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WO2025202359A1 - Anticorps anti-gpc1 et leurs utilisations - Google Patents

Anticorps anti-gpc1 et leurs utilisations

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Publication number
WO2025202359A1
WO2025202359A1 PCT/EP2025/058397 EP2025058397W WO2025202359A1 WO 2025202359 A1 WO2025202359 A1 WO 2025202359A1 EP 2025058397 W EP2025058397 W EP 2025058397W WO 2025202359 A1 WO2025202359 A1 WO 2025202359A1
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WIPO (PCT)
Prior art keywords
antibody
antigen
binding fragment
amino acid
human
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PCT/EP2025/058397
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English (en)
Inventor
Xueyuan JIANG
Yanan GUO
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Adcendo Aps
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Adcendo Aps
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Publication of WO2025202359A1 publication Critical patent/WO2025202359A1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Glypicans are a family of heparan sulfate proteoglycans (HSPGs) that interact with the plasma membrane through a glycosylphosphatidyl inositol anchor.
  • HSPGs heparan sulfate proteoglycans
  • GPC1, GPC2, GPC3, GPC4, GPC5 and GPC6 six GPC family members have been identified, including GPC1, GPC2, GPC3, GPC4, GPC5 and GPC6.
  • Glypicans are predominantly expressed during embryonic development and have been reported to play an important role in organ morphological development by influencing signaling pathways including Wnt, hedgehog, transforming growth factor-P, and fibroblast growth factor. Glypicans participate in many important processes, including cellular proliferation, migration, differentiation, extracellular matrix, and tumor microenvironment remodeling.
  • GPC1 has been proven to be a useful biomarker for multiple cancerous tissues, such as prostate, hepatocellular, and pancreatic carcinomas. Considering the important role of GPC1 in various tumors, there is a need to develop a therapeutic agent targeting GPC1.
  • This disclosure relates to anti-GPCl antibodies, antigen-binding fragment thereof, and the uses thereof.
  • the disclosure is related to an antibody or antigen-binding fragment thereof that binds to GPC1 (Glypican-1) comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2 and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 85% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 85% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 85% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2 and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 85% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 85% identical to a selected VL CDR2 amino acid sequence, and the VL CDR
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1-3, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4-6, respectively, according to Kabat definition.
  • the antibody or antigen-binding fragment thereof specifically binds to human, monkey, mouse, and/or rat GPC1.
  • the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof, or a single-chain variable fragment (scFv).
  • the antibody or antigen-binding fragment thereof is a human IgGl antibody or antigen-binding fragment thereof or a human IgG4 antibody or antigen-binding fragment thereof.
  • the disclosure is related to a nucleic acid comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof.
  • the nucleic acid is cDNA.
  • the disclosure is related to a vector comprising one or more of the nucleic acids described herein.
  • the disclosure is related to a cell comprising the vector described herein.
  • the cell is a CHO cell.
  • the disclosure is related to a cell comprising one or more of the nucleic acids described herein. In one aspect, the disclosure is related to a method of producing an antibody or an antigenbinding fragment thereof, the method comprising culturing the cell described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment; and collecting the antibody or the antigen-binding fragment produced by the cell.
  • the disclosure is related to an antibody or antigen-binding fragment thereof that binds to GPC1 comprising a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 10, and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 11.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment thereof specifically binds to human, monkey, mouse, and/or rat GPC1.
  • the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof, or a single-chain variable fragment (scFv).
  • the antibody or antigen-binding fragment is a human IgGl antibody or antigen-binding fragment thereof, or a human IgG4 antibody or antigen-binding fragment thereof.
  • the disclosure is related to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof described herein to the subject.
  • the cancer is gastric cancer, breast cancer, bladder cancer, pancreatic cancer, lung cancer, or colorectal cancer.
  • the disclosure is related to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein.
  • the disclosure is related to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the antibody or antigenbinding fragment thereof described herein.
  • the disclosure is related to a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein, and a pharmaceutically acceptable carrier.
