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

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to an anti-LIV-1 antibody or its antigen-binding fragment and its use. The present invention also relates to a preparation method of the anti-LIV-1 antibody or its antigen-binding fragment, a nucleic acid molecule encoding the anti-LIV-1 antibody or its antigen-binding fragment, a vector containing the nucleic acid molecule, and a host cell; a conjugate containing the anti-LIV-1 antibody or its antigen-binding fragment. The present invention further relates to the use of the anti-LIV-1 antibody or its antigen-binding fragment and a pharmaceutical composition and a conjugate containing the same. Background Art

[0002] LIV-1 (leucine-isoleucine-valine transport system 1) is one of the members of the LZT (LIV-1 subfamily of ZIP zinc transporters) subfamily in the zinc transporter ZIP (Zrt-, Irt-like proteins) family. Since ZIP is also known as SLC39A (solute carrier family 39A), LIV-1 is also called SLC39A6 or ZIP6.

[0003] The LIV-1 protein is mainly located in the cell membrane and may also be located in the membranes 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, which is responsible for transporting Zn2+ from outside the cell or organelles into the cytoplasm and mainly plays a role in maintaining intracellular zinc homeostasis.

[0004] Abnormalities of LIV-1 zinc transporter may lead to diseases such as cancer and may play a dual role in tumors. Research shows that inhibiting LIV-1 can inhibit the invasion of HeLa cells (cervical cancer cell line) by targeting the ERK1 / 2-Snail / Slug pathway; in prostate cancer cells (ARCaPE and ARCaPM cells), overexpression of LIV-1 leads to increased activities of MMP-2 and MMP-9, which in turn leads to the cleavage of heparin-binding epidermal growth factor, resulting in constitutive activation of the epidermal growth factor receptor and playing an important role in tumors. In addition, LIV-1 may be a mediator of key intracellular growth regulatory signaling pathways such as the mitogen-activated protein kinase pathway (MAPK), which is associated with abnormalities (such as carcinogenesis) in transformed cells. There is evidence that LIV1 interacts with the transcription factor STAT3 (Signal Transducer and Activator of Transcription 3) and snail, downregulates E-cadherin expression, promotes epithelial-mesenchymal transition (EMT), and thus promotes tumor metastasis.

[0005] Research has 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 and still highly expressed in breast cancer patients who have received hormone therapy (78%-92%). It is lowly expressed in ovarian cancer, uterine cancer, and lung cancer (10%-48%). The expression level is divided into 5 grades from low to high. In normal human tissues, the expression is limited. In breast tissue, 0%-50% of cells express, with a staining intensity of 1-2; in prostate tissue, 50%-100% of cells have a staining intensity of 2-4; in testicular tissue, about 50% of cells have the highest staining intensity of 1; and it is hardly expressed in other normal tissues. The differential expression between tumor tissues and normal tissues proves that LIV-1 is a suitable target for tumor treatment, especially 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 object of the present invention is to provide an anti-LIV-1 antibody or its antigen-binding fragment, and based on this antibody or its fragment, provide its uses. The "fragment" of the antibody molecule described in the present invention covers various functional fragments of the antibody, such as its antigen-binding part, such as Fab, F(ab') 2 or scFV fragments, etc.

[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. It is expected that the anti-LIV-1 antibody of the present invention has low immunogenicity in human subjects, and human subjects will have good tolerance to the anti-LIV-1 antibody.

[0010] The present invention provides an antibody targeting human LIV-1, which has the following advantages: (1) High affinity binding to human LIV-1 and target cells expressing human LIV-1; (2) Having good cross-reactivity in cynomolgus monkeys; (3) Being able to enter cells through endocytosis; (4) Suitable for constructing antibody-drug conjugates 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 and / or the three light-chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light-chain variable region as shown in SEQ ID NO: 8.

[0011] In some embodiments, the present invention provides an anti-LIV-1 antibody or an antigen-binding fragment thereof, which comprises a heavy-chain variable region and / or a light-chain variable region. 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: The heavy-chain variable region comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 7, 11, 12, 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, 17.

[0012] Based on the variable region amino acid sequences contained in the given antibody or fragment thereof of the present invention, those skilled in the art can routinely determine the CDRs contained therein. For example, according to the specific embodiments of the present invention, the CDRs in the variable region amino acid sequences are defined by the Kabat scheme, the IMGT scheme, the AbM scheme, the Chothia scheme or the Contact scheme.

[0013] In some embodiments, the present invention provides an anti-LIV-1 antibody or an antigen-binding fragment thereof, which comprises heavy-chain variable region CDRs (HCDR1, HCDR2, HCDR3) and light-chain variable region CDRs (LCDR1, LCDR2, 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.

[0014] In some embodiments, the present invention provides an anti-LIV-1 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and / or a light chain variable region, wherein: (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; (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; (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; (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; (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; (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; (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; (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; 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; (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; (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; (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 (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.

[0015] In particular, the antibody or antigen-binding fragment thereof of the present invention comprises at least a heavy chain variable region and / or a light chain variable region, both of which include the above CDRs and the intervening framework regions (FRs), and the arrangement of each domain is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further optionally, up to 10% differences in the amino acid sequence caused by the "at least 90% identity" 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 antibody or fragment thereof of the present invention. The differences may be caused by amino acid substitutions, deletions or insertions at any position.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a human or murine constant region, preferably comprising a human or murine heavy chain constant region and / or a light chain constant region; preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a light chain constant region of κ or λ type.

[0017] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain and / or a light chain constant region sequence from a human antibody germline consensus sequence. In some embodiments, the heavy chain constant region preferably comes from the 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 shown in SEQ ID NO: 9, or consists of the same. In a specific embodiment, the light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 10, or consists of the same.

[0018] It should be understood that sequence variants of these constant region domains can also be used, for example, those containing one or more amino acid modifications, where the amino acid positions are identified by the EU index system of Kabat et al. (1991).

[0019] In certain embodiments of the antibody 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.

[0020] In certain embodiments of the antibody of any of the foregoing, the antibody is a full-length antibody.

[0021] In some embodiments, the anti-LIV-1 antibody of the present invention is a complete antibody, such as an IgG1, IgG2, IgG3 or IgG4 antibody. In another embodiment, the anti-LIV-1 antibody of the present invention only covers its antigen-binding part, such as: Fab, Fab', Fab'-SH, (Fab') 2 , Fv, scFv, BsFv, dsFv or (dsFv) 2 fragment.

[0022] In some embodiments, the antigen-binding fragment is an antigen-binding fragment of an antibody or a half-antibody, such as Fab, Fab', Fab'-SH, (Fab') 2 , Fv, scFv, BsFv, dsFv or (dsFv) 2 fragment; more preferably, the antibody is IgG.

[0023] In a second aspect, the present invention provides a biomaterial, which comprises: (i) a nucleic acid molecule encoding the antibody or its antigen-binding fragment of the present invention; (ii) a vector containing the nucleic acid molecule of (i); and / or (iii) A host cell that comprises (i) the nucleic acid molecule described above and / or (ii) the vector described above, or the host cell is transformed or transfected with (i) the nucleic acid molecule described above and / or (ii) the vector described above.

[0024] In some embodiments, the nucleic acid molecule may be an isolated nucleic acid molecule.

