Antibodies targeting cd7 and uses thereof

Abstract

The application relates to an antibody targeting CD7 and application thereof, the antibody comprising a heavy chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3. The antibody targeting CD7 has high affinity and specificity, can efficiently target the CD7 antigen, and has simple structure and is easy to prepare. The CD7 blocking molecule can effectively reduce the expression of the cell surface CD7, and effectively inhibit the self-cannibalization of the CD7 chimeric antigen receptor cell.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an antibody targeting CD7 and its application. Background Technology

[0002] T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy, accounting for 10%-15% of ALL cases in children and approximately 20% of ALL cases in adults. Despite various chemotherapy regimens, less than 50% of adults and 75% of children with T-ALL survive for more than 5 years. Patients with relapsed or refractory (r / r) T-ALL are difficult to treat and have poor outcomes. Furthermore, the lack of salvage options such as targeted and immunotherapies contributes to a poor prognosis.

[0003] CD7 is a transmembrane glycoprotein expressed by T cells and natural killer (NK) cells and their precursors. It is also expressed in over 95% of T-cell lymphoblastic leukemia and T-cell lymphoma subsets. CD7 plays a co-stimulatory role in T-cell activation upon binding to its ligand K12 / SECTM1. However, CD7 appears to have no critical impact on T-cell development or function, as gene disruption of CD7 in mouse T-cell progenitor cells does not affect normal T-cell development and homeostasis, only causing minor alterations in T-cell effector function. Notably, CD7 is internalized upon antibody binding and has therefore been previously evaluated as a target for immunotoxin-loaded antibodies for the treatment of patients with T-cell malignancies. While no serious permanent adverse reactions associated with CD7 antibodies have been observed, its antitumor activity has been limited.

[0004] Chimeric antigen receptor T cell (CAR-T) therapy is a revolutionary cancer immunotherapy that has emerged in recent years. By transducing T cells to express chimeric antigen receptors, tumor-specific CAR-T cells are obtained. These cells specifically target and kill tumor cells, thus treating tumors. Although CD7 is an excellent target for T-ALL, the high expression of CD7 by both normal T cells and engineered CAR-T cells leads to cannibalism among CD7-CAR-T cells, significantly weakening their anti-tumor efficacy. Therefore, there is an urgent need to develop novel methods for preparing CD7-CAR-T cells. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an antibody targeting CD7 and its application.

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

[0007] In a first aspect, the present invention provides an antibody targeting CD7, the antibody comprising a heavy chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3;

[0008] The amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3;

[0009] The amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 4, SEQ ID No. 5, or SEQ ID No. 6;

[0010] The amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 7, SEQ ID No. 8, or SEQ ID No. 9.

[0011] SEQ ID No. 1: GYTYSSYY.

[0012] SEQ ID No. 2: GYISSSYC.

[0013] SEQ ID No. 3: GAIFTYKY.

[0014] SEQ ID No.4: IASDGST.

[0015] SEQ ID No. 5: IDSDGNT.

[0016] SEQ ID No. 6: IYTAADTT.

[0017] SEQ ID No. 7: AADQLGPAHFVVVAGFGY.

[0018] SEQ ID No. 8: AAEPLACVGDTWGYNY.

[0019] SEQ ID No. 9: ASAINTPAYYVPRLPLDFGN.

[0020] This invention screens antibodies targeting CD7. The CD7-targeting antibodies only include the heavy chain variable region, which has high affinity and specificity, can efficiently target the CD7 antigen, have a simple structure, are easy to prepare, and have important application value in the field of preparing drugs targeting CD7.

[0021] The amino acid sequence of the antibody's CDR1 includes the sequence shown in SEQ ID No. 1, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 4, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 7.

[0022] Preferably, the amino acid sequence of CDR1 of the antibody includes the sequence shown in SEQ ID No. 2, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 5, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 8.

[0023] Preferably, the amino acid sequence of CDR1 of the antibody includes the sequence shown in SEQ ID No. 3, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 6, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 9.

[0024] Preferably, the heavy chain variable region further includes frame regions FR1, FR2, FR3 and FR4.

[0025] Preferably, the amino acid sequence of FR1 includes the sequence shown in SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15 or SEQ ID No. 16.

[0026] SEQ ID No10: QVKLVESGGGSVQAGGSLTLSCAAS.

[0027] SEQ ID No. 11: QVQLVESGGGSVQAGGSLLRLSCAAS.

[0028] SEQ ID No. 12: QVQLVESGGGSVQAGGSLRLSCAGF.

[0029] SEQ ID No. 13: QVKLVQSGGGSVQAGGSLLRLSCAAS.

[0030] SEQ ID No. 14: EVQLVESGGGSVQAGGSLLRLSCAAS.

[0031] SEQ ID No. 15: QVKLVESGGGSVQGGGSLRLSCAFF.

[0032] SEQ ID No. 16: QVKLVESGGGSVQAGGSLLRLSCAAS.

[0033] Preferably, the amino acid sequence of FR2 includes the sequence shown in SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19 or SEQ ID No. 20.

[0034] SEQ ID No. 17: EVKLVQSGGGSVQAGGSLLRLSCAAS.

[0035] SEQ ID No. 18: MGWFRQASGKEREGVAA.

[0036] SEQ ID No. 19: MGWFRQAPGKEREGLAT.

[0037] SEQ ID No. 20: MAWFRQAPGKEREWVAS.

[0038] Preferably, the amino acid sequence of FR3 includes the sequence shown in SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28 or SEQ ID No. 29.

[0039] SEQ ID No. 21: SYADSVKGRFTISKDNAKNTLYLQMNSLKPEDTAMYYC.

[0040] SEQ ID No. 22: SYADSVKGRFTISKDNANNTLYLQMNSLKPEDTAMYYC.

[0041] SEQ ID No. 23: SYADSVKGRFTISKDKARNTLYLQMNSLKPEDTAMYYC.

[0042] SEQ ID No. 24: SYADSVKGRFTISKDNAKNTLYLQMNSLTPEDTAMYYC.

[0043] SEQ ID No. 25: SYADSVKGRFTISKDNAKNTLFLQMNSLKPEDTAMYYC.

[0044] SEQ ID No. 26:

[0045] HYADSVKGRFTIARDSTNNTVYLQMRNLKPEDTAMYYC.

[0046] SEQ ID No. 27: SYADPVKGRFTISKDNAKNTLYLQMNSLKPEDTAMYYC.

[0047] SEQ ID No. 28: SYTDSVKGRFTISKDNAKNTLYLQMNSLKPEDTAMYYC.

[0048] SEQ ID No. 29:

[0049] SYADSVKGRFTLSKDNANNTLYLQMNSLKPEDSAMYYC.

