ANTICORPO OU FRAGMENTO DE LIGAÇÃO AO ANTÍGENO DO MESMO, SEU MÉTODO DE PRODUÇÃO, SEUS USOS, COMPOSIÇÃO FARMACÊUTICA E IMUNOCONJUGADO

Abstract

The present invention relates to ctla-4 monoclonal antibodies, particularly humanized monoclonal antibodies specifically linked to ctla-4 with high affinity. The present invention also provides functional monoclonal antibodies cross-reacting to human, cynomolgus monkey, and mouse ctla-4. 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. Antibody epitopes are identified. Therapeutic compositions comprising the antibodies of the invention are also provided. The invention also provides methods for treating cancers and other diseases with anti-ctla-4 antibodies.

Description

Descriptive Report of the Invention Patent for ANTIBODY OR ANTIGEN-BINDING FRAGMENT, ITS PRODUCTION METHOD, ITS USES, PHARMACEUTICAL COMPOSITION AND IMMUNOCONJUGATE TECHNICAL FIELD

[01] The present invention relates generally to antibodies against CTLA-4 and their compositions, and to immunotherapy in the treatment of cancer, infections or other human diseases using anti-CTLA-4 antibodies. BACKGROUND OF THE INVENTION

[02] Cancer immunotherapy has become a high-profile research area in cancer treatment. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is one of the validated targets of immune checkpoints. After T cell activation, CTLA-4 is rapidly expressed on these T cells, usually within one hour of antigen engagement with the TCR. CTLA-4 can inhibit T cell signaling through competition with CD28. CD28 mediates one of the well-characterized T cell co-stimulatory signals: CD28 binding to its ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells leads to T cell proliferation, inducing the production of interleukin-2 and anti-apoptotic factors. Due to CTLA-4's much higher affinity binding to CD80 and CD86 than to CD28, CTLA-4 can compete with CD28's binding to CD80 and CD86, leading to the suppression of T cell activation.In addition to induced expression on activated T cells, CTLA-4 is constitutively expressed on the surface of regulatory T cells (Tregs), suggesting that CTLA-4 may be required for contact-mediated suppression and associated Treg production of cytokines. Petition 870260061236, dated 06 / 22 / 2026, p. 17 / 21 2 / 52 Transforming growth hormone beta and interleukin-10.

[003] CTLA-4 blockade can induce tumor regression, as 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 that has been approved as monotherapy for the treatment of advanced melanoma. The proposed mechanism of action for ipilimumab is interference with the interaction of CTLA-4, expressed on a subset of activated T cells, with CD80 / CD86 molecules on professional antigen-presenting cells. This results in T cell potentiation due to the blockade of the inhibitory modulation of T cell activation promoted by the interaction of CTLA-4 and CD80 / CD86. The resulting T cell activation, proliferation, and lymphocyte infiltration into tumors leads to tumor cell death. The commercial dosage form is a 5 mg / mL concentrate for solution for infusion.Ipilimumab is also under clinical investigation for other types of tumors, including prostate cancer and lung cancer. Another anti-CTLA-4 antibody, Tremelimumab, has been evaluated as monotherapy in melanoma and malignant mesothelioma. DESCRIPTION OF THE INVENTION

[004] The present invention provides isolated antibodies, in particular monoclonal antibodies or humanized monoclonal antibodies.

[005] 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.

[006] The antibody mentioned above or the antigen-binding fragment inhibits the binding of CTLA-4 to CD80 or CD86. Petition 870240025544, dated 03 / 25 / 2024, p. 10 / 68 3 / 52

[007] In the antibody mentioned above or in the antigen-binding fragment, the antibody-binding epitope or antigen-binding fragment comprises N145 or polysaccharide in N145 of CTLA-4.

[008] 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 and monkey CTLA-4, wherein the binding epitope of the antibody or antigen-binding fragment comprises CTLA-4 P138.

[009] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment:

[0010] a) binds to human CTLA-4 with a KD of 4.77E-10 M or less; and

[0011] b) binds to mouse CTLA-4 with a Kd of 1.39E09 M or less.

[0012] The antibody mentioned above, in which the antibody or antigen-binding fragment exhibits at least one of the following properties:

[0013] a) binds to human CTLA-4 with a KD between 4.77E-10 M and 2.08E-10 M, and to mouse CTLA-4 with a KD between 1.39E09 M and 9.06E-10 M;

[0014] b) improves the release of interleukin-2 from stimulated PBMCs.

[0015] c) does not bind substantially to any selected protein from the group consisting of Factor VIII, FGFR, PD-1, CD22, VEGF, CD3, HER3, OX40, and 4-1BB.

[0016] 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 a group consisting of SEQ ID NOs: Petition 870240025544, dated 03 / 25 / 2024, p. 11 / 68 4 / 52 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14

[0017] in which the antibody or antigen-binding fragment binds specifically to CTLA-4.

[0018] The present invention provides an antibody or an antigen-binding fragment thereof, comprising an amino acid sequence selected from a group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14,

[0019] in which the antibody or antigen-binding fragment binds specifically to CTLA-4.

[0020] The present invention provides an antibody or an antigen-binding fragment thereof, comprising:

[0021] a) a variable region of a heavy chain having an amino acid sequence that is at least 70%, 80%, 90% or 95% homologous to a sequence selected from a group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7; and

[0022] b) a variable region of a light chain having an amino acid sequence that is at least 70%, 80%, 90% or 95% homologous to a sequence selected from a group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13 and 14,

[0023] in which the antibody or antigen-binding fragment binds specifically to CTLA-4.

[0024] The present invention provides an antibody or an antigen-binding fragment thereof, comprising:

[0025] 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

[0026] 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,

[0027] in which the antibody or antigen-binding fragment Petition 870240025544, dated 03 / 25 / 2024, page 12 / 68 5 / 52 specifically links to CTLA-4.

[0028] In various embodiments, the antibody or an antigen-binding fragment thereof comprises:

[0029] a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; and

[0030] b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8,

[0031] in which the antibody or antigen-binding fragment binds specifically to CTLA-4;

[0032] or the antibody or an antigen-binding fragment thereof comprises:

[0033] a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 2; and

[0034] b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 9,

[0035] in which the antibody or antigen-binding fragment binds specifically to CTLA-4;

[0036] or the antibody or an antigen-binding fragment thereof comprises:

[0037] a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 3; and

[0038] b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 10,

[0039] in which the antibody or antigen-binding fragment Petition 870240025544, dated 03 / 25 / 2024, page 13 / 68 6 / 52 specifically links to CTLA-4;

[0040] or the antibody or an antigen-binding fragment thereof comprises:

[0041] a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4; and

[0042] b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 11,

[0043] in which the antibody or antigen-binding fragment binds specifically to CTLA-4;

[0044] or the antibody or an antigen-binding fragment thereof comprises:

[0045] a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 5; and

[0046] b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12,

[0047] in which the antibody or antigen-binding fragment binds specifically to CTLA-4;

[0048] or the antibody or an antigen-binding fragment thereof comprises:

[0049] a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6; and

[0050] b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 13,

[0051] in which the antibody or antigen-binding fragment Petition 870240025544, dated 03 / 25 / 2024, page 14 / 68 7 / 52 specifically links to CTLA-4;

[0052] or the antibody or an antigen-binding fragment thereof comprises:

[0053] a) a variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 7; and

[0054] b) a variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14,

[0055] in which the antibody or antigen-binding fragment binds specifically to CTLA-4;

[0056] The sequence of said antibody is shown in Table 1 and in Sequence Listing. Table 1 Amino acid sequences deduced from antibodies Clone ID SEQ ID NO Amino acid sequence W3162-1,1 01,2 Heavy chain 1 EEQLVESGGGLVQPGKSLKLSCSASGFTFR SSAMHWIRQPPGKGLDWVAFISSGGDTAYA DAVKGRFIVSRDNAENTLFLQLNSLKSEDTAI YYCVRMERIPTWGQGVMVTVSS Light chain 8 DIVLTQSPVLAVSLGQRATISCRASQSVSISS IN LIHWYQQRPGQQPKLLIYRTSN LASGIPAR FSGSGSGTDFTLSIDPVQADDVADYYCQQS RESPLTFGSGTKLEIK W3162-1. 145.10 Heavy chain 2 EVQLVESGGGLVQPGRSLKLSCAASDLTFS NYDMAWVRQTPTKGLEWVASISPNGGNTY YRDSVKGRFTVSRDNAKNSLYLQMDSLRSE DTATYYCARHLWFAYWGQGTLVTVSS Light chain 9 DIQMTQSPSSMSASLGDRVTISCQASQDIGS NLIWFQQKPGKSPRPMIYYATHLADGVPSR FSGSRSGSDYSLTISSLESEDVADYHCLQYK QYPRTFGGGTKLELK EVQLQESGPGLVKPSQSLSLTCSVTYHTITS Petition 870240025544, dated 03 / 25 / 2024, page 15 / 68 8 / 52 Clone ID SEQ ID NO Amino acid sequence W3162-1.1 46.19 Heavy chain 3 GYDWTWIRKFPGNQMEWMGYISYSGNTNY NPSLKSRISITRDTSKNQFFLHLNSVTSEDTA TYYCASM MVPHYYVM DAWGQGASVTVSS Light chain 10 DVVLTQTPPTSSATIGQSVSISCRSSQSLLN SDGNTYLYWYLQRPSQSPQLLIYLVSKLGS GVPNRFSGSGSGTDFTLKISGVEAEDLGLYY CVQGTHDPWTFGGGTKLELK W3162-1.1 54.8 Heavy chain 4 EVQLQQSGPEAGRPGSSVKISCKASGYTFT NYFMNWVKQSPGQGLEWIGRVDPENGRAD YAEKFKKKATLTADTTSNTAYIHLSSLTSEDT ATYFCARRAMDNYGFAYWGQGTLVTVSS Light chain 11 EIMLTQSPTIMAASLGEKITITCSANSSLSYM YWFQQKSGASPKLWVHGTSNLASGVPDRF SGSGSGTSYYLTINTM EAEDAATYFCHHWS NTQWTFGGGTKLELK W3162-1 .1 45.10-z7 Heavy chain 5 EVQLVESGGGLVQPGGSLRLSCAASDLTFS NYDMAWVRQAPGKGLEWVASISPSGGNTY YRDSVKGRFTISRDNAKNSLYLQMNSLRAE DTAVYYCARHLWFAYWGQGTLVTVSS Light chain 12 DIQMTQSPSSLSASVGDRVTITCQASQDIGS NLIWFQQKPGKAPKPMIYYATHLADGVPSRF SGSRSGTDYTLTISSLQPEDFATYYCLQYKQ YPRTFGGGTKVEIK W3162.1.1 4 6.-z12 Heavy chain 6 QVQLQESGPGLVKPSETLSLTCSVTYHTITS GYDWTWIRKPPGKGMEWIGYISYSGNTNYN PSLKSRVTISRDTSKNQFFLKLSSVTAADTA VYYCASMMVPHYYVMDAWGQGTLVTVSS Light chain 13 DIVMTQTPLSLSVTPGQPASISCRSSQSLLN SDGNTYLYWYLQKPGQSPQLLIYLVSKLGS GVPNRFSGSGSGTDFTLKISRVEAEDVGVY YCVQGTHDPWTFGGGTKVEIK QVQLVQSGAEVKKPGSSVKVSCKASGYTFT. Petition 870240025544, dated 03 / 25 / 2024, page 16 / 68 9 / 52 Clone ID SEQ ID NO Amino acid sequence W3162.1.1 5 4.8-z35 Heavy chain 7 NYFMNWVRQAPGQGLEWMGRVDPEQGRA DYAEKFKKRVTITADKSTSTAYMELSSLRSE DTAVYYCARRAMDNYGFAYWGQGTLVTVS S Light chain 14 EIVLTQSPDFQSVTPKEKVTITCSANSALSYM YWYQQKPDQSPKLWVHGTSNLASGVPSRF SGSGSGTDFTLTINSLEAEDAATYYCHHWS NTQWTFGGGTKVEIK

[0057] In another aspect, the invention provides an antibody or an antigen-binding fragment thereof, comprising a complementarity-determining region (CDR) with an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-41,

[0058] in which the antibody or antigen-binding fragment binds specifically to CTLA-4.

