Oretastatin derivatives and conjugates thereof

Abstract

The present invention provides oretastatin derivatives, oretastatin payloads, and oretastatin conjugates (e.g., single drug conjugates and / or dual drug conjugates), methods of making and using, and intermediates useful in their preparation. Also provided herein are methods of treating cancer and autoimmune diseases with the oretastatin conjugates described herein. Formula (I) imgabs0 #

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 392,806, filed on July 27, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Antibody-drug conjugates (ADCs) have attracted widespread interest as a new class of therapeutic agents. For example, ADCs can use monoclonal antibodies (mAbs) to deliver cytotoxic agents to tumor cells in a targeted manner, thereby enabling the use of highly cytotoxic drugs that cannot be used using traditional non-targeted treatment modalities. The design of ADCs (which are generally characterized by the connection of a cytotoxic agent to an antibody, usually via a linker) involves consideration of a variety of factors, including the presence of a coupling handle on the drug for connection to the linker, and linker technology that connects the drug to the antibody in a conditionally stable manner.

[0004] New cytotoxic agents comprising auristatin derivatives are described herein, which are microtubule inhibitors and can be combined with peptide linkers to form effective loads. The effective loads can be used to prepare single-drug conjugates and / or dual-drug conjugates when combined with cell-binding agents. The ADCs described herein can be used to treat cell proliferative diseases, such as cancer. Summary of the invention

[0005] New cytotoxic agents according to formula (I) are described herein. These cytotoxic agents can be combined with peptide linkers to form an effective load according to formula (II). The resulting effective load can then be used to prepare a single drug conjugate with a cell binding agent, formula (III), and / or for preparing a dual drug conjugate with a cell binding agent when combined with another effective load having a different formula (IV). Compounds of formula (III) and formula (IV) include ADCs that can be used to treat cell proliferative diseases (such as cancer). In addition, incorporating two types of effective loads [e.g., two types of effective loads with different mechanisms of action (MOA)] into an ADC can increase the efficacy of the drug and expand the therapeutic window of the drug.

[0006] Thus, in one aspect, the invention features a compound of formula (I),

[0007] D—Q (I),

[0008] or a pharmaceutically acceptable salt thereof, wherein:

[0009] D is represented by the following structural formula:

[0010] in

[0011] R1 is -H or -OH;

[0012] R 2 C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl;

[0013] Each R 3 and R 4 are independently -H or C1-C3 alkyl;

[0014] n is an integer from 1 to 6; and

[0015] Q is -H or -CH3.

[0016] In an embodiment, R 1 is -H.

[0017] In an embodiment, R 1 It is -OH.

[0018] In an embodiment, R 2 It is -CH3.

[0019] In an embodiment, R 2 It is -C(=O)OH.

[0020] In an embodiment, R 2 It is -C(=O)NHCH2CH2CH2OH.

[0021] In an embodiment, R 2 for

[0022] In an embodiment, R 1 -H and R 2 It is -C(=O)OH.

[0023] In an embodiment, R 1 -H and R 2 It is -C(=O)NHCH2CH2CH2OH.

[0024] In an embodiment, R 1 -H and R 2 for

[0025] In an embodiment, R 1 is -OH and R 2 It is -CH3.

[0026] In an embodiment, R 3 and R4 Both are -H.

[0027] In an embodiment, R 3 and R 4 Both are -CH3.

[0028] In an embodiment, n is 1.

[0029] In an embodiment, Q is -H.

[0030] In an embodiment, Q is -CH3.

[0031] In an embodiment, D is represented by one of the following structures:

[0032]

[0033]

[0034] In an embodiment, the compound has one of the following structures,

[0035]

[0036] or a pharmaceutically acceptable salt thereof.

[0037] In another aspect, the invention features a compound of formula (II),

[0038] D—CH2—NH—E—Z(II),

[0039] or a pharmaceutically acceptable salt thereof, wherein:

[0040] D is represented by the following structural formula:

[0041] in

[0042] R 1 is -H or -OH;

[0043] R 2 C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl;

[0044] Each R 3 and R 4 are independently -H or C1-C3 alkyl;

[0045] n is an integer from 1 to 6;

[0046] E is a peptide comprising 2 to 10 amino acids; wherein E is optionally substituted with one or more polyols; and wherein the N-terminus of the peptide is covalently linked to Z;

[0047] Z is -C(=O)-LY, Wherein m represents an integer from 1 to 10;

[0048] L is -(C1-C 10 Alkylene)-*, -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -、-(OCH2CH2) j -、-CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*; wherein j represents an integer of 1-10; and wherein * represents a site of covalent attachment to Y;

[0049] L1 is -(C1-C 10 Alkylene)-;

[0050] R 5 is -H or -CH3; and

[0051] Y is an electrophilic group or a nucleophilic group.

[0052] In an embodiment, R 1 is -H.

[0053] In an embodiment, R 1 It is -OH.

[0054] In an embodiment, R 2 It is -CH3.

[0055] In an embodiment, R 2 It is -C(=O)OH.

[0056] In an embodiment, R 2 It is -C(=O)NHCH2CH2CH2OH.

[0057] In an embodiment, R 2 for

[0058] In an embodiment, R 1 -H and R 2 It is -C(=O)OH.

[0059] In an embodiment, R 1 -H and R2 It is -C(=O)NHCH2CH2CH2OH.

[0060] In an embodiment, R 1 -H and R 2 for

[0061] In an embodiment, R 1 is -OH and R 2 It is -CH3.

[0062] In an embodiment, R 3 and R 4 Both are -H.

[0063] In an embodiment, R 3 and R 4 Both are -CH3.

[0064] In an embodiment, n is 1.

[0065] In an embodiment, E is a peptide of 2, 3 or 4 amino acids, wherein each amino acid in the peptide is an L amino acid, or at least one amino acid in the peptide is a D amino acid.

[0066] In an embodiment, E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with a polyol.

[0067] In an embodiment, E comprises an amino acid having the structure,

[0068]

[0069] Where R 6 It is -H or C1-C6 alkyl.

[0070] In an embodiment, E comprises an amino acid having the structure,

[0071]

[0072] In an embodiment, the following composition is composed of: -Ala-Val-*、-Val-Ala-*、-Gly-Gly-*、-Val-Cit-*、-Cit-Val-*、-Leu-Ala-*、-Ala-Leu-*、-Leu-Cit-*、-Cit-Leu-*、-Leu-Ala-*、-Ala-Leu-*、-Ly s-Lys-*、-Ala-Lys-*、-Lys-Ala-*、-Val-Lys-*、-Lys-Val-*、-Tyr-Arg-*、-Arg-Tyr-*、-Arg-Arg-*、-Ala-Ala-*、-Phe-Lys-*、-Lys-Phe-*、-Thr-Thr-*、-Thr-Met-*、-Met t-Thr-*、-Met-Tyr-*、-Tyr-Met-*、-Phe-Gln-*、-Gln-Phe-*、-Gly-Ser-*、-Leu-Gln-*、-Gln-Leu-*、-Ser-Ala-*、-Ser-Gly-*、-Val-Thr-*、-Thr-Val-*、-Val-Gln-*、-S er-Val-*、-Val-Ser-*、-Ala-Met-*、-Met-Ala-*、-Val-Arg-*、-Arg-Val-*、-Phe-Ala-*、-Ala-Phe-*、-Cit-Val-*、-Gln-Val-*、-Phe-Arg-*、-Arg-Phe-*、-Ala-Ala-* * 、-Gly-Gly-Gly- * 、-Ala-Val-Ala- * 、-Gly-Val-Gly- * 、-Ala-Val-Gly- * 、-Gly-Phe-Lys-*、-Lys-Phe-Gly-*、-Leu-Ala-Leu-*、-Val-Ala-Leu-*、-Leu-Ala-Val-*、-Val-Ala-Val-*、-Ala-Val-Ala-Gly- * 、-Gly-Phe-Gly-Gly- * 、-Gly-Gly-Phe-Gly-*、-Ala-Val-Gly-Gly-*、-Ala-Ala-Ala-Ala- * 、-Ala-Val-Ala-Ala- *, -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-*, wherein * indicates the N-terminus of the peptide covalently linked to Z.

[0073] In an embodiment, E is selected from the group consisting of: -L-Ala-L-Val-*, -L-Val-L-Ala-*, -L-Val-L-Lys-*, -L-Val-L-Arg-*, -L-Val-L-Cit-*, -L-Ala-L-Val-L-Glu-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-L-Val-L-Ala-*, -L-Ala-L-Ala-Gly-*, -L-Ala-L-Val-Gly-*, -Gly-Gly-L-Glu-*, -Gly-L-Phe-Gly-Gly-*, -Gly-L-Glu-Gly-Gly-*; wherein * indicates the N-terminus of the peptide covalently linked to Z.

[0074] In an embodiment, Z is -C(=O)-LY.

[0075] In an embodiment, Z is Here, m represents an integer from 1 to 10.

[0076] In an embodiment, Z is Here, m represents an integer from 1 to 10.

[0077] In an embodiment, L is -(C1-C 10 Alkylene)-.

[0078] In an embodiment, L is -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -or-(OCH2CH2) j -, wherein j represents an integer of 1 to 10; and wherein * represents a site for covalent bonding to Y.

[0079] In an embodiment, L is -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*, wherein j represents an integer of 1-10; and wherein * represents a site for covalent bonding to Y.

[0080] In an embodiment, L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, where * indicates the site of covalent attachment to Y.

[0081] In an embodiment, Y is a Michael acceptor group, succinimide, epoxy or halogen.

[0082] In an embodiment, Y is

[0083]

[0084] Where R 7 and R 8 Each is independently H or C1-C3 alkyl.

[0085] In an embodiment, Z is

[0086]

[0087] In the embodiments, -E-NH-CH 2- has one of the following structures, where * indicates the N-terminus of the peptide covalently linked to Z:

[0088]

[0089]

[0090] In an embodiment, ZE-NH-CH2- has one of the following structures,

[0091] In an embodiment, D is represented by one of the following structures:

[0092]

[0093] In an embodiment, the compound has one of the following structures,

[0094]

[0095]

[0096] or a pharmaceutically acceptable salt thereof.

[0097] In yet another aspect, the invention features a compound of formula (III),

[0098] {D—CH2—NH—E—Z'}p —C (III),

[0099] in:

[0100] D is represented by the following structural formula:

[0101] in

[0102] R 1 is -H or -OH;

[0103] R 2 C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl;

[0104] R 3 and R 4 are independently -H or C1-C3 alkyl;

[0105] n is an integer from 1 to 6;

[0106] E is a peptide comprising 2 to 10 amino acids; wherein E is optionally substituted with one or more polyols; and wherein the N-terminus of the peptide is covalently linked to Z';

[0107] Z' is -C(=O)-L-Y'-, wherein m represents an integer of 1-10, and * represents a site covalently linked to the C;

[0108] L is -(C1-C 10 Alkylene)-*, -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -、-(OCH2CH2) j -、-CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*; wherein j represents an integer from 1 to 10; and wherein * represents a site of covalent attachment to Y';

[0109] L1 is -(C1-C 10 Alkylene)-;

[0110] R 5 is -H or -CH3; and

[0111] C represents a cell-binding agent;

[0112] Y' is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on the cell binding agent;

[0113] p has a value between 1 and 18.

[0114] In an embodiment, R 1 is -H.

[0115] In an embodiment, R 1 It is -OH.

[0116] In an embodiment, R 2 It is -CH3.

[0117] In an embodiment, R 2 It is -C(=O)OH.

[0118] In an embodiment, R 2 It is -C(=O)NHCH2CH2CH2OH.

[0119] In an embodiment, R 2 for

[0120] In an embodiment, R 1 -H and R 2 It is -C(=O)OH.

[0121] In an embodiment, R 1 -H and R 2 It is -C(=O)NHCH2CH2CH2OH.

[0122] In an embodiment, R 1 -H and R 2 for

[0123] In an embodiment, R 1 is -OH and R 2 It is -CH3.

[0124] In an embodiment, R 3 and R 4 Both are -H.

[0125] In an embodiment, R 3 and R 4 Both are -CH3.

[0126] In an embodiment, n is 1.

[0127] In an embodiment, E is a peptide of 2, 3 or 4 amino acids, wherein each amino acid in the peptide is an L amino acid, or at least one amino acid in the peptide is a D amino acid.

[0128] In an embodiment, E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with a polyol.

[0129] In an embodiment, E comprises an amino acid having the structure,

[0130]

[0131] Where R 6 It is -H or C1-C6 alkyl.

[0132] In an embodiment, E comprises an amino acid having the structure,

[0133]

[0134] In an embodiment, E is selected from the group consisting of: -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Val-Cit-*, -Cit-Val-*, -Leu-Ala-*, -Ala-Leu-*, -Leu-Cit-*, -Cit-Leu-*, -Leu-Ala-*, -Ala-Leu-*, -Lys-Lys-*, -Ala-Lys-*, -Lys-Ala-*, -Val-Lys-*, -Lys-Val-*, -Tyr-Arg-*, -Arg-Tyr-*, -Arg-Arg-*, -Ala-Ala-*, -Phe-Lys-*, -Lys-Phe-*, -Thr-Thr-*, -Thr-Met-*, -Met- t-Thr-*, -Met-Tyr-*, -Tyr-Met-*, -Phe-Gln-*, -Gln-Phe-*, -Gly-Ser-*, -Leu-Gln -*, -Gln-Leu-*, -Ser-Ala-*, -Ser-Gly-*, -Val-Thr-*, -Thr-Val-*, -Val-Gln-*, -S er-Val-*, -Val-Ser-*, -Ala-Met-*, -Met-Ala-*, -Val-Arg-*, -Arg-Val-*, -Phe-Ala-*, -Ala-Phe-*, -Cit-Val-*, -Gln-Val-*, -Phe-Arg-*, -Arg-Phe-*, -Ala-Ala-Ala- *、-Gly-Gly-Gly- * 、-Ala-Val-Ala- * 、-Gly-Val-Gly- * 、-Ala-Val-Gly- * , -Gly-Phe-Lys-*, -Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Val-Ala-Leu-*, -Leu-Ala-Val-*, -Val-Ala-Val-*, -Ala-Val-Ala-Gly- * 、-Gly-Phe-Gly-Gly- * , -Gly-Gly-Phe-Gly-*, -Ala-Val-Gly-Gly-*, -Ala-Ala-Ala-Ala- * ,-Ala-Val-Ala-Ala- * , -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-*, wherein * indicates the N-terminus of the peptide covalently linked to Z'.

[0135] In an embodiment, E is selected from the group consisting of: -L-Ala-L-Val-*, -L-Val-L-Ala-*, -L-Val-L-Lys-*, -L-Val-L-Arg-*, -L-Val-L-Cit-*, -L-Ala-L-Val-L-Glu-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-L-Val-L-Ala-*, -L-Ala-L-Ala-Gly-*, -L-Ala-L-Val-Gly-*, -Gly-Gly-L-Glu-*, -Gly-L-Phe-Gly-Gly-*, -Gly-L-Glu-Gly-Gly-*; wherein * indicates the N-terminus of the peptide covalently linked to Z'.

[0136] In an embodiment, Z' is -C(=O)-L-Y'-.

[0137] In an embodiment, Z' is wherein m represents an integer of 1 to 10, and * represents a site for covalent bonding to the C.

[0138] In an embodiment, Z' is wherein m represents an integer of 1 to 10, and * represents a site for covalent bonding to the C.

[0139] In an embodiment, L is -(C1-C10 Alkylene)-.

[0140] In an embodiment, L is -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -or-(OCH2CH2) j -, wherein j represents an integer of 1 to 10; and wherein * represents a site of covalent bonding to Y'.

[0141] In an embodiment, L is -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*, wherein j represents an integer of 1 to 10; and wherein * represents a site of covalent bonding to Y'.

[0142] In an embodiment, L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, wherein * indicates the site of covalent attachment to Y'.

[0143] In embodiments, Y' is a group formed by reaction of an electrophilic group with a reactive nucleophilic group present on the cell binding agent.

[0144] In an embodiment, Y' is formed by

[0145]

[0146] Where R 7 and R 8 Each is independently -H or C1-C3 alkyl.

[0147] In an embodiment, Y' is

[0148]

[0149] Where R 7 and R 8 Each is independently -H or C1-C3 alkyl, and * indicates the site of covalent attachment to the C.

[0150] In an embodiment, Z' is formed by:

[0151]

[0152] In an embodiment, Z' is:

[0153]

[0154] Where * indicates the site of covalent attachment to C.

[0155] In the embodiments, -E-NH-CH 2- has one of the following structures, where * indicates the N-terminus of the peptide covalently linked to Z':

[0156]

[0157] In an embodiment, -Z'-E-NH-CH2- is formed by one of the following structures:

[0158]

[0159] In the embodiment, -Z'-E-NH-CH2- is one of the following structures, wherein * represents the connection with C

[0160] In an embodiment, D is represented by one of the following structures:

[0161]

[0162]

[0163] In an embodiment, D—CH2—NH—E—Z′— is formed by one of the following structures,

[0164]

[0165]

[0166] In an embodiment, {D—CH2—NH—E—Z'} p — C is one of the following structures, wherein C is a monoclonal antibody and p is the drug to antibody ratio (DAR), and p is an average of about 2-8, 4-8, or 7-8,

[0167]

[0168]

[0169] In embodiments, p is about 3-8 (eg, 3.2 to 8.0) or an average of 4-8.

[0170] In an embodiment, p is an average number of about 4.

[0171] In an embodiment, p is 4.

[0172] In an embodiment, p is an average number of about 7.5.

[0173] In an embodiment, p is an average number of about 8.

[0174] In an embodiment, p is 8.

[0175] In yet another aspect, the invention features a compound of formula (IV),

[0176] {D—CH2—NH—E—Z'} p' —C—{W} t (IV),

[0177] in:

[0178] D is represented by the following structural formula:

[0179] in

[0180] R 1 is -H or -OH;

[0181] R 2 C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl;

[0182] R 3 and R 4 are independently -H or C1-C3 alkyl;

[0183] n is an integer from 1 to 6;

[0184] E is a peptide comprising 2 to 10 amino acids; wherein E is optionally substituted with one or more polyols; and wherein the N-terminus of the peptide is covalently linked to Z';

[0185] Z' is -C(=O)-L-Y'-, wherein m represents an integer of 1-10, and * represents a site covalently linked to the C;

[0186] L is -(C1-C 10 Alkylene)-*, -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -、-(OCH2CH2) j -、-CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2)j N(R 5 )C(=O)-L1-*; wherein j represents an integer from 1 to 10; and wherein * represents a site of covalent attachment to Y';

[0187] L1 is -(C1-C 10 Alkylene)-;

[0188] R 5 is -H or -CH3; and

[0189] C represents a cell-binding agent;

[0190] Y' is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on the cell binding agent;

[0191] W is a group formed by the reaction of compound W' with a reactive nucleophilic group present on C; wherein W' is a cell-killing agent attached to a linker so that W' can be coupled to C;

[0192] p' and t have values ​​between 1 and 10; wherein p' and t may be the same or different numbers, and p':t is about 1:1, about 1:2 or about 2:1, and wherein p':t is 1:1 or 1:2 or 2:1.

[0193] In an embodiment, R 1 is -H.

[0194] In an embodiment, R 1 It is -OH.

[0195] In an embodiment, R 2 It is -CH3.

[0196] In an embodiment, R 2 It is -C(=O)OH.

[0197] In an embodiment, R 2 It is -C(=O)NHCH2CH2CH2OH.

[0198] In an embodiment, R 2 for

[0199] In an embodiment, R 1 -H and R 2 It is -C(=O)OH.

[0200] In an embodiment, R 1 -H and R 2 It is -C(=O)NHCH2CH2CH2OH.

[0201] In an embodiment, R 1 -H and R2 for

[0202] In an embodiment, R 1 is -OH and R 2 It is -CH3.

[0203] In an embodiment, R 3 and R 4 Both are -H.

[0204] In an embodiment, R 3 and R 4 Both are -CH3.

[0205] In an embodiment, n is 1.

[0206] In an embodiment, E is a peptide of 2, 3 or 4 amino acids, wherein each amino acid in the peptide is an L amino acid, or at least one amino acid in the peptide is a D amino acid.

[0207] In an embodiment, E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with a polyol.

[0208] In an embodiment, E comprises an amino acid having the structure,

[0209]

[0210] Where R 6 It is -H or C1-C6 alkyl.

[0211] In an embodiment, E comprises an amino acid having the structure,

[0212]

[0213] In an embodiment, the following composition is composed of: -Ala-Val-*、-Val-Ala-*、-Gly-Gly-*、-Val-Cit-*、-Cit-Val-*、-Leu-Ala-*、-Ala-Leu-*、-Leu-Cit-*、-Cit-Leu-*、-Leu-Ala-*、-Ala-Leu-*、-Ly s-Lys-*、-Ala-Lys-*、-Lys-Ala-*、-Val-Lys-*、-Lys-Val-*、-Tyr-Arg-*、-Arg-Tyr-*、-Arg-Arg-*、-Ala-Ala-*、-Phe-Lys-*、-Lys-Phe-*、-Thr-Thr-*、-Thr-Met-*、-Met t-Thr-*、-Met-Tyr-*、-Tyr-Met-*、-Phe-Gln-*、-Gln-Phe-*、-Gly-Ser-*、-Leu-Gln-*、-Gln-Leu-*、-Ser-Ala-*、-Ser-Gly-*、-Val-Thr-*、-Thr-Val-*、-Val-Gln-*、-S er-Val-*、-Val-Ser-*、-Ala-Met-*、-Met-Ala-*、-Val-Arg-*、-Arg-Val-*、-Phe-Ala-*、-Ala-Phe-*、-Cit-Val-*、-Gln-Val-*、-Phe-Arg-*、-Arg-Phe-*、-Ala-Ala-* * 、-Gly-Gly-Gly- * 、-Ala-Val-Ala- * 、-Gly-Val-Gly- * 、-Ala-Val-Gly- * 、-Gly-Phe-Lys-*、-Lys-Phe-Gly-*、-Leu-Ala-Leu-*、-Val-Ala-Leu-*、-Leu-Ala-Val-*、-Val-Ala-Val-*、-Ala-Val-Ala-Gly- * 、-Gly-Phe-Gly-Gly- * 、-Gly-Gly-Phe-Gly-*、-Ala-Val-Gly-Gly-*、-Ala-Ala-Ala-Ala- * 、-Ala-Val-Ala-Ala- *, -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-*, wherein * indicates the N-terminus of the peptide covalently linked to Z'.

[0214] In an embodiment, E is selected from the group consisting of: -L-Ala-L-Val-*, -L-Val-L-Ala-*, -L-Val-L-Lys-*, -L-Val-L-Arg-*, -L-Val-L-Cit-*, -L-Ala-L-Val-L-Glu-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-L-Val-L-Ala-*, -L-Ala-L-Ala-Gly-*, -L-Ala-L-Val-Gly-*, -Gly-Gly-L-Glu-*, -Gly-L-Phe-Gly-Gly-*, -Gly-L-Glu-Gly-Gly-*; wherein * indicates the N-terminus of the peptide covalently linked to Z'.

[0215] In an embodiment, Z' is -C(=O)-L-Y'-.

[0216] In an embodiment, Z' is wherein m represents an integer of 1 to 10, and * represents a site for covalent bonding to the C.

[0217] In an embodiment, Z' is wherein m represents an integer of 1 to 10, and * represents a site for covalent bonding to the C.

[0218] In an embodiment, L is -(C1-C 10 Alkylene)-.

[0219] In an embodiment, L is -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -or-(OCH2CH2) j -, wherein j represents an integer of 1 to 10; and wherein * represents a site of covalent bonding to Y'.

[0220] In an embodiment, L is -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*, wherein j represents an integer of 1 to 10; and wherein * represents a site of covalent bonding to Y'.

[0221] In an embodiment, L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, wherein * indicates the site of covalent attachment to Y'.

[0222] In embodiments, Y' is a group formed by reaction of an electrophilic group with a reactive nucleophilic group present on the cell binding agent.

[0223] In an embodiment, Y' is formed by

[0224]

[0225] Where R 7 and R 8 Each is independently -H or C1-C3 alkyl.

[0226] In an embodiment, Y' is

[0227]

[0228] Where R 7 and R 8 Each is independently -H or C1-C3 alkyl, and * indicates the site of covalent attachment to the C.

[0229] In an embodiment, Z' is formed by:

[0230]

[0231] In an embodiment, Z' is:

[0232]

[0233]

[0234] Where * indicates the site of covalent attachment to C.

[0235] In the embodiments, -E-NH-CH 2- has one of the following structures, where * indicates the N-terminus of the peptide covalently linked to Z':

[0236]

[0237] In an embodiment, -Z'-E-NH-CH2- is formed by one of the following structures:

[0238]

[0239] In an embodiment, -Z'-E-NH-CH2- is one of the following structures, wherein * represents the point of connection to C:

[0240]

[0241]

[0242] In an embodiment, D is represented by one of the following structures:

[0243]

[0244] In an embodiment, D—CH2—NH—E—Z′— is formed by one of the following structures,

[0245]

[0246]

[0247] In an embodiment, W is formed by covalently linking compound W' to C.

[0248] In an embodiment, W' is any molecule that can be covalently attached to C.

[0249] In an embodiment, D—CH2—NH—E—Z′— is formed from PL1, and W is formed from PL2.

[0250] In an embodiment, D—CH2—NH—E—Z′— is formed from PL3, and W is formed from PL4.

[0251] In an embodiment, D—CH2—NH—E—Z′— is formed from PL5, and W is formed from PL6.

[0252] In an embodiment, D—CH2—NH—E—Z′— is formed from PL7, and W is formed from PL8.

[0253] In an embodiment, D—CH2—NH—E—Z′— is formed from PL9, and W is formed from PL10.

[0254] In an embodiment, W' is any compound described in patent WO2021 / 173773 A1, which can be covalently linked to C.

[0255] In an embodiment, W' is any compound described in US2021 / 0283125 A1, which can be covalently linked to C.

[0256] In the embodiment, W' is meditecan described in patent WO2021 / 173773 A1.

[0257] In an embodiment, {D—CH2—NH—E—Z'} p' —C—{W} t is one of the following structures, wherein C is a monoclonal antibody, p' and t are drug to antibody ratio (DAR), and p':t is 1:1 or about 1:1, and p' and t are respectively an average of about 1-7, or an average of about 2, about 3, about 4, about 5, or about 6:

[0258]

[0259] In an embodiment, p' and t are both 4, or an average of 4, or about 4.

[0260] In embodiments, p':t is about 1:1, about 1:2, or about 2:1.

[0261] In embodiments, p':t is 1:1, 1:2 or 2:1.

[0262] In another aspect, the invention features a method of preparing a conjugate of formula (III) comprising a cell-binding agent and a drug, and the method comprises contacting the cell-binding agent with a compound of formula (II) such that a covalent bond is formed between the cell-binding agent and the compound of formula (II).

[0263] In yet another aspect, the invention features a method for preparing a two-drug conjugate of formula (IV) comprising a cell-binding agent and two different drugs, and the method comprises contacting the cell-binding agent with a compound of formula (II) and another compound of a different formula, such that a covalent bond is formed between the cell-binding agent and the compound of formula (II) and another compound having a formula different from formula (II).

[0264] In yet another aspect, the invention features a conjugate comprising a cell-binding agent and a drug. In embodiments, the conjugate is prepared according to any of the methods described herein.

[0265] In embodiments, the conjugate comprises a cell binding agent that is an antibody or antigen binding fragment thereof.

[0266] In embodiments, the conjugate comprises a cell binding agent that is a monoclonal antibody or an antigen binding fragment thereof.

[0267] In embodiments, the cell-binding agent is an antibody or antigen-binding fragment thereof; p is the drug-to-antibody ratio (DAR) and has a value between 1 and 18. In embodiments, p is an average of about 2-10, 4-8, or 7-8 (e.g., 3.2 to 8.0).

[0268] In embodiments, the cell-binding agent is a monoclonal antibody or an antigen-binding fragment thereof; p is the drug-to-antibody ratio (DAR) and has a value between 1 and 18. In embodiments, p is an average of about 2-10, 4-8, or 7-8 (e.g., 3.2 to 8.0).

[0269] In embodiments, the cell-binding agent is an antibody or antigen-binding fragment thereof; p' and t are drug-to-antibody ratios (DAR) and have values ​​between 1 and 10. In embodiments, p' is an average of about 2-6. In embodiments, t is an average of about 2-6. In embodiments, p':t is about 1:1. In embodiments, p':t is about 1:2. In embodiments, p':t is about 2:1. In embodiments, p':t is 1:1. In embodiments, p':t is 1:2. In embodiments, p':t is 2:1.

[0270] In embodiments, the cell-binding agent is a monoclonal antibody or an antigen-binding fragment thereof; p' and t are drug-to-antibody ratios (DAR) and have values ​​between 1 and 10. In embodiments, p' is an average of about 2-6. In embodiments, t is an average of about 2-6. In embodiments, p':t is about 1:1. In embodiments, p':t is about 1:2. In embodiments, p':t is about 2:1. In embodiments, p':t is 1:1. In embodiments, p':t is 1:2. In embodiments, p':t is 2:1.

[0271] In an embodiment, the compound of formula (IV) is trastuzumab-MB0324,

[0272]

[0273] In an embodiment, the compound of formula (IV) is trastuzumab-MB0326,

[0274]

[0275] In another aspect, the invention features a pharmaceutical composition including any of the conjugates described herein.

[0276] In yet another aspect, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, wherein the method comprises administering any of the conjugates described herein or any pharmaceutical composition comprising any of the conjugates described herein.

[0277] In another aspect, the invention features a pharmaceutical composition comprising a compound of any of Formula (III) as described herein.

[0278] In another aspect, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, the method comprising administering a compound of any of Formula (III) as described herein or a pharmaceutical composition comprising a compound of any of Formula (III) as described herein.

[0279] In another aspect, the invention features a pharmaceutical composition comprising a compound of any of Formula (IV) as described herein.

[0280] In another aspect, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, the method comprising administering a compound of any of Formula (IV) as described herein or a pharmaceutical composition comprising a compound of any of Formula (IV) as described herein.

[0281] In embodiments, the method is used to treat cancer.

[0282] In embodiments, the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, central nervous system tumor (CNS) tumor, colon cancer or colorectal cancer, diffuse intrinsic pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood cancer, Hodgkin's lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome (MDS), neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, pancreatic cancer, peritoneal cancer, prostate cancer, ovarian cancer, kidney cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, or Wilms tumor.

[0283] In embodiments, the cancer is breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0284] Figure 1 It is shown that all three ADCs (Trastuzumab-MB24-DAR4, Trastuzumab-MB3-DAR8, and Trastuzumab-MB0324) exhibited dose-dependent anti-tumor activity (0.3, 1, and / or 3 mg / kg).

[0285] Figure 2It is depicted that trastuzumab-MB26-DAR4, trastuzumab-MB3-DAR4, and trastuzumab-MB0326 all exhibited dose-dependent anti-tumor activity (1 and 3 mg / kg), with >90% TGI induced at the 3 mg / kg dose.

[0286] Figure 3 It is shown that all three ADCs (trastuzumab-MB24-DAR4, trastuzumab-MB3-DAR8, and trastuzumab-MB0324) exhibited dose-dependent anti-tumor activity (0.3, 1, and / or 3 mg / kg), with 3 mg / kg trastuzumab-MB0324 showing activity comparable to trastuzumab-MB24-DAR4.

[0287] Figure 4 It is shown that trastuzumab-MB26-DAR4, trastuzumab-MB3-DAR4, and trastuzumab-MB0326 all exhibited dose-dependent anti-tumor activity (1 and 3 mg / kg). DETAILED DESCRIPTION

[0288] definition

[0289] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings.When a trade name is used herein, unless the context indicates otherwise, the trade name includes product formulations of the trade name product, generic drugs, and one or more pharmaceutically active ingredients.

