (Scancell) (PCTWO01/88138), Ah coming organizes monoclonal antibody (alemutuzumab) (Campath, Millenium), and approval is at present used for the treatment of the Humanized monoclonal antibodies of B cell lymphocytic leukemia, the anti-cd 3 antibodies that Mo Luomona (muromonab)-CD3 (OrthocloneOKT3) is developed by OrthoBiotech/Johnson and Johnson, the anti-CD20 antibodies that ibritumomab tiuxetan (ibritumomabtiuxetan) (Zevalin) is developed by IDEC/ScheringAG, anti-CD 33 (p67 protein) antibody that lucky trastuzumab azoles rice star (gemtuzumabozogamicin) (Mylotarg) difficult to understand is developed by Celltech/Wyeth, the anti-LFA-3Fc fusions that A Laisaipu (alefacept) (Amevive) is developed by Biogen), ReoPro (ReoPro), developed by Centocor/Lilly, basiliximab (basiliximab) (Simulect) is developed by Novartis, palivizumab (palivizumab) (Synagis) is developed by Medimmune, the anti-TNF alpha antibodies that English husband monoclonal antibody (Remicade) is developed by Centocor, the anti-TNF alpha antibodies that adalimumab (adalimumab) (Humira) is developed by Abbott, Humicade, the anti-TNF alpha antibodies developed by Celltech, the anti-TNF alpha Fc fusions that etanercept (etanercept) (Enbrel) is developed by Immunex/Amgen, ABX-CBL, anti-CD147 antibody in being developed by Abgenix, ABX-IL8, anti-IL8 antibody in being developed by Abgenix, ABX-MA1, anti-MUC18 antibody in being developed by Abgenix, Pemtumomab (R1549, 90Y-muHMFG1), the anti-MUC1 developed by Antisoma, Therex (R1550), anti-MUC1 antibody in being developed by Antisoma, AngioMab (AS1405), in being developed by Antisoma, HuBC-1, in being developed by Antisoma, Thioplatin (AS1407) in being developed by Antisoma, Antegren (natalizumab (natalizumab)), anti alpha-4-β-1 (VLA-4) in being developed by Biogen and α-4-β-7 antibody, VLA-1mAb, anti-VLA-1 alpha 2 integrin antibodies in being developed by Biogen, LTBRmAb, anti-lymphotoyin beta receptor (LTBR) antibody in being developed by Biogen, CAT-152, anti-TGF-beta 2 antibody in being developed by CambridgeAntibodyTechnology, J695, anti-IL-12 antibody in being developed by CambridgeAntibodyTechnology and Abbott, CAT-192, anti-TGF beta 1 antibodies in being developed by CambridgeAntibodyTechnology and Genzyme, CAT-213, anti-CCL11 1 antibody during LymphoStat-B is developed by CambridgeAntibodyTechnology, by CambridgeAntibodyTechnology and HumanGenomeSciences, Inc. the anti-Blys antibody in exploitation, TRAIL-R1mAb, by CambridgeAntibodyTechnology and HumanGenomeSciences, Inc. anti-TRAIL-R 1 antibody in exploitation, Avastin rhuMAb-VEGF (bevacizumab), rhuMAb-VEGF), VEGF antibody in being developed by Genentech, anti-HER receptor family antibody in being developed by Genentech, anti-tissue factor (ATF), anti-tissue factor antibodies in being developed by Genentech, Xolair (Ao Mazuo monoclonal antibody (omalizumab)), anti-IgE antibodies in being developed by Genentech, Raptiva (efalizumab (efalizumab)), anti-CD11a antibody in being developed by Genentech and Xoma, MLN-02 antibody (being LDP-02 in the past), in being developed by Genentech and MilleniumPharmaceuticals, HuMaxCD4, anti-CD 4 antibodies in being developed by Genmab, HuMax-IL15, anti-IL15 antibody in being developed by Genmab and Amgen, HuMax-Inflam, in being developed by Genmab and Medarex, HuMax-Cancer, anti-heparanase (heparanase) I antibody in being developed by Genmab and Medarex and OxfordGcoSciences, HuMax-Lymphoma, in being developed by Genmab and Amgen, HuMax-TAC, in being developed by Genmab, IDEC-131, anti-CD40L antibodies in being developed by IDECPharmaceuticals, IDEC-151 (clenoliximab (clenoliximab)), anti-CD 4 antibodies in being developed by IDECPharmaceuticals, IDEC-114, Anti-CD80 McAb in being developed by IDECPharmaceuticals, IDEC-152, anti-CD23 in being developed by IDECPharmaceuticals, anti-scavenger cell shifter factor (MIF) antibody in being developed by IDECPharmaceuticals, BEC2, antiidiotypic antibody in being developed by Imclone, IMC-1C11, anti-KDR antibody in being developed by Imclone, DC101, anti-flk-1 antibody in being developed by Imclone, anti-VE cadherin antibody in being developed by Imclone, CEA-Cide (drawing shellfish pearl monoclonal antibody (labetuzumab)), anti-carcinoembryonic antigen (CEA) antibody in being developed by Immunomedics, LymphoCide (epratuzumab (epratuzumab)), anti-CD22 antibody in being developed by Immunomedics, AFP-Cide, in being developed by Immunomedics, MyelomaCide, in being developed by Immunomedics, LkoCide, in being developed by Immunomedics, ProstaCide, in being developed by Immunomedics, MDX-010, anti-CTLA 4 antibody in being developed by Medarex, MDX-060, AntiCD3 McAb 0 antibody in being developed by Medarex, MDX-070 in being developed by Medarex, MDX-018 in being developed by Medarex, Osidem (IDM-1), and the anti-Her2 antibody in being developed by Medarex and Immuno-DesignedMolecules, HuMax-CD4, anti-CD 4 antibodies in being developed by Medarex and Genmab, HuMax-IL15, anti-IL15 antibody in being developed by Medarex and Genmab, CNTO148, anti-TNF alpha antibodies in being developed by Medarex and Centocor/J & J, CNTO1275, anti-cytokine antibodies in being developed by Centocor/J & J, MOR101 and MOR102, adhesion molecule 1 (ICAM-1) (CD54) antibody between the anti-cell in being developed by MorphoSys, MOR201, anti-fibroblast growth factor receptor3 (FGFR-3) antibody in being developed by MorphoSys, Nuvion (tieing up western pearl monoclonal antibody (visilizumab)), anti-cd 3 antibodies in being developed by ProteinDesignLabs, HuZAF, anti-IFN-γ antibody in being developed by ProteinDesignLabs, anti-alpha 5 beta 1 integrin, in being developed by ProteinDesignLabs, anti-IL-12, in being developed by ProteinDesignLabs, ING-1, anti-Ep-CAM antibody in being developed by Xoma, Xolair (Ao Mazuo monoclonal antibody), the humanized anti-lgE antibodies developed by Genentech and Novartis and MLN01, anti-β 2 alpha 2 integrin antibodies in being developed by Xoma, in this paragraph, all above-cited reference are incorporated herein by reference all especially.
B. the structure of dual specific or polyspecific IgJR molecule
By the associated proteins comprising the first polypeptide chain using recombinant DNA technology (Figure 1B) to design dual specific or polyspecific, wherein said first polypeptide chain comprises VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3.Similarly, recombinant DNA technology design is used to comprise the second polypeptide chain and the 3rd polypeptide chain of VL1-CL or VH2-CH1 respectively.Optionally, can comprise X1 between CH1 and VL2, X1 is amino acid or polypeptide (joint).Joint sequence can be single amino acids or peptide sequence.Preferably, joint sequence is selected from EPKSCD; AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA (G 4s) 41sAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVTLFPP; AKTTPP; AKTTPPSVTPLAP; AKTTAP; AKTTAPSVYPLAP; ASTKGP; ASTKGPSVFPLAP, GGGGSGGGGSGGGGS; GENKVEYAPALMALS; GPAKELTPLKEAKVS and GHEAAAVMQVQYPAS.The selection of joint sequence is based on the crystal structure analysis of several Fab molecule.
Other joint sequences can comprise any sequence of the CL/CH1 structural domain of any length, but are not all residues of CL/CH1 structural domain; Front 5-12 amino-acid residue of such as CL/CH1 structural domain; Light chain joint can from C κ or C λ; And heavy chain joint can derive from the CH1 of any isotype, comprise C γ 1, C γ 2, C γ 3, C γ 4, C α 1, C α 2, C δ, C ε and C μ.Joint sequence can also derive from other protein such as Ig sample protein, (such as, TCR, FcR, KIR); Based on the sequence (such as, G4S repeats) of G/S; The sequence that hinge area is derivative; With other native sequences from other protein.
Fc district can be native sequences Fc district, or variant Fc district.In a preferred embodiment, Fc district Shi Ren Fc district.In another preferred embodiment, Fc district comprises the Fc district from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.
In conjunction with detailed description of the specificity IgJR molecule of particular target and preparation method thereof, can provide in Examples below part.
C. dual specific or the protein-bonded generation of polyspecific IgJR
Associated proteins of the present invention can be produced by any one in many technology known in the art.Such as, encoded packets is transfected in host cell by standard technique containing one or more expression vectors of the various polypeptide chains of VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3, VL1-CL and VH2-CH1.The various forms expection of term " transfection " comprises the extensively various technology being generally used for being introduced by foreign DNA in protokaryon or eukaryotic host cell, such as, and electroporation, calcium phosphate precipitation, deae dextran transfection etc.Although dual specific of the present invention or polyspecific IgJR protein may be expressed in protokaryon or eukaryotic host cell, but be preferred at eukaryotic expression dual specific or polyspecific IgJR protein, and be most preferred in mammalian host cell, because this kind of eukaryotic cell (and particularly mammalian cell) more than prokaryotic cell prokaryocyte may assemble and secrete dual specific or the polyspecific IgJR protein of correct folding and immunologic competence.
Preferred mammal host cell for expressing recombinant antibodies of the present invention comprises Chinese hamster ovary (Chinese hamster ovary celI) and (is included in Urlaub and Chasin, (1980) describe in Proc.Natl.Acad.Sci.USA77:4216-4220, the dhfr-CHO cell used together with DHFR selective marker, such as, as described in R.J.Kaufman and P.A.Sharp (1982) Mol.Biol.159:601-621), NS0 myeloma cell, COS cell, HEK293-F cell and SP2 cell.When encoding bispecific or polyspecific IgJR recombinant expression of proteins carrier are introduced in mammalian host cell, dual specific or polyspecific IgJR protein are by cultivating enough time section to produce by host cell, express in host cell to allow dual specific or polyspecific IgJR protein, or more preferably, dual specific or polyspecific IgJR protein secreting are in the substratum of wherein host cell growth.Dual specific or polyspecific IgJR protein can use Standard protein purification method to reclaim from substratum.
In the vote for recombinant expressed dual specific of the present invention or polyspecific IgJR protein, the transfection that the recombinant expression vector that encoded packets contains the polypeptide chain of VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3, VL1-CL and VH2-CH1 is mediated by 293fectin (Invitrogen) is introduced in HEK293-F cell.Cultivate selected transformant host cell to allow to express VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3, VL1-CL and VH2-CH1, and from substratum, reclaim complete dual specific or polyspecific IgJR protein.Use standard molecular biological technique to prepare recombinant expression vector, transfection host cell, selection transformant, cultivate host cell and reclaim dual specific or polyspecific IgJR protein from substratum.Further the invention provides synthesis dual specific of the present invention or polyspecific IgJR method of protein, it by cultivating host cell of the present invention until dual specific of the present invention or polyspecific IgJR protein are synthesized realization in suitable substratum.The method may further include and be separated dual specific or polyspecific IgJR protein from substratum.
Dual specific or the protein-bonded key character of polyspecific IgJR-Ig are that it can be produced in the mode similar to conventional antibody and purifying.Dual specific or the protein-bonded production of polyspecific IgJR-Ig cause having the homogeneous of required dual specific activity, single primary product.Produce " dual specific ", " polyspecific " and " multispecific multivalent " total length other previous described methods protein-bonded and do not cause single primary product, but the non-activity causing assembling, monospecific, polyspecific, multivalence, total length associated proteins and have the combination of different binding site the protein-bonded mixture of multivalence total length cell in or the production of secreting.Such as, based on by Miller and Presta, (design that the open WO2001/077342 (A1) of PCT describes, 16 kinds that there is heavy and light chain may be combined.Therefore the protein of 6.25% is only had may to be in required activity form.Use Standard chromatographic techniques that the protein separation of the protein of complete activity form and non-activity and amount of activated form is still remained to be confirmed, described Standard chromatographic techniques generally uses in extensive preparation.
II. the IgJR-Ig associated proteins of derivatize:
An embodiment provides the associated proteins of mark, wherein associated proteins of the present invention another kind of functional molecular (such as, another kind of peptide or protein) derivatize or connection.Such as, the associated proteins of mark of the present invention can derive by making functional be connected (by chemical coupling, genetic fusion, Non-covalent binding or other modes) of other molecular entities of associated proteins of the present invention and one or more, other molecular entities described such as another kind of antibody (such as, bi-specific antibody or double antibody), can detection reagent, cytotoxic agent, pharmaceutical agents and/or the protein or peptide that associated proteins and another kind of molecule (such as streptavidin core region or polyhistidine mark) combine can be mediated.
Associated proteins of the present invention can by the useful of derivatize detection reagent can comprise fluorescent chemicals.Exemplary fluorescence detection reagent can comprise fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalic sulfonic chloride, phycoerythrin etc.Associated proteins also can with detecting enzyme derivatize, such as alkaline phosphatase, horseradish peroxidase, notatin etc.When associated proteins is with when can detect enzyme derivatize, it is by adding enzyme for the production of can the other reagent of detection reaction product detect.Such as, when can detection reagent horseradish peroxidase exist time, add hydrogen peroxide and diaminobenzidine and cause detectable colored reaction product.Associated proteins also can use vitamin H derivatize, and is detected by the indirect inspection that avidin or streptavidin combine.
Another embodiment of the invention provides crystallized binding protein and comprises this crystal-like preparation and composition.In one embodiment, crystallized binding protein has the Half-life in vivo longer than protein-bonded soluble counterpart.In another embodiment, associated proteins retains biologic activity after crystallization.
Crystallized binding protein of the present invention can according to known in the art and disclosed in WO02072636 method produce, described patent is incorporated herein by reference.
Another embodiment of the invention provides glycosylated associated proteins, and wherein antibody or its antigen-binding portion thereof comprise one or more carbohydrate residue.Newborn body internal protein is produced can experience the further processing being called posttranslational modification.Especially, sugar (glycosyl) residue can add by enzymatic, and this process is called glycosylation.The protein with covalently bound oligosaccharide side chains obtained is called as glycosylated protein or glycoprotein.Antibody is the glycoprotein in Fc structural domain and variable domain with one or more carbohydrate residue.The effector function of carbohydrate residue in Fc structural domain to Fc structural domain plays an important role, the antigen of antagonist combines or there is MIN effect (R.Jefferis the transformation period, Biotechnol.Prog.21 (2005), 11-16 page).By contrast, the antigen-binding activity of the glycosylation possibility antagonist of variable domain has effect.Glycosylation in variable domain may have negative effect by antagonist binding affinity, may be due to sterically hindered (Co, M.S. people is waited, Mol.Immunol. (1993) 30:1361-1367), or cause increasing (Wallick to the avidity of antigen, S.C. people is waited, Exp.Med. (1988) 168:1099-1109; The people such as Wright, A., EMBOJ. (1991) 10:27172723).
One aspect of the present invention relates to generation glycosylation site mutation body, and wherein protein-bonded O-or N-glycosylation site suddenlys change.Those skilled in the art can use the well-known technology of standard to produce this type of mutant.Retaining biologic activity but having the glycosylation site mutation body of binding activities increased or reduce is another object of the present invention.
In another one embodiment, the glycosylation of antibody of the present invention or its antigen-binding portion thereof is modified.Such as, aglycosylated antibodies (i.e. this antibody deficiency glycosylation) can be prepared.Glycosylation can change, such as to increase the avidity of antibody to antigen.This kind of carbohydrate modification can have been come by the one or more glycosylation sites such as changed in antibody sequence.Such as, the one or more amino-acid substitutions causing one or more variable regions glycosylation site to be eliminated can be prepared, thus eliminate the glycosylation on that site.This kind not glycosyafated can increase the avidity of antibody to antigen.This kind of method is at the open WO2003016466A2 of PCT, and U.S. Patent number 5,714,350 and 6,350, and describe in further detail in 861, described patent separately entirety is incorporated herein by reference.
In addition or alternately, the associated proteins that the present invention that can prepare the type of glycosylation with change modifies, such as, have the not enough antibody of fucosylation of the fucosido residue of reduction, or have the antibody of decile GlcNAc structure of increase.The glycosylation pattern that this kind changes has shown the ADCC ability increasing antibody.This kind of carbohydrate modification can come by such as expressing antibody in the host cell of glycosylation machine with change.The cell with the glycosylation machine of change is described in this area, and can be used as the host cell of expressing recombinant antibodies of the present invention wherein, thus produces the glycosylated antibody with change.See, such as, the people such as Shields, R.L. (2002) J.Biol.Chem.277:26733-26740; The people such as Umana (1999) Nat.Biotech.17:176-1, and european patent number: EP1,176,195; The open WO03/035835 of PCT; WO99/5434280, described reference separately entirety is incorporated herein by reference.
Protein Glycosylation Overview depends on the aminoacid sequence of target protein matter, and the host cell of marking protein wherein.Different biology can produce different glycosylases (such as, glycosyltransferase and Glycosylase), and has different available substrates (nucleotide sugar).Due to this type of factor, Protein Glycosylation Overview pattern and glycosyl residue composition can depend on the host system of expressing specified protein wherein and different.Glycosyl residue useful in the present invention can include but not limited to, glucose, semi-lactosi, seminose, Fucose, n-acetylglucosamine and sialic acid.Preferably glycosylated associated proteins comprises glycosyl residue, thus makes glycosylation pattern be people.
Different Protein Glycosylation Overviews can cause different protein characteristics, and this is well known by persons skilled in the art.Such as, compare with the same protein expressed in mammalian cell such as Chinese hamster ovary celI system, produce in microorganism host such as yeast and utilize effect of the glycosylated treatment protein of yeast entogenous approach may be reduce.This kind of glycoprotein also can be immunogenic in people, and shows the transformation period reduced in vivo after application.People and the special receptor in other animals can identify specific glycosyl residue and promote that protein is removed fast from blood flow.Other adverse effects can comprise protein folding, solubility, susceptibility to proteolytic enzyme, transport, transhipment, compartmentation, secretion, by the change in other protein or factor identification, antigenicity or allergenicity.Therefore, practitioner may prefer the treatment protein with specific glycosylation composition and pattern, such as, be equal to or be at least similar to that glycosylation composition produced and pattern in the species specificity cell of people's cell or expection animal subject.
Be different from that glycosylated protein of host cell to express to have been come with expressing heterologous glycosylase by genetic modification host cell.Use technology known in the art, practitioner can produce the display glycosylated antibody of human protein or its antigen-binding portion thereof.Such as, yeast strain has carried out the glycosylase that genetic modification exists to express non-natural, thus makes glycosylated protein (glycoprotein) display of producing in these yeast strains be equal to the zooblast Protein Glycosylation Overview that particularly people's cell is that (U.S. Patent application 20040018590 and 20020137134 and PCT application WO2005100584A2).
Except associated proteins, the invention still further relates to antiidiotype (anti-Id) antibody special to this kind of associated proteins of the present invention.Anti-Id antibody is the antibody identifying unique determinants, and described unique determinants is general relevant to the antigen-binding portion thereof of another kind of antibody.Anti-Id can by preparing with associated proteins or Qi Han CDR district immune animal.The animal of immunity will identify, and reply the idiotypic determinant of immune antibody and produce anti-Id antibody.Anti-Id antibody also can be used as " immunogen " with induce immune response in another animal, thus produces so-called anti-Id antibody.
In addition, it will be appreciated by those skilled in the art that, target protein matter can use library of host cells to express, and described host cell carries out genetic engineering modified to express various glycosylase, thus makes member's host cell in library produce the target protein matter with variant glycosylation pattern.Practitioner can select subsequently and be separated the target protein matter with specific new glycosylation pattern.Preferably, the biological property that the protein display with regioselective new glycosylation pattern improves or changes.
III. the purposes of dual specific or polyspecific IgJR associated proteins (antibody)
The known ability that it is combined with 2 kinds or more antigens, associated proteins of the present invention may be used for detectable antigens (such as, in biological sample, such as serum or blood plasma), routine immunization is wherein used to measure, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry.Dual specific or polyspecific IgJR associated proteins detectable substance directly or indirectly mark, to promote the detection of combination or unconjugated antibody.Suitable detectable substance comprises various enzyme, prothetic group, fluorescent material, luminescent material and radio active material.The example of suitable enzymes comprises horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes or acetylcholinesterase; The example of suitable prosthetic group complexes comprises streptavidin/vitamin H and avidin/biotin; The example of suitable fluorescent materials comprises Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine base amine (dichlorotriazinylamine) fluorescein, dansyl chloride or phycoerythrin; The example of luminescent material comprises luminol,3-aminophthalic acid cyclic hydrazide; And the example of suitable radioactive material comprises 3h, 14c, 35s, 90y, 99tc, 111in, 125i, 131i, 177lu, 166ho or 153sm.
Associated proteins of the present invention preferably can in vitro and in vivo in and antigenic activity.Therefore, this kind of dual specific or polyspecific IgJR associated proteins can be such as, in the cell culture comprising antigen, for suppressing antigenic activity in people experimenter or in other mammalian subject of antigen with associated proteins of the present invention and its cross reaction.In another embodiment, the invention provides the method for reducing the antigenic activity in experimenter, it is harmful disease or illness that described experimenter suffers from wherein antigenic activity.Associated proteins of the present invention can be applied to people experimenter and be used for the treatment of object.