[0059] In another aspect, the invention provides an antibody, or an antigen-binding fragment thereof, comprising: a variable heavy chain region comprising the sequences CDR1, CDR2 and CDR3; and a variable light chain region comprising the sequences CDR1, CDR2 and CDR3,

[0060] wherein the CDR3 sequence of the variable heavy chain region comprises an amino acid sequence selected from a group consisting of SEQ ID NOs: 15, 16, 17 and 18, and their conservative modifications.

[0061] in which the antibody or antigen-binding fragment binds specifically to CTLA-4.

[0062] Preferably, wherein the CDR3 sequence of the antibody light chain variable region or an antigen-binding fragment thereof mentioned above comprises an amino acid sequence selected from a group consisting of SEQ ID NOs: 19, Petition 870240025544, dated 03 / 25 / 2024, page 17 / 68 10 / 52 20, 21 and 22, and their conservative modifications.

[0063] Preferably, wherein the CDR2 sequence of the antibody heavy chain variable region or its antigen-binding fragment mentioned above comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 23, 24, 25, 26, 27 and 28, and their conservative modifications.

[0064] Preferably, wherein the CDR2 sequence of the antibody light chain variable region or its antigen-binding fragment mentioned above comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 29, 30, 31 and 32, and their conservative modifications.

[0065] Preferably, wherein the CDR1 sequence of the antibody heavy chain variable region or its antigen-binding fragment mentioned above comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOS: 33, 34, 35 and 36, and their conservative modifications.

[0066] Preferably, the antibody of this invention, wherein the CDR1 sequence of the antibody light chain variable region or its antigen-binding fragment mentioned above comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOS: 37, 38, 39, 40 and 41, and their conservative modifications.

[0067] In a more preferred embodiment, the invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds specifically to CTLA-4 and comprises: a variable heavy chain region comprising the sequences CDR1, CDR2 and CDR3; Petition 870240025544, dated 03 / 25 / 2024, p. 18 / 68 11 / 52 and a variable light chain region comprising the sequences CDR1, CDR2 and CDR3, where:

[0068] a) the CDR1 sequence of the heavy chain variable region comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 33, 34, 35 and 36, and the CDR2 sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 23, 24, 25, 26, 27 and 28, the CDR3 sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 15, 16, 17 and 18;

[0069] b) and the CDR1 sequence of the variable light chain region comprises an amino acid sequence selected from a group consisting of amino acid sequences with SEQ ID NOs: 37, 38, 39, 40 and 41, and the CDR2 sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences with SEQ ID NOs: 29, 30, 31 and 32, the CDR3 sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences with SEQ ID NOs: 19, 20, 21 and 22,

[0070] in which the antibody or antigen-binding fragment binds specifically to CTLA-4.

[0071] An antibody or antigen-binding fragment of the same preferably comprises:

[0072] a) a CDR1 of the variable heavy chain region comprising SEQ ID NO: 15;

[0073] b) a CDR2 of the variable heavy chain region comprising SEQ ID NO: 23;

[0074] c) a CDR3 of the variable heavy chain region comprising SEQ ID NO: 33; Petition 870240025544, dated 03 / 25 / 2024, page 19 / 68 12 / 52

[0075] d) a CDR1 of the variable light chain region comprising SEQ ID NO: 19;

[0076] e) a CDR2 of the variable light chain region comprising SEQ ID NO: 29;

[0077] f) a CDR3 of the variable light chain region comprising SEQ ID NO: 37;

[0078] in which the antibody or antigen-binding fragment binds specifically to CTLA-4.

[0079] Another antibody or antigen-binding fragment thereof preferably comprises:

[0080] a) a CDR1 of the heavy chain variable region comprising SEQ ID NO: 16;

[0081] b) a CDR2 of the region comprising SEQ ID NO: 24; heavy chain variable

[0082] c) a CDR3 of the region comprising SEQ ID NO: 34; heavy chain variable

[0083] d) a CDR1 of the variable light chain region comprising SEQ ID NO: 20;

[0084] e) a CDR2 of the variable light chain region comprising SEQ ID NO: 30;

[0085] f) a CDR3 of the variable light chain region including SEQ ID NO: 38;

[0086] in which the antibody or antigen-binding fragment binds specifically to CTLA-4.

[0087] Another antibody or antigen-binding fragment thereof preferably comprises:

[0088] a) a CDR1 of the variable heavy chain region comprising SEQ ID NO: 17;

[0089] b) a CDR2 of the variable heavy chain region comprising SEQ ID NO: 25; Petition 870240025544, dated 03 / 25 / 2024, page 20 / 68 13 / 52

[0090] c) a CDR3 of the variable heavy chain region comprising SEQ ID NO: 35;

[0091] d) a CDR1 of the variable light chain region comprising SEQ ID NO: 19;

[0092] e) a CDR2 of the variable light chain region comprising SEQ ID NO: 31;

[0093] f) a CDR3 of the variable light chain region comprising SEQ ID NO: 39;

[0094] in which the antibody or antigen-binding fragment binds specifically to CTLA-4.

[0095] Another antibody or antigen-binding fragment thereof preferably comprises:

[0096] a) a CDR1 of the variable heavy chain region comprising SEQ ID NO: 18;

[0097] b) a CDR2 of the variable heavy chain region comprising SEQ ID NO: 26;

[0098] c) a CDR3 of the variable heavy chain region including SEQ ID NO: 36;

[0099] d) a CDR1 of the variable light chain region comprising SEQ ID NO: 22;

[00100] e) a CDR2 of the variable light chain region comprising SEQ ID NO: 32;

[00101] f) a CDR3 of the variable light chain region including SEQ ID NO: 40;

[00102] in which the antibody binds specifically to CTLA-4.

[00103] Another antibody or antigen-binding fragment thereof, preferably comprising:

[00104] a) a CDR1 of the variable heavy chain region comprising SEQ ID NO: 16;

[00105] b) a CDR2 of the variable heavy chain region Petition 870240025544, dated 03 / 25 / 2024, page 21 / 68 14 / 52 including SEQ ID NO: 27;

[00106] c) a CDR3 of the variable heavy chain region comprising SEQ ID NO: 34;

[00107] d) a CDR1 of the variable light chain region comprising SEQ ID NO: 20;

[00108] e) a CDR2 of the variable light chain region comprising SEQ ID NO: 30;

[00109] f) a CDR3 of the variable light chain region comprising SEQ ID NO: 38;

[00110] in which the antibody or antigen-binding fragment binds specifically to CTLA-4.

[00111] Another antibody or antigen-binding fragment thereof, preferably comprising:

[00112] a) a CDR1 of the variable heavy chain region comprising SEQ ID NO: 17;

[00113] b) a CDR2 of the variable heavy chain region comprising SEQ ID NO: 25;

[00114] c) a CDR3 of the variable heavy chain region comprising SEQ ID NO: 35;

[00115] d) a CDR1 of the variable light chain region comprising SEQ ID NO: 21;

[00116] e) a CDR2 of the variable light chain region comprising SEQ ID NO: 31;

[00117] f) a CDR3 of the variable light chain region comprising SEQ ID NO: 39;

[00118] in which the antibody or antigen-binding fragment binds specifically to CTLA-4.

[00119] Another antibody or antigen-binding fragment thereof, preferably comprising:

[00120] a) a CDR1 of the variable heavy chain region Petition 870240025544, dated 03 / 25 / 2024, page 22 / 68 15 / 52 including SEQ ID NO: 18;

[00121] b) a CDR2 of the variable heavy chain region comprising SEQ ID NO: 28;

[00122] c) a CDR3 of the variable heavy chain region comprising SEQ ID NO: 36;

[00123] d) a CDR1 of the variable light chain region comprising SEQ ID NO: 22;

[00124] e) a CDR2 of the variable light chain region comprising SEQ ID NO: 32;

[00125] f) a CDR3 of the variable light chain region comprising SEQ ID NO: 41;

[00126] in which the antibody or antigen-binding fragment binds specifically to CTLA-4.

[00127] The CDR sequences of said antibodies are shown in Table 2 and Sequence Listing. Table 2 CDR sequences of antibodies Clone ID SEQ ID NO CDR1 SEQ ID NO CDR2 SEQ ID NO CDR3 W31621.1 01.2 Heavy chain 33 SSAMH 23 FISSGGDTA YADAV KG 15 MERIPT Light chain 37 RASQSVSI SSINLIH 29 RTSNLAS 19 QQSRESPL T W31621.1 45.10 Heavy chain 34 NYDMA 24 SISPNGGNT YYRDSVKG 16 HLWFAY Light chain 38 QASQDIGS NLI 30 YATHLAD 20 LQYKQYPR T W31621.1 46.19 Heavy chain 35 SGYDWT 25 YISYSGNTN YNPSLKS 17 MMVPHYYV MDA Light chain 39 RSSQSLLN SDGNTYLY 31 LVSKLGS 21 VQGTHDP WT W31621.1 54.8 Heavy chain 36 NYFMN 26 RVDPENGRA DYAEKFKK 18 RAMDNYGF AY Light chain 40 SANSSLSY MY 32 GTSNLAS 22 HHWSNTQ WT W31621 .1 45.10-z7 Heavy chain 34 NYDMA 27 SISPSGGNT YYRDSVKG 16 HLWFAY Light chain 38 QASQDIGS NLI 30 YATHLAD 20 LQYKQYPR T Petition 870240025544, dated 03 / 25 / 2024, page 23 / 68 16 / 52 Clone ID SEQ ID NO CDR1 SEQ ID NO CDR2 SEQ ID NO CDR3 W3162.1 .14 6.-z12 Heavy chain 35 SGYDWT 25 YISYSGNTN YNPSLKS 17 MMVPHYYV MDA Light chain 39 RSSQSLLN SDGNTYLY 31 LVSKLGS 21 VQGTHDP WT W3162.1 ,15 4,8-z35 Heavy chain 36 NYFMN 28 RVDPEQGR ADYAEKFKK 18 RAMDNYGF AY Light chain 41 SANSALSY MY 32 GTSNLAS 22 HHWSNTQ WT

[00128] The antibodies of the invention may be chimeric antibodies.

[00129] The antibodies of the invention may be humanized antibodies.

[00130] The antibodies of the invention may be fully human antibodies.

[00131] The antibodies of the invention may be mouse antibodies.

[00132] The antibodies or their antigen-binding fragment of the invention may exhibit at least one of the following properties:

[00133] a) Bind to human CTLA-4 with a KD of 2.08E-09 M or lower, and / or to mouse CTLA-4 with a KD of 1.39E-09 M or lower;

[00134] b) Improve the release of interleukin-2 from stimulated PBMCs;

[00135] In another aspect, the invention provides a nucleic acid molecule that encodes the antibody, or its antigen-binding fragment.

