Spot universal CFR64-T cell prepared based on CRISPR / Cas9, and preparation method and application thereof

Abstract

The invention provides a spot universal CFR64-T cell prepared on the basis of CRISPR / Cas9 as well as a preparation method and application thereof. The CFR64-T cell is obtained by induced differentiation of recombinant human embryonic stem cells; in the recombinant human embryonic stem cell, a coding sequence of a CFR64 molecule is knocked into a TRAC site of the human embryonic stem cell at a fixed point through a CRISPR / Cas9 editing system; the CFR64 molecule comprises an antigen binding structural domain, a transmembrane structural domain and an intracellular costimulatory signal structural domain, the antigen binding domain is a human Fc gamma receptor extracellular domain, and the amino acid sequence of the antigen binding domain is shown in SEQ ID NO: 1. The CRISPR / Cas9 technology is adopted, gene integration sites are controllable and free of oncogene mutation risks, TCR genes are knocked out, the immunological rejection risk of universal CFR64-T cells to hosts is reduced, and meanwhile gene expression is more stable.

Description

Technical Field

[0001] The present invention belongs to the field of cell biology, and specifically relates to a readily available universal CFR64-T cell prepared based on CRISPR / Cas9, as well as a preparation method and application. Background Art

[0002] CAR-T therapy involves using gene editing technology to transduce coding sequences containing specific antigen recognition domains and T cell activation signals into T cells. The resulting CAR-T cells are activated by binding directly to specific antigens on the surface of tumor cells, releasing perforin, granzyme B, and other proteins to directly kill tumor cells. Simultaneously, the release of cytokines recruits endogenous immune cells to kill tumor cells, thereby achieving the goal of treating tumors. Because immune memory T cells can also be formed, a specific, long-lasting anti-tumor mechanism can be achieved.

[0003] CAR-T cells are considered one of the most promising therapies for curing cancer, demonstrating significant efficacy in treating hematologic malignancies. However, numerous challenges limit their clinical application. These include the high cost of adoptive immune cells, which are derived from the patient's own T cells. The production process is time-consuming, carries risks such as production failure and missed treatment windows. Advanced cancer patients undergoing chemotherapy and radiotherapy experience impaired immune cell activity and function, impacting the effectiveness of adoptive immune cell therapy. Adoptive therapy with allogeneic immune cells can trigger severe graft-versus-host disease, endangering the patient's life. The inefficiency of immunogenic gene knockout using allogeneic immune cells remains a challenge, hindering the source of adoptive immune cells. Furthermore, personalized immune cells cannot be mass-produced to reduce costs. Therefore, the development of universal CAR-T cells has become a key area of current CAR-T therapy research.

[0004] Switchable CAR-T cells indirectly recognize tumor cells through the mediation of various switch molecules, improving the versatility and controllability of CAR-T therapy. Furthermore, the combined use of multiple switch molecules allows a single switchable CAR-T cell to simultaneously target multiple tumor antigens, facilitating the targeted treatment of highly heterogeneous solid tumors. While the emergence of switchable CAR-T therapy has promoted the development of universal CAR-T, the switch molecules in switchable CAR-T cells contain amino acid epitopes not found in humans, resulting in a certain degree of immunogenicity. This may trigger an immune response in the body, thus affecting the long-term efficacy of switchable CAR-T therapy. Furthermore, batch effects in CAR-T cell production, the long production cycles, and the high cost all limit the large-scale clinical application of CAR-T.

[0005] Currently, CAR-T cells or CFR64-T cells are primarily derived from the patient's own T cells. These factors, such as long preparation cycles, poor cell viability, and the inability to mass-produce, limit their application. T cells differentiated from pluripotent stem cells represent a highly promising cell source for CAR-T therapy. By leveraging the self-renewal properties of pluripotent stem cells, it is theoretically possible to generate an unlimited supply of highly homologous T cells. This is crucial for addressing the significant batch effects, long preparation cycles, and high costs associated with traditional CAR-T production. Therefore, providing a readily available, universal CAR-T cell derived from induced differentiation of pluripotent stem cells would be of significant application value in tumor immunotherapy. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a generic off-the-shelf CFR64-T cell (full name: Chimeric antigen Fc receptor CD64-T cells, CFR64-T) prepared based on CRISPR / Cas9, as well as a preparation method and application. Based on the problems of CFR64-T cells in tumor immunotherapy, the present invention improves its preparation process, utilizes CRISPR / Cas9 technology to eliminate the endogenous TCR specificity of human embryonic stem cells while delivering CFR64 molecules to designated gene sites, constructs recombinant human embryonic stem cells, establishes an induced differentiation process system, and constructs a generic off-the-shelf CFR64-T cell. Compared with traditional CAR-T cells, the CFR64-T cell has stronger long-term anti-tumor activity and the ability to resist exhaustion, which provides a new idea for tumor immunotherapy.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a readily available universal CFR64-T cell prepared based on CRISPR / Cas9, wherein the CFR64-T cell is obtained by induced differentiation of recombinant human embryonic stem cells; the coding sequence of the CFR64 molecule is site-specifically knocked into the TRAC locus of the recombinant human embryonic stem cell using the CRISPR / Cas9 editing system;

[0009] The CFR64 molecule includes: an antigen binding domain, a transmembrane domain and an intracellular co-stimulatory signaling domain;

[0010] The antigen binding domain is the extracellular segment of a human Fcγ receptor, and the amino acid sequence thereof includes that shown in SEQ ID NO: 1.

