Combination therapy for the treatment of cancer

Abstract

Disclosed herein is a method for treating cancer in a human subject, the method comprising administering to the subject: i) an antibody-drug conjugate (ADC) comprising an antibody, a cleavable linker, and a cytotoxic agent; and ii) a bispecific checkpoint inhibitor. Further disclosed herein is a kit comprising: i) an antibody-drug conjugate (ADC) comprising an antibody, a cleavable linker, and a cytotoxic agent; and ii) a bispecific checkpoint inhibitor.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of priority of U.S. Provisional Application No. 63 / 369,718, filed Jul. 28, 2022, which is incorporated herein by reference in its entirety.

[0002] (Incorporation by Reference of Electronically Submitted Materials) A computer - readable nucleotide / amino acid sequence list, identified as one 137,421 - byte file named "ADCIO - 101 - WO - PCT.xml" created on Jul. 24, 2023 and submitted simultaneously with this specification, is incorporated herein by reference in its entirety.

[0003] The present disclosure provides a method of treating cancer in a human subject, including administering to the human subject an antibody - drug conjugate (ADC) and a bispecific binding protein. The present disclosure further provides a kit including an ADC and a bispecific binding protein.

Background Art

[0004] Surgery, radiotherapy, and chemotherapy are the most traditional and widely used treatment methods for cancer, either alone or in combination. These treatment methods are effective in removing or killing cancer cells, but often cause undesirable side effects such as hair loss, anemia, severe nausea, and the death of healthy cells in the patients being treated. These limitations present an urgent need for innovative and less harmful treatment methods for cancer. Antibody-based cancer therapy relies on the recognition and binding of antibody-drug conjugates to specific proteins on cancer cells. Antibody-drug conjugates (ADCs) can utilize the specificity of the antibody portion of the conjugate to directly deliver highly toxic agents to the cells to be killed. Using antibody or ADC cancer therapy in combination with other small molecule-based cancer therapies can improve treatment outcomes by attacking malignant cells and tumors in more than one way.

Summary of the Invention

[0005] The present disclosure provides a method of treating cancer in a human subject in need thereof, the method comprising administering to the human subject a) an antibody-drug conjugate (ADC) comprising i) an antibody or an antigen-binding fragment thereof that binds to a B7-H4 polypeptide, ii) a cleavable linker, and iii) a cytotoxic agent, and b) a bispecific binding protein having a first binding domain that specifically binds to Programmed cell death protein 1 (PD-1).

[0006] In some embodiments, the present disclosure provides a method of treating cancer in a human subject in need thereof, the method comprising administering to the human subject an antibody-drug conjugate (ADC) comprising a) i) an antibody or an antigen-binding fragment thereof, ii) a cleavable linker, and iii) a cytotoxic agent, wherein the cytotoxic agent is a compound of formula I:

[0007]

Chemical formula

[0008] [Chemical formula] (wherein Q is

[0009] [Chemical formula] and Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue, X is

[0010] [Chemical formula] and a = 0 to 5, b1 = 0 to 16, b2 = 0 to 16, c1 = 0 or 1, c2 = 0 or 1, d = 0 to 5, at least b1 or b2 = 0, and at least c1 or c2 = 0, G L is a linker for connecting to an antibody or antigen-binding fragment), and

[0011] [Chemical formula] (wherein R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached, form a cyclopropylene or cyclobutylene group, and e is 0 or 1), an ADC selected therefrom, and b) administering a bispecific binding protein having a first binding domain that specifically binds to PD-1.

[0012] In some embodiments of the method, the antibody or antigen-binding fragment thereof of the ADC comprises, respectively, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or functional variants thereof; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or functional variants thereof; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, or functional variants thereof; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, or functional variants thereof; or HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, or functional variants thereof, or HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in the first binding arm having the amino acid sequences of SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, and SEQ ID NO: 121, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in the second binding arm having the amino acid sequences of SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127.

[0013] In some aspects of the method, the antibody or antigen-binding fragment thereof of the ADC comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. In some aspects of the method, the antibody or antigen-binding fragment thereof of the ADC comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 within the first binding arm, each comprising the amino acid sequences of SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, and SEQ ID NO: 121, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 within the second binding arm, each comprising the amino acid sequences of SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127, and is a bispecific antibody or antigen-binding fragment thereof.In some aspects of the method, the antibody or antigen-binding fragment thereof of the ADC comprises a variable heavy (VH) chain and a variable light (VL) chain, or a functional variant thereof, each comprising the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 34, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO: 34, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 34, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequences of SEQ ID NO: 46 and SEQ ID NO: 34, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequences of SEQ ID NO: 47 and SEQ ID NO: 34, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 32, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 36, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 38, or a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 40, or a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequences of SEQ ID NO: 128 and SEQ ID NO: 130, within the first binding arm, and a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequences of SEQ ID NO: 132 and SEQ ID NO: 134, within the second binding arm.

[0014] In some aspects of the method, the antibody or antigen-binding fragment thereof of the ADC comprises a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 34. In some aspects of the method, the antibody or antigen-binding fragment thereof of the ADC binds to the OVCAR4 cell line. In some aspects of the method, the antibody or antigen-binding fragment thereof of the ADC comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 41.

[0015] In some embodiments of the method, the antibody or antigen-binding fragment thereof of the ADC is a bispecific antibody or antigen-binding fragment thereof, and includes a VH chain and a VL chain within a first binding arm, or functional variants thereof, each including the amino acid sequences of SEQ ID NO: 128 and SEQ ID NO: 130, and a VH chain and a VL chain within a second binding arm, or functional variants thereof, each including the amino acid sequences of SEQ ID NO: 132 and SEQ ID NO: 134.

[0016] In some embodiments of the method, the antibody or antigen-binding fragment thereof of the ADC includes a heavy chain and a light chain within a first binding arm, or functional variants thereof, each including the amino acid sequences of SEQ ID NO: 129 and SEQ ID NO: 131, and a heavy chain and a light chain within a second binding arm, or functional variants thereof, each including the amino acid sequences of SEQ ID NO: 133 and SEQ ID NO: 135.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC includes a heavy chain constant region including the amino acid sequence of SEQ ID NO: 52. In some embodiments, the antibody or antigen-binding fragment thereof of the ADC includes a light chain constant region including the amino acid sequence of SEQ ID NO: 42. In some embodiments, the antibody or antigen-binding fragment thereof of the ADC includes a heavy chain including the amino acid sequence of SEQ ID NO: 51 and a light chain including the amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody or antigen-binding fragment thereof of the ADC includes a heavy chain including the amino acid sequence of SEQ ID NO: 48 and a light chain including the amino acid sequence of SEQ ID NO: 44.

[0018] In some embodiments of the method, the antibody or antigen-binding fragment thereof of the ADC is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment thereof of the ADC is a humanized monoclonal antibody. In some embodiments of the method, the cleavable linker of the ADC is an mp-PEG8-val-ala linker. In some embodiments of the method, the cytotoxic agent of the ADC is a topoisomerase inhibitor (TOP1i), a tubulysin derivative, a pyrrolobenzodiazepine, or a combination thereof. In some embodiments, the cytotoxic agent of the ADC is a topoisomerase inhibitor.

[0019] In some embodiments, the cytotoxic agent is a topoisomerase inhibitor, and the ii) cleavable linker and iii) cytotoxic agent of the ADC are a compound of formula I:

[0020]

Chemical formula

[0021]

Chemical formula

[0022]

Chemical formula

[0023]

Chemical formula

[0024] [Chemical formula] (wherein R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached, form a cyclopropylene or cyclobutylene group, and e is 0 or 1).

[0025] In some embodiments, R L is of formula Ia.

[0026] In some embodiments, Q is (a) an amino acid residue selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, or (b) NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH Ala-Lys- C=O , NH -Val-Cit- C=O , NH -Phe-Cit- C=O , NH -Leu-Cit- C=O , NH -Ile-Cit- C=O , NH -Phe-Arg- C=O , NH -Trp-Cit- C=O , and NH -Gly-Val- C=O selected from the dipeptide residues, or (c) NH -Glu-Val-Ala- C=O , NH -Glu-Val-Cit- C=O ,NH -αGlu-Val-Ala- C=O 、and NH -αGlu-Val-Cit- C=O a tripeptide residue selected from, or (d) NH -Gly-Gly-Phe-Gly C=O 、and NH -Gly-Phe-Gly-Gly C=O a tetrapeptide residue selected from.

[0027] In some embodiments, a is (a) 0 to 3, or (b) 0 or 1, or (c) 0.

[0028] In some embodiments, b1 is (a) 0 to 8, or (b) 0, or (c) 2, or (d) 3, or (e) 4, or (f) 5, or (g) 8.

[0029] In some embodiments, b2 is (a) 0 to 8, or (b) 0, or (c) 2, or (d) 3, or (e) 4, or (f) 5, or (g) 8.

[0030] In some embodiments, (a) c1 is 0 or 1, (b) c2 is 0 or 1, and (c) at least one of c1 and c2 is 0.

[0031] In some embodiments, d is (a) 0 to 3, or (b) 1 or 2, or (c) 2, or (d) 5.

[0032] In some embodiments, (a) a is 0, b1 is 0, c1 is 1, c2 is 0, d is 2, and b2 is 0, 2, 3, 4, 5, or 8, or (b) a is 1, b2 is 0, c1 is 0, c2 is 0, d is 0, and b1 is 0, 2, 3, 4, 5, or 8, or (c) a is 0, b1 is 0, c1 is 0, c2 is 0, d is 1, and b2 is 0, 2, 3, 4, 5, or 8, or (d) b1 is 0, b2 is 0, c1 is 0, c2 is 0, one of a and d is 0, and the other of a and d is 1 or 5, or (e) a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 is 0, 2, 3, 4, 5, or 8.

[0033] In some embodiments, G L is selected from the following,

[0034] [Table 1] wherein Ar represents an arylene group, and X represents an alkyl. 5~6 wherein Ar represents an arylene group, and X represents an alkyl. 1~4 represents an alkyl.

[0035] In some embodiments, G L is L1-1 and G L1-2 selected from.

[0036] In some embodiments, R L is of formula Ib, (a) both R L1 and R L2 are H, or (b) R L1 is H and R L2 is methyl, or (c) both R L1 and R L2 are methyl, or (d) R L1 and R L2 together with the carbon atom to which they are attached form a cyclopropylene group, or (e) R L1 and R L2 together with the carbon atom to which they are attached form a cyclobutylene group.

[0037] In some embodiments, the ADC is of Formula IV: L-(D L ) p (IV) or a pharmaceutically acceptable salt or solvate thereof, wherein L is i) an antibody or an antigen-binding fragment thereof, and D L is ii) a cleavable linker and iii) a cytotoxic agent-containing drug linker unit, and D L is of Formula III,

[0038]

Chemical Formula

[0039]

Chemical Formula

[0040]

Chemical Formula

[0041] In some embodiments, G LL is selected from the following,

[0042]

Table 2

[0043] In some embodiments, G LL is G LL1-1 and G LL1-2 is selected from.

[0044] In some embodiments of the method, the drug loading (p) of the cytotoxic agent with respect to the antibody or antibody binding fragment is an integer from 1 to about 10.

[0045] In some embodiments of the method, the topoisomerase inhibitor is a compound of formula A as a single enantiomer or in an enantiomerically enriched form:

[0046]

Chemical formula

[0047] In some embodiments of the method, the topoisomerase inhibitor is a compound having formula VI,

[0048]

Chemical formula

[0049] In some embodiments of the method, ii) the linker and iii) the cytotoxic agent together comprise the following compound:

[0050]

Chemical formula

[0051] In some aspects of the method, the bispecific binding protein comprises: a) a first binding domain that specifically binds to PD-1; and b) a second binding domain that specifically binds to a T cell immunoreceptor with Ig and ITIM domains (TIGIT), T-cell immunoglobulin and mucin domain containing protein-3 (TIM-3), or cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). In some aspects of the method, the first binding domain comprises a heavy chain variable domain comprising HCDR1 having the amino acid sequence of SEQ ID NO: 56, HCDR2 having the amino acid sequence of SEQ ID NO: 57, and HCDR3 having the amino acid sequence of SEQ ID NO: 58, and a light chain variable domain comprising LCDR1 having the amino acid sequence of SEQ ID NO: 59, LCDR2 having the amino acid sequence of SEQ ID NO: 60, and LCDR3 having the amino acid sequence of SEQ ID NO: 61.

[0052] In some embodiments, the first binding domain that specifically binds to PD-1 comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 62 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 64. In some embodiments, the first binding domain that specifically binds to PD-1 comprises a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 64. In some embodiments, the first binding domain that specifically binds to PD-1 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 63 and a light chain having the amino acid sequence of SEQ ID NO: 65. In some embodiments, the first binding domain that specifically binds to PD-1 comprises a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 63 and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 65. In some embodiments of the method, the second binding domain that specifically binds to TIGIT comprises a heavy chain variable domain comprising HCDR1 having the amino acid sequence of SEQ ID NO: 66, HCDR2 having the amino acid sequence of SEQ ID NO: 67, and HCDR3 having the amino acid sequence of SEQ ID NO: 68, and a light chain variable domain comprising LCDR1 having the amino acid sequence of SEQ ID NO: 69, LCDR2 having the amino acid sequence of SEQ ID NO: 70, and LCDR3 having the amino acid sequence of SEQ ID NO: 71. In some embodiments, the second binding domain that specifically binds to TIGIT comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 72 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 74. In some embodiments, the second binding domain that specifically binds to TIGIT comprises a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 72 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the second binding domain that specifically binds to TIGIT comprises a heavy chain having the amino acid sequence of SEQ ID NO: 73 and a light chain having the amino acid sequence of SEQ ID NO: 75.

