CN101023102A

CN101023102A - Anti-OX40L antibodies

Info

Publication number: CN101023102A
Application number: CN200580031358.XA
Authority: CN
Inventors: 约瑟夫·恩德尔; 埃尔西·欧吉; 玛丽亚·芬特斯; 伊沃·格劳斯; 阿兰·拉布里吉恩; 马丁·兰岑德费尔; 保罗·帕伦; 弗兰克·雷贝阿斯; 拉尔夫·舒马赫; 泽贝尔·斯特凡; 扬·范德温克尔; 马丁·弗里斯玛-范武格特
Original assignee: F Hoffmann La Roche AG
Current assignee: F Hoffmann La Roche AG
Priority date: 2004-09-17
Filing date: 2005-09-16
Publication date: 2007-08-22
Anticipated expiration: 2025-09-16
Also published as: CN101023102B; ZA200701952B

Abstract

This invention relates to anti-OX40L antibodies and, in particular, to anti-OX40L antibodies and variants thereof that contain a Fc part derived from human origin and do not bind complement factor C1q. These antibodies have new and inventive properties causing a benefit for a patient suffering from inflammatory diseases.

Description

Anti-OX 40 L antibodies

The present invention relates generally to anti-OX 40 L antibodies, and relate to the anti-OX 40 L antibodies of debond complement factor C1q especially, relate to its pharmaceutical composition and application.Preferably, these antibody are the people's or humanized antibody.

When CD40/CD40L connects, human OX 40 L (gp34, SwissProt P23510) is expressed on activatory B cell and dendritic cell, and expresses (summary: Weinberg on the endotheliocyte of inflammation tissue, A.D., Trends Immunol.23 (2002) 102-109).It separates (these T cells are by producing the autokrine ring and infinite multiplication with OX40) first from the human leukemia cell that HTLV-1 infects.For example, OX40L and in following, mention: WO95/12673 at its antibody; WO 95/21915; WO 99/15200; Baum, P.R, etc., EMBO is (1994) 3992-4001 J.13; Imura, A., etc., Blood 89 (1997) 2951-2958; Imura, A., etc., J.Exp.Med.183 (1996) 2185-2195; Kjaergaard, J., etc., J.Immunol.167 (2001) 6669-6677; Lane, P., J.Exp.Med.191 (2000) 201-206; Mallett, S., and Barclay, A.N., Immunol.Today 12 (1991) 220-223; Mallett, S., etc., EMBO is (1990) 1063-1068 J.9; Ndhlovu, L.C., etc., J.Immunol.167 (2001) 2991-2999; Ohshima, Y., etc., J.Immunol.159 (1997) 3838-3848; Rogers, P.R., etc., Immunity 15 (2001) 445-455; St ü ber, E., and Strober, W., J.Exp.Med.183 (1996) 979-989; St ü ber, E, etc., Gastroenterology 115 (1998) 1205-1215; Takahashi, Y., etc., J.Virol.75 (2001) 6748-6757; Takasawa, N., etc., Jpn.J.Cancer Res.92 (2001) 377-382; Taylor, L., and Schwarz, H., J.Immunol.Meth.255 (2001) 67-72; Weinberg, A.D., etc., Nature Medicine 2 (1996) 183-189; Weinberg, A.D., etc., Semin.Immunol.10 (1998) 471-480; Weinberg, A.D., Trends Immunol.23 (2002) 102-109; Wu, T., etc., Transplant.Proc.33 (2001) 217-218; Higgins, L.M., etc., J.Immunol.162 (1999) 486-493; And Yoshioka, T, etc., Eur.J.Immunol.30 (2000) 2815-2823.Human OX 40 L is the part of the people OX40 (CD134) of transient expression on activatory CD4+T cell.Its part causes the common pungency signal of T cell activation in conjunction with OX40.Described the OX40/OX40L that produces two-way signaling and interact (Matsumura, Y., etc., J.Immunol.163 (1999) 3007-3011; Kotani, A., etc., Immunol.Lett.84 (2002) 1-7).In addition, the endotheliocyte of OX40/OX40L interaction regulation and control activated T cell and inflammation tissue sticks.Because OX40L is transient expression on activatory B cell, DC and endotheliocyte just, so, at the antibody of OX40L should be in inflammatory response optionally blocking t cell activation and endothelial cell adhesion, and make non-activated, periphery T cell is unaffected.Yoshioka, A., etc. (Eur.J.Immunol.30 (2000) 2815-2823) proved that neutralization resists-the treatment potentiality of mOX40L mAb in the rheumatoid arthritis mouse model.Using of it improves severity of disease significantly.In other relative disease model, for example, inflammatory dermatosis, tentative autoimmune disorders (EAE), GVHD, uropoiesis inflammatory bowel (urine inflammatory boweldisease), this antibody shows similar activity (Yoshioka, A., Deng., Eur.J.Immunol 30 (1999) 2815-2823; Salek-Ardakani, S., etc., J.Exp.Med.198 (2003) 315-324; Burgess, J.K., etc., J.Allergy Clin.Immunol.113 (2004) 683-689; Hoshino, A., etc., Eur.J.Immunol.33 (2003) 861-869; Arestides, R.S., etc., Eur.J Immunol.32 (2002) 2874-2880; Nohara, C., etc., J.Immunol.166 (2001) 2108-2115; Weinberg, A.D., etc., J.Immunol.162 (1999) 1818-1826; Higgins, L.M., etc., J.Immunol.162 (1999) 486-493; Humphreys, I.R., etc., J.Exp.Med.198 (2003) 1237-1242; Akiba, H., etc., J.Exp.Med.191 (2000) 375-380; Ishii, N., etc., Eur.J.Immunol.33 (2003) 2372-2381; Blazar, B.R., etc., Blood 101 (2003) 3741-3748; Tsukada, N., etc., Blood 95 (2000) 2434-2439; Akiba, H., etc., Biochem.Biophys.Res.Commun.251 (1998) 131-136).

After deliberation at the antiinflammation in the various diseases model of the antibody of OX40L (Sugamura, K., etc., Nat.Rev.Immunol.4 (2004) 420-431).

Tanaka, Y., etc., Int.J.Cancer 36, (1985) 549-555; Tozawa, H, etc., Int.J.Cancer 41 (1988) 231-238; And Miura, S., etc., Mol.Cell.Biol.11 (1991) 1313-1325 has described the mouse monoclonal antibody of called after TARM-34 and TAG-34, and its surface antigen with the human lymphocyte system of carrying human T-cell I type leukemia virus (HTLV-I) reacts.TAG-34 antibody can be purchased from MBL international corporation.TAG-34 also combines with OX40L.

Summary of the invention

The present invention relates to a kind of antibody, preferably a kind of monoclonal antibody is characterized in that described antibodies OX40L, contains the Fc fragment from the people source, and people complement factor C1q and/or the human Fc gamma receptor of debond on the NK cell.

The invention still further relates to a kind of antibody, preferably a kind of monoclonal antibody is characterized in that described antibody contains the Fc fragment from the people source, combines with the OX40L (in western blotting) of OX40L and sex change in the antibody concentration of 100ng.This antibodies to the OX40L polypeptide epitope of the identical epi-position of monoclonal antibody LC.001 bonded on.Such antibody is, for example, and LC.001, LC.033 and LC.060.These antibody are preferably human IgG1's type (wild-type), or people complement factor C1q and/or the human Fc gamma receptor of debond on the NK cell.

The invention still further relates to a kind of antibody in conjunction with OX40L, it is characterized in that comprising variable light chain and variable heavy chain, be characterised in that described variable heavy chain comprises CDR1, CDR2 and CDR3, CDR3 is characterised in that and is selected from SEQ ID NOs:33-38.Particularly preferably, CDR1 is selected from SEQ ID NOs:21-25, and CDR2 is selected from SEQ ID NOs:26-32 and CDR3 is selected from SEQ ID NOs:33-38.

Preferably to comprise that variable light chain and variable heavy chain are feature, be characterised in that described variable light chain comprises CDR1, CDR2 and CDR3 according to antibody of the present invention, CDR3 is characterised in that and is selected from SEQ IDNOs:51-57.Particularly preferably, CDR1 is selected from SEQ ID NOs:39-44, and CDR2 is selected from SEQID NOs:45-50 and CDR3 is selected from SEQ ID NOs:51-57.

According to antibody of the present invention preferably to comprise that variable light chain and variable heavy chain are feature, be characterised in that described variable heavy chain comprises CDR1, CDR2 and CDR3, CDR3 is characterised in that the CDR3 that the CDR3 of heavy chain is selected from SEQ ID NOs:33-38 and light chain is selected from SEQ ID NOs:51-57.Particularly preferably, variable heavy chain comprises the CDR1 that is selected from SEQ ID NOs:21-25, be selected from the CDR2 of SEQ ID NOs:26-32 and the CDR3 that is selected from SEQ ID NOs:33-38, and variable light chain comprises the CDR1 that is selected from SEQ ID NOs:39-44, is selected from the CDR2 of SEQ ID NOs:45-50 and the CDR3 that is selected from SEQ ID NOs:51-57.

All CDRs independently from selecting each other, still are the such modes with described antibodies OX40L naturally.Therefore, the light chain of identical LC antibody and the CDRs of heavy chain can make up, the perhaps heavy chain CDRs of the light chain CDRs of LC.001 and LC.001, LC.059 or LC.063 combination.CDRs on every chain is separated by framework amino acid.

Be characterised in that preferably that according to antibody of the present invention described antibody comprises the CDRs that is independently selected from by the following group of forming

A) light chain (V of aminoacid sequence SEQ ID NO:1 _L) heavy chain (V of variable C DRs and SEQ ID NO:2 _H) variable C DRs;

B) the weight chain variable CDRs of the light chain variable CDRs of aminoacid sequence SEQ ID NO:3 and SEQ ID NO:4;

C) the weight chain variable CDRs of the light chain variable CDRs of aminoacid sequence SEQ ID NO:5 and SEQ ID NO:6;

D) the weight chain variable CDRs of the light chain variable CDRs of aminoacid sequence SEQ ID NO:7 and SEQ ID NO:8;

E) the weight chain variable CDRs of the light chain variable CDRs of aminoacid sequence SEQ ID NO:9 and SEQ ID NO:10;

F) aminoacid sequence SEQ ID NO:11 or 16 light chain variable CDRs and the weight chain variable CDRs of SEQ ID NO:12;

G) light chain (V that defines by aminoacid sequence SEQ ID NO:1 _L) variable domains and by the heavy chain (V of SEQ IDNO:17 definition _H) variable domains;

H) by the light chain variable structural domain of aminoacid sequence SEQ ID NO:18 definition and the weight chain variable structural domain that defines by SEQ IDNO:19;

I) by the light chain variable structural domain of aminoacid sequence SEQ ID NO:1 definition and the weight chain variable structural domain that defines by SEQ IDNO:20;

Or its OX40L-binding fragment.

Be characterised in that preferably that according to antibody of the present invention described antibody comprises the variable region that is independently selected from by the following group of forming

A) light chain (V that defines by aminoacid sequence SEQ ID NO:1 _L) variable domains and by the heavy chain (V of SEQ IDNO:2 definition _H) variable domains;

B) by the light chain variable structural domain of aminoacid sequence SEQ ID NO:3 definition and the weight chain variable structural domain that defines by SEQ IDNO:4;

C) by the light chain variable structural domain of aminoacid sequence SEQ ID NO:5 definition and the weight chain variable structural domain that defines by SEQ IDNO:6;

D) by the light chain variable structural domain of aminoacid sequence SEQ ID NO:7 definition and the weight chain variable structural domain that defines by SEQ IDNO:8;

E) by the light chain variable structural domain of aminoacid sequence SEQ ID NO:9 definition and the weight chain variable structural domain that defines by SEQ IDNO:10;

F) by the light chain variable structural domain of aminoacid sequence SEQ ID NO:11 or 16 definition and the weight chain variable structural domain that defines by SEQID NO:12;

Or its OX40L-binding fragment.

Be characterised in that preferably according to antibody of the present invention that people's variable region of light chain comprises and be independently selected from NO:1, the aminoacid sequence of 3,5,7,9,11,16 and 18 groups of forming by SEQ ID.

Be characterised in that preferably according to antibody of the present invention that people's variable region of heavy chain comprises and be independently selected from NO:2, the aminoacid sequence of 4,6,8,10,12,17,19 and 20 groups of forming by SEQ ID.

The CDR district of heavy chain and light chain shows in SEQ ID NO:21-38 and 39-57.

Be characterised in that preferably that according to antibody of the present invention described antibody comprises by the light chain variable structural domain of aminoacid sequence SEQID NO:1 definition with by SEQ ID NO:2 the weight chain variable structural domain of 17 or 20 definition.

Be characterised in that preferably that according to antibody of the present invention people's CH comprises the aminoacid sequence that is independently selected from the group of being made up of SEQ ID NO:14 and 15 or the CH of SEQ ID NO:58.

Be characterised in that preferably that according to antibody of the present invention described antibody comprises κ-constant region of light chain or the SEQ ID NO:61 of SEQ ID NO:13,65 or 69 constant region of light chain.

Preferably, be characterised in that in conjunction with OX40L, and be human IgG1's kind (wild-type), and comprise gamma heavy chain SEQ ID NO:58,62 or 66 according to antibody of the present invention.Particularly preferably be such antibody, it comprises

A) gamma heavy chain SEQ ID NO:58 and κ light chain SEQ ID NO:61,

B) gamma heavy chain SEQ ID NO:62 and κ light chain SEQ ID NO:65 or

C) gamma heavy chain SEQ ID NO:66 and κ light chain SEQ ID NO:69.

Another embodiment of the invention is the antibody in conjunction with OX40L, it is characterized in that it is by clone hu-Mab＜hOX40L〉LC.001, hu-Mab＜hOX40L〉LC.005, hu-Mab＜hOX40L〉LC.010, hu-Mab＜hOX40L〉LC.019, hu-Mab＜hOX40L〉LC.029 or hu-Mab＜hOX40L〉the LC.033 generation.

According to antibody of the present invention preferably chimeric, the people's or humanized antibody.

Preferably be characterised in that with less than 10 according to antibody of the present invention ^-8M (10 ^-12-10 ^-8M) K _DValue is in conjunction with OX40L, more preferably is characterised in that in BIAcore detects 10 ^-12-10 ^-9The K of M _DScope.

According to the preferably interaction of inhibition OX40L and OX40 in use 0.5 μ g/ml wraps by the ELISA of the fixedly OX40L of concentration (being preferably the biotinylated OX40L that is fixed on the streptavidin surface) of antibody of the present invention, has the IC50 value that is not more than 4nM.More preferably, the IC50 value is in 1-4 nM scope.

Preferably be characterised in that according to antibody of the present invention, the debond of described antibody and complement factor C1q is meant that a kind of like this ELISA detects, promptly, compare with the Bmax of antibody LC.001, wherein concentration is that the maximum combined (Bmax) of antibody and the C1q of 10 μ g/ml is 30% or lower, preferably 20% or lower.

Preferably, described antibody debond people Fc γ RI, Fc γ RIIA and/or Fc γ RIIIA.Particularly preferably, the human Fc gamma receptor of described antibody debond on NK effector cell.

The debond that preferably is characterised in that the Fc γ acceptor on described antibody and the NK cell according to antibody of the present invention is meant such detection, promptly, compare with the Bmax of antibody LC.001, wherein concentration is that the antibody of 20 μ g/ml and the maximum combined (Bmax) of NK cell are 20% or lower, preferably 10% or lower.

Be characterised in that preferably that according to antibody of the present invention it does not combine with Fc γ RI.This means, in but the bonded that at detectable level is antibody in the 0.078-10 μ g/ml scope and the B-cell lymphoma cell that lacks Fc γ RIIA and Fc γ IIB express recombinant Fc γ RI detects, described antibody is characterised in that, compare with the EC50 value of LC.001, the EC50 value of described antibody is 5 times or higher, preferably 7 times or higher, such as 8 times or higher.

Preferably be characterised in that according to antibody of the present invention to be a kind of IgG4 antibody or IgG1 antibody, it comprises at least one amino acid mutation, and preferably in people Fc fragment, this makes and does not combine with complement factor C1q and/or do not combine with human Fc gamma receptor on the NK cell.

Preferably be characterised in that its not complement activation factor C3 according to antibody of the present invention.

Preferably be characterised in that according to antibody of the present invention and be people's subclass IgG4.In another preferred embodiment of the present invention, described antibody is characterised in that to any IgG kind, is preferably IgG1 or IgG4, it is included in E233, L234, L235, G236, D270, N297, E318, K320, K322, A327, A330, at least one sudden change of P331 and/or P329 (being numbered) according to the EU index.Particularly preferably be IgG1 sudden change PVA236, L234A/L235A and/or GLPSS331 and IgG4 sudden change L235E.In addition, preferably, IgG4 subclass antibody comprise sudden change S228P or sudden change S228P and L235E (Angal, S., etc., Mol.Immunol.30 (1993) 105-108).

Therefore, preferably be the antibody of people's subclass IgG1 according to antibody of the present invention, it contains one or more PVA236, the sudden change of GLPSS331 and/or L234A/L235A (being numbered according to the EU index).

Preferably, be characterised in that with OX40L according to antibody of the present invention to combine, be the IgG1 kind that contains the L234A/L235A that suddenlys change, and comprise gamma heavy chain SEQ ID NO:59,63 or 67.

Particularly preferably, antibody comprises

A) gamma heavy chain SEQ ID NO:59 and κ light chain SEQ ID NO:61,

B) gamma heavy chain SEQ ID NO:63 and κ light chain SEQ ID NO:65 or

C) gamma heavy chain SEQ ID NO:67 and κ light chain SEQ ID NO:69.

Preferably, be characterised in that it is the antibody that contains the IgG4 kind of the S228P that suddenlys change, comprise gamma heavy chain SEQ ID NO:60,64 or 68 according to antibody of the present invention.

Particularly preferably, antibody comprises

A) gamma heavy chain SEQ ID NO:60 and κ light chain SEQ ID NO:61,

B) gamma heavy chain SEQ ID NO:64 and κ light chain SEQ ID NO:65 or

C) gamma heavy chain SEQ ID NO:68 and κ light chain SEQ ID NO:69.

Be characterised in that preferably that according to antibody of the present invention it does not cause the cytotoxicity (CDC) that depends on complement.

Be characterised in that preferably that according to antibody of the present invention it does not cause the cytotoxicity (ADCC) of the cell that depends on antibody.

Therefore, the present invention includes anti-OX 40 L antibodies or be the single heavy chain or the light chain of feature with their CDRs, the variable region, complete amino acid sequence or hybridoma, and the Fc fragment that it does not comprise the Fc fragment or comprises any kind, preferably human IgG1 Fc or human IgG 4 Fc, come from people source unmodified or the Fc fragment of modifying by the above-mentioned sudden change of mentioning.

Therefore, the present invention also comprises antibody, monoclonal antibody preferably, be characterised in that described antibodies OX40L, comprise people source Fc fragment, and the human Fc gamma receptor on debond people complement factor C1q and/or the NK cell is characterised in that to be people IgG4 type or human IgG1 or human IgG 4 for being modified by the above-mentioned sudden change of mentioning.

Therefore, the present invention also comprises antibody, and preferably monoclonal antibody is characterised in that described antibody combines with the OX40L (in western blotting) of OX40L and sex change with the antibody concentration of 100ng.This antibodies is to the OX40L polypeptide epitope identical with monoclonal antibody LC.001 bonded epi-position.Described antibody does not comprise the Fc fragment, perhaps comprises the Fc fragment of any kind, preferably human IgG1 or human IgG 4, wild-type or modify by the above-mentioned sudden change of mentioning.

Have novelty and inventive features according to antibody of the present invention, it uses patient at the Antybody therapy of OX40L to needs, be in particular the patient who tormented by inflammatory disease, the patient who tormented by the GvHD in rheumatoid arthritis, allergic asthma and the transplanting brings benefit (also referring to Sugamura, K., Deng., Nat.Rev.Immunol.4 (2004) 420-431).

Another embodiment of the invention is a kind of nucleic acid molecule, and it is encoded according to antibody molecule of the present invention, its variable chains or CDR structural domain.

In a preferred embodiment of the invention, described antibody is Fab, F (ab ') ₂Or single-chain fragment.

Another embodiment of the invention is a kind of carrier, and it comprises according to nucleic acid molecule of the present invention.

Another embodiment of the invention is a kind of host cell, and it comprises according to carrier of the present invention.

Another embodiment of the invention is the method for a kind of preparation according to antibody of the present invention, and described method is included in and allows to cultivate according to host cell of the present invention under the described antibody molecule synthetic condition, and obtains described antibody molecule from described culture.

Another embodiment of the invention is a kind of composition, preferably according to the medicine or the diagnosis composition of antibody of the present invention.

