Monoclonal antibody recognizing cd7-car molecules and uses thereof

Abstract

The application provides a monoclonal antibody for recognizing a CD7-CAR molecule and application thereof, an amino acid sequence of a heavy chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:7, SEQ ID NO:21 or SEQ ID NO:35; an amino acid sequence of a light chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:14, SEQ ID NO:28 or SEQ ID NO:42, the monoclonal antibody for recognizing the CD7-CAR molecule can specifically recognize a CD7-CAR molecule on a CD7-CAR-T cell and has no cross reaction with other molecules; the monoclonal antibody for recognizing the CD7-CAR molecule has high binding activity to the CD7-CAR molecule, and a flow analysis method for detecting a CD7-CAR-T cell CAR transfection positive rate is developed and comprehensively methodologically verified, and the CD7-CAR molecule transfection efficiency can be accurately, specifically and sensitively detected.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a monoclonal antibody that recognizes CD7-CAR molecules and its applications. Background Technology

[0002] T-cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous hematologic malignancy, accounting for approximately 25% of adult acute lymphoblastic leukemia cases and 15% of childhood acute lymphoblastic leukemia cases. The human CD7 molecule is a cell surface glycoprotein with a molecular weight of approximately 40 kDa, belonging to the immunoglobulin superfamily. CD7 is highly expressed on most T-cell hematologic malignancies. Therefore, CD7 may be a very suitable therapeutic target for T-cell hematologic malignancies.

[0003] CN113933513A discloses a reagent composition for detecting acute T-lymphoblastic leukemia after targeted therapy and its application. Specifically, it is a reagent composition for flow cytometry detection of acute T-lymphoblastic leukemia after targeted therapy, comprising a first group of antibodies, a second group of antibodies, and a third group of antibodies. The first group of antibodies includes anti-CD99 antibody, anti-CD1a antibody, anti-CD34 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD8 antibody, anti-CD7 antibody, anti-CD45 antibody, and anti-CD2 antibody; the second group of antibodies includes anti-CD3 antibody, anti-CD7 antibody, anti-CD16 antibody, anti-CD56 antibody, and anti-CD45 antibody; the third group of antibodies includes anti-nuclear TdT antibody and anti-cytoplasmic CD3 antibody. This reagent composition can be used for flow cytometry detection of acute T-lymphoblastic leukemia after targeted therapy, reducing the probability of missed diagnoses, and can also perform simultaneous immunological evaluation.

[0004] Those skilled in the art have developed methods for CD7-CAR-T cell targeted therapy of T-cell acute lymphoblastic leukemia. However, currently available anti-CD7 antibodies can only recognize specific CD7 antibodies and cannot accurately detect CAR transfection efficiency. Therefore, providing a monoclonal antibody that can accurately detect CAR transfection efficiency is of great significance in targeted therapy of T-cell acute lymphoblastic leukemia. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a monoclonal antibody for recognizing CD7-CAR molecules and its applications. The monoclonal antibody for recognizing CD7-CAR molecules described in this invention can specifically recognize CD7-CAR molecules on CD7-CAR-T cells and exhibits no cross-reactivity with other molecules; the monoclonal antibody for recognizing CD7-CAR molecules has high binding activity to CD7-CAR molecules and can accurately detect CAR transfection efficiency.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a monoclonal antibody that recognizes CD7-CAR molecules, wherein the amino acid sequence of the heavy chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:7, SEQ ID NO:21 or SEQ ID NO:35;

[0008] The amino acid sequence of the light chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:14, SEQ ID NO:28 or SEQ ID NO:42.

[0009] Preferably, the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is as shown in SEQ ID NO:5, SEQ ID NO:19 or SEQ ID NO:33.

[0010] Preferably, the amino acid sequence of the heavy chain CDR2 of the monoclonal antibody is as shown in SEQ ID NO:6, SEQ ID NO:20 or SEQ ID NO:34.

