Monoclonal antibody for recognizing FLT3-CAR molecule and application thereof

Abstract

The invention provides a monoclonal antibody for recognizing FLT3-CAR molecules and application of the monoclonal antibody. The amino acid sequence of a heavy chain CDR3 of the monoclonal antibody is shown as 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 as shown in SEQ ID NO: 8, SEQ ID NO: 15 or SEQ ID NO: 22. The monoclonal antibody can specifically recognize an FLT3-CAR molecule on an FLT3-CAR-T cell, and has no cross reaction with other molecules; according to the present invention, the binding activity of the monoclonal antibody and the FLT3-CAR molecule is high, the flow type analysis method for the CAR transfection positive rate detection of the FLT3-CAR-T cell is developed, and the FLT3-CAR molecule transfection efficiency can be accurately and specifically detected.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a monoclonal antibody for identifying FLT3-CAR molecules and its applications. Background Art

[0002] Acute myeloid leukemia (AML) is a highly heterogeneous myeloid hematopoietic stem / progenitor cell malignancy and one of the most common types of leukemia in adults, accounting for about half of all leukemia patients. The main feature of AML is the abnormal proliferation of primitive and immature myeloid cells in the bone marrow and peripheral blood. The clinical symptoms mainly include anemia, bleeding, infection and fever, organ infiltration, metabolic abnormalities, etc. AML progresses rapidly and is highly invasive. Most cases are critically ill and have a poor prognosis. If not treated in time, it often endangers life.

[0003] In recent years, chimeric antigen receptor T cells (CAR-T) have shown significant clinical efficacy in the treatment of relapsed and refractory lymphoma. The CAR-T therapy activates the patient's T lymphocytes through gene modification technology and then inserts a specific tumor antigen receptor gene to form modified T cells, which can then specifically recognize and bind to the antigen on the surface of tumor cells to achieve specific killing of tumor cells. One of the keys to the success of this technology is to select a suitable target antigen, and by constructing the corresponding CAR molecule, the target can be accurately targeted to kill tumors. The best target antigen should be expressed only in tumor cells and not in normal cells.

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

[0005] Those skilled in the art have developed a method for targeted treatment of acute myeloid leukemia with FLT3-CAR-T cells. However, the currently disclosed FLT3 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 the CAR transfection efficiency is of great significance in the targeted treatment of acute myeloid leukemia. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a monoclonal antibody that recognizes the FLT3-CAR molecule and its applications. The monoclonal antibody that recognizes the FLT3-CAR molecule according to the present invention can specifically recognize the FLT3-CAR molecule on FLT3-CAR-T cells and has no cross-reaction with other molecules; the monoclonal antibody that recognizes the FLT3-CAR molecule has a high binding activity with the FLT3-CAR molecule and can accurately detect the CAR transfection efficiency.

[0007] To achieve the purpose of this invention, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a monoclonal antibody that recognizes the FLT3-CAR molecule, and the amino acid sequence of the heavy-chain CDR3 of the monoclonal antibody is shown as 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 as 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 shown as 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 shown as 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 shown as 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 as SEQ ID NO:2, the amino acid sequence of CDR2 is shown as SEQ ID NO:3, and the amino acid sequence of CDR3 is shown as SEQ ID NO:4; the amino acid sequence of the light-chain CDR1 of the monoclonal antibody is shown as SEQ ID NO:6, the amino acid sequence of CDR2 is GTS, and the amino acid sequence of CDR3 is shown as SEQ ID NO:7;

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

[0016] Alternatively, the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is as shown in SEQ ID NO:16, the amino acid sequence of CDR2 is as shown in SEQ ID NO:17, and the amino acid sequence of CDR3 is as shown in SEQ ID NO:18; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is as shown in SEQ ID NO:20, the amino acid sequence of CDR2 is GAS, and the amino acid sequence of CDR3 is as 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 as shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:12;

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

[0022] In the present invention, the monoclonal antibody that recognizes the FLT3-CAR molecule is an antibody against the epitope of the FLT3-CAR molecule. Three monoclonal antibodies are provided in the present invention, namely 1-B1, 1-B4, and 1-A1. Among them, 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; 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] In the present invention, rabbits are first immunized with the antigen, and the immune titer of the peripheral blood is detected. After successful immunization, spleen cells are collected, PCR is performed using amplification primers, the obtained VH-VL fragment is co-transformed with the yeast display vector pDispay into yeast competent cells, a single-domain antibody yeast display library is constructed and panned, and monoclonal yeasts are selected.

