Combination therapy with Anti-LIV-1 antibody-drug conjugate

Abstract

An anti-LIV-1 antibody, an anti-human LIV-1 antibody-drug conjugate, and the combined use thereof with another therapeutic agent, especially the combined use thereof with a PD-1 antagonist.

Description

Combination therapy of anti-LIV-1 antibody-drug conjugate TECHNICAL FIELD

[0001] The present disclosure relates to the field of antibody drugs, and in particular, the present disclosure discloses an anti-LIV-1 antibody, and an anti-LIV-1 antibody-drug conjugate, and the use thereof as an anticancer drug in combination with another therapeutic agent. BACKGROUND

[0002] The statements herein are provided only to aid in the understanding of the present disclosure, and do not necessarily constitute prior art.

[0003] All publications, patents, and patent applications cited in this disclosure are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. To the extent that any definition or usage of a term in such a incorporated publication, patent, or patent application is inconsistent or contrary to the definition of that term provided in this disclosure, the definition of that term provided in this disclosure applies and the definition of the term in the incorporated publication, patent, or patent application does not apply.

[0004] Zinc transporter ZIP6, also known as SLC39A6 or LIV-1, is a multiple transmembrane protein with zinc transporter and metalloproteinase activity. LIV-1 is regulated by estrogen and highly expressed in estrogen receptor-positive breast cancer (Taylor et al., Mol Med, 2007 13(7-8):396-406). Recent studies have found that LIV-1 is highly expressed in various tumor cells such as breast cancer, prostate cancer, pancreatic cancer, cervical cancer, and liver cancer, and is limitedly expressed in normal tissues, thus becoming a promising candidate target for ADC treatment, and is likely to become a prognostic and detection indicator for certain cancers. There are many variants of LIV-1, among which the variant numbered Swiss Prot Q13433 (SEQ ID NO: 25) is a representative for screening anti-LIV-1 antibodies.

[0005] An antibody-drug conjugate (ADC) is a technology that uses the specific recognition ability of antibodies to specific antigens on the surface of tumor cells to accurately deliver anti-tumor drugs (such as cytotoxic agents, cytostatic agents, small molecule chemotherapeutics, etc.) to tumor target cells, make them endocytose and release, and then accurately kill tumors. An antibody-drug conjugate generally consists of three parts: an antibody or antibody-like ligand, a small molecule drug, and a linker that couples the antibody or antibody-like ligand with the drug. Due to the appropriate molecular weight, high stability, strong targeting ability, and small side effects, antibody-drug conjugates have been considered as the most promising anti-tumor drugs.

[0006] There are studies using anti-LIV-1 ADC to treat cancer, for example, an anti-LIV-1 antibody is connected with an anti-cancer drug through a linker (see US RE48,959), but the connection mode disclosed in this patent has the disadvantages of easy falling off and poor stability, which limits the use of the drug and increases the side effects of the drug.

[0007] Therefore, there is an urgent need in the art to provide a new LIV-1-targeted antibody-drug conjugate with improved serum stability and reduced toxicity. SUMMARY

[0008] In one aspect, the present disclosure provides a method of treating a disease, comprising administering to a subject in need thereof an anti-LIV-1 antibody-drug conjugate (sometimes also referred to as "LIV-1-ADC" in the present disclosure) and another therapeutic agent, wherein the anti-LIV-1 antibody-drug conjugate comprises an anti-LIV-1 antibody and a drug.

[0009] In a second aspect, the present disclosure provides use of a combination of agents in the manufacture of a medicament for treating a disease, wherein the combination of agents comprises an anti-LIV-1 antibody-drug conjugate and another therapeutic agent, wherein the anti-LIV-1 antibody-drug conjugate comprises an anti-LIV-1 antibody and a drug.

[0010] In a third aspect, the present disclosure provides use of an anti-LIV-1 antibody-drug conjugate in the manufacture of a medicament for treating a disease in combination with another therapeutic agent.

[0011] In some embodiments, wherein the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0012] a) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 1, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 2;

[0013] b) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 4;

[0014] c) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 5, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 6; or

[0015] d) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 28, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 29;

[0016] The amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat, Chothia, AbM, or IMGT numbering system.

[0017] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0018] the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 1, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 2;

[0019] The amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat, Chothia, AbM, or IMGT numbering system, which are well known to one skilled in the art. In one embodiment, the amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat numbering system.

[0020] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0021] a-1) the heavy chain variable region comprises HCDR1 as set forth in SEQ ID NO: 7, HCDR2 as set forth in SEQ ID NO: 8, and HCDR3 as set forth in SEQ ID NO: 9, and the light chain variable region comprises LCDR1 as set forth in SEQ ID NO: 10, LCDR2 as set forth in SEQ ID NO: 11, and LCDR3 as set forth in SEQ ID NO: 12;

[0022] b-1) the heavy chain variable region comprises HCDR1 as set forth in SEQ ID NO: 13, HCDR2 as set forth in SEQ ID NO: 14, and HCDR3 as set forth in SEQ ID NO: 15, and the light chain variable region comprises LCDR1 as set forth in SEQ ID NO: 16, LCDR2 as set forth in SEQ ID NO: 17, and LCDR3 as set forth in SEQ ID NO: 18;

[0023] c-1) the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 19, HCDR2 set forth in SEQ ID NO: 20, and HCDR3 set forth in SEQ ID NO: 21, and the light chain variable region comprises LCDR1 set forth in SEQ ID NO: 22, LCDR2 set forth in SEQ ID NO: 23, and LCDR3 set forth in SEQ ID NO: 24; or

[0024] d-1) the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 7, HCDR2 set forth in SEQ ID NO: 89, and HCDR3 set forth in SEQ ID NO: 9, and the light chain variable region comprises LCDR1 set forth in SEQ ID NO: 10, LCDR2 set forth in SEQ ID NO: 11, and LCDR3 set forth in SEQ ID NO: 12.

[0025] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0026] the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 7, HCDR2 set forth in SEQ ID NO: 8, and HCDR3 set forth in SEQ ID NO: 9, and the light chain variable region comprises LCDR1 set forth in SEQ ID NO: 10, LCDR2 set forth in SEQ ID NO: 11, and LCDR3 set forth in SEQ ID NO: 12.

[0027] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0028] a-2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 32, or an amino acid sequence having at least 85% sequence identity thereto; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 85% sequence identity thereto;

[0029] b-2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 85% sequence identity thereto; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 85% sequence identity thereto;

[0030] c-2) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 5, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 5; and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 6, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 6; or

[0031] d-2) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 28, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 28; and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 29, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 29.

[0032] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 32, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 32; and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 2.

[0033] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 1; and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 2.

[0034] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 1; and the light chain variable region comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 2.

[0035] In the context of "at least 85%", it means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between any two of the foregoing, which can be an integer or a decimal.

[0036] In particular, the "at least 85% sequence identity" results in at most 15% difference in the amino acid sequence existing in any framework region in the heavy chain variable region or the light chain variable region. Alternatively, the at most 15% difference can exist in any domain or sequence other than CDR. The difference can be caused by deletion, addition or substitution of amino acid at any position, wherein the substitution can be conservative substitution or non-conservative substitution.

[0037] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 1 and a light chain variable region as set forth in SEQ ID NO: 2.

[0038] In some embodiments, the anti-LIV-1 antibody further comprises a constant region. In some embodiments, the heavy chain constant region is of IgGl or IgG4 subtype, and the light chain constant region is of kappa type. In some embodiments, the heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 26 or a variant thereof. In some embodiments, the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 27 or a variant thereof. A "variant of an amino acid sequence" refers to an amino acid sequence having at least 75% sequence identity to the amino acid sequence.

[0039] In some embodiments, the anti-LIV-1 antibody comprises a light chain as set forth in SEQ ID NO: 30 and a heavy chain as set forth in SEQ ID NO: 31.

[0040] In some embodiments, the anti-LIV-1 antibody is an antigen binding fragment, wherein the antigen binding fragment is selected from any one of Fab, scFv, Fv, Fab', F(ab')2, single domain antibody, scFab, linear antibody and multi-specific antibody.

[0041] In some embodiments, the anti-LIV-1 antibody-drug conjugate has a structure as set forth in [Formula I] below:

[0042] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0043] [Formula I]

[0044] wherein:

[0045] Ab is any of the aforementioned anti-LIV-1 antibody;

[0046] (AG)k is a conjugating group, wherein AG is selected from:

[0047] wherein the wavy line indicates the connection to Ab, and k is 0 or 1;

[0048] (L1)x is a first linker group, L1 is -(CH2-)t-C(=O)- or -(NH)j-(CH2CH2O)n-(CH2)q-C(=O)-, wherein x is 0 or 1, t is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; j is an integer from 0 to 4, e.g., 0, 1, 2, 3, and 4, n is an integer from 2 to 8, e.g., 2, 3, 4, 5, 6, 7, and 8, q is an integer from 1 to 6, e.g., 1, 2, 3, 4, 5, and 6; in some embodiments, t is an integer from 1 to 7;

[0049] (OP)m is an enzyme-cleavable oligopeptide, wherein m is 0 or an integer from 2 to 10; in some embodiments, (OP)m is selected from an oligopeptide formed from a combination of valine, citrulline, alanine, glycine, aspartic acid, tyrosine, phenylalanine, proline, isoleucine, lysine, serine, glutamic acid, threonine, or asparagine, optionally, the phenolic hydroxyl of tyrosine or the amide group of asparagine is glycosylated; in some embodiments, the phenolic hydroxyl of tyrosine or the amide group of asparagine is glycosylated with glucuronic acid, N-acetylglucosamine, glucose, or galactose; in some embodiments, (OP)m is a dipeptide, tripeptide, or tetrapeptide;

[0050] (L2)y is a second linker group, wherein L2 is -NH-Ph-CH2-O-C(=O)-, optionally containing a hydroxyl substituent on the phenyl ring (Ph), and y is 0 or 1; in some embodiments, L2 is p-aminobenzyloxy carbonyl or p-hydroxy-m-aminobenzyloxy carbonyl, optionally, the hydroxyl on the phenyl ring of L2 is glycosylated; in some embodiments, the hydroxyl on the phenyl ring of L2 is glycosylated with glucuronic acid, N-acetylglucosamine, glucose, or galactose;

[0051] D is a drug, e.g., a cytotoxic compound, an immunomodulator, an enzyme, or a hormone inhibitor, preferably a cytotoxic compound;

[0052] z is the ratio of drug to antibody, having a value that is an integer or decimal number from 1 to 24; in some embodiments, z is an integer or decimal number from 2 to 8; in some embodiments, z is 3.5; in some embodiments, z is 4; in some embodiments, z is from 3.5 to 4; in some embodiments, z is from 3.5 to 4.5.

[0053] In some embodiments, L1is -(CH2)t-C(=O)-, wherein t is an integer from 1 to 10, preferably an integer from 1 to 7, more preferably 2, 3, 4, or 5; or L1is -(NH)j-(CH2CH2O)n-(CH2)q-C(=O)-, wherein j is an integer from 0 to 4, preferably 0 or 1, n is an integer from 2 to 8, preferably 4, and q is an integer from 1 to 6, preferably 2.

[0054] In some embodiments, L1is -(CH2)2-C(=O)-, -(CH2)3-C(=O)-, -(CH2)4-C(=O)-, or -(CH2)5-C(=O)-.

[0055] In some embodiments, L1is -(CH2CH2O)4-(CH2)2-C(=O)- or -NH-(CH2CH2O)4-(CH2)2-C(=O)-.

[0056] In some embodiments, x is 0, i.e., L1is absent, and (AG)kand (OP)mare directly connected.

[0057] In some embodiments, the enzyme in the "enzyme-cleavable oligopeptide" is cathepsin or β-glucuronidase. In some embodiments, the enzyme in the "enzyme-cleavable oligopeptide" is cathepsin.

[0058] In some embodiments, (OP)mis an oligopeptide selected from the group consisting of valine, citrulline, alanine, glycine, aspartic acid, tyrosine, phenylalanine, proline, isoleucine, lysine, serine, glutamic acid, threonine, or asparagine, for example, a dipeptide, a tripeptide, or a tetrapeptide, optionally, the phenolic hydroxyl group of tyrosine or the amide group of asparagine is glycosylated, preferably glycosylated with glucuronic acid, N-acetylglucosamine (GlcNAc), glucose, or galactose.

[0059] The "glycosylation" refers to the transfer of a glycosyl or oligosaccharide group to a hydroxyl or an amino group of a compound, either by chemical or enzymatic means.

[0060] In some embodiments, glycosylation refers to the structural modification of an amino acid residue in the oligopeptide (OP)min which certain groups, such as the phenolic hydroxyl group of tyrosine or the amide group of asparagine, or a hydroxyl substituent on the benzene ring in L2, are reacted with a sugar.

[0061] In some embodiments, (OP)mis an oligopeptide formed from valine and citrulline. In some embodiments, (OP)mis -valine-citrulline-, a dipeptide.

[0062] In some embodiments, (OP)m is a tripeptide formed by glycine and tyrosine, optionally the phenolic hydroxyl of tyrosine is glycosylated. In some embodiments, (OP)m is a tripeptide of -glycine-glycine-tyrosine-, optionally the phenolic hydroxyl of tyrosine is glycosylated.

[0063] In some embodiments, (OP)m is a dipeptide formed by alanine and asparagine, wherein the amide group of asparagine is glycosylated.

[0064] In some embodiments, m is 0, i.e., (OP)m is absent, and (L1)x and (L2)y are directly linked.

[0065] In some embodiments, y in (L2)y is 1, and L2 is -NH-Ph-CH2-O-C(=O)-, optionally containing a hydroxyl substituent on the phenyl ring (Ph); preferably, L2 is p-aminobenzyloxy carbonyl or p-hydroxy-m-aminobenzyloxy carbonyl, optionally the hydroxyl on the phenyl ring thereof is glycosylated, preferably with glucuronic acid, N-acetylglucosamine (GlcNAc), glucose or galactose.

[0066] In some embodiments, L2 is p-hydroxy-m-aminobenzyloxy carbonyl, the hydroxyl on the phenyl ring thereof is glycosylated with glucuronic acid, N-acetylglucosamine (GlcNAc), glucose or galactose.

[0067] In some embodiments, L2 is p-hydroxy-m-aminobenzyloxy carbonyl.

[0068] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0069] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0070] [Formula I]

[0071] wherein Ab is any of the aforementioned anti-LIV-1 antibodies; AG is a succinimidyl group, L1 is -(CH2-)t-C(=O)-, wherein t is an integer from 1 to 7, (OP)m is -valine-citrulline- dipeptide, -glycine-glycine-tyrosine-tripeptide or -alanine-alanine-asparagine-tripeptide, optionally the phenolic hydroxyl of tyrosine or the amide group of asparagine is glycosylated with glucose, N-acetylglucosamine or galactose; L2 is p-aminobenzyloxy carbonyl (PAB); and k is 1, x is 1, y is 1, D and z are as defined above.

[0072] In some embodiments, AG is succinimidyl, L1 is -(CH2)t-C(=0)-, where t is 3 or 5, and (OP)m is -valine-citrulline-dipeptide.

[0073] In some embodiments, AG is succinimidyl, L1 is -(CH2)t-C(=0)-, where t is 3 or 5, and (OP)m is -valine-citrulline-dipeptide.

[0074] In some embodiments, AG is succinimidyl, L1 is -(CH2)t-C(=0)-, where t is 3 or 5, and (OP)m is -valine-citrulline-dipeptide.

[0075] In some embodiments, AG is succinimidyl, L1 is -(CH2)t-C(=0)-, where t is 3 or 5, and (OP)m is -valine-citrulline-dipeptide.

[0076] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0077] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0078] [Formula I]

[0079] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is -CH2-C(=0)-, L1 is -(NH)j-(CH2CH20)n-(CH2)q-C(=0)-, where j is an integer from 1 to 4, n is an integer from 2 to 8, and q is an integer from 1 to 6; (OP)m is -valine-citrulline-tripeptide or -glycine-glycine-tyrosine-tripeptide, optionally with the phenolic hydroxyl of the tyrosine glycosylated with glucose or galactose; L2 is p-aminobenzyloxycarbonyl (PAB) or hydroxy-m-aminobenzyloxycarbonyl, optionally with the hydroxyl on the phenyl ring glycosylated with glucuronic acid, N-acetylglucosamine (GlcNAc), glucose, or galactose; and k is 1, x is 0 or 1, y is 1, and D and z are as defined above.

[0080] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0081] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0082] [Formula I]

[0083] wherein Ab represents any of the foregoing anti-LIV-1 antibodies; AG is -CH2-C(=O)-, L1 is -(NH)j-(CH2CH2O)n-(CH2)q-C(=O)-, wherein j is an integer between 1 and 4, n is an integer between 2 and 8, and q is an integer between 1 and 6; m is 0; L2 is p-aminobenzyloxycarbonyl (PAB) or hydroxy-m-aminobenzyloxycarbonyl, optionally glycosylated with glucuronic acid, N-acetylglucosamine (GlcNAc), glucose or galactose at the hydroxyl group of the phenyl ring; and k is 1, x is 0 or 1, y is 1, and D and z are as defined above.

[0084] In some embodiments, L1 is -NH-(CH2CH2O)4-(CH2)2-C(=O)-.

[0085] In some embodiments, (OP)m is -valine-citrulline-dipeptide.

[0086] In some embodiments, (OP)m is -glycine-glycine-tyrosine-tripeptide.

[0087] In some embodiments, (OP)m is -glycine-glycine-tyrosine-tripeptide, wherein the phenolic hydroxyl group of the tyrosine is glycosylated with glucose.

[0088] In some embodiments, m is 0, i.e., (OP)m is absent, and (L1)x and (L2)y are directly linked.

[0089] In some embodiments, L2 is p-hydroxy-m-aminobenzyloxycarbonyl, which is glycosylated with glucuronic acid at the hydroxyl group of the phenyl ring.

[0090] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0091] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0092] [Formula I]

[0093] wherein Ab represents any of the foregoing anti-LIV-1 antibodies; AG is -C(=O)-, L1 is -(CH2-)t-C(=O)-, wherein t is an integer between 1 and 7, or L1 is -(NH)j-(CH2CH2O)n-(CH2)q-(C=O)-, wherein j is an integer between 0 and 4, n is an integer between 2 and 8, and q is an integer between 1 and 6; (OP)m is -valine-citrulline-dipeptide, L2 is PAB; and k is 1, x is 1, y is 1, and D and z are as defined above.

[0094] In some embodiments, L1 is -(CH2)2-C(=0)-.

[0095] In some embodiments, L1 is -(CH2)4-C(=0)-.

[0096] In some embodiments, L1 is -(CH2CH2O)4-(CH2)2-C(=0)-.

[0097] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0098] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0099] [Formula I]

[0100] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is -C(=0)-, k is 1; x is 0; (OP)m is -glycine-glycine-tyrosine tripeptide, the phenolic hydroxyl of which tyrosine is glycosylated with glucose; L2 is p-aminobenzyloxycarbonyl, y is 1; D is a drug; and z is a fraction or integer number from 1 to 10, preferably 3.5 to 4.5.

[0101] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0102] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0103] [Formula I]

[0104] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is -C(=0)-, k is 1; x is 0; (OP)m is -glycine-glycine-tyrosine tripeptide, the phenolic hydroxyl of which tyrosine is glycosylated with glucose; L2 is p-aminobenzyloxycarbonyl, y is 1; D is a drug; and z is a fraction or integer number from 1 to 10, preferably 3.5 to 4.5.

[0105] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0106] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0107] [Formula I]

[0108] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is -C(=O)-, k is 1; L1 is -NH-(CH2-CH2-O)4-CH2-CH2-C=O-, x is 1; m is 0; L2 is p-hydroxy-m- aminobenzyloxycarbonyl, the hydroxyl group on the benzene ring of which is glucuronosylated, y is 1; D is a drug; and z is a fraction or integer from 1 to 10, preferably 3.5 to 4.5.

[0109] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0110] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0111] [Formula I]

[0112] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is -C(=O)-, k is 1; L1 is -NH-(CH2-CH2-O)4-CH2-CH2-C=O-, x is 1; m is 0; L2 is p-hydroxy-m- aminobenzyloxycarbonyl, the hydroxyl group on the benzene ring of which is glucuronosylated, y is 1; D is a drug; and z is a fraction or integer from 1 to 10, preferably 3.5 to 4.5.

[0113] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0114] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0115] [Formula I]

[0116] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is -C(=O)-, k is 1; L1 is -NH-(CH2-CH2-O)4-CH2-CH2-C=O-, x is 1; m is 0; L2 is p-hydroxy-m- aminobenzyloxycarbonyl, the hydroxyl group on the benzene ring of which is glucuronosylated, y is 1; D is a drug; and z is a fraction or integer from 1 to 10, preferably 3.5 to 4.5.

[0117] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0118] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0119] [Formula I]

[0120] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is a succinimidyl group, k is 1; L1 is -(CH2)5-C(=0)-, x is 1; (OP)m is -valine-citrulline-dipeptide; L2 is p- aminobenzyloxycarbonyl, y is 1; D is a drug; and z is a fraction or integer number from 1 to 10, preferably 3.5 to 4.5.

[0121] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0122] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0123] [Formula I]

[0124] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is a succinimidyl group, k is 1; L1 is -(CH2)5-C(=0)-, x is 1; (OP)m is -glycine-glycine-tyrosine-tripeptide, wherein the phenolic hydroxyl group of tyrosine is glycosylated with a galactose; L2 is p- aminobenzyloxycarbonyl, y is 1; D is a drug; and z is a fraction or integer number from 1 to 10, preferably 3.5 to 4.5.

[0125] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0126] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0127] [Formula I]

[0128] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is a succinimidyl group, k is 1; L1 is -(CH2)5-C(=0)-, x is 1; (OP)m is -glycine-glycine-tyrosine-tripeptide; L2 is p- aminobenzyloxycarbonyl, y is 1; D is a drug; and z is a fraction or integer number from 1 to 10, preferably 3.5 to 4.5.

[0129] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0130] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0131] [Formula I]

[0132] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is a succinimidyl group, k is 1; L1 is -(CH2)5-C(=0)-, x is 1; (OP)m is a -glycine-glycine-tyrosine- tripeptide wherein the phenolic hydroxyl of the tyrosine is glycosylated with glucose; L2 is p-aminobenzyloxycarbonyl, y is 1; D is a drug; and z is a fraction or integer number from 1 to 10, preferably 3.5 to 4.5.

[0133] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0134] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0135] [Formula I]

[0136] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is a succinimidyl group, k is 1; L1 is -(CH2)3-C(=0)-, x is 1; (OP)m is a -alanine-alanine- asparagine- tripeptide wherein the amide group of the asparagine is glycosylated with glucose; L2 is p-aminobenzyloxycarbonyl, y is 1; D is a drug; and z is a fraction or integer number from 1 to 10, preferably 3.5 to 4.5.

