Anti-basal cell adhesion molecule antibody-drug conjugate

Abstract

The present disclosure relates to anti-basal cell adhesion molecule (BCAM) antibody-drug conjugates in which the anti-BCAM antibody is conjugated to an anti-tumor compound via a linker, pharmaceutical compositions comprising the anti-BCAM antibody-drug conjugates, and methods of treating cancer by administering an effective amount of the anti-BCAM antibody-drug conjugate to a subject in need thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Korean Patent Application No. 10-2022-0155122, entitled "ANTI-BASAL CELL ADHESION MOLECULE ANTIBODY-DRUG CONJUGATE," filed on November 18, 2022, the entire contents of which are incorporated herein by reference.

[0002] Technical field to which the invention belongs The present disclosure relates to an anti-basal cell adhesion molecule (BCAM) antibody-drug conjugate in which an anti-BCAM antibody is conjugated to an anti-tumor compound via a linker, a pharmaceutical composition comprising the anti-BCAM antibody-drug conjugate, and a method of treating cancer by administering an effective amount of the anti-BCAM antibody-drug conjugate to a subject in need thereof.

[0003] Background technology Basal cell adhesion molecule (BCAM) is a member of the immunoglobulin superfamily and a receptor for laminin, which promotes cell adhesion, migration, and invasion. BCAM plays an important role in tumor progression and has been reported to be overexpressed in certain cancers. It is also known as Lutheran antigen (LU). Antibody-drug conjugates (ADCs) consist of antibodies covalently linked to antitumor drugs via a linker, combining the specificity of antibodies with the cytotoxic properties of antitumor drugs using chemical linkers. There has been a need for anti-BCAM antibody-drug conjugates that can be used for therapeutic purposes in cancer treatment. Summary of the Invention

[0004] The present disclosure provides Ab-(L-(D) m ) nThe present invention provides an antibody-drug conjugate of formula (1) having the structure: or a pharmaceutically acceptable salt thereof. In formula (1), Ab is an anti-basal cell adhesion molecule (BCAM) antibody or an antigen-binding fragment thereof comprising (i) a heavy chain variable region comprising the VH CDR1 sequence of SEQ ID NO: 1, the VH CDR2 sequence of SEQ ID NO: 2, and the VH CDR3 sequence of SEQ ID NO: 3, and (ii) a light chain variable region comprising the VL CDR1 sequence of SEQ ID NO: 4, the VL CDR2 sequence of SEQ ID NO: 5, and the VL CDR3 sequence of SEQ ID NO: 6. Furthermore, D is an anti-tumor compound conjugated to the anti-BCAM antibody or antibody fragment via a linker, and L is a compound represented by formula (2): [ka] (wherein Y is a divalent group containing one or more atoms selected from C, N, O, P, and S; T is a (1+o)- or (2+o)-valent linking group; S is an atom or group optionally present to saturate the free valences of T; L' is a linker cleavable by cathepsin B; o is an integer from 1 to 5; * indicates covalent binding to an anti-BCAM antibody (Ab), and ** is a linker represented by (depicting a covalent bond to one or more antitumor compounds (D)).

[0005] In formula (1), n ​​is 1 to 10, and m is 1 to 5. In one embodiment, the anti-BCAM antibody has a heavy chain variable region comprising the sequence of SEQ ID NO: 7 and a light chain variable region comprising the sequence of SEQ ID NO: 8. In some embodiments, n is 3 to 8, and m is 1. In some embodiments, the drug (anti-tumor compound) to antibody (anti-BCAM antibody) ratio (DAR) is about 3 to about 8. In another embodiment, the DAR is about 4. The antigen-binding fragment may be an antibody fragment selected from the group consisting of a Fab fragment, a Fab' fragment, a Fab'-SH fragment, an Fv fragment, an ScFv fragment, a F(ab')2 fragment, a VL fragment, a VH fragment, an ScFv-Fc fragment, an (ScFv)2-Fc fragment, a diabody, a linear antibody, a fragment produced by a Fab expression library, an anti-idiotype (anti-Id) antibody, a complementary determining region (CDR), and an epitope-binding fragment. In another embodiment, the anti-BCAM antibody is a chimeric, humanized, or human antibody.

[0006] In one embodiment, the antibody according to the present invention may be IgG1, IgG2, IgG3, IgG4, or a variant thereof. In another embodiment, the antibody of the present invention may be IgG1, IgG4, or a variant thereof. The present disclosure also provides a pharmaceutical composition comprising the antibody-drug conjugate of formula (1) and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition is for treating cancer. In one embodiment, the cancer is one or more selected from the group consisting of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, renal cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, renal cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer, and urothelial cell carcinoma.

[0007] The present disclosure also provides a method for treating cancer, comprising administering an effective amount of an antibody-drug conjugate to a subject in need thereof. In one embodiment, the cancer is one or more selected from the group consisting of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, renal cancer, esophageal cancer, leukemia, hepatocellular carcinoma, renal cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer, and urothelial cell carcinoma.

[0008] In some aspects, the present disclosure includes the following embodiments (“items”): Item 1. Formula (1): [ka] (wherein Ab is an anti-basal cell adhesion molecule (BCAM) antibody or an antigen-binding fragment thereof, comprising: (i) a heavy chain variable region comprising the VH CDR1 sequence of SEQ ID NO: 1, the VH CDR2 sequence of SEQ ID NO: 2, and the VH CDR3 sequence of SEQ ID NO: 3; and (ii) a light chain variable region comprising the VL CDR1 sequence of SEQ ID NO: 4, the VL CDR2 sequence of SEQ ID NO: 5, and the VL CDR3 sequence of SEQ ID NO: 6; D is an anti-tumor compound conjugated to the anti-BCAM antibody via a linker; n is 1 to 10, m is 1 to 5; L is calculated using the formula (2): [ka] (wherein Y is a divalent group containing one or more atoms selected from C, N, O, P, and S; T is a (1+o)- or (2+o)-valent linking group; S is an atom or group optionally present to saturate the free valences of T; L' is a linker cleavable by cathepsin B; o is an integer from 1 to 5; * indicates covalent binding to an anti-BCAM antibody (Ab), and ** is a linker represented by:

[0009] Item 2. The antibody-drug conjugate of Item 1, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 7 and the light chain variable region comprises the sequence of SEQ ID NO: 8.

[0010] Item 3. The antibody-drug conjugate according to Item 1 or 2, wherein n is 3 to 8 and m is 1.

[0011] Item 4. The antibody-drug conjugate according to any one of Items 1 to 3, wherein the drug (anti-tumor compound) to antibody (anti-BCAM antibody) ratio (DAR) is about 3 to about 8.

[0012] Item 5. The antibody-drug conjugate of Item 4, having a DAR of about 4.

[0013] Item 6. The antibody-drug conjugate of any one of Items 1 to 5, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of a Fab fragment, a Fab' fragment, a Fab'-SH fragment, an Fv fragment, an scFv fragment, a F(ab')2 fragment, a VL fragment, a VH fragment, an ScFv-Fc fragment, and an (scFv)2-Fc fragment, a bispecific antibody, a linear antibody, a fragment produced by a Fab expression library, an anti-idiotype (anti-Id) antibody, a complementarity-determining region (CDR), and an epitope-binding fragment.

[0014] Item 7. The antibody-drug conjugate according to any one of Items 1 to 6, wherein the anti-BCAM antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0015] Item 8. The antibody-drug conjugate according to any one of items 1 to 7, wherein the linker is covalently attached to the antibody via a side chain of a cysteine ​​contained in the antibody.

[0016] Item 9. The linker (L') cleavable by cathepsin B is represented by formula (3) or formula (4): [ka] (In the formula, Axx is a trifunctional amino acid, provided that Axx in formula (3) is not an amino acid of the (D) configuration, Ayy in formulas (3) and (4) is an amino acid selected from Phe, Ala, Trp, Tyr, phenylglycine (Phg), Met, Val, His, Lys, Arg, citrulline (Cit), 2-aminobutyric acid (Abu), ornithine (Orn), Ser, Thr, Leu, and Ile, or Ayy in formula (3) is homo-tyrosine (homo-Tyr), homo-phenylalanine (homo-Phe), beta-phenylalanine (beta-Phe) and beta-homo-phenylalanine (beta-homo-Phe), Tyr(OR1) and homo-Tyr(OR1) (wherein R1 is -(CH2CHO) n1 -R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), with the proviso that Ayy in formula (4) is not an amino acid of the (D) configuration, Z is a group covalently bonded to the C-terminus of Ayy or Axx selected from -OH and -N(H)(R), where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; W is a drug-loading unit; ** represents a covalent bond to one or more moieties D, and *** indicates a covalent bond to T, When two or more linkers L' are present, each linker is independently selected from the aforementioned linkers of formula (3) and formula (4). The antibody-drug conjugate according to any one of items 1 to 8.

[0017] Item 10. At least one or both of Axx and Ayy are defined as follows: Axx, (a) Axx in formula (3) or (4) is an amino acid selected from Glu, 2-amino-pimelic acid (Apa), 2-aminoadipic acid (Aaa), 2,3-diamino-propionic acid (Dap), 2,4-diamino-butyric acid (Dab), Lys, Orn, Ser, amino-malonic acid (Ama), and homo-lysine (homo-Lys); Ayy, (b) Ayy in formula (3) is Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1) (wherein R1 is -(CH2CHO) n1 -R2, wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24, or (c) Ayy in formula (4) is an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, and Ser; Item 10. The antibody-drug conjugate according to Item 9, which satisfies the above.

[0018] Item 11. At least one or both of Axx and Ayy are defined as follows: Axx, (a) Axx in formula (3) or (4) is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; Ayy, (b) Ayy in formula (3) is an amino acid selected from Phe and Tyr, or (c) Ayy in formula (4) is an amino acid selected from Phe and Ser; 11. The antibody-drug conjugate according to Item 9 or 10, which satisfies the above condition.

[0019] Item 12. The drug-carrying unit (W) is represented by the formula (5): [ka] wherein Dxx is an amino acid that is absent or has a hydrophobic side chain; Dyy is absent, Phe, or an amino acid having a basic side chain, provided that when Dxx is an amino acid having a hydrophobic side chain, Dyy is Phe or an amino acid having a basic side chain, and when Dxx is a single covalent bond, Dyy is a single covalent bond, Phe, or an amino acid having a basic side chain; ** indicates a covalent bond to D, and ** 12. The antibody-drug conjugate according to any one of items 9 to 11, wherein ' represents a covalent bond to the N-terminus of Axx or Ayy).

[0020] Item 13. At least one of Dxx and Dyy, for example one or two, is defined as follows: (a) Dxx is an amino acid selected from Phe, Val, Tyr, homo-Phe, and Ala; (b) Dyy is absent or an amino acid selected from Arg, Lys, Cit, Orn, Dap, and Dab; Item 13. The antibody-drug conjugate according to Item 12, which satisfies the above.

[0021] Item 14. The drug-carrying unit (W) is represented by formula (6) or formula (7): [ka] (In the formula, A''xx is a trifunctional amino acid, provided that A''xx in formula (6) is not an amino acid of the (D) configuration, A'yy is an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn, with the proviso that A'yy in formula (7) is not an amino acid in the (D) configuration; when two or more A'yy are present, each A'yy is independently selected from the foregoing amino acids; A"yy is an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn, with the proviso that A"yy in formula (7) is not an amino acid in the (D) configuration; when more than one A"yy is present, each A"yy is independently selected from the foregoing amino acids; A'''yy is an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn, with the proviso that A'''yy in formula (7) is not an amino acid in the (D) configuration; when more than one A'''yy is present, each A'''yy is independently selected from the foregoing amino acids; A'xx is an amino acid, provided that A'xx in formula (6) is not an amino acid of the (D) configuration, A'''xx is an amino acid, provided that A'''xx in formula (6) is not an amino acid of the (D) configuration, p1 is an integer of 0 to 3, p2 is 0 or 1; p3 is an integer from 0 to 3, provided that when p2 is 0, p3 is not 0, p4 is an integer of 1 to 4, provided that p4 and o in formula (2) are selected so that m in formula (1) is an integer of 1 to 5; ** ' indicates a covalent bond to the N-terminus of Axx or Ayy, and ** indicates a covalent bond to the antitumor compound).

[0022] Item 15. At least one of A'xx, A''xx, A'''xx, A'yy, A''yy, and A'''yy, for example, one, two, three, four, five, or six, has the following definition: (a) A'xx is an amino acid selected from Arg, Lys, homo-Lys, Cit, Orn, Dap, and Dab; (b) A''xx is an amino acid selected from Lys, homo-Lys, Cit, Orn, Dap, and Dab; (c) A'''xx is an amino acid selected from Arg, Lys, homo-Lys, Cit, Orn, Dap, and Dab; (d) A'yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (e) A''yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (f) A'''yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; Item 15. The antibody-drug conjugate according to Item 14, which satisfies the above.

[0023] Item 16. The drug-carrying unit is represented by formula (8): [ka] wherein A''xx is a trifunctional amino acid selected from Glu, α-aminoadipic acid (Aaa), Dap, Ser, Thr, homo-serine (homo-Ser), homo-threonine (homo-Thr), and aminomalonic acid (Ama), with the proviso that A''xx is not an amino acid of the (D) configuration; Cxx is a single covalent bond unless A''xx is Ama; when A''xx is Ama, Cxx is Pro or an N-methyl amino acid, the N-terminus of Cxx is attached to the carboxyl terminus of Ama, and the C-terminus of Cxx is covalently attached to one moiety D; A'yy, A''yy, and A''yy are each independently an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn; A'xx and A'''xx are each independently an amino acid, provided that A'xx and A'''xx are not amino acids of configuration (D), p1 is 0 or 1, p2 is 0 or 1; p3 is an integer from 0 to 3, provided that when p2 is 0, p3 is not 0, p4 is an integer of 1 to 4, provided that p4 and o in formula (2) are selected so that m in formula (1) is an integer of 1 to 5; ** ' indicates a covalent bond to the N-terminus of Axx or Ayy, and ** indicates a covalent bond to the antitumor compound).

[0024] Item 17. At least one of A'xx, A''xx, A'''xx, A'yy, A''yy, and A'''yy, for example, one, two, three, four, five, or six, has the following definition: (a) A'xx is an amino acid selected from Arg, Lys, homo-Lys, Cit, Orn, Dap, and Dab; (b) A''xx is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; (c) A'''xx is an amino acid selected from Arg, Lys, homo-Lys, Cit, Orn, Dap, and Dab; (d) A'yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (e) A''yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (f) A'''yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; Item 17. The antibody-drug conjugate according to Item 16, which satisfies the above.

[0025] Item 18. The linking group (T) is of formula (9): [ka] wherein each AA is independently a moiety containing a trifunctional amino acid; α represents a covalent bond to Y at the N-terminus of AA, or a covalent bond to Y at the N-terminus of the first AA when o' is 2 to 5; o' is an integer from 1 to 5, provided that the other moiety L' is *** When attached to ', o' is 1 to 4, when o' is 1, the side chain of the trifunctional amino acid is covalently attached to S or L' and the C-terminus is covalently attached to a moiety different from that attached at the side chain, L' or S, respectively; When o' is 2, 3, 4 or 5, **** indicates a covalent bond to L', *** 18. The antibody-drug conjugate according to any one of items 1 to 17, wherein ' represents a covalent bond to S.

[0026] Item 19. The antibody-drug conjugate of Item 18, wherein each AA is independently a moiety comprising an amino acid selected from N-ε-propargyloxycarbonyl-L-lysine (Lys(Poc)), Asp, Glu, Orn, Lys, Dab, and Dap.

[0027] Item 20. The linking group (T) is represented by formula (10) or formula (11): [ka] (In the formula, each AA 1 and A.A. 2 are independently a moiety that contains a trifunctional amino acid; α represents a covalent bond to Y; In formula (11), the side chain of the trifunctional amino acid is covalently bonded to L' or S, and the C-terminus is covalently bonded to S or L', respectively, which is a moiety different from that bonded to the side chain; In formula (10), **** indicates a covalent bond to L', *** 18. The antibody-drug conjugate according to any one of items 1 to 17, wherein L′ represents a covalent bond to S or L′.

[0028] Item 21. Each AA 1 and A.A. 2is a moiety comprising an amino acid selected from Lys(Poc), Asp, Glu, Orn, Lys, Dab and Dap.

[0029] Item 22. The linking group (T) is represented by formula (12) or formula (13): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, n4 is an integer from 1 to 50, α represents a covalent bond to Y, and **** 18. The antibody-drug conjugate according to any one of items 1 to 17, wherein L′ represents a covalent bond to L′.

[0030] Item 23. Formula (1): [ka] (wherein Ab is an anti-basal cell adhesion molecule (BCAM) antibody or an antigen-binding fragment thereof, comprising: (i) a heavy chain variable region comprising the VH CDR1 sequence of SEQ ID NO: 1, the VH CDR2 sequence of SEQ ID NO: 2, and the VH CDR3 sequence of SEQ ID NO: 3; and (ii) a light chain variable region comprising the VL CDR1 sequence of SEQ ID NO: 4, the VL CDR2 sequence of SEQ ID NO: 5, and the VL CDR3 sequence of SEQ ID NO: 6; D is an anti-tumor compound conjugated to the anti-BCAM antibody via a linker; n is 1 to 10, m is 1 to 5, and The linker (L) is represented by formula (14) or formula (15): [ka] is expressed as Bxx in formulas (14) and (15) is a trifunctional amino acid, provided that Bxx in formula (14) is not in the (D) configuration, In formulas (14) and (15), By is Phe, homo-Phe, Ala, Trp, Tyr, Phg, Val, His, Lys, Abu, Met, Cit, Orn, Ser, Thr, Leu, Ile, Arg, and Tyr (OR1), where R1 is -(CH2CHO) n1 -R2, wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), or Byy in formula (14) is an amino acid selected from homo-Tyr, homo-Tyr(OR1) (wherein R1 is as defined for Tyr(OR1) above), homo-Phe, beta-Phe, and beta-homo-Phe; provided that when (q1)×(q3)>1 and q2=0, only the C-terminal Byy in formula (14) may be an amino acid selected from beta-Phe and beta-homo-Phe; provided that Byy in formula (15) is not in the (D) configuration, Bxx1 in formulas (14) and (15) is a single covalent bond or an amino acid having a hydrophobic or basic side chain; Bxx2 in formulas (14) and (15) is an amino acid having a hydrophobic or basic side chain, Bxx3 in formulas (14) and (15) is an amino acid, provided that Bxx3 in formula (14) is not in the (D) configuration, Bxx4 in formulas (14) and (15) is Phe, homo-Phe, Ala, Trp, Tyr, Phg, Val, His, Lys, Abu, Met, Cit, Orn, Ser, Thr, Leu, Ile, Arg, and Tyr (OR1) (wherein R1 is -(CH2CHO) n1-R2, wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), or Bxx4 in formula (14) is an amino acid selected from homo-Tyr, homo-Tyr(OR1), homo-Phe, beta-Phe, and beta-homo-Phe; provided that when (q2) × (q3) > 1, only the C-terminal Bxx4 in formula (14) may be an amino acid selected from beta-Phe and beta-homo-Phe; provided that Byy in formula (15) is not in the (D) configuration, Or the linker (L) is of formula (16): [ka] is expressed as Bxx in formula (16) is a carboxyamino acid or a trifunctional amino acid selected from Dap, Dab, Ser, Thr, Lys, Orn, homo-Lys, homo-Ser and homo-Thr, provided that Bxx is not in the (D) configuration; When Bxx is not Ama, Cxx is a single covalent bond; when Bxx is Ama, Cxx is Pro or an N-methyl amino acid, the N-terminus of Cxx is bonded to the carboxyl group of Ama, and the C-terminus of Cxx is covalently bonded to one moiety D; In formula (16), By is an amino acid selected from Phe, homo-Phe, Ala, Trp, Tyr, Phg, Val, His, Lys, Abu, Met, Cit, Orn, Ser, Thr, Leu, Ile, Arg, homo-Phe, beta-Phe, beta-homo-Phe, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1), and R1 is -(CH2CHO) n1 -R2, wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24; provided that when (q1) × (q3) > 1 and q2 = 0, only the C-terminal By may be an amino acid selected from beta-Phe and beta-homo-Phe; Bxx1 in formula (16) is a single covalent bond or an amino acid having a hydrophobic or basic side chain; Bxx2 in formula (16) is an amino acid having a hydrophobic or basic side chain, Bxx3 in formula (16) is an amino acid, provided that Bxx3 is not in the (D) configuration, Bxx4 in formula (16) is an amino acid selected from Phe, homo-Phe, Ala, Trp, Tyr, Phg, Val, His, Lys, Abu, Met, Cit, Orn, Ser, Thr, Leu, Ile, Arg, homo-Phe, beta-Phe, beta-homo-Phe, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1), wherein R1 is -(CH2CHO). n1 -R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24; provided that when (q2) × (q3) > 1, only Bxx4 at the C-terminus may be an amino acid selected from beta-Phe and beta-homo-Phe; S' is a divalent group containing one or more atoms selected from C, N, O, P, and S; Z' is a group covalently bonded to the C-terminus of Byy or Bxx4 in formula (14) and (16), or to the C-terminus of Bxx or Bxx3 in formula (15), and is selected from -OH and -N(H)(R) (wherein R represents a hydrogen atom, an alkyl group, or a cycloalkyl group); q1 is an integer of 0 to 5, q2 is an integer of 0 to 3, provided that when q1 is 0, q2 is not 0; q3 is an integer from 1 to 5, q1, q2, and q3 are selected so that m in formula (1) is an integer of 1 to 5; * indicates covalent binding to an anti-BCAM antibody (Ab), and each ** indicates a covalent bond to one moiety D), or a pharmaceutically acceptable salt thereof.

[0031] Item 24. At least one of Bxx, Byy, Bxx1, Bxx2, Bxx3 and Bxx4, for example, one, two, three, four, five or six, has the following definition: (a) Bxx is an amino acid selected from Dap, Dab, Lys, Orn, Ser, Glu, Ama, Thr, Tyr, Aaa, homo-Ser, and homo-Thr; (b) By is an amino acid selected from Cit, Phe, homo-Phe, Ser, Trp, Tyr, and Tyr(OR1), where R1 is -(CH2CH2O) n1 -R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24; (c) Bxx1 is a single covalent bond or an amino acid selected from Phe, homo-Phe, Phg, Val, Ser, Tyr, Ala, Leu, and Ile; (d) Bxx2 is an amino acid selected from Arg, Lys, Cit, Val, Leu, Ser, Ala, Gly, His, Gln, Phg, and Phe; (e) Bxx3 is an amino acid selected from Phe, homo-Phe, Phg, Val, Ser, Tyr, Ala, Leu, and Ile; (f) Bxx4 is an amino acid selected from Cit, Phe, homo-Phe, Ser, Trp, Tyr, and Tyr(OR1), wherein R1 is —(CH2CH2O) n1 -R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24; 24. The antibody-drug conjugate according to Item 23, which satisfies the above.

[0032] Item 25. Equation (17) or (18): [ka] (wherein Axx is an amino acid selected from Glu, Apa, Aaa, Dap, Dab, Lys, Orn, Ser, Ama, and homo-Lys, provided that Axx in formula (17) is not in the (D) configuration, In formula (17), Ayy is an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1), wherein R1 is -(CH2CHO) n1-R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24; Ayy in formula (18) is an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, and Ser, provided that Ayy in formula (18) is not in the (D) configuration, Dxx is an amino acid with a single covalent bond or a hydrophobic side chain, Dyy represents a single covalent bond, Phe, or an amino acid having a basic side chain, provided that when Dxx is an amino acid having a hydrophobic side chain, Dyy is an amino acid having a Phe or a basic side chain, and when Dxx is a single covalent bond, Dyy is an amino acid having a single covalent bond, Phe, or a basic side chain; Y is a divalent group containing one or more atoms selected from C, N, O, P, and S; T is a (2+m)-valent linking group; if S is absent, T is a (1+m)-valent linking group; S is an atom or group optionally present to saturate the free valences of T; Z represents a group covalently attached to the C-terminus of Ayy or Axx selected from -OH and -N(H)(R), where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; and 23. The antibody-drug conjugate according to any one of items 1 to 22, wherein Ab, D, m and n are as defined in any one of items 1 to 22.

[0033] Item 26. At least one of Axx, Ayy, Dxx, Dyy, D, Z, m, and T, for example, 1, 2, 3, 4, 5, 6, 7, or 8, is one of the following definitions: (a) Axx is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; (b) Ayy in formula (17) is an amino acid selected from Phe, homo-Phe, Tyr, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1); (c) Ayy in formula (18) is an amino acid selected from Phe, homo-Phe, or Ser; (d) Dxx is a moiety derived from an amino acid selected from Phe, Val, Tyr, homo-Phe, and Ala; (e) Dyy is a covalent bond or a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap, and Dab; (f) D is an antitumor compound selected from auristatin F (AF), MMAF, exatecan, maytansine, DM1, and DM4; (g) Z is —OH or —NH; (h) T is a compound represented by the formula (9'): [ka] wherein each AA is independently a moiety containing a trifunctional amino acid; α represents a covalent bond to Y; m is 1 to 5; when m is 1, the side chain of the trifunctional amino acid is covalently attached to S or Axx and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; When m is 2, 3, 4 or 5, **** indicates a covalent bond to Axx, *** ' denotes a covalent bond to S via the C-terminus of the chain of AA groups); (i) m is 2 and T is a group represented by the formula (10'): [ka] (In the formula, each AA 1 and A.A. 2 are independently a moiety that contains a trifunctional amino acid; α represents a covalent bond to Y; **** indicates a covalent bond to Axx, *** ' indicates a covalent bond to S); (j) m is 1 and T is the formula (11'): [ka] (In the formula, AA 1is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; The side chain of the trifunctional amino acid is covalently attached to Axx or S, and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; (k) m is 1, and T is a formula (12') or a formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, n4 is an integer from 1 to 50, α represents a covalent bond to Y, and **** indicates a covalent bond to Axx); 26. The antibody-drug conjugate according to Item 25, which satisfies the above.

[0034] Item 27. At least one of Axx, Ayy, Dxx, Dyy, D, Z, m, and T, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9, is one of the following definitions: (a) Axx is Lys; (b) Ayy in formula (17) is Tyr; (c) Ayy in formula (18) is Phe or Ser; (d) Dxx is Phe or Val; (e) Dyy is Arg or Cit; (f) D is an antitumor compound selected from AF, MMAF, exatecan, maytansine, DM1, and DM4; (g) Z is —OH or —NH; (h) m is 1 and T is the formula (11'): [ka] (In the formula, AA 1 is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; The side chain of the trifunctional amino acid is covalently attached to Axx or S, and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; (i) m is 1 and T is a formula (12') or a formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, n4 is an integer from 1 to 50, α represents a covalent bond to Y, and **** indicates a covalent bond to Axx); 27. The antibody-drug conjugate according to Item 25 or 26, which satisfies the above condition.

[0035] Item 28. wherein each Dxx-Dyy-Axx-Ayy is independently selected from Arg-Lys-Phe where Dxx is a covalent bond, Arg-Lys-homoPhe where Dxx is a covalent bond, Arg-Lys-Tyr where Dxx is a covalent bond, Cit-Lys-Phe where Dxx is a covalent bond, Cit-Lys-Tyr where Dxx is a covalent bond, Arg-Lys-homoTyr where Dxx is a covalent bond, Cit-Lys-homoTyr where Dxx is a covalent bond, Phe-Cit-Lys-Phe, Phe-Cit-Lys-Tyr, Phe-Arg-Lys-Tyr, Phe-Cit-Lys-homoTyr, Phe-Lys-Lys-Phe, homoPhe-Arg-Lys-Phe, homo-Phe-Cit-Lys-Tyr; and 28. The antibody-drug conjugate according to any of items 25 to 27, wherein in formula (18), each Dxx-Dyy-Ayy-Axx is independently selected from Arg-Phe-Lys in which Dxx is a covalent bond, Arg-Ser-Lys in which Dxx is a covalent bond, Cit-Phe-Lys in which Dxx is a covalent bond, Cit-Ser-Lys in which Dxx is a covalent bond, Cit-homoPhe-Lys in which Dxx is a covalent bond, Phe-Cit-Phe-Lys, homoPhe-Cit-Phe-Lys, and Phe-Arg-Phe-Lys.

[0036] Item 29. At least one of D, Z, m, and T, for example, one, two, three, or four, has the following definition: (a) D is an antitumor compound selected from AF, MMAF, exatecan, maytansine, DM1, and DM4; (b) Z is —OH or —NH; (c) m is 1 and T is the formula (11'): [ka] (In the formula, AA 1 is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; The side chain of the trifunctional amino acid is covalently attached to Axx or S, and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; (d) m is 1 and T is a formula (12') or a formula (13'): [ka] wherein each Azz is independently a moiety that includes one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, n4 is an integer from 1 to 50, α represents a covalent bond to Y, and **** indicates a covalent bond to Axx); 29. The antibody-drug conjugate according to any one of items 25 to 28, which satisfies the above.

[0037] Item 30. The following formula: [ka] (wherein Y is a divalent group containing one or more atoms selected from C, N, O, P, and S; T is a (2+m)-valent linking group; if S is absent, T is a (1+m)-valent linking group; S is an atom or group optionally present to saturate the free valences of T; Z represents a group covalently attached to the C-terminus of the amino acid selected from -OH and -N(H)(R), where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; and 29. The antibody-drug conjugate according to any one of items 1 to 22 and items 25 to 29, wherein Ab, D, m, and n are as defined in any one of items 1 to 22, with the proviso that Lys in the above formula is not in the (D) configuration.

[0038] Item 31. At least one of D, Z, m, and T, for example, one, two, three, or four, has the following definition: (a) D is an antitumor compound selected from AF, MMAF, exatecan, maytansine, DM1, and DM4; (b) Z is —OH or —NH; (c) m is 2 and T is the formula (10'): [ka] (In the formula, each AA 1 and A.A. 2 are independently a moiety that contains a trifunctional amino acid; α represents a covalent bond to Y; **** indicates a covalent bond to Lys, *** ' indicates a covalent bond to S); (d) m is 1 and T is the formula (11'): [ka] (In the formula, AA 1 is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; The side chain of the trifunctional amino acid is covalently attached to Lys or S, and the C-terminus is covalently attached to a moiety different from that attached to the side chain, either S or Lys, respectively; (e) m is 1 and T is a formula (12') or a formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, n4 is an integer from 1 to 50, α represents a covalent bond to Y, and **** indicates a covalent bond to Lys); 31. The antibody-drug conjugate according to Item 30, which satisfies the above.

[0039] Item 32. D, Z, m, and T are defined as follows: (a) D is DM1; (b) Z is —OH or —NH2, and (c) m is 1 and T is the formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, α represents a covalent bond to Y, and **** indicates a covalent bond to Lys).

[0040] Item 33. D, Z, m, and T are defined as follows: (a) D is AF; (b) Z is —OH or —NH2, and (c) m is 1 and T is the formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, α represents a covalent bond to Y, and **** 33. The antibody-drug conjugate according to any of items 30 to 32, wherein:

[0041] Item 34. Equation (19), (20), (21), or (22): [ka] (In the formula, Axx is a trifunctional amino acid, provided that Axx in formula (19) and formula (20) is not an amino acid of the (D) configuration, Ayy is an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, Orn, Ser, Thr, Leu, and Ile, or Ayy in formula (19) and formula (20) is an amino acid selected from homo-Tyr, homo-Phe, beta-Phe and beta-homo-Phe, Tyr(OR1) and homo-Tyr(OR1), wherein R1 is -(CH2CHO) n1 -R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24, provided that Ayy in formula (21) and formula (22) is not an amino acid of the (D) configuration, Each A''yy is independently an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn, with the proviso that A''yy in formula (20) and formula (22) is not an amino acid in the (D) configuration, Y is a divalent group containing one or more atoms selected from C, N, O, P, and S; T is a trivalent linking group; if S is absent, T is a divalent linking group; S is an atom or group optionally present to saturate the free valences of T; Z represents a group covalently bonded to the C-terminus of Ayy in formula (19) and formula (20), or to the C-terminus of Axx in formula (21) or formula (22), and is selected from -OH and -N(H)(R) (wherein R represents a hydrogen atom, an alkyl group, or a cycloalkyl group); D1 is an antitumor compound, m' is (m-1), where m is as defined in item 1, except that m' is not 0; When m' is 1, A''xx is a trifunctional amino acid, provided that A''xx in formula (19) and formula (21) is not an amino acid of the (D) configuration, and D2 is an antitumor compound; When m' is greater than 1, each D2 is independently selected from a hydrogen atom and an anti-tumor compound, and the multiple moieties D2 can be the same or different, provided that at least one D2 is not a hydrogen atom; when D2 is a hydrogen atom, A''xx is an amino acid, provided that A''xx in formula (19) and formula (21) is not an amino acid in the (D) configuration; when D2 is an anti-tumor compound, A''xx is a trifunctional amino acid, provided that A''xx in formula (19) and formula (21) is not an amino acid in the (D) configuration, 23. The antibody-drug conjugate according to any one of items 1 to 22, wherein Ab and n are as defined in any one of items 1 to 22.

