CN1056713A

CN1056713A - Anti-oncostatin m monoclonal antibodies

Info

Publication number: CN1056713A
Application number: CN 91102685
Authority: CN
Inventors: 苏珊·F·拉德卡; 彼得·S·林斯雷; 莫罕默德·肖亚伯
Original assignee: Bristol Myers Squibb Co
Current assignee: Bristol Myers Squibb Co
Priority date: 1990-03-29
Filing date: 1991-03-27
Publication date: 1991-12-04
Also published as: ZA912136B

Abstract

The present invention relates to the monoclonal antibody that detects oncostatin M.Monoclonal antibody of the present invention can be connected on the oncostatin M, suppresses the combination of oncostatin M acceptor, and/or suppresses the biological activity of oncostatin M.This antibody can be used in vivo or the existence of vitro examination oncostatin M and/or the biological activity of regulating oncostatin M.

Description

Anti-oncostatin M monoclonal antibodies

The present invention relates to the monoclonal antibody of anti-oncostatin M.Antibody of the present invention is characterised in that and can be attached on the oncostatin M, suppresses the oncostatin M receptors bind, and/or suppresses the biological activity of oncostatin M.Monoclonal antibody of the present invention can be used in vivo or the existence of vitro detection oncostatin M and/or the biological activity of regulating oncostatin M.

Generally acknowledge that at present somatic cell hybrid (" hybrid ") is an important source of the specific cells product that can not obtain from the original cultivation of short-term.Preferred example about this respect is the system of being hybridized myelomatosis by the generation of Milstein and other people foundation, can prepare the antigenic monoclonal antibody of a kind of non preference (Kohler and Milstein with this hybridization myelomatosis, 1985, Nature Journal (London) 256: 495; Galfre et al., 1977, Nature Journal (London) 266: 550).This hybridization product can constantly provide the monoclonal antibody of specific antigens.And these antibody can be used as medicament and are used for any method of using antibody in advance, but it has also been given high recognition power, low background and the preferred property of antibody sources can constantly be provided.

The generalized case MONOCLONAL ANTIBODIES SPECIFIC FOR at first comprises the separation of immunization, immune response cell, these cells are in polyoxyethylene glycol for example, and tumour cell (" not deactivation ") that can not immunoglobulin,exocrine with selected splitted constantly merges.The cell (hybridoma) that is produced being existed for example, HAT(6-oxypurine, aminopterin-induced syndrome, thymidine) growing in the substratum is identified.Each hybridoma all is the fusion product of a kind of single antibody forming cell and tumour cell.The ability of a kind of single specificity antibody of secretion and the latter permanent that this hybridoma has the former become viability, make it to breed continuously, thereby a kind of cell offspring that can forever produce single specificity antibody is provided.

The present invention relates to oncostatin M is had specific MONOCLONAL ANTIBODIES SPECIFIC FOR and utilization, oncostatin M is a kind of to multiple normal and novel cell mitogen (Cytokine) that transformant has the pleiotropy effect.Any monoclonal antibody with monoclonal anti body characteristics described herein all belongs within the scope of the present invention.For example, can competitive inhibition monoclonal antibody described herein combine and/or can regulate the bioactive a kind of mono-clonal of oncostatin with the immunologic opsonin of its oncostatin M antigenic determinant or mosaic antibody all belongs within the scope of the present invention.

Be used for describing embodiments of the invention and used hybridoma technology to produce anti-oncostatin M antibody of the present invention, still scope of the present invention is not limited to the utilization of these hybridoma techniques.According to whether can with natural oncostatin M, the sex change oncostatin M, perhaps the both can form immunoprecipitate these typical antibody are classified.Each class is also according to its growth inhibiting blocking ability and/or oncostatin M further characterization with its cell surface receptor bonded ability that oncostatin M is regulated.This antibody can be used to draw proteinic surperficial determinant of novel oncostatin M and functional site structure iron.

Following used term has implication as described below:

The two decisive enzyme-linked immunoassays of DDEIA-

The GIA-growth-inhibiting is measured

The HRP-horseradish peroxidase

Micro-EIA-trace enzyme-linked immunoassay

The MAb-monoclonal antibody

The OM-oncostatin M

The RRA-radioreceptor assay

The OM1-1R10F11 monoclonal antibody

The OM2-11R2F8 monoclonal antibody

The OM3-12R13D7 monoclonal antibody

The OM4-4R12C7 monoclonal antibody

The OM5-3R9D9 monoclonal antibody

The OM6-3R13F4 monoclonal antibody

The OM7-12R13B5 monoclonal antibody

The OM8-12R19E3 monoclonal antibody

Fig. 1 represents to obtain from the supernatant liquor that the Chinese hamster ovary celI of cDNA coding oncostatin M stable transfection is ³⁵The S-methionine(Met) and ³⁵The immunoprecipitation test of the oncostatin M of S-halfcystine mark.Figure 1A represents the series reaction of the oncostatin M (supernatant liquor of collecting) of anti-oncostatin M monoclonal antibodies and " natural " metabolic marker from the CHO transfection thing of metabolic mark.Figure 1B represents the reaction of these same antibody and the supernatant liquor collected from the Chinese hamster ovary celI of metabolic mark, these supernatant liquors are by use SDS, 2 mercapto ethanol processing and make it sex change before monoclonal antibody is cultivated boiling.Bands of a spectrum 1: negative control antibody; Bands of a spectrum 2:OM1; Bands of a spectrum 3:OM5; Bands of a spectrum 4:OM6; Bands of a spectrum 5:OM4; Bands of a spectrum 6:OM2; Bands of a spectrum 7:OM7; Bands of a spectrum 8:OM3; Bands of a spectrum 9:OM8.

Fig. 2 provide two kinds of different anti-oncostatin M monoclonal antibodies in GIA measures to A375 melanotic tumor clone in and the determination of activity data.Fig. 2 A represent different concns OM2 activity and Fig. 2 B represents the activity of different concns OM1.

It is right that Fig. 3 is illustrated in the radioreceptor assay various anti-oncostatin M monoclonal antibodies ¹²⁵I-oncostatin M and the influence of H2981 lung cancer cell line bonded.

