Anti-LIV-1 antibody and use thereof

Abstract

Provided are an antibody specifically binding to LIV-1 and a use thereof. Specifically provided are a murine-derived and humanized antibody that binds to LIV-1, a preparation method therefor and a use thereof, wherein the antibody has good affinity for LIV-1 protein, and therefore can be used in the preparation of drugs for treating tumors and the like.

Description

Antibodies against LIV-1 and their uses

[0001] This disclosure claims priority to Chinese patent application No. 202410022063.0, filed with the Patent Office of China on January 5, 2024, entitled “Anti-LIV-1 Antibodies and Uses Thereof,” the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present disclosure relates to the fields of bioengineering and biomedicine, and specifically to an anti-human LVI-1 antibody or an antigen-binding fragment thereof, its encoding nucleic acid, expression vector and expression cell, preparation method, pharmaceutical composition, and their use in treating diseases, such as treating tumors. Background Art

[0003] The zinc transporter LIV-1, also known as SLC39A6 or ZIP6, is encoded by a gene located on chromosome 18q12.2. It is a multi-transmembrane protein with both zinc transporter and metalloprotease activities. LIV-1, a member of the SLC39A family (ZIP family), primarily functions to facilitate the transport of extracellular zinc ions into the cytoplasm, thereby collaborating with other family members to maintain intracellular zinc homeostasis. In tumor tissues, LIV-1 has been shown to interact with the transcription factors STAT3 and Snail, thereby downregulating E-cadherin expression and promoting epithelial-mesenchymal transition (EMT), thereby playing a role in tumor cell trafficking and migration (Trends Endocrinol Metab 2004;15:461-3; Curr Opin Cell Biol 2005;17:548-58).

[0004] LIV-1 is not expressed in most normal tissues and is only positively expressed in the breast, testis, and prostate. Studies have shown that LIV-1 is significantly highly expressed in a variety of tumors, especially in breast cancer, with a positive expression rate of up to 93%. In addition to breast cancer, LIV-1 is also highly expressed in a variety of solid tumors, such as melanoma (82%), prostate cancer (72%), ovarian cancer (48%), uterine cancer, lung cancer, etc. (Molecular Cancer Therapeutics, 2014, 13 (12).). Currently, the ADC drug ladiratuzumab vdotin (SGN-LIV1A) targeting LIV-1 is in phase 2 clinical trials and has been proven to bring clinical benefits to patients with breast cancer, especially triple-negative breast cancer, further verifying that LIV-1 is a potential target for advanced solid tumors. Summary of the Invention

[0005] The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to LIV-1, nucleic acids encoding these antibodies and antigen-binding fragments thereof, pharmaceutical compositions and kits comprising the antibodies and antigen-binding fragments thereof, and the preparation of drugs that can be used to treat tumors, etc.

[0006] In a first aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to LIV-1, comprising: (a) HCDR1, HCDR2 and HCDR3 of the VH of any one of SEQ ID NOs: 11, 12, 13, 38, 45, 46, 47, 53, 54 or 55; and / or, (b) LCDR1, LCDR2 and LCDR3 of the VL of any one of SEQ ID NOs: 14, 15, 16, 35, 36, 37, 44 or 52; preferably, the HCDR1-3 and / or the LCDR1-3 are encoded according to the popular analysis method of Kabat or IMGT.

[0007] In a specific embodiment, the HCDR1, HCDR2 and HCDR3 have any sequence combination selected from the following or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence combination:

[0008] and,

[0009] (2) The LCDR1, LCDR2 and LCDR3 have a sequence combination selected from any of the following sequence combinations or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence combination:

[0010] Preferably, the substitution is a conservative amino acid substitution.

[0011] In another specific embodiment, it comprises a combination of heavy chain CDRs and light chain CDRs selected from the following: VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL2, VH5+VL2, VH6+VL4, VH7+VL4 or VH8+VL4, and a CDRs combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence of the heavy chain and light chain CDRs combination; preferably, the substitutions are substitutions of conservative amino acids.

[0012] In another specific embodiment, the framework regions of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof are derived from a human germline heavy chain template and a human germline light chain template, wherein:

[0013] (1) The framework region sequence is derived from the combined sequence of human germline heavy chains IGHV3-7*01 and IGHJ6*01; it comprises the FR1, FR2, and FR3 regions of IGHV3-7*01 as shown in SEQ ID NO: 42 and the FR4 region of IGHJ6*01 as shown in SEQ ID NO: 43;

[0014] (2) The framework region sequence is derived from the combined sequence of human germline heavy chains IGHV1-69-2*01 and IGHJ6*01; it comprises the FR1, FR2, and FR3 regions of IGHV1-69-2*01 as shown in SEQ ID NO: 49 and the FR4 region of IGHJ6*01 as shown in SEQ ID NO: 43;

[0015] (3) The framework region sequence is derived from the combined sequence of human germline heavy chains IGHV1-3*01 and IGHJ6*01; it comprises the FR1, FR2, and FR3 regions of IGHV1-3*01 as shown in SEQ ID NO: 57 and the FR4 region of IGHJ6*01 as shown in SEQ ID NO: 43;

[0016] (4) The framework region sequence is derived from the combined sequence of human germline light chains IGKV3-11*01 and IGKJ2*01; it comprises the FR1, FR2, and FR3 regions of IGKV3-11*01 as shown in SEQ ID NO: 39 and the FR4 region of IGKJ2*01 as shown in SEQ ID NO: 41;

[0017] (5) The framework region sequence is derived from the combined sequence of human germline light chains IGKV6-21*01 and IGKJ2*01; it comprises the FR1, FR2, and FR3 regions of IGKV6-21*01 as shown in SEQ ID NO: 40 and the FR4 region of IGKJ2*01 as shown in SEQ ID NO: 41;

[0018] (6) The framework region sequence is derived from the combined sequence of human germline light chains IGKV1-NL1*01 and IGKJ2*01; it comprises the FR1, FR2, and FR3 regions of IGKV1-NL1*01 as shown in SEQ ID NO: 48 and the FR4 region of IGKJ2*01 as shown in SEQ ID NO: 41;

[0019] (7) The framework region sequence is derived from the combined sequence of human germline light chains IGKV4-1*01 and IGKJ2*01; it includes the FR1, FR2, and FR3 regions of IGKV4-1*01 shown in SEQ ID NO: 56 and the FR4 region of IGKJ2*01 shown in SEQ ID NO: 41.

[0020] In another specific embodiment, the framework regions of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof further comprise one or more mutations selected from the following group, according to the Kabat or IMGT numbering system:

[0021] (1) The framework region of the heavy chain variable region includes: Q1E, V24T, M34I, W50R, N55Q, G56A, I70L, R72A, A76S, S77N, R98K, T98R or G101A; preferably includes R98K; or preferably includes T98R; or preferably includes V24T, G56A, I70L, T98R; or preferably includes V24T, G56A, I70L, T98R, G101A; or preferably includes Q1E, M34I, W50R, R72A; or preferably includes Q1E, M34I, W50R, G56A, R72A, S77N; or preferably includes Q1E, M34I, W50R, N55Q, R72A, A76S, S77N;

[0022] (2) The framework region of the light chain variable region includes: A40T, A43S, L46P, L47I, L48V, Y49K, V58I or T85M; preferably includes L46P, Y49K; or preferably includes L46P, L47I, V58I; or preferably includes L46P, L47I, V58I, T85M; or preferably includes A43S, L48V; or preferably includes A40T.

[0023] In a specific embodiment, the antibody or antigen-binding fragment comprises:

[0024] (1) the heavy chain variable region has the sequence shown in SEQ ID NO: 11, 12, 13, 38, 45, 46, 47, 53, 54 or 55;

[0025] (2) the light chain variable region has the sequence shown in SEQ ID NO: 14, 15, 16, 35, 36, 37, 44 or 52;

[0026] (3) An amino acid sequence that is at least 90% identical to the sequence of any one of (1) to (2) above, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of any one of (1) to (2) above.

[0027] In another specific embodiment, the heavy chain variable region and the light chain variable region are selected from the following groups:

[0028] (1) having the VH set forth in SEQ ID NO: 11 and the VL set forth in SEQ ID NO: 14;

[0029] (2) having the VH set forth in SEQ ID NO: 12 and the VL set forth in SEQ ID NO: 15;

[0030] (3) having the VH set forth in SEQ ID NO: 13 and the VL set forth in SEQ ID NO: 16;

[0031] (4) having the VH set forth in SEQ ID NO: 38 and the VL set forth in SEQ ID NO: 35;

[0032] (5) having the VH set forth in SEQ ID NO: 38 and the VL set forth in SEQ ID NO: 36;

[0033] (6) having the VH set forth in SEQ ID NO: 38 and the VL set forth in SEQ ID NO: 37;

[0034] (7) having the VH set forth in SEQ ID NO: 45 and the VL set forth in SEQ ID NO: 44;

[0035] (8) having the VH set forth in SEQ ID NO: 46 and the VL set forth in SEQ ID NO: 44;

[0036] (9) having the VH set forth in SEQ ID NO: 47 and the VL set forth in SEQ ID NO: 44;

[0037] (10) having the VH set forth in SEQ ID NO: 53 and the VL set forth in SEQ ID NO: 52;

[0038] (11) having the VH set forth in SEQ ID NO: 54 and the VL set forth in SEQ ID NO: 52;

[0039] (12) having the VH set forth in SEQ ID NO: 55 and the VL set forth in SEQ ID NO: 52;

[0040] (13) A VH and VL combination having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity compared to any of the sequence combinations of (1) to (12) above.

