Combination of antibody-drug conjugates and anti-pd-1 / tim-3 bispecific binding proteins

Abstract

A pharmaceutical product is provided for combined administration of an antibody-drug conjugate and an anti-PD-1 / TIM-3 bispecific binding protein. The antibody-drug conjugate is an antibody-drug conjugate wherein a drug-linker represented by the following formula (where A represents the linking position with the antibody) is conjugated to an antibody via a thioether bond, specifically an anti-TROP2 or anti-HER2 antibody. A therapeutic use and method are also provided, wherein the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein are administered in combination to a subject.

Description

[0001] This specification claims priority to U.S. Provisional Application No. 63 / 545,492, filed October 24, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0002] This disclosure relates to a pharmaceutical product for administering a combination of a specific antibody-drug conjugate and a bispecific binding protein (specifically, an anti-PD-1 / TIM-3 bispecific binding protein), and to therapeutic use and methods thereof in which the combination of the antibody-drug conjugate and the bispecific binding protein is administered to a subject, the specific antibody-drug conjugate having an antitumor drug conjugated to an antibody (specifically, an anti-TROP2 or anti-HER2 antibody) via a drug-linker structure. Background Technology

[0003] Cancer remains a major global health burden. Despite advances in immuno-oncology, the medical need for effective therapies remains unmet, particularly for patients with acquired resistance to immuno-oncology (IO).

[0004] Numerous molecular targets have been identified as potential targets for immunotherapy (IO) in cancer treatment. Some molecular targets under investigation for their therapeutic potential in immuno-oncology include cytotoxic T-lymphocyte antigen-4 (CTLA-4 or CD152), programmed death-ligand 1 (PD-L1 or B7-H1 or CD274), programmed death-1 (PD-1), OX40 (CD134 or TNFRSF4), and the T-cell inhibitory receptor T-cell immunoglobulin and mucin-containing domain-3 (TIM-3). However, not all patients respond to immunotherapy, and some cease to respond over time. The reasons for this acquired resistance to IO have puzzled researchers.

[0005] Therefore, it remains necessary to continue identifying candidate targets for immunotherapy (specifically, immunotherapies that overcome acquired resistance to immunotherapy and enhance patient responses beyond those currently evaluated in clinical practice).

[0006] In this regard, bispecific binding proteins that specifically bind to two immune checkpoint targets are under development. Examples include the anti-PD-1 / CTLA-4 bispecific antibody AK104 (canducurimab) and MEDI5752 (US Patent No. 10,457,732), which contain a first domain specifically binding to PD-1 and a second domain specifically binding to CTLA-4, and the anti-PD-1 / TIM-3 bispecific antibody AZD7789, which contains a first domain specifically binding to PD-1 and a second domain specifically binding to TIM-3. AZD7789 is undergoing clinical development for solid tumors, including squamous and non-squamous non-small cell lung cancer. For example, the anti-PD-1 / CTLA-4 and anti-PD-1 / TIM-3 bispecific antibodies are disclosed in WO2017 / 193032. AZD7789 is further disclosed in WO2022 / 221245.

[0007] Antibody-drug conjugates (ADCs), consisting of cytotoxic drugs conjugated to antibodies, can selectively deliver drugs to cancer cells and within the cancer cells, leading to cancer cell death (Ducry, L. et al., Bioconjugate Chem. (2010) 21, 5-13; Alley, SC et al., Current Opinion in Chemical Biology (2010) 14, 529-537; Damle NK Expert Opin.Biol.Ther. (2004) 4, 1445-1452; Senter PD et al., Nature Biotechnology (2012) 30, 631-637; Burris HA et al., J.Clin.Oncol. (2011) 29(4): 398-405).

[0008] One such antibody-drug conjugate is datopotamab deruxtecan (Dato-DXd, DS-1062), which consists of an antibody targeting TROP2 and a derivative of essanotecan. Specifically, WO2015 / 098099 and WO2020 / 240467 provide detailed descriptions of exemplary antibody-drug conjugates (including datopotamab) targeting TROP2. Datopotamab has demonstrated clinical efficacy in various tumor types, including lung cancer and breast cancer.

[0009] Another such antibody-drug conjugate is trastuzumab deruxtecan, which consists of an antibody targeting HER2 and a derivative of exatecan (Ogitani Y. et al., Clinical Cancer Research (2016) 22(20), 5097-5108; Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046).

[0010] Enhertu (trastuzumab) ® DS-8201 has demonstrated significant clinical efficacy in HER2-expressing solid tumors, including breast cancer, gastric cancer, colorectal cancer, and non-small cell lung cancer. Notably, among the aforementioned indications, DS-8201 has shown promising activity in tumors with low HER2 expression.

[0011] References that disclose the combined administration of antibody-drug conjugates and immune checkpoint inhibitors include Müller P. et al., Science Translational Medicine (2015) 7(315), 315ra188 (trastuzumab emtansine (T-DM1) in combination with anti-CTLA-4 and anti-PD-1 antibodies); and WO2018 / 110515 (trastuzumab dexamethasone (DS-8201) in combination with anti-PD-1, anti-PD-L1, anti-CD4 and anti-CD8 antibodies).

[0012] However, further identification of combination partners of antibody-drug conjugates (including anti-TROP2 antibody-drug conjugates such as DS-1062 and anti-HER2 antibody-drug conjugates such as DS-8201) is needed to enhance their therapeutic potential.

[0013] While antibody-drug conjugates such as DS-8201 and DS-1062 show therapeutic potential as monotherapy or in combination with checkpoint inhibitors, and bispecific checkpoint inhibitors also hold therapeutic potential, there remains a need for improved therapeutic compositions and methods that can enhance the efficacy of existing cancer therapies, increase the durability of treatment responses, improve patient tolerability, reduce dose-dependent toxicity, and / or provide alternative therapies for cancers that exhibit resistance or refractory behavior to prior cancer treatments. More specifically, there remains a need to further identify conjugates for combination with antibody-drug conjugates (specifically with anti-HER2 antibody-drug conjugates such as DS-1062 and anti-TROP2 antibody-drug conjugates such as DS-8201) to enhance their therapeutic potential. Therefore, it is desirable to provide a drug and treatment that achieves superior antitumor effects in the treatment of cancer, such as enhanced efficacy, increased durability of treatment responses, and / or reduced dose-dependent toxicity. Summary of the Invention

[0014] This disclosure provides a pharmaceutical product that exhibits superior antitumor efficacy in cancer treatment through the administration of an antibody-drug conjugate, specifically an anti-TROP2 antibody-drug conjugate or an anti-HER2 antibody-drug conjugate in combination with an anti-PD-1 / TIM-3 bispecific binding protein. This disclosure also provides a therapeutic use and method in which the antibody-drug conjugate and the bispecific binding protein are administered in combination to a subject.

[0015] Specifically, this disclosure relates to the following[1] to

[70] :

[0016] [1] A pharmaceutical product comprising an antibody-drug conjugate and an anti-PD-1 / TIM-3 bispecific binding protein for combined administration, wherein the antibody-drug conjugate is a drug linker represented by the following formula:

[0017]

[0018] Where A represents the linking site with the antibody, which is an antibody-drug conjugate to anti-TROP2 or anti-HER2 antibody via a thioether bond;

[0019] [2] The pharmaceutical product according to [1], wherein the drug-linker is conjugated to the anti-TROP2 antibody;

[0020] [3] According to the pharmaceutical product of [2], wherein the anti-TROP2 antibody is an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises CDRH1 composed of the amino acid sequence represented by SEQ ID NO: 3, CDRH2 composed of the amino acid sequence represented by SEQ ID NO: 4 and CDRH3 composed of the amino acid sequence represented by SEQ ID NO: 5, and the light chain comprises CDRL1 composed of the amino acid sequence represented by SEQ ID NO: 6, CDRL2 composed of the amino acid sequence represented by SEQ ID NO: 7 and CDRL3 composed of the amino acid sequence represented by SEQ ID NO: 8;

[0021] [4] According to the pharmaceutical product of [3], wherein the anti-TROP2 antibody is an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 9, and the light chain comprises a light chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 10;

[0022] [5] The pharmaceutical product according to [3] or [4], wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence represented by SEQ ID NO:12 and a light chain consisting of an amino acid sequence represented by SEQ ID NO:13;

[0023] [6] The pharmaceutical product according to [5] wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain;

[0024] [7] The pharmaceutical product according to any one of [2] to [6], wherein the average number of units of the drug-linker conjugated to each antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.5;

[0025] [8] The pharmaceutical product according to [7], wherein the anti-TROP2 antibody-drug conjugate is drutecan (DS-1062).

[0026] [9] The pharmaceutical product according to [1], wherein the drug-linker is conjugated to the anti-HER2 antibody;

[0027]

[10] According to the pharmaceutical product of [9], wherein the antiHER2 antibody is an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 16, CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 17 and CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 18, and the light chain comprises CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 19, CDRL2 consisting of the amino acid sequence consisting of amino acid residues 1 to 3 of SEQ ID NO: 20 and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 21;

[0028]

[11] According to the pharmaceutical product of

[10] , wherein the antiHER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 22, and the light chain comprising a light chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 23;

[0029]

[12] The pharmaceutical product according to

[11] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 14 and a light chain consisting of an amino acid sequence represented by SEQ ID NO: 15;

[0030]

[13] The pharmaceutical product according to

[11] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 24 and a light chain consisting of an amino acid sequence represented by SEQ ID NO: 15;

[0031]

[14] The pharmaceutical product according to any one of [9] to

[13] , wherein the average number of units of the drug-linker conjugated to each antiHER2 antibody molecule in the antibody-drug conjugate is in the range of 7 to 8;

[0032]

[15] The pharmaceutical product according to

[14] , wherein the anti-HER2 antibody-drug conjugate is trastuzumab (DS-8201).

[0033]

[16] The pharmaceutical product according to any one of [1] to

[15] , wherein the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain that specifically binds to the C'C'' and DE loops of the immunoglobulin variable (IgV) domain (SEQ ID NO: 45) of TIM-3;

[0034]

[17] The pharmaceutical product according to any one of [1] to

[16] , wherein the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain, the TIM-3 binding domain specifically binding to an epitope on the IgV domain of TIM-3 (SEQ ID NO:45), and the epitope includes N12, L47, R52, D53, V54, N55, Y56, W57, W62, L63, N64, G65, D66, F67, R68, K69, D71, T75 and E77;

[0035]

[18] The pharmaceutical product according to any one of claims [1] to

[17] , wherein the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain, the TIM-3 binding domain comprising a first set of CDRs: CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprising the amino acid sequences of SEQ ID NO: 35, 36, 37, 38, 39 and 40 or 35, 36, 37, 38, 39 and 45 respectively; and a PD-1 binding domain, the PD-1 binding domain comprising a second set of CDRs: CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprising the amino acid sequences of SEQ ID NO: 25, 26, 27, 28, 29 and 30 respectively;

[0036]

[19] The pharmaceutical product according to any one of [1] to

[18] , wherein the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 41 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 43, and has a PD-1 binding domain comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 31 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 33;

[0037]

[20] The pharmaceutical product according to any one of [1] to

[18] , wherein the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 42 and a light chain containing the amino acid sequence of SEQ ID NO: 43, and has a PD-1 binding domain comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 32 and a light chain containing the amino acid sequence of SEQ ID NO: 34;

[0038]

[21] The pharmaceutical product according to any one of [1] to

[20] , wherein the anti-PD-1 / TIM-3 bispecific binding protein is an antibody;

[0039]

[22] The pharmaceutical product according to

[21] , wherein the antibody is an IgG antibody;

[0040]

[23] The pharmaceutical product according to

[21] , wherein the antibody is an IgG1 antibody;

[0041]

[24] The pharmaceutical product according to

[22] or

[23] , wherein the antibody is human or humanized;

[0042]

[25] The pharmaceutical product according to any one of

[21] to

[24] , wherein the bispecific antibody is monovalent;

[0043]

[26] The pharmaceutical product according to any one of [1] to

[25] , wherein the anti-PD-1 / TIM-3 bispecific binding protein is DuetMab;

[0044]

[27] The pharmaceutical product according to any one of [1] to

[26] , wherein the anti-PD-1 / TIM-3 bispecific binding protein comprises a non-glycosylated Fc region;

[0045]

[28] The pharmaceutical product according to any one of [1] to

[26] , wherein the anti-PD-1 / TIM-3 bispecific binding protein comprises a deglycosylated Fc region;

[0046]

[29] The pharmaceutical product according to any one of [1] to

[26] , wherein the anti-PD-1 / TIM-3 bispecific binding protein comprises an Fc region having reduced fucosylation or being non-fucosylated;

[0047]

[30] The pharmaceutical product according to any one of [1] to

[29] , wherein the anti-PD-1 / TIM-3 bispecific binding protein is “AZD7789” or “sabestomig”;

[0048]

[31] The pharmaceutical product according to any one of [1] to

[30] , wherein the product is a composition comprising the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein for simultaneous administration;

[0049]

[32] The pharmaceutical product according to any one of [1] to

[30] , wherein the product is a combination formulation comprising the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein for sequential or separate simultaneous administration;

[0050]

[33] The pharmaceutical product according to any one of [1] to

[32] , wherein the product is used to treat cancer;

[0051]

[34] According to the pharmaceutical product of

[33] , the cancer is at least one of the following: breast cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, adenocarcinoma of the esophagogastric junction, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial carcinoma, prostate cancer, bladder cancer, endometrial cancer, gastrointestinal stromal tumor, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, sarcoma, and melanoma, cervical cancer, uterine cancer, testicular cancer and renal cell carcinoma;

[0052]

[35] The pharmaceutical product according to

[34] , wherein the cancer is lung cancer;

[0053]

[36] The pharmaceutical product according to

[35] , wherein the lung cancer is non-small cell lung cancer;

[0054]

[37] The pharmaceutical product according to

[34] , wherein the cancer is colorectal cancer;

[0055]

[38] The pharmaceutical product according to

[34] , wherein the cancer is breast cancer;

[0056]

[39] The pharmaceutical product according to

[38] , wherein the breast cancer is HER2-positive breast cancer;

[0057]

[40] The pharmaceutical product according to

[38] , wherein the breast cancer is HER2-low expression breast cancer;

[0058]

[41] The pharmaceutical product according to

[38] , wherein the breast cancer is triple-negative breast cancer;

[0059]

[42] The pharmaceutical product according to

[38] , wherein the breast cancer is hormone receptor (HR) positive, HER2 negative breast cancer;

[0060]

[43] The pharmaceutical product according to

[34] , wherein the cancer is gastric cancer;

[0061]

[44] The pharmaceutical product according to

[34] , wherein the cancer is pancreatic cancer;

[0062]

[45] The pharmaceutical product according to

[34] , wherein the cancer is ovarian cancer;

[0063]

[46] The pharmaceutical product according to

[34] , wherein the cancer is prostate cancer;

[0064]

[47] The pharmaceutical product according to

[34] , wherein the cancer is kidney cancer;

[0065]

[48] ​​The pharmaceutical product according to

[34] , wherein the cancer is bladder cancer;

[0066]

[49] The pharmaceutical product according to

[34] , wherein the cancer is endometrial cancer;

[0067]

[50] The pharmaceutical product according to

[34] , wherein the cancer is biliary tract cancer;