[0024] In addition, those skilled in the art can also easily obtain the structure of the antibody of the present invention (such as the heavy chain variable region and the light chain variable region of the antibody), and then the monoclonal antibody of the present invention can be prepared by recombinant methods. The antibody in the present invention can specifically recognize the FLT3-CAR molecule on FLT3-CAR-T cells, and such a 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 that recognizes the FLT3-CAR molecule according to the first aspect.

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

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

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

[0029] In a third aspect, the present invention provides the use of the monoclonal antibody that recognizes the FLT3-CAR molecule described in the first aspect in detecting the CAR molecule of FLT3-CAR-T cells.

[0030] In a fourth aspect, the present invention provides a pharmaceutical composition, which comprises the monoclonal antibody that recognizes the FLT3-CAR molecule described in the first aspect.

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

[0032] In a fifth aspect, the present invention provides a kit for detecting the FLT3-CAR molecule in a sample, which comprises the monoclonal antibody that recognizes the FLT3-CAR molecule described in the first aspect.

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

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

[0035] Table 1

[0036]

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

[0038] (1) 1-B1: Amino acid sequence of the heavy chain variable region: 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] Amino acid sequence of the light chain variable region: 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: Amino acid sequence of the heavy chain variable region: 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] Amino acid sequence of the light chain variable region: 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: Amino acid sequence of the heavy chain variable region: 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] Amino acid sequence of the light chain variable region: 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 the present invention are shown as follows:

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

[0070] SEQ ID NO: 22:

[0071] tgtcagtcggtgaaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagcctctggattctccctcactagttactacatgaactgggtccgccaggctccagggaaggggctggaatggatcggtatgattggtgctactggtaccacatactacgcgagctgggcgagaggccgattcaccatctccaaaacctcctcgaccacggtggatctgaaaatcaccagtccgacaaccgaggacacggccacctatttctgtgccagacatagtgatcctacttatattgatgccccttttcatccctggggcccaggcaccctggtcaccgtttccgca.

[0072] SEQ ID NO: 23:

[0073] atgacccagactccagcctctgtggaggcagctgtgggaggcacagtcaccatcaagtgccaggccagtcagaacatttacagcaatttagcctggtatcaacagaaaccagggcagcctcccaagctcctgatctatggtacatccactctggcatctggggtcccatcgcggttcagtggcggtggatctgggacagactacactctcaccatcagcggcgtgcagtgtgacgatgctgccacttactactgtcaaaactatcatggtattgctagttatgggaatgctttcggcggagggaccgaagtggtggtcaaacgaactgtg。

[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] tgtcagtcggtggcggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagcctctggattctccctcagtagcggctacatgagctgggtccgccaggctccagggaaggggctggaatggatcggaatcattaataatttggataacacatactacgcgagctgggcaaaaggccgattcaccatctccaaaacctcgaccacggtggatctcaaaatctccagtccgacaatcgaggacacggccacctatttctgtgccagagggacttatgtggactactttaatttgtggggccagggcaccctggtcaccgtctcgtca。

[0077] SEQ ID NO:25:

[0078] ctgacccagactccatcttccgtgtctgaacctgtgggaggcacagtcaccatcaattgccaggccagtgaaagcattagcaactacttgtcctggtatcagcagaaaccagggcagcctcccaagatcctgatctacggtgcatcaaaattggctgctggggtctcatcgcgattcagcggcagtggatctgggacggagttcactctcaccatcagcggcgtgcagtgtgacgatgctgccacttactactgtcaaggcggttattatagtagtggtgcgacttacgtggctttcggcggagggaccgaggtggtggtcaaaggtgatcca。

[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] tgtcagtcggtggaggagtccgggggagacctggtcaagcctggggcgtccctgacactcacctgcacagcctctggattctccttcagtgacagccactggatatcctgggtccgccaggctccagggaaggggctggagtggatcgcctccattcatactgatagttctggtagcacttactacgcgacctgggcgaaaggccgattcaccacctccaaagcctcgtcgaccacggtaacactgcaaatgaccagtctgacagccgcggacacggccacttatttctgtgcgagaggtggctatagtggtatttatcctggtcccttctacttgaacttgtggggcccaggcaccctggtcaccgtctcctcc。

[0082] SEQ ID NO:27:

[0083] atcagttgccagtccagtcagagtgtttataataagaatgccttatcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatggtgcatccactctggcatctggggtcccatcacggttcaaaggcagtggatctgggacacagttcactctcaccatcaacgacgtgcagtgtgccgatgctgccacttactactgtctaggcggttatagtagtactagtgatgatgctttcggcggagggaccgaggtggtggtcaaacgaactgtg。

[0084] The numerical ranges described in the present invention include not only the point values listed above, but also any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of brevity, the present invention does not exhaustively list the specific point values included in the described ranges.

