Anti-gpc1 antibodies and uses thereof

Abstract

The present disclosure relates to anti-GPC1 (glypican-1) antibodies, antigen-binding fragments thereof, and uses thereof, also including antibody-drug conjugate formats intended for cancer therapy.

Description

Technical Field

[0001] This disclosure relates to antibodies against GPC1 (phosphatidylinositol proteoglycan-1 (Glypican-1)), their antigen-binding fragments, and their uses. Background Technology

[0002] Phosphatidylinositol proteoglycans (GPCs) belong to the heparan sulfate proteoglycan (HSPG) family and interact with the plasma membrane via a glycosylphosphatidylinositol anchor. In humans, six GPC family members have been identified, including GPC1, GPC2, GPC3, GPC4, GPC5, and GPC6. Phosphatidylinositol proteoglycans are primarily expressed during embryonic development and have been reported to play important roles in organ morphology development by influencing signaling pathways including Wnt, Hedgehog, transforming growth factor-β, and fibroblast growth factor. Phosphatidylinositol proteoglycans are involved in many important processes, including cell proliferation, migration, differentiation, extracellular matrix remodeling, and tumor microenvironment remodeling. Studies have shown that aberrant expression of GPC1 has been detected in various cancers, and that GPC1 dysregulation can affect cancer progression. GPC1 has been shown to be a useful biomarker for various cancer tissues, such as prostate cancer, hepatocellular carcinoma, and pancreatic cancer. Given the important role of GPC1 in various tumors, there is a need to develop therapeutics targeting GPC1. Summary of the Invention

[0003] This disclosure relates to anti-GPC1 antibodies, their antigen-binding fragments, and their uses.

[0004] On one hand, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to GPC1 (phosphatidylinositol proteoglycan-1), comprising: The heavy chain variable region (VH) contains complementarity-determining regions (CDRs) 1, 2, and 3, wherein VH CDR1 contains an amino acid sequence that is at least 85% identical to the selected VH CDR1 amino acid sequence, VH CDR2 contains an amino acid sequence that is at least 85% identical to the selected VH CDR2 amino acid sequence, and VH CDR3 contains an amino acid sequence that is at least 85% identical to the selected VH CDR3 amino acid sequence; and The light chain variable region (VL) comprises CDRs 1, 2, and 3, wherein the VL CDR1 region contains an amino acid sequence that is at least 85% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region contains an amino acid sequence that is at least 85% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region contains an amino acid sequence that is at least 85% identical to the selected VL CDR3 amino acid sequence. The selected VH CDR 1, 2, and 3 amino acid sequences and the selected VL CDR 1, 2, and 3 amino acid sequences are one of the following: (1) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4-6, respectively; and (2) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 7-9, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4-6.

[0005] In some embodiments, according to the Kabat definition, the VH comprises CDR 1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 1-3, respectively, and the VL comprises CDR 1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 4-6, respectively.

[0006] In some embodiments, according to the definition of Chothia, the VH comprises CDR 1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 7-9, respectively, and the VL comprises CDR 1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 4-6, respectively.

[0007] In some embodiments, the antibody or its antigen-binding fragment specifically binds to human, monkey, mouse, and / or rat GPC1.

[0008] In some embodiments, the antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment, or a single-chain variable fragment (scFv).

[0009] In some embodiments, the antibody or its antigen-binding fragment is a human IgG1 antibody or its antigen-binding fragment, or a human IgG4 antibody or its antigen-binding fragment.

[0010] On the one hand, this disclosure relates to a nucleic acid comprising a polynucleotide encoding the antibody or an antigen-binding fragment thereof.

[0011] In some implementations, the nucleic acid is cDNA.

[0012] On the one hand, this disclosure relates to a vector that contains one or more nucleic acids described herein.

[0013] On one hand, this disclosure relates to a cell comprising the vector described herein. In one embodiment, the cell is an isolated cell, such as an isolated host cell. In some embodiments, the cell is a CHO cell.

[0014] On the one hand, this disclosure relates to a cell comprising one or more nucleic acids described herein.

[0015] On one hand, this disclosure relates to a method for producing an antibody or an antigen-binding fragment thereof, the method comprising culturing the cells described herein under conditions sufficient to cause the cells to produce the antibody or the antigen-binding fragment; and collecting the antibody or the antigen-binding fragment produced by the cells.

[0016] On one hand, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to GPC1, comprising a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 10 and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 11.

[0017] In some embodiments, the antibody or its antigen-binding fragment specifically binds to human, monkey, mouse, and / or rat GPC1.

[0018] In some embodiments, the antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment, or a single-chain variable fragment (scFv).

[0019] In some embodiments, the antibody or its antigen-binding fragment is a human IgG1 antibody or its antigen-binding fragment, or a human IgG4 antibody or its antigen-binding fragment.

[0020] On one hand, this disclosure relates to a method of treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein.

[0021] In some implementations, the cancer is stomach cancer, breast cancer, bladder cancer, pancreatic cancer, lung cancer, or colorectal cancer.

[0022] On one hand, this disclosure relates to a method for reducing the growth rate of tumors, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein.

[0023] On one hand, this disclosure relates to a method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein.

[0024] On the one hand, this disclosure relates to a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described herein, and a pharmaceutically acceptable carrier.

[0025] As used herein, the term “cancer” refers to cells capable of autonomous growth. Examples of such cells include cells exhibiting an abnormal state or condition characterized by rapid proliferative cell growth. The term is intended to include cancerous growth, such as tumors; carcinogenic processes, metastatic tissues, and cells, tissues, or organs undergoing malignant transformation, regardless of their histopathological type or stage of invasion. It also includes malignant tumors of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, hematologic, nervous, hepatic, gastrointestinal, and endocrine systems; and adenocarcinomas, including malignant tumors such as most colon cancers, renal cell carcinomas, prostate and / or testicular tumors, non-small cell lung cancer, and small bowel cancer. “Spontaneously occurring” cancer includes any cancer not experimentally induced by implanting cancer cells into a subject, and includes, for example, spontaneously occurring cancer, cancer caused by patient exposure to carcinogens, cancer resulting from the insertion of transgenic oncogenes or the knockout of tumor suppressor genes, and cancer caused by infection (e.g., viral infection). The term “carcinoma” is well-known in the art and refers to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcoma, which comprises malignant tumors composed of cancerous and sarcomatous tissue. "Adenocarcinoma" refers to cancer originating from glandular tissue or in which tumor cells form identifiable glandular structures. The term "sarcoma," as is well known in the art, refers to a malignant tumor of stromal origin. The term "hematopoietic neoplasia" includes diseases involving proliferative / neoplastic cells of hematopoietic origin. Hematopoietic neoplasia can originate from myeloid, lymphoid, or erythroid lineages or their precursor cells.

[0026] As used herein, the term "antibody" refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementarity-determining regions (CDRs) (e.g., any one of the three CDRs from the immunoglobulin light chain or any one of the three CDRs from the immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives, such as single-chain antibodies, biantibodies, and linear antibodies formed from antibody fragments.

[0027] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody that is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment comprises at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0028] As used herein, the term "human antibody" refers to an antibody encoded by an endogenous nucleic acid present in the human body (e.g., a rearranged heavy or light chain human immunoglobulin locus). In some embodiments, human antibodies are collected from the human body or generated in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies are generated in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are generated in bacterial or yeast cells. In some embodiments, human antibodies are generated in transgenic non-human animals (e.g., cattle) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0029] As used herein, the term "chimeric antibody" refers to an antibody that contains sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as humans and mice). A non-limiting example of a chimeric antibody is an antibody that contains all or part of a variable domain sequence (e.g., a light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and a constant domain of a human antibody. Other examples of chimeric antibodies are described herein and are known in the art.

[0030] As used herein, the term "humanized antibody" refers to a non-human antibody containing a minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and a sequence derived from a human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (receptor antibody) in which hypervariable (e.g., CDR) region residues of the receptor antibody are replaced by hypervariable (e.g., CDR) region residues of a non-human antibody (e.g., donor antibody) (e.g., mouse, rat, or rabbit antibody) having the desired specificity, affinity, and capability. In some embodiments, FR framework residues of a human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, the humanized antibody may contain residues not found in either the receptor antibody or the donor antibody. These modifications are made to further improve antibody performance. In some embodiments, the humanized antibody comprises substantially all of at least one, typically two, variable domains, wherein all or substantially all of the hypervariable loops (CDRs) correspond to those of non-human (e.g., mouse) immunoglobulins, and all or substantially all of the framework regions are those of human immunoglobulins. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of human immunoglobulins. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

[0031] As used herein, the term "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0032] As used herein, the term "multimeric antibody" refers to an antibody containing four or more (e.g., six, eight, or ten) immunoglobulin variable domains. In some embodiments, the multimeric antibody is capable of crosslinking a target molecule (e.g., GPC1) with at least one second target molecule on the surface of mammalian cells (e.g., human T cells).

[0033] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe animals, humans, or non-humans to whom the treatment according to the method of the invention is provided. The invention is contemplated for both veterinary and non-veterinary applications. Human patients can be adult humans or adolescent humans (e.g., humans under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. This includes, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domesticated, farm, and zoo animals.

[0034] As used herein, when referring to antibodies, the phrases "specifically binding" and "specifically binds" indicate that the antibody interacts preferentially with its target molecule (e.g., GPC1) over other molecules because this interaction depends on the presence of a specific structure on the target molecule (i.e., an antigenic determinant or epitope); in other words, the reagent recognizes and binds to molecules containing a specific structure, rather than binding to all molecules in general. Antibodies that specifically bind to a target molecule are called target-specific antibodies. For example, an antibody that specifically binds to the GPC1 molecule can be called a GPC1-specific antibody or an anti-GPC1 antibody.

[0035] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to an amino acid polymer of any length containing at least two amino acids.

[0036] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably to refer to a nucleotide polymer of any length containing at least two nucleotides, including but not limited to DNA, RNA, DNA / RNA hybrids, and their modifications.

[0037] 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 invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples described are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification (including definitions) shall prevail.

[0038] Other features and advantages of the invention will become apparent from the following detailed description and claims. Invention Details

[0040] This disclosure provides examples of antibodies that bind to GPC1 and their antigen-binding fragments.

[0041] GPC1

[0042] Heparan sulfate proteoglycans (HSPGs) are glycoproteins composed of a core protein covalently bound to several heparan sulfate (HS) glycosaminoglycan (GAG) chains. HSPGs are widely expressed on cell surfaces and in the extracellular matrix, where they interact with a wide range of ligands to mediate various cellular functions. HSPGs are classified into three groups based on their location: membrane HSPGs (e.g., cohesinic and phosphatidylinositol proteoglycans), secretory extracellular matrix HSPGs (synaptic proteoglycans, basement membrane proteoglycans, and type XVIII collagen), and secretory vesicle proteoglycans (serine proteoglycans).

[0043] Phosphatidylinositol proteoglycans of the HSPG family attach to the outer surface of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Six members of the human phosphatidylinositol proteoglycan family (GPC1 to GPC6) have been identified, which are divided into two broad subfamilies, phosphatidylinositol proteoglycans 1 / 2 / 4 / 6 and phosphatidylinositol proteoglycans 3 / 5, sharing ~25% amino acid identity. The three-dimensional structures of phosphatidylinositol proteoglycans are likely similar across the entire family because the positioning of 14 cysteine ​​residues is conserved in all family members. All phosphatidylinositol proteoglycans share attachment sites on the HS chain, particularly two sites close to the cell membrane, as well as the hydrophobic sequence necessary for the C-terminal GPI anchor.

[0044] GPC1 comprises 558 amino acids and consists of a secretory signal peptide, an N-terminal core protein, a C-terminal HS chain attachment region, and a GPI anchor attached to the cell membrane. The GPC1 protein is substituted by three HS chain clusters at Ser-486, Ser-488, and Ser-490, and further modified by two N-linked glycans at Asn-79 and Asn-116, these modifications affect GPC1 expression levels and HS substitution. The GPC1 structure with HS chains is associated with its biological function by modulating multiple signaling pathways through specific interactions with ligands and receptors in cell surface signaling complexes. The actual orientation and possible rotation of GPC1 (and other phosphatidylinositol proteoglycans) relative to the cell surface are unknown. The effects of these surface signaling complexes, whether activating or inhibiting, depend on the interaction of specific GPC1 domains with signaling components under specific physiological conditions.

[0045] FGF is a secreted glycoprotein that is readily chelated by the extracellular matrix and cell surface HPSG (including GPC1). GPC1 has been proposed as a co-receptor for FGF, enhancing FGF binding to its receptor and subsequently promoting FGF-FGFR activation and signaling. FGF binding to its receptor leads to receptor dimerization and transphosphorylation of the tyrosine kinase domain, resulting in the subsequent activation of various signaling pathways, including Ras-MAPK, PI3K-AKT-mTOR, and DAG-PKC. All signaling cascades ultimately lead to enhanced growth, survival, and angiogenesis. FGF2 is one of the heparin-binding growth factors (HBGFs) that drives tumor cell proliferation upon binding to its receptor and also plays a role in angiogenesis as an angiogenic growth factor. Increasing evidence suggests that changes in GPC1 expression have a profound impact on FGF2-induced cell proliferation or angiogenesis in tumors. For example, in breast cancer, GPC1 upregulation promotes tumor mitotic signaling by modulating heparin-binding growth factors (including FGF2). GPC1 has also been shown to work with type V collagen to concentrate FGF2 at the extracellular matrix (ECM) interface, thereby affecting ECM stability and breast tumor cell proliferation.

[0046] Similar to FGF2, VEGF-A is also angiogenic growth factor and plays an important role in angiogenesis. GPC1 acts as a co-receptor for VEGF-A, and this interaction is mediated by its HS chain. Removal of the HS chain via heparinase treatment eliminates GPC1's ability to bind VEGF. Cell binding assays using ECs also demonstrated that the addition of exogenous GPC1 enhances VEGF-A / VEGFR binding, suggesting a potential role for GPC1 in controlling angiogenesis.

[0047] Transforming growth factor-β (TGF-β) belongs to the cytokine superfamily and activates protein kinase receptors on the plasma membrane to regulate cell growth, death, differentiation, immune responses, angiogenesis, and inflammation. Dysregulation of this pathway leads to a wide range of pathologies, including cancer. However, TGF-β signaling is considered a challenging target due to its dual function and pleiotropic nature. In healthy cells and early-stage cancer cells, TGF-β acts as a tumor suppressor, while in late-stage cancer, it drives tumor progression and metastasis. GPC1 has been shown to interact with TGF-β and its receptor to stabilize their assembly, thereby enhancing Smad signaling.

[0048] Bone morphogenetic proteins (BMPs) play a crucial role in cell-cell communication during animal development and are potent growth factors that promote bone formation. Phosphatidylinositol proteoglycans have been shown to modulate BMP activity. GPC1 protein is primarily expressed in the human skeletal system and has also been identified in developing mouse skulls and skeletal structures. Therefore, it can be inferred that GPC1 is also involved in the regulation of the BMP signaling pathway. Based on currently available evidence, GPC1 appears to act as an inhibitor of BMP signaling regulation during osteogenic processes. Anchored GPC1 or soluble GPC1 on the cell surface can compete with BMP receptors for interaction with BMP ligands, resulting in a reduction in the amount of BMP available for binding to its receptors, thereby attenuating Smad-dependent or Smad-independent BMP signaling.

[0049] GPC1 has been reported to exist in two forms: a membrane-bound core protein and a secreted soluble form. It can be detected by immunoprecipitation in serum-free culture medium harvested from prostate cancer cells DU-145, possibly due to GPC1 shedding or proteolytic cleavage at the GPI anchor, a process also observed in GPC3. However, the specific cleavage mechanism or site remains unknown.