[0050] Preferably, the amino acid sequence of FR4 includes the sequence shown in SEQ ID No. 30 or SEQ ID No. 31.

[0051] SEQ ID No. 30: WGQGTQVTVSS.

[0052] SEQ ID No. 31: RGQGTQVTVSS.

[0053] Preferably, the amino acid sequence of the heavy chain variable region includes the sequence shown in SEQ ID No. 32, SEQ ID No. 33 or SEQ ID No. 34.

[0054] SEQ ID No. 32:

[0055] QVQLVESGGGSVQAGGSLRLSCAGFGYTYSSYYMGWFRQAPGKEREGVAAIASDGST SYADSVKGRFTISKDNAKNTLYLQMNSLKPEDTAMYYCAADQLGPAHFVVVAGFGYRGQ GTQVTVSS.

[0056] SEQ ID No. 33:

[0057] QVKLVESGGGSVQGGGSLRLSCAFFGYISSSYCMGWFRQAPGKEREGLATIDSDGNTS YADSVKGRFTISKDNAKNTLYLQMNSLTPEDTAMYYCAAEPLACVGDTWGYNYWGQGTQ VTVSS.

[0058] SEQ ID No. 34:

[0059] EVQLVESGGGSVQAGGSLRLSCAASGAIFTYKYMAWFRQAPGKEREWVASIYTAADT THYADSVKGRFTIARDSTNNTVYLQMRNLKPEDTAMYYCASAINTPAYYVPRLPLDFGNW GQGTQVTVSS.

[0060] In a second aspect, the present invention provides a nucleic acid molecule comprising the encoding gene of the antibody targeting CD7 described in the first aspect.

[0061] Preferably, the nucleotide sequence of the nucleic acid molecule includes the sequence shown in SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 37.

[0062] SEQ ID No. 35:

[0063] CAGGTCCAACTCGTCGAGTCAGGCGGCGGCTCCGTTCAAGCAGGCGGCTCACTCAGACTCAGCTGCGCAGGCTTCGGCTACACCTACAGCAGCTACTACATGGGCTGGTTCAGACAGGCCCCCGGCAAGGAGAGAGGGAGTGGCTGCCATTGCCAGCGACGGAAGCACCAGCTACGCCGACAGC GTGAAGGGCCGGTTCACCATCAGCAAGGACAACGCTAAGAACACCCTCTATCTCCAAATGAACAGCCTGAAGCCCGAAGATCCGCCATGTACTACTGCGCCGCCGACCAGCTGGGACCTGCCCATTTCGTGGTGGTGGCCGGATTCGGCTATAGAGGCCAAGGCACCCAAGTCACCGTGAGCAGC.

[0064] SEQ ID No. 36:

[0065] CAGGTCCAACTCGTCGAGTCAGGCGGCGGCTCCGTTCAAGCAGGCGGCTCACTCAGACTCAGCTGCGCAGGCTTCGGCTACACCTACAGCAGCTACTACATGGGCTGGTTCAGACAGGCCCCCGGCAAGGAGAGAGGGAGTGGCTGCCATTGCCAGCGACGGAAGCACCAGCTACGCCGACAGC GTGAAGGGCCGGTTCACCATCAGCAAGGACAACGCTAAGAACACCCTCTATCTCCAAATGAACAGCCTGAAGCCCGAAGATCCGCCATGTACTACTGCGCCGCCGACCAGCTGGGACCTGCCCATTTCGTGGTGGTGGCCGGATTCGGCTATAGAGGCCAAGGCACCCAAGTCACCGTGAGCAGC.

[0066] SEQ ID No. 37:

[0067] CAGGTCAAACTCGTCGAGAGCGGCGGCGGCAGCGTGCAAGGAGGAGGAAGCCTCAGACTCAGCTGCGCTTTCTTCGGCTACATCAGTTCATCATACTGCATGGGTTGGTTCAGACAAGCCCCTGGCAAAGAGAGAGAGGGACTGGCTACCATTGACAGCGACGGGAACACAAGCTACGCCGAC TCCGTTAAGGGAAGGTTCACCATTAGCAAGGACAACGCCAAGAACACACTGTATCTCCAGATGAACAGCCTGACCCCCGAAGACACCGCCATGTACTACTGCGCCGCCGAGCCCCTGGCCTGCGTTGGAGATACCTGGGGATACAACTACTGGGGACAGGGCACCCAAGTCACCGTGAGCAGC.

[0068] Thirdly, the present invention provides a CD7 blocking molecule, the blocking molecule comprising a signal peptide, an antigen-binding domain, and an intracellular localization domain.

[0069] Preferably, the signal peptide includes the CD8α signal peptide.

[0070] Preferably, the antigen-binding domain includes the antibody targeting CD7 described in the first aspect.

[0071] Preferably, the intracellular localization domain includes any one of the endoplasmic reticulum localization domain, the Golgi apparatus localization domain, or the proteasome localization domain.

[0072] Fourthly, the present invention provides a chimeric antigen receptor, the chimeric antigen receptor comprising a signal peptide, an antigen-binding domain, a hinge region, a transmembrane domain, and a signal transduction domain; the antigen-binding domain comprising the antibody targeting CD7 described in the first aspect.

[0073] Preferably, the signal peptide includes the CD8α signal peptide.

[0074] Preferably, the hinge region includes the CD8α hinge region.

[0075] Preferably, the transmembrane structural domain includes any one or a combination of at least two of the CD8α transmembrane region, CD28 transmembrane region, or DAP10 transmembrane region.

[0076] Preferably, the signal transduction domain includes an immune receptor tyrosine activation motif.

[0077] Preferably, the signal transduction domain further includes a co-stimulatory molecule, which includes any one or a combination of at least two of the following: 4-1BB, CD28 intracellular region, OX40, ICOS, or DAP10 intracellular region.

[0078] Preferably, the chimeric antigen receptor includes a CD8α signal peptide, an antibody targeting CD7 as described in the first aspect, a CD8α hinge region, a CD8α transmembrane region, and an immune receptor tyrosine activation motif.

[0079] Fifthly, the present invention provides an expression vector comprising the CD7 blocking molecule described in the third aspect and the encoding gene of the chimeric antigen receptor described in the fourth aspect.

[0080] Preferably, the expression vector is any one of a lentiviral vector, a retroviral vector, or an adeno-associated virus vector containing the CD7 blocking molecule described in the third aspect and the coding gene of the chimeric antigen receptor described in the fourth aspect, and more preferably a lentiviral vector.

[0081] In a sixth aspect, the present invention provides a recombinant lentivirus containing the expression vector described in the fifth aspect.