[0011] Preferably, the amino acid sequence of the light chain CDR1 of the monoclonal antibody is as shown in SEQ ID NO:12, SEQ ID NO:26 or SEQ ID NO:40.

[0012] Preferably, the amino acid sequence of the light chain CDR2 of the monoclonal antibody is as shown in SEQ ID NO:13, SEQ ID NO:27 or SEQ ID NO:41.

[0013] Preferably, the amino acid sequence of the heavy chain FR1 of the monoclonal antibody is as shown in SEQ ID NO:2, SEQ ID NO:16 or SEQ ID NO:30.

[0014] Preferably, the amino acid sequence of the heavy chain FR2 of the monoclonal antibody is as shown in SEQ ID NO:3, SEQ ID NO:17 or SEQ ID NO:31.

[0015] Preferably, the amino acid sequence of the heavy chain FR3 of the monoclonal antibody is as shown in SEQ ID NO:4, SEQ ID NO:18 or SEQ ID NO:32.

[0016] Preferably, the amino acid sequence of the light chain FR1 of the monoclonal antibody is as shown in SEQ ID NO:9, SEQ ID NO:23 or SEQ ID NO:37.

[0017] Preferably, the amino acid sequence of the light chain FR2 of the monoclonal antibody is as shown in SEQ ID NO:10, SEQ ID NO:24 or SEQ ID NO:38.

[0018] Preferably, the amino acid sequence of the light chain FR3 of the monoclonal antibody is as shown in SEQ ID NO:11, SEQ ID NO:25 or SEQ ID NO:39.

[0019] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO:1, SEQ ID NO:15 or SEQ ID NO:29.

[0020] Preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO:8, SEQ ID NO:22 or SEQ ID NO:36.

[0021] In this invention, the monoclonal antibody recognizing the CD7-CAR molecule is an antibody targeting the CD7-CAR molecule epitope. This invention provides three monoclonal antibodies: 10-B5-E7-B4, 10-B8-E7-F11, and 6-H2-D6-D9. Specifically, the amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody corresponding to 10-B5-E7-B4 are SEQ ID NO:1 and SEQ ID NO:8; the amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody corresponding to 10-B8-E7-F11 are SEQ ID NO:15 and SEQ ID NO:22; and the amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody corresponding to 6-H2-D6-D9 are SEQ ID NO:29 and SEQ ID NO:36.

[0022] This invention first immunizes mice with the CHO-K1-GeneD recombinant cell line, and then collects peripheral blood from the mice to test the immunogenicity. Mice with the highest immunogenicity are selected and subjected to a shock immunization using purified GeneD recombinant protein. Three days after immunization, the spleens of the mice are isolated for hybridoma fusion to generate hybridoma cells. Suitable hybridoma cells are then screened for appropriate cloning.

[0023] Furthermore, those skilled in the art can readily learn the structure of the antibodies of the present invention (such as the variable regions of the heavy chain and light chain), and then prepare the monoclonal antibodies of the present invention through recombinant methods. The antibodies of the present invention can specifically recognize CD7-CAR molecules on CD7-CAR-T cells, and these specific antibodies can be used to detect the transfection efficiency of CD7-CAR-T cells.

[0024] In a second aspect, the present invention provides a nucleic acid molecule that encodes the monoclonal antibody that recognizes the CD7-CAR molecule as described in the first aspect.

[0025] Preferably, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 10-B5-E7-B4 is shown in SEQ ID NO:43; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 10-B5-E7-B4 is shown in SEQ ID NO:44.

[0026] Preferably, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 10-B8-E7-F11 is shown in SEQ ID NO:45; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 10-B8-E7-F11 is shown in SEQ ID NO:46.

[0027] Preferably, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 6-H2-D6-D9 is shown in SEQ ID NO:47; and the nucleotide sequence encoding the light chain variable region of monoclonal antibody 6-H2-D6-D9 is shown in SEQ ID NO:48.

