Chimeric antigen receptor specifically binding to CD300C antigen or its receptor

Abstract

The present invention relates to a chimeric antigen receptor that specifically binds to a CD300c antigen or its receptor, an immune cell expressing the same, and the like. The chimeric antigen receptor that specifically binds to a CD300c antigen or its receptor of the present invention can not only specifically recognize cancer cells that express the CD300c antigen or the CD300c receptor and directly and effectively inhibit the growth and metastasis of cancer, but can also reduce side effects in vivo by inhibiting CD300c from activating T cells, and moreover, can also be effectively used in the immunotherapy of various cancers by increasing the therapeutic effect of cancer.

Description

Technical Field

[0001] The present invention relates to a chimeric antigen receptor that specifically binds to a CD300c antigen or its receptor, immune cells expressing the chimeric antigen receptor, and uses thereof. Background Art

[0002] Cancer is one of the leading causes of death in modern society. It is a disease caused by genetic mutations resulting from various factors, leading to changes in normal cells. It refers to malignant tumors that deviate from normal cell differentiation, proliferation, and growth patterns. Cancer is characterized by uncontrolled cell growth. This abnormal cell growth forms a cluster of cells called a tumor, which infiltrates surrounding tissues and, in severe cases, metastasizes to other organs. Cancer is a chronic, intractable disease. Even with treatments such as surgery, radiation, and medication, it is often incurable, causing suffering and ultimately death. In particular, the global incidence of cancer has been increasing by more than 5% annually in recent years due to factors such as an increasing elderly population and environmental degradation. According to the World Health Organization (WHO), the number of cancer cases will reach 30 million within the next 25 years, with 20 million deaths from cancer.

[0003] Cancer drug treatments, or anticancer drugs, are typically cytotoxic compounds that attack cancer cells and induce apoptosis. These drugs not only kill cancer cells but also damage normal cells, resulting in significant side effects. Therefore, targeted anticancer drugs have been developed to reduce side effects. However, while these targeted anticancer drugs can reduce side effects, they also have the limitation of a high probability of developing drug resistance. Consequently, there has been a recent surge in interest in immunotherapy drugs that utilize the body's immune system to mitigate the problems caused by toxicity and drug resistance. As an example of such immunotherapy drugs, immune checkpoint inhibitors have been developed that specifically bind to PD-L1 on the surface of cancer cells, inhibiting its binding to PD-1 on T cells, thereby activating T cells and enabling them to attack cancer cells. However, even these immune checkpoint inhibitors are effective against a limited number of cancer types, leading to an urgent need for the development of new immune checkpoint inhibitors that can achieve the same therapeutic effect across a wide range of cancers (Korean Patent Publication No. 10-2016-0016725).

[0004] On the other hand, chimeric antigen receptors (CARs) are artificial receptors designed to deliver antigen specificity to T cells. These CARs are composed of antigen-specific structural elements, transmembrane structural elements, and intracellular structural elements selected to activate T cells and provide specific immunity. Recently, research into treating cancer using cancer immunotherapy using cells transfected with genes encoding these CARs has been actively underway. Specifically, T cells are harvested from a patient, transduced with genes encoding the CARs, expanded, and then reinfused into the patient. Summary of the Invention

[0005] Technical issues

[0006] The present invention is proposed to solve the problems of the prior art as described above. An object of the present invention is to provide a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor, immune cells expressing the chimeric antigen receptor, and a composition for preventing or treating cancer using the chimeric antigen receptor.

[0007] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and ordinary technicians in the technical field to which the present invention belongs can clearly understand other technical problems not mentioned through the following contents.

[0008] Technical Solution

[0009] The present invention provides a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor.

[0010] In one embodiment of the present invention, the chimeric antigen receptor preferably comprises an antibody, single domain antibody, single chain variable fragment (scFv), or the like that specifically binds to the CD300c antigen, or comprises the CD300c antigen, and can specifically recognize and bind to the CD300c antigen or its receptor. To bind to the receptor, the CD300c antigen may comprise the entire sequence of the CD300c antigen, or may comprise only the extracellular domain (ECD) of the CD300c antigen sequence. The extracellular domain sequence of the CD300c antigen may comprise the amino acid sequence represented by SEQ ID NO: 14, preferably, an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 14, more preferably, an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 14, and most preferably, an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 14. Furthermore, the above-mentioned antibodies, single domain antibodies, single chain variable fragments, etc., in order to bind to the CD300c antigen, may comprise an amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, or 12. Preferably, the amino acid sequence may comprise an amino acid sequence having 90% or greater sequence identity to the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, or 12. More preferably, the amino acid sequence may comprise an amino acid sequence having 95% or greater sequence identity to the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, or 12. Most preferably, the amino acid sequence may comprise an amino acid sequence having 98% or greater sequence identity to the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, or 12. The "% sequence identity" for an amino acid sequence is determined by comparing a comparison region with an optimally aligned sequence. In the comparison region, a portion of the amino acid sequence may comprise additions or deletions (i.e., gaps) compared to the optimally aligned reference sequence (excluding additions or deletions). However, the sequence is not limited thereto as long as it is capable of specifically binding to the CD300c antigen or its receptor.

[0011] In another embodiment of the present invention, the chimeric antigen receptor may further include a signal peptide, a GS linker, a transmembrane domain, an intracytoplasmic domain, etc., but the chimeric antigen receptor is not limited thereto as long as the chimeric antigen receptor structural elements are generally known.

[0012] In another specific example of the present invention, preferably, the signal peptide may be a CD8α signal peptide sequence, the transmembrane domain may include sequences such as the CD8 hinge (hinge of cluster of differentiation 8), the CD28 transmembrane domain, and the intracytoplasmic domain may include sequences such as the CD28 intracellular domain and the CD3ζ intracellular domain.

[0013] In another embodiment of the present invention, the chimeric antigen receptor can be used to treat cancers expressing CD300c antigen on their surface by specifically recognizing and / or binding to CD300c antigen or its receptor.

[0014] Furthermore, the present invention provides a recombinant vector expressing the chimeric antigen receptor.

[0015] Furthermore, the present invention provides immune cells transformed by the above-mentioned recombinant vector.

[0016] In one embodiment of the present invention, the immune cells are preferably monocytes, macrophages, T cells, natural killer cells (NK cells), dendritic cells, etc., but are not limited thereto as long as they are immune cells used for cancer treatment.

[0017] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating cancer expressing CD300c antigen or CD300c receptor, comprising the above-mentioned immune cells as an active ingredient.

[0018] In one embodiment of the present invention, the cancer is selected from colorectal cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, blood cancer, etc., but the cancer is not limited thereto as long as the cancer expresses the CD300c antigen or CD300c receptor on its surface.

[0019] In another specific example of the present invention, the pharmaceutical composition may further include other existing anticancer drugs. Preferably, the anticancer drugs are doxorubicin, cisplatin, gemcitabine, oxaliplatin, 5-FU, cetuximab, panitumumab, nimotuzumab, necitumumab, cancer antigens, anticancer viruses, etc., but as long as they are substances currently used as anticancer drugs, they are not limited thereto. The cancer antigens are cancer vaccines specific to cancer species, preferably NY-ESO-1 as bladder cancer-specific cancer antigens, preferably HER2 as breast cancer-specific cancer antigens, preferably CEA as colorectal cancer-specific cancer antigens, and preferably VEGFR1 and VEGFR2 as lung cancer-specific cancer antigens, but as long as they are known anticancer drugs for cancer vaccines, they are not limited thereto. As an example of an anticancer virus, there are Imlygic, Pexa-Vec, etc., but as long as they are known anticancer viruses, they are not limited thereto. The aforementioned anticancer drugs can preferably be administered in combination with the monoclonal antibodies disclosed herein, or can be contained within the immune cells of the present invention, or can be bound to a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor, or can be contained together within an anticancer drug carrier. However, the present invention is not limited thereto.

