Anti-cd8 antibodies and uses thereof

Abstract

The application relates to the field of bioengineering antibody preparation, in particular to an anti-CD8 antibody and application thereof. The anti-CD8 antibody comprises a light chain constant region, a light chain variable region, a heavy chain constant region and a heavy chain variable region; the light chain variable region comprises an amino acid sequence as shown in SEQ ID No. 29 or SEQ ID No. 30; and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID No. 31 or SEQ ID No. 32. The preferred antibody screened by the application is coupled to a magnetic particle to obtain anti-CD8 antibody coupled magnetic beads. The coupled magnetic beads are used to enrich and sort CD8 T cells from human peripheral blood mononuclear cells, and a CD22-targeted chimeric antigen receptor gene is introduced into the CD8 T cells to prepare CD8 + CD22-CAR-T cells, which can effectively kill tumor cells expressing CD22.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to an anti-CD8 antibody and its applications. Background Technology

[0002] CD8 T cells help the body maintain homeostasis. When the body is infected by pathogens or invaded by cancer cells, quiescent naïve CD8 T cells can proliferate in large numbers and differentiate into cytotoxic effector CD8 T cells through interaction with antigen-presenting cells. Cytotoxic effector CD8 T cells can activate other immune cells by secreting a large number of cytokines and directly kill infected cells or cancer cells, thereby helping the body to restore homeostasis.

[0003] Adoptive transfer of chimeric antigen receptor T cells (CAR-T) modified to express tumor cell surface antigens is a revolutionary cancer immunotherapy. CARs are synthetic receptors that can redirect lymphocytes, most commonly T cells, to recognize and kill target cells expressing specific antigens. In recent years, rapidly developing clinical trials of CAR-T cell therapy have actively explored its potential applications. Due to its enormous potential for continuous optimization, such as better targets, better CAR structures, and more efficient manufacturing processes, CAR-T cell therapy has attracted more attention than traditional drugs.

[0004] The production of CAR-T cells involves multiple steps and requires quality control testing throughout the process. In simple terms, the process begins with removing blood from the patient / healthy individual using leukoablation to separate white blood cells. The remaining blood is then returned to the circulatory system. Next, the white blood cells are sorted for CD4 T cells and CD8 T cells to enrich T cells. These enriched T cells are then activated and genetically engineered to express specific CARs, allowing for further expansion to reach an appropriate number before finally being reinfused into the patient.

[0005] Antibodies are essential for cell sorting. Therefore, developing new anti-CD8 antibodies for sorting is of great significance. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this application is to provide an anti-CD8 antibody and its application, particularly using a screened preferred CD8 antibody to solve the problems in the prior art.

[0007] To achieve the above and other related objectives, a first aspect of this application provides the use of an anti-CD8 antibody for enriching and sorting CD8 T cells, wherein the anti-CD8 antibody comprises a light chain constant region, a light chain variable region, a heavy chain constant region, and a heavy chain variable region; the amino acid sequence of CDR1 in the light chain variable region comprises the sequence shown in SEQ ID No. 1 or SEQ ID No. 2; the amino acid sequence of CDR2 in the light chain variable region comprises the sequence shown in SEQ ID No. 3 or SEQ ID No. 4; the amino acid sequence of CDR3 in the light chain variable region comprises the sequence shown in SEQ ID No. 5 or SEQ ID No. 6; the amino acid sequence of CDR1 in the heavy chain variable region comprises the sequence shown in SEQ ID No. 7 or SEQ ID No. 8; the amino acid sequence of CDR2 in the heavy chain variable region comprises the sequence shown in SEQ ID No. 9 or SEQ ID No. 10; and the amino acid sequence of CDR3 in the heavy chain variable region comprises the sequence shown in SEQ ID No. 11 or SEQ ID No. 12.

[0008] A second aspect of this application provides a method for enriching and sorting CD8 T cells, comprising the following steps:

[0009] 1) The anti-CD8 antibody used in the above-mentioned application is coupled to the nanomagnetic particles to obtain anti-CD8 antibody-coupled magnetic beads;

[0010] 2) The anti-CD8 antibody-conjugated magnetic beads are co-incubated with mixed cells containing CD8 T cells, and then the incubated cells are placed in a magnetic field. The CD8 T cells are enriched and sorted by the binding of the magnetic field with the anti-CD8 antibody-conjugated magnetic beads.

[0011] The third aspect of this application provides the use of the method in the preparation of CAR-T cells.

[0012] Compared with the prior art, the beneficial effects of this application are as follows:

[0013] (1) The preferred anti-CD8 antibody obtained by screening in this invention can efficiently target the CD8 antigen and is easy to prepare.

[0014] (2) The CD22-CAR-T cells prepared from the CD8 T cells obtained by the present invention can be activated by antigens with high efficiency and specificity. Attached Figure Description

[0015] Figure 1A The graph shows the proportion of CD8 T cells before PBMC enrichment and sorting, and after enrichment and sorting using CD8-1 or CD8-4 coupled magnetic beads.

[0016] Figure 1BThis figure shows the proportion of CD8 T cells in the waste liquid after PBMC enrichment and sorting using anti-CD8-1 magnetic beads or anti-CD8-4 magnetic beads.

[0017] Figure 2A The results of recognizing the CD8 antigen using CD8-2 antibody, CD8-3 antibody, and CD8-7 antibody are shown in the figure.

[0018] Figure 2B This is a graph showing the results of using CD8-8 antibody to recognize the CD8 antigen.

[0019] Figure 3A The figure shows the proportion of CD8T cells in the cells before and after sorting and enrichment using anti-CD8-2 magnetic beads, anti-CD8-3 magnetic beads, and anti-CD8-7 magnetic beads, respectively.

[0020] Figure 3B The figure shows the proportion of CD8T cells in the cell waste fluid after sorting and enrichment using anti-CD8-2 magnetic beads, anti-CD8-3 magnetic beads, and anti-CD8-7 magnetic beads, respectively.

