WO2023072131A1 - 一种制备通用型car-t细胞的方法及其应用 - Google Patents

一种制备通用型car-t细胞的方法及其应用 Download PDF

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WO2023072131A1
WO2023072131A1 PCT/CN2022/127619 CN2022127619W WO2023072131A1 WO 2023072131 A1 WO2023072131 A1 WO 2023072131A1 CN 2022127619 W CN2022127619 W CN 2022127619W WO 2023072131 A1 WO2023072131 A1 WO 2023072131A1
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cells
car
binding molecule
cdr
amino acid
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PCT/CN2022/127619
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French (fr)
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王先进
彭亮
叶立军
黄倩
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深圳市菲鹏生物治疗股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to the technical field of CAR-T cell therapy, in particular to a method for preparing universal CAR-T cells and its application.
  • the efficiency of knocking out the ⁇ 2M gene in T cells has become higher and higher.
  • gene editing operations have a great impact on the state and growth rate of T cells.
  • the higher the knockout efficiency of the ⁇ 2M gene the worse the state of T cells after gene editing and the slower the cell growth rate. Therefore, in order to balance the two, the knockout efficiency of the ⁇ 2M gene will be controlled to a certain extent during the preparation of universal CAR-T cells. Therefore, if the prepared general-purpose CAR-T cells are to be used in clinical cell therapy, T cells that have not knocked out the ⁇ 2M gene must be removed. Otherwise, it will lead to graft-versus-host disease (GvHD) or host-versus-graft disease (HvGD) in clinical application.
  • GvHD graft-versus-host disease
  • HvGD host-versus-graft disease
  • the object of the present invention is to provide a ⁇ 2M-binding molecule.
  • the binding molecule can be used to remove T cells that have not knocked out the ⁇ 2M gene in general CAR-T cells, and realize the purification of general CAR-T cells, thereby reducing or avoiding graft-versus-host disease (GvHD) or host-versus-graft disease (HvGD).
  • GvHD graft-versus-host disease
  • HvGD host-versus-graft disease
  • Another object of the present invention is to provide the application of the above-mentioned ⁇ 2M-binding molecules in the detection and purification of universal CAR-T cells, as well as the application in the preparation of products for detection of ⁇ 2M or purification of universal CAR-T cells.
  • the products include commercially available preparations or kits.
  • Another object of the present invention is to provide a method for purifying universal CAR-T cells using the above preparation or kit.
  • the method has good purification effect, simple operation and is suitable for popularization.
  • Another object of the present invention is to provide a method for preparing universal CAR-T cells using the above-mentioned ⁇ 2M binding molecules. In order to directly obtain general-purpose CAR-T cells that meet the clinical purity requirements, avoiding the tedious purification process before application.
  • the present invention provides the following technical solutions:
  • the present invention provides a ⁇ 2M-binding molecule, which includes a module a that specifically binds to ⁇ 2M;
  • the module a includes a VH domain, and the VH domain includes CDR-H1 having the amino acid sequence shown in SEQ ID No.1, CDR-H2 having the amino acid sequence shown in SEQ ID No.2 and having the amino acid sequence shown in SEQ ID No. CDR-H3 of the amino acid sequence shown in 3.
  • the module a also includes a VL domain, and the VL domain includes a CDR-L1 having an amino acid sequence shown in SEQ ID No.4, a CDR-L2 having an amino acid sequence of AAA, and a CDR-L2 having an amino acid sequence of SEQ ID No.4. CDR-L3 of the amino acid sequence shown in ID No.5.
  • the present invention also provides a ⁇ 2M-binding molecule, which includes a module a that specifically binds to ⁇ 2M;
  • the module a comprises a VH domain and a VL domain, the VH domain comprising CDR-H1, CDR-H2 and CDR-H3;
  • the VL domain includes CDR-L1, CDR-L2 and CDR-L3;
  • amino acid sequences of the CDR-H1, the CDR-H2 and the CDR-H3 include the complementarity determining region sequence of SEQ ID No.6;
  • amino acid sequences of the CDR-L1, the CDR-L2 and the CDR-L3 include the complementarity determining region sequence of SEQ ID No.7.
  • amino acid sequences of the above-mentioned CDR-H1, the above-mentioned CDR-H2 and the above-mentioned CDR-H3 are defined by SEQ ID No.6 according to the Kabat, Chothia, or AbM numbering system;
  • amino acid sequences of the above-mentioned CDR-L1, the above-mentioned CDR-L2 and the above-mentioned CDR-L3 are defined by SEQ ID No.7 according to the Kabat, Chothia, or AbM numbering system.
  • the VH domain of the module a has the amino acid sequence shown in SEQ ID No.6.
  • the VL domain of the module a has the amino acid sequence shown in SEQ ID No.7.
  • the ⁇ 2M-binding molecules are selected from scFv molecules, Fv molecules, Fab molecules or intact antibody molecules that specifically bind to ⁇ 2M antigens.
  • the present invention provides the application of the ⁇ 2M-binding molecule described in any one of the foregoing embodiments in the preparation of ⁇ 2M detection products, or in the in vitro detection of ⁇ 2M not for the purpose of disease diagnosis or treatment.
  • the present invention provides the application of the ⁇ 2M binding molecule described in any one of the foregoing embodiments in purifying universal CAR-T cells or preparing products for purifying universal CAR-T cells.
  • the present invention provides a preparation for detecting ⁇ 2M or a preparation for purifying general-purpose CAR-T cells, the preparation includes the ⁇ 2M binding molecule described in any one of the preceding embodiments, and the ⁇ 2M binding molecule is preferably coupled to Linked with biotin.
  • the preparation for purifying universal CAR-T cells further includes a second binding molecule that specifically binds universal CAR-T cells.
  • the second binding molecule is preferably conjugated to biotin.
  • the second binding molecule is selected from other molecules capable of specifically binding to TCR-positive CAR-T cells.
  • the target protein that specifically binds to the second binding molecule is TCR or CD3.
  • the second binding molecule includes a module c that specifically binds to or CD3; the module c includes a VH domain having the amino acid sequence shown in SEQ ID No.8, and having the amino acid sequence shown in SEQ ID No. .9 the VL domain of the amino acid sequence shown.
  • the present invention provides a kit for purifying general-purpose CAR-T cells, the kit comprising the preparation for purifying general-purpose CAR-T cells described in the foregoing embodiments and optional consumables.
  • the present invention provides a kit for detecting ⁇ 2M, which includes the preparation for detecting ⁇ 2M described in the foregoing embodiments and optional consumables.
  • the present invention provides the preparation for purifying general-purpose CAR-T cells described in any of the preceding embodiments or the kit for purifying general-purpose CAR-T cells described in the preceding embodiments to purify general-purpose CAR-T cells.
  • a method for CAR-T cells comprising: after incubating the general-purpose CAR-T cells to be purified in a preparation for purifying general-purpose CAR-T cells, using anti-biotin-coupled magnetic beads for magnetic adsorption , take the cell suspension to obtain the purified universal CAR-T cells.
  • antibiotin-coupled magnetic beads refers to magnetic beads coupled with materials capable of specifically binding biotin, such as magnetic beads coupled with avidin.
  • the present invention provides a method for preparing universal CAR-T cells, comprising the following steps:
  • the present invention provides a method for purifying universal CAR-T cells, which includes: using the above-mentioned ⁇ 2M binding molecule to contact the universal CAR-T cells to be purified.
  • the VH domain and VL domain of the ⁇ 2M binding molecule provided by the present invention contain specific CDR sequences, which can specifically bind to ⁇ 2M, and the binding molecule is added to CAR-T cells with incomplete knockout of the ⁇ 2M gene.
  • the binding molecule can specifically bind to CAR-T cells without knockout of the ⁇ 2M gene, thereby separating CAR-T cells with knockout of the ⁇ 2M gene from CAR-T cells without knockout of the ⁇ 2M gene, realizing universal CAR-T cells of purification.
  • the general-purpose CAR-T cells can be significantly improved. - T cell purity. Especially after combined use with the second binding molecule whose target protein is CD3, the purity of the general-purpose CAR-T cells can be as high as 99.96%, and it has been proved by the characterization of the tumor-killing activity in vivo and in vitro that the general-purpose CAR-T cells provided by the present invention have The purification method did not cause adverse effects on the universal CAR-T cells.
  • the present invention also provides a method for preparing a universal CAR-T cell.
  • the preparation method uses the ⁇ 2M binding molecule provided by the present invention and an optional anti-CD3 antibody. After knocking out the ⁇ 2M gene and the TCR gene, the CAR-T If the cells are not separated, they can be directly co-cultured with them, and then isolated to obtain universal CAR-T cells, eliminating the need for an independent purification process and reducing the labor intensity of front-line staff.
  • Fig. 1 is the pCDHF plasmid map that uses in the experimental example 2 of the present invention
  • Fig. 2 is the kinetic affinity analysis fitting curve of anti-human ⁇ 2M antibody ⁇ 2M-2B1;
  • Figure 3 is a map of the pX330-spCAS9-HF1 plasmid used in Example 2 of the present invention.
  • Fig. 4 is the double knockout efficiency of TCR/ ⁇ 2M gene in Experimental Example 2 of the present invention.
