A method, kit and use for assessing the immunogenicity of a CAR molecule

Abstract

This invention belongs to the field of immunotherapy and relates to a method for evaluating the immunogenicity of CAR molecules. The method utilizes dendritic cells (DCs) loaded with CAR molecule peptides, which are then induced to mature and co-cultured with T lymphocytes to obtain CAR-specific cytotoxic T cells. By restimulating the peptides, the IFN-γ secreted by the specific cytotoxic T cells is detected to determine the immunogenicity of the CAR molecule. Compared with existing methods, the detection method and platform described in this invention have advantages such as shorter detection time, higher convenience, less experimental cell consumption, and lower detection cost, and can be used for in vitro evaluation of the immunogenicity of CAR molecules.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy and relates to a method, kit, and application for evaluating the immunogenicity of CAR molecules. Background Technology

[0002] Enzyme-linked immunospot assay (ELISPOT) is a cellular immunology technique that combines cell culture and ELISA technologies to detect antibody-secreting or cytokine-secreting cells in situ. It not only measures the amount of cytokines but also counts the frequency of cells secreting these cytokines. Furthermore, the capture antibodies used in the assay do not affect the secretion of cytokines by activated cells, making it most commonly used for the cellular immunoassay of vaccines.

[0003] In numerous studies, Elispot technology has been used to detect the number of antigen-specific cytotoxic T cells (CTLs) in peripheral blood lymphocytes of immunized animals. More recently, Elispot technology has also been used to analyze and evaluate pathogen-associated specific T lymphocytes from PBMCs (peripheral blood mononuclear cells) of patients with infectious diseases, and in vaccine trials that induce tumor-specific T cells in cancer patients.

[0004] Currently, there is no publicly available specific method for assessing the immunogenicity of CAR molecular cellular immunity using Elispot technology. Summary of the Invention

[0005] The purpose of this invention is to provide a method for evaluating the immunogenicity of chimeric antigen receptor (CAR) molecules, thereby filling the current gap in the detection of cellular immunogenicity.

[0006] The method includes the following steps: obtaining amino acid fragments of CAR and assembling a peptide library; loading the CAR peptide library onto DC cells; inducing maturation and co-culturing with T lymphocytes to obtain CAR molecules that specifically kill T cells; and detecting the IFN-γ secreted by the specific killing T cells through peptide library restimulation to assess the immunogenicity of CAR.

[0007] The immunogenicity is generated by the cellular immune pathway.

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

[0009] In one or more embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:1 or 2.

[0010] In one or more embodiments, the amino acid fragment of the CAR is an amino acid fragment of the antigen-binding domain.

[0011] In one or more embodiments, the CAR fragments are segmented according to the CAR's structural domains, such as 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 their structure; for example, when it is an scFv, it can be divided into light chain variable domain (VL) fragments and heavy chain variable domain (VH) fragments; when it is a conventional antibody, it can be divided into Fab fragments, Fc fragments, etc. Preferably, adjacent fragments have 0-11 amino acid overlaps, more preferably 5-11 amino acid overlaps.

[0012] In one or more embodiments, the CAR fragments are CAR segments of a specific length, with a segment length of 10-20 amino acids, and adjacent segments have amino acid overlap, for example, 0-11 amino acid overlap, more preferably 5-11 amino acid overlap.

[0013] In one or more embodiments, the peptide library contains 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 the CAR or the full-length amino acids of the antibody fragment in the CAR.

[0014] In one or more embodiments, the peptide library contains only one amino acid fragment of a CAR.

[0015] In one or more embodiments, the peptide library comprises one or more amino acid fragments of CARs as shown in SEQ ID NO:3-19.

[0016] In another or other embodiments, the peptide library comprises one or more amino acid fragments of CARs as shown in SEQ ID NO:20-53.

[0017] In one or more embodiments, the amino acid fragments of the CAR are obtained by biosynthesis, or may be directly synthesized in vitro by chemical synthesis.

[0018] In one or more embodiments, the detection is an ELISA detection or an ELISPOT detection, preferably an ELISPOT detection.