[0021] Figure 3C The figures show the proportion of CD8T cells in the cells before enrichment and the proportion of CD8T cells in the waste liquid after enrichment, obtained by sorting and enriching with anti-CD8-8 magnetic beads.

[0022] Figure 4 The total number of CD8 T cells on days 5, 8, 11, and 14 after viral infection.

[0023] Figure 5A , 5B 5C and 5D are graphs showing the expression rate of chimeric antigen receptor in CAR-T cells on days 5, 8, 11, and 14 after CD8 T cells are infected with the virus, respectively.

[0024] Figure 6A This image shows the killing effect of CAR-T cells on K562-luci cells in this invention.

[0025] Figure 6B This image shows the killing effect of CAR-T cells on K562-CD22-luci cells in this invention.

[0026] Figure 7 This is a graph showing the IFN-γ cytokine secretion levels in CAR-T cells. Detailed Implementation

[0027] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0028] Through extensive research and exploration, the inventors of this application screened a large number of antibody species and obtained two preferred CD8 antibody sequences. Anti-CD8 antibody-conjugated magnetic beads prepared using these antibodies can efficiently and with high purity enrich CD8 T cells from human PBMCs. The CD22 CAR-T cells constructed from the CD8 T cells enriched in this way can efficiently expand and kill tumor cells expressing CD22. Based on this, this application was completed.

[0029] This application provides, in one aspect, the use of anti-CD8 antibodies for enriching and sorting CD8 T cells, wherein the anti-CD8 antibody comprises a light chain constant region, a light chain variable region, a heavy chain constant region, and a heavy chain variable region; the amino acid sequence of CDR1 in the light chain variable region comprises the sequence shown in SEQ ID No. 1 or SEQ ID No. 2; the amino acid sequence of CDR2 in the light chain variable region comprises the sequence shown in SEQ ID No. 3 or SEQ ID No. 4; the amino acid sequence of CDR3 in the light chain variable region comprises the sequence shown in SEQ ID No. 5 or SEQ ID No. 6; the amino acid sequence of CDR1 in the heavy chain variable region comprises the sequence shown in SEQ ID No. 7 or SEQ ID No. 8; the amino acid sequence of CDR2 in the heavy chain variable region comprises the sequence shown in SEQ ID No. 9 or SEQ ID No. 10; and the amino acid sequence of CDR3 in the heavy chain variable region comprises the sequence shown in SEQ ID No. 11 or SEQ ID No. 12.

[0030] SEQ ID No. 1: QDITNY

[0031] SEQ ID No. 2: QDIGSN

[0032] SEQ ID No. 3: GAS

[0033] SEQ ID No. 4: HGT

[0034] SEQ ID No. 5: QQYNNYPLT

[0035] SEQ ID No. 6: VQFAQFPYT

[0036] SEQ ID No. 7: GFIFSNYG

[0037] SEQ ID No. 8: GYSFTNFG

[0038] SEQ ID No. 9: IWYDGSNK

[0039] SEQ ID No. 10: INTYTGEP

[0040] SEQ ID No.11: ARSYDMLTGYDGSYGLDV

[0041] SEQ ID No. 12: ARKDYAGFFDY

[0042] In the application of this application, the anti-CD8 antibody is used for enriching and sorting CD8 T cells. The amino acid sequence of the CDR1 of the light chain variable region includes the sequence shown in SEQ ID No. 1, the amino acid sequence of the CDR2 includes the sequence shown in SEQ ID No. 3, and the amino acid sequence of the CDR3 includes the sequence shown in SEQ ID No. 5.

[0043] In the application of this application, the anti-CD8 antibody is used for enriching and sorting CD8 T cells. The amino acid sequence of the CDR1 of the light chain variable region includes the sequence shown in SEQ ID No. 2, 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. 6.

[0044] In the application of this application, the anti-CD8 antibody is used for enriching and sorting CD8 T cells. The amino acid sequence of the CDR1 of the heavy chain variable region includes the sequence shown in SEQ ID No. 7, the amino acid sequence of the CDR2 includes the sequence shown in SEQ ID No. 9, and the amino acid sequence of the CDR3 includes the sequence shown in SEQ ID No. 11.

[0045] In the application of this application, the anti-CD8 antibody is used for enriching and sorting CD8 T cells. The amino acid sequence of the CDR1 of the heavy chain variable region includes the sequence shown in SEQ ID No. 8, the amino acid sequence of the CDR2 includes the sequence shown in SEQ ID No. 10, and the amino acid sequence of the CDR3 includes the sequence shown in SEQ ID No. 12.

[0046] In the application of this application, the anti-CD8 antibody is used for enriching and sorting CD8 T cells. The light chain variable region further includes framework regions FR1-FR4. The amino acid sequence of FR1 includes the sequence shown in SEQ ID No. 13 or SEQ ID No. 14. The amino acid sequence of FR2 includes the sequence shown in SEQ ID No. 15 or SEQ ID No. 16. The amino acid sequence of FR3 includes the sequence shown in SEQ ID No. 17 or SEQ ID No. 18. The amino acid sequence of FR4 includes the sequence shown in SEQ ID No. 19 or SEQ ID No. 20.

[0047] SEQ ID No.13: DIQMTQSPSSSLSASVGDRVTITCRAS

[0048] SEQ ID No.14: DILMTQSPSSMSVSLGDTVSITCHAS

[0049] SEQ ID No.15:LAWFQQKPGKAPKSLIY

[0050] SEQ ID No.16:MGWLQQKPGKSFKALIY

[0051] SEQ ID No.17: SLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYC

[0052] SEQ ID No.18: NLEYGVPSRFSGSGSGADYSLSISSLESEDFADYYC

[0053] SEQ ID No. 19: FGGGTKVEIK

[0054] SEQ ID No. 20: FGGGTSLEIK

[0055] In the application of this application, the anti-CD8 antibody is used for enriching and sorting CD8 T cells. The heavy chain variable region further includes the framework regions FR1-FR4. The amino acid sequence of FR1 includes the sequence shown in SEQ ID No. 21 or SEQ ID No. 22. The amino acid sequence of FR2 includes the sequence shown in SEQ ID No. 23 or SEQ ID No. 24. The amino acid sequence of FR3 includes the sequence shown in SEQ ID No. 25 or SEQ ID No. 26. The amino acid sequence of FR4 includes the sequence shown in SEQ ID No. 27 or SEQ ID No. 28.

