Antibody molecule binding to LIV-1 and use thereof

Abstract

The present disclosure relates to an antibody molecule binding to LIV-1 and having internalization activity or an antigen-binding fragment thereof, a derivative comprising the antibody or the antigen-binding fragment thereof, and an antibody-drug conjugate. In addition, the present disclosure also relates to related use of the antibody or the antigen-binding fragment thereof in the treatment of a LIV-1-associated disease and in detection and diagnosis.

Description

Antibody molecules that bind to LIV-1 and their applications

[0001] This application claims priority to Chinese patent application 202510191994.8, filed on February 20, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the field of immunology and relates to antibodies that bind to LIV-1 or antigen-binding fragments thereof, derivatives comprising said antibodies or antigen-binding fragments thereof, pharmaceutical compositions and their use in treating diseases such as breast cancer or prostate cancer. Background Technology

[0003] Human zinc ion transporter LIV-1 (also known as ZIP6, encoded by gene SLC39A6) is a member of the ZIP family of zinc ion transporters. It regulates zinc ion influx into cells and plays a crucial role in cellular zinc homeostasis. LIV-1 is a full-length 755-amino acid protein, an eight-transmembrane protein with a long extracellular N-terminus and a short intracellular C-terminus, exhibiting both zinc transporter and metalloproteinase activities. Furthermore, LIV-1 interacts with transcription factors STAT3 and Snail, downregulating E-cadherin expression and promoting epithelial-mesenchymal transition (EMT) (Yamashita S. et al, Nature 2004; 429:298-302).

[0004] LIV-1 is negative in most normal tissues, positive in breast, prostate, and testes, and highly expressed in various solid tumors. LIV-1 has an extremely high positivity rate in breast cancer, and also shows high expression rates in prostate cancer, melanoma, glioma, and other tumor tissues. According to Phase I clinical trial results from Seagen Inc., the positive rate of LIV-1 in breast cancer patients was as high as 98%, with 88% showing intermediate to high expression levels. The positive rate in primary breast cancer (100%) was higher than in metastatic breast cancer (89%). LIV-1 expression is also positively correlated with estrogen receptor expression, and high LIV-1 expression can still be detected in patient samples after hormone therapy. Tumor tissue sections from 20 triple-negative breast cancer patients showed that 65% of the tumors were LIV-1 positive (Mol Cancer Ther. 2014 Dec).

[0005] Based on the specific high expression of LIV-1 in tumors, Seagen INC. developed the LIV-1-targeting ADC drug Ladiratuzumab Vedotin (SGN-LIV1A), which uses the anti-LIV-1 antibody hLIV22 and MMAE as toxins (DAR value of 4). The hLIV22 antibody is linked to MMAE via a protease-degradable mc-val-cit-PABC linker. Published clinical data show that SGN-LIV-1A exhibits good anti-tumor activity in breast cancer with almost no target-mediated toxicity, and demonstrates good synergistic anti-tumor activity when combined with PD-1. The ORR in second-line breast cancer patients was 28%, thus LIV-1 is a clinically validated ADC target. However, the drug SGN-LIV-1A still faces the issue of non-response in patients with high LIV-1 expression.

[0006] Therefore, there is an urgent need to develop anti-human LIV-1 antibodies and their antibody-drug conjugates with high affinity and high endocytic activity to meet the clinical treatment needs of tumor patients with high LIV-1 expression.

[0007] Invention Overview

[0008] This disclosure provides an antibody against LIV-1 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof specifically binds to LIV-1 and does not significantly bind to ZIP10, ZIP4, ZIP5 and ZIP12.

[0009] In some implementations, the LIV-1 is a peptide (mRNA: NM_001099406.1) with GenBank accession number NP_001092876.1.

[0010] One technical solution provided in this disclosure is: an anti-LIV-1 antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is capable of specifically binding to LIV-1, and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively selected from the following group:

[0011] (1) SEQ ID NO: 1, 2, 3, 4, 5, 6;

[0012] (2) SEQ ID NO: 7, 8, 9, 10, 11, 12;

[0013] (3) SEQ ID NO: 13, 14, 15, 16, 17, 18; or

[0014] (4) SEQ ID NO: 19, 20, 21, 22, 23, 24.

[0015] Another technical solution provided in this disclosure is: an anti-LIV-1 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the following groups of heavy chain variable regions and light chain variable regions:

[0016] (1) SEQ ID NO: 25 and 26;

[0017] (2) SEQ ID NO: 27 and 28;

[0018] (3) SEQ ID NO: 29 and 30; or

[0019] (4) SEQ ID NO:31 and 32.

[0020] Preferably, it also has one or more of the following characteristics:

[0021] (1) It also includes a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region contains Fc; more preferably, Fc is derived from mice or humans; more preferably, the sequence of Fc is a natural or modified variant;

[0022] (2) It is a mouse-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody;

[0023] (3) It is a monoclonal antibody; or a full-length antibody, or its antigen-binding fragment is Fab, Fv, scFv, F(ab')2, linear antibody, or single-domain antibody;

[0024] (4) It is in the form of IgG1, IgG2, IgG3 or IgG4.