[0042] Item 35. At least one of Axx, Ayy, A''xx, A''yy, D1, D2, Z, m', and T, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9, is one of the following definitions: (a) Axx is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; (b) Ayy in formula (19) and formula (20) is an amino acid selected from Phe, homo-Phe, Tyr, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1); (c) A''xx is an amino acid selected from Lys, homo-Lys, Cit, Orn, Dap, and Dab; (d) A''yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (e) each D1 and D2 is independently an antitumor compound selected from AF, MMAF, exatecan, maytansine, DM1, and DM4; (f) Z is —OH or —NH; (g) m is 1 and T is the formula (11'): [ka] (In the formula, AA 1 is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; The side chain of the trifunctional amino acid is covalently attached to Axx or S, and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; (h) m is 1 and T is a formula (12') or a formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, n4 is an integer from 1 to 50, α represents a covalent bond to Y, and **** indicates a covalent bond to Axx); 35. The antibody-drug conjugate according to Item 34, which satisfies the above.

[0043] Item 36. At least one of Axx, Ayy, A''xx, A''yy, D1, D2, Z, m', and T, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9, is one of the following definitions: (a) Axx is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; (b) Ayy in formula (17) is an amino acid selected from Phe, homo-Phe, Tyr, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1); (c) A''xx is an amino acid selected from Lys, homo-Lys, Cit, Orn, Dap, and Dab; (d) A''yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (e) each of D1 and D2 is AF; (f) Z is —OH or —NH; (g) m' is 1 and T is the formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, α represents a covalent bond to Y, and **** indicates a covalent bond to Axx); 36. The antibody-drug conjugate according to Item 34 or 35, which satisfies the above.

[0044] Item 37. The antibody-drug conjugate according to any one of Items 36 to 37, wherein the antitumor compound (D) is selected from a DNA alkylating agent, a topoisomerase inhibitor, an RNA polymerase II inhibitor, a DNA cleaving agent, an antimitotic or microtubule-disrupting agent, an antimetabolite, a kinesin spindle protein inhibitor, a kinase inhibitor, a nicotinamide phosphoribosyltransferase inhibitor, a matrix metallopeptidase 9 inhibitor, a phosphatase inhibitor, or a radioactive isotope, and / or a pharmaceutically acceptable salt thereof; and when two or more Ds are present, each D is independently selected from the aforementioned compounds.

[0045] Item 38. The antibody-drug conjugate of any of Items 1 to 37, wherein the antitumor compound D is selected from amanitin, duocarmycin, auristatin F (AF), monomethylauristatin F (MMAF), maytansine, mertansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepines (PBDs) and dimers thereof, indilinobenzodiazepines (IBDs) and dimers thereof, or radioactive isotopes, and / or pharmaceutically acceptable salts thereof; and when two or more Ds are present, each D is independently selected from the foregoing compounds.

[0046] Item 39. The following compounds: [ka] (wherein each Ab and n is as defined in any of items 1 to 3 and / or items 6 to 8).

[0047] Item 40. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of Items 1 to 39 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0048] Item 41. The pharmaceutical composition according to Item 40, which is a pharmaceutical composition for treating or imaging cancer.

[0049] Item 42. The pharmaceutical composition for use according to Item 41, wherein the cancer is one or more selected from the group consisting of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain tumor, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, kidney cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, kidney cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer, and urothelial cell carcinoma.

[0050] Item 43. A method for treating cancer, comprising administering an effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of Items 1 to 39 to a subject in need thereof.

[0051] Item 44. The method of Item 43, wherein the cancer is one or more selected from the group consisting of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain tumor, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, kidney cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, kidney cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer, and urothelial cell carcinoma.

[0052] definition Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments described herein, several preferred methods, compositions, devices, and materials are described herein. However, before the materials and methods of the present invention are described, it should be understood that this disclosure is not limited to the particular molecules, compositions, methodologies, or protocols described herein, as these may vary according to routine experimentation and optimization. It should also be understood that the terminology used in the description is for the purpose of describing particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[0053] 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 belongs. However, in case of conflict, the present specification, including definitions, will prevail. Therefore, in the context of the embodiments described herein, the following definitions apply.

[0054] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0055] As used herein, the term "comprise" and its linguistic variations, such as "contain," indicate the presence of stated features, elements, method steps, etc., without excluding the presence of additional features, elements, method steps, etc. Conversely, the term "consisting of" and its linguistic variations indicate the presence of stated features, elements, method steps, etc., but excludes unrecited features, elements, method steps, etc., except for impurities normally associated therewith. The phrase "consisting essentially of" indicates the stated features, elements, method steps, etc., and any additional features, elements, method steps, etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments are described herein using the open phrase "comprising." Such embodiments encompass multiple closed "consisting of" and / or "consisting essentially of" embodiments, which may alternatively be claimed or described using such phrases. Additionally, the term "comprise" and its linguistic variations, insofar as technically meaningful, should be understood to disclose a more limited embodiment, as should the term "consisting of" such that there are no additional unrecited elements.

[0056] Where this specification refers to "preferred" embodiments / features, combinations of these "preferred" embodiments / features shall also be deemed to be disclosed, as long as this combination of "preferred" embodiments / features makes technical sense.

[0057] As used herein, the term "antibody-drug conjugate" refers to the conjugation of an antibody or antigen-binding fragment thereof with another anti-tumor compound, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, etc. The conjugation may be covalent.

[0058] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or an immunoreactive portion of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in this disclosure can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, as well as single-chain antibodies and humanized antibodies.

[0059] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a monoclonal cell line producing substantially homogeneous antibodies, i.e., the individual antibodies produced are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.

[0060] As used herein, the term "polyclonal antibody" refers to a mixture of antibodies secreted by different B-cell lineages. These antibodies are actually a collection of immunoglobulin molecules that react against a specific antigen.

[0061] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., the variable region sequences are derived from a rat antibody and the constant region sequences are derived from a human antibody.

[0062] As used herein, the term "humanized antibody" refers to forms of non-human (e.g., rat) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies can be human immunoglobulins in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0063] As used herein, the term "human antibody" refers to an antibody having variable regions in which both the framework and CDR regions are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences, or from antibodies containing consensus framework sequences derived from analysis of human framework sequences.

[0064] As used herein, the term "complementarity-determining region (CDR)" refers to the hypervariable regions of the light chain variable region (VL) and heavy chain variable region (VH). CDRs are the target protein-binding sites of antibody chains that have specificity for such target proteins. Each human VL or VH has three CDRs (CDRs 1 to 3, numbered sequentially from the N-terminus), which constitute approximately 15 to 20% of the variable domain. For example, the term "CDRH1" as used herein refers to the first CDR of the heavy chain variable region, and the term "CDRL1" as used herein refers to the first CDR of the light chain variable region. CDRs are structurally complementary to the epitope of the target protein and are therefore directly involved in binding specificity. The remaining portions of the VL or VH, known as the framework regions, have less variation in amino acid sequence.

[0065] Light and heavy chains are divided into structurally and functionally homologous regions. The terms "constant" and "variable" are used functionally. The variable domains of both the light (VL) and heavy (VH) chains determine antigen recognition and specificity. The constant domains of the light (CL) and heavy (CH1, CH2, or CH3) chains confer important biological properties, such as secretion, placental transport, Fc receptor binding, and complement binding. The more highly conserved portions of the variable regions are called framework regions (FRs). The variable regions of naturally occurring heavy and light chains each contain four FRs, primarily adopting a β-sheet structure and connected by three hypervariable regions. These regions form loops that connect and, in some cases, form part of the β-sheet structure. The hypervariable regions of each chain are held in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0066] As used herein, the term "antigen-binding fragment thereof" refers to a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab fragments, Fab' fragments, Fab'-SH fragments, Fv fragments, ScFv fragments, F(ab')2 fragments, VL fragments, VH fragments, ScFv-Fc fragments, (ScFv)2-Fc fragments, diabodies, linear antibodies, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity-determining regions), and epitope-binding fragments of any of the above that immunospecifically bind to a cancer cell antigen, a viral antigen, or a microbial antigen, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0067] As used herein, the term "anti-basal cell adhesion molecule (BCAM) antibody" refers to an antibody that specifically binds to a basal cell adhesion molecule (BCAM). As used herein, the term "specifically binds" with respect to an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more species. However, such cross-species reactivity does not, in itself, change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in itself, change the specific classification of the antibody. In some instances, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure, rather than proteins in general. If an antibody is specific for epitope "A," then in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0068] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a disclosed compound, such as an antibody-drug conjugate or an antitumor compound. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, acid tartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate. Pharmaceutically acceptable salts may include another molecule, such as an acetate ion, a succinate ion, or other counterion. Counter ions can be any organic or inorganic moiety that stabilizes the charge of the parent compound. Furthermore, pharmaceutically acceptable salts can have two or more charged atoms in their structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, it can have multiple counter ions. Therefore, pharmaceutically acceptable salts can have one or more charged atoms and / or one or more counter ions. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, PA, 1985, p. 1418, SM Berge, LM Bighley, and DC Monkhouse, "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66(1), 1-19; PH Stahl and CG Wermuth (eds.), Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zurich, Wiley-VCH, 2008, and AK Bansal et al., Pharmaceutical Technology, 3(32), 2008.Pharmaceutical salts can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Unless the context indicates otherwise, all references to the compounds of this disclosure (or the invention) should also be understood to refer to the pharmaceutically acceptable salts of the respective compound.

[0069] The term "linker," as used herein, refers to a moiety that covalently links two moieties, an antibody and an anti-tumor compound. In some instances, the term "linker," as used herein, can refer to a moiety that includes a cleavable element, as well as a moiety that includes additional elements, such as a linking group, a group that includes one or more solubilizing groups, etc. In some other instances, the term "linker," as used herein, can refer to a specifically defined element, such as a "cathepsin B-cleavable" linker (described further below).

[0070] As used herein, the term "cleavable" refers to a linker that covalently links two moieties, an antibody and an anti-tumor compound, but degrades under physiologically relevant conditions to sever the covalent bond between the moieties. Generally, cleavage liberates the anti-tumor compound from the antibody. As used herein, the term "cleavable" refers to a linker that is not easily degraded, particularly under physiological conditions. Such a linker is sufficiently resistant to degradation that it allows the anti-tumor compound to remain linked to the antibody or antigen-binding fragment until the antibody or antigen-binding fragment itself is at least partially degraded.

[0071] The phrase "capable of being cleaved by cathepsin B" characterizes any compound (or moiety that can be incorporated into a compound) that is cleaved upon contact with cathepsin B (Cat B) under appropriate conditions, e.g., as described in Section 2.3.5 below or in WO2019096867 (see Section 11.3.1). In preferred embodiments, said cleavage is (a) rapid and / or (b) cleavage is via the exopeptidase activity of Cat B. The above embodiment (b) regarding a compound or moiety "capable of being cleaved by the exopeptidase activity of Cat B" is defined in more detail in the following paragraph. The above-mentioned "fast" cleavage of embodiment (a) typically refers to a cleavage rate, T, of the subject compound, in which the corresponding unconjugated compound (i.e., a compound that does not include an antibody and that is quenched with a conjugation group, e.g., has a cysteine ​​covalently attached to the maleimide conjugation group) exhibits a cleavage rate, T, of 25 minutes or less, preferably 20 minutes or less, more preferably 18 minutes or less, even more preferably 16 minutes or less, and most preferably 14 minutes or less, in the conditions of the Cat B cleavage assay described in Section 2.3.5 below or in WO2019096867 (see Section 11.3.1). 1 / 2 The lower limit is not particularly limited. However, under the conditions of the Cat B cleavage assay described in Section 2.3.5 below or in WO2019096867 (see Section 11.3.1), the cleavage rate T is 0.2 minutes or more, typically 0.5 minutes or more, and even more typically 1 minute or more, for example 2 minutes or more. 1 / 2 It is realistic to expect

[0072] As used herein, the phrase "capable of cleavage by the exopeptidase activity of Cat B" indicates that each portion of the compound, particularly the linker, e.g., the C-terminal peptide unit, can be specifically recognized and cleaved by the exopeptidase (i.e., carboxydipeptidase) of cathepsin B. Such cleavage results in rapid release of the drug (or a modified drug having a group or moiety that remains attached thereto after cleavage by cathepsin B, "intra-drug") into the target cell. Cleavage of the linker, e.g., the C-terminal peptide unit, via the exopeptidase activity of Cat B can be assessed by in vitro enzymatic cleavage assays using recombinant human Cat B and UHPLC-MS / MS analysis, as further described below. Given that the exopeptidase activity of Cat B is typically associated with a higher cleavage rate compared to the endopeptidase activity of Cat B, in some embodiments, the phrase "a compound capable of cleavage by the exopeptidase activity of Cat B" can be confirmed by confirming a high Cat B cleavage rate. According to this aspect, a "compound that can be cleaved by the exopeptidase activity of Cat B" refers to a compound for which the following criteria are met: the corresponding unconjugated compound (i.e., a compound that does not include an antibody and is quenched with a conjugation group, e.g., having a cysteine ​​covalently attached to a maleimide conjugation group) exhibits a cleavage rate T of 25 minutes or less, preferably 20 minutes or less, more preferably 18 minutes or less, even more preferably 16 minutes or less, and most preferably 14 minutes or less, under the conditions of the Cat B cleavage assay described in Section 2.3.5 below or in WO2019096867 (see Section 11.3.1). 1 / 2 The lower limit is not particularly limited. However, under the conditions of the Cat B cleavage assay described in Section 2.3.5 below or in WO2019096867 (see Section 11.3.1), the cleavage rate T is 0.2 minutes or more, typically 0.5 minutes or more, and even more typically 1 minute or more, for example 2 minutes or more. 1 / 2 It is realistic to expect

[0073] As used herein, the term "peptide" refers to a compound comprising a consecutive sequence of at least two amino acids linked together via a peptide bond. The terms "dipeptide," "tripeptide," and "tetrapeptide" refer to compounds comprising a consecutive sequence of two, three, and four amino acids linked together via peptide bonds, respectively. In this context, the term "peptide bond" is meant to encompass (backbone) amide bonds as well as modified bonds that can be obtained when non-natural amino acids are introduced into a peptide sequence. In this case, the modified bond replaces the (backbone) amide bond formed in the consecutive peptide sequence by reacting the amino and carboxyl groups of two amino acid residues. For example, the modified bond may be an ester, thioester, carbamide, thiocarbamide, or triazole bond. Preferably, the amino acids forming the consecutive peptide sequence are linked together via backbone amide bonds. Peptides may be linear or branched. In a preferred embodiment, the peptide is a linear di-, tri-, or tetra-peptide, more preferably a linear tri- or tetra-peptide.

[0074] As used herein, the term "amino acid" refers to a compound containing or derived from at least one amino group and at least one acidic group, preferably a carboxyl group. The distance between the amino group and the acidic group is not particularly limited. α-, β-, and γ-amino acids are suitable, with α-amino acids, particularly α-aminocarboxylic acids, being particularly preferred. The term "amino acid" encompasses both naturally occurring amino acids, such as naturally occurring proteinogenic amino acids, and synthetic amino acids not found in nature. Hereinafter, amino acids may be represented by three-letter amino acid codes (Arg, Phe, Ala, Cys, Gly, Gln, etc.) or one-letter amino acid codes (R, F, A, C, G, Q, etc.). Unless otherwise specified, amino acids represented by three-letter amino acid codes refer to the corresponding (L)-amino acid or (D)-amino acid.

[0075] As used herein, amino acid sequences are written from N-terminus to C-terminus (left to right), and unless otherwise stated or dictated otherwise by context, all bonds between adjacent amino acid groups are formed by peptide (amide) bonds.

[0076] As used herein, the phrase "amino acid in the (D) configuration" refers to the (D) isomer of any naturally occurring or synthetic amino acid. This applies not only to α-amino acids, but also to β- and γ-amino acids. As used herein, the phrase "amino acid in the (D) configuration" is not meant to encompass non-chiral amino acids such as glycine or other non-chiral amino acids such as aminoisobutyric acid.

[0077] As used herein, the phrase "side chain of an amino acid" may refer to the moiety attached to the α-carbon of an amino acid. For example, the side chain of Ala is methyl, the side chain of Phe is phenylmethyl, the side chain of Cys is thiomethyl, the side chain of Tyr is 4-hydroxyphenylmethyl, etc. This definition includes both naturally occurring and non-naturally occurring side chains.

[0078] The term "functional group" refers to a group that can be attached to another functional group by forming at least one covalent bond without having to cleave a C—C or C—H covalent bond.

[0079] As used herein, the term "trifunctional" refers to a compound or moiety having three functional groups that can form or have formed three covalent bonds to adjacent moieties. Thus, the term "trifunctional amino acid" refers to a compound containing or derived from at least an amino group, an acid group (e.g., a carboxyl group), and another functional group such as an amino group or a carboxyl group. Non-limiting examples of trifunctional amino acids include Ser, Cys, Tyr, N-ε-propargyloxycarbonyl-L-lysine (Lys(Poc)), Asp, Glu, Orn, Lys, Dab, and Dap.

[0080] The term "C-terminus" as used herein refers to the C-terminus of an amino acid (peptide) chain. Attachment to the "C-terminus" means that a covalent bond is formed between an acid group in the backbone of the amino acid residue and the binding partner. For example, attachment of a group "X" to the C-terminus of an amino acid residue "Axx" forms an ester or amide type structural element. * -C(O)-X is produced, and the carbonyl group is derived from the acid group of Axx. * ) indicates the bond to the backbone. The term "C-terminal peptide unit" is used herein to characterize a peptide sequence of 2, 3 or 4 amino acids, where the C-terminal amino acid forms the C-terminus of the peptide sequence.

[0081] The term "N-terminus" as used herein refers to the N-terminus of an amino acid (peptide) chain. Attachment to the "N-terminus" means that a covalent bond is formed between the amino group in the backbone (skeleton) of the N-terminal amino acid residue and the binding partner (replacing one hydrogen atom). For example, attachment of the group "X" to the N-terminus of the amino acid residue "Axx" results in the structural element X-NH- * (wherein the amino group is derived from Axx, * ) indicates the bond to the main chain) is produced.

[0082] The term "hydrophobic" is used herein to characterize compounds, groups, or moieties that lack affinity for water. For example, the term "amino acid having a hydrophobic side chain" is used to characterize amino acids with hydrophobic or partially hydrophobic aliphatic side chains, or amino acids with aromatic side chains, such as Phe, Leu, Ile, Val, Tyr, Trp, and Ala. Naturally, any other amino acids exhibiting the same or higher degree of hydrophobicity should also be treated as hydrophobic within the meaning of the present disclosure. Comparison of the degree of hydrophobicity can be performed by measuring the n-octanol / water partition coefficient (at 25°C and pH 7): if the concentration ratio of other amino acids in n-octanol / water is equal to or higher than the concentration ratio of one or more of the amino acids Phe, Leu, Ile, Val, Tyr, Trp, and Ala, then such other amino acids should be treated as hydrophobic amino acids.

[0083] The term "amino acid having a basic side chain" is used herein to characterize natural or unnatural amino acids whose side chains contain one or more ionizable groups with a pKa value of 6 or greater. Examples of natural amino acids with basic side chains include Arg (guanidino group, pKa=12.5), Lys (amino group, pKa=10.5), and His (imidazole group, pKa=6). Examples of unnatural amino acids with basic side chains include citrulline (Cit), ornithine (Orn), 2,3-diamino-propionic acid (Dap), and 2,4-diamino-butyric acid (Dab).

[0084] As used herein, the term "alkyl group" refers to a linear (straight chain) or branched, saturated or unsaturated hydrocarbon group having 1 to 20 carbon atoms, preferably 1 to 5 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, vinyl, allyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl. More preferably, the alkyl group is a methyl or ethyl group.

[0085] The term "cycloalkyl group" as used herein refers to a substituted or unsubstituted cyclic hydrocarbon group having 3 to 20 carbon atoms, preferably 5 to 8 carbon atoms. A cycloalkyl group may consist of a single ring or may be formed by two or more condensed rings. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl. More preferably, the cycloalkyl group is a cyclopentyl or cyclohexyl group.

[0086] As used herein, the term "divalent maleimide derivative" (or, e.g., "divalent group derived from a compound selected from maleimides") refers to a divalent moiety derived from maleimide in which the double bond has been hydrogenated and two hydrogen atoms have been replaced by two covalent bonds that allow for bonding to adjacent moieties. For example, a divalent maleimide derivative can have the following structure: [ka] where R and R′ represent adjacent moieties to which the maleimide derivative is attached.

[0087] The moieties contain a chiral carbon atom (i.e., the atom bearing the sulfur atom). Unless otherwise specified, references to divalent maleimide derivatives should be understood to refer to pure stereoisomers as well as mixtures thereof, in particular racemic mixtures thereof.

[0088] The terms "divalent maleimide derivative" or "divalent group derived from a compound selected from maleimides" should be understood to further include any derivative of maleimide (as described above) that is further substituted at positions other than the 2- and 3-positions, as well as ring-opened hydrolyzed maleimide derivatives.

[0089] Divalent maleimide-type disulfide bridges (e.g., of the formula -SX 2 -S- / -SX 3 -S-(wherein, X 2 / X 3 represents a divalent radical derived from maleimide), which can be obtained, for example, by cyclization between the side chains in the presence of 2,3-dibromomaleimide or another suitable reagent as described by Kuan et al. in Chem. Eur. J. 2016, 22, 17112-17129.

[0090] In the context of the present disclosure, a "ring-opened hydrolyzed maleimide derivative" refers to a divalent moiety derived from maleimide in which the maleimide ring has been opened by hydrolysis. For example, hydrolysis of a divalent maleimide derivative RXS-R' (wherein X represents an unsubstituted divalent group derived from maleimide, and R / R' represent adjacent groups or moieties; the maleimide double bond at X is no longer present) results in a ring-opened hydrolyzed maleimide derivative of the formula R-NH-C(=O)-CH(S-R')-CH-COOH, or R-NH-C(=O)-CH-CH(S-R')-COOH, or a mixture thereof. Ring hydrolysis can be carried out, for example, under basic conditions. The following conditions are particularly suitable: at the end of the conjugation reaction (e.g., after the reaction of the maleimide moiety with the side chain of the cysteine ​​residue contained in the BCAM antibody or its antigen-binding fragment), the pH is adjusted to pH 8 by adding 10× pH 8 DPPS (0.2-0.5 times the reaction volume), and excess reactive drug linker and reducing agent (TCEP) are removed by gel filtration using a column suitable for gel filtration (e.g., a PF column, elution with pH 8 buffer). The eluate is then stirred overnight for 16 hours to complete the ring-opening, followed by a final buffer exchange with D-PBS and loading into an Amicon concentration unit. Unless otherwise specified, the term "ring-opened hydrolyzed maleimide derivative" should be understood to refer to only one of these structures or a mixture of any of these structures. Furthermore, the carbon bearing the sulfur atom is chiral. Unless otherwise specified, the term "ring-opened hydrolyzed maleimide derivative" should be understood to refer to pure stereoisomers as well as any mixtures thereof, particularly racemic mixtures thereof.

[0091] Furthermore, in the context of the present invention, the term "maleimide bond" refers to a divalent moiety derived from maleimide, as described above, that contains two covalent bonds that allow for attachment to adjacent groups or moieties. For example, in a maleimide derivative of the formula RXS-R', R / R' represent adjacent groups or moieties, and X represents a maleimide bond (a divalent group derived from maleimide where the maleimide double bond is no longer present). Thus, the term "maleimide bond" is synonymous with "maleimide derivative bond."

[0092] Similarly, in the context of the present invention, a "ring-opened hydrolyzed bond" refers to a divalent moiety derived from maleimide, as described above, that contains two covalent bonds that allow for attachment to adjacent groups or moieties. For example, in a ring-opened hydrolyzed maleimide derivative of the formula RXS-R', R / R' represent adjacent groups or moieties and X represents a ring-opened hydrolyzed maleimide bond. Thus, the term "ring-opened hydrolyzed bond" is synonymous with "ring-opened hydrolyzed derivative bond."

[0093] The expression "a divalent radical derived from a compound selected from...triazoles" is meant to characterize a divalent radical resulting from a 3+2 cycloaddition reaction of an alkyne and an azide. Such a divalent radical typically has the following structure: [ka] (wherein the single bond is characterized by a bond to an adjacent group, and there is no particular restriction as to which adjacent group is bonded to the nitrogen atom and which adjacent group is bonded to the carbon atom). In the context of the group Y, a divalent group can be formed by reacting an alkyne-containing group bonded to V with an azide-containing group bonded to T, or vice versa, in the presence or absence of a metal catalyst (e.g., as described by Becer et al. in "Click Chemistry beyond Metal-Catalyzed Cycloaddition," Angewandte Chemie Int. Ed. 2009, 48(27), 4900-4908). Examples of groups that can react in the absence of a metal catalyst include electron-deficient and strained alkynes such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]nonyne (BCN).

[0094] The expression "a divalent radical derived from a compound selected from hydrazones" is intended to characterize the divalent radical -CH=N-NH- resulting from the condensation of a carbonyl with a hydrazine group. In the context of the group Y, the divalent radical can be formed by reacting a carbonyl group attached to Ab with a hydrazine group attached to T, or vice versa.

[0095] The expression "... a divalent radical derived from a compound selected from carbonyl-containing compounds" is intended to characterize the divalent radical -C(=O)-X- (wherein X represents O, S, or NH) resulting from the reaction of an (activated) carbonyl with a nucleophilic group, such as the formation of an amide, ester, or thioester group. In the context of the group Y, the divalent radical can be formed by reacting a carbonyl-containing group (e.g., -C(=O)-Cl) attached to Ab with a nucleophilic group (e.g., -NH) attached to T, or vice versa. Unless otherwise specified, there are no particular restrictions on the orientation of the above-mentioned divalent radicals, i.e., on which partner the carbonyl -C is attached and which partner X is attached to.

[0096] In the above divalent groups, the term "derivatives thereof" means that any hydrogen atom may be replaced by a substituent as defined below, provided that the substitution does not prevent the formation of the divalent group.

[0097] Similarly, the term "derivative" is used to characterize moieties linked to adjacent moieties that differ from the original molecule from which they are derived only in the structural elements involved in the link to the adjacent moiety, which may include a covalent bond formed by an existing functional group, or a covalent bond and adjacent functional group newly introduced for this purpose.

[0098] Similarly, unless otherwise stated or the context dictates otherwise, the phrase "derived from" (e.g., "derived from a compound"), when used in connection with another group or moiety, is meant to describe a group or moiety that is identical to the referenced compound, etc., except for structural modifications necessary to join the group or moiety to one or more adjacent groups or moieties, typically by replacing a hydrogen atom or atomic group with a covalent bond (e.g., replacing an OH in a carboxyl group with a covalent bond upon amide bond formation with an amino group; further examples are shown at the end of the "Divalent Group (X)" section in the Table below).

[0099] As used herein, the term "carbonyl-containing group" refers to a divalent moiety that contains a carbonyl group (C=O) or a thiocarbonyl group (C=S). In preferred embodiments, the carbonyl-containing group has the following formula: -(CH2) a -(C=A)- (α) -(CH2) a -(C=A)-(CH2) b - (β) -(CH2) a -(C=A)-(C(CH3)2)-(CH2) b - (γ) -(C=A)-(NH) c -(CH2) b -(NH) d -(C=A)- (δ) (In the formula, a and b are each independently selected from 0 to 5, preferably 0, 1 or 2, more preferably 0 or 1, c and d are each selected from 0 or 1; in one embodiment, c is 0 and d is 1; in another particular embodiment, c is 1 and d is 0; in yet another particular embodiment, c and d are both 1; and in yet another particular embodiment, c and d are both 0; and Each A independently refers to a group represented by one of the following: O and S, preferably O.

[0100] In a more preferred embodiment, the carbonyl-containing group refers to a group represented by formula (α), where a is 0, 1, or 2, and A is O. Most preferably, the carbonyl-containing group is —(C═O)—.

[0101] In some embodiments, the term "amino-containing group" as used herein refers to a divalent moiety containing an amino group, e.g., -N(R)-, where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group. Preferably, the amino-containing group has the formula -(CH) a It is a —N(R)— moiety, in which R is a hydrogen atom, an alkyl group or a cycloalkyl group, and a is 0 to 6, preferably 0 or 1, and more preferably 0.

[0102] As used herein, the term "solubilizing group" refers to a hydrophilic group or moiety capable of increasing (improving) the water solubility of a moiety or compound to which it is attached. The solubilizing group can be, for example, a polyalkylene oxide group, such as a polyethylene oxide (PEO) or polypropylene oxide (PPO) group, preferably having 6 to 200 repeating units, more preferably 10 to 150 or 12 to 80 repeating units, e.g., 16 or 40 repeating units; a sugar group; or one or more ionic or ionizable groups, i.e., a moiety containing a charged (anionic or cationic) functional group at physiological pH (7.4), such as a moiety derived from an amino acid, e.g., Lys, Glu, Asp, His, Arg, diaminopropionic acid (Dap), diaminobutyric acid (Dab), 2-aminoadipic acid (Aad), carnitine, or Orn. ​​Examples of ionic or ionizable groups include ammonium, guanidinium, sulfate, phosphate, phosphonate, and sulfonate groups. Examples of sugar groups include monosaccharides, disaccharides, and linear or branched oligosaccharides, particularly linear or branched oligosaccharides having 3 to 10 monosaccharide units linked by glycosidic bonds, wherein each of the monosaccharide units in the monosaccharide, disaccharide, and oligosaccharide is independently selected from glucose, fructose, mannose, ribose, and galactose.

[0103] In the present disclosure, the expression "moiety comprising one or more solubilizing groups" preferably refers to a moiety derived from an amino acid comprising one, two, three or four, preferably one or two, ionic or ionizable groups selected from, for example, ammonium, guanidinium, sulfate, phosphate, phosphonate and sulfonate groups. Such moieties are preferably selected from Lys, Glu, Asp, His, Arg, Dap, Dab, Aad and Orn, more preferably Arg and His. In a further embodiment, the moiety may consist of an amino acid.

[0104] As used herein, the term "polyalkylene oxide" (or "polyalkylene glycol", "polyoxyalkylene") refers to a compound having the general structure HO-(XO) n-H, where X represents an alkylene group having 2 or 3 carbon atoms, and n represents the number of repeating units, for example, 6 to 200, 10 to 150, or 12 to 80 repeating units, for example, 16 or 40 repeating units, for example, 17, 18, 20, or 24 PEO repeating units. Thus, the term "polyalkylene oxide group" refers to a substance of the formula * -O-(XO) n - ** where X and n are as defined above, * and ** indicates a covalent bond to an adjacent moiety. In some embodiments, the term "polyalkylene oxide" can refer to polyethylene oxide (or polyethylene glycol, a C2-polyalkylene oxide) or polypropylene oxide (or polypropylene glycol, a C3-polyalkylene oxide). It is also possible to provide a polyalkylene oxide group in which two or more different alkylene groups as defined above are arranged randomly or in blocks.

[0105] Unless otherwise specified or the context dictates otherwise, the term "substituted" or "optionally substituted" groups includes F, Cl, Br, I, CN, NO, NH, NH-C 1~6 -Alkyl, N(C 1~6 -Alkyl)2, -XC 1~6 -Alkyl, -XC 2~6 -Alkenyl, -XC 2~6 -Alkynyl, -XC 6~14-aryl, -X-(5- to 14-membered heteroalkyl having 1 to 3 heteroatoms selected from N, O, S), where X represents a single bond, -(CH2)-, -O-, -S-, -S(O)-, -S(O)2-, -NH-, -CO-, or any combination thereof, e.g., -C(O)-NH-, -NH-C(O)-. The number of substituents is not particularly limited and can range from 1 to the maximum number of valences that can be saturated with substituents. This is typically 1, 2, or 3, usually 1 or 2, and most typically 1.

[0106] Unless otherwise specified, all valences of individual atoms of compounds or moieties described herein are saturated. Specifically, they are saturated by the designated bonding partners. If no bonding partners are designated or if too few bonding partners are designated, the remaining valences of each atom are saturated by the corresponding number of hydrogen atoms.

[0107] Unless otherwise specified, chiral compounds and moieties can exist in the form of pure stereoisomers or in the form of mixtures of stereoisomers (including 50:50 racemates). In the context of this disclosure, reference to a particular stereoisomer should be understood to refer to a compound or moiety in which the specified stereoisomer is present in at least 90% enantiomeric excess (ee), more preferably at least 95% ee, and most preferably 100% ee, where % ee is the product of (|RS|) / (R+S) * Defined as 100%, R and S represent the molar amounts of each enantiomer.

[0108] Unless the context dictates otherwise and / or unless otherwise expressly defined in this specification, all terms are intended to have their generally accepted meaning in the art as indicated by the IUPAC Gold Book (edition current as of November 1, 2022) or the Dictionary of Chemistry (Oxford, 8th Edition).

[0109] The term "anti-tumor compound" as used herein refers to a compound that has an anti-tumor effect and has a substituent or moiety that can be attached to a linker structure. When part or all of the linker is cleaved in a tumor cell, the anti-tumor compound portion is released, and the anti-tumor effect of the anti-tumor compound is exerted. When the linker is cleaved at the attachment site to the drug, the anti-tumor compound is released in its unmodified form and can exert its inherent anti-tumor effect.

[0110] The term "native drug" refers to a compound whose therapeutic effect has been established by in vitro and / or in vivo testing. In a preferred embodiment, the native drug is a compound whose therapeutic effect has been established by clinical trials. Most preferably, the native drug is a drug that is already on the market. The type of therapeutic effect to be established and the appropriate tests to be applied will, of course, depend on the type of medical indication to be treated.

