Anti-LIV-1 antibodies or antigen-binding fragments thereof and uses thereof

Abstract

The present invention relates to an anti-LIV-1 antibody or an antigen-binding fragment thereof and uses thereof. The anti-LIV-1 antibody or antigen-binding fragment provided by the present invention has high affinity for LIV-1, can block the binding of LIV-1 to its receptor and inhibit downstream signal transduction.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to anti-LIV-1 antibodies or antigen-binding fragments thereof and their uses. The present invention also relates to methods for preparing anti-LIV-1 antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the anti-LIV-1 antibodies or antigen-binding fragments thereof, vectors and host cells containing the nucleic acid molecules, and conjugates containing the anti-LIV-1 antibodies or antigen-binding fragments thereof. The present invention further relates to anti-LIV-1 antibodies or antigen-binding fragments thereof, pharmaceutical compositions containing the same, and uses of the conjugates. Background Art

[0002] LIV-1 (leucine-isoleucine-valine transport system 1) is a member of the LZT (LIV-1 subfamily of ZIP zinc transporters) within the ZIP (Zrt-, Irt-like) family of zinc transporters. Because ZIP is also known as SLC39A (solute carrier family 39A), LIV-1 is also known as SLC39A6 or ZIP6.

[0003] LIV-1 protein is mainly located in the cell membrane and may also be located in the membrane of intracellular organelles (such as the endoplasmic reticulum). It is rich in histidine residues and is a multi-transmembrane protein composed of 8 transmembrane domain proteins. It is responsible for transferring Zn2+ from outside the cell or organelles into the cytoplasm, and mainly plays a role in maintaining intracellular zinc homeostasis.

[0004] Abnormalities in the LIV-1 zinc transporter may contribute to diseases such as cancer and may play a dual role in tumorigenesis. Studies have shown that LIV-1 inhibition can inhibit the invasion of HeLa cells (a cervical cancer cell line) by targeting the ERK1 / 2-Snail / Slug pathway. In prostate cancer cells (ARCaPE and ARCaPM cells), LIV-1 overexpression leads to increased MMP-2 and MMP-9 activity, which in turn leads to cleavage of heparin-bound epidermal growth factor and constitutive activation of the epidermal growth factor receptor, playing a crucial role in tumorigenesis. Furthermore, LIV-1 may mediate key intracellular growth-regulating signaling pathways, such as the mitogen-activated protein kinase (MAPK) pathway, which are implicated in abnormalities in transformed cells, such as carcinogenesis. Evidence suggests that LIV-1 interacts with the transcription factor STAT3 (Signal Transducer and Activator of Transcription 3) and snail, downregulating E-cadherin expression and promoting epithelial-mesenchymal transition (EMT), thereby promoting tumor metastasis.

[0005] Studies have found that LIV-1 is highly expressed in breast cancer, prostate cancer, colorectal cancer, and melanoma (70%-90% in patient-derived tissues), with the highest expression in breast cancer. Expression remains high in breast cancer patients who have received hormone therapy (78%-92%). Low expression is observed in ovarian, uterine, and lung cancers (10%-48%). Expression is categorized into five levels, from low to high. Expression in normal human tissues is limited: in breast tissue, 0%-50% of cells express with an intensity of 1-2; in prostate tissue, 50%-100% of cells stain with an intensity of 2-4; and in testicular tissue, approximately 50% of cells stain with a maximum intensity of 1. Expression is virtually absent in other normal tissues. This differential expression between tumor and normal tissues demonstrates that LIV-1 is a suitable target for cancer therapy, particularly breast cancer.

[0006] Therefore, there is an urgent need to develop antibodies or related drugs targeting LIV-1 for tumor treatment. Summary of the Invention

[0007] The present invention provides an anti-LIV-1 antibody or antigen-binding fragment thereof, and uses thereof based on the antibody or fragment thereof. The "fragment" of the antibody molecule described herein encompasses various functional fragments of an antibody, such as its antigen-binding portion, such as Fab, F(ab')2, or scFV fragments.

[0008] The present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides an anti-LIV-1 antibody or an antigen-binding fragment thereof. The anti-LIV-1 antibody of the present invention is expected to have low immunogenicity in human subjects and be well tolerated by human subjects.

[0010] The present invention provides antibodies targeting human LIV-1, which have the following advantages:

[0011] (1) High affinity binding to human LIV-1 and target cells expressing human LIV-1;

[0012] (2) Have good cross-reactivity in cynomolgus monkeys;

[0013] (3) Ability to enter cells through endocytosis;

[0014] (4) Suitable for constructing an antibody-drug conjugate for effective treatment. The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 7, or the three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 8.

[0015] In some embodiments, the present invention provides an anti-LIV-1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises three heavy chain CDRs (HCDR1, HCDR2, HCDR3), and the light chain variable region comprises three light chain CDRs (LCDR1, LCDR2, LCDR3), wherein:

[0016] The heavy chain variable region comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 7, 11, 12, and 13; the light chain variable region comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 8, 14, 15, 16, and 17.

[0017] Based on the variable region amino acid sequence of a given antibody or fragment thereof of the present invention, a person skilled in the art can routinely determine the CDRs contained therein. For example, according to a specific embodiment of the present invention, the Kabat scheme, the IMGT scheme, the AbM scheme, the Chothia scheme or the Contact scheme are used to define the CDRs in the variable region amino acid sequence.

[0018] In some embodiments, the present invention provides an anti-LIV-1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region CDR (HCDR1, HCDR2, HCDR3) and a light chain variable region CDR (LCDR1, LCDR2, LCDR3), wherein:

[0019] HCDR1 consists of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 2, HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 3, LCDR1 consists of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 6.

[0020] In some embodiments, the present invention provides an anti-LIV-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and / or a light chain variable region, wherein:

[0021] (1) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 7; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 8;

[0022] (2) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 14;

[0023] (3) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 15;

[0024] (4) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 16;

[0025] (5) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

[0026] (6) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 14;

[0027] (7) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 15;

[0028] (8) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 16;

[0029] (9) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

[0030] (10) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 13; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 14;

[0031] (11) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 13; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 15;

[0032] (12) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 13; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 16; or

[0033] (13) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 13; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17.

[0034] In particular, the antibodies or antigen-binding fragments thereof of the present invention comprise at least a heavy chain variable region and / or a light chain variable region, both of which include the aforementioned CDRs and an intervening framework region (FR), with the respective domains arranged in the following manner: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Furthermore, optionally, the "at least 90% identity" resulting in a maximum of 10% difference in amino acid sequence may exist in any framework region of the heavy chain variable region or the light chain variable region, or in any domain or sequence other than the heavy chain variable region and the light chain variable region in the antibodies or fragments thereof of the present invention. Such differences may result from amino acid substitutions, deletions, or insertions at any position.

[0035] In some embodiments, the antibody or its antigen-binding fragment further comprises a human or mouse constant region, preferably a human or mouse heavy chain constant region and / or a light chain constant region; preferably, the antibody or its antigen-binding fragment comprises an IgG, IgA, IgM, IgD or IgE heavy chain constant region and / or a κ or λ type light chain constant region.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain and / or light chain constant region sequence derived from a human antibody germline consensus sequence. In some embodiments, the heavy chain constant region is preferably derived from a constant region sequence of human IgG1, IgG2, IgG3, or IgG4. In a specific embodiment, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9, or consists thereof. In a specific embodiment, the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10, or consists thereof.

[0037] It will be appreciated that sequence variants of these constant region domains may also be used, for example comprising one or more amino acid modifications, wherein the amino acid positions are identified by the EU index system of Kabat et al. (1991).

[0038] In certain embodiments of the antibodies of any of the foregoing, the antibody is in any form, such as a monoclonal antibody, a single-chain antibody, a bifunctional antibody, a single-domain antibody, a nanobody, a fully or partially humanized antibody, or a chimeric antibody. In a specific embodiment, the antibody is a monoclonal antibody. In a preferred embodiment, the antibody is a chimeric antibody. In another more preferred embodiment, the antibody is a humanized antibody.

[0039] In certain embodiments of the antibody of any of the foregoing, the antibody is a full-length antibody.