[0020] In another embodiment of the present invention, the pharmaceutical composition is characterized by inhibiting the proliferation, survival, metastasis, recurrence, and anticancer drug resistance of cancer or cancer stem cells, but the effects are not limited thereto as long as they are produced by the pharmaceutical composition of the present invention.

[0021] Furthermore, the present invention provides a method for treating cancer, comprising administering to an individual a composition comprising, as an active ingredient, immune cells expressing a chimeric antigen receptor that specifically binds to a CD300c antigen or its receptor.

[0022] Furthermore, the present invention provides a use of a composition comprising, as an active ingredient, immune cells expressing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor for preventing or treating cancer.

[0023] Furthermore, the present invention provides a use of immune cells expressing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor to prepare a drug for treating cancer.

[0024] Effects of the Invention

[0025] The chimeric antigen receptors of the present invention that specifically bind to the CD300c antigen or its receptor have a high affinity for the CD300c antigen or its receptor expressed on the surface of various cancer cells, binding to them and activating T cells while simultaneously inhibiting cancer cell proliferation. Therefore, they can be effectively used as immunotherapeutic agents for various cancers. Furthermore, immune cells expressing chimeric antigen receptors that specifically bind to the CD300c antigen or its receptor of the present invention not only have their own therapeutic effects but also inhibit cancer cell proliferation while activating T cells by inhibiting CD300c protein production, thereby effectively inhibiting cancer cell growth, development, and metastasis. Furthermore, when immune cells expressing chimeric antigen receptors that specifically bind to the CD300c antigen or its receptor of the present invention are used to treat resistant cancer cells that are resistant to apoptosis, this ability can be significantly reduced, potentially demonstrating excellent efficacy in preventing cancer recurrence. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The figure briefly shows the gene arrangement for preparing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor according to one embodiment of the present invention.

[0027] Figure 2 This figure shows a vector map for preparing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor according to one embodiment of the present invention.

[0028] Figure 3 This figure shows the results of Western blotting confirmation of a Jurkat cell line expressing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor according to one embodiment of the present invention.

[0029] Figure 4 This figure shows the results of confirming the anti-cancer effect of a Jurkat cell line expressing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor according to one embodiment of the present invention. DETAILED DESCRIPTION

[0030] The immune cells expressing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor of the present invention specifically bind to the CD300c antigen or its receptor on the surface of cancer cells, effectively inhibiting the growth of cancer cells and the progression of cancer by effectively inhibiting the CD300c mechanism. Therefore, they can be effectively used in the treatment of various cancers that express the CD300c antigen or CD300c receptor on their surface.

[0031] As used herein, the term "antibody" refers to an immunoglobulin molecule that immunologically reacts with a specific antigen, including polyclonal antibodies, monoclonal antibodies, and functional fragments thereof. Furthermore, the term may include forms produced through genetic engineering, such as chimeric antibodies (e.g., humanized murine antibodies) and heterologous conjugate antibodies (e.g., bispecific antibodies). A monoclonal antibody is an antibody that exhibits a single binding specificity and affinity for a single antigenic site. Unlike polyclonal antibodies, which contain antibodies specific for multiple different epitopes, a monoclonal antibody exhibits binding specificity and affinity for only a single epitope on an antigen, making it easier to control the quality of the therapeutic drug. In particular, the anti-CD300c monoclonal antibody of the present invention can not only specifically bind to cancer cells expressing CD300c, thereby exhibiting its own anti-cancer activity, but can also maximize cancer cell-dependent anti-cancer activity by stimulating immune cells. The structure of the above-mentioned antibodies includes the variable regions of the heavy and / or light chains. These variable regions include the portion of the primary structure that forms the antigen-binding site of the antibody molecule. The antibodies of the present invention can be composed of fragments containing these variable regions. Preferably, these variable regions can be replaced by soluble CD300c receptors. However, as long as the same effect as the anti-CD300c monoclonal antibody of the present invention can be produced, the antibodies are not limited thereto.

[0032] As used herein, a "single domain antibody" is an antibody specific for the CD300c antigen, wherein the CDR is an antibody that is part of a single domain polypeptide. Typically, the antibody is prepared using only two heavy chains and an antigen-binding site, but may include antibodies lacking all naturally occurring light chains, single domain antibodies derived from existing four-chain antibodies, modified antibodies, and single domain scaffolds other than those derived from antibodies.

[0033] In this specification, the term "single-chain variable fragment (scFv)" refers to a protein that connects the variable regions of the light chain and heavy chain of an antibody through a linker composed of a peptide sequence connected by about 15 amino acids. It can have the order of light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region, and has the same or similar antigenic specificity as the original antibody. The connection site is a hydrophilic flexible peptide chain mainly composed of glycine and serine, and the 15 amino acid sequence of "(Gly-Gly-Gly-Gly-Ser)3" or a similar sequence is mainly used. The above-mentioned antibody refers to an immunoglobulin molecule that immunologically reacts with a specific antigen, including polyclonal antibodies, monoclonal antibodies and their functional fragments. Furthermore, the above terms may include forms produced by genetic engineering, such as chimeric antibodies (e.g., humanized murine antibodies) and heterologous conjugate antibodies (e.g., bispecific antibodies). A monoclonal antibody is an antibody that exhibits a single binding specificity and affinity for a single antigenic site (epitope). Unlike polyclonal antibodies, which contain antibodies that exhibit specificity for multiple different epitopes, a monoclonal antibody exhibits binding specificity and affinity only for a single epitope on an antigen, making it easier to control the quality of therapeutic drugs. In particular, the anti-CD300c monoclonal antibody of the present invention can not only specifically bind to cancer cells expressing CD300c, thereby exhibiting its own anti-cancer activity, but can also maximize cancer cell-dependent anti-cancer activity by stimulating immune cells. The structure of the above-mentioned antibody includes the variable region of the heavy chain and / or light chain. The variable region includes the portion that forms the antigen binding site of the antibody molecule as the primary structure. The antibody of the present invention may be composed of several fragments comprising the variable region.

[0034] In the present specification, the term "prevention" refers to any action to inhibit a disease such as cancer or delay its onset by administering the pharmaceutical composition of the present invention.

[0035] In the present specification, the term "treatment" refers to any action to improve or beneficially change the symptoms of cancer or the like by administering the pharmaceutical composition of the present invention.

[0036] In the present specification, the term "individual" refers to a subject to which the pharmaceutical composition of the present invention can be administered, and the subject is not limited.

[0037] In this specification, the term "pharmaceutical composition" may be characterized as a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be characterized as being administered to humans. The pharmaceutical composition is not limited to these forms and can be prepared according to conventional methods into oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, as well as external preparations, suppositories, and sterile injectable solutions for use. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, and flavorings for oral administration; buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, and the like may be mixed for injection; and bases, excipients, lubricants, preservatives, and the like may be used for topical administration. The dosage form of the pharmaceutical composition of the present invention can be prepared in various forms by mixing with the above-mentioned pharmaceutically acceptable carriers. For example, in the case of oral administration, the drug can be prepared in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc.; in the case of injection, the drug can be prepared in the form of unit dose ampoules or multiple doses. In addition, the drug can be formulated into solutions, suspensions, tablets, capsules, sustained-release formulations, etc. In addition, the drug can be formulated into solutions, suspensions, tablets, capsules, sustained-release formulations, etc.