Two kinds of different anti-oncostatin M monoclonal antibodies that Fig. 4 represents to measure by EIA with combining between the secreted supernatant liquor of the COS cell of a series of sudden change oncostatin M structure transfection that contains aminoacid deletion or change.Total absorbance units when the data that provided are expression oD460.There is not the subtracting background combination.In this figure, last amino acid that " del " expression is removed from the C-end, and English alphabet represents that the amino acid that is carried out changes.

Fig. 5 represents different anti-oncostatin M monoclonal antibodies to by the parental generation structure, " SPOM ", or the immunoprecipitation ability of deletion mutant △ 44-47 excretory oncostatin M is relatively.Bands of a spectrum 1: negative control antibody; Bands of a spectrum 2:OM1; Bands of a spectrum 3:OM2; Bands of a spectrum 4:OM3.

Fig. 6 represent by two kinds of differences anti--location map of the surperficial determinant that oncostatin M monoclonal antibodies OM3 and OM4 measure.OM3 that will represent with absorbance units and the relative combination of OM4 are with negative control antibody with from plasmid △ 188-227, and the combination of the OM that obtains in the serum-free condition substratum of the COS cell of △ 188-227/L108S and GAG104 transfection compares.In conjunction with level compare with the level that combines of COS cell (" SPOM ") excretory OM (Linsley, et al., 1990, Mol.Cell.Biol.10:1882-1890).

Fig. 7 can influence a series of anti-oncostatin M monoclonal antibodies bonded oncostatin M sudden change diagrams.The OM boot sequence is positioned at residue-25 to-1.The undressed molecule of excretory is 227 amino acid longs from the COS cell, is cut into a kind of sophisticated 196 amino acid whose protein.（Linsley，et al.，1990，Mol.Cell.Biol.10：1882-1890）。At 184(△) lack the combination that can destroy OM1 and OM2 with interior with the C-end amino acid that comprises 184.In addition, can cancel the OM2 combination by the disappearance of residue 22-36 or 44-47.The surperficial determinant of antibody OM3 is positioned at and contains residue 108(arrow indication) the site because leucine is changed into the combination that Serine can destroy this mAb at this residue place.At residue 104(

) locate to insert glycine-L-Ala-glycine tripeptide and can cancel the OM4 combination.

The present invention relates to oncostatin M (a kind of novel cell mitogen (Cytokine) that multiple different normal and transformant is had pleiotropic effects) is had specific monoclonal antibody.And in conjunction with oncostatin M, suppress the oncostatin M receptors bind, and/or the bioactive monoclonal antibody of inhibition oncostatin M is described.

Described monoclonal antibody can be used for drawing the surperficial determinant figure of oncostatin M and define the structure-functional relationship in its field.This antibody can be used for diagnostic assay, for example, detects oncostatin M, or the existence of oncostatin M mutant form.Also can use this monoclonal antibody in vivo or the biological activity of external adjusting oncostatin M.By following chapters and sections the present invention is described in detail.

By characteristic to the defined monoclonal antibody of specificity of oncostatin M

Monoclonal antibody to the various surperficial determinants of the oncostatin M that defines natural or denatured form is described.And to oncostatin M biological activity inhibition ability and/or in conjunction with the ability of cell surface receptor monoclonal antibody is further classified according to them.Can the described monoclonal antibody of competitive inhibition and any monoclonal antibody of their oncostatin M surface determinant immunologic opsonin bonded, comprise chimeric antibody, all belong within the scope of the present invention.

The antigenic identification of oncostatin M

Originally oncostatin M is differentiated the inhibition effect of human cancer cell system with it, it at first is from phorbol 12-tetradecanoate 13-acetate " PMA ")-inductive human tissue cell lymphoma cell (Zarling et al., 1986, Proc.Natl.Acad.Sci.(USA) 83:9739-9743) with from activated T lymphocyte (Brown et al., 1987, separate in J.Immunol.139:2977-2983).This molecule is a kind of M of containing _rThe heat of=28,000 single polypeptide chain and acid acceptance protein.As other growth regulators that forms naturally, oncostatin M has various biological activitys.Find it to some, but be not whole that human cancer cell's cording has the growth system to press down effect.On the contrary, oncostatin M can stimulate some normal fibroblast, as the growth (Zarling et al., 1986, Proc.Natl.Acad.Sci(USA) 83:9739-9743) of human foreskin inoblast or WI-38 cell.Cloned the gene that goes out oncostatin M with sequencing, and in mammalian cell, given expression to a kind of active reorganization oncostatin M recently and (seen the U. S. application flowing water day .144 of application on January 15th, 1988,574, identical European patent application publication No. EPA0290948(1988 is open November 17), the present invention's reference also combines this patent full content).Mature form under the cutting behind the signal peptide is a kind of 227 amino acid whose glycoprotein that contain, and wherein five amino acid is a cysteine residues.This protein has the C-terminal district of a strongly hydrophilic, although oncostatin M is structurally uncorrelated with other known phytokinin, its mRNA contains a rich AU district at its 3 ' untranslated end.This district and many phytokinin, lymphokines and other growths-adjusting molecule homology in the oncostatin M messenger RNA(mRNA), the common method that this has pointed out a kind of regulatory gene to express.The cell receptor of having found oncostatin M in various mammalian cells exists.Main oncostatin M acceptor molecule is a kind of Mr=150,000-160,000 specific protein (Linsley et al., 1989, J.Biol.Chem.264:4282-4289).

Known technology by this area can obtain oncostatin M the cell source of composite reactive oncostatin M from various, this cell source comprises, for example, can produce the cell of oncostatin M and naturally with controlling recombinant DNA molecules cells transfected synthetic and/or the secretion oncostatin M.Also can synthesize oncostatin M including, but not limited to the solid-phase peptide synthetic method by chemical synthesis process.U.S. series application No.144 is seen in the method description for preparing oncostatin M, 574, on January 15 1988 applying date, it is open November 17 to be equal to disclosed European patent application EP A0290948(1988), this paper with reference to and combine the full content of this application.