[0025] The nucleic acid molecule of the present invention can be cloned into a vector and then used to transform or transfect a host cell. Accordingly, the present invention also provides a vector that comprises (i) the nucleic acid molecule described above. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, and the like.

[0026] The vector or nucleic acid molecule of the present invention can be used to transform or transfect a host cell for purposes such as preservation or antibody expression. Accordingly, the present invention also provides a host cell that comprises (ii) the vector described above or (i) the nucleic acid molecule described above, or the host cell is transformed or transfected with the nucleic acid molecule and / or vector of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, 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 preparing an antibody or an antigen-binding fragment thereof.

[0027] The antibody or antigen-binding fragment 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 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 with any other optional domains of the antibody to form 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 the host cell described herein under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0028] In a fourth aspect, the present invention provides a conjugate that comprises the antibody or antigen-binding fragment thereof of the present invention and a conjugate moiety, the conjugate moiety being another molecule; preferably, the conjugate moiety is a cytotoxic agent, an immunomodulator, an imaging agent, a fluorescent protein, a molecular marker, a therapeutic protein, a biopolymer, or an oligonucleotide, and the like.

[0029] Fifth aspect, the present invention provides an antibody-drug conjugate (ADC) comprising the antibody or antigen-binding fragment thereof described in the present invention, at least one therapeutic active substance or pharmaceutically active ingredient, and an optional linker; the antibody-drug conjugate has the structure shown in formula (I): Ab-(L-D)n Formula (I) Wherein, Ab is the antibody or antigen-binding fragment thereof described in the present invention; L is a linker; D is a therapeutic active substance or pharmaceutically active ingredient; n = 1-8, for example, n = 1, 2, 3, 4, 5, 6, 7 or 8.

[0030] The antibody-drug conjugate provided by the present invention has the following advantages: (1) Bind to target cells expressing human LIV-1 and have high affinity for it; (2) Can enter cells through endocytosis and kill target cells; in some embodiments, the ADC of the present invention has high endocytosis efficiency; (3) Treat and improve diseases related to abnormal function or expression of LIV-1 in a subject (such as cancer, such as ovarian cancer, colon cancer, triple-negative breast cancer, endometrial cancer, peritoneal cancer and lung cancer), or treat and improve one or more symptoms of the disease; (4) Reduce or inhibit tumor growth or progression in a subject (who has a tumor expressing LIV-1); (5) Exert cytotoxic activity in cells expressing LIV-1.

[0031] In one embodiment, n represents the number of drug-linker (L-D) moieties conjugated to a single antibody (Ab), and is preferably an integer from 1 to 8. In this case, the individual ADC conjugate may also be referred to as an ADC compound. In any embodiment 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 conjugated to a single antibody.

[0032] In another embodiment, n represents the average DAR of the prepared antibody-drug conjugate. In this case, n can be, for example, an integer or a decimal within 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.

[0033] IgG1 antibodies have 16 pairs of cysteine residues, which exist in the form of 12 intrachain and 4 interchain disulfide bonds. The interchain disulfide bonds have solvent accessibility and can be reduced by a reducing agent to form eight sulfhydryl groups, which then become the conjugation targets. In one embodiment, the linker is conjugated to the sulfhydryl group of cysteine on the anti-LIV-1 antibody.

[0034] In a specific embodiment, the therapeutic active substance or drug active ingredient is a cytotoxin, a phytotoxin, a small molecule toxin, a radioisotope, a maytansine alkaloid, etc. In a 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 therapeutic active substance or drug active ingredient is MMAE or DXD.

[0035] In one embodiment, the cytotoxin is covalently linked to the anti-LIV-1 antibody or its antigen-binding fragment directly or via a linker in a non-site-specific or site-specific manner.

[0036] In one embodiment, the linker is a protease-cleavable peptide linker. In a specific embodiment, the peptide linker contains a protease-cleavable peptide fragment. In a specific embodiment, the peptide linker may further contain, for example, a maleimide conjugated to an antibody cysteine residue.

[0037] In one embodiment, linker L and active substance D form Deruxtecan.

[0038] In a specific embodiment, the antibody-drug conjugate has the following structure: or

[0039] wherein, n = 1 - 8.

[0040] The antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate and / or antibody-drug conjugate provided by the present invention can be included in a pharmaceutical composition, and more particularly in a pharmaceutical formulation, so as to be 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 biological material of the present invention (including nucleic acid molecule, vector, host cell), conjugate, antibody-drug conjugate, and optionally a pharmaceutically acceptable carrier. In some embodiments of the present invention, the pharmaceutical composition further comprises a second therapeutic agent and / or optionally pharmaceutical excipients, and the second therapeutic agent is selected from cytokines, antibodies, chemotherapeutic agents, and small molecule drugs.

[0041] In a seventh aspect, the present invention provides the use of an anti-LIV-1 antibody or antigen-binding fragment thereof, or the biological material of the present invention (including nucleic acid molecule, vector, host cell), conjugate, antibody-drug conjugate or pharmaceutical composition in the preparation of any of the following products: (a) a product for detecting LIV-1; (b) a product for stimulating or enhancing an immune response; (c) a product for preventing and / or treating diseases associated with abnormal expression of LIV-1; (d) a product for killing cells expressing LIV-1 or inhibiting the growth of cells expressing LIV-1 in vitro or in vivo.

[0042] In one embodiment, the disease associated with abnormal expression of LIV-1 is a cancer with high expression of LIV-1, such as ovarian cancer, colon cancer, triple-negative breast cancer, endometrial cancer, peritoneal cancer, and lung cancer.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention, the antibody-drug conjugate of the present invention, biological material (including nucleic acid molecule, vector, host cell) and / or the pharmaceutical composition 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 uses described herein, such as for treating the related diseases or disorders mentioned herein.

[0044] In some embodiments, in (d), it includes the step of contacting the cells with an effective amount of the antibody or antigen-binding fragment thereof of the present invention, the antibody-drug conjugate of the present invention, biological material (including nucleic acid molecule, vector, host cell) and / or the pharmaceutical composition of the present invention.

[0045] 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, the method comprising administering to the subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of the present invention or the biological material of the present invention (including nucleic acid molecules, vectors, host cells), antibody-drug conjugate or pharmaceutical composition, etc. The disease associated with abnormal LIV-1 expression is a cancer with high LIV-1 expression, 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.

[0046] In a ninth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof of the present invention or the biological material of the present invention (including nucleic acid molecules, vectors, host cells) or pharmaceutical composition in the preparation of an antibody-drug conjugate for treating cancer.

[0047] In a tenth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof of the present invention or the biological material of the present invention (including nucleic acid molecules, vectors, host cells), antibody-drug conjugate or pharmaceutical composition in the preparation of a drug for treating cancer.

[0048] In some embodiments, the cancer is a cancer with high LIV-1 expression, such as ovarian cancer, colon cancer, triple-negative breast cancer, endometrial cancer, peritoneal cancer and lung cancer.

[0049] In an eleventh aspect, the present invention provides a method capable of blocking the binding of LIV-1 to its receptor and inhibiting downstream signal transduction, which comprises administering to a subject an effective amount of the antibody or antigen-binding fragment thereof disclosed in the present invention or the biological material of the present invention (including nucleic acid molecules, vectors, host cells), conjugate, antibody-drug conjugate or pharmaceutical composition.

