Monoclonal antibody for identifying FLT3-CAR molecule and application thereof

Abstract

This invention provides a monoclonal antibody for recognizing FLT3-CAR molecules and its applications. The amino acid sequence of the heavy chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:19; the amino acid sequence of the light chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:8, SEQ ID NO:15, or SEQ ID NO:22. The monoclonal antibody specifically recognizes FLT3-CAR molecules on FLT3-CAR-T cells and exhibits no cross-reactivity with other molecules. The monoclonal antibody has high binding activity to FLT3-CAR molecules, and a flow cytometry method for detecting the CAR transfection positivity rate of FLT3-CAR-T cells has been developed, which can accurately and specifically detect the FLT3-CAR molecule transfection efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a monoclonal antibody that recognizes FLT3-CAR molecules and its applications. Background Technology

[0002] Acute myeloid leukemia (AML) is a highly heterogeneous malignant tumor of myeloid hematopoietic stem / progenitor cells. It is one of the most common types of leukemia in adults, accounting for about half of all leukemia cases. The main characteristic of AML is the abnormal proliferation of primitive and immature myeloid cells in the bone marrow and peripheral blood. Clinical symptoms mainly include anemia, bleeding, infection and fever, organ infiltration, and metabolic abnormalities. AML progresses rapidly and is highly aggressive; most cases are acutely severe with a poor prognosis, and without timely treatment, it can often be life-threatening.

[0003] In recent years, chimeric antigen receptor T-cell (CAR-T) therapy has shown significant clinical efficacy in treating relapsed / refractory lymphoma. CAR-T therapy involves activating the patient's T lymphocytes through gene modification technology, then binding specific tumor antigen receptor genes to form modified T cells. These modified T cells can then specifically recognize and bind to antigens on the surface of tumor cells, achieving targeted killing of tumor cells. One of the keys to the success of this technology is selecting a suitable target antigen. By constructing the corresponding CAR molecule, the target can be precisely targeted to kill the tumor. Ideally, the target antigen should be expressed only in tumor cells and not in normal cells.

[0004] FLT3 (FMS-like tyrosine kinase 3) is a class III receptor tyrosine kinase. FLT3 is a transmembrane protein containing five immunoglobulin-like domains in its extracellular region. Numerous studies have confirmed that activating mutations in FLT3 play a crucial pathological role in the development and progression of AML. Mutant FLT3 induces abnormal activation of multiple intracellular signaling pathways, disrupting the proliferation, differentiation, and apoptosis of normal hematopoietic cells, leading to leukemia. Constitutive activation mutations of FLT3 have been observed in acute myeloid leukemia and acute lymphoblastic leukemia.

[0005] Those skilled in the art have developed methods for FLT3-CAR-T cell targeted therapy for acute myeloid leukemia. However, currently available FLT3-targeting antibodies can only recognize specific FLT3 antigens and cannot detect the CAR transfection efficiency of CAR-T cells. Therefore, providing a monoclonal antibody that can accurately detect CAR transfection efficiency is of great significance in targeted therapy for acute myeloid leukemia. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a monoclonal antibody for recognizing FLT3-CAR molecules and its applications. The monoclonal antibody for recognizing FLT3-CAR molecules described in this invention can specifically recognize FLT3-CAR molecules on FLT3-CAR-T cells and exhibits no cross-reactivity with other molecules. Furthermore, the monoclonal antibody for recognizing FLT3-CAR molecules exhibits high binding activity to FLT3-CAR molecules and can accurately detect CAR transfection efficiency.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a monoclonal antibody that recognizes an FLT3-CAR molecule, wherein the amino acid sequence of the heavy chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:4, SEQ ID NO:11 or SEQ ID NO:18;

[0009] The amino acid sequence of the light chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:7, SEQ ID NO:14 or SEQ ID NO:21.

[0010] Preferably, the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is as shown in SEQ ID NO:2, SEQ ID NO:9 or SEQ ID NO:16.

[0011] Preferably, the amino acid sequence of the heavy chain CDR2 of the monoclonal antibody is as shown in SEQ ID NO:3, SEQ ID NO:10 or SEQ ID NO:17.

