Anti-LIV-1 antibody-drug conjugate-based treatment of drug-resistant cancer

Abstract

A novel treatment method for a drug-resistant cancer, particularly an acquired drug-resistant cancer. Said method comprises administering an anti-LIV-1 antibody-drug conjugate to a subject in need thereof.

Description

Treatment of drug-resistant cancer based on anti-LIV-1 antibody-drug conjugate TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biological medicine, and relates to the treatment of drug-resistant cancer, especially acquired drug-resistant cancer, based on antibody-drug conjugate formed by linking anti-LIV-1 antibody with cytotoxic drug through linker structure. 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 disclosed herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually incorporated by reference. To the extent there is a contradiction or conflict between the definitions or uses of terms in an incorporated publication, patent, or patent application and the definitions provided herein, the definitions provided herein control.

[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 multiple 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] Cancer drug resistance is a major obstacle in the treatment of cancer. It is reported that about 15% of breast cancer patients overexpressing HER2 respond to trastuzumab after receiving extensive existing anti-cancer treatment, and about 85% of patients in this population have no response or only weak response to trastuzumab treatment. Such drug resistance mechanisms include drug efflux, acquisition of drug binding defective mutant of target, occupation of alternative survival pathways, and epigenetic changes. For example, RAF inhibitors are used to target malignant melanoma with B-raf V600E mutations, however, their clinical success is hindered by acquired drug resistance. Therefore, new treatment methods are needed to address the heterogeneity in cancer cell populations and drug resistance to drug treatment. SUMMARY

[0006] The inventors have found, through extensive and in-depth research, that anti-LIV-1 antibody-drug conjugates (sometimes also referred to herein as "anti-LIV-1-ADCs") can be used to treat cancer that is resistant to anti-cancer therapeutic agents, in particular to treat cancer that is resistant to treatment with a topoisomerase I inhibitor or a derivative thereof or an antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof.

[0007] In one aspect, the present disclosure provides a method of treating cancer, comprising administering an anti-LIV-1 antibody-drug conjugate to a subject in need thereof, wherein the subject has cancer that is resistant to an anti-cancer therapeutic agent. In some embodiments, the anti-cancer therapeutic agent is: (i) a topoisomerase I inhibitor or a derivative thereof; or (ii) an antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof (i.e., the drug moiety of the antibody-drug conjugate is a topoisomerase I inhibitor or a derivative thereof). In some embodiments, the anti-cancer therapeutic agent is DXD or a derivative thereof or SN-38 or a derivative thereof. In some embodiments, the anti-cancer therapeutic agent is an antibody targeting HER2 or TROP2 of an antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof. In some embodiments, the anti-cancer therapeutic agent is an anti-HER2 antagonist. In some embodiments, the anti-cancer therapeutic agent is an anti-HER2 antibody. In some embodiments, the anti-cancer therapeutic agent is an anti-HER2 antibody-drug conjugate. In some embodiments, the anti-cancer therapeutic agent is trastuzumab deruxtecan. In some embodiments, the anti-cancer therapeutic agent is Dato-DXD.

[0008] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0009] 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;

[0010] 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;

[0011] 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

[0012] 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;

[0013] The amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat, Chothia, AbM, or IMGT numbering system.

[0014] 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 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; 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 those 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.

[0015] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0016] 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;

[0017] 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;

[0018] 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

[0019] 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.

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

[0021] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0022] 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 comprises 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 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2;

[0023] b-2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 3, or comprises 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 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 4;

[0024] 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

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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 acids at any position, wherein the substitution can be conservative substitution or non-conservative substitution.

[0030] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0031] the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 1, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0032] In some embodiments, the anti-LIV-1 antibody 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 set forth in SEQ ID NO: 26 or a variant thereof. In some embodiments, the light chain constant region comprises an amino acid sequence 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.

[0033] In some embodiments, the anti-LIV-1 antibody comprises a light chain set forth in SEQ ID NO: 30 and a heavy chain set forth in SEQ ID NO: 31.

[0034] In some embodiments, the anti-LIV-1 antibody is an antigen-binding fragment, wherein the antigen-binding fragment is selected from the group consisting of: Fab, scFv, Fv, Fab', F(ab')2, single domain antibody, scFab, linear antibody, and multi-specific antibody.

[0035] In some embodiments, the anti-LIV-1 antibody-drug conjugate has a structure as set forth in [Formula I] below:

[0036] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0037] [Formula I]

[0038] wherein:

[0039] Ab is any of the aforementioned anti-LIV-1 antibodies;

[0040] (AG)k is a coupling group, wherein AG is selected from the group consisting of:

[0041] wherein the wavy line indicates attachment to Ab, and k is 0 or 1;

[0042] (L1)x is a first linker group, L1 is -(CH2-)t-C(=O)- or -(NH)j-(CH2CH2O)n-(CH2)q-C(=O)-, where 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;

[0043] (OP)m is an enzyme-cleavable oligopeptide, where 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;

[0044] (L2)y is a second linker group, where L2 is -NH-Ph-CH2-O-C(=O)-, optionally containing a hydroxyl substituent on the phenyl ring (Ph), y is 0 or 1, optionally, the hydroxyl on the phenyl ring is glycosylated; in some embodiments, L2 is p-aminobenzyloxy carbonyl, or p-hydroxy-m-aminobenzyloxy carbonyl; in some embodiments, the hydroxyl on the phenyl ring is glycosylated with glucuronic acid, N-acetylglucosamine, glucose, or galactose;

[0045] D is a drug, e.g., a cytotoxic compound, an immunomodulator, an enzyme, or a hormone inhibitor, preferably a cytotoxic compound;

[0046] 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.

[0047] 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. In some embodiments, 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.

[0048] In some embodiments, L1is -(CH2)2-C(=O)-, -(CH2)3-C(=O)-, -(CH2)4-C(=O)-, or -(CH2)5-C(=O)-.

[0049] In some embodiments, L1is -(CH2CH2O)4-(CH2)2-C(=O)- or -NH-(CH2CH2O)4-(CH2)2-C(=O)-.

[0050] In some embodiments, x is 0, i.e., L1is absent, and (AG)k and (OP)m are directly linked.

[0051] 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.

[0052] In some embodiments, (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, e.g., a dipeptide, tripeptide, or tetrapeptide formed therefrom; optionally, the phenolic hydroxyl of tyrosine or the amide group of asparagine is glycosylated, preferably glycosylated with glucuronic acid, N-acetylglucosamine (GlcNAc), glucose, or galactose.

[0053] The "glycosylation" refers to the transfer of a glycosyl or oligosaccharide group to a hydroxyl or amino group of a compound, accomplished by chemical or enzymatic means.

[0054] In some embodiments, glycosylation refers to the structural modification of an amino acid residue in the 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 sugar.

[0055] In some embodiments, (OP)m is an oligopeptide formed from valine and citrulline. In some embodiments, (OP)m is a -valine-citrulline- dipeptide.

[0056] 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 -glycine-glycine-tyrosine- tripeptide, optionally, the phenolic hydroxyl of tyrosine is glycosylated.

[0057] In some embodiments, (OP)m is a dipeptide formed by alanine and asparagine, wherein the amide group of asparagine is glycosylated.

[0058] In some embodiments, m is 0, i.e., (OP)m is absent, (L1)x and (L2)y are directly linked.

[0059] In some embodiments, y in (L2)y is 1, and L2 is -NH-Ph-CH2-O-C(=O)-, wherein the phenyl ring optionally contains a hydroxyl substituent; preferably, L2 is p-aminobenzyloxy carbonyl or p-hydroxy-m-aminobenzyloxy carbonyl; optionally, the hydroxyl on the phenyl ring is glycosylated, preferably glycosylated with glucuronic acid, N-acetylglucosamine (GlcNAc), glucose, or galactose.

[0060] In some embodiments, L2 is p-hydroxy-m-aminobenzyloxy carbonyl, wherein the hydroxyl on the phenyl ring is glycosylated with glucuronic acid, N-acetylglucosamine (GlcNAc), glucose, or galactose.

[0061] In some embodiments, L2 is p-aminobenzyloxy carbonyl.

[0062] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0063] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0064] [Formula I]

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0071] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0072] [Formula I]

[0073] 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-dipeptide 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 of L2 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.

[0074] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0075] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0076] [Formula I]

[0077] 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 from 1 to 4, n is an integer from 2 to 8, and q is an integer from 1 to 6; m is 0; L2 is p-aminobenzyloxycarbonyl (PAB) or hydroxy-m-aminobenzyloxycarbonyl, optionally wherein the hydroxyl on the phenyl ring of L2 is 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.

[0078] In some embodiments, L1 is -NH-(CH2CH2O)4-(CH2)2-C(=O)-.

[0079] In some embodiments, (OP)m is -valine-citrulline-dipeptide.

[0080] In some embodiments, (OP)m is -glycine-glycine-tyrosine-tripeptide.

[0081] In some embodiments, (OP)m is -glycine-glycine-tyrosine-tripeptide, wherein the phenolic hydroxyl of the tyrosine is glycosylated with glucose.

[0082] In some embodiments, m is 0, i.e., (OP)m is absent, and (L1)x and (L2)y are directly linked.

[0083] In some embodiments, L2 is p-hydroxy-m-aminobenzyloxycarbonyl, wherein the hydroxyl on the phenyl ring is glycosylated with glucuronic acid.

[0084] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0085] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0086] [Formula I]

[0087] 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 from 1 to 7, or L1 is -(NH)j-(CH2CH2O)n-(CH2)q-(C=O)-, wherein 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 -valine-citrulline-dipeptide, L2 is PAB; and k is 1, x is 1, y is 1, and D and z are as defined above.

[0088] In some embodiments, L1is -(CH2)2-C(=0)-.

[0089] In some embodiments, L1is -(CH2)4-C(=0)-.

[0090] In some embodiments, L1is -(CH2CH2O)4-(CH2)2-C(=0)-.

[0091] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0092] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0093] [Formula I]

[0094] 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, 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 between 1 and 10, preferably 3.5-4.5.

[0095] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0096] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0097] [Formula I]

[0098] 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, 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 between 1 and 10, preferably 3.5-4.5.

[0099] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0100] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0101] [Formula I]

[0102] 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 an integer from 1 to 10, preferably 3.5 to 4.5.

[0103] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0104] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0105] [Formula I]

[0106] 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 an integer from 1 to 10, preferably 3.5 to 4.5.

[0107] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0108] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0109] [Formula I]

[0110] 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 an integer from 1 to 10, preferably 3.5 to 4.5.

[0111] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0112] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0113] [Formula I]

[0114] 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.

[0115] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0116] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0117] [Formula I]

[0118] 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.

[0119] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0120] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0121] [Formula I]

[0122] 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.

[0123] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0124] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0125] [Formula I]

[0126] wherein Ab represents any of the foregoing 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 from 1 to 10, preferably 3.5 to 4.5.

[0127] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the structure of [Formula I] as follows:

[0128] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0129] [Formula I]

[0130] wherein Ab represents any of the foregoing 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 from 1 to 10, preferably 3.5 to 4.5.

[0131] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the structure of [Formula I] as follows:

[0132] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0133] [Formula I]

[0134] wherein Ab represents any of the foregoing 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 from 1 to 10, preferably 3.5 to 4.5.

[0135] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the structure of [Formula I] as follows:

[0136] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0137] [Formula I]

[0138] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is -C(=O)-, k is 1; L1 is -(CH2)4-C(=O)-, 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 integer number from 1 to 10, preferably 1 to 2.

[0139] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure [Formula I]:

[0140] Ab-[(AG)k-(L1)x-(OP)m-(L2)y-D]z

[0141] [Formula I]

[0142] wherein Ab represents any of the aforementioned anti-LIV-1 antibodies; AG is -C(=O)-, k is 1; L1 is -(CH2-CH2-O)4-(CH2)2-C(=O)-, 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 integer number from 1 to 10, preferably 1 to 2.

[0143] In some embodiments, the -(AG)k-(L1)x-(OP)p-(L2)y- in [Formula I] has the following structure, wherein "1" represents the connection to the anti-LIV-1 antibody Ab and "2" represents the connection to the drug D.

[0144] In some embodiments, the drug of the anti-LIV-1 antibody-drug conjugate is a tubulin inhibitor or a topoisomerase I inhibitor.

[0145] In some embodiments, the drug of the anti-LIV-1 antibody-drug conjugate is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoid (including but not limited to DM1, DM4), SN-38, or exatecan.

[0146] In some embodiments, the drug of the anti-LIV-1 antibody-drug conjugate is monomethyl auristatin E (MMAE).