[00136] The invention provides a cloning or expression vector comprising the nucleic acid molecule encoding the antibody or its antigen-binding fragment.

[00137] The invention also provides a host cell comprising one or more cloning or expression vectors.

[00138] In yet another aspect, the invention provides a process comprising cultivating the host cell of the invention and isolating the Petition 870240025544, dated 03 / 25 / 2024, page 24 / 68 17 / 52 antibody, wherein the antibody is prepared by immunization in an SD mouse with human CTLA-4 extracellular domain and mouse CTLA-4 extracellular domain.

[00139] The invention provides a transgenic animal, such as a mouse, comprising human immunoglobulin heavy and light chain transgenes, wherein the mouse expresses the antibody of this invention.

[00140] The invention provides a hybridoma prepared from the mouse of this invention, wherein the hybridoma produces said antibody.

[00141] In another aspect, the invention provides a pharmaceutical composition comprising the antibody or the antigen-binding fragment of said antibody in the invention, and one or more pharmaceutically acceptable excipients, diluents or vehicles.

[00142] The invention provides an immunoconjugate comprising said antibody or its antigen-binding fragment of this invention linked to a therapeutic agent.

[00143] In which the invention provides a pharmaceutical composition comprising said immunoconjugate and one or more pharmaceutically acceptable excipients, diluents or vehicles.

[00144] The invention also provides a method for preparing an anti-CTLA-4 antibody or an antigen-binding fragment thereof comprising:

[00145] (a) provide:

[00146] (i) an antibody sequence of the variable heavy chain region comprising a CDR1 sequence that is selected from a group consisting of SEQ ID NOs: 33-36, a CDR2 sequence that is selected from the group consisting of SEQ ID NOs: 23-28; and a CDR3 sequence that is selected from the group consisting of SEQ ID NOs: 15-18; and / or

[00147] (ii) an antibody sequence of the variable light chain region comprising a CDR1 sequence that is selected from the group Petition 870240025544, dated 03 / 25 / 2024, page 25 / 68 18 / 52 consisting of SEQ ID NOs: 37-41, a CDR2 sequence that is selected from the group consisting of SEQ ID NOs: 29-32, and a CDR3 sequence that is selected from the group consisting of SEQ ID NOs: 19-22; and

[00148] (b) express the altered antibody sequence as a protein.

[00149] The invention also provides a method of modulating an immune response in an individual, comprising administering to the individual the antibody or antigen-binding fragment of any of said antibodies of this invention.

[00150] The invention also provides the use of said antibody or its antigen-binding fragment in the manufacture of a medicament for the treatment or prophylaxis of an immune disorder or cancer.

[00151] The invention also provides a method for inhibiting the growth of tumor cells in an individual, comprising administering to the individual a therapeutically effective amount of said antibody or said antigen-binding fragment, to inhibit the growth of tumor cells.

[00152] Wherein, the invention provides a method in which tumor cells are from a cancer selected from a group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, and rectal cancer.

[00153] In which the invention provides a method in which the antibody is a chimeric antibody, humanized antibody, human antibody or mouse antibody. The aspects and advantages of this invention

[00154] The inventors generated humanized antibodies against CTLA-4 using proprietary hybridoma technology, in which the Petition 870240025544, dated 03 / 25 / 2024, page 26 / 68 19 / 52 antibodies inhibited CTLA-4 binding to its ligands CD80 and CD86. The antibodies reported in this invention have high binding affinity, specifically in binding to human and monkey CTLA-4 protein; and potent modulating immune responses and increasing interleukin-2 production.

[00155] One of the antibodies not only bound to human and monkey CTLA-4, but also to murine CTLA-4, which could greatly facilitate the preclinical validation of its efficacy in mouse tumor models. BRIEF DESCRIPTION OF THE DRAWINGS

[00156] Figure 1 shows graphs of chimeric antibodies that bind to human CTLA-4 in ELISA.

[00157] Figure 2 shows graphs of chimeric antibodies that bind to cynomolgus CTLA-4 in ELISA.

[00158] Figure 3 shows graphs of chimeric antibodies that bind to mouse CTLA-4 in ELISA.

[00159] Figure 4 shows graphs of chimeric antibodies that bind to human CTLA-4 in cells by FACS.

[00160] Figure 5 shows the result of chimeric antibodies that bind to human CTLA-4 by SPR.

[00161] Figure 6 shows the result of chimeric antibodies blocking ligand binding.

[00162] Figure 7 shows graphs of the chimeric binding of CTLA-4 inhibited by Abs in cells expressing CD80 or CD86.

[00163] Figure 8 shows the results of increased cytokine release by chimeric antibodies from SEB-stimulated PBMCs.

[00164] Figure 9 shows graphs of humanized antibodies binding to human, cynomolgus monkey, and mouse CTLA-4 in ELISA.

[00165] Figure 10a shows graphs of humanized antibodies. Petition 870240025544, dated 03 / 25 / 2024, p. 27 / 68 20 / 52 that bind to CTLA-4 in cells (FACS).

[00166] Figure 10b shows graphs of the affinity of humanized antibodies for FACS.

[00167] Figure 11 shows that humanized antibodies block ligand binding by ELISA.

[00168] Figure 12 shows that humanized antibodies block the binding of CTLA-4 to its ligands by FACS.

[00169] Figure 13 shows that humanized antibodies increase cytokine release in the SEB assay.

[00170] Figure 14 shows the SEC profile of W3162-1,146,19-Z12 or W3162-1,154,8-Z35 under different conditions.

[00171] Figure 15 shows the in vivo efficacy result of the W3162-146,19-z12 antibody.

[00172] Figure 16 shows that W3162 antibodies bind specifically to CTLA-4.

[00173] Figure 17 shows binding activity of antiCTLA-4 antibodies with CTLA-4 / human CTLA-4 mutants. (A) Ipilimumab, (B) W3162-1,146,19-z12 and (C) W3162-1,154.8-z35 antibodies were captured pre-coated with 2 pg / ml of anti-human-goat Fc IgG antibody and then incubated with diluted hCTLA4-His (WT) or its mutains (N113Q and N145Q), then HRP-antiHis antibody was added for detection.

[00174] Figure 18 shows the binding residues or epitopes mapped to human CTLA-4: (A) CD80 binding sites (PDB:1I8L), (B) CD86 (PDB:1I85), (C) tremelimumab (PDB: 5GGV), (D) Ipilimumab, (E) W3162-1146.19-z12 and (F) W3162-1154.8-z35, respectively. The CTLA-4 structure of IAH1 was used for DF to show the glycosylation structure. DETAILED DESCRIPTION

[00175] So that the present invention can be more easily Petition 870240025544, dated 03 / 25 / 2024, p. 28 / 68 21 / 52 included, certain terms are defined first. Additional definitions are provided throughout the detailed description.

[00176] The terms “cytotoxic T-lymphocyte-associated antigen-4”, “CTLA-4 protein”, “CTLA-4”, “CTLA4”, “CD152” are used interchangeably and include variants, isoforms, species homologs of human CTLA-4 or CTLA-4 from other species, and analogs having at least one epitope in common with CTLA-4.

[00177] The term “antibody,” as referred to herein, includes whole antibodies and any antigen-binding fragment (i.e., “antigen-binding portion”) or their single chains. An “antibody” refers to a protein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or an antigen-binding portion. Each heavy chain comprises a variable heavy chain region (abbreviated herein as VH) and a constant heavy chain region. The constant heavy chain region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a variable light chain region (abbreviated herein as VL) and a constant light chain region. The constant light chain region comprises one domain, CL.The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called structural regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminal to the carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[00178] The term “antibody,” as used in this description, refers to an immunoglobulin or a fragment or derivative thereof, and includes any polypeptide comprising a site of Petition 870240025544, dated 03 / 25 / 2024, page 29 / 68 22 / 52 antigen binding, regardless of whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and graft antibodies. The term “antibody” also includes antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e., the ability to bind specifically to CTLA-4. Typically, such fragments would comprise an antigen-binding fragment.

[00179] The terms “antigen-binding fragment,” “antigen-binding domain,” and “binding fragment” refer to a portion of an antibody molecule comprising amino acids responsible for the specific binding between the antibody and the antigen.In cases where an antigen is large, the antigen-binding fragment may only bind to a portion of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding fragment is referred to as an "epitope" or "antigenic determinant."

[00180] An antigen-binding fragment typically comprises a variable light chain (VL) region of the antibody and a variable heavy chain (VH) region of the antibody; however, it does not necessarily have to comprise both. For example, a so-called Fd antibody fragment consists only of a VH domain, but still retains some of the antibody's antigen-binding function intact.

[00181] According to the above, the term “epitope” defines an antigenic determinant that is specifically linked / identified by a binding fragment, as defined above. The binding fragment can specifically bind to / interact with conformational or continuous epitopes that are unique to the target structure, by Petition 870240025544, dated 03 / 25 / 2024, page 30 / 68 23 / 52 For example, human CTLA-4 and murine CTLA-4. A conformational or discontinuous epitope is characterized by polypeptide antigens having two or more distinct amino acid residues that are separated in the primary sequence but come together on the surface of the molecule when the polypeptide folds into the native protein / antigen. The two or more distinct amino acid residues contributing to the epitope are present in separate sections of one or more polypeptide chains. These residues come together on the surface of the molecule when the polypeptide chain(s) fold into a three-dimensional structure to form the epitope. In contrast, a continuous or linear epitope consists of two or more distinct amino acid residues that are present in a single linear segment of a polypeptide chain.

[00182] The term “binds to a CTLA-4 epitope” refers to antibodies that have specific binding to a particular epitope of CTLA-4, which can be defined by a linear amino acid sequence or by a tertiary, i.e., three-dimensional conformation in the CTLA-4 polypeptide portion. Binding means that the affinity of the antibodies for the CTLA-4 portion is substantially greater than their affinity for other related polypeptides. The term “substantially greater affinity” means that there is a measurable increase in affinity for the CTLA-4 portion 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, 103-fold, 104-fold, 105-fold, 106-fold or more for the particular CTLA-4 portion than for other proteins.Preferably, binding affinity is determined by enzyme-linked immunosorbent assay (ELISA), or by fluorescence-activated cell selection (FACS) or surface plasmon resonance (SPR) analysis. More preferably, binding specificity is... Petition 870240025544, dated 03 / 25 / 2024, page 31 / 68 24 / 52 obtained by fluorescence-activated cell selection (FACS) analysis.

[00183] The term “cross-reactivity” refers to the binding of an antigen fragment, described herein, to the same target molecule in humans, monkeys, and / or mice. Thus, “cross-reactivity” should be understood as interspecies reactivity to the same molecule X expressed in different species, but not to a molecule different from X. The cross-species specificity of a monoclonal antibody that recognizes, for example, human CTLA-4, monkey CTLA-4, and / or mouse CTLA-4 can be determined, for example, by FACS analysis.

[00184] As used herein, the term “individual” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except where cited, the terms “patient” or “individual” are used interchangeably.

[00185] The terms “treatment” and “therapeutic method” refer to both therapeutic treatment and prophylactic / preventive measures. Those in need of treatment may include individuals who already have a particular medical disorder, as well as those who may acquire the disorder.

