WO2021213245A1 - 抗体或其抗原结合片段、其制备方法及医药用途 - Google Patents

抗体或其抗原结合片段、其制备方法及医药用途 Download PDF

Info

Publication number
WO2021213245A1
WO2021213245A1 PCT/CN2021/087549 CN2021087549W WO2021213245A1 WO 2021213245 A1 WO2021213245 A1 WO 2021213245A1 CN 2021087549 W CN2021087549 W CN 2021087549W WO 2021213245 A1 WO2021213245 A1 WO 2021213245A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cancer
antibody
antigen
variable region
Prior art date
Application number
PCT/CN2021/087549
Other languages
English (en)
French (fr)
Inventor
花海清
余华星
何娟梅
包如迪
Original Assignee
上海翰森生物医药科技有限公司
江苏豪森药业集团有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海翰森生物医药科技有限公司, 江苏豪森药业集团有限公司 filed Critical 上海翰森生物医药科技有限公司
Priority to CN202180021548.2A priority Critical patent/CN115298216A/zh
Publication of WO2021213245A1 publication Critical patent/WO2021213245A1/zh

Links

Classifications

    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to an anti-GPC3 antibody, or an antigen-binding fragment thereof, which is specifically immunoreactive to human GPC3 receptor, a chimeric antibody containing the CDR region of the anti-GPC3 antibody, a humanized antibody, and a humanized antibody
  • Glypican 3 (Glypican 3, GPC3) is a 70kDa membrane protein, belonging to the Glypicans family that functions as an extracellular matrix in organ formation or as a receptor for cell growth factors. After GPC3 is expressed, it will be cleaved by furin enzyme to produce the N-terminal 40kd soluble part and the 30kd part is anchored to the C-terminal of the cell membrane by GPI molecules.
  • GPC3 is expressed in embryonic tissues, especially liver and kidney, and is an extracellular matrix protein related to organ formation. In adult tissues, GPC3 expression is not observed outside the placenta, but expression is observed in various cancer tissues such as hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, and lung squamous cell carcinoma. It can be seen that GPC3 is a protein expressed in embryonic tissues like alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and other proteins, so it is classified as an embryonic cancer antigen. That is, GPC3 exhibits the characteristics of not being expressed in normal tissue cells but specifically expressed in cancer cells, and therefore serves as a target molecule and tumor marker for cancer treatment.
  • AFP alpha-fetoprotein
  • CEA carcinoembryonic antigen
  • GPC3 plays an important role in maintaining the Wnt pathway and the activation of the Hedgehogs pathway.
  • heparan sulfate molecules coupled to GPC3 can enhance the binding of Wnts to their receptors and thus play an important role in maintaining the Wnt pathway.
  • GPC3 is expressed in the brain, digestive tract, bladder, gonads and skin and is highly expressed on the surface of hepatocellular carcinoma; Wnt pathway plays an important role in the development of liver cancer, such as 20% hepatocellular carcinoma ⁇ -Catenin pathway mutation and Frizzled-7 receptor Overexpression, so GPC3 may play a role in promoting the occurrence of some hepatocellular carcinomas.
  • an anti-GPC3 antibody or antigen-binding fragment thereof which comprises an antibody heavy chain variable region and an antibody light chain variable region, wherein the antibody heavy chain variable region comprises at least 1
  • One is selected from the HCDR shown in the following sequence: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15,
  • the antibody light chain variable region includes at least one LCDR selected from the following sequences: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.
  • the heavy chain variable region of the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 10, and SEQ ID NO: 13 shown in HCDR3.
  • the heavy chain variable region of the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: HCDR1 shown in SEQ ID NO: 8, HCDR2 shown in SEQ ID NO: 11, and SEQ ID NO: 14 shown in HCDR3.
  • the heavy chain variable region of the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 12, and SEQ ID NO: 15 shown in HCDR3.
  • the light chain variable region of the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 21 and SEQ ID NO: 24 shown in LCDR3.
  • the light chain variable region of the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention includes: LCDR1 shown in SEQ ID NO: 17, LCDR2 shown in SEQ ID NO: 22, and SEQ ID NO: 25 shown in LCDR3.
  • the light chain variable region of the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: LCDR1 shown in SEQ ID NO: 18, LCDR2 shown in SEQ ID NO: 23, and LCDR2 shown in SEQ ID NO: 26 shown in LCDR3.
  • the light chain variable region of the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 21, and LCDR2 shown in SEQ ID NO: 24 shown in LCDR3.
  • the light chain variable region of the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: LCDR1 shown in SEQ ID NO: 20, LCDR2 shown in SEQ ID NO: 23, and LCDR2 shown in SEQ ID NO: 26 shown in LCDR3.
  • the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: HCDR1 shown in SEQ ID NO: 7 and HCDR2 shown in SEQ ID NO: 10 and HCDR3 shown in SEQ ID NO: 13; And the antibody light chain variable region includes: LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 21, and LCDR3 shown in SEQ ID NO: 24.
  • the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: HCDR1 shown in SEQ ID NO: 8 and HCDR2 shown in SEQ ID NO: 11 and HCDR3 shown in SEQ ID NO: 14; And the antibody light chain variable region includes: LCDR1 shown in SEQ ID NO: 17, LCDR2 shown in SEQ ID NO: 22, and LCDR3 shown in SEQ ID NO: 25.
  • the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: HCDR1 shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:12, and HCDR3 shown in SEQ ID NO:15; And the antibody light chain variable region includes: LCDR1 shown in SEQ ID NO: 18, LCDR2 shown in SEQ ID NO: 23, and LCDR3 shown in SEQ ID NO: 26.
  • the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: the heavy chain variable region of the antibody comprises: HCDR1 shown in SEQ ID NO: 7 and HCDR2 shown in SEQ ID NO: 10 And the HCDR3 shown in SEQ ID NO: 13; and the antibody light chain variable region includes: LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 21 and shown in SEQ ID NO: 24 LCDR3.
  • the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention comprises: the heavy chain variable region of the antibody comprises: HCDR1 shown in SEQ ID NO: 9 and HCDR2 shown in SEQ ID NO: 12 And the HCDR3 shown in SEQ ID NO: 15; and the antibody light chain variable region includes: LCDR1 shown in SEQ ID NO: 20, LCDR2 shown in SEQ ID NO: 23 and shown in SEQ ID NO: 26 LCDR3.
  • the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention is selected from: murine antibody or antigen-binding fragment thereof, chimeric antibody or antigen-binding fragment thereof, human antibody or antigen-binding fragment thereof, humanized Antibodies or antigen-binding fragments thereof.
  • the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4 or a variant thereof ;
  • the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2 or IgG4;
  • the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO: 52.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention further comprises an IgG1 heavy chain constant region with enhanced ADCC toxicity after amino acid mutation.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention further comprises a light chain constant region derived from a human kappa chain, a lambda chain or a variant thereof;
  • the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain;
  • the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO:53.
  • the anti-GPC3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the following sequence, or compared with the following sequence A heavy chain variable region with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity: SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 37.
  • the anti-GPC3 antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the following sequence, or compared with the following sequence Light chain variable regions with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity: SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 38.
  • the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises:
  • the anti-GPC3 antibody or antigen-binding fragment thereof contains a heavy chain selected from the following sequences, or has at least 80% compared with the following sequences. %, 85%, 90%, 95% or 99% identity heavy chain: SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 49.
  • the anti-GPC3 antibody or antigen-binding fragment thereof contains a light chain selected from the following sequences, or has at least 80% compared with the following sequences. %, 85%, 90%, 95% or 99% identity light chain: SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO:50.
  • the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention wherein the anti-GPC3 antibody comprises:
  • a polynucleotide which encodes the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention.
  • an expression vector containing the polynucleotide of the present invention there is provided an expression vector containing the polynucleotide of the present invention.
  • a host cell into which or contains the expression vector of the present invention.
  • the host cell is bacteria, preferably Escherichia coli.
  • the host cell is yeast, preferably Pichia pastoris.
  • the host cell is a mammalian cell, preferably a CHO cell or HEK293 cell.
  • a method for producing an anti-GPC3 antibody including the steps of culturing the host cell of the present invention, isolating the antibody from the culture, and purifying the antibody.
  • a pharmaceutical composition which contains the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable excipient, diluent or carrier.
  • a detection or diagnosis kit which contains the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention and an excipient, diluent or carrier that can be used for detection or diagnosis.
  • a detection or diagnosis kit which contains the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention, and optionally, further comprises one or more kinds of detection of the anti-GPC3 antibody or A reagent that binds its antigen-binding fragment to GPC3 or its epitope.
  • an anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention, or the use of the above-mentioned pharmaceutical composition in the preparation of a kit, wherein the kit is used for detecting or diagnosing GPC3-mediated Disease or condition.
  • the disease or condition is cancer
  • the disease or condition is a cancer that expresses GPC3.
  • the cancer is selected from breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, Glioblastoma and melanoma.
  • the disease or condition is selected from the group consisting of hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, and lung squamous cell carcinoma.
  • a method for treating or preventing GPC3-mediated diseases including the steps of: providing a subject with a therapeutically effective amount or a prophylactically effective amount of the anti-GPC3 antibody or antigen binding thereof according to the present invention Fragment.
  • a method for treating or preventing GPC3-mediated diseases including the steps of: providing a subject with a therapeutically effective amount or a prophylactically effective amount of the pharmaceutical composition according to the present invention.
  • the subject is suspected of having, has suffered from, or is susceptible to a GPC3-mediated disease
  • the GPC3-mediated disease is selected from breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, and kidney cancer , Lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma and melanoma, preferably hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, lung squamous cell carcinoma .
  • the anti-GPC3 antibody or antigen-binding fragment of the present invention can specifically bind to GPC3 antigen (or its epitope) and GPC3-expressing cells, and has significant CDC activity and ADCC activity, and its tumor killing effect is significant.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention has good endocytosis and is suitable for coupling with drugs to construct ADC. While maintaining high specificity and killing activity, the anti-GPC3 antibody or antigen-binding fragment of the present invention has lower immunogenicity and higher stability, and is significantly better than Codrituzumab. The anti-GPC3 antibody or antigen-binding fragment of the present invention has better potential as an anti-cancer drug and ensures drug safety.
  • antibody in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the antibody light chain variable region of the present invention may further include a light chain constant region, and the light chain constant region includes human or murine ⁇ , ⁇ chains or variants thereof.
  • the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the number and position of the CDR amino acid residues of the VL and VH regions of the antibody or antigen-binding fragment of the present invention comply with the known Kabat numbering rules and Kabat or ABM definition rules (http://bioinf.org.uk/abs /).
  • APC antigen presenting cell
  • DC dendritic cells
  • PBMC topical blood mononuclear cells
  • monocytes B lymphoblasts
  • monocyte-derived dendritic cells monocyte-derived dendritic cells
  • antigen presentation refers to the process by which APC captures antigens and enables them to be recognized by T cells, for example as a component of MHC-I/MHC-II conjugates.
  • GPC3 includes any variant or isoform of GPC3 that is naturally expressed by the cell.
  • the antibodies of the present invention can cross-react with GPC3 derived from non-human species.
  • the antibody may also be specific for human GPC3, and may not show cross-reactivity with other species.
  • GPC3 or any variants or isoforms thereof can be isolated from cells or tissues that naturally express them, or can be produced by recombinant techniques using techniques commonly used in the art and those described herein.
  • the anti-GPC3 antibody targets human GPC3 with a normal glycosylation pattern.
  • recombinant human antibody includes human antibodies prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as:
  • Antibodies isolated from host cells transformed to express antibodies such as transfectomas;
  • Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies contain variable and constant regions, which utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.
  • murine antibody in the present invention refers to a monoclonal antibody to human GPC3 prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with GPC3 antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the murine GPC3 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, or further comprise murine IgG1 and IgG2. , IgG3 or IgG4 or its variant heavy chain constant region.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • the human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (ie, "humanized antibodies”) .
  • humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody)
  • CDR-grafted antibody refers to an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework.
  • Humanized antibodies can overcome the shortcomings of strong immune responses induced by chimeric antibodies that carry a large amount of mouse protein components.
  • the variable region of the human antibody can be subjected to minimal reverse mutation to maintain activity.
  • chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to select a hybridoma that secretes a murine-specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and change the mouse variable region gene.
  • the region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of a human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising human IgG1, IgG2 or IgG4 heavy chain constant region, or using amino acid mutations to enhance ADCC (antibody -dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1 heavy chain constant region.
  • ADCC antibody -dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity
  • antigen-binding fragment refers to antigen-binding fragments and antibody analogs of antibodies, which usually include at least part of the antigen-binding region or variable region (e.g., one or more CDRs) of a parental antibody.
  • Antibody fragments retain at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed on a mole basis, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target.
  • antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies.
  • Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.
  • the "Fab fragment” consists of the CH1 and variable regions of one light chain and one heavy chain.
  • the heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.
  • the "Fab' fragment” contains a light chain and a portion of a heavy chain that contains the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, so that it can be between the two heavy chains of the two Fab' fragments The formation of interchain disulfide bonds to form F(ab')2 molecules.
  • the "F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments held together by the disulfide bond between the two heavy chains.
  • the "Fv region” contains variable regions from both the heavy and light chains, but lacks the constant region.
  • multispecific antibody is used in its broadest sense to encompass antibodies with polyepitope specificity.
  • These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has polyepitope specificity; having two or more VL and VH regions The antibody, each VH-VL unit binds to a different target or a different epitope of the same target; an antibody with two or more single variable domains, each single variable domain with a different target Or binding to different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments that have been covalently or non-covalently linked together Wait.
  • single-chain antibody is a single-chain recombinant protein formed by connecting the heavy chain variable region VH and the light chain variable region VL of an antibody through a connecting peptide. It is the smallest antibody fragment with a complete antigen-binding site.
  • domain antibody fragment is an immunoglobulin fragment with immunological functions that only contains a heavy chain variable region or a light chain variable region chain.
  • two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment.
  • the two VH regions of the bivalent domain antibody fragment can target the same or different antigens.
  • binding to GPC3 refers to the ability to interact with human GPC3.