[0040] In some embodiments, the anti-LIV-1 antibodies of the present invention are complete antibodies, such as IgG1, IgG2, IgG3, or IgG4 antibodies. In another embodiment, the anti-LIV-1 antibodies of the present invention encompass only their antigen-binding portions, such as Fab, Fab', Fab'-SH, (Fab')2, Fv, scFv, BsFv, dsFv, or (dsFv)2 fragments.

[0041] In some embodiments, the antigen-binding fragment is an antigen-binding fragment of an antibody or half antibody, such as Fab, Fab', Fab'-SH, (Fab')2, Fv, scFv, BsFv, dsFv or (dsFv)2 fragment; more preferably, the antibody is IgG.

[0042] In a second aspect, the present invention provides a biomaterial comprising:

[0043] (i) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention;

[0044] (ii) a vector comprising the nucleic acid molecule described in (i); and / or

[0045] (iii) a host cell comprising the nucleic acid molecule described in (i) and / or the vector described in (ii), or the host cell is transformed or transfected with the nucleic acid molecule described in (i) and / or the vector described in (ii).

[0046] In some embodiments, the nucleic acid molecule can be an isolated nucleic acid molecule.

[0047] The nucleic acid molecules of the present invention can be cloned into vectors and then used to transform or transfect host cells. Therefore, the present invention also provides vectors comprising the nucleic acid molecule described in (i). In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a phage vector.

[0048] The vectors or nucleic acid molecules of the present invention can be used to transform or transfect host cells for purposes such as storage or antibody expression. Therefore, the present invention also provides host cells comprising the vector described in (ii) or the nucleic acid molecule described in (i), or the host cells transformed or transfected with the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant, or animal cell. In some embodiments, the host cell is prokaryotic, such as Escherichia coli. In other embodiments, the host cell is eukaryotic, such as 293 cells, CHO cells, yeast cells, or plant cells. In some embodiments, the host cell is other cells suitable for producing antibodies or antigen-binding fragments thereof.

[0049] The antibodies or antigen-binding fragments thereof provided by the present invention can be obtained by any method known in the art. For example, the heavy chain variable region and / or light chain variable region of the antibody can be first obtained from the nucleic acid molecule provided by the present invention, or the heavy chain and / or light chain of the antibody can be obtained, and then assembled into an antibody with the optional other domains of the antibody. Alternatively, in a third aspect, the present invention provides a method for preparing the anti-LIV-1 antibody or antigen-binding fragment thereof described herein, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0050] In a fourth aspect, the present invention provides a conjugate comprising the antibody or antigen-binding fragment thereof of the present invention and a conjugated portion, wherein the conjugated portion is another molecule; preferably, the conjugated portion is a cytotoxic agent, an immunomodulator, an imaging agent, a fluorescent protein, a molecular marker, a therapeutic protein, a biopolymer or an oligonucleotide, etc.

[0051] In a fifth aspect, the present invention provides an antibody-drug conjugate (ADC), comprising the antibody or antigen-binding fragment thereof of the present invention, at least one therapeutically active substance or pharmaceutically active ingredient, and an optional linker; the antibody-drug conjugate has a structure represented by formula (I):

[0052] Ab-(LD)n formula (I)

[0053] Wherein, Ab is the antibody or antigen-binding fragment thereof according to the present invention;

[0054] L is a linker;

[0055] D is a therapeutically active substance or a pharmaceutical active ingredient;

[0056] n=1-8, for example, n=1, 2, 3, 4, 5, 6, 7, or 8.

[0057] The antibody-drug conjugate provided by the present invention has the following advantages:

[0058] (1) Binds to target cells expressing human LIV-1 with high affinity;

[0059] (2) Ability to enter cells through endocytosis and kill target cells; in some embodiments, the ADC of the present invention has high endocytosis efficiency;

[0060] (3) treating or improving a subject's condition associated with abnormal LIV-1 function or expression (e.g., cancer, such as ovarian cancer, colon cancer, triple-negative breast cancer, endometrial cancer, peritoneal cancer, and lung cancer), or treating or improving one or more symptoms of the disease;

[0061] (4) reducing or inhibiting tumor growth or progression in a subject having a tumor expressing LIV-1;

[0062] (5) Exerts cytotoxic activity in cells expressing LIV-1.

[0063] In one embodiment, n represents the number of drug-linker (LD) moieties coupled to a single antibody (Ab), and is preferably an integer from 1 to 8. In this case, the single ADC conjugate may also be referred to as an ADC compound. In any of the embodiments herein, on the ADC compound according to the present invention, there may be 1, 2, 3, 4, 5, 6, 7, or 8 drug-linker moieties coupled to a single antibody.

[0064] In another embodiment, n represents the average DAR of the antibody-drug conjugate prepared. In this case, n can be, for example, an integer or decimal in the range of 1 to 8, 1.0 to about 8.0, 1.0 to about 7.0, or 1.0 to about 6.6, 2.0 to about 8.0, 2.0 to about 7.5, 2.0 to 6.5, or 2.0 to about 6.0, such as 3.0-4.0, 4.0-5.0, 7.0-8.0. In some aspects, n represents an average DAR of about 3. In some aspects, n represents an average DAR of about 4. In some aspects, n represents an average DAR of about 5. In some aspects, n represents an average DAR of about 6. In some aspects, n represents an average DAR of about 7. In some aspects, n represents an average DAR of about 8.

[0065] IgG1 antibodies have 16 pairs of cysteine residues, which are present as 12 intrachain and 4 interchain disulfide bonds. The interchain disulfide bonds are solvent-accessible and can be reduced by reducing agents to form eight sulfhydryl groups, which serve as conjugation targets. In one embodiment, the linker is conjugated to the sulfhydryl groups of cysteine residues on the anti-LIV-1 antibody.

[0066] In one specific embodiment, the therapeutically active substance or pharmaceutically active ingredient is a cytotoxin, a plant toxin, a small molecule toxin, a radioisotope, a maytansinoid, or the like. In one specific embodiment, the cytotoxin is dolastatin and its auristatin derivatives, such as monomethyl auristatin E (MMAE). In another specific embodiment, the cytotoxin is a topoisomerase I inhibitor, such as a camptothecin derivative. In a preferred embodiment, the cytotoxin is DXD. In another embodiment, the therapeutically active substance or pharmaceutically active ingredient is MMAE or DXD.

[0067] In one embodiment, the cytotoxin is covalently linked to the anti-LIV-1 antibody or antigen-binding fragment thereof directly or via a linker in a non-site-specific or site-specific manner.

[0068] In one embodiment, the linker is a protease-cleavable peptide linker. In a specific embodiment, the peptide linker comprises a protease-cleavable peptide fragment. In a specific embodiment, the peptide linker can also comprise, for example, a maleimide coupled to an antibody cysteine residue.

[0069] In one embodiment, the linker L and the active substance D constitute Deruxtecan.

[0070] In a specific embodiment, the antibody-drug conjugate has the following structure:

[0071] or

[0072]

[0073] Among them, n=1-8.

[0074] The antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, host cells, conjugates and / or antibody-drug conjugates provided by the present invention can be included in pharmaceutical compositions, more particularly in pharmaceutical preparations, and thus used for various purposes according to actual needs. Therefore, in a sixth aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the biomaterial of the present invention (including nucleic acid molecules, vectors, host cells), conjugates, antibody-drug conjugates and optional pharmaceutically acceptable carriers. In some embodiments of the present invention, the pharmaceutical composition further comprises a second therapeutic agent and / or optionally a pharmaceutical excipient, the second therapeutic agent being selected from cytokines, antibodies, chemotherapeutic agents and small molecule drugs.

[0075] In a seventh aspect, the present invention provides use of an anti-LIV-1 antibody or antigen-binding fragment thereof, or a biomaterial (including a nucleic acid molecule, a vector, a host cell), a conjugate, an antibody-drug conjugate, or a pharmaceutical composition of the present invention in the preparation of any of the following products:

[0076] (a) Products for detecting LIV-1;

[0077] (b) products that stimulate or enhance the immune response;

[0078] (c) products for preventing and / or treating diseases associated with abnormal LIV-1 expression;

[0079] (d) A product that kills LIV-1 expressing cells or inhibits the growth of LIV-1 expressing cells in vitro or in vivo.