[0038] Meanwhile, examples of suitable carriers, excipients and diluents for formulation may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil, etc. In addition, fillers, anticoagulants, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc. may also be included.

[0039] The route of administration according to pharmaceutical composition of the present invention includes but is not limited to, comprises oral cavity, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual or rectal.Preferred oral or parenteral administration.The term " parenteral " that uses in this application comprises subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intrabursa, intrasternal, intrathecal, intralesional and intracranial injection or infusion technology.Pharmaceutical composition of the present invention also can be used for rectal administration with the form of suppository.

[0040] The pharmaceutical composition of the present invention can vary according to various factors such as the activity of the specific compound used, age, weight, general health condition, sex, formulation, administration time, route of administration, excretion rate, drug combination, and the severity of the specific disease to be prevented or treated. The dosage of the above-mentioned pharmaceutical composition can be appropriately selected by a person of ordinary skill in the art according to the patient's condition, weight, degree of disease, drug form, route of administration, and cycle. The daily dosage can be 0.0001 mg / kg to 500 mg / kg or 0.001 mg / kg to 500 mg / kg. It can be administered once a day or in divided doses. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition of the present invention can be formulated into pills, dragees, capsules, liquids, gels, syrups, slurries, and suspensions.

[0041] Hereinafter, the following examples are provided to facilitate understanding of the present invention. However, the following examples are provided only to facilitate understanding of the present invention, and the content of the present invention is not limited to the following examples.

[0042] Example

[0043] Example 1: Preparation of a chimeric antigen receptor expression vector specifically binding to CD300c antigen or its receptor

[0044] In order to prepare a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor, a CD8α signal peptide (CD8α signal peptide, DNA sequence 1) containing the amino acid sequence of sequence 2 that enables the synthesized protein to move to the correct position after passing through the cell membrane, a single-chain variable fragment of CK1, CL6, CL7, CL10, or SL18 (anti-CD300c single chain variable fragment; aCD300c scFv, DNA sequences 3, 5, 7, 9, or 11, respectively) containing the amino acid sequence of sequence 4, 6, 8, 10, or 12 that specifically bind to the CD300c antigen or its receptor, or a CD300c scFv containing the amino acid sequence of sequence 14 were sequentially inserted into the pLVX-Puro vector (Addgene). Chimeric antigen receptor expression vectors (pLVX-Puro / aCD300c scFv or pLVX-Puro / CD300c ECD-CAR) that specifically bind to the CD300c antigen or its receptor were prepared using an ECD (extracellular domain) antigen (DNA sequence: SEQ ID NO: 13), a GS linker containing the amino acid sequence of SEQ ID NO: 16 (DNA sequence: SEQ ID NO: 15), a CD8 hinge containing the amino acid sequence of SEQ ID NO: 18 as a transmembrane domain (DNA sequence: SEQ ID NO: 17), a CD28 transmembrane domain containing the amino acid sequence of SEQ ID NO: 20 (DNA sequence: SEQ ID NO: 19), a CD28 intracellular domain containing the amino acid sequence of SEQ ID NO: 22 as an intracytoplasmic domain for transmitting macrophage activation signals (DNA sequence: SEQ ID NO: 21), and a CD3ζ intracellular domain containing the amino acid sequence of SEQ ID NO: 24 (DNA sequence: SEQ ID NO: 23). The gene and protein sequence combinations of each prepared vector are shown in Table 1. Furthermore, the schematic diagram of gene arrangement is shown in Figure 1 As shown, the example of the prepared vector map is as follows Figure 2 shown.

[0045] Table 1

[0046]

[0047] Example 2. Preparation of a recombinant protein for expressing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor Lentivirus

[0048] The HEK293T cell line (ATCC) required for the preparation of a recombinant lentivirus expressing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor is prepared as follows. The complete medium used for cell culture is a fresh Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) with the addition of heat-treated fetal bovine serum (FBS, fetalbovine serum) to a concentration of 10%, followed by the addition of 1X penicillin-streptomycin (Penicillin-Streptomycin) (Gibco), which is then mixed evenly by inversion and preheated to 37°C for use. Then, the HEK293T cell line is a frozen cell stock that is quickly moved to a 37°C constant temperature water bath before use, rapidly thawed within 2 to 3 minutes, and inoculated into 30mL of complete medium, and cultured in an incubator at 37°C and 5% CO2. Then, when the cell saturation reaches 80% or more, subculture is performed to maintain it. For lentiviral transfection, 1×10 6 cells~2×10 6 HEK293T cells were inoculated into 10 mL of complete culture medium and cultured for 16 hours before lentiviral transfection.

[0049] Lenti-X was used for transfection of lentivirus expressing chimeric antigen receptors that specifically bind to CD300c antigen or its receptor. TM Expression System (Takara) kit. Sterile water (Invitrogen) was added to 7 μg of pLVX-Puro / aCD300C scFv or pLVX-Puro / CD300c ECD-CAR expression vector prepared in the same manner as in Example 1 to make up to 600 μL, and then loaded into Lenti-X TM The nanoparticle complex solution was prepared by mixing the cells in the Lenti-X Packaging Single Shots tube within the Expression System. The nanoparticle complex solution, which had been reacted at room temperature for 10 minutes, was then added dropwise to the HEK293T cells while being shaken to mix. After incubation for 4 hours in an incubator, 6 mL of new complete medium was added and incubated for 48 hours. After incubation, the supernatant was collected, centrifuged to remove cell residues, and filtered through a 0.45 μm filter. The cells were then stored at -80°C until use.

[0050] Add 20 μL of the obtained lentiviral supernatant to the Lenti-X TM After the GoStix cassette sample well (S) in the expression system, 3 drops of Chase solution were added to the sample well and reacted at room temperature for 10 minutes. The presence of bands was confirmed to confirm whether 5×10 5 Recombinant lentivirus with an effective capacity above IFU / mL.

[0051] The results confirmed the production of recombinant lentiviruses expressing chimeric antigen receptors that specifically bind to the CD300c antigen or its receptor. The produced lentiviruses were confirmed to express chimeric antigen receptors containing amino acid sequences of sequence 4, 6, 8, 10, 12, or 14, respectively.

[0052] Example 3: Preparation of Jurkat cells expressing a chimeric antigen receptor that specifically binds to CD300c antigen or its receptor cell

[0053] In order to prepare Jurkat cells expressing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor, the recombinant chronic disease prepared in the same manner as in Example 2 was transfected into the Jurkat cell line. In more detail, 0.1MOI to 10MOI of the recombinant lentivirus was inoculated into a complete culture medium supplemented with 8μg / mL of polybrene (Merk Company) and evenly mixed by inverting it upside down. Then, 1mL of the mixture was added to each well of the Jurkat cell line prepared in the 6-well culture plate, and the mixture was centrifuged at a speed of 1800rmp for 45 to 90 minutes, and then cultured in a culture incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, 5×10 5 cells / mL concentration for subculture.

[0054] Then, in order to confirm whether the chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor is normally expressed in the transfected Jurkat cell line, the total protein of the cultured Jurkat cells was recovered using PRO-PREP solution (iNtRON). Then, after measuring the concentration of the obtained protein using a microBCA protein assay kit (Thermofisher), the same amount of protein was used for Western blotting. In more detail, the same amount of protein was electrophoresed in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Invitrogen), and the electrophoresed protein was moved using a nitrocellulose membrane (Invitrogen). Then, the protein-bound nitrocellulose membrane was blocked with a 5% skim milk (BD) solution to block the non-specific reaction of the antibody, and anti-CD3 antibody and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Cell Signaling Technologies, USA) were used as primary antibodies, and diluted with 5% skim milk to a concentration of 1:1000, respectively. After the nitrocellulose membrane was treated and reacted, the unbound antibody was removed. Then, horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technologies) was diluted to a concentration of 1:2000 as a secondary antibody for treatment. Then, after treating with an enhanced chemiluminescence reagent solution (ECL solution) (Thermofisher) to induce a color reaction, the amount of protein was quantified using a luminescent image analysis device iBright1500 (Invitrogen). The results of the western blotting method are shown in FIG. Figure 3 shown.