[00186] The term “conservative modifications” refers to modifications in the nucleotide and amino acid sequence that do not significantly affect or alter the binding characteristics of the antibody encoded by the nucleotide sequence or containing the amino acid sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions, and deletions. Modifications can be introduced into the sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions Petition 870240025544, dated 03 / 25 / 2024, page 32 / 68 25 / 52 of amino acids include those in which the amino acid residue is replaced by an amino acid residue that has a similar side chain. Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[00187] The experimental methods in the following examples are conventional methods, unless otherwise specified. EXAMPLES Example 1: Preparation of research materials 1. Expression and purification of soluble CTLA-4

[00188] Extracellular domain (ECD) genes of human and mouse CTLA-4 with hexahistidine (6xHis) - or Fc-label were cloned into the expression vector and then used for transfection of Expi293 cells using the Expi293 Expression System Kit. Cells were cultured in Expi293 Expression Medium on an orbital shaker platform rotating at 135 rpm in a 37 °C incubator containing a humidified atmosphere with 8% CO2. The harvested supernatant was used for protein purification. Hexahistidine-labeled proteins were purified using the Ni-NTA column, and Fc-labeled proteins were purified using the Protein A column. 2. Development of cell lines Petition 870240025544, dated 03 / 25 / 2024, page 33 / 68 26 / 52

[00189] The full-length human CTLA-4 gene was cloned into an expression vector for the development of a stable cell line. Briefly, a 30 mL volume of 293F cells at a density of 1x10⁶ / mL was transfected with 30 pg of DNA using Plasfect reagent. The transfected cells were placed in an incubator environment at 37°C, 8% CO₂, and 100 rpm agitation speed. 24–48 hours after transfection, blasticidin, at a final concentration of 4–6 pg / mL, was used to select stable clones. The selected clones were tested by FACS using an anti-CTLA-4 antibody.

[00190] To obtain cynomolgus monkey CTLA-4 expressing cells, the full-length cynomolgus monkey CTLA-4 gene was cloned into an expression vector for cell pool development. Briefly, a 30 mL volume of 293F cells at a density of 1x10⁶ / mL was transfected with 30 pg of DNA using Plasfect Reagent (Life Technology). The transfected cells were placed in an incubator environment at 37°C, 8% CO₂, and 100 rpm agitation speed. 24 hours after transfection, blasticidin at a final concentration of 4 pg / mL was used to select the cell pool. The selected cell pools were tested by FACS using an anti-CTLA4 antibody. Example 2: Antibody hybridoma generation 1. Immunization

[00191] Human CTLA-4 and murine CTLA-4 were used for immunization of SD mice. Specifically, three SD mice were immunized with 30 pg / animal of ECD of human and mouse CTLA-4 protein in adjuvant. The adjuvant included Titer-Max, AdjuPhos, and CpG-ODN. Mice were injected once weekly either into the soft tissue of the paw or subcutaneously. The titer of Petition 870240025544, dated 03 / 25 / 2024, p. 34 / 68 Antibody levels in serum (27 / 52) were measured by ELISA monthly. When the antibody titer was sufficiently high, the mouse with the highest titer received a final booster with ECD of human and mouse CTLA-4 protein in Dulbecco's Phosphate Buffered Saline (DPBS) without adjuvant. After several days, the spleen and lymph nodes were removed from the mouse, and the lymphocytes were separated for fusion. 2. Cell Fusion

[00192] Cell fusion was performed as follows: Myeloma cells, SP2 / 0 cells, were thawed the week prior to fusion and divided 1:2 daily until the day before fusion to maintain logarithmic growth. B lymphocytes isolated from immunized mouse lymph nodes and myeloma cells were treated with trypsin, respectively, and the reaction was stopped by the addition of FBS. B lymphocytes were combined with myeloma cells in a 1:1 ratio. The cell mixture was then washed and resuspended at 2x10⁶ cells / mL in an electrofusion solution containing 0.3 M sucrose, 0.1 mM magnesium acetate, and 0.1 mM calcium acetate. Electrofusion of cells was conducted using the Btx Electro Cell Manipulator (Ecm 2001) following the manufacturer's standard protocol. Next, the cell suspension from the fusion chamber was immediately transferred to a sterile flask containing fresh medium and incubated for 2 hours in an incubator at 37°C.The cell suspension was then mixed and transferred to 60 of the 96-well plates (1x10⁴ cells / well). The 96-well plates were cultured at 37°C and 5% CO₂ with periodic monitoring. When the clones were large enough (after 7-10 days), 180 pL / well of supernatant were removed, and then 200 pL of fresh medium were added per well. After 72 hours, 100 pL of supernatant were transferred from the tissue culture plates to 96-well assay plates. Petition 870240025544, dated 03 / 25 / 2024, page 35 / 68 28 / 52 screening. 3. Hybridoma scan

[00193] A large number of hybridoma clones were screened for binding to human, murine, and monkey CTLA-4 proteins, as well as engineered cells expressing human CTLA-4. Once specific CTLA-4 binding and blocking activity was verified through first and second screening, positive hybridoma cell lines were subcloned into 96-well plates using limited dilution. The plates were cultured at 37°C, 5% CO2 until positive clones were screened for competition with CTLA-4-binding ligands CD80 and CD86. Culture supernatants from selected positive clones were collected for antibody purification and subsequent characterization. Leading candidates were selected for VH and VL sequencing. 4. Determination of VH and VL hybridoma sequences

[00194] VH and VL genes from antibodies of selected hybridoma clones were isolated by RT-PCR or 5' RACE. Specifically, total RNA was isolated from hybridoma cells using RNeasy Plus Mini Kit (Qiagen). First-strand cDNA was reverse transcribed using oligo dT. VH and VL genes from antibodies were amplified from cDNA using sets of constant-region-3' and degenerate-5' primers. The 5' degenerate primers were designed based on the coding region of the signal sequence upstream of the variable Ig sequences. The PCR product was then ligated to the pMD18-T vector, and 10 pL of the ligation product were transformed into Top10 competent cells. The transformed cells were spread on 2xYT plates with carbenicillin and incubated overnight at 37°C. 15 positive colonies were randomly selected for DNA sequencing by Biosune. Alternatively, 5' RACE was Petition 870240025544, dated 03 / 25 / 2024, page 36 / 68 29 / 52 was used to identify the VH and VL sequences of selected hybridoma clones. First, RNA was reverse transcribed to cDNA using the 5'-RACE kit (Takara-28001488), followed by PCR using 3' degenerate primers and 3' adapter primers (ExTaq: Takara-RR001B). PCR fragments were inserted into the pMD18-T vector (Takara-D101C) and sent for sequencing (Biosune, Shanghai). Example 3: Production and characterization of chimeric antibodies 1. Production of chimeric antibodies

[00195] The amino acid sequences deduced from VH and VL are listed in Table 3. The underlined sequences are CDRs defined by the Kabat design system. The variable regions of these rat antibodies were fused with the constant region of human antibodies, and the chimeric antibodies were expressed from Expi293 cells and purified using Protein A chromatography. Table 3. Sequence of the variable region of anti-CTLA-4 mouse antibodies. Clone ID SEQ ID NO Amino acid sequence W3162- 1.101.2 VH 1 EEQLVESGGGLVQPGKSLKLSCSASAGFTFRSSAMHWIRQ PPGKGL DWVAFISSGGDTAYADAVKGRFIVSRDNAENTLFLQLNSL KSED TAIYYCVRMERIPTWGQGVMVTVSS VL 8 DIVLTQSPVLAVSLGQRATISCRASQSVSISSINLIHWYQQ RPGQQ PKLLIYRTSNLASGIPARFSGSGSGTDFTLSIDPVQADDVA DYYCQ QSRESPLTFGSGTKLEIK W3162- 1.145.10 VH 2 EVQLVESGGGLVQPGRSLKLSCAASDLTFSNYDMAWVR QTPTKG Petition 870240025544, dated 03 / 25 / 2024, page 37 / 68 30 / 52 ID do Clone SEQ ID NO Aminoacid Sequence LEWVASISPNGGNTYYRDSVKGRFTVSRDNAKNSLYLQM DSLRS EDTATYYCARHLWFAYWGQGTLVTVSS VL 9 DIQMTQSPSSMSASLGDRVTISCQASQDIGSNLIWFQQKP GKSPRP MIYYATHLADGVPSRFSGSRSGSDYSLTISSLESEDVADY HCLQY KQYPRTFGGGTKLELK W3162- 1.146.19 VH 3 EVQLQESGPGLVKPSQSLSLTCSVTYHTITSGYDWTWIRK FPGNQ MEWMGYISYSGNTNYNPSLKSRISITRDTSKNQFFLHLNS VTSED TATYYCASMMVPHYYVMDAWGQGASVTVSS VL 10 DVVLTQTPPTSSATIGQSVSISCRSSQSLLNSDGNTYLYW YLQRPS QSPQLLIYLVSKLGSGVPNRFSGSGSGTDFTLKISGVEAE DLGLYY CVQGTHDPWTFGGGTKLELK W3162- 1.154.8 VH 4 EVQLQQSGPEAGRPGSSVKISCKASGYTFTNYFMNWVKQ SPGQG LEWIGRVDPENGRADYAEKFKKKATLTADTTSNTAYIH LS SLTS EDTATYFCARRAMDNYGFAYWGQGTLVTVSS VL 11 EIMLTQSPTIMAASLGEKITITCSANSLSYMYWFQQKSGA SPKLW VHGTSNLASGVPDRFSGSGSGTSYYLTINTMEAEDAATYF CHHW SNTQWTFGGGTKLELK 2. Caracterização de anticorpos quimericos 2.1 Antibody tests for CTLA-4 humans, macaques and murines (ELISA, FACS and SPR)

[00196] Chimeric antibodies with a variable region from rats and a constant region from humans were expressed from mammalian cells and purified using Protein A affinity chromatography. Petition 870240025544, dated 03 / 25 / 2024, pp. 38 / 68 31 / 52

[00197] The antibodies were tested for CTLA-4 binding by ELISA. As shown in Figures 1, 2, and 3, all four antibodies bound to human and monkey CTLA-4 with EC50 comparable to Ipilimumab (WBP316-BMK1), however only one antibody W31621,146,19 also bound to murine CTLA-4 at an EC50 of 0.01 nM. To confirm that the antibodies were able to bind to CTLA-4 on the cell surface, a cell line expressing CTLA-4 was used in FACS assays. These antibodies also bound to CTLA-4 on the cell surface (Figure 4) with EC50 ranging from 1.14 nM to 9.42 nM. W3162-1.146.19 bound to CTLA-4 on the cell surface with an EC50 of 3.25 nM, and W3162-1.154.8 bound to CTLA-4 on the cell surface with an EC50 of 1.26 nM.