[0056] SEQ ID No.21: QVQLVESGGGVDQPGRSLRLSCAAS

[0057] SEQ ID No.22: QIQLVQSGPELRKPGETVRISCKAS

[0058] SEQ ID No.23: IHWVRQAPGKGLEWVAV

[0059] SEQ ID No.24: MIWVKQAPGKGLKWLGW

[0060] SEQ ID No.25: YFEDSVKGRFNISRDNSKNIVYLQMNSLRAEDTAVYFC

[0061] SEQ ID No.26: TYADDLKGRFAFSLETSANTAYLKINNFKNEDMATYFC

[0062] SEQ ID No. 27: WGQGTTVTVSS

[0063] SEQ ID No. 28: WGQGTTLTVSS

[0064] In the application of this application, the anti-CD8 antibody is used for enriching and sorting CD8 T cells. The amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID No. 29 or SEQ ID No. 30; the amino acid sequence of the heavy chain variable region includes the sequence shown in SEQ ID No. 31 or SEQ ID No. 32.

[0065] SEQ ID No. 29:

[0066] DIQMTQSPSSSLSASVGDRVTITCRASQDITNYLAWFQQKPGKAPKSLIYGASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPLTFGGGTKVEIK

[0067] SEQ ID No. 30:

[0068] DILMTQSPSSMSVSLGDTVSITCHASQDIGSNMGWLQQKPGKSFKALIYHGTNLEYGVPSRFSGSGSGADYSLSISSLESEDFADYYCVQFAQFPYTFGGGTSLEIK

[0069] SEQ ID No. 31:

[0070] QVQLVESGGGVDQPGRSLRLSCAASGFIFSNYGIHWVRQAPGKGLEWVAVIWYDGSNKYFEDSVKGRFNISRDNSKNIVYLQMNSLRAEDTAVYFCARSYDMLTGSGDYYGLDVWGQGTTVTVSS

[0071] SEQ ID No. 32:

[0072] QIQLVQSGPELRKPGETVRISCKASGYSFTNFGMIWVKQAPGKGLKWLGWINTYTGEPTYADDLKGRFAFSLETSANTAYLKINNFKNEDMATYFCARKDYAGFFDYWGQGTTLTVSS

[0073] In the application of this application, the anti-CD8 antibody is used for enriching and sorting CD8 T cells. The nucleotide sequence of the light chain variable region includes the sequence shown in SEQ ID No. 33 or SEQ ID No. 34, and the nucleotide sequence of the light chain constant region includes the sequence shown in SEQ ID No. 35 or SEQ ID No. 36. The nucleotide sequence of the heavy chain variable region includes the sequence shown in SEQ ID No. 37 or SEQ ID No. 38, and the nucleotide sequence of the heavy chain constant region includes the sequence shown in SEQ ID No. 39 or SEQ ID No. 40.

[0074] SEQ ID No. 33:

[0075] gacattcagatgacccagagccccagcagcctgagcgccagcgtgggagacagcgtgaccataacctgcagagccagccaagacataaccaactacctcgcctggtttcagcagaagcccggcaaagccccaaaaagcctcatctacggcgcctcctcct gcaaagcggcgtgccaagcaaattcagcggaagcggctccggaaccgacttcaccctgactatctccagcctccagcccgaagacttcgccacctactactgccaacaatacaacaactaccccctgaccttcggcggcggcacaaaggtggagatcaaa.

[0076] SEQ ID No. 34:

[0077] gacattctgatgacccagagccccagctccatgagcgtgagcctgggagacaccgtgagcatcacctgccacgccagccaagacataggaagcaacatgggctggctgcaacaaaaacccggcaaatcctttaaagctctcatctaccacggaaccaacctcgaatacggcgtgcccagcagattcagcggaagcggcagcggcgcagactacagcctgagcatcagctccctcgaaagcgaggacttcgccgactactactgcgtccagttcgcccaatttccttacacattcggcggcgggaccagcctggagatcaag。

[0078] SEQ ID No.35:

[0079] cgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagttcgcccgtcacaaagagcttcaacaggggagagtgt。

[0080] SEQ ID No.36:

[0081] agagcagatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt。

[0082] SEQ ID No.37:

[0083] caggtccaactcgtcgagtcaggcggcggcgtcgaccaacccggaagaagcctcagactcagctgcgcagcaagcggcttcatattttcaaactacggcatccactgggtcagacaagcccccggcaaaggcctggagtgggtggctgtgatttggtacgatggcagcaacaagtacttcgaggatagtgtgaaaggcaggttcaacataagcagggacaacagcaagaacatcgtgtacctgcagatgaacagcctgagagccgaggacacagcagtgtacttctgcgccaggagctacgacatgctgacaggaagcggagactactacggactggacgtgtggggccaggggaccaccgtgaccgtgagcagc。

[0084] SEQ ID No.38:

[0085] cagatccaactcgtccaaagcggccccgagctcagaaaacccggcgaaaccgtcagaataagctgcaaggcctccggctacagcttcaccaacttcggcatgatatgggtgaagcaagcacccggcaaaggcctgaagtggctgggatggataaacacctacaccggcgagcccacctacgccgacgacctcaaaggcagattcgccttctctctggagacaagcgctaacaccgcttacctgaaaatcaacaacttcaagaatgaggacatggccacctacttctgcgcccggaaggactacgccggcttctttgactactggggccagggcaccaccctgaccgtctcctcc。

[0086] SEQ ID No.39:

[0087] gctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccccgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa。

[0088] SEQ ID No.40:

[0089] gctaagaccacccccccttccgtgtatcccctggctcctggatctgccgcccagacaaactccatggtgaccctgggctgtctggtgaaaggctattttcctgaacccgtgaccgtgacctggaacagcggctctctgtctagcggcgtgcatacttttcccgccgtgctgcagtccgacctgtataccctgagttcctccgtgaccgtgccctcttccacctggcctagcgagaccgtgacctgcaatgtggcccaccccgcttccagcaccaaggtggataagaaaatcgtgccccgcgactgcggctgtaagccttgtatctgcaccgtgcccgaagtgagctcagtgttcattttcccccccaagcccaaagacgtgctgaccatcaccctgacccccaaagtgacctgcgtggtggtggatattagcaaggatgaccccgaagtgcagttttcttggttcgtggacgacgtggaagtgcacaccgcccagacccagcctagagaggagcagttcaactccacattcaggagtgtgagcgagctgcccattatgcaccaggattggctgaacgggaaggagttcaaatgtagggtgaacagcgccgccttccccgctcctattgaaaaaaccatcagcaagaccaagggcagacccaaggccccccaggtgtacaccatccccccccccaaggagcagatggccaaggacaaggtgagcctgacctgcatgatcaccgacttcttccccgaggacatcaccgtggagtggcagtggaacggccagcccgccgagaactacaagaacacccagcccatcatggacaccgacggcagctacttcgtgtacagcaagctgaacgtgcagaagagcaactgggaggccggaaacaccttcacctgcagcgtgctgcacgagggcctgcacaaccaccacaccgagaagagcctgagccacagccccggcaag。

[0090] This invention involves inserting the encoding gene of the anti-CD8 antibody into an expression vector to obtain a recombinant expression vector. The recombinant expression vector is then introduced into cells and cultured, followed by separation and purification to obtain the anti-CD8 antibody. The expression vector of this invention can accurately express the anti-CD8 antibody.

[0091] Specifically, the expression vector can be a viral vector or a non-viral vector. For example, non-viral vectors include plasmids, phagemids, Cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses. Viral vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). The vector may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site. The vector may also include components that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein coats.

[0092] Specifically, the introduced cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

[0093] More specifically, the introduced cells include many cell types, such as prokaryotic cells like Escherichia coli or Bacillus subtilis, fungal cells like yeast or Aspergillus, insect cells like S2 ​​Drosophila or Sf9, or animal cells like fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0094] This application also provides a method for enriching and sorting CD8 T cells, comprising the following steps:

[0095] 1) The anti-CD8 antibody sequence used in the above-mentioned application is coupled to the nanomagnetic particles to obtain anti-CD8 antibody-coupled magnetic beads;

[0096] 2) The anti-CD8 antibody-conjugated magnetic beads are co-incubated with mixed cells containing CD8 T cells, and then the incubated cells are placed in a magnetic field. The CD8 T cells are enriched and sorted by the binding of the magnetic field with the anti-CD8 antibody-conjugated magnetic beads.

[0097] In the method for enriching and sorting CD8 T cells provided in this application, in step 1), the mass ratio of the magnetic beads to the separated antibody is (1-10):1.

[0098] In the method for enriching and sorting CD8 T cells provided in this application, in step 1), the coupling reaction is carried out in a buffer solution. In a preferred embodiment, the buffer solution can be a BB solution. The buffer solution is added to adjust the pH value of the reaction solution.

[0099] In the method for enriching and sorting CD8 T cells provided in this application, step 1) requires blocking in a blocking solution for 1-2 hours. In a specific embodiment, the blocking time is 1 hour. The blocking solution can be a BSA aqueous solution with a concentration of 10-30 mg / mL. More specifically, in a preferred embodiment, the concentration of the BSA aqueous solution is 20 mg / mL.

[0100] In the method for enriching and sorting CD8 T cells provided in this application, step 2) includes mixed cells containing CD8 T cells, which include human peripheral blood mononuclear cells.

[0101] In the method for enriching and sorting CD8 T cells provided in this application, step 2) refers to incubation where the ratio of peripheral blood mononuclear cells to isolated antibodies is 16 × 10⁻⁶. 6 Cells: (0.5–1.5) μg. The incubation is performed in a flow cytometry buffer, which is a 1×PBS solution containing 1.8%–2.2% FBS and 1.8 mM–2.2 mM EDTA. Specifically, the flow cytometry buffer is a 1×PBS solution containing 2% FBS and 2 mM EDTA.

[0102] In the method for enriching and sorting CD8 T cells provided in this application, step 2) refers to passing the incubation solution through a sorting column, discarding the cells that flow down, rinsing the column away from the magnetic field with flow cytometry buffer, collecting the liquid that flows down, and obtaining the CD8 T cells.

[0103] This application also provides the use of the method in preparing CAR-T cells. Specifically, the CAR-T cells can be CD8+CD22-CAR-T cells. The CD8+CD22-CAR-T cells... + CD22-CAR-T cells can be used to treat cancer, adaptive immune diseases, autoimmune diseases, inflammatory diseases, or infectious diseases. Specifically, the cancer is selected from leukemia (e.g., chronic lymphocytic leukemia, relapsed or refractory B-cell precursor acute lymphoblastic leukemia) and lymphoma.

[0104] The present application will be further illustrated by the following examples, but these examples do not limit the scope of the present application.

[0105] In the following embodiments of this application:

[0106] For the HD SIN03 CD22(V29)41BBz(ka) lentiviral vector and K562-luci and K562-CD22-luci target cells, please refer to application number CN 114149506 A.

[0107] Example 1 Antibody Preparation

[0108] This invention identifies two preferred antibodies, named CD8-1 and CD8-4, by searching previous literature and patents and screening a large number of antibody databases.