[0137] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0138] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0139] [Formula I]

[0140] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is a succinimidyl group, k is 1; L1 is -(CH2)3-C(=0)-, x is 1; (OP)m is a -alanine-alanine- asparagine- tripeptide wherein the amide group of the asparagine is glycosylated with glucose; L2 is p-aminobenzyloxycarbonyl, y is 1; D is a drug; and z is a fraction or integer number from 1 to 10, preferably 3.5 to 4.5.

[0141] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0142] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0143] [Formula I]

[0144] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is -C(=0)-, k is 1; L1 is -(CH2)4-C(=0)-, x is 1; (OP)m is -valine-arginine-dipeptide; L2 is p- aminobenzyloxycarbonyl, y is 1; D is a drug; and z is a fraction or an integer from 1 to 10, preferably 1 to 2.

[0145] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0146] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0147] [Formula I]

[0148] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is -C(=0)-, k is 1; L1 is -(CH2-CH2-0)4-(CH2)2-C(=0)-, x is 1; (OP)m is -valine-arginine-dipeptide; L2 is p- aminobenzyloxycarbonyl, y is 1; D is a drug; and z is a fraction or an integer from 1 to 10, preferably 1 to 2.

[0149] In some embodiments, -(AG)k-(L1)x-(OP)p-(L2)y- in [Formula I] has the following structure, wherein "1" represents attachment to the anti-LIV-1 antibody Ab and "2" represents attachment to the drug D.

[0150] In some embodiments, the cytotoxic compound is a DNA alkylating agent, a topoisomerase-1 inhibitor, a topoisomerase-2 inhibitor, an RNA polymerase inhibitor, a microtubulin inhibitor (e.g., inhibits polymerization of microtubulin). Alternatively, the cytotoxic compound methylates DNA.

[0151] In some embodiments, the drug is a microtubulin inhibitor or a topoisomerase I inhibitor.

[0152] In some embodiments, the drug is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine (including, but not limited to, DM1, DM4), SN-38, and exatecan.

[0153] In some embodiments, the drug is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), DM1, DM4, SN-38, and exatecan.

[0154] In some embodiments, the drug is monomethyl auristatin E (MMAE).

[0155] In some embodiments, [(AG)k-(L1)x-(OP)m-(L2)y-D] in [Formula I] has the following structure:

[0156] wherein the wavy line indicates attachment to the anti-LIV-1 antibody Ab.

[0157] In some embodiments, [(AG)k-(L1)x-(OP)m-(L2)y-D] in [Formula I] has the following structure:

[0158] wherein the wavy line indicates attachment to the anti-LIV-1 antibody Ab.

[0159] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure:

[0160] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure:

[0161] In some embodiments, the heavy chain variable region of the Ab comprises HCDR1 set forth in SEQ ID NO: 7, HCDR2 set forth in SEQ ID NO: 8, and HCDR3 set forth in SEQ ID NO: 9, and the light chain variable region of the Ab comprises LCDR1 set forth in SEQ ID NO: 10, LCDR2 set forth in SEQ ID NO: 11, and LCDR3 set forth in SEQ ID NO: 12.

[0162] In some embodiments, the heavy chain variable region of the Ab comprises the amino acid sequence of SEQ ID NO: 1, and the light chain variable region of the Ab comprises the amino acid sequence of SEQ ID NO: 2.

[0163] In some embodiments, the Ab comprises a light chain as set forth in SEQ ID NO: 30 and a heavy chain as set forth in SEQ ID NO: 31, and z is a number from 2 to 8, either fractional or integral. In some embodiments, the antibody portion of the antibody-drug conjugate is a multispecific antibody comprising the foregoing anti-LIV-1 antibody. For example, the antibody is a bivalent tetramer comprising two light chains and two heavy chains, wherein the first pair of heavy and light chains comprises a heavy chain variable region and a light chain variable region of the foregoing anti-LIV-1 antibody or antigen-binding fragment thereof, and the second pair of heavy or light chains of the tetramer is capable of binding a non-LIV-1 antigen, wherein the non-LIV-1 antigen is selected from CD3, EGFR, HER2, HER3, PD-L1, c-MET, TROP-2, CEA5, B7-H3, SIRPa, PSMA, ROR1, or CD47, among others.

[0164] In some embodiments, the other therapeutic agent is an antibody or antibody-drug conjugate against a target selected from EGFR, VEGF, VEGFR2, CTLA-4, PD-L1, PD-1, HER2, CD20, CD47, SIRPa, CD73, LAG3, TIGIT, CD27, OX40, ICOS, BTLA, TIM3, BCMA, c-MET, TAA-1 / 2 / 3, HER3, B7H3, B7H4, Claudin 18.2, c-MET, Nectin-4, ROR1, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, PSMA, ROR1, folate receptor-a, mesothelin, ENPP3, Guanylate cyclase C, SLC44A4, NaPi2b, CD70, Mucin 1, STEAP1, Nectin 4, 5T4, SLTRK6, SC-16, P-cadherin, PSMA, Fibronectin extra domain B, Endothelin receptor ETB, Tenascin c, Collagen IV, Periostin, CD30, CD79b, CD22, CD138, CD37, CD33, CD74, CD19, and CD98.

[0165] In some embodiments, the other therapeutic agent is an anti-HER2 antibody or an anti-HER2 antibody-drug conjugate. In some embodiments, the drug of the anti-HER2 antibody-drug conjugate is a cytotoxic compound. In some embodiments, the cytotoxic compound is a microtubulin inhibitor or a topoisomerase I inhibitor. Anti-Her2 antibodies or anti-Her2 antibody-drug conjugates known in the art can be used in combination with the anti-LIV-1 antibody-drug conjugates of the present disclosure.

[0166] In some embodiments, the other therapeutic agent is an anti-Trop2 antibody or an anti-Trop2 antibody-drug conjugate. In some embodiments, the drug of the anti-Trop2 antibody-drug conjugate is a cytotoxic compound. In some embodiments, the cytotoxic compound is a microtubulin inhibitor or a topoisomerase I inhibitor. Anti-Trop2 antibodies or anti-Trop2 antibody-drug conjugates known in the art can be used in combination with the anti-LIV-1 antibody-drug conjugates of the disclosure.

[0167] In some embodiments, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 75; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 76. The amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat, Chothia, AbM, or IMGT numbering system.

[0168] In some embodiments, the anti-Trop2 antibody or anti-Trop2 antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 79; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 80. The amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat, Chothia, AbM, or IMGT numbering system.

[0169] In some embodiments, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as set forth in SEQ ID NO: 90, HCDR2 as set forth in SEQ ID NO: 91, and HCDR3 as set forth in SEQ ID NO: 92, and the light chain variable region comprises LCDR1 as set forth in SEQ ID NO: 93, LCDR2 as set forth in SEQ ID NO: 94, and LCDR3 as set forth in SEQ ID NO: 95.

[0170] In some embodiments, the anti-Trop2 antibody or anti-Trop2 antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 of SEQ ID NO: 83, HCDR2 of SEQ ID NO: 84, and HCDR3 of SEQ ID NO: 85, and the light chain variable region comprises LCDR1 of SEQ ID NO: 86, LCDR2 of SEQ ID NO: 87, and LCDR3 of SEQ ID NO: 88.

[0171] In some embodiments, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain variable region of SEQ ID NO: 75 and a light chain variable region of SEQ ID NO: 76.

[0172] In some embodiments, the anti-Trop2 antibody or anti-Trop2 antibody-drug conjugate comprises a heavy chain variable region of SEQ ID NO: 79 and a light chain variable region of SEQ ID NO: 80.

[0173] In some embodiments, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain of SEQ ID NO: 77 and a light chain of SEQ ID NO: 78.

[0174] In some embodiments, the anti-Trop2 antibody or anti-Trop2 antibody-drug conjugate comprises a heavy chain of SEQ ID NO: 81 and a light chain of SEQ ID NO: 82.

[0175] In some embodiments, the other therapeutic agent is an anti-HER2 antibody-drug conjugate, wherein the drug is a tubulin inhibitor or a topoisomerase I inhibitor. In some embodiments, the tubulin inhibitor includes, but is not limited to, maytansinoids (such as DM1 and DM4), auristatin derivatives (such as MMAE and MMAF). The topoisomerase I inhibitor includes, but is not limited to, camptothecin analogs (such as irinotecan and SN-38), calicheamicin analogs (such as calicheamicin γ1I and N-acetyl-γ1I calicheamicin), and anthramycin derivatives (such as PBD derivatives).

[0176] In some embodiments, the other therapeutic agent is an anti-Trop2 antibody-drug conjugate, wherein the drug is a tubulin inhibitor or a topoisomerase I inhibitor. In some embodiments, the tubulin inhibitor includes, but is not limited to, maytansinoids (such as DM1 and DM4), auristatin derivatives (such as MMAE and MMAF). The topoisomerase I inhibitor includes, but is not limited to, camptothecin analogs (such as irinotecan and SN-38), calicheamicin analogs (such as calicheamicin γ1I and N-acetyl-γ1I calicheamicin), and anthramycin derivatives (such as PBD derivatives).

[0177] In some embodiments, the other therapeutic agent is an anti-HER2 antibody-drug conjugate, wherein the drug is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoid, SN-38, irinotecan, topetecan, and exatecan.

[0178] In some embodiments, the other therapeutic agent is an anti-HER2 antibody-drug conjugate, wherein the drug is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), DM1, DM4, SN-38, irinotecan, topetecan, and exatecan.

[0179] In some embodiments, the other therapeutic agent is an anti-Trop2 antibody-drug conjugate, wherein the drug is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoid, SN-38, irinotecan, topetecan, and exatecan.

[0180] In some embodiments, the other therapeutic agent is an anti-Trop2 antibody-drug conjugate, wherein the drug is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), DM1, DM4, SN-38, irinotecan, topetecan, and exatecan.

[0181] In some embodiments, the anti-HER2 antibody-drug conjugate comprises a topoisomerase I inhibitor. In some embodiments, the topoisomerase I inhibitor is selected from irinotecan, topetecan, and exatecan. In some embodiments, the drug of the anti-HER2 antibody-drug conjugate is exatecan.

[0182] In some embodiments, the anti-Trop-2 antibody-drug conjugate comprises a topoisomerase I inhibitor. In some embodiments, the topoisomerase I inhibitor is selected from irinotecan, topetecan, and SN-38. In some embodiments, the drug of the anti-Trop-2 antibody-drug conjugate is SN-38.

[0183] In some embodiments, the anti-HER2 antibody-drug conjugate has the structure shown below:

[0184] wherein n is a number or an integer from 2 to 8, preferably a number or an integer from 3 to 8, more preferably a number or an integer from 7 to 8, further preferably a number or an integer from 7.5 to 8; most preferably, n is about 8.

[0185] In some embodiments, the anti-Trop2 antibody-drug conjugate has the structure shown below:

[0186] wherein n is a number or an integer from 2 to 8, preferably a number or an integer from 3 to 8, more preferably a number or an integer from 7 to 8, further preferably a number or an integer from 7.5 to 8; most preferably, n is about 8.

[0187] In some embodiments, the other therapeutic agent is an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody is selected from trastuzumab, pertuzumab, zanidatamab, and Zenocutuzumab. The above anti-HER2 antibodies include their respective biosimilar products.

[0188] In some embodiments, the other therapeutic agent is an anti-HER2 antibody-drug conjugate. In some embodiments, the anti-HER2 antibody-drug conjugate is selected from the group consisting of trastuzumab deruxtecan (DS-8201), disitamab vedotin, A166 (trastuzumab botin), T-DM1 (enheresumab), RC48-ADC, SHR-A1811, and combinations thereof. The above anti-HER2 antibody-drug conjugates include their respective biosimilar products.

[0189] In some embodiments, the other therapeutic agent is an anti-Trop2 antibody.

[0190] In some embodiments, the other therapeutic agent is an anti-Trop2 antibody-drug conjugate (Trop-2 ADC).

[0191] Exemplary Trop-2 ADCs useful in the methods provided herein are described in patent applications WO21225892 (Shanghai EscoGent Biotechnology Co., Ltd.; ESG-401, STI-3258), WO22010797 (BiOneCure Therapeutics; BIO-106), CN112237634 (Shanghai Fudan Zhangjiang Biopharmaceutical Co., Ltd.; FDA018-ADC), WO19114666 (Sichuan Kolon Pharmaceutical Co., Ltd.; KLA264), WO22078524 (Hangzhou DAC Biotechnology Co., Ltd.; DAC-002), WO15098999 (Daiichi Sankyo; datopotamab deruxtecan), WO21147993 (Jiangsu Hengrui Medicine Co., Ltd.; SHR-A1921), and WO21052402 (Sichuan Boli Pharmaceutical Co., Ltd.; BL-M02D1).

[0192] In some embodiments, the Trop-2 ADC is selected from sacituzumab govitecan (ImmunoMedics / Gilead), Datopotamab Deruxtecan (DS-1062, Dato-Dxd; Daiichi Sankyo / AstraZeneca), SKB-264 (KL-A264; Sichuan Kelun Pharmaceutical), ESG-401 (Shanghai Sijian Biotech / Levena Biopharma), JS-108 (DAC-002; Junshi Biosciences / Hangzhou Duoxi Biotech), FDA018-ADC (Shanghai Fudan-Zhangjiang Biopharma), BAT-8003 (BaoTaoTai Bio), STI-3258 (Sorrento), OXG-64 (Oncoxx), BDI-4702 (OBI Pharma), BL-M02D1 (Systimmune), Anti-Trop-2 Ab (Mediterrania Theranostic / Legochem), KD-065 (Nanjing Kaidi Biotech), Anti-Trop2 sdAb (Kisoli Biotech), Anti-Trop-2 ADC (Shandong Fangtans Biopharmaceutical), LIV-2008 (LivTech / Yakult Honsha), TROP2-TRACTr (BiTE; Janux), TROP-2-IR700 (Chiome, photosensitizer), TROP2-XPAT (Amunix), GQ-1003 (Genequantum Healthcare, Samsung BioLogics), El-3s (Immunomedics / Gilead, IBC Pharmaceuticals), Humanized Anti-Trop2-SN38 Antibody Conjugate (Shanghai Sijian Biotech, TOT Biopharma), Anti-Trop2 Antibody-CLB-SN-38 conjugate (Shanghai Fudan-Zhangjiang Biopharma), TROP2-Ab8 (Abmart), Trop2-IgG (Nanjing Medical University), 90Y-DTPA-AF650 (Peking University First Hospital), hRS7-CM (SynAffix), and 89Zr-DFO-AF650 (University of Wisconsin-Madison).

[0193] In some embodiments, the anti-Trop2 antibody-drug conjugate is selected from the group consisting of Sacituzumab govitecan, Datopotamab Deruxtecan, SKB-264 (Sichuan Kelun Pharmaceutical), OBI-992 (OBI Pharma), 9MW-2921 (Shanghai Maiwei Biological), MHB-036C (Shanghai Minghui Pharmaceutical), DB-1305 (Ying En Biological), BIO-106 (BiOneCure Therapeutics Inc), SHR-A1921 (Jiangsu Hengrui Medicine), GQ-1010 (Qidem Medicine), LCB-84 (LigaChem Biosciences / Janssen Biotech), JSKN-016 (Kangning Jierui), DXC-1002 (Hangzhou Duoxi Biological), IBI-130 (Sindabio), FZ-AD004 (Shanghai Fudan Zhangjiang Biological Medicine Co., Ltd.), BL-M02D1 (Sichuan Bailitianheng Pharmaceutical Co., Ltd. / Systimmune), RN927C / PF06664178, BAT8008 (Bai'etaibio), JS108 (DAC-002), FDA018, ESG-401 (Shanghai Poetry Jian Biological Technology Co., Ltd.), DB1305, or a combination thereof. The above anti-Trop2 antibody-drug conjugates include their respective biosimilars.

[0194] In some embodiments, the other therapeutic agent is Trastuzumab Deruxtecan (DS-8201), trade name Enhertu, which is an antibody-drug conjugate targeting HER2; the Trastuzumab Deruxtecan includes biosimilars thereof.

[0195] In some embodiments, the other therapeutic agent is Sacituzumab govitecan, trade name The Sacituzumab govitecan includes biosimilars thereof.

[0196] In some embodiments, the other therapeutic agent is an immune checkpoint inhibitor.

[0197] In some embodiments, the immune checkpoint inhibitor is a PD-1 binding antagonist, a PD-L1 binding antagonist, a CD47 binding antagonist, a SIRP a binding antagonist, or a CD73 binding antagonist.

[0198] In some embodiments, the PD-1 binding antagonist is an anti-PD-L1 antibody that inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising a PD-1 binding portion fused to a constant region (such as an Fc region of an immunoglobulin)).

[0199] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. Any of the anti-PD-1 antibodies known in the art can be used in conjunction with the anti-LIV-1 antibody-drug conjugates of the present disclosure.

[0200] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody that comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 35, HCDR2 set forth in SEQ ID NO: 36, and HCDR3 set forth in SEQ ID NO: 37, and the light chain variable region comprises LCDR1 set forth in SEQ ID NO: 38, LCDR2 set forth in SEQ ID NO: 39, and LCDR3 set forth in SEQ ID NO: 40.

[0201] In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 41 and a light chain variable region as set forth in SEQ ID NO: 42.

[0202] In some embodiments, the anti-PD-1 antibody comprises a heavy chain as set forth in SEQ ID NO: 43 and a light chain as set forth in SEQ ID NO: 44.

[0203] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 bispecific antibody. Any of the anti-PD-1 bispecific antibodies known in the art can be used in conjunction with the anti-LIV-1 antibody-drug conjugates of the present disclosure.

[0204] In some embodiments, the PD-1 binding antagonist is selected from pembrolizumab (KEYTRUDA MK-3475, SCH900475), zimberelimab (AB122, GLS-010, WBP-3055), nivolumab (OPDIVO BMS-936558, MDX-1106), cemiplimab (LIBTAYO), atezolizumab (TECENTRIQ, Cimiprizumab (rwlc, REGN-2810), pidilizumab (CT-011), AMG-404, MEDI0680 (AMP-514), spartalizumab (PDR001), tislelizumab (BGB-A317), toripalimab (JS-001), genolimzumab (CBT-501, APL-501, GB 226), camrelizumab (SHR-1210), sintilimab (… IBI-308), dostarlimab (TSR-042, WBP-285), lambrolizumab (MK-3475); sasanlimab (PF-06801591), cetrelimab (JNJ-63723283), serplulimab (HLX-10), retifanlimab (MGA-012), balstilimab (AGEN2034), prolgolimab (BCD 100), budigalimab (ABBV-181), vopratelimab (JTX-4014), Retifanlimab, Cadonilimab, BMS-986213 (Relatlimab + Nivolumab), ivonescimab, geptanolimab, QL-1604 (Iparomlimab), HX-008 (Pucotenlimab), AK-105, CS-1003, BI-754091, LZM-009, Sym-021, BAT-1306, PD1-PIK, tebotelimab (MGD013; PD-1 / LAG-3), RO-7247669 (PD-1 / LAG-3), FS-118 (LAG-3 / PD-L1), RO-7121661 (PD-1 / TIM-3), RG7769 (PD-1 / TIM-3), PF-06936308 (PD-1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), XmAb-20717 (PD-1 / CTLA4), AK-104 (CTLA4 / PD-1), MEDI-5752 (CTLA4 / PD-1), SSGJ-707 (PD-1 / VEGF), MK-2010 (PD-1 / VEGF), JS207 (PD-1 / VEGF), and CTX-10726 (PD-1 / VEGF). The above PD-1 binding antagonists include their respective biosimilar products.

[0205] In some embodiments, the PD-1 binding antagonist is Pembrolizumab. In some embodiments, the PD-1 binding antagonist is ivonescimab.

[0206] In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody that inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising a PD-L1 binding portion fused to a constant region (such as an Fc region of an immunoglobulin)).

[0207] In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. Any of the anti-PD-L1 antibodies known in the art can be used in conjunction with the anti-LIV-1 antibody-drug conjugates of the present disclosure.

[0208] In some embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region comprising HCDR1 set forth in SEQ ID NO: 45, HCDR2 set forth in SEQ ID NO: 46, and HCDR3 set forth in SEQ ID NO: 47, and a light chain variable region comprising LCDR1 set forth in SEQ ID NO: 48, LCDR2 set forth in SEQ ID NO: 49, and LCDR3 set forth in SEQ ID NO: 50.

[0209] In some embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 51 and a light chain variable region as set forth in SEQ ID NO: 52.

[0210] In some embodiments, the anti-PD-L1 antibody comprises a light chain as set forth in SEQ ID NO: 53 and a heavy chain as set forth in SEQ ID NO: 54.

[0211] In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 bispecific antibody. Any of the anti-PD-L1 bispecific antibodies known in the art can be used in conjunction with the anti-LIV-1 antibody-drug conjugates of the present disclosure.

[0212] In some embodiments, the PD-L1 binding antagonist is selected from the group consisting of atezolizumab (TECENTRIQ®) avelumab (BAVENCIO®, MSB0010718C), envafolimab (ASC22), durvalumab (IMFINZI®, MEDI-4736), adebrelimab, BMS-936559 (MDX1105), cosibelimab (CK-301), lodapolimab (LY 3300054), garivulimab (BGB A333), envafolimab (KN035), opucolimab (HLX 20), manelimab (BCD 135), CX-072, CBT-502 (TQB2450), MSB-2311, sugemalimab (CS-1001; WBP3155), A167 (KL-A167, HBM 9167), STI-A1015 (IMC-001), FAZ-053, BMS-936559 (MDX1105), INCB086550, GEN-1046 (PD-L1 / 4-1BB), FPT-155 (CTLA4 / PD-L1 / CD28), M7824 (PD-L1 / TGFβ-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM-3 / PDL1), INBRX-105 (4-1BB / PDL1), GNS-1480 (PD-L1 / EGFR), SCTB-14 (PD-L1 / VEGF), PM8002 (PD-L1 / VEGF), IMM2518 (PD-L1 / VEGF), YBL-008 (PD-L1 / VEGF), and HB0025 (PD-L1 / VEGF). The aforementioned PD-L1 binding antagonists include their respective biosimilar products.

[0213] In some embodiments, the PD-L1 binding antagonist is Atezolizumab.

[0214] In some embodiments, the SIRPα binding antagonist is an anti-SIRPα antibody that inhibits the binding of SIRPα to CD47. In some embodiments, the SIRPα binding antagonist is an immunoadhesin (e.g., an immunoadhesin of a SIRPα moiety or a SIRPα binding portion fused to an Fc region of an immunoglobulin).