[0082] In a seventh aspect, the present invention provides a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the CD7 blocking molecule described in the third aspect and the chimeric antigen receptor described in the fourth aspect.

[0083] Preferably, the chimeric antigen receptor immune cells comprise the expression vector described in the fifth aspect and / or the recombinant lentivirus described in the sixth aspect.

[0084] Preferably, the chimeric antigen receptor immune cells include any one or a combination of at least two of T lymphocytes, B lymphocytes, NK cells, mast cells, or macrophages.

[0085] In an eighth aspect, the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor immune cells described in the seventh aspect.

[0086] In a ninth aspect, the present invention provides the use of an antibody targeting CD7 according to the first aspect, a nucleic acid molecule according to the second aspect, a CD7 blocking molecule according to the third aspect, a chimeric antigen receptor according to the fourth aspect, an expression vector according to the fifth aspect, a recombinant lentivirus according to the sixth aspect, a chimeric antigen receptor immune cell according to the seventh aspect, or a pharmaceutical composition according to the eighth aspect in the preparation of a drug for treating tumors.

[0087] Preferably, the tumor includes a tumor expressing CD7.

[0088] Compared with the prior art, the present invention has the following beneficial effects:

[0089] (1) The anti-CD7 antibody of the present invention only includes the heavy chain variable region, which has high affinity and specificity, can efficiently target CD7 antigen, and has a simple structure and is easy to prepare;

[0090] (2) In this invention, a chimeric antigen receptor is constructed using the anti-CD7 antibody, which can efficiently target CD7;

[0091] (3) The CD7 blocking molecule of the present invention can effectively reduce the expression of CD7 on the cell surface and effectively inhibit the self-killing of CD7 chimeric antigen receptor cells;

[0092] (4) The chimeric antigen receptor cells of the present invention can specifically recognize CD7 positive tumor cells and kill them efficiently. Attached Figure Description

[0093] Figure 1A The image shows the results of ELISA detection of CD7-VHH-4 antibody EC50.

[0094] Figure 1B The image shows the results of ELISA detection of CD7-VHH-10 antibody EC50.

[0095] Figure 1C The image shows the results of ELISA detection of CD7-VHH-18 antibody EC50.

[0096] Figure 2 FACS detection results for anti-CD7 nanobodies recognizing CD7 antigen.

[0097] Figure 3 Map of lentiviral vector plasmids targeting the CD7 chimeric antigen receptor.

[0098] Figure 4 This is a schematic diagram of the structure of the chimeric antigen receptor expressing anti-CD7 and the CD7 blocking sequence in Example 4;

[0099] Figure 5 The graph shows the flow cytometry results of the expression rates of chimeric antigen receptors (CD7-4-20-PEBL1, CD7-10-20-PEBL1, CD7-18-20-PEBL1) on T lymphocytes.

[0100] Figure 6 The image shows the results of CD7 expression detection on the surface of CD7-CAR-T cells, where the dashed line represents the control group of T cells that were not infected with the virus.

[0101] Figure 7A This image shows the killing effect of CAR-T cells on K562-luci cells in this invention.

[0102] Figure 7B This image shows the killing effect of CAR-T cells on CCRF-CEM-luci cells according to the present invention.

[0103] Figure 7C This image shows the killing effect of CAR-T cells on Jurkat-luci cells according to the present invention.

[0104] Figure 8 This represents the level of IFNγ secreted by CAR-T cells in this invention. Detailed Implementation

[0105] 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.

[0106] Example 1

[0107] Constructing a phage nanobody library and performing panning and preliminary screening using ELISA.

[0108] 1. Construction of phage nanobody library

[0109] (1) Bactrian camels were immunized with CD7-Fc expressing the extracellular region. After verifying the titer by ELISA, 200 mL of peripheral blood was drawn.

[0110] (2) Sorting lymphocytes, obtaining peripheral blood mononuclear lymphocyte precipitate, and extracting RNA;

[0111] (3) Use III reverse transcriptase synthesizes first-strand cDNA using RNA as a template, and then amplifies the VHH gene using nested PCR.

[0112] (4) The VHH gene was inserted into the pMECS phage display vector. After electrotransformation into TG1 competent cells, the bacterial culture was used for library identification. All remaining cultures were evenly spread on LB / AMPGLU plates. After bacterial growth, the bacterial colony was collected, 1 / 3 volume of 50% glycerol was added, mixed well, dispensed, and stored at -80℃. A library with a capacity greater than 10 was successfully constructed. 9 Phage display of camel VHH immune library.

[0113] 2. Selection of phage nanobody libraries

[0114] The purified CD7-His recombinant protein was diluted to 4 μg / mL with PBS buffer. In a 96-well microplate, three replicates were selected, and 100 μL (400 ng / well) was added to each well. The plate was incubated overnight at 4°C, with PBS used as a negative control. The coating solution was discarded, and 150 μL of 2% skim milk powder was added to each well. The plate was blocked at 25°C for 1 h. The plate was washed four times with PBST. The prepared phage solution was then diluted to 5 × 10⁻⁶ with 2% skim milk powder. 11 Add pfu / mL to an ELISA plate, 100 μL / well, and incubate at 25°C for 2 h. Discard the phage sample, wash 10 times with PBST, then wash 5 times with PBS. Add 100 μL of freshly prepared 0.1 M triethylamine to each well, incubate at 25°C for 10 min, aspirate the eluent, and quickly neutralize with an equal volume of 1 M Tris-HCl (pH 7.4). Take a portion of the eluent to determine the phage titer. Take another 400 μL of the eluent and infect 4 mL of freshly cultured logarithmic-phase TG1 bacterial suspension (OD600 approximately 0.6). Incubate at 37°C for 30 min, add 16 mL of 2×YT / AMP-GLU, and incubate at 37°C and 200 rpm until the OD600 reaches 0.7. 100 μL of bacterial suspension was serially diluted and evenly spread onto 2×YT / ampicillin / glucose agar plates for library capacity and diversity determination. 100 μL of bacterial suspension (i.e., the phage display vector library) was inoculated into 2×YT / AMP-GLU medium and cultured to the logarithmic phase. Helper phages were added for library rescue. Phage particles were obtained, and their titers were measured. The phage particles were then concentrated and purified for the next round of screening. The remaining bacterial suspension was centrifuged and resuspended in an appropriate volume of 2×YT medium. The suspension was spread onto plates containing screening resistance and cultured overnight. Bacteria were scraped from the plates using an appropriate amount of liquid culture medium, resuspended in 2×YT medium containing 1 / 3 volume of 50% glycerol, and aliquoted. All bacteria were stored at -80°C.

[0115] Repeat the above filtering operation 3 times.