  • cancer refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • malignancies of the various organ systems such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, and cancer of the small intestine.
  • Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen(s), cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections.
  • a carcinogen e.g., a tumor suppressor gene
  • infections e.g., viral infections.
  • the term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • hematopoietic neoplastic disorders includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin.
  • a hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • antibody refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope.
  • CDR complementary determining region
  • Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies.
  • an antibody can contain an Fc region of a human antibody.
  • the term antibody also includes derivatives, e.g., single-chain antibodies, diabodies, linear antibodies formed from antibody fragments.
  • the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen.
  • the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain).
  • variable domains include, e.g., Fab, Fab’, F(ab’)2, and Fv fragments.
  • human antibody refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) present in a human.
  • a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells).
  • a human antibody is produced in a nonhuman cell (e.g., a mouse or hamster cell line).
  • a human antibody is produced in a bacterial or yeast cell.
  • a human antibody is produced in a transgenic non-human animal (e.g., a bovine) containing an unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus).
  • a transgenic non-human animal e.g., a bovine
  • human immunoglobulin locus e.g., heavy or light chain human immunoglobulin locus
  • the term “chimeric antibody” refers to an antibody that contains a sequence present in at least two different antibodies (e.g., antibodies from two different mammalian species such as a human and a mouse antibody).
  • a non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and/or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.
  • humanized antibody refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin.
  • humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody), e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity.
  • the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues.
  • humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance.
  • the humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., mouse) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin.
  • CDRs hypervariable loops
  • the humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically, that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • 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.
  • single-chain antibody refers to a single polypeptide that contains at least two immunoglobulin variable domains (e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain) that is capable of specifically binding to an antigen.
  • immunoglobulin variable domains e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain
  • single-chain antibodies are described herein.
  • multimeric antibody refers to an antibody that contains four or more (e.g., six, eight, or ten) immunoglobulin variable domains.
  • the multimeric antibody is able to crosslink one target molecule (e.g., GPC1) to at least one second target molecule on the surface of a mammalian cell (e.g., a human T-cell).
  • the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided.
  • Veterinary and non-veterinary applications are contemplated by the present invention.
  • Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old).
  • patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates.
  • non-human primates e.g., monkey, chimpanzee, gorilla, and the like
  • rodents e.g., rats, mice, gerbils, hamsters, ferrets, rabbits
  • lagomorphs e.g., swine (e.g., pig, miniature pig)
  • swine e.g., pig, miniature pig
  • equine canine
  • feline bovine
  • other domestic, farm, and zoo animals equine, canine, feline, bovine, and other domestic, farm, and zoo animals.
  • the phrases “specifically binding” and “specifically binds” mean that the antibody interacts with its target molecule (e.g., GPC1) preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general.
  • An antibody that specifically binds to the target molecule may be referred to as a target-specific antibody.
  • an antibody that specifically binds to a GPC1 molecule may be referred to as a GPC1 -specific antibody or an anti-GPCl antibody.
  • polypeptide As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.
  • nucleic acid molecule As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.
  • FIG. 1 lists heavy chain variable region and light chain variable region CDR sequences of anti- GPCl antibody as defined by Kabat definition and Chothia definition.
  • FIG. 2 lists selected amino acid sequences discussed in the disclosure. DETAILED DESCRIPTION
  • the present disclosure provides examples of antibodies, antigen-binding fragment thereof, that bind to GPC1.
  • Heparan sulfate proteoglycans are glycoproteins consisting of a core protein covalently bound to several heparan sulfate (HS) glycosaminoglycan (GAG) chains. HSPGs are expressed ubiquitously on the cell surface and in the extracellular matrix where they interact with a wide range of ligands to mediate various cellular functions. HSPGs are categorized into three groups based on their location: membrane HSPGs (e.g., syndecans and glypicans), secreted extracellular matrix HSPGs (agrin, perlecan, type XVIII collagen), and secretory vesicle proteoglycans (serglycin).