Can use any method in many method of immunity, select oncostatin M is had the monoclonal antibody of affinity by measuring its ability in conjunction with oncostatin M, these method of immunity are including, but not limited to enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, the Western engram analysis, the radioimmunity metric system is measured, competition and non-competitive immunoassay.For example, can easily use following solid phase micro enzyme testing method (" Micro EIA ").In a word, be by measuring it determines antibody in the hybridization product supernatant liquor to the binding ability that covers the oncostatin M on the solid surface of hole (Well) existence.Add after the supernatant liquor, in hole (Well), add the F(ab ' that peroxidase connects) ₂Sheep anti mouse Ig.After all unconjugated materials are rinsed out, represent bonded enzyme by adding a kind of substrate that a kind of colour-change occurs.This change in color has been represented the formation of monoclonal antibody/oncostatin M mixture in the hole indirectly.

The active inhibition of oncostatin M

Suppress the bioactive antibody of oncostatin M and in treatment is used, have special purposes.Can discern this antibody by following growth-inhibiting measuring method (" GIA ").Always, GIA can as a kind of test macro estimate in a kind of antibody and oncostatin M to the target cell growth with breed inhibiting ability.

Inhibition to the oncostatin receptors bind

Cell surface receptor generally has the affinity of height to its part.Part is the beginning of the various cellular activities of control with combining of specific cells surface receptor.Use U. S. application serial number .144 described below and application in 15 days January in 1988 of quoting in front, the method for radioreceptor assay described in 574 has shown combining of oncostatin M and a kind of membrane receptor.The human cancer cell who is measured comprises the A375(melanoma); The A875(melanoma); The Me1109(melanoma); The T24(bladder cancer); The A549(adenocarcinoma of lung); The H1477(melanoma); The Me180(melanoma); With MCF(mammary cancer). ¹²⁵The combination of I-oncostatin M has specificity and saturability, and is not suppressed by other known peptide growth regulators.With the Scatchard analysis revealed of various different clones in conjunction with the gained data, ¹²⁵The I-oncostatin M is about 10 with Kd ^-9M is attached to 1-2 * 10 of each cell ⁴On the individual binding site, use radioreceptor assay method described below, measure monoclonal antibody obstruction oncostatin M and its cell surface receptor bonded ability that hybrid cell line produces.

The MONOCLONAL ANTIBODIES SPECIFIC FOR method of anti-oncostatin M

Can use oncostatin M is expelled in the mammalian hosts as immunogen, as mouse, ox, goat, sheep, rabbits etc. are especially injected with a kind of adjuvant, as complete Freund ' s adjuvant, any preparation of the present invention resisting-oncostatin M antibody in the whole bag of tricks of aluminum hydroxide gel or analogue.With host's bloodletting, blood is used to separate polyclonal antibody then.Also can use peripheral blood lymphocyte, splenic lymphocyte (B-Cells), or lymph node lymphocyte and a kind of suitable myeloma cell merge, and forever survives so that mono-clonal is expressed the karyomit(e) of oncostatin M specific antibody.

Though used the embodiment of mouse monoclonal antibody when description is of the present invention, the present invention is not limited thereto, and comprise, for example the use of people's antibody.This antibody can be by utilization human body hybridoma (Cote et al., 1983, Proc.Natl.Acad.Sci(U.S.A), 80:2026-2030) or by with EBV(Epstein Barr virus) at vitro conversion human body B cell (Cole et al., 1985, in " Monclonal Antibodies and Cancer Therapy " Alan R.Liss PP.77-96) obtains.And can use the generation " chimeric antibody " of nearest foundation method (Morrison et al., 1984, Proc.Natl.Acad.Sci.(U.S.A), 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda etal., 1985, Nature 314:452-454).The latter's method comprises having the gene that obtains the specific mouse antibody molecule of suitable antigen and the gene that obtains fuse together from have suitable bioactive human antibody molecules from a kind of.

Can use recent disclosed a kind of method to prepare a kind of whole compositions that can define the monoclonal antibody of oncostatin M and (see Sastry, et al., 1989, Proc.Natl.Acad.Sci(U.S.A) 86:5728-5732, with Huse et al., 1989, Seience 246:1275-1281).Therefore, a kind of Fab segment cDNA storehouse that obtains from the animal spleen DNA of oncostatin M immunity can produce in bacterial host cell.

With suitable proteolytic enzyme, as, stomach en-, papoid, and analogue are handled and can be modified monoclonal antibody of the present invention, with produce can with oncostatin M immunologic opsonin bonded Fab, F(ab ') ₂Or Fv fragment.

Also can be) with whole antibody molecule or its Fab, F(ab ' ₂Or the Fv fragment is attached on a kind of compound, and these compounds include, but are not limited to, and signal produces compound such as fluorescence radiation agent, radioactively labelled substance, a kind of chromophoric group, enzyme, chemoluminescence or bioluminescent molecules etc.Also can be) whole antibody or its Fab, F(ab ' ₂Or the Fv fragment is connected to and can strengthens or suppress on a kind of somatomedin of oncostatin M biological activity, perhaps is attached on the toxin so that can selectively the cell of expressing the oncostatin M precursor on cell surface be killed.Be used for marker, protein, the method that toxin etc. are attached on antibody and the antibody fragment is well known in the art.See, for example, United States Patent (USP) .4,220,450; 4,235,869; 3,935,074; With 3,996,345.

Therefore, in another embodiment of the present invention, provide a kind of its antigen binding site of preparation can with oncostatin bonded monoclonal antibody, or its segmental method comprises

(a) cultivate a kind of clone, said clone can produce a kind of anti-oncostatin M antibody, or its fragment, and reclaims prepared product; Or

(b) from a kind of complete anti-oncostatin M antibody the preparation a kind of anti-oncostatin M antibody fragment and, if necessary,

(c) (a) or the antibody or the antibody fragment of preparation (b) be attached to a kind of marker, somatomedin, or on the toxin.