[0080] In one embodiment, the disease associated with abnormal expression of LIV-1 is a cancer that highly expresses LIV-1, such as ovarian cancer, colon cancer, triple-negative breast cancer, endometrial cancer, peritoneal cancer, and lung cancer.

[0081] In some embodiments, the antibodies or antigen-binding fragments thereof, the antibody-drug conjugates, biological materials (including nucleic acid molecules, vectors, host cells) and / or pharmaceutical compositions of the present invention can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the purposes described herein, for example, for the treatment of the relevant diseases or conditions mentioned herein.

[0082] In some embodiments, the step (d) includes contacting the cell with an effective amount of the antibody or antigen-binding fragment thereof of the present invention, the antibody-drug conjugate of the present invention, the biomaterial (including nucleic acid molecules, vectors, host cells) and / or the pharmaceutical composition of the present invention.

[0083] In an eighth aspect, the present invention provides a method for preventing and / or treating a disease associated with abnormal LIV-1 expression in a subject in need thereof, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof, or a biomaterial (including nucleic acid molecules, vectors, host cells), antibody-drug conjugate, or pharmaceutical composition described herein. The disease associated with abnormal LIV-1 expression is a cancer that overexpresses LIV-1, such as ovarian cancer, colon cancer, triple-negative breast cancer, endometrial cancer, peritoneal cancer, and lung cancer. Preferably, the subject is a mammal; more preferably, the subject is a human.

[0084] In a ninth aspect, the present invention provides use of the antibody or antigen-binding fragment thereof, or the biomaterial (including nucleic acid molecules, vectors, host cells), or pharmaceutical composition of the present invention in the preparation of an antibody-drug conjugate for treating cancer.

[0085] In a tenth aspect, the present invention provides use of the antibody or antigen-binding fragment thereof, or the biomaterial (including nucleic acid molecules, vectors, host cells), antibody-drug conjugate, or pharmaceutical composition of the present invention in the preparation of a drug for treating cancer.

[0086] In some embodiments, the cancer is a cancer that highly expresses LIV-1, such as ovarian cancer, colon cancer, triple-negative breast cancer, endometrial cancer, peritoneal cancer, and lung cancer.

[0087] In an eleventh aspect, the present invention provides a method for blocking the binding of LIV-1 to its receptor and inhibiting downstream signal transduction, comprising administering to a subject an effective amount of the antibody or antigen-binding fragment thereof disclosed in the present invention or the biomaterial (including nucleic acid molecules, vectors, host cells), conjugate, antibody-drug conjugate or pharmaceutical composition of the present invention.

[0088] In a twelfth aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof, conjugate or antibody-drug conjugate of the present invention.

[0089] In a thirteenth aspect, the present invention provides a method for detecting the presence or level of LIV-1 in a sample, comprising the steps of contacting the antibody or antigen-binding fragment or conjugate of the present invention with the sample, and detecting whether the antibody or antigen-binding fragment or conjugate forms a complex with LIV-1.

[0090] The present invention is further illustrated in the following drawings and specific embodiments. However, these drawings and specific embodiments should not be considered to limit the scope of the present invention, and changes that are readily apparent to those skilled in the art will be included within the spirit of the present invention and the protection scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 The results showed that the anti-LIV-1 hybridoma antibody had binding activity to the LIV1 protein.

[0092] Figure 2 The anti-LIV-1 hybridoma antibody showed binding activity to 293T-LIV1 cells.

[0093] Figure 3 The anti-LIV-1 hybridoma antibody showed binding activity to cynomolgus monkey LIV1 protein.

[0094] Figure 4A The anti-LIV-1 chimeric antibody showed binding activity to SKOV3 cells; Figure 4B The anti-LIV-1 chimeric antibody showed binding activity to HCT116 cells.

[0095] Figure 5 The cytotoxic activity of the anti-LIV-1 chimeric antibody against 293T-LIV1 cells using a secondary antibody method is shown.

[0096] Figure 6 The results showed that the anti-LIV-1 humanized antibody had binding activity to LIV1 protein.

[0097] Figure 7 The cytotoxicity of humanized anti-LIV-1 antibodies against 293T-LIV1 cells was demonstrated using a secondary antibody assay.

[0098] Figure 8 The binding activity of the anti-LIV-1 humanized antibody ADC to 293T-LIV1 cells was shown.

[0099] Figure 9 The cytotoxicity of anti-LIV-1 humanized antibody MMAE conjugate against 293T-LIV1 cells was shown. DETAILED DESCRIPTION

[0100] All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples described herein are illustrative only and are not intended to be limiting. Other features, objects and advantages of the present invention will be apparent from this specification and the accompanying drawings and from the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. For the purposes of the present invention, the following terms are defined below.

[0101] definition

[0102] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0103] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0104] The term "and / or" should be understood to mean any one of the options or a combination of any two or more of the options.

[0105] As used herein, the term "comprises" or "includes" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this article, when the term "comprises" or "includes" is used, unless otherwise indicated, it also covers the situation consisting of the stated elements, integers or steps.

[0106] The term "antibody" is used herein in the broadest sense to refer to a protein that contains an antigen binding site and is capable of specifically recognizing and binding to an antigen. An antibody may be a complete antibody, any antigen-binding fragment thereof, or a single chain thereof, and thus the term "antibody" includes any protein or polypeptide that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen.

[0107] The term "immunoglobulin" refers to a protein with the structure of a naturally occurring antibody and is generally used interchangeably with the term "antibody" in this application. IgG immunoglobulins are heterotetrameric glycoproteins composed of two disulfide-bonded light chains and two heavy chains. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also known as the heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also known as the light chain variable domain, followed by a light chain constant domain (CL). In an IgG molecule, the VH-CH1 heavy chain pair with the VL-CL light chain to form the Fab fragment, which specifically binds to the antigen. Therefore, an IgG immunoglobulin essentially consists of two Fab molecules connected by an immunoglobulin hinge region and two dimerized Fc regions. The heavy chains of immunoglobulins can be assigned to one of five classes based on the type of their constant regions, termed α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can also be assigned to one of two classes, termed κ and λ, based on the amino acid sequence of their constant domains.

[0108] The term "antibody fragment" refers to a molecule, distinct from an intact antibody, that comprises a portion of an intact antibody and is capable of binding to the same antigen as the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibodies (e.g., scFv), single-domain antibodies, bivalent or bispecific antibodies or fragments thereof, camelid antibodies (heavy-chain antibodies), and multispecific antibodies formed from antibody fragments.

[0109] The terms "antigen-binding site" and "antigen-binding domain" are used interchangeably to refer to the region of an antibody molecule that actually binds to an antigen. Antigen-binding sites include, but are not limited to, Fv, Fab fragments, Fab', Fab'-SH, F(ab')2, single-chain antibody molecules (e.g., scFv), VHH, and other formats.

[0110] The term "variable region" or "variable domain" of an antibody refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable region of an antibody can be further subdivided into hypervariable regions (i.e., complementarity determining regions (CDRs)) and relatively conserved regions (i.e., framework regions (FRs)) intervening between the hypervariable regions. In the case of IgG immunoglobulins, the heavy or light chain variable region comprises, from N-terminus to C-terminus, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, respectively. In the case of heavy chain antibodies (also referred to herein as nanobodies), such as those from Camelidae, the antigen-binding site is composed of a single VH domain (i.e., a "VHH" domain). The VHH of a native heavy chain antibody has a similar structure to the heavy chain variable region of a native IgG antibody, comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs).

[0111] "Complementarity determining regions," or "CDR regions," or "CDRs," or "hypervariable regions," are regions of an antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen-contacting residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. CDRs in a variable domain are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.

[0112] A variety of methods are known in the art for determining CDR sequences within a given VH or VL amino acid sequence: the Kabat complementarity determining region (CDR) is determined based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), while the Chothia method refers to the positions of structural loops (Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:877-883). The AbM CDR is a compromise between the Kabat CDR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. The "Contact" CDR is based on analysis of available complex crystal structures. The residues in each of these CDRs, according to different CDR determination methods, are described below.