[0050] Human GPC1 is expressed not only in the central nervous system (CNS) and skeletal system during development, but also in other tissues in adults. GPC1 is overexpressed in a variety of cancer types, including breast cancer, esophageal squamous cell carcinoma (ESCC), glioma, and pancreatic cancer. Its high expression is associated with poor prognosis, making it a potential target for cancer therapy.

[0051] A detailed review of GPC1 and its functions can be found in Pan J, Ho M. Role of glypican-1 inregulating multiple cellular signaling pathways. Am J Physiol Cell Physiol. 2021 Nov 1;321(5):C846-C858, which is incorporated herein by reference in its entirety.

[0052] This disclosure provides anti-GPC1 antibodies, their antigen-binding fragments, and methods for using these anti-GPC1 antibodies and antigen-binding fragments to inhibit tumor growth and treat cancer.

[0053] Antibody and antigen binding fragment

[0054] This disclosure provides an anti-GPC1 antibody and its antigen-binding fragment. Typically, antibodies (also called immunoglobulins) consist of two classes of polypeptide chains (light chains and heavy chains). The non-limiting antibody of this disclosure can be a complete four-component immunoglobulin chain antibody, comprising two heavy chains and two light chains. The heavy chain of the antibody can be any isotype, including IgM, IgG, IgE, IgA, or IgD, or subisotypes, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a κ light chain or a λ light chain. The antibody may contain two identical copies of the light chain and two identical copies of the heavy chain. Each contains a variable domain (or variable region, V...). H The antibody consists of heavy chains containing one variable domain (or variable region) and multiple constant domains (or constant regions), which are linked together by disulfide bonds within their constant domains to form the antibody's "stem." Each heavy chain contains one variable domain (or variable region). L A light chain and a constant structural domain (or constant region) are each bonded to a heavy chain via disulfide bonds. The variable region of each light chain is aligned with the variable region of the heavy chain it is bonded to. The variable regions of both the light and heavy chains contain three highly variable regions sandwiched between more conservative framework regions (FRs).

[0055] These hypervariable regions, known as complementarity-determining regions (CDRs), form loops containing the antibody's primary antigen-binding surface. The four framework regions primarily employ a β-sheet conformation, with CDRs forming loops that connect and, in some cases, form part of the β-sheet structure. CDRs in each chain remain closely adjacent through the framework regions and, together with CDRs from other chains, contribute to the formation of the antigen-binding region.

[0056] Identifying the antibody CDR region by analyzing the amino acid sequence of the antibody is a well-known method, and several CDR definitions are commonly used. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of the structural loop region. These methods and definitions are described in, for example, Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody Engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan et al., "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular Immunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med.132: 211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(1-3):9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2):269-94 (Jan. 1998); Chothia et al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated into this paper in its entirety by reference.

[0057] CDR is important for recognizing epitopes of antigens. As used in this article, an "epitaph" is the smallest part of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about three, four, five, six, or seven amino acids, but these amino acids do not have to be in a continuous linear sequence of the primary antigen structure, as the epitope can depend on the three-dimensional conformation of the antigen based on its secondary and tertiary structures.

[0058] In some implementations, antibodies are complete immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, but their constant regions, particularly their hinge region and upper CH2 domain, differ. The sequences and differences of IgG subclasses are known in the art and described, for example, in Vidarsson et al., "IgG subclasses and allotypes: from structure to effector functions." Frontiers in Immunology 5 (2014); Irani et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular Immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

[0059] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, camels). The antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies comprising an immunoglobulin-binding domain fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" refers to an antibody portion that retains the complete antibody-specific binding activity, i.e., any portion of an antibody capable of specifically binding to an epitope on a complete antibody target molecule. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Therefore, in some embodiments, an antibody or its antigen-binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibody, bispecific scFv, biantibody, linear antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising an antibody-binding domain or a binding domain homologous to an antibody-binding domain. Non-limiting examples of antigen-binding domains include, for example, heavy chain and / or light chain CDRs of an intact antibody, variable regions of heavy chain and / or light chain of an intact antibody, full-length heavy chain or light chain of an intact antibody, or a single CDR derived from the heavy chain or light chain of an intact antibody.

[0060] Anti-GPC1 antibody and antigen-binding fragment

[0061] This disclosure provides antibodies that specifically bind to GPC1 (e.g., human GPC1) and antigen-binding fragments thereof. The antibodies and antigen-binding fragments described herein are capable of binding to GPC1. These antibodies can be agonists or antagonists. In some embodiments, these antibodies can enhance an immune response. In some embodiments, these antibodies can block GPC1 activity.

[0062] This disclosure provides, for example, anti-GPC1 antibody 3G1, its chimeric antibodies, and human or humanized antibodies thereof.

[0063] The CDR sequences of 3G1 and antibodies derived from 3G1 (e.g., human or humanized antibodies) include the heavy chain variable domain CDR and light chain variable domain CDR as defined by Kabat and Chothia, as shown in the sequence overview herein.

[0064] The amino acid sequences of the heavy chain variable region and the light chain variable region of 3G1 are shown in the sequence overview in this article.

[0065] In some embodiments, the antibody or its antigen-binding fragment described herein may include one, two or three heavy chain variable regions (CDRs) selected from the groups SEQ ID NO:1-3 and SEQ ID NO:7-9, and / or one, two or three light chain variable regions (CDRs) selected from the groups SEQ ID NO:4-6.

[0066] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, 3, wherein CDR1 contains or is composed of an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to or constitutes the amino acid sequence of a selected VH CDR1, CDR2 contains or is composed of an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to or constitutes the amino acid sequence of a selected VH CDR2, and CDR3 contains or is composed of an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to or constitutes the amino acid sequence of a selected VH CDR3. In some embodiments, the antibody may have a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the CDR1 region contains or is composed of an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to or constitutes the selected VL CDR1 amino acid sequence, the CDR2 region contains or is composed of an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to or constitutes the selected VL CDR2 amino acid sequence, and the CDR3 region contains or is composed of an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to or constitutes the selected VL CDR3 amino acid sequence. The selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs 1, 2, and 3 amino acid sequences are as outlined in the sequence summary herein.

[0067] This disclosure also provides antibodies or antigen-binding fragments thereof that bind to GPC1. The antibodies or antigen-binding fragments thereof comprise: a heavy chain variable region (VH) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 10, and a light chain variable region (VL) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11.

[0068] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecule is identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. Taking into account the number of gaps introduced for optimal alignment and the length of each gap, the percentage identity between two sequences is a function of the number of shared positions. For example, a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5 can be used to perform sequence comparisons and determine the percentage identity between two sequences.

[0069] This disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises a CDR or has a sequence as shown in the sequence summary herein. When the polypeptide pairs with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to GPC1 (e.g., human GPC1).

[0070] Anti-GPC1 antibodies and antigen-binding fragments can also be antibody variants or antibody fragments. Other antibodies described herein are polyclonal, monoclonal, human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly generated antibodies (i.e., intracellular antibodies), and their antigen-binding fragments. The antibody or its antigen-binding fragment can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0071] As long as the desired affinity and specificity of the full-length antibody are preserved, antibody fragments are suitable for use with the methods presented herein. Therefore, fragments of antibodies binding to GPC1 will retain the ability to bind to GPC1. Fv fragments are antibody fragments containing complete antigen recognition and binding sites. This region consists of a tightly associated dimer of a heavy-chain variable domain and a light-chain variable domain, which can be covalent, for example, in scFv. It is in this configuration that the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. The six CDRs, or a subset thereof, collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only three antigen-specific CDRs) can have the ability to recognize and bind antigens, although typically with lower affinity than the entire binding site.

[0072] Single-chain Fv or (scFv) antibody fragments contain the VH and VL domains (or regions) of the antibody, which are located within a single polypeptide chain. Typically, scFv polypeptides also contain a polypeptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding.

[0073] The Fab fragment contains variable and constant domains of the light chain and a variable domain and a first constant domain (CH1) of the heavy chain. The F(ab')2 antibody fragment comprises a pair of Fab fragments, which are typically covalently linked near their carboxyl terminus by a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known in the art.

[0074] Linear antibodies consist of a pair of tandem Fd regions (VH-CH1-VH-CH1), which together with a complementary light chain polypeptide form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0075] The antibodies and antibody fragments disclosed herein can be modified in the Fc region to provide desired effector function or serum half-life. In some embodiments, the Fc region can be modified to silence or reduce complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the Fc region can be modified to increase complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC).

[0076] Antibody polymerization can be achieved through the natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, a certain percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody polymers.

[0077] Alternatively, antibody homodimers can be formed using chemical linking techniques known in the art. For example, heterobifunctional crosslinking agents, including but not limited to SMCC (succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylic acid ester) and SATA (N-succinimide-S-acetylthioacetate), can be used to form antibody polymers. Exemplary schemes for forming antibody homodimers are described in Ghetie et al. (…). Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997). Antibody homodimers can be converted into Fab'2 homodimers by pepsin digestion. Another way to form antibody homodimers is by using Zhao et al. ( J. Immunol. The homosexual T15 peptide described in 25:396-404, 2002.

[0078] Any antibody or antigen-binding fragment described herein may be conjugated to a stable molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stable molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation to a stable molecule may increase the half-life or prolong the biological activity of the antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0079] In one or more embodiments of this disclosure, the antibody or antigen-binding fragment thereof characterized by binding to GPC1 may be characterized by at least one of the following: a. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) Immunoglobulin light chain variable region (VL), comprising the amino acid sequence shown in SEQ ID NO:4 as VL complementarity-determining region 1 (CDR1), the amino acid sequence shown in SEQ ID NO:5 as VL CDR2, and the amino acid sequence shown in SEQ ID NO:6 as VL CDR3, and / or ii) Immunoglobulin heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 1 as VH complementarity-determining region 1 (CDR1), the amino acid sequence shown in SEQ ID NO: 2 as VH CDR2, and the amino acid sequence shown in SEQ ID NO: 3 as VH CDR3; or b. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) Immunoglobulin light chain variable region (VL), comprising the amino acid sequence shown in SEQ ID NO:4 as VL complementarity-determining region 1 (CDR1), the amino acid sequence shown in SEQ ID NO:5 as VL CDR2, and the amino acid sequence shown in SEQ ID NO:6 as VL CDR3, and / or ii) Immunoglobulin heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 7 as VH complementarity-determining region 1 (CDR1), the amino acid sequence shown in SEQ ID NO: 8 as VH CDR2, and the amino acid sequence shown in SEQ ID NO: 9 as VH CDR3; or c. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) The immunoglobulin light chain variable region (VL), comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11 or a sequence having at least 85% sequence identity with it; and / or ii) The immunoglobulin heavy chain variable region (VH), comprising or consisting of the amino acid sequence shown in SEQ ID NO: 10 or a sequence having at least 85% sequence identity with it; or d. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) Immunoglobulin light chains comprising, or having at least 85% sequence identity with, the amino acid sequence shown in SEQ ID NO: 11, and the amino acid sequence shown in SEQ ID NO: 15, or having at least 85% sequence identity with, or consisting of said sequences; and / or ii) An immunoglobulin heavy chain comprising, or having at least 85% sequence identity with, the amino acid sequence shown in SEQ ID NO: 10, and the amino acid sequence shown in SEQ ID NO: 14, or having at least 85% sequence identity with, or consisting of said sequences; or e. An antibody that competes with an antibody or antigen-binding fragment as defined in any of a to d.

[0080] Antibody characteristics

[0081] The antibodies or antigen-binding fragments thereof described herein may be GPC1 agonists or antagonists. In some embodiments, the antibodies can inhibit GPC1 activity by binding to GPC1. In some embodiments, the antibodies can upregulate or downregulate the immune response.

[0082] In some embodiments, the antibody or its antigen-binding fragment specifically binds to GPC1 (e.g., human GPC1, monkey GPC1 (e.g., rhesus monkey, cynomolgus monkey)). Macaca fascicularis ( ), mouse GPC1 and / or rat GPC1), with a dissociation rate (koff) of less than 0.1 s. - ¹, less than 0.01 s - ¹, less than 0.001 s - ¹, less than 0.0001 s - ¹, less than 0.00001 s - ¹, less than 0.000001 s - ¹or less than 0.0000001 s - ¹. In some implementations, the dissociation rate (koff) is greater than 0.01 s. - ¹, greater than 0.001 s - ¹, greater than 0.0001 s - ¹, Greater than 0.00001 s - ¹, Greater than 0.000001 s - ¹, greater than 0.0000001s - ¹ or greater than 0.00000001 s - ¹.

[0083] In some implementations, the kinetic binding rate (kon) is greater than 1 x 10⁻⁶. 2 / Ms, greater than 1 x 10 3 / Ms, greater than 1 x 10 4 / Ms, greater than 1 x 10 5 / Ms or greater than 1 x 10 6 / Ms. In some implementations, the kinetic binding rate (kon) is less than 1 x 10 5 / Ms, less than 1 x 10 6 / Ms or less than 1 x 10 7 / Ms.

[0084] The affinity can be derived from the quotient of the kinetic rate constant (KD = koff / kon). In some implementations, KD is less than 1 x 10⁻⁶. -6 M, less than 1 x 10 -7 M, less than 1 x 10 -8 M, less than 1 x 10 -9 M, less than 1 x 10 -10 M, less than 1 x 10 -11 M, less than 1 x 10 -12 M, less than 1 x 10 -13 M or less than 1 x 10 -14 M. In some embodiments, KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10⁻⁶. -7 M, greater than 1 x 10 -8 M, greater than 1 x 10 -9 M, greater than 1 x 10 -10 M, greater than 1 x 10 -11 M, greater than 1 x 10 -12 M, greater than 1 x 10 -13 M, greater than 1 x 10 -14 M.

[0085] Common techniques used to measure antibody affinity for antigens include, for example, ELISA, RIA, and surface plasmon resonance (SPR).

[0086] In some embodiments, the tumor growth inhibition percentage (TGI%) of the antibody or its antigen-binding fragment described herein is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the tumor growth inhibition percentage of the antibody or its antigen-binding fragment described herein is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TGI% can be determined, for example, at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, the percentage of tumor growth inhibition (TGI%) is calculated using the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)]×100% Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day zero. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day zero.

[0087] In some embodiments, the antibody or antigen-binding fragment described herein is a GPC1 antagonist. In some embodiments, the antibody or antigen-binding fragment described herein reduces GPC1 signaling in target cells expressing GPC1.

[0088] In some embodiments, the antibody or antigen-binding fragments described herein can bind to tumor cells expressing GPC1. In some embodiments, the antibody or antigen-binding fragments described herein bind to tumor-associated cells expressing GPC1. In some embodiments, the antibody or antigen-binding fragments described herein can induce complement-dependent cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) and kill tumor cells.

[0089] In some embodiments, the antibody or antigen-binding fragment described herein has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are both ADCC and phagocytosis.

[0090] In some implementations, the antibody or antigen-binding fragments described herein can induce complement-dependent cytotoxicity (CDC).

[0091] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody is a human IgG1 antibody, optionally carrying a SI mutation, LALA mutation, N297A mutation, YTE mutation, and / or FLAA mutation. In some embodiments, the antibody is a human IgG4 antibody, optionally carrying a SI mutation, LALA mutation, N297A mutation, YTE mutation, and / or FLAA mutation.