  • antigen-binding site refers to a three-dimensional site recognized by the antibody or antigen-binding fragment of the present invention.
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • Epitopes can be formed by adjacent amino acids or non-adjacent amino acids that are juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with the denaturing solvent.
  • Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include the techniques in the art and the techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
  • the terms “specific binding” and “selective binding” used in the present invention refer to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody is approximately 10 -7 M or even lower than the equilibrium dissociation smaller dissociation constant ( K D ) binds to a predetermined antigen, and its binding affinity to the predetermined antigen is at least twice its binding affinity to non-specific antigens (such as BSA, etc.) other than the predetermined antigen or closely related antigens.
  • K D equilibrium dissociation smaller dissociation constant
  • the term “antibody that recognizes an antigen” can be used interchangeably with the term “antibody that specifically binds” herein.
  • cross-reactivity refers to the ability of the antibody of the present invention to bind to GPC3 from different species.
  • the antibody of the present invention that binds to human GPC3 can also bind to GPC3 of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (such as SPR and ELISA), or binding or functional interaction with cells that physiologically express GPC3.
  • binding assays such as SPR and ELISA
  • Methods of determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • Inhibition or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with anti-GPC3 antibody compared to ligand not contacted with anti-GPC3 antibody.
  • inhibition of growth is intended to include any measurable decrease in cell growth.
  • inducing an immune response and “enhancing an immune response” are used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a specific antigen.
  • induction for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
  • ADCC namely antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity
  • Fc receptors are directly killed by recognizing the Fc segment of antibodies and are coated with antibodies.
  • the target cell The ADCC effect function of the antibody can be enhanced or reduced by modifying the Fc segment of IgG.
  • the modification refers to mutations in the constant region of the heavy chain of the antibody.
  • mice can be immunized with human GPC3 or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions.
  • the human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImmunoGeneTics (IMGT), or from the Journal of Immunoglobulin, 2001ISBN012441351.
  • the engineered antibody or antigen-binding fragment of the present invention can be prepared and purified by conventional methods.
  • the cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the FC region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • the resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • the antibody of the present invention refers to a monoclonal antibody.
  • the monoclonal antibody (mAb) of the present invention refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to a eukaryotic, prokaryotic or phage cloned cell line.
  • Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, synthesis technology (such as CDR-grafting), or other existing technologies.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as comprising any one of the antibodies of the present invention, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in the treated patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient.
  • any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms it can be evaluated whether the symptoms of the disease have been alleviated.
  • the embodiments of the present invention may be ineffective in alleviating the symptoms of the target disease that each patient has, according to any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.
  • naturally occurring refers to the fact that the object can be found in nature.
  • polypeptide sequences or polynucleotide sequences that exist in organisms (including viruses) that can be isolated from natural sources and have not been intentionally modified artificially in the laboratory are naturally occurring.
  • Effective amount includes an amount sufficient to improve or prevent the symptoms or conditions of medical conditions.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • Exogenous refers to substances that are produced outside organisms, cells, or humans according to the background.
  • Endogenous refers to a substance produced in a cell, organism, or human body according to the background.
  • “Homology” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the greatest percentage of homology.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably, and all such names include their progeny. Therefore, the words “transformant” and “transformed cell” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. Where a different name is meant, it is clearly visible from the context.
  • “Pharmaceutical composition” means containing one or more antibodies or antigen-binding fragments thereof described herein, and other components such as physiological/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • the following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention.
  • the experimental methods that do not specify specific conditions in the examples of the present invention usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experimental manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer.
  • the reagents without specific sources are the conventional reagents purchased on the market.
  • the protein antigen is human GPC3 recombinant protein (UniProt#P51654, Gln 25-His 559), with a His tag at the C terminal, that is, human GPC3His protein, purchased from AcroBiosystems (Cat#GP3-H52H4).
  • Example 2 Obtaining mouse hybridoma and antibody sequence
  • the indirect ELISA method as described in Example 3(1) was used for the immunized mouse serum to evaluate the serum titer and the ability to bind to cell surface antigens, and control the titer detection (more than 100,000-fold dilution) to determine the start of cell fusion.
  • the immunized mice with strong serum titer were selected for a final immunization and then the mice were sacrificed.
  • the spleen cells and SP2/0 myeloma cells were fused and plated to obtain hybridomas.
  • the target hybridomas were screened by indirect ELISA and established by the limiting dilution method.
  • the strain is a monoclonal cell strain.
  • the obtained positive antibody strains are further screened using indirect ELISA to select hybridomas that bind the recombinant protein.
  • the logarithmic growth phase hybridoma cells were collected, and RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript TM Reverse Transcriptase, Takara #2680A).
  • the cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sequenced, and finally the sequences of mouse antibodies M1, M2 and M3 were obtained.
  • TMB chromogenic substrate (Suzhou Yake Chemical Reagent Co., Ltd., cat#S0025), incubate at 25°C for 8-15 minutes in the dark, add 50 ⁇ L/well of 1M HCl to stop For the reaction, read the absorbance value at 450nm with a microplate reader (Thermo, Ascent), and analyze the data.
  • mice antibody has a good affinity for the human GPC3 antigen.
  • GPC3 high-expressing tumor cells HepG2 (ATCC deposit number: HB-8065), after trypsinization, the cells were collected by centrifugation, the cell density was adjusted with FACS buffer (1 ⁇ PBS containing 2% FBS) and then spread on a 96-well U bottom plate , 1 ⁇ 10 5 to 2 ⁇ 10 5 cells per well. Centrifugation: 1200g for 5 minutes, discard the supernatant, add 100 ⁇ L of antibody solution that has been serially diluted with FACS buffer, and incubate at 4°C for 1 hour.
  • Centrifugation 1200g for 5 minutes, discard the supernatant, wash the cells twice with PBS, add the fluorescent-labeled secondary antibody working solution PE anti-human IgG Fc Antibody (Biolegend, Cat#409304) or FITC anti-mouse IgG Antibody prepared in FACS buffer (Biolegend, Cat#406001), resuspend the cells in 100 ⁇ L per well, and incubate at 4°C for 1 hour. Centrifugation: 1200g, 5 minutes, discard the supernatant. The cells were washed twice with PBS, resuspended in PBS, using a flow cytometer DxFlex fluorescent signal is detected, and the concentration of EC 50 curve analysis for antibody binding cells.
  • the constant region of an exemplary chimeric antibody may be a human IgG1 heavy chain constant region and a kappa light chain Constant region.
  • the Biacore test method is as follows:
  • fixation reagent 10mM sodium acetate, pH 5.0
  • the antibody stock solution was diluted to 4 ⁇ g/mL with running reagents, and injected into the experimental channel (FC4) at a flow rate of 10 ⁇ L/min to capture about 200 RU.
  • the reference channel (FC3) does not require ligand capture.
  • the human GPC 3 protein was diluted by a 2-fold ratio with the running reagent, and the diluted samples were sequentially injected into the experimental channel and the reference channel at a flow rate of 30 ⁇ L/min according to the corresponding binding time and dissociation time.
  • the chip needs to be regenerated with 3M magnesium chloride at a flow rate of 20 ⁇ L/min for 30s to wash away the ligand and undissociated analyte.
  • the experimental channel needs to recapture the same amount of ligand.
  • GPC3 high-expressing cells (CHO-K1 cells overexpressing human or monkey GPC3, and human liver cancer cells JHH-7 expressing GPC3), after trypsinization, the cells were collected by centrifugation, and FACS buffer (1 containing 2% FBS) ⁇ PBS) after adjusting the cell density, spread on a 96-well U bottom plate, with 1 ⁇ 10 5 to 2 ⁇ 10 5 cells per well. Centrifugation: 1200g for 5 minutes, discard the supernatant, add 100 ⁇ L of antibody solution that has been gradually diluted with FACS buffer, and incubate at 4°C for 1 hour.
  • Centrifugation 1200g for 5 minutes, discard the supernatant, wash the cells twice with PBS, add the fluorescent-labeled secondary antibody working solution PE anti-human IgG Fc Antibody (Biolegend, Cat#409304) or FITC anti-mouse IgG prepared with FACS buffer Antibody (Biolegend, Cat#406001), resuspend cells in 100 ⁇ L per well, and incubate at 4°C for 1 hour. Centrifugation: 1200g, 5 minutes, discard the supernatant. After washing the cells twice with PBS, they were resuspended in PBS, the fluorescence signal was detected by the flow cytometer DxFlex, and the EC 50 concentration of the antibody-bound cells was analyzed by a curve.
  • PE anti-human IgG Fc Antibody Biolegend, Cat#409304
  • FITC anti-mouse IgG prepared with FACS buffer Antibody
  • the humanization of the mouse-derived anti-human GPC3 monoclonal antibody is carried out according to the methods published in many documents in the field. In short, human constant domains are used instead of parental (murine antibody) constant domains, and human antibody sequences are selected based on the homology of murine and human antibodies.
  • the present invention combines murine antibodies M1, M2, and M3 into humans. Sourced.
  • the heavy and light chain variable region sequences are compared with the human antibody germline database to obtain a human germline template with high homology.
  • the CDR region of the mouse antibody is transplanted to the selected corresponding humanized template. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important impact on the conformation of VL and VH are back-mutated, and the CDR region Chemically unstable amino acid residues were optimized. After expression testing and comparison of the number of back mutations, an antibody composed of humanized heavy chain variable region HCVR and light chain variable region LCVR sequences was selected. The sequence is as follows:
  • the antibody heavy chain constant region is selected from the human IgG1 natural constant region shown in SEQ ID NO: 52
  • the light chain constant region is selected from the constant region of the human kappa chain shown in SEQ ID NO: 53, and the heavy chain and light chain sequences are as follows:
  • SEQ ID NO: 31 SEQ ID NO: 43 SEQ ID NO: 28 SEQ ID NO: 40 H2-1 SEQ ID NO: 32 SEQ ID NO: 44 SEQ ID NO: 33 SEQ ID NO: 45 H2-2 SEQ ID NO: 32 SEQ ID NO: 44 SEQ ID NO: 34 SEQ ID NO: 46 H2-3 SEQ ID NO: 32 SEQ ID NO: 44 SEQ ID NO: 35 SEQ ID NO: 47 H2-4 SEQ ID NO: 32 SEQ ID NO: 44 SEQ ID NO: 36 SEQ ID NO: 48 H3-1 SEQ ID NO: 37 SEQ ID NO: 49 SEQ ID NO: 38 SEQ ID NO: 50
  • CDNA fragments were synthesized according to the amino acid sequences of the light and heavy chains of the above humanized antibodies, and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20).
  • the expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and incubated in a CO 2 incubator 4- 5 days.
  • Example 7 In vitro binding affinity experiment of humanized antibody:
  • the results show that the humanized antibody of the present invention has a high affinity with the human GPC3 antigen.
  • the results show that the humanized antibody has high affinity with stable cell lines overexpressing human or monkey GPC3 antigen, and can bind to human liver cancer cell JHH-7.
  • the antibody of the present invention can be endocytosed with human GPC3 after binding to GPC3, use the stable transfected cell line CHO-K1-human GPC3 and human liver cancer cell JHH-7 (CBP60204, purchased from Nanjing Kebai Biotechnology Co., Ltd.) And HepG2 (SCSP-510, purchased from the Cell Bank of the Chinese Academy of Sciences) for evaluation.
  • CBP60204 purchased from Nanjing Kebai Biotechnology Co., Ltd.
  • SCSP-510 purchased from the Cell Bank of the Chinese Academy of Sciences
  • the cells were digested with trypsin, and the cells were collected and resuspended in pre-cooled FACS buffer to adjust the cell concentration to 1 ⁇ 10 6 /mL.
  • Take the EP tube add 1 mL of cell suspension, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add 1 mL of the prepared antibody to be tested to resuspend the cells, the final concentration of the antibody is 20 ⁇ g/mL, and incubate for 1 hour at 4°C. Discard the supernatant by centrifugation (4°C, 1500 rpm ⁇ 5 minutes), wash twice with FACS buffer, and discard the supernatant.
  • Percentage of antibody endocytosis (fluorescence intensity value at each time point-average fluorescence intensity value of Blank group)/(average fluorescence intensity value at zero point-average fluorescence intensity value of Blank group)
  • the target cell of this experiment is the human GPC3 overexpression stable cell line CHO-K1-hGPC3.
  • the experimental medium is cell culture medium F12K (Gibco TM , Cat#21127022) without FBS, which is used for cell and antibody resuspension or dilution.
  • CHO-K1-hGPC3 cells were trypsinized, collected by centrifugation, resuspended in experimental medium to 1 ⁇ 10 5 cells/ml, spread 50 ⁇ L on a white 96-well plate (Corning, 3610), and added 25 ⁇ L 4 ⁇ working concentration of antibody (The initial working concentration of the antibody is 20nM, 5-fold dilution, 10 concentration points, and 0nM points), incubate at 37°C for 30 minutes, add 25 ⁇ L of 80% human serum (GemCell TM US, Cat#100-512), 37°C Incubate for 24 hours in an incubator. Add 50 ⁇ L CellTiter-Glo (Promega, Cat#G7573), mix well and react for 10 minutes in the dark at room temperature, and read with a multifunctional microplate reader (Thermofisher, Lux).
  • Antibody killing percentage (%) (E-S)/(E-M) ⁇ 100
  • E is the value of the well without antibody, that is, cell + culture medium + human serum
  • S is the value of the sample well, namely cell + antibody + human serum
  • M is the number of medium + human serum wells.
  • the panoramic isoelectric focusing (iCIEF) method was used to detect and compare the purity of the humanized antibody H3-1 at the starting 0 point and the charge heterogeneity after being placed at 25°C and 40°C for one month. This examines the stability of the antibody.
  • the carrier amphoteric electrolyte can form a certain range of pH gradient in the tube.
  • the sample composition According to its chargeability, it migrates to the cathode or anode, and when the pH value in the capillary is the same as the isoelectric point (pI) of the component, the net charge of the solute molecule is zero, and the component will aggregate at this point in a macroscopic view without further Migration, to achieve the purpose of separating each component in a complex sample.
  • the pI value and peak ratio main peak, acid peak, alkali peak
  • the sample is set with 3 conditions: the starting point is 0 o'clock; it is placed in a stability test chamber (Memmer, model HPP 1060) with a humidity of 65% and a temperature of 25°C for 1 month; The test box (Memmer, model HPP 1060) is placed for 1 month under the conditions of a humidity of 65% and a temperature of 40°C.
  • a stability test chamber Memmer, model HPP 1060
  • the test box (Memmer, model HPP 1060) is placed for 1 month under the conditions of a humidity of 65% and a temperature of 40°C.
  • Take 20 ⁇ l of the corresponding sample and add it to the EP tube containing 80 ⁇ l cIEF Master Mix solution in step 2), vortex to mix and centrifuge, transfer 80ul of the supernatant to a 96-well sample plate, and centrifuge for later use.
  • Example 11 Stability experiment of molecular variants of humanized antibodies
  • CE-SDS capillary electrophoresis
  • Sample preparation Samples are taken at the starting point 0; samples are placed in a stability test chamber (Memmer, model HPP 1060) with a humidity of 65% and a temperature of 25°C for 1 month; in the stability test chamber (Memmer) , Model HPP 1060) The humidity is 65% and the temperature is 40°C and placed for 1 month for sampling.
  • Non-reducing CE sample treatment add samples to the EP tube, the amount of protein for each sample is 50 ⁇ g, add 1 ⁇ L of 10kD internal standard (Protein Simple, Cat#046-144), add 2.5 ⁇ L of 250mM IAM (Sigma , Cat#I1149-5G), add 1 ⁇ sample buffer (Protein Simple, Cat#046-567) to a final volume of 50 ⁇ L.
  • Reduce CE sample treatment add samples to the EP tube, the amount of protein for each sample is 50 ⁇ g, add 1 ⁇ L of 10kD internal standard (Protein Simple, Cat#046-144), add 2.5 ⁇ L of ⁇ -mercaptoethanol ( Sigma, Cat#M3148-25ML), add 1 ⁇ sample buffer (Protein Simple, Cat#046-567) to a final volume of 50 ⁇ L.

