Novel chimeric antigen receptors (CARs) with enhanced function

Abstract

The present invention relates to a novel chimeric antigen receptor that uses a part of CD99L2, which is known to play an important role in cell adhesion and migration, as a backbone of the chimeric antigen receptor, immune cells containing the same, and uses thereof. CD99L2-based CAR-T cells show improved T cell activity and tumor treatment efficiency compared to existing CAR-T cells, and therefore can be usefully used in the treatment of immune cells for cancer treatment.

Description

[Technical Field]

[0001] The present invention relates to a novel chimeric antigen receptor that uses a partial region of CD99L2, which is known to play an important role in cell adhesion and migration, as the backbone of the chimeric antigen receptor, immune cells containing the same, and uses thereof. [Background technology]

[0002] CAR-T cells are gene-transduced T cells in which a fusion protein (i.e., a chimeric antigen receptor; CAR) consisting of a recombinant antibody (e.g., scFv) region that specifically binds to a cancer antigen on the surface of tumor cells linked to a T cell receptor signaling domain is artificially expressed in T cells isolated from a patient's blood (Kershaw MH, et al., Nat Rev Immunol. 2005;5(12):928-40). When the CAR protein gene is introduced into T cells in the form of a retrovirus or lentivirus, the high gene transfer efficiency allows more than 50% of T cells to express the CAR protein on their surface within two weeks, making it possible to rapidly produce large numbers of tumor-specific T cells.

[0003] When the antibody portion of the CAR protein recognizes tumors, the CAR-T cells activate and act as tumor-killing T cells. Clinical trials of CAR-T cell therapeutics have rapidly increased since the late 2000s (Jena B, et al., Blood. 2010;116(7):1035-44). In particular, CAR-T cell therapy targeting CD19, a blood tumor cancer antigen of the B lymphocyte system, has achieved remarkable results since early clinical trials. CD19 CAR-T cell therapy, which showed some efficacy in B-cell lymphomas around 2010, has grown rapidly, starting with a report by a research team at the University of Pennsylvania of complete remission in a patient with chronic lymphocytic leukemia who was resistant to existing treatments. Recently, the remarkable therapeutic effect of CD19 CAR-T cell therapy, in which 27 of 30 patients with acute lymphocytic leukemia who were resistant to all previous treatments achieved complete remission within one month and the 6-month overall survival rate reached 78%, has led to large-scale investments by numerous multinational pharmaceutical companies (Maude SL, et al., N Engl J Med. 2014;371(16):1507-17). As a result, two CD19-targeted CAR-T cell therapies were approved by the FDA at the end of 2017.

[0004] Currently, CAR-T cell development is focused primarily on hematological tumors, with expansion to several solid tumors in progress (Yip A, Webster RM, Nat Rev Drug Discov. 2018;17(3):161-2). Among hematological tumor targets, anti-BCMA CAR-T cell therapy is the most advanced, targeting multiple myeloma. CAR-T cell therapy targeting B lymphatic tumor antigens, such as CD20 and CD22, in addition to CD19, is also under development. Several clinical trials of CAR-T cell therapy for solid tumors, including GD2 (brain tumor) and mesothelin (pleural cancer), have been conducted, but no dramatic results have yet been reported. This is presumably due to several factors that hinder the efficacy of CAR-T cells in solid tumors. For example, in the case of solid tumors, unlike leukemias, where tumor cells are primarily distributed in the blood and do not adequately generate a tumor microenvironment, solid tumors create an immune-resistant tumor microenvironment by secreting immunosuppressive cytokines such as TGF-β and IL-10, recruiting immunosuppressive cells such as regulatory T cells and myeloid-derived suppressor cells (MDSCs), or expressing immunosuppressive ligands such as PD-L1 on the tumor surface (Rabinovich GA, et al., Annu Rev Immunol. 2007;25:267-96). Therefore, for the future widespread use of CAR-T cell therapy, it is essential to develop CAR-T cells with significantly increased T cell activity that can overcome the immunosuppressive environment and exert an effective effect.

[0005] One strategy to enhance CAR-T cell function is to enhance T cell activation by altering the structure of the CAR protein itself (Dotti G, et al., Immunol Rev. 2014;257(1):107-26). CAR proteins are designed to incorporate the variable region of an antibody (single-chain variable fragment; scFv) that recognizes a cancer antigen, linked to an intracellular signaling domain via a backbone region (extracellular space + transmembrane domain). The intracellular signaling domain is primarily based on the intracellular region of the CD3 zeta chain, a signaling subunit of the T cell receptor (first-generation CARs). Efforts to improve CAR-T cell function through CAR protein modification have continued, with most of these efforts taking the form of altering or adding the signaling domain of a costimulatory molecule (Morello A, et al., Cancer Discov. 2016;6(2):133-46). For example, two currently available CAR-T cell therapies use the intracellular portions of the costimulatory molecules CD28 and 41BB, respectively (second-generation CARs), and subsequent attempts have been made to develop CARs that contain both the CD28 and 41BB intracellular domains (third-generation CARs). Currently available Kymriah CAR-T cells from Novartis and Yescarta CAR-T cells from Gilead Sciences are second-generation CAR-T cells that use the 41BB and CD28 intracellular domains, respectively.

[0006] In contrast, the CAR backbone region has only been used for physical binding, with only a portion of CD8, CD28, IgG1, or IgG4 being used. Therefore, altering the CAR backbone region offers a new way to modify CAR-T cell function.

[0007] Under these technical circumstances, the present inventors explored the possibility of improving the tumor therapeutic efficacy of CAR-T cells by introducing a new CAR design using a region containing the transmembrane domain of the CD99L2 protein as the CAR backbone region. As a result, they confirmed that CD99L2-backbone CAR-T cells exhibit significantly improved antitumor effects compared to conventional CD8-backbone CAR-T cells, leading to the completion of the present invention.

[0008] The information set forth in this Background section is intended solely to enhance understanding of the background of the present invention, and as such may not include information that constitutes prior art known to those of ordinary skill in the art to which the present invention pertains. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a chimeric antigen receptor and an immune cell containing the same that exhibit improved tumor therapeutic effects.

