Novel monoclonal antibody against cytotoxic T lymphocyte-associated protein 4 (CTLA-4)

Abstract

To provide antibodies against CTLA-4 and compositions thereof, and immunotherapy in the treatment of cancer, infections or other human diseases using anti-CTLA-4 antibodies.SOLUTION: 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.SELECTED DRAWING: None

Description

Technical Field

[0001] Technical Field The present invention generally relates to antibodies against CTLA-4 and compositions thereof, and to immunotherapy in the treatment using anti-CTLA-4 antibodies for cancer, infection, or other human diseases.

Background Art

[0002] Background of the Invention Cancer immunotherapy is a recent area of research for treating cancer. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is one of the established targets of immune checkpoints. After activation of T cells, CTLA-4 is rapidly expressed on those T cells, usually within one hour after the antigen binds to the TCR. CTLA-4 can suppress T cell signaling through competition with CD28. CD28 mediates one of the well-characterized T cell co-stimulatory signals: binding of CD28 to its ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells results in T cell proliferation by inducing the production of interleukin-2 and anti-apoptotic factors. Since the affinity of CTLA-4 for binding to CD80 and CD86 is much higher than that of CD28, CTLA-4 can outcompete CD28 for binding to CD80 and CD86, resulting in suppression of T cell activation. In addition to its induced expression on activated T cells, CTLA-4 is constitutively expressed on the surface of regulatory T cells (Tregs), suggesting the possibility that CTLA-4 is required for contact-mediated suppression and its association with the production of immunosuppressive cytokines such as transforming growth factor beta and interleukin-10 by Tregs.

[0003] Inhibition of CTLA-4 can induce tumor regression, which has been demonstrated in numerous preclinical and clinical studies. Two antibodies against CTLA-4 are in clinical development. Ipilimumab (MDX-010, BMS-734016), a fully human anti-CTLA-4 monoclonal antibody of the IgG1 kappa isotype, is an immunomodulatory agent approved as a single-agent therapy for the treatment of advanced melanoma. The proposed mechanism of action of ipilimumab is the interference with the interaction of CTLA-4, which is expressed on a subset of activated T cells, with CD80 / CD86 molecules on professional antigen-presenting cells. This results in the enhancement of T cells by inhibiting the inhibitory regulation of T cell activation, which is promoted by the interaction of CTLA-4 with CD80 / CD86. The resulting activation, proliferation of T cells, and infiltration of lymphocytes into the tumor lead to tumor cell death. The commercially available dosage form is a 5 mg / mL concentrate for injection solution. Ipilimumab is also in clinical studies for other tumor types, including prostate cancer and lung cancer. Another anti-CTLA-4 antibody, tremelimumab, has been evaluated as a single-agent therapy in melanoma and malignant mesothelioma.

Summary of the Invention

[0004] Disclosure of the Invention The present invention provides an isolated antibody, particularly a monoclonal antibody or a humanized monoclonal antibody.

[0005] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds to human, monkey, and mouse CTLA-4.

[0006] The aforementioned antibody or antigen-binding fragment inhibits the binding of CTLA-4 to CD80 or CD86.

[0007] In the aforementioned antibody or antigen-binding fragment, the binding epitope of the antibody or antigen-binding fragment comprises a polysaccharide at N145 or N145 of CTLA-4.

[0008] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds to human CTLA-4 and cynomolgus CTLA-4, and wherein the binding epitope of the antibody or antigen-binding fragment comprises P138 of CTLA-4.

[0009] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment a) binds to human CTLA-4 with a K of 4.77E-10 M or less; and D b) binds to mouse CTLA-4 with a K of 1.39E-09 M or less. b) binds to mouse CTLA-4 with a K of 1.39E-09 M or less. D In the aforementioned antibody, the antibody or antigen-binding fragment exhibits at least one of the following characteristics:

[0010] In the aforementioned antibody, the antibody or antigen-binding fragment exhibits at least one of the following characteristics: a) binds to human CTLA-4 with a K of 4.77E-10 M to 2.08E-10 M and binds to mouse CTLA-4 with a K of 1.39E-09 M to 9.06E-10 M; D a) binds to human CTLA-4 with a K of 4.77E-10 M to 2.08E-10 M and binds to mouse CTLA-4 with a K of 1.39E-09 M to 9.06E-10 M; D b) enhances the release of interleukin-2 from stimulated PBMC; b) enhances the release of interleukin-2 from stimulated PBMC; c) does not substantially bind to any protein selected from the group consisting of factor VIII, FGFR, PD-1, CD22, VEGF, CD3, HER3, OX40, and 4-1BB.

[0011] The present invention provides 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 the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, wherein the antibody or antigen-binding fragment specifically binds to CTLA-4.

[0012] The present invention provides an antibody or an antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, Here, the antibody or antigen-binding fragment specifically binds to CTLA-4.

[0013] The present invention provides: a) a variable region of a heavy chain having an amino acid sequence that is at least 70%, 80%, 90%, or 95% identical to a sequence selected from the 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% identical to a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14 comprising an antibody or an antigen-binding fragment thereof, where the antibody or antigen-binding fragment specifically binds to CTLA-4.

[0014] The present invention provides: 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; 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, 11, 12, 13, and 14 comprising an antibody or an antigen-binding fragment thereof, where the antibody or antigen-binding fragment specifically binds to CTLA-4.

[0015] In various embodiments, the antibody or antigen-binding fragment thereof is: 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 comprising, where the antibody or antigen-binding fragment specifically binds to CTLA-4; or, the antibody or antigen-binding fragment thereof is: 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 selected from the group consisting of SEQ ID NO: 9 and comprising the antibody or antigen-binding fragment specifically binds to CTLA-4; or, the antibody or its antigen-binding fragment is: a) A variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 3; and b) A variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and comprising the antibody or antigen-binding fragment specifically binds to CTLA-4; or, the antibody or its antigen-binding fragment is: 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 and comprising the antibody or antigen-binding fragment specifically binds to CTLA-4; or, the antibody or its antigen-binding fragment is: 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 and comprising the antibody or antigen-binding fragment specifically binds to CTLA-4; or, the antibody or its antigen-binding fragment is: 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 and comprising the antibody or antigen-binding fragment specifically binds to CTLA-4; Or, the antibody or antigen-binding fragment thereof is: a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting 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 and the antibody or antigen-binding fragment specifically binds to CTLA-4.

[0016] The sequence of the antibody is shown in Table 1 and the Sequence Listing.

[0017] (Table 1) Putative Amino Acid Sequences of Antibodies TIFF0007717767000001.tif187151

[0018] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof comprising a complementarity-determining region (CDR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 41, wherein the antibody or antigen-binding fragment specifically binds to CTLA-4.

[0019] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof comprising a variable region of a heavy chain comprising CDR1, CDR2, and CDR3 sequences; and a variable region of a light chain comprising CDR1, CDR2, and CDR3 sequences, wherein the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, and 18, and conservative modifications thereof, wherein the antibody or antigen-binding fragment specifically binds to CTLA-4.

[0020] Preferably, herein, the light chain variable region CDR3 sequence of the aforementioned antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22, and conservative modifications thereof.

[0021] Preferably, here the CDR2 sequence of the heavy chain variable region of the aforementioned antibody or its antigen-binding fragment comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 23, 24, 25, 26, 27, and 28, and conservative modifications thereof.

[0022] Preferably, here the CDR2 sequence of the light chain variable region of the aforementioned antibody or its antigen-binding fragment comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 29, 30, 31, and 32, and conservative modifications thereof.

[0023] Preferably, here the CDR1 sequence of the heavy chain variable region of the aforementioned antibody or its antigen-binding fragment comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 33, 34, 35, and 36, and conservative modifications thereof.

[0024] Preferably, in the antibody of the present invention, the CDR1 sequence of the light chain variable region of the aforementioned antibody or its antigen-binding fragment comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 37, 38, 39, 40, and 41, and conservative modifications thereof.

[0025] In a more preferred embodiment, the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds to CTLA-4 and comprises: a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences; and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: a) the CDR1 sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 33, 34, 35, and 36, the CDR2 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 23, 24, 25, 26, 27, and 28, and the CDR3 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 15, 16, 17, and 18; b) and the light chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 37, 38, 39, 40, and 41, and the CDR2 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 29, 30, 31, and 32, and the CDR3 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 19, 20, 21, and 22, wherein the antibody or antigen-binding fragment specifically binds to CTLA-4.

[0026] Preferred antibodies or antigen-binding fragments thereof are: a) a heavy chain variable region CDR1 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 CDR1 comprising SEQ ID NO: 19; e) a light chain variable region CDR2 comprising SEQ ID NO: 29; f) a light chain variable region CDR3 comprising SEQ ID NO: 37; comprising wherein the antibody or antigen-binding fragment specifically binds to CTLA-4.

[0027] Another preferred antibody or antigen-binding fragment thereof is: a) a heavy chain variable region CDR1 comprising SEQ ID NO: 16; b) a heavy chain variable region CDR2 comprising SEQ ID NO: 24; c) a heavy chain variable region CDR3 comprising SEQ ID NO: 34; d) a light chain variable region CDR1 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; comprising The antibody or the antigen-binding fragment specifically binds to CTLA-4.

[0028] Another preferred antibody or its antigen-binding fragment is: a) Heavy chain variable region CDR1 containing SEQ ID NO: 17; b) Heavy chain variable region CDR2 containing SEQ ID NO: 25; c) Heavy chain variable region CDR3 containing SEQ ID NO: 35; d) Light chain variable region CDR1 containing SEQ ID NO: 19; e) Light chain variable region CDR2 containing SEQ ID NO: 31; f) Light chain variable region CDR3 containing SEQ ID NO: 39; and the antibody or the antigen-binding fragment specifically binds to CTLA-4.