Abstract

提供了抗体或其抗原结合片段、其制备方法及其医药用途。具体地，提供了包含抗GPC3抗体CDR区的嵌合抗体、人源化抗体，以及包含抗GPC3抗体或其抗原结合片段的药物组合物，及其作为抗癌药物的用途。尤其地，提供了一种人源化的抗GPC3抗体、及其在制备用于治疗GPC3介导的疾病或病症的药物中的用途，以及用于细胞生长抑制剂、用于肿瘤检测和诊断中的用途。

Description

抗体或其抗原结合片段、其制备方法及医药用途 技术领域
本发明涉及一种特异性地对人GPC3受体具有免疫反应性的抗GPC3抗体、或其抗原结合片段、包含所述抗GPC3抗体CDR区的嵌合抗体、人源化抗体，以及包含人抗GPC3抗体及其抗原结合片段的药物组合物，以及其作为细胞生长抑制剂和抗癌药物用途和检测或诊断肿瘤的用途。
背景技术
磷脂酰肌醇蛋白聚糖3(Glypican3，GPC3)是一种70kDa膜蛋白，属于作为器官形成中的细胞外基质发挥细胞粘附作用、或作为细胞生长因子的受体起作用的Glypicans家族。GPC3表达之后会被furin酶切割产生N端40kd可溶性部分以及30kd部分通过GPI分子锚定于细胞膜的C端。
GPC3在胚胎组织、特别是肝脏和肾脏中表达，是与器官形成相关的细胞外基质蛋白。在成人组织中，在胎盘以外观察不到GPC3的表达，但是在肝细胞癌、黑素瘤、卵巢透明细胞癌、肺鳞状细胞癌等各种癌组织中观察到表达。可见，GPC3与甲胎蛋白(α-fetoprotein；AFP)、癌胚抗原(Carcinoembryonic antigen；CEA)等蛋白质同样地是在胚胎组织中表达的蛋白质，因此被归类为胚胎性癌抗原。即，GPC3显示出在正常组织细胞中不表达、但在癌细胞中特异性表达的特征，因此作为癌症治疗的靶分子、肿瘤标志物。另外，基因组学和功能研究表明，GPC3对于维持Wnt通路，Hedgehogs通路的激活中有重要作用，例如GPC3偶联的硫酸乙酰肝素分子可以增强Wnts与其受体的结合从而对于维持Wnt通路有重要作用。GPC3表达于脑、消化道、膀胱，性腺和皮肤并高度表达于肝细胞癌表面；肝癌发生中Wnt通路起到了重要的作用，例如20％肝细胞癌β-Catenin通路突变以及Frizzled-7受体过度表达，因此GPC3在部分肝细胞癌发生过程中可能起到了促进作用。
发明内容
根据本发明的一些实施方案，提供了一种抗GPC3抗体或其抗原结合片段，其包含抗体重链可变区和抗体轻链可变区，其中所述的抗体重链可变区包含至少1个选自以下序列所示的HCDR：SEQ ID NO:7，SEQ ID NO:8，SEQ ID NO:9，SEQ ID NO:10，SEQ ID NO:11，SEQ ID NO:12，SEQ ID NO:13，SEQ ID NO:14或SEQ ID NO:15，
所述的抗体轻链可变区包含至少1个选自以下序列所示的LCDR：SEQ ID NO:16，SEQ ID NO:17，SEQ ID NO:18，SEQ ID NO:19，SEQ ID NO:20，SEQ ID NO:21，SEQ ID NO:22，SEQ ID NO:23，SEQ ID NO:24，SEQ ID NO:25或SEQ ID NO:26。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段的重链可变区包含：SEQ ID NO:7所示的HCDR1、SEQ ID NO:10所示的HCDR2和SEQ ID NO:13所示 的HCDR3。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段的重链可变区包含：SEQ ID NO:8所示的HCDR1、SEQ ID NO:11所示的HCDR2和SEQ ID NO:14所示的HCDR3。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段的重链可变区包含：SEQ ID NO:9所示的HCDR1、SEQ ID NO:12所示的HCDR2和SEQ ID NO:15所示的HCDR3。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段的轻链可变区包含：SEQ ID NO:16所示的LCDR1、SEQ ID NO:21所示的LCDR2和SEQ ID NO:24所示的LCDR3。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段的轻链可变区包含：SEQ ID NO:17所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:25所示的LCDR3。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段的轻链可变区包含：SEQ ID NO:18所示的LCDR1、SEQ ID NO:23所示的LCDR2和SEQ ID NO:26所示的LCDR3。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段的轻链可变区包含：SEQ ID NO:19所示的LCDR1、SEQ ID NO:21所示的LCDR2和SEQ ID NO:24所示的LCDR3。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段的轻链可变区包含：SEQ ID NO:20所示的LCDR1、SEQ ID NO:23所示的LCDR2和SEQ ID NO:26所示的LCDR3。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段包含：SEQ ID NO:7所示的HCDR1、SEQ ID NO:10所示的HCDR2和SEQ ID NO:13所示的HCDR3；以及所述的抗体轻链可变区包含：SEQ ID NO:16所示的LCDR1、SEQ ID NO:21所示的LCDR2和SEQ ID NO:24所示的LCDR3。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段包含：SEQ ID NO:8所示的HCDR1、SEQ ID NO:11所示的HCDR2和SEQ ID NO:14所示的HCDR3；以及所述的抗体轻链可变区包含：SEQ ID NO:17所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:25所示的LCDR3。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段包含：SEQ ID NO:9所示的HCDR1、SEQ ID NO:12所示的HCDR2和SEQ ID NO:15所示的HCDR3；以及所述的抗体轻链可变区包含：SEQ ID NO:18所示的LCDR1、SEQ ID NO:23所示的LCDR2和SEQ ID NO:26所示的LCDR3。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段包含：所述的抗体重链可变区包含：SEQ ID NO:7所示的HCDR1、SEQ ID NO:10所示的HCDR2和SEQ  ID NO:13所示的HCDR3；以及所述的抗体轻链可变区包含：SEQ ID NO:19所示的LCDR1、SEQ ID NO:21所示的LCDR2和SEQ ID NO:24所示的LCDR3。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段包含：所述的抗体重链可变区包含：SEQ ID NO:9所示的HCDR1、SEQ ID NO:12所示的HCDR2和SEQ ID NO:15所示的HCDR3；以及所述的抗体轻链可变区包含：SEQ ID NO:20所示的LCDR1、SEQ ID NO:23所示的LCDR2和SEQ ID NO:26所示的LCDR3。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段选自：鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、人抗体或其抗原结合片段、人源化抗体或其抗原结合片段。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段，所述抗GPC3抗体或其抗原结合片段进一步包含源自人IgG1、IgG2、IgG3或IgG4的重链恒定区或其变体；
优选地，所述抗GPC3抗体或其抗原结合片段进一步包含源自人IgG1、IgG2或IgG4的重链恒定区；
进一步选地，所述抗GPC3抗体或其抗原结合片段进一步包含如SEQ ID NO：52所示的重链恒定区。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段，所述抗GPC3抗体或其抗原结合片段进一步包含经氨基酸突变后而具有增强的ADCC毒性的IgG1重链恒定区。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段，所述抗GPC3抗体或其抗原结合片段进一步包含源自人κ链、λ链的轻链恒定区或其变体；
优选地，所述抗GPC3抗体或其抗原结合片段进一步包含源自人κ链的轻链恒定区；
进一步优选地，所述抗GPC3抗体或其抗原结合片段进一步包含如SEQ ID NO:53所示的轻链恒定区。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段包含选自如下序列所示的重链可变区，或与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区：SEQ ID NO:27，SEQ ID NO:29，SEQ ID NO:30，SEQ ID NO:31，SEQ ID NO:32或SEQ ID NO:37。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段包含选自如下序列所示的轻链可变区，或与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区：SEQ ID NO:28，SEQ ID NO:33，SEQ ID NO:34，SEQ ID NO:35，SEQ ID NO:36或SEQ ID NO:38。
在优选的实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段包含：
SEQ ID NO：27所示的重链可变区和SEQ ID NO：28所示的轻链可变区；
SEQ ID NO：29所示的重链可变区和SEQ ID NO：28所示的轻链可变区；
SEQ ID NO：30所示的重链可变区和SEQ ID NO：28所示的轻链可变区；
SEQ ID NO：31所示的重链可变区和SEQ ID NO：28所示的轻链可变区；
SEQ ID NO：32所示的重链可变区和SEQ ID NO：33所示的轻链可变区；
SEQ ID NO：32所示的重链可变区和SEQ ID NO：34所示的轻链可变区；
SEQ ID NO：32所示的重链可变区和SEQ ID NO：35所示的轻链可变区；
SEQ ID NO：32所示的重链可变区和SEQ ID NO：36所示的轻链可变区；或
SEQ ID NO：37所示的重链可变区和SEQ ID NO：38所示的轻链可变区。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段含有选自如下序列所示的重链，或与以下序列相比具有至少80％，85％，90％，95％或99％同一性的重链：SEQ ID NO:39，SEQ ID NO:41，SEQ ID NO:42，SEQ ID NO:43，SEQ ID NO:44或SEQ ID NO:49。
在一些实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段含有选自如下序列所示的轻链，或与以下序列相比具有至少80％，85％，90％，95％或99％同一性轻链：SEQ ID NO:40，SEQ ID NO:45，SEQ ID NO:46，SEQ ID NO:47，SEQ ID NO:48或SEQ ID NO:50。
在一个具体的实施方案中，根据本发明的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体包含：
SEQ ID NO：39所示的重链和SEQ ID NO：40所示的轻链；
SEQ ID NO：42所示的重链和SEQ ID NO：40所示的轻链；
SEQ ID NO：43所示的重链和SEQ ID NO：40所示的轻链；
SEQ ID NO：44所示的重链和SEQ ID NO：45所示的轻链；
SEQ ID NO：44所示的重链和SEQ ID NO：46所示的轻链；
SEQ ID NO：44所示的重链和SEQ ID NO：47所示的轻链；
SEQ ID NO：44所示的重链和SEQ ID NO：48所示的轻链；或，
SEQ ID NO：49所示的重链和SEQ ID NO：50所示的轻链。
根据本发明的一些实施方案，提供了一种多核苷酸，其编码本发明的抗GPC3抗体或其抗原结合片段。
根据本发明的一些实施方案，提供了一种表达载体，其含有本发明的多核苷酸。
根据本发明的一些实施方案，提供了一种宿主细胞，其导入或含有本发明的表达载体。在一个具体的实施方案中，所述的宿主细胞为细菌，优选为大肠杆菌。
