CN1997668A - Immunoglobulins - Google Patents

Abstract

The present invention concerns immunoglobulins, such as antibodies, which specifically bind Oncostatin M (OSM), particularly human OSM (hOSM) and modulate the interaction between OSM and gp130. In typical embodiments, OSM is glycosylated. The invention also concerns antibodies that modulate the interaction between both Site II and Site III of OSM and their respective interacting partners. Further disclosed are pharmaceutical compositions, screening and medical treatment methods.

Description

Immunoglobulin (Ig)

Invention field

The present invention relates to immunoglobulin (Ig), its specificity is in conjunction with system knurl albumen M (OSM), particularly people OSM (hOSM).More particularly, the present invention relates to the antibody of specificity in conjunction with hOSM.The invention still further relates to the method for described immunoglobulin therapy disease or obstacle, the medicinal compositions that contains described immunoglobulin (Ig) and production method.By the apparent others of the present invention of following description.

Background of invention

System knurl albumen M is the glycoprotein of a kind of 28KDa, interleukin 6 (IL-6) family that belongs to cytokine, this family comprises IL-6, leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), myocardial nutrition albumen-1 (CT-1) and the myocardial nutrition albumen-1 like cell factor (referring to (1995) Blood 86:1243-1254 such as Kishimoto T), their shared gp130 transmembrane signal transduction acceptors (referring to Taga T and Kishimoto T (1997) Annu.Rev.Immunol.15:797-819).OSM is found (referring to Malik N (1989) etc., Mol Cell Biol 9:2847-2853) at first because of it suppresses melanoma cell series A375 growth.Subsequently, found more multiaction, found that it is the multi-functional mediators that is similar to other member of IL-6 family.OSM produces in various cell types, comprises scavenger cell, activating T cell (referring to Zarling JM (1986) PNAS (USA) 83:9739-9743), polymorphonuclear neutrophisls (referring to (1999) Blood 93:1413-1421 such as GrenierA), eosinophilic granulocyte (referring to (2002) Dev.Dyn.225:327-31 such as Tamura S), dendritic cell (referring to (2002) Cytokine 17:335-340 such as Suda T).It is present in pancreas, kidney, testis, spleen, stomach and brain (referring to (2005) Anat Rec A Discov Mol Cell Evol Biol 283:182-186 such as Znoyko I) and the marrow (referring to (2003) Acta Haematol 109:68-75 such as Psenak O).Its main biological action comprises activation endothelium (referring to (1993) Blood 82:33-7 such as Brown TJ), activate acute phase reaction (referring to (1996) Blood 87:1851-1854 such as Benigni F), inducing cell propagation or differentiation, regulating the inflammatory mediators discharges and hemoposieis (referring to (2003) 102:3154-3162 such as Tanaka M), bone is rebuild (referring to de Hooge ASK (2002) Am J Pathol160:1733-1743) and is promoted vasculogenesis (referring to (1999) ArteriosclerThromb Vasc Biol19:1835-1842 such as Vasse M) and wound healing.

OSM acceptor (OSM acceptor β, " OSMR β ") extensively expressing on the cell, comprise epithelial cell, chondrocyte, inoblast (referring to (2003) J Immunol170:548-555 such as Langdon C), neurone, unstriated muscle, lymphoglandula, bone, heart, small intestine, lung and kidney (referring to (2002) Mech Dev 115:127-131 such as Tamura S) and endotheliocyte.The evidence prompting of some aspects, endotheliocyte is the main target of OSM.These cells show phenotypic alternation of far-reaching significance and secular (referring to (1997) J Clin Invest 100:158-168 such as Modur V) stimulate the back to express the 10-20 doubly high-affinity and the low affinity receptor of high quantity with OSM.In addition, OSM is the main autocrine growth factor of Kaposi sarcoma cell, and the Kaposi sarcoma cell is considered to endothelium origin (referring to (1995) J ClinInvest 96:1319-1327 such as Murakami-Mori K).

The same with other IL-6 family cytokine, OSM is in conjunction with transmembrane signal transduction glycoprotein gp130.The key feature of gp130 cytokine is to form the oligomerization receptor complex, and it comprises gp130 and one or more depend on the co-receptor (summarizing in (2003) Biochem such as Heinrich PC J.374:1-20) of part.Therefore, according to the composition of formation receptor complex, these cytokines all can mediate total and unique biological activity in vitro and in vivo.The difference of people OSM (hOSM) and other IL-6 cytokine is, its can with gp130 and two kinds of co-receptor LIFR or system knurl protein receptor (OSMR) in any form mixture.Fig. 1 illustrates the interaction between hOSM and gp130, LIFR and the OSMR.Cracked the crystalline structure of hOSM already, shown that this structure contained 4 α helical bundles and 2 potential glycosylation sites.By hOSM molecule site-directed mutagenesis being identified two independently ligand-binding site points (referring to (2000) Structural Fold Des.8:863-874 such as Deller MC).First is called site II (being sometimes referred to as " site 2 "), and itself and gp130 interact, and second site is called site III (being sometimes referred to as " site 3 "), in opposite molecular end, and itself and LIFR or OSMR interaction.Mutagenesis experiment shows, the binding site of LIFR and OSMR much at one, but the single amino acids sudden change can be distinguished the two.

OSM is synthesized as precursor protein, and precursor protein comprises hydrophobic N end signal sequence and 33 amino acid whose C end propetides of 25 amino acid (AA), and these two is cut, and produces ripe OSM.The certain biologically active of OSM precursor protein, but significantly increased active (referring to (1992) Prog.Growth Factor Res.4:157-170 such as Bruce A.G., (1989) Mol.Cell Biol.9:2847-2853 such as Malik N) by cutting C end propetide.Already OSM was described as " compact column type molecule ", had the dimension of about 20  * 27  * 56 .4 alpha helical regions (spiral C 105-131 AA and spiral D 159-185 AA, AA number and begin after removing signal sequence for spiral A 10-37 AA, spiral B 67-90 AA) are arranged.Spiral A and C comprise " kinking ".Spiral connects by two outstanding rings (AB ring 38-66 AA, CD ring 130-158 AA), is arranged in two antiparallels to (A-D and B-C).(referring to (2000) Structure 8 such as Deller M.C; 863-874).

OSM through site II in conjunction with as if gp130 can make another OSM molecule interact by site III in conjunction with gp130.OSM also passes through site III in conjunction with LIFR or OSMR.Therefore, OSM and its acceptor form mixture, and this mixture is made of 1 gp130,1 LIFR or OSMR and 2 OSM molecules.(referring to Sporeno E (1994) J.Biol.Chem.269:10991-10995, (1998) Prot.Engineer 11:1093-1102 and Gearing D.P (1992) Science 225:306-312 such as Staunton D).

Utilize mutagenesis to find, for site II OSM-gp130 combination, important residue is Gln20, Gly120, Gln16 and Asn124.For site III OSM-OSMR combination, important residue is Phe160 and Lys163.Therefore, OSM site II interacts and to depend on Gln20, Gly120, Asn124 on the hOSM, and than low degree depend on Gln16 on the hOSM.Having identified 3 complementary residues (Phe169, Tyr196 and Glu282) in gp130 merits attention in OSM and gp130 interaction especially.(referring to (2000) Structure 8:863-874 such as Deller M, (2002) J.Mol.Biol.315:637-646 such as Aasland D, (2000) FEBS Lett.468:120-124 such as Timmermann A).

SEQ.I.D.NO:13 has listed the hOSM aminoacid sequence by 1 beginning.

MGVLLTQRTLLSLVLALLFPSMASMAAIGSCSKEYRVLLG QLQK QTDLMQD

TSRLLDPYIRIQGLDVPKLREHCRERPGAFPSEETLRGLGRRGFLQTLNAT

LGCVLHRLADLEQRLPKAQDLERSGLNIEDLEKLQMARPNIL GLRN NIYCM

AQLLDNSDTAEPTKAGRGASQPPTPTPASDAFQRKLEGCRFLHGYHRFMHS

VGRVFSKWGESPNRSRRHSPHQALRKGVRRTRPSRKGKRLMTRGQLPR.

(SEQ.I.D.NO：13)

The site II residue that will note especially highlights with runic, and underlines.

SEQ.I.D.NO:14 has listed the cDNA of coding hOSM.

ATGGGGGTACTGCTCACACAGAGGACGCTGCTCAGTCTGGTCCTTGCACTC

CTGTTTCCAAGCATGGCGAGCATGGCGGCTATAGGCAGCTGCTCGAAAGAG

TACCGCGTGCTCCTTGGCCAGCTCCAGAAGCAGACAGATCTCATGCAGGAC

ACCAGCAGACTCCTGGACCCCTATATACGTATCCAAGGCCTGGATGTTCCT

AAACTGAGAGAGCACTGCAGGGAGCGCCCCGGGGCCTTCCCCAGTGAGGAG

ACCCTGAGGGGGCTGGGCAGGCGGGGCTTCCTGCAGACCCTCAATGCCACA

CTGGGCTGCGTCCTGCACAGACTGGCCGACTTAGAGCAGCGCCTCCCCAAG

GCCCAGGATTTGGAGAGGTCTGGGCTGAACATCGAGGACTTGGAGAAGCTG

CAGATGGCGAGGCCGAACATCCTCGGGCTCAGGAACAACATCTACTGCATG

GCCCAGCTGCTGGACAACTCAGACACGGCTGAGCCCACGAAGGCTGGCCGG

GGGGCCTCTCAGCCGCCCACCCCCACCCCTGCCTCGGATGCTTTTCAGCGC

AAGCTGGAGGGCTGCAGGTTCCTGCATGGCTACCATCGCTTCATGCACTCA

GTGGGGCGGGTCTTCAGCAAGTGGGGGGAGAGCCCGAACCGGAGCCGGAGA

CACAGCCCCCACCAGGCCCTGAGGAAGGGGGTGCGCAGGACCAGACCCTCC

AGGAAAGGCAAGAGACTCATGACCAGGGGACAGCTGCCCCGGTAG

(SEQ.I.D.NO：14)

Rheumatoid arthritis (RA) comprises one group of completely different but syndrome of inherent relevant pathologic process.These pathologic processes are: local and system inflammation, synovial cell breed, cause forming the vasculogenesis and the apposition of pannus tissue, and the invasion and attack of pannus tissue and destruction cartilage and bone produce distortion and deformity.The basis of this pathology is to discharge cytokine and the inflammatory mediators (is stated from Rheumatology.Hochberg referring to Firestein G (2003) by enter and stay on cell in the inflammation joint and endogenous joint tissue cell long-period, Silman, Smolen, Weinblatt and Weisman edit .Pub.Mosby.855-884).Initiation event among the RA is unknown, but a large amount of evidence shows, they relate to external source or from " self " antigen activates T of body lymphocyte (referring to Firestein G (2004) J Clin Invest 114:471-4).In case they are activated, the degree that then keeps the course of disease to carry out required T cell also is uncertain, although the therapeutical agent of selectively targeted T cell such as CTLA4Ig may be to terminal illness effective (referring to (2003) New Engl J Med 349:1907-15 such as Kremer JM, (2004) Annual meeting of the American College of RheumatologyAbstract 1475 such as Moreland L).

Comprise that in the developing incident the earliest of rheumatoid synovial monokaryon and polymorphonuclear cell raise, to pass the endothelium in synovial membrane-lining cell's layer kapillary.Polymorph migrates in the synovia (SF), and lymphocyte remains on the kapillary vicinity, and it can be organized subsequently and turn to the dystopy lymph follicle.After this immunocyte flows into inoblast sample synovial cell (FLS) propagation.Different with its normal counterpart, as if RA FLS has avoided causing the regulate process of propagation and apoptosis stagnation, causes it to continue accumulation (referring to (2004) Arthritis Res Ther 7:12-18 such as Yamanishi Y).And emerging pannus is organized and is born the neovascularity of being supported by extracellular matrix now, further expands to allow it.This process relates to fibroblast proliferation, matrix is rebuild and vasculogenesis, is very similar to not controlled wound healing incident.Monocyte migrates in the developing pannus tissue, and the experience differentiation, becomes the scavenger cell with chronic activation phenotype.Equally, the end differentiation eventually of B cell experience forms the long lifetime plasma cell, and its secretion comprises the antibody of Rheumatoid factors, polyclonal.The part that the ability that the synovial membrane of inflammation is kept the local differentiation of medullary cell and lymphocyte is based in part on somatomedin (for example GMCSF and IL-6) produces.FLS and inherent monocyte all discharge soluble factor, and it stimulates inflammatory cell further to be raised by blood, and in case of emergency drive in the course of disease next step-destroy joint cartilage and bone is rebuild.The pannus tissue is invasive.Destructive enzyme for example MMP and the cytokine that changes cell (it keeps cartilage and bone structure integrity) phenotype are secreted in its forward position.As a result, proteoglycan is lost, and the II collagen type irreversibly is cut, and causes cartilage reduction and loses.Bone also experiences many far-reaching variations, comprises that focus erosion, subchondral bone matter are loose.Finally, these variations cause late that the distortion of observed joint characteristic and subluxation (are stated from Rheumatology.Hochberg, Silman, Smolen referring to Gordon D and Hastings D (2003) among the RA, Weinblatt and Weisman edit, Pub.Mosby.765-780).

RA is a systemic disease, may be to produce owing to the inflammatory mediators is entered in the blood by the joint.This influences the intravital many tracts of machine, comprise skin, eye, liver, kidney, brain and blood vessel lining cell, cause M ﹠ M to increase and (be stated from Rheumatology.Hochberg referring to Matteson EL (2003), Silman, Smolen, Weinblatt and Weisman edit, Pub.Mosby.781-792).Most of mortality ratio is too high to be because the cardiovascular disorder that atherosclerosis causes all has atherosclerotic plaque formation because rheumatoid synovial develops related many pathologic processes.

The target of RA treatment is pain management, reduces inflammation and blocks the process that causes disorganization.Traditionally, with on-steroidal anti-inflammatory medicine (NSAIDS), low dosage steroid and so-called antirheumatic (DMARDS) the treatment RA that improves the state of an illness.Effort levels is low, outbreak is slow, toxicity, tolerance difference and the resistance increase that makes progress have in time influenced the application of these treatments (comprising methotrexate (MTX), sulfasalazine, golden preparation and leflunomide).Recently, the bio-pharmaceutical (Enbrel for example that introduce to suppress cytokine tumour necrosis factor (TNF) ^TM, Remicide ^TMAnd Humira ^TM) existing major progress (referring to Roberts L and McColl GJ (2004) Intern Med J 34:687-93).

Therefore, target of the present invention provides the methods of treatment of a kind of RA of treatment and other disease and obstacle, and described other disease and obstacle be chronic inflammatory disease and obstacle specifically, for example osteoarthritis and psoriatic.Specifically, target of the present invention provides immunoglobulin (Ig), particularly specificity is in conjunction with OSM (hOSM for example, its site II specifically) and in disease and treating dysfunction regulate the interactional antibody between (promptly suppressing or blocking-up) OSM and the gp130, wherein said disease and obstacle respond to this interactional adjusting.

Have more and more evidences to support following hypothesis: regulating the OSM-gp130 interaction may be useful to treating such disease and obstacle.

Clinical evidence

OSM is present among people RA patient's the SF (referring to (1997) 56:184-7 such as Hui W).These levels and following parameter correlation: the neutrophilic granulocyte number among the SF, (being " TNF " sometimes) level of the TNF α among the SF and cartilage destruction marker ((2000) ArthritisRheum 43:281-288 such as Manicourt DH).And RA patient's synovial tissue spontaneously exsomatizes and secretes OSM (referring to (1997) Arthritis and Rheumatism 40:1096-1105 such as Okamoto H).Also show, OSM is present in ((1998) ArthritisRheum 41:1760-1771 such as Cawston TE) in the synovia scavenger cell, as described in not long ago, OSM acceptor and gp130 express on endotheliocyte, synovioblast, chondrocyte and scleroblast.And, soaking into the cell expressing OSM of atherosclerotic plaque and aortic aneurysm, this points out this cytokine relevant with chronic inflammatory diseases (referring to (2001) Ann NY Acad Sci 936:621-4 such as Mirshahi F).

External evidence

The OSM acceptor quantity that endotheliocyte is expressed is 10-20 times (referring to (1991) J Immunol 147:2175-2180 such as Brown TJ, (1989) JBiol Chem 264:4282-4289 such as Linsley PS) of other cell type.OSM combines individually or with other cytokine and activates endothelium synergistically, to discharge cytokine and chemokine, and, mediate it and extravasate into (referring to (1997) J Clin Invest 100:158-168 such as Modur V) in the synovial tissue in conjunction with neutrophilic granulocyte, monocyte and lymphocyte.Also show, OSM is that vasculogenesis (referring to (1999) Aterioscler Thromb Vasc Biol 19:1835-1842 such as Vasse M) becomes the effective stimulus agent of (therefore helping forming the pannus tissue, discharging IL-6, MMP) of fiber (FLS) cell activation and propagation with synovial membrane, and with TNF and IL-1 co-action, induce this mediators to discharge (referring to (2000) Am J Pathol 157:1187-1196 such as Langdon C).Show that also OSM (with IL-1) induces cartilage to discharge collagen protein and proteoglycan (referring to (1995) Biochem Biophys Res Commun 215:377-385 such as Cawston T).And, OSM inducing hepatocyte acute phase protein discharges and produces the IL-6 acceptor (referring to (1997) J Immunol 159:5648-5643 such as Cichy J, therefore Kurash JK (2004) Exp Cell Res 292:342-58), have and help the rheumatoid inflammation systemic effect of (comprising fatigue).In addition, OSM is at external evoked osteoclast differentiation and active (referring to (2002) JImmunol 169:3353-3362 such as Palmqvist P).

Evidence in the body

Gland virus expression mouse OSM (mOSM) produces serious inflammation and aggressiveness sacroiliitis (referring to (2000) Am J Pathol 157:1187-1196 such as Langdon C) in the joint of normal mouse.Equally, in the knock-out mice that lacks TNF, IL-1, IL-6 and iNOS, after adenovirus is transmitted mOSM, also observe aggressiveness disease (referring to (2003) Arthritis and Rheumatism 48:1750-1761 such as de Hooge ASK), this shows that OSM can mediate all aspects of sacroiliitis pathology.Use the mOSM vector expression mouse OSM of gland virus expression to cause the distinctive growth plate damage of juvenile idiopathic arthritis (referring to (2003) Arthritis and Rheumatism 48:1750-1761 such as de Hooge ASK).In the arthritic experimental model of bringing out property of collagen protein, therapeutic gives mouse anti OSM antibody preventable disease whole further evolution.When having the anti-OSM of the arthritic mouse of bringing out property of pristane (making the people associate recurrence/restoration model of human disease), observe similar result (referring to (2001) Arthritis and Rheumatism 44:2697-2702 such as Plater-Zyberk C) when preventative.In monkey, subcutaneous injection OSM brings out acute phase reaction and local chronic inflammatory diseases (referring to (1999) Toxicol Pathol 27:151-155 such as Loy JK).Showed already that OSM was in being injected into the goat joint time, brought out that monokaryon and PMN soak into and proteoglycan discharges (referring to (1999) Arthritis Rheum 42:2543-2551 such as Bell MC).The transgenosis of mOSM in the mouse lymph knot crossed to express and caused T cell maturation outside the thymus gland, memory T cell propagation and can not get rid of autoimmunization T cell (referring to (2003) Blood 102:1397-1404 such as Louis I).The transgenosis of OSM in pancreas cross express cause with late period RA synovial membrane in the similar extensive fibrosis (referring to (1995) Mol Cell Biol 15:2349-2358 such as Malik N) of observed situation.

In WO99/48523, we disclose the purposes of OSM antagonist in inflammatory diseases and treating dysfunction.Its disclosure is used anti-mouse OSM antibody in the mouse arthritis model.

Disclosed in this manual all patents and reference all clearly and intactly are hereby incorporated by.

Summary of the invention

The present inventor's supposition, regulate (particularly blocking-up) the site II of hOSM and the interaction between the gp130 with specificity in conjunction with the antibody of hOSM, to regulate the multiple compound signal transduction of all potential OSM acceptors, the biological activity of this cytokine effectively will be neutralized to the treatment significance degree.However, but the present inventor finds, seals the site II of hOSM and the neutralization that site III promotes this cytokine astoundingly simultaneously.And the present inventor finds, the hOSM glycosylation plays beyond thought effect in hOSM and specificity in conjunction with the binding events between the antibody of hOSM.

The present invention therefore provide specificity in conjunction with hOSM and with interactional therapeutic antibodies 15E10 of the site II of hOSM or 10D3 (it can be chimeric antibody, people's antibody, humanized antibody, bi-specific antibody or its Fab).See table A.

In one embodiment of the invention, provide specificity in conjunction with hOSM and regulate interactional therapeutic antibodies or its Fab between the site II of (promptly suppressing or blocking-up) hOSM and the gp130.In certain embodiments, therapeutic antibodies or its Fab specificity are in conjunction with the site II of hOSM.

In another embodiment, provide therapeutic antibodies or its Fab of specificity in conjunction with hOSM, it comprises following CDRH3:SEQ.I.D.NO:3 or SEQ.I.D.NO:42.

In another embodiment of the invention, therapeutic antibodies or its Fab of specificity in conjunction with hOSM is provided, it comprises following CDR:

CDRH1：SEQ.I.D.NO：1

CDRH2：SEQ.I.D.NO：2

CDRH3：SEQ.I.D.NO：3

CDRL1：SEQ.I.D.NO：4

CDRL2：SEQ.I.D.NO：5

CDRL3：SEQ.I.D.NO：6

In another embodiment of the invention, therapeutic antibodies or its Fab of specificity in conjunction with hOSM is provided, it comprises following CDR:

CDRH1：SEQ.I.D.NO：40

CDRH2：SEQ.I.D.NO：41

CDRH3：SEQ.I.D.NO：42

CDRL1：SEQ.I.D.NO：43

CDRL2：SEQ.I.D.NO：44

CDRL3：SEQ.I.D.NO：45

In whole this specification sheets, term " CDR ", " CDRL1 ", " CDRL2 ", " CDRL3 ", " CDRH1 ", " CDRH2 ", " CDRH3 " follow at Kabat etc.; Sequencesof proteins of Immunological Interest NIH, the Kabat coding scheme that proposes in 1987.Therefore, with the CDR of the present invention that given a definition:

CDR: residue

CDRH1：31-35B

CDRH2：50-65

CDRH3：95-102

CDRL1：24-34

CDRL2：50-56

CDRL3：89-97

In another embodiment of the invention, mouse therapeutic antibodies or its Fab are provided, it comprises and has sequence: the V of SEQ.I.D.NO:7 _HStructural domain and V with sequence: SEQ.I.D.NO:8 _LStructural domain.

In another embodiment of the invention, mouse therapeutic antibodies or its Fab are provided, it comprises and has sequence: the V of SEQ.I.D.NO:46 _HStructural domain and V with sequence: SEQ.I.D.NO:47 _LStructural domain.

In one embodiment of the invention, provide humanization therapeutic antibodies or its Fab, it comprises the V with the listed sequence of SEQ.I.D.NO:9 _HChain and V with the listed sequence of SEQ.I.D.NO:10 _LStructural domain.

In one embodiment of the invention, provide humanization therapeutic antibodies or its Fab, it comprises the V with the listed sequence of SEQ.I.D.NO:48 _HChain and V with the listed sequence of SEQ.I.D.NO:49 _LStructural domain.

In another embodiment of the invention, the humanization therapeutic antibodies is provided, this antibody comprises heavy chain with the listed sequence of SEQ.I.D.NO:11 and the light chain with the listed sequence of SEQ.I.D.NO:12.

In another embodiment of the invention, the humanization therapeutic antibodies is provided, this antibody comprises heavy chain with the listed sequence of SEQ.I.D.NO:50 and the light chain with the listed sequence of SEQ.I.D.NO:51.

In another embodiment of the invention, provide humanization therapeutic antibodies or its Fab, the interaction between its adjusting (promptly suppressing or blocking-up) hOSM and the gp130.

In another embodiment of the invention, provide to contain SEQ.I.D.NO:7 or SEQ.I.D.NO:9 or SEQ.I.D.NO:46 or SEQ.I.D.NO:48 or basic isolating V by its antibody of forming _HStructural domain.

In another embodiment of the invention, provide to contain to be selected from following V _HThe therapeutic antibodies of structural domain or its Fab: SEQ.I.D.NO:7, SEQ.I.D.NO:9, SEQ.I.D.NO:46, SEQ.I.D.NO:48.

In another embodiment of the invention, therapeutic antibodies or its Fab are provided, its competitive inhibition contains the combination of therapeutic antibodies of the CDRH3 of SEQ.I.D.NO:3.

In another embodiment of the invention, therapeutic antibodies or its Fab are provided, its competitive inhibition contains SEQ.I.D.NO:1,2,3,4,5 and 6 the therapeutic antibodies of CDR and combining of hOSM.

In another embodiment, provide therapeutic antibodies or its Fab, its competitive inhibition contains SEQ.I.D.NO:11 heavy chain and the therapeutic antibodies of SEQ.I.D.NO:12 light chain and combining of hOSM.

In another embodiment of the invention, provide treatment to suffer from the people patient's of disease or obstacle method, wherein said disease or obstacle respond to interactional adjusting between hOSM and the gp130, and this method comprises the treatment gonosome described herein that gives described patient treatment significant quantity or the step of its Fab.

In another embodiment of the invention, people patient's the method that provides treatment to suffer from inflammatory diseases or obstacle, this method comprise the treatment gonosome described herein that gives described patient treatment significant quantity or the step of its Fab.

In another embodiment of the invention, the method that provides treatment to suffer from the people patient of arthritis disease (particularly rheumatoid arthritis, childhood morbidity property sacroiliitis or osteoarthritis), this method comprise the treatment gonosome described herein that gives described patient treatment significant quantity or the step of its Fab.

In another embodiment of the invention, be provided at and suffer from the method that alleviates or prevent this degraded among the people patient of (or quick in) cartilage degradation, this method comprises the treatment gonosome described herein that gives described patient treatment significant quantity or the step of its Fab.

In another embodiment of the invention, be provided at and reduce the method that TNF α produces among the patient who suffers from disease or obstacle, minimizing responds to TNF α for wherein said disease or obstacle, and this method comprises treatment gonosome described herein or its Fab that gives described patient treatment significant quantity.

In another embodiment of the invention, the method of performance outside the joint of treatment of arthritis disease or obstacle is provided, and this method comprises and gives to be showed the treatment gonosome described herein of the people's patient treatment significant quantity that is perplexed or the step of its Fab in the joint of arthritis disease or obstacle outside.

In another embodiment of the invention, people patient's the method that provides treatment to suffer from endotheliocyte source disease, this method comprise the treatment gonosome described herein that gives described patient treatment significant quantity or the step of its Fab.

The present invention also provides treatment gonosome described herein or the purposes of its Fab in the medicine of disease described herein and obstacle is produced.

In another embodiment of the invention, provide the production method of treatment gonosome described herein or its Fab.

In another embodiment of the invention, research OSM (particularly hOSM) is provided and does interactional experiment between the mating partner (for example gp130, LIFR, OSMR) (particularly ELISA experiment) mutually, this experiment comprise provide glycosylation OSM sample (utilizing vertebrate host cell (for example mammalian host cell) glycosylation usually, for example CHO glycosylation) be used for described research step.

In the further embodiment of the present invention, we provide specificity in conjunction with Natively glycosylated hOSM and regulate (promptly suppressing or blocking-up) Natively glycosylated hOSM and be selected from gp130, LIFR, OSMR β make interactional therapeutic antibodies between the mating partner mutually.

We further provide the production method of the medicinal compositions that contains therapeutic antibodies, and wherein said therapeutic antibodies specificity is in conjunction with the interaction between hOSM and adjusting (promptly suppressing or blocking-up) hOSM and the gp130, and the method includes the steps of:

(a) provide the candidate antibody;

(b) provide glycosylation OSM (particularly hOSM and/or the Natively glycosylated hOSM that produces by the mammalian host cell (for example Chinese hamster ovary celI of recombinant conversion) of recombinant conversion or transfection);

(c) allowing under the bonded condition antibody of step (a) to be contacted with the hOSM of step (b);

(d) whether the antibody of determination step (c) regulates the interaction between hOSM and the gp130;

(e) make step (a) or described antibody humanization (d) alternatively;

(f) step (d) or described antibody (e) are joined in the medicinal compositions.