[0116] Three rounds of solid-phase screening were performed on the immune nanobody library in vitro, resulting in the effective enrichment of phage clones with binding activity. After prokaryotic induction and expression of monoclonal phages, phage clones capable of binding to the extracellular region of the antigen were further screened by ELISA.

[0117] 3. Phage packaging

[0118] Take 100 μL of the frozen bacterial culture from the previous round of panning and add it to 100 mL of 2×YT / AMPGL culture medium. Incubate at 37°C with shaking (200 rpm) until the logarithmic development phase (OD600 value of 0.6). Add 90 μL of helper phage M13K07 (1.7×10⁻⁶). 13 The mixture (PFU / mL) was first incubated at 37°C for 30 min, centrifuged at 2800×g for 10 min to collect bacterial cells, resuspended in 200 mL of 2×YT / AMP-KAN medium, and cultured at 37°C with shaking (200 rpm) for 12 h. After centrifugation at 4°C and 3800×g for 30 min to remove bacterial cells, the supernatant was collected and 1 / 5 volume of pre-cooled PEG / NaCl was added and mixed well. The phage was precipitated for 2 h, and after centrifugation at 4°C and 3800×g for 30 min to collect the phage, a final volume of 2 mL was used. Resuspend the phage in PBS solution and transfer it to a 15 mL centrifuge tube. Centrifuge at 12000×g for 15 min at 4 °C and collect the supernatant. Add 1 / 5 volume of pre-cooled PEG / NaCl solution, mix by inverting, and incubate on ice for 2 h. Centrifuge at 10000×g for 10 min at 4 °C, discard the supernatant, resuspend the phage pellet in 1 mL PBS, and incubate overnight at 4 °C on a shaker to fully dissolve the phage particles. Mix the phage solution with an equal volume of 60% glycerol and aliquot into 1.5 mL EP tubes and store at -80 °C.

[0119] The phage library was panned three times using CD7 antigen. To avoid losing sequence diversity, the initial ELISA screening was performed on the panning products from the second and third rounds. Positive clones were randomly selected from the panning products and induced to express. The expression supernatant was the crude VHH antibody. The VHH antibody sequence of the monoclonal strain was determined by sequencing.

[0120] Example 2

[0121] Fluorescence-activated cell sorting (FACS) candidate clones

[0122] Cells were cultured according to standard cell culture protocols. CD7-positive and CD7-negative cell suspensions were prepared by trypsin digestion. After centrifugation (300×g, 5 min) to remove the culture medium, the cells were resuspended in Flow Buffer to a final volume of 2×10⁶ cells / mL. 6cell / mL, add 2×10⁻⁶ cells / mL to each well of a V-bottom 96-well plate. 5 Cell suspensions of 100 cells were centrifuged at 300×g for 5 min, the supernatant was removed, and the cells were resuspended with crude VHH antibody extract. The cells were incubated at 4℃ for 1 h, centrifuged at 300×g for 5 min, the supernatant was removed, and the cells were resuspended with Flow Buffer. The APC anti-his antibody was diluted to 2 μg / mL with Flow Buffer, and 100 μL of the solution was added to each well for cell resuspension. The cells were incubated at 4℃ for 1 h, and the cells were washed 3 times with Flow Buffer. The cells were then resuspended with 200 μL of Flow Buffer and analyzed by flow cytometry. Candidate antibodies were screened and named CD7-VHH-4, CD7-VHH-10, and CD7-VHH-18.

[0123] The amino acid sequence of the CD7-VHH-4 heavy chain variable region is shown in SEQ ID No. 32, the amino acid sequence of the CD7-VHH-10 heavy chain variable region is shown in SEQ ID No. 33, and the amino acid sequence of the CD7-VHH-18 heavy chain variable region is shown in SEQ ID No. 34.

[0124] Example 34

[0125] Expression, purification, and EC50 determination of VHH-mIgG2aFc nanobody.

[0126] 1. To further identify the antibodies obtained in Example 2, it is necessary to express the antibodies in mammalian cells. Therefore, a plasmid vector C-4pCP.Stuffer-mCg2a-FC expressing VHH with a mouse Fc tag was first constructed. The construction method includes the following steps:

[0127] (1) The VHH fragment was amplified by PCR. The reaction system and PCR reaction conditions are shown in Table 1 below.

[0128] Table 1

[0129]

[0130] (2) The enzyme digestion system and reaction conditions are shown in Table 2. The digested vector was used... The DNA was purified using a PCR purification kit, and the air-dried DNA was dissolved in 20 μL of water to determine the DNA concentration.

[0131] Table 2

[0132]

[0133] (3) The homologous recombination reaction system was 10 μL, as shown in Table 3;

[0134] Table 3

[0135]

[0136]

[0137] (4) Take the entire homologous recombination reaction system and add it to DH5α competent cells to transform DH5α competent cells. The transformation conditions are shown in Table 4.

[0138] Table 4

[0139]

[0140] (5) Transformation plates were selected for single-clone PCR pre-identification. The PCR identification system conditions are shown in Table 5. The samples were sent to a sequencing company for sequencing identification. The sequencing results met expectations, and a plasmid vector expressing VHH with mouse Fc tag was successfully constructed.

[0141] Table 5

[0142]

[0143] Approximately 24 hours before plasmid transfection, 293E cells were passaged to achieve a cell density of approximately 2.6 × 10⁻⁶. 6 Cells / mL, 0.15 mg scFV-mIgG1 / 100 mL 293E was transfected into 293E cells using the PEI method, DNA:PEI = 1:2. The cells were cultured at 37℃, 130 rpm, 8% CO2 for 6 days. The cell culture supernatant was collected at 3000 rpm for 30 min. The collected supernatant containing the target antibody was filtered through a Millex-GP Filter Unit 0.45 μm Sterile filter and then processed using MabSelect. TM Sure TM Centrifuge and concentrate, wash column with 1×PBS, elute protein with 0.1M (mol / L) Gly-HCl and neutralize with 1 / 10 volume of Tris-HCl at pH 8.5, dialyze protein overnight at 4°C, quantify using the A280 method with NanoDrop 2000, and determine antibody purity by SEC-HPLC.

[0144] 2. In addition, the purified CD7-VHH antibodies (CD7-VHH-4, CD7-VHH-10, CD7-VHH-18) were subjected to EC50 determination by ELISA.

[0145] The methods include:

[0146] (1) Coating antigen

[0147] Dilute CD7 antigen (hCD7-Fc, Kaika Biotechnology) to 2 μg / mL with PBS, add 50 μL to the ELISA plate, seal the plate with sealing film, and incubate overnight at 4C.