[0147] In some embodiments, the [(AG)k-(L1)x-(OP)m-(L2)y-D] in [Formula I] has the following structure:

[0148] wherein the wavy line represents the connection to the anti-LIV-1 antibody Ab.

[0149] In some embodiments, the [(AG)k-(L1)x-(OP)m-(L2)y-D] in [Formula I] has the following structure:

[0150] wherein the wavy line indicates attachment to the anti-LIV-1 antibody Ab.

[0151] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure:

[0152] In some embodiments, the anti-LIV-1 antibody-drug conjugate has the following structure:

[0153] In some embodiments, the heavy chain variable region of the Ab 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 of the Ab 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.

[0154] 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.

[0155] 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.

[0156] In some embodiments, the antibody portion of the anti-LIV-1 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 the heavy chain variable region and the light chain variable region of the foregoing anti-LIV-1 antibody, 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, etc.

[0157] In some embodiments, the anti-cancer therapeutic agent is:

[0158] (i) a topoisomerase I inhibitor or a derivative thereof; or

[0159] (ii) an antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof (i.e., the drug moiety of the antibody-drug conjugate is a topoisomerase I inhibitor or a derivative thereof).

[0160] In some embodiments, the topoisomerase I inhibitor includes, but is not limited to, DXD (Exatecan derivative), or a derivative thereof, SN-38 or a derivative thereof, Exatecan or a derivative thereof, Rubitecan or a derivative thereof, Topotecan or a derivative thereof, Irinotecan (Topotecan) or a derivative thereof, Camptothecin or a derivative thereof. In some embodiments, the topoisomerase I inhibitor is DXD or a derivative thereof or SN-38 or a derivative thereof.

[0161] In some embodiments, the antibody of the antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof is an anti-tumor associated antigen antibody. In some embodiments, the antibody is an anti-HER2 antibody and an anti-TROP2 antibody. In some embodiments, the anti-HER2 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0162] the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 41, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 42. The amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat, Chothia, AbM, or IMGT numbering system.

[0163] In some embodiments, the anti-HER2 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0164] the heavy chain variable region comprises HCDR1 as set forth in SEQ ID NO: 35, HCDR2 as set forth in SEQ ID NO: 36, and HCDR3 as set forth in SEQ ID NO: 37, and the light chain variable region comprises LCDR1 as set forth in SEQ ID NO: 38, LCDR2 as set forth in SEQ ID NO: 39, and LCDR3 as set forth in SEQ ID NO: 40.

[0165] In some embodiments, the anti-HER2 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.

[0166] In some embodiments, the anti-HER2 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.

[0167] In some embodiments, the antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof is Trastuzumab Deruxtecan.

[0168] In some embodiments, the anti-TROP2 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0169] the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 51, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 52. The amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat, Chothia, AbM, or IMGT numbering system.

[0170] In some embodiments, the anti-TROP2 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0171] the heavy chain variable region comprises HCDR1 as set forth in SEQ ID NO: 45, HCDR2 as set forth in SEQ ID NO: 46, and HCDR3 as set forth in SEQ ID NO: 47, and the light chain variable region comprises LCDR1 as set forth in SEQ ID NO: 48, LCDR2 as set forth in SEQ ID NO: 49, and LCDR3 as set forth in SEQ ID NO: 50.

[0172] In some embodiments, the anti-TROP2 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.

[0173] In some embodiments, the anti-TROP2 antibody comprises a heavy chain as set forth in SEQ ID NO: 53 and a light chain as set forth in SEQ ID NO: 54.

[0174] In some embodiments, the antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof is Dato-DXD.

[0175] In some embodiments, the anti-cancer therapeutic agent is selected from the group consisting of Trastuzumab Deruxtecan, Trastuzumab Rezetecan, JSKN-003 (Anbenitamab repodatecan), IBI-354, TQB-2101, BL-M07D1, BNT-323 (Trastuzumab Pamirtecan), FDA022, GQ1005, DAN-311, T-PL1, PRO1102, MTX-1000, Dato-DXD (Dato-DXD), Sacituzumab Govitecan, sacituzumab tirumotecan, SHR-A1921, ESG-401, FDA018, DB-1305, MHB036C, BAT8008, BL-M02D1, 9MW2921, HS-20105, GQ1010, OBI-992, FZ-AD004, and DXC1002.

[0176] In some embodiments, the anti-cancer therapeutic agent is selected from the group consisting of Trastuzumab Deruxtecan and Dato-DXD (Dato-DXD).

[0177] In some embodiments, the anti-cancer therapeutic agent is a HER2 antagonist. In some embodiments, the HER2 antagonist is an anti-HER2 antibody, an anti-HER2 antibody-drug conjugate, or a small molecule HER2 inhibitor.

[0178] 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:

[0179] the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 41, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 42. The amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat, Chothia, AbM, or IMGT numbering system.

[0180] 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:

[0181] the heavy chain variable region comprises HCDR1 as set forth in SEQ ID NO: 35, HCDR2 as set forth in SEQ ID NO: 36, and HCDR3 as set forth in SEQ ID NO: 37, and the light chain variable region comprises LCDR1 as set forth in SEQ ID NO: 38, LCDR2 as set forth in SEQ ID NO: 39, and LCDR3 as set forth in SEQ ID NO: 40.

[0182] In some embodiments, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate 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.

[0183] In some embodiments, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain as set forth in SEQ ID NO: 43 and a light chain as set forth in SEQ ID NO: 44. In some embodiments, the anti-cancer therapeutic agent is trastuzumab.

[0184] In some embodiments, the anti-cancer therapeutic agent is an anti-HER2 antibody-drug conjugate.

[0185] In some embodiments, the anti-cancer 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); the topoisomerase I inhibitor includes, but is not limited to, camptothecin analogs (such as exatecan, irinotecan and SN-38), calicheamicin analogs (such as calicheamicin γ1I and N-acetyl-γ1I calicheamicin) and anthramycin analogs (such as PBD derivatives).

[0186] In some embodiments, the anti-cancer therapeutic agent is an anti-HER2 antibody-drug conjugate, wherein the drug is selected from maytansinoids, exatecan, SN-38, irinotecan, topetecan or derivatives thereof. In some embodiments, the anti-cancer therapeutic agent is an anti-HER2 antibody-drug conjugate, wherein the drug is exatecan.

[0187] In some embodiments, the anti-HER2 antibody-drug conjugate has the following structure:

[0188] 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.

[0189] In some embodiments, the anti-cancer therapeutic agent is an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody is selected from the group consisting of trastuzumab, pertuzumab, zanidatamab, Zenocutuzumab, inetetamab, margetuximab, HLX22, IAH0968, BAT1006, B002T, HK001, TrasGEX (timigutuzumab), and FS102. The above-mentioned anti-HER2 antibodies include their respective biosimilars.

[0190] In some embodiments, the anti-cancer 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), vedicitumab, trastuzumab rezetecan, SYD985 (trastuzumab duocarmazine), trastuzumab botidotin, BAT8001, TAA013, MRG002, KADCYLA® (trastuzumab vedotin), LCB14-0110, SYA1501, DB-1303, JSKN-003 (Anbenitamab repodatecan), BL-M07D1, TQB2102, GQ1005, IBI354, NCB001 (anvatabart opadotin), MM-302, DX126-262, or a combination thereof. The above-mentioned anti-HER2 antibody-drug conjugates include their respective biosimilars.

[0191] In some embodiments, the anti-cancer therapeutic agent is a small molecule HER2 inhibitor. In some embodiments, the small molecule HER2 inhibitor is selected from the group consisting of neratinib, lapatinib canertinib, zongertinib, and irbinitinib.

[0192] In some embodiments, the anti-cancer therapeutic agent is Trastuzumab Deruxtecan (DS-8201), trade name Enhertu is an antibody-drug conjugate targeting HER2; said Trastuzumab Deruxtecan includes biosimilars thereof.

[0193] In some embodiments, the anti-cancer therapeutic agent is Dato-DXD (datopotamab deruxtecan, also known as Dato-DXd), trade name Datroway, is an antibody-drug conjugate targeting TROP2; said Dato-DXD includes biosimilars thereof.

[0194] In some embodiments, the subject has a cancer that is resistant to an anti-cancer therapeutic agent, wherein the cancer is selected from the group consisting of: breast cancer, gastric cancer, lung cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, endometrial cancer, pancreatic cancer, uterine cervical cancer, squamous cell cancer, small cell lung cancer, gastric / Esophagogastric junction (GEJ) adenocarcinoma, esophageal squamous cell carcinoma, esophageal cancer, bladder cancer, salivary gland cancer, biliary tract cancer, Paget’s disease, hepatocellular carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumor, colon cancer, rectal cancer, glioma, mesothelioma, head and neck cancer, skin cancer, uterine cancer, peritoneal cancer, liver cancer, vulvar cancer, melanoma, leukemia, malignant lymphoma, sarcoma, plasmacytoma, triple-negative breast cancer, triple-positive breast cancer, HER2-positive breast cancer, hormone receptor-positive breast cancer, and multiple myeloma. In some embodiments, the resistance is acquired resistance due to treatment with an anti-cancer therapeutic agent.

[0195] In some embodiments, the resistance is not acquired resistance due to treatment with an anti-cancer therapeutic agent.

[0196] In some embodiments, the resistance is acquired resistance due to treatment with an anti-HER2 antibody.

[0197] In some embodiments, the resistance is acquired resistance due to treatment with an anti-HER2 antibody-drug conjugate.

[0198] In some embodiments, the resistance is acquired resistance due to treatment with Trastuzumab Deruxtecan (DS8201).

[0199] In some embodiments, the resistance is acquired resistance due to treatment with Trastuzumab (Herceptin).

[0200] In some embodiments, the drug resistance is a drug resistance acquired due to treatment with an antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof.

[0201] In some embodiments, the drug resistance is a drug resistance acquired due to treatment with a topoisomerase I inhibitor or a derivative thereof.

[0202] In some embodiments, the topoisomerase I inhibitor comprises, but is not limited to, DXD (Exatecan derivative), SN-38, Exatecan, Rubitecan, Topotecan, Irinotecan, and Camptothecin.

[0203] In some embodiments, the drug resistance is not a drug resistance acquired due to treatment with an antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof.

[0204] In some embodiments, the drug resistance is a drug resistance acquired due to treatment with DXD, SN-38, Irinotecan, or Topotecan.

[0205] In some embodiments, the subject has a cancer that is resistant to an anti-cancer therapeutic agent, wherein the cancer is a hematological tumor.

[0206] In some embodiments, the subject has a cancer that is resistant to an anti-cancer therapeutic agent, wherein the cancer is selected from the group consisting of non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, and anaplastic large cell lymphoma.

[0207] In some embodiments, the subject has a cancer that is resistant to an anti-cancer therapeutic agent, wherein the cancer is a solid tumor.

[0208] In some embodiments, the subject has a cancer that is resistant to an anti-cancer therapeutic agent, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, gastric cancer, ovarian cancer, endometrial cancer, and prostate cancer.

[0209] In some embodiments, the subject has a cancer that is resistant to an anti-cancer therapeutic agent, wherein the cancer is breast cancer.

[0210] In some embodiments, the subject has a cancer that is resistant to an anti-cancer therapeutic agent, wherein the cancer is triple-negative breast cancer.

[0211] In some embodiments, the subject has a cancer that is resistant to an anti-cancer therapeutic agent, wherein the cancer is unresectable locally advanced or metastatic (LA / M) triple negative breast cancer (TNBC).

[0212] In some embodiments, the cancer is a HER2-expressing cancer. In some embodiments, the cancer is a HER2-overexpressing cancer. In some embodiments, the HER2-overexpressing cancer is a cancer in which the expression of HER2 is determined to be 3+ by immunohistochemistry.

[0213] In some embodiments, the cancer is a HER2-low expressing cancer. In some embodiments, the HER2-low expressing cancer is a cancer in which the expression of HER2 is determined to be 2+ by immunohistochemistry and the expression of HER2 is determined to be negative by in situ hybridization. In some embodiments, the HER2-low expressing cancer is a cancer in which the expression of HER2 is determined to be 1+ by immunohistochemistry.

[0214] In some embodiments, the foregoing method of treating a cancer, further comprising administering to the subject another therapeutic agent.

[0215] In some embodiments, the foregoing method of treating a cancer, wherein the another therapeutic agent is an immune checkpoint inhibitor.

[0216] In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-SIRPa antibody.

[0217] In some embodiments, the anti-PD-1 antibody 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, and Pucotenlimab. The aforementioned anti-PD-1 antibodies include their respective biosimilars.

[0218] In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of: atezolizumab, avelumab, envafolimab, durvalumab, adebrelimab, cosibelimab, lodapolimab, garivulimab, envafolimab, opucolimab, manelimab, and sugemalimab. The aforementioned anti-PD-L1 antibodies include their respective biosimilars.