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

[0086] The monoclonal antibody that recognizes the FLT3-CAR molecule of the present invention can specifically recognize the FLT3-CAR molecule on FLT3-CAR-T cells; the monoclonal antibody that recognizes the FLT3-CAR molecule has a high binding activity with the FLT3-CAR molecule, and the monoclonal antibody that recognizes the FLT3-CAR molecule can be used to detect the transfection efficiency of FLT3-CAR-T cells. Description of the Drawings

[0087] Figure 1 It is the detection result diagram of agarose gel electrophoresis in Example 2.

[0088] Figure 2 It is the flow cytometry result diagram of the yeast library in Example 3.

[0089] Figure 3 It is the EC of flow cytometry of clone 1-H10 antibody in Example 5 50 detection result diagram.

[0090] Figure 4 It is the EC of flow cytometry of clone 1-G6 antibody in Example 5 50 detection result diagram.

[0091] Figure 5 It is the EC of flow cytometry of clone 1-G1 antibody in Example 5 50 detection result diagram.

[0092] Figure 6 It is the EC of flow cytometry of Clone 1-B11 antibody in Example 5 50 Detection result graph.

[0093] Figure 7 It is the EC of flow cytometry of Clone 1-F11 antibody in Example 5 50 Detection result graph.

[0094] Figure 8 It is the EC of flow cytometry of Clone 1-E6 antibody in Example 5 50 Detection result graph.

[0095] Figure 9 It is the EC of flow cytometry of Clone 1-E4 antibody in Example 5 50 Detection result graph.

[0096] Figure 10 It is the EC of flow cytometry of Clone 1-A8 antibody in Example 5 50 Detection result graph.

[0097] Figure 11 It is the EC of flow cytometry of Clone 1-B4 antibody in Example 5 50 Detection result graph.

[0098] Figure 12 It is the EC of flow cytometry of Clone 1-A1 antibody in Example 5 50 Detection result graph.

[0099] Figure 13 It is the EC of flow cytometry of Clone 1-B1 antibody in Example 5 50 Detection result graph.

[0100] Figure 14 It is the detection result graph of flow cytometry after conjugating three candidate antibodies with FITC fluorescein in Example 6. Specific implementation manners

[0101] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0102] For those not specifying specific techniques or conditions in the examples, they shall be carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels of commercial purchase.

[0103] Example 1: Immunizing rabbits with antigens

[0104] Using the FLT3 VHH antigen (2-A5-(G4S)3-2-A5-His) prepared in the early stage of the project as an immunogen, 4 New Zealand white rabbits (Zhenhu Experimental Animal Technology Co., Ltd.) were immunized by multiple subcutaneous injections. A total of four immunizations were carried out at an interval of 14 days, and the single immunization dose was 100 μg immunogen / rabbit. After three immunizations, blood was collected from the ear vein, and after separating the serum, the immune titer was detected.

[0105] Method for detecting immune titer: 1 mL of peripheral blood was collected, and the centrifuge tube containing the blood sample was placed in an incubator at 37 °C for 1 hour; then the blood sample was transferred to 4 °C overnight and centrifuged at 2000 rpm and 18 °C for 20 min; the serum was separated and serially diluted according to the dilution gradient shown in Table 2. After serial dilution of the serum, it was added to a 96-well plate pre-coated with 1 μg / mL 2-A5-(G4S)3-2-A5-His antigen, and the enzyme-linked immunosorbent assay (ELISA) was used to detect the immune titer. The results are shown in Table 2, indicating that the immune serum binds to the target protein, and the OD 450 value changes in a gradient with the serum dilution gradient, 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 fragment

[0109] After collecting rabbit spleen cells, the cells were lysed with Trizol lysis solution to extract RNA. Reverse transcription was performed using PrimeScriptTM II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#: 6210B) to prepare cDNA. PCR was carried out using rabbit monoclonal antibody amplification primers, and 1% agarose was used for electrophoresis analysis of the PCR products. The results were as Figure 1 , and fragments with a molecular weight of about 300 - 400 bp were isolated. The PCR products were recovered using a gel extraction kit (Qiagen, CAT#: 28706), and the concentration was measured using NanoDrop. After obtaining the heavy chain VH and light chain VL respectively, the VH-VL single-chain antibody sequence was constructed by overlap PCR (overlap extension PCR). The VH-VL fragment was co-transformed with the yeast display vector pDisplay (iCarTab) into yeast competent cells to construct a single-domain antibody yeast display library.