在另一个具体的实施方案中，所述的宿主细胞为酵母菌，优选为毕赤酵母。
在另一个具体的实施方案中，所述的宿主细胞为哺乳动物细胞，优选为CHO细胞或HEK293细胞。
根据本发明的一些实施方案，提供了一种生产抗GPC3抗体的方法，包括步骤：培养本发明的宿主细胞、从培养物中分离抗体、以及对所述抗体进行纯化。
根据本发明的一些实施方案，提供了一种药物组合物，其含有本发明的抗GPC3抗 体或其抗原结合片段以及可药用的赋形剂、稀释剂或载体。
根据本发明的一些实施方案，提供了一种检测或诊断试剂盒，其含有本发明的抗GPC3抗体或其抗原结合片段以及可用于检测或诊断的赋形剂、稀释剂或载体。
根据本发明的一些实施方案，提供了一种检测或诊断试剂盒，其含有本发明的抗GPC3抗体或其抗原结合片段，任选地，还包含一种或多种检测所述抗GPC3抗体或其抗原结合片段与GPC3或其表位结合的试剂。
根据本发明的一些实施方案，提供了本发明的抗GPC3抗体或其抗原结合片段，或上述的药物组合物在制备药物中的用途，所述药物用于治疗或预防GPC3介导的疾病或病症。
根据本发明的一些实施方案，提供了根据本发明的抗GPC3抗体或其抗原结合片段，或上述的药物组合物在制备试剂盒中的用途，其中所述试剂盒用于检测或诊断GPC3介导的疾病或病症。
在一些实施方案中，根据本发明上述的用途，所述的疾病或病症为癌症。
在优选的实施方案中，所述的疾病或病症为表达GPC3的癌症。
在进一步优选的实施方案中，所述的癌症选自乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤和黑色素瘤。
在具体的实施方案中，所述的疾病或病症选自：肝细胞癌、黑素瘤、卵巢透明细胞癌、肺鳞状细胞癌。
根据本发明的一些实施方案，提供了一种治疗或预防GPC3介导的疾病的方法，包括步骤：向受试者提供治疗有效量或预防有效量的根据本发明的抗GPC3抗体或其抗原结合片段。
根据本发明的一些实施方案，提供了一种治疗或预防GPC3介导的疾病的方法，包括步骤：向受试者提供治疗有效量或预防有效量的根据本发明的药物组合物。
在一些实施方案中，所述受试者是疑似患有、已经患有、易感于GPC3介导的疾病，GPC3介导的疾病选自乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤和黑色素瘤，优选，肝细胞癌、黑色素瘤、卵巢透明细胞癌、肺鳞状细胞癌。
本发明的抗GPC3抗体或抗原结合片段，其能够特异性的与GPC3抗原(或其表位)、表达GPC3的细胞结合，且具有显著的CDC活性，ADCC活性，其对肿瘤的杀伤效果显著。
另外，本发明的抗GPC3抗体或其抗原结合片段具有良好的内吞作用，适于和药物偶联，构建ADC。本发明的抗GPC3抗体或抗原结合片段在保持高特异性和杀伤活性的同时，免疫原性更低，稳定性更高，并显著优于Codrituzumab。本发明抗GPC3抗体或抗原结合片段具有更好的作为抗癌药物的潜力，保证用药安全。
具体实施方式
发明详述
一、术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本发明所用氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。
本发明所述的术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
在本发明中，本发明所述的抗体轻链可变区可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本发明中，本发明所述的抗体重链可变区可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区指LCDR1、LCDR2，和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本发明所述的抗体或抗原结合片段的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则和Kabat或ABM定义规则(http://bioinf.org.uk/abs/)。
术语“抗原呈递细胞”或“APC”是在其表面上展示与MHC复合的外来抗原的细胞。T细胞利用T细胞受体(TCR)识别这种复合物。APC的实例包括但不限于树突细胞(DC)、外用血单个核细胞(PBMC)、单核细胞、B淋巴母细胞和单核细胞衍生的树突细胞。
术语“抗原呈递”是指APC捕获抗原和使它们能够被T细胞识别的过程，例如作为MHC-I/MHC-II偶联物的组分。
术语“GPC3”包括由细胞天然表达的GPC3的任何变体或同种型。本发明的抗体可与得自非人物种的GPC3交叉反应。作为另一种选择，该抗体也可以是人GPC3特异性的，可不表现出与其他物种的交叉反应性。GPC3或其任何变体或同种型可从天然表达它 们的细胞或组织中分离而得，或使用本领域通用以及本文所述的那些技术通过重组技术产生。优选地，抗GPC3抗体靶向具有正常糖基化模式的人源GPC3。
术语“重组人抗体”包括通过重组方法制备、表达、创建或分离的人抗体，所涉及的技术和方法在本领域中是熟知的，诸如：
1.从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；
2.从经转化以表达抗体的宿主细胞如转染瘤中分离的抗体；
3.从重组组合人抗体文库中分离的抗体；以及
4.通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。
此类重组人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的对人GPC3的单克隆抗体。制备时用GPC3抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本发明一个优选的实施方案中，所述的鼠源GPC3抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。
术语“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本发明的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架中产生的抗体。人源化抗体可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的免疫应答反应的缺点。为避免在免疫原性下降的同时引起活性的下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变后增强ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1重链恒定区。
术语“抗原结合片段”是指抗体的抗原结合片段及抗体类似物，其通常包括至少部 分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母体抗体的至少某些结合特异性。通常，当基于摩尔来表示活性时，抗体片段保留至少10％的母体结合活性。优选地，抗体片段保留至少20％、50％、70％、80％、90％、95％或100％或更多的母体抗体对靶标的结合亲和力。抗原结合片段实例包括但不限于：Fab、Fab’、F(ab’)2、Fv片段、线性抗体(linear antibody)、单链抗体、纳米抗体、结构域抗体和多特异性抗体。工程改造的抗体变体综述于Holliger和Hudson，2005，Nat.Biotechnol.23：1126-1136中。
“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。
“Fc”区含有包含抗体的CH2和CH3结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。
“Fab’片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’)2分子。
“F(ab’)2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此，F(ab’)2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。
“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区。
术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区VH和轻链可变区VL的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双抗体(diabodies)、双特异性双抗体和三抗体(triabodies)、己共价或非共价连接在一起的抗体片段等。
术语“单链抗体”是由抗体的重链可变区VH和轻链可变区VL通过一段连接肽连接而成的单链重组蛋白，它是具有完全抗原结合位点的最小抗体片段。
术语“结构域抗体片段”是仅含有重链可变区或轻链可变区链的具有免疫学功能的免疫球蛋白片段。在某些情况下，两个或多个VH区与肽接头共价连接以形成二价结构域抗体片段。二价结构域抗体片段的两个VH区可靶向相同或不同抗原。
本发明的术语“与GPC3结合”，指能与人GPC3相互作用。
本发明的术语“抗原结合位点”指本发明抗体或抗原结合片段识别的三维空间位点。
术语“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位由给定的 抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。
本发明所用的术语“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。通常，当使用人GPC3作为分析物并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原结合，并且其与预定抗原结合的亲和力是其与预定抗原或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。术语“识别抗原的抗体”在本文中可以与术语“特异性结合的抗体”互换使用。
术语“交叉反应”是指本发明的抗体与来自不同物种的GPC3结合的能力。例如，结合人GPC3的本发明的抗体也可以结合另一物种的GPC3。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与纯化抗原的特异性反应性，或与生理表达GPC3的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本文所述的标准结合测定，例如表面等离子体共振(SPR)分析，或流式细胞术。
术语“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。配体的抑制/阻断优选地降低或改变无抑制或阻断的情况下发生配体结合时出现活性的正常水平或类型。抑制和阻断也旨在包括与抗GPC3抗体接触时，与未与抗GPC3抗体接触的配体相比，任何可测量的配体结合亲和力降低。
术语“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
术语“诱导免疫应答”和“增强免疫应答”可互换使用，并指免疫应答对特定抗原的剌激(即，被动或适应性的)。针对诱导CDC或ADCC的术语“诱导”是指剌激特定的直接细胞杀伤机制。
本发明中所述的“ADCC”，即antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，增强或降低降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。如，小鼠可以用人GPC3或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。相应抗体的cDNA序列可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在FC区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳 性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
本发明的抗体指单克隆抗体。本发明所述的单克隆抗体(mAb)，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术、合成技术(如CDR-grafting)、或其它现有技术进行重组得到。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，诸如包含本发明的任一种抗体，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽本发明的实施方案(例如治疗方法或制品)在缓解每个患都有的目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
整个说明书和权利要求书中使用的术语“基本上由……组成”或其变形表示包括所有所述元件或元件组，并且任选包括与所述元件类似或不同性质的其它元件，所述其它元件非显著改变指定给药方案、方法或组合物的基本性质或新性质。