As used herein, term " wherein antigenic activity is harmful illness " expection comprises disease or other illnesss, and the existence wherein suffering from antigen in the experimenter of this illness has shown or suspected the physiopathology being responsible for illness or the factor promoting condition worse.Therefore, wherein antigenic activity is harmful illness is that wherein antigenic activity reduces the illness that expection alleviates condition symptoms and/or progress.This kind of illness can such as be confirmed by the increase increase of antigen concentration (in such as experimenter's serum, blood plasma, the synovia etc.) of antigen concentration in the experimenter's biological fluid suffering from illness.The non-limitative example of the illness can treated with associated proteins of the present invention comprises hereafter and about those illnesss discussed in the pharmaceutical composition part of antibody of the present invention.
Dual specific of the present invention or polyspecific IgJR associated proteins can in conjunction with a kind of antigen or plurality of antigens.This kind of antigen includes but not limited to, the target listed in following database, and described database is incorporated herein by reference.These target databases comprise following list:
Dual specific or polyspecific IgJR associated proteins are useful as therapeutical agent to block 2 kinds of different targets to strengthen effect/security and/or to increase patient's coverage simultaneously.This kind of target can comprise solubility target (IL-13 and TNF) and cell surface receptor target (VEGF-A and EGFR).It can also be used for changing nyctitropic cytotoxicity for cancer therapy between inducing tumor cell and T cell (HER2 and CD3), or change nyctitropic cytotoxicity for autoimmunization/transplanting between autoreaction cell or effector cell, or change nyctitropic cytotoxicity between any target cell and effector cell to eliminate the pathogenic cell in any given disease.
In addition, when being designed to 2 kinds of different epi-positions in target same receptor when dual specific IgJR associated proteins, it may be used for trigger receptor cluster and activation.This may have benefit in preparation excitability and the anti-GPCR therapeutical agent of Antagonism.In this case, dual specific IgJR associated proteins may be used for 2 kinds of different epi-positions on a kind of cell of target for cluster/intracellular signaling (2 kinds of cell surface molecules) or intracellular signaling (for a kind of molecule).Similarly, dual specific IgJR associated proteins can be designed by 2 kinds of different epi-positions (or 2 of identical epi-position copies) of target CTLA-4 extracellular domain, triggers CTLA-4 and connects and negative signal, thus cause immunne response to be lowered.CTLA-4 is the target confirmed clinically of the many immunological disorder of being used for the treatment of property process.CTLA-4/B7 interacts by weakening the T cell propagation negative regulator T cell activation after cell cycle progress, IL-2 production and activation, and CTLA-4 (CD152) linking can lower T cell activation and the induction of Promote immunity tolerance.But the strategy being weakened T cell activation by agonistic antibody linking CTLA-4 is still unsuccessful, because CTLA-4 activation needs to connect.As (Stamper2001Nature410:608) that confirmed by crystal structure analysis, the interaction of molecules of CTLA-4/B7 is " crooked slide fastener " arrangement.But CTLA-4 binding reagents available does not at present have one to have connection character, comprise anti-CTLA-4 monoclonal antibody.There are several trials addressed this problem.In one case, the membrane-bound single-chain antibody of cell is produced, and significantly suppresses the allo-rojection (Hwang2002JI169:633) in mouse.When separating, the single-chain antibody that the artificial APC surface for CTLA-4 connects is produced and shows weakens t cell response (Griffin2000JI164:4433).In 2 kinds of situations, CTLA-4 connects by making membrane-bound antibody closely be confined to come in manual system.Although these experiments provide by triggering the Proof of Concept (proof-of-concept) of CTLA-4 negative signal conduction for immune down regulation, the reagent used in these reports is not suitable for therepic use.For this reason, CTLA-4 connects and can realize by using dual specific IgJR associated proteins molecule, 2 kinds of different epi-positions (or 2 of identical epi-position copies) of described dual specific IgJR associated proteins target CTLA-4 extracellular domain.Ultimate principle is that the distance of leap IgG2 binding site is not for effectively connecting CTLA-4 (between 2 CTLA-4 homodimers) about too greatly.But the distance much shorter on dual specific IgJR associated proteins between 2 combining sites (1 arm), also in scope, thus allows CTLA-4 suitably to connect.
Similarly, dual specific IgJR associated proteins can 2 different members (such as IL-12R α and β) of target cell surface receptors mixture.In addition, dual specific IgJR associated proteins can target CR1 and soluble protein/pathogenic agent, to drive the quick removing of target soluble protein/pathogenic agent.
In addition, dual specific of the present invention or polyspecific IgJR associated proteins may be used for tissue-specific delivery, and (target tissue mark and disease medium are for strengthening local PK, thus reach higher effect and/or lower toxicity), comprise Intracellular delivery (target internalization acceptor and intracellular molecules), be delivered to (target TfR and CNS disease medium are used for through hemato encephalic barrier) in brain.Dual specific or polyspecific IgJR associated proteins also can serve as carrier proteins, to be combined by antigen delivery to specific position via with the non-neutralizing epitope of that antigen, and also increase the transformation period of antigen.In addition, dual specific or polyspecific IgJR associated proteins can be designed to and the medical apparatus physical connection in patients with implantation, or these medical apparatus of target are (see Burke, SandraE.; Kuntz, RichardE.; Schwartz, LewisB., Zotarolimus (ABT-578) elutingstents.AdvancedDrugDeliveryReviews (2006), 58 (3), 437-446; Surfacecoatingsforbiologicalactivationandfunctionalizati onofmedicaldevices, Hildebrand, H.F.; Blanchemain, N.; Mayer, G.; Chai, F.; Lefebvre, M.; Boschin, F., SurfaceandCoatingsTechnology (2006), 200 (22-23), 6318-6324; Drug/devicecombinationsforlocaldrugtherapiesandinfection prophylaxis, Wu, Peng; Grainger, DavidW., Biomaterials (2006), 27 (11), 2450-2467; Mediationofthecytokinenetworkintheimplantationoforthoped icdevices., Marques, A.P.; Hunt, J.A.; Reis, RuiL., BiodegradableSystemsinTissueEngineeringandRegenerativeMe dicine (2005), 377-397).In brief, can Promotive union and recovery healthy tissues function by suitable cell type guiding medical implant position.Alternately, the suppression of by the IgJR with device coupling or target device, device being implanted to the rear medium (including but not limited to cytokine) discharged is also provided.Such as, support uses for many years in Interventional Cardiology, to remove occluded artery and to be improved to myocardium blood flow.But conventional bare mental stents is known causes restenosis (narrowing again of therapeutic area medium sized artery) in some patient, and can cause blood clot.Recently, the support of AntiCD3 McAb 4 antibody bag quilt is described, and it reduces restenosis by catching the endothelial progenitor cells (EPC) circulated in blood everywhere and stop blood clot to occur.Endotheliocyte is the cell of blood vessel lining, allows blood smooth flow.EPCs and support crust adhere to and form smooth layer, described smooth layer not only Promotive union also stops restenosis and blood clot, described restenosis and clot used relevant complication people such as (, 2005JAmCollCardiol.45 (10): 1574-9) Aoji to support before being.Except improving and needing the result of the patient of support, also exist and need involving of the patient of cardiovascular by-pass operation.Such as, the needs using and be used for by-pass operation transplanting from patient's leg or brachial artery will be eliminated with the prosthese vascular canal (prosthetic aortic) of anti-EPC antibody bag quilt.This will reduce surgical operation and anesthesia duration, and this will reduce coronary artery surgery surgical death successively.Dual specific or polyspecific IgJR associated proteins design by this way, thus make it and cell surface marker (such as CD34) and protein (or the epi-position of any kind, include but not limited to lipid and polysaccharide) combine, to promote recruiting cells on the device that described cell surface marker and protein have been coated on implantation.Generally speaking this kind of method also can be applied to other medical implants.Alternately, dual specific or polyspecific IgJR associated proteins can be coated on medical apparatus, and implanting and (maybe may need other fresh dual specific or polyspecific IgJR any other needs protein-bonded discharge all IgJR from device after, comprise the dual specific or the protein-bonded aging and sex change of polyspecific IgJR that have loaded), device can by using fresh dual specific or polyspecific IgJR associated proteins loads again to entire patient, wherein dual specific or polyspecific IgJR associated proteins are designed to by one group of binding site and object target (cytokine, cell surface marker (such as CD34) etc.) combine, and (comprise protein by another group with the target be coated on device, the epi-position of any kind, include but not limited to lipid, polysaccharide and polymkeric substance) combine.This technology has the advantage of the implant availability of expanding packet quilt.
Dual specific or the purposes of polyspecific IgJR associated proteins in various disease
Dual specific of the present invention or polyspecific IgJR associated proteins also can be used as treatment molecule to treat various disease.This type of dual specific or polyspecific IgJR associated proteins can in conjunction with one or more targets relating to specified disease.The example of this kind of target in various disease is hereafter describing.
1. human autoimmune and inflammatory response
Numerous protein generally involves in autoimmunization and inflammatory response, comprises C5, CCL1 (I-309), CCL11 (CCL11), 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/ CCL11-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11 (I-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1 (CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1 (endothelial mononuclear cell 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, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z, CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11, CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2, BMPR1A, BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1, IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11, IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA, LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF, TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21, TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2 and RNF110 (ZNF144).In one aspect, providing can in conjunction with the specificity IgJR associated proteins of one or more targets listed above.
2. asthma
The feature of atopic asthma is to there is eosinophilia, goblet cell metaplasia, epithelial cell change, airway hyperreactivity (AHR) and Th2 and Th1 cytokine-expressing and serum IgE level rising.Generally accepting airway inflammation is at present the key factor becoming Pathogenesis of Asthma basis, the complexity that the medium relating to inflammatory cell and secretion thereof comprises cytokine and chemokine influences each other, and described inflammatory cell is T cell, B cell, eosinophilic granulocyte, mastocyte and scavenger cell such as.Reflunomide is at present for the most important anti-inflammatory treatment of asthma, but their mechanism of action is nonspecific, and in adolescent patient colony, particularly there is security care.Therefore prove that the treatment of developing more specificity and target has reasonable ground.Increasing evidence shows that the IL-13 in mouse simulates many asthma features, comprise AHR, Polyblennia and airway fibrosis, (the people such as Finotto of not depending on eosinophilic inflammation, InternationalImmunology (2005), 17 (8), 993-1007; The people such as Padilla, JournalofImmunology (2005), 174 (12), 8097-8105).
Imply that IL-13 has keying action in causing the pathology relevant with asthma to reply.Exploitation anti-il-13 mab treatment is breathtaking novel method to reduce the effect of IL-13 in lung, and it is provided as sizable hope that asthma is newly treated.But other media of difference immunization route also relate to Pathogenesis of Asthma, and except IL-13, block the treatment interests that these media may provide other.This kind of target to including but not limited to, IL-13 and pro-inflammatory cytokine, such as tumor necrosis factor alpha (TNF-α).TNF-α can amplify the inflammatory response in asthma and may associate (the people such as McDonnell with disease severity, ProgressinRespiratoryResearch (2001), 31 (NewDrugsforAsthma, AllergyandCOPD), 247-250.).This hint blocks IL-13 and TNF-α may have beneficial effect, particularly in serious airway disorders.In preferred embodiments, dual specific of the present invention or polyspecific IgJR associated proteins are used for the treatment of asthma in conjunction with target IL-13 and TNF α.
The asthma mouse model of the animal model that wherein inflammation and AHR can be evaluated such as OVA-induction is known in the art and may be used for determining the ability of various dual specific or polyspecific IgJR associated proteins treatment asthma.Animal model for studying asthma is open in following reference: the people such as Coffman, JournalofExperimentalMedicine (2005), 201 (12), 1875-1879; The people such as Lloyd, AdvancesinImmunology (2001), 77,263-295; The people such as Boyce, JournalofExperimentalMedicine (2005), 201 (12), 1869-1873; With people such as Snibson, JournaloftheBritishSocietyforAllergyandClinicalImmunolog y (2005), 35 (2), 146-52.Except the general safety assessment that these targets are right, specificity test about immunosuppression degree in the selection that best target is right can be have a reasonable ground with helpful (see, the people such as Luster, Toxicology (1994), 92 (1-3), 229-43; The people such as Descotes, Developmentsinbiologicalstandardization (1992), 7799-102; The people such as Hart, JournalofAllergyandClinicalImmunology (2001), 108 (2), 250-257).
Based on above-disclosed ultimate principle and use same evaluation model about effect and security, can determine dual specific or polyspecific IgJR associated proteins can in conjunction with and be used for the treatment of other targets pair of asthma.Preferably this kind of target is to including but not limited to, IL-13 and IL-1 β, because IL-1 β also relates to the inflammatory response in asthma; IL-13 and the cytokine and the chemokine that relate to inflammation, such as IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; And IL-13 and ADAM8.Present invention also offers can in conjunction with being selected from the following dual specific relating to one or more targets of asthma or polyspecific IgJR associated proteins: CSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNA1, IFNB1, IFNG, histamine and Histamine Receptors, IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL18, IL19, KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL18R1, TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CCL24, CX3CL1, CXCL1, CXCL2, CXCL3, XCL1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STAT6, TBX21, TGFB1, TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR and chitinase.
3. rheumatoid arthritis
The feature of systemic disease rheumatoid arthritis (RA) is the chronic inflammatory reaction in synovium of joint, and corrodes with cartilage degeneration and nearly articular bone.The many pro-inflammatory cytokines comprising TNF, cytokine and somatomedin are expressed in diseased joints.Showing to RA mouse model systemic administration anti-TNF antibodies or sTNFR fusion rotein is anti-inflammatory and joint protection.Wherein with English husband monoclonal antibody (the anti-TNF monoclonal antibody (mAB) of the mosaic type) (HarrimanG that intravenously is used, HarperLK, SchaibleTF.1999Summaryofclinicaltrialsinrheumatoidarthri tisusinginfliximab, ananti-TNFalphatreatment.AnnRheumDis58Suppl1:I61-4.) clinical study of the TNF activity of RA patient is blocked, provide following evidence: TNF regulates IL-6, IL-8, MCP-1 and VEGF produces, immunity and inflammatory cell recruitment are to intraarticular, blood vessel occur and matrix metallopeptidase 1 and 3 blood level minimizing.In rheumatoid arthritis, the better understanding of pathways of inflammation has caused the qualification of the other treatment target relating to rheumatoid arthritis.Treatment likely is interleukin-6 antagonist (MRA), CTLA4Ig (abatacept such as, the people such as GenoveseMc, 2005Abataceptforrheumatoidarthritisrefractorytotumornecr osisfactoralphainhibition.NEnglJMed.353:1114-23.), and anti-B cell treatment (the appropriate uncommon agate of profit, OkamotoH, KamataniN.2004Rituximabforrheumatoidarthritis.NEnglJMed. 351:1909), test in randomized controlled trial in the past year.Other cytokines are identified and to be presented in animal model be favourable, comprise Interleukin-15, Interleukin-17 and interleukin-18, and the clinical trial of these reagent are underway at present.The bispecific antibody therapy combining anti-TNF and another kind of medium has very large potential in enhancing clinical efficacy and/or patient's coverage.Such as, blocking TNF and VEGF and can eradicate inflammation and blood vessel generation potentially, there is the physiopathology all relating to RA in described inflammation and blood vessel.Also contemplate and block with specific dual specific or polyspecific IgJR associated proteins other targets pair relating to RA, include but not limited to, TNF and IL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1 β; TNF and MIF; TNF and IL-17; And TNF and IL-15.Except the general safety assessment that these targets are right, about immunosuppression degree specificity test in the selection that best target is right can be have reasonable ground with helpful (see people such as Luster, Toxicology (1994), 92 (1-3), 229-43; The people such as Descotes, Developmentsinbiologicalstandardization (1992), 7799-102; The people such as Hart, JournalofAllergyandClinicalImmunology (2001), 108 (2), 250-257).Whether IgJRIg molecule can be used for treatment rheumatoid arthritis can use preclinical animal RA model, and such as Collagen-Induced Arthritis mouse model is assessed.Other useful models are also (see BrandDD., CompMed. (2005) 55 (2): 114-22) well-known in the art.
4.SLE
The immunopathology mark of SLE is the activation of polyclone B cell, and this causes hyperglobulinemia, autoantibody to produce and immunocomplex is formed.Basic exception is seemingly due to popularity T cell dysregulation, and T cell can not suppress taboo B cell clone.In addition, the interaction of B and T cell is by several cytokine such as IL-10, and costimulatory molecules such as CD40 and CD40L, B7 and CD28 and CTLA-4 are promoted, described cytokine and the initial second signal of costimulatory molecules.These interactions engulf removing together with immunocomplex and the impaired of apoptosis material, the tissue injury's perpetuity making immunne response Yu produce.Following target may relate to SLE and can be used for the treatment of intervention for dual specific or the protein-bonded method of polyspecific IgJR potentially: the treatment of B cell target: CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2, IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGS1, SLA2, CD81, IFNB1, IL10, TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3, MS4A1, ST6GAL1, CD1C, CHST10, HLA-A, HLA-DRA and NT5E., costimulatory signal: CTLA4 or B7.1/B7.2, B cell survival suppresses: BlyS, BAFF, complement inactivation: C5, cytokine regulates: key principle is the clean biological answer-reply in any tissue is proinflammatory or the result (see people such as SfiakisPP, 2005CurrOpinRheumatol17:550-7) that balances between anti-inflammatory cytokines local horizontal.SLE is regarded as the disease having the serum IL-4 of documentary evidence, the Th-2 of IL-6, IL-10 rising drives.Also contemplating can in conjunction with dual specific or the polyspecific IgJR associated proteins being selected from one or more following targets: IL-4, IL-6, IL-10, IFN-a and TNF-a.The therapeutic efficiency that target combination discussed above will strengthen about SLE, described therapeutic efficiency can carry out testing (see PengSL (2004) MethodsMolMed. in many lupus preclinical models; 102:227-72).
5. multiple sclerosis
Multiple sclerosis (MS) is complex man's autoimmune type disease with main unknown etiology.Immunological destruction throughout neural myelin basic protein (MBP) is the key pathological of multiple sclerosis.MS has complicated pathological disease, and described pathology relate to via CD4 +and CD8 +response in the infiltration of T cell and central nervous system.Cytokine, active nitrogen kind and the expression of costimulatory molecules in CNS are described all in MS.Main consideration is the amynologic mechanism facilitating autoimmunization to develop.Especially, antigen presentation, cytokine and white corpuscle interact and help to balance/regulate the regulatory T cells of other T cell such as Th1 and Th2 cell, are the essential scope about therapeutic target qualification.
IL-12 is produced by APC and the pro-inflammatory cytokine promoting Th1 effector cell to break up.Produce in the animal that IL-12 affects in the developing disease stove of patient suffering from MS and by EAE.Previous display interference IL-12 approach effectively stops the EAE in rodent, and in common marmoset, has advantageous effect with IL-12p40 in the EAE model of myelin induction in using anti-IL-12mAb body interior.
TWEAK is TNF family member, constitutive expression in the central nervous system (CNS), depends on that cell type has proinflammatory, propagation or cells apoptosis.Its acceptor Fn14 is expressed in CNS by endotheliocyte, reactive astrocytes and neurone.TWEAK and Fn14mRNA expresses to be increased in spinal cord in experimental autoimmune encephalomyelitis (EAE) period.When mouse is at the laggard row relax of initial period, the EAE that anti-TWEAK antibody treatment myelin oligodendroglia glycoprotein (MOG) is induced in C57BL/6 mouse causes disease severity to reduce and leukocyte infiltration.
One aspect of the present invention relates to can in conjunction with being selected from following one or more, preferably the dual specific of 2 kinds of targets or polyspecific IgJR associated proteins: IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200, IFN γ, GM-CSF, FGF, C5, CD52 and CCR2.Preferred embodiment comprises the anti-IL-12/TWEAKIgJR of dual specific and treats favourable therapeutical agent as to MS.Several animal models for assessment of dual specific or polyspecific IgJR associated proteins treatment MS availability be known in the art (see people such as SteinmanL, (2005) TrendsImmunol.26 (11): 565-71; LublinFD. people is waited, (1985) SpringerSeminImmunopathol.8 (3): 197-208; The people such as GenainCP, (1997) JMolMed.75 (3): 187-97; The people such as TuohyVK, (1999) JExpMed.189 (7): 1033-42; The people such as OwensT, (1995) NeurolClin.13 (1): 51-73; With ' people such as tHartBA, (2005) JImmunol175 (7): 4761-8.Except the general safety assessment that these targets are right, about immunosuppression degree specificity test in the selection that best target is right can be have reasonable ground with helpful (see people such as Luster, Toxicology (1994), 92 (1-3), 229-43; The people such as Descotes, Developmentsinbiologicalstandardization (1992), 7799-102; JonesR.2000Rovelizumab (ICOSCorp) .IDrugs.3 (4): 442-6).
6. sepsis
The physiopathology of sepsis is initial by the outer membrane fraction of gram-negative biological (lipopolysaccharides [LPS], lipid A, intracellular toxin) and gram-positive organism (lipoteichoicacid, peptidoglycan).These outer membrane fraction can with the CD14 receptors bind on onthe surface of monocytes.Due to the toll sample acceptor described in the recent period, signal passes to cell subsequently, thus causes the final production of proinflammatory cytokine tumor necrosin & (TNF-α) and interleukin 1 (IL-I).Inundatory inflammation and immunne response are the essential characteristics of septic shock, and play an important role in the tissue injury of being induced by sepsis, multiple organ failure and dead pathogeny.Cytokine, particularly tumour necrosis factor (TNF) and interleukin-(IL)-1, having shown is the critical mediator of septic shock.These cytokines have direct toxic action to tissue; They also activate Phospholipase A2.These and other effect causes platelet activation factor concentration to increase, accelerating oxidation nitrogen synthase activity, promotes the tissue infiltration via neutrophilic granulocyte and the activity promoting neutrophilic granulocyte.