[0068]

[51] An antibody-drug conjugate for use in combination with an anti-PD-1 / TIM-3 bispecific binding protein in the treatment of a subject with cancer, wherein the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein are as defined in any one of [1] to

[30] ;

[0069]

[52] The antibody-drug conjugate for use according to

[51] , wherein the cancer is defined as in any one of

[34] to

[50] ;

[0070]

[53] The antibody-drug conjugate for use according to

[51] or

[52] , wherein the use comprises sequentially administering the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein;

[0071]

[54] The antibody-drug conjugate for use as described in

[51] or

[52] , wherein the use includes administering the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein alone and simultaneously;

[0072]

[55] The antibody-drug conjugate for use according to any one of

[51] to

[54] , wherein the anti-PD-1 / TIM-3 bispecific binding protein is administered at a dose of about 750 mg or about 1500 mg;

[0073]

[56] The antibody-drug conjugate for use according to

[55] is wherein the anti-PD-1 / TIM-3 bispecific binding protein is administered once per treatment cycle, wherein the treatment cycle is approximately three weeks;

[0074]

[57] The antibody-drug conjugate for use according to any one of

[51] to

[56] , wherein the cancer is non-small cell lung cancer, and wherein the subject is untreated, or wherein the subject has acquired resistance to CPI;

[0075]

[58] The antibody-drug conjugate for use as described in

[57] , wherein the subject has any PD-L1 status;

[0076]

[59] The antibody-drug conjugate for use according to

[58] , wherein the subject has 1% to 49% PD-L1 status, or wherein the subject has less than 1% PD-L1 status;

[0077]

[60] The antibody-drug conjugate for use according to any one of

[51] to

[59] , wherein the subject does not have an operable genomic alteration;

[0078]

[61] A method of treating cancer, the method comprising administering, to a subject in need, in combination, an antibody-drug conjugate and an anti-PD-1 / TIM-3 bispecific binding protein as described in any one of [1] to

[30] ;

[0079]

[62] The method according to

[61] , wherein the cancer is as defined in any one of

[34] to

[50] ;

[0080]

[63] The method according to

[61] or

[62] , wherein the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein are administered sequentially;

[0081]

[64] The method according to

[61] or

[62] , wherein the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein are administered alone and simultaneously;

[0082]

[65] The method according to any one of

[61] to

[64] , wherein the anti-PD-1 / TIM-3 bispecific binding protein is administered at a dose of about 750 mg to about 1500 mg;

[0083]

[66] The method according to

[65] , wherein the anti-PD-1 / TIM-3 bispecific binding protein is administered once per treatment cycle, wherein the treatment cycle is approximately three weeks;

[0084]

[67] The method according to any one of

[61] to

[66] , wherein the cancer is non-small cell lung cancer, and wherein the subject is untreated, or wherein the subject has acquired resistance to CPI;

[0085]

[68] The method according to

[67] , wherein the subject has any PD-L1 status;

[0086]

[69] According to the method of

[68] , wherein the subject has 1% to 49% PD-L1 status, or wherein the subject has less than 1% PD-L1 status; and

[0087]

[70] The method according to any one of

[61] to

[69] , wherein the subject does not have operable genomic alterations.

[0088] [Beneficial effects of this disclosure]

[0089] This disclosure provides a pharmaceutical product comprising a specific antibody-drug conjugate having an antitumor drug conjugated to an antibody (specifically an anti-TROP2 or anti-HER2 antibody) via a linker structure and an anti-PD-1 / TIM-3 bispecific binding protein for combination administration, and therapeutic use and methods thereof for combination administration of the antibody-drug conjugate and the bispecific binding protein to a subject. Therefore, this disclosure provides a medicament and treatment that can achieve excellent antitumor effects in the treatment of cancer. Attached Figure Description

[0090] [Anti-TROP2 antibody] :

[0091] Figure 1 This is a diagram showing the amino acid sequence (SEQ ID NO: 1) of the heavy chain of the anti-TROP2 antibody.

[0092] Figure 2 This is a diagram showing the amino acid sequence (SEQ ID NO: 2) of the light chain of the anti-TROP2 antibody.

[0093] Figure 3 This is a diagram showing the amino acid sequence of the heavy chain CDRH1 (SEQ ID NO: 3 [= amino acid residues 50 to 54 of SEQ ID NO: 1]).

[0094] Figure 4 This is a diagram showing the amino acid sequence of the heavy chain CDRH2 (SEQ ID NO: 4 [= amino acid residues 69 to 85 of SEQ ID NO: 1]).

[0095] Figure 5 This is a diagram showing the amino acid sequence of the heavy chain CDRH3 (SEQ ID NO: 5 [= amino acid residues 118 to 129 of SEQ ID NO: 1]).

[0096] Figure 6 This is a diagram showing the amino acid sequence of the light chain CDRL1 (SEQ ID NO: 6 [= amino acid residues 44 to 54 of SEQ ID NO: 2]).

[0097] Figure 7 This is a diagram showing the amino acid sequence of the light chain CDRL2 (SEQ ID NO: 7 [= amino acid residues 70 to 76 of SEQ ID NO: 2]).

[0098] Figure 8 This is a diagram showing the amino acid sequence of the light chain CDRL3 (SEQ ID NO: 8 [= amino acid residues 109 to 117 of SEQ ID NO: 2]).

[0099] Figure 9 This is a diagram showing the amino acid sequence of the variable region of the heavy chain (SEQ ID NO: 9 [= amino acid residues 20 to 140 of SEQ ID NO: 1]).

[0100] Figure 10 This is a diagram showing the amino acid sequence of the variable region of the light chain (SEQ ID NO: 10 [= amino acid residues 21 to 129 of SEQ ID NO: 2]).

[0101] Figure 11 This is a diagram showing the amino acid sequence of the heavy chain (SEQ ID NO: 11 [= amino acid residues 20 to 469 of SEQ ID NO: 1]).

[0102] [Anti-HER2 antibody] :

[0103] Figure 12 This is a diagram showing the amino acid sequence (SEQ ID NO: 14) of the heavy chain of the anti-HER2 antibody.

[0104] Figure 13 This is a diagram showing the amino acid sequence (SEQ ID NO: 15) of the light chain of the anti-HER2 antibody.

[0105] Figure 14 This is a diagram showing the amino acid sequence of the heavy chain CDRH1 (SEQ ID NO: 16 [= amino acid residues 26 to 33 of SEQ ID NO: 14]).

[0106] Figure 15 This is a diagram showing the amino acid sequence of the heavy chain CDRH2 (SEQ ID NO: 17 [= amino acid residues 51 to 58 of SEQ ID NO: 14]).

[0107] Figure 16 This is a diagram showing the amino acid sequence of the heavy chain CDRH3 (SEQ ID NO: 18 [= amino acid residues 97 to 109 of SEQ ID NO: 14]).

[0108] Figure 17 This is a diagram showing the amino acid sequence of the light chain CDRL1 (SEQ ID NO: 19 [= amino acid residues 27 to 32 of SEQ ID NO: 15]).

[0109] Figure 18 This is a diagram showing the amino acid sequence (SEQ ID NO: 20 [= amino acid residues 50 to 56 of SEQ ID NO: 15]) containing the light chain CDRL2 (SAS).

[0110] Figure 19 This is a diagram showing the amino acid sequence of the light chain CDRL3 (SEQ ID NO: 21 [= amino acid residues 89 to 97 of SEQ ID NO: 15]).

[0111] Figure 20 This is a diagram showing the amino acid sequence of the variable region of the heavy chain (SEQ ID NO: 22 [= amino acid residues 1 to 120 of SEQ ID NO: 14]).

[0112] Figure 21 This is a diagram showing the amino acid sequence of the variable region of the light chain (SEQ ID NO: 23 [= amino acid residues 1 to 107 of SEQ ID NO: 15]).

[0113] Figure 22 This is a diagram showing the amino acid sequence of the heavy chain (SEQ ID NO: 24 [= amino acid residues 1 to 449 of SEQ ID NO: 14]).

[0114] [Anti-PD-1 / TIM-3 bispecific binding protein] :

[0115] Figure 23 This is a diagram showing the amino acid sequence (SEQ ID NO: 24) of the AZD7789 anti-PD1 heavy chain CDRH1.

[0116] Figure 24 This is a diagram showing the amino acid sequence (SEQ ID NO: 26) of the AZD7789 anti-PD1 heavy chain CDRH2.

[0117] Figure 25 This is a diagram showing the amino acid sequence (SEQ ID NO: 27) of the AZD7789 anti-PD1 heavy chain CDRH3.

[0118] Figure 26 This is a diagram showing the amino acid sequence (SEQ ID NO: 28) of the AZD7789 anti-PD1 light chain CDRL1.

[0119] Figure 27 This is a diagram showing the amino acid sequence (SEQ ID NO: 29) of the AZD7789 anti-PD1 light chain CDRL2.

[0120] Figure 28 This is a diagram showing the amino acid sequence (SEQ ID NO: 30) of the AZD7789 anti-PD1 light chain CDRL3.

[0121] Figure 29 This is a diagram showing the amino acid sequence (SEQ ID NO: 31) of the variable region of the AZD7789 anti-PD1 heavy chain.

[0122] Figure 30 This is a diagram showing the amino acid sequence (SEQ ID NO: 32) of the AZD7789 anti-PD1 heavy chain.

[0123] Figure 31 This is a diagram showing the amino acid sequence (SEQ ID NO: 33) of the variable region of the AZD7789 anti-PD1 light chain.

[0124] Figure 32 This is a diagram showing the amino acid sequence (SEQ ID NO: 34) of the AZD7789 anti-PD1 light chain.

[0125] Figure 33 This is a diagram showing the amino acid sequence (SEQ ID NO: 35) of the AZD7789 anti-TIM-3 heavy chain CDRH1.

[0126] Figure 34 This is a diagram showing the amino acid sequence (SEQ ID NO: 36) of the AZD7789 anti-TIM-3 heavy chain CDRH2.

[0127] Figure 35 This is a diagram showing the amino acid sequence (SEQ ID NO: 37) of the AZD7789 anti-TIM-3 heavy chain CDRH3.

[0128] Figure 36 This is a diagram showing the amino acid sequence (SEQ ID NO: 38) of the AZD7789 anti-TIM-3 heavy chain CDRL1.

[0129] Figure 37 This is a diagram showing the amino acid sequence (SEQ ID NO: 39) of the AZD7789 anti-TIM-3 heavy chain CDRL2.

[0130] Figure 38 This is a diagram showing the amino acid sequence (SEQ ID NO: 40) of the AZD7789 anti-TIM-3 heavy chain CDRL3.

[0131] Figure 39 This is a diagram showing the amino acid sequence (SEQ ID NO: 41) of the variable region of the AZD7789 anti-TIM-3 heavy chain.

[0132] Figure 40 This is a diagram showing the amino acid sequence (SEQ ID NO: 42) of the AZD7789 anti-TIM-3 heavy chain.

[0133] Figure 41 This is a diagram showing the amino acid sequence (SEQ ID NO: 43) of the variable region of the AZD7789 anti-TIM-3 light chain.

[0134] Figure 42 This is a diagram showing the amino acid sequence (SEQ ID NO: 44) of the AZD7789 anti-TIM-3 light chain.

[0135] Figure 43 This is a diagram showing the amino acid sequence (SEQ ID NO: 45) of the human TIM-3 IgV domain.

[0136] [experiment] :

[0137] Figure 44 and Figure 45 The figure shows the tumor cell lysis activity of DS-1062, AZD7789 and their combinations in a tumor-immune cell co-culture model.

[0138] Figure 46 and 47 The figure shows the antitumor activity of DS-8201, AZD7789, and their combinations in humanized mice.

[0139] Figure 48 The graph shows the pharmacokinetic changes in humanized mice associated with DS-8201 alone or in combination with AZD7789. Detailed Implementation

[0140] To facilitate a better understanding of this disclosure, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0141] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the use of many terms in this disclosure.

[0142] Unless the context otherwise requires, singular terms should include plural terms and plural terms should include singular terms.

[0143] Units, prefixes, and symbols are represented in their internationally recognized (SI) form. Numerical ranges include the values ​​that define that range.

[0144] It should be understood that wherever the language “contains” is used to describe aspects in this document, other similar aspects described as “consisting of” and / or “substantially composed of” are also provided.

[0145] The terms “inhibit” and “inhibition” can refer to a reduction in biological activity of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Cell proliferation can be determined using techniques recognized in the art that measure the rate of cell division, and / or the fraction of cells within a cell population undergoing cell division, and / or the rate of cell loss from the cell population due to terminal differentiation or cell death (e.g., thymidine incorporation).

[0146] The term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, rodents, etc., who will be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably in this document in relation to human subjects.

[0147] The term "pharmaceutical product" refers to a formulation whose form allows the active ingredient to have biological activity, either as a composition containing all active ingredients (for simultaneous administration) or as a combination of individual compositions each containing at least one, but not all, active ingredients (for sequential or simultaneous administration), and which does not contain any additional components that would have unacceptable toxicity to the subject to whom the product will be administered. Such a product may be sterile. "Simultaneous administration" means that the active ingredients are administered simultaneously. "Sequential administration" means that the active ingredients are administered one after another in any order at intervals between administrations. The time interval may be, for example, less than 24 hours; in another example, less than 6 hours; in yet another example, less than 2 hours.

[0148] Terms such as “treatment” or “remission” refer to (1) therapeutic measures that cure, alleviate, reduce or relieve symptoms of a diagnosed pathological condition or symptom and / or stop its progression, and (2) preventive measures that prevent and / or slow the progression of a targeted pathological condition or symptom. Therefore, those who require treatment include those who already have the condition; those who are susceptible to the condition; and those who need to prevent the condition. In some respects, the method of this disclosure is considered to have successfully “treated” the subject’s cancer if the patient shows, for example, complete, partial or transient remission of a certain type of cancer.

[0149] The terms “cancer,” “tumor,” “cancerous,” and “malignant” refer to or describe a physiological condition in mammals typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, breast cancer, lung cancer, colorectal cancer, stomach cancer, esophageal cancer, head and neck cancer, adenocarcinoma of the esophagogastric junction, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial carcinoma, prostate cancer, bladder cancer, endometrial cancer, gastrointestinal stromal tumors, gastrointestinal stromal tumors, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, sarcoma, melanoma, cervical cancer, uterine cancer, testicular cancer, and renal cell carcinoma. Cancer includes blood malignancies such as acute myeloid leukemia, multiple myeloma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, and solid tumors such as breast cancer, lung cancer, neuroblastoma, and colon cancer.

[0150] As used herein, the term "cytotoxic drug" is broadly defined and refers to substances that inhibit or prevent cell function and / or cause cell destruction (cell death) and / or exert antitumor / antiproliferative effects. For example, cytotoxic drugs directly or indirectly prevent the development, maturation, or spread of neoplastic tumor cells. The term includes agents that cause only cellular inhibition. The term includes chemotherapeutic agents as described below.

[0151] The term "chemotherapeutic agents" is a subset of the term "cytotoxic drugs," which includes natural or synthetic compounds.