[0113]

[0114] When referring to antibodies defined by specific CDR sequences defined herein, the scope of said antibodies also encompasses antibodies whose variable region sequences comprise said specific CDR sequences, but whose declared CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination).

[0115] The CDRs of the antibodies of the present invention can be manually assessed to determine their boundaries according to any protocol or combination thereof in the art. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways.

[0116] "Heavy chain constant region domain" or "heavy chain constant region" refers to a constant region domain from, obtained from, or derived from an immunoglobulin heavy chain, comprising heavy chain constant regions CH1, CH2, CH3, and optionally heavy chain constant region CH4 covalently linked sequentially from N-terminus to C-terminus. In most cases, the heavy chain constant regions CH1 and CH2 are connected by a heavy chain hinge region, but may also be connected by a flexible linker when appropriate.

[0117] The term "EC 50 ”, also known as the “median effective concentration”, is the concentration of a drug, antibody, or toxic agent that induces a response that is 50% between baseline and maximum after a specified exposure time.

[0118] As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0119] "Affinity" or "binding affinity" refers to the intrinsic binding ability that reflects the interaction between members of a binding pair. The affinity of a molecule X for its binding partner Y can be expressed by the equilibrium dissociation constant (K D ) indicates that the equilibrium dissociation constant is the dissociation rate constant and the association rate constant (k dis and k on Binding affinity can be measured by common methods known in the art.

[0120] The antibodies or antigen-binding fragments thereof of the present invention may comprise amino acid mutations and / or conservative modifications. The antibodies or antigen-binding fragments thereof of the present invention also include sequences having greater than 80% identity to the specifically provided amino acid sequences. In addition, the antibodies or antigen-binding fragments thereof of the present invention also include variants thereof.

[0121] Amino acid mutations can be amino acid substitutions, deletions, insertions, and / or additions. In some embodiments, amino acid mutations are substitutions of one or more amino acids, such as single amino acid substitutions or combinations of multiple amino acid substitutions. Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxyl termini of a polypeptide sequence, as well as deletions and insertions within the polypeptide sequence. Amino acid substitutions of the present invention optionally include conservative amino acid substitutions.

[0122] The "percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variants may comprise conservative modifications.

[0123] With respect to polypeptide sequences, "conservative modifications" include substitutions, deletions, or additions to a polypeptide sequence that result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude the polymorphic variants, interspecies homologs, and alleles of the present invention. The following eight groups contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.

[0124] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0125] The term "antibody-drug conjugate" or "ADC" refers to an antibody or antibody fragment covalently coupled to a therapeutically active substance or active pharmaceutical ingredient, such that the therapeutically active substance or active pharmaceutical ingredient is targeted to the binding target of the antibody to exhibit its pharmacological function. The therapeutically active substance or active pharmaceutical ingredient may be a cytotoxin capable of killing cells (preferably cancer cells) targeted by the ADC. The covalent attachment of the therapeutically active substance, active pharmaceutical ingredient, or cytotoxin may be performed in a non-site-specific manner using a linker, or in a site-specific manner.

[0126] The term "site-specific conjugation" refers to a method of specifically linking a therapeutically active substance or active pharmaceutical ingredient to a specific site of an antibody. In one embodiment, the conjugation is accomplished with the aid of a linker.

[0127] The term "cytotoxic agent" can be used interchangeably with "cytotoxin" and refers to a substance that inhibits or disrupts cellular function and / or causes cell death or destruction. In one embodiment, the cytotoxic agent may include, but is not limited to, bacterial toxins, plant toxins, small molecule toxins, radioactive isotopes, etc.

[0128] Any antibody-drug conjugate of the present invention can be prepared by conjugating dolastatin and its auristatin derivatives to an antibody. Dolastatin and its auristatin derivatives are important cytotoxins used in antibody-drug conjugates (ADCs). They interfere with microtubule dynamics and cell division and exhibit anti-tumor and antifungal activities. In one aspect, dolastatin and its auristatin derivatives can be, for example, monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethylauristatin F (MMAF), and other auristatins (e.g., those described in U.S. Patent Publication No. 20130129753).

[0129] Monomethyl auristatin (MMAE), also known as demethyl-auristatin E, is a well-known member of the auristatin compound family. Its structural formula is as follows:

[0130]

[0131] MMAE is conjugated to a monoclonal antibody via a linker to form an ADC. Generally speaking, the linker is cleaved after the ADC enters tumor cells, releasing MMAE, which then exerts its cytotoxic effect and kills tumor cells.

[0132] DXD, a derivative of Exatecan, is a potent DNA topoisomerase I inhibitor and has been widely used in the ADC field. It is linked to cysteine residues on antibodies via a cleavable tetrapeptide linker (GGFG) via a maleimide. Currently, Deruxtecan, consisting of maleimide-GGFG-DXD, is commercially available as an ADC drug linker and has the following structure:

[0133]

[0134] The terms "linker" and "connector" are used interchangeably herein to refer to a chemical moiety that covalently links an antibody to a therapeutically active substance or active pharmaceutical ingredient in an ADC. In one embodiment, the linker may comprise amino acid residues that connect the antigen to the payload. The amino acid residues may form a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide unit. The amino acid residues include naturally occurring amino acid analogs such as citrulline or β-amino acids, such as β-alanine, or ω-amino acids such as 4-amino-butyric acid.

[0135] According to the property classification, the linkers suitable for the present invention can be protease-degradable linkers, non-cleavable linkers, acid-sensitive linkers, silicone-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared-sensitive linkers, UV-sensitive linkers, etc.

[0136] The linker of the present invention can also be a combination of one or more linkers. For example, a cathepsin-degradable linker can be combined with other types of linkers to form a new linker. Therefore, the "linker" described in the present invention encompasses a single type of linker or a combination of different types of linkers, as long as it is capable of conjugating the antibody of the present invention to the drug.

[0137] The term "load" or "drug load" or "payable load" refers to the average number of therapeutically active substances or active pharmaceutical ingredients per antibody within the ADC molecule ("payable load" is used interchangeably herein with "therapeutically active substance or active pharmaceutical ingredient"). Drug load can range from 1-20 therapeutically active substances or active pharmaceutical ingredients per antibody.

[0138] The term "drug / antibody ratio" or "DAR" refers to the ratio of the therapeutically active substance or active pharmaceutical ingredient (D) coupled to the antibody to the antibody. The ADCs described herein typically have a DAR of 1-8, and in certain embodiments have a DAR of 1-8, 2-8, 2-6, 2-5, 3-8, 4-6, and 2-4. Representative DAR values are, for example, 1, 2, 3, 4, 5, 6, 7, 8, typically expressed as the letter D or a combination of DAR and a number, wherein the number represents the numerical value of the DAR, for example, D2 / DAR2 represents a drug / antibody ratio with a DAR value of 2. In some embodiments, the DAR is the average DAR, i.e., the overall ratio of the small molecule drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety in the product as measured by a detection method (e.g., by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, electrophoresis, and / or HPLC).

[0139] The DAR may be limited by the number of attachment sites on the antibody. For example, where the attachment site is a cysteine thiol, the antibody may have only one or a few cysteine thiol groups or may have only one or a few sufficiently reactive thiol groups through which the linker unit can be attached.

[0140] In some embodiments, the average DAR value of the conjugates of the invention is from 0 to 8, e.g., 3.0-4.0, 4.0-5.0, 7.0-8.0, e.g., a range having two of these values as endpoints.

[0141] The terms "individual" and "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0142] The term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the antibody molecules of the present invention are used to delay the development of the disease or to slow the progression of the disease.

[0143] The term "preventing" includes the inhibition of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some embodiments, a subject having a disease associated with aberrant expression of LIV-1 is a candidate for a preventative regimen.

[0144] The term "effective amount" refers to an amount or dosage of an antibody or composition of the present invention that produces the desired effect in a patient in need of treatment or prevention after administration to the patient in a single or multiple doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as the species of the mammal; weight, age, and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0145] The term "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount of an antibody or antibody fragment or composition can vary according to factors such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or antibody portion to stimulate the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition are less than the therapeutically beneficial effects. Relative to an untreated subject, a "therapeutically effective amount" preferably suppresses a measurable parameter by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%.