[0215] In some embodiments, the SIRPα binding antagonist is an anti-SIRPα antibody. Any of the anti-SIRPα antibodies known in the art can be used in conjunction with the anti-LIV-1 antibody-drug conjugates of the present disclosure.

[0216] In some embodiments, the anti-SIRPa antibody comprises a heavy chain variable region comprising HCDR1 set forth in SEQ ID NO: 55, HCDR2 set forth in SEQ ID NO: 56, and HCDR3 set forth in SEQ ID NO: 57, and a light chain variable region comprising LCDR1 set forth in SEQ ID NO: 58, LCDR2 set forth in SEQ ID NO: 59, and LCDR3 set forth in SEQ ID NO: 60.

[0217] In some embodiments, the anti-SIRPa antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 61 and a light chain variable region as set forth in SEQ ID NO: 62.

[0218] In some embodiments, the anti-SIRPa antibody comprises a heavy chain as set forth in SEQ ID NO: 63 and a light chain as set forth in SEQ ID NO: 64.

[0219] In some embodiments, the SIRPa binding antagonist is an anti-SIRPa antibody, including but not limited to BR105, CC-95251, HCB-101, BI765063, GS-0189, IBI397, BI-770371, APX-700, ES-004, ADU1805, ELA-026, and BYON-4228. The above anti-SIRPa antibodies include their respective biosimilar products.

[0220] In some embodiments, the SIRPa binding antagonist is an anti-SIRPa antibody BR105, wherein BR105 can be prepared by reference to patent application WO2022121980A1.

[0221] Additional anti-SIRPa antibodies for use in the present disclosure include anti-SIRPa antibodies described in the following patent applications: WO200140307, WO2002092784, WO2007133811, WO2009046541, WO2010083253, WO2011076781, WO2013056352, WO2015138600, WO2016179399, WO2016205042, WO2017178653, WO2018026600, WO2018057669, WO2018107058, WO2018190719, WO2018210793, WO2019023347, WO2019042470, WO2019175218, WO2019183266, WO2020013170, WO2020068752, and WO2020088580.

[0222] In some embodiments, the SIRPa binding antagonist is a SIRPa-Fc fusion protein. Any SIRPa-Fc fusion protein known in the art can be used in conjunction with the anti-LIV-1 antibody-drug conjugates of the present disclosure. In some embodiments, the SIRPa-Fc fusion protein is selected from ALX-148 (evorpacept), timdarpacept, TTI-621, TTI-622, JMT601 (CPO107), SL-172154, SIRPa-F8, JMT601 (CPO107), SS002M91, SIRPa-lgG4-Fc-Fc, and hCD172a (SIRPa)-Fc-LIGHT. Exemplary SIRPa-Fc fusion proteins include ALX-148 (also known as evorpacept, described in WO2013109752), timdarpacept, TTI-621 or TTI-622 (described in WO2014094122). The above-mentioned SIRPa-Fc fusion proteins include their respective biosimilars.

[0223] In some embodiments, the SIRPa binding antagonist is selected from the group consisting of: BR105, CC-95251, HCB-101, BI765063, GS-0189, IBI397, BI-770371, APX-700, ES-004, ADU1805, ELA-026, BYON-4228, ALX-148 (evorpacept), timdarpacept, TTI-621, TTI-622, JMT601 (CPO107), SL-172154, SIRPa-F8, JMT601 (CPO107), SS002M91, SIRPa-lgG4-Fc-Fc, and hCD172a (SIRPa)-Fc-LIGHT; including their respective biosimilar products.

[0224] In some embodiments, the CD47 binding antagonist is an anti-CD47 antibody that inhibits the binding of SIRPa to CD47. In some embodiments, the CD47 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising a CD47 binding moiety fused to a constant region (e.g., an Fc region of an immunoglobulin)).

[0225] In some embodiments, the CD47 binding antagonist is an anti-CD47 antibody. Any of the anti-CD47 antibodies known in the art can be used in conjunction with the anti-LIV-1 antibody-drug conjugates of the present disclosure. In some embodiments, the anti-CD47 antibody is selected from the group consisting of IBI-188 (letaplimab), TJC-4 (lemzoparlimab), SHR-1603, HLX-24, LQ-001, IMC-002, ZL-1201, B6H12, GenSci-059 (gentulizumab), TAY-018, PT-240, 1F8-GMCSF, SY-102, and KD-015.

[0226] Anti-CD47 antibodies for use in the present disclosure include anti-CD47 antibodies described in the following patent applications: WO199727873, WO199940940, WO2002092784, WO2005044857, WO2009046541, WO2010070047, WO2011143624, WO2012170250, WO2013109752, WO2013119714, WO2014087248, WO2015191861, WO2016022971, WO2016023040, WO2016024021, WO2016081423, WO2016109415, WO2016141328, WO2016188449, WO2017027422, WO2017049251, WO2017053423, WO2017121771, WO2017194634, WO2017196793, WO2017215585, WO2018075857, WO2018075960, WO2018089508, WO2018095428, WO2018137705, WO2018233575, WO2019027903, WO2019034895, WO2019042119, WO2019042285, WO2019042470, WO2019086573, WO2019108733, WO2019138367, WO2019144895, WO2019157843, WO2019179366, WO2019184912, WO2019185717, WO2019201236, WO2019238012, WO2019241732, WO2020019135, WO2020036977, WO2020043188, and WO2020009725.

[0227] In some embodiments, the CD47 binding antagonist is an anti-CD47 bispecific antibody. Any anti-CD47 bispecific antibody known in the art can be used in conjunction with the anti-LIV-1 antibody-drug conjugates of the present disclosure. Exemplary bispecific antibodies targeting CD47 include, but are not limited to, simridarlimab (IBI-322, CD47 / PD-L1), IMM-0306 (CD47 / CD20), TJ-L1C4 (CD47 / PD-L1), HX-009 (CD47 / PD-1), PMC-122 (CD47 / PD-L1), PT-217, (CD47 / DLL3), IMM-26011 (CD47 / FLT3), IMM-0207 (CD47 / VEGF), IMM-2902 (CD47 / HER2), BH29xx (CD47 / PD-L1), IMM-03 (CD47 / CD20), IMM-2502 (CD47 / PD-L1), HMBD-004B (CD47 / BCMA), and HMBD-004A (CD47 / CD33).

[0228] In some embodiments, the CD47 binding antagonist is selected from the group consisting of magrolimab, lemzoparlimab, letaplimab, ligufalimab, AO-176, simridarlimab (IBI-322), gentulizumab, ZL-1201, IMC-002, SRF-231, CC-90002 (also known as INBRX-103), NI-1701 (also known as TG-1801), STI-6643, SHR-1603, HLX-24, LQ-001, B6H12, TAY-018, PT-240, 1F8-GMCSF, SY-102, and KD-015; including their respective biosimilar products.

[0229] In some embodiments, the CD73 binding antagonist is an anti-CD73 antibody that inhibits CD73 activity, such as 5’-ectonucleotidase activity. In some embodiments, the CD73 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising a CD73 binding moiety fused to a constant region (e.g., an immunoglobulin Fc region)).

[0230] In some embodiments, the CD73 binding antagonist is an anti-CD73 antibody. Any anti-CD73 antibody known in the art can be used in conjunction with the anti-LIV-1 antibody-drug conjugates of the present disclosure.

[0231] In some embodiments, the anti-CD73 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 65, HCDR2 set forth in SEQ ID NO: 66, and HCDR3 set forth in SEQ ID NO: 67, and the light chain variable region comprises LCDR1 set forth in SEQ ID NO: 68, LCDR2 set forth in SEQ ID NO: 69, and LCDR3 set forth in SEQ ID NO: 70.

[0232] In some embodiments, the anti-CD73 antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 71 and a light chain variable region as set forth in SEQ ID NO: 72.

[0233] In some embodiments, the anti-CD73 antibody comprises a heavy chain as set forth in SEQ ID NO: 73 and a light chain as set forth in SEQ ID NO: 74.

[0234] In some embodiments, the CD73-binding antagonist is an anti-CD73 bispecific antibody. Any anti-CD73 bispecific antibody known in the art can be used in combination with the anti-LIV-1 antibody-drug conjugates of the present disclosure.

[0235] In some embodiments, the CD73-binding antagonist is selected from the group consisting of: BR101, oleclumab, mupadolimab, HB-0045, JAB-BX102, AK-119, Sym-024, uliledlimab, and IBI-325; the aforementioned CD73-binding antagonists include their respective biosimilar products.

[0236] In some embodiments, the CD73-binding antagonist is BR101, which can be prepared by reference to WO2020253568A1.

[0237] In some embodiments, the additional therapeutic agent is a chemotherapeutic agent.

[0238] In some embodiments, the chemotherapeutic agent is a platinum, an anthracycline, a taxane, or a nucleoside analogue.

[0239] In some embodiments, the platinum is carboplatin, cisplatin, or oxaliplatin. In some embodiments, the platinum is carboplatin.

[0240] In some embodiments, the anthracycline is doxorubicin (also known as adriamycin). In some embodiments, the doxorubicin includes morpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrolino doxorubicin, liposomal doxorubicin, and deoxydoxorubicin.

[0241] In some embodiments, the taxane is paclitaxel. In some embodiments, the taxane is nab-paclitaxel (e.g., Abraxane®), paclitaxel, or docetaxel. In some embodiments, the taxane is nab-paclitaxel. ) or paclitaxel. In some embodiments, the taxane is nab-paclitaxel In some embodiments, the taxane is paclitaxel.

[0242] In some embodiments, the nucleoside analogue is gemcitabine.

[0243] In some embodiments, the chemotherapeutic agent is carboplatin, which is administered at a dose of between 200 mg / m 2 and 750 mg / m 2 .

[0244] In some embodiments, the chemotherapeutic agent is carboplatin, and is administered by intravenous injection.

[0245] In some embodiments, the chemotherapeutic agent is doxorubicin, which is administered at a dose of between 40 mg / m 2 and 80 mg / m 2 .

[0246] In some embodiments, the chemotherapeutic agent is doxorubicin, and is administered by intravenous injection.

[0247] In some embodiments, the chemotherapeutic agent is paclitaxel, which is administered at a dose of between 100 mg / m 2 and 260 mg / m 2 .

[0248] In some embodiments, the chemotherapeutic agent is paclitaxel, and is administered by intravenous injection.

[0249] In some embodiments, the chemotherapeutic agent is gemcitabine, and is administered by intravenous injection.

[0250] In some embodiments, the chemotherapeutic agent is gemcitabine, which is administered at a dose of between 1000 mg / m 2 and 1250 mg / m 2 .

[0251] In some embodiments, the additional therapeutic agent is an inhibitor of CDK4 and CDK6 (a CDK4 / 6 inhibitor).

[0252] In some embodiments, the CDK4 / 6 inhibitor is selected from the group consisting of ribociclib, abemaciclib, repotrectinib, trilaciclib, lerociclib, avosentan, roncitinib, pyroxicine, Trilaciclib, riviciclib, milciclib, RGB-286638, NSN3106729, PHA-793887, R547, indirubin, NU6102, bohemine, CDK9-IN-7, CGP60474, purvalanol A, PF-06873600, nimbolide, FN-1501, AG-024322, ON123300, G1T28, G1T38, AMG925, SHR-6390, BPI-1178, BPI-16350, FCN437, BEBT-209, Ty-302, TQB-3616, HS-10342, PF-06842874, CS-2002, MM-D37K, CDK4 / 6-IN-2, SU9516, and AT7519.

[0253] In some embodiments, the CDK4 / 6 inhibitor is ribociclib or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK4 / 6 inhibitor is repotrectinib or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK4 / 6 inhibitor is abemaciclib or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK4 / 6 inhibitor is Trilaciclib or a pharmaceutically acceptable salt thereof.

[0254] In some embodiments, the anti-LIV-1 antibody-drug conjugate and the other therapeutic agent are administered simultaneously.

[0255] In some embodiments, the anti-LIV-1 antibody-drug conjugate and the other therapeutic agent are administered sequentially.

[0256] In some embodiments, the anti-LIV-1 antibody-drug conjugate and the other therapeutic agent are administered separately.

[0257] In some embodiments, the disease is a tumor or a cancer.

[0258] In some embodiments, the tumor or cancer is selected from the group consisting of breast cancer, ovarian cancer, melanoma, prostate cancer, endometrial cancer, pancreatic cancer, lung cancer, cervical cancer, squamous cell carcinoma, non-small cell lung cancer, small cell lung cancer, gastric / gastroesophageal junction (GEJ) adenocarcinoma, esophageal squamous cell carcinoma, bladder cancer, hepatocellular cancer, gastric cancer, glioblastoma, renal cell carcinoma, gastrointestinal tumor, colorectal cancer, glioma, mesothelioma, head and neck cancer, head and neck squamous cell carcinoma, esophageal cancer, skin cancer, uterine cancer, triple negative breast cancer, triple positive breast cancer, HER2 positive breast cancer, hormone receptor positive breast cancer, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, and multiple myeloma.

[0259] In some embodiments, the tumor or cancer is selected from the group consisting of breast cancer, ovarian cancer, and melanoma.

[0260] In some embodiments, the tumor or cancer is a solid tumor. In some embodiments, the tumor or cancer is selected from the group consisting of breast cancer, ovarian cancer, melanoma, prostate cancer, endometrial cancer, pancreatic cancer, lung cancer, cervical cancer, squamous cell carcinoma, non-small cell lung cancer, small cell lung cancer, gastric / gastroesophageal junction (GEJ) adenocarcinoma, esophageal squamous cell carcinoma, bladder cancer, hepatocellular cancer, gastric cancer, glioblastoma, renal cell carcinoma, gastrointestinal tumor, colorectal cancer, glioma, mesothelioma, head and neck cancer, head and neck squamous cell carcinoma, esophageal cancer, skin cancer, uterine cancer, triple negative breast cancer, triple positive breast cancer, HER2 positive breast cancer, hormone receptor positive breast cancer, and multiple myeloma.

[0261] In some embodiments, the tumor or cancer is a hematological tumor.

[0262] In some embodiments, the tumor or cancer is selected from the group consisting of non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, acute lymphocytic leukemia, and anaplastic large cell lymphoma.

[0263] In some embodiments, the tumor or cancer is selected from the group consisting of lung cancer, breast cancer, and prostate cancer.

[0264] In some embodiments, the tumor or cancer is breast cancer, in particular triple negative breast cancer.

[0265] In some embodiments, the tumor or cancer is unresectable locally advanced or metastatic (LA / M) triple negative breast cancer (TNBC).

[0266] In a third aspect, the disclosure provides use of an anti-LIV-1 antibody-drug conjugate and another therapeutic agent as a medicament. In some embodiments, use as a medicament in the treatment of a tumor or cancer.

[0267] In a fourth aspect, the present disclosure provides use of an anti-LIV-1 antibody-drug conjugate and another therapeutic agent in the manufacture of a medicament for treating a disease.

[0268] In a fifth aspect, the present disclosure provides use of an anti-LIV-1 antibody-drug conjugate in the manufacture of a medicament for treating a disease in combination with another therapeutic agent.

[0269] In some embodiments, the subject has a tumor comprising one or more LIV-1 expressing cells. In some embodiments, at least about 0.01%, at least about 0.1%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the tumor cells express LIV-1.

[0270] In some embodiments, the subject has a tumor that expresses PD-1 or a ligand of PD-1, such as PD-L1 or PD-L2. Methods of measuring the amount of PD-1, PD-L1, or PD-L2 known in the art are contemplated herein to determine the amount in tumor cells, see, e.g., WO2017 / 210473. In some embodiments, the tumor has a PD-L1 expression level of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0271] In some embodiments, the subject has a tumor that expresses HER2. In some embodiments, the tumor has a HER2 expression amount of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0272] In some embodiments, the subject has a tumor that expresses Trop2. In some embodiments, the tumor has a Trop2 expression amount of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0273] In some embodiments, the subject has a tumor that expresses CD47. In some embodiments, the tumor has a CD47 expression amount of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0274] In some embodiments, the subject has a tumor that expresses CD73. In some embodiments, the tumor has a CD73 expression amount of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0275] Treatment with the combination therapy of an anti-LIVl-ADC and another therapeutic agent, such as an anti-PD-1 antibody, can be further combined with other treatments for the disorder, such as additional chemotherapy, radiation, stem cell therapy, surgery, and the like. Other classes of agents that can be administered with the combination therapy of the disclosure include, for example, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono-, di-, and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemosensitizers, duocarmycins, etoposides, fluoropyrimidines, ionophores, lexitropsins, nitrosoureas, cisplatin, proformers, purine antimetabolites, puromycins, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, aromatase inhibitors (such as fulvestrant), estrogen receptor modulators (such as tamoxifen and toremifene), and the like.

[0276] In some embodiments, treatment with the combination therapy of an anti-LIV-1-ADC and another therapeutic agent further comprises an additional chemotherapeutic agent, including but not limited to carboplatin, doxorubicin, or paclitaxel, mTOR inhibitors such as Everolimus, carboplatin (Bristol Myers Squibb, New York, NY).

[0277] The anti-LIV-1-ADC and another therapeutic agent, such as an anti-PD-1 antibody, are administered to a subject in combination in amounts that inhibit the growth of cancer cells while the subject tolerates. In some embodiments, the therapeutic effect of the combination of an anti-LIV1-ADC and another therapeutic agent, such as an anti-PD-1 antibody, is synergistic or additive. For some combinations, each agent in the combination can be effectively administered in lower amounts than when administered alone.

[0278] In a sixth aspect, the disclosure provides kits for treating a disease. The kits can include a container comprising an anti-LIV-1 antibody-drug conjugate of the disclosure and, optionally, containers comprising one or more of another therapeutic agent, such as an anti-PD-1 antibody, and, optionally, an additional chemotherapeutic agent. If desired, the kits can further include one or more of various conventional pharmaceutical kit components, such as containers with one or more pharmaceutically-acceptable carriers, additional containers, and the like, as will be readily apparent to those in the art. Printed instructions, either as an insert or a label, can also be included in the kits, indicating quantities of components to be administered, guidelines for administration, and / or guidelines for mixing components.

[0279] It should be understood that, in the scope of the present disclosure, each of the technical features of the present disclosure described above and each of the technical features specifically described hereinafter (e.g., in the examples) can be combined with each other to constitute a new or preferred technical solution. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0280] Figure 1A shows the binding activity of antibody A to human LIV-1 detected by ELISA.

[0281] Figure 1B shows the binding activity of antibody B and antibody C to human LIV-1 detected by ELISA.

[0282] Figure 2 shows the binding activity of antibody-drug conjugate A-BrAcMMAE to human LIV-1 detected by ELISA.

[0283] Figure 3 shows the inhibitory activity of ADC on the proliferation of cancer cell line Calu6 cells in vitro.

[0284] Figure 4 shows the inhibitory activity of ADC on the proliferation of human LIV-1 overexpressing cancer cell line MCF7-ATCC-hLIV-1-#7 cells in vitro.

[0285] Figure 5 shows the inhibitory activity of ADC on the proliferation of human LIV-1 overexpressing cancer cell line MCF7-ATCC-hLIV-1-#12 cells in vitro.

[0286] Figure 6 shows the inhibitory activity of ADC on the proliferation of human LIV-1 overexpressing cancer cell line MCF7-ATCC-hLIV-1-#17 cells in vitro.

[0287] Figure 7 shows the results of the experiment of xenotransplanting MCF7 breast cancer cell line into NSG mice. The dose and administration time are shown in the figure.

[0288] Figure 8 shows the results of the experiment of xenotransplanting HCC1806 breast cancer cell line into NSG mice. The dose and administration time are shown in the figure.

[0289] Figure 9 shows the results of the experiment of xenotransplanting PC3 prostate cancer cell line into nude mice. The dose and administration time are shown in the figure.

[0290] Figure 10 shows the results of the experiment of xenotransplanting Calu-6 lung cancer cell line into nude mice. The dose and administration time are shown in the figure.

[0291] Figure 11 shows the results of the experiment of xenotransplanting Calu-6 lung cancer cell line into nude mice. The dose and administration time are shown in the figure.

[0292] Figure 12 shows the results of the xenograft PC3 prostate cancer cell line to nude mice. The dose and administration time are shown.

[0293] Figure 13 shows the results of the xenograft PA-1 ovarian cancer cell line to nude mice. The dose and administration time are shown.

[0294] Figure 14 shows the inhibitory activity of anti-LIV-1 ADC combined with anti-PD-1 antibody on mouse A375 cell transplanted tumor.

[0295] Figure 15 shows the inhibitory activity of anti-LIV-1 ADC combined with anti-PD-L1 antibody on mouse A375 cell transplanted tumor.

[0296] Figure 16 shows the inhibitory activity of anti-LIV-1 ADC combined with anti-PD-1 antibody on mouse HCC1806 cell transplanted tumor.

[0297] Figure 17 shows the killing effect of anti-LIV-1-ADC combined with anti-CD73 antibody on breast cancer cells SKBR3.

[0298] Figure 18 shows the killing effect of anti-LIV-1-ADC combined with anti-SIRPa antibody on SKBR3 cells. * indicates that the combination administration group has statistical difference compared with the anti-LIV-1-ADC single drug group, *p<0.05, **p<0.01, ***p<0.001.

[0299] Figure 19 shows the killing effect of anti-LIV-1-ADC combined with CDK4 / 6 inhibitor Palbociclib on breast cancer cells.

[0300] Figure 20 shows the killing effect of anti-LIV-1-ADC combined with anti-Trop2-ADC on breast cancer cells.

[0301] Figure 21 shows the killing effect of anti-LIV-1-ADC combined with anti-Her2 antibody on breast cancer cells.

[0302] Figures 22A-22C show the killing effect of anti-LIV-1-ADC combined with doxorubicin, gemcitabine or carboplatin on breast cancer cells, respectively. DETAILED DESCRIPTION

[0303] The inventors have provided a combination use of an anti-LIV-1 antibody-drug conjugate and another therapeutic agent, in particular an immune checkpoint inhibitor, for treating a tumor or cancer. In vivo and in vitro experiments have proved that the drug combination of the present disclosure has better killing tumor cell activity and higher inhibitory activity of tumor growth compared with single drug.

[0304] To better appreciate the present disclosure, the following terms are defined.

[0305] Unless otherwise indicated, all singular terms will also include the plural, the present tense will also include the gerund and the past tense.

[0306] Unless otherwise indicated, the term "about" includes values within the standard deviation of the stated value.

[0307] The phrase "consisting essentially of means that the composition and method can include additional ingredients and / or steps, but only if the additional ingredients and / or steps do not materially alter the basic and novel characteristics of the claimed composition or method. The phrase "comprising" is intended to mean that the compositions and methods include the recited steps or components, and that additional steps or components can be included.

