[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2024114404A1 - Récepteur antigénique chimérique se liant spécifiquement à gpc3 et son utilisation - Google Patents

Récepteur antigénique chimérique se liant spécifiquement à gpc3 et son utilisation Download PDF

Info

Publication number
WO2024114404A1
WO2024114404A1 PCT/CN2023/132244 CN2023132244W WO2024114404A1 WO 2024114404 A1 WO2024114404 A1 WO 2024114404A1 CN 2023132244 W CN2023132244 W CN 2023132244W WO 2024114404 A1 WO2024114404 A1 WO 2024114404A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
seq
cdr
variant
variable region
Prior art date
Application number
PCT/CN2023/132244
Other languages
English (en)
Chinese (zh)
Inventor
蔡珍珍
侯攀燕
周阳
葛均友
卫立辛
Original Assignee
成都科伦精准生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 成都科伦精准生物科技有限公司 filed Critical 成都科伦精准生物科技有限公司
Publication of WO2024114404A1 publication Critical patent/WO2024114404A1/fr

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biomedicine, and in particular, the present invention relates to single-chain antibodies and chimeric antigen receptors (CARs) that specifically bind to GPC3.
  • CARs chimeric antigen receptors
  • the present invention also relates to immune cells expressing the CAR, nucleic acid molecules encoding such CARs or co-expressed molecules, and methods for preparing the modified immune cells.
  • the present invention also relates to methods for using these CARs and immune cells to prevent and/or treat GPC3-positive tumors such as hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma.
  • HCC hepatocellular carcinoma
  • melanoma melanoma
  • ovarian clear cell carcinoma GPC3-positive tumors
  • Glypican-3 (GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS and SGBS1) is a member of the heparan sulfate proteoglycan family. It is anchored on the cell surface through glycosylated phosphatidylinositol and is one of the representative liver cancer markers in preclinical studies. 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-related death in my country, which seriously threatens the life and health of the Chinese people.
  • a large number of patients with hepatocellular carcinoma still lack accurate and effective clinical treatment methods.
  • Most patients with liver cancer are already in the advanced or advanced 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%, and the overall 5-year survival rate is low, only 7% to 10%.
  • Chimeric antigen receptor (CAR)-T cell therapy is considered to be one of the most promising cancer treatment methods and has become a new hope for human beings to fight cancer. It cultivates immune cells collected from patients in vitro, transduces specific exogenous genes in vitro, and then amplifies them in vitro and then infuses them back into the patient's body to achieve the purpose of treating tumors in a non-MHC restricted manner.
  • CAR-T cell therapy has achieved remarkable therapeutic effects in the treatment of hematological malignancies, with a complete remission rate of over 90% for relapsed and refractory B-cell leukemia. Solid tumors account for about 90% of all malignant tumors, and there is a large demand for therapeutic drugs. However, the therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient, mainly because solid tumors have complex tumor microenvironments and high tumor heterogeneity.
  • the present invention provides a CAR targeting GPC3 or a modified immune cell co-expressing a CAR targeting GPC3.
  • the modified immune cell can be used for the treatment of GPC3-positive hepatocellular carcinoma (HCC), melanoma, ovarian clear cell carcinoma, etc.
  • HCC hepatocellular carcinoma
  • melanoma melanoma
  • ovarian clear cell carcinoma etc.
  • the inventors first developed a fully human antibody with low immunogenicity that can specifically recognize/bind to GPC3.
  • the present invention further designs and constructs a CAR targeting GPC3.
  • the CAR of the present invention can direct the specificity and reactivity of immune effector cells to cells expressing GPC3 (such as hepatocellular carcinoma (HCC), melanoma and ovarian clear cell carcinoma) in a non-MHC restricted manner so that they can be eliminated. Therefore, the CAR targeting GPC3 of the present invention has the potential to be used for the prevention and/or treatment of GPC3-positive tumors such as hepatocellular carcinoma (HCC), melanoma and ovarian clear cell carcinoma, and has great clinical value.
  • HCC hepatocellular carcinoma
  • melanoma and ovarian clear cell carcinoma a non-MHC restricted manner
  • the substitutions are conservative substitutions.
  • the CDRs are defined according to the Kabat, IMGT, Chothia, or AbM numbering systems.
  • the antigen binding molecules of the invention 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:
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 10 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 11 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 25 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 26 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with a sequence of SEQ ID NO: 54 or a variant thereof
  • CDR-H3 with a sequence of SEQ ID NO: 55 or a variant thereof
  • VL light chain variable region
  • the variant described in any one of (1a)-(1d) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.
  • the antigen binding molecules of the invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 16 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 17 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 31 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 32 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with a sequence of SEQ ID NO: 60 or a variant thereof
  • CDR-H3 with a sequence of SEQ ID NO: 61 or a variant thereof
  • VL light chain variable region
  • the variant described in any one of (2a)-(2d) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, The substitutions described are conservative substitutions.
  • the antigen binding molecules of the invention 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 having a sequence of SEQ ID NO: 21 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 11 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 21 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 26 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with a sequence of SEQ ID NO: 50 or a variant thereof
  • CDR-H3 with a sequence of SEQ ID NO: 40 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 65 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 55 or a variant thereof
  • VL light chain variable region
  • the variant described in any one of (3a)-(3d) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.
  • the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VH).
  • VH heavy chain variable region
  • VH light chain variable region
  • VL Chain variable region
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 23 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 11 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 37 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 26 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 52 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 40 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 67 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 55 or a variant thereof
  • VL light chain variable region
  • the variant described in any one of (4a)-(4d) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived.
  • the substitutions are conservative substitutions.
  • the antigen binding molecules of the invention comprise:
  • VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof and/or a VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof;
  • VH comprising the sequence shown in SEQ ID NO:5 or a variant thereof and/or a VL comprising the sequence shown in SEQ ID NO:6 or a variant thereof;
  • VH comprising the sequence shown in SEQ ID NO:7 or a variant thereof and/or a VL comprising the sequence shown in SEQ ID NO:8 or a variant thereof;
  • the 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 compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.
  • the antigen binding molecule described in any of the above embodiments may comprise a constant region from or derived from a human immunoglobulin.
  • the heavy chain of the antigen binding molecule comprises a heavy chain constant region from or derived from human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4).
  • the heavy chain of the antigen binding molecule comprises a wild-type Fc region, or comprises a mutated or chemically modified Fc region, which has an effector function (e.g., enhanced ADCC activity) that is changed compared to a wild-type Fc region.
  • the light chain of the antigen binding molecule comprises a light chain constant region from or derived from a human immunoglobulin (eg, kappa or lambda).
  • a human immunoglobulin eg, kappa or lambda
  • the antigen binding molecule described in any of the above embodiments is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the antigen binding molecule is selected from a full-length antibody, a Fab fragment, a Fab' fragment, a F(ab)' 2 fragment, a F(ab)' 3 fragment, a single-chain antibody (e.g., scFv, di-scFv or (scFv) 2 ), a miniantibody, a disulfide-stabilized Fv protein (dsFv) and a single domain antibody (sdAb, nanobody).
  • a single-chain antibody e.g., scFv, di-scFv or (scFv) 2
  • dsFv disulfide-stabilized Fv protein
  • sdAb single domain antibody
  • the VH and VL of the antigen binding molecules of the invention are linked by one or more linkers.
  • the linker is typically a peptide linker, such as a flexible and/or soluble peptide linker, such as a peptide linker rich in glycine, serine and/or threonine.
  • the linker also includes charged residues (such as lysine and/or glutamic acid), which can improve solubility.
  • the linker also includes one or more proline.
  • the linker comprises one or more (e.g., 1, 2, or 3) sequences as shown in ( GmS ) n , wherein 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 a sequence of SEQ ID NO: 76.
  • the antigen binding molecules of the invention are single chain antibodies, such as scFv, di-scFv or (scFv)2.
  • the single-chain antibody comprises, from its N-terminus to its C-terminus:
  • VH-linker comprising a sequence as shown in SEQ ID NO: 1 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 2 or a variant thereof; or, a VL-linker comprising a sequence as shown in SEQ ID NO: 2 or a variant thereof-a VH comprising a sequence as shown in SEQ ID NO: 1 or a variant thereof;
  • VH-linker comprising a sequence as shown in SEQ ID NO: 3 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 4 or a variant thereof; or, a VL-linker comprising a sequence as shown in SEQ ID NO: 4 or a variant thereof-a VH comprising a sequence as shown in SEQ ID NO: 3 or a variant thereof;
