Anti-LIV-1 antibodies and anti-LIV-1 antibody-drug conjugates and their pharmacokinetic use

Abstract

The invention provides an anti-human LIV-1 antibody or its antigen-binding fragment, and an antibody-drug conjugate comprising the anti-human LIV-1 antibody or its antigen-binding fragment, and further provides the use of the anti-LIV-1 antibody or its antigen-binding fragment, the antibody-drug conjugate, or a pharmaceutically acceptable salt or solvent compound thereof.

Description

[Technical Field]

[0001] The present invention relates to the field of antibody drugs, and more specifically, the present invention discloses anti-LIV-1 antibodies or their antigen-binding fragments, chimeric antibodies containing the CDR region of the above anti-LIV-1 antibody, humanized antibodies, and anti-LIV-1 antibody-drug conjugates or their pharmaceutically usable salts or solvent compounds, as well as their use as anticancer agents. [Background technology]

[0002] The information provided herein is intended solely to provide background information relevant to this disclosure and to aid in understanding the present invention, and does not necessarily constitute prior art.

[0003] All publications, patents, and patent applications described herein are incorporated by reference to the same extent as any individual publication, patent, or patent application is incorporated by reference in particular and individually. In the event of any inconsistency between the definition or use of a term in an incorporated publication, patent, or patent application and the definitions provided herein, the definitions provided herein shall prevail.

[0004] The zinc transporter protein ZIP6, also known as SLC39A6 or LIV-1, is a multi-pass transmembrane protein possessing zinc transporter and metalloproteinase activity. LIV-1 is regulated by estrogen and is highly expressed in estrogen receptor-positive breast cancer (Taylor et al., Mol Med, 2007 13(7-8):396-406). Recent studies have shown that LIV-1 is highly expressed in several types of tumor cells, including breast cancer, prostate cancer, pancreatic cancer, cervical cancer, and liver cancer, but its expression is limited in normal tissues. This makes it a promising candidate for ADC therapy and suggests it may be a prognostic and detection indicator for certain cancers. There are several variants of LIV-1, and among them, the variant with the number Swiss Prot Q13433 (SEQ ID NO: 25) is a representative target for screening anti-LIV-1 antibodies.

[0005] Antibody-drug conjugates (ADCs) are a technology that utilizes the specific recognition ability of antibodies against specific antigens on the surface of tumor cells to accurately deliver antitumor drugs (e.g., cytotoxic agents, cell inhibitors, small molecule chemotherapeutic drugs, etc.) to tumor target cells, where they are released via endocytosis, and further accurately kill the tumor. Antibody-drug conjugates generally consist of three parts: an antibody or antibody-like ligand, a small molecule drug, and a linker that couples the antibody or antibody-like ligand to the drug. Due to their appropriate molecular weight, high stability, strong targeting, and low toxicity and side effects, antibody-drug conjugates are considered to be the most promising antitumor drugs.

[0006] The use of anti-LIV-1 ADCs in cancer treatment has already been studied; for example, anti-LIV-1 antibodies are linked to anticancer drugs by maleimide ligators (see US RE48,959). However, this linking method has drawbacks such as being prone to detachment and having poor stability, which limits drug use and increases drug side effects.

[0007] Therefore, there is an urgent need in this field for research and development that provides novel antibody-drug conjugates targeting LIV-1 that improve serum stability and reduce toxicity. [Overview of the project]

[0008] In one embodiment, the present invention provides an anti-LIV-1 antibody or its antigen-binding fragment. The antibody or its antigen-binding fragment specifically binds to the extracellular domain (SEQ ID NO: 25) of the LIV-1 protein, consisting of amino acids at positions 29 to 325. Among these, the anti-LIV-1 antibody or its antigen-binding fragment is antibody A, antibody B, or antibody C.

[0009] Regarding the specific sequence, the above anti-LIV-1 antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region each include the heavy chain complementarity determining regions 1-3 (CDR-H1, CDR-H2, and CDR-H3) and light chain complementarity determining regions 1-3 (CDR-L1, CDR-L2, and CDR-L3) shown below, of which, The heavy chain variable region of antibody A includes CDR-H1, CDR-H2, and CDR-H3, whose amino acid sequences are shown in SEQ ID NOs. 7, 8, and 9, respectively, and the light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, whose amino acid sequences are shown in SEQ ID NOs. 10, 11, and 12, respectively. The heavy chain variable region of antibody B includes CDR-H1, CDR-H2, and CDR-H3, whose amino acid sequences are shown in SEQ ID NOs. 13, 14, and 15, respectively, and the light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, whose amino acid sequences are shown in SEQ ID NOs. 16, 17, and 18, respectively. The heavy chain variable region of antibody C includes CDR-H1, CDR-H2, and CDR-H3, whose amino acid sequences are shown in SEQ ID NOs. 19, 20, and 21, respectively, and the light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, whose amino acid sequences are shown in SEQ ID NOs. 22, 23, and 24, respectively.

[0010] The CDR sequence of the above antibody is obtained according to the Kabat numbering rules. However, those skilled in the art may obtain the CDR sequence of the antibody of the present invention according to other known numbering rules (e.g., IMGT, Chothia, etc.).

[0011] In some embodiments, the anti-LIV-1 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain and the light chain each comprising a heavy chain variable region (VH) and a light chain variable region (VL) as shown below, of which, Antibody A contains the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof in the VH sequence, and the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof in the VL sequence. Antibody B comprises a VH sequence that includes the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof, and a VL sequence that includes the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof. Antibody C comprises a VH sequence that includes the amino acid sequence shown in SEQ ID NO: 5 or a variant thereof, and a VL sequence that includes the amino acid sequence shown in SEQ ID NO: 6 or a variant thereof.

[0012] In some embodiments, the anti-LIV-1 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, and the heavy chain and the light chain each comprise a heavy chain variable region (VH) and a light chain variable region (VL) shown below: The antibody comprises a VH sequence that includes the amino acid sequence shown in SEQ ID NO: 32 or a variant thereof, and a VL sequence that includes the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof.

[0013] In the context of the present invention, "variant of an amino acid sequence" refers to an amino acid sequence having at least 75% sequence identity with the above amino acid sequence (e.g., at least 80%, preferably at least 85%, more preferably at least 90%, still more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity, i.e., any percentage identity of ≧75%).

[0014] In some examples, the anti-LIV-1 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, and the heavy chain and the light chain each comprise a heavy chain variable region (VH) and a light chain variable region (VL) shown below: The VH of the above antibody A comprises a sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 1, and the VL of the above antibody A comprises a sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 2. The VH of the above antibody A comprises a sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 32, and the VL of the above antibody A comprises a sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 2. The VH of antibody B contains a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, and the VL of antibody B contains a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4. The VH portion of antibody C contains a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, and the VL portion of antibody C contains a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 6.

[0015] In some examples, the anti-LIV-1 antibody or its antigen-binding fragment may be a tetramer containing two light chains and two heavy chains, an antibody containing one or more heavy chains or light chains, Fab, Fab', F(ab')2 and Fv, or a single-chain antibody.

[0016] In particular, the anti-LIV-1 antibody or its antigen-binding fragment of the present invention includes at least a heavy chain variable region and a light chain variable region, both of which include the above-mentioned CDR and spaced framework regions (FRs), and each domain is arranged as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Therefore, with respect to the heavy chain variable region and the light chain variable region contained in the anti-LIV-1 antibody or its fragment of the present invention, at most 25% of the difference in amino acid sequence due to the "at least 75% sequence identity" may be present in either the framework region of the heavy chain variable region or the light chain variable region. Alternatively, with respect to the entire anti-LIV-1 antibody or its fragment of the present invention, at most 25% of the difference may be present in any domain or sequence other than the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment of the present invention. The above difference can be caused by amino acid deletion, addition, or substitution at any position, of which substitutions may be conserved or non-conservative substitutions.

[0017] Preferably, the anti-LIV-1 antibody or antigen-binding fragment provided in the present invention may further include a constant region. Preferably, the anti-LIV-1 antibody or antigen-binding fragment further includes a human or mouse heavy chain constant region (CH) and / or a light chain constant region (CL), and more preferably includes a heavy chain constant region selected from IgG, IgA, IgM, IgD, or IgE and / or a κ or λ-type light chain constant region.

[0018] Preferably, the antibody is a monoclonal antibody, preferably a mouse, chimeric, or humanized monoclonal antibody, and more preferably, the monoclonal antibody has a heavy chain constant region of the IgG1 or IgG4 subtype and a light chain constant region of the κ type. Alternatively, for example, the antibody is an immunoglobulin, specifically IgA, IgD, IgE, IgG, or IgM, for example, a human subtype of IgA, IgD, IgE, IgG, or IgM, more preferably a human IgG1, IgG2, IgG3, or IgG4 subtype.

[0019] According to a specific embodiment of the present invention, the anti-LIV-1 antibody or its antigen-binding fragment comprises a heavy chain constant region, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 26 or a variant thereof. Alternatively, the anti-LIV-1 antibody or its binding fragment comprises a light chain constant region, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 27 or a variant thereof. As limited to the foregoing, "amino acid sequence variant" refers to an amino acid sequence having at least 75% sequence identity with the above amino acid sequence.

[0020] In some embodiments, the anti-LIV-1 antibody described above comprises a light chain represented by SEQ ID NO: 30 and a heavy chain represented by SEQ ID NO: 31.

[0021] According to the antibody provided in the present invention, the present invention provides a nucleic acid molecule encoding an anti-LIV-1 antibody or its antigen-binding fragment as described in the present invention.

[0022] The nucleic acid molecule of the present invention can be cloned into a vector and further transformed or transfected into a host cell. Accordingly, in a further embodiment, the present invention provides a vector comprising the nucleic acid molecule of the present invention. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a phage vector.

[0023] The vectors or nucleic acid molecules of the present invention can be used for the transformation or transfection of host cells or for entry into host cells by any means, for purposes such as the storage or expression of antibodies. Accordingly, in a further embodiment, the present invention provides a host cell comprising the nucleic acid molecules and / or vectors of the present invention, or the host cell is transformed or transfected with the nucleic acid molecules and / or vectors of the present invention. The host cell may be any prokaryotic or eukaryotic cell, for example, a bacterial or insect, fungal, plant or animal cell (e.g., a CHO cell).

[0024] The anti-LIV-1 antibody or its antigen-binding fragment provided in the present invention can be obtained by any method known in the art. For example, first, the heavy chain variable region and / or light chain variable region of the antibody may be obtained from the nucleic acid molecule provided in the present invention, or the heavy chain and / or light chain of the antibody molecule may be obtained, and then the antibody may be assembled by combining them with optionally other domains of the antibody molecule, or the host cells provided in the present invention may be cultured if the host cells enable the expression of the heavy chain variable region and / or light chain variable region of the antibody molecule or the heavy chain and / or light chain of the antibody molecule for assembly of the antibody. Optionally, the method further comprises the step of recovering the produced antibody molecule.

[0025] Furthermore, the present invention further provides a multispecific antibody, for example, a bispecific antibody, comprising the above-mentioned anti-LIV-1 antibody or its antigen-binding fragment. The multispecific antibody simultaneously targets LIV-1 antigen and non-LIV-1 antigen, the non-LIV-1 antigen being selected from CD3, EGFR, HER2, HER3, PD-L1, c-MET, TROP-2, CEA5, B7-H3, SIRPα, PSMA, ROR1, and CD47, or a combination thereof.

[0026] In another embodiment, the present invention provides an antibody-drug conjugate targeting LIV-1 or a pharmaceutically acceptable salt thereof, comprising an anti-LIV-1 antibody or an antigen-binding fragment thereof covalently bound to a drug, and a drug linked to the antibody by a linker. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the following structure (Formula I): [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z, among them, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment. AG is a coupling group, where k is 0 or 1, AG is linked to L1, and AG is selected from succinimidyl (SUCC), acetyl, or carbonyl groups. [ka] This corresponds to the structure, Among them, the wavy line indicates binding to the antibody. (L1)x is the first group of linkers, where x is 0 or 1, and L1 represents a chain of 1 to 18 groups selected from -CH2-, -C(=O)-, -NH-, -O-, and -S-, where each NH, O, and S is separated from another NH, O, or S by at least two carbon atoms derived from -CH2- and / or -C(=O)-. (OP)p is an enzymatically cleavable oligopeptide, where p is the number of condensed amino acids of the oligopeptide, and p is 0 or 2-10, preferably 0, 2 or 3. (L2)y is the second group of connectants, of which y is either 0 or 1.

[0027] In the first embodiment, L2 represents a chain of 1 to 18 groups selected from -CH2-, -C(=O)-, -NH-, -O-, and -S-, where each NH, O, and S is separated from another NH, O, or S by at least two groups selected from -CH2- and / or -C(=O)-.

[0028] In the second example, L2 represents an o-, m-, or p-hydroxyphenyl group or an o-, m-, or p-aminophenyl group.

[0029] In the third embodiment, L2 represents a combination of a chain from the first embodiment and a group from the second embodiment.

[0030] D is the drug of the antibody-drug conjugate represented by formula [Formula I], where D may be any biologically active part, such as a cytotoxic compound, an immunomodulator, or an enzyme or hormone inhibitor, and is preferably a cytotoxic compound.

[0031] Each antibody may be linked to one or more (AG)k-(L1)x-(OP)p-(L2)yD groups. The drug-to-antibody ratio is z, and its value is a decimal or integer between 1 and 24. In some embodiments, z is 3.5. In some embodiments, z is 4. In some embodiments, z is between 3.5 and 4. In some embodiments, z is between 2 and 8.

[0032] In another preferred example, L1 is selected from -(CH2-)oC(=O)-, where o is 1 to 10, preferably 1 to 7, more preferably 2, 3, 4, or 5, or is selected from -(NH)j-(CH2CH2O)n-(CH2)q-(C=O)-, where j is 0 to 4, preferably 0 or 1, n is 2 to 8, preferably 4, and q is 1 to 6, preferably 2.

[0033] In another preferred example, L1 is selected from -(CH2)2-C(=O)-, -(CH2)3-C(=O)-, -(CH2)4-C(=O)-, or -(CH2)5-C(=O)-.

[0034] In another preferred example, L1 is selected from -(CH2-CH2-O)4-CH2-CH2-C=O- or -NH-(CH2CH2O)4-(CH2)2-(C=O)-.