[0308] A "subject" or "patient" according to the present disclosure is an animal, including a human patient, in need of anti-cancer treatment or therapy. In certain aspects, the present disclosure can also be applied in veterinary practice to any mammal or other animal in need of such LIV-1 targeted anti-cancer treatment. This can include, for example, non-human primates, canines, felines, swine, equines, and any other animal for which an anti-cancer treatment against LIV-1 is indicated.

[0309] The term "antibody" as used herein is intended to include any known type of natural or engineered antigen binding protein or polypeptide, including at least one antigen-specific variable domain (VL or VH, or analogous domains of engineered functional polypeptide binding domains). The antibody can be polyclonal, monoclonal, or synthetically engineered, such as a humanized monoclonal antibody. Derivatives and fragments of polyclonal and / or monoclonal antibodies are also contemplated for use. These include Fc fragments, Fab fragments, single chain antibodies and polymers thereof, as well as synthetic polypeptides having two or more binding specificities, and the like.

[0310] Antibody fragments (or antigen binding fragments) will generally compete with full antibody for specific binding, and include separate heavy chains, light chains, Fab, Fab', F(ab')2, F(ab)c, diabodies, dabs, nanobodies, and Fv. Antibody fragments can be produced by DNA recombination techniques, or by enzymatic or chemical separation of intact immunoglobulins. The term "antibody" also includes diabodies (homo-dimeric Fv fragments) or minibodies (VL-VH-CH3), bispecific antibodies or the like. Bispecific or bifunctional antibodies are artificial hybrid antibodies having two different heavy / light chain pairs and two different binding sites (see Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). The term "antibody" includes the antibody itself (naked antibody) or the antibody conjugated to a cytotoxic or cytostatic drug, referred to herein as an antibody-drug conjugate or ADC.

[0311] Monoclonal antibodies are typically isolated and purified. This means that the monoclonal antibody is generally at least 50% pure of interfering proteins and other contaminants produced during production or purification, but does not exclude the possibility that the monoclonal antibody is combined with an excess of a pharmaceutically acceptable carrier or other carrier intended to facilitate its use. Sometimes the monoclonal antibody is at least 60%, 70%, 80%, 90%, 95%, or 99% w / w pure of interfering proteins and contaminants produced or purified.

[0312] The basic structural unit of a natural antibody is a tetrameric subunit structure. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region when expressed is associated with a cleavable signal sequence. The variable region without the signal sequence is sometimes referred to as the mature variable region. Thus, for example, light chain mature variable region means the light chain variable region without the light chain signal sequence. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0313] Specific binding of a monoclonal antibody to its target antigen means having at least 10 6 ,10 7 ,10 8 ,10 9 or 10 10 M -1Specific binding is detectable in magnitude to specific functional groups or a particular spatial fit (e.g., lock and key types) resulting in the formation of a bond between the binding site and the target, as opposed to non-specific binding which results from Van der Waals forces. However, specific binding does not necessarily mean that a monoclonal antibody binds one and only one target.

[0314] The light chains are classified as kappa or lambda. The heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. In the light chains and the heavy chains, the variable region and the constant region are joined by a "J" region of about 12 or more amino acids. The heavy chains also include a "D" region of about 10 or more amino acids. (See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989, the entire contents of which are incorporated by reference for all purposes).

[0315] The mature variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites, two binding sites are identical, bifunctional or bispecific antibodies differ. These chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N- to C-terminus, both light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 in the variable region. The assignment of amino acids to each domain follows the Kabat convention, Immunological Protein Sequences (National Institutes of Health, Bethesda, Md., 1987 and 1991, or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989)). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or different light chains are assigned the same number.

[0316] A "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs in a variable domain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The precise amino acid sequence boundaries of each CDR in a given variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment systems, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering with a large number of crystal structures. Correspondence between the various numbering systems is well known to those skilled in the art. For example, CDRs can be derived by using the AbYsis database (www.bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cg).

[0317] In other words, when a CDR sequence under one numbering system and its position in an antibody is provided, the skilled artisan has the ability to determine the corresponding CDR sequence under another numbering system and its position in an antibody. The technical solutions corresponding to different numbering systems are considered as equivalent technical solutions. In one embodiment, the CDRs of the antibodies of the present disclosure are determined according to the Kabat numbering scheme.

[0318] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed both by contiguous amino acids or noncontiguous amino acids juxtaposed in three-dimensional space. Epitopes formed from contiguous amino acids are typically linear epitopes, while epitopes formed by an amino acid sequence that is noncontiguous in sequence but finds its way into the same spatial structure are typically conformational epitopes. A "linear epitope" is formed by at least 3, and more usually, at least 5 or 8-10 amino acids, in a unique spatial conformation. Methods of determining the spatial conformation of an epitope include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0319] Antibodies that recognize the same or overlapping epitopes can be detected by simple immunoassay methods, i.e., by detecting the ability of one antibody to compete with another antibody for binding to the antigen of interest. The epitope to which an antibody binds can also be determined by x-ray crystallography to determine the interacting residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that cause a reduction or elimination of binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies have overlapping epitopes if some, but not all, amino acid mutations in the antigen that cause a reduction or elimination of binding of one antibody reduce or eliminate binding of the other antibody.

[0320] Competition between antibodies can be detected experimentally by measuring the ability of a test antibody to inhibit specific binding of a reference antibody to a common antigen (e.g., Junghans et al., Cancer Res. 50: 1495, 1990). In a competitive binding experiment, a test antibody inhibits binding of a reference antibody by at least 50% when the test antibody and the reference antibody are competing, e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of the test antibody over the reference antibody, but preferably 75%, 90%, or 99% in a competitive binding experiment. Antibodies identified by competition experiments (competing antibodies) include antibodies that bind to the same epitope as the reference antibody, as well as antibodies that bind to an adjacent epitope to the reference antibody.

[0321] To distinguish between conservative and non-conservative substitutions of amino acids, amino acids are classified as follows: Class I (hydrophobic side chains): Met, Ala, Val, Leu, He; Class II (neutral hydrophilic side chains): Cys, Ser, Thr; Class III (acidic side chains): Asp, Glu; Class IV (basic side chains): Asn, Gin, His, Lys, Arg; Class V (residues influencing chain orientation): Gly, Pro; Class VI (aromatic side chains): Trp, Tyr, Phe. Conservative substitutions include substitutions among amino acids within the same class. Non-conservative substitutions are those among amino acids of different classes.

[0322] The percent similarity of the compared sequences of the test antibody and the reference antibody can be determined by aligning the sequences of the antibodies to the greatest extent possible according to the Kabat numbering convention. After alignment, if a region of the test antibody structure (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of the reference antibody, the percent similarity of the compared sequences of the test antibody and the reference antibody is the number of positions in which the amino acids are identical divided by the total number of aligned positions in both regions, not counting gaps, multiplied by 100 to convert to a percentage.

[0323] A composition or method "comprising" one or more stated elements can include other elements not specifically listed. For example, a composition comprising an antibody can also contain the antibody alone or in combination with other ingredients.

[0324] The specification of a range of values includes all integers within or defining the range.

[0325] Antibody effector function refers to functions produced by the Fc domain of Ig. For example, these functions can be antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, or complement-dependent cellular cytotoxicity. For example, binding of the Fc effector domain to Fc receptors on immune cells with phagocytic or lytic activity, or binding of the Fc effector domain to components of the complement system, can produce effector functions. In general, the effects mediated by Fc-binding cells or complement components result in growth inhibition and / or apoptosis of LIV-1 target cell-expressing cells. The Fc region of an antibody can recruit Fc receptor (FcR)-expressing cells and draw them to the side of the target cell bound by the antibody. Cells that express FcRs on their membranes include FcyRIII (CD 16), FcyRII (CD32), and FcyRI (CD64), which can act as effector cells that kill IgG-bound cells. These effector cells include monocytes, macrophages, natural killer cells, neutrophils, and eosinophils. IgG contact with FcyR can activate antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). ADCC is mediated by CD16.sup.+ effector cells through the secretion of membrane pore-forming proteins and proteases, while phagocytosis is mediated by CD32.sup.+ and CD64.sup.+ effector cells (see Fundamental Immunology, 4th Ed., Paul ed., Lippincott-Raven, New York, 1997, Chapters 3, 17, and 30; Uchida et al., 2004, J. Exp. Med. 199:1659-69; Akewanlop et al., 2001, Cancer Res. 61:4061-65; Watanabe et al., 1999, Breast Cancer Res. Treat. 53:199-207). In addition to ADCC and ADCP, the Fc region of a cell-bound antibody can also activate the complement classical pathway, triggering complement-dependent cellular cytotoxicity (CDC). Upon formation of an antibody-antigen complex, Clq of the complement system binds to the Fc region of the antibody. Upon binding of Clq to antibody-bound cells, a cascade can be initiated, including proteolytic activation of C4 and C2 to generate C3 convertase. C3 convertase cleaves C3 to C3b, which can activate terminal complement components, including C5b, C6, C7, C8, and C9. Collectively, these proteins form a membrane attack complex pore on antibody-coated cells. These pores disrupt the integrity of the cell membrane, killing the target cell (see Immunobiology, 6th Ed., Janeway et al., Garland Science, New York, 2005, Chapter 2).

[0326] The term "antibody-dependent cellular cytotoxicity," or ADCC, is a mechanism of inducing cell death that relies on the interaction of antibody-coated target cells with immune cells (also known as effector cells) that have lytic activity. These effector cells include natural killer cells, monocyte / macrophages, and neutrophils. Effector cells attach to the Fc effector domain of Ig, while IgG binds to the target cell through the antigen-binding site. Effector cell activity results in the death of the antibody-coated target cell.

[0327] The term "antibody-dependent cellular phagocytosis," or ADCP, refers to the process by which antibody-coated cells are internalized, in whole or in part, by phagocytic immune cells (such as macrophages, neutrophils, and dendritic cells) that bind to the Fc effector domain of Ig.

[0328] The term "complement-dependent cytotoxicity," or CDC, refers to a mechanism of inducing cell death in which the Fc effector domain of an antibody bound to a target cell activates a series of enzymatic reactions that ultimately form pores in the target cell membrane. Typically, an antigen-antibody complex, such as that formed by an antibody-coated target cell, binds and activates complement component Clq, thereby activating the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of the target cell, promoting ADCC by binding to complement receptors (such as CR3) on leukocytes.

[0329] "Cytotoxic effect" refers to the depletion, elimination, and / or killing of target cells. "Cytotoxic agent" refers to an agent that has a cytotoxic effect on cells. Cytotoxic agents can be conjugated to antibodies or administered in conjunction with antibodies.

[0330] "Cytostatic effect" refers to the inhibition of cell proliferation. "Cytostatic agent" refers to an agent that has a cytostatic effect on cells, thereby inhibiting the growth and / or expansion of a particular cell subpopulation. Cytostatic agents can be conjugated to antibodies or administered in conjunction with antibodies.

[0331] The term "pharmaceutically acceptable" means approved or approvable by a regulatory agency or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "pharmaceutically compatible ingredient" refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or carrier with which an anti-LIV-1 antibody-drug conjugate is combined.

[0332] The phrase "pharmaceutically acceptable salt" refers to an organic or inorganic salt of an anti-LIV-1 antibody or conjugate thereof that is pharmaceutically acceptable, or a formulation with which the anti-LIV-1 antibody-drug conjugate is combined. Exemplary salts include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,l 'dimethylpyrrolidinium 2,3 naphthalenedisulfonate) salts. Pharmaceutically acceptable salts can include another molecule such as an acetate ion, a succinate ion, or other counter ion. The counter ion can be any organic or inorganic moiety that stabilizes the electric charge on the parent compound. In addition, the pharmaceutically acceptable salt can have more than one charged atom in its structure. An example of a pharmaceutically acceptable salt having multiple charged atoms can have more than one counter ion. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counter ions.

[0333] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. The progeny can not be completely identical to the parent cell both in genetic and protein expression makeup, but can contain mutations. The term as used herein includes mutant progeny which have the same functional or biological activity as the cells originally selected or screened for. Host cells include prokaryotic and eukaryotic host cells, with eukaryotic host cells including, but not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, canine, monkey, porcine, goat, bovine, equine, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp., Yarrowia lipolytica, and Neurospora crassa. Host cells of the present patent do not include objects that are not patentably distinct under patent law.

[0334] The term antibody-drug conjugate (ADC) refers to an antibody or antibody fragment linked to a biologically active toxic drug through a linker. The antibody or antibody fragment described in the present disclosure can be conjugated to an effector molecule in any manner. For example, the antibody or antibody fragment can be attached to a toxic drug by chemical or recombinant means. Chemical means of making fusions or conjugates are known in the art. The method for conjugating the antibody or antibody fragment and the drug must be able to link the antibody to the toxic drug without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.

[0335] A cytotoxic drug or cytotoxic compound refers to a substance that inhibits or prevents the function of cells, and / or causes the death or destruction of cells. Cytotoxic drugs can in principle kill tumor cells at sufficiently high concentrations, but due to lack of specificity, they also cause apoptosis of normal cells, leading to serious side effects. Cytotoxic drugs include toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g. At 211 131 125 90 186 188 153 212 32 and Lu), chemotherapeutic drugs, antibiotics and nucleolytic enzymes.

[0336] The antibody of the present disclosure can be conjugated to the cytotoxic drug through a conjugating agent. Examples of the conjugating agent can be any one or several of non-selective conjugating agents, conjugating agents utilizing carboxyl groups, peptide chains, conjugating agents utilizing disulfide bonds. The non-selective conjugating agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde and the like. The conjugating agent utilizing carboxyl groups can be any one or several of aconitic anhydride conjugating agents (such as aconitic anhydride), acylhydrazone conjugating agents (conjugating site is acylhydrazone).

[0337] Certain residues on the antibody (such as Cys or Lys, etc.) are used to link to a variety of functional groups, including imaging agents (e.g. chromophoric groups and fluorescent groups), diagnostic agents (e.g. MRI contrast agents and radioisotopes), stabilizing agents (e.g. ethylene glycol polymers) and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate, the functional agent (e.g. a drug, a detection agent, a stabilizing agent) is conjugated (covalently linked) to the antibody, the functional agent can be directly or indirectly linked to the antibody through a linker.

[0338] ​​​​​​​​Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC comprises a linker (or linker moiety) between the drug and the antibody. The term "linker moiety" or "linking fragment" or "linking unit" refers to a chemical moiety or bond that links one end to an antibody or antigen-binding fragment thereof and the other end to a drug, and can also be linked to other linkers before being linked to a drug. The linker can be a degradable linker or a non-degradable linker. Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g., the linker is degraded at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptide-based linkers that are degradable by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or saccharide linkers, e.g., glucuronide-containing linkers that are degradable by glucuronidases. Peptide-based linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine; or tripeptides, such as glycine-phenylalanine-glycine; or tetrapeptides, such as glycine-glycine-phenylalanine-glycine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that are degradable under reducing conditions (e.g., disulfide linker). Non-degradable linkers are typically released under conditions in which the antibody is hydrolyzed by proteases.

[0339] The linker has a reactive functional group capable of reacting with certain amino acid residues prior to attachment to the antibody, and the attachment is achieved through the reactive functional group. Thiol-specific reactive functional groups are preferred and include, for example, maleimides, haloamides (e.g., iodo-, bromo-, or chloro-); haloesters (e.g., iodo-, bromo-, or chloro-); halo-methyl ketones (e.g., iodo-, bromo-, or chloro-), benzyl halides (e.g., iodo-, bromo-, or chloro-); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-bis-(mercurymethyl) dioxane, and counterions such as acetate, chloride, or nitrate; and polymethylenedimethylthioether sulfonate. The linker can include, for example, a maleimide attached to the antibody via a thio-butyrimidate.

[0340] In the present disclosure, drug-linker compounds can be used to form ADCs in a single step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, a cysteine residue is reacted with a reactive portion of a linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug, thereby forming an ADC.

[0341] Typically, the functional group on the linker is chosen to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkyne group on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and the alkyne. Other useful functional groups include, for example, ketones and aldehydes (for reaction with hydrazides and alkoxy amines), phosphines (for reaction with azides); isocyanates and isothiocyanates (for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimidyl esters (for reaction with amines and alcohols). These and other ligation strategies, such as those described in Bioconjugate Techniques, 2nd Edition (Elsevier), are well known to those skilled in the art. It will be appreciated by those skilled in the art that for selective reaction of the drug moiety and the linker, each member of a complementary pair of reactive functional groups can be used on either the linker or the drug when the complementary pair is chosen.

[0342] The present disclosure also provides methods of making an ADC, which can further comprise: combining an antibody with a drug-linker compound (or linker-drug, LD) under conditions sufficient to form an antibody conjugate (ADC).

[0343] In certain embodiments, the methods of the present disclosure comprise: combining an antibody with a linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present disclosure further comprise: combining the antibody-linker conjugate with a drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0344] Drug loading, also referred to as drug-to-antibody ratio (DAR), is the average number of drug molecules conjugated per antibody in an ADC. It can range, for example, from about 1 to about 10 drug molecules conjugated per antibody, and in certain embodiments, from about 1 to about 8 drug molecules conjugated per antibody, preferably from the range of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8. Illustratively, the drug loading can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 on average. The general formula of the ADCs of the present disclosure includes a collection of antibody-drug conjugates within the aforementioned ranges. In embodiments of the present disclosure, the drug loading can be denoted as z, which is a decimal or an integer. Drug loading can be determined by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC.

[0345] In one embodiment of the present disclosure, the cytotoxic drug is conjugated to the antibody via a linking unit.

[0346] The loading of the ligand drug conjugate can be controlled by the following non-limiting methods, including:

[0347] (1) controlling the molar ratio of the drug linker fragment and the mAb,

[0348] (2) controlling the reaction time and temperature,

[0349] (3) selecting different reaction reagents.

[0350] The present disclosure is illustrated below with reference to specific examples. Those skilled in the art will appreciate that the examples are used to illustrate the present disclosure and do not limit the scope of the present disclosure in any way.

[0351] As used herein, "sugar" refers to a monovalent radical of a monosaccharide (e.g., a pyranose or furanose). The sugar can include a hemiacetal or carboxylic acid (from oxidation of the pendant -CH2OH group). In some embodiments, the sugar is in the beta-D configuration. In some embodiments, the sugar is glucose, glucuronic acid, N-acetylglucosamine, or galactose.

[0352] "glycosylation" refers to the transfer of a glycosyl or oligosaccharide group to a hydroxyl or amino group of a compound, accomplished either chemically or enzymatically. In some embodiments, glycosylation refers to the structural modification of an amino acid residue in an oligopeptide (OP)m by reaction of certain groups therein (e.g., the phenolic hydroxyl of tyrosine or the amide group of asparagine) or a hydroxyl substituent on the phenyl ring in L2 with a carbohydrate.

[0353] The term "hydroxyl" refers to -OH.

[0354] The term "amino" refers to -NH2.

[0355] Dichloromethane (DCM) is abbreviated as DCM.

[0356] N,N-Diisopropylethylamine (DIEA) is abbreviated as DIEA.

[0357] Dimethylformamide (DMF) is abbreviated as DMF.

[0358] O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate is abbreviated as HATU.

[0359] 1-hydroxybenzotriazole is abbreviated as HOBt.

[0360] Ph in the structure refers to a phenyl ring.

[0361] "Substituted" means that one or more hydrogen atoms, preferably 1 to 6, more preferably 1 to 3 hydrogen atoms, of a group are independently of each other replaced with the corresponding number of substituents. It is possible or not possible to substitute, as determined by one skilled in the art (experimentally or theoretically) without undue effort. For example, an amino group with a free hydrogen can not be stable when bound to a carbon atom with an unsaturated (e.g. olefinic) bond.

[0362] "Anti-HER2 antibody" or "HER2 antibody" is an antibody that binds to the HER2 receptor. Optionally, the HER2 antibody further interferes with the activation or function of HER2. Various anti-HER2 antibodies are known in the art. Preferably, such antibodies are monoclonal antibodies. They can be so-called chimeric antibodies, humanized antibodies or fully human antibodies. They can be full-length anti-HER2 antibodies, anti-HER2 antibody fragments with the same biological activity, including amino acid sequence variants and / or glycosylation variants of such antibodies or fragments. Examples of known humanized anti-HER2 antibodies include trastuzumab and pertuzumab.

[0363] Suitable anti-HER2 antibody-drug conjugates are, e.g., trastuzumab deruxtecan (DS-8201), which is an antibody-drug conjugate consisting of huMAb4D5-8 (HERCEPTIN™) and exatecan, which has been approved for the treatment of metastatic breast cancer. Other suitable anti-HER2 antibody-drug conjugates are T-DM1 (ado-trastuzumab emtansine), A166 (trastuzumab botidotin), RC48-ADC and SHR-A1811.

[0364] The terms "trastuzumab", "pertuzumab", "T-DM1" and "DS8201" encompass all corresponding anti-HER2 antibodies that meet the requirements to obtain marketing authorization as the same or a biosimilar product in the countries or regions selected from the group consisting of the United States, Europe and Japan. Trastuzumab has the CDR regions defined in U.S. Patent 5,821,337. Pertuzumab has the CDR regions defined in WO 01 / 00245.

[0365] The term "anti-TROP2 antibody" refers to an antibody that specifically binds to TROP2 (TACSTD2: tumor-associated calcium signal transducer 2; EGP-1) and preferably has internalization activity in TROP2-expressing cells by binding to TROP2. Examples of anti-TROP2 antibodies include hTINA1-H1L1 (International Publication No. WO 2015 / 098099).

[0366] The term“immune checkpoint inhibitor” refers to a therapeutic agent that targets at least one immune checkpoint protein to alter the regulation of an immune response, e.g., downregulate, inhibit, upregulate, or activate an immune response. Immune checkpoint proteins are known in the art and include, but are not limited to, cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), V-domain Ig Suppressor of T-cell Activation (VISTA), B7-H2, B7-H3, B7-H4, B7-H6, 2B4, ICOS, HVEM, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPa (CD47), CD73, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA, IDO, OX40, and A2aR. In some aspects, the immune checkpoint protein can be expressed on the surface of an activated T cell. Therapeutic agents that can be used as immune checkpoint inhibitors for the methods of the disclosure include, but are not limited to, therapeutic agents that target one or more of PD-1, PD-L1, PD-L2, SIRPa (CD47), and CD73. In some aspects, the immune checkpoint inhibitor enhances or inhibits the function of one or more targeted immune checkpoint proteins. In some aspects, the immune checkpoint inhibitor is a PD-L1 / PD-1 binding antagonist, such as Atezolizumab, Pembrolizumab, as described herein.

[0367] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1, PD-L2. In some aspects, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one aspect, a PD-1 binding antagonist reduces the negative co-stimulatory signal mediated by or through signaling through PD-1 mediated by a cell surface protein expressed on T lymphocytes, resulting in less dysfunctional T cell dysfunction (e.g., enhances the response of effectors to antigen recognition). In some aspects, a PD-1 binding antagonist is an anti-PD-1 antibody. In a specific aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab). In another specific aspect, the PD-1 binding antagonist is MK-3475 (pembrolizumab).