[0028] Thirdly, the present invention provides the use of the monoclonal antibody described in the first aspect for recognizing CD7-CAR molecules in the detection of CAR molecules in CD7-CAR-T cells.

[0029] Fourthly, the present invention provides a pharmaceutical composition comprising the monoclonal antibody that recognizes the CD7-CAR molecule as described in the first aspect;

[0030] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or diluent.

[0031] Fifthly, the present invention provides a kit for detecting CD7-CAR molecules in a sample, the kit comprising the monoclonal antibody for recognizing CD7-CAR molecules as described in the first aspect.

[0032] In a sixth aspect, the present invention provides the application of the kit for detecting CD7-CAR molecules in a sample as described in the fifth aspect in the determination of anti-drug antibody levels.

[0033] The amino acid sequences involved in this invention are shown in Table 1:

[0034] Table 1

[0035]

[0036] In the following amino acid sequences, the underlined sequences represent CDR regions;

[0037] (1)GeneD(10-B5-E7-B4)

[0038] The amino acid sequence of the heavy chain variable region is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, as shown in SEQ ID NO:1.

[0039] QVQLQQSGAELMKPGASVKISCKATGYTFS TYWIE WIKQRPGHGLEWIG EILHGSATNYNEKFKG KATFTADTSSNTAYMQLSSLTSEDSAVYYCAR GAYPYAMDY WGQGTSVTVSS.

[0040] SEQ ID NO:2 (Heavy chain FR1)

[0041] QVQLQQSGAELMKPGASVKISCKATGYTFS.

[0042] SEQ ID NO:3 (Heavy chain FR2)

[0043] WIKQRPGHGLEWIG.

[0044] SEQ ID NO:4 (Heavy chain FR3)

[0045] KATFTADTSSNTAYMQLSSLTSEDSAVYYCAR.

[0046] SEQ ID NO:5 (Heavy chain CDR1)

[0047] TYWIE .

[0048] SEQ ID NO:6 (Heavy chain CDR2)

[0049] EILHGSATNYNEKFKG .

[0050] SEQ ID NO:7 (Heavy chain CDR3)

[0051] GAYPYAMDY .

[0052] The amino acid sequence of the light chain variable region is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, as shown in SEQ ID NO:8.

[0053] DIVMTQSTSSLSASLGDRVTISC RSSQDINNYLN WYQQKPDGTVKLLIY YTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLRT FGGGTKLEIK.

[0054] SEQ ID NO:9 (Light chain FR1)

[0055] DIVMTQSTSSLSASLGDRVTISC.

[0056] SEQ ID NO:10 (Light chain FR2)

[0057] WYQQKPDGTVKLLIY.

[0058] SEQ ID NO:11 (Light chain FR3)

[0059] GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC.

[0060] SEQ ID NO:12 (Light chain CDR1)

[0061] RSSQDINNYLN.

[0062] SEQ ID NO:13 (Light chain CDR2)

[0063] YTSRLHS .

[0064] SEQ ID NO:14 (Light chain CDR3)

[0065] QQGNTLRT .

[0066] (2)GeneD(10-B8-E7-F11)

[0067] The amino acid sequence of the heavy chain variable region is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, as shown in SEQ ID NO:15.

[0068] EVLLQQSGPELIKHGAPVKITCKASGYTFT DYNMD WVKQSHGKSLEWIG DINPNNGGTIYNQKFKG KVTLTVDKFSSTAYMELRSLTAEDTAVYFCAR SGYYRYAMDY WGQGTSVTVSS.

[0069] SEQ ID NO:16 (Heavy chain FR1)

[0070] EVLLQQSGPELIKHGAPVKITCKASGYTFT.

[0071] SEQ ID NO:17 (Heavy chain FR2)

[0072] WVKQSHGKSLEWIG.

[0073] SEQ ID NO:18 (Heavy chain FR3)

[0074] KVTLTVDKFSSTAYMELRSLTAEDTAVYFCAR.

[0075] SEQ ID NO:19 (Heavy chain CDR1)

[0076] DYNMD .

