NZ788333A - Novel monoclonal antibodies to cytotoxic t-lymphocyte-associated protein 4 (ctla-4) - Google Patents

Abstract

The present invention provides CTLA-4 monoclonal antibodies, particularly humanized monoclonal antibodies specifically binding to CTLA-4 with high affinity. The present invention also provides functional monoclonal antibodies cross-reactive to CTLA-4 of human, cynomolgus monkey and mouse. The present invention further provides amino acid sequences of the antibodies of the invention, cloning or expression vectors, host cells and methods for expressing or isolating the antibodies. The epitopes of the antibodies are identified. Therapeutic compositions comprising the antibodies of the invention are also provided. The invention also provides methods for treating cancers and other diseases with anti-CTLA-4 antibodies. t invention further provides amino acid sequences of the antibodies of the invention, cloning or expression vectors, host cells and methods for expressing or isolating the antibodies. The epitopes of the antibodies are identified. Therapeutic compositions comprising the antibodies of the invention are also provided. The invention also provides methods for treating cancers and other diseases with anti-CTLA-4 antibodies.

Description

Novel onal antibodies to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) Technical Field The present invention relates generally to antibodies against CTLA—4 and compositions thereof, and immunotherapy in the treatment of cancer, infections or other human diseases using anti—CTLA—4 antibodies.

Background of the Invention Cancer immunotherapy has become a hot research area of treating cancer. xic T—lymphocyte—associated protein 4 (CTLA—4) is one of the validated targets of immune checkpoints. After T cell activation, CTLA—4 quickly expresses on those T cells, generally Within one hour of antigen engagement With TCR. CTLA—4 can inhibit T cell signaling h competition With CD28. CD28 es one of well terized T cell co—stimulatory signal: CD28 binding to its ligands CD80 (B7—1) and CD86 (B7—2) on antigen presenting cells leads to T cell proliferation by inducing production of eukin—2 and poptotic factors. Due to much higher affinity binding of CTLA—4 to CD80 and CD86 than that of CD28, CTLA—4 can out—compete With CD28 binding on CD80 and CD86, leading to suppression of T cell tion. In addition to induced expression on activated T cells, CTLA—4 is constitutively expressed on the surface of regulatory T cells (Treg), suggesting that CTLA—4 may be required for t—mediated suppression and associated With Treg production of immunosuppressive cytokines such as transforming growth factor beta and iterleukin— 10.

CTLA—4 blockade can induce tumor regression, demonstrating in a number of preclinical and al studies. Two antibodies t CTLA—4 are in clinical pment. Ipilimumab (MDX—OlO, EMS—734016), a fully human anti—CTLA—4 monoclonal antibody of IgGl—kappa isotype, is an modulatory agent that has been approved as monotherapy for treatment of advanced melanoma. The proposed mechanism of action for Ipilimumab is interference of the interaction of CTLA—4, sed on a subset of activated T cells, With CD80/CD86 les on professional antigen ting cells. This results in T—cell potentiation due to blockade of the inhibitory modulation of T—cell activation promoted by the CTLA—4 and CD80/CD86 interaction. The resulting T—cell activation, proliferation and lymphocyte infiltration into tumors, leads to tumor cell death. The commercial dosage form is a 5 mg/mL concentrate for solution for infusion.

Ipilimumab is also under clinical investigation of other tumor types, including prostate and lung cancers. Another anti—CTLA—4 antibody Tremelimumab was evaluated as monotherapy in melanoma and malignant mesothelioma.

Disclosure of the Invention The present invention provides isolated antibodies, in particular monoclonal antibodies or humanized monoclonal dies.

In one aspect, the present invention provides an antibody or an n binding—fragment thereof, wherein the antibody or the antigen binding—fragment binds to human, monkey and mouse CTLA—4.

The aforesaid antibody or the antigen binding—fragment inhibits CTLA—4 binding to CD80 or CD86.

In the aforesaid antibody or the antigen binding—fragment, binding epitope of the antibody or antigen binding—fragment comprises N145 or polysaccharide on N145 of CTLA—4.

In one aspect, the present invention provides an antibody or an antigen binding fragment thereof, wherein the antibody or the antigen binding fragment binds to human, monkey CTLA—4, wherein binding epitope of the antibody or antigen binding—fragment comprises P138 of CTLA—4.

In one aspect, the present invention provides an antibody or an antigen binding fragment thereof, wherein the antibody or the n binding—fragment a) binds to human CTLA—4 with a KB of 4.77E—10 M or less; and b) binds to mouse CTLA—4 with a KB of 1.39E—09 M or less.

The aforesaid antibody, wherein the antibody or the antigen binding—fragment exhibits at least one of the following properties: a) binds to human CTLA—4 with a KB of between 4.77E—10 M and 2.08E—10 M and to mouse CTLA—4 with a KB of between 1.39E—09 M and 9.06E—10 M; b) enhances eukin—2 e from stimulated PBMCs. c) does not ntially bind to any protein selected from a group ting of Factor VIII, FGFR, PD—l, CD22, VEGF, CD3, HER3, 0X40, and 4—1BB.

The present invention provides an antibody or an antigen binding nt thereof, comprising an amino acid ce that is at least 70%, 80%, 90% or 95% homologous to a sequence selected from a group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, wherein the antibody or the n binding—fragment specifically binds to CTLA—4.

The t invention provides an antibody or an n binding fragment thereof, comprising an amino acid sequence selected from a group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, wherein the antibody or the antigen binding—fragment specifically binds to CTLA—4.

The present invention provides an antibody, or an antigen—binding fragment thereof, comprising: a) a le region of a heavy chain having an amino acid sequence that is at least 70%, 80%, 90% or 95% homologous to a sequence selected from a group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7; and b) a variable region of a light chain having an amino acid sequence that is at least 70%, 80%, 90% or 95% homologous to a sequence selected from a group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14, wherein the dy or the antigen g—fragment specifically binds The present ion provides an antibody or an antigen binding fragment thereof, comprising: a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7; b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and wherein the antibody or the antigen binding—fragment specifically binds to CTLA—4.

WO 09701 In various ments, the antibody or an antigen binding fragment thereof comprises: a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; and b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, wherein the antibody or the antigen binding—fragment specifically binds or the antibody or an antigen binding fragment thereof comprises: a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 2; and b) a variable region of a light chain having an amino acid sequence ed from the group consisting of SEQ ID NO: 9, wherein the antibody or the antigen binding—fragment specifically binds to CTLA—4; or the antibody or an antigen binding fragment f comprises: a) a le region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 3; and b) a le region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, n the dy or the antigen binding—fragment specifically binds to CTLA—4; or the antibody or an antigen binding fragment thereof comprises: a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4; and b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 11, wherein the antibody or the antigen binding—fragment specifically binds to CTLA—4; or the antibody or an antigen binding fragment thereof comprises: a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 5; and b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, wherein the dy or the antigen binding—fragment specifically binds or the antibody or an antigen g fragment thereof comprises: a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6; and b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 13, wherein the antibody or the antigen binding—fragment specifically binds to CTLA—4; or the antibody or an antigen binding fragment thereof comprises: a) a variable region of a heavy chain having an amino acid sequence ed from the group ting of SEQ ID NO: 7; and b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, n the antibody or the antigen binding—fragment specifically binds to CTLA—4; The sequence of said antibody is shown in Table l and Sequence Listing.

Table l Deduced amino acid ces of the antibodies EEQLVESGGGLVQPGKSLKLSCSASGFTFRSSAMHWIRQ Heavy chain 1 PPGKGLDWVAFISSGGDTAYADAVKGRFIVSRDNAENTL W3162-1.1 FLQLNSLKSEDTAIYYCVRMERIPTWGQGVMVTVSS 01.2 -_ DIVLTQSPVLAVSLGQRATISCRASQSVSISSINLIHWYQQ Light chain RPGQQPKLLIYRTSNLASGIPARFSGSGSGTDFTLSIDPV QADDVADYYCQQSRESPLTFGSGTKLEIK EVQLVESGGGLVQPGRSLKLSCAASDLTFSNYDMAWVR W3162—1. Heavy chain 2 QTPTKGLEWVASISPNGGNTYYRDSVKGRFTVSRDNAK NSLYLQMDSLRSEDTATYYCARHLWFAYWGQGTLVTVS 145.10——— --DIQMTQSPSSMSASLGDRVTISCQASQDIGSNLIWFQQKPGKSPRPMIYYATHLADGVPSRFSGSRSGSDYSLTISSLESEDVADYHCLQYKQYPRTFGGGTKLELK SGPGLVKPSQSLSLTCSVTYHTITSGYDWTWIR MEWMGYISYSGNTNYNPSLKSRISITRDTSKN H h ' W3162—1.1 QFFLHLNSVTSEDTATYYCASMMVPHYYVMDAWGQG ASVTVSS 46.19 DVVLTQTPPTSSATIGQSVSISCRSSQSLLNSDGNTYLYW YLQRPSQSPQLLIYLVSKLGSGVPNRFSGSGSGTDFTLKI SGVEAEDLGLYYCVQGTHDPWTFGGGTKLELK EVQLQQSGPEAGRPGSSVKISCKASGYTFTNYFMNWVK QSPGQGLEWIGRVDPENGRADYAEKFKKKATLTADTTS H h ' W3162—1 .1 NTAYIHLSSLTSEDTATYFCARRAMDNYGFAYWGQGTL VTVSS 54.8 EIMLTQSPTIMAASLGEKITITCSANSSLSYMYWFQQKS GASPKLWVHGTSNLASGVPDRFSGSGSGTSYYLTINTM EAEDAATYFCHHWSNTQWTFGGGTKLELK EVQLVESGGGLVQPGGSLRLSCAASDLTFSNYDMAWVR ' LEWVASISPSGGNTYYRDSVKGRFTISRDNAK H h W3162—1 .1 NSLYLQMNSLRAEDTAVYYCARHLWFAYWGQGTLVTV 45.10—z7 DIQMTQSPSSLSASVGDRVTITCQASQDIGSNLIWFQQKP GKAPKPMIYYATHLADGVPSRFSGSRSGTDYTLTISSLQP EDFATYYCLQYKQYPRTFGGGTKVEIK QVQLQESGPGLVKPSETLSLTCSVTYHTITSGYDWTWIR KPPGKGMEWIGYISYSGNTNYNPSLKSRVTISRDTSKNQ Hea chain FFLKLSSVTAADTAVYYCASMMVPHYYVMDAWGQGTL VTVSS 6.—z12 DIVMTQTPLSLSVTPGQPASISCRSSQSLLNSDGNTYLY WYLQKPGQSPQLLIYLVSKLGSGVPNRFSGSGSGTDFTL AEDVGVYYCVQGTHDPWTFGGGTKVEIK QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYFMNWV ' RQAPGQGLEWMGRVDPEQGRADYAEKFKKRVTITADK H h STSTAYMELSSLRSEDTAVYYCARRAMDNYGFAYWGQ GTLVTVSS 4.8—z35 EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKP DQSPKLWVHGTSNLASGVPSRFSGSGSGTDFTLTINSLE AEDAATYYCHHWSNTQWTFGGGTKVEIK In another aspect, the invention provides an dy or an antigen binding fragment f, comprising a complementarity—determining region (CDR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15—41, wherein the antibody or the antigen binding—fragment specifically binds to CTLA—4.