[0368] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and B7-1. In some aspects, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some aspects, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and B7-1. In one aspect, a PD-L1 binding antagonist reduces the negative co-stimulatory signal mediated by or through signaling through PD-L1 mediated by a cell surface protein expressed on T lymphocytes, resulting in less dysfunctional T cell dysfunction (e.g., enhances the response of effectors to antigen recognition). In some aspects, a PD-L1 binding antagonist is an anti-PD-L1 antibody. In another specific aspect, the anti-PD-L1 antibody is MPDL3280A (atezolizumab). In a certain specific aspect, the anti-PD-L1 antibody is Atezolizumab.

[0369] A "SIRPa binding antagonist" refers to an agent that specifically binds to SIRPa or CD47 and prevents or reduces the activity of the SIRPa signaling pathway. Examples of "SIRPa binding antagonists" include, but are not limited to, anti-SIRPa antibodies, fusion proteins comprising SIRPa, such as SIRPa-Fc fusion proteins. Tumor cells highly express CD47, and when SIRPa expressed on phagocytic cells that have phagocytic activity binds to CD47 and they interact, a "don't eat me" signal is transmitted to the phagocytic cell. In this way, the tumor cell escapes phagocytosis by the phagocytic cell. Anti-SIRPa antibodies inhibit the binding between SIRPa and CD47, thereby inhibiting the transmission of the "don't eat me" signal from the tumor cell to the phagocytic cell, which enhances the phagocytosis of the phagocytic cell to phagocytose the tumor cell.

[0370] A "CD47 binding antagonist" refers to an agent that specifically binds to CD47 and prevents or reduces the activity of the CD47 signaling pathway. Examples of "CD47 binding antagonists" include, but are not limited to, anti-CD47 antibodies.

[0371] A "CD73 binding antagonist" refers to an agent that specifically binds to CD73 and prevents or reduces the activity of the CD73 signaling pathway, such as inhibiting its 5'-nucleotidase activity. Examples of "CD73 binding antagonists" include, but are not limited to, anti-C73 antibodies.

[0372] A "biosimilar" or "biosimilar product" refers to an antibody having the same primary amino acid sequence as a reference antibody (e.g., trastuzumab) and optionally can have detectable differences in post-translational modifications (e.g., glycosylation and / or phosphorylation) compared to the reference antibody. In some embodiments, a biosimilar is an antibody or antigen-binding fragment thereof having a light chain with the same primary amino acid sequence as a reference antibody (e.g., trastuzumab) and a heavy chain with the same primary amino acid sequence as the reference antibody. In some examples, a biosimilar is an antibody or antigen-binding fragment thereof whose light chain comprises the same light chain variable domain sequence as a reference antibody (e.g., trastuzumab) and whose heavy chain comprises the same heavy chain variable domain sequence as the reference antibody. In some embodiments, a biosimilar can have a similar glycosylation pattern compared to a reference antibody (e.g., trastuzumab). In other embodiments, a biosimilar can have a different glycosylation pattern compared to a reference antibody (e.g., trastuzumab).

[0373] A "chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, platinum, anthracycline antibiotics, nucleoside analogs (purines and pyrimidines), taxanes, camptothecins, epipodophyllotoxins, DNA alkylating agents, folic acid antagonists, vinca alkaloids, ribonucleotide inhibitors, estrogen inhibitors, progesterone inhibitors, androgen inhibitors, aromatase inhibitors, interferons, taxols, dox, 5-fluorouracil, and gemcitabine.

[0374] The term "platinum" as used herein refers to platinum-based chemotherapeutic agents, including but not limited to, cisplatin, carboplatin, and oxaliplatin.

[0375] An "anthracycline antibiotic" is a type of antibiotic that is derived from the fungus Streptococcus peucetius, examples of which include daunorubicin, doxorubicin, epirubicin, and any other anthracycline chemotherapeutic agent.

[0376] "Doxorubicin" is an anthracycline antibiotic. The chemical full name of doxorubicin is (8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)]-7,8,9,10-tetrahydro-6,8,11 - trihydroxy-8-(hydroxyacetyl)-methoxy-5,12-naphthacenedione.

[0377] A "taxane" as used herein is a diterpene that can bind tubulin, promote microtubule assembly and stabilization, and / or prevent microtubule disassembly. Included herein are taxanes that include taxoid 10-deacetylbaccatin III and / or derivatives thereof. Exemplary taxanes include, but are not limited to, paclitaxel (i.e., Taxol® CAS#33069-62-4), docetaxel (i.e., Taxotere® CAS#114977-28-5), larotaxel, cabazitaxel, milataxel, tesetaxel, and / or orataxel. In some embodiments, the taxane is an albumin-coated nanoparticle (e.g., nab-paclitaxel (i.e., Abraxane® ) and / or nab-docetaxel (ABI-008)). In some embodiments, the taxane is nab-paclitaxel In some embodiments, the taxane is formulated in Cremophor® (e.g., Cremophor® ) and / or Tween, such as polysorbate 80 (e.g., Tween® ). In some embodiments, the taxane is a liposome-encapsulated taxane. In some embodiments, the taxane is a prodrug form and / or conjugated form of taxane (e.g., DHA covalently conjugated to paclitaxel, polyglutamated paclitaxel, and / or linoleyl carbonate paclitaxel). In some embodiments, the paclitaxel is formulated substantially free of surfactants (e.g., in the absence of CREMOPHOR and / or TWEEN, such as paclitaxel).

[0378] The term "CDK4 / 6 inhibitor" refers to a compound capable of negatively regulating or inhibiting all or part of the enzymatic activity of CDK4 and / or 6. And the CDK4 / 6 is used throughout the specification to refer to both CDK4 and CDK6. As long as the compound is able to block the formation of CDK4 / 6-cyclin D complex and stop cell division, it can be used as a CDK4 / 6 inhibitor without limitation, exemplarily, it can be abemaciclib, ribociclib, palbociclib trilaciclib, PF-06873600 or a pharmaceutically acceptable salt thereof.

[0379] The experimental methods in the following examples are all routine methods unless otherwise specified. The raw materials, reagents and materials used in the following examples are all commercially available products unless otherwise specified.

[0380] Example 1 Preparation of Anti-LIV-1 Antibodies

[0381] The present disclosure relates to methods of preparing antibodies in mice immunized with a fragment of the extracellular region of human LIV-1, and methods of humanizing murine antibodies against human LIV-1. The murine antibodies are prepared by injecting mice with a composition comprising a fragment of the extracellular region of human LIV-1, identifying the presence of antibodies by taking serum samples. Lymphocytes in the lymph nodes of the mice are obtained, specific B cells against the human LIV-1 antigen are enriched, single B cell sequencing is performed and antibody sequences are analyzed.

[0382] After obtaining the antibody sequences, the antibodies can be prepared using recombinant technology, and chimerization and humanization of the murine antibodies can be performed. Specifically, the mouse complementarity determining regions are transferred from the heavy and light chain variable regions of the mouse immunoglobulin to the corresponding variable regions of the human antibody, and then human residues in the framework region are replaced with murine analogs to prepare humanized antibodies. During the preparation of humanized antibodies, the CDR regions can be optionally affinity matured or hotspot sites can be removed to improve certain aspects of the performance of the antibodies.

[0383] The antibodies prepared in this example include monoclonal antibodies A, B, and C, wherein antibody A is a humanized antibody, and antibodies B and C are chimeric antibodies. The mVH sequence of the murine antibody of antibody A is shown in SEQ ID NO: 28, and the mVL sequence is shown in SEQ ID NO: 29; the VH sequence of antibody A is shown in SEQ ID NO: 1, and the VL sequence is shown in SEQ ID NO: 2; antibody A-1 is obtained by humanizing the murine antibody of antibody A, and has the same CDR sequences as antibody A, the VH sequence of antibody A-1 is shown in SEQ ID NO: 32, and the VL sequence is shown in SEQ ID NO: 2; the VH sequence of antibody B is shown in SEQ ID NO: 3, and the VL sequence is shown in SEQ ID NO: 4; the VH sequence of antibody C is shown in SEQ ID NO: 5, and the VL sequence is shown in SEQ ID NO: 6. The heavy chain constant region sequence of each antibody is shown in SEQ ID NO: 26, and the light chain constant region sequence is shown in SEQ ID NO: 27.

[0384] Further, the binding affinity of the obtained antibodies to human LIV-1 antigen (with His tag, amino acid sequence see NP_036451.1, comprising amino acids at positions Phe229-Ile323) was detected. The results showed that the binding activity of antibody A to human LIV-1 was significantly improved after humanization, as shown in Table 1. The binding affinity was detected by ForteBio.

[0385] Table 1. Binding activity of anti-LIV-1 antibodies to LIV-1 protein

[0386] Example 2 Detection of the binding ability of anti-LIV-1 antibodies to LIV-1 protein and LIV-1 expressing cell lines

[0387] 2.1 Binding of anti-LIV-1 antibodies to LIV-1 protein

[0388] In this experiment, enzyme-linked immunosorbent assay (ELISA) was used to link LIV-1 antigen to a solid support, and the antibody to be detected in the sample was combined with it to form a solid-phase antigen-antibody complex, then an enzyme-labeled secondary antibody was combined with the antibody in the solid-phase immune complex to form a solid-phase antigen-antibody-enzyme-labeled secondary antibody complex, and the degree of color development after adding the substrate was determined, and the absorbance value was positively correlated with the binding activity of the antibody.

[0389] Specifically, hLIV-1 (with His tag, amino acid sequence see NP_036451.1, comprising amino acids at positions Phe229-Ile323) was diluted with coating buffer to 2 μg / mL, added to an enzyme-labeled plate, and coated at 4°C for 15-20 hours. Then 300 μL of blocking solution was added to each well, and blocked at room temperature for 1 hour. The antibody to be tested was diluted with diluent, and incubated at room temperature for 1 hour. The goat anti-human Fc domain secondary antibody (Jackson Immune, Cat#109-035-170) was diluted with diluent, and incubated at room temperature for 1 hour. After completion of the secondary antibody incubation, TMB color developing liquid was used to incubate at room temperature for 1-10 minutes. After color development was completed, 50 μL of stop solution (4 M sulfuric acid) was added to each well to terminate the substrate reaction. The absorbance was measured using an enzyme-labeled instrument, and the absorbance value of each well was read at a detection wavelength of 450 nm. The Prism software was used to analyze the data, with the concentration of the antibody to be tested as the abscissa, the average value of the absorbance as the ordinate, and the dose-response curve was plotted using the Sigmoidal, 4PL four-parameter equation. Equation:

[0390] Y = Bottom + (X^Hillslope)*(Top-Bottom) / (X^HillSlope+EC50^HillSlope).

[0391] The results are shown in FIGS. 1A and 1B, and the anti-LIV-1 antibody of the present disclosure can specifically bind to human LIV-1 antigen.

[0392] 2.2 Binding of anti-LIV-1 antibody to LIV-1-expressing cell lines

[0393] The flow cytometry was used to detect the specific binding ability of anti-hLIV-1 antibody A to cell surface-expressed LIV-1 antigen.

[0394] The cell lines selected in this experiment were: Calu-6 (ATCC HTB-56, anaplastic lung cancer), HCC1806 (ATCC CRL-2335, human breast cancer TNM stage IIB level 2), PC-3 (ATCC CRL-1435, prostate cancer), MCF7 (ATCC HTB-22, breast cancer metastatic site) overexpressing LIV-1, i.e., MCF7-ATCC-LIV-1 #7, #12, #17. The Calu6 and HCC1806 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, the PC-3 cells were cultured in F12K medium containing 10% fetal bovine serum, and the MCF7-ATCC-LIV-1 overexpression cell lines were cultured in EMEM medium containing 10% fetal bovine serum and 1 μg / mL puromycin at 37°C in a 5% CO2 incubator. After each cell line was dissociated and washed in staining buffer (Biolengend), the number of cells was counted and adjusted to 2 x 105 Cells / 100 μL staining buffer. Then, antibody A was added to a final concentration of 10 μg / mL, and the reaction was carried out at 4 °C for 15 minutes. After the reaction, the cells were washed in staining buffer, and then the PE-labeled constant region (Fc) specific antibody (rabbit anti-human IgG PE conjugate, BioLegend, 410707) was resuspended in 2 μL / 2 × 10⁻⁶ cells / 100 μL staining buffer. 5 Cells were incubated at 100 μL PBS and reacted at 4°C for 15 minutes. After the reaction, cells were washed in staining buffer and single-cell readings on the PE channel were analyzed using a Novocyte 3000 (Agilent) device. Negative controls were treated with a non-specific isotype control commercial antibody IgG (BioXCell, BE0297) followed by treatment with a PE-labeled constant region (Fc)-specific antibody. To compare the binding of anti-LIV-1 antibody A to different cancer cells, the quotient of the shifted reading of the experimental group treated with antibody A as disclosed herein divided by the shifted reading of the control group is expressed as the MFI ratio: MFI of anti-LIV-1 antibody / MFI of control antibody. The experimental results are shown in Table 2.

[0395] Experiments have demonstrated and confirmed that the anti-LIV-1 antibody disclosed herein specifically binds to LIV-1 expressed in various cancer cell lines of breast cancer, lung cancer, and prostate cancer.

[0396] Table 2. Staining readings of antibody A binding to various cell lines

[0397] Example 3: Synthesis of compounds used to prepare antibody-drug conjugates

[0398] In this embodiment, experimental methods without specific conditions are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Reagents without a specific source are commercially available, standard reagents.

[0399] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰ ohms. -6 The unit (ppm) is given. 1 The H NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer, with deuterated dimethyl sulfoxide as the solvent and tetramethylsilane (TMS) as the internal standard.

[0400] MS was determined using a Shimadzu LCMS-2020 Single Quadrupole liquid chromatography-mass spectrometry system (manufacturer: Shimadzu, MS model: 2020 Single Quadrupole MS).

[0401] RP-HPLC analysis was determined using Shimadzu LC-2030c Plus liquid chromatograph, preparation was using Shimadzu Nexera liquid preparation system, preparation column: Phenomenex Gemini NX 5μ, C18, 150x50mm, mobile phase: 0.1% trifluoroacetic acid in water / 0.1% trifluoroacetic acid in acetonitrile (ACN). 150x50mm, mobile phase: 0.1% trifluoroacetic acid in water / 0.1% trifluoroacetic acid in acetonitrile (ACN).

[0402] Silica gel column chromatography used SiliCycle (Canada) 200-300 mesh silica gel as carrier.

[0403] 3.1 Synthesis of compound 3

[0404] The synthesis process is as follows:

[0405] To a solution of compound 1 (110 mg, purchased from MedChemExpress, Catalog No: HY-100374) in anhydrous DMF (2 mL) was added glutaric anhydride (12 mg), followed by DIEA (0.04 mL). The reaction was stirred at room temperature for 1 h, then diluted with DCM (10 mL). Pentafluorophenol (90 mg) and (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) (100 mg) were added, and the mixture was stirred at room temperature. After 30 min, the reaction was concentrated under reduced pressure, and the residue was purified by RP-HPLC to give compound 3 as a white powder (102 mg).

[0406] MS determination: m / z 1403.6 [M+H] + .

[0407] 3.2 Synthesis of compound 4

[0408] The synthesis process is as follows:

[0409] To a solution of compound 1 (110 mg) in anhydrous DMF (2 mL) was added DIEA (40 μL) and adipic acid bis-pentafluorophenol ester (200 mg). The reaction was stirred at room temperature for 30 min, and the mixture was directly purified by RP-HPLC to give compound 4 as a white powder (89 mg).

[0410] MS determination: m / z 1417.6 [M+H] + .

[0411] 3.3 Synthesis of compound 5

[0412] The synthesis process is as follows:

[0413] To a solution of compound 1 (110 mg) in anhydrous DMF (2 mL) was added DIEA (40 pL) and PEG3 diacid bis pentafluorophenol ester (300 mg). The reaction was stirred at room temperature for 30 min, the mixture was directly purified by RP-HPLC to give compound 5 as a white powder (105 mg).

[0414] MS determination: m / z 1565.8 [M+H] + .

[0415] 3.4 Synthesis of compound 10

[0416] The synthesis proceeded as follows:

[0417] To a solution of compound 6 (65 mg) and MMAE (72 mg, purchased from MedChemExpress, Cat# HY-15162) in anhydrous DMF (2 mL) was added DIEA (0.02 mL) followed by 1-hydroxybenzotriazole (HOBt) (3 mg). The mixture was stirred at room temperature for 18 h, then diluted with water (20 mL). The mixture was extracted with ethyl acetate (40 mL), the organic layer was dried over Na2S04, concentrated to dryness under reduced pressure to give crude compound 7, which was dissolved in MeOH (2 mL). Zinc powder (200 mg) was added followed by formic acid (0.2 mL). The mixture was stirred at room temperature for 30 min. The solid was removed by filtration, the filtrate was directly purified by RP-HPLC to give compound 8 as a white powder (72 mg).

[0418] Compound 8 (70 mg) was dissolved in acetonitrile / water (6 / 4, v / v), to this solution was added NaOH (aq., 1 M, 0.35 mL) and stirred at room temperature. After 1 h, bromoacetic anhydride (52 mg) was added followed by sodium hydroxide (aq. 1 M, 0.2 mL). After 30 min, the mixture was directly purified by RP-HPLC to give compound 10 as a white powder (38 mg).

[0419] MS determination: m / z 1179.6 [M+H] + .

[0420] 3.5 Synthesis of compound 11

[0421] The synthesis proceeded as follows:

[0422] To a solution of compound 1 (25 mg) in acetonitrile / water (6 / 4, v / v, 2 mL) was added saturated NaHC03solution (0.02 mL) and bromoacetic anhydride (10 mg). The reaction was stirred at room temperature for 10 min, and the mixture was directly purified by RP-HPLC to give compound 11 as a white powder (21 mg).

[0423] MS determination: m / z 1243.6 [M+H] + .

[0424] 1 H NMR (400 MHz, DMSO-d6) δ 10.00 - 10.01 (m, 1H), 8.02 - 8.32 (m, 3H), 7.56 - 7.89 (m, 3H), 7.15 - 7.35 (m, 7H), 5.97 (t, J = 6.0 Hz, 1H), 5.33 - 5.41 (m, 3H), 4.96 - 5.09 (m, 2H), 4.63 - 4.73 (m, 1H), 4.48 - 4.50 (m, 1H), 4.36 - 4.45 (m, 2H), 4.23 - 4.28 (m, 2H), 3.91 - 4.04 (m, 4H), 3.47 - 3.79 (m, 2H), 3.11 - 3.36 (m, 9H), 2.85 - 3.08 (m, 7H), 2.39 - 2.43 (m, 1H), 2.26 - 2.30 (m, 1H), 1.17 - 2.14 (m, 15H), 0.73 - 1.05 (m, 30H).

[0425] 3.6 Synthesis of compound 22 (MC-VC-PAB-MMAE)

[0426] The synthesis procedure is as follows:

[0427] To a solution of compound 1 (62 mg) in anhydrous DMF (2 mL) was added maleimide caproic acid (12 mg), followed by DIEA (0.02 mL) and HATU (20 mg). The reaction mixture was stirred at room temperature (22 °C). After 15 min, the crude reaction mixture was directly purified by RP-HPLC to give compound 22 as a white solid (62 mg, TFA salt) after lyophilization.

[0428] MS determination: m / z 1316.8 [M+H] + .

[0429] 3.7 Synthesis of compound 29 (MC-GGY-PAB-MMAE)

[0430] The synthesis procedure is as follows:

[0431] To a solution of compound 23 (Combi Block, 460 mg) and p-aminobenzyl alcohol (130 mg) in DMF (5 mL) was added DIEA (0.35 mL) followed by HATU (390 mg). The reaction mixture was stirred at room temperature for 20 minutes and then diluted with EtOAc (100 mL). The mixture was washed with 0.5 M hydrochloric acid (50 mL) and water (50 mL). The organic layer was dried (over Na2S04) and evaporated to dryness under reduced pressure. The residue was triturated with diethyl ether / hexane (1 / 1, v / v, 100 mL) to give crude compound 24 as a light brown solid which was dissolved in DMF (5 mL). Diisopropylamine (5 mL) was added and the mixture was stirred at room temperature for 2 hours. The reaction was then concentrated under reduced pressure to about 4 mL. Fmoc-Gly-Gly-OH (fluorenylmethoxycarbonyl glycine-glycine, 360 mg) and DIEA (0.35 mL) were added followed by HATU (0.4 g) and the mixture was stirred at room temperature. After 20 minutes the reaction was diluted with EtOAc (120 mL). The mixture was washed with 0.5 M hydrochloric acid (50 mL) and water (50 mL). The organic layer was dried (over Na2S04) and evaporated to dryness under reduced pressure. The crude product was purified by RP-HPLC to give compound 25 as a white solid (550 mg).

[0432] Compound 25 (340 mg) was dissolved in DMF (4 mL). To this solution was added DIEA (0.08 mL) and bis-p-nitrophenyl carbonate (bis-PNP carbonate, 300 mg) and the mixture was stirred at room temperature for 16 hours. The reaction was purified directly by RP-HPLC to give compound 26 as a white solid (335 mg).

[0433] To a solution of compound 26 (84 mg) and MMAE (0.072 g) in DMF (3 mL) was added DIEA (0.02 mL) and the reaction was stirred at room temperature for 24 hours. Piperidine (0.15 mL) was added. After 30 minutes the mixture was purified directly by RP-HPLC to give compound 27 as a white solid (TFA salt, 92 mg) which was treated with TFA / DCM (1 / 2, v / v, 3 mL) at room temperature for 40 minutes. The mixture was concentrated under reduced pressure to give a crude product which was purified by RP-HPLC to give compound 28 as a white solid (TFA salt, 64 mg).

[0434] To a solution of compound 28 (25 mg, TFA salt) in DMF (2 mL) was added 6-maleimidohexanoic acid N-hydroxysuccinimidyl ester (Sigma Aldrich, 9 mg) followed by DIEA (0.007 mL). The mixture was stirred at room temperature. After 30 minutes, the mixture was purified by RP-HPLC to give compound 29 as a white solid (22 mg).

[0435] MS: m / z 1337.8 [M+H] + .

[0436] 3.8 Synthesis of compound 39 (MC-GGY(Gal)-PAB-MMAE)

[0437] The synthesis proceeded as follows:

[0438] To a stirred solution of acetyl bromide-a-D-galactose (compound 31, CombiBlocks, 2.3 g) and Fmoc-Tyr-OtBu (fluorenylmethoxycarbonyl-tyrosine tert-butyl ester, compound 30, 2.1 g) in dry acetonitrile (50 mL) was added Ag2O (8 mmol) at 0 °C under an argon atmosphere. The solution was stirred at room temperature for 4 hours. The mixture was filtered and the filtrate was evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 32 as a white solid (2.7 g).