[0029] Another preferred antibody or its antigen-binding fragment is: a) Heavy chain variable region CDR1 containing SEQ ID NO: 18; b) Heavy chain variable region CDR2 containing SEQ ID NO: 26; c) Heavy chain variable region CDR3 containing SEQ ID NO: 36; d) Light chain variable region CDR1 containing SEQ ID NO: 22; e) Light chain variable region CDR2 containing SEQ ID NO: 32; f) Light chain variable region CDR3 containing SEQ ID NO: 40; and the antibody specifically binds to CTLA-4.

[0030] Another preferred antibody or its antigen-binding fragment is: a) Heavy chain variable region CDR1 containing SEQ ID NO: 16; b) Heavy chain variable region CDR2 containing SEQ ID NO: 27; c) Heavy chain variable region CDR3 containing SEQ ID NO: 34; d) Light chain variable region CDR1 containing SEQ ID NO: 20; e) Light chain variable region CDR2 containing SEQ ID NO: 30; f) Light chain variable region CDR3 containing SEQ ID NO: 38; comprising the antibody or the antigen-binding fragment specifically binds to CTLA-4.

[0031] Another preferred antibody or its antigen-binding fragment is: a) Heavy chain variable region CDR1 containing SEQ ID NO: 17; b) Heavy chain variable region CDR2 containing SEQ ID NO: 25; c) Heavy chain variable region CDR3 containing SEQ ID NO: 35; d) Light chain variable region CDR1 containing SEQ ID NO: 21; e) Light chain variable region CDR2 containing SEQ ID NO: 31; f) Light chain variable region CDR3 containing SEQ ID NO: 39; comprising the antibody or the antigen-binding fragment specifically binds to CTLA-4.

[0032] Another preferred antibody or its antigen-binding fragment is: a) Heavy chain variable region CDR1 containing SEQ ID NO: 18; b) Heavy chain variable region CDR2 containing SEQ ID NO: 28; c) Heavy chain variable region CDR3 containing SEQ ID NO: 36; d) Light chain variable region CDR1 containing SEQ ID NO: 22; e) Light chain variable region CDR2 containing SEQ ID NO: 32; f) Light chain variable region CDR3 containing SEQ ID NO: 41; comprising the antibody or the antigen-binding fragment specifically binds to CTLA-4.

[0033] The CDR sequences of the above antibodies are shown in Table 2 and the Sequence Listing.

[0034] (Table 2) CDR sequences of the antibody TIFF0007717767000002.tif169151

[0035] The antibody of the present invention can be a chimeric antibody. The antibody of the present invention can be a humanized antibody. The antibody of the present invention can be a fully human antibody. The antibody of the present invention can be a rat antibody.

[0036] The antibody of the present invention or its antigen-binding fragment can exhibit at least one of the following characteristics: a) Binding to human CTLA-4 with a K of 2.08E-09 M or less and / or binding to mouse CTLA-4 with a K of 1.39E-09 M or less; D and / or binding to mouse CTLA-4 with a K of 1.39E-09 M or less; D and binding to mouse CTLA-4; b) Enhancing the release of interleukin-2 from stimulated PBMCs.

[0037] In a further aspect, the present invention provides a nucleic acid molecule encoding the antibody or its antigen-binding fragment.

[0038] The present invention provides a cloning vector or expression vector comprising a nucleic acid molecule encoding the antibody or its antigen-binding fragment.

[0039] The present invention also provides a host cell comprising one or more cloning vectors or expression vectors.

[0040] In yet another aspect, the present invention provides a method comprising culturing the host cell of the present invention and isolating the antibody, wherein the antibody is prepared by immunization with the extracellular domain of human CTLA-4 and the extracellular domain of mouse CTLA-4 in SD rats.

[0041] The present invention provides a transgenic animal, such as a rat, that contains transgenes for the heavy and light chains of human immunoglobulins, where the rat expresses the antibody of this invention.

[0042] The present invention provides a hybridoma prepared from the rat of this invention, where the hybridoma produces the antibody.

[0043] In a further aspect, the present invention provides a pharmaceutical composition comprising the antibody of the present invention or an antigen-binding fragment of said antibody and one or more of a pharmaceutically acceptable excipient, diluent, or carrier.

[0044] The present invention provides an immunoconjugate comprising the antibody of the present invention or an antigen-binding fragment thereof conjugated to a therapeutic substance.

[0045] Here, the present invention provides a pharmaceutical composition comprising the immunoconjugate and one or more of a pharmaceutically acceptable excipient, diluent, or carrier.

[0046] The present invention also provides a method for preparing an anti-CTLA-4 antibody or an antigen-binding fragment thereof, the method comprising the following steps: (a) (i) A heavy chain variable region antibody sequence comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 33 - 36, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 23 - 28; and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 15 - 18; and / or (ii) A light chain variable region antibody sequence comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 37 - 41, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 29 - 32, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 19 - 22 is provided; and (b) expressing the modified antibody sequence as a protein.

[0047] The present invention also provides a method for regulating an immune response in a subject, the method comprising administering to the subject any one of the antibodies or antigen-binding fragments thereof in this invention.

[0048] The present invention also provides the use of the antibody or its antigen-binding fragment in the manufacture of a medicament for the treatment or prevention of an immune disorder or cancer.

[0049] The present invention also provides a method for suppressing the growth of tumor cells in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody or the antigen-binding fragment to suppress the growth of tumor cells.

[0050] Here, the present invention provides the method, wherein the tumor cells are of cancer selected from the group consisting of melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or uveal malignant melanoma, uterine cancer, ovarian cancer, and rectal cancer.

[0051] Here, the present invention provides the method, wherein the antibody is a chimeric antibody, a humanized antibody, a human antibody, or a rat antibody.

[0052] Features and advantages of this invention The inventors prepared a humanized antibody against CTLA-4 by utilizing a proprietary hybridoma technology, where the antibody inhibited the binding of CTLA-4 to its ligands CD80 and CD86. The antibodies reported in this invention specifically bind to CTLA-4 proteins of both human and monkey with high binding affinity; and potently regulate the immune response and increase the production of interleukin-2.

[0053] One of the antibodies not only bound to human and monkey CTLA-4 but also to murine CTLA-4, which can greatly facilitate the preclinical verification of its efficacy in a mouse tumor model. [Invention 1001] An antibody or antigen-binding fragment thereof that binds to CTLA-4 of humans, monkeys, and mice. [Invention 1002] The antibody or antigen-binding fragment thereof of Invention 1001, wherein the antibody inhibits the binding of CTLA-4 to CD80 or CD86. [Invention 1003] The antibody or antigen-binding fragment thereof of Invention 1001 or 1002, wherein the binding epitope of the antibody or antigen-binding fragment contains a polysaccharide at N145 or N145 of CTLA-4. [Invention 1004] An antibody or antigen-binding fragment thereof that binds to CTLA-4 of humans and monkeys, and the binding epitope of the antibody or antigen-binding fragment contains P138 of CTLA-4. [Invention 1005] (a) Binds to human CTLA-4 with a K of 4.77E-10 M or less; and D (b) Binds to mouse CTLA-4 with a K of 1.39E-09 M or less, An antibody or antigen-binding fragment thereof. D [Invention 1006] (a) Binds to human CTLA-4 with a K of 4.77E-10 M to 2.08E-10 M and binds to mouse CTLA-4 with a K of 1.39E-09 M to 9.06E-10 M; (b) Exhibits at least one of the properties of enhancing the release of interleukin-2 from stimulated PBMCs An antibody or antigen-binding fragment thereof of Invention 1005. D [Invention 1007] D Contains an amino acid sequence that is at least 70%, 80%, 90%, or 95% homologous to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, Specifically binds to CTLA-4, An antibody or antigen-binding fragment thereof. [Invention 1008] Contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, Specifically binds to CTLA-4, An antibody or antigen-binding fragment thereof. [Invention 1009] (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 the 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% identical to a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14 comprising specifically binding to CTLA-4, an antibody or an antigen-binding fragment thereof. [Inventive Item 1010] (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; 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, 11, 12, 13, and 14 comprising specifically binding to CTLA-4, an antibody or an antigen-binding fragment thereof. [Inventive Item 1011] comprising a complementarity-determining region (CDR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 41, specifically binding to CTLA-4, an antibody or an antigen-binding fragment thereof. [Inventive Item 1012] A heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences; and A light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising an antibody or an antigen-binding fragment thereof, wherein the CDR3 sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, and 18, and conservative modifications thereof, the antibody or antigen-binding fragment specifically binds to CTLA-4, said antibody or an antigen-binding fragment thereof. [Inventive Item 1013] The antibody or an antigen-binding fragment thereof according to Inventive Item 1012, wherein the CDR3 sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22, and conservative modifications thereof. [Inventive Item 1014] The antibody or an antigen-binding fragment thereof according to Inventive Item 1012 or 1013, wherein the CDR2 sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, 27, and 28, and amino acid sequences of conservative modifications thereof. [Inventive Item 1015] The antibody or an antigen-binding fragment thereof according to any one of Inventive Items 1012 to 1014, wherein the CDR2 sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, and 32, and amino acid sequences of conservative modifications thereof. [Inventive Item 1016] An antibody or antigen-binding fragment thereof according to any one of Inventions 1012 to 1015, wherein the heavy-chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 33, 34, 35, and 36, and conservative modifications thereof. [Invention 1017] An antibody or antigen-binding fragment thereof according to any one of Inventions 1012 to 1016, wherein the light-chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 37, 38, 39, 40, and 41, and conservative modifications thereof. [Invention 1018] An antibody or antigen-binding fragment thereof according to any one of Inventions 1001 to 1017, wherein the antibody is a chimeric antibody, a humanized antibody, a fully human antibody, or a rat antibody. [Invention 1019] The antibody is (a) binds to human CTLA-4 with a K of 2.08E-09 M or less and / or binds to mouse CTLA-4 with a K of 1.39E-09 M or less; D (b) has the property of enhancing the release of interleukin-2 from stimulated PBMC D among at least one of the properties An antibody or antigen-binding fragment thereof according to any one of Inventions 1001 to 1004 or 1007 to 1018, which exhibits [Invention 1020] A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of Inventions 1001 to 1019. [Invention 1021] A cloning vector or expression vector containing the nucleic acid molecule of Invention 1020. [Invention 1022] A host cell containing one or more of the cloning vectors or expression vectors of Invention 1021. [Invention 1023] A method for producing an antibody according to any one of Inventions 1001 to 1019, comprising culturing the host cell of Invention 1022 and isolating the antibody. [Invention 1024] The method of Invention 1023, wherein the antibody is prepared by immunizing SD rats with the extracellular domain of human CTLA-4 and the extracellular domain of mouse CTLA-4. [Invention 1025] A transgenic rat containing transgenes of the heavy and light chains of human immunoglobulins and expressing an antibody according to any one of Inventions 1001 to 1019. [Invention 1026] A hybridoma prepared from the rat of Invention 1025 that produces an antibody according to any one of Inventions 1001 to 1019. [Invention 1027] A pharmaceutical composition comprising any one of the antibodies of the present invention 1001 to 1019 or an antigen-binding fragment thereof and one or more of a pharmaceutically acceptable excipient, diluent, and carrier. [The present invention 1028] An immunoconjugate comprising any one of the antibodies of the present invention 1001 to 1019 or an antigen-binding fragment thereof conjugated to a therapeutic substance. [The present invention 1029] A pharmaceutical composition comprising the immunoconjugate of the present invention 1028 and one or more of a pharmaceutically acceptable excipient, diluent, and carrier. [The present invention 1030] A method for preparing an anti-CTLA-4 antibody or an antigen-binding fragment thereof, comprising the following steps: (a) providing (i) a heavy-chain variable region antibody sequence comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 33 to 36, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 23 to 28, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 15 to 18; and / or (ii) a light-chain variable region antibody sequence comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 37 to 41, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 29 to 32, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 19 to 22 ; and (b) expressing the modified antibody sequence as a protein. [The present invention 1031] A method for regulating an immune response in a subject, comprising administering to the subject any one of the antibodies of the present invention 1001 to 1019 or an antigen-binding fragment thereof. [The present invention 1032] Use of any one of the antibodies of the present invention 1001 to 1019 or an antigen-binding fragment thereof in the manufacture of a medicament for the treatment or prevention of an immune disorder or cancer. [The present invention 1033] A method for suppressing the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of any one of the antibodies of the present invention 1001 to 1019 or an antigen-binding fragment thereof for suppressing the growth of tumor cells. [The present invention 1034] The method of the present invention 1033, wherein the tumor cells are of a cancer selected from the group consisting of melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or uveal malignant melanoma, uterine cancer, ovarian cancer, and rectal cancer. [The present invention 1035] The method of the present invention 1033 or 1034, wherein the antibody is a chimeric antibody, a humanized antibody, a human antibody, or a rat antibody.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0055] Detailed Description To make the present invention more readily understood, some terms are first defined. Additional definitions are set forth throughout the detailed description.

