Human peripheral blood cell immunotyping antibody combination and method based on flow cytometry

Abstract

The invention relates to a human peripheral blood cell immunotyping antibody combination and method based on flow cytometry. The antibody combination comprises an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-EGFR antibody, an anti-CD25 antibody, an anti-CD127 antibody, an anti-CD45RA antibody, an anti-CCR7 antibody, an anti-CD27 antibody, an anti-PD-1 antibody, an antiTIGIT antibody and an anti-41BB antibody. The method comprises the following steps: mixing the antibody combination with a sample to be detected, incubating, performing flow cytometry detection to obtain detection data, and further judging the proportion and / or the number of target cell populations. According to the present invention, the optimal cell surface marker antibody combination is designed and obtained, the CAR-T cells in the sample are subjected to immune typing by using the 12-color antibody combination through the flow cytometry, the detection result is rapidly obtained, the CAR-T cell proportion of each phenotype is efficiently identified, and the method has high precision and high stability.

Description

Technical Field

[0001] The present invention relates to the analysis and detection of biological samples, and particularly to an antibody combination and method for immunophenotyping human peripheral blood cells based on flow cytometry. Background Art

[0002] CAR-T therapy is Chimeric Antigen Receptor T-Cell Immunotherapy. It is a novel and precise targeted therapy for treating tumors. In recent years, through optimization and improvement, it has achieved good results in clinical tumor treatment. It is a very promising new tumor immunotherapy method that can be precise, fast, efficient, and may even cure cancer.

[0003] Studying how CAR-T cell preparations evolve in patients after infusion, that is, studying their cell kinetic characteristics, helps to understand the in vivo metabolism characteristics of CAR-T, explore the internal mechanisms affecting the effectiveness and safety of CAR-T, and guide the development of more efficient and safe CAR-T drugs.

[0004] Currently, there is no antibody combination and method for immunophenotyping human peripheral blood cells based on flow cytometry as in the present application. Summary of the Invention

[0005] The present invention first provides a method for immunophenotyping CAR-T cells based on flow cytometry. The method includes the steps of: mixing a fluorescently labeled antibody with a sample to be detected, incubating, detecting by flow cytometry to obtain detection data, and then determining the proportion and / or number of the target cell population; wherein: the fluorescently labeled antibody includes: anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-EGFR antibody, anti-CD25 antibody, anti-CD127 antibody, anti-CD45RA antibody, anti-CCR7 antibody, anti-CD27 antibody, anti-PD-1 antibody, anti-TIGIT antibody, anti-41BB antibody.

[0006] In some embodiments, the target cell population includes: CD3+CAR+ / EGFR+CD25+CD127-, CD3+CAR+ / EGFR+PD-1+, CD3+CAR+ / EGFR+TIGIT+, CD3+CAR+ / EGFR+CCR7+CD45RA-, CD3+CAR+ / EGFR+CD8+CCR7+CD27+.

[0007] In some embodiments, the CAR-T cells express a membrane-bound EGFR protein. In certain embodiments, the T cells are introduced with an expression cassette of the EGFR protein to express an exogenous EGFR protein. Preferably, the expression cassette of the EGFR protein is linked to the expression cassette of the CAR by a 2A peptide (such as P2A, T2A, E2A, F2A) and transferred into the T cells by gene transduction. Self-cleavage occurs during expression. Therefore, detecting the EGFR expression can indirectly indicate the expression of the CAR.

[0008] In some embodiments, the anti-CD3 antibody is UCHT1, the anti-CD4 antibody is RPA-T4, the anti-CD8 antibody is SK1, the anti-EGFR antibody is AY13, the anti-CD25 antibody is M-A251, the anti-CD127 antibody is HIL-7R-M21, the anti-CD45RA antibody is HI100, the anti-CCR7 antibody is 3d12, the anti-CD27 antibody is M-T271, the anti-PD-1 antibody is MIH4, the anti-TIGIT antibody is 741182, and / or the anti-41BB antibody is 4b4-1.