[0010] Another object of the present invention is to provide a nucleic acid encoding the chimeric antigen receptor, an expression vector containing the nucleic acid, and a virus containing the expression vector.

[0011] Another object of the present invention is to provide a cancer therapeutic composition comprising the immune cells, a cancer therapeutic method using the immune cells, uses of the immune cells for cancer therapy, and use of the immune cells for the manufacture of a drug for cancer therapy. [Means for solving the problem]

[0012] To achieve the above object, the present invention provides a chimeric antigen receptor comprising an extracellular domain and a transmembrane domain derived from the CD99L2 protein.

[0013] The present invention also provides a nucleic acid encoding the chimeric antigen receptor, an expression vector containing the nucleic acid, a virus containing the expression vector, and an immune cell expressing the chimeric antigen receptor.

[0014] The present invention also provides a cancer therapeutic composition comprising the immune cells, a cancer therapeutic method using the immune cells, use of the immune cells for cancer therapy, and use of the immune cells for the manufacture of a medicament for cancer therapy. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 shows the design of CD99L2-backbone CARs and the results of in vitro activity verification. Figure 1A shows a schematic diagram of the structural design of each CAR protein (hCD8 L: human CD8a leader, αCD19 scFv: single-chain variable fragment of anti-CD19 antibody (clone FMC63), EC: extracellular domain, TM: transmembrane domain, cyt: cytoplasmic domain). Figures 1B and 1E show the expression level of CAR proteins on the surface of CAR-T cells (numbers in the graph: percentage of cells (%)). Figures 1C and 1F are graphs showing the killing ability of each CAR-T cell against Raji-Luc lymphoma cells (relative light unit: luciferase activity value in surviving Raji-Luc cells after overnight culture with CAR-T cells; ET ratio (effector:target ratio): ratio of the number of co-cultured CAR-T cells (effector) to the number of Raji-Luc cells (target)). Figures 1D and 1G are graphs showing the amount of IFN-γ secreted as supernatant after co-culture with CAR-T cells and Raji cells. [Figure 2] Figure 2 shows the kinetic analysis of CD99L2-backbone CAR-T cell activation. The results are shown by flow cytometry (MFI: mean fluorescent intensity) of the expression of CD4-positive (Figure 2A) and CD8-positive (Figure 2B) CAR-T cell surface activation markers over time when CAR-T cells were co-cultured with Raji cells. [Figure 3]Figure 3 shows the in vivo tumor elimination promoting effect of CD99L2-backbone CAR-T cells. NSG mice were intravenously injected with Raji-Luc cells (day 0) and then with CAR-T cells on day 7. The images show representative images of the in vivo tumor cell proliferation measured by bioluminescence imaging at different times. DETAILED DESCRIPTION OF THE INVENTION

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0017] Chimeric antigen receptors (CARs) are artificial receptors that combine the antigen-recognition domain of an antibody with a cell membrane domain and an intracellular signaling domain. T cells expressing this receptor (CAR-T cells) are activated by the antibody domain, enabling them to specifically kill tumors. Therefore, CAR-T cells have been developed as antibody-gene cell therapies that combine the tumor-targeting ability of antibodies with the tumor-killing ability of T cells. They have demonstrated excellent therapeutic efficacy, particularly against hematologic tumors, and two or more CAR-T cell therapies have been launched. However, while CAR-T cell therapy has demonstrated high therapeutic efficacy against hematologic tumors, where tumor cells are frequently encountered in the blood, it has shown low efficacy against solid tumors. Therefore, for CAR-T cell therapy to be applicable to solid tumors, its function must be improved. As part of strategies to enhance CAR-T cell function, efforts are being made to engineer more efficient CAR proteins by modifying their structure.

[0018] The CAR backbone region contains a transmembrane domain, and the transmembrane domain of a novel cell membrane protein can be used to improve CAR function. In this study, CD99L2 was used as the target. CD99L2 (CD99 antigen-like 2) is a cell membrane protein belonging to the CD99 family, which is known to be expressed primarily in leukocytes and endothelial cells. Functionally, these proteins have been reported to promote cell adhesion and cell migration (Pasello M, et al., J Cell Commun Signal. 2018;12(1):55-68). In particular, CD99L2 has been reported to be involved in the extravasation of neutrophils, monocytes, T cells, and other cells under inflammatory conditions. Furthermore, it has been suggested that CD99L2 expressed on vascular endothelial cells may be involved in leukocyte extravasation (Seelige R, et al., J Immunol. 2013;190(3):892-6). CD99L2 forms a heterodimer with CD99 (Nam G, et al., J Immunol. 2013;191(11):5730-42). CD99 protein has been reported to be involved in T cell costimulation, suggesting that CD99L2 may contribute to T cell activation (Oh KI, et al., Exp Mol Med. 2007;39(2):176-84).

[0019] As a result, the present invention aims to present a new concept of CAR-T cells, which have improved function through T cell activation, by designing and producing a CAR protein into which the CD99L2 region has been introduced.

[0020] Thus, in one aspect, the present invention comprises: (a) an antigen binding domain; (b) a backbone comprising an extracellular binding domain and a transmembrane domain; (c) an intracellular signaling domain; A chimeric antigen receptor (CAR) comprising: The present invention relates to a chimeric antigen receptor (CAR), wherein the extracellular binding domain comprises an extracellular domain derived from CD99L2, and the transmembrane domain comprises a transmembrane domain derived from CD99L2.

[0021] In the present invention, the term "backbone" refers to a region comprising an extracellular spacer domain and a transmembrane domain.

[0022] In the present invention, the term "extracellular spacer domain" refers to the region that connects the antigen-binding domain and the transmembrane domain.

[0023] In the present invention, the extracellular binding domain may comprise all or a portion of the extracellular domain derived from CD99L2, preferably the extracellular domain derived from human CD99L2. The extracellular domain derived from CD99L2 may comprise all or a portion of the amino acid sequence represented by SEQ ID NO: 10, but is not limited thereto.

