GPC3 car and use thereof

Abstract

The present application relates to a GPC3 CAR and the use thereof, and specifically relates to a chimeric antigen receptor, comprising an antigen-binding domain, a hinge region and a transmembrane domain, the antigen-binding domain comprising a heavy chain variable region VH and a light chain variable region VL, and the VH and VL comprising at least one CDR. Further provided in the present application are a modified immune cell comprising the chimeric antigen receptor, a pharmaceutical composition, a nucleic acid molecule encoding the chimeric antigen receptor, a vector comprising the nucleic acid molecule, and a cell comprising the vector. Further provided in the present application is the use of the chimeric antigen receptor in the prevention and / or treatment of diseases.

Description

GPC3 CAR and uses thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a chimeric antigen receptor targeting GPC3. BACKGROUND

[0002] Glypican-3 (GPC3) is a cell surface protein belonging to the heparan sulfate proteoglycan family. Its core protein consists of 580 amino acids, with a size of about 70 kDa, and is anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor, with two heparan sulfate (HS) side chains near the C-terminus. After processing by furin protease, the protein can be cleaved into an approximately 40 kDa N-terminal soluble protein (sGPC3) and a 30 kDa C-terminal membrane protein.

[0003] GPC3 is a cancer-associated glycoprotein that is highly expressed in fetal liver but not in normal adult liver tissue, but is re-expressed in hepatocellular carcinoma, and is closely related to the development of hepatocellular carcinoma. Not only is the detection rate higher in the early stages of hepatocellular carcinoma, but as the hepatocellular carcinoma develops, the detection rate also increases. In addition, GPC3 is also expressed in tumors such as melanoma, ovarian clear cell carcinoma, and yolk sac tumor. Considering the specific high expression of GPC3 in hepatocellular carcinoma, melanoma, and other tumors, it is considered a candidate target for tumor immunotherapy. SUMMARY

[0004] The present application provides a chimeric antigen receptor targeting GPC3. In the present application, the chimeric antigen receptor has one or more of the following properties: (1) capable of binding to GPC3 with good binding activity; (2) capable of binding to GPC3 expressed on the surface of target cells; (3) capable of killing GPC3-positive tumors; (4) capable of being expressed on the surface of immune cells. The present application also provides a modified immune cell comprising the chimeric antigen receptor. In the present application, the modified immune cell has one or more of the following properties: (1) capable of binding to GPC3 with good binding activity; (2) capable of binding to GPC3 expressed on the surface of target cells; (3) capable of killing GPC3-positive tumors.

[0005] In one aspect, the present application provides a chimeric antigen receptor comprising an antigen binding domain, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising an antibody heavy chain variable region VH, the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the amino acid sequence of the HCDR1 being set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 being set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 3.

[0006] In certain embodiments, the chimeric antigen receptor comprises an intracellular signaling domain.

[0007] In certain embodiments, the chimeric antigen receptor comprises a costimulatory domain.

[0008] In certain embodiments, the chimeric antigen receptor comprises a signal peptide.

[0009] In certain embodiments, the antigen binding domain further comprises a light chain variable region VL, the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, the amino acid sequence of the LCDR1 being set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 being set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 being set forth in SEQ ID NO: 11.

[0010] In certain embodiments, the antigen binding domain comprises a VH comprising HCDR1, HCDR2, and HCDR3, and a VL comprising LCDR1, LCDR2, and LCDR3, the amino acid sequence of the HCDR1 being set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 being set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 being set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 being set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 being set forth in SEQ ID NO: 11.

[0011] In certain embodiments, the antigen binding domain comprises a VH having the amino acid sequence set forth in SEQ ID NO: 8 and a VL having the amino acid sequence set forth in SEQ ID NO: 16.

[0012] In certain embodiments, the antigen binding domain comprises a scFv comprising a VH having the amino acid sequence set forth in SEQ ID NO: 8 and a VL having the amino acid sequence set forth in SEQ ID NO: 16.

[0013] In certain embodiments, the antigen binding domain comprises a scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17.

[0014] In certain embodiments, the antigen binding domain is capable of binding to GPC3.

[0015] In certain embodiments, the chimeric antigen receptor comprises an antigen binding domain comprising a GPC3 scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17, a CD8 hinge region, the amino acid sequence of which is set forth in SEQ ID NO: 20, a CD28 transmembrane domain, the amino acid sequence of which is set forth in SEQ ID NO: 21, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0016] In another aspect, the present application provides a modified immune cell, wherein the immune cell comprises the chimeric antigen receptor.

[0017] In certain embodiments, the immune cell is selected from one or more of the following: a T cell, an NK cell, an NKT cell, an iNKT cell, a gd T cell, a dendritic cell, and a macrophage.

[0018] In certain embodiments, the immune cell further comprises a fusion protein.

[0019] In certain embodiments, the fusion protein comprises an IL-10, a CD8 hinge region, and a CD8 transmembrane domain.

[0020] In another aspect, the present application provides an isolated nucleic acid molecule encoding the chimeric antigen receptor.

[0021] In certain embodiments, the nucleic acid molecule further encodes a fusion protein.

[0022] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0023] In another aspect, the present application provides a cell comprising the nucleic acid molecule and / or the vector.

[0024] In another aspect, the present application provides a pharmaceutical composition comprising the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0025] On the other hand, this application provides the use of the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0026] On the other hand, this application provides a method for preventing and / or treating diseases and / or conditions, comprising administering the chimeric antigen receptor, the modified immune cells, the nucleic acid molecules, the carrier, the cells, and / or the pharmaceutical composition to a subject in need.

[0027] On the other hand, this application provides the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition for the prevention and / or treatment of diseases and / or conditions.

[0028] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0029] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0030] Figure 1 shows the chimeric antigen receptor structure design of the GPC3 scFv described in this application, which uses different hinge regions and transmembrane structural domains.

[0031] Figure 2 shows the expression level results of GPC3 CAR with different hinge regions as described in this application.

[0032] Figure 3 shows the cytokine secretion levels of GPC3 CARs with different hinge regions as described in this application.

[0033] Figure 4A shows the killing effect of GPC3 CAR with different hinge regions on target cells Hep3B as described in this application; Figure 4B shows the killing effect of GPC3 CAR with different hinge regions on target cells Huh7 as described in this application; Figure 4C shows the killing effect of GPC3 CAR with different hinge regions on non-target cells MB231 as described in this application.

[0034] Figure 5 shows the expression level results of GPC3 CARs with different transmembrane domains described herein.

[0035] Figure 6 shows the cytokine secretion level results of GPC3 CARs with different transmembrane domains described herein.

[0036] Figure 7A shows the killing ability results of GPC3 CARs with different transmembrane domains described herein against target cells Hep3B; Figure 7B shows the killing ability results of GPC3 CARs with different transmembrane domains described herein against non-target cells MB231.

[0037] Figure 8 shows the results of tumor-bearing animal experiments of GPC3 CARs described herein.

[0038] Figure 9 shows the expression level results of 8F8 CARs with 8F8 scFv described herein.

[0039] Figure 10 shows the cytokine secretion level results of 8F8 CARs with 8F8 scFv described herein.

[0040] Figure 11 shows the expression level results of ROR1 CARs with ROR1 scFv described herein.

[0041] Figure 12 shows the cytokine secretion level results of ROR1 CARs with ROR1 scFv described herein.

[0042] Figure 13 shows the expression level results of GPC3 CARs on iNKT cells described herein.

[0043] Figure 14A shows the killing ability results of GPC3 CAR-iNKT against target cells Hep3B described herein; Figure 14B shows the killing ability results of GPC3 CAR-iNKT against target cells Huh7 described herein.

[0044] Figure 15 shows the expression level results of GPC3 CARs on γδT cells described herein.

[0045] Figure 16 shows the killing ability results of GPC3 CAR-γδT against target cells Hep3B described herein.

[0046] Figure 17 shows the structural design of GPC3 CAR and membrane-bound IL-10-GPC3 CAR described herein.

[0047] Figure 18 shows the results of the expression levels of the GPC3 CAR and the membrane-bound IL-10-GPC3 CAR on T cells.

[0048] Figure 19 shows the results of the cytokine secretion levels of the GPC3 CAR and the membrane-bound IL-10-GPC3 CAR.

[0049] Figure 20A shows the results of the killing ability of the GPC3 CAR-T and the membrane-bound IL-10-GPC3 CAR-T on the target cell Huh7; Figure 20B shows the results of the killing ability of the GPC3 CAR-T and the membrane-bound IL-10-GPC3 CAR-T on the target cell Hep3B; Figure 20C shows the results of the killing ability of the GPC3 CAR-T and the membrane-bound IL-10-GPC3 CAR-T on the non-target cell MB231. DETAILED DESCRIPTION

[0050] The present application will be readily understood by the following detailed description in conjunction with the accompanying drawings, and the foregoing information. Other advantages of the application will be realized and appreciated by persons of ordinary skill in the art.

[0051] TERMS

[0052] In the present application, the terms "GPC3", "glypican 3", and "Glypican-3" can be used interchangeably, and generally refer to a cell surface protein belonging to the heparan sulfate proteoglycan family. In the present application, the GPC3 can be the intact GPC3 and functionally active fragments, homologues, analogues, variants, or derivatives thereof. For example, the GPC3 can be the full-length GPC3, or a truncated GPC3 retaining functional activity. In the present application, the GPC3 can be of any species origin. For example, the GPC3 can be human GPC3. In the present application, the GPC3 can be wild-type, or artificially modified. For example, the GPC3 can be a modified GPC3.

[0053] In the present application, the terms "chimeric antigen receptor" and "CAR" can be used interchangeably, generally refer to a recombinant polypeptide comprising at least an extracellular domain that specifically binds to an antigen or target, a transmembrane domain, and an intracellular domain. In the present application, the extracellular domain can comprise an antigen binding domain and a hinge region. In the present application, the extracellular domain can further comprise a signal peptide. In the present application, the intracellular domain can comprise an intracellular signaling domain. In the present application, the intracellular domain can further comprise one or more costimulatory domains. In the present application, the CAR can comprise, in order, an optional antigen binding domain, a hinge region, a transmembrane domain region, and / or an intracellular signaling domain. In the present application, the CAR can comprise, in order, an optional antigen binding domain, a hinge region, a transmembrane domain region, a costimulatory domain, and / or an intracellular signaling domain. In the present application, the CAR can comprise, in order, an optional signal peptide, an antigen binding domain, a hinge region, a transmembrane domain region, a costimulatory domain, and / or an intracellular signaling domain. In the present application, the CAR can be secreted by a cell or artificially synthesized.

[0054] In the present application, the term "signal peptide (SP)" generally refers to a peptide, polypeptide, or protein that can direct a nascent protein to the endoplasmic reticulum upon protein translation or post-translationally. In the present application, the signal peptide can comprise a signal peptide derived from CD8a.

[0055] In the present application, the term "antigen binding domain" generally refers to a protein or polypeptide that is capable of specifically binding to an antigen or target. In the present application, an antigen binding domain can comprise an antigen binding protein. In the present application, the antigen binding protein can comprise a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that facilitates binding of the antigen binding portion to an antigen. In the present application, the antigen binding protein can be wild-type, or artificially engineered. In the present application, the antigen binding protein can comprise a protein scaffold of, for example, antibody origin or an alternative or artificial scaffold with grafted CDRs or CDR derivatives. In the present application, the antigen binding protein can be an antibody or an antigen binding fragment thereof, as well as variants, homologues, derivatives or analogues thereof. For example, the antigen binding fragment can be a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH or dAb fragment. In the present application, the antigen binding protein can comprise a heavy chain variable region (VH). In the present application, the antigen binding protein can further comprise a light chain variable region (VL). In the present application, the VH and VL regions can comprise regions of hypervariability, termed complementarity determining regions (CDRs) or hypervariable regions (HVRs), alternating with regions that are more conserved, termed framework regions (FRs). In the present application, the VH and VL each comprise three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. For example, the variable domains of the heavy and light chains each comprise four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4). In the present application, the antigen binding domain can specifically bind to a tumor antigen or target. In the present application, the antigen binding domain can specifically bind to a GPC3 positive tumor. In the present application, the antigen binding domain can specifically bind to a solid tumor and / or a blood tumor. In the present application, the antigen binding domain can specifically bind to a liver cancer, an ovarian cancer, a melanoma, a pancreatic cancer, a lung cancer, a colon cancer, a breast cancer, a prostate cancer, a non-small cell lung cancer, a small cell lung cancer, a squamous cell carcinoma, a renal cell carcinoma, a colorectal cancer, a gastric cancer, a glioma, an ovarian clear cell carcinoma, an yolk sac tumor, and / or a hepatocellular carcinoma. In the present application, the antigen binding domain can comprise an antigen binding protein, which can be an scFv. In the present application, the scFv can comprise a VH and a VL. In the present application, the VH and VL of the scFv can be connected by a linker. For example, the linker can be a flexible linker. In the present application, the scFv can specifically bind to a tumor antigen or target. In the present application, the scFv can specifically bind to GPC3.