[0041] In a specific embodiment, the antibody or antigen-binding fragment thereof binds to human LIV-1 with a dissociation constant (KD) of no more than 2×10 -9 M.

[0042] In another specific embodiment, the antibody or antigen-binding fragment thereof is:

[0043] (1) Chimeric antibodies or fragments thereof;

[0044] (2) humanized antibodies or fragments thereof;

[0045] (3) fully human antibodies or fragments thereof;

[0046] Preferably, the antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, a diabody or a single domain antibody.

[0047] In a specific embodiment, the antibody comprises the sequence of the constant region of any one of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, it comprises the sequence of the constant region of human or murine antibodies IgG1, IgG2, IgG3 or IgG4.

[0048] In another specific embodiment, the antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibodies, nanobodies and the minimum recognition unit of an antibody.

[0049] In another specific embodiment, the antibody or antigen-binding fragment thereof is further conjugated with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic drug or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent or a photosensitizer.

[0050] In a second aspect, the present disclosure provides a multispecific antigen-binding molecule, comprising a first antigen-binding moiety and a second antigen-binding moiety, wherein the first antigen-binding moiety comprises the antibody or antigen-binding fragment thereof described in the first aspect above, and the second antigen-binding moiety specifically binds to an antigen other than LIV-1 or binds to an LIV-1 antigen epitope different from the LIV-1 antigen epitope bound by the first antigen-binding moiety; preferably, the other antigen is selected from CD3, CD28, CD137, CD134, CD27, ICOS, CD16, CD56, CD335, CD336, CD337, NKG2A, NKG2D, KIR, DNAM-1 or CD161; preferably, the multispecific antigen-binding molecule is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

[0051] In a third aspect, the present disclosure provides a chimeric antigen receptor (CAR), which comprises at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen binding domain comprises the antibody or antigen-binding fragment thereof described in the first aspect above.

[0052] In a fourth aspect, the present disclosure provides an immune effector cell, which comprises the chimeric antigen receptor described in the third aspect above or comprises a nucleic acid fragment encoding the chimeric antigen receptor described in the third aspect above; preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), monocytes, macrophages, dendritic cells or mast cells; the T cells can be selected from inflammatory T cells, cytotoxic T cells, regulatory T cells (Treg) or helper T cells; preferably, the immune effector cell is an allogeneic immune effector cell or an autologous immune cell.

[0053] In a fifth aspect, the present disclosure provides an isolated nucleic acid molecule encoding the antibody, antigen-binding fragment, or any combination thereof described in the first aspect, the multispecific antigen-binding molecule described in the second aspect, or the chimeric antigen receptor described in the third aspect.

[0054] In a sixth aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule of the fifth aspect.

[0055] In the seventh aspect, the present disclosure provides an isolated host cell of the nucleic acid molecule described in the fifth aspect, or the expression vector described in the sixth aspect; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and / or Bacillus subtilis; more preferably, the host cell is selected from Expi293 or CHO cells.

[0056] In the eighth aspect, the present disclosure provides a method for preparing the antibody or antigen-binding fragment thereof described in the first aspect, or the multispecific antigen-binding molecule described in the second aspect, culturing the host cell described in the seventh aspect under appropriate conditions, and isolating the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule.

[0057] In the ninth aspect, the present disclosure provides a method for preparing the immune effector cell described in the fourth aspect, the method comprising introducing a nucleic acid fragment encoding the CAR described in the third aspect into the immune effector cell, and optionally, the method further comprises initiating the immune effector cell to express the CAR described in the third aspect.

[0058] In the tenth aspect, the present disclosure provides a pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof described in the first aspect, the multispecific antigen-binding molecule described in the second aspect, the chimeric antigen receptor described in the third aspect, the immune effector cell described in the fourth aspect, the isolated nucleic acid molecule described in the fifth aspect, the expression vector described in the sixth aspect, the host cell described in the seventh aspect, or the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared by the method described in the eighth aspect, or the immune effector cell prepared by the method described in the ninth aspect; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; preferably, the pharmaceutical composition further comprises an additional anti-tumor agent.

[0059] In the eleventh aspect, the present disclosure provides the use of the antibody or antigen-binding fragment thereof described in the first aspect, the multispecific antigen-binding molecule described in the second aspect, the chimeric antigen receptor described in the third aspect, the immune effector cell described in the fourth aspect, the isolated nucleic acid molecule described in the fifth aspect, the expression vector described in the sixth aspect, the host cell described in the seventh aspect, the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared by the method described in the eighth aspect, the immune effector cell prepared by the method described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect in the preparation of a medicament for preventing and / or treating a tumor disease; preferably, the tumor disease is breast cancer, prostate cancer, cervical cancer, melanoma, ovarian cancer, endometrial cancer, invasive mammary duct carcinoma, non-small cell lung cancer, pancreatic cancer, lung cancer, or squamous cell carcinoma.

[0060] In the twelfth aspect, the present disclosure provides a method for preventing and / or treating a tumor disease, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment thereof described in the first aspect, the multispecific antigen-binding molecule described in the second aspect, the chimeric antigen receptor described in the third aspect, the immune effector cell described in the fourth aspect, the isolated nucleic acid molecule described in the fifth aspect, the expression vector described in the sixth aspect, the host cell described in the seventh aspect, the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared by the method described in the eighth aspect, the immune effector cell prepared by the method described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect; preferably, the tumor disease is breast cancer, prostate cancer, cervical cancer, melanoma, ovarian cancer, endometrial cancer, invasive mammary duct carcinoma, non-small cell lung cancer, pancreatic cancer, lung cancer, or squamous cell carcinoma.

[0061] In the thirteenth aspect, the present disclosure provides the antibody or antigen-binding fragment thereof described in the first aspect, the multispecific antigen-binding molecule described in the second aspect, the chimeric antigen receptor described in the third aspect, the immune effector cell described in the fourth aspect, the isolated nucleic acid molecule described in the fifth aspect, the expression vector described in the sixth aspect, the host cell described in the seventh aspect, the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared by the method described in the eighth aspect, the immune effector cell prepared by the method described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect for preventing and / or treating tumor diseases; preferably, the tumor disease is breast cancer, prostate cancer, cervical cancer, melanoma, ovarian cancer, endometrial cancer, invasive mammary duct carcinoma, non-small cell lung cancer, pancreatic cancer, lung cancer, or squamous cell carcinoma.

[0062] In the fourteenth aspect, the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof described in the first aspect, the multispecific antigen-binding molecule described in the second aspect, the chimeric antigen receptor described in the third aspect, the immune effector cell described in the fourth aspect, the isolated nucleic acid molecule described in the fifth aspect, the expression vector described in the sixth aspect, the host cell described in the seventh aspect, the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared by the method described in the eighth aspect, the immune effector cell prepared by the method described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect; optionally, further comprising instructions for use.

[0063] This disclosure aims to obtain antibody sequences targeting LIV-1 to generate more LIV-1-targeting drug forms. Testing has shown that the LIV-1 antibodies disclosed herein, obtained through screening of cells expressing high LIV-1 protein, exhibit excellent endocytosis, PK and PD activity, and anti-tumor effects, while maintaining low toxicity. These antibodies can be used to prepare ADC molecules, and the prepared ADC molecules exhibit superior binding, endocytosis, plasma stability, and anti-tumor effects compared to positive control agents.

[0064] Definitions and Explanations of Terms

[0065] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with a target antigen, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including, but not limited to, chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies, diabodies, triabodies, and tetrabodies, antibody conjugates), and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments). In addition, unless otherwise indicated, the term "monoclonal antibody" (mAb) is intended to include intact antibody molecules that are capable of specifically binding to a target protein, as well as incomplete antibody fragments (e.g., Fab and F(ab')2 fragments, which lack the Fc fragment of an intact antibody (which is cleared more rapidly from the animal circulation) and therefore lack Fc-mediated effector functions (see Wahl et al., J. Nucl. Med. 24:316, 1983; the contents of which are incorporated herein in their entirety).

[0066] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0067] The term "LIV-1" herein, also referred to as Zip6, refers to a zinc transporter belonging to a subfamily of ZIP zinc transporters known as LZTs. In GeneCard, LIV-1 is also referred to as SLC39A6 (solute carrier protein family 39 (zinc transporter), member 6). Although for the sake of brevity, this disclosure is primarily exemplified by LIV-1, it should be understood that the definitions and embodiments relating to LIV-1 may also be applied to other zinc transporters disclosed herein.

