Chimeric antigen receptor targeting AXL and use thereof

Abstract

The present application relates to a chimeric antigen receptor targeting AXL and the use thereof, and specifically relates to a chimeric antigen receptor, comprising an antigen-binding domain, a hinge region, a transmembrane domain and a costimulatory domain, the antigen-binding domain comprising a heavy chain variable region (VH), and the VH 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. The present application also provides the use of the chimeric antigen receptor in the prevention and / or treatment of diseases.

Description

Chimeric antigen receptors targeting AXL and uses thereof TECHNICAL FIELD

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

[0002] AXL is a member of TAM receptor tyrosine kinase family, and the TAM family includes Tyro3, AXL and MERTK. The extracellular region of AXL is composed of two immunoglobulin-like regions and two fibronectin III-like regions, the intracellular region has tyrosine kinase activity, and the transmembrane region connects the extracellular region and the intracellular region. AXL is highly expressed in various cancers, such as granulocytic leukemia, pyrethroid leukemia, megakaryocytic leukemia, endometrial cancer, gastric cancer, colon cancer, prostate cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, renal cell carcinoma, glioblastoma, melanoma, osteosarcoma, etc., and mediates the occurrence of epithelial-mesenchymal transition. Abnormal expression of AXL is associated with clinical pathological features and poor prognosis of cancer patients. AXL can also induce the occurrence of drug resistance by regulating signal pathways and affecting tumor microenvironment.

[0003] Therefore, immunotherapy targeting AXL has great research prospects. As an attractive target, AXL urgently needs to develop more effective treatment methods that can effectively bind to it to meet the needs of the majority of disease treatment. SUMMARY

[0004] The present application provides a chimeric antigen receptor targeting AXL. In the present application, the chimeric antigen receptor has one or more of the following properties: (1) capable of binding to AXL and having good binding activity; (2) capable of binding to AXL expressed on the surface of target cells; (3) capable of killing AXL-positive tumors; (4) capable of blocking the binding of AXL to its receptor. 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 AXL and having good binding activity; (2) capable of binding to AXL expressed on the surface of target cells; (3) capable of killing AXL-positive tumors; (4) capable of blocking the binding of AXL to its receptor.

[0005] In one aspect, the present application provides a chimeric antigen receptor comprising an antigen binding domain, a hinge region, a transmembrane domain, and a costimulatory domain, the antigen binding domain comprising a heavy chain variable region VH, the VH comprising a heavy chain complementarity determining region HCDR1, a HCDR2, and a 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, the HCDR3 having the amino acid sequence of SEQ ID NO: 3, the hinge region derived from a hinge region of CD28.

[0006] In certain embodiments, the amino acid sequence of the hinge region is set forth in SEQ ID NO: 19.

[0007] In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from any one of the following 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, and SLAM.

[0008] In certain embodiments, the transmembrane domain is derived from a transmembrane domain of CD28.

[0009] In certain embodiments, the amino acid sequence of the transmembrane domain is set forth in SEQ ID NO: 20.

[0010] In certain embodiments, 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 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.

[0011] In certain embodiments, the costimulatory domain is derived from a costimulatory domain of 4-1BB.

[0012] In some embodiments, the amino acid sequence of the costimulatory domain is set forth in SEQ ID NO: 21.

[0013] In some embodiments, the chimeric antigen receptor further comprises an intracellular signaling domain.

[0014] In some embodiments, the intracellular signaling region comprises an intracellular signaling region 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.

[0015] In some embodiments, the intracellular signaling domain is derived from an intracellular signaling domain of CD3 zeta.

[0016] In some embodiments, the chimeric antigen receptor further comprises a signal peptide.

[0017] In some embodiments, the signal peptide is derived from CD8 alpha.

[0018] In some embodiments, the amino acid sequence of the VH is set forth in SEQ ID NO: 8.

[0019] In some 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 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.

[0020] In some embodiments, the amino acid sequence of the VL is set forth in SEQ ID NO: 16.

[0021] In some embodiments, the antigen binding domain comprises an scFv, the scFv comprising a VH and a VL, the amino acid sequence of the VH is set forth in SEQ ID NO: 8, and the amino acid sequence of the VL is set forth in SEQ ID NO: 16.

[0022] In some embodiments, the antigen binding domain specifically binds to AXL.

[0023] In certain embodiments, the chimeric antigen receptor comprises an antigen binding domain comprising an AXL scFv, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of the AXL scFv is set forth in SEQ ID NO: 17.

[0024] In certain embodiments, the chimeric antigen receptor comprises an antigen binding domain comprising an AXL scFv, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of the AXL scFv is set forth in SEQ ID NO: 17.

[0025] In certain embodiments, the chimeric antigen receptor comprises an antigen binding domain comprising an AXL scFv, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of the AXL scFv is set forth in SEQ ID NO: 17.

[0026] In certain embodiments, the immune cell is selected from one or more of the following: a T cell, a NK cell, a NKT cell, an iNKT cell, a gd T cell, a dendritic cell, and a macrophage.

[0027] In certain embodiments, the immune cell is a T cell, an iNKT cell, and / or a gd T cell.

[0028] In certain embodiments, the immune cell is a T cell, an iNKT cell, and / or a gd T cell.

[0029] In certain embodiments, the immune cell is a T cell, an iNKT cell, and / or a gd T cell.

[0030] In certain embodiments, the immune cell is a T cell, an iNKT cell, and / or a gd T cell.

[0031] In certain embodiments, the immune cell is a T cell, an iNKT cell, and / or a gd T cell.

[0032] In certain embodiments, the immune cell is a T cell, an iNKT cell, and / or a gd T cell.

[0033] In certain embodiments, the disease and / or disorder is a tumor.

[0034] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0035] In certain embodiments, the tumor is granulocytic leukemia, pyrethroid leukemia, megakaryocytic leukemia, endometrial cancer, gastric cancer, colon cancer, prostate cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, renal cell carcinoma, glioblastoma, melanoma, osteosarcoma, pancreatic cancer, and / or lymphoma.

[0036] In another aspect, the present application provides a method of preventing and / or treating a disease and / or a disorder, comprising administering to a subject in need thereof the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition.

[0037] In certain embodiments, the disease and / or disorder is a tumor.

[0038] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0039] In certain embodiments, the tumor is granulocytic leukemia, pyrethroid leukemia, megakaryocytic leukemia, endometrial cancer, gastric cancer, colon cancer, prostate cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, renal cell carcinoma, glioblastoma, melanoma, osteosarcoma, pancreatic cancer, and / or lymphoma.