In another aspect, the ion provides an antibody, or an antigen binding fragment thereof, comprising: a heavy chain variable region comprising CDRl, CDR2, and CDR3 sequences; and a light chain variable region comprising CDRl, CDR2, and CDR3 sequences, wherein the heavy chain variable region CDR3 sequence comprises an amino acid sequence ed from a group consisting of SEQ ID NOs: 15, 16, 17, and 18, and conservative modifications f, wherein the antibody or the antigen g—fragment specifically binds ably, wherein the light chain variable region CDR3 sequence of the aforesaid antibody or antigen g fragment thereof comprises an amino acid sequence selected from a group consisting of SEQ ID NOs: 19, 20, 21, and 22, and conservative modifications thereof.

Preferably, wherein the heavy chain variable region CDR2 sequence of the aforesaid antibody or antigen g fragment thereof comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 23, 24, 25, 26, 27, and 28, and vative modifications thereof.

Preferably, wherein the light chain variable region CDR2 sequence of the aforesaid antibody or antigen binding fragment thereof comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 29, 30, 31, and 32, and conservative modifications f.

Preferably, wherein the heavy chain variable region CDRl sequence of the aforesaid antibody or antigen binding fragment thereof comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 33, 34, 35, and 36, and conservative modifications thereof.

Preferably, the antibody of this invention, wherein the light chain variable region CDRl sequence of the aforesaid antibody or antigen binding fragment thereof comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 37, 38, 39, 40, and 41, and conservative modifications thereof.

In more preferred embodiment, the invention provides an antibody, or an antigen binding fragment thereof, wherein the dy or antigen binding nt ically binds to CTLA—4 and comprises: a heavy chain variable region that comprises CDRl, CDR2, and CDR3 sequences; and a light chain variable region that comprises CDRl, CDR2, and CDR3 sequences, wherein: a) the heavy chain variable region CDRl sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 33, 34, 35, and 36, and CDR2 ce comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 23, 24, 25, 26, 27, and 28, CDR3 ce ses an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 15, 16, 17, and 18; b) and the light chain variable region CDRl sequence comprises an amino acid ce selected from a group consisting of amino acid sequences of SEQ ID NOs: 37, 38, 39, 40, and 41, and CDR2 sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 29, 30, 31, and 32, CDR3 sequence ses an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 19, 20, 21, and 22, wherein the antibody or the antigen binding—fragment ically binds to CTLA—4.

A preferred antibody or an antigen binding fragment thereof comprises: a) a heavy chain variable region CDRl comprising SEQ ID NO: 15; b) a heavy chain variable region CDR2 comprising SEQ ID NO: 23; C) a heavy chain variable region CDR3 comprising SEQ ID NO: 33; d) a light chain variable region CDRl comprising SEQ ID NOs: l9; e) a light chain variable region CDR2 comprising SEQ ID NOs: 29; f) a light chain variable region CDR3 comprising SEQ ID NOs: 37; wherein the antibody or the antigen binding—fragment specifically binds to CTLA—4.

Another preferred antibody or an antigen binding fragment thereof comprises: a) a heavy chain variable region CDRl comprising SEQ ID NO: 16; b) a heavy chain variable region CDR2 comprising SEQ ID NOs: 24; c) a heavy chain variable region CDR3 comprising SEQ ID NOs: 34; d) a light chain variable region CDRl comprising SEQ ID NOs: 20; e) a light chain variable region CDR2 comprising SEQ ID NO: 30; f) a light chain variable region CDR3 comprising SEQ ID NO: 38; n the dy or the antigen binding—fragment specifically binds Another preferred antibody or an antigen g fragment thereof comprises: a) a heavy chain variable region CDRl comprising SEQ ID NO: 17; b) a heavy chain variable region CDR2 sing SEQ ID NO: 25; c) a heavy chain variable region CDR3 comprising SEQ ID NO: 35; d) a light chain le region CDRl comprising SEQ ID NO: 19; e) a light chain variable region CDR2 comprising SEQ ID NO: 3 l; f) a light chain variable region CDR3 comprising SEQ ID NO: 39; wherein the antibody or the antigen binding—fragment ically binds to CTLA—4.

Another preferred antibody or an antigen binding fragment thereof comprises: WO 09701 a) a heavy chain variable region CDRl sing SEQ ID NO: 18; b) a heavy chain variable region CDR2 comprising SEQ ID NO: 26; C) a heavy chain variable region CDR3 comprising SEQ ID NO: 36; d) a light chain variable region CDRl comprising SEQ ID NO: 22; e) a light chain variable region CDR2 comprising SEQ ID NO: 32; f) a light chain variable region CDR3 comprising SEQ ID NO: 40; wherein the antibody specifically binds to CTLA—4.

Another red antibody or an antigen binding fragment thereof comprises: a) a heavy chain le region CDRl comprising SEQ ID NO: 16; b) a heavy chain variable region CDR2 comprising SEQ ID NO: 27; c) a heavy chain variable region CDR3 comprising SEQ ID NO: 34; d) a light chain variable region CDRl comprising SEQ ID NO: 20; e) a light chain variable region CDR2 comprising SEQ ID NO: 30; f) a light chain variable region CDR3 comprising SEQ ID NO: 38; wherein the antibody or the antigen binding—fragment specifically binds to CTLA—4.

Another preferred antibody or an n binding fragment thereof comprises: a) a heavy chain variable region CDRl comprising SEQ ID NO: 17; b) a heavy chain variable region CDR2 comprising SEQ ID NO: 25; c) a heavy chain variable region CDR3 comprising SEQ ID NO: 35; d) a light chain variable region CDRl comprising SEQ ID NO: 21; e) a light chain variable region CDR2 comprising SEQ ID NO: 3 l; f) a light chain variable region CDR3 comprising SEQ ID NO: 39; wherein the dy or the antigen binding—fragment specifically binds to CTLA—4.

Another red antibody or an antigen binding fragment thereof comprises: a) a heavy chain variable region CDRl comprising SEQ ID NO: 18; WO 09701 b) a heavy chain variable region CDR2 comprising SEQ ID NO: 28; C) a heavy chain variable region CDR3 comprising SEQ ID NO: 36; d) a light chain variable region CDRl comprising SEQ ID NO: 22; e) a light chain le region CDR2 comprising SEQ ID NO: 32; f) a light chain variable region CDR3 comprising SEQ ID NO: 41; wherein the antibody or the antigen binding—fragment specifically binds to CTLA—4.

The CDR sequences of said dies are shown in Table 2 and Sequence Listing.

Table 2 The CDR sequences of the dies SBQID SEQlD SEQlD Clone ID. CDRl CDR2 CDR3 NO NO NO W3162—1.1 Heavy HSSGGDTAYADAV 33 SSAMH 23 15 MERIPT 01.2 chain KG light 37 RASQSVSISSINLIH 29 RTSNLAS 19 QQSRESPLT chain W3 162—1 .1 Heavy SISPNGGNIYYRDS 34 NYDMA 24 16 HLWFAY 45.10 chain VKG light 38 QASQDIGSNLI YATHLAD LQYKQYPRT chain W3 162—1 .1 Heavy YISYSGNTNYNPSL lVflVIVPHYYVMD SGYDWT 25 17 46.19 chain KS A light RSSQSLLNSDGNTY 39 31 LVSKLGS 21 VQGTHDPWT chain LY W3162—1.1 Heavy RVDPENGRADYAE 36 NYFMN 26 18 RAMDNYGFAY 54.8 chain KFKK light SANSSLSYMY 32 GTSNLAS 22 HHWSNIQWT chain W3162—1 .1 Heavy SlSPSGGNTYYRDS 34 NYDMA 27 16 HLWFAY 45 . l 0—z7 chain VKG light 38 QASQDIGSNLI YATHLAD LQYKQYPRT chain W3 162. 1 .14 Heavy YISYSGN'INYNPSL MMVPHYYVMD SGYDWT 25 17 6.—z 12 chain KS A light LNSDGNTY 39 31 LVSKLGS 21 VQGTHDPWT chain LY W3 1 62. l . l 5 Heavy RVDPHQGRADYAE 36 NYFMN 28 18 RAMDNYGFAY 4.8—z3 5 chain KFKK WO 09701 light chain The antibodies of the ion can be chimeric antibody.

The dies of the invention can be humanized antibody.

The antibodies of the invention can be fully human antibody.

The antibodies of the invention can be rat antibody.

The antibodies or the antigen binding fragment thereof of the invention can exhibit at least one of the following properties: a) binds to human CTLA—4 With a KB of 2.08E—09 M or less and/or to mouse CTLA—4 With a KB of 1.39E—09 M or less; b) enhances interleukin—2 release from the ated PBMCs; In a further aspect, the invention provides a nucleic acid molecule encoding the antibody, or antigen binding fragment thereof.

The invention es a cloning or expression vector comprising the c acid molecule encoding the antibody, or antigen binding fragment thereof.

The invention also provides a host cell comprising one or more g or BXPI‘BSSlOH V€CtOI‘S.

In yet another aspect, the invention provides a process, comprising ing the host cell of the invention and isolating the antibody, wherein the antibody is prepared through immunization in a SD rat with human CTLA—4 extracellular domain and mouse CTLA—4 extracellular domain.

The invention es a transgenic animal such as rat comprising human immunoglobulin heavy and light chain transgenes, wherein the rat expresses the antibody of this invention.

The invention es hybridoma prepared from the rat of this invention, wherein the hybridoma produces said antibody.

In a further aspect, the invention provides pharmaceutical composition comprising the antibody, or the antigen binding nt of said antibody in the invention, and one or more of a pharmaceutically acceptable excipient, a diluent or a carrier.

The invention provides an immunoconjugate comprising said antibody, or antigen—binding fragment thereof in this invention, linked to a therapeutic agent.

Wherein, the invention provides a pharmaceutical composition comprising said immunoconjugate and one or more of a pharmaceutically acceptable excipient, a diluent or a carrier.

The invention also provides a method for ing an anti—CTLA—4 antibody or an antigen—binding fragment thereof comprising: (a) providing: (i) a heavy chain variable region antibody sequence comprising a CDRl sequence that is ed from a group consisting of SEQ ID NOs: 33—36, a CDR2 sequence that is selected from a group consisting of SEQ ID NOs: 23—28; and a CDR3 sequence that is ed from the group ting of SEQ ID NOs: 15—18; and/or (ii) a light chain variable region antibody sequence comprising a CDRl ce that is selected from the group consisting of SEQ ID NOs: 37—41, a CDR2 sequence that is selected from the group consisting of SEQ ID NOs: 29—32, and a CDR3 sequence that is selected from the group consisting of SEQ ID NOs: 19—22; and (b) sing the altered antibody sequence as a protein.