本发明所述的应用于某个对象的术语“天然存在的”是指这样的事实，即该对象可在自然界中发现。例如存在于可从自然界来源分离得到的生物体(包括病毒)、且未经人工在实验室中有意修饰的多肽序列或多核苷酸序列即是天然存在的。
“有效量”包含足以改善或预防医字病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指要据背景在生物、细胞或人体外产生的物质。
“内源性”指根据背景在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述抗体或其抗原结合片段，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
以下结合实施例用于进一步描述本发明，但这些实施例并非限制着本发明的范围。本发明实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1：抗原准备
蛋白抗原是人GPC3重组蛋白(UniProt#P51654,Gln 25-His 559)，C端带His标签，即人GPC3His蛋白，购买自AcroBiosystems公司(Cat#GP3-H52H4)。
表1.人GPC3(Gln 25-His 559)氨基酸序列
Figure PCTCN2021087549-appb-000001
Figure PCTCN2021087549-appb-000002
实施例2：鼠杂交瘤及抗体序列的获得
用人GPC3重组蛋白进行动物免疫，共5只C57bl/6和5只SJL小鼠，雌性，10周龄，小鼠均购自北京维通利华实验动物技术有限公司。使用Sigma完全弗氏佐剂(CFA)和Sigma不完全弗氏佐剂(IFA)，免疫原和免疫佐剂以1:1的比例充分混合乳化，制成稳定“油包水”液体；注射剂量25μg/200μL/小鼠。
免疫方案
第1天 第一次免疫，完全弗氏佐剂
第21天 第二次免疫，不完全弗氏佐剂
第35天 第三次免疫，不完全弗氏佐剂
第42天 采血和血清效价检测(3免血)
第49天 第四次免疫，不完全弗氏佐剂
第56天 采血和血清效价检测(4免血)
对免疫小鼠血清使用如实施例3(1)所述的间接ELISA法评估血清效价及结合细胞表面抗原的能力，对照效价检测情况(大于10万倍稀释度)决定启动细胞融合。选择血清效价强的免疫小鼠进行一次终免疫后处死小鼠，取脾细胞和SP2/0骨髓瘤细胞融合后铺板获得杂交瘤，通过间接ELISA筛选到目标杂交瘤，并通过有限稀释法建株为单克隆细胞株。得到的阳性抗体株进一步使用间接ELISA进行筛选，从而选定结合重组蛋白的杂交瘤。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,15596-018)提取RNA并反转录(PrimeScript TM Reverse Transcriptase，Takara#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen，TB326 Rev.B 0503)进行PCR扩增后测序，最终得到鼠源抗体M1、M2和M3的序列。
表2.鼠单抗的重链和轻链可变区序列
Figure PCTCN2021087549-appb-000003
Figure PCTCN2021087549-appb-000004
注：下划线表示鼠单抗CDR序列。
表3.鼠源抗体CDR区序列
抗体 HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3
M1 SEQ ID NO：7 SEQ ID NO：10 SEQ ID NO：13 SEQ ID NO：16 SEQ ID NO：21 SEQ ID NO：24
M2 SEQ ID NO：8 SEQ ID NO：11 SEQ ID NO：14 SEQ ID NO：17 SEQ ID NO：22 SEQ ID NO：25
M3 SEQ ID NO：9 SEQ ID NO：12 SEQ ID NO：15 SEQ ID NO：18 SEQ ID NO：23 SEQ ID NO：26
实施例3：抗体的体外结合活性检测方法
(1)体外间接ELISA结合实验：
用pH7.4的PBS将GPC3 His蛋白(AcroBiosystems，Cat#GP3-H52H4)稀释至0.5μg/ml浓度，以100μL/孔的体积加入96孔高亲和力酶标板中，于4℃冰箱孵育过夜(16-20小时)。用PBST(pH7.4 PBS含0.05％Tween-20)洗板3次后，加入用PBST稀释的1％牛血清白蛋白(BSA)封闭液200μL/孔，37℃孵育0.5小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板1次。
用含1％BSA的PBST稀释待测抗体，100nM起始，5倍梯度稀释，11个剂量，以100μL/孔加到酶标板中，放于37℃孵育1小时。孵育结束后用PBST洗板3次，加入200μL/孔用含1％BSA的PBST稀释的HRP标记羊抗鼠二抗(Jackson ImmunoResearch Laboratories，cat#115-035-071)或HRP标记羊抗人二抗(Rockland，cat#609-103-123)，37℃孵育0.5小时。用PBST洗板5次后，加入100μL/孔TMB显色底物(苏州亚科化学试剂股份有限公司，cat#S0025)，于25℃避光孵育8-15分钟，加入50μL/孔1M HCl 终止反应，用酶标仪(Thermo,Ascent)在450nm处读取吸收值，分析数据。
做浓度信号值曲线分析结果，如下表所示：
表4.鼠抗体对人GPC3抗原的亲和力(EC 50值)
鼠抗体 ELISA，EC 50(nM)
M1 0.1
M2 0.09
M3 0.11
结果显示：鼠抗体对人GPC3抗原的亲和力良好。
(2)体外FACS结合实验：
GPC3高表达肿瘤细胞HepG2(ATCC保藏号：HB-8065)，经胰酶消化后，离心收集细胞，用FACS buffer(含2％FBS的1×PBS)调节细胞密度后分铺于96孔U底板，每孔1×10 5至2×10 5个细胞。离心：1200g、5分钟，弃上清，加入100μL已用FACS buffer梯度稀释的抗体溶液，4℃孵育1小时。离心：1200g、5分钟，弃上清，PBS洗细胞2次后，添加FACS buffer配制的荧光标记二抗工作液PE anti-human IgG Fc Antibody(Biolegend,Cat#409304)或FITC anti-mouse IgG Antibody(Biolegend,Cat#406001)，100μL每孔重悬细胞，4℃孵育1小时。离心：1200g、5分钟，弃上清。PBS洗细胞2次后，再重悬于PBS，使用流式细胞仪DxFlex检测荧光信号，并作曲线分析抗体结合细胞的EC 50浓度。
表5.鼠抗体对人GPC3高表达细胞的亲和力(EC 50值)
鼠抗体 FACS，EC 50(nM)
M1 2.05
M2 0.06
M3 4.07
结果显示：鼠抗体对人GPC3高表达细胞的亲和力良好。
实施例4：小鼠抗体嵌合化实验
将筛选得到的鼠源抗体进行嵌合化，鼠源抗体可变区克隆在人抗体的恒定区得到嵌合抗体，示例性嵌合抗体的恒定区可以是人IgG1重链恒定区和kappa轻链恒定区。
(1)体外蛋白结合实验：
根据实施例3(1)的体外间接ELISA结合实验和Biacore方法，测试嵌合抗体与人GPC3抗原的亲和力。
Biacore测试方法如下：
a)芯片制备：
将鼠抗人IgG(Fc)抗体用固定试剂(10mM醋酸钠，pH 5.0)稀释到25μg/mL，芯片每个通道约使用100μL鼠抗人IgG(Fc)抗体，固定两个通道约使用190μL固定试剂加入10μL鼠抗人IgG(Fc)抗体。首先，CM5芯片的表面用400mM EDC and 100mM NHS以10μL/min的流速进行420s的活化。其次，将25μg/mL的鼠抗人IgG(Fc)抗体以10μL/min的流速注入到实验通道(FC4)约420s，固定量约为9000至14000RU。最后，芯片用1M 乙醇胺以10μL/min进行420s封闭。参比通道(FC3)与试验通道(FC4)进行相同的操作。
b)捕获配体:
将抗体原液用运行试剂分别稀释至4μg/mL，并以10μL/min的流速注入到实验通道(FC4)捕获约200RU。参比通道(FC3)不需要进行配体的捕获。
c)分析物多循环分析：
将人GPC 3蛋白用运行试剂进行2倍倍比稀释，将稀释后的样品依次以30μL/min的流速按照相应结合时间和解离时间注入到实验通道与参比通道。每一个浓度分析后，芯片需要用3M氯化镁以20μL/min的流速再生30s，洗掉配体以及未解离的分析物。进行下一个浓度分析时，实验通道需要重新捕获相同量的配体。
d)数据分析：
使用Biacore T200分析软件计算每个抗体的KD值。参比通道(FC3)用于背景的扣减。
表6.嵌合抗体对人GPC3抗原的亲和力
Figure PCTCN2021087549-appb-000005
ELISA和Biacore结果见表6，显示嵌合抗体与人GPC3抗原具有良好的亲和力。
(2)体外细胞结合实验：
GPC3高表达细胞(过表达人或猴GPC3的CHO-K1细胞，和表达GPC3的人肝癌细胞JHH-7)，经胰酶消化后，离心收集细胞，用FACS缓冲液(含2％FBS的1×PBS)调节细胞密度后分铺于96孔U底板，每孔1×10 5至2×10 5个细胞。离心：1200g、5分钟，弃上清，加入100μL已用FACS缓冲液梯度稀释的抗体溶液，4℃孵育1小时。离心：1200g、5分钟，弃上清，PBS洗细胞2次后，添加FACS缓冲液配制的荧光标记二抗工作液PE anti-human IgG Fc Antibody(Biolegend,Cat#409304)或FITC anti-mouse IgG Antibody(Biolegend,Cat#406001)，100μL每孔重悬细胞，4℃孵育1小时。离心：1200g、5分钟，弃上清。PBS洗细胞2次后，再重悬于PBS，使用流式细胞计数仪DxFlex检测荧光信号，并作曲线分析抗体结合细胞的EC 50浓度。
表7.嵌合抗体对表达GPC3细胞的亲和力(EC 50值)
Figure PCTCN2021087549-appb-000006
结果显示：嵌合抗体与表达GPC3的细胞均具有很高的亲和力。
实施例5：小鼠抗体人源化实验
鼠源抗人GPC3单克隆抗体人源化如本领域许多文献公示的方法进行。简言之，使用人恒定结构域替代亲本(鼠源抗体)恒定结构域，根据鼠源抗体和人抗体的同源性选择人种抗体序列，本发明将鼠源抗体M1、M2和M3进行人源化。
在所获得的鼠源抗体VH/VL CDR典型结构的基础上，将重、轻链可变区序列与人源抗体种系数据库比较，获得同源性高的人种系模板。
将鼠源抗体的CDR区移植到选择好的相应人源化模板上。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，并对CDR区化学不稳定氨基酸残基优化，经表达测试和回复突变数量对比，选择出人源化重链可变区HCVR和轻链可变区LCVR序列组合而成的抗体，序列如下：
表8.抗体重链和轻链可变区CDR序列
Figure PCTCN2021087549-appb-000007
表9.人源化抗体CDR区
抗体 HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3
H1-1 SEQ ID NO：7 SEQ ID NO：10 SEQ ID NO：13 SEQ ID NO：19 SEQ ID NO：21 SEQ ID NO：24
H1-2 SEQ ID NO：7 SEQ ID NO：10 SEQ ID NO：13 SEQ ID NO：19 SEQ ID NO：21 SEQ ID NO：24
H1-3 SEQ ID NO：7 SEQ ID NO：10 SEQ ID NO：13 SEQ ID NO：19 SEQ ID NO：21 SEQ ID NO：24
H1-4 SEQ ID NO：7 SEQ ID NO：10 SEQ ID NO：13 SEQ ID NO：19 SEQ ID NO：21 SEQ ID NO：24
H2-1 SEQ ID NO：8 SEQ ID NO：11 SEQ ID NO：14 SEQ ID NO：17 SEQ ID NO：22 SEQ ID NO：25
H2-2 SEQ ID NO：8 SEQ ID NO：11 SEQ ID NO：14 SEQ ID NO：17 SEQ ID NO：22 SEQ ID NO：25
H2-3 SEQ ID NO：8 SEQ ID NO：11 SEQ ID NO：14 SEQ ID NO：17 SEQ ID NO：22 SEQ ID NO：25
H2-4 SEQ ID NO：8 SEQ ID NO：11 SEQ ID NO：14 SEQ ID NO：17 SEQ ID NO：22 SEQ ID NO：25
H3-1 SEQ ID NO：9 SEQ ID NO：12 SEQ ID NO：15 SEQ ID NO：20 SEQ ID NO：23 SEQ ID NO：26
表10.人源化抗体的重链和轻链可变区序列
Figure PCTCN2021087549-appb-000008
Figure PCTCN2021087549-appb-000009
注：下划线表示单抗CDR序列。
将设计的重链和轻链可变区序列与IgG1重链恒定区和轻链恒定区序列连接，示例性的，抗体重链恒定区选自SEQ ID NO：52所示的人IgG1天然恒定区，轻链恒定区选自序列如SEQ ID NO：53所示的人κ链的恒定区，得到重链和轻链序列如下：