[0219] In some embodiments, the anti-SIRPa antibody 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, and BYON-4228. The aforementioned anti-SIRPa antibodies include their respective biosimilars.

[0220] In some embodiments, the anti-PD-1 antibody is Pembrolizumab.

[0221] In some embodiments, the anti-PD-L1 antibody is Atezolizumab.

[0222] In some embodiments, the anti-SIRPa antibody is BR105, which can be prepared by reference to WO2022121980A1.

[0223] In some embodiments, the anti-LIV-1 antibody-drug conjugate and the other therapeutic agent are administered simultaneously.

[0224] In some embodiments, the anti-LIV-1 antibody-drug conjugate and the other therapeutic agent are administered sequentially.

[0225] In some embodiments, the anti-LIV-1 antibody-drug conjugate and the other therapeutic agent are administered separately.

[0226] In another aspect, the disclosure provides an anti-LIV-1 antibody-drug conjugate for use as a medicament for treating a cancer that is resistant to an anti-cancer therapeutic agent.

[0227] In a third aspect, the disclosure provides use of an anti-LIV-1 antibody-drug conjugate in the manufacture of a medicament for treating a cancer that is resistant to an anti-cancer therapeutic agent.

[0228] In some embodiments, the anti-cancer therapeutic agent is a HER2 antagonist. In some embodiments, the anti-cancer therapeutic agent is an anti-HER2 antibody, an anti-HER2 antibody-drug conjugate, or a small molecule HER2 inhibitor. In some embodiments, the anti-cancer therapeutic agent is a topoisomerase I inhibitor. In some embodiments, the anti-cancer therapeutic agent is an antibody-drug conjugate comprising a topoisomerase I inhibitor. In some embodiments, the topoisomerase I inhibitor is DXD or SN-38. In some embodiments, the antibody of the antibody-drug conjugate comprising a topoisomerase I inhibitor targets a tumor-associated antigen. In some embodiments, the antibody of the antibody-drug conjugate comprising a topoisomerase I inhibitor targets HER2 or TROP2.

[0229] It should be understood that, within 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

[0230] Figure 1A shows the binding activity of antibody A to human LIV-1 detected by ELISA.

[0231] Figure 1B shows the binding activity of antibody B, antibody C to human LIV-1 detected by ELISA.

[0232] Figure 2 shows the binding activity of antibody-drug conjugate A-BrAcMMAE to human LIV-1 detected by ELISA.

[0233] Figure 3 shows the inhibitory activity of ADC on the proliferation of cancer cell line Calu-6 cells in vitro.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] Figure 7 shows the results of xenotransplantation of MCF7 breast cancer cell line into NSG mice, the dose and administration time are shown in the figure.

[0238] Figure 8 shows the results of xenotransplantation of HCC1806 breast cancer cell line into NSG mice, the dose and administration time are shown in the figure.

[0239] Figure 9 shows the results of xenotransplantation of PC3 prostate cancer cell line into nude mice, the dose and administration time are shown in the figure.

[0240] Figure 10 shows the results of xenotransplantation of Calu-6 lung cancer cell line into nude mice, the dose and administration time are shown in the figure.

[0241] Figure 11 shows the results of xenotransplantation of Calu-6 lung cancer cell line into nude mice, the dose and administration time are shown in the figure.

[0242] Figure 12 shows the results of the xenograft PC3 prostate cancer cell line to nude mice, the dose and administration time are shown in the figure.

[0243] Figure 13 shows the results of the xenograft PA-1 ovarian cancer cell line to nude mice, the dose and administration time are shown in the figure.

[0244] Figure 14 shows the inhibitory activity of anti-LIV-1 ADC combined with anti-PD-1 antibody on mouse A375 cell transplanted tumor.

[0245] Figure 15 shows the inhibitory activity of anti-LIV-1 ADC combined with anti-PD-L1 antibody on mouse A375 cell transplanted tumor.

[0246] Figure 16 shows the inhibitory activity of anti-LIV-1 ADC combined with anti-PD-1 antibody on mouse HCC1806 cell transplanted tumor.

[0247] Figure 17 shows the killing effect of anti-LIV-1-ADC combined with anti-SIRPa antibody on SK-BR-3 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.

[0248] Figure 18 shows the in vitro proliferation inhibition effect of deucalituzumab on NCI-H2170 and NCI-H2170 / Enhertu-R drug-resistant cells with a drug-resistant concentration of 5.23nM.

[0249] Figure 19 shows the in vitro proliferation inhibition effect of trastuzumab on NCI-H2170 and NCI-H2170 / Enhertu-R drug-resistant cells with a drug-resistant concentration of 5.23nM.

[0250] Figure 20 shows the in vitro proliferation inhibition effect of DXD on NCI-H2170 and NCI-H2170 / Enhertu-R drug-resistant cells with a drug-resistant concentration of 5.23nM.

[0251] Figure 21 shows the in vitro proliferation inhibition effect of deucalituzumab on NCI-H2170 and NCI-H2170 / Enhertu-R drug-resistant cells with a drug-resistant concentration of 13.1nM.

[0252] Figure 22 shows the in vitro proliferation inhibition effect of DXD on NCI-H2170 and NCI-H2170 / Enhertu-R drug-resistant cells with a drug-resistant concentration of 13.1nM.

[0253] Figure 23 shows the in vitro proliferation inhibition effect of anti-LIV-1 ADC and deucetaxel on NCI-H2170 / Enhertu-R resistant cells at a resistant concentration of 5.23 nM.

[0254] Figure 24 shows the in vitro proliferation inhibition effect of anti-LIV-1 ADC and deucetaxel on NCI-H2170 / Enhertu-R resistant cells at a resistant concentration of 13.1 nM.

[0255] Figure 25A shows the in vitro proliferation inhibition effect of deucetaxel on BT474 and BT474 / Enhertu-R resistant cells.

[0256] Figure 25B shows the in vitro proliferation inhibition effect of DXD on BT474 and BT474 / Enhertu-R resistant cells.

[0257] Figure 26 shows the in vitro proliferation inhibition effect of anti-LIV-1 ADC and deucetaxel on BT474 / Enhertu-R resistant cells.

[0258] Figure 27 shows the in vitro proliferation inhibition effect of deucetaxel on NCI-H2710, NCI-H2170 / Enhertu-R (no drug withdrawal) and NCI-H2170 / Enhertu-R (drug withdrawal treatment for 3 weeks) cells.

[0259] Figure 28 shows the in vitro proliferation inhibition effect of anti-LIV-1 ADC and deucetaxel on NCI-H2170 / Enhertu-R (drug withdrawal treatment for 3 weeks).

[0260] Figure 29A shows the in vitro proliferation inhibition effect of SN-38 on COLO205 and COLO205 / SN-38-R resistant cells.

[0261] Figure 29B shows the in vitro proliferation inhibition effect of irinotecan on COLO205 and COLO205 / SN-38-R resistant cells.

[0262] Figure 30A shows the in vitro proliferation inhibition effect of DXD on COLO205 and COLO205 / SN-38-R resistant cells.

[0263] Figure 30B shows the in vitro proliferation inhibition effect of exatecan on COLO205 and COLO205 / SN-38-R resistant cells.

[0264] Figure 31 shows the in vitro proliferation inhibition effect of Dato-DXD on COLO205 and COLO205 / SN-38-R resistant cells.

[0265] Figure 32 shows the in vitro proliferation inhibition effect of anti-LIV-1 ADC and SN-38 on COLO205 / SN-38-R resistant cells.

[0266] Figure 33A shows the in vitro proliferation inhibition effect of deucetaxel on HCC1954 and HCC1954 / Enhertu-R resistant cells.

[0267] Figure 33B shows the in vitro proliferation inhibition effect of DXD on HCC1954 and HCC1954 / Enhertu-R cells.

[0268] Figure 34 shows the in vitro proliferation inhibition effect of anti-LIV-1 ADC and deucetaxel on HCC1954 / Enhertu-R resistant cells. DETAILED DESCRIPTION

[0269] To better appreciate the present disclosure, the following terms are defined.

[0270] Unless otherwise indicated, all singular terms will also include the plural, the active to the passive, and vice versa.

[0271] Unless otherwise indicated, the term "about" includes values within the standard deviation of the stated value.

[0272] 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 ingredients, but do not exclude additional steps or ingredients.

[0273] A "subject" or "patient" of 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.

[0274] The term "resistant" is used to mean unresponsive to treatment with a therapeutic agent. The term can also be expressed as "non-responsive" or "non-responding." The term "resistant" can be "acquired resistance of a tumor or cancer due to treatment with a therapeutic agent (e.g., an anti-cancer agent)" or can be "intrinsic resistance of a tumor or cancer independent of treatment with a therapeutic agent (e.g., an anti-cancer agent)." A tumor that is resistant to a therapeutic agent (e.g., an anti-cancer agent) includes a tumor or cancer that is unresponsive to treatment with an anti-cancer agent and / or has a reduced ability to produce a significant response (e.g., a partial response and / or a complete response) to treatment with an anti-cancer agent.

[0275] Determining or assessing "drug resistance" is known in the art and described in the examples. Acquisition of drug resistance can be assessed by determining the growth of drug tolerant persister cells as described in Example 10. In some embodiments, the change in IC 50 , EC 50 can be indicative of drug resistance. In some embodiments, the change is greater than any of about 50%, 100%, and / or 200%. Further, changes in acquisition of drug resistance and / or maintenance of sensitivity can be assessed in vivo, for example, by assessing response to anti-cancer agent treatment, duration of response, and / or progression free time, e.g., partial response and complete response. Changes in acquisition of drug resistance and / or maintenance of sensitivity can be based on changes in the number of individuals in a population who respond to anti-cancer agent treatment, duration of response, and / or progression free time, e.g., partial response and complete response.

[0276] The term "HER2 antagonist resistant" is used to mean non-responsive to treatment with a HER2 antagonist.

[0277] The terms "HER2 receptor antagonist" and "HER2 antagonist" refer to a compound that inhibits the expression or function of a HER2 protein or gene. In the context of the present disclosure, a HER2 antagonist refers to a receptor tyrosine kinase inhibitor, in particular a HER2 receptor protein inhibitor, examples including antibodies that bind HER2, anti-HER2 antibody-drug conjugates, and small molecule inhibitors that inhibit HER2 activity.

[0278] An "anti-HER2 antibody" or "HER2 antibody" is an antibody that binds to a 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 having 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.

[0279] A suitable anti-HER2 antibody-drug conjugate is 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 (emtansine), A166 (trastuzumab botidotin), RC48-ADC, and SHR-A1811.

[0280] 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 countries or regions selected from 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.

[0281] The term "topoisomerase I inhibitor" refers to a small molecule compound that is capable of inhibiting the activity of DNA topoisomerase type I enzyme. Type I topoisomerases can catalyze changes in DNA topology via transient single-strand breaks in the DNA. Type I topoisomerases can be further classified into 1A and 1B subtypes. A description of type I topoisomerases can be found in, for example, Baker et al. (2009) Nucleic Acids Res 37(3), 693-701. Topoisomerase I inhibitors that can be used as payloads in the ADCs described herein include camptothecin (CPT)- and non-camptothecin-based inhibitors. Useful camptothecins include, for example, topotecan, irinotecan, belotecan, exatecan, and derivatives thereof. Useful non-camptothecins include, for example, indenoisoquinolines (e.g., indeno[l,2-c]isoquinoline, NSC 314622, indotecan (LMP-400), innotecan (LMP-776), phenanthridines (e.g., Topovale (ARC-111)), and indolocarbazoles (e.g., BE-13793C). In some embodiments, the topoisomerase I inhibitor is a camptothecin (e.g., irinotecan, topotecan, belotecan, or exatecan derivative, such as SN-38 or DXD). In some embodiments, the topoisomerase I inhibitor is SN-38. In some embodiments, the topoisomerase I inhibitor is DXD.

[0282] 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 (e.g., different glycoforms). 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).

[0283] The term "antibody" is intended to include any known type of natural or engineered antigen binding protein or polypeptide that includes 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, e.g., 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.

[0284] Generally, antibody fragments (or antigen binding fragments) will compete with intact antibodies for specific binding to the target, including independent 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.

[0285] Monoclonal antibodies are typically isolated and purified. This means that the antibody is 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 excess pharmaceutically acceptable carriers or other carriers 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.

[0286] 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.

[0287] Specific binding of a monoclonal antibody to its target antigen means having an affinity of at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M -1 . Specific binding is detectable in magnitude to non-specific binding, which binds to at least one unrelated target. Specific binding can be the result of a bond formed between a particular functional group or a particular spatial match (e.g., lock and key type), while non-specific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that the monoclonal antibody binds one and only one target.

