CN116874609A - Novel modularized universal CAR-T cell and preparation method thereof - Google Patents
Novel modularized universal CAR-T cell and preparation method thereof Download PDFInfo
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- CN116874609A CN116874609A CN202310788008.8A CN202310788008A CN116874609A CN 116874609 A CN116874609 A CN 116874609A CN 202310788008 A CN202310788008 A CN 202310788008A CN 116874609 A CN116874609 A CN 116874609A
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Abstract
The invention discloses a novel modularized universal CAR-T cell and a preparation method thereof. The invention discloses a chimeric antigen receptor, which comprises a component 1 and a component 2, wherein the component 1 and the component 2 are connected by a self-assembly system; wherein, component 1 is antigen recognition domain protein and component 2 is intracellular signaling domain protein. The invention splits the complete chimeric antigen receptor CAR into two parts through an SdCocher/GvTagOpti covalent coupling system, and transmits the part of SdDeltaN 17 connected with an intracellular signal domain to an allogeneic T cell; whereas the portion of Gv linked to the antigen recognition domain is purified in the form of a protein, the two portions can be covalently bound to form a complete CAR-T by spontaneously formed isopeptidic bonds between Sd Δn17/Gv. MU-CAR-T cells that bind to different scFV are able to specifically recognize the corresponding positive target cells for killing.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a novel modularized universal CAR-T cell and a preparation method thereof.
Background
Chimeric antigen receptor T cells (Chimeric antigen receptor-T cells, CAR-T) cells have definite therapeutic effects for treating hematological malignancies. At present, patient autologous T cells are often used for modifying CAR-T cells in clinical tests, but the preparation time, cost, functional dependence on patient T cells and the like are limited. Therefore, the method for treating the existing Universal allogeneic CAR-T cells (Universal CAR-T, UCA R-T) becomes a potential alternative method, and can overcome the uncertainty factors such as insufficient number of the self-T cells of a patient, the production cycle process and the like.
Currently, the hotspot of CAR-T cell therapy is to maintain long-term survival and proliferation capacity of T cells in vivo, looking for a subpopulation of T cells in a young, poorly differentiated state. T memory stem cells (T memory stem cell, T) SCM ) Is a rare memory lymphocyte subgroup with longer survival ability and strong immune reconstruction potential, has the characteristics of stem cells and memory T cells, and therefore in CAR-T cell treatment, the more the TSCM subgroup is, the better the treatment effect is.
Suppression of immune rejection is an important hurdle to be overcome by universal CAR-T. The donor T cell antigen receptor (TCR) recognizes human leukocyte antigens (hu man lymphocyte antigen, HLA) on the surface of the recipient cells, thereby causing graft versus host disease (Graft versus host disease, gvHD) to damage tissues and organs of the body. At the same time, HLA on the surface of donor T cells is also recognized by the immune system of the recipient and is attacked and cleared, which affects the survival and function of CAR-T, namely host versus graft reaction (Host versus graft reaction, hvGR). TRAC and B2M genes of T cells can be knocked out through CRISPR/Cas technology, so that the rejection problem of GvHD and allogeneic cells is avoided.
Because of the personalized nature of current CAR-T drugs, the drug must be specifically prepared and obtained from the cells of each patient, and is time consuming, costly, and risky to failure. Therefore, there is a need to develop new modular universal CAR-T cells (MU-CAR-T) that allow allogeneic CAR-T cells to selectively target a variety of antigens, more widely used in different tumors and in different patients, to address the current impediments.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a novel modularized universal CAR-T cell and a preparation method thereof.
It is a first object of the present invention to provide a chimeric antigen receptor.
A second object of the invention is to provide the use of said chimeric antigen receptor for the preparation of modular universal CAR-T cells.
A third object of the invention is to provide a method for preparing modular universal CAR-T cells.
The fourth object of the invention is to provide the modularized universal CAR-T cell prepared by the preparation method.
A fifth object of the invention is to provide the use of said modular universal CAR-T cells for the preparation of a medicament against HIV virus or T cell lymphoma.