[0111] The antitumor compound may be released in its unmodified (native) form or in a modified form, e.g., as a D-Dxx-Dyy moiety, as long as the group or moiety remains attached to it after cleavage by cathepsin B. This is advantageous if the remaining modified drug, D-Dxx-Dyy, is pharmacologically active. In this context, pharmacologically active means that the released modified drug, e.g., the D-Dxx-Dyy moiety, retains at least 20%, preferably at least 50%, and more preferably at least 80% of the pharmacological activity of the native drug when released intracellularly by the conjugate. To ensure realistic conditions, such activity testing should not be performed by comparing the cytotoxicity of the released modified drug with that of the native drug, because under these conditions, the modified drug must enter cells, which may introduce a cell permeability bias. The difference in permeability between these two substances is not relevant here, since the modified drug is released intracellularly. The activity of modified and native drugs can be compared in cell-free binding assays to determine the Ki (binding affinity) of the drug for the appropriate target receptor. If it is not possible to determine the Ki, the IC50 for cytotoxicity in HER2+ cell lines can be compared for two ADCs with identical linker systems, one designed to release the modified drug and the other designed to release the native drug.

[0112] When referring to particular classes of drug molecules, e.g., anti-tumor agents, topoisomerase inhibitors, RNA polymerase II inhibitors, DNA cleaving agents, anti-mitotic or microtubule disrupting agents, antimetabolites, kinase inhibitors, immunomodulators, or anti-infective agents, these terms shall have their generally accepted meaning in the medical field, as set forth, for example, in Mosby's Medical Dictionary, Mosby, Elsevier 10th Edition (2016), or Oxford Textbook of Oncology, David J. Kerr, OUP Oxford 3rd Edition (2016).

[0113] Thus, the drug used in the Ligand-Drug-Conjugates of the present disclosure may be a native drug (e.g., a drug that naturally contains one or more functional groups that allow for covalent attachment to the conjugate), or it may be a drug that has been chemically modified to incorporate a functional group (e.g., a group selected from hydroxyl, carboxyl, amino, and thiol groups) that allows for covalent attachment to an adjacent group or moiety, provided that the modified drug is pharmacologically active. In this context, pharmacologically active means at least 20%, preferably at least 50%, and more preferably at least 80% of the pharmacological activity of the native drug.

[0114] The term "auristatin" (or "auristatin analog") refers to a class of compounds structurally related to the naturally occurring pentapeptide dolastin 10. As used herein, an auristatin analog (auristatin) has the following formula: [ka] (wherein R3 represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, preferably a hydrogen atom or a methyl group; and R4 represents the side chain of any natural or unnatural amino acid).

[0115] In certain embodiments, compounds of the present disclosure utilize certain auristatin analogs, representative examples of which include monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). Hereinafter, the expression "auristatin analog", when characterizing an analog used in accordance with the present disclosure, refers in particular to auristatin X, where the C-terminal amino acid X (shown above) is selected from Phe (in which case the auristatin analog is auristatin Phe / F(AF)), Cit (in which case the auristatin analog is auristatin Cit(ACit)), Arg (in which case the auristatin analog is auristatin Arg(AArg)), Lys (in which case the auristatin analog is auristatin Lys(ALys)), Orn (in which case the auristatin analog is auristatin Orn(AOrn)), Dab (in which case the auristatin analog is auristatin Dab(ADab)), and Dap (in which case the auristatin analog is auristatin Dap(ADap)). As used herein, auristatin analogs (e.g., AF, ACit, AArg, ALys, AOrn, ADab, ADap) should be considered native drugs in addition to the native drugs defined above.

[0116] As used herein, the term "pharmaceutical composition" refers to a composition comprising an antibody-drug conjugate of the present disclosure, optionally together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers for use in pharmaceutical compositions of the present disclosure are well known to those skilled in the art and are selected based on several factors, such as the specific anti-tumor compound used and its concentration, stability, and intended bioavailability; the disease, disorder, or condition being treated with the composition; the subject, its age, size, and general condition; and the route of administration. For example, pharmaceutical compositions of the present disclosure can be prepared by mixing an antibody-drug conjugate of the present disclosure with a sterilized liquid (including water and oils (oils derived from petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.)), a solvent such as saline, aqueous dextrose, or aqueous glycerol solution, and additives such as humectants, emulsifiers, or pH buffering agents.

[0117] As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, alleviating, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. For example, "treating" cancer can refer to inhibiting the growth and / or spread of cancer cells, killing cancer cells, or shrinking a tumor. Treatment can be administered to subjects who do not show signs of the disease, disorder, and / or condition and / or who show only early symptoms of the disease, disorder, and / or condition, with the intent of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0118] As used herein, the terms "about," "approximate," "at or about," and "substantially" mean that the amount or value in question may be the exact value as claimed or taught herein, or a value that provides an equivalent result or effect. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as necessary, to reflect tolerances, conversion factors, rounding, measurement errors, and the like, as well as other factors known to those skilled in the art to provide equivalent results or effects. In some cases, a value that provides an equivalent result or effect cannot be reasonably determined. In such cases, "about" and "approximate," as used herein, are generally understood to mean a ±20% variation range of the nominal value, unless otherwise indicated or inferred. In general, a quantity, size, formulation, parameter, or other quantity or characteristic is "about," "approximate," or "approximate," whether or not expressly stated as such. Furthermore, when "about," "approximately," or "approximately" is used before a quantitative value, the parameter is understood to include the specific quantitative value itself unless otherwise specified. Conversely, reference to a quantitative value may refer to an "integer" of that value, if this is technically meaningful. For example, in formula (1), when reference is made to a designation "m" having a specific value (e.g., 1 to 5), m should be understood as an integer of the specified value (e.g., an integer from 1 to 5). Similarly, when reference is made to designations p1, p2, p3, p4, o', etc., these designations may be understood as integers of the specified value (as long as this is technically meaningful). However, while designations such as "m" may be understood as integers in a technically meaningful way when viewing individual molecules and / or compositions containing only a single molecular species, in the context of the present disclosure, providing such molecules in the form of a mixture containing two or more types of molecules of the present disclosure, which may differ from each other with respect to designations such as "m," does not exclude this. The average value resulting from a designation such as "m" is not necessarily an integer.Nevertheless, such mixtures are intended to be encompassed by the present disclosure, whether or not an "integer" is referenced. When an integer is referenced, it should be understood as characterizing an individual molecular species, which may be provided in pure form or in admixture with other molecules of the present disclosure.

[0119] The term "cancer" as used herein is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, kidney cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, renal cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer, and urothelial cell carcinoma.

[0120] The term "effective amount" of "therapeutically effective amount" refers to the amount of a therapeutic agent (e.g., antibody-drug conjugate) that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, alleviate, ameliorate, relieve, alleviate the symptoms of, prevent, delay the onset of, inhibit the progression of, reduce the severity of, and / or reduce the incidence of, the disease, disorder, and / or condition.

[0121] The terms "subject," "patient," "individual," and the like are used interchangeably herein and refer to any animal, any mammalian subject, or cells thereof, either in vitro or in situ, that are amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human. [Brief explanation of the drawings]

[0122] [Figure 1a]FIG. 1a shows the dose-dependent cytotoxic activity of Ab1-linker-DM1 against BCAM-positive human cancer cell lines. [Figure 1b] FIG. 1b shows the dose-dependent cytotoxic activity of Ab1-linker-AF against BCAM-positive human cancer cell lines. [Figure 1c] FIG. 1c shows the dose-dependent cytotoxic activity of Ab2-linker-AF against BCAM-positive human cancer cell lines. [Figure 1d] FIG. 1d shows the dose-dependent cytotoxic activity of Ab3-linker-MMAF against BCAM-positive human cancer cell lines. [Figure 2] FIG. 2 shows the A431 tumor growth curve after treatment with Ab1-linker-AF. [Figure 3] FIG. 3 shows the relative tumor growth rate 14 days after treatment with Ab1-linker-AF. [Figure 4a] 4a shows an exemplary structure of an antibody-drug conjugate of the present disclosure (antibody-drug conjugate No. 1 (Ab-linker-AF)). In FIG. 4a, Ab is an anti-BCAM antibody or an antigen-binding fragment thereof, as described below, and n is 1 to 10. [Figure 4b] Figure 4b shows an exemplary structure of an antibody-drug conjugate of the present disclosure (antibody-drug conjugate No. 2 (Ab-linker-MMAF)). In Figure 4b, Ab is an anti-BCAM antibody or an antigen-binding fragment thereof, as described below, and n is 1 to 10. [Figure 5] Figure 5 shows an exemplary structure of an antibody-drug conjugate of the present disclosure (antibody-drug conjugate No. 3 (Ab-linker-DM1)). In Figure 5, Ab is an anti-BCAM antibody or an antigen-binding fragment thereof, as described below, and n is 1 to 10. [Figure 6a] Figure 6a shows the results of SE-HPLC of Ab (Ab1), where the sample was loaded onto a size-exclusion column (TSKgel G3000SWXL, 7.8 x 300 mm (TOSOH)). [Figure 6b] FIG. 6b shows the results of SE-HPLC of the Abs (results for Ab2). [Figure 6c] FIG. 6c shows the results of SE-HPLC of the Abs (results for Ab3). [Figure 7a] Figure 7a shows the results of SDS-PAGE of Abs (results for Ab1), where the three lanes of samples and their loading amounts were, from left to right: M, marker, 5 μL; 1, non-reduced, 3 μg; and 2, reduced, 3 μg. [Figure 7b] FIG. 7b shows the results of SDS-PAGE of the Abs (results for Ab2). [Figure 7c] FIG. 7c shows the results of SDS-PAGE of Abs (results for Ab3). [Figure 8] FIG. 8 shows the SEC chromatogram (aggregate content assignment) of the HG4K IgG4-linker-DM1 conjugate. [Figure 9] FIG. 9 shows the SEC chromatogram (aggregate content assignment) of the HG4K IgG4-linker-AF conjugate. [Figure 10] FIG. 10 shows the SEC chromatogram (aggregate content assignment) of the Ab1-linker-DM1 conjugate. [Figure 11] FIG. 11 shows the SEC chromatogram (aggregate content assignment) of the Ab1-linker-AF conjugate. [Figure 12a] FIG. 12a shows the SEC chromatogram (aggregate content assignment) of the Ab2-linker-AF conjugate. [Figure 12b] FIG. 12b shows the SEC chromatogram (aggregate content assignment) of the Ab1-linker-MMAF conjugate. [Figure 12c] FIG. 12c shows the SEC chromatogram (aggregate content assignment) of the Ab3-linker-MMAF conjugate. [Figure 12d] Figure 12d shows the SEC chromatogram (aggregate content assignment) of the HG4 IgG4-linker-MMAF conjugate. [Figure 13] FIG. 13 shows MS native deglycosylation (DAR assignment) of HG4K IgG4-linker-DM1 conjugate. [Figure 14]Figure 14 shows MS native deglycosylation (DAR assignment) of HG4K IgG4-linker-AF conjugate. [Figure 15] FIG. 15 shows MS native deglycosylation (DAR assignment) of Ab1-linker-DM1 conjugate. [Figure 16] FIG. 16 shows MS native deglycosylation (DAR assignment) of Ab1-linker-AF conjugate. [Figure 17a] FIG. 17a shows MS native deglycosylation (DAR assignment) of Ab2-linker-AF conjugate. [Figure 17b] FIG. 17b shows MS native deglycosylation (DAR assignment) of the Ab1-linker-MMAF conjugate. [Figure 17c] FIG. 17c shows MS native deglycosylation (DAR assignment) of the Ab3-linker-MMAF conjugate. [Figure 17d] Figure 17d shows MS native deglycosylation (DAR assignment) of HG4K IgG4-linker-MMAF conjugate. [Figure 18] FIG. 18 shows the indirect ELISA binding affinity of Ab1 and Ab2 to recombinant human BCAM protein. [Figure 19] FIG. 19 shows the sandwich ELISA binding affinity of Ab1 and Ab3 to recombinant human BCAM protein. [Figure 20a] Figure 20a shows the binding affinity of Abs to BCAM-overexpressing HEK293 cells (Figure 20a: results for Ab1 and Ab2). [Figure 20b] Figure 20b shows the binding affinity of Abs to BCAM-overexpressing HEK293 cells (Figure 20b: results for Ab3). [Figure 21] FIG. 21 shows the internalization properties of Ab1 into MKN-1 cancer cells.

[0123] Detailed Description 1. Antibody-drug conjugates The present disclosure provides Ab-(L-(D)m)n The present invention provides an antibody-drug conjugate of formula (1) having the structure:

[0124] (1) Anti-BCAM antibody In formula (1), Ab is an anti-basal cell adhesion molecule (BCAM) antibody or an antigen-binding fragment thereof, comprising: (i) a heavy chain variable region comprising the VH CDR1 sequence of SEQ ID NO: 1, the VH CDR2 sequence of SEQ ID NO: 2, and the VH CDR3 sequence of SEQ ID NO: 3; and (ii) a light chain variable region comprising the VL CDR1 sequence of SEQ ID NO: 4, the VL CDR2 sequence of SEQ ID NO: 5, and the VL CDR3 sequence of SEQ ID NO: 6.

[0125] In one embodiment, in the anti-BCAM antibody, the heavy chain variable region comprises the sequence of SEQ ID NO: 7 and the light chain variable region comprises the sequence of SEQ ID NO: 8. In one embodiment, in the BCAM antibody, the heavy chain variable region consists of the sequence of SEQ ID NO: 7 and the light chain variable region consists of the sequence of SEQ ID NO: 8. In one embodiment, in the anti-BCAM antibody, the heavy chain comprises or consists of the sequence of SEQ ID NO: 9 and the light chain comprises or consists of the sequence of SEQ ID NO: 11. In one embodiment, in the anti-BCAM antibody, the heavy chain comprises or consists of the sequence of SEQ ID NO: 10 and the light chain comprises or consists of the sequence of SEQ ID NO: 11. In one embodiment, in the anti-BCAM antibody, the heavy chain comprises or consists of the sequence of SEQ ID NO: 12 and the light chain comprises or consists of the sequence of SEQ ID NO: 11.

[0126] Furthermore, the antigen-binding fragment may be an antibody fragment selected from the group consisting of a Fab fragment, a Fab' fragment, a Fab'-SH fragment, an Fv fragment, an scFv fragment, a F(ab')2 fragment, a VL fragment, a VH fragment, an ScFv-Fc fragment, and an (scFv)2-Fc fragment, a bispecific antibody, a linear antibody, a fragment produced by a Fab expression library, an anti-idiotype (anti-Id) antibody, a complementarity-determining region (CDR), and an epitope-binding fragment. In another embodiment, the anti-BCAM antibody is a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the antibody or fragment thereof according to the present disclosure is a mature version of any of the antibodies or fragments thereof disclosed herein. In one embodiment, the antibody according to the present disclosure may be of the human IgG1, IgG2, IgG3, or IgG4 type, or a variant thereof. In another embodiment, the antibody of the present disclosure may be of the human IgG1, IgG4 type, or a variant thereof. The sequence information referred to herein is as follows: TIFF2025539807000038.tif200170

[0127] Anti-BCAM antibodies and antigen-binding fragments thereof can bind with high affinity to both BCAM protein and BCAM-positive cancer cells, and can be efficiently internalized into BCAM-expressing cancer cells after binding.

[0128] Anti-BCAM antibodies and antigen-binding fragments thereof can be produced by any method known in the art, such as recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be carried out from any suitable host cell known in the art (e.g., mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.).

[0129] In one embodiment, the anti-BCAM antibody is Ab1, Ab2, or Ab3 prepared as described in Example 1 of the present disclosure. Ab1, Ab2, or Ab3 has a heavy chain variable region comprising the sequence of SEQ ID NO:7 and a light chain variable region comprising the sequence of SEQ ID NO:8. Ab1 has a heavy chain comprising or consisting of the sequence of SEQ ID NO:9 and a light chain comprising or consisting of the sequence of SEQ ID NO:11. Ab2 has a heavy chain comprising or consisting of the sequence of SEQ ID NO:10 and a light chain comprising or consisting of the sequence of SEQ ID NO:11. Ab3 has a heavy chain comprising or consisting of the sequence of SEQ ID NO:12 and a light chain comprising or consisting of the sequence of SEQ ID NO:11.

[0130] (2) Linker In formula (1), L is a linker, and the anti-tumor compound (D) is conjugated to the anti-BCAM antibody via the linker.

[0131] (2.1) Linker of formula (2) In one embodiment of the present invention, the linker (L) is of formula (2): [ka] (wherein Y is a divalent group containing one or more atoms selected from C, N, O, P, and S, preferably a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound, and derivatives thereof, more preferably a divalent group derived from maleimide and its derivatives, for example, a ring-opened hydrolyzed maleimide derivative; T is a (1+o)- or (2+o)-valent linking group; S is an atom or group optionally present to saturate the free valences of T; L' is a linker that can be cleaved by cathepsin B, o is an integer of 1 to 5, preferably 1 or 2, more preferably 1; * indicates covalent binding to an anti-BCAM antibody (Ab), and **indicates a covalent bond to one or more antitumor compounds (D).

[0132] In a preferred embodiment, the linker (L) is covalently attached to the anti-BCAM antibody or fragment thereof via a side chain of a cysteine ​​contained in the antibody or antibody fragment.

[0133] (2.1.1) Cathepsin B-cleavable linker (L') The linker of formula (2) contains a cleavable element (L') that serves as a substrate for specific recognition and cleavage by cathepsin B, particularly fast cleavage and / or cleavage by the exopeptidase activity of Cat B. The cleavable linker (L') is covalently attached to a linking group (T) and one or more antitumor compounds (D).

[0134] In one embodiment, the linker (L') that can be cleaved by cathepsin B has formula (3) or formula (4): [ka] (In the formula, Axx is a trifunctional amino acid, provided that Axx in formula (3) is not an amino acid of the (D) configuration, Ayy in formulas (3) and (4) is an amino acid selected from Phe, Ala, Trp, Tyr, phenylglycine (Phg), Met, Val, His, Lys, Arg, citrulline (Cit), 2-aminobutyric acid (Abu), ornithine (Orn), Ser, Thr, Leu, and Ile, or Ayy in formula (3) is homo-tyrosine (homo-Tyr), homo-phenylalanine (homo-Phe), beta-phenylalanine (beta-Phe) and beta-homo-phenylalanine (beta-homo-Phe), Tyr(OR1) and homo-Tyr(OR1) (wherein R1 is -(CH2CHO) n1 -R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), with the proviso that Ayy in formula (4) is not an amino acid of the (D) configuration, Z is a group covalently bonded to the C-terminus of Ayy or Axx selected from -OH and -N(H)(R), where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; W is a drug-loading unit; ** represents a covalent bond to one or more moieties D, and *** indicates a covalent bond to T, When more than one linker (L') is present, each linker is independently selected from the aforementioned linkers of formula (3) and formula (4).

[0135] Without wishing to be bound by theory, it is believed that Ayy confers structural features to the compounds of the present disclosure for specific recognition and cleavage by the exopeptidase activity of Cat B. As a result, the compounds can release drugs at a significantly higher rate than compounds that are cleaved by the endopeptidase activity of Cat B, such as compounds containing a Val-Cit-PABC linker system.

[0136] Furthermore, the sterically demanding moiety (Ab-YT(S) structure) present in the side chain of residue Axx in formula (3) / (4) does not adversely affect the binding affinity of the compounds of the present disclosure to Cat B, nor the cleavage rate of the compounds by the exopeptidase mechanism of Cat B. Without wishing to be bound by theory, it is believed that the sterically demanding moiety is oriented outside the Cat B binding groove (hydrophobic pocket), thus resulting in superior cleavage rate by the exopeptidase mechanism.

[0137] In formulas (3) and (4), at least one or both of Axx and Ayy have the following definitions: Axx, (a) Axx in formula (3) or (4) is an amino acid selected from Glu, 2-amino-pimelic acid (Apa), 2-aminoadipic acid (Aaa), 2,3-diamino-propionic acid (Dap), 2,4-diamino-butyric acid (Dab), Lys, Orn, Ser, amino-malonic acid (Ama), and homo-lysine (homo-Lys), preferably an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; Ayy, (b) Ayy in formula (3) is Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1) (wherein R1 is -(CH2CHO) n1 -R2, wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), preferably an amino acid selected from Phe, homo-Phe, Tyr, homo-Tyr, Tyr(OR1) and homo-Tyr(OR1), more preferably an amino acid selected from Phe and Tyr, or (c) Ayy in formula (4) is an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, and Ser, preferably an amino acid selected from Phe, homo-Phe, and Ser, more preferably an amino acid selected from Phe and Ser; It is preferable that the following is satisfied.

[0138] In formulas (3) and (4), at least one or both of Axx and Ayy have the following definitions: Axx, (a) Axx in formula (3) or (4) is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; Ayy, (b) Ayy in formula (3) is an amino acid selected from Phe and Tyr, or (c) Ayy in formula (4) is an amino acid selected from Phe and Ser; It is more preferable that the following be satisfied.

[0139] (2.1.2) Drug-carrying unit (W) The drug-carrying unit (W) is a moiety that links the C-terminal dipeptide contained in the cleavable linker (L'), such as the moiety Axx-Ayy of formula (3) or the moiety Ayy-Axx of formula (4), to one or more antitumor compounds (D).

[0140] In a preferred embodiment, the drug-carrying unit (W) is represented by formula (5): [ka] wherein Dxx is an amino acid that is absent or has a hydrophobic side chain; Dyy is absent, Phe, or an amino acid having a basic side chain, provided that when Dxx is an amino acid having a hydrophobic side chain, Dyy is Phe or an amino acid having a basic side chain, and when Dxx is a single covalent bond, Dyy is a single covalent bond, Phe, or an amino acid having a basic side chain; ** indicates a covalent bond to D, and ** ' represents a covalent bond to the N-terminus of Axx or Ayy).

[0141] In formula (5), at least one of Dxx and Dyy, for example, one or two of them, has the following definition: (a) Dxx is an amino acid selected from Phe, Val, Tyr, homo-Phe, and Ala; (b) Dyy is absent or an amino acid selected from Arg, Lys, Cit, Orn, Dap, and Dab; It is preferable that the following is satisfied.

[0142] In formula (5), at least one of Dxx and Dyy, for example, one or two of them, has the following definition: (a) Dxx is an amino acid selected from Phe and Val; (b) Dyy is absent or is an amino acid selected from Arg and Cit; It is more preferable that the following be satisfied.

[0143] In some embodiments, Dxx may include additional elements, such as a single covalent bond or an amino acid having a hydrophobic side chain, optionally linked to D through a divalent moiety selected from maleimides, triazoles, hydrazones, carbonyl-containing groups, and derivatives thereof, preferably through a divalent maleimide derivative, such as a ring-opened hydrolyzed maleimide derivative.

[0144] In another preferred embodiment, the drug-carrying unit (W) is represented by formula (6) or formula (7): [ka] (In the formula, A''xx is a trifunctional amino acid, provided that A''xx in formula (6) is not an amino acid of the (D) configuration, A'yy is an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn, with the proviso that A'yy in formula (7) is not an amino acid in the (D) configuration; when more than one A'yy is present, each A'yy is independently selected from the foregoing amino acids; A"yy is an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn, with the proviso that A"yy in formula (7) is not an amino acid in the (D) configuration; when more than one A"yy is present, each A"yy is independently selected from the foregoing amino acids; A'''yy is an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn, with the proviso that A'''yy in formula (7) is not an amino acid in the (D) configuration; when more than one A'''yy is present, each A'''yy is independently selected from the foregoing amino acids; A'xx is an amino acid, provided that A'xx in formula (6) is not an amino acid of the (D) configuration, A'''xx is an amino acid, provided that A'''xx in formula (6) is not an amino acid of the (D) configuration, p1 is an integer of 0 to 3, preferably 0; p2 is 0 or 1, preferably 1; p3 is an integer from 0 to 3, provided that when p2 is 0, p3 is not 0; p3 is preferably 0; p4 is an integer of 1 to 4, provided that p4 and o in formula (2) are selected so that m in formula (1) is an integer of 1 to 5; ** ' indicates a covalent bond to the N-terminus of Axx or Ayy, and ** indicates a covalent bond to an antitumor compound).

[0145] In formulas (6) and (7), at least one of A'xx, A''xx, A'''xx, A'yy, A''yy, and A'''yy, for example, one, two, three, four, five, or six, has the following definition: (a) A'xx is an amino acid selected from Arg, Lys, homo-Lys, Cit, Orn, Dap, and Dab; (b) A''xx is an amino acid selected from Lys, homo-Lys, Cit, Orn, Dap, and Dab; (c) A'''xx is an amino acid selected from Arg, Lys, homo-Lys, Cit, Orn, Dap, and Dab; (d) A'yy is an amino acid selected from Phe, Ala, Trp, Phg and Tyr, preferably an amino acid selected from Phe and Tyr; (e) A''yy is an amino acid selected from Phe, Ala, Trp, Phg and Tyr, preferably an amino acid selected from Phe and Tyr; (f) A'''yy is an amino acid selected from Phe, Ala, Trp, Phg and Tyr, preferably an amino acid selected from Phe and Tyr; It is preferable that the following is satisfied.

[0146] Peptides of formula (3) / (6), formula (3) / (7), formula (4) / (6), or formula (4) / (7) act as specific substrates for the exopeptidase activity of Cat B. That is, the linker of formula (3) or formula (4) described herein can be cleaved at its N-terminus by Cat B to release the drug-carrying unit (W). When W is a moiety of formula (6) or formula (7), it can then be cleaved by Cat B, thus releasing the antitumor compound (D) attached to the N-terminus of A'xx or A'yy, and one or more moieties comprising (A''xx(D)-A''yy) / (A''yy-A''xx(D)). In some embodiments of the present disclosure, moieties comprising (A''xx(D)-A''yy) / (A''yy-A''xx(D)) exhibit pharmacological (antitumor) activity.

[0147] In some embodiments of the invention, each moiety comprising (A''xx(D)-A''yy) / (A''yy-A''xx(D)) can be "self-immolative" insofar as it can undergo intramolecular aminolysis (i.e., formation of a 5- or 6-membered ring, or formation of a diketopiperazine (DKP)) to liberate moiety (D) as a product. If p2 ≥ 1, then the peptide (A''xx(D)-A''yy) p2 / (A''yy-A''xx(D) p2 ) can act as a substrate for Cat B to cleave the (p2-1) amide bond between amino acids A''yy-A''xx / A''yy-A''xx, thus liberating the p2 dipeptides (A''xx(D)-A''yy) / (A''yy-A''xx(D)). In some embodiments, each dipeptide can then undergo intramolecular aminolysis (A''xx(D)-A''yy) or DKP formation (A''yy-A''xx(D)) to liberate the p2 moiety (D) as a product.

[0148] Thus, when the drug-carrying unit (W) represents a peptide of formula (6) or formula (7), the linker can release two or more drug molecules (the same or different drugs), thereby achieving a high DAR and improving overall pharmacological activity. Drug release can occur according to a multi-step mechanism. For example, (W) is first released from the compound of formula (I), then acts as a substrate for Cat B to release moiety (D), and finally releases the p2 moiety-containing peptide (A"xx(D)-A"yy) / (A"yy-A"xx(D)), which is pharmacologically active as it is (e.g., intra-antitumor compounds) and / or can undergo intramolecular aminolysis, DKP formation, or hydrolysis to release the p2 moiety (D).

[0149] The compounds of the present disclosure are generally stable in the extracellular environment (e.g., in plasma) in the absence of Cat B (i.e., an enzyme capable of cleaving the linker). However, upon exposure to Cat B, the linker (L') is recognized and cleaved, ultimately initiating spontaneous self-immolative aminolysis, which cleaves the bond covalently linking the self-immolative moiety, e.g., A"xx-A"yy, to the drug, thereby liberating compound (D) in a pharmacologically active form. Self-immolative aminolysis can occur when A"xx represents an amino acid such as Glu, Aaa, Dap, Dab, Ser, Thr, homoSer, or homoThr.

[0150] In yet another preferred embodiment, the drug-carrying unit (W) is represented by formula (8): [ka] wherein A''xx is a trifunctional amino acid selected from Glu, α-aminoadipic acid (Aaa), Dap, Ser, Thr, homo-serine (homo-Ser), homo-threonine (homo-Thr), and aminomalonic acid (Ama), with the proviso that A''xx is not an amino acid of the (D) configuration; Cxx is a single covalent bond unless A''xx is Ama; when A''xx is Ama, Cxx is Pro or an N-methyl amino acid, the N-terminus of Cxx is attached to the carboxyl terminus of Ama, and the C-terminus of Cxx is covalently attached to one moiety D; A'yy, A''yy, and A''yy are each independently an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn; A'xx and A'''xx are each independently an amino acid, provided that A'xx and A'''xx are not amino acids of configuration (D), p1 is 0 or 1, preferably 0; p2 is 0 or 1, preferably 1; p3 is an integer from 0 to 3, provided that when p2 is 0, p3 is not 0; p3 is preferably 0; p4 is an integer of 1 to 4, provided that p4 and o in formula (2) are selected so that m in formula (1) is an integer of 1 to 5; ** ' indicates a covalent bond to the N-terminus of Axx or Ayy, and ** indicates a covalent bond to an antitumor compound).

[0151] In formula (8), at least one of A'xx, A''xx, A'''xx, A'yy, A''yy, and A'''yy, for example, one, two, three, four, five, or six, has the following definition: (a) A'xx is an amino acid selected from Arg, Lys, homo-Lys, Cit, Orn, Dap, and Dab; (b) A''xx is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; (c) A'''xx is an amino acid selected from Arg, Lys, homo-Lys, Cit, Orn, Dap, and Dab; (d) A'yy is an amino acid selected from Phe, Ala, Trp, Phg and Tyr, preferably an amino acid selected from Phe and Tyr; (e) A''yy is an amino acid selected from Phe, Ala, Trp, Phg and Tyr, preferably an amino acid selected from Phe and Tyr; (f) A'''yy is an amino acid selected from Phe, Ala, Trp, Phg and Tyr, preferably an amino acid selected from Phe and Tyr; It is preferable that the following is satisfied.

[0152] It is well known that peptides and proteins containing a Pro residue at the penultimate N-terminal position undergo nonenzymatic aminolysis to form DKPs. The mechanism of DKP formation involves nucleophilic attack of the N-terminal nitrogen on the carbonyl of the second amino acid. This intramolecular aminolysis proceeds readily and plays an important role in the biosynthetic pathway of biologically active cyclic dipeptides, such as c(His-Pro), which are found throughout the central nervous system, peripheral tissues, and body fluids. In the dipeptide (Ama(Cxx-D)-A''yy), the mechanism of DKP formation involves nucleophilic attack of the N-terminal nitrogen on the side chain of Cxx, thus liberating the moiety D.

[0153] Peptides of formula (3) / (8) or formula (4) / (8) can act as substrates for the exopeptidase activity of Cat B, liberating a peptide-containing moiety having formula (8), which can then be cleaved by Cat B to release a moiety comprising moieties (D) and (A"xx(Cxx-D)-A"yy). Peptides (A'xx(Cxx-D2)-A'yy) are "self-immolative" moieties and can undergo intramolecular aminolysis (i.e., formation of a 5- or 6-membered ring, or formation of a diketopiperazine (DKP)) to release moiety (D) as the product. If p2 >= 1, then peptide (A"xx(Cxx-D)-A"yy) p2can act as a substrate for Cat B and cleave the (p2-1) amide bond between amino acids A''yy and A''xx, thus liberating p2 peptides (A''xx(Cxx-D)-A''yy). Each peptide (A''xx(Cxx-D)-A''yy) can then undergo intramolecular aminolysis to liberate the p2 moiety (D) as a product.

[0154] Thus, when the drug-carrying unit (W) is a peptide of formula (8), drug release occurs according to a multi-step mechanism; for example, (W) can first be released from the compound of formula (I), then act as a substrate for Cat B to release moiety (D) and the p2 peptide (A"xx(Cxx-D)-A"yy), and finally undergo intramolecular aminolysis to release the p2 moiety (D). In the peptide (Ama(Cxx-D)-A"yy), the mechanism of DKP formation (intramolecular aminolysis) involves nucleophilic attack of the N-terminal nitrogen of Ama on the ester carbonyl of Cxx, thus liberating (D).

[0155] (2.1.3) Linking group (T) The linking group (T) represents a (1+o)valent or (2+o)valent linking group, for example, a divalent, trivalent, tetravalent, pentavalent, or hexavalent linking group. The linking group links the antibody or fragment thereof to the moiety (S), if present, and one or more (o) cleavable moieties (L') via the divalent group (Y), thereby forming a linear or branched structure. Preferably, the linking group (T) is a divalent, trivalent, or tetravalent group. More preferably, the linking group (T) is a divalent or trivalent group. T can be linked to Y, S, and L', for example, via a chemoselective ligation procedure for the formation of an amide bond, via "click chemistry" (e.g., azide-alkyne cycloaddition), or the like.

[0156] In some embodiments, T acts as a moiety for the attachment of multiple drugs. It can be, for example, a small organic group with two or more valences, with a molecular weight of 200 Da or less, or even only 100 Da or less, but it can also be a more complex and / or larger moiety derived from a functional polymer, copolymer, dendrimer, or synthetic structure containing multiple reactive groups for attachment to L'.