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

[0111] Construction of yeast display library

[0112] Step 1: Construct a yeast display vector: Digest the pDisplay vector with SfiI (NEB, CAT#: R0123L) at 50 °C overnight. Separate the display vector fragment using 1% agarose gel, cut out the ~5000 bp vector fragment for gel extraction; at the same time, use a DNA fragment recovery kit (TakaRa, CAT#: 9761) to purify the PCR digestion product, and measure the concentration with NanoDrop.

[0113] Step 2: Electrotransform yeast competent cells: Prepare the electroporation cuvette, ligation product, and electrotransformation competent cells and pre-cool them on ice; take the pre-cooled VH-VL fragment and the digested display vector and add them to the electrotransformation competent cells, place them on ice for 1 min, add 70 μL of the DNA / competent cell mixture to each electroporation cuvette, and place the electroporation cuvette on ice; perform electroporation at 1500 V, 5 ms; immediately after electroporation, add medium equilibrated to room temperature to resuspend the cells, and culture them on a shaker at 37 °C for 1 hour. Additionally, take 20 μL of the bacterial solution, add 980 μL of medium for dilution, then take 100 μL of the diluted product, add 900 μL of medium for the second dilution, take 50 μL and spread it evenly on an agar plate, and culture it at 37 °C for 3 - 5 days. Take out the plate, calculate the number of clones that can be produced by each ligation, and calculate the library capacity. At the same time, pick 20 monoclonal colonies on the plate, culture them overnight with shaking at 37 °C, send the bacterial solution for sequencing, and calculate the diversity of the library.

[0114] Panning of the yeast display library: Dilute the antigen to 50 μg / mL with PBS, conjugate it with biotin, and then add it to the yeast display library for incubation for 1 hour; after incubation, add magnetic beads pre-coated with streptavidin, and continue to incubate for 30 min; use a magnetic pole to adsorb the display library and remove the yeast that fails to bind to the magnetic beads. Take the yeast adsorbed on the magnetic beads and culture it overnight, then add induction medium and continue to induce for 18 hours. Take a part of the yeast library for flow cytometry analysis to detect the positive rate of clones that can bind to the target antigen. The results are as Figure 2 shown. The results show that after two rounds of magnetic sorting, 26% of the clones in the display library can bind to the target antigen ( Figure 2 D).

[0115] Figure 2 Flow cytometry results of the yeast library. A: The original library without induction; B: The original library induced for 48 hours; C: The library after one round of magnetic sorting induced for 48 hours; D: The library after two rounds of magnetic sorting induced for 48 hours; AlexaFluor 647: V5 tag; PE: Target antigen.

[0116] Repeat the above panning steps 2-3 times, coat the plates, pick yeast monoclonal colonies for flow cytometry detection. Pick monoclonal yeast cells from the yeast display library after one round of magnetic sorting. After induction of expression, incubate them with biotinylated antigen protein and PE-streptavidin respectively, and use flow cytometry to detect the positive rate of candidate clones. The results are shown in Table 3. Select the yeast monoclonal cells with positive flow cytometry results for Sanger sequencing analysis.

[0117] Table 3

[0118]

[0119]

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

[0121] Take out the LVTransm transfection reagent (iCarTab, Cat#LVTran100) and the antibody expression vector pcDNA3.4-hIgG1-Fc2 from the refrigerator, thaw them at room temperature, and mix well by pipetting up and down. Take out the PBS buffer and warm it to room temperature. Add 500 μL of PBS to a well of a 24-well plate, add 4 μg of pcDNA3.4-hIgG1-Fc2, mix well by pipetting up and down, then add 12 μL of LVTransm, and immediately mix well by pipetting. Let it stand at room temperature for 10 minutes. The mixture here is called the DNA / LVTransm complex; add the above 532 μL of DNA / LVTransm complex to 1.5 mL of 293F cells, and gently shake to mix well. Place the cells in a 37°C, 5% CO2 incubator and culture at 130 rpm for 6-8 hours, then add 1.5 mL of fresh 293 medium, and put the cells back into the incubator for continued culture; after continuous culture for 3 days, centrifuge to collect the culture medium supernatant, filter it through a 0.45 μm filter membrane, and transfer the filtrate to a sterile centrifuge tube for subsequent flow cytometry and ELISA detection.