Another embodiment of the invention is a kind of pharmaceutical composition, and it comprises according to antibody of the present invention and at least a pharmaceutical excipient.

Another embodiment of the invention is a kind of method for the treatment of the patient who needs treatment, it is characterized in that to described patient's administering therapeutic significant quantity according to antibody of the present invention.

Another embodiment of the invention is the application that is used for the treatment of according to antibody of the present invention, and it is preferably used for treating inflammatory disease, is used in particular for treating and/or preventing rheumatoid arthritis, asthma and GvHD (graft versus host disease (GVH disease)).

Another embodiment of the invention is the application that is used to prepare medicine according to antibody of the present invention, and described medicine is used to prevent and/or treat inflammatory disease, is preferably used for treating rheumatoid arthritis, asthma and GvHD.

Another embodiment of the invention is a kind of diagnostic kit, and it comprises according to antibody of the present invention, according to nucleic acid molecule of the present invention, according to carrier of the present invention or according to host cell of the present invention.

Detailed Description Of The Invention

Term " OX40L " is meant the II type membranin that belongs to the TNF-ligand family.Other called after ACT-4 acceptor, CD134L, gp34 or TNF4_ people.It has the molecular weight of 34 KDa, and is stored among the SwissProt, and registration number is P23510.

Term " OX40 " is meant and OX40L bonded acceptor.It is the I type membranin that belongs to the TNF receptor family.Other called after ACT-4, OX40L acceptor, CD134 antigen, ACT35 antigen, TNR4 people.It has the molecular weight of 50kDa, and is stored among the SwissProt, and registration number is P43489.

Term " antibody " comprises various forms of antibody, be preferably monoclonal antibody, it includes but not limited to the antibody (variant or sudden change antibody) of complete antibody, antibody fragment, people's antibody, chimeric antibody, humanized antibody and heredity processing, as long as keep according to characteristic feature of the present invention.Particularly preferably be people or humanized monoclonal antibody, particularly recombinant human antibody.

When being used for this paper, term " monoclonal antibody " or " monoclonal antibody combination " are meant the preparation of the antibody molecule of single amino acid composition.

Term " chimeric antibody " is meant a kind of monoclonal antibody, and it comprises the variable region from a kind of source or species, that is, land and derive from different sources or at least a portion constant region of species, this antibody prepares by recombinant DNA technology usually.Preferably include the chimeric antibody of mouse variable region and human constant region." chimeric antibody " of other preferred form that the present invention is included be, wherein constant region is modified or changed from the constant region of initial antibodies, to produce according to characteristic of the present invention, particularly about C1q in conjunction with and/or those of Fc acceptor (FcR) binding characteristic.Such chimeric antibody also is called " kind-conversion antibody ".Chimeric antibody is the expression product of immunoglobulin gene, and described immunoglobulin gene comprises the dna fragmentation of the immune globulin variable region of encoding and the dna fragmentation of coding constant region for immunoglobulin.The method that produces chimeric antibody comprises conventional recombinant DNA well known in the art and gene transfection technology.Referring to, for example, Morrison, S.L., etc., Proc.Natl.Acad.Sci.USA 81 (1984) 6851-6855; US Patent Nos.5,202,238 and 5,204,244.

Term " humanized antibody " is meant such antibody, compares with the CDR of parent's immunoglobulin (Ig), and wherein framework or " complementary determining region " (CDR) have been modified to and comprise not homospecific immunoglobulin (Ig) CDR.In a preferred embodiment, mouse CDR is transplanted to the framework region of people's antibody, described to prepare " humanized antibody ".Referring to, for example, Riechmann, L., etc., Nature332 (1988) 323-327; And Neuberger, M.S., etc., Nature 314 (1985) 268-270.Those of the corresponding representative identification of particularly preferred CDRs antigen sequence mentioned above, described antigen is at chimeric and bifunctional antibody." humanized antibody " of other form that the present invention includes is that wherein constant region is modified in addition or changed from the constant region of initial antibodies, to produce according to characteristic of the present invention, particularly about C1q in conjunction with and/or those antibody of Fc acceptor (FcR) bonded characteristic.

Term " people's antibody " when being used for this paper, is intended to comprise to have the antibody that the ethnic group of coming from is the variable and constant region of immunoglobulin sequences.(van Dijk, M.A. and van de Winkel, J.G., Curr.Opin.Chem.Biol.5 (2001) 368-374) that people's antibody is known in the art.People's antibody can also produce in transgenic animal (for example, mouse), and under the situation that lacks the endogenous immunoglobulin generation, in case immunity, described animal can produce the whole all the components or the selected part of people's antibody.When antigenic stimulation, with ethnic group be the immunoglobulin gene array transfer to will cause in such germ line mutation mouse people's production of antibodies (referring to, for example, Jakobovits, A., etc., Proc.Natl.Acad.Sci.USA 90 (1993) 2551-2555; Jakobovits, A., etc., Nature 362 (1993) 255-258; Bruggemann, M., etc., Year Immunol.7 (1993) 33-40).People's antibody can also produce (Hoogenboom, H.R., and Winter, G, J.Mol.Biol.227 (1992) 381-388 in phage display library; Marks, J.D., etc., J.Mol.Biol.222 (1991) 581-597).Cole etc. and Boemer etc. technology can also be used to prepare human monoclonal antibodies (Cole etc., Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); And Boemer, P., etc., J.Immunol.147 (1991) 86-95).Owing to mention according to chimeric and humanized antibody of the present invention, term " people's antibody ", when being used for this paper, also comprise such antibody, it is modified to produce according to characteristic of the present invention in constant region, particularly about C1q combination and/or FcR bonded characteristic, for example, by " kind conversion ", promptly, segmental change of Fc or sudden change (for example, from IgG1 to IgG4 and/or IgG1/IgG4 sudden change).In addition, the present invention includes the monoclonal human antibody at OX40L, it is in conjunction with C1q and/or FcR.Such people's antibody is characterised in that relative mouse OX40L is for the highly selective of human OX 40 L (combining low＞30 times with mouse OX40L binding ratio) with human OX 40 L, and when reaching 500nM concentration, do not show non-specific binding to TNF α or CD40L.Such antibody is effective to produce not and C1q and/or FcR bonded antibody.

Term " recombinant human antibody ", when being used for this paper, be intended to comprise by recombinant methods, expression, generation or isolating everyone antibody, such as from host cell such as NS0 or Chinese hamster ovary celI or from the genetically modified animal of human immunoglobulin gene (for example mouse) isolated antibody, perhaps use the antibody that transfection is expressed to the recombinant expression vector in the host cell.Such recombinant antibodies has the variable and constant region of rearrangement form.To carry out somatic hypermutation in the body according to recombinant human antibody of the present invention.Therefore, although derive from and be that VH is relevant with the VL sequence with ethnic group, the aminoacid sequence in described recombinant antibodies VH and VL district is that people's antibody kind is a naturally occurring sequence in all the components in vivo.

When being used for this paper, antibody and every pair of light chain of antigen bonded and heavy chain are participated in " variable region " (variable region of light chain (VL), variable region of heavy chain (VH)) expression directly.The structural domain of variable people's light chain and heavy chain has identical conventional structure, and each structural domain comprises 4 frameworks (FR) districts, and its sequence is generally guarded, and (or complementary determining region CDRs) connects by 3 " hypervariable regions ".Framework region adopts β-sheet conformation, and CDRs can form the ring that connects β-laminated structure.CDRs in every chain is had by framework region in their three-D space structure, and forms antigen binding site with the CDRs of other chain.Heavy chain of antibody and light chain CDR3 district play a part particularly important in the binding specificity/avidity according to antibody of the present invention, and therefore provide another object of the present invention.

When being used for this paper, term " hypervariable region " or " antigen-bound fraction of antibody " are meant is responsible for Antigen-Binding antibody amino-acid residue.Described hypervariable region comprises the amino-acid residue of " complementary determining region " or " CDRs "." framework " or " FR " district is those variable domains zones except that the hypervariable region residue of this paper definition.Therefore, the light chain of antibody and heavy chain are held from N-to C-and are comprised structural domain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.CDRs on every chain is separated by such framework amino acid.Especially, the CDR3 of heavy chain is in conjunction with the maximum zone of contribution to antigen.According to Kabat etc., Sequences of Proteins of Immunological Interest, the 5th edition., PublicHealth Service, National Institutes of Health, Bethesda, the standard definition of MD (1991) has been determined CDR and FR zone.

When being used for this paper, term " nucleic acid or nucleic acid molecule " is intended to comprise dna molecular and RNA molecule.Nucleic acid molecule can be strand or double-stranded, but double-stranded DNA preferably.

When it and another nucleotide sequence were in the function association, nucleic acid was " operability connection ".For example, if it is expressed as participating in albumen before the polypeptide excretory, so, the DNA of presequence or secretion property leader sequence is operably connected on the DNA of polypeptide; If it has influenced transcribing of described sequence, so promotor or enhanser are operably connected on the encoding sequence; Perhaps, if it is positioned at the position that promotes translation, so ribosome bind site is operably connected on the encoding sequence.Usually, " can be operatively connected " means, and the dna sequence dna that connect is adjacent, and, in the situation of secretion property leader sequence, be adjacent and in reading frame.Yet it is adjacent that enhanser needs not to be.Connection is by realizing in the connection of restriction site easily.Synthetic oligonucleotide adapter or joint according to conventional convention, are used in if there is no such site.

When being used for this paper, expression " cell ", " clone " and " cell culture " are used interchangeably, and all such titles all comprise the offspring.Therefore, word " transformant " and " cell transformed " comprise elementary subject cell and come from its culture, and do not consider the number of times that shifts.Be also to be understood that all the progeny can not be accurately identical owing to have a mind to or sudden change unintentionally on the DNA content.Comprise the identical functions or the bioactive variant offspring that have and in original transformant, screened.Under the situation of using different titles, it will be clearly from context.

" constant domain " do not participated in antibody directly and combined with antigenic, but shows different effector functions.The aminoacid sequence that depends on their CH is divided into following kind: IgA, IgD, IgE with antibody or immunoglobulin (Ig), IgG and IgM, and several in these can further be divided into subclass (isotype), for example, IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2.The CH corresponding with different types of immunoglobulin (Ig) is called α respectively, ε, γ and μ.Be preferably the IgG type according to antibody of the present invention.

Comprise the Fc fragment according to antibody of the present invention, preferably come from the Fc fragment in people source, and all other fragments of human constant region preferably.The Fc fragment of antibody is participated in complement activation, C1q combination, C3 activation and Fc receptors bind directly.Although antibody depends on certain conditions to the influence of complement system,, cause with combining of C1q by the binding site that defines in the Fc fragment.Such binding site is known in the art, and for example by Lukas, T.J., etc., J.Immunol.127 (1981) 2555-2560; Brunhouse, R., and Cebra, J.J., Mol.Immunol.16 (1979) 907-917; Burton, D.R., etc., Nature 288 (1980) 338-344; Thommesen, J.E., etc., Mol.Immunol.37 (2000) 995-1004; Idusogie, E.E., etc., J.Immunol.164 (2000) 4178-4184; Hezareh, M., etc., J.Virol.75 (2001) 12161-12168; Morgan, A., etc., Immunology 86 (1995) 319-324; Be described with EP 0 307 434.Such binding site such as L234, L235, D270, N297, E318, K320, K322, P331 and P329 (be numbered according to Kabat EU index, vide infra).The antibody of subclass IgG1, IgG2 and IgG3 shows complement activation, C1q combination and C3 activation usually, and IgG4 activating complement system does not combine and do not activate C3 with C1q.When being used for this paper, the such Fc fragment of expression that term " derives from the people's complement factor C1q on people source and the debond NK cell and/or the Fc fragment of human Fc gamma receptor ", promptly, it is people's antibody Fc fragment of subclass IgG4, perhaps people's antibody Fc fragment of subclass IgG1, IgG2 or IgG3, it is modified by this way, promptly can not detect C1q combination, C3 activation and/or the FcR combination of hereinafter definition." the Fc fragment of antibody " is the known term of technician, and the papoid that is based on antibody decomposes and defines.Preferably, described Fc fragment is a people Fc fragment, and particularly preferably from human IgG 4 subclass, preferably in hinge area sudden change (for example, S228P and/or L235E), or from the Fc fragment of the sudden change of human IgG1's subclass.It most preferably is the Fc fragment that comprises such CH, promptly, described CH is selected from SEQ ID NO:14 and 15 zones that show, perhaps be included in SEQ ID NO:58,59,60, have the SEQ ID NO:14 of sudden change L234A and L235A, perhaps have among the SEQ ID NO:15 of sudden change S228P or sudden change S228P and L235E.

The present invention relates to combine with OX40L and not with the antibody of complement factor C1q and/or Fc receptors bind.In a preferred embodiment of the invention, these antibody do not cause cytotoxicity (CDC) that depends on complement and/or the cytotoxicity (ADCC) that depends on the cell of antibody.Preferably, this antibody is characterised in that it comprises the Fc fragment that derives from the people source in conjunction with OX40L, and debond complement factor C1q.More preferably, this antibody is the people's or humanized monoclonal antibody.

Effector functions by the mediation of the Fc fragment in described antibody Fc zone is meant that antibody combines the effector functions (these functions comprise the activation of Fc acceptor to complement cascade and/or cell activation) that the back is moved with antigen.

The function of complement cascade can detect by CH50 and assess.To join with the sheep red blood cell (SRBC) of anti--erythrocyte antibody (EA) (EA) sensitization and detect in the serum, cause the classical pathway of globulolysis with activation.The required serum volume of cracking 50% red corpuscle is determined CH50 unit.AP-CH50 measures alternative and approach in latter stage.Except using rabbit erythrocyte, flow process is similar.When adding detection serum, alternative route is activated.

C1q and two kinds of serine proteases, C1r and C1s form mixture C1, are first compositions of cytotoxicity (CDC) approach that depends on complement.For the activating complement cascade effect, C1q is in conjunction with being attached to bimolecular at least IgG1 on the antigenicity target spot or the IgM (Ward, E.S., and Ghetie, V., Ther.Immunol.2 (1995) 77-94) of a part.Burton, D.R. has described (Mol.Immunol.22 (1985) 161-206) and has comprised that the heavy chain zone of amino-acid residue 318-337 participates in complement fixation(CF).Duncan, A.R., and Winter, G. (Nature 332 (1988) 738-740) uses site-directed mutagenesis, has reported that Glu318, Lys320 and Lys322 form and the binding site of C1q.Glu318, Lys320 and Lys322 residue are suppressing the cracked ability of complement-mediated with effect during C1q combines by the synthetic small peptide that contains these residues and are confirming.

Term " depends on the cytotoxicity (CDC) of complement " and is meant, in the presence of complement, according to the cracking of antibody of the present invention to the human endothelial cell of expression OX40L.Preferably, in the presence of complement, by using the human endothelial cell of expressing OX40L according to antibody treatment of the present invention, and measure CDC.Preferably that cell is fluorescein-labelled with calcium.If in the concentration of 30 μ g/ml, the cracking of described antibody induction 20% or more target cell can be found CDC so.The inventor finds that for the characteristic according to antibody of the present invention, it is important reducing with combining of complement factor C1q in ELISA detects.In such detection,, in flat board, add the people C1q or the human serum of purifying in theory with the dull and stereotyped antibody sandwich of ELISA with the different concns scope.By the antibody at C1q, then the conjugate of peroxidase-mark detects the combination of C1q.Bonded detect (maximum combined Bmax) as for peroxidase substrate ABTS  (2,2 '-azido--two-[3-ethyl benzo thiazole phenanthroline-6-sulphonate (6)] detect in the optical density (OD) (OD405) of 405nm.Therefore, the present invention relates to a kind of antibody, it is characterized in that, the debond of described antibody and complement factor C1q is meant such ELISA detection assay, wherein in the antibody concentration of 10 μ g/ml, C1q with according to the maximum combined (Bmax) of antibody of the present invention be with the observed Bmax of antibody LC.001 20% or lower, preferably, 10% or lower.

Further preferably, in detecting, ELISA shows the activation that complement factor C3 is reduced according to antibody of the present invention.Described detection is used with the identical mode of C1q detection and is carried out.In such detection, in theory, with the dull and stereotyped antibody sandwich of ELISA, to wherein adding human serum with the different concns scope.By the antibody at C3, then the conjugate of peroxidase-mark detects the combination of C3.Bonded detects (maximum combined Bmax) as detecting in the optical density (OD) (OD405) of 405nm for peroxidase substrate ABTS .Therefore, the present invention relates to a kind of antibody, it is characterized in that, the debond of described antibody and complement factor C3 is meant such ELISA detection assay, wherein in the antibody concentration of 10 μ g/ml, the maximum combined of C3 and described antibody (Bmax) be antibody LC.001 Bmax 10%, preferably, 5% or lower.

It is the function that is mediated by the Fc receptors bind that term " depends on the cytotoxicity (ADCC) of the cell of antibody ", and is meant in the presence of the effector cell, according to the cracking of antibody of the present invention to the target cell of expression OX40L.Preferably, by when having the effector cell, such as the PBMC (peripheral blood lymphocytes) of fresh separated or from the effector cell of dark yellow tectum (buffy coats) purifying, as monocyte or NK (NK cell) cell, use express OX40L according to antibody treatment of the present invention the class red corpuscle (for example, the K562 cell of express recombinant human OX 40 L) preparation, and measure ADCC.Target cell is used ⁵¹The Cr mark, and subsequently with the antibody incubation.The cell of mark with effector cell's incubation, and is analyzed that supernatant liquid discharges ⁵¹Cr.Contrast comprises with effector cell and need not described antibody incubation target spot endotheliocyte.By measuring them and express cell such as recombinant expressed Fc γ RI and/or the cell of Fc γ RIIA or the combining of NK cell (expressing Fc γ RIIIA basically) of Fc γ acceptor, and the ability of the initial step of research antibody induction regulation and control ADCC.Preferably measure with the NK cell on the combining of Fc γ R.

Fc receptors bind effector functions is subjected to the Fc district of antibody and the interactional regulation and control of Fc acceptor (FcRs), and the Fc acceptor is a distinctive cell surface receptor on the hematopoietic cell.Fc acceptor contactin, and shown that its mediation removes the pathogenic agent of antibody-Bao quilt by the phagolysis of immunocomplex, with the cytotoxicity (ADCC) of cell regulate and control by depending on antibody and will be with the cracking of red corpuscle and various other cell object (for example, tumour cell) of corresponding antibodies bag quilt.Van de Winkel, J.G, and Anderson, C.L., J.Leukoc.Biol.49 (1991) 511-524).FcRs by them to the specificity of immunoglobulin (Ig) isotype and define; The Fc acceptor of IgG antibody is called Fc γ R, and IgE is called Fc ε R, and IgA is called Fc α R, or the like.For example, the Fc receptors bind is at Ravetch, J.V., and Kinet, J.P., Annu.Rev.Immunol.9 (1991) 457-492; Capel, P.J., etc., Immunomethods 4 (1994) 25-34; De Haas, M., etc., J.Lab.Clin.Med.126 (1995) 330-341; And Gessner, J.E., etc., describe among Ann.Hematol.76 (1998) 231-248.

The crosslinked effector functions that causes broad variety for the acceptor (Fc γ R) of IgG antibody Fc structural domain comprises phagolysis, depends on cytotoxicity and the release of inflammatory regulatory factor and the regulation and control that immunocomplex is removed and antibody produces of the cell of antibody.In the people, distinguished 3 kinds of Fc γ R, they are:

-Fc γ RI (CD64), and expresses on scavenger cell, monocyte, neutrophilic granulocyte and eosinophilic granulocyte in conjunction with monomer I gG with high-affinity.At least a modification of E233-G236, P238, D265, N297, A327 and P329 reduces and the combining of Fc γ RI among the IgG.At the IgG2 of position 233-236 residue, substitution IgG1 and IgG4 will reduce 10 with combining of Fc γ RI ³-doubly, and the erythrocytic person monocytic cell who has eliminated antagonist-sensitization replys (Armour, K.L. wait ..Eur.J.Immunol.29 (1999) 2613-2624).

-Fc γ RII (CD32) with in by the time low-affinity and express widely in conjunction with compound IgG.These acceptors can be divided into 2 kinds of main types, Fc γ RIIA and Fc γ RIIB.As if the cell that Fc γ RIIA kills and wounds in many participations (for example, scavenger cell, monocyte, neutrophilic granulocyte) is gone up and is found, and can activate the process of killing and wounding.As if Fc γ RIIB works in process of inhibition, and is finding on B cell, the scavenger cell and on mastocyte and the eosinophilic granulocyte.On the B cell, seem that it exercises other immunoglobulin (Ig) generation of inhibition and isotype is changed as the function to the conversion of IgE kind.On scavenger cell, Fc γ RIIB effect suppresses the phagolysis by Fc γ RIIA regulation and control.On eosinophilic granulocyte and mastocyte, the b form can by IgE and its independently receptors bind help to suppress the activation of these cells.For example, for E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, at least a IgG of R292 and K414 sudden change is found to have reduced and the combining of Fc γ RIIA.