[0056] In the present application, the term "Hinge" generally refers to a peptide, polypeptide, or protein molecule. In the present application, the Hinge can be flexible, allowing independent movement of the antigen binding domain. In the present application, the Hinge can be subdivided into upper, middle, and lower hinge domains. In the present application, the Hinge can comprise a Hinge region derived from CD28, IgGl, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc epsilon R gamma, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. In the present application, the Hinge can comprise a Hinge region derived from CD8.

[0057] In the present application, the term "Transmembrane domain (TM)" generally refers to a domain of a peptide, polypeptide, or protein that is capable of spanning the plasma membrane of a cell. These domains can be used to anchor an extracellular domain to the cell membrane. For example, the Transmembrane domain can comprise a Transmembrane domain selected from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 epsilon, CD3 zeta, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc epsilon R gamma, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the Transmembrane domain can comprise a Transmembrane domain derived from CD28.

[0058] In the present application, the term "co-stimulatory domain" generally refers to a portion of a CAR that is capable of transducing an effector signal in the intracellular signaling region. For example, the co-stimulatory domain can include a co-stimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc epsilon RI gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ligand for CD83, CD40, and MyD88. For example, the co-stimulatory domain can be a co-stimulatory domain derived from 4-1BB.

[0059] In the present application, the term "intracellular signaling domain" generally refers to an intracellular region that can generate a signal that promotes the immune effector function of a CAR-containing cell (e.g., a CAR-T cell or a CAR-iNKT cell). For example, the intracellular signaling region can include an intracellular signaling region of one or more proteins selected from the group consisting of CD3 zeta, CD3 delta, CD3 gamma, CD3 epsilon, CD79a, CD79b, Fc epsilon RI gamma, Fc epsilon RI beta, Fc gamma RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (KSHV), DAP10, DAP-12, and a domain comprising at least one ITAM. For example, the intracellular signaling region can be a signaling domain derived from CD3 zeta.

[0060] The proteins and / or amino acid sequences referred to in the present application are also understood to encompass variants or homologues of the recited proteins having the same or similar function. In the present application, the variants can be proteins or polypeptides having one or more amino acid substitutions, deletions or additions in the amino acid sequence of the recited protein (e.g., the chimeric antigen receptors described herein). For example, the functional variants can include proteins or polypeptides that have been altered by at least 1, e.g., 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions and / or insertions. The functional variants can substantially retain the biological properties of the recited protein or the recited polypeptide prior to the alterations (e.g., substitutions, deletions or additions). For example, the functional variants can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the recited protein or the recited polypeptide prior to the alterations. For example, the substitutions can be conservative substitutions. In the present application, a portion of the amino acid sequence of the chimeric antigen receptor can be homologous to the corresponding amino acid sequence in an antibody from a particular species, or belong to a particular class. In the present application, the homologues can be proteins or polypeptides having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the recited protein and / or the recited polypeptide (e.g., the chimeric antigen receptors described herein).

[0061] In the present application, the term "immune cell" generally refers to a cell involved in an immune response. For example, the immune cell can be an immune cell that performs an effector function. For example, the performance of an effector function can include clearance of a foreign antigen or promotion of an immune effector response, etc. In the present application, the immune effector cell can include a T cell, a B cell, a natural killer cell (NK cell), a macrophage, an NKT cell, an iNKT cell, a γδ T cell, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte, a peripheral blood mononuclear cell, an embryonic stem cell, a lymphoid progenitor cell, and / or a pluripotent stem cell. For example, the immune effector cell can be a T cell. For example, the immune cell can be an iNKT cell. For example, the immune effector cell can be a γδ T cell.

[0062] In the present application, the term "fusion protein" generally refers to a protein composed of two or more polypeptides. In the present application, the fusion protein is generally a contiguous polypeptide formed by the joining together of two or more polypeptide components that do not normally associate in nature, through peptide bonds at the amino and carboxy termini. In the present application, the association refers to the two or more polypeptide components can be directly associated or indirectly associated. In the present application, the indirect association can be through a linker. For example, the fusion protein can be a membrane-bound cytokine. For example, the fusion protein can be a membrane-bound IL-10. For example, the fusion protein can comprise IL-10 and a transmembrane domain. For example, the fusion protein can comprise IL-10, a hinge region, and a transmembrane domain. For example, the fusion protein can comprise IL-10, a CD8 hinge region, and a CD8 transmembrane domain.

[0063] In the present application, the term "pharmaceutically acceptable carrier" generally refers to a nontoxic material that does not interfere with the effectiveness or the biological activity of the active ingredient. For example, the pharmaceutically acceptable carrier includes a pharmaceutically acceptable carrier, excipient, or stabilizer, which is nontoxic to the cells or mammals exposed to it at the dosages and concentrations employed. For example, the physiologically acceptable carrier can be water, salt, protein, polysaccharide, lipid, or inactivated viral particle.

[0064] In the present application, the term "preventing and / or treating" generally refers to preventing and / or treating a disease. For example, the preventing and / or treating can be preventing the onset of the disease, slowing or reversing the disease progression, preventing or slowing the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith, or preventing further increase in the severity of the disease and any symptoms associated therewith. In the present application, the disease can be a tumor disease. For example, preventing or alleviating the onset of one or more symptoms associated with the tumor, reducing the severity and duration of the tumor and symptoms associated therewith.

[0065] In the present application, the term "tumor" generally refers to any new pathological tissue proliferation, which presents tumor antigens recognizable by the immune system. In the present application, the tumor can include benign or malignant tumors (cancers). In the present application, the cancer can be metastatic cancer and non-metastatic cancer. In the present application, the tumor can include solid tumors and blood tumors. In the present application, the solid tumor generally refers to a tangible tumor that can be detected by clinical examination means. For example, the solid tumor can include a neoplasm or a solid lesion formed by abnormal cell growth. In the present application, the blood tumor generally refers to a type of hematopoietic system disease. In the present application, the blood tumor can include various types of leukemia, multiple myeloma or malignant lymphoma. In the present application, the tumor can be a GPC3-expressing tumor. For example, the tumor can be liver cancer, ovarian cancer, melanoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, glioma, ovarian clear cell carcinoma, yolk sac tumor and / or hepatocellular carcinoma.

[0066] In the present application, the term "subject" generally refers to a human or a non-human animal, including but not limited to a cat, a dog, a horse, a pig, a cow, a sheep, a rabbit, a mouse, a rat or a monkey.

[0067] In the present application, the term "comprising" generally means including the recited features but not excluding other elements.

[0068] In the present application, the term "about" generally means a variation within a range of 0.5-10% above or below the specified numerical value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified numerical value.

[0069] DETAILED DESCRIPTION

[0070] Chimeric antigen receptor

[0071] In one aspect, the present application provides a chimeric antigen receptor, which can comprise an antigen binding domain, a hinge region and a transmembrane domain, wherein the antigen binding domain is capable of binding to GPC3, the hinge region is derived from the hinge region of CD8, and the transmembrane domain is derived from the transmembrane domain of CD28.

[0072] In the present application, the chimeric antigen receptor has one or more of the following properties: (1) capable of binding to GPC3 with good binding activity; (2) capable of binding to GPC3 expressed on the surface of target cells; (3) capable of producing a killing effect on GPC3-positive tumors; (4) capable of being expressed on the surface of immune cells.

[0073] In the present application, the hinge region can be any species origin. For example, the hinge region can be murine, rabbit, goat, llama, or human origin. For example, the hinge region can be a human CD8 hinge region. In the present application, the hinge region can be a variant thereof. For example, the variant includes substitution, deletion, and / or addition of one or more amino acids to the amino acid sequence of the CD8 hinge region. For example, 1-30, 1-20, or 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions. In the present application, the hinge region can be a homolog thereof. For example, the homolog can be an amino acid sequence having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the CD8 hinge region. In the present application, the amino acid sequence of the hinge region can be as set forth in SEQ ID NO: 20.

[0074] In the present application, the transmembrane domain can be derived from a transmembrane domain of CD28. In the present application, the transmembrane domain can be any species origin. For example, the transmembrane domain can be murine, rabbit, goat, llama, or human origin. For example, the transmembrane domain can be a human CD28 transmembrane domain. In the present application, the transmembrane domain can be a variant thereof. For example, the variant includes substitution, deletion, and / or addition of one or more amino acids to the amino acid sequence of the CD28 transmembrane domain. For example, 1-30, 1-20, or 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions. In the present application, the transmembrane domain can be a homolog thereof. For example, the homolog can be an amino acid sequence having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the CD28 transmembrane domain. In the present application, the amino acid sequence of the transmembrane domain can be as set forth in SEQ ID NO: 21.

[0075] In the present application, CDRs, also known as complementarity determining regions, are portions of the variable region. The amino acid residues of this region can contact the antigen or the epitope of the antigen. The CDRs of an antigen binding protein can be determined by various encoding systems, such as CCG, Kabat, Chothia, IMGT, AbM, integrated consideration of Kabat / Chothia, etc. These encoding systems are known in the art, and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. A person skilled in the art can determine the CDR regions according to the sequence and structure of the antigen binding protein using different encoding systems. The CDR regions can be different using different encoding systems. In the present application, the CDRs encompass the CDR sequences determined according to any CDR partitioning method; also encompass variants thereof, which include substitution, deletion and / or addition of one or more amino acids of the CDR amino acid sequence. For example, 1-30, 1-20 or 1-10 amino acid substitutions, deletions and / or insertions; also encompass homologues thereof, which can be an amino acid sequence having at least about 85% (for example, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the CDR amino acid sequence. For example, the CDRs of the antigen binding protein described in the present application can be determined using IMGT. For example, the CDRs of the antigen binding protein described in the present application can be determined using Kabat.

[0076] In the present application, the chimeric antigen receptor can comprise an antigen binding domain, which can comprise an antigen binding protein, which can comprise at least one CDR in the variable region of the heavy chain. In the present application, the antigen binding domain can comprise an antigen binding protein, which can comprise a heavy chain variable region VH, which can comprise heavy chain complementarity determining regions HCDR1, HCDR2 and / or HCDR3. For example, the VH can comprise HCDR1, HCDR2 and HCDR3.

[0077] In the present application, the chimeric antigen receptor can comprise an antigen binding domain, which can comprise an antigen binding protein, which can comprise a VH, which can comprise HCDR1, HCDR2 and HCDR3, the amino acid sequence of HCDR1 is shown as SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown as SEQ ID NO: 2, and the amino acid sequence of HCDR3 is shown as SEQ ID NO: 3.

[0078] In the present application, the antigen binding domain can further comprise at least one CDR in the antibody heavy chain variable region. In the present application, the antigen binding domain can comprise an antigen binding protein, which can further comprise a heavy chain variable region VH, which can comprise a heavy chain complementarity determining region HCDR1, HCDR2 and / or HCDR3. For example, the VH can comprise HCDR1, HCDR2 and HCDR3.

[0079] In the present application, the antigen binding domain can comprise an antigen binding protein, which can further comprise a VL, which can comprise a LCDR1, a LCDR2 and a LCDR3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 9, the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 10, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 11.

[0080] In the present application, the antigen binding domain can comprise an antigen binding protein, which can further comprise a VH and a VL, the VH can comprise a HCDR1, a HCDR2 and a HCDR3, the VL can comprise a LCDR1, a LCDR2 and a LCDR3, the amino acid sequence of the HCDR1 is shown as SEQ ID NO: 1, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 2, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 9, the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 10, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 11.

[0081] In the present application, the VH can comprise H-FR1, H-FR2, H-FR3, and / or H-FR4. In the present application, the antigen binding protein can comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3, and H-FR4. In the present application, the C-terminus of the H-FR1 can be directly or indirectly connected to the N-terminus of the HCDR1. The H-FR2 can be located between the HCDR1 and the HCDR2. In the present application, the H-FR3 can be located between the HCDR2 and the HCDR3. In the present application, the N-terminus of the H-FR4 can be directly or indirectly connected to the C-terminus of the HCDR3. For example, the VH can comprise H-FR1, H-FR2, H-FR3, and H-FR4, the amino acid sequence of the H-FR1 is set forth in SEQ ID NO: 4, the amino acid sequence of the H-FR2 is set forth in SEQ ID NO: 5, the amino acid sequence of the H-FR3 is set forth in SEQ ID NO: 6, and the amino acid sequence of the H-FR4 is set forth in SEQ ID NO: 7.