  • VH-linker comprising a sequence as shown in SEQ ID NO: 5 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 6 or a variant thereof; or, a VL-linker comprising a sequence as shown in SEQ ID NO: 6 or a variant thereof-a VH comprising a sequence as shown in SEQ ID NO: 5 or a variant thereof;
  • VH-linker comprising a sequence as shown in SEQ ID NO: 7 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 8 or a variant thereof; or, a VL-linker comprising a sequence as shown in SEQ ID NO: 8 or a variant thereof-a VH comprising a sequence as shown in SEQ ID NO: 7 or a variant thereof;
  • the 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 compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions.
  • the single-chain antibody comprises a single-chain antibody as shown in any one of SEQ ID NOs: 68, 69, 70, 71.
  • the antigen binding molecules of the present invention further comprise a constant region derived from human immunoglobulin.
  • the heavy chain of the antigen binding molecules comprises a heavy chain constant region derived from human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antigen binding molecules comprises a light chain constant region derived from human immunoglobulin (e.g., ⁇ or ⁇ ).
  • the heavy chain of the antigen binding molecule comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which has one or more amino acid 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, deletions or additions of 1, 2, 3, 4 or 5 amino acids) compared to the wild-type sequence from which it is derived; and/or,
  • CH heavy chain constant region
  • the light chain of the antigen-binding molecule comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof, which has one or more amino acid 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, deletions or additions of 1, 2, 3, 4 or 5 amino acids) compared to the wild-type sequence from which it is derived.
  • CL light chain constant region
  • 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 IgG1, 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 antigen binding molecules of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, a DNA molecule encoding the antigen binding molecule is obtained by chemical synthesis or PCR amplification, the resulting DNA molecule is inserted into an expression vector, and then transfected into a host cell. Then, the host cell after transfection is cultured under specific conditions, and the antigen binding molecules of the present invention are expressed.
  • 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)). In addition, these antigen-binding fragments can also be directly produced by 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 directly obtained 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 culture medium of recombinant host cells. Other techniques for preparing these antigen-binding fragments are fully known to those skilled in the art.
  • the second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antigen binding molecule of the present invention.
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:89, (ii) a sequence substantially identical to SEQ ID NO:89 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:89, or a sequence having 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) the nucleotide sequence shown in SEQ ID NO:90, (v) a sequence substantially identical to SEQ ID NO:90 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:90, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v) above.
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:91, (ii) a sequence substantially identical to SEQ ID NO:91 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:91, or a sequence having 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) the nucleotide sequence shown in SEQ ID NO:92, (v) a sequence substantially identical to SEQ ID NO:92 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions compared to SEQ ID NO:92), or (vi) a degenerate sequence of the above (iv) or (v).
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:93, (ii) a sequence substantially identical to SEQ ID NO:93 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:93, or a sequence having 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) the nucleotide sequence shown in SEQ ID NO:94, (v) a sequence substantially identical to SEQ ID NO:94 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity with SEQ ID NO:94, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v) above.
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:95, (ii) a sequence substantially identical to SEQ ID NO:95 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:95, or a sequence having 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) the nucleotide sequence shown in SEQ ID NO:96, (v) a sequence substantially identical to SEQ ID NO:96 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity with SEQ ID NO:96, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v) above.
  • the isolated nucleic acid molecule comprises: (i) the nucleotide sequence shown in SEQ ID NO:72, (ii) a sequence substantially identical to SEQ ID NO:72 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:72, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above.
  • the isolated nucleic acid molecule comprises: (i) the nucleotide sequence shown in SEQ ID NO:73, (ii) a sequence substantially identical to SEQ ID NO:73 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:73, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above.
  • the isolated nucleic acid molecule comprises: (i) a nucleotide sequence as shown in SEQ ID NO: 74;
  • the invention relates to a sequence of the invention that is a sequence of the invention that is substantially identical to SEQ ID NO:74 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:74), or (iii) a degenerate sequence of (i) or (ii) above.
  • the isolated nucleic acid molecule comprises: (i) the nucleotide sequence shown in SEQ ID NO:75, (ii) a sequence substantially identical to SEQ ID NO:75 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:75, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above.
  • the third aspect of the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above.
  • the vector of the present invention is, for example, a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector or a viral vector; preferably, the vector is an expression vector; preferably, the vector is an episomal vector; preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector.
  • the fourth aspect of the present invention provides a host cell comprising an isolated nucleic acid molecule of the present invention or a vector of the present invention.
  • the host cell can be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., Escherichia coli).
  • Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.
  • Suitable insect cells include, but are not limited to, Sf9 cells.
  • the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).
  • the host cell of the present invention may be a chimeric antigen receptor T cell (CAR-T).
  • the isolated nucleic acid molecule contained in the host cell may include a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further includes a nucleotide sequence encoding an antigen binding molecule (eg, ScFv) of the present invention.
  • the isolated nucleic acid molecule contained in the host cell encodes a chimeric antigen receptor comprising an antigen binding molecule (eg, scFv) of the present invention.
  • the present invention also relates to a method for preparing the antigen-binding molecule of the present invention, comprising culturing the host cell as described above under conditions allowing protein expression, and recovering the antigen-binding molecule from the cultured host cell culture.
  • the present invention relates to a CAR targeting GPC3, which has the characteristics of non-MHC-restricted GPC3 recognition ability, which confers the ability of immune cells (e.g., T cells, NK cells, monocytes, macrophages or dendritic cells) expressing the CAR to recognize cells expressing GPC3 (e.g., tumor cells) independently of antigen processing and presentation.
  • immune cells e.g., T cells, NK cells, monocytes, macrophages or dendritic cells
  • the fifth aspect of the present invention provides a chimeric antigen receptor, which comprises an antigen binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain.
  • 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 the antigen binding molecule described in the first aspect.
  • the antigen binding domain comprises the antigen binding molecule as a first antigen binding domain, and further comprises a second antigen binding domain that does not bind to GPC3.
  • the antigen bound by the second antigen binding domain is selected from: PD-1, PD-L1, CTLA-, CD3, ASGPR1, CD19, MSLN, PSMA, MUC1, EGFR, HER2, CD276, GD2, BCMA, CD33 or Claudin18.2.
  • the antigen binding domain is a single chain antibody.
  • the first antigen binding domain comprises the sequence shown in any one of SEQ ID NO:68,69,70,71 or a variant thereof, wherein the variant has a sequence that is 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 to any one of SEQ ID NO:68,69,70,71, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.
  • the transmembrane domain included 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 a cell membrane (particularly a eukaryotic cell membrane).
  • the transmembrane domain suitable for the CAR of the present invention can be derived from a natural source.
  • the transmembrane domain can be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain can be a synthetic non-naturally occurring protein segment, such as a protein segment mainly comprising hydrophobic residues such as leucine and valine.
  • the transmembrane domain is a transmembrane region selected from the group consisting of the ⁇ , ⁇ or ⁇ chains of T cell receptors, 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 a transmembrane region selected from the following proteins: CD8, CD28, CD4, PD-1, CD152 and CD154.
  • the transmembrane domain comprises a CD8 transmembrane region whose sequence is shown in SEQ ID NO:77.
  • the chimeric antigen receptor of the present invention comprises 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 (e.g., IgG1 or IgG4).
  • an immunoglobulin e.g., IgG1 or IgG4
  • CH2 and CH3 extend the antigen binding domain of the CAR from the cell membrane of the cell expressing the CAR, and can more accurately mimic the size and domain structure of a natural TCR.
  • the spacer domain comprises a hinge domain.
  • a hinge domain can be an amino acid segment usually found between two domains of a protein, which can allow the protein to be flexible and allow one or two domains to move relative to each other. Therefore, the hinge domain can be any amino acid sequence as long as it can provide this flexibility of the extracellular antigen binding domain and its mobility relative to the transmembrane domain.
  • the hinge domain is a hinge region of a naturally occurring protein or a portion thereof.