[0012] Preferably, the amino acid sequence of the light chain CDR1 of the monoclonal antibody is as shown in SEQ ID NO:6, SEQ ID NO:13 or SEQ ID NO:20.

[0013] Preferably, the amino acid sequence of the light chain CDR2 of the monoclonal antibody is selected from GTS or GAS.

[0014] Preferably, the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:2, the amino acid sequence of CDR2 is shown in SEQ ID NO:3, and the amino acid sequence of CDR3 is shown in SEQ ID NO:4; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:6, the amino acid sequence of CDR2 is GTS, and the amino acid sequence of CDR3 is shown in SEQ ID NO:7.

[0015] Alternatively, the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:9, the amino acid sequence of CDR2 is shown in SEQ ID NO:10, and the amino acid sequence of CDR3 is shown in SEQ ID NO:11; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:13, the amino acid sequence of CDR2 is GAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO:14.

[0016] Alternatively, the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:16, the amino acid sequence of CDR2 is shown in SEQ ID NO:17, and the amino acid sequence of CDR3 is shown in SEQ ID NO:18; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:20, the amino acid sequence of CDR2 is GAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO:21.

[0017] 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:8 or SEQ ID NO:15.

[0018] Preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO:5, SEQ ID NO:12 or SEQ ID NO:19.

[0019] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:5;

[0020] Alternatively, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12;

[0021] Alternatively, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19.

[0022] In this invention, the monoclonal antibody recognizing the FLT3-CAR molecule is an antibody targeting the FLT3-CAR molecule epitope. This invention provides three monoclonal antibodies: 1-B1, 1-B4, and 1-A1. Specifically, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody corresponding to 1-B1 are SEQ ID NO:1 and SEQ ID NO:5; the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody corresponding to 1-B4 are SEQ ID NO:8 and SEQ ID NO:12; and the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody corresponding to 1-A1 are SEQ ID NO:15 and SEQ ID NO:19.

[0023] This invention first immunizes rabbits with an antigen, and peripheral blood is collected for immunotiter testing. After successful immunization, spleen cells are collected, and PCR is performed using amplification primers. The obtained VH-VL fragment is co-transformed with the yeast display vector pDispay into competent yeast cells to construct a single-domain antibody yeast display library, which is then panned to select monoclonal yeast cells.

[0024] 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 FLT3-CAR molecules on FLT3-CAR-T cells, and this specific antibody can be used to detect the transfection efficiency of FLT3-CAR-T cells.

[0025] In a second aspect, the present invention provides a nucleic acid molecule that encodes the monoclonal antibody for recognizing the FLT3-CAR molecule described in the first aspect.

[0026] Preferably, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1-B1 is shown in SEQ ID NO:22; and the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1-B1 is shown in SEQ ID NO:23.

[0027] Preferably, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1-B4 is shown in SEQ ID NO:24; and the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1-B4 is shown in SEQ ID NO:25.

[0028] Preferably, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1-A1 is shown in SEQ ID NO:26; and the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1-A1 is shown in SEQ ID NO:27.

[0029] Thirdly, the present invention provides the use of the monoclonal antibody described in the first aspect for recognizing FLT3-CAR molecules in the detection of CAR molecules in FLT3-CAR-T cells.

[0030] Fourthly, the present invention provides a pharmaceutical composition comprising the monoclonal antibody that recognizes the FLT3-CAR molecule as described in the first aspect.

[0031] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or diluent.

[0032] Fifthly, the present invention provides a kit for detecting FLT3-CAR molecules in a sample, the kit comprising the monoclonal antibody for recognizing FLT3-CAR molecules as described in the first aspect.

[0033] In a sixth aspect, the present invention provides the application of the kit for detecting FLT3-CAR molecules in a sample as described in the fifth aspect in the determination of anti-drug antibody levels.