By hereinafter describing apparent others of the present invention, target and advantage.

The accompanying drawing summary

Fig. 1 is a width of cloth synoptic diagram, illustrates the interaction between OSM and gp130, LIFR and the OSMR β.

Fig. 2 illustrates according to the scheme of the use 15E10 of hereinafter embodiment proposition and 10D3 chimeric antibody and uses the gp130 of hOSM (last figure) and cOSM (figure below) to suppress ELISA.About further detailed description, description vide infra.

Fig. 3 illustrates the KB cell experiment that uses hOSM (last figure) and cOSM (figure below) according to the embodiment scheme of 15E10 that uses embodiment and 10D3 chimeric antibody.

The gp130 that Fig. 4 illustrates anti-hOSM (last figure) and cOSM (figure below) suppresses ELISA, wherein the inhibition percentage mapping that the antibody concentration with 4 kinds of humanized antibodies (B1L1, B1L2, B4L1, B4L2) and chimeric antibody 15E10 is changed.

The gp130 that Fig. 5 illustrates embodiment suppresses ELISA, wherein contrasts various humanized antibodies (B2L2, B3L2, B4L2) and the chimeric antibody 15E10 combination to the hOSM of CHO generation.

Fig. 6 illustrates Fig. 5 experiment of using cOSM to replace hOSM.

Fig. 7 illustrates Fig. 5 experiment of the 25% people AB serum solution of the hOSM that uses CHO production.

Fig. 8 illustrates Fig. 7 experiment of using cOSM to replace hOSM.

The gp130 that Fig. 9 illustrates the neutrophilic granulocyte OSM of 4 kinds of different people samples that use humanized antibody B2L2, B3L2, B4L2 and chimeric antibody 15E10 suppresses ELISA.

Figure 10 illustrates and uses anti-separation to suppress ELISA from the 3 kinds of humanized antibodies (B2L2, B3L2 and B4L2) of the hOSM of people RA patient synovia and the gp130 of 15E10 chimeric antibody.

Figure 11-16 illustrates with the result under Fig. 5-10 condition of KB cell experiment replacement gp130 inhibition ELISA, and the exception part is the single human sample that the KB cell experiment of the neutrophilic granulocyte OSM of Figure 15 uses neutrophilic granulocyte OSM.Therefore, Figure 11 illustrates the KB experiment of the hOSM of CHO production, Figure 12 illustrates the KB experiment of the cOSM of CHO production, Figure 13 illustrates the KB experiment of the 25% people AB serum solution of the hOSM that CHO produces, Figure 14 illustrates the KB experiment of the 25% people AB serum solution of the cOSM that CHO produces, Figure 15 illustrates the KB experiment of neutrophilic granulocyte OSM, and Figure 16 illustrates separation from the KB of the OSM of RA patient SF cell experiment.

The gp130 of Fc cracking mutant that Figure 17 illustrates parental generation mouse 15E10, chimeric antibody 15E10, humanized antibody construction B3L2 and the B3L2 of the hOSM that anti-CHO produces suppresses ELISA.About more detailed situation, referring to description.

Figure 18 illustrates Figure 17 experiment of using cOSM.

Figure 19 illustrates the KB cell experiment of Fc cracking mutant of parental generation mouse 15E10,15E10 chimeric antibody, humanization construction B3L2 and the B3L2 of the hOSM that anti-CHO produces.

Figure 20 is a width of cloth synoptic diagram, illustrates the competitive assay of embodiment.

Figure 21 illustrates the inhibition of the mouse 10D3 competition antibody of embodiment to 15E10 (B3L2 humanization construction).10D3 etc. volumetric molar concentration (0.15 μ g/ml) competes the inhibition percentage of thing to 15E10: 62.3%.

Figure 22 a illustrates the representative standard curve in the gp130-OSM ELISA that uses non-glycosylated OSM, and wherein being used to wrap by the gp130 concentration of elisa plate is 1 μ g/ml.

Figure 22 b shows, when being used to wrap when being increased to 4 μ g/ml by the gp130 concentration of plate, the sensitivity of gp130-OSM ELISA increases.

Figure 22 c illustrates with glycosylation and the two gp130-OSMELISA that carries out of non-glycosylated OSM.Non-glycosylated OSM: filled circles; Glycosylation OSM: hollow triangle.Notice the ELISA sensitivity of non-glycosylated OSM higherly, this may be the result that the epi-position that used detection antibody discerned has been sheltered in glycosylation.

Figure 23 a illustrates OSM neutralizing antibody Mab295 (R﹠amp; D Sytems) effect in gp130-OSM ELISA.Independent OSM: open circles; OSM+Mab296: solid triangle; OSM+MAb295 but on elisa plate, do not have gp130: filled squares.

Figure 23 b is how Mab295 can strengthen the OSM signal in gp130-OSM ELISA a synoptic diagram.

Figure 24 illustrates the data of KB cell experiment, has shown among the Mab295 and the validity of OSM.With the independent OSM of 1ng/ml or before experiment with this concentration of Mab295 blended OSM irritation cell of various concentration.Independent OSM: solid triangle; OSM+Mab295: open circles; No OSM stimulates: filled squares.

Figure 25 illustrates the effect of OSM site III specific antibody OM4-11.31 in gp130-OSMELISA.Independent OSM: open circles; OSM+ isotype contrast IgG: solid inverted triangle; OSM+ site II OSM specific antibody: open squares; OSM+OM4-11.31: filled circles.

Figure 26 illustrates mixture and the gp130 bonded inhibition of site II specificity OSM antibody OM4-5.3 to OSM and site III specific antibody (OM4-11.17).12.5ng/ml independent OSM: solid bars rod; OSM+OM4-11.17: diagonal bars rod; OSM+OM4-11.17+ contrasts IgG: netted lines rod; OSM+OM4-11.17+OM4-5.3: strokes and dots bar rod.

Figure 27 illustrates site and the appearance situation of non-site II specificity OSM antibody in personnel selection OSM mice immunized serum of using gp130-OSM ELISA to detect.Strengthen (being respectively a, b and c) post analysis serum for the first time, for the second time and for the third time at personnel selection OSM.OSM+ preimmune serum: open circles; OSM+ immune mouse antiserum(antisera): solid inverted triangle; OSM+ immune mouse antiserum(antisera) but on elisa plate, do not have gp130: hollow inverted triangle.

Figure 28 illustrate between the site II OSM specific antibody (" hum 15E10 ", humanization 15E10) in the KB cell experiment, measured and the site III specificity OSM antibody (17H10) in OSM and aspect synergetic property.The OSM neutralization of independent 17H10 (a) and independent hum 15E10 (b): filled circles; The OSM neutralization of antibody combination: hollow triangle.

Figure 29 illustrates the OSM pungency IL-6 excretory effectiveness that humanization 15E10 antibody suppresses the RA synovioblast.Each symbol is all represented the inoblast by different patients' acquisitions.

Figure 30 illustrates anti-OSM antibody OM4-5.3 to the inhibition of OSM in conjunction with gp130.OSM (25ng/ml) with shown in the OM4-5.3 preincubation of concentration, join in the elisa plate then.Independent OSM: filled circles; OSM+OM4-5.3: open circles.

Figure 31 a illustrates OM4-41.5 and suppresses glycosylation and the non-glycosylated OSM effectiveness difference in conjunction with gp130.Non-glycosylated OSM: filled circles; Glycosylation OSM: hollow triangle.

Figure 31 b illustrates OM4-5.3.1 and suppresses glycosylation and the non-glycosylated OSM effectiveness difference in conjunction with gp130.Non-glycosylated OSM: filled circles; Glycosylation OSM: hollow triangle.

Figure 32 has shown two kinds of site II specificity OSM antibody (a:15E10, b:5H2) activity of anti-glycosylation (filled circles) and non-glycosylated (hollow triangle) in gp130-OSM ELISA.

Figure 33 illustrates the paired sera of taking from RA patient and the association between serum among the SF and the synovia [OSM].

Figure 34 a, 34b and 35 illustrate the OSM concentration that the OSM ELISA that uses embodiment detects in the OA synovia.Figure 34 b shows that two samples have extra high OSM synovia concentration.

Figure 36 illustrates the OSM concentration that existed in the OA patients serum in 12 months clinical experiment cycles.# number is patient's identifier.

Figure 37 illustrates the typical OSM typical curve in 25% people AB serum.

Detailed Description Of The Invention

1. antibody structure

1.1 whole antibody

Whole antibody is generally heteromultimers glycoprotein, contains at least two heavy chains and two light chains. Except IgM, whole antibody is the allos tetramer glycoprotein of about 150Kda, is comprised of two identical light (L) chain identical with two heavy (H) chains. Usually, every light chain is connected to heavy chain by a covalent disulfide bonds, and the disulfide bond number between the different Immunoglobulin Isotype heavy chain is different. Every heavy chain and light chain all also have intrachain disulfide bond. Every heavy chain all has variable domains (V at an end_H), after connect many constant regions. Every light chain all has variable domains (V_L), constant region is at its another end; First constant region of constant region of light chain and heavy chain is in juxtaposition, and light chain variable domain and weight chain variable domain are in juxtaposition. Based on the amino acid sequence of constant region, the light chain of most of vertebrate kind antibody can be divided into a kind of in two kinds of types that are called κ and λ. According to its CH amino acid sequence, people's antibody can be divided into 5 different types: IgA, IgD, IgE, IgG and IgM. IgG and IgA can further be further divided into hypotype: IgG1, IgG2, IgG3 and IgG4; And IgA1 and IgA2. Have the species variant, Mouse and rat has IgG2a, IgG2b at least. The variable domains of antibody is given antibody binding specificity, and some district shows particular variable, is called complementary determining region (CDR). The conservative part of variable region is called framework region (FR). The variable domains of complete heavy chain being connected with light chain respectively contains 4 FR that connected by 3 CDR. CDR in every chain is kept closely approaching by the FR district, and impels the antigen binding site that forms antibody with the CDR of another chain. Constant region is not participated in the combination of antibody and antigen directly, but show various effector functions, for example participate in the cytotoxicity (ADCC) of antibody dependent cellular mediation, by means of in conjunction with the phagocytosis of Fc γ acceptor, by means of the half life/clearance rate of neonatal Fc receptor (FcRn) with utilize the CDC of the Clq component of complement cascade.

Therefore, in one embodiment, we provide the therapeutic complete antibody of specific binding hOSM, and this antibody is regulated the interaction between hOSM and the gp130. But the site II of antibody specific binding hOSM, and suppress or blocking-up hOSM and its interaction between the interactional corresponding residue of gp130 participation OSM. The ELISA method of embodiment can be used for determining whether any specific antibodies or its Fab regulate the interaction between hOSM and the gp130. Therapeutic complete antibody can comprise the constant region (or heavy chain or light chain) of any isotype mentioned above or its hypotype. In one embodiment, antibody is IgG isotype, particularly IgG1. Antibody can be rat, mouse, rabbit, primate or people's antibody. In a typical embodiments, antibody is primate (for example macaque, old century monkey (Old World monkey) or Giantopithecus (Great Ape), referring to for example WO99/55369, WO93/02108) or people's antibody.

The therapeutic complete antibody of the CDRH3 that contains SEQ.I.D.NO:3 or SEQ.I.D.NO:42 is provided in another embodiment. In another embodiment, provide contain have SEQ. I.D.NO:1,2,3,4, the therapeutic complete antibody of the variable region of 5 and 6 CDR or SEQ.I.D.NO:40,41,42,43,44 and 45 variable region.

In another embodiment, provide and contain the V with SEQ.I.D.NO:7 sequence_HThe V of domain and SEQ.I.D.NO:8 sequence_LThe mouse therapeutic complete antibody of domain or its Fab.

In another embodiment, provide and contain the V with SEQ.I.D.NO:46 sequence_HThe V of domain and SEQ.I.D.NO:47 sequence_LThe mouse therapeutic complete antibody of domain or its Fab.

1.1.2 people's antibody

Can utilize several different methods well known by persons skilled in the art to produce people's antibody. Can prepare people's antibody by the hybridoma method of end user's myeloma or mouse-people's xenogenesis myeloma cell line, referring to Kozbor J.Immunol 133,3001, (1984) and Brodeur.Monoclonal Antibody Production Techniques and Applications, 51-63 page or leaf (Marcel Dekker Inc, 1987). Alternative method comprise use all adopt the phage library of people V district repertoire or transgenic mice (referring to Winter G, (1994), Annu.Rev. Immunol 12,433-455, Green LL (1999), J.Immunol.methods 231,11-23).

Can utilize at present the transgenic mice strain that several its mouse immunoglobulin genes seats have replaced by human immunoglobulin gene's section (referring to Tomizuka K, (2000) PNAS 97,722-727; Fishwild D.M (1996) Nature Biotechnol.14,845-851, Mendez MJ, 1997, Nature Genetics, 15,146-156). When antigen was attacked, such mouse can produce people's antibody repertoire, can therefrom select purpose antibody.

It should be noted that especially human lymphocyte is implanted into the Trimera in the raying mouse^TMSystem is (referring to Eren R etc., (1998) Immunology 93:154-161), the selective (SLAM of lymphocyte antibody system, referring to Babcook etc., PNAS (1996) 93:7843-7848) (in this system, people's (or other species) lymphocyte has realized that effectively the external antibody of extensive combination produces step, succeeded by going flatung, limiting dilution and selection step) and Xenomouse II^TM(Abgenix Inc). Alternative can be by using Morphodoma^TMThe Morphotek Inc of technology obtains.

Display technique of bacteriophage can be used for producing people's antibody (and fragment), referring to McCafferty; Nature, (1994) the EMBO 13:3245-3260 such as 348,552-553 (1990) and Griffiths AD. According to this technology, antibody V domain gene is cloned in the main or less important coat protein gene of filobactivirus (for example M13 or fd) by frame, and shows that as the function antibody fragment (usually by means of helper phage) is on the phage particle surface. The encoding gene that causes selecting the antibody with those characteristics based on the selection of antibody function characteristic. Display technique of bacteriophage can be used for by selecting antigen-specific antibodies (referring to Marks in the library of taking from the human B cell preparation of suffering from the individual of above-mentioned disease or obstacle or taking from not immune people's donor; J.Mol.Bio. 222,581-597,1991). Contain at needs in the situation of people's whole antibody of Fc domain, the fragment that must will be derived from phage display is cloned into and contains required constant region and set up in the mammalian expression vector of stable expression cell line.

Affinity maturation technology (Marks; Bio/technol 10,779-783 (1992)) can be used for promoting binding affinity, wherein by in succession replacing H and L chain V district with natural variant and selecting to promote the affinity of primary human antibody take the binding affinity that promotes as the basis. Also can utilize at present the variant of this technology, for example " the epi-position marking " is referring to WO93/06213. In addition referring to Waterhouse; Nucl.Acids Res 21,2265-2266 (1993).

Therefore, in another embodiment, provide human therapy whole antibody or its Fab of specific binding hOSM and adjusting (namely suppressing or blocking-up) hOSM and gp130 interaction. In another embodiment, provide the site II of specific binding hOSM and human therapy whole antibody or its Fab of adjusting (namely suppressing or blocking-up) hOSM and gp130 interaction.

On the other hand, provide human therapy whole antibody or its Fab of the CDRH3 that contains SEQ.I.D.NO:3 or SEQ.I.D.NO:42, its specific binding hOSM, and the interaction between adjusting (namely suppressing or blocking-up) hOSM and the gp130. In another embodiment, provide contain have SEQ.I.D.NO:1, the variable region of 2,3,4,5 and 6 CDR or have human therapy whole antibody or its Fab of SEQ.I.D.NO:40,41,42,43,44 and 45 variable region.

1.2 chimeric antibody and humanized antibody

Use inhuman whole antibody treatment human disease or obstacle with present well-established potential Immunogenicity, especially when repeating to give antibody, that is to say that patient's immune system can be identified as non-self antibody with inhuman whole antibody, and set up neutralization reaction. Except exploitation human antibody (referring to above), various technology have in these years also been developed, to overcome these problems, these technology generally comprise the composition that reduces inhuman amino acid sequence in the therapeutic complete antibody, keep simultaneously being obtained by immune animal (for example mouse, rat or rabbit) non-human antibody's relatively easy property. Put it briefly, use two approach to realize this target. First approach is chimeric antibody, and it generally comprises inhuman (for example rodent, for example mouse) variable domains that merges to human constant region. Because the antigen binding site of antibody is arranged in the variable region, so chimeric antibody has kept its binding affinity to antigen, but obtained the effector function of human constant region, therefore can carry out effector function for example mentioned above. Chimeric antibody uses the recombinant DNA method to produce usually. Use conventional method (for example use can specific binding antibody of the present invention H and L chain encoding gene (for example SEQ.I.D.NO:1 mentioned above, 2,3,4,5 with 6 coding DNA) oligonucleotide probe) separate and measure the sequence of antibody coding DNA (for example cDNA). Hybridoma is as the typical case source of such DNA. In case separated, just DNA is placed expression vector, then it is transfected in the host cell (for example Escherichia coli, COS cell, Chinese hamster ovary celI or myeloma cell) that can not produce in other cases immunoglobulin (Ig), synthetic to obtain antibody. Can replace corresponding inhuman (for example mouse) H and L constant region by the coded sequence of employment L and H chain and come modifying DNA, referring to for example Morrison; PNAS 81,6851 (1984).

Second approach comprises that generation is wherein by making the variable region humanization reduce the humanized antibody of the inhuman ratio of antibody. Two kinds of humanization technology are popularized. First is to utilize CDR grafting humanization. CDR constructs the ring near antibody N end, and the surface that is on the support that is provided by framework region is provided for they at this place. The antigen-binding specificity of antibody is mainly limited by topological structure and the chemical feature on its CDR surface. These features are again by the conformation of single CDR, relative distribution and the character that CDR consists of the residue side chain and the decision that distributes of CDR. Arrive on suitable the people's framework (" acceptor " framework) and constant region by inciting somebody to action the only CDR grafting of inhuman (for example mouse) antibody (" donor " antibody), can realize that immunogenic significantly reduction is (referring to (1986) Nature 321 such as Jones, (1988) Science 239 such as 522-525 and Verhoeyen M, 1534-1536). But, the CDR grafting itself may not can keep antigenic binding property fully, often find if recover significant antigen binding affinity, then some framework residue of donor antibody must keep (being sometimes referred to as " back mutation ") in the humanization molecule (referring to (1989) PNAS 86,10 such as Queen C, 029-10,033, Co, M etc. (1991) Nature 351,501-502). In the case, can be by selecting to have the people V district of maximal sequence homology (being generally more than 60%) with inhuman donor antibody in the database, in order to people's framework (FR) is provided. Can carry out people FR by people's consensus sequence or single people's antibody selects. Where necessary, the Key residues of donor antibody is replaced the people be subjected in the body frame, to keep the CDR conformation. The microcomputer modelling of antibody can be used for the auxiliary structurally very important residue of this kind of differentiating, referring to WO99/48523.

Perhaps, can realize humanization by " frosting " method. Statistical analysis to distinctive people and rat immune globulin heavy chain and variable region of light chain discloses, the definite pattern of exposed residue is variant in people and mouse-anti body, and most of each surface location has strong skewed popularity (referring to Padlan E.A. etc. to the different residues of minority; (1991) Mol.Immunol.28, (1994) J.Mol.Biol.235 such as 489-498 and Pedersen J.T.; 959-973). Therefore, might by replacing exposed residues different from common those residues that exist in people's antibody in its framework region, reduce the immunogenicity of inhuman Fv. Because protein antigenicity may be relevant with surperficial accessibility, so may being enough to make the mouse variable region to become human immune system " sightless ", the displacement of surface residue (is stated from Handbook of Experimental Pharmacology 113 volumes referring to (1994) such as Mark G.E. in addition: The pharmacology of monoclonal Antibodies, Springer-Verlag, 134 pages of 105-). This humanization method is called as " frosting ", because only the antibody surface is changed, supports that residue maintains the original state. Other alternative is set forth in WO04/006955.

Therefore, another embodiment of the invention provides chimeric therapeutic antibodies, it comprises and merges that (it can be the IgG isotype to human constant region, IgG1 for example) inhuman (for example rodent) variable domains, its specific binding hOSM, and regulate the site II of hOSM and the interaction between the gp130.

In another embodiment, provide and contain inhuman (for example rodent) variable region and human constant region (it can be the IgG isotype, IgG1 for example) chimeric therapeutic antibodies, its specific binding hOSM, this antibody further comprises the CDRH3 of SEQ.I.D.NO:3 or SEQ.I.D.NO:42. Such antibody can further comprise the human constant region of IgG isotype (for example IgG1).

In another embodiment, provide and contain inhuman (for example rodent) variable region and human constant region (it can be the IgG isotype, IgG1 for example) chimeric therapeutic antibodies, its specific binding hOSM has SEQ.I.D.NO:1,2,3,4,5 and 6 or SEQ.I.D.NO:40,41,42,43,44 and 45 CDR.

In another embodiment, provide specific binding hOSM and adjusting (namely suppressing or blocking-up) the site II of hOSM and the humanization therapeutic antibodies of gp130 interaction or its Fab.

In another embodiment, provide specific binding hOSM and comprise humanization therapeutic antibodies or its Fab of the CDRH3 of SEQ.I.D. NO:3 or SEQ.I.D.NO:42. Such antibody can comprise the human constant region of IgG isotype (for example IgG1).

In another embodiment, provide specific binding hOSM and comprise SEQ.I.D. NO:1,2,3,4,5 and 6 or humanization therapeutic antibodies or its Fab of SEQ.I.D.NO:40,41,42,43,44 and 45 CDR. Such antibody can comprise the human constant region of IgG isotype (for example IgG1).

In another embodiment, humanization therapeutic antibodies or its Fab are provided, its specific binding hOSM regulates the interaction between hOSM and the gp130, and comprise the light chain of the heavy chain of SEQ.I.D.NO:11 and SEQ.I.D.NO:12 or substantially consisting of.

In another embodiment, humanization therapeutic antibodies or its Fab are provided, its specific binding hOSM regulates the interaction between hOSM and the gp130, this antibody comprise the light chain of the heavy chain of SEQ.I.D.NO:50 and SEQ.I.D.NO:51 or substantially consisting of.

In another embodiment, humanization therapeutic antibodies or its Fab are provided, its specific binding hOSM, regulate the interaction between hOSM and the gp130, wherein said antibody or its fragment comprise the CDRH3 of SEQ.I.D.NO:3, further comprise alternatively SEQ.I.D.NO:1,2,4,5 and 6 CDR, wherein people's acceptor heavy chain framework region 28,29,30,71 and 94 and people's acceptor light chain framework 49 and 71 s' residue is by the corresponding residue displacement that exists in the donor antibody framework that is derived from CDRH3.

In another embodiment, humanization therapeutic antibodies or its Fab are provided, its specific binding hOSM, regulate the interaction between hOSM and the gp130, wherein said antibody or its fragment comprise the CDRH3 of SEQ.I.D.NO:42, further comprise alternatively SEQ.I.D.NO:40,41,43,44,45 CDR, wherein the residue of 36,38,46,47,71 of people's acceptor heavy chain framework region 28,44,48,67,69,71,73 and people's acceptor light chain frameworks is by the corresponding residue displacement that exists in the donor antibody framework that is derived from CDRH3.

It will be apparent for a person skilled in the art that term " is derived from " source that not only is intended to limit on the Material Physics origin meaning, and be used for being equal to this material on the limiting structure but the material of not rising in mentioned source. Therefore, " residue that exists in the donor antibody that CDRH3 is derived from " need not necessarily by the donor antibody purifying.

In another embodiment, humanization therapeutic antibodies or its Fab of specific binding hOSM are provided, wherein said antibody or its fragment comprise the CDRH3 of SEQ.I.D.NO:3, further comprise alternatively SEQ.I.D.NO:1,2,4,5 and 6 CDR, wherein people's heavy chain framework comprises one or more (for example whole) following residue (or its conservative substitution thing):

The position residue

28             S

29             L

30             T

71             K

94             K

People's light chain comprises any or two in the following residue (or its conservative substitution thing):

The position residue

49             E

71             Y

In another embodiment, humanization therapeutic antibodies or its Fab of specific binding hOSM are provided, wherein said antibody or its fragment comprise SEQ.I.D.NO:1,2,3,4,5 and 6 CDR, and wherein people's heavy chain framework comprises one or more (for example whole) following residue (or its conservative substitution thing):

The position residue

28            S

29            L

30            T

71            K

94            K

People's light chain comprises any or two in the following residue (or its conservative substitution thing):

The position residue

49            E

71            Y

In another embodiment, humanization therapeutic antibodies or its Fab of specific binding hOSM are provided, wherein said antibody or its fragment comprise the CDRH3 of SEQ.I.D.NO:42, further comprise alternatively SEQ.I.D.NO:40,41,43,44,45 CDR, wherein people's heavy chain framework comprises one or more (for example whole) following residue (or its conservative substitution thing):

The position residue

28         I

48         I

44         K

67 A

69 L

71 V

73 K

People's light chain comprises one or more (for example whole) following residue (or its conservative substitution thing):

The position residue

36 F

38 K

46 R

47 W

71 Y

In another embodiment, humanization therapeutic antibodies or its Fab of specificity in conjunction with hOSM is provided, wherein said antibody or its fragment comprise SEQ.I.D.NO:40,41,43,44,45 CDR, and wherein people's heavy chain framework comprises one or more (for example whole) following residue (or its conservative substitution thing):

The position residue

28 I

48 I

44 K

67 A

69 L

71 V

73 K

People's light chain comprises one or more (for example whole) following residue (or its conservative substitution thing):

The position residue

36 F

38 K

46 R

47 W

71 Y

Art-recognized can regarding some aminoacid replacement as " conservative ".Side chain characteristic based on total is divided into a plurality of classifications with amino acid, keeps the whole of antibody of the present invention or its Fab or basic all displacements of binding affinity to be counted as conservative substitution in these classifications, sees table:

Side chain	The member
		Hydrophobicity	met、ala、val、leu、ile
Neutral hydrophilic	cys、ser、thr
		Acid	asp、glu
Alkalescence	asn、gln、his、lys、arg
		Influence the residue of chain direction	gly、pro
Aromatic series	trp、tyr、phe

1.3 bi-specific antibody

Bi-specific antibody is the antibody that at least two different epi-positions is had binding specificity.The method for preparing such antibody is known in the art.Traditionally, the recombinant production of bi-specific antibody is based on two IgH chains-right coexpression of L chain, wherein two H chains have different binding specificities, referring to Millstein etc., EMBO such as Nature 305 537-539 (1983), WO93/08829 and Traunecker, 10,1991,3655-3659.Because the random assignment of H and L chain so produce the potential mixture of 10 kinds of different antibodies structures, wherein only has a kind and has required binding specificity.Alternative method comprises that the variable domains that will have required binding specificity merges to containing to the CH in small part hinge area, CH2 and CH3 district.Preferably have and be contained in the light chain that exists at least a fusion rotein CH1 district in conjunction with necessary site.DNA and (if necessary) L chain of these fusion roteins of coding are inserted in the expression vector separately, and cotransfection is gone in the appropriate host biology then.But it is possible being inserted into the encoding sequence of two or whole three chains in the expression vector.In a preferred method, bi-specific antibody by the H chain that in one arm, has first kind of binding specificity and H-L chain that second binding specificity is provided in other one arm to forming, referring to WO94/04690.In addition referring to Methods inEnzymology such as Suresh 121,210,1986.

In one embodiment of the invention, provide the dual specific therapeutic antibodies, at least a binding specificity of wherein said antibody is at hOSM, and wherein said antibody is regulated (promptly suppressing or blocking-up) the site II of hOSM and the interaction between the gp130.Such antibody can further comprise the constant region of human IgG isotype (for example IgG1).

In one embodiment of the invention, provide the dual specific therapeutic antibodies, at least a binding specificity of wherein said antibody is at hOSM, and wherein said antibody comprises at least one CDRH3 among SEQ.I.D.NO:3 or the SEQ.I.D.NO:42.Such antibody can further comprise the constant region of human IgG isotype (for example IgG1).

In one embodiment of the invention, the dual specific therapeutic antibodies is provided, at least a binding specificity of wherein said antibody is at hOSM, and wherein said antibody comprises SEQ.I.D.NO:1,2,3,4 at least, 5 and 6 or SEQ.I.D.NO:40,41,42,43,44 and 45 CDR.Such antibody can further comprise the constant region of human IgG isotype (for example IgG1).