  • Fig. 6 is a comparison diagram of the activation effect of CAR-T cells after purification in Experimental Example 3 of the present invention.
  • Fig. 7 is a picture of the in vitro tumor killing effect of purified CAR-T cells in Experimental Example 4 of the present invention.
  • Figure 8 is a comparison chart of the release of tumor-killing cytokines from CAR-T cells in vitro after purification in Experimental Example 4 of the present invention.
  • Fig. 9 is a diagram of the in vivo tumor killing effect of purified CAR-T cells in Experimental Example 5 of the present invention.
  • FIG. 10 is a survival curve drawn according to the survival conditions of mice in Experimental Example 5 of the present invention.
  • amino acid denotes a naturally occurring carboxy alpha-amino acid.
  • Naturally occurring amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), Cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile , I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P) , serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).
  • alanine three-letter code: Ala, one-letter code:
  • universal CAR-T cells generally refers to T cells obtained from healthy volunteers, whose related genes (mainly genes related to GVHD and HvGD diseases) have been knocked out, such as the expression of TCR (such as the coding of ⁇ chain and ⁇ chain) gene), HLA (such as the gene encoding ⁇ 2 microglobulin ( ⁇ 2M gene)), or the gene encoding molecules such as CD52), and CAR-T cells made after the transfer of the CAR (chimeric antigen receptor) gene.
  • TCR such as the coding of ⁇ chain and ⁇ chain
  • HLA such as the gene encoding ⁇ 2 microglobulin ( ⁇ 2M gene)
  • CD52 chimeric antigen receptor
  • the present invention uses the ⁇ 2M protein as an antigen and constructs a binding molecule that specifically binds to the ⁇ 2M protein, that is, a ⁇ 2M binding molecule, based on the antibody structure.
  • the present invention provides a module (a1) that specifically binds to ⁇ 2M, the module (a1) includes a VH domain, and the VH domain includes a compound having SEQ ID No. CDR-H1 having the amino acid sequence shown in 1, CDR-H2 having the amino acid sequence shown in SEQ ID No.2 and CDR-H3 having the amino acid sequence shown in SEQ ID No.3.
  • SEQ ID No.1-3 is defined by the heavy chain variable region sequence according to the IMGT numbering system.
  • the present invention provides a module (a2) that specifically binds to ⁇ 2M, the module (a2) includes a VL domain, and the VL domain includes amino acids shown in SEQ ID No.4
  • SEQ ID No.4, AAA of CDR-L2, and SEQ ID No.5 are defined by the light chain variable region sequence according to the IMGT numbering system.
  • the present invention provides a module (a3) that specifically binds to ⁇ 2M, the module (a3) includes a VH domain and a VL domain, and the VH domain includes a compound having SEQ ID No.
  • amino acid sequences of the CDR-H1, the CDR-H2 and the CDR-H3 include the complementarity determining region sequence of SEQ ID No.6;
  • amino acid sequences of the CDR-L1, the CDR-L2 and the CDR-L3 include the complementarity determining region sequence of SEQ ID No.7.
  • CDR-H1, CDR-H2 and CDR-H3 can also be defined by SEQ ID No.6 according to the Kabat, Chothia, or AbM numbering system.
  • amino acid sequences of the above-mentioned CDR-L1, the above-mentioned CDR-L2 and the above-mentioned CDR-L3 can also be defined by SEQ ID No.7 according to the Kabat, Chothia, or AbM numbering system.
  • sequences of the above complementarity determining regions are defined according to the Kabat, Chothia, or AbM numbering system, as shown in the following table:
  • IMGT is based on the numbering system of The international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev.Comparat.Immunol.27:5577, 2003.
  • IMGT The international ImMunoGeneTics information system
  • Chothia The immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196: 901 917; Chothia et al. (1989) Nature 342: 878 883).
  • the present invention provides the following modules:
  • VH domain comprises a VH domain, and said VH domain has the amino acid sequence shown in SEQ ID No.6.
  • Module (a21) comprises VL structural domain, and described VL structural domain has the aminoacid sequence shown in SEQ ID No.7.
  • the module (a31) includes a VH domain and a VL domain, the VH domain has the amino acid sequence shown in SEQ ID No.6, and the VL domain has the amino acid sequence shown in SEQ ID No.7.
  • the present invention provides scFV molecules, which include a VH domain and a VL domain connected by an elastic linker peptide (Linker); the VH domain has SEQ ID The amino acid sequence shown in No.6, the VL domain has the amino acid sequence shown in SEQ ID No.7.
  • Linker an elastic linker peptide
  • the amino acid residue composition and length of the Linker can be adjusted by those skilled in the art according to actual needs, including but not limited to glycine (Gly) and serine (Ser) with a length of 15-25 amino acid residues. constitute.
  • Exemplary Linkers include: (GGGGS)n(SEQ ID No.22), (GGGS)n(SEQ ID No.23), (GGS)n(SEQ ID No.24), or (GS)n(SEQ ID No.24) ID No.25) wherein n is selected from 1,2,3,4,5 or 6.
  • the present invention provides an Fv molecule, the Fv molecule comprising a VH domain and a VL domain connected by a short peptide; the VH domain has an amino acid sequence shown in SEQ ID No.6, and the VL domain It has the amino acid sequence shown in SEQ ID No.7.
  • the short peptides can be adjusted and obtained by those skilled in the art according to actual needs through conventional means, including but not limited to short peptide chains consisting of 3-9 amino acid residues.
  • the present invention provides a Fab molecule, the Fab molecule comprising the VH domain, the VL domain, a light chain constant region (CL) and a heavy chain constant region (CH1); the VH domain It has the amino acid sequence shown in SEQ ID No.6, and the VL domain has the amino acid sequence shown in SEQ ID No.7.
  • CH1 and CL those skilled in the art can select according to actual needs, for example, the CH1 is selected from any one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or IgM, and the CL is selected from From ⁇ chain or ⁇ chain, in addition, those skilled in the art can also adjust the sequence and modification of CH1 and CL selected above by conventional means according to actual needs.
  • the present invention also provides a complete antibody molecule, which includes two identical heavy chains and two identical light chains, the heavy chains include a VH domain and a heavy chain constant region, and the light chains include The chain includes a VL domain and a light chain constant region; the VH domain has an amino acid sequence shown in SEQ ID No.6, and the VL domain has an amino acid sequence shown in SEQ ID No.7.
  • the heavy chain constant region can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or Any one or several of IgM
  • the light chain constant region can be selected from ⁇ chain or ⁇ chain.
  • the heavy chain constant region and the light chain constant region of said intact antibody molecule are selected from murine IgG.
  • the present invention provides the application of each module, scFV molecule, Fv molecule, Fab molecule or complete antibody molecule described in the foregoing embodiments in the preparation of ⁇ 2M detection products, or in the in vitro detection of ⁇ 2M not for the purpose of disease diagnosis Applications.
  • the present invention provides each module, scFv molecule, Fv molecule, Fab molecule or complete antibody molecule described in the foregoing embodiments in purifying general-purpose CAR-T cells or preparing products for purifying general-purpose CAR-T cells Applications.
  • the genes knocked out of the universal CAR-T cells include at least the ⁇ 2M gene.
  • the genes knocked out in the universal CAR-T cells also include TCR genes.
  • it may be a gene encoding an ⁇ chain of a TCR molecule or a gene encoding a ⁇ chain thereof.
  • the present invention provides a preparation for purifying general-purpose CAR-T cells, the preparation includes each module, scFV molecule, Fv molecule, Fab molecule described in any one of the preceding embodiments Molecules or intact antibody molecules; each module, scFv molecule, Fv molecule, Fab molecule or intact antibody molecule is coupled with biotin.
  • biotin is a magnetic bead adsorption separation method commonly used in the art.
  • the present invention provides a kit for purifying general-purpose CAR-T cells, the kit including the preparation and consumables described in the foregoing embodiments.
  • the consumables include conventional kit components in the art, such as orifice plates, reaction containers, liquid-taking devices, and the like.
  • the knocked-out gene of the universal CAR-T cell also includes a TCR gene; the preparation or the kit further includes a second binding molecule, the The second binding molecule is coupled with biotin.
  • the second binding molecule can specifically bind to CAR-T cells positively expressed by TCR.
  • the target protein of the second binding molecule is TCR or CD3, for example, can be selected from CD3 antibody or TCR antibody.
  • the dosage ratio of the second binding molecule and the ⁇ 2M binding molecule those skilled in the art can routinely select according to actual needs.
  • the target protein specifically bound to the second binding molecule is TCR.
  • the target protein that specifically binds to the second binding molecule is CD3, and the second binding molecule includes module c that specifically binds to CD3.
  • the module c includes a VH domain with the amino acid sequence shown in SEQ ID No.8, and a VL domain with the amino acid sequence shown in SEQ ID No.9.
  • the present invention provides a method for purifying general-purpose CAR-T cells in a specific embodiment.
  • the method uses the above-mentioned preparation or kit, and the general-purpose CAR-T cells are incubated in the preparation Finally, use anti-biotin-coupled magnetic beads for magnetic adsorption, and take the cell suspension to obtain purified general-purpose CAR-T cells.