[0034] The amino acid sequences involved in this invention are shown in Table 1:

[0035] Table 1

[0036]

[0037] In the following amino acid sequences, the underlined sequences represent CDR regions;

[0038] (1) 1-B1: Heavy chain variable region amino acid sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, as shown in SEQ ID NO:1:

[0039] CQSVKESGGRLVTPGTPLTLTCTAS GFSLTSYY MNWVRQAPGKGLEWIGM IGATGTT YYASWARGRFTISKTSSTTVDLKITSPTTEDTATYFC ARHSDPTYIDAPFHP WGPGTLVTVSA.

[0040] SEQ ID NO:2 (Heavy chain CDR1): GFSLTSYY .

[0041] SEQ ID NO:3 (Heavy chain CDR2): IGATGTT .

[0042] SEQ ID NO:4 (Heavy chain CDR3): ARHSDPTYIDAPFHP .

[0043] The amino acid sequence of the light chain variable region is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, as shown in SEQ ID NO:5.

[0044] MTQTPASVEAAVGGTVTIKCQAS QNIYSN LAWYQQKPGQPPKLLIY GTS TLASGVPSRFSGGGSGTDYTLTISGVQCDDAATYYC QNYHGIASYGNA FGGGTEVVVKRTV.

[0045] SEQ ID NO:6 (Light chain CDR1): QNIYSN .

[0046] Light chain CDR2: GTS .

[0047] SEQ ID NO:7 (Light chain CDR3): QNYHGIASYGNA .

[0048] (2) 1-B4: Heavy chain variable region amino acid sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, as shown in SEQ ID NO:8:

[0049] CQSVAESGGRLVTPGTPLTLTCTAS GFSLSSGY MSWVRQAPGKGLEWIGI INNLDNT YYASWAKGRFTISKTSTTVDLKISSPTIEDTATYFC ARGTYVDYFNL WGQGTLVTVSS.

[0050] SEQ ID NO:9 (Heavy chain CDR1): GFSLSSGY .

[0051] SEQ ID NO:10 (Heavy chain CDR2): INNLDNT .

[0052] SEQ ID NO:11 (Heavy chain CDR3): ARGTYVDYFNL .

[0053] The amino acid sequence of the light chain variable region is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, as shown in SEQ ID NO:12.

[0054] LTQTPSSVSEPVGGTVTINCQAS ESISNY LSWYQQKPGQPPKILIY GASKLAAGVSSRFSGSGSGTEFTLTISGVQCDDAATYYC QGGYYSSGATYVA FGGGTEVVVKGDP.

[0055] SEQ ID NO:13 (Light chain CDR1): ESISNY .

[0056] Light chain CDR2: GAS .

[0057] SEQ ID NO:14 (Light chain CDR3): QGGYYSSGATYVA .

[0058] (3) 1-A1: Heavy chain variable region amino acid sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, as shown in SEQ ID NO:15:

[0059] CQSVEESGGDLVKPGASLTLTCTAS GFSFSDSHW ISWVRQAPGKGLEWIAS IHTDSSGST YYATWAKGRFTTSKASSTTVTLQMTSLTAADTATYFC ARGGYSGIYPGPFYLNL WGPGTLVTVSS.

[0060] SEQ ID NO:16 (Heavy chain CDR1): GFSFSDSHW .

[0061] SEQ ID NO:17 (Heavy chain CDR2): IHTDSSGST .

[0062] SEQ ID NO:18 (Heavy chain CDR3): ARGGYSGIYPGPFYLNL .

[0063] The amino acid sequence of the light chain variable region is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, as shown in SEQ ID NO:19.

[0064] ISCQSS QSVYNKNA LSWYQQKPGQPPKLLIY GAS TLASGVPSRFKGSGSGTQFTLTINDVQCADAATYYC LGGYSSTSDDA FGGGTEVVVKRTV.

[0065] SEQ ID NO:20 (Light chain CDR1): QSVYNKNA .

[0066] Light chain CDR2: GAS .

[0067] SEQ ID NO:21 (Light chain CDR3): LGGYSSTSDDA .