1.4 antibody fragment

In certain embodiments of the invention, provide interactional therapeutic antibodies fragment between adjusting OSM (particularly hOSM) and the gp130.Such fragment can be the functional antigen binding fragment of whole antibody and/or humanization and/or chimeric antibody, for example the Fab of antibody mentioned above, Fd, Fab ', F (ab ') ₂, Fv, ScFv fragment.Traditionally, such fragment digests whole antibody production (referring to for example WO94/29348) by for example papain digestion proteolysis, but can be directly by the host cell production of recombinant conversion.About the production of ScFv, referring to Bird etc.; (1988) Science, 242,423-426.In addition, can use various engineerings as mentioned below to produce antibody fragment.

As if the interaction energy of two chains of Fv fragment be lower than the Fab fragment.For stablizing V _HAnd V _LThe combination of structural domain is with peptide (Bird etc., (1988) Science 242,423-426, Huston etc., PNAS, 85,5879-5883), disulphide bridges (Glockshuber etc., (1990) Biochemistry, 29,1362-1367) with " pestle mortar structure (knob in hole) " sudden change (zhu etc. (1997), ProteinSci., 6,781-788) connect them.The ScFv fragment can be passed through the well-known method production of those skilled in the art, referring to (1991) Methods companion MethodsEnzymol such as Whitlow, and 2, (1993) Int.Rev.Immunol 10 such as 97-105 and Huston, 195-217.ScFv can produce in bacterial cell (for example intestinal bacteria), produces but be more typically in the eukaryotic cell.Unit price that the shortcoming of ScFv is a product and short half life thereof,, the unit price of product has been got rid of owing to multivalence increases in conjunction with the avidity that produces.For the trial that overcomes these problems comprises that (Adams etc. (1993) Can.Res 53, (1995) Protein Eng.8 such as 4026-4034 and McCartney are 301-314) by the ScFv production divalence (ScFv ') that contains additional C end halfcystine by chemical coupling ₂, or by contain in pairs the spontaneous locus specificity dimerization of the ScFv of C end cysteine residues (referring to (1995) Cell.Biophys 26 such as Kipriyanov, 187-204) production divalence (ScFv ') ₂Perhaps, can force ScFv to form polymer by peptide linker being foreshortened to 3-12 residue to form " double antibody (diabodies) ", referring to Holliger etc., PNAS (1993), 90,6444-6448.Dwindling joint can further produce the ScFV tripolymer (" three antibody (triabodies) " is referring to (1997) Protein Eng such as Kortt, 10,423-433) and the tetramer (" four antibody (tetrabodies) " referring to (1999) FEBS Lett such as Le Gall, 453,164-168).Also can by with albumen dimerization motif gene fusion to form " little antibody (miniantibodies) " (referring to (1992) Biochemistry 31 such as Pack, 1579-1584) and " miniantibody (minibodies) " (referring to (1996) such as Hu, Cancer Res.56,3055-3061), realize the structure of divalence ScFV molecule.Also can connect two ScFv unit, produce ScFv-Sc-Fv concatermer (tandem) ((ScFV) by utilizing the 3rd peptide linker ₂), referring to (1995) J.Immol.154 such as Kurucz, 4576-4582.Can by non-covalent in conjunction with two warm protein products of strand (by the V that utilizes short circuit head with a kind of antibody _HThe V of structural domain and another kind of antibody _LStructural domain connects and constitutes) produce the dual specific double antibody, referring to (1998) such as Kipriyanov, Int.J.Can 77,763-772.Can be by importing disulphide bridges or aforesaid " pestle mortar structure " sudden change or by forming wherein the stability that strand double antibody (ScDb) that two hybrid ScFv fragments connect through peptide linker strengthens such dual specific double antibody, referring to (1999) J.Immunol.Methods 226 179-188 such as Kontermann.By for example the ScFv fragment being merged CH3 structural domain or Fab fragment to the IgG molecule through hinge area, can obtain the tetravalence bispecific molecule, referring to (1997) Nature Biotechnol.15 such as Coloma, 159-163.Perhaps, by merge dual specific strand double antibody create the tetravalence bispecific molecule (referring to Alt etc., (1999) FEBS Lett 454,90-94).Also can (the DiBi molecular antibody referring to (1998) FEBSLett 432 such as Muller, 45-49) or to prevent that intermolecular paired direction from making contains 4 antibody variable territory (V by making ScFv-ScFv concatermer dimerization with the joint that contains helix-loop-helix motif _HAnd V _L) the single chain molecule dimerization (the series connection double antibody, referring to Kipriyanov etc., (1999) J.Mol.Biol.293 41-56), forms less tetravalence bispecific molecule.Can create dual specific F (ab ') by chemical coupling Fab ' fragment or by the assorted dimerization of leucine zipper ₂Fragment (referring to Shalaby etc., (1992) J.Exp.Med.175, (1992) such as 217-225 and Kostelny, J.Immunol.148,1547-1553).Also can utilize isolating V _HAnd V _LStructural domain (Domantis plc), referring to US 6,248,516, US 6,291,158, US 6,172,197.

In one embodiment, provide specificity in conjunction with hOSM and regulate interactional therapeutic antibodies fragment (ScFv for example mentioned above, Fab, Fd, Fab ', F (ab ') between the site II of (promptly suppressing or blocking-up) hOSM and the gp130 ₂) or the engineered antibody fragment).The therapeutic antibodies fragment can comprise the CDRH3 with SEQ.I.D.NO:3 sequence, the CDR that has SEQ.I.D.NO:1,2,4,5 and 6 listed sequences alternatively in addition, perhaps the therapeutic antibodies fragment can comprise the CDRH3 with SEQ.I.D.NO:42, has the CDR of SEQ.I.D.NO:40,41,43,44 and 45 listed sequences alternatively in addition.

1.5 the assorted antibody (Heteroconjugate antibody) of puting together

Assorted put together antibody and also constitute embodiment of the present invention.Assorted put together antibody and forms, use any cross-linking method formation easily by two covalently bound antibody.Referring to US4,676,980.

1.6 other modification

It is generally acknowledged, the effector function of the interaction mediate antibody between the Fc district of antibody and the various Fc acceptor (Fc γ R), the cytotoxic activity (ADCC), complement that comprises antibody dependent cellular in conjunction with, phagolysis and antibody the half life/removing.Can carry out various modifications to the Fc district of antibody of the present invention according to required effector characteristic.For example, specific sudden change in the Fc district makes in other cases to the antibody of cracking performance and becomes non-cracking performance, such sudden change is specified in EP0629 240B1 and EP 0307434B2, perhaps people can join in the antibody remedying the receptors bind epi-position, to increase the serum half life, referring to US 5,739,277.5 generally acknowledged human Fc gamma receptors are arranged at present: Fc γ R (I), Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa and neonatal Fc Rn.Shields etc., (2001) J.Biol.Chem 276,6591-6604 show that one group of general IgG1 residue participates in conjunction with all Fc γ R, and Fc γ RII and Fc γ RIII utilize this common group different loci in addition.When one group of IgG1 residue: Pro-238, Asp-265, Asp-270, Asn-297 and Pro-239 changed over L-Ala, it reduced the combination to all Fc γ R.All residue all is arranged in IgG CH2 structural domain, and clusters near the hinge area that connects CH1 and CH2.Fc γ RI only utilizes the combination of common group IgG1 residue, and except common group, and Fc γ RII and Fc γ RIII also interact with different residue.The change of some residue only reduces and the combining of Fc γ RII (for example Arg-292) or Fc γ RIII (for example Glu-293).Some variants shows the lifting that combines with Fc γ RII or Fc γ RIII, but does not influence and the combining of another acceptor (for example combining of Ser-267Ala lifting and Fc γ RII, but unaffected with combining of Fc γ RIII).Other variant shows the lifting that combines with Fc γ RII or Fc γ RIII, with another acceptor combine reduction (for example Ser-298Ala promotes and to reduce with combining of Fc γ RIII and the combining of Fc γ RII).For Fc γ RIIIa, best combination IgG1 variant has the L-Ala displacement of combination at Ser-298, Glu-333 and Lys-334.It is generally acknowledged that neonatal Fc Rn acceptor had both related to cleaning antibody, relate to again pass through tissue transcytosis (referring to (2000) Annu.Rev.Immunol.18 such as Junghans R.P (1997) Immunol.Res16.29-57 and Ghetie, 739-766).The human IgG1's residue that is determined with people FcRn direct interaction comprises Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435.Therefore the present invention relates to the antibody of the present invention that (or multiple) the above residue that describes in detail that has any changes, with improve the half life/removing and/or effector function, for example ADCC and/or complement cracking.Provide the humanization therapeutic antibodies more on the one hand of the present invention, its specificity is in conjunction with hOSM, and regulates the interaction between hOSM and the gp130 and have L-Ala (or other interference) at 235 (for example L235A) and 237 (for example G237A) and replace.In the further embodiment of the present invention, the humanization therapeutic antibodies is provided, its specificity is in conjunction with hOSM, and comprises the heavy chain of SEQ.I.D.NO:61 and the light chain of SEQ.I.D.NO:12.

Other modification comprises the glycosylation variant of antibody of the present invention.Known conservative position in antibody constant region makes antibody glycosylation antagonist function (those effector functions particularly for example mentioned above) have profound influence, referring to (1996) such as for example Boyd, Mol.Immunol.32,1311-1318.Imagined and wherein added, replace, lack or modify the therapeutic antibodies of the present invention of one or more sugar moieties or the glycosylation variant of its Fab.Introduce l-asparagine-X-Serine or l-asparagine-X-Threonine motif and created the potential site of using for enzyme connection sugar moieties, and therefore can be used for operating the glycosylation of antibody.At (2001) Biochemistry 40 such as Raju, among the 8868-8876, by using β-1,4-galactosyltransferase and/or α, the glycosylation again of 2,3 sialytransferases and/or sialylated again method have increased the terminal sialylated of TNFR-IgG immunoadhesin.It is generally acknowledged, increase the half life of terminal sialylated increase immunoglobulin (Ig).The same with most of glycoprotein, antibody produces as sugared type mixture in fact usually.When antibody produced in eukaryotic cell (particularly mammalian cell), this mixture was obvious especially.Developed the whole bag of tricks and produced the sugared type of qualification, referring to Science such as Zhang (2004), 303,371, Sears etc., Science, (2001) 291,2344, Wacker etc. (2002) Science, 298 1790, Davis etc. (2002) Chem.Rev.102,579, Hang etc. (2001) Acc.Chem.Res 34,727.Therefore, the present invention relates to multiple therapeutic as described herein (being generally mono-clonal) antibody (it can be IgG isotype, for example IgG1), its restricted number that comprises described antibody or its Fab is (for example below 7 kinds, for example below 5 kinds, as 2 kinds or a kind) sugared type.

Other embodiment of the present invention comprises therapeutic antibodies of the present invention or its Fab, and it is coupled to charged non-protein polymer, for example polyoxyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene.It is a kind of being used to of having established increasing the albumen half life and reduce protein antigenicity and immunogenic technology that albumen and PEG put together.Studied the application of employing different molecular weight and type (linearity or branch) PEGization already with whole antibody and Fab ' fragment, referring to (2000) Int.J.Pharmaceut.198:83-95 such as Koumenis I.L.

The existence of hemato encephalic barrier (BBB) has hindered human cytokines to be transmitted in the brain.Pass BBB at needs and transmit under the situation of antibody of the present invention or antibody fragment of the present invention, proposed various strategies and strengthened in this transmission that needs under the situation.

In order to obtain the desired nutritional thing and the factor by blood, BBB has some specific receptors, and it is transported to brain with compound by circulation blood.Research shows, some compound, as Regular Insulin (referring to (1989) Brain Res.420:32-38 such as Duffy KR), Transferrins,iron complexes (referring to (1987) J.Neurosci 18:299-304 such as Fishman JB) and type-1 insulin like growth factor and 2 (referring to (1986) Metabolism 37:136-140 such as Pardridge WM (1986) Endocrine Rev.7:314-330 and Duffy KR), pass BBB through receptor-mediated transcytosis.Therefore, the acceptor of these molecules provides a kind of potential method: use so-called " carrierization " antibody to make antibody of the present invention and/or antibody fragment of the present invention enter brain (referring to Pardridge WM (1999) Advanced Drug Delivery Review 36:299-321).For example, showed already that the antibody of TfR was gone in the brain essence (referring to (1993) Science 259:373-377 such as (1991) PNAS 88:4771-4775 such as Friden PM and Friden PM) by dynamic transhipment.Therefore, a potential method is to produce bi-specific antibody for example mentioned above or dual specific fragment, wherein (for example first specific specificity comprises the CDRH3 of SEQ.I.D.NO:3 to first specific specificity at the site II of hOSM, also have SEQ.I.D.NO:1,2,4,5 and 6 CDR alternatively, or comprise the CDRH3 of SEQ.I.D.NO:42, also have SEQ.I.D.NO:40,41,43,44,45 CDR alternatively), and second specific specificity is at the transhipment acceptor that is arranged in BBB, and for example second specific specificity is transported acceptor at Transferrins,iron complexes.

2. competitive immunization sphaeroprotein

The present invention also provides immunoglobulin (Ig), antibody and antigen-binding fragments of antibodies, and other albumen entity, immunoadhesin for example, their specificitys are in conjunction with hOSM, and combining between the therapeutic antibodies of the present invention of competitive inhibition hOSM and the light chain of heavy chain that contains SEQ.I.D.NO:11 and SEQ.I.D.NO:12 or its Fab.Competitive immunization sphaeroprotein, antibody and antigen-binding fragments of antibodies and other albumen entity (for example immunoadhesin) show at least 25% inhibition waiting under the volumetric molar concentration, and common 35% or higher inhibition, more generally at least 50% inhibition.

Therefore, in one embodiment of the invention, provide screening candidate's antibody or antibody fragment to determine whether candidate's antibody or antibody fragment are the method for competitive antibody as herein described, and this method may further comprise the steps:

(a) incubation candidate antibody or antibody fragment and contain the heavy chain of SEQ.I.D.NO:11 and therapeutic antibodies or its Fab of the light chain of SEQ.I.D.NO:12;

(b) candidate's antibody of determining step (a) or its antibody fragment whether combining between this therapeutic antibodies of competitive inhibition or its Fab and the OSM (particularly with hOSM between combine).Usually use the experiment of ELISA type, for example the ELISA that states in an embodiment.Usually, OSM and/or hOSM are by glycosylation.Usually, OSM and/or hOSM are by the mammalian cell glycosylation, and mammalian cell for example is CHO, NS0 cell or people's cell of recombinant conversion.In other embodiments, OSM and hOSM are by its n cell glycosylation of originating, and promptly hOSM is by the glycosylation of people's cell (for example hOSM is separable from people's antibody).

Therefore, also provide competitive therapeutic anti body of laws or its Fab, its competitive inhibition comprise have SEQ.I.D.NO:1, the combination of the therapeutic antibodies of the CDR of 2,3,4,5 and 6 listed sequences or its Fab.

Competitive therapeutic antibodies or its Fab also are provided, and its competitive inhibition comprises the therapeutic antibodies of light chain of the heavy chain of SEQ.I.D.NO:11 and SEQ.I.D.NO:12 or the combination of its Fab.

Competitive therapeutic antibodies or its Fab can have any in the above-mentioned antibody structure.For example, competitive therapeutic antibodies can be primate or people's whole antibody or humanized antibody, is generally the IgG isotype, for example IgG1 or IgG4.Competitive therapeutic antibodies fragment can be Fab, Fab ', Fd, F (ab ') ₂, ScFv etc.Competitive therapeutic antibodies can be produced according to this specification sheets disclosed method.

The typical scenario that is used for screening method mentioned above is set forth in following embodiment.

10D3 is an example of competitive antibody of the present invention.The Table A that vide infra.

2.1 other screening method

Play beyond thought effect in the binding events of glycosylation between anti-hOSM antibody and hOSM of the part of one side more of the present invention based on following discovery: hOSM.Therefore the present invention extends to the method for screening specificity in conjunction with the antibody of hOSM, and this method is included in and allows described antibody of incubation and glycosylation OSM (particularly hOSM) under the bonded condition, and detects the binding affinity of antibody.Hereinafter the ELISA method of Xiang Shuing makes such method become possibility.Can have the binding affinity (Kd) that is higher than 1 μ M (usually above 100nM, more generally being higher than 1nM, for example more than the 100pM) and select antibody (it can have any structure of above detailed description) for benchmark.

Can be that benchmark is selected antibody further with its ability in conjunction with non-glycosylated OSM (for example hOSM).Therefore, usually can be in conjunction with glycosylation OSM (for example hOSM) and further can also be with same or analogous degree (for example with Biacore with it ^TMExperiment detects, and has same or analogous binding affinity) select antibody in conjunction with non-glycosylated OSM (for example hOSM) for benchmark.

The antibody of selecting according to this paper method is through engineering approaches (for example, in case of necessity, the humanization by for example operating the antibody coding polynucleotide) further, and joins in the medicinal compositions.Polynucleotide by this paper method antibody of selecting and this antibody of encoding constitute embodiment of the present invention.Therefore, the invention provides the method that the antibody (for example antibody that produces at OSM/hOSM) in conjunction with OSM (particularly hOSM) is inferred in screening, this method comprises:

(a) allowing described antibody of incubation and glycosylation OSM (particularly glycosylation hOSM) under the bonded condition;

(b) binding affinity of the described antibody of detection;

(c) if the binding affinity that described antibody has is higher than 1 μ M,, then select described antibody usually above 100nM;

(d) provide the coded polynucleotide of the described antibody of step (c), and transform or the transfection mammalian host cell with the carrier that contains described polynucleotide;

(e) allowing described antibody-secreting to go into the described host cell of culturing step (d) under the condition in the substratum;

(f) substratum of purification step (e) alternatively;

(g) step (e) or antibody (f) are joined in the medicinal compositions.

The medicine of the antibody disease described in detail below differentiated by this method or the obstacle purposes in producing also is provided.

The purposes of specificity in producing in conjunction with Natively glycosylated hOSM (particularly in conjunction with Natively glycosylated hOSM site II) and the medicine of regulating (for example whole antibody, people's antibody, humanized antibody, chimeric antibody) disease described in detail below of interactional antibody between described Natively glycosylated hOSM and the gp130 or obstacle also is provided.Further be provided under the same experimental conditions with to the antibody of the same or analogous binding affinity specificity of non-glycosylated hOSM in conjunction with Natively glycosylated hOSM.One embodiment of the invention are the antibody of specificity in conjunction with glycosylation OSM, particularly in conjunction with those antibody of Natively glycosylated hOSM.Antibody 15E10 is the example of specificity in conjunction with the antibody of glycosylation hOSM.

In certain embodiments, this method is used by the glycosylated hOSM of mammalian cell (for example CHO or NS0).In other embodiments, this method is used by the glycosylated hOSM of the people's cell human host cell of transfection (for example heavily transform or) or by isolated natural hOSM in the human body (for example by the hOSM that is present in the cell preparation in sacroiliitis (for example RA) people patient's synovia).

3. production method

Antibody of the present invention can be used as the polyclone all living creatures and produces, but more generally produces as mono-clonal group basic all a groups of the same antibody of anti-specific antigens binding site (promptly as).Antibody of the present invention can be produced in genetically modified organism, for example goat is (referring to (1999) such as Pollock, J.Immunol.Methods 231:147-157), chicken (referring to Morrow KJJ (2000) Genet.Eng.News 20:1-55), mouse are (referring to Pollock etc., ibid) or plant (referring to Doran PM, (2000) Curr.Opinion Biotechnol.11,199-204, Ma JK-C (1998), Nat.Med.4; 601-606, Baez J etc., BioPharm (2000) 13:50-54, Stoger E etc.; (2000) Plant Mol.Biol.42:583-590).Also can produce antibody by chemosynthesis.But, use the well-known reconstitution cell culture technique of those skilled in the art to produce antibody of the present invention usually.Separate the polynucleotide of encoding antibody, and be inserted in the replicable vector (for example plasmid), be used for further clone (amplification) or expression.Useful expression system is NADPH-linked glutamate synthase system (for example Lonza Biologics sold system), particularly (vide infra) when host cell is CHO or NS0.Use ordinary method (for example oligonucleotide probe) to be easy to separate and the polynucleotide of the encoding antibody that checks order.Spendable carrier comprises plasmid, virus, phage, transposon, minute chromosome, and wherein plasmid is a typical embodiments.In general, such carrier further comprises signal sequence, replication orgin, one or more marker gene, enhancer element, promotor and the transcription termination sequence that effectively is connected to light chain and/or heavy chain polynucleotide, is beneficial to express.The polynucleotide of coding light chain and heavy chain can be inserted in the carrier separately, and import (for example passing through electroporation) in same host cell, if needs are perhaps arranged, heavy chain and light chain can be inserted in the identical carrier simultaneously, be used for being transfected into host cell.Therefore, according to one embodiment of the invention, the method of the carrier of the light chain that makes up coding therapeutic antibodies of the present invention or its Fab and/or heavy chain is provided, and this method comprises the light chain of code book invention therapeutic antibodies and/or the polynucleotide of heavy chain is inserted in the carrier.See table A.

In another embodiment of the present invention, the mouse V that provides coding to have SEQ.I.D.NO:15 or the listed sequence of SEQ.I.D.NO:52 _HThe polynucleotide of structural domain.

In another embodiment of the invention, the mouse V that provides coding to have SEQ.I.D.NO:16 or the listed sequence of SEQ.I.D.NO:53 _LThe polynucleotide of structural domain.

In another embodiment of the invention, the humanization V that provides coding to have SEQ.I.D.NO:17 or the listed sequence of SEQ.I.D.NO:54 _HThe polynucleotide of structural domain.

In another embodiment of the invention, the humanization V that provides coding to have SEQ.I.D.NO:18 or the listed sequence of SEQ.I.D.NO:55 _LThe polynucleotide of chain.

In another embodiment of the invention, provide coding to have the polynucleotide of the humanization heavy chain of SEQ.I.D.NO:19 or the listed sequence of SEQ.I.D.NO:56.

In another embodiment of the invention, provide coding to have the polynucleotide of the humanization light chain of SEQ.I.D.NO:20 or the listed sequence of SEQ.I.D.NO:57.

Those skilled in the art will understand immediately, owing to the Feng Yuxing of genetic code, also can obtain the alternative polynucleotide of the open polynucleotide of this paper of code book invention polypeptide.

3.1 signal sequence

Antibody of the present invention can be used as the fusion rotein production with allos signal sequence, and this allos signal sequence has the specificity cleavage site at the N of maturation protein end.This signal sequence should be discerned and process to host cell.For prokaryotic host cell, signal sequence can be alkaline phosphatase, penicillinase or thermally-stabilised enterotoxin 1 I leader sequence.For yeast secretary, signal sequence can be yeast invertase leader sequence, alpha factor leader sequence or acid phosphatase leader sequence, referring to for example WO90/13646.In the mammal cell line system, can utilize viral secretory leader sequence (for example herpes simplex gD signal) and native immunoglobulin signal sequence (for example people Ig heavy chain).Common signal sequence is pressed frame and is connected with the coding DNA of antibody of the present invention.

3.2 replication orgin

Replication orgin is well-known in this area, and pBR322 is suitable for most of gram negative bacteriums, and 2 μ plasmids are suitable for most of yeast, various viral starting points, and for example SV40, polyomavirus, adenovirus, VSV or BPV are suitable for most of mammalian cells.In general, mammalian expression vector does not need the replication orgin element, but can use SV40, because it comprises early promoter.

3.3 selective marker

The typical albumen (a) of selecting coded by said gene is given the resistance of microbiotic or other toxin (for example penbritin, Xin Meisu, methotrexate or tsiklomitsin) or (b) extra-nutrition defective or unavailable nutrition in the complex medium is provided.Selection scheme can comprise the cessation of growth cessation that makes host cell.Because the selective marker drug resistance of giving for example is so survive with therapeutic antibodies encoding gene success cell transformed of the present invention.Another example is so-called DHFR selective marker, and wherein transformant is cultivated in the presence of methotrexate.Chinese hamster ovary celI is to be used for the useful especially clone that DHFR selects.Use the amplification of DHFR system and select the method for host cell to continue to use for a long time in this area, referring to J.Mol.Biol. (1982) 159 such as Kaufman R.J., 601-621 is about summary, referring to Werner RG, Noe W, Kopp K, Schluter M, " Appropriate mammalian expression systems forbiopharmaceuticals ", Arzneimittel-Forschung.48 (8): 870-80,1998Aug.Another example is NADPH-linked glutamate synthase expression system (Lonza Biologics).Be applicable to that it is the trp1 gene that zymic is selected gene; Referring to Nature such as Stinchcomb 282,38,1979.

3.4 promotor

The promotor that will be suitable for expressing antibody of the present invention effectively is connected to the DNA/ polynucleotide of encoding antibody.The prokaryotic hosts promotor comprises phoA promotor, β-Nei Xiananmei and lactose promoter systems, alkaline phosphatase, tryptophane and hybrid promotor, for example Tac.Be suitable for that expression promoter comprises glycerol 3-phosphate acid kinase or other glycolytic ferment in yeast cell, for example enolase, glyceraldehyde 3 phosphate desaturase, hexokinase, pyruvic carboxylase, phosphofructokinase, glucose 6 phosphoric acid isomerases, 3-phoshoglyceric acid mutase and glucokinase.The induction type Yeast promoter comprises the enzyme of alcohol dehydrogenase 2, different cell pigment C, acid phosphatase, metallothionein(MT) and responsible nitrogen metabolism or maltose/galactose utilization.

Be used for comprising viral promotors in mammal cell line system expression promoter, for example polyomavirus, fowlpox virus and adenovirus (for example adenovirus 2), bovine papilloma virus, avian sarcomata virus, cytomegalovirus (particularly immediate early gene promotor), retrovirus, hepatitis B virus, Actin muscle, Rous sarcoma virus (RSV) promotor and simian virus 40 are early stage or late promoter.Certainly, the selection of promotor is based on the proper compatibility with the host cell that is used to express.

3.5 enhancer element

When suitable, for example when being suitable in higher eucaryote, expressing, can use the enhancer element that in carrier, effectively is connected to promoter element.Suitable Mammals enhancer sequence comprises the enhancer element of globin, elastoser, albumin, fetoprotein and Regular Insulin.Perhaps, people can use the enhancer element of eukaryotic cell virus, for example SV40 enhanser (being positioned at bp 100-270), the sub-enhanser of cytomegalovirus early promoter, polyomavirus enhanser, baculovirus enhanser or mouse IgG2a locus (referring to WO04/009823).Enhanser is usually located at the site of promotor upstream on the carrier.

3.6 host cell

For the clone or the expression vector of code book invention antibody, proper host cell is prokaryotic cell prokaryocyte, yeast cell or higher eucaryotic cells.Suitable prokaryotic cell prokaryocyte comprises eubacterium, enterobacteriaceae for example, as Escherichia (Escherichia) (intestinal bacteria (ATCC31 for example for example, 446,31,537,27,325)), enterobacter (Enterobacter), erwinia (Erwinia), Klebsiella (Klebsiella), proteus (Proteus), salmonella (Salmonella) (for example Salmonella typhimurium (Salmonella typhimurium)), serratia (Serratia) (for example serratia marcescens (Serratia marcescans)) and Shigella (Shigella) and bacillus (for example subtilis (B.subtilis) and Bacillus licheniformis (B.licheniformis)) (referring to DD 266 710), Rhodopseudomonas (for example Pseudomonas aeruginosa (P.aeruginosa)) and streptomyces (Streptomyces).In yeast host cell, also imagined yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), schizosaccharomyces pombe (schizosaccharomyces pombe), (for example ATCC 16 for genus kluyveromyces (Kluyveromyces), 045,12,424,24178,56,500), yarrowia (EP402,226), (EP 183 for pichia pastoris phaff (Pichia Pastors), 070, in addition referring to J.Biotechnol.108 such as Peng (2004) 185-192), Candida (Candida), Trichodermareesei (Trichodermareesia) (EP244,234), Penicillium (Penicillin), Tolypocladium and Aspergillus (Aspergillus) host, for example Aspergillus nidulans (A.nidulans) and aspergillus niger (A.niger).