[0056] The terms "cytotoxic T lymphocyte-associated antigen-4", "protein CTLA-4", "CTLA-4", "CTLA4", and "CD152" are used interchangeably and include human CTLA-4 or variants, isoforms, species homologs of CTLA-4 from other species, and analogs having at least one epitope common to CTLA-4.

[0057] As used herein, the term "antibody" includes full-length antibodies and any antigen-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 linked to each other by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), and between the CDRs are regions of more conserved sequence called framework regions (FRs). Each of VH and VL is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains include the binding domains that interact with an antigen.

[0058] As used in this disclosure, the term "antibody" refers to an immunoglobulin or fragment or derivative thereof, whether produced in vitro or in vivo, and includes any polypeptide containing an antigen-binding site. The term includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, heterospecific antibodies, humanized antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, variant antibodies, 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 the antigen-binding function, i.e., the ability to specifically bind to CTLA-4. Typically, such fragments may include antigen-binding fragments.

[0059] The terms "antigen-binding fragment", "antigen-binding domain", and "binding fragment" refer to a portion of an antibody molecule that contains the amino acids responsible for the specific binding between the antibody and the antigen. For example, if the antigen is large, the antigen-binding fragment may only bind to a portion of the antigen. A portion of the antigen molecule that plays a role in the specific interaction with the antigen-binding fragment is referred to as an "epitope" or "antigenic determinant".

[0060] Antigen-binding fragments typically include the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH), but do not necessarily have to include both. For example, the so-called Fd antibody fragment consists of only the VH domain but still retains some of the antigen-binding function of the intact antibody.

[0061] Along the above, the term "epitope" defines an antigenic determinant that is specifically bound / identified by the binding fragment defined above. The binding fragment can specifically bind / interact with a target structure, such as a conformational epitope or a continuous epitope that is unique to the structure of human CTLA-4 and murine CTLA-4. A conformational epitope or a discontinuous epitope is characterized as a polypeptide antigen by the presence of two or more separate amino acid residues that are separated in the primary sequence but come together on the surface of the molecule when the polypeptide folds into an unfolded protein / antigen. The two or more separate amino acid residues contributing to the epitope are present in distant regions of one or more polypeptide chains. These residues come together on the surface of the molecule when the polypeptide chain folds into a three-dimensional structure to form the epitope. In contrast, a continuous epitope or a linear epitope consists of two or more separate amino acid residues present in a single linear segment of the polypeptide chain.

[0062] The term "binds to an epitope of CTLA-4" may be defined by a linear amino acid sequence or may refer to an antibody having specific binding affinity for a specific epitope of CTLA-4 defined by the tertiary, i.e., three-dimensional, structure in a portion of the CTLA-4 polypeptide. Binding means that the antibody affinity for a 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 a portion of CTLA-4 compared to the affinity for other related polypeptides. Preferably the affinity is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold, 10 3 times, 10 4 times, 10 5 times, 10 6times or greater. Preferably, the binding affinity is determined by enzyme-linked immunosorbent assay (ELISA), or by fluorescence-activated cell sorting (FACS) analysis or surface plasmon resonance (SPR). More preferably, the binding specificity is obtained by fluorescence-activated cell sorting (FACS) analysis.

[0063] The term "cross-reactivity" as used herein refers to the binding of the antigen fragments described herein to the same target molecule in humans, monkeys, and / or rodents (mice or rats). Thus, "cross-reactivity" should be understood as interspecies reactivity that is directed against the same molecule X expressed in different species and not against other molecules other than X. For example, the cross-species specificity of a monoclonal antibody that recognizes human CTLA-4 against monkey and / or rodent (mouse or rat) CTLA-4 can be determined, for example, by FACS analysis.

[0064] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, which are, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Unless otherwise specified, the terms "patient" or "subject" are used interchangeably.

[0065] The terms "treatment" and "method of treatment" refer to both therapeutic treatment and prophylactic / preventive means. An individual in need of treatment may include not only an individual who will ultimately have a particular medical disorder, but also an individual who already has the disorder.

[0066] The term "conservative modification" refers to modifications of nucleotide and amino acid sequences that do not significantly affect and also do not alter the binding characteristics of the antibody encoded by the nucleotide sequence or the antibody comprising the amino acid sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions, and deletions. The modifications can be introduced into the sequence by standard techniques known in the art, such as site-directed mutagenesis and mutagenesis via PCR. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0067] Unless otherwise specified, the experimental procedures in the following examples are conventional procedures.

Example

[0068] Example 1 : Preparation of Research Materials 1. Expression and Purification of Soluble CTLA-4 Human and mouse CTLA-4 extracellular domain (ECD) genes with a hexahistidine (6xHis) tag or an Fc tag were cloned into expression vectors and then used for transfection of Expi293 cells using the Expi293 Expression System Kit. The cells were cultured in Expi293 Expression Medium on an orbital shaking platform rotating at 135 rpm in an incubator at 37 °C containing humidified air with 8% CO2. The harvested supernatant was used for protein purification. The hexahistidine-tagged protein was purified using an Ni-NTA column, and the Fc-tagged protein was purified using a protein A column.

[0069] 2. Development of Cell Lines The gene for full-length human CTLA-4 was cloned into an expression vector for the development of stable cell lines. Briefly, 30 mL of 293F cells at a density of 1 x 10 6 cells / mL were transfected with 30 μg of DNA using Plasfect reagent. The transfected cells were placed in an incubator set at 37 °C, 8% CO2, and a shaking speed of 100 rpm. 4 - 6 μg / mL of blasticidin at a final concentration was used to select stable clones 24 - 48 hours after transfection. The selected clones were tested by FACS using an anti-CTLA-4 antibody.

[0070] For the purpose of obtaining cells expressing cynomolgus monkey CTLA-4, the gene for full-length cynomolgus monkey CTLA-4 was cloned into an expression vector for the development of cell pools. Briefly, 1 x 10 6A 30 mL volume of 293F cells at a density of cells / mL was transfected with 30 μg of DNA using Plasfect reagent (Life Technology). The transfected cells were placed in an incubator set at 37 °C, 8% CO2, and a shaking speed of 100 rpm. Twenty-four hours after transfection, blasticidin at a final concentration of 4 μg / mL was used to select the cell pool. The selected cell pool was tested by FACS using an anti-CTLA-4 antibody.

[0071] Example 2 : Preparation of antibody hybridomas 1. Immunization Human CTLA-4 and murine CTLA-4 were used for immunization of SD rats. Specifically, three SD rats were immunized with 30 μg / animal of human and mouse CTLA-4 ECD proteins in adjuvant. The adjuvant contained Titer-Max, Adju-Phos, and CpG-ODN. The rats were injected both intradermally and subcutaneously from the plantar surface once a week. Antibody titers in the serum were measured monthly by ELISA. When the antibody titers were high enough, the rat with the highest titer was given a final boost with human and mouse CTLA-4 ECD proteins in adjuvant-free Dulbecco's phosphate-buffered saline (DPBS). After several days, the spleen and lymph nodes were harvested from the rat and lymphocytes were isolated for fusion.