-Fc γ RIII (CD16) with in by the time low-affinity and exist in conjunction with IgG with two types.

Fc γ RIIIA finds on NK cell, scavenger cell, eosinophilic granulocyte and some monocytes and T cell, and regulation and control ADCC.Fc γ RIIIB highly expresses on neutrophilic granulocyte.

For example, for E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, the sudden change that K338 and D376 are at least a is found to have reduced and the combining of Fc γ RIIIA.

At Shields, R.L. waits among .JBC 276 (2001) 6591-6604 and describes for the bonded method of the location in Fc acceptor binding site, mutational site mentioned above on the human IgG1 and mensuration and Fc γ RI and Fc γ RIIA.

When being used for this paper, term " Fc acceptor " is meant activated receptor, it is characterized in that existing with the mutually associating kytoplasm of described acceptor (cytoplasmatic) ITAM sequence (referring to, for example, Ravetch, J.V., and Bolland, S., Annu.Rev.Immunol.19 (2001) 275-290).Such acceptor is Fc γ RI, Fc γ RIIA and Fc γ RIIIA.Preferably show acceptor according to acceptor of the present invention, preferably with combining that Fc γ IIIA reduces with Fc γ.Preferably, term " does not combine with Fc γ R " and means, and in the antibody concentration of 10 μ g/ml, is the bonded 10% found for antibody LC.001 or still less according to antibody of the present invention and combining of NK cell.

Although IgG4 shows the FcR combination of minimizing, the antibody of other IgG subclass shows strong combination.Yet, if change Pro238, Asp265, Asp270, Asn297 (losing the Fc carbohydrate), Pro329 and 234,235,236 and 237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435 also provide the FcR bonded residue that reduces (Shields, R.L., wait .J.Biol.Chem.276 (2001) 6591-6604; Lund, J. waits J.9 (1995) 115-119 of .FASEB; Morgan, A., etc., Immunology 86 (1995) 319-324; With EP 0 307 434).Preferably, the antibody according to IgG1 of the present invention or IgG2 subclass comprises sudden change PVA236, GLPSS331 and/or L234A/L235A.Antibody according to IgG4 subclass of the present invention preferably includes sudden change L235E.Other preferred IgG4 sudden change is S228P or L235E and S228P (referring to table 1).

When being used for this paper, term " combine " and mean at BIAcore and detect with OX40L (PharmaciaBiosensor AB, Uppsala, Sweden) in the combining of antibody and human OX 40 L.For further confirmation, in wrapping by the ELISA of microtitration flat board, the OX40L that uses purifying determines that with combining also of OX40L perhaps determine, wherein the antibodies of direct or indirect mark is to the K562 cell of expression OX40L in FACS-detects.

In BIAcore detected, antibodies was to the surface, and the combination of OX40L is resonated by surperficial cytogene, and (Surface Plasmon Resonance SPR) measures.Bonded avidity is by term ka (the associating rate constant of antibody and antigen), kd (dissociated rate constant) and K _D(kd/ka) define.Show 10 according to antibody of the present invention ^-8Or littler, preferred about 10 ^-12-10 ^-9The K of M _D(referring to example).Therefore, the present invention relates to antibody mentioned above, wherein said antibody in BIAcore detects with less than 10 ^-8The K of M _DValue combines with OX40L, preferably K wherein _DScope be 10 ^-12-10 ^-9M.

The OX40L-specificity in conjunction with ELISA in, by to the microtitration flat board, and the anti-human IgG of puting together with HRP-and conventional ELISA step detect combining of antibody and OX40L with OX40L bag.EC in this detection ₅₀Value is preferably in the 3nM-8nM scope.

When being used for this paper, term " suppress OX40 combine with OX40L " is meant combining of antibody of the present invention and human OX 40 L, suppresses the OX40/OX40L interaction thus, and suppresses OX40L inductive signal thus and conduct.

Antibody of the present invention suppresses the interaction of hOX40L/OX40, preferably

I) on the external level shown in detecting by ELISA, by at the bag of (solid phase) 0.5 μ g/ml biotinylation OX40L by the interaction that the antibody of concentration blocks biotinylated, fixed OX40L and solvable OX40, have the IC50 value of 1nM-4nM scope,

Ii) on the external level shown in detecting by Biacore,, have the IC50 value of 1nM-10nM scope by the interaction that the antibody of 0.78-100nM antibody concentration blocks fixed OX40 and soluble OX40L (10nM is preferably hOX40L-His),

Iii) on the cell levels shown in being detected by FACS-, (concentration is 2 * 10 to the K562 cell (K562_OX40L) of wherein said antibody inhibition expression OX40L ⁵Individual cell/sample) with the interaction of OX40, have the IC50 value of 4nM-20nM scope,

Iv) detect by the conduction of OX40-signal, wherein said antibody retardance is transmitted to every duplicate samples 3 * 10 by K562_OX40L inductive OX40 signal ⁴In the HeLa-cell of individual expression OX40, the retardance that this causes the NF kB activation has the IC50 value of 1nM-5nM scope,

V) detect, wherein by being 1.5 * 10 in concentration by the T cell activation ⁵The K562_OX40L of individual cell/sample and concentration are the PHA of 0.75 μ g/ml, and described antibody blocks OX40L inductive t cell activation, have the IC50 value of 1nM-10nM scope, and/or

Vi) detect by the T cell activation, wherein by in concentration being the activatory B cell or the dendritic cell (Tetanus detection) of the antibody of 10 μ g/ml, described antibody retardance OX40L inductive t cell activation obtains the inhibition of 40%-60%.

Preferred such antibody, that is, it shows inhibition in ELISA detects, and is suppressed by the interaction of concentration retardance fixed OX40L and soluble OX40 by the bag at 0.5 μ g/ml OX40L, has the IC50 value of 1nM-4nM scope.

Therefore, another preferred embodiment of the present invention relates to a kind of antibody, it is characterized in that described antibody suppresses OX40/OX40L thus and interacts, and suppresses the conduction of OX40L inductive signal thus.

More preferably, reach TNF α or the CD40L of 500nM in concentration, do not show non-specific binding with TNF α and CD40L according to antibody of the present invention.

More preferably, compare with human OX 40 L, according to antibody of the present invention show be lower than at least 30 times with the combining of mouse OX40L.

More preferably, concentration be 10 μ g/ml do not induce the downward modulation that OX40L is expressed according to antibody of the present invention on the HUVEC cell.

In a preferred embodiment, antibody of the present invention is characterised in that they comprise the combination of variable domains, and described combination is independently selected from the group of being made up of following combination

A) by the light chain variable structural domain of the antibody LC.001 of aminoacid sequence SEQ ID NO:1 definition with by the weight chain variable structural domain of the antibody LC.001 of SEQ ID NO:2 definition;

B) by the light chain variable structural domain of the antibody LC.005 of aminoacid sequence SEQ ID NO:3 definition with by the weight chain variable structural domain of the antibody LC.005 of SEQ ID NO:4 definition;

C) by the light chain variable structural domain of the antibody LC.010 of aminoacid sequence SEQ ID NO:5 definition with by the weight chain variable structural domain of the antibody LC.010 of SEQ ID NO:6 definition;

D) by the light chain variable structural domain of the antibody LC.029 of aminoacid sequence SEQ ID NO:7 definition with by the weight chain variable structural domain of the antibody LC.029 of SEQ ID NO:8 definition;

E) by the light chain variable structural domain of the antibody LC.019 of aminoacid sequence SEQ ID NO:9 definition with by the weight chain variable structural domain of the antibody LC.019 of SEQ ID NO:10 definition;

F) by the light chain variable structural domain of the antibody LC.033 of aminoacid sequence SEQ ID NO:11 or 16 definition with by the weight chain variable structural domain of the antibody LC.033 of SEQ ID NO:12 definition;

I) by the light chain variable structural domain of aminoacid sequence SEQ ID NO:1 definition and the weight chain variable structural domain that defines by SEQ IDNO:20.

In a further preferred embodiment, antibody of the present invention is characterised in that they comprise the constant region that is independently selected from by the following group of forming

K) light chain/κ chain that defines by sequence SEQ ID NO:13;

L) has the heavy chain/γ chain of IgG1 isotype SEQ ID NO:14 of the sudden change of one or more L234A of being selected from and L235A, PVA236 or GLPSS331;

M) heavy chain of IgG4 isotype SEQ ID NO:15/γ chain;

N) has IgG4 isotype SEQ IDNO:15 heavy chain/γ chain of sudden change S228P or sudden change S228P and L235E;

O) be included in SEQ ID NO:61, the light chain constant chain in 65 or 69;

P) be included in SEQ ID NO:58, the heavy chain constant chain in 59,60,62,63,64,66,67 or 68.

More preferably be the combination of every kind of variable antibody structure territory a)-i) with γ chain l), m), n) or p), and preferably with κ chain k) or o) all combinations.Particularly preferably be following antibody: such antibody, it comprises antibody LC.001, LC.005, LC.010, LC.019, LC.029, LC.033, LC.059, the variable chains of LC.060 or LC.063 (wherein every kind of antibody has the κ chain of sequence SEQ IDNO:13 definition), or be included in SEQ ID NO:61, the light chain constant chain in 65 or 69 and have heavy chain/γ chain of the IgG1 isotype SEQ ID NO:14 of sudden change L234A and L235A, or be included in SEQ ID NO:59, the heavy chain constant chain in 63 or 67; Such antibody, it comprises antibody LC.001, LC.005, LC.010, LC.019, LC.029, LC.033, LC.059, the variable chains of LC.060 or LC.063 (wherein every kind of antibody has the κ chain that sequence SEQ ID NO:13 defines), or be included in SEQ ID NO:59, heavy chain/γ chain of heavy chain constant chain in 63 or 67 and IgG4 isotype SEQ IDNO:15, or be included in SEQ ID NO:60, and the heavy chain constant chain in 64 or 68, all these 3 kinds all do not have the S228P sudden change; Such antibody, it comprises antibody LC.001, LC.005, LC.010, LC.019, LC.029, LC.033, LC.059, the variable chains of LC.060 or LC.063 (wherein every kind of antibody has the κ chain of sequence SEQ ID NO:13 definition), or be included in SEQ ID NO:61, the light chain constant chain in 65 or 69 and have heavy chain/γ chain of the IgG4 isotype SEQ ID NO:15 of sudden change S228P, or be included in SEQ ID NO:60, the heavy chain constant chain in 64 or 68.

Preferably, described antibody comprises light chain variable CDR and the SEQ ID NO:2 of aminoacid sequence SEQ ID NO:1,17 or 20 weight chain variable CDR, or the weight chain variable CDR of the light chain variable CDR of aminoacid sequence SEQ ID NO:18 and SEQ ID NO:19.

Preferred antibody is characterised in that described antibody is human IgG 4 subclass or other people's subclass (preferred IgG1), and it comprises that at least one causes debond complement factor C1q and/or loses FCR bonded amino acid mutation.For example, so preferred variant antibody comprises the aminoacid sequence SEQ ID NO:14 with sudden change L234A and L235A, perhaps has or the SEQID NO:15 of the S228P that do not suddenly change.

According to preferred antibody of the present invention is to be defined as IgG1v1 (PVA-236; Be expressed as the GLPSS331 of E233P; L234V; L235A; △ G236; A327G; A330S; P331S), IgG1v2 (L234A; L235A) and IgG4v1 (S228P; L235E) and the antibody of IgG4x (S228P).

Budapest pact according to the microbial preservation international recognition that is used for the patented procedure purpose, according to hybridoma cell line hu-Mab＜hOX40L of the present invention〉LC.001 on July 27th, 2004 be deposited in Germany Germany microbial preservation center (Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH) (DSMZ), preserving number DSM ACC2672.

Budapest pact according to the microbial preservation international recognition that is used for the patented procedure purpose, according to hybridoma cell line hu-Mab＜hOX40L of the present invention〉LC.005 (DSM ACC 2685), hu-Mab＜hOX40L〉LC.010 (DSM ACC 2686), hu-Mab＜hOX40L〉LC.019, hu-Mab＜hOX40L〉LC.029 (DSM ACC 2688) and hu-Mab＜hOX40L LC.033 (DSM ACC 2689) on September 2nd, 2004 be deposited in Germany Germany microbial preservation center (DSMZ).

The antibody that can obtain from described clone is the preferred embodiment of the invention, and especially effectively as intermediate material, be used to produce debond complement factor C1q and/or debond human Fc gamma receptor according to antibody of the present invention.

Other preferred embodiment of the present invention is isolating anti-OX 40 L antibodies, and it is in conjunction with OX40L, and the monoclonal antibody LC.005 that produces with hybridoma cell line by institute's preservation, LC.010 or the LC.029 OX40L-epi-position combination that also bonded is identical with it.

Another embodiment of the invention is the method that is used to produce at the antibody of OX40L, and described antibody debond people complement factor C1q and/or human Fc gamma receptor at OX40L is characterized in that, coding is with less than 10 ^-8The KD value of M is modified by this way in conjunction with the nucleotide sequence of the heavy chain of the antibody of OX40L, so that complement factor C1q and/or human Fc gamma receptor on the antibody debond NK cell of described modification, the nucleic acid of described modification and the nucleic acid of encoding said antibody light chain are inserted in the expression vector, described carrier is inserted in protokaryon or the eukaryotic host cell, and coded albumen is expressed and is reclaimed from host cell or supernatant liquid.

Another embodiment of the invention is the method that is used to produce according to the antibody of debond complement factor C1q of the present invention and/or debond human Fc gamma receptor, be characterised in that, can modify by " kind conversion " from the antibody that one of described clone obtains, that is, change or sudden change preferably are defined as IgG1v1 (PVA-236; Be expressed as the GLPSS331 of E233P; L234V; L235A; Δ G236; A327G; A330S; P331S), IgG1v2 (L234A; L235A) and IgG4v1 (S228P; L235E) and the Fc fragment of IgG4x (S228P) (for example, from IgG1 to IgG4 and/or IgG1/IgG4 sudden change).

In a further preferred embodiment, these antibody also comprise and being selected from by Fab, F (ab ') ₂The antibody fragment of the group of forming with single-chain fragment.

Therefore, " variant " anti-OX 40 L antibodies is meant a kind of like this molecule at this paper, and it is by adding in the parental antibody sequence, delete and/or replacing one or more amino-acid residues and different with " parent " anti-OX 40 L antibodies aminoacid sequence in aminoacid sequence.In preferred embodiments, described variant is included in one or more constant regions of parental antibody or variable region, preferably at one or more aminoacid replacement of constant region.For example, described variant can be included at least one of one or more variable regions of parental antibody, for example, from about 1 to about 10, and preferably from about 2 to about 5 replacements.Usually, described variant has such aminoacid sequence, that is, itself and parental antibody is constant and/or the variable domains sequence has at least 90% aminoacid sequence homogeny, preferred at least 95% and more preferably at least 99% homogeny.

The present invention includes the method for initial aminoacid sequence be selected from the parental antibody heavy chain CDR of the group of forming by SEQ ID NOs:21-38 and/or be selected from the light chain of antibody CDR of the group of forming by SEQ ID NOs:39-57 of modifying, it is characterized in that, the nucleic acid of the described initial aminoacid sequence of coding is provided, modify described nucleic acid, in this, 1 amino acid is modified among the heavy chain CDR1,1-2 amino acid is modified among the heavy chain CDR2,1-2 amino acid is modified among the heavy chain CDR3,1-3 amino acid is modified among the light chain CDR1,1-3 amino acid is modified among the light chain CDR2, and/or 1-3 amino acid is modified among the light chain CDR3, in antibody structure, express the cdr amino acid sequence of described modification, measure described antibody whether with less than 10 ^-8The K of M _DIn conjunction with OX40L, and if described antibody with less than 10 ^-8The K of M _DIn conjunction with OX40L, select the CDR of described modification.Preferably, such modification is that conserved sequence is modified.

At the per-cent that this paper is defined as amino-acid residue identical with the parental antibody residue in candidate sequence, if necessary, after described sequence being compared and introduce breach, obtain maximum sequence homogeny percentage ratio about the homogeny of sequence or homology.N-end in the antibody sequence, C-end or inner prolongation, deletion or insertion should not be interpreted as influencing sequence homogeny or homology.Variant keeps the ability in conjunction with human OX 40 L, and preferably has the characteristic of those characteristics that are better than parental antibody.For example, because OX40L is transient expression on B cell, dendritic cell and scavenger cell not only, also at endotheliocyte (Kotani, A., Deng., Immunol.Lett.84 (2002) 1-7), asm cell (=ASM) (Burgess, J.K., J.Allergy Clin.Immunol 113 (2004) 683-689) and microglia (Weinberg, A.D., etc., J.Immunol.162 (1999) 1818-1826) last transient expression, so described variant can have the side effect that reduces in treatment rheumatoid arthritis and asthma process.Antibody at OX40L can cause cell injury with combining of endotheliocyte, ASM and microglia, and cause vascular leakage with combining of endotheliocyte, combine with the ASM cell and to cause lung to damage, cause little gelationus damage with combining of microglia.

In this article, " parent " antibody is by the amino acid sequences encoded a kind of antibody that is used to prepare variant.Preferably, described parental antibody has people's ramework region, and if exist, have people's antibody constant region.For example, described parental antibody can be humanized or people's antibody, is preferably the IgG1 type.

In addition, comprise that according to antibody of the present invention such antibody with " conserved sequence modification ", Nucleotide and amino acid sequence modifications, described modification do not influence or changes the above-mentioned feature of mentioning according to antibody of the present invention.Modification can be introduced by standard technique known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis technology.Conserved amino acid replaces those replacements comprise that amino-acid residue wherein replaces with the amino-acid residue with similar side chain.This area has defined the amino-acid residue family with similar side chain.These families comprise (for example having basic side chain, Methionin, arginine, Histidine), acid side-chain (for example, aspartic acid, L-glutamic acid), uncharged polar side chain (for example, glycine, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine, tryptophane), non-polar sidechain (for example, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met)), β-side-chain branching (for example, Threonine, Xie Ansuan, Isoleucine) and aromatic side chain (for example, tyrosine, phenylalanine, tryptophane, Histidine) amino acid.Therefore, the non-essential amino acid residue of the expectation of people's anti-OX 40 L antibodies can preferably use the another kind of amino-acid residue from same side chain family to replace.

Aminoacid replacement can pass through based on Riechmann, L., etc., Nature 332 (1988) 323-327 and Queen, C., etc., the mutagenesis of the making molecular model that Proc.Natl.Acad.Sci.USA 86 (1989) 10029-10033 describe is carried out.

The present invention also comprises the method that is used to produce antibody, it is characterized in that, to coding with less than 10 ^-8The KD value of M is modified by this way in conjunction with first nucleotide sequence of the heavy chain of the antibody of OX40L, so that the antibody modified does not combine with complement factor C1q and/or human Fc gamma receptor on the NK cell, first nucleic acid of described modification and second nucleic acid of encoding said antibody light chain are inserted in the expression vector, described carrier is inserted in protokaryon or the eukaryotic host cell, under allowing described antibody synthetic condition, cultivates described host cell and from described culture, reclaim described antibody.

The present invention also comprises the method that is used to produce according to the present invention and comprises the segmental antibody of Fc that derives from the people source, described method comprises the following steps: a) with encoding according to first nucleotide sequence of parent's human antibody light chain of the present invention and the described parent human antibody heavy chain's of coding the second dna sequence dna transformed host cell, wherein the Fc fragment is modified, and makes described Fc fragment debond complement factor C1q and/or Fc acceptor; B) express described first and second dna sequence dnas, so that produce described heavy chain of antibody and light chain; And c) from host cell or host cell culture, reclaims described antibody.

The present invention also comprise the antibody mentioned above of encoding nucleic acid molecule, comprise the corresponding carrier of these nucleic acid and be used for the corresponding host cell of these carriers.The present invention includes the method that is used to prepare described antibody, it is included in and cultivates corresponding host cell under the condition that allows synthetic described antibody, and reclaim described antibody from described culture, for example, by the nucleic acid of in protokaryon or eukaryotic host cell, expressing encoding heavy chain and the nucleic acid of light chain of encoding, and reclaim described polypeptide from described cell and realize.