[0024] In the present invention, the transmembrane domain (TM) may comprise all or a portion of a CD99L2-derived transmembrane domain, preferably a human CD99L2-derived transmembrane domain. The CD99L2-derived transmembrane domain may comprise all or a portion of the amino acid sequence set forth in SEQ ID NO: 11, but is not limited thereto.

[0025] Furthermore, in the present invention, the chimeric antigen receptor may further comprise an intracellular domain derived from CD99L2.

[0026] The CD99L2-derived intracellular domain may comprise all or part of the CD99L2-derived intracellular domain, and preferably comprises the amino acid sequence represented by SEQ ID NO: 12, but is not limited thereto.

[0027] In the present invention, the extracellular binding domain may further comprise a hinge domain.

[0028] The hinge domain may be composed of any oligopeptide or polypeptide and may contain 1 to 100 amino acid residues, preferably 10 to 70 amino acid residues, but is not limited thereto.

[0029] In the present invention, the intracellular signaling domain refers to a portion that is located inside the cell membrane of an immune cell, i.e., in the cytoplasm, and that transmits a signal into the cell to activate the immune response of the immune cell when the antigen-binding domain included in the extracellular domain binds to a target antigen.

[0030] In the present invention, the intracellular signaling domain is preferably, but not limited to, one or more intracellular signaling domains selected from the group consisting of CD3 zeta (ζ), CD3 gamma (γ), delta (δ), CD3 epsilon (ε), FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, and CD66d, and more preferably CD3 zeta (ζ). The intracellular signaling domain of CD3 zeta (ζ) of the present invention may have an amino acid sequence comprising, but not limited to, SEQ ID NO: 13 or the amino acid sequence of SEQ ID NO: 14 in which the glutamine (Q) residue at the 14th amino acid residue of SEQ ID NO: 13 is substituted with lysine (K).

[0031] Furthermore, the intracellular signaling domain of the present invention may additionally comprise, but is not limited to, a costimulatory domain, preferably one or more costimulatory domains selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), ICOS, LFA-1, GITR, MyD88, DAP1, PD-1, LIGHT, NKG2C, B7-H3, and CD83 ligand.

[0032] Preferably, the intracellular signaling domain of the present invention can comprise, but is not limited to, an intracellular signaling domain of CD3 zeta (ζ) comprising the amino acid sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14 and a costimulatory domain of 4-1BB comprising the amino acid sequence represented by SEQ ID NO: 15.

[0033] In particular, a chimeric antigen receptor according to the present invention can comprise one or more intracellular signaling domains and one or more costimulatory domains.

[0034] When a chimeric antigen receptor according to the present invention comprises one or more intracellular signaling domains and one or more costimulatory domains, the one or more costimulatory domains and the one or more intracellular signaling domains can be linked in tandem. In this case, the domains can be linked directly or, alternatively, via an oligopeptide or polypeptide linker consisting of 2 to 10 amino acid residues. Preferably, such a linker sequence is a glycine-serine contiguous sequence.

[0035] In the present invention, the chimeric antigen receptor may further comprise a T cell immune function enhancer, including, but not limited to, IL-7 (interleukin 7), IL-12, IL-15, IL-18, IL-21, or CCL-19. For details of T cell immune function enhancers, see WO2016 / 056228A.

[0036] In the present invention, the chimeric antigen receptor may further comprise an interleukin receptor chain containing a JAK-binding motif and a STAT3 / 5-binding motif, including, but not limited to, IL-2Rβ. In this regard, reference may be made to WO2016 / 127257A.

[0037] First-generation CARs comprise an extracellular domain containing an antigen-recognition region specifically expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain, and only CD3ζ is used as the signaling domain. However, they have problems with their therapeutic effect on cancer and their duration of action is short. Such first-generation CARs are specifically described in U.S. Patent No. 6,319,494, which is incorporated herein by reference.

[0038] Second-generation CARs have been produced by combining costimulatory domains (CD28 or CD137 / 4-1BB) with CD3ζ to enhance immune cell reactivity. Compared to first-generation CARs, the number of CAR-containing immune cells remaining in the body has been significantly increased. While second-generation CARs use a single costimulatory domain, third-generation CARs use two or more costimulatory domains. To achieve proliferation and persistence of CAR-containing immune cells in vivo, the costimulatory domain can be combined with 4-1BB, CD28, or OX40. Second-generation CARs are specifically described in U.S. Patent Nos. 7,741,465, 7,446,190, or 9,212,229, and third-generation CARs are specifically described in U.S. Patent No. 8,822,647, both of which are incorporated herein by reference.

[0039] Fourth-generation CARs include additional genes encoding cytokines such as IL-12 or IL-15, allowing for further expression of cytokine CAR-based immune proteins, while fifth-generation CARs further include an interleukin receptor chain, e.g., IL-2Rβ, to enhance immune cell expression. Fourth-generation CARs are specifically described in U.S. Patent No. 10,316,102, and fifth-generation CARs are specifically described in U.S. Patent No. 10,336,810, both of which are incorporated herein by reference.

[0040] In the present invention, the antigen-binding domain may include, but is not limited to, an antibody or antigen-binding fragment thereof that specifically binds to an antigen selected from the group consisting of:

[0041] 4-1BB, B cell maturation antigen (BCMA), B-cell activating factor (BAFF), B7-H3, B7-H6, carbonic anhydrase 9 (CA9; also known as CAIX or G250), cancer / testis antigen 1B (CTAG1B; also known as NY ESO-1 or LAGE2B), carcinoembryonic antigen (CEA), cyclin, cyclin A2, cyclin B1, CC Motif Chemokine Ligand 1(CCL-l), CCR4, CD3, CD4, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v 6, CD44v7 / 8, CD52, CD58, CD62, CD79A, CD79B, CD80, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), claudin-18 (CLDN18), CLDN6, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), tyrosine-protein kinase Met (c-Met), DLL3, epidermal growth factor receptor (EGFR), truncated epidermal growth factor receptor (tEGFR), type III epidermal growth factor receptor mutation (EGFRvIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHA2), estrogen receptor, Fc receptor, Fc receptor like 5 (FCRL5; also known as Fe receptor homolog 5 or FCRH5), fibroblast growth factor23 (FGF23), folate binding protein (FBP), folate receptor alpha (FOLR1), folate receptor beta (FOLR2), GD2 (ganglioside GD2, O-acetylated GD2 (OGD2)), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPCR5D), granulocyte-macrophage colony-stimulating factor (GM-CSF), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen (HBsAg), Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Ra), IL-13 receptor alpha 2 (IL-13Ra2), inducible T-cell costimulator (ICOS), insulin-like growth factor 1 receptor (IGF-1 receptor), integrin αvβ6, interferon receptor, IFNγ receptor (IFNγR), interleukin-2 receptor (IL-2R), interleukin-4 receptor (IL-4R), interleukin-5 receptor (IL-5R), interleukin-6 receptor (IL-6R), interleukin-17 receptor A (IL-17RA), interleukin-31 receptor (IL-31R), interleukin-36receptor (IL-36R), kinase insert domain receptor (KDR), L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM (CE7 epitope of L1-CAM), Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, lymphocyte-activation gene 3 (LAG3), Melanoma-associated antigen(MAGE)Al, MAGEA3, MAGEA6, MAGEAlO, mesothelin(MSLN), murine cytomegalovirus(CMV), mucin 1(MUC1), natural killer group 2 member D(NKG2D) ligands, melan A(MART-l), nerve growth factor(NGF), neural cell adhesion molecule (NCAM), neuropilin-1 (NRP-1), neuropilin-2 (NRP-2), carcinoembryonic antigen (oncofetal antigen) antigen), PD-L1, preferentially expressed antigen of melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor activator of nuclear factor kappa-β ligand (RANKL), receptor tyrosine kinase like orphan receptor 1 (ROR1), SLAM family member 7 (SLAMF7), survivin, trophoblast glycoprotein (TPBG; also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosine-related protein1 (TRP1; also known as TYRP1 or gp75), tyrosine related protein 2 (TRP2; also known as dopachrome tautomerase, dopachrome delta-isomerase, or DCT), and Wilms Tumor 1 (WT1).

[0042] In the present invention, the term "fragment" of an antibody means a fragment that has antigen-binding function, and is used to include scFv, Fab, F(ab')2, Fv, and nanobody fragments.

[0043] "Single-chain Fv" or "scFv (single chain variable fragment)" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide can further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0044] An "Fv" fragment is an antibody fragment that contains the complete antigen-recognition and binding region, which consists of a dimer of one heavy- and one light-chain variable domain in tight, essentially covalent association, e.g., as in an scFv.

[0045] A "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. A "F(ab')2" antibody fragment generally contains a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them.

[0046] A "nanobody" is a fragment containing a monomeric variable antibody domain. It is composed of low-molecular-weight fragments derived from antibody domains such as camelids, which primarily exhibit target specificity through the heavy monomeric antibody chain alone.

[0047] In the present invention, the antigen-binding fragment may be a single chain variable fragment (scFv) of an antibody or a nanobody.

[0048] In the present invention, the antigen-binding domain preferably comprises an anti-CD19 antibody or scFv, and the scFv of the anti-CD19 antibody may comprise, but is not limited to, the amino acid sequence represented by SEQ ID NO: 8.

[0049] In the present invention, the chimeric antigen receptor may additionally comprise a signal peptide (SP) at the N-terminus of the antigen-binding domain. In the present invention, the signal peptide may be derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, Ig-kappa, and IgG1 heavy chain, but is not limited thereto. Preferably, the signal peptide is the CD8α signal peptide, which may comprise the amino acid sequence set forth in SEQ ID NO:7.

[0050] As a preferred example, the chimeric antigen receptor according to the present invention is an extracellular domain derived from CD99L2 characterized by being represented by SEQ ID NO: 10, and a transmembrane domain derived from CD99L2 characterized by being represented by SEQ ID NO: 11; an intracellular domain derived from CD99L2, characterized in that it is represented by SEQ ID NO: 12; may include:

[0051] Furthermore, A 4-1BB costimulatory domain characterized by being represented by SEQ ID NO: 15; an intracellular signaling domain of CD3 zeta (ζ), characterized in that it is represented by SEQ ID NO: 13 or SEQ ID NO: 14, and / or It may include, but is not limited to, the CD8 signal peptide as set forth in SEQ ID NO:7.

[0052] In the present invention, for example, a chimeric antigen receptor comprising an antigen-binding region for CD19 may comprise the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3, or a variant thereof having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably at least 99% sequence identity to said amino acid sequence.

[0053] In another aspect, the present invention relates to a nucleic acid encoding the chimeric antigen receptor.

[0054] The term "nucleic acid" as used herein encompasses DNA (gDNA and cDNA) and RNA molecules, and the basic building blocks of nucleic acids, nucleotides, include not only naturally occurring nucleotides but also analogs in which the sugar or base region is modified. The nucleic acid sequence encoding the chimeric antigen receptor or each domain of the present invention can be modified. Such modifications include nucleotide additions, deletions, or non-conservative or conservative substitutions.

[0055] Nucleic acids (polynucleotides) encoding the chimeric antigen receptors of the present invention can be modified by codon optimization. This is due to codon degeneracy, and it should be well understood by those skilled in the art that there are many nucleotide sequences encoding polypeptides or variant fragments thereof. Some of these polynucleotides (nucleic acids) have minimal homology to the nucleotide sequence of any native gene. In particular, polynucleotides that are variable due to differences in codon usage, such as polynucleotides optimized for human, primate, and / or mammalian codon selection, are preferred.