  • the present invention provides a method for preparing universal CAR-T cells, comprising the following steps:
  • the present invention provides a method for purifying general-purpose CAR-T cells in a specific embodiment, which includes: using any of the above-mentioned modules, Fv molecules, Fab molecules, complete antibody molecules, or preparations to be treated Purify general-purpose CAR-T cell contacts to isolate CAR-T cells from which the ⁇ 2M gene has not been knocked out.
  • the purification method may also include using a second binding molecule to contact the universal CAR-T cells to be purified to separate and remove CAR-T cells without knockout of the TCR gene (i.e. CAR-T cells positive for TCR expression).
  • the target protein of the second binding molecule may be TCR or CD3.
  • TCR and CD3 exist in the form of complexes, and the removal of CAR-T cells without TCR gene knockout can be achieved by using binding molecules that bind to TCR or CD3.
  • the second binding molecule can be used after, before, or simultaneously with any of the modules, Fv molecules, Fab molecules, whole antibody molecules, or formulations described above.
  • Fv molecules, Fab molecules, complete antibody molecules or preparations to contact the universal CAR-T cells to be purified continue to use the second binding molecule to contact the universal CAR-T cells to be purified;
  • any of the above-mentioned modules, Fv molecules, Fab molecules, complete antibody molecules or preparations are used to contact the universal CAR-T cells to be purified.
  • said second binding molecule comprises module c that specifically binds to CD3;
  • the module c includes a VH domain with the amino acid sequence shown in SEQ ID No.8, and a VL domain with the amino acid sequence shown in SEQ ID No.9.
  • the modules, scFV molecules, Fv molecules, Fab molecules and complete antibody molecules provided in the first aspect above can be obtained by artificial synthesis, and complete antibody molecules can also be obtained by constructing hybridoma cells for expression and secretion.
  • This embodiment provides a method for preparing an anti-human ⁇ 2M antibody, and the specific steps are as follows:
  • mice On the third day after the last immunization of the mice, the spleens of the mice were taken out under aseptic conditions, placed in a plate, washed once with RPMI1640 (gibco, product number 12633012) basal culture medium, put on a nylon mesh in a small beaker and ground to make a cell suspension . Centrifuge, discard the supernatant, and resuspend in RPMI1640 culture medium.
  • RPMI1640 gibco, product number 12633012
  • splenocytes with the mouse myeloma cells SP20 prepared in advance at a ratio of 10:1, then use 1ml of 50% PEG1500 (sigma, product number 81210) for cell fusion, and add 15ml of RPMI1640 complete culture medium after 1 minute of fusion to stop Cell fusion. Centrifuge at 1000rpm for 5 minutes, discard the supernatant, gently resuspend with 50ml of RPMI1640 screening culture medium, divide equally into 10 96-well plates, culture at 100 ⁇ l/well, 37°C, 5% CO 2 . After culturing until the 6th day, the HAT culture medium was changed twice.
  • the cells corresponding to the positive wells were monoclonalized by the limiting dilution method; after three times of subcloning, several cell lines capable of stably secreting monoclonal antibodies were obtained, one of which was named ⁇ 2M-2B1 cell line.
  • PCR cycle condition is 95°C for 1 minute 1 cycle; 25 cycles at 95°C for 1 minute, 63°C for 1 minute and 72°C for 1 minute.
  • the resulting PCR products were cloned into T vector (InVitrogen) and sequenced.
  • Anti-human ⁇ 2M antibody ⁇ 2M-2B1 belongs to mouse IgG1 subtype, and its light chain is ⁇ chain;
  • the sequence of the heavy chain variable region is as follows:
  • the IMGT numbering system includes the heavy chain complementarity determining region 1 (CDR-H1): GYTFSSYV, that is, SEQ ID No.1, and the heavy chain complementarity determining region 2 (CDR-H2): FNPYNDGT, that is, SEQ ID No. .2 and heavy chain complementarity determining region 3 (CDR-H3): ARRGNTYDNFDY, ie SEQ ID No.3.
  • CDR-H1 GYTFSSYV
  • FNPYNDGT heavy chain complementarity determining region 2
  • CDR-H3 heavy chain complementarity determining region 3
  • CDR-L1 light chain complementarity determining region 1
  • CDR-L2 QTIGTW
  • CDR-L3 light chain complementarity determining Region 3
  • the anti-human ⁇ 2M antibody ⁇ 2M-2B1 obtained in Example 1 was biotin-coupled to prepare a ⁇ 2M-binding preparation.
  • the specific steps are as follows:
  • This example provides a method for preparing an anti-human CD3 antibody.
  • the overall steps are the same as in Example 1, except that the immune antigen is CD3.
  • One selected cell line was named CD3-2C1 cell line.
  • the CD3-2C1 antibody belongs to the mouse IgG1 subtype, and the light chain is a ⁇ chain; it has a VH domain with the amino acid sequence shown in SEQ ID No.8, and a VL domain with the amino acid sequence shown in SEQ ID No.9 .
  • the anti-human CD3 antibody CD3-2C1 obtained in Example 3 was biotin-coupled to prepare a CD3-binding preparation, and the coupling method was the same as that in Example 2.
  • the anti-human ⁇ 2M antibody ⁇ 2M-2B1 prepared in the above-mentioned Example 1 was used to detect the solution of recombinant human ⁇ 2M in vitro, and the detection method was as follows:
  • Buffer 1 ⁇ DPBS+0.02% Tween-20.
  • the kinetic affinity analysis fitting curve of the anti-human ⁇ 2M antibody ⁇ 2M-2B1 after 2-fold serial dilution is shown in Figure 2, and the affinity test results of the anti-human ⁇ 2M antibody ⁇ 2M-2B1 to human ⁇ 2M are shown in Table 2, and the results show that:
  • the anti-human ⁇ 2M antibody ⁇ 2M-2B1 has a high affinity to human ⁇ 2M (KD ⁇ 1.0E-12M), which proves that the anti-human ⁇ 2M antibody ⁇ 2M-2B1 provided in Example 1 can be used to detect ⁇ 2M, or to prepare an antibody for detecting ⁇ 2M product.
  • the ⁇ 2M-2B1-coupled biotin preparation prepared in Example 2 and the CD3-2C1-coupled biotin preparation prepared in Example 4 were used to purify the universal CAR-T cells with TCR and ⁇ 2M gene knockout , and the purification effect was investigated.
  • CART-CD19 cells CART cells targeting CD19.
  • 1 ⁇ 10 6 CART-CD19 cells were plated in the wells of a six-well plate, and 1 ⁇ g of the TRAC sgRNA expression plasmid pX330-spCAS9-HF1-TRAC (knockout of the T cell receptor ⁇ gene (MHC class II molecule), the nucleotide sequence for expressing sgRNA is shown in SEQ ID NO.10), the ⁇ 2M sgRNA expression plasmid of 1 ⁇ g pX330-spCAS9-HF1- ⁇ 2M (knockout the ⁇ 2 microglobulin gene of CAR-T cells (MHC class I Molecule), the nucleic acid sequence for expressing sgRNA is shown in SEQ ID NO.11) and mixed evenly, the pX330-spCAS9-HF1 plasmid map is shown in Figure 3, then add
  • SEQ ID NO.10 acaaaacugugcuagacaug;
  • SEQ ID NO. 11 cgcgagcacagcuaaggcca.
  • TCR/ ⁇ 2M gene double knockout efficiency is shown in Figure 4, and the results showed that the ratio of TCR/ ⁇ 2M gene KO cells was 71.35% by flow cytometry (FACS) analysis after changing the medium for 48 hours.
  • editing tools can also enter cells in the form of sgRNA and RNP through electroporation and virus infection.
  • the cells cultured for 4 days after electroporation were taken, centrifuged, and a certain amount of DPBS was resuspended to a density of 1 ⁇ 10 cells/ml, and treated with CD3-2C1-Biotin (anti-human CD3 antibody coupled with biotin, from Example 4) and ⁇ 2M -
  • CD3-2C1-Biotin anti-human CD3 antibody coupled with biotin, from Example 4
  • 2B1-Biotin anti-human ⁇ 2M antibody conjugated with biotin, from Example 2B1-Biotin (anti-human ⁇ 2M antibody conjugated with biotin, from Example 2) by adding 100 ⁇ l/ml CD3-2C1-Biotin and 100 ⁇ l/ml ⁇ 2M-2B1-Biotin and incubating on ice for 20 minutes in the dark , washed once with PBS, then added anti-Biotin Beads (purchased from Miltenyi Biotec, product number 130-090
  • CD3-2C1-Biotin and ⁇ 2M-2B1-Biotin can also be replaced by two-step purification using CD3-2C1-Biotin and ⁇ 2M-2B1-Biotin respectively, and the addition of the two purified antibodies The order had no significant effect on the purification results.
  • the effector cells are UCAR-T cells, CAR-T cells and T cells (the latter two are controls), in a 24-well plate, 5.0 ⁇ 10 5 cells are inoculated in each well, the volume is 500 ⁇ l, and the concentration of PHA-P is 2.5 ⁇ g/ml, after 48h, the membrane surface activation molecules CD25 and CD69 were detected.
  • FIG. 6 shows the comparison of the activation effect of CAR-T cells after purification. The results show that after the two-step purification, the universal CAR-T cannot be activated again, and the control is obviously activated.