[0050] In a twelfth aspect, the present invention provides a kit, which comprises the antibody or antigen-binding fragment thereof of the present invention, conjugate or antibody-drug conjugate.

[0051] In a thirteenth aspect, the present invention provides a method for detecting the presence or level of LIV-1 in a sample, which comprises the steps of contacting the antibody or antigen-binding fragment thereof or conjugate of the present invention with the sample and detecting whether the antibody or antigen-binding fragment thereof or conjugate forms a complex with LIV-1.

[0052] The present invention is further illustrated in the following drawings and specific embodiments. However, these drawings and specific embodiments should not be considered as limiting the scope of the present invention, and changes that are easily conceivable by those skilled in the art will be included within the spirit of the present invention and the scope of protection of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Shows the binding activity of anti-LIV-1 hybridoma antibody to LIV1 protein.

[0054] Figure 2 Shows the binding activity of anti-LIV-1 hybridoma antibody to 293T-LIV1 cells.

[0055] Figure 3 Shows the binding activity of anti-LIV-1 hybridoma antibody to cynomolgus monkey LIV1 protein. Figure 4A Shows the binding activity of anti-LIV-1 chimeric antibody to SKOV3 cells; Figure 4B Shows the binding activity of anti-LIV-1 chimeric antibody to HCT116 cells.

[0056] Figure 5 Shows the cytotoxic activity of anti-LIV-1 chimeric antibody against 293T-LIV1 cells using the secondary antibody method. Figure 6 Shows the binding activity of anti-LIV-1 humanized antibody to LIV1 protein.

[0057] Figure 7 Shows the killing activity of anti-LIV-1 humanized antibody against 293T-LIV1 cells using the secondary antibody method. Figure 8 Shows the binding activity of anti-LIV-1 humanized antibody ADC to 293T-LIV1 cells.

[0058] Figure 9 Shows the killing activity of anti-LIV-1 humanized antibody MMAE conjugate against 293T-LIV1 cells. Detailed implementation methods

[0059] 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, the drawings, and the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

[0060] Definitions Unless otherwise specified, the implementation of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are all within the scope of those skilled in the art.

[0061] The term "about", when used in conjunction with a numerical value, means a numerical value that encompasses 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.

[0062] The term "and / or" shall be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0063] As used herein, the term "comprising" or "including" means including the recited elements, integers or steps, but not excluding any other elements, integers or steps. In the present application, when the term "comprising" or "including" is used, unless otherwise specified, the case consisting of the recited elements, integers or steps is also encompassed.

[0064] The term "antibody" is used herein in the broadest sense to refer to a protein containing an antigen-binding site that is capable of specifically recognizing and binding to an antigen. An antibody can be a whole antibody and any antigen-binding fragment thereof or its single chain, and thus the term "antibody" includes any protein or polypeptide that contains at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen in the molecule.

[0065] The term "immunoglobulin" refers to a protein having the structure of a naturally occurring antibody and can generally be used interchangeably with the term "antibody" in the present application. An immunoglobulin of the IgG class is a heterotetrameric glycoprotein composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also referred to as a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also referred to as a light chain variable domain, followed by a light chain constant domain (CL). In an IgG molecule, generally VH-CH1 of the heavy chain pairs with VL-CL of the light chain to form a Fab fragment that specifically binds to an antigen. Thus, an IgG immunoglobulin is essentially composed of two Fab molecules and two dimerized Fc regions linked by an immunoglobulin hinge region. The heavy chain of an immunoglobulin can be assigned to one of 5 classes based on the type of its constant region, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and some of these classes can be further divided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an immunoglobulin can also be divided into one of two types based on the amino acid sequence of its constant domain, called κ and λ.

[0066] The term "antibody fragment" refers to a molecule that is different from a full antibody, which contains a part of the full antibody and is capable of binding to the antigen to which the full antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’) 2 , diabodies, linear antibodies, single-chain antibodies (such as scFv), single-domain antibodies, bivalent or bispecific antibodies or fragments thereof, camelid antibodies (heavy-chain antibodies), and multispecific antibodies formed from antibody fragments.

[0067] The terms "antigen-binding site" and "antigen-binding domain" are used interchangeably and refer to the region in 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 (such as scFv), VHH, and the like.

[0068] The "variable region" or "variable domain" of an antibody refers to the domain of the heavy or light chain of the antibody that participates in antibody-antigen binding. The variable region of an antibody can be further divided into hypervariable regions (i.e., complementarity-determining regions (CDRs)) and relatively conserved regions (i.e., framework regions (FRs)) interspersed between the hypervariable regions. In the case of IgG-class immunoglobulins, the heavy-chain variable region or the light-chain variable region sequentially includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. In the case of heavy-chain antibodies (also referred to herein as nanobodies), such as those from camelid heavy-chain antibodies, the antigen-binding site consists of a single VH domain (i.e., the "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, i.e., it contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs).

[0069] The "complementarity-determining region" or "CDR region" or "CDR" or "hypervariable region" is the region in the variable domain of an antibody that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen-contact residues ("antigen-contact points"). CDRs are mainly responsible for binding to antigen epitopes. The CDRs in the variable domain are usually referred to as CDR1, CDR2, and CDR3 and are numbered sequentially from the N-terminus.

[0070] There are various well-known schemes in the art for determining the CDR sequences in a given VH or VL amino acid sequence: Kabat complementarity-determining regions (CDRs) are determined based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), while Chothia 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), AbM CDRs are a compromise between Kabat CDRs and Chothia structural loops and are used by the AbM antibody modeling software of Oxford Molecular, and "Contact" CDRs are based on the analysis of available complex crystal structures. The residues of each of these CDRs are described below according to different CDR determination schemes.

[0071]

[0072] When referring to an antibody defined by the specific CDR sequences defined by the present invention, the scope of the antibody also encompasses such antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined by the present invention due to the application of different schemes (such as different assignment system rules or combinations).

[0073] The CDRs of the antibodies of the present invention can be artificially evaluated and determined for their boundaries according to any scheme or combination thereof in the art. Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses the CDR sequences determined in any of the above ways.

[0074] "Heavy chain constant region domain" or "heavy chain constant region" refers to the constant region domain derived from or obtained from or derived from the heavy chain of an immunoglobulin, including the heavy chain constant regions CH1, CH2, CH3, and optionally the heavy chain constant region CH4 covalently linked in sequence from the N-terminus to the C-terminus. In most cases, the heavy chain constant regions CH1 and CH2 are connected by a heavy chain hinge region, but when appropriate, they can also be connected by a flexible linker.

[0075] The term "EC 50 ", also known as "median effective concentration", refers to the concentration of a drug, antibody, or agent that induces a 50% response between the baseline and the maximum after a specific exposure time.

[0076] As used herein, the terms "bind" or "specifically bind" mean 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.

[0077] "Affinity" or "binding affinity" refers to the intrinsic binding ability that reflects the interaction between the members of a binding pair. The affinity of molecule X for its binding partner Y can be represented by the equilibrium dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (k dis and k on respectively). Binding affinity can be measured by common methods known in the art.