The invention also provides a method of modulating an immune response in a subject comprising administering to the subject the antibody, or n binding fragment of any one of said antibodies in this invention.

The invention also provides the use of said antibody or the antigen binding fragment f in the manufacture of a medicament for the treatment or prophylaxis of an immune disorder or cancer.

The invention also provides a method of inhibiting growth of tumor cells in a subject, comprising administering to the subject a eutically effective amount of said antibody, or said antigen—binding fragment to inhibit growth of the tumor cells.

Wherein, the invention provides the method, wherein the tumor cells are of a cancer selected from a group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone , pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, and rectal cancer.

Wherein, the invention provides the , wherein the dy is a chimeric dy, humanized antibody, human dy or rat antibody.

The features and advantages of this invention The inventors have generated humanized antibodies against CTLA—4 utilizing the proprietary hybridoma technology, wherein the antibodies inhibited CTLA—4 binding to its ligands CD80 and CD86. The antibodies reported in this ion have high binding affinity, specifically binding to both human and monkey CTLA—4 protein; and potent modulating immune responses and sing interleukin—2 production.

One of the antibodies not only bound to human and monkey CTLA—4, but also bound to murine CTLA—4, which could greatly facilitate preclinical validation of its efficacy in mouse tumor models.

Brief Description of the Drawings Figure 1 shows graphs of chimeric antibodies binding to human CTLA—4 in ELISA.

Figure 2 shows graphs of ic antibodies binding to cyno CTLA—4 in ELISA.

Figure 3 shows graphs of chimeric antibodies binding to mouse CTLA—4 in ELISA.

Figure 4 shows graphs of chimeric antibodies g to human CTLA—4 on cells by FACS.

Figure 5 shows the result of chimeric antibodies binding on human CTLA—4 by SPR.

Figure 6 shows the result of chimeric antibodies blocking ligand binding.

Figure 7 shows graphs of chimeric Abs inhibited CTLA—4 binding on CD80— or CD86— expressing cells.

Figure 8 shows the results of ic antibodies enhanced cytokine release from SEB stimulated PBMCs.

Figure 9 shows graphs of humanized antibodies binding to human, cynomolgus monkey and mouse CTLA—4 in ELISA.

Figure 10a shows graphs of humanized antibodies binding to CTLA—4 on cells (FACS).

Figure 10b shows graphs of affinity of humanized antibodies by FACS.

Figure 11 shows that humanized antibodies block ligand binding by ELISA.

Figure 12 shows that humanized antibodies block CTLA—4 g to its s by FACS.

Figure 13 shows that humanized antibodies enhance cytokine release in SEB assay.

Figure 14 shows the SEC profile of W3162—1.146.19—212 or W3162—1.154.8—Z35 at different conditions.

Figure 15 shows the result of in vivo efficacy of antibody W3162—146.19—z12.

Figure 16 shows that W3162 antibodies specifically bind to CTLA—4.

Figure 17. Binding activity of anti—CTLA4 antibodies with human CTLA—4/CTLA—4 mutants. (A) Ipilimumab, (B) W3162—1.146.19—z12 and (C) W3162—1.154.8—z35 antibodies were captured with pre—coated with 2 pig/ml goat—anti—human—IgG Fc antibody, and then incubated with diluted —His (WT) or its muteins (N113Q and N145Q), then HRP—anti—His antibody was added for detection.

Figure 18 shows the binding residues or epitopes mapped on human CTLA—4: (A) Binding sites of CD80 (PDB2118L), (B) CD86 (PDB21185), (C) tremelimumab (PDB: 5GGV), (D) umab, (E) W3162—1.146.19—z12 and (F) 1.154.8—z35, tively. The CTLA—4 structure of IAH1 was used for D—F to show the structure of glycosylation.

Detailed description In order that the present invention may be more readily understood, n terms are first defined. Additional definitions are set forth throughout the detailed description.

The terms oxic T lymphocyte—associated antigen—4”, in CTLA—4”, “CTLA—4”, “CTLA4”, “CD152” are used interchangeably, and include variants, isoforms, species homologs of human CTLA—4 or CTLA—4 of other species, and analogs having at least one common epitope with CTLA—4.

The term “antibody” as referred to herein includes whole antibodies and any n— binding fragment (i.e., "antigen—binding portion") or single chains thereof. An "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter—connected by disulfide bonds, or an antigen—binding n thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain nt region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework s (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino—terminus to carboxy—terminus in the following order: FRl, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a g domain that interacts with an antigen.

The term "antibody," as used in this disclosure, refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen—binding site, regardless whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, ecific, zed, single—chain, chimeric, WO 09701 synthetic, recombinant, hybrid, mutated, and grafted antibodies. The term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen—binding function, i.e., the ability to bind CTLA—4 specifically. Typically, such fragments would comprise an n—binding fragment.

The terms "antigen—binding nt," "antigen—binding domain," and "binding nt" refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between the antibody and the antigen. In instances, where an antigen is large, the antigen—binding nt may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen—binding nt is referred to as "epitope" or "antigenic determinant." An n—binding fragment typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it does not arily have to comprise both. For e, a so—called Fd dy fragment consists only of a VH domain, but still retains some antigen—binding function of the intact antibody.

In line with the above the term "epitope" defines an antigenic determinant, which is specifically bound/identified by a binding fragment as defined above. The binding fragment may specifically bind to/interact with conformational or continuous epitopes, which are unique for the target structure, e. g. the human CTLA—4 and murine CTLA—4. A conformational or discontinuous epitope is characterized for polypeptide antigens by the presence of two or more discrete amino acid residues which are ted in the primary sequence, but come together on the surface of the molecule when the polypeptide folds into the native protein/antigen. The two or more discrete amino acid residues contributing to the epitope are present on separate sections of one or more polypeptide chain(s). These residues come together on the surface of the molecule When the polypeptide chain(s) fold(s) into a three—dimensional structure to constitute the epitope. In contrast, a continuous or linear epitope consists of two or more discrete amino acid residues, Which are present in a single linear segment of a polypeptide chain.

The term "binds to an e of CTLA—4" refers to the antibodies have specific binding for a particular epitope of CTLA—4, Which may be d by a linear amino acid sequence, or by a tertiary, i.e., three—dimensional, conformation on part of the CTLA—4 polypeptide. Binding means that the dies affinity for the portion of CTLA—4 is substantially greater than their affinity for other related polypeptides. The term “substantially greater affinity” means that there is a measurable increase in the affinity for the portion of CTLA—4 as compared With the affinity for other related polypeptides. Preferably, the affinity is at least ld, , 5—fold d, ld, 103—fold, 104—fold, 105—fold, 106—fold or r for the particular portion of CTLA—4 than for other proteins. ably, the binding affinity is determined by enzyme—linked immunoabsorbent assay (ELISA), or by fluorescence—activated cell sorting (FACS) analysis or surface Plasmon nce (SPR). More preferably, the binding specificity is obtained by cence—activated cell sorting (FACS) analysis.

The term "cross—reactivity" refers to binding of an antigen fragment described herein to the same target molecule in human, monkey, and/or murine (mouse or rat). Thus, "cross—reactivity" is to be understood as an interspecies reactiVity to the same molecule X expressed in different species, but not to a molecule other than X. Cross—species icity of a monoclonal antibody recognizing e.g. human CTLA—4, to monkey, and/or to a murine (mouse or rat) CTLA—4, can be determined, for instance, by FACS analysis.

As used herein, the term "subject" includes any human or nonhuman animal.

The term "nonhuman animal" includes all vertebrates, e.g., mammals and non—mammals, such as an primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except when noted, the terms "patient" or "subject" are used interchangeably.

The terms "treatment" and "therapeutic method" refer to both therapeutic treatment and prophylactic/preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder.

The terms "conservative modifications" i.e., tide and amino acid sequence modifications which do not icantly affect or alter the binding characteristics of the antibody d by the nucleotide sequence or containing the amino acid sequence. Such conservative sequence modifications include nucleotide and amino acid tutions, ons and deletions. Modifications can be introduced into the sequence by standard techniques known in the art, such as site—directed mutagenesis and PCR—mediated mutagenesis. Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These es include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, ne, cysteine, tryptophan), ar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta—branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The experimental methods in the following examples are conventional methods, unless otherwise specified.

Examples e 1: ch materials preparation 1. Expression and purification of soluble CTLA-4 Human and mouse CTLA—4 extracellular domain (ECD) genes with hexahistidine (6xHis)— or Fc—tag were cloned into expression vector, and then used for ection of Expi293 cells using Expi293 Expression System Kit.

The cells were cultured in Expi293 sion Medium, on an orbital shaker rm ng at 135 rpm, in a 37 OC incubator containing a humidified atmosphere with 8% C02. The harvested atant was used for protein purification. Hexahistidine—tagged proteins were purified using Ni—NTA column and Fc—tagged proteins were purified using Protein A column. 2. Cell lines development The gene of full length Human CTLA—4 was cloned into an expression vector for development of stable cell line. Briefly, a volume of 30 mL 293F cells at a density of lxlO6/mL was transfected with 30 ug DNA using Plasfect Reagent. The transfected cells were put into in an incubator setting at 37 OC, 8% C02 and 100 rpm shaking speed. 24—48 hours after transfection, blasticidin at a final concentration of 4—6 ug/mL was used to select the stable clones. The selected clones were tested by FACS using an anti—CTLA—4 antibody.

In order to obtain cells expressing cynomolgus monkey CTLA—4, the gene of full length cynomolgus monkey CTLA—4 was cloned into an expression vector for development of cell pool. Briefly, a volume of 30 mL 293F cells at a density of lxlO6/mL was ected with 30 ug DNA using Plasfect Reagent (Life Technology). The transfected cells were put into in an incubator setting at 37 OC, 8% C02 and 100 rpm shaking speed. 24 hours after ection, blasticidin at a final concentration of 4 ug/ mL was used to select the cell pool. The selected cell pools were tested by FACS using an anti—CTLA—4 antibody Example 2: Antibody oma generation 1. Immunization Human CTLA—4 and murine CTLA—4 were used for immunization of SD rats.

Specifically, three SD rats were immunized with 30 pig/animal of human and mouse CTLA—4 ECD protein in adjuvant. The adjuvant included Titer—Max, Adju—Phos and CpG—ODN. The rats were injected once a week both from footpad and aneously. The antibody titer in serum was measured by ELISA every one month. When the antibody titer was sufficiently high, the rat with the highest titer was given a final boost with human and mouse CTLA—4 ECD protein in Dulbecco's Phosphate Buffered Saline (DPBS) t adjuvant. After several days, the spleen and lymph nodes were taken from the rat, and lymphocytes were separated for fusion. 2. Cell fusion The cell fusion was performed as following: myeloma cells SP2/0 cells were thawed the week before the , and were split at 1:2 every day until the day before the fusion to keep them in logarithmic growth. B lymphocytes isolated from lymph node of immunized rat and myeloma cells were respectively treated with trypsin and the reaction was stopped by adding FBS.