[0040] 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 preventing and / or treating a disease and / or a disorder.

[0041] In certain embodiments, the disease and / or disorder is a tumor.

[0042] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0043] In certain embodiments, the tumor is granulocytic leukemia, pyrethroid leukemia, megakaryocytic leukemia, endometrial cancer, gastric cancer, colon cancer, prostate cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, renal cell carcinoma, glioblastoma, melanoma, osteosarcoma, pancreatic cancer, and / or lymphoma.

[0044] Other aspects and advantages of the present application can be readily ascertained by one skilled in the art from the following detailed description. Only the preferred embodiments of the application are shown and described in the following detailed description. As will be realized by those skilled in the art, the application is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present application. Accordingly, the drawings and descriptions should be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0045] The specific features of the invention involved in the present application are shown in the appended claims. The features and advantages of the invention involved in the present application can be better understood by referring to the detailed description of exemplary embodiments and the attached drawings. The drawings are briefly described as follows:

[0046] Figure 1 shows a schematic diagram of the CAR structure described in the present application.

[0047] Figure 2 shows a graph of the expression efficiency of the CAR structure described in the present application on T cells.

[0048] Figure 3A shows a graph of the killing results of the CAR-T cells described in the present application on MDA-MB-231 target cells; Figure 3B shows a graph of the killing results of the CAR-T cells described in the present application on LCLC-103H target cells.

[0049] Figure 4A shows a graph of the change in fluorescence intensity over time of the killing of target cells by the CAR-T cells described in the present application; Figure 4B shows a graph of the killing efficiency of target cells by the CAR-T cells described in the present application.

[0050] Figure 5 shows a graph of the expression efficiency of the CAR structure described in the present application on iNKT cells.

[0051] Figure 6 shows a graph of the killing results of the CAR-iNKT cells described in the present application on target cells.

[0052] Figure 7 shows a graph of the expression efficiency of the CAR structure described in the present application on γδ T cells.

[0053] Figure 8 shows a graph of the killing results of the CAR-γδ cells described in the present application on target cells. DETAILED DESCRIPTION

[0054] The embodiments of the invention involved in the present application are illustrated by the following specific examples, and other advantages and effects of the invention involved in the present application can be easily understood by those skilled in the art from the disclosure of the present specification.

[0055] Definitions of terms

[0056] In the present application, the term "AXL" generally refers to a member of the TAM receptor tyrosine kinase family, which includes Tyro3, AXL, and MERTK. The AXL extracellular region consists of two immunoglobulin-like regions and two fibronectin III-like regions, the intracellular region has tyrosine kinase activity, and the transmembrane region connects the extracellular region with the intracellular region. In the present application, the AXL can be a complete AXL and a functionally active fragment, homolog, analog, variant or derivative thereof. For example, the AXL can be a full-length AXL or a truncated AXL that retains the activity of the complete AXL. In the present application, the AXL can be of any species origin. For example, the AXL can be a human AXL. In the present application, the AXL can be wild type or artificially modified. For example, the AXL can be a modified AXL.

[0057] In the present application, the terms "chimeric antigen receptor" and "CAR" can be used interchangeably, and 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 a costimulatory 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.

[0058] In the present application, the term "signal peptide (SP)" generally refers to a leader sequence at the amino-terminal (N-terminal) end of a nascent CAR, which can direct the nascent protein to the endoplasmic reticulum and express at the time of translation or after translation. In the present application, the signal peptide can comprise a signal peptide derived from CD8a.

[0059] 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 an AXL positive tumor. In the present application, the antigen binding domain can specifically bind to a solid and / or hematological tumor. In the present application, the antigen binding domain can specifically bind to a granulocytic leukemia, pyrethroid leukemia, megakaryocytic leukemia, endometrial cancer, gastric cancer, colon cancer, prostate cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, renal cell carcinoma, glioblastoma, melanoma, osteosarcoma, pancreatic cancer and / or lymphoma. 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 linked 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 AXL.

[0060] In the present application, the term "Hinge" generally refers to a peptide, polypeptide, or protein molecule. In the present application, the Hinge can be a dimeric molecule composed of two polypeptides with identical amino acid sequences. 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 CD28.

[0061] 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 employed to anchor an extracellular domain to the cell membrane. For example, the Transmembrane domain can comprise a Transmembrane domain of 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.

[0062] 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.

[0063] 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.

[0064] 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 antibodies 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).

[0065] 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 effector cell. For example, the immune effector cell can include clearing foreign antigens or promoting immune effector responses, etc. In the present application, the immune effector cell can include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, iNKT cells, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells, and / or pluripotent stem cells. For example, the immune effector cell can be a T cell. For example, the immune cell can be an iNKT cell.

[0066] In the present application, the term "pharmaceutically acceptable carrier" generally refers to a nontoxic material that does not interfere with the effectiveness or 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 particles.

[0067] In the present application, the term "preventing and / or treating" generally refers to preventing and / or treating a disease. For example, the prevention and / or treatment can be preventing the onset of the disease, slowing or reversing the progression of the disease, 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 a tumor, reducing the severity and duration of a tumor and symptoms associated therewith.

[0068] In the present application, the term "tumor" generally refers to any new pathological tissue proliferation in which tumor antigens recognizable by the immune system are present. In the present application, the tumor can include a benign or malignant tumor (cancer). In the present application, the cancer can be a metastatic cancer and a non-metastatic cancer. In the present application, the tumor can include a solid tumor and a blood tumor. 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 an AXL-expressing tumor. For example, the tumor can be a granulocytic leukemia, a pyrethroid leukemia, a megakaryocytic leukemia, an endometrial cancer, a stomach cancer, a colon cancer, a prostate cancer, a thyroid cancer, a lung cancer, a breast cancer, an ovarian cancer, a liver cancer, a renal cell carcinoma, a glioblastoma, a melanoma, an osteosarcoma, a pancreatic cancer, and / or a lymphoma.

[0069] 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.

[0070] In the present application, the term "comprising" generally means including the recited features but not excluding other elements.

[0071] In the present application, the term "about" generally means a variation of 0.5-10% above or below the specified numerical value, for example, a variation 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.

[0072] DETAILED DESCRIPTION

[0073] Chimeric antigen receptor

[0074] In an aspect, the present application provides a chimeric antigen receptor, which can comprise an antigen binding domain, a hinge region, a transmembrane domain, and a costimulatory domain, wherein the antigen binding domain is capable of binding to AXL, and the hinge region is derived from a hinge region of CD28.