The diagnosis and the treatment application of described antibody have been considered.In a kind of diagnostic use, the invention provides the method for determining that OX40L albumen exists, it comprises that the sample that suspection is contained OX40L is exposed to anti-OX 40 L antibodies, and the combining of definite antibody and sample.OX40L albumen can be inserted in the cytolemma of the cell of expressing OX40L by its membrane spaning domain, and the solubility ectodomain that perhaps can be used as by being discharged in the body fluid as the mechanism that comes off (shedding) or proteolysis discharges exists.For this application, the invention provides a kind of test kit, it comprises described antibody and uses the proteic directions for use of described antibody test OX40L.

Antibody of the present invention be effective to prevent and/or treat in the Mammals, preferably suspect the inflammatory disease suffer from or to suffer among the patient that such disease torments.Such disease comprises anaphylaxis, such as asthma.Other application is the treatment that autoimmune disorders comprises rheumatoid arthritis.

The present invention also is provided for the mammiferous method that treatment suffers the torment of inflammatory disease mentioned above, particularly asthma and rheumatoid arthritis.

Preferably, antibody of the present invention can be used for the treatment of the serious obstinate asthma among the patient, the control of the glucocorticosteroid that described patient's symptom is not fully sucked.Patient colony comprises the adult of the serious obstinate asthma of suffering from insufficient control and teenager (12 years old or age bigger).Antibody will be preferably by subcutaneous delivery every month once or twice.Main terminal point (endpoint) will preferably descend in acute exacerbation.Other terminal point comprises peak flow, wakes up at symptoms of asthma, night in the daytime, quality of life, emergency room follow up a case by regular visits to, do not have the fate of asthma, β-2 agonist to use, steroid reduces or weaken gradually and to the effect of height-responsiveness.

Also preferably will according to antibody of the present invention be used for single therapy or treat and suffer from Rheumatrex or other DMARDs (anti--rheumatoid medicine of disease modification, Disease ModifyingAnti-Rheumatic Drugs) combination in the grownup of serious acute (active) rheumatoid arthritis by the time.It will as per 2 or the subcutaneous injection in 4 weeks use.It will carry out chronic treatment in to the invalid patient of one or more DMARDs.Terminal point comprises sign and the minimizing of symptom and the inhibition of structural impairment progress in the adult patients of suffering from acute rheumatoid arthritis.The improvement of unable prevention, sign and symptom is by ACR standard (ACR20＞60%, ACR50＞35%, ACR70＞15%; Index derives from American College of Rheumatology; Www.rheumatology.com) measure.

Another embodiment of the invention is the application that is used to prepare the medicine for the treatment of these diseases according to antibody of the present invention.

The invention still further relates to antibody defined above and be used for the application of pharmaceutical compositions, and comprise pharmaceutical composition, it comprises according to antibody of the present invention, with medicine effective quantity, randomly with the buffer reagent and/or the adjuvant of the antibody that is used for the compounding pharmaceutical purpose.

The present invention also provides pharmaceutical composition, and it is included in the such antibody in the pharmaceutical carrier.In one embodiment, described pharmaceutical composition can be included in a cover product or the test kit.

Preferably produce according to antibody of the present invention by recombination method.Such method is well known in the art, and is included in the protein expression in protokaryon and the eukaryotic cell, separation antibody polypeptide subsequently, and be purified to pharmacy available purity usually.For protein expression, will encode light chain and heavy chain or its segmental nucleic acid are inserted in the expression vector by standard method.Be expressed in suitable protokaryon or eukaryotic host cell such as Chinese hamster ovary celI, NS0 cell, SP2/0 cell, HEK293 cell, COS cell, yeast or intestinal bacteria (E.coli) cell and carry out, and described antibody is reclaimed (supernatant liquid after the cracking or cell) from described cell.

The recombinant production of antibody is well known in the art, and for example, at Makrides, S.C., ProteinExpr.Purif.17 (1999) 183-202; Geisse, S., etc., Protein Expr.Purif.8 (1996) 271-282; Kaufman, R.J., Mol.Biotechnol.16 (2000) 151-161; Werner, R.G., etc., describe in the summary paper of Arzneimittelforschung 48 (1998) 870-880.

Described antibody may reside in the complete cell, in the cell lysate or with partial purification or basically the form of purifying exist.In order to remove other cellular constituent or other pollutent, for example, other nucleus or albumen by standard technique, comprise that alkali/SDS processing, column chromatography and other technology well known in the art carry out purifying.Referring to, Ausubel, F., etc., ed., Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

For example, the expression in the NS0 cell is by Barnes, L.M., etc., Cytotechnology 32 (2000) 109-123; And Barnes, L.M., etc., Biotech.Bioeng.73 (2001) 261-270 describes.For example, transient expression is by Durocher, Y., etc., Nucl.Acids.Res.30 (2002) E9 describes.The clone of variable domains is by Orlandi, R., etc., Proc.Natl.Acad.Sci.USA 86 (1989) 3833-3837; Carter, P., etc., Proc.Natl.Acad.Sci.USA 89 (1992) 4285-4289; And Norderhaug, L., etc., J.Immunol.Methods 204 (1997) 77-87 describe.Preferred transient expression system (HEK 293) is by Schlaeger, E.-J., and Christensen, K., by Schlaeger, E.-J. describes in J.Immunol.Methods 194 (1996) 191-199 in Cytotechnology 30 (1999) 71-83 neutralization.

For example, be applicable to that procaryotic control sequence comprises promotor, randomly operon sequence and ribosome bind site.Known genuine karyocyte application start, enhanser and polyadenylation signal.

When it was in the functional relationship with other nucleotide sequence, nucleic acid was " being operably connected ".For example, if it is expressed as participating in albumen before the polypeptide excretory, so, the DNA of presequence or secretion property leader sequence is operably connected on the polypeptid DNA; If it influences transcribing of described sequence, so promotor or enhanser are operably connected on the encoding sequence; If when perhaps it is positioned at the position that promotes translation, ribosome bind site is operably connected on the encoding sequence.Usually, " being operably connected " means the dna sequence dna that will connect is adjacent, and, in the situation of secretion property leader sequence, be adjacent and in reading frame.Yet it is adjacent that enhanser needs not to be.Connection is by realizing in the connection of restriction site easily.So, use according to routine in if there is no such site, uses synthetic oligonucleotide adapter or joint.

By routine immunization sphaeroprotein purification process, such as, for example, a-protein-agarose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography are suitably separated monoclonal antibody from substratum.Use ordinary method, be easy to DNA to the coding monoclonal antibody and separate with RNA and check order.Hybridoma can be used as the source of such DNA and RNA.In case separate, DNA can be inserted in the expression vector, then its transfection is not produced in addition the host cell of immunoglobulin (Ig), such as HEK 293 cells, Chinese hamster ovary celI or myeloma cell, in host cell, to obtain the synthetic of recombinant monoclonal antibodies.

The aminoacid sequence variant (or mutant) of human OX 40 L antibody prepares by suitable Nucleotide being changed introduce in the antibody dna or by Nucleotide is synthetic.Yet, only as indicated above carrying out like that in very limited scope of such modification.For example, described modification does not change the above-mentioned antibody characteristic of mentioning, such as IgG isotype and epi-position binding characteristic, but can improve output, the protein stability of recombinant production or promote purifying.

Can also not participate in keeping the cysteine residues of the correct conformation of anti-OX 40 L antibodies to replace with any, replace with Serine usually, improving the oxidative stability of described molecule, and prevent crosslinked unusually.On the contrary, can in described antibody, add the halfcystine key, to improve its stability (especially when described antibody is antibody fragment such as Fv fragment).

The nucleic acid molecule of amino acid variant that can be by prepared in various methods known in the art coding anti-OX 40 L antibodies.These methods comprise, but be not limited to, (or fixed point) mutagenesis, PCR mutagenesis and the cassette mutagenesis that separate the oligonucleotide-mediation of (the situation of naturally occurring aminoacid sequence variant) or variant by the preparation more early of humanization-OX40L antibody or non-variant translation from natural origin prepare.

The present invention is also about immunoconjugates, it comprises and is conjugated to cytotoxic agent, such as on chemotherapeutics, toxin (for example, the enzymatic activity toxin of bacterium, fungi, plant or animal-origin or its fragment), the radioactive isotope (that is radioactivity conjugate) according to antibody of the present invention.The conjugate of antibody and cytotoxic agent is used the difunctionality derivative of various bifunctional protein coupling agents such as N-succinimido-3-(2-pyridyl two mercaptan) propionic ester (SPDP), iminothiolane (IT), imido-ester; (such as dimethyl adipimidate HCL), active ester (such as two succinimido suberates), aldehyde (such as glutaraldehyde), two-azido cpd (such as two (right-the triazobenzene methyl) hexanediamines), two-tetroxide derivative (such as two-(right-the phenodiazine phenmethyl)-ethylenediatnine), vulcabond (such as tolyene 2, the 6-vulcabond) and two-active fluorine cpd (such as 1,5-two fluoro-2, the 4-dinitrobenzene) and prepare.For example, the ricin immunotoxin can be according at Vitetta, E.S., etc., Science 238 (1987) 1098-1104) described in be prepared like that.The l-isocyanide sulfenyl phenmethyl-3-methyl diethylene triaminepentaacetic acid(DTPA) (MX-DTPA) of carbon-14-mark is a kind of being used for the radioactive nuleus thuja acid to be coupled to exemplary sequestrant on the antibody.Referring to WO 94/11026.

The covalent modification of the another kind of type of described antibody comprises with in U.S. Patent number 4,640,835; 4,496,689; 4,301,144; 4,670,417; The mode that proposes in 4,791,192 or 4,179,337 is connected to antibody on a kind of in the various charged non-protein polymer, for example, and polyoxyethylene glycol, polypropylene glycol or polyoxyalkylene.

On the other hand, the invention provides from transgenosis non--the people animal isolating B cell of transgenic mice for example, described transgenosis is non--people's animal expression people anti-OX 40 L antibodies (for example, the parental antibody that produces by the clone that is selected from the group that generation forms according to the hybridoma of antibody of the present invention).Preferably, isolating B cell from transgenosis non--people animal transgenic mice and obtaining for example, described transgenosis is non--the people animal is with OX40L antigen purifying or recombinant forms and/or express the cellular immunization of OX40L.Preferably, described transgenosis is non--the people animal, for example transgenic mice has the people's of comprising heavy chain transgenosis and the genetically modified genome of people's light chain, its coding antibody of the present invention complete or part.Then, make the source (for example, hybridoma) of isolating B cell infinite multiplication so that people's anti-OX 40 L antibodies to be provided.Therefore, the present invention also provides the hybridoma that can produce according to human monoclonal antibodies of the present invention.In one embodiment, described hybridoma comprise from transgenosis non--the people animal B cell that obtains of transgenic mice for example, described transgenic animal have the people's of comprising heavy chain transgenosis and the genetically modified genome of people's light chain, its antibody of the present invention of encoding all or part of is fused on the cell of infinite multiplication.

In a specific embodiment, described transgenosis is non--the people animal is a transgenic mice, and it has the people's of comprising heavy chain transgenosis and the genetically modified genome of people's light chain, described genome encoding antibody of the present invention all or part of.Described transgenosis is non--the people animal can with the antigenic purifying of OX40L or enrichment preparation and/or the cell of expressing OX40L carry out immunity.Preferably, described transgenosis non--the people animal for example transgenic mice can produce isotype at the human monoclonal antibodies of OX40L.

According to human monoclonal antibodies of the present invention can by with the antigenic purifying of OX40L or enrichment preparation and/or the cell of expressing OX40L come immune transgenic non--the people animal for example transgenic mice produce, described transgenosis is non--and the people animal has the people's of comprising heavy chain transgenosis and the genetically modified genome of people's light chain, described genome encoding antibody of the present invention all or part of.Obtain the B cell (for example spleen B cell) of described animal then, and merge, to form the hybridoma of secretion at the infinite multiplication of the human monoclonal antibodies of OX40L with the myeloma cell.

In a preferred embodiment, can use the transgenic mice that carries groups of people's immunity system rather than mouse immune system at the human monoclonal antibodies of OX40L and produce.These transgenic mices, this paper is called " HuMAb " mouse, the human immunoglobulin gene's minigene seat (miniloci) (the described human immunoglobulin gene who does not reset comprises heavily (μ and γ) and κ light chain (constant region gene)) that contains the human immunoglobulin gene that coding do not reset, and the targeted mutagenesis (Lonberg that makes endogenous μ and κ chain gene seat inactivation, N., Deng., Nature 368 (1994) 856-859).Therefore, described mouse shows the mouse IgM of minimizing or the expression of K, and response immunity, the people who is introduced weighs and the light chain transgenosis has experienced kind conversion and somatic mutation, to produce the human IgG monoclonal antibody (Lonberg of high-affinity, N., etc., Nature 368 (1994) 856-859; At Lonberg, N. summarizes among Handbook ofExperimental Pharmacology 113 (1994) 49-101; Lonberg, N., and Huszar, D., Intern.Rev.Immunol.25 (1995) 65-93; And Harding, F., and Lonberg, N., Ann.N.Acad.Sci.764 (1995) 536-546).The preparation of HuMAb mouse is at Taylor, L., etc., Nucleic Acids Res.20 (1992) 6287-6295; Chen, J., etc., Int.Immunol.5 (1993) 647-656; Tuaillon, N., etc., Proc.Natl.Acad.Sci.USA 90 (1993) 3720-3724; Choi, TK., etc., Nat.Genet.4 (1993) 117-123; Chen, J., etc., EMBO is (1993) 821-830 J.12; Tuaillon, N., etc., J.Immunol.152 (1994) 2912-2920; Lonberg, N., etc., Nature 368 (1994) 856-859; Lonberg, N., Handbook of Experimental Pharmacology 113 (1994) 49-101; Taylor, L., etc., Int.Immunol.6 (1994) 579-591; Lonberg, N., and Huszar, D., Intern.Rev.Immunol.25 (1995) 65-93; Harding, F., and Lonberg, N., Ann.N.Acad.Sci.764 (1995) 536-546; Fishwild, D.M., etc., to describe among Nat.Biotechnol.14 (1996) 845-851, the content of all these is incorporated into this fully by reference.Can also referring to, U.S. Patent number 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,545,807; 5,770,429; WO 98/24884; WO 94/25585; WO 93/1227; WO 92/22645; With WO 92/03918.

In order to produce human monoclonal antibodies fully at OX40L, can be according to ordinary method, as by Lonberg, N., etc., Nature 368 (1994) 856-859; Fishwild, D.M., etc., Nat.Biotechnol.14 (1996) 845-851 and WO 98/24884 are described like that, and the HuMAb mouse is carried out immunity with the cell of the preparation OX40 antigen purification or enrichment and/or expression OX40L.Preferably, when immunity for the first time, mouse should be 6-16 age in week.For example, can use preparation purifying or enrichment that is coupled to solvable OX40L antigen on the KLH or in PBS (for example, from the cell purification of expressing OX40L) to come intraperitoneal immunity HuMAb mouse.This can make up by replacing immunity with isolating OX40L albumen with cell of expressing OX40L such as tumor cell line, to promote immunne response.Use various antigen cumulative experiences to show, when initially being used in antigen in the complete Freund's adjuvant and carrying out intraperitoneal (i.p.) immunity, thereafter be used in the antigen i.p. immunity (for example, reaching 6 times altogether) in the incomplete Freund's adjuvant week about, the HuMAb transgenic mice is replied best.Immunne response can be monitored the process of immune flow process with the plasma sample that obtains from the posterior orbit blood sampling.Blood plasma can screen by ELISA, and the mouse with enough anti-OX 40 L human normal immunoglobulin titres can be used to make corresponding B cell infinite multiplication.Before putting to death and removing spleen and lymphoglandula 3-4 days, can will strengthen in the mouse vein with antigen.Some mouse will carry out immunity for every kind of antigen.For example, 12 HuMAb mouse of HCo7 and HCo12 kind system can be by immunity altogether.

The HCo7 mouse has JKD in endogenous light chain (κ) gene within it and interrupts (as at Chen, J., Deng., EMBO is J.12 described in (1993) 821-830), the CMD in the heavy chain gene of source interrupts (as described in WO 01/14424 embodiment 1), KCo5 human kappa light chain transgenosis (as at Fishwild within it, D.M., etc., described in Nat.Biotechnol.14 (1996) 845-851) and HCo7 people's heavy chain transgenosis (as at U.S. Patent number 5, described in 770,429).

The HCo12 mouse has JKD in endogenous light chain (κ) gene within it and interrupts (as at Chen, J., Deng., EMBO is J.12 described in (1993) 821-830), the CMD in the heavy chain gene of source interrupts (as described in WO 01/14424 embodiment 1), KCo5 human kappa light chain transgenosis (as at Fishwild within it, D.M., Deng., described in Nat.Biotechnol.14 (1996) 845-851) and Hco 12 people's heavy chain transgenosiss (as described in WO 01/14424 embodiment 2).The lymphocyte of mouse can be separated, and use PEG and murine myeloma cell to merge, to produce hybridoma based on normal process.Then, the hybridoma that obtains of screening is about the generation of antigen-specific antibody.For example, use 50%PEG, will come from the spleen of immune mouse and the lymphocytic single cell suspension in lymphoglandula source and the SP 2/0 non-secretory murine myeloma cell (ATCC, CRL 1581) of 1/6 number and merge.With cell with about 2 * 10 ⁵Be seeded on the flat microtitration culture plate, wherein in selecting substratum, cultivate about 2 weeks.

Then, for people's anti-OX 40 L monoclonal igm and IgG antibody, screen single hole by ELISA.In case hybridoma growth widely takes place, after 10-14 days, analyze substratum usually.With the hybridoma renewed vaccination of secretory antibody, screening once more, and if be male for human IgG, anti-OX 40 L monoclonal antibody still, so can be by restricted dilution subclone at least 2 times.Then, stable subclone in vitro culture, is used for characterizing to produce antibody in tissue culture medium (TCM).

Detect tree mainly by about the non-specific detection (" IgG-ELISA ") of IgG, then detect and outward appearance (apparent) FACS detects and forms by specific ELISA, be used for determining and the OX40L albumen or the OX40L-express cell bonded antigen of purifying.Next step comprises functional detection, has wherein determined anti-OX 40 l antibodies interacting partner natural with it, for example for the OX40 of soluble, the purifying of the OX40L of purifying or the OX40L that on cell, expresses, competition, for example, competitive ELISA or FACS.Next step comprises functional detection, has wherein determined the ability of anti-OX 40 L antibodies retardance OX40-signal conduction, and for example, NF κ B-activates (=" NF κ B-detection ").Next step comprises functional detection, has wherein determined the retardance ability (" T-cell activation detection " and " TT-detection ") of anti-OX 40 L antibodies about the T cell activation.

Because the CDR sequence is responsible for antibody-antigenic interaction, so, may express according to recombinant antibodies of the present invention by construction of expression vector on the frame sequence that comes from different people's antibody, described expression vector comprise according to CDR sequence of the present invention (referring to, for example, Riechmann, L., Deng., Nature 332 (1998) 323-327; Jones, P., etc., Nature 321 (1986) 522-525; And Queen, C., etc., Proc.Natl.Acad.Sci.USA 86 (1989) 10029-10033).Such frame sequence can obtain from comprising kind of a public DNA database that is the human immunoglobulin gene sequence.Because being sequence, these kinds are not included in the variable gene of assembling fully that forms by V (D) J connection in the B cell mature process, so these kinds are that sequence is different from sophisticated antibody gene sequence.Planting is that gene order also runs through the sequence that the variable region is different from the secondary all the components antibody of high-affinity equably at individuality.

The present invention also comprises according to antibody of the present invention and is used for application at in-vitro diagnosis OX40L, preferably OX40L (soluble or film-bonded) by determining sample and bonded immunology detection according to antibody of the present invention.

On the other hand, the invention provides a kind of composition, for example, pharmaceutical composition, it comprises the combination of human monoclonal antibodies a kind of or of the present invention or its antigen-bound fraction, prepares with pharmaceutical carrier.More specifically, described composition is medicine or diagnosis composition, and even more specifically, described pharmaceutical composition comprises antibody defined above and at least a pharmaceutical excipient.Described composition must be aseptic, and is fluid to a certain extent, so that send described composition by syringe.

When being used for this paper, " pharmaceutical carrier " comprise any and all physical compatibilities solvent, disperse matrix, coating, antibacterium and anti-mycotic agent, etc. blend absorption delay agent etc.Preferably, described carrier is applicable to that intravenously, intramuscular, subcutaneous, parenteral, spinal cord or epidermis use (for example, by injection or perfusion).Preferably, such carrier is water-based pH buffered soln (for example, acetate, Citrate trianion, phosphoric acid salt or Histidine), preferably waits and oozes, and preferably contains inorganic salt, sugar, polyol and/or tensio-active agent in addition.Pharmaceutical carrier is still such as at Remington ' sPharmaceutical Sciences, and the 16th edition, those that describe among the Osol, A.Ed. (1980).