[0053] In some embodiments, the second binding domain that specifically binds to TIGIT comprises a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 73, and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 75.

[0054] In some embodiments, the second binding domain specifically binds to the C’C’’ and DE loops of the immunoglobulin variable (IgV) domain of TIM-3, or binds to the PS binding groove (FG and CC’ loops) of the IgV domain of TIM-3. In some embodiments, the second binding domain comprises CDR: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NOs: 84, 85, 86, 87, 88, and 89, respectively, or SEQ ID NOs: 84, 85, 86, 87, 88, and 90, respectively.

[0055] In some embodiments, the second binding domain specifically binds to an epitope on the IgV domain of TIM-3, and the epitope comprises N12, L47, R52, D53, V54, N55, Y56, W57, W62, L63, N64, G65, D66, F67, R68, K69, D71, T75, and E77 of TIM-3 (SEQ ID NO: 102).

[0056] In some embodiments, the first binding domain comprises a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each comprising the amino acid sequences of SEQ ID NOs: 56, 57, 58, 59, 60, and 61, respectively. In some embodiments, the first binding domain comprises a first heavy chain VH comprising the amino acid sequence of SEQ ID NO: 62 and a first light chain VL comprising the amino acid sequence of SEQ ID NO: 64, and the second binding domain comprises a second heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 91 and a second light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 94. In some embodiments, the first binding domain comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 63 and a first light chain comprising the amino acid sequence of SEQ ID NO: 65, and the second binding domain comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a second light chain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments of the method, the second binding domain specifically binds to CTLA-4. In some embodiments, the bispecific binding protein binds to human PD-1 and CTLA-4. In some embodiments, the bispecific binding protein comprises anti-PD-1 and anti-CTLA-4 heavy chain variable region (VH) CDR1, VH CDR2, VH CDR3, light chain variable region (VL) CDR1, VL CDR2, and VL CDR3 of the sequence of MEDI5752. In some embodiments, the second binding domain comprises VH CDR1 comprising the amino acid sequence of SEQ ID NO: 109, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 110, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 111, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 112, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 113, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 114. In some embodiments, the bispecific checkpoint inhibitor comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 105 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 104.

[0057] In some embodiments, the bispecific binding protein comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 106, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 107, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 108, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 59, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 61; and (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 109, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 110, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 111, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 112, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 113, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 114.

[0058] In some embodiments, the bispecific binding protein comprises (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 63 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 65; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 105 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 104.

[0059] In some embodiments of the method, the bispecific binding protein comprises an unglycosylated Fc region. In some embodiments, the bispecific binding protein comprises a deglycosylated Fc region. In some embodiments, the bispecific binding protein comprises an Fc region having reduced fucosylation or no fucosylation. In some embodiments, the bispecific binding protein is a humanized bispecific antibody or an antigen-binding fragment thereof. In some embodiments, the bispecific binding protein comprises an IgG heavy chain constant region. In some embodiments, the constant region comprises mutations at L234F, L235E, and P331S. In some embodiments, the IgG heavy chain constant region is an IgG1 heavy chain constant region. In some embodiments, the bispecific binding protein is an antibody. In some embodiments, the bispecific binding protein is a full-length antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is human or humanized. In some embodiments, the bispecific binding protein comprises a kappa light chain constant region. In some embodiments, the bispecific binding protein comprises a lambda light chain constant region.

[0060] In some embodiments of the method, the cancer comprises cancer cells that express a B7-H4 polypeptide. In some embodiments, the cancer is selected from ovarian cancer, breast cancer, uterine cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, renal cell cancer, pancreatic cancer, prostate cancer, cervical cancer, blood cancer, endometrial cancer, cholangiocarcinoma, NSCLC (squamous and / or adenocarcinoma), gastrointestinal cancers such as gastric cancer and colorectal cancer, and lung cancer. In some embodiments, the cancer is breast cancer selected from hormone receptor-positive (HR+) breast cancer, human epidermal growth factor receptor 2 positive (HER2+) breast cancer, and triple negative breast cancer (TNBC). In some embodiments, the cancer is homologous recombination deficient (HRD) cancer. In some embodiments, the cancer comprises one or more cells having a mutation in an HRD gene selected from BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L. In some embodiments, the mutant HRD gene is selected from BRCA1, BRCA2, and ATM.

[0061] The present disclosure also provides a pharmaceutical composition comprising 1) an antibody-drug conjugate and 2) a bispecific checkpoint inhibitor, wherein the antibody-drug conjugate comprises an antibody or an antigen-binding fragment thereof and a drug linker represented by the following formula:

[0062] [Chemical formula] and, The drug linker is conjugated to an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or an antigen-binding fragment thereof is an anti-B7H4 antibody. In some embodiments, the antibody or an antigen-binding fragment thereof binds to B7-H4 and comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a VH chain and a VL chain, or functional variants thereof, which comprise the amino acid sequences of SEQ ID NO:45 and SEQ ID NO:34, respectively. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:51 and a light chain comprising the amino acid sequence of SEQ ID NO:44.

[0063] In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:48 and a light chain comprising the amino acid sequence of SEQ ID NO:44. In some embodiments, the bispecific checkpoint inhibitor comprises: a) a first binding domain that specifically binds to PD-1 or PD-L1; and b) a second binding domain that specifically binds to a T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3), or cytotoxic T lymphocyte-associated antigen-4 (CTLA 4). In some embodiments, the first binding domain comprises a heavy chain variable domain that specifically binds to PD-1 and comprises HCDR1 having the amino acid sequence of SEQ ID NO:56, HCDR2 having the amino acid sequence of SEQ ID NO:57, and HCDR3 having the amino acid sequence of SEQ ID NO:58, and a light chain variable domain that comprises LCDR1 having the amino acid sequence of SEQ ID NO:59, LCDR2 having the amino acid sequence of SEQ ID NO:60, and LCDR3 having the amino acid sequence of SEQ ID NO:61.

[0064] In some embodiments, the first binding domain specifically binds to PD-1 and includes a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 62 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 64. In some embodiments, the first binding domain specifically binds to PD-1 and includes a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 62 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 64. In some embodiments, the first binding domain specifically binds to PD-1 and includes a heavy chain having the amino acid sequence of SEQ ID NO: 63 and a light chain having the amino acid sequence of SEQ ID NO: 65. In some embodiments, the first binding domain specifically binds to PD-1 and includes a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 63 and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 65.

[0065] In some embodiments, the second binding domain specifically binds to TIGIT and includes a heavy chain variable domain including HCDR1 having the amino acid sequence of SEQ ID NO: 66, HCDR2 having the amino acid sequence of SEQ ID NO: 67, and HCDR3 having the amino acid sequence of SEQ ID NO: 68, and a light chain variable domain including LCDR1 having the amino acid sequence of SEQ ID NO: 69, LCDR2 having the amino acid sequence of SEQ ID NO: 70, and LCDR3 having the amino acid sequence of SEQ ID NO: 71. In some embodiments, the second binding domain specifically binds to TIGIT and includes a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 72 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 74. In some embodiments, the second binding domain specifically binds to TIGIT and includes a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 72 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 74.

[0066] In some embodiments, the second binding domain specifically binds to TIGIT and comprises a heavy chain having the amino acid sequence of SEQ ID NO: 73 and a light chain having the amino acid sequence of SEQ ID NO: 75. In some embodiments, the second binding domain specifically binds to TIGIT and comprises a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 73 and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the second binding domain specifically binds to TIM-3. In some embodiments, the second binding domain specifically binds to TIM-3, and the second binding domain specifically binds to the C’C’’ and DE loops of the immunoglobulin variable (IgV) domain of TIM-3 or binds to the PS binding groove (FG and CC’ loops) of the IgV domain of TIM-3.

[0067] In some embodiments, the second binding domain comprises Complementarity-Determining Regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each comprising the amino acid sequences of SEQ ID NOs: 84, 85, 86, 87, 88, and 89, or each comprising the amino acid sequences of SEQ ID NOs: 84, 85, 86, 87, 88, and 90. In some embodiments, the second binding domain specifically binds to an epitope on the IgV domain of TIM-3, and the epitope comprises N12, L47, R52, D53, V54, N55, Y56, W57, W62, L63, N64, G65, D66, F67, R68, K69, D71, T75, and E77 of TIM-3 (SEQ ID NO: 102). In some embodiments, the second binding domain comprises a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 91 and a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 94.

[0068] In some embodiments, the second binding domain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the second binding domain specifically binds to CTLA-4. In some embodiments, the second binding domain comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 109, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 110, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 111, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 112, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 113, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 114. In some embodiments, the bispecific checkpoint inhibitor comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 105 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 104.

[0069] The present disclosure also encompasses kits containing any of the above pharmaceutical compositions. The present disclosure also encompasses the use of an antibody-drug conjugate in the manufacture of a medicament for use in combination with a bispecific checkpoint inhibitor, wherein the antibody-drug conjugate and the bispecific checkpoint inhibitor are as defined herein for treating cancer. The present disclosure also encompasses the use of a bispecific checkpoint inhibitor in the manufacture of a medicament for use in combination with an antibody-drug conjugate, wherein the bispecific checkpoint inhibitor and the antibody-drug conjugate are as defined herein for treating cancer. In some embodiments, the pharmaceutical composition is for use in the treatment of cancer. In some embodiments, the cancer is selected from ovarian cancer, breast cancer, uterine cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, renal cell cancer, pancreatic cancer, prostate cancer, cervical cancer, blood cancer, endometrial cancer, cholangiocarcinoma, NSCLC (squamous and / or adenocarcinoma), gastrointestinal cancers such as gastric cancer and colorectal cancer, and lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] The following drawings form a part of this specification and are included to further demonstrate exemplary embodiments of certain aspects of the present disclosure.

Figure 1A

Figure 1B

Figure 1C

Figure 2A

Figure 2B

Figure 2C

Figure 3A

Figure 3B

Figure 3C

Figure 4A

Figure 4B

Figure 4C

Figure 5A

Figure 5B

Figure 5C

Figure 6

Figure 7A

Figure 7B

Figure 8A

Figure 8B

Figure 8C

Figure 8D

Figure 9A

Figure 9B

Mode for Carrying Out the Invention

[0071] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular.

[0072] As used herein, "a" or "an" can mean one or more. As used herein, when used in conjunction with the word "comprising", the word "a" or "an" can mean one or more than one. As used herein, "another" or "further" can mean at least a second or more.

[0073] The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer only to alternatives or the alternatives are mutually exclusive, but this disclosure supports definitions that refer only to alternatives as well as "and / or".

[0074] As used herein, "comprising" (and any variations or forms of "comprising" such as "comprise" and "comprises"), "having" (and any variations or forms of "having" such as "have" and "has"), "including" (and any variations or forms of "including" such as "includes" and "include"), or "containing" (and any variations or forms of "containing" such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0075] Throughout this application, the term "about" is used to indicate that a value includes the variability of the inherent error of the method / device employed to determine that value, or the variability that exists between subjects under study. Typically, the term "about" is intended to include variability of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or less, or higher variability (either "above" or "below" the stated value), depending on the circumstances. In some embodiments, one of ordinary skill in the art will understand the level of variability indicated by the term "about" due to the context in which it is used herein. It should also be understood that the use of the term "about" includes the specifically recited values as well.

[0076] The use of the term "for example" and its corresponding abbreviation "e.g." (whether italicized or not) means that the specific terms described are representative examples and embodiments of the present disclosure not intended to be limited to the specific examples referenced or cited, unless otherwise expressly stated.

[0077] Any range provided in this specification includes all values within the specified range and values around the endpoints of the specified range. As used herein, "~(between)" is a range that includes the endpoints of the range. For example, a number from x~y explicitly includes the numbers x and y, and any number that falls within x and y.

[0078] The term "treating" or "treatment" refers to administering a compound or pharmaceutical composition to an animal to effect a change or improvement in a disease, disorder, or condition in the animal. In some embodiments, the methods of treatment of the present disclosure include one or more administration steps selected from oral, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal, inhalation, topical, or combinations thereof. In preferred embodiments, the administration is intravenous or intraarterial (e.g., by injection or infusion), or a combination thereof.

[0079] The term "dosage" means a specified amount of a compound or pharmaceutical provided in a single administration or over a specified period. In some embodiments, the dosage can be administered in more than one bolus, tablet, or injection. For example, in some embodiments, if subcutaneous administration is desired, the desired dosage may require a volume that cannot be easily accommodated in a single injection. In such embodiments, more than one injection can be used to achieve the desired dosage. In some embodiments, the dosage can be administered in more than one injection to minimize injection site reactions in an individual. In other embodiments, the compound or pharmaceutical is administered over a long period or by continuous infusion. The dosage can be described as the amount of the pharmaceutical per hour, per day, per week, or per month.

[0080] The terms "subject", "individual", and "patient" are used interchangeably herein to refer to a mammalian subject. In one embodiment, the "subject" is a human, domestic animal, farm animal, sport animal, and zoo animal, such as a human, non-human primate, dog, cat, guinea pig, rabbit, rat, mouse, horse, cow, etc. In one embodiment, the subject is a cynomolgus monkey (Macaca fascicularis). In a preferred embodiment, the subject is a human. In the methods of the present invention, the subject may not have been previously diagnosed as having cancer. Alternatively, the subject may have been previously diagnosed as having cancer. The subject can also be a subject exhibiting a disease risk factor or a subject asymptomatic for cancer. The subject can also be a subject suffering from cancer or at risk of developing cancer. Thus, in one embodiment, the methods of the present invention can be used to confirm the presence of cancer in a subject. For example, the subject may have been previously diagnosed with cancer by alternative means. In one embodiment, the subject has been previously administered cancer therapy.