[0009] In some embodiments, the fluorescent label of the anti-CD3 antibody is FITC, the fluorescent label of the anti-CD4 antibody is PerCP-Cy5.5, the fluorescent label of the anti-CD8 antibody is APC-Cy7, the fluorescent label of the anti-EGFR antibody is APC, the fluorescent label of the anti-CD25 antibody is BV421, the fluorescent label of the anti-CD127 antibody is PE, the fluorescent label of the anti-CD45RA antibody is BV510, the fluorescent label of the anti-CCR7 antibody is BV711, the fluorescent label of the anti-CD27 antibody is PE-CY7, the fluorescent label of the anti-PD-1 antibody is PE-CF594, the fluorescent label of the anti-TIGIT antibody is BV650, and / or the fluorescent label of the anti-41BB antibody is BV750.

[0010] In some embodiments, the sample to be detected is PBMC or lysed whole blood from an individual who has received CAR-T cell therapy. In certain embodiments, the PBMC is sorted from the peripheral blood of the individual. In certain examples, the method for obtaining the PBMC is as follows: adding anticoagulant (EDTA-K2) to the whole peripheral blood of the individual, mixing it with cell culture medium, adding a density separation solution, and sorting to obtain PBMC cells. In certain embodiments, the PBMC cells are cryopreserved or freshly prepared. In certain embodiments, the whole blood is cryopreserved or freshly prepared. In certain embodiments, the individual is a mammal, such as a human, rat, mouse, pig, rabbit, dog, monkey, or chimpanzee.

[0011] In some embodiments, the CAR-T binds to an antigen selected from the following: alpha-folate receptor, 5T4, alpha v beta 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD318, CD171, CEA, CSPG4, EGFR, the EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FR alpha, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11R alpha, IL-13R alpha 2, lambda, Lewis-Y, kappa, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM, Trop2, and VEGFR2.

[0012] In some embodiments, the fluorescently labeled antibody is first prepared into a mixture and then mixed with the sample; in other embodiments, the fluorescently labeled antibody is not premixed, but is directly added to the sample separately for mixing.

[0013] The present invention also provides an antibody combination for CAR-T cell immunophenotyping, the antibody combination comprising an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-EGFR antibody, an anti-CD25 antibody, an anti-CD127 antibody, an anti-CD45RA antibody, an anti-CCR7 antibody, an anti-CD27 antibody, an anti-PD-1 antibody, an anti-TIGIT antibody, and an anti-41BB antibody.

[0014] In some embodiments, the anti-CD3 antibody is UCHT1, the anti-CD4 antibody is RPA-T4, the anti-CD8 antibody is SK1, the anti-EGFR antibody is AY13, the anti-CD25 antibody is M-A251, the anti-CD127 antibody is HIL-7R-M21, the anti-CD45RA antibody is HI100, the anti-CCR7 antibody is 3d12, the anti-CD27 antibody is M-T271, the anti-PD-1 antibody is MIH4, the anti-TIGIT antibody is 741182, and / or the anti-41BB antibody is 4b4-1.

[0015] In some embodiments, the fluorescent label of the anti-CD3 antibody is FITC, the fluorescent label of the anti-CD4 antibody is PerCP-Cy5.5, the fluorescent label of the anti-CD8 antibody is APC-Cy7, the fluorescent label of the anti-EGFR antibody is APC, the fluorescent label of the anti-CD25 antibody is BV421, the fluorescent label of the anti-CD127 antibody is PE, the fluorescent label of the anti-CD45RA antibody is BV510, the fluorescent label of the anti-CCR7 antibody is BV711, the fluorescent label of the anti-CD27 antibody is PE-CY7, the fluorescent label of the anti-PD-1 antibody is PE-CF594, the fluorescent label of the anti-TIGIT antibody is BV650 and / or the fluorescent label of the anti-41BB antibody is BV750.

[0016] The present invention also provides a kit for immunophenotyping of CAR-T cells, and the kit contains the antibody combination as described in any one of the above.

[0017] In some embodiments, the antibody combination is a mixture or is dispensed in different containers.

[0018] In some embodiments, the kit further contains a cell viability dye, a flow cytometry staining buffer, an Fc blocker and / or a cell cryopreservation solution.

[0019] The present invention also provides the use of the antibody combination as described in any one of the above or the kit as described in any one of the above in the preparation of a product for immunophenotyping of CAR-T cells.