[0082] In the present application, the VL can comprise L-FR1, L-FR2, L-FR3, and / or L-FR4. In the present application, the antigen binding protein can comprise LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3, and L-FR4. In the present application, the C-terminus of the L-FR1 can be directly or indirectly connected to the N-terminus of the LCDR1. The L-FR2 can be located between the LCDR1 and the LCDR2. In the present application, the L-FR3 can be located between the LCDR2 and the LCDR3. In the present application, the N-terminus of the L-FR4 can be directly or indirectly connected to the C-terminus of the LCDR3. For example, the VL can comprise L-FR1, L-FR2, L-FR3, and L-FR4, the amino acid sequence of the L-FR1 is set forth in SEQ ID NO: 12, the amino acid sequence of the L-FR2 is set forth in SEQ ID NO: 13, the amino acid sequence of the L-FR3 is set forth in SEQ ID NO: 14, and the amino acid sequence of the L-FR4 is set forth in SEQ ID NO: 15.

[0083] In the present application, the directly or indirectly connected can be connected by intermolecular forces, or can be connected by a linker.

[0084] In the present application, the amino acid sequence of the FR can be any species origin FR. For example, the FR can be a murine, a rabbit, a goat, a llama, or a human origin FR. For example, the FR can be a murine origin FR. For example, the FR can be a human origin FR.

[0085] In the present application, the amino acid sequence of the FR can be adjusted as needed. For example, the FR can be a wild-type sequence. For example, the amino acid sequence of the FR can be mutated or optimized without reducing the binding activity / affinity of the chimeric antigen receptor to GPC3. For example, one or more amino acid sequences in the FR can be mutated or optimized without reducing the binding activity / affinity of the chimeric antigen receptor to GPC3. For example, the FR can be a variant thereof, which includes the amino acid sequence of the FR with one or more amino acids substituted, deleted, and / or added. For example, 1-30, 1-20, or 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions. For example, the FR can be a homolog, which can be an amino acid sequence having at least about 85% (for example, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the FR.

[0086] In the present application, the chimeric antigen receptor can comprise an antigen binding domain, which can comprise an antigen binding protein, which can comprise a VH, the amino acid sequence of which can be as set forth in SEQ ID NO: 8. In the present application, the antigen binding protein can further comprise a VL, the amino acid sequence of which can be as set forth in SEQ ID NO: 16. In the present application, the VH and / or VL can be a wild-type sequence. For example, one or more amino acid sequences in the VH and / or VL can be mutated or optimized without reducing the binding activity / affinity of the chimeric antigen receptor to GPC3. In the present application, the VH and / or VL can be a variant thereof. For example, the variant includes the amino acid sequence of the VH and / or VL with one or more amino acids substituted, deleted, and / or added. For example, the VH can further comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more homology to the amino acid sequence set forth in SEQ ID NO: 8. For example, the VL can further comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more homology to the amino acid sequence set forth in SEQ ID NO: 16.

[0087] In the present application, the chimeric antigen receptor can comprise an antigen binding domain, which can comprise an antigen binding protein, which can comprise a VH and a VL, the amino acid sequence of the VH can be as set forth in SEQ ID NO: 8, the amino acid sequence of the VL can be as set forth in SEQ ID NO: 16.

[0088] In the present application, the antigen binding protein can further comprise an immunoglobulin constant region. In the present application, the immunoglobulin constant region can be a heavy chain constant region and / or a light chain constant region of an antibody. In the present application, the immunoglobulin constant region can be a wild type sequence or can be mutated or optimized.

[0089] In the present application, the antigen binding protein can include, but is not limited to, an antibody, an antigen binding fragment, an immunoconjugate, a multispecific antibody, an antibody fragment, an antibody derivative, an antibody analog or a fusion protein, etc. In the present application, the antigen binding protein can be an antibody or an antigen binding fragment thereof. In the present application, the antigen binding fragment can be a Fab, a (Fab)2, a F(ab’)2, a scFv, a di-scFv, a Fv, a VHH or a dAb fragment of the antibody. For example, the antigen binding protein can be a scFv comprising a VH and a VL, the VH can comprise a HCDR1, a HCDR2 and a HCDR3, the amino acid sequence of the HCDR1 is as set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is as set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is as set forth in SEQ ID NO: 3, the VL can comprise a LCDR1, a LCDR2 and a LCDR3, the amino acid sequence of the LCDR1 is as set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is as set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is as set forth in SEQ ID NO: 11.

[0090] In the present application, the antigen binding protein can be a scFv comprising a VH and a VL, the amino acid sequence of the VH is as set forth in SEQ ID NO: 8, the amino acid sequence of the VL is as set forth in SEQ ID NO: 16. In the present application, the antigen binding protein can be a scFv comprising a VH and a VL, the amino acid sequence of the VH is as set forth in SEQ ID NO: 8, the amino acid sequence of the VL is as set forth in SEQ ID NO: 16, the VH and the VL can be connected by a linker. For example, the linker can be (GGGGS)n. For example, the linker can be (GGGGS)3. For example, the antigen binding protein can be a scFv, the amino acid sequence of which is as set forth in SEQ ID NO: 17.

[0091] In the present application, the antigen binding protein can be a chimeric antibody, a humanized antibody, or a fully human antibody. For example, the antigen binding protein can have mutations / optimizations in the FR and / or constant region without reducing the binding activity / affinity of the chimeric antigen receptor to GPC3. For example, the antigen binding protein can have lower immunogenicity without reducing the binding activity / affinity of the chimeric antigen receptor to GPC3.

[0092] In the present application, the antigen binding protein can be a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0093] In the present application, the antigen binding protein can be a monovalent antibody, a bivalent antibody, or a multivalent antibody.

[0094] In the present application, the chimeric antigen receptor can further comprise an intracellular signaling domain. In the present application, the intracellular signaling domain can comprise an intracellular signaling domain derived from CD3 zeta, CD3 delta, CD3 gamma, CD3 epsilon, CD79a, CD79b, Fc epsilon R1 gamma, Fc epsilon R1 beta, Fc gamma RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (KSHV), DAP10, DAP-12, or a domain comprising at least one ITAM. In the present application, the intracellular signaling domain can be derived from the intracellular signaling domain of CD3 zeta. In the present application, the intracellular signaling domain can be of any species origin. For example, the intracellular signaling domain can be of murine, rabbit, goat, llama, or human origin. For example, the intracellular signaling domain can be a human CD3 zeta intracellular signaling domain. In the present application, the intracellular signaling domain can be a variant thereof. For example, the variant comprises one or more amino acid substitutions, deletions, and / or additions to the amino acid sequence of the CD3 zeta intracellular signaling domain. For example, 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions. In the present application, the intracellular signaling domain can be a homolog thereof. For example, the homolog can have an amino acid sequence that has at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the CD3 zeta intracellular signaling domain. In the present application, the amino acid sequence of the intracellular signaling domain can be as set forth in SEQ ID NO: 25.

[0095] In the present application, the chimeric antigen receptor can further comprise a costimulatory domain. In the present application, the costimulatory domain can comprise a costimulatory domain derived from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc epsilon RI gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88. In the present application, the costimulatory domain can be derived from a costimulatory domain of 4-1BB. In the present application, the costimulatory domain can be of any species origin. For example, the costimulatory domain can be of murine, rabbit, goat, llama, or human origin. For example, the costimulatory domain can be a human 4-1BB costimulatory domain. In the present application, the costimulatory domain can be a variant thereof. For example, the variant comprises one or more amino acid substitutions, deletions, and / or additions to the amino acid sequence of the 4-1BB costimulatory domain. For example, 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions. In the present application, the costimulatory domain can be a homolog thereof. For example, the homolog can be an amino acid sequence having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the 4-1BB costimulatory domain. In the present application, the amino acid sequence of the costimulatory domain can be set forth in SEQ ID NO: 22.

[0096] In the present application, the chimeric antigen receptor can further comprise a signal peptide. In the present application, the signal peptide can be a signal peptide of a protein. For example, the signal peptide can be a signal peptide of a cytokine protein. For example, the signal peptide can be a signal peptide of a leukocyte differentiation antigen (CD molecule). In the present application, the signal peptide can be derived from CD8a. In the present application, the signal peptide can be a signal peptide of any species origin. For example, the signal peptide can be of murine, rabbit, goat, llama, or human origin. For example, the signal peptide can be a human CD8a signal peptide. In the present application, the signal peptide can be a variant thereof. For example, the variant comprises substitution, deletion, and / or addition of one or more amino acids to the amino acid sequence of the CD8a signal peptide. For example, 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions. In the present application, the signal peptide can be a homolog thereof. For example, the homolog can be an amino acid sequence having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the CD8a signal peptide. In the present application, the amino acid sequence of the signal peptide can be set forth as SEQ ID NO: 23.

[0097] In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 being set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 being set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 being set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 being set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 being set forth in SEQ ID NO: 11. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising a VH and a VL, the amino acid sequence of the VH being set forth in SEQ ID NO: 8, the amino acid sequence of the VL being set forth in SEQ ID NO: 16. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising a scFv, the amino acid sequence of the scFv being set forth in SEQ ID NO: 17.

[0098] In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region and a CD28 transmembrane domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2 and a HCDR3, the VL comprising a LCDR1, a LCDR2 and a LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and the CD28 transmembrane domain is set forth in SEQ ID NO: 26. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region and a CD28 transmembrane domain, the antigen binding domain comprising a VH and a VL, the amino acid sequence of the VH is set forth in SEQ ID NO: 8, the amino acid sequence of the VL is set forth in SEQ ID NO: 16, the amino acid sequence of the CD8 hinge region and the CD28 transmembrane domain is set forth in SEQ ID NO: 26. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region and a CD28 transmembrane domain, the amino acid sequence of the antigen binding domain, the CD8 hinge region and the CD28 transmembrane domain is set forth in SEQ ID NO: 27.

[0099] In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 being set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 being set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 being set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 being set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 being set forth in SEQ ID NO: 11. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the amino acid sequence of the VH being set forth in SEQ ID NO: 8, the amino acid sequence of the VL being set forth in SEQ ID NO: 16. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of the scFv being set forth in SEQ ID NO: 17.

[0100] In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 being set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 being set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 being set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 being set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 being set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and the CD28 transmembrane domain being set forth in SEQ ID NO: 26. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the amino acid sequence of the VH being set forth in SEQ ID NO: 8, the amino acid sequence of the VL being set forth in SEQ ID NO: 16, the amino acid sequence of the CD8 hinge region and the CD28 transmembrane domain being set forth in SEQ ID NO: 26. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of the antigen binding domain, the CD8 hinge region, and the CD28 transmembrane domain being set forth in SEQ ID NO: 27.

[0101] In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 being set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 being set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 being set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 being set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 being set forth in SEQ ID NO: 11. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the amino acid sequence of the VH being set forth in SEQ ID NO: 8, the amino acid sequence of the VL being set forth in SEQ ID NO: 16. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of the scFv being set forth in SEQ ID NO: 17.

[0102] In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and the CD28 transmembrane domain is set forth in SEQ ID NO: 26. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the amino acid sequence of the VH is set forth in SEQ ID NO: 8, the amino acid sequence of the VL is set forth in SEQ ID NO: 16, the amino acid sequence of the CD8 hinge region and the CD28 transmembrane domain is set forth in SEQ ID NO: 26. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of the antigen binding domain, the CD8 hinge region, and the CD28 transmembrane domain is set forth in SEQ ID NO: 27.

[0103] In the present application, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 being set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 being set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 being set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 being set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 being set forth in SEQ ID NO: 11. In the present application, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the amino acid sequence of the VH being set forth in SEQ ID NO: 8, the amino acid sequence of the VL being set forth in SEQ ID NO: 16. In the present application, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of the scFv being set forth in SEQ ID NO: 17.

[0104] In the present application, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 being set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 being set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 being set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 being set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 being set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and the CD28 transmembrane domain being set forth in SEQ ID NO: 26. In the present application, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the VH comprising the amino acid sequence set forth in SEQ ID NO: 8, the VL comprising the amino acid sequence set forth in SEQ ID NO: 16, the amino acid sequence of the CD8 hinge region and the CD28 transmembrane domain being set forth in SEQ ID NO: 26. In the present application, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain, the CD8 hinge region, and the CD28 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 27.