[0081] The term "efficacy" means the ability to produce a desired effect.

[0082] The term "side effect" means a physiological disease and / or condition resulting from treatment other than the desired effect. In some embodiments, side effects include injection site reactions, abnormal liver function tests, abnormal kidney function, liver toxicity, kidney toxicity, central nervous system abnormalities, myopathy, and fatigue. For example, an increase in aminotransferase levels in the serum may indicate liver toxicity or abnormal liver function. For example, an increase in bilirubin may indicate liver toxicity or abnormal liver function.

[0083] Disclosed herein are methods, combinations, and kits that include administering an antibody-drug conjugate (ADC) together with a bispecific binding protein. The methods, combinations, and kits can be used in the treatment of cancer in a subject described herein.

[0084] The methods, combinations, and kits herein are intended to provide improved cancer treatment by targeting cancer cells in different ways. In some embodiments, the ADCs described herein increase the sensitivity of tumor cells to death by another mechanism. In some embodiments, the ADCs described herein increase the antigenicity of tumor cells. In some embodiments, the bispecific binding protein targets tumor cells for killing by the subject's immune system. In some embodiments, while the ADC increases the antigenicity of tumor cells by increasing the availability of one or more cell surface marker antigens either on the tumor cells themselves or on antigen-presenting cells (such as dendritic cells), the bispecific binding protein binds to one or more cell surface marker antigens and induces an immune response that attacks the tumor cells.

[0085] Combination method In some aspects, the present disclosure provides a method of treating cancer in a human subject in need thereof, the method comprising administering to the human subject: a) an antibody-drug conjugate (ADC) comprising: i) an antibody or an antigen-binding fragment thereof that binds to a B7-H4 polypeptide, ii) a cleavable linker, and iii) a cytotoxic agent; and b) a bispecific checkpoint inhibitor. In some aspects, the checkpoint inhibitor is a bispecific binding protein having a first binding domain that specifically binds to programmed cell death protein 1 (PD-1).

[0086] In further aspects, the present disclosure provides a method of treating cancer in a human subject in need thereof, the method comprising administering to the human subject an antibody-drug conjugate (ADC) comprising: a) i) an antibody or an antigen-binding fragment thereof, ii) a cleavable linker, and iii) a cytotoxic agent, wherein the cytotoxic agent is a compound of formula I:

[0087]

Chemical formula

[0088]

Chemical formula

[0089]

Chemical formula

[0090]

Chemical formula

[0091] [Chemical formula] (wherein R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group, and e is 0 or 1), an ADC, and b) administering a bispecific checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a bispecific binding protein having a first binding domain that specifically binds to programmed cell death protein 1 (PD-1).

[0092] An antibody-drug conjugate that binds to B7-H4 The antibody or antigen-binding fragment of the ADC can be used to target cancer cells. In some embodiments, the antibody or antigen-binding fragment has a high affinity for the B7-H4 polypeptide both in vitro and in vivo, and thus can be advantageously used in methods for targeting or detecting B7-H4 epitopes.

[0093] B7-H4 (also known as V-set domain-containing T-cell activation inhibitor 1 and encoded by the VTCN1 gene) is a transmembrane polypeptide of the B7 family of costimulatory proteins. B7-H4 is understood to be expressed on the surface of antigen-presenting cells for interaction with ligands of immune cells (e.g., T lymphocytes, and CD28 is a potential ligand). B7-H4 has been observed to be highly expressed on cells of various cancer types and is considered to be a tumor-associated antigen. Furthermore, B7-H4 expression is not limited to specific cancer types such that it represents a target antigen for treating a wide range of cancer types.

[0094] In some embodiments, the cancer referred to herein is a cancer characterized by the expression (preferably overexpression) of the B7-H4 molecule. In other words, the cancer referred to herein may include cancerous cells that express the B7-H4 polypeptide. The cancerous cells may be contained within a tumor. In some embodiments, the B7-H4 polypeptide is contained within the B7-H4 polypeptide sequence or a fragment thereof. The "B7-H4 polypeptide" may include the full-length polypeptide sequence of B7-H4 (e.g., SEQ ID NO: 55), or any length fragment of the full-length polypeptide sequence of B7-H4 that contains an epitope that can bind (e.g., be bound by) the antibodies or antigen-binding fragments of the present disclosure (e.g., containing 5%, 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, or 95% of the polypeptide sequence of the full-length polypeptide sequence of B7-H4). The B7-H4 polypeptide may include a sequence having 75%, 80%, 85%, 90%, or 95% sequence identity to the sequence of SEQ ID NO: 55. Preferably, the B7-H4 polypeptide includes the sequence of SEQ ID NO: 55.

[0095] The term "antibody" encompasses monoclonal antibodies and their fragments (e.g., those exhibiting the desired biological activity). In a preferred embodiment, the antibodies of the present disclosure are monoclonal antibodies. In a more preferred embodiment, the antibodies are fully human monoclonal antibodies. In some embodiments, the methods of the present disclosure may employ polyclonal antibodies. In particular, an antibody is a protein comprising at least one or two heavy (H) chain variable regions (abbreviated herein as VH) and at least one or two light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into hypervariable regions termed "complementary determining regions" (CDRs) interspersed with more conserved regions termed "framework regions" (FRs). The ranges of the framework regions and CDRs are precisely defined. Preferably, each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The VH or VL chain of an antibody can further comprise all or part of a heavy or light chain constant region. In some embodiments, an antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, and the heavy and light immunoglobulin chains are interconnected, for example, by disulfide bonds.

[0096] The heavy chain constant region includes three domains, CH1, CH2, and CH3. The light chain constant region consists of one domain, CL. The variable regions of the heavy and light chains include binding domains that interact with antigens. The term "antibody" includes intact immunoglobulins of the IgA, IgG, IgE, IgD, IgM types (and their subtypes), and the light chains of the immunoglobulins can be of the kappa or lambda type. The term "antibody" also, as used herein, refers to a portion of an antibody that binds to one of the markers described above, e.g., a molecule in which one or more immunoglobulin chains are not full length but that binds to the marker. Examples of binding portions included within the term "antibody" include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains, (ii) an F(ab')2 fragment, which is a divalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region, (iii) an Fc fragment consisting of the VH and CH1 domains, (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of the VH domain (Ward et al., Nature 341:544-546, 1989), and (vi) an isolated complementarity-determining region (CDR), e.g., having a framework sufficient to bind to the antigen-binding portion of the variable region. The antigen-binding portions of the light chain variable region and the heavy chain variable region, e.g., the two domains of an Fv fragment, VL and VH, can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain (known as a single chain Fv (scFv), see, e.g., Bird et al. (1988) Science lAl-ATi-Alβ, and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) in which the VL and VH regions pair to form a monovalent molecule. Such single-chain antibodies are also included within the term "antibody". These can be obtained using conventional techniques known to those of skill in the art, and the portions are screened for utility in the same manner as intact antibodies.

[0097] In some embodiments, the antibody or antigen-binding fragment is one or more selected from murine antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, multispecific antibodies, or combinations thereof.

[0098] In some embodiments, the antigen-binding fragment is one or more selected from Fv fragments, Fab fragments, F(ab’)2 fragments, Fab’ fragments, dsFv fragments, scFv fragments, sc(Fv)2 fragments, or combinations thereof. In some embodiments, the antibody or antigen-binding fragment thereof (e.g., mAb) of the present disclosure is an scFV.

[0099] In some embodiments, the antibody or its antigen-binding fragment can bind to B7-H4 molecules across species, e.g., the antibody or fragment can bind to murine B7-H4, rat B7-H4, rabbit, human B7-H4, and / or cynomolgus monkey B7-H4. In some embodiments, the antibody or fragment can bind to human B7-H4 and cynomolgus monkey B7-H4. In some embodiments, the antibody or antigen-binding fragment can also bind to murine B7-H4.

[0100] In some embodiments, the antibody or its antigen-binding fragment can specifically bind to B7-H4, e.g., human B7-H4 and cynomolgus monkey B7-H4, but does not specifically bind to human B7-H1, B7-H2, and / or B7-H3.

[0101] In some embodiments, the antibody or its antigen-binding fragment of the ADC comprises, respectively, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) containing the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or functional variants thereof. The antibody or its antigen-binding fragment containing such sequences may be referred to herein as "ZY0EQD-E02" or "EQD-E02".

[0102] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC comprises, respectively, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each comprising the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. The antibody or antigen-binding fragment thereof comprising such sequences may be referred to herein as "ZY0EPQ-E02" or "EPQ-E02".

[0103] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC comprises, respectively, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18. The antibody or antigen-binding fragment thereof comprising such sequences may be referred to herein as "ZY0EOB-F05" or "EOB-F05".

[0104] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC comprises, respectively, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24. The antibody or antigen-binding fragment thereof comprising such sequences may be referred to herein as "ZY0EO5-E07" or "EO5-E07".

[0105] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC comprises, respectively, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each comprising the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30. The antibody or antigen-binding fragment thereof comprising such sequences may be referred to herein as "ZY0EP0-C07" or "EP0-C07".

[0106] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC is a bispecific antibody and includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 within a first binding arm, which include the amino acid sequences of SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, and SEQ ID NO: 121, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 within a second binding arm, which include the amino acid sequences of SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127.

[0107] In some embodiments, an antibody or antigen-binding fragment thereof having CDRs with the amino acid sequences disclosed above has beneficial advantages such as high target specificity or binding activity for the B7-H4 peptide on the surface of cancer cells.

[0108] In addition, or alternatively, the antibody or antigen-binding fragment thereof described herein may be described by its variable heavy (VH) chain and variable light (VL) chain.

[0109] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC comprises a variable heavy (VH) chain and a variable light (VL) chain, or a functional variant thereof, each comprising the amino acid sequence of SEQ ID NO: 45 and SEQ ID NO: 34, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequence of SEQ ID NO: 33 and SEQ ID NO: 34, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequence of SEQ ID NO: 43 and SEQ ID NO: 34, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequence of SEQ ID NO: 46 and SEQ ID NO: 34, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequence of SEQ ID NO: 47 and SEQ ID NO: 34, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequence of SEQ ID NO: 31 and SEQ ID NO: 32, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequence of SEQ ID NO: 35 and SEQ ID NO: 36, a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequence of SEQ ID NO: 37 and SEQ ID NO: 38, or a VH chain and a VL chain, or a functional variant thereof, each comprising the amino acid sequence of SEQ ID NO: 39 and SEQ ID NO: 40.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC comprises (i) a variable heavy chain, or a functional variant thereof, comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31, 33, 35, 37, or 39, and (ii) a variable light chain, or a functional variant thereof, comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32, 34, 36, 38, or 40.

[0111] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC comprises: (i) a variable heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 31, 33, 35, 37, 39, 43, 45, 46, or 47, or a functional variant thereof; and (ii) a variable light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 32, 34, 36, 38, or 40, or a functional variant thereof.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC may comprise: (i) a variable heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 33, or a functional variant thereof; and (ii) a variable light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 34, or a functional variant thereof.

[0113] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC may comprise: (i) a variable heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 45, or a functional variant thereof; and (ii) a variable light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 34, or a functional variant thereof.

[0114] Advantageously, the present disclosure demonstrates that the antibody or antigen-binding fragment of the claims can target a broader range of B7-H4-expressing cells compared to existing (commercially) available antibodies reported to target B7-H4. Thus, the present disclosure not only provides an antibody (or antigen-binding fragment thereof) having affinity and specificity for a clinically relevant target, but also demonstrates an associated unique advantage (e.g., an unexpected technical effect).

[0115] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are capable of binding to B7-H4 as an essential component of cancer cells (e.g., B7-H4 as an essential component of the cell membrane of cancer cells).

[0116] In some embodiments, the antibody of the ADC or an antigen-binding fragment thereof binds to the OVCAR4 cell line. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can bind to the OVCAR4 cell line and / or the CHO cell line (e.g., which may lack the exogenous nucleic acid encoding B7-H4). For example, the antibody or an antigen-binding fragment thereof binds to B7-H4 (e.g., a B7-H4 epitope) of the OVCAR4 cell line and / or the CHO cell line (e.g., which may lack the exogenous nucleic acid encoding B7-H4).

[0117] In some embodiments, the antibody or an antigen-binding fragment thereof binds to the OVCAR4 cell line and / or the CHO cell line (e.g., which may lack the exogenous nucleic acid encoding B7-H4) with a higher affinity compared to one or more antibodies selected from E Biosciences 14-5949 anti-human B7H4 mouse IgG, US biological B0000-35B anti-human B7H4 mouse IgG, R and D systems AF2514 anti-mouse B7H4 goat IgG1, Sigma SAB2500141 anti-B7H4 goat IgG1, isotype 1 CAT004 SP06-003, isotype 2 R and D normal goat IgG control (AB-108C), AdD serotec MCA2632, Epitomics 2516-1, eBiosciences, 145972-82, eBioscience 145970-85, or combinations thereof. The affinity (e.g., binding affinity) can be measured by any suitable method for measuring the binding affinity described herein.

[0118] The OVCAR4 cell line is a human ovarian cancer cell line, and the CHO cell line is an epithelial cell line derived from the ovary of Chinese hamster and is widely available.

[0119] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the antibody or antigen-binding fragment thereof of the ADC comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the antibody or antigen-binding fragment thereof of the ADC comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 42.

[0120] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising the amino acid sequence of SEQ ID NO: 44.