[0056] In the present invention, the nucleic acid encoding the chimeric antigen receptor is a nucleotide sequence encoding the extracellular domain from CD99L2, characterized in that it is set forth in SEQ ID NO: 19; a nucleotide sequence encoding a transmembrane domain from CD99L2, characterized in that it is represented by SEQ ID NO: 20; Including, Furthermore, a nucleotide sequence encoding an intracellular domain derived from CD99L2, characterized in that it is represented by SEQ ID NO: 21; A nucleotide sequence encoding a 4-1BB costimulatory domain characterized by being represented by SEQ ID NO: 25 or SEQ ID NO: 26; a nucleotide sequence encoding the intracellular signaling domain of CD3 zeta (ζ), characterized in that it is represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and / or and a nucleotide sequence encoding a CD8 signal peptide characterized by being represented by SEQ ID NO:16, but is not limited thereto.

[0057] Preferably, it may further comprise a nucleotide sequence encoding a single chain variable fragment (scFv) of an anti-CD19 antibody characterized by being represented by SEQ ID NO:17.

[0058] In one example of the present invention, the nucleic acid encoding the chimeric antigen receptor may comprise the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6, or a variant having at least 80%, preferably 90% or more, more preferably 95% or more, and most preferably at least 99% sequence identity to this nucleotide sequence.

[0059] In other aspects, the present invention relates to an expression vector containing such a nucleic acid, and a virus containing said expression vector.

[0060] The term "vector" as used herein refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. The vector may contain a sequence that directs autonomous replication in a cell, or may contain a sequence sufficient to allow integration into host cell DNA. The vector may be selected from the group consisting of, but is not limited to, DNA, RNA, plasmid, lentiviral vector, adenoviral vector, and retroviral vector.

[0061] In the present invention, the nucleic acid or vector is transfected into a packaging cell line. For "transfection" or "transfection," various techniques commonly used to introduce exogenous nucleic acid (DNA or RNA) into prokaryotic or eukaryotic host cells can be used, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection.

[0062] In the present invention, the virus produced from the virus-producing cells is transduced into immune cells. The nucleic acid of the virus "transduced" into the cells is either inserted into the genome of the cells or is used to produce a chimeric antigen receptor protein without being inserted.

[0063] In another aspect, the present invention relates to immune cells expressing the chimeric antigen receptor on their surface.

[0064] In the present invention, the immune cells may be, but are not limited to, T cells, NK cells, NKT cells, or macrophages, and are preferably T cells.

[0065] The immune cells expressing the chimeric antigen receptor according to the present invention may be CAR-T cells (chimeric antigen receptor T cells), CAR-NK cells (chimeric antigen receptor natural killer cells), CAR-NKT cells (chimeric antigen receptor natural killer T cells), or CAR-macrophages (chimeric antigen receptor macrophages).

[0066] In the present invention, the T cells are characterized in that they are selected from the group consisting of CD4-positive T cells, CD8-positive cytotoxic T lymphocytes (CTLs); gamma-delta T cells; tumor-infiltrating lymphocytes (TILs), and T cells isolated from peripheral blood mononuclear cells (PBMCs).

[0067] In yet another aspect, the present invention relates to a composition for treating cancer, comprising immune cells (e.g., T cells) that express the chimeric antigen receptor.

[0068] As used herein, "cancer" and "tumor" are used interchangeably to refer to or mean the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.

[0069] Cancers that can be treated with the CARs of the present invention include not only vascularized tumors, but also tumors that are not vascularized or have not yet been substantially vascularized. The cancers can include non-solid tumors (e.g., hematological tumors, such as leukemia and lymphoma) or solid tumors. Types of cancers that can be treated with the CARs of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas, and melanomas. Adult tumors and cancers and pediatric tumors and cancers are also included.

[0070] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological cancers (or hematopoietic malignancies) include leukemias, including acute leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, and myeloblastic, prolymphocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (e.g., chronic lymphocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (slow-onset and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplastic syndrome.

[0071] A solid tumor is an abnormal mass of tissue that generally does not contain cysts or liquid. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, rectal cancer, lymphoid malignancies, colon cancer, gastric cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, laryngeal cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver tumors, bile duct carcinoma, choriocarcinoma, Wilms' tumor, tumor, cervical cancer, testicular cancer, seminoma, bladder cancer, melanoma, and cancers of the central nervous system (CNS) (e.g., glioma (e.g., brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astroglioma, CNS lymphoma, germ cell tumor, medulloblastoma, schwannoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases).

[0072] The therapeutic composition of the present invention is a composition for preventing or treating cancer. The term "prevention" as used herein means any action of suppressing or delaying the progression of cancer by administering the composition of the present invention, and "treatment" means suppressing the progression of cancer and alleviating or eliminating symptoms.

[0073] Pharmaceutical compositions containing immune cells expressing a chimeric antigen receptor according to the present invention may further contain a pharmaceutically acceptable excipient, including, but not limited to, surfactants (preferably polysorbate-based nonionic surfactants), buffers such as neutral buffered saline and phosphate-buffered saline, sugars or sugar alcohols such as glucose, mannose, sucrose, dextran, or mannitol, amino acids such as glycine and histidine, proteins, or polypeptides, antioxidants, chelating agents such as EDTA or glutathione, and penetrating agents, auxiliary agents, and preservatives.

[0074] The compositions of the present invention can be formulated using methods known in the art so as to provide immediate, sustained or prolonged release of the active ingredient after administration to mammals other than humans. The dosage form may be in the form of powder, granules, tablets, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injection solution or sterile powder.

[0075] In yet another aspect, the present invention relates to a method for treating cancer, comprising the step of administering immune cells expressing the chimeric antigen receptor to a subject.

[0076] The present invention also relates to the use of said immune cells for cancer treatment.

[0077] The present invention also relates to the use of the immune cells for the manufacture of a medicament for treating cancer.

[0078] The subject may be a mammal having a tumor, and may specifically be, but is not limited to, a human.

[0079] The immune cells expressing the chimeric antigen receptor of the present invention or compositions containing the same can be administered by, but not limited to, oral administration, infusion, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrarectal administration, topical administration, intranasal injection, and the like.