[0078] 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 more 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.

[0079] Amino acid mutations can be amino acid substitutions, deletions, insertions, and / or additions. In some embodiments, the amino acid mutations are substitutions of one or more amino acids, such as a single amino acid substitution or a combination of multiple amino acid substitutions. Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxyl termini of the polypeptide sequence, as well as deletions and insertions within the polypeptide sequence. The amino acid substitutions of the present invention optionally include conservative substitutions of amino acids.

[0080] The "percent identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the specific amino acid sequence shown in this specification after aligning the candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps if necessary to achieve the maximum percent sequence identity, and without 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 relative to the antibody molecules and their sequences specifically disclosed herein, such as an identity of at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or higher. The variants may comprise conservative modifications.

[0081] For a polypeptide sequence, "conservative modification" includes substitution, deletion, or addition to the polypeptide sequence that results in the replacement of an amino acid with a chemically similar amino acid. Tables of conservative substitutions providing functionally similar amino acids are well known in the art. Variants of such conservative modifications are additional to and do not exclude polymorphic variants, interspecies homologs, and alleles of the present invention. The following 8 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 modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence.

[0082] As used herein, the term "therapeutic agent" encompasses any substance that is 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).

[0083] The term "antibody-drug conjugate" or "ADC" refers to an antibody or antibody fragment conjugated covalently 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 can be a cytotoxin capable of killing the cells (preferably cancer cells) targeted by the ADC. Covalent linkage of the therapeutically active substance, active pharmaceutical ingredient, or cytotoxin can be carried out in a non-site-specific manner using a linker, or in a site-specific manner.

[0084] The term "site-specific conjugation" refers to a mode of linkage that specifically attaches a therapeutically active substance or active pharmaceutical ingredient to a specific site on an antibody. In one embodiment, the conjugation is accomplished with the aid of a linker.

[0085] The term "cytotoxic agent" can be used interchangeably with "cytotoxin" and refers, in the context of the present invention, to a substance that inhibits or disrupts cell function and / or causes cell death or destruction. In one embodiment, cytotoxic agents can include, but are not limited to, bacterial toxins, plant toxins, small molecule toxins, radioisotopes, etc.

[0086] Any antibody-drug conjugate of the present invention can be prepared by conjugating dolastatin and its auristatin derivatives with an antibody. Dolastatin and its auristatin derivatives are important cytotoxins used in antibody-drug conjugates (ADCs), which interfere with microtubule dynamics, cell division, etc., and have anti-tumor and anti-fungal 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 (such as the auristatins described in U.S. Publication No. 20130129753), etc.

[0087] Monomethyl auristatin (MMAE), that is, desmethyl-auristatin E, is a well-known member of the auristatin compound family, and its structural formula is as follows:

[0088] MMAE is conjugated with a monoclonal antibody through a linker to form an ADC. Generally, the linker is cleaved after the ADC enters the tumor cells, thereby releasing MMAE to exert its cytotoxic effect and kill the tumor cells.

[0089] DXD, as a derivative of Exatecan, is an effective DNA topoisomerase I and is widely used in the ADC field. It is linked to the cysteine residue on the antibody through a cleavable tetrapeptide linker (GGFG) by means of maleimide. Currently, Deruxtecan composed of maleimide-GGFG-DXD is commercially available as an ADC drug linker and has the following structure:

[0090] The terms "linker" and "connector" can be used interchangeably in this application and refer to a chemical module that covalently links an antibody to a therapeutically active substance or an active pharmaceutical ingredient in an ADC. In one embodiment, the linker can include amino acid residues that link an antigen to a payload. The amino acid residues can form dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide units. The amino acid residues include those that occur naturally as well as non-naturally occurring amino acid analogs, such as citrulline or β-amino acids, such as β-alanine, or ω-amino acids such as 4-aminobutyric acid.

[0091] Classified according to properties, the linkers applicable to the present invention can be protease-degradable linkers, non-cleavable linkers, acid-sensitive linkers, linkers with silicone grease structures, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared-sensitive linkers, ultraviolet-sensitive linkers, etc.

[0092] 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 can conjugate the antibody of the present invention with a drug.

[0093] The term "payload" or "drug payload" or "active payload" refers to the average number of active payloads per antibody in the ADC molecule (in this article, "payload" can be used interchangeably with "therapeutic active substance or active pharmaceutical ingredient"). The range of the drug payload can be 1-20 therapeutic active substances or active pharmaceutical ingredients per antibody.

[0094] The term "drug / antibody ratio" or "DAR" refers to the ratio of the therapeutic active substance or active pharmaceutical ingredient (D) conjugated to the antibody to the antibody. The ADCs described herein typically have a DAR of 1-8, and in certain specific 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, usually represented as the letter D or a combination of DAR and a number, where the number represents the 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, that is, the overall ratio of the small molecule drug moiety (D) conjugated to the Ab moiety described herein in the product measured by a detection method (such as by conventional methods such as UV / visible light spectroscopy, mass spectrometry, ELISA assay, electrophoresis, and / or HPLC) to the Ab moiety.

[0095] The DAR may be limited by the number of conjugation sites on the antibody. For example, in the case where the conjugation 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 can be conjugated).

[0096] In some embodiments, the average DAR value of the conjugate of the present invention is 0 to 8, such as 3.0-4.0, 4.0-5.0, 7.0-8.0, for example, a range with two of these values as endpoints.

[0097] The terms "individual" or "subject" are used interchangeably and refer to a mammal. 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.

[0098] The term "treatment" refers to a clinical intervention that is intended to alter the natural course of a disease in an individual who is being treated. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and relieving or improving the prognosis. In some embodiments, the antibody molecules of the present invention are used to delay the development of a disease or to slow the progression of a disease.

[0099] The term "prevention" includes the inhibition of the occurrence or development of a disease or disorder or the symptoms of a particular disease or disorder. In some embodiments, a subject having a disease associated with abnormal LIV-1 expression is a candidate for a preventive regimen.

[0100] The term "effective amount" refers to such an amount or dose of an antibody or composition of the present invention that, when administered to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. The effective amount can be readily determined by an attending physician, who is a person skilled in the art, by considering a variety of factors such as the species of mammal; body weight, age, and general health; the specific disease involved; the degree or severity of the disease; the response of the individual patient; the specific antibody being administered; the mode of administration; the bioavailability characteristics of the administered formulation; the dosing regimen selected; and the use of any concomitant therapies.

[0101] The term "therapeutically effective amount" refers to an amount that effectively achieves the desired therapeutic result at the required dose and for the required period of time. The therapeutically effective amount of an antibody or antibody fragment or composition can vary depending on a variety of factors such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or antibody moiety to elicit the desired response in the individual. The therapeutically effective amount is also an amount in which any toxic or harmful effects of the antibody or antibody fragment or composition are less than the therapeutic beneficial effects. Relative to an untreated subject, the "therapeutically effective amount" preferably inhibits 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%.

[0102] The term "preventively effective amount" refers to an amount that effectively achieves the desired preventive result at the required dose and for the required period of time. Generally, since preventive doses are used in a subject before or at an earlier stage of a disease, the preventively effective amount will be less than the therapeutically effective amount.