[0157] In some embodiments of the present disclosure, the linking group (T) is selected to be hydrolytically stable, meaning that typically less than 20%, preferably less than 10%, of the test compound undergoes hydrolysis within 24 hours at 37° C. in phosphate buffered saline (PBS) solution at pH 7.4, as measured by HPLC, and the test compound is a compound based on the polyvalent group T, where all valencies of T are saturated with hydrogen atoms.

[0158] Ideally, the compounds of formula (I) having a linking group (T) exhibit such stability to hydrolysis when taken as a whole, i.e., less than 20%, more preferably less than 10%, of the compounds of formula (I) undergo hydrolysis within 24 hours at 37°C in phosphate buffered saline (PBS) at pH 7.4 as measured by HPLC.

[0159] In one embodiment, the linking group is a group containing at least one moiety derived from a trifunctional amino acid, such as Lys. The linking group may contain, in addition to the trifunctional amino acid, an additional (optional) linker and / or amino acid, provided that the additional amino acid is not a trifunctional amino acid or a moiety containing one or more ionic or ionizable groups. Examples of additional linkers include polyoxyalkylene oxides, particularly polyethylene oxides having 0 to 20, preferably 0 to 5 or 0 to 3, e.g., 1 to 3, ethylene oxide subunits. The additional amino acid may be selected, for example, from homo-Phe and Phe. When present, the additional linker and / or amino acid is preferably attached to the backbone of the trifunctional amino acid, i.e., to the carboxyl and / or amino groups of the trifunctional amino acid, such as Lys. When the moiety S is present, the additional linker preferably does not act as a solubilizing group (i.e., its effect on solubility is negligible compared to the moiety S). In some embodiments, the linking group consists of a moiety derived from a trifunctional amino acid (ie, does not include additional linkers and / or amino acids).

[0160] In one embodiment, the linking group (T) is represented by formula (9): [ka] wherein each AA is independently a moiety containing a trifunctional amino acid; α represents a covalent bond to Y at the N-terminus of AA, or a covalent bond to Y at the N-terminus of the first AA when o' is 2 to 5; o' is an integer from 1 to 5, provided that the other moiety L' is *** When attached to ', o' is 1 to 4, when o' is 1, the side chain of the trifunctional amino acid is covalently attached to S or L' and the C-terminus is covalently attached to a moiety different from that attached at the side chain, L' or S, respectively; When o' is 2, 3, 4 or 5, **** indicates a covalent bond to L', ***' indicates a covalent bond to S).

[0161] In formula (9), each AA is preferably a moiety containing an amino acid independently selected from N-ε-propargyloxycarbonyl-L-lysine (Lys(Poc)), Asp, Glu, Orn, Lys, Dab, and Dap. More preferably, each AA is a moiety containing an amino acid independently selected from Lys(Poc), Glu, Orn, and Lys. Most preferably, each AA is Lys.

[0162] In one embodiment, the linking group (T) is represented by formula (10), or formula (11): [ka] (In the formula, each AA 1 and A.A. 2 are independently a moiety that contains a trifunctional amino acid; α represents a covalent bond to Y; In formula (11), the side chain of the trifunctional amino acid is covalently bonded to L' or S, and the C-terminus is covalently bonded to S or L', respectively, which is a moiety different from that bonded to the side chain; In equation (10), **** indicates a covalent bond to L', *** ' represents a covalent bond to S or L').

[0163] In equations (10) and (11), each AA 1 and A.A. 2 are preferably moieties comprising amino acids independently selected from Lys(Poc), Asp, Glu, Orn, Lys, Dab and Dap. More preferably, each AA 1 and A.A. 2 are moieties comprising amino acids independently selected from Lys(Poc), Glu, Orn and Lys. Most preferably, each AA 1 and A.A. 2 is Lys.

[0164] In one embodiment, the linking group (T) is represented by formula (12) or formula (13): [ka] wherein Azz is a moiety comprising one or more solubilizing groups, preferably an amino acid selected from Arg, Dap, Dab, Orn, Lys and carnitine; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group such as -(C=O)-, or an amino-containing group such as -N(R)- (wherein R is a hydrogen atom, an alkyl group, or a cycloalkyl group); n2 is an integer of 0 to 5, preferably 0, 1, 2, or 3, more preferably 0 or 1, and most preferably 0; n3 is an integer of 1 to 50, preferably 2 to 24, more preferably 4 to 12, for example, 4 to 8, n4 is an integer of 1 to 50, preferably 2 to 20, and more preferably 2 to 12; α represents a covalent bond to Y, and **** indicates a covalent bond to L').

[0165] In a preferred embodiment, the linking group (T) is represented by formula (12') or formula (13'): [ka] (wherein Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group, preferably an amino-containing group, and more preferably -N(R)- (wherein R is a hydrogen atom, an alkyl group, or a cycloalkyl group), Y2 is a carbonyl-containing group such as -(C=O)- or an amino-containing group such as -N(R)- (wherein R is a hydrogen atom, an alkyl group, or a cycloalkyl group), preferably a carbonyl-containing group, more preferably -(C=O)-; n3 is an integer of 1 to 50, preferably 2 to 24, more preferably 4 to 12, for example, 4 to 8, n4 is an integer of 1 to 50, preferably 2 to 20, and more preferably 2 to 12; α represents a covalent bond to Y, and **** indicates a covalent bond to L').

[0166] In a preferred embodiment, the linking group (T) is represented by formula (10), formula (11), formula (13), or formula (13'). More preferably, the linking group (T) is represented by formula (10), formula (11), or formula (13'). Most preferably, the linking group (T) is represented by formula (11) or formula (13').

[0167] (2.1.4) Part (S) S is an atom or group optionally present to saturate the free valences of the linking group (T). For example, S may be a hydrogen atom, an alkyl group, or a cycloalkyl group. In one embodiment, S is a moiety that includes one or more, e.g., two, three, four, or five, solubilizing groups.

[0168] The attachment of a moiety S containing one or more solubilizing groups can reduce (or prevent) the aggregation tendency of the conjugate molecule, thus enabling excellent pharmacokinetic properties, e.g., biodistribution, hepatic clearance, even at high DARs (e.g., DAR=8). In some instances, aggregation of the conjugate molecule can be completely suppressed even at high DARs. Cleavage by the Cat B exopeptidase mechanism is possible even in the presence of sterically demanding solubilizing groups. Without being bound by any theory, it is believed that the moiety S is oriented outside the Cat B binding groove, thus enabling excellent selectivity and cleavage rates, e.g., via the exopeptidase mechanism. In some embodiments, the moiety S can compensate for the potential hydrophobicity of the antitumor compound, thereby maintaining excellent pharmacokinetic properties even when multiple antitumor compounds are attached to the linker (e.g., m>1).

[0169] In one embodiment, S represents a moiety comprising one or more, e.g., two, three, or four, solubilizing groups, each solubilizing group in (S) being: - a moiety comprising one or more ionic or ionizable groups, such as ammonium, guanidinium, sulfate or sulfonate groups, preferably derived from Arg, Dap, Dab, Orn, Lys, or carnitine; - a sugar moiety selected from monosaccharides, disaccharides and linear or branched oligosaccharides, in particular linear or branched oligosaccharides having 3 to 10 monosaccharide units linked by glycosidic bonds, wherein each of the monosaccharide units in the monosaccharides, disaccharides and oligosaccharides is independently selected from glucose, fructose, mannose, ribose and galactose; and - a polyalkylene oxide group, preferably C 2~3 C independently containing 6 to 200 repeating units, preferably 10 to 150 repeating units, more preferably 12 to 80 repeating units of a polyalkylene oxide group. 2~3 Polyalkylene oxide group are independently selected from the group consisting of:

[0170] There are no particular limitations regarding the general arrangement of the solubilizing groups in the moiety (S). Thus, the moiety (S) may have a linear structure (e.g., multiple solubilizing groups arranged randomly or in blocks), a cyclic structure, or a branched structure (e.g., multiple solubilizing groups grafted or dendrimer-like attached to a core molecule such as pentaerythritol or glycerol). The moiety (S) may also comprise multiple blocks, each of which may independently have a linear or branched structure.

[0171] In one embodiment, the moiety (S) comprises one or more solubilizing groups arranged in a linear block. For example, the moiety (S) may have the following formula: -(So 1 )-(So 2 )-[...]-(So n ), -(So) n’-, wherein each So 1 ~So n represents a solubilizing group such as a polyalkylene oxide group, e.g., a PEO group having 6 to 200 repeat units, or a moiety containing one or more ionic or ionizable groups such as Arg; and n' is an integer from 1 to 20, e.g., 1 to 10, with the proviso that directly attached polyalkylene oxide groups of the same structure are considered multiple repeat units of the same solubilizing group (and are not considered adjacent S0 groups). That is, adjacent polyalkylene oxide groups must be of different structure and / or linked via a functional group such as -C(O)-O- to be treated as separate S0 groups.

[0172] In another embodiment, the moiety (S) comprises one or more solubilizing groups attached to a core molecule, such as pentaerythritol or glycerol, either free or in the form of a graft or dendrimer. For example, the moiety (S) may be -((-Y'-X'(So m’ )) n’ -H: [ka] wherein X' is an (m'+2)-valent group, e.g., a trivalent or tetravalent group, Y' is a divalent group, each So is independently selected to be a solubilizing group such as a polyalkylene oxide group, e.g., a PEO group having 4 to 600 repeating units, or a moiety containing one or more ionizable groups, m' is 1, 2, 3, or more, preferably 1 or 2, and n' is an integer from 1 to 20, e.g., 1 to 10; or -X'(So) as exemplified in more detail below. n’ The dendritic dendrimer structure represented by: [ka] (Wherein, X' is an n-valent (branched) group, and each So 1 ~So nare independently selected to be a solubilizing group as described above, e.g., a polyalkylene oxide group, e.g., a PEO group having 4 to 600 repeating units, or a moiety containing one or more ionic groups, and n' is an integer from 1 to 20, e.g., 1 to 10.

[0173] In one embodiment, moiety (S) comprises one or more polyethylene oxide groups, preferably each polyethylene oxide group independently comprising from 6 to 200 repeat units, more preferably from 10 to 150 repeat units, and most preferably from 12 to 80 repeat units.

[0174] In a preferred embodiment, the moiety (S) is of formula (23): [ka] (wherein n5 is an integer of 6 to 200, preferably 10 to 150, more preferably 12 to 80, for example, 24, **** indicates a covalent bond to (T), X 1 is selected from a single covalent bond, —(C═O)—, and —N(R)—, where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; X 2 is an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group such as an acetyl group or a group of the formula -(CH2) n6 -CO2H group, a thiocarbonyl-containing group, formula -(CH2) n6 OR group, formula -(CH2) n6 A group of —SO3H, or an amino-containing group, such as a group of formula —(CH2) n6 -(C=A)-N(R)2 or -(CH2) n6 -N(R)2, where A is O or S, each R is independently selected from a hydrogen atom, an alkyl group, and a cycloalkyl group, and n6 is an integer from 1 to 6; X 2 is preferably —CH 3 , —CH 2 CH 2 OH, or a group of formula (24): [ka] (In the formula, each A is independently selected from O and S, preferably O; each R is independently selected from a hydrogen atom, an alkyl group, and a cycloalkyl group; n7 and n8 are each independently an integer of 1 to 6, preferably 1 or 2, and X 2 is most preferably —CH3).

[0175] When more than one (S) is present, each (S) is preferably a moiety of formula (23) as above.

[0176] (2.2) Linkers of Formula (14), Formula (15) and Formula (16) In one embodiment of the present invention, the linker (L) included in the compound of formula (1) is formula (14) or formula (15): [ka] (Wherein, Bxx in formulas (14) and (15) is a trifunctional amino acid, provided that Bxx in formula (14) is not in the (D) configuration, In formulas (14) and (15), By is Phe, homo-Phe, Ala, Trp, Tyr, Phg, Val, His, Lys, Abu, Met, Cit, Orn, Ser, Thr, Leu, Ile, Arg, and Tyr (OR1), where R1 is -(CH2CHO) n1 -R2, wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), or Byy in formula (14) is an amino acid selected from homo-Tyr, homo-Tyr(OR1) (wherein R1 is as defined for Tyr(OR1) above), homo-Phe, beta-Phe, and beta-homo-Phe; provided that when (q1)×(q3)>1 and q2=0, only the C-terminal Byy in formula (14) may be an amino acid selected from beta-Phe and beta-homo-Phe; provided that Byy in formula (15) is not in the (D) configuration, Bxx1 in formulas (14) and (15) is a single covalent bond or an amino acid having a hydrophobic or basic side chain; Bxx2 in formulas (14) and (15) is an amino acid having a hydrophobic or basic side chain, Bxx3 in formulas (14) and (15) is an amino acid, provided that Bxx3 in formula (14) is not in the (D) configuration, Bxx4 in formulas (14) and (15) is Phe, homo-Phe, Ala, Trp, Tyr, Phg, Val, His, Lys, Abu, Met, Cit, Orn, Ser, Thr, Leu, Ile, Arg, and Tyr (OR1) (wherein R1 is -(CH2CHO) n1 -R2, wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), or Bxx4 in formula (14) is an amino acid selected from homo-Tyr, homo-Tyr(OR1), homo-Phe, beta-Phe, and beta-homo-Phe; provided that when (q2) × (q3) > 1, only the C-terminal Bxx4 in formula (14) may be an amino acid selected from beta-Phe and beta-homo-Phe; provided that Byy in formula (15) is not in the (D) configuration, S' is a divalent group containing one or more atoms selected from C, N, O, P, and S, preferably 1 to 40 atoms, more preferably 1 to 20 atoms; preferably, S' is a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound, and derivatives thereof, more preferably a divalent group derived from maleimide and derivatives thereof, such as ring-opened hydrolyzed maleimide derivatives; Z' is a group covalently bonded to the C-terminus of Byy or Bxx4 in formula (14) or to the C-terminus of Bxx or Bxx3 in formula (15), and is selected from -OH and -N(H)(R) (wherein R represents a hydrogen atom, an alkyl group, or a cycloalkyl group); q1 is an integer of 0 to 5, q2 is an integer of 0 to 3, provided that when q1 is 0, q2 is not 0; q3 is an integer from 1 to 5, q1, q2, and q3 are selected so that m in formula (1) is an integer of 1 to 5; * indicates covalent binding to an anti-BCAM antibody (Ab), and ** represents a covalent bond to one moiety D).

[0177] In one embodiment of the present invention, the linker (L) included in the compound of formula (1) is represented by formula (16): [ka] (wherein Bxx in formula (16) is a carboxyamino acid or a trifunctional amino acid selected from Dap, Dab, Ser, Thr, Lys, Orn, homo-Lys, homo-Ser and homo-Thr, provided that Bxx is not in the (D) configuration, When Bxx is not Ama, Cxx is a single covalent bond; when Bxx is Ama, Cxx is Pro or an N-methyl amino acid, the N-terminus of Cxx is bonded to the carboxyl group of Ama, and the C-terminus of Cxx is covalently bonded to one moiety D; In formula (16), By is an amino acid selected from Phe, homo-Phe, Ala, Trp, Tyr, Phg, Val, His, Lys, Abu, Met, Cit, Orn, Ser, Thr, Leu, Ile, Arg, homo-Phe, beta-Phe, beta-homo-Phe, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1), and R1 is -(CH2CHO) n1 -R2, wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24; provided that when (q1) × (q3) > 1 and q2 = 0, only the C-terminal By may be an amino acid selected from beta-Phe and beta-homo-Phe; Bxx1 in formula (16) is a single covalent bond or an amino acid having a hydrophobic or basic side chain; Bxx2 in formula (16) is an amino acid having a hydrophobic or basic side chain, Bxx3 in formula (16) is an amino acid, provided that Bxx3 is not in the (D) configuration, Bxx4 in formula (16) is Phe, homo-Phe, Ala, Trp, Tyr, Phg, Val, His, Lys, Abu, Met, Cit, Orn, Ser, Thr, Leu, Ile, Arg, homo-Phe, beta-Phe, beta-homo-Phe, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1) (wherein R1 is -(CH2CHO) n1 -R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24; provided that when (q2) × (q3) > 1, only the C-terminal Bxx4 may be an amino acid selected from beta-Phe and beta-homo-Phe, S' is a divalent group containing one or more atoms selected from C, N, O, P, and S, preferably 1 to 40 atoms, more preferably 1 to 20 atoms; preferably, S' is a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound, and derivatives thereof, more preferably a divalent group derived from maleimide and derivatives thereof, such as ring-opened hydrolyzed maleimide derivatives; Z' is a group covalently bonded to the C-terminus of Byy or Bxx4 in formula (14) and (16), or to the C-terminus of Bxx or Bxx3 in formula (15), and is selected from -OH and -N(H)(R) (wherein R represents a hydrogen atom, an alkyl group, or a cycloalkyl group); q1 is an integer of 0 to 5, q2 is an integer of 0 to 3, provided that when q1 is 0, q2 is not 0; q3 is an integer from 1 to 5, q1, q2, and q3 are selected so that m in formula (1) is an integer of 1 to 5; * indicates covalent binding to an anti-BCAM antibody (Ab), and ** represents a covalent bond to one moiety D).

[0178] In formulas (14), (15) and (16), at least one of Bxx, Byy, Bxx1, Bxx2, Bxx3 and Bxx4, for example, one, two, three, four, five or six, has the following definition: (a) Bxx is an amino acid selected from Dap, Dab, Lys, Orn, Ser, Glu, Ama, Thr, Tyr, Aaa, homo-Ser and homo-Thr, preferably an amino acid selected from Lys and Dab, more preferably Lys; (b) By includes Cit, Phe, homo-Phe, Ser, Trp, Tyr, and Tyr(OR1), where R1 is -(CH2CH2O) n1 -R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), preferably an amino acid selected from Phe, Tyr, and Tyr(OR1), and when (q1) × (q3) > 1, By preferably represents Tyr or Tyr(OR1); (c) Bxx1 is a single covalent bond or an amino acid selected from Phe, homo-Phe, Phg, Val, Ser, Tyr, Ala, Leu, and Ile, preferably an amino acid selected from Phe, homo-Phe, Tyr, and Val, more preferably an amino acid selected from Phe, homo-Phe, and Tyr; (d) Bxx2 is an amino acid selected from Arg, Lys, Cit, Val, Leu, Ser, Ala, Gly, His, Gln, Phg and Phe, preferably an amino acid selected from Arg, Lys, Cit and Phe, more preferably an amino acid selected from Arg and Cit; (e) Bxx3 is an amino acid selected from Phe, homo-Phe, Phg, Val, Ser, Tyr, Ala, Leu, and Ile, preferably an amino acid selected from Phe, homo-Phe, Tyr, and Val, more preferably an amino acid selected from Phe, homo-Phe, and Tyr; (f) Bxx4 is Cit, Phe, homo-Phe, Ser, Trp, Tyr, and Tyr(OR1), where R1 is -(CH2CH2O) n1-R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), preferably an amino acid selected from Phe, Tyr, and Tyr(OR1), and when (q1) × (q3) > 1, By preferably represents Tyr or Tyr(OR1); It is preferable that the following is satisfied.

[0179] (3) Divalent group Y The conjugate of formula (1) has a divalent group Y containing one or more atoms selected from C, N, O, P, and S. The divalent group links a BCAM antibody or antigen-binding fragment thereof (Ab) to a branched group T. The divalent group is usually attached to the side chain of an amino acid contained in the Ab, such as Cys. Preferably, Y is a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound, and derivatives thereof. More preferably, Y is a divalent group derived from maleimide and its derivatives, such as ring-opened hydrolyzed maleimide derivatives. Most preferably, Y is a divalent group derived from ring-opened hydrolyzed maleimide.

[0180] Hydrolysis of the maleimide bond is usually carried out under basic conditions as the final step in the conjugation of the maleimide derivative to the Ab, for example, under the conditions described above.

[0181] In some instances, when n in a conjugate of formula (1) is greater than 1, the conjugate may comprise a mixture of (ring-closed) maleimide derivatives (Y) and ring-opened hydrolyzed maleimide derivatives (Y) attached to Ab. Thus, in conjugates described herein in which the group R is attached to the moiety Ab via a maleimide (shown below, on the left), when n is greater than 1, hydrolysis may be performed such that the conjugate of the invention may comprise both a ring-closed maleimide bond (A) and a ring-opened hydrolyzed maleimide bond (B) (shown below, on the left) to the Ab. [ka]

[0182] In conjugates of formula (1) where n is greater than 1, it is preferred that at least 50% of the Y bonds to Ab are ring-opening hydrolyzable maleimide bonds (B), with the remaining bonds being ring-closing maleimide bonds (A). In some examples, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, and preferably at least 98% of the Y bonds to Ab are ring-opening hydrolyzable maleimide bonds (B).

[0183] The presence of one or more ring-opening hydrolyzable maleimide bonds (even when present together with one or more ring-closing maleimide bonds) may contribute to the stability and therapeutic efficacy of the conjugates of the present invention. Without being bound by any theory, it is believed that the ring-opening hydrolyzable maleimide bond may, for example, prevent a retro-Michael reaction that would cause the release of reactive maleimides in the circulation and ultimately transfer the linker-payload to other thiol-containing molecules in the body, such as albumin. The ring-opening maleimide may also cooperate with the divalent group X and the solubilizing moiety S to achieve improved stability and therapeutic efficacy.

[0184] In a preferred embodiment, Y is a divalent group derived from maleimide and its derivatives, such as ring-opened hydrolyzed maleimide, and is preferably represented by the following formulae (26a) to (26c): [ka] (In the formula, R 3 is -(CH2) n9 -(C=A) n11 -or-(CH2CH2O) n10 -(C=A) n11 -, preferably -(CH2) n9 -(C=A) n11 - represents n9 is 1 to 6, preferably 1 or 2, more preferably 1; n10 is 1 to 6, preferably 1; n11 is 0 or 1, preferably 1, and A is O or S, preferably O; The methylene carbon atom is covalently bonded to the nitrogen atom of formulas (26a)-(26c) and the carbonyl or thiocarbonyl carbon is covalently bonded to T; β indicates covalent binding to Ab; and α' represents a covalent bond to T).

[0185] In a more preferred embodiment, Y is represented by formula (26b) or (26c), wherein R 3 is preferably of the formula -(CH2) n9 -(C=A) n11 -, wherein n9 is 1 or 2, n11 is 1, and A is O. Most preferably, R 3 is -CH2-C=O-.

[0186] (4) Antitumor compounds In formula (1), D is an antitumor compound (the drug moiety in the antibody-drug conjugate), which is conjugated to the anti-BCAM antibody via a linker. The antitumor compound is a compound that has an antitumor effect and has a substituent or partial structure that can be bound to the linker. When part or all of the linker is cleaved in tumor cells, the antitumor compound is released and exerts its antitumor effect. Because the linker is cleaved at the position where it is linked to the drug, the antitumor compound is released in its unmodified structure and can exert its inherent antitumor effect.

[0187] In formula (1), n ​​may be 1 to 10. In another embodiment, n may be 3 to 8. In one embodiment, n may be about 4. In another embodiment, n may be 8. In one embodiment, n may be 3 to 5. In one embodiment, n may be 7 to 9. Furthermore, in formula (1), m may be 1 to 5. In one embodiment, m may be 1.

[0188] The drug (anti-tumor compound) to antibody (anti-BCAM antibody) ratio (DAR) in an antibody-drug conjugate can be calculated by multiplying n by m in Equation (1). However, it is understood that when used to describe a sample containing many molecules, the drug-antibody ratio is often an average value due to the certain heterogeneity typically associated with the conjugation process. The average drug-antibody ratio may be, for example, in the range of about 1 to about 10, and may be about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10. In some embodiments, the DAR may be about 3 to about 8, and typically about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, or 9. In some embodiments, the DAR may be about 4. In some embodiments, the DAR may be about 8. In some embodiments, at least 50% by weight of the sample may be compounds having an average DAR of ± 2, and preferably at least 50% of the sample may be conjugates with an average DAR of ± 1. In some embodiments, a DAR of "about n" means that the measured DAR is within ± 20% of n (e.g., 80% of n to 120% of n).

[0189] In one embodiment, antitumor compounds may include tubulin filament-targeting toxins, DNA-targeting toxins, RNA-targeting toxins, nanocarriers, protein toxins, and the like. Tubulin filament-targeting toxins may include, for example, auristatins, maytansinoids, and taxoids. Auristatins are synthetic antitumor agents derived from the natural product dolastatin 10 and are 100 to 1000 times more toxic than the conventional cancer chemotherapy drug doxorubicin. Because dolastatin 10 is a nonspecific toxic substance, it is not used as an antitumor compound in the design of antibody-drug conjugates. However, synthetic analogs of this class of drugs, such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF or AF), can be used in antibody-drug conjugates as antitumor compounds. In particular, monomethyl auristatin E (MMAE) is an antimitotic agent that exerts its effects by blocking the tubulin polymerization process, causing cell cycle arrest and apoptosis. The main functions of monomethyl auristatin F (MMAF or AF) are the same as those of monomethyl auristatin E (MMAE), but MMAF is more hydrophilic, has a lower tendency to aggregate, and has lower systemic toxicity than MMAE.

[0190] Maytansinoids are microtubule-disrupting agents derived from the benzoansamacrolide maytansine. They inhibit tubulin polymerization, leading to mitotic arrest and cell death. Examples of maytansinoids include ansamitocin, mertansine / emtansine (DM1), and ravtansine / soravtansine (DM4).

[0191] Taxoids are antitumor drugs that promote microtubule assembly and inhibit disassembly, including, for example, paclitaxel (taxol) and docetaxel (taxotere).

[0192] DNA-targeting toxins can include compounds that modify DNA bases, intercalate between bases, or form crosslinks in DNA, such as the DNA alkylating agents cyclophosphamide, melphalan, and chlorambucil. DNA-targeting toxins can also include antimetabolites that mimic normal cellular molecules and interfere with DNA replication. These agents are typically DNA antagonists that block the nucleotide metabolic pathway, such as the pyrimidine analogs 5-fluorouracil (5-FU), fluoxuridine, gemcitabine, and purine analogs. DNA-targeting toxins can include, for example, calicheamicin, CC-1065 analogs, and duocarmycins.

[0193] Examples of toxins that target RNA include amatoxins.

[0194] Nanocarriers, ranging in size from 1 to 1000 nm, deliver therapeutic agents to disease sites for the treatment of various diseases. They can protect drugs from premature degradation and interaction with the biological environment, promote drug absorption into selected tissues, and / or improve intracellular penetration. Nanocarriers include, for example, liposomal nanocarriers and nonliposomal nanoparticles. Liposomes are small, spherical vesicles composed of one (unilamellar) or multiple (multilamellar) phospholipid bilayers, enclosing an aqueous interior space. Liposomes have the ability to retain hydrophilic macromolecules within their aqueous core and lipophilic macromolecules within their lipid membrane, making them suitable carriers for a wide range of drugs. Their self-assembly ability, biocompatibility, and large carrying capacity all contribute to their popularity as nanomedicines. Nanoparticles can also be fabricated from a wide range of biological and synthetic materials and can form a diverse array of structures. Such nanoparticles can include polymeric nanoparticles, which are solid particles, or particle dispersions, which can take the form of nanocapsules or nanospheres. Nanospheres are spherical polymer matrices in which therapeutic antitumor compounds are evenly distributed throughout, while nanocapsules encapsulate therapeutic cargo within a polymer membrane. Such nanocarriers, containing one or more of various types of antitumor compounds, can be used as the antitumor compounds in antibody-drug conjugates.

[0195] Anti-BCAM antibody-conjugated protein toxins may also be used as anti-tumor compounds in antibody-drug conjugates. These are produced by bacteria or plants and can consist of two parts: one that targets the cell surface and the other that enters the cytosol and inhibits protein synthesis. Bacterial toxins include, for example, Shiga toxin, Shiga-like toxin, Pseudomonas exotoxin, diphtheria toxin, and cholera toxin. Plant toxins include, for example, ricin, modeccin, abrin, volkensin, and viscumin.

[0196] In one embodiment, the anti-tumor compound (D) may be selected from a DNA alkylating agent, a topoisomerase inhibitor, an RNA polymerase II inhibitor, a DNA cleaving agent, an antimitotic or microtubule-disrupting agent, an antimetabolite, a kinesin spindle protein inhibitor, a kinase inhibitor, a nicotinamide phosphoribosyltransferase inhibitor, a matrix metallopeptidase 9 inhibitor, a phosphatase inhibitor, or a radioactive isotope, and / or a pharmaceutically acceptable salt thereof; when more than one D is present, each D is independently selected from the foregoing compounds.

[0197] In one embodiment, the antitumor compound may be a cytotoxic agent, including: (i) alkylating agents, such as aziridines (e.g., diazicon, mitomycin, and thiotepa), nitrogen mustards (e.g., mannomustine, mustine [mechlorethamine], aniline mustard, bendamustine, benzoic acid mustard, chlorambucil, C6-galactose mustard, melphalan, ocyclolin [nitromin], prednistine, uramustine, nitrogen mustard carbamates [e.g., estramustine], and oxazaphosphoryl). cyclophosphamide, ifosfamide, mafosfamide, and trofosfamide]), nitrosoureas (e.g., carmustine, fotemustine, lomustine, nimustine, N-nitroso-N-methylurea, ranimustine, semustine, and streptozotocin), platinum-containing compounds (e.g., cisplatin, carboplatin, and oxaliplatin), alkylsulfonates (e.g., busulfan, mannosulfan, and treosulfan), hydrazines (e.g., dacarbazine and procarbazine), imidazotetrazines (e.g., mitoxantrazine, thiazolinone ... zolomide and temozolomide), and triazines (e.g., hexamethylmelamine [altretamine]); (ii) cytotoxic antibiotics, such as anthracyclines (e.g., aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pirarubicin, and barbican), actinomycins (e.g., actinomycin D), bleomycins (e.g., bleomycin A2 and B2), mitomycins (e.g., mitomycin C), and plicamycin; (iii) antimetabolites, such as antifolates (e.g., , aminopterin, methotrexate, and pemetrexed), deoxynucleoside analogs (e.g., 5-azacytidine [azacytidine], 5-aza-2'-deoxycytidine [decitabine], cladribine, clofarabine, cytarabine, decitabine, fludarabine, gemcitabine, nelarabine, and pentostatin), fluoropyrimidines (e.g., 5-fluorouracil, 5-fluoro-5'-deoxyuridine [doxifluridine], and capecitabine), and thiopurines (e.g., thioguanine, azathioprine, and mercaptopurine);(iv) anti-microtubule agents, such as dolastatins (e.g., dolastatin 15), epothilones (e.g., epothilones A-F), taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinflunine, and vinorelbine), colchicine, nocodazole, podophyllotoxin, and rhizoxin; (v) histone deacetylase inhibitors, such as trichostatins (e.g., trichostatin A), romidepsin, and vorinostat; (vi) kinase inhibitors, such as curcumin, cysteine, and cysteine. clocreatine, deguelin, fostriecin, hispidin, tyrphostins (e.g., tyrphostins AG34 and AG879), bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib; (vii) topoisomerase I inhibitors, e.g., SN-38, exatecan, camptothecin, irinotecan, and topotecan; (viii) topoisomerase II targeting agents, e.g., topoisomerase II poisons (e.g., etoposide, tafluposide, teniposide, doxorubicin, and mitoxantrone) and topoisomerase I (ix) DNA or RNA synthesis inhibitors, such as 3-amino-1,2,4-benzotriazine-1,4-dioxide, cytosine β-D-arabinofuranoside, 5,6-dichlorobenzimidazole-1-β-D-ribofuranoside, ganciclovir, and hydroxyurea; (x) protein synthesis inhibitors, such as homoharringtonine; (xi) cell growth and differentiation regulators, such as retinoids (e.g., all-trans retinol [vitamin A], 11-cis retinol, o-to trans-retinal [vitamin A aldehyde], 11-cis-retinal, all-trans-retinoic acid [tretinoin], 9-cis-retinoic acid [alitretinoin], 11-cis-retinoic acid, 13-cis-retinoic acid [isotretinoin], all-trans-retinyl esters, etretinate, acitretin, adapalene, bexarotene, and tazarotene; (xii) cell proliferation inhibitors, such as mTOR inhibitors (e.g., rapamycin [sirolimus]), apigenin, cholecalciferol (vitamin D3), and sex hormone-binding globulin;(xiii) apoptosis inducers, such as 17-allylamino-17-demethoxygeldanamycin, melatonin, mevinolin, psoralens, thapsigargin, and troglitazone; (xiv) alkaloids, such as maytansinoids (e.g., DM1, DM2, DM3, DM4, maytansine, and ansamitocin), cryptophycins (e.g., cryptophycin 1 and cryptophycin 8), eleutherobin, discodermolide, bryostatin, auristatin (e.g., monomethylauristatin E, monomethylauristatin B, monomethylauristatin C, monomethylauristatin D, monomethylauristatin E, monomethylauristatin E, monomethylauristatin E, monomethylauristatin E, monomethylauristatin B ... Listatin F), tubulysin, cephalostatin, pancratistatin, sarcodictin; spongistatin; demecolcine; epipodophyllins (e.g., 9-aminocamptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate, metabolites of irinotecan such as SN-38, mitoxantrone, novantrone, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS2000)), mitomycins (e.g., mitomycin C); and their analogs, derivatives, and salts.