[0011] In the present invention, the off-the-shelf universal CFR64-T cells refer to T cells that have the chimeric antigen Fc receptor CD64 molecule directedly integrated into the TRAC gene site, have no endogenous TCR gene expression, and continuously and stably express the chimeric antigen Fc receptor CD64 molecule.

[0012] The present invention utilizes CRISPR / Cas9 technology to knock-in CFR64 molecules at the TRAC locus of human embryonic stem cells, eliminating endogenous TCR specificity while directionally delivering CFR64 molecules to designated gene loci, thereby constructing recombinant human embryonic stem cells (H1-TRAC-CFR64 cell line); by establishing a process system for differentiating recombinant human embryonic stem cells into CFR64-T cells, a readily available and universal CFR64-T cell was constructed.

[0013] The present invention overexpresses CFR64 molecules into human embryonic stem cells through targeted knock-in, and then efficiently induces the resulting recombinant human embryonic stem cells to differentiate into T cells, thereby obtaining off-the-shelf universal CFR64-T cells, which is expected to solve the problems of single source and universality of traditional CAR-T or CFR64-T cells. Compared with other transformation schemes, such as lentiviral transduction, the TRAC site-directed knock-in of the present invention has the following advantages: (1) the gene integration site is controllable and there is no risk of oncogene mutation; (2) the TCR gene is knocked out, reducing the risk of immune rejection of the universal CFR64-T cells to the host; (3) gene expression is more stable.

[0014] Preferably, the transmembrane domain is CD8αTM, and the amino acid sequence includes that shown in SEQ ID NO:2.

[0015] Preferably, the intracellular co-stimulatory signaling domain is 4-1BB, and the amino acid sequence includes that shown in SEQ ID NO: 3.

[0016] Preferably, the CFR64 molecule comprises CD64ECD, hinge sequence, CH3 Spacer, CD8αTM, 4-1BB and CD3ζ connected in sequence.

[0017] Preferably, the amino acid sequence of the CFR64 molecule includes that shown in SEQ ID NO:4.

[0018] Preferably, the human embryonic stem cells are derived from the hESC cell line H1.

[0019] Preferably, the CRISPR / Cas9 editing system comprises sgRNA, Cas9 and a homologous recombination fragment.

[0020] Preferably, the homologous recombination fragment comprises a homologous arm on the left side of the TRAC site, a nucleotide sequence encoding a CFR64 molecule, and a homologous arm on the right side of the TRAC site.

[0021] Preferably, the nucleotide sequence encoding the CFR64 molecule includes that shown in SEQ ID NO:5.

[0022] Preferably, the nucleotide sequence of the homology arm on the left side of the TRAC site includes that shown in SEQ ID NO:6.

[0023] Preferably, the nucleotide sequence of the homology arm on the right side of the TRAC site includes that shown in SEQ ID NO:7.

[0024] Preferably, the site targeted by the sgRNA is the first exon of the TRAC site of human embryonic stem cells.

[0025] Preferably, the DNA sequence corresponding to the sgRNA includes that shown in SEQ ID NO:8.

[0026] In the present invention, the sgRNA is used for TRAC site-directed integration, and the TRC gene knockout efficiency can reach more than 90%.

[0027] In a second aspect, the present invention provides a method for preparing off-the-shelf universal CFR64-T cells using CRISPR / Cas9, the method comprising: using the CRISPR / Cas9 editing system to transduce the coding sequence of the CFR64 molecule into human embryonic stem cells; sorting and amplifying the CFR64 molecule-positive human embryonic stem cells, and then inducing differentiation into CFR64-T cells.

[0028] In the present invention, the CFR64 molecule is directedly integrated into the TRAC site of human embryonic stem cells through CRISPR / Cas9 gene editing technology. The TRAC gene site forms a DNA double-strand break gap under the mediation of the TRAC-sgRNA / Cas9 protein complex in the CRISPR / Cas9 editing system. At the same time, the CFR64 molecule is integrated into the gap by gene homologous recombination. At this time, the TRAC gene site is inactivated due to frameshift mutation, while the CFR64 molecule is integrated into the TRAC genomic site and stably expressed. In the above gene editing method, the gene integration site is controllable, there is no risk of oncogene mutation, the TCR gene is knocked out, reducing the risk of immune rejection of the host by universal CFR64-T cells, and at the same time, gene expression is more stable.