[0055] like Figure 3 As shown, it was confirmed that the CD3ζ intracellular domain of the anti-CD3 antibody was bound, thereby confirming that a Jurkat cell line expressing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor was generated.

[0056] Subsequently, the expression level of the chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor was confirmed using flow cytometry (FACS). More specifically, Jurkat cells were treated with PE-fused anti-IgG antibody (Jackson Immunoresearch) and incubated at 4°C for 30 minutes for reaction, followed by confirmation using flow cytometry.

[0057] The results of flow cytometric analysis confirmed that a Jurkat cell line expressing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor had been generated.

[0058] Example 4: Confirmation of CD300c expression in cancer cells

[0059] To evaluate whether CD300c is expressed in various cancer cells, CD300c expression was assessed at the messenger RNA (mRNA) and protein levels by culturing various human solid cancer cell lines. Specifically, A549, a human lung cancer cell line, MDA-MB-231, a human breast cancer cell line, and CT26, a mouse colorectal cancer cell line, were cultured. Each cell line was fixed with 4% formaldehyde and blocked with 5% normal goat serum. Then, 1 μg of anti-CD300c antibody was treated and reacted, followed by staining with fluorescein isothiocyanate (FITC)-labeled anti-rabbit IgG antibody. Fluorescently labeled cells were then confirmed using flow cytometry.

[0060] The results confirmed that various cancer cells, including colorectal cancer, lung cancer, and breast cancer, have CD300c antigen on their cell surfaces.

[0061] Example 5: Jurkat cells expressing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor Anti-cancer effect

[0062] In order to confirm the anticancer effect of Jurkat cells expressing a chimeric antigen receptor that specifically binds to CD300c antigen or its receptor, the cancer cell killing effect was confirmed using A549 cell line. 5 A549 cell lines were seeded into 96-well culture plates at a concentration of 10 cells / mL. Then, Jurkat cells expressing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor prepared in the same manner as in Example 3 were treated with the cancer cells at a concentration of 20:1 and co-cultured. Jurkat cells not transfected with lentivirus were used as a control group. After 24 hours of co-culture, the co-cultured Jurkat cell lines were removed from each well, reacted with CCK-8 (Dojindo Co., Ltd.) for 1 hour, and the cells were cultured at OD 450nm The absorbance was measured to confirm the degree of killing of cancer cells. Figure 4 shown.

[0063] like Figure 4 As shown, Jurkat cell lines expressing a chimeric antigen receptor that specifically binds to the CD300c antigen or its receptor exhibited an anticancer effect on the A549 cell line, and it was confirmed that the anticancer effect was higher than that of Jurkat cell lines not transfected with a lentivirus.

[0064] These results confirm that the use of immune cell lines expressing chimeric antigen receptors that specifically bind to the CD300c antigen or its receptor can effectively treat cancer by enhancing the cancer therapeutic efficacy of immune cell lines. Furthermore, by specifically reacting only to cancer cells expressing the CD300c antigen on their surface, it has been confirmed that therapeutic effects can be maximized while minimizing side effects.

[0065] The above description is intended only to illustrate the present invention. Those skilled in the art will appreciate that the present invention can be readily modified into other specific forms without altering the technical concept or essential features of the present invention. Therefore, it should be understood that the above embodiments are intended to be illustrative only and not limiting.