[0019] In one or more embodiments, the ELISPOT detection includes the following steps:

[0020] 1) Preparation of T lymphocyte suspension after co-culturing with DC cells loaded with CAR peptide library;

[0021] 2) Dissolve and dilute the amino acid fragments of CAR in the peptide library;

[0022] 3) Clean and seal the ELISPOT pre-coated board;

[0023] 4) Add T lymphocyte suspension and dissolved and diluted peptide library to ELISPOT plate and incubate;

[0024] 5) After washing the plate, add the secondary antibody for incubation, develop color, and use the ELISPOT reader to read and analyze the plate.

[0025] In one or more embodiments, after mixing in step 4), the cell number density of the T lymphocyte suspension is 1 × 10⁻⁶. 4 -1×10 5 cells / well, preferably 3×10 4 -5×10 4 More preferably, 3 × 10 cells / well 4 cells / pores.

[0026] In one or more embodiments, after mixing in step 4), the final concentration of amino acid fragments in CAR is 1-10 μg / strip / ml, preferably 1-5 μg / strip / ml, and more preferably 1 μg / strip / ml.

[0027] In one or more embodiments, the ratio of T lymphocyte count to CAR amino acid fragment concentration is 3-5 × 10⁻⁶. 4 Cells: 1-5ug / strip / ml, preferably 3×10⁻⁵ 4 cells: 1-3ug / strip / ml, more preferably 3×10 4 cells: 1ug / cell / ml.

[0028] The present invention provides a method for assessing CAR-specific cellular immune responses. This method is not for diagnostic or therapeutic purposes. It assesses the immunogenicity of CAR-containing immune cells by evaluating the number of CAR molecule-specific cytotoxic T cells induced by CAR. It does not directly diagnose the immunogenicity of CAR-containing immune cells in the human body.

[0029] The present invention also provides a kit for assessing the immunogenicity level of chimeric antigen receptor (CAR), wherein the kit is used for assessment according to any embodiment of the present invention. The kit comprises one or more peptide libraries consisting 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 peptide library comprises one or more amino acid fragments of CARs as shown in SEQ ID NO:3-19.

[0031] In another or other embodiments, the peptide library comprises one or more amino acid fragments of CARs as shown in SEQ ID NO:20-53.

[0032] In one or more embodiments, the kit may further comprise sample processing reagents, such as DMSO diluent, for dissolving and diluting the peptide library. The kit may also comprise reagents for detecting IFN-γ secretion, such as ELISPOT assay reagents, including DPBS buffer, blocking solution, FBS, secondary antibody, etc. The kit may also use reagents for T cell culture or treatment, such as culture medium.

[0033] The kit can also be used in conjunction with a colorimetric reagent kit, such as the BCIP / NBT kit.

[0034] The method for assessing the immunogenicity of chimeric antigen receptor (CAR) molecules or the kit for assessing the immunogenicity level of CAR molecules described in this invention can be used in the preparation of any of the following products:

[0035] 1) Products used to compare the immunogenicity of CAR molecules selected for CAR-T therapy;

[0036] 2) Products used to screen CAR molecules with lower immunogenicity.

[0037] Compared with the prior art, the present invention has at least the following beneficial effects:

[0038] 1. Establish a systematic and short-cycle experimental procedure for determining the strength of CAR molecular cellular immunogenicity.

[0039] 2. The immunogenicity of CAR molecules can be compared by comparing the test results, thereby enabling screening of CAR molecules;

[0040] 3. It can verify the immunogenicity strength assessment results based on bioinformatics, and jointly establish the correlation between CAR molecule immunogenicity assessment and monitoring through "sequence-cell reactivity-clinical test results" to optimize the selection of CAR molecules. Attached Figure Description

[0041] Figure 1 This is a diagram of the experimental results from the ELISPOT enzyme-linked immunospot assay platform. Phytohemagglutinin (PHA) was used as a positive control. Detailed Implementation

[0042] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0044] The term "immunogenicity" refers to the property of an antigen to elicit an immune response, that is, the ability of an antigen to stimulate specific immune cells, thereby activating, proliferating, and differentiating the immune cells, ultimately producing immune effector substances such as antibodies and sensitized lymphocytes.

[0045] The term "cellular immunity" refers to an immune mechanism that uses a specific immune response mediated by T lymphocytes to produce immune effector cells and cytokines in order to eliminate infectious pathogens upon antigen recognition and activation.

[0046] The term "peptide library" refers to a mixture of peptide fragments of a certain length. In certain embodiments of the present invention, the peptide library may also contain only one type of peptide fragment of a certain length.