In order to achieve the above object, the present invention is realized by the following means:
a chimeric antigen receptor comprising component 1 and component 2, component 1 and component 2 being linked by a self-assembly system;
wherein, component 1 is antigen recognition structural domain protein, and the amino acid sequence is shown in SEQ ID NO: as shown in the figure 10 or 12,
component 2 is an intracellular signal domain protein, and the amino acid sequence of the intracellular signal domain protein is shown as SEQ ID NO:1 is shown in the specification;
the self-assembly system is a GvoTagOpti/SdCoatcher system, wherein the amino acid sequence of the GvoTagOpti is shown as SEQ ID NO:14, the amino acid sequence of SdCocher is shown as SEQ ID NO: 3.
Further, gvoTagOpti is disposed C-terminal to the antigen recognition domain protein.
Further, the SdCocher is disposed at the N-terminus of the intracellular signaling domain protein.
In the finally obtained chimeric antigen receptor, the amino acid sequence of the antigen recognition domain protein is shown as SEQ ID NO:10, the amino acid sequence of the chimeric antigen receptor VC-MU-CAR is shown in SEQ ID NO: shown at 22; the amino acid sequence of the antigen recognition domain protein is shown as SEQ ID NO:12, the amino acid sequence of the chimeric antigen receptor CD5-CD30-MU-CAR is shown in SEQ ID NO: shown at 24.
The invention also claims the use of said chimeric antigen receptor in the preparation of modular universal CAR-T cells.
The invention also provides a preparation method of the modularized universal CAR-T cell, which expresses intracellular signal domain protein in the T cell, and the amino acid sequence SEQ ID NO:1 or 2, obtaining a T cell expressing an intracellular signaling domain protein; and incubating and purifying the T cells expressing the intracellular signal domain protein and the antigen recognition domain protein to obtain the modularized universal CAR-T cells.
Preferably, the medium for culturing the T cells isT-SFM medium.
Preferably, the medium further comprises IL-7 and IL-15.
Further, the final concentration of IL-7 in the culture medium is 5-15 ng/mL, and the final concentration of IL-15 is 2-10 ng/mL.
Further, the final concentration of IL-7 in the medium was 10ng/mL and the final concentration of IL-15 was 5ng/mL.
Further, the T cells are T cells knocked out of the TRAC and B2M genes.
Further, TRAC and B2M genes in T cells are knocked out based on CRISPR/cas9 gene knockout technology, and the sgRNA sequences are respectively shown in SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO:19 and SEQ ID NO: shown at 20.
Further, the antigen recognition domain protein is packaged by using a lentivirus to obtain a virus loaded with the antigen recognition domain protein, and the virus is used for infecting T cells knocked out of TRAC and B2M genes to obtain the T cells containing the antigen recognition domain protein.
Further, T cells containing antigen recognition domain proteins are cultured with the medium for expansion and enrichment.
The invention also claims the modularized universal CAR-T cell prepared by the preparation method.
The invention also provides application of the modularized universal CAR-T cell in preparing medicines for resisting HIV virus or T cell lymphoma.
Compared with the prior art, the invention has the following beneficial effects:
the SdCocher/GvTagOpti covalent coupling system developed by the subject group (patent application number: 202110876301.0) is optimized to obtain a truncated body which does not influence the function, namely SdDeltaN 17/Gv, so as to reduce the immunogenicity. Splitting the complete chimeric antigen receptor CAR into two parts, and transducing the part of the Sd Δn17 linked to the intracellular signaling domain onto allogeneic T cells; the part of Gv connected with the antigen recognition domain is purified in the form of protein, and the two parts are covalently combined through an isopeptide bond formed spontaneously between SdDeltaN 17/Gv to form a complete CAR-T.
The invention screens and obtains T with higher amplification rate and stronger function SCM Dominant T cells with higher cell subgroup ratio, double knockout of TRAC and B2M genes of T cells is carried out by CRISPR/Cas gene editing technology, and TCR is obtained through TCR and HLA-I double negative enrichment - /HLA-I - The proportion of cells reaches 99%. Double knockout of TRAC and B2M genes does not affect the killing function of MU-CAR-T cells, and MU-CAR-T cells that bind to different scFvs can specifically recognize positive target cells corresponding to killing.
Drawings
FIG. 1 is a schematic representation of SdΔN17-28BBZ3-PA2-tCD19 lentiviral expression vector.
FIG. 2 is a diagram showing the results of expression and purification of VRC01scFV-GV-His and CD5-CD50 scFV-GV-His proteins.