[0105] In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 being set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 being set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 being set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 being set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 being set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region being set forth in SEQ ID NO: 20, the amino acid sequence of the CD28 transmembrane domain being set forth in SEQ ID NO: 21. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising a VH and a VL, the amino acid sequence of the VH being set forth in SEQ ID NO: 8, the amino acid sequence of the VL being set forth in SEQ ID NO: 16, the amino acid sequence of the CD8 hinge region being set forth in SEQ ID NO: 20, the amino acid sequence of the CD28 transmembrane domain being set forth in SEQ ID NO: 21. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising a scFv, the amino acid sequence of the scFv being set forth in SEQ ID NO: 17, the amino acid sequence of the CD8 hinge region being set forth in SEQ ID NO: 20, the amino acid sequence of the CD28 transmembrane domain being set forth in SEQ ID NO: 21. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the amino acid sequences of the antigen binding domain, the CD8 hinge region, and the CD28 transmembrane domain being set forth in SEQ ID NO: 27.

[0106] In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 being set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 being set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 being set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 being set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 being set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region being set forth in SEQ ID NO: 20, the amino acid sequence of the CD28 transmembrane domain being set forth in SEQ ID NO: 21. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the amino acid sequence of the VH being set forth in SEQ ID NO: 8, the amino acid sequence of the VL being set forth in SEQ ID NO: 16, the amino acid sequence of the CD8 hinge region being set forth in SEQ ID NO: 20, the amino acid sequence of the CD28 transmembrane domain being set forth in SEQ ID NO: 21. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of the scFv being set forth in SEQ ID NO: 17, the amino acid sequence of the CD8 hinge region being set forth in SEQ ID NO: 20, the amino acid sequence of the CD28 transmembrane domain being set forth in SEQ ID NO: 21.

[0107] In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 being set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 being set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 being set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 being set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 being set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region being set forth in SEQ ID NO: 20, the amino acid sequence of the CD28 transmembrane domain being set forth in SEQ ID NO: 21. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the amino acid sequence of the VH being set forth in SEQ ID NO: 8, the amino acid sequence of the VL being set forth in SEQ ID NO: 16, the amino acid sequence of the CD8 hinge region being set forth in SEQ ID NO: 20, the amino acid sequence of the CD28 transmembrane domain being set forth in SEQ ID NO: 21. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of the scFv being set forth in SEQ ID NO: 17, the amino acid sequence of the CD8 hinge region being set forth in SEQ ID NO: 20, the amino acid sequence of the CD28 transmembrane domain being set forth in SEQ ID NO: 21.

[0108] In the present application, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 being set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 being set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 being set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 being set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 being set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region being set forth in SEQ ID NO: 20, the amino acid sequence of the CD28 transmembrane domain being set forth in SEQ ID NO: 21. In the present application, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the amino acid sequence of the VH being set forth in SEQ ID NO: 8, the amino acid sequence of the VL being set forth in SEQ ID NO: 16, the amino acid sequence of the CD8 hinge region being set forth in SEQ ID NO: 20, the amino acid sequence of the CD28 transmembrane domain being set forth in SEQ ID NO: 21. In the present application, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of the scFv being set forth in SEQ ID NO: 17, the amino acid sequence of the CD8 hinge region being set forth in SEQ ID NO: 20, the amino acid sequence of the CD28 transmembrane domain being set forth in SEQ ID NO: 21.

[0109] Modified immune cells

[0110] In another aspect, the present application provides a modified immune cell, wherein the immune cell can comprise a chimeric antigen receptor. In the present application, the chimeric antigen receptor can be as previously described. In the present application, the modified immune cell has one or more of the following properties: (1) capable of binding to GPC3 with good binding activity; (2) capable of binding to GPC3 expressed on the surface of a target cell; (3) capable of killing GPC3-positive tumors.

[0111] In the present application, the immune cells can promote an immune response. In the present application, the immune cells can exert a killing effect on target cells. In the present application, the immune cells can be T cells, NK cells, NKT cells, dendritic cells, macrophages, TIL cells, iNKT cells, CIK cells, gd T cells, and / or DNT cells. For example, the immune cells can be immune effector cells. For example, the immune cells can be T cells. For example, the immune cells can be iNKT cells. For example, the immune cells can be gd T cells. For example, the immune cells can be a mixture, which can comprise different immune cell species, for example, the mixture can comprise one or more immune cells.

[0112] In the present application, the modified immune cells can be CAR-T cells. For example, the CAR-T cells can comprise a chimeric antigen receptor, which can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising a scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17. For example, the CAR-T cells can comprise a chimeric antigen receptor, which can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17. For example, the CAR-T cells can comprise a chimeric antigen receptor, which can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17. For example, the CAR-T cells can comprise a chimeric antigen receptor, which can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17.

[0113] In the present application, the modified immune cell can be a CAR-T cell. For example, the CAR-T cell can comprise a chimeric antigen receptor, which can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and CD28 transmembrane domain is set forth in SEQ ID NO: 26. For example, the CAR-T cell can comprise a chimeric antigen receptor, which can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and CD28 transmembrane domain is set forth in SEQ ID NO: 26.For example, the CAR-T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and CD28 transmembrane domain set forth in SEQ ID NO: 26. For example, the CAR-T cell can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and CD28 transmembrane domain set forth in SEQ ID NO: 26.

[0114] In the present application, the modified immune cell can be a CAR-T cell. For example, the CAR-T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the amino acid sequence of which is set forth in SEQ ID NO: 27. For example, the CAR-T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of which is set forth in SEQ ID NO: 27. For example, the CAR-T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of which is set forth in SEQ ID NO: 27. For example, the CAR-T cell can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of which is set forth in SEQ ID NO: 27.

[0115] In the present application, the modified immune cell can be a CAR-iNKT cell. For example, the CAR-iNKT cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising a scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17. For example, the CAR-iNKT cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17. For example, the CAR-iNKT cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17. For example, the CAR-iNKT cell can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17.

[0116] In the present application, the modified immune cell can be a CAR-iNKT cell. For example, the CAR-iNKT cell can comprise a chimeric antigen receptor, which can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and CD28 transmembrane domain is set forth in SEQ ID NO: 26. For example, the CAR-iNKT cell can comprise a chimeric antigen receptor, which can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and CD28 transmembrane domain is set forth in SEQ ID NO: 26.For example, the CAR-iNKT cells can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and CD28 transmembrane domain set forth in SEQ ID NO: 26. For example, the CAR-iNKT cells can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and CD28 transmembrane domain set forth in SEQ ID NO: 26.

[0117] In the present application, the modified immune cell can be a CAR-iNKT cell. For example, the CAR-iNKT cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the amino acid sequence of which is set forth in SEQ ID NO: 27. For example, the CAR-iNKT cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of which is set forth in SEQ ID NO: 27. For example, the CAR-iNKT cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of which is set forth in SEQ ID NO: 27. For example, the CAR-iNKT cell can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of which is set forth in SEQ ID NO: 27.

[0118] In the present application, the modified immune cell can be a CAR-gammadelta T cell. For example, the CAR-gammadelta T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising a scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17. For example, the CAR-gammadelta T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17. For example, the CAR-gammadelta T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17. For example, the CAR-gammadelta T cell can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a scFv, the amino acid sequence of which is set forth in SEQ ID NO: 17.

[0119] In the present application, the modified immune cell can be a CAR-gammadelta T cell. For example, the CAR-gammadelta T cell can comprise a chimeric antigen receptor, which can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and CD28 transmembrane domain is set forth in SEQ ID NO: 26. For example, the CAR-gammadelta T cell can comprise a chimeric antigen receptor, which can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and CD28 transmembrane domain is set forth in SEQ ID NO: 26.For example, the CAR-gammadelta T cells can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and CD28 transmembrane domain set forth in SEQ ID NO: 26. For example, the CAR-gammadelta T cells can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 set forth in SEQ ID NO: 11, the amino acid sequence of the CD8 hinge region and CD28 transmembrane domain set forth in SEQ ID NO: 26.

[0120] In the present application, the modified immune cell can be a CAR-gammadelta T cell. For example, the CAR-gammadelta T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the amino acid sequence of which is set forth in SEQ ID NO: 27. For example, the CAR-gammadelta T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of which is set forth in SEQ ID NO: 27. For example, the CAR-gammadelta T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of which is set forth in SEQ ID NO: 27. For example, the CAR-gammadelta T cell can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of which is set forth in SEQ ID NO: 27.

[0121] In the present application, the modified immune cell further comprises a fusion protein. For example, the fusion protein can be a membrane-bound cytokine. For example, the fusion protein can be a membrane-bound IL-10.

[0122] In the present application, the fusion protein can comprise IL-10 and a transmembrane domain. In the present application, the IL-10 can be human IL-10. In the present application, the amino acid sequence of the IL-10 can be set forth in SEQ ID NO: 32. In the present application, the transmembrane domain can be selected from the transmembrane domain of the following group of proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 epsilon, CD3 zeta, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc epsilon RI gamma, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, or SLAM. For example, the transmembrane domain can be the transmembrane domain of CD8. For example, the amino acid sequence of the transmembrane domain can be set forth in SEQ ID NO: 24. In the present application, the fusion protein can comprise IL-10 and a CD8 transmembrane domain. In the present application, the IL-10 and the transmembrane domain can be directly or indirectly linked. For example, the IL-10 and the transmembrane domain can be directly linked. For example, the IL-10 and the transmembrane domain can be indirectly linked. For example, the IL-10 and the transmembrane domain can be linked via a hinge region.

[0123] In the present application, the fusion protein can further comprise a hinge region. In the present application, the hinge region can be selected from the hinge region of the following group of proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc epsilon RI gamma, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, or LIGHT. For example, the hinge region can be the hinge region of CD8. For example, the amino acid sequence of the hinge region can be set forth in SEQ ID NO: 20. In the present application, the fusion protein can comprise IL-10, a hinge region, and a transmembrane domain. For example, the fusion protein can comprise IL-10, a CD8 hinge region, and a CD8 transmembrane domain.

[0124] In the present application, the modified immune cell can comprise a chimeric antigen receptor and a fusion protein. For example, the modified immune cell can comprise a chimeric antigen receptor and a fusion protein, the chimeric antigen receptor can comprise an antigen binding domain targeting GPC3, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 11, the fusion protein can comprise an IL-10, a CD8 hinge region, and a CD8 transmembrane domain. For example, the modified immune cell can comprise a chimeric antigen receptor and a fusion protein, the amino acid sequence of the chimeric antigen receptor comprising an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 11, the fusion protein can comprise an IL-10, a CD8 hinge region, and a CD8 transmembrane domain.

[0125] Nucleic acid molecules, vectors, and cells

[0126] In another aspect, the present application also provides an isolated nucleic acid molecule, which can encode the chimeric antigen receptor.

[0127] In the present application, the nucleic acid molecule also encodes a fusion protein. For example, the fusion protein can be a membrane-bound cytokine. For example, the fusion protein can be a membrane-bound IL-10.

[0128] In the present application, the fusion protein encoded by the nucleic acid molecule can comprise IL-10 and a transmembrane domain. In the present application, the IL-10 can be human IL-10. In the present application, the amino acid sequence of the IL-10 can be set forth in SEQ ID NO: 32. In the present application, the transmembrane domain can be selected from the group consisting of transmembrane domains of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 epsilon, CD3 zeta, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc epsilon RI gamma, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, or SLAM. For example, the transmembrane domain can be a transmembrane domain of CD8. For example, the amino acid sequence of the transmembrane domain can be set forth in SEQ ID NO: 24. In the present application, the fusion protein encoded by the nucleic acid molecule can comprise IL-10 and a CD8 transmembrane domain.

[0129] In the present application, the fusion protein encoded by the nucleic acid molecule can further comprise a hinge region. In the present application, the hinge region can be selected from the group consisting of hinge regions of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc epsilon RI gamma, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, or LIGHT. For example, the hinge region can be a hinge region of CD8. For example, the amino acid sequence of the hinge region can be set forth in SEQ ID NO: 20. In the present application, the fusion protein encoded by the nucleic acid molecule can comprise IL-10, a hinge region, and a transmembrane domain. For example, the fusion protein encoded by the nucleic acid molecule can comprise IL-10, a CD8 hinge region, and a CD8 transmembrane domain.