Antibody concentration is preferably from 0.1mg/ml to 50mg/ml.Preferably, the pH value of the buffered soln of 1mM-200mM buffer concentration is in the 4.0-8.0 scope.Preferred salt is the sodium-chlor and/or the sodium phosphate of 1mM-200mM scope.Preferred sugar is sucrose and/or the trehalose in 1%-15% (w/v) scope.The glycerol that preferred polyol is 1%-15% (w/v) scope, propylene glycol, liquid macrogol and/or similar.Tensio-active agent is polysorbate of 0.001%-0.5% (w/v) scope (for example polysorbate 20 or 80) and/or poloxamere preferably.Preferred pharmaceutical composition contains the antibody of 0.1mg/ml-50 mg/ml and the salt of 1mM-200mM pH 4.0-8.0 phosphoric acid buffer.

Composition of the present invention can be administered to the patient who needs it by the whole bag of tricks known in the art.Those skilled in the art should be appreciated that, approach of using and/or mode will depend on the result of needs and difference.

Pharmaceutical excipient or carrier comprise aseptic aqueous solution or sterile powder, are used for preparing aseptic injectable solution or dispersion liquid temporarily.The such medium of pharmaceutically active substance and the application of reagent are known in the art.

When being used for this paper, phrase " parenteral administration " and " use in the stomach other places " mean the mode of administration except intestines and topical application, usually pass through the mode of injection, and include but not limited to, in the intravenously, intramuscular, intra-arterial, sheath, in the capsule, in the eye socket, in the heart, under intracutaneous, intraperitoneal, transtracheal, subcutaneous, the epidermis, under the intraarticular, capsule, under the arachnoid membrane, in the backbone, exterior dura and intrasternal injection and perfusion.

The actual administration level of the activeconstituents in pharmaceutical composition of the present invention can be different, so that obtain a certain amount of activeconstituents, described a certain amount of activeconstituents is effectively realized the ideal therapeutic response for concrete patient, composition and mode of administration, and the patient is not had toxicity.The administration level of selecting will depend on many pharmacokinetics factors, comprise the used of the present invention concrete composition or the activity of its ester, salt or amine, the approach of using, the time of using, the particular compound discharge rate of using, the time length of treatment is with other medicines, compound and/or the material that used concrete combination of compositions is used, known factor in the medical fields such as the patient's age that treat, sex, body weight, situation, comprehensive health shape body and existing case history.Typical dosage weekly can be in the scope of the about 20mg/kg of about 0.1mg/kg-or more, and this depends on factor mentioned above.

Following example, reference, sequence table and accompanying drawing are provided, and with auxiliary the understanding of the present invention, their true scope is listed in the claim of enclosing.Should be appreciated that, can improve the method that proposes not deviate from spirit of the present invention.

The accompanying drawing summary

Fig. 1 a: show TAG-34, LC.001, LC.005, LC.010, LC.019, LC.029, " in conjunction with the ELISA " of LC.033.

Fig. 1 b: show TAG-34, LC.001, LC.005, LC.010, LC.019, LC.029, " retardance ELISA "+IC50 data of LC.033.

Fig. 2: show TAG-34, LC.001, " the sealing FACS " of LC.005.

Fig. 3: show TAG-34, LC.001, " the NFkB-detection " of LC.019 and LC.024 (uncombined antibody).

Figure 4 and 5: show TAG-34, " detection of T cell activation " of LC.001 and LC.005 and IC50-value (Fig. 4: IL-2 discharges, Fig. 5: suppress).

Fig. 6: show TAG-34, " TT-detection " data of LC.001 and LC.033.

Fig. 7: the cross reactivity that shows antibody of the present invention and mouse OX40L.A) for transfection with the WT cell on the control of the hOX40L that expresses, B) antibody and the K562 cell of expressing hOX40L combines, C) for transfection with the WT cell on express the control of mOX40L, D) combining of antibody and the K562 cell of expressing hOX40L, and E) combine (n=3) of antibody and WT K562 cell.

Fig. 8: the ability (n=3) that shows antibodies C1q of the present invention.

Fig. 9: show that antibody of the present invention activates the ability (n=3) of C3c.

Figure 10: the ability that shows antibodies Fc γ RI of the present invention (n=4), Fc γ RIIa (n=4) and Fc γ RIIb (n=4).

Figure 11: the ability (mean value of 6 donors ± SEM) that shows Fc γ RIIIa (CD16) on the antibodies NK cell of the present invention.

Figure 12: western blotting; Swimming lane 1,4,7: mark;

Swimming lane

2,5,8:100ng OX40L;

Swimming lane

3,6,9:40ng OX40L.

Sequence table is described

SEQ ID NO:1 κ light chain, the variable region of LC.001

The gamma heavy chain variable region of SEQ ID NO:2 LC.001

SEQ ID NO:3 κ light chain, the variable region of LC.005

The gamma heavy chain variable region of SEQ ID NO:4 LC.005

SEQ ID NO:5 κ light chain, the variable region of LC.010

The gamma heavy chain variable region of SEQ ID NO:6 LC.010

SEQ ID NO:7 κ light chain, the variable region of LC.029

The gamma heavy chain variable region of SEQ ID NO:8 LC.029

SEQ ID NO:9 κ light chain, the variable region of LC.019

The gamma heavy chain variable region of SEQ ID NO:10 LC.019

SEQ ID NO:11 κ light chain, the variable region of LC.033

The gamma heavy chain variable region of SEQ ID NO:12 LC.033

SEQ ID NO:13 κ constant region of light chain

SEQ ID NO:14 γ 1 CH

SEQ ID NO:15 γ 4 CH

SEQ ID NO:16 κ light chain, the sudden change variable region of LC.033

SEQ ID NO:1 κ light chain, the variable region of LC.059

The gamma heavy chain variable region of SEQ ID NO:17 LC.059

SEQ ID NO:18 κ light chain, the variable region of LC.060

The gamma heavy chain variable region of SEQ ID NO:19 LC.060

SEQ ID NO:1 κ light chain, the variable region of LC.063

The gamma heavy chain variable region of SEQ ID NO:20 LC.063

SEQ ID NO:21-57 CDR sequence

The gamma heavy chain (human IgG1's type) of SEQ ID NO:58 LC.001

The gamma heavy chain of SEQ ID NO:59 LC.001 (L234A, L235A human IgG1 mutant)

The gamma heavy chain of SEQ ID NO:60 LC.001 (S228P human IgG 4 mutant)

The κ light chain of SEQ ID NO:61 LC.001

The gamma heavy chain (human IgG1's type) of SEQ ID NO:62 LC.005

The gamma heavy chain of SEQ ID NO:63 LC.005 (L234A, L235A human IgG1 mutant)

The gamma heavy chain of SEQ ID NO:64 LC.005 (S228P human IgG 4 mutant)

The κ light chain of SEQ ID NO:65 LC.005

The gamma heavy chain (human IgG1's type) of SEQ ID NO:66 LC.060

The gamma heavy chain of SEQ ID NO:67 LC.060 (L234A, L235A human IgG1 mutant)

The gamma heavy chain of SEQ ID NO:68 LC.060 (S228P human IgG 4 mutant)

The κ light chain of SEQ ID NO:69 LC.060

Initialism:

Amino acid abbreviations is 3 (Leu) or 1 alphabetic coding (L).

S228P means in the Serine of IgG4 heavy chain position 228 and the displacement of proline(Pro).

L234 mean according to EU numbering (Kabat) in the position 234 amino acid leucine.

L234A means in the position 234 amino acid leucine and becomes L-Ala.

L235A means in the position 235 amino acid leucine and becomes L-Ala.

PVA236 means in 236 zones, and the ELLG of IgG1 or the EFLG of IgG4 are modified to PVA.

GLPSS331 means in 331 zones, and the ALPAP of IgG1 or the GLPAP of IgG2 become GLPSS.

It is deleted that Δ G236 means the amino acid of position 236.

IgG4x means the S228P that suddenlys change among the IgG4.

LC2010-001 is the different name of LC.001.

Fcg is the different name of Fcgamma (Fc γ).

Other sequence modification of antibody defines similarly.

Be fused to the soluble human OX 40 L of reorganization on the histidine mark	hOX40L-His
	hOX40L-His	Be fused to the soluble mouse OX40L of reorganization on the histidine mark	mOX40L-His
Be fused to the soluble human OX 40 L of reorganization on the Flag mark	hOX40L-Flag		mOX40L-His
	hOX40L-Flag	Be fused to the soluble mouse OX40L of reorganization on the Flag mark	mOX40L-Flag
Be fused to the soluble people OX40 of reorganization on the people Fc γ	hOX40-hFc		mOX40L-Flag
	hOX40-hFc	Rabbit resists-mouse Fc γ monoclonal antibody	Anti--mFc
Goat Anti-Human Fc γ monoclonal antibody	Anti--hFc	Rabbit resists-mouse Fc γ monoclonal antibody	Anti--mFc
Goat Anti-Human Fc γ monoclonal antibody	Anti--hFc	Mouse anti-Histidine monoclonal antibody	Anti--His
Be fused to the soluble people OX40 of reorganization on the mouse Fc γ	hOX40-mFc	Mouse anti-Histidine monoclonal antibody	Anti--His
	hOX40-mFc	Mouse anti-TNF alpha monoclonal antibodies	Anti-TNF alpha
Mouse anti-CD40L monoclonal antibody	Anti-CD 40 L	Mouse anti-TNF alpha monoclonal antibodies	Anti-TNF alpha
Mouse anti-CD40L monoclonal antibody	Anti-CD 40 L	Tumor necrosis factor alpha	TNFα
The CD40 part	CD40L	Tumor necrosis factor alpha	TNFα
The CD40 part	CD40L	Rabbit Anti-Human OX40L monoclonal antibody	TAG34
People Anti-Human OX40L monoclonal antibody	LC.001，LC.005，	Rabbit Anti-Human OX40L monoclonal antibody	TAG34

	LC.010，LC.019， LC.029，LC.033， LC.059，LC.060， LC.063
	LC.010，LC.019， LC.029，LC.033， LC.059，LC.060， LC.063	Phytohemagglutinin	PHA

Embodiment

Embodiment 1

Produce the hybridoma cell line of producing anti--0X40L antibody

The cultivation of hybridoma

At IMDM (Cambrex), tire is cloned 1 bovine serum (Perbio Science), the original hybridoma clone factor (Igen), Sodium.alpha.-ketopropionate, penicillin/streptomycin, 2 mercapto ethanol, in HAT (Sigma-Aldrich) and the kantlex (Invitrogen), at 37 ℃ and 5%CO ₂, cultivate the HuMab hybridoma.

The immune flow process of transgenic mice

LC2010-001: with 6 HCo7 (2 male and 4 female), planting is GG2201 (Medarex, San Jos é, CA, USA) and 4 HCo12 (4 are male), planting is GG2198 (Medarex, San Jos é, CA, USA) alternatively use transient transfection human OX 40 L (hOX40L) expression vector 1 * 10 ⁶Individual HEK293 cell and the soluble ectodomain of 20 μ g hOX40L carry out immunity.Carry out 8 immunity altogether, 4 times the cell with expression hOX40L carries out intraperitoneal (i.p.) immunity and carries out subcutaneous (s.c.) immunity with recombinant protein 4 times bottom tail.For immunity for the first time, with 100 μ l 1 * 10 ⁶Individual HEK293-hOX40L cell and 100 μ l complete Freund's adjuvant (CFA; Difco Laboratories, Detroit USA) mixes.The cell of 100 μ l in PBS used in other immunity for all, perhaps with recombinant protein and 100 μ l incomplete Freund's adjuvant (ICFA; Difco) mix.

When the serum titer of finding anti--hOX40L was enough, before fusion 4 and 3 days, the 15 other intravenouslys of μ g hOX40L ectodomain (i.v.) that mouse is used among the 200 μ l PBS strengthened 2 times.

LC2010-001, LC.059, LC.060 and LC.063 derive from the HCo12 mouse.

LC2010-005 ,-010 ,-019 ,-029 and-033: with 5 HCo7 (4 male and 1 female), kind is that (CA is USA) with the soluble ectodomain immunity of 20 μ ghOX40L for Medarex, San Jos é for GG2201.Carry out 7 immunity, 4 intraperitoneal (i.p.) and 3 subcutaneous (s.c.) immunity in the tail bottom altogether.For immunity for the first time, with 100 μ l recombinant proteins and 100 μ l complete Freund's adjuvant (CFA; Difco Laboratories, Detroit USA) mixes.Other immunity for all are with 100 μ l recombinant proteins and 100 μ l incomplete Freund's adjuvant (ICFA; Difco) mix.

The generation of hybridoma

Mouse is put to death, and collect spleen and at abdominal aorta and the lateral lymphoglandula of Vena cava.According to standard operating instructions, carry out the fusion of splenocyte and lymph-node cell with fusion partner SP 2.0 cells.

Antigen-specific ELISA

Determine anti-OX 40 L titre in the immune serum by antigen-specific ELISA.(96 flat ELISA flat boards Greiner) with the OX40L bag quilt that is dissolved in 0.1 μ g/ml purifying among the PBS, and at room temperature wrap and are spent the night with flat board.After this, at room temperature the hole was sealed 1 hour with PBSTC (PBS that contains 0.05% polysorbas20 (Sigma-Aldrich Chemie BV) and 2% chicken serum (Gibco)).

With serum sample (taps) dilution in 1: 50 in PBSTC that detects, and add in the hole.To be dissolved in PBSTC at 1: 100 from the serum that the preceding mouse of immunity obtains, and as negative control.To be dissolved in PBSTC at 1: 50 at the mouse antibodies of human OX 40 L, and as positive control.With flat board room temperature incubation 1 hour.Subsequently, flat board is washed 2 times with PBST (PBS that contains 0.05%Tween 20).Gt-α-huIgG-HRP (Jackson) is diluted in PBSTC at 1: 5000, and adds in the hole of containing specimen and negative control.Rb-α-mIgG (Jackson) is diluted in PBSTC at 1: 3000, and adds in the hole of containing positive control.With flat board incubation 1 hour at room temperature.At last, with flat board with PBST washing 3 times, and with the ABTS of prepared fresh ^Solution (1mg/ml) (ABTS:2,2 '-azido-two (3-ethylbenzene base thiazoline-6-sulfonic acid)) at room temperature dark place reaction 30 minutes.Measure absorption at the 405nm place.

κ-ELISA

In order to determine whether produce people's antibody by merging the hybridoma that produces, carried out κ-ELISA.Rat anti-human IgG κ-light chain the antibody (DAKO) that the ELISA flat board is used in 1/10000 dilution among the PBS wraps quilt, 4 ℃ of overnight incubation.After discarding cell, flat board is passed through at room temperature to seal in 1 hour with PBSTC (PBSC has replenished 0.05% tween 20 (PBSTC)) incubation.After this, with the hole with the 1/2 hybridoma culture supernatant liquid incubation that is diluted among the PBSTC.1/2 substratum that is diluted among the PBSTC is used as negative control, and the 1/100 positive mice serum of κ-light chain that is diluted among the PBSTC is used as positive control.Subsequently, with hole washing 3 times, and at 37 ℃, be diluted in rat anti-human IgG F that the HRP-among the PBSTC puts together (ab ') with 1/2000 ₂(DAKO) incubation 1h.With hole washing 3 times, and at room temperature (RT) dark place with ABTS of prepared fresh ^Solution (1mg/ml) detects 30 minutes.On the ELISA plate reader, measure the absorption of 405nm.

Embodiment 2

The clone and the order-checking of anti-OX 40 L HuMab variable domains

(κ-light chain and γ 1-heavy chain)

Synthesize/PCR method by standard cDNA, separate the variable region of light chain V of coding OX40L HuMabs _LWith variable region of heavy chain V _HNucleotide sequence.

Use GeneRacer ^TMTest kit (Invitrogen) is from 1 * 10 ⁶-1 * 10 ⁷The total RNA of preparation in the individual hybridoma.The RNA that derives from hybridoma synthesizes and GeneRacer as the 1st chain cDNA ^TMThe template that the Oligo-dT primer connects.Synthetic and the coding V of the 2nd chain cDNA _LWith V _HThe segmental further pcr amplification of cDNA use reverse light chain and heavy chain primer and 5 '-special GeneRacer respectively ^TMPrimer carries out, the nucleotide sequence complementation of described reverse light chain and heavy chain primer and κ-light chain and γ 1-CH.Use is from Invitrogen ^TMThe TOPO of Life Technologies ^TMTA clones test kit, and pCR4-TOPO ^TMAs cloning vector, and with the PCR product cloning.By using EcoRI digestion with the restricted drawing of suitable plasmid, and about respectively 740 and the V of 790 bp _LAnd V _HThe dna fragmentation size of expectation/calculating, and determine clone's PCR product.

Clone's the segmental dna sequence dna of PCR is determined by the two strands order-checking.

(Wisconsin) (InforMax Inc) is used for routine data and handles GCG for software package version 10.2 and carrier-NTI 8 for Genetics Computer Group, Madison.Use GCG mould CLUSTALW comparison DNA and protein sequence.Service routine GENEDOC (version 2 .1) carries out sequence alignment.

Embodiment 3

Make up the expression plasmid of anti-OX 40 L IgG1 HuMab

The encoding gene of anti-OX 40 L HuMab light chain and heavy chain is assembled in the mammalian cell expression vector respectively.

Therefore, the anti-OX 40 L HuMab variable region of light chain (V that will encode _L) and people's κ-constant region of light chain (C _L, SEQ ID NO:13, or from SEQ ID NO:61,65 or 69) gene fragment couple together, similarly with anti-OX 40 L HuMab variable region of heavy chain (V _H) and people γ 1-CH (C _H1-hinge-C _H2-C _H3, SEQ ID NO:14, or from SEQ ID NO:58,62 or 66) gene fragment couple together.

About the general information that therefrom can infer people's light chain that codon is used and heavy chain nucleotide sequence at Kabat, E.A., etc., Sequences of Proteins of Immunological Interest, the 5th edition., provide among the NIH Publication No.91-3242 (1991).

The transcription unit of anti-OX 40 L HuMab κ-light chain is made up of following elements:

The directly early stage enhanser and the promotor of-Human cytomegalic inclusion disease virus (HCMV),

-comprise the synthetic 5 '-UT of Kozak sequence,

-comprise the mouse immuning ball protein heavy chain signal sequence of signal sequence intron,

-clone's the variable light chain cdna of anti-OX 40 L HuMab, it is with the unique BsmI restriction site of 5 ' end, and donor splicing site, and 3 ' hold unique N otI restriction site and arrange,

-genome people κ-gene constant region, it comprise intron 2 mouse Ig-κ enhansers [Picard, D., and Schaffner, W., Nature 307 (1984) 80-82] and

-human normal immunoglobulin κ-polyadenylic acidization (" poly A ") signal sequence.

The transcription unit of anti-OX 40 L HuMab γ 1-heavy chain is made up of following elements:

-comprise the synthetic 5 '-UT of Kozak sequence,

The mouse immuning ball protein heavy chain signal sequence of-modification, it comprises the signal sequence intron,

-clone's anti-OX 40 L HuMab variable heavy chain cDNA, it is with the unique BsmI restriction site of 5 ' end, and donor splicing site, and 3 ' hold unique N otI restriction site and arrange,

-genome people γ 1-heavy chain gene constant region, it comprises mouse Ig μ-enhanser (Neuberger, M.S., EMBO be (1983) 1373-1378 J.2),

-people γ 1-immunoglobulin (Ig) polyadenylic acidization (" poly A ") signal sequence.

The functional element of anti-OX 40 L HuMab κ-light chain and γ 1-heavy chain expression plasmid: except anti-

Outside-OX40L HuMab κ-light chain or the γ 1-heavy chain expression box, these plasmids also comprise

-hygromycin gene

The replication orgin of-Ai Ba virus (EBV), oriP

The replication orgin of-carrier pUC18, its allow this plasmid in intestinal bacteria, to duplicate and

-in intestinal bacteria, give the β-Nei Xiananmei gene of amicillin resistance.

Embodiment 4

Make up the expression plasmid of anti-OX 40 L IgG4 HuMab

Anti-OX 40 L γ 4-heavy chain prototype expression plasmid is derived from anti-OX 40 L γ 1-heavy chain expression plasmid, it is by personnel selection genome γ 4-constant region (SEQ ID NO:15 or from SEQ ID NO:60,64 or 68 the not γ 4-constant region of sudden change) and γ 4-immunoglobulin (Ig) poly-adenosine-signal sequence replacement people's gene group γ 1-constant region and γ 1-immunoglobulin (Ig) poly-adenosine (" poly A ") signal sequence.

For the expression of anti-OX 40 L HuMab κ-light chain, use with being used for the described identical expression plasmid of IgG1 (referring to above).