[0121] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 and a light chain comprising the amino acid sequence of SEQ ID NO: 44.

[0122] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC is a monoclonal antibody.

[0123] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC is a humanized monoclonal antibody.

[0124] Linker In some embodiments, the antibody or antigen-binding fragment thereof is linked to the cytotoxic agent by a linker. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to the cytotoxic agent by a linker. As used herein, "conjugated" means linked via a covalent or ionic bond. In some embodiments, the linker binds (e.g., is conjugated) to an amino acid of the antibody or antigen-binding fragment of the ADC in a cleavable manner. In some embodiments, the cleavable linker of the ADC is an mp-PEG8-val-ala linker.

[0125] In some embodiments, the linker is represented by R L and is preferably (Ia):

[0126]

Chem.

[0127]

Chem.

[0128]

Chem.

[0129]

Chem.

[0130] In some embodiments, Q is an amino acid residue. The amino acid can be a natural amino acid or a non-natural amino acid. For example, Q can be selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, where Cit is citrulline.

[0131] In some embodiments, Q contains a dipeptide residue. The amino acids in the dipeptide can be any combination of natural and non-natural amino acids. In some embodiments, the dipeptide contains a natural amino acid. When the linker is a cathepsin labile linker, the dipeptide is a site of action for cathepsin-mediated cleavage. The dipeptide is then a recognition site for cathepsin.

[0132] In some embodiments, Q is NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , NH -Val-Cit- C=O , NH -Phe-Cit- C=O , NH -Leu-Cit- C=O , NH -Ile-Cit- C=O , NH -Phe-Arg- C=O , NH -Trp-Cit- C=O , and NH -Gly-Val- C=O selected from, where Cit is citrulline. Preferably, Q is NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , and NH -Val-Cit- C=Oselected from. More preferably, Q is NH -Phe-Lys- C=O , NH -Val-Cit- C=O , and NH -Val-Ala- C=O selected from.

[0133] Other suitable dipeptide combinations include NH -Gly-Gly- C=O , NH -Gly-Val- C=O , NH -Pro-Pro- C=O , and NH -Val-Glu- C=O can be mentioned. Other dipeptide combinations can be used, including those described in Dubowchik et al., Bioconjugate Chemistry, 2002, 13855 - 869, which is incorporated herein by reference.

[0134] In some embodiments, Q is a tripeptide residue. The amino acids in the tripeptide can be any combination of natural and non-natural amino acids. In some embodiments, the tripeptide contains natural amino acids. When the linker is a cathepsin-labile linker, the tripeptide is a site of action for cathepsin-mediated cleavage. The tripeptide is then a recognition site for cathepsin. Particularly interesting tripeptide linkers are NH -Glu-Val-Ala- C=O , NH -Glu-Val-Cit- C=O , NH -αGlu-Val-Ala- C=O , and NH -αGlu-Val-Cit- C=O .

[0135] In some embodiments, Q is a tetrapeptide residue. The amino acids in the tetrapeptide can be any combination of natural and non-natural amino acids. In some embodiments, the tetrapeptide contains natural amino acids. When the linker is a cathepsin-labile linker, the tetrapeptide is the site of cathepsin-mediated cleavage. The tetrapeptide is then the recognition site for cathepsin. Particularly interesting tetrapeptide linkers are NH -Gly-Gly-Phe-Gly C=O and NH -Gly-Phe-Gly-Gly C=O . Preferably, the tetrapeptide is NH -Gly-Gly-Phe-Gly C=O .

[0136] In the above representation of peptide residues, NH - represents the N-terminus of the residue, and - C=O represents the C-terminus. The C-terminus binds, for example, to the NH of the cytotoxic agent.

[0137] Glu is the residue of glutamic acid, i.e., represents the following,

[0138]

Chem.

[0139]

Chem.

[0140] In some embodiments, the amino acid side chains are chemically protected where appropriate. The side chain protecting groups can be the groups discussed above. The protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence containing a Boc side chain protected Lys residue is cleavable by cathepsin. In some embodiments, the protecting groups for the side chains of the amino acids are well-known, described in the Novabiochem Catalog, and as described above.

[0141] In some embodiments of X, a can be 0, 1, 2, 3, 4, or 5. Preferably, a is from 0 to 3. More preferably, a is 0 or 1. Even more preferably, a is 0. In some embodiments, b1 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. Preferably, b1 is from 0 to 12. More preferably, b1 is from 0 to 8 and can be 0, 2, 3, 4, 5, or 8. In some embodiments, b2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. Preferably, b2 is from 0 to 12. More preferably, b2 is from 0 to 8 and can be 0, 2, 3, 4, 5, or 8. In some embodiments, c1 can be 0 or 1. In some embodiments, c2 can be 0 or 1. Preferably, only one of c1 and c2 may not be 0. In some embodiments, d can be 0, 1, 2, 3, 4, or 5. Preferably, d is from 0 to 3. More preferably, d is 1 or 2. In one embodiment, d is 2. In one embodiment, d is 5.

[0142] In some embodiments of X, a is 0, b1 is 0, c1 is 1, c2 is 0, d is 2, and b2 can be from 0 to 8. In some embodiments, b2 can be 0, 2, 3, 4, 5, or 8. In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 0, d is 0, and b1 can be from 0 to 8. In some embodiments, b1 is 0, 2, 3, 4, 5, or 8. In some embodiments of X, a is 0, b1 is 0, c1 is 0, c2 is 0, d is 1, and b2 can be from 0 to 8. In some embodiments, b2 can be 0, 2, 3, 4, 5, or 8. In some embodiments of X, b1 is 0, b2 is 0, c1 is 0, c2 is 0, and one of a and d is 0. The other of a and d is from 1 to 5. In some embodiments, the other of a and d is 1. In some embodiments, the other of a and d is 5. In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 can be from 0 to 8. In some embodiments, b2 can be 0, 2, 3, 4, 5, or 8.

[0143] As described above, R L1 and R L2 can be independently selected from H and methyl, or together with the carbon atom to which they are attached, can form a cyclopropylene or cyclobutylene group.

[0144] In some embodiments, both R L1 and R L2 are H. In some embodiments, R L1 is H and R L2 is methyl. In some embodiments, both R L1 and R L2 are methyl. In some embodiments, R L1 and R L2 together with the carbon atom to which they are attached, form a cyclopropylene group. In some embodiments, R L1 and R L2Together with the carbon atoms to which they are attached, they form a cyclobutylene group.

[0145] In formula Ib, in some embodiments, e is 0. In some embodiments, e is 1 and the nitro group can be at any available position on the ring. In some embodiments, it is in the ortho position. In some embodiments, it is in the para position.

[0146] In some embodiments, G L can be selected from the following

[0147] [Table 3] In the formula, Ar represents a C 5~6 arylene group, for example, phenylene, and X represents a C 1~4 alkyl.

[0148] In some embodiments, G L is G L1-1 and G L1-2 selected from. In one embodiment, G L is G L1-1 is.

[0149] In some embodiments, R L is selected from the following.

[0150] [Table 4-1]

[0151] [Table 4-2]

[0152] In some embodiments, the linker is represented by R LL and preferably (Ia’):

[0153] [Chemical formula] (wherein Q and X are as defined above, and G LL is a linker (e.g., a ligand unit) attached to the antibody or antigen-binding fragment thereof described herein), and (Ib’):

[0154] [Chemical formula] (wherein R L1 and R L2 are as defined above), and p is an integer from 1 to 20.

[0155] In some embodiments, G LL may be selected from the following,

[0156] [Table 5] Ar represents a C 5~6 arylene group, e.g., phenylene, and X represents a C 1~4 alkyl.

[0157] In some embodiments, G LL is selected from G LL1-1 and G LL1-2 . In one embodiment, G LL is G LL1-1 .

[0158] In some embodiments, R LL is a group derived from the above R L group.

[0159] In some embodiments where the compounds described herein are provided in a single enantiomeric or enantiomerically enriched form, the enantiomerically enriched form has an enantiomer ratio of greater than 60:40, 70:30, 80:20, or 90:10. In some embodiments, the enantiomer ratio is greater than 95:5, 97:3, or 99:1.

[0160] Drug loading The average number of drugs per antibody in the preparation of an ADC from a conjugation reaction can be characterized by conventional means such as UV, reverse-phase HPLC, HIC, mass spectrometry, ELISA assay, and electrophoresis. The quantitative distribution of ADCs with respect to p can also be determined. By ELISA, the average value of p in a particular preparation of an ADC can be determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070, Sanderson et al (2005) Clin. Cancer Res. 11:843-852). However, the distribution of p (drug) values is not distinguishable by antibody-antigen binding and the detection limit of ELISA. Also, ELISA assays for the detection of antibody-drug conjugates do not determine where the drug moiety, such as a heavy or light chain fragment, or a specific amino acid residue, is attached to the antibody. In some cases, the isolation, purification, and characterization of homogeneous ADCs, where p is a particular value from ADCs with other drug loads, can be achieved by means such as reverse-phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.

[0161] Typically, fewer drug moieties than the theoretical maximum are conjugated to the antibody during the conjugation reaction. The antibody may contain, for example, many lysine residues that do not react with the drug linker. Only the most reactive lysine groups can react with the amine-reactive linker reagent. Also, only the most reactive cysteine thiol groups can react with the thiol-reactive linker reagent. Generally, antibodies contain few, if any, free and reactive cysteine thiol groups that can be linked to the drug moiety. Most cysteine thiol residues in the antibody of the compound exist as disulfide bridges and must be reduced using a reducing agent such as dithiothreitol (DTT) or TCEP under partial or complete reduction conditions. The loading (drug / antibody ratio) of the ADC can be controlled in several different ways, including (i) limiting the molar excess of the drug linker relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reduction conditions for cysteine thiol modification.

[0162] Certain antibodies have reducible interchain disulfides, i.e., cysteine bridges. The antibody can be made reactive for conjugation with the linker reagent by treatment with a reducing agent such as DTT (dithiothreitol). Thus, each cysteine bridge theoretically forms two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody through the reaction of lysine with 2-iminothiolane (Traut reagent), which results in the conversion of an amine to a thiol. Reactive thiol groups can be introduced into the antibody (or a fragment thereof) by engineering one, two, three, four, or more cysteine residues (e.g., by preparing a mutant antibody that includes one or more non-natural cysteine amino acid residues). U.S. Patent No. 7,521,541 teaches engineering antibodies by introducing reactive cysteine amino acids.

[0163] Cysteine amino acids can be engineered at reactive sites in antibodies and do not form intra-chain or inter-molecular disulfide bonds (Junutula, et al., 2008b Nature Biotech., 26(8):925-932, Dornan et al (2009) Blood 114(13):2721-2729, U.S. Patent No. 7,521,541, U.S. Patent No. 7,723,485, International Publication No. 2009 / 052249). Engineered cysteine thiols can react with the drug linkers of the present invention having thiol-reactive electrophilic groups such as maleimide or α-haloamide to form an ADC having a cysteine-engineered antibody. Thus, the position of the drug moiety can be designed, controlled, and known. Since engineered cysteine thiol groups typically react with drug-linker reagents in high yield, the drug loading can be controlled. By engineering IgG antibodies to introduce cysteine amino acids by substitution at a single site on the heavy or light chain, two new cysteines are obtained on the symmetric antibody.

[0164] Cytotoxic agent The cytotoxic agent of the ADC may be referred to herein as a "drug" or an "active agent". In some embodiments, the cytotoxic agent is a drug. In some embodiments, the cytotoxic agent of the ADC is a topoisomerase inhibitor, a tubulysin derivative, a pyrrolobenzodiazepine, or a combination thereof.

[0165] In some embodiments, the topoisomerase inhibitor is a compound of formula A as a single enantiomer or in an enantiomerically enriched form:

[0166]

Chemical formula

[0167] In some embodiments, the present disclosure provides a conjugate comprising the following topoisomerase inhibitor derivative (A * , drug moiety).

[0168]

Chemical formula

[0169] The present disclosure provides a cytotoxic agent (drug unit) of an ADC that is connected to the ligand unit of the ADC via the linker (linking unit) described above. The ligand unit is preferably an antibody or an antigen-binding fragment thereof. In some embodiments, the present disclosure also provides A to which the linker is attached * , and intermediates for their synthesis, as well as released warheads.

[0170] In some embodiments, the cytotoxic agent is a topoisomerase inhibitor, and the cleavable linker of the ADC and the cytotoxic agent together are a compound of formula I:

[0171]

Chemical formula

[0172] In some embodiments, the ADC is of formula IV: L-(D L ) p (IV) or a pharmaceutically acceptable salt or solvate thereof, wherein L is i) an antibody or an antigen-binding fragment thereof, and D L is ii) a drug linker unit comprising a cleavable linker and iii) a cytotoxic agent, and D L is of formula III,

[0173]

Chemical formula

[0174] In some embodiments, the drug loading (p) of the cytotoxic agent with respect to the antibody or antibody-binding fragment is an integer from 1 to about 10.

[0175] In some embodiments, the topoisomerase inhibitor is a compound having Formula VI,

[0176]

Chemical formula

[0177] In some embodiments, the linker and the cytotoxic agent together comprise the following compounds:

[0178]

Chemical formula

[0179] In some embodiments, the linker and the cytotoxic agent together comprise the following compounds:

[0180]

Chemical formula

[0181] The cytotoxic agent is typically conjugated to or "loaded onto" an antibody or antigen-binding fragment. The drug load (p) is the average number of drugs per antibody or antigen-binding fragment (e.g., ligand unit).

