WO2023246574A1 - Anticorps ciblant gpc3 et son utilisation - Google Patents

Anticorps ciblant gpc3 et son utilisation Download PDF

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WO2023246574A1
WO2023246574A1 PCT/CN2023/100096 CN2023100096W WO2023246574A1 WO 2023246574 A1 WO2023246574 A1 WO 2023246574A1 CN 2023100096 W CN2023100096 W CN 2023100096W WO 2023246574 A1 WO2023246574 A1 WO 2023246574A1
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seq
cdr
variant
sequence
variable region
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PCT/CN2023/100096
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English (en)
Chinese (zh)
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田海军
刘雪松
周阳
宋婷玉
蔡珍珍
王江漫
姜琳
葛均友
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四川科伦博泰生物医药股份有限公司
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Publication of WO2023246574A1 publication Critical patent/WO2023246574A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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

Definitions

  • the present application relates to the field of biomedicine. Specifically, the present application relates to antibodies or antigen-binding fragments thereof that specifically bind to GPC3.
  • the present invention also relates to the use of these antibodies or antigen-binding fragments thereof for the prevention and/or treatment of diseases related to the expression of GPC3, such as liver cancer, melanoma, ovarian cancer and other cancers, and the prevention and/or treatment of liver cancer, melanoma, ovarian cancer and other methods for GPC3-positive tumors.
  • Glypican-3 (Glypican-3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS and SGBS1) is a member of the heparan sulfate proteoglycan family. Phosphatidylinositol is anchored on the cell surface and is one of the representative liver cancer markers in current preclinical research. GPC3 is expressed in many human malignant tumor cells and serum, including hepatocellular carcinoma (HCC), melanoma and ovarian clear cell carcinoma, and is rarely expressed in other cancers and normal tissues. GPC3 is a potential biomarker for HCC. It forms a complex with WNT, activates downstream signaling pathways, promotes the proliferation of liver cancer cells, and participates in the regulation of multiple signaling pathways closely related to tumor occurrence and development.
  • HCC hepatocellular carcinoma
  • melanoma melanoma and ovarian clear cell carcinoma
  • liver cancer is the fourth most common malignant tumor and the third leading cause of cancer death in my country, seriously threatening the lives and health of our people.
  • a large number of patients with hepatocellular carcinoma still lack precise and effective clinical treatments.
  • Most liver cancer patients are already in the advanced or late stage when diagnosed. Only 30% of patients have the opportunity for surgical resection.
  • the metastasis and recurrence rate within 5 years after resection is as high as 60% to 70%.
  • the overall 5-year survival rate is low, only 7% to 10%. .
  • GPC3-positive hepatocellular carcinoma HCC
  • melanoma melanoma
  • ovarian clear cell carcinoma GPC3-positive hepatocellular carcinoma
  • the inventor developed a fully human antibody with low immunogenicity and high specificity for GPC3 that can specifically recognize/bind GPC3, which has the potential to prevent and/or treat diseases related to the expression of GPC3, such as liver cancer. , melanoma, ovarian cancer and other cancers, and has great clinical value.
  • the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to GPC3, wherein the antibody or an antigen-binding fragment thereof includes the following complementarity determining regions (CDRs):
  • CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:9; and/or, the variable light chain shown in SEQ ID NO:10 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
  • At least one CDR contains a mutation, which is a substitution, deletion or addition of one or several amino acids (for example, a substitution, deletion or addition of 1, 2 or 3 amino acids).
  • the substitutions are conservative substitutions.
  • the CDRs are defined according to the IMGT, Kabat, Chothia or AbM numbering system.
  • the antibodies of the invention or antigen-binding fragments thereof comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 whose sequence is SEQ ID NO: 26 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 27 or a variant thereof; The sequence is CDR-H3 of SEQ ID NO: 73 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: the sequence is SEQ ID CDR-L1 of NO: 74 or a variant thereof; CDR-L2 whose sequence is SEQ ID NO: 30 or a variant thereof; CDR-L3 whose sequence is SEQ ID NO: 75 or a variant thereof;
  • the variant described in any one of (1a)-(1f) has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids) compared to the sequence from which it is derived. substitution, deletion or addition). In certain embodiments, the substitutions are conservative substitutions.
  • the antibodies of the invention or antigen-binding fragments thereof comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined by the IMGT numbering system:
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 20 or a variant thereof
  • CDR-H3 whose sequence is SEQ ID NO: 21 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 33 or a variant thereof
  • CDR-H3 with the sequence SEQ ID NO: 34 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 46 or a variant thereof
  • CDR-H3 whose sequence is SEQ ID NO: 47 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 33 or a variant thereof
  • CDR-H3 with the sequence SEQ ID NO: 56 or a variant thereof
  • VL light chain variable region
  • a heavy chain variable region comprising the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 66 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 67 or a variant thereof; The sequence is SEQ ID NO: 68 or its CDR-H3 of the variant; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 69 or a variant thereof; whose sequence is SEQ ID NO: 70 or CDR-L2 of a variant thereof; CDR-L3 whose sequence is SEQ ID NO: 65 or a variant thereof; or,
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 33 or a variant thereof
  • CDR-H3 with the sequence SEQ ID NO: 76 or a variant thereof
  • VL light chain variable region
  • the variant described in any one of (2a)-(2f) has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids) compared to the sequence from which it is derived. Substitutions, deletions, or additions); in certain embodiments, the substitutions are conservative substitutions.
  • the antibodies of the invention or antigen-binding fragments thereof comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 25 or a variant thereof
  • CDR-H3 with the sequence SEQ ID NO: 15 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 38 or a variant thereof
  • CDR-H3 whose sequence is SEQ ID NO: 28 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 51 or a variant thereof
  • CDR-H3 whose sequence is SEQ ID NO: 41 or a variant thereof
  • VL light chain variable region
  • VH Heavy chain variable region containing the following 3 CDRs: the sequence is SEQ ID NO: 37 or a variant thereof CDR-H1; CDR-H2 whose sequence is SEQ ID NO: 38 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 52 or a variant thereof; and/or a light chain comprising the following 3 CDRs may Variable region (VL): CDR-L1 whose sequence is SEQ ID NO: 53 or a variant thereof; CDR-L2 whose sequence is SEQ ID NO: 54 or a variant thereof; CDR-L2 whose sequence is SEQ ID NO: 55 or a variant thereof CDR-L3;
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 72 or a variant thereof
  • CDR-H3 whose sequence is SEQ ID NO: 62 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 38 or a variant thereof
  • CDR-H3 with the sequence SEQ ID NO: 73 or a variant thereof
  • VL light chain variable region
  • the variant described in any one of (3a)-(3f) has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids) compared to the sequence from which it is derived. substitution, deletion or addition). In certain embodiments, the substitutions are conservative substitutions.
  • the antibodies of the invention or antigen-binding fragments thereof comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system:
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 81 or a variant thereof
  • CDR-H3 with the sequence SEQ ID NO: 15 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 79 or a variant thereof
  • CDR-H3 whose sequence is SEQ ID NO: 28 or a variant thereof
  • VL light chain variable region
  • VH Heavy chain variable region containing the following 3 CDRs: the sequence is SEQ ID NO: 96 or a variant thereof CDR-H1; a CDR-H2 whose sequence is SEQ ID NO: 97 or a variant thereof; a CDR-H3 whose sequence is SEQ ID NO: 41 or a variant thereof; and/or a light chain comprising the following 3 CDRs may Variable region (VL): CDR-L1 whose sequence is SEQ ID NO: 42 or a variant thereof; CDR-L2 whose sequence is SEQ ID NO: 43 or a variant thereof; CDR-L2 whose sequence is SEQ ID NO: 44 or a variant thereof CDR-L3;
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 79 or a variant thereof
  • CDR-H3 with the sequence SEQ ID NO: 73 or a variant thereof
  • VL light chain variable region
  • the variant described in any one of (4a)-(4f) has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids) compared to the sequence from which it is derived. substitution, deletion or addition). In certain embodiments, the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof includes framework regions (FRs) from human immunoglobulins.
  • the antibodies or antigen-binding fragments thereof of the invention comprise:
  • VH comprising the sequence shown in SEQ ID NO:1 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:2 or a variant thereof;
  • VH comprising the sequence shown in SEQ ID NO:3 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:4 or a variant thereof;
  • VH comprising a sequence as set forth in SEQ ID NO:5 or a variant thereof and/or a VH comprising a sequence as set forth in SEQ ID NO:6 VL of the sequence shown or a variant thereof;
  • VH comprising the sequence shown in SEQ ID NO:7 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:8 or a variant thereof;
  • VH comprising the sequence shown in SEQ ID NO:9 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:10 or a variant thereof;
  • VH comprising the sequence shown in SEQ ID NO:11 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:12 or a variant thereof;
  • said variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100% sequence identity, or having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, substitution, deletion or addition of 3, 4 or 5 amino acids). In certain embodiments, the substitutions are conservative substitutions.