  • the spacer domain is selected from a hinge domain and/or a CH2 and CH3 region of an immunoglobulin (e.g., IgG1 or IgG4).
  • the hinge domain comprises a hinge region of CD8, IgG4, PD-1, CD152, or CD154.
  • the hinge domain comprises a CD8 hinge region having a sequence as shown in SEQ ID NO: 78.
  • the CAR of the present invention may further include a signal peptide at its N-terminus.
  • a signal peptide is a polypeptide sequence that targets the sequence connected thereto to the desired site.
  • the signal peptide can target the CAR connected thereto to the secretory pathway of the cell and allow the CAR to be further integrated and anchored into the lipid bilayer.
  • Signal peptides that can be used for CAR are known to those skilled in the art.
  • the signal peptide comprises a heavy chain signal peptide (e.g., a heavy chain signal peptide of IgG1), a granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR2) signal peptide, an IL2 signal peptide, or a CD8 ⁇ signal peptide.
  • the signal peptide is selected from a CD8 ⁇ signal peptide.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO:82.
  • the CAR of the present invention can also be co-expressed with another biologically active molecule.
  • the other biologically active molecule can have its own signal peptide, which is named signal peptide to distinguish it from the signal peptide in the previous paragraph.
  • the signal peptide-2 guides the transport of other biologically active molecules to a specific site in 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 shown in SEQ ID NO: 84).
  • the intracellular signaling domain contained in the CAR of the present invention participates in the signal transduction generated by the binding of the CAR of the present invention to GPC3 into the interior of the immune effector cell, activates at least one normal effector function of the immune effector cell expressing CAR, or enhances the secretion of at least one cytokine (e.g., IL-2, IFN- ⁇ ) of the immune effector cell expressing CAR.
  • cytokine e.g., IL-2, IFN- ⁇
  • the intracellular signaling domain comprises a primary signaling domain and/or a co-stimulatory signaling domain.
  • the primary signaling domain may be any intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an intracellular signaling domain selected from the following proteins: CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CDS, CD22, CD79a, CD79b or CD66d. In certain embodiments, the primary signaling domain comprises an intracellular signaling domain of CD3 ⁇ .
  • the costimulatory signaling domain may be an intracellular signaling domain from a costimulatory molecule.
  • the costimulatory signaling domain comprises an intracellular signaling domain selected from the following proteins: CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS) or DAP10.
  • the co-stimulatory 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 of both.
  • the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains. In such embodiments, the two or more co-stimulatory signaling domains may be the same or different.
  • the intracellular signaling domain comprises a primary signaling domain and at least one co-stimulatory signaling domain.
  • the primary signaling domain and at least one co-stimulatory signaling domain can be connected in series to the carboxyl terminus of the transmembrane domain in any order.
  • the intracellular signaling domain may include the intracellular signaling domain of CD3 ⁇ and the intracellular signaling domain of CD137(4-1BB).
  • the intracellular signaling domain of CD3 ⁇ includes the amino acid sequence shown in SEQ ID NO:79.
  • the intracellular signaling domain of CD137(4-1BB) includes the amino acid sequence shown in SEQ ID NO:80.
  • the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO:81.
  • the present invention provides a chimeric antigen receptor capable of specifically binding to GPC3, wherein the chimeric antigen receptor comprises an antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain from its N-terminus to its C-terminus.
  • the intracellular signaling domain is a co-stimulatory signaling domain and a primary signaling domain from the N-terminus to the C-terminus.
  • the spacer domain comprises the hinge region of CD8 (e.g., CD8 ⁇ ) (e.g., the hinge region whose sequence is shown in SEQ ID NO:78).
  • the transmembrane domain comprises a transmembrane region of CD8 (e.g., CD8 ⁇ ) (e.g., a transmembrane region whose sequence is shown in SEQ ID NO:77).
  • CD8 e.g., CD8 ⁇
  • SEQ ID NO:77 a transmembrane region whose sequence is shown in SEQ ID NO:77.
  • the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3 ⁇ (e.g., the sequence shown in SEQ ID NO:79), and the co-stimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., the sequence shown in SEQ ID NO:80); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO:81.
  • the chimeric antigen receptor comprises the signal peptide, antigen binding domain, spacer domain, transmembrane domain, intracellular signaling domain (co-stimulatory signaling domain and primary signaling domain from N-terminus to C-terminus) in sequence.
  • the signal peptide comprises a heavy chain signal peptide of IgG1 or a CD8 ⁇ signal peptide (e.g., a signal peptide having a sequence as shown in SEQ ID NO: 82).
  • the CAR of the present invention comprises a sequence as shown in any one of SEQ ID NO: 85, 86, 87, 88, or a variant thereof, wherein the variant has at least 70%, 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 compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.
  • the CAR described in the fifth aspect of the present invention can also be co-expressed with other biologically active molecules.
  • the self-cleaving peptide can prevent the amino acids from forming covalent bonds during the translation process and maintain the translation to continue, so that the translation product is "self-cut", thereby separating the chimeric antigen receptor of the present invention from other biologically active molecules.
  • the chimeric antigen receptor that can specifically bind to GPC3 becomes an independent CAR having an extracellular antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, while other biologically active molecules can be secreted outside the cell or expressed as membrane-forming chimeric polypeptides or proteins.
  • the immune cells expressing CAR amplify and enrich in the tumor microenvironment
  • other biologically active molecules are enriched in the tumor microenvironment and synergize with CAR to exert anti-tumor effects.
  • the nucleic acid sequence encoding CAR is connected to the nucleic acid sequence of another biologically active molecule through the nucleic acid sequence of a self-cleaving peptide.
  • CAR can be at the N-terminus or C-terminus of another biologically active molecule.
  • CAR is at the 5' end of another biologically active molecule.
  • Any self-cleaving peptide that can cause the fusion protein to cleave into two independent proteins can be applied to the present invention.
  • the self-cleaving peptide is P2A, preferably having the sequence shown in SEQ ID NO: 130, and its nucleotide sequence can be optimized according to the needs of genetic recombination.
  • the fusion protein comprising CAR and another biologically active molecule has the following structure:
  • N'-signal peptide--extracellular antigen binding domain that specifically binds to GPC3--spacer domain--transmembrane domain--intracellular signal transduction domain--self-cleaving peptide--signal peptide-2--another biologically active molecule-C', wherein the signal peptide-2 is the same as or different from the N-terminal signal peptide.
  • the signal peptide-2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (e.g., as shown in SEQ ID NO:84).
  • the method of generating a chimeric antigen receptor and an immune effector cell (eg, T cell) comprising the chimeric antigen receptor is known in the art, and may include transfecting cells with at least one polynucleotide encoding CAR, and expressing the polynucleotide in the cell.
  • the nucleic acid molecule encoding the CAR of the present invention may be included in an expression vector (eg, a lentiviral vector), which can be expressed in a host cell such as a T cell to manufacture 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 an amino acid sequence includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence.
  • the nucleotide sequence encoding the chimeric antigen receptor described in the fifth aspect is selected from: (1) a sequence shown in any one of SEQ ID NOs: 72, 73, 74, 75, or a degenerate variant thereof; (2) a sequence substantially identical to the sequence described in (1), for example, a sequence having 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 compared to (1), or a sequence having one or more nucleotide substitutions compared to the sequence described in (1); and the sequence substantially retains at least one biological activity of the nucleotide sequence from which it is derived (for example, being able to encode the ability to direct the specificity and reactivity of immune effect
  • the CAR of the present invention can also be co-expressed with other biologically active molecules to synergistically exert anti-tumor effects.
  • the seventh aspect of the present invention also provides a nucleic acid construct, which comprises a first nucleic acid sequence encoding the chimeric antigen receptor described in the fifth aspect, and further comprises a second nucleic acid sequence encoding another biologically active molecule.
  • the additional biologically active molecule encoded by the second nucleic acid sequence has anti-tumor activity.
  • the additional biologically active molecule encoded by the second nucleotide sequence further comprises a signal peptide-2 at its N-terminus.
  • the first nucleotide sequence is located upstream of the second nucleotide sequence.
  • the first nucleic acid sequence and the second nucleic acid sequence are connected by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof).
  • a self-cleaving peptide e.g., P2A, E2A, F2A, T2A or any combination thereof.
  • the sequence encoding the self-cleaving peptide is connected to the 3' end of the first nucleotide sequence and to the 5' end of the second nucleotide sequence.
  • the self-cleaving peptide is P2A (e.g., as shown in SEQ ID NO:83).
  • the nucleic acid construct of the seventh aspect comprises, from its 5' end to its 3' end, in order: a nucleotide sequence encoding the signal peptide, a nucleotide sequence encoding the antigen binding domain, a nucleotide sequence encoding the spacer domain, a nucleotide sequence encoding the transmembrane domain, a nucleotide sequence encoding the intracellular signaling domain, a nucleotide sequence encoding the self-cleaving peptide sequence, a nucleotide sequence encoding the signal peptide-2 Acid sequences, nucleotide sequences encoding other biologically active molecules.