[0092] In some embodiments, the antibody or antigen-binding fragments described herein do not have a functional Fc region. For example, the antibody or antigen-binding fragments are Fab, Fab', F(ab')2, and Fv fragments. In some embodiments, the Fc region has an LALA mutation (according to EU numbers L234A and L235A mutations) or an LALA-PG mutation (according to EU numbers L234A, L235A, and P329G mutations). In some embodiments, the Fc region has a FLAA mutation (according to EU numbers F234A and L235A). In some embodiments, the Fc has an SI mutation (according to EU numbers S239D and I332E mutations). In some embodiments, the Fc has an N297A mutation according to EU numbers. In some embodiments, the Fc has a YTE mutation (according to EU numbers M252Y, S254T, and T256E).

[0093] The antibodies or antigen-binding fragments described herein are effectively internalized into GPC1-expressing cells. The internalization of 3G1 was evaluated in Example 5 described herein. Compared to a total of 22 other anti-GPC1 antibodies prepared according to Example 1, 3G1 was found to have the best internalization properties in GPC1-expressing cells. This property makes 3G1 an excellent candidate for antibody-drug conjugates (ADCs).

[0094] In other words, in one or more embodiments, the antibody or antigen-binding fragments described herein exhibit a favorable rate of endocytosis in GPC1-expressing cells. As used herein, endocytosis refers to the cellular process of bringing substances into the cell. In some embodiments, endocytosis as used herein can refer to different forms of endocytosis classified by mechanistic pathway—examples include clathrin-mediated endocytosis and caveolae-mediated endocytosis. For a more general description, endocytosis as used herein may also be simply referred to as receptor-mediated endocytosis.

[0095] Anti-GPC1 antibody-drug conjugate (anti-GPC1 ADC)

[0096] Antibodies exhibiting favorable receptor-mediated endocytosis rates against cancer-associated receptors and / or antigens are suitable candidates for developing antibody-drug conjugates (ADCs). ADCs are a class of potent biologics engineered for targeted therapy, particularly for cancer treatment. ADCs are complex molecules consisting of an antibody (intact mAb or antibody fragment) linked to an active agent (such as a bioactive drug, radiopharmaceutical, or cytotoxic compound) via a stable chemical linker (which may contain unstable bonds, such as enzyme-cleavable bonds or self-sacrificing parts). By combining the unique targeting capabilities of antibodies with the cell-killing capabilities of cytotoxic drugs, antibody-drug conjugates allow for sensitive differentiation between healthy and affected tissues based on antibody-antigen expression. This means that, unlike traditional non-targeted chemotherapy drugs such as cisplatin, ADCs actively target and attack cancer cells, thus minimizing the impact on healthy cells with little or no antigen expression.

[0097] In one embodiment, this disclosure relates to antibody-drug conjugates targeting GPC1, comprising an antibody or antigen-binding fragment as described herein and a payload (e.g., a cytotoxic drug), optionally linked together by a connector.

[0098] In one or more embodiments of this disclosure, for example when an ADC is introduced, the antibody or antigen-binding fragment thereof characterized by binding to GPC1 may be characterized by at least one of the following: a. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) Immunoglobulin light chain variable region (VL), comprising the amino acid sequence shown in SEQ ID NO:4 as VL complementarity-determining region 1 (CDR1), the amino acid sequence shown in SEQ ID NO:5 as VL CDR2, and the amino acid sequence shown in SEQ ID NO:6 as VL CDR3, and / or ii) Immunoglobulin heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 1 as VH complementarity-determining region 1 (CDR1), the amino acid sequence shown in SEQ ID NO: 2 as VH CDR2, and the amino acid sequence shown in SEQ ID NO: 3 as VH CDR3; or b. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) Immunoglobulin light chain variable region (VL), comprising the amino acid sequence shown in SEQ ID NO:4 as VL complementarity-determining region 1 (CDR1), the amino acid sequence shown in SEQ ID NO:5 as VL CDR2, and the amino acid sequence shown in SEQ ID NO:6 as VL CDR3, and / or ii) Immunoglobulin heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 7 as VH complementarity-determining region 1 (CDR1), the amino acid sequence shown in SEQ ID NO: 8 as VH CDR2, and the amino acid sequence shown in SEQ ID NO: 9 as VH CDR3; or c. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) The immunoglobulin light chain variable region (VL), comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11 or a sequence having at least 85% sequence identity with it; and / or ii) The immunoglobulin heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 10 or a sequence having at least 85% sequence identity with it, or consisting of said sequence; or d. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) Immunoglobulin light chains comprising, or having at least 85% sequence identity with, the amino acid sequence shown in SEQ ID NO: 11, and the amino acid sequence shown in SEQ ID NO: 15, or having at least 85% sequence identity with, or consisting of said sequences; and / or ii) An immunoglobulin heavy chain comprising, or having at least 85% sequence identity with, the amino acid sequence shown in SEQ ID NO: 10, and the amino acid sequence shown in SEQ ID NO: 14, or having at least 85% sequence identity with, or consisting of said sequences; or e. An antibody that competes with an antibody or antigen-binding fragment as defined in any of a to d.

[0099] In one or more embodiments of this disclosure, the anti-GPC1 antibody or its antigen-binding fragment comprises

[0100] i) Containing the amino acid sequences shown in SEQ ID NO: 11 and SEQ ID NO: 15, or sequences having at least 85% sequence identity with them, or immunoglobulin light chains composed of said sequences; and / or

[0101] ii) Containing the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 14, or sequences having at least 85% sequence identity with them, or an immunoglobulin heavy chain composed of said sequences. It should be interpreted as referring to an anti-GPC1 antibody or its antigen-binding fragment, comprising: an amino acid sequence having at least 85% sequence identity (e.g., 90% or higher, e.g., 95%, e.g., 96%, 97%, 98%, 99% or higher) with the cumulative (i.e. combined) length of SEQ ID NO: 11 and 15, or an immunoglobulin light chain composed of said amino acid sequence; and / or (similarly) an amino acid sequence having at least 85% sequence identity (e.g., 90% or higher, e.g., 95%, e.g., 96%, 97%, 98%, 99% or higher) with the cumulative (i.e. combined) length of SEQ ID NO: 10 and 14, or an immunoglobulin heavy chain composed of said amino acid sequence.

[0102] In one embodiment of this disclosure, an antibody-drug conjugate (ADC) as defined herein comprises: a. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) Immunoglobulin light chain variable regions (VLs) comprising VL complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VLCDR1 region contains an amino acid sequence that is at least 85% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region contains an amino acid sequence that is at least 85% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region contains an amino acid sequence that is at least 85% identical to the selected VL CDR3 amino acid sequence. ii) Variable regions (VH) of the immunoglobulin heavy chain containing VH complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region contains an amino acid sequence that is at least 85% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region contains an amino acid sequence that is at least 85% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region contains an amino acid sequence that is at least 85% identical to the selected VH CDR3 amino acid sequence. The selected VH CDR 1, 2, and 3 amino acid sequences and the selected VL CDR 1, 2, and 3 amino acid sequences are one of the following: (1) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4-6, respectively; and (2) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 7-9, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4-6, respectively; b. Surfactants; and c. Optionally, a connector that connects a) and b).

[0103] In one embodiment of this disclosure, an antibody-drug conjugate (ADC) as defined herein comprises: a. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) A VL comprising the amino acid sequence shown in SEQ ID NO: 4 or a sequence having at least 85% sequence identity as the complementarity-determining region 1 (CDR1) of the immunoglobulin light chain variable region (VL), the amino acid sequence shown in SEQ ID NO: 5 or a sequence having at least 85% sequence identity as VL CDR2, and the amino acid sequence shown in SEQ ID NO: 6 or a sequence having at least 85% sequence identity as VL CDR3, and / or ii) A VH comprising the amino acid sequence shown in SEQ ID NO: 1 or having at least 85% sequence identity as the complementarity-determining region 1 (CDR1) of the immunoglobulin heavy chain variable region (VH), the amino acid sequence shown in SEQ ID NO: 2 or having at least 85% sequence identity as the VH CDR2, and the amino acid sequence shown in SEQ ID NO: 3 or having at least 85% sequence identity as the VH CDR3; b. Surfactants; and c. Optionally, a connector that connects a) and b).

[0104] In one embodiment of this disclosure, an antibody-drug conjugate (ADC) as defined herein comprises: a. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, comprising: i) A VL comprising the amino acid sequence shown in SEQ ID NO: 4 as the complementarity-determining region 1 (CDR1) of the immunoglobulin light chain variable region (VL), the amino acid sequence shown in SEQ ID NO: 5 as VL CDR2, and the amino acid sequence shown in SEQ ID NO: 6 as VL CDR3, and / or ii) The amino acid sequence shown in SEQ ID NO: 1, which is the complementarity-determining region 1 (CDR1) of the immunoglobulin heavy chain variable region (VH), the amino acid sequence shown in SEQ ID NO: 2, which is the VH CDR2, and the amino acid sequence shown in SEQ ID NO: 3, which is the VH CDR3; b. Surfactants; and c. Optionally, a connector that connects a) and b).

[0105] In one embodiment of this disclosure, an antibody-drug conjugate (ADC) as defined herein comprises: a. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) A VL comprising the amino acid sequence shown in SEQ ID NO: 4 or a sequence having at least 85% sequence identity as the complementarity-determining region 1 (CDR1) of the immunoglobulin light chain variable region (VL), the amino acid sequence shown in SEQ ID NO: 5 or a sequence having at least 85% sequence identity as VL CDR2, and the amino acid sequence shown in SEQ ID NO: 6 or a sequence having at least 85% sequence identity as VL CDR3, and / or ii) A VH comprising the amino acid sequence shown in SEQ ID NO: 7 or having at least 85% sequence identity as the complementarity-determining region 1 (CDR1) of the immunoglobulin heavy chain variable region (VH), the amino acid sequence shown in SEQ ID NO: 8 or having at least 85% sequence identity as the VH CDR2, and the amino acid sequence shown in SEQ ID NO: 9 or having at least 85% sequence identity as the VH CDR3; b. Surfactants; and c. Optionally, a connector that connects a) and b).

[0106] In one embodiment of this disclosure, an antibody-drug conjugate (ADC) as defined herein comprises: a. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) A VL comprising the amino acid sequence shown in SEQ ID NO: 4 as the complementarity-determining region 1 (CDR1) of the immunoglobulin light chain variable region (VL), the amino acid sequence shown in SEQ ID NO: 5 as VL CDR2, and the amino acid sequence shown in SEQ ID NO: 6 as VL CDR3, and / or ii) A VH comprising the amino acid sequence shown in SEQ ID NO: 7 as complementarity-determining region 1 (CDR1) of the immunoglobulin heavy chain variable region (VH), the amino acid sequence shown in SEQ ID NO: 8 as VH CDR2, and the amino acid sequence shown in SEQ ID NO: 9 as VH CDR3; b. Surfactants; and c. Optionally, a connector that connects a) and b).

[0107] In one or more embodiments, the antibody or antigen-binding fragment thereof described herein may comprise a heavy chain variable region (VH) containing VH complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region contains or is composed of an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 100% identical to or constitutes thereof with SEQ ID NO: 1 (if defined according to Kabat) or SEQ ID NO: 7 (if defined according to Chothia). In one or more embodiments, the antibody or antigen-binding fragment thereof described herein may comprise a heavy chain variable region (VH) containing VH complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR2 region contains or is composed of an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 100% identical to or constitutes thereof with SEQ ID NO: 2 (if defined according to Kabat) or SEQ ID NO: 8 (if defined according to Chothia). In one or more embodiments, the antibody or antigen-binding fragment thereof described herein may comprise a heavy chain variable region (VH) comprising VH complementarity-determining regions (CDRs) 1, 2, 3, wherein the VH CDR3 region comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 100% identical to or constitutes the same as SEQ ID NO: 3 (if defined according to Kabat) or SEQ ID NO: 9 (if defined according to Chothia).

[0108] In one or more embodiments, the antibody or antigen-binding fragment thereof described herein may comprise a light chain variable region (VL) containing VL complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VL CDR1 region contains or is composed of an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 100% identical to or constitutes thereof with SEQ ID NO: 4. In one or more embodiments, the antibody or antigen-binding fragment thereof described herein may comprise a light chain variable region (VL) containing VL complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VL CDR2 region contains or is composed of an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 100% identical to or constitutes thereof with SEQ ID NO: 5. In one or more embodiments, the antibody or antigen-binding fragment thereof described herein may comprise a light chain variable region (VL) containing VL complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VL CDR3 region contains or is composed of an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 100% identical to or constitutes thereof with SEQ ID NO: 6.

[0109] In one embodiment of this disclosure, the amino acid sequences of SEQ ID NO: 1-3 and SEQ ID NO: 4-6 are defined according to Kabat. In one embodiment of this disclosure, the amino acid sequences of SEQ ID NO: 7-9 and SEQ ID NO: 4-6 are defined according to Chothia.

[0110] In one embodiment of this disclosure, an antibody-drug conjugate (ADC) as defined herein comprises: a. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) Containing the amino acid sequence shown in SEQ ID NO: 11 or a sequence having at least 85% sequence identity with it, or the variable region (VL) of the immunoglobulin light chain composed thereof; and / or ii) Contains the amino acid sequence shown in SEQ ID NO: 10 or a sequence having at least 85% sequence identity with it or an immunoglobulin heavy chain variable region (VH) composed of it; b. Surfactants; and c. Optionally, the connector that connects a) and b).

[0111] In one or more embodiments of this disclosure, the antibody or antigen-binding fragment thereof described herein may comprise a light chain variable region (VL), wherein the VL comprises or is composed of an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 100% identical to or composed of the same as SEQ ID NO: 11, and / or may comprise a heavy chain variable region (VH), wherein the VH comprises or is composed of an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 100% identical to or composed of the same as the same as SEQ ID NO: 10.

[0112] It is widely accepted and understood in the art that the CDR is important for antigen recognition and is primarily responsible for it. Therefore, in a preferred embodiment, any sequence identity variations within the heavy chain variable region (VH) or light chain variable region (VL) are located outside the CDR. All variant antibodies and antigen-binding fragments disclosed herein retain the ability to effectively bind to one or more of human GPC1 (SEQ ID NO: 16), monkey GPC1 (SEQ ID NO: 17), mouse GPC1 (SEQ ID NO: 18), and rat GPC1 (SEQ ID NO: 19).

[0113] In one embodiment of this disclosure, an antibody-drug conjugate (ADC) as defined herein comprises: a. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) Contains the amino acid sequence shown in SEQ ID NO: 11 or the variable region (VL) of the immunoglobulin light chain composed therefrom; and / or ii) Contains the amino acid sequence shown in SEQ ID NO: 10 or the variable region (VH) of the immunoglobulin heavy chain composed therefrom; b. Surfactants; and c. Optionally, the connector that connects a) and b).

[0114] In one embodiment of this disclosure, an antibody-drug conjugate (ADC) as defined herein comprises: a. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) The variable region (VL) of the immunoglobulin light chain, consisting of the amino acid sequence shown in SEQ ID NO: 11; and / or ii) The variable region (VH) of the immunoglobulin heavy chain, consisting of the amino acid sequence shown in SEQ ID NO: 10; b. Surfactants; and c. Optionally, the connector that connects a) and b).

[0115] In one embodiment of this disclosure, an antibody-drug conjugate (ADC) as defined herein comprises: a. Antibodies as defined herein, containing i) Contains the amino acid sequence shown in SEQ ID NO: 15 or the constant region of the immunoglobulin light chain composed thereof; and / or ii) Contains the amino acid sequence shown in SEQ ID NO: 14 or the immunoglobulin heavy chain constant region composed thereof; b. Surfactants; and c. Optionally, the connector that connects a) and b).