[0288] Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define a class of antibodies as IgG, IgM, IgA, IgD, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids. The heavy chain also includes 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, incorporated by reference in its entirety for all purposes).

[0289] The mature variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites, bispecific or bifunctional antibodies differ. These chains all have the same general structure of relatively conserved framework regions (FR) joined by more variable 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' variable regions consist of domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids in each domain follows the Kabat, Sequences of Proteins of Immunological Interest (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) conventions. Kabat also provides a widely used numbering convention (Kabat numbering), in which corresponding residues between different heavy chains or between different light chains are assigned the same number.

[0290] 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 obtained by using the AbYsis database (www.bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cg).

[0291] In other words, when a CDR sequence under one numbering system and its position in an antibody are provided, the skilled person 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 disclosure are determined according to the Kabat numbering scheme.

[0292] 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 harboring the proper three-dimensional structure. Epitopes formed from contiguous amino acids are typically conformationally independent, while epitopes formed by three-dimensional structure are typically conformationally dependent. An epitope usually includes at least 3, and more usually, at least 5 or 8-10 amino acids that form a unique spatial conformation. Methods of determining spatial conformation of epitopes 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).

[0293] To distinguish between conservative and non-conservative substitutions of amino acids, the following groups are useful: 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 (small, uncharged polar side chains): 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.

[0294] Sequence alignment identifies the maximum alignment of the antibody sequence according to the Kabat numbering convention. After alignment, if the structure region of the antibody to be tested (e.g., the entire mature variable region of the heavy chain or light chain) is compared with the same region of the reference antibody, the percentage of similarity of the compared sequences of the antibody to be tested and the reference antibody is the number of positions occupied by the same amino acids in the regions of the antibody to be tested and the reference antibody divided by the total number of aligned positions of the two regions, without counting gaps, multiplied by 100 to convert to percentage.

[0295] A composition or method "comprising" one or more stated elements can include other elements not specifically stated. For example, a composition comprising an antibody can also contain the antibody alone or in combination with other ingredients.

[0296] The specification of a range of values includes all integers or fractions within the range or defining the range.

[0297] 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 a complex between an antibody and antigen, Clq of the complement system binds to the Fc region of the antibody. Upon binding of Clq to cell-bound antibody, 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 the antibody-coated cell. 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).

[0298] 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.

[0299] 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.

[0300] 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.

[0301] "Cytotoxic effect" refers to an effect that depletes, eliminates, and / or kills 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.

[0302] "Cell inhibitory effect" refers to an inhibition of cell proliferation. "Cell inhibitor" refers to an agent that has an inhibitory effect on cell proliferation, thereby inhibiting the growth and / or expansion of a particular cell subpopulation. Cell inhibitors can be conjugated to antibodies or administered in conjunction with antibodies.

[0303] 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.

[0304] The phrase "pharmaceutically acceptable salt" refers to organic or inorganic salts of an anti-LIV-1 antibody or conjugate thereof that are pharmaceutically acceptable, or salts of a formulation with which the anti-LIV-1 antibody or conjugate thereof is normally combined in the manufacture of a medicament. Exemplary salts include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, acid phosphate, acid citrate, acid fumarate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,l' -dimethyl -2-hydroxy -3-naphthoate) salts. Pharmaceutically acceptable salts can include another molecule such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the electric charge on the parent compound. In addition, pharmaceutically acceptable salts can have more than one charged atom in its structure. Examples of pharmaceutically acceptable salts that are part of a multiple charged atom can have more than one counterion. Thus, pharmaceutically acceptable salts can have one or more charged atoms and / or one or more counterions.

[0305] The term antibody-drug conjugate (ADC) refers to an antibody or antibody fragment linked to a toxic drug having biological activity through a linker. The antibody or antibody fragment described in the present disclosure can be conjugated to an effector molecule by any means. For example, the antibody or antibody fragment can be attached to a toxic drug by chemical or recombinant means. Chemical means of preparing 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.

[0306] 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 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32and Lu's radioisotopes), chemotherapeutic drugs, antibiotics, and nucleolytic enzymes.

[0307] The antibodies of the present disclosure can be conjugated to the cytotoxic drugs via a coupling agent. Examples of the coupling agent can be any one or several of nonselective coupling agents, coupling agents utilizing carboxyl groups, peptide chains, and coupling agents utilizing disulfide bonds. The nonselective coupling agent refers to a compound that forms a covalent bond between an effector molecule and an antibody, such as glutaraldehyde or the like. The coupling agent utilizing carboxyl groups can be any one or several of aconitic anhydride-based coupling agents (such as aconitic anhydride), acylhydrazone-based coupling agents (coupling site: acylhydrazone).

[0308] Certain residues on the antibody (such as Cys or Lys, etc.) are used for attachment 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) being conjugated (covalently linked) to the antibody, the functional agent being linked to the antibody either directly or indirectly via a linker.

[0309] The antibody can be conjugated to a drug to form an antibody-drug conjugate (ADC). Typically, an ADC comprises a linker (or linker moiety) between the drug and the antibody. The term "linker moiety" or "linking fragment" or "linking moiety" or "linker" 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, or to another linker before linking to the drug. The linker can be a degradable linker or a non-degradable linker. A degradable linker is 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, e.g., 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, e.g., 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, e.g., 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). A non-degradable linker is typically susceptible to release of the drug under conditions in which the antibody is hydrolyzed by proteases.

[0310] The linker has a reactive functional group capable of reacting with certain amino acid residues prior to attachment to the antibody, and 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-(mercurimethyl)dioxane, and counterions are acetate, chloride, or nitrate; and polymethylenedimethylthioether sulfonate. The linker can include, for example, a maleimide attached to the antibody through a thiodiglycolic imide.

[0311] In the present disclosure, the drug-linker compounds can be used to form ADCs in a single step. In other embodiments, the bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, the cysteine residue is reacted with the reactive portion of the linker in a first step, and in a subsequent step, the functional group on the linker is reacted with the drug, thereby forming the ADC.

[0312] In general, the functional group on the linker is selected 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 through a 1,3-dipolar cycloaddition between the azide and the alkyne. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxy amines); phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimidyl esters (suitable 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. Those skilled in the art will appreciate that for selective reaction of the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used on either the linker or the drug.

[0313] The present disclosure also provides methods of making an ADC, which can further comprise: combining the antibody with the drug-linker compound (or linker-drug, LD) under conditions sufficient to form an antibody conjugate (ADC).

[0314] In certain embodiments, the methods of the 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 disclosure further comprise: combining the antibody-linker conjugate with a drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.

[0315] Drug loading, also referred to as drug-to-antibody ratio (DAR), is the average number of drug molecules conjugated to each antibody in an ADC. It can range, for example, from about 1 to about 10 drugs per antibody conjugated, and in certain embodiments, from about 1 to about 8 drugs per antibody conjugated, 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. Exemplarily, the drug loading can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 in average. The general formula of the ADCs of the disclosure includes a collection of antibody-drug conjugates within the aforementioned range. In embodiments of the disclosure, the drug loading can be denoted as z, which is a decimal or an integer. The drug loading can be determined by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, and HPLC.

[0316] In one embodiment of the disclosure, the cytotoxic drug is conjugated to the antibody through a linking unit.

[0317] The drug loading of the ligand-drug conjugate can be controlled by the following non-limiting methods, including:

[0318] (1) controlling the molar ratio of the drug linker fragment and the mAb,

[0319] (2) controlling the reaction time and temperature,

[0320] (3) selecting different reaction reagents.

[0321] 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.

[0322] "glycosylation" refers to the transfer of a glycosyl or oligosaccharide group to a hydroxyl or amino group of a compound, either by chemical or enzymatic means. 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 saccharide.

[0323] The term "hydroxyl" refers to -OH.

[0324] The term "amino" means -NH2.

[0325] Dichloromethane (DCM) is also referred to as DCM.

[0326] N,N-Diisopropylethylamine (DIEA) is also referred to as DIEA.

[0327] Dimethylformamide (DMF) is also referred to as DMF.

[0328] O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) is also referred to as HATU.

[0329] 1-Hydroxybenzotriazole (HOBt) is also referred to as HOBt.

[0330] Ph means a phenyl ring.

[0331] "Substituted" means that one or more hydrogen atoms, preferably 1 to 6, more preferably 1 to 3, of a group are independently of each other replaced with the corresponding number of substituents. It is possible or not possible to determine, without undue effort (experimentally or theoretically), whether a substitution is possible or not. For example, an amino or hydroxyl group with a free hydrogen can not be stable when bound to a carbon atom with an unsaturated (e.g., olefinic) bond.

[0332] The term“immune checkpoint inhibitor” refers to a therapeutic agent that targets at least one immune checkpoint protein to alter the regulation of the immune response, for example, downregulate, inhibit, upregulate, or activate the 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 of immune checkpoint inhibitors useful in 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, and SIRPa (CD47). 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 antibody, such as Atezolizumab, Pembrolizumab, as described herein.

[0333] An“anti-PD-1 antibody” or“PD-1 antibody” refers to an antibody that binds to PD-1. Such an antibody can decrease, block, inhibit, abrogate, or interfere with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and PD-L2.

[0334] The term“anti-PD-L1 antibody” refers to an antibody that binds to PD-L1. Such an antibody can decrease, 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.

[0335] The term“anti-SIRPa antibody” refers to an antibody that binds to SIRPa. Anti-SIRPa antibodies inhibit the binding between SIRPa and CD47, thereby inhibiting the“don’t eat me” signal from tumor cells to phagocytes, thereby enhancing phagocytosis by phagocytes to phagocytose tumor cells.

[0336] The present disclosure is illustrated below with reference to specific examples. Those skilled in the art will understand that the examples are used only for the purpose of illustration of the present disclosure and are not intended to limit the scope of the present disclosure in any way.

[0337] The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials, reagents and materials used in the following examples are all commercially available unless otherwise specified.

[0338] Example 1 Preparation of Anti-LIV-1 Antibodies

[0339] 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 obtained.

[0340] After the antibody sequences are obtained, the antibodies can be prepared using recombinant technology, and the murine antibodies can be chimerized and humanized. 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 regions are replaced with murine analogs to prepare humanized antibodies. During the preparation of the 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.

[0341] 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.

[0342] 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 was significantly improved after humanization, as shown in Table 1. The binding affinity was detected by ForteBio.

[0343] Table 1. Binding activity of anti-LIV-1 antibody to LIV-1 protein

[0344] Example 2 Detection of the binding ability of anti-LIV-1 antibody to LIV-1 protein and LIV-1 expressing cell lines

[0345] 2.1 Binding of anti-LIV-1 antibody to LIV-1 protein

[0346] In this experiment, enzyme-linked immunosorbent assay (ELISA) was used. LIV-1 antigen was connected to a solid carrier, and the antibody to be detected in the sample was combined to form a solid-phase antigen-antibody complex. Then, the 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. 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.

[0347] 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 and added to the 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 detected 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 the completion of the secondary antibody incubation, TMB color developing solution was incubated at room temperature for 1-10 minutes. After the color development was completed, 50 μL of stop solution (4M sulfuric acid) was added to each well to terminate the substrate reaction. The absorbance was measured by an enzyme-labeled instrument, and the absorbance value of each well was read at a detection wavelength of 450 nm. The data was analyzed by Prism software, with the concentration of the antibody to be detected as the abscissa and the average absorbance value as the ordinate. The dose-response curve was drawn by Sigmoidal, 4PL four-parameter equation. Equation:

[0348] Y = Bottom + (X^Hillslope) * (Top-Bottom) / (X^HillSlope + EC50^HillSlope).

[0349] The results are shown in Figures 1A and 1B, and the anti-LIV-1 antibody of the present disclosure can specifically bind to human LIV-1 antigen.

[0350] 2.2 Anti-LIV-1 antibody binding to LIV-1 expressing cell lines

[0351] The specific binding ability of anti-hLIV-1 antibody A to cell surface expressed LIV-1 antigen was detected using flow cytometry.