Although the present invention has specifically imagined protokaryon and yeast host cell, common host cell of the present invention is a vertebrate cells.Suitable vertebrate host cell comprises mammalian cell, COS-1 (ATCC No.CRL 1650) for example, COS-7 (ATCC CRL 1651), the human embryonic kidney cell is 293, baby hamster kidney cell (BHK) (ATCC CRL.1632), BHK570 (ATCC NO:CRL 10314), 293 (ATCC NO.CRL 1573), Chinese hamster ovary cell CHO (CHO-K1 for example, ATCC NO:CCL 61, DHFR-CHO clone (for example DG44) is (referring to Urlaub etc., (1986) Somatic Cell Mol.Genet.12,555-556)), particularly those are suitable for the Chinese hamster ovary celI system of suspension culture), the mouse sustenticular cell, monkey-kidney cells, African green monkey kidney cell (ATCC CRL-1587), the HELA cell, Madin-Darby canine kidney(cell line) (ATCC CCL 34), human pneumonocyte (ATCC CCL 75), Hep G2 and myelomatosis or lymphoma cell, for example NS0 is (referring to US 5,807,715), Sp2/0, Y0.

Therefore, in one embodiment of the invention, provide the host cell of stable conversion, it comprises coding therapeutic antibodies or the heavy chain of its Fab and/or the carrier of light chain as described herein.Usually, such host cell comprises first carrier of the light chain of encoding and second carrier of the described heavy chain of coding.

Fermentation using bacteria

Bacterial system is particularly suitable for the expressing antibodies fragment.Such fragment is arranged in born of the same parents or pericentral siphon.Insoluble periplasm protein can be extracted and refolding according to method known to those skilled in the art, to form activated protein, referring to (1999) J.Biotechnol.72 such as Sanchez, (1999) Lett Appl Microbiol such as 13-20 and Cupit PM, 29,273-277.

3.7 cell culture processes

Can cultivate carrier transformed host cells by any means well known by persons skilled in the art with coding therapeutic antibodies of the present invention or its Fab.Host cell can be cultivated in spindle-type flask, rolling bottle or tubular fibre system, but for scale operation, preferably uses stirred-tank reactor, particularly for suspension culture.Usually, stirring tank is suitable for using for example sparger, baffle plate or the ventilation of low shearing impeller.For bubble tower and airlift reactor, can use the directly ventilation of air or oxygen bubble.When cultivating host cell in serum free medium, preferred culture medium replenishes cytoprotective (for example Pluronic F-68), to help prevent the cell injury that produces owing to venting process.According to the host cell feature, perhaps can use the growth matrix of microcarrier as anchorage-dependent cell system, perhaps cell can be suitable for suspension culture (this is representational).The cultivation of host cell (particularly vertebrate host cell) can utilize various operator schemes, and for example batch feeding, repeated batch are processed (referring to (1994) cytotechnology 15:103-109 such as Drapeau), continued processing (extended batch processing) or perfusion culture in batches.Although the mammalian host cell of recombinant conversion can be grown in containing the substratum of serum (substratum that for example contains foetal calf serum (FCS)), preferred such host cell is at synthetic serum free medium (for example being disclosed in the substratum of (1995) Cytotechnology 17:153-163 such as Keen) or commercially available substratum (for example ProCHO-CDM or UltraCHO ^TMCultivate in (Cambrex NJ, USA)), can add the energy when needed, for example glucose and the synthetically grown factor, for example Recombulin.The serum-free culture of host cell may require those host cells to be suitable for growing under serum-free condition.An adaptive method is to cultivate this host cell in containing the substratum of serum, and repeatedly 80% substratum is replaced by serum free medium, so that adapting to serum-free condition, the host cell acquistion (is stated from Animal Celltechnology:Developments towards the 21st century editors such as () Beuvery E.C. referring to (1995) such as for example Scharfenberg K, the 619-623 page or leaf, Kluwer Academic publishers).

Can use various technology to reclaim and purifying secreted antibody of the present invention of going in the substratum, so that the purification degrees that is suitable for intended purpose to be provided by substratum.For example, compare with the substratum that contains therapeutic antibodies, the therapeutic antibodies of the present invention that is used for the treatment of people patient requires at least 95% purity, more generally 98% or 99% purity usually.At first, use centrifugally usually, then use for example supernatant liquor clarification steps of micro-filtration, ultrafiltration and/or Depth Filtration, by removing cell debris in the substratum.Can utilize various other technology (for example dialysis and gel electrophoresis) and chromatographic technique, for example hydroxylapatite (HA), affinity chromatography (comprising the affinity tag system alternatively, for example polyhistidyl) and/or hydrophobic interaction chromatography (HIC is referring to US 5,429,746).In one embodiment, after various clarification steps, use A albumen or G albumen affinity chromatography, then further use chromatographic step, for example ion-exchange and or HA chromatography, negatively charged ion or cationic exchange, size exclusion chromatography and ammonium phosphate precipitation, catch antibody of the present invention.Usually, also use various viruses to remove step (for example using for example nanofiltration of DV-20 filter).After these different steps, provide to contain purifying (the being generally mono-clonal) goods of (for example more than the 100mg/ml) antibody of the present invention or its Fab more than the 75mg/ml at least, therefore constitute embodiment of the present invention.Proportionately, these goods do not have the antibody of the present invention of aggregated forms substantially.

4. medicinal compositions

Aforesaid purifying antibody goods of the present invention (particularly mono-clonal goods) can be joined in the medicinal compositions, be used for the treatment of for example human disease and the obstacle of above-outlined.Usually, this composition further comprises pharmacy acceptable (the being inertia) carrier that qualified pharmacy practitioner is known and require, referring to for example Remingtons Pharmaceutical Sciences, and 16 editions, (1980), Mack Publishing Co.The example of this carrier comprises the sterilization carrier, for example is buffered to salt solution, Ringers solution or the glucose solution of pH5-8 with suitable reducing.Aptly, being used for the injection (for example by injecting in vein, intraperitoneal, intracutaneous, subcutaneous, intramuscular or the portal vein) or the medicinal compositions of continuous infusion does not have the visible particulate matter, and can comprise 0.1ng-100mg antibody, is generally 5mg-25mg antibody.The method for preparing this medicinal compositions is that those skilled in the art are known.In one embodiment, medicinal compositions comprises the therapeutic antibodies of the present invention of 0.1ng-100mg unit dosage, has working instructions alternatively.But medicinal compositions freeze-drying of the present invention, well-known or conspicuous method is made again according to those skilled in the art before giving.When embodiment of the present invention comprise the antibody of the present invention with IgG1 isotype, can in medicinal compositions, add copper chelator, for example Citrate trianion (for example Trisodium Citrate) or EDTA or Histidine are with the degree of this isotype antibody degraded of reducing the copper mediation, referring to EP0612251.

In general, the effective dose and the treatment plan that give antibody of the present invention are determined by rule of thumb, depend on the factor of patient's age, body weight and healthy state and the disease that will treat or obstacle and so on.Such factor is within attending doctor's ken.For example be found in (1977) Antibodies in human diagnosis andtherapy such as Smith about the guide of selecting suitable dose, Raven Press, New York, but generally between 1mg-1000mg.In one embodiment, people patient's the dosage regimen that RA is suffered from treatment be 100mg or about the antibody of the present invention (or its Fab) of (being 50mg-200mg), weekly or per two weeks subcutaneous giving once.Composition of the present invention also preventability uses.

According to the disease that will treat or obstacle, contain the antibody of the present invention for the treatment of significant quantity medicinal compositions can with the another kind of medicine of significant quantity simultaneously, independent or sequentially use, described another kind of medicine for example is an anti-inflammatory drug, for example NSAID, methotrexate, Bucillamine, mercaptosuccinic acid sodium or one or more anti-TNF alpha therapeutant, for example Enbrel ^TM(etanercept), Remicade ^TM(infliximab), Humira ^TM(A Da wood Dan Ke) and/or CDP870.Antibody of the present invention can with the anti-TNF alpha receptor antibody coupling of significant quantity, referring to (2000) Ann Rheum Dis 59 (suppl 1): 41-43 such as Davis MW.In other embodiments, antibody of the present invention can with the anti-IL-1/IL-1R of significant quantity (Kineret for example ^TM), CTLA4-Ig, IL-6 (referring to Choy etc., 54), IL-8, IL-15, VEGF, IL-17, the material of IL-18 (referring to (2001) Curr.Opin.Immunol.13:611-616 such as Taylor), anti--ICAM and/or anti-CD 4 antibodies, anti-MMP family member's (for example MMP-1,2,3 and/or 13) material coupling (2002) Ann.Rheum.Dis 61 (suppl 1):.Antibody of the present invention also can with the material coupling of the cell of removing known participation inflammatory process, for example use Mabthera ^TMRemove the positive B cell of CD20.Comprise angiogenesis inhibitor treatment, for example beta 2 integrin alpha with other treatment of antibody combination of the present invention _vβ ₃Antagonist Kringles 1-5 (referring to (2003) such as Sumariwalla P, Arthritis Res Ther 5:R32-R39), solubility Flt-1 (referring to Miotla etc., (2000) Lab.Invest.80:1195-1205) or anti-COX-2 material.For simplicity, the present invention has also imagined the assembly test kit that contains medicinal composition, and it contains antibody of the present invention or its Fab part, together with this another kind of medicine, and has working instructions alternatively.And, the invention provides and contain the mono-clonal therapeutic antibodies described herein for the treatment of significant quantity or the medicinal compositions of its Fab, be used for the treatment of site II that regulates OSM and the disease that the interaction between the gp130 responds.The present invention also provides the medicinal compositions that contains the mono-clonal therapeutic antibodies for the treatment of significant quantity, and this antibody comprises heavy chain with the listed sequence of SEQ.I.D.NO:11 and the light chain with the listed sequence of SEQ.I.D.NO:12.

The present invention also provides the medicinal compositions that contains the mono-clonal therapeutic antibodies for the treatment of significant quantity, and this antibody comprises heavy chain with the listed sequence of SEQ.I.D.NO:50 and the light chain with the listed sequence of SEQ.I.D.NO:51.

4.1 not only regulatory site II interacted but also the interactional medicinal compositions of regulatory site III

One aspect of the present invention to small part based on following beyond thought discovery: with its mating partner of doing mutually separately (is gp130 for site II promptly, be OSMR β and/or LIFR and/or be used for gp130 for site III in conjunction with second OSM molecule) regulate the interaction of site II and the site III of hOSM simultaneously, with regulate these two sites in separately any interaction compare, show synergy.

Therefore the present invention provides the interactional method between hOSM and gp130 and LIFR and/or the OSMR β of regulating, interactional site II antagonist between the site II of this method comprises to be provided and can regulate (promptly suppressing or blocking-up) hOSM and the gp130, and interactional site III antagonist between the site III that can regulate (promptly suppressing or blocking-up) hOSM and OSMR and/or LIFR and the gp130 (being used to combine second OSM molecule) is provided.With regulate these two sites in separately any interaction compare, this method has synergy.

Therefore the present invention provides the interactional method between hOSM and gp130 and LIFR and/or the OSMR β of regulating, interactional site II antagonist between the site II of this method comprises to be provided and can regulate (promptly suppressing or blocking-up) hOSM and the gp130, and interactional site III antagonist between the site III that can regulate (promptly suppressing or blocking-up) hOSM and OSMR and/or the LIFR is provided.

In one embodiment, the medicinal compositions that contains first therapeutic antibodies and second therapeutic antibodies is provided, the first therapeutic antibodies specificity is in conjunction with hOSM, and interaction (the site II antibody between adjusting hOSM and the gp130, the example is provided by this specification sheets), the second therapeutic antibodies specificity is in conjunction with hOSM, and interaction (the site III antibody between adjusting hOSM and OSMR and/or the LIFR, the example can MAB295, R﹠amp; D systems is commercially available).In the experiment of ELISA type or as embodiment, state interactional ability between adjusting (promptly suppressing or blocking-up) hOSM and OSMR β and/or the LIFR according to it, do not suppress OSM and gp130 bonded ability with OSM and in the ELSIA of embodiment experiment in promptly testing, can distinguish second therapeutic antibodies with its KB at embodiment.

Can in the ELISA of embodiment experiment, suppress OSM bonded ability according to it and distinguish site II antibody.Usually, first and second therapeutic antibodies are all monoclonal antibody.Certainly, it will be apparent for a person skilled in the art that, medicinal compositions not necessarily contains two kinds of antagonism entities (for example two kinds of therapeutic antibodies entities), because for example might provide specificity in conjunction with hOSM and regulatory site II and site III and its are made the interactional bi-specific antibody of mating partner separately mutually simultaneously.

In another embodiment, the assembly test kit is provided, it comprises first medicinal compositions and second medicinal compositions, have working instructions alternatively, first medicinal compositions comprise specificity in conjunction with hOSM and regulate the site II of hOSM and gp130 between interactional therapeutic antibodies, second medicinal compositions comprise specificity in conjunction with hOSM and regulate the site III of hOSM and OSMR β and/or LIFR between interactional therapeutic antibodies.

In another embodiment, also provide treatment to suffer from hOSM and do interaction between the mating partner (for example gp130 and OSMR β and/or LIFR) mutually and regulate the disease respond or obstacle (inflammatory diseases or obstacle (arthritis disease for example for example, for example rheumatoid arthritis or osteoarthritis)) people patient's method, this method comprises simultaneously, sequential or separately treat significant quantity the first therapeutic antagonist (for example antibody) and the treatment significant quantity second antagonist (for example antibody), the first therapeutic antagonist specificity is in conjunction with hOSM, and the site II of adjusting hOSM and the interaction between the gp130, the second antagonist specificity is in conjunction with hOSM, and the site III of adjusting hOSM and the interaction between OSMR β and/or the LIFR.

Certainly, it will be apparent for a person skilled in the art that the first following at least antagonist (for example antibody) can realize and identical target mentioned above: promptly in conjunction with gp130, and regulate (for example blocking-up) (a) between gp130 and the hOSM and (b) interactional first antagonist between OSMR β and/or LIFR and the hOSM.

5. clinical application

Antibody of the present invention can be used for treating to regulating various diseases or the obstacle that interactional treatment responds between hOSM site II and the gp130.To should be mentioned that especially and relate to disease or the obstacle (being alpha mediated disease of TNF or obstacle) that produces the horizontal TNF α of pathology and be characterised in that those diseases or the obstacle that cartilage (particularly joint cartilage) decomposes or damages.Describe in detail as mentioned, antibody of the present invention or can be used as single pharmacotherapy perhaps can be used for the treatment associating of this inflammatory arthropathy (for example RA) with another kind, is used for the treatment of this inflammatory arthropathy.Antibody of the present invention can be used for treating the clinical described disease of having made a definite diagnosis form, or is used for prevention and shows effect responsive patient, or is used to slow down or stops disease and develop to clinical significance.For the RA treatment, in case ameliorate disease symptom occurs, antibody then of the present invention can be used for preventing recurrence.When the patient suffered from the batch-type disease, antibody of the present invention can be used for prolonging the interval between the acute phase of disease.Antibody of the present invention also can be used for treating the formation of symptom (for example Feltys syndromes) outside the joint of RA and/or treatment atherosclerotic plaque.For the RA treatment, can use the combination of antibody of the present invention together with medicine mentioned above.Other can comprise morbidity property sacroiliitis childhood, psoriasis arthropathica and ankylosing spondylitis by the joint disease that gives antibody benefit of the present invention.

Osteoarthritis (OA) is a kind of chronic degenerative diseases of cause the unknown, it is characterized in that joint cartilage and function of joint lose gradually.It is divided into two classes at present.Primary OA can be partial or general, and the latter is more common in the postmenopausal women, the Heberdens tubercle occurs.Secondary cases OA has the basic cause of disease, for example wound, obesity, Paget disease or inflammatory arthritis.Losing of joint cartilage often is accompanied by the variation of plumpness bone, and spur forms, subchondral bone thickens and the synovial membrane inflammation.Be noted that the DB that tied down by weight-bearing joint (for example knee, hand and hip) especially.OA is the extremely weak disease of a kind of people of making, and needs joint replacement when it is the most serious, with reactivating property and termination arthralgia.The trend of big spur occurs according to the patient, hipbone sacroiliitis is divided into hypertrophic and atrophic type (referring to Solomon L (1976) J Bone Joint Surg 58,176); Other joint may be equally to the reaction that has of this disease.Hypertrophic OA may be relevant with the special property sent out of diffusivity bone matrix hyperplasia with the pyrophosphate salt crystal deposition.Present treatment comprises uses non-opium class anodyne (for example paracetamol and U-26225A (Tramadol)), NSAIDS (for example Cox-2 specific inhibitor, for example celecoxib, rofecoxib), opium class anodyne and glycosamine and chondroitin sulfate.Therefore, in one embodiment of the invention, be provided at the method for this disease of treatment among the people patient who suffers from osteoarthritis (for example primary or Secondary cases), this method comprises therapeutic antibodies of the present invention or its fragment that gives described patient treatment significant quantity as described herein.The invention still further relates to the combination of therapeutic antibodies of the present invention together with another kind of therapeutant (one or more particularly above-mentioned OA therapeutants).

Psoriatic is a kind of chronic dermatosis with remarkable sickness rate, influences about 2% white race crowd.Although for many people it is a kind of gentle relatively disease, it may have deep influence to those affected people.Showed already that the psoriatic's of hospital care DB was similar to patient with angina pectoris, and approached heart failure patient (Finlay etc., (1990); Br.J.Dermatol, 123,751).Psoriatic most common form is chronic spot shape disease.The red squamous spot that it presents sharp outline is distributed in scalp, lower back and limbs extensor surface usually.Clinical change disease comprises drips shape chronic eczema, fat leakage type chronic eczema and pustule type disease.Small number of patients also develops into the seronegativity inflammatory arthritis.At microscopically, affected skin demonstrates keratinocyte propagation and unusual differentiation increase, the auxiliary lymphocyte of activation T-and neutrophilic granulocyte soaks into and the activation of skin vasculature.These change corresponding to crossing of somatomedin and acceptor, pro-inflammatory cytokine and vasculogenesis peptide expresses.But although further investigate, etiology and pathogeny that should disease be still uncertain, even if the central role that has confirmed activating T cell in animal model system is (referring to (1999) Arch.Dermatol.135 such as Nickoloff, 546-552).Present treatment comprises topical therapeutic, for example novel vitamin D analogues, reflunomide, anthraline and retinoid, for example Tazarotene gel.Phototherapy comprises uses UV-B or psoralene and ultraviolet light,long wave and excimer laser irradiation.Systematicness retinoid treatment comprises etretinate and Acitretin, isotretinoin, liarozole.Other treatment comprises that methotrexate, hydroxyurea, S-Neoral and calcium are according to Phosphoric acid esterase antagonist, 6-thioguanine, azathioprine, sulfasalazine and fumarate.Recently, proposed or shown Ontak ^TM(denileukin-2 (Denileukin Diftitox)), Zenapax ^TM(daclizumab (Daclizumab)), basiliximab (Basiliximab), anti-CD 4 antibodies, pearl monoclonal antibody (Efalizumab), Alefacept in accordance with the law ^TM, Siplizumab, IDEC-114 and BMS 188667 (CTLA4Ig) and so on biotherapy useful to treating this disease.And, anti-TNF alpha treatment (Enbrel for example ^TM(etanercept), Remicade ^TM(infliximab), Humira ^TM(adalimumab) and/or CDP870) can with the antibody combined psoriatic (comprising its clinical change disease) that is used for the treatment of of the present invention.

The evidence of OSM in psoriatic lesions sees (1998) Arch.Dermatol.Res 290:9 such as Boifati, 13.System knurl albumen M is by the spontaneous secretion of short-term organ cultures of pathology psoriatic skin (referring to Bonifati C etc., ibid).And the composing type activation of the main signal transduction molecule STAT3 in OSM acceptor downstream in the mouse keratinocyte causes the spontaneous psoriatic lesions (referring to (2005) Nature Medicine 11:43-49 such as Sano S) that occurs.

Antibody of the present invention therefore can be used as single pharmacotherapy or with these therapeutants combinations mentioned above, be used for the treatment of psoriatic (chronic spot, drip shape chronic eczema, fat leakage type chronic eczema, pustule type disease, seronegativity inflammatory arthritis dependency psoriatic), atopic dermatitis/eczema, acne, ichthyosis, pemphigus, viral verruca.

Systemic lupus erythematous (SLE) is a kind of systemic autoimmune disease, it is characterized in that producing autoantibody, formation immunocomplex and immune-mediated tissue injury and (summarize .Hochberg in Rheumatology (2003), Silman, Smolen, Weinblatt and Weisman edit, Pub.Mosby.1291-1430).Pathological manifestations comprises fibrinoid necrosis, hematoxylin body basophilic, blood vessel injury and dermis of skin-epidermis joining region fracture, inflammatory arthritis and glomerulonephritis.SLE can come across any age, comprises appearing among the newborn infant.It is one of common disease of invasion and attack women's breeding time, and is obviously more common in the women than in the man, obviously more frequent than white people to African people's influence.Its sickness rate in the U.S. is estimated as the annual 1.8-7.6 example of per 100,000 people.SLE increases relevant with the mortality ratio that is mainly caused by infection and kidney and CNS complication.The treatment of lupus and complication thereof is by each patient's the decision that needs.Non-steroidal anti-inflammatory drugs is the seek peace important roentgenism x of warm nature serositis of muscle-bone conditions, constitutional disease.Anti-malaria medicaments (for example Plaquenil, chloroquine and Quinacrime) is used for the treatment of the muscle-bone conditions and the physique symptom of on-steroidal and low dosage steroid refractory.Most of clinical manifestation of SLE responds to steroid therapy, but these Side effects of pharmaceutical drugs may limit the dosage and the time-histories of treatment simultaneously.Immunosuppressive drug (especially azathioprine) can be used for more serious disease.Recently, SLE is shown promising achievement (summarizing in (2005) CurrDir Autoimmune 8:193-205 such as Looney RJ) with antibody Rituxan (Rituximab) treatment of eliminating the B cell.Have found that the level of system knurl albumen M in the SLE patients serum promotes, and shows its level relevant with disease activity (referring to (1997) Eur Cytokine Netw 8:281-286 such as Robak E).Therefore, the present invention relates to use antibody of the present invention (or as single pharmacotherapy, or with the current SLE therapeutant combined therapy SLE that describes in detail more than one or more.

Systemic sclerosis (SS) comprises sclerodermatous change disease and Raynauds phenomenon, is the systemic disease of a kind of skin and internal organs.It is characterized in that extracellular matrix accumulates in skin and internal organ.System knurl albumen M can stimulate excessive extracellular matrix accumulation (to roll up referring to (1997) J Mol Med Abstract such as Bamber B 76: 61-69).System knurl albumen M is by systemic sclerosis patient's the spontaneous generation of cultivation monocyte (referring to (1999) Rheumatology (Oxford) 38:612-617 such as Hasegawa M), and is present in the scleroderma and (summarizes in Atama SP and White B (2003) Cytokinegrowth Factor Rev 14:537-550) in the pulmonary fibrosis bronchoalveolar lavage fluid.Therefore, the present invention relates to the purposes that antibody of the present invention is treated SS as single pharmacotherapy or with another kind of drug regimen and become disease.

In acute lung injury (particularly under the pneumonia situation) patient's bronchoalveolar lavage fluid, detected OSM ((2002) Develop Dyman 225:327-331 such as Tamura S) already.Neutrophilic granulocyte is these patients' OSM cell source seemingly, and the OSM concentration in the BAL liquid is relevant with the PMN number.Because neutrophilic granulocyte is the OSM source, and at when activation secretion OSM, thus probably OSM to be present in neutrophilic granulocyte wherein be that airway inflammation (comprising COPD and severe asthma) is significantly in any patient's of component the lung.In addition, OSM is also expressed by (mouse) tissue eosinophilic granulocyte, and can be remarkable OSM source in inflammatory process, and referring to Tamura, ibid.

Use adenovirus carrier in airway of mice, to cross expression OSM, induced far-reaching eosinophilic granulocyte inflammation and apposition (referring to (2003) J.Immunol.170:548-555 such as Langdon C) and TIMP-1 to express (referring to (1999) J.Interfer.Cytokine Res. such as Kerr C, 19:1195-1205).Mouse lung inoblast contact OSM stimulates Eotaxin to discharge, and Eotaxin is a kind of effective eosinophilic granulocyte chemoattractant.And OSM stimulates proliferation, and induces collagen protein to produce, and prevents people's lung fibroblast apoptosis (referring to Scaffidi, A.K. etc. (2002) Brit.J.Pharamcol 136:793-801).Although in these observationss mechanism behind is unknown, apposition may partly be the result (referring to Cichy, J. etc. (1998) Biochem.J 329:335-339) that the synthetic strong specificity of α 1 proteinase inhibitor raises.Have been found that also OSM promotes inoblast dependent mast cells propagation and histamine content significantly to increase (referring to (2001) Arch.Dermatol.Res 293:508-514 such as Gyotoku E).Directly instillation OSM brings out fast and the IL-6 that continues secretion (referring to Li, H.L. (2002) J.Drug Targ 10:55-62) in the isolated rat lung.Therefore, the present invention relates to antibody of the present invention (as single pharmacotherapy or with the combination of another kind of medicine) purposes of treatment inflammatory lung disease (for example asthma and COPD (chronic obstructive pulmonary disease)).

Detected OSM already in multiple sclerosis (MS) patient's brain, OSM is arranged in microglia, stellate cell and infiltration white corpuscle (referring to Journalof Neuropathology ﹠amp such as Ruprecht K in brain; Experimental Neurology.60 (11): 1087-98,2001Nov).OSM induces brain endothelial cell secretion IL-6 and MCP-1, adds TNF α and OSM and produces concerted reaction.OSM also induces ICAMl to express on the brain micro blood vessel endothelium cell, and it can strengthen the leukocyte infiltration (Ruprecht K etc., ibid) in the cerebral tissue.The inflammation in promoting brain, OSM also can directly facilitate neuron loss.HIV patient's monocyte supernatant liquor produces significantly the neuroblast growth and suppresses, and also causes nerve cell death, has shown that the mediators of these effects is system knurl albumen M.OSM because suffering from neuronal cell, many HIV patients loses the encephalatrophy that causes, so may be a kind of mediators of this pathology.Obviously, OSM also can take place to work in other CNS disease of neuron loss therein.Interesting is, in alzheimer's disease (AD), and α ₁Antichymotrypsin (ACT) is a kind of in the amyloid associated protein, and it is expressed in the lesion sharply increases, and has to be beneficial to the deposition of paraprotein in amyloid plaque and neurofibrillary tangles.Known is effective inductor of ACT by soaking into activating T cell and monocyte the two and microglia excretory OSM, therefore can help AD pathology (referring to (1998) J Biol.Chem.273:4112-4118 and Kordula T Journal of Neuroscience.20 (20): 7510-6 such as Kordula T, 2000).

The work prompting of Tamura etc., OSM may relate to the development of neuropathic pain and keep (referring to (2003) Eur.J.Neurosci.17:2287-2298 such as Tamura S.).Their research discloses, the sexy feel of a part of nociception neuron expression OSM beta receptor.All OSM β R+ve neurones are also expressed VR1 and P2X3 acceptor, shown that the two development is conclusive (referring to (2003) J.Pharmacol.Exp.Ther 304 such as (2002) PNAS 99:179-184 such as Jarvis M.F. and Walker K.M, 56-62) to these acceptors to neuropathic pain and inflammatory pain.And, OSM-/-mouse reduced the adverse reaction to chemistry, heat, internal organ and mechanical pain, this and VR1 ^{+ ve}P2X3 ^{+ ve}The minimizing of small neuron relevant (referring to Morikawa, Y. etc. (2004): J Neurosci 24,1941-1947).

Therefore, the invention still further relates to antibody of the present invention (as single pharmacotherapy or with the combination of another kind of medicine) purposes in for example above-mentioned central nervous system disease or obstacle (for example multiple sclerosis (MS), alzheimer's disease (AD) and other dementia) treatment, and relate to its purposes in pain (particularly neuropathic pain and/or inflammatory pain) treatment.