[0029] During the differentiation of pluripotent stem cells into T cells, the chromatin of the cell genome undergoes drastic changes to regulate gene expression, thereby causing the cells to differentiate into cell types at specific stages. Traditional lentivirus-mediated target gene integration is random integration of genomic sites. Therefore, during T cell differentiation, many genomic integration sites will be silenced due to euchromatin-heterochromatin transition, resulting in instability in target gene expression. However, the expression of CFR64 molecules mediated by TRAC site-directed integration is not easily silenced, as the genomic integration site is single and clear, and the chromatin of the TCR gene site is in an open state during the T cell differentiation stage. Therefore, gene expression is more stable.

[0030] Preferably, the sorting and amplification steps include: after gene editing of human embryonic stem cells, culturing them under conditions of stem cell culture medium for 1-2 weeks, sorting and purifying CFR64 molecule-positive human embryonic stem cells by flow cytometry, and then passage-amplifying the purified embryonic stem cells in vitro.

[0031] In the present invention, wild-type H1 embryonic stem cells are gene-edited and then cultured under the conditions of Matrigel matrix gel and mTesR1 stem cell culture medium for 1-2 weeks. After the transient expression of the homologous recombination plasmid disappears, the CFR64 molecule-positive H1 embryonic stem cells continuously express the CFR64 target sequence that is integrated in the TRAC site. The CFR64 molecule-positive H1 embryonic stem cells are then sorted and purified by flow cytometry, and the purified H1-TRAC-CFR64 embryonic stem cells are then passaged and amplified in vitro and then frozen.

[0032] Preferably, the step of inducing differentiation comprises: inducing CFR64 molecule-positive human embryonic stem cells to differentiate into hematopoietic precursor cells; and then inducing the hematopoietic precursor cells to differentiate into T cells.

[0033] Preferably, the step of inducing differentiation of CFR64-positive human embryonic stem cells comprises: forming embryoid bodies from CFR64-positive human embryonic stem cells by centrifugation; inoculating the embryoid bodies into a medium without matrix cells and serum for culture, and adding cytokines VEGF, BMP4 and SB431542 to induce them to differentiate into CD34 + CD43 - Hematopoietic endothelial cell differentiation; cytokines SCF, TPO, FLT3-L, IL-3 and IL-6 were added to induce them to become CD34 + CD45 + Hematopoietic precursor cells.

[0034] In the present invention, the centrifugal method comprises the following steps: (1-2)×10 6H1-TRAC-CFR64 embryonic stem cells were inoculated into an AggreWell well containing 1 mL of mTesR1 medium containing 10 μM Thiazovivin. After the cells settled to the bottom of the well for 3 minutes, the AgreeWell well was placed in a centrifuge and centrifuged at 100 g for 3 minutes with a centrifuge speed of 1. After centrifugation, the AgreeWell well was placed in an incubator and cultured at 37°C for 24 hours.

[0035] In the present invention, the inoculation amount of the embryoid bodies is 100-150 per six-well plate.

[0036] Preferably, the culture medium comprises: mTesR1 culture medium or StemLine II culture medium.

[0037] In the present invention, the culture media used include mTesR1 (day 0-2) and StemLine II (day 2-6).

[0038] Preferably, the concentration of VEGF in the culture is 1-50 ng / mL; for example, it can be 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL or 50 ng / mL, etc.

[0039] Preferably, the concentration of BMP4 in the culture is 1-10 ng / mL; for example, it can be 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL or 10 ng / mL, etc.

[0040] Preferably, the concentration of SB431542 in the culture is 1-10 μM; for example, it can be 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM or 10 μM, etc.

[0041] As a preferred embodiment of the present invention, the concentrations of the cytokines VEGF, BMP4 and SB431542 in the culture are 50 ng / mL, 4 ng / mL and 6 μM, respectively.

[0042] In the present invention, after adding the cytokines VEGF, BMP4, and SB431542, differentiation induction conditions are as follows: on days 0-2, the culture medium used is mTesR1 supplemented with 1-50 ng / mL VEGF and 1-10 ng / mL BMP4; on days 2-4, the culture medium used is StemLine II supplemented with 1-50 ng / mL VEGF and 1-10 μM SB431542; and on days 4-6, the culture medium used is StemLine II supplemented with 1-50 ng / mL VEGF. The cells are incubated at 37°C with 5% CO2.

[0043] In the present invention, the ratio and number of CD34-positive and CD43-negative hemogenic endothelial cells obtained under the above-mentioned induction differentiation conditions after optimization are the highest.

[0044] Preferably, the concentration of the cytokines SCF, TPO, FLT3-L, IL-3 or IL-6 in the culture is independently 1-50 ng / mL; for example, it can be 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL or 50 ng / mL, etc.

[0045] In the present invention, after adding the cytokines SCF, TPO, FLT3-L, IL-3 and IL-6, the conditions for inducing differentiation are as follows: hematopoietic endothelial cells selected by CD34 magnetic beads are induced to differentiate in the presence of OP9-DL4 stromal cells using αMEM medium containing 20% FBS to which the above-mentioned cytokines are added for 8-12 days, and half of the medium is replaced every 2-3 days during the differentiation period.