[00198] The binding kinetics of four antibodies were measured using SPR. Antibodies were captured on immobilized anti-human-goat Fc, and then human CTLA-4 ECD at different concentrations was injected sequentially. Diagrams of the sensors for the reference channel and buffer channel were subtracted from the diagrams of the test sensor diagrams. The data were used for fitting in the 1:1 binding analysis. As shown in Figure 5 and Table 4, all four antibodies bound to the human CTLA-4 ECD domain with higher affinity than with Ipilimumab (WBP316BMK1), with Kd ranging from 2.08 E-09 nM to 6.80 E-11 nM. Table 4. Antibody binding kinetics at the ECD of human CTLA-4. Antibody ka (1 / Ms) kd (1 / s) Kd (M) W3162-1.101.2 xAb.IgGI 6.95E+05 6.97E-05 1.00E-10 W3162-1.145.10 xAb.IgG1 7.93E+06 1.65E-02 2.08E-09 W3162-1,146.19 xAb.IgG1 7.09E+05 1.48E-04 2.08E-10 3.46E-03 3.68E-09 2.2 Competition with chimeric antibody ligands

[00199] CTLA-4 was found to bind to both CD80 and CD86 with an affinity 20 to 50 times greater than CD28 [Krummel 1996]. Petition 870240025544, dated 03 / 25 / 2024, pp. 39 / 68 32 / 52 Therefore, anti-CTLA-4 antibodies were tested to see if they could compete with CD80 and CD86 binding to CTLA-4. Both ELISA and FACS were used as competition assays. In the ELISA-based competition assay, human CTLA-4 was coated onto plates and antibodies mixed with biotinylated ligands were added to the plate. The bound ligands were detected by HRP-conjugated streptavidin. As shown in Figures 6a and 6b, all four antibodies competed with CD80 (B71, L1) and CD86 (B7-2, L2) ligands in binding to CTLA-4, and three of them, except W3162-1,101,2, had EC50 comparable to Ipilimumab (WBP316BMK1). In a FACS assay, the antibody mixture and biotinylated human CTLA-4 was added to cells expressing CD80 or CD86, and bound human CTLA-4 was detected by streptavidin-conjugated PE.As shown in Figures 7a (top panel) and 7b (bottom panel), all four antibodies could effectively block CTLA-4 binding to ligand-expressing cells. Three antibodies, except W3162-1.154.8, were able to completely block CTLA4 binding to CD80 cells, while Ipilimumab WBP316-BMK could only partially block this binding, even at 200 nM, the highest concentration used (Figure 7a). In the FACS assay for blocking CTLA-4 binding to CD86 cells (Figure 7b), all four antibodies were able to completely block CTLA-4 binding to CD86 cells, while Ipilimumab could only partially block this binding, even at 200 nM, the highest concentration used. The kinetics of W3162-1.101.2 appeared different: the blockade was less effective than with Ipilimumab at low concentrations and more effective than with Ipilimumab at high concentrations.Three other antibodies were more effective than ipilimumab in blocking CTLA4 at all concentrations tested. 2.3 Role of chimeric antibodies in the SEB assay Petition 870240025544, dated 03 / 25 / 2024, p. 40 / 68 33 / 52

[00200] The function of anti-CTLA-4 antibodies with different concentrations of 1.34 nM, 3.35 nM, 8.71 nM, 21.4 nM, 53.6 nM, and 134 nM were tested in a modified T-cell stimulation assay (SEB assay). Staphylococcal enterotoxin B (SEB) was used as a stimulator of human T-cell activation, in which CTLA-4 was reported as an important participant. T-cell activation was measured by IL-2 secretion. As shown in Figure 8, all four antibodies promoted IL-2 secretion in a dose-dependent manner, comparable to or superior to ipilimumab. Example 4: Characterization of humanized antibodies 1. Humanization

[00201] The “best fit” approach was used to humanize light and heavy antibody chains.

[00202] Three anti-CTLA-4 antibodies (except W3162-1.101.2 due to its relatively weak binding activity in ELISA and FACS) were selected for humanization using the CDR grafting technique. The CDRs (underlined in Table 5) and FRs, from variable regions of the antibodies, were defined using the Kabat system. Based on sequence homology and structural similarity, the FR1-3 region of the mouse gene was replaced by the humanized FR1-3 region, while the FR4 region of the mouse gene was replaced by the humanized FR4 region derived from the JH and JK genes that had the most similar structures. Post-translational modification (PTM) hotspots of the variable regions were modified to reduce the risk of PTM. After verifying the template sequence and codon optimization, the heavy chain variable region and light chain variable region were synthesized and cloned into an expression vector and then used for the expression of humanized antibodies.Humanized antibodies were purified using Protein A chromatography, and binding kinetics at human, monkey, and murine CTLA-4 was measured using the... Petition 870240025544, dated 03 / 25 / 2024, page 41 / 68 34 / 52 SPR method. Table 5. Sequence of the variable region of humanized anti-CTLA-4 antibodies. ID do Clone SEQ ID NO Sequência de aminoácidos W3162-1 .1 45.10-z7 VH 5 EVQLVESGGGLVQPGGSLRLSCAASDLTFSNYDMAWVR QAPG KGLEWVASISPSGGNTYYRDSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARHLWFAYWGQGTLVTVSS VL 12 DIQMTQSPSSLSASVGDRVTITCQASQDIGSNLIWFQQKP GKAP KPMIYYATHLADGVPSRFSGSRSGTDYTLTISSLQPEDFA TYYCLQYKQYPRTFGGGTKVEIK W3162.1. 14 6.19-z12 VH 6 QVQLQESGPGLVKPSETLSLTCSVTYHTITSGYDWTWIR KPPGK GMEWIGYISYSGNTNYNPSLKSRVTISRDTSKNQFFLKLS SVTADTAVYYCASM MVPHYYVM DAWGQGTLVTVSS VL 13 DIVMTQTPLSLSVTPGQPASISCRSSQSLLNSDGNTYLY WYLQKPGQSPQLLIYLVSKLGSGVPNRFSGSGSGTDFTL KISRVEAEDVGVYYCVQGTHDPWTFGGGTKVEIK W3162.1. 15 4.8-z35 VH 7 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYFMNWVR QAP GQGLEWMGRVDPEQGRADYAEKFKKRVTITADKSTSTA YMELSSLRSEDTAVYYCARRAMDNYGFAYWGQGTLVTV SS VL 14 EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKP DQSP KLWVHGTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDA ATYYCHHWSNTQWTFGGGTKVEIK Petition 870240025544, on 03 / 25 / 2024, page. 42 / 68 35 / 52 Tabela 6. A variety of human anti-CTLA-4 antibodiesClone ID SEQ ID NO DNA Sequence W3162- 1.145 VH 42 GAGGTGCAGCTGGTGGAGAGCGGCGGAGGACTGGTGCAAC CTGGCGGAAGC CTGAGACTGAGCTGCGCCGCCAGCGACCTGACCTTCAGCAA CTACGACATGG CCTGGGTGAGACAGGCCCCTGGCAAGGGACTGGAGTGGGT GGCCAGCATCA GCCCCAGCGGCGGCAACACCTACTAGGGACAGCGTGAA GGGCAGGTTCA CCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAG ATGAACAGGCCCT GAGGCGGAGGACCAGGCCTAGGCCCT CTGTGGGTTCGCC TACTGGGGCCAGGGCACACTGGTGACCGTGAGCAGC .10-z7 VL 45 GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAG CGTGGGGCATAGGGTGACCATCACCAGCCAGGCCAGCCAG GACATCGCACCAGCTGAGCCAGGCCAGGCCAGCCAG CAAGGCCCCCAAGCCTATGATCTACTACGCCACCCACCTGG CCGATGGCGTGCCTAGCAGATTCAGCGGCAGCAGAAGCGG CACCGACTACCCCTGACCATCAGCAGCAGCCTGCAGCCCGAGG ACTTCGCCACCTACTACTGCCTGCAGTACAAGCAGTACCCCA GAACCTTCGGGCGAGGCGAGGAGGA16.146 VH 43 CAGGTGCAGCTGCAGGAGAGCGGACCCGGACTGGTGAAGC CCTCCGAGACC CTGAGCCTGACCTGCAGCGTGACCTACCACACCATCACCAG CGGCTACGACT GGACCTGGATCAGAAAGCCCCCCGGCAAAGGCATGGAGTG GATCGGCTACAT CAGCTACAGCGGCAACACCAACTACAACCCCAGCCTGAAGA GCAGGGTGAC CATCAGCAGGGACACCAGCAAGAACCAGTTCTTCCTGAAGC TGAGCAGCGTG ACAGCCGCCGATACCGCCGTGTACTACTGCGCCAGCATGAT GGTGCCCCACTA CTACGTGATGGACGCCTGGGGACAGGGCACCCTGGTGACA GTGAGCAGC . 19-z12 VL 46 GACATCGTGATGACCCAGACCCCCCTGAGCCTGAGCGTGAC ACCTGGACAGCCCGCCAGCATCAGCTGCAGGTCCAGCCAG AGCCTGCTGAACAGCGACGGCAACACCTACCTGTACTGGTA CCTGCAGAAGCCTGGCCAGAGCCCCCAGCTGCTGATCTACC TGGTGTCCAAGCTGGGCAGCGGCGTGCCTAACAGGTTTAGC GGCAGCGGCAGCGGCACCGATTTCACCCTGAAGATCAGCA GGGTGGAGGCCGAGGATGTGGGCGTGTACTACTGCGTGCA GGGCACCCACGATCCTTGGACCTTCGGCGGCGGAACCAAG GTGGAGATCAAG W3162- 1.154 VH 44 CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGC CCGGCAGCAGC GTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAA CTACTTCATGA ACTGGGTGAGGCAGGCCCCTGGACAAGGCCTGGAGTGGGGGGCAGAGAGTGG ATCCCGAGCAGGGCAGGGCCGACTACGCCGAGAAGTTCAA GAAGAGGGTGA CCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAG CTGAGCAGCCT. Petition 870240025544, of 25 / 03 / 2024, p. 43 / 68 36 / 52 Clone ID SEQ ID NO DNA Sequence .8-z35 GAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGAGA GCCATGGACAA CTACGGCTTCGCCTACTGGGGCCAGGGAACCCTGGTGACC GTGAGCAGC VL 47 GAGATTCGTGCTGACCCAGCCGACCGACCGGACC CCCCAAGGAGAAGGTGACCATCACCTGCAGCGCCAACAGC GCCCTGAGCTACATGTACTGGTACCAGCAGAAGCCCGACCA GAGCCCCAAGCTGTGGGTGCACGGCACCAGCAATCTGGCC AGCGGCGTGCCTAGCAGATTTAGCGGCAGCGGCAGCGGCA CCGATTTCACCCCAGCCAGGCCGACCGACCGCC GCCGCTACCTACTACTGCCACCACTGGAGCAACACCCAGTG GACCTTCGGCGGCGGCACCAAGGTGGA GATCAAG 2.Characterization of humanized antibodies 2.1 Human, macaque, and murine CTLA-4-bound antibodies 2.1.1 Linking to CTLA-4 in ELISA

[00203] Humanized antibodies were expressed from mammalian cells and purified using Protein A affinity chromatography. Ipilimumab was from a commercial source. Isotype control antibody, human CTLA-4 ECD with different markers (hFc or 6xHis) and murine CTLA-4.ECD-hFc were prepared by WuXi Biologics. Murine CTLA-4.ECD-6xHis and cynomolgus monkey CTLA-4.ECD-6xHis were acquired from Sino Biological. HRP-conjugated human-goat IgG Fc was acquired from Bethyl (Cat: A80304P).

[00204] ELISA was used to test the binding of anti-human CTLA-4 antibodies to human, murine, and cynomolgus monkey CTLA-4 protein. A 96-well plate was coated with CTLA-4.ECD6xHis (1.0 pg / mL), cynomolgus monkey CTLA-4.ECD-6xHis (0.5 pg / mL), or mouse CTLA-4.ECD-6xHis (0.5 pg / mL) at 4°C for 16–20 hours. After 1 hour of blocking with 2% BSA in DBPS, the test antibodies, as well as the positive and negative control antibodies, were added to the plates and incubated at room temperature for 1 hour. Antibody binding to the plates was detected by HRP-conjugated anti-human-goat IgG antibody (1:5000 dilution) with a 1-hour incubation. Color was developed by Petition 870240025544, dated 03 / 25 / 2024, p. 44 / 68 37 / 52 dispersion of 100 pL of TMB substrate for 8 minutes and then stopped by 100 pL of 2N HCl. Absorbance at 450 nM was measured using a microplate spectrophotometer.