[0352] The cell lines selected for this experiment were: Calu-6 (ATCC HTB-56, anaplastic lung cancer), HCC1806 (ATCC CRL-2335, human breast adenocarcinoma TNM stage IIB grade 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. Calu6, HCC1806 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, PC-3 cells in F12K medium containing 10% fetal bovine serum, MCF7-ATCC-LIV-1 overexpressing cell lines in EMEM medium containing 10% fetal bovine serum and 1 pg / mL puromycin at 37°C in a 5% CO2 incubator. After dissociating each cell line and washing in staining buffer (Biolengend), the number of cells was counted and adjusted to 2 x 10 5 antibody A was added to a final concentration of 10 pg / mL, then reacted for 15 minutes at 4°C. After the reaction, the cells were washed in staining buffer, then the PE-labeled constant region (Fc)-specific antibody (rabbit anti-human IgG PE conjugate, BioLegend, 410707) was suspended in 2 pL / 2 x 10 5 After the cell reaction, the cells were washed in staining buffer and the single cells were analyzed for reading in the PE channel using a Novocyte 3000 (Agilent) device. The negative control was treated with a non-specific isotype control commercial antibody IgG (BioXCell, BE0297) and then treated with the PE-labeled constant region (Fc)-specific antibody. To compare the degree of 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 in the present disclosure divided by the shifted reading of the control group was expressed (MFI ratio: MFI of anti-LIV-1 antibody / MFI of control antibody). The experimental results are shown in Table 2.

[0353] The experiments demonstrate and confirm that the anti-LIV-1 antibody in the present disclosure specifically binds to LIV-1 expressed in various cancer cell lines of breast cancer, lung cancer, and prostate cancer.

[0354] Table 2. Staining readouts of antibody A binding to each cell line

[0355] Example 3. Synthesis of compounds for preparing antibody-conjugated drugs

[0356] In this example, the experimental methods not specified in the specific conditions, usually in accordance with the conventional conditions, or according to the raw materials or the manufacturer's recommended conditions. The reagents not specified in the specific source, for the market to buy the conventional reagents.

[0357] The structure of the compound is determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). NMR shift (δ) is given in units of 10 -6 (ppm). 1 The determination of HNMR is measured by Bruker AVANCE-400 nuclear magnetic instrument, the determination of solvent is deuterated dimethyl sulfoxide, and the internal standard is tetramethylsilane (TMS).

[0358] MS is measured by Shimadzu LCMS-2020 Single Quaddrupole liquid chromatography-mass spectrometer (manufacturer: Shimadzu, MS model: 2020 Single Quadrupole MS).

[0359] RP-HPLC analysis is measured by Shimadzu LC-2030c Plus liquid chromatograph, and preparation is prepared by Shimadzu Nexera liquid preparation system. The preparation chromatographic column is Phenomenex Gemini NX 5μ, C18, 150×50mm, mobile phase: 0.1% trifluoroacetic acid in water solution / 0.1% trifluoroacetic acid in acetonitrile (ACN) solution.

[0360] Silica gel column chromatography uses SiliCycle (Canada) 200-300 mesh silica gel as carrier.

[0361] 3.1 Synthesis of compound 3

[0362] The synthesis process is as follows:

[0363] To a solution of compound 1 (110 mg, purchased from MedChemExpress, Cat# 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.HC1) (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).

[0364] MS determination: m / z 1403.6 [M+H] + .

[0365] 3.2 Synthesis of compound 4

[0366] The synthesis proceeded as follows:

[0367] To a solution of compound 1 (110 mg) in anhydrous DMF (2 mL) was added DIEA (40 pL) 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).

[0368] MS determination: m / z 1417.6 [M+H] + .

[0369] 3.3 Synthesis of compound 5

[0370] The synthesis proceeded as follows:

[0371] 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 and the mixture was directly purified by RP-HPLC to give compound 5 as a white powder (105 mg).

[0372] MS determination: m / z 1565.8 [M+H] + .

[0373] 3.4 Synthesis of compound 10

[0374] The synthesis proceeded as follows:

[0375] To a solution of compound 6 (65 mg) and MMAE (72 mg, purchased from MedChemExpress, Cat. No: 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).

[0376] 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).

[0377] MS determination: m / z 1179.6 [M+H] + .

[0378] 3.5 Synthesis of compound 11

[0379] The synthesis procedure is as follows:

[0380] 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, the mixture was directly purified by RP-HPLC to give compound 11 as a white powder (21 mg).

[0381] MS determination: m / z 1243.6 [M+H] + .

[0382] 1H 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).

[0383] 3.6 Synthesis of compound 22 (MC-VC-PAB-MMAE)

[0384] The synthesis procedure is as follows:

[0385] To a solution of compound 1 (62 mg) in anhydrous DMF (2 mL) was added maleimide hexanoic 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 (62 mg, TFA salt) as a white solid after lyophilization.

[0386] MS measurement: m / z 1316.8 [M+H] + .

[0387] 3.7 Synthesis of compound 29 (MC-GGY-PAB-MMAE)

[0388] The synthesis procedure is as follows:

[0389] 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), 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).

[0390] 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).

[0391] 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 the crude product which was purified by RP-HPLC to give compound 28 as a white solid (TFA salt, 64 mg).

[0392] 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).

[0393] MS: m / z 1337.8 [M+H] + .

[0394] 3.8 Synthesis of compound 39 (MC-GGY(Gal)-PAB-MMAE)

[0395] The synthesis procedure was as follows:

[0396] 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).

[0397] 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).

[0398] 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).

[0399] 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).

[0400] To a solution of compound 37 (28 mg, TFA salt) in DMF (2 mL) was added 6-maleimidocaproic 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).

[0401] MS: m / z 1499.9 [M+H] + .

[0402] 3.9 Synthesis of compound 48 (MC-GGY-(Glc)-PAB-MMAE)

[0403] The synthesis proceeded as follows:

[0404] 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).

[0405] 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).

[0406] 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).

[0407] 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).

[0408] 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).

[0409] MS: m / z 1499.9 [M+H] + .

[0410] 3.10 Synthesis of compound 55 (MC-AAN-(GlcNAc)-PAB-MMAE)

[0411] The synthesis proceeded as follows:

[0412] 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 (over 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 (over 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).

[0413] 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 (over 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).

[0414] To 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 (over 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).

[0415] 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).

[0416] MS: m / z 1519.9 [M+H] + .

[0417] 3.11 Synthesis of compound 56 (BrAc-GGY(Glc)-PAB-MMAE)

[0418] The synthesis procedure was as follows:

[0419] 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.

[0420] MS: 1426.80 [M+H] + .

[0421] 3.12 Synthesis of compound 17 (BrAc-PEG4-GlcA-MMAE)

[0422] The synthesis procedure was as follows:

[0423] 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.

[0424] MS: 1471.6 [M+H] + .

[0425] 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.

[0426] MS: 1426.7 [M+H] + .

[0427] 3.13 Synthesis of compound 20 (BrAc-PEG4-PAB-MMAE)

[0428] The synthesis was carried out as follows:

[0429] 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.

[0430] 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.

[0431] MS: 1491.0 [M+H] + .

[0432] Preparation and physicochemical characterization of antibody-drug conjugates

[0433] 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 with an electrophilic functional group, such as a maleimide.

[0434] 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.

[0435] 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 incubation at room temperature for about 1 hour, the ADC was purified by desalting with G25 resin, filtered under sterile conditions with a 0.2 pm filter, and stored frozen. The average DAR value of the ADC was determined to be between 3.5 and 4.0 by hydrophobic interaction chromatography-high performance liquid chromatography analysis.

[0436] 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 incubation at room temperature for about 1 hour, the ADC was purified by desalting with G25 resin, filtered under sterile conditions with a 0.2 pm filter, and stored frozen. The average DAR value of the ADC was determined to be between 3.5 and 4.0 by hydrophobic interaction chromatography-high performance liquid chromatography analysis.

[0437] Methods for detecting DAR values:

[0438] The DAR values of the ADCs of the disclosure were analyzed using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). The ADCs were separated in the 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 the initial conditions, using a linear gradient of 85% buffer solution A and 15% buffer solution B compared to 5% buffer solution A and 95% buffer solution B for 30 minutes, and an additional 5 minutes 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 profiles were detected at 214 nm and 280 nm for DAR value calculation.

[0439] Exemplary, partially antibody-drug conjugates (ADCs) prepared are shown in Table 3.

[0440] Table 3. ADCs prepared in the disclosure

[0441] Preparation of control L-MMMAE

[0442] 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.

[0443] Example 5 Antibody-drug conjugate binding activity with LIV-1 antigen

[0444] 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).

[0445] The results are shown in Figure 2, and the antibody-drug conjugate of the present disclosure can specifically bind to LIV-1 antigen.

[0446] Example 6 In vitro killing effect of antibody-drug conjugate

[0447] 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 ATP of metabolically active cells.

[0448] The cell lines selected in the experiment are: human lung cancer (undifferentiated) cell line Calu-6, human breast cancer cell MCF7-ATCC-LIV-1 overexpressing LIV-1 #7, #12, #17.

[0449] 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, and 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, with duplicate wells for each concentration, and solvent controls and cell-free medium wells of the corresponding concentration are set up, and 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, and the luminous value is measured, and the IC 50 values (nM) of the anti-LIV-1 antibody A-BrAcMMAE conjugate on various cells are calculated (Table 3). The calculation results are shown in Figures 3, 4, 5, 6 and Table 4-1 for the proliferation inhibition of the four tumor cells.

[0450] Table 4-1. IC values (nM) of antibody A and control antibody without binding function conjugated with MMAE for the proliferation inhibition of each cell line 50

[0451] 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.

[0452] In addition, the disclosure also detects the in vitro killing activity of ADCs of different linkers conjugated with antibody A on different cells, and the results are shown in Tables 4-2 and 4-3.

[0453] Table 4-2. IC values (nM) of ADCs for the proliferation inhibition of MCF7-LIV-1 #17 cells 50

[0454] Table 4-3. IC values (nM) of ADCs for the proliferation inhibition of Calu6 cells 50 ​​​

[0455] In summary, the anti-LIV-1 antibody-MMAE conjugate of the present disclosure has obvious anti-tumor activity and good targeting.

[0456] Example 7 In vivo efficacy study of antibody-drug conjugate

[0457] 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).

[0458] When the tumor growth reached 150 mm 3 , the administration of the 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 a 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 taken for each group until one or more animals were euthanized. All animal procedures were performed in accordance with an approved protocol by the Institutional Animal Care and Use Committee in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility. The formula for TGI is as follows:

[0459] TGI (%) = 1 - [(Td - T0) / (Cd - C0)] x 100%

[0460] 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.

[0461] 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.

[0462] In the HCC1806 xenograft efficacy study, the TGI was 102.99% on day 21 after administration.

[0463] In the PC-3 xenograft efficacy study, on day 11 after administration, the TGI was 101.5%.

[0464] 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. The above results show that A-BrAcMMAE exhibits significant anti-tumor efficacy in different tumor models.

[0465] In addition, the present disclosure also detected the tumor inhibition effect of A-BrAcMMAE and the prior art LIV-1 targeting ADC ladiratuzumab vedotin (referred to as: L-Mc-MMAE) in PC3 and PA-1 mouse tumor models, and the results are shown in Figure 12 and Tables 5-1, 5-2.

[0466] Table 5-1. Tumor inhibition rate of different ADCs on PC3 mouse subcutaneous tumors

[0467] Table 5-2. Tumor inhibition rate of ADCs on PA-1 mouse subcutaneous tumors

[0468] 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 is significantly superior to L-McMMAE in mouse tumor models. Therefore, the A-BrAcMMAE antibody conjugate can be used to treat various cancers expressing LIV-1.

[0469] Example 8 In vivo efficacy study of anti-LIV-1 antibody-drug conjugate in combination with anti-PD-(L)1 antibody

[0470] The anti-PD-1 antibody used in this example is Pembrolizumab (Keytruda), and the preparation of h409A11 antibody is referred to in WO2008156712A1; the anti-PD-L1 antibody used is Atezolizumab (Tecentriq), and the preparation is referred to in WO2023279092A2. The specific experimental scheme is as follows:

[0471] Tumor cells grown in NCG immunodeficient mice implanted into cultures: A375 melanoma cells from ATCC (5 × 10⁻⁶) 6 Cells), HCC1806 breast cancer cells from ATCC (5 × 10⁻⁶) 6 Cells in 50% matrix gel.

[0472] When the tumor grows to an average size of 100-150mm 3 Mice were randomly divided into four groups: IgG4 antibody control, anti-LIV1-ADC monotherapy group, anti-PD-(L)1 antibody monotherapy group, and anti-LIV1-ADC + anti-PD(L)1 antibody combination group. On day 0, each mouse received an intravenous infusion of 10 × 10⁻⁶ IgG antibodies. 6 Human PBMC cells were administered as follows: 3 mg / kg anti-LIV-1 ADC was administered intravenously on day 1, once; 10 mg / kg anti-PD-(L)1 antibody was administered intravenously every 5 days on days 1, 6, 11, and 16, for a total of 4 doses; or a combination of 3 mg / kg anti-LIV-1 ADC administered intravenously on day 1, once, and 10 mg / kg anti-PD-(L)1 antibody administered intravenously every 5 days on days 1, 6, 11, and 16, for a total of 4 doses. Tumor volume was monitored using calipers; when the tumor volume reached approximately 2000 mmHg... 3 Animals were euthanized as needed. Tumor volume was measured in each group until one or more animals were euthanized. All animal procedures were performed in facilities accredited by the Laboratory Animal Care Assessment and Accreditation Association, in accordance with protocols approved by the Institutional Animal Care and Use Committee.