The purposes of anti-oncostatin M monoclonal antibodies

The superiority of antibody of the present invention is can be used for detecting the nature or the reorganization oncostatin M of natural or denatured form or detect in the serum sample oncostatin M existing with free form or cooperative programs conjugated protein with it.Antibody of the present invention also can be used for suppressing in the body biological activity of oncostatin M.Polyclone or monoclonal antibody can be used for detecting in the sample, as cell or physiological fluid, as the oncostatin M in the blood.The discovery of oncostatin M also can be used as the indication of the existence of oncocyte in the body fluid.Thus, anti-oncostatin M antibody can be used for diagnosis and/or prognosis cancer and/or other cell growth phase related disorders.This antibody can also be used in the affinity chromatograph analysis, separation and purifying are from the oncostatin M in nature or synthetic source.Also can be used for these antibody in the control volume or the amount of the oncostatin M that substratum and cell combine, whereby by forming a strain specific antibodies: the oncostatin M mixture is with the competitive inhibition oncostatin M: the combination of oncostatin M acceptor, regulate the growth of cell.Therefore can be with antibody of the present invention as medicine, treating with the oncostatin M growth stimulating activity is the cell growth disorder disease of pathogenic factors.

Therefore, for this purpose, also provide a kind of pharmaceutical composition, it comprises the antibody of the present invention as active ingredient, or its fragment, and with pharmaceutically acceptable thinner, vehicle, or its carrier combination.

The generation of the anti-id AB of imitation oncostatin M effect

Also can prepare and to imitate the bioactive anti-id AB of oncostatin M with monoclonal antibody of the present invention.Anti-id AB or anti-idiotype are directly and resist the antigen binding domain of another kind of antibody molecule or the antibody of variable region (being called idiotype).Theoretically, according to network model (Jerne, N.K., 1974, the Ann.Immunol.(Paris) 125:373 of the idiotype of Jerne relation; Jerne, N.K.et al., 1982, EMBO 234), carry out immunization with a kind of antibody molecule of expressing corresponding to a certain given antigenic antibody binding site and should produce one group of anti-id AB, wherein partial antibody and this antigen all have a kind of and this antibody binding site complementary structure.Then with suppress oncostatin M and the monoclonal antibody of its receptors bind carry out immunization should produce imitate oncostatin M with and with the anti-idiotype of oncostatin M receptors bind.Therefore, the Monoclonal Antibody that these can enough direct anti-oncostatin Ms and can be in vivo all belong within the scope of the present invention with the anti-idiotype of external imitation oncostatin effect.Equally, be included within the range of definition of the monoclonal antibody of oncostatin M herein with oncostatin M bonded anti-id AB.

The antigenic determinant location of oncostatin M

To this growth regulator, the structural analysis of oncostatin M is an important preceding program process determining the biological action that rises in self balance or pathological state of this novel cell kinetin.Among the described in the back embodiment, the monoclonal antibody (OM1 to OM8) of a series of prepared anti-reorganization oncostatin Ms is analyzed, structural to determine them in conjunction with demand and antigenic surface determinant position.These antibody can detect linear (OM3 and OM4) also can detect folding (OM1 and OM2) antigenic surperficial determinant.Very approaching with the linear antigenic surface determinant position that OM3 and OM4 detect.Its antigenic surface determinant comprises that the OM3 of residue 108 and the oncostatin M with sex change that folds all can react.And MabOM4, its antigenic surface determinant can only combine with the oncostatin M of sex change by can be destroyed after inserting a tripeptides at amino-acid residue 104.

Monoclonal antibody OM2 can remove the functional effect of oncostatin M in a kind of growth-inhibiting is measured.The data that the back provided show that antibody OM2 is by the neutralized effect of OM of restriction OM and combining of its acceptor.By serological analysis, determined specific aminoacid insertion, disappearance or replaced the combination that sudden change can influence neutralizing antibody.The GIA of these experimental results and these mutating molecules and RRA activation analysis gained result are very closely related, thereby showing that this neutralizing antibody binding site is arranged in because of biological activity need carry out suitably folding OM molecule tertiary structure.

Acceptor and OM2 bonded destroy and have confirmed that effectively the important district of function of oncostatin M highly depends on this viewpoint of suitable tertiary structure in the mutant that has disappearance or change in the middle of non-vicinity (C-end, △ 22-36, △ 44-47) amino-acid residue.These data also show the N-end of ripe molecule and the district in the C-end, by direct formation receptor binding site or by stablizing its active necessary tertiary structure indirectly, are the elementary zones of oncostatin M functionally active perhaps.

For interleukin-6 similar discovery is arranged, interleukin-and oncostatin M have some identical functions characteristic, comprise the growth that suppresses external some cancerous cell line and induce plasminogen activator supressor (Brown, et al., 1990, and in " Molecular Biology of the Cardiovascular System, Elsevier; Amsterdam, PP.195-206).The amino acid whose companion's row of residue 31 and the first five C-terminal disappearance reduces its activity significantly behind the-terminal amino acid of IL-6, and the disappearance of the 16th C-terminal residue has fully phased out its functionally active.A series of neutralizing antibodies of IL-6 show as can be discerned by amino and the amino acids formed antigenic surface determinant of C-terminal, and think that equally 7 C one end amino acids of IL-6 are a kind of αLuo Xuanjiegou (Brakenhoff, et al., 1990, J.Immunol.145:561-568).As if resulting result is opposite with oncostatin M, and intramolecular disulfide bond is not the basic structure (Jambou, et al., 1988, Proc.Natl.Acad.Sci(U.S.A) 85:9462-9430) of the functionally active of IL-6.Prepared alpha-interferon (Cebrian, et al., 1987, J.Immunol.138:484-490) and beta-interferon (Redlich ﹠amp; Grossberg, 1989, J.Immunol 143:1887-1893) neutralizing monoclonal antibody, and the same with oncostatin M, and disulfide bond is necessary functional structure.The antigenic surface determinant location map of the plain molecule of mutation disturbance was not still disclosed.It is how to regulate the similar tertiary structure of these molecules and functional character that structure one functional analysis that gathers these and other phytokinin will help to describe different basic structures.Fig. 7 just uses OM2 and with the graph model figure in the important district of the determined oncostatin M molecular function of location map of OM3 and OM4 antigenic surface determinant.

Though the neutralizing antibody binding site does not exist, inner deletion mutation data show by non-neutral antibody, OM1, and the antigenic surface determinant of mensuration still keeps complete.Similar result that L-Ala screening by human growth hormone is mutagenic obtained, many therein sudden changes can influence the combination of acceptor, and do not influence combination (the Cunningham and Wells of those monoclonal antibodies of anti-surperficial determinant rather than anti-receptor binding domain, 1989, Science 244:1081-1085).These data show the local failure that can occur receptor binding site under the situation that does not cause the large-scale damage of oncostatin M tertiary structure.Up to the present utilization sudden change still can not draw the location map of OM1 antigenic surface determinant.OM1 antigenic surface determinant may depend on the terminal folding and stable tertiary structure of C-.