[0439] Compound 32 (2 g) was re-dissolved in trifluoroacetic acid / dichloromethane (TFA / DCM, 1 / 1, v / v, 40 mL). After 30 min, the mixture was diluted with DCM (100 mL) and washed with water (40 mL x 4). The organic layer was evaporated to dryness to give compound 33, which was dissolved in DMF (30 mL). To this solution was added p-aminobenzyl alcohol (0.34 g), followed by DIEA (1 mL) and HATU (1 g). The reaction mixture was stirred at room temperature for 20 min, then diluted with EtOAc (120 mL). The mixture was washed with 0.5 M hydrochloric acid (100 mL) and water (50 mL). The organic layer was dried (over Na2S04) and evaporated to dryness under reduced pressure. The residue was triturated with hexane / ether (1 / 1, 70 mL) to give crude compound 34 as a light yellow solid, which was dissolved in DMF (20 mL). Diisopropylamine (20 mL) was added, and the mixture was stirred at room temperature for 2 h. The reaction was then concentrated under reduced pressure to about 20 mL and diluted with DMF (20 mL). Fmoc-Gly-Gly-OH (0.9 g) and DIEA (0.9 mL) were added, followed by HATU (1.0 g), and the mixture was stirred at room temperature. After 20 min, the reaction mixture was diluted with EtOAc (80 mL). The mixture was washed with 0.5 M hydrochloric acid (80 mL) and water (100 mL). The organic layer was dried (over Na2S04) and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography to give compound 35 as a light yellow solid (1.7 g).

[0440] Compound 35 (1.2 g) was dissolved in DMF (10 mL). To this solution was added DIEA (0.2 mL) and bis-PNP carbonate (0.8 g), and the mixture was stirred at room temperature for 16 h. The reaction was then diluted with EtOAc (100 mL) and washed with water (3 x 50 mL). The organic layer was dried (over Na2S04) and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography to give compound 36 as a white solid (1.2 g).

[0441] To a solution of compound 36 (0.12 g) and MMAE (0.072 g) in DMF (2 mL) was added DIEA (0.018 mL) and the reaction was stirred at room temperature for 24 hours. The mixture was then diluted with EtOAc (30 mL) and washed with hydrochloric acid (0.5 M, 30 mL). The organic layer was washed with water (20 mL), dried (over Na2S04) and evaporated to dryness under reduced pressure to give crude compound 37 which was re-dissolved in MeOH (3 mL) and MeONa (4.4 M in MeOH, 0.1 mL) was added. The reaction mixture was stirred at room temperature for 2 hours and then neutralised with 1 N hydrochloric acid (0.5 mL). The mixture was purified directly by RP-HPLC to give compound 38 as a white solid (TFA salt, 75 mg).

[0442] To a solution of compound 37 (28 mg, TFA salt) in DMF (2 mL) was added 6-maleimidohexanoic acid N-hydroxysuccinimidyl ester (Sigma Aldrich, 8 mg) followed by DIEA (0.007 mL). The mixture was stirred at room temperature. After 30 minutes the mixture was purified by RP-HPLC to give compound 39 as a white solid (23 mg).

[0443] MS: m / z 1499.9 [M+H] + .

[0444] 3.9 Synthesis of compound 48 (MC-GGY-(Glc)-PAB-MMAE)

[0445] The synthesis proceeded as follows:

[0446] To a stirred solution of ethyl bromide-a-D-glucose (compound 40, 4.6 g) and Fmoc-Tyr-OtBu (fluorenylmethoxycarbonyl-tyrosine tert-butyl ester, compound 30, 4.1 g) in dry acetonitrile (100 mL) was added Ag20 (15 mmol) under argon pressure at 0 °C and the solution was stirred at room temperature for 4 hours. The mixture was filtered and the filtrate was evaporated to dryness under reduced pressure. Purification by silica gel column chromatography gave compound 41 as a white solid (5.3 g).

[0447] Compound 41 (4 g) was re-dissolved in TFA / DCM (1 / 1, v / v, 80 mL). After 30 min, the mixture was diluted with DCM (200 mL) and washed with water (50 mL x 4). The organic layer was evaporated to dryness to give the crude acid, compound 42, which was dissolved in DMF (50 mL). To this solution was added p-aminobenzyl alcohol (0.65 g), followed by DIEA (1.8 mL) and HATU (1.9 g). The reaction mixture was stirred at room temperature for 20 min, then diluted with EtOAc (200 mL). The mixture was washed with 0.5 M hydrochloric acid (100 mL) and water (150 mL). The organic layer was dried (over Na2S04) and evaporated to dryness under reduced pressure. The residue was triturated with hexane / ether (1 / 1, 100 mL) to give the crude compound 43 as a tan solid, which was dissolved in DMF (30 mL). Diisopropylamine (30 mL) was added, and the mixture was stirred at room temperature for 2 h. The reaction was then concentrated under reduced pressure to about 20 mL and diluted with DMF (30 mL). Fmoc-Gly-Gly-OH (1.8 g) and DIEA (1.8 mL) were added, followed by HATU (1.9 g), and the mixture was stirred at room temperature. After 20 min, the reaction mixture was diluted with EtOAc (150 mL). The mixture was washed with 0.5 M hydrochloric acid (150 mL) and water (200 mL). The organic layer was dried (over Na2S04) and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography to give compound 44 as a light yellow solid (3.2 g).

[0448] Compound 44 (2.4 g) was dissolved in DMF (20 mL). To this solution was added DIEA (0.5 mL) and bis(nitrophenyl)carbonate (1.5 g), and the mixture was stirred at room temperature for 16 h. The reaction was then diluted with EtOAc (200 mL) and washed with water (3 x 100 mL). The organic layer was dried (over Na2S04) and evaporated to dryness under reduced pressure. The residue was purified by column chromatography to give compound 45 as a white solid (2.3 g).

[0449] To a solution of compound 45 (0.12 g) and MMAE (0.072 g) in DMF (2 mL) was added DIEA (0.018 mL) and the reaction stirred at room temperature for 24 hours. The mixture was then diluted with EtOAc (30 mL) and washed with hydrochloric acid (0.5 M, 30 mL). The organic layer was washed with water (20 mL), dried (over Na2SO4) and evaporated to dryness under reduced pressure to give crude compound 46 which was re-dissolved in MeOH (3 mL) and MeONa (4.4 M in MeOH, 0.1 mL). The reaction mixture was stirred at room temperature for 2 hours then neutralised with 1 N hydrochloric acid (0.5 mL). The mixture was purified directly by RP-HPLC to give compound 47 as a white solid (TFA salt, 72 mg).

[0450] To a solution of compound 47 (28 mg, TFA salt) in DMF (2 mL) was added 6-maleimidohexanoic acid N-hydroxysuccinimidyl ester (Sigma Aldrich, 8 mg) followed by DIEA (0.007 mL). The mixture was stirred at room temperature. After 30 minutes the mixture was purified by RP-HPLC to give compound 48 as a white solid (24 mg).

[0451] MS: m / z 1499.9 [M+H] + .

[0452] 3.10 Synthesis of compound 55 (MC-AAN-(GlcNAc)-PAB-MMAE)

[0453] The synthesis proceeded as follows:

[0454] To a solution of compound 49 (AA Block, 68 mg) and p-aminobenzyl alcohol (13 mg) in DMF (2 mL) was added DIEA (0.035 mL) followed by HATU (40 mg). The reaction mixture was stirred at room temperature for 20 minutes then diluted with EtOAc (50 mL). The mixture was washed with 0.5 M hydrochloric acid (30 mL) and water (30 mL). The organic layer was dried (with Na2SO4) and evaporated to dryness under reduced pressure. The residue was triturated with diethyl ether (100 mL) to give crude compound 50 as a tan solid which was dissolved in DMF (4 mL). Diisopropylamine (4 mL) was added and the mixture stirred at room temperature for 2 hours. The reaction was then concentrated under reduced pressure to approximately 3 mL. Fmoc-Ala-Ala-OH (fluorenylmethoxycarbonyl-alanine-alanine, 40 mg) and DIEA (0.035 mL) were added followed by HATU (0.04 g) and the mixture stirred at room temperature. After 20 minutes the reaction was diluted with EtOAc (40 mL). The mixture was washed with 0.5 M hydrochloric acid (30 mL) and water (50 mL). The organic layer was dried (with Na2SO4) and evaporated to dryness under reduced pressure. The crude product was purified by RP-HPLC to give compound 51 as a pale yellow solid (82 mg).

[0455] Compound 51 (80 mg) was dissolved in DMF (2 mL). To this solution was added DIEA (0.01 mL) and bis-PNP carbonate (60 mg) and the mixture stirred at room temperature for 16 hours. The reaction was then diluted with EtOAc (50 mL) and washed with water (3 x 15 mL). The organic layer was dried (with Na2SO4) and evaporated to dryness under reduced pressure. The crude product was purified by RP-HPLC to give compound 52 as a white solid (78 mg).

[0456] To a solution of compound 52 (55 mg) and MMAE (0.036 g) in DMF (2 mL) was added DIEA (0.01 mL) and stirred at room temperature for 24 h. The mixture was then diluted with EtOAc (30 mL) and washed with hydrochloric acid (0.5 M, 30 mL). The organic layer was washed with water (20 mL), dried (with Na2SO4) and evaporated to dryness under reduced pressure to give crude compound 53 which was redissolved in MeOH (2 mL) and MeONa (4.4 M in MeOH, 0.05 mL). The reaction mixture was stirred at room temperature for 2 hours then neutralised with 1 N hydrochloric acid (0.5 mL). The mixture was purified directly by RP-HPLC to give compound 54 as a white solid (TFA salt, 56 mg).

[0457] To a solution of compound 54 (29 mg, TFA salt) in DMF (2 mL) was added 6-maleimidohexanoic acid N-hydroxysuccinimidyl ester (Sigma Aldrich, 8 mg) followed by DIEA (0.007 mL). The mixture was stirred at room temperature. After 30 min, the mixture was purified by RP-HPLC to give compound 55 as a white solid (21 mg).

[0458] MS: m / z 1519.9 [M+H] + .

[0459] 3.11 Synthesis of compound 56 (BrAc-GGY(Glc)-PAB-MMAE)

[0460] The synthesis procedure was as follows:

[0461] To a solution of compound 47 (28 mg) in acetonitrile / water (6 / 4, v / v, 2 mL) was added saturated NaHC03solution (0.04 mL) and bromoacetic anhydride (10 mg). After the reaction mixture was stirred at room temperature for 10 min, the crude mixture was purified by RP-HPLC to give compound 56 as a white solid (27 mg) after lyophilization.

[0462] MS: 1426.80 [M+H] + .

[0463] 3.12 Synthesis of compound 17 (BrAc-PEG4-GlcA-MMAE)

[0464] The synthesis procedure was as follows:

[0465] To a solution of compound 12 (65 mg, prepared as described in WO2022026915) and MMAE (72 mg) in dry DMF (2 mL) was added DIEA (0.02 mL) followed by HOBt (3 mg). After stirring the reaction mixture at room temperature (22 °C) for 18 h, it was diluted with water (20 mL). The reaction mixture was extracted with diethyl ether (40 mL) and the organic phase was dried over Na2S04and concentrated to dryness under reduced pressure to obtain crude compound 13, which was finally dissolved in methanol (2 mL). Zinc powder (200 mg) was added to the methanolic solution of compound 13 followed by formic acid (0.2 mL). It was stirred at room temperature for 30 min. The solid was removed by filtration and the filtrate was directly purified by RP-HPLC to obtain compound 14 (72 mg) as a white solid after lyophilization. To a solution of compound 14 (TFA salt, 66 mg) and Fmoc-NH-PEG4-COOH (purchased from PurePEG, cat. no. 433704, 25 mg) in dry DMF (2 mL) was added DIEA (0.025 mL) followed by HATU (20 mg). The mixture was stirred at room temperature. After 16 h, the crude mixture was purified by RP-HPLC to obtain compound 15 (72 mg) as a white powder after lyophilization.

[0466] MS: 1471.6 [M+H] + .

[0467] Compound 15 (70 mg) was dissolved in acetonitrile / water (6 / 4, v / v, 3 mL) and NaOH (aq., 1 M, 0.3 mL) was added. The reaction mixture was stirred at room temperature (22 °C) to obtain compound 16. After 8 h, to the crude of compound 16 was added hydrochloric acid (1 M, 0.12 mL) followed by bromoacetic anhydride (14 mg). The crude reaction mixture was directly purified by RP-HPLC to obtain compound 17 (46 mg) as a white solid after lyophilization.

[0468] MS: 1426.7 [M+H] + .

[0469] 3.13 Synthesis of compound 20 (BrAc-PEG4-PAB-MMAE)

[0470] The synthesis was carried out as follows:

[0471] To a solution of compound 1 (62 mg) and Fmoc-NH-PEG4-COOH (compound 18, 25 mg, purchased from PurePEG, Cat# 433704-1H) in anhydrous DMF (1 mL) was added DIEA (0.025 mL) followed by HATU (20 mg). The reaction mixture was stirred at room temperature (22 °C). After 15 min, piperidine (0.1 mL) was added and the reaction was continued for 30 min. The crude reaction mixture was directly purified by RP-HPLC to give compound 19 (61 mg, TFA salt) as a white solid after lyophilization.

[0472] To a solution of compound 19 (37 mg) in acetonitrile / water (6 / 4, v / v, 2 mL) was added saturated NaHC03solution (0.03 mL) and bromoacetic anhydride (7 mg). After the reaction mixture was stirred at room temperature for 10 min, the crude mixture was purified by RP-HPLC to give compound 20 (32 mg) as a white solid after lyophilization.

[0473] MS: 1491.0 [M+H] + .

[0474] Preparation and physicochemical characterization of antibody-drug conjugates

[0475] The antibody prepared in Example 1 was treated with a reducing agent, such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) or dithiothreitol (DTT), to reduce some or all of the cysteine disulfide residues to form highly nucleophilic cysteine thiol groups (-CH2SH). The partially or completely reduced antibody was then reacted with a drug linker or linker reagent having an electrophilic functional group, such as a maleimide.

[0476] For example, antibody A was dissolved in PBS at pH 7.2, 2 mM EDTA was added, and the antibody was reduced with TCEP (TCEP: antibody molar ratio of 2.8: 1). After incubation at 37 °C for about 120 min, drug linker compound 22, 29, 39, 48, or 55 was added to the reduced antibody (drug linker: antibody molar ratio of 5: 1), and 5% (v / v) DMSO was added. After about 1 h at room temperature, the ADC was purified and desalted by elution with G25 resin into phosphate buffer (pH 7.4) and filtered with a 0.2 μιη filter under sterile conditions and stored frozen. Analysis by hydrophobic interaction chromatography-high performance liquid chromatography determined that the average DAR value of the ADC was between 3.5 and 4.0.

[0477] For example, antibody A was dissolved in phosphate buffer at pH 8.0, 2 mM EDTA was added, and the antibody was reduced with TCEP (TCEP: antibody molar ratio of 2.8: 1). After incubation at 37 °C for about 120 minutes, drug linker compound 10, 11, 17, 56, or 20 was added to the reduced antibody (drug linker: antibody molar ratio of 5: 1), and 5% (v / v) DMSO was added. After about 1 hour at room temperature, the ADC was purified by desalting with G25 resin, filtered under sterile conditions with a 0.2 pm filter, and stored frozen. HIC-HPLC analysis determined the average DAR value of the ADC to be between 3.5 and 4.0.

[0478] For example, antibody A was dissolved in PBS at pH 7.2, drug linker compound 3, 4, or 5 was added to the antibody (drug linker: antibody molar ratio of 3: 1), and 10% (v / v) DMSO was added. After about 2 hours at room temperature, the drug linker was added again, and the reaction was allowed to proceed for another 2 hours at room temperature, for a final drug linker: antibody molar ratio of 6: 1. The ADC was purified by desalting with G25 resin, filtered under sterile conditions with a 0.2 pm filter, and stored frozen. HIC-HPLC analysis determined the average DAR value of the ADC to be between 1.5 and 1.9.

[0479] Methods for detecting DAR values:

[0480] HIC-HPLC analysis was used to analyze the DAR values of the ADCs of the disclosure. The ADCs were separated in a chromatographic column using a MabPac HIC-Butyl analytical column (4.6 x 100 mm, 5 pm, Cat. No. 088558, ThermoFisher, USA). 25 mM sodium phosphate buffer (pH 6.8) containing 1.5 M ammonium sulfate was used as buffer solution A, and 25 mM sodium phosphate buffer (pH 6.8) containing 25% acetonitrile was used as buffer solution B, and 85% buffer solution A and 15% buffer solution B were stabilized as initial conditions, using a linear gradient of 85% buffer solution A and 15% buffer solution B for 30 minutes compared to 5% buffer solution A and 95% buffer solution B, and an additional 5 minutes elution using 5% buffer solution A and 95% buffer solution B. The flow rate and temperature were set to 0.5 mL / min and 25 °C. The ADC drug profile was detected at 214 nm and 280 nm for DAR value calculation.

[0481] Exemplary, partially antibody-drug conjugates (ADCs) prepared are shown in Table 3.

[0482] Table 3. ADCs prepared in the disclosure

[0483] Preparation of control L-MMMAE

[0484] Ladiratuzumab antibody, abbreviated as L, was prepared according to the reference patent US2013259860A1, the heavy chain sequence of which is shown in SEQ ID NO: 33, and the light chain sequence of which is shown in SEQ ID NO: 34; the antibody L was coupled with MC-MMAE (compound 22) to obtain L-Mc-MMAE with a DAR of 3.5-4, which was used for subsequent in vivo efficacy test.

[0485] Example 5 Antibody-drug conjugate binding activity with LIV-1 antigen

[0486] hLIV-1 was diluted with coating buffer to 2 μg / mL, added to the enzyme-labeled plate, and coated at 4°C for 15-20 hours. 300 μL of blocking solution was added to each well, and blocked at room temperature for 1 hour. The antibody-drug conjugate to be tested was diluted with diluent, and incubated at room temperature for 1 hour. The monoclonal antibody of the mouse anti-toxin was diluted with diluent, and incubated at room temperature for 1 hour. The goat anti-mouse Fc domain secondary antibody was diluted with diluent, and incubated at room temperature for 1 hour. After the secondary antibody incubation was completed, TMB color developing solution was incubated at room temperature for 1-10 minutes. After the color development was completed, 50 μL of stop solution (4 M sulfuric acid) was added to each well to stop the substrate reaction. The absorbance was measured on an enzyme-labeled instrument, and the absorbance value of each well was read at a detection wavelength of 450 nm. Prism software was used to analyze the data, with the naked antibody A protein concentration as the abscissa, the average value of the corresponding absorbance as the ordinate, and the Sigmoidal, 4PL four-parameter equation to draw the dose-response curve. The equation is Y = Bottom + (X^Hillslope) * (Top-Bottom) / (X^HillSlope + EC50^HillSlope).

[0487] The results are shown in Figure 2, and the antibody-drug conjugate of the present disclosure can specifically bind to LIV-1 antigen.

[0488] Example 6 In vitro killing effect of antibody-drug conjugate

[0489] In this experiment, CellTiterGlo2 (Promega) reagent was used to evaluate the anti-proliferative effect of the drug. The heat-resistant luciferase contained in the reagent can catalyze the mono-oxygenation of luciferin in the presence of Mg2+, ATP produced by living cells, and molecular oxygen, to produce a stable “glowing” luminescent signal, thereby determining the number of living cells in the culture by quantifying the marker of metabolically active cells, ATP.

[0490] The cell lines selected in the experiment are: human lung cancer (undifferentiated) cell line Calu-6, human breast cancer cell MCF7-ATCC-LIV-1 #7, #12, #17 overexpressing LIV-1.

[0491] Calu-6 cells are cultured in RPMI-1640 medium containing 10% fetal bovine serum, and MCF7-ATCC-LIV-1 overexpressing cell lines are cultured in EMEM medium containing 10% fetal bovine serum and 1 μg / mL puromycin at 37°C in a 5% CO2 incubator. Four kinds of cells are inoculated into 96-well plates at a density of 2×10 3 -5×10 3 cells per well, 50 μL / well, after 24 h of culture, different concentrations of A-BrAc-MMAE or control IgG1-BrAc-MMAE diluted in culture medium are added, 100 μL / well, duplicate wells are set for each concentration, and solvent controls and cell-free medium wells of the corresponding concentration are set, after 96 h of culture at 37°C in a 5% CO2 incubator, 100 μL CellTiterGlo2 is added per well, mixed on an orbital shaker at room temperature for 15 min, the luminous value is measured, and the IC 50 value (nM) of the anti-LIV-1 antibody A-BrAcMMAE conjugate on various cells is calculated (Table 3). The calculation results are shown in Figures 3, 4, 5, 6 and Table 4-1.

[0492] Table 4-1. IC 50 value (nM) of the anti-LIV-1 antibody A-BrAcMMAE conjugate on various cells is calculated (Table 3). The calculation results are shown in Figures 3, 4, 5, 6 and Table 4-1.

[0493] As can be seen from Table 4-1 and Figures 3, 4, 5 and 6, A-BrAcMMAE has obvious killing effect on three tumor cells with different expression levels of LIV-1, and the killing effect is proportional to the expression level of LIV-1. And it has obvious proliferation inhibition effect on breast cancer and lung cancer cells.

[0494] In addition, the disclosure also detects the in vitro killing activity of ADCs coupled with different linkers and antibody A on different cells, and the results are shown in Tables 4-2 and 4-3.

[0495] Table 4-2. IC 50 value (nM) of the anti-LIV-1 antibody A-BrAcMMAE conjugate on various cells is calculated (Table 3). The calculation results are shown in Figures 3, 4, 5, 6 and Table 4-1.

[0496] Table 4-3. IC 50 value (nM) of the anti-LIV-1 antibody A-BrAcMMAE conjugate on various cells is calculated (Table 3). The calculation results are shown in Figures 3, 4, 5, 6 and Table 4-1.

[0497] In summary, the anti-LIV-1 antibody-MMAE conjugate of the present disclosure has obvious anti-tumor activity and good targeting.

[0498] Example 7 In vivo efficacy study of antibody-drug conjugate

[0499] CD1 athymic nude mice were implanted with tumor cells grown in culture: Calu-6 from ATCC (2.5 x 10 6 cells in 50% matrigel), PC-3 from ATCC (2.5 x 10 6 cells), PA-1 from ATCC (5 x 10 6 cells in 50% matrigel). NSG mice were implanted with tumor cells grown in culture: HCC1806 from ATCC (2.5 x 10 6 cells in 50% matrigel), MCF-7 from NCI (10 x 10 6 cells, for MCF-7 to grow in vivo, 8.5 μg / mL estradiol was added to the drinking water of female mice).