[0290] As used herein, the term "antibody" refers to an agent that specifically binds to a specific antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex including immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibodies include, but are not limited to, monoclonal antibodies or polyclonal antibodies. In some embodiments, antibodies may include one or more constant region sequences unique to mouse, rabbit, primate or human antibodies. In some embodiments, antibodies may include one or more humanized, primatized, chimeric, etc. sequence elements as known in the art. In many embodiments, the term "antibody" is used to refer to one or more constructs or forms known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, in an embodiment, the antibodies used according to the present invention are selected from the following but are not limited to the following forms: complete IgA, IgG, IgE or IgM antibodies; bispecific or multispecific antibodies (e.g., etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments and isolated CDRs or collections thereof; single-chain Fvs; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof); camel antibodies; shielded antibodies (e.g., ); Small modular immune drugs ( S mall M odular I mmuno P harmaceuticals, “SMIPs TM ”); single-chain or tandem diabodies VHH; Mini-antibodies; Ankyrin repeat protein or DART;TCR-like antibody; MicroProteins; and In some embodiments, the antibody may lack covalent modifications that it may have when it is naturally produced (e.g., linked to a glycan). In some embodiments, the antibody may include covalent modifications (e.g., linked to a glycan, a payload [e.g., a detectable portion, a therapeutic portion, a catalytic portion, etc.] or other pendant groups [e.g., polyethylene glycol, etc.]). In many embodiments, the antibody is or includes a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementary determining regions (CDRs); in some embodiments, the antibody is or includes a polypeptide whose amino acid sequence includes at least one CDR substantially identical to a CDR found in a reference antibody (e.g., at least one heavy chain CDR and / or at least one light chain CDR). In some embodiments, the antibody agent is or includes a polypeptide whose amino acid sequence includes a structural element recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain.

[0291] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single antigenic site. The modifier "monoclonal" indicates the characteristic of the antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method.

[0292] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions generated (or assembled) from human immunoglobulin sequences. In some embodiments, an antibody (or antibody component) may be considered "human" even if its amino acid sequence, for example in one or more CDRs and in particular CDR3, includes residues or elements that are not encoded by human germline immunoglobulin sequences (e.g., including sequence variations that may be (initially) introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0293] As known in the art, the term "humanized" is generally used to refer to an amino acid sequence that includes V or V sequences from a reference antibody produced in a non-human species (e.g., mouse). H and V L In some embodiments, "humanized" antibodies (or antibody components) are antibodies that immunospecifically bind to related antigens and have a framework (FR) region and a complementary determining region (CDR), wherein the framework region has an amino acid sequence substantially identical to a human antibody amino acid sequence, and the complementary determining region has an amino acid sequence substantially identical to a non-human antibody amino acid sequence. Humanized antibodies include substantially all at least one, and usually two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all CDR regions correspond to the CDR regions of non-human immunoglobulins (i.e., donor immunoglobulins), and all or substantially all framework regions are framework regions of human immunoglobulin consensus sequences. In some embodiments, humanized antibodies also include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. In some embodiments, humanized antibodies contain a light chain, and at least two variable domains of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and optionally CH4 regions of the heavy chain constant region. In some embodiments, the humanized antibody contains only humanized V L In some embodiments, the humanized antibody contains only humanized V H In some specific embodiments, the humanized antibody contains a humanized V H and V L district.

[0294] An "intact antibody" is one that contains an antigen-binding variable region and a light chain constant domain (C L ) and heavy chain constant domain C H 1. C H 2. C H 3 and C H4, depending on the antibody class. The constant domain may be a native sequence constant domain (eg, a human native sequence constant domain) or an amino acid sequence variant thereof.

[0295] "Antibody fragments" include a portion of an intact antibody, including its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments that immunospecifically bind to a target antigen (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen), diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed by one or more antibody fragments, one or more fragments produced by a Fab expression library, or an antigen epitope binding fragment of any of the above.

[0296] An "antigen" is the entity to which an antibody specifically binds.

[0297] It should be understood that as used herein, the term "binding" generally refers to a non-covalent association between or among two or more entities. "Direct" binding involves physical contact between entities or moieties; indirect binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can generally be evaluated in any of a variety of contexts, including where the interacting entities or moieties are studied independently or in the context of more complex systems (e.g., when covalently or otherwise associated with a carrier entity and / or in a biological system or cell). In some embodiments, "binding" refers to a type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction can be measured based on the dissociation constant (K) of the interaction. d ) indicates that the smaller K d =K indicates greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of formation and dissociation of antigen binding sites / antigen complexes, where these rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "association rate constant" (K on ) and the “dissociation rate constant” (K off ) can be determined by calculating the concentration and the actual association and dissociation rates. (See Nature 361: 186-87 (1993)). off / K on The ratio cancels out all parameters not related to affinity and is equal to the dissociation constant K d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473).

[0298] The terms "specific binding" and "specifically bind" mean that the antibody or antibody derivative will bind to the corresponding epitope of its target antigen in a highly selective manner and will not do so with many other antigens. Typically, the antibody or antibody derivative will bind to the corresponding epitope of its target antigen in a highly selective manner with a specificity of at least about 1×10 -7 M, and preferably 10 -8 M to 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 The term "specificity" refers to the ability of a cell-binding agent (e.g., such as an antibody or fragment thereof, as described herein) to specifically bind to (e.g., immunoreact with) a given target antigen, e.g., a human target antigen.

[0299] In general, a "protein" is a polypeptide (i.e., a string of at least two amino acids linked to each other by peptide bonds). A protein may include moieties other than amino acids (e.g., may be a glycoprotein) and / or may be otherwise processed or modified. One of ordinary skill in the art will appreciate that a "protein" may be an entire polypeptide chain (with or without a signal sequence) as produced by a cell, or may be a functional portion thereof. One of ordinary skill will further appreciate that a protein may sometimes include more than one polypeptide chain, which may be linked, for example, by one or more disulfide bonds or associated by other means.

[0300] The term "inhibit" or "inhibition of" means to reduce by a measurable amount or to prevent entirely.

[0301] The term "substantially" or "essentially" refers to a majority of a population, mixture or sample, i.e., >50%, preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more of the population.

[0302] The term "cytotoxic activity" refers to the cell-killing effect of a drug or auristatin conjugate or an intracellular metabolite of an auristatin conjugate. Cytotoxic activity may be expressed as IC 50 A value which is the concentration per unit volume (molar or mass) at which half of the cells survive.

[0303] The term "cytostatic activity" refers to the antiproliferative effect of a drug or an auristatin conjugate or an intracellular metabolite of an auristatin conjugate.

[0304] As used herein, the term "cytotoxic agent" refers to a substance that has cytotoxic activity and causes cell destruction. The term is intended to include chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.

[0305] As used herein, the term "cytostatic" refers to a substance that inhibits cellular function, including cell growth or proliferation. Cytostatics include inhibitors, such as protein inhibitors, such as enzyme inhibitors. Cytostatics have cytostatic activity.

[0306] As used herein, the term "payload" refers to a substance that can be attached to a cell-binding agent. The payload consists of a cytotoxic agent and a linker.

[0307] As defined herein, a "linker," "linker moiety," or "linking group" refers to a moiety that connects two groups, such as a cell-binding agent and a cytotoxic compound. Typically, a linker is substantially inert under the conditions under which the two groups to which it is connected are connected. A bifunctional cross-linking agent may comprise two reactive groups, one at each end of the linker moiety, such that one reactive group may first react with the cytotoxic compound to provide a compound with a linker moiety and a second reactive group, which may then react with the cell-binding agent. Alternatively, one end of the bifunctional cross-linking agent may first react with the cell-binding agent to provide a cell-binding agent with a linker moiety and a second reactive group, which may then react with the cytotoxic compound. The linking moiety may contain a chemical bond that allows the cytotoxic moiety to be released at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid-labile bonds, photolabile bonds, peptidase-labile bonds, and esterase-labile bonds (see, e.g., U.S. Pat. Nos. 5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026; and 7,414,073). Disulfide bonds, thioether bonds, and peptidase-labile bonds are preferred. Other linkers that can be used in the present invention include non-cleavable linkers, such as the linkers described in detail in U.S. Publication No. 20050169933, or charged linkers or hydrophilic linkers, and which are described in US2009 / 0274713, US2010 / 01293140 and WO 2009 / 134976, each of which is expressly incorporated herein by reference.

[0308] The terms "abnormal cell growth" and "proliferative disorder" are used interchangeably in this application. Unless otherwise specified, as used herein, "abnormal cell growth" refers to cell growth that is out of normal regulatory mechanisms (e.g., loss of contact inhibition). This includes, for example, the following abnormal growth: (1) tumor cells (tumors) that proliferate by expressing mutant tyrosine kinases or overexpressing receptor tyrosine kinases; (2) benign and malignant cells of other proliferative diseases with abnormal tyrosine kinase activation; (3) any tumor that proliferates through receptor tyrosine kinases; (4) any tumor that proliferates through abnormal serine / threonine kinase activation; and (5) benign and malignant cells of other proliferative diseases with abnormal serine / threonine kinase activation.

[0309] The terms "cancer" and "cancerous" refer to or describe the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancerous and / or benign or precancerous cells.

[0310] As used herein, "autoimmune disease" refers to a disease or condition caused by and directed against an individual's own tissues or proteins.

[0311] As used herein, the term "patient" or "subject" refers to any organism to which a provided compound or a compound described herein is administered according to the present invention, for example, for experimental, diagnostic, preventive and / or therapeutic purposes. Typical subjects include animals. The term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human being at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In specific embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal and / or a cloned animal. In embodiments, the animal is a mammal, such as a mouse, a rat, a rabbit, a non-human primate and a human; an insect; a worm, etc. In embodiments, the subject is a human. In some embodiments, the subject may suffer from and / or be susceptible to a disease, a disorder and / or a condition (e.g., cancer). As used herein, a "patient population" or "subject population" refers to a plurality of patients or subjects.

[0312] As used herein, the term "normal" when used to modify the term "individual" or "subject" refers to an individual or group of individuals who do not suffer from a particular disease or condition and are not carriers of the disease or condition. The term "normal" is also used herein to define a biological specimen or sample isolated from a normal or wild-type individual or subject, such as a "normal biological sample."

[0313] An individual "suffering from" a disease, disorder, and / or condition (eg, any of the cancers described herein) has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0314] An individual who is "susceptible" to a disease, disorder, and / or condition has not yet been diagnosed with and / or may not be showing symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer) may have one or more of the following characteristics: (1) a genetic mutation associated with developing the disease, disorder, and / or condition; (2) a genetic polymorphism associated with developing the disease, disorder, and / or condition; (3) an increased and / or decreased expression and / or activity of a protein associated with the disease, disorder, and / or condition; (4) habits and / or lifestyle associated with developing the disease, disorder, and / or condition; (5) a family history of the disease, disorder, and / or condition; (6) a reaction to certain bacteria or viruses; (7) exposure to certain chemicals. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0315] Unless otherwise indicated by the context, the term "treat" or "treatment" refers to any administration of a therapeutic molecule (e.g., any compound described herein) that partially or completely alleviates, improves, alleviates, inhibits, delays the onset, delays the progression, reduces the severity, and / or reduces the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition (e.g., cancer). The treatment may be directed to subjects who do not show signs of the relevant disease, disorder, and / or condition and / or subjects who only show early signs of the disease, disorder, and / or condition. Alternatively or in addition, the treatment may be directed to subjects who show one or more established signs of the relevant disease, disorder, and / or condition. Alternatively, the pharmacological and / or physiological effects may be preventive, that is, the effects of completely or partially preventing the disease or its symptoms (e.g., delaying the onset of the disease or its symptoms or slowing its progression). In this regard, the methods of the present invention include administering a "prophylactically effective amount" of a binding agent. "Prophylactically effective amount" refers to an amount that can effectively achieve the desired preventive result within the necessary dosage and time period. Therefore, the purpose of treatment (including preventive treatment) is to inhibit or slow down (mitigate) adverse physiological changes or diseases, such as the development or spread of cancer. For the purposes of the present invention, useful or desired clinical results include, but are not limited to, symptom relief, disease degree relief, disease state stabilization (i.e., no deterioration), disease progression delay or slowing, disease state improvement or alleviation, and relief (whether partial relief or complete relief), whether such results are detectable or undetectable. Treatment may also include an extension of survival compared to the expected survival without treatment. Patients in need of treatment include people who have already suffered from a condition or disease and people who are susceptible to a condition or disease.

[0316] In the context of cancer, the term "treat" includes any or all of the following: killing tumor cells; inhibiting the growth of tumor cells, cancer cells or tumors; inhibiting the replication of tumor cells or cancer cells; reducing the overall tumor burden or reducing the number of cancer cells; and ameliorating one or more symptoms associated with the disease.

[0317] In the context of autoimmune disease, the term "treating" includes any or all of the following: inhibiting the replication of cells associated with the autoimmune disease state (including but not limited to cells producing autoimmune antibodies), reducing the autoimmune antibody load, and ameliorating one or more symptoms of the autoimmune disease.

[0318] The term "therapeutically effective amount" or "effective amount" refers to an amount of a conjugate that is effective for treating or preventing a disease or disorder (e.g., as described herein) in a mammal. In the case of cancer, a therapeutically effective amount of a conjugate can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow down to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. To the extent that the drug can inhibit the growth of existing cancer cells and / or kill existing cancer cells, the drug can be cytostatic and / or cytotoxic. With regard to cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0319] As used herein, the term "pharmaceutically acceptable form" refers to a form of a disclosed compound, including but not limited to pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, isomers, prodrugs, and isotopically labeled derivatives thereof. In one embodiment, "pharmaceutically acceptable form" includes but is not limited to pharmaceutically acceptable salts, esters, prodrugs, and isotopically labeled derivatives thereof. In an embodiment, "pharmaceutically acceptable form" includes but is not limited to pharmaceutically acceptable isomers and stereoisomers, prodrugs, and isotopically labeled derivatives thereof.

[0320] In an embodiment, the pharmaceutically acceptable form is a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., auristatin, auristatin effective load or auristatin conjugate). In some aspects, the compound may contain at least one amino group, and therefore may form an acid addition salt with an amino group. Exemplary salts include, but are not limited to, sulfates, trifluoroacetates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, saccharates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). Pharmaceutically acceptable salts may involve including another molecule, such as acetate ions, succinate ions, or other counterions. Counterions may be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, pharmaceutically acceptable salts may have more than one charged atom in their structure. Where multiple charged atoms are part of a pharmaceutically acceptable salt there may be multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0321] As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent (e.g., a compound according to any of Formulas (I)-III) described herein) is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition can be specially formulated for administration in solid or liquid form, including pharmaceutical compositions suitable for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., tablets targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous, or epidural injection, for example, as a sterile solution or suspension or a sustained release formulation; topical administration, for example, as a cream, ointment, or controlled release patch or spray to the skin, lungs, or oral cavity; intravaginal or rectal, for example, as a vaginal suppository, cream, or foam; sublingual; ocular; transdermal; or nasal, pulmonary, and to other mucosal surfaces.

[0322] As used herein, "carrier" or "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle used with the composition. In some exemplary embodiments, the carrier may include sterile liquids, such as water and oils, including oils of petroleum, animal oils, vegetable oils or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or includes one or more solid components. In some embodiments, the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using a coating (such as lecithin), maintaining the desired particle size in the case of a dispersion, and using a surfactant. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In some cases, it may be desirable to include an isotonic agent in the composition, such as sugar, polyols (such as mannitol, sorbitol), sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0323] As used herein, the term "kit" refers to any delivery system for delivering materials. The delivery system may include a system that allows various diagnostic or therapeutic reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or support materials (e.g., buffers, written instructions for performing detection, etc.) to be stored, transported, or delivered from one location to another. For example, the kit includes one or more capsules (e.g., boxes, barrels, bottles, ampoules, etc.) containing relevant reaction reagents and / or support materials. As used herein, the term "bulk kit" refers to a delivery system comprising two or more independent containers each containing a sub-portion of a total kit assembly. The container can be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for detection, and a second container contains oligonucleotides. The term "bulk kit" is intended to cover a kit containing an analyte-specific reagent (Analyte Specific Reagent, ASR) regulated under Section 520 (e) of the Federal Food, Drug, and Cosmetic Act, but is not limited thereto. Indeed, any delivery system comprising two or more separate containers, each containing a sub-portion of the total kit components, is included in the term "bulk kit." In contrast, a "combination kit" refers to a delivery system that contains all components in a single container (e.g., in a single box containing each required part). The term "kit" includes both bulk kits and combination kits.

[0324] As used herein, the term "administering" generally refers to administering a composition to a subject or system to achieve delivery of a composition or a medicament included in the composition. One of ordinary skill in the art will appreciate the various routes that may be used to administer to a subject (e.g., human) in appropriate circumstances. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. For example, in some embodiments, administration may be through the eye, oral, parenteral, topical, etc. In an embodiment, administration is parenteral (e.g., intravenous administration). In an embodiment, intravenous administration is intravenous infusion. In some specific embodiments, administration can be transbronchial (e.g., by bronchial instillation), buccal, cutaneous (which can be or include, for example, one or more of dermal topical, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intra-organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous administration, etc.

[0325] As used herein, the term "nucleophilic" refers to a reactive group that is rich in electrons, has a non-shared electron pair that acts as a reactive site, and reacts with a positively charged or electron-deficient site. Examples of nucleophilic groups suitable for the present invention include, but are not limited to, amino groups (e.g., primary amines, secondary amines, hydroxylamines, and / or hydrazines), thiols, phenols, and alcohols. In an embodiment, the nucleophilic functional group comprises: amino, hydrazine, hydroxylamino, hydroxyl, or sulfhydryl. In an embodiment, the nucleophilic functional group is formamide, N-hydroxyformamide, carboxyhydrazide, or guanidinium. In an embodiment, the nucleophilic group is a thiol group or comprises a thiol group. Some nucleophilic groups must be activated with a base so that they can react with electrophilic groups. For example, when nucleophilic thiols and hydroxyls are present in a multifunctional compound, the compound must be mixed with an aqueous base in order to remove protons and provide thiolation or hydroxylation anions to achieve reaction with electrophilic groups. Unless a base is desired to participate in the reaction, a non-nucleophilic base is preferred. In some embodiments, the base may be present in the form of a buffer component.

[0326] As used herein, the term "electrophilic" refers to a reactive group susceptible to nucleophilic attack; that is, susceptible to reacting with a nucleophilic group to be attacked. The selection of electrophilic groups can be performed so that it is possible to react with the nucleophilic group of the paired reactant. For example, when the nucleophilic reactive group is an amino group, one or more electrophilic groups can be selected to react with the amino group. Similarly, when the nucleophilic reactive group is a thiol moiety, the corresponding electrophilic group can be a thiol reactive group, etc. Examples of electrophilic groups suitable for the present invention include, but are not limited to, carboxylate, acyl chloride group, anhydride, isocyanato, thioisocyanato, epoxy, activated hydroxyl, succinimidyl ester, sulfosuccinimidyl ester, maleimido and vinylsulfonyl. In embodiments, the electrophilic group is an aldehyde, an α-haloketone, a maleimide, a succinimide, a hydroxysuccinimide, an isothiocyanate, an isocyanate, an acyl azide, a sulfonyl chloride, a mesylate, a glyoxal, an epoxy group, an oxirane, a carbonate, an imidoester, an anhydride, a fluorophenyl ester, a hydroxymethylphosphine derivative, a carbonate, a haloacetyl group, a chlorotriazine, a haloacetyl group, an alkyl halide, an aziridine, an acryloyl derivative, a ketone, a carboxylic acid, an ester, acetyl chloride, or acetic anhydride. In embodiments, the electrophilic group is or comprises a maleimide or succinimide group. The carboxylic acid group may be activated for reaction with a nucleophile, including reaction with a suitable hydroxyl-containing compound in the presence of a dehydrating agent such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU). For example, carboxylic acids can react with alkoxy-substituted N-hydroxysuccinimide or N-hydroxysulfosuccinimide in the presence of DCC to form reactive electrophilic groups N-hydroxysuccinimide ester and N-hydroxysulfosuccinimide ester, respectively. Carboxylic acids can also be activated by reacting with acyl halides, such as acyl chlorides (e.g., acetyl chloride), to provide reactive anhydride groups. In another example, carboxylic acids can be converted to acyl chloride groups using, for example, thionyl chloride or acyl chlorides that are capable of undergoing an exchange reaction.

[0327] Unless otherwise indicated, the term "alkyl" by itself or as part of another term refers to a substituted or unsubstituted straight or branched chain, saturated or unsaturated hydrocarbon having the specified number of carbon atoms (e.g., "-C1-C8 alkyl" or "-C1-C8 alkyl"). 10"Alkyl refers to an alkyl group having 1 to 8 or 1 to 10 carbon atoms, respectively). When the number of carbon atoms is not indicated, the alkyl group has 1 to 8 carbon atoms. Representative straight-chain "-C1-C8 alkyl" includes, but is not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, and -n-octyl; while branched-C3-C8 alkyl includes, but is not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl; unsaturated-C2-C8 alkyl includes, but is not limited to In some embodiments, the alkyl group may be substituted with one or more groups. In some embodiments, the alkyl group may be substituted with one or more groups. In some embodiments, the alkyl group may be substituted with one or more groups. In some embodiments, the alkyl group may be substituted with one or more groups. In some embodiments, the alkyl group may be substituted with one or more groups. In some embodiments, the alkyl group may be substituted with one or more groups. In some embodiments, the alkyl group may be substituted with one or more groups. In some embodiments, the alkyl group may be substituted with one or more groups. In some embodiments, the alkyl group may be substituted with one or more groups. In some embodiments, the alkyl group may be substituted with one or more groups.

[0328] Unless otherwise indicated, "alkylene" by itself or as part of another term refers to a substituted or unsubstituted saturated, branched or straight chain or cyclic hydrocarbon radical having the specified number of carbon atoms (usually 1-10 carbon atoms) and having two monovalent radical centers derived by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene groups include, but are not limited to, methylene (—CH2—), 1,2-ethylene (—CH2CH2—), 1,3-propylene (—CH2CH2CH2—), 1,4-butylene (—CH2CH2CH2CH2—), and the like. In a preferred aspect, an alkylene group is a branched or straight chain hydrocarbon (i.e., it is not a cyclic hydrocarbon).

[0329] Unless otherwise indicated, "aryl" by itself or as part of another term means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical having a specified number of carbon atoms (usually 6-20 carbon atoms) derived by removing one hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented as "Ar" in the exemplary structures. Typical aryl groups include, but are not limited to, groups derived from benzene, substituted benzenes, naphthalene, anthracene, biphenyl, and the like. An exemplary aryl group is phenyl.

[0330] Unless otherwise stated, "arylene" by itself or as part of another term refers to an aryl group as defined above which has two covalent bonds (ie, it is divalent) and which may be in the ortho, meta, or para positions.

[0331] Unless otherwise specified, "C3-C8 heterocycle" by itself or as part of another term refers to a monovalent substituted or unsubstituted aromatic or non-aromatic monocyclic or bicyclic ring system having 3 to 8 carbon atoms (also referred to as ring members) and one to four heteroatom ring members independently selected from N, O, P or S, and derived by removing one hydrogen atom from a ring atom of a parent ring system. One or more N, C or S atoms in the heterocycle may be oxidized. The ring including the heteroatom may be aromatic or non-aromatic. A heterocycle in which all the ring atoms are associated with aromaticity is referred to as a heteroaryl, otherwise it is referred to as a heterocarbocycle. Unless otherwise specified, a heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Thus, a heteroaryl group may be bonded through an aromatic carbon of its aromatic ring system, referred to as a C-bonded heteroaryl, or through a non-double-bonded N atom (i.e., non-=N—) in its aromatic ring system, referred to as an N-bonded heteroaryl. Thus, nitrogen-containing heterocycles may be C-bonded or N-bonded, and include pyrrole moieties such as pyrrol-1-yl (N-bonded) and pyrrol-3-yl (C-bonded), and imidazole moieties such as imidazol-1-yl and imidazol-3-yl (both N-bonded), and imidazol-2-yl, imidazol-4-yl and imidazol-5-yl moieties (all of which are C-bonded).

[0332] As used herein, the term "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system having a total of six to fourteen ring members, wherein the ring system has a single point of attachment to the rest of the molecule, wherein at least one ring in the system is aromatic, wherein each ring in the system contains 4 to 7 ring members, and wherein at least one ring atom is a heteroatom such as, but not limited to, nitrogen and oxygen.

[0333] Unless otherwise indicated, "C3-C8 heteroaryl" is an aromatic C3-C8 heterocycle, wherein the subscript represents the total number of carbons of the ring system of the heterocycle or the total number of aromatic carbons of the aromatic ring system of the heteroaryl, and does not refer to the size of the ring system or the presence or absence of ring fusion. Representative examples of C3-C8 heterocycles include, but are not limited to, pyrrolidinyl, azetidinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolyl, thienyl (thiophene), furanyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolyl, and isoxazolyl. When explicitly given, the size of the heterocycle or heteroaryl ring system is indicated by the total number of atoms in the ring. For example, a designation as a 5-membered or 6-membered heteroaryl indicates the total number of aromatic atoms in the heteroaromatic ring system of the heteroaryl (i.e., 5 or 6), but does not imply the number of aromatic heteroatoms or aromatic carbons in the ring system. Fused heteroaryls are either explicitly specified or implied by the context, and are generally indicated by the number of aromatic atoms in each aromatic ring fused together to form the fused heteroaromatic ring system. For example, a 5,6-membered heteroaryl is an aromatic 5-membered ring fused to an aromatic 6-membered ring, wherein one or both of the rings have one or more aromatic heteroatoms, or wherein one heteroatom is shared between the two rings.

[0334] A heterocycle fused to an aryl or heteroaryl such that the heterocycle remains non-aromatic and becomes part of a larger structure by attachment to the non-aromatic portion of the fused ring system is an example of an optionally substituted heterocycle where the heterocycle is substituted by ring fusion to an aryl or heteroaryl. Likewise, an aryl or heteroaryl fused to a heterocycle or carbocycle that becomes part of a larger structure by attachment to the aromatic portion of the fused ring system is an example of an optionally substituted aryl or heterocycle where the aryl or heterocycle is substituted by ring fusion to a heterocycle or carbocycle.

[0335] Unless otherwise stated, "C3-C8 heterocyclyl (heterocyclo)" by itself or as part of another term refers to a C3-C8 heterocyclic ring (heterocyclic) as defined above, wherein one of the hydrogen atoms of the heterocyclic ring is replaced by a bond (i.e., it is divalent). Unless otherwise stated, "C3-C8 heteroarylene" by itself or as part of another term refers to a C3-C8 heteroaryl group as defined above, wherein one of the hydrogen atoms of the heteroaryl group is replaced by a bond (i.e., it is divalent).

[0336] Unless otherwise indicated, "C3-C8 carbocycle" by itself or as part of another term is a 3-, 4-, 5-, 6-, 7- or 8-membered monovalent, substituted or unsubstituted, saturated or unsaturated non-aromatic monocyclic or bicyclic carbocycle derived by removing one hydrogen atom from a ring atom of a parent ring system. Representative C3-C8 carbocycles 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.

[0337] Unless otherwise stated, "C3-C8 carbocyclo" by itself or as part of another term refers to a C3-C8 carbocycle group as defined above wherein another of the carbocycle's hydrogen atoms is replaced by a bond (ie, it is divalent).

[0338] Unless otherwise stated, the term "heteroalkyl", by itself or in combination with another term, unless otherwise stated, means a stable straight or branched chain hydrocarbon or combination thereof, which is fully saturated or contains from 1 to 3 degrees of unsaturation, consisting of the specified number of carbon atoms and from one to ten, preferably from one to three, heteroatoms selected from the group consisting of O, N, Si and S, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The one or more heteroatoms O, N and S may be located at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the rest of the molecule. The heteroatom Si may be located at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the rest of the molecule. Examples include —CH2—CH2—O—CH3, —CH2—CH2—NH—CH3, —CH2—CH2—N(CH3)—CH3, —CH2—S—CH2—CH3, —CH2—CH2—S(O)—CH3, —NH—CH2—CH2—NH—C(O)—CH2—CH3, —CH2—CH2—S(O)2—CH3, —CH═CH—O—CH3, —Si(CH3)3, —CH2—CH═N—O—CH3, and —CH═CH—N(CH3)—CH3. Up to two heteroatoms may be consecutive, for example, —CH2—NH—OCH3 and —CH2—O—Si(CH3)3. Typically, the C1 to C4 heteroalkyl or heteroalkylene group has 1 to 4 carbon atoms and 1 or 2 heteroatoms, and the C1 to C3 heteroalkyl or heteroalkylene group has 1 to 3 carbon atoms and 1 or 2 heteroatoms. In some aspects, the heteroalkyl or heteroalkylene group is saturated.

[0339] Unless otherwise indicated, the term "heteroalkylene" by itself or in combination with another term means a divalent radical derived from a heteroalkyl group (as discussed above), such as exemplified by -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene, heteroatoms may also occupy either or both of the chain termini. Additionally, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied.

[0340] Unless otherwise stated, "aminoalkyl" by itself or in combination with another term means a heteroalkyl group in which the alkyl moiety as defined herein is substituted with amino, alkylamino, dialkylamino or cycloalkylamino. Exemplary non-limiting aminoalkyl groups are -CH2NH2, -CH2CH2NH2, -CH2CH2NHCH3, and -CH2CH2N(CH3)2, and further include branched species such as -CH(CH3)NH2 and -C(CH3)CH2NH2 in either the (R)- or (S)-configuration. Alternatively, aminoalkyl is an alkyl moiety, group or substituent as defined herein in which sp 3 The carbon has been replaced by an amino or alkylamino moiety, wherein its sp 3 Nitrogen replaces the sp 3 Carbon, provided that at least one sp 3 The carbon remains. When the aminoalkyl moiety refers to a substituent of a larger structure or another moiety, the aminoalkyl group is covalently attached to the structure or moiety through the carbon radical of the alkyl portion of the aminoalkyl group.

[0341] Unless otherwise stated, "alkylamino" and "cycloalkylamino" by themselves or in combination with another term mean an alkyl or cycloalkyl group as described herein, wherein a radical carbon of the alkyl or cycloalkyl group has been replaced by a nitrogen group, provided that at least one sp 3 The carbon is retained. In those cases where an alkylamino group is substituted at its nitrogen with another alkyl moiety, the resulting substituted group is sometimes referred to as a dialkylamino moiety, group or substituent, wherein the alkyl moiety replacing the nitrogen is independently selected. Exemplary and non-limiting amino, alkylamino and dialkylamino substituents include those having the structure —N(R′) 2, wherein in these examples, R′ is independently selected from hydrogen or C 1-6 Alkyl, usually hydrogen or methyl, and in the cycloalkylamine included in the heterocycloalkyl, two R' together with the nitrogen to which they are attached define the heterocycle. When both R' are hydrogen or alkyl, the moieties are sometimes described as primary amino and tertiary amine groups, respectively. When one R' is hydrogen and the other is alkyl, the moieties are sometimes described as secondary amino. Primary and secondary alkylamino moieties are more reactive as nucleophiles for carbonyl-containing electrophilic centers, while tertiary amines are more basic.

[0342] "Substituted alkyl" and "substituted aryl" refer to alkyl and aryl groups, respectively, in which one or more hydrogen atoms (usually one) are independently replaced by a substituent. Typical substituents include, but are not limited to, -X, -R', -OH, -OR', -SR', -N(R')2, -N(R')3, =NR', -CX3, -CN, -NO2, -NR'C(=O)R', -C(=O)R', -C(=O)N(R')2, -S(=O)2R', -S(=O)2NR, -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3= , PO3H2, -C(=O)R', -C(=S)R', -CO2R', -CO2-, -C(=S)OR', -C(=O)SR', -C(=S)SR', -C(=O)N(R')2, -C(=S)N(R)2, and -C(=NR)N(R')2, wherein each X is independently selected from the group consisting of the following halogens: -F, -CI, -Br, and -I; and wherein each R' is independently selected from the group consisting of -H, -C1-C 20 Alkyl, -C6-C 20 Aryl, -C3-C 14 A group consisting of a heterocycle, a protecting group and a prodrug moiety.

[0343] More typically, the substituent is selected from the group consisting of —X, —R′, —OH, —OR′, —SR′, —N(R′)2, —N(R′)3, ═NR′, —NR′C(═O)R, —C(═O)R′, —C(═O)N(R′)2, —S(═O)2R′, —S(═O)2NR′, —S(═O)R′, —C(═O)R′, —C(═S)R, —C(═O)N(R′)2, —C(═S)N(R′)2, and —C(═NR)N(R′)2, wherein each X is independently wherein each X is -F; and each R' is independently selected from the group consisting of -F and -CI, or from the group consisting of -X, -R, -OH, -OR', -N(R')2, -N(R')3, -NR'C(=O)R', -C(=O)N(R')2, -S(=O)2R', -S(=O)2NR', -S(=O)R', -C(=O)R', -C(=O)N(R')2, -C(=NR)N(R')2, a protecting group, and a prodrug moiety, wherein each X is -F; and wherein each R' is independently selected from the group consisting of hydrogen, -C1-C 20 Alkyl, -C6-C 20 Aryl, -C3-C 14 In some aspects, the alkyl substituent is selected from the group consisting of —N(R′)2, —N(R′)3, and —C(═NR)N(R′)2, wherein R is selected from the group consisting of hydrogen and —C1-C 20In some embodiments, the alkyl group is substituted with a series of vinyloxy groups to define a PEG unit. Alkylene, carbocycle, carbocyclyl, arylene, heteroalkyl, heteroalkylene, heterocycle, heterocyclyl, heteroaryl and heteroarylene as described above may also be substituted similarly.