  • the eighth aspect of the present invention provides a vector comprising the isolated nucleic acid molecule described in the second aspect or the sixth aspect, or the nucleic acid construct described in the seventh aspect.
  • the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector, and a viral vector.
  • 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-defective retrovirus or lentivirus.
  • the ninth aspect of the present invention provides a host cell, which comprises the isolated nucleic acid molecule as described in the sixth aspect, the nucleic acid construct as described in the seventh aspect, or the vector as described in the eighth aspect.
  • the vector as described above can be introduced into the host cell 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 present invention.
  • the host cell comprises the nucleic acid construct of the seventh aspect or a vector comprising the nucleic acid construct, and the host cell expresses the chimeric antigen receptor of the present invention and another biologically active molecule.
  • the host cell is selected from the immune cells of mammals (such as humans).
  • the immune cells are derived from patients or healthy donors.
  • 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 a cell expressing a chimeric antigen receptor of the present invention, comprising: (1) providing a host cell; (2) introducing the isolated nucleic acid molecule according to the sixth aspect or a vector comprising the nucleic acid molecule into a host cell;
  • the invention also provides a method for producing a cell that co-expresses the chimeric antigen receptor of the present invention and another biologically active molecule, comprising: (1) providing a host cell; (2) introducing the nucleic acid construct of the seventh aspect or a vector comprising the nucleic acid construct into the host cell to obtain a host cell that co-expresses the chimeric antigen receptor and another biologically active molecule.
  • the host cell is selected from immune cells, such as T lymphocytes, NK cells, monocytes, dendritic cells, macrophages and any combination thereof.
  • the immune cell is selected from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof.
  • the host cells are provided from a patient or a healthy donor and are pretreated; the pretreatment includes sorting, activation and/or proliferation of immune cells; in certain embodiments, the pretreatment includes contacting the immune cells with anti-CD3 antibodies and anti-CD28 antibodies, 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 host cell by viral infection. In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the host cell by non-viral vector transfection, such as by transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method, electroporation method, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection.
  • non-viral vector transfection such as by transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method, electroporation method, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection.
  • the method further comprises: amplifying the host cells obtained in step (2).
  • the eleventh aspect of the present invention further provides a modified immune cell, which comprises and expresses the isolated nucleic acid molecule described in the second aspect and the sixth aspect of the present invention.
  • the modified immune cell expresses the chimeric antigen receptor described in the fifth aspect.
  • the twelfth aspect of the present invention further provides a modified immune cell, which comprises and expresses the nucleic acid construct of the seventh aspect of the present invention.
  • the modified immune cell expresses the chimeric antigen receptor of the fifth aspect and the additional biologically active molecule.
  • the immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably, the immune cells are obtained from patients; alternatively, the immune cells are obtained from healthy donors; preferably, the immune cells are derived from T lymphocytes or NK cells.
  • the engineered immune cells express genes related to immune rejection (e.g., TRAC, TRBC, B2M, HLA-A, HLA-B, or HLA-C) and genes of immune co-inhibitory pathways or signaling molecules (e.g., PD- 1, CTLA-4 or LAG-3) is inhibited; preferably, the transcription or expression of the target gene is inhibited by a method selected from gene knockout (e.g., CRISPR, CRISPR/Cas9), homologous recombination, and interfering RNA.
  • gene knockout e.g., CRISPR, CRISPR/Cas9
  • homologous recombination e.g., interfering RNA.
  • the present invention also provides a method for preparing modified immune cells, which comprises: (1) providing immune cells from a patient or a healthy donor; (2) introducing the isolated nucleic acid molecule described in the sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector containing them into the immune cells described in step (1) to obtain immune cells capable of expressing and optionally other biologically active molecules.
  • the immune cells are pretreated, and the pretreatment includes sorting, activation and/or proliferation of the immune cells; more preferably, the pretreatment includes contacting the immune cells with anti-CD3 antibodies and anti-CD28 antibodies, 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 cells by viral infection.
  • the nucleic acid molecule or vector in step (2), is introduced into the immune cell by non-viral vector transfection, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method.
  • non-viral vector transfection such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method.
  • the method further comprises the step of expanding the immune cells obtained in step (2) after step (2).
  • the present invention also provides an immune cell composition, which includes the modified immune cells of any of the foregoing aspects, and optional unmodified and/or unsuccessfully modified immune cells, which do not express the target CAR.
  • an immune cell composition which includes the modified immune cells of any of the foregoing aspects, and optional unmodified and/or unsuccessfully modified immune cells, which do not express the target CAR.
  • the engineered immune cells expressing the target CAR account for 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. Therefore, in another aspect, the present invention also provides an immune cell line containing the immune cell composition.
  • the present invention provides a pharmaceutical composition, which contains the antigen binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which a chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule described in the second aspect or the sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third aspect or the eighth aspect, the host cell described in the fourth aspect or the ninth aspect, the modified immune cell described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect, and a pharmaceutically acceptable carrier and/or excipient.
  • 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 pharmaceutical composition of the present invention comprises: the antigen binding molecule described in the first aspect.
  • the pharmaceutical composition of the present invention comprises: the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, or a vector comprising the same.
  • the pharmaceutical composition of the present invention comprises: the modified immune cell described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect.
  • the host cell described in the first or ninth aspect, the modified immune cell described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect can 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 composition of the present invention should be sterile and stable under production and storage conditions.
  • a preferred dosage form is an injection.
  • Such an injection can be a sterile injection solution.
  • the sterile injection solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for easy storage and use.
  • Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., 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
  • glucose solution e.g., 5% glucose
  • a solution containing a surfactant e.g., 0.01% polysorbate 20
  • a pH buffer solution e.g., phosphate buffer solution
  • Ringer's solution e.g., Ringer's solution, and any combination thereof.
  • the antigen binding molecules described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including the CAR construct co-expressed by the chimeric antigen receptor and another bioactive molecule), the nucleic acid molecules described in the second aspect or the sixth aspect, the nucleic acid construct described in the seventh aspect, the carrier described in the third aspect or the eighth aspect, the host cell described in the fourth aspect or the ninth aspect, the transformed immune cell described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect can be applied by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, leaf sheath, endocytoplasmic reticulum groove, groin, bladder, local (such as, powder, ointment or drops), or nasal route.
  • parenteral administration e.g., intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection. It should be understood by the technician that the route of administration and/or mode will change according to the intended purpose.
  • the pharmaceutical composition of the present invention may include a "therapeutically effective amount” or a "prophylactically effective amount” of the antigen binding molecule of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect (including a CAR construct co-expressing a chimeric antigen receptor and another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third aspect or the eighth aspect, the host cell of the fourth aspect or the ninth aspect, the modified immune cell of the eleventh aspect or the twelfth aspect, or the immune cell of the thirteenth aspect. Immune cell composition.
  • Preventive effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease.
  • “Therapeutically effective amount” refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients with a disease.
  • the antigen binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct co-expressed with a chimeric antigen receptor and another biologically active molecule), the isolated nucleic acid molecule described in the second aspect or the sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third aspect or the eighth aspect, the host cell described in the fourth aspect or the ninth aspect, the modified immune cell described in the eleventh aspect or the twelfth aspect, or the therapeutically effective amount of the immune cell composition described in the thirteenth aspect may vary according to the following factors: 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
