A method for preparing low chromosomal abnormality TCR-T cells based on CRISPR-mediated site-specific integration of TRAC locus and methotrexate screening

Abstract

The application discloses a low chromosomal abnormality TCR-T cell preparation method based on CRISPR-mediated TRAC locus site-directed integration and methotrexate screening, and belongs to the field of genetic engineering and immune cell therapy. The method comprises the following steps: delivering a dsDNA homologous template and an RNP complex into T cells through electroporation to perform homology-directed repair, to obtain electroporated T cells, wherein the RNP complex comprises a Cas9 protein and a gRNA, the gRNA targets a TRAC gene, and the homologous template comprises a target TCR gene and a mutant DHFR gene; and culturing the electroporated T cells, and using methotrexate enrichment screening during cell culture to obtain screened TCR-T cells. Through optimization of homologous template design and HDR repair regulation strategies, the site-directed integration of TCR-T cell phenotype uniformity and functional persistence is realized, the TCR expression is more stable, the exhaustion marker gene is down-regulated, TCR signal enrichment is realized, and more excellent anti-tumor potential is exhibited, the DHFR-FS / methotrexate enrichment strategy is innovatively introduced, TCR-T cells are effectively enriched, and guidance is provided for efficient preparation and production of TCR-T cells.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a method for preparing low-chromosome-abnormal TCR-T cells based on CRISPR-mediated TRAC locus-directed integration and methotrexate screening. Background Technology

[0002] Adoptive T-cell therapy has become an important strategy in the field of cancer treatment in recent years. Among them, chimeric antigen receptor T-cell (CAR-T) and T-cell receptor-engineered T-cell (TCR-T) therapies have shown significant efficacy in hematologic malignancies and some solid tumors. TCR-T therapy can overcome the inherent limitation of CAR-T therapy, which relies on tumor surface antigens, by recognizing intracellular antigenic epitopes presented by the major histocompatibility complex (MHC), showing unique advantages in targeting certain tumors without obvious surface targets. However, current methods for preparing TCR-T cells mainly rely on transduction with lentiviral (LV) or retroviral vectors. The random gene insertion mediated by these viruses poses several potential risks, including insertional mutations, interference with endogenous gene function, and even induction of clonal expansion and potential tumor formation. In fact, the U.S. Food and Drug Administration (FDA) recently issued a safety warning regarding secondary T-cell lymphoma following CAR-T therapy, and clinical studies have reported events where viral insertion in CAR-T cells led to the inactivation of tumor suppressor genes such as TET2 and TP53. In addition, the GMP-level production process of viral vectors is complex and costly, which severely restricts their large-scale clinical application.

[0003] With the development of CRISPR / Cas9 gene editing technology, T-cell engineering has entered a new stage of precision and controllability. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-mediated precise integration of exogenous TCR sequences into the TCRα constant region (TRAC) site not only achieves uniform and stable expression but also simultaneously knocks out endogenous TCRs, avoiding mismatches. Previous studies have shown that targeting the integration of CAR structures into the TRAC site can improve the persistence and anti-tumor ability of CAR-T cells, and this strategy provides important insights for TCR-T cell engineering. However, emerging evidence shows that CRISPR / Cas9-mediated double-strand breaks at the TRAC site may trigger mitotic abnormalities during T-cell proliferation, inducing chromosomal instability such as chromosome 14 loss. Since this site is located on chromosome 14, such chromosomal loss is likely due to DNA damage repair defects, seriously affecting the safety of gene-edited products. Therefore, CRISPR-based T-cell engineering currently faces two major challenges: firstly, the knock-in efficiency of large-sized TCR genes is relatively low; secondly, the increase in genomic instability events such as chromosome loss limits its clinical translation.

[0004] To address the aforementioned issues, this patent proposes a dual optimization strategy: firstly, it systematically optimizes CRISPR RNP (ribonucleoprotein) electroporation conditions to enhance the knock-in efficiency of the TRAC site; secondly, it introduces a methotrexate (MTX) selective drug screening mechanism, selectively enriching cells that have successfully integrated the TCR by co-expressing the DHFR-L22F / F31S resistant mutant, while simultaneously eliminating unedited cells and abnormal clones with chromosome 14 loss. Methotrexate, an FDA-approved antifolate drug, has had its safety verified clinically. This patent, through comprehensive functional verification using in vitro killing assays, cytokine detection, and in vivo xenograft models, demonstrates that this strategy not only significantly improves editing efficiency but also yields TRAC-DHFR-TCR-T cells whose anti-tumor function is comparable to that of traditional viral-based TCR-T cells. This patent provides a transferable solution to the safety and efficiency issues in T-cell therapy, advancing the clinical application of TCR-T. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for preparing low-chromosome-abnormal TCR-T cells based on CRISPR-mediated TRAC locus-directed integration and methotrexate screening, in order to solve the problems in the prior art.