[0109] The amino acid sequence of the light chain variable region of the CD8-1 antibody is SEQ ID No. 29, and the amino acid sequence of the heavy chain variable region is SEQ ID No. 31. The nucleic acid sequence of the light chain variable region of the CD8-1 antibody is SEQ ID No. 33, the nucleic acid sequence of the heavy chain variable region is SEQ ID No. 37, the nucleic acid sequence of the light chain constant region is SEQ ID No. 35, and the nucleic acid sequence of the heavy chain constant region is SEQ ID No. 39. The heavy chain constant region uses human IgG1, and the light chain constant region uses kappa.

[0110] The amino acid sequence of the light chain variable region of the CD8-4 antibody is shown in SEQ ID No. 30, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 32. The nucleic acid sequence of the light chain variable region of the CD8-4 antibody is SEQ ID No. 34, the nucleic acid sequence of the heavy chain variable region is SEQ ID No. 38, the nucleic acid sequence of the light chain constant region is SEQ ID No. 36, and the nucleic acid sequence of the heavy chain constant region is SEQ ID No. 40. The heavy chain constant region uses human IgG1, and the light chain constant region uses kappa.

[0111] Comparative antibodies:

[0112] This embodiment provides four contrasting CD8 antibodies: CD8-2, CD8-3, CD8-7, and CD8-8.

[0113] The heavy chain amino acid sequence of the CD8-2 antibody is as follows:

[0114] SEQ ID No. 41:

[0115] EVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWIGRIDPANDNTLYASKFQGRATITADTSSTAYLELSSLRSEDTAVYYCGRGYGYYVFDHWGQGTLVTVSS.

[0116] The light chain amino acid sequence of the CD8-2 antibody is as follows:

[0117] SEQ ID No. 42:

[0118] DVQITQSPSSSLSASVGDRVTITTCRTSRSISQYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNENPLTFGQGTKVEIK.

[0119] The heavy chain amino acid sequence of the CD8-3 antibody is as follows:

[0120] SEQ ID No. 43:

[0121] EVQLQQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGKGLEWMGRIDPANDNTLYASKFQGRVTITADTSNTAYMELSSLRSEDTAVYYCGRGYGYYVFDHWGQGTTVTVSS.

[0122] The light chain amino acid sequence of the CD8-3 antibody is as follows:

[0123] SEQ ID No. 44:

[0124] DIKMTQSPSSSLSASVGDRVTITTCRTSRSISQYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNENPLTFGAGTKLEIK.

[0125] The heavy chain amino acid sequence of the CD8-7 antibody is as follows:

[0126] SEQ ID No. 45:

[0127] EVQLQQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGKGLEWMGRIDPANDNTLYARKFQGRVTITADTSNTAYMELSSLRSEDTAVYYCTRGYGYYVFDTWGQGTTVTVSS.

[0128] The light chain amino acid sequence of the CD8-7 antibody is as follows:

[0129] SEQ ID No. 46:

[0130] DIKMTQSPSSSLSASVGDRVTITTCRTSRSISQYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVNEFPVTFGAGTKLEIK.

[0131] CD8-8 was purchased from Yiqiao Shenzhou, item number: 10980-MM28.

[0132] 1.1 Construction of recombinant expression vectors

[0133] Recombinant expression vectors can be constructed based on the nucleic acid / amino acid sequences of CD8-1 and CD8-4 antibodies. The nucleic acids of CD8-1, CD8-4 antibodies and contrast antibodies can be cloned into commonly used prokaryotic expression vectors using conventional methods, or they can be prepared by a professional vector preparation company. In this example, the antibody separation and expression vector were completed by Shanghai Baiying Biotechnology Co., Ltd. and the results were verified to be correct.

[0134] 1.2 Prokaryotic expression of isolated antibodies

[0135] 1.2.1 Culture of CHO cells

[0136] CHO cell lines were placed in RPMI 1640 medium containing 10% inactivated fetal bovine serum, 100 IU / mL penicillin, and 100 IU / mL streptomycin. The medium was then incubated at 37°C in a 5% CO2 incubator, with the medium being changed every 48 hours.

[0137] 1.2.2 Transfection and Expression

[0138] 1) Take 145M of the cells obtained in step 1.4.1 of this embodiment, centrifuge to remove the supernatant, and retain the precipitate.

[0139] 2) Add about 0.5 mL of electroporation buffer to the precipitate obtained in 1), mix well, and then add an appropriate amount of the recombinant expression vector (concentration 500 ng / μL) obtained in 1.3 to obtain a cell plasmid suspension.

[0140] 3) After thoroughly mixing the above cell plasmid suspension, take 1 mL and add it to a 1 mL electroporation tube. Place the electroporation tube into the electroporation apparatus for electroporation.

[0141] 4) After electroporation, aliquot the cells from the electroporation tube into a shaker containing 20 mL of culture medium and incubate for 40 min.

[0142] 5) After incubation, place the shake flasks in a 37°C, 270 rpm, 8% CO2 incubator. After 24 hours, add feed / sodium butyrate / double antibiotics and continue incubation for 3–7 days. On day 5, take samples for ELISA testing.

[0143] 1.2.3 Antibody purification

[0144] Experimental method: Protein A affinity chromatography column purification

[0145] (1) Equilibration column: l×PBS, flow rate 1mL / min, 20mL;

[0146] (2) Sample loading: flow rate 1 mL / min;

[0147] (3) Washing: 1×PBS, flow rate 1mL / min, 20mL;

[0148] (4) Elution: Citrate buffer (pH 3.4), 1 mL / min, collected in aliquots, approximately 500 μL per tube. A total of 10 tubes were collected, and the absorbance at 280 nm was read using a NanoDrop instrument;

[0149] (5) Dialysis: The high concentration of protein was aspirated into a dialysis bag and placed in a beaker containing 1×PBS for dialysis to obtain the purified isolated antibody.