Sepsis and septic shock treatment are still clinical problem, and use the recent expection test for the biologically conditioning agent (i.e. anti-TNF, anti-MIF) of inflammatory response only to show the clinical benefit of appropriateness.In the recent period, interest has turned to the treatment for reversing the adjoint immunosuppression time period.Research in laboratory animal and critical afflicted patient has confirmed that this immunosuppression, anergy and organ system dysfunction are facilitated in lymphoid organ and some parenchymal tissue's apoptosis increase.During sepsis syndrome, lymphocytic apoptosis can by IL-2 do not exist or by glucocorticosteroid, granzyme or so-called ' death ' cytokine: tumor necrosis factor alpha or FasL discharge and trigger.Apoptosis is in progress via the autoactivation of kytoplasm and/or plastosome Caspase, and described autoactivation can by the impact of the short of Bcl-2 family or anti-apoptotic members.In laboratory animal, use the process of apoptosis inhibitor not only can stop the apoptosis of lymphoidocyte; It also improves result.Although to a great extent owing to using with tissue target to relevant technical difficulty to it, use the clinical trial of anti-apoptotic agent still remote, lymphocytic apoptosis suppresses representative about the attracting therapeutic target of septic patient.Similarly, the dual specificity reagent of target inflammatory mediator and apoptotic mediators may have additional benefit.One aspect of the present invention relates to can in conjunction with being selected from following one or more targets relating to sepsis, the preferably IgJR molecule of 2 kinds of targets: 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, PTAFR, TLR2, TLR4, GPR44, HMOX1, Midkine, IRAK1, NFKB2, SERPINA1, SERPINE1 and TREM1.This kind of dual specific or polyspecific IgJR associated proteins can carry out assessing (see people such as BurasJA for effect of sepsis in preclinical animal model known in the art, (2005) NatRevDrugDiscov.4 (10): the people such as 854-65 and CalandraT, (2000) NatMed.6 (2): 164-70).
7. neurological disorder
7.1. neurodegenerative disease
The normally age-dependent disease of chronic neurodegenerative disease, is characterized in that the progressive loss (Neuronal cell death, demyelination) of neuronal function, and mobility is lost and the loss of memory.Become chronic neurodegenerative disease (such as, Alzheimer) the emerging knowledge of mechanism on basis shows complicated nosetiology, and many factors has been identified as promoting its development and progress, the such as age, glycemic (glycemic) state, amyloid produces and multimerization, the Advanced Glycation end product (AGE) combined with its acceptor RAGE (acceptor about AGE) gathers, brain oxidative stress increases, cerebral blood flow (CBF) reduces, neuroinflamation comprises inflammatory cytokine and chemokine release, neuron dysfunction and Activated Microglia.Therefore the complexity that these chronic neurodegenerative diseases represent between various kinds of cell type and medium interacts.Therapeutic strategy about this type of disease is limited, and main composition non-specific anti-inflammatory agent (such as reflunomide, COX inhibitor) or reagent block inflammatory processes, to stop neuron loss and/or synaptic function.These treatments can not stop progression of disease.Recent research implies the treatment of target more, such as, for the antibody of solubility A-b peptide (comprising A-b oligomerization form), not only helps to stop progression of disease also to help to maintain memory.These preliminary observations hint target exceedes the specific treatment of a kind of disease medium (such as A-b and pro-inflammatory cytokine such as TNF), can provide than use the single disease mechanisms of target (such as independent solubility A-b) observe the even better therapeutic efficiency to chronic neurodegenerative disease (see people such as C.E.Shepherd, NeurobiolAging.2005Oct24; NelsonRB., CurrPharmDes.2005; 11:3335; WilliamL.Klein.; NeurochemInt.2002; 41:345; The people such as MichelleCJanelsins, JNeuroinflammation.2005; 2:23; SolomanB., CurrAlzheimerRes.2004; 1:149; The people such as IgorKlyubin, NatMed.2005; 11:556-61; The people such as ArancioO, EMBOJournal (2004) 1-10; The people such as BornemannKD, AmJPathol.2001; 158:63; The people such as DeaneR, NatMed.2003; 9:907-13; With people such as EliezerMasliah, Neuron.2005; 46:857).
Dual specific of the present invention or polyspecific IgJR associated proteins can in conjunction with one or more targets relating to chronic neurodegenerative disease such as alzheimer's disease.This kind of target includes but not limited to, relate to the pathogenetic any medium of AD, solubility or cell surface, such as AGE (S100A, Amphiphilic proteins (amphoterin)), pro-inflammatory cytokine (such as IL-1), chemokine (such as MCP1), suppress the molecule (such as Nogo, RGMA) of neurotization, strengthen the molecule (neurotrophin) of neurite outgrowth.Dual specific or the protein-bonded effect of polyspecific IgJR can be verified in preclinical animal model, such as overexpression amyloid precursor protein or RAGE and the transgenic mice of development Alzheimer sample symptom.In addition, dual specific or polyspecific IgJR associated proteins can be built and in animal model test efficacy, and best treatment dual specific or polyspecific IgJR associated proteins can be selected for testing in people patient.Dual specific or polyspecific IgJR associated proteins also may be used for treating other neurodegenerative diseases such as Parkinson's disease.Alpha-synapse nucleoprotein (Synuclein) relates to Parkinson pathology.The dual specific of target alpha-synapse nucleoprotein and inflammatory mediator such as TNF, IL-1, MCP-1 or polyspecific IgJR associated proteins can prove for parkinsonian effective treatment, and be expected in the present invention.
7.2. neuron regeneration and Spinal injury
Although the knowledge of pathology increases, Spinal injury (SCI) is still destructive situation and representative feature is the medical indications that high medical science needs.Most of Spinal injury is dampened or compression, and primary injury is secondary lesion mechanism (inflammatory mediator such as cytokine and chemokine) usually subsequently, described secondary lesion mechanism makes initial damage worsen and cause for lesion area significantly to be amplified, sometimes more than 10 times.These primary in SCI and Secondary cases mechanism be very similar to by other modes such as in those in wind-induced brain injury.There is no gratifying treatment, and high dosage bolus injection methyl meticortelone (MP) is unique treatment used in damage latter 8 hours narrow time windows.But this treatment is only expected and is stoped secondary lesion and do not produce any significant functional rehabilitation.It is subject to fierce criticism in default of clear and definite effect and serious detrimental action, and the immunosuppression and gross tissue pathology muscle of described detrimental action as having Subsequent infection change.Do not ratify the other drug of stimulation of endogenous regeneration potential, biotechnological formulation or small molecules, but treatment principle likely and drug candidates show effect in SCI animal model in recent years.Functional rehabilitation in people SCI lacks to a great extent by suppressing the factor of neurite outgrowth to cause, the described factor at damaging part place, in scar tissue, in myelin and on damage relevant cell.This kind of factor is Nogo protein Nogo protein NogoA, OMgp and MAG, RGMA, the relevant CSPG of scar (chondroitin sulfate proteoglycan) and the supressor (sometimes joining albumen for brain signal albumen and liver) about reactive astrocytes.But, at damaging part place, not only find growth-inhibiting molecule, also found neurite outgrowth stimulating factor, as neurotrophin, ln, L1 etc.Neurite outgrowth suppresses and growth promotes this overall possible explanation of molecule, block monofactor such as NogoA or RGMA and cause significant functional rehabilitation in rodent SCI model, promote because the minimizing of inhibitory effect can make balance transfer to growth from growth-inhibiting.But, grow recovery that Inhibitory molecules observes and not exclusively for blocking single spinous process.In order to reach more fast and recover more significantly, blocking-up 2 kinds of spinous processes may be needed to grow Inhibitory molecules such as Nogo and RGMA, or block nerves is dashed forward to grow Inhibitory molecules and strengthen spinous process and is grown the function strengthening molecule such as Nogo and neurotrophin, or block nerves prominent grow Inhibitory molecules such as Nogo and pro-inflammatory molecular such as TNF (see people such as McGeeAW, TrendsNeurosci.2003; 26:193; The people such as MarcoDomeniconi, JNeurolSci.2005; 233:43; The people such as MilanMakwanal, FEBSJ.2005; 272:2628; BarryJ.Dickson, Science.2002; 298:1959; The people such as FeliciaYuHsuanTeng, JNeurosciRes.2005; 79:273; The people such as TaraKarnezis, NatureNeuroscience2004; 7,736; The people such as GangXu, J.Neurochem.2004; 91; 1018).
In one aspect, providing can in conjunction with the right dual specific of following target or polyspecific IgJR associated proteins, and described target is to such as NgR and RGMA; NogoA and RGMA; MAG and RGMA; OMGp and RGMA; RGMA and RGMB; CSPGs and RGMA; Aggrecan, Midkine, neurocan, versican, phosphoglycan, Te38 and TNF-a; With the A β globulomer-specific antibody promoting the Antibody Combination that dendron and aixs cylinder are sprouted.Dendron pathology are very early stage AD symptom, and the exsule length of known NOGOA constrained tree.This kind of ab type and any SCI candidate (myelin protein matter) Ab can combine by people.Other dual specifics or polyspecific IgJR associated proteins target can comprise any combination of NgR-p75, NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo, Lingo-Troy, Lingo-p75, MAG or Omgp.In addition, target can also comprise any medium relating to spinous process and suppress, solubility or cell surface, such as Nogo, Ompg, MAG, RGMA, brain signal albumen, liver join albumen, solubility A-b, pro-inflammatory cytokine (such as IL-1), chemokine (such as MIP1a), suppress the molecule of neurotization.The anti-RGMA of anti-nogo/ or similar dual specific or the protein-bonded effect of polyspecific IgJR can be verified in the preclinical animal model of Spinal injury.In addition, these dual specifics or polyspecific IgJR associated proteins can be built and in animal model test efficacy, and best treatment dual specific or polyspecific IgJR associated proteins can be selected for testing in people patient.In addition, the dual specific IgJR associated proteins of 2 different ligands binding sites on the single acceptor of target can be built, described acceptor such as in conjunction with the Nogo acceptor of 3 kinds of parts Nogo, Ompg and MAG, and in conjunction with the RAGE of A-b and S100A.In addition, spinous process grows inhibitor such as nogo and nogo acceptor, also stops in amynologic disease is as multiple sclerosis in neurotization and works.The suppression of nogo-nogo acceptor interaction has shown the recovery strengthened in multiple sclerosis animal model.Therefore, blocking immunity medium such as cytokine can growing dual specific or the polyspecific IgJR associated proteins of inhibitor molecules such as nogo or RGM function as IL-12 and spinous process, can providing than blocking independent immunity or spinous process grows the faster and larger effect of inhibitor molecules.
8. oncological disorders
Mab treatment has been revealed as the important treatment modality of cancer, and (vonMehrenM, waits people, 2003Monoclonalantibodytherapyforcancer.AnnuRevMed.; 54:343-69).Antibody can by following performance antitumor action: cell death inducing, change nyctitropic cytotoxicity, interference ligand-receptor interaction or stop the protein expression to knurl phenotype key.In addition, antibody can target tumor microenvironment component, the formation of interference important feature such as tumour associated vasculature.Antibody can also its part of target be the acceptor of somatomedin, such as EGF-R ELISA.Therefore antibody suppress the native ligand of stimulate cell growth to be combined with the tumour cell of target.Alternately, antibody can induced anti-idiotype network, the cytotoxicity of complement-mediated or antibody dependent cellular cytotoxicity (ADCC).Treat with monospecific and compare, the use of the bi-specific antibody of the tumour medium that target 2 kinds separates may produce additional benefit.Can in conjunction with following target to the dual specific for the treatment of oncology diseases or polyspecific IgJR associated proteins being also expection: 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.The combination of other targets comprises one or more members of EGF/erb-2/erb-3 family.Dual specific or polyspecific IgJR associated proteins other targets (one or more) combinable of relating to oncology diseases include but not limited to, are selected from following those: CD52, CD20, CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGF1, IGF2, IL12A, IL1A, IL1B, IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2, EGF, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1, IGF1R, IL2, VEGF, BCL2, CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1, IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3, IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL, TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1, IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3, NR2F6, NR4A3, ESR1, ESR2, NR0B1, NR0B2, NR1D2, NR1H2, NR1H4, NR1I2, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2, NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1, BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10, FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2, IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, INSL3, INSL4, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9, MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3, CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18, CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164, COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1, CDH12, CLDN3, CLN3, CYB5, CYC1, DAB2IP, DES, DNCL1, ELAC2, ENO2, ENO3, FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A, IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID, PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1, TPM2, TRPC6, ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR, LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB1, VEGF, VEGFC, ANGPTL3, BAI1, COL4A3, IL8, LAMA5, NRP1, NRP2, STAB1, ANGPTL4, PECAM1, PF4, PROK2, SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2, EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B (p27Kip1), CDKN2A (p16INK4a), COL6A1, CTNNB1 (b-catenin), CTSB (cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3, GSN (gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130), ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67), NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA), PTEN, SERPINB5 (mammary gland silk presses down albumen), SERPINE1 (PAI-1), TGFA, THBS1 (THBS1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A (topoisomerase I ia), TP53, AZGP1 (zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cip1), CLDN7 (close protein-7), CLU (CLU), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin), GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b4 integrin), KLF5 (GCBoxBP), KRT19 (Keratin 19), KRTHB6 (hair specificity II type Keratin sulfate), MACMARCKS, MT3 (metallothionein(MT)-III), MUC1 (Saliva Orthana), PTGS2 (COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipotropins B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2, THBS4 and TNFAIP2 (B94).
IV. pharmaceutical composition
Present invention also offers the pharmaceutical composition comprising associated proteins of the present invention and pharmaceutically acceptable carrier.Comprise protein-bonded pharmaceutical composition of the present invention for using in following, but be not limited to following aspect, Illnesses Diagnoses, detection or monitoring, the prevention of illness or its one or more symptoms, treatment, management or improvement and/or research.In a particular embodiment, composition comprises one or more associated proteins of the present invention.In another embodiment, pharmaceutical composition comprises one or more associated proteins of the present invention, and for sanatory one or more prevention or therapeutical agents except associated proteins of the present invention.Preferably, prevention or the known prevention in illness or its one or more symptoms of therapeutical agent, treatment, management or useful in improving, or use wherein or use just wherein at present.According to these embodiments, composition can comprise carrier, thinner or vehicle further.
Associated proteins of the present invention can mix in the pharmaceutical composition that is suitable for using to experimenter.Usually, pharmaceutical composition comprises associated proteins of the present invention and pharmaceutically acceptable carrier.As used herein, " pharmaceutically acceptable carrier " comprise any of physiological compatible and all solvents, dispersion medium, coating, antibacterial agent and anti-mycotic agent, etc. blend absorption delay agent etc.The example of pharmaceutically acceptable carrier comprise following one or more: water, salt solution, phosphate-buffered saline, glucose, glycerine, ethanol etc., and combination.In many cases, it will be preferred for comprising isotonic agent in the composition, and described isotonic agent such as sugar, polyhydric alcohols are as mannitol, Sorbitol Powder or sodium-chlor.Pharmaceutically acceptable carrier can comprise a small amount of auxiliary substance further, such as wetting agent or emulsifying agent, sanitas or damping fluid, and described auxiliary substance strengthens storage life or the effect of antibody or antibody moiety.
Various delivery system is known, and may be used for the combination of using one or more antibody of the present invention or one or more antibody of the present invention and preventive or therapeutical agent, for preventing, managing, treat or improve illness or its one or more symptoms, such as by encapsulated in liposome, particulate, microcapsule, the reconstitution cell that antibody or antibody fragment can be expressed, receptor-mediated endocytosis (see, such as, Wu and Wu, J.Biol.Chem.262:4429-4432 (1987)), as the nucleic acid construct etc. of retrovirus or other carrier parts.The method using prevention of the present invention or therapeutical agent includes but not limited to, parenteral administration (such as, intracutaneous, intramuscular, intraperitoneal, intravenously and subcutaneous), epidural is used, use in knurl and mucosal administration (such as, nose in and peroral route).In addition, lung can be used to use, such as, utilize sucker or atomizer and the preparation containing propellant.See, such as, U.S. Patent number 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 number WO92/19244, WO97/32572, WO97/44013, WO98/31346 and WO99/66903, described patent separately entirety is incorporated herein by reference.In one embodiment, associated proteins of the present invention, combination treatment or composition of the present invention use lung Drug delivery technology (Alkermes, Inc., Cambridge, Mass.) to use.In a particular embodiment, in prevention of the present invention or therapeutical agent intramuscular, intravenously, knurl, in per os, nose, lung or subcutaneous administration.Prevention or therapeutical agent can be used by any conventional route, such as by infusion or bolus injection, by absorbing via epithelium or mucocutaneous lining (such as, oral mucosa, rectum and intestinal mucosa etc.), and can use together with other biological promoting agent.Using can be whole body or local.
In a particular embodiment, the region that the topical application of prevention of the present invention or therapeutical agent is treated at needs may be needed; This can by such as but not limited to local infusion, injection or come by implant, described implant is porous or non-porous material, comprise film and matrix, such as silicon rubber (sialastic) film, polymkeric substance, fibre substrate (such as) or collagen stroma.In one embodiment, one or more antibody of the antagonist antagonises of significant quantity are locally applied to the affected area of experimenter, to prevent, to treat, to manage and/or to improve illness or its symptom.In another embodiment, one or more antibody of the present invention of significant quantity, with one or more therapies except associated proteins of the present invention of significant quantity (such as, one or more prevention or therapeutical agents) combination, be locally applied to the affected area of experimenter, to prevent, to treat, to manage and/or to improve illness or its one or more symptoms.
In another embodiment, prevention or therapeutical agent can be sent in controlled release or sustained release system.In one embodiment, pump can be used to reach controlled release or sustained release is (see Langer, the same; Sefton, 1987, CRCCrit.Ref.Biomed.Eng.14:20; The people such as Buchwald, 1980, Surgery88:507; The people such as Saudek, 1989, N.Engl.J.Med.321:574).In another embodiment, polymeric material may be used for reaching the controlled release of therapy of the present invention or sustained release (see such as, MedicalApplicationsofControlledRelease, Langer and Wise (eds.), CRCPres., BocaRaton, Fla. (1974); ControlledDrugBioavailability, DrugProductDesignandPerformance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, 1983, J., Macromol.Sci.Rev.Macromol.Chem.23:61; Also see people such as Levy, 1985, Science228:190; The people such as During, 1989, Ann.Neurol.25:351; The people such as Howard, 1989, J.Neurosurg.71:105); U.S. Patent number 5,679,377; U.S. Patent number 5,916,597; U.S. Patent number 5,912,015; U.S. Patent number 5,989,463; U.S. Patent number 5,128,326; PCT publication number WO99/15154; With PCT publication number WO99/20253.The example of the polymkeric substance used in extended release preparation includes but not limited to, polymethyl acrylic acid 2-hydroxyl ethyl ester, polymethylmethacrylate, polyacrylic acid, vinyl-vinyl acetate copolymer, polymethyl acrylic acid, polyglycolide (PLG), polyanhydride, PVP, polyvinyl alcohol, polyacrylamide, polyoxyethylene glycol, polylactide (PLA), RH502H (PLGA) and poe.In preferred embodiments, the polymkeric substance used in extended release preparation is inertia, containing can leaching impurity, storage-stable, aseptic and biodegradable.In another one embodiment, controlled release or sustained release system can be placed close to prevention or therapeutic target, thus only need body dose part (see, such as, Goodson, inMedicalApplicationsofControlledRelease, the same, 2nd volume, 115-138 page (1984)).
Controlled release system is discussed in the summary of Langer (1990, Science249:1527-1533).Any technology well known by persons skilled in the art may be used to produce the extended release preparation comprising one or more therapeutical agents of the present invention.See, such as, U.S. Patent number 4, 526, 938, the open WO91/05548 of PCT, the open WO96/20698 of PCT, the people such as Ning, 1996, " IntratumoralRadioimmunotheraphyofaHumanColonCancerXenogr aftUsingaSustained-ReleaseGel ", Radiotherapy & Oncology39:179-189, the people such as Song, 1995, " AntibodyMediatedLungTargetingofLong-CirculatingEmulsions ", PDAJournalofPharmaceuticalScience & Technology50:372-397, the people such as Cleek, 1997, " BiodegradablePolymericCarriersforabFGFAntibodyforCardiov ascularApplication ", the people such as Pro.Int ' l.Symp.Control.Rel.Bioact.Mater.24:853-854 and Lam, 1997, " MicroencapsulationofRecombinantHumanizedMonoclonalAntibo dyforLocalDelivery ", Proc.Int ' l.Symp.ControlRel.Bioact.Mater.24:759-760, described reference separately entirety is incorporated herein by reference.
In a particular embodiment, when composition of the present invention be coding prevention or the nucleic acid of therapeutical agent time, nucleic acid can use the expression of preventive or the therapeutical agent promoting it to encode in body, and this is by following realization, be configured to the part of suitable nucleic acid expression vector and used, thus make it enter in cell, such as utilize retroviral vector (see U.S. Patent number 4,980,286), or direct injection, or use microparticle bombardment (such as, particle gun; Biolistic, Dupont), or with lipid or cell surface receptor or transfection agents bag quilt, or to be connected with the known homeobox sample peptide entering core and to use (see, such as, the people such as Joliot, 1991, Proc.Natl.Acad.Sci.USA88:1864-1868).Alternately, nucleic acid can be introduced in cell and to be integrated in host cell DNA for expressing by homologous recombination.