[0103] The term "pharmaceutical composition" refers to a composition that exists 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 aids, etc.

[0104] Biomaterials (nucleic acid molecules, vectors, and host cells) The present invention provides nucleic acid molecules encoding any of the above 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 by genetic engineering methods using methods well known in the art. In addition, the polynucleotides and nucleic acids of the present invention may contain a segment encoding a secretion signal peptide and be operably linked to the segment encoding the antibody molecule or antigen-binding fragment thereof of the present invention, so as to direct the secretory expression of the antibody molecule or antigen-binding fragment thereof of the present invention.

[0105] The present invention also provides vectors containing the nucleic acid molecules of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector and a prokaryotic expression vector. An "expression vector" refers to a vector containing a recombinant polynucleotide that contains expression control sequences operably linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression can 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) incorporated into the recombinant polynucleotide.

[0106] The present invention further provides prokaryotic and eukaryotic host cells containing the nucleic acid molecule or the vector. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom. A host cell is any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells, and prokaryotic cells, such as Escherichia coli cells. Host cells include cultured cells and also cells within transgenic animals, transgenic plants, or cultured plant or animal tissues. Host cells suitable for replicating and supporting the expression of the antibody molecules or antigen-binding fragments thereof of the present invention are well known in the art. Such cells can be transfected or transduced with a specific expression vector, and a large number of vector-containing cells can be grown for inoculating a large-scale fermenter to obtain a sufficient amount of antibody molecules.

[0107] Compositions and Pharmaceutical Preparations The present invention provides a composition comprising the antibody molecule of the present invention or an antigen-binding fragment thereof. Preferably, the composition is a pharmaceutical composition.

[0108] In one embodiment, the composition of the present invention further comprises a pharmaceutically acceptable excipient, such as a pharmaceutically acceptable carrier and a pharmaceutically acceptable excipient known in the art. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises an anti-LIV-1 antibody of the present invention or an ADC molecule thereof, and a combination of one or more other therapeutic agents.

[0109] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, absorption delaying agents, and the like that are physiologically compatible.

[0110] For the use and application of pharmaceutically acceptable excipients, see also "Handbook of Pharmaceutical Excipients", Eighth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

[0111] The compositions of the present invention can be in various forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as powders or suspensions, liquid solutions (e.g., injectable solutions and infusible solutions), liposome formulations and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0112] The route of administration of the composition of the present invention is according to known methods, for example, oral, intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional routes; through sustained release systems or through implantable devices. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by implantable devices.

[0113] The subject can be a mammal, for example, a primate, for example, a human (e.g., an individual suffering from a disease described herein or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from a disease described herein (e.g., migraine) or is at risk of suffering from a disease described herein. In certain embodiments, the subject has received or has previously received other treatments.

[0114] A drug comprising the antibody described herein can be prepared by mixing an anti-LIV-1 antibody of the present invention or an ADC molecule thereof having the desired purity with one or more optional pharmaceutically acceptable excipients, preferably in the form of a lyophilized preparation or an aqueous solution.

[0115] The pharmaceutical composition or formulation of the present invention may also comprise more than one active ingredient which is required for the particular indication being treated, preferably those active ingredients having complementary activities which 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.

[0116] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, for example films or microcapsules.

[0117] Preparation of the antibodies and antibody-drug conjugates of the present invention In one embodiment, the present invention provides a method for preparing an anti-LIV-1 antibody, which method comprises culturing a host cell comprising a nucleic acid encoding an anti-LIV-1 antibody or an expression vector comprising said nucleic acid under conditions suitable for expressing 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).

[0118] To recombinantly produce the anti-LIV-1 antibody of the present invention, first the nucleic acid encoding the anti-LIV-1 antibody of the present invention is isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures, for example by using oligonucleotide probes capable of specifically binding to the nucleic acid encoding the anti-LIV-1 antibody of the present invention.

[0119] The anti-LIV-1 antibody of the present invention prepared as described herein can be purified by known prior art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these are obvious to those skilled in the art. The purity of the anti-LIV-1 antibody 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, etc.

[0120] The generation of antibody-drug conjugates can be accomplished by any technique known to those skilled in the art. In some aspects, the conjugation of the drug-linker to the antibody is accomplished by reacting with the amino acid residues of the antibody. In some embodiments, a heteroaryl linker L with a leaving group is applied to conjugate the drug D to the cysteine residue of the antibody to prepare the conjugate of formula (I) of the present invention. In some embodiments, the interchain disulfide bonds of the antibody can be disrupted by controlling the conditions of treating the antibody with a reducing agent such as tris(2-hydroxyethyl)phosphine (TCEP) to expose free thiol groups for conjugation with the heteroaryl linker-drug. For IgG1-type antibodies, up to four linked disulfide bonds can be reduced, thereby generating up to 8 reactive thiol groups for conjugation. The conjugates prepared by this method can contain zero, one, two, three, four, five, six, seven, or eight drugs in each antibody molecule.

[0121] When the conjugate prepared is a composition of conjugates with different drug conjugation sites and / or numbers, 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 drugs per antibody of the antibody-drug conjugate composition produced can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. In some other embodiments, the quantitative distribution of the antibody-drug conjugate represented by n can also be determined. Separation, purification, and characterization of homogeneous antibody-drug conjugates with n being a certain value from antibody-drug conjugates with other drug loadings can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0122] Examples The following examples further illustrate the present invention. However, it should be understood that the examples are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art.

[0123] Unless otherwise specified, the implementation of the present invention will employ conventional methods in the fields of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology. The experimental methods mentioned, unless otherwise specified, are all conventional methods in the art using default parameters, steps, etc.; the experimental materials used, unless otherwise specified, are all commercially available products. For those not specified in the examples regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the corresponding product specifications. For those reagents or instruments not specified regarding the manufacturer, they are all conventional products that can be obtained through regular channels.

[0124] Example 1: Preparation of a mouse hybridoma producing an anti-human LIV-1 antibody 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: Animal immunization: The LIV-1-Fc protein was used as the immunogen and diluted to 1 mg / ml and mixed with an equal volume of CFA (Sigma, F5881) or IFA (Sigma, F5506) (CFA was used for the primary immunization, and IFA was used for subsequent immunizations). Female Balb / c mice were immunized with the protein. The primary immunization was 50 μg / mouse, and subsequent immunizations were all 25 μg / mouse. Immunization was repeated more than 3 times at 2-week intervals. 14 days after the last immunization, a booster immunization was performed by intraperitoneal injection of LIV-1 protein. 3 days later, the spleens of the mice were taken for cell fusion.

[0125] 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 + ) and cultured in a 96-well culture plate with HAT (Sigma-Aldrich, catalog number: H0262) medium. After 10 days, the antibody screening of the hybridoma cell supernatant was carried out.

[0126] Screening of hybridoma positive clones: The culture cell supernatants were collected and subjected to ELISA or FACS binding experiments. The positive wells of the cell culture supernatants were selected and cloned in a 96-well culture plate with HT (Sigma-Aldrich, catalog number: H0137) medium. After 7-10 days, visible clones appeared. The supernatant was taken for antibody detection; observed under an inverted microscope, the positive wells with only single clone growth were selected, expanded and cryopreserved. After culturing with serum-free medium (SFM, Thermo, 12045076), the supernatant was collected and purified for subsequent detection.