[0047] The purpose of this invention is to provide a method for evaluating the immunogenicity of chimeric antigen receptor (CAR) molecules, thereby filling the current gap in the detection of cellular immunogenicity.

[0048] The first aspect of the present invention provides a method for evaluating the immunogenicity of chimeric antigen receptor (CAR) molecules, comprising the following steps: obtaining amino acid fragments of CAR and assembling a peptide library; loading the CAR peptide library onto dendritic cells (DCs); inducing maturation and co-culturing the DCs with T lymphocytes to obtain CAR molecules that specifically kill T cells; and detecting the IFN-γ secreted by the specific killing T cells through peptide library restimulation to evaluate the immunogenicity of CAR.

[0049] The immunogenicity is generated by the cellular immune pathway.

[0050] In one or more embodiments, the amino acid fragment of the CAR is a complete fragment or a partial fragment of the CAR.

[0051] In one or more embodiments, the CAR can be any publicly disclosed CAR, such as the CAR in CN202111681582.0. Herein, the CAR is a chimeric antigen receptor whose structure includes 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 includes an intracellular co-stimulatory domain and an intracellular signaling domain.

[0052] In one or more embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:1 or 2.

[0053] Considering that the signal peptide, transmembrane region, and intracellular region in the CAR can be selected as human sequences with low immunogenicity, or only non-human amino acid sequences can be evaluated, in one or more embodiments, the amino acid fragment of the CAR is an amino acid fragment of the antigen-binding domain.

[0054] In one or more embodiments, the CAR fragments are segmented according to the functional structure of the CAR, such as including signal peptide fragments, antibody fragments, hinge region fragments, transmembrane region fragments, and intracellular region fragments.

[0055] "Antibody fragment" refers to a portion of a complete antibody, preferably the antigen-binding region and / or variable region of the complete antibody. The antibody fragment is preferably an antigen-binding fragment of the antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; and VHH. Antibody fragments can be further segmented; for example, scFV fragments can be divided into light chain variable domain (VL) fragments and heavy chain variable domain (VH) fragments, while conventional antibodies can be divided into Fab fragments, Fc fragments, etc. Preferably, adjacent fragments have 0-11 amino acid overlaps, more preferably 5-11 amino acid overlaps.

[0056] In one or more embodiments, the CAR fragments are segmented into segments of a specific length, with each segment being 10-20 amino acids long. Adjacent segments overlap with each other, for example, with 0-11 amino acid overlaps, more preferably 5-11 amino acid overlaps. For example, if a segment is 12 amino acids long and there is a 5-amino acid overlap between adjacent segments, segment 1 consists of amino acids at positions 1-12, segment 2 consists of amino acids at positions 8-19, and so on. Segmenting into segments of a specific length allows for better identification of epitopes that trigger cellular immune responses; segments that are too short may disrupt the smallest epitope unit, while segments that are too long may fold. Similarly, the overlap of amino acids between adjacent segments also avoids the destruction of the smallest epitope unit due to artificial segmentation.

[0057] In one or more embodiments, the peptide library contains 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 the CAR or the full-length amino acids of the antibody fragment in the CAR, which can be used to assess whether the CAR will induce a cellular immune response.

[0058] In one or more embodiments, the peptide library contains only one amino acid fragment of a CAR. This is used to assess whether the amino acid fragment elicits a cellular immune response, and when using peptide libraries containing different amino acid fragments for detection, the amino acid fragment with the highest cellular immunogenicity can be identified by comparison.

[0059] In one or more embodiments, the peptide library comprises one or more amino acid fragments of CARs as shown in SEQ ID NO:3-19.

[0060] In one or more embodiments, the peptide library comprises amino acid fragments of one or more CARs as shown in SEQ ID NO:20-53.

[0061] In one or more embodiments, the amino acid fragments of the CAR are obtained by biosynthesis, or may be directly synthesized in vitro by chemical synthesis.

[0062] In one or more embodiments, the detection is an ELISA detection or an ELISPOT detection, preferably an ELISPOT detection.