[0066] Industrial applicability

[0067] Since the chimeric antigen receptor of the present invention that specifically binds to the CD300c antigen or its receptor specifically binds to the CD300c antigen or its receptor, it can not only be used to treat cancers that secrete the CD300c antigen, but can also reduce side effects by inhibiting CD300c activation of T cells in vivo. Furthermore, it can also be effectively used in the immunotherapy of various cancers by increasing the therapeutic effect of cancer. Sequence Listing <110> Centric Biotech <120> Chimeric antigen receptor specifically binding to CD300C antigen or its receptor <130> P22113418WP <150> KR1020190155027 <151> 2019-11-28 <150> KR1020200162200 <151> 2020-11-27 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 63 <212> DNA <213> Artificial sequence <220> <223> CD8α signal peptide <400> 1 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 2 <211> 21 <212> PRT <213> Synthetic sequence <220> <223> CD8α signal peptide <400> 2 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 3 <211> 714 <212> DNA <213> Synthetic sequence <220> <223> CK1 scFv <400> 3 gaagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60 agctgtgccg ccagcggatt caccttcagc cgctatgcca tgacctgggt tcgccaagca 120 cctggcaaag gcctggaatg ggtgagcagc atgagcggca ccggcggcac cacctattat 180 gccgatagcg tgaaaggtcg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240 ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtgc ccgcggcgcc 300 tatggctttg atcattgggg acaaggtact ctggtgaccg tgagcagcag tggaggaggt 360 agcggaggtg gtggatctgg aggtggaggt agtgaaatcg tgctgaccca gagccctggc 420 accctgagcc tgagccctgg cgaacgcgca acactgtcat gccgcgccag ccagagcatc 480 ggcaactatc tgaactggta tcagcagaaa ccaggtcagg ctccacgtct gctgatctat 540 gatgccagca acctggaaac cggcatccct gatcgcttct caggatctgg aagcggtacc 600 gattttaccc tgaccatcag ccgcctggaa cctgaggact ttgccgtgta ttattgtcag 660 cagagtagcg ccatccctta taccttcggt cagggcacta aagtggaaat caaa 714 <210> 4 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> CK1 scFv <400> 4 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Met Ser Gly Thr Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Tyr Gly Phe Asp His Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu 130 135 140 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile 145 150 155 160 Gly Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 165 170 175 Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr Gly Ile Pro Asp Arg 180 185 190 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 195 200 205 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Ala 210 215 220 Ile Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 225 230 235 <210> 5 <211> 729 <212> DNA <213> Artificial sequence <220> <223> CL6 scFv <400> 5 gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc tcttcgcctc 60 tcctgtgcag cctccggatt cactttcagc agctacggta tgcattgggt cagacaggca 120 ccaggtaagg gactggagtg ggtctctgca attagcggta gcggtggtag cacttactac 180 gcagacagcg tgaagggtcg cttcaccatc tcacgcgaca actccaagaa caccctgtac 240 ctgcagatga acagccttcg cgcagaggac actgccgtgt attactgcgc agtcagtggt 300 gcaggtcgtg gtttcttcga ctactgggga caaggtactc tggtcactgt ctcctcaggt 360 ggaggcggtt caggcggagg tggatctggc ggtggcggat cccagtctgt gctgactcag 420 ccaccttcag catctggtac tccaggtcag cgcgtcacca tcagctgcag cggtagcagc 480 agcaacattg gtagcaacta cgtgtactgg tatcagcaac tcccaggcac cgctcctaag 540 ctcctgattt acgaggacaa caagcgtcct agtggtgtgc ctgatcgctt ttctgggtcc 600 aagtctggca cctcagcctc tctggctatc agtggacttc gctccgagga cgaggctgac 660 tattactgca gcagctacac tagcagcagc actgtgatct tcggcggtgg gaccaaactg 720 accgtccta 729 <210> 6 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> CL6 scFv <400> 6 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Ser Gly Ala Gly Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala 130 135 140 Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser 145 150 155 160 Ser Asn Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly 165 170 175 Thr Ala Pro Lys Leu Leu Ile Tyr Glu Asp Asn Lys Arg Pro Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu 195 200 205 Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser 210 215 220 Ser Tyr Thr Ser Ser Ser Thr Val Ile Phe Gly Gly Gly Thr Lys Leu 225 230 235 240 Thr Val Leu <210> 7 <211> 720 <212> DNA <213> Artificial sequence <220> <223> CL7 scFv <400> 7 gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc tcttcgcctc 60 tcctgtgcag cctccggatt cactttcagc cgctacgcaa tgagctgggt cagacaggca 120 ccaggtaagg gactggagtg ggtctctgca attagcggta gcggtggtag cacttactac 180 gcagacagcg tgaagggtcg cttcaccatc tcacgcgaca actccaagaa caccctgtac 240 ctgcagatga acagccttcg cgcagaggac actgccgtgt attactgcgc acgtagcagc 300 cagggtatct tcgacatctg gggacaaggt actctggtca ctgtctcctc aggtggaggc 360 ggttcaggcg gaggtggatc tggcggtggc ggatcccagt ctgtgctgac tcagccacct 420 tcagcatctg gtactccagg tcagcgcgtc accatcagct gcagtggtaa caatatcggt 480 actagacgcg tgcattggta tcagcaactc ccagacaccg ctcctaagct cctgatttac 540 agtaagaaca accgtcctag tggtgtgcct gatcgctttt ctgggtccaa gtctggcacc 600 tcagcctctc tggctatcag tggacttcgc tccgaggacg aggctgacta ttactgcgca 660 gcatgggacg acagcctgag cggtcctgtg ttcggcggtg ggaccaaact gaccgtccta 720 <210> 8 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> CL7 scFv <400> 8 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Gln Gly Ile Phe Asp Ile Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly 130 135 140 Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Asn Asn Ile Gly 145 150 155 160 Thr Arg Arg Val His Trp Tyr Gln Gln Leu Pro Asp Thr Ala Pro Lys 165 170 175 Leu Leu Ile Tyr Ser Lys Asn Asn Arg Pro Ser Gly Val Pro Asp Arg 180 185 190 Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly 195 200 205 Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp 210 215 220 Ser Leu Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 225 230 235 240 <210> 9 <211> 720 <212> DNA <213> Synthetic sequence <220> <223> CL10 scFv <400> 9 gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc tcttcgcctc 60 tcctgtgcag cctccggatt cactttcagc agctacggta tgcattgggt cagacaggca 120 ccaggtaagg gactggagtg ggtctctgca attagcggta gcggtggtag cacttactac 180 gcagacagcg tgaagggtcg cttcaccatc tcacgcgaca actccaagaa caccctgtac 240 ctgcagatga acagccttcg cgcagaggac actgccgtgt attactgcgc aagcggttac 300 ggtctgatgg acgtgtgggg acaaggtact ctggtcactg tctcctcagg tggaggcggt 360 tcaggcggag gtggatctgg cggtggcgga tcccagtctg tgctgactca gccaccttca 420 gcatctggta ctccaggtca gcgcgtcacc atcagctgca ctcgtagcag cggtatcatc 480 gcaagcaact acgtgcagtg gtatcagcaa ctcccaggca ccgctcctaa gctcctgatt 540 taccgcaaca accagcgccc tagtggtgtg cctgatcgct tttctgggtc caagtctggc 600 acctcagcct ctctggctat cagtggactt cgctccgagg acgaggctga ctattactgc 660 agcagctacg caggtaacaa caacctggtg ttcggcggtg ggaccaaact gaccgtccta 720 <210> 10 <211> 240 <212> PRT <213> Artificial sequence <220> <223> CL10 scFv <400> 10 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Gly Tyr Gly Leu Met Asp Val Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 130 135 140 Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ile Ile 145 150 155 160 Ala Ser Asn Tyr Val Gln Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 165 170 175 Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp 180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 195 200 205 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala 210 215 220 Gly Asn Asn Asn Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 225 230 235 240 <210> 11 <211> 717 <212> DNA <213> Artificial sequence <220> <223> SL18 scFv <400> 11 cgagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60 agctgtgccg ccagcggatt caccttcagc gattatcata tgcattgggt tcgccaagca 120 cctggcaaag gcctggaatg ggtgagcacc atcagcagca gcggcggcta tacctattat 180 gccgaaagcg tgaaaagccg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240 ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtgc ccgatcgata 300 cgcctgcctc tggattattg gggacaaggt actctggtga ccgtgagcag cagtggagga 360 ggtagcggag gtggtggatc tggaggtgga ggtagtcaga gcgtgctgac ccagcctcct 420 agcgcctccg gtacaccagg acagcgcgtg actattagct gtagcggcaa caacatcggc 480 agcaaaggcg tgcattggta tcagcaactg cctggaactg cacctaagct gctgatctat 540 gaagatagca aacgccctag cggcgtgcgt gatcgcttta gcggtagcaa atcaggcacc 600 agcgccagcc tggccatcag cggccttcgc tccgaagatg aagccgatta ttattgtcag 660 agctatgata gcaccaaagg cgtggtgttt ggtggcggta ccaagctgac cgtgctg 717 <210> 12 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> SL18 scFv <400> 12 Arg Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 His Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Ser Ser Gly Gly Tyr Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ile Arg Leu Pro Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly 130 135 140 Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Asn Asn Ile Gly 145 150 155 160 Ser Lys Gly Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys 165 170 175 Leu Leu Ile Tyr Glu Asp Ser Lys Arg Pro Ser Gly Val Arg Asp Arg 180 185 190 Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly 195 200 205 Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 210 215 220 Thr Lys Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 225 230 235 <210> 13 <211> 489 <212> DNA <213> Artificial sequence <220> <223> CD300c ECD <400> 13 ggctattttc ctctgagcca ccccatgacc gtggcgggcc ccgtgggggg atccctgagt 60 gtgcagtgtc gctatgagaa ggaacacagg accctcaaca aattctggtg cagaccacca 120 cagattctcc gatgtgacaa gattgtggag accaaagggt cagcagggaa aaggaatggc 180 cgagtgtcca tcagggacag tcctgcaaac ctcagcttca cagtgaccct ggagaatctc 240 acagaggagg acgcaggcac ctactggtgt ggggtggata caccgtggct ccgagacttt 300 catgatccca ttgtcgaggt tgaggtgtcc gtgttcccgg ccgggacgac cacagcctcc 360 agcccccaga gctccatggg cacctcaggt cctcccacga agctgcccgt gcacacctgg 420 cccagcgtga ccagaaagga cagccccgaa cccagcccac accctggctc cctgttcagc 480 aatgtccgc 489 <210> 14 <211> 163 <212> PRT <213> Synthetic Sequence <220> <223> CD300c ECD <400> 14 Gly Tyr Phe Pro Leu Ser His Pro Met Thr Val Ala Gly Pro Val Gly 1 5 10 15 Gly Ser Leu Ser Val Gln Cys Arg Tyr Glu Lys Glu His Arg Thr Leu 20 25 30 Asn Lys Phe Trp Cys Arg Pro Pro Gln Ile Leu Arg Cys Asp Lys Ile 35 40 45 Val Glu Thr Lys Gly Ser Ala Gly Lys Arg Asn Gly Arg Val Ser Ile 50 55 60 Arg Asp Ser Pro Ala Asn Leu Ser Phe Thr Val Thr Leu Glu Asn Leu 65 70 75 80 Thr Glu Glu Asp Ala Gly Thr Tyr Trp Cys Gly Val Asp Thr Pro Trp 85 90 95 Leu Arg Asp Phe His Asp Pro Ile Val Glu Val Glu Val Ser Val Phe 100 105 110 Pro Ala Gly Thr Thr Thr Ala Ser Ser Pro Gln Ser Ser Met Gly Thr 115 120 125 Ser Gly Pro Pro Thr Lys Leu Pro Val His Thr Trp Pro Ser Val Thr 130 135 140 Arg Lys Asp Ser Pro Glu Pro Ser Pro His Pro Gly Ser Leu Phe Ser 145 150 155 160 Asn Val Arg <210> 15 <211> 30 <212> DNA <213> Artificial sequence <220> <223> GS linker <400> 15 ggtggcggag gttctggagg tggaggttcc 30 <210> 16 <211> 10 <212> PRT <213> Artificial sequence <220> <223> GS linker <400> 16 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 17 <211> 135 <212> DNA <213> Artificial sequence <220> <223> CD8 hinge <400> 17 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 18 <211> 45 <212> PRT <213> Artificial sequence <220> <223> CD8 hinge <400> 18 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 