[0344] As used herein, "protecting group" means a moiety that prevents or reduces the ability of the atom or functional group to which it is attached to participate in undesired reactions. Typical protecting groups for atoms or functional groups are described in Greene (1999), "Protective groups". ROTECTIVE G ROUPS I N O RGANIC S YNTHESIS , 3rd edition", Wiley Interscience. Protecting groups for heteroatoms such as oxygen, sulfur, and nitrogen are used in some cases to minimize or avoid undesired reactions with electrophilic compounds. In other cases, protecting groups are used to reduce or eliminate the nucleophilicity and / or basicity of the unprotected heteroatom. Non-limiting examples of protected oxygen are represented by -OR PR Given, where R PR is a protecting group for hydroxyl, wherein the hydroxyl is usually protected as an ester (e.g., acetate, propionate, or benzoate). Other protecting groups for hydroxyl avoid interfering with the nucleophilicity of organometallic reagents or other strongly basic reagents, wherein the hydroxyl is usually protected in the form of an ether, including alkyl or heterocycloalkyl ethers (e.g., methyl or tetrahydropyranyl ether), alkoxymethyl ethers (e.g., methoxymethyl or ethoxymethyl ether), optionally substituted aryl ethers, and silyl ethers (e.g., trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBS / TBDMS), triisopropylsilyl (TIPS), and [2-(trimethylsilyl)ethoxy]-methylsilyl (SEM)). Nitrogen protecting groups include, for example, in —NHR PR or—N(R PR )2- is a protecting group for a primary or secondary amine, wherein R PR At least one of them is a nitrogen atom protecting group, or two R PR Together they form a protecting group.

[0345] When the protecting group is capable of preventing or avoiding undesirable side reactions or premature loss of the protecting group under the reaction conditions required to achieve the desired chemical transformation elsewhere in the molecule and during the purification of the newly formed molecule when necessary, and can be removed under conditions that do not adversely affect the structural or stereochemical integrity of the newly formed molecule, the protecting group is suitable. As an example and not limitation, suitable protecting groups may include those protecting groups previously described for protecting functional groups. Suitable protecting groups are sometimes protecting groups used in peptide coupling reactions.

[0346] "Aromatic alcohol" by itself or as part of a larger structure refers to an aromatic ring system substituted with a hydroxyl functional group -OH. Thus, an aromatic alcohol refers to any aryl, heteroaryl, arylene and heteroarylene moiety as described herein, which has a hydroxyl functional group bound to an aromatic carbon of its aromatic ring system. An aromatic alcohol can be part of a larger moiety, such as when its aromatic ring system is a substituent of such moiety, or can be embedded in the larger moiety by ring fusion, and can be optionally substituted with a moiety as described herein including one or more other hydroxyl substituents. A phenolic alcohol is an aromatic alcohol having a phenolic group as the aromatic ring.

[0347] "Aliphatic alcohol" by itself or as part of a larger structure refers to a moiety having a non-aromatic carbon bonded to a hydroxyl functional group -OH. The carbon bearing the hydroxyl group may be unsubstituted (i.e., methanol), or may have one, two, or three optionally substituted branched or unbranched alkyl substituents to define a primary alcohol or a secondary or tertiary aliphatic alcohol within a linear or cyclic structure. When part of a larger structure, the alcohol may be a substituent of such structure by being bonded to such hydroxyl bearing carbon, via a carbon of an alkyl or other moiety as described herein, or via a substituent of such an alkyl or other moiety. Aliphatic alcohols encompass non-aromatic cyclic structures (i.e., optionally substituted carbocyclic and heterocarbocyclic rings) in which the hydroxyl functional group is bonded to a non-aromatic carbon of its cyclic ring system.

[0348] As used herein, "arylalkyl" or "heteroarylalkyl" means a substituent, moiety or group in which an aryl moiety is bonded to an alkyl moiety, i.e., aryl-alkyl-, in which the alkyl and aryl groups are as described above, for example, C6H5-CH2- or C6H5-CH(CH3)CH2-. An arylalkyl or heteroarylalkyl group is bonded to an alkyl moiety by sp 3 The carbon is associated with a larger structure or moiety.

[0349] As used herein, "succinimide moiety" refers to an organic moiety consisting of a succinimide ring system, which is present in a type of Y' in a compound of formula (III), which is generally further composed of an alkylene-containing moiety bound to the amide nitrogen of the ring system. The succinimide moiety is generally produced by the Michael addition reaction of a thiol group of a cell-binding agent with the maleimide ring system of an auristatin payload compound (Formula II). Thus, the succinimide moiety is composed of a sulfhydryl-substituted succinimide ring system, and when it is present in an auristatin conjugate, its amide nitrogen is substituted by the remainder of the cell-binding agent of the auristatin conjugate, and optionally by one or more substituents present on the maleimide ring system of the compound of Formula II.

[0350] As used herein, "acid-amide moiety" refers to a succinic acid with an amide substituent produced from a sulfenyl-substituted succinimide ring system of a succinimide moiety that breaks one of its carbonyl-nitrogen bonds by hydrolysis. The hydrolysis that produces the succinic acid-amide moiety provides a linker that is less likely to suffer premature loss of the linker to which it is bound by eliminating the antibody-sulfenyl substituent. It is expected that hydrolysis of the succinimide ring system of the sulfenyl-substituted succinimide moiety will provide regiochemical isomers of the acid-amide moiety due to differences in the reactivity of the two carbonyl carbons of the succinimide ring system, which can be at least partially attributed to any substituents present in the maleimide ring system of the compound of Formula II and to the sulfenyl substituent introduced by the targeting ligand.

[0351] As used herein, the term "prodrug" refers to a less biologically active compound or an inactive compound that is converted into a more biologically active compound via a chemical or biological process (i.e., a chemical reaction or an enzymatic biotransformation) in vivo. Typically, a biologically active compound exhibits less biological activity (i.e., is converted into a prodrug) by chemically modifying the compound with a prodrug moiety. In some aspects, the prodrug is a Type II prodrug that is bioactivated extracellularly (e.g., in digestive fluids) or in the body's circulatory system (e.g., in the blood). Exemplary prodrugs are esters and (β-D-pyranoglucosides.

[0352] In many cases, the components of the conjugates, linkers and components described herein will refer to reactive groups. "Reactive group" or RG is a component containing a linker that can form a bond with an auristatin effective load or an auristatin conjugate; or a group of a reactive site (RS) of a auristatin forming a bond. RS is a reactive site in a reactive group (RG). Reactive groups include thiol groups to form disulfide bonds or thioether bonds, aldehydes, ketones or hydrazines to form hydrazone bonds, carboxyl or amino groups to form peptide bonds, carboxyl or hydroxyl groups to form ester bonds, sulfonic acids to form sulfonamide bonds, alcohols to form carbamate bonds, and amines to form sulfonamide bonds or carbamate bonds. The following table illustrates reactive groups, reactive sites and exemplary functional groups that can be formed after the reaction of the reactive sites. The table is not restrictive. It will be understood by those skilled in the art that the R' and R" moieties referred to in the table are actually any organic moieties (e.g., alkyl, aryl, heteroaryl, or substituted alkyl, aryl, or heteroaryl) that are compatible with the bond formation provided in converting RG to one of the exemplary functional groups. It will also be understood that, as applicable to embodiments of the present invention, R' may represent one or more components of a self-stabilizing linker or an optional secondary linker, as the case may be, and R" may represent one or more components of an optional secondary linker, an auristatin, a stabilizing unit, or a detection unit, as the case may be.

[0353]

[0354] The combinations of substituents and variables contemplated by this invention are only those that result in the formation of stable compounds. As used herein, the term "stable" refers to compounds that have sufficient stability to allow manufacturing and maintain the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

[0355] The compounds of the invention are preferably isolated and purified following their preparation to obtain compositions containing an amount equal to or greater than 95% by weight ("substantially pure"), which are then used or formulated as described herein.

[0356] As used herein, the term "conjugate" refers to a compound described herein, or a derivative thereof, linked to a cell-binding agent.

[0357] As used herein, the term "linkable to a cell binding agent" refers to compounds described herein or derivatives thereof, which contain at least one linking group or a precursor thereof suitable for binding these compounds or derivatives thereof to a cell binding agent.

[0358] The term "precursor" of a given group refers to any group that can generate said group by any deprotection, chemical modification or coupling reaction.

[0359] The term "linked to a cell binding agent" refers to a conjugate molecule comprising at least one of the compounds described herein or their derivatives, which is bound to a cell binding agent via a suitable linking group or a precursor thereof.

[0360] "Therapeutic agent" encompasses both biologic agents (eg, antibodies, peptides, proteins, enzymes) or chemotherapeutic agents.

[0361] A "chemotherapeutic agent" is a chemical compound that is useful in treating cancer.

[0362] "Metabolites" are products produced by the metabolism of a particular compound, its derivative or its conjugate or its salt in vivo. Metabolites of a compound, its derivative or its conjugate can be identified using conventional techniques known in the art, and their activity can be determined using assays such as those described herein. The product can be produced, for example, by oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc. of the administered compound. Therefore, the present invention includes metabolites of the compounds, their derivatives or their conjugates of the present invention, including compounds, their derivatives or their conjugates produced by a method comprising contacting a compound, its derivative or its conjugate of the present invention with a mammal for a period of time sufficient to produce its metabolic product.

[0363] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code and those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, selenocysteine, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure (i.e., carbon bound to hydrogen, carboxyl, amino, and R groups) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. The analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. One amino acid that can be specifically used is citrulline, which is a derivative of arginine and is involved in the formation of urea in the liver. Amino acid mimetics refer to compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids. The term "non-natural amino acid" is intended to represent the "D" stereochemical form of the twenty naturally occurring amino acids described above. It should also be understood that the term non-natural amino acid includes a homologue of a natural amino acid or its D isomer, and a synthetic modified form of a natural amino acid. The form of synthetic modification includes, but is not limited to, an amino acid having a side chain shortened or extended to two carbon atoms, an amino acid comprising an optionally substituted aryl, and an amino acid comprising a halogenated group, preferably an amino acid halogenated alkyl and an aryl, and an amino acid in which N is substituted, such as N-methyl-alanine. An amino acid or peptide can be connected to a linker / spacer or a cell binding agent via a terminal amine or terminal carboxylic acid of an amino acid or peptide. An amino acid can also be connected to a linker / spacer or a cell binding agent via a side chain reactive group, such as, but not limited to, a thiol group of cysteine, an epsilon amine of lysine, or a side chain hydroxyl of serine or threonine.

[0364] In an embodiment, the amino acid is NH2-C(R aa 'R aaa )-C(=O)OH, wherein R aa and R aa ' are each independently H; an optionally substituted straight chain, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms; an aryl, heteroaryl or heterocyclic group, or R and the N-terminal nitrogen atom may together form a heterocycle (e.g., as in proline). The term "amino acid residue" refers to the corresponding residue when one hydrogen atom is removed from the amine terminal and / or the hydroxyl group is removed from the carboxyl terminal of an amino acid, such as -NH-C(R aa 'R aa )-C(=O)O-.

[0365] As used herein, amino acids can be L or D isomers. Unless otherwise specified, when referring to an amino acid, it can be L or D isomers or mixtures thereof. In embodiments, unless otherwise specified, when a peptide is referenced by its amino acid sequence, each of the amino acids can be L or D isomers. Unless otherwise specified, if one amino acid in a peptide is designated as a D isomer, the other one or more amino acids are L isomers. For example, the peptide D-Ala-Ala means D-Ala-L-Ala.

[0366] Amino acids and peptides can be protected by blocking groups. Blocking groups are atoms or chemical moieties that prevent the N-terminal of an amino acid or peptide from undergoing undesired reactions, and can be used in drug-ligand conjugate synthesis processes. It should remain connected to the N-terminal throughout the synthesis process, and can be removed by selectively achieving removal of chemistry or other conditions after completing the synthesis of the drug conjugate. Blocking groups suitable for N-terminal protection are well known in the field of peptide chemistry. Exemplary blocking groups include, but are not limited to, methyl esters, tert-butyl esters, 9-fluorenylmethyl carbamate (Fmoc) and benzyloxycarbonyl (Cbz).

[0367] The term "peptide cleavable by a protease" refers to a peptide containing a protease cleavage recognition sequence. As used herein, a protease is an enzyme that cleaves a peptide bond. The cleavage recognition sequence of a protease is a specific amino acid sequence that is recognized by the protease during proteolytic cleavage. Many protease cleavage sites are known in the art, and these and other cleavage sites may be included in the linker portion. See, e.g., Matayoshi et al., Science 247:954 (1990); Dunn et al., Meth. Enzymol. 241:254 (1994); Seidah et al., Meth. Enzymol. 244:175 (1994); Thornberry, Meth. Enzymol. 244:615 (1994); Weber et al., Meth. Enzymol. 244:595 (1994); Smith et al., Meth. Enzymol. 244:412 (1994); Bouvier et al., Meth. Enzymol. 248:614 (1995), Hardy et al., AMYLOID PROTEIN PRECURSOR IN DEVELOPMENT, AGING, AND ALZHEIMER'S DISEASE, Masters et al., eds., pp. 190-198 (1994).

[0368] The peptide sequence is selected based on its ability to be cleaved by proteases, non-limiting examples of which include cathepsins B, C, D, H, L and S and furin. Preferably, the peptide sequence is capable of being cleaved in vitro by an appropriate isolated protease, which can be determined using an in vitro protease cleavage assay known in the art.

[0369] In another embodiment, the peptide sequence is selected based on its ability to be cleaved by lysosomal proteases. Lysosomal proteases are proteases that are primarily located in the lysosome, but may also be located in the endosome. Examples of lysosomal proteases include, but are not limited to, cathepsin B, C, D, H, L, and S and furin.

[0370] In another embodiment, the peptide sequence is selected based on its ability to be cleaved by a tumor-associated protease, such as a protease found on the surface of a cancer cell or outside a cell near a tumor cell, non-limiting examples of which include phorate oligopeptidase (TOP), CD10 (neprilysin), matrix metalloproteinase (e.g., MMP2 or MMP9), type II transmembrane serine protease (e.g., Hepsin, testisin, TMPRSS4 or matriptase / MT-SP1), legumain, and enzymes described in the following references (Current Topics in Developmental Biology: Cell Surface Proteases, Vol. 54, Zucker S. 2003, Boston, MA). The ability of a peptide to be cleaved by a tumor-associated protease can be determined using an in vitro protease cleavage assay known in the art.

[0371] The term "cation" refers to an ion with a positive charge. The cation can be monovalent (e.g., Na + , K + etc.), divalent (e.g., Ca 2+ Mg 2+ etc.) or polyvalent (e.g., Al 3+ In an embodiment, the cation is monovalent.

[0372] Compound of formula (I)

[0373] In some aspects, the invention features compounds (eg, cytotoxic agents) comprising auristatin derivatives. The compounds can exhibit desired cytotoxic properties and can be generated from conjugates as described herein after cleavage of the linker.

[0374] In one aspect, provided herein are compounds having a structure according to Formula (I):

[0375] D—Q(I),

[0376] or a pharmaceutically acceptable salt thereof, wherein:

[0377] D is represented by the following structural formula:

[0378] in

[0379] R 1 is -H or -OH;

[0380] R 2 C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl;

[0381] Each R 3 and R 4 are independently -H or C1-C3 alkyl;

[0382] n is an integer from 1 to 6; and

[0383] Q is -H or -CH3.

[0384] In an embodiment, R 1 is -H.

[0385] In an embodiment, R 1 It is -OH.

[0386] In an embodiment, R 2 It is C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl.

[0387] In an embodiment, R 2 is a C1-C3 alkyl group. 2 It is -CH3.

[0388] In an embodiment, R 2 is -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH or -C(=O)NHCH2CH2CH2OH. In the embodiment, R 2 It is -C(=O)OH.

[0389] In an embodiment, R 2is heteroaryl (e.g., C3-C8 heteroaryl). 2 is a N- and / or S-containing heteroaryl. 2 for

[0390] In an embodiment, R 1 -H and R 2 It is -C(=O)OH.

[0391] In an embodiment, R 1 -H and R 2 It is -C(=O)NHCH2CH2CH2OH.

[0392] In an embodiment, R 1 -H and R 2 for

[0393] In an embodiment, R 1 is -OH and R 2 It is -CH3.

[0394] In an embodiment, each R 3 and R 4 are independently -H or C1-C3 alkyl.

[0395] In an embodiment, R 3 and R 4 Both are -H. In the embodiment, R 3 and R 4 Both are C1-C3 alkyl. 3 and R 4 Both are -CH3.

[0396] In an embodiment, n is an integer from 1 to 6. In an embodiment, n is 1. In an embodiment, n is 2. In an embodiment, n is 3. In an embodiment, n is 4. In an embodiment, n is 5. In an embodiment, n is 6. In an embodiment, n is 1.

[0397] In an embodiment, Q is -H.

[0398] In an embodiment, Q is -CH3.

[0399] In an embodiment, D is represented by one of the following structures:

[0400]

[0401] In an embodiment, D is (DI).

[0402] In an embodiment, D is (D-II).

[0403] In an embodiment, D is (D-III).

[0404] In an embodiment, D is (D-IV).

[0405] In an embodiment, the compound has one of the following structures,

[0406]

[0407] or a pharmaceutically acceptable salt thereof.

[0408] In an embodiment, the compound is Compound D1 or a pharmaceutically acceptable salt thereof.

[0409] In an embodiment, the compound is Compound D2 or a pharmaceutically acceptable salt thereof.

[0410] In an embodiment, the compound is Compound D3 or a pharmaceutically acceptable salt thereof.

[0411] In an embodiment, the compound is Compound D4 or a pharmaceutically acceptable salt thereof.

[0412] In an embodiment, the compound is Compound D5 or a pharmaceutically acceptable salt thereof.

[0413] In an embodiment, the compound is Compound D6 or a pharmaceutically acceptable salt thereof.

[0414] In an embodiment, the compound is Compound D7 or a pharmaceutically acceptable salt thereof.

[0415] In an embodiment, the compound is Compound D8 or a pharmaceutically acceptable salt thereof.

[0416] Compound of formula (II)

[0417] In some aspects, compounds comprising auristatin derivatives may include a peptide linker to form a payload: Such compounds may be used to prepare conjugates comprising a cell-binding agent as described herein.

[0418] In embodiments, the compound is formed from or comprises a structure according to any embodiment of Formula (I) as described herein.

[0419] In another aspect, the invention features a compound of formula (II),

[0420] D—CH2—NH—E—Z (II),

[0421] or a pharmaceutically acceptable salt thereof, wherein:

[0422] D is represented by the following structural formula:

[0423] in

[0424] R 1 is -H or -OH;

[0425] R 2 C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl;

[0426] Each R 3 and R 4 are independently -H or C1-C3 alkyl;

[0427] n is an integer from 1 to 6;

[0428] E is a peptide comprising 2 to 10 amino acids; wherein E is optionally substituted with one or more polyols; and wherein the N-terminus of the peptide is covalently linked to Z;

[0429] Z is -C(=O)-LY, Wherein m represents an integer from 1 to 10;

[0430] L is -(C1-C 10 Alkylene)-*, -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -、-(OCH2CH2) j -、-CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*; wherein j represents an integer of 1-10; and wherein * represents a site of covalent attachment to Y;

[0431] L1 is -(C1-C 10 Alkylene)-;

[0432] R 5 is -H or -CH3; and

[0433] Y is an electrophilic group or a nucleophilic group.

[0434] In an embodiment, R 1 is -H.

[0435] In an embodiment, R 1 It is -OH.

[0436] In an embodiment, R 2 It is C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl.

[0437] In an embodiment, R 2 is a C1-C3 alkyl group. 2 It is -CH3.

[0438] In an embodiment, R 2 is -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH or -C(=O)NHCH2CH2CH2OH. In the embodiment, R 2 It is -C(=O)OH.

[0439] In an embodiment, R 2 is heteroaryl (e.g., C3-C8 heteroaryl). 2 is a N- and / or S-containing heteroaryl. 2 for

[0440] In an embodiment, R 1 -H and R 2 It is -C(=O)OH.

[0441] In an embodiment, R 1 -H and R 2 It is -C(=O)NHCH2CH2CH2OH.

[0442] In an embodiment, R 1 -H and R 2 for

[0443] In an embodiment, R 1 is -OH and R 2 It is -CH3.

[0444] In an embodiment, each R 3 and R 4 are independently -H or C1-C3 alkyl.

[0445] In an embodiment, R 3 and R 4 Both are -H. In the embodiment, R 3 and R 4Both are C1-C3 alkyl. 3 and R 4 Both are -CH3.

[0446] In an embodiment, n is an integer from 1 to 6. In an embodiment, n is 1. In an embodiment, n is 2. In an embodiment, n is 3. In an embodiment, n is 4. In an embodiment, n is 5. In an embodiment, n is 6. In an embodiment, n is 1.

[0447] In an embodiment, E is a peptide of 2, 3 or 4 amino acids, wherein each amino acid in the peptide is an L amino acid, or at least one amino acid in the peptide is a D amino acid.

[0448] In an embodiment, E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with a polyol.

[0449] In an embodiment, E comprises an amino acid having the structure,

[0450]

[0451] Where R 6 It is -H or C1-C6 alkyl.

[0452] In an embodiment, E comprises an amino acid having the structure,

[0453]

[0454] In an embodiment, the following composition is composed of: -Ala-Val-*、-Val-Ala-*、-Gly-Gly-*、-Val-Cit-*、-Cit-Val-*、-Leu-Ala-*、-Ala-Leu-*、-Leu-Cit-*、-Cit-Leu-*、-Leu-Ala-*、-Ala-Leu-*、-Ly s-Lys-*、-Ala-Lys-*、-Lys-Ala-*、-Val-Lys-*、-Lys-Val-*、-Tyr-Arg-*、-Arg-Tyr-*、-Arg-Arg-*、-Ala-Ala-*、-Phe-Lys-*、-Lys-Phe-*、-Thr-Thr-*、-Thr-Met-*、-Met t-Thr-*、-Met-Tyr-*、-Tyr-Met-*、-Phe-Gln-*、-Gln-Phe-*、-Gly-Ser-*、-Leu-Gln-*、-Gln-Leu-*、-Ser-Ala-*、-Ser-Gly-*、-Val-Thr-*、-Thr-Val-*、-Val-Gln-*、-S er-Val-*、-Val-Ser-*、-Ala-Met-*、-Met-Ala-*、-Val-Arg-*、-Arg-Val-*、-Phe-Ala-*、-Ala-Phe-*、-Cit-Val-*、-Gln-Val-*、-Phe-Arg-*、-Arg-Phe-*、-Ala-Ala-* * 、-Gly-Gly-Gly- * 、-Ala-Val-Ala- * 、-Gly-Val-Gly- * 、-Ala-Val-Gly- * 、-Gly-Phe-Lys-*、-Lys-Phe-Gly-*、-Leu-Ala-Leu-*、-Val-Ala-Leu-*、-Leu-Ala-Val-*、-Val-Ala-Val-*、-Ala-Val-Ala-Gly- * 、-Gly-Phe-Gly-Gly- * 、-Gly-Gly-Phe-Gly-*、-Ala-Val-Gly-Gly-*、-Ala-Ala-Ala-Ala- * 、-Ala-Val-Ala-Ala- *, -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-*, wherein * indicates the N-terminus of the peptide covalently linked to Z.

[0455] In an embodiment, E is selected from the group consisting of: -L-Ala-L-Val-*, -L-Val-L-Ala-*, -L-Val-L-Lys-*, -L-Val-L-Arg-*, -L-Val-L-Cit-*, -L-Ala-L-Val-L-Glu-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-L-Val-L-Ala-*, -L-Ala-L-Ala-Gly-*, -L-Ala-L-Val-Gly-*, -Gly-Gly-L-Glu-*, -Gly-L-Phe-Gly-Gly-*, -Gly-L-Glu-Gly-Gly-*; wherein * indicates the N-terminus of the peptide covalently linked to Z.

[0456] In an embodiment, Z is -C(=O)-LY.

[0457] In an embodiment, Z is In the embodiment, m represents an integer of 1 to 10. In the embodiment, m is 1. In the embodiment, m is 2. In the embodiment, m is 3. In the embodiment, m is 4. In the embodiment, m is 5. In the embodiment, m is 6. In the embodiment, m is 7. In the embodiment, m is 8. In the embodiment, m is 9. In the embodiment, m is 10.

[0458] In an embodiment, Z is In the embodiment, m represents an integer of 1 to 10. In the embodiment, m is 1. In the embodiment, m is 2. In the embodiment, m is 3. In the embodiment, m is 4. In the embodiment, m is 5. In the embodiment, m is 6. In the embodiment, m is 7. In the embodiment, m is 8. In the embodiment, m is 9. In the embodiment, m is 10.

[0459] In an embodiment, L is -(C1-C 10 Alkylene)-.

[0460] In an embodiment, L is -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -or-(OCH2CH2) j -, wherein j represents an integer of 1 to 10; and wherein * represents a site for covalent bonding to Y.

[0461] In an embodiment, L is -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*, wherein j represents an integer of 1-10; and wherein * represents a site for covalent bonding to Y.

[0462] In an embodiment, L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, where * indicates the site of covalent attachment to Y.

[0463] In embodiments, Y is a Michael acceptor group, succinimide, epoxy, or halogen.

[0464] In an embodiment, Y is

[0465]

[0466] Where R 7 and R 8 Each is independently H or C1-C3 alkyl.

[0467] In an embodiment, Z is

[0468]

[0469] In the embodiments, -E-NH-CH 2- has one of the following structures, where * indicates the N-terminus of the peptide covalently linked to Z:

[0470]

[0471] In an embodiment, ZE-NH-CH2- has one of the following structures,

[0472]

[0473] In an embodiment, D is represented by one of the following structures:

[0474]

[0475] In an embodiment, D is (DI).

[0476] In an embodiment, D is (D-II).

[0477] In an embodiment, D is (D-III).

[0478] In an embodiment, D is (D-IV).

[0479] In an embodiment, the compound has one of the following structures,

[0480] or a pharmaceutically acceptable salt thereof.

[0481] In an embodiment, the compound is (PL1) or a pharmaceutically acceptable salt thereof.

[0482] In an embodiment, the compound is (PL2) or a pharmaceutically acceptable salt thereof.

[0483] In an embodiment, the compound is (PL3) or a pharmaceutically acceptable salt thereof.

[0484] In an embodiment, the compound is (PL4) or a pharmaceutically acceptable salt thereof.

[0485] In an embodiment, the compound is (PL5) or a pharmaceutically acceptable salt thereof.

[0486] In an embodiment, the compound is (PL6) or a pharmaceutically acceptable salt thereof.

[0487] In an embodiment, the compound is (PL7) or a pharmaceutically acceptable salt thereof.

[0488] In an embodiment, the compound is (PL8) or a pharmaceutically acceptable salt thereof.

[0489] In an embodiment, the compound is (PL9) or a pharmaceutically acceptable salt thereof.

[0490] In an embodiment, the compound is (PL10) or a pharmaceutically acceptable salt thereof.

[0491] Compounds of formula (III) and formula (IV)

[0492] Compound of formula (III)

[0493] In some aspects, the invention features a conjugate (e.g., a single-drug conjugate) comprising a cell-binding agent and a payload (e.g., a payload comprising an auristatin derivative and a linker). In embodiments, the portion of the conjugate comprising the auristatin derivative is formed by a structure according to any embodiment of formula (II) as described herein.

[0494] In yet another aspect, the invention features a compound of formula (III),

[0495] {D—CH2—NH—E—Z'} p —C(III),

[0496] in:

[0497] D is represented by the following structural formula:

[0498] in

[0499] R 1 is -H or -OH;

[0500] R 2 C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl;

[0501] R 3 and R 4 are independently -H or C1-C3 alkyl;

[0502] n is an integer from 1 to 6;

[0503] E is a peptide comprising 2 to 10 amino acids; wherein E is optionally substituted with one or more polyols; and wherein the N-terminus of the peptide is covalently linked to Z';

[0504] Z' is -C(=O)-L-Y'-, wherein m represents an integer of 1-10, and * represents a site covalently linked to the C;

[0505] L is -(C1-C 10 Alkylene)-*, -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -、-(OCH2CH2) j -、-CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*; wherein j represents an integer from 1 to 10; and wherein * represents a site of covalent attachment to Y';

[0506] L 1 =-(C1-C 10 Alkylene)-;

[0507] R 5 is -H or -CH3; and

[0508] C represents a cell-binding agent;

[0509] Y' is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on the cell binding agent;

[0510] p has a value between 1 and 18.

[0511] In an embodiment, R 1 is -H.

[0512] In an embodiment, R 1 It is -OH.

[0513] In an embodiment, R 2 It is C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl.

[0514] In an embodiment, R 2 is a C1-C3 alkyl group. 2 It is -CH3.

[0515] In an embodiment, R 2 is -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH or -C(=O)NHCH2CH2CH2OH. In the embodiment, R 2 It is -C(=O)OH.

[0516] In an embodiment, R 2 is heteroaryl (e.g., C3-C8 heteroaryl). 2 is a N- and / or S-containing heteroaryl. 2 for

[0517] In an embodiment, R 1 -H and R 2 It is -C(=O)OH.

[0518] In an embodiment, R 1 -H and R 2 It is -C(=O)NHCH2CH2CH2OH.

[0519] In an embodiment, R 1 -H and R 2 for

[0520] In an embodiment, R 1is -OH and R 2 It is -CH3.

[0521] In an embodiment, each R 3 and R 4 are independently -H or C1-C3 alkyl.

[0522] In an embodiment, R 3 and R 4 Both are -H. In the embodiment, R 3 and R 4 Both are C1-C3 alkyl. 3 and R 4 Both are -CH3.

[0523] In an embodiment, n is an integer from 1 to 6. In an embodiment, n is 1. In an embodiment, n is 2. In an embodiment, n is 3. In an embodiment, n is 4. In an embodiment, n is 5. In an embodiment, n is 6. In an embodiment, n is 1.

[0524] In an embodiment, E is a peptide of 2, 3 or 4 amino acids, wherein each amino acid in the peptide is an L amino acid, or at least one amino acid in the peptide is a D amino acid.

[0525] In an embodiment, E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with a polyol.

[0526] In an embodiment, E comprises an amino acid having the structure,

[0527]

[0528] Where R 6 It is -H or C1-C6 alkyl.

[0529] In an embodiment, E comprises an amino acid having the structure,

[0530]

[0531] In an example, the following group consists of: Ala-Val-*、-Val-Ala-*、-Gly-Gly-*、-Val-Cit-*、-Cit-Val-*、-Leu-Ala-*、-Ala-Leu-*、-Leu-Cit-*、-Cit-Leu-*、-Leu-Ala-*、-Ala-Leu-*、-Lys -Lys-*、-Ala-Lys-*、-Lys-Ala-*、-Val-Lys-*、-Lys-Val-*、-Tyr-Arg-*、-Arg-Tyr-*、-Arg-Arg-*、-Ala-Ala-*、-Phe-Lys-*、-Lys-Phe-*、-Thr-Thr-*、-Thr-Met-*、-Met t-Thr-*、-Met-Tyr-*、-Tyr-Met-*、-Phe-Gln-*、-Gln-Phe-*、-Gly-Ser-*、-Leu-Gln-*、-Gln-Leu-*、-Ser-Ala-*、-Ser-Gly-*、-Val-Thr-*、-Thr-Val-*、-Val-Gln-*、-S er-Val-*、-Val-Ser-*、-Ala-Met-*、-Met-Ala-*、-Val-Arg-*、-Arg-Val-*、-Phe-Ala-*、-Ala-Phe-*、-Cit-Val-*、-Gln-Val-*、-Phe-Arg-*、-Arg-Phe-*、-Ala-Ala-* * 、-Gly-Gly-Gly- * 、-Ala-Val-Ala- * 、-Gly-Val-Gly- * 、-Ala-Val-Gly- * 、-Gly-Phe-Lys-*、-Lys-Phe-Gly-*、-Leu-Ala-Leu-*、-Val-Ala-Leu-*、-Leu-Ala-Val-*、-Val-Ala-Val-*、-Ala-Val-Ala-Gly- * 、-Gly-Phe-Gly-Gly- * 、-Gly-Gly-Phe-Gly-*、-Ala-Val-Gly-Gly-*、-Ala-Ala-Ala-Ala- * 、-Ala-Val-Ala-Ala- *, -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-*, wherein * indicates the N-terminus of the peptide covalently linked to Z'.