[0146] The term "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, a prophylactic effective amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0147] The term "pharmaceutical composition" refers to a composition that is in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered. The pharmaceutical composition includes, but is not limited to, carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0148] Biological materials (nucleic acid molecules, vectors and host cells)

[0149] The present invention provides nucleic acid molecules encoding any of the above-mentioned antibody molecules or antigen-binding fragments thereof. Polynucleotide sequences encoding the antibody molecules or antigen-binding fragments thereof of the present invention can be generated by de novo solid-phase DNA synthesis or genetic engineering methods using methods well known in the art. In addition, the polynucleotides and nucleic acids of the present invention may include a segment encoding a secretory signal peptide, which can be operably linked to the segment encoding the antibody molecules or antigen-binding fragments thereof of the present invention, thereby directing the secretory expression of the antibody molecules or antigen-binding fragments thereof of the present invention.

[0150] The present invention also provides vectors comprising the nucleic acid molecules of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector or a prokaryotic expression vector. An "expression vector" refers to a vector comprising a recombinant polynucleotide, which comprises expression control sequences operably linked to the nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0151] The present invention also provides prokaryotic and eukaryotic host cells comprising the nucleic acid molecules or the vectors. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce antibody molecules of the present invention, including eukaryotic cells, for example, mammalian cells, insect cells, yeast cells, and prokaryotic cells, for example, E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues. Host cells suitable for replicating and supporting the expression of antibody molecules of the present invention or their antigen-binding fragments are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large amounts of vector-containing cells can be grown to inoculate large-scale fermenters, thereby obtaining sufficient amounts of antibody molecules.

[0152] Compositions and pharmaceutical preparations

[0153] The present invention provides a composition comprising the antibody molecule or antigen-binding fragment thereof of the present invention. Preferably, the composition is a pharmaceutical composition.

[0154] In one embodiment, the composition of the present invention further comprises a pharmaceutical excipient, such as a pharmaceutical carrier or a pharmaceutical excipient known in the art. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises an anti-LIV-1 antibody or ADC molecule thereof of the present invention and a combination of one or more other therapeutic agents.

[0155] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0156] For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and SC Owen, Pharmaceutical Press, London, Chicago.

[0157] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as powders or suspensions, liquid solutions (e.g., injectable solutions and infusible solutions), liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0158] The administration route of the composition of the present invention is according to known methods, for example, oral, intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional routes; by sustained release system or by implant device. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by implant device.

[0159] The subject can be a mammal, e.g., a primate, e.g., a human (e.g., an individual suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from or is at risk of suffering from a disease described herein (e.g., migraine). In certain embodiments, the subject is receiving or has received other treatments.

[0160] A medicament comprising the antibody described herein can be prepared by mixing the anti-LIV-1 antibody of the present invention or its ADC molecule having the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0161] The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient, the active ingredients being required for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents. The active ingredients are suitably combined in amounts effective for the intended use.

[0162] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0163] Preparation of Antibodies and Antibody-Drug Conjugates of the Invention

[0164] In one embodiment, the present invention provides a method for producing an anti-LIV-1 antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the anti-LIV-1 antibody or an expression vector comprising the nucleic acid under conditions suitable for expression of the nucleic acid encoding the anti-LIV-1 antibody, and optionally isolating the anti-LIV-1 antibody. In certain embodiments, the method further comprises recovering the anti-LIV-1 antibody from the host cell (or host cell culture medium).

[0165] To recombinantly produce an anti-LIV-1 antibody of the invention, nucleic acid encoding the anti-LIV-1 antibody of the invention is first isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acid is readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that specifically bind to the nucleic acid encoding the anti-LIV-1 antibody of the invention.

[0166] The anti-LIV-1 antibodies of the present invention, prepared as described herein, can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these will be apparent to those skilled in the art. The purity of the anti-LIV-1 antibodies of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0167] Antibody-drug conjugates can be produced by any technique known to those skilled in the art. In some aspects, drug-linker conjugation to the antibody is accomplished by reaction with an amino acid residue of the antibody. In some embodiments, a heteroaryl linker L with a leaving group is used to conjugate the drug D to a cysteine residue of the antibody to prepare the conjugate of Formula (I) of the present invention. In some embodiments, by controlling the conditions under which the antibody is treated with a reducing agent such as tris(2-hydroxyethyl)phosphine (TCEP), interchain disulfide bonds can be disrupted and free sulfhydryl groups exposed for conjugation with the heteroaryl linker-drug. For IgG1 antibodies, up to four interlinking disulfide bonds can be reduced, generating up to eight reactive sulfhydryl groups for conjugation. Conjugates prepared by this method can contain zero, one, two, three, four, five, six, seven, or eight drugs per antibody molecule.

[0168] When the prepared conjugate is a composition having different drug conjugation sites and / or numbers of conjugates, the drug loading of the conjugate is represented by the average DAR, which is the average number of drug molecules per antibody. The average number of drug per antibody of the prepared antibody-drug conjugate composition can be characterized by conventional means, such as mass spectrometry, ELISA assay, and HPLC. In other embodiments, the quantitative distribution of the antibody-drug conjugate, represented by n, can also be determined. Homogeneous antibody-drug conjugates where n is a certain value can be separated, purified, and characterized from antibody-drug conjugates having different drug loadings by means such as reversed-phase HPLC or electrophoresis.

[0169] Example

[0170] The present invention is further illustrated by the following examples; however, it should be understood that the examples are described in an illustrative rather than a limiting sense and that various modifications may be made by those skilled in the art.

[0171] Unless expressly stated to the contrary, the practice of the present invention will employ conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology within the art. The experimental methods described, unless otherwise specified, are conventional methods in the art using default parameters, steps, and the like; the experimental materials used, unless otherwise specified, are commercially available products. For examples in which specific techniques or conditions are not specified, the techniques or conditions described in the literature within the art or in accordance with the corresponding product instructions were used. For reagents or instruments used for which the manufacturer is not specified, all are conventional products that can be purchased through regular channels.

[0172] Example 1: Preparation of mouse hybridomas producing anti-human LIV-1 antibodies

[0173] In this example, mice were immunized with LIV-1 protein to obtain antibodies against human LIV-1. Specifically, the extracellular fragment of human LIV-1 (UNIPROT KB: Q13433) protein was used as the immunogen to immunize mice. The specific method is as follows:

[0174] Animal immunization: LIV-1-Fc protein was used as an immunogen, diluted to 1 mg / ml and mixed with an equal volume of CFA (Sigma, F5881) or IFA (Sigma, F5506) (CFA was used for the initial immunization, and IFA was used for subsequent immunizations). Balb / c female mice were immunized with protein at a dose of 50 μg / mouse for the initial immunization and 25 μg / mouse for subsequent immunizations. Immunizations were repeated for more than three times at intervals of two weeks. 14 days after the last immunization, LIV-1 protein was injected intraperitoneally for pulse immunization. Three days later, the mouse spleens were harvested for cell fusion.

[0175] Cell fusion: Mouse spleen cells and SP2 / 0 cells (ATCC No. CRL-1581) were electrofused at a ratio of 2:1 (BTX electrofusion instrument: ECM2001 + ) were cultured in 96-well culture plates using HAT medium (Sigma-Aldrich, Catalog No. H0262), and hybridoma cell supernatant antibody screening was performed after 10 days.

[0176] Screening for hybridoma-positive clones: Collect the cultured cell supernatant and perform an ELISA or FACS binding assay. Select the positive wells in the cell culture supernatant and culture them in 96-well culture plates using HT medium (Sigma-Aldrich, Catalog No. H0137). After 7-10 days, visible clones will appear, and the supernatant will be collected for antibody detection. Observe under an inverted microscope and select the positive wells with only a single clone. Expand the culture and freeze. After culturing in serum-free medium (SFM, Thermo, 12045076), collect the supernatant and purify it for subsequent testing.