[0035] In another preferred example, x is 0, i.e., L1 is absent, and the AG group and the (OP)p group are directly linked.

[0036] In another preferred example, the enzyme in the above-mentioned "enzyme-cleavable oligopeptide" is cathepsin or β-glucuronidase.

[0037] Preferably, the enzyme of the "enzyme-cleavable oligopeptide" is a cathepsin, and the cathepsin-cleavable oligopeptide is selected from the group consisting of ValCit, ValAla, AlaAlaAsn, GlyGlyPhe, GlyGlyTyr, GlyGlyPheGly and its derivatives, and the hydroxyl group of tyrosine or the terminal amino group of asparagine may be selectively substituted (glycosylated) with a galactosyl group, a glucosyl group, or a 1-deoxy-2-acetamido-2-deoxy-glucosyl group.

[0038] The term "glycosylation" above refers to the transfer of a glycosyl or oligosaccharide group to a hydroxyl or amino group of a compound, and is completed by chemical or enzymatic methods.

[0039] In one example, glycosylation refers to the structural modification of an amino acid residue in the oligopeptide (OP)p by a reaction between a sugar and a group within it (for example, the phenol hydroxyl group of tyrosine or the amide group of asparagine) or a hydroxyl substituent on the benzene ring in L2.

[0040] In another preferred example, (OP)p is selected from oligopeptides formed by combining valine, citrulline, alanine, glycine, asparagine, tyrosine, phenylalanine, proline, isoleucine, lysine, serine, glutamic acid, threonine, or asparagine, for example, the formed dipeptides, tripeptides, or tetrapeptides, and optionally, the phenol hydroxyl group of tyrosine or the amide group of asparagine is glycosylated, preferably with glucuronic acid, N-acetylglucosamine (GlcNAc), glucose, or galactose.

[0041] In another preferred example, (OP)p is a dipeptide formed from valine and citrulline. In some embodiments, (OP)p is a valine-citrulline-dipeptide.

[0042] In another preferred example, (OP)p is a tripeptide formed from glycine and tyrosine, and optionally, the phenol hydroxyl group of tyrosine is glycosylated. In some embodiments, (OP)p is a glycine-glycine-tyrosine tripeptide, and optionally, the phenol hydroxyl group of tyrosine is glycosylated.

[0043] In another preferred example, (OP)p is a dipeptide formed from alanine and asparagine, where the amide group of asparagine is glycosylated.

[0044] In another preferred example, p is 0, i.e., (OP)p does not exist, and the (L1)x group and the (L2)y group are directly linked.

[0045] In another preferred example, in (L2)y, y is 1 and L2 represents -NH-Ph-CH2-OC(=O)-, where the benzene ring selectively contains a hydroxy substituent, more preferably L is a p-aminobenzyloxycarbonyl group or a p-hydroxy-m-aminobenzyloxycarbonyl group, and optionally the hydroxyl group in the benzene ring is glycosylated, preferably with glucuronic acid, N-acetylglucosamine (GlcNAc), glucose, or galactose.

[0046] In one example, L2 represents a p-hydroxy-m-aminobenzyloxycarbonyl group, where the hydroxyl group in the benzene ring is glycosylated with glucuronic acid, N-acetylglucosamine (GlcNAc), glucose, or galactose.

[0047] In some examples, the antibody-drug conjugate and its pharmaceutically acceptable salt comprise the structure shown in [Formula I] below. [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Of these, Ab represents one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is a succinimidyl group, L1 is -(CH2-)oC(=O)-, where o is 1 to 7, (OP)p is -valine-citrulline-dipeptide, -glycine-glycine-tyrosine-tripeptide, or -alanine-alanine-asparagine-tripeptide, and optionally the phenol hydroxyl group of tyrosine or the amide group of asparagine is glycosylated with glucose, N-acetylglucosamine, or galactose, L2 is a p-aminobenzyloxycarbonyl group (PAB), and k is 1, x is 1, y is 1, and D and z are as defined above.

[0048] In some examples, AG is a succinimidyl group, L1 is -(CH2)oC(=O)-, where o is 3 or 5, and (OP)p is a valine-citrulline-dipeptide.

[0049] In some examples, AG is a succinimidyl group, L1 is -(CH2)oC(=O)-, where o is 3 or 5, and (OP)p is a -glycine-glycine-tyrosine-tripeptide.

[0050] In some examples, AG is a succinimidyl group, L1 is -(CH2)oC(=O)- where o is 3 or 5, and (OP)p is a -glycine-glycine-tyrosine-tripeptide where the phenol hydroxyl group of tyrosine is glycosylated with galactose or glucose.

[0051] In some examples, AG is a succinimidyl group, L1 is -(CH2)oC(=O)-, where o is 3 or 5, and (OP)p is an alanine-alanine-asparagine-tripeptide, where the amide group of asparagine is glycosylated with glucose.

[0052] In some examples, the antibody-drug conjugate and its pharmaceutically acceptable salt comprise the structure shown in [Formula I] below. [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Of these, Ab represents one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is -CH2-C(=O)-, L1 is -(NH)j-(CH2CH2O)n-(CH2)q-(C=O)-, where j is 1-4, n is 2-8, q is 1-6, (OP)p is -valine-citrulline-tripeptide or -glycine-glycine-tyrosine-tripeptide, and optionally the phenol hydroxyl group of tyrosine is glycerol The compound is glycosylated with coagulant or galactose, where L2 is a p-aminobenzyloxycarbonyl group (PAB) or a hydroxy-m-aminobenzyloxycarbonyl group, optionally, the hydroxyl group in the benzene ring 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.

[0053] In some examples, the antibody-drug conjugate and its pharmaceutically acceptable salt comprise the structure shown in [Formula I] below. [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Of these, Ab represents one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is -CH2-C(=O)-, L1 is -(NH)j-(CH2CH2O)n-(CH2)q-(C=O)-, where j is 1-4, n is 2-8, q is 1-6, p is 0, L2 is a p-aminobenzyloxycarbonyl group (PAB) or a hydroxy-m-aminobenzyloxycarbonyl group, where the hydroxyl group in the benzene ring is optionally 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.

[0054] In some examples, L1 is -NH-(CH2CH2O)4-(CH2)2-(C=O)-.

[0055] In some examples, (OP)p is a valine-citrulline dipeptide.

[0056] In some examples, (OP)p is a glycine-glycine-tyrosine tripeptide.

[0057] In some examples, (OP)p is a glycine-glycine-tyrosine tripeptide, in which the phenol hydroxyl group of tyrosine is glycosylated by glucose.

[0058] In some embodiments, p is 0, i.e., (OP)p does not exist, and the (L1)x group and the (L2)y group are directly linked.

[0059] In some examples, L2 is a p-hydroxy-m-aminobenzyloxycarbonyl group, and the hydroxyl group in the benzene ring is glycosylated by glucuronic acid.

[0060] In some examples, the antibody-drug conjugate and its pharmaceutically acceptable salt comprise the structure shown in [Formula I] below. [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Of these, Ab represents an antibody or antigen-binding fragment of any one of the above anti-human LIV-1, AG is -C(=O)-, L1 is -(CH2-)oC(=O)-, where o is 1-7, or L1 is -(NH)j-(CH2CH2O)n-(CH2)q-(C=O)-, where j is 0-4, n is 2-8, q is 1-6, (OP)p 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.

[0061] In some examples, L1 is -(CH2)2-C(=O)-.

[0062] In some examples, L1 is -(CH2)4-C(=O)-.

[0063] In some examples, L1 is -(CH2CH2O)4-(CH2)2-(C=O)-.

[0064] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the structure shown in [Formula I] below, [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Of these, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is -C(=O)-, k is 1, x is 0, (OP)p is -valine-citrulline-dipeptide, L2 is a p-aminobenzyloxycarbonyl group, y is 1, D is a drug, and Z is a decimal or integer between 1 and 10, preferably 3.5 to 4.

[0065] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the structure shown in [Formula I] below, [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z In this compound, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is -C(=O)-, k is 1, x is 0, (OP)p is a -glycine-glycine-tyrosine-tripeptide, where the phenol hydroxyl group of tyrosine is glycosylated by glucose, L2 is a p-aminobenzyloxycarbonyl group, y is 1, D is a drug, and Z is a decimal or integer between 1 and 10, preferably 3.5 to 4.

[0066] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the structure shown in [Formula I] below, [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Among these, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is -C(=O)-, k is 1, x is 0, p is 0, L2 is a p-hydroxy-m-aminobenzyloxycarbonyl group, the hydroxyl group in its benzene ring is glycosylated by glucuronic acid, y is 1, D is a drug, and Z is a decimal or integer between 1 and 10, preferably 3.5 to 4.

[0067] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the structure shown in [Formula I] below, [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Of these, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is -C(=O)-, k is 1, L1 is -NH-(CH2-CH2-O)4-CH2-CH2-C=O-, x is 1, (OP)p is -valine-citrulline-dipeptide, L2 is p-aminobenzyloxycarbonyl group, y is 1, D is a drug, and Z is a decimal or integer between 1 and 10, preferably between 3.5 and 4.

[0068] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the structure shown in [Formula I] below, [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Among these, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is -C(=O)-, k is 1, L1 is -NH-(CH2-CH2-O)4-CH2-CH2-C=O-, x is 1, p is 0, L2 is a p-hydroxy-m-aminobenzyloxycarbonyl group, the hydroxyl group in the benzene ring is glycosylated by glucuronic acid, y is 1, D is a drug, and Z is a decimal or integer between 1 and 10, preferably between 3.5 and 4.

[0069] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the structure shown in [Formula I] below, [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Among these, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is a succinimidyl group, k is 1, L1 is -(CH2)5-C(=O)-, x is 1, (OP)p is a valine-citrulline dipeptide, L2 is a p-aminobenzyloxycarbonyl group, y is 1, D is a drug, and Z is a decimal or integer between 1 and 10, preferably 3.5 to 4.

[0070] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the structure shown in [Formula I] below, [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Among these, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is a succinimidyl group, k is 1, L1 is -(CH2)5-C(=O)-, x is 1, (OP)p is a -glycine-glycine-tyrosine-tripeptide, where the phenol hydroxyl group of tyrosine is glycosylated with galactose, L2 is a p-aminobenzyloxycarbonyl group, y is 1, D is a drug, and Z is a decimal or integer between 1 and 10, preferably 3.5 to 4.

[0071] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the structure shown in [Formula I] below, [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Of these, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is a succinimidyl group, k is 1, L1 is -(CH2)5-C(=O)-, x is 1, (OP)p is a -glycine-glycine-tyrosine-tripeptide, L2 is a p-aminobenzyloxycarbonyl group, y is 1, D is a drug, and Z is a decimal or integer between 1 and 10, preferably 3.5 to 4.

[0072] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the structure shown in [Formula I] below, [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Among these, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is a succinimidyl group, k is 1, L1 is -(CH2)5-C(=O)-, x is 1, (OP)p is a -glycine-glycine-tyrosine-tripeptide, where the phenol hydroxyl group of tyrosine is glycosylated by glucose, L2 is a p-aminobenzyloxycarbonyl group, y is 1, D is a drug, and Z is a decimal or integer between 1 and 10, preferably 3.5 to 4.

[0073] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the structure shown in [Formula I] below, [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Among these, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is a succinimidyl group, k is 1, L1 is -(CH2)3-C(=O)-, x is 1, (OP)p is an alanine-alanine-asparagine-tripeptide, where the amide group of asparagine is glycosylated with glucose, L2 is a p-aminobenzyloxycarbonyl group, y is 1, D is a drug, and Z is a decimal or integer between 1 and 10, preferably 3.5 to 4.

[0074] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the structure shown in [Formula I] below, [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Of these, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is -C(=O)-, k is 1, L1 is -(CH2)2-C(=O)-, x is 1, (OP)p is -valine-citrulline-dipeptide, L2 is a p-aminobenzyloxycarbonyl group, y is 1, D is a drug, and Z is a decimal or integer between 1 and 10, preferably 1 to 2.

[0075] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the structure shown in [Formula I] below, [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Of these, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is -C(=O)-, k is 1, L1 is -(CH2)4-C(=O)-, x is 1, (OP)p is -valine-citrulline-dipeptide, L2 is p-aminobenzyloxycarbonyl group, y is 1, D is a drug, and Z is a decimal or integer between 1 and 10, preferably 1 to 2.

[0076] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises the structure shown in [Formula I] below, [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)yD]z Of these, Ab represents any one of the above anti-human LIV-1 antibodies or its antigen-binding fragment, AG is -C(=O)-, k is 1, L1 is -(CH2-CH2-O)4-(CH2)2-C(=O)-, x is 1, (OP)p is -valine-citrulline-dipeptide, L2 is p-aminobenzyloxycarbonyl group, y is 1, D is a drug, and Z is a decimal or integer between 1 and 10, preferably 1 to 2.

[0077] In several preferred embodiments, -(AG)k-(L1)x-(OP)p-(L2)y- has the following structure, where "1" indicates binding to antibody Ab and "2" indicates binding to drug D.

[0078] [Table 1] JPEG2026509871000003.jpg80169

[0079] In another preferred example, the cytotoxic compound is a compound that alkylates DNA, or a compound that inhibits the polymerization of topoisomerase-1, topoisomerase-2, tubulin, or RNA polymerase. Alternatively, the cytotoxic compound methylates DNA. The drug is selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine, SN-38, or exatecan.

[0080] In another preferred example, [(AG)k-(L1)x-(OP)p-(L2)yD] described in [Formula I] is, [ka] It has the following structure, and the wavy line indicates binding to the antibody Ab.

[0081] In another preferred example, [(AG)k-(L1)x-(OP)p-(L2)yD] described in [Formula I] is, [ka] It has the following structure, and the wavy line indicates binding to the antibody Ab.

[0082] In some examples, the antibody-drug conjugate described in [Formula I] and its pharmaceutically acceptable salts are: [ka] JPEG2026509871000007.jpg85169 It has a structure selected from among them.