[0006] To achieve the above and other related objectives, the first aspect of the present invention provides a method for preparing TCR-T cells mediated by the CRISPR / Cas9 system, comprising the following steps:

[0007] a) The dsDNA homologous template and the RNP complex are delivered into T cells via electroporation for homologous targeted repair to obtain electroporated T cells. The RNP complex contains Cas9 protein and gRNA, the gRNA targets the TRAC gene, and the homologous template contains the target TCR gene and the mutant DHFR gene.

[0008] b) Culture electroporated T cells and use methotrexate for enrichment and screening during cell culture to obtain screened TCR-T cells.

[0009] A second aspect of the present invention provides a method for reducing the level of chromosome loss in CRISPR / Cas9 system-mediated TCR-T cell products, wherein the TCR-T cell products are prepared using the above-described TCR-T cell preparation method, so that the level of chromosome 14 loss in the TCR-T cell products is not significantly different from the level of chromosome 14 loss in unedited T cells.

[0010] A third aspect of the present invention provides a TCR-T cell prepared using the above method.

[0011] The fourth aspect of this invention provides the application of the above-mentioned TCR-T cells in the preparation of antitumor drugs.

[0012] As described above, the method for preparing low-chromosome-abnormal TCR-T cells based on CRISPR-mediated TRAC locus-directed integration and methotrexate screening of the present invention has the following beneficial effects:

[0013] This study focuses on the systematic optimization of CRISPR / Cas9 non-viral site-directed integration technology. The electroporation gene editing process, homologous template design, and HDR repair regulation strategy were systematically optimized to improve the efficiency of CRISPR / Cas9 non-viral gene editing, achieving efficient and safe TCR-T cell construction. By analyzing the phenotypic uniformity and functional durability of site-directed integration TCR-T cells, it was found that their TCR expression is more stable, exhaustion marker genes are downregulated, and TCR signaling is enriched, exhibiting superior anti-tumor potential. Simultaneously, this study innovatively introduced a DHFR-FS / methotrexate enrichment strategy. Methotrexate screening addressed the risk of chromosome loss derived from gene editing, further improving gene editing efficiency and effectively enriching TCR-T cells. In vivo and in vitro experiments validated the complete effector function and good safety of TCR-T cells, providing a safer, more efficient, and more economical treatment option for cancer patients, laying the foundation for clinical translation, and demonstrating strong innovation. Attached Figure Description

[0014] Figure 1 This diagram illustrates the EGFP knock-in strategy for the TRAC gene locus.

[0015] Figure 2 The diagram shows the optimal time window for electroporation after T cell activation; A, schematic diagram of electroporation operation; BC, analysis of GFP positivity rate (B) and number of GFP-positive cells (C) on day 4 after electroporation with pMAX-EGFP plasmid at different time points; DE, analysis of GFP positivity rate (D) and number of GFP-positive cells (E) on day 4 after electroporation with EGFP homology-directed repair template (HDRT) targeting the TRAC gene site at different time points; HDRT stands for homology-directed repair template.

[0016] Figure 3 The graph shows the optimal gene knock-in efficiency achieved by combining B1 mix buffer with the EO138 electroporation program; A, a representative flow cytometry plot of EGFP targeted insertion; B, EGFP targeted insertion efficiency and total number of gene knock-in (KI) cells.

[0017] Figure 4 This diagram shows the CMVpp65-A*02:01-TCR gene box knock-in strategy for the TRAC gene locus.

[0018] Figure 5 The diagram shows the effect of homologous template quality and truncated Cas9 target sequence on TCR gene knock-in efficiency; A, a representative flow cytometry (FACS) plot of TCR targeted insertion using CMV pp65-A*02:01 for different electroporation parameters (homologous template quality and tCTS / conventional homologous arm); BC. Analysis of cell knock-in (KI) efficiency (B) and KI cell number (C) on day 4 after electroporation using specified homologous template quality; Reg.HA: regular homologous arm.

[0019] Figure 6 The image shows the effect of HDR enhancer v2 on TCR gene knock-in efficiency; A, representative flow cytometry images of CMV pp65-A*02:01-TCR targeted insertion at different concentrations of HDR enhancer v2; BC, analysis of cell knock-in (KI) efficiency (B) and KI cell number (C) on day 4 after electroporation following treatment with different concentrations of HDR enhancer v2 for 24 hours.

[0020] Figure 7The diagram shows a comparison of the in vitro cell-killing capabilities of LV-TCR-T and TRAC-TCR-T cells; A, a schematic diagram of the T cell and target cell co-culture system; B, the in vitro killing efficiency (Effective-to-target ratio: E:T ratio) of K562-A*02:01-pp65 cells as determined by flow cytometry.

[0021] Figure 8 The analysis shows the activation markers of LV-TCR-T and TRAC-TCR-T cells; A, C, representative flow cytometry plots of CD69(A) and CD137(C) expression; B, D, positive rate analysis of CD69(B) and CD137(D) expression in T cells after co-culturing with K562-A*02:01-pp65 cells for 24 hours (using CD8+ and TCR+ cells as the analysis gate).