[0046] In the present invention, the ratio and number of CD34-positive CD45-positive hematopoietic precursor cells obtained under the above-mentioned induction differentiation conditions after optimization are the highest.

[0047] Preferably, the hematopoietic precursor cells are cultured at a temperature of 35-42°C, for example, 35°C, 37°C, 40°C or 42°C.

[0048] Preferably, the hematopoietic precursor cells are cultured for 6-12 days, for example, 6 days, 8 days, 10 days or 12 days.

[0049] Preferably, the step of inducing differentiation of hematopoietic precursor cells comprises: inoculating hematopoietic precursor cells onto OP9-DL1 / 4 stromal cells for culture, and adding cytokines IL-7 and FLT3-L required for T cell differentiation to induce the hematopoietic precursor cells to differentiate into T cells.

[0050] In the present invention, the inoculation amount of the hematopoietic precursor cells is 1 million to 1.2 million per six-well plate.

[0051] Preferably, the concentrations of the cytokines IL-7 and FLT3-L in the culture are each independently 1-50 ng / mL, for example, 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL or 50 ng / mL, etc.

[0052] In the present invention, after adding the cytokines IL-7 and FLT3-L, the conditions for inducing differentiation are as follows: the differentiated hematopoietic progenitor stem cells are induced to differentiate in the presence of OP9-DL4 stromal cells using αMEM medium containing 20% FBS and the addition of the above-mentioned cytokines for 35-42 days, and half of the medium is replaced every 2-3 days during the differentiation period.

[0053] In the present invention, under the above-mentioned induction differentiation conditions after optimization, the proportion and number of CD4 and CD8 double-positive progenitor T cells are the highest.

[0054] Preferably, the culture temperature during the differentiation induction process is 35-42°C, for example, 35°C, 37°C, 40°C or 42°C.

[0055] Preferably, the culture time for the differentiation induction process is 35-42 days, for example, 35 days, 37 days, 40 days or 42 days.

[0056] In a third aspect, the present invention provides a CRISPR / Cas9 editing system, which includes sgRNA, Cas9 and a homologous recombination fragment.

[0057] Preferably, the homologous recombination fragment comprises a homologous arm on the left side of the TRAC site, a nucleotide sequence encoding a CFR64 molecule, and a homologous arm on the right side of the TRAC site.

[0058] Preferably, the nucleotide sequence encoding the CFR64 molecule includes that shown in SEQ ID NO:5.

[0059] Preferably, the nucleotide sequence of the homology arm on the left side of the TRAC site includes that shown in SEQ ID NO:6.

[0060] Preferably, the nucleotide sequence of the homology arm on the right side of the TRAC site includes that shown in SEQ ID NO:7.

[0061] Preferably, the site targeted by the sgRNA is the first exon of the TRAC site of human embryonic stem cells; the DNA sequence corresponding to the sgRNA includes that shown in SEQ ID NO:8.

[0062] In a fourth aspect, the present invention provides a recombinant human embryonic stem cell, in which the coding sequence of the CFR64 molecule is site-specifically knocked into the TRAC site of the human embryonic stem cell through the CRISPR / Cas9 editing system; the amino acid sequence of the CFR64 molecule includes that shown in SEQ ID NO:4.

[0063] In a fifth aspect, the present invention provides a recombinant CD34 + CD43 - Hematopoietic endothelial cells are obtained by inducing differentiation of the recombinant human embryonic stem cells described in the fourth aspect.

[0064] In a sixth aspect, the present invention provides a recombinant CD34 + CD45 + Hematopoietic precursor cells, said cells are composed of the recombinant CD34 + CD43 - Hematopoietic endothelial cells were induced to differentiate.

[0065] In the seventh aspect, the present invention provides the off-the-shelf universal CFR64-T cells prepared based on CRISPR / Cas9 as described in the first aspect, the recombinant human embryonic stem cells as described in the fourth aspect, and the recombinant CD34 T cells as described in the fifth aspect. + CD43 - Hematopoietic endothelial cells or the recombinant CD34 described in the sixth aspect + CD45 + The application of any one or a combination of at least two of the hematopoietic precursor cells in the preparation of tumor therapeutic drugs.

[0066] The numerical range described in the present invention includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.

[0067] Compared with the prior art, the present invention has the following beneficial effects:

[0068] (1) The present invention uses CRISPR / Cas9 gene editing technology to integrate CFR64 molecules into the TRAC site of human embryonic stem cells. The TRAC gene site forms a DNA double-strand break gap under the mediation of the TRAC-sgRNA / Cas9 protein complex in the CRISPR / Cas9 editing system. At the same time, the CFR64 molecule is integrated into the gap by gene homologous recombination. At this time, the TRAC gene site is inactivated due to frameshift mutation, and the CFR64 molecule is integrated into the TRAC genomic site and stably expressed. In the above gene editing method, the gene integration site is controllable, there is no risk of oncogene mutation, the TCR gene is knocked out, and the risk of immune rejection of the host by universal CFR64-T cells is reduced. At the same time, gene expression is more stable. During the differentiation of pluripotent stem cells into T cells, the chromatin of the cell genome undergoes drastic changes to regulate gene expression so that the cells differentiate into cell types at a specific stage. Traditional lentivirus-mediated integration of target genes occurs randomly at genomic loci. Therefore, during T cell differentiation, many genomic integration sites are silenced due to euchromatin-heterochromatin transitions, resulting in unstable target gene expression. However, TRAC site-directed integration of CFR64 molecules, due to the single, clear genomic integration site and the open chromatin state of the TCR gene locus during T cell differentiation, is less likely to be silenced, resulting in more stable gene expression.