[0130] In the present application, the nucleic acid molecule can comprise a sequence encoding a signal peptide. For example, the signal peptide can be a CD8 alpha signal peptide.

[0131] In the present application, the nucleic acid molecule can be produced or synthesized by (i) amplification in vitro, for example, produced by polymerase chain reaction (PCR) amplification, (ii) produced by recombination by cloning, (iii) purified, for example, fractionated by enzyme digestion and gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis.

[0132] In the present application, the nucleic acid molecule can be DNA and / or RNA. In the present application, the nucleic acid molecule can be an artificially synthesized nucleic acid analogue.

[0133] In the present application, the nucleic acid molecule can be a modified nucleic acid molecule.

[0134] In the present application, the nucleic acid molecule can be a mixture. For example, the nucleic acid molecule can encode the chimeric antigen receptor and the fusion protein, respectively.

[0135] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0136] In the present application, the vector can comprise one or more of the nucleic acid molecules. In the present application, the vector can comprise one or more of the nucleic acid molecules. For example, the vector can comprise a nucleic acid molecule encoding the chimeric antigen receptor. For example, the vector can comprise nucleic acid molecules encoding the chimeric antigen receptor and the fusion protein.

[0137] In the present application, the nucleic acid molecule further comprises a sequence encoding a self-cleaving peptide. In the present application, the sequence encoding the self-cleaving peptide can be located between the sequences encoding the chimeric antigen receptor and the fusion protein. In the present application, the self-cleaving peptide can comprise a 2A peptide. For example, the self-cleaving peptide can be P2A, T2A, E2A, or F2A. For example, the self-cleaving peptide can be P2A. For example, the self-cleaving peptide can be GSGP2A.

[0138] In the present application, the vector can be an expression vector or a cloning vector. In the present application, the vector can be a viral vector or a non-viral vector. In the present application, the vector can be a viral vector, a plasmid vector, a phage vector, or other vectors commonly used in, for example, genetic engineering. For example, the viral vector can be an adenovirus, an adeno-associated virus, a retrovirus (including lentivirus). In the present application, the vector can be a fusion type vector or a non-fusion type vector.

[0139] In the present application, the vector can further comprise other genes. For example, the other genes can be marker genes.

[0140] In the present application, the vector can contain various elements that control expression. For example, the vector can include a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and / or a reporter gene. For example, the vector can also contain a replication initiation site. For example, the vector can include components that assist in entry into a cell. To enable the nucleic acid molecule to replicate in the vector, the 5' end and the 3' end of the nucleic acid molecule can also contain long terminal repeat sequences.

[0141] In the present application, the nucleic acid molecule can contain, in order from the 5' end to the 3' end, nucleotide sequences of a gene encoding an antigen binding domain, a gene encoding a CD8 hinge region, and a gene encoding a CD28 transmembrane domain, the antigen binding domain comprising an amino acid sequence represented by SEQ ID NO: 17.

[0142] In the present application, the nucleic acid molecule can contain, in order from the 5' end to the 3' end, nucleotide sequences of a gene encoding a CD8a signal peptide, a gene encoding an antigen binding domain, a gene encoding a CD8 hinge region, a gene encoding a CD28 transmembrane domain, a gene encoding a 4-1BB costimulatory domain, and a gene encoding a CD3 zeta intracellular signaling domain, the antigen binding domain comprising an amino acid sequence represented by SEQ ID NO: 17.

[0143] In the present application, the nucleic acid molecule can contain, in order from the 5' end to the 3' end, nucleotide sequences of a gene encoding a CD8a signal peptide, a gene encoding an antigen binding domain, a gene encoding a CD8 hinge region, a gene encoding a CD28 transmembrane domain, a gene encoding a 4-1BB costimulatory domain, a gene encoding a CD3 zeta intracellular signaling domain, a gene encoding a GSGP2A self-cleavage peptide, a gene encoding a CD8a signal peptide, a gene encoding IL-10, a gene encoding a CD8 hinge region, and a gene encoding a CD8 transmembrane domain, the antigen binding domain comprising an amino acid sequence represented by SEQ ID NO: 17.

[0144] In another aspect, the present application provides a cell comprising the nucleic acid molecule and / or the vector.

[0145] In the present application, the cell can include progeny of a single cell. The progeny can not necessarily be identical to the original parent cell (in morphology of total DNA complement or on the genome) due to natural, accidental, or intentional mutation.

[0146] In the present application, the cell can be a prokaryotic cell (e.g., a bacterial cell), a CHO cell, an NS / 0 cell, a HEK293T cell, or a HEK293A cell, or other eukaryotic cells such as fungal or yeast cells, etc. In the present application, the cell can also be an immune cell.

[0147] In the present application, the cell can comprise one or more of the nucleic acid molecules and / or one or more vectors. In the present application, the vector can comprise one or more of the nucleic acid molecules and / or one or more vectors.

[0148] In the present application, the vector can be introduced into the cell by methods known in the art. For example, the method can be electroporation, Lipofectine transfection or Lipofectamine transfection.

[0149] In another aspect, the present application provides a method of making the chimeric antigen receptor, the method comprising culturing the cell under conditions such that the chimeric antigen receptor is expressed.

[0150] Pharmaceutical composition

[0151] In another aspect, the present application also provides a pharmaceutical composition, which can comprise the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0152] In the present application, the pharmaceutical composition can comprise one or more suitable formulations of (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. Acceptable ingredients of the composition are preferably non-toxic to the recipient at the doses and concentrations employed. The pharmaceutical composition of the present application can include liquid, frozen and lyophilized compositions.

[0153] In the present application, the pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents which are compatible with pharmaceutical administration, generally safe, non-toxic and neither biologically nor otherwise undesirable.

[0154] In the present application, the pharmaceutical composition can comprise parenteral, transdermal, intracavitary, intraarterial, intrathecal and / or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition can be performed by different ways, for example intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0155] Pharmaceutical combination

[0156] In another aspect, the present application provides a pharmaceutical combination comprising the chimeric antigen receptor and / or the modified immune cell, the pharmaceutical combination can further comprise one or more active ingredients other than the chimeric antigen receptor and / or the modified immune cell.

[0157] For example, the pharmaceutical combination can further comprise a substance related to immune response. For example, the pharmaceutical combination can further comprise a drug related to immune response.

[0158] In another aspect, the present application provides a pharmaceutical combination comprising the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule and / or the cell and a therapeutic agent. In the present application, the therapeutic agent can be selected from one or more of the following group: an anti-tumor drug, a chemotherapeutic agent, a radioisotope or an immune checkpoint inhibitor.

[0159] In another aspect, the present application also provides a regimen in which the chimeric antigen receptor and / or the modified immune cell is combined with other active ingredient(s). For example, the chimeric antigen receptor and / or the modified immune cell is combined with other therapeutic agent(s). In the present application, the pharmaceutical combination can be administered separately, simultaneously or sequentially. In the present application, the pharmaceutical combination can be administered at the same or different dose or administration route. For example, the active ingredients in the pharmaceutical combination are administered to the patient as separate entities at the same / different dose, administration route. In the present application, the ingredients in the pharmaceutical combination can be administered to the patient simultaneously as a single entity or dose. For example, the ingredients in the pharmaceutical combination are administered to the patient as separate entities simultaneously, jointly or sequentially. In the present application, the specific administration route can be determined according to the category of the active ingredient, and the specific administration dose can be adjusted according to the severity of the disease, the physical condition of the subject, etc.

[0160] In the present application, the different active ingredients in the pharmaceutical combination can be mixed or placed separately. For example, the active ingredients can be placed in the same container. For example, the active ingredients can be placed in different containers.

[0161] Preparation method

[0162] In another aspect, the present application provides a method for preparing the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition. For example, the method can comprise culturing the cell under conditions such that the chimeric antigen receptor is expressed. For example, the method can comprise introducing the vector into the immune cell.

[0163] Use

[0164] In another aspect, the present application also provides the use of the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a medicament, which can be used for preventing, diagnosing and / or treating a disease and / or a disorder.

[0165] In another aspect, the present application also provides a method of preventing, diagnosing and / or treating a disease and / or a disorder, which can comprise administering the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition to a subject in need thereof.

[0166] In another aspect, the present application also provides the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition for use in preventing, diagnosing and / or treating a disease and / or a disorder.

[0167] In the present application, the disease and / or disorder can be a GPC3-related disease and / or disorder.

[0168] In the present application, the disease and / or disorder can be a tumor. In the present application, the tumor can be a solid tumor and / or a hematological tumor. In the present application, the tumor can be a GPC3-positive tumor. In the present application, the tumor can be a liver cancer, an ovarian cancer, a melanoma, a pancreatic cancer, a lung cancer, a colon cancer, a breast cancer, a prostate cancer, a non-small cell lung cancer, a small cell lung cancer, a squamous cell carcinoma, a renal cell carcinoma, a colorectal cancer, a gastric cancer, a glioma, an ovarian clear cell carcinoma, a yolk sac tumor and / or a hepatocellular carcinoma.

[0169] In the present application, the prevention, diagnosis and / or treatment can be preventing the onset of the disease, slowing down or reversing the disease progression, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith or preventing further increase in the severity of the disease and any symptoms associated therewith.

[0170] In another aspect, the present application provides the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition for use in diagnosing a disease and / or a disorder associated with the expression of GPC3.

[0171] In another aspect, the present application provides the use of the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition for the preparation of a diagnostic agent, wherein the diagnostic agent is for diagnosing a disease and / or a disorder associated with the expression of GPC3.

[0172] In the present application, the diagnostic agent can be used alone or in combination with an instrument, device, apparatus or system. The diagnostic agent can be used for in vitro detection of human samples (e.g., various body fluids, cells, tissue samples, etc.) in the process of prevention, diagnosis, treatment monitoring, prognosis observation, health status evaluation and prediction of genetic diseases.

[0173] In another aspect, the present application also provides a detection kit, which can comprise the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition, and the detection kit is used for detecting the presence and / or content of GPC3 in a sample or a subject. For example, the detection kit can be used for preventing, diagnosing and / or treating diseases and / or disorders.

[0174] In another aspect, the present application provides the use of the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a detection kit, and the detection kit is used for diagnosis.

[0175] In another aspect, the present application also provides a method for detecting the presence and / or content of GPC3, which can comprise using the chimeric antigen receptor and / or the modified immune cell.

[0176] In another aspect, the present application provides a method for detecting GPC3 in a sample or a subject, which comprises administering the chimeric antigen receptor and / or the modified immune cell. In the present application, the administration can be performed in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0177] Without wishing to be bound by any theory, the examples below are merely intended to illustrate the chimeric antigen receptor, the preparation method and the use of the present application, and are not intended to limit the scope of the present application.

[0178] Examples

[0179] Experimental materials

[0180] In the technical solutions given in the examples, the exemplary amino acid sequences used are as follows:

[0181] CD8αSP: SEQ ID NO:23; GPC3 scfv: SEQ ID NO:17, wherein the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; CD8 hinge region: SEQ ID NO:20; CD28 hinge region: SEQ ID NO:18; IgG4 hinge region: SEQ ID NO:19; CD8 transmembrane domain: SEQ ID NO:24; CD28 transmembrane domain: SEQ ID NO:21; 4-1BB co-stimulatory domain: SEQ ID NO:22; CD3ζ: SEQ ID NO:25.

[0182] Example 1: Preparation and Expression of Chimeric Antigen Receptors

[0183] 1.1 Cell Culture

[0184] 293T human embryonic kidney cells, Hep3B and Huh7 liver cancer cells, MDA-MB-231 breast cancer cells, and MEC1 chronic lymphocytic leukemia cell line were all purchased from ATCC, USA. The MEC1-ROR1 cell line is an internally generated, stably expressing ROR1 line. GFP was stably expressed in all target cells for easy detection of cell killing. 293T, Hep3B, and Huh7 cell lines were maintained in complete medium (DMEM containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine). MDA-MB-231, MEC1, and MEC1-ROR1 cell lines were maintained in complete medium (IMDM containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine). PBMCs were isolated from whole blood of healthy donors using Ficoll-Paque and cultured at 2 × 10⁻⁶ cells / mL. 7 The sample was aliquoted into 1 mL at a concentration of 10 cells / mL and frozen in a liquid nitrogen tank. The culture medium was heat-inactivated FBS with 10% DMSO (vol / vol).