[0077] SEQ ID NO:20 (Heavy chain CDR2)

[0078] DINPNNGGTIYNQKFKG .

[0079] SEQ ID NO:21 (Heavy chain CDR3)

[0080] SGYYRYAMDY .

[0081] The amino acid sequence of the light chain variable region is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, as shown in SEQ ID NO:22.

[0082] DIQMTQSPSSLSASLGGNVTITC KASQDINKYVA WYQHKPGKGLRRLIH YSSTLQP GIPSRFSGSGSGRDYSFSIRNLEPEDIATYYC LQYDNFLRT FGGGTKLEIK.

[0083] SEQ ID NO:23 (Light chain FR1)

[0084] DIQMTQSPSSLSASLGGNVTITC.

[0085] SEQ ID NO:24 (Light chain FR2)

[0086] WYQHKPGKGLRRLIH.

[0087] SEQ ID NO:25 (Light chain FR3)

[0088] GIPSRFSGSGSGRDYSFSIRNLEPEDIATYYC.

[0089] SEQ ID NO:26 (Light chain CDR1)

[0090] KASQDINKYVA .

[0091] SEQ ID NO:27 (Light chain CDR2)

[0092] YSSTLQP .

[0093] SEQ ID NO:28 (Light chain CDR3)

[0094] LQYDNFLRT .

[0095] (3)GeneD(6-H2-D6-D9)

[0096] The amino acid sequence of the heavy chain variable region is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, as shown in SEQ ID NO:29.

[0097] EVLLQQSGPELIKHGAPVKITCKASGYTFT DYNMD WVKQSHGKSLEWIG DINPNNGGTIYNQKFKG KVTLTVDKFSSTAYMELRSLTAEDTAVYFCAR SGYYRYAMDY WGQGTSVTVSS.

[0098] SEQ ID NO:30 (Heavy chain FR1)

[0099] EVLLQQSGPELIKHGAPVKITCKASGYTFT.

[0100] SEQ ID NO:31 (Heavy chain FR2)

[0101] WVKQSHGKSLEWIG.

[0102] SEQ ID NO:32 (Heavy chain FR3)

[0103] KVTLTVDKFSSTAYMELRSLTAEDTAVYFCAR.

[0104] SEQ ID NO:33 (Heavy chain CDR1)

[0105] DYNMD .

[0106] SEQ ID NO:34 (Heavy chain CDR2)

[0107] DINPNNGGTIYNQKFKG .

[0108] SEQ ID NO:35 (Heavy chain CDR3)

[0109] SGYYRYAMDY .

[0110] The amino acid sequence of the light chain variable region is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, as shown in SEQ ID NO:36.

[0111] DIVMTQSPSSLSASLGGNVTITC KASQDINKYVA WYQHKPGKGLRRLIH YSSTLQP GIPSRFSGSGSGRDYSFSVRNLEPEDIATYYC LQYDNFLRT FGGGTKLELK.

[0112] SEQ ID NO:37 (Light chain FR1)

[0113] DIVMTQSPSSLSASLGGNVTITC.

[0114] SEQ ID NO:38 (Light chain FR2)

[0115] WYQHKPGKGLRRLIH.

[0116] SEQ ID NO:39 (Light chain FR3)

[0117] GIPSRFSGSGSGRDYSFSVRNLEPEDIATYYC.

[0118] SEQ ID NO:40 (Light chain CDR1)

[0119] KASQDINKYVA .

[0120] SEQ ID NO:41 (Light chain CDR2)

[0121] YSSTLQP .

[0122] SEQ ID NO:42 (Light chain CDR3)

[0123] LQYDNFLRT .

[0124] The nucleotide sequences involved in this invention are shown below:

[0125] (1)GeneD(10-B5-E7-B4)

[0126] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 10-B5-E7-B4 is shown in SEQ ID NO:43; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 10-B5-E7-B4 is shown in SEQ ID NO:44.