[0025] Another technical solution provided in this disclosure is: an antibody-drug conjugate, which is formed by conjugating the anti-LIV-1 antibody or its antigen-binding fragment as described in this disclosure with other bioactive molecules. Preferably, the other bioactive molecules are small molecule drugs, such as antitumor drugs, and the antitumor drugs are preferably antitumor compounds. Preferably, the antibody and other bioactive molecules are linked by a linker.

[0026] Another technical solution provided in this disclosure is: a conjugate formed by conjugating the anti-LIV-1 antibody or its antigen-binding fragment as described in this disclosure with a capture marker or a detection marker. Preferably, the detection marker includes, but is not limited to, radionuclides, luminescent substances (e.g., fluorescein), colored substances, or enzymes.

[0027] Another technical solution provided in this disclosure is: a fusion protein, wherein one of the fused portions contains the anti-LIV-1 antibody or its antigen-binding fragment as described in this disclosure.

[0028] Another technical solution provided in this disclosure is: a bispecific antibody or a multispecific antibody, wherein one of its antigen-binding domains contains the anti-LIV-1 antibody or its antigen-binding fragment as described in this disclosure.

[0029] Another technical solution provided in this disclosure is: nucleic acid encoding the antibody or antigen-binding fragment of the anti-LIV-1 described in this disclosure, the fusion protein described in this disclosure, or the bispecific antibody or multispecific antibody described in this disclosure.

[0030] Another technical solution provided in this disclosure is a recombinant vector containing the nucleic acid described in this disclosure.

[0031] Another technical solution provided in this disclosure is: a host cell containing the recombinant vector or nucleic acid described in this disclosure.

[0032] Another technical solution provided in this disclosure is: the host cell as described in this disclosure is a prokaryotic cell, such as Escherichia coli; or a eukaryotic cell, such as yeast or a mammalian cell, such as CHO cells or HEK293 cells.

[0033] Another technical solution provided in this disclosure is: a method for preparing the anti-LIV-1 antibody or its antigen-binding fragment, the fusion protein, or the bispecific or multispecific antibody described in this disclosure, comprising: culturing the host cells described in this disclosure under suitable conditions, and purifying the expression product from the cells.

[0034] Another technical solution provided in this disclosure is: a method for detecting LIV-1 in a sample, comprising:

[0035] (1) Contact the sample with the anti-LIV-1 antibody or its antigen-binding fragment described in this disclosure, the fusion protein described in this disclosure, or the bispecific antibody or multispecific antibody described in this disclosure;

[0036] (2) Detect the formation of a complex of an anti-LIV-1 antibody or its antigen-binding fragment, fusion protein, bispecific antibody or multispecific antibody with LIV-1; optionally, the anti-LIV-1 antibody or its antigen-binding fragment, fusion protein, bispecific antibody or multispecific antibody is detectably labeled.

[0037] Another technical solution provided in this disclosure is: a pharmaceutical composition comprising an effective amount of the anti-LIV-1 antibody or its antigen-binding fragment as described in this disclosure, or comprising an effective amount of the antibody-drug conjugate as described in this disclosure, or comprising an effective amount of the fusion protein as described in this disclosure, or comprising an effective amount of the bispecific antibody or multispecific antibody as described in this disclosure, or comprising an effective amount of the nucleic acid as described in this disclosure, or comprising an effective amount of the recombinant vector as described in this disclosure, or comprising an effective amount of the host cell as described in this disclosure.

[0038] Preferably, it also contains a pharmaceutically acceptable carrier.

[0039] Preferably, it also contains one or more additional therapeutic agents.

[0040] Another technical solution provided in this disclosure is: a method for treating a subject with a disease related to LIV-1 expression, the method comprising administering the pharmaceutical composition described in this disclosure to the subject in need.

[0041] Preferably, the disease is a tumor, especially a solid tumor.

[0042] Preferably, the treatment also includes administering additional therapeutic agents to the subject.

[0043] It should be noted that the division of the CDR and FR in the antibody variable region of this disclosure is determined according to the Kabat definition. Other nomenclature and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies based on the antibody sequence of this disclosure that contain one or more CDRs derived from any nomenclature system are explicitly kept within the scope of this disclosure. Attached Figure Description

[0044] The accompanying drawings further illustrate the novel features disclosed herein. A better understanding of the features and advantages disclosed herein will be achieved by referring to these drawings; however, it should be understood that these drawings are for illustrating specific embodiments of the principles disclosed herein and are not intended to limit the scope of the appended claims.

[0045] Figures 1A and 1B show the binding of chimeric antibodies CH1-16 to human LIV-1. Figure 1A shows the binding activity of CH1-6 with human LIV-1, and Figure 1B shows the binding activity of CH1, 2, 9-16 with human LIV-1.