[0069] (2) The present invention overexpresses CFR64 molecules into human embryonic stem cells by targeted knock-in, and efficiently induces the differentiation of recombinant human embryonic stem cells (H1-TRAC-CFR64 cell line) into TCR-knockout CFR64-T cells (iTRAC-CFR64-T), thereby obtaining off-the-shelf universal CFR64-T cells, which is expected to solve the problems of single source and universality of traditional CAR-T or CFR64-T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is a schematic diagram of constructing a CFR64 molecule for site-directed knock-in at the TRAC gene locus.

[0071] Figure 2 Figure 1 is the detection result of CFR64 molecule-positive H1 cells. Figure A is the result of genomic PCR identification, and Figure B is the expression of CFR64 molecules detected by flow cytometry.

[0072] Figure 3 Schematic diagram of the process of induced differentiation.

[0073] Figure 4 This is the result of differentiation of the H1-TRAC-CFR64 cell line.

[0074] Figure 5 The killing effect of iTRAC-CFR64-T cells on SKOV3 HER2-positive tumor cell line in vitro.

[0075] Figure 6 The figure shows the killing effect of iTRAC-CFR64-T cells on Hela-Claudin18.2-positive tumor cell lines in vitro.

[0076] Figure 7 The killing effect of iTRAC-CFR64-T cells on MKN45-Claudin18.2-positive tumor cell lines in vitro. DETAILED DESCRIPTION

[0077] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0078] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0079] The sources of the experimental materials used in this invention are as follows:

[0080] 1. Cytokines: IL-3, IL-6, IL-7, TPO, SCF, VEGF, and FLT3-L were purchased from Peprotech; BMP4 was purchased from R&D.

[0081] 2. Cell culture medium and related reagents: mTesR1 medium and Thiazovivin were from Stemcell; StemLine II was from Sigma; αMEM medium was from Thermo Fisher; SB431542 was from Tocris.

[0082] 3. Experimental consumables: AggreWell comes from Stemcell; various culture dishes come from Greiner.

[0083] 4. Gene editing reagents: TRAC-sgRNA was synthesized by Genscript, Cas9 protein was from ThermoFisher; electroporation kit was from Lonza Human Stem cell Nucleofector TM Kit 2, electroporation program B-016, electroporator Lonza 2b.

[0084] Example 1

[0085] In this embodiment, a CFR64 homologous recombination vector of the TRAC site is constructed. The chimeric antigen receptor includes an antigen binding domain, a transmembrane domain, and an intracellular co-stimulatory signaling domain. The antigen binding domain of CFR64 is the extracellular segment of the human Fcγ receptor (CD64a). According to the immune characteristics of T cells, the appropriate combination of intracellular co-stimulatory domains is selected through screening, combination, and verification of the intracellular co-stimulatory domains, thereby designing a new CFR64 molecule. Homologous sequences are designed for the natural TRAC site sequence, so that directional knock-in of the TRAC site can be achieved by using homologous recombination repair, such as Figure 1 The specific steps are as follows:

[0086] The target gene fragment of CFR64 molecule and the homologous arm sequences on both sides of the TRAC site were integrated into the pUC19 vector by molecular cloning to obtain the pUC19-TRAC HDR-CFR64 homologous recombination vector.

[0087] DNA sequence of the homology arm on the left side of the TRAC site, SEQ ID NO: 6:

[0088] gaattcgagctcggtacccggggatccctattaaataaaagaataagcagtattattaagtagccctgcatttcaggtttccttgagtggcaggccaggcctggcc gtgaacgttcactgaaatcatggcctcttggccaagattgatagcttgtgcctgtccctgagtcccagtccatcacgagcagctggtttctaagatgctatttcccg tataaagcatgagaccgtgacttgccagccccacagagccccgcccttgtccatcactggcatctggactccagcctgggttggggcaaagagggaaatgagatcat gtcctaaccctgatcctcttgtcccacagatatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcacc.

[0089] DNA sequence of the homology arm on the right side of the TRAC site, SEQ ID NO: 7:

[0090] gcggccgcgttaaattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaaca gtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccaggtaagggcagctttggtgcctt cgcaggctgtttccttgcttcaggaatggccaggttctgcccagctctggtcaatgatgtctaaaactcctctgattggtggtctcggccttatccattgccaccaaaaccct ctttttaactaagaaacagtgagccttgttctggcagtccagagaatgacacgggaaaaaagcagatggatcctctagagtcgacctgcaggcatgcaagcttggcgtaatcatg.