[0116] In one embodiment of this disclosure, an antibody-drug conjugate (ADC) as defined herein comprises: a. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) Containing the amino acid sequences shown in SEQ ID NO: 11 and SEQ ID NO: 15, or sequences having at least 85% sequence identity with them, or immunoglobulin light chains composed of such sequences; and / or ii) Contains the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 14, or sequences having at least 85% sequence identity with them, or immunoglobulin heavy chains composed of them; b. Surfactants; and c. Optionally, the connector that connects a) and b).

[0117] In one embodiment of this disclosure, an antibody-drug conjugate (ADC) as defined herein comprises: a. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) An immunoglobulin light chain comprising or consisting of the amino acid sequences shown in SEQ ID NO: 11 and SEQ ID NO: 15; and / or ii) An immunoglobulin heavy chain comprising or consisting of the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 14; b. Surfactants; and c. Optionally, the connector that connects a) and b).

[0118] Surfactant

[0119] The ADC disclosed herein comprises an active agent, such as a drug, which can be delivered intracellularly to cells expressing GPC1. The active agent may be, for example, a therapeutic agent, a radioisotope, or a detectable marker. In a preferred embodiment, the active agent is a therapeutic agent.

[0120] In one embodiment, the active agent may be or comprises a radioactive isotope. The radioactive isotope can be used as a radiation emitter for treating affected tissue or for diagnostic purposes. In one embodiment, the radioactive isotope may contain... 60 Co、 89 Sr、 90 Y、 99m Tc、¹³¹I、¹³ 7 Cs、¹ 5 ³Sm or²²³Rd or by 60 Co、 89 Sr、 90 Y、 99m Tc、¹³¹I、¹³ 7 Cs、¹ 5 It consists of ³Sm or ²²³Rd. In one embodiment of this disclosure, the radioactive isotope may be used in combination with a chelating agent such as DOTA or EDTA or other chelating agents known in the art.

[0121] In one embodiment, the active agent is a therapeutic agent. Therapeutic agent categories include DNA cross-linking agents, DNA alkylating agents, DNA strand-breaking agents, anthracyclines, antimetabolites, antimicrotubule / antimitotic agents, histone deacetylase inhibitors, kinase inhibitors, metabolic inhibitors, peptide antibiotics, immune checkpoint inhibitors, platinum-based antitumor drugs, topoisomerase inhibitors, DNA or RNA polymerase inhibitors, nucleotide-based agents, and cytotoxic antibiotics.

[0122] In a preferred embodiment, the active agent is a cytotoxic agent that allows for the efficient killing of cells expressing GPC1.

[0123] In one embodiment, the active agent is a chemotherapeutic agent.

[0124] In one embodiment, the active agent is a DNA cross-linking agent, such as cisplatin, carboplatin, oxaliplatin, mitomycin C (MMC), pyrrolobenzodiazepines, dimer pyrrolobenzodiazepines, or derivatives or prodrugs of any of the foregoing, such as SGD-1882.

[0125] In one embodiment of this disclosure, the active agent is a DNA alkylating agent, such as a nitrogen mustard, tri(2-chloroethyl)amine, pyridobenzodiazepines, indolinebenzodiazepine dimer, or docamycin SA, or a derivative or prodrug of any of the foregoing substances.

[0126] In one embodiment, the active agent is a DNA strand-breaking agent, such as calicheamicin or hamiltrone or a derivative thereof.

[0127] In one embodiment, the active agent is an anthracycline drug, such as daunorubicin, doxorubicin, epirubicin, idarubicin, or PNU-159682, or a derivative or prodrug of any of the foregoing substances.

[0128] In one embodiment, the active agent is an antimetabolite, such as a folic acid antagonist, methotrexate, a purine antimetabolite, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, cladribine, a pyrimidine antimetabolite, 5-fluorouracil, 5-fluorodeoxyuridine, cytarabine, gemcitabine, or a derivative or prodrug of any of the foregoing.

[0129] In one embodiment, the active agent is an antimitotic agent, such as auristatin, dolastatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), taxanes, paclitaxel, docetaxel, vinblastine, vincristine, vindesine, vinorelbine, maytansine, colchicine, podophyllotoxin, or derivatives or prodrugs of any of the foregoing substances.

[0130] In one embodiment, the active agent is monomethylaurestatin E (MMAE) or a derivative thereof.

[0131] Due to its high toxicity, MMAE inhibits cell division by blocking microtubule polymerization and therefore cannot be used as a single chemotherapy drug. However, the combination of MMAE with an anti-CD30 monoclonal antibody (Brentuximab Vedotin, trade name Adcetris™) has been shown to be stable in the extracellular fluid, cleavable by cathepsins, and safe for therapeutic use.

[0132] In one embodiment, the active agent is a histone deacetylase inhibitor, such as trichostatin A, vorinostat, belinostat, panabiostat, givinostat, resminostat, abexinostat, quisinostat, rocilinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entinostat, tacedinaline, 4SC202, mocetinostat, romidepsin, nicotinamide, sirtinol, cambinol, or EX-527, or a derivative or prodrug of any of the foregoing substances.

[0133] In one embodiment, the active agent is a kinase inhibitor, such as genistein, lavendustin C, PP1-AG1872, PP2-AG1879, SU6656, CGP77675, PD166285, imatinib, erlotinib, gefitinib, lavendustin A, cetuximab, UCS15A, herbimycin A, or radicicol, or a derivative or prodrug of any of the foregoing substances.

[0134] In one embodiment, the active agent is a metabolic inhibitor, such as a NAMPT inhibitor. Examples of NAMPT inhibitors include, but are not limited to, APO866, GMX-1777, GMX-1778, ATG-019, or OT-82, or derivatives or prodrugs of any of the foregoing substances.

[0135] In one embodiment, the active agent is an immune checkpoint inhibitor, such as a PD-1 inhibitor or a PD-L1 inhibitor. Examples of PD-1 inhibitors include, but are not limited to, pembrolizumab, nivolumab, cimiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, AMP-224, or AMP-514, or any derivative thereof. Examples of PD-L1 inhibitors include, but are not limited to, atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, or BMS-986189, or derivatives or prodrugs of any of the foregoing substances.

[0136] In one embodiment, the active agent is a platinum-based antitumor drug, such as lipoplatin, cisplatin, carboplatin, oxaliplatin, nedaplatin, picoplatin, phenanthriplatin, satraplatin, or triplatin tetranitrate, or a derivative or prodrug of any of the foregoing substances.

[0137] In one embodiment, the active agent is a topoisomerase inhibitor, such as a topoisomerase-I (Topo1) inhibitor. Examples of topoisomerase inhibitors include, but are not limited to, camptothecin, topotecan, belototecan, lurtotecan, irinotecan, SN-38, exatecan, Dxd, or derivatives or prodrugs of any of the foregoing substances.

[0138] In one embodiment, the active agent is a Topo1 inhibitor, such as ethanotecan or a derivative thereof.

[0139] In one embodiment, the active agent is a DNA polymerase inhibitor or an RNA polymerase inhibitor, such as amanitin, alpha-amanitin, actinomycin D, aphidicolin, or a derivative or prodrug of any of the foregoing.

[0140] In one embodiment, the active agent is a nucleotide-based drug, such as an RNA oligonucleotide or a DNA oligonucleotide, such as siRNA or miRNA.

[0141] Each antibody molecule may have one or more drug units. The ratio of the number of drug molecules in each antibody is called the drug-antibody ratio (DAR). In one embodiment, the DAR is between 1 and 10, for example, between 2 and 8, for example, between 2 and 6, for example, 2 or 4. In another embodiment, the DAR is 2, 3, 4, 5, 6, 7 or 8.

[0142] connector

[0143] Stable linker between antibody and active agent is a crucial aspect of ADC technology. Linkers can be based on chemical motifs, including disulfides, hydrazones, or peptides (cleavable), or thioethers (non-cleavable), and control the distribution and delivery of the cytotoxic agent to target cells. Both cleavable and non-cleavable linkers have been proven safe in preclinical and clinical trials. For example, brentuximab vedotin contains an enzyme-sensitive cleavable linker that delivers the potent and highly toxic antimicrotubule agent monomethylaurestatin E (MMAE) (a synthetic antitumor agent) to cells.

[0144] Another approved ADC, trastuzumab emtansine, is a combination of the microtubule formation inhibitor mertansine (DM-1) (a maytansine derivative) and the antibody trastuzumab (Herceptin™, Genentech / Roche) linked by a stable, non-cleavable linker.

[0145] The type of cleavable or non-cleavable linker imparts specific properties to the delivered drug. For example, a cleavable linker can be cleaved, for instance, by enzymes in the target cell, leading to the effective intracellular release of the active agent (e.g., a cytotoxic agent). Conversely, ADCs containing non-cleavable linkers lack a drug release mechanism and must rely on mechanisms such as the degradation of the targeting antibody to achieve drug release. Furthermore, as those skilled in the art will understand, linker composition can influence key factors such as the overall solubility and pharmacokinetic properties of the ADC.

[0146] For both types of connectors, drug release is crucial for achieving cellular effects. Drugs capable of freely diffusing across the cell membrane can escape from the target cell and, in a process known as “bystander killing,” also attack neighboring cells, such as cancer cells near target cells expressing GPC1. In one embodiment of this disclosure, the ADC described herein may contain a payload that allows bystander killing of neighboring cells, such as adjacent tumors and / or cancer cells. This bystander killing is particularly desirable in cases where the adjacent tumors and / or cancer cells do not express any GPC1. For other cases, bystander killing is preferably not observed, such as when the neighboring cells are healthy cells / tissues. Therefore, in one or more embodiments of this disclosure, the ADC described herein contains a payload that does not provide any significant bystander killing of neighboring cells.

[0147] In a preferred embodiment of this disclosure, the GPC1-targeting ADC disclosed herein includes a linker that connects an antibody to an active agent.

[0148] In one embodiment of this disclosure, the connector may be pyrolytic or non-pyrolytic.

[0149] Cleavable groups include disulfide bonds, amide bonds, substituted amide bonds in the form of peptide bonds, thioamide bonds, ester bonds, thioester bonds, vicinal diol bonds, or hemiacetals. These or other cleavable bonds may include enzyme-cleavable bonds, such as peptide bonds (cleaved by peptidases), phosphate ester bonds (cleaved by phosphatases), nucleic acid bonds (cleaved by endonucleases), and sugar bonds (cleaved by glycosidases).

[0150] In another embodiment of this disclosure, the connector is a cleavable connector that allows the active agent to be released intracellularly into the target cell.

[0151] In another embodiment, the linker is a peptide linker. The choice of peptide sequence is crucial for the success of the conjugate. In some embodiments, the linker is stable to serum proteases but can be cleaved by lysosomal enzymes in target cells.

[0152] In another embodiment, the linker is a linker containing an enzyme-cleavable peptide, such as a linker containing a cathepsin-cleavable peptide. The cathepsin can be one of several types of cathepsins, which is a group of lysosomal proteases.

[0153] In one or more embodiments of this disclosure, the connector comprises a dipeptide, tripeptide, tetrapeptide, or pentapeptide, or is composed of a dipeptide, tripeptide, tetrapeptide, or pentapeptide.

[0154] In one or more embodiments of this disclosure, the linker comprises or is composed of a tetrapeptide, wherein each amino acid of the tetrapeptide is independently selected from glycine or phenylalanine. In one or more embodiments of this disclosure, the tetrapeptide is GGFG (SEQ ID NO: 20) and has the structure of formula (I): , Where 'L' represents the connectivity with the remaining linker (towards the antibody or antibody-binding attachment group of the ADC), and 'D' represents the connectivity with the active agent (directly or in the form of a spacer).

[0155] In another embodiment of this disclosure, the connector comprises or is composed of a dipeptide, such as valine-citrulline (VC) or valine-alanine (VA).

[0156] In one embodiment, the linker comprises or is composed of a dipeptide, such as valine-citrulline (VC) or valine-alanine (VA), which may be further linked to other structural elements via amide bonds. Valine-citrulline-based linkers can be cleaved by lysosomal cathepsins, wherein the citrulline carboxyl functional group is modified to a substituted amide, while valine-alanine-based linkers can be cleaved by other lysosomal proteases (including other cathepsins), wherein the alanine carboxyl functional group is modified to a substituted amide.

[0157] In another embodiment of this disclosure, the antibody-drug conjugate, as defined herein, further comprises a spacer, such as a spacer comprising p-aminobenzyl (PAB), p-aminobenzylcarbamate, p-aminobenzyloxycarbonyl (PABC), or polyethylene glycol (PEG). Spacers such as PABC are sometimes also referred to as self-sacrificing linkers. Self-sacrificing linkers (or spacers) are well known in the art.

[0158] In one embodiment of this disclosure, the antibody-drug conjugate, as defined herein, comprises a p-aminobenzyloxycarbonyl group (PABC). PABC is sometimes used interchangeably with PAB in the art. For clarity, PABC is intended to be interpreted in this disclosure as a motif having the following structure: , Where 'L' represents the connectivity with the remaining linker (towards the antibody in the ADC direction), and 'D' represents the connectivity with the active agent (directly or in the form of a spacer).

[0159] In another embodiment of this disclosure, the antibody-drug conjugate, as defined herein, further comprises an attachment group, such as an attachment group comprising or consisting of: maleimide and hexanoic acid (MC, sometimes also called maleimide hexanoyl), N-hydroxysuccinimide, a reactive attachment group of a protein-bound carbohydrate that is targeted to a modified or unmodified protein, a peptide sequence required for an enzymatic reaction, an azide or alkyne (e.g. for SPAAC or CuAAC), or derived from any of the foregoing groups by reacting with an antibody or a chemically or enzymatically generated derivative thereof.

[0160] In one embodiment of this disclosure, the ADC further comprises an attachment entity. The attachment entity may, for example, link an antibody and a cleavable linker, wherein the attachment entity is a reaction product between an antibody amino acid side chain and a reactive attachment group in a linker precursor. In one embodiment, the reactive attachment group comprises or is composed of maleimide and hexanoic acid (MC), wherein the maleimide preferably reacts with cysteine ​​thiol during coupling to the antibody. In other embodiments, the attachment group comprises or is composed of: N-hydroxysuccinimide, a reactive attachment group of a protein-bound carbohydrate that is targeted for modification or non-modification, a peptide sequence required for an enzymatic reaction, an azide or alkyne (e.g., for SPAAC or CuAAC), or is derived from any of the foregoing groups by reacting with an antibody or a chemically or enzymatically generated derivative thereof.

[0161] Within the meanings of both attachment group and attachment entity as described above, MC refers to the motif known in the field of ADCs, and is sometimes also referred to as the maleimide hexanoyl entity. The acid terminus of hexanoic acid (in the MC group) is typically attached to the nitrogen of a subsequent group (the activator / cytotoxic agent of the ADC, or another spacer / connector) by forming an amide bond, such as the amine terminus of a 2-6 residue peptide contained in an ADC.

[0162] In one embodiment of this disclosure, the ADC comprises an antibody targeting GPC1 as defined herein, and a linker-drug complex, Vedotin. Vedotin is a linker-drug complex comprising the cytotoxic agent MMAE, a spacer (p-aminobenzyloxycarbonyl), a cathepsin-cleavable linker (valine-citrulline dipeptide), and an attachment group consisting of hexanoic acid and maleimide. Vedotin is MC-VC-PABC-MMAE.

[0163] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit comprising VC-PABC-MMAE.

[0164] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit comprising VC-PABC-MMAF.

[0165] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit comprising VC-PABC-ethanotecan.

[0166] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit comprising VA-PABC-ethanotecan.

[0167] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit comprising VC-PABC-Dxd.

[0168] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit comprising VA-PABC-Dxd.

[0169] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit comprising GGFG-PABC-MMAE.