[0072] 2. Cell fusion Cell fusion was performed as follows: SP2 / 0 cells, which are myeloma cells, were thawed the week before fusion and divided 1:2 daily until the day before fusion to maintain them in a logarithmic growth state. B lymphocytes isolated from the lymph nodes of immunized rats and myeloma cells were each treated with trypsin, and the reaction was stopped by the addition of FBS. B lymphocytes were combined with myeloma cells at a ratio of 1:1. The cell mixture was then washed and resuspended in an electrofusion solution containing 0.3 M sucrose, 0.1 mM magnesium acetate, and 0.1 mM calcium acetate at 2 x 10 6 cells / ml. Electro cell fusion was performed using a Btx Electro Cell Manipulator (Ecm 2001) according to the manufacturer's standard protocol. Subsequently, the cell suspension from the fusion chamber was promptly transferred into a sterile flask containing fresh medium and incubated in an incubator at 37°C for 2 hours. The cell suspension was then mixed and transferred to 60 96-well plates (1 x 10 4 cells / well). The 96-well plates were cultured at 37°C and 5% CO2 and monitored regularly. When the clones had grown sufficiently large (after 7 - 10 days), 180 μL / well of the supernatant was removed, and then 200 μL of fresh medium was added per well. After 72 hours, 100 μL of the supernatant was transferred from the tissue culture plate to a 96-well assay plate for screening.

[0073] 3. Screening of Hybridomas Multiple hybridoma clones were screened for binding not only to genetically engineered human CTLA-4 expressing cells, but also to CTLA-4 proteins from human, murine, and monkey. Once specific CTLA-4 binding and inhibitory activity were confirmed through the first and second screenings, the positive hybridoma strains were subcloned into 96-well plates using limiting dilution. The plates were cultured at 37 °C, 5% CO2 until the positive clones were further screened for competition with the binding of the ligands CD80 and CD86 to CTLA-4. The culture supernatants of the selected positive clones were collected for antibody purification and further characterization. Lead candidates were selected for VH and VL sequencing.

[0074] 4. Determination of VH and VL Sequences from Hybridomas The VH and VL genes of the antibody of the selected hybridoma clone were isolated by RT-PCR or 5’ RACE. Specifically, total RNA was isolated from hybridoma cells using the RNeasy Plus Mini Kit (Qiagen). First-strand cDNA was reverse transcribed using oligo dT. The VH and VL genes of the antibody were amplified from the cDNA using a set of 3’ constant region degenerate primers and 5’ degenerate primers. The 5’ degenerate primers were designed based on the coding region of the upstream signal sequence of the Ig variable sequence. The PCR products were then ligated into the pMD18-T vector, and 10 μL of the ligation product was transformed into Top10 competent cells. The transformed cells were plated on 2xYT plates containing carbenicillin and incubated overnight at 37 °C. 15 positive colonies were randomly picked for DNA sequencing by Biosune. Alternatively, 5’ RACE was used to identify the VH and VL sequences of the selected hybridoma clone. First, RNA was first reverse transcribed into cDNA using the 5’-RACE kit (Takara-28001488), followed by PCR using a 3’ degenerate primer and a 3’ adapter primer (ExTaq: Takara-RR001B). The PCR fragment was inserted into the pMD18-T vector (Takara-D101C) and sent for sequencing (Biosune, Shanghai).

[0075] Example 3 : Generation and Characterization of Chimeric Antibodies 1. Generation of Chimeric Antibodies The deduced amino acid sequences of VH and VL are listed in Table 3. The underlined sequences are the CDRs defined by the Kabat notation system. The variable regions of these rat antibodies were fused with the constant regions of human antibodies, and the chimeric antibodies were expressed from Expi293 cells and purified using protein A chromatography.

[0076] (Table 3) Variable Region Sequences of Rat Anti-CTLA-4 Antibodies TIFF0007717767000003.tif146152

[0077] 2. Characterization of Chimeric Antibodies 2.1 The antibodies bound to human, monkey, and murine CTLA-4 (ELISA, FACS, and SPR). Chimeric antibodies with rat variable regions and human constant regions were expressed from mammalian cells and purified using protein A affinity chromatography.

[0078] The antibodies were tested in a CTLA-4 binding ELISA. As shown in Figures 1, 2, and 3, all four antibodies bound to human and monkey CTLA-4 with EC50 values comparable to ipilimumab (WBP316-BMK1), but only one antibody, W3162-1.146.19, also bound to murine CTLA-4 with an EC 50 of 0.01 nM. To confirm that the antibodies could bind to CTLA-4 on the cell surface, a CTLA-4 expressing cell line was used in a FACS assay. These antibodies also bound to CTLA-4 on the cell surface (Figure 4) with EC 50 values in the range of 1.14 nM to 9.42 nM. W3162-1.146.19 bound to CTLA-4 on the cell surface with an EC 50 of 3.25 nM, and W3162-1.154.8 bound to CTLA-4 on the cell surface with an EC 50 of 1.26 nM.

[0079] The binding kinetics of the four antibodies were measured using SPR. The antibodies were captured on immobilized goat anti-human Fc, and then different concentrations of human CTLA-4 ECD were injected in sequence. Sensorgrams for the reference channel and buffer channel were subtracted from the test sensorgram. The data were used by fitting to a 1:1 binding analysis. As shown in Figure 5 and Table 4, all four antibodies had higher affinities than ipilimumab (WBP316-BMK1), with K values in the range of 2.08E-09 nM to 6.80E-11 nM. Dand bound to the human CTLA-4 ECD domain.

[0080] (Table 4) Kinetics of antibody binding to human CTLA-4 ECD TIFF0007717767000004.tif52128

[0081] 2.2 Competition of chimeric antibodies with ligands CTLA-4 is known to bind to both CD80 and CD86 with an affinity 20 - 50-fold higher than CD28 [Krummel 1996]. Therefore, it was tested whether anti-CTLA-4 antibodies could compete with their binding to CD80 and CD86 to CTLA-4. Both ELISA and FACS were used as competition assays. In the ELISA-based competition assay, human CTLA-4 was coated on the plate, and the antibody mixed with biotinylated ligand was added into the plate. The bound ligand was detected by HRP-conjugated streptavidin. As shown in Figures 6a and 6b, all four antibodies competed with the ligands CD80 (B7-1, L1) and CD86 (B7-2, L2) in binding to CTLA-4, and three of them, excluding W3162-1.101.2, had an EC comparable to ipilimumab (WBP316-BMK1). 50It had. In the FACS assay, a mixture of antibody and biotinylated human CTLA-4 was added to CD80-expressing cells or CD86-expressing cells, and the bound human CTLA-4 was detected by PE-conjugated streptavidin. As shown in Figure 7a (upper panel) and Figure 7b (lower panel), all four antibodies were able to effectively inhibit the binding of CTLA-4 to ligand-expressing cells. Three antibodies except W3162-1.154.8 were able to completely inhibit the binding of CTLA-4 to CD80 cells, while ipilimumab WBP316-BMK was only able to partially inhibit this binding even at the maximum concentration of 200 nM used (Figure 7a). In the FACS assay that inhibits the binding of CTLA-4 to CD86 cells (Figure 7b), all four antibodies were able to completely inhibit the binding of CTLA-4 to CD86 cells, while ipilimumab was only able to partially inhibit this binding even at the maximum concentration of 200 nM used. The kinetics of W3162-1.101.2 seemed to be different: the inhibition was less effective than ipilimumab at low concentrations and more effective than ipilimumab at high concentrations. The other three antibodies were more effective than ipilimumab in inhibiting CTLA-4 at all concentrations tested.

[0082] 2.3 Function of the chimeric antibody in the SEB assay The function of anti-CTLA-4 antibodies at different concentrations of 1.34 nM, 3.35 nM, 8.71 nM, 21.4 nM, 53.6 nM, and 134 nM was tested in a modified T cell stimulation assay (SEB assay). Staphylococcal enterotoxin B (SEB) was used as a stimulator of the activation of human T cells, where CTLA-4 has been reported to play an important role. The activation of T cells was measured by the secretion of IL-2. As shown in Figure 8, all four antibodies promoted the secretion of IL-2 in a dose-dependent manner and were comparable to or better than ipilimumab.

[0083] Example 4 Characterization of Humanized Antibodies 1. Humanization The "Best Fit" approach was used to humanize the light and heavy chains of the antibody.

[0084] Three anti-CTLA-4 antibodies (except for W3162-1.101.2 due to its relatively low binding activity in ELISA and FACS) were selected for humanization using CDR grafting technology. The CDRs (underlined in Table 5) and FRs of the variable regions of the antibodies were defined using the Kabat system. Based on sequence homology and structural similarity, the genes of the rat FR1-3 regions were replaced by humanized FR1-3 regions, while the FR4 region of the rat gene was replaced by a humanized FR4 region derived from the most similar JH and JK genes with the same structure. The hotspots of post-translational modification (PTM) in the variable regions were modified to reduce the risk of PTM. After confirming the template sequence and codon optimization, the heavy chain variable region and the light chain variable region were synthesized, cloned into the expression vector, and then used for the expression of the humanized antibody. The humanized antibody was purified using protein A chromatography, and the kinetics of binding to human, monkey, and murine CTLA-4 were measured using the SPR technique.

[0085] (Table 5) Variable Region Sequences of Humanized Anti-CTLA-4 Antibodies TIFF0007717767000005.tif99149

[0086] (Table 6) Variable Regions of Humanized Anti-CTLA-4 Antibodies TIFF0007717767000006.tif191149

[0087] 2. Characterization of Humanized Antibodies 2.1 The antibody bound to human, monkey, and murine CTLA-4 2.1.1 CTLA-4 Binding ELISA The humanized antibodies were expressed from mammalian cells and purified using protein A affinity chromatography. Ipilimumab was from a commercial source. Isotype control antibodies, 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 cynomolgus CTLA-4 ECD-6xHis were purchased from Sino Biological. HRP-conjugated goat anti-human IgG Fc was purchased from Bethyl (Catalog: A80-304P).