Wherein A is VRC01scFV-GV-His protein; b is CD5-CD50 scFV-GV-His protein.
FIG. 3 shows the expansion rate and cell subsets of T cells in different media and under different cytokine conditions; wherein A is the expansion condition of T cells under different culture medium conditions; b is the expansion condition of T cells under different cell factors; c is CD8 under different cytokines + T in T cells SCM The ratio of the subpopulations.
FIG. 4 is a graph of the results of a selection of dominant T cells, wherein A is the amplification rate detection; b is the detection of effector functions.
FIG. 5 is a graph of the results of flow assays after TRAC and HLA-I knockdown and double negative enrichment.
FIG. 6 is a schematic diagram of the construction of MU-CAR-T based on SDcatcher-GvoptiTag platform.
FIG. 7 is a graph showing the statistics of the killing ratio of MU-CAR-T cells. Wherein A is Jurkat gp160 Cell line as positive target cell B is Karpas-299 cell as positive target cell.
FIG. 8 is a process for preparing MU-CAR-T cells.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
EXAMPLE 1 construction of pHR-SFFV-SdΔN17-28BBZ3-PA2-tCD19 vector
1. The pHR-SFFV-SdΔN17-28BBZ3-PA2-tCD19 vector was synthesized.
28BBZ3 is a third generation CAR structure, is an intracellular signaling of CAR-T, and is composed of CD28 (nucleotides 460-660,GenBank NM_006139.3), CD137 (nucleotides 640-765,GenBank NM_001561.5) and CD3 zeta (nucleotides 160-492,Genbank NM_198053.2) intracellular domain linkages. 28BBZ3 has the amino acid sequence shown in SEQ ID NO:1, the nucleotide sequence of the expressed gene is shown as SEQ ID NO: 2.
28BBZ3 is connected with SdΔN17 through a linker to obtain SdΔN17-28BBZ3. SdDeltaN 17 is an SdCocher truncate, and the amino acid sequence of the SdDeltaN 17 is shown as SEQ ID NO:3, the nucleotide sequence of the expressed gene is shown as SEQ ID NO: 4. The amino acid sequence of SdΔN17-28BBZ3 is shown as SEQ ID NO:5, the nucleotide sequence of the gene expressing the gene is shown as SEQ ID NO: shown at 6.
As shown in FIG. 1, a schematic diagram of SdΔN17-28BBZ3-PA2-tCD19 lentiviral expression vector is shown. SdDeltaN 17-28BBZ3 is connected with tCD19 through P2A, and inserted into pHR-SFFV-KRAB-dCAS9-P2A-mCherry lentiviral vector (Addgene # 60954) through double restriction sites of MluI and NotI, thus obtaining pHR-SFFV-SdDeltaN 17-28BBZ3-PA2-tCD19 lentiviral vector. Wherein P2A is located at the C-terminal end of SdΔN17-28BBZ3 and at the N-terminal end of tCD 19. P2A is transcription element sequence, tCD19 (truncated form of CD19 molecule) gene sequence is used as screening marker or safety switch, PHR is carrier, SFFV is promoter.
2. Lentivirus package
(1) Cell culture dishes were pretreated with L-polylysine K (poly-K), HEK293T cells were plated, cells were required to be individually and evenly distributed, and cultured for 24h to a cell density of 70-80%.
(2) mu.L of Opti-MEM medium and 45. Mu.L of PEI-MAX transfection reagent were mixed and incubated at room temperature (15-25 ℃) for 5min to give diluted PEI-MAX transfection reagent. Mixing 500 mu L of Opti-MEM medium, 3 mu g of VSVG, 6 mu g of psPAX2, 6 mu g of pHR-SFFV-SdΔN17-28BBZ3-PA2-tCD19 and diluted PEI-MAX transfection reagent, and incubating for 20min at room temperature to obtain the PEI-MAX-DNA mixed solution.
Adding PEI-MAX-DNA mixed solution into the cell culture medium with the cell density reaching 70-80%. After transfection for 6-8 h, 7mL of fresh DMEM culture medium is replaced, after culture for 48h, virus supernatant is collected, 3000g is centrifuged for 5min, cell debris is removed, virus concentrated mixed solution is added, and after inversion and mixing, the virus culture solution is obtained after standing overnight (12 h) at 4 ℃. The virus concentrate mixture consisted of 2.5mL 50% (m/v) PEG 6000, 1.06mL 4M NaCl and 1.14mL PBS.