19 <211> 81 <212> DNA <213> Artificial sequence <220> <223> CD28 transmembrane domain <400> 19 ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60 gcctttatta ttttctgggt g 81 <210> 20 <211> 27 <212> PRT <213> Artificial sequence <220> <223> CD28 transmembrane domain <400> 20 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 21 <211> 123 <212> DNA <213> Artificial sequence <220> <223> CD28 intracellular domain <400> 21 aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60 gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120 tcc 123 <210> 22 <211> 41 <212> PRT <213> Artificial sequence <220> <223> CD28 intracellular domain <400> 22 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 23 <211> 339 <212> DNA <213> Artificial sequence <220> <223> CD3zeta intracellular domain <400> 23 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339 <210> 24 <211> 112 <212> PRT <213> Artificial sequence <220> <223> CD3zeta intracellular domain <400> 24 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 25 <211> 1485 <212> DNA <213> Artificial sequence <220> <223> CK1 CAR <400> 25 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggaagtgc agctgctgga aagtggaggt ggactggtgc agcctggcgg cagcctgcgc 120 ctgagctgtg ccgccagcgg attcaccttc agccgctatg ccatgacctg ggttcgccaa 180 gcacctggca aaggcctgga atgggtgagc agcatgagcg gcaccggcgg caccacctat 240 tatgccgata gcgtgaaagg tcgctttacc atcagccgcg ataacagcaa aaacaccctg 300 tatctgcaga tgaacagcct gcgcgccgag gacaccgcag tctactactg tgcccgcggc 360 gcctatggct ttgatcattg gggacaaggt actctggtga ccgtgagcag cagtggagga 420 ggtagcggag gtggtggatc tggaggtgga ggtagtgaaa tcgtgctgac ccagagccct 480 ggcaccctga gcctgagccc tggcgaacgc gcaacactgt catgccgcgc cagccagagc 540 atcggcaact atctgaactg gtatcagcag aaaccaggtc aggctccacg tctgctgatc 600 tatgatgcca gcaacctgga aaccggcatc cctgatcgct tctcaggatc tggaagcggt 660 accgatttta ccctgaccat cagccgcctg gaacctgagg actttgccgt gtattattgt 720 cagcagagta gcgccatccc ttataccttc ggtcagggca ctaaagtgga atcaaggt 780 ggcggaggtt ctggaggtgg aggttccacc acgacccg cgccgcgacc accacccg 840 ggcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 900 gggggcgcag tgcacacgag ggggctggac tcgcctgtg attttgtt gctggtggtg 960 gttggtggag tcctggcttg ctatagcttg ctagtacag tggcctttat tattttctgg 1020 gtgaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac tccccgccgc 1080 cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt cgcagcctat 1140 cgctccagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1200 cagctctata acgagctca tctaggacga agaggagt acgatgtttt ggacagagaga 1260 cgtggccgggg accctgagat gggggaag ccgagaagga agaccctca gggaggctg 1320 tacaatgaac tgcagaaaga tagatggcg gaggcctaca gtgagattgg gatgaaggc 1380 gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccag 1440 gacacctacg acgcccttca catgcaggcc ctgccccctc gctaa 1485 <210> 26 <211> 494 <212> PRT <213> Artificial Sequence <220> <223> CK1 CAR <400> 26 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Arg Tyr Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ser Ser Met Ser Gly Thr Gly Gly Thr Thr Tyr 65 70 75 80 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Gly Ala Tyr Gly Phe Asp His Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Ser Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro 145 150 155 160 Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 165 170 175 Ala Ser Gln Ser Ile Gly Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro 180 185 190 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr 195 200 205 Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 210 215 220 Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 225 230 235 240 Gln Gln Ser Ser Ala Ile Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val 245 250 255 Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Thr Thr 260 265 270 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 275 280 285 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 290 295 300 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val 305 310 315 320 Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe 325 330 335 Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp 340 345 350 Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr 355 360 365 Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val 370 375 380 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 385 390 395 400 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 405 410 415 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 420 425 430 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 435 440 445 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 450 455 460 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 465 470 475 480 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 27 <211> 1500 <212> DNA <213> Artificial sequence <220> <223> CL6 CAR <400> 27 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggaggtgc agctgttgga gtctggtgga ggcttggtac agcctggagg ttctcttcgc 120 ctctcctgtg cagcctccgg attcactttc agcagctacg gtatgcattg ggtcagacag 180 gcaccaggta agggactgga gtgggtctct gcaattagcg gtagcggtgg tagcacttac 240 tacgcagaca gcgtgaaggg tcgcttcacc atctcacgcg acaactccaa gaacaccctg 300 tacctgcaga tgaacagcct tcgcgcagag gacactgccg tgtattactg cgcagtcagt 360 ggtgcaggtc gtggtttctt cgactactgg ggacaaggta ctctggtcac tgtctcctca 420 ggtggaggcg gttcaggcgg aggtggatct ggcggtggcg gatcccagtc tgtgctgact 480 cagccacctt cagcatctgg tactccaggt cagcgcgtca ccatcagctg cagcggtagc 540 agcagcaaca ttggtagcaa ctacgtgtac tggtatcagc aactcccagg caccgctcct 600 aagctcctga tttacgagga caacaagcgt cctagtggtg tgcctgatcg cttttctggg 660 tccaagtctg gcacctcagc ctctctggct atcagtggac ttcgctccga ggacgaggct 720 gactattact gcagcagcta cactagcagc agcactgtga tcttcggcgg tgggaccaaa 780 ctgaccgtcc taggtggcgg aggttctgga ggtggaggtt ccaccacgac gccagcgccg 840 cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 900 tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatttt 960 tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 1020 tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 1080 atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 1140 gacttcgcag cctatcgctc cagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1200 cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1260 gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 1320 cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1380 attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1440 agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgctaa 1500 <210> 28 <211> 499 <212> PRT <213> Synthetic Sequence <220> <223> CL6 CAR <400> 28 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr 65 70 75 80 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Val Ser Gly Ala Gly Arg Gly Phe Phe Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr 145 150 155 160 Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser 165 170 175 Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr Trp Tyr 180 185 190 Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Glu Asp Asn 195 200 205 Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly 210 215 220 Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala 225 230 235 240 Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Val Ile Phe Gly 245 250 255 Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly 260 265 270 Gly Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 275 280 285 Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 290 295 300 Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe 305 310 315 320 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 325 330 335 Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg 340 345 350 Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 355 360 365 Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 370 375 380 Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 385 390 395 400 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 405 410 415 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 420 425 430 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 435 440 445 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 450 455 460 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 465 470 475 480 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 485 490 495 Pro Pro Arg <210> 29 <211> 1491 <212> DNA <213> Artificial Sequence <220> <223> CL7 CAR <400> 29 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggaggtgc agctgttgga gtctggtgga ggcttggtac agcctggagg ttctcttcgc 120 ctctcctgtg cagcctccgg attcactttc agccgctacg caatgagctg ggtcagacag 180 gcaccaggta agggactgga gtgggtctct gcaattagcg gtagcggtgg tagcacttac 240 tacgcagaca gcgtgaaggg tcgcttcacc atctcacgcg acaactccaa gaacaccctg 300 tacctgcaga tgaacagcct tcgcgcagag gacactgccg tgtattactg cgcacgtagc 360 agccagggta tcttcgacat ctggggacaa ggtactctgg tcactgtctc ctcaggtgga 420 ggcggttcag gcggaggtgg atctggcggt ggcggatccc agtctgtgct gactcagcca 480 ccttcagcat ctggtactcc aggtcagcgc gtcaccatca gctgcagtgg taacaatatc 540 ggtactagac gcgtgcattg gtatcagcaa ctcccagaca ccgctcctaa gctcctgatt 600 tacagtaaga acaaccgtcc tagtggtgtg cctgatcgct tttctgggtc caagtctggc 660 acctcagcct ctctggctat cagtggactt cgctccgagg acgaggctga ctattactgc 720 gcagcatggg acgacagcct gagcggtcct gtgttcggcg gtgggaccaa actgaccgtc 780 ctaggtggcg gaggttctgg aggtggaggt tccaccacga cgccagcgcc gcgaccacca 840 acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 900 gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgattt ttgggtgctg 960 gtggtggttg gtggagtcct ggcttgctat agcttgctag taacagtggc ctttattatt 1020 ttctgggtga ggagtaagag gagcaggctc ctgcacagtg actacatgaa catgactccc 1080 cgccgccccg ggcccacccg caagcattac cagccctatg ccccaccacg cgacttcgca 1140 gcctatcgct ccagagtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc 1200 cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1260 aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1320 ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1380 aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1440 accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta a 1491 <210> 30 <211> 496 <212> PRT <213> Artificial Sequence <220> <223> CL7 CAR <400> 30 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Arg Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr 65 70 75 80 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Ser Ser Gln Gly Ile Phe Asp Ile Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro 145 150 155 160 Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser 165 170 175 Gly Asn Asn Ile Gly Thr Arg Arg Val His Trp Tyr Gln Gln Leu Pro 180 185 190 Asp Thr Ala Pro Lys Leu Leu Ile Tyr Ser Lys Asn Asn Arg Pro Ser 195 200 205 Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser 210 215 220 Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 225 230 235 240 Ala Ala Trp Asp Asp Ser Leu Ser Gly Pro Val Phe Gly Gly Gly Thr 245 250 255 Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr 260 265 270 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 275 280 285 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 290 295 300 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu 305 310 315 320 Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 325 330 335 Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His 