  • membrane HSPGs e.g., syndecans and glypicans
  • secreted extracellular matrix HSPGs agrin, perlecan, type XVIII collagen
  • secretory vesicle proteoglycans secretory ves
  • Glypicans of the HSPGs family, are attached to the outer surface of the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor.
  • GPI glycosyl-phosphatidylinositol
  • Six human glypican family members (GPC1 to GPC6) have been identified and fall into two broad subfamilies, glypicans 1/2/4/6 and glypicans 3/5, both sharing -25% amino acid identity.
  • the three-dimensional structure of glypicans might be similar across the family, as the localization of 14 cysteine residues is conserved in all family members. All glypicans share attachment sites for HS chains, in particular, the two close to the cell membrane, and a hydrophobic sequence necessary for the GPI anchor in the C-terminal tail.
  • GPC1 contains 558 amino acids and is composed of a secretory signal peptide, an N-terminal core protein, a C-terminal HS chain attachment region, and a GPI anchor attached to cell membrane.
  • GPC1 protein is substituted with a cluster of three HS chains at positions Ser-486, Ser-488, and Ser-490 and is also decorated with two N-linked glycans at Asn-79 and Asn-116 that affect the GPC1 expression level, as well as HS substitution.
  • the structure of GPC1 with the HS chains is related to its biological function by modulating various signaling pathways through specific interactions with ligands and receptors in cell surface signaling complexes. The actual orientation and possible rotation of GPC1 (and other glypicans) relative to the cell surface is unknown. The impact of these surface signaling complexes, either activating or inhibitory, is dependent upon specific GPC1 domain interaction with signaling components under specific physiological conditions.
  • FGFs are secreted glycoproteins that are readily sequestered by the extracellular matrix and the cell surface HPSGs, including GPC1.
  • GPC1 has been proposed to act as a coreceptor for FGFs that enhances the binding of FGF to its receptor, subsequently promoting FGF-FGFR activation and signaling.
  • the binding of FGF to its receptor leads to receptor dimerization and transphosphorylation of tyrosine kinase domains, resulting in subsequent activation of various signaling pathways, including Ras-MAPK, PI3K-AKT-mTOR, and DAG-PKC. All signaling cascades ultimately result in enhanced growth, survival, and angiogenesis.
  • FGF2 is one of the heparin-binding growth factors (HBGFs) that can drive tumor cell proliferation upon binding to its receptors, and also functions as an angiogenic growth factor in angiogenesis.
  • HBGFs heparin-binding growth factors
  • Mounting evidence indicates that changes in GPC1 expression have profound consequences on FGF2 -induced cell proliferation or angiogenesis in tumors.
  • GPC1 is upregulated and promotes tumor mitogenic signaling by modulating heparin-binding growth factors, including FGF2.
  • GPC1 has also been shown to cooperate with type V collagen to concentrate FGF2 at the extracellular cell matrix (ECM) interface, thereby affecting ECM stability and breast tumor cell proliferation.
  • ECM extracellular cell matrix
  • VEGF-A is also an angiogenic growth factor and plays an essential role in angiogenesis.
  • GPC1 acts as a coreceptor of VEGF-A, and this interaction is mediated by its HS chains, as HS removal by heparinase treatment abolished the ability of GPC1 to bind to VEGF.
  • Cell binding assays using ECs also demonstrated that the addition of exogenous GPC1 could potentiate the VEGF-A/VEGFR binding, suggesting the potential role of GPC1 in the control of angiogenesis
  • TGF- The transforming growth factor-P
  • the transforming growth factor-P belongs to a superfamily of cytokines that activate protein kinase receptors on the plasma membrane to regulate cell growth, death, differentiation, immune response, angiogenesis, and inflammation. Dysregulation of this pathway contributes to a broad variety of pathologies, including cancer.
  • TGF- signaling is considered a challenging target due to its dual functions and pleiotropic nature.
  • TGF-P acts as a tumor suppressor, whereas in the late stage of cancer, it drives tumor progression and metastasis.