B lymphocytes were combined with myeloma cells at 1 : 1 ratio. The cell mixture was then washed and re—suspended at 2><106 ml in electric fusion solution containing 0.3 M sucrose, 0.1 mM magnesium acetate and 0.1mM calcium acetate. The electric cell fusion was conducted using th Electro Cell Manipulator (Ecm 2001) following the manufacturer’s standard protocol. Then the cell suspension from the fusion chamber was immediately transferred into a sterile flask containing fresh medium, and incubated for 2 hours in 37 OC incubator. The cell suspension was then mixed and transferred into 60 of 96—well plates (1x104 cells/well). The 96—well plates were ed at 37 OC and 5% C02 with periodically monitoring. When the clones were big enough (after 7—10 days), 180 ML /well of supernatant were d and then 200 ML fresh medium per well was add. After 72 hours, 100 ML of supernatant were transferred from the tissue culture plates to 96—well assay plates for screening. 3. Hybridoma screening A large number of hybridoma clones were screened on binding to human, murine and monkey CTLA—4 proteins as well as ered human CTLA—4 expressing cells. Once specific CTLA—4 binding and ng activity were verified through first and second screening, the positive hybridoma lines were subcloned into 96—well plates using limited dilution. The plates were cultured at 37 OC, 5% C02 until the positive clones were further screened for competition with ligands CD80 and CD86 binding to CTLA—4. The cultural supernatants of selected positive clones were collected for dies purification and further characterization. The lead candidates were ed for VH and VL sequencing. 4. Determination of VH and VL seguences from hybridoma The VH and VL genes of the antibodies of selected hybridoma clones were isolated by RT—PCR or 5’ RACE. Specifically, total RNA was isolated from hybridoma cells by using RNeasy Plus Mini Kit (Qiagen). The first strand cDNA was reverse ripted using oligo dT. VH and VL genes of the antibodies were amplified from cDNA using 3’— nt region degenerated primer and 5’— degenerated primer sets. The 5’ degenerated primers were ed based on the upstream signal sequence—coding region of Ig variable sequences. The PCR product was then d into T vector and 10 ML of the ligation product was transformed into ToplO competent cells.

Transformed cells were plated on 2XYT plates with carbenicillin and incubated ght at 37 0C. 15 positive colonies were randomly picked for DNA sequencing by Biosune. Alternatively, 5’ RACE was used to identify the VH and VL sequences of selected hybridoma clones. First, RNA was first e transcribed into cDNA using 5’—RACE kit (Takara—28001488), followed by PCR using enerated primers and 3’—adaptor primers (ExTaq: Takara—RROOlB). PCR fragments was inserted into pMDl 8—T vector (Takara—DlOlC) and sent for cing ne, Shanghai).

Example 3: Chimeric antibodies production and characterization 1. Chimeric antibody production The deduced amino acid sequences of VH and VL are listed in the Table 3.

Underlined sequences are CDRs defined by Kabat delineation system. The variable regions of these rat antibodies were fused with constant region of human antibody, and the chimeric antibodies were expressed from Expi293 cells and ed using Protein A chromatography.

Table 3. The variable region sequence of rat anti—CTLA—4 antibodies Clone ID Amino ac1d sequence. .

ID NO EEQLVESGGGLVQPGKSLKLSCSASGFTFRSSAMHWIRQPPGKGL l DWVAFISSGGDTAYADAVKGRFIVSRDNAENTLFLQLNSLKSED W3162-1. ITAIYYCVRMERIPTWGQGVMVTVSS 101.2 DIVLTQSPVLAVSLGQRATISCRASQSVSISSINLIHWYQQRPGQQ PKLLIYRTSNLASGIPARFSGSGSGTDFTLSIDPVQADDVADYYCQ MFGSGTKLEIK EVQLVESGGGLVQPGRSLKLSCAASDLTFSNYDMAWVRQTPTKG 2 LEWVASISPNGGNTYYRDSVKGRFTVSRDNAKNSLYLQMDSLRS W3 162-1 EDTATYYCARHLWFAYWGQGTLVTVSS . 145. 10 DIQMTQSPSSMSASLGDRVTISCQASQDIGSNLIWFQQKPGKSPRP MIYYATHLADGVPSRFSGSRSGSDYSLTISSLESEDVADYHCLJE KQYPRTFGGGTKLELK EVQLQESGPGLVKPSQSLSLTCSVTYHTITSGYDWTWIRKFPGNQ MEWMGYISYSGNTNYNPSLKSRISITRDTSKNQFFLHLNSVTSED W3162-1. TATYYCASMMVPHYYVMDAWGQGASVTVSS 146.19 DVVLTQTPPTSSATIGQSVSISCRSS! QSLLNSDGNTYLYWYLQRPS QSPQLLIYLVSKLGSGVPNRFSGSGSGTDFTLKISGVEAEDLGLYY CV!QGTHDPWTFGGGTKLELK EVQLQQSGPEAGRPGSSVKISCKASGYTFTNYFMNWVKQSPGQG VH 4 LEWIGRVDPENGRADYAEKFKKKATLTADTTSNTAYIHLSSLTS W3162-1. EDTATYFCARRAMDNYGFAYWGQGTLVTVSS 154.8 EIMLTQSPTIMAASLGEKITITCSANSSLSYMYWFQQKSGASPKLW VHGTSNLASGVPDRFSGSGSGTSYYLTINTMEAEDAATYFCm SNT!QWTFGGGTKLELK 2. Characterization of chimeric antibodies 2.1 Antibodies bound to human, monkey and murine CTLA—4 (ELISA, FACS and SPR) Chimeric antibodies with rat variable region and human constant region were expressed from mammalian cells and purified using Protein A ty chromatography.

The antibodies were tested on CTLA—4—binding ELISA. As shown in Figure 1, 2 and 3, all the four antibodies bound to human and monkey CTLA—4 with EC50 comparable to Ipilimumab (WBP316—BMK1), but only one antibody W3162—1.146.19 also bound to murine CTLA—4 at ECso of 0.01 nM. In order to confirm that the antibodies were able to bind CTLA—4 on cell e, a CTLA—4— expressing cell line was used in FACS . These antibodies also bound to CTLA—4 on cell surface (Figure 4) with EC50 ranging from 1.14 nM to 9.42 nM. W3162—1.146.19 bounds to CTLA—4 on cell surface with EC50 of 3.25 nM and W3162—1.154.8 bounds to CTLA—4 on cell surface with ECso of 1.26 nM.

The binding kinetics of four antibodies were measured using SPR. The antibodies were ed on immobilized goat uman PC, and then human CTLA—4 ECD at different concentration was ed orderly. The sensorgrams for reference channel and buffer channel were subtracted from the test sensorgrams. The data was used to fit in 1:1 binding analysis. As show in the Figure 5 and Table 4, all the four dies bound to human CTLA—4 ECD domain with higher affinity than Ipilimumab 6—BMK1), with KD range of 2.08 E—09 nM to 6.80E—ll nM.

Table 4. Kinetic of antibody binding on human CTLA—4 ECD Antibody ka (1/Ms) kd (1/s) K1) (M) W3162-1.101.2 xAb.IgG1 6.95E+05 6.97E-05 1.00E-10 W3162-1.145.10 xAb.IgG1 7.93E+06 1.65E-02 2.08E-09 W3162-1.146.19 xAb.IgG1 7.09E+05 04 2.08E-10 W3162-1.154.8 xAb.IgG1 l.85E+06 1.25E-04 6.80E-11 WBP316-BMK1 9.42E+05 3.46E-03 3.68E-09 2.2 Competition with ligands of chimeric antibodies CTLA—4 was found binding to both CD80 and CD86 at 20 to 50 folds higher affinity than CD28 el 1996]. Therefore, the anti—CTLA—4 antibodies were tested whether they can compete with CD80 and CD86’s binding on CTLA—4. Both ELISA and FACS were used as the competition assays. In ELISA based competition assay, human CTLA—4 was coated on the plates, and the antibodies mixed with biotinylated s were added into the plate. The bound ligands were detected by HRP ated streptaVidin. As shown in Figure 6a and 6b, all four antibodies competed with ligands CD80 (B7—l, L1) and CD86 (B7—2, L2) in CTLA—4 g, and three of them except W3l62—l.101.2 had comparable EC50 with Ipilimumab (WBP3l6—BMK1). In a FACS assay, the mixture of antibodies and biotinylated human CTLA—4 was added to CD80 or CD86 expressing cells, and the bound human CTLA—4 was detected by PE conjugated streptaVidin.

As shown in Figure 7a (upper panel) and 7b (lower panel), all the four antibodies could effectively block CTLA—4’s binding on the ligand—expressing cells. Three dies except W3162—1.154.8 could completely block CTLA—4 binding on CD80 cells, whereas Ipilimumab WBP316—BMK could only partially block this g even at 200 nM, the highest concentration used (Figure 7a). In the FACS assay of blocking CTLA—4 binding on CD86 cells (Figure 7b), All of 4 antibodies could completely block CTLA—4 binding on CD86 cells, whereas Ipilimumab could only partially block this binding even at 200 nM, the highest concentration used. The kinetics of W3162—1.101.2 appeared differently: the blocking was less effective than Ipilimumab at low concentration and more effective than Ipilimumab at high concentration. Other three dies were more effective than Ipilimumab in blocking CTLA—4 at all the concentration tested. 2.3 on in SEB assay of chimeric antibodies The function of anti—CTLA—4 antibodies with different concentration of 1.34 nM, 3.35 nM, 8.71 nM, 21.4 nM, 53.6 nM, 134 nM were tested in a modified T cell stimulation assay (SEB assay). Staphylococcal enterotoxin B (SEB) was used as a stimulator of human T cell activation, in which CTLA—4 was ed as an important player. The T cell activation was measured by secretion of IL—2. As shown in the Figure 8, all the four antibodies ed IL—2 secretion in a dose dependent manner, comparable with or superior to Ipilimumab.

Example 4: Characterization of humanized antibody 1. zation “Best Fit” approach was used to humanize antibody light and heavy chains.

Three anti—CTLA—4 antibodies (except W3162—1.101.2 due to its relatively low binding ty in ELISA and FACS) were selected for humanization, using CDR—grafting technique. The CDRs (underlined in Table 5) and FRs of variable s of the antibodies were defined using Kabat system. Based on the sequence homology and structural similarity, the gene of rat region FRl —3 was replaced by humanized region FRI—3, while region FR4 of the rat gene was replaced by humanized FR4 region derived from JH and JK genes that had the most similar ures. The hot spots of post—translational modification (PTM) of variable s were modified to reduce the PTM risk. After verifying the template sequence and codon optimization, the heavy chain variable region and light chain variable region were synthesized and cloned into an expression vector, and then used for expression of the humanized antibodies. The humanized antibodies were purified using Protein A chromatography, and the kinetics binding on human, monkey and murine CTLA—4 were measured using SPR method.