[0088] ELISA was used to test the binding of anti-human CTLA-4 antibodies to human, murine, and cynomolgus CTLA-4 proteins. 96-well plates were coated with human CTLA-4.ECD-6xHis (1.0 μg / mL), cynomolgus CTLA-4.ECD-6xHis (0.5 μg / mL), or mouse CTLA-4.ECD-6xHis (0.5 μg / mL) at 4 °C for 16 - 20 hours. After 1-hour blocking with 2% BSA in DBPS, test antibodies as well as positive and negative control antibodies were added to the plates and incubated at room temperature for 1 hour. Binding of antibodies to the plates was detected by HRP-conjugated goat anti-human IgG antibody (1:5000 dilution) with 1-hour incubation. Color was developed by preparing 100 μL of TMB substrate for 8 minutes and then stopped by 100 μL of 2N HCl. Absorbance at 450 nM was measured using a microplate spectrophotometer.

[0089] As shown in Figure 9, two antibodies, W3162-1.146.19-z12-IgGk and W3162-1.154.8-z35-IgGk, bound to human CTLA-4 with EC 50 of 0.03 nM and 0.04 nM, respectively, which was 50Slightly higher (Figure 9A). The two antibodies also bound to cynomolgus CTLA-4 with an EC50 of 0.05 nM (Figure 9B), however, only W3162-1.146.19-z12-IgGk bound to murine CTLA-4 with an EC50 of 0.19 nM. Neither W3162-1.154.8-z35-IgGk nor ipilimumab bound to murine CTLA-4 (Figure 9C).

[0090] 2.1.2 CTLA-4 Binding FACS The human CTLA4-expressing 293F cell line was developed by WuXi Biologics. The PE-conjugated goat anti-human IgG Fc fragment was purchased from Jackson (Catalog No. 109-115-098). A number of 1 x 10 5 cells per well were 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 the test antibody, positive control, and negative control were added to the resuspended cells and incubated at 4°C for 1 hour. The cells were washed twice with 200 μL of 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. An additional washing step was performed twice using 200 μL of DPBS containing 1% BSA, followed by centrifugation at 1500 rpm for 4 minutes at 4°C. Finally, the cells were resuspended in 100 μL of DPBS containing 1% BSA, and the fluorescence values were measured by flow cytometry and analyzed by FlowJo.

[0091] These antibodies were also able to bind to human CTLA-4 on the cell surface in the FACS assay. As shown in Figure 11 (Figures 10a and 10b), W3162-1.146.19-z12-IgGk, W3162-1.154.8-z35-IgGk, and ipilimumab had slightly different EC 50 values of 1.58 nM, 0.66 nM, and 0.83 nM, respectively.

[0092] 2.2 Binding kinetics of these antibodies 2.2.1 The binding kinetics of these antibodies were measured using SPR The experiment was based on SPR technology to measure the on-rate constant (ka) and off-rate constant (kd) of the antibodies against CTLA-4 ECD. The affinity constant (K D ) was determined as a result.

[0093] 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 (catalog number 109-005-098). In the immobilization step, the activation buffer was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection. The CM5 sensor chip was activated with the activation buffer for 420 seconds. 30 μg / mL of goat anti-human IgG Fcγ antibody in 10 mM NaAc (pH 4.5) was then injected into the Fc1-Fc4 channels at a flow rate of 5 μL / min for 200 seconds. The chip was inactivated with 1 M ethanolamine-HCl (GE). Then, the antibody was captured on the chip. Briefly, 4 μg / mL of the antibody in running buffer (HBS-EP+) was individually injected into the Fc3 channel at a flow rate of 10 μL / min for 30 seconds. 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 the analyte CTLA-4 (WBP316.hCTLA-4.ECD-6xHis), as well as blank running buffer, were sequentially injected into the Fc1-Fc4 channels at a flow rate of 30 μL / min during a 120-second binding step, followed by a 2400-second dissociation step. The regeneration buffer (10 mM glycine, pH 1.5) was injected at 10 μL / min for 30 seconds after each dissociation step.

[0094] The binding kinetics of these antibodies were measured using SPR. The antibodies were captured by immobilized anti-human Fc and different concentrations of CTLA-4-ECD were injected in sequence. The sensorgrams for the reference channel and the buffer channel were subtracted from the test sensorgram. The data were used for 1:1 binding analysis against human, monkey, and mouse CTLA-4.ECD-6xHis. As shown in Table 7, the humanized antibodies W3162-1.146.19-Z12, W145, and W3162-1.154.8-Z35 bound to the human CTLA-4-ECD domain with affinities of 0.477 nM, 1.84 nM, and 0.0968 nM, respectively. When compared with the rat antibody, the humanized antibodies had comparable affinities. W3162-1.146.19-Z12, and W3162-1.154.8-Z35 had significantly higher affinities than ipilimumab (K D = 3.68 nM). Antibody W3162-1.146.19-Z12 was also able to bind to murine CTLA-4, and its affinity before and after humanization is shown in Table 9. Its affinity of 1.39 nM after humanization was slightly lower than that of its parental antibody of 0.906 nM.

[0095] The affinities of W3162-1.146.19-Z12, W3162-1.145.10-z7, and W3162-1.154.8-Z35 for cynomolgus monkey CTLA-4-ECD were 1.92 nM, 0.598 nM, 0.131 nM, respectively (Table 8).

[0096] (Table 7) Kinetics of antibody binding to human CTLA-4 ECD TIFF0007717767000007.tif44148

[0097] (Table 8) Kinetics of antibody binding to monkey CTLA-4 ECD TIFF0007717767000008.tif38148

[0098] (Table 9) Kinetics of Antibody Binding to Mouse CTLA-4 ECD TIFF0007717767000009.tif19148

[0099] 2.2.2 Affinity Test by FACS FITC-conjugated goat anti-human IgG Fc was purchased from Jackson Immunoresearch Lab (Catalog No. 109-095-098), and BD CantoII was used for this assay. Briefly, HEK293 cells expressing human CTLA-4 were seeded in a 96-well U-bottom plate (BD) at a density of 5 x 10 4 cells / well. Test antibodies were serially diluted 1:2-fold in PBS containing 1% BSA and incubated with the cells at 4°C for 1 hour. After centrifugation at 1500 rpm for 4 minutes, the supernatant was discarded. The secondary antibody, FITC-conjugated goat anti-human IgG Fc (3.2 FITCs per 1 IgG, Jackson Immunoresearch Lab), was added to the resuspended cells at a final concentration of 14 μg / ml and incubated in the dark at 4°C for 30 minutes. The cells were then washed once and resuspended in PBS containing 1% BSA and analyzed by flow cytometry (BD). Fluorescence intensity was converted to bound molecules / cell based on quantitative beads (Quantum™ MESF Kits, Bangs Laboratories). K D was calculated using Graphpad Prism5.

[0100] The affinity of the humanized antibody binding to CTLA-4 on the cell surface was measured by a flow cytometry method modified from Benedict's method [Benedict 1997 JIM]. After measuring the fluorescence of antibody binding to CTLA-4-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 the equation, the calculated affinity constant K DIt is shown in Table 10. The affinities of the humanized antibodies W3162-1.146.19-Z12 and W3162-1.154.8-Z35 were 5.05 nM and 0.35 nM, respectively, while the affinity of ipilimumab was 0.97 nM.

[0101] (Table 10) Affinity test by FACS TIFF0007717767000010.tif28139

[0102] 2.3 Competition with ligands To test whether the humanized antibodies maintain their ability to inhibit the binding of CTLA-4 to CD80 and CD86, both ELISA and FACS were used in competition assays. Two CTLA-4 ligands, CD80 and CD86, were purchased from Sino Biological (catalog numbers 10698-H08H and 10699-H08H). The biotinylated anti-His tag antibody was purchased from Genscript (catalog number A00613). The HRP-conjugated streptavidin was purchased from Invitrogen (catalog number SNN1004).

[0103] 2.3.1 Competition assay based on ELISA ELISA was used to test whether an antibody 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 μg / mL) at 4 °C for 16 - 20 hours. After 1 hour of blocking with 2% BSA in DBPS, the test antibody as well as positive and negative control antibodies were pre-mixed with 0.25 μg / mL of CD80-6xHis or CD86-6xHis and then added to the plates and incubated at room temperature for 1 hour. After washing 3 times with PBS containing 0.05% Tween 20, the biotinylated anti-His tag antibody was diluted 1:2000 and added. The plates were incubated at room temperature for 1 hour. The bound ligand was detected with HRP-conjugated streptavidin (1:20000). Color was developed by preparing 100 μL of TMB substrate for 8 minutes and then stopped with 100 μL of 2N HCl. Absorbance at 450 nM was measured using a microplate spectrophotometer.

[0104] As shown in Figure 11, W3162-1.146.19-z12-IgGk and W3162-1.154.8-z35-IgGk had an effect similar to ipilimumab in inhibiting ligand binding to coated CTLA-4, and the IC 50 for CD80 was 0.87 nM, 0.63 nM, and 0.40 nM, and for CD86 was 0.71 nM, 0.50 nM, and 0.42 nM.

[0105] 2.3.2 FACS assay To test whether the antibody can inhibit the binding of CTLA-4 to CD80 and CD86 on the cell surface, the inventors used FACS to test this competition. The CD80-expressing CHO cell line and the CD86-expressing CHO cell line were developed by WuXi Biologics. Biotinylated CTLA-4.ECD.hFc was produced by WuXi Biologics. PE-conjugated streptavidin was purchased from eBioscience (catalog number 12-4317).

[0106] CD80-expressing cells or CD86-expressing cells were added to each well of a 96-well plate at 1 x 10 5 per well and centrifuged at 1500 rpm for 4 minutes at 4°C before removing the supernatant. Serial dilutions of the test antibody, positive control, and negative control were mixed with biotinylated human CTLA4.ECD.hFc. Since the density of the ligand on the cell surface varies, 0.02 μg / mL of hCTLA-4.ECD.hFc-biotin was used for human CD80 cells and 0.08 μg / mL of hCTLA-4.ECD.hFc-biotin was used for human CD86 cells. Then, the mixture of the antibody and CTLA-4 was added to the cells and incubated at 4°C for 1 hour. The cells were washed twice with 200 μL of FACS buffer (DPBS containing 1% BSA). Streptavidin PE diluted 1:333 in FACS buffer was added to the cells and incubated at 4°C for 1 hour. An additional washing step was performed twice using 200 μL of FACS buffer, followed by centrifugation at 1500 rpm for 4 minutes at 4°C. Finally, the cells were resuspended in 100 μL of FACS buffer, and the fluorescence values were measured by flow cytometry and analyzed by FlowJo.