Pharmaceutical composition of the present invention is formulated as with its expection route of administration compatible.The example of route of administration includes but not limited to, parenteral, such as, (such as, sucks), through skin (such as, locally), across mucous membrane and rectal administration in intravenously, intracutaneous, subcutaneous, per os, nose.In a particular embodiment, composition is formulated as pharmaceutical composition according to conventional procedure, and described pharmaceutical composition is suitable for intravenously, subcutaneous, intramuscular, per os, nose interior or be locally applied to the mankind.Usually, the composition used for intravenously is the solution in sterile isotonic water-based damping fluid.If desired, composition can also comprise solubilizing agent and local anesthetic such as lignocaine (lignocamne), to alleviate the pain at injection site place.
If composition of the present invention is by topical application, so composition can be formulated as ointment, emulsifiable paste, transdermal skin patches, lotion, gel, shampoo, sprays, aerosol, solution, emulsion form or other forms well known to the skilled person.See, such as, Remington ' sPharmaceuticalSciencesandIntroductiontoPharmaceuticalDo sageForms, the 19th edition, MackPub.Co., Easton, Pa. (1995).For non-sprayable topical formulations, general use comprises the carrier compatible with topical application or one or more vehicle, and the viscosity with the dynamic viscosity being preferably greater than water is extremely semi-solid or solid form.Suitable preparation includes but not limited to, solution, suspension, emulsion, emulsifiable paste, ointment, powder, liniment, ointment etc., carry out sterilizing if desired or be mixed for affecting various character with auxiliary agent (such as sanitas, stablizer, wetting agent, damping fluid or salt), such as, osmotic pressure.Other suitable topical formulations comprise sprayable aerosol preparations, wherein activeconstituents, preferably combines with solid or liquid inert carrier, with pressurized volatile (such as, gaseous propellant, such as freonll-11) pack in the mixture or be packaged in squeeze bottle.Wetting Agent for Printing Inks or wetting agent also can be added in pharmaceutical composition and formulation if desired.The example of this kind of other composition is well-known in the art.
If method of the present invention comprises the intranasal administration of composition, so composition can be formulated as aerosol form, sprays, mist or drop form.Especially, with the aerosol spray form conventional delivery presented from compression wrap or atomizer, suitable propelling agent (such as Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas) can be used for prevention used according to the invention or therapeutical agent.When pressurised aerosol, dose unit can by providing valve to determine, to send the amount of metering.Capsule and the cartridge (being made up of such as gelatin) of the powdered mixture of inclusion compound and suitable powder base such as lactose or starch can be prepared, for using in sucker or insufflator.If method of the present invention comprises oral administration, so composition can be formulated as the oral form such as tablet, capsule, flat jelly, granular capsule, solution, suspension.Tablet or capsule can by the acceptable vehicle preparations of ordinary method pharmacy, and described excipients is as tackiness agent (such as, pregelatinized corn starch, polyvinylpyrrolidone or Vltra tears); Filling agent (such as, lactose, Microcrystalline Cellulose or secondary calcium phosphate); Lubricant (such as, Magnesium Stearate, talcum or silica); Disintegrating agent (such as, yam starch or sodium starch glycollate); Or wetting agent (such as, sodium lauryl sulphate).Tablet can carry out bag quilt by method well-known in the art.Liquid preparation for oral administration can take following form, but is not limited to following form, solution, syrup or suspension, or they can be rendered as drying products, for before use with water or other suitable vector constructions.This type of liquid preparation can by the acceptable additive preparation of ordinary method pharmacy, and described additive is suspension agent (such as, sorbitol syrup, derivatived cellulose or hydrogenated edible fats) such as; Emulsifying agent (such as, Yelkin TTS or gum arabic); Nonaqueous carrier (such as, Prunus amygdalus oil, grease, ethanol or fractionated vegetable oil); With sanitas (such as, methyl p-hydroxybenzoate or propylparaben or Sorbic Acid).Time suitable, preparation can also comprise buffering salt, seasonings, tinting material and sweeting agent.Preparation for oral administration can suitably be prepared, for slow releasing, controlled release or sustained release one or more prevention or therapeutical agents.
Method of the present invention can comprise to be used with the lung of the composition of aerosol agent preparation, such as, utilize sucker or atomizer.See, such as, U.S. Patent number 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; With PCT publication number WO92/19244, WO97/32572, WO97/44013, WO98/31346 and WO99/66903, described patent separately entirety is incorporated herein by reference.In a particular embodiment, associated proteins of the present invention, combination treatment and/or composition of the present invention use lung Drug delivery technology (Alkermes, Inc., Cambridge, Mass) to use.
Method of the present invention can comprise preparation using for the composition by injection (such as by bolus injection or continuous infusion) parenteral administration.Preparation for injecting can be presented with unit dosage (such as, in ampoule or multi-dose containers) together with the sanitas added.Composition can take this kind of form, as the suspension in oiliness or aqueous carrier, solution or emulsion, and can comprise that reagent preparation such as suspends, stable and/or dispersion agent.Alternately, activeconstituents can be used for building with suitable carrier (such as, aseptic without pyrogeneous substance water) before use for powder type.
Method of the present invention can comprise using of the composition being formulated as accumulation preparation in addition.This type of prolonged action preparation can by implanting (such as subcutaneous or intramuscular) or being used by intramuscularly.Therefore, such as, composition with suitable polymerization or hydrophobic material (such as, the emulsion as in acceptable oil) or ion exchange resin preparation, or can be formulated as sparing soluble derivative (such as, as slightly soluble salt).
Method of the present invention comprises using of the composition being formulated as neutrality or salt form.Pharmacologically acceptable salts comprises those that formed by negatively charged ion, such as derive from those of hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartrate etc., and those formation by positively charged ion, such as derive from sodium hydroxide, potassium, ammonium, calcium, iron, Isopropylamine, triethylamine, 2-ethylaminoethyl alcohol, Histidine, those of PROCAINE HCL, PHARMA GRADE etc.
Usually, the composition of composition separates or mixes to be provided with unit dosage, such as, as the dry lyophilize powder in sealed vessel or anhydrous enriching agent, and described sealed vessel such as ampoule or wafer, the amount of its instruction promoting agent.When method of application is infusion, composition can distribute by the infusion bottle comprising other water of sterile pharmaceutical grade or salt solution.When method of application is injection, sterile water for injection or salt solution ampoule can be provided, thus composition can be mixed before administration.
Especially, present invention also offers one or more prevention or therapeutical agent or pharmaceutical compositions of the present invention of being packaged in sealed vessel, described sealed vessel such as ampoule or wafer, the amount of its instruction promoting agent.In one embodiment, one or more prevention or therapeutical agent or pharmaceutical compositions of the present invention, there is provided as the dry sterile cryo dried powder in sealed vessel or anhydrous enriching agent, and (such as, with water or salt solution) to suitable concn can be rebuild use for experimenter.Preferably, one or more prevention or therapeutical agent or pharmaceutical compositions of the present invention, there is provided as the dry sterile cryo dried powder in sealed vessel, its unitary dose is at least 5mg, more preferably at least 10mg, at least 15mg, at least 25mg, at least 35mg, at least 45mg, at least 50mg, at least 75mg or at least 100mg.Cryodesiccated prevention of the present invention or therapeutical agent or pharmaceutical composition should be stored in 2 DEG C-8 DEG C in its former container, and prevention of the present invention or therapeutical agent or pharmaceutical composition should be used in 1 week after reconstruction, preferably in 5 days, in 72 hours, in 48 hours, in 24 hours, in 12 hours, in 6 hours, in 5 hours, in 3 hours or in 1 hour.In alternative embodiment, one or more prevention or therapeutical agent or pharmaceutical compositions of the present invention, provide in liquid form in the amount of indicator and the sealed vessel of concentration.Preferably, the composition used of liquid form provides in sealed vessel, its amount is at least 0.25mg/ml, more preferably at least 0.5mg/ml, at least 1mg/ml, at least 2.5mg/ml, at least 5mg/ml, at least 8mg/ml, at least 10mg/ml, at least 15mg/kg, at least 25mg/ml, at least 50mg/ml, at least 75mg/ml or at least 100mg/ml.Liquid form should be stored in 2 DEG C-8 DEG C in its former container.
Associated proteins of the present invention can mix and be applicable in the pharmaceutical composition of parenteral administration.Preferably, antibody or antibody moiety will be prepared as and comprise the protein-bonded Injectable solution of 0.1-250mg/ml.Injectable solution can be made up of the liquid in flint or amber vial, ampoule or prefilled syringe or lyophilize formulation.Damping fluid can be L-Histidine (1-50mM), best 5-10mM, pH5.0-7.0 (Optimal pH 6.0).Other suitable damping fluids include but not limited to, sodium succinate, Trisodium Citrate, sodium phosphate or potassiumphosphate.Sodium-chlor may be used for the toxicity of the solution modifying concentration 0-300mM (for the best 150mM of liquid dosage form).Can cryoprotectant be comprised for lyophilize formulation, be mainly 0-10% sucrose (best 0.5-1.0%).Other suitable cryoprotectants comprise trehalose and lactose.Can swelling agent be comprised for lyophilize formulation, be mainly 1-10% mannitol (best 2-4%).Stablizer can use in liquid and lyophilize formulation, is mainly 1-50mML-methionine(Met) (best 5-10mM).Other suitable swelling agents comprise glycine, arginine, can comprise as 0-0.05% polysorbate80 (best 0.005-0.01%).Other tensio-active agent includes but not limited to, polysorbate20 and BRIJ tensio-active agent.Be prepared as Injectable solution for parenteral administration, comprise protein-bonded pharmaceutical composition of the present invention and can comprise reagent as adjuvant further, such as absorb for increasing treatment protein (such as, antibody) or those of dispersion.Useful especially adjuvant is Unidasa, such as (recombinant human Unidasa).In Injectable solution, add Unidasa improve parenteral administration, the people's bioavailability particularly after subcutaneous administration.It also allows larger injection site volume (being namely greater than 1ml) and the MIN injection site reaction incidence (WO2004078140 and US2006104968 see being incorporated herein by reference) with less pain and discomfort
Composition of the present invention can be various ways.These comprise such as, liquid, semisolid and solid dosage, such as liquor (such as, injectable and can infusion solution), dispersion or suspension, tablet, pill, powder, liposome and suppository.Preferred form depends on expection method of application and treatment use.General preferred composition is injectable and can infusion solution form, such as, be similar to those the composition used by other antibody passive immunizations people.Preferred method of application is parenteral (such as, intravenously, subcutaneous, intraperitoneal, intramuscular).In preferred embodiments, antibody is used by intravenous infusion or injection.In a further preferred embodiment, antibody is used by intramuscular or subcutaneous injection.
Therapeutic composition must be generally aseptic and be stable under preparation and storage requirement.Composition can be formulated as solution, microemulsion, dispersion, liposome or is suitable for other ordered structures of high drug level.Sterile injectable solution can by following preparation: mix in suitable solvent together with being combined with a kind of composition enumerated or composition by the active compound (that is, antibody or antibody moiety) of requirement above, carry out filtration sterilization subsequently if desired.Usually, dispersion is prepared by being mixed in sterile carrier by active compound, and described sterile carrier comprises basic dispersion medium and from enumerating those other required compositions above.When aseptic, the lyophilize powder for the preparation of sterile injectable solution, preferred preparation method produces by the solution of its previous sterile filtration vacuum-drying and the spraying dry that activeconstituents adds any powder of required composition in addition.The correct mobility of solution can be maintained by following, such as, utilize coating such as Yelkin TTS, maintains required granular size in the case of a dispersion and utilizes tensio-active agent.The prolongation of Injectable composition absorbs and can cause by comprising the reagent postponing to absorb in the composition, described reagent such as Monostearate and gelatin.
Associated proteins of the present invention can be used by multiple method known in the art, although for many treatment use, preferred route of administration/pattern is subcutaneous injection, intravenous injection or infusion.As should be understood as technical staff, route of administration and/or pattern will become according to results needed.In certain embodiments, active compound can be prepared together with carrier, and protection compound is avoided quick release by described carrier, such as controlled release preparation, comprises implant, transdermal skin patches and microencapsulated delivery systems.Biodegradable, biocompatible polymkeric substance can be used, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, poe and poly(lactic acid).Many methods for the preparation of this kind of preparation are that patented protection or those skilled in the art are generally known.See, such as, SustainedandControlledReleaseDrugDeliverySystems, J.R.Robinson, ed., MarcelDekker, Inc., NewYork, 1978.
In certain embodiments, associated proteins of the present invention such as, maybe can assimilate oral administration together with edible carrier with inert diluent.Compound (with if desired, other compositions) also can load in hard or soft shell gelatin capsules, be compressed into tablet, or directly mix in the diet of experimenter.Use for through oral area treatment, compound can with vehicle fusion, and to use with the form of tablet of can ingesting, buccal tablets, lozenge, capsule, elixir, suspension, syrup, thin slice (wafel) etc.In order to by using compound of the present invention except parenteral administration, may must with the combined thing of material bag or compound and material be used altogether, to prevent its inactivation.
Complementarity active compound also can mix in composition.In certain embodiments, associated proteins of the present invention and one or more other therapeutical agents are prepared altogether and/or use altogether, and described therapeutical agent is useful to treating that wherein IL-12 activity is harmful illness.Such as, associated proteins of the present invention can be prepared with one or more the other antibody (such as, in conjunction with the antibody of other cytokines or cell surface binding molecule) in conjunction with other targets and/or use altogether altogether.In addition, one or more antibody of the present invention can combinationally use with 2 kinds or more aforementioned therapies agent.This type of combination treatment advantageously can utilize the therapeutical agent used compared with low dosage, thus avoids the possible toxicity relevant to various monotherapy or complication.
In certain embodiments, associated proteins is connected with Increased Plasma Half-life carrier known in the art.Examples of such carriers includes but not limited to, Fc structural domain, polyoxyethylene glycol and dextran.Examples of such carriers describes in such as U.S. Application Serial Number 09/428,082 and disclosed PCT application WO99/25044, and described patent is hereby incorporated by order to any object.
In a particular embodiment, the nucleotide sequence of encode associated proteins of the present invention or another kind prevention of the present invention or therapeutical agent is used, to treat via gene therapy, to prevent, manage or improve illness or its one or more symptoms.Gene therapy refer to by use expression to experimenter or therapy that expressible nucleic acid carries out.In this embodiment of the present invention, nucleic acid is produced the antibody of its coding or is mediated prevention of the present invention or the therapeutical agent of prevention or therapeutic action.
This area can any method about gene therapy can be used according to the invention.About the general summary of gene therapy method, see people such as Goldspiel, 1993, ClinicalPharmacy12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann.Rev.Pharmacol.Toxicol.32:573-596; Mulligan, Science260:926-932 (1993); And Morgan and Anderson, 1993, Ann.Rev.Biochem.62:191-217; May, 1993, TIBTECH11 (5): 155-215.In spendable recombinant DNA technology field, usually known method is described in the people such as Ausubel (eds.), CurrentProtocolsinMolecularBiology, JohnWiley & Sons, NY (1993); And Kriegler, GeneTransferandExpression, ALaboratoryManual, StocktonPress, NY (1990).Being described in detail in the US20050042664A1 be incorporated herein by reference of various gene therapy method is open.
Associated proteins of the present invention can be used for treating wherein by the various diseases that the target of associated proteins identification is harmful.This kind of disease includes but not limited to, rheumatoid arthritis, osteoarthritis, JCA, septic arthritis, Lyme arthritis, arthritic psoriasis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, CrohnShi is sick, ulcerative colitis, inflammatory bowel, insulin-dependent diabetes, thyroiditis, asthma, allergic disease, psoriasis, dermatitis scleroderma, graft versus host disease (GVH disease), organ-graft refection, acute or the chronic immunological disorders relevant to organ transplantation, sarcoidosis, atherosclerosis, disseminated inravascular coagulation, KawasakiShi is sick, GraveShi is sick, nephrotic syndrome, Chronic Fatigue Syndrome, Wei Genashi granulomatosis, anaphylactoid purpura, kidney microvascular is scorching, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, emaciation, transmissible disease, parasitosis, acquired immune deficiency syndrome (AIDS), acute transverse myelitis, huntington's chorea, Parkinson's disease, Alzheimer, apoplexy, primary biliary cirrhosis, hemolytic anemia, malignant tumour, in heart failure, myocardial infarction, AddisonShi is sick, sporadic pluriglandular I type lacks and pluriglandular II type lacks, Schmidt Cotard, adult's (acute) respiratory distress syndrome, bald head, alopecia areata, seronegative arthropathy, joint disease, ReiterShi is sick, arthropathia psoriatica, ulcerative colitis inflammatory joint disease, enteropathic synovitis, chlamydozoan, Yersinia and Salmonellas dependency joint disease, spondyloarthropathy, atheromatous disease/arteriosclerosis, atopic allergology, autoimmunity bullous diseases, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune hemolytic anemia, Coombs positive hemolytic anemias, acquired pernicious anemia, juvenile pernicious anemia, myalgia encephalitis/RoyalFree disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerotic hepatitis, hidden originality autoimmune hepatitis, acquired immunodeficiency disease syndrome, acquired immunodeficiency related diseases, hepatitis B, hepatitis C, common various immune deficiencies (common variable hypogammag lobulinemia), dilated cardiomyopathy, atocia, ovarian failure, premature ovarian failure, fibrotic lung disease, CFA, interstitial lung disease after inflammation, interstitial pneumonia, Connective Tissue Disease interstitial lung disease, mixed connective tissue disease associated pulmonary diseases, systemic scleroderma dependency interstitial lung disease, Arthritis and Rheumatoid Arthritis interstitial lung disease, systemic lupus erythematosus associated pulmonary diseases, dermatomyositis/polymyositis associated pulmonary diseases, the sick associated pulmonary diseases of family name, ankylosing spondylitis associated pulmonary diseases, symptomica dispersivity tuberculosis, haemosiderosis associated pulmonary diseases, drug-induced interstitial lung disease, fibrosis, radioactive fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrate tuberculosis, interstitial lung disease after infecting, urarthritis, autoimmune hepatitis, 1 type autoimmune hepatitis (traditional autoimmunity or lupoid hepatitis), 2 type autoimmune hepatitis (anti-LKM antibody hepatitis), the hypoglycemia of autoimmunization mediation, there is the Type B insulin resistance of acanthosis nigricans, hypoparathyroidism, the acute immune disease relevant to organ transplantation, the chronic immunological disorders relevant to organ transplantation, osteoarthritis, primary sclerosing cholangitis, 1 type psoriasis, 2 type psoriasiss, idiopathic oligoleukocythemia, autoimmunity neutropenia, nephropathy NOS, glomerulonephritis, kidney microvascular is scorching, Lyme disease, discoid lupus erythematosus, idiopathic male infertility disease or NOS, Sperm autoimmunity, multiple sclerosis (all hypotypes), sympathetic ophthalmia, the pulmonary hypertension of connective tissue disease (CTD) secondary, Goodpasture Cotard, the lung performance of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, StillShi is sick, systemic scleroderma, Cotard, TakayasuShi disease/arteritis, AT, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, thyrocele property Autoimmune Thyroid hypofunction (HashimotoShi is sick), the hypofunction of atrophic Autoimmune Thyroid, primary myxedema, lens induced uveitis, primary angiitis, vitiligo acute hepatopathy, chronic hepatopathy, alcoholic cirrhosis, the liver injury of alcohol induction, choleosatatis, atopy hepatopathy, drug-induced hepatitis, nonalcoholic fatty liver disease, transformation reactions and asthma, B group streptococcus (GBS) infects, mental disorder (such as, depressed and schizophrenia), the disease of Th2 type and the mediation of Th1 type, acute and chronic pain (multi-form pain), and cancer such as lung, mammary gland, stomach, bladder, colon, pancreas, ovary, prostate gland and the rectum cancer and hematopoietic malignancies (leukemia and lymphoma), abetalipoproteinemia, acrocyanosis, acute and chronic parasitism or course of infection, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, gland cancer, atrial ectopic beat, AIDS dementia complex, the hepatitis of alcohol induction, allergic conjunctivitis, allergic contact dermatitis, rhinallergosis, allograft rejection, alpha-1-Antitrypsin deficiency, amyotrophic lateral sclerosis, anaemia, stenocardia, anterior horn cell sex change, anti-cd3 treatment, antiphospholipid syndrome, anti-acceptor allergy, to advocate peace peripheral aneurysm, aortic dissection is formed, arterial hypertension, arteriosclerosis, arterio venous fistula, ataxia, atrial fibrillation (persistence or paroxysmal), auricular flutter, atrioventricular block, B cell lymphoma, bone graft repels, bone marrow transplantation (BMT) is repelled, bundle branch block, Burkitt lymphomas, burn, irregular pulse, the dizzy syndrome of heart shake, cardiac tumor, myocardosis, cardiopulmonary bypass inflammatory response, cartilage transplantation repels, cerebellar cortical degeneration, cerebellum illness, irregularity or multifocal atrial tachycardia, chemotherapy associated conditions, chronic granulocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathology, lymphocytic leukemia (CLL), chronic obstructive disease of lung (COPD), chronic poisoning by salicylic acid salt, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob is sick, culture negative sepsis, cystic fibrosis, cytokine therapy associated conditions, dementia pugilistica, demyelinating disease, dengue hemorrhagic fever, dermatitis, dermatological conditions, polyuria, diabetes, diabetic arteriosclerotic disease, diffusivity Lewy corpusculum is sick, DCMP, basal ganglion illness, middle age mongolism, by the drug-induced drug-induced dyskinesia blocking CNS Dopamine Receptors, drug susceptibility, eczema, encephalomyelitis, endocarditis, incretopathy, epiglottitis, ebv infection, erythromelalgia, extrapyramidal tract and cerebellum illness, familial Observation on Specificity of Blood-sucking lymphohistocysis disease, Fetal Thymus Transplant is repelled, FriedreichShi ataxia, functional peripheral disorder of artery, fungoid sepsis, gas gangrene, stomach ulcer, glomerulonephritis, the transplant rejection of any organ or tissue, Gram-negative sepsis, gram positive sepsis, due to the granuloma of intracellular biological, hairy cell, Hallerrorden-Spatz is sick, hashimotoShi thyroiditis, spring fever, cardiac transplant rejection episode, hemochromatosis, hemodialysis, hemolytic uremic syndrome/thrombolysis thrombopenic purpura, hemorrhage, hepatitis (A), Xinier reservoir irregular pulse, HIV/HIV neuropathy, Hokdkin disease, hyperkinetic dyskinesia, allergy, hypersensitivity pneumonitis, hypertension, hypokinetic dyskinesia, hypothalmus-pituitary-adrenal axis is evaluated, idiopathic AddisonShi is sick, idiopathic pulmonary fibrosis, antibody-mediated cytotoxicity, weak, infantile spinal muscular atrophy, aorta inflammation, influenza a, ionization radiation irradiation, iridocyclitis/uveitis/optic neuritis, ischemic damage and reperfusion damage, ishemic stroke, juvenile rheumatoid arthritis, JSMA, Kaposi sarcoma, renal transplant rejection, Legionnella, leishmaniasis, leprosy, cortex spinal cord system injury, lipedema, liver transplantation is repelled, lymphedema, malaria, malignant lymphoma, malignant histocytosis, malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic, migraine, plastosome multisystem illness, MCTD, MG, multiple myeloma, multisystem sex change (MencelDejerine-ThomasShi-Drager and Machado-Joseph), myasthenia gravis, mycobacterium in bird born of the same parents, Mycobacterium tuberculosis, myelodysplastic syndrome, myocardial infarction, myocardial ischemia illness, nasopharyngeal carcinoma, newborn infant's chronic lung disease, ephritis, nephrosis, neurodegenerative disease, neurogenicity I myatrophy, Neutropenic is had a fever, non Hodgkin lymphoma, aorta abdominalis and branch's obturation thereof, Occlusive arterial illness, okt3 treats, testitis/epididymitis, testitis/vasotomy reverses operation, organomegaly, osteoporosis, pancreas transplant rejection, pancreas cancer, tumour related syndromes/the hypercalcemia of malignant tumour, parathyroid transplantation repels, inflammatory pelvic disease, perennial rhinitis, pericardial disease, periphery property atheromatosis, peripheral blood vessel illness, peritonitis, pernicious anemia, pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, incretopathy, MG and change of skin syndrome), postperfusion syndrome, syndrome after pump, syndrome after MI cardiotomy, preeclampsia, Progressive symmetric erythrokeratodermia core is benumbed, primary pulmonary hypertension, radiotherapy, RaynaudShi phenomenon and disease, RaynoudShi is sick, RefsumShi is sick, conventional narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcoma, scleroderma, senile chorea, Lewy small body type senile dementia, seronegative arthropathy, apoplexy, sicklemia, skin allograft rejection, change of skin syndrome, small intestine transplantation repels, solid tumor, specific arrhythmia, spinal ataxia, spinocerebellar degeneration, suis myositis, cerebellum structural impairment, subacute sclerosing panencephalitis, faint, cardiovascular systems syphilis, systemic anaphylaxis, systemic inflammatory response syndrome, generalized seizure juvenile rheumatoid arthritis, T cell or FABALL, telangiectasis, thromboangiitis obliterans, thrombocytopenia, toxicity, graft, wound/hemorrhage, type III allergy, the allergy of IV type, unstable angina, uremia, urosepsis, urticaria, valvular heart disease, varix, vasculitis, venous disease, venous thrombosis, ventricular fibrillation, virus and fungi infestation, viral encephalitis/aseptic meningitis, virus associated erythrophage syndrome, Wernicke-Korsakoff syndrome, WilsonShi is sick, the xenograft rejection (see people such as the people PCT publication number WO2002097048A2 such as Peritt, Leonard, the people such as PCT publication number WO9524918A1 and Salfeld, PCT publication number WO00/56772A1) of any organ or tissue.