(3) Centrifuging at 4deg.C and 3600rpm for 30min to obtain white precipitate on the tube wall, carefully removing the whole supernatant, adding 1mL fresh culture medium, suspending the precipitate to obtain virus concentrate, and immediately using or packaging and storing at-80deg.C.
EXAMPLE 2 obtaining scFV-Gv-His protein
1. Experimental method
1. Construction of scFV-Gv-His expression vector
VRC01scFV-Gv-His (nucleotide sequence shown as SEQ ID NO: 7) and CD5-CD30scFV-Gv-His (nucleotide sequence shown as SEQ ID NO: 8) are synthesized, and a Signal Peptide (SP) is connected at the N end of the sequence to guide the synthesized protein to transfer to the secretory pathway. VRC01scFV-Gv-His and CD5-CD30 linked to a Signal peptide by double XhoI and XbaI cleavage sites
The scFV-Gv-His is respectively inserted into pCDNA3.1-INTRON-IRES-GFP mammal expression vectors (the independent construction and preservation in the laboratory are carried out, based on the original vector pCDNA3.1 (+), an INTRON-IRES-GFP-WPER sequence is inserted at the double enzyme cutting sites of XbaI and ApaI to enhance the expression of a target gene), and recombinant pCDNA3.1-INTRON-SP-VRC01 scFV-Gv-His-IRES-GFP expression vectors and pCDNA3.1-INTRON-SP-CD5-CD30 scFV-Gv-His-IRES-GFP expression vectors are respectively obtained, thus obtaining the scFV-Gv-His expression vectors. The nucleotide and amino acid sequences of VRC01 are shown in SEQ ID NO:9 and 10, the nucleotide and amino acid sequences of CD5-CD30 are shown in SEQ ID NO:11 and 12, gv is GvoTagOpti, and the nucleotide and amino acid sequences thereof are respectively shown in SEQ ID NOs: 13 and 14.
2. Purification of scFV-Gv-His protein
(1) 20mL of Opti-MEM medium and 1.875mL of PEI-MAX transfection reagent were mixed and incubated at room temperature (15-25 ℃) for 5min to give diluted PEI-MAX transfection reagent. Mixing 20mL of Opti-MEM medium, 625 mug of scFV-GV-His expression vector and diluted PEI-MAX transfection reagent, and incubating for 20min at room temperature to obtain PEI-MAX-DNA mixed solution.
(2) Adding PEI-MAX-DNA mixed liquor into 500mL cell density of 2-3X 10 6 To culture medium of HEK-293F cells, 3.5mL of valproic acid sodium salt was added simultaneously to promote protein expression and inhibit cell growth at 5% CO 2 Is cultured in a sterile cell shaker incubator at a constant temperature of 37 ℃. After 24h and 72h of transfection, respectively, 12% PFF05 supplement was added to the medium to supplement glucose and the like.
(3) Cell supernatants were collected on days 7-10 of transfection, centrifuged at 4000rpm for 10min, the supernatants were collected, and the supernatants were filtered with a 0.45 μm filter to thoroughly remove cell debris, to obtain filtered supernatants.
(4) The filtered supernatant was passed through a three pass nickel column to ensure that more protein was bound to the upper nickel column.
The nickel column was eluted with different concentrations of Elutation Buffer (30 mM imidazole+ 50mM NaCl+20mM Tris-HCl,50mM imidazole+ 50mM NaCl+20mM Tris-HCl,500mM imidazole+ 50mM NaCl+20mM Tris-HCl), and the eluate was collected. Ultrafiltration tubes of the corresponding protein sizes (VRC 01scFv-GV-His protein 10KD ultrafiltration tube, CD5-CD30scFv-Gv-His protein 30KD ultrafiltration tube) were selected, and the collected eluate was ultrafiltered, and imidazole in the solution of the absorption Buffer was replaced with Binding Buffer (50mM NaCl+20mM Tris-HCl), to obtain purified scFV-Gv-His proteins, namely VRC01scFv-Gv-His (amino acid sequence shown as SEQ ID NO: 15) and CD5-CD30scFv-Gv-His (amino acid sequence shown as SEQ ID NO: 16).