[0152] According to the methods or uses of this disclosure, the compounds of this disclosure can be administered to a patient to promote an active therapeutic response to cancer. The term "active therapeutic response" in relation to cancer treatment refers to improvement in disease-related symptoms. For example, improvement in disease can be characterized as complete remission. The term "complete remission" means the absence of clinically detectable disease and the normalization of any prior test results. Alternatively, improvement in disease can be classified as partial remission. "Active therapeutic response" encompasses a reduction or inhibition of cancer progression and / or duration, a reduction or improvement in cancer severity, and / or improvement of one or more of its symptoms resulting from the administration of the compounds of this disclosure. In a specific aspect, such terms refer to one, two, three, or more outcomes following the administration of the compounds of this disclosure:

[0153] (1) Stabilization, reduction or elimination of cancer cell populations;

[0154] (2) Stabilization or reduction of cancer growth;

[0155] (3) Damage to cancer formation;

[0156] (4) Eradication, removal or control of primary, regional and / or metastatic cancers;

[0157] (5) Decrease in mortality rate;

[0158] (6) No disease, no relapse, no progression and / or an increase in overall survival, duration or rate;

[0159] (7) An increase in remission rate, duration of remission, or number of patients in remission or in remission;

[0160] (8) Decrease in hospitalization rate,

[0161] (9) Reduced hospital stay

[0162] (10) Maintain tumor size and not increase or increase by less than 10%, or less than 5%, or less than 4%, or less than 2%, and

[0163] (11) An increase in the number of patients in remission.

[0164] (12) The reduction in the number of adjuvant therapies (e.g., chemotherapy or hormone therapy) that would otherwise be required to treat cancer.

[0165] Clinical responses can be assessed using screening techniques such as PET, magnetic resonance imaging (MRI), X-ray imaging, computed tomography (CT), flow cytometry or fluorescence activated cell sorting (FACS) analysis, histology, gross pathology, and blood chemistry, including but not limited to changes detectable by ELISA, RIA, chromatography, etc. In addition to these positive treatment responses, subjects undergoing treatment may experience beneficial effects such as improvement in disease-related symptoms.

[0166] As used herein, the term "antibody" refers to a protein that recognizes and specifically binds to an antigen. Common or conventional mammalian antibodies comprise tetramers, which typically consist of two pairs of identical polypeptide chains, each pair consisting of a "light" chain (typically having a molecular weight of about 25 kDa) and a "heavy" chain (typically having a molecular weight of about 50 kDa–70 kDa). As used herein, the terms "heavy chain" and "light chain" refer to any immunoglobulin polypeptide having a sufficient sequence of variable domains to confer specificity against a target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids, which is typically responsible for antigen recognition. As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. The carboxyl-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in naturally occurring antibodies, full-length heavy chain immunoglobulin polypeptides include a variable domain (V... H ) and three constant structural domains (C H1 C H2 and C H3 ) and C H1 and C H2 The hinge region between, where the VH domain is at the amino terminus of the polypeptide and C H3 The domain is at the carboxyl terminus, and the full-length light chain immunoglobulin polypeptide includes a variable domain (V). L ) and constant structural domain (C L ), where V L The domain is located at the amino terminus of the polypeptide and C L The domain is located at the carboxyl terminus. However, those skilled in the art will understand that the position of the domain in naturally occurring antibodies can be modified in certain antibody-like binding protein forms without losing antigen-binding ability. Human light chains are classified as κ and λ light chains.

[0167] Within both the full-length light and heavy chains, variable and constant domains are typically linked by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. The variable regions of each light / heavy chain pair typically form antigen-binding sites. The variable domains of naturally occurring antibodies generally exhibit the same general structure as a relatively conserved backbone region (FR) linked by three hypervariable regions (also known as complementarity-determining regions or CDRs). The CDRs from the two chains of each pair are typically aligned via the backbone region, enabling binding to specific epitopes. From the amino terminus to the carboxyl terminus, the variable domains of both the light and heavy chains typically include domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0168] The term "antibody fragment" refers to a complete or full-length chain or a portion of an antibody, typically a target-binding or variable region. Examples of antibody fragments include, but are not limited to, F. ab F ab' F (ab')2 and F v Fragment. As used herein, the term "functional fragment" is generally synonymous with "antibody fragment" and, relative to an antibody, can refer to substances such as F... v F ab F (ab')2 Antibody fragments.

[0169] The references to the amino acid residue numbers in this article are based on the EU numbering system (also described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0170] "Monoclonal" antibodies or their antigen-binding fragments refer to a group of homogeneous antibodies or antigen-binding fragments that participate in the highly specific binding of a single antigenic determinant or epitope. This contrasts with polyclonal antibodies, which typically include different antibodies targeting different antigenic determinants. The term "monoclonal" antibody or its antigen-binding fragment includes full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal" antibodies or their antigen-binding fragments refer to such antibodies and their antigen-binding fragments prepared, including but not limited to, through hybridoma, phage selection, recombinant expression, and transgenic animals.

[0171] As used herein, the term "human antibody" includes antibodies having variable and constant regions that substantially correspond to the sequences of human immunoglobulins. In some respects, human antibodies are produced in non-human mammals, including but not limited to rodents (such as mice and rats) and rabbits (such as rabbits). In other respects, human antibodies are produced in hybridoma cells. In yet another respect, human antibodies are recombinantly produced. In some respects, bispecific binding proteins are human antibodies or humanized antibodies.

[0172] As used herein, the term "antigen" or "target antigen" refers to a molecule or part of a molecule that can be recognized and bound by the binding proteins of this disclosure. Target antigens can be used in animals to generate antibodies that can bind to epitopes of that antigen. Target antigens may have one or more epitopes.

[0173] As used herein, the term "epitope" refers to a local region or structural element of an antigen that is recognized and bound by a binding protein of this disclosure, such as an antibody or an antigen-binding fragment thereof. More precisely, an epitope is a specific structure bound by the CDR of a binding protein. Epitopes may include protein structural elements, portions of carbohydrates, or even lipid structures found in membranes. A binding protein is said to specifically bind an antigen when it preferentially recognizes its antigenic target in a complex mixture of proteins and / or macromolecules. The term "specific binding" refers to a binding protein that specifically binds to a molecule or a fragment thereof (e.g., an antigen). A binding protein that specifically binds to a molecule or a fragment thereof may bind to other molecules with lower affinity, as determined by, for example, an immunoassay, BIAcore, or other assays known in the art. Specifically, an antibody or fragment that specifically binds to at least one molecule or a fragment thereof may compete for non-specific binding to molecules. This disclosure particularly covers antibodies with multispecificity (e.g., antibodies specific to two or more discrete antigens). For example, a bispecific binding protein may bind to two adjacent epitopes on a single target antigen or may bind to two different antigens.

[0174] As used herein, the term "antigen binding site" refers to a site formed on the surface of a binding protein of the present disclosure, wherein an antigen or an epitope on an antigen is bound. Antigen binding sites of binding proteins are typically described by reference to loop structures formed by the complementarity-determining region (CDR) of the binding protein.

[0175] The term "chimeric" antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment whose amino acid sequence originates from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies or antigen-binding fragments derived from one mammal (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and ability, while the constant regions are homologous to sequences in antibodies or antigen-binding fragments derived from another mammal (typically human) to avoid triggering an immune response in that species.

[0176] The term “humanized” antibody or its antigen-binding fragment refers to a form of non-human (e.g., mouse) antibody or antigen-binding fragment that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing a minimal non-human (e.g., mouse) sequence. Typically, humanized antibodies or their antigen-binding fragments are human immunoglobulins in which residues from the complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) with the desired specificity, affinity, and capability (“CDR transplantation”) (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some instances, certain Fv framework region (FR) residues of a human immunoglobulin are replaced by corresponding residues from an antibody or fragment of a non-human species with the desired specificity, affinity, and capability. Humanized antibodies or their antigen-binding fragments can be further modified by substituting additional residues within the Fv framework region and / or non-human CDR residues to improve and optimize the specificity, affinity, and / or capability of the antibody or its antigen-binding fragment. Generally, humanized antibodies or their antigen-binding fragments will contain variable domains containing all or substantially all of the CDR regions corresponding to non-human immunoglobulins, while all or substantially all of the FR regions are those common sequences of human immunoglobulins. Humanized antibodies or their antigen-binding fragments may also contain at least a portion of the immunoglobulin constant region or domain (Fc), typically at least a portion of the constant region or domain of human immunoglobulins. Examples of methods for generating humanized antibodies are described in U.S. Patent 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994) and Roguska et al., ProteinEng. 9(10):895-904 (1996). In some aspects of this disclosure, “humanized antibody” is an antibody with surface remodeling.

[0177] The term "human" antibody or its antigen-binding fragment refers to an antibody or its antigen-binding fragment having an amino acid sequence derived from a human immunoglobulin gene locus, wherein such antibody or antigen-binding fragment is made using any technique known in the art. This definition of human antibody or its antigen-binding fragment includes complete antibodies or full-length antibodies and fragments thereof. "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or its antigen-binding fragment) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to the inherent binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody or its antigen-binding fragment and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (KD). Affinity can be measured and / or expressed using techniques known in the art, including but not limited to the equilibrium dissociation constant (KD) and the equilibrium association constant (KA). KD is calculated by the quotient of kdissociation / kassociation, while KA is calculated by k 解离 / k 缔合 The quotient is calculated. K 缔合 This refers to, for example, the association rate constant between an antibody or its antigen-binding fragment and an antigen, and k 解离 This refers to, for example, the dissociation rate constant of an antibody or its antigen-binding fragment from the antigen. k 缔合 and k 解离 Techniques known to those skilled in the art, such as BIAcore, can be used. ® Alternatively, KinExA can be used for measurement.

[0178] Epitopes can be, for example, continuous amino acids of a polypeptide (linear or continuous epitopes), or epitopes can be aggregated, for example, from two or more discontinuous regions of one or more polypeptides (conformational, nonlinear, discontinuous, or non-discontinuous epitopes). In some aspects of this disclosure, epitopes specifically bound to antibodies or their antigen-binding fragments can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen / deuterium exchange combined mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be performed using any of the methods known in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody / its antigen-binding fragments: Antigen crystals can be studied using well-known X-ray diffraction techniques, and can be further refined using computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, for example, MethEnzymol (1985) Vol. 114 and 115, edited by Wyckoff HW et al.; US 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, edited by Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323). Mutagenesis mapping studies can be performed using any methods known to those skilled in the art. See, for example, Champe M et al., (1995) JBiol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085, which describe mutagenesis techniques, including alanine scanning mutagenesis.

[0179] An antibody that “binds to the same epitope” as the reference antibody is an antibody that binds to the same amino acid residue as the reference antibody. The ability of an antibody to bind to the same epitope as the reference antibody can be determined by hydrogen / deuterium exchange assay (see, Coales et al., Rapid Commun. Mass Spectrom. 2009; 23: 639–647) or X-ray crystallography.

[0180] If an antibody preferentially binds to a given epitope or overlapping epitope, thereby partially blocking the binding of a reference antibody to that epitope, the antibody is considered to "competitively inhibit" or "cross-competitively inhibit" the binding of the reference antibody to that epitope. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. It can be considered that the antibody competitively inhibits the binding of the reference antibody to the given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0181] "Isolated" polypeptides, antibodies, polynucleotides, carriers, cells, or compositions are polypeptides, antibodies, polynucleotides, carriers, cells, or compositions in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, carriers, cells, or compositions include those that have been purified to the point that they no longer exist in a form found in nature. In some aspects of this disclosure, isolated antibodies, polynucleotides, carriers, cells, or compositions are substantially pure. As used herein, "substantially pure" means material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0182] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acids of any length. This polymer may be linear or branched, may include modified amino acids, and may be interrupted by non-amino acid components. These terms also cover polymers of naturally modified or intervened amino acids; such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art. It should be understood that because the polypeptides of this disclosure are antibody-based, in some aspects of this disclosure, the polypeptide may exist as a single chain or an associated chain.

[0183] [Description of the implementation plan]

[0184] The following describes a specific pattern for implementing this disclosure. The embodiments described below are given merely as an example of a typical implementation of this disclosure and are not intended to limit the scope of this disclosure.

[0185] 1. Antibody-drug conjugates

[0186] The antibody-drug conjugates used in this disclosure are antibody-drug conjugates in which a drug linker represented by the following formula is conjugated to an antibody, particularly an anti-TROP2 or anti-HER2 antibody, via a thioether bond:

[0187]

[0188] Where A represents the binding site with the antibody.

[0189] In this disclosure, the portion of the antibody-drug conjugate consisting of a linker and a drug is referred to as the "drug-linker". The drug-linker is attached to a thiol group (in other words, the sulfur atom of a cysteine ​​residue) formed at interchain disulfide bond sites in the antibody (two sites between the heavy chain and two sites between the heavy chain and the light chain).

[0190] The drug-linker disclosed herein comprises essanotecan (IUPAC name: (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-10,13-dione, also represented by the chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-10,13(9H,15H)-dione), which is a topoisomerase I inhibitor. Exanotecan is a camptothecin derivative with antitumor effects, represented by the following formula:

[0191]

[0192] The antibody-drug conjugates used in this disclosure can also be represented by the following formula:

[0193]

[0194] Here, the drug-linker is conjugated to the antibody (“antibody-”) via a thioether bond, specifically an anti-TROP2 or anti-HER2 antibody. The meaning of n is the same as the so-called drug-to-antibody ratio (DAR) and represents the average number of drug-linker units conjugated to each antibody molecule.

[0195] After migrating into cancer cells, the antibody-drug conjugate used in this disclosure is cleaved at the linker portion to release a compound represented by the following formula:

[0196]

[0197] 2. Antibodies in antibody-drug conjugates

[0198] The antibodies in the antibody-drug conjugates used in this disclosure are anti-TROP2 or anti-HER2 antibodies and can be derived from any species, such as humans, rats, mice, or rabbits. In cases where the antibody originates from a species other than humans, it may be chimeric or humanized, for example, using resin techniques. The antibody can be a polyclonal antibody or a monoclonal antibody, and is, for example, a monoclonal antibody.

[0199] The antibodies used in the antibody-drug conjugates in this disclosure are, for example, antibodies that have the characteristic of being able to target cancer cells, and are, for example, antibodies that have, for example, the characteristic of recognizing cancer cells, the characteristic of binding to cancer cells, the characteristic of internalizing in cancer cells, and / or the characteristic of cytotoxic activity against cancer cells.

[0200] The binding activity of antibodies to cancer cells can be confirmed using flow cytometry. Antibody internalization into cancer cells can be confirmed by: (1) visualizing the incorporation of antibodies into cells under a fluorescence microscope using a secondary antibody (fluorescently labeled) bound to a therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761); (2) measuring the fluorescence intensity incorporation into cells using a secondary antibody (fluorescently labeled) bound to a therapeutic antibody (Molecular Biology of the Cell, Vol. 15, 5268-5282, December 2004); or (3) using a Mab-ZAP assay of an immunotoxin bound to a therapeutic antibody, wherein the toxin is released upon incorporation into the cell to inhibit cell growth (BioTechniques 28: 162-165, January 2000). As an immunotoxin, a recombinant complex of diphtheria toxin catalytic domain and protein G can be used.

[0201] The antitumor activity of antibodies can be confirmed in vitro by measuring their inhibitory activity on cell growth. For example, cancer cell lines overexpressing the antibody's target protein can be cultured, and different concentrations of the antibody can be added to the culture system to measure their inhibitory activity on lesion formation, colony formation, and globular growth. Antitumor activity can also be confirmed in vivo, for example, by administering the antibody to nude mice with transplanted cancer cell lines that highly express the target protein and measuring changes in the cancer cells.