[0083] In some embodiments, the VH of Ab above includes three CDRs: CDR-H1, CDR-H2, and CDR-H3, of which, CDR-H1 contains the amino acid sequence shown in SEQ ID NO: 7, CDR-H2 contains the amino acid sequence shown in SEQ ID NO: 8, CDR-H3 contains the amino acid sequence shown in SEQ ID NO: 9, Furthermore, the above Ab VL includes three CDRs: CDR-L1, CDR-L2, and CDR-L3, of which, CDR-L1 contains the amino acid sequence shown in SEQ ID NO: 10, CDR-L2 contains the amino acid sequence shown in SEQ ID NO: 11, CDR-L3 contains the amino acid sequence shown in SEQ ID NO: 12.

[0084] In some embodiments, VH of Ab contains the amino acid sequence of SEQ ID NO: 1, and VL of Ab contains the amino acid sequence of SEQ ID NO: 2.

[0085] In some examples, Ab comprises the light chain indicated by SEQ ID NO: 30 and the heavy chain indicated by SEQ ID NO: 31, and z is a decimal or integer between 2 and 8. In another preferred example, the antibody portion of the antibody-drug conjugate or salt thereof is a multispecific antibody comprising the anti-LIV-1 antibody or its antigen-binding fragment. For example, the antibody is a bivalent tetramer comprising two light chains and two heavy chains, of which the first pair of heavy and light chains comprises the heavy chain variable region and light chain variable region of the anti-LIV-1 antibody or its antigen-binding fragment, and the second pair of heavy or light chains of the tetramer can bind to a non-LIV-1 antigen, of which the non-LIV-1 antigen is selected from CD3, EGFR, HER2, HER3, PD-L1, c-MET, TROP-2, CEA5, B7-H3, SIRPα, PSMA, ROR1, or CD47.

[0086] The LIV-1-targeting antibody-drug conjugates or pharmaceutically acceptable salts thereof, anti-LIV-1 antibodies or antigen-binding fragments, nucleic acid molecules, vectors, or host cells provided in the present invention may be further included in the composition, and in particular in the drug formulation, thereby being used for various purposes as required in practice. Accordingly, in a further embodiment, the present invention further provides compositions comprising the LIV-1-targeting antibody-drug conjugates or pharmaceutically acceptable salts thereof, anti-LIV-1 antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, and / or host cells provided in the present invention. Preferably, the above composition is a pharmaceutical composition further comprising selectively pharmaceutically acceptable carriers, additives, or excipients.

[0087] The present invention provides for the use of the above-mentioned LIV-1-targeting antibody-drug conjugate or pharmaceutically acceptable salt thereof, anti-LIV-1 antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell and / or composition as a drug (i.e., for therapeutic use).

[0088] In further embodiments, the present invention further provides the use of the above-mentioned LIV-1-targeting antibody-drug conjugate or pharmaceutically acceptable salt thereof, anti-LIV-1 antibody or antigen-binding fragment thereof, nucleic acid molecules, vectors, host cells and / or compositions in the preparation of drugs for treating tumors or cancer. Alternatively, the present invention further provides a method for treating a tumor, the method comprising administering to a subject in need thereof the above-mentioned LIV-1-targeting antibody-drug conjugate or pharmaceutically acceptable salt thereof, anti-LIV-1 antibody or antigen-binding fragment thereof, nucleic acid molecules, vectors, host cells and / or compositions provided in the present invention, where the subject is a mammal, preferably a primate, and more preferably a human.

[0089] In some embodiments, the antibody-drug conjugates of the present invention or pharmaceutically acceptable salts thereof, anti-LIV-1 antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, host cells and / or compositions can be used as drugs, and in some embodiments, they are used as drugs for treating tumors or cancer.

[0090] In some embodiments, the tumor or cancer is a tumor or cancer associated with high LIV-1 expression. In some embodiments, the tumor or cancer is any one selected from bladder cancer, breast cancer, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, colorectal cancer, glioma, mesothelioma, cervical cancer, triple-negative breast cancer, lung cancer, head and neck cancer, esophageal cancer, skin cancer, and uterine cancer.

[0091] In some embodiments, the above-mentioned tumor or cancer is a solid tumor.

[0092] In some manner, the above tumors or cancers are selected from breast cancer, bladder cancer, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, colorectal cancer, glioma, mesothelioma, cervical cancer, triple-negative breast cancer, lung cancer, head and neck cancer, esophageal cancer, skin cancer, and uterine cancer.

[0093] In some embodiments, the tumor or cancer described above is a hematological malignancy.

[0094] In some embodiments, the tumor or cancer described above is selected from non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, and anaplastic large cell lymphoma.

[0095] In some embodiments, the above-mentioned tumor or cancer is selected from lung cancer, breast cancer, and prostate cancer.

[0096] In some embodiments, the tumor or cancer described above is breast cancer, and more particularly, triple-negative breast cancer.

[0097] In a further aspect, the present invention provides an immune complex, said immune complex (a) The antibody or antigen-binding fragment thereof described in the present invention, (b) A coupling moiety selected from the group consisting of a detectable marker, a radionuclide, a cytokine, an enzyme, gold nanoparticles / nanorods, nanomagnetic particles, a virus-coated protein or VLP, or a combination thereof.

[0098] In another preferred example, the radioactive nuclide is: (i) Diagnostic isotopes selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188 or combinations thereof, and / or (ii) A therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0099] In another preferred example, the detectable label is selected from fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0100] Accordingly, the immune complex of the present invention can be used for the diagnosis of tumors or cancer by being used to detect LIV-1 in a sample. Based on this, the present invention further provides a method (including diagnostic and non-diagnostic) for detecting LIV-1 in a sample, the method comprising: (1) contacting a sample with the immune complex described in the present invention; and (2) detecting whether or not an antigen-antibody complex has been formed, and if a complex has been formed, indicating that LIV-1 is present in the sample.

[0101] Within the scope of the present invention, it should be understood that any combination of the above-described technical features of the present invention and the technical features specifically described below (for example, in the examples) can constitute a new or preferred technical solution. Due to space limitations, a detailed explanation is omitted here. [Brief explanation of the drawing]

[0102] [Figure 1A] This shows the detection of antibody A's binding activity to human LIV-1 using the ELISA method. [Figure 1B]This shows the detection of the binding activity of antibodies B and C to human LIV-1 using the ELISA method. [Figure 2] This shows the detection of the binding activity of the antibody-drug conjugate A-BrAcMMAE to human LIV-1 using the ELISA method. [Figure 3] This demonstrates the in vitro detection of ADC's growth inhibitory activity against the cancer cell line Calu6. [Figure 4] This demonstrates the in vitro detection of ADC proliferation inhibitory activity against the human LIV-1 overexpressing cancer cell line MCF7-ATCC-hLIV-1-#7. [Figure 5] This demonstrates the in vitro detection of ADC proliferation inhibitory activity against the human LIV-1 overexpressing cancer cell line MCF7-ATCC-hLIV-1-#12. [Figure 6] This demonstrates the in vitro detection of ADC proliferation inhibitory activity against the human LIV-1 overexpressing cancer cell line MCF7-ATCC-hLIV-1-#17. [Figure 7] The results of a xenotransplantation study of the MCF7 breast cancer cell line into NSG mice are shown. The dosage and administration time are as shown in the figure. [Figure 8] The results of a xenotransplantation study of the HCC1806 breast cancer cell line into NSG mice are shown. The dosage and administration time are as shown in the figure. [Figure 9] The results of a xenotransplantation study of the PC3 prostate cancer cell line into nude mice are shown. The dosage and administration time are as shown in the figure. [Figure 10] The results of a xenotransplantation study of the Calu-6 lung cancer cell line into nude mice are shown. The dosage and administration time are as shown in the figure. [Figure 11] The results of a xenotransplantation study of the Calu-6 lung cancer cell line into nude mice are shown. The dosage and administration time are as shown in the figure. [Figure 12] The results of a xenotransplantation study of the PC3 prostate cancer cell line into nude mice are shown. The dosage and administration time are as shown in the figure. [Figure 13] The results of a xenotransplantation study of the PA-1 ovarian cancer cell line into nude mice are shown. The dosage and administration time are as shown in the figure. [Modes for carrying out the invention]

[0103] The inventors, through extensive and detailed research, have provided an antibody that specifically binds to human LIV-1 and obtained a series of anti-human LIV-1 antibody-drug conjugates by coupling the antibody to a drug load. The above anti-human LIV-1 antibody and anti-human LIV-1 antibody-drug conjugates can be used to treat tumors or cancer, particularly tumors or cancers that express LIV-1, by being used in the preparation of drugs to treat tumors or cancer. In vivo and in vitro experiments have demonstrated that the anti-human LIV-1 antibody-drug conjugates of the present invention have good targeting ability against tumor cells and exhibit excellent antitumor activity. Furthermore, the anti-human LIV-1 antibody of the present invention can also be used for the diagnosis of tumors or cancer by being coupled to a detectable marker and used to detect the presence of LIV-1 in a sample. Based on this, the present invention is completed.

[0104] To better understand the present invention, the following terms are defined.

[0105] Unless otherwise specified, all singular terms include their plural forms, active voice, and past tense.

[0106] Unless otherwise specified in the context, the term "approximately" includes values ​​within the standard deviation range of the above values.

[0107] The phrase "basically from..." indicates that the composition and method may include additional components and / or steps, but only if the additional components and / or steps do not substantially alter the basic and new properties of the requested composition or method. The phrase "consisting of..." is intended to exclude all inactive components.

[0108] The “subject” or “patient” according to the present invention is an animal, and includes human patients requiring anti-cancer treatment or treatment. In some embodiments, the present invention may also be used in veterinary practice for any mammal or other animal requiring such LIV-1 targeted anti-cancer treatment. This may include, for example, non-human primates, dogs, felines, pigs, horses, and any other animals subject to anti-cancer treatment against LIV-1.

[0109] As used herein, the term “antibody” is intended to include any known type of natural or engineered antigen-binding protein or polypeptide comprising at least one antigen-specific variable domain (such as a VL or VH domain, or an engineered functional polypeptide-binding domain). Such antibody may be a polyclonal antibody, a monoclonal antibody, or an engineered synthetic antibody, such as a humanized monoclonal antibody. The use of derivatives and fragments of polyclonal antibodies and / or monoclonal antibodies is also conceivable. These include Fc fragments, Fab fragments, single-chain antibodies and their polymers, and synthetic polypeptides having two or more binding specificities.

[0110] Typically, antibody fragments (or antigen-binding fragments) compete with the complete antibody to obtain specific binding to the target and include independent heavy chains, light chains, Fab, Fab', F(ab')2, F(ab)c, diabodies, dabs, nanobodies, and Fv. Antibody fragments may be produced by DNA recombination technology or by enzymatic or chemical separation of complete immunoglobulins. The term "antibody" further includes diabodies (homodimerated Fv fragments) or minibodies (VL-VH-CH3), bispecific antibodies, or analogues. Bispecific or bifunctional antibodies are artificial mixed antibodies having two different heavy / light chain pairs and two different binding sites (see Songsivilai & Lachmann, Clin. Exp. Immunol., 79:315-321 (1990), Kostelny et al., J. Immunol., 148:1547-53 (1992)). The term "antibody" includes antibodies themselves (naked antibodies) or antibodies coupled to cytotoxic or cell growth inhibitory drugs, which are referred to here as antibody-drug conjugates or ADCs.

[0111] Monoclonal antibodies typically require isolation and purification. This means that the purity of interfering proteins and other contaminants produced during antibody production or purification is usually at least 50%, but this does not rule out the possibility that the monoclonal antibody may bind to an excess of drug-acceptable carriers or other carriers to facilitate its use. Monoclonal antibodies may have a w / w purity of at least 60%, 70%, 80%, 90%, 95%, or 99% of the produced or purified interfering proteins and contaminants.

[0112] The basic structural unit of natural antibodies is the tetrameric subunit structure. Each tetramer contains two pairs of similar polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids, primarily responsible for antigen recognition. This variable region is ligated to a cleavable signal peptide during expression. A variable region without a signal peptide is sometimes called a mature variable region. Therefore, for example, the light chain mature variable region refers to a light chain variable region without a light chain signal peptide. The carboxyl-terminal portion of each chain defines a single constant region, primarily responsible for effector function.

[0113] The specific binding of a monoclonal antibody to its target antigen is at least 10 6 , 10 7 , 10 8 , 10 9 or 10 10 M -1 This means that it has affinity for a particular target. Specific binding is detectable by an order of magnitude higher than nonspecific binding, which involves binding to at least one unrelated target. Specific binding can result from the formation of a bond between specific functional groups or specific spatial matchings (e.g., lock-and-key types), while nonspecific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that a monoclonal antibody binds to only one target.

[0114] The light chain is divided into κ or λ. The heavy chain is divided into γ, μ, α, δ, or ε, and the antibody isotypes are defined as IgG, IgM, IgA, IgD, and IgE, respectively. In the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain further includes a "D" region of approximately 10 or more amino acids. (Generally, refer to "Basic Immunology," Paul, W., ed., 2nd ed., Raven Press, NY, 1989, Ch. 7, which is incorporated in its entirety by citation for various purposes).

[0115] The maturation variable region of each light / heavy chain pair forms the antibody binding site. Therefore, a complete antibody has two binding sites, and these two binding sites are identical, whereas bifunctional or bispecific antibodies have different binding sites. Both of these chains exhibit the same, relatively conserved general structure of the framework region (FR), which is linked by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs, derived from the two chains of each pair, can bind to specific epitopes by being sequenced according to the framework region. From the N-terminus to the C-terminus, both the light and heavy chains consist of domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments for each domain conform to the Kabat immunological protein sequences (National Institutes of Health, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J.Mol.Biol.196:901-917 (1987), Chothia et al., Nature 342:878-883 (1989). Kabat further provides a widely used numbering convention (Kabat numbering), in which the same number is assigned to corresponding residues between different heavy chains or between different light chains.

[0116] The term "epitope" refers to a single site on an antigen to which an antibody binds. Epitopes can be formed from adjacent amino acids or from discontinuous amino acids resulting from the tertiary folding of one or more proteins. Epitopes formed from adjacent amino acids are usually retained in denaturing solvents, while epitopes formed by tertiary folding are usually lost in denaturing solvents. A single epitope typically contains at least three, more commonly, at least five or eight to ten amino acids to form a unique spatial conformation. Methods for determining epitope conformations include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0117] Antibodies that recognize the same or overlapping epitopes can be detected by a simple immunoassay method, namely by detecting the competitive ability of one antibody and the other antibody to bind to the target antigen. The epitopes of antibodies that bind to the antigen can also be measured by X-ray crystallography to determine the interaction residues. Alternatively, if all the amino acid mutations in the antigen that cause a decrease or elimination of binding for one antibody also cause a decrease or elimination of binding for the other antibody, then the two antibodies have the same epitope. If some of the amino acid mutations that cause a decrease or elimination of binding for one antibody in the antigen also cause a decrease or elimination of binding for the other antibody, then the two antibodies have overlapping epitopes.