[0148] (2) Washing

[0149] Discard the liquid in the microplate, add 200 μL of PBS to each well, discard the liquid, and pat dry on absorbent paper.

[0150] (3) Closed

[0151] Add 300 μL of PBS + 2% FBS buffer to each well, incubate at room temperature for 1 hour, discard the liquid, and pat dry on absorbent paper.

[0152] (4) Incubation of primary antibody

[0153] Dilute the primary antibody (purified CD7-VHH antibody) to 4 μg / mL with PBS + 2% FBS buffer, and then perform a 4-fold serial dilution to obtain 11 concentrations, 50 μL per well. Add an equal volume of PBS + 2% FBS buffer to the blank wells as a control. Seal the plate with sealing film, incubate at room temperature for 1 hour, discard the liquid, and pat dry on absorbent paper.

[0154] (5) Washing

[0155] Add 350 μL of TBST to each well for cleaning, soak for 30 seconds, pour out the liquid, pat dry on absorbent paper, and repeat the washing process 5 times.

[0156] (6) Incubation of secondary antibodies

[0157] Dilute the secondary antibody (Goat anti mouse IgG (HRP), Abcam) to 1 μg / mL with PBS + 2% FBS buffer, add 50 μL to each well, seal with sealing film, incubate at room temperature for 1 hour, discard the liquid, and pat dry on absorbent paper.

[0158] (7) Washing

[0159] Add 350 μL of TBST to each well for cleaning, soak for 30 seconds, pour out the liquid, pat dry on absorbent paper, and repeat the washing process 5 times.

[0160] (8) Add substrate

[0161] Add 50 μL of HRP substrate to each well and incubate at room temperature in the dark for 30 minutes.

[0162] (9) Add stop solution

[0163] Add 50 μL of stop solution to each well to terminate the substrate reaction. Measure the absorbance at 450 nm using a microplate reader.

[0164] (10) Data Analysis

[0165] The EC50 value was calculated using GraphPad Prism software.

[0166] The measurement results are shown in Table 6 and Figure 1A , Figure 1B , Figure 1C As shown in the results, the CD7 VHH antibody has high affinity.

[0167] Table 6

[0168] VHH antibody EC50 (ng / mL) CD7-VHH-4 4.524 CD7-VHH-10 2.429 CD7-VHH-18 6.773

[0169] Example 4

[0170] CD7 single-chain antibody was analyzed by flow cytometry.

[0171] 293T-CD7 tumor cells were incubated with purified recombinant anti-CD7 VHH antibody on ice for 30 min. The blank control group received no anti-CD7 VHH antibody. Cells were then incubated with APC-labeled goat anti-mouse IgG antibody for 30 min. Flow cytometry analysis was performed, and the results are shown below. Figure 2 As shown, the anti-CD7 antibody prepared by the present invention can recognize the CD7 antigen on the cell surface.

[0172] Example 5

[0173] This embodiment describes the preparation of a lentiviral vector expressing a chimeric antigen receptor targeting CD7 (CD7 CAR).

[0174] First, a lentiviral vector, HD-SIN03CD7CAR-PEBL, carrying a CD7 CAR chimeric antigen receptor and a CD7 blocking molecule was constructed. The vector map is shown below. Figure 3 As shown, a schematic diagram of the chimeric antigen receptor and the CD7 blocking molecule is as follows. Figure 4 As shown, it includes the CD8α signal peptide, anti-CD7 antibody (anti-CD7 VHH), CD8α hinge region, transmembrane region and immune receptor tyrosine activation motif (CD3ζ), 2A peptide, CD8α signal peptide, anti-CD7 antibody (anti-CD7 VHH), and endoplasmic reticulum localization domain.

[0175] The amino acid sequence of the signal peptide is shown in SEQ ID No. 38.

[0176] SEQ ID No. 38: MALPVTALLLPLALLLHAARP.

[0177] The amino acid sequences of anti-CD7 VHH are shown in SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20 or SEQ ID No. 21.

[0178] The amino acid sequences of the CD8α hinge region and transmembrane region are shown in SEQ ID No. 39.

[0179] SEQ ID No. 39:

[0180] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC.

[0181] The amino acid sequence of the 4-1BB intracellular region is shown in SEQ ID No. 40.

[0182] SEQ ID No. 40:

[0183] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0184] The CD3ζ amino acid sequence is shown in SEQ ID No. 41.

[0185] SEQ ID No. 41:

[0186] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0187] The amino acid sequence of the endoplasmic reticulum localization domain is shown in SEQ ID No. 42.

[0188] SEQ ID No. 42: EQKLISEEDLKDEL.

[0189] The specific preparation method is as follows:

[0190] 1. First, construct CD7-targeting CAR lentiviral vectors pSIN03 CD7-4-41BBz, pSIN03 CD7-10-41BBz, and pSIN03 CD7-18-41BBz without CD7 blocking molecules.

[0191] (1) Prepare the PCR reaction system according to Table 8, amplify the CD7-VHH fragment, and use the primers shown in Table 7.

[0192] Table 7

[0193]

[0194] Table 8

[0195] reagents Volume (μL) 10x buffer 5 2mM dNTP 5 <![CDATA[25mM MgSO4]]> 3 10μM primer F 1 10μM primer R 1 Template DNA (cDNA clone) 1 PCR-grade pure water 33 KOD-Plus-Neo 1

[0196] The above reagents are from TOYOBO Inc.

[0197] After preparation, the PCR reaction was carried out according to the procedure shown in Table 9.

[0198] Table 9

[0199]

[0200]

[0201] After the reaction was completed, the PCR products were subjected to 1% agarose gel electrophoresis, and the fragment of about 450 bp was recovered and quantified by ultraviolet absorption method.

[0202] (2) Prepare the PCR reaction system according to Table 11, add CD8a signal peptide before CD7-VHH fragment, and use primers as shown in Table 10.

[0203] Table 10

[0204]

[0205] Table 11

[0206] reagents Volume (μL) 10x buffer 5 2mM dNTP 5 <![CDATA[25mM MgSO4]]> 3 10μM primer F 1 10μM primer R 1 Template DNA (CD7-VHH from step (1)) 1 PCR-grade pure water 33 KOD-Plus-Neo 1

[0207] The above reagents are from TOYOBO Inc.

[0208] After preparation, the PCR reaction was carried out according to the procedure shown in Table 9.

[0209] After the reaction was completed, the PCR products were subjected to 1% agarose gel electrophoresis, and fragments of about 500 bp were recovered and quantified by ultraviolet absorption method.

[0210] (3) Prepare the PCR reaction system according to Table 13, amplify the CD8a hinge-TM-41BB-CD3Z fragment, and use the primers shown in Table 12.