[0150] 1.2.4 Basic Antibody Quality Control

[0151] 1) Concentration detection;

[0152] 2) Purity detection (SEC-HPLC);

[0153] Experimental materials: High performance liquid chromatograph, gel chromatography column, deionized water, mobile phase (Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl);

[0154] Experimental methods:

[0155] SEC experiments were performed using a high-performance liquid chromatograph (LC-20AT) and a gel chromatography column. The experimental conditions were as follows:

[0156] Flow rate: 1 mL / min; Injection volume: 20 μL

[0157] Column temperature: 35℃

[0158] Detection wavelengths: 214nm, 280nm

[0159] Data collection time: 15 min

[0160] Replace the water with the mobile phase, and slowly increase the flow rate to 1,000 mL / min until the baseline stabilizes. Transfer 50 μL of antibody to the corresponding numbered vial, place the vial in the appropriate position on the instrument, and inject the sample for 15 min. Analyze, process, and save the data. Replace the mobile phase with deionized water and rinse for 1.5 h.

[0161] The results showed that the antibody purity was >90%.

[0162] Example 2: Preparation of Antibody Conjugation to Magnetic Nanoparticles

[0163] In this embodiment, the antibody prepared in Example 1 is conjugated to magnetic nanoparticles to prepare antibody-conjugated magnetic beads. The experimental steps are as follows:

[0164] (1) Take 1 mg of nano-magnetic particles, add 1 mg of EDC and NHS (MES dissolved at pH 5.5), and react with shaking at 37 degrees Celsius for 0.5 h; centrifuge at 20000 g for 30 min to remove NHS and EDC.

[0165] (2) Add 0.5 mg of antibody to the precipitate, and then add 500 μL of pH 8.0 BB solution to adjust the pH of the buffer solution. Shake at room temperature for 2.5 h.

[0166] (3) Centrifuge at 20000g for 30min, and retain the supernatant (centrifuge the supernatant together with the next sample to remove interference) for BCA testing;

[0167] (4) Block the reaction with an aqueous solution containing 20 mg / mL BSA for 1 h, and shake the reaction at 37 degrees Celsius. Centrifuge at 20000g for 30 min and discard the supernatant.

[0168] (5) Wash with DEPC again, resuspend to 1 mL, and take 20 μL to 1 mL of pure water to test for DLS.

[0169] (6) Centrifuge at 20000g for 30 min, discard the supernatant, and resuspend in the preservation solution to 500 μL. Filter through a 0.22 μm filter membrane.

[0170] Example 3: PBMC Enrichment and Sorting

[0171] This embodiment uses the antibody-conjugated magnetic beads prepared in Example 2 to sort PBMCs. The experimental steps are as follows:

[0172] (1) Resuscitate PBMCs and let them rest for 2 hours at 37°C and 5% CO2.

[0173] (2) Collect PBMC into a 15mL centrifuge tube, centrifuge at 500g for 5min, discard the supernatant, and wash once with 2mL flow cytometry buffer (PBS + 2% FBS);

[0174] (3) Resuspend the PBMCs using flow cytometry buffer, count them, and take 2.5 × 10⁻⁶ samples. 6 Cells were centrifuged, resuspended in 50 μL of flow cytometry buffer, and 5 μL of antibody-conjugated magnetic beads were added. After mixing, the cells were incubated at room temperature for 15 min.

[0175] (4) Add 500 μL of flow cytometry buffer to terminate the reaction, transfer to MS Columns (MS Columns need to be moistened in advance according to the instructions), and after the cell suspension has been dripped, wash the MS Columns twice with 500 μL of flow cytometry buffer and collect the cell waste liquid;

[0176] (5) Add 1 mL of flow cytometry buffer to MS Columns and use the stopcock to collect the sorted and enriched cells into a 1.5 mL centrifuge tube.

[0177] The efficiency and purity of sorting and enriching CD8 T cells were determined by the following steps:

[0178] (1) Take 3 × 10⁻⁶ cells from the cell waste liquid, the enriched cells, and the PBMCs before enrichment and sorting. 5 Centrifuge each cell at 4°C and 500×g for 5 min, discard the supernatant, and wash once with flow cytometry buffer.

[0179] (2) Resuspend the cells in 100 μL of flow cytometry buffer, add 1 μg of PE-Cy7-anti-CD8 antibody and 1 μg of PE-anti-CD3 antibody, incubate on ice for 30 min, wash twice with flow cytometry buffer, and resuspend the cells in 300 μL of flow cytometry buffer. Use flow cytometer to detect the proportion of CD8 T cells in the waste liquid and enriched cells before and after sorting.

[0180] Depend on Figure 1A It can be seen that before enrichment and sorting, the proportion of CD8 T cells in PBMCs was 26.6%. After enrichment and sorting, the proportion of CD8 T cells increased significantly, exceeding 90%, indicating that anti-CD8-1 magnetic beads or anti-CD8-4 magnetic beads can enrich CD8 T cells from PBMCs with high purity. Figure 1B It can be seen that only a small number of CD8 T cells remain in the waste liquid after enrichment and sorting. As shown in the figure, more than 90% of CD8 T cells are enriched, indicating that anti-CD8-1 magnetic beads or anti-CD8-4 magnetic beads can efficiently enrich CD8 T cells from PBMCs.

[0181] The four comparative antibodies provided in Example 1 were used to recognize the CD8 antigen.

[0182] As shown in Figure 2, all four contrast antibodies can recognize the CD8 antigen on the cell surface.

[0183] Depend on Figure 3A It can be seen that before sorting and enrichment using anti-CD8-2 magnetic beads, anti-CD8-3 magnetic beads, and anti-CD8-7 magnetic beads, the proportion of CD8 T cells in PBMCs was 20.5%; after sorting and enrichment, the proportions of CD8 T cells were 78.2%, 77.7%, and 92.2%, respectively.

[0184] Depend on Figure 3B It can be seen that after sorting and enriching with anti-CD8-2 magnetic beads, anti-CD8-3 magnetic beads and anti-CD8-7 magnetic beads, the proportions of CD8 T cells in the cell waste liquid were 9.03%, 7.42% and 10.3%, respectively.

[0185] Depend on Figure 3C It can be seen that before sorting and enrichment using anti-CD8-8 magnetic beads, the proportion of CD8 T cells in PBMCs was 25.7%; after sorting and enrichment, the proportion of CD8 T cells in the waste liquid was 25.6%. This indicates that CD8 T cells were basically not enriched, making it impossible to perform flow cytometry detection of the enriched cells.