Inspection oncostatin M structure one emic several mutual survey methods have been narrated in the present invention and other documents.To represent its neutralization active because antibody OM2 is seemingly by checking oncostatin and combining of its acceptor, therefore might prepare a kind of can with the direct bonded anti-id AB of the neutralizing antibody internal structure that can discern the oncostatin M acceptor.Successfully use this method to prepare and directly acted on some not antibody of isoacceptor, as insulin receptor (Sege and Peterson, 1978,75:2443-2444) and B-adrenergic receptor (Schreiber Proc.Natl.Acad.Sci.(U.S.A), et al., 1980, Proc.Natl.Acad.Sci.(U.S.A) 77:7385-7389; Homey, et al., 1982, J.Clin.Invest.69:1147-1154).Some other (Gaulton and Green, 1986, Ann.Rev.Immunol.4:253-280; Vaux, et.al., 1990, Nature.345:495-502).Location with the detected surface antigen decision of monoclonal antibody described herein sieve can be used as important tool to be used for checking the biological function and the tissue distribution of oncostatin M.

Following non-limiting example and method are to be used for the present invention is done further invention.

The generation of oncostatin M monoclonal antibodies

A. immunization and fusion

By the reorganization oncostatin M of the expressing cho cell of plasmid pBOM transfection the 4Balb/C mouse is carried out immunization with preparation hybridoma (Linsley with 5 μ g or 10 μ g, et al., 1989, J.Biol.Chem.264:4282-4289) and by the described purifying (Zarling that carries out of document, et al., 1986, Proc.Natl.Acad.Sci.(USA) 83:9739-9743).The Chinese hamster ovary celI excretory oncostatin M of transfection be a kind of from 227 residue precursor molecules process have 196 amino acid whose ripe molecules (Linsley, et al., 1990, Mol.Cell.Biol.10:1882-1890).

Generally, in PBS, and emulsification in a kind of isopyknic complete Freund's adjuvant is subcutaneously injected into the emulsion of 25 μ g in the claw pad of a back leg with the oncostatin M resuspending.After two weeks, in same back leg claw pad, carry out the immunization of same procedure, and used oncostatin concentration is identical during with immunization for the first time.For the second time immunization is after two weeks, on the claw pad of same back with oncostatin M and non-complete Freund's adjuvant emulsive for the third time preparation inject to mouse.Last immunization is after three days, Qu Xia popliteal portion lymphoglandula, then with lymph-node cell and myeloma cell's ratio be 1: 1 ratio with 40% polyoxyethylene glycol as fusogen, rat bone marrow tumour 653/AG8 cell and lymph-node cell are merged.With 5 * 10 ⁴The concentration of cells/well is cultivated fusion product containing on the 96-hole flat board of xanthoglobulin-aminopterin-induced syndrome-thymidine agent alternatively, substratum is for containing 10% foetal calf serum, and the Dulbecco ' s that the Iscoves of Sodium.alpha.-ketopropionate and L-glutaminate revises revises Eagle ' s substratum (" IDMEM ").After one week, change original substratum with the fresh culture that contains selective agent.When as seen microscopically hybridizes product, the hybridization product is screened by following solid phase micro enzyme translocation fixed (" Micro EIA ").

B. enzyme-linked immunoassay

Show feature with following Micro EIA method with the antibodies specific of the oncostatin M of hybridoma preparation.In the hybridoma supernatant liquor, screen active ingredient with a kind of solid phase EIA of trace test flat board (Robbins Scientific, San Francisco) that is applicable to.The purifying oncostatin of 2 to 4 μ g/ml concentration is carried out the flat board cultivation with the amount of every hole 5 μ l, and make it air-dry overnight.After checking 1 hour, add the hybridoma supernatant liquor of 1 μ l volume, and at room temperature be incubated 1 hour with 5% skim-milk PBS solution and 0.1% sodiumazide.By the method for in PBS, flooding flat board is washed 6 times.Each is 1: 1500 peroxidase-conjugated F(ab ' with 5 μ l amount adding concentration in every hole) ₂The PBS+2%BSA solution of sheep anti-mouse igg (Pel-Freez, Rogers AK).After hatching 1 hour, by method washing plate as previously mentioned 6 times; The substrate that adds 10 μ l, 2,2 '-azino-(st.Louis MO.) and after 20 minutes carries out the measured value reading with OD=660nm to flat board to two-(3-ethylbenzene thiazoline-6-sulfonic acid) for " ABTS ", Sigma Chemical Co..According to determining positive hole with the background signal of the sero-fast signal correction of rabbit anti-oncostatin M for preparing previously.On soft agar, positive hybridization product is cloned.

Use a kind of couple of determinant EIA(DDEIA) method estimates the binding ability of its monoclonal antibody with tertiary structure antigenic surface determinant and a series of sudden change oncostatin M molecules.These mutant have insertion, disappearance or the replacement of amino-acid residue in the different loci of molecule.Before the analysis of carrying out DDEIA, measure the concentration of mutating molecule in the supernatant liquor earlier with the direct EIA method of a kind of mAb of utilization OM3.Analyzing for carrying out DDEIA, is the 0.05M carbonate buffer solution of protein G (Pharmacia) the protein affinity purification monoclonal antibody OM3 of 10 μ g/ml with 100 μ l concentration, and PH9.6 joins in the flat flat board in 96 holes, hatches down in 4 ℃ then and spends the night.Take out antibody, using PBS, 1%BSA after 0.05% polysorbas20 checked 1 hour, adds the purifying oncostatin M of specific concentrations or the supernatant liquor of a series of different weaker concns that adding obtains from the COS cell of the plasmid transfection of encoding mutant form OM.Should flat board 37 ℃ of insulations 2 hours down, with PBS washing 5 times, be that biotinylated (biotinylated) monoclonal antibody (OM1 or OM2) of 200ng/ml is incubated 1 hour down in 37 ℃ with 100 μ l concentration again.After 37 ℃ are incubated 1 hour down, with flat board washing 5 times; Add 1: 10 of 100 μ l HRP-bonded streptavidins (vector), 000 diluent, and dull and stereotyped in 37 ℃ of insulations 30 minutes down.After the washing, with 3,3 ', 5,5 ' tetramethyl reacts with it for p-diaminodiphenyl (" TMB "), stops this reaction with 1N sulfuric acid, and in A ₄₅₀Reading.