[0211] Table 12

[0212] Primers serial number sequence CD8H2-F SEQ ID No. 50 CGACGCCAGCGCCGCGACCACC Vector-R SEQ ID No. 51 TCGATAAGCTTGATATCG

[0213] Table 13

[0214] reagents Volume (μL) 10x buffer 5 2mM dNTP 5 <![CDATA[25mM MgSO4]]> 3 10μM primer F 1 10μM primer R 1 Template DNA (HD CD19 CAR) 1 PCR-grade pure water 33 KOD-Plus-Neo 1

[0215] The above reagents are from TOYOBO Inc.

[0216] After preparation, PCR reaction was carried out according to the PCR procedure shown in Table 9. After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis, and the fragment of about 700bp was recovered and quantified by ultraviolet absorption method.

[0217] (4) 5 μg of the laboratory-constructed HD SIN03 CD19 41BBz(ka) plasmid was digested with BamHI and EcoRI, reacted in a water bath at 37℃ for 2 h, and then the vector was recovered.

[0218] (5) The two fragments recovered in steps 2 and 3 above and the vector obtained in step 4 were ligated with recombinase. The recombinant reaction system is shown in Table 14. After preparation, the reaction was carried out in a water bath at 37°C for 0.5 h. The cells were then transformed into Escherichia coli stbl3 competent cells using conventional methods. Single clones were selected from the solid culture medium, cultured overnight, and identified by PCR. The PCR reaction preparation is shown in Table 15, and the PCR procedure is shown in Table 16. After PCR, positive clones were selected for further sequencing identification. The sequencing results were in line with expectations.

[0219] Table 14

[0220] reagents Dosage HD SIN03 CD19 41BBz(ka) 150ng CD8a signal CD7 VHH 15ng CD8a hinge-TM-41BB-CD3Z 15ng 5 x CE MultiS buffer 2μL Exnase MultiS 1μL PCR-grade pure water Up to 10μL Total volume 10μL

[0221] Table 15

[0222] reagents Volume (μL) Taq PCR Master Mix 10 10μM LV-F2 1 10μM LV-R 1 Template DNA bacterial culture 1 PCR-grade pure water 7 Total volume 20

[0223] Table 16

[0224]

[0225] 2. Construct vectors containing CD7 blocking molecules in HD SIN03 CD7-4-41BBz-CD7-20-PEBL1, HD SIN03 CD7-10-41BBz-CD7-20-PEBL1, and HD SIN03 CD7-18-41BBz-CD7-20-PEBL1.

[0226] (1) Prepare the PCR reaction system according to Table 18, amplify the signal peptide-CD7-VHH fragment, and use the primers shown in Table 17.

[0227] Table 17

[0228]

[0229] Table 18

[0230]

[0231]

[0232] The above reagents are from TOYOBO Inc.

[0233] After preparation, the PCR reaction was carried out according to the procedure shown in Table 9.

[0234] After the reaction was completed, the PCR products were subjected to 1% agarose gel electrophoresis, and fragments of about 500 bp were recovered and quantified by ultraviolet absorption method.

[0235] (2) Prepare the PCR reaction system according to Table 13, amplify the CD8a hinge-TM-41BB-CD3Z-P2A fragment, and use primers as shown in Table 19.

[0236] Table 19

[0237]

[0238] The above reagents are from TOYOBO Inc.

[0239] After preparation, PCR reaction was carried out according to the PCR procedure shown in Table 9. After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis, and the fragment of about 700bp was recovered and quantified by ultraviolet absorption method.

[0240] (3) Prepare the PCR reaction system according to Table 21, amplify the P2A-CD8a leader-CD7-10 / 20-PEBL1 fragment, and use the primers shown in Table 20.

[0241] Table 20

[0242]

[0243] Table 21

[0244] reagents Volume (μL) 10x buffer 5 2mM dNTP 5 <![CDATA[25mM MgSO4]]> 3 10μM primer F 1 10μM primer R 1 Template DNA (the product of step (1)) 1 PCR-grade pure water 33 KOD-Plus-Neo 1

[0245] The above reagents are from TOYOBO Inc.

[0246] After preparation, PCR reaction was carried out according to the PCR procedure shown in Table 9. After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis, and the fragment of about 550 bp was recovered and quantified by ultraviolet absorption method.

[0247] (4) 5 μg of the laboratory-constructed HD SIN03 CD19 41BBz(ka) plasmid was digested with BamHI and EcoRI, reacted in a water bath at 37℃ for 2 h, and then the vector was recovered.

[0248] (5) The three fragments recovered in steps 1, 2 and 3 above and the vector obtained in step 4 were ligated with recombinase. The recombinant reaction system is shown in Table 22. After preparation, the reaction was carried out in a water bath at 37°C for 0.5 h. The cells were then transformed into Escherichia coli stbl3 competent cells using conventional methods. Single clones were selected from the solid culture medium, cultured overnight, and identified by PCR. The PCR reaction preparation is shown in Table 15, and the PCR program is shown in Table 16. After PCR, positive clones were selected for further sequencing identification. The sequencing results were in line with expectations.

[0249] Table 22

[0250] reagents Dosage HD SIN03 CD19 41BBz(ka) 150ng CD8a signal CD7 VHH 15ng CD8a hinge-TM-41BB-CD3Z-P2A 15ng P2A-CD8a leader-CD7-10 / 20-PEBL1 15ng 5 x CE MultiS buffer 2μL Exnase MultiS 1μL PCR-grade pure water Up to 10μL Total volume 10μL

[0251] Example 6

[0252] This embodiment describes the packaging of lentiviruses, including the following steps:

[0253] (1) With 1.6×10 7 293T cells were seeded in 15cm culture dishes and cultured overnight at 37°C with 5% CO2 to prepare for virus packaging. The culture medium was DMEM with 10% fetal bovine serum (FBS) added.

[0254] (2) Dissolve 30 μg of the lentiviral vector constructed in Example 5, 12.5 μg of the helper plasmid gag / pol, and 10 μg of the envelope plasmid VSVg in 2000 μL of serum-free DMEM culture medium and mix well.

[0255] (3) Dissolve 157.5 μg PEI (1 μg / μL) in 2000 μL of serum-free DMEM culture medium, vortex at 1000 rpm for 5 seconds, and incubate at 25℃ for 5 min;

[0256] (4) Formation of transfection complex: Add PEI mixture to DNA mixture, vortex mix or gently mix immediately after addition, and incubate at 25°C for 20 min;

[0257] (5) Add 4 mL of the transfection complex to a 15 cm culture dish containing 25 mL of DMEM medium. After 4 hours, replace with fresh medium.

[0258] (6) After 48 hours, the viral supernatant was collected to obtain lentiviruses expressing chimeric antigen receptors with different structures.