[0170] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit comprising GGFG-PABC-MMAF.

[0171] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit comprising GGFG-PABC-ethanotecan.

[0172] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit comprising GGFG-ethanotecan.

[0173] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit comprising GGFG-PABC-Dxd.

[0174] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit comprising GGFG-Dxd.

[0175] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit of formula (I), wherein D represents PABC-MMAE.

[0176] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit of formula (I), wherein D represents PABC-MMAF.

[0177] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin unit of formula (I), wherein D represents PABC-ethanotecan.

[0178] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin of formula (I), wherein D represents essanotecan.

[0179] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin of formula (I), wherein D represents PABC-Dxd.

[0180] In one embodiment, the GPC1-targeting ADC of this disclosure comprises an antibody as defined herein, and a linker-spacer-toxin of formula (I), where D represents Dxd.

[0181] In one embodiment, the ADC targeting GPC1 disclosed herein comprises or consists of the following: a. Antibodies as defined herein include: i) Contains the amino acid sequence shown in SEQ ID NO: 11 or the variable region of an immunoglobulin light chain composed thereof, and / or ii) Contains the amino acid sequence shown in SEQ ID NO: 10 or the variable region of an immunoglobulin heavy chain composed therefrom. b. VC or VA dipeptide linker, c. MC attachment group, d. Optionally, PAB or PABC spacers, and e. MMAE as an active agent.

[0182] In one embodiment, the ADC targeting GPC1 disclosed herein comprises or consists of the following: a. Antibodies as defined herein include: i) An immunoglobulin light chain comprising or consisting of the amino acid sequences shown in SEQ ID NO: 11 and SEQ ID NO: 15, and / or ii) An immunoglobulin heavy chain comprising or consisting of the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 14, b. VC or VA connector, c. MC attachment group, d. Optionally, PAB or PABC spacers, and e. MMAE as an active agent.

[0183] In one embodiment, the ADC targeting GPC1 disclosed herein comprises or consists of the following: a. Antibodies as defined herein include: i) Contains the amino acid sequence shown in SEQ ID NO: 11 or the variable region of an immunoglobulin light chain composed thereof, and / or ii) Contains the amino acid sequence shown in SEQ ID NO: 10 or the variable region of an immunoglobulin heavy chain composed therefrom. b. VC or VA dipeptide linker, c. MC attachment group, d. Optionally, PAB or PABC spacers, and e. Exanotecan or Dxd as active agents.

[0184] In one embodiment, the ADC targeting GPC1 disclosed herein comprises or consists of the following: a. Antibodies as defined herein include: i) An immunoglobulin light chain comprising or consisting of the amino acid sequences shown in SEQ ID NO: 11 and SEQ ID NO: 15, and / or ii) An immunoglobulin heavy chain comprising or consisting of the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 14, b. VC or VA connector, c. MC attachment group, d. Optionally, PAB or PABC spacers, and e. Exanotecan or Dxd as active agents.

[0185] In one embodiment, the ADC targeting GPC1 disclosed herein comprises or consists of the following: a. Antibodies as defined herein include: i) Contains the amino acid sequence shown in SEQ ID NO: 11 or the variable region of an immunoglobulin light chain composed thereof, and / or ii) Contains the amino acid sequence shown in SEQ ID NO: 10 or the variable region of an immunoglobulin heavy chain composed therefrom. b. Tetrapeptide linkers, such as the linker of formula (I), c. MC attachment group, d. Optionally, PAB or PABC spacers, and e. MMAE as an active agent.

[0186] In one embodiment, the ADC targeting GPC1 disclosed herein comprises or consists of the following: a. Antibodies as defined herein include: i) An immunoglobulin light chain comprising or consisting of the amino acid sequences shown in SEQ ID NO: 11 and SEQ ID NO: 15, and / or ii) An immunoglobulin heavy chain comprising or consisting of the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 14, b. Tetrapeptide linkers, such as the linker of formula (I), c. MC attachment group, d. Optionally, PAB or PABC spacers, and e. MMAE as an active agent.

[0187] In one embodiment, the ADC targeting GPC1 disclosed herein comprises or consists of the following: a. Antibodies as defined herein include: i) Contains the amino acid sequence shown in SEQ ID NO: 11 or the variable region of an immunoglobulin light chain composed thereof, and / or ii) Contains the amino acid sequence shown in SEQ ID NO: 10 or the variable region of an immunoglobulin heavy chain composed therefrom. b. Tetrapeptide linkers, such as the linker of formula (I), c. MC attachment group, d. Optionally, PAB or PABC spacers, and e. Exanotecan or Dxd as active agents.

[0188] In one embodiment, the ADC targeting GPC1 disclosed herein comprises or consists of the following: a. Antibodies as defined herein include: i) An immunoglobulin light chain comprising or consisting of the amino acid sequences shown in SEQ ID NO: 11 and SEQ ID NO: 15, and / or ii) An immunoglobulin heavy chain comprising or consisting of the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 14, b. Tetrapeptide linkers, such as the linker of formula (I), c. MC attachment group, d. Optionally, PAB or PABC spacers, and e. Exanotecan or Dxd as active agents.

[0189] Methods for producing anti-GPC1 antibodies

[0190] Isolated fragments of human GPC1 can be used as immunogens to generate antibodies using standard polyclonal and monoclonal antibody preparation techniques. Polyclonal antibodies can be generated in animals through multiple injections (e.g., subcutaneous or intraperitoneal injections) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected together with at least one adjuvant. In some embodiments, the antigenic peptide or protein may be conjugated to an immunogenic agent of the species to be immunized. Animals may be injected with the antigenic peptide or protein more than once (e.g., two, three, or four times).

[0191] Full-length peptides or proteins can be used, or alternatively, their antigenic peptide fragments can be used as immunogens. The antigenic peptide of a protein contains at least eight (e.g., at least 10, 15, 20, or 30) amino acid residues of the GPC1 amino acid sequence and covers the epitope of the protein, such that antibodies generated against the peptide form specific immune complexes with the protein. As mentioned above, the full-length sequence of human GPC1 is known in the art. In some embodiments, Fc-tagged or His-tagged human GPC1 proteins are used as immunogens.

[0192] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic formulations may contain, for example, recombinantly expressed or chemically synthesized peptides (e.g., fragments of human GPC1). The formulation may also contain adjuvants, such as Freund's complete or incomplete adjuvants, or similar immunostimulants.

[0193] As described above, polyclonal antibodies can be prepared by immunizing suitable subjects with a GPC1 peptide or its antigenic peptide (e.g., a fraction of GPC1) as an immunogen. Antibody titers in immunized subjects can be monitored over time using standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using a fixed GPC1 peptide or peptide. If desired, antibody molecules can be isolated from mammals (e.g., from blood) and further purified using known techniques (e.g., protein A or protein G chromatography) to obtain IgG fractions. At an appropriate time following immunization, such as when the specific antibody titer is highest, antibody-producing cells can be obtained from the subject and purified using standard techniques (e.g., hybridoma techniques originally described by Kohler et al.). Nature 256:495-497, 1975), human B-cell hybridoma technology (Kozbor et al., Immunol. Today Monoclonal antibodies are prepared using hybridoma technology (4:72, 1983), EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or trimomas technology. The techniques for producing hybridomas are well-known (see, Generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells that produce monoclonal antibodies are detected by screening for antibodies that bind to peptides or epitopes of interest in the supernatant of hybridoma cultures, for example, using a standard ELISA assay.

[0194] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human, humanized, or chimeric antibodies or their antigen-binding fragments, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence constituting the antigen-binding site of the antibody or antigen-binding domain. Within this population of variants, some antibodies or antigen-binding fragments will have increased affinity for target proteins (e.g., GPC1). Any combination of deletions, insertions, and / or substitutions can be performed to obtain antibodies or their antigen-binding fragments with enhanced binding affinity to the target. Introducing amino acid changes into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as altering (e.g., increasing or decreasing) the number of glycosylation sites, altering the type of glycosylation sites (e.g., changing the amino acid sequence so that enzymes present in the cell link different sugars), or introducing new glycosylation sites.

[0195] The antibodies disclosed herein may be derived from any animal species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camels (e.g., camels and alpacas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including genetically engineered rodents to produce human antibodies.

[0196] Human and humanized antibodies include antibodies having variable and constant regions derived from human immunoglobulin sequences (or having the same amino acid sequences as those derived from human immunoglobulin sequences). Human antibodies may contain amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or somatic mutations in vivo), such as in CDRs.

[0197] Humanized antibodies typically possess a human frame (FR) with a transplanted non-human CDR. Therefore, a humanized antibody has one or more amino acid sequences introduced from a non-human source. These non-human amino acid residues are often referred to as “input” residues, which are usually derived from the “input” variable domain. Humanization is essentially performed by, for example, replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. These methods are described, for example, in Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); each of which is incorporated herein by reference in its entirety. Thus, a “humanized” antibody is a chimeric antibody in which essentially less than the intact human V domain is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are often mouse antibodies, in which some CDR residues and some FR residues are replaced with residues from similar sites in human antibodies.

[0198] Selecting the human VH and VL domains for preparing humanized antibodies is crucial for reducing immunogenicity. Following the so-called "best fit" method, the V domain sequence of mouse antibodies was screened against an entire library of known human domain sequences. The human sequence closest to the mouse sequence was accepted as the human FR for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)).

[0199] Importantly, antibody humanization must retain high specificity and affinity for the antigen, as well as other advantageous biological properties. To achieve this, humanized antibodies can be prepared using three-dimensional models of the parental and humanized sequences, employing methods that analyze parental sequences and various conceptual humanization products. Three-dimensional immunoglobulin models are universally available and familiar to those skilled in the art. Computer programs are available for illustrating and displaying possible three-dimensional conformations of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues affecting the candidate immunoglobulin's ability to bind its antigen. In this way, FR residues can be selected and combined from the receptor and input sequences to achieve desired antibody properties, such as increased affinity for the target antigen.

[0200] Typically, amino acid sequence variants of human, humanized, or chimeric anti-GPC1 antibodies will contain an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence present in the light or heavy chain of the original antibody.

[0201] In some implementations, mice with humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci (e.g., RenMab™ mice) are used to generate antibodies. A heavy chain immunoglobulin locus is a region of a chromosome containing genes encoding the antibody heavy chain. This locus may include, for example, the human IGHV (variable) gene, the human IGHD (diversity) gene, the human IGHJ (connection) gene, and the mouse heavy chain constant domain gene. A κ chain immunoglobulin locus is a region of a chromosome containing genes encoding the antibody light chain (κ chain). The κ chain immunoglobulin locus may include, for example, the human IGKV (variable) gene, the human IGKJ (connection) gene, and the mouse light chain constant domain gene. A detailed description of the RenMab™ mouse can be found in PCT / CN2020 / 075698 or US20200390073A1, which are incorporated herein by reference in their entirety.

[0202] In some implementations, mice with humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci (e.g., RenLite™ mice) are used to generate antibodies. A heavy chain immunoglobulin locus is a region of a chromosome containing genes encoding antibody heavy chains. This locus may include, for example, human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (connection) genes, and mouse heavy chain constant domain genes. A κ chain immunoglobulin locus is a region of a chromosome containing genes encoding universal light chains. κ chain immunoglobulin loci may include, for example, human IGKV (variable) genes, human IGKJ (connection) genes, and mouse light chain constant domain genes. A detailed description of RenLite™ mice can be found in PCT / CN2021 / 097652, which is incorporated herein by reference in its entirety.

[0203] The antibodies produced by these mice have a fully human VH, a fully human VL, and a mouse constant region. In some embodiments, the human VH and human VL are linked to the human IgG constant region (e.g., IgG1, IgG2, IgG3, and IgG4).

[0204] Identity or homology relative to the original sequence is typically the percentage of amino acid residues in the candidate sequence that are identical to those in human, humanized, or chimeric anti-GPC1 antibodies or fragments. It is calculated after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, without taking into account any conserved substitutions as part of the sequence identity.

[0205] Additional modifications can be made to the anti-GPC1 antibody or antigen-binding fragment. For example, one or more cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in that region. The resulting homodimeric antibody can have an extended in vitro and / or in vivo half-life. Homodimeric antibodies with extended in vitro and / or in vivo half-lives can also be used as described, for example, by Wolff et al. (… Cancer Res Preparation of heterobifunctional cross-linking agents (e.g., 53:2560-2565, 1993). Alternatively, antibodies with dual Fc regions can be engineered (see, for example, Stevenson et al., 53:2560-2565, 1993). Anti-Cancer Drug Design 3:219-230, 1989).

[0206] In some embodiments, the anti-GPC1 antibody or its antigen-binding fragment can be covalently modified. These covalent modifications can be performed by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications to the antibody or antibody fragment are introduced into the molecule by reacting the target amino acid residues of the antibody or fragment with an organic derivatizing agent capable of reacting with selected side chains or N- or C-terminal residues.

[0207] In some embodiments, antibody variants are provided that have a carbohydrate structure lacking fucose linked (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the glycan chain at Asn297 relative to the sum of all glycan structures linked at Asn297 (e.g., complex, hybrid, and high-mannose structures), for example, by measuring by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 of the Fc region (EU number of the Fc region residue; or position 314 of the Kabat number); however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucoidylated variants may possess improved ADCC function. In some implementations, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0208] In some implementations, to improve production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further engineered to replace serine at position 228 (EU number) of IgG4 with proline (S228P). For a detailed description of the S228 mutation, see, for example, Silva et al., “The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.” Journal of Biological Chemistry 290.9 (2015): 5462-5469, which is incorporated herein by reference in its entirety.

[0209] Recombinant vector

[0210] This disclosure also provides a recombinant vector (e.g., an expression vector) containing the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), a host cell into which the recombinant vector is introduced (i.e., a host cell containing the polynucleotide and / or a vector containing the polynucleotide), and the production of recombinant antibody polypeptides or fragments thereof by recombinant technology.

[0211] As used herein, a “vector” is any construct capable of delivering one or more polynucleotides of interest to a host cell when introduced into the host cell. An “expression vector” is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell to which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by operably linked regulatory elements (e.g., promoters, enhancers, and / or polyadenylate tails) located within the vector or in the host cell genome at or near the integration site of the polynucleotide of interest, such that the polynucleotide of interest will be translated in the host cell to which the expression vector has been introduced.

[0212] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-glucan), transformation, transfection, and infection and / or transduction (e.g., with recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, granules, phage vectors, and DNA or RNA expression vectors associated with cationic concentrators.

[0213] In some embodiments, the polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia virus or other poxviruses, retroviruses, or adenoviruses), which may involve the use of a non-pathogenic (deficient) reproducible virus, or possibly a replication-deficient virus. In the latter case, viral replication typically occurs only in complementary viral packaging cells. Suitable systems are disclosed in, for example, Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NY Acad. Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Patent Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89 / 01973; U.S. Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO91 / 02805; Berkner-Biotechniques, 6:616-627. 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for integrating DNA into such expression systems are well known to those skilled in the art. DNA can also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. The uptake of naked DNA can be increased by coating DNA onto biodegradable beads that are efficiently transported into cells.

[0214] For expression, a DNA insert containing a polynucleotide encoding an antibody or polypeptide disclosed herein can be operatively linked to a suitable promoter (e.g., a heterologous promoter), for example, the phage λPL promoter, E. coli (… E. coli The expression construct may include promoters such as lac, trp, and tac, early and late SV40 promoters, and promoters of retroviral LTRs. Other suitable promoters are known to those skilled in the art. The expression construct may also contain transcription initiation and termination sites, as well as ribosome binding sites for translation within the transcription region. The coding portion of the mature transcript expressed by the construct may include a translation initiation codon and a stop codon (UAA, UGA, or UAG) located at an appropriate position at the end of the polypeptide to be translated.