  • the antibodies of the invention are murine antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.
  • the antibodies of the invention or antigen-binding fragments thereof are selected from the group consisting of full-length antibodies, single-chain antibodies (e.g., scFv, di-scFv, or (scFv) 2 ), Fab, Fab', Fab'-SH, (Fab') 2 , F(ab)' 3 fragment, Fv fragment, minibody, disulfide-linked Fv (dsFv), single domain antibody (sdAb, nanobody), diabody, bispecific Antibodies and multispecific antibodies.
  • single-chain antibodies e.g., scFv, di-scFv, or (scFv) 2
  • Fab Fab', Fab'-SH, (Fab') 2 , F(ab)' 3 fragment, Fv fragment, minibody, disulfide-linked Fv (dsFv), single domain antibody (sdAb, nanobody), diabody, bispecific Antibodies and multispecific antibodies.
  • the VH and VL of an antibody of the invention, or antigen-binding fragment thereof are linked by one or more linkers.
  • the linker is typically a peptide linker, for example a flexible and/or soluble peptide linker, for example a glycine, serine and/or threonine rich peptide linker.
  • the linker also includes charged residues (such as lysine and/or glutamic acid), which can improve solubility.
  • the linker further includes one or more prolines.
  • the linker comprises one or several (eg, 1, 2, or 3) sequences represented by (GmS)n, where m is selected from an integer of 1-6 and n is selected from An integer of 1-6; preferably, m is 3, 4, or 5; preferably, n is 1 or 2.
  • the linker has the sequence of SEQ ID NO: 110.
  • the antibodies of the invention or antigen-binding fragments thereof are single chain antibodies, e.g., scFv, di- scFv or (scFv) 2 .
  • the single-chain antibody sequentially includes from its N-terminus to its C-terminus:
  • VH-linker comprising the sequence shown in SEQ ID NO:1 or its variant - VL comprising the sequence shown in SEQ ID NO:2 or its variant;
  • VH-linker comprising the sequence shown in SEQ ID NO:3 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:4 or a variant thereof;
  • VH-linker comprising the sequence shown in SEQ ID NO:5 or its variant - VL comprising the sequence shown in SEQ ID NO:6 or its variant;
  • VH-linker comprising the sequence shown in SEQ ID NO:7 or its variant - VL comprising the sequence shown in SEQ ID NO:8 or its variant;
  • a VH-linker comprising the sequence shown in SEQ ID NO:9 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:10 or a variant thereof;
  • VH-linker comprising the sequence shown in SEQ ID NO: 11 or a variant thereof - VL comprising a sequence shown in SEQ ID NO: 12 or a variant thereof;
  • VL-linker comprising the sequence shown in SEQ ID NO:2 or its variant - VH comprising the sequence shown in SEQ ID NO:1 or its variant;
  • VL-linker comprising the sequence shown in SEQ ID NO:4 or its variant - VH comprising the sequence shown in SEQ ID NO:3 or its variant;
  • VL-linker comprising the sequence shown in SEQ ID NO:6 or its variant - VH comprising the sequence shown in SEQ ID NO:5 or its variant;
  • VL-linker comprising the sequence shown in SEQ ID NO:8 or a variant thereof - VH comprising a sequence shown in SEQ ID NO:7 or a variant thereof;
  • a VL-linker comprising the sequence shown in SEQ ID NO:10 or a variant thereof - a VH comprising a sequence shown in SEQ ID NO:9 or a variant thereof; or
  • VL-linker comprising the sequence shown in SEQ ID NO:12 or its variant - VH comprising the sequence shown in SEQ ID NO:11 or its variant;
  • said variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100% sequence identity, or having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, Substitution, deletion or deletion of 3, 4 or 5 amino acids Add to). In certain embodiments, the substitutions are conservative substitutions.
  • the single-chain antibody comprises an amino acid sequence selected from the following: (1) the amino acid sequence shown in any one of SEQ ID NOs: 86, 88, 90, 92, 94, and 82; (2) Compared with the amino acid sequence shown in any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82, it has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91% , a sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; or (3) with SEQ ID NOs: Compared with the amino acid sequence shown in any one of 86, 88, 90, 92, 94 and 82, one or several amino acids are substituted, deleted or added (for example, 1, 2, 3, 4, 5, Substitution, deletion or addition of 6, 7, 8, 9, or 10 amino acids). In certain embodiments, the substitutions are conservative
  • the antibodies of the invention, or antigen-binding fragments thereof further comprise a constant region derived from a human immunoglobulin.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4), the antibody or antigen-binding fragment thereof
  • the light chain includes a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises the heavy chain constant region (CH) of a human immunoglobulin or a variant thereof as compared to the wild-type sequence from which it is derived.
  • CH heavy chain constant region
  • amino acids e.g., up to 20, up to 15, up to 10, or up to 5 substitutions, deletions, or additions; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; and/or,
  • the light chain of the antibody or antigen-binding fragment thereof comprises the light chain constant region (CL) of a human immunoglobulin or a variant thereof that has one or more amino acid differences compared to the wild-type sequence from which it is derived.
  • CL light chain constant region
  • Substitutions, deletions or additions e.g., substitutions, deletions, or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., substitutions of 1, 2, 3, 4 or 5 amino acids , missing or added).
  • the heavy chain constant region is an IgG, IgM, IgE, IgD or IgA heavy chain constant region.
  • the heavy chain constant region is an IgG heavy chain constant region, such as an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region.
  • the light chain constant region is a kappa or lambda light chain constant region. In certain preferred embodiments, the light chain constant region is a human kappa light chain constant region.
  • the antibodies of the invention or antigen-binding fragments thereof may be derivatized, for example linked to another molecule (e.g. another a polypeptide or protein).
  • another molecule e.g. another a polypeptide or protein.
  • derivatization e.g, labeling
  • the antibodies or antigen-binding fragments thereof of the invention are also intended to include such derivatized forms.
  • an antibody of the invention or an antigen-binding fragment thereof can be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming Bispecific antibodies), detection reagents, pharmaceutical reagents, and/or proteins or polypeptides capable of mediating the binding of an antibody or antigen-binding fragment to another molecule (e.g., avidin or polyhistidine tags).
  • another antibody e.g., forming Bispecific antibodies
  • detection reagents e.g., pharmaceutical reagents, and/or proteins or polypeptides capable of mediating the binding of an antibody or antigen-binding fragment to another molecule (e.g., avidin or polyhistidine tags).
  • the present invention provides a conjugate, which includes the antibody of the present invention or its antigen-binding fragment and a coupling part.
  • the coupling moiety is selected from detectable labels.
  • the detectable label of the present invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means.
  • labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides fluorescein (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin ( PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g.
  • enzymes e.g., horseradish peroxidas
  • such labels can be adapted for immunological detection (eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.).
  • the detectable label is selected from a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.
  • detectable labels as described above can be linked to the antibodies or antigen-binding fragments thereof of the invention via linkers of varying lengths to reduce potential steric hindrance.
  • the coupling moiety is selected from therapeutic agents.
  • the therapeutic agent is preferably an antineoplastic agent, such as a cytotoxic agent, cytokine, toxin, or radionuclide.
  • the coupling moiety is selected from substances capable of improving the biological properties of the antibody (e.g., increasing serum half-life), for example, may be a chemical group such as polyethylene glycol (PEG), methyl, or ethylene glycol. base, or sugar base.
  • PEG polyethylene glycol
  • methyl methyl
  • ethylene glycol. base or sugar base.
  • the present invention provides a multispecific antibody, which contains the antibody of the present invention or its antigen-binding fragment.
  • the multispecific antibody comprises an antibody of the invention, or an antigen-binding fragment thereof, as a first antigen-binding domain, and further comprises at least one second antigen-binding domain directed against another target.
  • each antigen-binding domain of the multispecific antibody retains its respective original binding specificity.
  • the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.
  • the antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology.
  • DNA molecules encoding the heavy chain and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibody of the invention.
  • the antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be produced directly from recombinant host cells (Reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000 )).
  • Fab′ fragments can be obtained directly from host cells; Fab′ fragments can be chemically coupled to form F(ab′) 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from the recombinant host cell culture medium. Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.
  • a second aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody of the invention or an antigen-binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof.