[0046] Figures 2A, 2B, and 2C show the internalization of the chimeric antibody CH1-16 on LIV-1 expressing cells. Specifically, Figure 2A shows the internalization activity of CH9-16 on MCF7 cells, Figure 2B shows the internalization activity of CH1-8 on CHOK1-hLIV1 cells, and Figure 2C shows the internalization activity of CH9-16 on CHOK1-hLIV1 cells.

[0047] Figures 3A, 3B, and 3C show the binding and internalization activities of humanized antibodies Ab1–4. Specifically, Figure 3A shows the binding of Ab1–3 to Jurkat, Figure 3B shows the binding of Ab1, Ab2, and Ab4 to MCF-7, and Figure 3C shows the internalization activities of Ab1–3 on MCF-7. Detailed Implementation

[0048] the term

[0049] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if specifically and individually indicated that each individual publication, patent or patent application is incorporated by reference.

[0050] Before this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0051] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. For example, a description of a range from 1 to 6 should be considered as specifically disclosing subranges from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and specific numerical points within those ranges, such as 1, 2, 3, 4, 5, 6. The above principles apply equally regardless of the breadth of the numerical values. When a range description is used, the range includes the endpoints of the range.

[0052] When referring to measurable values ​​such as quantities, temporary durations, etc., the term “about” means a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0053] The three-letter and single-letter codes for amino acids used in this article are as described in J. Biol. Chem, 243, p3558 (1968).

[0054] Human-derived LIV-1 is referred to as hLIV-1. Therefore, expressions such as "antibody against human LIV-1" and "antibody against hLIV-1" specifically refer to antibodies that can bind to hLIV-1 with sufficient affinity so that the antibodies can be used as diagnostic and / or therapeutic agents targeting hLIV-1.

[0055] LIV-1 derived from cynomolgus monkeys is designated as cyno LIV-1.

[0056] As used in this article, the term "antibody" typically refers to a Y-type tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies possess this structure. Each light chain consists of a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain contains a heavy chain variable domain (VH) and a heavy chain constant domain (CH), or heavy chain constant region (CH).

[0057] Five main classes of antibodies are known in this art: IgA, IgD, IgE, IgG, and IgM, with their corresponding heavy chain constant domains designated α, δ, ε, γ, and μ, respectively. IgG and IgA can be further subdivided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. The light chain of antibodies from any vertebrate species can be identified as one of two distinctly different types based on the amino acid sequence of their constant domains, termed κ and λ.

[0058] In the case of IgG, IgA, and IgD antibodies, this heavy chain constant region contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable-length segment rich in proline and cysteine. Each class of antibody further contains interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0059] The term "variable region" or "variable domain" indicates a significant change in the amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable region of each light / heavy chain pair forms the antigen-binding site, giving the complete IgG antibody two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, referred to as hypervariable regions (HVR), or more commonly, complementarity-determining regions (CDR). Each VH and VL has four backbone regions (FR), denoted as FR1, FR2, FR3, and FR4, respectively. Therefore, the CDR and FR sequences typically appear in the following sequence of the heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0060] The term "Fc" is used herein to define the C-terminal region of the immunoglobulin heavy chain, which comprises at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. Unless otherwise stated, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0061] As used herein, the broad category of "antibody" may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primate-derived antibodies, CDR-grafted antibodies, human antibodies (including recombinant human antibodies), recombinant antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-individual genotype antibodies, synthetic antibodies (including mutant proteins and their variants), etc.

[0062] The terms “full-length antibody,” “complete antibody,” and “intact antibody” may be used interchangeably in this document to refer to antibodies whose structure is substantially similar to that of natural antibodies or that contain an Fc region.

[0063] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced from a single cell clone that targets only a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or combinations of the above.

[0064] The term "chimeric antibody" is a construct in which a portion of the heavy and / or light chains is identical or homologous to a corresponding sequence in an antibody from a specific species or belonging to a specific antibody class or subclass, while the remainder of this or these chains is identical or homologous to a corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, or in fragments of such antibodies. In a narrower sense, a chimeric antibody comprises all or most of selected murine heavy and light chain variable regions operatively linked to human light and heavy chain constant regions. Constant region sequences, or variants or derivatives thereof, can be operatively associated with the disclosed heavy and light chain variable regions using standard molecular biology techniques to provide a full-length anti-LIV-1 antibody that can be used on its own or incorporated into this disclosure.

[0065] The term "humanized antibody" refers to a hybrid immunoglobulin, immunoglobulin chain, or fragment thereof containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) where residues of the receptor's core sequence (CDR) are replaced by residues of a CDR from a non-human species (donor antibody) possessing the desired specificity, affinity, and performance, such as mice, rats, rabbits, or primates. In some cases, framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. In certain circumstances, "reversion mutations" can be introduced into humanized antibodies where residues in one or more frame regions (FRs) of the variable region of the recipient human antibody are replaced by corresponding residues from a non-human species donor antibody. Such reversion mutations can help maintain the appropriate three-dimensional conformation of one or more grafted CDRs and thus improve affinity and antibody stability. Antibodies from a variety of donor species can be used, including but not limited to mice, rats, rabbits, or non-human primates. Additionally, humanized antibodies may contain novel residues not found in the recipient antibody or the donor antibody to further improve antibody performance.