  • K562 cells and K562-CD19 cells were used as target cells, and the purified effector cell UCAR-T used in this experiment was named UCART-19, and CAR-T cells without gene knockout were selected.
  • CART-19 and T cells as a control, first use cytocalceinTM violet 550 to stain the target cells, and then adjust the effector cell density to 5 ⁇ 10 6 cells/ml, and the target cell density to 5 ⁇ 10 5 cells/ml, The above three effector cells and target cells were added to the 96-well plate according to the ratio of (0:1), (0.25:1), (1:1), (5:1) and (10:1) respectively.
  • Figure 7 and Figure 8 show the in vitro tumor killing effect of purified CAR-T cells and the comparison of cytokine release. The results show that the in vitro tumor killing effect of purified general-purpose CAR-T cells is equivalent to that of conventional CAR-T cells .
  • NPG mice were injected with 5.0 ⁇ 10 5 raji-luc cells in vivo, and imaged with a small animal in vivo fluorescence imager 5 days later. According to the imaging results, they were divided into 5 groups and injected with PBS, T-cell, CAR-T, and UCART-19 1 respectively #cells, UCART-19 2# cells, the number is 5.0 ⁇ 10 6 , and then imaged with a small animal in vivo fluorescence imager every week, as shown in Figure 9, the results show: CAR-T, UCART-19 1#, UCART- The three groups of 19 2# had similar tumor killing effects in vivo; and the survival curve was drawn according to the survival of the mice, as shown in Figure 10.
  • mice The results showed that: the three groups of CAR-T, UCART-19 1#, and UCART-19 2# The survival rate of the mice was the same; it can be seen that the purified general-purpose CAR-T cells and conventional CAR-T cells have the same tumor-killing effect in vivo.
  • the anti-human ⁇ 2M antibody ⁇ 2M-2B1 in Example 2 was replaced by a commercially available anti-human ⁇ 2M antibody (Biolegend, 395702), coupled with PE, to prepare a ⁇ 2M-binding preparation; in addition, the anti-human CD3 antibody CD3 in Example 4 -2C1 was replaced with a commercially available anti-human CD3 antibody (Biolegend, 317302), coupled with PE to prepare a CD3-binding preparation; the coupling method was the same as in Example 2. Among them, the principle of action of PE and biotin is the same.
  • the universal CAR-T cells with TCR and ⁇ 2M gene knockout were purified, and the purification effect was investigated.

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Abstract

属于CAR-T细胞治疗技术领域，提供一种制备通用型CAR-T细胞的方法及其应用。提供的β2M结合分子的VH结构域和VL结构域中含有CDR序列，能够与β2M实现特异性结合，将该结合分子加入到CAR-T细胞中，该结合分子能够与β2M基因未敲除CAR-T细胞发生特异性结合，从而将敲除β2M基因的CAR-T细胞与未敲除β2M基因的CAR-T细胞分离，实现通用型CAR-T细胞的纯化。

Description

一种制备通用型CAR-T细胞的方法及其应用
优先权声明
本申请要求申请号为202111253059.8，申请日为2021年10月27日，发明名称为“一种制备通用型CAR-T细胞的方法及其应用”的中国发明专利申请的优先权，其全部内容通过引用并入本文中。
技术领域
本发明涉及CAR-T细胞治疗技术领域，尤其是涉及一种制备通用型CAR-T细胞的方法及其应用。
背景技术
2017年8月以来美国FDA先后批准靶向CD19的两个CAR-T产品上市(诺华制药的kyriamh和凯特制药的yescarta)，因此CAR-T细胞在血液病肿瘤治疗的需求不断升高。但是目前过继性细胞治疗还是基于自体细胞的回输治疗方式，每个患者都有单独制备CAR-T细胞，操作复杂，生产制备成本非常昂贵，诺华制药kyriamh产品的上市价格为每例47.5万美元，凯特制药的yescarta价格也要37.5万美元。大部分肿瘤患者在进行CAR-T细胞治疗前均经过反复多次的其他方式治疗，尤其是放化疗，这就导致从其外周血获得的T淋巴细胞非常少，且这些T淋巴细胞在体外非常难扩增，制备自体CAR-T细胞也非常困难。另外，不少婴幼儿患者和重症患者在制备CAR-T细胞前无法从患者外周血获得足够的T淋巴细胞。因此，同种异体CAR-T细胞(即通用型CAR-T细胞)既能极大的降低CAR-T细胞的生 产成本，也能为无法制备自体CAR-T细胞的患者提供治疗机会。通用型CAR-T细胞治疗比自体CAR-T细胞治疗有天然的优势，因此其研究开发越来越受到关注。
随着基因编辑技术的不断完善，敲除T细胞β2M基因的效率已越来越高。但基因编辑操作对T细胞的状态和生长速度有很大的影响，一般β2M基因的敲除效率越高，基因编辑后的T细胞状态越差、细胞生长速度越慢。因此为了兼顾二者平衡，在通用型CAR-T细胞制备过程中会将β2M基因敲除效率控制在一定程度。因此，制备的通用型CAR-T细胞如果要用于临床细胞治疗，就必须去除未敲除β2M基因的T细胞。否则，临床应用时会导致移植物抗宿主病(GvHD)或宿主抗移植物病(HvGD)。
鉴于此，特提出本发明。
发明内容
本发明的目的在于，提供一种β2M结合分子。该结合分子能够用于去除通用型CAR-T细胞中未敲除β2M基因的T细胞，实现通用型CAR-T细胞的纯化，从而减轻或避免移植物抗宿主病(GvHD)或宿主抗移植物病(HvGD)的发生。
本发明的另一个目的在于，提供上述β2M结合分子在β2M检测和纯化通用型CAR-T细胞的应用，以及在制备用于检测β2M或纯化通用型CAR-T细胞的产品中的应用。所述产品包括能够推广市售的制剂或试剂盒。
本发明另一个目的在于，提供应用上述制剂或试剂盒进行通用型CAR-T细胞纯化的方法。该方法纯化效果好，操作简便，适宜推广。
本发明还有一个目的在于，提供应用上述β2M结合分子制备通用型CAR-T细胞的方法。以期能够直接获得符合临床纯度需求的通用型CAR-T细胞，避免了应用前繁琐的纯化过程。
为了解决上述技术问题，实现上述目的，本发明提供以下技术方案：
第一方面，本发明提供一种β2M结合分子，其包括与β2M特异性结合的模块a；
所述模块a包括VH结构域，所述VH结构域包括具有SEQ ID No.1所示氨基酸序列的CDR-H1，具有SEQ ID No.2所示氨基酸序列的CDR-H2和具有SEQ ID No.3所示氨基酸序列的CDR-H3。