[0091] The antigen binding domain is the extracellular segment of a human Fcγ receptor, and the amino acid sequence is shown in SEQ ID NO: 1:

[0092] MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKF FHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFH.

[0093] The transmembrane domain is CD8αTM, and the amino acid sequence is shown in SEQ ID NO: 2;

[0094] IYIWAPLAGTCGVLLLSLVITLYC.

[0095] The amino acid sequence of 4-1BB is shown in SEQ ID NO:3;

[0096] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0097] The amino acid sequence of the CFR64 molecule, SEQ ID NO:4:

[0098] MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFHGGGSSGGGSGGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFEIYIWAPLAGTCGVLLLSLVITLYCTRTSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0099] The nucleotide sequence of the CFR64 molecule, SEQ ID NO:5:

[0100]

[0101] Example 2

[0102] In this example, the CFR64 molecule was site-specifically knocked into the TRAC site of the hESC cell line H1 in vitro, and the CAR-positive H1 cells were sorted and amplified to obtain the H1-CFR64 cell line. The expression of the CFR64 molecule in the H1-TRAC-CFR64 cell line was detected by flow cytometry. The specific steps include the following:

[0103] (1) Preparation of RNP complexes required for electroporation transfection

[0104] The RNP complex consists of a sgRNA targeting the TRAC site and Cas9 protein. Mix 0.1 nmol of sgRNA and 1 μg of Cas9 protein thoroughly, then incubate at 37°C for 40 minutes. After incubation, add 4 μg of the pUC19-TRAC HDR-CFR64 homologous recombination vector, mix thoroughly, and then add to 100 μL of electroporation buffer.

[0105] sgRNA sequence, SEQ ID NO:8:AGAGTCTCTCAGCTGGTACA.

[0106] (2) Electroporation transfection of pUC19-TRAC HDR-CFR64 and RNP complex

[0107] After thoroughly mixing 1 million H1 cells with 100 μL of electroporation buffer containing the pUC19-TRAC HDR-CFR64 and RNP complex, the cell suspension was gently transferred to an electroporation cuvette and electroporated using program B-016. After the program concluded, the cell suspension was removed and transferred to a six-well plate lined with Matrigel. The cells were then covered with 2 mL of mTesR1 medium supplemented with 10 μM thiazovivin to recover. After 12 hours, the medium was replaced with 1 μM thiazovivin and continued to culture and subculture. Five days after electroporation, CD64 expression was assessed by flow cytometry. The cell genome was extracted and PCR amplified using designed primers to confirm the targeted knock-in of CFR64 at the TRAC locus. CD64-positive H1 cells were sorted, expanded through multiple rounds of subculture, and cryopreserved.

[0108] PCR amplification test results are shown in Figure 2 Left, from Figure 2 As can be seen on the left, compared with the untreated H1 cell line, the expected size of the inserted gene band can be amplified.

[0109] PCR primer in the F direction: CCCAGTCACGACGTTGTAAAACGACG, SEQ ID NO: 9.

[0110] PCR R direction primer: TCTAGAGTCTAACAAAAAAGCCAAAAACGGCCA, SEQ ID NO: 10.

[0111] Flow cytometry results are shown in Figure 2 Right, from Figure 2 As can be seen on the right, compared with the isotype control, the sorted H1-TRAC-CFR64 cells all expressed positive CD64 molecules.

[0112] Example 3

[0113] In this example, the H1-TRAC-CFR64 cell line was induced to differentiate into CFR64-T cells in vitro. The differentiation induction process was as follows: Figure 3 As shown, the specific steps include:

[0114] H1-TRAC-CFR64 cells were digested with TrypLE, and 1 million H1-TRAC-CFR64 cells were placed in an AgreeWell well and 1 mL of mTesR1 medium containing 10 μM Thiazovivin was added. The AgreeWell plate was placed in a centrifuge and centrifuged at 100 g for 3 minutes to sediment the cells. The cells were then placed in a 37°C incubator for 24 hours to form embryoid bodies (EBs).

[0115] After 24 hours, EBs were gently lifted up with a 1 mL pipette tip and passed through a 40 μm mesh to remove the culture medium. EBs on the mesh were then rinsed with 1 mL of mTesR1 medium containing 10 μM Thiazovivin, 50 ng / mL VEGF, and 4 ng / mL BMP4 and evenly seeded into a six-well plate.

[0116] After 48 hours, the culture medium was replaced with StemLine II medium containing 50 ng / mL VEGF and 6 μM SB431542.

[0117] After 48 hours, the culture medium was replaced with StemLine II medium containing only 50 ng / mL VEGF. + CD43 - Differentiation of hematopoietic endothelial cells (HE).