[0063] In one or more embodiments, the ELISPOT detection includes the following steps:

[0064] 1) Preparation of T lymphocyte suspension after co-culturing with DC cells loaded with CAR peptide library;

[0065] 2) Dissolve and dilute the amino acid fragments of CAR in the peptide library;

[0066] 3) Clean and seal the ELISPOT pre-coated board;

[0067] 4) Add T lymphocyte suspension and dissolved and diluted peptide library to ELISPOT plate and incubate;

[0068] 5) After washing the plate, add the secondary antibody for incubation, develop color, and use the ELISPOT reader to read and analyze the plate.

[0069] In one or more embodiments, after mixing in step 4), the cell number density of the T lymphocyte suspension is 1 × 10⁻⁶. 4 -1×10 5 cells / well, e.g., 1×10 4 cells / well, 2×10 4 cells / well, 3×10 4 cells / well, 4×10 4 cells / well, 5×10 4 cells / well, 6×10 4 cells / well, 7×10 4cells / well, 8×10 4 cells / well, 9×10 4 cells / well, 1×10 5 cells / pores.

[0070] In one or more embodiments, the cell number density of the T lymphocyte suspension is preferably 3 × 10⁻⁶. 4 -5×10 4 cells / pores.

[0071] In one or more specific embodiments, the cell number density of the T lymphocyte suspension is preferably 3 × 10⁻⁶. 4 cells / pores.

[0072] In one or more embodiments, after mixing in step 4), the final concentration of amino acid fragments of CAR is 1-10 ug / segment / ml, for example 1 ug / segment / ml, 2 ug / segment / ml, 3 ug / segment / ml, 4 ug / segment / ml, 5 ug / segment / ml, 6 ug / segment / ml, 7 ug / segment / ml, 8 ug / segment / ml, 9 ug / segment / ml, 10 ug / segment / ml.

[0073] In one or more embodiments, the final concentration of the amino acid fragment of CAR is preferably 1-5 μg / segment / ml.

[0074] In one or more specific embodiments, the final concentration of the amino acid fragment in the CAR is preferably 1 μg / fragment / ml.

[0075] In one or more embodiments, the ratio of T lymphocyte count to CAR amino acid fragment concentration is 3-5 × 10⁻⁶. 4 Cells: 1-5ug / strip / ml, e.g., 3×10 4 cells / well: 1ug / strip / ml, 3×10 4 cells / well: 2ug / strip / ml, 3×10 4 Cells / well: 3ug / strip / ml, 3×10 4 Cells / well: 4ug / strip / ml, 3×10 4 Cells / well: 5ug / strip / ml, 4×10 4 cells / well: 1ug / strip / ml, 4×10 4 Cells / well: 3ug / strip / ml, 4×10 4 Cells / well: 5ug / strip / ml, 5×10 4 cells / well: 1ug / strip / ml, 5×10 4 Cells / well: 3ug / strip / ml, 5×104 cells / well: 5ug / strip / ml.

[0076] In one or more embodiments, the ratio of T lymphocyte count to CAR amino acid fragment concentration is preferably 3 × 10⁻⁶. 4 cells: 1-3ug / strip / ml.

[0077] In one or more specific embodiments, the ratio of T lymphocyte count to CAR amino acid fragment concentration is preferably 3 × 10⁻⁶. 4 cells: 1ug / cell / ml.

[0078] The present invention provides a method for assessing CAR-specific cellular immune responses. This method is not for diagnostic or therapeutic purposes. It assesses the immunogenicity of CAR-containing immune cells by evaluating the number of CAR-generated CAR-specific cytotoxic T cells, rather than directly diagnosing the immunogenicity of CAR-containing immune cells in the human body.

[0079] The present invention also provides a kit for assessing the level of chimeric antigen receptor (CAR) immunogenicity, which is evaluated by the method described in any embodiment of this invention.

[0080] The kit comprises one or more peptide libraries consisting of amino acid fragments of a CAR, wherein the peptide library is the peptide library described in any embodiment of the present invention. The kit may contain one peptide library containing 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 the CAR or the full-length amino acids of the antibody fragment in the CAR. Alternatively, the kit may contain multiple peptide libraries, each containing only a partial fragment of a CAR, and the combination of partial fragments of CARs from multiple peptide libraries covers the full-length amino acids of the CAR or the full-length amino acids of the antibody fragment in the CAR.

[0081] In one or more embodiments, the peptide library comprises one or more amino acid fragments of CARs as shown in SEQ ID NO:3-19.

[0082] In another or other embodiments, the peptide library comprises one or more amino acid fragments of CARs as shown in SEQ ID NO:20-53.