[0020] The present invention also provides a system for immunophenotyping of CAR-T cells, and the system includes a detection part and an analysis part, wherein:

[0021] The detection part includes the antibody combination as described in any one of the above or the kit as described in any one of the above, and is used for detecting the antigen expression level of a sample to be tested by flow cytometry;

[0022] The analysis part is used for analyzing the detection result of the detection part and classifying CAR-T cells.

[0023] In some embodiments, the classification includes CD3+CAR+ / EGFR+CD25+CD127-, CD3+CAR+ / EGFR+PD-1+, CD3+CAR+ / EGFR+TIGIT+, CD3+CAR+ / EGFR+CCR7+CD45RA-, CD3+CAR+ / EGFR+CD8+CCR7+CD27+.

[0024] In some embodiments, the CAR-T cells express membrane-bound EGFR protein.

[0025] The beneficial effects of the present invention are as follows: The present invention designs and obtains an optimal antibody combination for cell surface markers. Using a 12-color antibody combination, through flow cytometry, immunophenotyping of CAR-T cells in blood samples (such as PBMC, lysed whole blood, etc.) is performed, and the detection results are obtained quickly, and the proportions of various phenotypic CAR-T cells are efficiently identified. The method has high precision and stability. Brief Description of the Drawings

[0026] Figure 1 : Analytical batch information.

[0027] Figure 2 : Precision of CD3+EGFR+CD25+CD127-.

[0028] Figure 3 : Precision of CD3+EGFR+PD-1+.

[0029] Figure 4 : Precision of CD3+EGFR+TIGIT+.

[0030] Figure 5 : Precision of CD3+EGFR+CCR7+CD45RA-.

[0031] Figure 6 : Precision of CD3+EGFR+CD8+CCR7+CD27+.

[0032] Figure 7 : Robustness of the method. Detailed Embodiments

[0033] The inventors of the present application have conducted extensive and in-depth research. For the immunophenotyping detection of CAR-T cells, a 12-color antibody combination is designed, and through flow cytometry, the proportions of various CAR-T target cell populations can be obtained quickly and efficiently.

[0034] In the present invention, "+" represents positive, that is, it means that the antigen is expressed on the cell surface; "++" represents strongly positive, that is, it means that the antigen is highly expressed on the cell surface; "-" represents negative, that is, it means that the antigen is not expressed on the cell surface.

[0035] In the present invention, CD3+CAR+ / EGFR+CD25+CD127- represents CAR-Treg cells; CD3+CAR+ / EGFR+PD-1+ represents PD-1 positive CAR-T exhausted cells; CD3+CAR+ / EGFR+TIGIT+ represents TIGIT positive CAR-T exhausted cells; CD3+CAR+ / EGFR+CCR7+CD45RA- represents CAR-T memory cells; CD3+CAR+ / EGFR+CD8+CCR7+CD27+ represents CAR-T poorly differentiated cells.

[0036] In the present invention, the EGFR is the well-known EGFR in the art, such as human EGFR. EGFR contains N-terminal extracellular domains I and II, extracellular domain III, extracellular domain IV, transmembrane region, juxtamembrane domain, and tyrosine kinase domain. Preferably, the present invention uses truncated EGFR ("tEGFR", i.e., the fragment of EGFR described herein), especially the truncated EGFR that does not include its intracellular region (juxtamembrane domain and tyrosine kinase domain). In certain embodiments, the EGFR that does not include the intracellular region can be further truncated to not include extracellular domains I and II. Therefore, in certain embodiments, the tEGFR of the present invention contains the extracellular domain III, extracellular domain IV, and transmembrane region of EGFR, or consists of the extracellular domain III, extracellular domain IV, and transmembrane region of EGFR. In certain embodiments, the tEGFR contains the amino acid sequence of positions 310-646 of human EGFR, or consists of the amino acid sequence of positions 310-646 of human EGFR, wherein the amino acid sequence of positions 310-480 is the extracellular domain III of human EGFR, the amino acid sequence of positions 481-620 is the extracellular domain IV of human EGFR, and the amino acid sequence of positions 621-646 is the transmembrane region of human EGFR. In certain embodiments, the amino acid sequence of the tEGFR is as shown in SEQ ID NO:1.