[0185] 1.2 Clone Construction

[0186] The chimeric antigen receptors all adopted the structure of second-generation CARs. The gene structure encoding the CAR was inserted into the pALD expression plasmid through the BamHI and SalI cloning sites. Different combinations of hinge regions and transmembrane domains were designed, as shown in Figure 1. After double enzyme digestion and sequencing verification, the expression plasmid was amplified in large quantities and used for lentiviral packaging.

[0187] 1.3 Preparation of lentivirus

[0188] All lentiviruses were prepared from 293T. After washing the freshly resuspended 293T cells in PBS buffer, the cells were seeded in 10 cm dishes at 70-80% confluency. The mixed lentivirus packaging plasmids and transfection plasmids were mixed with the transfection agent Lipofectamine-3000 and transiently transfected into the seeded HEK293T cells. After 48 hours of culture, the supernatant was collected and filtered using a 0.45 pm filter, and the samples were aliquoted at 1 ml per tube and stored at -80 °C.

[0189] 1.4 Activation and transduction of T cells

[0190] To transduce the lentivirus encoding the corresponding structure into PBMC, the frozen PBMC was thawed and added to a 24-well plate, and CD3 and CD28 conjugated magnetic beads and IL2 (500 IU / ml) were added for 24-72 hours of activation. After the activated PBMC cells were distributed into multiple wells of a 24-well plate, the corresponding lentivirus and polybrene (8 g / ml) were added, and after centrifugation at 2000 g for 2 hours in a benchtop centrifuge, the cells were transferred to a 37 °C and 5% CO2 incubator for culture. The control group was replaced with complete culture medium instead of lentivirus, and the rest of the steps were the same. The transfected cells were further cultured in X-VIVO-15 cell culture medium containing IL-2 (500 IU / ml) for 48-72 hours before subsequent experiments and analysis.

[0191] 1.5 Detection of CAR expression level

[0192] The structure containing CAR was successfully cloned and packaged into lentivirus. After 72 hours and 168 hours of infection with different lentivirus structures, a small amount of sample was taken, stained with GPC3 protein fused with FITC fluorophore, and the CAR expression level was detected by flow cytometry.

[0193] 1.6 Flow analysis

[0194] Flow cytometry was performed using a flow cytometer (Beckman Coulter) in semi-automatic or plate mode, and data were analyzed using FlowJo software. Cells were washed once with FACS buffer (0.5% BSA, 0.1% NaN3, 2 mM EDTA, pH 7.0 in PBS) and resuspended to 1-5 x 10 6Cells were stained with 7-AAD before FACS analysis. Cells were stained with fluorophore-labeled antibodies or recombinant proteins, and incubated in the dark at 4°C for 40 min. Then 7-AAD was added for 5 min. After staining, unbound antibodies were washed away with FACS buffer, and the cells were centrifuged at 300 x g for 3 min. The supernatant was removed. After washing and resuspension with FACS buffer, the cells were analyzed by flow cytometry. Flow cytometry data were analyzed and processed using FlowJo software.

[0195] Experimental results:

[0196] CAR expression level results are shown in Figure 2. Compared with the non-transfected control (NTC), different designs can detect different levels of CAR structure expression. Under the same virus volume, the expression level of the GPC3 scFv-CD8H-CD28TM structure is higher than that of other CAR structure designs, and can maintain a substantially consistent CAR expression rate at day 3 and day 7, having a certain expression advantage. The experimental results show that the combination of the CAR structure prepared in the application has a good positive expression rate, and has a good CAR persistent expression ability.

[0197] Example 2: In vitro functional comparison of CAR structures with different hinge regions of GPC3 ScFv

[0198] 2.1 Tumor cell lines

[0199] Hep3B and Huh7 liver cancer cells were detected to express GPC3 endogenously. Human breast cancer cells MDA-MB-231 do not express GPC3. The three tumor cell lines stably express GFP after transfection and are used for subsequent experiments.

[0200] 2.2 Co-culture of CAR-T cells and tumor cells

[0201] Different hinge region GPC3 scFv-IgG4H-CD28TM, GPC3 scFv-CD8H-CD28TM and GPC3 scFv-CD28H-CD28TM structures were respectively transduced into T cells and cultured in an incubator for 72 hours. A small amount of sample was used for GPC3-FITC fluorescence staining to determine the CAR expression level of different structures. The uninfected T cells and CAR-T cells were accurately counted, adjusted to the same cell concentration of 10 6 . Based on the structure with the lowest CAR expression, the groups with higher expression were diluted and standardized using uninfected T cells, so that the cell concentration and CAR expression level of different groups were consistent. The standardized CAR-T cells were re-stained with GPC3-FITC to determine the equivalent CAR expression level. The target cells were also accurately counted, and the effector-target ratio was 1:1 and 5 x 10 4Cells were resuspended in 200 μl of cell culture medium and seeded in round-bottom 96-well plates. After 24 hours of culture, the supernatant was centrifuged and used to measure the secretion of γ-interferon or IL-2 by enzyme-linked immunosorbent assay.

[0202] 2.3 Measurement of the secretion level of cytokines

[0203] The supernatant was carefully aspirated after simple centrifugation, and after appropriate dilution, the ELISA MAX Deluxe Set Human IFN-γ Kit and the ELISA MAX Deluxe Set Human IL-2 Kit were used to measure the secretion level of cytokines in the culture medium. TM Deluxe Set human IFN-γ kit and ELISA MAX Deluxe Set human IL-2 kit TM Standard Set human IL-2 kit

[0204] 2.4 CAR-T multiple rounds of killing experiments on target cells (IncuCyte real-time imaging method)

[0205] First, the target cells Hep3B-GFP, Huh7-GFP and MB231-GFP were counted and plated in flat-bottom 96-well plates at 10,000 cells per well. At the same time, the uninfected T cells and the CAR-T cells were accurately counted and standardized for expression rate, and then added to the 96-well plates at an effector-to-target ratio of 1:8, i.e. 1250 T cells per well. The 96-well plates were placed in the IncuCyte for real-time imaging, which can monitor the growth curve of the GFP-labeled target cells. After 48 hours, the 96-well plates were removed, 5000 corresponding target cells were added to each well, and real-time imaging was continued to detect the persistence and multiple rounds of killing ability of the CAR-T cells.

[0206] Experimental results:

[0207] The results of the cytokine secretion level are shown in Figure 3. After co-culturing the CAR-T with the target cells Hep3B and Huh7, the secretion level of γ-interferon in the supernatant of the CD8 hinge region CAR co-culture system was significantly higher than that of the IgG4 and CD28 hinge region groups. In the co-culture with the non-target cells MB231 that do not express GPC3, the CD8 hinge region group and the other groups had similar γ-interferon secretion levels. The experimental results show that the CAR structure prepared in the present application can improve the secretion of cytokines, and the excellent activation ability of the CAR is caused by the specific binding of GPC3-positive cells.

[0208] The killing results of target cells are shown in FIGS. 4A-C. In the co-culture of GPC3-positive target cells Hep3B and Huh7, GPC3 scFv-CD8H-CD28TM can rapidly kill target cells even at a very low target ratio of 1:8, and also shows better cytotoxicity than the other two groups in the second round of target cell killing, with the ability to persistently kill target cells. In the killing of MB231 non-target cells, there is no difference between the groups, showing good specificity and safety. The experimental results show that the CAR structure prepared in the application can specifically kill target cells, has good persistence, and has the ability of multiple rounds of killing.

[0209] The experimental results show that when IgG4, CD8 and CD28 hinge regions are used, the GPC3 scFv CAR uses the CD8 hinge region structure, the cytokine secretion level is higher, and the cell activation is stronger and the cell killing ability is more effective and persistent when facing target cells.

[0210] Example 3: Comparison of in vitro functions of CAR structures with different transmembrane domains of GPC3 scFv

[0211] 3.1 Detection of CAR expression level

[0212] The structures of GPC3 scFv-CD8H-CD28TM and GPC3 scFv-CD8H-CD8TM were respectively transduced into T cells, and FITC-GPC3 protein fluorescent staining was performed at 72 hours, and the expression rate was determined by flow cytometry.

[0213] 3.2 Detection of cytokine secretion level

[0214] The uninfected T cells and CAR-T cells were accurately counted and adjusted to the same cell concentration. The group with higher expression was diluted and standardized using uninfected T cells, so that the cell concentrations of different groups and the CAR expression levels were consistent. The target cells were also accurately counted, resuspended in 200 μl of cell culture medium at a target ratio of 1:1 and a cell density of 5x10 4 After 24 hours of culture, the supernatant was centrifuged and used for enzyme-linked immunosorbent assay to determine the secretion of γ-interferon or IL-2.

[0215] 3.3 Multiple rounds of target cell killing experiment of CAR-T

[0216] Target cells Hep3B-GFP and MB231-GFP were counted and plated in flat-bottom 96-well plates at 10,000 cells per well. Meanwhile, uninfected T cells were precisely counted and standardized for expression rate, and then added to the 96-well plates at an effector-to-target ratio of 1:8, i.e. 1250 T cells per well. The 96-well plates were placed in the IncuCyte for real-time imaging, which can monitor the growth curve of the GFP-labeled target cells. After 48 hours and 92 hours, the 96-well plates were taken out twice, and real-time imaging was continued after 5000 corresponding target cells were added to each well to detect the persistence and multi-round killing ability of CAR-T cells.

[0217] Experimental results:

[0218] The CAR expression level results are shown in FIG. 5, and the CD28 transmembrane domain group and the CD8 transmembrane domain group obtained CAR positive rates of 45.5% and 28.3%, respectively. The expression level of the GPC3 scFv-CD8H-CD28TM structure is higher than that of other CAR structure designs, and has a certain expression advantage.

[0219] The cytokine secretion level results are shown in FIG. 6. After adjusting the CAR expression rate to be consistent and co-culturing with tumor cells, the CD28 transmembrane domain group can secrete a higher level of interferon-γ when encountering GPC3-positive target cells Hep3B, and has no special reaction to GPC3-negative cells MB231. The experimental results show that the CAR structure prepared in the present application can improve the level of cytokine secretion.

[0220] The target cell killing results are shown in FIGS. 7A-7B. In the in vitro target cell Hep3B killing experiment, the difference between the GPC3 scFv-CD8H-CD28TM and CD8TM groups in the first round of killing is small. After adding target cells for the second time at 48 hours, the CD28TM group has a more rapid and effective target cell clearance ability. After adding target cells for the third time at 92 hours, the CD28TM group can continuously and effectively kill target cells compared with the CD8TM group, and the CD8TM can only inhibit the growth of Hep3B. In the non-target cell MB231 killing, the difference between the two groups is small. The experimental results show that the CAR structure prepared in the present application can specifically kill target cells, has good persistence, and has the ability of multi-round killing.

[0221] The experimental results show that when CD8 and CD28 transmembrane domains are used, the GPC3 scFv CAR adopts the CD28 transmembrane domain structure, the cytokine secretion level is higher, and the cell activation and effective and persistent cell killing ability are stronger when facing target cells.

[0222] Example 4 GPC3 target cell Hep3B animal tumor-bearing experiment

[0223] NCG mice were inoculated with Hep3B cells (1 x 10 6 / each) three days later, and the experimental group was injected with 4 x 10 6 / each GPC3 scFv-CD8H-CD28TM structure GPC3 CAR-T cells, and 6000 IU IL-2 was injected intraperitoneally three times a week, and then the tumor growth was observed and measured continuously.

[0224] Experimental results:

[0225] The animal tumor-bearing test results are shown in Figure 8, and the GPC3 scFv-CD8H-CD28TM structure can rapidly exert an effective inhibition and killing effect on tumors. After 17 days of CAR-T injection, the tumor has been rapidly reduced and has been inhibited in growth thereafter. The experimental results show that the CAR structure prepared in the present application has an effective and persistent anti-tumor effect.

[0226] Example 5: Comparison of the in vitro functions of CAR structures using different scFvs of the same target or different scFvs of different targets

[0227] 5.1 Construction of CAR structure

[0228] GPC3 scFv was replaced by 8F8 scFv (SEQ ID NO: 29) of the same GPC3 target and ROR1 scFv (SEQ ID NO: 30) of a different target, respectively. Different hinge regions were designed for expression and functional comparison.

[0229] 5.2 Detection of cytokine secretion level

[0230] Different structures were also packaged with lentivirus and infected T cells, and the expression rate of CAR was detected using fluorescently labeled GPC3 or ROR1 protein at 72 hours and subsequent time points. After uniform expression, the target cells were precisely counted and co-cultured at a ratio of 1:1 and a cell density of 5 x 10 4 After 24 hours, the cells were centrifuged and the supernatant was used for enzyme-linked immunosorbent assay to determine IL-2 secretion.