  • GPC1 has been shown to interact with TGF-P and its receptors to stabilize their assembly for enhanced Smad signaling.
  • Bone morphogenetic proteins play substantial roles in cell-cell communication during animal development and are potent growth factors promoting bone formation. Glypicans have been shown to regulate BMP activity. GPC1 protein is mainly expressed in the skeletal system in humans, and Gpcl expression was also identified in the developing murine calvarium and skeletal structures. Thus, it may be inferred that GPC1 is also involved in the regulation of the BMP signaling pathway. Based on currently available evidence, GPC1 seems to act as an inhibitor in BMP signaling regulation in osteogenesis.
  • GPC1 has been reported to be present in two forms, a membrane bound core protein and secreted soluble forms , which can be detected in serum-free media harvested from prostate cancer cells DU- 145 by immunoprecipitation, probably due to GPC1 shedding or proteolytic cleavage at GPI anchor that was also found in GPC3.
  • the specific cleavage mechanism or site has been unknown.
  • Human GPC1 is expressed not only in the central nervous system (CNS) and skeletal system during development but also in other tissues in the adult. GPC1 is overexpressed in multiple types of cancers, including breast cancer, esophageal squamous cell carcinoma (ESCC), glioma, and pancreatic cancer. Its high expression is corelated with poorer prognosis, making it a potential target for cancer therapy.
  • the present disclosure provides anti-GPCl antibodies, antigen-binding fragments thereof, and methods of using these anti-GPCl antibodies and antigen-binding fragments to inhibit tumor growth and treat cancers.
  • antibodies also called immunoglobulins
  • a non-limiting antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains.
  • the heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgEl, IgE2, etc.
  • the light chain can be a kappa light chain or a lambda light chain.
  • CDRs complementary determining regions
  • the four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.
  • Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs are commonly used.
  • the Kabat definition is based on sequence variability
  • the Chothia definition is based on the location of the structural loop regions.
  • the CDRs are important for recognizing an epitope of an antigen.
  • an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody.
  • the minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.
  • the antibody is an intact immunoglobulin molecule (e.g., IgGl, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA).
  • the IgG subclasses (IgGl, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains.
  • the sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions.” Frontiers in immunology 5 (2014); Irani, et al.
  • the antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, camelid).
  • Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide.
  • the term “antigen binding domain” or “antigen binding fragment” is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab', F(ab')2, and variants of these fragments.
  • an antibody or an antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multi-specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain.
  • Non-limiting examples of antigen binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.
  • the disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to GPC1 (e.g., human GPC1).
  • GPC1 e.g., human GPC1
  • 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 increase immune response. In some embodiments, these antibodies can block GPC1 activity.
  • the disclosure provides e.g., anti-GPCl antibody 2C7, the chimeric antibodies thereof, and the human or humanized antibodies thereof.
  • the antibodies can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to a selected VH CDR3 amino acid sequence.
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • the antibody can have a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to a selected VL CDR3 amino acid sequence.
  • the selected VH CDRs 1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in FIG. 1.
  • Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions.
  • Linear antibodies can be bispecific or monospecific.
  • Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.
  • the Fc region can be modified to silence or decrease complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC).
  • the Fc region can be modified to increase complement-dependent cytotoxicity (CDC) or antibody -dependent cellular cytotoxicity (ADCC).
  • Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgGl molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.
  • purified antibody preparations e.g., purified IgGl molecules
  • antibody homodimers may be formed through chemical linkage techniques known in the art.
  • heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane-l -carboxylate) and SATA (N-succinimidyl S- acethylthio-acetate) can be used to form antibody multimers.
  • SMCC succinimidyl 4-(maleimidomethyl)cyclohexane-l -carboxylate
  • SATA N-succinimidyl S- acethylthio-acetate
  • An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997).
  • Antibody homodimers can be converted to Fab’ 2 homodimers through digestion with pepsin. Another way to form antibody homod
  • any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution).
  • stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin).
  • the conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human).