Associated proteins of the present invention may be used for treating the people suffering from autoimmune disorder, those diseases that described autoimmune disorder is particularly relevant to inflammation, comprise rheumatoid arthritis, spondylitis, transformation reactions, autoimmune diabetes, Autoimmune uveitis.
Preferably, associated proteins of the present invention or its antigen-binding portion thereof are used for the treatment of rheumatoid arthritis, CrohnShi disease, multiple sclerosis, insulin-dependent diabetes and psoriasis.
Associated proteins of the present invention also can be used together with one or more other therapeutical agents useful in various disease treatment.
Associated proteins of the present invention can be used alone or in combination to treat this kind of disease.Be to be understood that associated proteins can use separately or with other reagent such as therapeutic combination, described other reagent is selected according to its expection object by technician.Such as, other reagent can be art-recognized for treatment is by the disease of Antybody therapy of the present invention or the useful therapeutical agent of situation.Other reagent also can be the reagent giving therapeutic composition advantageous attributes, such as, affect the reagent of composition viscosity.
Should be further understood that the combination be included in the present invention it is those combinations useful to its expection object.Hereinafter described reagent is illustrative object and desirably not restrictive.Combination as part of the present invention can be antibody of the present invention and be selected from the other reagent of following at least one.Combination can also comprise and exceed a kind of other reagent, and such as, 2 kinds or 3 kinds of other reagent, if combination is such, thus make the composition formed can perform its expectation function.
The preferably combination for the treatment of autoimmunization and inflammatory diseases is non-steroidal anti-inflammatory drug, and also referred to as NSAIDS, it comprises medicine as Ibuprofen BP/EP.Other preferably combination are reflunomides, comprise Ultracortene-H; When with dual specific of the present invention or polyspecific IgJR associated proteins combined therapy patient, by reducing required steroid dosage gradually, can reduce or even eliminate the well-known side effect that steroid uses.The non-limitative example that antibody of the present invention or antibody moiety can combine with it for the therapeutical agent of rheumatoid arthritis comprises following: cell factor inhibiting antiphlogistic drug (CSAIDs); For antibody or the antagonist of other people cytokine or somatomedin, such as, 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, rabbit, EMAP-II, GM-CSF, FGF and PDGF.Associated proteins of the present invention or its antigen-binding portion thereof can with the Antibody Combination comprising CD154 (gp39 or CD40L) for cell surface molecule or its part, described cell surface molecule such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA.
The preferably combination of therapeutical agent can the difference in autoimmunization and subsequent inflammation cascade be disturbed; Preferred example comprises TNF antagonist, as chimeric, humanization or people TNF antibody, and D2E7, (PCT publication number WO97/29131), CA2 (Remicade tM), CDP571 and solubility p55 or p75TNF acceptor, its derivative (p75TNFR1gG (Enbrel tM) or p55TNFR1Gg (Lenercept), and TNF α converting Enzyme (TACE) inhibitor; Similarly because same cause IL-1 inhibitor (interleukin 1 converting enzyme inhibitor, IL-1RA etc.) can be effective.Other preferably combination comprise interleukin-11.Another preferably combination comprises the crucial partner (player) of autoimmune response, and described crucial partner and IL-12 function parallel action, depend on IL-12 function or consistent with IL-12 function; Particularly preferably be IL-18 antagonist, comprise IL-18 antibody or solubility IL-18 acceptor, or IL-18BP.Shown IL-12 and IL-18 and there is overlapping but different functions, and the antagonist-combination for the two may be the most effective.Another preferably combination is the anti-CD4 inhibitor of non-exhaustive.Another preferably combination comprises common stimulation approach CD80 (B7.1) or CD86 (B7.2) antagonist, comprises antibody, soluble receptors or antagonist ligands.
Associated proteins of the present invention can also and agent combination, described reagent such as methotrexate, 6-MP, azathioprine sulfasalazine, mesalazine, Olsalazine chloroquine (chloroquinine)/Oxychloroquine, Trolovol, sulfuration oxysuccinic acid gold (intramuscular and per os), azathioprine, colchicine, reflunomide (per os, suck and local injection), beta 2 adrenoreceptor agonists (salbutamol, terbutaline, Salmeterol), xanthine (theophylline, aminophylline), cromoglycate, Nedocromil, ketotifen, Rinovagos and second east alkali, S-Neoral, FK506, rapamycin, mycophenlate mofetil, Leflunomide, NSAIDs is Ibuprofen BP/EP such as, reflunomide is Ultracortene-H such as, phosphodiesterase inhibitor, adenosine agonists, antithrombotics, complement inhibitor, adrenergic agent, disturb reagent (the such as IRAK via the pro-inflammatory cytokine such as intracellular signaling of TNF α or IL-1, NIK, IKK, p38 or map kinase inhibitor), IL-1 β converting enzyme inhibitor, TNF α converting Enzyme (TACE) inhibitor, T cell signal transduction inhibitor such as kinase inhibitor, inhibitors of metalloproteinase, sulfasalazine, azathioprine, Ismipur, angiotensin converting enzyme inhibitor, soluble cytokine receptor and derivative thereof (such as, solubility p55 or p75TNF acceptor and derivative p75TNFRIgG (Enbrel tMwith p55TNFRIgG (Lenercept)), sIL-1RI, sIL-1RII, sIL-6R), anti-inflammatory cytokines (such as, IL-4, IL-10, IL-11, IL-13 and TGF β), celecoxib, folic acid, hydroxychloroquine sulfate, VIOXX, etanercept, English husband monoclonal antibody, Naproxen Base, valdecoxib, sulfasalazine, methyl meticortelone, meloxicam, methyl Ultracortene-H, disodium aurothiomalate, acetylsalicylic acid, Triamcinolone Acetonide, dextropropoxyphene napsilate/Paracetamol, folate, Maxicom, diclofenac, piroxicam, R-ETODOLAC, Diclofenac Sodium, oxaprozin, OXYCODONE HYDROCHLORIDE, dihydrocodeinone bitartrate/Paracetamol, Diclofenac Sodium/Misoprostol, fentanyl, Kineret (anakinra), people's recombinant chou, tramadol hydrochloride, sasapyrin, sulindac, cyanocobalamin/fa/ pyridoxol, Paracetamol, alendronate sodium, Ultracortene-H, morphine sulfate, Xylotox, indomethacin, Glucosamine Sulphate (glucosaminesulf)/chrondroitin, Warner), Sulphadiazine Sodium, OXYCODONE HYDROCHLORIDE/Paracetamol, Olopatdine Hydrochloridez, Misoprostol, naproxen sodium, omeprazole, endoxan, the appropriate uncommon agate of profit, IL-1TRAP, MRA, CTLA4-IG, IL-18BP, anti-IL-18, anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740, roflumilast (Roflumilast), IC-485, CDC-801 and America and Soviet Union's handkerchief agate (Mesopram).Preferably combination comprises methotrexate or Leflunomide, and when medium or severe rheumatoid arthritis, S-Neoral.
The nonrestrictive other reagent that also can combinationally use to treat rheumatoid arthritis with associated proteins includes but not limited to, following: non-steroidal anti-inflammatory drug (NSAIDs); Cell factor inhibiting antiphlogistic drug (CSAIDs); CDP-571/BAY-10-3356 (humanization anti-TNF alpha antibodies; Celltech/Bayer); CA2/ English husband monoclonal antibody (chimeric anti-TNF alpha antibodies; Centocor); 75kdTNFR-IgG/ etanercept (75kDTNF receptor-IgG fusion protein; Immunex; See such as, Arthritis & Rheumatism (1994) the 37th volume, S295; J.Invest.Med. (1996) the 44th volumes, 235A); 55kdTNF-IgG (55kDTNF receptor-IgG fusion protein; Hoffmann-LaRoche); IDEC-CE9.1/SB210396 (non-exhaustive primatized (primatized) anti-CD 4 antibodies; IDEC/SmithKline; See, such as Arthritis & Rheumatism (1995) the 38th volume, S185); DAB486-IL-2 and/or DAB389-IL-2 (IL-2 fusion rotein; Seragen; See such as, Arthritis & Rheumatism (1993) the 36th volume, 1223); Anti-Tac (the anti-IL-2R α of humanization; ProteinDesignLabs/Roche); IL-4 (anti-inflammatory cytokines DNAX/Schering); IL-10 (SCH52000; Recombinant il-10, anti-inflammatory cytokines; DNAX/Schering); IL-4; IL-10 and/or IL-4 agonist (such as, agonistic antibody); IL-1RA (IL-1 receptor antagonist; Synergen/Amgen); Kineret (/Amgen); TNF-bp/s-TNF (soluble TNF binding protein; See such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S284; Amer.J.Physiol.-HeartandCirculatoryPhysiology (1995) the 268th volume, 37-42 page); R973401 (phosphodiesterase IN type inhibitor; See such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S282); MK-966 (cox 2 inhibitor; See such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S81); Ilomedin (see such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S82); Methotrexate; Thalidomide (see such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S282) and thalidomide related drugs (such as, Celgen); Leflunomide (anti-inflammatory and cytokine inhibitor; See such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S131; InflammationResearch (1996) the 45th volume, 103-107 page); Tranexamic acid (plasminog en-activating inhibitor; See such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S284); T-614 (cytokine inhibitor; See such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S282); Prostaglandin E1 (see such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S282); Tenidap (non-steroidal anti-inflammatory drug; See such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S280); Naproxen Base (non-steroidal anti-inflammatory drug; See such as, NeuroReport (1996) the 7th volume, 1209-1213 page); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen BP/EP (non-steroidal anti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalazine (see such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S281); Azathioprine (see such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S281); ICE inhibitor (interleukin 1 converting enzyme inhibitor); Zap-70 and/or lck inhibitor (Tyrosylprotein kinase zap-70 or lck inhibitor); VEGF inhibitor and/or VEGF-R inhibitor (vascular endothelial growth factor or vascular endothelial growth factor receptor inhibitor; Angiogenesis inhibitor); Reflunomide antiphlogistic drug (such as, SB203580); TNF-converting enzyme inhibitor; Anti-IL-12 antibody; Anti-IL-18 antibody; Interleukin 11 (see such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S296); Interleukin-13 (see such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S308); Interleukin-17 inhibitor (see such as, Arthritis & Rheumatism (1996) the 39th volume, No.9 (supplementary issue), S120); Gold; Trolovol; Chloroquine; Chlorambucil; Oxychloroquine; S-Neoral; Endoxan; Total lymphoid irradiation; Anti-anti-thymocyte globulin; Anti-CD 4 antibodies; CD5-toxin; The peptide of oral administration and collagen; Lobenzarit Disodium; Cytokine modulators (CRAs) HP228 and HP466 (HoughtenPharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate ester oligodeoxynucleotide (ISIS2302; IsisPharmaceuticals, Inc.); SCR1 (TP10; TCellSciences, Inc.); Prednisone; Proteins, orgoteins; The many vitriol of glycosaminoglycan; Minocycline HCl; Anti-IL2R antibody; Hai Sheng and vegetable lipid (fish and plant seed lipid acid; See such as, the people such as DeLuca (1995) Rheum.Dis.Clin.NorthAm.21:759-777); Auranofin; Phenylbutazone; Meclofenamic acid; Flufenamic Acid; Intravenous immunoglobuin; Zileuton; Triazure; Mycophenolic acid (RS-61443); Tacrolimus (FK-506); Sirolimus (rapamycin); Therafectin (amiprilose) (therafectin); CldAdo (2-chlorodeoxyadenosine); Methotrexate; Antiviral agent; With immune modulator.
In one embodiment, associated proteins or one of its antigen-binding portion thereof and following reagent combined administration are used for the treatment of rheumatoid arthritis: the micromolecular inhibitor (ABT-123) of KDR, the micromolecular inhibitor of Tie-2; Methotrexate; Prednisone; Celecoxib; Folic acid; Hydroxychloroquine sulfate; VIOXX; Etanercept; English husband monoclonal antibody; Leflunomide; Naproxen Base; Valdecoxib; Sulfasalazine; Methyl meticortelone; Ibuprofen BP/EP; Meloxicam; Methyl Ultracortene-H; Disodium aurothiomalate; Acetylsalicylic acid; Azathioprine; Triamcinolone Acetonide; Dextropropoxyphene napsilate/Paracetamol; Folate; Maxicom; Diclofenac; Piroxicam; R-ETODOLAC; Diclofenac Sodium; Oxaprozin; OXYCODONE HYDROCHLORIDE; Dihydrocodeinone bitartrate/Paracetamol; Diclofenac Sodium/Misoprostol; Fentanyl; Kineret, people's recombinant chou; Tramadol hydrochloride; Sasapyrin; Sulindac; Cyanocobalamin/fa/ pyridoxol; Paracetamol; Alendronate sodium; Ultracortene-H; Morphine sulfate; Xylotox; Indomethacin; Glucosamine Sulphate/chrondroitin; S-Neoral; Warner); Sulphadiazine Sodium; OXYCODONE HYDROCHLORIDE/Paracetamol; Olopatdine Hydrochloridez; Misoprostol; Naproxen sodium; Omeprazole; Mycophenlate mofetil; Endoxan; The appropriate uncommon agate of profit; IL-1TRAP; MRA; CTLA4-IG; IL-18BP; ABT-874; ABT-325 (anti-IL18); Anti-IL15; BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485; CDC-801; With America and Soviet Union's handkerchief agate.
The non-limitative example that associated proteins of the present invention can combine with it for the therapeutical agent of inflammatory bowel comprises following: budesonide; Urogastron; Reflunomide; S-Neoral, sulfasalazine; Aminosalicylate; Ismipur; Azathioprine; Metronidazole; Lipoxygenase inhibitors; Mesalazine; Olsalazine; Balsalazide; Antioxidant; Thromboxane inhibitors; IL-1 receptor antagonist; Anti-IL-1 β monoclonal antibody; Anti-IL-6 monoclonal antibody; Somatomedin; Elastase inhibitor; Pyridyl-imidazolium compounds; For antibody or its antagonist of other people cytokine or somatomedin, described cytokine or somatomedin such as TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-17, IL-18, EMAP-II, GM-CSF, FGF and PDGF.Antibody of the present invention or its antigen-binding portion thereof can with the Antibody Combination for cell surface molecule or its part, described cell surface molecule such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90.Antibody of the present invention or its antigen-binding portion thereof can also and agent combination, described reagent such as methotrexate, S-Neoral, FK506, rapamycin, mycophenlate mofetil, Leflunomide, NSAIDs is Ibuprofen BP/EP such as, reflunomide is Ultracortene-H such as, phosphodiesterase inhibitor, adenosine agonists, antithrombotics, complement inhibitor, adrenergic agent, disturb reagent (the such as IRAK via the pro-inflammatory cytokine such as intracellular signaling of TNF α or IL-1, NIK, IKK, p38 or map kinase inhibitor), IL-1 β converting enzyme inhibitor, TNF α converting enzyme inhibitor, T cell signal transduction inhibitor such as kinase inhibitor, inhibitors of metalloproteinase, sulfasalazine, azathioprine, Ismipur, angiotensin converting enzyme inhibitor, soluble cytokine receptor and derivative (such as solubility p55 or p75TNF acceptor, sIL-1RI, sIL-1RII, and anti-inflammatory cytokines (such as, IL-4 sIL-6R), IL-10, IL-11, IL-13 and TGF β).
The preferred example that wherein associated proteins can combine for the therapeutical agent of CrohnShi disease comprises following: TNF antagonist, such as anti-TNF antibodies, D2E7 (PCT publication number WO97/29131; HUMIRA), CA2 (REMICADE), CDP571, TNFR-Ig construct, (p75TNFRIgG (ENBREL) and p55TNFRIgG (LENERCEPT)) inhibitor and PDE4 inhibitor.Antibody of the present invention or its antigen-binding portion thereof can with corticosteriods, such as budesonide and dexamethasone.Associated proteins of the present invention or its antigen-binding portion thereof can also and agent combination, described reagent is sulfasalazine, 5-aminosalicylic acid and Olsalazine such as, and the reagent of interference pro-inflammatory cytokine such as IL-1 synthesis or effect, such as IL-1 β converting enzyme inhibitor and IL-1ra.Antibody of the present invention or its antigen-binding portion thereof can also use together with T cell signal transduction inhibitor, such as, and tyrosine kinase inhibitor Ismipur.
Associated proteins of the present invention or its antigen-binding portion thereof can combine with IL-11.Associated proteins of the present invention or its antigen-binding portion thereof can with following agent combination: mesalazine, prednisone, azathioprine, purinethol, English husband monoclonal antibody, methyl meticortelone sodium succinate, diphenoxylate/Tropintran, loperamide hydrochloride, methotrexate, omeprazole, folate, Ciprofloxacin/glucose-water, dihydrocodeinone bitartrate/Paracetamol, tetracycline hydrochloride, fluocinolone acetonide, metronidazole, Thiomersalate/boric acid, QUESTRAN/sucrose, ciprofloxacin HCl, hyoscyamine sulfate, pethidine hydrochloride, Midazolam Hydrochloride, OXYCODONE HYDROCHLORIDE/Paracetamol, promethazine hydrochloride, sodium phosphate, sulfanilamide (SN) Jia isoxazole/trimethoprim, celecoxib, polycarbophil, dextropropoxyphene napsilate, hydrocortisone, multivitamin, balsalazide disodium, codeine phosphate/Paracetamol, colesevelam hydrocholoride (colesevelamhcl), cyanocobalamin, folic acid, levofloxacin, methyl meticortelone, natalizumab and interferon-gamma.