[0083] The kit may also include sample processing reagents, such as DMSO diluent, for dissolving and diluting the peptide library. The kit also includes reagents for detecting IFN-γ secretion, such as ELISPOT assay reagents, including DPBS buffer, blocking solution, FBS, secondary antibody, etc. The kit may also include reagents for T cell culture or treatment, such as culture medium.

[0084] The kit can also be used in conjunction with a colorimetric reagent kit, such as the BCIP / NBT kit.

[0085] The method for assessing the immunogenicity of chimeric antigen receptor (CAR) molecules or the kit for assessing the immunogenicity level of CAR molecules described in this invention can be used in the preparation of any of the following products:

[0086] 1) Products used to compare the immunogenicity of CAR molecules selected for CAR-T therapy;

[0087] 2) Products used to screen CAR molecules with lower immunogenicity.

[0088] I. Preparation of Test Samples

[0089] The PBMC samples from healthy individuals were obtained from apheresis blood purchased from Maishun Company. The PBMCs were separated using the Ficoll method. The separated PBMCs can be used directly in experiments or frozen and then thawed for use.

[0090] The peptide library consists of peptide segments of 12 amino acids each, covering the entire amino acid sequence of the CAR molecule. Adjacent peptide segments overlap by 5 amino acids. The peptide library was synthesized by a third-party company. The amino acid sequence of the CAR molecule is shown in Table 1. The synthesized peptide library powder was stored at -80℃ and dissolved in DMSO to a working solution concentration of 1 μg / strip / µl before use. The working solution can be stored briefly at -80℃.

[0091] FMC63 is a murine monoclonal antibody targeting human CD19, while 1444 is an alpaca antibody targeting human MSLN. Alpaca antibodies have higher homology with human antibodies and theoretically possess lower immunogenicity than murine antibodies. [1][2] .

[0092] Table 1

[0093]

[0094]

[0095]

[0096] Table 2

[0097]

[0098]

[0099] Example 1: Obtaining Specific T Cells

[0100] 1.1 Obtaining and culturing T cells:

[0101] (1) Day 1: 7.5 × 107 The isolated human peripheral blood mononuclear cells (PBMCs) were resuspended in AIM V medium free of any cytokines and stimulating factors, and the density was adjusted to 5 × 10⁶ cells / mL. 6 Cells / ml were seeded in three T25 culture flasks and incubated overnight at 37°C for cell adhesion.

[0102] (2) Day 2: Remove the T25 culture flask from the laminar flow hood, gently shake the flask, and then use a pipette to transfer the upper cell suspension into a 50ml centrifuge tube. Add preheated DPBS to the T25 culture flask, gently shake to further wash, and then transfer the DPBS into the 50ml centrifuge tube. Repeat this process 2-3 times. Observe under a microscope to ensure that lymphocytes have been largely cleared. Then, remove the DPBS, add 5ml of AIM V medium, 1000 IU / ml GM-CSF, and 500 IU / ml IL-4, mix thoroughly, and incubate at 37℃ for 48 hours. Count the cells collected in the 50ml centrifuge tube, centrifuge, discard the supernatant, and resuspend in 1ml of cryopreservation medium for cryopreservation. This portion of cells is designated as T supernatant cells and will be used for subsequent incubation with DC cells.

[0103] (3) Day 4: Add peptide library working solution at a concentration of 1 μg / strip / ml of culture medium, i.e., add 5 μl of the relevant peptide library solution to the T25 culture flask, and add the same volume of DMSO to the control group. Shake the flask to mix well, then transfer it to a 37℃ incubator and let it stand for 24 h to load the peptide library onto the DC cells.

[0104] (4) Day 5: After 24 hours, TNF-α (10 ng / ml), IFN-γ (1000 U / ml), PGE2 (250 ng / ml), R848 (1 μg / ml) and poly(I:C) at a concentration of 20 ng / ml were added to induce immature DCs to mature for 24 hours.

[0105] (5) Day 6: Collect peptide-loaded dendritic cells (DCs) from T25 culture flasks and co-culture them with revived T supernatant cells (cells frozen in step 2) at a ratio of 1:10 in AIM V + 5% FBS medium for 4 days to induce the cells to become specific T cells. The cell density was 1.5 × 10⁻⁶ cells / year. 6 Cells / ml, seeded in 12-well plates, 1 ml per well.