[0198] In another embodiment, the anti-tumor compound may be an agent that stimulates the immune system, including, but not limited to: (i) agonists / activators of tumor necrosis factor receptor superfamily member 4 (TNFRSF4, OX40, or CD134); (ii) agonists / activators of TNFRSF member 5 (TNFRSF5 or CD40); (iii) agonists / activators of TNFRSF member 9 (TNFRSF9, 4-1BB, or CD137); (iv) agonists / activators of TNFRSF member 18 (TNFRSF18, glucocorticoid-inducible TNFR-related protein [GITR], or CD357); (v) agonists / activators of Toll-like receptors (TLRs); and analogs, derivatives, fragments, and salts thereof.

[0199] In another embodiment, the anti-tumor compound may be an agent that inhibits an immune checkpoint, including: (i) an inhibitor of the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) receptor or its ligand; (ii) an inhibitor of the killer cell immunoglobulin-like receptor (KIR) or its ligand; (iii) an inhibitor of the lymphocyte activation gene 3 (LAG-3) receptor or its ligand; (iv) an inhibitor of indoleamine 2,3-dioxygenase (IDO or IDO1), such as indoximod (1 -methyl-D-tryptophan or NLG-8189), NLG-919, INCB024360, α-methyl-tryptophan, β-carbolines (9H-pyrido[3,4-b]indole or norharman), and cyclooxygenase-2 (COX-2) inhibitors (e.g., coxibs [such as apricoxib, celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, and valdecoxib] that downregulate IDO expression); and analogs, derivatives, fragments, and salts thereof.

[0200] In another embodiment, the anti-tumor compound may be an angiogenesis inhibitor, including inhibitors of vascular endothelial growth factor (VEGF) (e.g., squalamine) or its receptor (VEGFR) (e.g., axitinib, pazopanib, sorafenib, and sunitinib), inhibitors of platelet-derived growth factor (PDGF) (e.g., squalamine) or its receptor (PDGFR) (e.g., axitinib, pazopanib, sorafenib, and sunitinib), inhibitors of fibroblast growth factor (FGF) (e.g., squalamine) or its receptor (FGFR). inhibitors of angiopoietin or its receptor, inhibitors of integrins (e.g., ALG-1001 and JSM-6427), anecortave (anecortave acetate), angiostatins (e.g., angiostatin K1-3), αVβ3 inhibitors (e.g., etaracizumab), berberine, bleomycin, borrelidin, carboxyamidotriazole, cartilage-derived angiogenesis inhibitors (e.g., chondromodulin I and troponin I), castanospermine, CM101, cyclopropene fatty acids (e.g., sterculic acid), α-difluoromethylornithine steroids, endostatin, everolimus, fumagillin, genistein, interferon-α, interleukin-12, itraconazole, linomide, matrix metalloproteinase (MMP) inhibitors (e.g., batimastat, cipemastat, ilomastat, marimastat, prinomastat, levimastat, tanomastat, and tetracyclines [e.g., doxycycline, incyclinide, and minocycline]), 2-methoxyestradiol, pigment epithelium-derived factor (PEDF), platelet factor-4, PPAR-γ agonists (e.g., Thiazolidinediones (e.g., ciglitazone, lobeglitazone, netoglitazone, pioglitazone, rivoglitazone, rosiglitazone, and troglitazone), prolactin, sphingosine-1-phosphate inhibitors, squalene, staurosporine, antiangiogenic steroids (e.g., tetrahydrocortisol), as well as heparin, stilbenoids, suramin, SU5416, tasquinimod, tecogalan, tetrathiomolybdate, thalidomide and its derivatives (e.g., lenalidomide and pomalidomide), thiabendazole, thrombospondins (e.g.,Thrombospondin 1), TNP-470, tranilast, withaferin A, and their analogs, derivatives, fragments, and salts.

[0201] In another embodiment, the anti-tumor compound may be a drug including (i) drug efflux pump inhibitors, such as P-glycoprotein inhibitors (e.g., mifepristone and verapamil); (ii) cell adhesion inhibitors, such as cimetidine; (iii) Golgi apparatus disrupting agents, such as brefeldins (e.g., brefeldin A); (iv) ionizing radiation, such as X-rays; (v) radiosensitizers of tumor cells, such as poly(ADP-ribose) polymerase (PARP) inhibitors (e.g., 4-amino-1,8-naphthalimide), berberine, and indomethacin; (vi) promoters of cell survival after treatment with cytotoxic drugs or radiation, such as pifithrin-α; (vii) vaccines, such as those that recognize proteins produced by tumor cells and thereby stimulate the immune system to attack tumor cells; and analogs, derivatives, and salts thereof.

[0202] Drugs as used herein may also contain radioactive isotopes as substitutions for atoms contained therein. Examples of radioactive isotopes (radionuclides) include, for example: 3 H, 11 C. 14 C. 18 F, 32 P, 35 S, 64 Cu, 68 Ga, 86 Y, 99 Tc, 111 In, 123 I, 124 I, 125 I, 131 I, 177 Lu, 186 Re, 188 Re, 211 At, 212 Bi, 213 Bi or 225Ac. Radioisotope-labeled drugs can be used in targeted imaging experiments or targeted therapy (Wu et al., Nat. Biotech. 2005, 23, 1137-1146). Imaging can be performed by known computed tomography techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT); for reviews of these techniques and applications, see, for example, Shankar Vallabhajosula (ed.), Molecular Imaging, Radiopharmaceuticals for PET and SPECT, Springer Verlag, or Lucia Martiniova et al., Gallium-68 in Medical Imaging, Current Radiopharmaceuticals, 2016, 9, 187-207. Thus, in some embodiments, the conjugates of the present disclosure can be used to diagnose the progression and / or status of cancer.

[0203] In one embodiment, the antitumor compound (D) is selected from a DNA alkylating agent, a topoisomerase inhibitor, an RNA polymerase II inhibitor, a DNA cleaving agent, an antimitotic or microtubule-disrupting agent, an antimetabolite, a kinesin spindle protein inhibitor, a kinase inhibitor, a nicotinamide phosphoribosyltransferase inhibitor, a matrix metallopeptidase 9 inhibitor, a phosphatase inhibitor, or a radioisotope, and / or a pharmaceutically acceptable salt thereof; when more than one D is present, each D is independently selected from the foregoing compounds. Nevertheless, when more than one (D) is present in a compound of formula (I) (n>1 and / or m>1), it is preferred that the multiple moieties (D) are identical to one another.

[0204] In a preferred embodiment, the antitumor compound (D) is selected from amanitin, duocarmycin, auristatin, auristatin F (AF), monomethylauristatin F (MMAF), maytansine, mertansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepines (PBDs) and dimers thereof, indilinobenzodiazepines (IBDs) and dimers thereof, or radioisotopes, and / or pharmaceutically acceptable salts thereof; when more than one (D) is present, each (D) is independently selected from the foregoing compounds. Most preferably, the moieties (D) are identical to each other.

[0205] More preferably, the antitumor compound (D) is selected from auristatin, MMAF, exatecan, maytansine, DM1, and DM4; even more preferably, the antitumor compound is selected from auristatin and DM1. When more than one (D) is present, each (D) is independently selected from the foregoing compounds. Most preferably, the moieties (D) are identical to one another.

[0206] (5) Exemplary Antibody-Drug Conjugates In one embodiment, the antibody-drug conjugate may have the structure shown in Figure 4a (Ab-linker-AF), where Ab is prepared as described in Example 1 and conjugated to AF. Furthermore, n may be 1 to 10. In one embodiment, n may be about 4. In another embodiment, n may be about 8.

[0207] In another embodiment, the antibody-drug conjugate may have the structure shown in Figure 4b (Ab-linker-MMAF), where Ab is prepared as described in Example 1 and conjugated to MMAF. Furthermore, n may be 1 to 10. In one embodiment, n may be about 4. In another embodiment, n may be about 8.

[0208] In yet another embodiment, the antibody-drug conjugate may have the structure shown in Figure 5 (Ab-linker-DM1), where Ab is prepared as described in Example 1 and conjugated to DM1. Furthermore, n may be 1 to 10. In one embodiment, n may be about 4. In another embodiment, n may be about 8.

[0209] (6) Preferred antibody-drug conjugates In a preferred embodiment, the antibody-drug conjugate of the present disclosure has the formula (17), or the formula (18): [ka] (wherein Axx is an amino acid selected from Glu, Apa, Aaa, Dap, Dab, Lys, Orn, Ser, Ama, and homo-Lys, provided that Axx in formula (17) is not in the (D) configuration, In formula (17), Ayy is an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1), wherein R1 is -(CH2CHO) n1 -R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24; Ayy in formula (18) is an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, and Ser, provided that Ayy in formula (18) is not in the (D) configuration, Dxx is an amino acid with a single covalent bond or a hydrophobic side chain, Dyy represents a single covalent bond, Phe, or an amino acid having a basic side chain, provided that when Dxx is an amino acid having a hydrophobic side chain, Dyy is an amino acid having a Phe or a basic side chain, and when Dxx is a single covalent bond, Dyy is an amino acid having a single covalent bond, Phe, or a basic side chain; Y is a divalent group containing one or more atoms selected from C, N, O, P, and S, preferably a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound, and derivatives thereof, more preferably a divalent group derived from maleimide and its derivatives, such as ring-opened hydrolyzed maleimide derivatives; T is a (2+m)-valent linking group; if S is absent, T is a (1+m)-valent linking group; S is an atom or group optionally present to saturate the free valences of T; Z represents a group covalently attached to the C-terminus of Ayy or Axx selected from -OH and -N(H)(R), where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; and Ab, D, m and n are as defined in formula (1).

[0210] In formulas (17) and (18), at least one of Axx, Ayy, Dxx, Dyy, D, Z, m, and T, for example, 1, 2, 3, 4, 5, 6, 7, or 8, is selected from the following definitions: (a) Axx is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; (b) Ayy in formula (17) is an amino acid selected from Phe, homo-Phe, Tyr, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1); (c) Ayy in formula (18) is an amino acid selected from Phe, homo-Phe, or Ser; (d) Dxx is a moiety derived from an amino acid selected from Phe, Val, Tyr, homo-Phe, and Ala; (e) Dyy is a covalent bond or a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap, and Dab; (f) D is an antitumor compound selected from AF, MMAF, exatecan, maytansine, DM1 and DM4, preferably an antitumor compound selected from auristatin and DM1; (g) Z is —OH or —NH; (h) T is a compound represented by the formula (9'): [ka] wherein each AA is independently a moiety containing a trifunctional amino acid; α represents a covalent bond to Y; m is as defined in item 1; when m is 1, the side chain of the trifunctional amino acid is covalently attached to S or Axx and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; When m is 2, 3, 4 or 5, **** indicates a covalent bond to Axx, *** ' denotes a covalent bond to S via the C-terminus of the chain of AA groups); (i) m is 2 and T is a group represented by the formula (10'): [ka] (In the formula, each AA 1 and A.A. 2 are independently a moiety that contains a trifunctional amino acid; α represents a covalent bond to Y; **** indicates a covalent bond to Axx, *** ' indicates a covalent bond to S); (j) m is 1 and T is the formula (11'): [ka] (In the formula, AA 1 is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; The side chain of the trifunctional amino acid is covalently attached to Axx or S, and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; (k) m is 1, and T is a formula (12') or a formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer of 0 to 5, preferably 0, 1, 2, or 3, more preferably 0 or 1, and most preferably 0; n3 is an integer of 1 to 50, preferably 2 to 24, more preferably 4 to 12, for example, 4 to 8, n4 is an integer of 1 to 50, preferably 2 to 20, and more preferably 2 to 12; α represents a covalent bond to Y, and **** indicates a covalent bond to Axx); It is preferable that the following is satisfied.

[0211] In formulas (17) and (18), at least one of Axx, Ayy, Dxx, Dyy, D, Z, m, and T, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9, is selected from the following definitions: (a) Axx is Lys; (b) Ayy in formula (17) is Tyr; (c) Ayy in formula (18) is Phe or Ser; (d) Dxx is Phe or Val; (e) Dyy is Arg or Cit; (f) D is an antitumor compound selected from AF, MMAF, exatecan, maytansine, DM1 and DM4, preferably an antitumor compound selected from auristatin and DM1; (g) Z is —OH or —NH; (h) m is 1 and T is the formula (11'): [ka] (In the formula, AA 1 is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; The side chain of the trifunctional amino acid is covalently attached to Axx or S, and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; (i) m is 1 and T is a formula (12') or a formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer of 0 to 5, preferably 0, 1, 2, or 3, more preferably 0 or 1, and most preferably 0; n3 is an integer of 1 to 50, preferably 2 to 24, more preferably 4 to 12, for example, 4 to 8, n4 is an integer of 1 to 50, preferably 2 to 20, and more preferably 2 to 12; α represents a covalent bond to Y, and **** indicates a covalent bond to Axx); It is more preferable that the following be satisfied.

[0212] In formulas (17) and (18), at least one of Axx, Ayy, Dxx, Dyy, D, Z, m, and T, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9, is selected from the following definitions: (a) Axx is Lys; (b) Ayy in formula (17) is Tyr; (c) Ayy in formula (18) is Phe or Ser; (d) Dxx is Phe or Val; (e) Dyy is Arg or Cit; (f) D is an antitumor compound selected from AF and DM1; (g) Z is —OH or —NH; (h) m is 1 and T is the formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is 0, 1, 2 or 3, preferably 0 or 1, and most preferably 0; n3 is an integer of 2 to 24, preferably 4 to 12, for example, 4 to 8, α represents a covalent bond to Y, and **** indicates a covalent bond to Axx); It is even more preferable that

[0213] In a preferred embodiment, each Dxx-Dyy-Axx-Ayy in formula (17) is selected from the group consisting of Arg-Lys-Phe (wherein Dxx is a covalent bond), Arg-Lys-homoPhe (wherein Dxx is a covalent bond), Arg-Lys-Tyr (wherein Dxx is a covalent bond), Cit-Lys-Phe (wherein Dxx is a covalent bond), Cit-Lys-Tyr (wherein Dxx is a covalent bond), Arg-Lys- Independently selected from s-homoTyr (wherein Dxx is a covalent bond), Cit-Lys-homoTyr (wherein Dxx is a covalent bond), Phe-Cit-Lys-Phe, Phe-Cit-Lys-Tyr, Phe-Arg-Lys-Tyr, Phe-Cit-Lys-homoTyr, Phe-Lys-Lys-Phe, homoPhe-Arg-Lys-Phe, homo-Phe-Cit-Lys-Tyr.

[0214] In another preferred embodiment, each Dxx-Dyy-Ayy-Axx in formula (18) is independently selected from Arg-Phe-Lys (wherein Dxx is a covalent bond), Arg-Ser-Lys (wherein Dxx is a covalent bond), Cit-Phe-Lys (wherein Dxx is a covalent bond), Cit-Ser-Lys (wherein Dxx is a covalent bond), Cit-homoPhe-Lys (wherein Dxx is a covalent bond), Phe-Cit-Phe-Lys, homoPhe-Cit-Phe-Lys, and Phe-Arg-Phe-Lys.

[0215] In formulas (17) and (18), at least one of D, Z, m, and T, for example, one, two, three, or four, has the following definition: (a) D is an antitumor compound selected from AF, MMAF, exatecan, maytansine, DM1, and DM4; (b) Z is —OH or —NH; (c) m is 1 and T is the formula (11'): [ka] (In the formula, AA 1 is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; The side chain of the trifunctional amino acid is covalently attached to Axx or S, and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; (d) m is 1 and T is a formula (12') or a formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer of 0 to 5, preferably 0, 1, 2, or 3, more preferably 0 or 1, and most preferably 0; n3 is an integer of 1 to 50, preferably 2 to 24, more preferably 4 to 12, for example, 4 to 8, n4 is an integer of 1 to 50, preferably 2 to 20, and more preferably 2 to 12; α represents a covalent bond to Y, and **** indicates a covalent bond to Axx); It is even more preferred that

[0216] In a more preferred embodiment, the antibody-drug conjugate of the present disclosure has the following formula: [ka] (wherein Y is a divalent group containing one or more atoms selected from C, N, O, P, and S, preferably a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound, and derivatives thereof, more preferably a divalent group derived from maleimide and its derivatives, for example, a ring-opened hydrolyzed maleimide derivative; T is a (2+m)-valent linking group; if S is absent, T is a (1+m)-valent linking group; S is an atom or group optionally present to saturate the free valences of T; Z represents a group covalently attached to the C-terminus of the amino acid selected from -OH and -N(H)(R), where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; and Ab, D, m and n are as defined in formula (1), with the proviso that Lys is not in the (D) configuration.

[0217] In the above formula, at least one of D, Z, m, and T, for example, one, two, three, or four, has the following definition: (a) D is an antitumor compound selected from AF, MMAF, exatecan, maytansine, DM1, and DM4; (b) Z is —OH or —NH; (c) m is 2 and T is the formula (10'): [ka] (In the formula, each AA 1 and A.A. 2 are independently a moiety that contains a trifunctional amino acid; α represents a covalent bond to Y; **** indicates a covalent bond to Lys, *** ' indicates a covalent bond to S); (d) m is 1 and T is the formula (11'): [ka] (In the formula, AA 1 is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; The side chain of the trifunctional amino acid is covalently attached to Lys or S, and the C-terminus is covalently attached to a moiety different from that attached to the side chain, either S or Lys, respectively; (e) m is 1 and T is a formula (12') or a formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer of 0 to 5, preferably 0, 1, 2, or 3, more preferably 0 or 1, and most preferably 0; n3 is an integer of 1 to 50, preferably 2 to 24, more preferably 4 to 12, for example, 4 to 8, n4 is an integer of 1 to 50, preferably 2 to 20, and more preferably 2 to 12; α represents a covalent bond to Y, and **** indicates a covalent bond to Lys); It is preferable that the following is satisfied.

[0218] In the above formula, D, Z, m and T are more preferably defined as follows: (a) D is DM1; (b) Z is —OH or —NH2, and (c) m is 1 and T is the formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer of 0 to 5, preferably 0, 1, 2, or 3, more preferably 0 or 1, and most preferably 0; n3 is an integer of 1 to 50, preferably 2 to 24, more preferably 4 to 12, for example, 4 to 8, α represents a covalent bond to Y, and **** indicates a covalent bond to Lys).

[0219] In the above formula, D, Z, m and T are most preferably defined as follows: (a) D is an auristatin; (b) Z is —OH, and (c) m is 1 and T is the formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is 0, 1, 2 or 3, preferably 0 or 1, and most preferably 0; n3 is an integer of 2 to 24, preferably 4 to 12, for example, 4 to 8, α represents a covalent bond to Y, and **** indicates a covalent bond to Lys).

[0220] In a preferred embodiment, the antibody-drug conjugate of the present disclosure has the formula (19), (20), (21), or (22): [ka] (In the formula, Axx is a trifunctional amino acid, provided that Axx in formula (19) and formula (20) is not an amino acid of the (D) configuration, Ayy is an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, Orn, Ser, Thr, Leu, and Ile, or Ayy in formula (19) and formula (20) is an amino acid selected from homo-Tyr, homo-Phe, beta-Phe and beta-homo-Phe, Tyr(OR1) and homo-Tyr(OR1), wherein R1 is -(CH2CHO) n1 -R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24, provided that Ayy in formula (21) and formula (22) is not an amino acid of the (D) configuration, Each A''yy is independently an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn, with the proviso that A''yy in formula (20) and formula (22) is not an amino acid in the (D) configuration, Y is a divalent group containing one or more atoms selected from C, N, O, P, and S, preferably a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound, and derivatives thereof, more preferably a divalent group derived from maleimide and its derivatives, such as ring-opened hydrolyzed maleimide derivatives; T is a trivalent linking group; if S is absent, T is a divalent linking group; S is an atom or group optionally present to saturate the free valences of T; Z represents a group covalently bonded to the C-terminus of Ayy in formula (19) and formula (20), or to the C-terminus of Axx in formula (21) or formula (22), and is selected from -OH and -N(H)(R) (wherein R represents a hydrogen atom, an alkyl group, or a cycloalkyl group); D1 is an antitumor compound, m' is (m-1), where m is as defined in item 1, except that m' is not 0; When m' is 1, A''xx is a trifunctional amino acid, provided that A''xx in formula (19) and formula (21) is not an amino acid of the (D) configuration, and D2 is an antitumor compound; When m' is greater than 1, each D2 is independently selected from a hydrogen atom and an anti-tumor compound, and the multiple moieties D2 can be the same or different, provided that at least one D2 is not a hydrogen atom; when D2 is a hydrogen atom, A''xx is an amino acid, provided that A''xx in formula (19) and formula (21) is not an amino acid in the (D) configuration; when D2 is an anti-tumor compound, A''xx is a trifunctional amino acid, provided that A''xx in formula (19) and formula (21) is not an amino acid in the (D) configuration, Ab and n are as defined in formula (1).

[0221] In formula (19), formula (20), formula (21), and formula (22), at least one of Axx, Ayy, A''xx, A''yy, D1, D2, Z, m', and T, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9, is defined as follows: (a) Axx is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; (b) Ayy in formula (19) and formula (20) is an amino acid selected from Phe, homo-Phe, Tyr, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1); (c) A''xx is an amino acid selected from Lys, homo-Lys, Cit, Orn, Dap, and Dab; (d) A''yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (e) each D1 and D2 is independently an antitumor compound selected from AF, MMAF, exatecan, maytansine, DM1 and DM4, preferably an antitumor compound selected from auristatin and DM1; (f) Z is —OH; (g) m' is 1 and T is the formula (11'): [ka] (In the formula, AA 1 is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; The side chain of the trifunctional amino acid is covalently attached to Axx or S, and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; (h) m' is 1 and T is a formula (12') or a formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer of 0 to 5, preferably 0, 1, 2, or 3, more preferably 0 or 1, and most preferably 0; n3 is an integer of 1 to 50, preferably 2 to 24, more preferably 4 to 12, for example, 4 to 8, n4 is an integer of 1 to 50, preferably 2 to 20, and more preferably 2 to 12; α represents a covalent bond to Y, and **** indicates a covalent bond to Axx); Meet the following.

[0222] In formula (19), formula (20), formula (21), and formula (22), at least one of Axx, Ayy, A''xx, A''yy, D1, D2, Z, m', and T, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9, is defined as follows: (a) Axx is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; (b) Ayy in formula (17) is an amino acid selected from Phe, homo-Phe, Tyr, homo-Tyr, Tyr(OR1), and homo-Tyr(OR1); (c) A''xx is an amino acid selected from Lys, homo-Lys, Cit, Orn, Dap, and Dab; (d) A''yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (e) each of D1 and D2 is an auristatin; (f) Z is —OH; (g) m' is 1 and T is the formula (13'): [ka] wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is 0, 1, 2 or 3, preferably 0 or 1, and most preferably 0; n3 is an integer of 2 to 24, preferably 4 to 12, for example, 4 to 8, α represents a covalent bond to Y, and **** indicates a covalent bond to Axx); Meet the following.

[0223] In a more preferred embodiment, the antibody-drug conjugate of the present disclosure comprises the following compound: [ka] and [ka] and [ka] (In the formula, The Ab is an anti-BCAM antibody or antigen-binding fragment thereof comprising: (i) a heavy chain variable region comprising the VH CDR1 sequence of SEQ ID NO: 1, the VH CDR2 sequence of SEQ ID NO: 2, and the VH CDR3 sequence of SEQ ID NO: 3; and (ii) a light chain variable region comprising the VL CDR1 sequence of SEQ ID NO: 4, the VL CDR2 sequence of SEQ ID NO: 5, and the VL CDR3 sequence of SEQ ID NO: 6; and n is an integer of 1 to 10, and preferably an integer of 3 to 8.

[0224] In a preferred embodiment, in the above compound, Ab is an anti-BCAM antibody or an antigen-binding fragment thereof, the heavy chain variable region comprises the sequence of SEQ ID NO:7, and the light chain variable region comprises the sequence of SEQ ID NO:8.

[0225] In one embodiment, in the above compounds, the ring-opened maleimide bond to Ab may be replaced with a ring-closed maleimide bond. In some cases where n is greater than 1, the compound may contain a mixture of (ring-closed) maleimide derivatives and ring-opened hydrolyzed maleimide derivatives attached to Ab, whereby preferably at least 50% of the bonds to Ab are ring-opened hydrolyzed maleimide bonds, e.g., maleimide bonds represented by formula (26b) or (26c).

[0226] 2. Pharmaceutical Compositions Comprising Antibody-Drug Conjugates The present disclosure also provides a pharmaceutical composition comprising an antibody-drug conjugate of the present disclosure and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier can be selected based on the specific anti-tumor compound used, its concentration, stability, and intended bioavailability, the disease, disorder, or condition being treated with the composition, the subject, its age, size, and general condition, and the route of administration. For example, the antibody-drug conjugate of the present disclosure can be mixed with a sterilized liquid (including water and oils (oils derived from petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.)), a solvent such as saline, aqueous dextrose, or aqueous glycerol solution, and additives such as humectants, emulsifiers, or pH buffering agents to prepare a pharmaceutical composition of the present disclosure. Pharmaceutically acceptable carriers for solid dosage forms can include sugars, starches, and other conventional materials, including polysorbates, histidine, lactose, talc, sucrose, gelatin, carboxymethylcellulose, agar, mannitol, sorbitol, calcium phosphate, calcium carbonate, sodium carbonate, kaolin, alginic acid, acacia, corn starch, potato starch, sodium saccharin, magnesium carbonate, tragacanth, microcrystalline cellulose, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, and stearic acid. Additionally, such solid dosage forms can be uncoated or can be coated (e.g., to delay disintegration or absorption) by known techniques. Additionally, pharmaceutically acceptable carriers used in formulating liquid dosage forms for oral or parenteral administration include, for example, non-aqueous pharmaceutically acceptable polar solvents such as oils, alcohols, amides, esters, ethers, ketones, hydrocarbons, and mixtures thereof, as well as water, saline, dextrose solution, electrolyte solutions, or any other aqueous pharmaceutically acceptable liquid.

[0227] In one embodiment, the pharmaceutical composition is for treating cancer. The cancer may be a solid tumor (e.g., a cancer of epithelial origin) or a liquid tumor.

[0228] In one embodiment, the cancer may be one or more selected from the group consisting of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, renal cancer, esophageal cancer, leukemia, hepatocellular carcinoma, renal cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer, and urothelial cell carcinoma.

[0229] 3. Compound of formula (25), kit for modifying anti-BCAM antibody, and method for modifying anti-BCAM antibody In some aspects, the present disclosure relates to compounds that can be used to modify vector molecules, such as antibodies, that can interact with target cells as described above. Thus, the present disclosure relates to compounds of formula (25): [ka] wherein D and m have the same meanings as above for compounds of formula (1), and L 1 is expressed as equation (26): [ka] is a linker represented by L', T, S, o and ** has the same meaning as above for compounds of formula (I), and and Y' represents a moiety containing a conjugation group capable of forming a covalent bond to an anti-BCAM antibody or antigen-binding fragment thereof, or a pharmaceutically acceptable salt thereof.

[0230] In one embodiment, Y' is a moiety comprising a conjugation group selected from: an optionally substituted maleimide, preferably one capable of reacting with one or two thiol groups contained in an anti-BCAM antibody or antigen-binding fragment thereof; an optionally substituted haloacetamide, preferably one capable of reacting with a thiol group contained in an anti-BCAM antibody or antigen-binding fragment thereof; an ester, preferably one capable of reacting with a side chain of an amino acid contained in an anti-BCAM antibody or antigen-binding fragment thereof, such as an acyl halide, an N-hydroxysuccinimide ester (structure shown on the left below, where σ represents a covalent bond to the remainder of a compound of formula (25)), or a phenolic ester (structure shown on the right below, where σ represents a covalent bond to the remainder of a compound of formula (25) and each R is independently selected from H, F, NO, and CN). [ka] - carbonates, preferably those capable of reacting with the side chains of amino acids contained in the anti-BCAM antibody or antigen-binding fragment thereof, such as haloformates, or carbonates containing leaving groups, such as N-hydroxysuccinimide or phenol derivatives; an isocyanate or isothiocyanate, preferably one capable of reacting with the side chains of amino acids contained in the anti-BCAM antibody or antigen-binding fragment thereof; an azide, preferably one capable of reacting with an alkyne group contained in an anti-BCAM antibody or antigen-binding fragment thereof (in this case, the anti-BCAM antibody or antigen-binding fragment thereof can be modified by antibody engineering or other conjugation techniques to introduce an alkyne functional group), an alkyne, preferably one that can react with an azide group contained in an anti-BCAM antibody or antigen-binding fragment thereof (in this case, the anti-BCAM antibody or antigen-binding fragment thereof can be modified by antibody engineering or other conjugation techniques to introduce an azide functional group), and an amino group, for example a primary or secondary amino group, preferably one that can react with an anti-BCAM antibody or antigen-binding fragment thereof in the presence of an enzyme such as transglutaminase.

[0231] In one embodiment, the present disclosure therefore provides a compound of formula (25): [ka] wherein D and m have the meanings given above for compounds of formula (1), and L 2 is expressed by equation (27), equation (28), or equation (29): [ka] is expressed as In the formula, Bxx, Byy, Cxx, Bxx1, Bxx2, Bxx3, Bxx4, q1, q2, q3, Z' and ** has the same meaning as above for compounds of formula (I), and Y' has the same meaning as above for the compound of formula (25) or a pharmaceutically acceptable salt thereof.

[0232] In a preferred embodiment, the compound has formula (30) or formula (31): [ka] (In the formula, D, Dxx, Dyy, Ayy, Axx, T, S, Z, and m have the same meanings as in formulas (17) and (18), Y' has the same meaning as above for the compound of formula (25).

[0233] In a further preferred embodiment, each Dxx-Dyy-Axx-Ayy in formula (30) is selected from the group consisting of Arg-Lys-Phe (wherein Dxx is a covalent bond), Arg-Lys-homoPhe (wherein Dxx is a covalent bond), Arg-Lys-Tyr (wherein Dxx is a covalent bond), Cit-Lys-Phe (wherein Dxx is a covalent bond), Cit-Lys-Tyr (wherein Dxx is a covalent bond), Arg- independently selected from Lys-homoTyr (wherein Dxx is a covalent bond), Cit-Lys-homoTyr (wherein Dxx is a covalent bond), Phe-Cit-Lys-Phe, Phe-Cit-Lys-Tyr, Phe-Arg-Lys-Tyr, Phe-Cit-Lys-homoTyr, Phe-Lys-Lys-Phe, homoPhe-Arg-Lys-Phe, homo-Phe-Cit-Lys-Tyr; Each Dxx-Dyy-Ayy-Axx in formula (31) is independently selected from Arg-Phe-Lys (wherein Dxx is a covalent bond), Arg-Ser-Lys (wherein Dxx is a covalent bond), Cit-Phe-Lys (wherein Dxx is a covalent bond), Cit-Ser-Lys (wherein Dxx is a covalent bond), Cit-homoPhe-Lys (wherein Dxx is a covalent bond), Phe-Cit-Phe-Lys, homoPhe-Cit-Phe-Lys, and Phe-Arg-Phe-Lys.

[0234] In yet another preferred embodiment, the compound has the following formula: [ka] (wherein D, T, S, Y', Z and m have the same meanings as above).

[0235] In some aspects, the present disclosure relates to kits comprising the aforementioned compound (i.e., the compound of formula (25)) and a buffer that can be used to modify an anti-BCAM antibody or antigen-binding fragment thereof.

[0236] The compound and buffer (which together comprise the kit) can be provided individually, for example, in separate primary containers (which may be shipped to the customer in one box) that can be stored for long periods of time without degradation. The compound and buffer can be formulated and fractionated for a given amount of antibody or fragment thereof to be modified. In some embodiments, the compounds of the present disclosure are provided as solids (e.g., as lyophilized powders, or non-covalently adsorbed or covalently bound to a solid phase matrix as described further below) or as solutions in a suitable solvent, such as a water-miscible polar aprotic solvent (e.g., DMF, DMSO), that can be mixed with the buffer immediately prior to modification of the antibody or antibody fragment.

[0237] The buffer solution used in the kit of the present disclosure is not particularly limited. Preferably, the pH of the buffer solution is 6.0 to 10, more preferably 6.5 to 8.0. The buffer solution may be selected from, for example, 2-bis(2-hydroxyethyl)aminoacetic acid (bicine), carbonate-bicarbonate, tris(hydroxylmethyl)methylaminopropanesulfonic acid (TAPS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

[0238] In some aspects, the compounds of the present disclosure can be used in methods for the modification of anti-BCAM antibodies or antigen-binding fragments thereof, thus producing antibody-drug conjugates as described above.

[0239] In one embodiment, the method includes a step of reacting (contacting) an anti-BCAM antibody or its antigen-binding fragment. For example, the reaction can be carried out in a PBS buffer containing 3-10%, preferably 5-10%, DMSO. An activating agent such as tris(2-carboxyethyl)phosphine (TCEP) can be used. The reaction temperature is preferably 5-40°C, preferably 20-40°C. The reaction mixture can be purified using a suitable solvent by techniques known in the art, such as diafiltration or gel permeation chromatography. Examples of suitable stationary phases for isolating purified antibody-drug conjugates include polyacrylamide gels, such as Bio-Gel® P-30, and cross-linked dextran, such as Sorbadex®, Zetadex®, or Sephadex®. The method can be applied to any anti-BCAM antibody or its antigen-binding fragment.