3. Eluted proteins were visualized by SDS-PAGE polyacrylamide gel electrophoresis using Coomassie blue staining and western-blot labelling with anti-His-specific antibodies, respectively.
2. Experimental results
scFV-Gv-His proteins eluted by different concentrations of imidazole were electrophoresed by SDS-PAGE and verified with His-specific antibodies using Coomassie brilliant blue staining and Western-Blot, respectively: the first column is a protein standard, as a protein size reference standard; the second, third and fourth columns are 30mM imidazole eluent, 50mM imidazole eluent and 500mM imidazole protein complete eluent, respectively. As shown in FIG. 2A, VRC01scFv-Gv-His and as shown in FIG. 2B, CD5-CD50 scFv-Gv-Hi, each protein band corresponds to the predicted molecular weight of the corresponding protein.
EXAMPLE 3 determination of Medium for culturing T cells
1. Experimental method
1. Screening of different media
The serum-free and xeno-free culture medium greatly reduces the risk of introducing heterologous infectious agents into cells in the culture process, and simultaneously avoids cell activation or inactivation and the like caused by the unknown ingredients in the serum.
The invention selects RPMI 1640, KBM581, X-VIVO15,T-SFM and ImmunoCurt-XFThe five culture mediums are respectively used for CD8 + T cells are expanded and cultured, and the separated Peripheral Blood Mononuclear Cells (PBMCs) are mixed with 0.5 to 1 multiplied by 10 6 The density of the solution/mL is respectively resuspended in different culture media, and the anti-human CD3 antibody and the anti-human CD28 antibody with the final concentration of 1 mug/mL are added to activate lymphocytes to obtain CD8 + T lymphocytes, additional IL-2 was added to maintain cell growth at a final concentration of 10ng/mL, and a mixture containing 5% CO was added 2 Culturing in a sterile incubator at 37 ℃. The cell morphology was observed and counted every 3 days from 72h after lymphocyte activation, while detection by anti-human CD8 flow antibody was performed, fluid replacement was performed in a half-fluid-change manner, and the cell density was maintained at 0.5 to 1X 10 6 In the range of/mL, the cell counter is used for measuring each group three times for 15 days continuously, so that the culture medium with the fastest amplification rate is determined, and the method is favorable for clinical experiments and large-scale industrialization.
2. Screening of different cytokines
CD8 pairs using IL-2 alone, IL-7 in combination with IL-15 + T lymphocytes are expanded in culture and the detection of cell subsets. The PBMCs are sorted according to the proportion of 0.5 to 1 multiplied by 10 6 Density per mL resuspended in OptiVitro T-SFM cell serum-free Medium, and activated lymphocytes were added with anti-human CD3 antibody and anti-human CD28 antibody at a final concentration of 1 μg/mL to obtain CD8 + T lymphocytes and IL-2 or IL-7 and IL-15 were added to maintain cell growth at a final concentration of 10ng/mL and 5ng/mL, respectively, and 5% CO was added 2 Culturing in a sterile incubator at 37 ℃. Cell morphology was observed and counted every 2-3 days starting 72h after activation, while the through-flow antibody was directed against CD8 + T cells and the sub-population thereof are detected, the fluid is supplemented in a semi-fluid exchange mode, and the cell density is maintained at 0.5 to 1 multiplied by 10 6 the/mL range. The cells were transferred to shake flasks for culture at day 5 after cell activation, with a shake flask radius of 19mm and a rotational speed of 88rpm/min.
T memory stem cells (T memory stem cell, T) SCM ) Is a rare memory lymphocyte subgroup with longer survival ability and strong immune reconstruction potential, and has the characteristics of stem cell sample and the likeHas the characteristics of memory T cells, thus in CAR-T cell treatment, T in T lymphocytes infused back into the body after expansion SCM The ratio of the sub-populations is related to the therapeutic effect. According to CD8 + Expression of CD45RA and CCR7 in T lymphocytes, CD8 + T cells are divided into four sub-populations: naive T cells (Tn: CD45 RA) + CCR7 + ) Central memory T cells (Tcm: CD45RA - CCR7 + ) Effector memory T cells (Tem: CD45RA-CCR 7-) and terminal effector cells (Te: CD45RA + CCR7-)；T SCM The cells share a common phenotypic characteristic with naive T cells, but T SCM Cells can highly express CD95 and CD122, thus distinguishing them from naive T cells.