[0118] Antibody competition can be experimentally detected when an antibody awaiting measurement inhibits the specific binding of the reference antibody to a common antigen (e.g., Junghans et al., Cancer Res. 50:1495, 1990). In competitive binding experiments, if the antibody awaiting measurement is in excess of the reference antibody (e.g., at least 2x, 5x, 10x, 20x, or 100x), the binding of the reference antibody will be inhibited by at least 50% when the antibody awaiting measurement competes with the reference antibody, although 75%, 90%, or 99% are most preferable in competitive binding tests. Antibodies identifiable by competitive experiments (competing antibodies) include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to epitopes adjacent to the reference antibody.

[0119] To distinguish between conserved and non-conserved amino acid substitutions, amino acids are classified as follows: Class I (hydrophobic side chains): Met, Ala, Val, Leu, Ile; Class II (neutral hydrophilic side chains): Cys, Ser, Thr; Class III (acidic side chains): Asp, Glu; Class IV (basic side chains): Asn, Gln, His, Lys, Arg; Class V (residues affecting chain orientation): Gly, Pro; Class VI (aromatic side chains): Trp, Tyr, Phe. Conserved substitutions include substitutions between amino acids of the same class. Non-conservative substitutions are substitutions between amino acids of different classes.

[0120] Sequence alignment identification can be determined by aligning the antibody sequence to the maximum extent possible according to the Kabat numbering convention. After alignment, when comparing the structural region of the antibody awaiting measurement (e.g., the entire maturation variable region of the heavy or light chain) with the same region of the reference antibody, the percentage of similarity between the comparison sequences of the antibody awaiting measurement and the reference antibody is calculated by dividing the number of positions of the same amino acid in the antibody awaiting measurement and the reference antibody region by the total number of alignment positions in the two regions, without calculating the gap, and multiplying by 100 to convert it to a percentage.

[0121] A composition or method "containing" one or more of the above elements may also contain other elements not specifically described. For example, a composition containing an antibody may contain the antibody alone or in combination with other components.

[0122] Specifying a range of values ​​includes all integers within or defining that range.

[0123] The effector function of an antibody refers to the function produced by the Fc domain of Ig. For example, these functions may be antibody-dependent cytotoxicity, antibody-dependent cytophagocytosis, or complement-dependent cytotoxicity. For example, the Fc effector domain can produce effector function by binding to Fc receptors on immune cells that have phagocytic or lytic activity, or by binding to components of the complement system. Typically, the effects mediated by Fc-binding cells or complement components cause growth inhibition and / or apoptosis of target cells expressing LIV-1. The Fc region of an antibody can recruit Fc receptor (FcR)-expressing cells and attract them to the vicinity of target cells that bind to the antibody. Cells that membrane-express FcRs include FcγRIII(CD16), FcγRII(CD32), and FcγRIII(CD64), and may be effector cells that kill IgG-binding cells. These effector cells include monocytes, macrophages, natural killer cells, neutrophils, and eosinophils. Contact between IgG and FcγR can activate antibody-dependent cytotoxicity (ADCC) or antibody-dependent cytophagocytosis (ADCP). ADCC is mediated by the secretion of pore-forming proteins and proteases by CD16.sup.+ effector cells, while phagocytosis is mediated by CD32.sup.+ and CD64.sup.+ effector cells (see Basic Immunology, 4th edition, 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 cell-binding antibodies can also activate the classical complement pathway, inducing complement-dependent cytotoxicity (CDC). When an antibody forms a complex with an antigen, C1q of the complement system binds to the Fc region of the antibody.When C1q binds to an antibody on a binding cell, it initiates a cascade reaction involving the activation of C4 and C2 protein hydrolysis, thereby producing C3 invertase. C3 invertase can activate terminal complement components, including C5b, C6, C7, C8, and C9, by cleaving C3 into C3b. In general, these proteins form membrane attack complex pores in antibody-coated cells. These pores disrupt the integrity of the cell membrane, leading to the death of target cells (see Chapter 2 of Immunobiology, 6th edition, Janeway et al., Garland Science, New York, 2005).

[0124] The term "antibody-dependent cytotoxicity" or ADCC refers to a mechanism of cell death that relies on the interaction between antibody-coated target cells and immune cells with lytic activity (also called effector cells). These effector cells include natural killer cells, monocytes / macrophages, and neutrophils. Effector cells attach to the Fc effector domain of Ig, while IgG binds to target cells via its antigen-binding site. The effector cells exert their activity, causing the death of antibody-coated target cells.

[0125] The term "antibody-dependent cell phagocytosis" or ADCP refers to the process by which antibody-coated cells are whole or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells), which bind to the Fc effector domain of Ig.

[0126] The term "complement-dependent cytotoxicity" or CDC refers to a mechanism by which the Fc effector domain of an antibody that binds to a target cell activates a series of enzymatic reactions, ultimately forming pores in the target cell membrane, thereby inducing cell death. Typically, antigen-antibody complexes, such as those formed by coating target cells with an antibody, activate the complement cascade reaction by binding to and activating complement component C1q, leading to target cell death. Complement activation can also cause the accumulation of complement components on the target cell surface, promoting ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0127] "Cytotoxic effect" refers to the depletion, removal, and / or death of target cells. "Cytotoxic preparation" refers to a preparation that has a cytotoxic effect on cells. Cytotoxic preparations can be administered either coupled to an antibody or in combination with an antibody.

[0128] "Cell inhibitory effect" refers to the inhibition of cell proliferation. "Cell inhibitor" refers to a drug that has a cell inhibitory effect on cells, thereby inhibiting the growth and / or amplification of a specific cell subgroup. Cell inhibitors can be administered coupled to antibodies or in combination with antibodies.

[0129] The term "pharmaceutically acceptable" refers to a substance approved or eligible for approval by a regulatory body, or registered in a pharmacopoeia or another generally accepted pharmacopoeia for use in animals, particularly humans. The term "pharmaceutically compatible component" refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or carrier that conjugates to an anti-LIV-1 antibody.

[0130] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt or complex thereof of an anti-LIV-1 antibody, or a preparation used in combination with an anti-LIV-1 antibody. Exemplary salts include sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, citrates, tartrates, oleates, tannates, pantothenates, tartrates, ascorbicates, succinates, maleates, gentisinates, fumarates, glucons, glucurons, sugarates, formates, benzoates, glutamates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, and pamoates (i.e., 1,1'dimethylbis(2-hydroxy-3-naphthalene) salts). A pharmaceutically acceptable salt may contain other molecules such as acetate ions, succinate ions, or other counterions. The counterion may be any organic or inorganic part that stabilizes the charge in the parent compound. Furthermore, a pharmaceutically acceptable salt may have one or more charged atoms in its structure. Examples of pharmaceutically acceptable salts having multiple charged atoms may also have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0131] The terms “host cell,” “host cell line,” and “host cell culture” may be used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” and without regard to passage number, include primary transformed cells and their resulting offspring. Offspring may contain mutations, but may not be exactly the same as the parent cells in nucleic acid content. In this specification, the term includes mutant offspring having the same function or biological activity as cells screened or selected from primary transformed cells. Host cells include prokaryotic and eukaryotic host cells, of which eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include, but are not limited to, human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, such as Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, and Pichia stiptis. Pichia methanolica, Pichia genus, Saccharomyces cerevisiae, Saccharomyces genus, Hansenula polymorpha, Kluyveromyces genus, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum This includes *gramineum*, *Fusarium venenatum*, *Physcomitrella patens*, and *Neurospora crassa*.It includes Pichia, any Saccharomyces, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Chrysosporium lucknowense, any Fusarium, Yarrowia lipolytica, and Neurospora crassa. The host cells of this patent do not include objects not recognized by the patent law.

[0132] The term "antibody-drug conjugate" (ADC) refers to a conjugate in which a monoclonal antibody or antibody fragment is linked to a toxic drug having biological activity by a linking unit. The antibody or antibody fragment described in this disclosure may be coupled to an effector molecule by any method. For example, the antibody or antibody fragment may be attached to a toxic drug chemically or recombinantly. Chemical methods for preparing fusions or conjugates are known in the art. The method for coupling an antibody or antibody fragment to a 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.

[0133] A cytotoxic drug or cytotoxic compound refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at a sufficiently high concentration, but due to the lack of specificity, while killing tumor cells, they also cause apoptosis of normal cells and cause serious side effects. Cytotoxic drugs include toxins such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants or animals, radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153, Bi 212 , P 32 This includes radioactive isotopes of Lu, chemotherapeutic agents, antibiotics, and nucleases.

[0134] The antibody of the present invention and the cytotoxic drug can be coupled with a coupling agent. Examples of the coupling agent include one or more of the following: a non-selective coupling agent, a coupling agent using a carboxyl group, a coupling agent using a peptide chain, or a coupling agent using a disulfide bond. The non-selective coupling agent refers to a compound that causes a covalent bond to form between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent using a carboxyl group may be one or more of the following: a cis-aconitic anhydride coupling agent (e.g., cis-aconitic anhydride) or an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0135] Certain residues in an antibody (e.g., Cys or Lys) are used to link to multiple functional groups, including imaging reagents (e.g., chromogenic and fluorescent groups), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent complexes. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently bonded) to antibodies. Functional agents can be linked to antibodies directly or indirectly via linkers.

[0136] Antibodies can form antibody-drug conjugates (ADCs) by being coupled to drugs. Typically, ADCs include a linker (or linker) between the drug and the antibody. The terms “linker unit,” “linking fragment,” or “linking unit” refer to a chemical structural fragment or conjugate in which one end is linked to an antibody or its antigen-binding fragment and the other end is linked to a drug, and which may be linked to a drug after being linked to another linker. Linkers may be degradable or non-degradable. Degradable linkers are typically readily degraded in the intracellular environment, for example, by degradation of the linker at a target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptide group-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidases. The peptide linker may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine, or tripeptides such as glycine-phenylalanine-glycine, or tetrapeptides such as glycine-glycine-phenylalanine-glycine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that are hydrolyzed when the pH is less than 5.5, e.g., hydrazone linkers) and linkers that are degraded under reducing conditions (e.g., disulfide linker). Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by a protease.

[0137] Before being linked to the antibody, the linker has an active reactive group that can react with a certain amino acid residue, and linkage is achieved by the active reactive group. Mercapto-specific active reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodine, bromo, or chloro), haloesters (e.g., iodine, bromo, or chloro), halomethyl ketones (e.g., iodine, bromo, or chloro), benzyl halides (e.g., iodine, bromo, or chloro), vinyl sulfones, pyridyl disulfide, mercury derivatives such as 3,6-di-(mercurymethyl)dioxane whose counterion is an acetate ion, chloride ion, or nitrate ion, and polymethylene dimethyl sulfide thiosulfonates. The linker may also include, for example, a maleimide linked to the antibody via thiosuccinimide.

[0138] In the present invention, a drug-linker compound can be used to form an ADC in one simple step. In other embodiments, a bifunctional linker compound can be used to form an ADC in two or more steps. For example, an ADC may be formed by a cysteine ​​residue reacting with the reactive moiety of the linker in a first step, and then by a functional group in the linker reacting with the drug in a subsequent step.

[0139] Typically, the functional groups in the linker are selected to facilitate specific reactions with appropriate reactive groups in the drug moiety. As a non-limiting example, a moiety based on an azide compound can be used for specific reactions with a reactive alkynyl group in the drug moiety. The drug is covalently bonded to the linker by 1,3-dipolar cyclic addition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reactions with hydrazides and alkoxyamines), phosphines (suitable for reactions with azides), isocyanates and isothiocyanates (suitable for reactions with amines and alcohols), and activated esters such as N-hydroxysuccinimide esters (suitable for reactions with amines and alcohols). These and other coupling strategies are described, for example, in *Biocoupling Techniques*, 2nd edition (Elsevier) and are well known to those skilled in the art. Those skilled in the art will understand that, with respect to the selective reaction between the drug portion and the linker, if a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used as both the linker and the drug.

[0140] The present invention further provides a method for preparing an ADC, which may further include conjugating an antibody and a drug-linker compound (or a drug-linker compound (linker-drug, LD) under conditions sufficient for the formation of an antibody complex (ADC).

[0141] In one embodiment, the method of the present invention includes binding an antibody to a linker compound under conditions sufficient for the formation of an antibody-linker complex. In these embodiments, the method of the present invention further includes binding the antibody-linker complex to a drug moiety under conditions sufficient for the drug moiety to be covalently bonded to the antibody by the linker.

[0142] "Drug load," also known as the drug-to-antibody ratio (DAR), is the average number of drugs coupled to each antibody in the ADC. It may be, for example, in a range where each antibody is coupled to about 1 to about 10 drugs, and in a given example, in a range where each antibody is coupled to about 1 to about 8 drugs, preferably selected from the ranges 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 load may be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The general formula for the ADC in this disclosure includes a set of antibody-drug conjugates within the above-mentioned ranges. In embodiments of this disclosure, the drug load may be denoted by z and may be a decimal or an integer. The drug load can be measured by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, and HPLC.

[0143] In one embodiment of the present disclosure, a cytotoxic drug is coupled to an antibody by a coupling unit.

[0144] The amount of ligand-drug conjugates loaded is (1) Controlling the molar ratio of drug linker fragment to monoclonal antibody, (2) Controlling the reaction time and temperature, (3) Selecting different reaction reagents, It can be controlled by non-restrictive methods, including [specific methods].

[0145] The present invention will be described below with reference to specific examples. Those skilled in the art will understand that these examples are merely illustrative and do not limit the scope of the present invention in any way.

[0146] As used herein, “sugar” refers to a monovalent group of a monosaccharide (e.g., pyranose or furanose). The sugar may include a hemiacetal or a carboxylic acid (derived from the oxidation of an overhang-CH2OH group). In some examples, the sugar exhibits a β-D configuration. In some examples, the sugar is glucose, glucuronic acid, N-acetylglucosamine, or galactose.