  • the present invention provides a method for preventing and/or treating a disease associated with the expression of GPC3 in a subject (e.g., a human), the method comprising administering to a subject in need thereof an effective amount of the antigen binding molecule of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect (including a CAR construct in which a chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third aspect or the eighth aspect, the host cell of the fourth aspect or the ninth aspect, the modified immune cell of the eleventh aspect or the twelfth aspect, or the immune cell composition of the thirteenth aspect, or the pharmaceutical composition of the fifteenth aspect.
  • a subject e.g., a human
  • the method comprising administering to a subject in need thereof an effective amount of the antigen binding molecule of the first aspect of the present invention, the chimeric
  • the disease associated with the expression of GPC3 is selected from a proliferative disease, such as a tumor. In certain embodiments, the disease associated with the expression of GPC3 is a non-tumor-related indication associated with the expression of GPC3.
  • the tumor is a GPC3-positive tumor.
  • the tumor is selected from solid tumors (e.g., liver cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, ovarian clear cell carcinoma, melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, glioma, one or a combination thereof.
  • the tumor is selected from blood tumors (e.g., leukemia, lymphoma, etc.).
  • the method comprises administering to the subject an effective amount of the antigen binding molecule of the first aspect.
  • the method comprises administering to the subject an effective amount of the engineered immune cell of the eleventh or twelfth aspect, or the immune cell composition of the thirteenth aspect.
  • the method comprises the following steps: (1) providing the subject with the immune cells required (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 second aspect or the sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector containing them into the immune cells described in step (1); (3) administering the immune cells obtained in step (2) to the subject for treatment.
  • the immune cells required e.g., T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells
  • the isolated nucleic acid molecule described in the second aspect or the sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector containing them into the immune cells described in step (1)
  • administering the immune cells obtained in step (2) to the subject for treatment.
  • the method administers immune cells expressing a CAR of interest to the subject by dividing the dose into fractions, e.g., one, two, three or more times, administering a partial dose separately, e.g., administering a first percentage of the total dose on the first day of treatment, administering a second percentage of the total dose on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later) treatment day, e.g., administering a third percentage (e.g., the remaining percentage) of the total dose on a subsequent (e.g., third, fourth, fifth, sixth, seventh, eighth, ninth, tenth day or later) treatment day.
  • a partial dose separately, e.g., administering a first percentage of the total dose on the first day of treatment, administering a second percentage of the total dose on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later) treatment day, e.g., administering a third percentage (e.
  • 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 50% of the total dose of cells is administered on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh or later) treatment day.
  • 1/3 of the total dose of cells is administered on the first day of treatment, 1/3 of the total dose of cells is administered on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later) treatment day, and 1/3 of the total dose of cells is administered on a subsequent (e.g., third, fourth, fifth, sixth, seventh, eighth, ninth, tenth day or later) treatment day.
  • the total cell dose comprises 1 ⁇ 10 7 to 10 ⁇ 10 8 target CAR-positive immune cells, for example, comprises (1-5) ⁇ 10 7 to (5-10) ⁇ 10 8 target CAR-positive immune cells.
  • the physician may adjust the dosage or treatment regimen based on clinical circumstances such as the patient's condition, tumor size and stage, or combination therapy drugs.
  • the other agents include (i) increasing cells containing CAR nucleic acids or CAR polypeptides (e.g., immune cells expressing CAR of the present invention, modified immune cells of the present invention). (ii) an agent for improving the efficacy of cells (e.g., immune cells expressing the CAR of the present invention, modified immune cells or immune cell compositions) comprising CAR nucleic acids or CAR polypeptides; (iii) another pharmaceutically active agent with anti-tumor activity.
  • CAR nucleic acids or CAR polypeptides e.g., immune cells expressing CAR of the present invention, modified immune cells of the present invention.
  • an agent for improving the efficacy of cells e.g., immune cells expressing the CAR of the present invention, modified immune cells or immune cell compositions
  • another pharmaceutically active agent with anti-tumor activity e.g., another pharmaceutically active agent with anti-tumor activity.
  • agents can be administered before, simultaneously or after the antigen binding molecules described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a chimeric antigen receptor and a CAR construct co-expressed with another biologically active molecule), the isolated nucleic acid molecules described in the second aspect or the sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third aspect or the eighth aspect, the host cell described in the fourth aspect or the ninth aspect, the modified immune cell described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect.
  • the above method further comprises administering to the subject a second therapy, which can be any therapy known for 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 can be any therapy known for 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 can be applied separately or in combination with the above-described method; or, the second therapy can be applied simultaneously or sequentially with the above-described method.
  • the subject can be a mammal, such as a human.
  • the antigen binding molecule of the first aspect of the present invention the chimeric antigen receptor of the fifth aspect (including the CAR construct co-expressing the chimeric antigen receptor and another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third aspect or the eighth aspect, the host cell of the fourth aspect or the ninth aspect, the modified immune cell of the eleventh aspect or the twelfth aspect, or the immune cell composition of the thirteenth aspect, or the pharmaceutical composition of the fifteenth aspect are provided for the preparation of a drug for preventing and/or treating a disease associated with the expression of GPC3.
  • the dosage, dosage form, administration route, indication, combination therapy and other aspects of the aforementioned treatment methods can be applied to the use of the drug.
  • the present invention provides the antigen binding molecule of the first aspect, the chimeric antigen receptor of the fifth aspect (including the CAR construct co-expressing the chimeric antigen receptor and another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third aspect or the eighth aspect, the host cell of the fourth aspect or the ninth aspect, the modified immune cell of the eleventh aspect or the twelfth aspect, or the immune cell composition of the thirteenth aspect, or the drug of the fifteenth aspect.
  • the drug composition is used for preventing and/or treating diseases related to the expression of GPC3.
  • the dosage, dosage form, administration route, indications, combined therapy and other aspects of the aforementioned treatment methods can be applied to the use of the drug.
  • antigen binding molecule is an antibody molecule or an antigen binding fragment thereof.
  • antibody refers to an immunoglobulin molecule that can specifically bind 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.
  • a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.
  • the term includes not only complete polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chains (such as scFv, di-scFv, (scFv) 2 ) and domain antibodies (including, for example, shark and camel antibodies), as well as fusion proteins including antibodies, and immunoglobulin molecules of any other modified configuration including antigen recognition sites.
  • the antibodies of the present invention are not limited by any specific method for producing antibodies.
  • Antibodies include antibodies of any type, such as IgG, IgA or IgM (or its subclass), and antibodies do not need to belong to any specific type.
  • immunoglobulins can be assigned to different types. There are five major 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 ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • Antibody light chains can be classified as ⁇ (kappa) and ⁇ (lambda) light chains.
  • the heavy chain constant region consists of four 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 binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • VH and VL regions of antibodies can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four 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 form an antigen binding site, respectively.
  • the allocation of amino acids to each region or domain can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.
  • CDR complementarity determining region
  • the variable regions of the heavy and light chains each contain three CDRs, designated 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, such as According to the definition in 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), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol.
  • the CDRs contained in the antibodies or antigen-binding fragments thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia or IMGT numbering systems.
  • framework region or "FR” residues refers to those amino acid residues in the variable region of an antibody 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 polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion".
  • an 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 antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Non-limiting examples of antigen-binding fragments include camelid Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)' 2 fragment, F(ab)' 3 fragment, Fd, Fv, scFv, di-scFv, (scFv) 2 , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (“dsFv”) and single domain antibodies (sdAb, nanobodies) and polypeptides that contain 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.
  • 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 comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • the term “Fab'fragment” means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd fragment (consisting of VH and CH1 domains) of the heavy chain.