  • the antibodies or antigen-binding fragments thereof as described herein can be GPC1 agonist or antagonist.
  • the antibody by binding to GPC1, the antibody can inhibit GPC1 activity.
  • the antibody can upregulate immune response or downregulate immune response.
  • the antibody or antigen-binding fragments thereof specifically binds to GPC1 (e.g., human GPC1, monkey GPC1 (e.g., rhesus macaques, 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 1 or less than 0.0000001 s 1 .
  • GPC1 e.g., human GPC1, monkey GPC1 (e.g., rhesus macaques, Macaca fascicularis), mouse GPC1, and/or rat GPC1
  • 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
  • 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 0.0000001 s 1 or greater than 0.00000001 s 1 .
  • kinetic association rates (kon) is greater than 1 x 10 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 embodiments, kinetic association rates (kon) is less than 1 x 10 5 /Ms, less than 1 x 10 6 /Ms, or less than 1 x 10 7 /Ms.
  • KD is less than 1 x 10’ 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.
  • the antibody or antigen-binding fragment thereof described herein has a tumor growth inhibition percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%.
  • TGI% tumor growth inhibition percentage
  • the antibody or antigen-binding fragment thereof described herein has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%.
  • the TGI% can be determined, e.g., 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 treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts.
  • TGI% is calculated using the following formula:
  • TGI (%) [l-(Ti-T0)/(Vi-V0)]xl00%
  • Ti is the average tumor volume in the treatment group on day i.
  • TO is the average tumor volume in the treatment group on day zero.
  • Vi is the average tumor volume in the control group on day i.
  • V0 is the average tumor volume in the control group on day zero.
  • the antibodies or antigen-binding fragments thereof described herein are GPC1 antagonist. In some embodiments, the antibodies or antigen binding fragments described herein decrease GPC1 signal transduction in a target cell that expresses GPC1.
  • the antibodies or antigen binding fragments described herein can bind to tumor cells that express GPC1. In some embodiments, the antibody or antigen binding fragment described herein binds to tumor associated cells that express GPC1. In some embodiments, the antibodies or antigen binding fragments described herein can induce complement-dependent cytotoxicity (CDC) and/or antibody dependent cellular cytoxicity (ADCC), and kill the tumor cell.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody dependent cellular cytoxicity
  • Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.
  • Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis.
  • Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an antigen-binding domain.
  • some antibodies or antigen-binding fragments will have increased affinity for the target protein, e.g., GPC1.
  • any combination of deletions, insertions, and/or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target.
  • the amino acid changes introduced 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 changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell), or introducing new glycosylation sites.
  • Antibodies disclosed herein can be derived from any species of animal, including mammals.
  • Nonlimiting examples of native antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits), including transgenic rodents genetically engineered to produce human antibodies.
  • Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences.
  • Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.
  • a humanized antibody typically has a human framework (FR) grafted with non-human CDRs.
  • FR human framework
  • a humanized antibody has one or more amino acid sequence introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • Humanization can be essentially performed by e.g., substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. These methods are described in e.g., 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 by reference herein in its entirety. Accordingly, “humanized” antibodies are chimeric antibodies wherein substantially less than an intact human V domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.
  • VH and VL domains are very important for reducing immunogenicity.
  • the sequence of the V domain of a mouse antibody is screened against the entire library of known human-domain sequences.
  • the human sequence which is closest to that of the mouse is then 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)).
  • humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • amino acid sequence variants of the human, humanized, or chimeric anti-GPCl antibody will contain an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% percent identity with a sequence present in the light or heavy chain of the original antibody.
  • a mouse with a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus is used to generate antibodies.
  • the heavy chain immunoglobulin locus is a region on the chromosome that contains genes for the heavy chains of antibodies.
  • the locus can include e.g., human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy chain constant domain genes.
  • the kappa chain immunoglobulin locus is a region on the chromosome that contains genes that encode the light chains of antibodies (kappa chain).
  • the kappa chain immunoglobulin locus can include e.g., human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light chain constant domain genes.