[0240] 4. Preparation of Compounds of the Present Disclosure Below, methods for producing linkers, drug-linkers, and antibody-drug conjugates are provided. The compounds of the present disclosure can be synthesized using standard organic chemistry reactions, including peptide coupling and convergent strategies in solution and on resin, or Fmoc-based solid-phase peptide synthesis (SPPS). The introduction of various maleimide derivatives followed by chemoselective ligation to an anti-BCAM antibody or its antigen-binding fragment is also exemplified below. General strategies and methodologies that can be used to produce the compounds of the present disclosure are well known to those skilled in the art.

[0241] 5. Methods of treating cancer by administering pharmaceutical compositions The present disclosure also provides a method for treating cancer, comprising administering to a subject in need thereof an effective amount of an antibody-drug conjugate of the present disclosure.

[0242] In one embodiment, an effective amount refers to the amount of a therapeutic agent (e.g., an antibody-drug conjugate) that, when administered to a subject suffering from cancer, is sufficient to treat, alleviate, ameliorate, relieve, alleviate symptoms, prevent, delay the onset of, inhibit the progression of, reduce the severity of, and / or reduce the incidence of cancer. For example, the appropriate dosage of an antibody, antigen-binding fragment, or antibody-drug conjugate depends on various factors, such as the type of cancer being treated, the severity and course of the cancer, the responsiveness of the cancer, previous treatments, and the patient's medical history. The pharmaceutical composition can be administered once or over a series of treatments lasting from several days to several months, or until a cure is achieved or a reduction in the cancerous state (e.g., a decrease in tumor size) is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the patient's body and vary depending on the relative potency of the individual antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate. In certain embodiments, the dosage is 0.01 mg to 10 mg (e.g., 0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 7 mg, 8 mg, 9 mg, or 10 mg) per kg of body weight per day, and may be administered one or more times daily, one or more times weekly, one or more times monthly, or one or more times yearly. In certain embodiments, an antibody, antibody fragment (e.g., an antigen-binding fragment), or antibody-drug conjugate of the present disclosure is administered once every two weeks or once every three weeks. The treating physician can estimate the repetition frequency of administration based on the measured residence time and concentration of the drug in bodily fluids or tissues.

[0243] The pharmaceutical agent may be administered to a subject by any method. Such methods are well known to those skilled in the art and include, but are not limited to, oral, transdermal, inhalation, nasal, topical, intravaginal, ophthalmic, intraaural, intracerebral, rectal, sublingual, buccal, and parenteral administration (including injectables such as intravenous, intraarterial, intramuscular, and subcutaneous administration). Administration may be continuous or intermittent. The pharmaceutical composition may be administered therapeutically (e.g., to treat an existing disease or condition) or for imaging. However, the pharmaceutical composition may also be administered prophylactically (e.g., to prevent cancer, such as a blood cancer or solid tumor).

[0244] The pharmaceutical compositions of the present disclosure can also be administered (e.g., simultaneously) with additional therapeutic agents known in the art, such as cytokines, steroids, chemotherapeutic agents, antibiotics, or radiation. Such additional therapeutic agents (e.g., prophylactic or therapeutic agents) that can be administered in combination with the pharmaceutical compositions of the present disclosure can be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, about 1 hour apart, about 1 to about 2 hours apart, about 2 to about 3 hours apart, about 3 to about 4 hours apart, about 4 to about 5 hours apart, about 5 to about 6 hours apart, about 6 to about 7 hours apart, about 7 to about 8 hours apart, or about 8 to about 9 hours apart from the pharmaceutical composition of the present disclosure. The two or more therapeutic agents may be administered about 9 to 10 hours apart, about 10 to 11 hours apart, about 11 to 12 hours apart, about 12 to 18 hours apart, 18 to 24 hours apart, 24 to 36 hours apart, 36 to 48 hours apart, 48 to 52 hours apart, 52 to 60 hours apart, 60 to 72 hours apart, 72 to 84 hours apart, 84 to 96 hours apart, or 96 to 120 hours apart. Two or more therapeutic agents may be administered using the same or different modes of administration. Two or more therapeutic agents may be administered within the same patient visit.

[0245] Example Example 1 Example 1.1 - Preparation of anti-BCAM antibodies Anti-BCAM antibodies Ab1 (human IgG4 S228P isotype), Ab2 (human IgG1 isotype), and Ab3 (human IgG1 LALA isotype) were prepared under the same conditions as described below.

[0246] Ab1, Ab2, and Ab3 each have a heavy chain variable region having the sequence of SEQ ID NO:7 and a light chain variable region having the sequence of SEQ ID NO:8. The heavy chain variable region comprises the VH CDR1 sequence of SEQ ID NO:1, the VH CDR2 sequence of SEQ ID NO:2, and the VH CDR3 sequence of SEQ ID NO:3. The light chain variable region comprises the VL CDR1 sequence of SEQ ID NO:4, the VL CDR2 sequence of SEQ ID NO:5, and the VL CDR3 sequence of SEQ ID NO:6. Ab1 has a heavy chain comprising the sequence of SEQ ID NO:9 and a light chain comprising the sequence of SEQ ID NO:11. Ab2 has a heavy chain comprising the sequence of SEQ ID NO:10 and a light chain comprising the sequence of SEQ ID NO:11. Ab3 has a heavy chain comprising the sequence of SEQ ID NO:12 and a light chain comprising the sequence of SEQ ID NO:11.

[0247] The sequence information referenced herein is as follows: TIFF2025539807000086.tif224170

[0248] To generate anti-BCAM antibodies, their light and heavy chain variable regions were fused to the respective constant regions of human antibodies and cloned into separate expression DNA vectors. The prepared light and heavy chain DNA vectors were transiently transfected into ExpiCHO-S™ cells (ThermoFisher Scientific), which were then cultured in a CO2 incubator to express the monoclonal antibodies using the ExpiCHO® Expression Kit. After 8 days of culture, the supernatant was collected from the culture medium by ultracentrifugation and clarified using depth filtration or microfiltration.

[0249] Each harvested clarified culture medium was purified on Protein A resin using an FPLC system to remove culture components and impurities. Specifically, a column pre-packed with the resin was equilibrated with 50 mM Tris solution, 150 mM HCl, pH 7.5. Each harvested sample was then loaded onto the column and allowed to bind to the Protein A resin. After washing with the equilibration buffer and a high-salt buffer, the purified sample was eluted using a step gradient elution with an elution buffer (100 mM glycine in a low pH buffer). The pH of the eluted sample was then adjusted to 6.5 and subsequently loaded onto a cation exchange chromatography column equilibrated with 20 mM histidine, pH 6.5. A linear salt gradient was applied over 20 column volumes to elute the product with high resolution.

[0250] After purification, the pooled samples were adjusted to a pH range of 7.0–7.5 with 1 M Tris solution and then transferred to a 0.22 μm filter to remove aggregates. Finally, the buffer solution for each product was changed to 1× PBS (Corning, phosphate-buffered saline, pH 7.4) by ultrafiltration and diafiltration using Amicon® ultracentrifugal filters (Millipore). To confirm the concentration of the final formulated product, the A280 / extinction coefficient was analyzed by SoloVPE (Repligen). To confirm the purity of the product, samples were analyzed by size-exclusion HPLC (SE-HPLC) and SDS-PAGE.

[0251] First, the sample was loaded onto a size-exclusion column (TSKgel G3000SWXL 7.8 × 300 mm, TOSOH) and analyzed with a phosphate-chloride buffer. The SE-HPLC chromatograms are shown in Figure 6 ((A): Ab1, (B): Ab2, (C): Ab3). The SDS-PAGE results are shown in Figure 7 ((A): Ab1, (B): Ab2, (C): Ab3). The three lanes of samples and their loading amounts, from left to right, are: M, marker, 5 μL; 1, non-reduced sample, 3 μg; and 2, reduced sample, 3 μg.

[0252] The concentration and purity analysis results of the prepared anti-BCAM antibodies (Ab1, Ab2, Ab3) are summarized below. [Table 1A]

[0253] Example 1.2 - Binding affinity of Ab1 and Ab2 to human BCAM recombinant protein by indirect ELISA The binding affinity of Ab1 and Ab2 to human BCAM recombinant protein was assessed using indirect ELISA (enzyme-linked immunosorbent assay). Human BCAM (SinoBiological, USA) diluted to 25 nM in PBS and 50 μL of the prepared protein were added to a 96-well half-plate (Costar, USA) and incubated overnight at 4°C. The next day, the human BCAM solution was removed, and the plate was washed three times with 150 μL of PBS-T (PBS containing 0.1% Tween-20). The human BCAM-coated plate was blocked with 150 μL of 3% BSA blocking buffer at 37°C for 1 h. The blocking buffer was removed, and 50 μL of Ab1 and Ab2 (6 pM–1 μM, 12-fold serial dilutions) diluted in blocking buffer, as well as HG4K IgG4 (SinoBiological, USA) and HG1K IgG1 (SinoBiological, USA), were added to each well and incubated at 37°C for 2 h. The plate was washed three times with 150 μL of PBS-T, and secondary antibody (HRP-conjugated anti-human IgG Fc antibody; ThermoFisher Scientific, USA) diluted in blocking buffer (1:100,000) was added to each well and incubated at 37°C for 1 hour. The plate was washed three times with 150 μL of PBS-T, and 50 μL of TMB-ELISA substrate solution (ThermoFisher Scientific, USA) was added to each well and incubated at room temperature for 10 minutes. The absorbance (optical density at 450 nm, OD 450) was measured at 450 nm using a Synergy H1 (BioTek, Winooski, VT). Graphs were plotted as sigmoidal dose-response curves, and EC 50 The values ​​were calculated by nonlinear regression with a four-parameter logistic equation using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). Ab1 and Ab2 bound to human BCAM in a dose-dependent manner, and the EC 50 The values ​​were 0.112 nM and 0.078 nM, respectively. See Figure 18 and Table 1B. [Table 1B]

[0254] Example 1.3 – Binding affinity of Ab1 and Ab3 to human BCAM recombinant protein by sandwich ELISA The binding affinity of Ab1 and Ab3 to human BCAM recombinant protein was assessed using a sandwich ELISA (enzyme-linked immunosorbent assay). Anti-His antibody (Biolegend, USA) diluted to 4 μg / mL in PBS was added to a 96-well plate (ThermoFisher Scientific, USA) and incubated overnight at 4°C. The next day, the anti-His antibody solution was removed, and the plate was washed three times with 300 μL of PBS-T (PBS containing 0.1% Tween-20). The anti-His antibody-coated plate was blocked with 300 μL of 3% skim milk blocking buffer at room temperature for 1 hour. Human BCAM (SinoBiological, USA) diluted to 25 nM in blocking buffer and 100 μL of the prepared protein were added to the 96-well plate and incubated at room temperature for 1 hour. The plate was washed three times with 300 μL of PBS-T, and 100 μL of Ab1 and Ab3 (6.4 pM to 500 nM, 5-fold serial dilutions, 8 points) diluted in blocking buffer was added to each well and incubated at room temperature for 1 hour. The plate was washed three times with 300 μL of PBS-T, and secondary antibodies (HRP-conjugated anti-human IgG Fc antibody; Jackson ImmunoResearch, USA) diluted in blocking buffer (1:50,000) were added to each well and incubated at room temperature for 1 hour. The plate was washed three times with 300 μL of PBS-T, and 100 μL of TMB-ELISA substrate solution (ThermoFisher Scientific, USA) was added to each well and incubated at room temperature for 10 minutes. Absorbance (optical density at 450 nm, OD ) was measured. 450 ) was measured at 450 nm using a Synergy H1 (BioTek, Winooski, VT). Graphs were plotted as sigmoidal dose-response curves, and EC 50 The values ​​were calculated by nonlinear regression with a three-parameter logistic equation using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). Ab1 and Ab3 bound to human BCAM in a dose-dependent manner, and the EC 50The values ​​were 1.28 nM and 1.31 nM, respectively. See Figure 19 and Table 1C. [Table 1C]

[0255] Example 1.4 Cell Binding Affinities of Ab1, Ab2, and Ab3 to HEK293FT Cells Overexpressing Human BCAM by Flow Cytometry The binding affinities of Ab1, Ab2, and Ab3 to HEK293FT cells overexpressing human BCAM (HEK293FT / hBCAM) were assessed by flow cytometry. HEK293FT cells (3.0 × 10 6 ) were seeded onto a 10 cm cell culture dish and incubated at 37°C in a 5% CO2 incubator for 24 hours. The next day, jetPRIME transfection reagent (20 μL) was mixed with human BCAM expression plasmid (10 μg) prepared in jetPRIME transfection buffer (200 μL) and incubated at room temperature for 10 minutes. The mixture was added dropwise to HEK293FT cells and incubated at 37°C in a 5% CO2 incubator for 4 hours. After 4 hours, the mixture from the HEK293FT cells was replaced with fresh complete medium and incubated overnight. HEK293FT / hBCAM cells were cultured at 1.0 × 10 in FACS buffer. 5The cells were prepared at a concentration of 100 μL per 100 μL. 100 μL of Ab1, Ab2 (3 pM to 56 nM, serial 3-fold dilutions, 10-point series), and Ab3 (1.524 pM to 30 nM, serial 3-fold dilutions, 10-point series) diluted in FACS buffer, as well as HG4K IgG4 (SinoBiological, USA) and HG1K IgG1 (SinoBiological, USA), were added to the cells and incubated for 30 minutes at 4°C. The cells were washed twice with 200 μL of FACS buffer, and then 1 μg / mL of secondary antibody (AlexaFlour® 647 AffiniPure Goat Anti-Human IgG (H+L), Jackson ImmunoResearch, USA) diluted in FACS buffer was added to the cells and incubated for 30 minutes at 4°C in the dark. After washing twice with 200 μL of FACS buffer, 7-AAD (1:100) diluted in FACS buffer was added to the cells and transferred to FACS tubes for analysis. At least 1.0 × 10 cells per sample were collected using a FACSCanto II (BD, USA). 4 Viable cells were read. Geometric mean fluorescence intensity was measured to assess binding of Ab1, Ab2, and Ab3 to hBCAM on HEK293FT / hBCAM cells. Graphs were plotted as sigmoidal dose-response curves, and EC 50 The values ​​were calculated by nonlinear regression with a four-parameter logistic equation using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). Ab1, Ab2, and Ab3 bound to HEK293FT / hBCAM cells in a dose-dependent manner, and the EC values ​​of Ab1, Ab2, and Ab3 were 50 The values ​​were 0.184 nM, 0.232 nM and 0.167 nM, respectively. See Figures 20a and 20b and Tables 1D and 1E. [Table 1D] [Table 1E]

[0256] Example 1.5 – In vitro cell internalization characteristics of Ab1 on the MKN-1 cell line, which endogenously expresses BCAM The in vitro cellular internalization of Ab1 by receptor-mediated endocytosis in MKN-1 cells, which endogenously express BCAM, was evaluated by immunocytochemical analysis using a confocal laser scanning microscope. MKN-1 (human gastric adenosquamous carcinoma cell line, KCLB) cells were plated at 5.0 × 10 cells per well on a chamber slide (SPL, Korea). 4Cells were seeded and incubated overnight at 37°C and 5% CO2. The next day, the chamber slides were washed with 1 mL of PBS, and 500 μL of Ab1 (40 μg / mL) or HG4K IgG4 (SinoBiological, USA) (40 μg / mL) diluted in PBS was added to the indicated wells. The Ab1-treated chamber slides were divided into two groups: one group was used as a negative control for cellular internalization, and the other group was used to induce cellular internalization. In the negative control group, Ab1-treated cells in the chamber slides were incubated at 4°C for 1 hour, after which unbound antibody was removed and washed three times with 1 mL of PBS. The chamber slides were fixed with 500 μL of 4% PFA (paraformaldehyde; Sigma, USA) solution at room temperature for 10 minutes. For the cell internalization induction group, Ab1-treated chamber slides were incubated under the negative control conditions, then incubated at 4°C for 1 hour to wash off unbound antibody, followed by an additional 1 hour at 37°C. After removing unbound antibody and washing three times with 1 mL of PBS, the chamber slides were fixed with 500 μL of 4% PFA solution for 10 minutes at room temperature. The fixed chamber slides were washed twice with 1 mL of PBS, and the cells were then permeabilized with 1 mL of 0.1% Triton X-100 solution for 15 minutes at room temperature. After washing twice with PBS, the chamber slides were blocked with 1 mL of 3% BSA blocking solution for 20 minutes at room temperature. 500 μL of secondary antibody (10 μg / mL Alexa 488-conjugated goat anti-human IgG; Invitrogen, USA) diluted in blocking buffer was added to each well, and the chamber slides were incubated at 4°C for 1 hour. After washing three times with PBS, 500 μL of Hoechst reagent (1:5000) diluted in PBS was added to each well, and the chamber slide was incubated for 3 minutes at room temperature in the dark. After washing three times with PBS, Dako fluorescent mounting solution was added to the slide, and the slide was sealed with a clear cover glass. The slide was observed under a confocal laser scanning microscope (LSM 710 confocal laser scanning microscope, Carl Zeiss, Germany). In the negative control group (no internalization induction, 1 hour incubation at 4°C, Figure 21), Ab1 was detected on the cell surface of MKN-1 cells.In the cell internalization induction group (after incubation at 4°C for 1 hour, the cells were transferred to a 37°C incubator and then incubated at 37°C for 1 hour), 40 μg / mL of Ab1 was detected in the intracellular region of MKN-1 (Figure 21).

[0257] Example 2 – Preparation of antibody-drug conjugates The antibody-drug conjugates Ab-linker-DM1, Ab-linker-MMAF, and Ab-linker-AF were prepared as follows.

[0258] 2.1 List of abbreviations used in this example: Ac: Acetyl ADC: Antibody-drug conjugate ACN: acetonitrile AF: Auristatin F AMAS: N-α-maleimidoaceto-oxysuccinimide ester Cit: Citrulline CTC: chlorotrityl chloride Da: Dalton DIEA: Diisopropylethylamine DM1: Mertansine DMF: dimethylformamide DMSO: dimethyl sulfoxide EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ELSD: Evaporative Light Scattering Detector Et: Ethyl eq.: equivalent FA: Formic acid g: grams h:hour HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide HOBT: Hydroxybenzotriazole HPLC: High-performance liquid chromatography HRMS: High resolution mass spectrometry IgG: immunoglobulin IvDde: 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)isovaleryl K: kilo L: Liter Lys: Lysine m: millimeter ma: Maleimidoacetic acid Me: Methyl min:minutes MMAF: Monomethyl auristatin F mol: mole MS: Mass spectrometry m / z: mass-to-charge ratio NHS: N-hydroxysuccinimide nm: nanometer PBS: phosphate buffered saline PEG: polyethylene glycol PF: CentriPure PF filtration column pH: Hydrogen ion concentration quant.: quantitative RP: Reverse phase rt: room temperature Rt: retention time SEC: Size Exclusion Chromatography t: third TCEP: Tris(2-carboxyethyl)phosphine TFA: Trifluoroacetic acid Tyr: Tyrosine UPLC: Ultra-high performance liquid chromatography UV: Ultraviolet light V: Volume ℃: Celsius m:micro

[0259] 2.2 Starting Materials and Chemicals: The main starting materials and chemicals used in this example are listed below: > Synthesis solvents and deprotection reagents from Merck or Fischer Scientific AG (Switzerland); > TFA, triethylamine, DIEA, N-hydroxysuccinimide and HOSu from Sigma-Aldrich (Switzerland); > AMAS manufactured by Astatech Inc (USA); > Solvents and chemicals for high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) manufactured by Biosolve (France); > HATU.HPF6 made by Combi-Blocks (USA); > EDC.HCl manufactured by Apollo Chemical (USA); > DM1 manufactured by Immunogen Inc. (USA); > TCEP.HCl from Fluorochem (UK); > Bromoacetic acid and diisopropylmethandiimine and Pd / C from Acros Organics (Belgium); > H-Cit-Lys(PEG5-ma)-Tyr-OH.TFA from Ambiopharm (USA) (synthesis described in Section 2.8 below); > PBS (D8537) from Sigma-Aldrich (USA); > AF and MMAF manufactured by Angel Pharma Ltd (China).

[0260] 2.3 Method: The following methods were used to evaluate the compounds of the present disclosure.

[0261] 2.3.1 Purity measurement The purity of the compounds was determined by a UPLC-MS system.

[0262] Method 1: A Waters Acquity UPLC system was connected to a Waters SQD mass spectrometer equipped with a CSH C18 column (130 Å, 1.7 μm, 2.1 mm × 50 mm) heated to 40 °C, using solvent systems A (water + 0.1% FA) and B (ACN + 0.1% FA) with a flow rate of 0.9 mL / min and a 5–100% gradient of B over 2.7 min.

[0263] Method 2: A Waters Acquity UPLC system was connected to a Waters SQD mass spectrometer equipped with a CSH fluoro-phenyl column (130 Å, 1.7 μm, 2.1 mm × 50 mm) heated to 40 °C, using solvent systems A (water + 0.1% FA) and B (ACN + 0.1% FA) with a flow rate of 0.9 mL / min and a 5–100% gradient of B over 2.9 min.

[0264] Method 3: A Waters Acquity UPLC system was connected to a Waters SQD mass spectrometer equipped with a BEH C18 1.7 μm 50 × 2.1 mm column heated to 40 °C and a 2 μm insert filter precolumn (available from Waters) using solvent systems A1 (water + 0.1% FA) and B1 (ACN + 0.1% FA) with a flow rate of 0.9 mL / min and a 5–100% gradient of B1 over 2.9 min.

[0265] 2.3.2 Aggregates: Size Exclusion Chromatography (SEC) The aggregate content of the conjugate was measured using the following method and equipment: Apparatus 1 UPLC Waters Acquity Apparatus 2 UPLC Waters Acquity H-Class plus Bio Detector: Variable UV detector (TUV) Detector cell titanium Precolumn: Agilent AdvanceBio SEC 300Å 2.7μm 4.6 * 50mm Column: Agilent AdvanceBio SEC 300Å 2.7μm 4.6 * 150mm Mobile phase: Potassium phosphate 50mM pH 6.8 / 250mM KCl Wavelength 280nm Injection volume 10μL Column temperature ambient Sample manager temperature 25℃ Run time: 10 minutes Flow rate 0.35mL / min - isocratic mode Weak wash H2O / ACN (90 / 10v / v) Strong wash H2O / ACN (10 / 90v / v)

[0266] Mobile phase preparation: Potassium phosphate 50mM pH 6.8 / 250mM KCl Weigh 3.61 g of KH2PO4 and 4.09 g of K2HPO4 into a 1 L volumetric flask. Fill with milliQ water. Adjust the pH with 1 mol / L HCl or NaOH, if necessary. Add 18.64 g of KCl. Sample preparation: Prepare a 1-2.5 mg / mL ADC solution in milliQ water.

[0267] 2.3.3 Concentration: UV method · Equipment: UV spectrophotometer BioTek Synergy HT Buffer preparation: pH 7.4 PBS: Weigh 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4·2H2O, and 0.24 g KH2PO4 into a 1 L volumetric flask. Add 900 mL of milliQ water. Once all salts are dissolved, mix and adjust the pH to 7.35-7.44 with 1 mol / L HCl. Drain to 1 L with milliQ water. Sample preparation: Prepare a 1 mg / mL ADC solution in PBS, pH 7.4. Measure the absorbance at 252 nm and 280 nm using a UV reader. · Formula 1:

number

number

[0268] 2.3.4 DAR:MS method DAR was measured by native MS of the deglycosylated ADC using the following procedure and instrumentation. Sample preparation: EndoS reaction: Glycans were cleaved by incubation with EndoS enzyme at acidic pH for 1 hour at 37°C. The resulting samples were diluted twice with 50 mM ammonium acetate and 25 μg was injected for each sample. · UPLC: Separation was performed using a MAbPac™ SEC-1 column (Thermo Scientific) using 50 mM ammonium acetate at pH 7 as the mobile phase at 0.3 mL / min. MS: MS was performed using a QExactive HF Orbitrap operating in the high mass range. MS spectra were acquired from 1800 to 8000 m / z with the resolution set at 15k and SID 50 eV. Mass spectra were deconvoluted using Protein Deconvolution (Thermo Scientific).

[0269] 2.3.5 Cat B Cutting Cleavage of antibody-drug conjugates by Cat B can be assessed by an in vitro enzymatic cleavage assay using recombinant human cathepsin B (commercially available in precursor form from R&D Systems, Bio-Techne AG, catalog number 953-CY-010) and UHPLC-MS / MS analysis.

[0270] Briefly, the enzyme is reconstituted in 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer adjusted to pH 5.0 with 1 M NaOH solution, and then activated with 20 nM dithiothreitol (DDT) solution at room temperature for at least 15 minutes. In vitro enzyme assays are performed at 37°C in the presence of 2 μg / mL of activated recombinant human cathepsin B enzyme in 25 mM MES buffer, pH 5.0, using a test compound at a concentration of 10 μM (when the test compound is a linker according to the present invention in which the maleimide group is quenched in the presence of acetyl-cysteine ​​(Ac-Cys-OH)) or 2.5 μM (when the test compound is an antibody-drug conjugate). The enzyme cleavage reaction is stopped at each specified time point by mixing acetonitrile + 0.1% formic acid (FA) containing an internal standard (0.5 μM warfarin) in a 1 / 10 volume ratio. Analyses were performed using a Waters Acquity UPLC System connected to a Waters Xevo TQ triple quadrupole mass spectrometer. UHPLC was performed using an HSS T3 1.8 μm 50 × 2.1 mm column (Waters) heated to 45 °C and equipped with a 2 μm insert filter precolumn (Waters), and solvent systems A1 (HO + 0.1% FA) and B1 (acetonitrile + 0.1% FA) with a flow rate of 0.6 mL / min and a 5-95% gradient of B1 over 2.0 min. MS / MS was performed using a positive-mode electrospray ionization (ESI) interface and specific MRM transitions for each compound.

[0271] 2.4 Preparation and Characterization of Linker-Antitumor Compounds: The linker-antitumor compounds prepared are shown in Table 2 below. [Table 2]

[0272] When purified, compounds were purified by preparative reverse-phase HPLC on a Buchi C-835 using a Waters column XSelect CSH130 C18 5 μm 19 × 150 mm OBD and the indicated solvent system at a flow rate of 25 mL / min. Elution was monitored by UV at a wavelength of 220 nm and ELSD. Compound purity was determined by the previously described UPLC method (see Section 2.3.4).

[0273] 2.5 Preparation of DM1-Ac-Cit-Lys(PEG5-ma)-Tyr (DM1-linker) [ka]

[0274] Step 1. DIEA (0.28 mL, 1.63 mmol, 6.0 equiv) was added to a solution of bromoacetic acid (64.8 mg, 0.47 mmol, 1.7 equiv) and DM1 (200 mg, 0.27 mmol, 1.0 equiv) in DMF (2 mL) at room temperature. After stirring at room temperature for 1 hour, a mixture of HATU.HPF6 (113.3 mg, 0.30 mmol, 1.1 equiv) and 1-hydroxypyrrolidine-2,5-dione (34.3 mg, 0.30 mmol, 1.1 equiv) was added at room temperature. After stirring at room temperature for 30 minutes, TFA was added until an acidic pH was reached. Purification by preparative HPLC (30-60% ACN + 0.1% TFA in water + 0.1% TFA) and lyophilization afforded DM1-Ac-NHS ester (134.7 mg, 0.12 mmol, UV purity 80%, yield 45%) as a white powder. UPLC-MS (Method 2): Rt = 1.57 min, m / z = 891 [MH]. - .

[0275] Step 2. DIEA (31 mL, 0.18 mmol, 4.0 equiv.) was added to a mixture of DM1-Ac-NHS (50.0 mg, 44.8 mmol, 1.0 equiv.) and H-Cit-Lys(PEG5-ma)-Tyr. TFA (45.2 mg, 44.8 mmol, 1.0 equiv.) in DMF (1 mL) at room temperature. After stirring for 30 min at room temperature, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20-50% ACN + 0.1% TFA in water + 0.1% TFA) and lyophilization afforded DM1-Ac-Cit-Lys(PEG5-ma)-Tyr (74.9 mg, 44.8 mmol, UV purity 100%, quantitative) as a white powder. UPLC-MS (Method 1): Rt = 1.31 min, m / z = 1672 [MH]. - .

[0276] 2.6a Preparation of AF-Cit-Lys(PEG5-ma)-Tyr (AF-Linker) [ka]

[0277] H-Cit-Lys(PEG5-ma)-Tyr.TFA (120.0 mg, 0.12 mmol, 1.0 equiv.) was added to a mixture of DIEA (83 mL, 0.48 mmol, 4.0 equiv.), HATU.HPF6 (45.2 mg, 0.12 mmol, 1.0 equiv.), and AF.TFA (102.3 mg, 0.12 mmol, 1.0 equiv.) in DMF (2.5 mL) at room temperature. After stirring for 3.5 h at room temperature, TFA was added until an acidic pH was reached. Purification by preparative HPLC (10-50% ACN + 0.1% TFA in water + 0.1% TFA) and lyophilization afforded AF-Cit-Lys(PEG5-ma)-Tyr (31.7 mg, 19.5 mmol, 100% UV purity, 16% yield) as a white powder. UPLC-MS (method 3): Rt=1.29 min, m / z=1624[M+H] + , 1622[MH] - .

[0278] 2.6b Preparation of MMAF-Cit-Lys(ma)-Tyr-OH (MMAF-linker) [ka] [ka]

[0279] Step 1. DIEA (8.0 mL, 47.1 mmol, 3.0 equiv) was added to a mixture of tert-butyl L-tyrosinate (3.98 g, 16.6 mmol, 1.06 equiv), N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(diphenyl(p-tolyl)methyl)-L-lysine (10.0 g, 15.7 mmol, 1.0 equiv) and HATU (7.16 g, 18.8 mmol, 1.2 equiv) in DMF (150 mL) at 0° C. After stirring at room temperature for 30 minutes, the reaction mixture was poured into EtOAc (500 mL) and washed with ½ saturated brine (2×300 mL). The organic layer was dried over MgSO, filtered, and concentrated under reduced pressure to give tert-butyl (2S)-2-[[(2S)-6-[[diphenyl(p-tolyl)methyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-3-(4-hydroxyphenyl)propanoate (15.46 g, 15.22 mmol, UV purity 83%, yield 97%) as a yellow oil, which was used in the next step without further purification. UPLC-MS (Method 1): Rt = 1.34 min, m / z = 844 [M+H] + .

[0280] Step 2. tert-Butyl (2S)-2-[[(2S)-6-[[diphenyl(p-tolyl)methyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-3-(4-hydroxyphenyl)propanoate (15.46 g, 15.22 mmol, 1.0 equivalent) was dissolved in a mixture of DMF / piperidine (70 mL, 9:1 V / V) at room temperature. After stirring at room temperature for 25 minutes, the reaction mixture was concentrated to dryness. Purification by flash chromatography (n-heptane: EtOAc 100:0 to 0:100, then EtOAc: MeOH 95:5 to 90:10) afforded tert-butyl (2S)-2-[[(2S)-2-amino-6-[[diphenyl(p-tolyl)methyl]amino]hexanoyl]amino]-3-(4-hydroxyphenyl)propanoate (9.80 g, 15.10 mmol, UV purity 96%, yield 99%) as a yellow oil. UPLC-MS (Method 2): Rt = 1.44 min, m / z = 622 [M+H] + ,620[MH] - .

[0281] Step 3. tert-Butyl (2S)-2-[[(2S)-2-amino-6-[[diphenyl(p-tolyl)methyl]amino]hexanoyl]amino]-3-(4-hydroxyphenyl)propanoate (6.00 g, 9.25 mmol, 1.0 equiv), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5- To a mixture of ureidopentanoic acid (2.94 g, 7.40 mmol, 0.8 equiv.) and 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-1-amine, hydrochloride (2.13 g, 11.1 mmol, 1.2 equiv.) was added 1H-benzo[d][1,2,3]triazol-1-ol hydrate (1.70 g, 11.1 mmol, 1.2 equiv.) at room temperature. After stirring at room temperature for 2 h, the reaction mixture was poured into EtOAc (500 mL) and washed twice with ½ saturated brine (400 mL). The organic layer was concentrated under reduced pressure and placed on a high-vacuum rotavapor to remove excess pyridine. The crude product was purified by flash chromatography (n-heptane: EtOAc 90:10 to plain EtOAc) to give tert-butyl (2S)-2-[[(2S)-6-[[diphenyl(p-tolyl)methyl]amino]-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-ureido-pentanoyl]amino]hexanoyl]amino]-3-(4-hydroxyphenyl)propanoate (3.30 g, 3.26 mmol, UV purity 99%, yield 35%) as an off-white solid. UPLC-MS (Method 2): Rt = 1.99 min, m / z = 1001 [M+H] + .

[0282] Step 4. DBU (83 mL, 0.54 mmol, 1.1 equiv.) was added to a solution of tert-butyl (2S)-2-[[(2S)-6-[[diphenyl(p-tolyl)methyl]amino]-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-ureido-pentanoyl]amino]hexanoyl]amino]-3-(4-hydroxyphenyl)propanoate (500 mg, 0.49 mmol, 1.0 equiv.) in DMF (3 mL) at room temperature. After stirring at room temperature for 20 minutes, the reaction mixture was added to cold ether (60 mL). The rapidly precipitating off-white viscous product was collected by centrifugation and then purified by preparative HPLC (30-70% ACN + 0.1% TFA in water + 0.1% TFA) to give, after lyophilization, tert-butyl (2S)-2-[[(2S)-2-[[(2S)-2-amino-5-ureido-pentanoyl]amino]-6-[[diphenyl(p-tolyl)methyl]amino]hexanoyl]amino]-3-(4-hydroxyphenyl)propanoate; 2,2,2-trifluoroacetic acid (405 mg, 0.45 mmol, UV purity 100%, yield 92%) as a white powder. UPLC-MS (Method 2): Rt = 1.35 min, m / z = 780 [M+H] + , 778[MH] - .