[0075] In the present application, the chimeric antigen receptor has one or more of the following properties: (1) is capable of binding to AXL with good binding activity; (2) is capable of binding to AXL expressed on the surface of a target cell; (3) is capable of killing tumor; and (4) is capable of blocking the binding of AXL to its receptor.

[0076] In the present application, the hinge region can be a hinge region of any species origin. For example, the hinge region can be of murine, rabbit, goat, llama, or human origin. For example, the hinge region can be a human CD28 hinge region. In the present application, the hinge region 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 CD28 hinge region. 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 hinge region can be a homolog thereof. For example, the homolog can be an amino acid sequence 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 CD28 hinge region. In the present application, the amino acid sequence of the hinge region can be as set forth in SEQ ID NO: 19.

[0077] In the present application, the transmembrane domain can comprise a transmembrane domain derived from any one of the following 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, and SLAM. 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 of any species origin. For example, the transmembrane domain can be of 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 comprises one or more amino acid substitutions, deletions, and / or additions to the amino acid sequence of the CD28 transmembrane 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 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: 20.

[0078] In the present application, the co-stimulatory domain can comprise a co-stimulatory 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 co-stimulatory domain can be a co-stimulatory domain derived from 4-1BB. In the present application, the co-stimulatory domain can be a co-stimulatory domain of any species origin. For example, the co-stimulatory domain can be of murine, rabbit, goat, llama, or human origin. For example, the co-stimulatory domain can be a human 4-1BB co-stimulatory domain. In the present application, the co-stimulatory 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 co-stimulatory 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 co-stimulatory 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 4-1BB co-stimulatory domain. In the present application, the amino acid sequence of the co-stimulatory domain can be as set forth in SEQ ID NO: 21.

[0079] 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 an antibody heavy chain variable region. 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.

[0080] 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 epitope of the antigen. The CDRs of an antigen binding protein can be determined by various numbering systems, such as CCG, Kabat, Chothia, IMGT, AbM, integrated consideration of Kabat / Chothia, etc. These numbering systems are known in the art, and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. One of skill in the art can determine the CDR regions using different numbering systems based on the sequence and structure of the antigen binding protein. The CDR regions can differ using different numbering systems. In the present application, the CDRs encompass the CDR sequences determined according to any CDR assignment; also encompass variants thereof, which include substitution, deletion, and / or addition of one or more amino acids of the CDRs. 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% (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 CDRs. For example, the CDRs of the antigen binding proteins described herein can be determined using Kabat.

[0081] 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 a HCDR1, a HCDR2, and a HCDR3, 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, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3.

[0082] In the present application, the antigen binding domain can further comprise at least one CDR in the variable region of the light chain of an antibody. In the present application, the antigen binding domain can comprise an antigen binding protein, which can further comprise a VL in the variable region of the light chain, which can comprise a LCDR1, a LCDR2, and / or a LCDR3 in the light chain complementarity determining region. For example, the VL can comprise a LCDR1, a LCDR2, and a LCDR3.

[0083] 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.

[0084] In the present application, the VH can comprise a H-FR1, a H-FR2, a H-FR3 and / or a H-FR4. In the present application, the antigen binding protein can comprise a HCDR1, a HCDR2, a HCDR3, a H-FR1, a H-FR2, a H-FR3 and a 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.

[0085] In the present application, the VL can comprise a L-FR1, a L-FR2, a L-FR3 and / or a L-FR4. In the present application, the antigen binding protein can comprise a LCDR1, a LCDR2, a LCDR3, a L-FR1, a L-FR2, a L-FR3 and a 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.

[0086] In the present application, the direct or indirect connection can be connected by intermolecular forces, or can be connected by a linker.

[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, which can comprise a HCDR1, a HCDR2, and a HCDR3, 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, and a VL, which can comprise a LCDR1, a LCDR2, and a 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.

[0088] In the present application, the amino acid sequence of the FR can be of any species origin. For example, the FR can be of murine, rabbit, goat, llama, or human origin. For example, the FR can be of murine origin. For example, the FR can be of human origin.

[0089] In the present application, the amino acid sequence of the FR can be adjusted as desired. For example, the FR can be of wild-type sequence. For example, the amino acid sequence of the FR can be altered without reducing the binding activity / affinity of the chimeric antigen receptor to AXL. 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 AXL. For example, the FR can be a variant thereof, which comprises substitution, deletion, and / or addition of one or more amino acids to the amino acid sequence of the FR. 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% (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 FR.

[0090] 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 AXL. In the present application, the VH and / or VL can be a variant thereof. For example, the variant comprises one or more amino acids being substituted, deleted and / or added to the amino acid sequence of the VH and / or VL. 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.

[0091] 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, and the amino acid sequence of the VL can be as set forth in SEQ ID NO: 16.

[0092] 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.

[0093] 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 analogue 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 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 VL 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, the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 11. For example, the antigen binding protein can be a scFv comprising a VH and a VL, the amino acid sequence of the VH is shown as SEQ ID NO: 8, the amino acid sequence of the VL is shown as SEQ ID NO: 16. For example, the antigen binding protein can be a scFv, the amino acid sequence of which is shown as SEQ ID NO: 17.

[0094] 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 be mutated / optimized for FR and / or constant region without reducing the binding activity / affinity of the chimeric antigen receptor to AXL. For example, the antigen binding protein can have lower immunogenicity without reducing the binding activity / affinity of the chimeric antigen receptor to AXL.

[0095] In the present application, the antigen binding protein can be a monospecific antibody, a bispecific antibody or a multispecific antibody. For example, the antigen binding protein can further comprise a second domain. For example, the second domain can target AXL or other antigens. In the present application, the antigen binding protein can further comprise a third domain. For example, the third domain can target AXL or other antigens.

[0096] In the present application, the antigen binding protein can be a monovalent antibody, a bivalent antibody or a multivalent antibody. For example, the antigen binding protein has one antigen binding site. For example, the antigen binding protein has two antigen binding sites. For example, the antigen binding protein has multiple antigen binding sites.

[0097] 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 region 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. 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 set forth in SEQ ID NO: 22.

[0098] In the present application, the chimeric antigen receptor can further comprise a signal peptide. In the present application, the signal peptide can be derived from CD8 alpha. In the present application, the signal peptide can be of any species origin. For example, the signal peptide can be a human CD8 alpha signal peptide. In the present application, the signal peptide can be a variant thereof. In the present application, the signal peptide can be a homolog thereof. In the present application, the amino acid sequence of the signal peptide can be set forth in SEQ ID NO: 18.