  • the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.
  • the Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site. It is generally believed that six CDRs confer antigen binding specificity to an antibody. However, even a variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.
  • Fc means an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain of an antibody bound via a disulfide bond.
  • the Fc fragment of an antibody has a variety of different functions but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are 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 may be used, but variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol.
  • a disulfide bond may also be present between the VH and VL of the scFv.
  • the VH and VL domains may be positioned relative to each other in any suitable arrangement.
  • scFv comprising NH 2 -VH-VH-COOH, NH 2- VL-VL-COOH.
  • the scFv can form any possible engineering 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 single scFvs connected in series to form an antibody.
  • scFv can form (scFv) 2 , which refers to two or more single scFvs connected in parallel to form an antibody.
  • single-domain antibody has the meaning generally understood by those skilled in the art, which refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody.
  • sdAb single-domain antibody
  • Single domain antibodies are also called nanobodies.
  • Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody and/or the ability to compete with the full-length antibody for specific binding to the antigen.
  • Antibody antigen-binding fragments can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.
  • antibody includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • the expression “specific binding” or “specifically directed against” refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed.
  • the strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant ( KD ) of the interaction.
  • KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the 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 method involves measuring the speed of formation and dissociation of antigen binding sites/antigen complexes.
  • association rate constants ka or kon
  • dissociation rate constants kdis or koff
  • K D dissociation constant
  • kon and kdis values can be measured by any effective method.
  • the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR).
  • the dissociation constant can be measured using bioluminescence interferometry or Kinexa.
  • identity is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in two compared sequences is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine)
  • the molecules are identical at that position.
  • the "percent identity” between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared ⁇ 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (a total of 100). 6 positions match).
  • the two sequences are compared when aligned for maximum identity.
  • Such an alignment can be achieved 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.).
  • the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • conservative substitution means an amino acid substitution that does not adversely affect or change 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 substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids 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
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • a vector may include a sequence that replicates directly and autonomously in a cell, or may include a sequence sufficient to allow integration into the host cell DNA.
  • an expression vector When a vector enables the expression of a protein encoded by an inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell.
  • Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and viral vectors, etc.
  • plasmids such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • bacteriophages 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, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).
  • a vector may contain a variety of elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene.
  • the vector may also contain a replication initiation site.
  • the term "episomal vector” means that the vector is capable of replicating without being integrated into the host's chromosomal DNA and being gradually lost by dividing host cells, and also means that the vector replicates extrachromosomally or episomally.
  • viral vector is broadly used to refer to a nucleic acid molecule (e.g., a transfer plasmid) that includes a virally derived nucleic acid element that typically facilitates transfer or integration of the nucleic acid molecule into the genome of a cell, or a viral particle that mediates nucleic acid transfer.
  • the viral particle typically will include various viral components and sometimes host cell components.
  • viral vector can 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 primarily derived from viruses.
  • retroviral vector refers to a viral vector or plasmid that contains structural and functional genetic elements primarily derived from retroviruses, or portions thereof.
  • lentiviral vector refers to a viral vector or plasmid containing structural and functional genetic elements derived primarily from a lentivirus or portions thereof (including LTRs).
  • the terms "lentiviral vector”, “lentiviral expression vector” may be used to refer to a lentiviral transfer plasmid and/or an infectious lentiviral particle.
  • elements e.g., cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.
  • an "integration-defective" retrovirus or lentivirus refers to a retrovirus or lentivirus that has an integrase that cannot integrate the viral genome into the genome of a host cell.
  • the integrase protein is mutated to specifically reduce its integrase activity.
  • An integration-defective lentiviral vector can be obtained by modifying the pol gene encoding the integrase protein to produce a mutant pol gene encoding an integration-defective integrase.
  • the integration-defective viral vector has been described in patent application WO 2006/010834, which is incorporated herein by reference in its entirety.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, 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, NK cells, monocytes, macrophages or dendritic cells, etc.).
  • Host cells can include single cells or cell groups.
  • chimeric antigen receptor refers to a recombinant polypeptide construct comprising at least one extracellular antigen binding domain, a spacer domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain”), which combines antibody-based specificity for a target antigen (e.g., GPC3) with an immune effector cell activation intracellular domain to exhibit specific immune activity for cells expressing the target antigen (e.g., GPC3).
  • a target antigen e.g., GPC3
  • an immune effector cell activation intracellular domain e.g., GPC3
  • the expression "immune effector cell expressing CAR” refers to an immune effector cell expressing CAR and having an antigen-specificity determined by the targeting domain of the CAR.
  • Methods for making CARs are known in the art, see, for example, 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 U.S. Patent Publication 2012/0213783, all of which are incorporated herein by reference in their entirety.
  • extracellular antigen binding domain refers to a polypeptide that is capable of specifically binding to an antigen or receptor of interest.
  • the domain will be capable of interacting with cell surface molecules.
  • an extracellular antigen binding domain can be selected to recognize an antigen that is a target cell surface marker associated with a particular disease state.
  • intracellular signaling domain refers to the portion of a protein that transmits effector signaling function signals and directs cells to perform specialized functions.
  • the effector function of the T cell can be cytolytic activity or helper activity, including the secretion of cytokines.
  • primary signaling domain refers to a portion of a protein that is capable of regulating the primary activation of a TCR complex in a stimulatory or inhibitory manner.
  • Primary signaling domains that act in a stimulatory manner typically contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , 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 that provide the second signal required for the efficient activation and function of T lymphocytes after binding to an antigen, except for antigen receptors or Fc receptors.
  • Non-limiting examples of the 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” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: sterile water, saline, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives.
  • pH regulators include, but are not limited to, phosphate buffers.
  • 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, and the like.
  • Agents that maintain osmotic pressure include, but are not limited to, sugars, 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), etc.
  • 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 meanings generally understood by those skilled in the art, which are capable of stabilizing the desired activity of the active ingredient in the drug, 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 dried whey, albumin or casein) or their degradation products (such as lactalbumin
  • the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
  • such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., a 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
  • glucose solution e.g., 5% glucose
  • a surfactant e.g., 0.01% polysorbate 20
  • a pH buffer solution e.g., a phosphate buffer solution
  • Ringer's solution e.g., Ringer's solution, and any combination thereof.
  • prevention refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor) in a subject.
  • treatment refers to a method implemented in order to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable.
  • treatment can also refer to, compared to the expected survival period (if not receiving treatment), extending the survival period.
  • the term “subject” refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the term “subject” refers to a living organism in which an immune response can be elicited. In certain embodiments, the subject (e.g., a human) has a tumor (e.g., a tumor associated with GPC3), or is at risk of having the above-mentioned disease.
  • a tumor e.g., a tumor associated with GPC3
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount for preventing a disease e.g., a tumor
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on 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 mode of administration of the drug, and other treatments administered simultaneously, etc.
  • immune cell refers to a cell involved in an immune response, such as a cell involved in promoting immune effector functions.
  • immune cells include T cells (e.g., ⁇ / ⁇ T cells and ⁇ / ⁇ T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages.