[0182] The average number of drugs per antibody (or antigen-binding fragment) in a preparation of an ADC from a conjugation reaction can be characterized by conventional means such as UV, reverse-phase HPLC, HIC, mass spectrometry, ELISA assays, and electrophoresis. The quantitative distribution of the ADC with respect to p can also be determined. By ELISA, the average value of p in a particular preparation of the ADC can be determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070, Sanderson et al (2005) Clin. Cancer Res. 11:843-852). In some cases, the isolation, purification, and characterization of homogeneous ADCs, where p is a specific value from ADCs with other drug loads, can be achieved by means such as reverse-phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.

[0183] Cysteine amino acids can be engineered at reactive sites in the antibody (or antigen-binding fragment thereof), preferably not forming intra-chain or inter-molecular disulfide bonds (Junutula, et al., 2008b Nature Biotech., 26(8):925-932, Dornan et al (2009) Blood 114(13):2721-2729, US Patent No. 7521541, US Patent No. 7723485, International Publication No. 2009 / 052249). The engineered cysteine thiols can react with linkers in drugs having thiol-reactive electrophilic groups such as maleimide or α-haloamide (e.g., drugs of formula I below) to form an ADC having a cysteine-engineered antibody. Thus, the position of the drug unit can be designed, controlled, and known. Since the engineered cysteine thiol groups typically react with drug-linker reagents in high yields, the drug load can be controlled. By engineering IgG antibodies to introduce cysteine amino acids by substitution at a single site on the heavy or light chain, two new cysteines are obtained on the symmetric antibody. A drug load close to 2 can be achieved with near-homogeneity of the conjugation product ADC.

[0184] If more than one nucleophilic or electrophilic group of an antibody or its antigen-binding fragment reacts with a drug, the resulting product can be a mixture of ADC compounds having a distribution of drug units attached to the antibody, e.g., 1, 2, 3, etc. Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) can separate the compounds in the mixture by drug loading value. Preparations of ADCs having a single drug loading value (p) can be isolated.

[0185] Accordingly, the antibody-drug conjugate compositions of the present disclosure can include a mixture of antibody-drug conjugates in which the antibody or its antigen-binding fragment has one or more drug moieties, and the drug moieties can bind to the antibody or its antigen-binding fragment at various amino acid residues.

[0186] In some embodiments, the average number of drugs per antibody (or its antigen-binding fragment) is in the range of 1 to 20. In some embodiments, the range is selected from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10. In some embodiments, one drug is present per antibody (or its antigen-binding fragment). In some embodiments, the number of drugs per antibody (or its antigen-binding fragment) can be expressed as the ratio of drug (i.e., medicament) to antibody. This ratio is referred to as the Drug to Antibody Ratio (DAR). The DAR is the average number of drugs (i.e., medicaments) linked to each antibody. In some embodiments of the present disclosure, the DAR is in the range of 1 to 20. In some embodiments, the range of the DAR is selected from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10. In some embodiments, the DAR is from about 1 to about 8. In certain embodiments of the present disclosure, the DAR is about 8. In certain embodiments of the present disclosure, the DAR is 8.

[0187] In some embodiments, the ADC is AZD8205. AZD 8205 is an anti-B7H4 Ab conjugated to a topoisomerase inhibitor (TOPO) warhead. In some embodiments, AZD 8205 is targeted to ovarian cancer and cholangiocarcinoma (CCA). In some embodiments, AZD 8205 is targeted to triple-negative breast cancer (TNBC). In some embodiments, the ADC is the ADC described in International Publication No. WO 2022 / 053650, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the antibody has specific CDRs and binding specificities. In some embodiments, the antibody sequence is found in International Publication No. WO 2022 / 053650, and this antibody or its antigen-binding fragment comprises the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and / or the VH and VL chains comprising the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 34 (germline), and / or the heavy chain comprising the amino acid sequence of SEQ ID NO: 51, and the light chain comprising the amino acid sequence of SEQ ID NO: 44 (germline). In some embodiments, the ADC is named E02-GL-SG3932.

[0188] Bispecific binding protein The term "bispecific binding protein" means a protein having binding specificities for at least two distinct antigens (or targets) or different epitopes within the same antigen. Exemplary bispecific binding proteins can bind to two different epitopes of a target or to two different targets. Other such binding proteins can combine a first target binding site with a second binding site for a different target. In some embodiments, the bispecific binding protein is a bispecific antibody. In some embodiments, the bispecific binding protein is a bispecific checkpoint inhibitor.

[0189] In the present disclosure, the term "bispecific checkpoint inhibitor" refers to a checkpoint inhibitor that is bispecific. As used herein, a bispecific checkpoint inhibitor has binding specificity for at least two independent antigens (or targets) or different epitopes within the same antigen. Exemplary bispecific binding proteins can bind to two different epitopes of a target or to two different targets. Other such binding proteins can combine a first target binding site with a second binding site for a different target. A checkpoint inhibitor (or "immune-oncology (IO) agent") is an agent that inhibits the immunosuppressive system and activates antitumor immunity (Menon S. et al., Cancers (2016) 8, 106; Pardoll DM., Nat Rev Cancer (2012) 12, 252 - 264, Wolchok JD., Cell (2015) 162, 937). Targets for immune-mediated therapies in oncology include PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T lymphocyte-associated protein 4). TIGIT (T cell immunoreceptor with Ig and ITIM domains), an immunoreceptor present on some T cells and natural killer cells, is another target, along with leukemia inhibitory factor (LIF). Examples of known immune checkpoint inhibitors include the anti-PD-1 antibodies nivolumab (International Publication No. WO 2006 / 121168) and pembrolizumab (International Publication No. WO 2008 / 156712), the anti-PD-L1 antibodies atezolizumab (International Publication No. WO 2010 / 077634), durvalumab (International Publication No. WO 2011 / 066389), and avelumab (International Publication No. WO 2013 / 079174), as well as the anti-CTLA-4 antibodies ipilimumab (International Publication No. WO 2001 / 014424) and tremelimumab (International Publication No. WO 2000 / 037504), and anti-LIF antibodies such as h5D8 (U.S. Patent No. 10,583,191), each of which is incorporated herein by reference in its entirety.In some embodiments, the bispecific antibody provides additive and / or synergistic therapeutic effects resulting from the simultaneous targeting of two antigens by administration of a single manufactured molecule.

[0190] In some embodiments, the antibodies provided herein are monovalent bispecific antibodies (MBabs). The monovalent bispecific antibody scaffolds described herein provide an excellent platform for generating bispecific antibodies that meet all of the benefits associated with bispecific antibodies while reducing the above potential therapeutic risks due to their monovalent nature. Furthermore, the MBabs provided herein are readily expressed, stable, and likely to have low immunogenicity. As used herein, the term "monovalent bispecific," which may be abbreviated as "MBab," refers to a bispecific antibody in which each arm can specifically bind to a different target antigen and, for a given pair of different target antigens (A and B), the MBab can bind to one of each. In some embodiments, the monovalent bispecific antibody can specifically bind to two independent antigens (or targets) or two independent epitopes on the same antigen. Typically, the monovalent bispecific antibody comprises two different variable regions. In some embodiments, the binding affinities for the two independent antigens are approximately the same. In some embodiments, the binding affinities for the two independent antigens are different.

[0191] In some embodiments, the bispecific binding protein comprises (a) a first binding domain that specifically binds to PD-1 and (b) a second binding domain that specifically binds to a T cell immunoreceptor with Ig and ITIM domains (TIGIT), a T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3), or a cytotoxic T lymphocyte-associated antigen-4 (CTLA-4).

[0192] PD-1 and PD-L1 binding domains The term "programmed cell death protein 1 (PD-1)" refers to a type I membrane protein of approximately 31 kD that is a member of the extended proliferation CD28 / CTLA4 family of T cell regulators (see Ishida et al., "Induced Expression of PD-1, A Novel Member of the Immunoglobulin Gene Superfamily, Upon Programmed Cell Death," EMBO J. 11:3887-95 (1992)). PD-1 is expressed on activated T cells, B cells, and monocytes, and at low levels on natural killer (NK) T cells. Programmed death ligand 1 (PD-L1) is a ligand for PD-1. When PD-L1 binds to PD-1 on T cells, it prevents the T cells from killing the cells presenting PD-L1 (see Han et al., "PD-1 / PD-L1 pathway: current researches in cancer," Am J. Cancer Res., 10(3):727-742 (2020)). PD-L1 is a type I transmembrane glycoprotein of approximately 33 kD that is a member of the B7 family (see Sanmamed et al., "Inducible expression of B7-H1 (PD-L1) and its selective role in tumor site immune modulation," Cancer J. 20:256-261 (2014)). Antagonistic inhibition of the PD-1 / PD-L1 interaction increases T cell activation and enhances the recognition and elimination of tumor cells by the host immune system. Such antagonistic inhibition of the PD-1 / PD-L1 interaction can be achieved using agents that bind to either PD-1 or PD-L1 and block the interaction. The amino acid sequence of the human PD-1 protein is provided as UNIPROT ID number Q15116, while the amino acid sequence of human PD-L1 is provided as UNIPROT ID number Q9NZQ7.

[0193] Programmed cell death ligand 1 (PD-L1) is also part of a receptor and ligand complex involved in controlling T cell activation. In normal tissues, PD-L1 is expressed on T cells, B cells, dendritic cells, macrophages, mesenchymal stem cells, bone marrow-derived mast cells, and various non-hematopoietic cells. Its normal function is to regulate the balance between T cell activation and tolerance through its interaction with two receptors, namely programmed death 1 (also known as PD-1 or CD279) and CD80 (also known as B7-1 or B7.1). PD-L1 is also expressed by tumors and acts at multiple sites to help tumors avoid detection and elimination by the host immune system. PD-L1 is highly expressed in a wide range of cancers. In some cancers, the expression of PD-L1 has been associated with reduced survival and an unfavorable prognosis. Antibodies that block the interaction between PD-L1 and its receptor can reduce the PD-L1-dependent immunosuppressive effect and enhance the cytotoxic activity of anti-tumor T cells in vitro. Durvalumab is a human monoclonal antibody against human PD-L1 that is capable of blocking the binding of PD-L1 to both the PD-1 and CD80 receptors. The use of anti-PD-L1 antibodies to treat infections and tumors and enhance the adaptive immune response has been proposed (see U.S. Patent Nos. 8,779,108 and 9,493,565, which are incorporated herein by reference in their entirety).

[0194] As used herein, the term "MEDI5752" refers to an anti-PD-1 / CTLA-4 bispecific antibody comprising the light chain of SEQ ID NO: 45 and the heavy chain of SEQ ID NO: 46 (PD-1), and the light chain of SEQ ID NO: 47 and the heavy chain of SEQ ID NO: 48 (CTLA-4). MEDI5752 is disclosed in U.S. Patent No. 10,457,732, which is incorporated herein by reference in its entirety.

[0195] In some embodiments, the first binding domain comprises a heavy chain variable domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 56, an HCDR2 having the amino acid sequence of SEQ ID NO: 57, and an HCDR3 having the amino acid sequence of SEQ ID NO: 58, and a light chain variable domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 59, an LCDR2 having the amino acid sequence of SEQ ID NO: 60, and an LCDR3 having the amino acid sequence of SEQ ID NO: 61.

[0196] In some embodiments, the first binding domain that specifically binds to PD-1 comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 62 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 64.

[0197] In some embodiments, the first binding domain that specifically binds to PD-1 comprises a heavy chain variable domain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the first binding domain that specifically binds to PD-1 comprises a light chain variable domain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 64.

[0198] In some embodiments, the first binding domain that specifically binds to PD-1 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 63 and a light chain having the amino acid sequence of SEQ ID NO: 65.

[0199] In some embodiments, the first binding domain that specifically binds to PD-1 comprises a heavy chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the first binding domain that specifically binds to PD-1 comprises a light chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 65.

[0200] In some embodiments, the first binding domain that specifically binds to PD-1 comprises a heavy chain variable domain encoded by the nucleic acid sequence of SEQ ID NO: 76 and a light chain variable domain encoded by the nucleic acid sequence of SEQ ID NO: 78.

[0201] In some embodiments, the first binding domain that specifically binds to PD-1 comprises a heavy chain variable domain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 76 and a light chain variable domain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 78.

[0202] In some embodiments, the first binding domain that specifically binds to PD-1 comprises a heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 77 and a light chain encoded by the nucleic acid sequence of SEQ ID NO: 79.

[0203] In some embodiments, the first binding domain that specifically binds to PD-1 comprises a heavy chain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 77 and a light chain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 79.

[0204] TIGIT binding domain The term T cell immunoreceptor with Ig and ITIM domains (TIGIT) refers to an immunoreceptor present on some T cells and natural killer (NK) cells. TIGIT is upregulated by immune cells, including activated T cells, natural killer cells, and regulatory T cells.

[0205] In some embodiments, a second binding domain that specifically binds to TIGIT. In some embodiments, the second binding domain comprises a heavy chain variable domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 66, an HCDR2 having the amino acid sequence of SEQ ID NO: 67, and an HCDR3 having the amino acid sequence of SEQ ID NO: 68, and a light chain variable domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 69, an LCDR2 having the amino acid sequence of SEQ ID NO: 70, and an LCDR3 having the amino acid sequence of SEQ ID NO: 71.

[0206] In some embodiments, the second binding domain that specifically binds to TIGIT comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 72 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 74.

[0207] In some embodiments, the second binding domain that specifically binds to TIGIT comprises a heavy chain variable domain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the second binding domain that specifically binds to TIGIT comprises a light chain variable domain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 74.

