Method for evaluating generation of cellular immune response to car

Abstract

The present invention relates to a method for evaluating generation of cellular immune response to a CAR. The present invention specifically provides a method for evaluating CAR-specific cellular immune response, comprising the following steps: obtaining amino acid fragments of a CAR and forming the amino acid fragments into a peptide library, taking the peptide library as an antigen to stimulate a PBMC, detecting the condition of IFN-γ secretion in the PBMC, and evaluating the level of CAR-specific cellular immune response. By means of the method of the present invention, a specific site capable of causing the generation of CAR-specific cellular immunity can be identified, and the clinical response condition after CAR-T infusion can be predicted by determining the CAR-specific cellular immunity.

Description

A method for evaluating cellular immune responses generated by CAR Technical Field

[0001] The present invention belongs to the field of immunotherapy, and specifically relates to a method for evaluating CAR-induced cellular immune responses. Background Art

[0002] Genetically engineered chimeric antigen receptor cells (CAR-T, CAR-NK, etc.) primarily derive their genes from non-human sequences such as alpacas or mice. These non-human sequences or structures can trigger the production of CAR-specific humoral immune responses (anti-drug antibodies, ADA) and cellular immune responses (anti-CAR cytotoxic T cells, anti-CAR CTLs). ADA production is well-established, and it can weaken CAR-T cell activation, promote CAR-T cell exhaustion, and be associated with treatment failure.

[0003] However, the detection and research of CAR-specific cellular immune responses are still very limited, and are mainly limited to the evaluation of the therapeutic effects of hematological tumors. For example, a few studies have shown that the generation of CAR-specific cellular immune responses is related to the failure of treatment of B-cell acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). CN110824158A discloses an enzyme-linked immunosorbent assay (ELISPOT) to detect the ability of CART cells to secrete cytokines INF-γ in response to CD19 antigen stimulation. US20230123128A1 discloses the use of an ELISPOT analyzer to quantitatively detect INF-γ secreted by isolated cytotoxic T cells to evaluate the immune response of cytotoxic T cells to Tapasin genetically engineered cells.

[0004] Therefore, there is an urgent need to develop a detection method that can identify and evaluate the level of anti-CAR cytotoxic T cells produced by patients after solid tumor CAR-T cell infusion.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a detection method that can identify and evaluate the level of anti-CAR (chimeric antigen receptor) cytotoxic T cells produced by patients after solid tumor CAR-T cell infusion, and predict the therapeutic effect of CAR-containing immune cells based on the level of anti-CAR cytotoxic T cells.

[0007] The present invention provides a method for evaluating CAR-specific cellular immune responses, comprising the following steps: obtaining amino acid fragments (also referred to as peptide segments) of CAR and forming a peptide library, using the peptide library as an antigen to stimulate PBMCs, detecting the secretion of IFN-γ in PBMCs, and evaluating the level of CAR-specific cellular immune responses.

[0008] In one or more embodiments, the amino acid fragment of CAR is a complete fragment or a partial fragment of CAR.

[0009] In one or more embodiments, the CAR can be any CAR currently disclosed.

[0010] In one or more embodiments, the antigen binding domain of the CAR comprises the sequence shown in SEQ ID NO: 3. Preferably, the CAR further comprises an optional signal peptide, hinge region, transmembrane region, intracellular region fragment, etc.

[0011] In one or more embodiments, the sequence of the CAR is as shown in SEQ ID NO: 1.

[0012] In one or more embodiments, the amino acid fragment of the CAR is an amino acid fragment of an antigen binding domain.

[0013] In one or more embodiments, the partial fragments of the CAR are segmented according to the structural domain of the CAR, for example, they are divided into signal peptide fragments, antigen binding domain fragments, hinge region fragments, transmembrane region fragments, intracellular region fragments, etc. The antigen binding domain fragments can be conventional antibodies, scFv, single domain antibodies (VHH), etc. The antigen binding domain fragments can also be further segmented according to the structure, for example, when it is scFv, it can be divided into light chain variable domain (VL) fragments, heavy chain variable domain fragments (VH), and when it is a conventional antibody, it can be divided into Fab fragments, Fc fragments, etc. Preferably, there is 0-10 amino acid overlap between adjacent fragments, more preferably 1-9, 2-8, 3-7, 4-6 or 5 amino acid overlaps.