The non-limitative example that associated proteins of the present invention can combine with it for the therapeutical agent of multiple sclerosis comprises following: reflunomide; Ultracortene-H; Methyl meticortelone; Azathioprine; Endoxan; S-Neoral; Methotrexate; 4-aminopyridine; Tizanidine; Interferon-beta 1a (AVONEX; Biogen); Interferon-beta 1b (BETASERON; Chiron/Berlex); Alferon N) (InterferonSciences/Fujimoto), interferon-' alpha ' (AlfaWassermann/J & J), interferon beta 1A-IF (Serono/InhaleTherapeutics), Peg-Intron (Peginterferon) α 2b (Enzon/Schering-Plough), copolymer 1 (Cop-1; COPAXONE; TevaPharmaceuticalIndustries, Inc.); Hyperbaric oxygen; Intravenous immunoglobuin; CldAdo (clabribine); For antibody or its antagonist of other people cytokine or somatomedin and acceptor thereof, such as, 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.Associated proteins of the present invention can with the Antibody Combination for cell surface molecule or its part, described cell surface molecule such as CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90.Associated proteins of the present invention can also and agent combination, described reagent such as methotrexate, S-Neoral, FK506, rapamycin, mycophenlate mofetil, Leflunomide, NSAIDs is Ibuprofen BP/EP such as, reflunomide is Ultracortene-H such as, phosphodiesterase inhibitor, adenosine agonists, antithrombotics, complement inhibitor, adrenergic agent, disturb reagent (the such as IRAK via the pro-inflammatory cytokine such as intracellular signaling of TNF α or IL-1, NIK, IKK, p38 or map kinase inhibitor), IL-1 β converting enzyme inhibitor, tace inhibitor, T cell signal transduction inhibitor such as kinase inhibitor, inhibitors of metalloproteinase, sulfasalazine, azathioprine, Ismipur, angiotensin converting enzyme inhibitor, soluble cytokine receptor and derivative (such as solubility p55 or p75TNF acceptor, sIL-1RI, sIL-1RII, sIL-6R) and anti-inflammatory cytokines (such as, IL-4, IL-10, IL-11, IL-13 and TGF β).
The preferred example that wherein associated proteins of the present invention can combine for the therapeutical agent of multiple sclerosis comprises interferon-beta, such as IFN β 1a and IFN β 1b; Kao Pasong, reflunomide, caspase inhibitors, such as Caspase-1 inhibitor, IL-1 inhibitor, tnf inhibitor, and for the antibody of CD40L and CD80.
Associated proteins of the present invention can also and agent combination, described reagent such as Ah coming organizes monoclonal antibody, dronabinol, You Mai (Unimed), daclizumab (daclizumab), mitoxantrone, hydrochloric acid xaliproden (xaliprodenhydrochloride), the amino pyrrole of 4-is fixed, acetic acid glatiramer that, natalizumab, Xin Nabi alcohol (sinnabidol), a-immune factor (a-immunokine) NNSO3, ABR-215062, AnergiX.MS, chemokine receptor anagonists, BBR-2778, the ancient woods (calagualine) of OK a karaoke club, CPI-1189, LEM (liposome is by the mitoxantrone of encapsulated), THC.CBD (cannabinoid agonists), MBP-8298, America and Soviet Union's handkerchief agate (PDE4 inhibitor), MNA-715, anti-IL-6 receptor antibody, neural element (neurovax), pirfenidone allotrap1258 (RDP-1258), sTNF-R1, Talampanel (talampanel), Teriflunomide (teriflunomide), TGF-β 2, tiplimotide (tiplimotide), VLA-4 antagonist (such as, TR-14035, VLA4Ultrahaler, Antegran-ELAN/Biogen), interferon-gamma antagonist and IL-4 antagonist.
The non-limitative example that associated proteins of the present invention can combine with it for anginal therapeutical agent comprises following: acetylsalicylic acid, pannonit, isosorbide mononitrate, metroprolol succinate, atenolol USP 23, metoprolol tartrate, Amlodipine Besylate, hydrochloric acid ground that sorbide nitrate, heavy Clopidogrel Hydrogensulfate, nifedipine, atorvastatincalcuim, Repone K, Furosemide, simvastatin, verapamil hydrochloride, digoxin, propranolol hydrochloride, carvedilol, lisinopril, spironolactone, hydrochlorothiazide, enalapril maleate, nadolol, Ramipril, Enoxaparin Sodium, heparin sodium, valsartan, Sotalol hydrochloride, fenofibrate, ezetimibe (ezetimibe), bumetanide, Losartan Potassium, lisinopril/hydrochlorothiazide, felodipine, captopril, bisoprolol fumarate.
The non-limitative example that associated proteins of the present invention can combine with it for the therapeutical agent of ankylosing spondylitis comprises following: Ibuprofen BP/EP, diclofenac and Misoprostol, Naproxen Base, meloxicam, indomethacin, diclofenac, celecoxib, VIOXX, sulfasalazine, methotrexate, azathioprine, Minocycline HCl, prednisone, etanercept, English husband monoclonal antibody.
The non-limitative example that associated proteins of the present invention can combine with it for the therapeutical agent of asthma comprises following: salbutamol, Salmeterol/fluticasone, Menglusitena, fluticasone propionate, budesonide, prednisone, salmeterol xinafoate (sameterolxinafoate), Xopenex, salbutamol sulfate/Rinovagos, prednisolone phosphate sodium, Triamcinolone Acetonide, beclomethasone dipropionate, ipratropium bromide, Azythromycin, pirbuterol acetate, Ultracortene-H, Theophylline Anhydrous, methyl meticortelone sodium succinate, clarithromycin, Zafirlukast, Formoterol Fumarate, influenza virus vaccine, methyl meticortelone, Utimox, flunisolide, transformation reactions is injected, Sodium Cromoglicate, hydrochloric acid Fexofenadine, flunisolide/menthol, amoxicillin/clavulanate potassium, levofloxacin, sucker supplementary unit, guaiacol glycerol ether, dexamethasone sodium phosphate, Moxifloxacin hydrochloride, Doxycycline Hyclate, guaiacol glycerol ether/d-methorphan, p-racephedrine/cod/ chlorphenamine (chlorphenir), Gatifloxacin, cetirizine hydrochloride, Mometasone Furoate, salmeterol xinafoate, benzonatate, Cephalexin Monohydrate Micro/Compacted, pe/ dihydrocodeinone/chlorphenamine, cetirizine hydrochloride/pseudoephedrine (pseudoephed), phenylephrine/cod/ promethazine, morphine monomethyl ether/promethazine, cefprozil, dexamethasone, guaiacol glycerol ether/pseudoephedrine, chlorphenamine/dihydrocodeinone, sodium nedocromil, bricalin, suprarenin, methyl meticortelone, metaproterenol sulfate.
The non-limitative example that associated proteins of the present invention can combine with it for the therapeutical agent of COPD comprises following: salbutamol sulfate/Rinovagos, ipratropium bromide, Salmeterol/fluticasone, salbutamol, salmeterol xinafoate, fluticasone propionate, prednisone, Theophylline Anhydrous, methyl meticortelone sodium succinate, Menglusitena, budesonide, Formoterol Fumarate, Triamcinolone Acetonide, levofloxacin, guaiacol glycerol ether, Azythromycin, beclomethasone dipropionate, Xopenex, flunisolide, ceftriaxone sodium, Utimox, Gatifloxacin, Zafirlukast, amoxicillin/clavulanate potassium, flunisolide/menthol, chlorphenamine/dihydrocodeinone, metaproterenol sulfate, methyl meticortelone, Mometasone Furoate, p-racephedrine/cod/ chlorphenamine, pirbuterol acetate, p-racephedrine/Loratadine, bricalin, tiotropium bromide (tiotropiumbromide), (R, R)-formoterol, TgAAT, cilomilast (Cilomilast), roflumilast.
The non-limitative example that associated proteins of the present invention can combine with it for the therapeutical agent of HCV comprises following: interferon-' alpha '-2a, interferon-' alpha '-2b, interferon-' alpha ' con1, interferon-' alpha '-n1, Peg-Intron-α-2a, Peg-Intron-α-2b, ribavirin, peg-interferon α-2b+ribavirin, Ursodeoxycholic Acid (UDCA), Potenlini, Thymosin-Alpha1, mark's amine (Maxamine), VX-497 and any compound by disturbing following target to be used for the treatment of HCV: HCV polysaccharase, HCV proteolytic enzyme, HCV helicase, HCVIRES (internal ribosome entry site).
The non-limitative example that associated proteins of the present invention can combine with it for the therapeutical agent of idiopathic pulmonary fibrosis comprises following: prednisone, azathioprine, salbutamol, colchicine, salbutamol sulfate, digoxin, IFN-γ, methyl meticortelone sodium succinate (sodsucc), lorazepam, Furosemide, lisinopril, pannonit, spironolactone, endoxan, ipratropium bromide, actinomycin d, alteplase, fluticasone propionate, levofloxacin, metaproterenol sulfate, morphine sulfate, OXYCODONE HYDROCHLORIDE, Repone K, Triamcinolone Acetonide, anhydrous tacrolimus, calcium, interferon-' alpha ', methotrexate, mycophenlate mofetil, interferon-γ-1 β.
The non-limitative example that associated proteins of the present invention can combine with it for the therapeutical agent of myocardial infarction comprises following: acetylsalicylic acid, pannonit, metoprolol tartrate, Enoxaparin Sodium, heparin sodium, heavy Clopidogrel Hydrogensulfate, carvedilol, atenolol USP 23, morphine sulfate, metroprolol succinate, Warnerin, lisinopril, isosorbide mononitrate, digoxin, Furosemide, simvastatin, Ramipril, for Nip's enzyme, enalapril maleate, torasemide (torsemide), reteplase, Losartan Potassium, quinapril hydrochloride/magcarb, bumetanide, alteplase, enalaprilat, L-3428, Tirofiban hydrochloride monohydrate, hydrochloric acid ground that captopril, irbesartan, valsartan, propranolol hydrochloride, fosinopril sodium, Xylotox, Integrilin, cephazolin sodium, Tropintran, hexosamine, spironolactone, Interferon, rabbit, Sotalol hydrochloride, Repone K, Docusate Sodium, Dobutamine Hydrochloride USP, alprazolam, Pravastatin Sodium, atorvastatincalcuim, Midazolam Hydrochloride, pethidine hydrochloride, isosorbide mononitrate, suprarenin, dopamine hydrochloride, Bivalirudin, superstatin (rosuvastatin), ezetimibe/simvastatin, avasimibe (avasimibe), cariporide (cariporide).
The non-limitative example that associated proteins of the present invention can combine with it for psoriasis treatment agent comprises following: the micromolecular inhibitor of KDR (ABT-123), the micromolecular inhibitor of Tie-2, calcipotriol (calcipotriene), clobetasol propionate, Triamcinolone Acetonide, halobetasol propionate, tazarotene, methotrexate, fluocinolone acetonide, enhancement type Sch-11460, fluocinonide, Acitretin, tar (tar) shampoo, Valisone, Mometasone Furoate, KETOKONAZOL, pramoxine/fluocinolone acetonide, valerate cortisone, Cordran, urea, Betamethasone Valerate, clobetasol propionate/lubricant (emoll), fluticasone propionate, Azythromycin, hydrocortisone, moistening formula, folic acid, desonide, pimecrolimus (pimecrolimus), coal tar, Upjohn), folic acid etanercept, lactic acid, 8-methoxypsoralen, hc/ gallic acid bismuth (bismuthsubgal)/znox/resor, methyl Ultracortene-H, prednisone, opalizer, halcinonidedcorten, Whitfield's ointment, dithranol, clocortolone, coal extract, coal tar/Whitfield's ointment, coal tar/Whitfield's ointment/sulphur, desoximetasone, diazepam, lubricant, fluocinolone acetonide/lubricant, mineral oil/Viscotrol C/nalact, mineral oil/peanut oil, oil/Isopropyl myristate, psoralene, Whitfield's ointment, soap/tribromsalan, Thiomersalate/boric acid, celecoxib, English husband monoclonal antibody, S-Neoral, A Laisaipu, efalizumab, tacrolimus, pimecrolimus, PUVA, UVB, sulfasalazine.
The non-limitative example that associated proteins of the present invention can combine with it for the therapeutical agent of arthritic psoriasis comprises following: methotrexate, etanercept, VIOXX, celecoxib, folic acid, sulfasalazine, Naproxen Base, Leflunomide, methyl Ultracortene-H, indomethacin, hydroxychloroquine sulfate, prednisone, sulindac, enhancement type Sch-11460, English husband monoclonal antibody, methotrexate, folate, Triamcinolone Acetonide, diclofenac, dimethyl sulfoxide (DMSO), piroxicam, Diclofenac Sodium, Ketoprofen BP 93, meloxicam, methyl meticortelone, Maxicom, Tolmetin sodium, calcipotriol, S-Neoral, Diclofenac Sodium/Misoprostol, fluocinolone acetonide, Glucosamine Sulphate, disodium aurothiomalate, dihydrocodeinone bitartrate/Paracetamol, Ibuprofen BP/EP, risedronate sodium, Sulphadiazine Sodium, Tioguanine, valdecoxib, A Laisaipu, efalizumab.
The non-limitative example that associated proteins of the present invention can combine with it for the therapeutical agent of restenosis comprises following: sirolimus, taxol, everolimus (everolimus), tacrolimus, ABT-578, Paracetamol.
The non-limitative example that associated proteins of the present invention can combine with it for the therapeutical agent of sciatica comprises following: dihydrocodeinone bitartrate/Paracetamol, VIOXX, cyclobenzaprine hydrochloride, methyl meticortelone, Naproxen Base, Ibuprofen BP/EP, OXYCODONE HYDROCHLORIDE+/ Paracetamol, celecoxib, valdecoxib, methyl Ultracortene-H, prednisone, codeine phosphate/Paracetamol, tramadol hydrochloride/Paracetamol, Metaxolone, meloxicam, methocarbamol, Xylotox, Diclofenac Sodium, gabapentin, dexamethasone, carisoprodol, ketorolac amino fourth three acid alcohol salt, indomethacin, Paracetamol, diazepam, Maxicom, OXYCODONE HYDROCHLORIDE, tizanidine hydrochloride, Diclofenac Sodium/Misoprostol, dextropropoxyphene napsilate/Paracetamol, asa/oxycod/ oxycodone ter, Ibuprofen BP/EP/dihydrocodeinone bit, tramadol hydrochloride, R-ETODOLAC, regretol, Warner), carisoprodol/codeine phosphate/asa, morphine sulfate, multivitamin, naproxen sodium, citric acid orphenadrine, temazepam.
The preferred example that wherein associated proteins of the present invention can combine for the therapeutical agent of SLE (lupus) comprises following: NSAIDS, such as, and diclofenac, Naproxen Base, Ibuprofen BP/EP, piroxicam, indomethacin; COX2 inhibitor, such as, celecoxib, VIOXX, valdecoxib; Antimalarial drug, such as, Oxychloroquine; Steroid, such as, prednisone, Ultracortene-H, budesonide, dexamethasone; Cytotoxin, such as, azathioprine, endoxan, mycophenlate mofetil, methotrexate; PDE4 inhibitor or purine synthetic inhibitor, such as Cellcept.Associated proteins of the present invention can also and agent combination, described reagent such as sulfasalazine, 5-aminosalicylic acid, Olsalazine, according to lily magnolia, and the reagent that interference pro-inflammatory cytokine such as IL-1 synthesizes, produces or act on, such as caspase inhibitors, as IL-1 β converting enzyme inhibitor and IL-1ra.Associated proteins of the present invention can also use together with T cell signal transduction inhibitor, such as tyrosine kinase inhibitor; Or the molecule of targeting T-cells activated molecule, such as CTLA-4-IgG or anti-B7 family antibody, anti-PD-1 family antibody.Associated proteins of the present invention can combine with IL-11 or anti-cytokine antibodies, such as fragrant prominent sharp pearl monoclonal antibody (fonotolizumab) (anti-IFNg antibody), or anti-receptor receptor antibody, such as anti-IL-6 receptor antibody and the antibody for B cell surface molecular.Antibody of the present invention or its antigen-binding portion thereof can also use together with following reagent: LJP394 (abetimus (abetimus)), exhaust or the reagent of deactivation B cell, such as sharp appropriate uncommon agate (anti-CD20 antibodies), drench Fu Site (lymphostat)-B (anti-BlyS antibody), TNF antagonist, such as, anti-TNF antibodies, D2E7 (PCT publication number WO97/29131; HUMIRA), CA2 (REMICADE), CDP571, TNFR-Ig construct (p75TNFRIgG (ENBREL) and p55TNFRIgG (LENERCEPT)).
Pharmaceutical composition of the present invention can comprise the associated proteins of the present invention of " treatment significant quantity " or " prevention significant quantity "." treatment significant quantity " refers to the amount effectively reaching required treatment result at required dosage and time period.Protein-bonded treatment significant quantity can be determined by those skilled in the art, and can become according to following factor: individual morbid state, age, sex and weight, and associated proteins causes the ability of required response in individuality.Treatment significant quantity is also wherein treat advantageous effect to be greater than any toxicity of antibody or antibody moiety or the amount of deleterious effect." prevention significant quantity " refers to the amount effectively reaching required prevention result at required dosage and time period.Usually, because preventive dose uses before disease or when disease is early stage in experimenter, so prevention significant quantity will be less than treatment significant quantity.
Dosage can carry out adjusting to provide best required response (such as, treatment or prevention response).Such as, can use single bolus, several broken dose can be used in the past along with the time, or dosage can as treat situation the emergency state indicated by minimizing in proportion or increase.Consistent with dosage for ease of using, be particularly advantageous with unit dosage preparation parenteral composition.As used herein, unit dosage refers to be suitable as the physically discontinuous unit of single dose for mammalian subject to be treated; The active compound that each unit comprises the predetermined amount of calculating with produce relevant to required pharmaceutical carriers needed for curative effect.Directly depend on following about the detailed description of unit dosage of the present invention by following instruction: the limitation that the specific characteristic of (a) active compound and concrete treatment to be achieved or prophylactic effect and (b) coordinate this field being used for the treatment of the active compound of susceptibility in individuality intrinsic.
Exemplary, non-limiting scope about protein-bonded treatment of the present invention or prevention significant quantity is 0.1-20mg/kg, more preferably 1-10mg/kg.Should be understood that dose value can become according to condition type to be alleviated and seriousness.Should be further understood that for any particular subject; according to individual need with use or the professional judgement of people that supervision group compound is used; concrete dosage should adjust in the past along with the time; and dosage range as herein described is only exemplary, and do not wish the scope or the practice that limit claimed composition.
Sequence information used in this application is as follows:
SEQIDNO:1. mankind VEGF-A
SEQIDNO:2. mankind ANG-2
SEQIDNO:3. mankind HER-2
SEQIDNO:4. human TNF aphla
SEQIDNO:5. mankind IL-17A
SEQIDNO:6. VEGF antibody heavy chain
SEQIDNO:7. VEGF antibody light chain
SEQIDNO:8. anti-ANG-2 heavy chain of antibody
SEQIDNO:9. anti-ANG-2 light chain of antibody
SEQIDNO:10. anti-vegf/ANG-2 dual specific IgJR antibody HC1 chain
SEQIDNO:11. anti-vegf/ANG-2 dual specific IgJR antibody LC1 chain
SEQIDNO:12. anti-vegf/ANG-2 dual specific IgJR antibody HC2 chain
SEQIDNO:13. Anti-HER 2 heavy chain
SEQIDNO:14. Anti-HER 2 light chain
SEQIDNO:15. anti-vegf/HER2 dual specific IgJR antibody HC1 chain
SEQIDNO:16. anti-vegf/HER2 dual specific IgJR antibody LC1 chain
SEQIDNO:17. anti-vegf/HER2 dual specific IgJR antibody HC2 chain
SEQIDNO:18. anti-TNFalpha heavy chain of antibody
SEQIDNO:19. anti-TNFalpha light chain of antibody
SEQIDNO:20. anti-IL-17A heavy chain of antibody
SEQIDNO:21. anti-IL-17A light chain of antibody
SEQIDNO:22. anti-TNFalpha/IL-17A dual specific IgJR antibody HC1 chain
SEQIDNO:23. anti-TNFalpha/IL-17A dual specific IgJR antibody LC1 chain
SEQIDNO:24. anti-TNFalpha/IL-17A dual specific IgJR antibody HC2 chain
SEQIDNO:25. heavy chain signal peptide
SEQIDNO:26. light chain signal peptide
Associated proteins according to the present invention has demonstrated the following three valuable characteristics in aspect,
1. be easy to preparation, facilitate extraction purification.Due to the bi-specific antibody structure design of uniqueness of the present invention, in cell expressing dual specific IgJR antibody process, LV1-CL strand only has a kind of may pairing with the VH1-CH1 in long heavy chain, forms natural stabilisation structure land 1 (BR1) VH2-CH1 strand and also only has one may form natural stabilisation structure land 2 (BR2) with VL2-CL in long heavy chain.Therefore, in cell expressing process, only may produce a kind of IgJR bi-specific antibody structure, the antibody mixture of various arrangement combination can not be formed, thus be easy to separation and purification.Completely different from CN01810372 patent multivalent antibody structure design, this Patent design 2 heavy chains light chain different from 2 that link together matches and produces multiple dual anti-mixture (such as, light chain 1 can match with heavy chain 1, also can match with heavy chain 2) thus be difficult to the bi-specific antibody that separation and purification goes out monomer.And in the embodiment of the present invention 1.4 (Fig. 5), confirming only to produce dual specific IgJR antibody (unimodal) a kind of structure in cell expressing, molecular weight is about 248KD.
2. compare can find out with DVD-lg antibody structure, in two basic change protein structure (BR1 and BR2) of the present invention, variable region is all direct to be directly connected with constant region, therefore can retain conformation and the character of natural antibody land to greatest extent, cause dual specific IgJR antibody tertiary structure complete and stable.Dual specific IgJR antibody dsc analysis TM value in embodiment 1.5 (Fig. 5) is about 75 DEG C, and this value is apparently higher than other type antibodies.Stability analysis experiment in embodiment 1.5 can determine that dual specific IgJR Antibody stability is better than other type antibodies (Fig. 6).