[0202] Since the compounds conjugated in the antibody-drug conjugate exert antitumor effects, in one embodiment, the antibody itself should have antitumor effects. In order to specifically and selectively exert the cytotoxic activity of the antitumor compound against cancer cells, in one embodiment, the antibody should have the property of internalization to migrate into cancer cells.

[0203] The anti-TROP2 or anti-HER2 antibodies in the antibody-drug conjugates used in this disclosure can be obtained through procedures known in the art. For example, the antibodies of this disclosure can be obtained using methods conventionally performed in the art, including immunizing an animal with an antigenic peptide and collecting and purifying the antibodies produced in vivo. The source of the antigen is not limited to humans, and animals can be immunized with antigens derived from non-human animals such as mice, rats, etc. In this case, the cross-reactivity of the antibody bound to the obtained heterologous antigen with the human antigen can be tested to screen for antibodies suitable for human diseases.

[0204] Alternatively, antibody-producing cells that produce antibodies against an antigen may be fused with myeloma cells to create a hybridoma, from which monoclonal antibodies may be obtained, according to methods known in the art (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; and Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)).

[0205] Antigens can be obtained by genetically engineering host cells to produce genes encoding antigen proteins. Specifically, a vector that allows antigen gene expression is prepared and transferred into host cells, thereby expressing the gene. The expressed antigen can then be purified. Antibodies can also be obtained by immunizing animals with cells expressing the genetically engineered antigen or cell lines expressing the antigen as described above.

[0206] The anti-TROP2 or anti-HER2 antibodies used in the antibody-drug conjugates in this disclosure are, for example, recombinant antibodies obtained through artificial modification for the purpose of reducing heteroantigenicity against humans, such as chimeric antibodies or humanized antibodies, or, for example, antibodies that only have gene sequences derived from human antibodies, i.e., human antibodies. These antibodies can be prepared using known methods.

[0207] As examples of chimeric antibodies, chimeric antibodies whose variable and constant regions are derived from different species can be cited, such as chimeric antibodies whose variable regions are linked to the constant regions of human antibodies (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0208] Examples of humanized antibodies include antibodies obtained by integrating only the complementarity-determining region (CDR) of a heterologous antibody into a human-derived antibody (Nature (1986) 321, pp. 522-525), antibodies obtained by grafting a portion of the amino acid residues of the heterologous antibody framework and the CDR sequence of the heterologous antibody into a human antibody using a CDR grafting method (WO90 / 07861), and antibodies humanized using a gene transformation mutagenesis strategy (US Patent No. 5,821,337).

[0209] Examples of human antibodies include those generated from mice using human antibodies containing segments of human chromosomes with genes that contain both the heavy and light chains of human antibodies (see Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects, Vol. 10, pp. 69-73 (edited by Kitagawa, Y., Matsuda, T. and Iijima, S.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.). As an alternative, examples can be given of antibodies obtained through phage display, which are selected from human antibody libraries (see Wormstone, IM et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), pp. 189-203; Siriwardena, D. et al., Ophthalmology (2002) 109 (3), pp. 427-431, etc.).

[0210] The antibodies in the antibody-drug conjugates used in this disclosure also include modified variants of the antibodies. Modified variants refer to variants obtained through chemical or biological modifications to the antibodies according to this disclosure. Examples of chemically modified variants include variants comprising the linking of a chemical moiety to an amino acid backbone, variants comprising the linking of a chemical moiety to an N-linked or O-linked glycan chain, etc. Examples of biologically modified variants include variants obtained through post-translational modifications (such as N-linked or O-linked glycosylation, N-terminal or C-terminal processing, deamidation, isomerization of aspartic acid, or oxidation of methionine), and variants by adding methionine residues to the N-terminus through expression in prokaryotic host cells. Furthermore, antibodies labeled to enable the detection or separation of antibodies or antigens according to this disclosure, such as enzyme-labeled antibodies, fluorescently labeled antibodies, and affinity-labeled antibodies, are also included in the meaning of modified variants. Such modified variants of the antibodies according to this disclosure can be used to improve antibody stability and blood retention, reduce their antigenicity, detect or separate antibodies or antigens, etc.

[0211] Furthermore, antibody-dependent cytotoxic activity can be enhanced by modulating the glycans linked to the antibodies according to this disclosure (glycosylation, defucosylation, etc.). Techniques for modulating the glycan modification of antibodies, such as those disclosed in WO99 / 54342, WO00 / 61739, WO02 / 31140, WO2007 / 133855, and WO2013 / 120066, are known. However, the technique is not limited thereto. The anti-TROP2 or anti-HER2 antibodies according to this disclosure also include antibodies in which glycan modification is modulated.

[0212] It is known that antibodies produced in cultured mammalian cells have a lysine residue deletion at the C-terminus of the heavy chain (Journal of Chromatography A, 705: 129-134 (1995)), and it is also known that antibodies produced in cultured mammalian cells have a two-amino acid residue (glycine and lysine) deletion at the C-terminus of the heavy chain, and that a proline residue newly located at the C-terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletions and modifications of the heavy chain sequence do not affect the antigen-binding affinity and effector functions (complement activation, antibody-dependent cytotoxicity, etc.) of the antibody. Therefore, the anti-TROP2 or anti-HER2 antibody according to this disclosure also includes the antibody and the functional fragment of the antibody subjected to such modification, and also includes deletion variants in which one or two amino acids are missing at the C-terminus of the heavy chain, variants obtained by amidation of the deletion variant (e.g., the heavy chain in which the C-terminal proline residue has been amidated), etc. The heavy chains of the antibodies according to this disclosure may be of a type not limited to those described above, provided that antigen-binding affinity and effector function are conserved. The two heavy chains constituting the antibodies according to this disclosure may be one type selected from the group consisting of full-length heavy chains and the aforementioned deletion variants, or a combination of two types selected therefrom. The ratio of the amounts of each deletion variant may be influenced by the type of mammalian cells cultured to produce the antibodies according to this disclosure; however, examples can be given of antibodies in which one amino acid residue at the carboxyl terminus is missing in both heavy chains of the antibody according to this disclosure.

[0213] As isotypes of the anti-TROP2 or anti-HER2 antibodies according to this disclosure, examples include IgG (IgG1, IgG2, IgG3, IgG4), and IgG1 is an example.

[0214] In this disclosure, the term "anti-TROP2 antibody" refers to an antibody that specifically binds to TROP2 (TACSTD2: tumor-associated calcium signaling transducer 2; EGP-1) and, for example, has the activity of internalization in TROP2-expressing cells by binding to TROP2.

[0215] Examples of anti-TROP2 antibodies include hTINA1-H1L1 (WO2015 / 098099), and datopotamab can be cited as an example.

[0216] In this disclosure, the term "anti-HER2 antibody" refers to an antibody that specifically binds to HER2 (human epidermal growth factor receptor type 2; ErbB-2) and, for example, has the activity of internalization in HER2-expressing cells by binding to HER2.

[0217] Examples of anti-HER2 antibodies include trastuzumab (US Patent 5,821,337) and pertuzumab (WO01 / 00245), and trastuzumab can be used as an example.

[0218] 3. Preparation of antibody-drug conjugates

[0219] The drug-linker intermediate used to prepare antibody-drug conjugates according to this disclosure is represented by the following formula:

[0220]

[0221] The drug-linker intermediate can be represented by the chemical name N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycamide, and can be prepared with reference to the descriptions in WO2014 / 057687, WO2015 / 098099, WO2019 / 044947, etc.

[0222] The antibody-drug conjugates used in this disclosure can be prepared by reacting the above-mentioned drug-linker intermediate with an anti-TROP2 or anti-HER2 antibody having a thiol group (also known as a mercapto group).

[0223] Anti-TROP2 or anti-HER2 antibodies with thiol groups can be obtained by methods well known in the art (Hermanson, G. T, Bioconjugate Techniques, pp. 56–136, 456–493, Academic Press (1996)). For example, anti-TROP2 or anti-HER2 antibodies with thiol groups of interchain disulfides within each antibody can be obtained by reacting the antibody with a reducing agent such as tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) containing 0.3 to 3 molar equivalents of the interchain disulfide within each antibody in a buffer solution containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA).

[0224] In addition, antibody-drug conjugates with 2 to 20 molar equivalents of each anti-TROP2 or anti-HER2 antibody containing a thiol group can be prepared.

[0225] The average number of conjugated drug molecules per anti-TROP2 or anti-HER2 antibody molecule in the prepared antibody-drug conjugate can be determined, for example, by a calculation method based on measuring the UV absorbance of the antibody-drug conjugate and its conjugated precursor at two wavelengths of 280 nm and 370 nm (UV method), or by a calculation method based on quantification by HPLC measurement of fragments obtained by treating the antibody-drug conjugate with a reducing agent (HPLC method).

[0226] The calculation of the conjugation between anti-TROP2 or anti-HER2 antibodies and drug-linker intermediates, as well as the average number of drug molecules conjugated per antibody molecule in antibody-drug conjugates, can be performed with reference to the descriptions in WO2014 / 057687, WO2015 / 098099, WO2017 / 002776, WO2022 / 014698, etc.

[0227] In this disclosure, the term "anti-TROP2 antibody-drug conjugate" refers to an antibody-drug conjugate such that the antibody in the antibody-drug conjugate according to this disclosure is an anti-TROP2 antibody.

[0228] Anti-TROP2 antibodies, for example, are antibodies comprising a heavy chain and a light chain. The heavy chain comprises CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 3 [= the amino acid sequence consisting of amino acid residues 50 to 54 of SEQ ID NO: 1], CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 4 [= the amino acid sequence consisting of amino acid residues 69 to 85 of SEQ ID NO: 1], and CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 5 [= the amino acid sequence consisting of amino acid residues 118 to 129 of SEQ ID NO: 1]. The light chain comprises CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 6 [= the amino acid sequence consisting of amino acid residues 44 to 54 of SEQ ID NO: 2], CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 7 [= the amino acid sequence consisting of amino acid residues 70 to 76 of SEQ ID NO: 2], and CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 8 [= the amino acid sequence consisting of amino acid residues 44 to 54 of SEQ ID NO: 1]. The amino acid sequence consisting of amino acid residues 109 to 117 of 2 forms CDRL3.

[0229] In one embodiment, the anti-TROP2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain comprising a variable region consisting of the amino acid sequence represented by SEQ ID NO: 9 [= an amino acid sequence consisting of amino acid residues 20 to 140 of SEQ ID NO: 1], and the light chain comprising a variable region consisting of the amino acid sequence represented by SEQ ID NO: 10 [= an amino acid sequence consisting of amino acid residues 21 to 129 of SEQ ID NO: 2]. In another embodiment, the antibody comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 12 [= the amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO: 1] and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 13 [= the amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 2]; or the antibody comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 11 [= the amino acid sequence consisting of amino acid residues 20 to 469 of SEQ ID NO: 1] and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 13 [= the amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 2].

[0230] In anti-TROP2 antibody-drug conjugates, the average number of drug-linker units conjugated to each antibody molecule is 2 to 8, or 3 to 5, or 3.5 to 4.5, or about 4.

[0231] Anti-TROP2 antibody-drug conjugates can be prepared according to the descriptions in WO2015 / 098099, WO2017 / 002776 and WO2022 / 014698.

[0232] In some implementations, the anti-TROP2 antibody-drug conjugate is drutecan (DS-1062).

[0233] In this disclosure, the term "anti-HER2 antibody-drug conjugate" refers to an antibody-drug conjugate such that the antibody in the antibody-drug conjugate according to this disclosure is an anti-HER2 antibody.

[0234] Anti-HER2 antibodies are, for example, antibodies comprising a heavy chain and a light chain, wherein the heavy chain comprises CDRH1 consisting of an amino acid sequence of amino acid residues 26 to 33 of SEQ ID NO: 14, CDRH2 consisting of an amino acid sequence of amino acid residues 51 to 58 of SEQ ID NO: 14, and CDRH3 consisting of an amino acid sequence of amino acid residues 97 to 109 of SEQ ID NO: 14, and the light chain comprises CDRL1 consisting of an amino acid sequence of amino acid residues 27 to 32 of SEQ ID NO: 15, CDRL2 consisting of an amino acid sequence of amino acid residues 50 to 52 of SEQ ID NO: 15, and CDRL3 consisting of an amino acid sequence of amino acid residues 89 to 97 of SEQ ID NO: 15; and in another example, antibodies comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region consisting of an amino acid sequence of amino acid residues 1 to 120 of SEQ ID NO: 14, and the light chain comprises an amino acid sequence of amino acid residues 27 to 32 ... The variable region of the light chain, consisting of amino acid residues 1 to 107 of SEQ ID NO: 15; and in another example, an antibody comprising a heavy chain and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 14 and the light chain consisting of the amino acid sequence represented by SEQ ID NO: 15, or an antibody comprising a heavy chain and a light chain, the heavy chain consisting of amino acid residues 1 to 449 of SEQ ID NO: 14 and the light chain consisting of the amino acid sequence consisting of all amino acid residues 1 to 214 of SEQ ID NO: 15.

[0235] In anti-HER2 antibody-drug conjugates, the average number of drug-linker units conjugated to each antibody molecule is 2 to 8, or 3 to 8, or 7 to 8, or 7.5 to 8, or about 8.

[0236] The anti-HER2 antibody-drug conjugates used in this disclosure can be prepared with reference to the descriptions in WO2015 / 115091, etc.

[0237] In some implementations, the anti-HER2 antibody-drug conjugate is trastuzumab (DS-8201).

[0238] 4. Anti-PD-1 / TIM-3 bispecific binding protein

[0239] As used herein, a bispecific binding protein has binding specificity against at least two independent antigens (or targets) or different epitopes within the same antigen. Exemplary bispecific binding proteins may bind to two different epitopes of a target, or may bind to two different targets. Other such binding proteins may combine a first target binding site with a second target binding site. In some aspects, the binding protein is a bispecific antibody, the term "bispecific antibody" referring to an antibody that binds to two different epitopes. The epitopes may be on the same target antigen or on different target antigens.

[0240] In some respects, bispecific antibodies provide synergistic therapeutic effects derived from simultaneously targeting two antigens by administering a single manufactured molecule.

[0241] In some respects, the antibodies presented herein are monovalent bispecific antibodies (MBabs). The monovalent bispecific antibody scaffold described herein provides an excellent platform for generating bispecific antibodies that satisfy all the benefits associated with bispecific antibodies while reducing the aforementioned potential therapeutic risks due to their monovalent nature. Furthermore, the MBabs presented herein are readily expressible, stable, and may 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 specifically binds to a different target antigen, and for a given pair of different target antigens (A and B), the MBab binds to one of each. In some respects, monovalent bispecific antibodies can specifically bind to two independent antigens (or targets) or two independent epitopes on the same antigen. Typically, monovalent bispecific antibodies comprise two distinct variable regions. In some respects, the binding affinity against the two independent antigens is substantially the same. In some respects, the binding affinity against the two independent antigens is different.

[0242] The bispecific binding protein contains a first binding domain that specifically binds to PD-1 and a second binding domain that specifically binds to TIM-3.