  • the isolated nucleic acid molecule comprises a nucleotide sequence selected from:
  • the nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO: 100, (ii) a sequence substantially identical to SEQ ID NO: 100 (for example, A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO: 100, or a sequence having one or more nucleotide substitutions), or (iii )
  • the degenerate sequence of (i) or (ii) above; and/or, the nucleic acid molecule encoding the antibody light chain variable region includes: (iv) the nucleotide sequence shown in SEQ ID NO: 101, (v) ) A sequence that is substantially identical to SEQ ID NO: 101 (e.g., a sequence that has at least about 85%, 90%, 95%, 99% or greater sequence identity compared to SEQ ID NO: 101, or has a or more nucleotide substitutions), or (vi) the above (iv) or (
  • the nucleic acid molecule encoding the variable region of the antibody heavy chain includes: (i) the nucleotide sequence shown in SEQ ID NO: 102, (ii) a sequence that is substantially the same as SEQ ID NO: 102 (for example, A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO: 102, or a sequence having one or more nucleotide substitutions), or (iii ) The degenerate sequence of (i) or (ii) above; and/or, the nucleic acid molecule encoding the antibody light chain variable region includes: (iv) the nucleotide sequence shown in SEQ ID NO: 103, (v) ) A sequence that is substantially identical to SEQ ID NO:103 (e.g., a sequence that has at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO:103, or has a or more nucleotide substitutions), or (
  • the nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO: 104, (ii) a sequence that is substantially the same as SEQ ID NO: 104 (for example, A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO: 104, or a sequence having one or more nucleotide substitutions), or (iii )
  • the degenerate sequence of the above (i) or (ii); and/or, the nucleic acid molecule encoding the antibody light chain variable region includes: (iv) the nucleotide sequence shown in SEQ ID NO: 105, (v) ) A sequence that is substantially identical to SEQ ID NO:105 (e.g., a sequence that has at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO:105, or has a or more nucleotide substitutions), or (vi
  • the nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO: 106, (ii) a sequence that is substantially the same as SEQ ID NO: 106 (for example, A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO: 106, or a sequence having one or more nucleotide substitutions), or (iii ) The degenerate sequence of (i) or (ii) above; and/or, the nucleic acid molecule encoding the antibody light chain variable region includes: (iv) the nucleotide sequence shown in SEQ ID NO: 107, (v) ) A sequence that is substantially identical to SEQ ID NO:107 (e.g., a sequence that has at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO:107, or has a or more nucleotide substitutions), or (vi)
  • the nucleic acid molecule encoding the variable region of the antibody heavy chain comprises: (i) the nucleotide sequence shown in SEQ ID NO:108 Column, (ii) a sequence that is substantially identical to SEQ ID NO: 108 (e.g., a sequence that has at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO: 108 , or a sequence with one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above; and/or the nucleic acid molecule encoding the antibody light chain variable region comprises (iv) a nucleotide sequence represented by SEQ ID NO:109, (v) a sequence substantially identical to SEQ ID NO:109 (e.g., having at least about 85%, 90 %, 95%, 99% or higher sequence identity, or a sequence with one or more nucleotide substitutions), or (vi) a degenerate sequence of (
  • the nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO:84, (ii) a sequence that is substantially the same as SEQ ID NO:84 (e.g., A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO: 84, or a sequence having one or more nucleotide substitutions), or (iii )
  • the degenerate sequence of the above (i) or (ii); and/or, the nucleic acid molecule encoding the antibody light chain variable region includes: (iv) the nucleotide sequence shown in SEQ ID NO: 85, (v) ) A sequence that is substantially identical to SEQ ID NO:85 (e.g., a sequence that has at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO:85, or has a or more nucleotide substitutions), or (vi)
  • the isolated nucleic acid molecule comprises a nucleotide sequence selected from: (1) a nucleotide sequence represented by any one of SEQ ID NO: 87, 89, 91, 93, 95 and 83 Sequence; (2) Compared with the nucleotide sequence shown in any one of SEQ ID NO: 87, 89, 91, 93, 95 and 83, it has at least 50%, at least 55%, at least 60%, at least 65%, At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98 %, at least 99%, or 100% sequence identity.
  • a third aspect of the invention provides a vector (eg a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above.
  • vectors of the invention are, for example, DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR/Cas9 vectors, or viral vectors.
  • the vector is an expression vector.
  • the vector is an episomal vector.
  • the vector is a viral vector.
  • the viral vector is a lentiviral vector, an adenoviral vector, or a retroviral vector.
  • a fourth aspect of the invention provides a host cell comprising an isolated nucleic acid molecule or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • the present invention also relates to a method for preparing the antibody or antigen-binding fragment thereof of the present invention, which includes culturing the host cell as described above under conditions that allow the expression of the antibody or antigen-binding fragment thereof, and from The antibody or antigen-binding fragment thereof is recovered from the cultured host cell culture.
  • the present invention relates to a CAR targeting GPC3, the characteristics of which include non-MHC restricted GPC3 recognition ability, which confer an immune cell (for example, T cell, NK cell, monocyte, macrophage or dendritic cell) expressing the CAR The ability to recognize GPC3-expressing cells (such as tumor cells) independent of antigen processing and presentation.
  • an immune cell for example, T cell, NK cell, monocyte, macrophage or dendritic cell
  • the fifth aspect of the present invention provides a chimeric antigen receptor (CAR) capable of specifically binding to GPC3, which includes an extracellular antigen-binding domain (anti-GPC3 binding domain), a spacer domain, and a transmembrane domain. and intracellular signaling domains.
  • CAR chimeric antigen receptor
  • the antigen-binding domain contained in the chimeric antigen receptor of the present invention confers the ability of the CAR to recognize GPC3.
  • the antigen binding domain comprises an anti-GPC3 binding domain comprising an antibody or antigen-binding fragment thereof capable of specifically binding to GPC3 (eg, human GPC3).
  • the antibody or antigen-binding fragment thereof is selected from the antibodies or antigen-binding fragments thereof of the first aspect.
  • the antibody or antigen-binding fragment thereof is a single chain antibody, such as scFv, di-scFv, or (scFv) 2 .
  • the VH and VL of the antibody or antigen-binding fragment thereof are linked by a linker.
  • the linker comprises one or several (eg, 1, 2, or 3) sequences represented by (G m S) n , where m is selected from an integer from 1 to 6, n An integer selected from 1-6. In certain embodiments, m is 3, 4, or 5. In certain embodiments, n is 1 or 2. In certain embodiments, the linker has the sequence of SEQ ID NO: 110.
  • the antigen-binding domain comprises an amino acid sequence selected from the following: (1) the amino acid sequence shown in any one of SEQ ID NOs: 86, 88, 90, 92, 94, and 82; (2) ) has at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical Sequence; or (3) Compared with the amino acid sequence shown in any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82, it has one or several amino acid substitutions, deletions or additions (for example, 1, 2 substitution, deletion or addition of 1, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). In certain embodiments, the substitutions are conservative substitutions.
  • the transmembrane domain contained in the chimeric antigen receptor of the present invention can be any protein structure known in the art, as long as it can be thermodynamically stable in the cell membrane (especially the eukaryotic cell membrane).
  • the transmembrane domains of CARs suitable for use in the present invention can be derived from natural sources.
  • the transmembrane domain may be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain may be a synthetic non-naturally occurring protein segment, such as a protein segment containing primarily hydrophobic residues such as leucine and valine.
  • the transmembrane domain is selected from the transmembrane region of the following proteins: alpha, beta or zeta chain of T cell receptor, CD28, CD45, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD-1, and any combination thereof.
  • the transmembrane domain is selected from the transmembrane regions of CD8, CD4, PD-1, CD152 and CD154.
  • the transmembrane domain comprises the transmembrane region of CD8.
  • the transmembrane domain comprises the CD8 transmembrane region whose sequence is shown in SEQ ID NO: 111.
  • the chimeric antigen receptor of the present invention includes a spacer domain located between the extracellular antigen-binding domain and the transmembrane domain.
  • the spacer domain comprises the CH2 and CH3 regions of an immunoglobulin (eg, IgG1 or IgG4).
  • an immunoglobulin eg, IgG1 or IgG4
  • CH2 and CH3 extend the antigen-binding domain of the CAR away from the membrane of the CAR-expressing cell and more accurately mimic the size and domain structure of the native TCR structure.
  • the spacer domain comprises a hinge domain.
  • a hinge domain may be a stretch of amino acids typically found between two domains of a protein that may allow flexibility of the protein and movement of one or both domains relative to each other. Therefore, the hinge domain can be any amino acid sequence as long as it This flexibility of the extracellular antigen binding domain and its mobility relative to the transmembrane domain can be provided.