[0532] In an embodiment, E is selected from the group consisting of: -L-Ala-L-Val-*, -L-Val-L-Ala-*, -L-Val-L-Lys-*, -L-Val-L-Arg-*, -L-Val-L-Cit-*, -L-Ala-L-Val-L-Glu-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-L-Val-L-Ala-*, -L-Ala-L-Ala-Gly-*, -L-Ala-L-Val-Gly-*, -Gly-Gly-L-Glu-*, -Gly-L-Phe-Gly-Gly-*, -Gly-L-Glu-Gly-Gly-*; wherein * indicates the N-terminus of the peptide covalently linked to Z'.

[0533] In an embodiment, Z' is -C(=O)-L-Y'-.

[0534] In an embodiment, Z' is and * represents a site covalently attached to said C. In an embodiment, m represents an integer of 1-10. In an embodiment, m is 1. In an embodiment, m is 2. In an embodiment, m is 3. In an embodiment, m is 4. In an embodiment, m is 5. In an embodiment, m is 6. In an embodiment, m is 7. In an embodiment, m is 8. In an embodiment, m is 9. In an embodiment, m is 10.

[0535] In an embodiment, Z' is and * represents a site covalently attached to said C. In an embodiment, m represents an integer of 1-10. In an embodiment, m is 1. In an embodiment, m is 2. In an embodiment, m is 3. In an embodiment, m is 4. In an embodiment, m is 5. In an embodiment, m is 6. In an embodiment, m is 7. In an embodiment, m is 8. In an embodiment, m is 9. In an embodiment, m is 10.

[0536] In an embodiment, L is -(C1-C 10 Alkylene)-.

[0537] In an embodiment, L is -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -or-(OCH2CH2) j-, wherein j represents an integer of 1 to 10; and wherein * represents a site of covalent bonding to Y'.

[0538] In an embodiment, L is -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*, wherein j represents an integer of 1 to 10; and wherein * represents a site of covalent bonding to Y'.

[0539] In an embodiment, L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, wherein * indicates the site of covalent attachment to Y'.

[0540] In embodiments, Y' is a group formed by reaction of an electrophilic group with a reactive nucleophilic group present on the cell binding agent.

[0541] In an embodiment, Y' is formed by

[0542]

[0543] Where R 7 and R 8 Each is independently -H or C1-C3 alkyl.

[0544] In an embodiment, Y' is

[0545]

[0546] Where R 7 and R 8 Each is independently -H or C1-C3 alkyl, and * indicates the site of covalent attachment to the C.

[0547] In an embodiment, Z' is formed by:

[0548]

[0549] In an embodiment, Z' is:

[0550]

[0551] Where * indicates the site of covalent attachment to C.

[0552] In the embodiments, -E-NH-CH 2-has one of the following structures, where * indicates the N-terminus of the peptide covalently linked to Z':

[0553]

[0554] In an embodiment, -Z'-E-NH-CH2- is formed by one of the following structures:

[0555]

[0556] In an embodiment, Z'-E-NH-CH2- is one of the following structures, wherein * represents the point of connection to C:

[0557]

[0558] In an embodiment, D is represented by one of the following structures:

[0559]

[0560] In an embodiment, D is (DI).

[0561] In an embodiment, D is (D-II).

[0562] In an embodiment, D is (D-III).

[0563] In an embodiment, D is (D-IV).

[0564] In an embodiment, D—CH2—NH—E—Z′— is formed by one of the following structures,

[0565]

[0566]

[0567] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL1) or a pharmaceutically acceptable salt thereof.

[0568] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL2) or a pharmaceutically acceptable salt thereof.

[0569] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL3) or a pharmaceutically acceptable salt thereof.

[0570] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL4) or a pharmaceutically acceptable salt thereof.

[0571] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL5) or a pharmaceutically acceptable salt thereof.

[0572] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL6) or a pharmaceutically acceptable salt thereof.

[0573] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL7) or a pharmaceutically acceptable salt thereof.

[0574] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL8) or a pharmaceutically acceptable salt thereof.

[0575] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL9) or a pharmaceutically acceptable salt thereof.

[0576] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL10) or a pharmaceutically acceptable salt thereof.

[0577] In an embodiment, {D—CH2—NH—E—Z'} p — C is one of the following structures, wherein C is a monoclonal antibody and p is the drug to antibody ratio (DAR), and p is an average of about 2-8, 4-8, or 7-8,

[0578]

[0579]

[0580] In an embodiment, p has a value between 1 and 18.

[0581] In embodiments, p is about 3-8 (eg, 3.2 to 8.0) or an average of 4-8.

[0582] In an embodiment, p is an average number of about 4.

[0583] In an embodiment, p is 4.

[0584] In an embodiment, p is an average number of about 7.5.

[0585] In an embodiment, p is an average number of about 8.

[0586] In an embodiment, p is 8.

[0587] In an embodiment, {D—CH2—NH—E—Z'} p - C is (PL1'), wherein C is a monoclonal antibody, and p is the drug to antibody ratio (DAR), and p is an average of about 2-8, 4-8, or 7-8 (eg, 3.2 to 8.0).

[0588] In an embodiment, {D—CH2—NH—E—Z'} p- C is (PL2'), wherein C is a monoclonal antibody, and p is the drug to antibody ratio (DAR), and p is an average of about 2-8, 4-8, or 7-8 (eg, 3.2 to 8.0).

[0589] In an embodiment, {D—CH2—NH—E—Z'} p - C is (PL3'), wherein C is a monoclonal antibody, and p is the drug to antibody ratio (DAR), and p is an average of about 2-8, 4-8, or 7-8 (eg, 3.2 to 8.0).

[0590] In an embodiment, {D—CH2—NH—E—Z'} p - C is (PL4'), wherein C is a monoclonal antibody, and p is the drug to antibody ratio (DAR), and p is an average of about 2-8, 4-8, or 7-8 (eg, 3.2 to 8.0).

[0591] In an embodiment, {D—CH2—NH—E—Z'} p - C is (PL5'), wherein C is a monoclonal antibody, and p is the drug to antibody ratio (DAR), and p is an average of about 2-8, 4-8, or 7-8 (eg, 3.2 to 8.0).

[0592] In an embodiment, {D—CH2—NH—E—Z'} p - C is (PL6'), wherein C is a monoclonal antibody, and p is the drug to antibody ratio (DAR), and p is an average of about 2-8, 4-8, or 7-8 (eg, 3.2 to 8.0).

[0593] In an embodiment, {D—CH2—NH—E—Z'} p - C is (PL7'), wherein C is a monoclonal antibody, and p is the drug to antibody ratio (DAR), and p is an average of about 2-8, 4-8, or 7-8 (eg, 3.2 to 8.0).

[0594] In an embodiment, {D—CH2—NH—E—Z'} p - C is (PL8'), wherein C is a monoclonal antibody, and p is the drug to antibody ratio (DAR), and p is an average of about 2-8, 4-8, or 7-8 (eg, 3.2 to 8.0).

[0595] In an embodiment, {D—CH2—NH—E—Z'} p - C is (PL9'), wherein C is a monoclonal antibody, and p is the drug to antibody ratio (DAR), and p is an average of about 2-8, 4-8, or 7-8 (eg, 3.2 to 8.0).

[0596] In an embodiment, {D—CH2—NH—E—Z'} p - C is (PL10'), wherein C is a monoclonal antibody, and p is the drug to antibody ratio (DAR), and p is an average of about 2-8, 4-8, or 7-8 (eg, 3.2 to 8.0).

[0597] Compounds of formula (IV)

[0598] In some aspects, the invention features a conjugate (e.g., a dual-drug conjugate) comprising a cell-binding agent, a payload (e.g., a payload comprising an auristatin derivative and a linker) and another payload (e.g., another payload comprising a compound of a different formula). In an embodiment, the portion of the conjugate comprising the auristatin derivative is formed by formula (II) as described herein.

[0599] In addition, considering that there are many types of cancer cells, incorporating two types of payloads with different mechanisms of action (MOA) in ADC can increase the efficacy of the drug and expand the therapeutic window of the drug. For example, in a xenograft mouse model, anti-Her2 dual-drug ADC is more effective than the corresponding single-drug ADC and two single-drug variants co-administered [see, for example, Yamazaki, CM and Tsuchikama, K. et al., Antibody-drug conjugates with dual payloads for combating breast tumor heterogeneity and drug resistance. Nat Commun 12, 3528 (2021). https: / / doi.org / 10.1038 / s41467-021-23793-7].

[0600] In yet another aspect, the invention features a compound of formula (IV),

[0601] {D—CH2—NH—E—Z'} p' —C—{W} t (IV),

[0602] in:

[0603] D is represented by the following structural formula:

[0604] in

[0605] R 1 is -H or -OH;

[0606] R 2C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl;

[0607] R 3 and R 4 are independently -H or C1-C3 alkyl;

[0608] n is an integer from 1 to 6;

[0609] E is a peptide comprising 2 to 10 amino acids; wherein E is optionally substituted with one or more polyols; and wherein the N-terminus of the peptide is covalently linked to Z';

[0610] Z' is -C(=O)-L-Y'-, wherein m represents an integer of 1-10, and * represents a site covalently linked to the C;

[0611] L is -(C1-C 10 Alkylene)-*, -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -、-(OCH2CH2) j -、-CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*; wherein j represents an integer from 1 to 10; and wherein * represents a site of covalent attachment to Y';

[0612] L1 is -(C1-C 10 Alkylene)-;

[0613] R 5 is -H or -CH3; and

[0614] C represents a cell-binding agent;

[0615] Y' is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on the cell binding agent;

[0616] W is a group formed by the reaction of compound W' with a reactive nucleophilic group present on C; wherein W' is a cell-killing agent attached to a linker so that W' can be coupled to C;

[0617] p' and t have values ​​between 1 and 10; wherein p' and t may be the same or different numbers, and p':t is about 1:1, about 1:2 or about 2:1, and wherein p':t is 1:1 or 1:2 or 2:1.

[0618] In an embodiment, R 1 is -H.

[0619] In an embodiment, R 1 It is -OH.

[0620] In an embodiment, R 2 It is C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl.

[0621] In an embodiment, R 2 is a C1-C3 alkyl group. 2 It is -CH3.

[0622] In an embodiment, R 2 is -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH or -C(=O)NHCH2CH2CH2OH. In the embodiment, R 2 It is -C(=O)OH.

[0623] In an embodiment, R 2 is heteroaryl (e.g., C3-C8 heteroaryl). 2 is a N- and / or S-containing heteroaryl. 2 for

[0624] In an embodiment, R 1 -H and R 2 It is -C(=O)OH.

[0625] In an embodiment, R 1 -H and R 2 It is -C(=O)NHCH2CH2CH2OH.

[0626] In an embodiment, R 1 -H and R 2 for

[0627] In an embodiment, R 1 is -OH and R2 It is -CH3.

[0628] In an embodiment, each R 3 and R 4 are independently -H or C1-C3 alkyl.

[0629] In an embodiment, R 3 and R 4 Both are -H. In the embodiment, R 3 and R 4 Both are C1-C3 alkyl. 3 and R 4 Both are -CH3.

[0630] In an embodiment, n is an integer from 1 to 6. In an embodiment, n is 1. In an embodiment, n is 2. In an embodiment, n is 3. In an embodiment, n is 4. In an embodiment, n is 5. In an embodiment, n is 6. In an embodiment, n is 1.

[0631] In an embodiment, E is a peptide of 2, 3 or 4 amino acids, wherein each amino acid in the peptide is an L amino acid, or at least one amino acid in the peptide is a D amino acid.

[0632] In an embodiment, E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with a polyol.

[0633] In an embodiment, E comprises an amino acid having the structure,

[0634]

[0635] Where R 6 It is -H or C1-C6 alkyl.

[0636] In an embodiment, E comprises an amino acid having the structure,

[0637]

[0638] In an example, the following group consists of: Ala-Val-*、-Val-Ala-*、-Gly-Gly-*、-Val-Cit-*、-Cit-Val-*、-Leu-Ala-*、-Ala-Leu-*、-Leu-Cit-*、-Cit-Leu-*、-Leu-Ala-*、-Ala-Leu-*、-Lys -Lys-*、-Ala-Lys-*、-Lys-Ala-*、-Val-Lys-*、-Lys-Val-*、-Tyr-Arg-*、-Arg-Tyr-*、-Arg-Arg-*、-Ala-Ala-*、-Phe-Lys-*、-Lys-Phe-*、-Thr-Thr-*、-Thr-Met-*、-Met t-Thr-*、-Met-Tyr-*、-Tyr-Met-*、-Phe-Gln-*、-Gln-Phe-*、-Gly-Ser-*、-Leu-Gln-*、-Gln-Leu-*、-Ser-Ala-*、-Ser-Gly-*、-Val-Thr-*、-Thr-Val-*、-Val-Gln-*、-S er-Val-*、-Val-Ser-*、-Ala-Met-*、-Met-Ala-*、-Val-Arg-*、-Arg-Val-*、-Phe-Ala-*、-Ala-Phe-*、-Cit-Val-*、-Gln-Val-*、-Phe-Arg-*、-Arg-Phe-*、-Ala-Ala-* * 、-Gly-Gly-Gly- * 、-Ala-Val-Ala- * 、-Gly-Val-Gly- * 、-Ala-Val-Gly- * 、-Gly-Phe-Lys-*、-Lys-Phe-Gly-*、-Leu-Ala-Leu-*、-Val-Ala-Leu-*、-Leu-Ala-Val-*、-Val-Ala-Val-*、-Ala-Val-Ala-Gly- * 、-Gly-Phe-Gly-Gly- * 、-Gly-Gly-Phe-Gly-*、-Ala-Val-Gly-Gly-*、-Ala-Ala-Ala-Ala- * 、-Ala-Val-Ala-Ala- *, -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-*, wherein * indicates the N-terminus of the peptide covalently linked to Z'.

[0639] In an embodiment, E is selected from the group consisting of: -L-Ala-L-Val-*, -L-Val-L-Ala-*, -L-Val-L-Lys-*, -L-Val-L-Arg-*, -L-Val-L-Cit-*, -L-Ala-L-Val-L-Glu-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-L-Val-L-Ala-*, -L-Ala-L-Ala-Gly-*, -L-Ala-L-Val-Gly-*, -Gly-Gly-L-Glu-*, -Gly-L-Phe-Gly-Gly-*, -Gly-L-Glu-Gly-Gly-*; wherein * indicates the N-terminus of the peptide covalently linked to Z'.

[0640] In an embodiment, Z' is -C(=O)-L-Y'-.

[0641] In an embodiment, Z' is and * represents a site covalently attached to said C. In an embodiment, m represents an integer of 1-10. In an embodiment, m is 1. In an embodiment, m is 2. In an embodiment, m is 3. In an embodiment, m is 4. In an embodiment, m is 5. In an embodiment, m is 6. In an embodiment, m is 7. In an embodiment, m is 8. In an embodiment, m is 9. In an embodiment, m is 10.

[0642] In an embodiment, Z' is and * represents a site covalently attached to said C. In an embodiment, m represents an integer of 1-10. In an embodiment, m is 1. In an embodiment, m is 2. In an embodiment, m is 3. In an embodiment, m is 4. In an embodiment, m is 5. In an embodiment, m is 6. In an embodiment, m is 7. In an embodiment, m is 8. In an embodiment, m is 9. In an embodiment, m is 10.

[0643] In an embodiment, L is -(C1-C 10 Alkylene)-.

[0644] In an embodiment, L is -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -or-(OCH2CH2) j-, wherein j represents an integer of 1 to 10; and wherein * represents a site of covalent bonding to Y'.

[0645] In an embodiment, L is -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*, wherein j represents an integer of 1 to 10; and wherein * represents a site of covalent bonding to Y'.

[0646] In an embodiment, L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, wherein * indicates the site of covalent attachment to Y'.

[0647] In embodiments, Y' is a group formed by reaction of an electrophilic group with a reactive nucleophilic group present on the cell binding agent.

[0648] In an embodiment, Y' is formed by

[0649]

[0650] Where R 7 and R 8 Each is independently -H or C1-C3 alkyl.

[0651] In an embodiment, Y' is

[0652]

[0653] Where R 7 and R 8 Each is independently -H or C1-C3 alkyl, and * indicates the site of covalent attachment to the C.

[0654] In an embodiment, Z' is formed by:

[0655]

[0656] In an embodiment, Z' is:

[0657]

[0658] Where * indicates the site of covalent attachment to C.

[0659] In the embodiments, -E-NH-CH 2-has one of the following structures, where * indicates the N-terminus of the peptide covalently linked to Z':

[0660]

[0661] In an embodiment, -Z'-E-NH-CH2- is formed by one of the following structures:

[0662]

[0663] In an embodiment, -Z'-E-NH-CH2- is one of the following structures, wherein * represents the point of connection to C:

[0664]

[0665] In an embodiment, D is represented by one of the following structures:

[0666]

[0667] In an embodiment, D is (DI).

[0668] In an embodiment, D is (D-II).

[0669] In an embodiment, D is (D-III).

[0670] In an embodiment, D is (D-IV).

[0671] In an embodiment, D—CH2—NH—E—Z′— is formed by one of the following structures:

[0672]

[0673]

[0674] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL1) or a pharmaceutically acceptable salt thereof.

[0675] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL2) or a pharmaceutically acceptable salt thereof.

[0676] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL3) or a pharmaceutically acceptable salt thereof.

[0677] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL4) or a pharmaceutically acceptable salt thereof.

[0678] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL5) or a pharmaceutically acceptable salt thereof.

[0679] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL6) or a pharmaceutically acceptable salt thereof.

[0680] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL7) or a pharmaceutically acceptable salt thereof.

[0681] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL8) or a pharmaceutically acceptable salt thereof.

[0682] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL9) or a pharmaceutically acceptable salt thereof.

[0683] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL10) or a pharmaceutically acceptable salt thereof.

[0684] In an embodiment, W is formed by compound W'. In an embodiment, W is formed by covalently linking compound W' to C.

[0685] In an embodiment, W' is a compound that can be covalently linked to C. In an embodiment, W' is a compound described in International Patent Publication No. WO2021 / 173773 A1 (e.g., a compound that can be covalently linked to C). In an embodiment, W' is a compound described in International Patent Publication No. WO2021 / 173773 A1, Medinotecan. In an embodiment, W' is a compound described in U.S. Patent Publication No. US2021 / 0283125 A1 (e.g., a compound that can be covalently linked to C).

[0686] In an embodiment, W' is a compound according to formula (A)

[0687] D w —L w —Q w '—CH2—NH—E w —Z w (A)

[0688] or a pharmaceutically acceptable salt thereof, wherein:

[0689] Dw Represented by the following structural formula:

[0690] in

[0691] R 1w is -H, -OH or -OMe;

[0692] R 2w is a C1-C5 alkyl group;

[0693] R 3wand R 4w are independently -H, C1-C3 alkyl or C3-C6 cycloalkyl;

[0694] nw is an integer from 1 to 6;

[0695] L w does not exist, is -(C1-C5 alkylene)-, -CH2OCH2CH2- or -OCH2CH2OCH2CH2-;

[0696] Q w ' is -O- or -S-;

[0697] E w is a peptide comprising 2 to 10 amino acids; wherein E w is optionally substituted with one or more polyols; and wherein the N-terminus of the peptide is covalently linked to Z w ;

[0698] Z w -C(=O)-L 1w -Y, Where mw represents an integer from 1 to 10;

[0699] L 1w =-(C1-C 10 Alkylene)-*, -CH2(OCH2CH2) jw -*、-CH2CH2(OCH2CH2) jw -、-(OCH2CH2) jw -、-CH2CH2(OCH2CH2) jw N(R 5w )C(=O)-L 2w -* or -CH2(OCH2CH2) jw N(R 5w )C(=O)-L 2w -*; wherein jw represents an integer from 1 to 10; and wherein * represents covalent bonding to Y w The location of

[0700] L 2w =-(C1-C 10 Alkylene)-;

[0701] R 5w is -H or -CH3; and

[0702] Y w It is an electrophilic group or a nucleophilic group.

[0703] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL1), and W is formed from (PL2).

[0704] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL3) and W is formed from (PL4).

[0705] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL5) and W is formed from (PL6).

[0706] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL7) and W is formed from (PL8).

[0707] In an embodiment, D—CH2—NH—E—Z′— is formed from (PL9), and W is formed from (PL10).

[0708] In an embodiment, {D—CH2—NH—E—Z'} p' —C—{W} t is one of the following structures, wherein C is a monoclonal antibody, p' and t are drug to antibody ratio (DAR), and p':t is 1:1 or about 1:1, and p' and t are respectively an average number in the range of about 1-7, or an average number of about 2, about 3, about 4, about 5, or about 6:

[0709]

[0710] In an embodiment, p' and t are both 4, or an average of 4, or about 4.

[0711] In embodiments, p':t is about 1:1, about 1:2, or about 2:1.

[0712] In embodiments, p':t is 1:1, 1:2 or 2:1.

[0713] In an embodiment, {D—CH2—NH—E—Z'} p' —C—{W} t is (PL1"), wherein C is a monoclonal antibody, p' and t are drug to antibody ratio (DAR), and p':t is 1:1 (or about 1:1), and p' and t are an average of about 1-7 (e.g., an average of about 2, about 3, about 4, about 5, or about 6).

[0714] In an embodiment, {D—CH2—NH—E—Z'} p' —C—{W} t is (PL2"), wherein C is a monoclonal antibody, p' and t are drug to antibody ratio (DAR), and p':t is 1:1 (or about 1:1), and p' and t are an average of about 1-7 (e.g., an average of about 2, about 3, about 4, about 5, or about 6).

[0715] In another aspect, the invention features a method of preparing a conjugate of formula (III) comprising a cell-binding agent and a drug, and the method comprises contacting the cell-binding agent with a compound of formula (II) such that a covalent bond is formed between the cell-binding agent and the compound of formula (II).

[0716] In yet another aspect, the invention features a method for preparing a two-drug conjugate of formula (IV) comprising a cell-binding agent and two different drugs, and the method comprises contacting the cell-binding agent with a compound of formula (II) and another compound of a different formula, such that a covalent bond is formed between the cell-binding agent and the compound of formula (II) and another compound having a formula different from formula (II).

[0717] In yet another aspect, the invention features a conjugate comprising a cell-binding agent and a drug. In embodiments, the conjugate is prepared according to any of the methods described herein.

[0718] In embodiments, the conjugate comprises a cell binding agent that is an antibody or antigen binding fragment thereof.

[0719] In embodiments, the conjugate comprises a cell binding agent that is a monoclonal antibody or an antigen binding fragment thereof.

[0720] In embodiments, the cell-binding agent is an antibody or antigen-binding fragment thereof; p is the drug-to-antibody ratio (DAR) and has a value between 1 and 18. In embodiments, p is an average of about 2-10, 4-8, or 7-8 (e.g., 3.2 to 8.0).

[0721] In embodiments, the cell-binding agent is a monoclonal antibody or an antigen-binding fragment thereof; p is the drug-to-antibody ratio (DAR) and has a value between 1 and 18. In embodiments, p is an average of about 2-10, 4-8, or 7-8 (e.g., 3.2 to 8.0).

[0722] In embodiments, the cell-binding agent is an antibody or antigen-binding fragment thereof; p' and t are drug-to-antibody ratios (DAR) and have values ​​between 1 and 10. In embodiments, p' is an average of about 2-6. In embodiments, t is an average of about 2-6. In embodiments, p':t is about 1:1. In embodiments, p':t is about 1:2. In embodiments, p':t is about 2:1. In embodiments, p':t is 1:1. In embodiments, p':t is 1:2. In embodiments, p':t is 2:1.

[0723] In embodiments, the cell-binding agent is a monoclonal antibody or an antigen-binding fragment thereof; p' and t are drug-to-antibody ratios (DAR) and have values ​​between 1 and 10. In embodiments, p' is an average of about 2-6. In embodiments, t is an average of about 2-6. In embodiments, p':t is about 1:1. In embodiments, p':t is about 1:2. In embodiments, p':t is about 2:1. In embodiments, p':t is 1:1. In embodiments, p':t is 1:2. In embodiments, p':t is 2:1.

[0724] In another aspect, the invention features a pharmaceutical composition including any of the conjugates described herein.

[0725] In yet another aspect, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, wherein the method comprises administering any of the conjugates described herein or any pharmaceutical composition comprising any of the conjugates described herein.

[0726] In another aspect, the invention features a pharmaceutical composition comprising a compound of any of Formula (III) as described herein.

[0727] In another aspect, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, the method comprising administering a compound of any of Formula (III) as described herein or a pharmaceutical composition comprising a compound of any of Formula (III) as described herein.

[0728] In another aspect, the invention features a pharmaceutical composition comprising a compound of any of Formula (IV) as described herein.

[0729] In another aspect, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, the method comprising administering a compound of any of Formula (IV) as described herein or a pharmaceutical composition comprising a compound of any of Formula (IV) as described herein.

[0730] In embodiments, the method is used to treat cancer.

[0731] In embodiments, the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, a central nervous system tumor (CNS) tumor, colon cancer or colorectal cancer, diffuse intrinsic pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood cancer, Hodgkin's lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome (MDS), neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, pancreatic cancer, peritoneal cancer, prostate cancer, ovarian cancer, kidney cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, or Wilms' tumor.

[0732] In embodiments, the cancer is breast cancer.

[0733] Subscripts "p", "p'", and "t"

[0734] The conjugates described herein [e.g., any compound according to formula (III) or formula (IV)] may comprise a covalent linker of at least an auristatin derivative (e.g., any compound according to formula (II) described herein, such as those formed from any compound according to formula (I) described herein).

[0735] In embodiments, the subscript p (or p') represents the number of auristatin payload moieties [e.g., formed by a compound according to formula (II)] on a cell binding agent, and has a value of 1 to 18, 1 to 12, 1 to 10, or 1 to 8. Individual auristatin conjugates may also be referred to as auristatin conjugate compounds. In embodiments herein, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 auristatin payload moieties conjugated to a cell binding agent of an individual auristatin conjugate.

[0736] In embodiments, the subscript t represents the number of other payload moieties (e.g., W payload moieties). In embodiments herein, there may be 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) W payload moieties coupled to the cell binding agent of an individual auristatin conjugate.

[0737] In an embodiment, except for the number of auristatin effective load parts (and / or the number of W effective load parts) bound to each cell binding agent (i.e., auristatin conjugate composition), the group of individual auristatin conjugates is substantially consistent. In an embodiment, p and / or p' represent the average number of auristatin effective load parts bound to the cell binding agent of the auristatin conjugate composition. In the embodiment of the group, p (or p') is an average of 1 to about 18, 1 to about 10, 1 to about 8 or 1 to about 7, 2 to about 6, 3 to about 5 or 6 to about 8. In an embodiment, p (or p') is an average of about 2-10, 2-8, 4-8 or 7-8 (e.g., 3.2 to 8.0). In an embodiment, p (or p') is about 2. In an embodiment, p (or p') is about 4. In an embodiment, p (or p') is about 6. In an embodiment, p (or p') is about 8. In an embodiment, p (or p') is about 10. In embodiments, p (or p') is about 12. In embodiments, p (or p') is 2. In embodiments, p (or p') is 4. In embodiments, p (or p') is 8. In embodiments, p (or p') has a value of 3 to 4. In embodiments, p (or p') has a value of 4 to 5. In embodiments, p (or p') has a value of 5 to 6. In embodiments, p (or p') has a value of 6 to 7. In embodiments, p (or p') has a value of 7 to 8. In embodiments, p (or p') has a value of 7.4 to 8. In embodiments, p (or p') values ​​refer to the average drug load in the composition as well as the drug load of the primary ADC.

[0738] In an embodiment, except for the number of W effective load parts (and / or the number of Auristatin effective load parts) bound to each cell binding agent (i.e., Auristatin conjugate composition), the group of individual Auristatin conjugates is substantially consistent. In an embodiment, t represents the average number of W effective load parts bound to the cell binding agent of the Auristatin conjugate composition. In the embodiment of the group, t is an average of 1 to about 10, 1 to about 8 or 1 to about 7, 2 to about 6, 3 to about 5 or 6 to about 8. In an embodiment, t is an average of about 1-10 or 1-7. In an embodiment, t is about 2. In an embodiment, t is about 3. In an embodiment, t is about 4. In an embodiment, t is about 5. In an embodiment, t is about 6. In an embodiment, t is 2. In an embodiment, t is 3. In an embodiment, t is 4. In an embodiment, t is 5. In an embodiment, t is 6. In an embodiment, t is 4. In an embodiment, t value refers to the average drug load in the composition and the drug load of the main ADC.

[0739] In an embodiment, p' and t independently have a value between 1 and 10 or between 1 and 7 (e.g., about 2, 3, 4, 5, 6, or 7). In an embodiment, the value is an average. In an embodiment, p' and t are the same. In an embodiment, p' and t are different. In an embodiment, p':t is about 1:1, about 1:2, or about 2:1. In an embodiment, p':t is about 1:1. In an embodiment, p' and t are both about 4.

[0740] In embodiments, conjugation [(e.g., as found in any compound according to formula (III) described herein] will occur via reduced interchain disulfide bonds, and there may be about 1-18, 1-12, 1-10, 1-8, 2-8, 4-8, or 7-8 auristatin payload compounds conjugated to a cell-binding agent (e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) as described herein).

[0741] In embodiments, conjugation (e.g., as found in any compound according to formula (IV) described herein) will occur via reduced interchain disulfide bonds, and there may be about 1-10 or 1-7 auristatin payload compounds [e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) as described herein] conjugated to the cell binding agent, and about 1-10 or 1-7 W' payload compounds.

[0742] In embodiments, conjugation [e.g., as found in any compound according to formula (III) described herein] will occur via introduced cysteine ​​residues and reduced interchain disulfide bonds, and there may be about 1-18, 1-12, 1-10, 1-8, 2-8, 4-8, or 7-8 auristatin payload compounds [e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) as described herein] conjugated to a cell binding agent.

[0743] In embodiments, conjugation [e.g., as found in any compound according to formula (IV) described herein] will occur via introduced cysteine ​​residues and reduced interchain disulfide bonds, and there may be about 1-10 or 1-7 auristatin payload compounds [e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) as described herein] conjugated to the cell binding agent, and about 1-10 or 1-7 W' payload compounds.

[0744] In embodiments, conjugation [e.g., as found in any compound according to formula (III) described herein] will occur via an introduced cysteine ​​residue, and there may be about 1-18, 1-12, 1-10, 1-8, 2-8, 4-8, or 7-8 auristatin payload compounds [e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) as described herein] conjugated to a cell binding agent.

[0745] In embodiments, conjugation [e.g., as found in any compound according to formula (IV) described herein] will occur via an introduced cysteine ​​residue, and there may be about 1-10 or 1-7 auristatin payload compounds [e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) as described herein] conjugated to the cell binding agent, and about 1-10 or 1-7 W' payload compounds.

[0746] In embodiments, conjugation [e.g., as found in any compound according to formula (III) described herein] will occur via a lysine residue, and there may be about 1-18, 1-12, 1-10, 1-8, 2-8, 4-8, or 7-8 auristatin payload compounds [e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) as described herein] conjugated to a cell binding agent.

[0747] In embodiments, conjugation [e.g., as found in any compound according to formula (IV) described herein] will occur via a lysine residue, and there may be about 1-10 or 1-7 auristatin payload compounds [e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) as described herein] conjugated to the cell binding agent, and about 1-10 or 1-7 W' payload compounds.

[0748] Reactive groups on cell binding agents for covalent attachment

[0749] In an embodiment, the cell binding agent is bound to the peptide in the compound of formula (II) to release the linker to form a conjugate, such as a conjugate according to formula (III) or formula (IV). As described above, other linking components in formula (II) may be present in the conjugates described herein to provide additional space between the auristatin compound and the cell binding agent. In an embodiment, the cell binding agent is bound to the linker unit in formula (II) via a heteroatom of the cell binding agent.