[0099] In the present application, the chimeric antigen receptor can comprise a signal peptide, an antigen binding domain targeting AXL, a CD28 hinge region, a transmembrane domain, a costimulatory domain, and / or an intracellular signaling domain. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a transmembrane domain, and a costimulatory domain. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a transmembrane domain, and a costimulatory domain, which can comprise an antigen binding protein, which can comprise a VH, which can comprise a HCDR1, a HCDR2, and a HCDR3. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a transmembrane domain, and a costimulatory domain, which can comprise an antigen binding protein, which can comprise a VH, which can comprise a HCDR1, a HCDR2, and a HCDR3, 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. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a transmembrane domain, and a costimulatory domain, which can comprise an antigen binding protein, which can comprise a VH, the amino acid sequence of the VH is set forth in SEQ ID NO: 8.

[0100] For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a transmembrane domain, and a costimulatory domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can comprise a VH, the VH can comprise a HCDR1, a HCDR2, and a HCDR3, the antigen binding protein can comprise a VL, the VL can comprise a LCDR1, a LCDR2, and a LCDR3. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a transmembrane domain, and a costimulatory domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can comprise a VH, the VH can comprise a HCDR1, a HCDR2, and a HCDR3, the antigen binding protein can comprise a VL, the VL can comprise 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. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a transmembrane domain, and a costimulatory domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can comprise a VH, the amino acid sequence of the VH is set forth in SEQ ID NO: 8, the antigen binding protein can comprise a VL, the amino acid sequence of the VL is set forth in SEQ ID NO: 16. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a transmembrane domain, and a costimulatory domain, the antigen binding domain can comprise a scFv, the scFv can comprise a VH, the amino acid sequence of the VH is set forth in SEQ ID NO: 8, the scFv can comprise a VL, the amino acid sequence of the VL is set forth in SEQ ID NO: 16. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a transmembrane domain, and a costimulatory domain, the antigen binding domain can comprise a scFv, the amino acid sequence of the scFv is set forth in SEQ ID NO: 17.

[0101] In embodiments, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, which can comprise an antigen binding protein, which can comprise a VH, which can comprise a HCDR1, a HCDR2, and a HCDR3. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, which can comprise an antigen binding protein, which can comprise a VH, which can comprise a HCDR1, a HCDR2, and a HCDR3, 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. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, which can comprise an antigen binding protein, which can comprise a VH, the amino acid sequence of the VH is set forth in SEQ ID NO: 8.

[0102] In embodiments, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, which can comprise an antigen binding protein, which can comprise a VH, which can comprise a HCDR1, a HCDR2, and a HCDR3. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, which can comprise an antigen binding protein, which can comprise a VH, which can comprise a HCDR1, a HCDR2, and a HCDR3, 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. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, which can comprise an antigen binding protein, which can comprise a VH, the amino acid sequence of the VH is set forth in SEQ ID NO: 8.

[0103] For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can 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. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can 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 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. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can comprise 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. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise a scFv, the scFv comprises 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. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise a scFv, the amino acid sequence of the scFv is set forth in SEQ ID NO: 17.

[0104] In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can comprise a VH, the VH can comprise a HCDR1, a HCDR2, and a HCDR3, the HCDR1 has an amino acid sequence of SEQ ID NO: 1, the HCDR2 has an amino acid sequence of SEQ ID NO: 2, and the HCDR3 has an amino acid sequence of SEQ ID NO: 3. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can comprise a VH, the VH has an amino acid sequence of SEQ ID NO: 8.

[0105] For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can 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 HCDR1 amino acid sequence is set forth in SEQ ID NO: 1, the HCDR2 amino acid sequence is set forth in SEQ ID NO: 2, the HCDR3 amino acid sequence is set forth in SEQ ID NO: 3, the LCDR1 amino acid sequence is set forth in SEQ ID NO: 9, the LCDR2 amino acid sequence is set forth in SEQ ID NO: 10, the LCDR3 amino acid sequence is set forth in SEQ ID NO: 11. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can comprise a VH and a VL, the VH amino acid sequence is set forth in SEQ ID NO: 8, the VL amino acid sequence is set forth in SEQ ID NO: 16. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain can comprise a scFv, the scFv comprises a VH and a VL, the VH amino acid sequence is set forth in SEQ ID NO: 8, the VL amino acid sequence is set forth in SEQ ID NO: 16. For example, the chimeric antigen receptor can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain can comprise a scFv, the scFv amino acid sequence is set forth in SEQ ID NO: 17.

[0106] In the present application, the chimeric antigen receptor can comprise a signal peptide, an antigen binding domain targeting AXL, a CD28 hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. For example, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can comprise a VH, the VH can comprise a HCDR1, a HCDR2, and a HCDR3, the HCDR1 has an amino acid sequence of SEQ ID NO: 1, the HCDR2 has an amino acid sequence of SEQ ID NO: 2, and the HCDR3 has an amino acid sequence of SEQ ID NO: 3. For example, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can comprise a VH, the VH has an amino acid sequence of SEQ ID NO: 8. For example, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can 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 HCDR1 has an amino acid sequence of SEQ ID NO: 1, the HCDR2 has an amino acid sequence of SEQ ID NO: 2, the HCDR3 has an amino acid sequence of SEQ ID NO: 3, the LCDR1 has an amino acid sequence of SEQ ID NO: 9, the LCDR2 has an amino acid sequence of SEQ ID NO: 10, and the LCDR3 has an amino acid sequence of SEQ ID NO: 11.For example, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can comprise a VH, the amino acid sequence of the VH is set forth in SEQ ID NO: 8, and a VL, the amino acid sequence of the VL is set forth in SEQ ID NO: 16. For example, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain can comprise an scFv, the scFv comprises a VH, the amino acid sequence of the VH is set forth in SEQ ID NO: 8, and a VL, the amino acid sequence of the VL is set forth in SEQ ID NO: 16. For example, the chimeric antigen receptor can comprise a CD8a signal peptide, an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain can comprise an scFv, the amino acid sequence of the scFv is set forth in SEQ ID NO: 17.

[0107] In another aspect, the present application provides a modified immune cell, wherein the immune cell can comprise the chimeric antigen receptor. In the present application, the modified immune cell has one or more of the following properties: (1) is capable of binding to AXL and has good binding activity; (2) is capable of binding to AXL expressed on the surface of a target cell; (3) is capable of killing AXL-positive tumors; (4) is capable of blocking the binding of AXL to its receptor.