[0037] In the present invention, the CAR of the CAR-T cell can target any antigen, wherein the CAR-T cell simultaneously expresses a membrane-bound EGFR protein. In certain embodiments, the CAR targets alpha folate receptor, 5T4, alpha v beta 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD318, CD171, CEA, CSPG4, EGFR, the EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FR alpha, GD2, GD3, glycosylphosphatidylinositol-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11R alpha, IL-13R alpha 2, lambda, Lewis-Y, kappa, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM, Trop2, and / or VEGFR2. In a specific embodiment, the CAR targets BCMA. In a specific embodiment, the CAR is as described in the patent document CN201610932365.7.

[0038] The present invention will be illustrated by way of specific embodiments below. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the embodiments are conventional materials and methods in the art unless otherwise specified.

[0039] Examples

[0040] Example 1: Flow cytometry-based immunophenotyping method for human peripheral blood cells of the present invention

[0041] This example provides a flow cytometry-based immunophenotyping method for human peripheral blood cells, specifically detecting the proportions of the following subtypes of cells in the peripheral blood of a subject after BCMA CAR-T infusion treatment.

[0042] Lymphocyte subsets Cell type CAR-Treg CD3+CAR+ / EGFR+CD25+CD127- CAR-T Exhausted CD3+CAR+ / EGFR+PD-1+ CAR-T Exhausted CD3+CAR+ / EGFR+TIGIT+ CAR-T memory CD3+CAR+ / EGFR+CCR7+CD45RA- CAR-T less differentiated CD3+CAR+ / EGFR+CD8+CCR7+CD27+

[0043] 1. Method principle

[0044] This method is based on the flow cytometry platform to detect the proportions of CD3+CAR+ / EGFR+CD25+CD127-, CD3+CAR+ / EGFR+PD-1+, CD3+CAR+ / EGFR+TIGIT+, CD3+CAR+ / EGFR+CCR7+CD45RA-, and CD3+CAR+ / EGFR+CD8+CCR7+CD27+ cell populations in human PBMCs after infusion treatment with BCMA CAR-T (as described in the patent document CN201610932365.7). After extracting and separating PBMCs from EDTA-K2 anticoagulated whole blood, adding a fluorescent antibody mixture for incubation, washing, and then detecting on the machine. The target cell population is circled through a scatter plot, and the corresponding proportion (%Parent) is recorded.

[0045] 2. Main reagents

[0046]

[0047] 3. Instruments

[0048]

[0049] 4. Instrument parameters 4.1 Flow cytometer Instrument model: Aurora; Supporting software: SpectroFlo; Manufacturer: CYTEK.

[0050] 4.2 Fluorescent channels

[0051] FITC, PERCP-CY5.5, APC-CY7, APC, BV421, PE, BV510, BV711, PE-CY7, PE-CF594, BV650, BV750

[0052] 4.3 Flow rate

[0053] High speed (60 μL / min).

[0054] 4.4 Threshold

[0055] 300,000 (can be adjusted according to the actual situation).

[0056] 4.5 Gating strategy

[0057] In the FSC-A - FSC-H scatter plot, single cells are circled with the "singlet" gate;

[0058] In the CD3 FITC - SSC scatter plot (GATE = singlet), CD3+ T lymphocytes are circled with "CD3+";

[0059] In the CD8 PC7 scatter plot (GATE = CD3+), circle the CD3+CD8+ lymphocytes with "CD8+".

[0060] In the CAR+ / EGFR+-SSC scatter plot (GATE = CD3+), circle the CD3+CAR+ / EGFR+ lymphocytes with "CAR+ / EGFR+".

[0061] In the CD25+CD127- scatter plot (GATE = CD3+CAR+ / EGFR+), circle the CD3+CAR+ / EGFR+CD25+CD127- CAR-Treg cells with "CD25+CD127-".

[0062] In the PD1+-SSC scatter plot (GATE = CD3+CAR+ / EGFR+), circle the CD3+ / EGFR+PD-1+ CAR-T Exhausted cells with "PD1+".