[0283] Step 5. DIEA (0.14 mL, 0.82 mmol, 4.0 equiv.) was reacted with tert-butyl (2S)-2-[[(2S)-2-[[(2S)-2-amino-5-ureido-pentanoyl]amino]-6-[[diphenyl(p-tolyl)methyl]amino]hexanoyl]amino]-3-(4-hydroxyphenyl)propanoate; 2,2,2-trifluoroacetic acid (201.3 mg, 0.23 mmol, 1.1 equiv.), ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S) To a mixture of N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (153.1 mg, 0.20 mmol, 1.0 equiv.) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (79.9 mg, 0.20 mmol, 1.0 equiv.) was added at room temperature. After stirring at room temperature for 40 minutes, the reaction mixture was added to cold ether (60 mL). Centrifugation afforded tert-butyl(2S)-2-[[(2S)-6-[[diphenyl(p-tolyl)methyl]amino]-2-[[(2S)-2-[[(2S)-2-[[(2R,3R)-3-methoxy-3-[(2S)-1-[(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl-[(2S)-3-methyl-2-[[(2S)-3-methyl-2-(methylamino)

[0047] 3-(4-hydroxyphenyl)propanoate (345 mg, 0.21 mmol, UV purity 90%, quantitative) was obtained as a yellow oil. UPLC-MS (Method 2): Rt = 1.66 min, m / z = 1494 [M+H]. + , 1492[MH] - .

[0284] Step 6. tert-Butyl (2S)-2-[[(2S)-6-[[diphenyl(p-tolyl)methyl]amino]-2-[[(2S)-2-[[(2S)-2-[[(2R,3R)-3-methoxy-3-[(2S)-1-[(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl-[(2S)-3-methyl-2-[[(2S)-3-methyl-2-(methylamino)butanoyl]amino]

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[0098] [009 ... Centrifugation identified (2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2R,3R)-3-methoxy-3-[(2S)-1-[(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl-[(2S)-3-methyl-2-[[(2S)-3-methyl-2-(methylamino)butanoyl]amino]butanoyl]amino]heptanoyl]pyrrolyl

[0047] 2,2,2-Trifluoroacetic acid (290 mg, 0.18 mmol, UV purity 86%, yield 85%) was obtained as a white solid, which was used in the next step without further purification. UPLC-MS (Method 2): Rt = 1.07 min, m / z = 1181 [M+H]. + , 1179[MH] - .

[0285] Step 7. DIEA (0.26 mL, 1.59 mmol, 8.0 equiv.) was dissolved in DMF (4 mL) to prepare a solution of (2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2R,3R)-3-methoxy-3-[(2S)-1-[(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl-[(2S)-3-methyl-2-[[(2S)-3-methyl-2-(methylamino)butanoyl]amino]butano [I]yl]amino]heptanoyl]pyrrolidin-2-yl]-2-methyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-ureido-pentanoyl]amino]hexanoyl]amino]-3-(4-hydroxyphenyl)propanoic acid; 2,2,2-trifluoroacetic acid (280.0 mg, 0.20 mmol, 1.0 equiv.) and AMAS (58.1 mg, 0.22 mmol, 1.1 equiv.) were added at room temperature. After stirring at room temperature for 20 min, the reaction mixture was acidified with TFA (80 mL). Purification by preparative HPLC (20-70% ACN + 0.1% TFA in water + 0.1% TFA) and lyophilization afforded MMAF-Cit-Lys(ma)-Tyr-OH (86.0 mg, 57.3 mmol, UV purity 95%, yield 29%) as a white powder. UPLC-MS (method 3): Rt=1.03 min, m / z=1430[MH] - .

[0286] 2.7 Preparation and Characterization of Antibody-Drug Conjugates (ADCs): 2.7.1 Preparation of ADC Preparation of Ab1-Linker-DM1 Conjugate: A solution of TCEP·HCl (38 μg, 4.0 equiv.) in PBS (38 μL) was added to a solution of Ab1 (5 mg, 1.0 equiv.) in PBS (0.77 mL) at room temperature. The reaction mixture was flushed with nitrogen and then stirred at 40 °C. After stirring for 120 min at 40 °C, a solution of DM1-Ac-Cit-Lys(PEG5-ma)-Tyr (0.56 mg, 10.0 equiv.) in DMSO (50 μL) was added. The reaction mixture was stirred for 60 min at room temperature and then diluted to V = 2.5 mL with pH 8 PBS. Purification using a PF25 column and pH 8 PBS as the eluent yielded a fraction (3.5 mL) containing the desired ADC. The latter fraction was stirred for 16 h at room temperature, then centrifuged (10 min, 4000 rpm), and the supernatant was finally transferred to an Amicon concentration cell (4 mL, 50 kDa). The mixture was concentrated to V = 0.5 mL by centrifugation (15 min, 4500 rpm, 3800 g). PBS (4 mL) was added, and the mixture was concentrated to V = 0.5 mL by centrifugation (15 min, 4500 rpm, 3800 g). The final volume was adjusted to V = 1.0 mL with PBS. The solution was filtered using a 25 mm PES 0.22 mm Millex filter, then aliquoted (2 x 300 μL, 1 x 80 μL, 1 x 50 μL, 1 x 40 μL) and stored at -80°C. DAR (MS): 3.79. Concentration (UV280 nm): 3.20 mg / mL or 21.39 mmol / L. Aggregate content: 1.15%. Monomer content: 98.01%. V = 770 μL. m = 2.46 mg.

[0287] Preparation of HG4K IgG4-linker-DM1 conjugate: A solution of TCEP·HCl (115 μg, 6.0 equiv.) in PBS (115 μL) was added to a solution of HG4K (10 mg, 1.0 equiv.) in buffer (1.142 mL) at room temperature. The reaction mixture was flushed with nitrogen and then stirred at 40 °C. After stirring at 40 °C for 8 h, a solution of DM1-Ac-Cit-Lys(PEG5-ma)-Tyr (1.56 mg, 14.0 equiv.) in DMSO (100 μL) was added. The reaction mixture was stirred at room temperature for 60 min and then diluted to V = 2.5 mL with pH 8 PBS. Purification using a PF25 column and pH 8 PBS as the eluent yielded a fraction (3.5 mL) containing the desired ADC. The latter fraction was stirred at room temperature for 16 h, then centrifuged (10 min, 4000 rpm), and the supernatant was finally transferred to an Amicon concentration cell (4 mL, 50 kDa). The mixture was concentrated to V = 0.5 mL by centrifugation (10 min, 4500 rpm, 3800 g). PBS (4 mL) was added, and the mixture was concentrated to V = 0.5 mL by centrifugation (10 min, 4500 rpm, 3800 g). The final volume was adjusted to V = 1.0 mL with PBS. The solution was filtered using a 25 mm PES 0.22 mm Millex filter, then aliquoted (4 x 200 μL, 2 x 50 μL) and stored at -80°C. DAR (MS): 1.74. Concentration (UV280 nm): 7.00 mg / mL or 48.04 mmol / L. Aggregate content: 2.80%. Monomer content: 95.3%. V = 900 μL. m = 6.30 mg.

[0288] Preparation of Ab1-linker-AF conjugate: A solution of TCEP·HCl (0.755 mg, 4.0 equiv.) in PBS (755 μL) was added to a solution of Ab1 (98.8 mg, 1.0 equiv.) in PBS (19 mL) at room temperature. The reaction mixture was flushed with nitrogen and then stirred at 40 °C. After stirring at 40 °C for 120 min, a solution of AF-Cit-Lys(PEG5-ma)-Tyr (10.7 mg, 10.0 equiv.) in DMSO (1 mL) was added. The reaction mixture was stirred at room temperature for 60 min and then diluted to V = 30 mL with pH 8 PBS. Purification using three PF100 columns and pH 8 PBS as the eluent yielded a fraction (42 mL) containing the desired ADC. The latter fraction was stirred at room temperature for 16 h, then centrifuged (10 min, 4000 rpm), and the supernatant was finally transferred to an Amicon concentration cell (15 mL, 50 kDa). The mixture was concentrated to V = 1 mL by centrifugation (60 min, 4500 rpm, 3800 g). PBS (14 mL) was added, and the mixture was concentrated to V = 1 mL by centrifugation (30 min, 4500 rpm, 3800 g). The final volume was adjusted to V = 10 mL with PBS. The solution was filtered using a 25 mm PES 0.22 mm Millex filter and then aliquoted (18 x 500 μL, 2 x 250 μL, 1 x 150 μL, 2 x 100 μL, 4 x 50 μL) and stored at -80 °C. DAR (MS): 2.92. Concentration (UV280 nm): 8.16 mg / mL or 55.17 mmol / L. Aggregate content: 2.48%. Monomer content: 96.69%. V = 10.05 mL, m = 82.01 mg.

[0289] Preparation of HG4K IgG4-linker-AF-conjugate: A solution of TCEP·HCl (459 μg, 6.0 equiv) in PBS (459 μL) was added to a solution of HG4K (40 mg, 1.0 equiv) in buffer (4.45 mL) at room temperature. The reaction mixture was flushed with nitrogen and then stirred at 40 °C. After stirring at 40 °C for 8 h, a solution of AF-Cit-Lys(PEG5-ma)-Tyr (6.06 mg, 14.0 equiv) in DMSO (400 μL) was added. The reaction mixture was stirred at room temperature for 70 min and then diluted to V = 10 mL with pH 8 PBS. Purification using a PF100 column and pH 8 PBS as the eluent yielded a fraction (14 mL) containing the desired ADC. The latter fraction was stirred at room temperature for 16 h, then centrifuged (10 min, 4000 rpm), and the supernatant was finally transferred to an Amicon concentration cell (15 mL, 50 kDa). The mixture was concentrated to V = 0.5 mL by centrifugation (20 min, 4500 rpm, 3800 g). PBS (14 mL) was added, and the mixture was concentrated to V = 0.5 mL by centrifugation (20 min, 4500 rpm, 3800 g). The final volume was adjusted to V = 4.5 mL with PBS. The solution was filtered using a 25 mm PES 0.22 mm Millex filter, then aliquoted (14 x 250 μL, 2 x 200 μL, 4 x 50 μL) and stored at 80 °C. DAR (MS): 1.92. Concentration (UV280 nm): 6.89 mg / mL or 47.21 mmol / L. Aggregate content: 1.13%. Monomer content: 95.22%. V = 4500 mL. m = 31.00 mg.

[0290] Preparation of Ab2-linker-AF conjugate: A solution of TCEP·HCl (103 μg, 2.3 equiv) in PBS (103 μL) was added to a solution of Ab2 (22.38 mg, 1.0 equiv) in PBS (2 mL) at room temperature. The reaction mixture was flushed with nitrogen and then stirred at 40 °C. After stirring at 40 °C for 60 min, a solution of AF-Cit-Lys(PEG5-ma)-Tyr (1.87 mg, 7.4 equiv) in DMSO (200 μL) was added. The reaction mixture was stirred at room temperature for 60 min and then diluted to V = 5 mL with pH 8 PBS. Purification using a PF50 column and pH 8 PBS as the eluent yielded a fraction (7 mL) containing the desired ADC. The latter fraction was stirred at room temperature for 16 h, then centrifuged (10 min, 4000 rpm), and the supernatant was finally transferred to an Amicon concentration cell (15 mL, 50 kDa). The mixture was concentrated to V = 0.5 mL by centrifugation (17 min, 4500 rpm, 3800 g). PBS (14 mL) was added, and the mixture was concentrated to V = 0.5 mL by centrifugation (20 min, 4500 rpm, 3800 g). The final volume was adjusted to V = 2.4 mL with PBS. The solution was filtered using a 25 mm PES 0.22 mm Millex filter, then aliquoted (8 x 250 μL, 1 x 50 μL, 1 x 20 μL) and stored at -80°C. DAR (MS): 4.46. Concentration (UV280 nm): 8.45 mg / mL or 56.08 mmol / L. Aggregate content: 3.92%. Monomer content: 96.08%. V = 2.07 mL. m = 17.51 ​​mg.

[0291] Preparation of Ab1-linker-MMAF conjugate: A solution of TCEP·HCl (2.66 mg, 4.5 equiv) in PBS (1.86 mL) was added to a solution of Ab1 (300 mg, 1.0 equiv) in PBS (28.13 mL) at room temperature. The reaction mixture was purged with nitrogen and then stirred at 40 °C. After stirring at 40 °C for 4 h 15 min, a solution of MMAF-Cit-Lys(ma)-Tyr-OH (38.66 mg, 25.8 mmol, 12.5 equiv) in DMSO (2.58 mL) was added. The reaction mixture was stirred at room temperature for 1 h and then diluted with 3.6 mL of 10× pH 8 PBS. Purification was performed according to the manufacturer's instructions using three PF100 columns and one PF50 column with pH 8 PBS as the eluent to afford the desired ADC (49 mL). The ADC was stirred at room temperature for 19 hours, then centrifuged (4 min, 4000 rpm). Finally, the supernatant was transferred to six Amicon-15 concentration cells (15 mL, 50 kDa). The mixture was concentrated to V = 5 mL by centrifugation (4500 rpm, 3800 G). PBS buffer (10 mL) was added, and the mixture was again concentrated to V = 5 mL by centrifugation (4500 rpm, 3800 G) (this step was repeated twice). The final volume was adjusted to V = 30 mL with PBS buffer. The solution was filtered using a 13 mm PES 0.22 μm Millex filter, then aliquoted and stored at -80 °C. DAR (MS): 3.95. Concentration (UV280 nm): 9.29 mg / mL or 61.58 mmol / L. Aggregate content: 2.38%. Monomer content: 97.62%. V = 30.48 mL. m = 283.01 mg.

[0292] Preparation of Ab3-linker-MMAF conjugate: A solution of TCEP·HCl (0.11 mg, 2.5 equiv.) in PBS (77.3 μL) was added to a solution of Ab3 (22.5 mg, 1.0 equiv.) in PBS (4.25 mL) at room temperature. The reaction mixture was purged with nitrogen and then stirred at 40 °C. After stirring at 40 °C for 1 h 30 min, a solution of MMAF-Cit-Lys(ma)-Tyr-OH (1.85 mg, 8 equiv.) in DMSO (247 μL) was added. The reaction mixture was stirred at room temperature for 1 h and then diluted with 0.43 mL of 10× pH 8 PBS. Purification was performed according to the manufacturer's instructions using a PF50 column and pH 8 PBS as the eluent to obtain the desired ADC (7 mL). The ADC was stirred at room temperature for 18 h, then centrifuged (4 min, 4000 rpm), and the supernatant was finally transferred to an Amicon-15 concentration cell (15 mL, 50 kDa). The mixture was concentrated to V = 2 mL by centrifugation (4500 rpm, 3800 G), PBS buffer (13 mL) was added, and the mixture was again concentrated to V = 2 mL by centrifugation (4500 rpm, 3800 G) (this step was repeated twice). The final volume was adjusted to V = 2 mL with PBS buffer. The solution was filtered using a 13 mm PES 0.22 μm Millex filter, then aliquoted and stored at -80 °C. DAR (MS): 3.71. Concentration (UV280 nm): 10.61 mg / mL or 70.44 mmol / L. Aggregate content: 0.65%. Monomer content: 99.35%. V = 1.96 mL. m = 20.79 mg.

[0293] Preparation of HG4K IgG4-linker-MMAF conjugate: A solution of TCEP·HCl (3.9 mg, 14 equiv.) in PBS (2.7 mL) was added to a solution of HG4K IgG4 (140 mg, 1.0 equiv.) in PBS (16.7 mL) at room temperature. The reaction mixture was purged with nitrogen and then stirred at 40 °C. After stirring at 40 °C for 5 h, a solution of MMAF-Cit-Lys(ma)-Tyr-OH (4.18 mg, 8 equiv.) in DMSO (1.45 mL) was added. The reaction mixture was stirred at room temperature for 1 h and then diluted with 2.32 mL of 10x pH 8 PBS. Purification was performed according to the manufacturer's instructions using two PF100 columns, a PF25 column, and a PF10 column with pH 8 PBS as the eluent to obtain the desired ADC (33 mL). The ADC was stirred at room temperature for 19 hours, then centrifuged (4 min, 4000 rpm). Finally, the supernatant was transferred to three Amicon-15 concentration cells (15 mL, 50 kDa). The mixture was concentrated to V = 3 mL by centrifugation (4500 rpm, 3800 G). PBS buffer (12 mL) was added, and the mixture was again concentrated to V = 3 mL by centrifugation (4500 rpm, 3800 G) (this step was repeated twice). The final volume was adjusted to V = 14 mL with PBS buffer. The solution was filtered using a 13 mm PES 0.22 μm Millex filter, then aliquoted and stored at -80 °C. DAR (MS): 4.41. Concentration (UV280 nm): 9.54 mg / mL or 63.1 mmol / L. Aggregate content: 3.21%. Monomer content: 96.79%. V = 14.21 mL. m = 135.54 mg.

[0294] 2.7.2 Overview of Characterization of Prepared ADCs All characteristics are shown in Table 3 below. [Table 3]

[0295] The SEC chromatograms (aggregate content assignments) and mass spectra (MS) (DAR assignments) of the deglycosylated ADCs are shown in Figures 8-17b.

[0296] 2.8 Synthesis of H-Cit-Lys(PEG5-ma)-Tyr-OH·TFA The steps of the manufacturing process are carried out by classical chemical reactions and do not involve general engineering or biochemical processes associated with fermentation. All methods used throughout the production of peptides are based on well-documented organic reactions frequently used in peptide chemistry.

[0297] All amino acids and related products are purchased from authorized manufacturers or distributors. Manufacturer's Certificate of Analysis is obtained for each amino acid.

[0298] Peptide synthesis is carried out on 2-CTC resin according to the common Fmoc-t-butyl (Fmoc / tBu) strategy of solid-phase synthesis, with activation of the carboxyl group achieved by an appropriate combination of activating agents, such as diisopropylcarbodiimide / HOBT. Starting with the C-terminal amino acid, each amino acid is sequentially coupled to the peptide chain. Linear stepwise solid-phase peptide synthesis (SPPS) utilizes the principle of fully coupling a temporarily α-N-protected amino acid, such as Fmoc (9-fluorenyloxycarbonyl), onto a derivatized polymer support. After the first amino acid is fully incorporated, the α-N-protection of the incorporated residue is removed by an orthogonal deprotection solution, without affecting either the peptide-resin bond or the side-chain protecting groups. This results in a resin-anchored, fully side-chain-protected peptide segment containing only one free nucleophilic primary amine to which the next amino acid is attached. Following this procedure, all amino acids are incorporated stepwise as activated esters. The final amino acid in the sequence is coupled with an N-terminally protected Boc group. Lys derivatives were incorporated with side-chain protection orthogonal to Fmoc, such as ivDde groups, which were removed with 2% hydrazine in DMF. After removal of ivDde, the side chains were derivatized with maleimide-PEG5-OH using an activated ester. The peptides were subsequently treated with a TFA-based acidolysis cocktail, which cleaved them from the resin and deprotected the side chain groups. The peptides were then purified by liquid chromatography (RP-HPLC). The purified peptide TFA salts were lyophilized to yield a white to off-white powder.

[0299] The identity and purity of H-Cit-Lys(PEG5-ma)-Tyr-OH.TFA were analyzed by several physicochemical methods: visual inspection, purity = 95% by reversed-phase HPLC (standard method) and mass spectrometry (894.98 Da).

[0300] Example 3 Example 3.1 – Cytotoxic activity of Ab1-linker-DM1, Ab1-linker-AF, and Ab2-linker-AF against BCAM-expressing human cancer cell lines The in vitro cytotoxic activity of Ab1-linker-DM1, Ab1-linker-AF, and Ab2-linker-AF was evaluated in multiple human cancer cell lines (SK-BR-3, T47D, MCF-7, A431, or Huh7) expressing different levels of BCAM using the CellTiter-Glo® assay (Promega). Cells were seeded in 96-well clear flat-bottom plates (5,000 cells / well) in RPMI-1640 supplemented with 10% FBS and 1% penicillin / streptomycin and maintained at 37°C and 5% CO2. After overnight incubation, cells were treated with different concentrations of Ab1-linker-DM1, Ab1-linker-AF, or the negative control for each test substance (HG4K IgG4-linker-DM1 for Ab1-linker-DM1, and HG4K IgG4-linker-AF for Ab1-linker-AF and Ab2-linker-AF, respectively). After 72 hours, CellTiter-Glo® reagent was added to each well, and luminescence was measured using Synergy H1 (BioTek, Winooski, VT). All experiments were performed in triplicate. The percentage of viable cells was calculated using the following formula: (T-DO) / (V-DO) x 100 (T = luminescence of drug-treated cells, V = luminescence of PBS (vehicle)-treated cells, DO = luminescence of untreated cells on day 0). All data were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA). As a result, Ab1-linker-DM1 and Ab1-linker-AF significantly inhibited the proliferation of BCAM-high-expressing cell lines SK-BR-3, T47D, MCF-7, A431, and Huh7 in a dose-dependent manner. See Figure 1A (for Ab1-linker-DM1) and Figure 1B (for Ab1-linker-AF). Furthermore, the activity of Ab1-linker-DM1 and Ab1-linker-AF showed a strong positive correlation with the expression level of BCAM in the cancer cell lines. The GI of Ab1-linker-DM1 on SK-BR3, T47D, MCF-7, A431, and Huh7 cells was significantly inhibited. 50The GI values ​​(doses that inhibited cell proliferation by 50%) were 2.8 nM, 16 nM, 34 nM, 42 nM, and 22 nM, respectively (Table 4.1). 50 The values ​​were 0.9 nM, 31 nM, 59 nM, 9.1 nM, and 193 nM, respectively (Table 4.1). See Table 4.1 below. Next, the cytotoxicity of Ab2-linker-AF was examined using SK-BR3 and A431 cells. As a result, Ab2-linker-AF showed significant cytotoxic activity against the tested cells compared to HG4K IgG4-linker-AF, and GI 50 was 0.15 nM in SK-BR-3 and 1.7 nM in A431, but Ab2 itself did not induce cytotoxicity against the cells tested (Figure 1C and Table 4.1). [Table 4.1]

[0301] Example 3.2 – Cytotoxic activity of Ab1-linker-MMAF and Ab3-linker-MMAF against BCAM-expressing human cancer cell lines The in vitro cytotoxic activity of Ab1-linker-MMAF and Ab3-linker-MMAF was evaluated in multiple human cancer cell lines (KURAMOCHI, OVCAR3, SK-BR-3, VCaP, or RERF-GC-1B) expressing different levels of BCAM using the CellTiter-Glo® assay (Promega). Cells were seeded in 96-well clear flat-bottom plates (5,000 cells / well) in RPMI-1640 (KURAMOCHI, OVCAR3, and SK-BR-3) or DMEM (VCaP and RERF-GC-1B) supplemented with 10% FBS and 1% penicillin / streptomycin and maintained at 37°C and 5% CO. After overnight incubation, cells were treated with different concentrations of Ab1-linker-MMAF, Ab3-linker-MMAF, or negative controls for each test substance (HG4K IgG4-linker-MMAF for Ab1-linker-MMAF and Ab3-linker-MMAF, respectively). After 72 hours, CellTiter-Glo® reagent was added to each well, and luminescence was measured using an Envision 2105 (PerkinElmer, Waltham, MA). All experiments were performed in triplicate. The percentage of viable cells was calculated using the following formula: (T-DO) / (V-DO) x 100 (T = luminescence of drug-treated cells, V = luminescence of PBS (vehicle)-treated cells, DO = luminescence of untreated cells on day 0). All data were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA). As a result, Ab1-linker-MMAF and Ab3-linker-MMAF significantly inhibited the proliferation of the BCAM-high-expressing cell lines KURAMOCHI, OVCAR3, SK-BR-3, VCaP, and RERF-GC-1B in a dose-dependent manner (see Figure 1d). The cytotoxic activity of Ab1-linker-MMAF and Ab3-linker-MMAF showed a strong positive correlation with the expression level of BCAM in the cancer cell lines. The GI activity of Ab1-linker-MMAF against KURAMOCHI, OVCAR3, SK-BR-3, VCaP, and RERF-GC-1B cells was significantly inhibited (Figure 1d). 50The GI values ​​(doses that inhibited cell proliferation by 50%) were 5.3 nM, 5.1 nM, 0.31 nM, 14 nM, and 1.5 nM, respectively (Table 4.2). 50 The values ​​were 4.4 nM, 3.1 nM, 0.24 nM, 24 nM, and 1.4 nM, respectively (Table 4.2). Ab1-linker-MMAF and Ab3-linker-MMAAF showed significant cytotoxic activity against the cancer cell lines tested compared to HG4K IgG4-linker-MMAF, whereas Ab1 and Ab3 by themselves did not induce cytotoxicity. [Table 4.2]

[0302] Example 4: In vivo antitumor activity of Ab1-linker-AF in the A431 xenograft model The in vivo antitumor activity of Ab1-linker-AF was evaluated in a BCAM-positive A431 xenograft model. A431 cells (KCLB, Korea) were cultured in RPMI supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2. To establish the tumor model, 5 × 10 6 A431 cells were subcutaneously injected into the right flank of 6-week-old female BALB / c nude mice (Orientbio, Korea). Tumors were 75–150 mm 3 At the time of reaching 100 mg / kg, mice were randomly assigned to the following treatment groups (n=8 / group): PBS (vehicle), HG4K IgG4-linker-AF (20 mg / kg), Ab1 (20 mg / kg), and Ab1-linker-AF (5 mg / kg, 10 mg / kg, and 20 mg / kg). Each control or test substance was administered intravenously once a week for 2 weeks. Tumor diameters were measured 2-3 times a week, and tumor volumes were calculated using the following formula: [tumor volume = length × width] 2× 0.5]. As a result, administration of Ab1-linker-AF once a week for two weeks significantly suppressed A431 tumor growth in a dose-dependent manner. See Figure 2. On day 14 after the start of treatment, the mean tumor growth rate of the vehicle control group was set as 100% and compared with the other control groups (20 mg / kg HG4K IgG4-linker-AF and 20 mg / kg Ab1) and Ab1-linker-AF (5 mg / kg, 10 mg / kg, and 20 mg / kg) treatment groups. The mean tumor growth rates of Ab1-linker-AF at 5 mg / kg, 10 mg / kg, and 20 mg / kg were 34.2%, 11.4%, and 7.3%, respectively (see Figure 3), while the mean tumor growth rates of HG4K IgG4-linker-AF and Ab1 were 135.2% and 83.4%, respectively (see Figure 3). Since neither HG4K IgG4-linker-AF nor the parent Ab1 antibody exhibited potent antitumor activity, this study suggested that the antitumor activity of Ab1-linker-AF was due to a synergistic effect between the specific binding of Ab1 to BCAM and the cytotoxic activity of the antitumor compound AF.

[0303] Reference example: Cat B cleavage study The reference compounds shown in Table 5 below were prepared in the Reference Examples according to the methods described above and in WO 2019 / 096867. [Table 5]

[0304] The compounds were prepared by standard Fmoc-based SPPS using an Activo P-11 automated peptide synthesizer (available from Activotec) and Fmoc-Xxx-Wang resin (Xxx: C-terminal amino acid; loading: 0.60 mmol / g; Bachem). The coupling reaction for amide bond formation was carried out at room temperature for 30 min using 3 equivalents of Fmoc-amino acid, Fmoc-NH-PEG4-COOH, or Fmoc-NH-PEG5-COOH activated with HBTU (2.9 equivalents) in the presence of DIEA (7 equivalents). Fmoc deprotection was carried out using a solution of 20% piperidine in DMF. Selective removal of the Mtt side chain protecting group (Lys) was achieved using DCM / TFA / TIS (94 / 1 / 5, v / v / v).

[0305] For the synthesis of compounds 1-4 and 8, Fmoc was removed by fragment condensation (3 equiv. AF, 2.9 equiv. HBTU, 7 equiv. DIEA) over 30 min, followed by AF coupling. For the synthesis of compounds 9 and 10, Fmoc was removed under the same conditions (3 equiv. ACit, 2.9 equiv. HBTU, 7 equiv. DIEA) followed by auristatin Cit (ACit) coupling.

[0306] For the synthesis of compounds 1-4 and 8-10, the Mtt was removed with DCM / TFA / TIS (94 / 1 / 5, v / v / v), followed by the addition of the derivative Mal-PEG4-NHS to the resin for 30 min (3 equivalents of Mal-PEG4-NHS, 7 equivalents of DIEA). For compounds 1, 3, 8, 9, and 10, the maleimide residue on the PEG chain was then reacted with acetyl-cysteine ​​(Ac-Cys-OH) by chemoselective ligation (3 equivalents of Ac-Cys-OH, DIEA, 7 equivalents) on the resin for 20 min. TFA / TIS a / water (95 / 2.5 / 2.5, v / v / v; a The peptide was cleaved from the resin with simultaneous side-chain deprotection by treatment with TIS (triisopropylsilane) for 60 min. After concentration of the cleavage mixture, the crude peptide was precipitated with cold diethyl ether and centrifuged.

[0307] For the synthesis of compounds 5–7, after removal of Mtt with DCM / TFA / TIS (94 / 1 / 5, v / v / v), the derivative Mal-PEG4-NHS was added to the resin for 30 min (3 equiv. Mal-PEG4-NHS, 7 equiv. DIEA). Next, the maleimide residue on the PEG chain was reacted with acetyl-cysteine ​​(Ac-Cys-OH) on the resin for 20 min via chemoselective ligation between the maleimide and a thiol (3 equiv. Ac-Cys-OH, 7 equiv. DIEA). After Fmoc deprotection, the Mal derivative was inserted by adding the partial Mal-NHS to the N-terminus of Phe. The peptide was cleaved from the resin with TFA / TIS / water (95 / 2.5 / 2.5, v / v / v) for 60 min, simultaneously deprotecting the side chain. After concentration of the cleavage mixture, the crude peptide was precipitated with cold diethyl ether and centrifuged. Mertansine (DM1, 1.45 equivalents) was then reacted with the terminal maleimide group by chemoselective ligation in PBS buffer at pH 7.4 and acetonitrile (2:1 ratio).

[0308] For the synthesis of compounds 11–13, after Fmoc removal, the derivative Ma-NHS was added to the resin for 30 min (3 equiv. Ma-NHS, 7 equiv. DIEA). The maleimide residue was then reacted with acetyl-cysteine ​​(Ac-Cys-OH) on the resin for 20 min by chemoselective ligation (3 equiv. Ac-Cys-OH, 7 equiv. DIEA). The peptide was cleaved from the resin by treatment with TFA / TIS / water (95 / 2.5 / 2.5, v / v / v) for 60 min, simultaneously deprotecting the side chains. After concentration of the cleavage mixture, the crude peptide was precipitated with cold diethyl ether and centrifuged. Following their purification, the derivative DM1-smcc (1.1 equiv.) was reacted with the N-terminus of the linker in a solution of DMF and 4-methylmorpholine (6 equiv.) for 4 h.

[0309] Peptide purification was performed on a Waters Autopurification HPLC system connected to an SQD mass spectrometer equipped with an XSelect Peptide CSH C18 OBD Prep column (130 Å, 5 μm, 19 mm × 150 mm) using solvent systems A (0.1% TFA in water) and B (0.1% TFA in acetonitrile) at a flow rate of 24 mL / min and a 20–60% gradient of B over 30 min.

[0310] The appropriate fractions were concentrated and lyophilized. Purity was determined on a Waters Acquity UPLC System connected to an SQD mass spectrometer equipped with a CSH C18 column (130 Å, 1.7 μm, 2.1 mm × 50 mm) using solvent system A (0.1% FA in water) and B (0.1% FA in acetonitrile) at a flow rate of 0.6 mL / min with a 5-85% gradient of B over 5 min, or a CSH Floro-Phenyl column (130 Å, 1.7 μm, 2.1 mm × 50 mm) using solvent system A (0.1% FA in water) and B (0.1% FA in acetonitrile) at a flow rate of 0.9 mL / min with a 5-95% gradient of B over 2.9 min.