[0108] In the present application, the immune cell can be a T cell, an NK cell, an NKT cell, a dendritic cell, a macrophage, a TIL cell, an iNKT cell, a CIK cell, a gd T cell, and / or a DNT cell. For example, the immune cell can be a T cell, an iNKT cell, and / or a gd T cell. For example, the immune cell can be an immune effector cell. For example, the immune cell can be an iNKT cell. For example, the immune cell can be a T cell. For example, the immune cell can be a gd T cell. For example, the immune cell can be a mixture, the mixture can comprise different immune cell species, for example, the mixture can comprise one or more immune cells.

[0109] 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. For example, the CAR-iNKT cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can 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 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. For example, the CAR-iNKT cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can comprise 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. For example, the CAR-iNKT cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise a scFv, the amino acid sequence of the scFv is set forth in SEQ ID NO: 17.

[0110] For example, the CAR-iNKT cells can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting AXL, which can comprise an antigen binding protein, which can comprise a VH and a VL, which VH can comprise a HCDR1, a HCDR2, and a HCDR3, which VL can comprise 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 CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the CAR-iNKT cells can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting AXL, which can comprise an antigen binding protein, which can comprise 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, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the CAR-iNKT cells can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting AXL, which can comprise a scFv, the amino acid sequence of the scFv is set forth in SEQ ID NO: 17, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0111] 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. For example, the CAR-T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can 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 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. For example, the CAR-T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can comprise 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. For example, the CAR-T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise a scFv, the amino acid sequence of the scFv is set forth in SEQ ID NO: 17.

[0112] For example, the CAR-T cell can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting AXL, which can comprise an antigen binding protein, which can comprise a VH and a VL, which VH can comprise a HCDR1, a HCDR2, and a HCDR3, which VL can comprise 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 CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the CAR-T cell can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting AXL, which can comprise an antigen binding protein, which can comprise 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, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the CAR-T cell can comprise a chimeric antigen receptor that can comprise a CD8a signal peptide, an antigen binding domain targeting AXL, which can comprise a scFv, the amino acid sequence of the scFv is set forth in SEQ ID NO: 17, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0113] 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. For example, the CAR-gammadelta T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can 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 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. For example, the CAR-gammadelta T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise an antigen binding protein, the antigen binding protein can comprise 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. For example, the CAR-gammadelta T cell can comprise a chimeric antigen receptor that can comprise an antigen binding domain targeting AXL, a CD28 hinge region, a CD28 transmembrane domain, and a 4-1BB costimulatory domain, the antigen binding domain can comprise a scFv, the amino acid sequence of the scFv is set forth in SEQ ID NO: 17.

[0114] 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 AXL, which can comprise an antigen binding protein, which can comprise a VH and a VL, the VH of which can comprise a HCDR1, a HCDR2, and a HCDR3, the VL of which can comprise 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, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. 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 AXL, which can comprise an antigen binding protein, which can comprise 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, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. 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 AXL, which can comprise a scFv, the amino acid sequence of the scFv being set forth in SEQ ID NO: 17, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0115] Nucleic acid molecules, vectors, and cells

[0116] In another aspect, the present application also provides an isolated nucleic acid molecule, which can encode the chimeric antigen receptor.

[0117] In the present application, the nucleic acid molecule can comprise a sequence encoding a signal peptide.

[0118] 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 cloning recombination, (iii) purified, for example, by enzyme digestion and gel electrophoresis fractionation, or (iv) synthesized, for example, by chemical synthesis.

[0119] 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.

[0120] In the present application, the nucleic acid molecule can be a modified nucleic acid molecule.

[0121] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0122] 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.

[0123] 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.

[0124] In the present application, the vector can further comprise other genes. For example, the other genes can be marker genes.

[0125] 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 further contain a replication initiation site. For example, the vector can include components that assist in entering cells. In order for the nucleic acid molecule to replicate in the vector, the 5' end and the 3' end of the nucleic acid molecule can further comprise long terminal repeat sequences.

[0126] In another aspect, the present application provides a cell comprising the nucleic acid molecule and / or the vector.

[0127] 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 the morphology of total DNA complement or on the genome) due to natural, accidental, or intentional mutations.

[0128] 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.

[0129] 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.

[0130] 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 Lipofectamin transfection.

[0131] In another aspect, the present application provides a method of preparing the chimeric antigen receptor, the method comprising culturing the cell under conditions such that the chimeric antigen receptor is expressed.

[0132] Pharmaceutical composition

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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, administration of the pharmaceutical composition can be performed in different ways, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0137] Pharmaceutical combination

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] Preparation method

[0144] 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.

[0145] Use

[0146] 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.

[0147] 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.

[0148] 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.

[0149] In the present application, the disease and / or disorder can be an AXL-related disease and / or disorder.

[0150] 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 an AXL-positive tumor. In the present application, the tumor can be a granulocytic leukemia, a pyrethroid leukemia, a megakaryocytic leukemia, an endometrial carcinoma, a gastric carcinoma, a colon carcinoma, a prostate carcinoma, a thyroid carcinoma, a lung carcinoma, a breast carcinoma, an ovarian carcinoma, a liver carcinoma, a renal cell carcinoma, a glioblastoma, a melanoma, an osteosarcoma, a pancreatic carcinoma and / or a lymphoma.

[0151] 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.

[0152] 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 manufacture of a diagnostic agent for diagnosing a disease and / or a disorder associated with the expression of AXL.

[0153] 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 manufacture of a diagnostic agent for diagnosing a disease and / or a disorder associated with the expression of AXL.

[0154] In the present application, the diagnostic agent can be used alone or in combination with an instrument, an apparatus, a device or a 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.

[0155] In the present application, the diagnostic agent can be selected from the group consisting of a reagent, a kit, a calibrator and a quality control.

[0156] In the present application, the method of in vitro detection can be selected from the group consisting of Western Blot, ELISA and immunohistochemistry.

[0157] For example, the reagent can include a reagent capable of measuring the expression amount of AXL.

[0158] For example, the reagent can be selected from the group consisting of a reagent for performing Western Blot, a reagent for performing ELISA and a reagent for performing immunohistochemistry.

[0159] In another aspect, the present application also provides a detection kit, which can include the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition, for detecting the presence and / or amount of AXL in a sample or a subject. For example, the detection kit can be used for preventing, diagnosing and / or treating a disease and / or a disorder.

[0160] For example, the present application relates to an immunodetection kit for use in combination with the chimeric antigen receptor and / or the modified immune cell of the present application, using an immunodetection method such as ELISA, immunohistochemistry, Western Blot, flow cytometry, etc.

[0161] In another aspect, 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 of the present application in the manufacture of a detection kit for diagnosis.