OSM is present in the tissue macrophages of atherosclerotic lesion (referring to J.Clin Invest.100 such as Modur V., 158-168), and can promote the neovascularization feature of atherosclerotic plaque as angiogenesis factor, this feature is considered to cause vessel wall fragility.React as vasculogenesis, OSM had both induced IL-6 to secrete (effect of OSM and IL-1 and TNF α are respectively additive propertieies or synergitic in endotheliocyte) in endotheliocyte, induced COX-2 to express (referring to (1991) J.Immunol.147:2175-2180 such as Brown J.T) again.It is the vasculogenesis characteristic necessary (referring to Brown J.T etc., ibid) of OSM that endotheliocyte is induced COX2.But, OSM also induces other angiogenesis factor VEGF (Vasse, M etc. (1999) Arterioscler Thromb Vasc Biol.19:1835-1842) and bFGF (WijelahE.S. etc. (1997) J.Cell Sci 110:871-879) to express in endotheliocyte.Interesting is, the OSM Rd that human endothelial cell had is about 10-20 doubly (referring to Modur V. etc., ibid) of other cell.

Except the effect to endothelium, OSM also induces IL-6 and COX-2 to express in vascular smooth muscle cell (VSMC), and the noticeable change ((1999) Circ.Res.85:1124-1131 such as Bernard C.) that causes morphocytology.The calcium settling often is present in wherein, and scavenger cell is in the late arterial atheromatous lesions of main inflammatory cell.Scavenger cell is the main source of OSM, and interesting is, this cytokine can be induced bone type alkaline phosphatase and calcium deposition (2002) Circ.Res.91:9-16 such as () Shioi A. in the VSMC culture.OSM also induces respectively and suppresses the VSMC secretory tissue factor (TF) and TF channel inhibitor (TFPI), produces effective procoagulant activity (2002) Blood Coag.Fibrinol.13:449-455 such as () Mirshahi F. in the VSMC culture supernatant.And, OSM influences the endotheliocyte secretion von-Willebrand factor, histiotype plasminogen activator and PAI-1 slightly, this prompting " OSM may play a crucial role in atherosclerotic lesion development " ((1998) Blood Coag.Fibrinol.9 such as Portau J, 609-615).

The fibrinogenemia pulp-water is flat to be a kind of important vessel risk parameters, in the research of carrying out with hepatoma cell line, OSM be a kind of effective Fibrinogen secretion inductor (Vasse.M etc. (1996) Haemostasis 26, suppl4,331-339).But when high density (50ng/ml), OSM also increases human ldl receptor and expresses (Liu etc. (2003) Aterio.Thromb.Vasc.Biol.23:90-96).At last, OSM promotes the cholesterol esterification in J774 monocyte-scavenger cell, therefore may in the foam cell development of atherosclerotic lesion, promote this process ((1996) Biochem.Biophys Acta 1300 such as Maziere C, 30-34).

Therefore, the present invention relates to the purposes of antibody of the present invention in cardiovascular system diseases or treating dysfunction.Imagined the purposes of antibody of the present invention in the disease treatment of atherosclerosis and endotheliocyte origin in addition.Further imagined the purposes of antibody of the present invention in the treatment of HIV infected patient, especially for treating by the disease due to the virus infection (for example Kaposi sarcoma).

Antibody of the present invention also can be used for the Cycle Regulation disease, for example cancer (for example prostate cancer), myelomatosis.

Although with regard to the big volume description of treatment of human diseases or obstacle the present invention, the present invention also can be used for treating similar disease or the obstacle in the non-human mammal.

Table A

Albumen or polynucleotide (PN)	Antibody 15E10	Antibody 10D3
			CDRH1	SEQ.I.D.NO：1	SEQ.I.D.NO：40
CDRH2	SEQ.I.D.NO：2	SEQ.I.D.NO：41
			CDRH3	SEQ.I.D.NO：3	SEQ.I.D.NO：42
CDRL1	SEQ.I.D.NO：4	SEQ.I.D.NO：43
			CDRL2	SEQ.I.D.NO：5	SEQ.I.D.NO：44
CDRL3	SEQ.I.D.NO：6	SEQ.I.D.NO：45
			V HStructural domain (mouse)	SEQ.I.D.NO：7	SEQ.I.D.NO：46
V LStructural domain (mouse)	SEQ.I.D.NO：8	SEQ.I.D.NO：47
			V HStructural domain (humanization, B3)	SEQ.I.D.NO：9	SEQ.I.D.NO：48
V LStructural domain (humanization, L2)	SEQ.I.D.NO：10	SEQ.I.D.NO：49
			Heavy chain (humanization)	SEQ.I.D.NO：11	SEQ.I.D.NO：50
Light chain (humanization)	SEQ.I.D.NO：12	SEQ.I.D.NO：51
			V HStructural domain (mouse, PN)	SEQ.I.D.NO：15	SEQ.I.D.NO：52
V LStructural domain (mouse, PN)	SEQ.I.D.NO：16	SEQ.I.D.NO：53
			V HStructural domain (humanization, PN, B3)	SEQ.I.D.NO：17	SEQ.I.D.NO：54
V LStructural domain (humanization, PN, L2)	SEQ.I.D.NO：18	SEQ.I.D.NO：55
			Heavy chain (humanization, PN)	SEQ.I.D.NO：19	SEQ.I.D.NO：56
Light chain (humanization, PN)	SEQ.I.D.NO：20	SEQ.I.D.NO：57
			V HStructural domain (B4, humanization)	SEQ.I.D.NO：21	N/A
Heavy chain (humanization, Fc sudden change)	SEQ.I.D.NO：61	N/A
			Heavy chain (humanization, the Fc sudden change, PN)	SEQ.I.D.NO：62	N/A

Only utilize embodiment to describe the present invention now.Appended claims can comprise the summary of one or more following examples.

Embodiment

Embodiment 1-6 relates to production and the through engineering approaches of antibody 15E10.Embodiment 7 relates to production and the through engineering approaches of antibody 10D3.

1. the generation of monoclonal antibody

Generally according to E Harlow and D Lane, Antibodies a Laboratory Manual, Cold Spring Harbor Laboratory, the method for being stated in 1988 is by hybridoma manufacture order clonal antibody.The bone-marrow-derived lymphocyte of murine myeloma cell and target antigen immune mouse merges.The myelomatosis fusion partner makes the hybridoma immortalization, provides the ability that produces antibody by bone-marrow-derived lymphocyte simultaneously.

RIBI adjuvant (Sigma) suspension of the glycosylated human OSM (hOSM) that in Chinese hamster ovary celI, produces by peritoneal injection, 4 SJL mouse of immunity.After 2 weeks, with independent hOSM booster immunization mouse, then, after 2 weeks, with using anti-site III monoclonal antibody (OM4/11.17; 1: 1.5 weight of OSM: Mab: weight) neutral hOSM booster immunization mouse, with site II that immunne response is led, then, after other 2.5 weeks, use OSM-MAb mixture booster immunization mouse again, back in 5 weeks at last with independent OSM booster immunization mouse.Behind initial immunity 3 months, take out spleen, use PEG1500 (Boehringer) that bone-marrow-derived lymphocyte and the murine myeloma cell that derives from the P3X cell are merged, to produce hybridoma.By the single hybridoma cell line of limited dilution cloning (E Harlow and D Lane).Microscopically is differentiated the hole that contains single colony, the activity of test supernatant liquor.Amplification is used for low temperature storage, antibody producing etc. from activity the highest clone's cell.Initial OSM antibody is selected to be based on during gp130 suppresses to estimate in ELISA and the KB cell experiment and specificity and the effectiveness of people's glycosylation OSM, and (vide infra), back one experiment provides the inspection of OSM specificity.After identifying enough effectiveness and correct specific antibody, use further choice criteria:

The cross reactivity of 1/ couple of macaque OSM

2/ keeps the activity of anti-people OSM in the presence of blended people AB serum

3/ keeps the activity in anti-people's neutrophilic granulocyte OSM library and the activity of anti-RA synovial fluid cell source OSM

Suppress 1920 hybridomas of screening among the ELISA at gp130.43 generations surpass 50% inhibition, carry out limited dose response experiments to 15, select wherein 6 to be used for further research.Carry out subclone to these 6, and select main clone.

Select two antibody according to rendeing a service: clone 15E10 and clone 10D3 (referring to embodiment 7).The 15E10 murine antibody suppresses more effective always among the ELISA at gp130, but when people OSM was target antigen, the effectiveness that is had in the KB cell experiment was similar to 10D3.But in two experiments, the effectiveness of the anti-macaque OSM of 15E10 murine antibody all far surpasses 10D3.

2. clone the clone of 15E10 variable region

Extract total RNA by clone 15E10 hybridoma, use mouse leader sequence Auele Specific Primer and, produce the cDNA of heavy chain and variable region of light chain structural domain by reverse transcription according to the antibody constant region of being scheduled to isotype (IgG2a/ κ).CDNA with heavy chain and light chain structural domain is cloned among the carrier pCR2.1 then, is used for order-checking.

2.1RNA extract

Use the total RNA separation system of SV of Promega, according to manufacturer's explanation, by 10 of hybridoma clone 15E10 ⁶Extract total RNA in the precipitation of cell.

2.2 reverse transcription

Use is to mouse leader sequence and the specific primer reverse transcription of mouse IgG γ 2a/ κ constant region RNA, to produce the cDNA of heavy chain and light chain variable structural domain.The mixture of the primer that uses is set forth in Jones ST and Bendig MM Bio/technology 9:88-89 (1991).

Prepare 50 μ M mouse V _HAnd V _LThe mixture of leader sequence forward primer.The solution that also prepares 50 μ M mouse γ 2a and κ constant region reverse primer.

2.3 reverse transcription PCR (RT-PCR)

Use the Access RT-PCR system of Promega,, the RNA of encoding heavy chain and variable region of light chain is carried out reverse transcription with double according to manufacturer's explanation.V _HAnd V _LForward and reverse primer are as mentioned above.

3.2.3 the clone of PCR product

3.1 gel-purified

With RT-PCR product (2 * V _HWith 2 * V _L) gel-like fluid on to preparation type 1% sepharose that contains 0.01% ethidium bromide, and in the TAE damping fluid with 100V electrophoresis 1 hour, downcut V district band.With dna ladder electrophoresis on gel of 100bp, differentiate V in addition with permission _HAnd V _LBand.

Use the QIAquick of Qiagen ^TMGel extraction kit is according to manufacturer's explanation, by gel extraction and purifying DNA fragment.

3.2 connect

Use the TA clone test kit of Invitrogen, according to manufacturer's explanation, with the RT-PCR fragment (2 * V of purifying _HWith 2 * V _L) be cloned in the pCR2.1 carrier.

3.3 transform

According to the explanation of TA clone test kit the plasmid that connects is transformed in TOP10F ' cell.The transformant of 50 μ l and 200 μ l is coated on the L-agar plate, and this agar plate contains 100 μ g/ml penbritins, and covers the DMF solution of 8 μ l 500mM IPTG solution and 16 μ l 50mg/mlX-Gal.In 37 ℃ of incubation flat boards that spend the night.

3.4 order-checking

Add in the LB substratum of 100 μ g/ml penbritins in 5 white colonies of 37 ℃ of incubated overnight at 5ml.

Use Qiagen QIAprep Spin Miniprep Kit, according to manufacturer's explanation, extraction and purifying contain 15E10 V _HAnd V _LThe pCR2.1 plasmid of structural domain.

Use primer T7, M13 for and M13 rev to V _HAnd V _LThe structural domain order-checking.

15E10 V _HStructural domain aminoacid sequence (10 clones' of 2 RT-PCR reactions consensus sequences): SEQ.I.D.NO:7

15E10V _LStructural domain aminoacid sequence (10 clones' of 2 RT-PCR reactions consensus sequences): SEQ.I.D.NO:8

4. chimeric antibody

The chimeric antibody that design is made up of 3.4 the parental generation mouse V district of grafting in human IgG1/κ wild-type C district, to confirm the clone in correct mouse V district, when test humanization construction, this chimeric antibody is again as reference.Chimeric antibody is expressed in Chinese hamster ovary celI, the purifying chimeric antibody, and in gp130 inhibition ELISA and KB cell experiment, test its avidity (vide infra) to OSM site II.

Mouse V district by the pcr amplification clone is cloned into mammalian expression vector Rld and the required restriction site of Rln with importing.Hind III and Spe I site are designed for confines V _HStructural domain, and allow to be cloned in the improvement Rld carrier that contains people γ 1 wild-type C district.Hind III and BsiW I site are designed for confines V _LStructural domain, and allow to be cloned in the improvement Rln carrier that contains people κ C district.

4.1PCR amplification

V _HForward primer: 5 '-GAT GAA GCT TGC CAC CAT GGC TGT CCTAGG GCT ACT C-3 ' (SEQ.I.D.NO:22)

Hind III restriction site underlines, and the Kozak sequence is a runic.

V _HReverse primer: 5 '-GAT GG A CTA GTG TCC CTG TGC CCC AGAC-3 ' (SEQ.I.D.NO:23)

Spe I restriction site underlines.

V _LForward primer: 5 '-GAT G AA GCT TGC CAC CAT GGA TTT TCAGGT GCA GAT T-3 ' (SEQ.I.D.NO:24)

Hind III restriction site underlines, and the Kozak sequence is a runic.

V _LReverse primer: 5 '-GAT G CG TAC GTT TGA TTT CCA ACT TTGTCC C-3 ' (SEQ.I.D.NO:25)

BsiW I restriction site underlines

PCR reaction: water 66 μ l

10 * PCR damping fluid, 10 μ l

dNTP(2 mM) 10μl

Primer 1 (5 μ M) 4 μ l

Primer 2 (5 μ M) 4 μ l

AmpliTaq polysaccharase 2 μ l

The plasmid 4 μ l of purifying

Cumulative volume 100 μ l

Primer 1:V _HOr V _LForward primer

Primer 2: V _HOr V _LReverse primer

The plasmid of purifying: by the pCR2.1V of Qiagen Minipreps purifying _HOr V _LPlasmid (dilution 200X)

The PCR circulation: 1-95 ℃, 4 minutes

2-95 ℃, 1 minute

3-55 ℃, 1 minute

4-72 ℃, 1 minute

5-72 ℃, 7 minutes

Step 2-4: repeat 30 times

4.2 be cloned into mammalian expression vector

Use the MinElute PCR purification kit of Qiagen, according to manufacturer's explanation, purified pcr product.

4.2.1 restrictive diges-tion

With Hind III-Spe I digestion V _HPCR product and Rld hC γ 1wt mammalian expression vector:

10 * damping fluid (NEBuffer2), 5 μ l

BSA 100x(NEB) 0.5μl

DNA 5μl

Hind III(Promega) 2μl

Spe I(NEB) 2μl

Water 35.5 μ l

Cumulative volume 50 μ l

DNA: the V of purifying _HPCR product or Rld hC γ 1wt carrier (with 0.25 mg/ml) were in 37 ℃ of incubations 2 hours.

With Hind III-BsiW I digestion V _LPCR product and Rln hC κ mammalian expression vector:

10 * damping fluid (NEBuffer2), 5 μ l

DNA 5μl

Hind III(Promega) 2μl

Water 38 μ l

Cumulative volume 50 μ l

DNA: the V of purifying _LPCR product or Rln hC κ carrier (with 0.25 mg/ml) were in 37 ℃ of incubations 2 hours.

Add 2 μ l BsiW I (NEB), and in 55 ℃ of incubations 2 hours.

4.2.2 gel-purified

With on the gel-like fluid of restrictive diges-tion product to preparation type 1% sepharose that contains 0.01% ethidium bromide, and in the TAE damping fluid with 100V electrophoresis 1 hour, downcut Rld and Rln carrier and V _HAnd V _LPCR fragment band.With dna ladder electrophoresis on gel of 100bp, differentiate V in addition with permission _H, V _LAnd carrier strap.

Use the QIAquick gel extraction kit of Qiagen, according to manufacturer's explanation, by gel extraction and purify DNA.

4.2.3 connect

V with Hind III-Spe I digestion _HThe PCR fragment is connected in the Rld hC γ 1wt carrier of Hind III-Spe I digestion.

V with Hind III-BsiW I digestion _LThe PCR fragment is connected in the Rln hC κ carrier of Hind III-BsiW I digestion.

Use the LigaFast rapid DNA connected system of Promega, connect, obtain according to manufacturer's explanation:

V _H: carrier: the Rld hC γ 1wt of Hind III-Spe I digestion

Insert fragment: the V of Hind III-Spe I digestion _HThe PCR fragment

V _L: carrier: the Rln hC κ of Hind III-BsiW I digestion

Insert fragment: the V of Hind III-BsiW I digestion _LThe PCR fragment

4.2.4 transform

To connect product is transformed in the DH5 α competent cell:

Melt 200 μ l DH5 α bottles on ice.

Preparation 50 μ l equal portions in conversion tube.

Add 2 μ l and connect mixture, and mix gently, then incubation on ice 30 minutes with pipette tip.

Under the situation of not vibration with mixture in 42 ℃ of incubations 45 seconds.

Then it is transferred to and reaches 2 minutes in the ice.

Add 450 μ l SOC substratum, with pipe on the shaking culture case in 37 ℃ of incubations 1 hour.

100 μ l cultures are coated on the L-agar plate of adding 100 μ g/ml penbritins, and in 37 ℃ of incubations that spend the night.

4.2.5 order-checking

Add in the LB substratum of 100 μ g/ml penbritins in 37 ℃ of incubated overnight V at 5ml _HAnd V _LThe clone.

Use the QIAprep Spin Miniprep Kit of Qiagen, according to manufacturer's explanation, extraction and purifying contain V respectively _HAnd V _LThe Rld of structural domain and Rln plasmid.

Use in the Rld carrier forward primer and signal sequence and the reverse primer in people C γ 1 district to V _HDistrict's order-checking.

Use in the Rln carrier forward primer and signal sequence and the reverse primer in people C κ district to V _LDistrict's order-checking.

Discriminating has correct V _HAnd V _LThe clone of sequence, and preparation is used for the plasmid of expressing at Chinese hamster ovary celI.

4.3 the expression of chimeric antibody in Chinese hamster ovary celI

15E10V will be contained respectively _HAnd V _LThe Rld of structural domain and Rln plasmid transient cotransfection are gone in the Chinese hamster ovary celI and are expressed.The chimeric antibody that is produced by the cell culture supernatant purifying by the affinity chromatography on reorganization A Protein S epharose, and suppress to estimate in ELISA and the KB cell experiment its avidity (vide infra) to OSM at gp130.

4.3.1 plasmid purification

To contain Rld-15E10V _HAnd Rln-15E10V _LThe DH5 α cell of plasmid is added in the LB substratum of 100 μ g/ml penbritins in the shaking culture case 37 ℃ at 5ml and was cultivated 8 hours.

Add the LB substratum of 100 μ g/ml penbritins with the 8 times each workday culture of 1ml inoculation 200ml, and in the shaking culture case in 37 ℃ of incubations that spend the night.

Use the QIAfilter Plasmid Maxi Kit of Qiagen,, extract and plasmid purification according to manufacturer's explanation.Ethanol sedimentation is resuspended in the 200 μ l TE damping fluids, and behind 100 times of dilution mother liquors with the absorbance measurement plasmid concentration of 260nm.

4.3.2 transfection

At 4 * 175cm ²In the BD Falcon tissue culture flasks, cultivate Chinese hamster ovary celIs to being paved with adding containing in Glutamax-1 Dulbecco MEM (DMEM) substratum of ultralow foetal calf serum and 1% penicillin-Streptomycin sulphate in 37 ℃.

For each bottle, in 50ml Falcon pipe, add following material and under vortex, mix:

The 8ml Optimem 1 that contains Glutamax-1

20 μ g Rld-15E10V _HPlasmid purification

20 μ g Rln-15E10V _LPlasmid purification

240 μ l TransFast transfection reagents

With mixture in room temperature (RT) incubation 10-15 minute.Remove the DMEM substratum in the bottle, this mixture of vortex mixed then, and join in the bottle.

With mixture in 37 ℃ of incubations 1 hour.

In bottle, add 32ml Optimem, and in 37 ℃ of incubation 48-72 hours.

4.3.3 the purifying of chimeric antibody

Merge all 175cm ²The substratum of bottle, and on MSE Mistral 2000 with 1500rpm centrifugal 3 minutes, supernatant liquor is by the Filter System 0.22 μ m CA of 500mL.

On the Amersham Biosciences Akta Explorer that uses Unicorn software by clarifying supernatant liquor antibody purification.

The post that uses is 1ml HiTrap rProtein A Sepharose FF.

Flow velocity is 1ml/ minute.

Pillar loads clarifying supernatant liquor by pump A then with 10 CV (column volume) Dulbecco PBS balance.

Wash post with 20 CV Dulbecco PBS streams, pump A stream is washed till no supernatant liquor, make 10 CV Dulbecco PBS again by post, to guarantee that supernatant liquor is removed fully.

With 10 CV ImmunoPure IgG elution buffer (Pierce) wash-out antibody, and collect with the 1ml fraction that contains 100 μ l 1M Trizma-HCl pH8.0 neutralization buffer.

With 5 CV Dulbecco PBS reequilibrate posts.

By read absorbancy in 280nm with respect to blank solution, antibody in the quantitative elutriated fraction, blank solution contains 10 volume ImmunoPure IgG elution buffers+1 volume 1MTrizma-HCl pH8.0, merges to have the fraction of the pure antibody of capacity, and is stored in-20 ℃ with 100 μ l equal portions.

4.4 chimeric antibody analysis

Suppress to analyze in ELISA and the KB cell experiment in the 15E10 of purifying and 10D3 (vide infra) chimeric antibody and the two effectiveness of people and macaque OSM (hOSM and cOSM) at gp130.Stated that hereinafter gp130 suppresses the method for ELISA and KB cell experiment.

IC50 (μ g/ml) value of table 1:15E10 and 10D3 murine antibody and chimeric antibody

	gp130 ELISA	The KB cell experiment
			15E10 murine antibody 15E10 chimeric antibody 10D3 murine antibody 10D3 chimeric antibody	0.059 0.036 0.107 0.057	0.195 0.110 0.114 0.107

Suppress in ELISA (Fig. 2) and the KB cell experiment (Fig. 3) at gp130,15E10 and 10D3 chimeric antibody all in hOSM and cOSM.Chimeric antibody 15E10 than chimeric antibody 10D3 height, also is the same with the viewed result of parental generation murine antibody to the avidity of macaque OSM.Two kinds of chimeric antibodies all have curve profile and the IC50 value (table 1) that is similar to the parental generation murine antibody.Aminoacid sequence and the cDNA sequence of macaque OSM (cOSM) are set forth in SEQ.I.D.NO:63 and 64 respectively;

SEQ.I.D.NO：63：

MGVPLTRRTLLSLILALLFPSMASMAAMGSCSKEYRMLLGQLQKQTDLMQDTSR

LLDPYIRIQGLDIPKLREHCRESPGAFPSEETLRGLGRRGFLQTLNATLGCVLH

RLADLEQHLPKAQDLERSGLNIEDLEKLQMARPNVLGLRNNVYCMAQLLDNSDM

TEPTKAGRGTPQPPTPTPTSDVFQRKLEGCSFLRGYHRFMHSVGRIFSKWGESP

NRSRRHSPHQALRKGVRRTRPSRKGNRLMPRGQLPR

SEQ.I.D.NO：64：

ATGGGGGTACCGCTCACACGGAGGACGCTGCTCAGTCTGATCCTTGCACTCCTG

TTTCCAAGCATGGCAAGCATGGCGGCTATGGGCAGCTGCTCGAAAGAGTACCGC

ATGCTCCTTGGCCAGCTCCAGAAGCAGACAGATCTCATGCAGGACACCAGCAGG

CTCCTGGACCCCTATATACGTATCCAAGGCCTGGATATTCCTAAACTGAGAGAG

CACTGCAGAGAGAGCCCTGGGGCCTTCCCCAGCGAGGAGACCCTGAGGGGGCTG

GGCAGGCGGGGCTTCCTACAGACGCTCAATGCCACACTGGGCTGCGTCCTGCAC

AGACTGGCCGACTTAGAGCAGCATCTCCCCAAGGCCCAGGACTTGGAGAGGTCT

GGGCTGAACATAGAGGACTTAGAGAAGCTGCAGATGGCGAGGCCGAATGTCCTC

GGGCTCAGGAACAACGTCTACTGCATGGCCCAGCTGCTGGACAACTCAGACATG

ACTGAGCCCACGAAGGCCGGCCGGGGGACCCCTCAGCCGCCCACCCCCACCCCT

ACCTCAGATGTTTTTCAGCGCAAGCTGGAGGGCTGCAGTTTCCTGCGTGGCTAC

CATCGCTTCATGCACTCAGTGGGGCGGATCTTCAGCAAGTGGGGGGAGAGCCCG

AACCGGAGCCGGAGACACAGCCCCCACCAGGCCCTGCGGAAGGGGGTGCGCAGG

ACGAGACCCTCCAGGAAAGGCAATAGACTCATGCCCAGGGGACAGCTGCCCCGG

TAG

These results confirm, have successfully cloned correct 15E10 variable region, and generation can be in conjunction with the two antigen binding chimeric antibody of people and macaque OSM site II.But present humanization 15E10 heavy chain and light chain variable structural domain.

5.1.1 search mouse database

By the search peptide database, identify 15 and 15E10V _HAminoacid sequence has mouse sequence and 10 and the V of highest homology _LAminoacid sequence has the mouse sequence of highest homology.

Contrast 15E10V _HAminoacid sequence and whole 15 the mouse sequences that obtained by database search identify following important framework residue:

Position 15E10V _HThe mouse occurrence rate

75 R K 15/15

105 T Q 14/15

The numbering system that position sign closes civilian described Kabat etc.Contrast 15E10V _LAminoacid sequence and 10 mouse sequences that obtained by database search identify following important framework residue:

Position 15E10V _LThe mouse occurrence rate

9 T A 8/10

38 E Q 10/10

49 E Y 10/10

60 A V 10/10

5.1.2. Seeker's database

Use EasyBlast in peptide database, to differentiate and 15E10V _HAnd V _LFramework has people's frame sequence of highest homology.

To 15E10V _HIdentify two groups of human sequences:

Group A, select following framework wherein to carry out humanization:

QVQLQESGPGLVKPSETLSLTCTVSGGSIS SYYWSWIRQPPGKGLEWIG YIYYS

GSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR SPSSGSYYYYY

YGMDVWGQGTTVTVSS (SEQ.I.D.NO：26)

CDR underlines.

Group B, select following framework wherein to carry out humanization:

QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMHWVRQAPGKGLEWVA VIWYD

GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DLGGPLYWYF

DLWGRGTLVTVSS(SEQ.I.D.NO：27)

CDR underlines.

Following framework residue is differentiated to may be very important to recovering avidity, and may be needed reverse mutation:

Position (Kabat numbering) 15E10V _HGroup A group B

27 F G F

28 S S T

29 L I F

30 T S S

48 L I V

49 G G A

67 L V F

71 K V R

73 N T N

78 V F L

94 K R R

Design 8 humanization V with different reverse mutations _HConstruction, 4 people's frameworks (A1, A2, A3 and A4), 4 people's frameworks (B1, B2, B3 and B4) based on group B based on group A.

For 15E10V _L, identify lineup's sequence, select following sequence wherein to carry out humanization:

EIVLTQSPATLSLSPGERATLSC RASQSVSKYLAWYQQKPGQAPRLLIY DASNR ATGIPARFSGSGSGTDFTLTISNLEPEDFAVYYC QQRSNWPPTFGQGTKLEI

(SEQ.I.D.NO：28)

CDR underlines.

Following residue is differentiated to may be very important to recovering avidity, and may be needed reverse mutation:

Position (Kabat numbering) 15E10V _LPeople V _L

49 E Y

71 Y F

Design 2 constructions, one is direct grafting (L1), and another all has reverse mutation (L2) at two residues.