[0063] In the TIGIT+-SSC scatter plot (GATE = CD3+CAR+ / EGFR+), circle the CD3+CAR+ / EGFR+TIGIT+ CAR-T Exhausted cells with "TIGIT+".

[0064] In the CCR7+CD45RA- scatter plot (GATE = CD3+CAR+ / EGFR+), circle the CD3+CAR+ / EGFR+CCR7+CD45RA- CAR-T memory cells with "CCR7+CD45RA-".

[0065] In the CAR+ / EGFR+-SSC scatter plot (GATE = CD8+), circle the CD3+CD8+CAR+ / EGFR+ cells with "CAR+ / EGFR+".

[0066] In the CCR7+CD27+ scatter plot (GATE = CD3+CD8+CAR+ / EGFR+), circle the CD3+CAR+ / EGFR+CD8+CCR7+CD27+ CAR-T less differentiated cells with "CCR7+CD27+".

[0067] Record the proportions of T cells (CD3+CAR+ / EGFR+CD25+CD127-, CD3+CAR+ / EGFR+PD-1+, CD3+CAR+ / EGFR+TIGIT+, CD3+CAR+ / EGFR+CCR7+CD45RA-, CD3+CAR+ / EGFR+CD8+CCR7+CD27+). The specific calculation methods for the proportions of each cell are shown in the following table:

[0068]

[0069]

[0070] 4.6 Number of collected events

[0071] Set to collect 20,000 events for "CD3+" (can be changed according to actual situation).

[0072] 5. Preparation of reagents

[0073] The relevant preparations listed in this method are only for clarifying the preparation method, and the specific preparation volume can be adjusted proportionally according to the actual situation. If the corresponding index is not detected, the volume of the corresponding fluorescent antibody is made up with Stain Buffer (flow cytometry staining buffer).

[0074] 5.1 Preparation of fluorescent antibody mixture

[0075] Name Aspiration volume ratio (μL) FITC anti-human CD3 1 PerCP-Cy5.5 anti-human CD4 1 APC-Cy7 anti-human CD8 1 APC anti-human EGFR 1 BV421 anti-human CD25 1 PE anti-human CD127 1 BV510 anti-human CD45RA 1 BV711 anti-human CCR7 1 PE-CY7 anti-human CD27 1 PE-CF594 anti-human PD-1 1 BV750 anti-human 41BB 1 BV650 anti-human TIGIT 1

[0076] Mix well and use immediately after preparation.

[0077] 5.2 Preparation of cell cryopreservation solution

[0078] Add 200 μL of DMSO to 800 μL of RPMI Medium 1640 (1X), and the prepared solution is valid for 6 months.

[0079] 6. Experimental procedure

[0080] The following experiments are carried out at room temperature except those with incubation requirements.

[0081]

[0082]

[0083] 7. Remarks

[0084] When the cell morphology is significantly abnormal, reject the data.

[0085] 7.2 The decimal retention rules in the report are as follows:

[0086] Item Number of reserved digits Ratio (%) 2 digits after the decimal point CV% 2 digits after the decimal point RE% 2 digits after the decimal point

[0087] Example 2 Methodological investigation of the method for immunophenotyping of human peripheral blood cells based on flow cytometry of the present invention

[0088] The purpose of this example is to conduct methodological verification on aspects such as precision, method robustness, stability, and residue of the human peripheral blood cell immunophenotyping method based on flow cytometry described in Example 1, providing a basis for bioanalysis.

[0089] 1. Verification content

[0090] 1.1 Preparation of PBMC containing BCMA CAR-T reference product

[0091] After thawing, the BCMA CAR-T reference product was counted by a cell counter, and the total cell concentration was adjusted to approximately 3.5 - 5.0×10 6 cells / mL. The collected blank whole blood was taken, and an equal volume of the BCMA CAR-T reference product was added, and then PBMC was extracted according to the method in Example 1. This was used as the verification sample and denoted as "CAR-T PBMC".

[0092] 1.2 Precision

[0093] Take the CAR-T PBMC sample, and conduct within-batch precision and between-batch precision investigations on (CD3+CAR+ / EGFR+CD25+CD127-, CD3+CAR+ / EGFR+PD-1+, CD3+CAR+ / EGFR+TIGIT+, CD3+CAR+ / EGFR+CCR7+CD45RA-, CD3+CAR+ / EGFR+CD8+CCR7+CD27+) respectively: The precision (CV%) of the proportion and cell number of the above cell populations for each individual should not be greater than 30%.