[00205] As shown in Figure 9, the two antibodies W3162-1,146.19-z12-IgGk and W3162-1,154.8-z35-IgGk bound to human CTLA-4 with EC50s of 0.03 nM and 0.04 nM, respectively, slightly higher than the EC50 of Ipilimumab (WBP316-BMK1) 0.01 nM (Figure 9A). Both antibodies also bound to monkey CTLA-4 with an EC50 of 0.05 nM (Figure 9B), but only W3162-1,146.19-z12-IgGk bound to murine CTLA-4 with an EC50 of 0.19 nM. Neither W3162-1,154,8z35-IgGk nor Ipilimumab bound to murine CTLA-4 (Figure 9C). 2.1.2 Connection to CTLA-4 in FACS

[00206] The 293F human CTLA-4 expressing cell line was developed by WuXi Biologics. The PE-conjugated anti-human-goat IgG Fc fragment was acquired from Jackson (Catalog number 109-115-098). A number of 1x10⁵ cells per well was added to each well of a 96-well plate and centrifuged at 1500 rpm for 4 minutes at 4°C before removing the supernatant. Serial dilutions of test antibodies, positive and negative controls, were added to the resuspended cells and incubated for 1 hour at 4°C. The cells were washed twice with 200 µL of DPBS containing 1% BSA. Anti-human-goat IgG conjugated with PE (1:100) diluted in DPBS containing 1% BSA was added to the cells and incubated at 4°C for 1 hour. Additional washing steps were performed twice with 200 pL of DPBS containing 1% BSA followed by centrifugation at 1500 rpm for 4 minutes at 4°C.Finally, the cells were resuspended in 100 pL of DPBS containing 1% BSA, and fluorescence values ​​were measured by flow cytometry and analyzed by FlowJo.

[00207] These antibodies were also able to bind to CTLA Petition 870240025544, dated 03 / 25 / 2024, p. 45 / 68 38 / 52 human on the cell surface in the FACS assay. As shown in Figure 11 (Figure 10a and Figure 10b), W3162-1.146.19-z12-IgGk, W3162-1.154.8-z35-IgGk and Ipilimumab had slightly different EC50 values ​​of 1.58 nM, 0.66 nM and 0.83 nM, respectively. 2.2 The binding kinetics of these antibodies 2.2.1 The binding kinetics of these antibodies were measured using SPR

[00208] The experiment was to measure the in-rate (ka) and out-rate (KD) constants of the antibodies to the CTLA-4 ECD based on SPR technology. The affinity constant (KD) was consequently determined.

[00209] Biacore T200, CM5 Sensor Chip, S series, amine coupling kit, and 10x HBS-EP were purchased from GE Healthcare. Anti-human-goat Fc IgG antibody was purchased from Jackson ImmunoResearch Lab (Catalog number 109-005-098). In the immobilization step, activation buffer was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection. The CM5 sensor chip was activated for 420s with the activation buffer. 30 pg / mL of anti-human-goat Fc IgG antibody in 10 mM NaAc (pH 4.5) were then injected into the Fc1-Fc4 channels for 200s at a flow rate of 5 pL / min. The chip was deactivated with 1 M ethanolamine-HCl (GE). The antibodies were then captured on the chip. In summary, antibodies at 4 pg / ml in the execution buffer (HBS-EP+) were injected individually into the Fc3 channel for 30s at a flow rate of 10 pL / min. Eight different concentrations (20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, 0.3125 nM and 0.15625 nM) of the analyte CTLA-4 (WBP316.hCTLA-4) were used.ECD-6xHis) and blank running buffer were sequentially injected into the Fc1-Fc4 channels at a flow rate of 30 pL / min for a 120s association phase, followed by a 2400s dissociation phase. Regeneration buffer (10 mM Glycine pH 1.5) was injected at 10 pL / min for 30s following each dissociation phase. Petition 870240025544, dated 03 / 25 / 2024, pp. 46 / 68 39 / 52

[00210] The binding kinetics of these antibodies were measured using SPR. Antibodies were captured on immobilized anti-human Fc, and CTLA-4-ECD at different concentrations was injected sequentially. Sensor diagrams for the reference channel and buffer channel were subtracted from test sensor diagrams. The data were used for 1:1 binding analysis in CTLA4.ECD-6xHis from humans, monkeys, and mice. As shown in Table 7, the humanized antibodies W3162-1146.19-Z12, W145, and W3162-1154.8-Z35 bound to the human CTLA-4-ECD domain with affinity at 0.477 nM, 1.84 nM, and 0.0968 nM, respectively. Compared to mouse antibodies, the humanized antibodies had comparable affinity. W3162-1.146.19-Z12 and W3162-1.154.8Z35 exhibit significantly higher affinity than Ipilimumab (Kd=3.68 nM). The W3162-1.146.19-Z12 antibody can also bind to murine CTLA-4, and its affinity before and after humanization is shown in Table 9.After humanization, its affinity of 1.39 nM is slightly lower than the affinity of 0.906 nM of its parental antibody.

[00211] The binding affinity of W3162-1.146.19-Z12, W3162-1.145.10-Z7 and W3162-1.154.8-Z35 to cynomolgus monkey CTLA-4-ECD was 1.92 nM, 0.598 nM, 0.131 nM, respectively (Table 8). Table 7. Antibody binding kinetics in the ECD of human CTLA-4. Antibodies ka (1 / Ms) kd (1 / s) Kd (M) W3162-1.146.19-z12-uAb.IgG1K 2.06E+05 9.82E-05 4.77E-10 W3162-1.146.19 xAb.IgG1 7.09E+05 1.48E-04 2.08E-10 W3162_1.145.10-z7-uAb.IgG1K 7.37E+06 1.35E-02 1.84E-09 W3162-1.145.10 xAb.IgG1 7.93E+06 1.65E-02 2.08E-09 W3162_1.154.8-z35-uAb.IgG1K 1.23E+06 1.19E-04 9.68E-11 W3162-1.154.8 xAb.IgG1 1.85E+06 1.25E-04 6.80E-11 Ipilimumab 9.42E+05 3.46E-03 3.68E-09 Table 8. Antibody binding kinetics in the ECD of monkey CTLA-4. Petition 870240025544, dated 03 / 25 / 2024, pp. 47 / 68 40 / 52 Antibodies ka (1 / Ms) kd (1 / s) Kd (M) W3162-1.146.19-z12-uAb.IgG1K 1.73E+05 3.31E-04 1.92E-09 W3162-1.146.19 xAb.IgG1 2.91E+05 1.07E-04 3.69E-10 W3162_1.145.10-z7-uAb.IgG1K 4.52E+06 2.71E-03 5.98E-10 W3162-1.145.10 xAb.IgG1 1.06E+07 3.77E-03 3.55E-10 W3162_1.154.8-z35-uAb.IgG1K 9.32E+05 1.22E-04 1.31E-10 W3162-1.154.8 xAb.IgG1 1.25E+06 1.09E-04 8.72E-11 Table 9. Antibody binding kinetics in the mouse CTLA-4 ECD. Antibodies ka (1 / Ms) kd (1 / s) Kd (M) W3162-1.146.19-z8- 1.72E+05 2.39E-04 1.39E-09 W3162-1.146.19 xAb.IgG1 2.51E+05 2.28E-04 9.06E-10 2.2.2 FACS affinity test

[00212] IgG Fc anti-human-goat conjugated with FITC was acquired from Jackson Immunoresearch Lab (catalog number 109-095-098), and BD CantoII was used in this assay. Briefly, HEK293 cells expressing human CTLA-4 were transferred to 96-well U-bottom plates (BD) at a density of 5x10⁴ cells / well. Test antibodies were serially diluted 1:2-times in PBS with 1% BSA and incubated with cells at 4°C for 1 hour. After centrifugation at 1500 rpm for 4 min, the supernatant was discarded. The secondary antibody, FITC-conjugated anti-human-goat Fc IgG (3.2 FITC per IgG, Jackson Immunoresearch Lab), was added to resuspend the cells to a final concentration of 14 pg / ml and incubated at 4 °C in the dark for 30 min. The cells were then washed once and resuspended in PBS with 1% BSA and analyzed by flow cytometry (BD).Fluorescence intensity was converted into bound molecules / cells based on quantitative spheres (Quantum™ MESF Kits, Bangs Laboratories). Kd was calculated using Graphpad Prism5.

[00213] The affinity of humanized antibodies that bind to the cell surface of CTLA-4 was measured by flow cytometry, modified from the Benedict method [Benedict 1997 JIM]. Petition 870240025544, dated 03 / 25 / 2024, pages 48 / 68 41 / 52 After measuring the fluorescence of antibodies binding to CTLA-4-expressing CHO cells, the bound and free antibodies were analyzed and fitted to the equation, as shown in Figure 5. Based on the data and the formula, the calculated affinity constant KD is shown in Table 10. The affinity of the humanized antibodies W3162-1146.19-Z12 and W3162-1154.8-Z35 was high at 5.05 and 0.35 nM, respectively, while the affinity of Ipilimumab was 0.97 nM. Table 10. FACS affinity test Sample Kd (M) Bmax R2 W3162-1.146.19-z12-IgG1K 5.0E-09 1.2E-10 0.99 W3162-1.154.8-z35-IgG1K 3.5E-10 1.2E-10 0.98 Ipilimumab 9.7E-10 7.2E-11 0.99 2.3 Competition with ligands

[00214] To test whether the humanized antibodies retained their ability to block CTLA-4 binding to CD80 and CD86, both ELISA and FACS were used in the competition assay. Two CTLA-4 CD80 and CD86 ligands were acquired from Sino Biological (Catalog number 10698-H08H and 10699-H08H). The labeled biotinylated anti-His antibody was acquired from Genscript (Catalog number A00613). Streptavidin conjugated with HRP was acquired from Invitrogen (Catalog number SNN1004). 2.3.1 The ELISA-based competition assay

[00215] ELISA was used to test whether antibodies could inhibit the binding of human CTLA-4 to its human ligands CD80 and CD86. Plates were coated with human CTLA-4.ECD-hFc (0.5 pg / mL) at 4°C for 16–20 hours. After 1 hour of blocking with 2% BSA in DBPS, the test antibodies, as well as the positive and negative control antibodies, were premixed with 0.25 pg / mL of CD80-6xHis or CD86-6xHis and then added to the plates and incubated at room temperature for 1 hour. After Petition 870240025544, dated 03 / 25 / 2024, pp. 49 / 68 42 / 52 plates were washed three times with PBS containing 0.05% Tween 20, and the labeled biotinylated anti-His antibodies were diluted 1:2000 and added. Plates were incubated at room temperature for 1 hour. Bound ligands were detected by HRP-conjugated streptavidin (1:20000). Color was developed by dispersing 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.

[00216] As shown in Figure 11, W3162-1.146.19-z12-IgGk and W3162-1.154.8-z35-IgGk had a similar effect to Ipilimumab in blocking ligand binding with coated CTLA-4, with IC50 values ​​of 0.87 nM, 0.63 nM and 0.40 nM for CD80, and 0.71 nM, 0.50 nM and 0.42 nM for CD86. 2.3.2 The FACS test

[00217] To test whether antibodies could block CTLA-4 binding to CD80 and CD86 on the cell surface, FACS were used to test this competition. CHO cell lines expressing CD80 and CD86 were developed by WuXi Biologics. Biotinylated CTLA-4.ECD-hFc was produced by WuXi Biologics. PE-conjugated streptavidin was acquired from eBiocience (Catalog number 12-4317).