2. Experimental results
1. The different media showed significantly different expansion rates, starting from day 12 with OptiVitro T-SFM media, which showed significant advantages for long-term culture and expansion of T cells (FIG. 3A).
2. The combined IL-7+IL-15 cytokines amplified at significantly higher rates than the IL-2 group alone (FIG. 3B) and induced a greater proportion of TSCM cell subsets (FIG. 3C).
EXAMPLE 4 cultivation of dominant T cells
1. Experimental method
1. Screening and expansion culture of dominant T cells with faster expansion rate and stronger effector function:
blood samples were obtained by recruiting different healthy young persons (Donor 1-20), and Peripheral Blood Mononuclear Cells (PBMCs) were isolated from the blood samples at a rate of 0.5 to 1X 10 6 Density per mL resuspended in OptiVitro T-SFM cell serum-free Medium, and anti-human CD3 and CD28 antibodies were added at a final concentration of 1 μg/mL to activate lymphocytes to obtain CD8 + T lymphocytes for the maintenance of CD8 + Survival and proliferation of T lymphocytes, additional IL-7 and IL-15 were added at a final concentration of 10ng/mL and 5ng/mL, and a mixture containing 5% CO was added 2 Culturing in a sterile incubator at 37 ℃. After 72 hours of activation, the liquid is supplemented by a half-liquid-changing mode, and the cell density is maintained at 0.5 to 1 multiplied by 10 6 the/mL range. After activation of cellsOn day 5, the cells cultured by standing are transferred into shake flasks for culturing, the radius of the shake flasks is 19mm, the rotation speed is 88rpm/min, the cell morphology is observed and counted every 2-3 days, and the expansion rate of the cells is detected by the human anti-CD 8 flow antibody.
2. Five Donor-derived T cells, donor7, donor8, donor11, donor15, and Donor17, were sorted by immunomagnetic bead enrichment and subjected to co-energy validation.
The SdΔN17-28BBZ3 in the pHR-SFFV-SdΔN17-28BBZ3-PA2-tCD19 vector of example 1 was replaced with CD5-CDC30, and after constructing the pHR-SFFV-CD5-CDC30-PA2-tCD19 vector, a virus concentrate expressing CD5-CDC30 was prepared as in example 1, and five donor-derived CD8 was used + The T lymphocytes are respectively added with a virus concentrate of CD5-CDC30 to infect viruses, and the CD5-CD30-CAR-T cells are obtained. CD5-CD30-CAR-T cells and target cells karpas-299 in a quantitative ratio of 4:1, mixed culture was performed in 96-well PVDF plates pre-coated with IFN- γ antibody.
The secretion of IFN-gamma by CD5-CD30-CAR-T cells was examined using ELISPot assay.
2. Experimental results
1. Five Donor-derived T cells, donor7, donor8, donor11, donor15, and Donor17, proliferate at the fastest rate (a in fig. 4).
2. Under target cell stimulation, the levels of the secreted effector function cytokines IFN-gamma were significantly different, and the cell spot numbers of Donor7 and Donor17 were significantly higher than that of Donor8, donor11 and Donor15, indicating that Donor7 and Donor 17-derived CD5-CD30 CAR-T cells were activated and secreted higher levels of IFN-gamma killer cytokines after co-culture with positive target cells (FIG. 4B). Thus, the Donor7 and Donor17 Donor sources CD8 + T cells are the dominant T cells selected.
EXAMPLE 5 preparation of MU-CAR-T cells
1. Experimental method
1. TRAC and B2M gene knockout and TCR-/HLA - Cell sorting
The dominant T cells of example 4 were subjected to gene editing using electroporation, and the sgRNA sequences used were determined as set forth in SEQ ID NOs: 17. SEQ ID NO: 18. SEQ ID NO:19 and SEQ ID NO: shown at 20.