[0066] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or its antigen-binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including single epitopes, multiple epitopes, single domains, multiple domains, or intact extracellular domains (ECDs) or proteins. Peptides, proteins, glycoproteins, polysaccharides, and lipids, as well as portions thereof, can constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that elicits an immune response. Any form of antigen, or cells or preparations containing that antigen, can be used to generate antibodies specific to the antigenic determinant. An antigen can be an isolated full-length protein, a cell surface protein (e.g., used for immunization with cells expressing at least a portion of the antigen on their surface), or a soluble protein (e.g., used for immunization with only the ECD portion of the protein), or a protein construct (e.g., an Fc antigen). The antigen can be produced in genetically modified cells. Any of the foregoing antigens can be used alone or in combination with one or more immunogenic adjuvants known in the art. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector can be used to transform cells expressing the antigen, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0067] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear, cyclic, or branched, may contain modified amino acids, particularly conserved modified amino acids, and may be interrupted by non-amino acid components. The term also includes amino acid polymers that have been modified, for example, by glycosylation, esterification, acetylation, phosphorylation, methylation, or any other manipulation such as conjugation with a labeled component. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimics. “Derived from” a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a specified nucleic acid sequence.

[0068] The sequence identity or homology between the variable region of the humanized antibody and the variable region of the human receptor can be determined as discussed herein, and when such a determination is made, preferably at least 60% or 65% sequence identity will be shared, more preferably at least 70%, 75%, 80%, 85%, or 90% sequence identity, and even more preferably at least 93%, 95%, 98%, or 99% sequence identity. Preferably, the different residue positions are due to conserved amino acid substitutions. A “conserved substitution” is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of the protein. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region or frame region of the disclosed antibody can be replaced with amino acid residues of other similar side chains. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the sequence identity percentage or degree of similarity can be adjusted upwards to correct for the conservatism of the substitution.

[0069] During monoclonal antibody production, various physicochemical factors can easily generate post-translational modification (PTM) variants, such as glycosylation, oxidation, glycation, deamidation, isomerization, and terminal cyclization. These PTMs can cause changes in the physicochemical properties of antibodies, alter their interaction with the antibody Fc receptor, and affect their binding activity to the target antigen. Some PTMs can even reduce antibody stability and induce immunogenicity (JARASCH et al., JOURNAL OF PHARMACEUTICAL SCIENCES, 2015). The negative effects of PTMs can be eliminated by modifying the amino acid sites, such as through conserved substitutions. Amino acid substitutions of antibody CDRs for the purpose of modifying PTMs are also explicitly kept within the scope of this disclosure.

[0070] The antibodies disclosed herein may also include substitutions or modifications to constant regions (e.g., Fc), including but not limited to amino acid residue substitutions, mutations, and / or modifications, which produce compounds having preferred characteristics, including but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, decreased immunogenicity, increased yield, altered binding to Fc receptors (FcRs), enhanced or weakened ADCC or CDC, altered glycosylation and / or disulfide bonds, and modified binding specificity.

[0071] The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The term "KD" refers to the dissociation constant of a specific antibody-antigen interaction. Binding affinity can be determined using a variety of techniques known in the art, such as surface plasmon resonance, biolayer interferometry, bipolar interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0072] The term "pharmaceutical composition" refers to a formulation or combination of formulations containing one, two, or more active ingredients, wherein the active ingredients contained herein are present in a biologically effective form and do not contain any additional ingredients that would have unacceptable toxicity to a subject administering the formulation. When a "pharmaceutical composition" exists as a combination of individual formulations containing two or more different active ingredients, it can be administered simultaneously, sequentially, separately, or at intervals, with the aim of exerting the biological activity of multiple active ingredients together for the treatment of a disease.

[0073] The term "antibody-drug conjugate" (ADC) refers to an antibody covalently conjugated to a bioactive molecule, such as a therapeutic active substance or active pharmaceutical ingredient (API), so that the therapeutic active substance or active pharmaceutical ingredient (API) can target the antibody's binding target to exhibit its pharmacological function. The therapeutic active substance or active pharmaceutical ingredient can be a cytotoxic agent capable of killing cells targeted by the ADC, preferably malignant or cancerous cells. The covalent conjugation of the therapeutic active substance, active pharmaceutical ingredient, or cytotoxic agent can be performed in a non-site-specific manner using standard chemical linkers that conjugate the payload to lysine or cysteine ​​residues, or preferably, the conjugation is performed in a site-specific manner, which allows complete control over the conjugation site and the drug-to-antibody ratio of the resulting ADC. The ADCs described herein can be used to deliver cytotoxic agents or other payloads to target sites (e.g., tumorigenic cells and / or cells expressing LIV-1). As used herein, the terms "drug" and "warhead" are used interchangeably and refer to a biologically active or detectable molecule or compound, including anticancer agents. A "payload" can comprise a drug or warhead in combination with an optional linker compound. The warhead can contain peptides, polypeptides, proteins, precursor drugs that are metabolized into active agents in the body, polymers, nucleic acid molecules, small molecules, binders, mimics, synthetic drugs, inorganic molecules, organic molecules, and radioactive isotopes.