  • the hinge domain is the hinge region of a naturally occurring protein, or a portion thereof.
  • the hinge domain comprises the hinge region of CD8, or a portion thereof, eg, a fragment containing at least 15 (eg, 20, 25, 30, 35, or 40) contiguous amino acids of the hinge region of CD8.
  • the hinge domain comprises the hinge region of CD8, IgG4, PD-1, CD152 or CD154.
  • the spacer domain comprises the amino acid sequence set forth in SEQ ID NO: 112.
  • the CAR of the invention may further comprise a signal peptide at its N-terminus.
  • a signal peptide is a polypeptide sequence that targets the sequence to which it is linked to a desired site.
  • the signal peptide can target the CAR to which it is linked to the secretory pathway of the cell and allow further integration and anchoring of the CAR into the lipid bilayer.
  • Signal peptides useful for CARs are known to those skilled in the art.
  • the signal peptide comprises a heavy chain signal peptide (eg, a heavy chain signal peptide of IgG1), a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide, an IL2 signal peptide, or CD8 ⁇ signal peptide.
  • the signal peptide is selected from the group consisting of CD8 ⁇ signal peptides.
  • the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 116.
  • the CARs of the invention are co-expressed with additional biologically active molecules.
  • the additional bioactive molecule may have its own proprietary signal peptide, which is named signal peptide-2 to distinguish it from the signal peptide in the previous paragraph.
  • Signal peptide-2 guides the transport of additional bioactive molecules to specific sites within the cell or outside the cell membrane.
  • the signal peptide-2 may be the same as or different from the signal peptide described in the previous paragraph.
  • the signal peptide-2 may be different from the signal peptide described in the previous paragraph.
  • the signal peptide-2 is an IL2 signal peptide (e.g., the amino acid sequence is set forth in SEQ ID NO: 129).
  • the intracellular signaling domain contained in the CAR of the present invention is involved in transmitting the signal generated by the combination of the CAR of the present invention and GPC3 into the immune effector cells, activating at least one normal effector function of the immune effector cells expressing the CAR , or enhance the secretion of at least one cytokine (e.g., IL-2, IFN- ⁇ ) by CAR-expressing immune effector cells.
  • cytokine e.g., IL-2, IFN- ⁇
  • the intracellular signaling domain comprises a primary signaling domain and/or a costimulatory signaling domain.
  • the primary signaling domain can be any intracellular signaling domain comprising an immunoreceptor tyrosine activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an immunoreceptor tyrosine activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an intracellular signaling domain of a protein selected from CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CDS, CD22, CD79a, CD79b, or CD66d. In certain embodiments, the primary signaling domain comprises the intracellular signaling domain of CD3 ⁇ .
  • the costimulatory signaling domain can be an intracellular signaling domain from a costimulatory molecule.
  • the costimulatory signaling domain comprises an intracellular signaling domain of a protein selected from: CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4- 1BB), CD150(SLAMF1), CD270(HVEM), CD278(ICOS) or DAP10.
  • the costimulatory signaling domain is selected from the intracellular signaling domain of CD28, or the intracellular signaling domain of CD137(4-1BB), or a combination of fragments thereof.
  • the intracellular signaling domain comprises a costimulatory signaling domain. In certain embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains. In such embodiments, the two or more costimulatory signaling domains may be the same or different.
  • the intracellular signaling domain includes a primary signaling domain and at least one costimulatory signaling domain.
  • the primary signaling domain and the at least one costimulatory signaling domain can be concatenated in any order to the carboxyl terminus of the transmembrane domain.
  • the intracellular signaling domain can comprise the intracellular signaling domain of CD3 ⁇ and the intracellular signaling domain of CD137.
  • the intracellular signaling domain of CD3 ⁇ comprises the amino acid sequence set forth in SEQ ID NO: 113.
  • the intracellular signaling domain of CD137 comprises the amino acid sequence set forth in SEQ ID NO: 114.
  • the intracellular signaling domain of the chimeric antigen receptor has the sequence set forth in SEQ ID NO: 115.
  • the invention provides a chimeric antigen receptor that can specifically bind to GPC3.
  • the chimeric antigen receptor sequentially includes an antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signal from its N-terminus to its C-terminus. conductive domain.
  • the intracellular signaling domain from N-terminus to C-terminus is costimulatory signaling domain and primary signaling domain.
  • the signal peptide comprises a heavy chain signal peptide of IgG1 or a CD8 ⁇ signal peptide (e.g., a signal peptide of the sequence set forth in SEQ ID NO: 116).
  • the spacer domain comprises the hinge region of CD8 (e.g., CD8 ⁇ ) (e.g., the hinge region of the sequence set forth in SEQ ID NO: 112).
  • the transmembrane domain comprises the transmembrane region of CD8 (e.g., CD8 ⁇ ) (e.g., the transmembrane region of the sequence set forth in SEQ ID NO: 111).
  • the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises an intracellular signaling domain of CD3 ⁇ (e.g., as The sequence shown in SEQ ID NO: 113), the costimulatory signaling domain includes the intracellular signaling domain of CD137 (4-1BB) (for example, the sequence shown in SEQ ID NO: 114).
  • the intracellular signaling domain of the chimeric antigen receptor has the sequence set forth in SEQ ID NO: 115.
  • the chimeric antigen receptor includes the signal peptide, antigen-binding domain, spacer domain, transmembrane domain, and intracellular signaling domain in order from its N-terminus to its C-terminus. (From N-terminus to C-terminus are the costimulatory signaling domain and the primary signaling domain).
  • the signal peptide comprises the heavy chain signal peptide of IgG1 or the CD8 ⁇ signal peptide. In certain exemplary embodiments, the signal peptide comprises a CD8 ⁇ signal peptide having the sequence set forth in SEQ ID NO: 116.
  • the CAR of the invention comprises an amino acid sequence selected from:
  • Methods of generating chimeric antigen receptors and immune effector cells comprising the chimeric antigen receptors are known in the art and may include transfecting the cells with at least one polynucleotide encoding a CAR and in Polynucleotides are expressed in cells.
  • a nucleic acid molecule encoding a CAR of the invention can be included in an expression vector (eg, a lentiviral vector) capable of expression in a host cell, such as a T cell, to produce the CAR.
  • the sixth aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor according to the fifth aspect.
  • nucleotide sequence encoding a chimeric antigen receptor of the invention can have a variety of different sequences. Therefore, unless otherwise stated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence.
  • the seventh aspect of the present invention also provides a nucleic acid construct comprising a nucleic acid sequence encoding the chimeric antigen receptor described in the fifth aspect.
  • the eighth aspect of the present invention provides a vector comprising the isolated nucleic acid molecule described in the sixth aspect, or the nucleic acid construct described in the seventh aspect.
  • the vector is selected from the group consisting of DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR/Cas9 vectors, viral vectors.
  • the vector is an expression vector.
  • the vector is an episomal vector.
  • the vector is a viral vector.
  • the viral vector is a lentiviral vector, an adenoviral vector, or a retroviral vector.
  • the vector is an episomal or non-integrating viral vector, such as an integration-deficient retrovirus or lentivirus.
  • a ninth aspect of the present invention provides a host cell comprising the isolated nucleic acid molecule described in the sixth aspect, the nucleic acid construct described in the seventh aspect, or the vector described in the eighth aspect.
  • the vectors as described above can be introduced into host cells by various suitable means, such as calcium phosphate transfection, DEAE-dextran mediated transfection, Microinjection, electroporation, TALEN method, ZFN method, non-viral vector-mediated transfection (such as liposome) or viral vector-mediated transfection (such as lentiviral infection, retroviral infection, adenoviral infection), and other physical, chemical or biological means for transfer into host cells, such as transposon technology, CRISPR-Cas9 and other technologies.
  • the host cell comprises the isolated nucleic acid molecule of the sixth aspect or a vector comprising the nucleic acid molecule, and the host cell expresses the chimeric antigen receptor of the invention.
  • the host cell comprises the nucleic acid construct of the seventh aspect or a vector comprising the nucleic acid construct, the host cell expresses the chimeric antigen receptor of the invention and additional biologically active molecules .
  • the host cells are selected from mammalian (eg, human) immune cells.
  • the immune cells are derived from a patient or healthy donor.
  • the immune cells are selected from T lymphocytes, natural killer (NK) cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably, the immune cells are derived from T lymphocytes or NK cells.