在可选实施方式中，所述模块a还包括VL结构域，所述VL结构域包括具有SEQ ID No.4所示氨基酸序列的CDR-L1，具有氨基酸序列为AAA的CDR-L2和具有SEQ ID No.5所示氨基酸序列的CDR-L3。
另一方面，本发明还提供一种β2M结合分子，其包括与β2M特异性结合的模块a；
该模块a包括VH结构域和VL结构域，该VH结构域包括CDR-H1，CDR-H2和CDR-H3；
该VL结构域包括CDR-L1，CDR-L2和CDR-L3；
其中，所述CDR-H1、所述CDR-H2和所述CDR-H3的氨基酸序列包括SEQ ID No.6的互补决定区序列；
所述CDR-L1、所述CDR-L2和所述CDR-L3的氨基酸序列包括SEQ ID No.7的互补决定区序列。
在可选实施方式中，上述CDR-H1、上述CDR-H2和上述CDR-H3的氨基酸序列由SEQ ID No.6根据Kabat、Chothia、或AbM编号系统定义；
上述CDR-L1、上述CDR-L2和上述CDR-L3的氨基酸序列由SEQ ID No.7根据Kabat、Chothia、或AbM编号系统定义。
在可选实施方式中，所述模块a的VH结构域具有SEQ ID No.6所示氨基酸序列。
在可选的实施方式中，所述模块a的VL结构域具有SEQ ID No.7所示氨基酸序列。
在可选的实施方式中，所述β2M结合分子选自与β2M抗原特异性结合的scFv分子、Fv分子、Fab分子或完整抗体分子。
另一方面，本发明提供前述实施方式任一项所述的β2M结合分子在制备β2M检测产品中的应用，或者在非以疾病诊断或治疗为目的的β2M体外检测中的应用。
另一方面，本发明提供前述实施方式任一项所述的β2M结合分子在纯化通用型CAR-T细胞或者制备用于纯化通用型CAR-T细胞的产品中的应用。
另一方面，本发明提供用于检测β2M的制剂或用于纯化通用型CAR-T细胞的制剂，所述制剂包括前述实施方式任一项所述的β2M结合分子，所述β2M结合分子优选偶联有生物素。
在可选的实施方式中，所述用于纯化通用型CAR-T细胞的制剂还包括特异性结合通用型CAR-T细胞的第二结合分子。
在可选的实施方式中，所述第二结合分子优选偶联有生物素。
在可选的实施方式中，所述第二结合分子是选自能够与TCR阳性表达的CAR-T细胞特异性结合的其他分子。
对于第二结合分子与β2M结合分子的用量比例，本领域技术人员能够根据实际需求进行常规选择。
在可选的实施方式中，与所述第二结合分子特异性结合的靶蛋白为TCR或CD3。
在可选的实施方式中，所述第二结合分子包括与或CD3特异性结合的模块c；所述模块c包括具有SEQ ID No.8所示氨基酸序列的VH结构域，和具有SEQ ID No.9所示氨基酸序列的VL结构域。
另一方面，本发明提供用于纯化通用型CAR-T细胞的试剂盒，所述试剂盒包括前述实施方式所述的用于纯化通用型CAR-T细胞的制剂和任选的耗材。
另一方面，本发明提供用于检测β2M的试剂盒，所述试剂盒包括前述实施方式所述的用于检测β2M的制剂和任选的耗材。
另一方面，本发明提供采用前述任一项实施方式所述的用于纯化通用型CAR-T细胞的制剂或者采用前述实施方式所述的用于纯化通用型CAR-T细胞的试剂盒纯化通用型CAR-T细胞的方法，其包括：将待纯化的通用型CAR-T细胞置于用于纯化通用型CAR-T细胞的制剂中孵育后，使用抗生物素偶联的磁珠进行磁性吸附，取细胞悬液，得到纯化后的通用型CAR-T细胞。
本发明中，术语“抗生物素偶联的磁珠”是指偶联了能够特异性结合生物素材料的磁珠，例如偶联亲和素的磁珠。
另一方面，本发明提供了一种制备通用型CAR-T细胞的方法，包括以下步骤：
(1)制备前述实施方式所述的β2M结合分子和任选的抗CD3抗体；
(2)所述β2M结合分子和抗CD3抗体分别偶联生物素；
(3)敲除CAR-T细胞的β2M基因和TCR基因；
(4)偶联生物素后的β2M结合分子和抗CD3抗体与基因敲除后的CAR-T细胞共培养；
(5)使用抗生物素偶联的磁珠进行磁性吸附，取细胞悬液，即得纯化后的通用型CAR-T细胞。
第九方面，本发明提供一种纯化通用型CAR-T细胞的方法，其包括：使用如上所述的β2M结合分子与待纯化通用型CAR-T细胞接触的步骤。
本发明提供的β2M结合分子的VH结构域和VL结构域中含有特定的CDR序列，能够与β2M实现特异性结合，将该结合分子加入到β2M基因敲除不完全的CAR-T细胞中，该结合分子能够与β2M基因未敲除的CAR-T细胞发生特异性结合，从而将敲除β2M基因的CAR-T细胞与未敲除β2M基因的CAR-T细胞分离，实现通用型CAR-T细胞的纯化。
本发明提供的采用含有上述β2M结合分子的制剂或试剂盒对通用型CAR-T细胞进行纯化的方法，通用型CAR-T细胞经本发明提供的β2M结合分子纯化后，能够显著提高通用型CAR-T细胞纯度。尤其是与靶蛋白为CD3的第二结合分子联合使用后，通用型CAR-T细胞纯度可高达99.96％，且经过体内外杀瘤活性表征证明了，本发明提供的通用型CAR-T细胞的纯化方法未对通用型CAR-T细胞造成不良影响。
本发明还提供了一种通用型CAR-T细胞的制备方法，该制备方法使用本发明提供的β2M结合分子和任选的抗CD3抗体，在敲除β2M基因和TCR基因后，在CAR-T细胞未分离的情况下，直接与其共培养，而后分离得到通用型CAR-T细胞，省去了独立的纯化过程，减轻了一线工作人员的劳动强度。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实验例2中使用的pCDHF质粒图谱；
图2是抗人β2M抗体β2M-2B1的动力学亲和力分析拟合曲线；
图3为本发明实施例2中使用的pX330-spCAS9-HF1质粒图谱；
图4为本发明实验例2中TCR/β2M基因双敲除效率；
图5为本发明实验例2中纯化前后CAR-T细胞纯度对比图；
图6为本发明实验例3中纯化后CAR-T细胞被激活效果对比图；
图7为本发明实验例4中纯化后CAR-T细胞体外杀瘤效果图；
图8为本发明实验例4中纯化后CAR-T细胞体外杀瘤细胞因子释放对比图；
图9为本发明实验例5中纯化后CAR-T细胞体内杀瘤效果图；
图10为本发明实验例5中根据小鼠存活情况绘制的生存曲线。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。通常在此处附图中描述和示出的本发明实施例的组件可以以各种不同的配置来布置和设计。
因此，以下对在附图中提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
应注意到：相似的标号和字母在下面的附图中表示类似项，因此，一旦某一项在一个附图中被定义，则在随后的附图中不需要对其进行进一步定义和解释。
术语“氨基酸”表示天然存在的羧基α-氨基酸。天然存在的氨基酸包括丙氨酸(三字母密码：Ala，单字母密码：A)，精氨酸(Arg，R)，天冬酰胺(Asn，N)，天冬氨酸(Asp，D)，半胱氨酸(Cys，C)，谷氨酰胺(Gln，Q)，谷氨酸(Glu，E)，甘氨酸(Gly，G)，组氨酸(His，H)，异亮氨酸(Ile，I)，亮氨酸(Leu，L)，赖氨酸(Lys，K)，甲硫氨酸(Met，M)，苯丙氨酸(Phe，F)，脯氨酸(Pro，P)，丝氨酸(Ser，S)，苏氨酸(Thr，T)，色氨酸(Trp，W)，酪氨酸(Tyr，Y)，和缬氨酸(Val，V)。
术语“通用型CAR-T细胞”一般是指从健康志愿者获取的T细胞，敲除了其相关基因(主要是与GVHD和HvGD疾病相关的基因，例如表达TCR(如α链、β链的编码基因)、HLA(如β2微球蛋白的编码基因(β2M基因))、或CD52等分子的编码基因)以及转入了CAR(嵌合抗原受体)基因后制成的CAR-T细胞。
为了去除未敲除β2M基因的CAR-T细胞，本发明以β2M蛋白为抗原，从抗体结构出发，构建了具有特异性结合β2M蛋白的结合分子即β2M结合分子。
第一方面，在一次具体的实施方式中，本发明提供一种与β2M特异性结合的模块(a1)，所述模块(a1)包括VH结构域，所述VH结构域包括具有SEQ ID No.1所示氨基酸序列的CDR-H1，具有SEQ ID No.2所示氨基酸序列的CDR-H2和具有SEQ ID No.3所示氨基酸序列的CDR-H3。
其中，SEQ ID No.1-3由重链可变区序列根据IMGT编号系统定义。
在另一次具体实施方式中，本发明提供一种与β2M特异性结合的模块(a2)，所述模块(a2)包括VL结构域，所述VL结构域包括具有SEQ ID No.4所示氨基酸序列的CDR-L1，具有氨基酸序列为AAA的CDR-L2和具有SEQ ID No.5所示氨基酸序列的CDR-L3。
其中，SEQ ID No.4、CDR-L2的AAA、SEQ ID No.5由轻链可变区序列根据IMGT编号系统定义。
在另一次具体实施方式中，本发明提供一种与β2M特异性结合的模块(a3)，所述模块(a3)包括VH结构域和VL结构域，所述VH结构域包括具有SEQ ID No.1所示氨基酸序列的CDR-H1，具有SEQ ID No.2所示氨基酸序列的CDR-H2和具有SEQ ID No.3所示氨基酸序列的CDR-H3，所述VL结构域包括具有SEQ ID No.4所示氨基酸序列的CDR-L1，具有氨基酸序列为AAA的CDR-L2和具有SEQ ID No.5所示氨基酸序列的CDR-L3。