[0750] Heteroatoms that may be present on the cell-binding agent for such binding include sulfur (in one embodiment, from a thiol group of the targeting ligand), oxygen (in one embodiment, from a carboxyl or hydroxyl group of the targeting ligand), and optionally substituted nitrogen (in one embodiment, from a primary or secondary amine functional group of the targeting ligand, or in another embodiment, from an optionally substituted amide nitrogen). These heteroatoms may be present on the targeting ligand in the natural state of the cell-binding agent, such as in a naturally occurring antibody, or may be introduced into the targeting ligand by chemical modification or bioengineering.

[0751] In one embodiment, the cell binding agent has a thiol functional group, such that the cell binding agent is bound to the auristatin payload compound [e.g., any compound according to formula (II) described herein or formed from a compound according to formula (I) as described herein] via the sulfur atom of the thiol functional group.

[0752] In another embodiment, the cell-binding agent has one or more lysine residues capable of reacting with an activated ester (such as, but not limited to, N-hydroxysuccinimide, pentafluorophenyl, and p-nitrophenyl esters) of an auristatin payload compound [e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) as described herein] and thereby providing an amide bond consisting of the nitrogen atom of the cell-binding agent and the C=O group of the compound of formula (II).

[0753] In yet another aspect, the cell binding agent has one or more lysine residues that can be chemically modified to introduce one or more thiol groups. In these embodiments, the cell binding agent is covalently linked to the auristatin payload compound (e.g., any compound according to formula (II) described herein or formed by a compound of formula (I) as described herein) via the sulfur atom of the thiol functional group. Reagents that can be used to modify lysine in the described manner include, but are not limited to, S-acetylthioacetic acid N-succinimidyl ester (SATA) and 2-iminothiolane hydrochloride (Traut's Reagent).

[0754] In another embodiment, the cell binding agent has one or more carbohydrate groups that can be modified to provide one or more thiol functional groups. The chemically modified cell binding agent in the auristatin conjugate is bound to the auristatin payload compound [e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) as described herein] via the sulfur atom of the thiol functional group.

[0755] In yet another embodiment, the cell binding agent has one or more carbohydrate groups that can be oxidized to provide an aldehyde (—CHO) functional group [see, e.g., Laguzza et al., 1989, J. Med. Chem. 32(3):548-55]. In these embodiments, the corresponding aldehyde interacts with a reactive site on the auristatin payload compound [e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) as described herein] to form a bond between the auristatin payload compound [e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) as described herein] and the cell binding agent. Reactive sites on the auristatin payload compound [e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) as described herein] that are capable of interacting with a reactive carbonyl-containing functional group on the targeting ligand include, but are not limited to, hydrazine and hydroxylamine.

[0756] In some aspects, the cell binding agent is capable of forming a bond by interacting with a reactive functional group Y in an auristatin payload compound [e.g., in any compound according to formula (II)] to form a covalent bond between Y' in formula (III) or formula (IV) and the cell binding agent corresponding to the targeting ligand. The functional group Y having the ability to interact with the targeting ligand will depend on the nature of the cell binding agent. In an embodiment, the reactive group is a maleimide present on the auristatin payload compound before being connected to form the cell binding agent. The covalent connection of the cell binding agent to the auristatin payload compound is achieved by the interaction of the thiol functional group of the cell binding agent with the maleimide functional group Y of the payload compound [e.g., in any compound according to formula (II) described herein or formed by a compound of formula (I) as described herein] to form a succinimide substituted by a thiol group. The thiol functional group may be present on the cell binding agent in its native state, such as in a naturally occurring residue, or may be introduced into the cell binding agent by chemical modification or by bioengineering.

[0757] In another embodiment, the cell binding agent is an antibody and the thiol group is generated by reducing the interchain disulfide bonds of the antibody. Thus, in an embodiment, the auristatin payload compound is conjugated to a cysteine ​​residue from one or more reduced interchain disulfide bonds.

[0758] In another embodiment, the cell-binding agent is an antibody and the thiol functional group is chemically introduced into the antibody, for example, by introducing a cysteine ​​residue. Thus, in an embodiment, the auristatin payload compound is coupled to the cell-binding agent via the introduced cysteine ​​residue of the cell-binding agent.

[0759] For bioconjugates, it has been observed that the drug conjugation site can affect multiple parameters, including ease of conjugation, drug-linker stability, the effect on the biophysical properties of the resulting bioconjugate and in vitro cytotoxicity. Regarding drug-linker stability, the conjugation site of the drug-linker part and the cell binding agent can affect the ability of the conjugated drug-linker part to undergo elimination reactions, resulting in premature release of free drugs in some cases. The conjugation site on the targeting ligand includes, for example, reduced interchain disulfide bonds and cysteine ​​residues selected at the engineered site. In an embodiment, compared with the conjugation method using the thiol residue from the reduced disulfide bond, the conjugation method for forming the auristatin conjugate as described herein uses a thiol residue at a genetically engineered site (e.g., according to the position 239 of the EU index as shown in Kabat) that is not prone to elimination reactions. In other embodiments, the conjugation method for forming the auristatin conjugate as described herein uses a thiol residue at a site that is more prone to elimination reactions (e.g., generated by reduction of interchain disulfide bonds).

[0760] Cell binding agent (C)

[0761] In an embodiment of the present invention, a cell-binding agent is present. The cell-binding agent is used to target and present auristatin or a pharmaceutical composition containing auristatin to a specific target cell population, and the cell-binding agent interacts with the target cell population due to the presence of its targeting component or molecule and causes the subsequent release of free drugs in the target cell (i.e., intracellular) or in the vicinity of the target cell (i.e., extracellular).

[0762] In embodiments, the cell binding agent may be a ligand that binds to a moiety on the target cell, such as a cell surface receptor. In embodiments, the ligand may be a growth factor or a fragment thereof that binds to a growth factor receptor. In embodiments, the ligand may be a cytokine or a fragment thereof that binds to a cytokine receptor. In embodiments, the growth factor receptor or cytokine receptor is a cell surface receptor.

[0763] Thus, by appropriate selection of the cell binding agent, the therapeutic use of an auristatin conjugate [eg, a compound according to formula (III) or formula (IV) as described herein] can be followed.

[0764] Cell binding agents include, but are not limited to, proteins, polypeptides, and peptides. Suitable cell binding agents include, for example, antibodies (e.g., full-length antibodies and antigen-binding fragments thereof, including polyclonal antibodies and monoclonal antibodies), interferons, lymphokines, hormones, growth factors, colony stimulating factors, vitamins (e.g., folic acid), nutrient transport molecules (e.g., but not limited to transferrin), or any other cell binding molecules or substances. In an embodiment, the cell binding agent is an antibody or non-antibody protein targeting agent.

[0765] Antigens targeted by cell-binding agents

[0766] In embodiments, exemplary antigens or ligands include renin; growth hormone (eg, human growth hormone and bovine growth hormone); growth hormone releasing factor; parathyroid hormone; or thyroid stimulating hormone, or fragments thereof.

[0767] In the Examples, exemplary antigens or ligands include lipoprotein; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; or glucagon, or a fragment thereof.

[0768] In an embodiment, exemplary antigens or ligands include coagulation factors (e.g., factor vmc, factor IX, tissue factor, and von Willebrands factor); anticoagulant factors (e.g., protein C); atrial natriuretic factor; pulmonary surfactant factor; plasminogen activator (e.g., urokinase, human urine, or tissue-type plasminogen activator); bombesin; thrombin; or hematopoietic growth factor, or a fragment thereof.

[0769] In the Examples, exemplary antigens or ligands include tumor necrosis factor-α and tumor necrosis factor-β, or fragments thereof.

[0770] In the embodiments, exemplary antigens or ligands include enkephalinase; RANTES (i.e., regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein-1-alpha; serum albumin (human serum albumin); Muellerian-inhibiting substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; microbial protein (β-lactamase); deoxyribonuclease (DNase); IgE; inhibin; or activin; or a fragment thereof.

[0771] In embodiments, exemplary antigens or ligands include cytotoxic T lymphocyte-associated antigens (eg, CTLA-4) or fragments thereof.

[0772] In the Examples, exemplary antigens or ligands include vascular endothelial growth factor or a fragment thereof.

[0773] In an embodiment, exemplary antigens or ligands include receptors for hormones or growth factors; protein A or D; rheumatoid factor; neurotrophic factors (e.g., bone-derived neurotrophic factor, neurotrophic factor-3, neurotrophic factor-4, neurotrophic factor-5 or neurotrophic factor-6), nerve growth factors (e.g., NGF-b); platelet-derived growth factor; fibroblast growth factors (e.g., aFGF and bFGF); fibroblast growth factor receptor 2; epidermal growth factor; transforming growth factors (e.g., TGF-α, TGF-bI, TGF-p2, TGF-p3, TGF-p4 and TGF-p5); insulin-like growth factor-I and insulin-like growth factor-II; des(l-3)-IGF-I (brain IGF-I); or insulin-like growth factor binding protein, or a fragment thereof.

[0774] In the embodiments, exemplary antigens or ligands include melanin transferrin; CA6, CAK1, CALLA, CAECAM5, GD3; FLT3; PSMA; PSCA; MUC1; STEAP; CEA; TENB2; EphA receptor; EphB receptor; folate receptor; FOLR1; mesothelin; teratoma-derived growth factor (cripto); α v β6; or integrin, or a fragment thereof.

[0775] In the Examples, exemplary antigens or ligands include VEGF or VEGFR, or fragments thereof.

[0776] In embodiments, exemplary antigens or ligands include EGFR, or a fragment thereof.

[0777] In the Examples, exemplary antigens or ligands include FGFR3; LAMP1, p-cadherin or transferrin receptor, or fragments thereof.

[0778] In an embodiment, exemplary antigens or ligands include IRTA1; IRTA2; IRTA3; IRTA4; IRTA5, or fragments thereof.

[0779] In the embodiments, exemplary antigens or ligands include tyrosine-protein kinase transmembrane receptors (eg, ROR1 and ROR2), or fragments thereof.

[0780] In an embodiment, exemplary antigens or ligands include CD proteins (e.g., CD2, CD3, CD4, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD123, CD134, CD137, CD138, CD152, and CD276), or fragments thereof.

[0781] In embodiments, exemplary antigens or ligands include one or more tumor-associated antigens or cell surface receptors (see U.S. Publication No. 2008 / 0171040 or U.S. Publication No. 2008 / 0305044, each of which is incorporated by reference in its entirety), or fragments thereof.

[0782] In embodiments, exemplary antigens or ligands include erythropoietin or a fragment thereof.

[0783] In embodiments, exemplary antigens or ligands include osteoinductive factors or fragments thereof.

[0784] In the Examples, exemplary antigens or ligands include immunotoxins or fragments thereof.

[0785] In embodiments, exemplary antigens or ligands include bone morphogenetic proteins or fragments thereof.

[0786] In embodiments, exemplary antigens or ligands include interferons (eg, interferon-α, interferon-β, and interferon-γ).

[0787] In embodiments, exemplary antigens or ligands include colony stimulating factors (eg, M-CSF, GM-CSF, and G-CSF) or fragments thereof.

[0788] In embodiments, exemplary antigens or ligands include interleukins (eg, IL-1 to IL-10) or fragments thereof.

[0789] In the Examples, exemplary antigens or ligands include superoxide dismutase or a fragment thereof.

[0790] In embodiments, exemplary antigens or ligands include T cell receptors or fragments thereof.

[0791] In embodiments, exemplary antigens or ligands include surface membrane proteins or fragments thereof.

[0792] In embodiments, exemplary antigens or ligands include decay accelerating factors or fragments thereof.

[0793] In embodiments, exemplary antigens or ligands include viral antigens (eg, a portion of the HIV envelope) or fragments thereof.

[0794] In embodiments, exemplary antigens or ligands include transporters or fragments thereof.

[0795] In embodiments, exemplary antigens or ligands include homing receptors or fragments thereof.

[0796] In an embodiment, exemplary antigens or ligands include addressins or fragments thereof.

[0797] In the Examples, exemplary antigens or ligands include regulatory proteins or fragments thereof.

[0798] In an embodiment, exemplary antigens or ligands include integrins (e.g., CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM) or fragments thereof.

[0799] In embodiments, exemplary antigens or ligands include tumor-associated antigens (eg, HER2, HER3, and HER4 receptors) or fragments thereof.

[0800] In an embodiment, exemplary antigens or ligands include endoglin; c-Met; c-kit; 1GF1R; PSGR; NGEP; PSMA; PSCA; TMEFF2; LGR5; B7H4; TROP-2, DLL-3, CDH6, AXL, SLITRK6, ENPP3, BCMA, tissue factor or CD352, or a fragment thereof.

[0801] In embodiments, the cell-binding agent targets Apo2, BAFF-R, bone morphogenetic protein receptor, IGF-IR, CA125, CanAg, E16, ErbB2, MUC1, MUC16, Napi3b, TF, EpCAM, FcRH2, C242, CD2, CD3, CD4, CD5, CD6, CD11, CD18, CD19, CD20, CD21, CD22, CD26, CD30, CD33, CD37, CD38, CD40, CD44, CD56, CD70, CD72, CD79, CD90, CD 138, CRIPTO, CXCR5, LY64, TDGF1, endothelin B receptor, EphA receptor, EphB receptor, endothelin, FCRH1, HER2, HER2 / neu, HER3, class II MHC molecule Ia antigen, integrin, IRTA2, LIV-1, MPF, NaPi2b, PDL1, FLJ10372, KIAA1445, Mm42015, SEMA5B, SEMAG, prostate six-transmembrane epithelial antigen 1, IPCA-1, PCANP1, STMP, prostate antigen; insulin growth factor receptor, or folate receptor.

[0802] In an embodiment, the cell-binding agent targets GPNMB, NCAM (CD56), TACSTD2 (TROP-2), folate receptor alpha, tissue factor, ENPP3, CD70, P-cadherin, mesothelin, STEA1, CEACAM5, mucin 1, connexin 4, guanylyl cyclase C, SLC44A4, PSMA, LIV1 (ZIP6), SLITRK6, 5T4 or SC-16.

[0803] In embodiments, the cell-binding agent targets HER2 or EGFR.

[0804] In embodiments, the cell-binding agent targets fibronectin extradomain B (EDB), endothelial receptor ETB, PSMA, VEGFR2 (CD309), tissue factor, or ROB04.

[0805] In embodiments, the cell-binding agent targets type IV collagen, periostin, or tenascin c.

[0806] In embodiments, the cell-binding agent targets CD30, CD22, CD79b, CD19, CD138, CD74, CD37, CD33, CD19, or CD98.

[0807] In embodiments, the cell-binding agent targets HER2.

[0808] In embodiments, the cell-binding agent targets EGFR.

[0809] In embodiments, the cell-binding agent targets CD70.

[0810] In embodiments, the cell-binding agent targets CD33.

[0811] In embodiments, the cell-binding agent targets CD30.

[0812] In embodiments, the cell-binding agent targets CD22.

[0813] In embodiments, the cell-binding agent targets CD19.

[0814] In embodiments, the cell-binding agent targets Mucl.

[0815] In embodiments, the cell-binding agent targets CD37.

[0816] In embodiments, the cell-binding agent targets CD123.

[0817] Non-protein cell binding agents

[0818] In an embodiment, the cell binding agent is not a protein. For example, in an embodiment, the cell binding agent may be a vitamin that binds to a vitamin receptor, such as a cell surface receptor. In this regard, vitamin A binds to retinol binding protein (RBP) to form a complex that then binds to the STRA6 receptor with high affinity and increases vitamin A absorption. In another example, folic acid / folate / vitamin B9 binds to a cell surface folate receptor (FR), such as FRa, with high affinity. Folic acid or antibodies bound to FRa can be used to target folate receptors expressed on ovarian tumors and other tumors. In addition, vitamin D and its analogs bind to the vitamin D receptor.

[0819] Protein and Peptide Cell Binding Agents

[0820] In other embodiments, the cell-binding agent is a protein or polypeptide, or a compound comprising a protein or polypeptide (including an antibody, a non-antibody protein or polypeptide).

[0821] In embodiments, the cell-binding agent may be a lymphokine, a hormone, a growth factor, a colony stimulating factor, or a nutrient transport molecule.

[0822] In an embodiment, GM-CSF (a ligand / growth factor that binds myeloid cells) can be used as a cell binding agent against diseased cells from acute myeloid leukemia.

[0823] In embodiments, IL-2 that binds to activated T cells can be used to prevent transplant rejection, treat and prevent graft-versus-host disease, and treat acute T-cell leukemia.

[0824] In an embodiment, MSH that binds to melanocytes can be used to treat melanoma, and antibodies against melanoma can also be used to treat melanoma.

[0825] In embodiments, epidermal growth factor can be used to target squamous cell carcinomas, such as those of the lung and head and neck and gingiva.

[0826] In embodiments, somatostatin can be used to target neuroblastoma and other tumor types.

[0827] In embodiments, estrogen (or an estrogen analog) can be used to target breast cancer.

[0828] In embodiments, androgens (or androgen analogs) may be used to target the testicles.

[0829] In embodiments, the cell-binding agent is an antibody mimetic, such as an ankyrin repeat protein, Centyrin, or adnectin / monomer.

[0830] In an embodiment, the auristatin conjugate comprises a non-immunoreactive protein, polypeptide or peptide as its cell binding agent. Therefore, in an embodiment, the cell binding agent is a non-immunoreactive protein, polypeptide or peptide. Examples include but are not limited to transferrin, epidermal growth factor ("EGF"), bombesin, gastrin, gastrin releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factor ("TGF") (such as TGF-α and TGF-β), vaccinia virus growth factor ("VGF"), insulin and insulin-like growth factor I and II, somatostatin, lectin and apolipoprotein from low-density lipoprotein.

[0831] Antibodies and Related Cell Binding Agents

[0832] In embodiments, the cell-binding agent is an antibody or an antigen-binding fragment thereof. In any of the embodiments described herein, the cell-binding agent may be an antibody.

[0833] In embodiments where the cell-binding agent is an antibody or antigen-binding portion thereof (including antibody derivatives) or certain antibody mimetics, the cell-binding agent can bind to a ligand on the target cell, such as a cell surface ligand, including a cell surface receptor.

[0834] Suitable antibodies also include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, conjugated antibodies (i.e., antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), small modular immunopharmaceuticals ( S mall M odular I mmuno Pharmaceuticals, “SMIPs TM ”) and antibody fragments.

[0835] For example, antibodies include immunoglobulins (Ig) and fragments thereof that are specifically reactive to a specified protein or peptide or a fragment thereof. In an embodiment, antibodies include complete monoclonal antibodies, polyclonal antibodies, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNAR or a fragment thereof)) and antibody fragments, as long as they exhibit the desired biological activity. In an embodiment, the antibody is IgG, IgA, IgE, IgD or IgM. In an embodiment, the antibody is IgG1, IgG2, IgG3 or IgG4. In an embodiment, the antibody is IgAl or IgA2.

[0836] In embodiments, the cell-binding agent is a resurfaced antibody, a resurfaced single chain antibody, a resurfaced antibody fragment (or "antigen-binding portion"), or a bispecific antibody.

[0837] In embodiments, the cell-binding agent is a minibody, an avibody, a diabody, a triabody, a tetrabody, a nanobody, a probody, a domain antibody, or a monobody.

[0838] Antibody fragments may include a portion of an intact antibody, such as the antigen binding region or variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules. Antibody fragments may also be any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments may include isolated fragments, "Fv" fragments consisting of the variable regions of the heavy and light chains, recombinant single-chain polypeptide molecules ("ScFv proteins") in which the variable regions of the light and heavy chains are connected by a peptide linker, and minimal recognition units consisting of amino acid residues that mimic the hypervariable region.

[0839] Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from immunized animal sera. Useful monoclonal antibodies are homogeneous populations of antibodies directed against specific antigenic determinants (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids or fragments thereof). Monoclonal antibodies (mAbs) directed against antigens of interest can be prepared using any technique known in the art that provides antibody molecule production by continuous cell lines in culture.

[0840] In embodiments, the cell-binding agent is a monoclonal antibody or an antigen-binding fragment thereof.

[0841] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, or chimeric human-mouse (or other species) monoclonal antibodies. Antibodies include full-length antibodies and antigen-binding fragments thereof. Human monoclonal antibodies can be prepared by any of a number of techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80: 7308-7312; Kozbor et al., 1983, Immunology Today 4: 72-79; and Olsson et al., 1982, Meth. Enzymol. 92: 3-16).

[0842] In an embodiment, antibodies suitable for the present invention may include humanized or human antibodies. The humanized form of a non-human antibody is a chimeric Ig, Ig chain or fragment containing a minimal sequence derived from a non-human Ig [such as Fv, Fab, Fab', F(ab')2 or other antigen binding subsequences of an antibody]. Generally, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from the "import" variable domain. Humanization is achieved by replacing the corresponding sequence of a human antibody with a rodent complementary determining region (CDR) or CDR sequence [Riechmann et al., Nature 332 (6162): 323-7, 1988; Verhoeyen et al., Science. 239 (4847): 1534-6, 1988]. The "humanized" antibody is a chimeric antibody (U.S. Pat. No. 4,816,567, 1989), in which substantially less than a complete human variable domain has been replaced by a corresponding sequence from a non-human species. In an embodiment, the CDR of a non-human antibody (e.g., mouse) targeting a human antigen is transplanted to the framework region of the variable domain of human Ig. Various techniques known in the art are suitable for CDR transplantation, including, for example, site-directed mutagenesis. In an embodiment, a humanized antibody is generally a human antibody in which some CDR residues and possible FR residues are replaced by residues from similar sites in rodent antibodies. Humanized antibodies include human Ig (acceptor antibody) in which the residues from the CDR of the acceptor are replaced by residues from non-human species (such as mouse, rat or rabbit) (donor antibody) CDR, which has the desired specificity, affinity and ability. In an embodiment, monospecific and bispecific antibodies described herein have cross-reactivity with non-human primate common antigens. In some cases, corresponding non-human residues replace the Fv framework residues of human Ig. Humanized antibodies may be included in the acceptor antibody and in the CDR or framework sequences introduced that do not exist. In general, humanized antibodies include substantially all or at least one and usually two variable domains, wherein most (or even all) CDR regions correspond to those of non-human Ig, and most (or even all) FR regions are those of human Ig consensus sequences. Humanized antibodies also optimally include an Ig constant region (Fc), usually at least a portion of a human Ig constant region [Riechmann et al., Nature 332 (6162): 323-7, 1988; Verhoeyen et al., Science. 239 (4847): 1534-6, 1988].

[0843] Human antibodies can also be produced using a variety of techniques, including phage display libraries [Hoogenboom et al., Mol Immunol. (1991) 28(9): 1027-37; Marks et al., J Mol Biol. (1991) 222(3): 581-97] and preparation of human monoclonal antibodies [Reisfeld and Sell, 1985, Cancer Surv. 4(1): 271-90]. Similarly, human antibodies can be synthesized by introducing human Ig genes into transgenic animals in which endogenous Ig genes have been partially or completely inactivated. Upon stimulation, human antibody production was observed that was very similar to that seen in humans in all aspects, including gene rearrangement, assembly, and antibody repertoires [Fishwild et al., High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice, Nat Biotechnol. 1996 Jul;14(7):845-51; Lonberg et al., Antigen-specific human antibodies from mice comprising four distinct genetic modifications, Nature, 1994 Apr 28;368(6474):856-9; Lonberg and Huszar, Human antibodies from transgenic mice, Int. Rev. Immunol. 1995;13(1):65-93; Marks et al., By-passing immunization: building high affinity human antibodies by chain shuffling. Biotechnology (NY). 1992 Jul;10(7):779-83].

[0844] The antibody may be an antibody that immunospecifically binds to a target cell (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen) or a functionally active fragment, derivative, or analog of another antibody that binds to a tumor cell or matrix. In this context, "functional activity" means that the fragment, derivative, or analog is able to immunospecifically bind to a target cell. In order to determine which CDR sequences bind to an antigen, synthetic peptides containing CDR sequences may be used in a binding analysis with an antigen by any binding analysis method known in the art (e.g., BIAcore analysis) [see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md.; Kabat E et al., 1980, J. Immunology 125 (3): 961-969].

[0845] Other useful antibodies include antibody fragments such as, but not limited to, F(ab')2 fragments, Fab fragments, Fv, single chain antibodies, diabodies, triabodies, tetrabodies, scFv, scFv-FV, or any other molecule with the same specificity as an antibody.

[0846] Another form of antibody fragment is a peptide encoding a single CDR. CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDRs of the antibodies of interest. The genes are prepared, for example, by synthesizing the variable regions of RNA from antibody-producing cells using polymerase chain reaction. See, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," Monoclonal Antibodies: Production, Engineering And Clinical Application, Ritter et al. (eds.), pp. 166-179 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," Monoclonal Antibodies: Principles And Applications, Birch et al. (eds.), pp. 137-185 (Wiley-Liss, Inc. 1995).

[0847] In addition, recombinant antibodies (such as chimeric and humanized monoclonal antibodies) comprising human and non-human parts that can be prepared using standard recombinant DNA techniques are useful antibodies. Chimeric antibodies are molecules in which different parts are derived from different animal species, such as molecules with monoclonal variable regions derived from murines and human immunoglobulin constant regions. (See, for example, U.S. Patent No. 4,816,567; and No. 4,816,397, both of which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species that have one or more complementary determining regions (CDRs) from non-human species and framework regions from human immunoglobulin molecules. (See, e.g., U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) The chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, using the methods described in International Publication No. WO 87 / 02671; European Patent Publication No. 0 184 187; European Patent Publication No. 0 171 496; European Patent Publication No. 0 173494; International Publication No. WO 86 / 01533; U.S. Pat. No. 4,816,567; European Patent Publication No. 012 023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morison, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al., 1988, Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060; each of which is incorporated herein by reference in its entirety.

[0848] In some cases (e.g., when immunogenicity to non-human or chimeric antibodies may exist), fully human antibodies are more desirable and can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes but can express human heavy and light chain genes.

[0849] Antibodies include modified analogs and derivatives, i.e., modified by covalent attachment of any type of molecule, as long as the covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, but not limited to, derivatives and analogs of antibodies include those that have been further modified, such as by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, attachment to cell antibody units or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. In addition, analogs or derivatives may contain one or more non-natural amino acids.

[0850] The antibody may have modifications (e.g., substitutions, deletions, or additions) in amino acid residues that interact with Fc receptors. In particular, the antibody may have modifications in amino acid residues identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., International Publication No. WO 97 / 34631, which is incorporated herein by reference in its entirety).

[0851] Antibodies can be produced using methods well known in the art. For example, protocols for antibody production are described in Harlow and Lane, Antibodies: A Laboratory Manual, (1988). Typically, antibodies can be produced in mice, rats, guinea pigs, hamsters, camels, llamas, sharks or other appropriate hosts. Alternatively, antibodies can be prepared in chickens to produce IgY molecules [Schade et al., (1996) ALTEX 13 (5): 80-85]. In an embodiment, antibodies suitable for the present invention are subhuman primate antibodies. For example, general techniques for producing therapeutic antibodies in baboons can be found in, for example, Goldenberg et al., International Patent Publication No. WO 91 / 11465 (1991), and Losman et al., Int. J. Cancer 46: 310 (1990). In an embodiment, monoclonal antibodies can be prepared using the hybridoma method [Milstein and Cuello, (1983) Nature 305 (5934): 537-40]. In embodiments, monoclonal antibodies can also be produced by recombinant methods (US Pat. No. 4,166,452, 1979).

[0852] Many difficulties associated with producing monoclonal antibodies by immortalization of B cells can be overcome by engineering and expressing antibody fragments using phage display technology in Escherichia coli. To ensure the recovery of high-affinity monoclonal antibodies, combinatorial immunoglobulin libraries must usually contain larger pedigree sizes. A typical strategy is to synthesize cDNA by reverse transcriptase using mRNA obtained from lymphocytes or splenocytes of immunized mice. Heavy chain and light chain genes are amplified separately by PCR and connected to phage cloning vectors. Two different libraries are produced, one containing heavy chain genes and one containing light chain genes. Phage DNA is isolated from each library, and heavy chain and light chain sequences are connected together and packaged to form a combinatorial library. Each phage contains a pair of random heavy chain and light chain cDNAs, and guides the expression of antibody chains in infected cells after infection with Escherichia coli. In order to identify antibodies that recognize the antigen of interest, phage libraries are spread, and the antibody molecules present in the plaque are transferred to filters. The filter is cultivated with radiolabeled antigens, followed by washing to remove excess unbound ligands. Radioactive spots on the autoradiograph identify plaques containing antibody bound to the antigen.Cloning and expression vectors useful for generating human immunoglobulin phage libraries are available, for example, from STRATAGENE Cloning Systems (La Jolla, Calif.).

[0853] Similar strategies can be used to obtain high affinity scFvs. See, for example, Vaughn et al., Nat. Biotechnol., 14:309 314 (1996). A scFv library with a large repertoire can be generated by using a set of proteins corresponding to all known V H 、V k and V λ The PCR primers for the gene family were used to isolate V genes from unimmunized human donors. k and V λ The pools were combined to form one pool. These fragments were ligated into a phagemid vector. The scFv linker (Gly4, Ser)3 was then ligated into the phagemid V L Upstream of the fragment. H and Linker-V L The fragments were amplified and assembled on the JH region. H Linker-V L The fragments were ligated into the phagemid vector. The phagemid library could be prepared using filters as described above or using immunotubes (Nunc; Maxisorp TM) for panning. Similar results can be achieved by constructing a combinatorial immunoglobulin library from lymphocytes or spleen cells of immunized rabbits and by expressing scFv constructs in Pichia pastoris. See, for example, Ridder et al., Biotechnology, 13:255260 (1995). In addition, after isolating the appropriate scFv, antibody fragments with higher binding affinity and slower dissociation rates can be obtained by affinity maturation processes (such as CDR3 mutation induction and chain shuffling). See, for example, Jackson et al., Br. J. Cancer, 78:181 188 (1998); Osbourn et al., Immunotechnology, 2:181 196 (1996).

[0854] In embodiments, a conjugate described herein [eg, any compound according to formula (III) or formula (IV)] comprises a cell-binding agent that is an antibody that targets an antigen overexpressed in cancer cells.

[0855] In embodiments, a conjugate described herein (eg, any compound according to formula (III) or formula (IV)) comprises a cell-binding agent that is an antibody that recognizes a specific tumor-associated antigen (TAA).

[0856] Antibodies specifically immunospecific for cancer cell antigens are commercially available or produced by any method known to those skilled in the art (such as recombinant expression techniques). Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or a database similar thereto, literature publications, or by conventional cloning and sequencing.

[0857] In one embodiment, antibodies known to be useful in treating cancer may be used.

[0858] In another specific embodiment, the compositions and methods according to the invention use antibodies for treating autoimmune diseases.

[0859] In an embodiment, useful antibodies can bind to a receptor or receptor complex expressed on activated lymphocytes. The receptor or receptor complex can include an immunoglobulin gene superfamily member, a TNF receptor superfamily member, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein.

[0860] In embodiments, the conjugates described herein [e.g., any compound according to formula (III) or formula (IV)] comprise a cell-binding agent (e.g., an antibody or fragment thereof) targeting Apo2, BAFF-R, bone morphogenetic protein receptor, IGF-IR, CA125, CanAg, E16, ErbB2, MUC1, MUC16, Napi3b, TF, EpCAM, FcRH2, C242, CD2, CD3, CD4, CD5, CD6, CD11, CD18, CD19, CD20, CD21, CD22, CD26, CD30, CD33, CD37, CD38, CD40, CD44, C D56, CD70, CD72, CD79, CD90, CD138, CRIPTO, CXCR5, LY64, TDGF1, endothelin B receptor, EphA receptor, EphB receptor, endothelin, FCRH1, HER2, HER2 / neu, HER3, class II MHC molecule Ia antigen, integrin, IRTA2, LIV-1, MPF, NaPi2b, PDL1, FLJ10372, KIAA1445, Mm42015, SEMA5B, SEMAG, prostate six-transmembrane epithelial antigen 1, IPCA-1, PCANP1, STMP, prostate antigen; insulin growth factor receptor, or folate receptor.

[0861] In embodiments, the conjugate described herein [e.g., any compound according to formula (III) or formula (IV)] comprises a cell-binding agent (e.g., an antibody or fragment thereof) targeting GPNMB, NCAM (CD56), TACSTD2 (TROP-2), folate receptor alpha, tissue factor, ENPP3, CD70, P-cadherin, mesothelin, STEA1, CEACAM5, mucin 1, connexin 4, guanylyl cyclase C, SLC44A4, PSMA, LIV1 (ZIP6), SLITRK6, 5T4 or SC-16.