Humanization V _HConstruction A1:

QVQLQESGPGLVKPSETLSLTCTVSGFSLTNYGVHWIRQPPGKGLEWIGVIWRG

GSTDYNAAFMSRVTISVDTSKNQVSLKLSSVTAADTAVYYCAKSPNSNFYWYFD

VWGQGTTS (SEQ.I.D.NO：29)

Humanization V _HConstruction A2:

QVQLQESGPGLVKPSETLSLTCTVSGFSLTNYGVHWIRQPPGKGLEWIGVIWRG

GSTDYNAAFMSRVTISKDTSKNQVSLKLSSVTAADTAVYYCAKSPNSNFYWYFD

VWGQGTTS(SEQ.I.D.NO：30)

Humanization V _HConstruction A3:

QVQLQESGPGLVKPSETLSLTCTVSGFSLTNYGVHWIRQPPGKGLEWIGVIWRG

GSTDYNAAFMSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKSPNSNFYWYFD

VWGQGTTS(SEQ.I.D.NO：31)

Humanization V _HConstruction A4:

QVQLQESGPGLVKPSETLSLTCTVSGFSLTNYGVHWIRQPPGKGLEWIGVIWRG

GSTDYNAAFMSRLTISKDNSKNQVSLKLSSVTAADTAVYYCAKSPNSNFYWYFD

VWGQGTTS (SEQ.I.D.NO：32)

Humanization V _HConstruction B1:

QVQLVESGGGVVQPGRSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWVAVIWRG

GSTDYNAAFMSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPNSNFYWYFD

VWGRGTLV(SEQ.I.D.NO：33)

Humanization V _HConstruction B2:

QVQLVESGGGVVQPGRSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWVAVIWRG

GSTDYNAAFMSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPNSNFYWYFD

VWGRGTLV(SEQ.I.D.NO：34)

Humanization V _HConstruction B3:

QVQLVESGGGVVQPGRSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWVAVIWRG

GSTDYNAAFMSRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAKSPNSNFYWYFD

VWGRGTLV(SEQ.I.D.NO：35)

Humanization V _HConstruction B4:

QVQLVESGGGVVQPGRSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWVAVIWRG

GSTDYNAAFMSRLTISKDNSKNTLYLQMNSLRAEDTAVYYCAKSPNSNFYWYFD

VWHGRGTLV(SEQ.I.D.NO：36)

Humanization V _LConstruction L1:

EIVLTQSPATLSLSPGERATLSCSGSSSVSYMYWYQQKPGQAPRLLIYDTSNLA

SGIPARFSGSGSGTDFTLTISNLEPEDFAVYYCQQWSSYPPTFGQGTKLEIK

(SEQ.I.D.NO：37)

Humanization V _LConstruction L2:

EIVLTQSPATLSLSPGERATLSCSGSSSVSYMYWYQQKPGQAPRLLIEDTSNLA

SGIPARFSGSGSGTDYTLTISNLEPEDFAVYYCQQWSSYPPTFGQGTKLEIK

(SEQ.I.D.NO：38)

5.215E10 humanization

From the beginning prepare humanization V by making up overlapping oligonucleotide _HAnd V _LConstruction, described overlapping oligonucleotide contain restriction site and the people's signal sequence that is used for being cloned into Rld and Rln mammalian expression vector.Introduce Hind III and Spe I restriction site, to confine the V that contains people's signal sequence _HStructural domain is used for being cloned into the Rld that contains people γ 1 wild-type constant region.Introduce Hind III and BsiW I restriction site, to confine the V that contains people's signal sequence _LStructural domain is used for being cloned into the Rln that contains people κ constant region.

People's signal sequence: MGWSCIILFLVATATGVHS (SEQ.I.D.NO:39).Design 8 humanization V _HConstruction and 2 humanization V _LConstruction.This can produce 16 kinds of different chain combinations.Because it is consuming time that the oligonucleotide of variable region makes up, so initial decision only prepares V _HMinimum and maximum reverse mutation constructions (A1, A4, B1 and B4) of structural domain, and produce and two humanization V _LThe humanized antibody of construction combination.

Design 10 oligonucleotide and be used for making up, these 10 oligonucleotide length are 60 bases, and it is overlapping to have minimum 18 bases.

5.2.1 oligonucleotide makes up

Each oligonucleotide mother liquor by 5 μ l100 μ M prepares the oligonucleotide mixing solutions.Generally, use the software that is described in (2003) Methods 31:199-206 such as Ertl PF, carry out humanization V by making up overlapping oligonucleotide according to (1995) Gene 164 (1): 49-53 such as Stemmer WP _HAnd V _LSynthesizing of gene.

5.2.1.1 assembling PCR reaction:

Water 41.5 μ l

10 * ProofStart PCR damping fluid, 5 μ l

dNTP(10mM) 1.5μl

Oligonucleotide mixture 1 μ l

ProofStart archaeal dna polymerase 1 μ l

Cumulative volume 50 μ l

Assembling PCR circulation:

1-94 ℃, 2 minutes

2-94 ℃, 30 seconds

3-40 ℃, 2 minutes

4-72 ℃, 10 seconds

5-94 ℃, 15 seconds

6-40 ℃, 30 seconds

7-72 ℃, 20 seconds+3 seconds/circulation

Step 4-7 repeats 25 times.

5.2.1.2 recover PCR

Primer 1 and 2 is first upstream and downstream oligonucleotide that are used to assemble PCR.Recover PCR and allow the complete V gene of amplification.

Recover the PCR reaction:

Water 42 μ l

10 * ProofStart PCR damping fluid, 4 μ l

dNTP(10mM) 1.5μl

Primer 1 (100 μ M) 0.5 μ l

Primer 2 (100 μ M) 0.5 μ l

Assembling PCR reactant 1 μ l

ProofStart archaeal dna polymerase 0.5 μ l

Cumulative volume 50 μ l

Primer 1 primer 2

15E10-A1/A4 15E10-A4-U1 15E10-A4-L1

15E10-B1 15E10-B1-U1 15E10-B1-L1

15E10-B4 15E10-B1-U1 15E10-B4-L1

15E10-L1/L2 15E10-L1-U1 15E10-L1-L1

Recover the PCR circulation:

1-94 ℃, 2 minutes

2-94 ℃, 45 seconds

3-60 ℃, 30 seconds

4-72 ℃, 2 minutes

5-72 ℃, 4 minutes

Step 2-4 repeats 25 times.

Use the MinElute PCR purification kit of Qiagen, according to manufacturer's explanation, purifying recovers the PCR product.

5.2.2 restrictive diges-tion

As described in 4.2.1, with Hind III-Spe I digestion humanization 15E10V _HConstruction A1, A4, B1 are with two humanization 15E10V of Hind-III-BsiW I digestion _L

5.2.3 gel-purified

Product as 4.2.2 purifying restrictive diges-tion.

5.2.4 connect

15E10 humanization V with Hind III-Spe I digestion _HFragment is connected in the Rld hC γ 1wt carrier of Hind III-SpeI digestion.

15E10 humanization V with Hind III-BsiW I digestion _LFragment is connected in the Rln hC κ carrier of Hind III-BsiW I digestion.

Use the LigaFast rapid DNA connected system of Promega, connect according to manufacturer's explanation.

5.2.5 transform

As described in 4.2.5.

5.2.6 order-checking

With the bacterium colony of each reaction plate in the 5ml LB substratum of adding 100 μ g/ml penbritins in 37 ℃ of incubated overnight.

Use the QIAprep Spin Miniprep Kit of Qiagen,, extract and plasmid purification, and use described primer order-checking as 4.2.5 according to manufacturer's explanation.

Discriminating has correct humanization V _HAnd V _LThe clone of sequence, and preparation is used for the plasmid of expressing at Chinese hamster ovary celI.

6. the expression of humanized antibody in Chinese hamster ovary celI

4 humanization V of preparation in Rld hC γ 1wt and Rln hC κ mammalian expression vector _HConstruction (A1, A4, B1 and B4) and 2 humanization V _LConstruction (L1 and L2).8 kinds of plasmid heavy chains-light chain combination (A1L1, A1L2, A4L2, B1L2, B4L1 and B4L2) transient cotransfection is gone in the Chinese hamster ovary celI, and to express on a small scale, obtained 8 different humanized antibodies.Suppress the antibody (vide infra) that elisa assay produces with gp130 in supernatant liquor.

6.1 plasmid purification

The DH5 α cell that will contain one of plasmid of the 6th joint is added in the LB substratum of 100 μ g/ml penbritins in the shaking culture case 37 ℃ at 5ml and was cultivated 8 hours.

Add the 200ml LB substratum of 100 μ g/ml penbritins with 1ml 8 times each workday culture inoculation, and in the shaking culture case in 37 ℃ of incubations that spend the night.

Use the QIAfilter Plasmid Maxi Kit of Qiagen,, extract and plasmid purification according to manufacturer's explanation.Ethanol sedimentation is resuspended in the 200 μ l TE damping fluids, and behind 100 times of dilution mother liquors with the absorbance measuring plasmid concentration of 260nm.

6.2 transfection

With 10 ⁶9 holes of Chinese hamster ovary celI inoculation Coming Costar 3506 6-orifice plates, and in adding the DulbeccoMEM that contains Glutamax-l (DMEM) substratum of ultralow foetal calf serum and 1% penicillin-Streptomycin sulphate in 37 ℃ of incubated overnight.

For each hole, under vortex, be added in the following material among the 5ml Bijou:

1ml contains the Optimem 1 of Glutamax-1

5 μ g carry humanization V _HPlasmid

5 μ g carry humanization V _LPlasmid

30 μ g TransFast transfection reagents

So that each transfection all comprises different light chains and heavy chain combination.Carry out 10-15 minute incubation in room temperature.By taking out the DMEM substratum in the hole, this mixture of vortex mixed then, and join in the suitable hole.

Incubation carried out 1 hour in 37 ℃.

Every hole adds 2ml Optimem, and in 37 ℃ of incubation 48-72 hours.

6.3 analysis humanized antibody

Reclaim substratum by each hole, and on Eppendorf 5415R desk centrifuge centrifugal 1 minute with 13000rpm, with supernatant liquor by 0.2 μ m Pall Acrodisc 25mm needle-based strainer.

Suppress in 8 humanized antibodies of elisa assay (4 based on group A people framework, 4 based on group B people framework) and the 15E10 chimeric antibody the two effectiveness (referring to Fig. 4) with gp130 with hOSM and cOSM.

Table 2: suppress humanized antibody among the ELISA at gp130

The IC50 value of B1L1, B1L2, B4L1 and B4L2

	People OSM	Macaque OSM
			B1L1 B1L2 B4L1 B4L2 15E10 chimeric antibody	NA 0.334 NA 0.048 0.070	NA 0.110 0.167 0.040 0.060

The IC50 value is represented with μ g/ml

NA: suppress to be lower than 50%

In gp130 suppressed ELISA, the reverse mutation level in the humanized antibody of expression pair had a direct impact with the avidity of people and macaque OSM.The antibody of minimum reverse mutation (B1L1) can not detect the avidity of macaque OSM, to the avidity of people OSM only more than background.On the other hand, the antibody (B4L2) of reverse mutation is identical with chimeric 15E10 antibody at least to the avidity of people and macaque OSM at most.Contain the avidity that 2 humanized antibodies of reverse mutation light chain have and be higher than 2 humanized antibodies that contain direct grafted light chain.

4 humanized antibody nones organizing the A framework based on the people produce the inhibition signal in gp130 ELISA experiment.In fact, these antibody nones can be detected human IgG1's whole antibody (wherein the polyclonal antibody of capture antibody for producing at people's gamma heavy chain is the polyclonal antibody that produces at human kappa light chain and detect antibody) in goat in goat in ELISA.

Further analyze the supernatant liquor that contains these 4 kinds of antibody in human IgG heavy chain specific ELISA and light chain specific ELISA, two experiments all produce positive signal.Two ELISA use in goat the capture antibody that produces at human IgG heavy chain and light chain, and for the heavy chain specific ELISA, detect antibody to produce at human IgG γ chain, for the light chain specific ELISA, detect antibody and produce at human IgG κ chain.

These results suggest, wherein heavy chain is expressed heavy chain and light chain simultaneously by the humanized antibody of group A people frame design, but two chains variable antibody of combination results not.

Organize the V of maximum reverse mutations of B framework structure based on the people _HConstruction (B4) makes up with the light chain (L2) of reverse mutation, is proved to be the most effective humanized antibody.Production, purifying are also analyzed to contain and are organized B V _H3 kinds of humanized antibodies (B2L2, B3L2 and B4L2), to determine to be suitable for most the humanized antibody that material standed for is selected.

6.4: the people source V of preparation 6.3 _HConstruction

As preparation 2 kinds of humanization constructions B2 and B3 as described in the 5.2.1 to 5.2.6.

6.5 the expression of humanized antibody in Chinese hamster ovary celI

Contain humanization V with 3 kinds _HPlasmid (B2, B3 and B4) with the 6th the joint the humanization V that contains maximum reverse mutations _LThe combination transient cotransfection of plasmid (L2) go in the Chinese hamster ovary celI and express.3 kinds of humanized antibodies that produced by the cell culture supernatant purifying by the affinity chromatography on rProtein A Sepharose, and use the 15E10 chimeric antibody to suppress to estimate in ELISA and the KB cell experiment its avidity at gp130 to OSM as reference.

As described in 4.3.1, carry out plasmid purification.As described in 4.3.2, carry out transfection.As carrying out the purifying of humanized antibody as described in the 4.3.3.

6.6 analyze the humanized antibody of 6.5 joints

Suppress to analyze in ELISA and the KB cell experiment (vide infra) in the humanized antibody of purifying of 6.5 joints the two effectiveness at gp130 with people and macaque OSM.Experiment is carried out with the people OSM in various sources, comprises the people OSM+25% people AB serum of people OSM, the CHO generation that CHO produces, RA patient's neutrophilic granulocyte and synovia.

Gp130 suppresses ELISA: experimental data is shown in Fig. 5-10.

The KB cell experiment: experimental data is shown in Figure 11-16.

These results show that the effectiveness that humanized antibody (B3L2 and B4L2) is had is equal to the 15E10 chimeric antibody, but is higher than humanized antibody B2L2.This shows that humanization strategy (the especially selection of reverse mutation) obtains to recover fully antigen avidity.

B4 V _HThe aminoacid sequence of chain is:

QVQLVESGGGVVQPGRSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWVAVIWRG

GSTDYNAAFMSRLTISKDNSKNTLYLQMNSLRAEDTAVYYCAKSPNSNFYWYFD

VWGRGTLVTVSS(SEQ.I.D.NO：21)

V _LChain is SEQ.I.D.NO:12.

The V that contains SEQ.I.D.NO:21 _HThe V of chain and SEQ.I.D.NO:12 _LThe therapeutic antibodies of chain or its Fab can be considered to competitive antibody of the present invention, therefore constitute embodiment of the present invention.

6.7 the contrast of B3L2 humanized antibody and chimeric antibody and parental generation murine antibody

End user and macaque OSM contrast humanized antibody B3L2 as target antigen in experiment of gp130 inhibition and KB cell experiment (vide infra) and 15E10 is chimeric and the parental generation murine antibody.Design has the humanization B3L2 antibody (Leu that the Ala replacement is 235 and 237 Gly) of 2 point mutation in CH, express and purifying in Chinese hamster ovary celI.Sudden change has reduced the ability of antibody enforcement effector function (especially complement factor is raised).Humanized antibody material standed for B3L2 with complete heavy chain is called as B3L2 wt (wild-type), and mutant B3L2 antibody is called B3L2 mut in Figure 17-19.

Table 4: suppress humanization B3L2 wild-type in ELISA and the KB cell experiment at gp130 IC50 value contrast with parental generation murine antibody and chimeric antibody

	gp130 ELISA	The KB cell experiment
			The chimeric 15E10 B3L2 of mouse 15E10 wt	0.009 0.019 0.035	0.053 0.079 0.123

The IC50 value is in μ g/ml

These results confirm that humanization B3L2 antibody has the effectiveness that is equal to parental generation murine antibody 15E10.

The aminoacid sequence of humanization B3L2 heavy chain is set forth in SEQ.I.D.NO:11, and the aminoacid sequence of humanization B3L2 light chain is set forth in SEQ.I.D.NO:12.

Embodiment 7-antibody 10D3

7.1. the generation of monoclonal antibody

Detailed description as above embodiment 1 produces hybridoma 10D3.

7.2. the clone of clone 10D3 variable region

Extract total RNA by clone 10D3 hybridoma, use the antibody constant region of mouse leader sequence Auele Specific Primer and predetermined isotype (IgG1/ κ), produce the cDNA of heavy chain and light chain variable structural domain by reverse transcription.Then the cDNA of heavy chain and light chain variable structural domain is cloned among the carrier pCR2.1 and checks order.

7.2.1RNA extract

Use the total RNA separation system of SV of Promega, according to manufacturer's explanation, by 10 of hybridoma clone 10D3 ⁶Extract total RNA in the precipitation of cell.

7.2.2 reverse transcription

Use is to mouse leader sequence and the specific primer reverse transcription of mouse IgG γ 2a/ κ constant region RNA, to produce the cDNA of heavy chain and light chain variable structural domain.The mixture of the primer that uses is set forth in Jones ST and Bendig MM Bio/technology 9:88-89 (1991).

The mouse V for preparing 50 μ M _HAnd V _LThe mixture of leader sequence forward primer.Other prepares the mouse γ 2a of 50 μ M and the solution of κ constant region reverse primer.

7 2.3 reverse transcription PCRs (RT-PCR)

Use the Access RT-PCR system of Promega,, carry out the reverse transcription of the RNA of encoding heavy chain and variable region of light chain with double according to manufacturer's explanation.V _HAnd V _LForward and reverse primer are as mentioned above.

7.3.7.2.3 the clone of PCR product

7.3.1 gel-purified

With RT-PCR product (2 * V _HWith 2 * V _L) gel-like fluid on to preparation type 1% sepharose that contains 0.01% ethidium bromide, and in the TAE damping fluid with 100V electrophoresis 1 hour, downcut V district band.With dna ladder electrophoresis on gel of 100bp, differentiate V in addition with permission _HAnd V _LBand.

Use the QIAquick of Qiagen ^TMGel extraction kit is according to manufacturer's explanation, by gel extraction and purifying DNA fragment.

7.3.2 connect

Use the TA clone test kit of Invitrogen, according to manufacturer's explanation, with the RT-PCR fragment (2 * V of purifying _HWith 2 * V _L) be cloned in the pCR2.1 carrier.

7.3.3 transform

According to the explanation of TA clone test kit the plasmid that connects is transformed in TOP10F ' cell.The transformant of 50 μ l and 200 μ l is coated on the L-agar plate that contains 100 μ g/ml penbritins, and covers with the DMF solution of 8 μ l500mM IPTG solution and 16 μ l 50mg/ml X-Gal.In 37 ℃ of incubation flat boards that spend the night.

7.3.4 order-checking

Add in the LB substratum of 100 μ g/ml penbritins in 5 white colonies of 37 ℃ of incubated overnight at 5ml.

Use Qiagen QIAprep Spin Miniprep Kit, according to manufacturer's explanation, extraction and purifying contain 10D3V _HAnd V _LThe pCR2.1 plasmid of structural domain.

Use primer T7, M13 for and M13 rev to V _HAnd V _LThe structural domain order-checking.

10D3V _HStructural domain aminoacid sequence (10 clones' of 2 RT-PCR reactions consensus sequences): SEQ.I.D.NO:46

10D3V _LStructural domain aminoacid sequence (10 clones' of 2 RT-PCR reactions consensus sequences): SEQ.I.D.NO:47

7.4. chimeric antibody

The chimeric antibody that design is made up of the parental generation mouse V district of the 7.3.4 of grafting in human IgG1/κ wild-type C district, to confirm correct mouse V district clone, when test humanization construction, this chimeric antibody is again as reference.Chimeric antibody is expressed in Chinese hamster ovary celI, the purifying chimeric antibody, and in gp130 inhibition ELISA and KB cell experiment, test its avidity to OSM site II.

Mouse V district by the pcr amplification clone is cloned into mammalian expression vector Rld and the required restriction site of Rln with introducing.Design Hind III and Spe I site are used to confine V _HStructural domain, and allow to be cloned in the improvement Rld carrier that contains people γ 1 wild-type C district.Design Hind III and BsiW I site are used to confine V _LStructural domain, and allow to be cloned in the improvement Rln carrier that contains people κ C district.

7.4.1PCR amplification

V _HForward primer:

Hind III restriction site underlines, and the Kozak sequence is a runic.

V _HForward primer: 5 '-GAT G AA GCT TGC CAC CAT GGG ATG GAGCTG GGT CTT T-3 ' (SEQ.I.D.NO:58)

V _HReverse primer: 5 '-GAT GG A CTA GTG TGC CTT GGC CCC AATA-3 ' (SEQ.I.D.NO:65)

Spe I restriction site underlines.

V _LForward primer:

V _LForward primer: 5 '-GAT G AA GCT TGC CAC CAT GGA TTT ACAGGT GCA GAT T-3 ' (SEQ.I.D.NO:59)

Hind III restriction site underlines, and the Kozak sequence is a runic.

V _LReverse primer: 5 '-GAT G CG TAC GTT TCA GCT CCA GCT TGGTCC C-3 ' (SEQ.I.D.NO:60)

BsiW I restriction site underlines

PCR reaction: water 66 μ l

10 * PCR damping fluid l0 μ l

dNTP(2mM) 10μl

Primer 1 (5 μ M) 4 μ l

Primer 2 (5 μ M) 4 μ l

AmpliTaq polysaccharase 2 μ l

The plasmid 4 μ l of purifying

Cumulative volume 100 μ l

Primer 1:V _HOr V _LForward primer

Primer 2: V _HOr V _LReverse primer

The plasmid of purifying: by the pCR2.1 V of Qiagen Minipreps (dilution 200X) purifying _HOr V _LPlasmid

The PCR circulation: 1-95 ℃, 4 minutes

2-95 ℃, 1 minute

3-55 ℃, 1 minute

5-72 ℃, 7 minutes

Step 2-4: repeat 30 times

7.4.2 be cloned into mammalian expression vector

Use the MinElute PCR purification kit of Qiagen, according to manufacturer's explanation, purified pcr product.

7.4.2.1 restrictive diges-tion

With Hind III-Spe I digestion V _HPCR product and Rld hC γ 1wt mammalian expression vector:

10 * damping fluid (NEBufier2), 5 μ l

BSA 100x(NEB) 0.5μl

DNA 5μl

Hind III(Promega) 2μl

Spe I(NEB) 2μl

Water 35.5 μ l

Cumulative volume 50 μ l

DNA: the V of purifying _HPCR product or Rld hC γ 1wt carrier (with 0.25 mg/ml) were in 37 ℃ of incubations 2 hours.

With Hind III-BsiW I digestion V _LPCR product and Rln hC κ mammalian expression vector:

10 * damping fluid (NEBuffer2), 5 μ l

DNA 5μl

Hind III(Promega) 2μl

Water 38 μ l

Cumulative volume 50 μ l

DNA: the V of purifying _LPCR product or Rln hC κ carrier (with 0.25mg/ml) were in 37 ℃ of incubations 2 hours.

Add 2 μ l BsiW I (NEB), and in 55 ℃ of incubations 2 hours.

7.4.2.2 gel-purified

With on the gel-like fluid of restrictive diges-tion product to preparation type 1% sepharose that contains 0.01% ethidium bromide, and in the TAE damping fluid with 100V electrophoresis 1 hour, downcut Rld and Rln carrier and V _HAnd V _LPCR fragment band.With dna ladder electrophoresis on gel of 100 bp, differentiate V in addition with permission _H, V _LAnd carrier strap.

Use the QIAquick gel extraction kit of Qiagen, according to manufacturer's explanation, by gel extraction and purify DNA.

7.4.2.3 connect

V with Hind III-Spe I digestion _HThe PCR fragment is connected in the Rld hC γ 1wt carrier of Hind III-Spe I digestion.

V with Hind III-BsiW I digestion _LThe PCR fragment is connected in the Rln hC κ carrier of Hind III-BsiW I digestion.

Use the LigaFast rapid DNA connected system of Promega, connect, obtain according to manufacturer's explanation:

V _H: carrier: the Rld hC γ 1wt of Hind III-Spe I digestion

Insert fragment: the V of Hind III-Spe I digestion _HThe PCR fragment

V _L: carrier: the Rln hC κ of Hind III-BsiW I digestion

Insert fragment: the V of Hind III-BsiW I digestion _LThe PCR fragment

7.4.2.4 transform

The product that connects is transformed in the DH5 α competent cell:

Melt 200 μ l DH5 α bottles on ice.

Preparation 50 μ l equal portions in conversion tube.

Add 2 μ l and connect mixture, and mix gently, then incubation on ice 30 minutes with pipette tip.

Under the situation of not vibration with mixture in 42 ℃ of incubations 45 seconds.

Then it is transferred to and reaches 2 minutes in the ice.

Add 450 μ l SOC substratum, with pipe on the shaking culture case in 37 ℃ of incubations 1 hour.

100 μ l cultures are coated on the L-agar plate of adding 100 μ g/ml penbritins, and in 37 ℃ of incubations that spend the night.

7.4.2.5 order-checking

Add in the LB substratum of 100 μ g/ml penbritins in 37 ℃ of incubated overnight V at 5ml _HAnd V _LThe clone.

Use the QIAprep Spin Miniprep Kit of Qiagen, according to manufacturer's explanation, extraction and purifying contain V respectively _HAnd V _LThe Rld of structural domain and Rln plasmid.

Use in Rld carrier and signal sequence forward primer and the reverse primer in people C γ 1 district to V _HDistrict's order-checking.

Use in the Rln carrier forward primer and signal sequence and the reverse primer in people C κ district to V _LDistrict's order-checking.

Discriminating has correct V _HAnd V _LThe clone of sequence, and preparation is used for the plasmid of expressing at Chinese hamster ovary celI.

7.4.3 the expression of chimeric antibody in Chinese hamster ovary celI

10D3V will be contained respectively _HAnd V _LThe Rld of structural domain and Rln plasmid transient cotransfection are gone in the Chinese hamster ovary celI and are expressed.By the chimeric antibody of the affinity chromatography on reorganization A Protein S epharose, and in gp130 inhibition ELISA and KB cell experiment (vide infra), estimate its avidity to OSM by the generation of cell culture supernatant purifying.

7.4.3.1 plasmid purification

To contain Rld-10D3V _HAnd Rln-10D3V _LThe DH5 α cell of plasmid is added in the LB substratum of 100 μ g/ml penbritins in the shaking culture case 37 ℃ at 5ml and was cultivated 8 hours.

Add the LB substratum of 100 μ g/ml penbritins with 1ml 8 times each workday culture inoculation 200ml, and in the shaking culture case in 37 ℃ of incubations that spend the night.

Use the QIAfilter Plasmid Maxi Kit of Qiagen,, extract and plasmid purification according to manufacturer's explanation.Ethanol sedimentation is resuspended in the 200 μ l TE damping fluids, and behind 100 times of dilution mother liquors with the absorbance measuring plasmid concentration of 260nm.

7.4.3.2 transfection

At 4 * 175cm ²In the BD Falcon tissue culture flasks, in adding Duibecco MEM (DMEM) substratum that contains Glutamax-1 of ultralow foetal calf serum and 1% penicillin-Streptomycin sulphate, cultivate Chinese hamster ovary celI to being paved with in 37 ℃.

For each bottle, in 50ml Falcon pipe, under vortex, add following material and mixing:

8ml contains the Optimem 1 of Glutamax-1

20 μ g Rld-10D3V _HPlasmid purification

20 μ g Rln-10D3V _LPlasmid purification

240 μ l TransFast transfection reagents,

With mixture in room temperature (RT) incubation 10-15 minute.

Remove the DMEM substratum in the bottle, this mixture of vortex mixed then, and join in the bottle.

With mixture in 37 ℃ of incubations 1 hour.

In bottle, add 32ml Optimem, and in 37 ℃ of incubation 48-72 hours.

7.4.3.3 the purifying of chimeric antibody

Merge all 175cm ²The substratum of bottle, and on MSE Mistral 2000 with 1500rpm centrifugal 3 minutes, supernatant liquor is by the Filter System 0.22 μ m CA of 500mL.

On the Amersham Biosciences Akta Explorer that uses Unicorn software by clarifying supernatant liquor antibody purification.

The post that uses is 1ml HiTrap rProtein A Sepharose FF.

Flow velocity is 1ml/ minute.

Pillar loads clarified supernatant by pump A then with 10 CV Dulbecco PBS balances.

Wash post with 20 CV Dulbecco PBS streams, pump A stream is washed till no supernatant liquor, make 10 CV Dulbecco PBS again by post, to guarantee that supernatant liquor is removed fully.

With 10 CV ImmunoPure IgG elution buffer (Pierce) wash-out antibody, and collect with the 1ml fraction that contains 100 μ l 1M Trizma-HCl pH8.0 neutralization buffer.