[0186] Therefore, when PBMCs are sorted and enriched using anti-CD8-2, anti-CD8-3, anti-CD8-7 and CD8-8 antibodies, a large number of other contaminating cells are present in the enriched cells, and a large number of unenriched CD8 T cells remain in the waste liquid.

[0187] In summary, the present invention prepares anti-CD8 antibodies, preferably CD8-1 and CD8-4 antibodies. Compared with other CD8 antibodies such as CD8-2, CD8-3, CD8-7, and CD8-7, the CD8 antibody-conjugated magnetic beads prepared by the preferred anti-CD8 antibodies of the present invention can efficiently and with high purity sort and enrich CD8 T cells from PBMCs.

[0188] Example 4: Construction of CD8+CD22-CAR-T cells

[0189] 4.1 Packaging of lentiviruses, including the following steps:

[0190] (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.

[0191] (2) Dissolve 30 μg of HD SIN03 CD22(V29)41BBz(ka) lentiviral vector, 12.5 μg of helper plasmid gag / pol, and 10 μg of envelope plasmid VSVg in 2000 μL of serum-free DMEM culture medium and mix well.

[0192] (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;

[0193] (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;

[0194] (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.

[0195] (6) After 48 hours, the viral supernatant was collected to obtain lentivirus.

[0196] 4.2 Lentiviral Concentration

[0197] The viral supernatant prepared in 4.1 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 inverted and mixed thoroughly. The mixture was then incubated overnight at 4 °C. The mixture was centrifuged at 3500 rpm for 30 min at 4 °C. The supernatant was discarded, 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.

[0198] 4.3 Lentiviral titer detection

[0199] 500 μL of Jurkat cells (1 × 10⁻⁶) 5 (Number of cells) were seeded into 24-well culture plates; concentrated lentivirus (4.2) was added to the cell suspension at doses of 1 μL, 0.2 μL, and 0.04 μL, respectively, and polybrene was added to a final concentration of 5 μg / mL; after incubation at 37°C and 5% CO2 overnight, the medium was replaced with fresh medium; 72 h after 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 PE-anti-VHH 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) / (number of cells / number ... 5 ) × Positive rate / Viral volume (mL).

[0200] 4.4 Lentiviral transduction of CD8 T cells.

[0201] 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. The CD8 T cells enriched and sorted with anti-CD8-1 magnetic beads or anti-CD8-4 magnetic beads were adjusted to a density of 1 × 10⁻⁶ cells 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 CD8 T cells were collected and the cell density was adjusted to 1×10⁶. 6 / mL, add the lentivirus prepared in 4.3 according to the multiplicity of infection (MOI) = 10, and add polybrene to a final concentration of 5 μg / mL; incubate overnight at 37℃ and 5% CO2, then replace with fresh medium, and passage every 2 days.

[0202] 4.5 CD8 T cell chimeric antigen receptor expression includes the following steps:

[0203] (1) Count the number of infections 5, 8, 11, and 14 days after infection. Figure 4 (The day of infection is recorded as day 0) and 3 × 10⁻⁶ samples are collected. 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.

[0204] (2) Resuspend the cells in 100 μL PBS + 2% FBS, add 1 μg of AF488-anti-VHH antibody, and incubate on ice for 30 min; wash twice with PBS + 2% FBS, then resuspend the cells in 300 μL PBS + 2% FBS. Use uninfected T cells as a control and detect the infection efficiency by flow cytometry (see Figure 5).

[0205] Depend on Figure 4 It can be seen that CD8 T cells proliferated nearly 1000 times on the 14th day after infection with the virus compared to the day of infection, indicating that enriched and sorted CD8 T cells can proliferate rapidly under normal T cell culture conditions.

[0206] As shown in Figure 5, the chimeric antigen receptor was expressed relatively stably on days 5, 8, 11 and 14 after CD8 T cells were infected with the virus.

[0207] Example 5 In vitro toxicity test

[0208] This embodiment uses the CAR-T cells prepared in Example 4 to conduct an in vitro toxicity experiment, including the following steps:

[0209] (1) Target cell seeding:

[0210] Using CD22-negative cells and CD22-positive tumor cells as target cells, in this embodiment, the tumor cells are K562, and the CD22-negative cells are named tumor cells K562-luci(CD22) — CD22-positive tumor cells were named K562-CD22-luci(CD22) + As target cells, the target cell concentration was adjusted to 2 × 10⁻⁶. 5 / mL, take 50μL and inoculate it into a 96-well plate;

[0211] (2) Effector cell seeding:

[0212] CAR-T cells containing CD22 antibody prepared in Example 4 and control T cells (not infected with lentivirus) were used as effector cells; CAR-T cells and control T cells were added to 96-well plates at effector-to-target ratios of 0.3:1, 1:1 and 3:1.

[0213] 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:

[0214] Experimental group: CAR-T+ target cells;

[0215] Control group: Control T cells + target cells;

[0216] (3) After co-culturing effector cells and target cells for 18 hours, use The luciferase assay kit is used for detection; please refer to the specific detection steps. According to the instructions for the luciferase assay kit, the results are shown in Figure 6. The CAR-T cells constructed in this invention have highly efficient killing activity against CD22-positive tumor cells and no killing effect on CD22-negative cells, indicating that the CAR-T cells constructed in this invention also have high specificity.

[0217] Example 6 Detection of CAR-T cytokine secretion

[0218] 1. Cell culture supernatant

[0219] The cell culture with an effect-to-target ratio of 1:1 in Example 5 was centrifuged at 400×g for 10 min to remove the precipitate, and the supernatant was stored at -80℃ for testing.

[0220] 2. Reagent preparation

[0221] 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.

[0222] 3. Preparation of Standards and Samples

[0223] Standards: The stock solution of the standard was diluted 2 times with 5% 1640 medium, with a total of 8 dilution gradients, including zero concentration.