[0231] Experimental results:

[0232] The CAR expression level results of 8F8 scFv CAR-T are shown in FIG. 9. The 8F8-IgG4H-CD28TM can maintain a substantially consistent CAR expression rate at day 3 and day 8, and the CAR structure cannot be stably expressed by using CD8 or CD28 hinge region, which is decreased from 77.5% to 59.2% and from 59.3% to 41.5%, respectively. The experimental results show that the combination of the CAR structure of the application has a good positive expression rate, and the CAR persistent expression ability will be lost after replacing the antibody.

[0233] The cytokine secretion level results of 8F8 scFv CAR-T are shown in FIG. 10. In the co-culture system, the interleukin 2 secreted by the CD8 hinge region group is lower than that of the IgG4 and CD28 hinge group. The experimental results show that the cytokine secretion level effect is not good after the combination of the CAR structure of the application is replaced by other antibodies.

[0234] The CAR expression level results of ROR1 scFv CAR-T are shown in FIG. 11. The ROR1 CAR of the CD8 hinge plus CD28 transmembrane domain combination is basically not detected for CAR expression, and the expression rate of IgG4 and CD28 hinge is better. The experimental results show that the CAR expression level is low after the CAR combination of the application is replaced by the antibody of other target points, and the CAR structure combination prepared by the application has an outstanding effect.

[0235] The cytokine secretion level results of ROR1 scFv CAR-T are shown in FIG. 12. In the interleukin 2 determination, the interleukin 2 secretion level of the CD8 hinge region group is almost not detected. The experimental results show that the cytokine secretion level effect is not good after the combination of the CAR structure of the application is replaced by other antibodies.

[0236] The experimental results show that the combination of the antibody, the CD8 hinge region and the CD28 transmembrane domain of the application makes the CAR have a good effect, which is better than other CAR structures after replacing the antibody part.

[0237] Example 6 GPC3 ScFv and CD8H-CD28 are suitable for constructing CAR-iNKT

[0238] 6.1 iNKT cell culture and virus transfection

[0239] The PBMC is obtained by Ficoll density gradient centrifugation, and the iNKT cells in the PBMC are expanded by stimulating with a-galactosylceramide (aGalcer). The cells are counted by using trypan blue counting method during the culture process, and the complete culture medium containing IL-7 and IL-15 is added every 1-2 days according to the cell expansion to pass the passage.

[0240] 6.2 Detection of CAR expression efficiency

[0241] After transfection for 3-7 days, 3-5 x 105iNKT cells were taken from the above-mentioned amplified and transduced virus, washed once with buffer (staining buffer, DPBS containing 1% FBS), resuspended with 100 μL buffer, added with 1 μL PE-labeled recombinant GPC3 protein, His tag (PE-Labeled Recombinant GPC3 protein, His Tag (Site-specific conjugation)), and incubated at 4°C in the dark for 30 min. After washing twice with buffer, resuspend with 100 μL buffer, and read with CytoFLEX, the data analysis was performed using FlowJo 10.0. 5

[0242] 6.3 Co-culture of tumor cells and iNKT-CAR

[0243] After transfection of iNKT cells for 5-7 days, the killing efficiency was detected. The tumor cell line stably expressing GFP was inoculated in a 96-well plate at 1 x 105cells / well. After the tumor cells adhered, they were added to the 96-well plate containing the corresponding tumor cells at E / T = 1:1 for co-culture, and the tumor cells were re-supplemented for stimulation after 24-48 h of culture. The fluorescence intensity of the tumor cells was recorded in real time using xCELLigence RTCA, the data was processed using RTCA software, and the fluorescence intensity-time curve and killing efficiency chart were plotted using GraphPad prism 9. 4

[0244] Experimental results:

[0245] The CAR expression level results on iNKT cells are shown in Figure 13. The combination of GPC3 scFv and CD8H-CD28TM had an expression efficiency of up to 39.4% on iNKT cells, and the expression efficiency 6 days after transfection was close to that 3 days after transfection. Compared with the control construction GPC3 scFv-CD28H-CD28TM, although the expression efficiency was 45.8% 3 days after transfection, it decreased to 6.45% 6 days after transfection. The results showed that GPC3 scFv-CD8H-CD28TM could achieve higher transfection efficiency and maintain stable expression on iNKT cells, and could be used for the development of universal GPC3-CAR-iNKT.

[0246] ​​The killing results of CAR-iNKT on target cells are shown in Figure 14. GPC3 scFv-CD8H-CD28TM-CAR-iNKT has a significant killing effect on Huh7 and Hep3B with high expression of GPC3, and still has a strong and significant killing ability against target cells after multiple stimulations.

[0247] These results show that the combination of GPC3 ScFv-CD8H-CD28TM is also applicable on iNKT cells, and the CAR structure prepared in this application has good effect after being prepared as CAR-iNKT.

[0248] Example 7 GPC3 ScFv combined with CD8H-CD28 is suitable for constructing CAR-γδT

[0249] 7.1 γδT cell culture and virus transfection

[0250] PBMC was obtained by Ficoll density gradient centrifugation, and the separated PBMC was activated by adding CD3 / CD28 magnetic beads within 0-5 days of culture. The culture medium was X-vivo15 containing SR (5-10%), IPP (50uM), HEPES, IL-15 (10ng / ml), IL-21 (10ng / ml) and IL-2 (1000IU / ml). The adherent non-target cells were removed during culture. After 5 days of culture, the magnetic beads were removed, and the serum replacement was reduced to 5% for continued culture.

[0251] 7.2 Detection of CAR expression efficiency

[0252] After 3-7 days of transfection, 3-5x10 5 The above expanded and transduced γδT cells were washed once with buffer (DPBS containing 1% FBS), resuspended with 100 μL buffer, added with 1 μL PE-labeled recombinant GPC3 protein, His tag (PE-Labeled Recombinant GPC3 protein, His Tag (Site-specific conjugation)), and incubated at 4°C for 30 min. After washing twice with buffer, resuspend with 100 μL buffer, and read with CytoFLEX, the data analysis uses FlowJo10.0.

[0253] 7.3 Co-culture of tumor cells with iNKT-CAR

[0254] The killing efficiency of γδT cells was detected 5-7 days after transfection. The tumor cell line stably expressing GFP was added to the culture medium at a concentration of 1x10 4 / wells were seeded into 96-well plates. After tumor cells adhered, they were added to 96-well plates containing the corresponding tumor cells at an E / T ratio of 1:1 for co-culture. The fluorescence intensity of tumor cells was recorded in real time using xCELLigence RTCA, and the data were processed using RTCA software. GraphPad Prism 9 was used to plot graphs of fluorescence intensity versus time and killing efficiency.

[0255] Experimental results:

[0256] Figure 15 shows the CAR expression level results on γδT cells. The combination of GPC3 scFv and CD8H-CD28TM achieved an expression efficiency of up to 38.2% on γδT cells. The results indicate that the combination of GPC3 scFv-CD8H-CD28TM can also achieve high transfection efficiency on γδT cells.

[0257] The cell-killing results of CAR-γδT on target cells are shown in Figure 16. GPC3 scFv-CD8H-CD28TM-CAR-γδT showed a significant killing effect on Hep3B cells with high GPC3 expression. After two days of co-culture, GPC3 scFv-CD8H-CD28TM-CAR-γδT demonstrated a sustained and effective ability to kill target cells. The experimental results indicate that the CAR structure prepared in this application, after being prepared as CAR-γδT, can specifically kill target cells and has good killing persistence.

[0258] These results indicate that the combination of GPC3 scFv-CD8H-CD28TM is also applicable to γδT cells, and the CAR structure prepared in this application has good performance after being prepared as CAR-γδT.

[0259] Example 8: Membrane-bound IL-10-GPC3 CAR-T cells

[0260] The GPC3 CAR encoding GPC3 scFv-CD8H-CD28™ and the membrane-binding IL-10-GPC3 CAR structures were constructed as shown in Figure 17. All related gene structures were synthesized using GeneWiz. The GPC3 CAR encoding the GPC3 scFv-CD8H-CD28™ structure was induced to express by linking the CD8α signal peptide sequence to the Xho1 and SpeI cloning sites, followed by linking IL-10 (SEQ ID NO:32). Finally, the CD8 hinge region and CD8 transmembrane domain were linked to localize IL-10 expression to the cell membrane.

[0261] 8.1 Flow cytometry assays to detect the expression of GPC3 CAR and membrane-bound IL-10-GPC3 CAR in PBMC cells

[0262] Activated PBMCs, which were pre-activated for 24 hours with CD3 and CD28 conjugated magnetic beads and IL-2 (500 IU / mL), were distributed into multiple wells (1.0 mL, 1.0 x 10 6 / mL) of a 12-well tissue culture plate, and then GPC3 CAR, membrane-bound IL-10-GPC3 CAR encoding or control lentivirus was added. After 24 hours of incubation, the culture supernatant was removed and 2.0 mL of fresh X-VIVO culture solution was added to each well to resuspend the lentivirus-transduced PBMC cells. After another 72 hours of incubation at 37°C, 5% CO2, 0.3 x 10 6 cells were taken and centrifuged at 1300 rpm for five minutes, resuspended in 100 μl FASC buffer (containing 0.5% BSA, 0.1% NaN3, 2 mM EDTA, pH 7.0 PBS) and stained with anti-CD3-PB antibody to isolate T cells, while GPC3-FITC protein was used to detect the expression of CAR. Then flow cytometry was performed using a flow cytometer (Beckman Coulter) in plate mode, and the data was analyzed using FlowJo software.

[0263] Experimental results:

[0264] The CAR expression level results are shown in Figure 18. The positive rate of the membrane-bound IL-10-GPC3 CAR group using the GPC3 scFv-CD8H-CD28TM structure was 82.8%, which was higher than that of the GPC3 CAR group without IL-10 binding, and had a certain expression advantage. The experimental results show that the CAR structure constructed in the present application also has a good positive expression rate when combined with cytokines.

[0265] 8.2 Membrane-bound IL-10 can enhance the activation of GPC3 CAR on T cells

[0266] 8.2.1 Co-culture of CAR-T cells and tumor cells

[0267] The activation potency of the prepared GPC3 CAR-T and membrane-bound IL-10-GPC3 CAR-T was determined by co-culturing them with tumor cells. Lentivirus-transduced PBMCs (0.3 x 10 4 ) were co-cultured with negative expression cells (MB231 (GPC3-HBSAG-), 1.5 x 10 4 ), or with target tumor cells (Huh7 (expressing GPC3+HBSAG+), Hep3B (expressing GPC3+HBSAG+), 1.5 x 10 4) were co-cultured in a round-bottom 96-well tissue culture plate with an effector-to-target ratio of 1:5, and IMDM medium was added to a final volume of 200 μL per well. After centrifugation at 300 x g for 5 minutes, the tissue culture plate was incubated at 37°C and 5% CO2 for 24 hours. 100 μL of supernatant was transferred to a new round-bottom 96-well tissue culture plate. PBMCs untransduced with lentivirus were co-cultured with target tumor cells as a negative control group.

[0268] 8.2.2 Enzyme-linked immunosorbent assay

[0269] The cytokine secretion level was detected by using an EILSA kit to determine IFN-γ in the culture supernatant (Biolegend, CAT#430104). The concentration of IFN-γ in the culture supernatant was calculated according to the standard curve of the known standard.

[0270] Experimental results:

[0271] The results of the cytokine secretion level are shown in FIG. 19. After co-culturing the PBMCs after lentivirus transduction with target tumor cells (CAR-T cells + Huh7 / Hep3B), the concentration of IFN-γ secreted by the PBMCs in the cell culture solution increased, and the concentration of IFN-γ secreted after co-culturing the membrane-bound IL-10-GPC3 CAR-T with the target tumor cells was higher than that of the GPC3 CAR-T cell group. The experimental results show that after co-culturing with the target tumor cells, the T cells in the GPC3 CAR-T group and the membrane-bound IL-10-GPC3 CAR-T group can be effectively activated, and the activation effect of the membrane-bound IL-10-GPC3 CAR-T group is stronger. The CAR structure prepared in the present application also has a good cytokine secretion level after being combined with the cytokine.