[0147] The term "hydroxyl group" refers to the -OH group.

[0148] The term "amino group" refers to -NH2.

[0149] Dichloromethane is abbreviated as DCM.

[0150] N-ethyldiisopropylamine (N,N-Diisopropylethylamine) is abbreviated as DIEA.

[0151] N,N-dimethylformamide is abbreviated as DMF.

[0152] O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate is abbreviated as HATU.

[0153] 1-Hydroxybenzotriazole is abbreviated as HOBt.

[0154] "Substituting" means that one or more hydrogen atoms in a group, preferably 1 to 6, more preferably 1 to 3, are substituted by a number of substituents that correspond to each other independently. Those skilled in the art can determine possible or impossible substitutions (experimentally or theoretically) with little effort. For example, an amino or hydroxyl group with free hydrogen can become unstable when bonded to a carbon atom with an unsaturated (e.g., olefin) bond.

[0155] Unless otherwise specified, the experimental methods in the following examples are all standard procedures. Unless otherwise specified, the raw materials, reagents, etc., used in the following examples are all commercially available products.

[0156] Example 1: Preparation of anti-LIV-1 antibody This invention relates to a method for preparing antibodies by immunizing mice with human LIV-1 extracellular region fragments, and to humanizing anti-human LIV-1 antibodies against mouse antibodies. The mouse antibodies were prepared by injecting mice with a composition containing human LIV-1 extracellular region fragments and identifying the presence of antibodies by collecting serum samples. Lymphocytes were obtained from mouse lymph nodes, enriched with B cells specific to the human LIV-1 antigen, and antibody sequences were obtained by sequencing and analyzing single B cells.

[0157] After obtaining the antibody sequence, antibodies can be prepared using recombinant technology, and mouse antibodies can be chimeric and humanized. Specifically, humanized antibodies were prepared by transferring the mouse complementarity-determining region from the heavy and light chain variable regions of mouse immunoglobulin to the human variable region, and then substituting human residues in the framework region with a mouse analog. During the preparation of humanized antibodies, affinity maturation of the CDR region or removal of hotspot sites can be selectively performed to improve the performance of certain aspects of the antibody.

[0158] The antibodies prepared in this embodiment include monoclonal antibodies A, B, and C, of ​​which antibody A is a humanized antibody, and antibodies B and C are chimeric antibodies. The mouse antibody of antibody A has its mVH sequence shown in SEQ ID NO: 28 and its mVL sequence shown in SEQ ID NO: 29. The VH sequence of antibody A is shown in SEQ ID NO: 1 and its VL sequence is shown in SEQ ID NO: 2. Another antibody A-1, obtained by humanizing based on the mouse antibody of antibody A, has its CDR sequence the same as antibody A. The VH sequence of antibody A-1 is shown in SEQ ID NO: 32 and its VL sequence is shown in SEQ ID NO: 2. The VH sequence of antibody B is shown in SEQ ID NO: 3 and its VL sequence is shown in SEQ ID NO: 4. The VH sequence of antibody C is shown in SEQ ID NO: 5 and its VL sequence is shown in SEQ ID NO: 6. For each antibody, please refer to SEQ ID NO: 26 for the heavy chain constant region sequence and SEQ ID NO: 27 for the light chain constant region sequence.

[0159] Furthermore, the binding affinity of the prepared antibody to the human LIV-1 antigen (His-tagged, with amino acid sequence NP_036451.1, including the amino acid at position Phe229-Ile323) was detected. As shown in Table 1, antibody A demonstrated a significant increase in binding activity to human LIV-1 after humanization. Binding affinity was detected by ForteBio.

[0160] [Table 2]

[0161] Example 2: Detection of the binding ability of anti-LIV-1 antibody to LIV-1 protein and LIV-1 expressing cell lines. 2.1 Binding of anti-LIV-1 antibody to LIV-1 protein The binding ability of the anti-LIV-1 antibodies obtained through screening to LIV-1 was measured. Binding analysis can be performed in solution, suspension, or on a solid support. For example, the target antigen can be immobilized on a solid support (e.g., carbon or plastic surface or chip) and contacted with the antibody. After washing away unbound antibody or target protein, the bound complex can be detected. Binding assays were performed under conditions that reduced nonspecific binding, for example, using a high ionic strength buffer (e.g., 0.3-0.4 M NaCl) and a nonionic washing agent (e.g., 0.1% Triton X-100 or Tween 20) and / or a blocking protein (e.g., bovine serum albumin or gelatin). The analysis must include a negative control. Binding affinity can be measured by ELISA, Gator, BIACore, or other methods.

[0162] In this experiment, LIV-1 antigen was linked to a solid support using enzyme-linked immunosorbent assay (ELISA), and antibodies awaiting measurement in the sample were bound to it to form a solid-phase antigen-test antibody complex. Furthermore, an enzyme-labeled secondary antibody was bound to the antibody in the solid-phase immunocomplex to form a solid-phase antigen-test antibody-enzyme-labeled secondary antibody complex. The degree of color development after adding a substrate was measured, and a positive correlation was observed between the absorbance value and the antibody binding activity.

[0163] Specifically, hLIV-1 (His-tagged, amino acid sequence NP_036451.1, including the amino acid at position Phe229-Ile323) was diluted to 2 μg / mL with coating buffer, added to a microplate, 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. Antibodies awaiting measurement were diluted with diluent and incubated at room temperature for 1 hour. Sheep 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 incubation of the secondary antibody was complete, it was incubated with TMB chromogenic solution at room temperature for 1-10 minutes. After color development was complete, 50 μL of stop solution (4M sulfuric acid) was added to each well to stop the substrate reaction. Absorbance was measured by reading the absorbance of each well using a microplate reader at a detection wavelength of 450 nm. The data was analyzed using Prism software, and a dose-response curve was plotted using a sigmoidal, 4PL four-parameter equation, with the antibody concentration awaiting measurement on the x-axis and the corresponding average absorbance on the y-axis. Equation: Y=Bottom+(X^Hillslope)*(Top-Bottom) / (X^HillSlope+EC50^HillSlope).

[0164] As a result, as shown in Figures 1A and 1B, the anti-LIV-1 antibody of the present invention specifically bound to the human LIV-1 antigen.

[0165] 2.2 Binding of anti-LIV-1 antibodies to LIV-1 expressing cell lines The specific binding ability of anti-hLIV-1 antibody A to the LIV-1 antigen expressed on the cell surface was detected using a flow cytometer.

[0166] The cell lines selected for this experiment were Calu-6 (ATCC HTB-56, undifferentiated lung cancer), HCC1806 (ATCC CRL-2335, human breast cancer TNM stage IIB grade 2), PC-3 (ATCC CRL-1435, prostate cancer), and LIV-1 overexpressing MCF7 (ATCC HTB-22, metastatic breast cancer site), specifically MCF7-ATCC-LIV-1#7, #12, and #17. Calu6 and HCC1806 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, PC-3 cells in F12K medium containing 10% fetal bovine serum, and MCF7-ATCC-LIV-1 overexpressing cell lines in EMEM medium containing 10% fetal bovine serum and 1 μg / mL, at 37°C in a 5% CO2 incubator. After dissociating each cell line and washing it in staining buffer (Biolengend), the number of cells was counted and then measured in 2 × 10⁻¹⁴⁻¹ 5 Cells were prepared in 100 μL / 10⁴ cells in staining buffer. Antibody A was then added to a final concentration of 10 μg / mL, and the mixture was reacted at 4°C for 15 minutes. After the reaction, the cells were washed with staining buffer, and then 2 μL / 2 × 10⁶ PE-labeled constant region (Fc) specific antibody (rabbit anti-human IgG PE conjugate, BioLegend, 410707) was added. 5 Cells were suspended in 100 μL of PBS and reacted at 4°C for 15 minutes. After the cell reaction, the cells were washed with staining buffer, and the readings from single-cell PE channels were analyzed using a Novocyte 3000 (Agilent) instrument. Negative controls were treated with a nonspecific isotype control commercially available antibody IgG (BioXCell, BE0297), and then further treated with a PE-labeled constant region (Fc) specific antibody. To compare the degree of LIV-1 expression between cancer cell lines, the quotient obtained by dividing the shifted readings of the experimental group treated with antibody A in this invention by the shifted readings of the control group (MFI ratio: MFI of anti-LIV-1 / MFI of control antibody) was used. The experimental results are shown in Table 2.

[0167] Experiments have demonstrated and confirmed that the anti-LIV-1 antibody in this invention specifically binds to LIV-1 expressed in various cancer cell lines, including breast cancer, lung cancer, and prostate cancer.

[0168] [Table 3]

[0169] Example 3 Compound synthesis for preparing antibody-drug conjugates In this embodiment, experimental methods for which specific conditions are not specified generally follow normal conditions or conditions suggested by the raw material or product manufacturer. Reagents for which the specific source is not specified are commercially available, standard reagents.

[0170] The structure of the compound is determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The NMR shift (δ) is 10 -6 It is expressed in units of (ppm). 1 For 1HNMR measurements, a Bruker AVANCE-400 nuclear magnetic resonance spectrometer was used, with deuterated dimethyl sulfoxide as the measurement solvent and tetramethylsilane (TMS) as the internal standard.

[0171] For MS measurements, a Shimadzu LCMS-2020 Single Quadrupole Liquid Chromatograph Mass Spectrometer (Manufacturer: Shimadzu, MS Model: 2020 Single Quadrupole MS) is used.

[0172] For RP-HPLC analysis, a Shimadzu LC-2030c Plus liquid chromatograph was used, and for preparative sampling, a Shimadzu Nexera liquid preparative system was used. The preparative column was a Phenomenex Gemini NX 5μ, C18, 110Å, 150×50mm, and the mobile phase was an aqueous solution of 0.1% trifluoroacetic acid / a solution of 0.1% trifluoroacetic acid in acetonitrile (ACN).

[0173] Silica gel column chromatography uses 200-300 mesh silica gel from SiliCycle (Canada) as the support material.

[0174] 3.1 Synthesis of Compound 3 [ka] The synthesis process is as follows:

[0175] [ka] Compound 1 (110 mg, purchased from MedChemExpress, trade number: HY-100374) was dissolved in anhydrous DMF (2 mL) and glutaric anhydride (12 mg) was added, followed by the addition of DIEA (0.04 mL). The reaction was stirred at room temperature for 1 hour, and then diluted with DCM (10 mL). Pentafluorophenol (90 mg) and (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) (100 mg) were added, and the mixture was stirred at room temperature. After 30 minutes, the reaction was concentrated under reduced pressure, and the residue was purified by RP-HPLC to obtain compound 2, a white powder (102 mg). MS assay: m / z 1403.6 [M+H] + .

[0176] 3.2 Synthesis of Compound 4 [ka] The synthesis process is as follows:

[0177] [ka] Compound 1 (110 mg) was dissolved in anhydrous DMF (2 mL) and DIEA (40 μL) and bispentafluorophenol adipic acid (200 mg) were added. The reaction was stirred at room temperature for 30 minutes, and the mixture was directly purified by RP-HPLC to obtain compound 4, a white powder (89 mg). MS assay: m / z 1417.6 [M+H] + .

[0178] 3.3 Synthesis of Compound 5 [ka] The synthesis process is as follows:

[0179] [ka] Compound 1 (110 mg) was dissolved in anhydrous DMF (2 mL) and DIEA (40 μL) and PEG3 dicarboxylic acid bispentafluorophenol ester (300 mg) were added. The reaction was stirred at room temperature for 30 minutes, and the mixture was directly purified by RP-HPLC to obtain compound 5, a white powder (105 mg). MS assay: m / z 1565.8 [M+H] + .

[0180] 3.4 Synthesis of Compound 10 [ka] The synthesis process is as follows:

[0181] [ka] Compound 6 (65 mg) and MMAE (72 mg, purchased from MedChemExpress, trade number: HY-15162) were dissolved in anhydrous DMF (2 mL) and DIEA (0.02 mL) was added, followed by the addition of 1-hydroxybenzotriazole (HOBt) (3 mg). The mixture was stirred at room temperature for 18 hours and then diluted with water (20 mL). The mixture was extracted with ethyl acetate (40 mL), the organic layer was dried over Na2SO4, and concentrated under reduced pressure until dry to obtain crude compound 7, which was dissolved in MeOH (2 mL). Zinc powder (200 mg) was added, followed by the addition of formic acid (0.2 mL). The mixture was stirred at room temperature for 30 minutes. The solid was removed by filtration, and the filtrate was directly purified by RP-HPLC to obtain compound 8, a white powder (72 mg).

[0182] Compound 8 (70 mg) was dissolved in acetonitrile / water (6 / 4, v / v). NaOH (aq., 1 M, 0.35 mL) was added to the solution and stirred at room temperature. After 1 hour, bromoacetic anhydride (52 mg) was added, followed by sodium hydroxide (aq., 1 M, 0.2 mL). After 30 minutes, the mixture was directly purified by RP-HPLC to obtain compound 10, a white powder (38 mg). MS assay: m / z 1179.6 [M+H] + .

[0183] 3.5 Synthesis of Compound 11 [ka] The synthesis process is as follows:

[0184] [ka] To a solution of compound 1 (25 mg) in acetonitrile / water (6 / 4, v / v, 2 mL), saturated NaHCO3 solution (0.02 mL) and bromoacetic anhydride (10 mg) were added. The reaction was stirred at room temperature for 10 minutes, and the mixture was directly purified by RP-HPLC to obtain compound 11, a white powder (21 mg). MS assay: m / z 1243.6 [M+H] + . 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).

[0185] 3.6 Synthesis of Compound 22 (MC-VC-PAB-MMAE) [ka] The synthesis process is as follows:

[0186] [ka] A solution of compound 1 (62 mg) in anhydrous DMF (2 mL) was mixed with maleimidohexanoic acid (12 mg), followed by the addition of DIEA (0.02 mL) and HATU (20 mg). The reaction mixture was stirred at room temperature (22 °C). After 15 minutes, the crude reaction mixture was directly purified by RP-HPLC and lyophilized to obtain compound 22 (62 mg, TFA salt), a white solid. MS assay: m / z 1316.8 [M+H] + .