[0127] ELISA binding experiment: 50 μL of hybridoma supernatant, positive control (BR2-22a, Shanghai Hongcheng) and negative control (mIgG1, Shanghai Hongcheng) were added to each well of a 96-well flat-bottom detection plate (Corning, 9018) coated with human LIV-1 protein or cynomolgus monkey LIV-1 protein. Incubated at 37°C for 60 minutes, washed three times with PBST, and secondary antibody (Anti-Mouse IgG, Sigma, A0168) was added and incubated at 37°C for 30 minutes. Washed three times with PBST, 100 μL of TMB (Yingchuang Bio, EL0009) was added to each well, and the reaction was terminated by adding 50 μL of sulfuric acid after 15 minutes of color development. The OD value was read on an enzyme-linked immunosorbent assay (ELISA) reader, and the results are shown in Table 1.

[0128] FACS binding assay: Human LIV-1 293T cells were added to a 96-well plate (Corning, 3799), and then the hybridoma cell supernatant, positive control (BR2-22a, Shanghai Hongcheng), and negative control (mIgG1, Shanghai Hongcheng) were added to the 96-well plate containing the above four types of cells, respectively, and incubated at 4 °C for about 1 hour. After washing three times with FACS buffer, the cells were resuspended in 100 μL of secondary antibody (Invitrogen, B118301) dilution per well and incubated at 4 °C for about 1 hour. After washing three times with FACS buffer, the cells were resuspended in 100 μL of FACS buffer per well, and the mean fluorescence intensity (Median Fluorescence Intensity, MFI) was read using a flow cytometer (BECKMAN COULTER cytoFLEX or BD FACSCelesta™).

[0129] Hybridoma subcloning (limiting dilution method): The cells in the culture well of the hybridoma positive clone with binding activity to human LIV-1 overexpressing 293T cells and the extracellular domain protein of LIV-1 were mixed and aspirated into a centrifuge tube. An appropriate amount of medium was added, and after mixing, a small amount of cells was aspirated for counting. According to the counting results, the hybridoma cells were diluted to 5 cells per mL. 0.2 mL of the above cell suspension was added to each well of a 96-well plate. After culturing for one week, the culture supernatant containing a single cell colony was selected and detected again according to the above method.

[0130] Example 2: Preparation and identification of murine monoclonal antibody against human LIV-1 After culturing the hybridoma cells of the positive subclone in serum-free medium for 10 days, the supernatant was collected, and the murine monoclonal antibody was purified using a Protein A column (Bogelong (Shanghai) Biotechnology Co., Ltd., product number: AA0272). The binding activity of the anti-human LIV-1 antibody was detected by ELISA and flow cytometry.

[0131] 1. Binding activity of anti-human LIV-1 antibody to human LIV-1 Experiment on the binding of anti-LIV-1 antibody to human LIV-1 extracellular domain protein: The protein coated on the plate is human LIV-1 extracellular domain protein or cynomolgus monkey LIV-1 extracellular domain protein, 1 μL / ml, 50 μL / well, and incubated overnight at 4°C; incubated with 2% BSA at 150 μL / well for 1 hour at room temperature; washed three times with 200 μL / well PBST; prepared a mother liquor of 300 μL and 10 μg / ml for the sample or positive antibody; the dilution factor is 3.16 (100 μL mother liquor + 216 μL blocking solution), with a total of 11 gradients; 50 μL / well of the sample or positive antibody, and an equal volume of mIgG1 was added to column H12 as a negative control, and incubated at room temperature for 1 hour; washed three times with 200 μL / well PBST; added the secondary antibody (Anti-Mouse IgG, Sigma, A0168) to the hybridoma sample and incubated at 37°C for 30 minutes. Washed three times with PBST, added 100 μL of TMB (Yingchuang Biotech, EL0009) to each well, and terminated the reaction by adding 50 μL of sulfuric acid to each well after 15 minutes of color development. Read the OD450 value on an enzyme-linked immunosorbent assay reader. Analyze the experimental data using Graphpad Prism 8.0 software. With the logarithm of the antibody concentration as the x-axis and the corresponding OD450 value as the y-axis, select a four-parameter equation regression model to fit the antibody dose-effect curve and calculate the EC 50 。

[0132] The results are shown in Table 1, Figure 1 and Figure 2 as follows.

[0133] Table 1 Binding activity of anti-human LIV-1 hybridoma antibodies NT: No binding.

[0134]

[0135] 2. Binding activity of anti-human LIV-1 antibody to human 293T LIV-1 cells Adjust the cell concentration of human LIV-1-expressing 293T cells to 2×10 6Cells / mL, 50 μL / well were placed in a 96-well U-bottom plate, and the supernatant was discarded after centrifugation. 100 μL / well was added to the 96-well U-bottom plate, and the supernatant was discarded after centrifugation. The anti-LIV-1 antibody, control antibody (BR2-22a, Shanghai Hongcheng) were diluted to the initial working concentration with FACS buffer (PBS solution containing 1% FBS), and then serially diluted with FACS buffer solution. 100 μL of the antibody concentration gradient dilution solution diluted with FACS buffer (PBS solution containing 1% FBS) was added to each well and incubated at 4°C for about 1 hour. Washed three times with FACS buffer, 100 μL of the secondary antibody (Invitrogen, catalog number: B118301) diluted with FACS buffer was added to each well and incubated at 4°C for about 1 hour. 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 was 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 equation regression model was selected to fit the antibody dose-effect curve and calculate the EC 50 , and the results are shown in Table 1 and Figure 3 as follows.

[0136] As shown in Table 1, according to the ELISA binding and FACS binding results, monoclonal 161E5B8 was sequenced.

[0137] Example 3: Sequencing of anti-LIV-1 antibody and preparation of chimeric antibody and its functional identification 1. Sequencing, expression and purification of anti-LIV-1 antibody The above hybridoma positive clones were sequenced, and the sequences are shown in Table 2. The light and heavy chain variable regions obtained by sequencing (Table 2) were constructed onto the human constant regions (IgG1 / K, Table 3) to construct the corresponding chimeric antibodies, and the sequences were verified by sequencing. The chimeric antibodies were named by adding the prefix ch to the corresponding hybridoma clone number. For example, the chimeric antibody obtained by using hybridoma clone 161E5B8 through this example was named ch161E5B8 and was used for in vitro functional identification or in vivo pharmacodynamic studies.