[0272] 8.3 Membrane-bound IL-10 can enhance the tumor killing effect of GPC3 CAR

[0273] 8.3.1 Co-culture of CAR-T cells and tumor cells

[0274] The tumor killing potency of GPC3 CAR-T cells and membrane-bound IL-10-GPC3 CAR-T cells was determined by co-culturing them with tumor cells. Lentivirus-transduced PBMCs (0.3 x 10 4 ) were co-cultured with negative expression cells (MB231 (GPC3-HBSAG-), 1.5 x 10 4 ), or with target tumor cells (Huh7 (expressing GPC3+HBSAG+), Hep3B (expressing GPC3+HBSAG+), 1.5 x 10 4The cells were co-cultured in 96-well round-bottomed tissue culture plates with an effector cell to target cell ratio of 1:5. IMDM culture medium was added, and the final volume was 200 μL per well.

[0275] 8.3.2 CAR-T cell rechallenge assay against tumor cells (Incucyte instrument detection method)

[0276] Insert SX1 live cell analyzer ( In the SX1 Live-Cell Analysis System, after incubation at 37°C and 5% CO2 for 48 hours, T cells were subjected to drug resistance and then challenged. The cell culture plate was centrifuged at 300g for 5 minutes, and 100μl of supernatant was collected. Then, 5kJ of tumor cells were added, resulting in a final volume of 200μl. The cells were incubated for another 24 hours in an SX1 live cell analyzer. The killing effect of lentiviral transfection of PBMCs on target tumor cells was analyzed using Incucyte 2022A Rev1 software.

[0277] Experimental results:

[0278] [Corrected according to Rule 91, 19.11.2025] The results of target cell killing are shown in Figures 20A-C. In the CAR-T cell co-culture group with HUH7-GFP / Hep3B-GFP, compared with the untransduced T cell group, both the GPC3 scfv CAR T cell group and the GPC3 scfv CAR-membrane bound IL-10 T cell group showed significant tumor killing effects before the rechallenge experiment. The tumor cell rechallenge (5×10⁻⁶ cells / year) resulted in a tumor killing effect. 3 After [number] transductions, both the GPC3 CAR-T group and the membrane-bound IL-10-GPC3 CAR-T group showed effective killing of tumor cells, with the membrane-bound IL-10-GPC3 CAR-T group exhibiting a faster killing rate compared to the GPC3 CAR-T group. In the negative cell group (CAR-T cells + MB231-GFP cells), the number of tumor cells in the transduced PBMC group was not significantly different from that in the untransduced group, and neither type of CAR-T cell produced significant cell-killing effects. Even after rechallenge experiments, neither type of CAR-T cell produced significant killing effects. The experimental results indicate that the CAR structure prepared in this application, after binding to cytokines, also has a good killing effect on target cells and exhibits good persistence.

Claims

1. A chimeric antigen receptor comprising an antigen binding domain, a CD8 hinge region, and a CD28 transmembrane domain, the antigen binding domain comprising an antibody heavy chain variable region VH, the VH comprising a heavy chain complementarity determining region HCDR1, HCDR2, and HCDR3, the HCDR1 having the amino acid sequence of SEQ ID NO: 1, the HCDR2 having the amino acid sequence of SEQ ID NO: 2, and the HCDR3 having the amino acid sequence of SEQ ID NO:

3.

2. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises an intracellular signaling domain.

3. The chimeric antigen receptor of claim 2, wherein the intracellular signaling domain comprises an intracellular signaling domain derived from any one of the following proteins: CD3 zeta, CD3 delta, CD3 gamma, CD3 epsilon, CD79a, CD79b, Fc epsilon Rl gamma, Fc epsilon Rl beta, Fc gamma RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (KSHV), DAP10, DAP-12, and a domain comprising at least one ITAM.

4. The chimeric antigen receptor of any one of claims 2-3, wherein the intracellular signaling domain is derived from an intracellular signaling domain of CD3 zeta.

5. The chimeric antigen receptor of any one of claims 2-4, wherein the intracellular signaling domain has the amino acid sequence of SEQ ID NO:

25.

6. The chimeric antigen receptor of any one of claims 1-5, wherein the chimeric antigen receptor comprises a costimulatory domain.

7. The chimeric antigen receptor of claim 6, wherein the costimulatory domain comprises a costimulatory domain derived from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc epsilon Rl gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

8. The chimeric antigen receptor of any one of claims 6-7, wherein the costimulatory domain is derived from a costimulatory domain of 4-1BB.

9. The chimeric antigen receptor of any one of claims 6-8, wherein the costimulatory domain has the amino acid sequence of SEQ ID NO:

22.

10. The chimeric antigen receptor of any one of claims 1-9, wherein the chimeric antigen receptor comprises a signal peptide.

11. The chimeric antigen receptor of claim 10, wherein the signal peptide is derived from CD8 alpha.

12. The chimeric antigen receptor of any one of claims 10-11, wherein the amino acid sequence of the signal peptide is set forth in SEQ ID NO:

23.

13. The chimeric antigen receptor of any one of claims 1-12, wherein the amino acid sequence of the CD8 hinge region is set forth in SEQ ID NO:

20.

14. The chimeric antigen receptor of any one of claims 1-13, wherein the amino acid sequence of the CD28 transmembrane domain is set forth in SEQ ID NO:

21.

15. The chimeric antigen receptor of any one of claims 1-14, wherein the amino acid sequence of the VH is set forth in SEQ ID NO:

8.

16. The chimeric antigen receptor of any one of claims 1-15, wherein the antigen binding domain further comprises a light chain variable region, VL, which comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO:

11.

17. The chimeric antigen receptor of claim 16, wherein the amino acid sequence of the VL is set forth in SEQ ID NO:

16.

18. The chimeric antigen receptor of any one of claims 1-17, wherein the antigen binding domain comprises a VH comprising HCDR1, HCDR2, and HCDR3, and a VL comprising LCDR1, LCDR2, and LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO:

11.

19. The chimeric antigen receptor of any one of claims 1-18, wherein the antigen binding domain comprises a VH having the amino acid sequence set forth in SEQ ID NO: 8 and a VL having the amino acid sequence set forth in SEQ ID NO:

16.

20. The chimeric antigen receptor of any one of claims 1-19, wherein the antigen binding domain comprises a scFv comprising a VH having the amino acid sequence set forth in SEQ ID NO: 8 and a VL having the amino acid sequence set forth in SEQ ID NO:

16.

21. The chimeric antigen receptor of any one of claims 1-20, wherein the antigen binding domain comprises a scFv having the amino acid sequence set forth in SEQ ID NO:

17.

22. The chimeric antigen receptor of any one of claims 1-21, wherein the antigen binding domain is capable of binding GPC3.

23. The chimeric antigen receptor of any one of claims 1-22, comprising an antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 11, a CD8 hinge region, and a CD28 transmembrane domain.

24. The chimeric antigen receptor of any one of claims 1-23, comprising an antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 11, a CD8 hinge region, and a CD28 transmembrane domain, the amino acid sequence of the CD8 hinge region is set forth in SEQ ID NO: 20, the amino acid sequence of the CD28 transmembrane domain is set forth in SEQ ID NO:

21.

25. The chimeric antigen receptor of any one of claims 1-24, comprising an antigen binding domain comprising a VH and a VL, the VH comprising a HCDR1, a HCDR2, and a HCDR3, the VL comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 11, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

26. The chimeric antigen receptor of any one of claims 1-25, comprising an antigen binding domain comprising a GPC3 scFv, the amino acid sequence of the GPC3 scFv is set forth in SEQ ID NO: 17, a CD8 hinge region, the amino acid sequence of the CD8 hinge region is set forth in SEQ ID NO: 20, a CD28 transmembrane domain, the amino acid sequence of the CD28 transmembrane domain is set forth in SEQ ID NO: 21, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

27. A modified immune cell, wherein the immune cell comprises the chimeric antigen receptor of any one of claims 1-26.

28. The modified immune cell of claim 27, wherein the immune cell is selected from one or more of the following cells: a T cell, a NK cell, a NKT cell, an iNKT cell, a gd T cell, a dendritic cell, and a macrophage.

29. The modified immune cell of any one of claims 27-28, wherein the immune cell is a T cell.

30. The modified immune cell of any one of claims 27-29, wherein the immune cell is an iNKT cell.

31. The modified immune cell of any one of claims 27-30, wherein the immune cell is a gd T cell.

32. The modified immune cell of any one of claims 27-31, wherein the immune cell further comprises a fusion protein.

33. The modified immune cell of claim 32, wherein the fusion protein comprises an IL-10 and a transmembrane domain.

34. The modified immune cell of any one of claims 32-33, wherein the fusion protein comprises an IL-10, a hinge region, and a transmembrane domain.

35. The modified immune cell of any one of claims 32-34, wherein the fusion protein comprises an IL-10, a CD8 hinge region, and a CD8 transmembrane domain.

36. An isolated nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1-26.

37. The nucleic acid molecule of claim 36, further encoding a fusion protein.

38. The nucleic acid molecule of any one of claims 36-37, wherein the fusion protein comprises IL-10 and a transmembrane domain.

39. The nucleic acid molecule of any one of claims 36-38, wherein the fusion protein comprises IL-10, a hinge region, and a transmembrane domain.

40. The nucleic acid molecule of any one of claims 36-39, wherein the fusion protein comprises IL-10, a CD8 hinge region, and a CD8 transmembrane domain.

41. A vector comprising the nucleic acid molecule of any one of claims 36-40.

42. A cell comprising the nucleic acid molecule of any one of claims 36-40 and / or the vector of claim 41.

43. A pharmaceutical composition comprising the chimeric antigen receptor of any one of claims 1-26, the modified immune cell of any one of claims 27-35, the nucleic acid molecule of any one of claims 36-40, the vector of claim 41, and / or the cell of claim 42, and optionally a pharmaceutically acceptable carrier.

44. Use of the chimeric antigen receptor of any one of claims 1-26, the modified immune cell of any one of claims 27-35, the nucleic acid molecule of any one of claims 36-40, the vector of claim 41, the cell of claim 42, and / or the pharmaceutical composition of claim 43 in the manufacture of a medicament for the prevention and / or treatment of a disease and / or disorder.

45. The use of claim 44, wherein the disease and / or disorder is a tumor.

46. The use of claim 45, wherein the tumor is a solid tumor and / or a hematological tumor.

47. The use of any one of claims 45-46, wherein the tumor is a hepatocarcinoma, an ovarian carcinoma, a melanoma, a pancreatic carcinoma, a lung carcinoma, a colon carcinoma, a breast carcinoma, a prostate carcinoma, a non-small cell lung carcinoma, a small cell lung carcinoma, a squamous cell carcinoma, a renal cell carcinoma, a colorectal carcinoma, a gastric carcinoma, a glioma, an ovarian clear cell carcinoma, an yolk sac tumor, and / or a hepatocellular carcinoma.

48. A method of preventing and / or treating a disease and / or disorder, comprising administering to a subject in need thereof the chimeric antigen receptor of any one of claims 1-26, the modified immune cell of any one of claims 27-35, the nucleic acid molecule of any one of claims 36-40, the vector of claim 41, the cell of claim 42, and / or the pharmaceutical composition of claim 43.

49. The method of claim 48, wherein the disease and / or disorder is a tumor.

50. The method of claim 49, wherein the tumor is a solid tumor and / or a hematological tumor.

51. The method of any one of claims 49-50, wherein the tumor is a liver cancer, an ovarian cancer, a melanoma, a pancreatic cancer, a lung cancer, a colon cancer, a breast cancer, a prostate cancer, a non-small cell lung cancer, a small cell lung cancer, a squamous cell carcinoma, a renal cell carcinoma, a colorectal cancer, a gastric cancer, a glioma, an ovarian clear cell carcinoma, a yolk sac tumor, and / or a hepatocellular carcinoma.

52. The chimeric antigen receptor of any one of claims 1-26, the modified immune cell of any one of claims 27-35, the nucleic acid molecule of any one of claims 36-40, the vector of claim 41, the cell of claim 42, and / or the pharmaceutical composition of claim 43, for use in the prevention and / or treatment of a disease and / or a disorder.

53. The chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition of claim 52, wherein the disease and / or disorder is a tumor.

54. The chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition of claim 53, wherein the tumor is a solid tumor and / or a hematological tumor.

55. The chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition of any one of claims 53-54, wherein the tumor is a liver cancer, an ovarian cancer, a melanoma, a pancreatic cancer, a lung cancer, a colon cancer, a breast cancer, a prostate cancer, a non-small cell lung cancer, a small cell lung cancer, a squamous cell carcinoma, a renal cell carcinoma, a colorectal cancer, a gastric cancer, a glioma, an ovarian clear cell carcinoma, a yolk sac tumor, and / or a hepatocellular carcinoma.

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