[0068] The nucleotide sequences involved in this invention are shown below:

[0069] (1) 1-B1: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1-B1 is shown in SEQ ID NO:22; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1-B1 is shown in SEQ ID NO:23;

[0070] SEQ ID NO:22:

[0071] tgtcagtcggtgaaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagcctctggattctccctcactagttaactacatgaactgggtccgccaggctccagggaaggggctggaatggatcggtatgattggtgctactggtaccacatactacgcg agctgggcgagaggccgattcaccatctccaaaacctcctcgaccacggtggatctgaaaatcaccagtccgacaaccgaggacacggccacctatttctgtgccagacatagtgatcctacttatattgatgccccttttcatccctggggcccaggcaccctggtcaccgtttccgca.

[0072] SEQ ID NO:23:

[0073] atgacccagactccagcctctgtggaggcagctgtgggaggcacagtcaccatcaagtgccaggccagtcagaacatttacagcaatttagcctggtatcaacagaaaccagggcagcctcccaagctcctgatctatggtacatccactctggcatctggggtc ccatcgcggttcagtggcggtggatctgggacagactacactctcaccatcagcggcgtgcagtgtgacgatgctgccacttactactgtcaaaactatcatggtattgctagttatgggaatgctttcggcggagggaccgaagtggtggtcaaacgaactgtg.

[0074] (2) 1-B4: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1-B4 is shown in SEQ ID NO:24; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1-B4 is shown in SEQ ID NO:25;

[0075] SEQ ID NO:24:

[0076] tgtcagtcggtggcggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagcctctggattctccctcagtagcggctacatgagctgggtccgccaggctccagggaaggggctggaatggatcggaatcattaataatttggataacacata ctacgcgagctgggcaaaaggccgattcaccatctccaaaacctcgaccacggtggatctcaaaatctccagtccgacaatcgaggacacggccacctatttctgtgccagagggacttatgtggactactttaatttgtggggccagggcaccctggtcaccgtctcgtca.

[0077] SEQ ID NO:25:

[0078] ctgacccagactccatcttccgtgtctgaacctgtgggaggcacagtcaccatcaattgccaggccagtgaaagcattagcaactacttgtcctggtatcagcagaaaccagggcagcctcccaagatcctgatctacggtgcatcaaaattggctgctggggtctc atcgcgattcagcggcagtggatctgggacggagttcactctcaccatcagcggcgtgcagtgtgacgatgctgccacttactactgtcaaggcggttattatagtagtggtgcgacttacgtggctttcggcggagggaccgaggtggtggtcaaaggtgatcca.

[0079] (3) 1-A1: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1-A1 is shown in SEQ ID NO:26; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1-A1 is shown in SEQ ID NO:27;

[0080] SEQ ID NO:26:

[0081] tgtcagtcggtggaggagtccgggggagacctggtcaagcctggggcgtccctgacactcacctgcacagcctctggattctccttcagtgacagccactggatatcctgggtccgccaggctccagggaaggggctggagtggatcgcctccattcatactgatagttctggtagcacttactacgc gacctgggcgaaaggccgattcaccacctccaaagcctcgtcgaccacggtaacactgcaaatgaccagtctgacagccgcggacacggccacttatttctgtgcgagaggtggctatagtggtatttatcctggtcccttctacttgaacttgtggggcccaggcaccctggtcaccgtctcctcc.

[0082] SEQ ID NO:27:

[0083] atcagttgccagtccagtcagagtgtttataataagaatgccttatcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatggtgcatccactctggcatctggggtcccatcacggttcaaaggcagt ggatctgggacacagttcactctcaccatcaacgacgtgcagtgtgccgatgctgccacttactactgtctaggcggttatagtagtactagtgatgatgctttcggcggagggaccgaggtggtggtcaaacgaactgtg.

[0084] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0085] Compared with the prior art, the present invention has the following beneficial effects:

[0086] The monoclonal antibody that recognizes FLT3-CAR molecules described in this invention can specifically recognize FLT3-CAR molecules on FLT3-CAR-T cells; the monoclonal antibody that recognizes FLT3-CAR molecules has high binding activity to FLT3-CAR molecules, and the monoclonal antibody that recognizes FLT3-CAR molecules can be used to detect the transfection efficiency of FLT3-CAR-T cells. Attached Figure Description

[0087] Figure 1 This is a graph showing the results of agarose gel electrophoresis in Example 2.