[0118] CD34 positive HE cells were isolated using CD34 positive selection magnetic beads and seeded into six-well plates pre-plated with OP9-DL4 stromal cells. αMEM medium containing 50 ng / mL SCF, 20 ng / mL TPO, 20 ng / mL FLT3-L, 20 ng / mL IL-3, 20 ng / mL IL-6 and other cytokines was added to differentiate for 10 days to induce CD34 HE cells. + CD45 + Hematopoietic Progenitor Cells (HPCs).

[0119] 1.2 million HPCs were seeded into a six-well plate of OP9-DL1 / 4 stromal cells for co-culture, and then αMEM medium containing 20 ng / mL IL-7 and 10 ng / mL FLT3-L cytokines was added for differentiation for 35-42 days to induce their differentiation into progenitor T cells ( Figure 4 ), and the cells were transferred to new OP9-DL4 six-well plates every week. Flow cytometry showed that this T cell differentiation system can efficiently induce CD4 T cells in the H1-TRAC-CFR64 cell line. + CD8 + Double positive T cells ( Figure 4 ).

[0120] The double-positive T cells were stimulated with trastuzumab to transform into mature CD8 single-positive T cells, and mature CD8 iCFR64-T cells were obtained. Flow cytometry confirmed that they had a molecular phenotype similar to that of mature CD8 T cells in human peripheral blood ( Figure 4 ).

[0121] Example 4

[0122] Verify the anti-tumor effectiveness of iTRAC-CFR64-T cells

[0123] After iTRAC-CFR64-T cells were co-cultured with various tumor cell lines in a 96-well plate at the corresponding effector-target ratio for 24 hours, the luminescence value of each killing well was detected in a microplate reader through a luciferase-luciferase substrate reaction. The fewer the surviving tumor cells, the smaller the fluorescence signal. The killing efficiency can be calculated by comparing the fluorescence value with that of the control well containing only the corresponding tumor cells.

[0124] Figure 5-Figure 7Schematic diagram demonstrating the anti-tumor efficacy of iTRAC-CFR64-T cells. Under the guidance of specific monoclonal antibodies targeting HER2 (trastuzumab) and claudin18.2 (zolbetuximab), iTRAC-CFR64-T cells were able to effectively kill the SKOV3 HER2-positive tumor cell line in vitro, as well as claudin18.2-overexpressing Hela and MKN45 cell lines. Compared with the iTRAC-CFR64-T cell control group supplemented with IgG, iTRAC-CFR64-T cells demonstrated superior tumor-killing ability.

[0125] In summary, the present invention provides a method for overexpressing CFR64 molecules in the hESC cell line H1 through CRISPR / Cas9 technology, and efficiently inducing the differentiation of the H1-TRAC-CFR64 cell line into T cells, as well as the obtained universal CFR64-T cells. The universal CFR64-T cells, when used in combination with monoclonal antibody drugs, have stronger anti-tumor heterogeneity and antigen escape capabilities. At the same time, by targeted knock-in of the CFR64 molecule through the TRAC gene site, the universal CFR64-T cells have a lower risk of immune rejection and tumorigenicity. The universal CFR64-T cells are expected to solve the problems of the single source and versatility of traditional CAR-T or CFR64-T cells, and have important application value in tumor treatment.

[0126] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A generic off-the-shelf CFR64-T cell prepared based on CRISPR / Cas9, characterized in that: The CFR64-T cells are obtained by inducing differentiation of recombinant human embryonic stem cells; the coding sequence of the CFR64 molecule is site-specifically knocked into the TRAC site of the recombinant human embryonic stem cells using the CRISPR / Cas9 editing system; The CFR64 molecule includes: an antigen binding domain, a transmembrane domain and an intracellular co-stimulatory signaling domain; The antigen binding domain is the extracellular segment of a human Fcγ receptor, and the amino acid sequence thereof includes that shown in SEQ ID NO:

1.

2. The off-the-shelf universal CFR64-T cell prepared based on CRISPR / Cas9 according to claim 1, characterized in that The transmembrane domain is CD8αTM, and the amino acid sequence includes that shown in SEQ ID NO: 2; Preferably, the intracellular costimulatory signaling domain is 4-1BB, and the amino acid sequence includes that shown in SEQ ID NO: 3; Preferably, the CFR64 molecule comprises CD64ECD, hinge sequence, CH3 Spacer, CD8αTM, 4-1BB and CD3ζ connected in sequence; Preferably, the amino acid sequence of the CFR64 molecule comprises SEQ ID NO: 4; Preferably, the human embryonic stem cells are derived from the hESC cell line H1; Preferably, the CRISPR / Cas9 editing system comprises sgRNA, Cas9 and homologous recombination fragments; Preferably, the homologous recombination fragment comprises a homologous arm to the left of the TRAC site, a nucleotide sequence encoding a CFR64 molecule, and a homologous arm to the right of the TRAC site; Preferably, the nucleotide sequence encoding the CFR64 molecule includes SEQ ID NO: 5; Preferably, the nucleotide sequence of the homology arm on the left side of the TRAC site includes SEQ ID NO: 6; Preferably, the nucleotide sequence of the homology arm on the right side of the TRAC site includes SEQ ID NO: 7; Preferably, the site targeted by the sgRNA is the first exon of the TRAC site of human embryonic stem cells; Preferably, the DNA sequence corresponding to the sgRNA includes that shown in SEQ ID NO:

8.