[0127] SEQ ID NO:43:

[0128] caggttcagctgcagcagtctggagctgagctgatgaagcctggggcctcagtgaagatatcctgcaaggctactggctacacattcagtacctactggatagagtggataaagcagaggcctggacatggccttgagtggattggagagattttacatggaagtgctactaacta caatgaaaagttcaagggcaaggccacattcactgcagatacatcctccaacacagcctacatgcaactcagcagcctgacatctgaggactctgccgtctattattgtgcaagaggggcctacccctatgccatggactactggggtcaaggaacctcagtcaccgtctcctca.

[0129] SEQ ID NO:44:

[0130] gatattgtgatgacgcagtctacatcctccctgtctgcctctctggggagacagtcaccatcagttgcaggtcaagtcaggacattaacaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaaga ttacactcaggagtcccctcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttcggacgttcggtggagggacaaagttggaaataaaa.

[0131] (2)GeneD(10-B8-E7-F11)

[0132] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 10-B8-E7-F11 is shown in SEQ ID NO:45; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 10-B8-E7-F11 is shown in SEQ ID NO:46.

[0133] SEQ ID NO:45:

[0134] gaggtcctgctgcaacagtctggacctgagctaataaaagcatggggctccagtgaagataacctgcaaggcctctggatacacattcactgactacaacatggactgggtgaagcagagccatggaaagagccttgagtggattggagatattaatcctaacaatggtggaactatcta caaccagaagttcaagggcaaggtcacattgactgtagacaaattctccagcacagcctacatggagctccgcagcctgacagctgaggacactgcagtctatttctgtgcaagatcaggctactataggtatgctatggactactggggtcaaggaacctcagtcaccgtctcctca.

[0135] SEQ ID NO:46:

[0136] gatattcagatgactcagtctccatcctcactgtctgcatctctgggaggcaatgtcaccatcacttgcaaggcaagccaagacattaacaaatatgtagcttggtaccaacacaagcctggaaaaggtcttaggcggctcatacattactcatctacatt acagccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccttcagcatcaggaacctggagcctgaagatattgcaacgtattattgtctacagtatgataattttctacggacgttcggtggagggacaaagttggaaataaaa.

[0137] (3)GeneD(6-H2-D6-D9)

[0138] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 6-H2-D6-D9 is shown in SEQ ID NO:47; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 6-H2-D6-D9 is shown in SEQ ID NO:48.

[0139] SEQ ID NO:47:

[0140] gaggtcctgctgcaacagtctggacctgagctaataaaagcatggggctccagtgaagataacctgcaaggcctctggatacacattcactgactacaacatggactgggtgaagcagagccatggaaagagccttgagtggattggagatattaatcctaacaatggtggaactatcta caaccagaagttcaagggcaaggtcacattgactgtagacaaattctccagcacagcctacatggagctccgcagcctgacagctgaggacactgcagtctatttctgtgcaagatcaggctactataggtatgctatggactactggggtcaaggaacctcagtcaccgtctcctca.

[0141] SEQ ID NO:48:

[0142] gatattgtgatgacccagtctccatcctcactgtctgcatctctgggaggcaatgtcaccatcacttgcaaggcaagccaagacattaacaaatatgtagcttggtaccaacacaagcctggaaaaggtcttaggcggctcatacattactcatctacatt acagccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccttcagcgtcaggaacctggagcctgaagatattgcaacgtattattgtctacagtatgataattttctacggacgttcggtggagggaccaagctggagctgaaa.