[0014] In one or more embodiments, the partial fragments of the CAR are CAR segments of a specific length, the fragment length is 10-20 amino acids, and the amino acids between adjacent fragments overlap, for example, there is 0-10 amino acid overlap, more preferably 1-9, 2-8, 3-7, 4-6 or 5 amino acid overlap.

[0015] In one or more embodiments, the peptide library comprises a combination of multiple partial fragments of CAR; preferably, the combination of multiple partial fragments of CAR in the peptide library covers the full-length amino acids of CAR or the full-length amino acids of the antibody fragment in CAR.

[0016] In one or more embodiments, the peptide library comprises amino acid fragments of only one CAR.

[0017] In one or more embodiments, the peptide library comprises one or more amino acid fragments of CAR as shown in SEQ ID NOs: 1-6.

[0018] In one or more embodiments, the peptide library comprises one or more amino acid fragments of CAR as shown in SEQ ID NOs: 7-18.

[0019] In one or more embodiments, the amino acid fragment of the CAR is obtained by PCR amplification, or can also be directly chemically synthesized in vitro.

[0020] In one or more embodiments, the assay is an ELISA assay or an ELISPOT assay, preferably an ELISPOT assay.

[0021] In one or more embodiments, the ELISPOT assay comprises the following steps:

[0022] (1) Dissolving the amino acid fragments of CAR in the diluted peptide library;

[0023] (2) Washing and blocking the ELISPOT pre-coated plate;

[0024] (3) Add PBMC cell suspension to the ELISPOT plate, dissolve the diluted peptide library and incubate;

[0025] (4) After washing, add secondary antibody for incubation, develop color, and analyze using an ELISPOT reader.

[0026] In one or more embodiments, after dilution in step (1), the concentration of the amino acid fragment of CAR is 0.65-6.5 nM, preferably 1-6 nM, 1.3-6 nM, or 2-3 nM.

[0027] In one or more embodiments, the evaluation comprises determining whether the CAR-specific cellular immune response is positive and evaluating the level of the CAR-specific cellular immune response, and / or evaluating the level of the cellular immune response of each amino acid fragment in the CAR, and determining the epitope that triggers the cellular immune response.

[0028] The method of evaluating CAR-specific cellular immune responses of the present invention is a method for non-diagnostic or therapeutic purposes. It predicts the therapeutic effect of CAR-containing immune cells by evaluating the number of anti-CAR CTLs produced by CAR, and does not directly diagnose the therapeutic effect of the human body.

[0029] The present invention also provides a kit for evaluating a CAR-specific cellular immune response, wherein the kit is evaluated using the method described in any embodiment of the present invention. The kit comprises: one or more peptide libraries composed of amino acid fragments of CAR, wherein the peptide library is the peptide library described in any embodiment of the present invention.

[0030] In one or more embodiments, the kit may further comprise sample processing reagents, such as the diluent DMSO, for dissolving and diluting the peptide library. The kit may further comprise reagents for detecting IFN-γ secretion, such as reagents for ELISPOT detection, such as DPBS buffer, blocking solution, FBS, secondary antibodies, etc. The kit may further comprise reagents for culturing or treating PBMCs, such as culture medium.

[0031] The kit can also be used in conjunction with a color development kit, such as a BCIP / NBT kit.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] (1) It can identify specific sites that can cause the production of CAR-specific cellular immunity;

[0034] (2) It can provide guidance for CAR structure optimization;

[0035] (3) The clinical response after CAR-T transfusion can be predicted by judging the strength of specific CAR cell immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 shows the effect of different peptide concentrations on the number of ELISPOT detection spots;

[0037] Figure 2 shows the ELISPOT test results of different patients on days 0 and 28 after and without peptide library stimulation;

[0038] Figure 3 shows the ELISPOT test results of different CAR peptides for patient 1.

[0039] FIG4 shows the ELISPOT test results of different CAR peptides for patient 5. ... DETAILED DESCRIPTION

[0040] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0041] The term "immune response" refers to the overall body response to a target, such as an antigen, and preferably refers to a cellular immune response or a cellular and humoral immune response.

[0042] The term "cellular immune response" refers to an immune response against cells characterized by antigen presentation by class I or class II MHC, including helper T cells and killer T cells (i.e., CTLs), which secrete the cytokine INF-γ. The present invention assesses the level of CAR (chimeric antigen receptor)-specific cellular immune response by detecting the number of cells secreting INF-γ and evaluating the number of anti-CAR CTLs produced.

[0043] The term "peptide library" refers to a mixture of peptide fragments of a certain length. In the present invention, in certain embodiments, the peptide library may also contain only one peptide fragment of a certain length.