[0106] (6) Day 10: Repeatedly pipet and transfer the mixed incubated cells in the 12-well plates, and re-seed them in new 12-well plates. Transfer to an incubator for 4 hours to allow for re-attachment, the purpose being to isolate adherent cells such as dendritic cells (DCs). After attachment, collect the upper layer cells from different groups in the 12-well plates, and gently wash once with pre-warmed DPBS. Then, centrifuge the collected suspensions of the three groups of specific cytotoxic T cells, resuspend them in co-culture medium for counting, and adjust the cell density to 3 × 10⁻⁶ cells / well. 5 cells / ml.

[0107] 1.2 Enzyme-linked immunospot assay (ELISPOT) and colorimetric reaction

[0108] (1) Take out the enzyme-linked immunospot assay strip from the kit, place the strip on the plate provided with the kit, let it stand to warm up, and then wash it with 200ul / well DPBS. Repeat 4 times, and pat dry each time.

[0109] (2) Add blocking solution (AIM V + 10% FBS) at 200 μL / well and block at room temperature for 30 min.

[0110] (3) After ELISPOT plate blocking, remove the blocking solution and pat dry. Add different groups of cell suspension to the wells according to the experimental plate arrangement (Table 3), adding 100 μL of each well and 100 μL of co-culture medium containing 1 μL of 0.2 μg / strip / μL peptide library solution, finally reaching 3 × 10⁻⁶ cells / well. 4 The cell / 200µl culture system corresponded to a peptide library concentration of 1µg / cell / ml. The positive control was a cell suspension stimulated with PHA (500X stock solution, final concentration 1X working solution). After gentle aspiration and mixing, the cells were incubated in a 37℃ CO2 incubator for 20 hours, avoiding any movement of the plate during this time. Elispot plate placement is shown in Table 3.

[0111] (4) After incubation, wash the plate 5 times with DPBS, pat dry each time, and 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 for secondary antibody incubation.

[0112] (5) After incubation, wash the plate 5 times with DPBS and pat dry each time. Filter the BCIP / NBT substrate with a filter membrane, add 100 μL of the filtered substrate to each well, and develop the color for 5-10 min, observing every 2 min. If the color development is too strong, the color development can be stopped early.

[0113] (6) After the color development is complete, rinse gently with tap water 10 times, pat dry, wipe the bottom of the hole with absorbent paper, and read the plate under ELISPOTreader.

[0114] 1.3 Experimental Results:

[0115] Each group consisted of two replicates. DC cells loaded with different peptide libraries were co-cultured with T cells (supernatant) for 4 days, and then stimulated with DMSO, 1444 peptide library, or FMC63 peptide library, respectively. The obtained Elispot spot data are shown in Table 4. Figure 1 The difference between the number of spots obtained from restimulation of the 1444 peptide library and the number of spots obtained from the addition of DMSO represents the number of specific T cells targeting the 1444 peptide library, reflecting the cellular immunogenicity of the 1444 molecule. The experimental results suggest that the cellular immunogenicity of the 1444 molecule is weaker than that of FMC63, which is consistent with the higher homology between the 1444 molecule and human antibodies. This demonstrates that the method described in the invention for assessing the immunogenicity of CAR molecules is accurate and reliable.

[0116] Table 3

[0117] Group Elispot fabric board A Co-culture T(DC-DMSO) + DMSO B Co-culture T(DC-DMSO) + 1444 C Co-culture T(DC-DMSO) + FMC63 D Co-culture T(DC-1444) + DMSO E Co-culture T(DC-1444)+1444 F Co-culture T(DC-FMC63) + DMSO G Co-culture T(DC-FMC63)+FMC63 H Co-culture T(DC-DMSO) + PHA

[0118] Note: The 1444 peptide library is loaded during the DC stage. After maturation, it is co-cultured with T and denoted as co-cultured T(DC-1444). When it is stimulated again, the 1444 peptide library is used and denoted as co-cultured T(DC-1444)+1444, and so on.

[0119] Table 4

[0120]

[0121] References:

[0122] [1]EMA.Assessment report,Kymriah International non-proprietary name:tisagenlecleucel(EMA / 485563 / 2018)[R].Committee for Medicinal Products for Human Use(CHMP).Amsterdam:European Medicines Agency,2018;Procedure No.EMEA / H / C / 004090 / 0000.