  • the immune cells of the present invention can be self/autologous ("self") or non-self ("non-self", e.g., allogeneic, isogenic, or xenogeneic).
  • self refers to cells from the same subject
  • allogeneic refers to cells of the same species that are genetically different from the comparison cells
  • isogenic refers to cells from a different subject that are genetically identical to the comparison cells
  • xenogeneic refers to cells from a different subject that are genetically identical to the comparison cells
  • xenogeneic refers to cells from a different subject that are genetically identical to the comparison cells
  • “of” refers to cells that are 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 well known in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes.
  • T cells can 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; CD4T cells) CD4T cells, cytotoxic T cells (CTL; CD8T cells), CD4CD8T cells, CD4CD8T cells or any other T cell subsets.
  • T cells may include primary T cells and memory T cells.
  • 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 in vivo or in vitro to differentiate into immune cells.
  • immune cells include progenitor cells of immune cells, such as hematopoietic stem cells (HSCs) contained in CD34+ cell populations derived from umbilical cord blood, bone marrow or flowing peripheral blood, which differentiate into mature immune cells after administration in a subject, or they can be induced in vitro to differentiate into mature immune cells.
  • HSCs hematopoietic stem cells
  • modified immune cell refers to an immune cell expressing any CAR described herein, or an immune cell into which any isolated nucleic acid or vector described herein is introduced.
  • 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 for introducing polynucleotide constructs into cells are known in the art. In some embodiments, a stable transformation method can be used to integrate the polynucleotide construct into the genome of the cell.
  • a transient transformation method can be used to transiently express a polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell.
  • a virus-mediated method can be used.
  • Polynucleotides can be introduced into cells by any suitable method, such as recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc.
  • Transient transformation methods include, for example, but not limited to microinjection, electroporation, or microparticle bombardment.
  • Polynucleotides may be included in a vector, such as a plasmid vector or a viral vector.
  • immune effector function refers to the function or response of immune effector cells to enhance or promote immune attack on target cells (e.g., killing of target cells, or inhibiting their growth or proliferation).
  • target cells e.g., killing of target cells, or inhibiting their growth or proliferation.
  • the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines.
  • the terms "about” or “approximately” when used with a numerical variable generally means that the value of the variable is within experimental error (e.g., within a 95% confidence interval for the mean) or within ⁇ 10% or wider of the specified value.
  • the therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient, mainly because solid tumors have complex tumor microenvironments and high tumor heterogeneity.
  • the present invention provides a CAR targeting GPC3 or an immune cell comprising the CAR, which improves the killing of cells expressing tumor antigens and reduces their off-target toxicity to a certain extent by specifically targeting GPC3, thereby enhancing the tumor killing effect of CAR-T cells.
  • Figure 1A shows the results of the killing activity test of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T) against HepG2 target cells.
  • Figure 1B shows the results of the killing activity test of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T) against Huh7 target cells.
  • Figure 2A shows the results of the detection of IFN- ⁇ secretion levels in HepG2 target cells after activation of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T).
  • Figure 2B shows the results of the detection of TNF- ⁇ secretion levels in HepG2 target cells after activation of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T).
  • Figure 2C shows the results of the IFN- ⁇ secretion level detection in Huh7 target cells after activation of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T).
  • Figure 2D shows the results of the detection of TNF- ⁇ secretion levels in Huh7 target cells after activation of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, and AB9-T).
  • the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Compiled Molecular Biology Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present invention.
  • the full human phage library was screened using biotinylated GPC3 and SV magnetic beads, and the screening products were titrated by phage plating.
  • the first round of panning products were mixed with PBST and the second and third rounds of panning were performed according to the above steps.
  • a total of 4 fully human anti-GPC3 monoclonal antibodies CB6, AB9, CE3 and CH6 were screened. After sequencing and analyzing the above monoclonal antibodies, the sequences of VH and VL were obtained, and the sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 were obtained according to the Kabat, IMGT, Chothia and AbM numbering systems (see Tables 1 and 2 for specific sequences).
  • VH and VL of the above-mentioned fully human antibody were connected through a linker (SEQ ID NO: 76) to obtain scFv.
  • the sequence information of each scFv is shown in the following table.
  • the candidate scFv sequences and the positive control scFv sequence (P7D4) were constructed in the TGEX-KAL vector, and then transfected into expi293 cells for expression and purification of scFv-Fc protein.
  • the results of SEC analysis experiments showed (Table 4) that the main peak area of the four candidate scFv sequences (CB6, AB9, CE3, CH6) was greater than 90%.
  • GPC3 scFv-Fc protein a cell line expressing GPC3 (293T/GPC3+) was selected for cell binding assay.
  • Mouse IgG isotype antibody (Mouse IgG Isotype Control, from Thermo Fisher Sci.) was used as a negative control, and anti-P7D4 antibody (for related sequences, see WO2017020812) was used as a positive control.
  • a CAR lentiviral expression vector was further constructed.
  • the intracellular domain of CD137 (4-1BB) and the ITAM region of CD3 ⁇ were used as activation signals, fused with the above scFv, and CD8 ⁇ signal peptide, CD8 hinge region, CD8 transmembrane region were added to construct a chimeric antigen receptor expression vector.
  • the chimeric antigen receptor structure is shown in Table 6 below.
  • Human PBMC cells were isolated using lymphocyte separation medium (GE), cultured in an incubator at 37°C and 5% CO2 , 100 ⁇ L/mL of CD3 antibody and CD28 antibody were added, mixed thoroughly, and incubated at room temperature for 15 minutes.
  • GE lymphocyte separation medium
  • the tube remains in the magnet and is gently inverted to pour out the cells.
  • the cell density was adjusted to 1 ⁇ 10 6 cells/mL, and cytokine and antibody complexes (configured at a final concentration of 300 U/mL of IL-2, 10 ng/mL of IL-7, 5 ng/mL of IL-15, 500 ng/mL of Anti-CD3 (OKT3), and 2 ⁇ g/mL of Anti-CD28) were added and cultured for 48 hours.
  • CAR-T cells expressing the CARs described in Example 2 (CB6-T, AB9-T, CE3-T, and CH6-T) were obtained by the above method.
  • the nucleic acid sequence encoding CAR is expressed under the drive of the promoter, and the T cells transfected with lentivirus are labeled with GPC3 antigen and measured by flow cytometry to reflect the expression level of CAR on the surface of T cells.
  • the CAR positive rate of the CAR-T cells obtained in Example 3 was detected by the above method, and the FACS test results are shown in Table 7 below.
  • HEPG2-luc cells and Huh7-luc cells were digested with 0.25% trypsin, and digestion was terminated with 1640 medium containing 10% FBS. After centrifugation, the cells were resuspended, and the cell density was adjusted to 1 ⁇ 10 5 /mL.
  • the target cells HEPG2-luc were inoculated in a 96-well plate at 100 ⁇ L/well, and the cells were placed in a 5% CO 2 37°C incubator for 30 minutes. CAR-T was collected by centrifugation and resuspended in 10% FBS 1640 medium.
  • GPC3-CAR and blank T cells (UTD) without CAR transfection were used as effector cells, and then added to a 96-well plate containing HEPG2-luc at different E/T (effector cell/target cell) ratios, 100 ⁇ L/well, and the final volume was supplemented to 200 ⁇ L/well, and cultured in a 5% CO 2 37°C incubator for 18 to 24 hours. After the culture was completed, the well plate was removed from the incubator, 20ul of fluorescence detection reagent was added, and the fluorescence reading was detected using an enzyme reader.
  • the results of the CAR-T killing activity test are shown in Figure 1.
  • the four CAR-T cells (CB6-T, AB9-T, CE3-T, CH6-T) constructed in this application can effectively lyse tumor cells in different cell lines (HepG2, Huh7) and different E/T ratios.
  • the effector cell/target cell ratio is 1, the lysis rate of tumor cells is as high as about 99%.
  • HepG2-luc and Huh7-luc cells were collected, and the cell density was adjusted to 1 ⁇ 10 5 /mL using culture medium.
  • the target cells were inoculated in a 96-well plate at 100 ⁇ L/well, and the CAR-T cells, GPC3-CAR and blank T cells without CAR transfection were resuspended in culture medium as effector cells, and then added to the 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 was supplemented to 200 ⁇ L/well, and cultured in a 5% CO 2 37°C incubator overnight.
  • E/T effector cell/target cell
  • CB6-T, AB9-T, CE3-T, and CH6-T The four types of CAR-T cells constructed in the present application can kill tumor cells to varying degrees and release IFN- ⁇ (with HepG2 as the target cell, the test results are shown in FIG. 2A ; with Huh7 as the target cell, the test results are shown in FIG. 2C ) and TNF- ⁇ (with HepG2 as the target cell, the test results are shown in FIG. 2B ; with Huh7 as the target cell, the test results are shown in FIG. 2D ).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un récepteur antigénique chimérique se liant spécifiquement à GPC3, une cellule immunitaire modifiée exprimant le récepteur antigénique chimérique, et un procédé de préparation de la cellule immunitaire modifiée. La présente invention concerne également un procédé d'utilisation dudit antigène chimérique et de ladite cellule immunitaire pour prévenir et/ou traiter des maladies associées à l'expression de GPC3.
PCT/CN2023/132244 2022-11-28 2023-11-17 Récepteur antigénique chimérique se liant spécifiquement à gpc3 et son utilisation WO2024114404A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211499852.0 2022-11-28
CN202211499852 2022-11-28