C. immunoprecipitation

Each is with 4ml200 μ Ci's in the petri plate of a kind of 60 * 15cm ³⁵The S-methionine(Met) and ³⁵The Chinese hamster ovary celI of S-halfcystine and oncostatin-M-code cDNA transfection (Linsley et al., 1989, J.Biol.Chem.264:4282-4289) cultivate 4-8 hour together.Collect supernatant liquor and filtration; Add 10mM phenylmethyl sulfonylfluoride (" PMSF ") and 100mM tolylsulfonyl-Methionin-chloromethyl ketone (" TLCK ") to restrain proteolytic enzyme.In order to carry out immunoprecipitation, exhaust (spent) supernatant liquor and 100-200 μ l mark supernatant liquor 4 ℃ of concussion overnight incubation down from the hybridization product of secrete monoclonal antibody with what 200 μ l obtained.By separating immune complex with a kind of the cultivation with the covalently bound mono-clonal mouse-anti mouse of Reactigel particle (Pierce Chemicals) k light chain monoclonal antibody.After the flushing, in the sample buffer that contains 1%SDS and 5%2-mercaptoethanol, boil the wash-out immune complex.The material of wash-out is carried out electrophoresis in 15%SDS-PAGE.Use Amplify ^TM(Amersham Corp., Arlington Heights IL) carry out photoradiography to gel, and drying is carried out radioautogram with the X-AR5 X-of Kodak line film.

D. growth-inhibiting is measured

Measure antibody with determine whether can both in and the retarding effect of oncostatin M in growth-inhibiting test (" GIA ").In GIA, use A375 melanoma cells (4 * 10 ³Cell/50 μ l) as indicator cells.(Costar 3595 in flat 96 hole tissue culture plate with the A375 cell, Cambridge, MA) upward carrying out inferior being commissioned to train in the growth medium of the Eagle substratum (" DMEM ") that contains the Dulbecco modification of adding 10% heat-inactivated fetal bovine serum and penicillin/streptomycin supported 4 hours.Oncostatin M is diluted in growth medium, and the diluted sample that adds 50 μ l in each hole is measured its growth-inhibiting in triplicate.Then this cell was cultivated 72 hours down at 37 ℃.After this incubation period finishes, each hole with 100 μ l contain ( ¹²⁵I) iodo-2 '-((IL)) growth medium effect is 24 hours for Amersham Corp., Arlington Heights in 0.05 μ Ci/ hole for deoxyuridine.Monolayer is washed in phosphate buffered saline (PBS) (" PBS "), in 95% methyl alcohol, fix, then dry air.With the cell bonded ( ¹²⁵I)-iodo-deoxyuridine is dissolved in the 1N sodium hydroxide of 200 μ l, according to be attached among the active grown cell DNA ( ¹²⁵I)-amount of iodo-deoxyuridine measures the cell growth population.One units activity is defined as the growth of for untreated cell A375 cell and is subjected to 50% amount that suppresses needed oncostatin M.

E. oncostatin M radioreceptor assay

With a kind of radioreceptor assay method measure antibody for ¹²⁵I-mark oncostatin M and human cancer cell are the inhibition ability that H2981 goes up the respective fine cellular surface receptors bind of oncostatin M.With the H2981 cell with 2 * 10 ⁵The density of cells/well places on the 48 hole flat boards, and keeps 16-24 hour down in 37 ℃ before measuring beginning.Use binding buffer liquid (Linsley et al., 1986, Biochemistry 25:2978-2986) pair cell individual layer to wash once then.For measuring total binding, adding concentration is 0.5-100ng/ml's ¹²⁵The I-oncostatin M.In order to measure non-specific binding, adding ¹²⁵Handle is higher than in the time of the I-oncostatin M ¹²⁵The unlabelled oncostatin M of 20 to 100 times of concentration of I-oncostatin M concentration joins in the solid flat board.Under 23 ℃, make in conjunction with carrying out 2-5 hour, wash monolayer 4 times with binding buffer liquid then.Cell bonded radioactive activity composition is dissolved among the 1N NaoH and with gamma counter counts.Calculate the specificity combination by from total binding, deducting non-specific binding.Utilization Scatchard analyzes to determine separation constant (Kd) and combination active (Scatchard, 1949, Ann.N.Y.Acal.Sci 51:660).

F. oncostatin M mutant

Detect carrying out the bonded anti-oncostatin M monoclonal antibodies with a series of reorganization oncostatin M mutant that on dna level, insert, lack or replace.According to the method antagonist that (Spent) supernatant liquor carries out EIA that exhausts that obtains from the COS cell cultures of various parental generations and the of short duration transfection of mutation structure is measured.

G. use the oncostatin M in the monoclonal antibody measuring serum

The measuring method that oncostatin M exists in the serum is as follows: at first the human serum sample is placed on a kind of pillar of S-300 size according to molecular weight separation of serum albumen.The GIA activity of the stream part of from the S-300 pillar, collecting at serum sample acidifying before and after look.Before acidifying, in void volume stream part, most of serum do not show the GIA activity, and before joining GIA, carry out acidifying, in the serum sample part that neutralized again then, molecular weight shows that greater than the activity that stream parts of 200 kilodaltons represents the oncostatin in the serum generally is to combine with a kind of binding factor greater than 200kd, and is non-activity in this state.Check various serum samples with the Western engram analysis.The oncostatin M monoclonal antibody specific with corresponding to the combination of proteins in the high molecular of the oncostatin M molecular weight stream part, proved should stream part in the existence of oncostatin M.Be purified into the oncostatin M that is present in serum, and-terminal amino acid shows that in proper order it is identical with the repetition oncostatin M with the natural result who before obtains.