[0243] As used herein, the terms “PD-1,” “programmed cell death 1,” and “programmed cell death 1” are used interchangeably. The complete PD-1 sequence can be found at NCBI Reference Sequence: NG_012110.1. Programmed cell death 1 (“PD-1”) is an approximately 31 kD type I membrane protein member of the extended CD28 / CTLA-4 family of T cell regulators (see Ishida, Y. et al., (1992) Induced Expression Of PD-1, A Novel Member Of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death,” EMBO J. 11:3887-3895). PD-1 is expressed on activated T cells, B cells, and monocytes (Agata, Y. et al., (1996) “Expression of the PD-1 Antigen on the Surface of Stimulated Mouse T and B Lymphocytes,” Int. Immunol. 8(5):765-772; Martin-Orozco, N. et al., (2007) “Inhibitory Costimulation and Anti-Tumor Immunity,” Semin. Cancer Biol. 17(4):288-298). PD-1 is a receptor responsible for downregulating the immune system after activation by binding to PDL-1 or PDL-2 (Martin-Orozco, N. et al., (2007) "Inhibitory Costimulation and Anti-Tumor Immunity," Semin. Cancer Biol. 17(4):288-298), and is used as a cell death inducer (Ishida, Y. et al., (1992) "Induced Expression of PD-1, A Novel Member of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death," EMBO J. 11: 3887-3895; Subudhi, SK et al., (2005) "The Balance of Immune Responses: Costimulation Verse Coinhibition," J. Molec. Med. 83: 193-202).This process is utilized in many tumors through the overexpression of PD-L1, thereby suppressing the immune response.

[0244] PD-1 is a well-validated target for immuno-mediated therapy in oncology, with positive results in clinical trials for the treatment of melanoma and non-small cell lung cancer (NSCLC). Antagonistic inhibition of the PD-1 / PD-L1 interaction increases T cell activation, enhancing the host immune system's recognition and elimination of tumor cells. The use of anti-PD-L1 antibodies to treat infections and tumors and enhance adaptive immune responses has been proposed (see U.S. Patent Nos. 7,521,051, 7,563,869, and 7,595,048).

[0245] As used herein, the terms “protein-3 containing T-cell immunoglobulin and mucin domains” and “TIM-3” are used interchangeably and include variants, isotypes, and species homologs of human TIM-3. TIM-3 refers to a type I cell surface glycoprotein comprising an N-terminal immunoglobulin (Ig)-like domain, an O-linked glycosylated mucin domain with an N-linked glycosylated domain 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: SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIK (SEQ ID NO: 45). The T-cell inhibitory receptor TIM-3 plays a role in regulating anti-tumor immunity because it is expressed on IFN-γ-producing CD4+ helper 1 (Th1) and CD8+ T-cytotoxic 1 (Tc1) T cells. Initially identified as a T-cell inhibitory receptor, it acts as an immune checkpoint receptor, specifically limiting the duration and magnitude of Th1 and Tc1 T-cell responses. Further research has determined that the TIM-3 pathway can synergize with the PD-1 pathway to promote the development of severely dysfunctional phenotypes in CD8+ T cells in cancer. It is also present in regulatory T cells (T cells) in certain cancers. reg TIM-3 is expressed in cells of the innate immune system, including mouse mast cells, macrophages, dendritic cell (DC) subsets, NK and NKT cells, and human monocytes, as well as in mouse primary bronchial epithelial cell lines. TIM-3 can generate inhibitory signals that lead to apoptosis of Th1 and Tc1 cells and can mediate phagocytosis of apoptotic cells and cross-presentation of antigens.

[0246] The crystal structure of the IgV domain of TIM-3 reveals two antiparallel β-sheets tethered by disulfide bonds. Two additional disulfide bonds, formed by four non-classical cysteine ​​residues, stabilize the IgV domain and redirect the CC' ring toward the FG ring, forming a "crack" structure that is thought to be involved in ligand binding and is not found in other IgSF members. Instead, this "crack" assembly is a characteristic structure identified in all TIM family proteins, including TIM-1 and TIM-4. The binding of the IgV domain to appropriate ligands has been found to be important for the immunomodulatory effects of TIM-3 and contributes to the induction of peripheral tolerance and the suppression of antitumor immunity. The C'C" ring of TIM-3 involves amino acids after the β-chain C' and before the β-chain C" ring, for example, amino acids 50 to 54. The DE ring consists of amino acids 64 to 73, while the CC' and FG rings contain amino acids 35 to 43 and 92 to 99, respectively.

[0247] TIM-3 possesses several known ligands, such as galactolectin-9, phosphatidylserine, CEACAM1, and HMGB1. Galactolectin-9 is an S-type lectin with two distinct carbohydrate recognition domains linked by a long, flexible linker, and exhibits enhanced affinity for larger structures containing poly-N-acetylglucosamine. Galactolectin-9 lacks a signaling sequence and is located in the cytoplasm. However, it can be secreted and exerts its function by binding to glycoproteins on the surface of target cells via its carbohydrate chains (Freeman GJ et al., Immunol Rev. 2010 Can; 235(1): 172-89). Based on binding studies, mutagenesis, and cocrystal structures, TIM-3 in both humans and mice has been shown to be a receptor for phosphatidylserine, and cells expressing TIM-3 have been shown to bind to and / or phagocytose apoptotic cells expressing phosphatidylserine. The interaction between TIM-3 and phosphatidylserine does not preclude interaction with galactolectin-9, as the binding site has been found to be on the opposite side of the IgV domain. Given that the TIM-3 pathway is involved in key immunosuppressed immune cell populations in some cancers, it represents an attractive candidate for immuno-oncology therapy. See Anderson, AC, Cancer Immunol Res., (2014) 2:393-398; and Ferris, RL et al., J Immunol. (2014) 193:1525-1530.

[0248] As used herein, the term "AZD7789" refers to an anti-TIM-3 / PD-1 bispecific antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 42, a first light chain containing the amino acid sequence of SEQ ID NO: 44, a second heavy chain containing the amino acid sequence of SEQ ID NO: 32, and a second light chain containing the amino acid sequence of SEQ ID NO: 34. AZD7789 is disclosed in U.S. Patent No. 11,279,759, the entire contents of which are incorporated herein by reference. AZD7789 is a monovalent bispecific humanized immunoglobulin G1 (IgG1) monoclonal antibody (mAb) that specifically binds to TIM-3 and PD-1 and targets a unique TIM-3 epitope. AZD7789 is constructed on the backbone of the DuetMab molecule. The DuetMab design is described in Mazor et al., MAbs.7(2): 377–389 (March to April 2015), which is incorporated herein by reference in its entirety. The DuetMab design incorporates a kilt-hammer (KIH) technique for the heterodimerization of two different heavy chains and increases the efficiency of homologous heavy and light chain pairing by replacing the native disulfide bonds in one of the CH1-CL interfaces with engineered disulfide bonds.

[0249] AZD7789 contains: a pestle mutation in the heavy chain containing the variable region of TIM-3, and a mortar mutation in the heavy chain containing the variable region of PD-1.

[0250] In some aspects of this disclosure, the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain that specifically binds to the C'C'' and DE loops of the immunoglobulin variable (IgV) domain of TIM-3.

[0251] In some aspects of this disclosure, the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain that specifically binds to an epitope on the IgV domain of TIM-3 (SEQ ID NO: 45), 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.

[0252] In some aspects of this disclosure, the bispecific binding protein is an anti-PD-1 / TIM-3 bispecific antibody or its antigen-binding fragment, which specifically binds to human TIM-3 and human PD-1 and contains a CDR of the AZD7789 antibody.

[0253] In some aspects of this disclosure, the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain comprising a first set of CDRs: CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprising the amino acid sequences of SEQ ID NO: 35, 36, 37, 38, 39, and 40, or 35, 36, 37, 38, 39, and 45, respectively; and a PD-1 binding domain comprising a second set of CDRs: CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprising the amino acid sequences of SEQ ID NO: 25, 26, 27, 28, 29, and 30, respectively.

[0254] In some aspects of this disclosure, the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain and a PD-1 binding domain, wherein the TIM-3 binding domain comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:41 and a light chain variable domain containing the amino acid sequence of SEQ ID NO:43, and the PD-1 binding domain comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:31 and a light chain variable domain containing the amino acid sequence of SEQ ID NO:33.

[0255] In some aspects of this disclosure, the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain and a PD-1 binding domain, the TIM-3 binding domain comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 42 and a light chain containing the amino acid sequence of SEQ ID NO: 44, and the PD-1 binding domain comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 32 and a light chain containing the amino acid sequence of SEQ ID NO: 34.

[0256] In some aspects of this disclosure, the anti-PD-1 / TIM-3 bispecific binding protein is an antibody.

[0257] In some aspects of this disclosure, the anti-PD-1 / TIM-3 bispecific binding protein is an IgG antibody. In some aspects of this disclosure, the anti-PD-1 / TIM-3 bispecific binding protein is an IgG1 antibody.

[0258] In some aspects of this disclosure, the anti-PD-1 / TIM-3 bispecific binding protein is a humanized antibody.

[0259] In some aspects of this disclosure, the anti-PD-1 / TIM-3 bispecific binding protein is monovalent.

[0260] In some aspects of this disclosure, the anti-PD-1 / TIM-3 bispecific binding protein is DuetMab.

[0261] In some respects, the TIM-3-binding protein, a bispecific binding protein, includes a non-glycosylated Fc region. In some respects, the TIM-3-binding protein includes a deglycosylated Fc region. In some respects, the TIM-3-binding protein includes an Fc region with reduced fucosylation or that is non-fucosylated.

[0262] In some implementations, the anti-PD-1 / TIM-3 bispecific antibody is AZD7789 or sabestomig.

[0263] Anti-PD-1 / TIM-3 bispecific antibodies, including AZD7789, can be produced by methods disclosed in WO2017 / 193032 and WO2022 / 221245.

[0264] 5. Combination of antibody-drug conjugates and anti-PD-1 / TIM-3 bispecific binding protein

[0265] In the first combined embodiment of this disclosure, the antibody-drug conjugate combined with the anti-PD-1 / TIM-3 bispecific binding protein is an antibody-drug conjugate wherein the antibody is an anti-TROP2 antibody.

[0266] In one embodiment of the first combined embodiment described above, the anti-TROP2 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 3 [= amino acid residues 50 to 54 of SEQ ID NO: 1], CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 4 [= amino acid residues 69 to 85 of SEQ ID NO: 1], and CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 5 [= amino acid residues 118 to 129 of SEQ ID NO: 1], and the light chain comprises CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 6 [= amino acid residues 44 to 54 of SEQ ID NO: 2], CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 7 [= amino acid residues 70 to 76 of SEQ ID NO: 2], and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 8 [= amino acid residues 109 to 117 of SEQ ID NO: 2]. In another embodiment of the first combined embodiment described above, the anti-TROP2 antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region consisting of an amino acid sequence represented by amino acid residues 20 to 140 of SEQ ID NO: 9 [= amino acid residues 20 to 140 of SEQ ID NO: 1], and the light chain comprising a light chain variable region consisting of an amino acid sequence represented by amino acid residues 21 to 129 of SEQ ID NO: 2. In another embodiment of the first combined embodiment described above, the anti-TROP2 antibody comprises a heavy chain consisting of an amino acid sequence represented by amino acid residues 20 to 470 of SEQ ID NO: 1 and a light chain consisting of an amino acid sequence represented by amino acid residues 21 to 234 of SEQ ID NO: 2. In another embodiment of the first combined embodiment described above, the anti-TROP2 antibody comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 11 [= amino acid residues 20 to 469 of SEQ ID NO: 1] and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 13 [= amino acid residues 21 to 234 of SEQ ID NO: 2]. In another embodiment of the first combined embodiment described above, anti-TROP2 is delutecan (DS-1062).

[0267] In the second embodiment of this disclosure, the antibody-drug conjugate combined with the anti-PD-1 / TIM-3 bispecific binding protein is an antibody-drug conjugate wherein the antibody is an anti-HER2 antibody.

[0268] In one embodiment of the second combined embodiment described above, the anti-HER2 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 16, CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 17, and CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 18, and the light chain comprises CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 19, CDRL2 consisting of the amino acid sequence consisting of amino acid residues 1 to 3 of SEQ ID NO: 20, and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 21. In another embodiment of the second combined embodiment described above, the anti-HER2 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 22, and the light chain comprises a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 23. In yet another embodiment of the second combined embodiment described above, the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 14 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 15. In another embodiment of the second combined embodiment described above, the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 24 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 15. In another embodiment of the second combined embodiment described above, the anti-HER2 antibody is trastuzumab (DS-8201).

[0269] In a specific embodiment of the first combination of embodiments described above, the anti-TROP2 antibody-drug conjugate is drutecan (DS-1062), and the anti-PD-1 / TIM-3 bispecific binding protein is AZD7789.

[0270] In a specific embodiment of the second combination of embodiments described above, the anti-HER2 antibody-drug conjugate is trastuzumab (DS-8201), and the anti-PD-1 / TIM-3 bispecific binding protein is AZD7789, also known as Sabestomig.

[0271] In some aspects of the above combined implementation schemes, the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein are further combined with one or more chemotherapeutic agents.

[0272] 6. Combined Uses and Methods of Treatment

[0273] The following describes a pharmaceutical product and a therapeutic use and method thereof, wherein an anti-TROP2 or anti-HER2 antibody-drug conjugate and an anti-PD-1 / TIM-3 bispecific binding protein are administered in combination according to this disclosure.

[0274] The pharmaceutical products disclosed herein, as well as their therapeutic uses and methods, may be characterized in that the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein are individually contained as active components in different formulations and administered simultaneously or at different times, or may be characterized in that the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein are contained as active components in a single formulation and administered.

[0275] In the pharmaceutical products and treatment methods disclosed herein, the single anti-PD-1 / TIM-3 bispecific binding protein used herein may be administered in combination with an antibody-drug conjugate, or two or more different anti-PD-1 / TIM-3 bispecific binding proteins may be administered in combination with an antibody-drug conjugate.

[0276] The presence or absence of tumor markers such as TROP2 or HER2 can be determined, for example, by collecting tumor tissue from cancer patients to prepare formalin-fixed paraffin-embedded (FFPE) specimens and testing the specimens for gene products (proteins) using, for example, immunohistochemistry (IHC), flow cytometry, or Western blotting; or by testing the specimens for gene transcription using in situ hybridization (ISH), quantitative PCR (q-PCR), or microarray analysis; or by collecting cell-free circulating tumor DNA (ctDNA) from cancer patients and testing the ctDNA using methods such as next-generation sequencing (NGS).

[0277] The pharmaceutical products and treatments disclosed herein can be used in cancers expressing HER2 when comprising an anti-HER2 antibody-drug conjugate, which may be cancers that overexpress HER2 (high or intermediate) or cancers that express HER2 low.

[0278] In this disclosure, the term "HER2-overexpressing cancer" is not particularly limited, provided that it is recognized by those skilled in the art as cancer overexpressing HER2. Certain examples of HER2-overexpressing cancer may include cancers with a HER2 expression score of 3+ in IHC methods, and cancers with a HER2 expression score of 2+ in IHC methods and identified as HER2-positive in in situ hybridization (ISH). The in situ hybridization methods of this disclosure include fluorescence in situ hybridization (FISH) and two-color in situ hybridization (DISH).