[0208] In some embodiments, the second binding domain that specifically binds to TIGIT comprises a heavy chain having the amino acid sequence of SEQ ID NO: 73 and a light chain having the amino acid sequence of SEQ ID NO: 75.

[0209] In some embodiments, the second binding domain that specifically binds to TIGIT comprises a heavy chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the second binding domain that specifically binds to TIGIT comprises a light chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 75.

[0210] In some embodiments, the second binding domain that specifically binds to TIGIT comprises a heavy chain variable domain encoded by a nucleic acid sequence of SEQ ID NO: 80 and a light chain variable domain encoded by a nucleic acid sequence of SEQ ID NO: 82.

[0211] In some embodiments, the second binding domain that specifically binds to TIGIT comprises a heavy chain variable domain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 80 and a light chain variable domain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 82.

[0212] In some embodiments, the second binding domain that specifically binds to TIGIT comprises a heavy chain encoded by a nucleic acid sequence of SEQ ID NO: 81 and a light chain encoded by a nucleic acid sequence of SEQ ID NO: 83.

[0213] In some embodiments, the second binding domain that specifically binds to TIGIT comprises a heavy chain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 26 and a light chain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 28.

[0214] TIM-3 binding domain The term "T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3)" refers to a type I cell surface glycoprotein that includes an N-terminal immunoglobulin (Ig)-like domain, a mucin domain with O-linked glycosylation and N-linked glycosylation near the membrane, a single transmembrane domain, and a cytoplasmic region with a tyrosine phosphorylation motif. TIM-3 is a member of the T cell / transmembrane, immunoglobulin, and mucin (TIM) gene family. The amino acid sequence of the IgV domain of human TIM-3 is shown as SEQ ID NO: 102. The amino acid sequence of the human TIM-3 protein including the signal peptide is shown as SEQ ID NO: 103.

[0215] In some embodiments, the disclosure provides an anti-TIM-3 / PD-1 bispecific binding protein named "AZD7789" that includes the sequences of the monoclonal antibodies O13-1 (TIM-3) and LO115 (PD-1) described in U.S. Patent No. 10,457,732. The bispecific binding protein includes a first heavy chain that includes the amino acid sequence of SEQ ID NO: 92, a first light chain that includes the amino acid sequence of SEQ ID NO: 95, a second heavy chain that includes the amino acid sequence of SEQ ID NO: 63, and a second light chain that includes the amino acid sequence of SEQ ID NO: 65. The sequences of the anti-TIM-3 monoclonal antibodies O13-1 and clone 62 are also disclosed in the same U.S. Patent No. 10,457,732.

[0216] In some embodiments, the second binding domain specifically binds to the C’C’’ and DE loops of the immunoglobulin variable (IgV) domain of TIM-3, or binds to the PS binding groove (FG and CC’ loops) of the IgV domain of TIM-3. In some embodiments, the second binding domain that specifically binds to TIM-3 comprises complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having the amino acid sequences of SEQ ID NOs: 84, 85, 86, 87, 88, and 89, respectively, or SEQ ID NOs: 84, 85, 86, 87, 88, and 90, respectively. In some embodiments, the second binding domain that specifically binds to TIM-3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequences of SEQ ID NOs: 84, 85, 86, 87, 88, and 89, respectively, or SEQ ID NOs: 84, 85, 86, 87, 88, and 90, respectively. In some embodiments, the second binding domain specifically binds to an epitope on the IgV domain of TIM-3, and the epitope comprises N12, L47, R52, D53, V54, N55, Y56, W57, W62, L63, N64, G65, D66, F67, R68, K69, D71, T75, and E77 of TIM-3 (SEQ ID NO: 102).

[0217] In some embodiments, the second binding domain that specifically binds to TIM-3 comprises a second variable heavy domain (VH) comprising the amino acid sequence of SEQ ID NO: 91 and a second variable light domain (VL) comprising the amino acid sequence of SEQ ID NO: 94. In some embodiments, the second binding domain that specifically binds to TIM-3 comprises a second variable heavy domain (VH) that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 91 and a second variable light domain (VL) that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the second binding domain comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a second light chain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the second binding domain comprises a second heavy chain that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 92 and a second light chain that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 95.

[0218] In some embodiments, the second binding domain comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 96 and a first light chain comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, the second binding domain comprises a first heavy chain that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 96 and a first light chain that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the second binding domain comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 98 and a first light chain comprising the amino acid sequence of SEQ ID NO: 99. In some embodiments, the second binding domain comprises a first heavy chain that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 98 and a first light chain that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 99.

[0219] CTLA-4 binding domain As used herein, the terms "cytotoxic T-lymphocyte associated antigen-4", "CTLA-4", "CD152", and "hCTLA-4" are used interchangeably and include variants, isoforms, and species homologs of human CTLA-4. The complete CTLA-4 sequence can be found at NCBI reference sequence: NG_011502.1. The amino acid sequence of the human CTLA-4 protein is MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 115). Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) is expressed on activated T cells and functions as a co-inhibitor to maintain suppression of T cell responses after CD28-mediated T cell activation. CTLA-4 is thought to be part of a central inhibitory pathway that regulates the magnitude of initial activation of naive and memory T cells after TCR ligation and affects both anti-tumor immunity and autoimmunity. CTLA-4 is expressed only on T cells, and the expression of its ligands CD80 (B7.1) and CD86 (B7.2) is mainly restricted to antigen-presenting cells, T cells, and other immune mediator cells. Antagonist anti-CTLA-4 antibodies that block the CTLA-4 signaling pathway have been reported to enhance T cell activation.

[0220] In some embodiments, the second binding domain specifically binds to CTLA-4. In some embodiments, the bispecific binding protein is MEDI5752. The term "MEDI5752" refers to an anti-PD-1 / CTLA-4 bispecific antibody comprising a light chain of SEQ ID NO: 65 and a heavy chain of SEQ ID NO: 63 (PD-1), and a light chain of SEQ ID NO: 104 and a heavy chain of SEQ ID NO: 105 (CTLA-4). MEDI5752 is disclosed in U.S. Patent No. 10,457,732, which is incorporated herein by reference in its entirety.

[0221] In some embodiments, the second binding domain comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 109, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 110, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 111, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 112, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 113, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 114. In some embodiments, the second binding domain comprises a VH CDR1 that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 109, a VH CDR2 that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 110, a VH CDR3 that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 111, a VL CDR1 that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 112, a VL CDR2 that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 113, and a VL CDR3 that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 114.

[0222] In some embodiments, the second binding domain comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 105 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 104.

[0223] In some embodiments, the second binding domain comprises a heavy chain that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 105, and a light chain that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 104.

[0224] In some embodiments, the bispecific binding protein comprises an IgG heavy chain constant region. In some embodiments, the IgG heavy chain constant region is an IgG1 heavy chain constant region. In some embodiments, the constant region comprises mutations at L234F, L235E, and P331S. In some embodiments, the constant region comprises a knob mutation and a hole mutation, and optionally, the knob mutation is in the heavy chain comprising the variable region that binds CTLA-4, and the hole mutation is in the heavy chain comprising the variable region that binds PD-1.

[0225] In some embodiments, the bispecific binding protein is a humanized bispecific antibody or an antigen-binding fragment thereof. In some embodiments, the bispecific binding protein binds to human PD-1 and CTLA-4. In some embodiments, the bispecific binding protein comprises the anti-PD-1 and anti-CTLA-4 heavy chain variable region (VH) CDR1, VH CDR2, VH CDR3, light chain variable region (VL) CDR1, VL CDR2, and VL CDR3 of the sequence of MEDI5752.

[0226] Embodiments of the bispecific protein that binds to PD-1 Bispecific binding proteins having a PD-1 domain and one of the TIGIT, TIM-3, and CTLA-4 domains can be diverse. In some embodiments, the bispecific binding protein comprises an unglycosylated Fc region. In some embodiments, the bispecific binding protein comprises a deglycosylated Fc region. In some embodiments, the bispecific binding protein comprises an Fc region having reduced fucosylation or no fucosylation. In some embodiments, the bispecific binding protein is a full-length antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is human or humanized. In some embodiments, the bispecific binding protein comprises a kappa light chain constant region. In some embodiments, the bispecific binding protein comprises a lambda light chain constant region.

[0227] In some embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding a bispecific binding protein described herein. In some embodiments, the disclosure provides a host cell comprising a nucleic acid described herein. In some embodiments, the disclosure provides a host cell comprising a vector comprising a nucleic acid described herein. In some embodiments, the disclosure provides a host cell having a nucleic acid described herein integrated into its genome. In some embodiments, the disclosure provides a bispecific binding protein produced by a host cell described herein.

[0228] Cancer type In some embodiments of any of the methods disclosed herein, the method is used to treat cancer. In some embodiments, the cancer is one or more selected from ovarian cancer, breast cancer, uterine cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, renal cell cancer, pancreatic cancer, prostate cancer, cervical cancer, blood cancer, endometrial cancer, cholangiocarcinoma, NSCLC (squamous and / or adenocarcinoma), gastrointestinal cancers such as gastric cancer and colorectal cancer, and lung cancer. In some embodiments, the cancer is breast cancer selected from hormone receptor positive (HR+) breast cancer, human epidermal growth factor receptor 2 positive (HER2+) breast cancer, and triple negative breast cancer (TNBC). In some embodiments, the cancer is homologous recombination defective (HRD) cancer. In some embodiments, the cancer comprises one or more cells having a mutation in an HRD gene selected from BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L. In some embodiments, the mutant HRD gene is selected from BRCA1, BRCA2, and ATM.

[0229] Sequence

[0230]

Table 6

[0231] SEQ ID NO: 31 (ZY0EPQ-E02, variable heavy chain) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWNWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTILVDTSKNQFSLKLSSVTAADTAVYYCARNLYNWNLDSWGQGTLVTVSS

[0232] SEQ ID NO: 32 (ZY0EPQ-E02, variable light chain) DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGRAPKRLIYVASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPRTFGQGTKVEIK

[0233] Accession No. 33 (ZY0EQD-E02, variable heavy chain, e.g., pre-germ cell lineage) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWNWIRQPPGKGLEWIGEINHSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSS

[0234] Accession No. 34 (ZY0EQD-E02, variable light chain) DIQMTQSPSSLSASVGDRVTITCRASQDIRNDVGWYQQKPGKAPKRLIYAASRLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPRTFGQGTKVEIK

[0235] Accession No. 35 (ZY0EOB-F05, variable heavy chain) QVQLQESGPGLVKPSQTLSLTCTVSDGSISSGGYYWSWIRQHPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCATEKALATVTPSGYENYYTVDVWGQGTTVTVSS

[0236] Accession No. 36 (ZY0EOB-F05, variable light chain) DIQLTQSPSFLSASVGDRVTITCWASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHLNSYPLTFGGGTKVEIK

[0237] Accession No. 37 (ZY0EO5-E07, variable heavy chain) QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREKALASVIPSGYENYYVVDVWGQGTTVTVSS

[0238] Accession number 38 (ZY0EO5-E07, variable light chain): DIQLTQSPSFLSASVGGRVTITCWASQGIAGYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHLNSYPLTFGGGTKVEIK

[0239] Accession number 39: (ZY0EP0-C07, variable heavy chain) QVQLVESGGVLVKPGGSLRLSCAASGFTLSDYYMSWIRQAPGMGLEWVSYISSSGSTIYYTDSVKGRFTISRDSAKNSLYLQMNSLRAEDTAVYYCARDGVGFDYWGQGTLVTVSS

[0240] Accession number 40 (ZY0EP0-C07, variable light chain): EIVLTQSPGTLSLFPGERATLSCRASQSVSSSYLAWYQQKPGQSPRLLIYAASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLYTFGQGTKLEIK

[0241] Accession number 41 (Maia heavy chain constant region, cysteine insertions are underlined): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS C VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0242] Accession number 42 (light chain constant region) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0243] Sequence number 43 (ZY0EQD-E02, variable heavy chain, e.g., pre-germline serialization, e.g., variant of sequence number 33 / sequence number 45) QVQLQQWGAGLLKPSETLSLTCTVYGGSFSGYYWNWIRQPPGRGLEWIGEINHSGSTSYNPSLKSRITISIDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSS

[0244] Sequence number 44 (ZY0EQD-E02, light chain): DIQMTQSPSSLSASVGDRVTITCRASQDIRNDVGWYQQKPGKAPKRLIYAASRLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0245] Sequence number 45 (EQD-E02_GL, variable heavy chain, GL = germline-serialized) QVQLQQWGAGLLKPSETLSLACTVYGGSFSGYYWNWIRQPPGKGLEWIGEINHSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSS

[0246] Sequence number 46 (EQD-E02-GLY, variable heavy chain, GLY = germline-serialized with Y substitution) QVQLQQWGAGLLKPSETLSLACTVYGGSFSGYYWNWIRQPPGKGLEWIGEI YHSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSS

[0247] Sequence number 47 (EQD-E02-GLQ, variable heavy chain, germline-serialized with GLQ = Q substitution) QVQLQQWGAGLLKPSETLSLACTVYGGSFSGYYWNWIRQPPGKGLEWIGEI Q HSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSS

[0248] Sequence number 48 (E02-GL-Maia-heavy chain, cysteine insertion underlined) QVQLQQWGAGLLKPSETLSLACTVYGGSFSGYYWNWIRQPPGKGLEWIGEINHSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS C VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0249] Sequence number 49 (E02-GLY-Maia-heavy chain, germline-serialized with GLY = Y substitution) QVQLQQWGAGLLKPSETLSLACTVYGGSFSGYYWNWIRQPPGKGLEWIGEIY HSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0250] Sequence number 50 (E02-GLQ-Maia-heavy chain, germline-serialized with GLQ = Q substitution) QVQLQQWGAGLLKPSETLSLACTVYGGSFSGYYWNWIRQPPGKGLEWIGEI Q HSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0251] Accession No. 51 (E02-GL-WT-heavy chain) QVQLQQWGAGLLKPSETLSLACTVYGGSFSGYYWNWIRQPPGKGLEWIGEINHSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0252] Accession No. 52 (heavy chain constant region) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0253] Accession No. 53 (human B7H4 nucleic acid sequence including 5' and 3' UTRs) GCCACCatggcttccctggggcagatcctcttctggagcataattagcatcatcattattctggctggagcaattgcactcatcattggctttggtatttcagggagacactccatcacagtcactactgtcgcctcagctgggaacattggggaggatggaatcctgagctgcacttttgaacctgacatcaaactttctgatatcgtgatacaatggctgaaggaaggtgttttaggcttggtccatgagttcaaagaaggcaaagatgagctgtcggagcaggatgaaatgttcagaggccggacagcagtgtttgctgatcaagtgatagttggcaatgcctctttgcggctgaaaaacgtgcaactcacagatgctggcacctacaaatgttatatcatcacttctaaaggcaaggggaatgctaaccttgagtataaaactggagccttcagcatgccggaagtgaatgtggactataatgccagctcagagaccttgcggtgtgaggctccccgatggttcccccagcccacagtggtctgggcatcccaagttgaccagggagccaacttctcggaagtctccaataccagctttgagctgaactctgagaatgtgaccatgaaggttgtgtctgtgctctacaatgttacgatcaacaacacatactcctgtatgattgaaaatgacattgccaaagcaacaggggatatcaaagtgacagaatcggagatcaaaaggcggagtcacctacagctgctaaactcaaaggcttctctgtgtgtctcttctttctttgccatcagctgggcacttctgcctctcagcccttacctgatgctaaaaTAATAA

[0254] SEQ ID NO: 54 (Human B7H4 Nucleic Acid Sequence, Coding Sequence) atggcttccctggggcagatcctcttctggagcataattagcatcatcattattctggctggagcaattgcactcatcattggctttggtatttcagggagacactccatcacagtcactactgtcgcctcagctgggaacattggggaggatggaatcctgagctgcacttttgaacctgacatcaaactttctgatatcgtgatacaatggctgaaggaaggtgttttaggcttggtccatgagttcaaagaaggcaaagatgagctgtcggagcaggatgaaatgttcagaggccggacagcagtgtttgctgatcaagtgatagttggcaatgcctctttgcggctgaaaaacgtgcaactcacagatgctggcacctacaaatgttatatcatcacttctaaaggcaaggggaatgctaaccttgagtataaaactggagccttcagcatgccggaagtgaatgtggactataatgccagctcagagaccttgcggtgtgaggctccccgatggttcccccagcccacagtggtctgggcatcccaagttgaccagggagccaacttctcggaagtctccaataccagctttgagctgaactctgagaatgtgaccatgaaggttgtgtctgtgctctacaatgttacgatcaacaacacatactcctgtatgattgaaaatgacattgccaaagcaacaggggatatcaaagtgacagaatcggagatcaaaaggcggagtcacctacagctgctaaactcaaaggcttctctgtgtgtctcttctttctttgccatcagctgggcacttctgcctctcagcccttacctgatgctaaaa

[0255] Sequence number 55 (human B7H4 polypeptide sequence, UniProt accession number Q7Z7D3) MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK

[0256]

Table 7-1

[0257]

Table 7-2

[0258]

Table 7-3

[0259]

Table 7-4

[0260]

Table 7-5

[0261]

Table 8-1

[0262]

Table 8-2

[0263]

Table 8-3

[0264]

Table 9

[0265]

Table 10

[0266]

Table 11

[0267]

Table 12

[0268]

Table 13

[0269]

Table 14-1

[0270]

Table 14-2

Examples

[0271] Example 1: TOP1i-ADC induces tumor antigenicity and ligand expression in multiple in vitro model systems In vitro studies were performed to determine whether an antibody-drug conjugate (ADC) increases tumor antigenicity and the expression of certain cancer immunotherapy (IO) ligands. MX1 tumor cells were cultured in vitro for 24 hours at 37 °C and 5% CO2 in DMEM:F-12 medium containing 10% fetal bovine serum (FBS). The cultured cells were exposed to an ADC having the same warhead targeting TROP2 (antibody clone hRS7, hRS7(TROP2)-TOP1i) and a control ADC having a topoisomerase 1 inhibitor warhead (iso-TOP1i) at a concentration of 1 μg / ml for 5 days. The cells were harvested by trypsinization, stained with fluorescently labeled antibodies against various cell surface expression ligands, and evaluated by flow cytometry for each ligand measured. The statistical significance of the comparisons shown was * shown as p < 0.05, ** p < 0.01. The fold increase in geometric mean fluorescence intensity of expression compared to the control is plotted in FIGS. 1A-1C. As shown in FIG. 1A, the expression of immunogenicity and the immunogenic cell death markers MHC-I and calreticulin are significantly increased by hRS7(TROP2)-TOP1i ADC (MHC-I, p = 0.008; calreticulin, p = 0.008). As shown in FIG. 1B, the expression of the PD-1 ligand PD-L1 and the TIM3 ligand phosphatidylserine, as indicated by binding of fluorescently labeled Annexin-V (Ann-V) protein, are significantly increased by both ADCs (PD-L1, p = 0.029; calreticulin, p = 0.0029). As shown in FIG. 1C, the expression of the TIGIT ligands CD155, nectin-2, and nectin-4 are increased by both ADCs, and a significant increase was observed for CD155 expression by hRS7(TROP2)-TOP1i (CD155, p = 0.008).

[0272] The same set of ADCs was tested for their ability to affect ligand expression on dendritic cells. Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of HLA-A * 02:01-positive healthy donors using a RoboSep(™)-S automated cell separator (StemCell Technologies, Cambridge, MA) in combination with an EasySep(™) Direct Human PBMC isolation kit (StemCell Technologies) according to the manufacturer's instructions. Subsequently, monocytes were isolated from PBMCs using a negative selection magnetic microbead-based assay kit (Miltenyi Biotec, Gaithersburg, MD). Monocytes were cultured for 6 days in RPMI-1640 medium supplemented with 10% FBS, 50 ng / mL of recombinant human interleukin 4, and 50 ng / mL of recombinant human GM-CSF, and the medium was replenished on day 3 to generate immature monocyte-derived dendritic cells. The immature dendritic cells were then co-cultured at a 1:1 ratio for 6 hours in RPMI-1640 medium supplemented with 10% FBS with PC9 lung adenocarcinoma cells engineered to express melanoma antigen MART-1. The cultured cells were exposed to the control and hRS7(TROP2)-TOP1i ADCs at a concentration of 3 μg / mL for 72 hours. Ligand expression was measured by flow cytometry as described in Example 1. The statistical significance of the comparisons shown is * p < 0.05, ** shown as p < 0.01. As shown in Figure 2A, hRS7(TROP2)-TOP1i ADC significantly increased the expression of the CTLA4 ligand CD80 on dendritic cells (p = 0.044). As shown in Figure 2B, co-culture of tumor cells and dendritic cells resulted in a significant increase in TIM3 expression on dendritic cells (p = 0.007). As shown in Figure 2C, hRS7(TROP2)-TOP1i significantly increased the expression of the TIGIT ligand nectin 2 / CD112 on dendritic cells (p = 0.006). These results demonstrate that TOP1i ADCs are capable of regulating the expression of antigens that can be targeted using cancer immunotherapy.

[0273] Example 2: TOP1i ADC combined with AZD2936 enhances in vitro T cell-mediated killing of tumor cells in a tumor immune cell co-culture model A co-culture assay system was developed using melanoma antigen Melan A (MART-1) reactive T cells and PC9 lung adenocarcinoma cells engineered to express both the MART-1 antigen and Green Fluorescent Protein (GFP). Tumor cell lysis was evaluated using the Sartorius Incucyte Live Cell Analysis System, whereby loss of GFP signal intensity was used as a surrogate marker of cell death. Briefly, PC9-MART1-GFP cells were harvested from cell culture flasks using trypsin, washed once with complete RPMI medium as defined in Example 1, resuspended, and added to each well of a 96-well clear-bottomed, opaque-wall optical plate at 37 °C in 5% CO2 for 24 h according to the manufacturer's instructions. The PD-1-TIGIT (AZD2936), PD-1-TIM3 (AZD7789), or PD-1-CTLA4 (MEDI5752) bispecific antibodies were used at a concentration of 2 nM. The hRS7 (TROP2)-TOP1i ADC was used at 0.05 μg / ml. Immediately prior to incubation, the treatments were added to the wells either alone or in combination, and antigen-specific CD8+ T cells were added at an effector to target (E:T) ratio of 4:1 or 5:1 effector T cells to target tumor cells, depending on the donor. Tumor cell death and cell lysis rates were measured by image analysis of GFP intensity per well 5 days after T cell addition. The data are from experiments using three individual donors. The statistical significance of the comparisons shown is * p < 0.05, ** shown as p < 0.01, p < 0.001. As seen in Figures 3A - C, each bispecific antibody and the hRS7 (TROP2)-TOP1i ADC enhanced T cell-mediated cell lysis of PC9 tumor cells. In all cases, the combination of the bispecific antibody and the hRS7 (TROP2)-TOP1i ADC reached the highest level of cell lysis.

[0274] Antigen-specific CD8+ T cells secrete IFN-γ upon recognition of tumor antigens. To evaluate the amount of IFN-γ detected in the supernatant of co-cultures containing PC9-MART1-GFP tumor cells and tumor-reactive CD8+ T cells, separate parallel co-cultures were established in Nunc 96-well plates (Sigma Aldrich) in parallel with the Incucyte optical plates. For the evaluation of IFN-γ release, the supernatant was harvested from the co-cultures in the Nunc 96-well plates at 24, 72, and 144 hours. Mesoscale Discovery multi-spot ELISA for human IFN-γ was used according to the manufacturer's instructions to measure IFN-γ. As seen in Figure 4A, T cells strongly induced IFN-γ secretion during co-culture of MART-1 antigen-specific T cells and antigen-expressing tumor cells, while IFN-γ secretion was not measured in the absence of T cells. For the MART-1 tumor antigen-reactive T cell-PC9 lung adenocarcinoma co-culture, IFN-γ secretion by T cells was maintained in the presence of both hRS7(TROP2)-TOP1i ADC alone, AZD2936 and MEDI5752 alone, and combinations with ADC (Figures 4C-4D). AZD7789 enhanced IFN-γ, which was maintained in combination with hRS7(TROP2)-TOP1i ADC, Figure 4E.

[0275] Example 3: In Vivo Combination Efficacy Observed with hHER2-TOP1i ADC and Anti-PD-L1 in the EMT6-hHER2 Model EMT6 mouse mammary cancer cells engineered to express human HER2 were subcutaneously implanted into the flanks of female Balb / C mice on study day 0. Mice were housed in groups of no more than 6 animals per cage with continuous access to food and water. Nine mice were tested for each therapy. Monotherapy mice were administered the following: vehicle only, NIP228 negative control antibody (10 mg / kg) and negative control ADC (NIP228-SG3932, 10 mg / kg), anti-HER2 ADC (anti-HER2-SG3932, 10 mg / kg), anti-PD-L1 antibody (10 mg / kg), and anti-PD-1 / TIGIT bispecific antibody (10 mg / kg). With the exception of the vehicle only control, all combination therapy mice were administered anti-HER2 ADC in combination with vehicle, anti-PD-L1 antibody, and anti-PD-1 / TIGIT bispecific antibody (each at 10 mg / kg). Administration was carried out according to the schedule outlined schematically in Figure 5A, with ADC and IO therapy administered on days 7 and 14, and IO therapy alone administered on days 10 and 17. Mean tumor volume was measured using digital calipers and the means were plotted in Figure 5B (monotherapy) and Figure 5C (combination). As can be seen by comparing Figure 5B and Figure 5C, combination therapy significantly reduced mean tumor volume and growth. Mice were sacrificed on day 72.

[0276] Plots of individual mouse tumor volumes for each therapy are shown in Figure 6. As shown for some of the plots, there were complete responders (CR) to PD1 / TIGIT bispecific monotherapy and ADC + PD-L1 antibody and ADC + PD1 / TIGIT bispecific antibody combination therapies, indicated by the fractions next to the plots.

[0277] Example 4: AZD8205 enhances efficacy in combination with anti-PD-L1 in a mouse B7H4-overexpressing colon cancer mouse model CT26 colon cancer cells overexpressing murine B7H4 were subcutaneously implanted into the flanks of Balb / C mice on day 0 of the study. The mice were housed and cared for as described in Example 3. Therapy was administered as schematically shown in Figure 7A, with the ADC administered on days 10 and 17, and the anti-PD-L1 antibody administered on days 10, 13, 17, and 20. The ADC targeting B7H4 with a TOP1i warhead and linker (SG3932) is designated AZD8205. The mice were administered the following: untreated control (UT), anti-PD-L1 antibody alone (10 mg / kg), isotype monomethyl auristatin E (MMAE) warhead and TOP1i (SG3932) ADC alone (each 7 mg / kg), ADC targeting B7H4 with either MMAE or TOP1i (SG3932) warhead alone (each 7 mg / kg), or a combination of anti-PD-L1 antibody and one of the ADCs (10 mg / kg anti-PD-L1, 7 mg / kg ADC). Mean tumor volume was measured and plotted in Figure 7B. As seen in the figure, the combination therapy enhanced the efficacy against tumor cell proliferation in the model compared to monotherapy.