  • the tenth aspect of the present invention provides a method for preparing cells expressing the chimeric antigen receptor of the present invention, which includes: (1) providing a host cell; (2) converting the isolated nucleic acid molecule as described in the sixth aspect or containing the The vector of the nucleic acid molecule is introduced into the host cell to obtain a host cell capable of expressing the chimeric antigen receptor.
  • Also provided is a method for cells that co-express the chimeric antigen receptor of the present invention and other biologically active molecules which includes: (1) providing a host cell; (2) converting the nucleic acid construct described in the seventh aspect or containing the The vector of the nucleic acid construct is introduced into the host cell to obtain a host cell capable of co-expressing the chimeric antigen receptor and other biologically active molecules.
  • the host cells are selected from immune cells, such as T lymphocytes, NK cells, monocytes, dendritic cells, macrophages, and any combination thereof.
  • the immune cells are selected from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination of these cells.
  • the immune cells are provided from a patient or a healthy donor and undergo pretreatment; the pretreatment includes sorting, activation and/or proliferation of immune cells;
  • the pretreating includes contacting the immune cells with an anti-CD3 antibody and an anti-CD28 antibody, thereby stimulating the immune cells and inducing their proliferation, thereby generating pretreated immune cells.
  • the nucleic acid molecule or vector in step (2), is introduced into the immune cell via viral infection. In some embodiments, in step (2), the nucleic acid molecule or vector is introduced into the immune cell by non-viral vector transfection, such as through transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method, Electric penetration well method, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection.
  • non-viral vector transfection such as through transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method, Electric penetration well method, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection.
  • the method further includes: amplifying the immune cells obtained in step (2).
  • immune cells derived from patients or healthy donors can be transformed into immune cells expressing a CAR that specifically binds GPC3 and optionally additional bioactive molecules.
  • the eleventh aspect of the present invention also provides a modified immune cell expressing the CAR of the present invention that specifically binds GPC3.
  • the engineered immune cells comprise the isolated nucleic acid molecule of the sixth aspect or a vector comprising the nucleic acid molecule.
  • the engineered immune cell comprises the nucleic acid construct of the seventh aspect or a vector comprising the nucleic acid construct.
  • the immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells of a patient or a healthy donor, and any combination thereof. These immune cells are prepared into modified immune cells by introducing the isolated nucleic acid molecule described in the sixth aspect, the nucleic acid construct described in the seventh aspect, or the vector described in the eighth aspect through the method provided in the tenth aspect.
  • the modified immune cells include genes involved in immune rejection (e.g., TRAC, TRBC, B2M, HLA-A, HLA-B, or HLA-C) and genes for immune co-suppressive pathways or signaling molecules (for example, the transcription or expression of one or two target genes in PD-1, CTLA-4 or LAG-3) is inhibited, such that target gene-mediated signaling is blocked in the modified immune cells. Interruption; Preferably, the transcription or expression of the target gene is inhibited by a method selected from the group consisting of gene knockout (for example, CRISPR, CRISPR/Cas9), homologous recombination, and interfering RNA.
  • gene knockout for example, CRISPR, CRISPR/Cas9
  • homologous recombination for example, interfering RNA.
  • the present invention also provides an immune cell composition, which includes the aforementioned modified immune cells, and optionally unmodified and/or unsuccessfully modified immune cells.
  • These unmodified and /or the unsuccessfully engineered immune cells do not express CAR specifically targeting GPC3.
  • the immune cell composition can contain immune cells that express or do not express CAR specific for GPC3, and the immune cell composition can still meet the needs of clinical application.
  • the engineered immune cells expressing a CAR specific for GPC3 comprise approximately 10%-100%, preferably 40%-80%, of the total cell number of the immune cell composition.
  • the immune cell composition is cultured into an immune cell line, and thus, in another aspect, the invention also provides immune cell lines containing the immune cell composition.
  • the invention provides for the preparation of chimeric antigen receptors that specifically bind to GPC3, or for the preparation of cells expressing said chimeric antigen receptors or for co-expressing said chimeric antigen receptors and additional biological activities.
  • Molecular immune cell kit includes an isolated nucleic acid molecule as described in the sixth aspect, a nucleic acid construct as described in the seventh aspect or a vector as described in the eighth aspect, or a host as described in the ninth aspect cells, and necessary solvents, such as sterile water, physiological saline, or cell culture medium, such as LB culture medium, such as EliteCell primary T lymphocyte culture system (product number: PriMed-EliteCell-024), and optionally, Also includes instruction manual.
  • the invention provides the aforementioned kit for preparing a chimeric antigen receptor capable of specifically binding to GPC3, or a cell expressing the chimeric antigen receptor, or co-expressing the chimeric antigen receptor, and Applications of additional bioactive molecules to immune cells.
  • the present invention provides a pharmaceutical composition, which contains the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the chimeric antigen receptor (including bispecific chimeric antigen receptor) described in the fifth aspect. combined antigen receptor or CAR construct co-expressed with another biologically active molecule), the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the third or eighth aspect
  • the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as a drug with anti-tumor activity (e.g., anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti- CD3 antibody, anti-ASGPR1 antibody, sorafenib or its derivatives, regorafenib or its derivatives, pemetrexed, cisplatin, paclitaxel, gemcitabine, capecitabine or FOLFIRINOX).
  • a drug with anti-tumor activity e.g., anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti- CD3 antibody, anti-ASGPR1 antibody, sorafenib or its derivatives, regorafenib or its derivatives, pemetrexed, cisplatin, paclitaxel, gemcitabine, capecitabine or FOLFIRINOX.
  • the antibody or antigen-binding fragment thereof according to the first aspect of the present invention the chimeric antigen receptor according to the fifth aspect, the isolated nucleic acid molecule according to the second or sixth aspect, the The core mentioned in the seven aspects acid construct, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the modified immune cell described in the eleventh aspect, or the immune cell described in the twelfth aspect
  • the cellular composition and the additional pharmaceutically active agent may be administered simultaneously, separately, or sequentially.
  • the pharmaceutical composition of the present invention includes: the isolated nucleic acid molecule described in the sixth aspect, the nucleic acid construct described in the seventh aspect or the vector described in the eighth aspect, or the ninth aspect of host cells.
  • compositions of the invention comprise: a modified immune cell or immune cell composition of the invention.
  • the substance may be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including Injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the pharmaceutical compositions of the present invention should be sterile and stable under the conditions of production and storage.
  • One preferred dosage form is an injection.
  • Such injections may be sterile injectable solutions.
  • sterile injectable solutions may be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for ease of storage and use.
  • Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), Glucose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution such as 0.9% (w/v) NaCl
  • Glucose solutions eg 5% glucose
  • surfactant containing solutions eg 0.01% polysorbate 20
  • pH buffer solutions eg phosphate buffer solution
  • Ringer's solution any combination thereof.
  • the substance may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, eyeball, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical , topical (eg, powder, ointment, or drops), or nasal route.
  • the preferred route/mode of administration is parenteral (eg intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection).
  • parenteral eg intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection.
  • the route and/or mode of administration will vary depending on the intended purpose.
  • the host cells described in the fourth or ninth aspect, the modified immune cells described in the eleventh aspect, or the immune cell composition described in the twelfth aspect are administered by intravenous injection or bolus injection.
  • the pharmaceutical composition of the present invention may include a "therapeutic effective amount” or a "preventive effective amount” of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect, the second aspect Or the isolated nucleic acid molecule described in the sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the eleventh aspect
  • “Prophylactically effective amount” refers to an amount sufficient to prevent, prevent, or delay the occurrence of disease.
  • a “therapeutically effective amount” means an amount sufficient to cure or at least partially prevent disease and its complications in a patient who is already suffering from the disease.
  • the therapeutically effective amount of a drug may vary depending on factors such as: the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other concomitantly administered Treatment and more.
  • the invention provides a method for preventing and/or treating a disease associated with expression of GPC3 in a subject (e.g., a human), said method comprising administering to a subject in need thereof An effective amount of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the chimeric antigen receptor according to the fifth aspect, the isolated nucleic acid molecule according to the second or sixth aspect, the seventh aspect
  • the disease associated with expression of GPC3 is selected from proliferative diseases, such as tumors. In certain embodiments, the disease associated with expression of GPC3 is a non-tumor-related indication associated with expression of GPC3.
  • the tumor is a GPC3-positive tumor.
  • the tumor is selected from solid tumors.
  • the tumor is selected from liver cancer (e.g., hepatocellular carcinoma), melanoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma cancer, Renal cell carcinoma, colorectal cancer, gastric cancer, glioma, and ovarian cancer (eg, ovarian clear cell carcinoma).