[0074] As used in this article, the term "transfection" refers to the introduction of exogenous nucleic acids into eukaryotic cells. Transfection can be achieved through a variety of techniques known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and biolistics.

[0075] The term "stable transfection" or "stable transformation" refers to the introduction and integration of exogenous nucleic acids, DNA, or RNA into the genome of transfected cells. The term "stable transfectant" refers to a cell in which foreign DNA is stably integrated into the genomic DNA.

[0076] The terms "isolated polynucleotide" or "isolated nucleic acid" refer to nucleic acid molecules, DNA, or RNA that have been removed from their natural environment. For example, for the purposes of this disclosure, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or (partially or substantially) purified polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain such polynucleotide molecules, but which are present outside the chromosome or at a chromosomal location other than their natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this disclosure, as well as positive-stranded, negative-stranded, and double-stranded forms.

[0077] The terms "nucleic acid molecule encoding," "encoding DNA sequence," and "encoding DNA" refer to the sequence of deoxyribonucleotides along a deoxyribonucleic acid (DNA) chain. This sequence of deoxyribonucleotides determines the sequence of amino acids along a polypeptide (protein) chain. Therefore, a nucleic acid sequence encodes an amino acid sequence.

[0078] Methods for producing and purifying antibodies and antigen-binding fragments are well-known and available in the prior art, such as in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. The antibodies or antigen-binding fragments described in this invention are genetically engineered to add one or more human FR regions to a non-human CDR region. Human FR germline sequences are available from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr or from the journal Immunoglobulins, (2001) ISBN: 012441351.

[0079] The engineered antibodies or antigen-binding fragments thereof disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving and ion exchange.

[0080] As used herein, the terms “disease,” “symptom,” or “disorder,” etc., refer to any alteration or dysregulation that impairs or interferes with the normal function of cells, tissues, or organs. For example, “disease” includes, but is not limited to: tumors, pathogen infections, autoimmune diseases, T-cell dysfunction disorders, or deficiencies in immune tolerance (such as transplant rejection).

[0081] As used in this article, the term "treatment" refers to a clinical intervention in an attempt to alter an individual's or treat a disease caused by cells, which can be preventative or intervention in a clinicopathological process. Treatment effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and alleviating or improving prognosis.

[0082] As used herein, the term "combination" refers to a treatment regimen that provides at least two or more different therapies to achieve a specified therapeutic effect. These therapies can be physical, such as radiation therapy, or chemical, such as administering a drug to the subject, including combination drugs. "Combination drugs" refers to a combination of two or more pharmaceutical preparations, each containing an active ingredient, that are administered to a subject in combination. The active ingredients may be mixed together to form a single dosing unit or may be administered separately as independent dosing units; during administration, the different pharmaceutical preparations may be administered substantially synchronously, simultaneously, or sequentially.

[0083] Example

[0084] The present disclosure is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or according to the manufacturer's recommendations.

[0085] The positive control antibody hLIV22 in the following examples has a sequence derived from WO2012078688A2. Specifically, its heavy chain sequence is shown in SEQ ID NO:33, and its light chain sequence is shown in SEQ ID NO:34. This antibody was synthesized by Shanghai Baiying Biotechnology Co., Ltd.

[0086] Example 1: Animal Immunization

[0087] Balb / c mice, female, 6 weeks old (Shanghai Lingchang Biotechnology Co., Ltd.), were used in the experiment. The housing environment was SPF grade. After purchase, the mice were housed in a laboratory environment for one week with a 12 / 12-hour light / dark cycle, temperature 20-25℃, and humidity 40-60%. Immunization was performed via protein administration; the human LIV1 antigen was purchased from Acro Biotechnology (catalog number: LV1-H5223). Immunization was performed on days 0, 14, and 28, with a booster immunization 3 days before spleen cell fusion. During this period, mouse serum antibody titers were measured using ELISA and FACS methods. After the third immunization, mice with high and plateauing antibody titers were selected for spleen cell fusion. An optimized electrofusion procedure was used to fuse spleen lymphocytes with myeloma Sp2 / 0 cells (…). CRL-8287 TM Hybridoma cells are obtained by fusing them together.

[0088] Example 2: Screening of murine anti-LIV-1 monoclonal antibodies

[0089] After culturing the fused hybridoma cells for 7-14 days, the culture supernatant was collected. The supernatant was screened for antibodies using LIV1-Fc recombinant protein via ELISA. Positive antibody strains were further screened using CHOK1 cells stably expressing LIV1, compared with blank CHOK1 cells to exclude non-specific antibody-binding hybridoma strains. Flow cytometry was used to select hybridomas that bound both the recombinant protein and the cell antigen. Hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript). TM Reverse Transcriptase (Takara#2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev.B0503) and then sequenced.