[0279] In this disclosure, the term "cancer with low HER2 expression" is not particularly limited, provided that it is recognized by those skilled in the art as cancer with low HER2 expression. Certain examples of cancer with low HER2 expression may include cancers with a HER2 expression score of 2+ in IHC methods and HER2 expression negative in in situ hybridization methods, and cancers with a HER2 expression score of 1+ in IHC methods.

[0280] Methods for scoring the degree of HER2 expression using IHC or for determining the positivity or negativity of HER2 expression using in situ hybridization are not particularly limited, provided they are acceptable to those skilled in the art. Examples of such methods may include those described in the 4th edition of the HER2 testing guidelines for breast cancer (developed by the Japanese Pathology Board for Optimal Use of HER2 for Breast Cancer).

[0281] Cancer, particularly breast cancer, can be breast cancer that overexpresses HER2 (high or intermediate) or low HER2 expression, or triple-negative breast cancer, and / or may have a HER2 status score of IHC 3+, IHC 2+, IHC 1+, or IHC >0 and <1+.

[0282] The pharmaceutical products and treatments disclosed herein may be used in mammals, such as humans.

[0283] The antitumor efficacy of the drug products and treatment methods disclosed herein can be confirmed by establishing a model by transplanting cancer cells into test animal subjects and measuring the reduction in tumor volume or the extension of life following administration of the drug products and treatment methods disclosed herein. The effectiveness of the combined use of the antibody-drug conjugate and the bispecific binding protein used in this disclosure can then be confirmed by comparing the antitumor efficacy of administering the antibody-drug conjugate used in this disclosure alone with that of administering the bispecific binding protein alone.

[0284] The antitumor efficacy of the drug products and treatments disclosed herein can be confirmed in clinical trials using any of the following evaluation methods: Evaluation Criteria for Solid Tumor Response (RECIST), WHO evaluation method, Macdonald evaluation method, weight measurement, and other methods. It can also be determined based on indicators such as complete response (CR), partial response (PR), disease progression (PD), objective response rate (ORR), duration of response (DoR), progression-free survival (PFS), and overall survival (OS).

[0285] By using the methods described above, it can be demonstrated that the antitumor effects of the disclosed pharmaceutical products and treatment methods are superior to those of existing pharmaceutical products and treatment methods for cancer treatment.

[0286] The disclosed pharmaceutical products and treatments can delay the development of cancer cells, inhibit their growth, and further kill cancer cells. These effects can relieve cancer patients of cancer-related symptoms or improve their quality of life (QOL), and achieve therapeutic effects by sustaining their lives. Even if the disclosed pharmaceutical products and treatments cannot kill cancer cells, they can still achieve higher QOL and longer survival for cancer patients by inhibiting or controlling their growth.

[0287] The disclosed pharmaceutical product is expected to exert therapeutic effects when administered to patients as a systemic therapy and additionally when applied topically to cancerous tissue.

[0288] On the other hand, the pharmaceutical products and treatment methods disclosed herein provide for use as adjuvants in cancer therapies using ionizing radiation or other chemotherapeutic agents. For example, in the treatment of cancer, the treatment may include administering a therapeutically effective amount of the pharmaceutical product to a subject requiring treatment, simultaneously or sequentially administering ionizing radiation or other chemotherapeutic agents.

[0289] The pharmaceutical products and treatment methods disclosed herein can be used as adjuvant chemotherapy in combination with surgery. The pharmaceutical products disclosed herein can be administered before surgery for the purpose of reducing tumor size (referred to as preoperative adjuvant chemotherapy or neoadjuvant therapy), or can be administered after surgery for the purpose of preventing tumor recurrence (referred to as postoperative adjuvant chemotherapy or adjuvant therapy).

[0290] In some implementations, cancer cells may exhibit a BRCA1 and / or BRCA2 deficiency phenotype, meaning that BRCA1 and / or BRCA2 activity is reduced or eliminated in the cancer cells. Cancer cells with this phenotype may lack BRCA1 and / or BRCA2, i.e., the expression and / or activity of BRCA1 and / or BRCA2 can be reduced or eliminated in the cancer cells, for example, through mutations or polymorphisms in the encoding nucleic acids, or through amplification, mutations, or polymorphisms in genes encoding regulatory factors (e.g., the EMSY gene encoding a BRCA2 regulator) (Hughes-Davies et al., Cell, 115, 523-535). BRCA1 and BRCA2 are known tumor suppressor factors, and their wild-type alleles are frequently lost in tumors of heterozygous carriers (Jasin M., Oncogene, 21(58), 8981-93 (2002); Tutt et al., Trends Mol Med., 8 (12), 571-6, (2002)). The association between BRCA1 and / or BRCA2 mutations and breast cancer is well characterized in the art (Radice, PJ, Exp ClinCancer Res., 21(3 Supplement), 9-12 (2002)). Amplification of the EMSY gene, encoding the BRCA2 binding factor, is also known to be associated with breast and ovarian cancer. Carriers of mutations in BRCA1 and / or BRCA2 are also at high risk for certain cancers, including breast, ovarian, pancreatic, prostate, hematologic malignancies, gastrointestinal, and lung cancers. In some implementations, individuals are heterozygous for one or more variants (such as mutations and polymorphisms) in BRCA1 and / or BRCA2 or their regulators. The detection of variants in BRCA1 and BRCA2 is well known in the art and described, for example, in EP 699 754, EP 705 903, Neuhausen, SL and Ostrander, EA, Genet. Test, 1, 75-83 (1992); Chappnis, PO and Foulkes, WO, Cancer Treat Res, 107, 29-59 (2002); Janatova M. et al., Neoplasia, 50(4), 246-505 (2003); Janarkova, N., Ceska Gynekol., 68{1}, 11-6 (2003). The assay of amplification of the BRCA2 binding factor EMSY is described in Hughes-Davies et al., Cell, 115, 523-535.

[0291] Cancer-related mutations and polymorphisms can be detected at the nucleic acid level by detecting the presence of variant nucleic acid sequences, or at the protein level by detecting the presence of variant (i.e., mutant or allelic variant) peptides.

[0292] The pharmaceutical products of this disclosure containing at least one pharmaceutically suitable ingredient can be administered. Depending on the dosage, administration concentration, etc., of the antibody-drug conjugate and bispecific binding protein used in this disclosure, a pharmaceutically suitable ingredient can be appropriately selected and applied from pharmaceutical additives and the like commonly used in the art. The antibody-drug conjugate used in this disclosure can be administered, for example, as a pharmaceutical product containing a buffer (such as a histidine buffer), a mediator (such as sucrose and trehalose), and a surfactant (such as polysorbate 80 and 20). The antibody-drug conjugate used in the pharmaceutical products of this disclosure can be used as an injection, for example, as an aqueous injection or a lyophilized injection, and can be used, for example, as a lyophilized injection. In the case where the pharmaceutical product containing the antibody-drug conjugate used in this disclosure is an aqueous injection, the aqueous injection can be diluted with a suitable diluent and then administered via intravenous infusion. Examples of diluents may include glucose solution and physiological saline; in one embodiment, a glucose solution is exemplified, and in another embodiment, a 5% glucose solution is exemplified. In the case where the pharmaceutical product disclosed herein is a lyophilized injection, the required amount of the lyophilized injection pre-dissolved in water for injection can be diluted with a suitable diluent and then administered via intravenous infusion. Examples of diluents may include glucose solution and physiological saline; in one embodiment, a glucose solution is exemplified, and in another embodiment, a 5% glucose solution is exemplified.

[0293] Examples of routes of administration suitable for administering the pharmaceutical products of this disclosure may include intravenous, intradermal, subcutaneous, intramuscular, and intraperitoneal routes. In one embodiment, the route of administration is intravenous.

[0294] The dosage required to treat a specific disease state will necessarily vary depending on the subject being treated, the route of administration, and the severity of the disease. Further information regarding the route of administration and dosage regimens can be found in Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press, 1990, Volume 5, Chapter 25.2.

[0295] The anti-TROP2 antibody-drug conjugate used in this disclosure can be administered to humans at intervals from 1 day to 180 days, and can be administered, for example, once weekly, once every 2 weeks, once every 3 weeks, or once every 4 weeks, and in a particular example, once every 3 weeks. Furthermore, the antibody-drug conjugate used in this disclosure can be administered at doses from about 0.001 mg / kg to 100 mg / kg, and can be administered at doses from 0.8 mg / kg to 12.4 mg / kg. For example, the anti-TROP2 antibody-drug conjugate can be administered once every 3 weeks at doses of 0.27 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 6.0 mg / kg, or 8.0 mg / kg, and in a particular example, can be administered once every 3 weeks at a dose of 4.0 mg / kg or 6.0 mg / kg.

[0296] The anti-HER2 antibody-drug conjugate used in this disclosure can be administered to humans at intervals from 1 day to 180 days, at intervals of one week, two weeks, three weeks, or four weeks, and in a particular example, at intervals of three weeks. The anti-HER2 antibody-drug conjugate used in this disclosure can be administered at doses from about 0.001 mg / kg to 100 mg / kg per administration, and at doses from 0.8 mg / kg to 12.4 mg / kg per administration. For example, the anti-HER2 antibody-drug conjugate can be administered every three weeks at doses of 0.8 mg / kg, 1.6 mg / kg, 3.2 mg / kg, 5.4 mg / kg, 6.4 mg / kg, 7.4 mg / kg, or 8 mg / kg, and in a particular example, at doses of 5.4 mg / kg or 6.4 mg / kg per three weeks.

[0297] Bispecific binding proteins can be administered at appropriate doses via any suitable route of administration.

[0298] In some respects, AZD7789 or its antigen-binding fragment is administered to the subject at a dose of about 750 mg or about 1500 mg. In some respects, the dose used for administration is about 1500 mg. In some respects, the dose used for administration is about 750 mg.

[0299] In some cases, a dose of AZD7789 or its antigen-binding fragment is administered to the subject once per treatment cycle. In some cases, the treatment cycle is three weeks. In some cases, a dose of AZD7789 or its antigen-binding fragment is administered every three weeks for approximately 12 months, approximately 24 months, approximately 36 months, or approximately 48 months.

[0300] In some respects, sabestomig and delutecanza are used in subjects with non-small cell lung cancer, wherein sabestomig is administered at a dose of 750 mg or 1500 mg. In some respects, sabestomig and delutecanza are used in subjects with non-small cell lung cancer who have previously been treated with checkpoint inhibitors (CPIs), or who are treatment-naïve, and wherein sabestomig is administered at a dose of 750 mg or 1500 mg. In some respects, sabestomig and delutecanza are used in subjects with non-small cell lung cancer who have previously been treated with checkpoint inhibitors (CPIs), and wherein sabestomig is administered at a dose of 750 mg or 1500 mg, and wherein the subject with non-small cell lung cancer has acquired resistance to CPIs. In some respects, acquired resistance to CPI is defined as exposure to anti-PD-1 / PD-L1 monotherapy for 4 months or more (and 2 cycles or more) or exposure to anti-PD-1 / PD-L1 plus chemotherapy for 6 months or more. In some respects, subjects with non-small cell lung cancer (NSCLC) have any PD-L1 status. In some respects, subjects with NSCLC have less than 1% PD-L1 status. In some respects, subjects with NSCLC have more than 1% PD-L1 status. In some respects, subjects with NSCLC have more than 50% PD-L1 status. In some respects, subjects with NSCLC have 1% to 49% PD-L1 status. In some respects, PD-L1 status is determined by methods known in the art, such as the Ventana PD-L1 (SP263) assay. In some cases, non-small cell lung cancer (NSCLC) subjects did not have operable genomic alterations (i.e., genetic alterations performed with approved therapies, such as epidermal growth factor receptor [EGFR], anaplastic lymphoma kinase [ALK], ROS proto-oncogene 1 [ROS1], neurotrophic tyrosine receptor kinase [NTRK], proto-oncogene Braf [BRAF], transfection rearrangement [RET], mesenchymal-epithelial transition factor [MET], or other operable driver kinases). In some cases, a bispecific binding protein, antibody, or its antigen-binding fragment was administered in combination with one or more chemotherapeutic agents. In some cases, the chemotherapeutic agent was carboplatin. In some cases, carboplatin was administered for up to four cycles.

[0301] In some respects, the bispecific binding proteins disclosed herein can be formulated into pharmaceutical compositions with pharmaceutically acceptable carriers, excipients, or stabilizers. In some respects, such pharmaceutical compositions are suitable for administration to humans or non-human animals via any one or more routes of administration using methods known in the art. The term "pharmaceutically acceptable carrier" means one or more non-toxic materials that do not interfere with the effectiveness of the bioactivity of the active ingredient. Such formulations may conventionally include salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also include compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans. Other contemplated carriers, excipients, and / or additives that may be used in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients such as serum albumin, gelatin, casein, and salt-forming counterions such as sodium. These and additional known drug carriers, excipients, and / or additives suitable for the formulations described herein are known in the art, for example, as in "Remington: The Science & Practice of Pharmacy," 21st edition, Lippincott Williams & Wilkins, (2005) and "Physician's Desk Reference," 60th edition, Medical Economics, Montvale, NJ (2005). Pharmaceutically acceptable carriers suitable for the desired or required administration modality, solubility, and / or stability may be selected.

[0302] In some respects, therapeutic compositions can be formulated for specific routes of administration, such as oral, nasal, pulmonary, local (including oral and sublingual), rectal, vaginal, and / or parenteral administration. As used herein, the terms "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and local administration (usually by injection), and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Formulations of this disclosure suitable for local or transdermal application include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalers. Antibodies and other active substances can be mixed under sterile conditions with pharmaceutically acceptable carriers and with any preservatives, buffers or propellants that may be required (see, for example, U.S. Patents 7,378,110; 7,258,873; and 7,135,180; U.S. Patent Application Publications 2004 / 0042972 and 2004 / 0042971).

[0303] The formulation may exist in unit dosage form and may be prepared by any method known in the pharmaceutical field. The actual dose level of the active ingredient in the pharmaceutical compositions of this disclosure may vary to obtain an amount of active ingredient (e.g., a “therapeuticly effective amount”) that is effective in achieving the desired therapeutic response for a particular patient, composition, and administration method without toxicity to the patient. The selected dose level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition used, route of administration, time of administration, excretion rate of the particular compound used, duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health status, and prior medical history of the patient being treated, and similar factors well known in the medical field. These doses may be administered daily, weekly, bi-weekly, monthly, or less frequently (e.g., semi-annually), depending on the dose, method of administration, condition or symptom to be treated, and individual subject characteristics. The dose may also be administered via continuous infusion (such as by a pump). The administered dose may also depend on the route of administration. For example, subcutaneous administration may require a higher dose than intravenous administration. As stated above, any commonly used dosing regimen (e.g., 1 mg / kg–10 mg / kg, administered daily or twice weekly by injection or infusion) can be adapted and adapted to methods relevant to the treatment of human cancer patients.

[0304] [Example]

[0305] The present disclosure is described in detail with reference to the embodiments shown below. However, the present disclosure is not limited thereto. Furthermore, it is in no way intended to be limiting.