表11.人源化抗体的重链和轻链序列，及重链和轻链恒定区序列
Figure PCTCN2021087549-appb-000010
Figure PCTCN2021087549-appb-000011
Figure PCTCN2021087549-appb-000012
Figure PCTCN2021087549-appb-000013
表12.抗体及其重链、轻链、可变区的序列编号
人源化抗体编号 HCVR HC LCVR LC
H1-1 SEQ ID NO：27 SEQ ID NO：39 SEQ ID NO：28 SEQ ID NO：40
H1-2 SEQ ID NO：29 SEQ ID NO：41 SEQ ID NO：28 SEQ ID NO：40
H1-3 SEQ ID NO：30 SEQ ID NO：42 SEQ ID NO：28 SEQ ID NO：40
H1-4 SEQ ID NO：31 SEQ ID NO：43 SEQ ID NO：28 SEQ ID NO：40
H2-1 SEQ ID NO：32 SEQ ID NO：44 SEQ ID NO：33 SEQ ID NO：45
H2-2 SEQ ID NO：32 SEQ ID NO：44 SEQ ID NO：34 SEQ ID NO：46
H2-3 SEQ ID NO：32 SEQ ID NO：44 SEQ ID NO：35 SEQ ID NO：47
H2-4 SEQ ID NO：32 SEQ ID NO：44 SEQ ID NO：36 SEQ ID NO：48
H3-1 SEQ ID NO：37 SEQ ID NO：49 SEQ ID NO：38 SEQ ID NO：50
实施例6：人源化抗体的表达和纯化
根据以上各人源化抗体轻链和重链的氨基酸序列合成cDNA片段，插入到pcDNA3.1表达载体(Life Technologies Cat.No.V790-20)中。将表达载体和转染试剂PEI(Polysciences，Inc.Cat.No.23966)以1:2的比例转染HEK293细胞(Life Technologies Cat.No.11625019)，并置于CO 2孵育箱中孵育4-5天。收取细胞培养液，离心过滤后上样到抗体纯化亲和柱，经磷酸缓冲液洗柱、甘氨酸盐酸缓冲液(pH2.7 0.1M Gly-HCl)洗脱、1M Tris盐酸pH 9.0中和、以及磷酸缓冲液透析，得到本发明的人源化抗体蛋白，其浓度和纯度如下表。
表13.人源化抗体的浓度和纯度
人源化抗体编号 浓度(mg/ml) 纯度(％)
H1-1 0.68 98.1％
H1-2 0.52 98.8％
H1-3 0.55 96.5％
H1-4 0.98 97.6％
H2-1 2.19 98.1％
H2-2 0.35 98.8％
H2-3 0.92 98.8％
H2-4 0.49 98.7％
H3-1 1.12 98.4％
实施例7：人源化抗体的体外结合亲和力实验：
(1)体外间接ELISA结合实验：
使用实施例3(1)体外间接ELISA结合实验测定的各人源化抗体对人GPC3抗原的亲和力(EC 50)，结果见下表14：
表14.人源化抗体对人GPC3抗原的亲和力(EC 50)
人源化抗体编号 亲和力EC 50(nM)
H1-1 0.019
H1-2 0.021
H1-3 0.019
H1-4 0.021
H2-1 0.016
H2-2 0.007
H2-3 0.016
H2-4 0.013
H3-1 0.010
结果显示：本发明人源化抗体与人GPC3抗原均有很高的亲和力。
(2)体外FACS结合实验：
使用实施例4(2)体外FACS结合实验，测定各人源化抗体对GPC3抗原表达细胞的亲和力(EC 50)，结果如下表15所示：
表15.人源化抗体对人GPC3高表达细胞的亲和力(EC 50值)
Figure PCTCN2021087549-appb-000014
结果表明：人源化抗体与过表达人或猴GPC3抗原的稳定细胞株均有很高的亲和力，并且可以结合人肝癌细胞JHH-7。
实施例8：人源化抗体的内吞作用
检测本发明抗体结合GPC3后是否能和人GPC3共同内吞入细胞内，用稳转细胞株CHO-K1-human GPC3以及人肝癌细胞JHH-7(CBP60204，购自南京科佰生物科技有限公司)和HepG2(SCSP-510，购自中国科学院细胞库)进行评估。
细胞使用胰酶消化，收集细胞并用预冷的FACS缓冲液重悬，调整细胞浓度为1×10 6/mL。取EP管，加入1mL细胞悬液，1500rpm离心5分钟后弃上清，加入1mL已经配制好的待测抗体重悬细胞，抗体的终浓度均为20μg/mL，4℃摇床孵育1小时，离心弃上清(4℃、1500rpm×5分钟)，FACS缓冲液洗涤两次，弃上清。每管加入100μL荧光标记二抗工作液PE anti-human IgG Fc Antibody(Biolegend,Cat#409304)或FITC anti-mouse IgG Antibody(Biolegend,Cat#406001)，重悬细胞，4℃摇床孵育30分钟，离心弃上清(4℃、1500rpm×5分钟)，FACS buffer洗涤两次，弃上清。每管加入1mL预热的细胞培养基重悬细胞并混匀，分装为4管，每管200μL，分别为0分钟组，blank组，30分钟组和2小时组，取出0分钟及blank置于冰上，其余放置于37℃培养箱，分别内吞30分钟、2小时，在相应时间点取出EP管，置于冰上预冷5分钟，所有处理组离心弃上清(4℃、1500rpm×5分钟)，用FACS缓冲液洗涤一次，弃上清。0分钟组外所有处理组EP管中加入250μL strip buffer，室温孵育8分钟，离心弃上清(4℃、1500rpm ×5分钟)，FACS buffer洗涤两次，弃上清。所有处理组加入100μL PBS重悬细胞，用流式细胞仪DxFlex进行检测。
抗体内吞百分比＝(各个时间点荧光强度值-Blank组的平均荧光强度值)/(零点时的平均荧光轻度值-Blank组的平均荧光强度值)
表16.人源化抗体的内吞作用
Figure PCTCN2021087549-appb-000015
结果显示：本发明人源化抗体在CHO-K1-human GPC3、JHH-7和HepG2细胞中均有良好的内吞作用。
实施例9：人源化抗体的CDC实验
本实验的靶细胞为人GPC3过表达稳定细胞株CHO-K1-hGPC3。实验培养基为不含FBS的细胞培养基F12K(Gibco TM，Cat#21127022)，用于细胞和抗体重悬或稀释。CHO-K1-hGPC3细胞经胰酶消化，离心收集，用实验培养基重悬成1×10 5个/ml，50μL铺于白色96孔板(Corning，3610)，加入25μL 4×工作浓度的抗体(抗体起始工作浓度为20nM，5倍稀释，10个浓度点，并设立0nM点)，37℃孵育30分钟，加入25μL 80％人血清(GemCell TM US,Cat#100-512)，37℃培养箱培养24小时。添加50μL CellTiter-Glo(Promega，Cat#G7573)，混匀室温避光反应10分钟，多功能酶标仪(Thermofisher，Lux)读数。
抗体杀伤百分比(％)＝(E-S)/(E-M)×100
E为不加抗体孔数值，即细胞+培养基+人血清；
S为样品孔数值，即细胞+抗体+人血清；
M是培养基+人血清孔数值。
以抗体浓度为横坐标，杀伤百分比为纵坐标，使用GraphPad Prism 8.3.0中的log(inhibitor)vs.response--Variable slope进行曲线拟合，分析得到抗体杀伤细胞的IC 50浓度，结果见下表。
表17.人源化抗体的CDC作用
Figure PCTCN2021087549-appb-000016
结果显示：人源化抗体H1-3、H2-3、H3-1均具有显著的CDC作用。
实施例10：人源化抗体的电荷异质稳定性实验
本实验通过全景式等电聚焦(iCIEF)的方法，检测并比较了人源化抗体H3-1在起始0点、以及分别在25℃和40℃放置一个月的电荷异质体纯度，以此考察抗体的稳定性。
将带有两性基团的样品、两性电解质、缓冲剂和辅助添加剂的混合物注入毛细管内，当在毛细管两端加上直流电压时，载体两性电解质可以在管内形成一定范围的pH梯度，样品组分依据其所带电性向阴极或阳极泳动，毛细管内pH值与该组分的等电点(pI)相同时，溶质分子的净电荷为零，宏观上该组分将聚集在该点不再进一步迁移，达到使复杂样品中各组分分离的目的。采集图谱后，根据marker pI值与色谱峰的迁移时间的线性关系，从而得到供试品的pI值、峰比值(主峰、酸峰、碱峰)。主要步骤如下：
1)系统适应性样品准备：将Maurice cIEF System Suitability Kit(Protein Simple，Cat#046-044)里的适应性样品管取出，加入40μl去离子水和160μl System Suitability Test Mix，混匀后转移到1.5ml离心管中，涡旋震荡后离心，取160ul上清转移至96孔样品板待用。
2)配制cIEF Master Mix solution：包含37μl超纯水，35μl的1％MC(Protein Simple，Cat#101876)，4μl Pharmalyte pH 3–10(Protein Simple，Cat#17-0456-01)，2μl 500mM arginine(Protein Simple，Cat#042-691)，以及相应的两个pI Marker6.14(protein simple,cat:046-031)和9.99(protein simple,cat:046-034))各1μl，总体积为80μl。
3)供试品制备：样品设置3个条件：起始0点；在稳定性试验箱(Memmer,型号HPP 1060)湿度为65％和温度为25℃的条件下放置1个月；在稳定性试验箱(Memmer,型号HPP 1060)湿度为65％和温度为40℃的条件下放置1个月。取20μl相应样品，加至步骤2)中含80μl cIEF Master Mix solution的EP管中，涡旋振荡混匀后离心，取80ul上清转移至96孔样品板，离心待用。
4)上机检测：打开毛细管电泳仪(Protein Simple，maurice)以及软件，按照仪器操作步骤，进行仪器自检，安装毛细管卡盒(Protein Simple，Cat#PS-MC02-C)，将96孔样品板放入仪器的相应位置，进行cIEF分析。
表18.人源化抗体H3-1的iCIEF检测
Figure PCTCN2021087549-appb-000017
结果显示：与起始0点相比，H3-1在25℃放置一个月后的主峰比例变化不大，稳定性良好，在强制降解条件40℃放置一个月，抗体出现了预期的降解现象，体现在主峰比 例降为22.3％。
实施例11：人源化抗体的分子变异体稳定性实验
本实验通过毛细管电泳(CE-SDS)的方法，检测并比较了人源化抗体H3-1在起始0点、以及分别在25℃和40℃放置一个月的分子变异体的纯度。CE-SDS是基于蛋白样品在变性条件下凝胶电泳中的迁移，根据不同分子量的蛋白迁移时间的差异完成分离，从而得到非还原和还原处理后样品纯度的检测结果。
主要步骤如下：
1)样品准备：样品在起始0点取样；在稳定性试验箱(Memmer，型号HPP 1060)湿度为65％和温度为25℃的条件下放置1个月取样；在稳定性试验箱(Memmer,型号HPP 1060)湿度为65％和温度为40℃的条件下放置1个月取样。
2)非还原CE样品处理：向EP管中加入样品，每个样品的蛋白取用量为50μg，加入1μL的10kD内标(Protein Simple，Cat#046-144)，加入2.5μL的250mM IAM(Sigma，Cat#I1149-5G)，加入1×sample buffer(Protein Simple，Cat#046-567)至终体积为50μL。
3)还原CE样品处理：向EP管中加入样品，每个样品的蛋白取用量为50μg，加入1μL的10kD内标(Protein Simple，Cat#046-144)，加入2.5μL的β-巯基乙醇(Sigma，Cat#M3148-25ML)，加入1×sample buffer(Protein Simple，Cat#046-567)至终体积为50μL。
4)振荡混匀后，在干式恒温器(杭州奥盛仪器有限公司，型号MK20001)中70℃加热孵育10min，之后取出，冰上孵育5min，冷却后12000rpm离心5min。离心后取35μL上清转移至仪器匹配的96孔中，之后于1000rpm离心5min。
5)样品检测：将96孔样品板放入毛细管电泳仪(Protein Simple，Maurice)中，打开仪器以及软件，按照仪器操作步骤，进行仪器自检，安装毛细管卡盒(Protein Simple，Cat#090-157)，准备相应的试剂放入仪器的相应位置。根据仪器操作步骤，设置相应的参数，进行还原或者非还原CE分析。
6)数据处理：样品检测完成后，设置相应的积分参数，通过仪器自带软件进行计算分析，得到样品纯度。
表19.人源化抗体H3-1的CE-SDS检测
Figure PCTCN2021087549-appb-000018
非还原CE结果显示，人源化抗体H3-1在25℃放置一个月的主峰比例与0点的相近，40℃放置一个月主峰有所下降；在还原CE中，与0点相比，无论是25℃还是40℃放置 一个月，抗体的重轻链比例之和均变化不大。综上所述，人源化抗体H3-1的稳定性良好。