[0044] The purpose of the present invention is to provide a detection method that can identify and evaluate the level of anti-CAR cytotoxic T cells produced by patients after solid tumor CAR-T cell infusion, and predict the therapeutic effect of CAR-containing immune cells based on the level of anti-CAR cytotoxic T cells.

[0045] The first aspect of the present invention provides a method for evaluating CAR-specific cellular immune responses, comprising the following steps: obtaining amino acid fragments (also referred to as peptide segments) of CAR and forming a peptide library, using the peptide library as an antigen to stimulate PBMC, detecting the secretion of IFN-γ in PBMC, and evaluating the level of CAR-specific cellular immune responses.

[0046] In one or more embodiments, the amino acid fragment of CAR is a complete fragment or a partial fragment of CAR.

[0047] In one or more embodiments, the CAR can be any CAR disclosed in the prior art, such as the CAR in CN202111681582.0. Herein, CAR is a chimeric antigen receptor, the structure of which comprises: an extracellular region, a hinge region, a transmembrane region, and an intracellular region. The N segment of the CAR may also have a signal peptide. The intracellular region comprises an intracellular costimulatory domain and an intracellular signaling domain. In an exemplary embodiment, the antigen binding domain of the CAR comprises a sequence as shown in SEQ ID NO: 3.

[0048] In one or more embodiments, the sequence of the CAR is as shown in SEQ ID NO: 1.

[0049] Considering that the signal peptide, transmembrane region, and intracellular region in CAR can be selected as human sequences with lower immunogenicity, it is also possible to evaluate only non-human amino acid sequences. In one or more embodiments, the amino acid fragment of the CAR is an amino acid fragment of the antigen binding domain.

[0050] In one or more embodiments, the partial fragments of the CAR are segmented according to the functional structure of the CAR, for example, comprising a signal peptide fragment, an antibody fragment, a hinge region fragment, a transmembrane region fragment, and an intracellular region fragment.

[0051] "Antibody fragment" refers to a portion of an intact antibody, preferably the antigen binding region and / or variable region of an intact antibody. An antibody fragment is preferably an antigen binding fragment of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; VHH. Among them, the antibody fragment can be further segmented. For example, in the case of ScFV, it can be divided into a light chain variable domain (VL) fragment and a heavy chain variable domain fragment (VH); in the case of a conventional antibody, it can be divided into a Fab fragment, an Fc fragment, etc. Preferably, there is 0-10 amino acid overlap between adjacent fragments, more preferably 1-9, 2-8, 3-7, 4-6 or 5 amino acid overlaps.

[0052] In one or more embodiments, the partial fragments of the CAR are segmented according to a specific length, and the fragment length is 10-20 amino acids, and the amino acids overlap between adjacent fragments, for example, there is a 0-10 amino acid overlap, more preferably 1-9, 2-8, 3-7, 4-6 or 5 amino acid overlap. For example, when the fragment length is 15 amino acids and there is a 5 amino acid overlap between adjacent fragments, partial fragment 1 is the amino acids at positions 1-15, partial fragment 2 is 11-25, and so on. According to the segmentation of a specific length, the epitope that triggers a cellular immune response can be better identified; too short a length may destroy the minimum unit of the epitope, and too long a length may cause folding. Similarly, the overlapping of amino acids in adjacent fragments is also to avoid artificial segmentation and destroy the minimum unit of the epitope.

[0053] In one or more embodiments, the peptide library comprises a combination of partial fragments of multiple CARs; preferably, the combination of partial fragments of multiple CARs in the peptide library covers the full-length amino acids of CAR or the full-length amino acids of the antibody fragment in CAR, which can be used to evaluate whether CAR will induce a cellular immune response.

[0054] In one or more embodiments, the peptide library contains only one amino acid fragment of CAR. At this time, it is used to evaluate whether the amino acid fragment will cause a cellular immune response, and when a peptide library containing different amino acid fragments is used for detection, the amino acid fragment with the highest cellular immunogenicity can be determined by comparison.

[0055] In one or more embodiments, the peptide library comprises one or more amino acid fragments of CAR as shown in SEQ ID NOs: 1-6.

[0056] In one or more embodiments, the peptide library comprises one or more amino acid fragments of CAR as shown in SEQ ID NOs: 7-18.

[0057] In one or more embodiments, the amino acid fragment of the CAR is obtained by biosynthesis, or can also be directly chemically synthesized in vitro.