[0107] The results are shown in Figure 12. The two humanized antibodies were able to inhibit CTLA-4 / ligand binding more effectively than ipilimumab. At the highest concentration used, ipilimumab only inhibited 32% of CTLA-4 binding to CD80 and 40% of CTLA-4 binding to CD86. In comparison, antibody W3162-1.146.19-Z12 inhibited 71% of CTLA-4 binding to CD80 and 73% of CTLA-4 binding to CD86, and antibody W3162-1.154.8-Z35 inhibited 89% of CTLA-4 binding to CD80 and 98% of CTLA-4 binding to CD86. The IC 50 for ipilimumab, W3162-1.146.19-Z12, and W3162-1.154.8-Z35 against CD80 was 3.23 nM, 6.60 nM, and 0.07 nM, respectively. The IC 50 for ipilimumab, W3162-1.146.19-Z12, and W3162-1.154.8-Z35 against CD86 was 2.52 nM, 5.15 nM, and 0.28 nM, respectively.

[0108] Cytokine release from PBMCs stimulated with 2.4 SEB Anti-CTLA4 antibodies were tested to see if they could enhance cytokine release from human PBMCs after stimulation with SEB (from The Second Military Medical University). Peripheral blood from healthy donors was obtained and cells were isolated by Ficoll GE Healthcare, 17-1440-02) density gradient centrifugation. After removal of the buffy coat, platelets were removed by several washes with medium. A number of 1 x 10 5 human PBMC cells were added to each well of a 96-well plate. Serial dilutions of the test antibodies, positive control, and negative control were mixed with SEB (10 ng / mL) and then added to the pelleted cells and incubated at 37°C for 3 days. Supernatants were collected to measure human IL-2 concentration.

[0109] For the human IL-2 assay, plates were pre-coated with 1.0 μg / ml of human IL-2 antibody (R&D System MAB602) at 4 °C for 16 - 20 hours. After blocking for 1 hour with 2% BSA (BovoGen) in DBPS, the supernatant containing IL-2 was added to the plates and incubated at room temperature for 2 hours. After washing three times with PBST (containing 0.05% Tween 20), the biotinylated human IL-2 antibody (R&D system, BAF202) was diluted and added at a concentration of 0.5 μg / mL. The plates were incubated at room temperature for 1 hour. The bound biotinylated antibody was detected by streptavidin conjugate HRP (Invitrogen, SNN1004) diluted 1:20000. After 1 hour of incubation, the color was developed by preparing 100 μL of TMB substrate and then stopped with 100 μL of 2M HCl. Absorbance at 450 nm and 540 nm was measured using a microplate spectrophotometer.

[0110] In the cell-based assay, humanized antibodies (8.60 nM, 21.4 nM, 53.6 nM, 134 nM, 335 nM) were tested for their ability to enhance human PBMCs stimulated with the superantigen SEB. After 3 days of stimulation, IL-2 from PBMCs was measured using ELISA. Compared to the isotype control antibody, both of the two humanized antibodies (W3162-1.146.19-Z12, W3162-1.154.8-Z35), and ipilimumab were able to enhance the release of IL-2 from PBMCs in a dose-dependent manner (Figure 13).

[0111] 2.5 Thermal stability The stability of the lead antibody was tested at different temperatures. Briefly, 100 μL of each antibody sample was pipetted into individual tubes and the samples were incubated at 4 °C or 37 °C for 20 hours, or at 45 °C or 50 °C for 2 hours. The samples were then centrifuged at 12,000 rpm for 10 minutes. These samples were observed to detect any resulting precipitate and the samples were analyzed by SEC-HPLC for purity and elution time.

[0112] The SEC profiles of W3162-1.146.19-Z12 under different conditions are shown in Figures 14a - 14d. Neither the dilution time nor the percentage of the main peak (92.39% - 92.48%) under high temperature conditions changed significantly compared to that under low temperature (92.24%). The SEC profiles of W3162-1.154.8-Z35 under different high temperature conditions are shown in Figures 14e - 14h. Neither the dilution time nor the percentage of the main peak (97.14% - 97.17%) changed significantly compared to that under low temperature (96.84%). This set of data indicates that the antibody was stable under the high temperature conditions tested.

[0113] 2.6 Non-specific binding Both FACS assay and ELISA assay were used to test whether the antibody binds to other targets. In the 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-K1) were seeded at 1 x 10 cells per well 5Individually adjusted. The test antibody and the isotype control antibody were diluted to 10 μg / ml in PBS containing 1% BSA and incubated with the cells at 4°C for 1 hour. The cells were washed twice with 180 μL of PBS containing 1% BSA. The PE-conjugated goat anti-human IgG Fc fragment (Jackson, catalog number 109-115-098) was diluted to a final concentration of 5 μg / ml in PBS containing 1% BSA, then added to the resuspended cells and incubated at 4°C for 30 minutes in the dark. An additional washing step was performed twice with 180 μL of PBS containing 1% BSA, followed by centrifugation at 1500 rpm for 4 minutes at 4°C. Finally, the cells were resuspended in 100 μL of PBS containing 1% BSA, and the fluorescence values were measured by flow cytometry (BD CantoII) and analyzed by FlowJo.

[0114] In the ELISA assay, the test antibody and the isotype control antibody were tested for binding to 10 different target antigens, including factor VIII, FGFR-ECD, PD-1, CTLA-4.ECD, VEGF, HER3.ECD, OX40.ECD, 4-1BB.ECD, CD22.ECD, CD3e.ECD. The 96-well plates were coated with individual antigens (2 μg / mL) overnight at 4°C. After blocking for 1 hour with 2% BSA in PBS, the plates were washed 3 times with 300 μL of PBST. Both the test antibody and the isotype control antibody were diluted to 10 μg / mL in PBS containing 2% BSA, then added to the plates and incubated at room temperature for 2 hours. After washing 3 times with 300 μL of PBST, the HRP-conjugated goat anti-human IgG antibody (diluted 1:5000 in 2% BSA) was added to the plates and incubated at room temperature for 1 hour. Finally, the plates were washed 6 times with 300 μL of PBST. Color was developed by preparing 100 μL of TMB substrate for 12 minutes and then stopped with 100 μL of 2M HCl. The absorbance at 450 nM was measured using a microplate spectrophotometer.

[0115] In addition to CTLA-4, other unrelated proteins were used to test whether antibodies W3162-1.146.19-Z12 and W3162-1.154.8-Z35 could bind to these antigens. As shown in Figure 16, only CTLA-4 among the panel of antigens was detected by the two antibodies. Other antigens did not produce a signal in this ELISA assay. In contrast, the anti-OX40 antibody actually bound to OX40, indicating that this antigen was coated on the plate.

[0116] The specificity of the two antibodies was also tested on a panel of different cell lines in a FACS assay. The antibodies did not produce a detectable signal against any of these cell lines (data not shown).

[0117] 2.7 In Vivo Efficacy Since antibody W3162-1.146.19-Z12 cross-reacts with CTLA-4 in both humans and mice, the antitumor effect of this antibody was tested in a syngeneic mouse model. Mouse cancer cell line CT26 was used to construct a xenograft mouse model to test anti-CTLA-4 antibody W3162-1.146.19-Z12. An anti-mouse CTLA-4 antibody purchased from BioXCell was used as a positive control (BioXCell - BE0131). Tumor cells were maintained as a monolayer culture in vitro in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37 °C in a 5% CO2 atmosphere. Tumor cells were passaged twice a week as per the standard method after detaching the cells by trypsin-EDTA treatment. Growing cells in the exponential growth phase were harvested and counted for tumor inoculation. Female Balb / C mice were purchased from Beijing Vital River Laboratory Animal Co., Ltd. Mice 6 - 8 weeks old with a body weight of approximately 18 - 22 g were used in the study. Each mouse was injected with 1 x 10 in 0.1 mL of PBS mixed with 50 μL of Matrigel. 5Individual tumor cells were subcutaneously inoculated in the right axilla. When the average tumor volume reached 60 - 80 mm 3 3, the animals were randomly grouped. Anti - CTLA - 4 antibody and isotype control were used for treatment: injected intravenously into the mice twice a week. The size of the tumor was measured twice a week with vernier calipers, and the tumor volume was calculated by the formula a x b 2 x π / 6, where a is the length and b is the width (a > b).

[0118] When the average tumor volume reached approximately 70 mm 3 3, W3162 - 1.146.19 - Z12 (1 mg / kg, 3 mg / kg, 10 mg / kg), and control antibody (10 mg / kg) were injected twice a week for two weeks. The animals were monitored over time for tumor growth and body weight. As shown in Figure 15, W3162 - 1.146.19 - Z12 significantly inhibits tumor growth in a dose - dependent manner. At a dose of 1 mg / kg, W3162 - 1.146.19 - Z12 inhibited tumor growth compared to the control group. At a dose of 3 mg / kg, W3162 - 1.146.19 - Z12 suppressed the tumor volume to 160 mm3 on the 19th day, while 10 mg / kg of W3162 - 1.146.19 - Z12 induced tumor regression at the end of the study period.

[0119] 2.8 Epitope mapping Alanine scanning was used to identify the CTLA-4 epitopes of the antibody. In this experiment, alanine residues on hCTLA4 were mutated to glycine residues, and all other residues were mutated to alanine. For each residue of the human CTLA4 extracellular domain (ECD), point amino acid substitutions were generated using two consecutive PCR steps. The pcDNA3.3-hCTLA4_ECD.His plasmid encoding the ECD and C-terminal His tag of human CTLA4 was used as a template, and a set of mutagenic primers was used in the 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 the synthesis reaction of the mutant strand. In the second-step PCR, a linear DNA expression cassette composed of the CMV promoter, the mutant ECD of CTLA4, the His tag, and the polyadenylation of herpes simplex virus thymidine kinase (TK) was amplified and transiently expressed in HEK293F cells (Life Technologies, Gaithersburg, MD). In addition, three plasmid vectors were constructed to test the epitopes of glycans: pcDNA3.3-hCTLA4_ECD.His(N113Q), pcDNA3.3-hCTLA4_ECD.His(N145Q), and pcDNA3.3-hCTLA4_ECD.His(N113Q, N145Q). These three mutants were transiently expressed in HEK293F cells (Life Technologies, Gaithersburg, MD).