Table 5. The variable region sequence of zed anti—CTLA—4 antibodies SE ID . .

EVQLVESGGGLVQPGGSLRLSCAASDLTFSNYDMAWVRQAPG KGLEWVASISPSGGNTYYRDSVKGRFTISRDNAKNSLYLQMNS W3l62-l .l LRAEDTAVYYCARHLWFAYWGQGTLVTVSS DIQMTQSPSSLSASVGDRVTITCS 2ASS 2DIGSNLIWFQQKPGKAP KPMIYYATHLADGVPSRFSGSRSGTDYTLTISSLQPEDFATYYCL g QYKS QYPRTFGGGTKVEIK QVQLQESGPGLVKPSETLSLTCSVTYHTITSGYDWTWIRKPPGK GMEWIGYISYSGNTNYNPSLKSRVTISRDTSKNQFFLKLSSVTA W3l62.l.l4 ADTAVYYCASMMVPHYYVMDAWGQGTLVTVSS DIVMTQTPLSLSVTPGQPASISCRSSQSLLNSDGNTYLYWYLQK LLIYLVSKLGSGVPNRFSGSGSGTDFTLKISRVEAEDVG VYYCVQGTHDPWTFGGGTKVEIK QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYFMNWVRQAP GQGLEWMGRVDPEQGRADYAEKFKKRVTITADKSTSTAYMEL W3l62.l.15 SSLRSEDTAVYYCARRAMDNYGFAYWGQGTLVTVSS EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKPDQSP KLWVHGTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYC HHWSNTS 2WTFGGGTKVEIK WO 09701 Table 6. The variable region of humanized anti—CTLA—4 antibodies SEQ ID Clone ID DNA sequence GAGGTGCAGCTGGTGGAGAGCGGCGGAGGACTGGTGCAACCTGGCGGAAGC CTGAGACTGAGCTGCGCCGCCAGCGACCTGACCTTCAGCAACTACGACATGG CCTGGGTGAGACAGGCCCCTGGCAAGGGACTGGAGTGGGTGGCCAGCATCA GCCCCAGCGGCGGCAACACCTACTACAGGGACAGCGTGAAGGGCAGGTTCA CCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCT GAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCACCTGTGGTTCGCC W3162—1.145 TACTGGGGCCAGGGCACACTGGTGACCGTGAGCAGC .10—z7 GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGCGATA GGGTGACCATCACCTGCCAGGCCAGCCAGGACATCGGCAGCAACCTGATCTG GTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCCTATGATCTACTACGCCACCC ACCTGGCCGATGGCGTGCCTAGCAGATTCAGCGGCAGCAGAAGCGGCACCGA CTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACT GCCTGCAGTACAAGCAGTACCCCAGAACCTTCGGCGGCGGCACCAAGGTGGA GATCAAG CAGGTGCAGCTGCAGGAGAGCGGACCCGGACTGGTGAAGCCCTCCGAGACC CTGAGCCTGACCTGCAGCGTGACCTACCACACCATCACCAGCGGCTACGACT GGACCTGGATCAGAAAGCCCCCCGGCAAAGGCATGGAGTGGATCGGCTACAT VH CAGCGGCAACACCAACTACAACCCCAGCCTGAAGAGCAGGGTGAC CATCAGCAGGGACACCAGCAAGAACCAGTTCTTCCTGAAGCTGAGCAGCGTG GCCGATACCGCCGTGTACTACTGCGCCAGCATGATGGTGCCCCACTA “3162-1146 CTACGTGATGGACGCCTGGGGACAGGGCACCCTGGTGACAGTGAGCAGC GACATCGTGATGACCCAGACCCCCCTGAGCCTGAGCGTGACACCTGGACAGC . 19—z12 CCGCCAGCATCAGCTGCAGGTCCAGCCAGAGCCTGCTGAACAGCGACGGCAA CACCTACCTGTACTGGTACCTGCAGAAGCCTGGCCAGAGCCCCCAGCTGCTGA TCTACCTGGTGTCCAAGCTGGGCAGCGGCGTGCCTAACAGGTTTAGCGGCAG CGGCAGCGGCACCGATTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGAT GTGGGCGTGTACTACTGCGTGCAGGGCACCCACGATCCTTGGACCTTCGGCG GCGGAACCAAGGTGGAGATCAAG CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCCGGCAGCAGC GTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAACTACTTCATGA ACTGGGTGAGGCAGGCCCCTGGACAAGGCCTGGAGTGGATGGGCAGAGTGG ATCCCGAGCAGGGCAGGGCCGACTACGCCGAGAAGTTCAAGAAGAGGGTGA CCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCT GAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGAGAGCCATGGACAA W3162—1.154 CTACGGCTTCGCCTACTGGGGCCAGGGAACCCTGGTGACCGTGAGCAGC .8—235 GTGCTGACCCAGAGCCCCGACTTCCAGAGCGTGACCCCCAAGGAGA AGGTGACCATCACCTGCAGCGCCAACAGCGCCCTGAGCTACATGTACTGGTAC CAGCAGAAGCCCGACCAGAGCCCCAAGCTGTGGGTGCACGGCACCAGCAAT CTGGCCAGCGGCGTGCCTAGCAGATTTAGCGGCAGCGGCAGCGGCACCGATT TCACCCTGACCATCAACAGCCTGGAGGCCGAGGACGCCGCTACCTACTACTG CCACCACTGGAGCAACACCCAGTGGACCTTCGGCGGCGGCACCAAGGTGGA GATCAAG 2. Characterization of humanized antibodies 2.1 Antibodies bound to human, monkey and murine CTLA—4 2.1.1 CTLA—4—Binding ELISA Humanized antibodies were expressed from mammalian cells and purified using n A affinity chromatography. Ipilimumab was from commercial source. Isotype control antibody, human CTLA—4 ECD with different tags (hFc or 6xHis) and murine CTLA—4.ECD—hFc were prepared by WuXi Biologics. Murine CTLA—4.ECD—6xHis and lgus monkey CTLA—4 ECD—6xHis were purchased from Sino Biological. HRP—conjugated goat anti—human IgG Fc was sed from Bethyl (Cat: A80—304P).

ELISA was used to test binding of anti—human CTLA—4 antibodies to human, murine and cynomolgus monkey CTLA—4 protein. A 96—well plate was coated with human CTLA—4.ECD—6XHis (1.0 ug/mL), cynomoguls monkey CTLA—4.ECD—6XHis (0.5 ug/mL) or mouse CTLA—4.ECD—6XHis (0.5 ug/mL) at 4 0C for 16—20 hours. After 1 hour blocking with 2% BSA in DBPS, testing antibodies, as well as positive and ve control antibodies were added to the plates and incubated at room temperature for 1 hour. The binding of the antibodies to the plates were detected by HRP—conjugated goat anti—human IgG antibody (1:5000 dilution) with 1 hour incubation. The color was developed by dispensing 100 ML of TMB substrate for 8 mins, and then stopped by 100 ML of 2N HCl. The absorbance at 450 nM was ed using a microplate spectrophotometer.

As shown in Figure 9, the two antibodies W3162—1.146.19—zl2—IgGk and W3162—1.154.8—z35—IgGk bound to human CTLA—4 with EC50 of 0.03 nM and 0.04 nM, tively, slightly higher than EC50 of Ipilimumab (WBP316—BMK1) 0.01 nM (Figure 9A). The two antibodies also bound to monkey CTLA—4 with EC50 of 0.05 nM (Figure 9B), but only W3162—1.146.19—zl2—IgGk bound to murine CTLA—4 with EC50 0.19 nM.

Neither W3162—1.154.8—Z35—IgGk nor Ipilimumab bound to murine CTLA—4 (Figure 9C). 2.1.2 CTLA—4—binding FACS Human CTLA4 expression 293F cell line was developed by WuXi Biologics.

PE conjugated goat uman IgG Fc fragment was purchased from n (Catalog number 109—115—098). A number of 1 x 105 cells per well was added to each well of a 96—well plate and centrifuged at 1500 rpm for 4 minutes at 4 °C before removing the supernatant. Serial dilutions of test antibodies, positive and negative controls were added to the resuspended cells and incubated for 1 hour at 4 oC. The cells were washed two times with 200 ML DPBS containing 1% BSA. PE conjugated goat anti—human IgG (1:100) diluted in DPBS containing 1% BSA was added to the cells and incubated at 4 °C for 1 hour. Additional washing steps were performed two times with 200 ML DPBS containing 1% BSA followed by centrifugation at 1500 rpm for 4 minutes at 4 °C. y, the cells were resuspended in 100 ML DPBS containing 1% BSA and fluorescence values were measured by flow cytometry and analyzed by FlowJo.

These antibodies were also able to bound human CTLA—4 on cell surface in FACS assay. As shown in Figure 11 (Figure 10a and Figure 10b), W3162—1.146.19—zl2—IgGk, W3162—1.154.8—z35—IgGk and Ipilimumab had slightly different ECso of 1.58 nM, 0.66 nM and 0.83 nM, tively. 2.2 The binding kinetics of these antibodies 2.2.1 The binding kinetics of these antibodies were ed using SPR The experiment was to measure the on—rate constant (ka) and off—rate constant (kd) of the antibodies to CTLA—4 ECD based on SPR technology.

The affinity constant (KD) was consequently determined.

Biacore T200, Series S Sensor Chip CM5, Amine Coupling Kit, and 10x HBS—EP were purchased from GE Healthcare. Goat anti—human IgG Fc antibody was purchased from Jackson ImmunoResearch Lab og number 109—005—098). In immobilization step, the activation buffer was prepared by mixing 400 mM EDC and 100 mM NHS immediately prior to injection. The CM5 sensor chip was activated for 420 s with the activation . 30 ug/mL of goat anti—human IgG Fcy antibody in 10 mM NaAc (pH 4.5) was then injected to Fc1—Fc4 channels for 200s at a flow rate of 5 uL/min. The chip was deactivated by 1 M lamine—HCl (GE). Then the dies were captured on the chip. Briefly, 4 ug/mL antibodies in running buffer (HBS—EP+) was injected individually to Fc3 channel for 30 s at a flow rate of 10 uL/min. Eight different concentrations (20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, 0.3125 nM and 0.15625 nM) of e CTLA—4 6.hCTLA—4.ECD—6xHis) and blank running buffer were ed orderly to Fc1—Fc4 channels at a flow rate of 30 uL/min for an association phase of 120 s, followed by 2400 s dissociation phase. Regeneration buffer (10 mM Glycine pH 1.5) was injected at 10 uL/ min for 30 5 following every dissociation phase.