[0138] Table 2 CDR sequences and their numbers of anti-LIV-1 hybridoma antibodies (determined according to the Kabat scheme)

[0139] Table 3 Constant region sequences of chimeric antibodies and humanized antibodies

[0140] The corresponding nucleic acids encoding the antibodies were transfected into Expi293 cells (Gibco, catalog number: A14635) for antibody expression, and purified using a Protein A column. The specific method is as follows: Expi293 cells (Gibco, catalog number: A14635) at a density of 1.5×10 6 cells / mL were cultured in a shaker at 37°C and 8% CO 2 at 120 rpm. The cell density and viability were measured the next day. The density should be 3×10 6 cells / mL and the viability should be greater than 95%. The plasmid containing the coding nucleic acid was diluted with OPM-293 CD05 Medium (OPM, catalog number: 81075-001) at a total plasmid amount of 1 μg / mL. The volume of the medium for diluting the plasmid was 1 / 20 of the transfection volume, and the light chain to heavy chain ratio was 1:1.5. PEI (1 mg / mL, polysciences, catalog number: 24765-1) was inverted and mixed well, then diluted with OPM-293 CD05 Medium. The volume of the medium for diluting PEI was 1 / 20 of the transfection volume, and it was incubated at room temperature for 5 minutes. The diluted PEI was added to the diluted plasmid and mixed well. After incubating at room temperature for 15 minutes, the PEI / plasmid complex was added to a shake flask containing Expi293 cells (Gibco, catalog number: A14635). After culturing at 37°C, 8% CO 2 and 120 rpm for 24 hours, 10% OPM-293 ProFeed (OPM, catalog number: F081918) was added, and the culture was continued for 5 - 7 days. Then the supernatant was collected.

[0141] The gasket was placed at the bottom of the gravity chromatography column and pressed tightly. The volume of the Protein A (Cytiva, catalog number: 17549801) suspension = target packing volume / suspension ratio of the packing. The packing was vortexed thoroughly and added to the bottom of the gravity chromatography column. PBS was added until the outlet pH reached the target pH. The sample was added to the chromatography column, at least 10 column volumes (CV) of washing buffer was added, and 5 CV of elution buffer (20 mM NaAc, pH 3.0) was added. After incubating for 3 - 5 minutes, the eluate was collected. The pH was adjusted to 7.2 with neutralization buffer (1M Tris), the protein concentration was measured by Nanodrop, and the buffer was exchanged to PBS using a Millipore ultrafiltration centrifugal tube (50 kDa).

[0142] 2. Binding activity of anti-LIV-1 chimeric antibody to human ovarian cancer cell line SK-OV-3 and human colon cancer cell line HCT116 Human ovarian cancer cells SK-OV-3 and human colon cancer cells HCT116 were added to a 96-well U-bottom plate at 100 μL per well, and the supernatant was discarded by centrifugation. Antibody concentration gradient dilutions prepared with 100 μL of FACS buffer (PBS solution containing 1% FBS) were added to each well, and incubated at 4°C for about 1 hour. Washed three times with FACS buffer, and 100 μL of secondary antibody diluted with FACS buffer (Invitrogen, catalog number: A21445) was added to each well, and incubated at 4°C for about 1 hour. 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 a flow cytometer, and the data was analyzed using Graphpad Prism 8.0 software. With the logarithm of the antibody concentration as the x-axis and the corresponding MFI value as the y-axis, a four-parameter equation regression model was selected to fit the antibody dose-effect curve, and the EC50 was calculated.

[0143] The results are shown in Table 4 and Figure 4A and Figure 4B as shown, the chimeric antibody ch161E5B8 has strong binding activity to human ovarian cancer cells SK-OV-3 and human colon cancer cells HCT116, which is superior to PC (BR2-22a-hLIV22).

[0144] Table 4 Binding activity of anti-LIV-1 chimeric antibodies to human LIV-1 (EC 50 , nM)

[0145] N / A: No binding activity or poor binding activity, and no effective EC 50 value was obtained.

[0146] 3. αhFc-CL-MMAE and LIV1 antibody-induced cytotoxicity 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 with MMAE through a cleavable linker. αHFc-CL-MMAE recognizes the Fc end of the anti-human LIV-1 chimeric antibody, causes endocytosis on cells expressing LIV-1, and releases MMAE intracellularly to kill cells. 293T LIV-1 cells were obtained by trypsin digestion, and the cell density was adjusted to 4×10 4cells / mL. Add 25 μL of the antibody concentration gradient dilution solution diluted with the medium (the initial concentration is 20 nM, 4-fold dilution) and 50 μL of the cell suspension into each well of the 96-well plate with a white bottom and transparent walls. After incubating for 10 minutes, add 25 μL of the αHFc-CL-MMAE working solution (Moradec, AH-102AE-50) prepared with the medium into each well, and place the 96-well plate in an incubator at 37 °C with 5% CO₂ for culturing. After 3 days, add 50 μL of CTG (Adamas Life, RA-GL11-A) into each well, read the relative fluorescence units with an enzyme-linked immunosorbent assay (ELISA) reader, fit the RLU with a four-parameter model, and calculate the IC 50 value, and the results are shown in Figure 5 and Table 5.

[0147] Table 5 Cell killing activity of anti-LIV-1 chimeric antibody and αHFc-CL-MMAE conjugate

[0148] Example 4: Humanization of anti-human LIV-1 antibody and expression and purification of humanized antibody Determine the CDRs using the Kabat numbering, select the human germline gene with the highest homology to the murine sequence as the receptor framework, and transplant the CDRs of the murine sequence into the human framework. According to the importance of amino acids, perform back mutations, that is, mutate some key amino acids in the framework region after transplantation back to the corresponding murine amino acids, and design several variants for the heavy and light chains respectively.

[0149] 1. Humanization of anti-LIV-1 antibody 161E5B8 Transplant the HCDR and LCDR of the murine antibody 161E5B8 onto the human germline genes IGHV3-23*04 and IGKV3-20*02 respectively. The obtained sequences are shown in Table 6, and the variable region combinations corresponding to the constructed humanized antibodies are shown in Table 7.

[0150] Table 6 Humanized sequence of anti-LIV-1 antibody 161E5B8

[0151] Table 7 Heavy chain variable region sequence and light chain variable region sequence corresponding to the constructed humanized antibody

[0152] 2. Expression and purification of anti-LIV-1 humanized antibody The light and heavy chain variable regions of the humanized antibody in Table 6 were respectively constructed onto the human constant region (hIgG1 / K, Table 3) according to the combinations in Table 7, followed by gene synthesis. Sequencing confirmed consistency with the designed sequence. Expression was carried out in Expi293 cells (Gibco, catalog number: A14635) using the method in Example 2, and purification was performed using a Protein A column.

[0153] Example 5: Functional identification of anti-LIV-1 humanized antibody The binding activities of the humanized antibodies to human LIV-1 were detected respectively.

[0154] 1. Anti-LIV-1 humanized antibody ELISA binding experiment The binding activities of the anti-LIV-1 humanized antibodies to human LIV-1 were detected using the method disclosed in Example 3. The results of the antibody binding to human and cynomolgus monkey LIV-1 are as Figure 6 shown in Table 8. The humanized antibodies have ELISA binding affinities comparable to the positive control (BR2-22a-hLIV22).

[0155] Table 8 Anti-LIV-1 humanized antibody binding to human LIV-1 ELISA EC 50

[0156] 2. Anti-LIV-1 humanized antibody and αhFc-CL-MMAE induced cytotoxicity The method in Example 3 was used. αHFc-CL-MMAE (Moradec, AH-102AE) recognizes the Fc end of the anti-human LIV-1 humanized antibody, causes internalization on cells expressing LIV1, and releases MMAE intracellularly to kill cells. 293T LIV-1 cells were obtained by trypsin digestion, and the cell density was adjusted by centrifugation to 4×10 4 cells / mL. To each well of a white-bottom clear 96-well plate, 25 μL of a gradient dilution of the antibody diluted in medium (starting concentration of 20 nM, 4-fold dilution) and 50 μL of the cell suspension were added. After incubating for 10 minutes to induce cytotoxicity, 25 μL of the αHFc-CL-MMAE working solution (Moradec, AH-102AE-50) prepared in medium was added to each well. The 96-well plate was placed in an incubator at 37 °C and 5% CO₂ for 3 days to induce cytotoxicity. Then, 50 μL of CTG (Adamas Life, RA-GL11-A) was added to each well, and 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 50 value of each antibody. The results are as Figure 7 shown in Table 9.