3. A method for preparing off-the-shelf universal CFR64-T cells using CRISPR / Cas9, characterized in that: The method comprises: using the CRISPR / Cas9 editing system to transduce the coding sequence of the CFR64 molecule into human embryonic stem cells; sorting and amplifying the CFR64 molecule-positive human embryonic stem cells, and then inducing differentiation into CFR64-T cells.

4. The method for preparing off-the-shelf universal CFR64-T cells using CRISPR / Cas9 according to claim 3, characterized in that: The steps of sorting and amplifying include: After gene editing, human embryonic stem cells are cultured in stem cell culture medium for 1-2 weeks. CFR64-positive human embryonic stem cells are sorted and purified by flow cytometry, and the purified embryonic stem cells are then passaged and expanded in vitro. Preferably, the step of inducing differentiation comprises: inducing CFR64 molecule-positive human embryonic stem cells to differentiate into hematopoietic precursor cells; and then inducing the hematopoietic precursor cells to differentiate into T cells.

5. The method for preparing off-the-shelf universal CFR64-T cells using CRISPR / Cas9 according to claim 3 or 4, characterized in that: The steps of inducing differentiation of CFR64-positive human embryonic stem cells include: forming embryoid bodies from CFR64-positive human embryonic stem cells by centrifugation; inoculating the embryoid bodies into a medium without matrix cells and serum for culture, and adding cytokines VEGF, BMP4 and SB431542 to induce them to differentiate into CD34 + CD43 - Hematopoietic endothelial cell differentiation; cytokines SCF, TPO, FLT3-L, IL-3 and IL-6 were added to induce them to become CD34 + CD45 + hematopoietic precursor cells; Preferably, the culture medium comprises: mTesR1 medium or StemLine II medium; Preferably, the concentration of VEGF in the culture is 1-50 ng / mL; Preferably, the concentration of BMP4 in the culture is 1-10 ng / mL; Preferably, the concentration of SB431542 in the culture is 1-10 μM; Preferably, the concentration of the cytokine SCF, TPO, FLT3-L, IL-3 or IL-6 in the culture is independently 1-50 ng / mL; Preferably, the temperature of the embryoid body culture is 35-42°C; Preferably, the embryoid body culture time is 6-12 days; Preferably, the step of inducing differentiation of hematopoietic precursor cells comprises: inoculating the hematopoietic precursor cells onto OP9-DL1 / 4 stromal cells for culture, and adding cytokines IL-7 and FLT3-L required for T cell differentiation to induce the hematopoietic precursor cells to differentiate into T cells; Preferably, the concentrations of the cytokines IL-7 and FLT3-L in the culture are independently 1-50 ng / mL; Preferably, the culture temperature during the differentiation induction process is 35-42°C; Preferably, the culture time of the differentiation induction process is 35-42 days.

6. A CRISPR / Cas9 editing system, characterized in that The CRISPR / Cas9 editing system includes sgRNA, Cas9 and homologous recombination fragments; Preferably, the homologous recombination fragment comprises a homologous arm to the left of the TRAC site, a nucleotide sequence encoding a CFR64 molecule, and a homologous arm to the right of the TRAC site; Preferably, the nucleotide sequence encoding the CFR64 molecule includes SEQ ID NO: 5; Preferably, the nucleotide sequence of the homology arm on the left side of the TRAC site includes SEQ ID NO: 6; Preferably, the nucleotide sequence of the homology arm on the right side of the TRAC site includes SEQ ID NO: 7; Preferably, the site targeted by the sgRNA is the first exon of the TRAC site of human embryonic stem cells; the DNA sequence corresponding to the sgRNA includes that shown in SEQ ID NO:

8.

7. A recombinant human embryonic stem cell, characterized in that: The coding sequence of the CFR64 molecule is site-specifically knocked into the recombinant human embryonic stem cells at the TRAC site of the human embryonic stem cells using the CRISPR / Cas9 editing system; the amino acid sequence of the CFR64 molecule includes that shown in SEQ ID NO:

4.

8. A recombinant CD34 + CD43 - Hematopoietic endothelial cells, characterized in that The cells are obtained by inducing differentiation of the recombinant human embryonic stem cells according to claim 7.

9. A recombinant CD34 + CD45 + Hematopoietic precursor cells, characterized in that The cells are composed of the recombinant CD34 + CD43 - Hematopoietic endothelial cells were induced to differentiate.

10. The off-the-shelf universal CFR64-T cells prepared based on CRISPR / Cas9 according to claim 1 or 2, the recombinant human embryonic stem cells according to claim 7, the recombinant CD34 + CD43 - Hematopoietic endothelial cells or the recombinant CD34 according to claim 9 + CD45 + The application of any one or a combination of at least two of the hematopoietic precursor cells in the preparation of tumor therapeutic drugs.

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