  • the tumor is selected from the group consisting of hematological tumors; preferably, the hematological tumor is selected from the group consisting of leukemias and lymphomas.
  • the method includes administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of the first aspect.
  • the method includes administering to the subject an effective amount of the chimeric antigen receptor of the fifth aspect, the isolated nucleic acid molecule of the sixth aspect, the seventh aspect Nucleic acid construct, the vector described in the eighth aspect, the host cell described in the ninth aspect, the modified immune cell described in the eleventh aspect, or the immune cell composition described in the twelfth aspect.
  • the host cell is an immune cell (eg, a human immune cell).
  • the method includes the following steps: (1) providing the immune cells required by the subject (e.g., T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells); (2) introducing the isolated nucleic acid molecule described in the sixth aspect of the present invention, the nucleic acid construct described in the seventh aspect, or the vector described in the eighth aspect into step (1) immune cells to obtain cells expressing the chimeric antigen receptor and optionally additional biologically active molecules; (3) administering the immune cells obtained in step (2) to the subject for treatment.
  • the immune cells required by the subject e.g., T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells
  • immune cells required by the subject e.g., T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells
  • immune cells required by the subject e.g., T lymphocytes, NK cells, monocytes, macrophag
  • the method administers immune cells expressing a CAR of the invention to the subject by dose-fractionation, such as one, two, three or more divided administrations of portions of the dose, e.g., in treatment
  • dose-fractionation such as one, two, three or more divided administrations of portions of the dose, e.g., in treatment
  • the first percent of the total dose is administered on the first day of treatment and the second hundredth of the total dose is administered on subsequent (e.g., the second, third, fourth, fifth, sixth or seventh day or later) treatment days proportion, such as administering a third percent of the total dose (e.g., on a subsequent (e.g., third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or later) treatment day) , remaining percentage).
  • 10% of the total dose of cells is administered on the first day of treatment, 30% of the total dose of cells is administered on the second day, and the remaining 60% of the total dose of cells is administered on the third day.
  • 50% of the total dose of cells is administered on the first day of treatment and on subsequent (e.g., second, third, fourth, fifth, sixth or seventh or later) treatment days. Apply 50% of the total dose to cells.
  • 1/3 of the total dose of cells is administered on the first day of treatment, and on subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later)
  • the total cell dose includes 1 ⁇ 10 7 to 10 ⁇ 10 8 CAR-positive immune cells, for example, includes (1-5) ⁇ 10 7 to (5-10) ⁇ 10 8 CAR-positive immune cells .
  • physicians may decide based on patient status, tumor size and stage, or combination therapy Drugs and other clinical circumstances to adjust dosage or treatment regimen.
  • the antibody or antigen-binding fragment thereof according to the first aspect of the present invention the chimeric antigen receptor according to the fifth aspect, the isolated nucleic acid molecule according to the second or sixth aspect,
  • the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the modified immune cell described in the eleventh aspect or the tenth aspect is administered in combination with another agent.
  • the additional agents include (i) increasing cells comprising a CAR nucleic acid or CAR polypeptide (e.g., an immune cell expressing a CAR of the invention, a modified immune cell, or an immune cell composition of the invention) an agent that improves the efficacy of; (ii) improves a or Agents with multiple side effects; (iii) Additional pharmaceutically active agents with anti-tumor activity.
  • a CAR nucleic acid or CAR polypeptide e.g., an immune cell expressing a CAR of the invention, a modified immune cell, or an immune cell composition of the invention
  • reagents can be used in the administration of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect, the isolated nucleic acid molecule described in the second or sixth aspect, the seventh aspect
  • the nucleic acid construct, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the modified immune cell described in the eleventh aspect or the twelfth aspect The immune cell composition or pharmaceutical composition is administered before, at the same time or after.
  • the methods described above further include administering to the subject a second therapy, which may be any therapy known for use in tumors, such as surgery, chemotherapy, radiotherapy, immunotherapy, Gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof.
  • a second therapy which may be any therapy known for use in tumors, such as surgery, chemotherapy, radiotherapy, immunotherapy, Gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof.
  • the second therapy may be used separately or in combination with the methods described above; or, the second therapy may be used simultaneously or sequentially with the methods described above.
  • the subject can be a mammal, such as a human.
  • the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the chimeric antigen receptor according to the fifth aspect, the isolated nucleic acid molecule according to the second or sixth aspect, the The nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the modified immune cell described in the eleventh aspect or the twelfth aspect Use of the immune cell composition or pharmaceutical composition described in the aspect in the preparation of medicaments for preventing and/or treating tumors.
  • the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the chimeric antigen receptor according to the fifth aspect, the isolated nucleic acid molecule according to the second or sixth aspect, the Nucleic acids described in seven aspects
  • FR Antibody framework region Amino acid residues in the antibody variable region other than CDR residues
  • Kabat Immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991 ).
  • IMGT is based on The international ImMunoGeneTics information system initiated by Lefranc et al. (IMGT)), please refer to Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.
  • Chothia Immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. Man (1989) Nature 342:878-883).
  • the term “antibody” refers to an immunoglobulin molecule capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • Globulin molecules As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g. Fab, Fab', F(ab') 2 , Fv), single chain (e.g.
  • antibodies of the invention are not limited to any particular method of producing the antibodies.
  • Antibodies include antibodies of any type, such as IgG, IgA or IgM (or subclasses thereof), and the antibodies need not be of any particular type.
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins There are five main types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the heavy chain constant regions corresponding to the different types of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
  • the subunit structures and three-dimensional configurations of different types of immunoglobulins are well known.
  • the heavy chain constant region consists of 4 domains (CH1, hinge region, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL.
  • the constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q).
  • VH and VL regions of antibodies can also be subdivided into highly denaturing regions called complementarity-determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity-determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site.
  • the assignment of amino acids to each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901- 917; Chothia et al. (1989) Nature 342:878-883 definition.
  • CDR complementarity determining region
  • the variable regions of the heavy chain and light chain each contain three CDRs, named CDR1, CDR2 and CDR3.
  • CDR1, CDR2 and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • the CDRs contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art.
  • the CDRs contained in the antibodies of the invention, or antigen-binding fragments thereof are preferably determined by the Kabat, Chothia, or IMGT numbering systems.
  • framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, such as a fragment of a full-length antibody, that retains the ability to specifically bind the same antigen to which the full-length antibody binds, and/ or compete with the full-length antibody for specific binding to the antigen, which is also referred to as the "antigen-binding portion.” See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes.
  • antigen-binding fragments of the antibody are generated by enzymatic or chemical cleavage of the intact antibody.
  • Non-limiting examples of antigen-binding fragments include camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)' 2 fragment, F(ab )' 3 fragments, Fd, Fv, scFv, di-scFv, (scFv) 2 , minibodies, diabodies, tribodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv”) and single structures Domain antibodies (sdAb, Nanobodies) and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136 middle.
  • the term “Fd” means an antibody fragment consisting of VH and CH1 domains
  • the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 ( 1989));
  • the term “Fab fragment” means an antibody fragment consisting of VL, VH, CL and CH1 domains;
  • the term “F(ab') 2 fragment” means an antibody fragment consisting of two fragments connected by a disulfide bridge on the hinge region An antibody fragment of a Fab fragment;
  • the term “Fab'fragment” means the fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd of the heavy chain. Fragment (consisting of VH and CH1 domains).
  • Fv means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. generally recognized For, six CDRs confer the antigen-binding specificity of the antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.
  • Fc means a region formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain.
  • Antibody fragments The Fc fragment of an antibody has many different functions but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising VL and VH domains connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al.
  • a disulfide bond may also exist between VH and VL of scFv.
  • the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, scFv containing NH2-VH-VH-COOH, NH2-VL-VL-COOH.
  • the scFv can form any engineering possible structure, single chain antibody (scFv), tandem antibody (tandem di-scFvs), bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide bond stabilized Fv protein, camel Ig , IgNAR, etc.
  • scFv can form di-scFv, which refers to two or more individual scFvs connected in series to form an antibody.
  • scFv can form (scFv) 2 , which refers to two or more individual scFvs joining in parallel to form an antibody.
  • the term "diabody” refers to an antibody fragment having two antigen-binding sites that comprise a light chain variable domain (VL) in the same polypeptide chain (VH-VL). ) of the heavy chain variable domain (VH).
  • VL light chain variable domain
  • VH-VL heavy chain variable domain
  • linker that is too short to allow pairing between two domains on the same chain, the domain is forced to pair with the complementary domain of the other chain and two antigen-binding sites are created.