[0120] To test how the mutations affect antibody binding, a capture ELISA was performed. Briefly, the monoclonal antibodies ipilimumab, W3162-1.146.19-z12, and W3162-1.154.8-z35 (2 μg / mL) were captured on plates by goat anti-human IgG Fc (Bethyl Laboratories, Montgomery, TX) precoated at 2 μg / mL. After interaction with supernatants containing the quantified CTLA4 mutants, HRP-conjugated anti-His antibody (1:5000; Rockland Immunochemicals, Pottstown, PA) was added as the detection antibody. TMB was used as the substrate for HRP. Absorbance was normalized according to the mean of the control variant. After setting an additional cut-off (< 0.55) for fold change in binding, the finally determined epitope residues were identified.

[0121] The binding activities of antibodies W3162-1.146.19-z12, W3162-1.154.8-z35, and ipilimumab (W316-BMK1) to human CTLA4 were performed and all three antibodies were found to bind to human CTLA4 (Figure 17).

[0122] The point mutations tested that affected antibody binding to CTLA-4 are shown in Table 11. In light of the crystal structure of human CTLA4 (PDB code 1AH1), for none of the antibodies was it likely that several amino acid residues (e.g., Met38, Val40, Tyr60, Val71, Val73, Arg75, Val84, Cys85, Cys129, Ile149) directly contacted. The observed decrease in binding was highly likely to have resulted from the instability or rather the collapse of the CTLA4 structure after alanine substitution. The finally determined epitope residues are listed in Table 12 and marked in Figure 18.

[0123] As shown in FIGS. 18D and E, the epitopes of ipilimumab and W3162-1.146.19-z12 overlap with each other, except for a few residues such as N145 and P138. In comparison, W3162-1.154.8-z35 bound to a smaller region of CTLA-4 (FIG. 18F) than the other two antibodies. All three antibodies bound to the ligand-binding domain of CTLA-4 (FIGS. 18A and B) that contains the MYPPPY motif.

[0124] The overlapping epitopes of ipilimumab and W3162-1.146.19-z12 did not account for the unique cross-species binding of antibody W3162-1.146.19-z12. Since the N145 mutation on CTLA-4 only affected the binding of W3162-1.146.19-z12 to CTLA-4 and not the other two antibodies, the inventors further focused on the N-glycosylation site as a potential epitope. The effect of mutations at the two glycosylation sites of CTLA4 on antibody binding activity is shown in FIG. 17. The binding of ipilimumab or W3162-1.154.8-z35 to mutant CTLA-4 did not change significantly (FIGS. 17A and C). In contrast, the binding of W3162-1.146.19-z12 to mutant CTLA-4 N145Q decreased significantly, while the binding of this antibody to CTLA-4 with N113Q did not change. This set of data indicates that the glycan at N145 of CTLA-4 (FIG. 18E) could be the epitope of W3162-1.146.19-z12. The N145 residue is conserved in cynomolgus monkey and mouse CTLA-4.

[0125] The description of the present invention has been made by way of examples above. However, it is understood by those skilled in the art that the present invention is not limited to the examples. The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description, and it is intended that all changes which come within the meaning and range of equivalency of the claims are embraced therein.

[0126] (Table 11) Influence of point mutations of CTLA4 on antibody binding TIFF0007717767000011.tif216152 a The fold change in binding is related to the binding of some silent alanine substitutions.