[0090] Example 3: Evaluation of the binding activity of mouse-derived anti-LIV-1 chimeric antibody

[0091] After sequencing, chimeric antibodies were prepared by replacing the parental (mouse-derived antibody) constant domain with the human constant domain. The heavy and light chain genes were synthesized and cloned into expression plasmids, and the correctly sequenced heavy and light chain expression plasmids were co-transfected into 293F cells for expression and purification. The concentration of purified antibodies was measured at 280 nm absorbance, and antibody purity was detected by SDS-PAGE and SEC-HPLC. A total of 16 chimeric antibodies were obtained, labeled CH1–16. Their binding activity at the cellular level was evaluated, and the SPR method was used to assess the antibody affinity for human LIV-1 and cynomolgus monkey LIV-1 proteins. Specifically, CHOK1-hLIV1 cells were digested with EDTA, neutralized with fresh culture medium, centrifuged at 1000 rpm, and then a cell suspension of 1 × 10^6 / mL was prepared using FACS buffer (2% fetal bovine serum FBS). 100 μL / well was added to each well of a 96-well round-bottom plate. After centrifugation to remove the supernatant, add 100 μL of antibody (200 nM), dilute 5-fold to 8 wells, incubate at 4°C for 60 minutes, and wash 3 times. Add 100 μL of working concentration secondary antibody per well, incubate at 4°C for 60 minutes, wash 3 times, resuspend in 100 μL FACS buffer, and read the data on a flow cytometer. Finally, use GraphPad Prism 8.0 software for data processing, and select 4 chimeric antibodies with good performance. The results are shown in Figures 1A and 1B and Table 1. The EC50 values ​​for the binding of CH1, CH13, and CH2 on CHOK1-hLIV1 cells were 0.23 nM, 0.45 nM, and 0.30 nM, respectively.

[0092] Table 1 Summary of the binding activity of chimeric antibodies

[0093] Example 4 Evaluation of the internalization activity of mouse anti-LIV-1 chimeric antibody

[0094] Simultaneously, the internalization activity of the aforementioned chimeric antibodies was evaluated using the DT3C method in LIV-1 overexpressing cell lines and tumor cell lines. Specifically, well-grown MCF7 and CHOK1-hLIV1 cells were collected by centrifugation and seeded into 96-well white transparent plates at a certain cell density for overnight culture. The candidate antibody and positive control were mixed with DT3C at a 1:6 ratio and incubated at 37°C for 30 minutes to form mAb-DT3C conjugates. The mAb-DT3C conjugates were then serially diluted. The diluted mAb-DT3C conjugates were added to 96-well white transparent plates and incubated at 37°C in a 5% CO2 incubator for 6 days. After incubation, the 96-well plates were removed and equilibrated at room temperature for 30 minutes. A certain amount of [unspecified ingredient] was added to each well. Reagent was incubated at room temperature with shaking for 10 minutes, and the luminescence signal was detected using a microplate reader. The results are shown in Figures 2A, 2B, 2C and Table 2.

[0095] Table 2 Summary of the internalization activity of chimeric antibodies

[0096] Example 5: Evaluation of the binding and internalization activity of humanized antibodies

[0097] The selected chimeric leads CH1, CH2, CH7, and CH13 were humanized: chimeric antibodies were prepared by replacing the constant domain of the parent (mouse antibody) with a human constant domain, and then human antibody sequences were selected based on the homology between the parent and human antibodies for humanization. The heavy and light chain genes of each humanized variant were synthesized and cloned into expression plasmids, and the correctly sequenced heavy and light chain expression plasmids were co-transfected into 293F cells for expression and purification. The concentration of purified antibodies was measured by absorbance at 280 nm, and antibody purity was detected by SDS-PAGE and SEC-HPLC. After screening, the most active molecules from the purified humanized variants were named Ab1–4, where Ab1 was obtained from the humanization of CH1, Ab2 from the humanization of CH2, Ab3 from the humanization of CH7, and Ab4 from the humanization of CH13. Specific sequences are shown in Table 3. The CDR division of the antibody variable region was determined according to the Kabat definition.

[0098] Table 3. Amino acid sequences of each part of the variable region of humanized antibodies.

[0099] The binding and internalization activities of the humanized antibodies described above were determined using the methods of Examples 3 and 4. The results are shown in Figures 3A, 3B, 3C, and Table 4. The EC values ​​were calculated using the data processing software GraphPad Prism 8.0. 50 Values, Ab1~3, and the EC internalized on MCF7 under these experimental conditions. 50 The values ​​were 27.19 nM, 56.36 nM, and 78.68 nM, respectively, while hLIV22 EC 50 The molecular weight was 40.56 nM. Among them, Ab1 performed the best and was further tested.