[0162] In another aspect, the present application also provides a method of detecting the presence and / or amount of AXL, which can include using the chimeric antigen receptor and / or the modified immune cell.

[0163] In another aspect, the present application provides a method of detecting AXL in a sample or a subject, which can include 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.

[0164] Without wishing to be bound by any theory, the examples below are merely to illustrate the chimeric antigen receptors, methods of making and uses of the present application and are not intended to limit the scope of the present application.

[0165] Examples

[0166] Materials and methods

[0167] HEK293T was purchased from ATCC, AXL high expression cell line MDA-MB-231 and LCLC-103H, AXL weak expression cell line H460 and AXL negative expression cell line MDA-MB-453 were purchased from Keygen Biotech. MDA-MB-231, LCLC-103H, H460 and MDA-MB-453 stably expressing GFP were made in the laboratory. PBMC was isolated from whole blood of healthy donors using Ficoll-Paque and 1 mL samples were aliquoted at a concentration of 2 x 10 7 cells / mL and stored frozen in liquid nitrogen tanks, the culture medium was 10% DMSO (vol / vol) added in heat-inactivated FCS.

[0168] In the technical solutions given in the examples, the exemplary amino acid sequences used are as shown below:

[0169] CD8aSP: SEQ ID NO: 18; AXL scfv: SEQ ID NO: 17; CD28 hinge region: SEQ ID NO: 19; CD28 transmembrane domain: SEQ ID NO: 20; 4-1BB costimulatory domain: SEQ ID NO: 21; CD3 zeta: SEQ ID NO: 22; CD8 hinge region: SEQ ID NO: 24; CD28 costimulatory domain: SEQ ID NO: 25; CD8 transmembrane domain: SEQ ID NO: 23.

[0170] Example 1 lentiviral plasmid construction

[0171] A CAR structure #0 (SEQ ID NO: 26) was designed containing a CD8a signal peptide (SEQ ID NO: 18), a humanized AXL scfv (SEQ ID NO: 17), a CD28 hinge region (SEQ ID NO: 19), a CD28 transmembrane domain (SEQ ID NO: 20), a 4-1BB stimulatory domain (SEQ ID NO: 21), and a CD3 zeta (SEQ ID NO: 22) connected in order. The synthetic sequence was ligated to a pALD vector with an SFFV promoter by BamH1 and Spel. Based on the construction of #0, the CD28 hinge region was replaced with a CD8 hinge region (SEQ ID NO: 24) by overlapping PCR and ligated to the pALD vector to obtain a construction named #1 (SEQ ID NO: 27). Based on the construction of #0, the CD28 TM was replaced with a CD8 TM (SEQ ID NO: 23) by overlapping PCR and ligated to the pALD vector to obtain a construction named #2 (SEQ ID NO: 28). Based on the construction of #0, the CD28 hinge region was replaced with an IgG4 hinge region (SEQ ID NO: 32) by overlapping PCR and ligated to the pALD vector to obtain a construction named #3 (SEQ ID NO: 31). A control construction FDZD (SEQ ID NO: 29) was set. The schematic diagram of the constructed structures is shown in Figure 1.

[0172] Example 2 Expression of AXL-CAR on T cells

[0173] 2.1 T cell activation and transduction

[0174] Freshly isolated PBMCs were added with CD3 / CD28 magnetic beads (Thermofisher, Cat. No. 11131D) for T cell sorting, cell / magnetic bead = 1 / 3.

[0175] After the sorted T cells were activated by CD3 / CD28 magnetic beads for 48 h, they were transferred to a centrifuge tube, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and resuspended with serum-free X-VIVO 15 (Lonza, Cat. No. BEBP02-054Q). The cell density was adjusted to 1 x 10 6 cells / mL, and added to a 12-well plate, 1 mL / well.

[0176] The lentivirus was taken out from the -80 °C refrigerator and quickly thawed in a 37 °C water bath. The virus was added to the cells and mixed gently, and the amount of virus added was 100 ng p24 / 1 x 10 6 cells.

[0177] The next day after transduction, fetal bovine serum (FBS, Gibco) was added to a final concentration of 10%.

[0178] The cell density was maintained at 1-2 x 10 6 The culture medium was X-VIVO 15 containing 10% FBS and 300 IU / mL.

[0179] 2.2 Detection of CAR expression efficiency

[0180] After 5 days, 3-5 x 10 5 After removing the magnetic beads and washing once with stainning buffer (DPBS containing 1% FBS), the T cells activated and transduced with the virus were used for detection of CAR+ expression efficiency. After removing the magnetic beads and washing, the T cells were resuspended with 100 μL of buffer, 1 μL of PE-labeled recombinant protein L, His tag (PE-Labeled Recombinant Protein L, His Tag (Site-specific conjugation) (ACRO, L-PP2H2)) was added, and incubated at 4°C in the dark for 30 min. After washing twice with buffer, the T cells were resuspended with 100 μL of buffer and read on a CytoFLEX. Data analysis was performed using FlowJo 10.0.

[0181] The CAR expression efficiency results are shown in FIG. 2. Compared with #FDZD, #2 and #3, #0 and #1 had higher expression efficiency on T cells (positive cell percentage > 45%) and better expression stability, and the negative and positive cell populations were clearly separated, which was conducive to the subsequent development of CAR-T. The experimental results showed that the CAR structure constructed in the present application had a very good positive expression rate when expressed on T cells, and had very good expression stability.

[0182] Example 3 Killing of AXL-positive target cells by CAR-T

[0183] After 5-7 days of T cell transfection, the killing efficiency was detected. The tumor cell line stably expressing GFP was seeded at 1 x 10 4 / wells were seeded into 96-well plates. After tumor cells adhered, the positivity rates of #0-CAR-T and FDZD-CAR-T were adjusted to be consistent using untransfected T cells (Mock-T), and added to 96-well plates containing the corresponding tumor cells at an E / T ratio (effective cells calculated based on the total number of cells) of 1:1. 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 the fluorescence intensity over time and the killing efficiency, and one-way ANOVA was used for statistical analysis. In addition, we compared the killing effect of #0-CAR-T with the original FDZD-CAR-T.

[0184] The target cell killing results are shown in Figures 3A-B. For MDA-MB-231 and LCLC103H, which are highly expressed in AXL, #0 showed the strongest killing effect due to its high CAR expression efficiency and good CAR expression stability. It is suitable for cell therapy product development. #0 is preferred for further validation.