H. use the specificity of oncostatin M is selected the monoclonal antibody result

Carry out two groups of fusion experiments, be made up of four kinds of fusions for every group, every kind merges use a lymphoglandula that obtains from each mouse.In first group, four kinds of hybridization products are checked more widely.From second group, this group is more successfully to merge group (number with the positive that identifies in Micro EIA hybridization product is represented), can identify to 20 other anti-oncostatin M monoclonal acceptors.In the middle of these according to the coherent signal its EIA with immunoprecipitation is natural or the ability of sex change metabolic marker oncostatin M selects following hybrid to do further research: OM2, OM7, OM3, and OM8.According to the immunoprecipitation data that describe below, according to itself and natural oncostatin M, sex change oncostatin M, perhaps the response capacity of the two (table I) is referred in the class in the middle of three classes with these hybrids.Check then in these monoclonal antibodies 5 kinds with determine whether whether to have in the middle of them a kind of can in and the retarding effect of oncostatin M in the growth-inhibiting test.These results are as described below.

I. the immunoprecipitation of monoclonal antibody and oncostatin M

The immunoprecipitation data results identifies three kinds of monoclonal antibodies (OM2, OM2 and OM7), and they can only be with oozy from the CHO transfectant ³⁵The oncostatin M form of S-methionine mark reacts and (organizes 1(bands of a

spectrum

2,6,7; Fig. 1; See the table I that is used to discern antibody).Other three kinds of monoclonal antibodies (OM4, OM5 and OM6) can only with handle at first reduction and sex change with SDS and reductive agent and boil at 100 ℃ again ³⁵S-methionine mark oncostatin M reacts, but does not react (group 2, bands of a

spectrum

3,4,5) with natural oncostatin M.Other two kinds of monoclonal antibodies (OM3 and OM8) though can with biosynthetic labelling be natural or the oncostatin M of sex change react (group 3, bands of a spectrum 8,9).

J. in growth-inhibiting test and the monoclonal antibody of oncostatin M

Antibody purified can concentration rely in the mode (Fig. 2 A) and the effect of oncostatin M from hybrid OM2.The antibody OM1 of anti-natural oncostatin M does not show the growth inhibitory effect (Fig. 2 B) that checks oncostatin M.Measure by CIA, do not have a kind of antibody of discerning the sex change determinant can in and oncostatin M.As described below, further test the inhibition ability of neutralizing antibody OM2 to the oncostatin M receptors bind.

K. in the radioreceptor test, suppress oncostatin M bonded monoclonal antibody

OM2 antibody can suppress the combination of oncostatin M acceptor in concentration dependence mode, this with anti-natural non-in and the OM1 antibody in site, or the third antibody OM4 of antitypy oncostatin M antigenic surface determinant opposite (Fig. 3).

L. the analysis of the functional site of oncostatin M and antigenic surface determinant figure

Following a part of content description prepared can with the transfection CHO cell supernatant liquor in be purified into reorganization OM bonded monoclonal anti body characteristics (Linsley et al., 1989, J.Biol.Chem.264:4282-4289).By the product of COS emiocytosis by the plasmid transfection of a series of mutant form OM of coding is carried out serological analysis, we have determined the position of the antigenic surface determinant that some monoclonal antibody discerns, and have determined antibodies and needed some tertiary structure of biological activity of this OM molecule.

In two kinds of antibody can discerning folding antigenic surface determinant, in growth-inhibiting mensuration (GIA), cancel the active ability of OM and restriction OM bonded ability in radioreceptor assay (" RRA ") according to it, identify OM2, rather than OM1, be a kind of neutralizing antibody.The serological analysis of sudden change OM molecule shows that the binding site of OM2 is influenced by the non-proximity of OM, and shows two disulfide linkage (C ₄₉-C ₁₆₇) one of existence be neutralizing antibody bonded basic structure.In addition, some sudden change can cancel OM2 in conjunction with and do not cause the spherical false folding of OM molecule.

These data show the binding site of the antigenic surface determinant determined by OM2 and the OM dependency of having living space, and by OM1, those surperficial determinants that OM3 and OM4 discern are then different with them.Antibody described herein will help to determine physiological function and the distribution of OM as the immunology probe of checking OM in meaningful tissue and the body fluid.

M. draw the figure of the antigenic surface determinant of oncostatin M with EIA

Growth regulator oncostatin M (OM) is a kind of novel cell mitogen that normal and transformation cell lines is widely had multi-directional effect.In order to determine some physiological function of OM, we have prepared a series of monoclonal antibodies (OM1, OM2, OM3 and OM4) of anti-recombinant molecule as described below and have shown their feature.

Antibody OM1 and OM2 combine with natural OM, but do not combine with sex change OM, and this shows that they discern the antigenic surface determinant by the tertiary structure form.The third antibody OM3 combines with natural or sex change OM, and antibody OM4 only combines with sex change OM.By measuring antibody combines (mAb) antigenic surface determinant that can determine to be applicable to these monoclonal antibodies with the OM of one group of mutant form position.The OM3 binding site contains residue 108, and the binding site of OM4 is destroyed by 104 aminoacid insertion.

In order to determine the position of the antigenic surface determinant that these antibody is discerned, we to these antigenic surface determinants in the serum-free condition substratum with from having the combining of OM that obtains in the COS cell of the plasmid transfection of different loci upper amino acid residue OM mutant code this molecule and test with a series of.With direct EIA method the antibody OM3 that discerns linear antigenic surface determinant and OM4 and a series of sudden change OM combination of proteins are checked, in these sudden changes OM protein, utilize the insertion (Fig. 6) of terminator codon in a sequential manner with C one terminal amino group acid deletion.Mutant △ 188-227 can be by the OM3 combination, at 108 residue places leucine is changed into Serine, and △ 188-227/L108S is then not combined.The site, location of antibody OM4 at 104 (Fig. 6) since the insertion of glycine-L-Ala-glycine order destroy.Antibody OM5 and OM6 also are main and sex change OM reaction, and not same with OM4 antigenic surface determinant reaction because they and do not reacted by the bonded GAG104 of OM4 institute mutant.Up to the present there is not a kind of antigenic surface determinant locating information that these mutant can be provided in prepared a series of OM mutant.Antibody OM7 may with neutralizing antibody, antibody OM is equal to because it has identical Ig homotype and OM sudden change combination with OM, and antibody OM8 and OM3 may also be equal to by the same token.