[0157] Table 9 Cell killing activity of anti-LIV-1 humanized antibody and αhFc-CL-MMAE conjugate

[0158] Note: NT indicates not tested.

[0159] Example 6: Preparation of conjugate of anti-human LIV-1 humanized antibody and MMAE 6.1 Conjugate of anti-human LIV-1 humanized antibody and MMAE Add a certain amount of antibody and PBS buffer to make the concentration of the antibody in the reaction mixture about 1 mg / mL. Add a certain amount of EDTA solution to make the EDTA concentration in the mixture about 2 mM. Add 1M Tris salt solution to make the pH of the reaction system between 6.6 - 7.5. Add 2 - 6 equivalents of TCEP for reduction, mix well on a shaker at 37°C, and gently shake for 1 - 3 hours. Then adjust the temperature of the shaker to about 4°C to cool the reaction solution. Add 5 - 10 equivalents of Vc-MMAE (MCE, HY-15575) dissolved in DMSO and react at 4°C for 1 - 2 hours. Finally, add the reaction solution to a 30K ultrafiltration tube, exchange the solution 6 - 8 times with 10 mM histidine hydrochloride solution at pH 6.0, concentrate to an appropriate volume, add to a pre-equilibrated desalting column, and centrifuge to obtain the purified ADC product. Detect the concentration and DAR value, and its DAR value is 4. The structure of the conjugate of anti-human LIV1 antibody and MMAE is shown below.

[0160]

[0161] 6.2 FACS binding experiment of anti-human LIV-1 humanized antibody ADC Add 293T cells expressing human LIV-1 to a 96-well plate (Corning, 3799). Add 100 μL of serially diluted antibody ADC solution prepared with FACS buffer (PBS solution containing 1% FBS) to each well and incubate at 4°C for about 1 hour. Wash three times with FACS buffer. Add 100 μL of secondary antibody (Invitrogen, catalog number: A21445) diluted with FACS buffer to each well and incubate at 4°C for about 1 hour. Wash three times with FACS buffer. Add 100 μL of FACS buffer to resuspend the cells in each well. Read the MFI with a flow cytometer, analyze the data using Graphpad Prism 8.0 software. Use the logarithm of the antibody concentration as the x-axis and the corresponding MFI value as the y-axis, select a four-parameter equation regression model to fit the antibody dose-effect curve, and calculate the EC 50 .

[0162] The results are shown in Table 10 and Figure 8As shown, the LIV-1 humanized antibody ADC has strong binding activity with 293T LIV1, and its binding activity is comparable to that of the control (BR2-22a-hLIV22).

[0163] 6.3 Detection of the cytotoxicity of the ADC of the anti-human LIV-1 humanized antibody The 293T LIV-1 cells were digested with trypsin, and the cell concentration was adjusted to 4×10 4 cells / mL, and 50 μL / well was inoculated into a white-bottom transparent 96-well plate. 50 μL of the antibody concentration gradient dilution solution diluted with the culture medium was added to each well of the 96-well plate, and the 96-well plate was placed in an incubator at 37°C and 5% CO 2 for 3 days. 50 μL of CTG reagent was added to each well, and it was incubated at room temperature in the dark for 5 minutes, and the relative light unit value was read with an enzyme-linked immunosorbent assay 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 equation regression model was selected to fit the dose-effect curve of the antibody. The results are as Figure 9 shown in and Table 10. The ADCs of all the antibodies to be tested have strong cytotoxic activity against 293T LIV-1 cells, and their activity is comparable to that of the control (BR2-22a-hLIV22).

[0164] Table 10 Cytotoxic activity of the MMAE conjugate of the anti-LIV-1 humanized antibody

[0165] Sequence information: Sequence information of the humanized anti-LIV-1 antibody and the positive control antibody BR2-22a-hLIV22 (HGLG)<hLiv1 mAb2 HG; PRT / 1; artificial> (SEQ ID NO:18): QVQLVQSGAEVKKPGASVKVSCKASGLTIEDYYMHWVRQAPGQGLEWMGWIDPENGDTEYGPKFQGRVTMTRDTSINTAYMELSRLRSDDTAVYYCAVHNAHYGTWFAYWGQGTLVTVSS BR2-22a-hLIV22 (HGLG)<hLiv1 mAb2 LG; PRT / 1; artificial> (SEQ ID NO:19): DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWYQQRPGQSPRPLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIKR BR2-22a (murine) VH (SEQ ID NO: 20): EVQLQQSGAELVRSGASVKLSCTASGLNIEDYYMHWVKQRPEQGLEWIGWIDPENGDTEYGPKFQGKATMTADTSSNTAYLQLSSLTSGDTAVYYCTVHNAHYGTWFAYWGQGTLVTVSA BR2-22a (murine) VL (SEQ ID NO: 21): 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 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; (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; (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; (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; (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; (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; (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; (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; (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; (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; (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; (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 (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.

3. The LIV-1-binding antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The antibody is any form of a monoclonal antibody, a single-chain 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, such as Fab, Fab', Fab'-SH, (Fab')2, Fv, scFv, BsFv, dsFv or (dsFv)2 fragment.

4. The LIV-1-binding antibody or antigen-binding fragment thereof according to claim 3, wherein: The antibody or antigen-binding fragment thereof comprises a heavy chain constant region and / or a light chain constant region of human or mouse origin; The antibody or antigen-binding fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ 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.

5. The LIV-1-binding antibody or antigen-binding fragment thereof according to claim 4, 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.

6. A biomaterial comprising: (i) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; (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).

7. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, at least one therapeutically active substance or pharmaceutically active ingredient, and an optional linker; the antibody-drug conjugate has a structure as shown in formula (I): Ab-(LD)n formula (I) in, Ab is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; L is a linker; D is a therapeutically active substance or a pharmaceutically active ingredient; n=1-8。 8. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the biomaterial according to claim 6 and / or the antibody-drug conjugate according to claim 7, and an optional pharmaceutically acceptable carrier.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the biomaterial according to claim 6, the antibody-drug conjugate according to claim 7 and / or the pharmaceutical composition according to claim 8 in the preparation of any of the following products: (a) Products for testing LIV-1; (b) products that stimulate or enhance the immune response; (c) products for preventing and / or treating diseases associated with abnormal expression of LIV-1, including ovarian cancer, colon cancer, triple-negative breast cancer, endometrial cancer, peritoneal cancer and lung cancer; (d) A product that kills cells expressing LIV-1 or inhibits the growth of cells expressing LIV-1 in vitro or in vivo.

10. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the biomaterial according to claim 6, the antibody-drug conjugate according to claim 7 and / or the pharmaceutical composition according to claim 8.

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