[0185] The results of tumor cell fluorescence intensity and tumor cell killing efficiency are shown in Figures 4A-B. #0-CAR-T achieved an 80% killing effect against MDA-MB-231 and LCLC103H highly expressed in AXL cells, with a significant decrease in tumor cell fluorescence intensity and a marked reduction in tumor cell number. The killing effect of #0-CAR-T on tumor cells was significantly stronger than that of the control structure FDZD-CAR-T (p<0.01). #0-CAR-T achieved a 41.5% killing efficiency against H460 poorly expressed in AXL cells, with a decrease in tumor cell fluorescence intensity, and its killing effect was higher than that of FDZD-CAR-T (31%). However, #0-CAR-T showed no significant killing effect against AXL-negative MDA-MB-231, with no significant difference compared to FDZD-CAR-T. Compared with the control structure FDZD, #0 can improve clinical treatment efficacy without significantly increasing off-target effects.

[0186] Experimental results show that when the CAR structure constructed in this application is expressed on T cells, the resulting CAR-T cells have higher expression efficiency and tumor killing ability than CAR-T cells constructed with other CAR structures, which is more conducive to the development of subsequent products.

[0187] Example 4: Detection of CAR-iNKT expression efficiency

[0188] 4.1 iNKT cell culture

[0189] PBMC was obtained by Ficoll density gradient centrifugation, and iNKT cells in PBMC were expanded by stimulating with a-Galcer; trypan blue counting method was used to count the cells during the culture process, and according to the cell expansion, complete culture medium containing IL-7 and IL-15 was added every 1-2 days to subculture, and the virus can be transduced on the 2nd day to the 10th day of culture.

[0190] 4.2Detection of CAR expression efficiency

[0191] After 5 days, 3-5x10 5 The above activated and transduced iNKT cells were washed once with buffer (DPBS containing 1% FBS), resuspended with 100 μL buffer, added with 1 μL PE-labeled recombinant protein L, His tag (PE-labeled recombinant protein L, His tag (site-specific conjugation) (ACRO, L-PP2H2)), 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, and data analysis uses FlowJo 10.0.

[0192] The results are shown in Figure 5, and the expression efficiency of #0 on iNKT cells is as high as 42.6%. The experimental results show that the CAR structure constructed in the present application can achieve higher transfection efficiency on iNKT cells compared with the control construction (positive cell percentage of 6.2%), and the CAR structure constructed in the present application can provide a universal AXL-CAR-iNKT.

[0193] Example 5CAR-iNKT killing of AXL-positive target cells

[0194] After 5-7 days of iNKT cell transfection, the killing efficiency was detected. The tumor cell line stably expressing GFP was inoculated in a 96-well plate at 1x10 4 / well. After the tumor cells adhered, they were added to the 96-well plate containing the corresponding tumor cells at E / T=1:1. Similarly, the xCELLigence RTCA was used to record the fluorescence intensity of the tumor cells in real time, and the RTCA software was used for data processing, and GraphPad prism 9 was used to draw the fluorescence intensity-time curve and the killing efficiency chart, and One-way ANOVA was used for statistical analysis.

[0195] The results are shown in Figure 6. The #0-CAR-iNKT was significantly stronger than the FDZD-CAR-iNKT against the AXL high-expressing MDA-MB-231, and the fluorescence intensity of the tumor cells did not increase significantly, and the growth of the tumor cells was significantly inhibited. The #0-CAR-iNKT and the FDZD-CAR-iNKT had no killing effect on the AXL negative MDA-MB-231. Compared with the FDZD, the expression efficiency and the killing effect on the iNKT cells of the #0 were significantly better. The experimental results show that the CAR structure constructed in the present application has a higher tumor killing ability than the CAR-iNKT cells constructed by other CAR structures when expressed on the iNKT cells.

[0196] Example 6: Detection of expression efficiency of CAR-γδT

[0197] PBMC was obtained by Ficoll density gradient centrifugation, activated by CD3 / CD28 magnetic beads (Thermofisher, item number 11131D) from day 0 to day 5, and the culture medium was GT-T551, and 5-10% SR, IPP 50 μM, HEPES, IL-15 (10 ng / ml), IL-21 (10 ng / ml) and IL-2 (1000 IU / ml) were added to the culture medium. During the culture period, the non-target cells adhered to the wall were removed. On day 5, the magnetic beads were removed, and the virus was added for transfection.

[0198] The expression efficiency of CAR was detected. After 5 days of virus transduction, 3-5 x 10 5 The activated and virus-transduced γδT cells were washed once with buffer (DPBS containing 1% FBS), resuspended with 100 μL buffer, added with 1 μL PE-labeled recombinant protein L, His tag (PE-labeled recombinant protein L, His tag (site-specific conjugation) (ACRO, L-PP2H2)), 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, and the data analysis was performed using FlowJo 10.0.

[0199] The CAR expression efficiency results are shown in Figure 7. The transfection efficiency of the #0-γδT was greater than 26%, and the construction of the #0 was also suitable for γδT. The experimental results show that the CAR structure constructed in the present application is suitable for γδT cells, and has a higher expression efficiency.

[0200] Example 7: Killing of CAR-γδT against AXL positive target cells

[0201] The killing efficiency detection was performed 5-7 days after the transfection of γδ T cells. The tumor cell line stably expressing GFP was inoculated in a 96-well plate at 1×10 4 After the tumor cells adhered, they were added to the 96-well plate containing the corresponding tumor cells at E / T = 1:1. Similarly, the fluorescence intensity of the tumor cells was recorded in real time using xCELLigence RTCA, and the data was processed using RTCA software. GraphPad prism 9 was used to draw the fluorescence intensity-time curve and the killing efficiency chart, and One-way ANOVA was used for statistical analysis.

[0202] The target cell killing results are shown in FIG. 8. #0-γδT has a significant killing effect on AXL high expression MDA-MB-231, and #0-γδT has a stronger killing effect than γδT on AXL low expression. The experimental results show that the CAR structure constructed in the application applied to γδT also has a significant killing effect, has a higher tumor killing ability, and has no non-specific killing.

Claims

1. A chimeric antigen receptor comprising an antigen-binding domain, a hinge region, a transmembrane domain, and a co-stimulatory domain, wherein the antigen-binding domain comprises a heavy chain variable region VH, the VH comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, the amino acid sequence of HCDR1 being shown in SEQ ID NO:1, the amino acid sequence of HCDR2 being shown in SEQ ID NO:2, the amino acid sequence of HCDR3 being shown in SEQ ID NO:3, and the hinge region being derived from the hinge region of CD28.

2. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the hinge region is shown in SEQ ID NO:

19.