[0500] When the tumor growth reached 150 mm 3 , the administration of anti-LIV-1 ADC or non-binding control ADC (1, 3, 6 or 10 mg / kg) was started: intravenous injection once every four days for a total of four times (Q4D x 4), intravenous injection once a week for a total of two times (Q1W x 2), or single intravenous injection. The tumor volume was monitored using a caliper, and the animals were euthanized when the tumor volume reached about 2000 mm 3 . Tumor volume measurements were performed for each group until one or more animals were euthanized. All animal procedures were performed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, according to protocols approved by the Institutional Animal Care and Use Committee. The formula for calculating TGI is as follows:

[0501] TGI (%) = 1 - [(Td - T0) / (Cd - C0)] x 100%

[0502] where Td and Cd are the average tumor volumes of the treatment group and the control group on the day of tumor volume determination, and T0 and C0 are the average tumor volumes of the treatment group and the control group on day 0.

[0503] The results are shown in Figures 7-11. In the MCF7 xenograft efficacy study, the tumors of 4 mice completely regressed on day 22 after administration. The TGI was 126.3% on day 36 after administration.

[0504] In the HCC1806 xenograft efficacy study, the TGI was 102.99% on day 21 after administration.

[0505] In the PC-3 xenograft efficacy study, on day 11 after administration, the TGI was 101.5%.

[0506] In the Calu-6 xenograft efficacy study, on day 25 after administration, the TGI of the 3 mg / kg group was 75.14% and the TGI of the 6 mg / kg group was 111.56% in the multiple administration group. On day 28 after administration, the tumors of 7 mice in the 6 mg / kg group completely regressed. On day 25 after administration, the TGI of the 6 mg / kg group was 52.72% and the TGI of the 10 mg / kg group was 76.96% in the single administration group.

[0507] The above results show that A-BrAcMMAE exhibits significant anti-tumor efficacy in different tumor models.

[0508] In addition, the present disclosure also detected the tumor inhibition effect of A-BrAcMMAE and the prior art ADC ladiratuzumab vedotin (abbreviated as: L-Mc-MMAE) targeting LIV-1 in PC3 and PA-1 mouse tumor models, and the results are shown in Figure 12 and Tables 5-1, 5-2.

[0509] Table 5-1. Tumor inhibition rate of different ADCs on PC3 mouse subcutaneous tumors

[0510] Table 5-2. Tumor inhibition rate of ADCs on PA-1 mouse subcutaneous tumors

[0511] The above results show that in the prostate cancer (PC-3) and ovarian cancer (PA-1) models, A-BrAcMMAE can delay or prevent the growth of LIV-1 expressing tumors, showing significant anti-tumor activity. Although L-McMMAE reported in the previous literature also has anti-tumor activity, A-BrAcMMAE has significantly better efficacy than L-McMMAE in mouse tumor models. Therefore, the A-BrAcMMAE antibody conjugate can be used to treat various cancers expressing LIV-1.

[0512] Example 8 In vivo efficacy study of antibody-drug conjugate in combination with anti-PD-(L)1 antibody

[0513] The anti-PD-1 antibody used in this example is Pembrolizumab (Keytruda), and the preparation of h409A11 antibody is referred to in WO2008156712A1; the PD-L1 antibody used is Atezolizumab (Tecentriq), and the preparation is referred to in WO2023279092A2. The specific experimental scheme is as follows:

[0514] NCG immunodeficient mice implanted with tumor cells grown in culture: melanoma cells A375 (5 x 10 6 cells from ATCC, breast cancer cells HCC1806 (5 x 10 6 cells from ATCC in 50% Matrigel).

[0515] When the tumor growth reached an average of 100-150 mm 3 , the mice were randomly divided into groups: IgG4 antibody control group, anti-LIV1-ADC monotherapy group, anti-PD-(L)1 antibody monotherapy group, anti-LIV1-ADC + anti-PD(L)1 antibody combination group. Each mouse was intravenously infused with 10 x 10 6 human PBMC cells, administered as follows: 3 mg / kg anti-LIV-1 ADC was administered by intravenous injection on day 1, a total of 1 time; 10 mg / kg anti-PD-(L)1 antibody was administered by intravenous injection every 5 days for a total of 4 times on days 1, 6, 11, 16; or in combination with 3 mg / kg anti-LIV-1 ADC was administered by intravenous injection on day 1, a total of 1 time, and 10 mg / kg anti-PD-(L)1 antibody was administered by intravenous injection every 5 days for a total of 4 times on days 1, 6, 11, 16. The tumor volume was monitored using a caliper, and the animals were euthanized when the tumor volume reached about 2000 mm 3 . Tumor volume detection was performed for each group until one or more animals were euthanized. All animal procedures were performed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, according to protocols approved by the Institutional Animal Care and Use Committee.

[0516] The formula for calculating TGI is as follows:

[0517] TGI (%) = 1 - [(Td-T0) / (Cd-C0)] x 100%, where Td and Cd are the average tumor volumes of the treatment group and the control group on the day of tumor volume measurement, and T0 and C0 are the average tumor volumes of the treatment group and the control group on day 0.

[0518] The results are shown in Figures 14-16 and Tables 6-1 to 6-3.

[0519] Table 6-1. Tumor inhibition rate of different administration groups on A375 mouse subcutaneous tumors

[0520] Table 6-2. Tumor inhibition rate of different administration groups on A375 mouse subcutaneous tumors

[0521] Table 6-3. Tumor inhibition rate of different administration groups on HCC1806 mouse subcutaneous tumors

[0522] Results showed that in the A375 tumor transplantation efficacy study, the tumor inhibition rate of the LIV-1-ADC combined with anti-PD-1 antibody group was 95.18% on the 31st day after PBMC infusion. The tumor inhibition rate of the anti-LIV-1-ADC combined with anti-PD-L1 antibody group was 105.03%, and all the tumors were completely regressed from the 14th day.

[0523] In the HCC1806 tumor transplantation efficacy study, on the 20th day after administration, the tumor inhibition rate of the anti-LIV-1-ADC single-agent group was 38.88%, the anti-PD-1 antibody single-agent group had no tumor inhibition effect, and the tumor inhibition rate of the anti-LIV-1-ADC combined with anti-PD-1 group was 73.99%, which was better than that of the anti-LIV-1-ADC single-agent group.

[0524] Example 9 Killing effect of antibody-drug conjugate combined with anti-CD73 antibody on breast cancer SKBR3 cells

[0525] The anti-CD73 antibody used in this example was prepared by referring to the B11 antibody in WO2020253568A1, and the specific experimental scheme is as follows:

[0526] CD3 / CD28 antibody (purchased from Invitrogen, item number 16-0037-81 and 16-0289-81) was coated on the culture dish at a concentration of 5 μg / mL one day in advance, and was placed in the refrigerator at 4°C overnight. Fresh human PBMC (purchased from Australian Energy Biology) was counted after centrifugation, and was plated in the culture dish at a concentration of 1 × 10 6 4 SKBR3 cells (purchased from Chinese Academy of Sciences) were digested, centrifuged, and counted, and then 1 × 10 4 ​The A-BrAcMMAE was diluted into different concentration gradients (30, 5, 1.2, 0.24, 0.048 μg / mL) with 30 μg / mL as the starting concentration, and the anti-CD73 antibody B11 was administered at a concentration of 50 μg / mL. Different concentrations of A-BrAcMMAE and anti-CD73 antibody B11 were both prepared into 10x administration concentration, and 10 μL was added to the 96-well plate co-cultured with PBMC and SKBR3 cells. After incubation in a 37°C incubator for 72 hours, 100 μL of CellTiter-Glo reagent (purchased from Promega, product number G7572) was added to each well, and the plate was placed on a orbital shaker for 3 minutes, then the plate was incubated at room temperature for 10 minutes, and the luminescence signal was detected by a microplate reader. The killing effect of A-BrAcMMAE combined with anti-CD73 antibody B11 on SKBR3 cells was calculated. The results are shown in Figure 17.

[0527] The results show that compared with the A-BrAcMMAE monotherapy group, the combination of anti-CD73 antibody B11 significantly enhances the killing effect of A-BrAcMMAE on SKBR3.

[0528] Example 10 Killing of breast cancer SKBR3 cells by antibody drug conjugate combined with anti-SIRPα antibody

[0529] The anti-SIRPα antibody used in this example was prepared according to antibody 14# in WO2022121980A1, and the specific experimental scheme is as follows:

[0530] (1) Preparation of effector cells:

[0531] Fresh human PBMC (purchased from Shanghai Heyou Life Sciences) 400g was centrifuged for 10 min, the cell pellet was collected and resuspended in basal RPMI 1640 medium (Sigma, product number R8758) to a cell density of 1E8 / mL, and cultured at 37°C and 5% CO2 for 2h to adhere. Remove the culture supernatant and wash once, replace with complete RPMI 1640 medium containing 80 ng / mL human M-CSF (purchased from MedChemExpress, product number HY-P7050) for induction, replace the medium every 3 days, a total of 7 days of induction, and finally induce macrophages M0.

[0532] (2) Cell killing experiment:

[0533] On day 1, SKBR3 breast cancer cells and M0 macrophages were seeded at 90 μL per well in 96-well plates, with 8000 SKBR3 cells and 4000 M0 macrophages per well, in triplicate. The plates were cultured at 37°C and 5% CO2 for 16 h. A-BrAcMMAE was diluted to a working concentration of 3.125 μg / mL with complete RPMI 1640 medium, and anti-SIRPα antibody 14# was diluted to a working concentration of 10 μg / mL. 10 μL of the drug or blank control PBS was added to each well. After culturing at 37°C and 5% CO2 for 96 h, 100 μL of CellTiter-Glo reagent (Promega, catalog number G7572) was added to each well. The plates were shaken for 4 minutes at room temperature, incubated for 20 minutes at room temperature, and the luminescence value was measured using a microplate reader. The cytotoxic effect of A-BrAcMMAE combined with anti-SIRPα antibody 14# on SKBR3 cells was calculated. The results are shown in Figure 18.

[0534] Experimental results showed that both A-BrAcMMAE and anti-SIRPα antibody 14# exhibited certain killing effects. When A-BrAcMMAE and anti-SIRPα antibody were administered in combination, the combined administration group significantly enhanced the killing ability against tumor cells compared to the single-drug group.

[0535] Example 11: In vivo pharmacodynamic study of antibody-drug conjugate combined with anti-SIRPα antibody

[0536] The anti-SIRPα antibody used in this embodiment was prepared according to antibody 14# in WO2022121980A1, and the specific experimental procedure is as follows:

[0537] JIMT-1 cells (breast cancer cells, purchased from Procell) resuspended in PBS were mixed with an equal volume of matrix gel at a 1:1 ratio and then incubated at 5 × 10⁻⁶ ppm. 6 0.1 mL / mouse was injected subcutaneously into the right back of B-NDG hSIRPA mice. The tumor volume was increased to 120–180 mm². 3 Mice were randomly assigned to four groups: a PBS control group, an anti-SIRPα antibody 14# monotherapy group, an A-BrAcMMAE monotherapy group, and an A-BrAcMMAE + antibody 14# combination group, with six mice in each group. Administered as follows: 3 mg / kg A-BrAcMMAE was administered intraperitoneally on day 0, once daily; 20 mg / kg anti-SIRPα antibody 14# was administered intraperitoneally twice weekly (BTW) on days 1, 4, 8, 11, 15, 18, and 22, for a total of seven weeks. Tumor volume was monitored using calipers, and observation continued until day 24 after the first administration, calculating the tumor inhibition rate.

[0538] The formula for calculating the tumor inhibition rate (TGI) is as follows:

[0539] TGI(%) = [1 - (Ti - To) / (Ci - Co)] x 100%

[0540] Wherein, Ti is the mean tumor volume of the treatment group on the i-th day of administration, To is the mean tumor volume of the treatment group on the 0-th day of administration; Ci is the mean tumor volume of the control group on the i-th day of administration, Co is the mean tumor volume of the control group on the 0-th day of administration. The results are shown in Table 7.

[0541] Table 7. Tumor inhibition rate of different administration groups on JIMT-1 mouse subcutaneous tumors

[0542] The results show that on the 24th day after grouping administration, the tumor inhibition rate of the combination of A-BrAcMMAE and anti-SIRPa antibody 14# is 84.4%, which is significantly better than that of SIRPa antibody 14# single drug group (tumor inhibition rate 10.9%) and A-BrAcMMAE single drug group (tumor inhibition rate 59.4%), indicating that anti-SIRPa antibody and A-BrAcMMAE have synergistic effect.

[0543] Example 12 Inhibitory activity of antibody-drug conjugate combined with CDK4 / 6 inhibitor on tumor proliferation

[0544] After the breast cancer cells MCF-7 were digested, centrifuged and counted, 2000 / well was added to the 96-well plate, and the next day A-BrAcMMAE was diluted to different concentrations (8nM, 4.44nM, 2.47nM, 1.37nM, 0.76nM) and added to the 96-well plate, and at the same time CDK4 / 6 inhibitor Palbociclib (purchased from Selleck, S1116) was added to the corresponding 96-well plate at a concentration of 24.6nM. After incubating the culture plate in a 37°C incubator for 5 days, 100μL of CellTiter-Glo reagent (purchased from Promega, G7572) was added to each well, the culture plate was placed on a orbital shaker for 3 minutes, and then the culture plate was incubated at room temperature for 15 minutes. The luminescence signal was detected by a microplate reader, and the survival rate of cells was calculated to explore the killing effect of A-BrAcMMAE combined with Palbociclib on MCF-7 cells, and the results are shown in Figure 19.

[0545] The results show that the killing effect of A-BrAcMMAE combined with Palbociclib on MCF-7 cells is significantly better than that of the single drug group on MCF-7, i.e. A-BrAcMMAE combined with Palbociclib can enhance the killing effect on breast cancer cells MCF-7.

[0546] Example 13 Killing effect of antibody-drug conjugate combined with anti-Trop2-ADC on breast cancer cells MCF-7

[0547] After the breast cancer cells MCF-7 were digested, centrifuged, counted, 2000 cells were added to each well of a 96-well plate, and the next day A-BrAcMMAE was diluted to different concentrations (8, 4.44, 2.47, 1.37, 0.76 nM) and added to the 96-well plate, and Dato-DXd (purchased from Selleck, item number D4050) was added to the corresponding 96-well plate at a concentration of 0.3 nM. After incubating the plates in a 37°C incubator for 5 days, 100 μL of CellTiter-Glo reagent (purchased from Promega, item number G7572) was added to each well, the plates were mixed on an orbital shaker for 3 minutes, and then the plates were incubated at room temperature for 15 minutes. The luminescence signal was detected using a microplate reader, and the survival rate of the cells was calculated to investigate the killing effect of A-BrAcMMAE combined with Dato-DXd on MCF-7 cells. The results are shown in Figure 20.

[0548] The results show that the killing effect of A-BrAcMMAE combined with Dato-DXd on MCF-7 cells is better than that of the single drug group on MCF-7, i.e., A-BrAcMMAE combined with Dato-DXd has a certain synergistic effect on the killing of breast cancer cells MCF-7.

[0549] Example 14 In vitro killing activity of antibody-drug conjugate combined with anti-Her2 antibody on breast cancer cells

[0550] (1) Preparation of effector cells:

[0551] Fresh human PBMC (purchased from Shanghai Heyou Life Sciences) was centrifuged at 400g for 10 min, and the cell pellet was collected and resuspended in basal RPMI 1640 medium (Sigma, item number R8758) to a cell density of 1E8 / mL. The cells were cultured at 37°C and 5% CO2 for 2 h to adhere. The culture supernatant was removed and washed once, and replaced with complete RPMI 1640 medium containing 80 ng / mL human M-CSF (purchased from MedChemExpress, item number HY-P7050) for induction. The medium was replaced every 3 days, and the cells were induced for a total of 7 days. The final induction was macrophage M0.

[0552] (2) Cell killing experiment:

[0553] Breast cancer cells SKBR3 and macrophages M0 were plated in 96-well plates at 90 μL per well, with 8000 target cells per well and 4000 macrophages M0 per well, triplicate, and incubated at 37 °C, 5% CO2 for 16 h. A-BrAcMMAE was diluted with complete RPMI 1640 medium to a final working concentration of 3.125 ug / ml, and anti-Her2 antibody trastuzumab was diluted to a final working concentration of 0.5 μg / mL. 10 μl of drug or blank control PBS was added to each well, and the plates were incubated at 37 °C, 5% CO2 for 96 h. Then 100 μL of CellTiter-Glo reagent (purchased from Promega, Catalog No. G7572) was added to each well, and the plates were shaken on an orbital shaker at room temperature for 4 min, and then incubated at room temperature for 20 min. The luminescence value was measured by a microplate reader. The killing effect of A-BrAcMMAE combined with trastuzumab on SKBR3 cells was calculated, and the results are shown in Figure 21.

[0554] The experimental results show that A-BrAcMMAE monotherapy and trastuzumab monotherapy both show a certain killing effect. When A-BrAcMMAE and trastuzumab are combined, the combination group significantly improves the killing ability of tumor cells compared with the monotherapy group.

[0555] Example 15 Killing effect of antibody-drug conjugate combined with chemotherapeutic agents on breast cancer cells MCF-7

[0556] Breast cancer cells MCF-7 were digested, centrifuged, counted, and then added to 96-well plates at 2000 cells per well. The next day, A-BrAcMMAE was diluted to different concentrations (8, 4.44, 2.47, 1.37, 0.76 nM) and added to 96-well plates, and chemotherapeutic agents (8.8 nM of Doxorubicin (purchased from Selleck, Catalog No. E2516), 1.82 nM of Gemcitabine (purchased from Selleck, Catalog No. S1714), and 9.9 uM of Carboplatin (purchased from Selleck, Catalog No. C805203)) were added to corresponding 96-well plates. The plates were incubated in a 37 °C incubator for 5 days, 100 μL of CellTiter-Glo reagent (purchased from Promega, Catalog No. G7572) was added to each well, the plates were mixed on an orbital shaker for 3 min, and then the plates were incubated at room temperature for 15 min. The luminescence signal was detected by a microplate reader, the survival rate of cells was calculated, and the killing effect of A-BrAcMMAE combined with different chemotherapeutic agents on MCF-7 cells was investigated, and the results are shown in Figures 22A-22C.

[0557] The experimental results show that A-BrAcMMAE and Doxorubicin monotherapy can significantly kill MCF-7 cells, and A-BrAcMMAE combined with Doxorubicin enhances the killing effect on MCF-7 cells compared with the single drug group. A-BrAcMMAE combined with Gemcitabine has a better killing effect on MCF-7 cells than A-BrAcMMAE or Gemcitabine monotherapy, that is, A-BrAcMMAE combined with Gemcitabine has a certain synergistic effect on the killing of breast cancer cells MCF-7. A-BrAcMMAE and Carboplatin monotherapy both show a certain killing effect on MCF-7 cells, and A-BrAcMMAE combined with Carboplatin enhances the killing effect on MCF-7 cells compared with the single drug group.

[0558] Sequence information of the present disclosure:

[0559] SEQ ID NO: 1 (VH of antibody A):

[0560] SEQ ID NO: 2 (VL of antibody A):

[0561] SEQ ID NO: 3 (VH of antibody B):

[0562] SEQ ID NO: 4 (VL of antibody B):

[0563] SEQ ID NO: 5 (VH of antibody C):

[0564] SEQ ID NO: 6 (VL of antibody C):

[0565] SEQ ID NO: 7 (antibody A VH CDR-H1):

[0566] SEQ ID NO: 8 (antibody A VH CDR-H2):

[0567] SEQ ID NO: 9 (antibody A VH CDR-H3):

[0568] SEQ ID NO: 10 (antibody A VL CDR-L1):

[0569] SEQ ID NO: 11 (antibody AVL CDR-L2):

[0570] SEQ ID NO: 12 (antibody AVL CDR-L3):

[0571] SEQ ID NO: 13 (antibody B VH CDR-H1):

[0572] SEQ ID NO: 14 (antibody B VH CDR-H2):

[0573] SEQ ID NO: 15 (antibody B VH CDR-H3):

[0574] SEQ ID NO: 16 (antibody B VL CDR-L1):

[0575] SEQ ID NO: 17 (antibody B VL CDR-L2):

[0576] SEQ ID NO: 18 (antibody B VL CDR-L3):

[0577] SEQ ID NO: 19 (antibody C VH CDR-H1):

[0578] SEQ ID NO: 20 (antibody C VH CDR-H2):

[0579] SEQ ID NO: 21 (antibody C VH CDR-H3):

[0580] SEQ ID NO: 22 (antibody C VL CDR-L1):

[0581] SEQ ID NO: 23 (antibody C VL CDR-L2):

[0582] SEQ ID NO: 24 (antibody C VL CDR-L3):

[0583] SEQ ID NO: 25 (LIV-1 antigen amino acid sequence):

[0584] SEQ ID NO: 26 (heavy chain constant region):

[0585] SEQ ID NO: 27 (light chain constant region):

[0586] SEQ ID NO: 28 (murine antibody mVH of Antibody A, with CDRs underlined):

[0587] SEQ ID NO: 29 (murine antibody mVL of Antibody A, with CDRs underlined):

[0588] SEQ ID NO: 30 (light chain of Antibody A):

[0589] SEQ ID NO: 31 (heavy chain of Antibody A):

[0590] SEQ ID NO: 32 (heavy chain variable region of Antibody A-1):

[0591] SEQ ID NO: 33 (heavy chain of Ladiratuzumab):

[0592] SEQ ID NO: 34 (light chain of Ladiratuzumab):

[0593] SEQ ID NO: 35 (HCDR1 of anti-PD-1 antibody:

[0594] SEQ ID NO: 36 (HCDR2 of anti-PD-1 antibody):

[0595] SEQ ID NO: 37 (HCDR3 of anti-PD-1 antibody):

[0596] SEQ ID NO: 38 (LCDR1 of anti-PD-1 antibody:

[0597] SEQ ID NO: 39 (LCDR2 of anti-PD-1 antibody:

[0598] SEQ ID NO: 40 (LCDR3 of anti-PD-1 antibody):

[0599] SEQ ID NO: 41 (VH of anti-PD-1 antibody):

[0600] SEQ ID NO: 42 (VL of anti-PD-1 antibody):

[0601] SEQ ID NO: 43 (heavy chain of anti-PD-1 antibody):

[0602] SEQ ID NO: 44 (light chain of anti-PD-1 antibody):

[0603] SEQ ID NO: 45 (HCDR1 of anti-PD-L1 antibody):