[0068] The term "monospecific" as used herein refers to having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.

[0069] The term "multispecific" herein refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody / antigen-binding molecule can bind.

[0070] "Full-length antibody" and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody.

[0071] As used herein, the term "antigen-binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. An antibody fragment can be a Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb comprising both VH and VL domains; (vi) a dAb fragment consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) isolated complementarity determining regions (CDRs); and (ix) combinations of two or more isolated CDRs, which may optionally be linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, the two domains can be joined using recombinant methods via a linker that enables them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art. (The foregoing is incorporated herein in its entirety).

[0072] As used herein, the terms "single-domain antibody" (sdAb), "VHH," and "nanobody" have the same meaning and are used interchangeably. They refer to a single-domain antibody (sdAb) constructed by cloning the variable region of an antibody heavy chain to construct a single-domain antibody consisting of only a single heavy chain variable region (VH), which is the smallest fully functional antigen-binding fragment. The heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0073] The term "minimum recognition unit of an antibody" refers to an antibody that contains only one complementarity determining region (CDR) polypeptide, also known as a hypervariable region polypeptide. The molecular mass of this unit is only about 1% of that of a complete antibody, but it can still bind to the corresponding antigen.

[0074] As used herein, the term "bispecific antibody" refers to an antibody, typically a human or humanized antibody, that has monoclonal binding specificities for at least two different antigens. In the present disclosure, one of the binding specificities can be detected for an antigenic epitope of LIV1, and the other can be detected for another antigenic epitope of LIV1 or any other antigen other than LIV1, such as a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a viral protein, a virally encoded envelope protein, a bacterial protein, or a bacterial surface protein.

[0075] As used herein, the term "chimeric" antibody refers to an antibody having variable sequences of an immunoglobulin from one source organism (e.g., rat or mouse) and constant regions of an immunoglobulin from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art.

[0076] As used herein, the term "complementarity determining region" (CDR) refers to the hypervariable region found in both the light chain and heavy chain variable domains. The more highly conserved portion of the variable domain is referred to as a framework region (FR). As understood in the art, the amino acid position representing the hypervariable region of an antibody can vary according to context and various definitions known in the art. Some positions within the variable domain can be considered as hybrid hypervariable positions, because these positions can be considered to be within the hypervariable region under one set of standards (such as IMGT or Kabat), and are considered to be outside the hypervariable region under different sets of standards (such as Kabat or IMGT). One or more of these positions can also be found in the hypervariable region of extension. The present disclosure includes antibodies comprising modifications in these hybrid hypervariable positions. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a lamellae configuration, which are connected by three CDRs (CDR1, CDR2, and CDR3), which form a loop connecting the lamellae structure and, in some cases, form a part for the lamellae structure. The CDRs in each chain are closely held together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contribute to the formation of the antigen binding site of the antibody with the CDRs from other antibody chains. For example, in this article, CDR1-VH, CDR2-VH, and CDR3-VH refer to the first CDR, the second CDR, and the third CDR of the heavy chain variable region (VH), respectively, and these three CDRs constitute the CDR combination (VHCDR combination) of the heavy chain (or its variable region); CDR1-VL, CDR2-VL, and CDR3-VL refer to the first CDR, the second CDR, and the third CDR of the light chain variable region (VL), respectively, and these three CDRs constitute the CDR combination (VLCDR combination) of the light chain (or its variable region).

[0077] As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), without limitation to the method by which the antibody is produced.

[0078] As used herein, the term "VH" refers to the variable region of an immunoglobulin heavy chain (including the heavy chain of Fv, scFv or Fab) of an antibody. The term "VL" refers to the variable region of an immunoglobulin light chain (including the light chain of Fv, scFv, dsFv or Fab).

[0079] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in antibody-antigen binding but exhibits effector functions, such as interactions with Fc receptors. It has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" comprises at least one of the following: a CHI domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant regions" include "full-length heavy chain constant regions" and "heavy chain constant region fragments." The former has a structure substantially similar to that of a native antibody constant region, while the latter only comprises "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CHI domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CHI domain. For example, a typical "heavy chain constant region fragment" can be selected from the CHI, Fc, or CH3 domains.

[0080] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0081] The term "Fc" herein refers to the antibody carboxyl terminal portion of a complete antibody that is hydrolyzed by papain, and typically, it comprises the CH3 and CH2 domains of an antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at Cys226 position or from Pro230 to its carboxyl terminal. The C-terminal lysine (according to the residue 447 of the EU numbering system) in the Fc region can, for example, be present in the production or purification process of an antibody, or removed by recombinant engineering of a nucleic acid encoding an antibody heavy chain. Therefore, the Fc region may include or may not include Lys447.

[0082] The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0083] The term "fully human antibody" herein refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein are not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been transplanted to human framework sequences.

[0084] The term "naked antibody" herein refers to an antibody that is not connected, fused or conjugated to another agent or molecule (e.g., a label or drug), peptide or polypeptide. In specific embodiments, naked antibodies expressed by mammalian host cells can be glycosylated by the glycosylation machinery (e.g., glycosylase) of the host cell. In certain embodiments, naked antibodies are not glycosylated when expressed by host cells that do not have their own glycosylation machinery (e.g., glycosylase). In certain embodiments, naked antibodies are intact antibodies, while in other embodiments, naked antibodies are antigen-binding fragments of intact antibodies, such as Fab antibodies.

[0085] The term "conjugated antibody" herein refers to an antibody that can be associated with a pharmaceutically acceptable carrier or diluent, which can be a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody.

[0086] The term "diabody" herein refers to a bivalent, bispecific antibody that can bind to different epitopes on the same or different antigens.

[0087] As used herein, the term "percent (%) sequence identity" refers to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to the amino acid (or nucleotide) residues of a reference sequence after aligning sequences and introducing gaps (if necessary) to achieve maximum percent sequence identity (e.g., for optimal alignment, gaps can be introduced into one or both of the candidate and reference sequences, and for the purpose of comparison, non-homologous sequences can be ignored). For the purpose of determining percent sequence identity, alignment can be achieved in a variety of ways well known to those skilled in the art, such as using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that requires maximum alignment over the full length of the compared sequences. For example, a reference sequence aligned for comparison with a candidate sequence can show that the candidate sequence exhibits from 50% to 100% sequence identity over the full length of the candidate sequence or a selected portion of the continuous amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes can be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0088] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0089] (1) Acidic amino acids: Asp (D) and Glu (E);

[0090] (2) Basic amino acids: Lys (K), Arg (R), and His (H);

[0091] (3) hydrophilic uncharged amino acids: Ser (S), Thr (T), Asn (N), and Gln (Q);

[0092] (4) Aliphatic uncharged amino acids: Gly (G), Ala (A), Val (V), Leu (L) and Ile (I);

[0093] (5) Non-polar uncharged amino acids: Cys (C), Met (M), and Pro (P);

[0094] (6) Aromatic amino acids: Phe (F), Tyr (Y) and Trp (W).

[0095] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat.

[0096] As used herein, the term "specific binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules that are specifically recognized, for example, by an antibody or its antigen-binding fragment. An antibody or its antigen-binding fragment that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or its antigen-binding fragment that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (e.g., between 1 pM and 100 nM). An antibody or its antigen-binding fragment that does not exhibit specific binding to a specific antigen or epitope thereof will exhibit a KD of greater than 100 nM (e.g., greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM) for that specific antigen or epitope. Various immunoassays can be used to select antibodies that specifically immunoreact with a specific protein or carbohydrate. For example, solid phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with proteins or carbohydrates.

[0097] As used herein, the term "antibody conjugate" refers to a coupling / conjugate formed by chemically bonding an antibody molecule directly or through a linker to another molecule, such as an antibody-drug conjugate (ADC), wherein the drug molecule is the other molecule.

[0098] The term "chimeric antigen receptor (CAR)" herein refers to a recombinant protein comprising at least (1) an extracellular antigen binding domain, such as a variable heavy or light chain of an antibody, (2) a transmembrane domain that anchors CAR into immune effector cells, and (3) an intracellular signaling domain. In certain embodiments, the extracellular antigen binding domain of CAR comprises scFv. scFv can be derived from the variable heavy and light regions of a fusion antibody. Alternatively or in addition, scFv can be derived from Fab (rather than an antibody, e.g., obtained from a Fab library). In certain embodiments, scFv is fused to a transmembrane domain and then fused to an intracellular signaling domain.

[0099] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derived sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as vectors for direct expression of antibodies of the present disclosure in vitro and / or in vivo, such as in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP 2 101 823B1). (the foregoing is incorporated herein in its entirety).