[0123] [2] Bernd Potthoff, Fraser McBlane, Sebastian Spindeldreher, et al. Acell-based immunogenicity assay to detect antibodies against chimeric antigen receptor expressed by tisagenlecleucel[J]. J Immunol Methods. 2020;476:112692.

[0124] The sequences in this article

[0125]

[0126]

Claims

1. A method of assessing the immunogenicity of a chimeric antigen receptor (CAR) molecule, characterized in that, It includes the following steps: Obtain amino acid fragments of CAR and assemble them into a peptide library. By loading CAR peptide libraries onto dendritic cells (DCs) and co-culturing them with T lymphocytes, CAR molecules can be used to specifically kill T cells. The immunogenicity of CAR was assessed by detecting the cytokines secreted by specific cytotoxic T cells through peptide library restimulation. Preferably, the cytokine includes IFN-γ.

2. The method of claim 1, wherein, The amino acid fragment of the CAR is a complete fragment or a partial fragment of the CAR; Preferably, the CAR fragments are segmented according to the CAR domains, with 0-11 amino acid overlaps between adjacent fragments; or the CAR fragments are segmented according to a specific length, with a fragment length of 10-20 amino acids and 0-11 amino acid overlaps between adjacent fragments. More preferably, there is an overlap of 5-11 amino acids between adjacent segments.

3. The method of claim 1 or 2, wherein, The peptide library contains 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 the CAR.

4. The method according to any one of claims 1 to 3, characterized in that, The CAR includes the amino acid sequence shown in SEQ ID NO:1 or 2; preferably, the peptide library contains amino acid fragments of one or more CARs as shown in SEQ ID NO:3-19 or the peptide library contains amino acid fragments of one or more CARs as shown in SEQ ID NO:20-53.

5. The method according to any one of claims 1 to 4, wherein The detection is either ELISA or ELISPOT, with ELISPOT being preferred.

6. The method as described in claim 5, characterized in that, The ELISPOT detection includes the following steps: 1) Preparation of T lymphocyte suspension after co-culturing with DC cells loaded with CAR peptide library; 2) Dissolve and dilute the amino acid fragments of CAR in the peptide library; 3) Clean and seal the ELISPOT pre-coated board; 4) Add T lymphocyte suspension and dissolved and diluted peptide library to ELISPOT plate and incubate; 5) After washing the plate, add the secondary antibody for incubation, develop color, and use the ELISPOT reader to read and analyze the plate.

7. The method as described in claim 6, characterized in that, Step 4) The mixture has one or more of the following characteristics: (1) After mixing, the cell number density of the T lymphocyte suspension is 1 x 10 4 -1 x 10 5 cells / well, preferably 3 x 10 4 -5 x 10 4 cells / well, more preferably 3 x 10 4 cells / well; (2) After mixing, the final concentration of amino acid fragments in CAR is 1-10ug / strip / ml, preferably 1-5ug / strip / ml, and more preferably 1ug / strip / ml; (3) After mixing, the ratio of the number of T lymphocytes to the concentration of the amino acid fragment of the CAR is 3-5 x 10 4 cells: 1-5 ug / streak / ml, preferably 3 x 10 4 cells: 1-3 ug / streak / ml, more preferably 3 x 10 4 cells: 1 ug / streak / ml.

8. A kit for assessing the immunogenicity level of chimeric antigen receptor (CAR) molecules, characterized in that, The kit comprises one or more peptide libraries consisting of amino acid fragments of CAR, and optionally, the kit further comprises one or more of (1)-(3): (1) Peptide library dilution reagent; (2) A reagent for detecting cytokine secretion, preferably, the cytokines include IFN-γ; (3) Reagents for T cell culture or treatment.

9. The reagent kit as described in claim 8, characterized in that, The peptide library contains amino acid fragments of one or more CARs as shown in SEQ ID NO:3-19 or the peptide library contains amino acid fragments of one or more CARs as shown in SEQ ID NO:20-53.

10. The use of the method according to any one of claims 1-7 or the kit according to any one of claims 8-9 in the preparation of any of the following products: 1) Products used to compare the immunogenicity of CAR molecules selected for CAR-T therapy; 2) Products used to screen CAR molecules with lower immunogenicity.

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