[0604] SEQ ID NO: 46 (HCDR2 of anti-PD-L1 antibody):

[0605] SEQ ID NO: 47 (HCDR3 of anti-PD-L1 antibody):

[0606] SEQ ID NO: 48 (LCDR1 of anti-PD-L1 antibody):

[0607] SEQ ID NO: 49 (LCDR2 of anti-PD-L1 antibody):

[0608] SEQ ID NO: 50 (LCDR3 of anti-PD-L1 antibody)

[0609] SEQ ID NO: 51 (VH of anti-PD-L1 antibody)

[0610] SEQ ID NO: 52 (VL of anti-PD-L1 antibody)

[0611] SEQ ID NO: 53 (heavy chain of anti-PD-L1 antibody)

[0612] SEQ ID NO: 54 (light chain of anti-PD-L1 antibody)

[0613] SEQ ID NO: 55 (HCDR1 of anti-SIRPa antibody 14#):

[0614] SEQ ID NO: 56 (HCDR2 of anti-SIRPa antibody 14#):

[0615] SEQ ID NO: 57 (HCDR3 of anti-SIRPa antibody 14#):

[0616] SEQ ID NO: 58 (LCDR1 of anti-SIRPa antibody 14#):

[0617] SEQ ID NO: 59 (LCDR2 of anti-SIRPa antibody 14#):

[0618] SEQ ID NO: 60 (LCDR3 of anti-SIRPa antibody 14#):

[0619] SEQ ID NO: 61 (VH of anti-SIRPa antibody 14#):

[0620] SEQ ID NO: 62 (VL of anti-SIRPa antibody 14#):

[0621] SEQ ID NO: 63 (heavy chain of anti-SIRPa antibody 14#):

[0622] SEQ ID NO: 64 (light chain of anti-SIRPa antibody 14#):

[0623] SEQ ID NO: 65 (HCDR1 of anti-CD73 antibody B11):

[0624] SEQ ID NO: 66 (HCDR2 of anti-CD73 antibody B11):

[0625] SEQ ID NO: 67 (HCDR3 of anti-CD73 antibody B11):

[0626] SEQ ID NO: 68 (LCDR1 of anti-CD73 antibody B11):

[0627] SEQ ID NO:69 (LCDR2 of anti-CD73 antibody B11):

[0628] SEQ ID NO:70 (LCDR3 of anti-CD73 antibody B11):

[0629] SEQ ID NO:71 (VH of anti-CD73 antibody B11):

[0630] SEQ ID NO:72 (VL of anti-CD73 antibody B11):

[0631] SEQ ID NO:73 (heavy chain of anti-CD73 antibody B11):

[0632] SEQ ID NO:74 (light chain of anti-CD73 antibody B11):

[0633] SEQ ID NO:75 (heavy chain variable region of an anti-HER2 antibody):

[0634] SEQ ID NO:76 (light chain variable region of an anti-HER2 antibody):

[0635] SEQ ID NO:77 (heavy chain of an anti-HER2 antibody):

[0636] SEQ ID NO:78 (light chain of an anti-HER2 antibody):

[0637] SEQ ID NO:79 (heavy chain variable region of an anti-Trop2 antibody):

[0638] SEQ ID NO:80 (light chain variable region of an anti-Trop2 antibody):

[0639] SEQ ID NO:81 (heavy chain of an anti-Trop2 antibody):

[0640] SEQ ID NO:82 (light chain of an anti-Trop2 antibody):

[0641] SEQ ID NO:83 (HCDR1 of an anti-Trop2 antibody):

[0642] SEQ ID NO: 84 (anti-Trop2 antibody HCDR2):

[0643] SEQ ID NO: 85 (anti-Trop2 antibody HCDR3):

[0644] SEQ ID NO: 86 (anti-Trop2 antibody LCDR1:

[0645] SEQ ID NO: 87 (anti-Trop2 antibody LCDR2):

[0646] SEQ ID NO: 88 (anti-Trop2 antibody LCDR3):

[0647] SEQ ID NO: 89 (HCDR2 of murine antibody of antibody A):

[0648] SEQ ID NO: 90 (HCDR1 of anti-HER2 antibody):

[0649] SEQ ID NO: 91 (HCDR2 of anti-HER2 antibody):

[0650] SEQ ID NO: 92 (HCDR3 of anti-HER2 antibody):

[0651] SEQ ID NO: 93 (LCDR1 of anti-HER2 antibody):

[0652] SEQ ID NO: 94 (LCDR2 of anti-HER2 antibody):

[0653] SEQ ID NO: 95 (LCDR3 of anti-HER2 antibody):

[0654] All documents mentioned in this disclosure are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it is understood that various modifications or alterations can be made to the above-disclosed teachings without departing from the scope of the disclosure. Such equivalent forms are considered to be within the scope of the application as defined in the appended claims.

Claims

1. A method of treating a disease, comprising administering to a subject in need thereof an anti-LIV-1 antibody-drug conjugate and another therapeutic agent, wherein the anti-LIV-1 antibody-drug conjugate comprises an anti-LIV-1 antibody and a drug; preferably, the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein: a) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 1, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 2; b) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 4; c) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 5, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 6; or d) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 28, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 29; the amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat, Chothia, AbM, or IMGT numbering system; more preferably, a-1) the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 7, HCDR2 as shown in SEQ ID NO: 8, and HCDR3 as shown in SEQ ID NO: 9, and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 10, LCDR2 as shown in SEQ ID NO: 11, and LCDR3 as shown in SEQ ID NO: 12; b-1) the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 13, HCDR2 as shown in SEQ ID NO: 14, and HCDR3 as shown in SEQ ID NO: 15, and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 16, LCDR2 as shown in SEQ ID NO: 17, and LCDR3 as shown in SEQ ID NO: 18; c-1) the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 19, HCDR2 as shown in SEQ ID NO: 20, and HCDR3 as shown in SEQ ID NO: 21, and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 22, LCDR2 as shown in SEQ ID NO: 23, and LCDR3 as shown in SEQ ID NO: 24; or d-1) the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 25, HCDR2 as shown in SEQ ID NO: 26, and HCDR3 as shown in SEQ ID NO: 27, and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 28, LCDR2 as shown in SEQ ID NO: 29, and LCDR3 as shown in SEQ ID NO:

30. ​ ​ ​ ​ ​ ​ ​ ​ d-1) the heavy chain variable region comprises HCDR1 as set forth in SEQ ID NO: 7, HCDR2 as set forth in SEQ ID NO: 89, and HCDR3 as set forth in SEQ ID NO: 9, and the light chain variable region comprises LCDR1 as set forth in SEQ ID NO: 10, LCDR2 as set forth in SEQ ID NO: 11, and LCDR3 as set forth in SEQ ID NO:

12.

2. The method of claim 1, wherein the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein: a-2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 32, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 32; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2; b-2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 4; c-2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 5; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 6; or d-2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 28; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 29; Preferably, the anti-LIV-1 antibody comprises a heavy chain constant region as set forth in SEQ ID NO: 26 and / or a light chain constant region as set forth in SEQ ID NO: 27; more preferably, the anti-LIV-1 antibody comprises a light chain as set forth in SEQ ID NO: 30 and a heavy chain as set forth in SEQ ID NO:

31.

3. The method of claim 1 or 2, wherein the anti-LIV-1 antibody is an antigen binding fragment, wherein the antigen binding fragment is selected from any one of the following: Fab, scFv, Fv, Fab’, F(ab’)2, single domain antibody, scFab, linear antibody, and multispecific antibody.

4. The method of any one of claims 1 to 3, wherein the anti-LIV-1 antibody-drug conjugate has the structure of [Formula I] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z [Formula I] wherein: Ab is the anti-LIV-1 antibody of any one of claims 1 to 3; (AG)k is a conjugation group, wherein AG is selected from the group consisting of ###0001### wherein the wavy line indicates the point of attachment to Ab, and k is 0 or 1 ; (L1)x is a first linker group, wherein L1 is -(CH2-)t-C(=0)- or -(NH)j-(CH2CH20)n-(CH2)q-C(=0)-, x is 0 or 1, t is an integer from 1 to 10, preferably from 1 to 7, j is an integer from 0 to 4, n is an integer from 2 to 8, and q is an integer from 1 to 6; (OP)m is an enzyme-cleavable oligopeptide, wherein m is 0 or an integer from 2 to 10; preferably, (OP)m is an oligopeptide selected from the group consisting of valine, citrulline, alanine, glycine, aspartic acid, tyrosine, phenylalanine, proline, isoleucine, lysine, serine, glutamic acid, threonine or asparagine; optionally, the phenolic hydroxyl group of tyrosine or the amide group of asparagine of the oligopeptide is glycosylated, preferably with glucuronic acid, N-acetylglucosamine, glucose or galactose; more preferably, (OP)m is a dipeptide, tripeptide or tetrapeptide; (L2)y is a second linker group, wherein L2 is -NH-Ph-CH2-0-C(=0)-, which phenyl ring (Ph) optionally contains a hydroxyl substituent, and y is 0 or 1 ; preferably, L2 is p-aminobenzyloxy carbonyl or p-hydroxy-m-aminobenzyloxy carbonyl, and optionally, the hydroxyl group on the phenyl ring of L2 is glycosylated, preferably with glucuronic acid, N-acetylglucosamine, glucose or galactose; D is a drug, wherein the drug is a cytotoxic compound, an immunomodulator, an enzyme or a hormone inhibitor; and z is the ratio of drug to antibody, selected from an integer or a decimal number from 1 to 24.

5. The method of claim 4, wherein: (AG)k is a conjugation group, AG is selected from the group consisting of ###0002### the wavy line indicates the point of attachment to Ab, and k is 1 ; (L1)x is a first linker group, wherein L1 is -(NH)j-(CH2CH20)n-(CH2)q-C(=0)-, wherein x is 0 or 1, j is an integer from 0 to 4, n is an integer from 2 to 8, and q is an integer from 1 to 6; (OP)m is an enzyme-cleavable oligopeptide, wherein m is 0 or an integer from 2 to 10; preferably, (OP)m is an oligopeptide selected from the group consisting of valine, citrulline, alanine or tyrosine; optionally, the phenolic hydroxyl group of tyrosine of the oligopeptide is glycosylated with glucuronic acid, N-acetylglucosamine, glucose or galactose. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ (L2)y is a second linker group, wherein L2 is p-aminobenzyloxycarbonyl or p-hydroxy-m- aminobenzyloxycarbonyl, optionally the hydroxyl on the phenyl ring of L2 is glycosylated with a glucuronic acid, N-acetylglucosamine, glucose or galactose, and y is 1; D is a drug, wherein the drug is a cytotoxic compound; and z is an integer or a decimal number from 1 to 10.

6. The method of any one of claims 1 to 5, wherein the drug of the anti-LIV-1 antibody-drug conjugate is a microtubulin inhibitor or a topoisomerase I inhibitor; preferably, the drug is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoid, SN-38 and exatecan.

7. The method of claim 6, wherein the anti-LIV-1 antibody-drug conjugate has a structure selected from the following: Preferably, the Ab comprises a light chain as set forth in SEQ ID NO: 30 and a heavy chain as set forth in SEQ ID NO: 31, and z is a decimal number or an integer from 2 to 8.

8. The method of any one of claims 1 to 7, wherein the other therapeutic agent is an antibody or antibody-drug conjugate against a target selected from the group consisting of EGFR, VEGF, VEGFR2, CTLA-4, PD-L1, PD-1, HER2, CD20, CD47, SIRPalpha, CD73, LAG3, TIGIT, CD27, OX40, ICOS, BTLA, TIM3, BCMA, c-MET, TAA-1 / 2 / 3, HER3, B7H3, B7H4, Claudin 18.2, c-MET, Nectin-4, ROR1, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, PSMA, ROR1, folate receptor-a, mesothelin, ENPP3, guanylyl cyclase C, SLC44A4, NaPi2b, CD70, Mucin 1, STEAP1, Nectin 4, 5T4, SLTRK6, SC-16, P-cadherin, PSMA, Fibronectin extra domain B, Endothelin receptor ETB, Tenascin c, Collagen IV, Periostin, CD30, CD79b, CD22, CD138, CD37, CD33, CD74, CD19 and CD98; Preferably, the other therapeutic agent is an anti-HER2 antibody, an anti-HER2 antibody-drug conjugate, an anti-Trop2 antibody or an anti-Trop2 antibody-drug conjugate.

9. The method of claim 8, wherein: the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 75, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 76; the anti-Trop2 antibody or anti-Trop2 antibody-drug conjugate comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 79, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 80; Preferably, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain variable region as set forth in SEQ ID NO: 75 and a light chain variable region as set forth in SEQ ID NO: 76; the anti-Trop2 antibody or anti-Trop2 antibody-drug conjugate comprises a heavy chain variable region as set forth in SEQ ID NO: 79 and a light chain variable region as set forth in SEQ ID NO: 80; More preferably, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain as set forth in SEQ ID NO: 77 and a light chain as set forth in SEQ ID NO: 78; the anti-Trop2 antibody or anti-Trop2 antibody-drug conjugate comprises a heavy chain as set forth in SEQ ID NO: 81 and a light chain as set forth in SEQ ID NO:

82.

10. The method of claim 8 or 9, wherein the other therapeutic agent is an anti-HER2 antibody-drug conjugate or an anti-Trop2 antibody-drug conjugate, and the drug is a tubulin inhibitor or a topoisomerase I inhibitor; preferably, wherein the drug is selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoid, SN-38, irinotecan, topetecan, and exatecan.

11. The method of claim 8, wherein the anti-HER2 antibody or anti-HER2 antibody-drug conjugate is selected from trastuzumab, pertuzumab, zanidatamab, Zenocutuzumab, Trastuzumab Deruxtecan, disitamab vedotin, A166 (trastuzumab botin), T-DM1 (enhercevatamab), RC48-ADC, SHR-A1811, or a combination thereof. wherein the anti-Trop2 antibody or anti-Trop2 antibody-drug conjugate is selected from the group consisting of Sacituzumab govitecan, Datopotamab Deruxtecan, SKB-264, OBI-992, 9MW-2921, MHB-036C, DB-1305, BIO-106, SHR-A1921, GQ-1010, LCB-84, JSKN-016, DXC-1002, IBI-130, FZ-AD004, BL-M02D1, RN927C / PF06664178, BAT8008, JS108 (DAC-002), FDA018, ESG-401, DB1305, or a combination thereof.

12. The method of any one of claims 1 to 7, wherein the additional therapeutic agent is an immune checkpoint inhibitor; preferably, the immune checkpoint inhibitor is a PD-1 binding antagonist, a PD-L1 binding antagonist, a CD47 binding antagonist, a SIRPa binding antagonist, or a CD73 binding antagonist.

13. The method of claim 12, wherein: the PD-1 binding antagonist is an anti-PD-1 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, and HCDR3 of SEQ ID NO: 37, and the light chain variable region comprises LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 40; the PD-L1 binding antagonist is an anti-PD-L1 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 of SEQ ID NO: 45, HCDR2 of SEQ ID NO: 46, and HCDR3 of SEQ ID NO: 47, and the light chain variable region comprises LCDR1 of SEQ ID NO: 48, LCDR2 of SEQ ID NO: 49, and LCDR3 of SEQ ID NO: 50; the SIRPa binding antagonist is an anti-SIRPa antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 of SEQ ID NO: 55, HCDR2 of SEQ ID NO: 56, and HCDR3 of SEQ ID NO: 57, and the light chain variable region comprises LCDR1 of SEQ ID NO: 58, LCDR2 of SEQ ID NO: 59, and LCDR3 of SEQ ID NO: 60; The CD73 binding antagonist is an anti-CD73 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 of SEQ ID NO: 65, HCDR2 of SEQ ID NO: 66, and HCDR3 of SEQ ID NO: 67, and the light chain variable region comprises LCDR1 of SEQ ID NO: 68, LCDR2 of SEQ ID NO: 69, and LCDR3 of SEQ ID NO: 70; Preferably, the anti-PD-1 antibody comprises a heavy chain variable region of SEQ ID NO: 41 and a light chain variable region of SEQ ID NO: 42; the anti-PD-L1 antibody comprises a heavy chain variable region of SEQ ID NO: 51 and a light chain variable region of SEQ ID NO: 52; the anti-SIRPa antibody comprises a heavy chain variable region of SEQ ID NO: 61 and a light chain variable region of SEQ ID NO: 62; the anti-CD73 antibody comprises a heavy chain variable region of SEQ ID NO: 71 and a light chain variable region of SEQ ID NO: 72; More preferably, the anti-PD-1 antibody comprises a heavy chain of SEQ ID NO: 43 and a light chain of SEQ ID NO: 44; the anti-PD-L1 antibody comprises a heavy chain of SEQ ID NO: 53 and a light chain of SEQ ID NO: 54; the anti-SIRPa antibody comprises a heavy chain of SEQ ID NO: 63 and a light chain of SEQ ID NO: 64; the anti-CD73 antibody comprises a heavy chain of SEQ ID NO: 73 and a light chain of SEQ ID NO:

74.

14. The method of claim 12, wherein the PD-1 binding antagonist is selected from the group consisting of pembrolizumab, zimberelimab, nivolumab, cemiplimab, pidilizumab, AMG-404, MEDI0680, spartalizumab, tislelizumab, toripalimab, genolimzumab, camrelizumab, sintilimab, dostarlimab, lambrolizumab, sasanlimab, cetrelimab, serplulimab, retifanlimab, balstilimab, prolgolimab, budigalimab, vopratelimab, Retifanlimab, Cadonilimab, BMS-986213 (Relatlimab + Nivolumab), ivonescimab, geptanolimab, Iparomlimab, Pucotenlimab, AK-105, CS-1003, BI-754091, LZM-009, Sym-021, BAT-1306, PD1-PIK, tebotelimab, RO-7247669 (PD-1 / LAG-3), FS-118 (LAG-3 / PD-L1), RO-7121661 (PD-1 / TIM-3), RG7769 (PD-1 / TIM-3), PF-06936308 (PD-1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), XmAb-20717 (PD-1 / CTLA4), AK-104 (CTLA4 / PD-1), MEDI-5752 (CTLA4 / PD-1), SSGJ-707 (PD-1 / VEGF), MK-2010 (PD-1 / VEGF), JS207 (PD-1 / VEGF), and CTX-10726 (PD-1 / VEGF); the PD-L1 binding antagonist is selected from the group consisting of atezolizumab, avelumab, envafolimab, durvalumab, adebrelimab, BMS-936559 (MDX1105), cosibelimab, lodapolimab, garivulimab, envafolimab, opucolimab, manelimab, CX-072, CBT-502 (TQB2450), MSB-2311, sugemalimab, A167 (KL-A167), STI-A1015 (IMC-001), FAZ-053, BMS-936559 (MDX1105), INCB086550, GEN-1046 (PD-L1 / 4-1BB), FPT-155 (CTLA4 / PD-L1 / CD28), M7824 (PD-L1 / TGFβ-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM-3 / PDL1), INBRX-105 (4-1BB / PDL1), GNS-1480 (PD-L1 / EGFR), SCTB-14 (PD-L1 / VEGF), PM8002 (PD-L1 / VEGF), IMM2518 (PD-L1 / VEGF), YBL-008 (PD-L1 / VEGF), and HB0025 (PD-L1 / VEGF); the SIRPa binding antagonist is selected from the group consisting of BR105, CC-95251, HCB-101, BI765063, GS-0189, IBI397, BI-770371, APX-700, ES-004, ADU1805, ELA-026, BYON-4228, ALX-148 (evorpacept), timdarpacept, IMM01, TTI-621, TTI-622, JMT601 (CPO107), SL-172154, SIRPa-F8, JMT601 (CPO107), SS002M91, SIRPa-lgG4-Fc-Fc, and hCD172a (SIRPa)-Fc-LIGHT; The CD47-binding antagonist is selected from the group consisting of magrolimab, lemzoparlimab, letaplimab, ligufalimab, AO-176, simridarlimab (IBI-322), gentulizumab, ZL-1201, IMC-002, SRF-231, CC-90002 (also known as INBRX-103), NI-1701 (also known as TG-1801), STI-6643, SHR-1603, HLX-24, LQ-001, B6H12, TAY-018, PT-240, 1F8-GMCSF, SY-102, and KD-015. The CD73-binding antagonist is selected from the group consisting of BR101, oleclumab, mupadolimab, HB-0045, JAB-BX102, AK-119, Sym-024, uliledlimab, and IBI-325.

15. The method of any one of claims 1 to 7, wherein the additional therapeutic agent is a chemotherapeutic agent; preferably, the chemotherapeutic agent is a platinum, an anthracycline, a taxane, or a nucleoside analogue; more preferably, the platinum is carboplatin, the anthracycline is doxorubicin, the taxane is paclitaxel, and the nucleoside analogue is gemcitabine.

16. The method of any one of claims 1 to 7, wherein the additional therapeutic agent is an inhibitor of CDK4 and CDK6 (CDK4 / 6 inhibitor); preferably, the CDK4 / 6 inhibitor is selected from the group consisting of ribociclib, abemaciclib, ribociclib, trilaciclib, roscovitine, avosentan, ronablin, pyroxicil, Trilaciclib, riviciclib, milciclib, RGB-286638, NSN3106729, PHA-793887, R547, indirubin, NU6102, bohemine, CDK9-IN-7, CGP60474, purvalanol A, PF-06873600, nimbolide, FN-1501, AG-024322, ON123300, G1T28, G1T38, AMG925, SHR-6390, BPI-1178, BPI-16350, FCN437, BEBT-209, Ty-302, TQB-3616, HS-10342, PF-06842874, CS-2002, MM-D37K, CDK4 / 6-IN-2, SU9516, and AT7519.

17. The method of any one of claims 1 to 16, wherein the anti-LIV-1 antibody-drug conjugate and the other therapeutic agent are administered simultaneously, sequentially or separately.

18. The method of any one of claims 1 to 17, wherein the disease is a tumor or cancer; Preferably, the tumor or cancer is selected from the group consisting of breast cancer, ovarian cancer, melanoma, prostate cancer, endometrial cancer, pancreatic cancer, lung cancer, cervical cancer, squamous cell carcinoma, non-small cell lung cancer, small cell lung cancer, gastric / gastroesophageal junction (GEJ) adenocarcinoma, esophageal squamous cell carcinoma, bladder cancer, hepatocellular cancer, gastric cancer, glioblastoma, renal cell carcinoma, gastrointestinal tumor, colorectal cancer, glioma, mesothelioma, head and neck cancer, head and neck squamous cell carcinoma, esophageal cancer, skin cancer, uterine cancer, triple negative breast cancer, triple positive breast cancer, HER2 positive breast cancer, hormone receptor positive breast cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphoblastic leukemia, anaplastic large cell lymphoma, and multiple myeloma; More preferably, the tumor or cancer is selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, and melanoma.

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