[0100] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (such as plasmids), RNA vectors, viruses or other suitable replicons (such as viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. The expression vectors disclosed herein contain polynucleotide sequences and, for example, additional sequence elements for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences (such as promoters and enhancer regions) that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of the mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct the efficient transcription of the genes carried on the expression vector. The expression vectors disclosed herein may also contain the following polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

[0101] The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0102] The term "pharmaceutical composition" herein refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0103] As used herein, the terms "subject," "object," and "patient" refer to an organism that is being treated for a particular disease or condition, such as cancer or an infectious disease, as described herein. Examples of subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovidae family (such as cattle, bison, buffalo, elk, and yaks, etc.), sheep, and horses, etc., that are being treated for a disease or condition, such as a cell proliferative disorder, such as cancer or an infectious disease.

[0104] As used herein, the term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as the progression of a cell proliferative disorder (such as cancer or an infectious disease). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a disease or disease, as well as subjects susceptible to a disease or disease, or subjects intending to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0105] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0106] The term "appropriate conditions" herein refers to conditions suitable for culturing various host cells, wherein the host cells include eukaryotic cells and prokaryotic cells.

[0107] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Included within this definition are both benign and malignant cancers.

[0108] The term "tumor" herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" as used herein are not mutually exclusive.

[0109] The term "anti-tumor agent" herein refers to anti-tumor drugs, which are a class of drugs for treating tumor diseases, such as chemotherapy drugs, biological agents, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] FIG1 shows the binding reaction between hLIV1-1 humanized antibody and human LIV1 protein detected by ELISA.

[0111] FIG2 shows the binding reaction between hLIV1-2 humanized antibody and human LIV1 protein detected by ELISA.

[0112] FIG3 shows the binding reaction between hLIV1-3 humanized antibodies and human LIV1 protein detected by ELISA.

[0113] FIG4 shows the binding reaction between hLIV1-1 humanized antibody and NCI-H838 cells detected by FACS.

[0114] FIG5 is a FACS analysis of the binding reaction between hLIV1-2 humanized antibody and NCI-H838 cells.

[0115] FIG6 shows the binding reaction of hLIV1-3 humanized antibodies to NCI-H838 cells detected by FACS. DETAILED DESCRIPTION

[0116] The present disclosure is further described below with reference to specific examples, and the advantages and features of the present disclosure will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or those recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0117] The embodiments of the present disclosure are merely exemplary and do not constitute any limitation on the scope of the present disclosure. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present disclosure may be modified or replaced without departing from the spirit and scope of the present disclosure, but such modifications and replacements shall fall within the scope of protection of the present disclosure.

[0118] Example 1: Preparation of LIV-1 antigen and stable transgenic strain

[0119] 1.1 Preparation of Antigens

[0120] The amino acid sequence of the extracellular domain (ECD) of human LIV-1 protein (specific sequence as shown in SEQ ID NO: 1) and the amino acid sequence of the extracellular domain of cynomolgus macaque LIV-1 protein (specific sequence as shown in SEQ ID NO: 4) were coupled to hFc (humanized Fc fragment, specific sequence as shown in SEQ ID NO: 3) or mFc (murine Fc fragment, specific sequence as shown in SEQ ID NO: 2) tags and cloned into the pTT5 vector (Ubao Bio, Catalog No. VT2202). Plasmids were prepared according to established standard molecular biology methods. For specific methods, see Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). The expression vector and transfection reagent PEI (Polysciences, 24765-1) were added to OPTI-MEM (Gibco, Cat. No. 11058021), mixed thoroughly, and allowed to stand for 15 minutes. The mixture was then added to Expi293F cells (Thermofisher, A14527) and incubated in a shaking incubator at 37°C with 5% CO2 and 120 rpm. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopuma, F081918-001) and 6 g / L glucose (Sigma, G7528) were added. On the sixth day of transfection, the cell supernatant was collected. The culture supernatant was loaded onto a protein A chromatography column (protein A filler AT Protein A Diamond and chromatography column BXK16 / 26 were purchased from Bogelon), washed with PBS phosphate buffer (pH 7.4) and then with 20 mM PB, 1 M NaCl, pH 7.2, and finally eluted with pH 3.4 citric acid buffer. The Fc-tagged antigen eluted from the protein A chromatography column was collected and neutralized with 1 / 10 volume of 1 M Tris, pH 8.0, and dialyzed with PBS at 4°C overnight. The concentration of the dialyzed antigen was determined using Nanodrop, the purity of the antigen was determined using HPLC-SEC, and the endotoxin content of the antigen was detected using an endotoxin detection kit (purchased from Andus). Finally, the control antigen was sterile filtered through 0.22 μm and packaged and stored at -80°C.

[0121] Human LIV-1 extracellular region fragment: SEQ ID NO: 1

[0122] Mouse IgG2a Fc fragment: SEQ ID NO: 2

[0123] Human Fc segment: SEQ ID NO: 3

[0124] Cynomolgus monkey LIV-1 extracellular domain fragment: SEQ ID NO: 4 Note: The underlined part is the signal peptide.

[0125] 1.2 Preparation of control antibodies

[0126] The amino acid sequences of the anti-human LIV-1 monoclonal antibody ladiratuzumab (VH and VL sequences are shown in Table 1, SEQ ID NOs: 5-6; antibody sequences are from US20200165335A) and the anti-FITC isotype control antibody (VH and VL sequences are SEQ ID NOs: 7-8, respectively) were cloned into the pTT5 vector (Ubao Bio, Cat. No. VT2202). The antibody expression method was the same as the antigen preparation process described in Section 1.1 above.

[0127] Table 1. Sequence information of anti-human LIV-1 antibody Ladiratuzumab and isotype control antibody

[0128] 1.3 Preparation of CHO-K1 recombinant cell line expressing human LIV-1 protein

[0129] The nucleotide sequence encoding the full-length amino acid sequence of human LIV-1 (NCBI: NP_036451.4) was cloned into the pLVX-IRES-Puro vector (purchased from U-Bio, Catalog No. VT1464) and lentiviral production was performed in HEK293T cells (Cell Bank, Chinese Academy of Sciences, Catalog No. SCSP-502). Flow cytometry was used to detect the expression of lentivirus in CHO-K1 cells (Cell Bank, Chinese Academy of Sciences, Catalog No. SCSP-507) 72 hours after infection. Transfected cells were selectively cultured for two weeks in Advanced DMEM / F12 medium (Gibco, 12634-010) supplemented with 5 μg / ml puromycin (Gibco, Cat. No. A1113802). Positive monoclonal cells were sorted using a FACSAria II flow cytometer (BD Biosciences) using an anti-human LIV-1 control antibody (Ladiratuzumab, prepared in Section 1.2) and a goat anti-human IgG (H+L) antibody (Jackson, 109605088) into 96-well plates. The plates were incubated at 37°C in 5% (v / v) CO₂. After approximately two weeks, a subset of wells containing monoclonal clones were selected for expansion. Expanded clones were screened by flow cytometry, and clones with high expression levels were selected for expansion. These clones were then successfully used in subsequent experiments, including antibody screening.

[0130] Example 2: Preparation of anti-human LIV-1 mouse monoclonal antibodies

[0131] 2.1 Mouse immunization and serum titer detection

[0132] The animal immunization experiment was divided into six groups. The first group was immunized with human LIV-1-hFc protein (ACRO, #LV1-H5254); the second group was immunized with human LIV-1-his protein (ACRO, #LV1-H5223) and monkey LIV-1-his protein (ACRO, #LV1-C52H5); the third group was immunized with human LIV-1-hFc protein (prepared in Section 1.1 of Example 1) and monkey LIV-1-hFc protein (prepared in Section 1.1 of Example 1); the fourth, fifth, and sixth groups were immunized with human LIV-1-hFc protein (prepared in-house in Section 1.1 of Example 1) and CHOK1-hLIV-1 cells (prepared in Section 1.3 of Example 1). The experimental animals were 6-8 week old female Balb / c, SJL, and C57 BL / 6J mice (purchased from Shanghai Slake Co., Ltd.), housed in an SPF-grade environment. Blood was collected from the mice's orbitals before immunization to provide negative serum.

[0133] For the first immunization group, the antigen was emulsified with Alum (Thermo, 77161) and CpG (synthesized by a contract manufacturer, catalog number: ODN1826) and injected intraperitoneally (0.1 ml). Furthermore, the antigen was emulsified with TiterMax (Sigma, T2684) and CpG and injected subcutaneously and at multiple sites in the paw, for a total of 50 μg per mouse. Subsequently, booster immunizations were administered weekly, with 25 μg of the antigen emulsified with Alum or TiterMax injected subcutaneously or at multiple sites in the paw, for a total of 4-6 booster immunizations. For the second immunization group, the human LIV-1-his protein was administered as in the first group. For the second immunization, the monkey LIV-1-his protein was emulsified with Alum and CpG and injected subcutaneously at multiple sites, for a total of 25 μg. Subsequently, booster immunizations were administered weekly, alternating between human and monkey LIV-1-his proteins, for a total of 4-6 booster immunizations. The third immunization group received the same initial immunization with human LIV-1-hFc protein as the first group. For the second immunization, monkey LIV-1-hFc protein emulsified with Alum and CpG was injected subcutaneously at multiple sites, totaling 25 μg of the immunogen. Thereafter, booster immunizations were administered weekly, alternating between human and monkey LIV-1-hFc proteins, for a total of 4-6 booster immunizations. The fourth immunization group received the same initial immunization with human LIV-1-hFc protein as the first group. For the second immunization, 0.1 ml of CHOK1-hLIV-1 cells were injected intraperitoneally, with 5E6 cells injected per mouse. Thereafter, booster immunizations were administered weekly, alternating between protein and cell-based immunizations, for a total of 4-6 booster immunizations. Groups 5 and 6 received their first immunization with human LIV-1-hFc protein, using the same protocol as group 1. For the second immunization, mice were intraperitoneally injected with 0.1 ml of CHOK1-hLIV-1 cells, with 5E6 cells injected per mouse. Booster immunizations were administered every two weeks, alternating between protein and cell immunizations, for a total of 2-4 booster immunizations.