[0143] Compared with the prior art, the present invention has the following beneficial effects:

[0144] The monoclonal antibody that recognizes CD7-CAR molecules described in this invention can specifically recognize CD7-CAR molecules on CD7-CAR-T cells; the monoclonal antibody that recognizes CD7-CAR molecules has high binding activity to CD7-CAR molecules, and the monoclonal antibody that recognizes CD7-CAR molecules can be used to detect the transfection efficiency of CD7-CAR-T cells. Attached Figure Description

[0145] Figure 1 This is a graph showing the SDS-PAGE electrophoresis detection results in Example 1;

[0146] Figure 2 This is a flow cytometry analysis of the three monoclonal antibodies purified in Example 1;

[0147] Figure 3A This is a flow cytometry graph of the first screening of monoclonal antibodies 10-B5-E7-B4 (batch 1), 10-B8-E7-F11, and 10-B8-E7-B4 (batch 2) on LZ cells in Example 2.

[0148] Figure 3B This is a flow cytometry graph showing the first screening of monoclonal antibodies 6-H2-D6-D9 and 10-B8-E7-F11 on LZ cells in Example 2.

[0149] Figure 4A This is a flow cytometry graph of the first screening of monoclonal antibodies 10-B5-E7-B4 (batch 1), 10-B8-E7-F11, and 10-B5-E7-B4 (batch 2) on MXY cells in Example 2.

[0150] Figure 4B This is a flow cytometry graph showing the first screening of monoclonal antibodies 6-H2-D6-D9 and 10-B8-E7-F11 on MXY cells in Example 2.

[0151] Figure 5A This is the flow cytometry analysis of the second screening of monoclonal 10-B5-E7-B4 in Example 3;

[0152] Figure 5B This is the flow cytometry result of the second screening of monoclonal 6-H2-D6-D9 in Example 3;

[0153] Figure 6 This is a flow cytometry result of the commercial Fc-APC antibody in Example 3.

[0154] Figure 7A This is a flow cytogram for the specificity verification of the monoclonal antibody 6-H2-D6-D9 in Example 4.

[0155] Figure 7BThis is a flow cytogram for sensitivity verification of the monoclonal antibody 6-H2-D6-D9 in Example 4. Detailed Implementation

[0156] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0157] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0158] Example 1

[0159] Step 1: Hybridoma cell screening

[0160] Balb / c mice were immunized with the CHO-K1-GeneD recombinant cell line (selected by Aikon, project number: MH1807121; CHO-K1 empty cells, sourced from the Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-507). Peripheral blood was collected, and mice with the highest immunogenicity were selected by immunotiter assay. These mice were then subjected to a shock immunization with purified GeneD recombinant protein. Three days after immunization, spleen cells were extracted from the mice and fused with myeloma cells to form hybridoma cells. The hybridoma cells were cultured in HAT medium (Sigma, CAT#H0262) for 10 days, then transferred to HT medium (Bio-Long, CAT#BF08002). The supernatant was used to detect specific antibodies, and the cells were identified by ELISA. Wells with higher OD values ​​were selected for subcloning.

[0161] After two rounds of subcloning, flow cytometry results showed that three hybridoma cell lines (denoted as A3, A8, and B3) exhibited strong binding to K562-GeneD and could be used to prepare purified antibodies from ascites fluid. Clones A3, A8, and B3 correspond to the monoclonal pairs 10-B5-E7-B4, 10-B8-E7-F11, and 6-H2-D6-D9, respectively.

[0162] Step 2: Antibody preparation and purification

[0163] To prepare hybridoma cells for subclonal titer testing, under sterile conditions, approximately 1 × 10⁻⁶ cells were added. 6 Add one hybridoma cell to a 1.5 mL centrifuge tube, centrifuge at 250 g / min, 4 °C for 10 min, and discard the supernatant. Wash three times with 1×PBS, resuspend the hybridoma cells in 500 μL of 1×PBS, inject the cells into the abdomen of mice using a 1 mL syringe, collect ascites fluid after 7-10 days, precipitate with ammonium sulfate, and purify the antibody using Protein G affinity chromatography.