The serological analysis of N.OM1 and OM2 antigenic determinant

In order to analyze OM and antibody OM1 and OM2 bonded topology requirement, they only with the OM reaction of folded form, we have set up a kind of pair of determinant EIA method.This method use not only can with natural OM but also with sex change OM bonded antibody OM3, from COS transfectant mutant supernatant liquor, seek OM.To derive from mutant molecule by biotinylated (biotinylated) OM1 or OM2 bonded OM() concentration with compare by the typical curve of corresponding antibodies bonded purifying natural OM.

As the OM mutating molecule concentration of these antibodies, the ng/ml of unit, the comparing result that combines purifying OM concentration with them is listed in the table II.Therefore because in this measuring method, OM is present in saturation concentration in the undiluted supernatant liquor of the transfectant that suddenlys change, and needs a series of diluents to measure OM in the linear portion among the DDEIA.In the situation of inferred value of all checked OM greater than 200ng/ml, absorption value corresponding to the serial dilutions of this molecule different mutants has identical slope, and this shows that biotinylated (biotinylated) antibody combines with these mutating molecules with identical relevant affinity.

Show for the structural analysis of OM molecule and to have an intensive amphipathic molecule/pair have a liking for molecular regime between near C-end amino acid 168-196.OM1 and OM2 antibody are checked with the combining of a series of mutant with succession C-terminal deletion.OM1 and OM2 can both have the OM(that obtains the mutant of succession C-terminal disappearance from those from 186 amino acids with high affinity identification and represent with the protein binding of high density).For △ 185-227C-terminal deletion mutant, two kinds of antibody are all with lower affinity combination; Neutralizing antibody OM2 in conjunction with level than OM1 low in conjunction with level.Two kinds of antibody completely loses in conjunction with activity in C-terminal deletion mutant △ 184-227.In the both sexes district of a series of mutant, hydrophobic group or hydrophilic group are changed over neutral amino acids and can cause more complicated antibodies mode.In the replacement of three phenylalanines and glycine, only the change on 176 can cause completely losing of two kinds of antibodies, and can reduce the combination of neutralizing antibody OM2 184 change, and OM1 is not reduced.All there be not influence with the replacement of glycine for any combining among both at 169 phenylalanines.Two kinds of isolated proteins, H174G and H1786 change the wetting ability Histidine into glycine 174 and 178 respectively, can be with high affinity by OM1 and OM2 combination.

In natural oncostatin M molecule, have two intramolecular disulfide bonds.The C6-C167 disulfide linkage does not influence the local tertiary structure of OM1 and OM2 surface determinant, because the halfcystine on 6 can not reduce antibodies when changing into Serine.Cancelling the surperficial determinant that two disulfide linkage (C6S/C167S) can destroy two kinds of antibody, may be owing to caused the spherical false folding of this molecule.

Two kinds of sudden change OM molecules, △ 44-47 and △ 22-36, can provide information with distinguish antibody OM1 and OM2 in conjunction with demand.These deletion mutants can be by nonneutralizing antibody OM1 combination, but can not be neutralized antibody OM2 combination.Fig. 7 is with OM2 with the schematic diagram in the definite important district of oncostatin M molecular function of the location map of the surperficial determinant of OM3 and OM4.

M. depositing as the representational following clone of the present invention of microorganism has been deposited at American type culture collection, Rockville, and Maryland, and obtained the following number of asking for:

Clone is gone into Tibetan antibody

11R2F8.9 ATCCHB10398 OM2

12R13D7.2 ATCCHB10396 OM3

1R10F11.34.16 ATCCHB10397 OM1

Because the concrete clone of institute's preservation is for certain aspect of the present invention is illustrated individually, thereby scope of the present invention is by the clone of these preservations restriction, and anyly all belongs within the scope of the present invention in clone that is equal on the function or antibody.Really, according to the description and the accompanying drawing of front, except described those embodiment, can also carry out various modifications to the present invention, this is conspicuous for the skilled person in this area.This modification should belong within the claim scope of the present invention.

Claims

1, a kind of preparation monoclonal antibody, or its segmental method, the antigen binding site of this antibody can combine with oncostatin M, and method comprises

(a) cultivate a kind of clone, said clone can produce a kind of anti-oncostatin M antibody, or its fragment, and reclaims the product that is produced, or,

(b) a kind of anti-oncostatin M antibody fragment of preparation from a kind of complete anti-oncostatin M antibody; With, if necessary,

(c) with (a) or the antibody or the antibody fragment of preparation (b) be attached to a kind of marker, somatomedin, or on the toxin.

2, a kind of the method for claim 1, wherein prepared monoclonal antibody or fragment can combine with oncostatin M, and suppress the biological activity of oncostatin M.

3, a kind of method as claimed in claim 1 or 2, wherein prepared monoclonal antibody or segmental antigen binding site can combine with the avtive spot of oncostatin M.

4, a kind of the method for claim 1, wherein prepared monoclonal antibody or fragment competitive inhibition monoclonal antibody OM2, OM3, or OM1 combines with the immunologic opsonin of its target antigen.

5, a kind of the method for claim 1, this method prepares monoclonal antibody OM2; OM3; Or, OM1; Or its a kind of fragment.

6, a kind of as any method in the claim 1 to 5, wherein prepare a kind of Fab of monoclonal antibody, F(ab ') ₂, or the Fv fragment.

7, a kind of any method as claim 1 to 6 wherein with a kind of monoclonal antibody, or its fragment, is attached on a kind of marker that can produce detectable signal.

8, a kind of method as claimed in claim 7, wherein this marker comprises a kind of fluorescence radiation agent, a kind of radio-labeled thing, a kind of chromophoric group, or a kind of enzyme.

9, a kind of any method as claim 1 to 6, wherein with monoclonal antibody, or its fragment, be attached on a kind of somatomedin or the toxin.

10, a kind of clone, can prepare a kind of as the defined any monoclonal antibody of claim 1 to 6, or its antibody fragment.

11, hybridoma cell line 11R2F8,12R13D7, or 1R10F11.

12, a kind of method of pharmaceutical compositions comprises a kind of as the defined any monoclonal antibody of claim 1 to 9, or its fragment, and with a kind of pharmaceutically acceptable carrier, thinner or vehicle are mixed together.