[0187] 3.7 Synthesis of Compound 29 (MC-GGY-PAB-MMAE) [ka] The synthesis process is as follows:

[0188] [ka] To a solution of compound 23 (Combi Block, 460 mg) and p-aminobenzyl alcohol (130 mg) in DMF (5 mL), DIEA (0.35 mL) was added, followed by HATU (390 mg). The reaction mixture was stirred at room temperature for 20 minutes and then diluted with ELISA (100 mL). The mixture was washed with 0.5 M hydrochloric acid (50 mL) and water (50 mL). The organic layer was dried (with Na2SO4) and evaporated to dryness under reduced pressure. The residue was polished with diethyl ether / hexane (1 / 1, v / v, 100 mL) to obtain crude compound 24, 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 to approximately 4 mL under reduced pressure. Fmoc-Gly-Gly-OH (fluorenylmethoxycarbonylglycine-glycine, 360 mg) and DIEA (0.35 mL) were added, followed by the addition of HATU (0.4 g), and the mixture was stirred at room temperature. After 20 minutes, the reaction product was diluted with ₹ (120 mL). The mixture was washed with 0.5 M hydrochloric acid (50 mL) and water (50 mL). The organic layer was dried (with Na2SO4) and evaporated to dryness under reduced pressure. The crude product was purified by RP-HPLC to obtain compound 25 (550 mg), a white solid.

[0189] Compound 25 (340 mg) was dissolved in DMF (4 mL). DIEA (0.08 mL) and bis-p-nitrophenyl carbonate (bis-PNP carbonate, 300 mg) were added to the solution, and the mixture was stirred at room temperature for 16 hours. The reaction product was directly purified by RP-HPLC to obtain compound 26, a white solid (335 mg).

[0190] DIEA (0.02 mL) was added to a solution of compound 26 (84 mg) and MMAE (0.072 g) in DMF (3 mL), and the reaction was stirred at room temperature for 24 hours. Piperidine (0.15 mL) was added. After 30 minutes, the mixture was directly purified by RP-HPLC to obtain compound 27, 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 obtain a crude product, which was purified by RP-HPLC to obtain compound 28 (TFA salt, 64 mg), a white solid.

[0191] To a solution of compound 28 (25 mg, TFA salt) in DMF (2 mL), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (Sigma Aldrich, 9 mg) was added, followed by the addition of DIEA (0.007 mL). The mixture was stirred at room temperature. After 30 minutes, the mixture was purified by RP-HPLC to obtain compound 29, a white solid (22 mg). MS:m / z 1337.8 [M+H] + .

[0192] 3.8 Synthesis of Compound 39 (MC-GGY(Gal)-PAB-MMAE) [ka] The synthesis process is as follows:

[0193] [ka] Under an argon gas atmosphere, at 0°C, Ag2O (8 mmol) was added to a 50 mL solution of acetylbromo-α-D-galactose (compound 31, CombiBlocks, 2.3 g) and Fmoc-Tyr-OtBu (fluorenyl methoxycarbonyl tyrosine tert-butyl ester, compound 30, 2.1 g) in anhydrous acetonitrile while stirring. 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 obtain compound 32 (2.7 g), a white solid.

[0194] Compound 32 (2 g) was redissolved in trifluoroacetic acid / dichloromethane (TFA / DCM, 1 / 1, v / v, 40 mL). After 30 minutes, the mixture was diluted with DCM (100 mL) and washed with water (40 mL x 4). The organic layer was evaporated to dryness to obtain crude acid, i.e., compound 33, which was dissolved in DMF (30 mL). p-aminobenzyl alcohol (0.34 g) was added to the solution, followed by the addition of DIEA (1 mL) and HATU (1 g). The reaction mixture was stirred at room temperature for 20 minutes and then diluted with ELISA (120 mL). The mixture was washed with 0.5 M hydrochloric acid (100 mL) and water (50 mL). The organic layer was dried (with Na2SO4) and evaporated to dryness under reduced pressure. The residue was polished with hexane / diethyl ether (1 / 1, 70 mL) to obtain crude compound 34, a pale yellow solid, which was dissolved in DMF (20 mL). Diisopropylamine (20 mL) was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was then concentrated under reduced pressure to approximately 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 minutes, the reaction mixture was diluted with RINKAN (80 mL). The mixture was washed with 0.5 M hydrochloric acid (80 mL) and water (100 mL). The organic layer was dried (with Na2SO4) and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography to obtain compound 35, a pale yellow solid (1.7 g).

[0195] Compound 35 (1.2 g) was dissolved in DMF (10 mL). DIEA (0.2 mL) and bisPNP carbonate (0.8 g) were added to the solution, and the mixture was stirred at room temperature for 16 h. Then, the reaction was diluted with EtOAc (100 mL) and washed with water (3 × 50 mL). The organic layer was dried (with Na2SO4) and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography to give compound 36 (1.2 g) as a white solid.

[0196] DIEA (0.018 mL) was added to a solution of compound 36 (0.12 g) and MMAE (0.072 g) in DMF (2 mL), and the reaction was stirred at room temperature for 24 h. Then, the mixture was diluted with EtOAc (30 mL) and washed with hydrochloric acid (0.5 M, 30 mL). The organic layer was washed with water (20 mL), dried (with Na2SO4), and evaporated to dryness under reduced pressure to give crude compound 37, which was redissolved in MeOH (3 mL) and MeONa (4.4 M MeOH solution, 0.1 mL) was added. After the reaction mixture was stirred at room temperature for 2 h, it was neutralized with 1 N hydrochloric acid (0.5 mL). The mixture was purified directly by RP-HPLC to give compound 38 (75 mg) as a white solid (TFA salt).

[0197] 6-Maleimidocaproic acid N-hydroxysuccinimide ester (Sigma Aldrich, 8 mg) was added to a solution of compound 47 (28 mg, TFA salt) in DMF (2 mL), and then DIEA (0.007 mL) was added. The mixture was stirred at room temperature. After 30 min, the mixture was purified by RP-HPLC to give compound 39 (23 mg) as a white solid. MS: m / z 1499.9 [M+H] + .

[0198] 3.9 Synthesis of compound 48 (MC-GGY-(Glc)-PAB-MMAE)

Chemical Structure

[0199] [ka] Under argon pressure at 0°C, ethyl bromo-α-D-glucose (compound 40, 4.6 g) and Fmoc-Tyr-OtBu (fluorenyl methoxycarbonyl tyrosine tert-butyl ester, compound 30, 4.1 g) were mixed in 100 mL of anhydrous acetonitrile. Ag2O (15 mmol) was added to the mixture, 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. The compound was purified by silica gel column chromatography to obtain compound 41, a white solid (5.3 g).

[0200] Compound 41 (4 g) was redissolved in TFA / DCM (1 / 1, v / v, 80 mL). After 30 minutes, the mixture was diluted with DCM (200 mL) and washed with water (50 mL x 4). The organic layer was evaporated to dryness to obtain crude acid, i.e., compound 42, which was dissolved in DMF (50 mL). p-aminobenzyl alcohol (0.65 g) was added to the solution, followed by DIEA (1.8 mL) and HATU (1.9 g). The reaction mixture was stirred at room temperature for 20 minutes and then diluted with ELISA (200 mL). The mixture was washed with 0.5 M hydrochloric acid (100 mL) and water (150 mL). The organic layer was dried (with Na2SO4) and evaporated to dryness under reduced pressure. The residue was polished with hexane / diethyl ether (1 / 1, 100 mL) to obtain crude compound 43, a yellowish-brown solid, which was dissolved in DMF (30 mL). Diisopropylamine (30 mL) was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was then concentrated under reduced pressure to approximately 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 minutes, the reaction mixture was diluted with RINKAN (150 mL). The mixture was washed with 0.5 M hydrochloric acid (150 mL) and water (200 mL). The organic layer was dried (with Na2SO4) and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography to obtain compound 44, a pale yellow solid (3.2 g).

[0201] Compound 44 (2.4 g) was dissolved in DMF (20 mL). DIEA (0.5 mL) and bis(nitrophenyl) carbonate (1.5 g) were added to the solution, and the mixture was stirred at room temperature for 16 hours. The reaction was then diluted with SiO2 (200 mL) and washed with water (3 × 100 mL). The organic layer was dried (with Na2SO4) and evaporated to dryness under reduced pressure. The residue was purified by column chromatography to obtain compound 45 (2.3 g), a white solid.

[0202] 0.018 mL of DIEA was added to a solution of compound 45 (0.12 g) and MMAE (0.072 g) in DMF (2 mL), and the reaction was stirred at room temperature for 24 hours. The mixture was then diluted with RINKAN (30 mL) and washed with hydrochloric acid (0.5 M, 30 mL). The organic layer was washed with water (20 mL), dried (with Na2SO4), and evaporated to dryness under reduced pressure to obtain crude compound 46, which was then redissolved in MeOH (3 mL) and MeONa (4.4 M MeOH solution, 0.1 mL). The reaction mixture was stirred at room temperature for 2 hours and then neutralized with 1 N hydrochloric acid (0.5 mL). The mixture was directly purified by RP-HPLC to obtain compound 47 (TFA salt, 72 mg) as a white solid.

[0203] To a solution of compound 47 (28 mg, TFA salt) in DMF (2 mL), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (Sigma Aldrich, 8 mg) was added, followed by the addition of DIEA (0.007 mL). The mixture was stirred at room temperature. After 30 minutes, the mixture was purified by RP-HPLC to obtain compound 48, a white solid (24 mg). MS:m / z 1499.9 [M+H] + .

[0204] 3.10 Synthesis of Compound 55 (MC-AAN-(GlcNAc)-PAB-MMAE) [ka] The synthesis process is as follows:

[0205] [ka] To a solution of compound 49 (AA Block, 68 mg) and p-aminobenzyl alcohol (13 mg) in DMF (2 mL), DIEA (0.035 mL) was added, followed by HATU (40 mg). The reaction mixture was stirred at room temperature for 20 minutes and then diluted with ELISA (50 mL). The mixture was washed with 0.5 M hydrochloric acid (30 mL) and water (30 mL). The organic layer was dried (with Na2SO4) and evaporated to dryness under reduced pressure. The residue was polished with diethyl ether (100 mL) to obtain crude compound 50, a yellowish-brown solid, which was dissolved in DMF (4 mL). Diisopropylamine (4 mL) was added, and the mixture was stirred at room temperature for 2 hours. The reaction was then concentrated to approximately 3 mL under reduced pressure. Fmoc-Ala-Ala-OH (fluorenylmethoxycarbonyl-alanine-alanine, 40 mg) and DIEA (0.035 mL) were added, followed by the addition of HATU (0.04 g), and the mixture was stirred at room temperature. After 20 minutes, the reaction product was diluted with ₹ (40 mL). The mixture was washed with 0.5 M hydrochloric acid (30 mL) and water (50 mL). The organic layer was dried (with Na2SO4) and evaporated to dryness under reduced pressure. The crude product was purified by RP-HPLC to obtain compound 51 (82 mg), a pale yellow solid.

[0206] Compound 51 (80 mg) was dissolved in DMF (2 mL). DIEA (0.01 mL) and bis-PNP carbonate (60 mg) were added to the solution, and the mixture was stirred at room temperature for 16 hours. The reaction was then diluted with RINKAN (50 mL) and washed with water (3 × 15 mL). The organic layer was dried (with Na₂SO₄) and evaporated to dryness under reduced pressure. The crude product was purified by RP-HPLC to obtain compound 52, a white solid (78 mg).

[0207] To a solution of compound 52 (55 mg) and MMAE (0.036 g) in DMF (2 mL) was added DIEA (0.01 mL), and the reaction mixture was stirred at room temperature for 24 h. Then, the mixture was diluted with EtOAc (30 mL) and washed with hydrochloric acid (0.5 M, 30 mL). The organic layer was washed with water (20 mL), dried (with Na2SO4), and evaporated to dryness under reduced pressure to obtain crude compound 53, which was redissolved in MeOH (2 mL) and MeONa (4.4 M MeOH solution, 0.05 mL). After the reaction mixture was stirred at room temperature for 2 h, it was neutralized with 1 N hydrochloric acid (0.5 mL). The mixture was purified directly by RP-HPLC to obtain compound 54 as a white solid (TFA salt, 56 mg).

[0208] To a solution of compound 54 (29 mg, TFA salt) in DMF (2 mL) was added 6-maleimidocaproic acid N-hydroxysuccinimide ester (Sigma Aldrich, 8 mg), followed by addition of DIEA (0.007 mL). The mixture was stirred at room temperature. After 30 min, the mixture was purified by RP-HPLC to obtain compound 55 as a white solid (21 mg). MS: m / z 1519.9 [M+H] + .

[0209] 3.11 Synthesis of Compound 56 (BrAc-GGY(Glc)-PAB-MMAE)

Chemical Structure

[0210]

Chemical Structure

[0211] 3.12 Synthesis of Compound 17 (BrAc-PEG4-GlcA-MMAE) [ka] The synthesis process is as follows:

[0212] [ka] DIEA (0.02 mL) was added to a solution of anhydrous DMF (2 mL) containing compound 12 (65 mg, prepared according to WO2022026915) and MMAE (72 mg), followed by the addition of HOBt (3 mg). The reaction mixture was stirred at room temperature (22 °C) for 18 hours and then diluted with water (20 mL). The reaction mixture was extracted with diethyl ether (40 mL), the organic phase was dried over Na₂SO₄, and 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 methanol solution containing compound 13, followed by the addition of formic acid (0.2 mL). The reaction was stirred at room temperature for 30 minutes. The solid was removed by filtration, and the filtrate was directly purified by RP-HPLC. After lyophilization, compound 14 (72 mg), a white solid, was obtained. Compound 14 (TFA salt, 66 mg) and Fmoc-NH-PEG4-COOH (purchased from PurePEG, product number 433704, 25 mg) were dissolved in anhydrous DMF (2 mL), to which DIEA (0.025 mL) was added, followed by the addition of HATU (20 mg). The mixture was stirred at room temperature. After 16 hours, the crude mixture was purified by RP-HPLC and lyophilized to obtain compound 15 (72 mg), a white powder. MS:1471.6 [M+H] + .