[0862] In embodiments, a conjugate described herein (eg, any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets HER2 or EGFR.

[0863] In an embodiment, the conjugate described herein (e.g., any compound according to formula (III) or formula (IV)) comprises a cell-binding agent (e.g., an antibody or fragment thereof) targeting fibronectin extracellular domain B (EDB), endothelial receptor ETB, PSMA, VEGFR2 (CD309), tissue factor or ROBO4.

[0864] In embodiments, a conjugate described herein (eg, any compound according to formula (III) or formula (IV)) comprises a cell binding agent (eg, an antibody or fragment thereof) that targets type IV collagen, periostin or tenascin c.

[0865] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (e.g., an antibody or fragment thereof) that targets CD30, CD22, CD79b, CD19, CD138, CD74, CD37, CD33, CD19, or CD98.

[0866] In embodiments, a conjugate described herein (eg, any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets HER2.

[0867] In embodiments, a conjugate described herein (eg, any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets ROR1 or ROR2.

[0868] In embodiments, a conjugate described herein (eg, any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets FOLR1.

[0869] In embodiments, a conjugate described herein (eg, any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets Connexin-4.

[0870] In embodiments, a conjugate described herein (eg, any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets Trop2.

[0871] In embodiments, a conjugate described herein (eg, any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets EGFR.

[0872] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (e.g., an antibody or fragment thereof) that targets CD70.

[0873] In embodiments, a conjugate described herein (eg, any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets CD33.

[0874] In embodiments, a conjugate described herein (eg, any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets CD30.

[0875] In embodiments, a conjugate described herein (eg, any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets CD22.

[0876] In embodiments, a conjugate described herein (eg, any compound according to formula (III) or formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets CD19.

[0877] In embodiments, a conjugate described herein (eg, any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets Mucl.

[0878] In embodiments, a conjugate described herein (eg, any compound according to Formula (III) or Formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets CD37.

[0879] In embodiments, a conjugate described herein (eg, any compound according to formula (III) or formula (IV)) comprises a cell-binding agent (eg, an antibody or fragment thereof) that targets CD123.

[0880] Synthesis method

[0881] The compounds described herein [eg, compounds according to any of Formula (I), Formula (II), Formula (III), or Formula (IV)] can be prepared according to methods known in the art.

[0882] In the Examples, Scheme 1 provides an exemplary synthesis of the described compound D1 (MB-25).

[0883] Solution 1

[0884]

[0885] In the Examples, Scheme 2 provides an exemplary synthesis of the described compound D2 (MB-23).

[0886] Solution 2

[0887]

[0888] In the Examples, Scheme 3 provides an exemplary synthesis of the described compound PL1 (MB-26).

[0889] Solution 3

[0890]

[0891] In the Examples, Scheme 4 provides an exemplary synthesis of the described compound D5.

[0892] Solution 4

[0893]

[0894] In the Examples, Scheme 5 provides an exemplary synthesis of the described compound D6.

[0895] Solution 5

[0896]

[0897] In the Examples, Scheme 6 provides an exemplary synthesis of the described compound PL2 (MB-24).

[0898] Solution 6

[0899]

[0900] In the Examples, Scheme 7 provides an exemplary general method for preparing a conjugate (PL'):

[0901] Solution 7

[0902]

[0903] In the Examples, Scheme 8 provides an exemplary general method for preparing a dual drug conjugate (PL"):

[0904] Solution 8

[0905]

[0906] In the Examples, an exemplary experimental procedure for preparing a conjugate (PL') having a drug to antibody ratio (DAR) between 7-8 or 8:

[0907] Antibody C was treated with 8 equivalents (2 equivalents / disulfide bond) of 50mM pH 7.4 phosphate buffer (coupling buffer) containing tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and 10mM DTPA (diethylenetriaminepentaacetic acid) at 25°C for 2 hours, followed by the addition of 12 equivalents of DMSO containing the effective load (PL) (the volume of DMSO was about 12-15% of the volume of the phosphate buffer). The resulting reaction solution was rotated on a tube rotator at 25°C for 1 hour. The reaction mixture was then purified for several cycles using an ultrafiltration tube (30KD) with formulation buffer. The resulting conjugate (PL') typically has a drug to antibody ratio (DAR) between 7-8 or 8, and is measured as>95% monomer by size exclusion chromatography.

[0908] In the Examples, an exemplary experimental procedure for preparing a conjugate (PL') having a drug to antibody ratio (DAR) of about 4:

[0909] Antibody C was treated with 4 equivalents of 50mM pH 7.4 phosphate buffer (coupling buffer) containing tris(2-carboxyethyl)phosphine hydrochloride (TCEP) containing 2 equivalents of ZnCl2 at 25°C for 3 hours (references: JI, Ao; Sun, Chuchu; He, Wenxu, "Process for preparing antibody-drug conjugates with improved homogeneity", WO2020164561), followed by the addition of 6 equivalents of DMSO containing the payload PL (the volume of DMSO was approximately 12-15% of the volume of the phosphate buffer). The resulting reaction solution was rotated on a tube rotator at 25°C for 1 hour. The reaction was terminated with 4 equivalents of cysteine, followed by the addition of 10mM DTPA (4 equivalents) and 10mM DHAA (8 equivalents). It was purified for several cycles using an ultrafiltration tube (30KD) with a formulation buffer. The resulting conjugates typically had a drug to antibody ratio (DAR) of approximately 4 and were >95% monomeric as measured by size exclusion chromatography.

[0910] In an embodiment, the antibody-drug conjugate is trastuzumab-MB24. In an embodiment, Scheme 9 provides an exemplary synthesis method of the described antibody-drug conjugate trastuzumab-MB24:

[0911] Solution 9

[0912]

[0913] In an embodiment, the antibody-drug conjugate is trastuzumab-MB26. In an embodiment, Scheme 10 provides an exemplary synthesis method of the described antibody-drug conjugate trastuzumab-MB26:

[0914] Solution 10

[0915]

[0916] In the examples, an exemplary experimental procedure for preparing a dual drug conjugate (PL") is as follows:

[0917] Antibody C was treated with 4 equivalents of 50mM pH 7.4 phosphate buffer (coupling buffer) containing tris(2-carboxyethyl)phosphine hydrochloride (TCEP) containing 2 equivalents of ZnCl2 at 25°C for 3 hours (references: JI, Ao; Sun, Chuchu; He, Wenxu, "Process for preparing antibody-drug conjugates with improved homogeneity", WO2020164561), followed by the addition of 6 equivalents of DMSO containing the first payload PL (the volume of DMSO was approximately 12-15% of the volume of the phosphate buffer). The resulting reaction solution was rotated on a tube rotator at 25°C for 1 hour. The reaction was terminated with 4 equivalents of cysteine, followed by the addition of 10mM DTPA (4 equivalents) and 10mM DHAA (8 equivalents). It was purified for several cycles using an ultrafiltration tube (30KD) with coupling buffer. The resulting conjugate typically has a drug-to-antibody ratio (DAR) of about 4, which is treated with 5 equivalents of 50mM pH 7.4 phosphate buffer containing TCEP and 10mM DTPA at 25°C for 2 hours, followed by the addition of 6 equivalents of DMSO containing a second payload W' (the volume of DMSO is approximately 12-15% of the volume of the phosphate buffer). The resulting reaction solution is rotated on a tube rotator at 25°C for 1 hour. The reaction mixture is then purified using an ultrafiltration tube (30KD) with formulation buffer for several cycles to obtain a dual-drug conjugate (PL"). The DAR ratio of PL" corresponding to the two payloads is typically about 1:1, and the dual-drug conjugate is measured by size exclusion chromatography to be >95% monomeric.

[0918] In an embodiment, the antibody dual drug conjugate is trastuzumab-MB0324. In an embodiment, Scheme 11 provides an exemplary synthesis method of the described antibody dual drug conjugate trastuzumab-MB0324:

[0919] Solution 11

[0920]

[0921] In an embodiment, the antibody dual-drug conjugate is trastuzumab-MB0326. In an embodiment, Scheme 12 provides an exemplary synthesis method of the described antibody dual-drug conjugate trastuzumab-MB0326:

[0922] Solution 12

[0923]

[0924] Auristatin conjugate mixtures and compositions

[0925] The present invention provides a conjugate mixture and a pharmaceutical composition comprising any conjugate described herein (Formula III or Formula IV). The mixture and the pharmaceutical composition comprise a variety of conjugates. In some aspects, each conjugate in the mixture or composition is identical or substantially identical; however, the distribution of the drug-linker on the cell binding agent in the mixture or composition can be changed as the drug load. For example, a coupling technique for coupling the drug-linker to an antibody as a targeting ligand can produce a composition or mixture that is heterogeneous in terms of the distribution of the effective load compound on the antibody (cell binding agent) in the mixture and / or composition. In some aspects, the load of the effective load compound on each antibody molecule in the mixture or composition of the antibody molecule is an integer from 1 to 18.

[0926] In these aspects, when referring to the composition as a whole, the loading of the drug-linker is a value between 1 and about 18. A small percentage of unconjugated antibody may also be present in the composition or mixture. The average number of drug-linkers per cell-binding agent in the mixture or composition (i.e., the average drug loading) is an important attribute because it determines the maximum amount of drug that can be delivered to the target cell. The average drug loading can be about 1, 2 or about 2, 3 or about 3, 4 or about 4, 5 or about 5, 6 or about 6, 7 or about 7, 8 or about 8, 9 or about 9, 10 or about 10, 11 or about 11, 12 or about 12, 13 or about 13, 14 or about 14, 15 or about 15, 16 or about 16, 17 or about 17, 18 or about 18.

[0927] In some aspects, the mixture and pharmaceutical composition comprises a plurality of conjugates (i.e., a conjugate population); however, the conjugates are identical or substantially identical and are substantially homogeneous in terms of the distribution of drug-linkers on ligand molecules within the mixture and / or composition, and in terms of the loading of drug-linkers on cell binding agent molecules within the mixture and / or composition. In some of the aspects, the loading of drug-linkers on antibodies is 2, or 3, or 4, or 5, or 6, or 7, or 8. In the composition or mixture, there may also be a small percentage of unconjugated antibodies. In the embodiments, the average drug loading is about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 7.5, or about 8. Typically, the compositions and mixtures are produced using site-specific conjugation techniques and the conjugation is due to introduced cysteine ​​residues.

[0928] The average amount of effective load compound per cell binding agent in the conjugate reaction preparation can be characterized by conventional methods (e.g., HIC, UV, LC-MS, ELISA analysis). The quantitative distribution of the conjugate in terms of p or p' or t can also be determined. In some cases, separation, purification and characterization of homogeneous conjugates can be achieved by methods such as reverse phase HPLC or electrophoresis.

[0929] In some aspects, the composition is a pharmaceutical composition comprising a conjugate as described herein and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition is in liquid form. In some aspects, the pharmaceutical composition is a solid. In some aspects, the pharmaceutical composition is a lyophilized powder.

[0930] Compositions, including pharmaceutical compositions, may be provided in purified form. As used herein, "purified" means that when isolated, the isolate contains at least 95%, and in another aspect at least 98%, of the conjugate by weight of the isolate.

[0931] How to use

[0932] Compositions and methods of administration

[0933] In another aspect, the invention features a pharmaceutical composition comprising any compound described herein [e.g., any compound of Formula (I), Formula (II), Formula (III), or Formula (IV)] or a pharmaceutically acceptable salt thereof as described herein. In embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0934] In an embodiment, the pharmaceutical composition comprises the conjugate according to formula (III).

[0935] In an embodiment, the pharmaceutical composition comprises the conjugate according to formula (IV).

[0936] In an embodiment, the present invention provides a pharmaceutical composition comprising an auristatin conjugate as described herein and a pharmaceutically acceptable carrier. The auristatin conjugate can be in any form that allows the compound to be administered to a patient to treat a condition associated with the expression of an antigen bound by a cell binding agent. For example, the conjugate can be in the form of a liquid or a solid. The preferred route of administration is parenteral. Parenteral administration includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. In one aspect, the composition is parenteral. In one aspect, the conjugate is administered intravenously. Administration can be by any convenient route, such as by infusion or bolus injection.

[0937] The pharmaceutical composition may be formulated so that the compound is bioavailable upon administration of the composition to a patient.The composition may take the form of one or more dosage units.

[0938] The materials used to prepare the pharmaceutical composition may be non-toxic in the amount used. It is apparent to one of ordinary skill in the art that the optimal dosage of one or more active ingredients in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, but are not limited to, the type of animal (e.g., human), the specific form of the compound, the mode of administration, and the composition used.

[0939] The composition may be in the form of a liquid, for example. The liquid may be used for delivery by injection. In a composition administered by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer and an isotonic agent may also be included.

[0940] Liquid compositions (whether they are solutions, suspensions or other similar forms) may also include one or more of the following: sterile diluents, such as water for injection, saline solutions (preferably physiological saline), Ringer's solution, isotonic sodium chloride, fixed oils (such as synthetic monoglycerides or diglycerides that can be used as solvents or suspension media), polyethylene glycols, glycerol, cyclodextrins, propylene glycol or other solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as amino acids, acetates, citrates or phosphates; detergents, such as nonionic surfactants, polyols; and agents for adjusting tonicity, such as sodium chloride or glucose. Parenteral compositions may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass, plastic or other materials. Physiological saline is an exemplary adjuvant. Injectable compositions are preferably sterile.

[0941] The amount of conjugate effective to treat a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify the optimal dosage range. The precise dose to be used in the composition will also depend on the route of administration and the severity of the disease or condition and should be determined according to the judgment of the practitioner and the circumstances of each patient.

[0942] The composition comprises an effective amount of the compound such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of the compound by weight of the composition.

[0943] For intravenous administration, the composition may include about 0.01 to about 100 mg of auristatin conjugate per kilogram of animal body weight. In one aspect, the composition may include about 1 to about 100 mg of auristatin conjugate per kilogram of animal body weight. On the other hand, the amount administered will be about 0.1 to about 25 mg of compound per kilogram of body weight. Depending on the drug used, the dosage can be even lower, such as 1.0 μg / kg to 5.0 mg / kg, 4.0 mg / kg, 3.0 mg / kg, 2.0 mg / kg or 1.0 μg / kg based on the subject's body weight, or 1.0 μg / kg to 500.0 μg / kg.

[0944] Typically, the dose of the conjugate administered to the patient is typically about 0.01 mg / kg to about 100 mg / kg based on the subject's body weight or 1.0 μg / kg to 5.0 mg / kg based on the subject's body weight. In an embodiment, the dose administered to the patient is about 0.01 mg / kg to about 15 mg / kg based on the subject's body weight. In an embodiment, the dose administered to the patient is between about 0.1 mg / kg and about 15 mg / kg based on the subject's body weight. In an embodiment, the dose administered to the patient is between about 0.1 mg / kg and about 20 mg / kg based on the subject's body weight. In an embodiment, the dose administered is about 0.1 mg / kg to about 5 mg / kg or about 0.1 mg / kg to about 10 mg / kg based on the subject's body weight. In an embodiment, the dose administered is about 1 mg / kg to about 15 mg / kg based on the subject's body weight. In an embodiment, the dose administered is about 1 mg / kg to about 10 mg / kg based on the subject's body weight. In embodiments, the dosage administered during a treatment cycle is about 0.1 to 4 mg / kg, even more preferably, 0.1 to 3.2 mg / kg, or even more preferably, 0.1 to 2.7 mg / kg based on the subject's body weight.

[0945] The term "carrier" refers to a diluent, adjuvant or excipient that is administered with a compound. The pharmaceutical carrier may be a liquid, such as water, and an oil, including oils of petroleum, animal oils, vegetable oils or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil. The carrier may be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants may be used. In one embodiment, when administered to a patient, the compound or composition and the pharmaceutically acceptable carrier are sterile.

[0946] When intravenously administering the compound, water is an exemplary carrier. Saline solutions and dextrose and glycerol aqueous solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients, such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol. If desired, the compositions of the present invention may also contain a small amount of wetting agent or emulsifier or pH buffer.

[0947] In one embodiment, the conjugate is formulated into a pharmaceutical composition suitable for intravenous administration to animals (particularly humans) according to conventional procedures. Typically, the carrier or vehicle for intravenous administration is a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer. The composition for intravenous administration may optionally include a local anesthetic (such as lignocaine) to relieve pain at the injection site. Typically, such ingredients are provided separately or mixed together in unit dosage forms, for example, in a dry lyophilized powder or anhydrous concentrate supplied in an airtight sealed container (such as an ampoule or a medicine bag) indicating the active drug dose. When the conjugate is administered by infusion, it can be distributed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When the conjugate is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed before administration.

[0948] Pharmaceutical compositions are typically formulated to be sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0949] Cancer Treatment

[0950] The compounds described herein [e.g., any compound according to any of Formula (I), Formula (II), Formula (III), or Formula (IV)] can be effective in selectively inducing cell death in certain populations, for example, cells that overexpress certain antigens (including the antigens described herein, such as tumor-associated antigens).

[0951] In vitro cytotoxicity assay:

[0952] The cytotoxic potency of the compounds was evaluated using a cell counting kit-8 (CCK-8) assay (Shanghai Life Lab Biotech Co., Ltd.) in a flat-bottom 96-well cell culture plate (Corning Costar). Briefly, human tumor cells (1,000-10,000 cells / well, depending on the cell line) were cultured with compounds or conjugates in appropriate culture media at 37°C, 5% CO2 for 120 hours in the presence or absence of excess corresponding unconjugated antibodies.

[0953] For example, for any compound according to Formula (III) or Formula (IV), appropriate selection of a cell-binding agent can provide effective, highly selective targeting of cancer cells, which would be useful in the treatment of cancer.

[0954] The auristatin conjugates described herein [e.g., any compound according to formula (III) or formula (IV)] can be used to inhibit abnormal cell proliferation (e.g., abnormal cell proliferation of tumor cells or cancer cells, causing apoptosis of tumor or cancer cells), or for treating cancer in patients. Thus, provided herein is a method for treating cancer in a subject in need thereof, the method comprising administering to the subject one or more auristatin conjugates described herein.

[0955] In an embodiment, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, the method comprising administering any compound of Formula (III) or Formula (IV) as described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any compound of Formula (III) or Formula (IV) as described herein, or a pharmaceutically acceptable salt thereof.

[0956] Therefore, the compounds described herein [e.g., any compound according to formula (III) or formula (IV)] can be used to treat a variety of cancers accordingly. In an embodiment, the auristatin conjugate can be used to deliver drugs to tumor cells or cancer cells. Without being bound by theory, in one embodiment, the cell binding agent of the auristatin conjugate binds or associates with a cancer cell or tumor cell-associated antigen, and the auristatin conjugate can be absorbed (internalized) into the tumor cell or cancer cell by receptor-mediated endocytosis or other internalization mechanisms. The antigen may be attached to the tumor cell or cancer cell, or may be an extracellular matrix protein associated with the tumor cell or cancer cell. Once in the cell, the drug is released via intracellular peptide cleavage. In an alternative embodiment, the free drug is released from the auristatin conjugate outside the tumor cell or cancer cell, and the free drug then penetrates the cell.

[0957] In one embodiment, the cell-binding agent binds to a tumor cell or a cancer cell.

[0958] In another embodiment, the cell-binding agent binds to a tumor cell or cancer cell antigen on the surface of a tumor cell or cancer cell.

[0959] In another embodiment, the cell-binding agent binds to a tumor cell or cancer cell antigen, which is an extracellular matrix protein associated with a tumor cell or cancer cell.

[0960] The specificity of a cell-binding agent for a particular tumor cell or cancer cell can be important in determining the tumor or cancer that is most effectively treated.

[0961] Cancers that can be treated with auristatin conjugates include, but are not limited to, hematopoietic cancers, such as lymphomas (Hodgkin's lymphoma and non-Hodgkin's lymphoma) and leukemias and solid tumors. Examples of hematopoietic cancers include follicular lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, acute myeloblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma and multiple myeloma. Examples of solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and Adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial cancer, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma and retinoblastoma.

[0962] In embodiments, the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, a central nervous system tumor (CNS) tumor, colon cancer or colorectal cancer, diffuse intrinsic pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood cancer, Hodgkin's lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome (MDS), neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, pancreatic cancer, peritoneal cancer, prostate cancer, ovarian cancer, kidney cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, or Wilms' tumor.

[0963] In embodiments, the cancer is breast cancer.

[0964] Multimodal therapy for cancer

[0965] Cancer, including but not limited to tumors, metastases, or other diseases or disorders characterized by uncontrolled cell growth can be treated or inhibited by administering an auristatin conjugate.

[0966] In other embodiments, a method of treating cancer is provided, comprising administering an effective amount of an auristatin conjugate and a chemotherapeutic agent to a patient in need thereof. In one embodiment, the chemotherapeutic agent is a therapeutic agent that has not been found to be difficult to use for treating cancer. In another embodiment, the chemotherapeutic agent is a therapeutic agent that has been found to be difficult to use for treating cancer. Auristatin conjugates can be administered to patients who have also undergone surgery to treat cancer.

[0967] In an embodiment, the patient also receives additional treatment, such as radiation therapy. In a specific embodiment, the auristatin conjugate is administered simultaneously with the chemotherapeutic agent or with the radiation therapy. In another embodiment, the chemotherapeutic agent or the radiation therapy is administered before or after the administration of the auristatin conjugate.

[0968] The chemotherapeutic agent can be administered over a series of treatments.Any one or a combination of chemotherapeutic agents, such as one or more standard of care chemotherapeutic agents, can be administered.

[0969] In addition, methods of treating cancer with auristatin conjugates are provided as an alternative to chemotherapy or radiotherapy, where chemotherapy or radiotherapy has been or can be shown to be too toxic to the treated subject, e.g., produces unacceptable or intolerable side effects. The treated patient can optionally be treated with another cancer treatment, such as surgery, radiotherapy or chemotherapy, depending on which treatment is acceptable or tolerable.

[0970] Treatment of autoimmune diseases

[0971] Auristatin conjugates can be used to kill or inhibit the undesirable replication of cells that produce autoimmune diseases or to treat autoimmune diseases.

[0972] Auristatin conjugates can be used to treat autoimmune diseases of patients in a variety of situations accordingly. Auristatin conjugates can be used to deliver drugs to target cells. Not bound by theory, in one embodiment, the auristatin conjugates associate with antigens on the surface of proinflammatory or improperly stimulated immune cells, and then through receptor-mediated endocytosis, the auristatin conjugates are absorbed into the target cells. Once inside the cell, the cell binding agent is cleaved so that the auristatin is released. Then, the released auristatin is free to migrate in the cytosol and induce cytotoxic or cytostatic activity. In an alternative embodiment, the drug is cleaved from the auristatin conjugate outside the target cell, and then the auristatin penetrates the cell.

[0973] In one embodiment, the cell binding agent binds an autoimmune antigen.In one aspect, the antigen is located on the surface of a cell associated with an autoimmune disease.

[0974] In one embodiment, the cell-binding agent binds activated lymphocytes associated with an autoimmune disease state.

[0975] In another embodiment, the auristatin conjugate kills or inhibits the proliferation of cells that produce autoimmune antibodies associated with a particular autoimmune disease.

[0976] Specific types of autoimmune diseases that may be treated with auristatin conjugates include, but are not limited to, Th2 lymphocyte-associated diseases (e.g., atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, and graft-versus-host disease); Th1 lymphocyte-associated diseases (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis, and tuberculosis); and activated B lymphocyte-associated diseases (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes).

[0977] Method for preparing auristatin conjugate

[0978] The auristatin conjugates described herein can be prepared in a sequential construction of the antibody, linker and drug units, or in a convergent manner by assembling parts followed by a complete assembly step.

[0979] In one set of embodiments, an auristatin payload compound as provided herein is combined with a suitable cell binding agent to facilitate covalent attachment of the auristatin payload compound to the cell binding agent. In embodiments, the cell binding agent is an antibody having at least 2, at least 4, at least 6, or 8 thiols that can be used to attach the auristatin payload compound due to reduced interchain disulfide bonds. In embodiments, the auristatin payload compound is attached to the cell binding agent via a cysteine ​​moiety introduced by the antibody.

[0980] Therapeutic kits

[0981] In some aspects, kits for cancer treatment and autoimmune disease treatment are provided. The kits may include a pharmaceutical composition comprising an auristatin conjugate as described herein.

[0982] In an embodiment, the kit may include instructions for any of the methods of treatment described herein. The included instructions may provide a description of administering the pharmaceutical composition to a subject to achieve the desired activity, such as a disease or condition (e.g., cancer) for treating the subject. In an embodiment, instructions related to the use of the pharmaceutical composition described herein may include information about the dosage, dosing schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. The instructions provided in the kit of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical composition is used to treat, delay onset, and / or alleviate a disease or condition of the subject.

[0983] In an embodiment, the kit provided herein is contained in a suitable package. Suitable packaging includes, but is not limited to, vials, bottles, jars, soft packaging, etc. Also contemplated are packages for use in combination with specific devices (e.g., inhalers, nasal administration devices, or infusion devices). In an embodiment, the kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle).

[0984] In embodiments, the kits provided herein include an additional therapeutic agent useful for treating cancer of an autoimmune disease as described herein.

[0985] Examples

[0986] Although certain compounds, compositions and methods described herein have been specifically described according to certain embodiments, the following examples are merely illustrative of the compounds of the present invention and are not intended to limit the compounds.

[0987] One skilled in the art can easily ascertain the essential characteristics of this invention from the present description, and without departing from the spirit and scope of the invention, can make various changes and modifications of the invention to adapt it to various usages and conditions.

[0988] All references, patents or applications in the United States or other countries cited in this application are incorporated herein by reference as if they were written in their entirety. In the event of any inconsistency, the textual material disclosed herein shall prevail.

[0989] The following abbreviations are used for the following terms:

[0990] ADC Antibody-Drug Conjugate

[0991] ACN Acetonitrile

[0992] DAR drug to antibody ratio

[0993] DCC N,N'-Dicyclohexylcarbodiimide

[0994] DCM Dichloromethane

[0995] DHAA Dehydroascorbic acid

[0996] DIPA Diisopropylamine

[0997] DIPEA Diisopropylethylamine

[0998] DMF N,N-Dimethylformamide

[0999] DMSO Dimethyl sulfoxide

[1000] DMTMM 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride

[1001] DMTMMT 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate

[1002] DTPA Diethylenetriaminepentaacetic acid

[1003] EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

[1004] HATU 3-Oxidized 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate

[1005] HIC Hydrophobic Interaction Chromatography

[1006] HOBt Hydroxybenzotriazole

[1007] HOPO 2-Hydroxypyridine-N-oxide

[1008] iv intravenous

[1009] LC-MS liquid chromatography-mass spectrometry

[1010] M Moore

[1011] nM nanomolar

[1012] NMM N-Methylmorpholine

[1013] PPTS Pyridinium p-toluenesulfonate

[1014] PTSA 4-Methylbenzenesulfonic acid

[1015] PyBOP Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate

[1016] SEC Size Exclusion Chromatography

[1017] TBAF Tetrabutylammonium fluoride

[1018] TBS tert-Butyldimethylsilyl

[1019] TBSCl tert-Butyldimethylsilyl chloride

[1020] TCEP 3,3',3"-Phosphinetriyltripropionate hydrochloride

[1021] TEA Triethylamine

[1022] TFA Trifluoroacetic acid

[1023] THF Tetrahydrofuran

[1024] TLC Thin layer chromatography

[1025] TrOH Triphenylmethanol

[1026] Tr triphenylmethyl

[1027] p-TsOH p-Toluenesulfonic acid

[1028] Example 1: Exemplary Synthesis of Compound MB-25 (D1)

[1029] General preparation procedure of 2-(tritylthio)acetaldehyde (2)

[1030]

[1031] 1,4-dithiane-2,5-diol (compound 1, 17.0g, 111mmol) and trifluoroacetic acid (12.3g, 108mmol, 8.02mL) were added to a solution of triphenylmethanol (29.0g, 111mmol) in dichloromethane (170mL) in sequence. The reactant was stirred at 25 ° C for 2 hours. TLC (petroleum ether / ethyl acetate=10 / 1, Rf=0.3) showed that the reaction was complete. The reaction mixture was quenched by adding NaHCO3 aqueous solution (100mL) and extracted with dichloromethane (3×300mL). The combined organic layer was washed with H2O (2×300mL), dried over Na2SO4 and filtered, and the filtrate was concentrated in vacuo to obtain a residue, which was purified by column chromatography (SiO2, petroleum ether / ethyl acetate, from 1 / 0 to 15 / 1) to obtain compound 2 (8.5g, 8.0mmol, yield: 7.2%) as a white solid. 1 H NMR (400MHz, DMSO-d6), δppm 3.15 (d, J = 2.25Hz, 2H), 7.14-7.39 (m, 15H), 8.90 (t, J = 2.38Hz, 1H).

[1032] General procedure for the preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(2-(tritylthio)ethyl)amino)butyrylamino)butyramide (3).

[1033]

[1034] To a mixture of MMAE (5.0 g, 6.95 mmol) and compound 2 (9.25 g, 13.9 mmol) in methanol (100 mL) was added acetic acid (2.09 g, 34.8 mmol). After stirring at 25 ° C for 30 minutes under nitrogen protection, sodium cyanoborohydride (2.18 g, 34.8 mmol) was added to the reaction mixture in one go. The reactants were stirred at 25 ° C for 12 hours. LCMS showed that the reaction was complete. The reaction mixture was purified by preparative HPLC [column: Welch Xtimate C18 (250×100 mm×10 μm); mobile phase, A: water (NH4HCO3), B: acetonitrile; B%: 75%-99%, 20 minutes] to give compound 3 (4.0 g, yield: 56.2%) as a white solid. 1H NMR (400MHz, methanol-d4), δppm 0.79-0.91 (m, 6H), 0.92-1.07 (m, 13H), 1.16 (br t, J = 7.52Hz, 3H), 1.21 (br d,J=5.87Hz,3H),1.30-1.52(m,2H),1.54-1.67(m,1H),1.68-2.10(m,6H),2.14(br d,J=10.76Hz,3H),2.21-2.47(m,4H),2.48-2.56(m,2H),2.57-2.69(m,2H),3.14(s,1H),3.23(dt,J=11.62,7.21Hz,1H),3.33(br d,J=4.28Hz,5H),3.38(s,4H),3.40-3.49(m,1H),3.66-3.81(m,1H),4.18-4.32(m,2H),4.50-4.67(m,3H),4.71(br d,J=8.19Hz,1H),4.80(br s,1H),7.20-7.46(m,20H).

[1035] General procedure for the preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((S)-2-((2-mercaptoethyl)(methyl)amino)-3-methylbutyrylamino)-N,3-dimethylbutyramide (MB-25) (D1).

[1036]

[1037] To a solution of compound 3 (150 mg, 147 μmol) in trifluoroacetic acid (2.23 g, 19.5 mmol) was added triisopropylsilyl chloride (384 mg, 2.43 mmol) and H2O (402 mg, 22.3 mmol). The reaction mixture was stirred at 25 ° C for 1 hour. LCMS showed that the reaction was complete. It was diluted with acetonitrile (0.20 mL) and filtered. The filtrate was purified by preparative HPLC [column: C18 (150×30 mm×5 μm); mobile phase, A: water (NH4HCO3), B: acetonitrile; B%: 60%-90%, 10 minutes] to give compound MB-25 (D1) (23.0 mg, yield: 20.1%) as a white solid. MS (ESI+): m / z calculated value 778.52 (M+H) + , the measured value is 778.56. 1H NMR (400MHz, methanol-d4), δppm 0.77-0.91(m,6H),0.93-1.09(m,13H),1.09-1.16(m,3H),1.18(br d,J=6.44Hz,3H),1.31-1.64(m,3H),1.65-2.27(m,7H),2.32(br d,J=11.92Hz,3H),2.45-2.85(m,7H),3.11-3.24(m,2H),3.34-3.46(m,6H), 3.52-3.92(m,2H),4.05-4.31(m,2H),4.45-4.81(m,5H),7.18-7.44(m,5H).

[1038] Example 2: Exemplary Synthesis of Compound MB-23 (D2)

[1039] General procedure for the preparation of (S)-2-((2R,3R)-3-((S)-1-((6S,9S,12S,13R)-12-((S)-sec-butyl)-6,9-diisopropyl-13-methoxy-5,11-dimethyl-7,10-dioxo-1,1,1-triphenyl-2-thia-5,8,11-triazapentadecan-15-yl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoylamino)-3-phenylpropanoic acid (4).