[0177] ELISA binding assay: 50 μL of hybridoma supernatant, positive control (BR2-22a, Shanghai Hongcheng), and negative control (mIgG1, Shanghai Hongcheng) were added to a 96-well flat-bottom assay plate (Corning, 9018) coated with human LIV-1 protein or cynomolgus macaque LIV-1 protein, respectively. The plates were incubated at 37°C for 60 minutes, washed three times with PBST, and then a secondary antibody (Anti-Mouse IgG, Sigma, A0168) was added and incubated at 37°C for 30 minutes. The plates were washed three times with PBST, and 100 μL of TMB (Inc. Biotech, EL0009) was added to each well. After color development for 15 minutes, the reaction was terminated by adding 50 μL of sulfuric acid to each well. OD values were read on a microplate reader. The results are shown in Table 1.

[0178] FACS binding assay: Human LIV-1-producing 293T cells were plated in a 96-well plate (Corning, 3799). Hybridoma supernatant, a positive control (BR2-22a, Shanghai Hongcheng), and a negative control (mIgG1, Shanghai Hongcheng) were then added to the 96-well plate containing the four cells and incubated at 4°C for approximately 1 hour. The cells were washed three times with FACS buffer, and 100 μL of secondary antibody (Invitrogen, B118301) diluted in buffer was added to each well to resuspend the cells. The cells were incubated at 4°C for approximately 1 hour. After washing three times with FACS buffer, 100 μL of FACS buffer was added to each well to resuspend the cells. The mean fluorescence intensity (MFI) was measured using a flow cytometer (BECKMAN COULTER cytoFLEX or BD FACSCelesta™).

[0179] Hybridoma subcloning (limiting dilution method): Mix the cells in the culture wells of hybridoma-positive clones that have binding activity to human LIV-1-overexpressing 293T cells and LIV-1 extracellular domain protein, then pipette them into a centrifuge tube. Add an appropriate amount of culture medium, mix thoroughly, aspirate a small amount of cells, and count them. Based on the count results, dilute the hybridoma cells to 5 cells per mL. Add 0.2 mL of the above cell suspension to each well of a 96-well plate. After culturing for one week, select the culture supernatant containing single cell colonies and retest as described above.

[0180] Example 2: Preparation and identification of mouse monoclonal antibodies against human LIV-1

[0181] Hybridoma cells from positive subclones were cultured in serum-free medium for 10 days, and the supernatant was collected. Mouse monoclonal antibodies were purified using a Protein A column (Bioglone (Shanghai) Biotechnology Co., Ltd., Catalog No. AA0272). The binding activity of the anti-human LIV-1 antibodies was determined by ELISA and flow cytometry.

[0182] 1. Binding activity of anti-human LIV-1 antibodies to human LIV-1

[0183] Binding experiment of anti-LIV-1 antibody to human LIV-1 extracellular domain protein: The coating protein was human LIV-1 extracellular domain protein or cynomolgus monkey LIV-1 extracellular domain protein, 1 μL / ml, 50 μL / well, and coated at 4°C overnight; incubated with 150 μL / well 2% BSA at room temperature for 1 hour; washed three times with 200 μL / well PBST; prepared 300 μL of 10 μg / ml stock solution of sample or positive antibody; the dilution factor was 3.16 (100 μL stock solution + 216 μL blocking solution), with a total of 11 gradients; 50 μL / well of sample or positive antibody, and an equal volume of mIgG1 as a negative control was added to column H12, and incubated at room temperature for 1 hour; washed three times with 200 μL / well PBST; hybridoma samples were added with secondary antibody (Anti-Mouse IgG, Sigma, A0168) and incubated at 37°C for 30 minutes. The cells were washed three times with PBST, and 100 μL of TMB (Inc. Biotech, EL0009) was added to each well. After 15 minutes of color development, 50 μL of sulfuric acid was added to each well to terminate the reaction. OD450 values were read on a microplate reader. Graphpad Prism 8.0 software was used to analyze the experimental data. The logarithm of the antibody concentration was used as the x-axis, and the corresponding OD450 value was used as the y-axis. A four-parameter regression model was used to fit the antibody dose-effect curve and calculate the EC 50 .

[0184] The results are shown in Table 1. Figure 1 and Figure 2 shown.

[0185] Table 1 Binding activity of anti-human LIV-1 hybridoma antibodies

[0186] NT: No binding.

[0187]

[0188] 2. Binding activity of anti-human LIV-1 antibodies to human 293T LIV-1 cells

[0189] The cell concentration of 293T cells expressing human LIV-1 was adjusted to 2 × 10 6 Cells were plated at 50 μL / well in a 96-well U-bottom plate at 100 μL / well and centrifuged. The supernatant was discarded after centrifugation. Anti-LIV-1 antibody and control antibody (BR2-22a, Shanghai Hongcheng) were diluted to their starting working concentrations in FACS buffer (PBS containing 1% FBS) and then serially diluted in FACS buffer. 100 μL of the antibody serial dilutions diluted in FACS buffer (PBS containing 1% FBS) were added to each well and incubated at 4°C for approximately 1 hour. Wash cells three times with FACS buffer and add 100 μL of secondary antibody (Invitrogen, Cat. No. B118301) diluted in FACS buffer to each well and incubate at 4°C for approximately 1 hour. Wash cells three times with FACS buffer and resuspend cells in 100 μL of FACS buffer per well. The MFI was read by flow cytometry and analyzed using Graphpad Prism 8.0 software. The logarithm of the antibody concentration was used as the x-axis and the corresponding MFI value was used as the y-axis. A four-parameter regression model was used to fit the antibody dose-effect curve and calculate the EC. 50 The results are shown in Table 1 and Figure 3 shown.

[0190] As shown in Table 1 , based on the ELISA binding and FACS binding results, the monoclonal 161E5B8 was sequenced.

[0191] Example 3: Anti-LIV-1 Antibody Sequencing and Preparation of Chimeric Antibodies and Their Functional Identification

[0192] 1. Sequencing, Expression, and Purification of Anti-LIV-1 Antibodies

[0193] The positive hybridoma clones were sequenced, and the sequences are shown in Table 2. The sequenced light and heavy chain variable regions (Table 2) were then incorporated into human constant regions (IgG1 / κ, Table 3) to construct the corresponding chimeric antibodies, and the sequences were verified by sequencing. Chimeric antibodies are designated by appending the prefix "ch" to the corresponding hybridoma clone number. For example, the chimeric antibody obtained in this example using hybridoma clone 161E5B8 was named ch161E5B8 and used for in vitro functional characterization or in vivo efficacy studies.

[0194] Table 2 CDR sequences and their numbers of anti-LIV-1 hybridoma antibodies (determined according to the Kabat scheme)

[0195]

[0196] Table 3 Constant region sequences of chimeric and humanized antibodies

[0197]

[0198] The corresponding nucleic acid encoding the antibody was transfected into Expi293 cells (Gibco, Catalog No. A14635) for antibody expression and purified using a Protein A column as follows:

[0199] 1.5×10 6 Expi293 cells (Gibco, Cat. No. A14635) were cultured at 37°C, 8% CO2, and 120 rpm in a shaker. The next day, the cell density and viability were measured. The density should be around 3 × 10 6 cells / mL, with a viability greater than 95%. Dilute the plasmid containing the encoding nucleic acid in OPM-293 CD05 Medium (OPM, Catalog No. 81075-001) to a total volume of 1 μg / mL. The volume of medium used to dilute the plasmid should be 1 / 20 of the transfection volume, with a light chain to heavy chain ratio of 1:1.5. Invert and mix thoroughly, then dilute with OPM-293 CD05 Medium (1 mg / mL, Polysciences, Catalog No. 24765-1) to 1 / 20 of the transfection volume. Incubate at room temperature for 5 minutes. Add the diluted PEI to the diluted plasmid, mix thoroughly, and incubate at room temperature for 15 minutes. The PEI / plasmid complex was added to a shake flask containing Expi293 cells (Gibco, Catalog No. A14635). After culturing at 37°C, 8% CO2, and 120 rpm for 24 hours, 10% OPM-293 ProFeed (OPM, Catalog No. F081918) was added. The culture was continued for 5-7 days, and the supernatant was collected.