[0088] Figure 2 This is a flow cytometry result of the yeast cell bank in Example 3.

[0089] Figure 3 EC2 cells obtained by flow cytometry using the cloned 1-H10 antibody in Example 5 50 Detection results image.

[0090] Figure 4 EC2 cells obtained by flow cytometry using the clone 1-G6 antibody in Example 5 50 Detection results image.

[0091] Figure 5 EC2 cells obtained by flow cytometry using the clone 1-G1 antibody in Example 5 50 Detection results image.

[0092] Figure 6 EC2 cells obtained by flow cytometry using the clonal 1-B11 antibody in Example 5 50 Detection results image.

[0093] Figure 7 EC2 cells obtained by flow cytometry using the clone 1-F11 antibody in Example 5 50 Detection results image.

[0094] Figure 8 EC2 cells obtained by flow cytometry using the clone 1-E6 antibody in Example 5 50 Detection results image.

[0095] Figure 9 EC2 cells obtained by flow cytometry using clone 1-E4 antibody in Example 5 50 Detection results image.

[0096] Figure 10 EC200 using flow cytometry with clone 1-A8 antibody in Example 5 50 Detection results image.

[0097] Figure 11 The EC of clone 1-B4 antibody flow cytometry in Example 5 50 Detection results image.

[0098] Figure 12 EC200 using clonal 1-A1 antibody flow cytometry in Example 5 50 Detection results image.

[0099] Figure 13 It is the EC of the clone 1-B1 antibody flow cytometry in Example 5. 50 Detection results image.

[0100] Figure 14 This is a flow cytometry result of the three candidate antibodies conjugated with FITC fluorescein in Example 6. Detailed Implementation

[0101] 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 in any way.

[0102] 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.

[0103] Example 1: Immunizing rabbits with antigen

[0104] The FLT3 VHH antigen (2-A5-(G4S)3-2-A5-His) prepared in the previous stage of the project was used as an immunogen to immunize four New Zealand white rabbits (Zhenhu Experimental Animal Technology Co., Ltd.). Multiple subcutaneous immunizations were administered, with a total of four immunizations given at 14-day intervals. The single immunization dose was 100 μg immunogen per rabbit. After the three immunizations were completed, blood was collected from the ear vein, and the serum was separated to determine the immunogenicity.

[0105] Methods for detecting immunogenicity: 1 mL of peripheral blood was collected, and the centrifuge tube containing the blood sample was placed in a 37°C incubator for 1 hour. The blood sample was then transferred to 4°C and incubated overnight, followed by centrifugation at 2000 rpm and 18°C ​​for 20 min. Serum was separated and subjected to limiting dilution according to the dilution gradient shown in Table 2. The diluted serum was then added to a 96-well plate pre-coated with 1 μg / mL 2-A5-(G4S)3-2-A5-His antigen. Immunogenicity was detected using enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 2. The results indicate that the immune serum binds to the target protein, and the OD value increases with the serum dilution gradient. 450 The values ​​showed a gradient change, indicating successful immunization. Spleen cells were collected to establish a rabbit monoclonal antibody yeast display library.

[0106] Table 2

[0107]

[0108] Example 2: Amplification of VH-VL antibody fragments

[0109] After collecting rabbit spleen cells, the cells were lysed using Trizol lysis buffer, RNA was extracted, and reverse transcription was performed using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#: 6210B) to prepare cDNA. PCR was then performed using rabbit monoclonal antibody amplification primers, and the PCR products were analyzed by electrophoresis using 1% agarose gel. The results are as follows: Figure 1 Fragments with a molecular weight of approximately 300-400 bp were isolated. PCR products were recovered using a gel extraction kit (Qiagen, CAT#:28706), and concentrations were determined using NanoDrop. Heavy chain (VH) and light chain (VL) were obtained separately, and VH-VL single-chain antibody sequences were constructed using overlap PCR. The VH-VL fragments were co-transformed into competent yeast cells using the yeast display vector pDisplay (iCarTab) to construct a single-domain antibody yeast display library.