[00218] Cells expressing CD80 or CD86 were added to each well of a 96-well plate at 1x10⁵ per well and centrifuged at 1500 rpm for 4 minutes at 4 °C before supernatant removal. Serial dilutions of test antibodies, positive and negative controls, were mixed with biotinylated human CTLA-4.ECD-hFc. Due to the different densities of the ligands on the cell surface, 0.02 pg / mL of hCTLA-4.ECD-hFc-Biotin was used in human CD80 cells and 0.08 pg / mL of hCTLA-4.ECD-hFc-Biotin in human CD86 cells. The antibody and CTLA mixtures were then... Petition 870240025544, dated 03 / 25 / 2024, pp. 50 / 68 43 / 52 were added to the cells and incubated for 1 hour at 4°C. The cells were washed twice with 200 μL of FACS buffer (DPBS containing 1% BSA). Streptavidin PE, 1 to 333 diluted in FACS buffer, was added to the cells and incubated at 4°C for 1 hour. Additional washing steps were performed twice with 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 fluorescence values ​​were measured by flow cytometry and analyzed by FlowJo.

[00219] The results are shown in Figure 12. The two humanized antibodies could more effectively block CTLA-4 / ligand binding than Ipilimumab. At the highest concentration used, Ipilimumab blocked only 32% of CTLA-4 binding on CD80 and 40% of CTLA-4 binding on CD86. In comparison, the W3162-1.146.19-Z12 antibody blocked 71% of CTLA-4 binding on CD80 and 73% of CTLA-4 binding on CD86, and the W3162-1.146.19-Z12 antibody blocked 89% of CTLA-4 binding on CD80 and 98% of CTLA-4 binding on CD86. The IC50 values ​​of ipilimumab, W3162-1146.19-Z12, and W3162-1154.8-Z35 directly against CD80 were 3.23, 6.60, and 0.07 nM, respectively. The IC50 values ​​of ipilimumab, W3162-1146.19-Z12, and W3162-1154.8-Z35 directly against CD86 were 2.52, 5.15, and 0.28 nM, respectively. 2.4 Cytokine release from PBMCs stimulated by SEB

[00220] Anti-CTLA-4 antibodies were tested to see if they could increase cytokine release from human PBMCs after stimulation with SEB (from the Second Military Medical University). Peripheral blood was obtained from healthy donors and cells were isolated by density gradient centrifugation (Ficoll GE Healthcare, 171440-02). After the floating layer was removed, platelets were removed by multiple washes with medium. A number of 1x105 Petition 870240025544, dated 03 / 25 / 2024, pp. 51 / 68 44 / 52 human PBMC cells were added to each well of a 96-well plate. Serial dilutions of test antibodies, positive and negative controls, were mixed with SEB (10 ng / mL) and then added to the granulated cells and incubated for 3 days at 37°C. Supernatants were collected to measure the concentration of human IL-2.

[00221] For the human IL-2 test, plates were pre-coated with 1.0 pg / ml of human IL-2 antibody (R&D System MAB602) at 4°C for 16-20 hours. After 1 hour of blocking with 2% BSA (BovoGen) in DBPS, the IL-2-containing supernatants were added to the plates and incubated at room temperature for 2 hours. After washing three times with PBST (containing 0.05% Tween 20), biotinylated human IL-2 antibody (R&D system, BAF202) was diluted and added at a concentration of 0.5 g / mL. The plates were incubated at room temperature for 1 hour. The bound biotinylated antibody was detected by streptavidin conjugated with HRP diluted 1:20000 (Invitrogen, SNN1004). After 1 hour of incubation, the color was developed by dispersing 100 pL of TMB substrate and then stopped with 100 pL of 2M HCl. Absorbance at 450 nM and 540 nM was measured using a microplate spectrophotometer.

[00222] In a cell-based assay, humanized antibodies (8.60 nM, 21.4 nM, 53.6 nM, 134 nM, 335 nM) were tested to see if they could increase human PBMCs stimulated by SEB superantigen. After 3 days of stimulation, PBMC IL-2 was measured using ELISA. Compared to an isotype control antibody, both humanized antibodies (W3162-1146.19-Z12, W3162-1154.8Z35) and Ipilimumab were able to increase IL-2 release from PBMCs in a dose-dependent manner (Figure 13). 2.5 Thermostability

[00223] The stability of the main antibodies was tested in Petition 870240025544, dated 03 / 25 / 2024, pages 52 / 68 45 / 52 different temperatures. Briefly, 100 pL of each antibody sample was pipetted into individual tubes and the samples were incubated at 4°C or 37°C for 20 hours, or 45°C or 50°C for 2 hours. Then, the samples were centrifuged at 12,000 rpm for 10 minutes. These samples were observed for possible precipitation, and the samples were analyzed by SEC-HPLC for purity and elution time.

[00224] The SEC profile of W3162-1.146.19-Z12 under different conditions was shown in Figure 14 ad. Neither the dilution time nor the main peak percentage (92.39% to 92.48%) under high temperature conditions changed significantly compared to low temperature conditions (92.24%). The SEC profile of W31621.154.8-Z35 under different high temperature conditions was shown in Figure 14 eh. Neither the dilution time nor the main peak percentage (97.14% - 97.17%) changed significantly compared to low temperature conditions (96.84%). This dataset indicates that the antibodies were stable under the tested high temperature conditions. 2.6 Non-specific link

[00225] FACS and ELISA assays were used to test whether antibodies bind 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, CHOK1) were set to 1x10⁵ cells per well. Test antibodies and isotype control antibodies were diluted to 10 pg / ml in PBS containing 1% BSA and incubated with cells at 4°C for 1 hour. Cells were washed twice with 180 pL of PBS containing 1% BSA. The PE-conjugated anti-human-goat IgG Fc fragment (Jackson, Catalog number 109-115-098) was diluted to a final concentration of 5 pg / ml in PBS with 1% BSA, then added to Petition 870240025544, dated 03 / 25 / 2024, pp. 53 / 68 46 / 52 resuspended the cells and incubated at 4°C in the dark for 30 min. Additional washing steps were 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 fluorescence values ​​were measured by flow cytometry (BD CantolI) and analyzed by FlowJo.

[00226] In the ELISA assay, test antibodies and isotype control antibodies were tested by 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, and CD3e.ECD. A 96-well plate was coated with the individual antigens (2 μg / mL) at 4 °C overnight. After 1 hour of blocking with 2% BSA in PBS, the plate was washed 3 times with 300 μL of PBST. The test antibodies, as well as the isotype control antibodies, were diluted to 10 μg / mL in PBS containing 2% BSA, then added to the plate and incubated at room temperature for 2 hours. After 3 washes with 300 μL of PBST, the HRP-conjugated anti-human-goat IgG antibody (1:5000 diluted in 2% BSA) was added to the plate and incubated at room temperature for 1 hour. Finally, the plates were washed six times with 300 μL of PBST.The color was developed by dispersing 100 μL of TMB substrate for 12 min and then stopping with 100 μL of 2M HCl. The absorbance at 450 nM was measured using a microplate spectrophotometer.

[00227] In addition to CTLA-4, other irrelevant proteins were used to test whether the W3162-1.146.19-Z12 and W3162-1.154.8-Z35 antibodies were able to bind to these antigens. As shown in Figure 16, among the panel of antigens, only CTLA-4 was detected by both antibodies. Other antigens did not generate a signal in this ELISA assay. In contrast, the anti-OX40 antibody bound to OX40, suggesting that this antigen was coated on the plate. Petition 870240025544, dated 03 / 25 / 2024, pages 54 / 68 47 / 52

[00228] The specificity of the two antibodies was also tested in a panel of different cell lines in the FACS assay. The antibodies did not generate a detectable signal in any of these cell lines (data not shown). 2.7 In vivo efficacy

[00229] Because the W3162-1.146.19-Z12 antibody cross-reacts with human and murine CTLA-4, the antitumor efficacy of this antibody was tested in syngeneic mouse models. The CT26 mouse cancer cell line was used to establish the mouse xenograft model to test the anti-CTLA-4 antibody W3162-1.146.19-Z12. An anti-murine CTLA-4 antibody acquired from BioXCell was used as a positive control (BioXCell-BE0131). Tumor cells were maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 pg / mL streptomycin at 37 °C in a 5% CO2 atmosphere. Tumor cells were routinely subcultured twice weekly after cell separation by trypsin-EDTA treatment. Cells growing in an exponential growth phase were harvested and counted for tumor inoculation.Female Balb / C mice were acquired from Beijing Vital River Laboratory Animal Co., Ltd. Mice aged 6-8 weeks and weighing approximately 18-22 g were used for the study. Each mouse was subcutaneously inoculated in the right helpers with 1x10⁵ tumor cells in 0.1 mL of PBS mixed with 50 µL of Matrigel. When the average tumor volume reached 60-80 mm³, the animals were randomly grouped. Anti-CTLA-4 antibodies and an isotype control were used for treatment: injected intravenously into mice twice weekly. Tumor size was measured twice weekly using a caliper, and tumor volume was... Petition 870240025544, dated 03 / 25 / 2024, pages 55 / 68 48 / 52 calculated using the formula a*b²xn / 6, where a is length and b is width (a>b).

[00230] When the mean tumor volume reached approximately 70 mm3, W3162-1.146.19-Z12 (1 mg / kg, 3 mg / kg, 10 mg / kg) and control antibodies (10 mg / kg) were injected twice weekly for two weeks. Animals were monitored for tumor growth and body weight over time. As shown in Figure 15, W3162-1.146.19-Z12 significantly inhibits tumor growth in a dose-dependent manner. At the 1 mg / kg dose, 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 inhibited tumor volume to 160 mm3 on day 19, while at 10 mg / kg, W3162-1.146.19-Z12 induced tumor regression at the end of the study period. 2.8 Epitope Mapping

[00231] Alanine screening was used to identify the CTLA-4 epitope of the antibodies. In this experiment, alanine residues in hCTLA-4 were mutated to glycine residues, and all other residues were mutated to alanines. For each residue of the extracellular domain (ECD) of human CTLA-4, amino acid point substitutions were made using two sequential PCR steps. A pcDNA3.3-hCTLA4_ECD.His plasmid encoding the ECD of human CTLA-4 and a C-terminal His marker were used as a template, and a set of mutagenic primers was used for the first PCR step using the QuikChange Lightning multi-site targeted mutagenesis kit (Agilent Technologies, Palo Alto, CA). The Dpn I endonuclease was used to digest the parental template after the mutant strand synthesis reaction. In the second-step PCR, the linear DNA expression cassette, composed of a CMV promoter, CTLA-4 mutant ECD, a His-marker, and thymidine polyadenylation, was used. Petition 870240025544, dated 03 / 25 / 2024, pages 56 / 68 Herpes simplex virus 49 / 52 kinase (TK) was amplified and transiently expressed in HEK293F cells (Life Technologies, Gaithersburg, MD). Additionally, three plasmid vectors were constructed to test the epitope of the glycans: pcDNA3,3-hCTLA4_ECD.His (N113Q), pcDNA3.3-hCTLA4_ECD.His (N145Q), and pcDNA3.3-hCTLA4_ECD.His (N113Q, N145Q). These three muteins were transiently expressed in HEK293F cells (Life Technologies, Gaithersburg, MD).