[0100] Table 4 Summary of Binding and Internalization Activities of Humanized Antibodies

[0101] Example 6: Cross-species activity of humanized antibody Ab1

[0102] The cross-species activity assay for humanized antibody Ab1 assesses antibody binding activity by detecting the fluorescence signal of antibody binding on the cell surface and evaluating the intensity of the fluorescence signal. Specifically, serially diluted antibody molecules and control molecules are mixed with 1 x 10⁻⁶... 5Cells were incubated at 4°C for 1 hour, excess antibody was washed away, and murine Alexa Flour 647-labeled anti-human Fc antibody was added. The cells were incubated at 4°C for 30 minutes, excess antibody was washed away, and the cells were resuspended in 200 μL of 1% BSA / PBS buffer. Fluorescence signals on the cell surface were read by flow cytometry. The cells used were HEK293 stably expressing cynomolgus monkey LIV-1, rat LIV-1, and mouse LIV-1. Data processing software GraphPad Prism 8.0 was used for fitting and calculation of EC50. 50 The values ​​and results are shown in Table 5. Under these experimental conditions, Ab1 showed EC values ​​similar to those of LIV-1 cynomolgus monkeys. 50 The concentration was 0.41 nM, and it showed virtually no binding to rat LIV-1 and mouse LIV-1.

[0103] Table 5. Cross-species activity of humanized antibody Ab1

[0104] Example 7: Binding specificity of humanized antibody Ab1

[0105] The binding activity of Ab1 with other LIV-1 family members (ZIP10, ZIP4, ZIP5, ZIP12) was detected. Specifically, 100 μL / well of antigen (LIV-1, ZIP10, ZIP4, ZIP5, ZIP12: 2 μg / mL) was coated and incubated overnight at 4°C. Blocking was performed at room temperature for 1 hour with 100 μL of blocking buffer (2% BSA in PBS). Washing was repeated three times with PBST using a BioTek automated plate washer. 100 μL of diluted antibody (starting concentration 200 nM, serially diluted 1:5) was added to each well. Incubation was performed at room temperature for 2 hours. Washing was repeated three times with PBST. 100 μL of diluted HRP anti-human secondary antibody was added to each well and incubated at room temperature for 1 hour. Washing was repeated six times with PBST. 100 μL of TMB substrate was added to each well and incubated at room temperature for 5 minutes, followed by 100 μL of stop solution to stop the reaction. The OD values ​​at a wavelength of 450 nm were read using an Envision microplate reader. The results are shown in Table 6.

[0106] Table 6. Binding specificity of humanized antibody Ab1

[0107] The half-binding concentration (EC50) was calculated using GraphPad Prism 8.0 software. 50 Ab1 showed no significant binding activity with other members of the ZIP family (ZIP10, ZIP4, ZIP5, ZIP12), indicating that the antibody obtained in this disclosure has good selectivity.

[0108] Example 8: ADCC and CDC activities of humanized antibody Ab1

[0109] Both naked anti-Ab1 and hLIV22 Fc isoforms are IgG1, and ADCC activity was evaluated on MCF7 tumor cells using CTG. Specifically, MCF7 target cells were first plated. The viability of target cells used in the ADCC assay should be higher than 95%. After collecting MCF7 cells, the cell suspension concentration was adjusted to 1.5E5 / mL in MEM + 2% FBS. 100 μL was added to the wells of a 96-well plate (3799). NK cells were counted, and the suspension was adjusted to 7.5E5 / mL, with 50 mL added to each well (E:T ratio of 5:1). The antibody was diluted and added to the corresponding wells, 50 mL / well. The mixture was gently mixed and incubated at 37°C for 24 hours with 5% CO2. The lymphocyte culture supernatant was removed, 50 μL of CTG was added to each well, and the cells were incubated for 10 minutes. The chemiluminescence value was then read using a microplate reader. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 8.0, and the results are shown in Table 7. Both Ab1 and hLIV22 can induce ADCC effects in MCF7 cells in a dose-dependent manner, and the ADCC effect of Ab1 is slightly stronger than that of the control antibody hLIV22.

[0110] Table 7 ADCC activity of humanized antibody Ab1

[0111] Simultaneously, CDC activity was evaluated on MCF7 tumor cells using CTG. Specifically, MCF7 target cells were first plated. The viability of target cells used in the CDC assay should be higher than 95%. After collecting MCF7 cells, the cell suspension concentration was adjusted to 6E5 / mL in MEM + 10% FBS. 50 μL was added to the wells of a 96-well plate (Corning, 3903) and incubated at 37°C and 5% CO2. 4× antibody stock solution was prepared, diluted 5-fold with MEM medium, and added to the corresponding wells. 25 μL of antibody was added to each well and incubated at 4°C. Human complement and MEM medium (FBS-free) were diluted 1:2.5, and 25 μL / well was added to the 96-well plate and incubated at 37°C and 5% CO2 for 6 h or 24 h. Finally, 50 μL of CTG reagent was added to each well, and the plate was incubated at room temperature for 10 min in the dark. The plate was shaken at 300 rpm and the chemiluminescence value was read using a microplate reader. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 8.0 for fitting. 50 The results showed that neither Ab1 nor hLIV22 induced the CDC effect in MCF7 cells.