[0259] Example 7

[0260] This embodiment involves lentivirus concentration.

[0261] The viral supernatant prepared in Example 6 was filtered through a 0.45 μm filter membrane and collected into a 50 mL centrifuge tube. 1 / 4 of the PEG-NaCl viral concentrate was added, and the mixture was mixed by inverting. The mixture was then incubated overnight at 4 °C. The tube was centrifuged at 3500 rpm for 30 min at 4 °C. The supernatant was removed, and RPMI 1640 medium (containing 10% FBS) was added to dissolve and resuspend the viral precipitate. The concentrated lentivirus suspension was aliquoted into 50 μL portions and stored in finished tubes at -80 °C.

[0262] Example 8

[0263] This embodiment performs lentivirus titer detection.

[0264] 500 μL of Jurkat cells (1 × 10⁻⁶) 5 Cells were seeded in 48-well plates; the concentrated lentivirus from Example 7 was added to the cell suspension at concentrations of 1 μL, 0.2 μL, and 0.04 μL, respectively, and polybrene was added to a final concentration of 5 μg / mL; the cells were cultured overnight at 37°C with 5% CO2, and then the medium was replaced with fresh medium; after 72 h of infection, the cells were centrifuged at 400 × g for 5 min, the supernatant was discarded, and the cells were collected. 100 μL of PBS + 2% FBS was added to resuspend the cells, and 1 μg of iF488-anti-VHH cocktail antibody was added. The cells were incubated on ice for 30 min; after washing twice with PBS + 2% FBS, 300 μL of PBS + 2% FBS was added to resuspend the cells, and the infection efficiency was detected by flow cytometry; a cell sample with a positive rate of 15% was preferred, and the titer (TU / mL) was calculated as: number of cells (102) / (102) = number of cells ... 5 ) × Positive rate / Viral volume (mL).

[0265] Example 9

[0266] This embodiment utilizes lentivirus transduction of T lymphocytes.

[0267] Anti-human CD3 antibody and anti-human CD28 antibody were diluted with PBS to final concentrations of 1 μg / mL and 0.5 μg / mL, respectively, and coated with the solutions in well plates. The plates were then incubated overnight at 4°C. The antibody coating solution in the well plates was discarded, and the plates were washed twice with 1 mL of PBS. Human PBMCs were then adjusted to a density of 1 × 10⁻⁶ cells / well using T cell culture medium (X-VIVO + 10% FBS + IL-2 (300 U / mL)). 6 / mL, and then seeded into CD3 and CD28 antibody-coated well plates for activation for 48h; the activated T cells were collected and the cell density was adjusted to 1×10⁶. 6 / mL, add the lentivirus prepared in Example 7 according to the multiplicity of infection (MOI) = 10, add polybrene to the final concentration of 5 μg / mL; incubate overnight at 37°C and 5% CO2, then replace with fresh medium, and passage every 2 days.

[0268] Example 10

[0269] This embodiment demonstrates T-lymphocyte chimeric antigen receptor expression, including the following steps:

[0270] 1. Five days after infection, collect 3×10⁻⁶ samples. 5 T cells were centrifuged at 400×g for 5 min at 4℃, the supernatant was discarded, and the cells were washed once with PBS + 2% FBS.

[0271] 2. Resuspend cells in 100 μL PBS + 2% FBS, add 1 μg of iF488-anti-VHH cocktail antibody, and incubate on ice for 30 min; wash twice with PBS + 2% FBS, then resuspend cells in 300 μL PBS + 2% FBS. Use uninfected T cells as a control. Flow cytometry was used to detect infection efficiency. Results are shown below. Figure 5 As shown, the infected CAR-T cells showed a significant positive cell population, indicating that the present invention successfully constructed CAR-T cells expressing chimeric antigen receptors with different structures, labeled HDSIN03 CD7-4-41BBz-CD7-20-PEBL1, HD SIN03 CD7-10-41BBz-CD7-20-PEBL1, and HD SIN03 CD7-18-41BBz-CD7-20-PEBL1 (abbreviated as CD7-4-20-PEBL1, CD7-10-20-PEBL1, and CD7-18-20-PEBL1, respectively).

[0272] Example 11

[0273] This embodiment describes the expression of CD7 on the surface of chimeric antigen receptor T cells, including the following steps:

[0274] 1. Five days after infection, collect 3×10⁻⁶ samples. 5 T cells were centrifuged at 400×g for 5 min at 4℃, the supernatant was discarded, and the cells were washed once with PBS + 2% FBS.

[0275] 2. Resuspend cells in 100 μL PBS + 2% FBS, add 1 μg of APC-anti-CD7 antibody, and incubate on ice for 30 min; wash twice with PBS + 2% FBS, then resuspend cells in 300 μL PBS + 2% FBS. Use uninfected T cells as a control. Detect CD7 expression on the cell surface using flow cytometry. Results are as follows: Figure 6 As shown, the expression of CD7 on the surface of infected CAR-T cells was significantly reduced, indicating that the CAR-T cells constructed in this invention, which contain CD7 blocking molecules and chimeric antigen receptors with different expression structures, successfully blocked the expression of CD7 on the cell surface.

[0276] Example 12

[0277] In this embodiment, an in vitro toxicity experiment was conducted on CAR-T cells.

[0278] 1. Target cell seeding:

[0279] K562-luci (CD7-), CCRF-CEM-luci (CD7+), and Jurkat-luci (CD7+) were used as target cells, and the target cell concentration was adjusted to 2 × 10⁻⁶. 5 / mL, take 50μL and inoculate it into a white opaque 96-well plate;

[0280] 2. Effector cell seeding:

[0281] CD7 CAR-T cells and control T cells were used as effector cells. CAR-T cells and control T cells were added to 96-well plates at effector-to-target ratios of 0.5:1, 1:1 and 2:1.

[0282] Each group had two replicates, and the average value of the two replicates was taken. The experimental group and the control group are as follows:

[0283] Experimental group: CAR-T+ target cells;

[0284] Control group: Control T cells + target cells;

[0285] 3. After co-culturing effector cells and target cells for 18 hours, Steady- The luciferase assay kit is used for detection; please refer to Steady- for specific detection steps. The instructions for the luciferase assay kit are as follows: Figures 7A-7C As shown, the constructed CAR-T cells had no significant killing effect on CD7-negative cells, but exhibited strong killing activity against CD7-positive tumor cells.

[0286] Example 13

[0287] This embodiment detects the secretion of CAR-T cytokines.

[0288] 1. Cell culture supernatant

[0289] The cell cultures of CAR-T cells and control T cells from Example 12 were centrifuged at 400×g for 10 min to remove the precipitate. The supernatant was then stored at -80℃ for later testing.