[0110] Example 3: Construction and selection of yeast display library

[0111] Construction of yeast display library

[0112] Step 1: Construction of the yeast display vector: The pDisplay vector was digested with SfiI (NEB, CAT#: R0123L) overnight at 50°C. The display vector fragment was separated using a 1% agarose gel, and a ~5000bp fragment was extracted for gel recovery. Simultaneously, the PCR digestion products were purified using a DNA fragment recovery kit (TakaRa, CAT#: 9761), and the concentration was determined using NanoDrop.

[0113] Step 2: Electroporation of Yeast Competent Cells: Prepare electroporation cuvettes, ligation products, and electroporation competent cells, and pre-chill them on ice. Add the pre-chilled VH-VL fragment and the enzyme-digested display vector to the electroporation competent cells, place them on ice for 1 min, add 70 μL of DNA / competent cell mixture to each cuvette, and place the cuvettes on ice. Perform electroporation at 1500V for 5 ms. After electroporation, immediately resuspend the cells in culture medium equilibrated to room temperature and incubate at 37°C with shaking for 1 hour. Separately, take 20 μL of the bacterial culture, dilute it with 980 μL of culture medium, take 100 μL of the diluted product, dilute it again with 900 μL of culture medium, and take 50 μL to spread evenly on an agar plate. Incubate at 37°C for 3-5 days. Remove the plate, calculate the number of clones that can be generated by each ligation, and calculate the library capacity. At the same time, pick 20 single clones from the plate, incubate them overnight at 37°C with shaking, and send them for sequencing to calculate the library diversity.

[0114] Panning of the yeast display library: The antigen was diluted to 50 μg / mL with PBS, conjugated with biotin, and added to the yeast display library for incubation for 1 hour. After incubation, magnetic beads pre-coated with streptavidin were added, and incubation continued for 30 minutes. The display library was then adsorbed using magnetic poles, and yeast cells that failed to bind to the magnetic beads were removed. The yeast cells adsorbed on the magnetic beads were cultured overnight, followed by induction medium for another 18 hours. A portion of the yeast library was analyzed by flow cytometry to detect the positive rate of clones that could bind to the target antigen. The results are as follows: Figure 2 As shown, the results indicate that after two rounds of magnetic sorting, 26% of the clones in the display library could bind to the target antigen. Figure 2 D).

[0115] Figure 2 For flow cytometry results of yeast libraries: A: Original library without induction; B: Original library induced for expression for 48 hours; C: Library induced for expression for 48 hours after one round of magnetic sorting; D: Library induced for expression for 48 hours after two rounds of magnetic sorting; Alexa Fluor 647: V5 tag; PE: Target antigen.

[0116] Repeat the above selection steps 2-3 times, plate the cells, select yeast monoclonal cells for flow cytometry detection, select monoclonal yeast cells from the yeast display library after one round of magnetic sorting, induce expression, and incubate with biotinylate antigen protein and PE-streptavidin respectively. Flow cytometry is used to detect the positive rate of candidate clones. The results are shown in Table 3. Select yeast monoclonal cells with positive flow cytometry results for Sanger sequencing analysis.

[0117] Table 3

[0118]

[0119]

[0120] Example 4: Transient transfection expression of single-domain antibodies

[0121] Remove the LVTransm transfection reagent (iCarTab, Cat#LVTran100) and antibody expression vector pcDNA3.4-hIgG1-Fc2 from the freezer. After thawing at room temperature, mix thoroughly by pipetting. Remove the PBS buffer and warm to room temperature. Add 500 μL of PBS to one well of a 24-well plate, add 4 μg of pcDNA3.4-hIgG1-Fc2, mix thoroughly by pipetting, then add 12 μL of LVTransm and immediately mix by pipetting. Let stand at room temperature for 10 minutes. This mixture is called the DNA / LVTransm complex. Add 532 μL of the DNA / LVTransm complex to 1.5 mL of 293F cells and gently shake to mix thoroughly. Cells were placed in a 37°C, 5% CO2 incubator and cultured at 130 rpm for 6–8 hours. Then, 1.5 mL of fresh 293 medium was added, and the cells were returned to the incubator for further culture. After 3 consecutive days of culture, the supernatant was collected by centrifugation, filtered through a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube for subsequent flow cytometry and ELISA detection.