[0122] Example 5: Detection of binding affinity between candidate antibody and target protein

[0123] ELISA experimental procedure: Take a 96-well ELISA plate, dilute the antigen with CBS buffer to 2 μg / mL, add 100 μL to each well, seal it, and incubate it overnight at 4°C; take out the coated ELISA plate, remove the coated antigen, and wash it 3 times with PBST; add 200 μL / well of 5% BSA and block it at room temperature for 1 hour; after removing the blocking buffer, wash it 3 times with PBST, add the serially diluted candidate antibody, and incubate it at room temperature for 1 hour; wash it 3 times with PBST, add the diluted HRP-Protein A, and incubate it at room temperature for 30 minutes; after washing it 3 times with PBST, add the TMB chromogenic solution; read the OD value with an ELISA reader450 The numerical values are shown in Table 4.

[0124] Table 4

[0125]

[0126]

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

[0128] Flow cytometry experimental procedure: Resuscitate the cell line highly expressing the target gene and the negative control cell line from liquid nitrogen, and adjust the cell state to the logarithmic growth phase; Divide the two types of cells into several portions, with the number of cells in each portion being 5×10 5 cells; Incubate the expressed antibodies with the target cells respectively, mix well, and incubate at room temperature for 1 hour. Centrifuge at 800g for 5 minutes at room temperature, discard the supernatant containing the antibody, and wash the cells 3 times with PBS; Add 1 μL of PE-labeled Anti-human IgG (eBioscience, Cat#: 12-4998-82), mix well, and incubate in the dark at room temperature for 30 minutes; Centrifuge at 800g for 5 minutes at room temperature, discard the supernatant containing the secondary antibody, and wash the cells 3 times with PBS; Resuspend the cells with 500 μL of PBS for flow cytometry analysis. After gradient dilution of the candidate antibodies, incubate them with the cell line expressing the target gene, and detect the binding of the candidate antibodies to the target cells under different concentrations by flow cytometry. The results show that all antibodies can bind to the target antigen protein with high affinity.

[0129] Figures 3 - 13 For the EC 50 detection of the candidate antibody by flow cytometry, where Figure 3 is the EC 50 detection result graph of the clone 1-H10 antibody by flow cytometry; Figure 4 is the EC 50 detection result graph of the clone 1-G6 antibody by flow cytometry; Figure 5 is the EC 50 detection result graph of the clone 1-G1 antibody by flow cytometry; Figure 6 is the EC 50 detection result graph of the clone 1-B11 antibody by flow cytometry; Figure 7 is the EC 50 detection result graph of the clone 1-F11 antibody by flow cytometry; Figure 8 is the EC 50 detection result graph of the clone 1-E6 antibody by flow cytometry; Figure 9 is the EC 50Detection result graph; Figure 10 is the EC of clone 1-A8 antibody by flow cytometry 50 Detection result graph; Figure 11 is the EC of clone 1-B4 antibody by flow cytometry 50 Detection result graph; Figure 12 is the EC of clone 1-A1 antibody by flow cytometry 50 Detection result graph; Figure 13 is the EC of clone 1-B1 antibody by flow cytometry 50 Detection result graph.

[0130] Experimental procedure for detecting the affinity of recombinant antibodies: For the 2 selected candidate clones, the target protein was immobilized on the chip, and the prepared candidate single-domain antibodies were used as the mobile phase respectively to detect the binding ability of the candidate single-domain antibodies to the target protein. The affinity detection 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] The affinity detection results show that the 2 selected candidate clones (1-A1, 1-B1) have a high affinity for the target antigen protein.