Embodiment 5

Make up the expression plasmid of mutant (variant) anti-OX 40 L IgG1 and IgG4 based on LC.001

Use QuickChange ^TMSite-directed mutagenesis test kit (Stratagene) generates the expression plasmid of encoding mutant body anti-OX 40 L γ 1-and γ 4-heavy chain by site-directed mutagenesis wild-type expression plasmid.According to the EU numbering (Edelman, G.M., etc., Proc.Natl.Acad.Sci.USA 63 (1969) 78-85; Kabat, E.A., etc., Sequences of Proteins of Immunological Interest, the 5th edition., Public Health Service, NIH Publication No.91-3242, Bethesda, MD (1991)), amino acid is numbered.

Table 1

Isotype	Abbreviation	Sudden change	Describe
Isotype	Abbreviation	Sudden change	Describe	IgG1	IgG1v1	PVA-236; The GLPSS331 that represents by E233P; L234V; L235A; △ G236; A327G; A330S; P331S	Aminoacid sequence Glu with people γ 1-heavy chain ₂₃₃Leu ₂₃₄Leu ₂₃₅Gly ₂₃₆The aminoacid sequence Pro of personnel selection γ 2-heavy chain ₂₃₃Val ₂₃₄Ala ₂₃₅Replace.Aminoacid sequence Ala with people γ 1-heavy chain ₃₂₇Leu ₃₂₈Pro ₃₂₉Ala ₃₃₀Pro ₃₃₁The aminoacid sequence Gly of personnel selection γ 4-heavy chain ₃₂₇Leu ₃₂₈Pro ₃₂₉Ser ₃₃₀Ser ₃₃₁Replace.
IgG1	IgG1v2	L234A; L235A	Aminoacid sequence Leu with people γ 1-heavy chain ₂₃₄Leu ₂₃₅Use aminoacid sequence Ala ₂₃₄Ala ₂₃₅Replace	IgG1	IgG1v1
IgG1	IgG1v2	L234A; L235A		IgG4	IgG4v1	S228P; L235E	Ser with people γ 4-heavy chain ₂₂₈Use Pro ₂₂₈Replace, and with the Leu of people γ 4-heavy chain ₂₃₅Use Glu ₂₃₅Replace
IgG4	IgG4x	S228P	Ser with people γ 4-heavy chain ₂₂₈Use Pro ₂₂₈Replace	IgG4	IgG4v1	S228P; L235E

Embodiment 6

The production of reorganization anti-OX 40 l HuMabs

Reorganization HuMabs produces by the transient transfection of adherent HEK293-EBNA cell (ATTC CRL-10852), and described HEK293-EBNA cell cultures is in the DMEM (Gibco) that has replenished 10% ultralow IgG FCS (Gibco), 2mM glutamine (Gibco), 1%v/v non-essential amino acid (Gibco) and 250 μ g/mlG418 (Roche).For transfection, with 3: 1-6: the reagent of 1 scope (μ l) uses Fugene than the ratio of DNA (μ g) ^TM6 (Roche) transfection reagent.Use 1: 2-2: the plasmid of the light chain proportion chain encoding of 1 molar ratio, light chain immunoglobulin is expressed from two different plasmids with heavy chain.After transfection 4-11 days, collect the cell culture supernatant liquid that contains HuMab.Before purifying, supernatant liquid is kept at-20 ℃.

About people's antibody in HEK293 for example recombinant expressed general information at Meissner, P., etc., provide among Biotechnol.Bioeng.75 (2001) 197-203..

Embodiment 7

Antibody TAG34, LC.001, LC.005, LC.010, LC.019, LC.029, the avidity analysis of LC.033

Instrument: Biacore 3000, operation and reaction buffer: HBS-P (10mM HEPES, 150mM NaCl, 0.005% polysorbas20, ph 7.4), 25 ℃.7 concentration that are injected between 0.78nM and the 100nM of analyte were carried out 3 minutes, and washed 5 minutes with HBS-P.(carboxymethylated dextran surface, regeneration CM) is undertaken by 2 injections that each 10mM glycine pH 2.0 continues 1min on the surface.The order of chip, test format and injection and dynamics data are corresponding with the description in the following table.Dynamics data is by with dynamics data and 1: 1 suitable calculating of Langmuir combination model.

Table 2

Chip	Catch	Part	Analyte	ka(1/Ms)	kd(1/s)	KD(M)
Chip	Catch	Part	Analyte	ka(1/Ms)	kd(1/s)	KD(M)	CM5	Anti--mFcg	TAG34	hOX40L-His	8.84×10 ⁴	3.32×10 ^-5	3.75×10 ^-10
CM5	Anti--hFcg	LC.001	hOX40L-His	9.01×10 ⁴	7.16×10 ^-9	＜1.1×10 ^-11	CM5	Anti--mFcg	TAG34	hOX40L-His	8.84×10 ⁴	3.32×10 ^-5	3.75×10 ^-10
CM5	Anti--hFcg	LC.001	hOX40L-His	9.01×10 ⁴	7.16×10 ^-9	＜1.1×10 ^-11	CM5	Anti--hFcg	LC.005	hOX40L-His	6.84×10 ⁴	2，02×10 ^-7	＜1.5×10 ^-11
CM5	Anti--hFcg	LC.010	hOX40L-His	6.25×10 ⁴	2.5×10 ^-5	3.99×10 ^-10	CM5	Anti--hFcg	LC.005	hOX40L-His	6.84×10 ⁴	2，02×10 ^-7	＜1.5×10 ^-11
CM5	Anti--hFcg	LC.010	hOX40L-His	6.25×10 ⁴	2.5×10 ^-5	3.99×10 ^-10	CM5	Anti--hFcg	LC.019	hOX40L-His	7.89×10 ⁴	7.53×10 ^-8	＜1.2×10 ^-11
CM5	Anti--hFcg	LC.029	hOX40L-His	1，41×10 ⁵	2，4×10 ^-8	＜7，1×10 ^-12	CM5	Anti--hFcg	LC.019	hOX40L-His	7.89×10 ⁴	7.53×10 ^-8	＜1.2×10 ^-11
CM5	Anti--hFcg	LC.029	hOX40L-His	1，41×10 ⁵	2，4×10 ^-8	＜7，1×10 ^-12	CM5	Anti--hFcg	LC.033	hOX40L-His	7.01×10 ⁴	2.09×10 ^-7	＜1.4×10 ^-11

Not interacting between TAG34 and mOX40L can be determined.

Preserve for data evaluation and data that all Biacore detect

From the sample curve, deduct negative control data (for example, the damping fluid curve), with baseline wander of corrective system inherent and minimizing noise signal.BiaEvaluation edition 4 .01 is used for the Sensorgrams analysis and affinity data calculates.

Embodiment 8

Suppress hOX40L and fixed hOX40 interactional anti--the inhibition competition detection of hOX40L antibody

Instrument: Biacore 3000, and operation and reaction buffer: HBS-P (10mM HEPES, 150mM NaCl, 0.005% polysorbas20, ph7.4), 25 ℃.Before the injection, with analyte (10nM) and competitor (8 concentration between 0.78nM and 100nM) at 22 ℃ of incubation 20min at least in advance.Analyte+/-injection of competitor carried out 3 minutes, and with HBS-P washing 3 minutes.The regeneration on surface is undertaken by 2 injections that each 10mM glycine pH 2.0 continues 1min.The order of chip, test format and injection and dynamics data are corresponding with the description in the following table 3.

Table 3

Chip	Part	Analyte	Competitor	IC50(M)
Chip	Part	Analyte	Competitor	IC50(M)	CM5	OX40-hFc	hOX40L-His	TAG34		7×10 ^-9
CM5	OX40-hFc	hOX40L-His	LC.001	4×10 ^-9	CM5	OX40-hFc	hOX40L-His	TAG34		7×10 ^-9
CM5	OX40-hFc	hOX40L-His	LC.001	4×10 ^-9	CM5	OX40-hFc	hOX40L-His	LC.005	3×10 ^-9

All antibody suppresses combine (the solution avidity) of OX40L and OX40 in the solution.LC.001 and LC.005 show the IC50-value lower than TAG34.

Embodiment 9

Anti-OX 40 L antibodies TAG34, LC.001, LC.005, LC.010, LC.01 9, LC.029, LC.033, the epi-position characteristic description of LC.060

Instrument: Biacore 3000, and operation and reaction buffer: HBS-P (10mM HEPES, 150mM NaCl, 0.005% polysorbas20, ph7.4), 25 ℃.Determine the epi-position group by the cross competition between the antibody of listing.Before the injection, with analyte (50nM) and competitor (100nM) at 22 ℃ of incubation 20min at least in advance.Analyte+/-injection of competitor carried out 2 minutes, and with HBS-P washing 3 minutes.The regeneration on surface is undertaken by 2 injections that each 10mM glycine pH 2.0 continues 1min.The order of chip, test format and injection and dynamics data are corresponding with the description in the following table 4.

Table 4

Chip	Catch	Part	Analyte	Competitor	Epi-position
Chip	Catch	Part	Analyte	Competitor	Epi-position	CM5	Anti--hFcg	Anti-OX 40 L (A, B, C)	hOX40L-His	TAG34	A
CM5	Anti--hFcg	Anti-OX 40 L (A, B, C)	hOX40L-His	LC.001	A	CM5	Anti--hFcg	Anti-OX 40 L (A, B, C)	hOX40L-His	TAG34	A
CM5	Anti--hFcg	Anti-OX 40 L (A, B, C)	hOX40L-His	LC.001	A	CM5	Anti--hFcg	Anti-OX 40 L (A, B, C)	hOX40L-His	LC.005	B
CM5	Anti--hFcg	Anti-OX 40 L (A, B, C)	hOX40L-His	LC.010	B	CM5	Anti--hFcg	Anti-OX 40 L (A, B, C)	hOX40L-His	LC.005	B
CM5	Anti--hFcg	Anti-OX 40 L (A, B, C)	hOX40L-His	LC.010	B	CM5	Anti--hFcg	Anti-OX 40 L (A, B, C)	hOX40L-His	LC.019	A/B
CM5	Anti--hFcg	Anti-OX 40 L (A, B, C)	hOX40L-His	LC.029	B	CM5	Anti--hFcg	Anti-OX 40 L (A, B, C)	hOX40L-His	LC.019	A/B
CM5	Anti--hFcg	Anti-OX 40 L (A, B, C)	hOX40L-His	LC.029	B	CM5	Anti--hFCg	Anti-OX 40 L (A, B, C)	hOX40L-His	LC.033	A
CM5	Anti--hFCg	Anti-OX 40 L (A, B, C)	hOX40L-His	LC.060	A	CM5	Anti--hFCg	Anti-OX 40 L (A, B, C)	hOX40L-His	LC.033	A

The OX40L epi-position of TAG34 identification is defined as epi-position A.Yet TAG34 debond antibody concentration is the OX40L (in western blotting detects) of the sex change of 100ng.Antibody in an epi-position group (A or B) shows and intersect to suppress active, and on the same group antibody does not show additional binding signal.The LC.019 neutralization is from other antibody of group A and group B.

Embodiment 10

TAG34, the binding specificity of LC.001 and LC.005 and CD40L and TNF α

Instrument: Biacore 3000, operation and reaction buffer: HBS-P (10mM HEPES, 150mM NaCl, 0.005% polysorbas20, ph 7.4), 25 ℃.Analyte be injected at 100nM and 500nM carried out 3 minutes, and with HBS-P washing 2 minutes.The regeneration on surface is undertaken by 2 injections that each 100mMHCl continues 1min.The order of chip, test format and injection and dynamics data are corresponding with the description in the following table 5.

Table 5

Chip	Catch	Part	Analyte
Chip	Catch	Part	Analyte	CM5	Anti--mFcg resists-hFcg	TAG34 LC.001 LC.005 anti-TNF alpha anti-CD 40 L	TNFα CD40L OX40L

In this detection, CD40L shows to all antibody or to certain non-specific binding of chip surface, but behind the subtracting background signal, this detection shows, there are not TNF α and CD40L (reaching 500nM) and fixed antibody TAG34, the non-specific binding of LC.001 and LC.005.

Embodiment 11

The avidity analysis of antibody LC.001-IgG1 and LC.001-IgG4x

Instrument: Biacore 3000, operation and reaction buffer: HBS-P (10mM HEPES, 150mM NaCl, 0.005% polysorbas20, ph 7.4), 25 ℃.8 concentration that are injected at 0.78nM-100nM of analyte were carried out 3 minutes, and washed 5 minutes with HBS-P.The regeneration on surface is undertaken by 2 injections that each 100mM HCl continues 1min.The order of chip, test format and injection and dynamics data are corresponding with the description in the following table.Dynamics data is by with dynamics data and 1: 1 suitable calculating of Langmuir combination model.

Table 6

Chip	Catch	Part	Analyte	ka(1/Ms)	kd(1/s)	K _D(M)
Chip	Catch	Part	Analyte	ka(1/Ms)	kd(1/s)	K _D(M)	CM5	Anti--mFcg	LC.001	hOX40L-His	4.27×10 ⁴	3.46×10 ^-8	＜2,3×10 ^-11
CM5	Anti--mFcg	LC.001-IgG4×	hOX40L-His	4.85×10 ⁴	7.72×10 ^-8	＜2,06×10 ^-11	CM5	Anti--mFcg	LC.001	hOX40L-His	4.27×10 ⁴	3.46×10 ^-8	＜2,3×10 ^-11

LC.001 shows the avidity identical to hOX40L-His with LC.001-IgG4x.

Embodiment 12

Detect ELISA detection with OX40L bonded antibody

SA-is wrapped the flat board of quilt, and (96 flat ELISA flat boards Microcoat) at room temperature wrap by 1 hour with the biotinylated OX40L of 0.5 μ g/ml that is dissolved in the incubation buffering liquid (IB=contains the PBS of 0.1% polysorbas20 (Serva) and 1% closed protein).Then, flat board is washed 2 times with lavation buffer solution (WB=contains the salt solution of 0.1% polysorbas20).

With sample (cell culture supernatant liquid or antibody purified) serial dilution in IB, and add in the hole.With flat board incubation 1 hour at room temperature.Subsequently, flat board is washed 2 times with WB.Subsequently, will in IB, be diluted to 50ng/ml at the goat antibody of human IgG and the conjugate of POD (Dianova), and add in the hole.Flat board is incubation 1 hour at room temperature.At last, flat board is washed 2 times with WB, and dark is located with ready-made available ABTS under room temperature (RT) ^Solution reaction.When the absorption of maximum concentration reaches enough OD, measure at 405nm and to absorb that (Fig. 1 a).Obtain the EC50 value in the 3nM-8 nM scope.

Embodiment 13

Detecting the ELISA that suppresses the interactional antibody of people's OX40/ human OX 40 L detects

The flat board (96 flat ELISA flat boards, Microcoat, Germany) that SA-is wrapped quilt at room temperature wraps by 1 hour with the biotinylated OX40L of 0.5 μ g/ml that is dissolved among the IB.Then, with flat board WB (PBS damping fluid, 0.1% (w/v) tween ^TM20) washing is 2 times.

Sample is diluted to 1 μ g/ml concentration in IB, and adds in the hole with serial dilution.In order to obtain the maximum combined of OX40 and OX40L, in some hole, only add IB.Then, in each hole, add concentration and be 0.2 μ g/ml with digoxigenin (Roche Diagnostics GmbH, the people OX40 solution of DE) puting together.With flat board incubation 1 hour at room temperature.Subsequently, flat board is washed 2 times with WB.With sheep＜digoxigenin 〉-POD (Roche) is diluted to 50mU/ml, and adds in the hole in IB.Flat board is incubation 1 hour at room temperature.At last, flat board is washed 2 times with WB, and dark is located with ready-made available ABTS under room temperature (RT) ^Solution reaction.After 10-20 minute, measure absorption (Fig. 1 b) at 405nm.Obtain the IC50 value in the 1nM-4nM scope.

Embodiment 14

The FACS-that detects the interactional HuMabs of human OX 40 L that suppresses people OX40 and go up expression at K562 cell (K562 OX40L cell) detects

Purpose: be used for determining the hOX40:hFc fusion rotein of HuMab hOX40L retardance Dig-mark and the detection of the interactional characteristic of cell line k562 hOX40L of expressing hOX40L.

Method: described detection is carried out as " competitor " as " detection agent " and HuMabhOX40L with the hOX40:hFc of Dig mark.

Detection agent: liquid storage 0.5 μ g/ μ l-1: 10 are diluted among the PBS, and 100 μ l resist-digoxigenin-FLUOS.Be diluted at 1: 25 the PBS/0.5%BSA/1% closed reagent (Roche DiagnosticsGmbH, DE) in.

With 2 * 10 ⁵K562 OX40L cell (growing in ISF-0) washs in 2ml PBS, and is resuspended among the 100 μ l PBS.(competitor/reagent concerns 0: 1/1: 1/1.5: 1/2: 1/2.5: 2/5: 1) to be added in competitor among the PBS after this.After this is the 30Min incubation time, RT and daylight.Then, add reagent (in PBS); Incubation time: 30Min, RT and daylight.Cell is washed in 2ml PBS, and by centrifugation.Add and be used for painted secondary antibody (anti--digoxigenin-fluorescein, Fab-fragment (Roche, 1207741)), and incubation 30Min, 4 ℃ at the dark place.Cell is washed in 2ml PBS, and by centrifugation.Afterwards cell is resuspended among the 0.5ml PBS.(Fig. 2) carried out in being determined at of sample among the FACS-Scan.

Embodiment 15

Determine antibody to the functional detection of the inhibition ability of hOX40/hOX40L signal conduction (,, NF κ B-detects ")

The HeLa cell (HeLa_OX40) of Hela wild-type (wt) and expressing human OX40 is grown in minimum essential medium (MEM), 1 * Sodium.alpha.-ketopropionate, 1 * non-essential amino acid (Gibco) is among the 10%FCS, and in the situation of reconstitution cell ,+600 μ g/ml G418.The K562 of K562 and expression OX40L is grown in the ISF-O substratum, and in the situation of reconstitution cell, adds 200 μ g/mlG418.

With HeLa_wt or HeLa_OX40 cell with 3 * 10 ⁴The cell density of individual cell/100 μ l is seeded in the flat board of 96-hole, w/o G418, and at CO ₂Overnight incubation in the-incubator.Add K562_wt or K562_OX40L cell with cell-ratio of 1: 1-cell relation.Formalin-fixed or the K562 cell (frozen at-70 ℃) that do not have formalin-fixed to express OX40L are thawed, and dilution in 1: 10 in MEM/10%FCS; With the K562_OX40L cell with at antibody preincubation 30min under RT of OX40L.The stimulation time of K562_OX40L cell is between 30-150min.According to supplier's directions for use of Active Motif NE-test kit, carry out extracting albumen from nucleus.The TransAM NF κ B-ELISA of Active Motif (directions for use that detects according to the supplier carries out) is used for determining the conduction of OX40-signal, and it causes the NF kB activation.Described detection is carried out (Fig. 3) in wavelength 450/620nm absorption place in Tecan MTP-readout instrument.Obtain the IC50 value in the 0.6-5nM scope for all LC antibody.

Embodiment 16

The T-cell activation detects

Detect principle:

Human peripheral blood mononuclear cell (PBL) is activated with the T-cell mitogen phytohemagglutinin (PHA) that is lower than optimum concn, and stimulate jointly with the K562 cell of overexpression OX40L.Under testing conditions, produce IL-2 at 37 ℃ of activated T-cells of cultivating 24hr.Use ELISA and detect, be determined at the cytokine in the supernatant liquid.In order to determine the retardation of Mab, with the PBL co-cultivation before, with K562 OX40L cell with the suitable diluent of antibody incubation 1 hour in advance.