[0164] The purified antibody was analyzed by SDS-PAGE. The SDS-PAGE electrophoresis results are shown in the figure below. Figure 1 As shown, three monoclonal antibodies recognizing the CD7-CAR molecule were obtained. Figure 1 In the assay, lane M was the Protein Marker; lane 1 was the 6-H2-D6-D9 GeneD monoclonal antibody; lane 2 was the 10-B5-E7-B4 GeneD monoclonal antibody; and lane 3 was the 10-B8-E7-F11 GeneD monoclonal antibody. The antibodies were FITC-conjugated, and flow cytometry was performed on K562-GeneD and control K562-CD8a cells to identify their specificity. The flow cytometry results are shown below. Figure 2 As shown, from Figure 2 The results show that the obtained antibody has good specificity and can recognize K562-GeneD cells.

[0165] Example 2

[0166] The first screening of monoclonal antibodies recognizing the CD7-CAR molecule involved flow cytometry analysis of the three purified monoclonal antibodies on produced CD7-CAR-T cells, T cells, and CD7-negative T (CD7-T) cells. Specific details are shown below:

[0167] The experimental procedure is as follows: Divide the cells into several portions, with each portion containing 5 × 10⁶ cells. 5 Add 1 mL of 1×PBS to each cell line and centrifuge at 3500 rpm for 3 min. Discard the supernatant, resuspend the cells in 100 μL of PBS, add 5 μL of CD3-PE antibody (BD, CAT#555333) and 2 μL of monoclonal antibody recognizing CD7-CAR molecules (Aikon, CAT#MH1807121), and incubate at room temperature in the dark for 12 min. Wash the cells with 1 mL of 1×PBS and centrifuge at 3500 rpm for 3 min. Discard the supernatant, resuspend the cells in 200 μL of 1×PBS, add 2 μL of 7-AAD (BD, CAT#559925), and perform flow cytometry analysis. Figure 3A -B is the flow cytometry result of the first screening of three monoclonal antibodies on LZ cells (Bosen Ji'anke, LOT#LZ012019082701). Figure 3A This is a flow cytometry graph of the first screening of monoclonal antibodies 10-B5-E7-B4 (batch 1), 10-B8-E7-F11, and 10-B8-E7-B4 (batch 2) on LZ cells. Figure 3B This is a flow cytogram of the first screening of monoclonal antibodies 6-H2-D6-D9 and 10-B8-E7-F11 on LZ cells. Figure 4A-B is the flow cytometry result of the first screening of three monoclonal antibodies on MXY cells (Bosen Ji'anke, LOT#MXY12019082601). Figure 4A This is a flow cytometry graph of the first screening of monoclonal antibodies 10-B5-E7-B4 (batch 1), 10-B8-E7-F11, and 10-B5-E7-B4 (batch 2) on MXY cells; Figure 4B This is a flow cytometry graph showing the first screening of monoclonal antibodies 6-H2-D6-D9 and 10-B8-E7-F11 on MXY cells. Considering both non-specific binding and transfection efficiency, the two monoclonal antibodies 6-H2-D6-D9 and 10-B5-E7-B4, which recognize the CD7-CAR molecule, showed better performance.

[0168] Example 3

[0169] A second screening of monoclonal antibodies that recognize CD7-CAR molecules was conducted, with further screening of monoclonal antibodies 6-H2-D6-D9 and 10-B5-E7-B4. Flow cytometry analysis was performed on the produced CD7-CAR-T cells, T cells, and CD7-negative T (CD7-T) cells, with a commercially available Fc-APC antibody as a control.

[0170] The experimental procedure is as follows: Divide the cells into several portions, with each portion containing 5 × 10⁶ cells. 5 Add 1 mL of 1×PBS to each cell line and centrifuge at 3500 rpm for 3 min. Discard the supernatant. Dilute Fc-APC antibody (Jackson Immuno Research, CAT#109-605-008) 1:2000 with PBS to obtain Fc-APC antibody dilution buffer. For the experimental group, resuspend the cells in 100 μL of antibody dilution buffer and add 5 μL of CD3-PE antibody. For the other groups, resuspend the cells in 100 μL of PBS, add 5 μL of CD3-PE antibody, and 2 μL or 1 μL of monoclonal antibody recognizing CD7-CAR molecules. Incubate at room temperature in the dark for 12 min. Wash the cells with 1 mL of 1×PBS and centrifuge at 3500 rpm for 3 min. Discard the supernatant. Resuspend the cells in 200 μL of 1×PBS and add 2 μL of 7-AAD. Analyze by flow cytometry.