[0306] Example 1A: Preparation of anti-TROP2 antibody-drug conjugate (DS-1062)

[0307] According to the preparation methods described in WO2015 / 098099, WO2017 / 002776 and WO2022 / 014698, and using an anti-TROP2 antibody (an antibody comprising a heavy chain consisting of an amino acid sequence represented by amino acid residues 20 to 470 of SEQ ID NO: 1 and a light chain consisting of an amino acid sequence represented by amino acid residues 21 to 234 of SEQ ID NO: 2), an anti-TROP2 antibody-drug conjugate (DS-1062: delutecan) is prepared, wherein the drug-linker represented by the following formula is conjugated to the anti-TROP2 antibody via a thioether bond:

[0308]

[0309] Where A represents the binding site with the antibody. The DAR of the antibody-drug conjugate is approximately 4.0.

[0310] Example 1B: Preparation of anti-HER2 antibody-drug conjugate (DS-8201)

[0311] According to the preparation method described in WO2015 / 115091, and using an anti-HER2 antibody (an antibody comprising a heavy chain consisting of an amino acid sequence represented by amino acid residues 1 to 449 of SEQ ID NO: 24 (SEQ ID NO: 14) and a light chain consisting of an amino acid sequence consisting of all amino acid residues 1 to 214 of SEQ ID NO: 15), an anti-HER2 antibody-drug conjugate (DS-8201: trastuzumab) was prepared, wherein the drug-linker represented by the following formula is conjugated to the anti-HER2 antibody via a thioether bond:

[0312]

[0313] Where A represents the binding site with the antibody.

[0314] The DAR of the antibody-drug conjugate was 7.7 or 7.8.

[0315] Example 2: Preparation of anti-PD-1 / TIM-3 bispecific antibody (AZD7789)

[0316] AZD7789 (anti-PD-1 / TIM-3 bispecific antibody) can be prepared according to the methods disclosed in WO2017 / 193032, WO2022 / 221245 and Mazor et al., MAbs. 7(2): 377–389 (March to April 2015).

[0317] Example 3: Anti-tumor test: In vitro study in a tumor-immune cell co-culture model

[0318] The combination of antibody-drug conjugate DS-1062 and AZD7789.

[0319] A co-culture assay system was developed utilizing melanoma antigen Melan A (MART-1)-responsive T cells and PC9 lung adenocarcinoma cells engineered to express both MART-1 antigen and green fluorescent protein (GFP). Briefly, PC9-MART1-GFP cells were harvested from cell culture flasks using trypsin, washed once with complete RPMI medium, resuspended, and added to each well of a 96-well clear-bottom, opaque-walled optical plate at 37°C in 5% CO2 for 24 hours according to the manufacturer's instructions. Anti-PD-1 / TIM3 bispecific antibody (AZD7789) was used at a concentration of 10 μg / mL. DS-1062 was used at 10 μg / mL. Individual or combined treatments were added to the wells immediately before resting, with antigen-specific CD8+ T cells added at a 1:1 effector to target (E:T) ratio (effector T cells to target tumor cells). Tumor cell lysis was evaluated using the Sartorius Incucyte live-cell analysis system, with loss of GFP signal intensity used as a surrogate marker of cell death. Five days after T cell addition, the percentage of tumor cell death and cell lysis was measured by image analysis of GFP intensity in each well. The activities of DS-1062, AZD7789, and their combinations in a tumor-immune cell co-culture model were also investigated. Figure 44 and Figure 45 As shown. Figure 45 The statistical significance of the comparisons shown is expressed as **p < 0.01.

[0320] result :

[0321] like Figure 44 and Figure 45 As shown, AZD7789 and DS-1062, as monotherapy, enhanced T cell-mediated cytolysis of PC9-MART1 tumor cells. The combination of AZD7789 and DS-1062 achieved the highest degree of tumor cell cytolysis.

[0322] Therefore, the combination of DS-1062 and AZD2936 enhanced in vitro T cell-mediated tumor cell killing in a tumor-immune cell co-culture model.

[0323] Example 4: In participants with advanced or metastatic non-small cell lung cancer (Tropion-Lung04), the efficacy of [a certain treatment] was evaluated. Lutecan (Dato-DXd) in combination with immunotherapy with or without carboplatin was investigated in a phase 1b, multicenter, 2-part, open-label trial. Label Research .

[0324] research group :

[0325] Participants with advanced or metastatic non-small cell lung cancer (NSCLC) but without operable genomic alterations (i.e., genetic alterations performed with approved therapies, such as epidermal growth factor receptor [EGFR], anaplastic lymphoma kinase [ALK], ROS proto-oncogene 1 [ROS1], neurotrophic tyrosine receptor kinase [NTRK], proto-oncogene Braf [BRAF], transfection rearrangement [RET], mesenchymal-epithelial transition factor [MET], or other operable driver kinases). Participants with non-operable driver kinase alterations and who are not receiving available targeted therapies should be considered for this study.

[0326] Groups 12 and 13 (Part 1 and Part 2) will recruit participants with any PD-L1 status of acquired resistance to checkpoint inhibitors (CPIs) following one or two prior series of systemic therapies for advanced or metastatic NSCLC, where at least one prior series of therapies should contain an approved anti-PD-1 / PD-L1.

[0327] Group 14 will recruit participants with any PD-L1 status (untreated) who have not previously received systemic anticancer therapy for advanced or metastatic NSCLC.

[0328] Research Design :

[0329] This is a multicenter, open-label, multi-dose, and regimen-phase 1b study of Dato-DXd in combination with immunotherapy (AZD7789[sabestomig]) in participants with advanced or metastatic NSCLC who do not have operable genomic alterations (i.e., genetic alterations performed with approved therapies, such as EGFR, ALK, ROS1, NTRK, BRAF, RET, MET, or other operable driver kinases), with or without up to 4 cycles of carboplatin. Participants with non-operable driver kinase alterations and who are not receiving available targeted therapy, or those with tumors containing KRAS mutations, should be considered for this study. The primary objective is to evaluate the safety and tolerability of Dato-DXd in combination with immunotherapy (with or without up to 4 cycles of carboplatin) in participants with advanced or metastatic NSCLC. The study population has been described above. In each study group, the combination of two dose levels of Dato-DXd (4 mg / kg and 6 mg / kg) with AZD7789 (750 mg or 1500 mg) with or without carboplatin for up to four cycles will be investigated:

[0330] Group 12: In participants with CPI-acquired resistance to NSCLC, a combination of 6 mg / kg Dato-DXd and 1500 mg AZD7789, Q3W;

[0331] Group 13: In participants with CPI-acquired resistance to NSCLC, a combination of 4 mg / kg Dato-DXd with 1500 mg AZD7789, Q3W, or a combination of 6 mg / kg Dato-DXd with 750 mg AZD7789, Q3W (triggered only if downgrade is required).

[0332] Group 14: In participants with untreated NSCLC, a combination of 4 mg / kg or 6 mg / kg Dato-DXd with 750 mg or 1500 mg AZD7789, Q3W. The doses of Dato-DXd and AZD7789 will be those considered to be tolerable in CPI-acquired resistance participants from Group 12 or 13.

[0333] Groups 12 and 13 will recruit participants with acquired resistance to CPIs following one or two prior series of systemic therapies for advanced or metastatic NSCLC. Groups 12 and 13 will recruit participants with any PD-L1 status, including 12 to 20 participants with PD-L1 <1%. Group 14 will recruit participants with any PD-L1 status, including approximately 20 participants with PD-L1 levels ranging from 1% to 49%.

[0334] Objectives, outcome measurement, and endpoints :

[0335] The safety and tolerability, efficacy (e.g., objective response rate, duration of response, disease control rate, progression-free survival, response time, best percentage change in measurable tumor SoD, overall survival, pharmacokinetic profile, and immunogenicity) of combination therapy with Dato-DXd and Sabestomig (with or without up to 4 cycles of carboplatin) will be evaluated.

[0336] Example 5: Anti-tumor test: In vivo study in humanized mice

[0337] The combination of antibody-drug conjugate DS-8201 and AZD7789.

[0338] At Jackson Labs, using CD34 + Umbilical cord blood stem cell transplantation was performed on female immunocompromised NOD.Cg-PPrkdcscid Il2rgtm1Wjl / SzJ (NSG) mice. Humanized mice were obtained 12 weeks post-implantation, with a 7-day adaptation period allowed. At 14 weeks post-implantation, 1e6 Caki-1 cells were subcutaneously injected into the right abdomen. Tumor volume was measured twice weekly using electronic calipers and calculated using the formula (width...). 2 Calculated as (×length) / 2. For efficacy assessment, when the tumor reaches 125mm... 3 Treatment was initiated when the tumor reached an average volume. To assess changes in tumor pharmacokinetics (PD) by flow cytometry, treatment was initiated when the tumor reached 250 mm. 3 Treatment began when the tumor volume was at its average size. Mice were randomly assigned to the treatment groups shown in Table 1 based on tumor size and umbilical cord blood donor.

[0339] Table 1

[0340]

[0341] Preparation of 10mg / kg DS-8201

[0342] The DS-8201 dosing solution (batch HA306) was prepared by diluting the DS-8201 stock solution (20.1 mg / mL) to 2 mg / mL in a medium (25 mM histidine buffer, 9% sucrose, pH 5.5) and then administered via intravenous (IV) injection at a dosing volume of 5 mL / kg.

[0343] AZD7789 formulation 10 mg / kg

[0344] The dosing solution of AZD7789 (batch SP22-031) was prepared by diluting the stock solution (50.3 mg / mL) to 2 mg / mL in PBS and then administered via intraperitoneal (IP) injection at a dosing volume of 5 mL / kg.

[0345] Flow cytometry staining

[0346] Tumors were excised and dissociated into single-cell suspensions using the Miltenyi Human Tumor Dissociation Kit. Single-cell suspensions were counted on a CellaMX high-throughput automated cell counter and seeded at 1e6 to 2e6 cells / sample in 96-well V-shaped plates. Cells were stained with Live Blue / Dead Blue at room temperature for 30 minutes, followed by mouse and human Fc blocking at room temperature for 10 minutes. The fully stained extracellular master mixture was applied on ice for 30 minutes, and then the cells were stored overnight in fixation buffer at 4°C. After each staining step, the plates were washed with staining buffer (PBS + 2% BSA). The next morning, permeabilization buffer was added on ice for 30 minutes. The human and mouse Fc blocking steps were repeated, with the intracellular master mixture applied on ice for 30 minutes. After a final wash with staining buffer, the samples were resuspended in 120 μL of FACS buffer. The plates were filtered and run on a Cytek Aurora system.

[0347] The tumor growth rate for each animal was calculated by fitting the growth curve of each tumor to an exponential model log10(tumor volume) = a + b · time + error, where a and b are parameters corresponding to the logarithmic initial volume and growth rate, respectively. The Mann-Whitney U test was used to evaluate the statistical significance of differences in tumor growth rates between groups.

[0348] For differences in pharmacokinetics obtained by flow cytometry, a standard one-way ANOVA was performed. P-values ​​are reported as follows: p ≥ 0.05 (ns, not significant); *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0349] result :

[0350] As summarized in Table 1, humanized Caki-1 tumor-bearing mice were treated with DS-8201 and AZD7789 in single-agent and combined formulations. Figure 46 (Tumor volume over time (±SEM)) Figure 47 (Tumor growth rate (±SEM)) and the results shown in Table 2 show that, compared with the single agent DS-8201 (5.5%) or AZD7789 (18.7%), the combination of DS-8201+AZD7789 (107.7%) showed enhanced tumor growth rate inhibition relative to the mediator.

[0351] Table 2

[0352]

[0353] Pharmacodynamic changes

[0354] Caki-1 cells were subcutaneously implanted into CD34 cells. + The right ventral region of NSG mice with umbilical cord blood was used for treatment with DS-8201 alone or in combination with 10 mg / kg AZD7789, according to the dosing regimens outlined in Table 1. Tumors were collected 8 days after the start of treatment, and pharmacokinetic changes were assessed by flow cytometry.

[0355] As from Figure 48 The results shown indicate that, compared to AZD7789 monotherapy, the DS-8201+AZD7789 combination revealed tumor-invasive CD8 infiltration throughout the entire tumor obtained 8 days after treatment initiation. + A significant increase in T cells was observed in the combination of DS-8201 and AZD7789 compared to other groups, with activation of CD69 (cdc69) also noted. + ), proliferation (ki67) + ) and effector (granzyme B) + CD8 + An increasing trend in the proportion of T cells was observed in this combination. Additionally, NK (CD56) cells were also observed. + Increased abundance of CD33 cells. In the myeloid compartment (CD33... + In cells, granzyme B expression was significantly increased compared to the single therapy DS-8201.

[0356] The foregoing written description is considered sufficient to enable those skilled in the art to practice these embodiments. The foregoing description and examples detail certain embodiments and describe the best mode anticipated by the inventors. However, it should be understood that, however detailed the foregoing may appear in the text, the embodiments can be practiced in many ways, and the claims include any equivalents thereof.

[0357] Free text of sequence lists

[0358] SEQ ID NO: 1 - Amino acid sequence of the heavy chain of anti-TROP2 antibody

[0359] SEQ ID NO: 2 - Amino acid sequence of the light chain of anti-TROP2 antibody

[0360] The amino acid sequence of SEQ ID NO: 3-heavy chain CDRH1 [= amino acid residues 50 to 54 of SEQ ID NO: 1]

[0361] The amino acid sequence of SEQ ID NO: 4-heavy chain CDRH2 [= amino acid residues 69 to 85 of SEQ ID NO: 1]

[0362] The amino acid sequence of SEQ ID NO: 5-heavy chain CDRH3 [= amino acid residues 118 to 129 of SEQ ID NO: 1]

[0363] The amino acid sequence of the light chain CDRL1 in SEQ ID NO: 6 [= amino acid residues 44 to 54 of SEQ ID NO: 2]

[0364] The amino acid sequence of the light chain CDRL2 in SEQ ID NO: 7 [= amino acid residues 70 to 76 of SEQ ID NO: 2]

[0365] The amino acid sequence of the light chain CDRL3 of SEQ ID NO: 8 [= amino acid residues 109 to 117 of SEQ ID NO: 2]

[0366] SEQ ID NO: 9 - Amino acid sequence of the heavy chain variable region [= amino acid residues 20 to 140 of SEQ ID NO: 1]

[0367] SEQ ID NO: 10 - Amino acid sequence of the light chain variable region [= amino acid residues 21 to 129 of SEQ ID NO: 2]

[0368] SEQ ID NO: 11 - Amino acid sequence of the heavy chain [= amino acid residues 20 to 469 of SEQ ID NO: 1]

[0369] SEQ ID NO: 12 - Amino acid sequence of the heavy chain [= amino acid residues 20 to 470 of SEQ ID NO: 1]

[0370] SEQ ID NO: 13 - Amino acid sequence of the light chain [= Amino acid residues 21 to 234 of SEQ ID NO: 2]

[0371] SEQ ID NO: 14 - Amino acid sequence of the heavy chain of the anti-HER2 antibody

[0372] SEQ ID NO: 15 - Amino acid sequence of the light chain of the anti-HER2 antibody

[0373] The amino acid sequence of heavy chain CDRH1 in SEQ ID NO: 16 [= amino acid residues 26 to 33 of SEQ ID NO: 14]

[0374] The amino acid sequence of the heavy chain CDRH2 of SEQ ID NO: 17 [= amino acid residues 51 to 58 of SEQ ID NO: 14]

[0375] The amino acid sequence of SEQ ID NO: 18-heavy chain CDRH3 [= amino acid residues 97 to 109 of SEQ ID NO: 14]

[0376] SEQ ID NO: 19 - Amino acid sequence of light chain CDRL1 [= Amino acid residues 27 to 32 of SEQ ID NO: 15]

[0377] SEQ ID NO: 20 - Amino acid sequence containing the light chain CDRL2 (SAS) [= Amino acid residues 50 to 56 of SEQ ID NO: 15]

[0378] The amino acid sequence of the light chain CDRL3 of SEQ ID NO: 21 [= amino acid residues 89 to 97 of SEQ ID NO: 15]

[0379] SEQ ID NO: 22 - Amino acid sequence of the heavy chain variable region [= amino acid residues 1 to 120 of SEQ ID NO: 14]

[0380] SEQ ID NO: 23 - Amino acid sequence of the light chain variable region [= amino acid residues 1 to 107 of SEQ ID NO: 15]

[0381] SEQ ID NO: 24 - Amino acid sequence of the heavy chain [= amino acid residues 1 to 449 of SEQ ID NO: 14]

[0382] SEQ ID NO: 25 - Amino acid sequence of anti-PD1 heavy chain CDRH1 of anti-PD-1 / TIM-3 bispecific antibody

[0383] SEQ ID NO: 26 - Amino acid sequence of anti-PD1 heavy chain CDRH2 of anti-PD-1 / TIM-3 bispecific antibody

[0384] SEQ ID NO: 27 - Amino acid sequence of anti-PD1 heavy chain CDRH3 of anti-PD-1 / TIM-3 bispecific antibody

[0385] SEQ ID NO: 28 - Amino acid sequence of the anti-PD1 light chain CDRL1 of the anti-PD-1 / TIM-3 bispecific antibody

[0386] SEQ ID NO: 29 - Amino acid sequence of the anti-PD1 light chain CDRL2 of the anti-PD-1 / TIM-3 bispecific antibody

[0387] SEQ ID NO: 30 - Amino acid sequence of the anti-PD1 light chain CDRL3 of the anti-PD-1 / TIM-3 bispecific antibody.