Claims (22)

  1. 一种抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中，所述重链可变区包含：
    SEQ ID NO:7所示的HCDR1、SEQ ID NO:10所示的HCDR2和SEQ ID NO:13所示的HCDR3，或，
    SEQ ID NO:8所示的HCDR1、SEQ ID NO:11所示的HCDR2和SEQ ID NO:14所示的HCDR3，或，
    SEQ ID NO:9所示的HCDR1、SEQ ID NO:12所示的HCDR2和SEQ ID NO:15所示的HCDR3；
    所述轻链可变区包含：
    SEQ ID NO:16所示的LCDR1、SEQ ID NO:21所示的LCDR2和SEQ ID NO:24所示的LCDR3，或，
    SEQ ID NO:17所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:25所示的LCDR3，或，
    SEQ ID NO:18所示的LCDR1、SEQ ID NO:23所示的LCDR2和SEQ ID NO:26所示的LCDR3，或，
    SEQ ID NO:19所示的LCDR1、SEQ ID NO:21所示的LCDR2和SEQ ID NO:24所示的LCDR3，或，
    SEQ ID NO:20所示的LCDR1、SEQ ID NO:23所示的LCDR2和SEQ ID NO:26所示的LCDR3。
  2. 如权利要求1所述的抗GPC3抗体或其抗原结合片段，其中：
    所述重链可变区包含SEQ ID NO:7所示的HCDR1、SEQ ID NO:10所示的HCDR2和SEQ ID NO:13所示的HCDR3；所述轻链可变区包含SEQ ID NO:16所示的LCDR1、SEQ ID NO:21所示的LCDR2和SEQ ID NO:24所示的LCDR3；或，
    所述重链可变区包含SEQ ID NO:8所示的HCDR1、SEQ ID NO:11所示的HCDR2和SEQ ID NO:14所示的HCDR3；所述轻链可变区包含SEQ ID NO:17所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:25所示的LCDR3；或，
    所述重链可变区包含SEQ ID NO:9所示的HCDR1、SEQ ID NO:12所示的HCDR2和SEQ ID NO:15所示的HCDR3；所述轻链可变区包含SEQ ID NO:18所示的LCDR1、SEQ ID NO:23所示的LCDR2和SEQ ID NO:26所示的LCDR3，或，
    所述重链可变区包含SEQ ID NO:7所示的HCDR1、SEQ ID NO:10所示的HCDR2和SEQ ID NO:13所示的HCDR3；所述轻链可变区包含SEQ ID NO:19所示的LCDR1、SEQ ID NO:21所示的LCDR2和SEQ ID NO:24所示的LCDR3；或，
    所述重链可变区包含SEQ ID NO:9所示的HCDR1、SEQ ID NO:12所示的 HCDR2和SEQ ID NO:15所示的HCDR3；所述轻链可变区包含SEQ ID NO:20所示的LCDR1、SEQ ID NO:23所示的LCDR2和SEQ ID NO:26所示的LCDR3。
  3. 如权利要求1或2所述的抗GPC3抗体或其抗原结合片段，其选自鼠源抗体或其或其抗原结合片段，嵌合抗体或其或其抗原结合片段，人抗体或其抗原结合片段，或者人源化抗体或其或其抗原结合片段。
  4. 如权利要求3所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段进一步包含源自人IgG1、IgG2、IgG3或IgG4的重链恒定区或其变体；
    优选地，所述抗GPC3抗体或其抗原结合片段进一步包含源自人IgG1、IgG2或IgG4的重链恒定区；
    进一步选地，所述抗GPC3抗体或其抗原结合片段进一步包含如SEQ ID NO：52所示的重链恒定区。
  5. 如权利要求3所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段进一步包含源自人κ链、λ链的轻链恒定区或其变体；
    优选地，所述抗GPC3抗体或其抗原结合片段进一步包含源自人κ链的轻链恒定区；
    进一步优选地，所述抗GPC3抗体或其抗原结合片段进一步包含如SEQ ID NO:53所示的轻链恒定区。
  6. 如权利要求3所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段包含选自如下序列所示的重链可变区，或与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区：SEQ ID NO:27，SEQ ID NO:29，SEQ ID NO:30，SEQ ID NO:31，SEQ ID NO:32或SEQ ID NO:37；
    和/或，选自如下序列所示的轻链可变区，或与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区：SEQ ID NO:28，SEQ ID NO:33，SEQ ID NO:34，SEQ ID NO:35，SEQ ID NO:36或SEQ ID NO:38。
  7. 如权利要求6所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段包含：
    SEQ ID NO：27所示的重链可变区和SEQ ID NO：28所示的轻链可变区；
    SEQ ID NO：29所示的重链可变区和SEQ ID NO：28所示的轻链可变区；
    SEQ ID NO：30所示的重链可变区和SEQ ID NO：28所示的轻链可变区；
    SEQ ID NO：31所示的重链可变区和SEQ ID NO：28所示的轻链可变区；
    SEQ ID NO：32所示的重链可变区和SEQ ID NO：33所示的轻链可变区；
    SEQ ID NO：32所示的重链可变区和SEQ ID NO：34所示的轻链可变区；
    SEQ ID NO：32所示的重链可变区和SEQ ID NO：35所示的轻链可变区；
    SEQ ID NO：32所示的重链可变区和SEQ ID NO：36所示的轻链可变区；或
    SEQ ID NO：37所示的重链可变区和SEQ ID NO：38所示的轻链可变区。
  8. 如权利要求6所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段含有选自如下序列所示的重链，或与以下序列相比具有至少80％，85％，90％，95％或99％同一性的重链：SEQ ID NO:39，SEQ ID NO:41，SEQ ID NO:42，SEQ ID NO:43，SEQ ID NO:44或SEQ ID NO:49；
    和/或，选自如下序列所示的轻链，或与以下序列相比具有至少80％，85％，90％，95％或99％同一性的轻链：SEQ ID NO:40，SEQ ID NO:45，SEQ ID NO:46，SEQ ID NO:47，SEQ ID NO:48或SEQ ID NO:50。
  9. 如权利要求8所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体包含：
    SEQ ID NO：39所示的重链和SEQ ID NO：40所示的轻链；
    SEQ ID NO：42所示的重链和SEQ ID NO：40所示的轻链；
    SEQ ID NO：43所示的重链和SEQ ID NO：40所示的轻链；
    SEQ ID NO：44所示的重链和SEQ ID NO：45所示的轻链；
    SEQ ID NO：44所示的重链和SEQ ID NO：46所示的轻链；
    SEQ ID NO：44所示的重链和SEQ ID NO：47所示的轻链；
    SEQ ID NO：44所示的重链和SEQ ID NO：48所示的轻链；或，
    SEQ ID NO：49所示的重链和SEQ ID NO：50所示的轻链。
  10. 一种多核苷酸，其编码权利要求1-9任一项所述的抗GPC3抗体或其抗原结合片段。
  11. 一种表达载体，其含有权利要求10所述的多核苷酸。
  12. 一种宿主细胞，其导入或含有权利要求11所述的表达载体。
  13. 如权利要求12所述的宿主细胞，其中所述的宿主细胞选自细菌、酵母菌或哺乳动物细胞；其中，细菌优选为大肠杆菌；酵母菌优选毕赤酵母；哺乳动物细胞优选CHO细胞或HEK293细胞。
  14. 一种生产抗GPC3抗体的方法，包括步骤：
    1)培养权利要求12-13中任一项所述的宿主细胞；
    2)从培养物中分离抗体；以及
    3)对所述抗体进行纯化。
  15. 一种药物组合物，其含有权利要求1-9任一项所述的抗GPC3抗体或其抗原结合片段、以及可药用的赋形剂、稀释剂或载体。
  16. 一种检测或诊断试剂盒，其含有权利要求1-9任一项所述的抗GPC3抗体或其抗原结合片段，任选地，还包含一种或多种检测所述抗GPC3抗体或其抗原结合片段与GPC3或其表位结合的试剂。
  17. 权利要求1-9任一项所述的抗GPC3抗体或其抗原结合片段或权利要求15所述的药物组合物在制备用于治疗或预防GPC3介导的疾病或病症的药物中的用途。
  18. 权利要求1-9任一项所述的抗GPC3抗体或其抗原结合片段或权利要求15所述的药物组合物在制备试剂盒中的用途，其中，所述试剂盒用于检测、诊断、预后GPC3介导的疾病或病症。
  19. 根据权利要求17或18所述的用途，其中：
    所述疾病或病症为癌症；
    优选地，所述疾病或病症为表达GPC3的癌症；
    更优选地，所述癌症选自乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤和黑色素瘤。
  20. 根据权利要求1-9任一项所述的抗GPC3抗体或其抗原结合片段或权利要求15所述的药物组合物，其用于治疗或预防GPC3介导的疾病；
    所述疾病选自乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤和黑色素瘤。
  21. 根据权利要求1-9任一项所述的抗GPC3抗体或其抗原结合片段或权利要求15所述的药物组合物，其用于检测、诊断、预后GPC3介导的疾病；
    所述疾病选自乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤和黑色素瘤，优选肝癌、黑色素瘤、卵巢癌、非小细胞肺癌、前列腺癌。
  22. 一种治疗或预防GPC3介导的疾病的方法，包括步骤：
    向受试者提供治疗有效量或预防有效量的权利要求1-9任一项所述的抗GPC3抗体或其抗原结合片段；或者
    向受试者提供治疗有效量或预防有效量的权利要求15所述的药物组合物；
    其中所述GPC3介导的疾病选自乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤和黑色素瘤。
PCT/CN2021/087549 2020-04-20 2021-04-15 抗体或其抗原结合片段、其制备方法及医药用途 WO2021213245A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202180021548.2A CN115298216A (zh) 2020-04-20 2021-04-15 抗体或其抗原结合片段、其制备方法及医药用途