[0213] Compound 15 (70 mg) was dissolved in acetonitrile / water (6 / 4, v / v, 3 mL) solution, 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 hours, hydrochloric acid (1 M, 0.12 mL) was added to the crude compound 16, followed by the addition of bromoacetic anhydride (14 mg). The crude reaction mixture was directly purified by RP-HPLC and lyophilized to obtain compound 17 (46 mg), a white solid. MS:1426.7 [M+H] + .

[0214] 3.13 Synthesis of Compound 20 (BrAc-PEG4-PAB-MMAE) [ka] The synthesis process is as follows:

[0215] [ka] To a solution of anhydrous DMF (1 mL) containing compound 1 (62 mg) and Fmoc-NH-PEG4-COOH (compound 18, 25 mg, purchased from PurePEG, product number 433704-1H), DIEA (0.025 mL) was added, followed by HATU (20 mg). The reaction mixture was stirred at room temperature (22°C). After 15 minutes, piperidine (0.1 mL) was added, and the reaction was continued for 30 minutes. The crude reaction mixture was directly purified by RP-HPLC and lyophilized to obtain compound 19 (61 mg, TFA salt), a white solid.

[0216] To an acetonitrile / water (6 / 4, v / v, 2 mL) solution containing compound 19 (37 mg), saturated NaHCO3 solution (0.03 mL) and bromoacetic anhydride (7 mg) were added. The reaction mixture was stirred at room temperature for 10 minutes, and the crude mixture was purified by RP-HPLC. After lyophilization, compound 20 (32 mg) was obtained as a white solid. MS:1491.0 [M+H] + .

[0217] Example 4 Preparation and physicochemical characterization of antibody-drug conjugates Treating the antibody prepared in Example 1, for example, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) or dithiothreitol (DTT), with a reducing agent reduced some or all of the cysteine ​​disulfide residues, forming a highly nucleophilic cysteine ​​mercapto group (-CH2SH). As a result, the partially or completely reduced antibody reacted with an electrophilic functional group (e.g., maleimide or α-carbonyl group) in the drug conjugate or conjugate reagent.

[0218] For example, antibody A was dissolved in PBS at pH 7.2, 2 mM EDTA was added, and the antibody was reduced with TCEP (the molar ratio of TCEP to Ab was 2.8:1). After incubation at 37°C for approximately 120 minutes, drug linker compounds 22, 29, 39, 48, or 55 were added to the reduced antibody (the molar ratio of drug linker to antibody was 5:1), and 5% (v / v) DMSO was added. After approximately 1 hour at room temperature, the mixture was eluted and purified with G25 resin, desalted in phosphate buffer (pH 7.4), filtered through a 0.2 μm filter under sterile conditions, and frozen for storage. Analysis by hydrophobic interaction chromatography-high-performance liquid chromatography confirmed that the mean DAR value of ADC was between 3.5 and 4.0.

[0219] For example, antibody A was dissolved in a phosphate buffer at pH 8.0, 2 mM EDTA was added, and the antibody was reduced with TCEP (TCEP:Ab molar ratio was 2.8:1). After incubation at 37°C for approximately 120 minutes, drug linker compounds 10, 11, 17, 56, or 20 were added to the reduced antibody (drug linker:antibody molar ratio was 5:1), and 5% (v / v) DMSO was added. After approximately 1 hour at room temperature, the mixture was eluted, purified, and desalted using G25 resin, filtered through a 0.2 μm filter under sterile conditions, and stored frozen. Analysis by hydrophobic interaction chromatography-high-performance liquid chromatography confirmed that the mean DAR value of ADC was between 3.5 and 4.0.

[0220] For example, antibody A was dissolved in PBS at pH 7.2, drug linker compounds 3, 4, or 5 were added to the antibody (the molar ratio of drug linker to antibody was 3:1), and 10% (v / v) DMSO was added. After approximately 2 hours at room temperature, the drug linker was repeatedly added, and the mixture was reacted again at room temperature for another 2 hours, resulting in a final drug linker to antibody molar ratio of 6:1. The mixture was eluted, purified, and desalted using G25 resin, filtered through a 0.2 μm filter under sterile conditions, and stored frozen. Analysis by hydrophobic interaction chromatography-high-performance liquid chromatography confirmed that the average DAR value of the ADC was between 1.5 and 1.9.

[0221] Method for detecting DAR values: The DAR values ​​of the ADCs of the present invention were analyzed by hydrophobic interaction chromatography-high-performance liquid chromatography (HIC-HPLC). The ADCs were isolated in a MabPac HIC-Butyl analytical column (4.6 × 100 mm, 5 μm, product number 088558, ThermoFisher, USA). A 25 mM sodium phosphate buffer (pH 6.8) containing 1.5 M ammonium sulfate was used as buffer solution A, and a 25 mM sodium phosphate buffer (pH 6.8) containing 25% acetonitrile was used as buffer solution B. 85% buffer solution A and 15% buffer solution B were stabilized as initial conditions. Linear gradient elution was performed for 30 minutes using 85% buffer solution A and 15% buffer solution B compared to 5% buffer solution A and 95% buffer solution B, followed by an additional elution for 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. To calculate the DAR value, ADC drug distribution was detected at 214 nm and 280 nm.

[0222] Examples of prepared antibody-drug conjugates (ADCs) are shown in Table 3.

[0223] [Table 4] JPEG2026509871000037.jpg210169 JPEG2026509871000038.jpg54169

[0224] Preparation of control L-MMMAE The ladiratuzumab antibody, abbreviated as L, was prepared with reference to patent US2013259860A1, its heavy chain sequence with reference to SEQ ID NO: 33, and its light chain sequence with reference to SEQ ID NO: 34. Antibody L was coupled to MC-MMAE to obtain L-Mc-MMAE with a DAR of 3.5-4, which was used in subsequent in vivo efficacy studies.

[0225] Example 5 Binding activity of antibody-drug conjugate to LIV-1 antigen hLIV-1 was diluted to 2 μg / mL with coating buffer and added to a microplate, where it was coated at 4°C for 15-20 hours. Furthermore, 300 μL of blocking solution was added to each well, and the wells were blocked at room temperature for 1 hour. The antibody-drug conjugates awaiting measurement were diluted with the diluent and incubated at room temperature for 1 hour. The monoclonal antibody of the mouse antibody toxin was diluted with the diluent and incubated at room temperature for 1 hour. The sheep anti-mouse Fc domain secondary antibody was diluted with the diluent and incubated at room temperature for 1 hour. After incubation of the secondary antibody was complete, it was incubated with TMB chromogenic solution at room temperature for 1-10 minutes. After color development was complete, 50 μL of stop solution (4M sulfuric acid) was added to each well to stop the substrate reaction. Absorbance was measured by reading the absorbance of each well with a microplate reader at a detection wavelength of 450 nm. The data was analyzed using Prism software, and a dose-response curve was plotted using a sigmoidal, 4PL four-parameter equation with the naked anti-protein concentration of antibody A on the x-axis and the corresponding mean absorbance on the y-axis. The equation is Y = Bottom + (X^Hillslope) * (Top - Bottom) / (X^HillSlope + EC50^HillSlope).

[0226] As a result, as shown in Figure 2, the antibody-drug conjugate of the present invention specifically bound to the LIV-1 antigen.

[0227] Example 6: In vitro killing effect of antibody-drug conjugates The efficacy and selectivity of the ADC according to the present invention were tested in vitro by measuring their cytotoxicity in related cancer cell lines, such as cancer cell lines expressing the antigen corresponding to the antibody portion of the ADC and similar cancer cell lines lacking the antigen.

[0228] This experiment investigated the inhibitory effects of anti-Liv1 antibody complexes, such as A-BrAcMMAE, on the proliferation of various tumor cell lines.

[0229] This experiment evaluated the antiproliferative activity of drugs using the CellTiterGlo2 (Promega) reagent. The thermostable luciferase contained in this reagent catalyzes the monooxygenation of fluorescein in the presence of Mg2+, ATP produced by living cells, and molecular oxygen, producing a stable "glow-type" luminescence signal. By quantifying ATP, a marker of metabolically active cells, the number of living cells in the culture was measured.

[0230] The cell lines selected for this experiment included the human lung cancer cell line (undifferentiated) Calu6, and LIV-1 overexpressing human breast cancer cells MCF7-ATCC-LIV-1#7, #12, and #17.

[0231] Calu6 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, and an MCF7-ATCC-LIV-1 overexpressing cell line was cultured in EMEM medium containing 10% fetal bovine serum and 1 μg / mL, at 37°C in a 5% CO2 incubator. Each of the four cell types yielded 2 × 10⁶ cells. 3 ~5×10 3Cells were inoculated at a density of 1 cell / well into a 96-well plate at 50 μL / well and incubated for 24 hours. Then, 100 μL / well of antibody A-MMAE conjugate or control antibody-MMAE conjugate (IgG1-BrAc-MMAE), diluted with different concentrations of culture medium, was added. Parallel wells were created for each concentration, and corresponding solvent control and cell-free medium wells were also prepared. After incubation for 96 hours at 37°C in a 5% CO2 incubator, 100 μL of CellTiterGlo2 was added to each well, and the mixture was placed in an orbital shaker at room temperature for 15 minutes. The glow value was measured, and the IC50 of the anti-LIV-1 antibody A-BrAcMMAE conjugate against various cells was measured. 50 The values ​​(nM) (Table 3) were calculated. The calculation results and the inhibitory effects on the proliferation of the four types of tumor cells are shown in Figures 3, 4, 5, 6 and Table 4-1.

[0232] [Table 5] As is clear from Table 4-1 and Figures 3, 4, 5, and 6, A-BrAcMMAE exhibits clear killing activity against all three types of tumor cells with different LIV-1 expression levels, and this killing effect is directly proportional to the LIV-1 expression level. Furthermore, it exhibits clear inhibitory activity against cancer cell lines derived from two different sources, breast cancer and lung cancer.

[0233] Furthermore, the present invention further detects the in vitro killing activity of ADCs (antibody denatures) coupled to antibody A using different linkers against different cells, and the results are shown in Tables 4-2 and 4-3.

[0234] [Table 6] [Table 7]

[0235] As described above, the anti-LIV-1 antibody-MMAE conjugate described in the present invention has clear antitumor activity and good targeting ability, and can deliver small molecule toxic drugs to tumor sites, providing a novel antibody-drug conjugate for the treatment of LIV-1 positive breast cancer.

[0236] Example 7: Study of the in vivo efficacy of antibody-drug conjugates In CD1 athymoid nude mice, tumor cells grown in culture, namely Calu-6 derived from ATCC (2.5 × 10⁶ cells in 50% Matrigel), were used. 6 Cells), PC-3 derived from ATCC (2.5 × 10 6 Cells), PA-1 derived from ATCC (5 × 10⁶ in 50% Matrigel) 6 NSG mice were transplanted with tumor cells grown in culture, namely HCC1806 derived from ATCC (2.5 × 10⁶ cells in 50% Matrigel). 6 Cells), MCF-7 derived from NCI (10 × 10 6 In the cell culture, MCF-7 cells were transplanted into female mice (with 8.5 μg / mL of estradiol added to their drinking water to promote in vivo growth).

[0237] The tumor is 150 mm 3 When the tumors grew, humanized LIV-1 ADC or unbound control ADC (1, 3, 6, or 10 mg / kg) was initiated. The administration methods included intravenous injection once every 4 days for a total of 4 times (q4d×4), intravenous injection once a week for a total of 2 times (qw×2), or a single intravenous injection. Tumor volume was monitored using calipers, and when the tumor volume reached approximately 2000 mm³, 3 When the TGI was reached, the animals were euthanized. Tumor volume graphs were plotted for each group until one or more animals were euthanized. All animal experiment procedures were performed in facilities certified by the Laboratory Animal Care Evaluation and Accreditation Association, following protocols approved by the Facility Animal Care Use Committee. The formula for calculating TGI is as follows:

[0238] (Math 1) TGI(%)=1-[(Td-T0) / (Cd-C0)]×100% Of these, Td and Cd represent the average tumor volume on the day of tumor volume measurement for the treatment group and the control group, while T0 and C0 represent the average tumor volume on day 0 for the treatment group and the control group.

[0239] As a result, as shown in Figures 7-11, in the MCF7 efficacy study, the tumors of 4 mice completely regressed on day 22 after administration. On day 36 after administration, the TGI was 126.3%. In the HCC1806 efficacy study, the TGI was 102.99% on day 21 after administration. In the PC-3 efficacy study, the TGI was 101.5% on day 11 after administration. In the Calu-6 efficacy study, in the multiple-dose group, the TGI on day 25 after administration was 75.14% for the 3 mg / kg group and 111.56% for the 6 mg / kg group, and on day 28 after administration, the tumors of 7 mice in the 6 mg / kg group completely regressed. In the single-dose group, the TGI on day 25 after administration was 52.72% for the 6 mg / kg group and 76.96% for the 10 mg / kg group. A-BrAcMMAE demonstrated significant antitumor therapeutic effects in various tumor models.

[0240] Furthermore, the present invention further detects the tumor inhibitory effects of A-BrAcMMAE and the conventional Liv-1-targeting ADC ladiratuzumab vedotin (abbreviated as L-Mc-MMAE) in PC3 and PA-1 mouse tumor models, and the results are shown in Figure 12 and Tables 5-1 and 5-2.

[0241] [Table 8] [Table 9]

[0242] These results demonstrate that A-BrAcMMAE can delay or inhibit the growth of LIV-1 expressing tumors in prostate cancer (PC-3) and ovarian cancer (PA-1) models, exhibiting significant antitumor activity. While L-McMMAE, reported in previous literature, also possesses antitumor activity, A-BrAcMMAE showed clearly superior efficacy compared to L-McMMAE in mouse tumor models. Therefore, A-BrAcMMAE antibody-drug conjugates can be used to treat multiple types of cancer expressing LIV-1.