[0290] 2. Reagent preparation

[0291] The detection was performed using the Linko Bio ELISA kit (catalog number: Human Gamma Interferon ELISA Kit: EK180-96). Before the detection, all reagents and samples were brought to 25°C. 1× washing buffer and 1× detection buffer were prepared according to the instructions for use, and the antibody was detected.

[0292] 3. Preparation of Standards and Samples

[0293] Standards: The stock solution of the standard was diluted twice using 5% 1640 culture medium, with a total of 8 dilution gradients, including zero concentration.

[0294] Samples: Dilute the samples using 5% 1640 medium.

[0295] 4. Testing Steps

[0296] (1) Soaking the microplate: Add 300 μL of 1× washing solution and let it stand for 30 seconds. After discarding the washing solution, pat the microplate dry on absorbent paper.

[0297] (2) Add standard: Add 100 μL of 2-fold serially diluted standard to the standard wells and add 100 μL of 5% 1640 culture medium to the blank wells;

[0298] (3) Add sample: Add 100 μL of cell culture supernatant to the sample well;

[0299] (4) Add detection antibody: Add 50 μL of diluted detection antibody (1:100 dilution) to each well;

[0300] (5) Incubation: Seal the plate with sealing film, shake at 300 rpm, and incubate at 25°C for 2 hours;

[0301] (6) Washing: Discard the liquid, add 300 μL of washing solution to each well and wash the plate 6 times;

[0302] (7) Enzyme incubation: Add 100 μL of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well;

[0303] (8) Incubation: Seal the plate with a new sealing film, shake at 300 rpm, and incubate at 25°C for 45 min;

[0304] (9) Washing: Repeat step (6);

[0305] (10) Adding substrate for color development: Add 100 μL of TMB substrate to each well, incubate in the dark at 25°C for 15 min;

[0306] (11) Add stop solution: Add 100 μL of stop solution to each well and mix thoroughly;

[0307] (12) Detection reading: The OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm was measured using an ELISA reader. The calibrated OD value is the measured value at 450nm minus the measured value at 630nm.

[0308] IFN-γ factor secretion results as follows Figure 8 As shown, only trace amounts of IFN-γ factor were detected in the CAR-T cell culture alone, indicating that the CD7-CAR-T cells containing CD7 blocking molecules constructed in this invention effectively inhibited cannibalism and abnormal activation caused by cannibalism.

[0309] The applicant declares that this invention illustrates a CD7-targeting antibody and its application through the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials of this invention, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0310] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0311] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A nanobody targeting CD7, characterized in that, The nanobody includes a heavy chain variable region, which includes CDR1, CDR2 and CDR3; The amino acid sequence of CDR1 of the nanobody is shown in SEQ ID No. 1, the amino acid sequence of CDR2 is shown in SEQ ID No. 4, and the amino acid sequence of CDR3 is shown in SEQ ID No.

7.

2. The CD7-targeting nanobody according to claim 1, characterized in that, The heavy chain variable region also includes frame regions FR1, FR2, FR3 and FR4.

3. The CD7-targeting nanobody according to claim 2, characterized in that, The amino acid sequence of FR1 includes the sequences shown in SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15 or SEQ ID No.

16.

4. The CD7-targeting nanobody according to claim 2, characterized in that, The amino acid sequence of FR2 includes the sequence shown in SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19 or SEQ ID No.

20.

5. The CD7-targeting nanobody according to claim 2, characterized in that, The amino acid sequence of FR3 includes the sequences shown in SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28 or SEQ ID No.

29.

6. The CD7-targeting nanobody according to claim 2, characterized in that, The amino acid sequence of FR4 includes the sequence shown in SEQ ID No. 30 or SEQ ID No.

31.

7. The CD7-targeting nanobody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region includes the sequence shown in SEQ ID No. 32, SEQ ID No. 33 or SEQ ID No.

34.

8. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the CD7-targeting nanobody as described in any one of claims 1-7.

9. The nucleic acid molecule according to claim 8, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No.

37.

10. A CD7 blocking molecule, characterized in that, The blocking molecule consists of a signal peptide, an antigen-binding domain, and an intracellular localization domain. The signal peptide is the CD8α signal peptide; The antigen-binding domain is the CD7-targeting nanobody as described in any one of claims 1-7; The intracellular localization domain is the endoplasmic reticulum localization domain. The amino acid sequence of the endoplasmic reticulum localization domain is SEQ ID No.

42.

11. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor is composed of a signal peptide, an antigen-binding domain, a hinge region, a transmembrane domain, and a signal transduction domain. The antigen-binding domain is the CD7-targeting nanobody as described in any one of claims 1-7; The signal peptide is the CD8α signal peptide; The hinge region is the CD8α hinge region; The transmembrane domain is the CD8α transmembrane region; The signal transduction domain is a co-stimulatory molecule and an immune receptor tyrosine activation motif. The co-stimulatory molecule is 4-1BB; The tyrosine activation motif of the immune receptor is CD3ζ.

12. An expression carrier, characterized in that, The expression vector includes a gene encoding the CD7 blocking molecule of claim 10 and the chimeric antigen receptor of claim 11.

13. The expression vector according to claim 12, characterized in that, The expression vector is a lentiviral vector or an adeno-associated virus vector.

14. The expression vector according to claim 13, characterized in that, The expression vector is a lentiviral vector.

15. A recombinant lentivirus, characterized in that, The recombinant lentivirus contains the expression vector as described in claim 12.

16. A chimeric antigen receptor immune cell, characterized in that, The chimeric antigen receptor immune cells express the CD7 blocking molecule of claim 10 and the chimeric antigen receptor of claim 11.

17. The chimeric antigen receptor immune cell according to claim 16, characterized in that, The chimeric antigen receptor immune cells comprise the expression vector of claim 12 and / or the recombinant lentivirus of claim 15.

18. The chimeric antigen receptor immune cell according to claim 16, characterized in that, The chimeric antigen receptor immune cells include any one or a combination of at least two of the following: T lymphocytes, B lymphocytes, NK cells, mast cells, or macrophages.

19. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the chimeric antigen receptor immune cells as described in claim 16.

20. The use of the CD7-targeting nanobody according to any one of claims 1-7, the nucleic acid molecule according to claim 8, the CD7 blocking molecule according to claim 10, the chimeric antigen receptor according to claim 11, the expression vector according to claim 12, the recombinant lentivirus according to claim 15, the chimeric antigen receptor immune cell according to claim 16, or the pharmaceutical composition according to claim 19 in the preparation of a drug for treating K562, CCRF-CEM, and Jurkat tumor cells.

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