[0134] In each of the above immunization groups, orbital blood was collected from mice after the second booster immunization and every one to two subsequent booster immunizations. Enzyme-linked immunosorbent assay (ELISA) was used to measure the binding titer of antibodies to the human LIV-1-his protein in mouse serum. The results showed that the sera from the immunized mice all showed varying degrees of binding to the immunogen, demonstrating an antigen-antibody reaction.

[0135] 2.2 Spleen cell fusion and hybridoma screening

[0136] Mice with high antibody titers in their serum were selected for spleen cell fusion. Three days before spleen cell fusion, booster immunization was performed by subcutaneous, plantar and intraperitoneal injection of 50 μg / mouse antigen solution prepared in physiological saline.

[0137] After mice were sacrificed, splenocytes and lymphocytes were collected. After centrifugation at 1500 rpm, the supernatant was discarded, and ACK lysis buffer (Gibco, A1049201) was added to the cells to lyse the erythrocytes contaminating the cells to obtain a cell suspension. The cells were washed three times with DMEM basal medium (Gibco, 10569044) at 1500 rpm and then mixed with mouse myeloma SP2 / 0 cells (purchased from ATCC, Cat. CRL-1581) at a viable cell ratio of 2:1. Cell fusion was performed using the BTX ECM2001+ high-efficiency electrofusion method (see METHODS IN ENZYMOLOGY, Vol. 220). The fused cells were diluted into DMEM medium containing 20% ​​(w / w) fetal bovine serum (ExCellBio, FND500), 1× HAT (Sigma, H0262-10VL), bovine insulin (Yeason, 40107ES25), and NEAA (Gibco, 11140050), and then cultured at 5×10 4 200 μL of each cell culture medium was added to each well of a 96-well cell culture plate and cultured in a 5% (v / v) CO2, 37°C incubator.

[0138] After 7 days, the supernatants from the fusion plates were screened using ELISA to determine binding activity against human LIV-1 protein. Supernatants from positive clones with high binding activity were further evaluated for binding activity against monkey LIV-1 protein and cells overexpressing human LIV-1 using ELISA. Based on the screening results, eligible positive clones were selected and subcloned using semi-solid culture medium (purchased from stemcell, Cat. 03810). After 7 days, the clones were individually transferred to 96-well culture plates and expanded in DMEM medium supplemented with 10% (w / w) fetal bovine serum and 1×HT (Sigma, H0137-10VL). One day later, preliminary screening was performed using ELISA. Single clones with positive binding activity against human LIV-1 protein were selected and expanded to 24-well plates for further culture. Three days later, the culture supernatants were further assayed to evaluate their binding activity against monkey LIV-1 and mouse LIV-1 proteins.

[0139] Based on the test results of the 24-well plate samples, the best clone was selected and expanded in DMEM medium containing 10% FBS at 37°C and 5% CO2. After 7 days, the supernatant was collected and purified with protein A to obtain purified hybridoma monoclonal antibodies.

[0140] The hybridoma antibodies identified above were identified by ELISA, FACS, etc., and three mouse candidate antibodies LIV1-1, LIV1-2 and LIV1-3 with good binding and blocking activities were obtained.

[0141] Example 3: Construction and preparation of anti-LIV-1 chimeric antibodies

[0142] Using molecular biological techniques, DNA sequences encoding heavy and light chain immunoglobulins were obtained from the hybridoma mouse antibodies, and chimeric antibodies were constructed after sequencing.

[0143] The nucleic acids encoding the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the hybridoma antibody were codon-optimized according to the codon usage preference of Homo sapiens, and then fully synthesized. The heavy chain variable region was cloned into the vector PTT5-huIgG4CH1-CH2-CH3 containing the human heavy chain constant region (SEQ ID NO: 9) and regulatory elements to express the complete IgG heavy chain in mammalian cells. Similarly, the light chain variable region was cloned into the vector PTT5-huIgGLC(Kappa) containing the human light chain constant region (SEQ ID NO: 10) and regulatory elements to express the complete IgG light chain in mammalian cells. After correct sequencing, the protein was transfected into Expi-293 mammalian cells, and the expressed protein was secreted into the culture medium. The supernatants were collected, filtered, and purified. Protein A chromatography was used to purify the protein. The culture supernatant was loaded onto a Protein A column of appropriate size and eluted with a high-salt eluent (20 mM phosphate buffer, 1 M NaCl, pH 7.4) for 3 to 5 column volumes. The eluent was eluted with an eluent (50 mM NaAc-HAc, pH 3.5). The protein was concentrated by ultrafiltration using a concentrator (Millipore) and the OD was measured. 280nm The protein concentration was determined by spectrophotometry. SDS-PAGE was used to analyze the aggregation or degradation of the purified protein. Chimeric antibodies CHI-LIV1-1, CHI-LIV1-2, and CHI-LIV1-3, which have the same sequences as the murine VH and VL, were obtained. The murine / chimeric antibody VH, VL, and CDR sequences (according to Kabat classification) are shown in Tables 2 and 3 below.

[0144] Table 2. Murine / chimeric antibody VH and VL sequences

[0145] Table 3. CDRs of murine / chimeric antibody molecules (Kabat analysis)

[0146] Example 4: Identification of LIV-1 chimeric antibodies

[0147] 4.1 Enzyme-linked immunosorbent assay (ELISA) was used to detect the binding of chimeric antibodies to human / monkey LIV-1 proteins.

[0148] Human LIV1-mFc (produced in-house as described in Section 1.1 of the Examples) and monkey LIV1-mFc (produced in-house as described in Section 1.1 of the Examples) were diluted to 2 μg / ml and 50 μl per well was added to a 96-well plate (coning, 9018). The plates were incubated overnight at 4°C, washed three times with PBST, and then blocked with 0.5% skim milk (Sanggong, A600669-0250) at room temperature for 2 hours. The blocking solution was discarded and the plates were washed three times with PBST. The chimeric antibody CHI-LIV1- 1. CHI-LIV1-2 and CHI-LIV1-3 were diluted to 100 nM, and 10-fold serial dilutions were performed at 8 points. 50 μl / well was added to a blocked 96-well plate. After incubation at room temperature for 1 hour, the plate was washed 5 times with PBST. HRP (horseradish peroxidase)-labeled secondary antibody (purchased from Jackson, 109-035-088) was added. After incubation at room temperature for 1 hour, the plate was washed 5 times with PBST. TMB substrate 50 μl / well was added. After incubation at room temperature for 5-10 minutes, stop solution (1.0 N HCl) 50 μl / well was added. OD was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). 450nm The results of the chimeric antibody and protein detection are shown in Table 4, which shows that the chimeric antibody can bind to human LIV1 protein and monkey LIV1 protein. The isotype control antibody is anti-FITC antibody (produced in-house), and the data in the table are OD 450nm value.

[0149] Table 4. ELISA detection of the binding reaction between chimeric antibodies and human / monkey LIV-1 protein Note: “ / ” means no combination

[0150] 4.2 Flow cytometry (FACS) assay to detect the binding activity of chimeric antibodies to endogenous cells MCF-7 and NCI-H838

[0151] MCF-7 cells (an invasive ductal mammary carcinoma cell line, obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai) and NCI-H838 cells (a human non-small cell lung cancer cell line, obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai) that overexpress human LIV-1 protein were cultured in T-75 cell culture flasks to the logarithmic growth phase. The supernatant was discarded by centrifugation, and the cell pellet was washed once with PBS. The cells were seeded in a 96-well plate at a density of 2E5 cells / well and centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. Chimeric antibodies CHI-LIV1-1, CHI-LIV1-2, and CHI-LIV1-3 were diluted to 100 nM, 5-fold serial dilutions were made into 8 points, and 50 μl of secondary antibody (Alexa Fluor 500, 50 μl) was added to each well. The cells were resuspended, incubated at 4°C for 1 hour, washed twice with PBS, and 50 μl of secondary antibody (Alexa Fluor 500, 50 μl) was added to each well. Incubation was continued at 4°C for 1 hour with 647AffiniPure Goat Anti-Human IgG (H+L) (Jackson Immuno, 109-605-088). After washing twice with PBS, the cells were detected and analyzed by FACS (FACS Canto™, purchased from BD Biosciences). The results are shown in Table 5, which demonstrate that the chimeric antibodies CHI-LIV1-1, CHI-LIV1-2, and CHI-LIV1-3 specifically bind to MCF-7 and NCI-H838 cells to varying degrees. The data in the table are MFI values ​​and EC50 values ​​(nM).