The binding kinetics of these antibodies were measured using SPR. The antibodies were captured on immobilized anti—human Fc and CTLA—4—ECD at different tration was injected orderly. The sensorgrams for reference channel and buffer channel were subtracted from the test sensorgrams. The data was used for 1:1 binding analysis on human, monkey and mouse .ECD—6xHis. As show in Table 7, humanized antibodies W3162—1.146.19—Z12, W145 and W3162—1.154.8—Z35 bound to human CTLA—4—ECD domain with affinity at 0.477 nM, 1.84 nM and 0.0968 nM, respectively. Comparing with rat antibodies, the humanized antibodies had comparable affinity. W3162—1.146.19—Z12 and W3162—1.154.8—Z35 have significantly higher affinity than umab (KD=3.68 nM). The antibody W3162—1.146.19—Zl2 could also bind to murine CTLA—4, and its affinity before and after humanization is shown in Table 9. After humanization, its affinity 1.39 nM is slightly lower than affinity 0.906 nM of its parental antibody.

The affinity of W3162—1.146.19—Z12, W3162—1.145.10—z7 and 1.154.8—Z35 binding to cynomolgus monkey CTLA—4—ECD was 1.92 nM, 0.598 nM, 0.131 nM, respectively (Table 8).

Table 7. Kinetic of dy—binding on human CTLA—4 ECD Antibodies ka (1M5) kd (1/s) KD (M) W3162-1.146.l9-zl2-uAb.IgGlK 2.06E+05 9.82E-05 4.77E-10 W3162-1.146.19 XAb.IgGl 7.09E+05 1.48E-04 2.08E-10 W3162_1.145.10-z7-uAb.IgGlK 7.37E+06 1.35E-02 1.84E-09 W3162-1.145.10 Gl 7.93E+06 1.65E-02 2.08E-09 W3l62_l.154.8-z35-uAb.IgGlK 1.23E+06 1.19E-04 9.68E-11 W3162-1.154.8 XAb.IgGl 1.85E+06 1.25E-04 6.80E-11 Ipilimumab 9.42E+05 3.46E-03 3.68E-09 Table 8. Kinetic of antibody—binding on monkey CTLA—4 ECD Antibodies ka (1M8) kd (1/s) KD (M) W3162-1.146.l9-zl2-uAb.IgGlK 1.73E+05 3.31E-04 1.92E-09 W3162-1.146.19 XAb.IgGl 2.91E+05 04 3.69E-10 W3162_1.145.10-z7-uAb.IgGlK 4.52E+06 2.71E-03 10 W3162-1.145.10 XAb.IgGl 1.06E+07 3.77E-03 3.55E-10 W3l62_l.154.8-z35-uAb.IgGlK 9.32E+05 1.22E-04 10 W3162-1.154.8 XAb.IgGl 1.25E+06 1.09E-04 8.72E-11 Table 9. Kinetic of antibody—binding on mouse CTLA—4 ECD dies ka (1M5) kd (1/s) KD (M) W3162-1.146.l9-z8-uAb.IgGlK 1.72E+05 2.39E-04 1.39E-09 W3162-1.146.19 XAb.IgGl 2.51E+05 2.28E-04 9.06E-10 2.2.2 Affinity test by FACS FITC conjugated goat anti—human IgG Fc was purchased from Jackson Immunoresearch Lab (catalog number 109—095—098), and BD CantoII was used for this assay. Briefly, HEK293 cells expressing human CTLA—4 were transferred in to 96—well U—bottom plates (BD) at a density of 5x104 cells/well. Testing dies were 1:2—fold serially diluted in PBS with 1%BSA and ted with cells at 4 0C for 1 hour. After centrifugation at 1500 rpm for 4 min, the supernatant was discarded. The ary antibody, FITC conjugated goat uman IgG Fc (3.2 FITC per IgG, Jackson Immunoresearch Lab), was added to re—suspend cells to final concentration 14 ug/ml, and incubated at 4 0C in the dark for 30 min. The cells were then washed once and re—suspended in PBS with 1%BSA, and analyzed by flow cytometery (BD). Fluorescence intensity was converted to bound molecules/cell based on the quantitative beads (QuantumTM MESF Kits, Bangs Laboratories). K1) was calculated using ad Prism5.

The affinity of humanized antibodies binding to cell surface CTLA—4 was measured by flow cytometry method, modified from Benedict’s method [Benedict 1997 JIM]. After measure fluoresce of antibodies binding to —expressing CHO cells, the bound antibody and free antibody were analyzed and fitted to the equation, as shown in Figure 5. Based on the data and formula, calculated ty constant K1) is shown in the Table 10.

Affinity of humanized antibody W3 162— 1 . 146. 19—Z 1 2 and W3162—1.154.8—Z35 had high affinity at 5.05 and 0.35 nM, respectively, whereas the affinity of umab is 0.97 nM.

Table 10. Affinity test by FACS Sample KD (M) Bmax R2 W3162-1.146.19-z12-IgG1K 5.0E-09 1.2E-10 0.99 W3162-1.154.8-z35-IgG1K 3.5E-10 1.2E-10 0.98 Ipilimumab 9.7E-10 7.2E-11 0.99 2.3 Competition with ligands In order to test whether the humanized antibodies ined its y of blocking CTLA—4 binding on CD80 and CD86, both ELISA and FACS were used in competition assay. Two CTLA—4 ligands CD80 and CD86 were purchased from Sino Biological (Catalog number H08H and 10699—H08H). Biotinylated is tag antibody was purchased from Genscript (Catalog number A00613). HRP conjugated streptavidin was purchased from Invitrogen (Catalog number SNN1004). 2.3.1 The ELISA—based competition assay ELISA was used to test r the antibodies could inhibit the binding of human CTLA—4 to its ligands human CD80 and CD86. Plates were coated with human CTLA—4.ECD.hFc (0.5 ug/mL) at 4 0C for 16—20 hours. After 1 hour blocking with 2% BSA in DBPS, testing dies, as well as positive and negative control antibodies were pre—mixed with 0.25 ug/mL of CD80—6xHis or CD86—6xHis, and then added to the plates and incubated at room temperature for 1 hour. After washing three times with PBS containing 0.05% Tween 20, biotinylated anti—His tag antibody was 122000 diluted and added. The plates were incubated at room ature for 1 hour. The bound ligands were detected by HRP conjugated streptavidin (1220000). The color was developed by dispensing 100 ML of TMB substrate for 8 mins, and then d by 100 ML of 2N HCl. The absorbance at 450 nM was measured using a microplate ophotometer.

As shown in Figure 11, W3162—1.146.19—zl2—IgGk and W3162—1.154.8—Z35—IgGk had similar effect as Ipilimumab in blocking ligands binding with coated CTLA—4, with ICso of 0.87 nM, 0.63 nM and 0.40 nM for CD80, and , 0.50 nM and 0.42 nM for CD86. 2.3.2 The FACS assay To test whether the antibodies could block CTLA—4 binding to CD80 and CD86 on cell surface, we used FACS to test this competition. The CD80— and CD86— expressing CHO cell lines were developed by WuXi Biologics.

Biotinylated CTLA—4.ECD.hFc was made by WuXi ics. PE conjugated Streptavidin was purchased from eBioscience (Catalog number 12—4317).

CD80— or CD86—expressing cells were added to each well of a 96—well plate at 1><105 per well and centrifuged at 1500 rpm for 4 minutes at 4 °C before ng the supernatant. Serial dilutions of test antibodies, positive and negative controls were mixed with biotinylated human CTLA4.ECD.hFc.

Due to different density of ligands on cell surface, 0.02 ug/mL of hCTLA—4.ECD.hFc—Biotin was used for human CD80 cells and 0.08 ug/mL of hCTLA—4.ECD.hFc—Biotin for human CD86 cells. Then the mixtures of antibody and CTLA—4 were added to the cells and incubated for 1 hour at 4 oC. The cells were washed two times with 200 ML FACS buffer (DPBS containing 1% BSA). avidin PE 1 to 333 diluted in FACS buffer was added to the cells and incubated at 4 °C for 1 hour. Additional g steps were performed two times with 200 ML FACS buffer followed by centrifugation at 1500 rpm for 4 minutes at 4 oC. Finally, the cells were resuspended in 100 ML FACS buffer and fluorescence values were measured by flow cytometry and analyzed by FlowJo.

The results are shown in Figure 12. The two humanized antibodies could more effectively block CTLA—4/ligand g than Ipilimumab. At the highest concentration used, Ipilimumab only blocked 32% CTLA—4 binding to CD80 and 40% of CTLA—4 binding to CD86. In comparison, antibody W3162—1.146.19—Zl2 d 71% of CTLA—4 g on CD80 and 73% of CTLA—4 binding on CD86, and antibody W3162—1.154.8—Z35 blocked 89% of CTLA—4 binding on CD80 and 98% of CTLA—4 binding on CD86. ICso of Ipilimumab, W3162—1.146.19—Zl2 and W3162—1.154.8—Z35 directing against CD80 were 3.23, 6.60 and 0.07 nM respectively. ICso of umab, W3162—1.146.19—Zl2 and W3162—1.154.8—Z35 directing t CD86 were 2.52, 5.15 and 0.28 nM respectively. 2.4 Cytokine release of SEB stimulated PBMCs Anti—CTLA4 antibodies were tested whether they could enhance cytokines release of human PBMC after SEB (from The Second Military Medical University) stimulation. Peripheral blood from healthy donors was obtained and cells were isolated by Ficoll GE Healthcare, 17—1440—02) density gradient centrifugation. After the buoyant layer was removed, the platelets were removed by several washes with medium. A number of 1><105 human PBMC cells were added to each well of a l plate. Serial dilutions of test antibodies, ve and negative controls were mixed with SEB (10 ng/mL), and then added to the pelleted cells and incubated for 3 days at 37 oC. The supernatants were collected to measure human IL—2 tration.

For human IL—2 test, plates were pre—coated with 1.0 ug/ml human IL—2 antibody (R&D System ) at 4 °C for 16—20 hours. After 1 hour blocking with 2% BSA (BovoGen) in DBPS, the supernatants containing IL—2 were added to the plates and incubated at room temperature for 2 hours.

After washing three times with PBST ining 0.05% Tween 20), biotinylated human IL—2 antibody (R&D , BAF202) was diluted and added at concentration of 0.5 g/mL. The plates were incubated at room temperature for 1 hour. The bound biotinylated antibody was detected by 1220000 diluted streptavidin conjugated HRP (Invitrogen, SNN1004). After 1 hour incubation, the color was developed by sing 100 ML of TMB substrate, and then stopped by 100 ML of 2M HCl. The absorbance at 450 nm and 540 nm was measured using a microplate spectrophotometer.