Electrotransformation experiments were performed to knock out TRAC gene (NCBI accession number: NG_ 001332.3) and B2M gene (NCBI accession number: NM_ 004048.4) according to the P3 Primary Cell 4D-NucleofectrTMX kit instructions: the support was prepared as per 4.5:1 to prepare a 1 to 5X 10 mixed solution by adding the mixed solution into NucleofectorTMSOLUTION 6 washing/mL dominant T cells with PBS, re-suspending the cells with the mixed solution, adding sgRNA and Cas9 mRNA respectively, transferring into an electric rotating cup, placing into a 4D-NucleofectorTMXUnit instrument, and selectingElectrotransformation of procedure E0-115, transferring cells to a pre-warmed medium, and adding a medium containing 5% CO 2 Incubate in a sterile incubator at 37 ℃. After 24h, a second electrotransformation is performed to improve the knockout efficiency, and CD8 of the TRAC and B2M genes is obtained + T lymphocytes.
CD8 after electric conversion + T lymphocytes were cultured for 48h at 1X 10 intervals 6 5. Mu.L of Bio-TCR and 5. Mu.L of Bio-HLA-I antibody, respectively, were added, incubated on ice for 15min, washed with 10 volumes of PBS, and the cells were resuspended in PBS, and Streptavidin Nanobeads was added in a volume ratio to the primary antibodies (Bio-TCR and Bio-HLA-I antibody) of 1:1, after incubation on ice for 15min, 10 volumes of PBS were added for washing, and the cells were resuspended in 2.5mL of PBS. Attaching to sorting magnet for 5min, pouring the separated cell suspension into new test tube, repeating twice to improve sorting purity, thereby obtaining higher purity TCR - HLA-I double negative CD8 + T lymphocytes.
2. Preparation of MU-CAR-T cells
(1) Taking a proper amount of TCR-/HLA-I with good growth state - Double negative CD8 + T lymphocytes were placed in a centrifuge tube and centrifuged at 500g for 5min. The supernatant was discarded and 1mL: 1X 10 6 The virus concentrate prepared in example 1 was added in proportion to individual cells, and Polybrene was added at a final concentration of 8. Mu.g/mL, and gently stirred and mixed to obtain a cell suspension.
(2) The cells of step (1) are subjected toTransferring the suspension into a culture dish, centrifuging at 37deg.C and 350 Xg for 90min, and returning to 5% CO 2 Continuously culturing in a sterile incubator at 37 ℃. After 12h of incubation, the cells were centrifuged, changed, and the cells were further expanded with fresh medium containing 10ng/mL IL-7 and 5ng/mL IL-15.
(3) And (3) after 2 days of adding the virus concentrate, detecting the expression condition of the cells CD19 in the step (2) by using a flow so as to determine the infection efficiency, and carrying out positive enrichment on the cells by using CD19 magnetic beads to obtain the enriched and amplified cells. Specific scFV-Gv-His proteins were added to the culture supernatant enriched for expanded cells and incubated for 12h to obtain modular universal CAR-T cells (MU-CAR-T cells). The specific scFV-Gv-His proteins were VRC01scFV-Gv-His protein and CD5-CD30scFV-Gv-His protein obtained in example 2, respectively.
After the VRC01scFV-Gv-His protein is incubated, a specific HIV-1 targeted CAR-T cell (called VC-MU-CAR-T cell for short) is obtained, wherein the chimeric antigen receptor VC-MU-CAR is contained, and the amino acid sequence is shown as SEQ ID NO: 21; after the CD5-CD30scFV-Gv-His protein is incubated, a CAR-T cell (called CD5-CD30-MU-CAR-T cell for short) of the specific targeted T cell lymphoma is obtained, wherein the chimeric antigen receptor CD5-CD30-MU-CAR is contained, and the amino acid sequence is shown as SEQ ID NO: shown at 22.
2. Experimental method
As shown in fig. 5, TCR - The cell population of/HLA-I-is enriched to more than 99%. FIG. 6 is a schematic diagram showing the structure of a chimeric antigen receptor MU-CAR-T constructed based on the SDcatcher-GvoptiTag platform. It contained SdΔN17-28BBZ3 of example 1 as an antigen recognition domain protein, and scFV-Gv-His protein of example 2 as an intracellular signaling domain protein.