[0080] The dosage of the active ingredient can be selected appropriately depending on various factors such as the administration route, the age, sex, weight, and severity of the patient, and the therapeutic composition according to the present invention can be administered in parallel with known compounds that are effective in preventing, ameliorating, or treating cancer symptoms.

[0081] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0082] Example 1: Materials and Methods

[0083] Example 1-1: Mice and cell lines

[0084] Immunodeficient NSG mice were purchased from Jackson Laboratory, Inc. Raji lymphoma cells were purchased from ATCC.

[0085] Example 1-2: Construction of lentiviral vector for CAR expression

[0086] The CD19-targeting CD8-backbone CAR (h19BBz) ORF cDNA was prepared by DNA synthesis (Integrated DNA Technologies) according to the previously published sequence (US Patent Publication US2013 / 0287748A1). The CD19-targeting CD99L2-backbone CAR ORF cDNA (FL2LBBz, FL2PBBz) was prepared by extracting the sequences of several CD99L2 extracellular, transmembrane, and intracellular domains from the human CD99L2 ORF sequence (NM_031462.4) in the NCBI database, linking them to the human 41BB intracellular domain and human CD3 zeta chain intracellular domain sequences through codon optimization and DNA synthesis (Integrated DNA Technologies), and then linking them again to the anti-CD19 scFv (clone FMC63) by PCR. The lentiviral vector for CAR expression was a modified version of the pCDH-EF1 (Addgene #72266) vector, and each CAR ORF cDNA was cloned into the BamHI / SalI restriction enzyme site. The amino acid and nucleotide sequences of each CAR protein are listed in Tables 1 and 2 below.

[0087] [Table 1]

[0088] [Table 2] JPEG0007764068000003.jpg253161

[0089] The amino acid and nucleotide sequences of each domain constituting the CAR protein are as shown in Tables 3 and 4 below.

[0090] [Table 3]

[0091] [Table 4] JPEG0007764068000006.jpg166147

[0092] Example 1-3: Production of lentivirus for CAR expression

[0093] Each lentiviral plasmid was transfected into 293T cells (ATCC) together with three types of packaging DNA (pMD.2G, pMDLg / pRRE, pRSV-rev) using Lipofectamin 3000 (Invitrogen). After 24-48 hours, the culture supernatant containing the secreted lentivirus was collected and filtered (0.45 μm filter) to remove any particles remaining within the cells. The supernatant was then concentrated 100-fold using an ultra-high speed centrifuge and used as the lentiviral concentrate for CAR-T cell generation.

[0094] Examples 1-4: Preparation of CAR-T cells

[0095] Leukocytes obtained from healthy donors by leukapheresis were supplemented with TransAct reagent (10 μl / ml, Miltenyi) and then cultured for 24 hours in a medium (Miltenyi) containing human IL-7 (12.5 ng / ml, Miltenyi) and human IL-15 (12.5 ng / ml, Miltenyi) to activate T cells. After washing twice, the activated T cells were supplemented with a lentivirus concentrate and cultured for two days in a medium containing human IL-7 and human IL-15 for lentiviral transduction. The transduced T cells were washed twice and then transferred to fresh medium containing human IL-7 and human IL-15. They were then expanded for nine days, with medium changes every 2–3 days, and used as CAR-T cells. The expression of CAR protein on the cell surface was measured by staining the final expanded CAR-T cells with biotin-labeled anti-FMC63 antibody (ACROBiosystems) and PE-labeled streptavidin (BD Biosciences) and then measuring it by flow cytometry (FACS-Canto II, BD Biosciences).

[0096] Example 1-5: Preparation of luciferase-expressing Raji cells (Raji-Luc)

[0097] To artificially express luciferase in cells, we constructed a lentiviral vector capable of co-expressing luciferase and GFP. pLECE3 (Lee SH, et al., PLoS One. 2020;15(1):e0223814) is a bicistronic lentiviral vector containing a multi-cloning site under the EF1α promoter and GFP cloned under the CMV promoter. The firefly luciferase ORF cDNA extracted from pGL3-basic plasmid (Promega) was cloned into the multi-cloning site to create the pLECE3-Luc vector. The pLECE3-luc plasmid was transduced into a lentivirus packaging cell line (293FT cells, Invitrogen) together with three lentivirus packaging plasmids (pMDLg / pRRE, pRSVrev, and pMD.G) using Lipofectamin 2000 reagent. After 24–48 hours, the culture supernatant containing the secreted lentivirus was collected and concentrated 10-fold using a centrifugal filter. The lentivirus concentrate was added to Raji cells, and transduction was performed by centrifugation at 2500 rpm for 90 minutes at room temperature in the presence of polybrene (6 μg / ml, Sigma-Aldrich). GFP-positive cells from the transduced Raji cells were isolated and purified using a flow cytometer (FACS-Aria II, BD Biosciences) and used as Raji-Luc cells.

[0098] Example 1-6: Measurement of tumor-killing ability and IFN-γ secretion ability of CAR-T cells

[0099] CAR-T cells (1.2 × 103–7.5 × 105 cells / 100 100 μL / well), grown for 9 days after lentiviral transduction, were added to Raji-Luc cells (3 × 104 cells / 50 100 μL / well) at various ratios (0.2–25:1) in a 96-well plate and co-cultured overnight. After adding 50 μL of D-luciferin (600 μg / ml, Promega) and incubating at 37°C for 10 minutes, luciferase activity in the viable Raji-Luc cells was induced. The luminescence of these cells was measured using a Tecan luminometer. The tumor-killing activity of CAR-T cells was measured by calculating the tumor cell survival rate relative to that of untreated Raji-Luc cells.

[0100] To measure the activation level of CAR-T cells, CAR-T cells and Raji cells were mixed at equal numbers (3 × 10 cells) and co-cultured in a 96-well plate for 24 hours. The culture supernatant was then collected and the amount of IFN-γ secreted in the supernatant was measured using ELISA (human IFN-γ ELISA kit, BD Biosciences).