With 5 CV Dulbecco PBS reequilibrate posts.

By read absorbancy in 280nm with respect to blank solution, antibody in the quantitative elutriated fraction, blank solution contains 10 volume ImmunoPure IgG elution buffers+1 volume 1MTrizma-HCl pH8.0, merges to have the fraction of the pure antibody of capacity, and is stored in-20 ℃ with 100 μ l equal portions.

7.4.4 the analysis of chimeric antibody

Suppress to analyze in ELISA and the KB cell experiment (vide infra) in the 10D3 chimeric antibody the two effectiveness at gp130 with people and macaque OSM.Stated that hereinafter gp130 suppresses the method for ELISA and KB cell experiment.

Suppress in ELISA and the KB cell experiment at gp130, in the 10D3 chimeric antibody and OSM.These results confirm, have successfully cloned correct variable region, and generation can be in conjunction with the two antigen binding chimeric antibody of people and macaque OSM site II.But present humanization 10D3 heavy chain and light chain variable structural domain.

Clone mouse variable region and to its order-checking, grafting is on people γ 1/ κ constant region, to produce chimeric antibody then.Anti-people that chimeric 10D3 antibody demonstrates in gp130 ELISA and KB cell experiment (vide infra) and the effectiveness of macaque OSM are equal to the parental generation murine antibody.

Use " optimum matching " strategy with the murine antibody humanization.For the variable heavy chain structural domain, select to have the sequence of 65% identity, with mouse CDR grafting on people's framework.Design has various reverse mutations in a large number to recover the construction of avidity in framework.These constructions are:

The construction reverse mutation

A T28I

B T28I、R71V、T73K

C T28I、V67A、M69L、R71V、T73K

D T28I、M48I、G44K、V67A、M69L、R71V、T73K

For variable light chain structural domain, select to have the sequence of 60.0% identity, with mouse CDR grafting on people's framework.Design has various reverse mutations in a large number to recover the construction of avidity in framework.These constructions are:

The construction reverse mutation

LA does not have (directly grafting)

LB L46R、L47W

LC Y36F、Q38K

LD Y36F、Q38K、L46R、L47W

LE Y36F、Q38K、L46R、L47W、F71Y

By making up the only synthetic construction (A, D, LA, LE) of overlapping oligonucleotide with minimum and maximum reverse mutations.Express 4 kinds of humanized antibodies combinations (ALA, ALE, DLA, DLE) at the Chinese hamster ovary celI middle and small scale, the affinity of antibody of clear liquid analytically in gp130 ELISA.

Only humanized antibody ALE and DLE demonstrate inhibition in gp130 ELISA, but ALE suppresses not enough owing to the antibody concentration in supernatant liquor is low, so select DLE.In Chinese hamster ovary celI, amplify and produce humanized antibody DLE, antibody purification, and use the 10D3 chimeric antibody in gp130 ELISA and KB cell experiment, to analyze antibody in contrast.

IC50 value (gp130 ELISA) (μ g/ml):

hOSM cOSM

Chimeric antibody 0.032 0.246

DLE 0.021 0.059

In gp130 ELISA, even if the effectiveness of anti-people OSM of humanized antibody 10D3 DLE and macaque OSM does not surpass chimeric antibody, at least equivalence yet.

In the KB cell experiment, analyze humanization 10D3 DLE and 10D3 chimeric antibody.10D3DLE obtains the anti-people OSM IC50 value of 0.205 μ g/ml and the anti-macaque OSMIC50 value of 0.07 μ g/ml.

In a word, successfully will resist the antibody 10D3 humanization of people OSM site II, the effectiveness of its demonstration is equal to the parental generation murine antibody.

Material

The total RNA separation system of SV: Promega Z3100

Access RT-PCR system: Promega A1250

QIAquick gel extraction kit: Qiagen 28704

Gel-like fluid: Sigma G7654

Agarose: invitrogen 15510-019

Ethidium bromide: Sigma E1510

TAE damping fluid: self-control

100bp dna ladder: New England BioLabs N3231S

TA clones test kit: Invitrogen 45-0046

TOP10F ' cell: Invitrogen 44-0300

L-agar+100 μ g/ml penbritins: self-control

X-Ga1, the DMF solution of 50mg/ml: Promega V394A

AmpliTaq archaeal dna polymerase: Applied Biosystems

10x PCR damping fluid: Applied Biosystems

E-Gel 1.2% agarose: Invitrogen G501801

LB substratum+100 μ g/ml penbritins: self-control

QIAprep Spin Miniprep Kit：Qiagen 27106

MinElute PCR purification kit: Qiagen 28004

NEBuffer2 10x concentrated solution: New England Biolabs B7002S

The BSA 100x concentrated solution of purifying: New England Biolabs B9001S

BsiW I：New England Biolabs R0553L

Hind III：Promega R604A

Spe I：New England Biolabs R0133S

LigaFast Rapid DNA connected system: Promega M8225

MAX Efficiency DH5 α chemoreception attitude cell: Invitrogen 18258-012

SOC substratum: self-control

QIAfilter Plasmid Maxi Kit：Qiagen 12263

The Dulbecco MEM:InVitrogen 31966-O21 that contains Glutamax-1

The Optimem 1:Invitrogen 51985-026 that contains Glutamax-1

TransFast transfection reagent: Promega E2431

1ml HiTrap reorganization A Protein S epharose FF:Amersham Biosciences17-5079-01

Dulbecco PBS：Sigma D8537

ImmunoPure IgG elution buffer: Pierce 21009

1M Trizma-HCl pH 8.0：Sigma T2694

ProofStart archaeal dna polymerase: Qiagen 1016816

ProofStart PCR damping fluid: Qiagen 1016961

Embodiment 8.gp130 suppresses ELISA

OSM is in regular turn in conjunction with any of gp130 and OSM acceptor or LIF acceptor.Experiment described here allows to measure the OSM (for example hOSM) in conjunction with the gp130 on the elisa plate.In addition, this experiment allows to measure the inhibition of the antibody of anti-OSM site II to OSM and gp130 receptors bind.

8.1 material

1.Nunc Immunoplate 1 F96 Maxisorp(Life Technologies，4-39454A)

2. people gp130-Fc 100 μ g/ml (R﹠amp; D Systems, 671-GP-100)

3.PBS

4.BSA(Sigma A7030)

5. people OSM 10 μ g/ml (R﹠amp that recombinate; D Systems, non-glycosylated)

6. anti-people OSM 50 μ g/ml (R﹠amp of biotinylation; D Systems, BAF295)

7. streptavidin HRP (Amersham RPN4401)

8.3,3 ', 5,5 '-tetramethyl benzidine (TMB) is (Sigma)

9. sulfuric acid

10.Tween 20(Sigma P7949)

8.2 the preparation of reagent

1. the preparation of plate: with PBS dilution people gp130-Fc to 1 μ g/ml, add, cover and in 4 ℃ of incubations that spend the night with 50 μ l/ holes.

2. rinsing damping fluid: in 1L PBS, add 500 μ l Tween 20 (0.05%)

3. blocking-up damping fluid: in 500ml PBS, add 5g BSA (1%)

8.3 method

1. use standard plate washing method rinsing plate and pat dry.

2. the blocking-up damping fluid that adds 200 μ l holes, and in room temperature incubation 1 hour.

3. as step 1 rinsing.

4. add 50 μ l/ hole OSM standard substance or samples.Cover and in stirring at room 2 hours.(per sample, with blocking-up damping fluid or tissue culture medium (TCM) with OSM be diluted to 100,50,25,12.5,6.25,3.125,1.563 and 0ng/ml)

5. as step 1 rinsing.

6. add 50 μ l/ holes to block the anti-people OSM of biotinylation that damping fluid is diluted to 30ng/ml.Cover and in stirring at room 1 hour.

7. as step 1 rinsing.

8. add 50 μ l/ holes and be diluted to 1/4000 streptavidin HRP with the blocking-up damping fluid.Cover and in stirring at room 30 minutes.

9. as step 1 rinsing.

10. the tmb substrate that adds 100 μ l/ holes.Cover and in stirring at room 30 minutes.

11. add the 1M H in 50 μ l/ holes ₂SO ₄

12. read 450 _NmOD.

8.4 be used to analyze the application of antibody-mediated gp130-OSM in conjunction with the experiment that suppresses

1) 25ng/ml OSM is mixed with the anti--OSM antibody of various concentration or the antiserum(antisera) of the various dilution OSM of containing antibody.In room temperature incubation 1 hour.

2) 96 orifice plates that contain bonded gp130 to as above preparation add the antibody-OSM mixture in 50 μ l/ holes.

3) proceed aforesaid experiment.

9.KB experiment

Introduction

KB cell (a kind of human epithelial cell is) is expressed gp130 together with LIF and OSM receptor mRNA (Mosley, J.Biol Chem., 271 (50) 32635-32643).OSM and LIF the two all induce the KB cell to discharge IL-6.This clone had been used for differentiating interactional monoclonal antibody between adjusting OSM and the gp130 already.

9.1 method

The KB cell derives from ECACC (preserving number 94050408), and remains among the DMEM+10% heat inactivation FCS (" KB substratum ") that adds glutamine.Cell divides bottle twice weekly with monolayer growth.Use Sigma non-enzymatic cell dissociation substratum or Versene isolated cell.

1. add 2 * 10 ⁴Cell/100 μ l hole/96 orifice plates, and the incubation that spends the night (37 ℃, 5%CO ₂).

2. prepare the OSM standard substance in substratum.

3. prepare 1ng/ml OSM+ antibody/serum dilution.In room temperature incubation 1 hour.

4. remove the substratum of KB cell plate carefully, add OSM standard substance and OSM-mixtures of antibodies.

5. in 37 ℃ of about 16-18 of incubation hours.

6. remove substratum and analyze IL-6.

Attention:

It is freezing that substratum can keep, until preparing to begin analysis.

Substratum should dilute about 20 times and be used for experiment.

When the screening hybridoma, the ratio of clone's substratum and KB substratum should be constant, and the OSM standard substance should prepare in this mixture.

Stimulate the KB cell to produce maximum IL-6 output with about 100ng/ml OSM, but 1ng/ml is enough to observe in the antibody and activity.

10. competitive experiment

This experiment allows to measure specificity and in conjunction with the candidate non-human antibody of hOSM site II the humanized antibody (for present embodiment, with the 15E10-B3L2 representative) of the light chain of heavy chain with SEQ.I.D.NO:11 and the SEQ.I.D.NO:12 bonded with solubility glycosyl hOSM is suppressed.Illustrating of this embodiment experiment in Figure 20.

Monoclonal antibody (being referred to herein as OM4-11.31) with anti-site III is wrapped by plate.

For typical curve: will begin the standard substance of 15E10-B3L2 purifying of serial dilution and the solubility glycosyl people OSM incubation of 50ng/ml by 1 μ g/ml.Antibody, resists by one of anti-site III then mixture is caught onboard in conjunction with OSM by site II.

Test for competitiveness: will begin candidate's antibody and the solubility glycosyl people OSM of 50ng/ml and the 15E10-B3L2 incubation of 150ng/ml of serial dilution by 1 μ g/ml.

By the anti-people γ chain two anti-existence that detect compound 15E10-B3L2.

Method:

1/ bag quilt

PBS solution with the anti-people OSM of 4 μ g/ml site III antibody (OM4-11.31, self-control) wraps by Nunc Maxisorp Immunoplate with 50 μ l/ holes.With plate in 4 ℃ of incubations that spend the night.

2/ blocking-up

With PBS+0.05%Tween (PBST) rinsing plate 3 times.The PBS solution that in every hole, adds 100 μ l1%BSA (Sigma A7030).Under vibration with plate in room temperature incubation 2 hours.

3/ preincubation

The 15E10B3L2 standard substance:

Prepare the solution of 1 μ g/ml 15E10-B3L2 antibody in the blocking-up damping fluid of 50ng/ml people OSM, and 67 μ l are joined in two capable holes of non-adsorptivity 96 orifice plate A.The capable antibody of blocking-up damping fluid serial dilution B-G with the 50ng/ml people OSM of 50 μ l.

Competitive antibody:

Prepare the solution of 1 μ g/ml competitive antibody in the blocking-up damping fluid of 150ng/ml 15E10-B3L2+50ng/mlhOSM, and 100 μ l are joined in two capable holes of non-adsorptivity 96 orifice plate A.The capable antibody of blocking-up damping fluid serial dilution B-G with the 150ng/ml 15E10-B3L2+50ng/ml people OSM of 50 μ l.Diluent incubation with two holes and uncontested property antibody.

The preincubation plate under static conditions in room temperature incubation 1 hour.

4/ incubation

With PBST rinsing bag by plate 3 times.

Various standard substance and the sample of 45 μ l are transferred to bag by in the same holes on the plate by the preincubation plate.In blank well, add PBS.

Under vibration with plate in room temperature incubation 2 hours.

5/ 2 is anti-

With PBST rinsing plate 3 times.

In every hole, add 50 μ l goats anti-people γ chain-peroxidase (Sigma A6029) with 2000 times of blocking-up damping fluid dilutions.

Under vibration with plate in room temperature incubation 1 hour.

6/ substrate

With PBST rinsing plate 3 times.

The aqueous solution for preparing OPD substrate (Sigma P9187) according to manufacturer's explanation.

In every hole, add 50 μ l.

Plate is in the room temperature incubation.

7/ stops

In case colour developing fully occurs, then by adding 10 μ l 3M H ₂SO ₄/ hole stops producing colour response.

Use blank well as 0 absorbancy, with plate reader in 490nm to the plate reading.

Draw the typical curve of 490nm absorbancy to 15El0 concentration.

Read the concentration of compound 15El0 in the sample that contains competitive antibody by typical curve.Following calculating suppresses percentage:

15E10 concentration ng/ml ÷ 150ng/ml in the 100-[(sample) * 100]

Draw to suppress the curve of percentage to competitive antibody concentration, by under the volumetric molar concentration competitive antibody such as curve reads out in (150ng/ml competitive antibody) to the inhibition percentage of 15E10.

Embodiment 10.1:10D3 is as competitive antibody

Mouse 10D3 clone's E9 antibody (mother liquor) of 267 μ g/ml is as the competitor of 15E10.10D3 has light chain and the heavy chain CDR that above Table A is listed.

The result:

10D3(μg/ml)	Compound 15E10 (μ g/ml)	Suppress percentage
			1 0.5 0.25 0.125 0.062 0.031 0.016	0.019 0.029 0.044 0.062 0.092 0.132 0.146	87.3 80.7 70.7 58.7 38.7 12.0 2.7

Etc. the 10D3 competitor of volumetric molar concentration (0.15 μ g/ml) inhibition percentage: 62.3% to 15E10.Referring to Figure 21.

Embodiment 11-differentiates in conjunction with OSM and is that OSM site II or site III are specific anti- Body

With regard to biological function, OSM must both interact with gp130, interacts with LIFR or OSMR β again.The prima facies mutual effect of OSM and gp130 relates to OSM site II, and the interaction of OSM and OSMR β or LIFR takes place through site III.Infer the antibody of target site II or site III OSM sequence or enough make antibodies can cover in the epi-position energy in these sites and the OSM activity thus near these sites.

Measure the experiment of OSM-gp130 bonded and be set forth in embodiment 8.Typical typical curve (in 1 μ g/ml, gp130) is shown in Figure 22.

By changing experiment condition (to 4 μ g/ml), sensitivity can greatly promote, shown in Figure 22 b.

And, although above data use non-glycosylated OSM to produce, glycosylation OSM in this experiment also in conjunction with gp130.Referring to Figure 22 c.

In this experiment, use commercially available neutralization anti-OSM antibody (Mab295, R﹠amp; DSystems), whether can block the OSM-gp130 interaction to observe it.Surprisingly, it strengthens the OSM signal, as shown in figure 23.

When Mab 295 (30 μ g/ml) when joining among the OSM, is compared with the independent OSM of OSM concentration＞10ng/ml, it will roughly be doubled by the OD reading that ELISA obtains.If gp130 is removed by the plate part omitted, then the signal of OSM+Mab295 generation is reduced to background values.The present inventor supposes following annotation: Mab295 and debond or blocking-up OSM site II.When low OSM concentration, the MAB295 antibody molecule is only in conjunction with an OSM, but this OSM is also free in conjunction with gp130, because site II can use.When higher concentration, antibody molecule is in conjunction with two OSM molecules, and wherein any all can be used in conjunction with gp130, therefore draw the molecule that has 2 OSM, these 2 OSM are all in conjunction with the gp130 molecule, and one directly in conjunction with gp130, and another two valency characteristics owing to antibody are to connect.Estimate that any non-site II antibody all should have this effect, but because Mab 295 is neutralizing antibody (referring to Figure 24), so its certain combination or blocking-up OSM site III.Therefore, be that site II is specific or site III is specific with OSM antibody during the KB cell experiment of the gp130-OSM ELISA of Application Example 8 experiment and embodiment 9 can identify.More particularly, site III antibody in the KB cell experiment should in and OSM, but in ELISA experiment or not and the OSM-gp130 combination.Site II antibody in ELISA and KB experiment all in and OSM.

Gp130-OSM ELISA experiment is as elementary hybridoma screening, to detect the interactional antibody of inhibition gp130-OSM that produces in embodiment 1.In addition, also hybridoma is screened, to detect OSM in conjunction with activity.Test among the OSM that demonstrates high OSM combination but in the ELISA of embodiment 8 experiment, do not suppress OSM-gp130 bonded hybridoma supernatant liquor and situation with the KB cell experiment of embodiment 9.This identifies many sites III specificity OSM antibody.Such antibody is called as OM4-11.31.

When using site III OSM specific antibody in gp130-OSM ELISA, it has greatly strengthened the OSM signal, as shown in figure 25.

Site II antibody 1B5 (1 μ g/ml) suppresses the OSM-gp130 combination fully.But site III OSM antibody OM4-11.3.1 causes that OSM bonded two-stage dose-dependently strengthens.When using the highest OM4-11.3.1 concentration, signal is approximately 2 times of independent OSM signal, but along with antibody concentration reduces, signal strengthens, and until reaching peak value, signal strengthens infers it is that form can be in conjunction with the result of antibody-OSM mixture of gp130.The isotype contrast IgG of OM4-11.3.1 does not act in conjunction with having OSM-gp130.Figure 25 shows that this ELISA distinguishes the highly sensitive of site II and non-site II specific antibody, because the former suppresses the OSM combination, but the latter strengthens the OSM combination.

Embodiment 11.1-site II and the anti-OSM antibody of site III specificity are at ELISA OSM- Effect in the gp130 experiment

When site II and site III OSM specific antibody mixed, site II antibody had remarkable effect in the gp130-OSM of embodiment 8 ELISA, as shown in figure 26.

Site III specificity OSM antibody OM4-11.17 has strengthened independent OSM signal greatly.Do not influence though this enhancement is not subjected to add contrast IgG, add angle of striking II specificity OSM antibody OM4-5.3 and greatly reduce signal.A small amount of detectable signal of believing Figure 26 rightmost side post should be owing to the inferior good incubation time of site II mAb before being added to the gp130 plate and site III-OSM mixture.

The site II specificity OSM antibody (referring to embodiment 1) that gp130-OSM ELISA allows monitoring to occur in people OSM immune mouse antiserum(antisera) is shown in Figure 27 a, 27b and 27c.

After strengthening for the first time, mainly produce non-site II antibody, but after strengthening for the second time, begin to occur site II specific antibody, after strengthening for the third time, obviously to observe site II antibody and preponderate, serum-concentration is higher.

Embodiment 11.2-OSM site II and site III specific antibody are assisted the OSM neutral Same-action

Because OSM site II and site III are that the OSM function is necessary, so the combination of the antibody in two sites of target can be in OSM and middle co-action.OSM site III not only is used for and OSMR β and LIFR interact, and is used for combining of second OSM molecule and gp130, compares with those antibody of anti-site II, and this has the effectiveness that helps increase site III specific antibody.

Figure 28 a and 28b illustrate the KB experiment, wherein, compare with any independent antibody, and the combination of site II specific antibody and site III specific antibody has increased the OSM neutral greatly and renderd a service.

The concentration that is used for the antibody of this combination is shown in following table.

[17H100]	[people 15E10]
		ng/ml	ng/ml
20	120
		7	40
2.2	13.3
		0.7	4.4
0.3	1.5
		0.082	0.5
0.027	0.165
		0.0091	0.55

The contrast of the most activated site II and site III specific antibody shows, the latter in OSM and in more effective.But, the cross reactivity of observing site II antibody and site III antibody and the OSM of other kind is different, because all effective site II antibody all in and macaque OSM (in gp130-OSM ELISA and KB cell experiment), and site III antibody can not be so (in only in the KB experiment with macaque OSM).

Infer OSM and gp130 and with the two interactional blocking-up of OSMR β or LIFR be site II antibody and site III antibody in OSM with in synergistic basis.But, also might be that being combined with of antibody is beneficial to the combination at the another kind of antibody of different loci.

In the combination of embodiment 11.3-site II and site III specificity OSM antibody and OSM Optimization

Because having strengthened neutralization greatly, the combination of site II and site III OSM antibody renders a service, so can be used to develop the strategy of optimal concentration based on the binding affinity imagination of different antibodies.

Embodiment 11.3 is theoretical properties.

At first, should use plasma resonance commercial measurement site II or site III specific antibody to previous respectively by the avidity of site III or site II specific antibody bonded OSM.If binding constant (Kd) is significantly different with combining of OSM with single antibody, then in the combination of site II and site III antibody cooperative interaction appears.

Based on the data of these antibodies researchs, can use site II and the site III antibody concentration of 10 times of Kd values of double dilution preparation to the 1/10Kd value.In addition, the combination of two kinds of antibody of preparation, so that the combination of the site III antibody of the site II antibody of each concentration and each concentration, the feasible combination that equates that can study site II or site III antibody and OSM, and the advantage in site II and the site III antibodies.Test the OSM neutralization of all antibody dilutions and combination with the KB cell experiment.The data of this experiment make can be chosen among the OSM and in the most effective antibody combination.

The anti-site of embodiment 12-II OSM specific antibody suppresses OSM stimulates the RA synovial membrane to become fine The ability of dimension cell

Before, we showed that site II and site III specificity OSM antibody can suppress OSM stimulated the KB cell.But these cells are that epithelial cell, process transform, and can not represent the cell that is present in the rheumatoid synovial membrane.Therefore, we have studied the ability of site II specificity OSM antibody inhibition OSM stimulation RA synovioblast.

With inoblast with 2 * 10 ⁴Cells/well is seeded in 96 orifice plates, and cultivates in containing the DMEM of 10%FCS, and until being paved with, a week is changed 3 subcultures.Then substratum is replaced by fresh culture, this fresh culture or do not contain OSM perhaps contains 1ng/mlOSM, perhaps contain with substratum in 1 hour the 1ng/ml OSM of anti-OSM antibody preincubation of various concentration.After 48 hours, take out culture supernatant, and be stored in-20 ℃, until using elisa assay IL-6 concentration.

Figure 29 illustrates the representative data of 4 RA synovioblasts strain.OSM antibody causes that the IL-6 excretory that OSM is stimulated suppresses fully, shows a little difference although antibody is renderd a service between different cell strains.

Embodiment 13-OSM glycosylation resists in the OSM antibody and the effect of rendeing a service

Use previously described method, by producing anti-OSM antibody with non-glycosylated OSM immune mouse.Screen these antibody and cause identifying the interference OSM effective neutralizing antibody of gp130 bonded (OM4-5.3), as shown in figure 30.

Estimate that OM5-5.3 should have the ability of similar anti-glycosylation OSM (Chinese hamster ovary celI glycosylation).But, when the subclone (OM4-5.3.1) of measuring this antibody suppresses glycosylation OSM (the glycosylated hOSM of Chinese hamster ovary celI) in conjunction with the ability of gp130, observe and render a service significantly forfeiture, shown in Figure 31 a.In addition, in deriving from other site II specific antibody of non-glycosylated OSM immune mouse, also observe the effectiveness of this anti-glycosylation OSM and compare forfeiture, shown in Figure 31 b with non-glycosylated OSM.

And in the KB cell experiment, the effectiveness that the site III antibody that derives from non-glycosylated OSM immunity also demonstrates anti-glycosylation OSM is about 1/10 times of non-glycosylated OSM-see table 1.

Table 1

Antibody	Non-glycosylated OSM	Glycosylation OSM
				IC50ng/ml	IC50ng/ml
OM4-11.17	4.1	45.5
			OM4-11.31	7.7	89.6

Because the antibody with more effective anti-non-glycosylated OSM of non-glycosylated OSM immunity generation rather than glycosylation form can produce the higher antibody of effectiveness that resists this form OSM so we think with glycosylation OSM immunity.Actual result is exactly like this.Figure 32 a and 32b illustrate the anti-glycosylation of two the site II specificity OSM antibody (15E10 and 5H2) that derive from glycosylation OSM immunity among the gp130-OSM ELISA and the activity of non-glycosylated OSM.

Association among the embodiment 14-RA patient between serum and the synovia OSM level

One of main position that OSM produces among the RA patient is arranged in arthritis knuckle, because can detect high OSM level in synovia.On the contrary, RA patient's serum OSM level is very low, only along with the exploitation of following examples 16 disclosed highly sensitive ELISA just might accurately be measured these levels.We have studied the possibility association between the OSM concentration in arthritis knuckle and the circulation by the synovia and the serum sample of measure R A patient coupling.

Serum and the synovia OSM level measured with the ELISA experiment (OM4-11.31 antibody capture OSM) of hereinafter statement are shown in following table, and Figure 33 illustrates two associations between the measuring result.Freezing sample after sampling only just melts before these are measured.The relation conefficient of these two parameters of measuring by linear regression is 0.9447.

The patient	Serum [OSM]	SF[OSM]
				pg/ml	pg/ml
1	9.8	43.24
			2	13.7	101.445
3	0	0
			4	88.56	397
5	22.64	142.12
			6	18	147.4
7	13	9.2
			8	13.8	29.88
9	10.68	14.76
			10	13.8	15.96

The prompting of good correlation between serum and the SF OSM level, the OSM production site beyond the arthritis knuckle is less relatively to the influence of circulation OSM level, perhaps these positions with in this joint, produce the mode that is associated and regulate OSM production.In any case the present inventor infers that this dependency can allow by serum OSM measuring result prediction joint OSM level, and can be used for dosage setting be used for the treatment of RA patient in and OSM antibody.

The measurement of OSM in embodiment 15-OA patient synovia (SF) and the serum

Because cartilage degradation is the feature of osteoarthritis, OSM (when particularly working in coordination with IL-1 and other cytokine) can induce chondrolysis, so we have measured the OSM level in OA patient's synovia and the serum.

By the cell in the centrifugal removal SF sample.With 0.1U/ml Unidasa (Fluka, 53725) in room temperature treatment supernatant liquor 1 hour, after this with 4000rpm centrifugal 10 minutes to it.Take out supernatant liquor, and be divided into equal portions, be stored in-80 ℃, until analysis.

In two experiments that are shown in Figure 34 a and b and 35, use the ELSIA of embodiment 16 to detect, analyze the OSM concentration among the OA SF.

Although 13 no detectable OSM are arranged among 46 OA SF, manyly contain relatively high level (＞200pg/ml) OSM detects OSM concentration＞1000pg/ml in 3 samples.

OSM concentration in the embodiment 15.1-OA serum

High density OSM in the OA synovia is astonishing, often is lower than level among the RA SF (referring to (2000) Arthritis Rheum.43 (2) such as Manicourt DH: 281-88) because previous report shows OSM level in the OA synovia.We again in 12 months cycles in several different time points use following examples 16 the ELSIA experiment measuring be in the serum OSM level of the OA patient in the clinical experiment.Figure 36 shows that the serum OSM concentration among these patients or lower perhaps can not detect.But, OA patient's serum and synovia OSM level are not carried out related because sample does not match.

Embodiment 16-is used for the sensitive ELISA of detection of biological sample lower concentration OSM

We have developed and have used site III OSM specificity capture antibody OM4-11.31 to measure the sensitive ELISA of the OSM in the biological sample.As shown in figure 37, this ELISA allows to detect and is low to moderate＜OSM of 2pg/ml, has been used for serum analysis and synovia sample.

Below provided the scheme of using this ELISA and serum sample or synovia.