Method:

By the density gradient centrifugation in Histopaque -1077 (Sigma), human peripheral blood mononuclear cell (PBL) is separated from the whole blood of heparinization.With after the Hanks washing, use Turk ' s solution counting cells, and with cell with 10 ⁶The concentration of/ml is resuspended among the RPMI 1640 (Gibco) that has replenished penicillin, Streptomycin sulphate and glutamine (Gibco 10378-016) and 10%FBS.K562 control cells (wild-type) remains in the identical RPMI substratum that has replenished above-mentioned substance.With transfection the K562 cell of OX40L remain on that to have replenished final concentration be that (G418 is in identical substratum Gibco) for the Geneticin of 50mg/ml.Is 1.5 * 10 with K562 cell (WT or OX40L+) with identical substratum dilution ⁵Individual cell/ml, and with 50 μ l/ holes (0.75 * 10 ⁴/ hole) branch installs in each hole of 96 hole tissue culture plate.The volume of suitable Mab diluent with 20 μ l/ holes added in the hole, and at 37 ℃ of incubation 1hr.Each part diluent detects in bipartite hole.PBL is with 100 μ l/ holes (10 ⁵Individual cells/well) volume adds.PBL than the final ratio of K562 cell be～13: 1.Add PHA (10 *) (Sigma L-9132) with 20 μ l/ holes (final concentration 0.75 μ g/ml).Make the cumulative volume in each hole reach 200 μ l with RPMI/10%FCS.With flat board at 37 ℃ of 5%CO ₂Incubation 24 hrs in the-moistening incubator.With flat board centrifugal after, collect supernatant liquid, and according to producer's specification sheets, (CA CatNo2627KI) detects IL-2 (Fig. 4) for BD, San Diego by ELISA.In order to calculate IC50 (50% the Mab concentration that the IL-2 of the PBL that retardance OX40L-stimulates discharges), the background IL-2 concentration that will produce in control cultures (PBL+PHA+K562WT) deducts (Fig. 5) from the total IL-2 by the PBL generation of K562 * OX40L+ cytositimulation.IC ₅₀ TAG34：0.07μM；LC.001：2nM；LC.005：10nM。IC ₅₀Value is in the 2-10nM scope.

Embodiment 17

Tetanus detects (' TT-detections ') and detects antibody to the restraining effect by the peripheral blood lymphocyte of tetanus toxin stimulation

By Ficoll Hypaque peripheral blood lymphocytes (PBMC) is separated from the blood of heparinization.In most of situations, the PBMC of fresh separated is used for this detection.In some cases, also use the PBMC of refrigeration.The substratum that is used for this detection is to contain 10% people male sex AB serum (Sigma-Aldrich); 2mM glutamine and Pen/Strep (the ready-made available mixture of microbiotic penicillin and Streptomycin sulphate (Roche Diagnostics GmbH DE); Lyophilize thing with the 20ml reconstruction; Every 1000ml substratum 2ml) RPMI.

In order to be attached on the plastics, with every hole 300.000 PBMC pre-overnight incubation on the flat flat board in 96 holes.

Second day, (Chiron Behring) joined in the hole with tetanus toxin (TT), final concentration 2-5 μ g/ml.TT is only contained in positive control hole (maximum propagation/stimulation), and antibody (as the IgG of purifying) adds in all other the hole, final concentration 10 μ g/ml.Mouse Mab TAG-34 is included in (final concentration 10 μ g/ml) in the detection.Use independent non-irritating background control medium.All detections are carried out in triplicate.

Continue to cultivate (37 ℃, 5%CO ₂, 95% humidity) after 6 days, add ³H-thymidine, final concentration are 1 μ Curie/ml, and after between extra 16h incubation period, collect dull and stereotyped and determine bonded in beta-counter ³H-thymidine (Fig. 6).

Embodiment 18

The cross reactivity of OX40L antibody and mouse OX40L

In order to determine the cross reaction ability of antibody of the present invention and mouse OX40L, with the antibody of serial dilution and control antibodies K562-mOX40L cell incubation with stably express mOX40L.Also assessed and the combining of the K562-hOX40L cell of K562 WT cell and stably express hOX40L.(the HuMab antibody of α-KLH) is as negative control at keyhole limpet hemocyanin.Comprise antibody RM134L, rat anti-mOX40L (eBioscience, San Diego, CA) positive control of expressing as mOX40L.Comprise antibody TAG-34, mouse anti-mOX40L (MBL, Nagoya, Japan) positive control of expressing as hOX40L.In order to detect bonded people antibody, use the goat Anti-Human IgG antibody of fluorescein (FITC)-put together.In order to detect bonded RM134L, with biotinylated rabbit anti--(DAKO, Glostrup is Denmark) with the streptavidin applied in any combination of (DAKO) puting together with phycoerythrin (PE) for rat IgG antibody.In order to detect bonded TAG-34, the rabbit of using FITC-to put together resists-mouse IgG antibody.Use Graphpad Prism software to determine about in the EC50 value of the HuMabs of 20 μ g/ml detection or the calculating of maximum combined (Bmax) with non-linear regression (S type dose-response) with variable slope.

The result:

Can be according to LC.001 of the present invention in conjunction with hOX40L, this is shown by the EC50 value of 5.16 ± 2.93 μ g/ml and 385.22 Bmax (MFI) value, but debond mOX40L or WT cell, this is shown by 11.41 and 9.67 Bmax (MFI) value respectively.In addition, also can be according to LC.001 of the present invention (IgG4) effectively in conjunction with hOX40L, this is shown by the EC50 value of 8.19 ± 1.05 μ g/ml and 311.30 Bmax (MFI) value, but debond mOX40L or WT cell, and this is shown by 13.47 and 9.58 Bmax (MFI) value respectively.As expected, negative control α-KLH does not combine (Fig. 7) with any cell.Therefore, compare with human OX 40 L, according to OX40L antibody of the present invention show at least 30 times lower with the combining of mouse OX40L.

Embodiment 19

The potentiality of OX40L HuMabs activating complement system

C1q and C3c are in conjunction with ELISA

In order to determine antibody induction C1q of the present invention combination and C3 activatory ability, with ELISA dull and stereotyped antibody and control antibodies bag quilt with serial dilution.Very weakly (The Binding Site, Birmingham is England) as negative control in conjunction with the human IgG 4 of C1q.Comprise human IgG1 (TheBinding Site) and α-KLH (IgG1) as positive control.Subsequently, with the antibody of bag quilt with the C1q of reorganization or as the human serum incubation of the collection in C3 source.In order to detect bonded C1q, use with puted together horseradish peroxidase (HRP) pig (DAKO) anti--the rabbit antibody (DAKO) at C1q of rabbit igg antibody combination.In order to detect activatory C3c (producing) by C3 activation, use with puted together HRP (rabbit PA) is anti-for Jackson ImmunoResearch Laboratories, West Grove-the mouse Anti-Human C3c antibody (DAKO) of mouse IgG antibody combination.In order to assess bag, use the goat Anti-Human IgG antibody of puting together with HRP to manifest the antibody of bag quilt by the difference of efficient.Use Graphpad Prism software to determine about in the EC50 value of the HuMabs of 10 μ g/ml detection or the calculating of maximum combined (Bmax) with non-linear regression (S type dose-response) with variable slope.

The result:

Can be according to LC.001 of the present invention effectively in conjunction with C1q, this is shown by the EC50 value of 2.19 ± 0.42 μ g/ml and 3.089 Bmax (OD405) value.In addition, positive control human IgG1 and anti--KLH can both be effectively in conjunction with C1q, and this is by the EC50 value that is respectively 4.17 ± 1.08 μ g/ml and 2.57 ± 1.51 μ g/ml and be respectively 2.685 and 3.306 Bmax (OD405) value and show.As expected, negative control human IgG 4 debond C1q, this OD405 Bmax value by 0.353 shows.And, having lost ability according to LC.001IgG4x of the present invention in conjunction with C1q, this OD405 Bmax value by 0.357 shows.

When arranging with C1q bonded ability, C3c takes place in the mode that depends on antibody concentration by the deposition of LC.001, has the EC50 value of 2.67 ± 0.16 μ g/ml and 2.614 Bmax (OD405) value.In addition, positive control human IgG1 and anti--KLH can both deposit C3c effectively, and this is the EC50 value of 5.45 ± 0.36 μ g/ml and 2.16 ± 0.26 μ g/ml and is respectively 2.543 and 2.633 Bmax (OD405) value and shows by being respectively.As expected, negative control human IgG 4 does not deposit C3c, and this OD405 Bmax value by 0.095 shows.And, having lost the ability that deposits C3c according to LC.001IgG4x of the present invention, this OD405 Bmax value by 0.090 shows (Fig. 8 and 9).

Embodiment 20

OX40L HuMabs is in conjunction with the potentiality of Fc γ acceptor I, IIa and IIb

The cytotoxicity (ADCC) that the IgG-inductive depends on the cell of antibody is subjected to the regulation and control of the Fc γ acceptor (Fc γ R) on the effector cell.In order to determine the ability of antibodies Fc γ Rs of the present invention, with transfection stably the IIA1.6 cell of people Fc γ RI, Fc γ RIIa, Fc γ RIIb (derive from the restricted dilution of IIA1 cell; Jones, B., etc., J.Immunol.136 (1986) 348-356) and wild-type cell with the antibody and the control antibodies incubation of serial dilution.(The BindingSite Ltd., UK) human IgG 4 (The Binding Site) with debond Fc γ RII is used as negative control to the human IgG2 of debond Fc γ RI.Comprise and be used for Fc γ RI bonded human IgG1 (The Binding Site) and be used for Fc γ RII bonded human IgG 3 (The Binding Site) as positive control.Use the antibody of puting together with phycoerythrin (PE), detect bonded antibody by FACS at human IgG.Use Graphpad Prism software usefulness non-linear regression curve adaptive (variable slope) is determined about the EC50 value of the HuMabs that detects at 10 μ g/ml or the calculating of maximum combined (Bmax).

LC.001 can be effectively in conjunction with Fc γ RI (suitable with contrast IgG1 antibody), this is shown by the EC50 value of 0.11 ± 0.03 μ g/ml and 8041.54 Bmax (MFI) value, but debond Fc γ RIIa and Fc γ RIIb, and this is by being respectively 25,06 and 21,18 Bmax (MFI) value shows.

Compare with LC.001, LC.001IgG4x is more effectively in conjunction with Fc γ RI, and suitable with contrast IgG4 antibody, has the EC50 value of 0.86 ± 0.12 μ g/ml and 6030.07 Bmax (MFI) value.Do not observe combine (Bmax (MFI) value is respectively 21.40 and 19.27) of LC.001 IgG4x and Fc γ RIIa and Fc γ RIIb, and contrast IgG3 antibody can be with it in conjunction with (Bmax (MFI) value is respectively 536.65 and 418.59) (Figure 10).Therefore, comparing with the EC50 value of antibody LC.001, is 8 times for LC.001IgG4x in conjunction with the EC50 value of Fc γ RI.

Embodiment 21

Fc γ RIIIa bonded potentiality on OX40L HuMabs and the NK cell

In order to determine Fc γ RIIIa (CD16) the bonded ability on antibody of the present invention and NK cell (NK) cell, peripheral blood lymphocytes (PBMCs) is separated, and, (resist-CD16 clone 3G8, RDI at the sealing mouse antibodies that exists or do not exist 20 μ g/ml at Fc γ RIIIa, Flanders, NJ) time, with 20 μ g/ml HuMab antibody and control antibodies incubations, to confirm combination by Fc γ RIIIa.Use not with Fc γ RIIIa bonded human IgG2 and IgG4 (The Binding Site) as negative control.Comprise human IgG1 and IgG3 (The Binding Site) conduct for Fc γ RIIIa bonded positive control.Use and FITC-labelled goat F (ab) ₂Anti-Human IgG (Fc) antibody (Protosimmunoresearch, Burlingame, CA) mouse Anti-Human CD56 (NK-cell surface marker) antibody (the BD Biosciences Pharmingen of Zu He PE-mark, San Diego, CA), detect bonded antibody on the NK cell by facs analysis.Determined the maximum combined (Bmax) of the HuMab that 20 μ g/ml detect.

LC.001 can be effectively in conjunction with Fc γ RIIIa (suitable with contrast IgG1 antibody), and this Bmax (MFI) value by 641.37 shows.Adding has been eliminated the combination (with 145.38 background dyeing compare, Bmax (MFI) value be 194.61) of LC.001 with the NK cell at the blocking antibody of Fc γ RIIIa.LC.001 IgG4x debond Fc γ RIIIa, and performance is equivalent to contrast IgG4 antibody, has 170.52 Bmax (MFI) value, this causes approximately only is the Bmax of 10% LC.001IgG4x of LC.001 Bmax.Adding does not influence (Bmax (MFI) value 174.26) (Figure 11) to LC.001 IgG4x in conjunction with having at the blocking antibody of Fc γ RIIIa.

Embodiment 22

HMab_hOX40L and Mab TAG-34 and HUVEC (primary human huve cell/juvenile cell) bonded effect

Description endotheliocyte expression hOX40L (Kotani, A., etc., Immunol.Lett.84 (2002) 1-7).The natural expression hOX40L of Human umbilical vein endothelial cells (HUVEC), and therefore can be used as " endotheliocyte model ".The purpose of this detection is the destiny of determining combine the hOX40L on the HUVEC cell of back with antibody TAG-34 and LC.001.

HUVEC is thawed, and in T175-flask (Sarstedt), in ECG-M substratum+2%FCS, expanded 4 days.With cell inoculation (10.000 cells/well) in 24 hole flat boards.

After 3 days, change substratum into ECG-M+0.5%FCS.The antibody (＜KLH〉(at the antibody of keyhole limpet hemocyanin), TAG-34 or LC.001 are used to induce downward modulation) that adds 10 μ g/ml, and incubation 2,5h or 24h.With TAG-34 or LC.001 the HUVEC cell is dyeed again.

With every kind is that the secondary antibody at human IgG (=＜h 〉) at mouse IgG (=＜m 〉) or Alexa488 mark of the Alexa488 mark of 10 μ g/ml is carried out FACS-dyeing.FACS-is determined in the FACS-scanner (Becton Dickinson) and carries out, and calculates mean fluorecence density (MFI).

＜KLH〉antibody is as nonspecific negative control.

Table 7 shows, add LC.001 2,5 or 24h after all do not cause the downward modulation that OX40L expresses (relatively) with the 4th row and the 5th and 6 row on the HUVEC cell.Yet, add TAG 34 2, behind the 5h and in strong (the about 3 times) downward modulation (the 10th row is gone relatively with the 11st and 12) that all shows behind the 24h hOX40L on the HUVEC cell.

Concentration be 10 μ g/ml according to the fail downward modulation of inducing OX40L on the HUVEC cell, to express of antibody of the present invention.

Table 7

The Mab that is used to reduce	Be used for painted Mab	The secondary Mab that is used for FACS	MFI
			MFI		2.5h	24h
			1. substratum contrast	-	2.5h	24h		<h>	5.17	5.39
2. substratum contrast	LC.001	<h>	1. substratum contrast	-	28.52	24.99		<h>	5.17	5.39
2. substratum contrast	LC.001	<h>	3.<KLH>	-	28.52	24.99	<h>	4.76	4.74
4.<KLH>	LC.001	<h>	3.<KLH>	-	31.44	23.07	<h>	4.76	4.74
4.<KLH>	LC.001	<h>	5.LC.001	-	31.44	23.07	<h>	36.52	30.78
6.LC.001	LC.001	<h>	5.LC.001	-	38.58	38.69	<h>	36.52	30.78
6.LC.001	LC.001	<h>	7. substratum-contrast	-	38.58	38.69	<m>	3.66	3.18
8. substratum-contrast	TAG-34	<m>	7. substratum-contrast	-	31.81	25.32	<m>	3.66	3.18
8. substratum-contrast	TAG-34	<m>	9.<KLH>	-	31.81	25.32	<m>	3.68	3.43
10.<KLH>	TAG-34	<m>	9.<KLH>	-	30.79	31.58	<m>	3.68	3.43
10.<KLH>	TAG-34	<m>	11.TAG-34	-	30.79	31.58	<m>	9.44	7.39
12.TAG34	TAG-34	<m>	11.TAG-34	-	8.97	14.89	<m>	9.44	7.39

Embodiment 23

TAG34, the western blot analysis of LC.001 and LC.005

Preparation 40 and 100ng hOx40L-His (R﹠amp; D system has the theoretical size of 28-34kDa) and molecular weight marker Magik Mark XP (Invitrogen; 20,30,40,50,60,80,100,120,220kDa) be used for gel-electrophoresis.Therefore, with x μ l protein, 2.5 μ l NuPage LDS (lithium dodecyl sulfate salt) sample buffers (4 *), 1 μ l NuPage reductive agent (10 *), and H ₂O adds 10 μ l and mixes, and at 70 ℃ of sex change 10min.After this, with (the Novex to the NuPage gel of sample on the sample; 10%Bis-Tris), electrophoresis 1h and in 150V 1 * MOPS electrophoretic buffer (Novex).

In half-dried chamber, use 1 * NuPage transfering buffering liquid (1 * damping fluid, 0.1% antioxidant, 10% methyl alcohol) to continue 1h/50mA, described gel is passed through half-dried trace (Semi-Dry-Blot) trace (Millipore to pvdf membrane; By incubation 5min in methyl alcohol and in 1 * transfering buffering liquid incubation 10min and with film activation).Under RT, shake 1h, film is sealed in 1 * PBS/5% milk/0,5% tween.One-level antibody (pAB) is diluted in 1 * PBS/1% milk/0.5% tween, add and be incubated overnight at 4 ℃.

LC.001:1.9 μ l (1.6 μ g) is in the cumulative volume of 4ml

LC.005：1.1μl(1.6μg)/4ml

TAG34：1.6μl(1.6μg)/4ml

Film is washed 3 in 1 * PBS/0.5% tween *.Secondary antibody (sAB) is diluted in 1 * PBS/1% milk/0.5% tween, add and at RT incubation 1.5h.For LC.001 and LC.005, with the polyclonal antibody at human IgG (Pierce) of dilution in 1: 10000 as sAB; For TAG34, with dilution in 1: 400 from Lumi-Light western blotting test kit (Roche) at the polyclonal antibody of mouse IgG as sAB.Film is washed 2 * lasting 30min with 1 * PBS/0.5% tween.Directions for use according to the producer uses Lumi-Light western blotting test kit (Roche) to be used for detecting.The result of western blotting shows in Figure 12.LC.001 can detect the OX40L of (dodecyl sulphate) sex change, and LC.005 and TAG34 can not be in conjunction with the OX40L of sex change.