[0473] The formula for calculating TGI is as follows:

[0474] TGI (%) = 1 - [(Td - T0) / (Cd - C0)] × 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. The results are shown in Figures 14-16 and Tables 6-1 to 6-3.

[0475] Table 6-1. Tumor inhibition rate of different drug administration groups against subcutaneous tumors of mouse A375

[0476] Table 6-2. Tumor inhibition rate of different drug administration groups against subcutaneous tumors of mouse A375.

[0477] Table 6-3. Tumor inhibition rate of different drug administration groups on subcutaneous tumors in HCC1806 mice

[0478] The results show that in the A375 transplanted tumor efficacy study, on the 31st day after PBMC infusion, the tumor inhibition rate of the anti-LIV-1-ADC combined with anti-PD-1 antibody group was 95.18%. 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.

[0479] In the HCC1806 transplanted tumor efficacy study, on the 20th day after administration, the tumor inhibition rate of the anti-LIV-1-ADC single drug group was 38.88%, the anti-PD-1 antibody single drug group had no tumor inhibition effect, and the tumor inhibition rate of the anti-LIV-1-ADC combined with anti-PD-1 antibody group was 73.99%, which was better than that of the anti-LIV-1-ADC single drug group.

[0480] Example 9 Killing effect of anti-LIV-1 antibody-drug conjugate combined with anti-SIRPa antibody on breast cancer SKBR3 cells

[0481] The anti-SIRPa antibody used in this example was prepared by referring to antibody 14# in WO2022121980A1, and the specific experimental scheme is as follows:

[0482] (1) Preparation of effector cells:

[0483] 400g of human fresh PBMC (purchased from Shanghai Heyou Life Science) was centrifuged for 10 min, the cell precipitate was collected and resuspended in the basic RPMI 1640 medium (Sigma, item number R8758) to make the cell density 1E8 / mL, and cultured at 37°C and 5% CO2 for 2h to make it adhere. Remove the culture supernatant and wash once, and replace with complete RPMI 1640 medium containing 80ng / mL human M-CSF (purchased from MedChemExpress, item number HY-P7050) for induction, and replace the medium every 3 days, a total of 7 days of induction, and the final induction is macrophage M0.

[0484] (2) Cell killing experiment:

[0485] SK-BR-3 breast cancer cells and macrophages M0 were plated in 96-well plates at 90 μL per well, with 8000 breast cancer cells per well and 4000 macrophages M0 per well in 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 μg / mL, and anti-SIRPa antibody was diluted to a final working concentration of 10 μg / mL, 10 μL of drug or blank control PBS was added to each well, and after incubation at 37°C, 5% CO2 for 96 h, 100 μL of CellTiter-Glo reagent (purchased from Promega, product number G7572) was added to each well, and the plate was shaken on an orbital shaker at room temperature for 4 min, and then incubated at room temperature for 20 min, and the luminescence value was measured on a microplate reader. The killing effect of A-BrAcMMAE combined with anti-SIRPa antibody 14# on SK-BR-3 cells was calculated. The results are shown in Figure 17.

[0486] The results show that both A-BrAcMMAE and anti-SIRPa antibody 14# show killing effects on SKBR3, but when A-BrAcMMAE and anti-SIRPa antibody are administered in combination, the killing ability on breast cancer cells is significantly improved.

[0487] Example 10 Construction of a cell line resistant to HER2 antagonists

[0488] A cell line resistant to Enhertu was constructed using human lung cancer cells NCI-H2170 (purchased from Nanjing Keyeibio Co., Ltd.).

[0489] The resistance curve of NCI-H2170 to Enhertu was detected, and IC 30 was used as the initial drug concentration, and then increased to IC 50 was used as the drug concentration, and the concentration of Enhertu was increased continuously for more than 6 months until NCI-H2170 cells that could grow normally at a concentration of 13.1 nM of Enhertu were screened, i.e., NCI-H2170 / Enhertu-R cells, and the NCI-H2170 / Enhertu-R cells could still maintain the resistance trait after continuous passage and cryopreservation and recovery.

[0490] Resistance detection:

[0491] NCI-H2170 and NCI-H2170 / Enhertu-R cells were trypsinized, counted and seeded in 96-well plates at a density of 4000 / 5000 cells per well. The next day, DS-8201 (i.e., trastuzumab, prepared according to WO2015115091A1), trastuzumab (purchased from Selleck, Cat. No. A2007) and DXD (Exatecan derivative, purchased from Selleck, Cat. No. E2891) were administered. The initial concentration of DS-8201 was 333.3 nM, and 9 points were obtained by serial dilution with a 5-fold gradient;

[0492] In NCI-H2170 / Enhertu-R cells with a resistant concentration of 5.23 nM of DS-8201, the initial concentration of trastuzumab was 343.6 nM, and 9 points were obtained by dilution with a 5-fold gradient; the initial concentration of DXD was 100 μM, and 9 points were obtained by dilution with a 5-fold gradient.

[0493] In NCI-H2170 / Enhertu-R cells with a resistant concentration of 13.1 nM of DS-8201, the initial concentration of DXD was 100 nM, and 9 points were obtained by dilution with a 3-fold gradient.

[0494] Blank control groups were also set up, with 3 replicate wells per group. After 5-6 days of treatment with DS-8201 and DXD, 100 μL of Cell Titer-Glo reagent (purchased from Promega, Cat. No. G7572) was added to each well. The plates were mixed on a orbital shaker for 3 minutes, and then incubated at room temperature for 15 minutes. The luminescent signal was detected using a microplate reader, and the in vitro proliferation inhibition curves of DS-8201, trastuzumab and DXD on NCI-H2170 and NCI-H2170 / Enhertu-R cells were drawn and the IC50 values were calculated. The results are shown in Table 7 and Figures 18, 19, 20, 21 and 22.

[0495] Table 7. In vitro proliferation inhibition IC50 of cells 50 (nM)

[0496] Results show that trastuzumab has obvious inhibitory effect on the proliferation of NCI-H2170 cells in vitro, while NCI-H2170 / Enhertu-R drug-resistant cells have consistent and stable resistance to trastuzumab. At the same time, the toxin DXD of trastuzumab has no obvious difference in inhibiting the proliferation of NCI-H2170 and NCI-H2170 / Enhertu-R cells, but trastuzumab has significant inhibitory effect on the proliferation of NCI-H2170 cells, and has no inhibitory effect on the in vitro proliferation of NCI-H2170 / Enhertu-R drug-resistant cells. It is proved that the drug-resistant strain NCI-H2170 / Enhertu-R is resistant to the antibody part of the trastuzumab drug, i.e. trastuzumab, but has no resistance to the toxin DXD.

[0497] Example 11 Killing of HER2 antagonist-resistant cancer cells by anti-LIV-1 antibody-drug conjugate

[0498] NCI-H2170 and NCI-H2170 / Enhertu-R cells were trypsinized, counted, and seeded in 96-well plates at a density of 4000 / 5000 cells per well. The next day, trastuzumab and A-BrAcMMAE were administered at an initial concentration of 333.3 nM, and 9 points were obtained by serial dilution with a 5-fold gradient. A blank control group was also set up, with 3 replicate wells per group. After 5-6 days of trastuzumab and A-BrAcMMAE treatment, 100 μL of Cell Titer-Glo reagent (purchased from Promega, product number G7572) was added to each well. The plates were mixed on a orbital shaker for 3 minutes, and then incubated at room temperature for 15 minutes. The luminescence signal was detected using a microplate reader, and the in vitro proliferation inhibition curves of trastuzumab and A-BrAcMMAE on NCI-H2170 / Enhertu-R cells were drawn and the IC50 values were calculated. The results are shown in Table 8, Figure 23 and Figure 24.

[0499] Table 8. In vitro proliferation inhibition IC50 of A-BrAcMMAE on NCI-H2170 / Enhertu-R cells 50 (nM)

[0500] Results show that the in vitro proliferation inhibition IC50 of trastuzumab on NCI-H2170 / Enhertu-R cells resistant to 5.23 nM and 13.1 nM trastuzumab is 195.8 nM and 230.4 nM, respectively, indicating that the drug-resistant cells have obvious resistance to trastuzumab; the in vitro proliferation inhibition IC50 of A-BrAcMMAE on drug-resistant cell lines is 0.8 nM and 1.1 nM, respectively, indicating that the drug-resistant cells have no resistance to the toxin DXD of trastuzumab. 50 50 ​3.58nM and 4.44nM, respectively, indicating that A-BrAcMMAE can significantly inhibit the proliferation of NCI-H2170 / Enhertu-R cells in the case of resistance to trastuzumab.

[0501] Example 12 Killing of HER2 antagonist-resistant cancer cells by anti-LIV-1 antibody-drug conjugate

[0502] 12.1 Construction of BT474 / Enhertu-R:

[0503] Referring to Example 10, BT474 / Enhertu-R cells resistant to trastuzumab were constructed based on BT474 (breast cancer cells, purchased from Nanjing Keyeibio Technology Co., Ltd.) using the in vitro concentration gradient method, and the resistant concentration of trastuzumab was 0.52nM.

[0504] 12.2 Detection of killing activity of Enhertu on BT474 / Enhertu-R cells

[0505] After trypsin digestion, centrifugation and counting, BT474 and BT474 / Enhertu-R cells were seeded in a 96-well plate at a cell density of 5000 cells / well, and trastuzumab was administered the next day. The administration concentration of trastuzumab was 1000nM as the highest administration concentration, followed by 300nM for 5-fold gradient continuous dilution to obtain 8 points; DXD (purchased from Selleck, item number E2891) was diluted at a starting concentration of 100nM, with 3-fold gradient continuous dilution to obtain 9 points, and a blank control group was set up, with 3 duplicate wells in each group. After 5 days of trastuzumab and DXD treatment, 100μL of CellTiter-Glo reagent (purchased from Promega, item number G7572) was added to each well, the culture plate was placed on a orbital shaker for 3 minutes, then the culture plate was incubated at room temperature for 15 minutes, and the luminescence signal was detected by a microplate reader. The in vitro proliferation inhibition curves of trastuzumab / DXD on BT474 and BT474 / Enhertu-R cells were drawn, and the results are shown in Figures 25A and 25B.

[0506] The results show that trastuzumab has a significant proliferation inhibitory effect on BT474 cells, and compared with BT474, trastuzumab does not have a significant proliferation inhibitory effect on the resistant cell line BT474 / Enhertu-R, even at a trastuzumab administration concentration of 1000nM, the proliferation inhibition rate of BT474 / Enhertu-R cells is only 15.6%, i.e. BT474 / Enhertu-R has developed resistance to trastuzumab.

[0507] DXD also has an IC50 The IC50value of DXD against BT474 / Enhertu-R cell proliferation inhibition was 22.05 nM, while the inhibition rate of DXD at the maximum concentration of 100 nM was only 39.65%, which fully demonstrated that BT474 / Enhertu-R also developed resistance to DXD. 50 The IC50value of DXD against BT474 / Enhertu-R cell proliferation inhibition was 22.05 nM, while the inhibition rate of DXD at the maximum concentration of 100 nM was only 39.65%, which fully demonstrated that BT474 / Enhertu-R also developed resistance to DXD.

[0508] 12.3A-BrAcMMAE against BT474 / Enhertu-R cell killing activity detection

[0509] When the resistant concentration of BT474 / Enhertu-R cells to trastuzumab was 0.52 nM, the killing activity of anti-LIV-1 ADC against it was detected.

[0510] BT474 and BT474 / Enhertu-R cells were trypsinized, counted, and seeded in a 96-well plate at a cell density of 5000 cells per well. The next day, trastuzumab and A-BrAcMMAE were administered. The administration concentration of trastuzumab was 1000 nM as the highest administration concentration, and 300 nM was used for 5-fold gradient continuous dilution to obtain 8 points. The initial concentration of A-BrAcMMAE was 1000 nM, and 5-fold gradient continuous dilution was used to obtain 9 points. A blank control group was also set up, and each group had 3 duplicate wells. After 5 days of trastuzumab and A-BrAcMMAE treatment, 100 μL of CellTiter-Glo reagent (purchased from Promega, product number G7572) was added to each well. The culture plate was placed on a orbital shaker for 3 minutes, and then incubated at room temperature for 15 minutes. The luminescent signal was detected by a microplate reader, and the in vitro proliferation inhibition curve of trastuzumab and A-BrAcMMAE against BT474 / Enhertu-R cells was drawn. The results are shown in FIG. 26.