340 345 350 Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 355 360 365 His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 370 375 380 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 385 390 395 400 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 405 410 415 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 420 425 430 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 435 440 445 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 450 455 460 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 465 470 475 480 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 495 <210> 31 <211> 1491 <212> DNA <213> Artificial sequence <220> <223> CL10 CAR <400> 31 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggaggtgc agctgttgga gtctggtgga ggcttggtac agcctggagg ttctcttcgc 120 ctctcctgtg cagcctccgg attcactttc agcagctacg gtatgcattg ggtcagacag 180 gcaccaggta agggactgga gtgggtctct gcaattagcg gtagcggtgg tagcacttac 240 tacgcagaca gcgtgaaggg tcgcttcacc atctcacgcg acaactccaa gaacaccctg 300 tacctgcaga tgaacagcct tcgcgcagag gacactgccg tgtattactg cgcaagcggt 360 tacggtctga tggacgtgtg gggacaaggt actctggtca ctgtctcctc aggtggaggc 420 ggttcaggcg gaggtggatc tggcggtggc ggatcccagt ctgtgctgac tcagccacct 480 tcagcatctg gtactccagg tcagcgcgtc accatcagct gcactcgtag cagcggtatc 540 atcgcaagca actacgtgca gtggtatcag caactcccag gcaccgctcc taagctcctg 600 atttaccgca acaaccagcg ccctagtggt gtgcctgatc gcttttctgg gtccaagtct 660 ggcacctcag cctctctggc tatcagtgga cttcgctccg aggacgaggc tgactattac 720 tgcagcagct acgcaggtaa caacaacctg gtgttcggcg gtgggaccaa actgaccgtc 780 ctaggtggcg gaggttctgg aggtggaggt tccaccacga cgccagcgcc gcgaccacca 840 acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 900 gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgattt ttgggtgctg 960 gtggtggttg gtggagtcct ggcttgctat agcttgctag taacagtggc ctttattatt 1020 ttctgggtga ggagtaagag gagcaggctc ctgcacagtg actacatgaa catgactccc 1080 cgccgccccg ggcccacccg caagcattac cagccctatg ccccaccacg cgacttcgca 1140 gcctatcgct ccagagtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc 1200 cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1260 aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1320 ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1380 aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1440 accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta a 1491 <210> 32 <211> 496 <212> PRT <213> Artificial Sequence <220> <223> CL10 CAR <400> 32 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr 65 70 75 80 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Ser Gly Tyr Gly Leu Met Asp Val Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro 145 150 155 160 Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Arg 165 170 175 Ser Ser Gly Ile Ile Ala Ser Asn Tyr Val Gln Trp Tyr Gln Gln Leu 180 185 190 Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro 195 200 205 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala 210 215 220 Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr 225 230 235 240 Cys Ser Ser Tyr Ala Gly Asn Asn Asn Leu Val Phe Gly Gly Gly Thr 245 250 255 Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr 260 265 270 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 275 280 285 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 290 295 300 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu 305 310 315 320 Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 325 330 335 Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His 340 345 350 Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 355 360 365 His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 370 375 380 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 385 390 395 400 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 405 410 415 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 420 425 430 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 435 440 445 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 450 455 460 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 465 470 475 480 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 495 <210> 33 <211> 1488 <212> DNA <213> Artificial Sequence <220> <223> SL18 CAR <400> 33 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccgcgagtgc agctgctgga aagtggaggt ggactggtgc agcctggcgg cagcctgcgc 120 ctgagctgtg ccgccagcgg attcaccttc agcgattatc atatgcattg ggttcgccaa 180 gcacctggca aaggcctgga atgggtgagc accatcagca gcagcggcgg ctatacctat 240 tatgccgaaa gcgtgaaaag ccgctttacc atcagccgcg ataacagcaa aaacaccctg 300 tatctgcaga tgaacagcct gcgcgccgag gacaccgcag tctactactg tgcccgatcg 360 atacgcctgc ctctggatta ttggggacaa ggtactctgg tgaccgtgag cagcagtgga 420 ggaggtagcg gaggtggtgg atctggaggt ggaggtagtc agagcgtgct gacccagcct 480 cctagcgcct ccggtacacc aggacagcgc gtgactatta gctgtagcgg caacaacatc 540 ggcagcaaag gcgtgcattg gtatcagcaa ctgcctggaa ctgcacctaa gctgctgatc 600 tatgaagata gcaaacgccc tagcggcgtg cgtgatcgct ttagcggtag caaatcaggc 660 accagcgcca gcctggccat cagcggcctt cgctccgaag atgaagccga ttattattgt 720 cagagctatg atagcaccaa aggcgtggtg tttggtggcg gtaccaagct gaccgtgctg 780 ggtggcggag gttctggagg tggaggttcc accacgacgc cagcgccgcg accaccaaca 840 ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 900 gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatttttg ggtgctggtg 960 gtggttggtg gagtcctggc ttgctatagc ttgctagtaa cagtggcctt tattattttc 1020 tgggtgagga gtaagaggag caggctcctg cacagtgact acatgaacat gactccccgc 1080 cgccccgggc ccacccgcaa gcattaccag ccctatgccc caccacgcga cttcgcagcc 1140 tatcgctcca gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag 1200 aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 1260 agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1320 ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1380 ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1440 aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgctaa 1488 <210> 34 <211> 495 <212> PRT <213> Synthetic sequence <220> <223> SL18 CAR <400> 34 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Arg Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Asp Tyr His Met His Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ser Thr Ile Ser Ser Ser Gly Gly Tyr Thr Tyr 65 70 75 80 Tyr Ala Glu Ser Val Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Ser Ile Arg Leu Pro Leu Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro 145 150 155 160 Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser 165 170 175 Gly Asn Asn Ile Gly Ser Lys Gly Val His Trp Tyr Gln Gln Leu Pro 180 185 190 Gly Thr Ala Pro Lys Leu Leu Ile Tyr Glu Asp Ser Lys Arg Pro Ser 195 200 205 Gly Val Arg Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser 210 215 220 Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 225 230 235 240 Gln Ser Tyr Asp Ser Thr Lys Gly Val Val Phe Gly Gly Gly Thr Lys 245 250 255 Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Thr 260 265 270 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 275 280 285 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 290 295 300 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val 305 310 315 320 Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala 325 330 335 Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser 340 345 350 Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His 355 360 365 Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg 370 375 380 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 385 390 395 400 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 405 410 415 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 420 425 430 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 435 440 445 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 450 455 460 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 465 470 475 480 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 495 <210> 35 <211> 1260 <212> DNA <213> Artificial sequence <220> <223> CD300c ECD CAR <400> 35 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccgggctatt ttcctctgag ccaccccatg accgtggcgg gccccgtggg gggatccctg 120 agtgtgcagt gtcgctatga gaaggaacac aggaccctca acaaattctg gtgcagacca 180 ccacagattc tccgatgtga caagattgtg gagaccaaag ggtcagcagg gaaaaggaat 240 ggccgagtgt ccatcaggga cagtcctgca aacctcagct tcacagtgac cctggagaat 300 ctcacagagg aggacgcagg cacctactgg tgtggggtgg atacaccgtg gctccgagac 360 tttcatgatc ccattgtcga ggttgaggtg tccgtgttcc cggccgggac gaccacagcc 420 tccagccccc agagctccat gggcacctca ggtcctccca cgaagctgcc cgtgcacacc 480 tggcccagcg tgaccagaaa ggacagcccc gaacccagcc cacaccctgg ctccctgttc 540 agcaatgtcc gcggtggcgg aggttctgga ggtggaggtt ccaccacgac gccagcgccg 600 cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 660 tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatttt 720 tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 780 tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 840 atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 900 gacttcgcag cctatcgctc cagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 960 cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1020 gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 1080 cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1140 attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1200 agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgctaa 1260 <210> 36 <211> 419 <212> PRT <213> Artificial Sequence <220> <223> CD300c ECD CAR <400> 36 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Tyr Phe Pro Leu Ser His Pro Met Thr Val 20 25 30 Ala Gly Pro Val Gly Gly Ser Leu Ser Val Gln Cys Arg Tyr Glu Lys 35 40 45 Glu His Arg Thr Leu Asn Lys Phe Trp Cys Arg Pro Pro Gln Ile Leu 50 55 60 Arg Cys Asp Lys Ile Val Glu Thr Lys Gly Ser Ala Gly Lys Arg Asn 65 70 75 80 Gly Arg Val Ser Ile Arg Asp Ser Pro Ala Asn Leu Ser Phe Thr Val 85 90 95 Thr Leu Glu Asn Leu Thr Glu Glu Asp Ala Gly Thr Tyr Trp Cys Gly 100 105 110 Val Asp Thr Pro Trp Leu Arg Asp Phe His Asp Pro Ile Val Glu Val 115 120 125 Glu Val Ser Val Phe Pro Ala Gly Thr Thr Thr Ala Ser Ser Pro Gln 130 135 140 Ser Ser Met Gly Thr Ser Gly Pro Pro Thr Lys Leu Pro Val His Thr 145 150 155 160 Trp Pro Ser Val Thr Arg Lys Asp Ser Pro Glu Pro Ser Pro His Pro 165 170 175 Gly Ser Leu Phe Ser Asn Val Arg Gly Gly Gly Gly Ser Gly Gly Gly 180 185 190 Gly Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 195 200 205 Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 210 215 220 Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe 225 230 235 240 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 245 250 255 Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg 260 265 270 Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 275 280 285 Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 290 295 300 Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 305 310 315 320 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 325 330 335 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 340 345 350 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 355 360 365 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 370 375 380 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 385 390 395 400 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 405 410 415 Pro Pro Arg