  • Bifunctional antibodies can be bivalent or bispecific. Bifunctional antibodies are more fully described in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., PNAS USA 90:6444-6448 (1993). Trifunctional and tetrafunctional antibodies are also described in Hudson et al., Nature Medicine 9:129-134 (2003).
  • Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
  • Antigen-binding fragments of an antibody can be obtained from a given antibody (e.g., the antibodies provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods) ), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as for intact antibodies.
  • antibody includes not only intact antibodies but also antigen-binding fragments of the antibodies, unless the context clearly indicates otherwise.
  • the expression “specific binding” or “specific targeting” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction.
  • KD equilibrium dissociation constant
  • the term “KD” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the specific binding properties between two molecules can be determined using methods known in the art.
  • One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate.
  • Both the "association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187).
  • the ratio kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990;59:439-473).
  • KD, kon and kdis values can be measured by any valid method.
  • dissociation constants can be measured in Biacore using surface plasmon resonance (SPR).
  • bioluminescence interferometry or Kinexa can be used to measure dissociation constants.
  • identity is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or two A certain position in each polypeptide is occupied by lysine)
  • Percent identity between two sequences is a function of the number of matching positions common to the two sequences divided by the number of positions compared ⁇ 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have There is 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6 positions).
  • comparisons are made when two sequences are aligned to yield maximum identity.
  • alignment can be accomplished using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the PAM120 weight residue table using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0).
  • the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithm can be used using the Blossum 62 matrix or PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector may include sequences that replicate directly and autonomously in the cell, or may include sequences sufficient to permit integration into the host cell DNA.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and viral vectors, etc.
  • Non-limiting examples of viral vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, vesicle viruses (such as SV40).
  • a vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the term "episomal vector” means that the vector is capable of replicating without integrating into the chromosomal DNA of the host and is not gradually lost by dividing host cells. It also means that the vector is extrachromosomally or episomally. copy.
  • viral vector is used broadly to refer to a nucleic acid molecule (eg, a transfer plasmid) that includes a virus-derived nucleic acid element that typically facilitates the transfer or integration of the nucleic acid molecule into the genome of a cell, or mediates the transfer of nucleic acid of virus particles.
  • viral particles will typically include various viral components and sometimes host cell components.
  • viral vector may refer to a virus or viral particle capable of transferring nucleic acid into a cell, or to the transferred nucleic acid itself.
  • Viral vectors and transfer plasmids contain structural and/or functional genetic elements derived primarily from viruses.
  • retroviral vector refers to a viral vector or plasmid containing structural and functional genetic elements derived primarily from retroviruses, or portions thereof.
  • lentiviral vector refers to a viral vector or plasmid containing structural and functional genetic elements derived primarily from lentiviruses, or portions thereof (including LTRs).
  • elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells, immune cells (such as T lymphocytes cells, NK cells, monocytes, macrophages or dendritic cells, etc.). Host cells can include single cells or populations of cells.
  • chimeric antigen receptor refers to a receptor that contains at least one extracellular antigen-binding domain, spacer domain, transmembrane domain, and cytoplasmic signaling domain (also referred to herein as "Intracellular signaling domain”) recombinant polypeptide construct that combines antibody-based specificity for an antigen of interest (e.g., GPC3) with an immune effector cell-activating intracellular domain to demonstrate resistance to expression of the antigen of interest (e.g., GPC3 ) cell-specific immune activity.
  • the expression “CAR-expressing immune effector cells” refers to immune effector cells that express CAR and have antigen specificity determined by the targeting domain of the CAR.
  • CARs for cancer treatment
  • Methods of making CARs are known in the art, see, e.g., Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368 :1509-1518, 2013; Han et al., J. Hematol. Oncol., 6:47, 2013; PCT patent publications WO2012/079000, WO2013/059593; and US patent publication 2012/0213783, all of which are incorporated by reference in their entirety Incorporated herein.
  • extracellular antigen-binding domain refers to a polypeptide capable of specifically binding to an antigen or receptor of interest. This domain will be able to interact with cell surface molecules. For example, the extracellular antigen-binding domain can be selected to recognize an antigen that is a cell surface marker on a target cell associated with a particular disease state.
  • intracellular signaling domain refers to the portion of a protein that conducts effector signaling functions and directs the cell to perform specialized functions. Therefore, the intracellular signaling domain has the ability to activate at least one normal effector function of the CAR-expressing immune effector cell.
  • the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines.
  • primary signaling domain refers to a portion of a protein capable of modulating primary activation of a TCR complex in a stimulatory manner or in an inhibitory manner.
  • Primary signaling domains that act in a stimulatory manner often contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • costimulatory signaling domain refers to the intracellular signaling domain of a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes after binding to an antigen.
  • Non-limiting examples of costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278(ICOS), DAP10.
  • the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: sterile water, physiological saline, pH adjusters, surfactants , adjuvants, ionic strength enhancers, diluents, reagents to maintain osmotic pressure, reagents to delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc.
  • the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions).
  • such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% (w/v) NaCl
  • dextrose solutions eg 5% glucose
  • surfactant containing solutions eg 0.01% polysorbate 20
  • pH buffer solutions eg phosphate buffer solution
  • Ringer's solution any combination thereof.
  • prevention refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom (eg, tumor) in a subject.
  • treatment is Refers to methods performed to obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no further worsening) of the disease state, delaying or slowing the progression of the disease, ameliorating or alleviating the symptoms of the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable.
  • treatment may also refer to prolonging survival compared to expected survival if not receiving treatment.
  • the term “subject” refers to a mammal, such as a primate mammal, such as a human.
  • the term “subject” is meant to include living organisms in which an immune response can be elicited.
  • the subject eg, a human
  • has a tumor eg, a GPC3-related tumor
  • the term "effective amount" refers to an amount sufficient to obtain, at least in part, the desired effect.
  • a disease-preventing (e.g., tumor) effective amount refers to an amount that is sufficient to prevent, prevent, or delay the occurrence of a disease (e.g., a tumor);
  • a disease-treating effective amount refers to an amount that is sufficient to cure or at least partially prevent an existing disease. The patient's disease and the amount of its complications. Determining such effective amounts is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.
  • immune cell refers to a cell involved in an immune response, such as in promoting immune effector function.
  • immune cells include T cells (eg, alpha/beta T cells and gamma/delta T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages.
  • Immune cells of the invention may be self/autologous ("self") or non-self ("non-self", eg allogeneic, syngeneic or allogeneic).
  • autologous refers to cells from the same subject;
  • allogeneic refers to cells of the same species that are genetically different from the comparison cells;
  • isogenic means cells that are genetically different from the comparison cells.
  • allogeneic means cells from a different species than the comparison cells.
  • the cells of the invention are allogeneic.
  • T lymphocytes and/or NK cells.
  • T cell or “T lymphocyte” is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • the T cells may be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells.
  • T cells can be helper T cells (HTL; CD4 T cells) CD4 T cells, cytotoxic T cells (CTL; CD8 T cells), CD4CD8 T cells, CD4CD8 T cells, or any other subset of T cells.
  • T cells can include naive T cells and memory T cells. cell.
  • immune cells also include NK cells, monocytes, macrophages or dendritic cells, NKT cells, neutrophils, and macrophages.
  • Immune cells also include progenitor cells of immune cells, wherein the progenitor cells can be induced to differentiate into immune cells in vivo or in vitro.
  • the immune cells include progenitor cells of immune cells, such as hematopoietic stem cells (HSCs) contained within a population of CD34+ cells derived from cord blood, bone marrow, or circulating peripheral blood, which are administered in a subject Then differentiate into mature immune cells, or they can be induced to differentiate into mature immune cells in vitro.
  • HSCs hematopoietic stem cells
  • modified immune cell refers to an immune cell that expresses any of the CARs described herein, or has been introduced with any of the isolated nucleic acids or vectors described herein.
  • the CAR polypeptide can also be synthesized in situ in the cell. Alternatively, the CAR polypeptide can be produced extracellularly and then introduced into the cell. Methods of introducing polynucleotide constructs into cells are known in the art. In some embodiments, stable transformation methods can be used to integrate the polynucleotide construct into the genome of the cell.
  • transient transformation methods can be used to transiently express the polynucleotide construct without the polynucleotide construct being integrated into the genome of the cell.
  • virus-mediated methods may be used.
  • Polynucleotides can be introduced into cells by any suitable method, such as recombinant viral vectors (eg, retroviruses, adenoviruses), liposomes, and the like.
  • Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or microparticle bombardment.
  • the polynucleotide may be included in a vector, such as a plasmid vector or a viral vector.