  • human IGKV variable
  • human IGKJ joining
  • mouse light chain constant domain genes e.g., mouse light chain constant domain genes.
  • RenMabTM mice can be found in PCT/CN2020/075698 or US20200390073A1, which is incorporated herein by reference in its entirety.
  • a mouse with a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus is used to generate antibodies.
  • the heavy chain immunoglobulin locus is a region on the chromosome that contains genes for the heavy chains of antibodies.
  • the locus can include e.g., human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy chain constant domain genes.
  • the kappa chain immunoglobulin locus is a region on the chromosome that contains genes that encode a common light chain.
  • the kappa chain immunoglobulin locus can include e.g., a human IGKV (variable) gene, a human IGKJ (joining) gene, and mouse light chain constant domain genes.
  • IGKV variable
  • IGKJ joining
  • mouse light chain constant domain genes e.g., a human IGKV (variable) gene, a human IGKJ (joining) gene, and mouse light chain constant domain genes.
  • RenLiteTM mice can be found in PCT/CN2021/097652, which is incorporated herein by reference in its entirety.
  • Identity or homology with respect to an original sequence is usually the percentage of amino acid residues present within the candidate sequence that are identical with a sequence present within the human, humanized, or chimeric anti-GPCl antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • a cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have any increased half-life in vitro and/or in vivo.
  • Homodimeric antibodies with increased half-life in vitro and/or in vivo can also be prepared using heterobifunctional cross-linkers as described, for example, in Wolff et al. (Cancer Res. 53:2560-2565, 1993).
  • an antibody can be engineered which has dual Fc regions (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).
  • a covalent modification can be made to the anti-GPCl antibody or antigen-binding fragment thereof.
  • These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage.
  • Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N- or C-terminal residues.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • the Fc region of the antibodies was further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P).
  • S228P serine at position 228
  • a detailed description regarding S228 mutation is described, e.g., in 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 by reference in its entirety.
  • the present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein), host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide), and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.
  • recombinant vectors e.g., an expression vectors
  • an isolated polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide)
  • a “vector” is any construct capable of delivering one or more polynucleotide(s) of interest to a host cell when the vector is introduced to the host cell.
  • An “expression vector” is capable of delivering and expressing the one or more polynucleotide(s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced.
  • the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
  • regulatory elements such as a promoter, enhancer, and/or a poly-A tail
  • a vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and/or transduction (e.g., with recombinant virus).
  • vectors include viral vectors (which can be used to generate recombinant virus), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
  • a polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • a viral expression system e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • vaccinia or other pox virus, retrovirus, or adenovirus
  • viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci.
  • the DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter), such as the phage lambda PE promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral ETRs, to name a few.
  • a heterologous promoter such as the phage lambda PE promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral ETRs, to name a few.
  • Other suitable promoters are known to the skilled artisan.
  • the expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors can include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E.
  • coli Streptomyces, and Salmonella typhimurium cells
  • fungal cells such as yeast cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells
  • plant cells Appropriate culture mediums and conditions for the host cells described herein are known in the art.
  • Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
  • Non-limiting bacterial promoters suitable for use include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
  • Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
  • Introduction of the construct into the host cell can be effected by 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.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretion signals may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide (e.g., antibody) can be expressed in a modified form, such as a fusion protein e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • the antibodies or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes.
  • the antibody or antigen binding fragment disclosed herein may be used to treat a disease characterized by cells expressing GPC1.
  • the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject.
  • the treatment can halt, slow, retard, or inhibit progression of a cancer.
  • the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.
  • the disclosure provides the antibody or pharmaceutical composition as disclosed herein for use as a medicament.
  • the disclosure provides a use of the antibody or pharmaceutical composition as disclosed herein for the manufacture of a medicament for treatment of a disease, e.g. cancer.
  • the disclosure features methods that include administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a cancer), e.g., breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancy.