[0200] Place the gasket at the bottom of the gravity chromatography column and press firmly. The volume of the Protein A (Cytiva, Cat. No. 17549801) filler suspension equals the target filler volume / filler suspension ratio. Vortex the filler thoroughly and add it to the bottom of the gravity chromatography column. Add PBS until the outlet pH reaches the target pH. Add the sample to the column, add at least 10 column volumes (CV) of wash buffer, and add 5 CV of elution buffer (20 mM NaAc, pH 3.0). Incubate for 3-5 minutes, and collect the eluate. Adjust the pH to 7.2 with neutralization buffer (1 M Tris). Measure the protein concentration using a Nanodrop filter and exchange the buffer with PBS using a Millipore ultrafiltration centrifuge tube (50 kDa).

[0201] 2. Binding activity of anti-LIV-1 chimeric antibodies to human ovarian cancer cells SK-OV-3 and human colon cancer cells HCT116

[0202] Human ovarian cancer cells SK-OV-3 and human colon cancer cells HCT116 were added to a 96-well U-bottom plate at a concentration of 100 μL per well, centrifuged, and the supernatant discarded. 100 μL of a serial dilution of the antibody prepared in FACS buffer (PBS containing 1% FBS) was added to each well and incubated at 4°C for approximately 1 hour. The cells were washed three times with FACS buffer, and 100 μL of secondary antibody (Invitrogen, Catalog No. A21445) diluted in FACS buffer was added to each well and incubated at 4°C for approximately 1 hour. The cells were washed three times with FACS buffer, and 100 μL of FACS buffer was added to each well to resuspend the cells. The MFI was measured by flow cytometry, and the data were analyzed using Graphpad Prism 8.0 software. The logarithm of the antibody concentration was used as the x-axis, and the corresponding MFI value was used as the y-axis. A four-parameter regression model was used to fit the antibody dose-effect curve, and the EC50 was calculated.

[0203] The results are shown in Table 4 and Figure 4A and Figure 4B As shown, the chimeric antibody ch161E5B8 has strong binding activity with human ovarian cancer cells SK-OV-3 and human colon cancer cells HCT116, which is better than PC (BR2-22a-hLIV22).

[0204] Table 4 Binding activity of anti-LIV-1 chimeric antibodies to human LIV-1 (EC 50 , nM)

[0205]

[0206] N / A: No binding activity or poor binding activity, no effective EC 50 value.

[0207] 3. Cytotoxicity Induced by αhFc-CL-MMAE and LIV1 Antibody

[0208] The anti-human IgG Fc-MMAE antibody with a cleavable linker (αHFc-CL-MMAE (Moradec, AH-102AE)) is an anti-human IgG Fc-specific antibody conjugated to MMAE via a cleavable linker. αHFc-CL-MMAE recognizes the Fc terminus of the anti-human LIV-1 chimeric antibody, leading to endocytosis and intracellular release of MMAE in LIV-1-expressing cells, which kills the cells. 293T LIV-1 cells were obtained by trypsinization and centrifuged to adjust the cell density to 4 × 10 4 cells / mL. Add 25 μL of antibody concentration gradient dilutions diluted in culture medium (starting and ending concentration is 20 nM, 4-fold dilution) and 50 μL of cell suspension to each well of a white bottom transparent 96-well plate. After incubation for 10 minutes, add 25 μL of αHFc-CL-MMAE working solution (Moradec, AH-102AE-50) prepared in culture medium to each well, and place the 96-well plate in an incubator at 37°C and 5% CO. After 3 days, add 50 μL of CTG (Adamas Life, RA-GL11-A) to each well, read the fluorescence relative light units with a microplate reader, and use a four-parameter model to fit the RLU to calculate the IC of each antibody. 50 Value, the result is Figure 5 and as shown in Table 5.

[0209] Table 5 Cytotoxic activity of anti-LIV-1 chimeric antibody and αHFc-CL-MMAE conjugate

[0210]

[0211] Example 4: Humanization of anti-human LIV-1 antibodies and expression and purification of humanized antibodies

[0212] Using Kabat numbering to identify CDRs, the human germline gene with the highest homology to the mouse sequence was selected as the acceptor framework, and the mouse CDRs were transplanted into the human framework. Based on the importance of amino acids, backmutations were performed, remutating key amino acids in the transplanted framework region to their corresponding mouse counterparts. Several variants were designed for each heavy and light chain.

[0213] 1. Humanization of the anti-LIV-1 antibody 161E5B8

[0214] The HCDR and LCDR of the 161E5B8 mouse antibody were transplanted into the human genes IGHV3-23*04 and IGKV3-20*02, respectively. The obtained sequences are shown in Table 6, and the corresponding variable region combinations of the constructed humanized antibodies are shown in Table 7.

[0215] Table 6 Humanized sequence of anti-LIV-1 antibody 161E5B8

[0216]

[0217] Table 7 Heavy chain variable region sequences and light chain variable region sequences corresponding to the constructed humanized antibodies

[0218]

[0219] 2. Expression and Purification of Humanized Anti-LIV-1 Antibodies

[0220] The light and heavy chain variable regions of the humanized antibodies listed in Table 6 were constructed onto human constant regions (hIgG1 / κ, Table 3) according to the combinations listed in Table 7. Gene synthesis was performed and sequencing confirmed consistency with the designed sequences. Expression was performed in Expi293 cells (Gibco, Catalog No. A14635) using the method described in Example 2 and purification was performed using a Protein A column.

[0221] Example 5: Functional characterization of anti-LIV-1 humanized antibodies

[0222] The binding activity of the humanized antibodies to human LIV-1 was tested.

[0223] 1. Anti-LIV-1 humanized antibody ELISA binding assay

[0224] The binding activity of the anti-LIV-1 humanized antibody to human LIV-1 was detected using the method disclosed in Example 3. The results of the antibody binding to human and cynomolgus monkey LIV-1 are shown in Figure 3. Figure 6 and as shown in Table 8. The humanized antibody had an ELISA binding affinity comparable to that of the positive control (BR2-22a-hLIV22).

[0225] Table 8 Anti-LIV-1 humanized antibodies binding to human LIV-1 ELISA EC 50

[0226]

[0227] 2. Cytotoxicity Induced by Humanized Anti-LIV-1 Antibody and αhFc-CL-MMAE

[0228] The method described in Example 3 was used. αHFc-CL-MMAE (Moradec, AH-102AE) recognizes the Fc terminus of the anti-human LIV-1 humanized antibody, leading to endocytosis of LIV1-expressing cells and the release of MMAE to kill the cells. 293T LIV-1 cells were obtained by trypsinization and centrifuged to adjust the cell density to 4 × 10 4Cells / mL. 25 μL of antibody concentration gradient diluted in culture medium (starting and ending at 20 nM, 4-fold dilution) and 50 μL of cell suspension were added to each well of a white bottom transparent 96-well plate to induce cytotoxicity. After incubation for 10 minutes, 25 μL of αHFc-CL-MMAE working solution (Moradec, AH-102AE-50) prepared in culture medium was added to each well. The 96-well plate was placed in an incubator at 37°C and 5% CO to induce cytotoxicity for 3 days. After that, 50 μL of CTG (Adamas Life, RA-GL11-A) was added to each well. The relative light units of fluorescence were read using a microplate reader. The RLU was fitted using a four-parameter model to calculate the IC of each antibody. 50 Value, the result is Figure 7 and as shown in Table 9.

[0229] Table 9 Cytotoxicity of anti-LIV-1 humanized antibody and αhFc-CL-MMAE conjugate

[0230]

[0231] Note: NT means not tested.