[0215] As noted, the expression vector may include at least one optional biomarker. Such biomarkers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in *E. coli* and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as *E. coli*, *Streptomyces*, etc. Streptomyces ) and Salmonella typhimurium ( Salmonella typhimurium The host cells described herein include: fungal cells, such as yeast cells; insect cells, such as Drosophila S2 and Fall Armyworm Sf9 cells; animal cells, such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture media and conditions for the host cells described herein are known in the art.

[0216] Non-limiting vectors for bacteria include pQE70, pQE60, and pQE-9, available from Qiagen; pBS, Phagescript, Bluescript, pNH8A, pNH16a, pNH18A, and pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5, available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG, available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL, available from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

[0217] Suitable non-restrictive bacterial promoters include the Escherichia coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoters, λPR and PL promoters, and trp promoters. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, promoters of retroviral LTRs (e.g., the Rous sarcoma virus (RSV) promoter), and metallothionein promoters (e.g., the mouse metallothionein-I promoter).

[0218] In *Saccharomyces cerevisiae*, various vectors containing constitutive or inducible promoters (such as α-factor, alcohol oxidase, and PGH) can be used. See Ausubel et al. (1989), *Current Protocols in Molecular Biology*, John Wiley & Sons, New York, NY, and Grant et al., *MethodsEnzymol.*, 153: 516-544 (1997).

[0219] The construct can be introduced into host cells via calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0220] By inserting enhancer sequences into vectors, transcription of DNA encoding the disclosed antibodies can be increased in higher eukaryotes. Enhancers are cis-acting elements of DNA, typically about 10 to 300 bp, that increase the transcriptional activity of promoters in a given host cell type. Examples of enhancers include the SV40 enhancer (located late on the origin of replication, at base pairs 100 to 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer late on the origin of replication, and the adenovirus enhancer.

[0221] To facilitate the secretion of translated proteins into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment, an appropriate secretion signal can be integrated into the expressed polypeptide. This signal can be endogenous to the polypeptide or a heterologous signal.

[0222] The peptide (e.g., an antibody) may be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or a histidine-tagged peptide, and may include not only secretion signals but also additional heterologous functional regions. For example, additional amino acid regions (especially charged amino acids) may be added to the N-terminus of the peptide to improve its stability and persistence in host cells, during purification, or subsequent processing and storage. Furthermore, peptide moieties may be added to the peptide to facilitate purification. These regions can be removed prior to the final preparation of the peptide. Adding peptide moieties to peptides to guide secretion or excretion, improve stability, and facilitate purification are techniques well-known and conventional in the art.

[0223] Treatment

[0224] The antibodies or antigen-binding fragments disclosed herein can be used for a variety of therapeutic purposes. For example, the antibodies or antigen-binding fragments or antibody-drug conjugates disclosed herein can be used to treat diseases characterized by cells expressing GPC1.

[0225] On the one hand, this disclosure provides methods for treating cancer in a subject, methods for reducing the rate of increase in tumor volume over time in a subject, methods for reducing the risk of metastasis, or methods for reducing the risk of additional metastasis in a subject. In some embodiments, the treatment can prevent, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment can lead to a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0226] On the one hand, this disclosure provides antibodies, antibody-drug conjugates or pharmaceutical compositions disclosed herein for use as pharmaceuticals.

[0227] On the one hand, this disclosure provides the use of the antibodies, antibody-drug conjugates or pharmaceutical compositions disclosed herein in the preparation of medicaments for treating diseases (e.g., cancer).

[0228] The scope of this application also includes antibody-drug conjugates as described above, which are used as drugs.

[0229] One or more embodiments of this disclosure relate to an antibody-drug conjugate as described above, for use in treating a disease or condition characterized by cells expressing GPC1, more preferably said disease or condition being a cancer characterized by GPC1 expression (e.g., GPC1 overexpression).

[0230] One or more embodiments of this disclosure relate to methods for treating diseases or conditions characterized by cells expressing GPC1, more preferably said disease or condition being cancer characterized by GPC1 expression (e.g., GPC1 overexpression), said method comprising administering a therapeutically effective amount of the antibody-drug conjugate as described above to a subject in need.

[0231] One or more embodiments of this disclosure relate to the use of the antibody-drug conjugate as described above in the preparation of a medicament for treating a disease or condition characterized by cells expressing GPC1, more preferably said disease or condition being cancer characterized by GPC1 expression (e.g., GPC1 overexpression).

[0232] In one or more embodiments of this disclosure, the disease or condition characterized by cells expressing GPC1 is cancer. In one or more embodiments, the cancer may be one or more of the following: breast cancer, carcinoid tumor, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer (including hepatocellular carcinoma), lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancies.

[0233] In one or more embodiments, the cancer is breast cancer, including TNBC, HER2-positive breast cancer, ER-positive breast cancer, and PR-positive breast cancer.

[0234] In one or more embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer (also known as castrate-resistant prostate cancer).

[0235] In one or more embodiments, the cancer is NSCLC, ovarian cancer, melanoma, colorectal cancer, breast cancer, head and neck cancer, gastrointestinal cancer, bladder cancer, or bone cancer.

[0236] In one or more embodiments, the cancer may be a solid tumor. In some embodiments, the cancer is esophageal squamous cell carcinoma, cervical squamous cell carcinoma, cutaneous squamous cell carcinoma, or head and neck squamous cell carcinoma. In one or more embodiments, the cancer is head and neck squamous cell carcinoma (SCCHN, also known as head and neck squamous cell carcinoma (HNSCC)), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal cancer.

[0237] In one or more embodiments, the cancer may be triple-negative breast cancer (TNBC), gastric cancer, urothelial carcinoma (including upper-tract urothelial cancer), Merkel-cell carcinoma, or head and neck cancer.

[0238] In one or more embodiments, the cancer may be melanoma, pancreatic cancer, mesothelioma, or an advanced solid tumor. In one or more embodiments, the cancer may be Hodgkin's lymphoma.

[0239] In one or more embodiments, the cancer is esophageal cancer, gastric cancer, glioblastoma, breast cancer, ovarian cancer, bladder cancer, pancreatic cancer, lung cancer, colorectal cancer, gastric cancer, prostate cancer, prostate cancer bone metastasis, kidney cancer, multiple myeloma, or cholangiocarcinoma (including intrahepatic cholangiocarcinoma).

[0240] In one or more embodiments of this disclosure, the cancer is esophageal cancer, head and neck cancer, cervical cancer, lung cancer, bladder cancer, or breast cancer, including any of the subtypes of the foregoing.

[0241] In one or more embodiments of this disclosure, the cancer is a sarcoma, such as gastric / gastric adenocarcinoma, NSCLC adenocarcinoma, esophageal adenocarcinoma, gastroesophageal junction adenocarcinoma, pancreatic ductal adenocarcinoma, thymoma, uterine cancer, osteosarcoma, or soft tissue sarcoma (STS), or a subtype thereof.

[0242] In one or more embodiments of this disclosure, the soft tissue sarcoma (STS) is selected from epithelioid sarcoma (including epithelial ovarian cancer), clear cell sarcoma, alveolar soft part sarcoma, extraosseous myxoid chondrosarcoma, epithelioid hemangioendothelioma, inflammatory myofibroblastic tumor, undifferentiated embryonal sarcoma, alveolar soft part sarcoma (ASPS), angiosarcoma, chondrosarcoma, dermatofibrosarcoma protuberans (DFSP), desmoid sarcoma, and Ewing's sarcoma. Sarcoma, fibrosarcoma, myxofibrosarcoma, gastrointestinal stromal tumor (GIST), non-uterine leiomyosarcoma, uterine leiomyosarcoma, liposarcoma, malignant fibrohistiocytoma (MFH), malignant peripheral nerve sheath tumor (MPNST), rhabdomyosarcoma, synovial sarcoma, and / or leiomyosarcoma (LMS).

[0243] In one or more embodiments of this disclosure, the disease or condition characterized by cells expressing GPC1 is fibrosis, such as fibrosis of the liver, lungs, heart, kidneys, skin, or bones (including bone marrow).

[0244] On the one hand, this disclosure provides methods including administering a therapeutically effective amount of the disclosed antibody or its antigen-binding fragment (including antibody-drug conjugates) to a subject in need (e.g., a subject who has, or has been identified or diagnosed with, cancer).

[0245] In some embodiments, the compositions and methods disclosed herein can be used to treat subjects at risk of cancer. Patients with cancer can be identified using various methods known in the art.

[0246] As used herein, “effective amount” means an amount or dose sufficient to produce a beneficial or desired outcome, including stopping, slowing, delaying, or inhibiting the progression of a disease (e.g., an autoimmune disease or cancer). Effective amounts will vary depending on factors such as the age and weight of the subject, the severity of symptoms, and the route of administration of the antibody to be administered, the antigen-binding fragment, the polynucleotide encoding the antibody, the carrier containing the polynucleotide, and / or a combination thereof, and therefore can be determined on a case-by-case basis.

[0247] An effective amount can be administered via a single or multiple doses. For example, an effective amount of an antibody or antigen-binding fragment is sufficient to improve, stop, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or sufficient to improve, stop, stabilize, reverse, slow, and / or delay the in vitro proliferation of cells (e.g., biopsy cells, any cancer cells or cell lines described herein, e.g., cancer cell lines)). As understood in the art, the effective amount of an antibody or antigen-binding fragment can vary, particularly depending on the patient's medical history and other factors such as the type (and / or dose) of the antibody used.

[0248] The effective amount and regimen for administering the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein can be determined empirically, and such determination is within the competence of those skilled in the art. Those skilled in the art will understand that the dosage that must be administered will vary, for example, depending on the mammal receiving the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein, the route of administration, the specific type of antibody, antibody-encoding polynucleotide, antigen-binding fragment, and / or composition used, and other drugs administered to the mammal. Guidance on the selection of appropriate dosages of antibodies or antigen-binding fragments can be found in the literature on the therapeutic use of antibodies and antigen-binding fragments, such as Handbook of Monoclonal Antibodies, edited by Ferrone et al., Noges Publications, Park Ridge, NJ, 1985, Chapter 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, edited by Haber et al., Raven Press, New York, 1977, pp. 365-389.

[0249] Pharmaceutical Compositions and Routes of Administration

[0250] This document also provides pharmaceutical compositions containing antibodies, antigen-binding fragments thereof, or antibody-drug conjugates (ADCs) targeting GPC1 as described herein. These pharmaceutical compositions can be formulated in any manner known in the art.

[0251] The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0252] Compositions containing antibodies, antigen-binding fragments thereof, or antibody-drug conjugates (ADCs) targeting GPC1 as described herein may be formulated for parenteral (e.g., intravenous, intra-arterial, intra-muscular, intradermal, subcutaneous, or intraperitoneal) administration in unit dosage forms (i.e., physically discrete units containing a predetermined amount of the active compound, facilitating administration and dose uniformity).

[0253] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. The pharmaceutical composition may be provided in unit dosage forms (i.e., single-dose doses). The pharmaceutical composition may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants.

[0254] Excipients intended for formulation with antibodies, their antigen-binding fragments, or antibody-drug conjugates should generally be pharmaceutically acceptable. "Pharmaceutically acceptable" should be understood as non-toxic materials that do not diminish the effectiveness of the ADC. Such pharmaceutically acceptable buffers, carriers, or excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, Ed., Mack Publishing Company (1990) and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), the disclosures of which are incorporated herein by reference).

[0255] The formulation depends on the chosen route of administration. For injection, the antibody or ADC can be formulated in an aqueous solution, preferably in a physiologically compatible buffer to minimize injection site discomfort. The solution may contain formulations such as suspending agents, stabilizers, and / or dispersants. Alternatively, the antibody or ADC may be in lyophilized form for reconstitution with a suitable medium (e.g., sterile pyrogen-free water) prior to use.

[0256] The toxicity and therapeutic efficacy of the composition can be determined using standard pharmaceutical procedures in cell cultures or laboratory animals (e.g., monkeys). For example, the LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective to 50% of the population) can be determined: the therapeutic index is the ratio of LD50 to ED50. Agents exhibiting a high therapeutic index are preferred. If an agent exhibits undesirable side effects, care should be taken to minimize potential harm (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined using other standard pharmaceutical procedures.

[0257] As will be understood by those skilled in the art, the amount of a compound can vary depending on its specific activity. A suitable dosage may contain a predetermined amount of the active composition, which, in combination with the desired diluent, is calculated to produce the desired therapeutic effect.

[0258] The pharmaceutical composition may be contained in a container, package, or dispenser, and accompanied by instructions for administration. This disclosure also provides methods for producing antibodies or antigen-binding fragments thereof for the various uses described herein.

[0259] sequence

[0260] Example

[0261] The present invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0262] Example 1. Generation of human anti-GPC1 antibody

[0263] Human GPC1 protein (Kactus Biosystems, catalog number: GPC-HM111) or the DNA encoding this protein was emulsified with an adjuvant and used to immunize RenLite™ mice (Biocytogen, combining a fully human heavy chain variable domain with a universal light chain in situ substitution). RenLite™ mice are described, for example, in PCT / CN2021 / 097652, which is incorporated herein by reference in its entirety. Retroorbital blood was collected as a negative control prior to immunization.

[0264] A total of three immunizations were administered. The first and second immunizations were three weeks apart, and the second and third immunizations were two weeks apart. One week after the third immunization, retroorbital blood was collected, and serum antibody titers were detected by fluorescence-activated cell sorting (FACS). One week later, mice with high titers were given a booster immunization via intraperitoneal injection of GPC1 protein.

[0265] Antigen-specific immune cells are isolated from immunized mice to further obtain anti-GPC1 antibodies or the light and heavy chain variable region sequences of anti-GPC1 antibodies. For example, plasma cells secreting antigen-specific monoclonal antibodies are screened and identified using single-cell technology (such as the Beacon® Optofluidic System, Berkeley Lights Inc.), and then the antibody variable region sequence is obtained using reverse transcription and PCR sequencing. The obtained variable region sequence is cloned into a vector containing the sequence encoding the human IgG1 constant region for antibody expression. The binding of the expressed antibody to GPC1 is verified by FACS.

[0266] Exemplary antibodies obtained by this method include 3G1. The VH and VL CDR 1-3 sequences of 3G1, as well as the VH and VL regions, are outlined in this document. Several other anti-GPC1 antibodies were obtained following the same procedure. A total of 23 antibodies were obtained and evaluated as part of a candidate selection process, among which 3G1 demonstrated overall superiority over the remaining 22 control antibodies in terms of GPC1 binding properties, internalization properties, specificity and off-target reactivity, cross-species reactivity, and stability.

[0267] In the following experiments, a reference antibody (named Ref1) specific to GPC1 was used, which was synthesized based on publicly available amino acid sequence information (WO2016168885A1). Specifically, the VH and VL sequences shown in SEQ ID NO: 12-13 were linked to the human IgG1 constant region to form Ref1.

[0268] Example 2. Binding specificity of anti-GPC1 antibody

[0269] The binding specificity of the anti-GPC1 antibody to his-tagged human GPC1 (hGPC1-His, ACRO Biosystems, catalog number: GP1-H52H9), his-tagged human GPC2 (hGPC2-His, ACRO Biosystems, catalog number: GP2-H52H3), his-tagged human GPC3 (hGPC3-His, ACRO Biosystems, catalog number: GP3-H52H4), or his-tagged human GPC4 (hGPC4-His, ACRO Biosystems, catalog number: GP4-H52H3) was validated using the Biacore™ (Biacore, Inc., Piscataway NJ) 8K biosensor (equipped with a pre-immobilized Protein A sensor chip).