In a cell—based assay, the humanized antibodies (8.60 nM, 21.4 nM, 53.6 nM, 134 nM, 335 nM) were tested r they could enhance superantigen SEB stimulated human PBMCs. After 3 days stimulation, IL—2 from the PBMC was measure using ELISA. Compared with an isotype control antibody, both the two humanized antibodies (W3162—1.146.19—Z12, 1.154.8—Z35) and Ipilimumab could enhance IL—2 release from the PBMCs in a dose—dependent manner (Figure 13). 2.5 Thermostability The stability of the lead antibodies was tested at different temperature.

Briefly, 100 uL each antibody sample was pipetted into dual tubes, and the samples were incubated at 4 °C or 37 °C for 20 hours, or 45 °C or 50 °C for 2 hours. Then the samples were centrifuged at 12,000 rpm for 10 minutes.

Those samples were observed to find possible precipitation, and the samples were analyzed by SEC—HPLC for purity and elution time.

The SEC profile of W3162—1.146.19—Zl2 at different conditions was shown in Figure 14 a—d. Neither dilution time nor main peak percentage (92.39% to 92.48%) at high ature conditions significantly changed, comparing with that at low ature (92.24%). The SEC profile of W3162—1.154.8—Z35 at ent high temperature conditions was shown in Figure 14 e—h. Neither dilution time nor main peak percentage (97.14% — 97.17%) significantly changed, compared with at low temperature (96.84%).

This set of data indicates that the antibodies were stable in tested high temperature conditions. 2.6 Nonspecific binding Both FACS and ELISA assays were used to test r the dies binding to other targets. In FACS assay, different cell lines (Ramos, Raji, MDA—MB—453, BT474, Jurkat, Hut78, A431, A204, CaLu—6, A375, HepG2, BxPC—3, HT29, FaDu, 293F, CHO—Kl) were ed to 1 x 105 cells per well. Testing antibodies and Isotype control antibodies were diluted to 10 ug/ml in PBS containing 1%BSA and incubated with cells at 4 0C for 1 hr.

The cells were washed twice with 180 ML PBS containing 1%BSA. PE conjugated goat anti—human IgG Fc fragment (Jackson, Catalog number 109—115—098) was diluted to final concentration 5 ug/ml in PBS with 1%BSA, then added to re—suspend cells and incubated at 4 0C in the dark for min. Additional washing steps were med twice with 180 ML PBS containing 1% BSA followed by centrifugation at 1500 rpm for 4 minutes at 4 oC. Finally, the cells were re—suspended in 100 ML PBS containing 1% BSA and fluorescence values were measured by flow cytometry (BD Canto II) and analyzed by FlowJo.

In the ELISA assay, the testing antibodies, e control antibodies were tested binding to 10 different target antigens including Factor VIII, FGFR—ECD, PD—1, CTLA—4.ECD, VEGF, CD, OX40.ECD, 4—1BB.ECD, CD22.ECD, CD3e.ECD. A 96—well plate was coated with the individual antigens (2 ug/mL) at 4 °C overnight. After 1 hour blocking with 2% BSA in PBS, wash plate 3 times with 300 ML PBST. Testing antibodies, as well as isotype control antibodies were d to 10 ug/mL in PBS containing 2%BSA, then were added to the plate and incubated at room ature for 2 hours. After 3 times washing with 300 ML PBST, HRP—conjugated goat anti—human IgG antibody (1:5000 diluted in 2%BSA) was added to the plate and incubated at room temperature for 1 hours. Finally, the plates were washed siX times with 300 ML PBST. The color was developed by dispensing 100 ML of TMB substrate for 12 min, and then stopped by 100 ML of 2M HCl. The absorbance at 450 nM was measured using a microplate spectrophotometer.

In addition to CTLA—4, other irrelevant proteins were used to test whether the antibody W3162—1.146.19—Z12 and W3162—1.154.8—Z35 were able to bind to these antigens. As shown in Figure 16, among the panel of antigens, only CTLA—4 was detected by the two antibodies. Other ns did not te signal in this ELISA assay. On contrast, X40 antibody did bind to 0X40, suggesting this antigen was coated on the plate.

The specificity of the two antibodies were also tested on a panel of different cell lines in FACS assay. The antibodies did not generate detectable signal to any of these cell lines (data not shown). 2.7 In vivo efficacy Due to antibody W3l62—l.l46.l9—212 cross—reacts to both human and murine CTLA—4, the umor efficacy of this antibody was tested in a syngeneic mouse models. Mouse cancer cell line CT26 was used to establish xenograft mouse model to test anti—CTLA—4 antibody l.l46.l9—212.

An anti—murine CTLA—4 antibody sed from BioXCell was used as a positive control (BioXCell—BE0131). The tumor cells were maintained in vitro as a monolayer culture in RPMI—l640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 ug/mL streptomycin at 37 °C in an atmosphere of 5% C02. The tumor cells were routinely tured twice weekly after detaching the cells by trypsin—EDTA ent. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Female Balb/C mice were purchased from Beijing Vital River Laboratory Animal Co., Ltd. The mice at age 6—8 weeks with weight approximately 18—22 g were used for the study. Each mouse was inoculated subcutaneously at the right axillaries with l><105 tumor cells in 0.1 mL of PBS mixed with 50 ML matrigel. When the average tumor volume reaches 60—80 mm3, the animals were ly grouped. The anti—CTLA—4 antibodies and isotype control were used for treatment: intravenously injected into mice twice a week. The tumor size was measured twice weekly by a vernier caliper, and tumor volume was calculated by the formula a><b2 ><71:/6 where a is length and b is width (a>b).

When e tumor volume reached approximately 70 mm3, W3l62—l.l46.l9—212 (1 mg/kg, 3 mg/kg, 10 mg/kg) and l antibodies (10 mg/kg) were injected twice a week for two weeks. The animals were monitored for tumor growth and body weight over time. As shown in Figure , W3l62—l.l46.l9—Zl2 significantly ting tumor growth in a dose—dependent manner. At 1 mg/kg dose, W3l62—l.l46.l9—Zl2 inhibited tumor growth, compared with control group. At 3 mg/kg dose, W3l62—l.l46.l9—Zl2 inhibit tumor volume to 160 mm3 at day 19, s mg/kg W3l62—l.l46.l9—Zl2 induced tumor regression at the end of the study period. 2.8 Epitope mapping Alanine scanning was used to identify CTLA—4 epitope of the antibodies. In this experiment, alanine residues on hCTLA4 were mutated to glycine residues, and all other residues were mutated to alanines. For each residue of human CTLA4 extracellular domain (ECD), point amino acid substitutions were made using two sequential PCR steps. A .3—hCTLA4_ECD.His plasmid that encodes ECD of human CTLA4 and a inal His—tag was used as template, and a set of mutagenic primers were used for first step PCR using the QuikChange lightning multi site—directed mutagenesis kit (Agilent technologies, Palo Alto, CA). Dpn I endonuclease was used to digest the parental template after mutant strand synthesis on. In the second—step PCR, linear DNA expression cassette, composed of a CMV promoter, mutated ECD of CTLA4, a g and a herpes simplex Virus ine kinase (TK) polyadenylation, was amplified and ently expressed in HEK293F cells (Life Technologies, Gaithersburg, MD). In addition, three plasmid vectors were constructed to test the epitope of glycans: pcDNA3.3—hCTLA4_ECD.His (N113Q), pcDNA3.3—hCTLA4_ECD.His (N145Q), and pcDNA3.3—hCTLA4_ECD.His (N113Q, . These three muteins were transiently expressed in HEK293F cells (Life Technologies, Gaithersburg, MD).

In order to test how the mutations affect antibody—binding, a e ELISA was conducted. Briefly, ipilimumab, W3162—1.146.19—zl2 and 1.154.8—z35 (2 ug/mL) monoclonal antibodies were captured by pre—coated with 2 ug/mL goat—anti—human—IgG Fc (Bethyl Laboratories, Montgomery, TX) in plates. After interacting with the supernatant that contains quantified CTLA4 muteins, HRP conjugated anti—His antibody (1:5000; Rockland Immunochemicals, Pottstown, PA) was added as detection dy. TMB was used as substance of HRP. Absorbance was normalized ing to the average of control mutants. After g an additional cutoff to the binding fold change (<0.55), the final determined epitope es were identified.

The binding activities of antibodies W3162—1.146.19—zl2, W3162—1.154.8—Z3 5 and ipilimumab (W316—BMK1) to human CTLA4 were conducted, all three antibodies were found binding to human CTLA4 e The tested point mutations that affect antibody binding to CTLA—4 was shown in Table 11. According to human CTLA4 crystal structures (PDB code 1AH1), some amino acid residues (e.g. Met38, Val40, Tyr60, Val71, Val73, Arg75, Val84, Cys85, Cysl29, Ilel49) were unlikely to directly contact any antibodies. The observed binding reductions most probably resulted from the instability or even collapse of CTLA4 structure after alanine substitutions. The final determined epitope residues were listed in Table 12 and marked on Figure 18.

As shown on Figure 18D and E, the epitopes of Ipilimumab and 1.146.19—zl2 overlap to each other, except a few residues such as N145 and P138. In comparison, W3162—1.154.8—z35 bound to a smaller area of CTLA—4 (Figure 18 F) than other two antibodies. All the three antibodies bound to ligand binding domain of CTLA—4 (Figure 18 A and B), which involve the MYPPPY motif.

The overlapped epitopes of Ipilimumab and W3l62—l.l46.l9—212 did not explain the unique cross—species binding of antibody W3l62—l.l46.l9—212. As N145 mutation on CTLA—4 only affected W3l62—l.l46.l9—212 binding to CTLA—4, not affecting other two dies, we further looked at the N—glycosylation sites as potential epitopes. The effect of mutations on two glycosylation sites of CTLA4 on antibody—binding activity is shown on Figure 17. The binding of umab or W3l62—l .154.8—235 on mutated CTLA—4 was not significantly changed (Figure 17 A and C). In contract, the g of W3l62—l.l46.l9—212 on mutated CTLA—4 Nl45Q significantly reduced whereas this antibody’s binding on CTLA—4 with N1 l3Q did not change. This set of data indicates that the glycan (Figure 18E) on N145 of CTLA—4 could be the epitope of l.l46.l9—212. The N145 residue is conserved in CTLA—4 of guls monkey and mouse.

The ption of the present invention has been made above by the examples.

However, it is tood by the skilled in the art that the present invention is not limited to the examples. The invention may be embodied in other specific forms without departing form the spirit or essential characteristics thereof.

Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of lency of the claims are intended to be embraced therein.