3. associated proteins district (BR1 and BR2) of the present invention only has a polypeptide or Amino acid linker in long heavy chain, be different from DVD-lg antibody and need two polypeptide or the Amino acid linker two basic change protein region (VD1 and VD2) respectively on fixed length heavy chain and light chain, the unique texture of dual specific IgJR antibody list joint makes dual specific IgJR antibody BR1 land rotary freedom large, be easy to multi-angle conjugated antigen, do not affect the avidity of the synchronous conjugated antigen of bi-specific antibody.Avidity measurement result display in the embodiment of the present application 1.7, the avidity of dual specific IgJR antibody and parental antibody is basically identical.(ClarissaG.Jakob, 1, * RohintonEdalji, 2, Structurerevealsfunctionofthedualvariabledomainimmunoglo bulin (DVD-Ig is pointed out in DVD-lg affinity of antibody and structural research document tM) molecule, mAbs5:3,358 – 363; May/June2013), VD1 is fixed by two polypeptide using in light chain and heavy chain or Amino acid linker, and rotary freedom is restricted, may reduce two special DVD-lg affinity of antibody.The unique design of dual specific IgJR antibody of the present invention more perfectly retains the antigen-antibody avidity of parental antibody.
In a preferred embodiment, associated proteins of the present invention is its part, such as fragment.Preferably implement in archives at another, associated proteins of the present invention is antibody.The invention still further relates to a kind of test kit, it comprises associated proteins of the present invention or described pharmaceutical composition or drug regimen.It is evident that for those skilled in the art, other suitable modifications of the inventive method described herein and adaptation are obvious, and the scope without the need to deviating from the present invention or embodiment disclosed herein uses suitable equivalence to carry out.Although the present invention is described in detail at present, more clearly will understand the present invention by reference to following embodiment, described embodiment be included only for illustrative object and do not wish limit the present invention.
Embodiment
Embodiment 1: the anti-VEGF/generation of HER2 dual specific IgJR antibody and the analysis of binding property
Antigen:
VEGF-A has 232 amino acid whose sequences (UniProtKB-P15692).HER2 (ERBB2) (ANGP2) has 1255 amino acid whose sequences (UniProtKB-P04626).
Antibody sources:
Exemplary anti-VEGF/HER2 bi-specific antibody is by people's anti-vegf-A antibody, rhuMAb-VEGF ( genentech) and people's Anti-HER 2, the bi-specific antibody that Herceptin (trastuzumab) (Herceptin, Genentech) is formed.The aminoacid sequence of rhuMAb-VEGF at US7,297, open in 334.The aminoacid sequence of Herceptin at US7,152,72 and WO92/22653 in open.
Embodiment 1.1, the structure of anti-VEGF/HER2 dual specific IgJR antibody
Specifically, all gene fragments of coding anti-VEGF/HER2 dual specific IgJR light chain of antibody and heavy chain are synthesized, and the DNA sequence encoding leading peptide (MSVPTQVLGLLLLWLTDARC (light chain signal peptide) (SEQIDNO:26) that the 5' of described gene fragment holds, MEWSWVFLFFLSVTTGVHS (heavy chain signal peptide) (SEQIDNO:25)), the described secretion of leading peptide targeting proteins matter in eukaryotic cell (Fig. 3).According to specification (givenspecification) the synthetic gene fragment that DNA2.0 (California, USA) is given, its coding corresponds to the amino acid of construct #A1, construct #A2 and construct #A3.
By side for limiting cleavage sites, the gene fragment of 5 ' HindIII and 3 ' EcoRI, is connected in HindIII and the EcoRI restriction site of pcDNA3.1 expression vector (lifetechnology) (Fig. 4).For further detailed description, see the materials and methods finally listed.DNA sequence dna is confirmed by DNA sequencing.The cysteine residues (C) held at the C of the VL2 of the first polypeptide chain JRHC1 (construct #B1) does not participate in maintaining the protein-bonded correct conformation of dual specific IgJR, therefore can lack or be substituted, usually Serine is replaced to, to improve the protein-bonded oxidation-resistance of dual specific IgJR and to prevent abnormal crosslinked.Further, can one or more halfcystine key be joined in this antibody, to improve its stability.In the structure of IgJR bi-specific antibody, add halfcystine at the C-end of JRHC2 (construct #B3), form disulfide linkage (Fig. 3) with the halfcystine of the hinge area EPKSCD with the first polypeptide chain JRHC1.
InvitrogenVectorNTAdvancesuite version 11 is used for sequence to produce, map, analyze, annotate and explanation.
Embodiment 1.2, the generation of anti-VEGF/HER2 dual specific IgJR antibody
Use as in CurrentProtocolsinCellBiology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J.andYamada, K.M. (eds.), JohnWiley & Sons, the standard cell culture techniques described in Inc..At FREESTYLE tMtransient transfection is carried out in 293 expression systems (INVITROGEN, CALIFORNIA, USA).
According to the explanation of manufacturers, by using HEK293-Freestyle system (Invitrogen, California, USA) transient transfection difference encoded packets is containing VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3 (construct #B1), three plasmids of VL1-CL (construct #B2) and VH2-CH1 (construct #B3), produce antibody.X1 is short circuit head, such as EPKSCD (also can not having).In brief, with the mixture transfection HEK293-FreeStyle cell (HEK293-F cell) (Invitrogen) of above-mentioned three expression plasmids and 293fectin (Invitrogen), described cell expresses suspension growth in the shaking flask of substratum (Invitrogen) or the fermentor tank of stirring at the FreeStyle293 containing serum-free.Such as, with 1.0x10 6hEK293-F cell is inoculated in the 2L shaking flask (Corning) of the substratum containing 600mL by the density of cell/mL, and cultivates under 120rpm, 8%CO2.Latter second day of cell inoculation, at about 1.5x10 6under the cell density of individual cell/mL, with about 42mL mixture transfectional cell, described mixture comprises A) 20mLOpti-MEM (Invitrogen), wherein there are 600 μ g with total plasmid DNA (1 μ g/mL) of equimolar ratio rate coding VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3, VL1-CL and VH2-CH1 and B) 20mlOpti-MEM+1.2mL293fectin or fectin (2 μ Ι/mL).According to the consumption of glucose, add glucose solution during the fermentation.5-10 days results containing the supernatant liquor of secretory antibody, and direct antibody purification or by supernatant liquor freezen protective from supernatant liquor.
Embodiment 1.3, the purifying of anti-VEGF/HER2 dual specific IgJR antibody
Reference standard scheme is from the cell culture supernatant purifying anti-VEGF/HER2 dual specific IgJR antibody filtered.In brief, antibody is applied on albumin A Sepharose post (GEHealthcare), and washs with PBS.The eluate of antibody is that (pH is 3.5 to acid pH, immediately and eluate.At the 20mM Histidine of pH6.0, by size exclusion chromatography (Superdex200, GEHealthcare), separating monomer JR antibody is separated in 140mMNaCl.Such as MILLIPOREAmiconUltra (30MWCO) Centrifugal concentrators is used to concentrate monomeric igg as required.
By being determined at the optical density(OD) (OD) at 280nm place, use according to people such as Pace, ProteinScience, the molar extinction coefficient of the aminoacid sequence calculating of 1995,4,2411-1423, determines the protein concn of the antibody of purifying.
Embodiment 1.4, the size exclusion chromatography of anti-VEGF/HER2 dual specific IgJR antibody and SDS-PAGE analyze
By size exclusion chromatography (SEC) for according to molecular weight isolated protein.In aqueous mobile phase, carry protein, and make it pass through the porous stationary phase resin be filled in cylinder.The residence time in cylinder is the function of the size in hole in the hydrokinetics size of protein and potting resin bed.Compared to larger molecule, less molecule can pass less hole in resin thus stop the longer time.Under the following conditions, use GEAKETAexployer to carry out SEC, and detect based on UV.
Cylinder: GEHiTrapSPHP prepacked column
Flow velocity: 5mL/min
Cleaning (Sanitization): 1MNaOH15min
Neutralization: 20mM citric acid/phosphoric acid salt pH5.4
Balance: 20mM citric acid/phosphoric acid salt pH5.4
Wash-out: with 20mM citric acid/phosphate buffered saline buffer pH5.4+1MNaCl gradient elution more than 20CV
Measure the absorbancy at wavelength 280nm place.For the chromatogram of gained, by the distribution (Fig. 5 A) of the kind (aggregate, monomer and fragment) of the percentage analysis different molecular weight size of the signal total area.In fig. 5, in SEC chromatogram, only show a single Absorption Spectroscopy, confirm in cell expressing process, only produce a kind of expression product and IgJR antibody.It is 248KD that SDS-PAGE in figure 5b shows anti-VEGF/HER2 dual specific IgJR antibody molecule amount.
Embodiment 1.5, the stability of anti-VEGF/HER2 dual specific IgJR antibody and preparation
(1) dsc (DSC)
Use the thermostability of Nano-DSC (TAinstrument, NewCastle, DE, USA) evaluating protein quality sample.Anti-VEGF/HER2 dual specific IgJR antibody that the 1mg/ml be dissolved in PBS amounts to 300ug is applied to Nano-DSC at 25 DEG C with the sweep velocity of 1 DEG C/min between 100 DEG C.Obtain thermal map, calculating Tm is about 75 DEG C (Fig. 6).
The condition of DSC:
TA device: Nano-DSC
Temperature range: 25 DEG C to 100 DEG C
Sweep velocity (Ramprate): 1 DEG C/min
Sample: PBS, pH7.2
Sample concentration: 1mg/ml
Sample size: 300ug
(2) preparation and stability analysis
Analyze the physical stability of anti-VEGF/HER2 dual specific IgJR antibody molecule prepared according to table 1 under different conditions: 25 DEG C of (with 350rpm vibration) storages 7 days, 40 DEG C of storages 14 days with from-20 DEG C to room temperature freeze-thaw cycle 10 times.The room temperature of 25 DEG C be sample for the production of and typical temperature condition in preparation and storage.
Think that in 40 DEG C of storages 14 days and 10 freeze-thaw cycle are violent stable conditions, this stable condition provides the indication of permanent stability.In container sample aliquot sealed to 0.1ml, then make pill with periodic intervals, and take out a small amount of part and be used for size exclusion chromatography analysis.Use WatersBEH200 (WatersInc, Massachusetts, USA), carry out size exclusion chromatography in following condition: 1.7um post, PBS moving phase, 0.25ml/min, 35 DEG C of column temperatures, setting point ~ 4ug load.Analyze the per-cent of aggregate, for the stability (Fig. 7) of instruction anti-VEGF/HER2 dual specific IgJR antibody.It is strong that Fig. 7 shows anti-VEGF/HER2 dual specific IgJR antibody stability in following preparation: pH5.5,10mM Histidine, 280mM sucrose, 0.02% Polysorbate 20.
Table 1: the preparation comprising 20mg/mL anti-VEGF/HER2 dual specific IgJR antibody
Embodiment 1.6, the analysis of anti-VEGF/HER2 dual specific IgJR antibodies character
Use is equipped with biosensor ((the fort é BIO that anti-hIgGFc catches (AHC) biosensor tips (anti-hIgGFccapture (AHC) biosensortip) and streptavidin bag quilt, MenloPark, CA)) fort é BIO qK esystem carries out avidity measurement.AHC avidity is measured, tests the anti-VEGF/HER2 dual specific IgJR antibodies Antibodies of purifying and the binding ability at AHC sensor tip.Load most advanced and sophisticated with 20 μ g/ml anti-VEGFs/HER2 dual specific IgJR antibodies Antibodies.Load and within 300 seconds, cause catching level between 1.8 to 2nm.By being diluted to next VEGF-A (the R & d system) antigen for the preparation of binding analysis of 300nM in 1XPBS.Initial association also monitors 200 seconds, is transferred at tip in 1XPBS damping fluid (Gibco, PBSpH7.2) afterwards, dissociates 300 seconds to monitor.Then tip is transferred in the second antigen (HER2), and monitor 200 seconds.Wash 300 seconds in 1 × PBS afterwards.Also carried out first loading HER2 antigen, loaded the experiment (Fig. 8 C and D) of VEGF-A antigen subsequently.Then biostrome interference analysis (bio-layerinterferometryanalysis) is used to monitor the binding activities of anti-VEGF/HER2 dual specific IgJR antibodies Antibodies in real time.Based on measured association rate constant (" K on") and dissociation rate constant (" K off") calculate antigen-binding affinity, be expressed as dissociation constant (K d).Use data analysis software 6.4 (fort é BIO) process also analytical data (Fig. 8).Octet binding curve shows two kinds of antigens and combines continuously, is not when being exposed to the second antigen, and the first antigen is corresponding to be dissociated.This curve shows that dual specific IgJR antibody can in the mode of noncompetitive association simultaneously in conjunction with two kinds of antigen VEGF and HER2 (Fig. 8).The association rate constant (" K of anti-VEGF/HER2 dual specific IgJR antibodies Antibodies and two kinds of antigen VEGF and HER2 is shown in table 2 on"), dissociation rate constant (" K off"), and avidity, be expressed as dissociation constant (K d).These experiments show, anti-VEGF/HER2 dual specific IgJR antibodies Antibodies remains the avidity that parental antibody is combined with two kinds of antigen VEGF and HER2.
Table 2, the association rate constant (" K of anti-VEGF/HER2 dual specific IgJR antibody and two kinds of antigen VEGF and HER2 on"), dissociation rate constant (" K off"), and avidity, be expressed as dissociation constant (K d).
Loading sample ID Sample ID K D(M) K on(1/Ms) K off(1/s)
Ig JR VEGF/HER2 VEGF 1.40x10 -10 1.65x10 5 2.31x10 -5
Ig JR VEGF/HER2 HER2 3.46x10 -10 4.91x10 5 1.72x10 -4
Embodiment 1.7, anti-VEGF/HER2 dual specific IgJR antibody in vitro pharmaceutical research
(1) HUVEC cell proliferating determining
In order to measure cell proliferation, thaw HUVEC, and with 2 × 10 5cell/ml is resuspended in ice-cold endothelial basal growth medium (EBM), and described substratum is not containing somatomedin and FBS additive.By the cell suspension inoculation of about 50ul in each hole of 96 hole tissue culturing plates.By the test antibody of total 50 μ l prescribed concentration and 15ng/mlVEGF (R & d system, California, USA) preincubation 1 hour, then joined in 96 porocyte plates.At 37 DEG C, 5%CO 2lower lasting culture plate 4 days.At the end of cultivation, 25ulAlamarBlue (BioSourceInternational, Camarillo, CA) is joined in each hole, under the same conditions culture plate 6 hours again.Then on fluorescence plate reader (MolecularDevice, Sunnyvale, CA) to the excitation/emission photoreading of 530/590nm.All research carries out twice, and the concentration of each sample has three parts.Fig. 9 shows anti-VEGF/HER2 dual specific IgJR antibody and rhuMAb-VEGF without significant difference in the effect of HUVEC cell proliferating determining, and anti-VEGF/HER2 dual specific IgJR antibody remains rhuMAb-VEGF effect in vitro.
(2) SKBR-3 cell proliferating determining
There is 5%CO 2damp atmosphere, in perfect medium, cultivate the MCF-7 SKBr3 available from American type culture collection (Manassas, VA) at 37 DEG C, described perfect medium contains RPMI1640 (LifeTechnologies, Inc., GrandIsland, NY), and be supplemented with 10% foetal calf serum (SigmaChemicalCo., St.Louis, MO), 2mML-glutamine, 100 units/ml penicillin and 100 μ g/ml Streptomycin sulphates.With every hole 10 4individual cell to seed cells in 96 orifice plates and from the different extent of dilution of the Anti-HER 2 of test incubation 72 hours in triplicate.For assessment cell survival, add 150 μ lMTT reagent [3-(4,5-lutidine (dimethylthiazol)-2-base)-2,5-diphenyltetrazolium bromide, 0.5-1mgml; SigmaChemicalCo., St.Louis, MO] and incubation 4 hours.Use spectrophotometer to carry out chromaticity evaluation at 570nm, and the Growth of Cells per-cent calculated from the absorbance of untreated and treated cell is as follows: untreated OD570 × 100 of OD570/ of % Growth of Cells=treated.Figure 10 shows anti-VEGF/HER2 dual specific IgJR antibody and Trastuzumab (Herceptin) without significant difference in SKBr-3 cell proliferating determining, and anti-VEGF/HER2 dual specific IgJR antibody retains Trastuzumab vitro efficacy.Also do not predict the external model based on cell of the synergistic effect of anti-vegf/HER2.In body, mouse xenograft tumor model will be used for the synergy of showing anti-VEGF/HER2 dual specific IgJR antibody, i.e. the effect of anti-VEGF/HER2 dual specific IgJR antibody is better than the effect of only rhuMAb-VEGF or Trastuzumab.
Embodiment 2: the generation of anti-VEGF/ANG-2 bi-specific antibody and property analysis
Embodiment 2 and embodiment 3 are only that the sequence of antibody is different from what distinguish in embodiment 1, and the expression vector of use, position, other materials are identical with experimental technique.Below just sketch embodiment 2 and embodiment 3, specific experiment step please refer to embodiment 1.
Antigen:
VEGF-A has 232 amino acid whose sequences (UniProtKB-P15692).ANG-2 (ANGP2) has 496 amino acid whose sequences (UniProtKB-O15123).
Antibody sources:
Exemplary anti-vegf/ANG-2 bi-specific antibody is by people's anti-vegf-A antibody, rhuMAb-VEGF (bevacizumab) ( and the bi-specific antibody that formed of the anti-ANG-2 antibody of people Genentech).The aminoacid sequence of rhuMAb-VEGF at US7,297, open in 334.The aminoacid sequence of the anti-ANG-2 antibody of people is open in US2013/0129722.
Embodiment 2.1, the structure of anti-VEGF/ANG-2 bi-specific antibody
As people such as Sambrook, J., Molecularcloning:Alaboratorymanual; ColdSpringHarborLaboratoryPress, ColdSpringHarbor, NewYork, 1989 descriptions, use standard recombinant dna technique method to operate DNA.Explanation according to manufacturers uses molecular biology reagents.
Specifically, all gene fragments of coding anti-VEGF/ANG-2 bi-specific antibody light chain and heavy chain are synthesized, and the DNA sequence encoding leading peptide (MSVPTQVLGLLLLWLTDARC (light chain signal peptide) (SEQIDNO:26) that the 5' of described gene fragment holds, MEWSWVFLFFLSVTTGVHS (heavy chain signal peptide) (SEQIDNO:25), the described secretion of leading peptide targeting proteins matter in eukaryotic cell (Figure 12).According to specification (givenspecification) the synthetic gene fragment that DNA2.0 (California, USA) is given, its coding corresponds to the amino acid of construct #A1, construct #A2 and construct #A3.
By side for limiting cleavage sites, the gene fragment of 5 ' HindIII and 3 ' EcoRI, is connected in HindIII and the EcoRI restriction site of pcDNA3.1 expression vector (lifetechnology) (Fig. 4).For detailed step, see the detailed description of the construction process of the JR antibody expression vector finally listed.DNA sequence dna is confirmed by DNA sequencing.The cysteine residues (C) held at the C of the VL2 of the first polypeptide chain JRHC1 (construct #A1) does not participate in maintaining the protein-bonded correct conformation of dual specific IgJR, therefore can lack or be substituted, usually Serine is replaced to, to improve the protein-bonded oxidation-resistance of dual specific IgJR and to prevent abnormal crosslinked.Further, can one or more halfcystine key be joined in this antibody, to improve its stability.In the structure of IgJR bi-specific antibody, add halfcystine at the C-end of JRHC2 (construct #A3), form disulfide linkage (Figure 12) with the halfcystine of the hinge area EPKSCD with the first polypeptide chain JRHC1.
InvitrogenVectorNTAdvancesuite version 11 is used for sequence to produce, map, analyze, annotate and explanation.
Embodiment 2.2, the generation of anti-VEGF/ANG-2 bi-specific antibody
Use as in CurrentProtocolsinCellBiology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J.andYamada, K.M. (eds.), JohnWiley & Sons, the standard cell culture techniques described in Inc..At FREESTYLE tMtransient transfection is carried out in 293 expression systems (INVITROGEN, CALIFORNIA, USA).
According to the explanation of manufacturers, by using HEK293-Freestyle system (Invitrogen, California, USA) transient transfection difference encoded packets is containing VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3 (construct #A1), three plasmids of VL1-CL (construct #A2) and VH2-CH1 (construct #A3), produce antibody.X1 is short circuit head, such as EPKSCD (also can not having).In brief, with the mixture transfection HEK293-FreeStyle cell (HEK293-F cell) (Invitrogen) of above-mentioned three expression plasmids and 293fectin (Invitrogen), described cell expresses suspension growth in the shaking flask of substratum (Invitrogen) or the fermentor tank of stirring at the FreeStyle293 containing serum-free.Such as, with 1.0x10 6hEK293-F cell is inoculated in the 2L shaking flask (Corning) of the substratum containing 600mL by the density of cell/mL, and cultivates under 120rpm, 8%CO2.Latter second day of cell inoculation, at about 1.5x10 6under the cell density of individual cell/mL, with about 42mL mixture transfectional cell, described mixture comprises A) 20mLOpti-MEM (Invitrogen), wherein there are 600 μ g with total plasmid DNA (1 μ g/mL) of equimolar ratio rate coding VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3, VL1-CL and VH2-CH1 and B) 20mlOpti-MEM+1.2mL293fectin or fectin (2 μ Ι/mL).According to the consumption of glucose, add glucose solution during the fermentation.5-10 days results containing the supernatant liquor of secretory antibody, and direct antibody purification or by supernatant liquor freezen protective from supernatant liquor.