[0058] In one or more embodiments, the assay is an ELISA assay or an ELISPOT assay, preferably an ELISPOT assay.

[0059] In one or more embodiments, the ELISPOT assay comprises the following steps:

[0060] (1) Dissolving the amino acid fragments of CAR in the diluted peptide library;

[0061] (2) Washing and blocking the ELISPOT pre-coated plate;

[0062] (3) Add PBMC cell suspension to the ELISPOT plate, dissolve the diluted peptide library and incubate;

[0063] (4) After washing, add secondary antibody for incubation, develop color, and analyze using an ELISPOT reader.

[0064] In one or more embodiments, after dilution in step (1), the concentration of the amino acid fragment of CAR is 0.65-6.5 nM, preferably 1-6 nM, 1.3-6 nM, or 2-3 nM.

[0065] The number of spots detected by ELISPOT represents the number of cells in PBMC that secrete the cytokine IFN-γ, that is, the number of anti-CAR CTLs. The level of CAR-specific cellular immune response is evaluated based on the changes in the number of anti-CAR CTL cells.

[0066] In one or more embodiments, the evaluating comprises determining whether the CAR-specific cellular immune response is positive and evaluating the level of the CAR-specific cellular immune response.

[0067] In one or more embodiments, the positive criterion is: the number of ELISPOT detection spots using the peptide library as antigen stimulation is 1.5 times or more than the number of ELISPOT detection spots without antigen stimulation, which is positive, and less than 1.5 times is negative. The criterion for evaluating the level of cellular immune response is: the more ELISPOT detection spots, the higher the level of cellular immune response. Preferably, the peptide library comprises a combination of partial fragments of multiple CARs, and the combination of partial fragments of multiple CARs covers the full-length amino acids of CAR or the full-length amino acids of antibody fragments in CAR.

[0068] In one or more embodiments, the evaluation includes evaluating the level of cellular immune response of each amino acid fragment in CAR, and determining the epitope that triggers the cellular immune response. In one or more embodiments, the peptide library contains only one amino acid fragment of CAR, and the peptide libraries containing amino acid fragments of different CARs are used as antigen stimulation, and the cellular immune response of each amino acid fragment in CAR is positive and the immunogenicity strength according to the above-mentioned judgment criteria. Preferably, the combination of the amino acid fragments of CAR contained in the different peptide libraries covers the full-length amino acids of CAR or the full-length amino acids of the antibody fragment in CAR. By combining the test results of different peptide libraries, it is possible to determine whether the CAR-specific cellular immune response is positive. In one or more embodiments, the test results of different peptide libraries are compared to determine the amino acid fragments with higher immunogenicity in CAR.

[0069] The method of evaluating a CAR-specific cellular immune response of the present invention is not intended for diagnosis or treatment. It predicts the therapeutic efficacy of CAR-containing immune cells by evaluating the number of anti-CAR CTLs produced by the CAR, but does not directly diagnose the therapeutic efficacy of the human body. A greater number of anti-CAR CTLs produced by the CAR indicates a poorer therapeutic efficacy of the CAR-containing immune cells. Based on the test results, the CAR can be modified to reduce the level of the cellular immune response, thereby improving the therapeutic efficacy of the immune cells.

[0070] The present invention also provides a kit for evaluating CAR-specific cellular immune responses, wherein the kit is evaluated according to the method described in any embodiment of the present invention.

[0071] The kit comprises: one or more peptide libraries composed of amino acid fragments of CAR, wherein the peptide library is the peptide library described in any embodiment of the present invention. The kit may comprise a peptide library comprising a combination of partial fragments of multiple CARs, and the combination of partial fragments of multiple CARs in the peptide library covers the full-length amino acids of CAR or the full-length amino acids of the antibody fragment in CAR. The kit comprises multiple peptide libraries, the peptide library contains only partial fragments of one type of CAR, and the combination of partial fragments of CAR in multiple peptide libraries covers the full-length amino acids of CAR or the full-length amino acids of the antibody fragment in CAR.

[0072] The kit may also include sample processing reagents, such as the diluent DMSO, for dissolving and diluting the peptide library. The kit may also include reagents for detecting IFN-γ secretion, such as reagents for ELISPOT assay, such as DPBS buffer, blocking solution, FBS, secondary antibodies, etc. The kit may also include reagents for PBMC culture or treatment, such as culture medium.

[0073] The kit can also be used in conjunction with a color development kit, such as a BCIP / NBT kit.