[0134] Example 6: Flow cytometry for antibody screening

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

[0136] Resuscitate the cell line with high expression of the target gene (FLT3-CAR-Jurkat cells) and the negative control cell line (Jurkat cells) from liquid nitrogen, and adjust the cell state to the logarithmic growth phase; divide the two types of cells into several parts respectively, with the number of cells in each part being 5×10 5 cells. Add 1 mL of PBS to each, centrifuge at 1200 g at room temperature for 3 min, discard the supernatant, add 100 μL of PBS to resuspend the cells, add 1 μg of each of the three monoclonal antibodies, and add 10 mL of CD3-PE (BD, CAT#555333) to each. Incubate at room temperature in the dark for 12 min. After incubation, add 1 mL of 1×PBS to wash the cells respectively, and centrifuge at 1200 g for 3 min. Discard the supernatant, add 200 μL of 1×PBS to resuspend the cells respectively, then add 2 μL of 7-AAD (BD, CAT#559925), and perform flow cytometry detection. The results are as Figure 14As shown, the results indicate that the positive rate of the antibody and the negative control Jurkat cell line is <1%, without non-specific binding. The binding activities of the three monoclonal antibodies to FLT3-CAR-Jurkat cells are >90%. Among them, the antibodies with clone numbers 1-B1 and 1-B4 have relatively high binding activities to FLT3-CAR-Jurkat cells. The antibodies with clone numbers 1-B1 and 1-B4 can be used as antibodies for detecting the CAR molecule of FLT3-CAR-T cells.

[0137] In summary, in the present invention, rabbits are first immunized with an antigen, and the immune titer of the peripheral blood is detected. After successful immunization, spleen cells are collected, PCR is performed using amplification primers, the obtained VH-VL fragment is co-transformed with the yeast display vector pDispay into yeast competent cells, a single-domain antibody yeast display library is constructed and screened, and monoclonal yeasts are selected to obtain monoclonal antibodies that recognize the FLT3-CAR molecule. The monoclonal antibody can specifically recognize the FLT3-CAR molecule on FLT3-CAR-T cells, and such a 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 implementation manner 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 thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the 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 CDR3 of the monoclonal antibody is shown as SEQ ID NO:4, SEQ ID NO:11 or SEQ ID NO:18; The amino acid sequence of the light chain CDR3 of the monoclonal antibody is shown as SEQ ID NO:7, SEQ ID NO:14 or SEQ ID NO:

21.

2. The monoclonal antibody that recognizes the FLT3-CAR molecule according to claim 1, characterized in that, The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown as SEQ ID NO:2, SEQ ID NO:9 or SEQ ID NO:16; Preferably, the amino acid sequence of the heavy chain CDR2 of the monoclonal antibody is shown as SEQ ID NO:3, SEQ ID NO:10 or SEQ ID NO:17; Preferably, the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown as SEQ ID NO:6, SEQ ID NO:13 or SEQ ID NO:20; Preferably, the amino acid sequence of the light chain CDR2 of the monoclonal antibody is selected from GTS or GAS.

3. The monoclonal antibody that recognizes the FLT3-CAR molecule according to claim 1 or 2, characterized in that, The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown as SEQ ID NO:2, the amino acid sequence of CDR2 is shown as SEQ ID NO:3, and the amino acid sequence of CDR3 is shown as SEQ ID NO:4; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown as SEQ ID NO:6, the amino acid sequence of CDR2 is GTS, and the amino acid sequence of CDR3 is shown as SEQ ID NO:7; Or, the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown as SEQ ID NO:9, the amino acid sequence of CDR2 is shown as SEQ ID NO:10, and the amino acid sequence of CDR3 is shown as SEQ ID NO:11; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown as SEQ ID NO:13, the amino acid sequence of CDR2 is GAS, and the amino acid sequence of CDR3 is shown as SEQ ID NO:14; Or, the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown as SEQ ID NO:16, the amino acid sequence of CDR2 is shown as SEQ ID NO:17, and the amino acid sequence of CDR3 is shown as SEQ ID NO:18; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown as SEQ ID NO:20, the amino acid sequence of CDR2 is GAS, and the amino acid sequence of CDR3 is shown as SEQ ID NO:

21.

4. The monoclonal antibody that recognizes 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 as SEQ ID NO:1, SEQ ID NO:8 or SEQ ID NO:15; Preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO:5, SEQ ID NO:12 or SEQ ID NO:

19.

5. The monoclonal antibody that recognizes the FLT3-CAR molecule according to any one of claims 1-4, characterized in that, 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; or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 12; or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

19.

6. 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 1-5.

7. Use of the monoclonal antibody that recognizes the FLT3-CAR molecule according to any one of claims 1-5 in detecting the CAR molecule of FLT3-CAR-T cells.

8. 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 1-5; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or diluent.

9. A kit for detecting the FLT3-CAR molecule in a sample, characterized in that, The kit comprises the monoclonal antibody that recognizes the FLT3-CAR molecule according to any one of claims 1-5.

10. Application of the kit for detecting the FLT3-CAR molecule in a sample according to claim 9 in the determination of the level of drug-resistant antibodies.

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