[0511] The results showed that A-BrAcMMAE could significantly inhibit the proliferation of BT474 / Enhertu-R cells in BT474 cells resistant to trastuzumab, with an IC50value of 1.11 nM, indicating that A-BrAcMMAE had good proliferation inhibition effect on BT474 / Enhertu-R cells. That is, the A-BrAcMMAE of the present disclosure can be used for the treatment of HER2 antagonist or DXD resistant cancer. 50 The results showed that A-BrAcMMAE could significantly inhibit the proliferation of BT474 / Enhertu-R cells in BT474 cells resistant to trastuzumab, with an IC50value of 1.11 nM, indicating that A-BrAcMMAE had good proliferation inhibition effect on BT474 / Enhertu-R cells. That is, the A-BrAcMMAE of the present disclosure can be used for the treatment of HER2 antagonist or DXD resistant cancer.

[0512] Example 13 Killing of HER2 antagonist resistant cancer cells after drug withdrawal for 3 weeks by anti-LIV-1 antibody-drug conjugate

[0513] After 3-week withdrawal of deucaliximab treatment on NCI-H2170 / Enhertu-R cells, NCI-H2170 and NCI-H2170 / Enhertu-R (non-withdrawal), NCI-H2170 / Enhertu-R (withdrawal for 3 weeks) cells were trypsinized, counted, and seeded in 96-well plates at a density of 3000 cells per well. The next day, deucaliximab and A-BrAcMMAE were administered at an initial concentration of 1000 nM, and 9 points were obtained by serial dilution with a 5-fold gradient. A blank control group was set up at the same time, and 3 duplicate wells were set up for each group. After 5 days of deucaliximab and A-BrAcMMAE treatment, 100 μL of CellTiter-Glo reagent (purchased from Promega, product number G7572) was added to each well. The culture plate was placed on a orbital shaker for 3 minutes, and then incubated at room temperature for 15 minutes. The luminescence signal was detected by an enzyme-labeled instrument, and the in vitro proliferation inhibition curve of deucaliximab and A-BrAcMMAE on NCI-H2170 and NCI-H2170 / Enhertu-R cells was drawn, and the IC 50 values were calculated. The results are shown in Table 9, Table 10, Figure 27 and Figure 28.

[0514] Table 9. IC values of deucaliximab on cell proliferation inhibition 50

[0515] Table 10. IC values of A-BrAcMMAE on cell proliferation inhibition 50

[0516] The results show that NCI-H2170 / Enhertu-R (withdrawal for 3 weeks) cells still have obvious drug resistance to deucaliximab, and there is no difference in drug resistance to deucaliximab between NCI-H2170 / Enhertu-R (non-withdrawal) cells.

[0517] A-BrAcMMAE still has a significant killing effect on NCI-H2170 / Enhertu-R (withdrawal for 3 weeks) drug-resistant cells, and the IC 50 value is 2.03 nM.

[0518] Example 14 Killing of topoisomerase I inhibitor-resistant cancer cells by anti-LIV-1 antibody-drug conjugate

[0519] ​​SN-38 is the main active metabolite of irinotecan (CPT-11), which has 100-1000 times of in vitro anti-tumor activity of irinotecan, and is a typical topoisomerase I inhibitor by inhibiting the function of DNA topoisomerase I to hinder the synthesis of DNA. In order to study whether the anti-LIV-1 antibody-drug conjugate of the present disclosure can be used to treat cancer resistant to various topoisomerase I inhibitors, a cell line resistant to SN-38 was constructed.

[0520] 14.1 Construction of COLO205 / SN-38-R:

[0521] Human colon cancer cells COLO205 cells (purchased from the Cell Resource Center of Shanghai Life Sciences Institute, Chinese Academy of Sciences) were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The cells were induced for drug resistance using the method of increasing drug concentration in vitro. The in vitro cell proliferation inhibition curve of COLO205 cells to SN38 was detected, and IC 30 was used as the initial drug concentration, and then increased to IC 50 As the drug concentration, the SN-38 drug induction concentration was continuously increased, and the cells were cultured for more than 6 months, until COLO205 cells that could still grow stably under the condition of 15nM SN-38 were screened out, and named COLO205 / SN-38-R.

[0522] 14.2 Detection of killing activity of SN-38 and irinotecan on COLO205 / SN-38-R cells

[0523] COLO205 and COLO205 / SN-38-R cells were trypsinized, counted, and seeded in a 96-well plate at a density of 5000 cells per well. The next day, SN-38 or irinotecan was added for treatment, with an initial concentration of SN-38 of 100nM and 9 points obtained by 3-fold gradient dilution, and an initial concentration of irinotecan of 100μM and 9 points obtained by 3-fold gradient dilution. A blank control group was set up, and 3 replicate wells were set up for each group. After 5 days of SN-38 treatment, 100μL of CellTiter-Glo reagent (purchased from Promega, product number G7572) was added to each well. The plate was placed on a orbital shaker for 3 minutes, and then incubated at room temperature for 15 minutes. The luminescence signal was detected by a microplate reader, and the in vitro proliferation inhibition curve of SN-38 on COLO205 and COLO205 / SN-38-R cells was drawn and the IC 50 value was calculated. The results are shown in FIGS. 29A, 29B and Table 11.

[0524] Table 11. In vitro proliferation inhibition effect of SN-38 and irinotecan on cells

[0525] The results showed that COLO205 / SN-38-R cells had significant resistance to SN-38, and the resistant concentration was 15 nM. When the administration concentration of SN-38 was 100 nM, the proliferation inhibition rate of COLO205 / SN-38-R cells was only 30.2%. In addition, the drug-resistant cell strain COLO205 / SN-38-R also developed corresponding resistance to irinotecan.

[0526] 14.3 Detection of the killing activity of topoisomerase I inhibitors DXD and Exatecan on drug-resistant cells

[0527] COLO205 and COLO205 / SN-38-R cells were trypsinized, counted, and seeded in a 96-well plate at a density of 5000 cells per well. The next day, DXD (purchased from Selleck, item number E2891) or Exatecan was administered. The initial concentration of DXD was 300 nM, which was serially diluted by 5-fold to obtain 9 points. The initial concentration of Exatecan was 100 μM, which was diluted by 3-fold to obtain 9 points. A blank control group was set up, and 3 replicate wells were set up for each group. After 5 days of DXD and Exatecan treatment, 100 μL of CellTiter-Glo reagent (purchased from Promega, item number G7572) was added to each well. The culture plate was placed on a orbital shaker for 3 minutes, and then incubated at room temperature for 15 minutes. The luminescent signal was detected using a microplate reader, and the in vitro proliferation inhibition curves of DXD and Exatecan on COLO205 and COLO205 / SN-38-R cells were drawn and the IC 50 values were calculated. The results are shown in FIGS. 30A, 30B and Table 12.

[0528] Table 12. In vitro proliferation inhibition of cells by DXD and Exatecan

[0529] The results showed that COLO205 / SN-38-R cells developed resistance to DXD and Exatecan. The IC 50 values of DXD and Exatecan on COLO205 cells were 1.3 nM and 1.5 nM, respectively, and the IC 50 values of DXD and Exatecan on COLO205 / SN-38-R cells were 196.9 nM and 45.6 nM, respectively, indicating that COLO205 / SN-38-R cells developed resistance to both DXD and Exatecan.

[0530] 14.4 Detection of the killing activity of Dato-DXD on COLO205 and COLO205 / SN-38-R cells

[0531] COLO205 and COLO205 / SN-38-R cells were trypsinized, counted, and seeded in 96-well plates at a density of 5000 cells per well. The next day, Dato-DXD (purchased from Selleck, item number D4050) was administered at an initial concentration of 300 nM, with 5-fold serial dilution to obtain 9 points. A blank control group was set up, with 3 replicate wells per group. After 5 days of Dato-DXD treatment, 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 incubated at room temperature for 15 minutes. The luminescent signal was detected using a microplate reader, and the in vitro proliferation inhibition curve of Dato-DXD on COLO205 and COLO205 / SN-38-R cells was plotted, with the IC50 value calculated. The results are shown in Figure 31 and Table 13.

[0532] Table 13. Inhibition of cell proliferation in vitro by Dato-DXD

[0533] IC50 of Dato-DXD on COLO205 cell proliferation 50 The IC50 of Dato-DXD on COLO205 / SN-38-R cells was only 22.41% at the maximum administration concentration of 300 nM, indicating that COLO205 / SN-38-R cells were resistant to Dato-DXD.

[0534] 14.3 Detection of the killing activity of A-BrAcMMAE on COLO205 / SN-38-R cells

[0535] COLO205 / SN-38-R cells were trypsinized, counted, and seeded in 96-well plates at a density of 5000 cells per well. The next day, A-BrAcMMAE and SN-38 were administered at an initial concentration of 300 nM and 100 nM, respectively, with 5-fold and 3-fold serial dilution to obtain 9 points. A blank control group was set up, with 3 replicate wells per group. After 5 days of A-BrAcMMAE treatment, 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 incubated at room temperature for 15 minutes. The luminescent signal was detected using a microplate reader, and the in vitro proliferation inhibition curve of A-BrAcMMAE on COLO205 / SN-38-R cells was plotted, with the IC50 value calculated. The results are shown in Figure 32.

[0536] The results show that the inhibition rate of SN-38 at the maximum concentration of 100 nM is 30.2%, while the IC 50 of A-BrAcMMAE on the proliferation of COLO205 / SN-38-R cells is 4 nM, indicating that A-BrAcMMAE has obvious proliferation inhibition activity on COLO205 / SN-38-R cells. That is, the A-BrAcMMAE of the present disclosure can be used for the treatment of SN-38-resistant cancer.

[0537] Example 15 Killing of HER2 antagonist-resistant cancer cells by anti-LIV-1 antibody-drug conjugate

[0538] 15.1 Construction of HCC1954 / Enhertu-R:

[0539] HCC1954 cells (purchased from the Cell Resource Center of Shanghai Life Science Research Institute, Chinese Academy of Sciences) were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% double antibiotics (penicillin and streptomycin) and cultured in a 37°C, 5% CO2 incubator. The HCC1954 cells were digested, counted, and then plated at a density of 5000 cells / well in a 96-well plate. The next day, trastuzumab was diluted to different concentrations for treatment of HCC1954 cells. Trastuzumab was diluted at a starting concentration of 333.3 nM, with a 5-fold gradient, to obtain 9 different concentrations, and a blank control group was set up, with 3 replicate wells in each group. After 6 days of trastuzumab treatment, 100 μL of CellTiter-Glo reagent (purchased from Promega, product number G7572) was added to each well. The culture plate was placed on a orbital shaker for 3 minutes, and then incubated at room temperature for 15 minutes. The luminescence signal was detected using a microplate reader to obtain the in vitro proliferation inhibition curve of trastuzumab on HCC1954 cells and the IC 50 value was 0.44 nM.

[0540] The HCC1954 cells were digested, counted, and then plated at a density of 7 x 10 5 cells in a 10 cm culture dish. After overnight culture, the medium was replaced, and different concentrations of trastuzumab were added for treatment of the cells. The IC 50 value was used as the highest induction concentration, and the concentration of trastuzumab was gradually increased until the cells could grow normally in a culture medium containing 6.5 nM trastuzumab, obtaining the drug-resistant cell strain HCC1954 / Enhertu-R.

[0541] 15.2 In vitro proliferation inhibition experiment of trastuzumab on HCC1954 and HCC1954 / Enhertu-R cells

[0542] HCC1954 and HCC1954 / Enhertu-R cells in the logarithmic growth phase were trypsinized and counted, then seeded at a density of 2000 cells / well in 96-well plates and cultured overnight at 37°C in a 5% CO2 incubator. The next day, the cells were treated with trastuzumab and DXD. Trastuzumab and DXD were serially diluted 5-fold starting at 300 nM to obtain 9 concentrations, with each concentration set in 3 replicates. After 5 days of drug treatment, 100 μL of CellTiter-Glo reagent (purchased from Promega, catalog number G7572) was added to each well, and the culture plate was mixed on a microplate shaker for 3 minutes. The culture plate was then incubated at room temperature for 15 minutes, and the luminescence signal was detected using a microplate reader. The in vitro proliferation inhibition curves of trastuzumab and DXD on HCC1954 and HCC1954 / Enhertu-R cells were obtained. The results are shown in Table 14, Figure 33A, and Figure 33B.