Publications (1)

Publication Number Publication Date
WO2024114404A1 true WO2024114404A1 (fr) 2024-06-06

Family

ID=91322969

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/132244 WO2024114404A1 (fr) 2022-11-28 2023-11-17 Récepteur antigénique chimérique se liant spécifiquement à gpc3 et son utilisation

Country Status (1)

Country Link
WO (1) WO2024114404A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103168104A (zh) * 2010-07-22 2013-06-19 加利福尼亚大学董事会 抗肿瘤抗原抗体及其使用方法
CN109021108A (zh) * 2018-08-27 2018-12-18 南京医科大学 抗gpc3全人源化抗体、其嵌合抗原受体细胞及应用
US20220056408A1 (en) * 2018-12-13 2022-02-24 Carsgen Therapeutics Co., Ltd. Immune effector cell targeting gpc3 and application thereof
WO2022166876A1 (fr) * 2021-02-03 2022-08-11 江苏先声药业有限公司 Anticorps monoclonal pour reconnaître de manière spécifique le glypicane-3 et son utilisation
WO2022171100A1 (fr) * 2021-02-10 2022-08-18 江苏先声药业有限公司 Anticorps gpc3 humanisé et son utilisation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103168104A (zh) * 2010-07-22 2013-06-19 加利福尼亚大学董事会 抗肿瘤抗原抗体及其使用方法
CN109021108A (zh) * 2018-08-27 2018-12-18 南京医科大学 抗gpc3全人源化抗体、其嵌合抗原受体细胞及应用
US20220056408A1 (en) * 2018-12-13 2022-02-24 Carsgen Therapeutics Co., Ltd. Immune effector cell targeting gpc3 and application thereof
WO2022166876A1 (fr) * 2021-02-03 2022-08-11 江苏先声药业有限公司 Anticorps monoclonal pour reconnaître de manière spécifique le glypicane-3 et son utilisation
WO2022171100A1 (fr) * 2021-02-10 2022-08-18 江苏先声药业有限公司 Anticorps gpc3 humanisé et son utilisation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU QIAN-YONG; MARK GILBERT; LI YI-PING: "Recent Progress in the Research and Development of CAR-T Products", CHINESE JOURNAL OF NEW DRUGS, GAI-KAN BIANJIBU, BEIJING, CN, vol. 30, no. 19, 31 December 2021 (2021-12-31), CN , pages 1759 - 1767, XP009555269, ISSN: 1003-3734 *
WU, X.Q. ET AL.: "Combined Antitumor Effects of Sorafenib and GPC3-CAR T Cells in Mouse Models of Hepatocellular Carcinoma", MOLECULAR THERAPY, vol. 27, no. 8, 31 August 2019 (2019-08-31), XP093016416, DOI: 10.1016/j.ymthe.2019.04.020 *

Similar Documents

Publication Publication Date Title
JP7425604B2 (ja) 抗ｃｔｌａ４－抗ｐｄ－１二機能性抗体、その医薬組成物および使用
JP7578938B2 (ja) Ｂ７－ｈ３に対するモノクローナル抗体および細胞治療におけるその使用
JP7280828B2 (ja) Ｂｃｍａを標的とする抗体およびその使用
JP7510246B2 (ja) 抗グリピカン３抗体およびその使用
JP7082620B2 (ja) 抗ｐｄ１モノクローナル抗体、その医薬組成物およびその使用
CN114401989B (zh) 靶向bcma的抗体及嵌合抗原受体
JP2022515487A (ja) クローディン１８．２結合部分およびその利用
US20230203178A1 (en) Chimeric antigen receptor car or car construct targeting bcma and cd19 and application thereof
CN116234911A (zh) 特异性结合msln的嵌合抗原受体及其应用
CN113754780A (zh) 靶向cldn18.2的嵌合抗原受体、其组合物及用途
CN115232209B (zh) 靶向gprc5d的抗体及其用途
EP3712179A1 (fr) Unité de liaison ciblant la protéine d'activation des fibroblastes et application associée
CN111454358A (zh) 一种嵌合抗原受体及其应用
EP3730519B1 (fr) Anticorps ou fragment de liaison à l'antigène de celui-ci reconnaissant spécifiquement des tumeurs malignes de lymphocytes b, récepteur antigénique chimérique le comprenant, et utilisations associées
KR20240046224A (ko) 이중특이성 항체 및 그 용도
CN109970859B (zh) Glypican-3特异性抗体及其特异性CAR-T细胞
WO2024114404A1 (fr) Récepteur antigénique chimérique se liant spécifiquement à gpc3 et son utilisation
WO2023246578A1 (fr) Récepteur antigénique chimérique se liant spécifiquement à gpc3 et son utilisation
WO2024114410A1 (fr) Anticorps ciblant gpc3 et son utilisation
CN116063527A (zh) 靶向间皮素的抗体及其用途
WO2023246574A1 (fr) Anticorps ciblant gpc3 et son utilisation
TW202204419A (zh) 嵌合抗原受體
CN118234755A (zh) 特异性结合msln的嵌合抗原受体及其应用
WO2023274355A1 (fr) Combinaison de cellules souches mésenchymateuses modifiées et de cellules effectrices immunitaires pour le traitement de tumeurs
CN114478767A (zh) 特异性结合cd33的嵌合抗原受体及其应用

Legal Events

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

Ref document number: 23896556

Country of ref document: EP

Kind code of ref document: A1