[00232] In order to test how mutations affect antibody binding, a capture ELISA was conducted. Briefly, the monoclonal antibodies Ipilimumab, W3162-1.146.19-z12 and W3162-1.154.8-z35 (2 pg / mL) were captured and pre-coated with 2 pg / mL of anti-human-goat Fc IgG (Bethyl Laboratories, Montgomery, TX) in plates. After interacting with the supernatant containing quantified CTLA-4 mutants, HRP-conjugated anti-His antibody (1:5000; Rockland Immunochemicals, Pottstown, PA) was added as a detection antibody. TMB was used as the HRP substance. Absorbance was normalized to the mean of the control mutants. After defining an additional cutoff for the "foldchange" of the linkage (<0.55), certain terminal epitope residues were identified.

[00233] The binding activities of antibodies W3162-1.146.19z12, W3162-1.154.8-z35 and ipilimumab (W316-BMK1) to human CTLA-4 were conducted, all three antibodies were found to bind to human CTLA-4 (Figure 17).

[00234] The tested point mutations affecting antibody binding to CTLA-4 are shown in Table 11. According to the crystal structures of human CTLA-4 (PDB code 1AH1), some amino acid residues (e.g., Met38, Val40, Tyr60, Val71, Val73, Arg75, Val84, Cys85, Cys129, Ile149) would likely not directly contact any antibody. Binding reductions Petition 870240025544, dated 03 / 25 / 2024, pp. 57 / 68 The 50 / 52 observed likely resulted from instability or even collapse of the CTLA-4 structure after alanine substitutions. The determined final epitope residues are listed in Table 12 and marked in Figure 18.

[00235] As shown in Figures 18 D and E, the epitopes of Ipilimumab and W3162-1146.19-z12 overlap, except for some residues, such as N145 and P138. In comparison, W3162-1154.8-z35 bound to a smaller area of ​​CTLA-4 (Figure 18F) than the other two antibodies. All three antibodies bound to the ligand-binding domain of CTLA-4 (Figure 18 A and B), which involves the MYPPPY motif.

[00236] The overlapping epitopes of Ipilimumab and W3162-1.146.19z12 did not explain the unique cross-species binding of the W3162-1.146.19-z12 antibody. Since the N145 mutation in CTLA-4 only affected the binding of W3162-1.146.19-z12 to CTLA-4, not affecting the other two antibodies, the N-glycosylation sites were also observed as potential epitopes. The effect of mutations in two CTLA-4 glycosylation sites on antibody binding activity is shown in Figure 17. The binding of Ipilimumab or W3162-1.154.8-z35 to mutated CTLA4 was not significantly altered (Figure 17 A and C). In contrast, the binding of W3162-1.146.19-z12 to mutated CTLA-4 N145Q was significantly reduced, while the binding of this antibody to CTLA-4 N113Q was not altered. This data set indicates that the glycan (Figure 18E) in CTLA-4 N145 could be the epitope of W3162-1.146.19-z12. The N145 residue is conserved in cynomolgus monkey and mouse CTLA-4.

[00237] The description of the present invention has been given above by way of example. However, it is understood by those skilled in the art that the present invention is not limited to the examples. The invention can be embodied in other specific forms without deviation from the spirit or scope of the invention. Petition 870240025544, dated 03 / 25 / 2024, pp. 58 / 68 51 / 52 its essential characteristics. The scope of the invention is further indicated by the appended claims and not by the preceding description, and all modifications that fall within the meaning and range of equivalence of the claims shall be adopted herein. Table 11. The effect of CTLA-4 point mutations on antibody binding. Ipilimumab W3162-1.146.19-z12-IgG1K W3162-1.154.8-z35-IgG1K CTLA4 Residue Position aFoldchange SD CTLA4 Residue Position *aFoldchange SD CTLA4 Residue Position aFoldchange SD P 136 0.191 0.00 G 146 0.166 0.00 P 136 0.155 0.00 V 40 0.201 0.00 V 40 0.181 0.00 V 40 0.187 0.00 G 146 0.208 0.00 C 129 0.182 0.00 G 146 0.201 0.00 C 129 0.210 0.00 M 38 0.182 0.00 C 129 0.211 0.00 C 85 0.229 0.00 I 149 0.183 0.00 C 85 0.218 0.00 V 84 0.233 0.01 V 81 0.191 0.01 V 84 0.232 0.00 Y 60 0.236 0.04 N* 145 0.196 0.00 M 38 0.238 0.00 M 134 0.240 0.01 Q 76 0.200 0.00 Y 60 0.240 0.07 I 149 0.244 0.01V 84 0.201 0.00 R 75 0.298 0.00 M 38 0.253 0.02 Y 60 0.223 0.05 I 149 0.319 0.00 V 81 0.268 0.03 P 136 0.231 0.00 P 138 0.339 0.00 R 75 0.273 0.00 M 134 0.254 0.00 V 71 0.347 0.10 I 143 0.278 0.00 I 128 0.256 0.00 K 65 0.351 0.00 I 128 0.286 0.00 C 85 0.256 0.03T 88 0.433 0.02 V 71 0.335 0.08 I 143 0.264 0.00 M 134 0.455 0.00 K 130 0.364 0.00 R 75 0.278 0.00 E 83 0.479 0.00 G 142 0.369 0.01 V 73 0.338 0,01 V 73 0,498 0,01 G 144 0,375 0,00 V 71 0,343 0,06 R 70 0,515 0,04 T 88 0,377 0,06 K 130 0,348 0,00 A 66 0,519 0,02 R 70 0,378 0,10 A 66 0,395 0,02 L 74 0,520 0,00 V 73 0,380 0,00 L 74 0,398 0,00 P 137 0,534 0,00 A 66 0,395 0,03 L 126 0,398 0,02 G 64 0,543 0,10 E 83 0,396 0,02 E 83 0,405 0,01 H 39 0,549 0,01 A 86 0,401 0,03 T 88 0,423 0,06 P 137 0,407 0,00 P 137 0,425 0,00 L 74 0,410 0,00 Y 139 0,431 0,00 L 126 0,412 0,01 T 82 0,436 0,00 H 39 0,420 0,01 P 138 0,445 0,00 Y 139 0,434 0,00 E 132 0,457 0,01 T 82 0,450 0,01 G 144 0,478 0,00 E 132 0,470 0,04 T 147 0,486 0,03 M 90 0,479 0,02 L 141 0,486 0,00 T 147 0,508 0,03 M 90 0,492 0,00 Y 127 0,508 0,01 Q 148 0,500 0,02 Q 80 0,517 0,02 V 131 0,504 0,02 L 141 0,518 0,03 R 70 0,507 0,08 E 68 0,529 0,00 Y 89 0,512 0,05 Q 148 0,532 0,04 Y 127 0,529 0,02, Petição 870240025544, de 25 / 03 / 2024, pág. 59 / 68 52 / 52 Ipilimumab W3162-1.146.19-z12-lgG1K W3162-1.154.8-z35-lgG1K CTLA4 aFold- Residue Position change SD CTLA4 aFo / d- Residue Position change SD CTLA4 aFold- Residue Position change SD G 125 0.537 0.02 Q 76 0.545 0.00 G 92 0.548 0.00 H 39 0.529 0.00 T 72 0.541 0.01 G 142 0.543 0.02 C 103 0.544 0.03 The fold-change in linkage refers to the linkage of various silent alanine substitutions. Table 12. Identified epitopes of three antibodies Ipilimumab W3162 1.146.19-1 MsGlK W316- -l.lF4.B-E3F-IgGlK CTLA4 CTLA4 CTLA4 Portion Location Position Locotiou Positioii Location Residue Residue Residue H 39 A Ή 39 AH 39 AA ¢6 BC loop A 66 El C loop G 64 El C loop E <58 CR 70 CK 65 BC loop X 70 c T 72 CA 66 BC loop L 74 c L 74 CX 70 CQ 76 c Q 76 CL 74 c Q SO c V SI C' E £3 c V SI c T E2 C' I BE c T E2 c E S3 c M 134 FE S3 c T SS c P 136 FG loop A E6 c M 90 CC loop P 137 FG loop T E8 c G 92 CP 138 FG loop M 90 CC loop L 126 FG 146 GG 92 CI 128 FG 125 f K 130 FL 126 f E 132 F 1 128 f 134 FK 130 f I 136 FG loop E 132 f P 137 FG loop M 134 FP 138 FG loop P 136 EG loop y 139 FG loop P 137 FG loop L 141 GY 139 FG loop G 142 GL 141 GI 143 GG 142 GG 144 G 1 143 G 14? GG 144 GG 146 GG 146 GQ 148 GQ 148 G Table legend 12 : CTLA-4 residue - Position - Localization

Claims

1. An antibody or antigen-binding fragment thereof, characterized in that it comprises a variable light chain region comprising CDR1, CDR2 and CDR3 sequences and a variable heavy chain region comprising CDR1, CDR2 and CDR3 sequences; and a variable light chain region comprising a light chain complementarity-determining region 1 (LCDR1) defined by SEQ ID NO: 41, LCDR2 defined by SEQ ID NO: 32 and LCDR3 defined by SEQ ID NO: 22, and a variable heavy chain region comprising a heavy chain complementarity-determining region 1 (HCDR1) defined by SEQ ID NO: 36, HCDR2 defined by SEQ ID NO: 28 and HCDR3 defined by SEQ ID NO: 18; wherein the antibody or antigen-binding fragment binds specifically to CTLA-4.

2. Antibody or antigen-binding fragment thereof, according to claim 1, characterized in that the variable region of the light chain has the amino acid sequence SEQ ID NO:

14.

3. Antibody or antigen-binding fragment thereof, according to claim 1 or 2, characterized in that the variable region of the heavy chain has the amino acid sequence SEQ ID NO:

7.

4. Antibody or antigen-binding fragment thereof, according to claim 1, characterized in that the variable region of the light chain has the amino acid sequence SEQ ID NO: 14 and the variable region of the heavy chain has the amino acid sequence SEQ ID NO:

7.

5. Antibody or antigen-binding fragment thereof, according to any one of claims 1 to 4, characterized in that the antibody inhibits the binding of CTLA-4 to CD80 or CD86.

6. Antibody or antigen-binding fragment thereof, according to any one of claims 1 to 5, characterized in that the antibody-binding epitope or antigen-binding fragment comprises N145, polysaccharide in N145 or P138 of CTLA-4.

7. Antibody or antigen-binding fragment thereof, according to any one of claims 1 to 6, characterized in that the antibody or antigen-binding fragment a) binds to human CTLA-4 with a KD of 4.77E-10 M or less; and b) binds to mouse CTLA-4 with a KD of 1.39E-09 M or less.

8. An antibody or antigen-binding fragment thereof, according to any one of claims 1 to 7, characterized in that the antibody is a chimeric, humanized, fully human or mouse antibody.

9. Pharmaceutical composition, characterized in that it comprises the antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 8, and one or more pharmaceutically acceptable excipients, diluents and carriers.

10. Immunoconjugate, characterized in that it comprises the antibody or an antigen-binding fragment thereof, as defined in any one of claims 1 to 8, linked to a therapeutic agent.

11. Method for preparing an anti-CTLA-4 antibody or an antigen-binding fragment thereof, characterized in that it comprises: (a) providing the antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 8; and (b) expressing the altered antibody sequence as a protein.

12. Antibody or antigen-binding fragment thereof, according to any one of claims 1 to 8, characterized in that it is for use as a medicament in modulating the immune response in an individual.

13. Use of the antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 8, characterized in that it is for the production of a medicament in inhibiting the growth of tumor cells. Petition 870260061236, dated 06 / 22 / 2026, page 14 / 21 3 / 3 14. Use of the antibody or antigen-binding fragment thereof, according to claim 13, characterized in that the tumor cells are from a selected cancer from a group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, and rectal cancer.

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