[0278] Example 5: The combination of AZD8205 + anti-PD-L1 significantly increases tumor T cell and TIGIT expression in the mB7-H4 CT26 model A separate cohort of mice from the study outlined in Example 4 was administered the same treatments as those used in Example 4, according to the dosing schedule outlined schematically in FIG. 8A. The ADC and IO combination therapy was co-administered on days 10 and 17, while IO therapy alone was administered on days 13 and 20. This cohort of mice was terminated on day 18 of the study for pharmacodynamic (PD) evaluation of the immune cell composition of the tumors and tumor-draining lymph nodes. Tumors and lymph nodes were dissociated into single cell suspensions, stained with fluorescently labeled antibodies specific for cell surface identity markers of mouse immune cell populations, and analyzed by flow cytometry. The percentage of CD45+ immune cells was determined by dividing the number of CD45+ cells by the total number of viable single cells. The statistical significance of the comparisons shown was * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, as shown. As shown in FIG. 8B, the combination of AZD8205 and anti-PD-L1 significantly increased the percentage of CD3+ T cells within the total CD45+ immune cell population (p = 0.048 AZD8205 + anti-PD-L1 vs. untreated). The percentage of TIGIT+ CD4 and TIGIT+ CD8 T cells was determined by dividing the number of CD4 or CD8 and TIGIT double-positive cells by the total number of CD4 or CD8 positive T cells. As shown in FIG. 8C, the combination of AZD8205 and anti-PD-L1 significantly increased the percentage of TIGIT+ T cells for both CD4+ and CD8+ T cells. The survival of mice in each group was plotted in FIG. 8D. There was a significant increase in the median survival for the combination of AZD8205 and anti-PD-L1 compared to AZD8205 alone.

[0279] Example 6: Robust Antitumor Activity Using a Combination of B7-H4 ADC and PD-1-TIGIT Bispecificity in the mB7-H4 CT26 Model CT26 colon cancer cells overexpressing mouse B7-H4 were subcutaneously implanted into the flanks of Balb / C mice on day 0 of the study as described in Example 4. Therapies were administered as outlined in Figure 9A, with the ADC administered at 7 mg / kg on days 10 and 17, and the anti-PD-1 or PD-1-TIGIT bispecific antibody administered at 10 mg / kg on days 10, 13, 17, and 20. Plots of the individual mouse tumor volumes for each therapy are shown in Figure 9B. As shown for some of the plots, there were complete responders (CR) to the PD-1-TIGIT bispecific monotherapy and the AZD8205 monotherapy, but more complete responders were seen for the anti-PD1 / TIGIT bispecific antibody in combination with AZD8205, as indicated by the fractions next to the plots.

Claims

1. A pharmaceutical composition comprising an antibody-drug conjugate (ADC) for treating cancer in a human subject in combination with a bispecific checkpoint inhibitor, The ADC, i) an antibody or its antigen-binding fragment, ii) A linker that can be cut, iii) A cytotoxic agent, The cytotoxic agent is a compound of formula I: 【Chemistry 1】 as well as its salts and solvates, where R Ｌ However, it is a linker that can be cut, and the linker that can be cut is 【Chemistry 2】 (In the formula, Q is, 【Transformation 3】 Q Ｘ This is a case where Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue. X is, 【Chemistry 4】 Therefore, a = 0 to 5, b1 = 0 to 16, b2 = 0 to 16, c1 = 0 or 1, c2 = 0 or 1, d = 0 to 5, at least b1 or b2 = 0, at least c1 or c2 = 0, G Ｌ (This is a linker for connecting to the antibody or antigen-binding fragment), and 【Transformation 5】 (In the formula, R Ｌ１ and R Ｌ２ A pharmaceutical composition in which ( is independently selected from H and methyl, or together with the carbon atom to which they are bonded, to form a cyclopropylene or cyclobutylene group, and e is selected from 0 or 1).

2. A pharmaceutical composition comprising an antibody-drug conjugate (ADC) for treating cancer in a human subject in combination with a bispecific checkpoint inhibitor, The ADC, i) An antibody or its antigen-binding fragment that binds to the B7-H4 polypeptide, ii) A linker that can be cut, iii) A pharmaceutical composition comprising a cytotoxic agent.

3. The antibody or antigen-binding fragment of the ADC, Each of these comprises the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, including heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), or their functional variants. Each of these includes the amino acid sequences of SEQ ID NOs. 7, 8, 9, 10, 11, and 12, respectively, representing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or their functional variants. Each of these includes the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or their functional variants. Each of these includes the amino acid sequences of SEQ ID NOs. 19, 20, 21, 22, 23, and 24, respectively, and comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or their functional variants, Each of these includes the amino acid sequences of SEQ ID NOs. 25, 26, 27, 28, 29, and 30, respectively, and may be HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or their functional variants, or The pharmaceutical composition according to claim 1 or 2, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in a first binding arm, comprising the amino acid sequences of SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, and SEQ ID NO: 121, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in a second binding arm, comprising the amino acid sequences of SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO:

127.

4. The antibody or antigen-binding fragment of the ADC, Variable heavy (VH) chains and variable light (VL) chains, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 34, respectively. Variable heavy (VH) chains and variable light (VL) chains, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO: 34, respectively. Variable heavy (VH) chains and variable light (VL) chains, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 34, respectively. Variable heavy (VH) chains and variable light (VL) chains, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 46 and SEQ ID NO: 34, respectively. Variable heavy (VH) chains and variable light (VL) chains, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 47 and SEQ ID NO: 34, respectively. Each contains the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 32, respectively, a VH chain and a VL chain, or functional variants thereof. Each contains the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 36, respectively, a VH chain and a VL chain, or functional variants thereof. Each contains the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 38, respectively, a VH chain and a VL chain, or functional variants thereof, Each contains the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 40, respectively, including the VH chain and VL chain, or functional variants thereof, The pharmaceutical composition according to claim 1 or 2, comprising a VH chain and a VL chain in a first binding arm, or functional variants thereof, each comprising the amino acid sequences of SEQ ID NO: 128 and SEQ ID NO: 130, respectively, and a VH chain and a VL chain in a second binding arm, or functional variants thereof, each comprising the amino acid sequences of SEQ ID NO: 132 and SEQ ID NO: 134, respectively.

5. i) The antibody of the ADC or the antigen-binding fragment thereof comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 51 and a light chain containing the amino acid sequence of SEQ ID NO: 44, or ii) The antibody of the ADC or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 48 and a light chain containing the amino acid sequence of SEQ ID NO: 44, or iii) The antibody or antigen-binding fragment of the ADC is a bispecific antibody or antigen-binding fragment, each comprising a heavy chain and a light chain in the first binding arm, or a functional variant thereof, each containing the amino acid sequences of SEQ ID NO: 129 and SEQ ID NO: 131, and a heavy chain and a light chain in the second binding arm, or a functional variant thereof, each containing the amino acid sequences of SEQ ID NO: 133 and SEQ ID NO: 135, The pharmaceutical composition according to claim 1 or 2.

6. The pharmaceutical composition according to claim 1 or 2, wherein the cleavable linker of the ADC is an mp-PEG8-val-ala linker.

7. The cytotoxic agent is a topoisomerase inhibitor, and the ii) cleavable linker and iii) cytotoxic agent of the ADC are compounds of formula I: 【Transformation 6】 It also includes salts and solvates thereof, in which R Ｌ However, the linker is cuttable, and the linker is cuttable 【Transformation 7】 (In the formula, Q is, 【Transformation 8】 Q Ｘ This is a case where Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue. X is, 【Chemistry 9】 Therefore, a = 0 to 5, b1 = 0 to 16, b2 = 0 to 16, c1 = 0 or 1, c2 = 0 or 1, d = 0 to 5, at least b1 or b2 = 0, at least c1 or c2 = 0, G Ｌ (This is a linker for connecting to the antibody or antigen-binding fragment), and 【Chemistry 10】 (wherein R Ｌ１ and R Ｌ２ are each independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group, and e is 0 or 1), the pharmaceutical composition according to claim 2.

8. The following compounds combine the ii) cleavable linker and iii) cytotoxic agent: 【Chemistry 11】 A pharmaceutical composition according to claim 1 or 2, comprising:

9. The aforementioned bispecific checkpoint inhibitor a) A first binding domain that specifically binds to PD-1 or PD-L1, b) The pharmaceutical composition according to claim 1 or 2, comprising a second binding domain that specifically binds to a T cell immune receptor (TIGIT) having an Ig and an ITIM domain, a T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3), or a cytotoxic T lymphocyte-associated antigen-4 (CTLA 4).

10. The pharmaceutical composition according to claim 9, wherein the first binding domain of the bispecificity checkpoint inhibitor comprises a heavy chain variable domain that specifically binds to PD-1 and includes HCDR1 having the amino acid sequence of SEQ ID NO: 56, HCDR2 having the amino acid sequence of SEQ ID NO: 57, and HCDR3 having the amino acid sequence of SEQ ID NO: 58, and a light chain variable domain that includes LCDR1 having the amino acid sequence of SEQ ID NO: 59, LCDR2 having the amino acid sequence of SEQ ID NO: 60, and LCDR3 having the amino acid sequence of SEQ ID NO:

61.

11. The pharmaceutical composition according to claim 10, wherein the first binding domain of the bispecificity checkpoint inhibitor comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 62 and a light chain variable domain having the amino acid sequence of SEQ ID NO:

64.

12. The pharmaceutical composition according to claim 10, wherein the first binding domain of the bispecificity checkpoint inhibitor comprises a heavy chain having the amino acid sequence of SEQ ID NO: 63 and a light chain having the amino acid sequence of SEQ ID NO:

65.

13. i) The second binding domain of the bispecificity checkpoint inhibitor comprises a heavy chain variable domain that specifically binds to TIGIT and includes HCDR1 having the amino acid sequence of SEQ ID NO: 66, HCDR2 having the amino acid sequence of SEQ ID NO: 67, and HCDR3 having the amino acid sequence of SEQ ID NO: 68, and a light chain variable domain that includes LCDR1 having the amino acid sequence of SEQ ID NO: 69, LCDR2 having the amino acid sequence of SEQ ID NO: 70, and LCDR3 having the amino acid sequence of SEQ ID NO: 71, or ii) The second binding domain of the bispecific checkpoint inhibitor specifically binds to TIM-3 and includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each containing the amino acid sequence of SEQ ID NOs: 84, 85, 86, 87, 88, and 89, or each containing the amino acid sequence of SEQ ID NOs: 84, 85, 86, 87, 88, and 90, or iii) The second binding domain of the bispecific checkpoint inhibitor specifically binds to CTLA-4 and includes VH CDR1 containing the amino acid sequence of SEQ ID NO: 109, VH CDR2 containing the amino acid sequence of SEQ ID NO: 110, VH CDR3 containing the amino acid sequence of SEQ ID NO: 111, VL CDR1 containing the amino acid sequence of SEQ ID NO: 112, VL CDR2 containing the amino acid sequence of SEQ ID NO: 113, and VL CDR3 containing the amino acid sequence of SEQ ID NO:

114. The pharmaceutical composition according to claim 9.

14. i) The second binding domain of the bispecific checkpoint inhibitor comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 72 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 74, or ii) The second binding domain of the bispecificity checkpoint inhibitor specifically binds to TIM-3 and includes a second heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 91 and a second light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 94, or iii) The second binding domain of the bispecific checkpoint inhibitor specifically binds to CTLA-4 and includes a heavy chain containing the amino acid sequence described in SEQ ID NO: 105 and a light chain containing the amino acid sequence described in SEQ ID NO:

104. The pharmaceutical composition according to claim 13.

15. The pharmaceutical composition according to claim 14, wherein the second binding domain specifically binds to TIGIT and comprises a heavy chain having the amino acid sequence of SEQ ID NO: 73 and a light chain having the amino acid sequence of SEQ ID NO:

75.

16. The pharmaceutical composition according to claim 1 or 2, wherein the cancer is selected from ovarian cancer, breast cancer, uterine cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, renal cell carcinoma, pancreatic cancer, prostate cancer, cervical cancer, hematological cancer, endometrial cancer, cholangiocarcinoma, NSCLC (squamous cell carcinoma and / or adenocarcinoma), gastrointestinal cancers such as stomach cancer and colorectal cancer, and lung cancer.

17. The pharmaceutical composition according to claim 1 or 2, wherein the cancer is a breast cancer selected from hormone receptor-positive (HR+) breast cancer, human epidermal growth factor receptor 2-positive (HER2+) breast cancer, and triple-negative breast cancer (TNBC).

18. The aforementioned cancer is homologous recombination deficiency (HRD) cancer. The pharmaceutical composition according to claim 1 or 2, wherein the cancer comprises one or more cells having a mutation in the HRD gene selected from BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L.

19. A pharmaceutical composition comprising 1) an antibody-drug conjugate and 2) a bispecificity checkpoint inhibitor, wherein the antibody-drug conjugate comprises an antibody or its antigen-binding fragment and a drug linker represented by the following formula: 【Chemistry 12】 and, A pharmaceutical composition in which the drug linker is conjugated to the antibody or its antigen-binding fragment.

20. A pharmaceutical composition according to claim 19 for use in the treatment of cancer.

21. A kit comprising 1) an antibody-drug conjugate and 2) a bispecificity checkpoint inhibitor, wherein the antibody-drug conjugate comprises an antibody or an antigen-binding fragment thereof and a drug linker represented by the following formula: 【Chemistry 13】 and, A kit in which the drug linker is conjugated to the antibody or its antigen-binding fragment.

Images