[0152] Table 5. FACS detection of the binding activity of chimeric antibodies to endogenous cells MCF-7 and NCI-H838 Note: “ / ” means no combination

[0153] 4.3 Biacore Detection of Affinity of Chimeric Antibodies to Human LIV-1 Protein

[0154] This experiment used a Biacore 8K (GE) instrument to determine the affinity of the test antibody for the antigen human LIV-1-His (purchased from Acro, cat#LV1-H5223) using multi-cycle kinetics. The running buffer used was 1× HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (Cat.#BR-1006-69, Cytiva). The flow-through cell temperature was set at 25°C, and the sample chamber temperature was set at 16°C. Both were pre-treated with running buffer. A specific amount of the test antibody was affinity-captured using a Protein A biosensor chip (Cat.#29127556, Cytiva). A specific concentration of antigen was then passed over the chip surface. The reaction signal was monitored in real time on the Biacore 8K instrument (GE) to generate association and dissociation curves. First, the antigen human LIV-1 was diluted 1:1 with running buffer from a starting concentration (see detailed results for actual concentrations tested). A concentration gradient was established, and binding was monitored by injecting varying concentrations of antigen for 240 seconds at a flow rate of 30 μL / min and a dissociation time of 600 seconds. After each dissociation cycle, the antigen-antibody complex was washed clean with glycine-HCl regeneration solution (Cat. #BR-1003-54, Cytiva) at pH 1.5 for 30 seconds at a flow rate of 30 μL / min, completing chip surface regeneration.

[0155] The experimental data were fitted with a (1:1) Langmuir model using Cytiva Biacore 8K Evaluation version 2.0 software to obtain the association rate (Ka), dissociation rate (Kd), and affinity value (KD). The chimeric antibody showed superior binding activity to human LIV-1 protein compared to the positive control antibody ladirtuzumab, as shown in Table 6 below.

[0156] Table 6. Biacore detection of the binding activity of chimeric antibodies to human LIV-1 protein

[0157] Example 5: Design of humanized LIV-1 antibody

[0158] By comparing the IMGT (http: / / imgt.cines.fr) database of human antibody heavy and light chain variable region germline genes, we selected heavy and light chain variable region germline genes with high homology to mouse antibodies as templates. The mouse antibody CDRs were then transplanted into the corresponding human templates, forming a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on the antibody's three-dimensional structure, we backmutated buried residues, residues that directly interact with the CDR regions, and residues in the framework regions that have a significant impact on VL and VH conformation, resulting in humanized monoclonal antibodies.

[0159] 5.1 Humanization of LIV1-1

[0160] The humanized light chain templates for the murine antibody LIV1-1 were IGKV3-11*01 / IGKV6-21*01 and IGKJ2*01, and the humanized heavy chain templates were IGHV3-7*01 and IGHJ6*01. The CDRs of the murine antibody were transplanted into their respective humanized templates to generate the corresponding humanized version, hLIV1-1. As needed, key amino acids in the FR region sequence of the humanized hLIV1-1 antibody were backmutated to their murine counterparts to maintain the original affinity. The CDR amino acid residues were assigned and annotated using the Kabat numbering system. Detailed mutation design is shown in Table 7.

[0161] Table 7. Humanized antibody mutation design of hLIV1-1 (natural sequence numbering) Note: L46P means the 46th L is mutated to P, and so on. The mutated amino acids are numbered in natural order.

[0162] The specific sequence of the variable region of hLIV1-1 humanized antibody is as follows:

[0163] The amino acid sequence of hLIV1-1.VL1 is shown in SEQ ID NO: 35:

[0164] The amino acid sequence of hLIV1-1.VL4 is shown in SEQ ID NO: 36:

[0165] The amino acid sequence of hLIV1-1.VL6 is shown in SEQ ID NO: 37:

[0166] The amino acid sequence of hLIV1-1.VH1 is shown in SEQ ID NO: 38:

[0167] The amino acid sequence of the humanized light chain template IGKV3-11*01 is shown in SEQ ID NO: 39:

[0168] The amino acid sequence of the humanized light chain template IGKV6-21*01 is shown in SEQ ID NO: 40:

[0169] The amino acid sequence of the humanized light chain template IGKJ2*01 is shown in SEQ ID NO: 41:

[0170] The amino acid sequence of the humanized heavy chain template IGHV3-7*01 is shown in SEQ ID NO: 42:

[0171] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 43:

[0172] Note: The mutation points are bolded and underlined.

[0173] The present disclosure selects different light chain and heavy chain sequences from the backmutation designs of the light chain and heavy chain variable regions of the hLIV1-1 humanized antibody described above for cross-combination, ultimately obtaining a variety of hLIV1-1 humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0174] Table 8. Amino acid sequences corresponding to the variable regions of hLIV1-1 antibodies

[0175] According to the Kabat numbering system, the results of sequence analysis of the above humanized antibody VH and VL are shown in Table 9.

[0176] Table 9. Kabat analysis results of hLIV1-1 humanized antibody VH and VL sequences

[0177] 5.2 Humanization of LIV1-2

[0178] The humanized light chain templates for the murine antibody LIV1-2 were IGKV1-NL1*01 and IGKJ2*01, and the humanized heavy chain templates were IGHV1-69-2*01 and IGHJ6*01. The CDRs (based on the Kabat classification) of the murine antibody LIV1-2 were transplanted into their humanized templates to obtain the corresponding humanized versions. As needed, key amino acids in the FR region sequences of the humanized LIV1-2 antibody were backmutated to their corresponding mouse counterparts to maintain the original affinity. The CDR amino acid residues were identified and annotated using the Kabat numbering system. LIV1-2 contains sites susceptible to chemical modification; point mutations were performed to eliminate these modifications. Specific mutation designs are shown in Table 10.

[0179] Table 10. Humanized antibody mutation design of LIV1-2 (natural sequence numbering) Note: A43S indicates that the 43rd amino acid position is mutated to S, and so on. The mutated amino acids are numbered in natural order.

[0180] The specific sequence of the hLIV1-2 humanized antibody variable region is as follows:

[0181] The amino acid sequence of hLIV1-2.VL1 is shown in SEQ ID NO: 44:

[0182] The amino acid sequence of hLIV1-2.VH1 is shown in SEQ ID NO: 45:

[0183] The amino acid sequence of hLIV1-2.VH3a is shown in SEQ ID NO: 46:

[0184] The amino acid sequence of hLIV1-2.VH3b is shown in SEQ ID NO: 47:

[0185] The amino acid sequence of the humanized light chain template IGKV1-NL1*01 is shown in SEQ ID NO: 48:

[0186] The amino acid sequence of the humanized light chain template IGKJ2*01 is shown in SEQ ID NO: 41:

[0187] The amino acid sequence of the humanized heavy chain template IGHV1-69-2*01 is shown in SEQ ID NO: 49:

[0188] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 43:

[0189] Note: The mutation points are bolded and underlined.

[0190] The present disclosure selects different light chain and heavy chain sequences from the backmutation designs of the light chain and heavy chain variable regions of the hLIV1-2 humanized antibody described above for cross-combination, ultimately obtaining a variety of hLIV1-2 humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0191] Table 11. Amino acid sequences corresponding to the variable regions of hLIV1-2 antibodies

[0192] According to the Kabat numbering system, the results of sequence analysis of the above humanized antibodies VH and VL are shown in Table 12.

[0193] Table 12. Kabat analysis results of hLIV1-2 humanized antibody VH and VL sequences

[0194] 5.3 Humanization of LIV1-3

[0195] The humanized light chain templates for the murine antibody LIV1-3 were IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates were IGHV1-3*01 and IGHJ6*01. The CDRs of the murine antibody LIV1-3 were transplanted into their humanized templates to generate the corresponding humanized versions. As needed, key amino acids in the FR region sequences of the humanized LIV1-3 antibody were backmutated to their murine counterparts to maintain the original affinity. The CDR amino acid residues of the antibody were identified and annotated using the IMGT numbering system. LIV1-3 contains sites susceptible to chemical modification; we performed point mutations at these sites to eliminate the risk of modification. Detailed mutation design is shown in Table 13.

[0196] Table 13. Humanized antibody mutation design of hLIV1-3 (natural sequence numbering) Note: A40T means the 40th A is mutated to T, and so on. The mutated amino acids are numbered in natural order.