[0388] SEQ ID NO: 31 - Amino acid sequence of the anti-PD1 heavy chain variable region of the anti-PD-1 / TIM-3 bispecific antibody

[0389] SEQ ID NO: 32 - Amino acid sequence of the anti-PD1 heavy chain of the anti-PD-1 / TIM-3 bispecific antibody

[0390] SEQ ID NO: 33 - Amino acid sequence of the anti-PD1 light chain variable region of the anti-PD-1 / TIM-3 bispecific antibody

[0391] SEQ ID NO: 34 - Amino acid sequence of the anti-PD1 light chain of the anti-PD-1 / TIM-3 bispecific antibody

[0392] SEQ ID NO: 35 - Amino acid sequence of anti-TIM-3 heavy chain CDRH1 of anti-PD-1 / TIM-3 bispecific antibody

[0393] SEQ ID NO: 36 - Amino acid sequence of anti-TIM-3 heavy chain CDRH2 of anti-PD-1 / TIM-3 bispecific antibody

[0394] SEQ ID NO: 37 - Amino acid sequence of anti-TIM-3 heavy chain CDRH3 of anti-PD-1 / TIM-3 bispecific antibody

[0395] SEQ ID NO: 38 - Amino acid sequence of the anti-TIM-3 light chain CDRL1 of the anti-PD-1 / TIM-3 bispecific antibody.

[0396] SEQ ID NO: 39 - Amino acid sequence of the anti-TIM-3 light chain CDRL2 of the anti-PD-1 / TIM-3 bispecific antibody.

[0397] SEQ ID NO: 40 - Amino acid sequence of the anti-TIM-3 light chain CDRL3 of the anti-PD-1 / TIM-3 bispecific antibody.

[0398] SEQ ID NO: 41 - Amino acid sequence of the anti-TIM-3 heavy chain variable region of the anti-PD-1 / TIM-3 bispecific antibody

[0399] SEQ ID NO: 42 - Amino acid sequence of the anti-TIM-3 heavy chain of the anti-PD-1 / TIM-3 bispecific antibody

[0400] SEQ ID NO: 43 - Amino acid sequence of the anti-TIM-3 light chain variable region of the anti-PD-1 / TIM-3 bispecific antibody

[0401] SEQ ID NO: 44 - Amino acid sequence of the anti-TIM-3 light chain of the anti-PD-1 / TIM-3 bispecific antibody

[0402] Amino acid sequence of SEQ ID NO: 45-human TIM-3 IgV

Claims

1. A pharmaceutical product comprising an antibody-drug conjugate and an anti-PD-1 / TIM-3 bispecific binding protein for combined administration, wherein the antibody-drug conjugate is a drug linker represented by the following formula: Where A represents the linking site with the antibody, which is an antibody-drug conjugate to the anti-TROP2 or anti-HER2 antibody via a thioether bond.

2. The pharmaceutical product of claim 1, wherein the drug-linker is conjugated to the anti-TROP2 antibody.

3. The pharmaceutical product according to claim 2, wherein the anti-TROP2 antibody is an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises CDRH1 composed of the amino acid sequence represented by SEQ ID NO: 3, CDRH2 composed of the amino acid sequence represented by SEQ ID NO: 4, and CDRH3 composed of the amino acid sequence represented by SEQ ID NO: 5, and the light chain comprises CDRL1 composed of the amino acid sequence represented by SEQ ID NO: 6, CDRL2 composed of the amino acid sequence represented by SEQ ID NO: 7, and CDRL3 composed of the amino acid sequence represented by SEQ ID NO:

8.

4. The pharmaceutical product according to claim 3, wherein the anti-TROP2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 9, and the light chain comprising a light chain variable region consisting of an amino acid sequence represented by SEQ ID NO:

10.

5. The pharmaceutical product according to claim 3 or 4, wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 12 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

13.

6. The pharmaceutical product of claim 5, wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

7. The pharmaceutical product according to any one of claims 1 to 6, wherein the average number of units of the drug-linker conjugated to each antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.

5.

8. The pharmaceutical product according to claim 7, wherein the anti-TROP2 antibody-drug conjugate is drutecan (DS-1062).

9. The pharmaceutical product of claim 1, wherein the drug-linker is conjugated to the anti-HER2 antibody.

10. The pharmaceutical product of claim 9, wherein the anti-HER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain comprising CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 16, CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 17, and CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 18, and the light chain comprising CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 19, CDRL2 consisting of the amino acid sequence consisting of amino acid residues 1 to 3 of SEQ ID NO: 20, and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO:

21.

11. The pharmaceutical product of claim 10, wherein the anti-HER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 22, and the light chain comprising a light chain variable region consisting of an amino acid sequence represented by SEQ ID NO:

23.

12. The pharmaceutical product of claim 11, wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 14 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

15.

13. The pharmaceutical product of claim 11, wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 24 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

15.

14. The pharmaceutical product according to any one of claims 9 to 13, wherein the average number of units of the drug-linker conjugated to each anti-HER2 antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

15. The pharmaceutical product of claim 14, wherein the anti-HER2 antibody-drug conjugate is trastuzumab (DS-8201).

16. The pharmaceutical product according to any one of claims 1 to 15, wherein the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain, the TIM-3 binding domain specifically binding to the C'C'' loop and DE loop of the immunoglobulin variable (IgV) domain of TIM-3 (SEQ ID NO: 45).

17. The pharmaceutical product according to any one of claims 1 to 16, wherein the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain, the TIM-3 binding domain specifically binding to an epitope on the IgV domain of TIM-3 (SEQ ID NO: 45), and the epitope includes N12, L47, R52, D53, V54, N55, Y56, W57, W62, L63, N64, G65, D66, F67, R68, K69, D71, T75, and E77.

18. The pharmaceutical product according to any one of claims 1 to 17, wherein the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain, the TIM-3 binding domain comprising a first set of CDRs: CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprising the amino acid sequences of SEQ ID NO: 35, 36, 37, 38, 39, and 40 or 35, 36, 37, 38, 39, and 45, respectively; and a PD-1 binding domain, the PD-1 binding domain comprising a second set of CDRs: CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprising the amino acid sequences of SEQ ID NO: 25, 26, 27, 28, 29, and 30, respectively.

19. The pharmaceutical product according to any one of claims 1 to 18, wherein the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:41 and a light chain variable domain containing the amino acid sequence of SEQ ID NO:43, and has a PD-1 binding domain comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:31 and a light chain variable domain containing the amino acid sequence of SEQ ID NO:

33.

20. The pharmaceutical product according to any one of claims 1 to 18, wherein the anti-PD-1 / TIM-3 bispecific binding protein has a TIM-3 binding domain comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 42 and a light chain containing the amino acid sequence of SEQ ID NO: 44, and has a PD-1 binding domain comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 32 and a light chain containing the amino acid sequence of SEQ ID NO:

34.

21. The pharmaceutical product according to any one of claims 1 to 20, wherein the anti-PD-1 / TIM-3 bispecific binding protein is an antibody.

22. The pharmaceutical product according to claim 21, wherein the antibody is an IgG antibody.

23. The pharmaceutical product according to claim 21, wherein the antibody is an IgG1 antibody.

24. The pharmaceutical product according to claim 22 or 23, wherein the antibody is human or humanized.

25. The pharmaceutical product according to any one of claims 21 to 24, wherein the bispecific antibody is monovalent.

26. The pharmaceutical product according to any one of claims 1 to 25, wherein the anti-PD-1 / TIM-3 bispecific binding protein is DuetMab.

27. The pharmaceutical product according to any one of claims 1 to 26, wherein the anti-PD-1 / TIM-3 bispecific binding protein comprises a non-glycosylated Fc region.

28. The pharmaceutical product according to any one of claims 1 to 26, wherein the anti-PD-1 / TIM-3 bispecific binding protein comprises a deglycosylated Fc region.

29. The pharmaceutical product according to any one of claims 1 to 26, wherein the anti-PD-1 / TIM-3 bispecific binding protein comprises an Fc region having reduced fucosylation or being non-fucosylated.

30. The pharmaceutical product according to any one of claims 1 to 29, wherein the anti-PD-1 / TIM-3 bispecific binding protein is "AZD7789".

31. The pharmaceutical product according to any one of claims 1 to 30, wherein the product is a composition comprising the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein for simultaneous administration.

32. The pharmaceutical product according to any one of claims 1 to 30, wherein the product is a combination formulation comprising the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein, to be administered sequentially or separately.

33. The pharmaceutical product according to any one of claims 1 to 32, wherein the product is used to treat cancer.

34. The pharmaceutical product of claim 33, wherein the cancer is at least one selected from the group consisting of: breast cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, adenocarcinoma of the esophagogastric junction, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial carcinoma, prostate cancer, bladder cancer, endometrial cancer, gastrointestinal stromal tumor, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, sarcoma, and melanoma, cervical cancer, uterine cancer, testicular cancer, and renal cell carcinoma.

35. The pharmaceutical product of claim 34, wherein the cancer is lung cancer.

36. The pharmaceutical product according to claim 35, wherein the lung cancer is non-small cell lung cancer.

37. The pharmaceutical product according to claim 34, wherein the cancer is colorectal cancer.

38. The pharmaceutical product of claim 34, wherein the cancer is breast cancer.

39. The pharmaceutical product of claim 38, wherein the breast cancer is HER2-positive breast cancer.

40. The pharmaceutical product of claim 38, wherein the breast cancer is HER2-low expression breast cancer.

41. The pharmaceutical product of claim 38, wherein the breast cancer is triple-negative breast cancer.

42. The pharmaceutical product of claim 38, wherein the breast cancer is hormone receptor (HR) positive, HER2 negative breast cancer.

43. The pharmaceutical product according to claim 34, wherein the cancer is gastric cancer.

44. The pharmaceutical product of claim 34, wherein the cancer is pancreatic cancer.

45. The pharmaceutical product according to claim 34, wherein the cancer is ovarian cancer.

46. ​​The pharmaceutical product of claim 34, wherein the cancer is prostate cancer.

47. The pharmaceutical product of claim 34, wherein the cancer is kidney cancer.

48. The pharmaceutical product of claim 34, wherein the cancer is bladder cancer.

49. The pharmaceutical product according to claim 34, wherein the cancer is endometrial cancer.

50. The pharmaceutical product according to claim 34, wherein the cancer is biliary tract cancer.

51. An antibody-drug conjugate for use in combination with an anti-PD-1 / TIM-3 bispecific binding protein in the treatment of a subject with cancer, wherein the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein are as defined in any one of claims 1 to 30.

52. The antibody-drug conjugate for use according to claim 51, wherein the cancer is defined as any one of claims 34 to 50.

53. The antibody-drug conjugate for use according to claim 51 or 52, wherein the use comprises sequentially administering the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein.

54. The antibody-drug conjugate for use according to claim 51 or 52, wherein the use comprises administering the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein alone and simultaneously.

55. The antibody-drug conjugate for use according to any one of claims 51 to 54, wherein the anti-PD-1 / TIM-3 bispecific binding protein is administered at a dose of about 750 mg or about 1500 mg.

56. The antibody-drug conjugate of claim 55, wherein the anti-PD-1 / TIM-3 bispecific binding protein is administered once per treatment cycle, wherein the treatment cycle is approximately three weeks.

57. The antibody-drug conjugate for use according to any one of claims 51 to 56, wherein the cancer is non-small cell lung cancer, and wherein the subject is untreated, or wherein the subject has acquired resistance to CPI.

58. The antibody-drug conjugate for use according to claim 57, wherein the subject has any PD-L1 status.

59. The antibody-drug conjugate for use according to claim 58, wherein the subject has 1% to 49% PD-L1 status, or wherein the subject has less than 1% PD-L1 status.

60. The antibody-drug conjugate for use according to any one of claims 51 to 59, wherein the subject does not have operable genomic alterations.

61. A method of treating cancer, the method comprising administering, to a subject in need, a combination of an antibody-drug conjugate and an anti-PD-1 / TIM-3 bispecific binding protein as defined in any one of claims 1 to 30.

62. The method of claim 61, wherein the cancer is defined as in any one of claims 34 to 50.

63. The method according to claim 61 or 62, wherein the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein are administered sequentially.

64. The method according to claim 61 or 62, wherein the antibody-drug conjugate and the anti-PD-1 / TIM-3 bispecific binding protein are administered individually and simultaneously.

65. The method according to any one of claims 61 to 64, wherein the anti-PD-1 / TIM-3 bispecific binding protein is administered at a dose of about 750 mg to about 1500 mg.

66. The method of claim 65, wherein the anti-PD-1 / TIM-3 bispecific binding protein is administered once per treatment cycle, wherein the treatment cycle is approximately three weeks.

67. The method according to any one of claims 61 to 66, wherein the cancer is non-small cell lung cancer, and wherein the subject is untreated, or wherein the subject has acquired resistance to CPI.

68. The method of claim 67, wherein the subject has any PD-L1 status.

69. The method of claim 68, wherein the subject has 1% to 49% PD-L1 status, or wherein the subject has less than 1% PD-L1 status.

70. The method according to any one of claims 61 to 69, wherein the subject does not have operable genomic alterations.

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