  • immune effector function refers to a function or response of an immune effector cell that enhances or promotes an immune attack on a target cell (eg, killing of the target cell, or inhibiting its growth or proliferation).
  • the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines.
  • the present invention provides antibodies targeting GPC3 or antigen-binding fragments thereof, which can be used to prepare chimeric antigen receptors.
  • Immune effector cells expressing chimeric antigen receptors of the present invention have improved effector functions (eg, tumor killing activity and cytokine releasing activity).
  • effector functions eg, tumor killing activity and cytokine releasing activity.
  • in vivo experiments have also proven that immune effector cells expressing chimeric antigen receptors prepared based on the antibodies of the present invention or antigen-binding fragments thereof have stronger tumor killing effects.
  • Figure 1 shows the detection results of the killing activity of CAR-T cells (blank T, CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, CH3-T) on target cells.
  • Figure 2A shows the detection results of IFN- ⁇ secretion levels after activation of CAR-T cells (blank T, CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, CH3-T).
  • Figure 2B shows the detection results of IL-2 secretion levels after activation of CAR-T cells (blank T, CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, CH3-T).
  • Figure 3A shows the tumor volume change curve of B-NDG mice treated with PBS, blank T cells, CA3-T, CC8-T, and CH3-T.
  • Figure 3B shows the body weight change curve of B-NDG mice treated with PBS, blank T cells, CA3-T, CC8-T, and CH3-T.
  • Biotinylated GPC3 and SV magnetic beads were used to screen the fully human phage library, and the screened products were tested for phage titration by plating. Mix the product of the first round of sifting with PBST and perform the second and third rounds of sifting according to the above steps.
  • a single colony was inoculated from the phage panning product titer plate into a 96-deep well plate, and monoclonal phage were detected by ELISA.
  • the candidate scFv sequence was constructed in the TGEX-KAL vector, and then transfected into expi293 cells for expression and purification of scFv-Fc protein.
  • SEC analysis experimental results show (Table 4) that 6 candidate scFv sequence monomer peaks (main peak area) are greater than 80%.
  • GPC3 scFv-Fc protein binding affinity To characterize GPC3 scFv-Fc protein binding affinity, a GPC3-expressing cell line (293T/GPC3+) was selected for cell binding assay.
  • Mouse IgG Isotype Control (from Thermo Fisher Sci.) was used as a negative control, and anti-GPC3 antibody GC33 (Ishiguro et al, 2008; for GC33 related sequences, see US20150259417A1) was used as a positive control.
  • the results in Table 5 show that the affinity of the six candidate scFvs to GPC3-positive cells 293T/GPC3+ was significantly better than that of the control group.
  • Example 2 Construction and preparation of chimeric antigen receptor (CAR) lentiviral expression vector
  • a CAR lentiviral expression vector was further constructed.
  • the intracellular domain of CD137 (4-1BB) and the ITAM region of CD3 ⁇ are used as activation signals and fused with the above scFv.
  • the CD8 ⁇ signal peptide, CD8 hinge region, and CD8 transmembrane region are added to construct a chimeric antigen receptor expression Vector and constructed chimeric antigen receptor structure are shown in Table 6 below.
  • Isolation of primary T cells Separate human PBMC cells using lymphocyte separation medium (GE), culture them in an incubator at 37°C and 5% CO2, add 100 ⁇ l/mL CD3 antibodies and CD28 antibodies, and mix thoroughly. After homogenization, incubate at room temperature for 15 minutes. Take out the magnetic beads, pipet up and down at least 5 times with a pipette, and mix thoroughly. Pipette 50 ⁇ l magnetic beads/mL into the above sample, mix thoroughly, and incubate at room temperature for 10 minutes. Add complete culture medium to a total volume of 2.5 mL in the tube, insert the tube (open the cap) into the magnetic pole, and let stand at room temperature for 5 minutes.
  • GE lymphocyte separation medium
  • cytokines and antibody complexes (IL-2, 10ng/mL IL-7, 5ng/mL IL-15, 500ng at a final concentration of 300U/mL) into the six-well plate.
  • IL-2 10ng/mL IL-7, 5ng/mL IL-15, 500ng at a final concentration of 300U/mL
  • /mL Anti-CD3 OKT3
  • 2 ⁇ g/mL Anti-CD28 configuration 2 ⁇ g/mL Anti-CD28 configuration
  • required virus amount (mL) (MOI*number of cells)/virus titer.
  • CAR-T cells expressing the CAR (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T and CH3-T) described in Example 2 and the positive control GC33-T were obtained respectively by the above method. cell.
  • the nucleic acid sequence encoding the CAR is expressed under the drive of a promoter.
  • T cells transfected with lentivirus are labeled using GPC3 antigen and measured by flow cytometry to reflect the expression level of CAR on the surface of T cells.
  • the CAR positivity rate of the CAR-T cells obtained in Example 3 was detected by the above method, and the FACS detection results are shown in Table 7 below.
  • HEPG2-luc was placed in a 96-well plate in a 5% CO 2 37°C incubator for 30 minutes.
  • CAR-T centrifuge and resuspend CAR-T cells, GPC3-CAR and untransfected CAR blank T cells (UTD) in 1640 medium with 10% FBS as effector cells, and then follow different E/T (effector cells/target cells) were added to a 96-well plate containing HEPG2-luc at 100 ⁇ L/well, and the final volume was added to 200 ⁇ L/well, and cultured in a 37°C incubator with 5% CO2 for 18 to 24 hours. After the culture is completed, take the well plate out of the incubator, add 20ul of fluorescence detection reagent, and use a microplate reader to detect the fluorescence reading.
  • E/T effector cells/target cells
  • the killing activity test results of CAR-T are shown in Figure 1.
  • the 6 types of CAR-T cells (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, CH3-T) constructed in this application were detected in different It can effectively lyse tumor cells at any E/T ratio.
  • CA3-T, CC8-T, CH3-T and CE9-T have particularly outstanding effects, with the ratio of effector cells/target cells When the ratio is 10, the lysis rate of tumor cells is as high as 98%.
  • Collect HepG2-luc cells use culture medium to adjust the cell density to 1 ⁇ 10 5 cells/mL, inoculate target cells in a 96-well plate at 100 ⁇ L/well, and resuspend CAR-T cells, GPC3-CAR and Blank T cells that have not been transfected with CAR are used as effector cells, and then added to a 96-well plate containing target cells at an E/T (effector cell/target cell) ratio of 1:1, 100 ⁇ L/well, and the final volume is filled to 200 ⁇ L/ Wells were incubated overnight in a 37 °C incubator with 5% CO 2 .
  • the test results are shown in Figure 2.
  • the six types of CAR-T cells (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, CH3-T) constructed in this application can kill tumors to varying degrees. cells, and release IFN- ⁇ (the detection results are shown in Figure 2A) or IL-2 (the detection results are shown in Figure 2B).
  • Example 7 In vivo model to evaluate the killing ability of CAR-T cells against target cells
  • mice were subcutaneously inoculated with 5 ⁇ 10 6 HepG2 tumor cells on the right side or right scapula. When the average tumor volume reached 100-150 mm 3 , they were randomly divided into 6 groups. Each mouse was given cyclophosphate intraperitoneally. amide 100 mg/kg, and 5 ⁇ 10 6 CAR-T cells and blank T cells not transfected with CAR were reinfused into the tail vein the next day. Tumor diameters were measured with vernier calipers and mice were weighed twice a week. The results of the killing ability of CAR-T cells on target cells are shown in Figure 3A, and the changes in mouse body weight are shown in Figure 3B.
  • the CAR-T cells (CA3-T, CC8-T, CH3-T) constructed in this application have good inhibitory effects on tumor cells. There were no animal deaths or significant weight loss in all treatment groups during the observation period, and no obvious drug toxic reactions were observed. The mice were well tolerated during the treatment period.

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Abstract

L'invention concerne un anticorps se liant de manière spécifique à GPC3 ou un fragment de liaison à l'antigène de celui-ci, et un récepteur antigénique chimérique (CAR) comprenant l'anticorps ou le fragment de liaison à l'antigène de celui-ci. La présente invention concerne en particulier une cellule immunitaire modifiée exprimant le CAR, et un procédé de préparation de la cellule immunitaire modifiée. La présente invention concerne également l'utilisation de ces anticorps, du CAR et de la cellule immunitaire pour prévenir et/ou traiter des maladies liées à l'expression de GPC3, telles que le cancer du foie, le mélanome, le cancer de l'ovaire, etc, et une méthode de prévention et/ou de traitement de tumeurs positives à GPC3 telles que le cancer du foie, le mélanome, le cancer de l'ovaire, etc.
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