Claims

1. An antibody against LIV-1 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment being specifically capable of binding to LIV-1, comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, said light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the amino acid sequences of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively selected from the group consisting of: (1) SEQ ID NO: 1, 2, 3, 4, 5, 6; (2) SEQ ID NO: 7, 8, 9, 10, 11, 12; (3) SEQ ID NO: 13, 14, 15, 16, 17, 18; or (4) SEQ ID NO: 19, 20, 21, 22, 23, 24.

2. An antibody against LIV-1 or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 80% sequence identity with a heavy chain variable region and a light chain variable region of any one of the following groups: (1) SEQ ID NO: 25 and 26; (2) SEQ ID NO: 27 and 28; (3) SEQ ID NO: 29 and 30; or (4) SEQ ID NO:31 and 32.

3. The anti-LIV-1 antibody or its antigen-binding fragment as described in claim 1 or 2, further comprising one or more of the following characteristics: (1) It also includes a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region contains Fc; more preferably, Fc is derived from mice or humans; more preferably, the sequence of Fc is a natural or modified variant; (2) It is a mouse-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; (3) It is a monoclonal antibody; or a full-length antibody, or its antigen-binding fragment is Fab, Fv, scFv, F(ab')2, linear antibody, or single-domain antibody; (4) It is in the form of IgG1, IgG2, IgG3 or IgG4.

4. An antibody-drug conjugate formed by conjugating an anti-LIV-1 antibody or its antigen-binding fragment as described in any one of the preceding claims with other bioactive molecules, preferably, the other bioactive molecules being small molecule drugs, such as antitumor drugs, preferably antitumor compounds, and preferably, the antibody and other bioactive molecules being linked by a linker.

5. A conjugate formed by conjugating an antibody against LIV-1 as described in any one of the preceding claims or an antigen-binding fragment thereof with a capture marker or a detection marker, preferably, the detection marker including but not limited to a radionuclide, a luminescent substance (e.g., fluorescein), a colored substance, or an enzyme.

6. A fusion protein, wherein one fused portion comprises an antibody against LIV-1 as described in any one of claims 1-3 or an antigen-binding fragment thereof.

7. A bispecific antibody or a multispecific antibody, wherein one antigen-binding domain comprises the anti-LIV-1 antibody or its antigen-binding fragment as described in any one of claims 1-3.

8. Nucleic acid encoding the antibody against LIV-1 as described in any one of claims 1-3 or its antigen-binding fragment, the fusion protein as described in claim 6, or the bispecific antibody or multispecific antibody as described in claim 7.

9. A recombinant vector comprising the nucleic acid of claim 8.

10. A host cell comprising the recombinant vector of claim 9 or the nucleic acid of claim 8.

11. The host cell of claim 10, wherein it is a prokaryotic cell, such as Escherichia coli; or a eukaryotic cell, such as yeast or a mammalian cell, such as a CHO cell or a HEK293 cell.

12. A method for preparing the anti-LIV-1 antibody or its antigen-binding fragment according to any one of claims 1-3, the fusion protein according to claim 6, or the bispecific antibody or multispecific antibody according to claim 7, comprising: The host cells of claim 10 or 11 are cultured under suitable conditions, and the expression product is purified from said cells.

13. A method for detecting LIV-1 in a sample, comprising: (1) Contact the sample with the anti-LIV-1 antibody or its antigen-binding fragment as described in any one of claims 1-3, the fusion protein as described in claim 6, or the bispecific antibody or multispecific antibody as described in claim 7; (2) Detect the formation of a complex of an anti-LIV-1 antibody or its antigen-binding fragment, fusion protein, bispecific antibody or multispecific antibody with LIV-1; optionally, the anti-LIV-1 antibody or its antigen-binding fragment, fusion protein, bispecific antibody or multispecific antibody is detectably labeled.

14. A pharmaceutical composition comprising an effective amount of any anti-LIV-1 antibody of claim 1-3 or an antigen-binding fragment thereof, or an effective amount of an antibody-drug conjugate of claim 4, or an effective amount of a fusion protein of claim 6, or an effective amount of a bispecific or multispecific antibody of claim 7, or an effective amount of a nucleic acid of claim 8, or an effective amount of a recombinant vector of claim 9, or an effective amount of a host cell of claim 10 or 11.

15. The pharmaceutical composition of claim 14, further comprising a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 14 or 15, further comprising one or more additional therapeutic agents.

17. A method of treating a subject with a disease associated with LIV-1 expression, the method comprising administering to a subject in need of the pharmaceutical composition of any one of claims 14-16.

18. The method of claim 17, wherein the disease is a tumor, particularly a solid tumor.

19. The method of claim 17 or 18, further comprising administering additional therapeutic agents to the subject.

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