[0074] Example 1: Exploring the concentration of amino acid fragments of CAR

[0075] A method for evaluating CAR-specific cellular immune responses, the experimental steps are as follows:

[0076] 1. Peptide fragments and dilution:

[0077] (1) Peptide source: The peptide library covering the VHH in MSLN CAR was commissioned to be synthesized by GenScript. The peptide library contains 12 peptides, each with 15 amino acids, and there are 5 amino acid repeats between adjacent peptides;

[0078] The amino acid sequences of MSLN CAR, VHH, and peptide are shown in the table below;

[0079] (2) Dilution of single peptide: Dissolve each peptide in 1 mL of DMSO, vortex to mix, aliquot, and store in a -80°C refrigerator for later use.

[0080] (3) Dilution of mixed peptides: First dissolve peptide 1 in 1 mL of DMSO. After fully dissolving and mixing, dissolve peptide 2 in the same solution. Repeat this process until all 12 peptides are completely dissolved. Mix well and then divide the mixture into smaller pieces. Store in a -80°C refrigerator for later use.

[0081] 2. Prepare complete medium: Use a pipette to transfer 47 mL of AIM-V medium to a 50 mL sterile centrifuge tube. Add 2.5 mL of FBS, 500 μL of GlutMAX, and 2.5 μL of IL-2 (stock concentration 100,000 IU / mL). Mix by pipetting and preheat in a 37°C water bath for 10 min.

[0082] 3. Preparation of blocking solution: The blocking solution is DPBS containing 10% FBS.

[0083] 4. Wash the plate: Wash the ELISPOT pre-coated plate with 200uL / well sterile DPBS 4 times, patting dry each time.

[0084] 5. Blocking: Add 200 μL / well of blocking solution and block at room temperature for 30 minutes.

[0085] 6. PBMC Thawing: Remove frozen PBMCs and thaw them in a 37°C water bath. Shake the PBMCs constantly to accelerate thawing. Remove them from the water bath when only a small amount of ice remains.

[0086] 7. Wash PBMCs: Slowly transfer the revived PBMCs into a 15 mL centrifuge tube pre-added with 8 mL of complete culture medium. Centrifuge at 1500 rpm for 5 min. Discard the supernatant and resuspend in 1 mL of complete culture medium. Gently pipette to mix.

[0087] 8. Cell counting: Take out 20 μL of cell suspension and mix it with 20 μL of trypan blue dye. Take 20 μL of the mixture for counting and adjust the cell concentration to 2.5E6 / mL (i.e., 5E5 / 200 μL).

[0088] 9. Cell plating: After the ELISPOT plate is blocked, add 200 μL of cell suspension to each well. Do not add mixed peptides to wells 1-2, add peptides to a final concentration of 6.5 nM in well 3, add peptides to a final concentration of 2.6 nM in wells 4-5, add peptides to a final concentration of 1.3 nM in wells 6-7, and add peptides to a final concentration of 0.65 nM in well 8. Gently pipette to mix and place in a 37°C CO2 incubator for 24 hours. Avoid moving the plate during this period.

[0089] 10. Washing: After incubation, wash the plate 5 times with DPBS, patting dry each time.

[0090] 10. Secondary antibody incubation: Prepare DPBS containing 0.5% FBS and 0.5% secondary antibody (7-B6-ALP) according to the required amount, add 100uL to each well, and incubate at room temperature for 2h.

[0091] 11. Washing: After incubation, wash the plate 5 times with DPBS, patting dry each time.

[0092] 12. Color development: Filter the BCIP / NBT substrate with a filter membrane, add 100uL of the filtered substrate to each well, and develop color for 15 minutes. Observe every 5 minutes. If the color is too heavy, stop the color development early.

[0093] 13. Reading the plate: After color development is complete, gently rinse with tap water 10 times, pat dry, wipe the bottom of the wells with absorbent paper, and read the plate in an ELISPOT reader (reading the plate is more accurate after leaving it at room temperature for 24 hours).

[0094] The results are shown in Figure 1. No peptide was added as a negative background control. The results show that when the final concentration was greater than 1.3 nM, the average number of spots was more than twice the background level, and the number of spots gradually increased with increasing peptide concentration. Considering that DMSO has certain toxicity to cells and damages the PVDF membrane, the higher DMSO concentration (1.25%) at a final concentration of 6.5 nM has greater cytotoxicity and PVDF membrane damage. Therefore, a lower concentration of 2.6 nM (DMSO concentration of 0.5%) was selected as the optimal concentration for this experiment.