[0311] MS analysis was performed using an electrospray ionization (ESI) interface in positive and negative modes. The analytical results of the compounds obtained in the Reference Examples are shown in Table 6 below. [Table 6]

[0312] The propensity of compounds 1-13 to be cleaved by cathepsin B was assessed according to an in vitro enzymatic cleavage assay using recombinant human cathepsin B and UHPLC-MS / MS analysis, as described above in Section 2.3.5. The results are shown in Table 7 below. [Table 7]

[0313] These results clearly demonstrate that exo-Cat B cleavage and drug release (AF-Arg, AF-Cit, ACit, DM1-Mal-Phe-Lys, DM1-Mal-Phe-Cit, and DM1-Mcc-Phe-Cit) in compounds 1, 3, 5, 6-8, 11, and 12 were simultaneous and very rapid. For example, Cat B-mediated drug release from compound 5 occurred 20-fold faster compared to the reference PABC compound Cys-MC-Val-Cit-PABC-MMAF. The rapid cleavage rates achieved by compounds 1-8 and 11-12 demonstrate that compounds of the present invention containing linkers of formula (3) / (4) exhibit high selectivity and binding affinity for the exopeptidase activity of Cat B. Furthermore, surprisingly, the presence of the Ac-Cys-PEG4 moiety on the side chain of the Lys residue was found not to adversely affect the binding affinity of the compounds for Cat B. These results also show that, in contrast to PABC linker systems (e.g., as in the reference compound Cys-MC-Val-Cit-PABC-MMAF), cleavage of Cat B by an endopeptidase-based mechanism occurs at a significantly slower rate. As particularly notable examples, compounds 11 and 12 are spontaneously cleaved by exo-Cat B (T 1 / 2 <1 min), demonstrating the excellent binding properties of substrates based on linkers of formula (3) / (4); the favorable interaction between the C-terminal Tyr and the occluding loop of Cat B likely contributes strongly to the fast cleavage rates observed with these compounds.

Claims

1. The following formula (1): 【Chemistry 1】 wherein Ab is an anti-basal cell adhesion molecule (BCAM) antibody or antigen-binding fragment thereof, comprising: (i) a heavy chain variable region comprising the VH CDR1 sequence of SEQ ID NO: 1, the VH CDR2 sequence of SEQ ID NO: 2, and the VH CDR3 sequence of SEQ ID NO: 3; and (ii) a light chain variable region comprising the VL CDR1 sequence of SEQ ID NO: 4, the VL CDR2 sequence of SEQ ID NO: 5, and the VL CDR3 sequence of SEQ ID NO: 6; D is an antitumor compound conjugated to the Ab via a linker; n is 1 to 10; m is 1 to 5; L is represented by the formula (2): 【Chemistry 2】 (wherein Y is a divalent group containing one or more atoms selected from C, N, O, P, and S; T is a (1+o)- or (2+o)-valent linking group; S is an atom or group optionally present to saturate the free valences of T; L' is a linker cleavable by cathepsin B; o is an integer from 1 to 5; ＊ indicates a covalent bond to the Ab, and ＊＊ is a linker represented by:

2. 2. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 7 and the light chain variable region comprises the sequence of SEQ ID NO:

8.

3. 2. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein n is 3 to 8 and m is 1.

4. 2. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the drug (the anti-tumor compound) to antibody (the anti-BCAM antibody or antigen-binding fragment thereof) ratio (DAR) is from about 3 to about 8.

5. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 4, wherein the DAR is about 4.

6. The antigen-binding fragment thereof may be a Fab fragment, a Fab' fragment, a Fab'-SH fragment, an Fv fragment, an scFv fragment, an F(ab') ２ fragment, VL fragment, VH fragment, ScFv-Fc fragment, and (scFv) ２ The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt thereof, which is an antibody fragment selected from the group consisting of an Fc fragment, a bispecific antibody, a linear antibody, a fragment produced by an Fab expression library, an anti-idiotypic (anti-Id) antibody, a complementarity-determining region (CDR), and an epitope-binding fragment.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the anti-BCAM antibody is a chimeric antibody, a humanized antibody, or a human antibody.

8. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the linker is covalently bound to the anti-BCAM antibody or antigen-binding fragment thereof via a side chain of a cysteine ​​contained in the anti-BCAM antibody or antigen-binding fragment thereof.

9. The linker (L′) that can be cleaved by cathepsin B is represented by formula (3) or formula (4): 【Transformation 3】 (Wherein, Axx is a trifunctional amino acid, provided that Axx in formula (3) is not an amino acid of the (D) configuration, Ayy in formulas (3) and (4) is an amino acid selected from Phe, Ala, Trp, Tyr, phenylglycine (Phg), Met, Val, His, Lys, Arg, citrulline (Cit), 2-aminobutyric acid (Abu), ornithine (Orn), Ser, Thr, Leu, and Ile, or Ayy in formula (3) is homo-tyrosine (homo-Tyr), homo-phenylalanine (homo-Phe), beta-phenylalanine (beta-Phe), and beta-homo-phenylalanine (beta-homo-Phe), Tyr (OR １ ) and homo-Tyr(OR １ ) (wherein, R １ is -(CH ２ CH ２ O) ｎ１ -R ２ and R ２ is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), with the proviso that Ayy in formula (4) is not an amino acid of the (D) configuration, Z is a group covalently bonded to the C-terminus of Ayy or Axx selected from —OH and —N(H)(R), where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; W is a drug-carrying unit; ＊＊ represents a covalent bond to one or more moieties D, and ＊＊＊ indicates a covalent bond to T), The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein when two or more linkers L' are present, each linker is independently selected from the aforementioned linkers of formula (3) and formula (4).

10. At least one or both of Axx and Ayy are defined as follows: Axx, (a) Axx in formula (3) or (4) is an amino acid selected from Glu, 2-amino-pimelic acid (Apa), 2-aminoadipic acid (Aaa), 2,3-diamino-propionic acid (Dap), 2,4-diamino-butyric acid (Dab), Lys, Orn, Ser, amino-malonic acid (Ama), and homo-lysine (homo-Lys); Ayy, (b) Ayy in formula (3) is Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, homo-Tyr, Tyr (OR １ ) and homo-Tyr(OR １ ) (wherein, R １ Ha-(CH ２ CH ２ O) ｎ１ -R ２ and R ２ is a hydrogen atom or a methyl group, and n1 is an integer from 2 to 24; or (c) Ayy in formula (4) is an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, and Ser; The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 9, which satisfies the above.

11. At least one or both of Axx and Ayy are defined as follows: Axx, (a) Axx in formula (3) or (4) is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; Ayy, (b) Ayy in formula (3) is an amino acid selected from Phe and Tyr; or (c) Ayy in formula (4) is an amino acid selected from Phe and Ser; The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 9, which satisfies the above.

12. The drug-carrying unit (W) is represented by formula (5): 【Chemistry 4】 wherein Dxx is an amino acid that is absent or has a hydrophobic side chain; Dyy is absent, Phe, or an amino acid having a basic side chain, with the proviso that when Dxx is an amino acid having a hydrophobic side chain, Dyy is Phe or an amino acid having a basic side chain, and when Dxx is a single covalent bond, Dyy is a single covalent bond, Phe, or an amino acid having a basic side chain; ＊＊ indicates a covalent bond to D, and ＊＊ 10. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 9, wherein Axx or Ayy is a group represented by the formula:

13. At least one of Dxx and Dyy has the following definition: (a) Dxx is an amino acid selected from Phe, Val, Tyr, homo-Phe, and Ala; (b) Dyy is absent or an amino acid selected from Arg, Lys, Cit, Orn, Dap, and Dab; The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 12, which satisfies the above.

14. The drug-carrying unit (W) is represented by formula (6) or formula (7): 【Transformation 5】 (Wherein A''xx is a trifunctional amino acid, provided that A''xx in formula (6) is not an amino acid of the (D) configuration, A'yy is an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn, with the proviso that A'yy in formula (7) is not an amino acid in the (D) configuration; when more than one A'yy is present, each A'yy is independently selected from the foregoing amino acids; A″yy is an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn, with the proviso that A″yy in formula (7) is not an amino acid in the (D) configuration; when more than one A″yy is present, each A″yy is independently selected from the foregoing amino acids; A'''yy is an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn, with the proviso that A'''yy in formula (7) is not an amino acid in the (D) configuration; when more than one A'''yy is present, each A'''yy is independently selected from the foregoing amino acids; A'xx is an amino acid, provided that A'xx in formula (6) is not an amino acid of the (D) configuration, A'''xx is an amino acid, provided that A'''xx in formula (6) is not an amino acid of the (D) configuration, p1 is an integer from 0 to 3, p2 is 0 or 1; p3 is an integer from 0 to 3, provided that when p2 is 0, p3 is not 0; p4 is an integer from 1 to 4, provided that p4 and o in formula (2) are selected such that m in formula (1) is an integer from 1 to 5; ＊＊ ' denotes a covalent bond to the N-terminus of Axx or Ayy, and ＊＊ The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 9, wherein:

15. The following (a) to (f): (a) A'xx is an amino acid selected from Arg, Lys, homo-Lys, Cit, Orn, Dap, and Dab; (b) A''xx is an amino acid selected from Lys, homo-Lys, Cit, Orn, Dap, and Dab; (c) A'''xx is an amino acid selected from Arg, Lys, homo-Lys, Cit, Orn, Dap, and Dab; (d) A'yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (e) A″yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (f) A'''yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 14, which satisfies at least one of the following definitions:

16. The drug-carrying unit is represented by formula (8): 【Transformation 6】 wherein A″xx is a trifunctional amino acid selected from Glu, α-aminoadipic acid (Aaa), Dap, Ser, Thr, homo-serine (homo-Ser), homo-threonine (homo-Thr), and aminomalonic acid (Ama), with the proviso that A″xx is not an amino acid of the (D) configuration; Cxx is a single covalent bond unless A''xx is Ama; when A''xx is Ama, Cxx is Pro or an N-methyl amino acid, the N-terminus of Cxx is attached to the carboxyl terminus of Ama, and the C-terminus of Cxx is covalently attached to one moiety D; A'yy, A''yy, and A'''yy are each independently an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn; A'xx and A'''xx are each independently an amino acid, with the proviso that A'xx and A'''xx are not amino acids of the (D) configuration; p1 is 0 or 1; p2 is 0 or 1; p3 is an integer from 0 to 3, provided that when p2 is 0, p3 is not 0; p4 is an integer from 1 to 4, provided that p4 and o in formula (2) are selected such that m in formula (1) is an integer from 1 to 5; ＊＊ ' denotes a covalent bond to the N-terminus of Axx or Ayy, and ＊＊ The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 9, wherein:

17. The following (a) to (f): (a) A'xx is an amino acid selected from Arg, Lys, homo-Lys, Cit, Orn, Dap, and Dab; (b) A''xx is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; (c) A'''xx is an amino acid selected from Arg, Lys, homo-Lys, Cit, Orn, Dap, and Dab; (d) A'yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (e) A″yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (f) A'''yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; 17. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 16, which satisfies at least one of the following definitions:

18. The linking group (T) is represented by the formula (9): 【Transformation 7】 wherein each AA is independently a moiety comprising a trifunctional amino acid; α represents a covalent bond of the N-terminus of AA to Y, or, when o′ is 2 to 5, a covalent bond of the N-terminus of the first AA to Y; o' is an integer from 1 to 5, provided that the other moiety L' is ＊＊＊ When attached to ', o' is 1 to 4; when o' is 1, the side chain of the trifunctional amino acid is covalently attached to S or L' and the C-terminus is covalently attached to L' or S, respectively, a moiety different from that attached at the side chain; When o' is 2, 3 or 4, ＊＊＊＊ represents a covalent bond to L', ＊＊＊ ' represents a covalent bond to S or L'; The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein when o' is 5, **** represents a covalent bond to L', and ***' represents a covalent bond to S.

19. The antibody-drug conjugate of claim 18, or a pharmaceutically acceptable salt thereof, wherein each AA is independently a moiety comprising an amino acid selected from N-ε-propargyloxycarbonyl-L-lysine (Lys(Poc)), Asp, Glu, Orn, Lys, Dab, and Dap.

20. The linking group (T) is represented by formula (10) or formula (11): 【Transformation 8】 (In the formula, each AA １ and A.A. ２ are independently a moiety that contains a trifunctional amino acid; α represents a covalent bond to Y; In formula (11), the side chain of the trifunctional amino acid is covalently bonded to L' or S, and the C-terminus is covalently bonded to S or L', which is a moiety different from that bonded to the side chain, respectively; In formula (10), ＊＊＊＊ represents a covalent bond to L′, ＊＊＊ 2. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, wherein:

21. Each AA １ and A.A. ２ is a moiety comprising an amino acid selected from Lys(Poc), Asp, Glu, Orn, Lys, Dab, and Dap, or a pharmaceutically acceptable salt thereof.

22. The linking group (T) is represented by formula (12) or formula (13): 【Chemistry 9】 wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, n4 is an integer from 1 to 50, a represents a covalent bond to Y, and ＊＊＊＊ The antibody-drug conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein:

23. The following formula (1): 【Chemistry 10】 wherein Ab is an anti-basal cell adhesion molecule (BCAM) antibody or antigen-binding fragment thereof, comprising: (i) a heavy chain variable region comprising the VH CDR1 sequence of SEQ ID NO: 1, the VH CDR2 sequence of SEQ ID NO: 2, and the VH CDR3 sequence of SEQ ID NO: 3; and (ii) a light chain variable region comprising the VL CDR1 sequence of SEQ ID NO: 4, the VL CDR2 sequence of SEQ ID NO: 5, and the VL CDR3 sequence of SEQ ID NO: 6; D is an antitumor compound conjugated to the Ab via a linker; n is 1 to 10; m is 1 to 5, and L is a linker, The linker (L) is represented by formula (14) or formula (15): 【Chemistry 11】 is expressed as Bxx in formulas (14) and (15) is a trifunctional amino acid, provided that Bxx in formula (14) is not in the (D) configuration, Byy in formulas (14) and (15) is Phe, homo-Phe, Ala, Trp, Tyr, Phg, Val, His, Lys, Abu, Met, Cit, Orn, Ser, Thr, Leu, Ile, Arg, and Tyr (OR １ ) (wherein, R １ is -(CH ２ CH ２ O) ｎ１ -R ２ wherein R ２ is a hydrogen atom or a methyl group, and n1 is an integer from 2 to 24), or Byy in formula (14) is an amino acid selected from homo-Tyr, homo-Tyr (OR １ ) (wherein, R １ is the above Tyr(OR １ )), an amino acid selected from homo-Phe, beta-Phe and beta-homo-Phe; provided that when (q1)×(q3)>1 and q2=0, only the C-terminal Byy in formula (14) may be an amino acid selected from beta-Phe and beta-homo-Phe; provided that Byy in formula (15) is not in the (D) configuration, Bxx in formulas (14) and (15) １ is an amino acid having a single covalent bond or a hydrophobic or basic side chain, Bxx in formulas (14) and (15) ２ is an amino acid with a hydrophobic or basic side chain, Bxx in formulas (14) and (15) ３ is an amino acid, provided that Bxx in formula (14) ３ is not (D) placement, Bxx in formulas (14) and (15) ４ are Phe, Homo-Phe, Ala, Trp, Tyr, Phg, Val, His, Lys, Abu, Met, Cit, Orn, Ser, Thr, Leu, He, Arg and Tyr (OR １ ) (wherein, R １ is -(CH ２ CH ２ O) ｎ１ -R ２ wherein R ２ is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), or Bxx in formula (14) ４ is homo-Tyr, homo-Tyr (OR １ ), homo-Phe, beta-Phe, and beta-homo-Phe; provided that when (q2)×(q3)>1, the C-terminal Bxx in formula (14) is an amino acid selected from ４ may be an amino acid selected from beta-Phe and beta-homo-Phe; provided that Byy in formula (15) is not in the (D) configuration, Or, the linker (L) is represented by formula (16): 【Chemistry 12】 is expressed as Bxx in formula (16) is a carboxyamino acid or a trifunctional amino acid selected from Dap, Dab, Ser, Thr, Lys, Orn, homo-Lys, homo-Ser and homo-Thr, provided that Bxx is not in the (D) configuration; When Bxx is not Ama, Cxx is a single covalent bond; when Bxx is Ama, Cxx is Pro or an N-methyl amino acid, the N-terminus of Cxx is bonded to the carboxyl group of Ama, and the C-terminus of Cxx is covalently bonded to one moiety, D; In formula (16), Byy is Phe, homo-Phe, Ala, Trp, Tyr, Phg, Val, His, Lys, Abu, Met, Cit, Orn, Ser, Thr, Leu, Ile, Arg, homo-Phe, beta-Phe, beta-homo-Phe, homo-Tyr, Tyr (OR １ ) and homo-Tyr(OR １ ) and R １ is -(CH ２ CH ２ O) ｎ１ -R ２ and R ２ represents a hydrogen atom or a methyl group, and n1 represents an integer of 2 to 24, provided that when (q1)×(q3)>1 and q2=0, only the C-terminal Byy may be an amino acid selected from beta-Phe and beta-homo-Phe; Bxx in formula (16) １ is an amino acid having a single covalent bond or a hydrophobic or basic side chain, Bxx in formula (16) ２ is an amino acid with a hydrophobic or basic side chain, Bxx in formula (16) ３ is an amino acid, except for Bxx ３ is not (D) placement, Bxx in formula (16) ４ are Phe, homo-Phe, Ala, Trp, Tyr, Phg, Val, His, Lys, Abu, Met, Cit, Orn, Ser, Thr, Leu, Ile, Arg, homo-Phe, beta-Phe, beta-homo-Phe, homo-Tyr, Tyr (OR １ ) and homo-Tyr(OR １ ) wherein R １ Ha-(CH ２ CH ２ O) ｎ１ -R ２ and R ２ is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24; provided that if (q2) × (q3) > 1, then Bxx at the C-terminus ４ may be only amino acids selected from beta-Phe and beta-homo-Phe; S′ is a divalent group containing one or more atoms selected from C, N, O, P, and S; Z' is Byy or Bxx in formulas (14) and (16). ４ or Bxx or Bxx in formula (15) ３ is a group covalently attached to the C-terminus of q1 is an integer from 0 to 5, q2 is an integer from 0 to 3, provided that when q1 is 0, q2 is not 0; q3 is an integer from 1 to 5, q1, q2, and q3 are selected so that m in formula (1) is an integer from 1 to 5; ＊ indicates a covalent bond to the Ab, and each ＊＊ indicates a covalent bond to one moiety D), or a pharmaceutically acceptable salt thereof.

24. The following (a) to (f): (a) Bxx is an amino acid selected from Dap, Dab, Lys, Orn, Ser, Glu, Ama, Thr, Tyr, Aaa, homo-Ser and homo-Thr; (b) Byy is Cit, Phe, homo-Phe, Ser, Trp, Tyr, and Tyr (OR １ ) wherein R １ is -(CH ２ CH ２ O) ｎ１ -R ２ and R ２ is a hydrogen atom or a methyl group, and n1 is an integer from 2 to 24; (c) Bxx １ is a single covalent bond or an amino acid selected from Phe, homo-Phe, Phg, Val, Ser, Tyr, Ala, Leu, and Ile; (d) Bxx ２ is an amino acid selected from Arg, Lys, Cit, Val, Leu, Ser, Ala, Gly, His, Gln, Phg and Phe; (e) Bxx ３ is an amino acid selected from Phe, homo-Phe, Phg, Val, Ser, Tyr, Ala, Leu, and Ile; (f) Bxx ４ are Cit, Phe, homo-Phe, Ser, Trp, Tyr and Tyr (OR １ ) wherein R １ is -(CH ２ CH ２ O) ｎ１ -R ２ and R ２ is a hydrogen atom or a methyl group, and n1 is an integer from 2 to 24; 24. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 23, which satisfies at least one of the following definitions:

25. Formula (17) or Formula (18): 【Chemistry 13】 (Wherein, Axx is an amino acid selected from Glu, Apa, Aaa, Dap, Dab, Lys, Orn, Ser, Ama, and homo-Lys, provided that Axx in formula (17) is not in the (D) configuration, Ayy in formula (17) is Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, homo-Tyr, Tyr (OR １ ) and homo-Tyr(OR １ ) wherein R １ Ha-(CH ２ CH ２ O) ｎ１ -R ２ and R ２ is a hydrogen atom or a methyl group, n1 is an integer from 2 to 24, Ayy in formula (18) is an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, and Ser, provided that Ayy in formula (18) is not in the (D) configuration, Dxx is an amino acid with a single covalent bond or a hydrophobic side chain; Dyy represents a single covalent bond, Phe, or an amino acid having a basic side chain, provided that when Dxx is an amino acid having a hydrophobic side chain, Dyy is an amino acid having Phe or a basic side chain, and when Dxx is a single covalent bond, Dyy is an amino acid having a single covalent bond, Phe, or a basic side chain; Y is a divalent group containing one or more atoms selected from C, N, O, P, and S; T is a (2+m)-valent linking group; if S is absent, T is a (1+m)-valent linking group; S is an atom or group optionally present to saturate the free valences of T; Z represents a group covalently bonded to the C-terminus of Ayy or Axx selected from -OH and -N(H)(R), where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; and The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Ab, D, m, and n are as defined in claim 1.

26. The following (a) to (k): (a) Axx is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; (b) Ayy in formula (17) is Phe, homo-Phe, Tyr, homo-Tyr, Tyr (OR １ ) and homo-Tyr(OR １ ) is an amino acid selected from (c) Ayy in formula (18) is an amino acid selected from Phe, homo-Phe, or Ser; (d) Dxx is a moiety derived from an amino acid selected from Phe, Val, Tyr, homo-Phe, and Ala; (e) Dyy is a covalent bond or a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap, and Dab; (f) D is a compound selected from a drug selected from auristatin F (AF), MMAF, exatecan, maytansine, DM1, and DM4; (g) Z is —OH or —NH ２ is; (h) T is represented by the formula (9'): 【Chemistry 14】 wherein each AA is independently a moiety comprising a trifunctional amino acid; α represents a covalent bond to Y; m is as defined in claim 1; when m is 1, the side chain of the trifunctional amino acid is covalently attached to S or Axx and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; When m is 2, 3, 4 or 5, ＊＊＊＊ indicates a covalent bond to Axx, ＊＊＊ ' denotes a covalent bond to S via the C-terminus of the chain of AA groups; (i) m is 2 and T is a group represented by the formula (10'): 【Chemistry 15】 (In the formula, each AA １ and A.A. ２ are independently a moiety that contains a trifunctional amino acid; α represents a covalent bond to Y; ＊＊＊＊ indicates a covalent bond to Axx, ＊＊＊ ' denotes a covalent bond to S); (j) m is 1 and T is a group represented by the formula (11′): 【Chemistry 16】 (In the formula, AA １ is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; the side chain of the trifunctional amino acid is covalently attached to Axx or S, and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; (k) m is 1 and T is represented by formula (12′) or formula (13′): 【Chemistry 17】 wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, n4 is an integer from 1 to 50, a represents a covalent bond to Y, and ＊＊＊＊ indicates a covalent bond to Axx; 26. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 25, which satisfies at least one of the following definitions:

27. ​​The following (a) to (i): (a) Axx is Lys; (b) Ayy in formula (17) is Tyr; (c) Ayy in formula (18) is Phe or Ser; (d) Dxx is Phe or Val; (e) Dyy is Arg or Cit; (f) D is a compound selected from a drug selected from AF, MMAF, exatecan, maytansine, DM1, and DM4; (g) Z is —OH or —NH ２ is; (h) m is 1 and T is a group represented by the formula (11'): [Chemistry 18] (In the formula, AA １ is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; the side chain of the trifunctional amino acid is covalently attached to Axx or S, and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; (i) m is 1 and T is a group represented by formula (12′) or formula (13′): 【Chemistry 19】 wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, n4 is an integer from 1 to 50, a represents a covalent bond to Y, and ＊＊＊＊ indicates a covalent bond to Axx; 26. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 25, which satisfies at least one of the following definitions:

28. In formula (17), each Dxx-Dyy-Axx-Ayy is independently Arg-Lys-Phe in which Dxx is a covalent bond, Arg-Lys-homoPhe in which Dxx is a covalent bond, Arg-Lys-Tyr in which Dxx is a covalent bond, Cit-Lys-Phe in which Dxx is a covalent bond, Cit-Lys-Tyr in which Dxx is a covalent bond, Arg- is selected from Lys-homoTyr, Cit-Lys-homoTyr in which Dxx is a covalent bond, Phe-Cit-Lys-Phe, Phe-Cit-Lys-Tyr, Phe-Arg-Lys-Tyr, Phe-Cit-Lys-homoTyr, Phe-Lys-Lys-Phe, homoPhe-Arg-Lys-Phe, homo-Phe-Cit-Lys-Tyr; and The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 25, wherein in formula (18), each Dxx-Dyy-Ayy-Axx is independently selected from Arg-Phe-Lys in which Dxx is a covalent bond, Arg-Ser-Lys in which Dxx is a covalent bond, Cit-Phe-Lys in which Dxx is a covalent bond, Cit-Ser-Lys in which Dxx is a covalent bond, Cit-homoPhe-Lys in which Dxx is a covalent bond, Phe-Cit-Phe-Lys, homoPhe-Cit-Phe-Lys, and Phe-Arg-Phe-Lys.

29. The following (a) to (d): (a) D is a compound selected from a drug selected from AF, MMAF, exatecan, maytansine, DM1, and DM4; (b) Z is —OH or —NH ２ is; (c) m is 1 and T is a group represented by the formula (11'): 【Chemistry 20】 (In the formula, AA １ is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; the side chain of the trifunctional amino acid is covalently attached to Axx or S, and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; (d) m is 1 and T is a group represented by formula (12′) or formula (13′): 【Chemistry 21】 wherein Azz is a single covalent bond or a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, n4 is an integer from 1 to 50, a represents a covalent bond to Y, and ＊＊＊＊ indicates a covalent bond to Axx; 26. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 25, which satisfies at least one of the following definitions:

30. The following formula: 【Chemistry 22】 (In the formula, Y is a divalent group containing one or more atoms selected from C, N, O, P, and S; T is a (2+m)-valent linking group; if S is absent, T is a (1+m)-valent linking group; S is an atom or group optionally present to saturate the free valences of T; Z represents a group covalently attached to the C-terminus of the amino acid selected from -OH and -N(H)(R), where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; and The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein Ab, D, m, and n are as defined in claim 1, with the proviso that Lys is not in the (D) configuration.

31. (a) to (e) of the following: (a) D is a compound selected from a drug selected from AF, MMAF, exatecan, maytansine, DM1, and DM4; (b) Z is —OH or —NH ２ is; (c) m is 2 and T is a group represented by the formula (10'): 【Chemistry 23】 (In the formula, each AA １ and A.A. ２ are independently a moiety that contains a trifunctional amino acid; α represents a covalent bond to Y; ＊＊＊＊ indicates a covalent bond to Lys, ＊＊＊ ' denotes a covalent bond to S); (d) m is 1 and T is a group represented by the formula (11'): 【Chemistry 24】 (In the formula, AA １ is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; the side chain of the trifunctional amino acid is covalently attached to Lys or S, and the C-terminus is covalently attached to a moiety different from that attached at the side chain, either S or Lys, respectively; (e) m is 1 and T is a group represented by formula (12′) or formula (13′): 【Chemistry 25】 wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, n4 is an integer from 1 to 50, a represents a covalent bond to Y, and ＊＊＊＊ indicates a covalent bond to Lys); 31. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 30, which satisfies at least one of the following definitions:

32. D, Z, m and T are defined as follows: (a) D is DM1; (b) Z is —OH or —NH ２ and (c) m is 1 and T is a group represented by the formula (13'): 【Chemistry 26】 wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, a represents a covalent bond to Y, and ＊＊＊＊ The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 30, wherein:

33. D, Z, m and T are defined as follows: (a) D is AF; (b) Z is —OH or —NH ２ and (c) m is 1 and T is a group represented by the formula (13'): 【Chemistry 27】 wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, a represents a covalent bond to Y, and ＊＊＊＊ The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 30, wherein:

34. Formula (19), Formula (20), Formula (21), or Formula (22): 【Chemistry 28】 (In the formula, Axx is a trifunctional amino acid, provided that Axx in formula (19) and formula (20) is not an amino acid of the (D) configuration, Ayy is an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, Orn, Ser, Thr, Leu, and Ile, or Ayy in formula (19) and formula (20) is homo-Tyr, homo-Phe, beta-Phe, beta-homo-Phe, Tyr (OR １ ) and homo-Tyr(OR １ ) wherein R １ is -(CH ２ CH ２ O) ｎ１ -R ２ and R ２ is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24, provided that Ayy in formula (21) and formula (22) is not an amino acid of the (D) configuration, Each A″yy is independently an amino acid selected from Phe, Ala, Trp, Tyr, Phg, Met, Val, His, Lys, Arg, Cit, Abu, and Orn, with the proviso that A″yy in formula (20) and formula (22) is not an amino acid in the (D) configuration, Y is a divalent group containing one or more atoms selected from C, N, O, P, and S; T is a trivalent linking group; if S is absent, T is a divalent linking group; S is an atom or group optionally present to saturate the free valences of T; Z represents a group covalently bonded to the C-terminus of Ayy in formula (19) and formula (20), or to the C-terminus of Axx in formula (21) or formula (22), and is selected from -OH and -N(H)(R) (wherein R represents a hydrogen atom, an alkyl group, or a cycloalkyl group); D1 is an antitumor compound, m' is (m-1), where m is as defined in claim 1, with the proviso that m' is not 0; When m' is 1, A"xx is a trifunctional amino acid, provided that A"xx in formula (19) and formula (21) is not an amino acid of the (D) configuration, and D2 is an antitumor compound; When m' is greater than 1, each D2 is independently selected from a hydrogen atom and an anti-tumor compound, and the multiple moieties D2 can be the same or different, with the proviso that at least one D2 is not a hydrogen atom; when D2 is a hydrogen atom, A''xx is an amino acid with the proviso that A''xx in formula (19) and formula (21) is not an amino acid in the (D) configuration; when D2 is an anti-tumor compound, A''xx is a trifunctional amino acid with the proviso that A''xx in formula (19) and formula (21) is not an amino acid in the (D) configuration, The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Ab and n are as defined in claim 1.

35. The following (a) to (h): (a) Axx is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; (b) Ayy in formula (19) and formula (20) is Phe, homo-Phe, Tyr, homo-Tyr, Tyr (OR １ ) and homo-Tyr(OR １ ) is an amino acid selected from (c) A''xx is an amino acid selected from Lys, homo-Lys, Cit, Orn, Dap, and Dab; (d) A″yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (e) each D1 and D2 is independently a compound selected from AF, MMAF, exatecan, maytansine, DM1, and DM4; (f) Z is —OH or —NH ２ is; (g) m′ is 1 and T is a group represented by the formula (11′): 【Chemistry 29】 (In the formula, AA １ is a moiety containing a trifunctional amino acid, α represents a covalent bond to Y; the side chain of the trifunctional amino acid is covalently attached to Axx or S, and the C-terminus is covalently attached to S or Axx, respectively, a moiety different from that attached at the side chain; (h) m′ is 1, and T is a group represented by formula (12′) or formula (13′): 【Transformation 30】 wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, n4 is an integer from 1 to 50, a represents a covalent bond to Y, and ＊＊＊＊ indicates a covalent bond to Axx; 35. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 34, which satisfies at least one of the following definitions:

36. The following (a) to (g): (a) Axx is an amino acid selected from Dap, Dab, Lys, Orn, and homo-Lys; (b) Ayy in formula (17) is Phe, homo-Phe, Tyr, homo-Tyr, Tyr (OR １ ) and homo-Tyr(OR １ ) is an amino acid selected from (c) A''xx is an amino acid selected from Lys, homo-Lys, Cit, Orn, Dap, and Dab; (d) A″yy is an amino acid selected from Phe, Ala, Trp, Phg, and Tyr; (e) each D1 and D2 is AF; (f) Z is —OH or —NH ２ is; (g) m′ is 1 and T is a group represented by the formula (13′): 【Chemistry 31】 wherein Azz is a moiety containing one or more solubilizing groups; Y1 is a single covalent bond, an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group, or an amino-containing group; Y2 is a single covalent bond, a carbonyl-containing group, or an amino-containing group; n2 is an integer from 0 to 5, n3 is an integer from 1 to 50, a represents a covalent bond to Y, and ＊＊＊＊ indicates a covalent bond to Axx; 35. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 34, which satisfies at least one of the following definitions:

37. 24. The antibody-drug conjugate of claim 1 or 23, or a pharmaceutically acceptable salt thereof, wherein the anti-tumor compound (D) is selected from a DNA alkylating agent, a topoisomerase inhibitor, an RNA polymerase II inhibitor, a DNA cleaving agent, an antimitotic or microtubule-disrupting agent, an antimetabolite, a kinesin spindle protein inhibitor, a kinase inhibitor, a nicotinamide phosphoribosyltransferase inhibitor, a matrix metallopeptidase 9 inhibitor, a phosphatase inhibitor, or a radioactive isotope, and / or a pharmaceutically acceptable salt thereof; and when more than one D is present, each D is independently selected from the foregoing compounds.

38. 24. The antibody-drug conjugate of claim 1 or 23, or a pharmaceutically acceptable salt thereof, wherein the anti-tumor compound D is selected from amanitin, duocarmycin, auristatin F (AF), monomethylauristatin F (MMAF), maytansine, mertansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepines (PBDs) and dimers thereof, indilinobenzodiazepines (IBDs) and dimers thereof, or radioactive isotopes, and / or pharmaceutically acceptable salts thereof; and when more than one D is present, each D is independently selected from the foregoing compounds.

39. The following compounds: 【Chemistry 32】 and 【Transformation 33】 and 【Transformation 34】 2. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt thereof, selected from: (wherein each Ab and n is as defined in claim 1).

40. A pharmaceutical composition comprising the antibody-drug conjugate of claim 1 or 23 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

41. 41. The pharmaceutical composition according to claim 40, which is a pharmaceutical composition for treating or imaging cancer.

42. 42. The pharmaceutical composition of claim 41, wherein the cancer is one or more selected from the group consisting of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain tumor, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, kidney cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, renal cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer, and urothelial cell carcinoma.

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