Table ll. The effect of CTLA4 point mutations on antibody binding Ipilimumab W3162-1.146.19-z12-IgG1K W3162-1.154.8-z35-IgG1K CTLA4 Fold CTLA4 Fold CTLA4 Fold p0s1 10_t_ Residu positio chang SD Residu positio chang SD Residu chang SD a a e n e e e e QDQDWWD%%KWHMMWWWWWW<WHQQM<HHWKZHEK<m0m<w 136 0.191 0.00 Q 136 0.155 0.00 40 0.201 0.00 V 40 0.187 0.00 146 0.208 0.00 C 146 0.201 0.00 129 0.210 0.00 M 129 0.211 0.00 85 0.229 0.00 I 85 0.218 0.00 84 0.233 0.01 V 84 0.232 0.00 60 0.236 0.04 N>l< 38 0.238 0.00 134 0.240 0.01 Q 60 0.240 0.07 149 0.244 0.01 V 75 0.298 0.00 38 0.253 0.02 Y 149 0.319 0.00 81 0.268 0.03 E 0.00 138 0.339 75 0.273 0.00 M 71 0.347 0.10 143 0.278 0.00 l IEQIFUIFIDHFU<|m|§H|W<WH>U~<Z<OOQ<IFU 65 0.351 0.00 128 0.286 0.00 C 88 0.433 0.02 71 0.335 0.08 l 134 0.455 0.00 130 0.364 0.00 R 83 0.479 0.00 142 0.369 0.01 V 73 0.498 0.01 144 0.375 0.00 V 70 0.515 0.04 88 0.377 0.06 g 66 0.519 0.02 70 0.378 0.10 A 74 0.520 0.00 73 0.380 0-00 L 137 0.534 0-00 66 0.395 0.03 L 64 0.543 0.10 83 0.396 0.02 g 39 0.549 0.01 86 0.401 0.03 I 137 0.407 0.00 E 74 0.410 0.00 Y 126 0.412 0.01 I 39 0.420 0.01 E 139 0.434 0.00 g 82 0.450 0.01 G 132 0.470 0.04 I 90 0.479 0.02 L 147 0.508 0.03 M 127 0.508 0.01 Q 80 0.517 0.02 V 141 0.518 0.03 3 68 0.529 0.00 Y 148 0.532 0.04 Y 125 0.537 0.02 E 76 0.545 0.00 I 92 0.548 0.00 G a Fold Change in g is relative to the binding of several silent alanine substitutions.

Table 12. Identified es of three antibodies Ipilimumab W3162-1.146.19-z12-IgG1K W3162-1.154.8-z35-IgG1K CTLA4 CTLA4 Position Location Position Location Position Location Residue Residue roo§%>ma<oorwm>m 39 I 39 A 66 A 64 B C loop 68 R 65 B C loop 70 T 66 B C loop 74 L 70 76 Q 74 80 V 83 C' 81 T 88 C' 82 E 134 83 T 136 FG loop 86 M C' C" loop 137 FG loop 88 G C" rw>wo 138 FG loop 90 C' C" loop L 146 92 C" ormwamWH 128 139 orm-u-w-uZt-HWH 141 >—4 >—4 143 00C) 144 WO 09701

Claims (35)

1. Claims 1. An antibody or an antigen binding fragment thereof, wherein the antibody or the antigen binding fragment binds to human, monkey and mouse CTLA—4.
2. 2. The antibody or the antigen binding—fragment thereof according to claim 1, wherein the antibody inhibits CTLA—4 g to CD80 or CD86.
3. 3. The antibody or the antigen binding—fragment thereof according to claim 1 or 10 2, wherein binding epitope of antibody or antigen binding—fragment ses N145 or polysaccharide on N145 of CTLA—4.
4. 4. An antibody or an antigen binding fragment thereof, wherein the dy or the antigen binding fragment binds to human, monkey CTLA—4, n 15 binding epitope of antibody or antigen binding fragment comprises P138 of
5. 5. An antibody or an antigen binding fragment thereof, wherein the antibody or the antigen binding—fragment 20 a) binds to human CTLA—4 with a KB of 4.77E—10 M or less; and b) binds to mouse CTLA—4 with a KB of 1.39E—09 M or less.
6. 6. The antibody or an antigen binding fragment thereof according to claim 5, n the antibody or the antigen binding—fragment exhibits at least one of 25 the following properties: a) binds to human CTLA—4 with a KB of between 10 M and 2.08E—10 M and to mouse CTLA—4 with a KB of between 1.39E—09 M and 9.06E—10 b) enhances interleukin—2 release from stimulated PBMCs.
7. 7. An antibody or an antigen binding fragment thereof, comprising an amino acid sequence that is at least 70%, 80%, 90% or 95% homologous to a sequence selected from a group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, 13, and 14, n the antibody or the antigen binding fragment specifically binds to
8. 8. An antibody or an antigen binding fragment thereof, comprising an amino acid sequence selected from a group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 10 7, 8, 9,10,11,12, l3, and 14, wherein the antibody or the n binding fragment specifically binds to CTLA—4.
9. 9. An antibody, or an n—binding fragment thereof, comprising: 15 a) a variable region of a heavy chain having an amino acid sequence that is at least 70%, 80%, 90% or 95% homologous to a sequence selected from a group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, and 7; and b) a le region of a light chain having an amino acid sequence that is at least 70%, 80%, 90% or 95% gous to a sequence selected from a group 20 consisting of SEQ ID NOs: 8, 9, 10, ll, l2, l3, and 14, wherein the antibody or the antigen binding fragment specifically binds to CTLA—4.
10. 10. An antibody or an antigen binding fragment thereof, comprising: 25 a) a variable region of a heavy chain having an amino acid sequence ed from the group ting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, and 7; and b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, ll, l2, l3, and 14, wherein the antibody or the antigen binding fragment specifically binds to 30 CTLA—4.
11. 11. An antibody or an antigen binding fragment thereof, comprising a complementarity—determining region (CDR) having an amino acid sequence selected from the group ting of SEQ ID NOs: 15—41, wherein the antibody or the antigen binding fragment specifically binds to CTLA—4.
12. 12. An antibody, or an antigen binding fragment thereof, comprising: a heavy chain variable region comprising CDRl, CDR2, and CDR3 10 sequences; and a light chain variable region comprising CDRl, CDR2, and CDR3 sequences, n the heavy chain variable region CDR3 sequence comprises an amino acid sequence ed from a group consisting of SEQ ID NOs: 15, 16, 15 17, and 18, and conservative modifications thereof, wherein the antibody or the antigen binding fragment specifically binds to CTLA—4.
13. 13. The dy or the n binding fragment thereof according to claim 12, 20 wherein the light chain variable region CDR3 sequence comprises an amino acid sequence ed from a group consisting of SEQ ID NOs: 19, 20, 21, and 22, and vative modifications thereof.
14. 14. The antibody or the antigen binding fragment thereof according to claim 12 25 or 13, wherein the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 23, 24, 25, 26, 27, and 28, and conservative modifications thereof. 30
15. 15. The antibody or the antigen g fragment thereof according to any of claims 12 to 14, wherein the light chain variable region CDR2 sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 29, 30, 31, and 32, and vative modifications thereof.
16. 16. The antibody or the n binding fragment thereof according to any of claims 12 to 15, wherein the heavy chain variable region CDRl sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 33, 34, 35, and 36, and conservative 10 modifications thereof.
17. 17. The antibody or the n g fragment thereof according to any of claims 12 to 16, wherein the light chain variable region CDRl sequence comprises an amino acid sequence selected from a group consisting of amino 15 acid sequences of SEQ ID NOs: 37, 38, 39, 40, and 41, and vative modifications thereof.
18. 18. The antibody or the antigen binding fragment f according to any one of claims 1 to 17, wherein the antibody is chimeric, humanized, fully human, or 20 rat antibody.
19. 19. The antibody or the antigen binding fragment thereof according to any one of claim 1 to 4, or 7 to 18, wherein the antibody exhibits at least one of the following properties: 25 a) binds to human CTLA—4 with a KB of 2.08E—09 M or less and/or to mouse CTLA—4 with a KB of 1.39E—09 M or less; b) enhances interleukin—2 release from stimulated PBMCs.
20. 20. A nucleic acid molecule ng the antibody or the antigen g 30 fragment thereof according to any one of claims 1 to 19.
21. 21. A cloning or expression vector comprising the nucleic acid molecule of claim 20.
22. 22. A host cell comprising one or more cloning or expression vectors of claim
23. 23. A process for the production of the antibody of any one of claims 1 to 19, sing culturing the host cell of claim 22 and isolating the antibody.
24. 24. The process of claims 23, wherein the dy is prepared through immunization in a SD rat with human CTLA—4 extracellular domain and mouse CTLA—4 extracellular domain. 15
25. 25. A transgenic rat comprising human immunoglobulin heavy and light chain transgenes, wherein the rat expresses the antibody of any one of claims 1 to 19.
26. 26. A hybridoma ed from the rat of claim 25, wherein the hybridoma produces the antibody of any one of claims 1 to 19.
27. 27. A pharmaceutical composition comprising the antibody or the antigen binding fragment f according to any one of claims 1 to 19, and one or more of a ceutically acceptable excipient, a diluent and a carrier. 25
28. 28. An immunoconjugate comprising the antibody or the antigen binding fragment thereof according to any one of claims 1 to 19, linked to a therapeutic agent.
29. 29. A pharmaceutical composition comprising the immunoconjugate of claim 30 28 and one or more of a ceutically acceptable excipient, a diluent and a carrier.
30. 30. A method for preparing an anti—CTLA—4 antibody or an antigen—binding fragment thereof comprising: (a) providing: (i) a heavy chain variable region antibody sequence comprising a CDRl sequence that is selected from a group consisting of SEQ ID NOs: 33—36, a CDR2 sequence that is selected from a group consisting of SEQ ID NOs: 23—28; and a CDR3 sequence that is selected from the group consisting of SEQ ID 10 NOs: 15—18;and/or (ii) a light chain variable region antibody sequence comprising a CDRl sequence that is ed from the group ting of SEQ ID NOs: 37—41, a CDR2 ce that is selected from the group consisting of SEQ ID NOs: 29—32, and a CDR3 sequence that is selected from the group consisting of SEQ 15 ID NOs: 19—22; and (b) expressing the altered antibody sequence as a n.
31. 31. A method of modulating an immune response in a subject comprising administering to the subject the dy or antigen binding fragment thereof 20 according to any one of claims 1 to 19.
32. 32. Use of the antibody or antigen binding fragment thereof according to any one of claims 1 to 19 in the manufacture of a medicament for the treatment or prophylaxis of an immune disorder or cancer.
33. 33. A method of inhibiting growth of tumor cells in a subject, comprising administering to the subject a eutically effective amount of the antibody or the n—binding fragment thereof according to any one of claims 1 to 19, to inhibit growth of the tumor cells.
34. 34. The method of claim 33, wherein the tumor cells are of a cancer selected from a group consisting of melanoma, renal , prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin , cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, and rectal cancer.
35. 35. The method of any one of claims 33 or 34, wherein the antibody is a chimeric antibody, humanized dy, human antibody or rat antibody. W3162—l.101.2 XAb.IgGl W3162—l.l45.10XAb.|gGl W3162—l.l46.19 Gl OD450 W3162—l.154.8 XAb.IgGl Ipilimumab Isotype of ic Ab \axxx‘s“‘:‘¢““‘i‘ v 0.0001 0.01 l 100 Ab (nM)