另一方面，在另一次具体实施方式中，所述CDR-H1、所述CDR-H2和所述CDR-H3的氨基酸序列包括SEQ ID No.6的互补决定区序列；
所述CDR-L1、所述CDR-L2和所述CDR-L3的氨基酸序列包括SEQ ID No.7的互补决定区序列。
在另一次具体实施方式中，上述CDR-H1、CDR-H2和CDR-H3还可以是由SEQ ID No.6根据Kabat、Chothia、或AbM编号系统定义。
在另一次具体实施方式中，上述CDR-L1、上述CDR-L2和上述CDR-L3的氨基酸序列还可以是由SEQ ID No.7根据Kabat、Chothia、或AbM编号系统定义。
上述互补决定区根据Kabat、Chothia、或AbM编号系统定义的序列，如下表所示：
Figure PCTCN2022127619-appb-000001
IMGT基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information system(IMGT))的编号系统，可参阅Lefranc et al.，Dev.Comparat.Immunol.27：55 77，2003。
Kabat由ElvinA.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，Md.，1991)。
Chothia由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196：901 917；Chothia等人(1989)Nature 342：878 883)。
AbM定义方式来源于Martin的相关研究(Martin ACR，Cheetham JC，Rees AR(1989)Modelling antibody hypervariable loops：A combined algorithm.Proc Natl Acad Sci USA 86：9268-9272)，此定义方法整合了Kabat及Chothia两者的部分定义。
结合第一方面所述的各个模块中含有CDR的氨基酸序列，第二方面，在一次具体的实施方式中，本发明提供了如下各模块：
模块(a11)包括VH结构域，所述VH结构域具有SEQ ID No.6所示氨基酸序列。
模块(a21)包括VL结构域，所述VL结构域具有SEQ ID No.7所示氨基酸序列。
模块(a31)包括VH结构域和VL结构域，所述VH结构域具有SEQ ID No.6所示氨基酸序列，所述VL结构域具有SEQ ID No.7所示氨基酸序列。
结合第二方面提供的各模块，第三方面，本发明提供了scFV分子，所述scFV分子包括由弹性连接肽(Linker)连接的VH结构域和VL结构域；所述VH结构域具有SEQ ID No.6所示氨基酸序列，所述VL结构域具有SEQ ID No.7所示氨基酸序列。
对于所述Linker的氨基酸残基组成和长度本领域技术人员能够根据实际需求，通过常规手段调整获得，包括但不限于由长度为15～25个氨基酸残基的甘氨酸(Gly)和丝氨酸(Ser)构成。
作为示例性的Linker包括：(GGGGS)n(SEQ ID No.22)、(GGGS)n(SEQ ID No.23)、(GGS)n(SEQ ID No.24)、或(GS)n(SEQ ID No.25)其中n选自1，2，3，4，5或6。第四方面，本发明提供了Fv分子，所述Fv分子包括由短肽连接的VH结构域和VL结构域；所述VH结构域具有SEQ ID No.6所示氨基酸序列，所述VL结构域具有SEQ ID No.7所示氨基酸序列。
所述短肽本领域技术人员能够根据实际需求，通过常规手段调整获得，包括但不限于由3～9个氨基酸残基组成的短肽链。
第五方面，本发明提供了Fab分子，所述Fab分子包括所述VH结构域、所述VL结构域、轻链恒定区(CL)和一个重链恒定区(CH1)；所述VH结构域具有SEQ ID No.6所示氨基酸序列，所述VL结构域具有SEQ ID No.7所示氨基酸序列。
对于CH1和CL，本领域技术人员能够根据实际需求进行选择，例如所述CH1选自IgG1、IgG2、IgG3、IgG4、IgA、IgD、IgE或IgM中的任一种或几种，所述CL选自κ链或λ链，此外，本领域技术人员还可以实际需求，通过常规手段对上述选用的CH1和CL的序列及修饰进行调整。
第六方面，本发明还提供了完整抗体分子，所述完整抗体分子包括2条相同的重链和2条相同的轻链，所述重链包括VH结构域和重链恒定区，所述轻链包括VL结构域和轻链恒定区；所述VH结构域具有SEQ ID No.6所示氨基酸序列，所述VL结构域具有SEQ ID No.7所示氨基酸序列。
对于重链恒定区和轻链恒定区的具体序列组成，本领域技术人员能够根据实际需求进行选择，例如所述重链恒定区可以选自IgG1、IgG2、IgG3、IgG4、IgA、IgD、IgE或IgM中的任一种或几种，所述轻链恒定区可以选自κ链或λ链。
在一次具体的实施方式中，所述完整抗体分子的重链恒定区和轻链恒定区选自鼠IgG。
此外，本领域技术人员还可以实际需求，通过常规手段对上述选用的重链恒定区和轻链恒定区的序列或者修饰进行调整。
第七方面，本发明提供前述实施方式所述的各模块、scFV分子、Fv分子、Fab分子或完整抗体分子在制备β2M检测产品中的应用，或者在非以疾病诊断为目的的β2M体外检测中的应用。
第八方面，本发明提供前述实施方式所述的各模块、scFV分子、Fv分子、Fab分子或完整抗体分子在纯化通用型CAR-T细胞或者制备用于纯化通用型CAR-T细胞的产品中的应用。
在一次具体的实施方式中，所述通用型CAR-T细胞被敲除的基因至少包括β2M基因。
在一次具体的实施方式中，所述通用型CAR-T细胞被敲除的基因还包括TCR基因。例如可以是TCR分子的α链编码基因或其β链编码基因。
第九方面，在一次具体的实施方式中，本发明提供用于纯化通用型CAR-T细胞的制剂，所述制剂包括前述实施方式任一项所述的各模块、scFV分子、Fv分子、Fab分子或完整抗体分子；所述各模块、scFV分子、Fv分子、Fab分子或完整抗体分子偶联有生物素。可以理解的，所述偶联生物素为本领域常用的磁珠吸附分离手段。
第十方面，在一次具体的实施方式中，本发明提供用于纯化通用型CAR-T细胞的试剂盒，所述试剂盒包括前述实施方式所述制剂和耗材。可以理解的，所述耗材包括本领域常规的试剂盒组成，例如孔板、反应容器、取液装置等。
第十一方面，在一次具体的实施方式中，当所述通用型CAR-T细胞被敲除的基因还包括TCR基因时；所述制剂或所述试剂盒还包括第二结合分 子，所述第二结合分子偶联有生物素。第二结合分子能够与TCR阳性表达的CAR-T细胞特异性结合。
第二结合分子的靶蛋白为TCR或CD3，例如可以选自CD3抗体或TCR抗体。对于第二结合分子与β2M结合分子的用量比例，本领域技术人员能够根据实际需求进行常规选择。
在另一次具体实施方式中，与所述第二结合分子特异性结合的靶蛋白为TCR。
在另一次具体实施方式中，与所述第二结合分子特异性结合的靶蛋白为CD3，所述第二结合分子包括与CD3特异性结合的模块c。
。
所述模块c包括具有SEQ ID No.8所示氨基酸序列的VH结构域，和具有SEQ ID No.9所示氨基酸序列的VL结构域。
第十二方面，本发明在一次具体的实施方式中提供了一种纯化通用型CAR-T细胞的方法，该方法使用上述的制剂或者试剂盒，将通用型CAR-T细胞置于制剂中孵育后，使用抗生物素偶联的磁珠进行磁性吸附，取细胞悬液，得到纯化后的通用型CAR-T细胞。
在另一次具体实施方式中，本发明提供了一种制备通用型CAR-T细胞的方法，包括以下步骤：
(1)制备前述实施方式所述的β2M结合分子和任选的抗CD3抗体；
(2)所述β2M结合分子和抗CD3抗体分别偶联生物素；
(3)敲除CAR-T细胞的β2M基因和TCR基因；
(4)偶联生物素后的β2M结合分子和抗CD3抗体与基因敲除后的CAR-T细胞共培养；
(5)使用抗生物素偶联的磁珠进行磁性吸附，取细胞悬液，即得纯化后的通用型CAR-T细胞。
第十三方面，本发明在一次具体的实施方式中提供了一种纯化通用型CAR-T细胞的方法，其包括：使用上述任意模块、Fv分子、Fab分子、完整抗体分子、或制剂与待纯化通用型CAR-T细胞接触，以从中分离去除未敲除β2M基因的CAR-T细胞。
在另一次具体实施方式中，当需要的通用型CAR-T细胞还敲除了TCR基因时，该纯化方法还可以包括使用第二结合分子与待纯化通用型CAR-T细胞接触，以从中分离去除未敲除TCR基因的CAR-T细胞(即TCR表达阳性的CAR-T细胞)。
在另一次具体实施方式中，第二结合分子的靶蛋白可以是TCR，也可以是CD3。未敲除TCR基因的CAR-T细胞上，TCR和CD3以复合物形式存在，使用结合TCR或CD3的结合分子均能实现未敲除TCR基因的CAR-T细胞的去除。
在另一次具体实施方式中，第二结合分子的使用顺序可以在使用上述任意模块、Fv分子、Fab分子、完整抗体分子、或制剂之后使用，之前使用，或二者同时使用。
例如，使用上述任意模块、Fv分子、Fab分子、完整抗体分子或制剂，和第二结合分子同时与待纯化通用型CAR-T细胞接触；
例如，在使用上述任意模块、Fv分子、Fab分子、完整抗体分子或制剂与待纯化通用型CAR-T细胞接触后，再继续使用第二结合分子与待纯化通用型CAR-T细胞接触；
例如，在使用第二结合分子与待纯化通用型CAR-T细胞接触后，再使用上述任意模块、Fv分子、Fab分子、完整抗体分子或制剂，与待纯化通用型CAR-T细胞接触。