[0101] Examples 1-7: Analysis of activation markers of CAR-T cells

[0102] To compare the degree of activation of each CAR-T cell, CAR-T cells (1 × 105 cells / 200 40 μL / well) grown for 9 days after lentiviral transduction were mixed with Raji cells (2 × 104 cells / 200 100 μL / well) whose growth had been suppressed by irradiation (2000 rad) and co-cultured for 3 days in a 96-well plate. During coculture, cells were harvested every 24 hours, and the cell surface was stained with anti-CD69 antibody (FN50, BD Horizon), anti-CD44 antibody (IM7, Invitrogen), anti-CD25 antibody (M-A251, BioLegend), anti-CD4 antibody (RPA-T4, BD Pharmigen), anti-CD8 antibody (RPA-T8, BD Pharmigen), and the anti-FMC63 scFv antibody (Y45, ACROBiosystems). Fluorescence intensity was measured by flow cytometry (FACS-LSRII, BD Bioscience).

[0103] Examples 1-8: Evaluation of the in vivo effects of CAR-T cells

[0104] Immunodeficient NSG mice were intravenously injected with Raji-Luc cells (5 × 10 cells per mouse) 7 days after which CAR-T cells (1 × 10 cells per mouse) expanded for 9 days after lentiviral transduction were intravenously injected. Changes in tumor burden were monitored by measuring bioluminescence intensity using a bioluminescence imaging system (IVIS, Perkin Elmer) after periodic intraperitoneal injection of D-luciferin (2 mg per mouse, Promega).

[0105] Example 2: Generation of CD99L2 backbone CAR-T cells and activity analysis

[0106] We engineered CAR proteins by substituting the CD8 extracellular and transmembrane domains of human CD19-targeting CD8-backbone CARs with portions of CD99L2. We used either the extracellular and transmembrane domains of CD99L2 (FL2PBBz) or the intracellular domain (FL2LBBz) (Figure 1A). Lentiviruses carrying the cDNAs of these CD19-targeting CD99L2-backbone CARs were then transfected into T cells isolated from human peripheral blood to generate CAR-T cells. Flow cytometry analysis revealed that the FL2LBBz CAR protein showed significantly higher expression than the FL2PBBz CAR protein, both in CD4 T cells and CD8 T cells (Figure 1B, lower panel: CD4-negative T cells). Next, to confirm the tumor-killing ability of these CAR-T cells, we co-cultured them with Raji cells, a human CD19 benign lymphoma cell line, and confirmed that the tumor-killing ability of FL2LBBz CAR-T cells was superior to that of FL2PBBz CAR-T cells (Figure 1C). Consistent with this, we measured the amount of IFN-γ secreted upon activation of CAR-T cells when co-cultured with tumor cells and confirmed that FL2LBBz CAR-T cells secreted significantly more IFN-γ than FL2PBBz CAR-T cells (Figure 1D). Therefore, we selected FL2LBBz CAR-T cells as a CD99L2-backbone CAR for further studies.

[0107] To compare the in vitro tumor-killing and IFN-γ production of FL2LBBz CAR-T cells with those of existing CD8-backbone CAR-T cells (h19BBz), we generated two CAR-T cells. We found that the CAR expression rate (mean fluorescence intensity) per cell in FL2LBBz CAR-T cells was slightly lower than that in h19BBz CAR-T cells (Figure 1E). However, both CAR-T cells exhibited similar tumor cell-killing activity, and FL2LBBz CAR-T cells exhibited slightly improved IFN-γ secretion compared to h19BBz (Figures 1F and 1G). Therefore, CD99L2-backbone CAR-T cells exhibited similar or slightly improved in vitro activity compared to existing CD8-backbone CAR-T cells.

[0108] Example 3: Analysis of activation markers of CD99L2 backbone CAR-T cells

[0109] To observe the degree of tumor activation of CD99L2-backbone CAR-T cells in more detail, the time course of expression of cell surface activation markers (CD69, CD44, CD25), which increase upon T cell activation, was measured by flow cytometry.

[0110] As a result, the time-dependent increase in expression of CD69, CD44, and CD25 in CD99L2-backbone CAR-T cells was significantly higher than that in CD8-backbone CAR-T cells in both CD4 (Figure 2A) and CD8 (Figure 2B) CAR-T cells. Therefore, CD99L2-backbone CAR-T cells demonstrated a significantly superior level of activation over time after antigen stimulation compared to CD8-backbone CAR-T cells.

[0111] Example 4: Analysis of the in vivo antitumor effect of CD99L2-backbone CAR-T cells

[0112] To test the in vivo efficacy of CD99L2-backbone CAR-T cells, we intravenously injected luciferase-expressing Raji lymphoma cells into immunodeficient mice (NSG mice) 7 days after intravenous injection of equal numbers of CD8-backbone CAR-T cells and CD99L2-backbone CAR-T cells. The therapeutic efficacy of both CAR-T cells was analyzed by bioluminescence imaging.

[0113] As a result, it was confirmed that CD8-backbone CAR-T cells showed low efficacy at cell doses, while CD99L2-backbone CAR-T cells showed significant tumor elimination efficacy (Figure 3).

[0114] In other words, CD99L2-backbone CAR-T cells were confirmed to exhibit significantly improved activity and in vivo antitumor efficacy compared to existing CAR-T cells, suggesting the development of a novel CAR construct that imparts new activation functionality to the CAR backbone region. [Industrial Applicability]

[0115] In this study, we confirmed the T cell activation function of CD99L2 (CD99 antigen-like 2), a cell membrane protein belonging to the CD99 family, and constructed a new chimeric antigen receptor (CAR) containing the extracellular domain and transmembrane domain of CD99L2 as a backbone. These CD99L2-based CAR-T cells exhibit improved T cell activity and tumor therapeutic efficacy compared to CAR-T cells with existing backbones, and therefore can be useful for immune cell therapy for cancer treatment.