[0232] Example 6: Preparation of a conjugate of anti-human LIV-1 humanized antibody and MMAE

[0233] 6.1 Anti-human LIV-1 humanized antibody MMAE conjugate

[0234] A certain amount of antibody and PBS buffer were added to the reaction mixture to achieve an antibody concentration of approximately 1 mg / mL. An amount of EDTA solution was added to achieve an EDTA concentration of approximately 2 mM. 1M Tris saline was added to adjust the pH of the reaction system to between 6.6 and 7.5. 2-6 equivalents of TCEP were added for reduction, and the mixture was gently shaken on a homogenizer at 37°C for 1-3 hours. The homogenizer temperature was then adjusted to approximately 4°C. The reaction mixture was cooled, and 5-10 equivalents of DMSO-dissolved Vc-MMAE (MCE, HY-15575) were added. The mixture was reacted at 4°C for 1-2 hours. Finally, the reaction mixture was transferred to a 30K ultrafiltration tube and replaced with 10 mM, pH 6.0, histidine hydrochloride solution 6-8 times. The mixture was concentrated to an appropriate volume and applied to a pre-equilibrated desalting column. The purified ADC product was centrifuged and the concentration and DAR value were determined. The DAR value was 4. The structure of the anti-human LIV1 antibody-MMAE conjugate is shown below.

[0235]

[0236] 6.2 FACS Binding Assay of Anti-Human LIV-1 Humanized Antibody ADC

[0237] Human LIV-1 293T cells were added to a 96-well plate (Corning, 3799), and 100 μL of FACS buffer (PBS solution containing 1% FBS) prepared with a gradient dilution of the antibody ADC was added to each well, and the cells were incubated at 4°C for about 1 hour. The cells were washed three times with FACS buffer, and 100 μL of secondary antibody diluted in FACS buffer (Invitrogen, Cat. No.: A21445) was added to each well, and the cells were incubated at 4°C for about 1 hour. The cells were washed three times with FACS buffer, and 100 μL of FACS buffer was added to each well to resuspend the cells. The MFI was read by flow cytometry, and the data were analyzed using Graphpad Prism 8.0 software. The logarithm of the antibody concentration was used as the x-axis, and the corresponding MFI value was used as the y-axis. A four-parameter regression model was used to fit the antibody dose-effect curve, and the EC was calculated. 50 .

[0238] The results are shown in Table 10 and Figure 8 As shown, the LIV-1 humanized antibody ADC has strong binding activity to 293T LIV1, and its binding activity is comparable to that of the control (BR2-22a-hLIV22).

[0239] 6.3 Cytotoxicity Testing of Humanized Antibody ADC Against Human LIV-1

[0240] 293T LIV-1 cells were obtained by trypsinization and the cell concentration was adjusted to 4 × 10 4 Cells / mL, 50 μL / well were inoculated in a white bottom transparent 96-well plate. 50 μL of antibody concentration gradient dilution diluted in culture medium was added to each well of the 96-well plate, and the 96-well plate was placed in a 37°C, 5% CO2 incubator for 3 days. 50 μL of CTG reagent was added to each well, incubated in the dark at room temperature for 5 minutes, and the relative light unit value was read using a microplate reader. The experimental data were analyzed using Graphpad Prism 8.0 software, with the logarithm of the antibody concentration as the x-axis and the corresponding RLU value as the y-axis. A four-parameter regression model was used to fit the dose-effect curve of the antibody. The results are shown in the figure. Figure 9 As shown in Table 10, all the ADCs of the tested antibodies had strong cytotoxic activity against 293T LIV-1 cells, which was comparable to that of the control (BR2-22a-hLIV22).

[0241] Table 10 Cytotoxicity of anti-LIV-1 humanized antibody MMAE conjugates

[0242]

[0243] Sequence information:

[0244] Sequence information of humanized anti-LIV-1 antibody and positive control antibody

[0245] BR2-22a-hLIV22 (HGLG)<hLiv1 mAb2 HG; PRT / 1; artificial> (SEQ ID NO:18):

[0246] QVQLVQSGAEVKKPGASVKVSCKASGLTIEDYYMHWVRQAPGQGLEWMGWIDPENGDTEYGPKFQGRVTMTRDTSINTAYMELSRLRSDDTAVYYCAVHNAHYGTWFAYWGQGTLVTVSS

[0247] BR2-22a-hLIV22 (HGLG)<hLiv1 mAb2 LG; PRT / 1; artificial> (SEQ ID NO:19):

[0248] DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWYQQRPGQSPRPLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIKR

[0249] BR2-22a (mouse origin) VH (SEQ ID NO: 20):

[0250] EVQLQQSGAELVRSGASVKLSCTASGLNIEDYYMHWVKQRPEQGLEWIGWIDPENGDTEYGPKFQGKATMTADTSSNTAYLQLSSLTSGDTAVYYCTVHNAHYGTWFAYWGQGTLVTVSA

[0251] BR2-22a (mouse origin) VL (SEQ ID NO: 21):

[0252] DVLMTQTPLSLPVSLGDQASISCRSSQSLLHSSGNTYLEWYLQRPGQSPKPLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYYCFQGSHVPYTFGGGTKLEIKR

Claims

1. An antibody or antigen-binding fragment thereof that binds to LIV-1, comprising heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein: HCDR1 consists of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 2, HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 3, LCDR1 consists of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:

6.

2. The LIV-1-binding antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein: (1) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8; (2) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14; (3) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15; (4) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16; or (5) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

16.

3. The LIV-1-binding antibody or antigen-binding fragment thereof according to claim 2, wherein The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein: (1) The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 7; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 8; (2) The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 14; (3) The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 15; (4) the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 16; or (5) The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 13; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:

16.

4. The LIV-1-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antibody is any form of a monoclonal antibody, a bifunctional antibody, a fully or partially humanized antibody, or a chimeric antibody; and / or The antigen-binding fragment is an antigen-binding fragment of an antibody.

5. The LIV-1-binding antibody or antigen-binding fragment thereof according to claim 4, wherein The antigen-binding fragment is a Fab, Fab', Fab'-SH, (Fab')2, Fv, scFv, BsFv, dsFv or (dsFv)2 fragment.

6. The LIV-1-binding antibody or antigen-binding fragment thereof according to claim 4, wherein The antibody or antigen-binding fragment thereof comprises a heavy chain constant region and / or a light chain constant region of human or murine origin; The antibody or antigen-binding fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a kappa or lambda type light chain constant region; or The antibody is a monoclonal antibody, and the heavy chain constant region of the monoclonal antibody is of IgG1, IgG2, IgG3 or IgG4 subtype.

7. The LIV-1-binding antibody or antigen-binding fragment thereof according to claim 6, wherein: The heavy chain constant region of the monoclonal antibody comprises or consists of the amino acid sequence shown in SEQ ID NO: 9; and / or the light chain constant region of the monoclonal antibody comprises or consists of the amino acid sequence shown in SEQ ID NO:

10.

8. A biomaterial comprising: (i) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7; (ii) a vector comprising the nucleic acid molecule described in (i); and / or (iii) a host cell comprising the nucleic acid molecule described in (i) and / or the vector described in (ii), or the host cell is transformed or transfected with the nucleic acid molecule described in (i) and / or the vector described in (ii).

9. An antibody-drug conjugate comprising the antibody of any one of claims 1-4 and 6-7 and at least one therapeutically active substance or pharmaceutically active ingredient; the antibody-drug conjugate has the structure represented by formula (I): Ab-(LD)n formula (I) in, Ab is the antibody according to any one of claims 1-4, 6-7; L is a linker; D is dolastatin and its auristatin derivatives, or a topoisomerase I inhibitor; n=1-8。 10. The antibody-drug conjugate according to claim 9, wherein The antibody-drug conjugate further comprises a linker.

11. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the host cell according to claim 8 and / or the antibody-drug conjugate according to claim 9.

12. The pharmaceutical composition according to claim 11, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

13. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the host cell according to claim 8, the antibody-drug conjugate according to claim 9 or 10, and / or the pharmaceutical composition according to claim 11 or 12 in the preparation of a product for detecting LIV-1.

14. Use of the antibody-drug conjugate according to claim 9 or 10 in the preparation of a product for preventing and / or treating diseases associated with abnormal LIV-1 expression, wherein the associated diseases are ovarian cancer, colon cancer, triple-negative breast cancer, cervical cancer, and lung cancer.

15. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the biomaterial according to claim 8, the antibody-drug conjugate according to claim 9 or 10, and / or the pharmaceutical composition according to claim 11 or 12.

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