[0127] (Table 12) Identified epitopes of three antibodies TIFF0007717767000012.tif180165Sequence information SEQUENCE LISTING <110> WuXi Biologics (Shanghai) Co.,Ltd. <120> Novel monoclonal antibodies to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) <160> 47 <170> PatentIn version 3.3 <210> 1 <211> 114 <212> PRT <213> Artificial Sequence <400> 1 Glu Glu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Arg Ser Ser 20 25 30 Ala Met His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Asp Trp Val 35 40 45 Ala Phe Ile Ser Ser Gly Gly Asp Thr Ala Tyr Ala Asp Ala Val Lys 50 55 60 Gly Arg Phe Ile Val Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu 65 70 75 80 Gln Leu Asn Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Val 85 90 95 Arg Met Glu Arg Ile Pro Thr Trp Gly Gln Gly Val Met Val Thr Val 100 105 110 Ser Ser <210> 2 <211> 115 <212> PRT <213> Artificial Sequence <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Asp Leu Thr Phe Ser Asn Tyr 20 25 30 Asp Met Ala Trp Val Arg Gln Thr Pro Thr Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Pro Asn Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg His Leu Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 3 <211> 120 <212> PRT <213> Artificial Sequence <400> 3 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Tyr His Thr Ile Thr Ser Gly 20 25 30 Tyr Asp Trp Thr Trp Ile Arg Lys Phe Pro Gly Asn Gln Met Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu His Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Ser Met Met Val Pro His Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105 110 Gly Ala Ser Val Thr Val Ser Ser 115 120 <210> 4 <211> 119 <212> PRT <213> Artificial Sequence <400> 4 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Ala Gly Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Phe Met Asn Trp Val Lys Gln Ser Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Val Asp Pro Glu Asn Gly Arg Ala Asp Tyr Ala Glu Lys Phe 50 55 60 Lys Lys Lys Ala Thr Leu Thr Ala Asp Thr Thr Ser Asn Thr Ala Tyr 65 70 75 80 Ile His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Arg Ala Met Asp Asn Tyr Gly Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 5 <211> 115 <212> PRT <213> Artificial Sequence <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Asp Leu Thr Phe Ser Asn Tyr 20 25 30 Asp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Pro Ser Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 His Leu Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 6 <211> 120 <212> PRT <213> Artificial Sequence <400> 6 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Tyr His Thr Ile Thr Ser Gly 20 25 30 Tyr Asp Trp Thr Trp Ile Arg Lys Pro Pro Gly Lys Gly Met Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Met Met Val Pro His Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 7 <211> 119 <212> PRT <213> Artificial Sequence <400> 7 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Phe Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Val Asp Pro Glu Gln Gly Arg Ala Asp Tyr Ala Glu Lys Phe 50 55 60 Lys Lys Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Ala Met Asp Asn Tyr Gly Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 8 <211> 110 <212> PRT <213> Artificial Sequence <400> 8 Asp Ile Val Leu Thr Gln Ser Pro Val Leu Ala Val Ser Leu Gly Gln 1 5 10 15 Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Ser Ser 20 25 30 Ile Asn Leu Ile His Trp Tyr Gln Gln Arg Pro Gly Gln Gln Pro Lys 35 40 45 Leu Leu Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asp Pro 65 70 75 80 Val Gln Ala Asp Asp Val Ala Asp Tyr Tyr Cys Gln Gln Ser Arg Glu 85 90 95 Ser Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 9 <211> 107 <212> PRT <213> Artificial Sequence <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Gln Ala Ser Gln Asp Ile Gly Ser Asn 20 25 30 Leu Ile Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Pro Met Ile 35 40 45 Tyr Tyr Ala Thr His Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Val Ala Asp Tyr His Cys Leu Gln Tyr Lys Gln Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 10 <211> 112 <212> PRT <213> Artificial Sequence <400> 10 Asp Val Val Leu Thr Gln Thr Pro Pro Thr Ser Ser Ala Thr Ile Gly 1 5 10 15 Gln Ser Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Tyr Trp Tyr Leu Gln Arg Pro Ser Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Val Ser Lys Leu Gly Ser Gly Val Pro 50 55 60 Asn Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Gly Val Glu Ala Glu Asp Leu Gly Leu Tyr Tyr Cys Val Gln Gly 85 90 95 Thr His Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 11 <211> 106 <212> PRT <213> Artificial Sequence <400> 11 Glu Ile Met Leu Thr Gln Ser Pro Thr Ile Met Ala Ala Ser Leu Gly 1 5 10 15 Glu Lys Ile Thr Ile Thr Cys Ser Ala Asn Ser Ser Leu Ser Tyr Met 20 25 30 Tyr Trp Phe Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp Val His 35 40 45 Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Tyr Leu Thr Ile Asn Thr Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Phe Cys His His Trp Ser Asn Thr Gln Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 12 <211> 107 <212> PRT <213> Artificial Sequence <400> 12 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 Gln Ala Ser Gln Asp Ile Gly Ser Asn 20 25 30 Leu Ile Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Met Ile 35 40 45 Tyr Tyr Ala Thr His Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Lys Gln Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 13 <211> 112 <212> PRT <213> Artificial Sequence <400> 13 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Val Ser Lys Leu Gly Ser Gly Val Pro 50 55 60 Asn Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Gly 85 90 95 Thr His Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 14 <211> 106 <212> PRT <213> Artificial Sequence <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ala Leu Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Trp Val His 35 40 45 Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His His Trp Ser Asn Thr Gln Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 15 <211> 6 <212> PRT <213> Artificial Sequence <400> 15 Met Glu Arg Ile Pro Thr 1 5 <210> 16 <211> 6 <212> PRT <213> Artificial Sequence <400> 16 His Leu Trp Phe Ala Tyr 1 5 <210> 17 <211> 11 <212> PRT <213> Artificial Sequence <400> 17 Met Met Val Pro His Tyr Tyr Val Met Asp Ala 1 5 10 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <400> 18 Arg Ala Met Asp Asn Tyr Gly Phe Ala Tyr 1 5 10 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <400> 19 Gln Gln Ser Arg Glu Ser Pro Leu Thr 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <400> 20 Leu Gln Tyr Lys Gln Tyr Pro Arg Thr 1 5 <210> 21 <211> 9 <212> PRT <213> Artificial Sequence <400> 21 Val Gln Gly Thr His Asp Pro Trp Thr 1 5 <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <400> 22 His His Trp Ser Asn Thr Gln Trp Thr 1 5 <210> 23 <211> 16 <212> PRT <213> Artificial Sequence <400> 23 Phe Ile Ser Ser Gly Gly Asp Thr Ala Tyr Ala Asp Ala Val Lys Gly 1 5 10 15 <210> 24 <211> 17 <212> PRT <213> Artificial Sequence <400> 24 Ser Ile Ser Pro Asn Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val Lys 1 5 10 15 Gly <210> 25 <211> 16 <212> PRT <213> Artificial Sequence <400> 25 Tyr Ile Ser Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 26 <211> 17 <212> PRT <213> Artificial Sequence <400> 26 Arg Val Asp Pro Glu Asn Gly Arg Ala Asp Tyr Ala Glu Lys Phe Lys 1 5 10 15 Lys <210> 27 <211> 17 <212> PRT <213> Artificial Sequence <400> 27 Ser Ile Ser Pro Ser Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val Lys 1 5 10 15 Gly <210> 28 <211> 17 <212> PRT <213> Artificial Sequence <400> 28 Arg Val Asp Pro Glu Gln Gly Arg Ala Asp Tyr Ala Glu Lys Phe Lys 1 5 10 15 Lys <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <400> 29 Arg Thr Ser Asn Leu Ala Ser 1 5 <210> 30 <211> 7 <212> PRT <213> Artificial Sequence <400> 30 Tyr Ala Thr His Leu Ala Asp 1 5 <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <400> 31 Leu Val Ser Lys Leu Gly Ser 1 5 <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <400> 32 Gly Thr Ser Asn Leu Ala Ser 1 5 <210> 33 <211> 5 <212> PRT <213> Artificial Sequence <400> 33 Ser Ser Ala Met His 1 5 <210> 34 <211> 5 <212> PRT <213> Artificial Sequence <400> 34 Asn Tyr Asp Met Ala 1 5 <210> 35 <211> 6 <212> PRT <213> Artificial Sequence <400> 35 Ser Gly Tyr Asp Trp Thr 1 5 <210> 36 <211> 5 <212> PRT <213> Artificial Sequence <400> 36 Asn Tyr Phe Met Asn 1 5 <210> 37 <211> 10 <212> PRT <213> Artificial Sequence <400> 37 Arg Ala Ser Gln Ser Val Ser Ile Ser Ser Ile Asn Leu Ile His 1 5 10 15 <210> 38 <211> 11 <212> PRT <213> Artificial Sequence <400> 38 Gln Ala Ser Gln Asp Ile Gly Ser Asn Leu Ile 1 5 10 <210> 39 <211> 16 <212> PRT <213> Artificial Sequence <400> 39 Arg Ser Ser Gln Ser Leu Leu Asn Ser Asp Gly Asn Thr Tyr Leu Tyr 1 5 10 15 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <400> 40 Ser Ala Asn Ser Ser Leu Ser Tyr Met Tyr 1 5 10 <210> 41 <211> 10 <212> PRT <213> Artificial Sequence <400> 41 Ser Ala Asn Ser Ala Leu Ser Tyr Met Tyr 1 5 10 <210> 42 <211> 345 <212> DNA <213> Artificial Sequence <400> 42 gaggtgcagc tggtggagag cggcggagga ctggtgcaac ctggcggaag cctgagactg 60 agctgcgccg ccagcgacct gaccttcagc aactacgaca tggcctgggt gagacaggcc 120 cctggcaagg gactggagtg ggtggccagc atcagcccca gcggcggcaa cacctactac 180 agggacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa cagcctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caggcacctg 300 tggttcgcct actggggcca gggcacactg gtgaccgtga gcagc 345 <210> 43 <211> 360 <212> DNA <213> Artificial Sequence <400> 43 caggtgcagc tgcaggagag cggacccgga ctggtgaagc cctccgagac cctgagcctg 60 acctgcagcg tgacctacca caccatcacc agcggctacg actggacctg gatcagaaag 120 ccccccggca aaggcatgga gtggatcggc tacatcagct acagcggcaa caccaactac 180 aaccccagcc tgaagagcag ggtgaccatc agcagggaca ccagcaagaa ccagttcttc 240 ctgaagctga gcagcgtgac agccgccgat accgccgtgt actactgcgc cagcatgatg 300 gtgccccact actacgtgat ggacgcctgg ggacagggca ccctggtgac agtgagcagc 360 <210> 44 <211> 357 <212> DNA <213> Artificial Sequence <400> 44 caggtgcagc tggtgcagag cggagccgag gtgaagaagc ccggcagcag cgtgaaggtg 60 agctgcaagg ccagcggcta caccttcacc aactacttca tgaactgggt gaggcaggcc 120 cctggacaag gcctggagtg gatgggcaga gtggatcccg agcagggcag ggccgactac 180 gccgagaagt tcaagaagag ggtgaccatc accgccgaca agagcaccag caccgcctac 240 atggagctga gcagcctgag gagcgaggac accgccgtgt actactgcgc caggagagcc 300 atggacaact acggcttcgc ctactggggc cagggaaccc tggtgaccgt gagcagc 357 <210> 45 <211> 321 <212> DNA <213> Artificial Sequence <400> 45 gacatccaga tgacccagag ccctagcagc ctgagcgcca gcgtgggcga tagggtgacc 60 atcacctgcc aggccagcca ggacatcggc agcaacctga tctggttcca gcagaagccc 120 ggcaaggccc ccaagcctat gatctactac gccacccacc tggccgatgg cgtgcctagc 180 agattcagcg gcagcagaag cggcaccgac tacaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgcctgcag tacaagcagt accccagaac cttcggcggc 300 ggcaccaagg tggagatcaa g 321 <210> 46 <211> 336 <212> DNA <213> Artificial Sequence <400> 46 gacatcgtga tgacccagac ccccctgagc ctgagcgtga cacctggaca gcccgccagc 60 atcagctgca ggtccagcca gagcctgctg aacagcgacg gcaacaccta cctgtactgg 120 tacctgcaga agcctggcca gagcccccag ctgctgatct acctggtgtc caagctgggc 180 agcggcgtgc ctaacaggtt tagcggcagc ggcagcggca ccgatttcac cctgaagatc 240 agcagggtgg aggccgagga tgtgggcgtg tactactgcg tgcagggcac ccacgatcct 300 tggaccttcg gcggcggaac caaggtggag atcaag 336 <210> 47 <211> 318 <212> DNA <213> Artificial Sequence <400> 47 gagatcgtgc tgacccagag ccccgacttc cagagcgtga cccccaagga gaaggtgacc 60 atcacctgca gcgccaacag cgccctgagc tacatgtact ggtaccagca gaagcccgac 120 cagagcccca agctgtgggt gcacggcacc agcaatctgg ccagcggcgt gcctagcaga 180 tttagcggca gcggcagcgg caccgatttc accctgacca tcaacagcct ggaggccgag 240 gacgccgcta cctactactg ccaccactgg agcaacaccc agtggacctt cggcggcggc 300 accaaggtgg agatcaag 318

Claims

**Claim 1** An antibody or antigen-binding fragment thereof that includes a variable light (VL) region and a variable heavy (VH) region and binds to human and monkey CTLA-4, wherein the VL region includes a light chain complementarity-determining region 1 (LCDR1) defined by SEQ ID NO: 41, an LCDR2 defined by SEQ ID NO: 32, and an LCDR3 defined by SEQ ID NO: 22, and the VH region includes a heavy chain complementarity-determining region 1 (HCDR1) defined by SEQ ID NO: 36, an HCDR2 defined by SEQ ID NO: 28, and an HCDR3 defined by SEQ ID NO: 18, the antibody or antigen-binding fragment thereof. **Claim 2** The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody inhibits the binding of CTLA-4 to CD80 or CD86. **Claim 3** The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the binding epitope of the antibody or antigen-binding fragment includes P138 of CTLA-4. **Claim 4** The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which binds to human CTLA-4 with a KD of 4.77E-10 M or less. **Claim 5** (a) The property of binding to human CTLA-4 with a KD of 4.77E-10 M to 9.68E-11 M; (b) The property of enhancing the release of interleukin-2 from stimulated PBMC The antibody or antigen-binding fragment thereof according to claim 4, which exhibits at least one of the above. **Claim 6** The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is a chimeric antibody, a humanized antibody, or a rat antibody. **Claim 7** A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. **Claim 8** A cloning vector or expression vector containing the nucleic acid molecule according to claim 7. **Claim 9** A host cell containing one or more of the cloning vectors or expression vectors according to claim 8. **Claim 10** A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and one or more of a pharmaceutically acceptable excipient, diluent, and carrier. **Claim 11** An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 linked to a therapeutic substance. **Claim 12** A pharmaceutical composition comprising the immunoconjugate according to claim 11 and one or more of a pharmaceutically acceptable excipient, diluent, and carrier. **Claim 13** A method for preparing an anti-CTLA-4 antibody or an antigen-binding fragment thereof, comprising the following steps: (a) (i) A heavy-chain variable region antibody sequence comprising a CDR1 sequence defined by SEQ ID NO: 36, a CDR2 sequence defined by SEQ ID NO: 28, and a CDR3 sequence defined by SEQ ID NO: 18; and (ii) A light-chain variable region antibody sequence comprising a CDR1 sequence defined by SEQ ID NO: 41, a CDR2 sequence defined by SEQ ID NO: 32, and a CDR3 sequence defined by SEQ ID NO: 22 Providing step; and (b) Expressing the antibody sequence of (a) as a protein.

14. Use of the antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6 in the manufacture of a medicament for the treatment or prevention of immune disorders or cancer.

15. A medicament for suppressing the growth of tumor cells in a subject, comprising a therapeutically effective amount of the antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6.

16. The medicament according to claim 15, wherein the tumor cells are of a cancer selected from the group consisting of melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or uveal malignant melanoma, uterine cancer, ovarian cancer, and rectal cancer.

17. The medicament according to claim 15 or 16, wherein the antibody is a humanized antibody.

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