Claims

1. A chimeric antigen receptor that specifically binds to CD300c antigen, characterized in that: The chimeric antigen receptor comprises a single-chain variable fragment having an amino acid sequence as represented by SEQ ID NO:

8.

2. The chimeric antigen receptor according to claim 1, characterized in that The chimeric antigen receptor further comprises a signal peptide, a GS linker, a transmembrane domain and an intracytoplasmic domain.

3. The chimeric antigen receptor according to claim 2, characterized in that The above signal peptide is CD8α signal peptide.

4. The chimeric antigen receptor according to claim 2, characterized in that The above-mentioned transmembrane domains are CD8 hinge (hinge of cluster of differentiation 8) and CD28 transmembrane domain (CD28 transmembranedomain).

5. The chimeric antigen receptor according to claim 2, characterized in that The above-mentioned intracytoplasmic domains are CD28 intracellular domain (CD28 intracellular domain) and CD3ζ intracellular domain (CD3ζ intracellular domain).

6. The chimeric antigen receptor according to claim 1, characterized in that Use of the chimeric antigen receptor for treating cancer expressing CD300c antigen.

7. A recombinant vector, characterized in that: Expressing the chimeric antigen receptor according to any one of claims 1 to 6.

8. An immune cell, characterized in that Transformed by the recombinant vector according to claim 7.

9. The immune cell according to claim 8, characterized in that The immune cells are at least one selected from the group consisting of monocytes, macrophages, T cells, natural killer cells (NK cells) and dendritic cells.

10. A pharmaceutical composition for treating cancer expressing CD300c antigen, characterized in that: Comprising the immune cell according to claim 8 as an active ingredient; The above-mentioned cancer is one or more selected from the group consisting of colorectal cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer and blood cancer.

11. The pharmaceutical composition according to claim 10, characterized in that The above-mentioned pharmaceutical composition also contains other anticancer drugs.

12. The pharmaceutical composition according to claim 10, characterized in that The pharmaceutical composition inhibits cancer proliferation, survival, metastasis, recurrence or anticancer drug resistance.

13. Use of the chimeric antigen receptor according to any one of claims 1 to 6, the recombinant vector according to claim 7, the immune cell according to claim 8 or 9, or the pharmaceutical composition according to any one of claims 10 to 12 in the preparation of a drug for treating cancer; The above cancer is lung cancer.

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