Claims

1. (a) an antigen binding domain; (b) a backbone comprising an extracellular binding domain and a transmembrane domain; (c) an intracellular signaling domain; and A chimeric antigen receptor (CAR) comprising: the chimeric antigen receptor comprises an intracellular domain derived from CD99L2; a chimeric antigen receptor (CAR), wherein the extracellular binding domain comprises a CD99L2-derived extracellular domain, and the transmembrane domain comprises a CD99L2-derived transmembrane domain.

2. The chimeric antigen receptor according to claim 1, wherein the extracellular domain derived from CD99L2 comprises the amino acid sequence represented by SEQ ID NO:

10.

3. The chimeric antigen receptor according to claim 1, wherein the CD99L2-derived transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

11.

4. The chimeric antigen receptor according to claim 1, wherein the CD99L2-derived intracellular domain comprises the amino acid sequence represented by SEQ ID NO:

12.

5. The intracellular signaling domain comprises: an intracellular signaling domain selected from the group consisting of CD3 zeta (ζ), CD3 gamma (γ), delta (δ), CD3 epsilon (ε), FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, and CD66d; and / or a co-stimulatory domain selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), ICOS, LFA-1, GITR, MyD88, DAP1, PD-1, LIGHT, NKG2C, B7-H3, and CD83 ligand; The chimeric antigen receptor of claim 1, comprising:

6. The chimeric antigen receptor according to claim 5, wherein the intracellular signaling domain of CD3 zeta (ζ) comprises the amino acid sequence represented by SEQ ID NO: 13 or SEQ ID NO:

14.

7. 2. The chimeric antigen receptor of claim 1, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to an antigen selected from the group consisting of: 4-1BB, B cell maturation antigen (BCMA), B-cell activating factor (BAFF), B7-H3, B7-H6, carbonic anhydrase 9 (CA9; also known as CAIX or G250), cancer / testis antigen 1B (CTAG1B; also known as NY ESO-1 or LAGE2B), carcinoembryonic antigen (CEA), cyclin, cyclin A2, cyclin B1, C-C Motif Chemokine Ligand 1 (CCL-l), CCR4, CD3, CD4, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v 6, CD44v7 / 8, CD52, CD58, CD62, CD79A, CD79B, CD80, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), claudin-18 (CLDN18), CLDN6, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), tyrosine-protein kinase Met (c-Met), DLL3, epidermal growth factor receptor (EGFR), truncated epidermal growth factor receptor (tEGFR), type III epidermal growth factor receptor mutation (EGFRvIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHA2), estrogen receptor receptor), Fc receptor, Fc receptor like 5 (FCRL5; also known as Fe receptor homolog 5 or FCRH5), fibroblast growth factor23 (FGF23), folate binding protein (FBP), folate receptor alpha (FOLR1), folate receptor beta (FOLR2), GD2 (ganglioside GD2, O - acetylated GD2 (OGD2)), ganglioside GD3, glycoprotein 100 (gp100), glypican - 3 (GPC3), G Protein Coupled Receptor 5D (GPCR5D), granulocyte - macrophage colony - stimulating factor (GM - CSF), Her2 / neu (receptor tyrosine kinase erb - B2), Her3 (erb - B3), Her4 (erb - B4), erbB dimers, Human high molecular weight melanoma - associated antigen (HMW - MAA), hepatitis B surface antigen (HBsAg), Human leukocyte antigen A1 (HLA - A1), Human leukocyte antigen A2 (HLA - A2), IL - 22 receptor alpha (IL - 22Ra), IL - 13 receptor alpha 2 (IL - 13Ra2), inducible T - cell costimulator (ICOS), insulin - like growth factor 1 receptor (IGF - 1 receptor), integrin αvβ6, interferon receptor, IFNγ receptor (IFNγR), interleukin - 2 receptor (IL - 2R), interleukin - 4 receptor (IL - 4R), interleukin - 5 receptor (IL - 5R), interleukin - 6 receptor (IL - 6R), interleukin - 17 receptor A (IL - 17RA), interleukin - 31 receptor (IL - 31R), interleukin - 36receptor (IL-36R), kinase insert domain receptor (KDR), L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM (CE7 epitope of L1-CAM), Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, lymphocyte-activation gene 3 (LAG3), Melanoma-associated antigen (MAGE) Al, MAGEA3, MAGEA6, MAGEAlO, mesothelin (MSLN), murine cytomegalovirus (CMV), mucin 1 (MUC1), natural killer group 2 member D (NKG2D) ligands, melan A (MART-l), nerve growth factor (NGF), neural cell adhesion molecule (NCAM), neuropilin-1 (NRP-1), neuropilin-2 (NRP-2), oncofetal antigen antigen), PD-L1, preferentially expressed antigen of melanoma (PRAME), progesterone receptor, prostate-specific antigen (prostate specific antigen), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), receptor activator of nuclear factor kappa-β ligand (RANKL), receptor tyrosine kinase like orphan receptor 1 (ROR1), SLAM family member 7 (SLAMF7), survivin, trophoblast glycoprotein (TPBG; also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosine-related protein1 (TRP1; also known as TYRP1 or gp75), tyrosine related protein 2 (TRP2; also known as dopachrome tautomerase, dopachrome delta-isomerase or DCT), and Wilms Tumor 1 (WT1).

8. The chimeric antigen receptor of claim 7, wherein the antigen-binding fragment is an antibody single chain variable fragment (scFv) or a nanobody.

9. The chimeric antigen receptor of claim 1, further comprising a signal peptide at the N-terminus of the antigen-binding domain.

10. The chimeric antigen receptor of claim 9, wherein the signal peptide is a CD8α signal peptide comprising the amino acid sequence of SEQ ID NO:

7.

11. The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO:

3.

12. A nucleic acid encoding a chimeric antigen receptor described in any one of claims 1 to 11.

13. An expression vector comprising the nucleic acid described in claim 12.

14. A virus comprising the expression vector described in claim 13.

15. An immune cell expressing a chimeric antigen receptor described in any one of claims 1 to 11 on its surface.

16. The immune cell of claim 15, wherein the immune cell is a T cell, a NK cell, a NKT cell, or a macrophage.

17. A composition for cancer treatment comprising the immune cells described in claim 16.

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