[0094] Within-batch precision: Take 3 individuals, and conduct precision investigations with 3 replicates for each individual in the same analysis batch.

[0095] Between-batch precision: Take 3 individuals, and conduct 3 independent analysis batch determinations on 2 days to investigate precision.

[0096] Investigation of different operators: Conduct precision investigations of 2 different operators.

[0097] Note: When the verification result does not meet the acceptance criteria, report the final result in the report, and the population data is for reference only.

[0098] 1.3 Method robustness

[0099] Method robustness is used to investigate the stability of the method. The investigation includes flow cytometry detection after incubating the cell suspension with fluorescent antibodies for 35 minutes (analysis batch 22914MV04-002), 30 minutes (analysis batch 22914MV04-004), and 30 minutes (analysis batch 22914MV04-005). The robustness of the method is detected through the dark incubation time of different fluorescent antibody mixtures.

[0100] 2. Data processing

[0101] All the measurement results were collected by SpectroFlo software, and the data was calculated using SpectroFlo software and Microsoft Excel.

[0102] CV% = standard deviation / average value × 100%

[0103] 3. Main software systems

[0104] The main computerized systems used in this validation include:

[0105]

[0106] 4. Results and discussion

[0107] 4.1 Analytical batch description

[0108] A total of 12 analytical batches were carried out in this validation, and the tests were performed on 9 different dates. See the detailed description of each analytical batch in Figure 1 .

[0109] 4.2 Precision

[0110] The results of the precision validation are shown in Figures 2 to 6 .

[0111] The within-batch, between-batch, and precision among different operators of the ratios of CD3+CAR+ / EGFR+CD25+CD127-, CD3+CAR+ / EGFR+PD-1+, CD3+CAR+ / EGFR+TIGIT+, CD3+CAR+ / EGFR+CCR7+CD45RA-, and CD3+CAR+ / EGFR+CD8+CCR7+CD27+ met the acceptance criteria of CV% not exceeding 30%, and the validation items passed.

[0112] 4.3 Method robustness

[0113] The results of the method robustness validation are shown in Figure 7 . Among them, based on the method of Example 1, the time windows for dark incubation after adding the fluorescent antibody mixture were 35 minutes (22914MV04-002 analytical batch), 30 minutes (22914MV04-004 analytical batch), and 30 minutes (22914MV04-005 analytical batch). The method robustness was detected by the dark incubation time of different fluorescent antibody mixtures.

[0114] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for immunophenotyping of CAR-T cells, characterized in that, Comprising the steps of: mixing a fluorescently labeled antibody with a sample to be detected, incubating, and detecting by flow cytometry to obtain detection data, and further determining the proportion and / or number of the target cell population; wherein: The fluorescently labeled antibody includes: anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-EGFR antibody, anti-CD25 antibody, anti-CD127 antibody, anti-CD45RA antibody, anti-CCR7 antibody, anti-CD27 antibody, anti-PD-1 antibody, anti-TIGIT antibody, anti-41BB antibody; The target cell population includes: CD3+CAR+ / EGFR+CD25+CD127-, CD3+CAR+ / EGFR+PD-1+, CD3+CAR+ / EGFR+TIGIT+, CD3+CAR+ / EGFR+CCR7+CD45RA-, CD3+CAR+ / EGFR+CD8+CCR7+CD27+; The CAR-T cells express membrane-bound EGFR protein.

2. The method according to claim 1, characterized in that, The anti-CD3 antibody is UCHT1, the anti-CD4 antibody is RPA-T4, the anti-CD8 antibody is SK1, the anti-EGFR antibody is AY13, the anti-CD25 antibody is M-A251, the anti-CD127 antibody is HIL-7R-M21, the anti-CD45RA antibody is HI100, the anti-CCR7 antibody is 3d12, the anti-CD27 antibody is M-T271, the anti-PD-1 antibody is MIH4, the anti-TIGIT antibody is 741182 and / or the anti-41BB antibody is 4b4-1.