[0171] Flow cytometry results as follows Figure 5A , Figure 5B and Figure 6 As shown, Figure 5A This is a flow cytometry result from the second screening of monoclonal 10-B5-E7-B4. Figure 5B This is a flow cytometry result from the second screening of monoclonal 6-H2-D6-D9. Figure 6This is a flow cytometry result of a commercially available Fc-APC antibody. The results showed that the two monoclonal antibodies recognizing CD7-CAR molecules had slightly higher transfection efficiency in detecting CD7-CAR-T cells than the Fc-APC antibody, and the former produced more distinct cell clustering than the latter. The transfection efficiencies of monoclonal antibodies 6-H2-D6-D9 and 10-B5-E7-B4 were essentially equivalent, but the nonspecificity of 6-H2-D6-D9 was slightly lower than that of 10-B5-E7-B4. Ultimately, the monoclonal antibody with clone number 6-H2-D6-D9 was selected as the antibody for detecting CAR molecules in CD7-CAR-T cells.

[0172] Example 4

[0173] The monoclonal antibody with clone number 6-H2-D6-D9 was selected as the antibody for detecting the CAR transfection positivity rate. A flow cytometry method for detecting the CAR transfection positivity rate of CD7-CAR-T cells was developed and fully validated. The experimental steps are as described in Example 3.

[0174] Figure 7A This is a flow cytometry plot verifying the specificity of monoclonal 6-H2-D6-D9. Figure 7B This is a flow cytogram for sensitivity validation. Specificity validation results show that the antibody specifically recognizes CD7-CAR, with the negative control showing lower sensitivity, indicating the method's specificity. Sensitivity validation results show that the method's sensitivity is 1.03% (the mean of two detection values ​​of 0.93% and 1.13%).

[0175] In summary, this invention first immunizes mice with the CHO-K1-GeneD recombinant cell line, and then collects peripheral blood from the mice for immunotiter testing. Mice with the highest immunotiter are selected and subjected to a pulse immunization using purified GeneD recombinant protein. Three days after immunization, the mouse spleens are isolated for hybridoma fusion to generate hybridoma cells. Suitable hybridoma cells are then screened and cloned to obtain monoclonal antibodies that recognize CD7-CAR molecules. These monoclonal antibodies specifically recognize CD7-CAR molecules on CD7-CAR-T cells, and this specific antibody can be used to detect the transfection efficiency of CD7-CAR-T cells.

[0176] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A monoclonal antibody that recognizes the CD7-CAR molecule, characterized in that, The amino acid sequence of the heavy chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:35; The amino acid sequence of the light chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:42; The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:33; The amino acid sequence of the heavy chain CDR2 of the monoclonal antibody is shown in SEQ ID NO:34; The amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:40; The amino acid sequence of the light chain CDR2 of the monoclonal antibody is shown in SEQ ID NO:

41.

2. The monoclonal antibody recognizing the CD7-CAR molecule according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:

29.

3. The monoclonal antibody for recognizing CD7-CAR molecules according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

36.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the monoclonal antibody that recognizes the CD7-CAR molecule according to any one of claims 1-3.

5. Use of the monoclonal antibody recognizing the CD7-CAR molecule according to any one of claims 1-3 for non-disease diagnostic purposes in detecting the CAR molecule in CD7-CAR-T cells.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the monoclonal antibody that recognizes the CD7-CAR molecule according to any one of claims 1-3.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or diluent.

8. A kit for detecting CD7-CAR molecules in a sample, characterized in that, The kit comprises the monoclonal antibody that recognizes the CD7-CAR molecule according to any one of claims 1-3.

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