[0095] Example 2 Positive sample screening

[0096] The experimental steps were the same as those in Example 1, except that the final concentration of the added peptide was 2.6 nM. PBMCs were obtained from three patients, and the peptide library stimulation and unstimulation on day 0, as well as the peptide library stimulation and unstimulation on day D28 were tested.

[0097] The experimental results are shown in Figure 2.

[0098] Judging criteria:

[0099] Positive and negative judgment: if the number of spots in the stimulated group is more than 1.5 times that of the non-stimulated group, it is positive; if it is less than 1.5 times, it is negative; the number of spots in the stimulated group of patient 1 and patient 3 is 1.8 times and 1.6 times that of the non-stimulated group, respectively, and they are positive patients; patient 2 has no spots in both the stimulated and non-stimulated groups, showing a negative result.

[0100] Example 3 Identification of Immunogenic Peptides

[0101] The experimental steps were the same as those in Example 1, except that the final concentration of the added peptides was 2.6 nM, single peptides 1-12 were added, and the PBMCs were from patients 1 and 5, respectively.

[0102] The experimental results are shown in Figures 3-4, with Figure 3 showing the results for Patient 1 and Figure 4 showing the results for Patient 5. For Patient 1, peptides 1, 3, 4, and 5 showed strong immunogenicity, with the number of spots in the peptide-stimulated group changing by a factor of 1.5 relative to the number in the non-peptide group. In Patient 5, peptide 6 exhibited strong immunogenicity, with the number of spots in the peptide-stimulated group changing by a factor of 2 relative to the number in the non-peptide group.

Claims

1. A method for evaluating a CAR (chimeric antigen receptor)-specific cellular immune response, characterized in that: The method comprises the following steps: obtaining amino acid fragments of CAR and forming a peptide library, using the peptide library as an antigen to stimulate PBMC, detecting the secretion of IFN-γ in PBMC, and evaluating the level of CAR-specific cellular immune response.

2. The method according to claim 1, characterized in that The amino acid fragment of CAR is a complete fragment or a partial fragment of CAR; Preferably, the partial fragments of the CAR are segmented according to the structural domains of the CAR, and there is an overlap of 0-10 amino acids between adjacent fragments; or the partial fragments of the CAR are segmented according to a specific length of the CAR, the fragment length is 10-20 amino acids, and there is an overlap of 0-10 amino acids between adjacent fragments.

3. The method according to claim 1 or 2, characterized in that The peptide library comprises a combination of partial fragments of multiple CARs; preferably, the combination of partial fragments of multiple CARs in the peptide library covers the full-length amino acids of CAR.

4. The method according to any one of claims 1 to 3, characterized in that: The CAR contains the sequence shown in SEQ ID NO: 3; preferably, the peptide library contains one or more amino acid fragments of CAR as shown in SEQ ID NO: 1-18.

5. The method according to any one of claims 1 to 4, characterized in that: The detection is ELISA detection or ELISPOT detection, preferably ELISPOT detection.

6. The method according to claim 5, characterized in that The ELISPOT assay comprises the following steps: (1) dissolving the amino acid fragments of CAR in the diluted peptide library; (2) washing and blocking the ELISPOT pre-coated plate; (3) Add PBMC cell suspension to the ELISPOT plate, dissolve the diluted peptide library and incubate; (4) After washing, add secondary antibody for incubation, develop color, and use ELISPOT reader for analysis.

7. The method according to claim 6, characterized in that After dilution in step (1), the concentration of the amino acid fragment of CAR is 0.65-6.5 nM, preferably 1-6 nM, 1.3-6 nM, or 2-3 nM.

8. The method according to claims 1 to 7, characterized in that The evaluation comprises determining whether the CAR-specific cellular immune response is positive and evaluating the level of the CAR-specific cellular immune response, and / or evaluating the level of the cellular immune response of each amino acid fragment in the CAR, and determining the epitope that triggers the cellular immune response.

9. A kit for evaluating CAR (chimeric antigen receptor)-specific cellular immune response, characterized in that: The kit comprises one or more peptide libraries composed of amino acid fragments of CAR, and optionally, the kit further comprises one or more of (1)-(3): (1) Peptide library dilution reagent; (2) Reagents for detecting IFN-γ secretion; (3) PBMC culture reagents.

10. The kit according to claim 9, characterized in that The peptide library comprises one or more amino acid fragments of CAR as shown in SEQ ID NOs: 1-18.

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