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202010314338.X 2020-04-20
CN202010314338 2020-04-20
CN202010936093 2020-09-08
CN202010936093.4 2020-09-08

Publications (1)

Publication Number Publication Date
WO2021213245A1 true WO2021213245A1 (zh) 2021-10-28

Family

ID=78270294

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/087549 WO2021213245A1 (zh) 2020-04-20 2021-04-15 抗体或其抗原结合片段、其制备方法及医药用途

Country Status (3)

Country Link
CN (1) CN115298216A (zh)
TW (1) TW202144433A (zh)
WO (1) WO2021213245A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114478771A (zh) * 2021-12-14 2022-05-13 英诺湖医药(杭州)有限公司 Ox40抗体及其医药用途
WO2023246574A1 (zh) * 2022-06-24 2023-12-28 四川科伦博泰生物医药股份有限公司 靶向gpc3的抗体及其用途

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016036973A1 (en) * 2014-09-04 2016-03-10 The Trustees Of The University Of Pennsylvania Glypican-3 antibody and uses thereof
WO2016191675A2 (en) * 2015-05-27 2016-12-01 La Jolla Biologics, Inc. Antibodies against glypican-3 and their uses in cancer diagnosis and treatment
WO2017020812A1 (zh) * 2015-08-03 2017-02-09 科济生物医药（上海）有限公司 抗磷脂酰肌醇蛋白多糖-3的抗体及其应用
CN108164600A (zh) * 2016-12-07 2018-06-15 上海吉倍生物技术有限公司 一种抗gpc3抗体及其制备方法和用途
WO2019059411A1 (en) * 2017-09-20 2019-03-28 Chugai Seiyaku Kabushiki Kaisha DOSAGE FOR POLYTHERAPY USING PD-1 AXIS BINDING ANTAGONISTS AND GPC3 TARGETING AGENT

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016036973A1 (en) * 2014-09-04 2016-03-10 The Trustees Of The University Of Pennsylvania Glypican-3 antibody and uses thereof
WO2016191675A2 (en) * 2015-05-27 2016-12-01 La Jolla Biologics, Inc. Antibodies against glypican-3 and their uses in cancer diagnosis and treatment
WO2017020812A1 (zh) * 2015-08-03 2017-02-09 科济生物医药（上海）有限公司 抗磷脂酰肌醇蛋白多糖-3的抗体及其应用
CN108164600A (zh) * 2016-12-07 2018-06-15 上海吉倍生物技术有限公司 一种抗gpc3抗体及其制备方法和用途
WO2019059411A1 (en) * 2017-09-20 2019-03-28 Chugai Seiyaku Kabushiki Kaisha DOSAGE FOR POLYTHERAPY USING PD-1 AXIS BINDING ANTAGONISTS AND GPC3 TARGETING AGENT

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ABOU-ALFA GHASSAN K; PUIG OSCAR; DANIELE BRUNO; KUDO MASATOSHI; MERLE PHILIPPE; PARK JOONG-WON; ROSS PAUL; PERON JEAN-MARIE; EBERT: "Randomized Phase II Placebo Controlled Study of Codrituzumab in Previously Treated Patients with Advanced Hepatocellular Carcinoma", JOURNAL OF HEPATOLOGY, vol. 65, no. 2, 13 April 2016 (2016-04-13), pages 289 - 295, XP029642041, ISSN: 0168-8278, DOI: 10.1016/j.jhep.2016.04.004 *
FENG MINGQIAN , HO MITCHELL: "Glypican-3 Antibodies: A New Therapeutic Target for Liver Cancer", FEBS LETTERS, vol. 588, no. 2, 15 October 2013 (2013-10-15), NL, pages 377 - 382, XP028669969, ISSN: 0014-5793, DOI: 10.1016/j.febslet.2013.10.002 *
WANG SHENGHAO;MUHAMMAD KALIM;ZHAN JINBIAO: "The Structure and Function of GPC3 and Its Application in the Diagnosis and Treatment of Hepatocellular Carcinoma", CHINESE JOURNAL OF CELL BIOLOGY, vol. 40, no. 2, 29 January 2018 (2018-01-29), pages 275 - 281, XP055859827, DOI: 10.11844/cjcb.2018.02.0237 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114478771A (zh) * 2021-12-14 2022-05-13 英诺湖医药(杭州)有限公司 Ox40抗体及其医药用途
WO2023246574A1 (zh) * 2022-06-24 2023-12-28 四川科伦博泰生物医药股份有限公司 靶向gpc3的抗体及其用途

Also Published As

Publication number Publication date
TW202144433A (zh) 2021-12-01
CN115298216A (zh) 2022-11-04

Similar Documents

Publication Publication Date Title
CN110366560B (zh) 抗b7-h4抗体、其抗原结合片段及其医药用途
TWI673287B (zh) 抗b7-h3抗體、其抗原結合片段及其醫藥用途
WO2018177393A1 (zh) B7-h3抗体、其抗原结合片段及其医药用途
WO2018219327A1 (zh) 抗cd40抗体、其抗原结合片段及其医药用途
WO2019141268A1 (zh) 抗4-1bb抗体、其抗原结合片段及其医药用途
WO2020228604A1 (zh) 抗trop-2抗体、其抗原结合片段及其医药用途
WO2019184935A1 (zh) 抗cd27抗体、其抗原结合片段及其医药用途
WO2021213245A1 (zh) 抗体或其抗原结合片段、其制备方法及医药用途
WO2021110095A1 (zh) 抗gpc3抗体、其抗原结合片段及其医药用途
TWI685504B (zh) 抗gitr抗體、其抗原結合片段及其醫藥用途
WO2021018168A1 (zh) 抗bcma抗体、其抗原结合片段及其医药用途
WO2019238074A1 (zh) 一种高亲和力高生物活性的lag-3抗体及其应用
WO2022078490A1 (zh) 抗erbb3抗体或其抗原结合片段及其医药用途
WO2021209066A1 (zh) 特异性抗原结合分子，其制备方法及医药用途
TWI836070B (zh) 抗trop-2抗體、其抗原結合片段及其醫藥用途
TWI840399B (zh) 結合人il-4r的抗體、其抗原結合片段及其醫藥用途
WO2021160153A1 (zh) 一种靶向人cd47的单域抗体及其用途
WO2022078424A1 (zh) 抗trop-2抗体、其抗原结合片段或其突变体、及其医药用途
WO2023198138A1 (zh) 抗体或其抗原结合片段及其医药用途
WO2022144025A1 (zh) 一种抗erbb3受体的抗体或其抗原结合片段及其医药用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21792918

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21792918

Country of ref document: EP

Kind code of ref document: A1