Embodiment 2.3, the purifying of anti-VEGF/ANG-2 dual specific IgJR antibody
Reference standard scheme is from the cell culture supernatant purifying anti-VEGF/ANG-2 bi-specific antibody filtered.In brief, antibody is applied on albumin A Sepharose post (GEHealthcare), and washs with PBS.The eluate of antibody is acid pH (pH is 3.5), immediately and eluate.At the 20mM Histidine of pH6.0, by size exclusion molecule chromatography (Superdex200, GEHealthcare), collectin matter is separated with monomeric igg in 140mMNaCl.Converge monomeric igg fraction, use such as MILLIPOREAmiconUltra (30MWCO) Centrifugal concentrators to concentrate monomeric igg fraction as required, and be stored in-80 DEG C.
By being determined at the optical density(OD) (OD) at 280nm place, use according to people such as Pace, ProteinScience, the molar extinction coefficient of the aminoacid sequence calculating of 1995,4,2411-1423, determines the protein concn of the antibody of purifying.
Embodiment 2.4: the analysis of anti-VEGF/ANG-2 dual specific IgJR antibodies character
Use is equipped with biosensor ((the fort é BIO that anti-hIgGFc catches (AHC) biosensor tips (anti-hIgGFccapture (AHC) biosensortip) and streptavidin bag quilt, MenloPark, CA)) fort é BIO qK esystem carries out avidity measurement.AHC avidity is measured, tests the anti-vegf/ANG-2 dual specific IgJR antibody of purifying and the binding ability at AHC sensor tip.Load most advanced and sophisticated with 20 μ g/ml anti-vegf/ANG-2 dual specific IgJR antibody.Load and within 300 seconds, cause catching level between 1.8 to 2nm.By being diluted to next VEGF-A (the R & d system) antigen for the preparation of binding analysis of 300nM in 1XPBS.Initial association also monitors 200 seconds, is transferred at tip in 1XPBS damping fluid (Gibco, PBSpH7.2) afterwards, dissociates 300 seconds to monitor.Then tip is transferred in the second antigen (VEGF-A), and monitor 200 seconds.Wash 300 seconds in 1 × PBS afterwards.Also carried out first loading VEGF-A antigen, loaded the experiment (Figure 13 A) of ANG-2 antigen subsequently.Then biostrome interference analysis (bio-layerinterferometryanalysis) is used to monitor the binding activities of anti-vegf/ANG-2 dual specific IgJR antibody in real time.Based on measured association rate constant (" K on") and dissociation rate constant (" K off") calculate antigen-binding affinity, be expressed as dissociation constant (K d).Use data analysis software 6.4 (fort é BIO) process also analytical data (Figure 13).Octet binding curve shows two kinds of antigens and combines continuously, is not when being exposed to the second antigen, and the first antigen is corresponding to be dissociated.This curve shows that dual specific IgJR antibody can associate the mode of (non-competitiveassociation) simultaneously in conjunction with two kinds of antigen VEGF and ANG-2 (Figure 13) with noncompetitive.The association rate constant (" K of anti-vegf/ANG-2 dual specific IgJR antibody and two kinds of antigen VEGF and ANG-2 is shown in table 3 on"), dissociation rate constant (" K off"), and avidity, be expressed as dissociation constant (K d).These experiments show, anti-vegf/ANG-2 dual specific IgJR antibody capable is combined with two kinds of antigen VEGF and ANG-2 with very high avidity.
Table 3, the association rate constant (" K of anti-VEGF/ANG-2 dual specific IgJR antibody and two kinds of antigen VEGF and ANG-2 on"), dissociation rate constant (" K off"), and avidity, be expressed as dissociation constant (K d).
Loading sample ID Sample ID K D(M) K on(1/Ms) K off(1/s)
Ig JR VEGF/ANG-2 VEGF 3.80x10 -10 2.10x10 5 7.98x10 -5
Ig JR VEGF/ANG-2 ANG-2 7.01x10 -12 1.91x10 5 1.34x10 -6
Embodiment 3: the generation of anti-TNF alpha/IL-17A bi-specific antibody and the analysis of binding property
Antigen:
TNFalpha has 233 amino acid whose sequences (UniProtKB – P01375).IL-17A (ANGP2) has 155 aminoacid sequences (UniProtKB-Q16552).
Antibody sources:
Exemplary anti-TNF alpha/IL-17A bi-specific antibody is by the anti-TNFalpha antibody of people, adalimumab (adalimumab) ( abbvie) and the anti-IL-17A antibody of people, the bi-specific antibody that Secukinumab (Cosentyx, Novartis) is formed.The aminoacid sequence of adalimumab is open in US599226/US31476.The aminoacid sequence of Secukinumab is open in US763673.
Embodiment 3.1, the structure of anti-TNF alpha/IL-17A bi-specific antibody
Specifically, all gene fragments of coding anti-TNF alpha/IL-17A bi-specific antibody light chain and heavy chain are synthesized, and the DNA sequence encoding leading peptide (MSVPTQVLGLLLLWLTDARC (light chain signal peptide) (SEQIDNO:26) that the 5' of described gene fragment holds, MEWSWVFLFFLSVTTGVHS (heavy chain signal peptide) (SEQIDNO:25)), the described secretion of leading peptide targeting proteins matter in eukaryotic cell (Figure 15).According to specification (givenspecification) the synthetic gene fragment that DNA2.0 (California, USA) is given, its coding corresponds to the amino acid of construct #C1, construct #C2 and construct #C3.
By side for limiting cleavage sites, the gene fragment of 5 ' HindIII and 3 ' EcoRI, is connected in HindIII and the EcoRI restriction site of pcDNA3.1 expression vector (lifetechnology) (Fig. 4).For further detailed description, see the materials and methods finally listed.DNA sequence dna is confirmed by DNA sequencing.The cysteine residues (C) held at the C of the VL2 of the first polypeptide chain JRHC1 (construct #C1) does not participate in maintaining the protein-bonded correct conformation of dual specific IgJR, therefore can lack or be substituted, usually Serine is replaced to, to improve the protein-bonded oxidation-resistance of dual specific IgJR and to prevent abnormal crosslinked.Further, can one or more halfcystine key be joined in this antibody, to improve its stability.In the structure of IgJR bi-specific antibody, add halfcystine at the C-end of JRHC2 (construct #C3), form disulfide linkage (Figure 15) with the halfcystine of the hinge area EPKSCD with the first polypeptide chain JRHC1.
InvitrogenVectorNTAdvancesuite version 11 is used for sequence to produce, map, analyze, annotate and explanation.
Embodiment 3.2, the generation of anti-TNF alpha/IL-17A bi-specific antibody
Use as in CurrentProtocolsinCellBiology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J.andYamada, K.M. (eds.), JohnWiley & Sons, the standard cell culture techniques described in Inc..At FREESTYLE tMtransient transfection is carried out in 293 expression systems (INVITROGEN, CALIFORNIA, USA).
According to the explanation of manufacturers, by using HEK293-Freestyle system (Invitrogen, California, USA) transient transfection difference encoded packets is containing VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3 (construct #C1), three plasmids of VL1-CL (construct #C2) and VH2-CH1 (construct #C3), produce antibody.X1 is short circuit head, such as EPKSCD, optionally also can not have.In brief, with the mixture transfection HEK293-FreeStyle cell (HEK293-F cell) (Invitrogen) of above-mentioned three expression plasmids and 293fectin (Invitrogen), described cell expresses suspension growth in the shaking flask of substratum (Invitrogen) or the fermentor tank of stirring at the FreeStyle293 containing serum-free.Such as, with 1.0x10 6hEK293-F cell is inoculated in the 2L shaking flask (Corning) of the substratum containing 600mL by the density of cell/mL, and cultivates under 120rpm, 8%CO2.Latter second day of cell inoculation, at about 1.5x10 6under the cell density of individual cell/mL, with about 42mL mixture transfectional cell, described mixture comprises A) 20mLOpti-MEM (Invitrogen), wherein there are 600 μ g with total plasmid DNA (1 μ g/mL) of equimolar ratio rate coding VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3, VL1-CL and VH2-CH1 and B) 20mlOpti-MEM+1.2mL293fectin or fectin (2 μ Ι/mL).According to the consumption of glucose, add glucose solution during the fermentation.5-10 days results containing the supernatant liquor of secretory antibody, and direct antibody purification or by supernatant liquor freezen protective from supernatant liquor.
Embodiment 3.3, the purifying of anti-TNF alpha/IL-17A dual specific IgJR antibody
Reference standard scheme is from the cell culture supernatant purifying anti-TNF alpha/IL-17A bi-specific antibody filtered.In brief, antibody is applied on albumin A Sepharose post (GEHealthcare), and washs with PBS.The eluate of antibody is acid pH (pH is 3.5), immediately and eluate.At the 20mM Histidine of pH6.0, by size exclusion chromatography (Superdex200, GEHealthcare), collectin matter is separated with monomeric igg in 140mMNaCl.Converge monomeric igg fraction, use such as MILLIPOREAmiconUltra (30MWCO) Centrifugal concentrators to concentrate monomeric igg fraction as required, and be stored in-80 DEG C.
By being determined at the optical density(OD) (OD) at 280nm place, use according to people such as Pace, ProteinScience, the molar extinction coefficient of the aminoacid sequence calculating of 1995,4,2411-1423, determines the protein concn of the antibody of purifying.
Embodiment 3.4, the analysis of anti-TNF alpha/IL-17A dual specific IgJR antibodies character
Use is equipped with biosensor ((the fort é BIO that anti-hIgGFc catches (AHC) biosensor tips (anti-hIgGFccapture (AHC) biosensortip) and streptavidin bag quilt, MenloPark, CA)) fort é BIO qK esystem carries out avidity measurement.AHC avidity is measured, tests the anti-TNFalpha/IL-17A dual specific IgJR antibody of purifying and the binding ability at AHC sensor tip.Load most advanced and sophisticated with 20 μ g/ml anti-TNFalpha/IL-17A dual specific IgJR antibody.Load and within 300 seconds, cause catching level between 1.8 to 2nm.By being diluted to next TNFalpha (the R & d system) antigen for the preparation of binding analysis of 300nM in 1XPBS.Initial association also monitors 200 seconds, is transferred at tip in 1XPBS damping fluid (Gibco, PBSpH7.2) afterwards, dissociates 300 seconds to monitor.Then tip is transferred in the second antigen (IL-17A), and monitor 200 seconds.Wash 300 seconds in 1 × PBS afterwards.Then biostrome interference analysis (bio-layerinterferometryanalysis) is used to monitor the binding activities of anti-TNFalpha/IL-17A dual specific IgJR antibody in real time.Based on measured association rate constant (" K on") and dissociation rate constant (" K off") calculate antigen-binding affinity, be expressed as dissociation constant (K d).Use data analysis software 6.4 (fort é BIO) process also analytical data (Figure 16).Octet binding curve shows two kinds of antigens and combines continuously, is not when being exposed to the second antigen, and the first antigen is corresponding to be dissociated.This curve shows that dual specific IgJR antibody can in the mode of noncompetitive association simultaneously in conjunction with two kinds of antigen TNFalpha and IL-17A (Figure 16).The association rate constant (" K of anti-TNF alpha/IL-17A dual specific IgJR antibody and two kinds of antigen TNFalpha and IL-17A is shown in table 4 on"), dissociation rate constant (" K off"), and avidity, be expressed as dissociation constant (K d).These experiments show, anti-TNFalpha/IL-17A dual specific IgJR antibody capable is combined with two kinds of antigen TNFalpha and IL-17A with very high avidity.
Table 4, the association rate constant (" K of anti-TNF alpha/IL-17A dual specific IgJR antibody and two kinds of antigen TNFalpha and IL-17A on"), dissociation rate constant (" K off"), and avidity, be expressed as dissociation constant (K d).
The detailed description of the construction process of JR antibody expression vector
Gene expression dose depends on that the adaptation selected the codon of mammalian cell selected by the codon of open gene.Use patented and precalculate the codon selection technique (DNA2.0 of (pre-computed), Inc) DNA sequence dna of VH1-CH1-(the X1)-VL2-CL-X2-CH2-CH3 chain (JRHC1 chain) in the aminoacid sequence of coding JR bi-specific antibody or VL1-CL chain (JRLC1 chain) or VH2-CH1 (JRHC2 chain) chain is optimized, under described codon selection technique carrys out comfortable strong translational selection, in the prediction group of the gene that mammalian cell camber is expressed.
Synthesized corresponding gene fragment (JRHCl, JRLC1 and JRHC2 chain) at DNA2.0, Inc, the side of described gene fragment is restriction cleavage sites, 5 ' HindIII and 3 ' EcoRI.
Show the collection of illustrative plates of three kinds of JR antibody expression vectors in the diagram.Substantially, the cDNA fragment of the 2.086Kb of coding HC1 chain is cloned into HindIII and the EcoRI restriction site in pCDNA3.1 carrier, produces plasmid, i.e. construct #1 carrier (Fig. 4).The cDNA fragment of the 0.714Kb of coding LC1 chain is cloned into the HindIII/EcoRI restriction site in pCDNA3.1 carrier, produces plasmid, i.e. construct #2 carrier.The cDNA fragment of the 0.732Kb of coding HC2 chain is cloned in the HindIII/EcoRI restriction site in pCDNA3.1 carrier, produces plasmid, i.e. construct #3 carrier.
By order-checking company, these three kinds of plasmids are checked order.
Experimental technique in plasmid construction adopts the conventional test method in this area to build.Particularly can see description below:
(1) restrictive diges-tion
A. below combining in 1.5ml centrifuge tube:
5uLpcDNA3.1 carrier
2uL10X damping fluid 4
1uLBSA
1uLHindIII
1uLEcoRI
10uLH 2O
B. mixed gently by pressure-vaccum.
C. incubation pipe 1 hour at 37 DEG C.
D. reactant is loaded in 1% sepharose.
1% sepharose preparation
Weigh 1g agarose;
Agarose powder is poured into there is 100ml1xTAE can in the flask of microwave heating, microwave heating 3min;
Agarose solution is made to cool 5min;
Add the final concentration of ethidium bromide (EtBr) to about 0.2-0.5 μ g/mL.At 4 DEG C, the gel newly poured into is placed 10-15 minute, until it solidifies completely;
Sample-loading buffer is joined often in the sample of digestion;
Carefully molecular weight ladder is loaded in the first swimming lane of gel;
Carefully sample is loaded in other holes of gel;
Gel is run, until dye line is greatly about being gel 75-80% in downward direction at 80-150V;
Powered-down, the electrode of deenergization, then removes gel carefully from gel box;
Use visual and the analyzing DNA fragment of imager (Imager);
Use QIAquick gel extraction kit (QIAquickGelExtractionKit) purifying DNA fragment from 1% sepharose.
(2) connect
A. below combining in an Eppendorf pipe:
The pcDNA3.1 carrier of 25ng restriction digest
75ngHC1 or LC1 or HC2 chain DNA fragment
Ligase enzyme damping fluid (for 10X damping fluid, 10 μ L reactants add 1 μ L damping fluid)
0.5-1 μ LT4DNA ligase enzyme
Add H 2o is 10 μ L extremely altogether
B. at room temperature incubation 2hr, carries out conversion below afterwards.
(3) transform
The DNA in falcon pipe, 5 μ L connected and 50 μ LDH5 α tMcompetent cell mixes.Mixed gently several times by the bottom of flicking pipe with finger;
Competent cell/DNA mixture is placed into 20-30min on ice;
By by 1/2 to 2/3 bottom pipe to being placed in 42 DEG C of water baths, each conversion tube of heat-shocked 45 seconds;
Pipe is put back into and continues 2min on ice;
Add 250 μ LSOC substratum and cultivate 45min in 37 DEG C of shaking culture casees;
Whole conversion product is applied on the 10cmLB agar plate containing penbritin;
Spend the night at 37 DEG C of culture plates;
Select bacterial colony, and be inoculated in the 2mlLB substratum in 10mlfalcon pipe, containing penbritin in described substratum;
37 DEG C of overnight incubation, carry out the small-sized preparation of following plasmid.
(4) the small-sized preparation of plasmid
QIAprep.kit is used to carry out the small-sized preparation of plasmid;
1.5ml bacterial cultures is transferred in 2ml Eppendorf tube, and at the centrifugal 1min of 13,000rpm.Abandon supernatant liquor;
The bacterial cell of precipitation is resuspended in 250 μ l damping fluid P1, and transfers in Eppendorf tube;
Add 250 μ l damping fluid P2, and put upside down test tube 4-6 mixing gently;
Add 350 μ l damping fluid N3, and put upside down test tube 4-6 time gently immediately;
In small table whizzer with 13,000rpm centrifugal 10 minutes;
By pressure-vaccum, the supernatant liquor from step 4 is applied on QIAprep centrifugal column;
Centrifugal 60s.Abandon and flow through liquid (flow-through);
QIAprep post is washed by adding 0.75ml damping fluid PE, and centrifugal 30-60s;
Abandon and flow through liquid, and more centrifugal 1min to remove residual lavation buffer solution;
QIAprep post is placed in clean 1.5ml Eppendorf tube.In order to eluted dna, 50 μ l water are joined the center of each QIAprep centrifugal column, leave standstill 1 minute, and centrifugal 1min.Namely the plasmid obtained can be used for cell transfecting.
Be understandable that, description is above only example of the present invention, and the scope that thus the claims in the present invention are protected not merely limits with particular disclosed herein.Any equivalent embodiment will be deemed to be within the scope of the present invention.In fact, according to description above, carrying out relevant modifications and variations to the present invention will be all possible for those skilled in the art, and thus, this modifications and variations are also by within the scope of claim that drops on appended by the present invention.

Claims (10)

1. the associated proteins comprising the first polypeptide chain of a dual specific or polyspecific, wherein said first polypeptide chain comprises VH1-CH1-(X1)-VL2-CL-X2-CH2-CH3, wherein VH1 is the first variable region of heavy chain, CH1 is CH1, and VL2 is the second variable region of light chain, and CL is constant region of light chain, X2 represents amino acid or the polypeptide of hinge area, CH2 and CH3 forms Fc district, and X1 is optional, and representative is used as amino acid or the polypeptide of joint.
2. associated proteins according to claim 1, wherein said associated proteins also comprise the second polypeptide chain and or the 3rd polypeptide chain, wherein said second polypeptide chain comprises VL1-CL, wherein VL1 is the first variable region of light chain, CL is constant region of light chain, and described 3rd polypeptide chain comprises VH2-CH1, wherein VH2 is the second variable region of heavy chain, and CH1 is CH1.
3. an associated proteins conjugate, it comprises the associated proteins described in aforementioned any one claim, and described associated proteins conjugate comprises further and is selected from following reagent: immunoadhesin molecule, developer, therapeutical agent and cytotoxic agent.
4. the nucleic acid be separated, the protein-bonded aminoacid sequence of its coding claim 1 or 2.
5. a carrier, it comprises the nucleic acid of separation according to claim 4.
6. a host cell, it comprises carrier according to claim 5.
7. produce a protein-bonded method, it cultivates the host cell of claim 6 under being included in and being enough to produce described protein-bonded condition in the medium.
8. a protein, its method according to claim 7 is produced.
9. a pharmaceutical composition, it comprises protein according to any one of claim 1-3 and 8 and pharmaceutically acceptable carrier.
10. any one of claim 1-3 and 8 protein for the preparation for the treatment of the disease of experimenter or the medicine of illness in purposes.
CN201510796803.7A 2015-11-18 2015-11-18 Bispecific or multispecific Ig JR binding protein Pending CN105542011A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106248971A (en) * 2016-08-19 2016-12-21 合肥瀚科迈博生物技术有限公司 For detecting the ELISA kit of HER2 content, using method and purposes in human serum
CN108218993A (en) * 2018-01-05 2018-06-29 李华顺 It is a kind of using ROBO1 as the bispecific antibody of target spot and its preparation and application
WO2019120245A1 (en) * 2017-12-22 2019-06-27 Chimagen Biosciences, Ltd. Covalent multi-specific antibodies
CN110382549A (en) * 2017-02-20 2019-10-25 Y生物股份有限公司 Novel multi-specific binding protein
CN113631574A (en) * 2019-01-31 2021-11-09 努玛治疗有限公司 Multispecific antibodies specific for TNF alpha and IL-17A, antibodies targeting IL-17A, and methods of use thereof

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CN102369215A (en) * 2009-04-02 2012-03-07 罗切格利卡特公司 Multispecific antibodies comprising full length antibodies and single chain fab fragments
CN103068847A (en) * 2010-08-24 2013-04-24 罗切格利卡特公司 Activatable bispecific antibodies

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN102369215A (en) * 2009-04-02 2012-03-07 罗切格利卡特公司 Multispecific antibodies comprising full length antibodies and single chain fab fragments
CN103068847A (en) * 2010-08-24 2013-04-24 罗切格利卡特公司 Activatable bispecific antibodies

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106248971A (en) * 2016-08-19 2016-12-21 合肥瀚科迈博生物技术有限公司 For detecting the ELISA kit of HER2 content, using method and purposes in human serum
CN110382549A (en) * 2017-02-20 2019-10-25 Y生物股份有限公司 Novel multi-specific binding protein
WO2019120245A1 (en) * 2017-12-22 2019-06-27 Chimagen Biosciences, Ltd. Covalent multi-specific antibodies
CN108218993A (en) * 2018-01-05 2018-06-29 李华顺 It is a kind of using ROBO1 as the bispecific antibody of target spot and its preparation and application
CN113631574A (en) * 2019-01-31 2021-11-09 努玛治疗有限公司 Multispecific antibodies specific for TNF alpha and IL-17A, antibodies targeting IL-17A, and methods of use thereof

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