  • a subject in need thereof e.g., a subject having, or identified or diagnosed as having, a cancer
  • breast cancer e.g., triple-negative breast cancer
  • carcinoid cancer e.g., cervical cancer, endometrial cancer, glioma, head and neck cancer,
  • the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer.
  • NSCLC non-small cell lung carcinoma
  • SCLC small cell lung cancer
  • the cancer is NSCLC, ovarian cancer, melanoma, colorectal cancer, breast cancer, a hematological malignancy, head and neck cancer, gastrointestinal cancer, bladder cancer, or bone cancer.
  • the subject has a solid tumor.
  • the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal carcinoma.
  • the subject has Hodgkin's lymphoma.
  • the subject has triple-negative breast cancer (TNBC), gastric cancer, urothelial cancer, Merkel-cell carcinoma, or head and neck cancer.
  • TNBC triple-negative breast cancer
  • the cancer is melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies, especially Non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.
  • the cancer is oesophageal cancer, gastric cancer, breast cancer, ovarian cancer, bladder cancer, pancreatic cancer, lung cancer, colorectal cancer, stomach cancer, prostate cancer, kidney cancer, multiple myeloma, cholangiocarcinoma, or Non-Hodgkin Lymphoma.
  • compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer.
  • Patients with cancer can be identified with various methods known in the art.
  • an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., an autoimmune disease or a cancer.
  • An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the antibody, antigen binding fragment, antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
  • an effective amount can be administered in one or more administrations.
  • an effective amount of an antibody or an antigen binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of an autoimmune disease or a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)) in vitro.
  • a cell e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)
  • an effective amount of an antibody or antigen binding fragment may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of antibody used.
  • Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments, and/or compositions disclosed herein used and other drugs being administered to the mammal.
  • compositions that contain the antibodies or antigenbinding fragments described herein.
  • the pharmaceutical compositions may be formulated in any manner known in the art.
  • compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).
  • intended route of administration e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal.
  • compositions containing the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage).
  • parenteral e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal
  • dosage unit form i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage.
  • compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions.
  • Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration).
  • Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen.
  • antibodies can be formulated in aqueous solutions, preferably in physiologically-compatible buffers to reduce discomfort at the site of injection.
  • the solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys).
  • One can, for example, determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population): the therapeutic index being the ratio of LD50:ED50.
  • Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects).
  • Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • disclosure also provides methods of manufacturing the antibodies or antigen binding fragments thereof for various uses as described herein.
  • RenLiteTM mice Human GPC1 protein (Kactus Biosystems, Cat #: GPC-HM111) or DNA encoding this protein was emulsified with adjuvants, and was used to immunize RenLiteTM mice (Biocytogen, complete human heavy chain variable domain combined with a common light chain substitution in situ). The RenLiteTM mice are described, e.g., in PCT/CN2021/097652, which is incorporated herein by reference in its entirety. Before immunization, retro-orbital blood was collected as a negative control.
  • a total of three immunizations were performed.
  • the first and second immunizations were separated by three weeks, and the second and third immunizations were separated by two weeks.
  • retro-orbital blood was collected, and the antibody titer of serum was detected by fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • Antigen-specific immune cells were isolated from the immunized mice to further obtain anti- GPCl antibodies or to obtain the light chain and heavy chain variable region sequences of the anti-GPCl antibodies.
  • single cell technology e.g., Beacon® Optofluidic System, Berkeley Lights Inc.
  • reverse transcription and PCR sequencing were used to obtain antibody variable region sequences.
  • the obtained variable region sequences were cloned into a vector containing a sequence encoding the human IgGl constant region for antibody expression. Binding of the expressed antibodies to GPC1 was verified by FACS.
  • Exemplary antibody obtained by this method included 2C7.
  • the VH and VL CDR 1-3 sequences of 2C7 are shown in FIG. 1, and the VH and VL region are shown in FIG. 2.
  • Refl One reference antibody with specificity for GPC1, synthesized from published amino acid sequence information, were used in the following experiments (designated Refl). Specifically, The VH and VL sequences set forth in SEQ ID NOs: 12-13 were linked to the human IgGl constant region to form Refl .