[0122] Example 5: Detection of the binding affinity between candidate antibodies and target proteins

[0123] ELISA experimental procedure: Take a 96-well microplate, dilute the antigen to 2 μg / mL with CBS buffer, add 100 μL to each well, seal, and incubate overnight at 4°C; remove the coated microplate, remove the coated antigen, and wash 3 times with PBST; add 200 μL / well of 5% BSA and block at room temperature for 1 hour; remove the blocking buffer, wash 3 times with PBST, add serially diluted candidate antibodies, and incubate at room temperature for 1 hour; wash 3 times with PBST, add diluted HRP-Protein A, and incubate at room temperature for 30 minutes; wash 3 times with PBST, add TMB chromogenic solution; read the OD using a microplate reader.450 The numerical values ​​and results are shown in Table 4.

[0124] Table 4

[0125]

[0126]

[0127] The results in Table 4 show that, except for one clone (1-G8) which binds weakly to the target antigen, all other clones can bind to the target antigen protein.

[0128] Flow cytometry experimental procedure: Resuscitate the cell line expressing the target gene and the negative control cell line from liquid nitrogen and adjust the cell state to the logarithmic growth phase; divide both cell lines into several fractions, with each fraction containing 5 × 10⁻⁶ cells. 5 Cells were incubated with the expressed antibody separately, and after thorough mixing, the cells were incubated at room temperature for 1 hour. After centrifugation at 800g for 5 minutes at room temperature, the supernatant containing the antibody was removed, and the cells were washed three times with PBS. 1 μL of PE-labeled Anti-human IgG (eBioscience, Cat#:12-4998-82) was added, and after thorough mixing, the cells were incubated at room temperature in the dark for 30 minutes. After centrifugation at 800g for 5 minutes at room temperature, the supernatant containing the secondary antibody was removed, and the cells were washed three times with PBS. The cells were resuspended in 500 μL of PBS for flow cytometry analysis. Serially diluted candidate antibodies were incubated with cell lines expressing the target gene, and flow cytometry was used to detect the binding of candidate antibodies to target cells at different concentrations. The results showed that all antibodies could bind to the target antigen protein with high affinity.

[0129] Figure 3-13 EC for candidate antibody flow cytometry 50 The detection, among which, Figure 3 It is an EC by flow cytometry of clonal 1-H10 antibody. 50 Test result image; Figure 4 It is an EC by flow cytometry of clonal 1-G6 antibody. 50 Test result image; Figure 5 It is an EC by flow cytometry of clonal 1-G1 antibody. 50 Test result image; Figure 6 It is an EC by flow cytometry of clonal 1-B11 antibody. 50 Test result image; Figure 7 It is EC by flow cytometry of Cloning 1-F11 antibody. 50 Test result image; Figure 8 It is an EC cell culture of clonal 1-E6 antibody flow cytometry. 50 Test result image; Figure 9 It is an EC by flow cytometry of clonal 1-E4 antibody. 50Test result image; Figure 10 It is an EC by flow cytometry of clone 1-A8 antibody. 50 Test result image; Figure 11 It is an EC by flow cytometry of clonal 1-B4 antibody. 50 Test result image; Figure 12 It is an EC by flow cytometry of clonal 1-A1 antibody. 50 Test result image; Figure 13 It is an EC by flow cytometry of clonal 1-B1 antibody. 50 Detection results image.

[0130] Recombinant antibody affinity assay procedure: Two candidate clones were selected, and the target protein was immobilized on a chip. The binding ability of the candidate single-domain antibody to the target protein was detected using the prepared candidate single-domain antibody as the mobile phase. The affinity assay results of 1-A1 and 1-B1 antibodies are shown in Table 5.