3. The method according to claim 1, characterized in that, The fluorescent label of the anti-CD3 antibody is FITC, the fluorescent label of the anti-CD4 antibody is PerCP-Cy5.5, the fluorescent label of the anti-CD8 antibody is APC-Cy7, the fluorescent label of the anti-EGFR antibody is APC, the fluorescent label of the anti-CD25 antibody is BV421, the fluorescent label of the anti-CD127 antibody is PE, the fluorescent label of the anti-CD45RA antibody is BV510, the fluorescent label of the anti-CCR7 antibody is BV711, the fluorescent label of the anti-CD27 antibody is PE-CY7, the fluorescent label of the anti-PD-1 antibody is PE-CF594, the fluorescent label of the anti-TIGIT antibody is BV650 and / or the fluorescent label of the anti-41BB antibody is BV750.

4. The method according to claim 1, characterized in that, The sample to be detected is PBMC or lysed whole blood from an individual who has received CAR-T cell therapy; Preferably, the PBMC is sorted from the peripheral blood of an individual; Preferably, the PBMC cells are cryopreserved or freshly prepared; Preferably, the whole blood is cryopreserved or freshly prepared.

5. An antibody combination for immunophenotyping of CAR-T cells, characterized in that, The antibody combination includes anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-EGFR antibody, anti-CD25 antibody, anti-CD127 antibody, anti-CD45RA antibody, anti-CCR7 antibody, anti-CD27 antibody, anti-PD-1 antibody, anti-TIGIT antibody and anti-41BB antibody; Preferably, the anti-CD3 antibody is UCHT1, the anti-CD4 antibody is RPA-T4, the anti-CD8 antibody is SK1, the anti-EGFR antibody is AY13, the anti-CD25 antibody is M-A251, the anti-CD127 antibody is HIL-7R-M21, the anti-CD45RA antibody is HI100, the anti-CCR7 antibody is 3d12, the anti-CD27 antibody is M-T271, the anti-PD-1 antibody is MIH4, the anti-TIGIT antibody is 741182, and / or the anti-41BB antibody is 4b4-1; Preferably, the fluorescent label of the anti-CD3 antibody is FITC, the fluorescent label of the anti-CD4 antibody is PerCP-Cy5.5, the fluorescent label of the anti-CD8 antibody is APC-Cy7, the fluorescent label of the anti-EGFR antibody is APC, the fluorescent label of the anti-CD25 antibody is BV421, the fluorescent label of the anti-CD127 antibody is PE, the fluorescent label of the anti-CD45RA antibody is BV510, the fluorescent label of the anti-CCR7 antibody is BV711, the fluorescent label of the anti-CD27 antibody is PE-CY7, the fluorescent label of the anti-PD-1 antibody is PE-CF594, the fluorescent label of the anti-TIGIT antibody is BV650, and / or the fluorescent label of the anti-41BB antibody is BV750.

6. A kit for immunophenotyping of CAR-T cells, characterized in that, The kit contains the antibody combination according to claim 5.

7. The kit according to claim 6, characterized in that, The antibody combination is a mixture or is dispensed in different containers.

8. The application according to claim 6, characterized in that, The kit further contains a cell viability identification dye, a flow cytometry staining buffer, an Fc blocker, and / or a cell cryopreservation solution.

9. The application of the antibody combination according to claim 5 or the kit according to any one of claims 6-8 in the preparation of a product for immunophenotyping of CAR-T cells.

10. A system for immunophenotyping of CAR-T cells, characterized in that, The system includes a detection part and an analysis part, wherein: The detection part includes the antibody combination according to claim 5 or the kit according to any one of claims 6-8, and is used to detect the antigen expression level of a sample to be tested by flow cytometry; The analysis part is used to analyze the detection results of the detection part and classify CAR-T cells. Preferably, the classification includes CD3+CAR+ / EGFR+CD25+CD127-, CD3+CAR+ / EGFR+PD-1+, CD3+CAR+ / EGFR+TIGIT+, CD3+CAR+ / EGFR+CCR7+CD45RA-, CD3+CAR+ / EGFR+CD8+CCR7+CD27+; Preferably, the CAR-T cells express membrane-bound EGFR protein.

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