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Sequence table

＜110〉Hoffman-Laluoai Ltd

＜120〉anti-OX 40 L antibodies

<130>22672 WO

<150>EP 04022158

<151>2004-09-17

<150>EP 04030546

<151>2004-12-23

<160>69

<170>PatentIn version 3.2

<210>1

<211>107

<212>PRT

＜213〉artificial

<220>

<223>light chain，variable region of LC.001，LC.059 and LC.063

<400>1

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>2

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain, the variable region of LC.001

<400>2

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ile Ile Ser Gly Ser Gly Gly Phe Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Val Ala Pro Gly Thr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Leu Val Thr Val Ser Ser

115 120

<210>3

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉light chain, the variable region of LC.005

<400>3

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210>4

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain, the variable region of LC.005

<400>4

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ala Ile Trp Tyr Asp Gly His Asp Lys Tyr Tyr Ser Tyr Tyr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ser Ser Ser Trp Tyr Arg Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>5

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉light chain, the variable region of LC.010

<400>5

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210>6

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain, the variable region of LC.010

<400>6

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ala Ile Trp Tyr Asp Gly His Asp Lys Tyr Tyr Ala Tyr Tyr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ser Ser Ser Trp Tyr Arg Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>7

<211>58

<212>PRT

＜213〉artificial

<220>

＜223〉light chain, the variable region of LC.029

<400>7

Met Leu His Pro Leu Cys Lys Val Gly Ser His Gln Gly Ser Val Ala

1 5 10 15

Val Asp Leu Gly Gln Ile Ser Leu Ser Pro Ser Ala Ala Cys Ser Leu

20 25 30

Lys Ile Leu Gln Leu Ile Thr Val Asn Ser Ile Ile Val Ser Leu Thr

35 40 45

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

50 55

<210>8

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain, the variable region of LC.029

<400>8

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ala Ile Trp Tyr Asp Gly His Asp Lys Tyr Tyr Ser Tyr Tyr Val

50 55 60

Lys Gly Arg Phe Thr Ile Sar Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ser Ser Ser Trp Tyr Arg Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>9

<211>57

<212>PRT

＜213〉artificial

<220>

＜223〉light chain, the variable region of LC.019

<400>9

Met Pro Pro Val Trp Lys Val Gly Ser His Gln Gly Ser Ala Ala Val

1 5 10 15

Asp Leu Gly Gln Ile Ser Leu Ser Pro Ser Ala Ala Cys Ser Leu Lys

20 25 30

Ile Leu Gln Leu Ile Thr Val Asn Ser Leu Ile Val Thr Leu Thr Phe

35 40 45

Gly Gly Gly Thr Lys Val Glu Ile Lys

50 55

<210>10

<211>116

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain, the variable region of LC.019

<400>10

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Lys Asn Trp Ser Phe Asp Phe Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<2l0>11

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉light chain, the variable region of LC.033 (a)

<400>11

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Ser Arg Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Val Ser Gly

50 55 60

Ser Gly Pro Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Asp Tyr Cys Gln Gln Arg Ser Asn Trp Gln Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105

<210>12

<211>121

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain, the variable region of LC.033

<400>12

Gln Lys Gln Leu Val Glu Phe Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Asn Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Met Gly Ile Tyr Tyr Tyr Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>13

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉light chain, constant region

<400>13

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>14

<211>330

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain, constant region (γ 1)

<400>14

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>15

<211>327

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain, constant region (γ 4)

<400>15

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr I1e Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>16

<211>104

<212>PRT

＜213〉artificial

<220>

＜223〉light chain, the variable region of LC.033 (b)

<400>16

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Thr Phe

85 90 95

Gly Gln Gly Thr Lys Val Glu Ile

100

<210>17

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain, the variable region of LC.059

<400>17

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ile Ile Ser Gly Ser Gly Gly Phe Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Arg Leu Arg Ala Glu Asp Thr Ala Ile Tyr Phe Cys

85 90 95

Ala Lys Asp Asp Ile Pro Ala Ala Gly Thr Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>18

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉light chain, the variable region of LC.060

<400>18

Ala Ile Gln Leu Thr GlnSer Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Val Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>19

<211>119

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain, the variable region of LC.060

<400>19

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Ile Ser Gly Ser Gly Gly Leu Thr Lys Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ils Ser Arg Asp Asn Ser Lys Arg Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Leu Val Thr Gly Ala Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>20

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain, the variable region of LC.063

<400>20

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ile Ile Ser Gly Ser Gly Gly Phe Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Arg Leu Arg Ala Glu Asp Thr Ala Ile Tyr Phe Cys

85 90 95

Ala Lys Asp Asp Ile Pro Ala Ala Gly Thr Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>21

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>21

Ser Tyr Thr Met His

1 5

<210>22

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>22

Ser Tyr Ala Met Ser

1 5

<210>23

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>23

Asn Phe Gly Met His

1 5

<210>24

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>24

Asn Tyr Gly Met His

1 5

<210>25

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>25

Ser Tyr Ala Met Asn

1 5

<210>26

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>26

Ile Ile Ser Gly Ser Gly Gly Phe Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>27

<2ll>17

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>27

Ala Ile Trp Tyr Asp Gly His Asp Lys Tyr Tyr Ser Tyr Tyr Val Lys

1 5 10 15

Gly

<210>28

<2ll>17

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>28

Ala Ile Trp Tyr Asp Gly His Asp Lys Tyr Tyr Ala Tyr Tyr Val Lys

1 5 10 15

Gly

<210>29

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>29

Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val Lys

1 5 10 15

Gly

<2l0>30

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>30

Val Ile Trp Asn Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val Lys

1 5 10 15

Gly

<210>3l

<211>18

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>31

Ile Ile Ser Gly Ser Gly Gly Phe Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly Arg

<210>32

<211>18

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>32

Leu Ile Ser Gly Ser Gly Gly Leu Thr Lys Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly Arg

<210>33

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>33

Asp Ser Ser Ser Trp Tyr Arg Tyr Phe Asp Tyr

1 5 10

<210>34

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>34

Asp Arg Leu Val Ala Pro Gly Thr Phe Asp Tyr

1 5 10

<210>35

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>35

Lys Asn Trp Ser Phe Asp Phe

1 5

<210>36

<211>12

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>36

Asp Arg Met Gly Ile Tyr Tyr Tyr Gly Met Asp Val

1 5 10

<210>37

<211>12

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>37

Lys Asp Asp Ile Pro Ala Ala Gly Thr Phe Asp Pro

1 5 10

<210>38

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>38

Lys Asp Ile Leu Val Thr Gly A1a Leu Asp Tyr

1 5 10

<210>39

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>39

Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala

1 5 10

<210>40

<211>12

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>40

Arg ALa Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala

1 5 10

<210>41

<211>12

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>41

Arg Ala Ser Gln Ser Val Ser Ser Asn Tyr Leu Ala

1 5 10

<210>42

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>42

Arg Ala Ser Gln Gly Val Ser Arg Tyr Leu Ala

1 5 10

<210>43

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>43

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210>44

<211>24

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>44

Leu Ser Ala Ser Val Gly Asp Arg Va1 Thr Ile Thr Cys Arg Ala Ser

1 5 10 15

Gln Gly Ile Ser Ser Ala Leu Ala

20

<210>45

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>45

Gly Ala Ser Ser Arg Ala Thr

1 5

<210>46

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>46

Ala Ala Ser Ser Leu Gln Ser

1 5

<210>47

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>47

Met Pro Pro Val Trp Lys Val

1 5

<210>48

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>48

Asp Ala Ser Asn Arg Ala Thr

1 5

<210>49

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>49

Leu His Pro Leu Cys Lys Val

1 5

<210>50

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>50

Asp Val Ser Ser Leu Glu Ser

1 5

<210>51

<211>8

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>51

Asn Ser Leu Ile Val Thr Leu Thr

1 5

<210>52

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>52

Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr

1 5

<210>53

<211>8

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>53

Gln Gln Tyr Gly Ser Ser Phe Thr

1 5

<210>54

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>54

Gln Gln Arg Ser Asn Trp Gln Tyr Thr

1 5

<210>55

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>55

Gln Gln Arg Ser Asn Trp Thr

1 5

<210>56

<211>8

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>56

Asn Ser Ile Ile Val Ser Leu Thr

1 5

<210>57

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉CDR/ antibody fragment

<400>57

Gln Gln Phe Asn Ser Tyr Trp Thr Phe

1 5

<210>58

<211>450

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain of LC.001 (human IgG1's type)

<400>58

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ile Ile Ser Gly Ser Gly Gly Phe Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Val Ala Pro Gly Thr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>59

<211>450

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain of LC.001 (L234A, L235A human IgG1 mutant)

<400>59

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ile Ile Ser Gly Ser Gly Gly Phe Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Val Ala Pro Gly Thr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr LysAsn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>60

<211>447

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain of LC.001 (S228P human IgG 4 mutant)

<400>60

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ile Ile Ser Gly Ser Gly Gly Phe Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Val Ala Pro Gly Thr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210>61

<211>214

<212>PRT

＜213〉artificial

<220>

＜223〉light chain of LC.001

<400>61

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr

85 90 95

Thr Phs Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>62

<211>450

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain of LC.005 (human IgG1's type)

<400>62

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ala Ile Trp Tyr Asp Gly His Asp Lys Tyr Tyr Ser Tyr Tyr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ser Ser Ser Trp Tyr Arg Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>63

<211>450

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain of LC.005 (L234A, L235A human IgG1 mutant)

<400>63

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ala Ile Trp Tyr Asp Gly His Asp Lys Tyr Tyr Ser Tyr Tyr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ser Ser Ser Trp Tyr Arg Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>64

<211>447

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain of LC.005 (s228P human IgG 4 mutant)

<400>64

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe

20 25 30

Gly Mer His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ala Ile Trp Tyr Asp Gly His Asp Lys Tyr Tyr Ser Tyr Tyr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ser Ser Ser Trp Tyr Arg Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210>65

<211>214

<212>PRT

＜213〉artificial

<220>

＜223〉light chain of LC.005

<400>65

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Als Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Ash Phe Tyr Pro Arg Glu A1a

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>66

<211>449

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain of LC.060 (human IgG1's type)

<400>66

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Als Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Ile Ser Gly Ser Gly Gly Leu Thr Lys Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Arg Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Leu Val Thr Gly Ala Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro G11n Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

55 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210>67

<211>449

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain of LC.060 (L234A, L235A human IgG1 mutant)

<400>67

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Ile Ser Gly Ser Gly Gly Leu Thr Lys Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Arg Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Leu Val Thr Gly Ala Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His G1n Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210>68

<211>446

<212>PRT

＜213〉artificial

<220>

＜223〉heavy chain of LC.060 (S228P human IgG 4 mutant)

<400>68

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Ile Ser Gly Ser Gly Gly Leu Thr Lys Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Arg Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Leu Val Thr Gly Ala Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210>69

<211>213

<212>PRT

＜213〉artificial

<220>

＜223〉light chain of LC.060

<400>69

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Val Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Ash Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

The Chinese translation of PCT/RO/134 table

Relate to the microorganism of preservation or the indication of other biomaterial

(PCT detailed rules and regulations 13bis)

A. the indication of making below relates at specification sheets 24Page or leaf the 9-20Row (is annotated: explanation in Chinese book the 21Page or leaf the 12-22The microorganism of the preservation of mentioning OK) or other biomaterial
		B. preservation is identified more to be deposited in and is identified on the attached sheet
Preservation organization names Germany microbial preservation center (DSMZ)
		Mascheroder Weg 1b D-38124 Brunswick, preservation mechanism address (comprising postcode and country) Germany
Preservation date 27.07.2004 ^/ 02.09.2004 ^	Preserving number DSMACC2672 ^*... ....
Preservation date 27.07.2004 ^/ 02.09.2004 ^	Preserving number DSMACC2672 ^*... ....	C. this information of other indication (if inapplicable leaving a blank) continues on attached sheet
Biomaterial DSM ACC2672 about preservation ^, DSM ACC2685 ^, DSM ACC2686 ^, DSM ACC2688 ^With DSM ACC2689 ^*, relate to the explanation of expert's solution
		D. make the appointment office (if its indication is not at all appointment offices) of indication
CA (Canada), EP (EUROPEAN PATENT OFFICE), SG (Singapore)
CA (Canada), EP (EUROPEAN PATENT OFFICE), SG (Singapore)		E. Zhi Shi independent explanation (if inapplicable leaving a blank)
Below will submit to international office (the regulation indication is the general aspects of " preservation registration number " for example) after the listed indication
		Only use this page or leaf of to receive with international application for the office of accepting	Only use this page or leaf of to be received by international office on December 16th, 2005 for international office.
Authorize official	Authorize official Wagner, Valhalie

C. additional indication (attached sheet)

The applicant: Hoffman-Laluoai Ltd, etc.

Applicant's document reference number: 22672 WO-SR

International application no: PCT/EP2005/009968

The indication that the indication that expert's solution of the preservation biomaterial of quoting about specification sheets is relevant relates to the biomaterial of preservation all is included in the specification sheets.Following other indication does not require it is the part of specification sheets, and should treat as " indication that separates ".They only relate to expert's solution.The other indication of below making relates in the 24th page in specification sheets (annotating: Chinese the 21st page) and being called:

hu Mab<hOX40L>LC.001 DSMACC2672

hu Mab<hOX40L>LC.005 DSMACC2685

hu Mab<hOX40L>LC.010 DSMACC2686

hu Mab<hOX40L>LC.019 DSMACC2688

hu Mab<hOX40L>LC.033 DSMACC2689

The biomaterial of preservation.

Indication in addition is:

Specify for CA (Canada):

For Canadian appointment, according to Canadian Patent bill patent detailed rules and regulations the 107th and 108 regulations, have only by sample being signed and issued to Independent Expert's (detailed rules and regulations 10 (4)) of chief's nomination, up to authorizing Canadian Patent, or, just can provide the sample of the biomaterial of preservation up to applying for the date out of court or that abandoned and no longer require recovery or be withdrawn.

Specify for EP (Ou Zhuanju):

Appointment about EPO, regulation according to the detailed rules and regulations 28 (3) of EPC detailed rules for the implementation, have only by sample being signed and issued to the expert (detailed rules and regulations 28 (4) EPC) of claimant's nomination, open up to the European patent authorization notification, if perhaps apply for out of court or recall or look and remove, until from 20 years applyings date, just can provide the sample of the biomaterial of preservation.

Specify for SG (Singapore):

The applicant draws attention to thus, and we were intended to according to nineteen ninety-five the 3rd period of patent rule catalogue four, and above-mentioned culture only can offer the expert.

Claims

1. an antibody is characterized in that described antibodies OX40L, contains the Fc fragment and the debond complement factor Clq that derive from the people source.

2. according to the antibody of claim 1, it is characterized in that the human Fc gamma receptor on the described antibody debond NK cell.

3. according to the antibody of claim 1 or 2, it is characterized in that described antibody is people's antibody.

4. according to the antibody of claim 1 or 2, it is characterized in that described antibody is chimeric or humanized antibody.

5. according to each antibody among the claim 1-4, wherein said antibody is with less than 10 ^-8The K of M _DValue combines with OX40L.

6. according to the antibody of claim 5, wherein said K _D10 ^-12-10 ^-9The M scope.

7. according to each antibody among the claim 1-6, it is characterized in that described antibody is the antibody of people's subclass IgG1, comprise one or more sudden changes from PVA236, GLPSS331 and/or L234A/L235A (being numbered) according to the EU index.

8. according to each antibody among the claim 1-6, it is characterized in that described antibody is the antibody of people's subclass IgG4.

9. according to the antibody of claim 8, it is characterized in that containing sudden change S228P.

10. according to the antibody of claim 8 or 9, it is characterized in that containing sudden change L235E.

11., it is characterized in that not activating complement factor C3 of described antibody according to each antibody among the claim 1-10.

12., it is characterized in that it does not cause the cytotoxicity (CDC) that depends on complement according to each antibody among the claim 1-11.

13., it is characterized in that it does not cause the cytotoxicity of the cell that depends on antibody (ADCC) according to each antibody among the claim 1-12.

14. according to each antibody among the claim 1-13, it is characterized in that it shows inhibition in ELISA detects,, suppressed by concentration at the bag of 0.5 μ g/ml OX40L, have the IC50 value of 1nM-4nM scope by the interaction between retardance fixed OX40L and the soluble OX40.

15. an antibody is characterized in that described antibodies OX40L, and described antibody comprises the variable region combination that is independently selected from the group of being made up of following combination

A) by the light chain variable structural domain of aminoacid sequence SEQ ID NO:1 definition and the weight chain variable structural domain that defines by SEQ IDNO:2;

F) by the light chain variable structural domain of aminoacid sequence SEQ ID NO:11 or 16 definition and the weight chain variable structural domain that defines by SEQ ID NO:12;

G) light chain (V that defines by aminoacid sequence SEQ ID NO:1 _L) variable domains and by the heavy chain (V of SEQID NO:17 definition _H) variable domains;

16., it is characterized in that described people's variable region of light chain comprises to be independently selected from NO:1, the aminoacid sequence of 3,5,7,9,11,16 and 18 groups of forming by SEQ ID according to each antibody among the claim 1-15.

17., it is characterized in that described people's variable region of heavy chain comprises to be independently selected from NO:2, the aminoacid sequence of 4,6,8,10,12,17,19 and 20 groups of forming by SEQ ID according to each antibody among the claim 1-16.

18., it is characterized in that described antibody comprises by the light chain variable structural domain of aminoacid sequence SEQ ID NO:1 definition with by SEQ ID NO:2 the weight chain variable structural domains of 17 or 20 definition according to each antibody among the claim 1-16.

19., it is characterized in that described antibody comprises that in the Fc fragment at least 1 makes not and complement factor Clq bonded amino acid mutation according to each antibody among the claim 1-18.

20., it is characterized in that described antibody comprises κ constant region of light chain or the SEQ ID NO:61 by SEQID NO:13 definition, 65 or 69 constant region of light chain according to each antibody among the claim 1-19.

21., it is characterized in that described antibody comprises by SEQID NO:14 or the CH of SEQ ID NO:15 definition or the CH of SEQ ID NO:58 according to each antibody among the claim 1-20.

22. one kind in conjunction with OX40L, comprise and the antibody of variable light chain and variable heavy chain it is characterized in that described variable heavy chain comprises that the CDR3 and/or the described variable light chain that are selected from SEQ ID NOs:33-38 comprise the CDR3 that is selected from SEQ ID NOs:51-57.

23. antibody according to claim 22, it is characterized in that described variable heavy chain comprises CDR1 that is selected from SEQ IDNOs:21-25 and the CDR2 that is selected from SEQ ID NOs:26-32, and/or described variable light chain comprises CDR1 that is selected from SEQ ID NOs:39-44 and the CDR2 that is selected from SEQ ID NOs:45-50.

24. an antibody is characterized in that it is produced by the clone that is selected from the group of being made up of following clone:

hu-Mab<hOX40L>LC.001，

hu-Mab<hOX40L>LC.005，hu-Mab<hOX40L>LC.010，

hu-Mab<hOX40L>LC.019，hu-Mab<hOX40L>LC.029

And hu-Mab＜hOX40L〉LC.033.

25., it is characterized in that described antibody is Fab, F (ab ') according to each antibody among the claim 1-24 ₂Or single-chain fragment.

26. be used to produce method, it is characterized in that coding is with less than 10 according to the antibody of claim 1-25 ^-8The K of M _DValue is modified by this way in conjunction with first nucleotide sequence of the heavy chain of the antibody of OX40L, so that complement factor Clq and/or human Fc gamma receptor on the antibody debond NK cell of described modification, first nucleic acid of described modification and second nucleic acid of encoding said antibody light chain are inserted in the expression vector, described carrier is inserted in protokaryon or the eukaryotic host cell, described host cell is cultivated under the condition that allows synthetic described antibody, and reclaimed described antibody from described culture.

27. according to the method that is used to produce antibody of claim 26, the sequence encoding of described first nucleic acid that it is characterized in that the encoding said antibody heavy chain is according to the heavy chain of the antibody of claim 22.

28. the nucleic acid molecule of each antibody molecule among the claim 1-25 that encodes.

29. carrier that comprises the nucleic acid molecule of claim 28.

30. host cell that comprises the carrier of claim 29.

31. one kind is used for preparing each the method for antibody molecule of claim 1-25, it comprises cultivates the host cell of claim 30 allowing under the described antibody molecule synthetic condition, and reclaims described antibody molecule from described culture.

32. a composition, it comprises among the claim 1-25 antibody molecule that each antibody molecule or the method by claim 26 or 27 produce.

33. the composition of claim 32, it is a kind of pharmaceutical composition or diagnosis composition.

34. a pharmaceutical composition, it comprises among the claim 1-25 each antibody and at least a pharmaceutical excipient.

35. be used for the treatment of the patient's of needs treatments method, it is characterized in that to described patient's administering therapeutic significant quantity according to each antibody among the claim 1-23.

36. the application that the antibody that defines among the claim 1-23 is used for the treatment of.

37. the antibody of each definition is used to prevent and treat the application of inflammatory disease among the claim 1-23.

38. the application that the antibody of each definition is used to prepare prevention and treats the medicine of inflammatory disease among the claim 1-23.

39. test kit, it comprises among the claim 1-25 each antibody molecule, the nucleic acid molecule of claim 28, the carrier of claim 29 or the host cell of claim 30.

40. modify the method for initial aminoacid sequence be selected from the parental antibody heavy chain CDR of the group of forming by SEQ ID NOs:21-38 and/or be selected from the parental antibody light chain CDR of the group of forming by SEQ ID NOs:39-57, it is characterized in that, the nucleic acid of the described initial aminoacid sequence of coding is provided, modify described nucleic acid, wherein, 1 amino acid is modified among the heavy chain CDR1,1-2 amino acid is modified among the heavy chain CDR2,1-2 amino acid is modified among the heavy chain CDR3,1-3 amino acid is modified among the light chain CDR1,1-3 amino acid is modified among the light chain CDR2, and/or 1-3 amino acid is modified among the light chain CDR3, in antibody structure, express the cdr amino acid sequence of described modification, measure described antibody whether with less than 10 ^-8The K of M _DIn conjunction with OX40L, and if described antibody with less than 10 ^-8The K of M _DIn conjunction with OX40L, select the CDR of described modification.

41. an antibody is characterized in that described antibodies OX40L, is human IgG1's kind and comprises gamma heavy chain SEQ ID NO:58,62 or 66.

42. according to the antibody of claim 41, it comprises

A) gamma heavy chain SEQ ID NO:58 and κ light chain SEQ ID NO:61,

B) gamma heavy chain SEQ ID NO:62 and κ light chain SEQ ID NO:65 or

C) gamma heavy chain SEQ ID NO:66 and κ light chain SEQ ID NO:69.

43. an antibody is characterized in that described antibodies OX40L, is the IgG1 kind that comprises the L234A/L235A that suddenlys change, and comprises gamma heavy chain SEQ ID NO:59,63 or 67.

44. according to the antibody of claim 43, it comprises

A) gamma heavy chain SEQ ID NO:59 and κ light chain SEQ ID NO:61,

B) gamma heavy chain SEQ ID NO:63 and κ light chain SEQ ID NO:65 or

C) gamma heavy chain SEQ ID NO:67 and κ light chain SEQ ID NO:69.

45. an antibody is characterized in that described antibodies OX40L, is the IgG4 kind that contains the S228P that suddenlys change, and comprises gamma heavy chain SEQ ID NO:60,64 or 68.

46. according to the antibody of claim 45, it comprises

A) gamma heavy chain SEQ ID NO:60 and κ light chain SEQ ID NO:61,

B) gamma heavy chain SEQ ID NO:64 and κ light chain SEQ ID NO:65 or

C) gamma heavy chain SEQ ID NO:68 and κ light chain SEQ ID NO:69.