[0131] Table 5

[0132] Antibody <![CDATA[Kon(M -1 s -1 )]]> <![CDATA[Koff(s -1 )]]> KD(M) 1-A1 <![CDATA[6.113×10 4 ]]> <![CDATA[1.880×10 -7 ]]> <![CDATA[3.075×10 -12 ]]> 1-B1 <![CDATA[3.012×10 4 ]]> <![CDATA[1.87010 -7 ]]> <![CDATA[6.208×10 -12 ]]>

[0133] Affinity testing results showed that the two selected candidate clones (1-A1, 1-B1) had high affinity for the target antigen protein.

[0134] Example 6: Antibody Screening Flow Cytometry Detection

[0135] Three candidate antibodies (1-B1, 1-B4, and 1-A1) were selected, labeled with FITC, and flow cytometry was used to detect the binding of the candidate antibodies to recombinant cells overexpressing the target gene.

[0136] Cell lines expressing the target gene (FLT3-CAR-Jurkat cells) and negative control cell lines (Jurkat cells) were resuscitated in liquid nitrogen and adjusted to the logarithmic growth phase. Each cell type was then divided into several fractions, with each fraction containing 5 × 10⁻⁶ cells. 5 Cells were collected, each with 1 mL PBS, centrifuged at 1200g for 3 min at room temperature, the supernatant was discarded, and the cells were resuspended in 100 μL PBS. 1 μg of each of the three monoclonal antibodies was added, followed by 10 mL of CD3-PE (BD, CAT#555333). Cells were incubated at room temperature in the dark for 12 min. After incubation, 1 mL of 1×PBS was added to wash the cells, and the cells were centrifuged at 1200g for 3 min. The supernatant was discarded, and the cells were resuspended in 200 μL of 1×PBS. Then, 2 μL of 7-AAD (BD, CAT#559925) was added, and the cells were analyzed by flow cytometry. Results are as follows: Figure 14As shown, the results indicate that the positive rate of the antibody with the negative control Jurkat cell line is <1%, with no non-specific binding. The three monoclonal antibodies have a binding activity of >90% with FLT3-CAR-Jurkat cells. Among them, the antibodies of clones 1-B1 and 1-B4 have higher binding activity with FLT3-CAR-Jurkat cells. The antibodies of clones 1-B1 and 1-B4 can be used as antibodies to detect CAR molecules in FLT3-CAR-T cells.

[0137] In summary, this invention first immunizes rabbits with an antigen, and then uses peripheral blood for immunotiter testing. After successful immunization, spleen cells are collected, and PCR is performed using amplification primers. The obtained VH-VL fragment is co-transformed with the yeast display vector pDispay into competent yeast cells to construct a single-domain antibody yeast display library. This library is then panned to select monoclonal yeast cells, yielding a monoclonal antibody that recognizes the FLT3-CAR molecule. This monoclonal antibody specifically recognizes the FLT3-CAR molecule on FLT3-CAR-T cells, and this specific antibody can be used to detect the transfection efficiency of FLT3-CAR-T cells.

[0138] 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 FLT3-CAR molecule, characterized in that, The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:2, the amino acid sequence of CDR2 is shown in SEQ ID NO:3, and the amino acid sequence of CDR3 is shown in SEQ ID NO:4; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:6, the amino acid sequence of CDR2 is GTS, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

7.

2. The monoclonal antibody for recognizing FLT3-CAR molecules 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:

1.

3. The monoclonal antibody for recognizing FLT3-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:

5.

4. The monoclonal antibody recognizing the FLT3-CAR molecule according to any one of claims 1-3, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

5.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the monoclonal antibody that recognizes the FLT3-CAR molecule according to any one of claims 1-4.

6. Use of the monoclonal antibody recognizing the FLT3-CAR molecule according to any one of claims 1-4 in the preparation of a reagent for detecting the CAR molecule in FLT3-CAR-T cells.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the monoclonal antibody that recognizes the FLT3-CAR molecule according to any one of claims 1-4.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

9. A kit for detecting FLT3-CAR molecules in a sample, characterized in that, The kit comprises the monoclonal antibody that recognizes the FLT3-CAR molecule according to any one of claims 1-4.

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