[0197] The specific sequence of the hLIV1-3 humanized antibody variable region is as follows:

[0198] The amino acid sequence of hLIV1-3.VL1 is shown in SEQ ID NO: 52:

[0199] The amino acid sequence of hLIV1-3.VH1 is shown in SEQ ID NO: 53:

[0200] The amino acid sequence of hLIV1-3.VH2a is shown in SEQ ID NO: 54:

[0201] The amino acid sequence of hLIV1-3.VH3a is shown in SEQ ID NO: 55:

[0202] The amino acid sequence of the humanized light chain template IGKV4-1*01 is shown in SEQ ID NO: 56:

[0203] The amino acid sequence of the humanized light chain template IGKJ2*01 is shown in SEQ ID NO: 41:

[0204] The amino acid sequence of the humanized heavy chain template IGHV1-3*01 is shown in SEQ ID NO: 57:

[0205] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 43:

[0206] Note: The mutation points are bolded and underlined.

[0207] The present disclosure selects different light chain and heavy chain sequences from the backmutation designs of the light chain and heavy chain variable regions of the hLIV1-3 humanized antibodies described above for cross-combination, ultimately obtaining a variety of hLIV1-3 humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0208] Table 14. Amino acid sequences corresponding to the variable regions of hLIV1-3 antibodies

[0209] According to the IMGT numbering system, the results of the VH and VL sequence analysis of the above humanized antibodies are shown in Table 15.

[0210] Table 15. IMGT analysis results of hLIV1-3 humanized antibody VH and VL sequences

[0211] Example 6: Identification of humanized LIV-1 antibodies

[0212] 6.1 ELISA assay to detect binding of humanized antibodies to human / monkey LIV-1 protein

[0213] In order to detect the binding activity of LIV-1 humanized antibodies to human LIV-1 protein, the same enzyme-linked immunosorbent assay (ELISA) as in Example 4.1 was used to detect the binding of humanized antibodies to human / monkey LIV-1 protein. The test results are shown in Table 16 and Figures 1 to 3, indicating that the purified humanized antibodies have a binding ability to human LIV1 at the protein level comparable to that of positive antibodies. The data in the table are OD 450nm value.

[0214] Table 16. ELISA detection of binding reaction between LIV-1 humanized antibodies and human LIV1 protein

[0215] 6.2 Flow cytometry (FACS) assay to detect the binding activity of humanized antibodies to endogenous cells NCI-H838

[0216] In order to detect the binding activity of LIV-1 humanized antibodies to endogenous cells expressing LIV-1 protein, the same FACS detection method as in Example 4.2 was used to detect the binding of LIV-1 humanized antibodies to endogenous cells NCI-H838 expressing LIV-1 protein. The detection results are shown in Table 17 and Figures 4-6, indicating that the humanized antibodies have different degrees of specific binding to NCI-H838 cells. The data in the table are MFI values.

[0217] Table 17. FACS detection of binding reaction between LIV-1 humanized antibody and NCI-H838 cells

[0218] 6.3 Biacore Detection of Affinity of Humanized Antibodies to Human / Monkey LIV-1 Protein

[0219] This experiment used a Biacore 8K (GE) instrument to determine the affinity of the test antibodies for the antigens human LIV-1-mFc (produced in-house, as described in Example 1.1) and monkey LIV-1-mFc (produced in-house, as described in Section 1.1 of Example 1) using multi-cycle kinetics. The running buffer used was 1× HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (Cat. #BR-1006-69, Cytiva). The flow cell temperature was set at 25°C, and the sample chamber temperature was set at 16°C. Both were pre-treated with running buffer. A certain amount of the test antibody was affinity-captured using a Protein A biosensor chip (Cat. #29127556, Cytiva). A certain concentration of antigen was then passed over the chip surface. The reaction signal was monitored in real time on the Biacore 8K instrument (GE) to generate association and dissociation curves. First, human LIV-1 and monkey LIV-1 antigens were diluted 1:1 with running buffer from a starting concentration (actual concentrations tested are detailed in the results). A concentration gradient was established, and binding was monitored by injecting varying concentrations of antigen for 240 seconds at a flow rate of 30 μL / min and a dissociation time of 600 seconds. After each dissociation cycle, the antigen-antibody complex was washed clean with glycine-HCl regeneration solution (Cat.# BR-1003-54, Cytiva) at pH 1.5 for 30 seconds at a flow rate of 30 μL / min to regenerate the chip surface.

[0220] The data were fitted using Cytiva Biacore 8K Evaluation version 2.0 software using a (1:1) Langmuir model to determine the association rate (Ka), dissociation rate (Kd), and affinity (KD). The results showed that the humanized antibody had comparable binding activity to the positive molecule. See Table 18 below for details.

[0221] Table 18. Biacore detection of affinity of humanized antibodies to human / monkey LIV-1 protein

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to LIV-1, characterized in that, The antibody or its antigen-binding fragment comprises: (a) The HCDR1, HCDR2 and HCDR3 of VH shown in any one of SEQ ID NOs: 11, 12, 13, 38, 45, 46, 47, 53, 54 or 55; and / or, (b) The LCDR1, LCDR2 and LCDR3 of VL shown in any one of SEQ ID NOs: 14, 15, 16, 35, 36, 37, 44 or 52; Preferably, the HCDR1-3 and / or the LCDR1-3 are encoded according to the general analysis methods of Kabat or IMGT.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The HCDR1, HCDR2, and HCDR3 have any sequence combination selected from the following or a sequence combination having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions as compared to the said sequence combination: And, (2) The LCDR1, LCDR2, and LCDR3 have any sequence combination selected from the following or a sequence combination having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions as compared with the said sequence combination: Preferably, the substitution is a conservative amino acid substitution.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein, It comprises a combination of heavy chain CDRs and light chain CDRs selected from: VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL2, VH5+VL2, VH6+VL4, VH7+VL4 or VH8+VL4, and a combination of CDRs with 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence of the combination of the heavy chain and light chain CDRs; Preferably, the substitution is a conservative amino acid substitution.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The framework regions of the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment are derived from germline heavy chain templates and germline light chain templates, wherein: (1) The framework region sequence is derived from the combined sequence of germline heavy chain IGHV3-7*01 and IGHJ6*01; it comprises the FR1, FR2, FR3 regions of IGHV3-7*01 shown in SEQ ID NO: 42 and the FR4 region of IGHJ6*01 shown in SEQ ID NO: 43; (2) The framework region sequence is derived from the combined sequence of germline heavy chain IGHV1-69-2*01 and IGHJ6*01; it comprises the FR1, FR2, FR3 regions of IGHV1-69-2*01 shown in SEQ ID NO: 49 and the FR4 region of IGHJ6*01 shown in SEQ ID NO: 43; (3) The framework region sequence is derived from the combined sequence of germline heavy chain IGHV1-3*01 and IGHJ6*01; it comprises the FR1, FR2, FR3 regions of IGHV1-3*01 shown in SEQ ID NO: 57 and the FR4 region of IGHJ6*01 shown in SEQ ID NO: 43; (4) The framework region sequence is derived from the combined sequence of germline light chain IGKV3-11*01 and IGKJ2*01; it comprises the FR1, FR2, FR3 regions of IGKV3-11*01 shown in SEQ ID NO: 39 and the FR4 region of IGKJ2*01 shown in SEQ ID NO: 41; (5) The framework region sequence is derived from the combined sequence of germline light chain IGKV6-21*01 and IGKJ2*01; it comprises the FR1, FR2, FR3 regions of IGKV6-21*01 shown in SEQ ID NO: 40 and the FR4 region of IGKJ2*01 shown in SEQ ID NO: 41; (6) The framework region sequence is derived from a combined sequence of the human germline light chain IGKV1-NL1*01 and IGKJ2*01; it includes the FR1, FR2, and FR3 regions of IGKV1-NL1*01 shown in SEQ ID NO: 48 and the FR4 region of IGKJ2*01 shown in SEQ ID NO: 41; (7) The framework region sequence is derived from a combined sequence of the human germline light chain IGKV4-1*01 and IGKJ2*01; it includes the FR1, FR2, and FR3 regions of IGKV4-1*01 shown in SEQ ID NO: 56 and the FR4 region of IGKJ2*01 shown in SEQ ID NO:

41.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, Numbered according to the Kabat or IMGT numbering system, the framework regions of the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment further include one or more mutations selected from the following groups, where: (1) The framework region of the heavy chain variable region includes: Q1E, V24T, M34I, W50R, N55Q, G56A, I70L, R72A, A76S, S77N, R98K, T98R, or G101A; preferably includes R98K; or preferably includes T98R; or preferably includes V24T, G56A, I70L, T98R; or preferably includes V24T, G56A, I70L, T98R, G101A; or preferably includes Q1E, M34I, W50R, R72A; or preferably includes Q1E, M34I, W50R, G56A, R72A, S77N; or preferably includes Q1E, M34I, W50R, N55Q, R72A, A76S, S77N; (2) The framework region of the light chain variable region includes: A40T, A43S, L46P, L47I, L48V, Y49K, V58I, or T85M; preferably includes L46P, Y49K; or preferably includes L46P, L47I, V58I; or preferably includes L46P, L47I, V58I, T85M; or preferably includes A43S, L48V; or preferably includes A40T.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, characterized in that, The antibody or its antigen-binding fragment described above comprises: (1) The heavy chain variable region has the sequence shown in SEQ ID NO: 11, 12, 13, 38, 45, 46, 47, 53, 54, or 55; (2) The light chain variable region has the sequence shown in SEQ ID NO: 14, 15, 16, 35, 36, 37, 44, or 52; (3) An amino acid sequence having at least 90% identity compared to any one of the sequences in (1) to (2) above, preferably an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein The heavy chain variable region and the light chain variable region described above are selected from the following groups: (1) Having VH shown in SEQ ID NO: 11 and VL shown in SEQ ID NO: 14; (2) Having VH shown in SEQ ID NO: 12 and VL shown in SEQ ID NO: 15; (3) having a VH as shown in SEQ ID NO: 13 and a VL as shown in SEQ ID NO: 16; (4) having a VH as shown in SEQ ID NO: 38 and a VL as shown in SEQ ID NO: 35; (5) having a VH as shown in SEQ ID NO: 38 and a VL as shown in SEQ ID NO: 36; (6) having a VH as shown in SEQ ID NO: 38 and a VL as shown in SEQ ID NO: 37; (7) having a VH as shown in SEQ ID NO: 45 and a VL as shown in SEQ ID NO: 44; (8) having a VH as shown in SEQ ID NO: 46 and a VL as shown in SEQ ID NO: 44; (9) having a VH as shown in SEQ ID NO: 47 and a VL as shown in SEQ ID NO: 44; (10) having a VH as shown in SEQ ID NO: 53 and a VL as shown in SEQ ID NO: 52; (11) having a VH as shown in SEQ ID NO: 54 and a VL as shown in SEQ ID NO: 52; (12) having a VH as shown in SEQ ID NO: 55 and a VL as shown in SEQ ID NO: 52; (13) having a VH and VL combination with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity compared to any of the sequences in (1) - (12) above.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, characterized in that Its dissociation constant (KD) for binding to human LIV-1 is not greater than 2×10 -9 M.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, characterized in that The antibody or its antigen-binding fragment is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; (3) a fully human antibody or a fragment thereof; Preferably, the antibody or its antigen-binding fragment is selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies), monovalent antibodies, multivalent antibodies, full-length antibodies, antibody fragments, naked antibodies, conjugated antibodies, humanized antibodies, fully human antibodies, Fab, Fab’, F(ab’)2, Fd, Fv, scFv, diabody or single-domain antibodies.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, characterized in that, The antibody comprises the sequence of any one of the constant regions of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably comprises the sequence of the constant region of human or murine antibodies IgG1, IgG2, IgG3 or IgG4.

11. An antibody or antigen-binding fragment thereof according to any one of claims 1-10, characterized in that, The antigen-binding fragment is selected from one or more of F(ab)2, Fab’, Fab, Fv, scFv, bispecific antibodies, nanobodies and the minimal antibody recognition unit.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1-11, characterized in that The antibody or its antigen-binding fragment is further conjugated with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioactive isotopes, chemotherapeutic drugs or immunomodulators, and the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents or photosensitizers.

13. A multispecific antigen-binding molecule, characterized in that, The multispecific antigen-binding molecule comprises a first antigen-binding module and a second antigen-binding module. The first antigen-binding module comprises the antibody or its antigen-binding fragment according to any one of claims 1-12. The second antigen-binding module specifically binds to an antigen other than LIV-1 or binds to an LIV-1 antigen epitope different from the LIV-1 antigen epitope bound by the first antigen-binding module; Preferably, the other antigen is selected from CD3, CD28, CD137, CD134, CD27, ICOS, CD16, CD56, CD335, CD336, CD337, NKG2A, NKG2D, KIR, DNAM-1 or CD161; Preferably, the multispecific antigen-binding molecule is a bispecific antibody, a trispecific antibody or a tetra-specific antibody.

14. A chimeric antigen receptor (CAR), characterized in that, The chimeric antigen receptor comprises at least an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain. The extracellular antigen-binding domain comprises the antibody or its antigen-binding fragment according to any one of claims 1-12.

15. An immune effector cell, characterized in that, The immune effector cell comprises the chimeric antigen receptor according to claim 14 or comprises a nucleic acid fragment encoding the chimeric antigen receptor according to claim 14; Preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), monocytes, macrophages, dendritic cells or mast cells; The T cells can be selected from inflammatory T cells, cytotoxic T cells, regulatory T cells (Tregs) or helper T cells; Preferably, the immune effector cell is an allogeneic immune effector cell or an autologous immune cell.

16. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody, antigen-binding fragment, or any combination thereof according to any one of claims 1-12, the multispecific antigen-binding molecule according to claim 13 or the chimeric antigen receptor according to claim 14.

17. An expression vector comprising the isolated nucleic acid molecule according to claim 16.

18. An isolated host cell comprising the isolated nucleic acid molecule according to claim 16 or the expression vector according to claim 17; Preferably, the host cell is a eukaryotic cell or a prokaryotic cell; More preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and / or Bacillus subtilis; More preferably, the host cell is selected from Expi293 or CHO cells.

19. A method for preparing an antibody or an antigen-binding fragment thereof according to any one of claims 1-12 or a multispecific antigen-binding molecule according to claim 13, characterized in that, Culturing the host cell according to claim 18 under appropriate conditions and isolating the antibody or its antigen-binding fragment or the multispecific antigen-binding molecule.

20. A method for preparing the immune effector cells as claimed in claim 15, characterized in that, The method comprises introducing a nucleic acid fragment encoding the CAR according to claim 14 into an immune effector cell. Optionally, the method further comprises initiating the expression of the CAR according to claim 14 in the immune effector cell.

21. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody or its antigen-binding fragment according to any one of claims 1-12, the multispecific antigen-binding molecule according to claim 13, the chimeric antigen receptor according to claim 14, the immune effector cell according to claim 15, the isolated nucleic acid molecule according to claim 16, the expression vector according to claim 17, the cell according to claim 18, or the antibody or its antigen-binding fragment or multispecific antigen-binding molecule prepared by the method according to claim 19, the immune effector cell prepared by the method according to claim 20; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; preferably, the pharmaceutical composition further comprises an additional anti-tumor agent.

22. Use of the antibody or its antigen-binding fragment according to any one of claims 1-12, the multispecific antigen-binding molecule according to claim 13, the chimeric antigen receptor according to claim 14, the immune effector cell according to claim 15, the isolated nucleic acid molecule according to claim 16, the expression vector according to claim 17, the cell according to claim 18, the antibody or its antigen-binding fragment or multispecific antigen-binding molecule prepared by the method according to claim 19, the immune effector cell prepared by the method according to claim 20, or the pharmaceutical composition according to claim 21 in the preparation of a medicament for preventing and / or treating tumor diseases; Preferably, the tumor diseases are breast cancer, prostate cancer, cervical cancer, melanoma, ovarian cancer, endometrial cancer, invasive ductal carcinoma of breast, non-small cell lung cancer, pancreatic cancer, lung cancer, or squamous cell carcinoma.

23. A method for preventing and / or treating tumor diseases, comprising administering to a patient in need thereof an effective amount of the antibody or its antigen-binding fragment according to any one of claims 1-12, the multispecific antigen-binding molecule according to claim 13, the chimeric antigen receptor according to claim 14, the immune effector cell according to claim 15, the isolated nucleic acid molecule according to claim 16, the expression vector according to claim 17, the cell according to claim 18, the antibody or its antigen-binding fragment or multispecific antigen-binding molecule prepared by the method according to claim 19, the immune effector cell prepared by the method according to claim 20, or the pharmaceutical composition according to claim 21; Preferably, the tumor diseases are breast cancer, prostate cancer, cervical cancer, melanoma, ovarian cancer, endometrial cancer, invasive ductal carcinoma of breast, non-small cell lung cancer, pancreatic cancer, lung cancer, or squamous cell carcinoma.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1-12, the multispecific antigen-binding molecule according to claim 13, the chimeric antigen receptor according to claim 14, the immune effector cell according to claim 15, the isolated nucleic acid molecule according to claim 16, the expression vector according to claim 17, the cell according to claim 18, the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared by the method according to claim 19, the immune effector cell prepared by the method according to claim 20, or the pharmaceutical composition according to claim 21, characterized in that, For preventing and / or treating tumor diseases; Preferably, the tumor diseases are breast cancer, prostate cancer, cervical cancer, melanoma, ovarian cancer, endometrial cancer, invasive ductal carcinoma of breast, non-small cell lung cancer, pancreatic cancer, lung cancer, or squamous cell carcinoma.

25. A kit, which comprises the antibody or its antigen-binding fragment according to any one of claims 1-12, the multispecific antigen-binding molecule according to claim 13, the chimeric antigen receptor according to claim 14, the immune effector cell according to claim 15, the isolated nucleic acid molecule according to claim 16, the expression vector according to claim 17, the cell according to claim 18, the antibody or its antigen-binding fragment or multispecific antigen-binding molecule prepared by the method according to claim 19, the immune effector cell prepared by the method according to claim 20, or the pharmaceutical composition according to claim 21; optionally, it further comprises instructions for use.

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