在另一次具体实施方式中，所述第二结合分子包括与CD3特异性结合的模块c；
该模块c包括具有SEQ ID No.8所示氨基酸序列的VH结构域，和具有SEQ ID No.9所示氨基酸序列的VL结构域。下面结合附图，对本发明的一些实施方式作详细说明。在不冲突的情况下，下述的实施例及实施例中的特征可以相互组合。
上述第一方面提供的各模块、scFV分子、Fv分子、Fab分子和完整抗体分子可以通过人工合成获得，完整抗体分子还可以通过构建杂交瘤细胞，表达分泌获得。
实施例1
本实施例提供了一种抗人β2M抗体的制备方法，具体步骤如下：
将重组β2M蛋白抗原(深圳市菲鹏生物股份有限公司生产，货号BA-PAB-MU0001)等量与弗氏佐剂(sigma，货号F5881)混合进行乳化。背部皮下多点免疫注射健康BALB/C小鼠(广东省实验动物中心，6周龄，雌性)；14天后腹腔加强免疫；四次加强免疫后采小鼠尾血进行效价检测，选取效价最高的小鼠脾脏与小鼠骨髓瘤细胞SP2/0进行细胞融合。
小鼠末次免疫后第三天，在无菌条件取出小鼠脾脏，置于平皿中，RPMI1640(gibco，货号12633012)基础培养液冲洗一次，放于小烧杯的尼龙网上研磨，制成细胞悬液。离心，弃上清，RPMI1640培养液重悬。将脾细胞与提前准备好的小鼠骨髓瘤细胞SP20以10∶1比例混合均匀，然后用1ml50％的PEG1500(sigma，货号81210)进行细胞融合，融合1分钟后加入15ml的RPMI1640完全培养液终止细胞融合。1000rpm，离心5分钟，弃上清，用50ml的RPMI1640筛选培养液轻轻重悬，平分于10块96孔板，100μl/孔，37℃，5％CO 2培养。培养至第6天，换HAT培养液两次。第二天取细胞上清，用人β2M抗原采用ELISA方法检测。检测阳性孔对应的细胞采取 有限稀释法进行单克隆化；经过3次亚克隆，筛选获得多株能稳定分泌单抗的细胞株，其中1株命名为β2M-2B1细胞株。
使用QIAGEN RNAeasy Mini试剂盒将1到500万个β2M-2B1细胞株的亚克隆杂交瘤细胞用来提取总RNA，然后使用SuperScript III RT试剂盒(InVitrogen)产生第一链cDNA，随后使用来自小鼠IgG重链(IgG1、IgG2a、IgG2b)和轻链(κ或λ)恒定区的常规引物，通过PCR反应并扩增抗人β2M IgG可变区的双链cDNA，PCR循环条件为95℃1分钟1个循环；在95℃1分钟、63℃1分钟和72℃1分钟下进行25个循环。将得到的PCR产物克隆到T载体(InVitrogen)中并进行测序。
测序结果如下：
抗人β2M抗体β2M-2B1属于小鼠IgG1亚型，轻链为κ链；
其重链可变区序列如下：
Figure PCTCN2022127619-appb-000002
其中，根据IMGT编号系统定义，其包含有重链互补决定区1(CDR-H1)：GYTFSSYV，即SEQ ID No.1，重链互补决定区2(CDR-H2)：FNPYNDGT，即SEQ ID No.2和重链互补决定区3(CDR-H3)：ARRGNTYDNFDY，即SEQ ID No.3。
其轻链可变区序列如下：
Figure PCTCN2022127619-appb-000003
Figure PCTCN2022127619-appb-000004
其中，根据IMGT编号系统定义，其包含有轻链互补决定区1(CDR-L1)：QTIGTW，即SEQ ID No.4，轻链互补决定区2(CDR-L2)：AAA和轻链互补决定区3(CDR-L3)：QQLYSSPLT，即SEQ ID No.5。
实施例2
本实施例对实施例1得到的抗人β2M抗体β2M-2B1进行生物素偶联，制备得到β2M结合制剂，具体步骤如下：
将抗人β2M抗体β2M-2B1分别用0.1mol/L碳酸氢钠(pH8.0)稀释到1mg/ml，用1ml DMSO(二甲基亚砜)溶解BNHS 1mg，向1ml抗体(即1mg)加入120μl BNHS溶液(即含BNHS 120μg)，将生物素和抗体按照质量比为1∶8.3进行混合，室温(20～25℃)搅拌2小时(搅拌时间不宜过长，抗体会失活)，混合液装入透析袋，用0.01mol/L，pH＝7.4的PBS溶液于4℃下透析过夜，次日再透析4～6h，去除游离的生物素，根据透析完的混合液终体积加等体积的缓存液，4℃避光保存，缓存液配比(体积比)为：PBS+0.01％防腐剂+1％BSA。
实施例3
本实施例提供了抗人CD3抗体的制备方法，整体步骤与实施例1相同，区别在于，免疫抗原为CD3。选取得到的1株细胞株命名为CD3-2C1细胞株。
经测序，CD3-2C1抗体属于小鼠IgG1亚型，轻链为κ链；具有SEQ ID No.8所示氨基酸序列的VH结构域，和具有SEQ ID No.9所示氨基酸序列的VL结构域。
实施例4
本实施例对实施例3得到的抗人CD3抗体CD3-2C1进行生物素偶联，制备得到CD3结合制剂，偶联方法与实施例2相同。
实验例1
本实验例应用上述实施例1制备得到的抗人β2M抗体β2M-2B1对重组人β2M的溶液进行体外检测，检测方法如下：
将5μg/ml Ag(重组人β2M，His标签)、mIgG(10μg/ml，2×)和抗人β2M抗体β2M-2B1(10μg/ml，2×)共培养，2倍梯度稀释，然后采用HIS1K生物传感器监测β2M-2B1与β2M的结合情况，并利用Octet HIS1K测定抗人β2M抗体β2M-2B1的亲和力。
实验条件设定如表1所示：
表1实验例1中HIS1K生物传感器检测的实验条件
N Data Name(数据名称) Assay Time(sec)分析时间 Flow Rate流速
l Baseline(基线) 60 1000
2 Association(结合) 180 1000
3 Dissociation(分离) 300 1000
4 Baseline1(基线1) 90 1000
5 Loading(负载) 120 1000
6 Custom(习惯) 5 1000
缓冲液：1×DPBS+0.02％Tween-20。
2倍梯度稀释后的抗人β2M抗体β2M-2B1的动力学亲和力分析拟合曲线如图2所示，抗人β2M抗体β2M-2B1对人β2M的亲和力实验结果如表2所示，结果显示：抗人β2M抗体β2M-2B1对人β2M具有很高的亲和力 (KD＜1.0E-12M)，证明实施例1提供的抗人β2M抗体β2M-2B1能够用于检测β2M，或用于制备检测β2M的产品。
表2抗人β2M抗体β2M-2B1对人β2M的亲和力结果
Figure PCTCN2022127619-appb-000005
实验例2
本实验例采用实施例2制备得到的β2M-2B1偶联生物素制剂和实施例4制备得到的CD3-2C1偶联生物素制剂，对敲除TCR和β2M基因的通用型CAR-T细胞进行纯化，并对纯化效果进行考察。
1.1 TCR/β2M基因敲除
将图1的目的质粒制备的慢病毒感染T细胞，得到CART-CD19细胞(靶向CD19的CART细胞)。在六孔板的孔中铺1×10 6CART-CD19细胞，在200μL的OPTI-MEM中加入1μg pX330-spCAS9-HF1-TRAC的TRAC sgRNA表达质粒(敲除T细胞受体的α基因(MHC II类分子)，表达sgRNA的核酸序列如SEQ ID NO.10所示)，1μg pX330-spCAS9-HF1-β2M的β2M sgRNA表达质粒(敲除CAR-T细胞的β2微球蛋白基因(MHC I类分子)，表达sgRNA的核酸序列如SEQ ID NO.11所示)混合均匀，pX330-spCAS9-HF1质粒图谱如图3所示，再加入4μL PEI或者6μL lipo2000试剂，混合均匀后室温静置15min，滴入孔中，6～8h后将细胞换回新鲜培养基培养。
其中，SEQ ID NO.10：acaaaacugugcuagacaug；
SEQ ID NO.11：cgcgagcacagcuaaggcca。
TCR/β2M基因双敲除效率如图4所示，结果显示：换液48h后通过流式细胞术(FACS)分析TCR/β2M基因KO细胞比例为71.35％。此外，编辑工具还可以以sgRNA及RNP形式通过电转及病毒感染的方式进入细胞。
1.2KO细胞(UCAR-T细胞)纯化
取电转后培养4天后的细胞，离心，一定量DPBS重悬密度至1×10 8个/ml，经CD3-2C1-Biotin(偶联生物素的抗人CD3抗体，来自实施例4)和β2M-2B1-Biotin(偶联生物素的抗人β2M抗体，来自实施例2)一步纯化，纯化方法为：加入100μl/ml CD3-2C1-Biotin和100μl/mlβ2M-2B1-Biotin冰上避光孵育20min，PBS洗一遍，再加入anti-Biotin Beads(购自Miltenyi Biotec，货号130-090-485)冰上孵育15min后，放入磁铁中5min，收集包含UCAR-T细胞的细胞悬液。取2×10 5个/ml细胞利用APC-anti-human TCRa/β(Biolegend，B259839)和PE-anti-humanβ2M(Biolegend，B226121)抗体孵育15min流式检测细胞纯度。
纯化前后CAR-T细胞纯度对比图如图5所示，结果显示：TCR/β2M基因敲除及细胞纯度，纯度99.96％。
需要说明的是，上述CD3-2C1-Biotin和β2M-2B1-Biotin一步纯化方法，也可以替换为分别使用CD3-2C1-Biotin和β2M-2B1-Biotin进行两步纯化，且两种纯化抗体的加入顺序对纯化结果无明显影响。