[0270] Antibodies were captured on a Protein A chip for detection. Purified antibodies (2 μg / mL) were loaded at a flow rate of 10 μL / min to bind recombinant histagged human proteins (400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, and 0 nM). The flow rate was 30 μL / min, and the binding and dissociation times were set to 180 s and 400 s, respectively. After the last injection of each titration, the chip was regenerated for 30 s with glycine solution (pH 2.0) at a flow rate of 30 μL / min.

[0271] Using Biacore™ 8K evaluation software, the data were globally fitted to a 1:1 Langmuir combined model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994). Methods Enzymology 6. 99-110), simultaneously obtaining the kinetic binding rate (kon) and dissociation rate (koff). Affinity is derived from the quotient of the kinetic rate constant (KD=koff / kon). As will be understood by those skilled in the art, the same method is used for testing antibodies with appropriate adjustments to parameters (e.g., antibody concentration).

[0272] The results are summarized in the table below, which shows that 3G1 specifically recognizes human GPC1, but does not recognize other phosphatidylinositol proteoglycan members.

[0273] Table 1

[0274] Example 2b. Binding specificity of anti-GPC1 antibody (off-target screening)

[0275] The Retrogenix Cell Microarray Technology Platform (Charles River) was used to screen for specific off-target binding interactions of the anti-GPC1 antibody (3G1). The binding of the anti-GPC1 antibody to immobilized human embryonic kidney (HEK) 293 cells expressing 6105 different full-length human plasma membrane proteins, secretory or cell surface-anchored secretory proteins, and an additional 400 human heterodimers was screened at 20 μg / mL.

[0276] Compared to the control anti-GPC1 clone, the 3G1 antibody exhibited significant specific interactions with its intended target, GPC1, on both immobilized and live-cell microarrays. Furthermore, no other specific interactions were identified. Here, significant specific interactions were evaluated as interactions with a signal-to-noise ratio (SNR) greater than 1.

[0277] Example 2c. Binding specificity of anti-GPC1 antibody (KO cell line)

[0278] TE-8 is a recognized cell line model system for studying esophageal cancer. 3G1 has been evaluated in native GPC1-positive TE-8 cell models and GPC1 knockout (KO) negative TE-8 clones. Flow cytometry confirmed that 3G1 binds only to unmodified cells, but not to GPC1-knockout cells, demonstrating GPC1-specific binding of the antibody in this cancer model. Furthermore, 3G1 was evaluated in GPC1-positive FaDu head and neck cancer (HNSCC) cells and GPC1-negative Burkitt lymphoma Raji cells. Flow cytometry revealed that it binds to GPC1-positive FaDu cells in nanomolar ranges, but not to GPC1-negative Raji cells.

[0279] The binding affinity of the GPC1-targeting 3G1 antibody to GPC1-positive Fadu cells and GPC1-negative Raji cells was also detected by flow cytometry. A non-targeting isotype of human immunoglobulin G1 (hIgG1) (Ultra-LEAF™ purified human IgG1 isotype control recombinant antibody, clone QA16A12, #403502, BioLegend, San Diego, CA, USA) was used as a negative control. The antibody was labeled with Alexa Fluor™ 647 (AF-647) using the GlyCLICK Fluorophore 647 kit (L1-F03, Genovis, Kävlinge, Sweden).

[0280] Human FaDu head and neck squamous cell carcinoma cell line (HTB-43™, lot number 70044484) and Burkitt lymphoma Raji cell line (CCL-86™, lot number 70053718) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in EMEM (C0005-01, AddexBio, San Diego, CA, USA) or RPMI-1640 medium (A10491-01, Gibco®, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; #S0615, Sigma) and 1% penicillin-streptomycin (#15070, Gibco®), respectively. Cells were maintained in a humidified incubator at 37°C and 5% CO2.

[0281] Cells were washed with phosphate-buffered saline (PBS, 10010031, Gibco®) and, for Fadu, dissociated from the culture flasks using StemPro™ Accutase™ cell dissociation reagent (A1110501, Gibco®). Cells were collected by centrifugation (300 xg, 5 min, 4°C), washed in cold FACS (fluorescence-activated cell sorting) buffer (PBS containing 2% FBS), and then stained on ice for 30 min with the corresponding AF-647 fluorescently labeled antibody diluted in FACS buffer. Finally, cells were washed again with ice-cold FACS buffer and resuspended in ice-cold FACS buffer containing DAPI (4',6-diamidinyl-2-phenylindole) (1 µg / mL, D3571, Invitrogen™) for analysis using a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA). The analysis employed a three-step gating strategy to analyze live cells and obtain their mean fluorescence intensity (MFI) of AF-647: cell sorting by cell morphology (step #1), including single cells (step #2), and selection of live cells by DAPI staining (step #3). Binding curves were created using GraphPad Prism software version 10.0.3 (GraphPad Software Inc., La Jolla, CA, USA). The half-maximum effective concentration (EC50) for each test item was determined using four-parameter logistic regression (4PL) analysis. 50 )value.

[0282] Binding in Fadu cells was evaluated in three independent experiments, and the results are shown below. In each case, the EC50 of 3G1 was in the nanomolar range.

[0283]

[0284] In summary, 3G1 was shown to bind to GPC-1 in a variety of different cancer cell lines, but showed no interaction in GPC-1-negative cell lines. Using the non-targeted allotype control hIgG1 as a negative control, it showed no binding affinity to any of the evaluated cell lines.

[0285] Example 3. Cross-species binding of anti-GPC1 antibody

[0286] CHO-hGPC1 cells, CHO-fasGPC1 cells, CHO-mGPC1 cells, or CHO-ratGPC1 cells were respectively inoculated at 5 × 10⁻⁶. 4Cells were transferred to 96-well plates at a density of [number] cells / well. Serially diluted anti-GPC1 antibody was added to the 96-well plates and incubated at 4°C for 30 min. Then, cells were incubated with the secondary antibody anti-hIgG-Fc-Alexa Fluor 647 (RL1-H) (Jackson ImmunoResearchLaboratories, Inc., Catalog No.: 109-606-170) at 4°C in the dark for 15 min, followed by flow cytometry analysis. EC50 was calculated.

[0287] CHO-hGPC1 cells, CHO-fasGPC1 cells, CHO-mGPC1 cells, and CHO-ratGPC1 cells were obtained by transfecting CHO-S cells with vectors expressing human GPC1 (SEQ ID NO: 16), cynomolgus monkey GPC1 (SEQ ID NO: 17), mouse GPC1 (SEQ ID NO: 18), and rat GPC1 (SEQ ID NO: 19), respectively.

[0288] The test results are shown in the table below. 3G1 can bind to human GPC1, monkey GPC1, mouse GPC1, and rat GPC1. Notably, Ref1 does not bind to monkey GPC1.

[0289] Table 2

[0290] Example 3a. Cross-species binding of anti-GPC1 antibody (flow cytometry)

[0291] The cross-species binding affinity of 3G1 was assessed in HEK293 cells transiently transfected with plasmids that resulted in the expression of human, cynomolgus monkey, rat, or mouse GPC1 protein and the green fluorescent protein (GFP) reporter gene. A non-targeted isotype of human immunoglobulin G1 (hIgG1) (Ultra-LEAF™ purified human IgG1 isotype control recombinant antibody, clone QA16A12, 403502, BioLegend, San Diego, CA, USA) was used as a negative control.

[0292] Species-specific plasmids were generated from the pLenti-P2A-GFP backbone (PS100108, Origene Technologies, Inc., Rockville, MD, USA). Vectors containing human, cynomolgus monkey, rat, or mouse GPC1 copy DNA (cDNA) integrated into the plasmid multiple cloning site were also produced by Origene Technologies, Inc., Rockville, MD, USA.

[0293] HEK293 cells (CRL-1573, American Type Culture Collection (ATCC), Manassas, VA, USA) were cultured in EMEM (C0005-01, AddexBio, San Diego, CA, USA) supplemented with 10% FBS (fetal bovine serum, F4135, Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin mixture (10000 U / mL, Gibco®, Thermo Fisher Scientific, Waltham, MA, USA).

[0294] For transient transfection of human, cynomolgus monkey, rat, or mouse GPC1 and GFP encoding plasmids, cells were transfected using FuGENE® HD transfection reagent (Promega, Madison, WI, USA). Plasmid DNA was mixed with FuGENE® HD transfection reagent (E2312, Promega, Madison, WI, USA) at a 4:1 ratio (transfection reagent: DNA) in Opti-MEM™ I serum-depleted medium (51985034, Gibco®) and incubated at room temperature for 15 minutes. Subsequently, the transfection reagent / DNA mixture was added to a T75 culture flask (90076, TPP, Trasadingen, Switzerland) containing HEK293 cells in 20 mL of growth medium.

[0295] After applying the transfection mixture, cells were incubated in a humidified incubator at 37°C and 5% CO2 for 72 hours. Cells were then washed with phosphate-buffered saline (PBS, 10010031, Gibco®, Thermo Fisher Scientific) and dissociated from the culture flasks using StemPro™ Accutase™ cell dissociation reagent (A1110501, Gibco®). Cells were collected by centrifugation (300x g, 5 min, 4°C), washed in fluorescence-activated cell sorting (FACS) buffer (PBS containing 2% FBS), and stained on ice for 30 min with the appropriate AF-647 fluorescently labeled antibody (10 µg / mL, dissolved in FACS buffer). Finally, the cells were washed again with ice-cold FACS buffer and resuspended in ice-cold FACS buffer containing 4',6-diamidinyl-2-phenylindole (DAPI) (1 µg / mL, D3571, Invitrogen™) before analysis using a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA). The analysis involved a four-step gating strategy to obtain the mean fluorescence intensity (MFI) of AF-647 in GFP-positive cells: cell selection was performed through the following steps: cell sorting by cell shape (step #1), including single cells (step #2), selection of viable cells by DAPI staining (step #3), and high GFP-positive cells (step #4).

[0296] Combination curves were created using GraphPad Prism software version 10.0.3 (GraphPad Software Inc., La Jolla, CA, USA). The half-maximal effective concentration (EC50) for each test item was determined using four-parameter logistic regression (4PL) nonlinear regression analysis. 50 )value.

[0297] The EC50 values ​​reported in Table 1a represent the mean ± standard deviation (SD) of five independent experiments. In all experiments, the 3G1 antibody showed high affinity in the nanomolar range for cells transfected with human, cynomolgus monkey, rat, or mouse GPC1.

[0298] Table 1a

[0299] Specifically, cross-species reactivity with GPC1 in both humans and monkeys (cynomolgus monkeys) is desirable because it allows for easier extrapolation / transfer (credibly) of effects observed in non-human primate studies to anticipated effects in humans, due to the biological similarities between humans and monkeys. Notably, Ref1 (a known anti-GPC1 antibody from WO2016168885A1) does not bind to cynomolgus monkey GPC1, thus giving 3G1 an advantage in providing a better candidate antibody for development for anticipated human medical treatments.

[0300] Example 4. Binding activity of anti-GPC1 antibody to tumor cells

[0301] The binding activity of the anti-GPC1 antibody to cancer cell lines was verified by flow cytometry. In short, human lung cancer cells NCI-H1792 overexpressing GPC1 were subjected to flow cytometry at a rate of 1 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 10 cells / well in 96-well plates. Serially diluted antibody (maximum concentration: 150 µg / mL, 2-fold dilution) was added to each well and incubated at 4°C for 30 min. After washing once with PBS, cells were incubated with the secondary antibody Alexa Fluor® 647 anti-human IgG Fcγ (Jackson Immuno Research, catalog number: 109-606-170) at 4°C for 15 min, followed by flow cytometry analysis. EC50 was calculated. The results showed that the EC50 value of 3G1 binding to NCI-H1792 was 0.7518 µg / mL.

[0302] Example 5. Internalization of anti-GCP1 antibody

[0303] Anti-GPC1 antibody (working concentration 2.5 μg / mL) was added to human lung cancer cells NCI-H1792 along with Fab fragment goat anti-human IgG (Jackson Immuno Research, catalog number: 109-007-008) labeled with pHAb amine-reactive dye (Promega, catalog number: G9845) and incubated for up to 24 hours. Cells were centrifuged and washed with FACS buffer, then measured using flow cytometry. Human IgG1 protein was used as the isotype control (ISO). Data showed that 3G1 exhibited a good rate of endocytosis in NCI-H1792 cells. Evaluation of 3G1 relative to Ref1 and 22 other selected clones (see Example 1) indicated that 3G1 showed optimal overall internalization in GPC1-expressing NCI-H1792 cells.

[0304] Example 6. Epitope determination of anti-GPC1 antibody

[0305] Epitope binding assays were performed using Biacore™ to determine whether the two anti-GPC1 antibodies targeted the same or overlapping epitopes. The entire experiment used 1× HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM EDTA, and 0.05% P20, pH 7.4) diluted with HBS-EP+ buffer (10×) as the run buffer. Approximately 50 RU of hGPC1-His protein (ACRO Biosystems, catalog number: GP1-H52H9) was captured at a flow rate of 10 µL / min, and 200 nM of the test antibody (analyte 1) was injected at a flow rate of 30 µL / min to bind the ligand. Another control antibody (analyte 2) was injected under the same conditions to determine whether the binding of the different antibodies interfered with each other. The binding time for each antibody was 200 seconds.

[0306] Binding values ​​for each antibody were obtained using Biacore 8K evaluation software. To quantify the interference of one antibody binding to another, binding ratios were calculated to compare each antibody pair. The binding ratio was defined as the binding value of the second antibody (analyte 2) divided by the binding value of the first antibody (analyte 1). The binding ratios for each antibody pair are summarized in the table below. More specifically, if analyte 1 exhibits a blocking effect on analyte 2, the binding ratio is less than 0.5. If analyte 1 does not exhibit a blocking effect on analyte 2, the binding ratio is greater than 0.5. Typically, interfering antibody pairs have the same or overlapping epitopes. The results showed that 3G1 and Ref1 recognize different epitopes.

[0307] Table 3

[0308] Example 7. Generation of antibody-drug conjugates (ADCs) targeting GPC1

[0309] The ADCs were prepared using common conjugation methods previously described in the art (Doronina et al., 2003 Nature biotechnology 21(7):778-84; Francisco et al., 2003. Blood 102(4):1458-65; Hamblett et al., 2004. Clinical cancer research 10(20):7063-70), the contents of which are incorporated herein by reference.

[0310] The antibody is mildly reduced by dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) before conjugation, and then contacted with an excess of the intended linker-payload with maleimide hexanoyl attachment groups.

[0311] Exemplarily, 3G1-MC-VC-PABC-MMAE can be prepared by incubating 3G1 at a concentration of 5 mg / mL in a buffer of 50 mM sodium borate, 50 mM NaCl, and pH 8.0 containing 10 mM DTT at 37°C for 10 min. Then, the DTT is removed by buffer exchange using a 30 kDa NMWL centrifuge filter, replacing it with fresh PBS containing 1 mM EDTA at pH 7.4, and the concentration is adjusted to 2 mg / mL. Subsequently, it is immediately conjugated with 5-10 molar excess of maleimide hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl monomethyl auratestatin E (MC-VC-PAB-MMAE, i.e., Vittin) dissolved in anhydrous DMSO, with a final DMSO concentration of 10% v / v during conjugation, and reacted at 37°C for 2 h. The resulting ADC is purified by gel filtration on a PD-10 desalting column.

[0312] The following ADCs were prepared using well-known concatenation methods, as exemplarily listed above.