[0224] Samples: Dilute the samples using 5% 1640 medium as directed.

[0225] 4. Testing Steps

[0226] (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.

[0227] (2) Add standard: Add 100 μL of 2× diluted standard to the standard wells and add 100 μL of 5% 1640 medium to the blank wells;

[0228] (3) Add sample: Add 100 μL of cell culture supernatant to the sample well;

[0229] (4) Add detection antibody: Add 50 μL of diluted detection antibody (1:100 dilution) to each well;

[0230] (5) Incubation: Seal the plate with sealing film, shake at 300 rpm, and incubate at 25°C for 2 hours;

[0231] (6) Washing: Discard the liquid, add 300 μL of washing solution to each well and wash the plate 6 times;

[0232] (7) Enzyme incubation: Add 100 μL of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well;

[0233] (8) Incubation: Seal the plate with a new sealing film, shake at 300 rpm, and incubate at 25°C for 45 min;

[0234] (9) Washing: Repeat step (6);

[0235] (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;

[0236] (11) Add stop solution: Add 100 μL of stop solution to each well and mix thoroughly;

[0237] (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.

[0238] IFN-γ factor secretion results as follows Figure 7 As shown, spontaneous CAR-T cell culture alone was detected in spontaneous culture, as well as in K562-luci and CAR-T culture. Higher levels of IFN-γ were detected in K562-CD22-luci and CAR-T culture. This indicates that the CAR-T cells constructed in this invention can also release cytokines to CD22-positive tumor cells to exert a killing function, and have high specificity. They do not have significant cytokine secretion on CD22-negative cells.

[0239] Depend on Figure 7 It is known that by using the sorted CD8 T cells to further prepare chimeric antigen receptor cells, the chimeric antigen receptor cells can efficiently kill tumor cells and secrete cytokines to exert their killing function, and have high specificity.

[0240] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.

Claims

1. The use of anti-CD8 antibodies for enriching and sorting CD8 T cells, among which, The anti-CD8 antibody includes a light chain constant region, a light chain variable region, a heavy chain constant region, and a heavy chain variable region; The amino acid sequence of CDR1 in the light chain variable region is the sequence shown in SEQ ID No. 1, the amino acid sequence of CDR2 is GAS, and the amino acid sequence of CDR3 is the sequence shown in SEQ ID No. 5; the amino acid sequence of CDR1 in the heavy chain variable region is the sequence shown in SEQ ID No. 7, the amino acid sequence of CDR2 in the heavy chain variable region is the sequence shown in SEQ ID No. 9, and the amino acid sequence of CDR3 in the heavy chain variable region is the sequence shown in SEQ ID No.

11. Alternatively, the amino acid sequence of CDR1 in the light chain variable region is the sequence shown in SEQ ID No. 2, the amino acid sequence of CDR2 is HGT, and the amino acid sequence of CDR3 is the sequence shown in SEQ ID No. 6; the amino acid sequence of CDR1 in the heavy chain variable region is the sequence shown in SEQ ID No. 8, the amino acid sequence of CDR2 in the heavy chain variable region is the sequence shown in SEQ ID No. 10, and the amino acid sequence of CDR3 in the heavy chain variable region is the sequence shown in SEQ ID No.

12.

2. The use according to claim 1, characterized in that, The light chain variable region further includes framework regions FR1-FR4; the amino acid sequence of FR1 includes the sequence shown in SEQ ID No. 13 or SEQ ID No. 14; the amino acid sequence of FR2 includes the sequence shown in SEQ ID No. 15 or SEQ ID No. 16; the amino acid sequence of FR3 includes the sequence shown in SEQ ID No. 17 or SEQ ID No. 18; and the amino acid sequence of FR4 includes the sequence shown in SEQ ID No. 19 or SEQ ID No.

20. And / or, the heavy chain variable region further includes framework regions FR1-FR4; the amino acid sequence of FR1 includes the sequence shown in SEQ ID No. 21 or SEQ ID No. 22; the amino acid sequence of FR2 includes the sequence shown in SEQ ID No. 23 or SEQ ID No. 24; the amino acid sequence of FR3 includes the sequence shown in SEQ ID No. 25 or SEQ ID No. 26; and the amino acid sequence of FR4 includes the sequence shown in SEQ ID No. 27 or SEQ ID No.

28.

3. The use according to claim 1, characterized in that, The nucleotide sequence of the light chain constant region of the anti-CD8 antibody is selected from the sequence shown in SEQ ID No. 35, and the nucleotide sequence of the heavy chain constant region is selected from the sequence shown in SEQ ID No. 39; or, the nucleotide sequence of the light chain constant region of the anti-CD8 antibody is selected from the sequence shown in SEQ ID No. 36, and the nucleotide sequence of the heavy chain constant region is selected from the sequence shown in SEQ ID No.

40.

4. The use according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the anti-CD8 antibody is SEQ ID No. 29, and the amino acid sequence of the heavy chain variable region is SEQ ID No. 31; or, the amino acid sequence of the light chain variable region of the anti-CD8 antibody is SEQ ID No. 30, and the amino acid sequence of the heavy chain variable region is SEQ ID No.

32.

5. A method for enriching and sorting CD8 T cells, comprising the following steps: 1) The anti-CD8 antibody of any one of claims 1-4 is coupled to the magnetic nanoparticles to obtain anti-CD8 antibody-coupled magnetic beads; 2) The anti-CD8 antibody-conjugated magnetic beads are co-incubated with mixed cells containing CD8 T cells, and then the incubated cells are placed in a magnetic field. The CD8 T cells are enriched and sorted by the binding of the magnetic field with the anti-CD8 antibody-conjugated magnetic beads.

6. The method according to claim 5, characterized in that, The mixed cells containing CD8 T cells include human peripheral blood mononuclear cells.

7. Use of the method according to claim 5 or 6 in the preparation of CAR-T cells.

8. The use according to claim 7, wherein the CAR-T cells are CD8+CD22-CAR-T cells.

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