实验例3
对实验例2步骤1.2纯化后的通用型CAR-T细胞的激活活性进行检测，步骤如下：
本实验例中效应细胞为UCAR-T细胞，CAR-T细胞及T细胞(后两者为对照)，24孔板，每孔接种5.0×10 5个细胞，体积500μl，加PHA-P浓度2.5μg/ml，48h后检测膜表面活化分子CD25和CD69。
纯化后CAR-T细胞被激活效果对比图如图6所示，结果显示：通过两步纯化后通用型CAR-T不能被再次激活，对照明显被激活。
实验例4
对实验例2步骤1.2纯化后的通用型CAR-T细胞的体外杀瘤活性进行检测，步骤如下：
本实验以K562细胞、K562-CD19细胞(记为K19)为靶细胞，命名本实验例中使用的纯化后的效应细胞UCAR-T为UCART-19，并选择未敲除基因的CAR-T细胞(命名为CART-19)及T细胞作为对照，先利用cytocalceinTM violet 550对靶细胞进行染色，其次调整效应细胞密度为5×10 6个/ml，靶细胞密度为5×10 5个/ml，将上述三种效应细胞与靶细胞分别按照(0∶1)、(0.25∶1)、(1∶1)、(5∶1)和(10∶1)的数量比，加入96孔板中混匀共培养6h及24h，在上述时间段显微镜下观察凋亡情况，并将混合细胞离心，上清利用Human IL-2 Ready-SET-Go与Human IFN gamma ELISA Ready-SET-GoELISA试剂盒检测IL-2及IFN-γ，沉淀部分用100μl binding buffer重悬，300g离心5min，添加1.2μl APC-Annexin V和1.2μl PI染料，避光孵育15min，添加100μl binding buffer流式检测凋亡效率。
纯化后CAR-T细胞体外杀瘤效果图和细胞因子释放对比图如图7和图8所示，结果显示：纯化后的通用型CAR-T细胞与常规CAR-T细胞的体外杀瘤效果相当。
实验例5
对实验例2步骤1.2纯化后的通用型CAR-T细胞的体内杀瘤活性进行检测，步骤如下：
20只NPG小鼠体内注射raji-luc细胞5.0×10 5，5天后采用小动物活体荧光成像仪成像，根据成像结果分成5组，分别注射PBS、T-cell、CAR-T、UCART-19 1#细胞、UCART-19 2#细胞，数量5.0×10 6个，然后每周采用小动物活体荧光成像仪成像，如图9所示，结果显示：CAR-T、UCART-19 1#、UCART-19 2#这3组的体内杀瘤效果相当；并根据小鼠存活情况绘制生存曲线，如图10所示，结果显示：CAR-T、UCART-19 1#、UCART-19 2#这3组小鼠的存活率相同；可见，纯化后的通用型CAR-T细胞与常规CAR-T细胞的体内杀瘤效果相当。
对比例1
将实施例2中的抗人β2M抗体β2M-2B1替换为市售抗人β2M抗体(Biolegend，395702)，偶联PE，制备得到β2M结合制剂；另外，将实施例4中的抗人CD3抗体CD3-2C1替换为市售抗人CD3抗体(Biolegend，317302)，偶联PE，制备得到CD3结合制剂；偶联方法与实施例2相同。其中，PE与生物素的作用原理相同。
采用以上制备得到的β2M结合制剂和CD3结合制剂，对敲除TCR和β2M基因的通用型CAR-T细胞进行纯化，并对纯化效果进行考察。
结果显示：以上β2M结合制剂和CD3结合制剂对通用型CAR-T细胞的纯化效果仅为72.73％，说明与市售抗人β2M抗体和抗人CD3抗体相比， 本发明抗人β2M抗体β2M-2B1和抗人CD3抗体CD3-2C1对通用型CAR-T细胞具有显著的纯化效果。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (16)

  1. 一种β2M结合分子，其特征在于，其包括与β2M特异性结合的模块a；
    所述模块a包括VH结构域，所述VH结构域包括具有SEQ ID No.1所示氨基酸序列的CDR-H1，具有SEQ ID No.2所示氨基酸序列的CDR-H2和具有SEQ ID No.3所示氨基酸序列的CDR-H3；
    所述模块a还包括VL结构域，所述VL结构域包括具有SEQ ID No.4所示氨基酸序列的CDR-L1，具有氨基酸序列为AAA的CDR-L2和具有SEQ ID No.5所示氨基酸序列的CDR-L3。
  2. 一种β2M结合分子，其特征在于，其包括与β2M特异性结合的模块a；
    所述模块a包括VH结构域和VL结构域，所述VH结构域包括CDR-H1，CDR-H2和CDR-H3；
    所述VL结构域包括CDR-L1，CDR-L2和CDR-L3；
    其中，所述CDR-H1、所述CDR-H2和所述CDR-H3的氨基酸序列包括SEQ ID No.6的互补决定区序列；
    所述CDR-L1、所述CDR-L2和所述CDR-L3的氨基酸序列包括SEQ ID No.7的互补决定区序列；
    优选地，其中，所述CDR-H1、所述CDR-H2和所述CDR-H3的氨基酸序列由SEQ ID No.6根据Kabat、Chothia、或AbM编号系统定义；
    所述CDR-L1、所述CDR-L2和所述CDR-L3的氨基酸序列由SEQ ID No.7根据Kabat、Chothia、或AbM编号系统定义。
  3. 根据权利要求1或2所述的β2M结合分子，其特征在于，所述VH结构域具有SEQ ID No.6所示氨基酸序列。
  4. 根据权利要求1-3任一所述的β2M结合分子，其特征在于，所述VL结构域具有SEQ ID No.7所示氨基酸序列。
  5. 根据权利要求1～4任一项所述的β2M结合分子，其特征在于，所述β2M结合分子选自与β2M抗原特异性结合的scFv分子、Fv分子、Fab分子或完整抗体分子。
  6. 权利要求1～5任一项所述的β2M结合分子在制备β2M检测产品中的应用，或者在非以疾病诊断或治疗为目的的β2M体外检测中的应用。
  7. 权利要求1～5任一项所述的β2M结合分子在纯化通用型CAR-T细胞或者制备用于纯化通用型CAR-T细胞的产品中的应用。
  8. 用于检测β2M的制剂或用于纯化通用型CAR-T细胞的制剂，其特征在于，所述制剂包括第一结合分子，所述第一结合分子为权利要求1～5任一项所述的β2M结合分子；
    优选地，所述β2M结合分子偶联有生物素。
  9. 根据权利要求8所述的用于纯化通用型CAR-T细胞的制剂，其特征在于，当所述通用型CAR-T细胞被敲除的基因还包括TCR基因时；所述用于纯化通用型CAR-T细胞的制剂还包括特异性结合TCR阳性表达的CAR-T细胞的第二结合分子；
    优选地，与所述第二结合分子特异性结合的靶蛋白为TCR或CD3；
    优选地，所述第二结合分子偶联有生物素。
  10. 根据权利要求9所述的用于纯化通用型CAR-T细胞的制剂，其特征在于，所述第二结合分子包括与CD3特异性结合的模块c；
    所述模块c包括具有SEQ ID No.8所示氨基酸序列的VH结构域，和具有SEQ ID No.9所示氨基酸序列的VL结构域。
  11. 用于纯化通用型CAR-T细胞的试剂盒，其特征在于，所述试剂盒包括权利要求8～10任一项所述的用于纯化通用型CAR-T细胞的制剂和任选的耗材。
  12. 用于检测β2M的试剂盒，其特征在于，所述试剂盒包括权利要求8所述的用于检测β2M的制剂和任选的耗材。
  13. 采用权利要求8～10任一项所述的用于纯化通用型CAR-T细胞的制剂，或者采用权利要求11所述的用于纯化通用型CAR-T细胞的试剂盒纯化通用型CAR-T细胞的方法，其特征在于，将待纯化通用型CAR-T细胞置于用于纯化通用型CAR-T细胞的制剂中孵育后，使用抗生物素偶联的磁珠进行磁性吸附，取细胞悬液，得到纯化后的通用型CAR-T细胞。
  14. 一种制备通用型CAR-T细胞的方法，其特征在于，包括以下步骤：
    (1)制备权利要求1～5任一项所述的β2M结合分子，并制备抗CD3抗体；
    (2)所述β2M结合分子和抗CD3抗体分别偶联生物素；
    (3)CAR-T细胞敲除β2M基因和TCR基因；
    (4)所述偶联生物素后的β2M结合分子和抗CD3抗体与基因敲除后的CAR-T细胞共培养；
    (5)使用抗生物素偶联的磁珠进行磁性吸附，取细胞悬液，即得纯化后的通用型CAR-T细胞。
  15. 一种纯化通用型CAR-T细胞的方法，其特征在于，其包括：使用权利要求1～5任一项所述的β2M结合分子与待纯化通用型CAR-T细胞接触的步骤。
  16. 根据权利要求15所述的方法，其特征在于，包括步骤：使用所述β2M结合分子和第二结合分子与所述待纯化通用型CAR-T细胞接触，
    或者，在使用第二结合分子与所述待纯化通用型CAR-T细胞接触后，再使用所述β2M结合分子与待纯化通用型CAR-T细胞接触，
    或者，在使用所述β2M结合分子与所述待纯化通用型CAR-T细胞接触后，再使用第二结合分子与所述待纯化通用型CAR-T细胞接触；
    其中，所述第二结合分子特异性结合TCR阳性表达的CAR-T细胞；
    优选地，所述第二结合分子的靶蛋白为TCR或CD3；
    优选地，所述第二结合分子包括与CD3特异性结合的模块c；
    所述模块c包括具有SEQ ID No.8所示氨基酸序列的VH结构域，和具有SEQ ID No.9所示氨基酸序列的VL结构域。
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