[0243] Sequence information of the present invention: Sequence ID 1 (VH of antibody A) EVQLVQSGAEVKKPGATVKISCKASGLNIEDYYMHWVQQAPGKGLEWMGWIDPENGDTEYAEKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCTVHNAHYGTWFAYWGQGTTVTVSS Sequence ID 2 (VL of antibody A) DIVMTQTPLSLSVTPGQPASISCRSSQTLVRSDGNTYLEWYLQKPGQSPQLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKLEIK Sequence ID 3 (VH of antibody B) EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQGLEWIGWIDPENGDTEYAPKFQGKATLTADTSSNTAYLLLSSLTSEDTAVYYCKRRFYSMDYWGQGTSVTVSS Sequence ID 4 (VL of antibody B) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK Sequence ID No. 5 (VH of antibody C) DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCAREGRLRPFAYWGQGTLVTVSA Sequence ID 6 (VL of antibody C) NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSWTFGGGTKLEIK Sequence ID 7 (Antibody A VH CDR-H1) DYYMH Sequence ID No. 8 (Antibody A VH CDR-H2) WIDPENGDTEYAEKFQG SEQ ID NO: 9 (Antibody A VH CDR-H3) HNAHYGTWFAY Sequence ID No. 10 (Antibody A VL CDR-L1) RSSQTLVRSDGNTYLE Sequence ID 11 (Antibody A VL CDR-L2) RVSNRFS Sequence ID No. 12 (Antibody A VL CDR-L3) FQGSHVPYT Sequence ID 13 (Antibody B VH CDR-H1) DYYMH Sequence ID No. 14 (Antibody B VH CDR-H2) WIDPENGDTEYAPKFQG SEQ ID NO: 15 (Antibody B VH CDR-H3) RFYSMDY Sequence ID No. 16 (Antibody B VL CDR-L1) RSSQSIVHSNGNTYLE Sequence ID 17 (Antibody B VL CDR-L2) KVSNRFS Sequence ID No. 18 (Antibody B VL CDR-L3) FQGSHVPLT Sequence ID 19 (Antibody C VH CDR-H1) SDYAWN Sequence ID No. 20 (Antibody C VH CDR-H2) YITYSGSTSYNPSLKS SEQ ID NO: 21 (Antibody C VH CDR-H3) EGRLRPFAY Sequence ID 22 (Antibody C VL CDR-L1) KSSQSVLYSSNQKNYLA Sequence ID 23 (Antibody C VL CDR-L2) WASTRES Sequence ID No. 24 (Antibody C VL CDR-L3) HQYLSSWT Sequence ID 25 (LIV-1 antigen amino acid sequence) MARKLSVILILTFALSVTNPLHELKAAAFPQTTEKISPNWESGINVDLAISTRQYHLQQLFYRYGENNSLSVEGFRKLLQNIGIDKIKRIHIHHDHDHHSDHEHHSDHERHSDHEHHSEHEHHSDHDHHSHHNHAASGKNKRKALCPDHDSDSSGKDPRNSQGKGAHRPEHASGRRNVKDSVSASEVTSTVYNTVSEGTHFLETIETPRPGKLFPKDVSSSTPPSVTSKSRVSRLAGRKTNESVSEPRKGFMYSRNTNENPQECFNASKLLTSHGMGIQVPLNATEFNYLCPAIINQIDARSCLIHTSEKKAEIPPKTYSLQIAWVGGFIAISIISFLSLLGVILVPLMNRVFFKFLLSFLVALAVGTLSGDAFLHLLPHSHASHHHSHSHEEPAMEMKRGPLFSHLSSQNIEESAYFDSTWKGLTALGGLYFMFLVEHVLTLIKQFKDKKKKNQKKPENDDDVEIKKQLSKYESQLSTNEEKVDTDDRTEGYLRADSQEPSHFDSQQPAVLEEEEVMIAHAHPQEVYNEYVPRGCKNKCHSHFHDTLGQSDDLIHHHHDYHHILHHHHHQNHHPHSHSQRYSREELKDAGVATLAWMVIMGDGLHNFSDGLAIGAAFTEGLSSGLSTSVAVFCHELPHELGDFAVLLKAGMTVKQAVLYNALSAMLAYLGMATGIFIGHYAENVSMWIFALTAGLFMYVALVDMVPEMLHNDASDHGCSRWGYFFLQNAGMLLGFGIMLLISIFEHKIVFRINF Sequence number 26 (heavy chain constant region) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 27 (Light chain steady region) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID No. 28 (Mouse antibody mVH of antibody A, of which the underlined portion is CDR) EVQLQQSGAELVRSGASVKVSCKASGLNIE DYYMH WVKQRPEQGLE WIGWIDPENGDTEYGPKFQG KATMTADTSSNTAYLQLSSLTSGDTAVYYCTV HNAHYGTWFAY WGQGTLVTVSS Sequence ID No. 29 (mVL of mouse antibody A, of which the underlined portion is CDR) DVLMTQTPLSLPVSLGDQASISC RSSQTLVRSDGNTYLE WYLQKPGQSPKLLIY RVSNRFS GVPDRFSGSGSGTDFTLRISRVEAEDLGLYYC FQGSHVPYT FGGGTKLEIK Sequence ID 30 (Light chain of antibody A) DIVMTQTPLSLSVTPGQPASISCRSSQTLVRSDGNTYLEWYLQKPGQSPQLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 31 (Heavy chain of antibody A) EVQLVQSGAEVKKPGATVKISCKASGLNIEDYYMHWVQQAPGKGLEWMGWIDPENGDTEYAEKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCTVHNAHYGTWFAYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 32 (Heavy chain variable region of antibody A-1): EVQLVQSGAEVKKPGATVKISCKASGLNIE DYYMH WVQQAPGKGLE WMGWIDPENGDTEYAEKFQG RVTITADTSTDTAYMELSSLRSEDTAVYYCTV HNAHYGTWFAY WGQGTTVTVSS Sequence ID 33 (Ladiratuzumab heavy chain): QVQLVQSGAEVKKPGASVKVSCKASGLTIEDYYMHWVRQAPGQGLEWMGWIDPENGDTEYGPKFQGRVTMTRDTSINTAYMELSRLRSDDTAVYYCAVHNAHYGTWFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 34 (Ladiratuzumab light chain): DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWYQQRPGQSPRPLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVE IKRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC All documents relating to the present invention are cited by reference in this application, so that each document is cited independently. Furthermore, after reading the above-described aspects of the present invention, those skilled in the art can make various changes and modifications to the invention, and these equivalent forms should be understood to be similarly limited in scope to the claims attached to this application.

Claims

1. An anti-human LIV-1 antibody or its antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), The antibody is selected from antibody A, antibody B, and antibody C, among which, The VH of antibody A contains three CDRs: CDR-H1, CDR-H2, and CDR-H3, of which, CDR-H1 contains the amino acid sequence shown in SEQ ID NO: 7, CDR-H2 contains the amino acid sequence shown in SEQ ID NO: 8, CDR-H3 contains the amino acid sequence shown in Sequence ID No. 9, Furthermore, the VL of antibody A contains three CDRs: CDR-L1, CDR-L2, and CDR-L3, of which, CDR-L1 contains the amino acid sequence shown in SEQ ID NO: 10, CDR-L2 contains the amino acid sequence shown in SEQ ID NO: 11, CDR-L3 contains the amino acid sequence shown in SEQ ID NO: 12, The VH of antibody B contains three CDRs: CDR-H1, CDR-H2, and CDR-H3, of which, CDR-H1 contains the amino acid sequence shown in SEQ ID NO: 13, CDR-H2 contains the amino acid sequence shown in SEQ ID NO: 14, CDR-H3 contains the amino acid sequence shown in SEQ ID NO: 15, Furthermore, the VL of antibody B contains three CDRs: CDR-L1, CDR-L2, and CDR-L3, of which, CDR-L1 contains the amino acid sequence of SEQ ID NO:

16. CDR-L2 contains the amino acid sequence of SEQ ID NO:

17. CDR-L3 contains the amino acid sequence of SEQ ID NO: 18, and The VH of the aforementioned antibody C contains three CDRs: CDR-H1, CDR-H2, and CDR-H3, of which, CDR-H1 contains the amino acid sequence shown in SEQ ID NO: 19, CDR-H2 contains the amino acid sequence shown in SEQ ID NO: 20, CDR-H3 contains the amino acid sequence shown in SEQ ID NO: 21, Furthermore, the VL of antibody C contains three CDRs: CDR-L1, CDR-L2, and CDR-L3, of which, CDR-L1 contains the amino acid sequence shown in SEQ ID NO: 22, CDR-L2 contains the amino acid sequence shown in SEQ ID NO: 23, CDR-L3 contains the amino acid sequence shown in SEQ ID NO:

24. Anti-human LIV-1 antibody or its antigen-binding fragment.

2. The VH of antibody A contains a sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 1, and the VL of antibody A contains a sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO:

2. The VH of antibody B contains a sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 3, and the VL of antibody B contains a sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

4. The VH of antibody C contains a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, and the VL of antibody C contains a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 6, or The VH of antibody A contains a sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 32, and the VL of antibody A contains a sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

2. The anti-human LIV-1 antibody or its antigen-binding fragment according to claim 1.

3. The anti-human LIV-1 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a humanized antibody, a chimeric antibody, or a mouse antibody.

4. The antibody or antigen-binding fragment thereof is a tetramer comprising two light chains and two heavy chains, an antibody comprising one or more heavy chains or light chains, Fab, Fab', F(ab')2, Fv, or a single-chain antibody, preferably the antibody comprising a heavy chain constant region indicated by SEQ ID NO: 26 and / or a light chain constant region indicated by SEQ ID NO: 27, and more preferably the antibody comprising a light chain indicated by SEQ ID NO: 30 and a heavy chain indicated by SEQ ID NO: 31, according to claim 1, an anti-human LIV-1 antibody or antigen-binding fragment thereof.

5. An antibody-drug conjugate (ADC) comprising an anti-human LIV-1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 4, and a pharmaceutically acceptable salt thereof.

6. The antibody-drug conjugate and its pharmaceutically acceptable salt comprise the structure shown in [Formula I] below. [Formula I] Ab-[(AG)k-(L1)x-(OP)p-(L2)y-D]z Eventually, Ab is an anti-human LIV-1 antibody or its antigen-binding fragment according to any one of claims 1 to 4. AG is a coupling group, and among them, k is 0 or 1, and AG is 【Chemistry 1】 Selected based on this principle, Among them, the dashed line indicates the connection point to Ab. (L1)x is the first linking group, and of which, x is 0 or 1, and L1 is -CH ２ The expression represents a chain containing 1 to 18 groups selected from -, -C(=O)-, -NH-, -O-, and -S-, wherein each NH, O, and S contains at least two carbon atoms between another NH, O, or S, preferably at least two -CH ２ - including, (OP)p is an oligopeptide that can be cleaved by enzymes, and of which p is 0 or 2-10. (L2)y is the second linking group, and of which, y is 0 or 1, and L2 is -CH ２ This represents a chain containing 1 to 18 groups selected from -, -C(=O)-, -NH-, -O-, and -S-, wherein at least two CH groups are between each NH, O, and S and another NH, O, or S. ２ Includes a base and / or It represents one o-, m-, or p-hydroxybenzyl group or an o-, m-, or p-aminobenzyl group, Preferably, y is 1 and L2 is -NH-Ph-CH ２ The compound is -O-C(=O)-, where the benzene ring selectively contains a hydroxy substituent, more preferably L2 is a p-aminobenzyloxycarbonyl group or a p-hydroxy-m-aminobenzyloxycarbonyl group, and optionally the hydroxyl group in the benzene ring is glycosylated, preferably with glucuronic acid, N-acetylglucosamine, glucose or galactose. D is an antibody-drug conjugate drug, of which the drug is a cytotoxic compound, an immunomodulator, an enzyme or a hormone inhibitor, and z is the ratio of drug to antibody, and its value is an integer or decimal between 1 and 24. The antibody-drug conjugate and a pharmaceutically acceptable salt thereof according to claim 5.

7. L1 is -(CH ２ -)o-C(=O)- or -(NH)j-(CH ２ CH ２ O)n-(CH ２ )q-(C=O)-, where o is 1 to 10, preferably 1 to 7, j is 0 to 4, n is 2 to 8, q is 1 to 6, (OP)p is selected from oligopeptides formed by combining valine, citrulline, alanine, glycine, asparagine, tyrosine, phenylalanine, proline, isoleucine, lysine, serine, glutamic acid, threonine, or asparagine, and p is 2 to 10, more preferably a dipeptide, tripeptide, or tetrapeptide, and optionally the phenol hydroxyl group of tyrosine or the amide group of asparagine is glycosylated, preferably with glucuronic acid, N-acetylglucosamine, glucose, or galactose. Preferably, The antibody-drug conjugate described in [Formula I] and its pharmaceutically acceptable salts are, 【Chemistry 2】 【change】 It has a structure selected from, More preferably, Ab includes the light chain indicated by SEQ ID NO: 30 and the heavy chain indicated by SEQ ID NO: 31, and z is a decimal or integer between 2 and 8. The antibody-drug conjugate and a pharmaceutically acceptable salt thereof according to claim 6.

8. The antibody-drug conjugate and pharmaceutically acceptable salt thereof according to claim 6, wherein the drug is selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine, SN-38, or exatecan.

9. The antibody is a bivalent tetramer comprising two light chains and two heavy chains, wherein the first pair of heavy and light chains comprises a heavy chain variable region and a light chain variable region of the anti-human LIV-1 antibody or its antigen-binding fragment described in any one of claims 1 to 4, and the second pair of heavy or light chains of the tetramer can bind to 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, SIRPα, PSMA, ROR1, and CD47, and the antibody-drug conjugate and pharmaceutically acceptable salt thereof described in any one of claims 5 to 8.

10. The use of an anti-human LIV-1 antibody or its antigen-binding fragment according to any one of claims 1 to 4, an antibody-drug conjugate according to any one of claims 5 to 9, and a pharmaceutically acceptable salt thereof in the preparation of a drug for treating a tumor or cancer, Preferably, the tumor or cancer is selected from breast cancer, bladder cancer, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, colorectal cancer, glioma, mesothelioma, cervical cancer, triple-negative breast cancer, lung cancer, head and neck cancer, esophageal cancer, skin cancer, and uterine cancer. use.

11. It is an immune complex, (a) an anti-human LIV-1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, (b) A coupling moiety selected from the group consisting of a detectable marker, a radionuclide, a cytokine, an enzyme, gold nanoparticles / nanorods, nanomagnetic particles, a virus-coated protein or VLP, or a combination thereof, Immune complex.

12. A method for detecting LIV-1 molecules in a sample, comprising: (1) contacting the sample with the immunocomplex described in claim 11; and (2) detecting whether or not an antigen-antibody complex has been formed, and if a complex has been formed, indicating that LIV-1 is present in the sample. method.

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