  • the binding specificity of the anti-GPCl antibody to his-tagged human GPC1 was verified using BiacoreTM (Biacore, Inc., Piscataway N.J.) 8K biosensor equipped with pre-immobilized Protein A sensor chips.
  • Antibodies was captured on the Protein A chip for the detection. 2 pg/mL purified antibodies were loaded at 10 pL/min to bind to the recombinant his-tagged 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 pL/min, the binding and dissociation time were set to 180 s and 400 s, respectively. The chip was regenerated after the last injection of each titration with a glycine solution (pH 2.0) at 30 pL/min for 30 seconds.
  • a glycine solution pH 2.0
  • Example 3 Cross-species binding of anti-GPCl antibody CHO-hGPCl cells, CHO-fasGPCl cells, CHO-mGPCl cells, or CHO-ratGPCl cells were transferred to a 96-well plate at a density of 1X10 5 5X10 4 cells/well respectively. Series diluted anti-GPCl antibodies were added to the 96-well plate, and incubated at 4°C for 30 min. Then, the cells were incubated with the secondary antibody anti-hlgG-Fc-Alex Flour 647 (RL1-H) (Jackson ImmunoResearch Laboratories, Inc., Cat#: 109-606-170) at 4°C in the dark for 15 minutes before flow cytometry analysis. EC50 was calculated.
  • 2C7 can bind to human GPC1, monkey GPC1, mouse GPC1, and rat GPC1.
  • Anti-GPCl antibody (work concentration 2.5 pg/mL) together with Fab Fragment Goat AntiHuman IgG (Jackson Immuno Research, Cat #: 109-007-008) labeled by pHAb Amine Rescyive Dye (Promega, Cat #: G9845) were added to human lung cancer cells NCI-H1792 (IxlO 5 cells/well), and incubated for 24 hours. The cells were centrifuged and washed with FACS buffer, and then measured using a flow cytometer. For isotype control (ISO), human IgGl protein was used. Datas show that 2C7 has good endocytosis rate in NCI-H1792 cells.
  • ISO isotype control
  • Epitope binding assays were performed using BiacoreTM to determine whether two anti-GPCl antibodies target the same or overlapping epitopes, lx HBS-EP+ buffer (10 rnM 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 rnM ethylenediaminetetraacetic acid (EDTA) and 0.05% P20, pH7.4) diluted from HBS-EP+ buffer (lOx) was used as the running buffer throughout the experiment.
  • HBS-EP+ buffer 10 rnM 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 rnM ethylenediaminetetraacetic acid (EDTA) and 0.05% P20, pH7.4
  • hGPCl-His protein (ACRO Biosystems, Cat #: GP1-H52H9) was captured at a flow rate of 10 pL/min, and 200 nM of the tested antibody (Analyte 1) was injected at a flow rate of 30 pL/min to bind the ligand.
  • Another comparison antibody (Analyte 2) was injected under the same conditions to determine whether the binding of different antibodies interfered with each other. The binding time was 200 s for each antibody.
  • the binding value of each antibody was obtained using Biacore 8K Evaluation Software. To quantify the interference of one antibody binding to another, a binding ratio was calculated to compare each pair of antibodies. The binding ratio is defined as the binding value of the second antibody (Analyte 2), divided by the binding value of the first antibody (Analyte 1). The binding ratio of each antibody pair was summarized in the table below. More specifically, the binding ratio was less than 0.5, if analyte 1 exhibited a blocking effect to analyte 2. The binding ratio was over 0.5, if analyte 1 did not exhibit a blocking effect to analyte 2. In general, antibody pairs that interfere with each other have the same or overlapping epitopes.

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Abstract

La présente divulgation concerne des anticorps anti-GPC1 (glypican-1), des fragments de liaison à l'antigène de ceux-ci, et leurs utilisations.
PCT/EP2025/058397 2024-03-27 2025-03-27 Anticorps anti-gpc1 et leurs utilisations Pending WO2025202359A1 (fr)

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