[1040]

[1041] To a suspension of MMAF (5.0 g, 6.85 mmol) and compound 2 (10.0 g, 20.5 mmol) in methanol (100 mL) was added Molecular sieves (5.0 g) and acetic acid (2.05 g, 34.1 mmol). After stirring at 25 ° C for 30 minutes, sodium cyanoborohydride (2.15 g, 34.1 mmol) was added to the reaction mixture in one go. The reactants were stirred at 25 ° C for 12 hours. LCMS showed that the reaction was complete. It was quenched with water (500 mL) and extracted with ethyl acetate (3×500 mL). The organic phase was dried over sodium sulfate, filtered and the filtrate was concentrated to give a residue, which was purified by preparative HPLC [column: Welch Xtimate C18 (250×70 mm×10 μm); mobile phase, A: water (NH4HCO3), B: acetonitrile; B%: 45%-75%, 16 minutes] to give compound 4 (4.0 g, yield: 56.6%) as a white solid. MS (ESI+) m / z, calculated value 1034.60 (M+H) + , the measured value is 1034.70. 1H NMR (400MHz, methanol-d4), δppm 0.74-0.88(m,6H),0.90-1.09(m,13H),1.18(brdd,J=18.30,5.54Hz,3H),1.27-1.69(m,4H),1.71-1.98(m,4H),2.10(br d,J=3.34Hz,3H),2.15-2.52(m,6H),2.59(br dd,J=9.54,4.77Hz,2H),2.89-3.04(m,1H),3.08-3.28(m,5H),3.35(br d,J=7.15Hz,3H),3.38-3.45(m,1H),3.46-3.58(m,1H),3.62-3.76(m,1H),3.84(br d,J=3.58Hz,1H),4.17(br s,1H),4.59(br d,J=8.70Hz,1H),4.73(br d,J=7.75Hz,1H),7.16-7.32(m,14H),7.35-7.45(m,6H).

[1042] General procedure for the preparation of (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((2-mercaptoethyl)(methyl)amino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropanoic acid (MB-23) (D2).

[1043]

[1044] To a suspension of compound 4 (2.0 g, 1.93 mmol, 1.0 equiv.) in trifluoroacetic acid (29.2 g, 256 mmol, 19.0 mL) was added triisopropylsilyl chloride (5.05 g, 31.9 mmol, 6.55 mL) and H2O (5.29 g, 293 mmol). The reaction mixture was stirred at 25 °C for 1 hour under N2 protection. LCMS showed that the reaction was complete. The reaction mixture was concentrated in vacuo to give a residue, which was purified by preparative HPLC [column: Phenomenex Luna C18 (80×30 mm×3 μm); mobile phase, A: water, B: acetonitrile; B%: 15%-45%, 8 minutes] to give the product MB-23 (D2) (1.50 g, yield: 77.0%) as a white solid. HRMS (ESI+), m / z calculated value 792.49 (M+H) + , the measured value is 792.4944. 1H NMR (400 MHz, methanol-d4), δ ppm 0.75-0.91 (m, 6H), 0.93-1.07 (m, 11H), 1.07-1.22 (m, 5H), 1.26-1.47 (m, 2H), 1.50-1.67 (m, 1H), 1.70-1.94 (m, 3H), 1.98-2.18 (m, 2H), 2.19-2.53 (m, 6H), 2.75-3.03 (m, 5H), 3. 14(s,1H),3.18-3.29(m,4H),3.33-3.45(m,6H),3.46-3.56(m,1H),3.62-3.76(m,1H), 3.85(dd,J=8.50,1.65Hz,1H),4.02-4.18(m,1H),4.63-4.76(m,4H),7.13-7.31(m,5H).

[1045] Example 3: Exemplary Synthesis of Compound MB-26 (PL1)

[1046] General procedure for the preparation of (9H-fluoren-9-yl)methyl((3R,4S,7S,10S,18S,21S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11,18,22-tetramethyl-6,9,17,20-tetraoxo-2-oxa-14-thia-5,8,11,16,19-pentaazatricos-21-yl)carbamate (6).

[1047]

[1048] To a solution of compound MB-25 (1.95 g, 2.15 mmol) and compound 5 (2.48 g, 3.87 mmol) in N,N-dimethylformamide (19.5 mL) was added HCl / diethyl ether (6.40 M, 1.32 mL). The reactants were stirred at 25 ° C for 12 hours. LCMS showed that the reaction was complete. It was concentrated under reduced pressure to remove HCl / diethyl ether. The residue was purified by preparative HPLC [column: Phenomenex luna C18 (250×50 mm×10 μm); mobile phase, A: water (TFA), B: acetonitrile; B%: 30%-70%, 10 minutes] to give compound 6 (1.65 g, yield: 52.3%) as a white solid. 1H NMR (400 MHz, methanol-d4), δ ppm 0.82-1.21 (m, 17H), 1.31-1.49 (m, 3H), 1.52-2.25 (m, 4H), 2.33-2.58 (m, 2H), 2.78-3.06 (m, 3H), 3.09-3.14 (m, 1H), 3.20 (dt, J = 11.63, 7.25 Hz, 1H), 3.33-3.38 (m, 2H), 3.38-3.50 (m, 1H), 3.50- 3.58(m,1H),3.64-3.94(m,2H),4.02-4.11(m,1H),4.14-4.33(m,3H),4.34-4.42(m,1H),4.46-4 .58(m,1H),4.59-4.64(m,1H),4.66-4.72(m,1H),4.73-4.80(m,1H),7.17-7.45(m,5H),7.67(br d,J=7.50Hz,1H),7.81(d,J=7.50Hz,1H),7.95-8.03(m,1H),8.20-8.28(m,1H),8.50-8.62(m,1H).

[1049] General procedure for the preparation of (S)-2-((2S,10S,13S)-13-amino-2-isopropyl-3,10,14-trimethyl-9,12-dioxo-6-thia-3,8,11-triazapentadecanoylamino)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N,3-dimethylbutanamide (7).

[1050]

[1051] To a solution of compound 6 (1.65 g, 1.38 mmol) in N,N-dimethylformamide (15.0 mL) was added morpholine (599 mg, 6.88 mmol, 605 μL). The reaction mixture was stirred at 25 ° C for 12 hours under N2 protection. LCMS showed that the reaction was complete. It was concentrated under reduced pressure to give a residue, which was purified by preparative HPLC [column: C18 (250×50 mm×10 μm); mobile phase, A: water (NH4HCO3), B: acetonitrile; B%: 35%-65%, 10 minutes] to give compound 7 (880 mg, yield: 65.4%) as a white solid. 1H NMR (400MHz, methanol-d4), δppm0.74-1.04(m,24H),1.05-1.17(m,6H),1.27-1.45(m,4H),1.48-1.60(m,1H),1.61-2.23(m,8H),2.29(br d,J=12.35Hz,3H),2.38-2.55(m,2H),2.59-2.89(m,5H),3.06-3.18(m,3H),3.27(br d,J=1.22Hz,7H),3.39(br d,J=9.29Hz,1H),3.47-3.74(m,2H),3.75-3.91(m,1H),3.96-4.41(m,5H),4.45-4.64(m,3H),4.71(br dd,J=18.28,8.99Hz,2H),7.07-7.45(m,5H).

[1052] (S)-N1-((3R,4S,7S,10S,18S,21S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11,18,22 General procedure for the preparation of 5-tetramethyl-6,9,17,20-tetraoxo-2-oxa-14-thia-5,8,11,16,19-pentaazatricos-21-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)-N5-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)glutaramide (MB-26) (PL1).

[1053]

[1054] To a solution of compound 8 (245 mg, 0.487 mmol) and dicyclohexylcarbodiimide (201 mg, 0.974 mmol) in N, N-dimethylformamide (4.0 mL) was added N-hydroxysuccinimide (112 mg, 0.974 mmol). After stirring at 25 ° C for 6 hours, the reaction mixture was filtered to remove the precipitate. The filtrate was added to a solution of compound 7 (500 mg, 0.511 mmol) in N, N-dimethylformamide (7.50 mL). The resulting reaction mixture was stirred at 25 ° C for another 12 hours. LCMS showed that the reaction was complete. It was filtered, and the filtrate was directly loaded onto preparative HPLC (column: Phenomenex C18 (75×30 mm×3 μm); mobile phase, A: water, B: acetonitrile; B%: 30%-65%, 8 minutes) for purification to obtain the product MB-26 (PL1) (85.0 mg, yield: 11.6%) as a white solid. HRMS (ESI+): m / z calculated value 1462.85 (M+H) + , the measured value is 1462.8763. 1 HNMR (400MHz, methanol-d4), δppm 0.80-0.91(m,6H),0.92-1.08(m,18H),1.09-1.20(m,6H),1.22-1.49(m,10H),1.61(ddd,J=14.87,11.77,7.45Hz,5H),2.15(br d,J=3.81Hz,10H),2.20-2.36(m,7H),2.45-2.55(m,2H),2.66-2.85(m,5H),3.11-3.17(m,2H),3.35(s,5H),3.43(br dd,J=9.83,4.11Hz,3H),3.47-3.51(m,3H),3.60-3.81(m,7H),3.88(br dd,J=7.81,4.35Hz,1H),4.15-4.29(m,3H),4.31-4.37(m,3H),4.51-4.57(m,1H),4.60-4.67(m,1H),4.74(br d,J=8.46Hz,1H),6.74-6.84(m,2H),7.13-7.43(m,5H).

[1055] Example 4: Exemplary Synthesis of Compound MB-24 (PL2)

[1056] General procedure for the preparation of (S)-2-((2R,3R)-3-((S)-1-((5S,8S,16S,19S,22S,23R)-22-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-5,16,19-triisopropyl-23-methoxy-8,15,21-trimethyl-3,6,9,17,20-pentaoxo-2-oxa-12-thia-4,7,10,15,18,21-hexaazapentacos-25-yl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropanoic acid (9).

[1057]

[1058] To a mixture of compound MB-23 (900 mg, 1.13 mmol) and compound 5 (1.09 g, 1.70 mmol) in N,N-dimethylformamide (3 mL) was added HCl / diethyl ether (6.40 M, 236 μL). The reaction mixture was stirred at 25 °C for 12 hours. LCMS showed that the reaction was complete. It was concentrated under reduced pressure to remove diethyl ether. The residue was purified by preparative HPLC [column: Phenomenex luna C18 (250×50 mm×10 μm); mobile phase, A: water (TFA), B: acetonitrile; B%: 30%-60%, 10 minutes] to give compound 9 (450 mg, yield: 32.6%) as a white solid. LCMS (ESI+): m / z calculated value 1213.7 (M+H) + , the measured value is 1213.8. 1H NMR: (400MHz, methanol-d4), δppm, 0.81-0.89 (m, 3H), 0.90-1.05 (m, 18H), 1.06-1.17 (m, 6H), 1.20 (d, J = 6.72Hz, 2H), 1.24-1.47 (m ,6H),1.49-1.65(m,1H),1.69-1.94(m,3H),1.99-2.14(m,2H),2.15-2.28(m,1H),2.29-2.40(m,1H),2.45(brs,2H),2.89(br s,6H),3.09-3.14(m,1H),3.16-3.29(m,4H),3.34(d,J=4.40Hz,4H),3.37-3.52(m,3H),3.62-3.74(m,1H),3.88(br d,J=7.09Hz,3H),4.03-4.18(m,1H),4.24(br d,J=6.24Hz,3H),4.32-4.46(m,2H),4.51(s,2H),4.67-4.78(m,2H),7.14-7.35(m,7H),7.36-7.43(m,2H),7.67(br d,J=7.09Hz,2H),7.77-7.85(m,2H).

[1059] General procedure for the preparation of (S)-2-((2R,3R)-3-((S)-1-((3S,6S,14S,17S,20S,21R)-3-amino-20-((S)-sec-butyl)-14,17-diisopropyl-21-methoxy-2,6,13,19-tetramethyl-4,7,15,18-tetraoxo-10-thia-5,8,13,16,19-pentaazatricosyl-23-yl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoylamino)-3-phenylpropanoic acid (10).

[1060]

[1061] To a solution of compound 9 (800 mg, 0.659 mmol) in N,N-dimethylformamide (20.0 mL) was added morpholine (373 mg, 4.28 mmol). The reaction mixture was stirred at 25 ° C for 12 hours. LCMS showed that the reaction was complete. It was concentrated in vacuo to give a residue, which was purified by preparative HPLC [column: C18 (250×50 mm×10 μm); mobile phase, A: water (NH4HCO3), B: acetonitrile; B%: 20%-50%, 10 minutes] to give the product 10 (625 mg, yield: 87.0%) as a white solid. LCMS (ESI+): m / z calculated value 991.6 (M+H)+ , the measured value is 991.7. 1 HNMR (400MHz, methanol-d4), δppm 0.77-0.91(m,5H),0.93-1.10(m,18H),1.16(br s,1H),1.19(br d,J=6.60Hz,2H),1.24-1.33(m,1H),1.39(br d,J=7.70Hz,1H),1.42(br d,J=7.09Hz,2H),1.46-1.68(m,2H),1.72-1.94(m,3H),2.00-2.13(m,2H),2.15-2.27(m,2H),2.31(br s,1H),2.33(s,2H),2.38-2.53(m,2H),2.65-2.77(m,3H),2.79(br d,J=9.78Hz,1H),2.83-3.02(m,2H),3.25(br s,4H),3.33-3.38(m,5H),3.38-3.54(m,2H),3.69(br d,J=5.87Hz,2H),3.74-3.89(m,1H),4.07-4.21(m,1H),4.25-4.33(m,1H),4.38(br s,2H),4.52-4.73(m,3H),4.79(br d,J=8.19Hz,3H),7.09-7.30(m,5H).

[1062] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,7S,10S,18S,21S,24S,30S,31R,32R,33R)-4-((S)-sec-butyl)-24-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)-30,31,32,33,34-pentahydroxy-7 General procedure for the preparation of (MB-24) (PL2) : (1,0,21-triisopropyl-3-methoxy-5,11,18-trimethyl-6,9,17,20,23,27-hexaoxo-14-thia-5,8,11,16,19,22,28-heptaazatriacontria-1-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropanoic acid (MB-24) (PL2).

[1063]

[1064] N-hydroxysuccinimide (22.8 mg, 198 μmol) was added to a solution of compound 8 (50.0 mg, 99.3 μmol) and dicyclohexylcarbodiimide (40.9 mg, 198 μmol) in DMF (0.40 mL). After stirring for 6 hours at 25 ° C, a solution of compound 10 (103 mg, 104 μmol) in DMF (0.40 mL) was added to the above reaction mixture, and it was continued to be stirred for another 12 hours at 25 ° C. LCMS showed that the reaction was complete. It was filtered, and the filtrate was directly loaded onto a preparative HPLC [column: Phenomenex Luna C18 (80×30 mm×3 μm); mobile phase, A: water, B: acetonitrile; B%: 15%-45%, 8 minutes] for purification to obtain the product MB-24 (PL2) (200.6 mg, yield: 13.9%) as a white solid. HRMS (ESI+), m / z, calculated value 1476.83 (M+H) + , the measured value is 1476.8430. 1 H NMR (400MHz, methanol-d4), δppm 0.82-0.91(m,6H),0.93-1.06(m,17H),1.13(dd,J=16.03,6.74Hz,3H),1.20(d,J=6.79Hz,2H),1.23-1.48(m,8H),1.49-1.69 (m,5H),,1.70-2.00(m,4H),2.01-2.18(m,4H),2.19-2.36(m,5H),2.37-2.54(m,5H),2.61-3.08(m,7H),3.14(s,1H),3.27(br d,J=14.90Hz,4H),3.33-3.56(m,9H),3.59-3.91(m,8H),4.03-4.22(m,2H),4.25-4.44(m,4H),4. 64-4.71(m,1H),4.75-4.82(m,2H),6.80(d,J=1.19Hz,2H),7.13-7.20(m,1H),7.21-7.30(m,4H).

[1065] Example 5: Exemplary Synthesis of Compound D5

[1066] General procedure for the preparation of 2-(methylthio)acetaldehyde.

[1067]

[1068] A solution of 1,1-dimethoxy-2-methylsulfanyl-ethane (2.00 g, 14.7 mmol, 1.96 mL) in aqueous HCl (0.32 M, 5.70 mL) was stirred at 50° C. for 0.5 h. The mixture was concentrated under reduced pressure to give a pale yellow oil, which was distilled at 60° C. to give 2-methylsulfanyl acetaldehyde (550 mg, 6.10 mmol, yield: 41.5%) as a pale yellow oil. 1 H NMR (400MHz, CDCl3) δ2.03 (s, 3H), 3.15 (d, J = 3.50Hz, 2H), 9.46 (t, J = 3.56Hz, 1H).

[1069] General procedure for the preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(2-(methylthio)ethyl)amino)butyrylamino)butyramide (D5).

[1070]

[1071] A mixture of (2S)-N-[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]-3-methyl-2-(methylamino)butanamide (MMAE, 100 mg, 139 μmol), 2-methylsulfanyl acetaldehyde (25.1 mg, 279 μmol) and acetic acid (41.8 mg, 696 μmol) was stirred at 25° C. for 20 minutes, then sodium cyanoborohydride (43.8 mg, 696 μmol) was added in one portion. The reaction mixture was stirred at 25 ° C for 12 hours. LCMS showed that the reaction was complete. The reaction mixture was filtered and the filtrate was purified by preparative HPLC (column: Waters Xbridge BEH C18 100×30mm×10μm; mobile phase: [H2O(10mMNH4HCO3)-acetonitrile]; gradient: 40%-75% B in 8.0 minutes) to give compound D5 (21.9 mg, yield: 19.9%) as a white solid. LCMS (ESI + ): m / z 792.3[M+H]+ . 1 H NMR(400MHz,DMSO-d6)δ0.67-0.80(m,6H),0.80-0.95(m,13H),0.96-1.09(m,6H),1.20-1.38(m,1H),1.42-1.61(m,2H),1.61-1 .86(m,3H),1.86-2.01(m,2H),2.02-2.08(m,3H),2.08-2.18(m,1H),2.19-2.34(m,4H),2.36-2.46(m,1H),2.52-2.79(m,5H),2. 95-3.11(m,2H),3.13-3.28(m,8H),3.32-3.39(m,1H),3.41-3.63(m,1H),3.78(dd,J=9.38,1.75Hz,1H),3.88-4.11(m,2H),4.38 -4.61(m,2H),4.61-4.83(m,1H),5.27-5.48(m,1H),7.08-7.21(m,1H),7.23-7.39(m,4H),7.53-7.99(m,1H),8.07-8.26(m,1H).

[1072] Example 6: Exemplary Synthesis of Compound D6

[1073] General procedure for the preparation of ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(2-(methylthio)ethyl)amino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (D6).

[1074]

[1075] To a mixture of ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (MMAF, 110 mg, 150 μmol) in methanol (2.00 mL) was added acetic acid (45.2 mg, 751 μmol) and 2-methylsulfanyl acetaldehyde (27.1 mg, 301 μmol, 2.00 equiv) followed by sodium cyanoborate (47.2 mg, 751 μmol) in one portion. The reaction mixture was stirred at 25 °C for 12 hours. LCMS showed the reaction was complete. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H 2 O (10 mM NH 4 HCO 3 )-acetonitrile]; gradient: 40%-75% B in 8.0 minutes) to give compound D6 (57.4 mg, yield: 46.9%) as a white solid. LCMS (ESI + ): m / z 806.4[M+H] + . 1 H NMR (400 MHz, methanol-d4) δ 0.81-0.92 (m, 6H), 0.95-1.06 (m, 11H), 1.08-1.23 (m, 4H), 1.12-1.14 (m, 1H), 1.26-1.47 (m, 2H), 1.49-1.67 (m, 1H), 1.70-1.94 (m, 3H), 1.99-2.18 (m, 5H), 2.21-2.34 (m, 1H), 2.31-2.33 (m, 1H), 2.35-2.53 (m, 5H), 2.59-2.79 (m, 3H), 2.80 -2.98(m,3H),3.14(s,1H),3.19(dt,J=11.91,7.30Hz,1H),3.24-3.28(m,2H),3.29(s,1H),3.32-3.45(m,7H),3.47-3.56(m,1H ),3.61-3.78(m,1H),3.85(dd,J=8.50,2.13Hz,1H),4.01-4.21(m,1H),4.60-4.71(m,1H),4.72-4.84(m,2H),7.13-7.31(m,5H).

[1076] Example 7: Exemplary Synthesis of Antibody-Drug Conjugate Trastuzumab-MB24

[1077]

[1078] 50 mM coupling buffer (pH 7.4): 1 L of the solution contains 6.86 g of Na2HPO4·2H2O and 1.58 g of NaH2PO4·H2O.

[1079] 10 mM DTPA aqueous solution: 1 L of the solution contains 3.90 g DTPA and 1.20 g NaOH.

[1080] 10 mM TCEP aqueous solution: 1 L of the solution contains 2.866 g TCEP.

[1081] Preparation buffer (pH 6.0) (10 mM His / His-HCl): 1 L of the solution contains 0.73 g His and 1.12 g His-HCl.

[1082] Antibody preparation: 627 mg of freeze-dried trastuzumab powder was dissolved in 22 mL of purified water. The resulting antibody solution was dialyzed using an ultrafiltration tube (30KD) with 50 mM coupling buffer for 4 cycles to obtain an antibody concentration of 11.97 mg / ml (using the extinction coefficient of trastuzumab ε 280 =213380M -1 cm -1 ).

[1083] Reduction of trastuzumab: 3.4 mL of 50 mM coupling buffer was added to a tube containing 2.5 mL (30 mg, 0.000207 mmol of trastuzumab) of the trastuzumab solution prepared above, followed by 87 μl of 10 mM TCEP (0.00087 mmol, 4.2 equivalents) and 41.4 μl of 10 mM ZnCl2 solution (0.000414 mmol, 2 equivalents). The tube was placed in a thermal mixer and the reduction reaction was carried out at 25°C for 3 hours.

[1084] Coupling between antibody and payload: 5.68 mg (0.00385 mmol) of payload MB-24 (PL2) was dissolved in 769 μL of DMSO to obtain a 5 mM solution. 331 μL (0.00166 mmol, 8 equivalents) of DMSO solution containing 5 mM MB-24 was added to the above trastuzumab reduction solution. The tube was placed in a thermomixer and the coupling reaction was carried out at 25°C for 1 hour, and then 5 mM cysteine ​​solution (166 μL) was added. Then, 10 mM DTPA solution (83 μL) and 10 mM DHAA solution (166 μL) were added to remove zinc ions.

[1085] Purification: The coupling reaction solution was purified for 8 cycles using an ultrafiltration tube (30KD) with 10 mM His / His-HCl preparation buffer to obtain 2.6 mL (8.2 mg / mL) of preparation buffer containing trastuzumab-MB24 (21.3 mg, yield = 68%, DAR = 4.3).

[1086] Physicochemical characterization of trastuzumab-MB24 (Extinction coefficient of payload MB-24: ε 280 =764.4M -1 cm -1 and ε 260 =1280.1M -1 cm -1 ). Additional characterization data are provided in Table 1.

[1087] Table 1

[1088]

[1089] Example 8: Exemplary Synthesis of Antibody-Drug Conjugate Trastuzumab-MB26

[1090]

[1091] Reduction of trastuzumab: 4 mL of 50 mM coupling buffer was added to a tube containing 2.9 mL (35 mg, 0.000240 mmol of trastuzumab) of trastuzumab solution (11.97 mg / mL), followed by 92 μl of 10 mM TCEP (0.00092 mmol, 3.8 equivalents) and 48 μl of 10 mM ZnCl2 solution (0.00048 mmol, 2 equivalents). The tube was placed in a thermal mixer and subjected to reduction reaction at 25°C for 3 hours.

[1092] Coupling between antibody and payload: 8.43 mg (0.00576 mmol) of payload MB-26 (PL1) was dissolved in 1153 μL of DMSO to obtain a 5 mM solution. 384 μL (0.00192 mmol, 8 equivalents) of DMSO solution containing 5 mM MB-24 was added to the above trastuzumab reduction solution. The tube was placed in a thermal mixer and the coupling reaction was carried out at 25°C for 1 hour, and then 5 mM cysteine ​​solution (192 μL) was added. Then, 10 mM DTPA solution (96 μL) and 10 mM DHAA solution (192 μL) were added to remove zinc ions.

[1093] Purification: The coupling reaction solution was purified for 8 cycles using an ultrafiltration tube (30KD) with 10 mM His / His-HCl preparation buffer to obtain 3.7 mL (5.7 mg / mL) of preparation buffer containing trastuzumab-MB26 (21.1 mg, yield = 58%, DAR = 4.3).

[1094] Physicochemical characterization of trastuzumab-MB26 (Extinction coefficient of payload MB-26: ε 280 =639.5M -1 cm -1 and ε 260 =1006.3M -1 cm -1 ). Additional characterization data are provided in Table 2.

[1095] Table 2

[1096]

[1097] Example 9: Exemplary Synthesis of the Dual-Drug Conjugate Trastuzumab-MB0324

[1098]

[1099] Reduction of trastuzumab: To a tube containing 4.1 mL (9.83 mg / mL, 40.3 mg of trastuzumab) of trastuzumab-containing coupling buffer, 3.7 mL of 50 mM coupling buffer was added, followed by 116 μl of 10 mM TCEP (0.00116 mmol, 4.2 equivalents) and 55 μl of 10 mM ZnCl2 (2 equivalents). The tube was placed in a thermal mixer and the reduction reaction was carried out at 25°C for 3 hours.

[1100] Coupling between antibody and first payload: 4.02 mg (0.00272 mmol) of payload MB-24 was dissolved in 544 μl of DMSO to obtain a 5 mM solution. 442 μl of DMSO solution containing 5 mM MB-24 (0.00221 mmol, 8 equivalents / antibody molecule) was added to the above trastuzumab reduction solution. The tube was placed in a thermal mixer and the coupling reaction was carried out at 25° C. for 1 hour, followed by the addition of 221 μl of cysteine ​​solution (5 mM), 110 μl of DTPA solution (10 mM), and 221 μl of DHAA solution (10 mM).

[1101] Purification: The coupling reaction solution was purified by 4 cycles of coupling buffer (containing 10% 10 mM DTPA) using an ultrafiltration tube (30 KD) to obtain 3.8 mL (8.48 mg / mL) of coupling buffer containing trastuzumab-MB24 (32.2 mg). Based on HIC-HPLC, the DAR value was 4.2.

[1102] Reduction of trastuzumab-MB24: 4.1 mL of 50 mM coupling buffer (containing 10% of 10 mM DTPA solution) and 110 μl of 10 mM TCEP solution (5 equivalents) were added to the tube containing the above-mentioned coupling buffer containing trastuzumab-MB24 (3.8 mL, 8.48 mg / mL). The tube was placed in a thermomixer and the reduction reaction was carried out at 25° C. for 2 hours.

[1103] Coupling with the second payload: 4.33 mg (0.0081 mmol) of payload Medinotecan (MB-3) was dissolved in 761 μL of DMSO to obtain a 5 mM solution. 264 μL (0.00132 mmol, 6 equivalents) of 5 mM Medinotecan in DMSO was added to the above Trastuzumab-MB24 reduction solution. The tube was placed in a thermomixer and the coupling reaction was carried out at 25°C for 1 hour.

[1104] Purification: The coupling reaction solution was purified for 8 cycles using an ultrafiltration tube (30KD) with 10 mM His / His-HCl preparation buffer to obtain 1.6 mL (15.5 mg / mL) of preparation buffer containing trastuzumab-MB0324 (24.8 mg, yield = 62%, DAR of effective load meditecan = 3.9, DAR of effective load MB-24 = 4.1).

[1105] Physicochemical characterization of trastuzumab-MB0324 (extinction coefficient of meditecan: ε 280 =4546M -1 cm -1 and ε 360 =17513M -1 cm -1 ; Extinction coefficient of MB-24: ε 280 =764.4M -1 cm -1 and ε 260 =1280.1M -1 cm -1 ), and more characterization data are provided in Table 3.

[1106] Table 3

[1107]

[1108] Example 10: Exemplary Synthesis of the Dual-Drug Conjugate Trastuzumab-MB0326

[1109]

[1110] Reduction of trastuzumab: To a tube containing 3.2 mL (the concentration of trastuzumab in the coupling buffer was 15.6 mg / mL) of trastuzumab-containing coupling buffer, 3.7 mL of 50 mM coupling buffer was added, followed by 131 μl (3.8 equivalents) of 10 mM TCEP and 69 μl (2 equivalents) of 10 mM ZnCl 2. The tube was placed in a thermomixer and the reduction reaction was carried out at 25° C. for 3 hours.

[1111] Coupling between antibody and first payload: 40.8 mg (0.0279 mmol) of payload MB-26 was dissolved in 5.6 mL of DMSO to obtain a 5 mM solution. 552 μL of DMSO solution containing 5 mM MB-26 (0.00276 mmol, 8 equivalents / antibody molecule) was added to the above trastuzumab reduction solution. The tube was placed in a thermal mixer and the coupling reaction was carried out at 25° C. for 1 hour, followed by the addition of 276 μl of cysteine ​​solution (5 mM), 138 μl of DTPA solution (10 mM), and 276 μl of DHAA solution (10 mM).

[1112] Purification: The coupling reaction solution was purified by 4 cycles of coupling buffer (containing 10% 10 mM DTPA) using an ultrafiltration tube (30 KD) to obtain 7.8 mL (6 mg / mL) of coupling buffer containing trastuzumab-MB26 (46.8 mg). Based on HIC-HPLC, the DAR value was 4.4.

[1113] Reduction of trastuzumab-MB26: 1.4 mL of 50 mM coupling buffer (containing 10% of 10 mM DTPA solution) and 129 μl of 10 mM TCEP solution (4 equivalents) were added to the tube containing the above-mentioned coupling buffer containing trastuzumab-MB26 (7.8 mL, 6 mg / mL). The tube was placed in a thermomixer and the reduction reaction was carried out at 25°C for 2 hours.

[1114] Coupling with the second payload: 15.6 mg (0.0137 mmol) of payload Medinotecan (MB-3) was dissolved in 2740 μL of DMSO to obtain a 5 mM solution. 371 μL (0.00185 mmol, 6 equivalents) of 5 mM Medinotecan in DMSO was added to the above reduced solution of Trastuzumab-MB26 (0.000309 mmol). The tube was placed in a thermomixer and the coupling reaction was carried out at 25°C for 1 hour.

[1115] Purification: The above coupling reaction solution was purified by 8 cycles of ultrafiltration tube (30KD) with 10mM His / His-HCl formulation buffer to obtain 4mL (10.1mg / mL) of formulation buffer containing trastuzumab-MB0326 (40.4mg, yield = 75%, DAR of effective load Meditec = 3.6, DAR of effective load MB-26 = 4.4). 80μL of 1% polysorbate 80 was added to the solution for final storage.

[1116] Physicochemical characterization of trastuzumab-MB0326 (extinction coefficient of meditecan: ε 280 =4546M -1 cm -1 and ε 360 =17513M -1 cm -1 ; Extinction coefficient of MB-26: ε 280 =639.5M -1 cm -1 and ε 260 =1006.3M -1 cm -1 ), and more characterization data are provided in Table 4.

[1117] Table 4

[1118]

[1119] Example 11: In vitro analysis of toxins and ADCs

[1120] 175 μL of cell suspension was dispensed into a 96-well plate at 1500 cells / well and incubated in a humidified incubator (37°C, 5% CO2) for 24 hours. 25 μL of compounds at different concentrations were added to the cell culture medium (fetal bovine serum, Invitrogen) in the plate as a 5× solution and incubated in the incubator for 120 hours. CCK-8 was thawed on the bench or in a 37°C water bath, and 10 μL of CCK-8 was added to each well of the incubation plate (be careful not to introduce bubbles into the wells as they will interfere with OD readings), followed by further incubation in the incubator for 1-4 hours. The absorbance at 450 nm was measured using a SpectraMax i3x microplate reader and the cell inhibition rate was calculated. IC was generated by using GraphPad Prism software 50 Curve and IC 50 value.

[1121] The toxins (expected metabolites of ADC) and the in vitro cytotoxicity of ADC are described in Table 5.