3. The chimeric antigen receptor according to any one of claims 1-2, wherein the transmembrane domain comprises a transmembrane domain derived from any of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, 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.

4. The chimeric antigen receptor according to any one of claims 1-3, wherein the transmembrane domain is derived from the transmembrane domain of CD28.

5. The chimeric antigen receptor according to any one of claims 1-4, wherein the amino acid sequence of the transmembrane domain is as shown in SEQ ID NO:

20.

6. The chimeric antigen receptor according to any one of claims 1-5, wherein the co-stimulatory domain comprises a co-stimulatory 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, FcεRIγ, 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.

7. The chimeric antigen receptor according to any one of claims 1-6, wherein the co-stimulatory domain is derived from the co-stimulatory domain of 4-1BB.

8. The chimeric antigen receptor according to any one of claims 1-7, wherein the amino acid sequence of the co-stimulatory domain is as shown in SEQ ID NO:

21.

9. The chimeric antigen receptor according to any one of claims 1-8, wherein the chimeric antigen receptor further comprises an intracellular signal transduction domain.

10. The chimeric antigen receptor of claim 9, wherein the intracellular signaling region comprises an intracellular signaling region derived from any of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpesvirus (KSHV), DAP10, DAP-12, and a domain comprising at least one ITAM.

11. The chimeric antigen receptor according to any one of claims 9-10, wherein the intracellular signal transduction domain is derived from the intracellular signal transduction domain of CD3ζ.

12. The chimeric antigen receptor according to any one of claims 1-11, wherein the chimeric antigen receptor further comprises a signal peptide.

13. The chimeric antigen receptor of claim 12, wherein the signal peptide is derived from CD8α.

14. The chimeric antigen receptor according to any one of claims 1-13, wherein the amino acid sequence of the VH is as shown in SEQ ID NO:

8.

15. The chimeric antigen receptor according to any one of claims 1-14, wherein 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 LCDR1 being as shown in SEQ ID NO:9, the amino acid sequence of LCDR2 being as shown in SEQ ID NO:10, and the amino acid sequence of LCDR3 being as shown in SEQ ID NO:

11.

16. The chimeric antigen receptor according to claim 15, wherein the amino acid sequence of the VL is as shown in SEQ ID NO:

16.

17. The chimeric antigen receptor according to any one of claims 1-16, wherein the antigen-binding domain comprises scFv, the scFv comprises VH and VL, the amino acid sequence of VH is shown in SEQ ID NO:8, and the amino acid sequence of VL is shown in SEQ ID NO:

16.

18. The chimeric antigen receptor according to any one of claims 1-17, wherein the antigen-binding domain specifically binds to AXL.

19. The chimeric antigen receptor according to any one of claims 1-18, comprising an antigen-binding domain, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signal transduction domain, wherein the antigen-binding domain comprises AXL scFv, and the amino acid sequence of the AXL scFv is shown in SEQ ID NO:

17.

20. The chimeric antigen receptor according to any one of claims 1-19, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:

26.

21. A modified immune cell, wherein the immune cell comprises a chimeric antigen receptor according to any one of claims 1-20.

22. The modified immune cell according to claim 21, wherein the immune cell is selected from one or more of the following cells: T cells, NK cells, NKT cells, iNKT cells, γδT cells, dendritic cells, and macrophages.

23. The modified immune cell according to any one of claims 21-22, wherein the immune cell is a T cell, an iNKT cell, and / or a γδT cell.

24. An isolated nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1-20.

25. A vector comprising the nucleic acid molecule of claim 24.

26. A cell comprising the nucleic acid molecule of claim 24 and / or the vector of claim 25.

27. A pharmaceutical composition comprising a chimeric antigen receptor according to any one of claims 1-20, a modified immune cell according to any one of claims 21-23, a nucleic acid molecule according to claim 24, a carrier according to claim 25 and / or a cell according to claim 26, and optionally a pharmaceutically acceptable carrier.

28. Use of the chimeric antigen receptor of any one of claims 1-20, the modified immune cell of any one of claims 21-23, the nucleic acid molecule of claim 24, the carrier of claim 25, the cell of claim 26, and / or the pharmaceutical composition of claim 27 in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

29. The use according to claim 28, wherein the disease and / or condition is a tumor.

30. The use according to claim 29, wherein the tumor is a solid tumor and / or a hematoma.

31. The use according to any one of claims 29-30, wherein the tumor is granulocytic leukemia, pyrethroid leukemia, megakaryocytic leukemia, endometrial cancer, gastric cancer, colon cancer, prostate cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, renal cell carcinoma, glioblastoma, melanoma, osteosarcoma, pancreatic cancer and / or lymphoma.

32. A method for preventing and / or treating diseases and / or conditions, comprising administering to a subject in need the chimeric antigen receptor of any one of claims 1-20, the modified immune cell of any one of claims 21-23, the nucleic acid molecule of claim 24, the carrier of claim 25, the cell of claim 26, and / or the pharmaceutical composition of claim 27.

33. The method of claim 32, wherein the disease and / or symptom is a tumor.

34. The method of claim 33, wherein the tumor is a solid tumor and / or a hematoma.

35. The method according to any one of claims 33-34, wherein the tumor is granulocytic leukemia, pyrethroid leukemia, megakaryocytic leukemia, endometrial cancer, gastric cancer, colon cancer, prostate cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, renal cell carcinoma, glioblastoma, melanoma, osteosarcoma, pancreatic cancer and / or lymphoma.

36. The chimeric antigen receptor of any one of claims 1-20, the modified immune cell of any one of claims 21-23, the nucleic acid molecule of claim 24, the carrier of claim 25, the cell of claim 26, and / or the pharmaceutical composition of claim 27, for the prevention and / or treatment of diseases and / or conditions.

37. The chimeric antigen receptor, modified immune cell, nucleic acid molecule, carrier, cell and / or pharmaceutical composition according to claim 36, wherein the disease and / or condition is a tumor.

38. The chimeric antigen receptor, modified immune cell, nucleic acid molecule, carrier, cell and / or pharmaceutical composition according to claim 37, wherein the tumor is a solid tumor and / or hematologic malignancy.

39. The chimeric antigen receptor, modified immune cell, nucleic acid molecule, carrier, cell and / or pharmaceutical composition according to any one of claims 37-38, wherein the tumor is granulocytic leukemia, pyrethroid leukemia, megakaryocytic leukemia, endometrial cancer, gastric cancer, colon cancer, prostate cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, renal cell carcinoma, glioblastoma, melanoma, osteosarcoma, pancreatic cancer and / or lymphoma.

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