[0313] 3G1-MC-VC-PABC-MMAE (ADC-A)

[0314] 3G1-MC-VA-PABC-MMAE (ADC-B)

[0315] 3G1-MC-VC-PABC-MMAE (ADC-C)

[0316] 3G1-MC-GGFG-PABC-MMAE (ADC-D)

[0317] 3G1-MC-VA-PABC-Ecinotecan (ADC-E)

[0318] 3G1-MC-VC-PABC-Eciticon (ADC-F)

[0319] 3G1-MC-GGFG-PABC-Ecitabine (ADC-G)

[0320] 3G1-MC-GGFG-Ecitenetane (ADC-H)

[0321] 3G1-MC-VA-PABC-Dxd (ADC-I)

[0322] 3G1-MC-VC-PABC-Dxd (ADC-J)

[0323] 3G1-MC-GGFG-PABC-Dxd (ADC-K)

[0324] 3G1-MC-GGFG-Dxd (ADC-L)

[0325] Other implementation plans

[0326] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to phosphatidylinositol proteoglycan-1 (GPC1), comprising: a. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) Immunoglobulin light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 4 as VL complementarity-determining region 1 (CDR1), the amino acid sequence shown in SEQ ID NO: 5 as VL CDR2, and the amino acid sequence shown in SEQ ID NO: 6 as VL CDR3; and / or ii) The immunoglobulin heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 1 as VH complementarity-determining region 1 (CDR1), the amino acid sequence shown in SEQ ID NO: 2 as VH CDR2, and the amino acid sequence shown in SEQ ID NO: 3 as VH CDR3; or b. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) Immunoglobulin light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 4 as VL complementarity-determining region 1 (CDR1), the amino acid sequence shown in SEQ ID NO: 5 as VL CDR2, and the amino acid sequence shown in SEQ ID NO: 6 as VL CDR3; and / or ii) The immunoglobulin heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 7 as VH complementarity-determining region 1 (CDR1), the amino acid sequence shown in SEQ ID NO: 8 as VH CDR2, and the amino acid sequence shown in SEQ ID NO: 9 as VH CDR3; or c. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) The immunoglobulin light chain variable region (VL), comprising the amino acid sequence shown in SEQ ID NO: 11 or a sequence having at least 85% sequence identity with it, or consisting of the above sequence; and / or ii) The immunoglobulin heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 10 or a sequence having at least 85% sequence identity with it, or consisting of the above sequence; or d. Anti-GPC1 antibodies or their antigen-binding fragments as defined herein, containing i) Immunoglobulin light chains comprising the amino acid sequence shown in SEQ ID NO: 11 or a sequence having at least 85% sequence identity with it, and the amino acid sequence shown in SEQ ID NO: 15 or a sequence having at least 85% sequence identity with it, or consisting of the above sequences; and / or ii) Immunoglobulin heavy chain comprising the amino acid sequence shown in SEQ ID NO: 10 or a sequence having at least 85% sequence identity with it, and the amino acid sequence shown in SEQ ID NO: 14 or a sequence having at least 85% sequence identity with it, or consisting of the above sequences.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR 1, 2, 3 of the amino acid sequences shown in SEQ ID NO: 1-3, respectively, and the VL comprises CDR 1, 2, 3 of the amino acid sequences shown in SEQ ID NO: 4-6, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the definition of Chothia, the VH comprises CDR 1, 2, 3 of the amino acid sequences shown in SEQ ID NO: 7-9, respectively, and the VL comprises CDR 1, 2, 3 of the amino acid sequences shown in SEQ ID NO: 4-6, respectively.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, It comprises: a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 10 or composed thereof, and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 11 or composed thereof.

5. The antibody or antigen-binding fragment thereof according to claim 4, comprising a heavy chain variable region (VH) containing the same VH CDR1, VH CDR2, and VH CDR3 as those in SEQ ID NO: 10; and a light chain variable region (VL) containing the same VLCDR1, VL CDR2, and VL CDR3 as those in SEQ ID NO:

11.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof specifically binds to human, monkey, mouse and / or rat GPC1.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof, or a single-chain variable fragment (scFv).

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof is a human IgG1 antibody or antigen-binding fragment thereof, or a human IgG4 antibody or antigen-binding fragment thereof.

9. A nucleic acid comprising a polynucleotide encoding an anti-GPC1 antibody or an antigen-binding fragment thereof as described in any one of claims 1-8.

10. The nucleic acid according to claim 9, wherein the nucleic acid is cDNA.

11. A vector comprising one or more nucleic acids as described in claim 9 or claim 10.

12. A cell comprising the vector of claim 11 or the nucleic acid of claim 9 or 10.

13. The cell of claim 12, wherein the cell is a CHO cell.

14. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising: (a) The cells are cultured under conditions sufficient to cause the cells of any one of claims 12-13 to produce the antibody or the antigen-binding fragment; as well as (b) Collect the antibody or antigen-binding fragment produced by the cells.

15. An antibody-drug conjugate (ADC) targeting GPC1, comprising: a. The antibody or antigen-binding fragment thereof as described in any one of claims 1-8; b. Surfactants; and c. Optionally, a connector that connects a and b.

16. The antibody-drug conjugate of claim 15, wherein the active agent is a therapeutic agent, a radioisotope, or a detectable marker.

17. The antibody-drug conjugate according to any one of claims 15-16, wherein the active agent is a cytotoxic agent.

18. The antibody-drug conjugate according to any one of claims 15-17, wherein the active agent is a therapeutic agent, such as a DNA cross-linking agent, DNA alkylating agent, DNA strand breaking agent, anthracycline drug, antimetabolite, antimicrotubule agent / antimitotic agent, histone deacetylase inhibitor, kinase inhibitor, metabolism inhibitor, peptide antibiotic, immune checkpoint inhibitor, platinum-based antitumor drug, topoisomerase inhibitor, DNA polymerase inhibitor or RNA polymerase inhibitor, nucleotide-based agent or cytotoxic antibiotic.

19. The antibody-drug conjugate according to any one of claims 15-18, wherein the active agent is a cytotoxic agent that allows for effective killing of cells expressing GPC1.

20. The antibody-drug conjugate according to any one of claims 15-19, wherein the active agent is a chemotherapeutic agent.

21. The antibody-drug conjugate according to any one of claims 15-20, wherein the active agent is a DNA cross-linking agent.

22. The antibody-drug conjugate of claim 21, wherein the DNA cross-linking agent is cisplatin, carboplatin, oxaliplatin, mitomycin C (MMC), pyrrolobenzodiazepines (PDB), dimer pyrrolobenzodiazepines, or a derivative or prodrug of any of the foregoing substances, such as SGD-1882.

23. The antibody-drug conjugate according to any one of claims 15-20, wherein the active agent is a DNA alkylating agent.

24. The antibody-drug conjugate according to claim 23, wherein the DNA alkylating agent is a nitrogen mustard, tri(2-chloroethyl)amine, pyridobenzodiazepines, indolinobenzodiazepine dimer, or docamycin SA, or a derivative or prodrug of any of the foregoing substances.

25. The antibody-drug conjugate according to any one of claims 15-20, wherein the active agent is a DNA strand breaking agent.

26. The antibody-drug conjugate of claim 25, wherein the DNA strand-breaking agent is chachiomycin or hamiltrone or a derivative thereof.

27. The antibody-drug conjugate according to any one of claims 15-20, wherein the active agent is an anthracycline drug.

28. The antibody-drug conjugate according to claim 27, wherein the anthracycline drug is daunorubicin, doxorubicin, epirubicin, idarubicin, or PNU-159682, or a derivative or prodrug of any of the foregoing substances.

29. The antibody-drug conjugate according to any one of claims 15-20, wherein the active agent is an antimetabolite.

30. The antibody-drug conjugate according to claim 29, wherein the antimetabolite is a folic acid antagonist, methotrexate, a purine antimetabolite, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, cladribine, a pyrimidine antimetabolite, 5-fluorouracil, 5-fluorodeoxyuridine, cytarabine, gemcitabine, or a derivative or prodrug of any of the foregoing substances.

31. The antibody-drug conjugate according to any one of claims 15-20, wherein the active agent is an antimitotic agent.

32. The antibody-drug conjugate according to claim 31, wherein the antimitotic agent is aurestatin, dolalastatin, monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), taxanes, paclitaxel, docetaxel, vinblastine, vincristine, vinblastine, vinorelbine, maytansine, colchicine, podophyllotoxin, or a derivative or prodrug of any of the foregoing substances.

33. The antibody-drug conjugate according to any one of claims 15-20, wherein the active agent is a histone deacetylase inhibitor.

34. The antibody-drug conjugate according to claim 33, wherein the histone deacetylase inhibitor is trogostatin A, vorinostat, belinostat, pabistat, gilvestat, remistat, abexistat, quistat, rosilistat, pramistat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entenolol, tasidarin, 4SC202, moxistat, romidesin, nicotinamide, sertinol, canbinol, or EX-527, or a derivative or prodrug of any of the foregoing substances.

35. The antibody-drug conjugate according to any one of claims 15-20, wherein the active agent is a kinase inhibitor.

36. The antibody-drug conjugate according to claim 35, wherein the kinase inhibitor is genistein, fumonisin C, PP1-AG1872, PP2-AG1879, SU6656, CGP77675, PD166285, imatinib, erlotinib, gefitinib, fumonisin A, cetuximab, UCS15A, atrazine A or rhizobacterin, or a derivative or prodrug of any of the foregoing substances.

37. The antibody-drug conjugate according to any one of claims 15-20, wherein the active agent is a metabolic inhibitor.

38. The antibody-drug conjugate according to claim 37, wherein the metabolic inhibitor is a NAMPT inhibitor, APO866, GMX-1777, GMX-1778, ATG-019 or OT-82, or a derivative or prodrug of any of the foregoing substances.

39. The antibody-drug conjugate according to any one of claims 15-20, wherein the active agent is an immune checkpoint inhibitor.

40. The antibody-drug conjugate of claim 39, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

41. The antibody-drug conjugate of claim 40, wherein the PD-1 inhibitor is pembrolizumab, nivolumab, cimiprimab, JTX-4014, spartazumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostalimab, AMP-224 or AMP-514, or any derivative thereof.

42. The antibody-drug conjugate of claim 40, wherein the immune checkpoint inhibitor is a PD-L1 inhibitor.

43. The antibody-drug conjugate according to claim 42, wherein the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170 or BMS-986189, or a derivative or prodrug of any of the foregoing substances.

44. The antibody-drug conjugate according to any one of claims 15-20, wherein the active agent is a platinum-based antitumor drug.

45. The antibody-drug conjugate according to claim 44, wherein the platinum-based antitumor drug is liposomal cisplatin, cisplatin, carboplatin, oxaliplatin, nedaplatin, pyridineplatin, phenanthreneplatin, saxaplatin or triplatin tetranitrate, or a derivative or prodrug of any of the foregoing substances.

46. ​​The antibody-drug conjugate according to any one of claims 15-20, wherein the active agent is a topoisomerase inhibitor, such as a topoisomerase-I (Topo1) inhibitor.

47. The antibody-drug conjugate according to claim 46, wherein the topoisomerase inhibitor is camptothecin, topotecan, belotetane, rubotecan, irinotecan, SN-38, eczetane, Dxd, or a derivative or prodrug of any of the foregoing substances.

48. The antibody-drug conjugate according to any one of claims 15-20, wherein the active agent is a DNA polymerase inhibitor or an RNA polymerase inhibitor.

49. The antibody-drug conjugate according to claim 48, wherein the DNA polymerase inhibitor or RNA polymerase inhibitor is amanita, α-amanita, actinomycin D, apigenin, or a derivative or prodrug of any of the foregoing substances.

50. The antibody-drug conjugate according to any one of claims 15-49, wherein the active agent comprises a radioisotope.

51. The antibody-drug conjugate according to any one of claims 15-50, wherein the drug-antibody ratio (DAR) of the antibody-drug conjugate is between 1 and 10, for example between 2 and 8.

52. The antibody-drug conjugate according to any one of claims 15-51, wherein the antibody-drug conjugate is characterized in that the DAR is 2, 3, 4, 5, 6, 7 or 8.

53. The antibody-drug conjugate according to any one of claims 15-52, wherein the antibody-drug conjugate comprises a cleavable linker or a non-cleavable linker.

54. The antibody-drug conjugate according to any one of claims 15-53, wherein the connector comprises an attachment group, such as a maleimide group, for attachment to a sulfur atom on the antibody.

55. The antibody-drug conjugate according to any one of claims 15-54, wherein the linker comprises a dipeptide, tripeptide, tetrapeptide, or pentapeptide, or is composed of a dipeptide, tripeptide, tetrapeptide, or pentapeptide.

56. The antibody-drug conjugate according to any one of claims 15-55, wherein the linker comprises or is composed of a dipeptide, such as valine-citrulline (VC) or valine-alanine (VA).

57. The antibody-drug conjugate according to any one of claims 15-55, wherein the linker comprises or is composed of a tetrapeptide, wherein each amino acid is independently selected from glycine and phenylalanine, for example, wherein the linker comprises a portion of formula (I): Equation (I) Wherein 'L' represents the connectivity with the remaining linker in the direction of the antibody or antibody-binding attachment group of the ADC, and where 'D' represents the connectivity with the active agent directly or in the form of a spacer.

58. The antibody-drug conjugate according to any one of claims 15-57, wherein the antibody-drug conjugate is further characterized by comprising a spacer, for example, a spacer comprising p-aminobenzyl (PAB), p-aminobenzylcarbamate, p-aminobenzyloxycarbonyl (PABC), or polyethylene glycol (PEG).

59. The antibody-drug conjugate according to any one of claims 15-58, wherein the antibody-drug conjugate comprises a p-aminobenzyloxycarbonyl group (PABC) as a spacer.

60. The antibody-drug conjugate according to any one of claims 15-59, wherein the antibody-drug conjugate comprises an attachment group for covalent bonding with the antibody, preferably wherein the attachment group comprises a maleimide moiety, more preferably wherein the attachment group comprises a maleimide hexanoyl (MC) moiety.

61. A pharmaceutical composition comprising an anti-GPC1 antibody according to any one of claims 1-8 or an ADC targeting GPC1 according to any one of claims 15-60, and one or more excipients.

62. A kit comprising an anti-GPC1 antibody according to any one of claims 1-8, or an ADC targeting GPC1 according to any one of claims 15-60, or a pharmaceutical composition according to claim 61.

63. The anti-GPC1 antibody according to any one of claims 1-8, or the GPC1-targeting ADC according to any one of claims 16-60, or the pharmaceutical composition according to claim 61, used as a drug.

64. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-8 or an ADC targeting GPC1 according to any one of claims 15-60, in a method of treating a subject with cancer.

65. The composition for use according to claim 64, wherein the cancer is characterized by cells expressing GPC1, such as tumor cells expressing GPC1.

66. The composition for use according to any one of claims 64-65, wherein the cancer is breast cancer, triple-negative breast cancer, carcinoid, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, stomach cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancy.

67. The composition for use according to any one of claims 64-65, wherein the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-resistant prostate cancer.

68. The composition for use according to any one of claims 64-65, wherein the cancer is esophageal cancer, gastric cancer, breast cancer, ovarian cancer, bladder cancer, pancreatic cancer, lung cancer, colorectal cancer, gastric cancer, prostate cancer, kidney cancer, multiple myeloma, or cholangiocarcinoma.

69. The composition for use according to any one of claims 64-65, wherein the cancer is pancreatic cancer, colorectal cancer, gastric cancer, breast cancer, bladder cancer, or lung cancer.

70. A method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-8 or an ADC targeting GPC1 as described in any one of claims 15-60.

71. A method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-8 or an ADC targeting GPC1 as described in any one of claims 15-60.

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