[1122] Table 5

[1123]

[1124] Example 12: In vivo efficacy of ADC in the NCI-N87 CDX model Each mouse (female Balb / c-nude mice from Vital Rivers) was subcutaneously inoculated with NCI-N87 tumor cells (5×10 6 ), these cells were mixed with Matrigel (50:50) in 0.2 mL PBS and used to generate tumors. On day 6 after tumor inoculation, when the average tumor volume reached approximately 160 mm 3 At 14:00, the animals were randomly divided into groups and then treatment was started for efficacy study. Each group had 8 mice. The test and control articles were administered to tumor-bearing mice via the tail vein at a volume of 5 mL / kg.

[1125] Tumor size was measured twice weekly in two dimensions using calipers and expressed in mm using the following formula: 3 Indicates volume: V = 0.5a × b 2 , where a and b are the long and short dimensions of the tumor, respectively. The results are presented as mean ± SEM.

[1126] Statistical analysis: Two-way ANOVA was performed to compare the tumor volume between the two groups. All data were analyzed using Graphpad Prism 6.0, and P < 0.05 was considered statistically significant. Both statistical analysis and biological observations were taken into account.

[1127] Tumor growth inhibition: T / C values ​​were calculated using tumor size. T / C (%) of the relative tumor proliferation rate was calculated using the following formula: T / C (%) = (Ti / T0) / (Vi / V0) × 100%. Relative tumor growth inhibition was calculated using the following formula: TGI (%) = [1-(Ti / T0) / (Vi / V0)] × 100%. Ti refers to the average tumor volume of the treatment group measured at each specified time point after treatment; T0 refers to the tumor volume of the treatment group at grouping; Vi refers to the average tumor volume of the vehicle control group measured at each specified time point after treatment; V0 refers to the tumor volume of the vehicle control group at grouping. If T / C>40%, there is no therapeutic effect; if T / C=<40% and p value<0.05, there is tumor inhibition.

[1128] The anti-tumor effect of ADC in the NCI-N87 CDX model Figure 1 and 2 and as shown in Tables 6 and 7. Figure 1As shown, all three ADCs (trastuzumab-MB24-DAR4, trastuzumab-MB3-DAR8, and trastuzumab-MB0324) exhibited dose-dependent antitumor activity (0.3, 1, and / or 3 mg / kg). Similarly, trastuzumab-MB26-DAR4, trastuzumab-MB3-DAR4, and trastuzumab-MB0326 all exhibited dose-dependent antitumor activity (1 and 3 mg / kg), with >90% TGI induced at a dose of 3 mg / kg, as shown in FIG. Figure 2 As shown in Figure 4, the dual-drug ADC (trastuzumab-MB0326) showed comparable activity to the single-drug ADCs (trastuzumab-MB03-DAR4 and trastuzumab-MB26-DAR4).

[1129] Table 6

[1130]

[1131]

[1132] Table 7

[1133]

[1134] Example 13: In vivo efficacy of ADC in the JIMT-1CDX model

[1135] Each mouse (Scid-Beige from Shanghai Lingchang Biotech) was inoculated subcutaneously with JIMT-1 tumor cells (1×10 7 ), these cells were mixed with Matrigel (50:50) in 0.2 mL PBS and used to generate tumors. On day 6 after tumor inoculation, when the average tumor volume reached approximately 175 mm 3 At 14:00, the animals were randomly divided into groups and then treatment was started for efficacy study. Each group had 8 mice. The test and control articles were administered to tumor-bearing mice via the tail vein at a volume of 5 mL / kg.

[1136] Tumor size was measured twice weekly in two dimensions using calipers and expressed in mm using the following formula: 3 Indicates volume: V = 0.5a × b 2 , where a and b are the long and short dimensions of the tumor, respectively. The results are presented as mean ± SEM.

[1137] Statistical analysis: Two-way ANOVA was performed to compare tumor volumes between the two groups. All data were analyzed using Graphpad Prism 6.0, and P < 0.05 was considered statistically significant. Both statistical analysis and biological observations were taken into account.

[1138] Tumor growth inhibition: T / C values ​​were calculated using tumor size. T / C (%) of the relative tumor proliferation rate was calculated using the following formula: T / C (%) = (Ti / T0) / (Vi / V0) × 100%. Relative tumor growth inhibition was calculated using the following formula: TGI (%) = [1-(Ti / T0) / (Vi / V0)] × 100%. Ti refers to the average tumor volume of the treatment group measured at each specified time point after treatment; T0 refers to the tumor volume of the treatment group at grouping; Vi refers to the average tumor volume of the vehicle control group measured at each specified time point after treatment; V0 refers to the tumor volume of the vehicle control group at grouping. If T / C>40%, there is no therapeutic effect; if T / C=<40% and p value<0.05, there is tumor inhibition.

[1139] The anti-tumor effect of ADC in JIMT-1CDX model Figure 3 and 4 and as shown in Tables 8 and 9. Figure 3 As shown, all three ADCs (trastuzumab-MB24-DAR4, trastuzumab-MB3-DAR8, and trastuzumab-MB0324) exhibited dose-dependent antitumor activity (0.3, 1, and / or 3 mg / kg), with 3 mg / kg trastuzumab-MB0324 showing comparable activity to trastuzumab-MB24-DAR4. Similarly, trastuzumab-MB26-DAR4, trastuzumab-MB3-DAR4, and trastuzumab-MB0326 all exhibited dose-dependent antitumor activity (1 and 3 mg / kg), as shown in FIG. Figure 4 In this study, the dual-drug ADC (trastuzumab-MB0326) showed slightly superior tumor growth inhibition compared to the single-drug ADCs (trastuzumab-MB3-DAR4 and trastuzumab-MB26-DAR4) at 1 or 3 mg / kg.

[1140] Table 8

[1141]

[1142] Table 9

[1143]

[1144]

[1145] Equivalent

[1146] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.The scope of the present invention is not limited to the above description, but is instead set forth as in the following claims.

Claims

1. A compound of formula (I), D—Q (I), or a pharmaceutically acceptable salt thereof, wherein: D is represented by the following structural formula: in R 1 is -H or -OH; R 2 C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl; Each R 3 and R 4 are independently -H or C1-C3 alkyl; n is an integer from 1 to 6; and Q is -H or -CH3.

2. The compound according to claim 1, wherein R 1 is -H.

3. The compound according to claim 1, wherein R 1 It is -OH.

4. A compound according to any one of claims 1 to 3, wherein R 2 It is -CH3.

5. A compound according to any one of claims 1 to 3, wherein R 2 It is -C(=O)OH.

6. A compound according to any one of claims 1 to 3, wherein R 2 It is -C(=O)NHCH2CH2CH2OH.

7. A compound according to any one of claims 1 to 3, wherein R 2 for 8. The compound according to claim 1, wherein R 1 -H and R 2 It is -C(=O)OH.

9. The compound according to claim 1, wherein R 1 -H and R 2 It is -C(=O)NHCH2CH2CH2OH.

10. The compound according to claim 1, wherein R 1 -H and R 2 for 11. The compound according to claim 1, wherein R 1 is -OH and R 2 It is -CH3.

12. A compound according to any one of claims 1 to 11, wherein R 3 and R 4 Both are -H.

13. A compound according to any one of claims 1 to 11, wherein R 3 and R 4 Both are -CH3.

14. The compound according to any one of claims 1 to 13, wherein n is 1.

15. The compound according to any one of claims 1 to 14, wherein Q is -H.

16. A compound according to any one of claims 1 to 14, wherein Q is -CH3.

17. The compound according to claim 1, wherein D is represented by one of the following structures:

18. The compound according to claim 1, wherein the compound has one of the following structures: or a pharmaceutically acceptable salt thereof.

19. A compound of formula (II), D—CH2—NH—E—Z (II), or a pharmaceutically acceptable salt thereof, wherein: D is represented by the following structural formula: in R 1 is -H or -OH; R 2 C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl; Each R 3 and R 4 are independently -H or C1-C3 alkyl; n is an integer from 1 to 6; E is a peptide comprising 2 to 10 amino acids; wherein E is optionally substituted with one or more polyols; and wherein the N-terminus of the peptide is covalently linked to Z; Z is -C(=O)-LY, Wherein m represents an integer from 1 to 10; L is -(C1-C 10 Alkylene)-*, -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -、-(OCH2CH2) j -、-CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*; wherein j represents an integer of 1-10; and wherein * represents a site of covalent attachment to Y; L1 is -(C1-C 10 Alkylene)-; R 5 is -H or -CH3; and Y is an electrophilic group or a nucleophilic group.

20. The compound according to claim 19, wherein R 1 is -H.

21. The compound according to claim 19, wherein R 1 It is -OH.

22. A compound according to any one of claims 19 to 21, wherein R 2 It is -CH3.

23. A compound according to any one of claims 19 to 21, wherein R 2 It is -C(=O)OH.

24. A compound according to any one of claims 19 to 21, wherein R 2 It is -C(=O)NHCH2CH2CH2OH.

25. A compound according to any one of claims 19 to 21, wherein R 2 for 26. The compound according to claim 19, wherein R 1 -H and R 2 It is -C(=O)OH.

27. The compound according to claim 19, wherein R 1 -H and R 2 It is -C(=O)NHCH2CH2CH2OH.

28. The compound according to claim 19, wherein R 1 -H and R 2 for 29. The compound according to claim 19, wherein R 1 is -OH and R 2 It is -CH3.

30. A compound according to any one of claims 19 to 29, wherein R 3 and R 4 Both are -H.

31. A compound according to any one of claims 19 to 29, wherein R 3 and R 4 Both are -CH3.

32. A compound according to any one of claims 19 to 31, wherein n is 1.

33. A compound according to any one of claims 19 to 32, wherein E is a peptide of 2, 3 or 4 amino acids; each amino acid in the peptide is an L amino acid, or at least one amino acid in the peptide is a D amino acid.

34. according to the compound described in any one of claims 19 to 33, wherein E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein said glutamine or glutamic acid is optionally substituted by a polyol.

35. according to the compound described in any one of claims 19 to 34, wherein E comprises the amino acid having the following structure, Where R 6 It is -H or C1-C6 alkyl.

36. The compound of claim 35, wherein E comprises an amino acid having the structure, 37. The compound of any one of claims 19 to 33, wherein E is selected from the group consisting of: -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Val-Cit-*, -Cit-Val-*, -Leu-Ala-*, -Ala-Leu-*, -Leu-Cit-*, -Cit-Leu-*, -Leu-Ala-*, -Ala-Leu-*, -Lys-Lys-*, -Ala-Lys-*, -Lys-Ala-*, -Val-Lys-*, -Lys-Val-*, -Tyr-Arg-*, -Arg-Tyr-*, -Arg-Arg-*, -Ala-Ala-*, -Phe-Lys-*, -Lys-Phe-*, -Thr-Thr-*, -Th r-Met-*, -Met-Thr-*, -Met-Tyr-*, -Tyr-Met-*, -Phe-Gln-*, -Gln-Phe-*, -Gly-Ser-*, -Leu-Gln-*, -Gln-Leu-*, -Ser-Ala-*, -Ser-Gly-*, -Val-Thr-*, -Thr-Val-*, -Val-Gln -*, -Ser-Val-*, -Val-Ser-*, -Ala-Met-*, -Met-Ala-*, -Val-Arg-*, -Arg-Val-*, -Phe-Ala-*, -Ala-Phe-*, -Cit-Val-*, -Gln-Val-*, -Phe-Arg-*, -Arg-Phe-*, -Ala-Ala-Ala- * 、-Gly-Gly-Gly- * 、-Ala-Val-Ala- * 、-Gly-Val-Gly- * 、-Ala-Val-Gly- * , -Gly-Phe-Lys-*, -Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Val-Ala-Leu-*, -Leu-Ala-Val-*, -Val-Ala-Val-*, -Ala-Val-Ala-Gly- * 、-Gly-Phe-Gly-Gly- * , -Gly-Gly-Phe-Gly-*, -Ala-Val-Gly-Gly-*, -Ala-Ala-Ala-Ala- * ,-Ala-Val-Ala-Ala- * , -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-*, wherein * indicates the N-terminus of the peptide covalently linked to Z.

38. The compound of claim 37, wherein E is selected from the group consisting of: -L-Ala-L-Val-*, -L-Val-L-Ala-*, -L-Val-L-Lys-*, -L-Val-L-Arg-*, -L-Val-L-Cit-*, -L-Ala-L-Val-L-Glu-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-L-Val-L-Ala-*, -L-Ala-L-Ala-Gly-*, -L-Ala-L-Val-Gly-*, -Gly-Gly-L-Glu-*, -Gly-L-Phe-Gly-Gly-*, -Gly-L-Glu-Gly-Gly-*; wherein * represents the N-terminus of the peptide covalently linked to Z.

39. The compound according to any one of claims 19 to 38, wherein Z is -C(=O)-LY.

40. A compound according to any one of claims 19 to 38, wherein Z is Here, m represents an integer from 1 to 10.

41. A compound according to any one of claims 19 to 38, wherein Z is Here, m represents an integer from 1 to 10.

42. A compound according to any one of claims 19 to 39, wherein L is -(C1-C 10 Alkylene)-.

43. A compound according to any one of claims 19 to 39, wherein L is -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -or-(OCH2CH2) j -, wherein j represents an integer of 1 to 10; and wherein * represents a site for covalent bonding to Y.

44. A compound according to any one of claims 19 to 39, wherein L is -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*, wherein j represents an integer of 1-10; and wherein * represents a site for covalent bonding to Y.

45. according to the compound described in any one of claims 19 to 39 and 44, wherein L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-* or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, wherein * represents the site of covalent attachment to Y.

46. ​​A compound according to any one of claims 19 to 39, wherein Y is a Michael acceptor group, succinimide, epoxy or halogen.

47. The compound according to claim 46, wherein Y is Where R 7 and R 8 Each is independently H or C1-C3 alkyl.

48. A compound according to any one of claims 19 to 38, wherein Z is 49. A compound according to any one of claims 19 to 32, wherein -E-NH-CH 2- has one of the following structures, where * indicates the N-terminus of the peptide covalently linked to Z:

50. A compound according to any one of claims 19 to 32, wherein ZE-NH-CH2- has one of the following structures:

51. The compound of claim 19, wherein D is represented by one of the following structures:

52. The compound of claim 19, wherein the compound has one of the following structures, or a pharmaceutically acceptable salt thereof.

53. A compound of formula (III), {D—CH2—NH—E—Z'} p —C (III), in: D is represented by the following structural formula: in R 1 is -H or -OH; R 2 C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl; R 3 and R 4 are independently -H or C1-C3 alkyl; n is an integer from 1 to 6; E is a peptide comprising 2 to 10 amino acids; wherein E is optionally substituted with one or more polyols; and wherein the N-terminus of the peptide is covalently linked to Z'; Z' is -C(=O)-L-Y'-, wherein m represents an integer of 1-10, and * represents a site covalently linked to the C; L is -(C1-C 10 Alkylene)-*, -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -、-(OCH2CH2) j -、-CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*; wherein j represents an integer from 1 to 10; and wherein * represents a site of covalent attachment to Y'; L1 is -(C1-C 10 Alkylene)-; R 5 is -H or -CH3; and C represents a cell-binding agent; Y' is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on the cell binding agent; p has a value between 1 and 18.

54. The compound according to claim 53, wherein R 1 is -H.

55. The compound according to claim 53, wherein R 1 It is -OH.

56. A compound according to any one of claims 53 to 55, wherein R 2 It is -CH3.

57. A compound according to any one of claims 53 to 55, wherein R 2 It is -C(=O)OH.

58. A compound according to any one of claims 53 to 55, wherein R 2 It is -C(=O)NHCH2CH2CH2OH.

59. according to the compound described in any one of claims 53 to 55, wherein R 2 for 60. The compound according to claim 53, wherein R 1 -H and R 2 It is -C(=O)OH.

61. The compound according to claim 53, wherein R 1 -H and R 2 It is -C(=O)NHCH2CH2CH2OH.

62. The compound according to claim 53, wherein R 1 -H and R 2 for 63. The compound according to claim 53, wherein R 1 is -OH and R 2 It is -CH3.

64. according to the compound described in any one of claims 53 to 63, wherein R 3 and R 4 Both are -H.

65. according to the compound described in any one of claims 53 to 63, wherein R 3 and R 4 Both are -CH3.

66. A compound according to any one of claims 53 to 65, wherein n is 1.

67. A compound according to any one of claims 53 to 66, wherein E is a peptide of 2, 3 or 4 amino acids; each amino acid in the peptide is an L amino acid, or at least one amino acid in the peptide is a D amino acid.

68. according to the compound described in any one of claims 53 to 67, wherein E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein said glutamine or glutamic acid is optionally substituted by a polyol.

69. according to the compound described in any one of claims 53 to 67, wherein E comprises the amino acid with following structure, Where R 6 It is -H or C1-C6 alkyl.

70. The compound of claim 69, wherein E comprises an amino acid having the structure, 71. The compound of any one of claims 53 to 67, wherein E is selected from the group consisting of: -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Val-Cit-*, -Cit-Val-*, -Leu-Ala-*, -Ala-Leu-*, -Leu-Cit-*, -Cit-Leu-*, -Leu-Ala-*, -Ala-Leu-*, -Lys-Lys-*, -Ala-Lys-*, -Lys-Ala-*, -Val-Lys-*, -Lys-Val-*, -Tyr-Arg-*, -Arg-Tyr-*, -Arg-Arg-*, -Ala-Ala-*, -Phe-Lys-*, -Lys-Phe-*, -Thr-Thr-*, -Th r-Met-*, -Met-Thr-*, -Met-Tyr-*, -Tyr-Met-*, -Phe-Gln-*, -Gln-Phe-*, -Gly-Ser-*, -Leu-Gln-*, -Gln-Leu-*, -Ser-Ala-*, -Ser-Gly-*, -Val-Thr-*, -Thr-Val-*, -Val-Gln -*, -Ser-Val-*, -Val-Ser-*, -Ala-Met-*, -Met-Ala-*, -Val-Arg-*, -Arg-Val-*, -Phe-Ala-*, -Ala-Phe-*, -Cit-Val-*, -Gln-Val-*, -Phe-Arg-*, -Arg-Phe-*, -Ala-Ala-Ala- * 、-Gly-Gly-Gly- * 、-Ala-Val-Ala- * 、-Gly-Val-Gly- * 、-Ala-Val-Gly- * , -Gly-Phe-Lys-*, -Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Val-Ala-Leu-*, -Leu-Ala-Val-*, -Val-Ala-Val-*, -Ala-Val-Ala-Gly- * 、-Gly-Phe-Gly-Gly- * , -Gly-Gly-Phe-Gly-*, -Ala-Val-Gly-Gly-*, -Ala-Ala-Ala-Ala- * ,-Ala-Val-Ala-Ala- * , -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-*, wherein * indicates the N-terminus of the peptide covalently linked to Z'.

72. The compound of claim 71, wherein E is selected from the group consisting of: -L-Ala-L-Val-*, -L-Val-L-Ala-*, -L-Val-L-Lys-*, -L-Val-L-Arg-*, -L-Val-L-Cit-*, -L-Ala-L-Val-L-Glu-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-L-Val-L-Ala-*, -L-Ala-L-Ala-Gly-*, -L-Ala-L-Val-Gly-*, -Gly-Gly-L-Glu-*, -Gly-L-Phe-Gly-Gly-*, -Gly-L-Glu-Gly-Gly-*; wherein * indicates the N-terminus of the peptide covalently linked to Z'.

73. The compound of any one of claims 53 to 72, wherein Z' is -C(=O)-L-Y'-.

74. according to the compound described in any one of claims 53 to 72, wherein Z 'is wherein m represents an integer of 1 to 10, and * represents a site for covalent bonding to the C.

75. according to the compound described in any one of claims 53 to 72, wherein Z 'is wherein m represents an integer of 1 to 10, and * represents a site for covalent bonding to the C.

76. according to the compound described in any one of claims 53 to 73, wherein L is -(C1-C 10 Alkylene)-.

77. A compound according to any one of claims 53 to 73, wherein L is -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -or-(OCH2CH2) j -, wherein j represents an integer of 1 to 10; and wherein * represents a site of covalent bonding to Y'.

78. A compound according to any one of claims 53 to 73, wherein L is -CH2CH2(OCH2CH2) j N(R5)C(=O)-L1-*or -CH2(OCH2CH2) j N(R5)C(=O)-L1-*, wherein j represents an integer of 1-10; and wherein * represents a site of covalent bonding to Y'.

79. according to the compound described in any one of claims 53 to 73 and 78, wherein L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-* or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, wherein * represents the site of covalent attachment to Y'.

80. A compound according to any one of claims 53 to 79, wherein Y' is a group formed by reaction of an electrophilic group with a reactive nucleophilic group present on the cell binding agent.

81. according to the compound described in claim 80, wherein Y 'is formed by following Where R 7 and R 8 Each is independently H or C1-C3 alkyl.

82. The compound according to claim 80, wherein Y' is Where R 7 and R 8 Each is independently -H or C1-C3 alkyl, and * indicates the site of covalent attachment to the C.

83. A compound according to any one of claims 53 to 72, wherein Z' is formed by:

84. A compound according to any one of claims 53 to 72, wherein Z' is: Where * indicates the site of covalent attachment to C.

85. according to the compound described in any one of claim 53 to 66, wherein -E-NH-CH 2- has one of the following structures, where * indicates the N-terminus of the peptide covalently linked to Z':

86. A compound according to any one of claims 53 to 66, wherein -Z'-E-NH-CH2- is formed by one of the following structures:

87. according to the compound described in any one of claims 53 to 66, wherein -Z '-E-NH-CH2- is one of the following structures, wherein * represents the connection point with the C:

88. The compound of claim 53, wherein D is represented by one of the following structures:

89. The compound of claim 53, wherein D—CH2—NH—E—Z'— is formed by one of the following structures:

90. The compound according to claim 53, wherein {D—CH2—NH—E—Z'} p — C is one of the following structures, wherein C is a monoclonal antibody and p is the drug to antibody ratio (DAR), and p is an average of about 2-8, 4-8, or 7-8, 91. The compound of any one of claims 53 to 90, wherein p is an average of about 3-8 or 4-8.

92. The compound of any one of claims 53 to 90, wherein p is 8 or an average of about 4, about 7.5, or about 8.

93. A compound of formula (IV), {D—CH2—NH—E—Z'} p' —C—{W} t (IV), in: D is represented by the following structural formula: in R 1 is -H or -OH; R 2 C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH or heteroaryl; R 3 and R 4 are independently -H or C1-C3 alkyl; n is an integer from 1 to 6; E is a peptide comprising 2 to 10 amino acids; wherein E is optionally substituted with one or more polyols; and wherein the N-terminus of the peptide is covalently linked to Z'; Z' is -C(=O)-L-Y'-, wherein m represents an integer of 1-10, and * represents a site covalently linked to the C; L is -(C1-C 10 Alkylene)-*, -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -、-(OCH2CH2) j -、-CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*; wherein j represents an integer from 1 to 10; and wherein * represents a site of covalent attachment to Y'; L1 is -(C1-C 10 Alkylene)-; R 5 is -H or -CH3; and C represents a cell-binding agent; Y' is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on the cell binding agent; W is a group formed by the reaction of compound W' with a reactive nucleophilic group present on C; wherein W' is a cell-killing agent attached to a linker so that W' can be coupled to C; p' and t have values ​​between 1 and 10; wherein p' and t may be the same or different numbers, and p':t is about 1:1, about 1:2 or about 2:1, and wherein p':t is 1:1 or 1:2 or 2:

1.

94. The compound according to claim 93, wherein R 1 is -H.

95. The compound according to claim 93, wherein R 1 It is -OH.

96. according to the compound described in any one of claims 93 to 95, wherein R 2 It is -CH3.

97. according to the compound described in any one of claims 93 to 95, wherein R 2 It is -C(=O)OH.

98. according to the compound described in any one of claims 93 to 95, wherein R 2 It is -C(=O)NHCH2CH2CH2OH.

99. according to the compound described in any one of claims 93 to 95, wherein R 2 for 100. The compound according to claim 93, wherein R 1 -H and R 2 It is -C(=O)OH.

101. The compound according to claim 93, wherein R 1 -H and R 2 It is -C(=O)NHCH2CH2CH2OH.

102. The compound according to claim 93, wherein R 1 -H and R 2 for 103. The compound according to claim 93, wherein R 1 is -OH and R 2 It is -CH3.

104. according to the compound described in any one of claims 93 to 103, wherein R 3 and R 4 Both are -H.

105. according to the compound described in any one of claims 93 to 103, wherein R 3 and R 4 Both are -CH3.

106. A compound according to any one of claims 93 to 105, wherein n is 1.

107. A compound according to any one of claims 93 to 106, wherein E is a peptide of 2, 3 or 4 amino acids; each amino acid in the peptide is an L amino acid, or at least one amino acid in the peptide is a D amino acid.

108. according to the compound described in any one of claims 93 to 107, wherein E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein said glutamine or glutamic acid is optionally substituted by a polyol.

109. according to the compound described in any one of claims 93 to 107, wherein E comprises the amino acid with following structure, Where R 6 It is -H or C1-C6 alkyl.

110. The compound of claim 109, wherein E comprises an amino acid having the structure, 111. The compound of any one of claims 93 to 107, wherein E is selected from the group consisting of: -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Val-Cit-*, -Cit-Val-*, -Leu-Ala-*, -Ala-Leu-*, -Leu-Cit-*, -Cit-Leu-*, -Leu-Ala-*, -Ala-Leu-*, -Lys-Lys-*, -Ala-Lys-*, -Lys-Ala-*, -Val-Lys-*, -Lys-Val-*, -Tyr-Arg-*, -Arg-Tyr-*, -Arg-Arg-*, -Ala-Ala-*, -Phe-Lys-*, -Lys-Phe-*, -Thr-Thr-*, -Thr- hr-Met-*, -Met-Thr-*, -Met-Tyr-*, -Tyr-Met-*, -Phe-Gln-*, -Gln-Phe-*, -Gly-Ser-* , -Leu-Gln-*, -Gln-Leu-*, -Ser-Ala-*, -Ser-Gly-*, -Val-Thr-*, -Thr-Val-*, -Val-Gl n-*, -Ser-Val-*, -Val-Ser-*, -Ala-Met-*, -Met-Ala-*, -Val-Arg-*, -Arg-Val-*, -Phe-Ala-*, -Ala-Phe-*, -Cit-Val-*, -Gln-Val-*, -Phe-Arg-*, -Arg-Phe-*, -Ala-Ala-Ala- * 、-Gly-Gly-Gly- * 、-Ala-Val-Ala- * 、-Gly-Val-Gly- * 、-Ala-Val-Gly- * , -Gly-Phe-Lys-*, -Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Val-Ala-Leu-*, -Leu-Ala-Val-*, -Val-Ala-Val-*, -Ala-Val-Ala-Gly- * 、-Gly-Phe-Gly-Gly- * , -Gly-Gly-Phe-Gly-*, -Ala-Val-Gly-Gly-*, -Ala-Ala-Ala-Ala- * ,-Ala-Val-Ala-Ala- * , -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-*, wherein * indicates the N-terminus of the peptide covalently linked to Z'.

112. The compound of claim 111, wherein E is selected from the group consisting of: -L-Ala-L-Val-*, -L-Val-L-Ala-*, -L-Val-L-Lys-*, -L-Val-L-Arg-*, -L-Val-L-Cit-*, -L-Ala-L-Val-L-Glu-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-L-Val-L-Ala-*, -L-Ala-L-Ala-Gly-*, -L-Ala-L-Val-Gly-*, -Gly-Gly-L-Glu-*, -Gly-L-Phe-Gly-Gly-*, -Gly-L-Glu-Gly-Gly-*; wherein * indicates the N-terminus of the peptide covalently linked to Z'.

113. The compound of any one of claims 93 to 112, wherein Z' is -C(=O)-L-Y'-.

114. according to the compound described in any one of claims 93 to 112, wherein Z 'is wherein m represents an integer of 1 to 10, and * represents a site for covalent bonding to the C.

115. according to the compound described in any one of claims 93 to 112, wherein Z 'is wherein m represents an integer of 1 to 10, and * represents a site for covalent bonding to the C.

116. according to the compound described in any one of claims 93 to 113, wherein L is -(C1-C 10 Alkylene)-.

117. A compound according to any one of claims 93 to 113, wherein L is -CH2(OCH2CH2) j -*、-CH2CH2(OCH2CH2) j -or-(OCH2CH2) j -, wherein j represents an integer of 1 to 10; and wherein * represents a site of covalent bonding to Y'.

118. A compound according to any one of claims 93 to 113, wherein L is -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1-* or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1-*, wherein j represents an integer of 1 to 10; and wherein * represents a site of covalent bonding to Y'.

119. according to the compound described in any one of claims 93 to 113 and 118, wherein L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, - CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-* or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, wherein * indicates the site of covalent attachment to Y'.

120. A compound according to any one of claims 93 to 119, wherein Y' is a group formed by reaction of an electrophilic group with a reactive nucleophilic group present on the cell binding agent.

121. The compound of claim 120, wherein Y' is formed by: Where R 7 and R 8 Each is independently H or C1-C3 alkyl.

122. The compound according to claim 120, wherein Y' is Where R 7 and R 8 Each is independently -H or C1-C3 alkyl, and * indicates the site of covalent attachment to the C.

123. A compound according to any one of claims 93 to 112, wherein Z' is formed by:

124. A compound according to any one of claims 93 to 112, wherein Z' is: Where * indicates the site of covalent attachment to C.

125. according to the compound described in any one of claims 93 to 106, wherein -E-NH-CH 2- has one of the following structures, where * indicates the N-terminus of the peptide covalently linked to Z':

126. A compound according to any one of claims 93 to 106, wherein -Z'-E-NH-CH2- is formed by one of the following structures:

127. A compound according to any one of claims 93 to 106, wherein -Z'-E-NH-CH2- is one of the following structures, wherein * represents the point of connection to the C:

128. The compound of claim 93, wherein D is represented by one of the following structures:

129. The compound of claim 93, wherein D—CH2—NH—E—Z'— is formed by one of the following structures:

130. The compound of any one of claims 93 to 129, wherein W is formed by covalently linking compound W' to C.

131. A compound according to any one of claims 93 to 129, wherein W' is any molecule that can be covalently attached to C.

132. The compound of claim 93, wherein D—CH2—NH—E—Z'— is formed by PL1 and W is formed by PL2.

133. The compound of claim 93, wherein D—CH2—NH—E—Z′— is formed from PL3 and W is formed from PL4.

134. The compound of claim 93, wherein D—CH2—NH—E—Z'— is formed from PL5 and W is formed from PL6.

135. The compound of claim 93, wherein D—CH2—NH—E—Z'— is formed from PL7 and W is formed from PL8.

136. The compound of claim 93, wherein D—CH2—NH—E—Z′— is formed from PL9 and W is formed from PL10.

137. The compound according to claim 93, wherein {D—CH2—NH—E—Z'} p' —C—{W} t is one of the following structures, wherein C is a monoclonal antibody, p' and t are drug to antibody ratio (DAR), and p':t is 1:1 or about 1:1, and p' and t are respectively an average of about 1-7, or an average of about 2, about 3, about 4, about 5, or about 6:

138. A compound according to any one of claims 93 to 137, wherein both p' and t are an average of 4.

139. The compound of any one of claims 93 to 137, wherein p':t is about 1:1, about 1:2, or about 2:

1.

140. A compound according to any one of claims 93 to 137, wherein p':t is 1:1 or 1:2 or 2:

1.

141. A compound according to any one of claims 53 to 140, wherein the cell binding agent is an antibody or an antigen binding fragment thereof.

142. The compound of claim 141, wherein the cell binding agent is a monoclonal antibody or an antigen binding fragment thereof.

143. The compound of claim 93, wherein the compound is trastuzumab-MB0324, 144. The compound of claim 93, wherein the compound is trastuzumab-MB0326, 145. A pharmaceutical composition comprising a compound according to any one of claims 53 to 144.

146. A method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, the method comprising administering to a subject in need thereof a compound according to any one of claims 53 to 144 or a pharmaceutical composition according to claim 145.

147. The method of claim 146, wherein the method is used to treat cancer.

148. The method of claim 147, wherein the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, a central nervous system tumor (CNS) tumor, colon cancer or colorectal cancer, diffuse intrinsic pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood cancer, Hodgkin's lymphoma, cancer of the pancreas, peritoneum, prostate, ovary, kidney, rhabdomyosarcoma, salivary gland, sarcoma, skin, small intestine, squamous cell carcinoma, testicular cancer, thyroid cancer, uterus, or Wilms tumor.

149. The method of claim 148, wherein the cancer is breast cancer.

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