OSM ELISA scheme

Material and reagent

11.Nunc Immunoplate F96 maxisorp(Life Technologies 4-39454A)

12. monoclonal anti-human OSM (OM4-11.31 GSK)

13. glycosylation hOSM, 420 μ g/ml (Chinese hamster ovary celI glycosylation)

14. the anti-people OSM 50 μ g/ml (R﹠amp of biotinylated goat; D Systems BAF295)

15. streptavidin HRP (Amersham RPN4401)

16.PBS(SIGM AD8537 1L)

17.BSA(SIGMA A7888 500g)

18. phenol red solution 0.5% (SIGMA P0290 100ml)

19.TMB(SIGMA T-8665 1L)

20. blended AB normal male serum contrast (SIGMA H4522) lot number #043K0500

21. sulfuric acid, 1M

22.PBS tablet (100 of SIGMA P4417)

23.Tween 20(Sigma P7949)

24. plate sealer

The preparation of reagent

The plate preparation-with PBS monoclonal anti-human OSM is diluted to 4 μ g/ml and adds 50 μ l/ holes, cover with sealed strip, in 4 ℃ of incubations that spend the night.

The rinsing damping fluid-25 PBS tablet+2.5mlTween 20 (0.05%) of adding in the 5L deionized water.

The blocking-up damping fluid-adding 10g BSA (2%) in 500ml PBS.(adding the phenol red and 5M NaOH of 800 μ l, is neutral until pH)

The contrast of AB serum

In the Sorvall whizzer with 16K, 30 minutes rotation 100ml (using the Oakridge pipe of 4 * balance) to 0.02g.

Make supernatant liquor pass through sterile gauze (still muddy but do not have particle).

Be divided into equal portions and freezing.

Detect the same day, melting AB serum, 13K microcentrifugation 5 minutes, and with PBS dilution 1 → 4 (serum is opaque, but fine concerning using)

The standard substance preparation

For serum analysis, preparation standard product in the serum of PBS dilution 1 → 4.

Analyze preparation standard product in containing the PBS solution of 1%BSA for SF.

Maximum sensitivity if desired:

The OSM of use 112,56,28,14,7,3.5,1.75 and 0pg/ml.

Method

5. with rinsing damping fluid rinsing plate 4 * and pat dry.

6. the blocking-up damping fluid that adds 200 μ l holes, sealing plate and in room temperature vibration 2 hours, or in+4 ℃ of static spending the night.

7. as step l rinsing.

8. add 50 μ l/ hole standard substance or samples.Cover and in stirring at room 2 hours.(if the serum analysis sample then is diluted in standard substance in the 25% blended AB serum)

5. as step 1 rinsing.

6. add 50 μ l/ holes and be diluted to the anti-people OSM of biotinylation of 50ng/ml with the blocking-up damping fluid that contains 1% lowlenthal serum.Cover and in stirring at room 1 hour.

7. as step 1 rinsing.

8. add 50 μ l holes and be diluted to 1/4000 streptavidin HRP with the blocking-up damping fluid.Cover and in stirring at room 30 minutes.

9. as step 1 rinsing.

10. add 100 μ lTMB substrates.Cover and in stirring at room 40 minutes.

11., add the 1M H in 50 μ l/ holes for stopping experiment ₂SO ₄

12. the vibration plate after immediately in the 450nm reading.

Sequence table

SEQ.I.D.NO：1

NYGVH

SEQ.I.D.NO：2

VIWRGGSTDYNAAFMS

SEQ.I.D.NO：3

SPNSNFYWYFDV

SEQ.I.D.NO：4

SGSSSVSYMY

SEQ.I.D.NO：5

DTSNLAS

SEQ.I.D.NO：6

QQWSSYPPT

SEQ.I.D.NO：7

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWRGG

STDYNAAFMSRLSITKDNSRSQVFFKMNSLQADDTAIYYCAKSPNSNFYWYFDVW

GTGTTVTVSS

SEQ.I.D.NO：8

QIVLTQSPTIMSASPGEKVTMTCSGSSSVSYMYWYQEKPGSSPRLLIEDTSNLAS

GVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGSGTKLEIK

SEQ.I.D.NO：9

QVQLVESGGGVVQPGRSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWVAVIWRGG

STDYNAAFMSRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAKSPNSNFYWYFDVW

GRGTLVTVSS

SEQ.I.D.NO：10

EIVLTQSPATLSLSPGERATLSCSGSSSVSYMYWYQQKPGQAPRLLIEDTSNLAS

GIPARFSGSGSGTDYTLTISNLEPEDFAVYYCQQWSSYPPTFGQGTKLEIK

SEQ.I.D.NO：11

QVQLVESGGGVVQPGRSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWVAVIWRGG

STDYNAAFMSRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAKSPNSNFYWYFDVW

GRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA

LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP

KSCDKTHTCPPCPAPELLGGPEVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

QKSLSLSPGK

SEQ.I.D.NO：12

EIVLTQSPATLSLSPGERATLSCSGSSSVSYMYWYQQKPGQAPRLLIEDTSNLAS

GIPARFSGSGSGTDYTLTISNLEPEDFAVYYCQQWSSYPPTFGQGTKLEIKRTVA

APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ.I.D.NO：15

CAGGTGCAACTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGT

CCATAACCTGCACAGTCTCTGGTTTCTCATTAACTAATTATGGTGTACACTGGGT

TCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGAGGTGGA

AGCACAGACTACAATGCAGCTTTCATGTCCAGACTGAGCATCACCAAGGACAACT

CCAGGAGCCAAGTTTTCTTTAAAATGAACAGTCTACAAGCTGATGACACTGCCAT

ATACTACTGTGCCAAAAGTCCGAATAGTAACTTTTACTGGTATTTCGATGTCTGG

GGCACAGGGACCACGGTCACCGTCTCCTCA

SEQ.I.D.NO：16

CAAATTGTTCTCACCCAGTCTCCAACAATCATGTCTGCATCTCCAGGGGAGAAGG

TCACCATGACCTGCAGTGGCAGCTCAAGTGTAAGTTACATGTATTGGTACCAGGA

GAAGCCAGGATCCTCCCCCAGACTCCTGATTGAAGACACATCCAACCTGGCTTCT

GGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAA

TCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGAGTAG

TTATCCACCCACGTTCGGCTCGGGGACAAAGTTGGAAATCAAA

SEQ.I.D.NO：17

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGA

GACTCTCCTGTGCAGCGTCTGGATTCTCATTAACTAATTATGGTGTACACTGGGT

CCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTGATATGGAGAGGTGGA

AGCACAGACTACAATGCAGCTTTCATGTCCCGATTCACCATCTCCAAGGACAATT

CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGT

GTATTACTGTGCGAAAAGTCCGAATAGTAACTTTTACTGGTATTTCGATGTCTGG

GGCCGTGGCACACTAGTCACAGTCTCCTCA

SEQ.I.D.NO：18

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAG

CCACCCTCTCCTGCAGTGGCAGCTCAAGTGTAAGTTACATGTATTGGTACCAACA

GAAACCTGGCCAGGCTCCCAGGCTCCTCATCGAAGACACATCCAACCTGGCTTCT

GGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTACACTCTCACCA

TCAGCAACCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAACAGTGGAGTAG

TTATCCACCCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAA

SEO.I.D.NO：19

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGA

GACTCTCCTGTGCAGCGTCTGGATTCTCATTAACTAATTATGGTGTACACTGGGT

CCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTGATATGGAGAGGTGGA

AGCACAGACTACAATGCAGCTTTCATGTCCCGATTCACCATCTCCAAGGACAATT

CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGT

GTATTACTGTGCGAAAAGTCCGAATAGTAACTTTTACTGGTATTTCGATGTCTGG

GGCCGTGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCT

TCCCCCTGGCACCcTCCtCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG

CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC

CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT

CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACAT

CTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCC

AAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG

GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTC

CCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG

GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC

CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT

GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC

CTCCCAGCCCCCAtCGAGAAAACCaTCTCCAAAGCCAAAGGGCAGCCCCGAGAAC

CACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGaACCAGGTCAG

CCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG

AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG

ACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA

GGGGAACGTcTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACG

CAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

SEQ.I.D.NO：20

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAG

CCACCCTCTCCTGCAGTGGCAGCTCAAGTGTAAGTTACATGTATTGGTACCAACA

GAAACCTGGCCAGGCTCCCAGGCTCCTCATCGAAGACACATCCAACCTGGCTTCT

GGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTACACTCTCACCA

TCAGCAACCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAACAGTGGAGTAG

TTATCCACCCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTGGCT

GCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTG

CCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTG

GAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG

GACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAG

ACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTC

GCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

SEQ.I.D.NO：21

QVQLVESGGGVVQPGRSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWVAVIWRG

GSTDYNAAFMSRLTISKDNSKNTLYLQMNSLRAEDTAVYYCAKSPNSNFYWYFD

VWGRGTLVTVSS

SEQ.I.D.NO：40

DYNMD

SEQ.I.D.NO：41

DINPNNGGTIDNQKFKD

SEQ.I.D.NO：42

GIYYYGSHYFDY

SEQ.I.D.NO：43

SATSSVSVMH

SEQ.I.D.NO：44

DTSKLAS

SEQ.I.D.NO：45

QQWSSNPLT

SEQ.I.D.NO：46

EVQLQQSGPELVKPGASVKISCKASGYIFTDYNMDWVKQSHGKKLEWIGDINPNN

SGTIDNQKFKDKATLTVDKSSSTAYMELRSLTSEDTAVYYCARGIYYYGSHYFDY

WGQGTTLTVSS

SEQ.I.D.NO：47

QIVLTQSPAIMSASPGEKVTMTCSATSSVSVMHWFQKKSGTSPKRWIYDTSKLAS

GVPTRFSGSGSGTSYSLTISSMEAEDTATYYCQQWSSNPLTFGSGTKLELK

SEQ.I.D.NO：48

EVQLVQSGAEVKKPGASVKVSCKASGYIFTDYNMDWVRQAPGQKLEWIGDINPNN

GGTIDNQKFKDRATLTVDKSTSTVYMELSSLRSEDTAVYYCARGIYYYGSHYFDY

WGQGTLVTVSS

SEQ.I.D.NO：49

EIVLTQSPSSLSASVGDRVTITCSATSSVSVMHWFQKKPGKAPKRWIYDTSKLAS

GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPLTFGGGTKVDIK

SEQ.I.D.NO：50

EVQLVQSGAEVKKPGASVKVSCKASGYIFTDYNMDWVRQAPGQKLEWIGDINPNN

GGTIDNQKFKDRATLTVDKSTSTVYMELSSLRSEDTAVYYCARGIYYYGSHYFDY

WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG

ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE

PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP

EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK

ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW

ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY

TQKSLSLSPGK

SEQ.I.D.NO：51

EIVLTQSPSSLSASVGDRVTITCSATSSVSVMHWFQKKPGKAPKRWIYDTSKLAS

GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPLTFGGGTKVDIKRTVA

APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ.I.D.NO：52

GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGA

AGATATCCTGCAAGGCCTCTGGATACATATTCACTGACTACAACATGGACTGGGT

GAAGCAGAGCCATGGAAAGAAACTTGAGTGGATTGGAGATATTAATCCTAATAAT

GGTGGTACTATCGACAACCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACA

AGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACACTGC

AGTCTATTACTGTGCAAGAGGGATTTATTACTACGGTAGTCACTACTTTGACTAT

TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

SEQ.I.D.NO：53

CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCAC

CATGACCTGCAGTGCCACCTCAAGTGTAAGTGTCATGCACTGGTTCCAGAAGAAGTCAG

GTACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTACT

CGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGTAGCATGGAGGC

TGAAGATACTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCACTCACGTTCGGTT

CTGGGACCAAGCTGGAGCTGAAA

SEQ.I.D.NO：54

GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGT

TTCCTGCAAGGCATCTGGATACATATTCACCGACTACAACATGGACTGGGTGCGACAGG

CCCCTGGACAAAAACTTGAGTGGATTGGAGATATTAATCCTAATAATGGTGGTACTATC

GACAACCAGAAGTTCAAGGACAGAGCCACCTTGACCGTAGACAAGTCCACGAGCACAGT

CTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAG

GGATTTATTACTACGGTAGTCACTACTTTGACTATTGGGGCCAGGGAACACTAGTCACA

GTCTCCTCA

SEQ.I.D.NO：55

GAAATTGTGTTGACGCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCAC

CATCACTTGCAGTGCCACCTCAAGTGTAAGTGTCATGCACTGGTTCCAGAAGAAACCAG

GGAAAGCCCCTAAGAGATGGATCTATGACACATCCAAACTGGCTTCTGGGGTCCCATCA

AGGTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTCACCATCAGCAGTCTGCAACC

TGAAGATTTTGCAACTTATTACTGCCAGCAGTGGAGTAGTAACCCACTCACGTTCGGCG

GAGGGACCAAAGTGGATATCAAA

SEQ.I.D.NO：56

GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGT

TTCCTGCAAGGCATCTGGATACATATTCACCGACTACAACATGGACTGGGTGCGACAGG

CCCCTGGACAAAAACTTGAGTGGATTGGAGATATTAATCCTAATAATGGTGGTACTATC

GACAACCAGAAGTTCAAGGACAGAGCCACCTTGACCGTAGACAAGTCCACGAGCACAGT

CTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAG

GGATTTATTACTACGGTAGTCACTACTTTGACTATTGGGGCCAGGGAACACTAGTCACA

GTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCcTCCtCCAAGAG

CACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG

TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTC

CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT

GGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA

AGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT

GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT

GATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTG

AGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG

CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA

GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCC

CCAtCGAGAAAACCaTCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC

CTGCCCCCaTCCCGGGATGAGCTGACCAAGaACCAGGTCAGCCTGACCTGCCTGGTCAA

AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA

ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAG

CTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTcTTCTCATGCTCCGTGATGCA

TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

SEQ.I.D.NO：57

GAAATTGTGTTGACGCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCAC

CATCACTTGCAGTGCCACCTCAAGTGTAAGTGTCATGCACTGGTTCCAGAAGAAACCAG

GGAAAGCCCCTAAGAGATGGATCTATGACACATCCAAACTGGCTTCTGGGGTCCCATCA

AGGTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTCACCATCAGCAGTCTGCAACC

TGAAGATTTTGCAACTTATTACTGCCAGCAGTGGAGTAGTAACCCACTCACGTTCGGCG

GAGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCG

CCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTT

CTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACT

CCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC

CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCA

TCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

SBQ.I.D.NO：61

QVQLVESGGGVVQPGRSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWVAVIWRG

GSTDYNAAFMSRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAKSPNSNFYWYFD

VWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN

SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK

KVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC

KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH

EALHNHYTQKSLSLSPGK

SEQ.I.D.NO：62

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTG

AGACTCTCCTGTGCAGCGTCTGGATTCTCATTAACTAATTATGGTGTACACTGG

GTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTGATATGCAGAGGT

GGAAGCACAGACTACAATGCAGCTTTCATGTCCCGATTCACCATCTCCAAGGAC

AATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACG

GCTGTGTATTACTGTGCGAAAAGTCCGAATAGTAACTTTTACTGGTATTTCGAT

GTCTGGGGCCGTGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCA

TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC

CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAAC

TCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA

GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACC

CAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAG

AAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCA

CCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGAC

ACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC

CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT

AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTC

AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC

AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCC

AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAG

CTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGC

GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC

ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC

GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT

GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

Claims (73)

1. therapeutic antibodies, its specificity be in conjunction with OSM, hOSM particularly, and regulate interaction between OSM and the gp130.

2. the antibody of claim 1, it comprises the CDRH3 of SEQ.I.D.NO:3.

3. the antibody of claim 2, it further comprises:

The CDRH1 of SEQ.I.D.NO:1

The CDRH2 of SEQ.I.D.NO:2

The CDRL1 of SEQ.I.D.NO:4

The CDRL2 of SEQ.I.D.NO:5

The CDRL3 of SEQ.I.D.NO:6.

4. the antibody of claim 1, it comprises the CDRH3 of SEQ.I.D.NO:42.

5. the antibody of claim 4, it further comprises:

The CDRH1 of SEQ.I.D.NO:40

The CDRH2 of SEQ.I.D.NO:41

The CDRL1 of SEQ.I.D.NO:43

The CDRL2 of SEQ.I.D.NO:44

The CDRL3 of SEQ.I.D.NO:45.

6. each antibody of claim 1-5, wherein said antibody is selected from whole antibody, chimeric antibody, humanized antibody, bi-specific antibody, the assorted antibody (heteroconjugate) of puting together.

7. the antibody of claim 6, wherein said antibody is whole antibody.

8. the antibody of claim 7, wherein said whole antibody is mouse, rat, rabbit, primate or people's antibody.

9. the antibody of claim 8, wherein said whole antibody behaviour antibody.

10. the antibody of claim 6, wherein said antibody is chimeric antibody or humanized antibody.

11. the antibody of claim 8, wherein said antibody are humanized antibody.

12. the humanized antibody of claim 2, wherein the residue 28,29,30,71 of people's acceptor variable heavy chain framework region and 94 and 49 and 71 of the variable light chain framework of people's acceptor replaced by the corresponding residue in the donor antibody framework that CDRH3 was derived from.

13. the antibody of claim 12, wherein said people's heavy chain framework comprise following residue (or its conservative substitution thing):

The position residue

28 S

29 L

30 T

71 K

94 K

Described people's light chain comprises following residue (or its conservative substitution thing):

The position residue

49 E

71 Y

14. a specific specificity in conjunction with hOSM and regulate hOSM and gp130 between interactional humanization therapeutic antibodies, it comprises the VH structural domain of SEQ.I.D.NO:9 and the V of SEQ.I.D.NO:10 _LStructural domain.

15. a specific specificity in conjunction with hOSM and regulate hOSM and gp130 between interactional humanization therapeutic antibodies, it comprises the heavy chain of SEQ.I.D.NO:11 and the light chain of SE Q.I.D.NO:12.

16. a specific specificity in conjunction with hOSM and regulate hOSM and gp130 between interactional humanization therapeutic antibodies, it comprises the V of SEQ.I.D.NO:48 _HThe V of structural domain and SEQ.I.D.NO:49 _LStructural domain.

17. a specific specificity in conjunction with hOSM and regulate hOSM and gp130 between interactional humanization therapeutic antibodies, it comprises the heavy chain of SEQ.I.D.NO:50 and the light chain of SEQ.I.D.NO:51.

18. the therapeutic antibodies of aforementioned each claim, it further comprises the people's CH that is selected from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgM.

19. the therapeutic antibodies of claim 18, wherein said constant region are the IgG isotype, for example the constant region of IgG1 or IgG4.

20. the therapeutic antibodies of claim 19, wherein said constant region are IgG1.

21. the therapeutic antibodies of claim 20, wherein said constant region sudden change makes antibody become non-cracking performance.

22. the therapeutic antibodies of aforementioned each claim, wherein said antibody is regulated the site II of hOSM and the interaction between the gp130.

23. the therapeutic antibodies of claim 22, wherein said antibody suppresses described interaction.

24. the therapeutic antibodies of claim 23, the described interaction of wherein said antibody blocking.

25. a Fab, it is the Fab of the therapeutic antibodies of aforementioned each claim.

26. the fragment of claim 25, wherein said fragment is selected from: Fab, Fab ', Fd, F (ab) ₂, ScFv.

27. a medicinal compositions, it comprises therapeutic antibodies or its Fab of aforementioned each claim.

28. the method to the people patient who regulates interact between hOSM and the gp130 disease that responds or obstacle is suffered from a treatment, described method comprises the step of the composition of the claim 27 that gives described patient treatment significant quantity.

29. the people patient's of chronic inflammatory disease or obstacle method is suffered from a treatment, described method comprises the step of the composition of the claim 27 that gives described patient treatment significant quantity.

30. the people patient's of arthritis disease or obstacle method is suffered from a treatment, described method comprises the step of the composition of the claim 27 that gives described patient treatment significant quantity.

31. the method for claim 30, wherein said patient suffers from rheumatoid arthritis and/or osteoarthritis.

32. for example people patient's of asthma or COPD method of inflammatory lung disease is suffered from a treatment, described method comprises the step of the composition of the claim 27 that gives described patient treatment significant quantity.

33. psoriatic people patient's method is suffered from a treatment, described method comprises the step of the composition of the claim 27 that gives described patient treatment significant quantity.

34. for example atherosclerotic people patient's method of cardiovascular disorder or obstacle is suffered from a treatment, described method comprises the step of the composition of the claim 27 that gives described patient treatment significant quantity.

35. the people patient's of Kaposi sarcoma method is suffered from a treatment, described method comprises the step of the composition of the claim 27 that gives described patient treatment significant quantity.

36. each therapeutic antibodies or the purposes of its Fab in drug manufacture among the claim 1-26, wherein said medicine is used for the treatment of the disease that interacts and respond between hOSM and the gp130 regulating, for example rheumatoid arthritis, osteoarthritis, psoriatic, asthma, COPD.

37. a medicine, it comprises among the claim 1-20 each therapeutic antibodies or its Fab.

38. each therapeutic antibodies or the heavy chain of its Fab and/or the carrier (for example plasmid) of light chain among the claim 1-26 that encodes, for example described carrier comprise among the claim 39-46 each polynucleotide.

39. the V of the SEQ.I.D.NO:9 that encodes _HThe polynucleotide of structural domain, described polynucleotide comprise SEQ.I.D.NO:17 or are made up of it substantially.

40. the V of the SEQ.I.D.NO:10 that encodes _LThe polynucleotide of structural domain, described polynucleotide comprise SEQ.I.D.NO:18 or are made up of it substantially.

41. the polynucleotide of the heavy chain of the SEQ.I.D.NO:11 that encodes, described polynucleotide comprise SEQ.I.D.NO:19 or are made up of it substantially.

42. the polynucleotide of the light chain of the SEQ.I.D.NO:12 that encodes, described polynucleotide comprise SEQ.I.D.NO:20 or are made up of it substantially.

43. the V of the SEQ.I.D.NO:48 that encodes _HThe polynucleotide of structural domain, described polynucleotide comprise SEQ.I.D.NO:54 or are made up of it substantially.

44. the V of the SEQ.I.D.NO:49 that encodes _LThe polynucleotide of structural domain, described polynucleotide comprise SEQ.I.D.NO:55 or are made up of it substantially.

45. the polynucleotide of the heavy chain of the SEQ.I.D.NO:50 that encodes, described polynucleotide comprise SEQ.I.D.NO:56 or are made up of it substantially.

46. the polynucleotide of the light chain of the SEQ.I.D.NO:51 that encodes, described polynucleotide comprise SEQ.I.D.NO:57 or are made up of it substantially.

47. the recombinant host cell of stable conversion or transfection, it comprises the carrier of claim 38.

48. the recombinant host cell of stable conversion or transfection, second carrier that it comprises first carrier of the polynucleotide that contain SEQ.I.D.NO:17 and contains the polynucleotide of SEQ.I.D.NO:18.

49. the recombinant host cell of stable conversion or transfection, second carrier that it comprises first carrier of the polynucleotide that contain SEQ.I.D.NO:19 and contains the polynucleotide of SEQ.I.D.NO:20.

50. the recombinant host cell of stable conversion or transfection, second carrier that it comprises first carrier of the polynucleotide that contain SEQ.I.D.NO:54 and contains the polynucleotide of SEQ.I.D.NO:55.

51. the recombinant host cell of stable conversion or transfection, second carrier that it comprises first carrier of the polynucleotide that contain SEQ.I.D.NO:56 and contains the polynucleotide of SEQ.I.D.NO:57.

52. each host cell among the claim 47-51, wherein said host cell are vertebrate cells.

53. the host cell of claim 52, wherein said cell are mammalian cell.

54. the host cell of claim 53, wherein said cell are CHO or NSO.

55. a manufacture of therapeutic antibody or method, it comprises the step of cultivating each host cell among the claim 47-51.

56. the therapeutic antibodies of a competitive inhibition claim 15 in ELISA type experiment and OSM, particularly hOSM, more especially site II bonded antibody or the Fab of hOSM, prerequisite is the CDRH3 that described competitive antibody does not comprise SEQ.I.D.NO:42.

57. a medicinal compositions, it comprises the competitive antibody of claim 56.

58. a treatment is suffered from regulating interact between hOSM and the gp130 disease that responds or obstacle (such as arthritis disease, for example rheumatoid arthritis and/or osteoarthritis) people patient's method, described method comprises the step of the composition of the claim 57 that gives described patient treatment significant quantity.

59. specificity is in conjunction with the purposes of the therapeutic antibodies of the albumen skeleton of glycosylation hOSM (for example claim 15 or 17 antibody) in drug manufacture, wherein said medicine is used for the treatment of and is selected from following disease or obstacle: arthritis disease, for example rheumatoid arthritis, childhood morbidity property sacroiliitis, psoriasis arthropathica, ankylosing spondylitis; Psoriatic, for example chronic spot disease; Inflammatory lung disease, for example COPD or severe asthma; MS; Dementia, for example alzheimer's disease; Pain, for example nervosa or inflammatory pain; Atherosclerosis; Cycle Regulation disease, for example cancer (for example prostate cancer), myelomatosis.

60. medicinal compositions that contains first therapeutic antibodies and second therapeutic antibodies, wherein the first therapeutic antibodies specificity is in conjunction with hOSM and regulate the site II of hOSM and the interaction between the gp130, and the second therapeutic antibodies specificity is in conjunction with hOSM and regulate the site III of hOSM and the interaction between OSMR β and/or the LIFR.

61. medicinal compositions that contains the dual specific therapeutic antibodies, wherein said dual specific therapeutic antibodies is in conjunction with hOSM, and regulates between the site II of (a) hOSM and the gp130 and (b) the site III of hOSM and the interaction between OSMR β and/or the LIFR.

62. a medicinal compositions that contains first antagonist at least, wherein said first antagonist be in conjunction with hOSM, and regulate between the site II of (a) hOSM and the gp130 and (b) the site III of hOSM and the interaction between OSMR β and/or the LIFR.

63. one kind contains first antagonist at least (such as the protein antagonist, antibody for example) medicinal compositions, wherein said first antagonist is in conjunction with gp130 and/or OSMR β and/or LIFR, and regulates (for example suppressing or blocking-up) (a) between gp130 and the hOSM and (b) interaction between OSMR β and/or LIFR and the hOSM.

64. the method for the antibody (for example antibody that produces at OSM/hOSM) in conjunction with OSM, particularly hOSM is inferred in a screening, this method comprises;

(a) allowing the described antibody of incubation and glycosylation OSM, particularly glycosylation hOSM under the bonded condition;

(b) binding affinity of the described antibody of measurement;

(c) if the binding affinity that described antibody has is higher than 1 μ M,, then select described antibody usually above 100nM;

(d) provide the coded polynucleotide of the described antibody of step (c), and transform or the transfection mammalian host cell with the carrier that contains described polynucleotide;

(e) allowing described antibody-secreting to go into the described host cell of culturing step (d) under the condition in the substratum;

(f) substratum of purification step (e) alternatively;

(g) step (e) or antibody (f) are joined in the medicinal compositions.

65. the method for the antibody (for example antibody that produces at OSM/hOSM) in conjunction with OSM, particularly hOSM is inferred in a screening, this method comprises;

(a) allowing the described antibody of incubation and glycosylation OSM, particularly glycosylation hOSM under the bonded condition;

(b) binding affinity of the described antibody of measurement;

(c) if the binding affinity that described antibody has is higher than 1 μ M,, then select described antibody usually above 100nM.

66. claim 64 or 65 each methods, wherein said OSM is by for example CHO glycosylation of mammalian host cell.

67. the method for claim 64 or 65, wherein said hOSM is Natively glycosylated hOSM.

68. the method for claim 67, wherein said hOSM particularly suffers from arthritis disease by the people and for example separates in people's synovia of RA.

69. an antibody, its antibody for identifying by each method of claim 65-68.

70. a medicinal compositions, it comprises the antibody and the medicinal inert carrier of claim 69.

71. the therapeutic antibodies of claim 1, it can also be in conjunction with cOSM except can be in conjunction with the hOSM.

72. being included in the experiment of ELISA type, the method for the hOSM in a detection of biological sample, particularly people's synovia or the human serum, this method use site III antibody as capture antibody.

73. the method for claim 72, the experiment of wherein said ELISA type are substantially as described in the embodiment 16.

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