Example 6 MU in vitro functional verification of CAR-T cells
1. Experimental method
Taking a proper amount of TCR-/HLA-I-double negative CD8 with good growth state + T lymphocytes, 1mL: 1X 10 6 Ratio of cells to be infected with VRC01-CAR virus concentrate and CD5-CD30-CAR virus concentrate prepared in example 4, respectively, and after 12 hr, centrifuging to change the solution and continuing culturing to prepare VRC01-CAR-T fineCells and CD5-CD30 CAR-T cells as positive controls, which were enriched for CD19 magnetic beads to obtain T cells highly expressing CAR, cell populations enriched by more than 90% according to the method of example 5. The preparation method of the VRC01-CAR virus concentrate comprises the following steps: sdΔN17-28BBZ3 in pHR-SFFV-SdΔN17-28BBZ3-PA2-tCD19 vector of example 1 is replaced with VRC01, and after pHR-SFFV-VRC01-PA2-tCD19 vector is constructed, a virus concentrate expressing VRC01 is prepared according to the method of example 1.
Selection of Jurkat expressing HIV-1 protein gp160 gp160 The cell line served as a positive target cell for VC-MU-CAR-T. T cell lymphoma cell line Karpas-299 cells highly expressing CD5 and CD30 are used as positive target cells of CD5-CD 30-MU-CAR-T.
Positive target cells per well 10 4 The cell number of the cell is respectively added into a 96-well plate at the bottom of U, and the MU-CAR-T cell is 10 according to the effective target ratio: 1. 5: 1. 2.5: 1. 1.25: 1. 0.625:1 and culturing in a mixed mode, and determining the killing toxicity of MU-CAR-T cells to specific target cells by detecting the release amount of LDH after 24 hours.
2. Experimental results
As shown in fig. 7 a and B, MU-CAR-T exhibited similar killing effects as CAR-T, and at a higher effective target ratio of 10: the killing effect in the case of 1 is almost the same as that of CAR-T; whereas T cells transduced with SD.DELTA.N17-28 BBZ3 alone (enriched expanded cells obtained in step (3) of example 5, which were not incubated with scFV-Gv-His protein) did not show specific killing, it was demonstrated that only scFV binding to specific targets activated CAR-T cell signaling, thus exerting killing target cell function.
As shown in fig. 8, in the whole preparation process of MU-CAR-T, MU-CAR-T selectively targets various antigens by covalently binding scFv, and allogeneic T cells are transformed to be transformed into a stable and universal cell bank, thereby realizing the construction of modular and universal CAR-T cells.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (10)
1. A chimeric antigen receptor comprising a component 1 and a component 2, wherein the component 1 and the component 2 are linked by a self-assembly system;
wherein, component 1 is antigen recognition structural domain protein, and the amino acid sequence is shown in SEQ ID NO: as shown in the figure 10 or 12,
component 2 is an intracellular signal domain protein, and the amino acid sequence of the intracellular signal domain protein is shown as SEQ ID NO:1 is shown in the specification;
the self-assembly system is a GvoTagOpti/SdCoatcher system, wherein the amino acid sequence of the GvoTagOpti is shown as SEQ ID NO:14, the amino acid sequence of SdCocher is shown as SEQ ID NO: 3.
2. The chimeric antigen receptor according to claim 1, wherein GvoTagOpti is disposed C-terminal to the antigen recognition domain protein.
3. The chimeric antigen receptor according to claim 1, wherein the SdCatcher is disposed N-terminal to the intracellular signaling domain protein.
4. Use of the chimeric antigen receptor of any one of claims 1 to 3 for the preparation of a modular universal CAR-T cell.
5. A method of making a modular universal CAR-T cell, comprising expressing in the T cell the intracellular signaling domain protein of claim 1 having the amino acid sequence of SEQ ID NO:1 or 2, obtaining a T cell expressing an intracellular signaling domain protein; and incubating and purifying the T cells expressing the intracellular signal domain protein and the antigen recognition domain protein in claim 1 to obtain the modularized universal CAR-T cells.
6. The method according to claim 5, wherein the medium for culturing the T cells isT-SFM medium.
7. The method according to claim 6, wherein the medium further comprises IL-7 and IL-15.
8. The method of claim 5, wherein the T cells are TRAC and B2M gene knocked-out T cells.
9. The modular universal CAR-T cell prepared by the method of claims 5-8.
10. Use of the modular universal CAR-T cell of claim 9 in the manufacture of a medicament against HIV virus or T cell lymphoma.
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