[0543] Table 14: Inhibitory effects of trastuzumab and DXD on in vitro cell proliferation

[0544] The results showed that HCC1954 / Enhertu-R cells exhibited significant resistance to trastuzumab. Furthermore, DXD showed an IC50 inhibitory effect on the proliferation of HCC1954 and HCC1954 / Enhertu cells. 50 The values ​​were 2.01 nM and 55.51 nM, respectively, indicating that HCC1954 / Enhertu cells developed some resistance to DXD.

[0545] 15.3A-BrAcMMAE cytotoxic activity assay against HCC1954 / Enhertu-R cells

[0546] HCC1954 / Enhertu-R cells in logarithmic growth phase were trypsinized and counted, then seeded at a density of 2000 cells / well in 96-well plates and cultured overnight at 37°C in a 5% CO2 incubator. The next day, the cells were treated with A-BrAcMMAE and trastuzumab. A-BrAcMMAE and trastuzumab were serially diluted 5-fold at a starting concentration of 300 nM to obtain 9 concentrations, with each concentration in triplicate. After 5 days of drug treatment, 100 μL of CellTiter-Glo reagent (purchased from Promega, catalog number G7572) was added to each well, and the plate was mixed on a microplate shaker for 3 minutes. The plate was then incubated at room temperature for 15 minutes, and the luminescence signal was detected using a microplate reader. The in vitro proliferation inhibition curves of A-BrAcMMAE and trastuzumab on HCC1954 / Enhertu-R cells were obtained. The results are shown in Figure 34.

[0547] Results show that A-BrAcMMAE can significantly inhibit the in vitro proliferation of HCC1954 / Enhertu-R cells, IC 50 was 4.53 nM.

[0548] Sequence information of the present disclosure:

[0549] SEQ ID NO: 1 (VH of antibody A):

[0550] SEQ ID NO: 2 (VL of antibody A):

[0551] SEQ ID NO: 3 (VH of antibody B):

[0552] SEQ ID NO: 4 (VL of antibody B):

[0553] SEQ ID NO: 5 (VH of antibody C):

[0554] SEQ ID NO: 6 (VL of antibody C):

[0555] SEQ ID NO: 7 (antibody A VH CDR-H1):

[0556] SEQ ID NO: 8 (antibody A VH CDR-H2):

[0557] SEQ ID NO: 9 (antibody A VH CDR-H3):

[0558] SEQ ID NO: 10 (antibody A VL CDR-L1):

[0559] SEQ ID NO: 11 (antibody A VL CDR-L2):

[0560] SEQ ID NO: 12 (antibody A VL CDR-L3):

[0561] SEQ ID NO: 13 (antibody B VH CDR-H1):

[0562] SEQ ID NO: 14 (antibody B VH CDR-H2):

[0563] SEQ ID NO: 15 (antibody B VH CDR-H3):

[0564] SEQ ID NO: 16 (antibody B VL CDR-L1):

[0565] SEQ ID NO: 17 (antibody B VL CDR-L2):

[0566] SEQ ID NO: 18 (antibody B VL CDR-L3):

[0567] SEQ ID NO: 19 (antibody C VH CDR-H1):

[0568] SEQ ID NO: 20 (antibody C VH CDR-H2):

[0569] SEQ ID NO: 21 (antibody C VH CDR-H3):

[0570] SEQ ID NO: 22 (antibody C VL CDR-L1):

[0571] SEQ ID NO: 23 (antibody C VL CDR-L2):

[0572] SEQ ID NO: 24 (antibody C VL CDR-L3):

[0573] SEQ ID NO: 25 (LIV-1 antigen amino acid sequence):

[0574] SEQ ID NO: 26 (heavy chain constant region):

[0575] SEQ ID NO: 27 (light chain constant region):

[0576] SEQ ID NO: 28 (murine antibody mVH of antibody A, with CDRs underlined):

[0577] SEQ ID NO: 29 (murine antibody mVL of antibody A, with CDRs underlined):

[0578] SEQ ID NO: 30 (light chain of antibody A):

[0579] SEQ ID NO: 31 (heavy chain of antibody A):

[0580] SEQ ID NO: 32 (heavy chain variable region of antibody A-1):

[0581] SEQ ID NO: 33 (heavy chain of ladiratuzumab):

[0582] SEQ ID NO: 34 (light chain of ladiratuzumab):

[0583] SEQ ID NO: 35 (HCDR1 of trastuzumab):

[0584] SEQ ID NO: 36 (HCDR2 of trastuzumab):

[0585] SEQ ID NO: 37 (HCDR3 of trastuzumab):

[0586] SEQ ID NO: 38 (LCDR1 of trastuzumab):

[0587] SEQ ID NO: 39 (LCDR2 of trastuzumab):

[0588] SEQ ID NO: 40 (LCDR3 of trastuzumab):

[0589] SEQ ID NO: 41 (heavy chain variable region of trastuzumab):

[0590] SEQ ID NO: 42 (light chain variable region of trastuzumab):

[0591] SEQ ID NO: 43 (heavy chain of trastuzumab):

[0592] SEQ ID NO: 44 (light chain of trastuzumab):

[0593] SEQ ID NO: 45 (HCDR1 of denintuzumab):

[0594] SEQ ID NO: 46 (HCDR2 of denintuzumab):

[0595] SEQ ID NO: 47 (HCDR3 of denintuzumab):

[0596] SEQ ID NO: 48 (LCDR1 of denintuzumab):

[0597] SEQ ID NO: 49 (LCDR2 of denintuzumab):

[0598] SEQ ID NO: 50 (LCDR3 of denintuzumab):

[0599] SEQ ID NO: 51 (heavy chain variable region of denintuzumab):

[0600] SEQ ID NO: 52 (light chain variable region of denintuzumab):

[0601] SEQ ID NO: 53 (heavy chain of denintuzumab):

[0602] SEQ ID NO: 54 (light chain of denintuzumab):

[0603] All documents mentioned in this disclosure are incorporated herein by reference as if each document were individually incorporated by reference. It is also to be understood that various modifications or alterations can be made to this disclosure by those skilled in the art once given the benefit of the foregoing description, and that such modifications or alterations are intended to fall within the scope of the claims appended hereto.

Claims

1. A method of treating cancer, comprising administering to a subject in need thereof an anti-LIV-1 antibody-drug conjugate, wherein the subject has a cancer that is resistant to an anti-cancer therapeutic agent; 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 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 according to 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 according to 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, 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)-, optionally containing a hydroxyl substituent on the phenyl ring (Ph), 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 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### wherein 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)-, 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, 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 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 group consisting of: 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 anti-cancer therapeutic agent is: (i) a topoisomerase I inhibitor or a derivative thereof; or (ii) an antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof; preferably, the topoisomerase I inhibitor is selected from DXD, SN-38, exatecan, rubitecan, topotecan, irinotecan, camptothecin, or a derivative thereof; more preferably, the topoisomerase I inhibitor is DXD, SN-38, or a derivative thereof.

9. The method of claim 8, wherein the antibody of the antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof is an anti-tumor associated antigen antibody; preferably, the antibody is an anti-HER2 antibody and an anti-TROP2 antibody.

10. The method of claim 9, wherein the anti-HER2 antibody comprises a heavy chain variable region and a light chain variable region, wherein: i) 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; preferably, the anti-HER2 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; more preferably, the anti-HER2 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.

11. The method of claim 9, wherein the anti-TROP2 antibody comprises a heavy chain variable region and a light chain variable region, wherein: i) the heavy chain variable region comprises 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 the light chain variable region comprises 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; preferably, the anti-TROP2 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; more preferably, the anti-TROP2 antibody comprises a heavy chain as set forth in SEQ ID NO: 53 and a light chain as set forth in SEQ ID NO:

54. the heavy chain variable region comprises HCDR1 as set forth in SEQ ID NO: 45, HCDR2 as set forth in SEQ ID NO: 46, and HCDR3 as set forth in SEQ ID NO: 47, and the light chain variable region comprises LCDR1 as set forth in SEQ ID NO: 48, LCDR2 as set forth in SEQ ID NO: 49, and LCDR3 as set forth in SEQ ID NO: 50; preferably, the anti-TROP2 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; more preferably, the anti-TROP2 antibody comprises a heavy chain as set forth in SEQ ID NO: 53 and a light chain as set forth in SEQ ID NO:

54.

12. The method of claim 8, wherein the anti-cancer therapeutic agent is selected from the group consisting of Trastuzumab Deruxtecan, Trastuzumab Rezetecan, JSKN-003 (Anbenitamab repodatecan), IBI-354, TQB-2101, BL-M07D1, BNT-323 (Trastuzumab Pamirtecan), FDA022, GQ1005, DAN-311, T-PL1, PRO1102, MTX-1000, Dato-DXD, Sacituzumab Govitecan, sacituzumab tirumotecan, SHR-A1921, ESG-401, FDA018, DB-1305, MHB036C, BAT8008, BL-M02D1, 9MW2921, HS-20105, GQ1010, OBI-992, FZ-AD004, and DXC1002; preferably, the anti-cancer therapeutic agent is selected from the group consisting of Trastuzumab Deruxtecan and Dato-DXD.

13. The method of any one of claims 1-7, wherein the anti-cancer therapeutic agent is a HER2 antagonist; preferably, the HER2 antagonist is an anti-HER2 antibody, an anti-HER2 antibody-drug conjugate, or a small molecule HER2 inhibitor.

14. The method of claim 13, 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 as set forth in SEQ ID NO: 35, HCDR2 as set forth in SEQ ID NO: 36, and HCDR3 as set forth in SEQ ID NO: 37, and the light chain variable region comprises LCDR1 as set forth in SEQ ID NO: 38, LCDR2 as set forth in SEQ ID NO: 39, and LCDR3 as set forth in SEQ ID NO: 40; preferably, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate 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; more preferably, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain as set forth in SEQ ID NO: 43 and a light chain as set forth in SEQ ID NO:

44.

15. The method of claim 13 or 14, wherein the drug of the anti-HER2 antibody-drug conjugate is a tubulin inhibitor or a topoisomerase I inhibitor; preferably, the drug is selected from the group consisting of maytansinoids, exatecan, SN-38, irinotecan, topetecan, or derivatives thereof.

16. The method of claim 13, wherein the anti-HER2 antibody or anti-HER2 antibody-drug conjugate is selected from the group consisting of trastuzumab, pertuzumab, zanidatamab, Zenocutuzumab, enherumab, inetetamab, margetuximab, vedicitumab, trastuzumab rezetecan, SYD985 (trastuzumab duocarmazine), trastuzumab botidotin, BAT8001, TAA013, MRG002 (trastuzumab vedotin), LCB14-0110, SYA1501, DB-1303, JSKN-003 (Anbenitamab repodatecan), BL-M07D1, HLX22, TQB2102, GQ1005, IBI354, NCB001 (anvatabart opadotin), MM-302, IAH0968, BAT1006, B002T, DX126-262, HK001, TrasGEX (timigutuzumab), and FS102; The small molecule HER2 inhibitor is selected from the group consisting of Neratinib, Lapatinib canertinib, zongertinib, and irbinitinib. the anti-HER2 antibody or anti-HER2 antibody-drug conjugate 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; more preferably, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain as set forth in SEQ ID NO: 43 and a light chain as set forth in SEQ ID NO:

44.

17. The method of any one of claims 1 to 16, wherein the subject has a cancer that is resistant to an anti-cancer therapeutic agent, wherein the cancer is selected from the group consisting of: breast cancer, gastric cancer, lung cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, endometrial cancer, pancreatic cancer, uterine cervical cancer, squamous cell carcinoma, small cell lung cancer, gastric / Esophagogastric junction (GEJ) adenocarcinoma, esophageal squamous cell carcinoma, esophageal cancer, bladder cancer, salivary gland cancer, biliary tract cancer, Paget's disease, hepatocellular carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumor, colon cancer, rectal cancer, glioma, mesothelioma, head and neck cancer, skin cancer, uterine cancer, peritoneal cancer, liver cancer, vulvar cancer, melanoma, leukemia, malignant lymphoma, sarcoma, plasmacytoma, triple negative breast cancer, triple positive breast cancer, HER2 positive breast cancer, hormone receptor positive breast cancer, and multiple myeloma; preferably, the resistance is acquired resistance due to treatment with an anti-cancer therapeutic agent.

18. The method of any one of claims 1 to 17, further comprising administering to the subject another therapeutic agent; preferably, the other therapeutic agent is an immune checkpoint inhibitor; more preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-SIRPa antibody.

19. The method of claim 18, wherein: the anti-PD-1 antibody 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, and Pucotenlimab; the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, envafolimab, durvalumab, adebrelimab, cosibelimab, lodapolimab, garivulimab, envafolimab, opucolimab, manelimab, and sugemalimab; the anti-SIRPa antibody 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, and BYON-4228.

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