[0022] Figure 9 The analysis shows the cytokine secretion of LV-TCR-T and TRAC-TCR-T cells; the positive rate of granzyme B (GZMB) expression in CD8+ and TCR+ (mTCR+) T cells after co-culturing with K562-A*02:01-pp65 cells for 16 hours; the positive rate of CD and IFN-γ expression; and the positive rate of EF and TNF-α expression.

[0023] Figure 10 The image shows the deletion of chromosome 14 in CRISPR / Cas9-induced primary T cells; A, a schematic diagram of detection using commercial fluorescence in situ hybridization (FISH) probes, with red probes binding to the proximal chromosomal region of the centriole and green probes binding to the distal chromosomal region of the centriole; B, FISH analysis results of untreated (control), lentiviral engineered (LV-TCR-T), and CRISPR engineered (TRAC-TCR-T) cells, with images showing typical signal patterns detected in each group of cells and the frequency of each signal pattern marked below; # p<0.05 ## p<0.01 (Fisher exact test, comparing the total frequency of missing signals); * P<0.05 (Fisher exact test, comparing the frequencies of the two signals); Sample size: control group n=299, LV-TCR-T group n=300, TRAC-TCR-T group n=297.

[0024] Figure 11The transcriptome shows the differential analysis between LV-TCR-T and TRAC-TCR-T cells; A. Gene expression heatmap of CD8+mTCR+ T cell populations in TRAC-TCR-T and LV-TCR-T cells; B. Pathway enrichment analysis of differentially expressed genes (DEGs).

[0025] Figure 12 The knock-in strategy is shown as CMVpp65-A02:01-DHFR-TCR gene cassette.

[0026] Figure 13 The diagram shows the efficient enrichment of TRAC-DHFR-TCR-T cells with methotrexate; A, the experimental flowchart for optimizing MTX enrichment conditions; B, the concentration gradient screening of MTX in primary human T cells (n=3); C, representative flow cytometry plots of TRAC-DHFR-TCR-T cells in the 100 nM MTX-treated and untreated groups; D, the analysis of the change in the positive rate of modified TCR+ (mTCR+) population in CD8+ cells over time after 100 nM MTX treatment (n=6).

[0027] Figure 14 The results show that MTX enrichment significantly reduces chromosome loss; A, Fluorescence in situ hybridization (FISH) analysis was used to detect untreated cells (control group), TRAC-TCR-T cells, and MTX-treated TRAC-DHFR-TCR-T cells; The figure shows examples of all signal patterns detected in each group of cells, with the frequency of each signal pattern marked below. #p<0.05, ##p<0.01 (Fisher's exact test, comparing the frequency of distal signal deletion); *P<0.05 (Fisher's exact test, comparing the frequency of simultaneous deletion of two signals or only distal signal deletion); Sample size: control group n=208, TRAC-DHFR-TCR-T group n=203, TRAC-DHFR-TCR-T group n=213; B, Chromosome deletion rate analysis of 3 independent donors in each group.

[0028] Figure 15 The image shows TRAC-TCR-T cells achieving tumor clearance in vivo; A, a schematic diagram of a metastatic K562-A*02:01-pp65-luciferase xenograft model; B, bioluminescence imaging of treated mice over time (color-coded radiation intensity, unit: p / sec / cm). 2 / sr); CF, Quantitative analysis of tumor burden: photon number intensity (C, D, E) and mean photon number intensity (F); G, Kaplan-Meier analysis of mouse survival. Detailed Implementation

[0029] This invention provides a method for preparing low-chromosomal-abnormality TCR-T cells based on CRISPR-mediated TRAC locus-directed integration and methotrexate selection. Utilizing the pUC57 mini-HDR vector system, this invention introduces a TCR expression cassette and the L22F / F31S double-mutant DHFR into TCR-T cells via site-directed integration technology. The introduction of the L22F / F31S double-mutant DHFR confers methotrexate (MTX) resistance to the edited cells, achieving efficient enrichment of CRISPR-edited cells. Methotrexate treatment can increase the editing efficiency from 13% to 70% while reducing the chromosomal abnormality rate. MTX selectively eliminates chromosomally abnormal cells by inhibiting endogenous DHFR activity, reducing the proportion of T cells lost due to chromosome 14 abnormalities. In vivo and in vitro experiments have verified the complete effector function and good safety of TCR-T cells, laying the foundation for subsequent clinical translation.

[0030] TCRs (T cell receptors) are heterodimeric cell surface proteins of the immunoglobulin superfamily, associated with invariant proteins of the CD3 complex involved in mediating signal transduction. TCRs exist in αβ and γδ forms, which are structurally similar but have distinctly different structural locations and potentially vastly different functions. The α and β chains of the native heterodimeric αβTCR are transmembrane proteins, each containing two extracellular domains: a near-membrane constant domain and a distal-membrane variable domain. Each constant and variable domain includes an intrachain disulfide bond. The variable domain contains a highly polymorphic loop similar to the complementarity-determining region (CDR) of an antibody.

[0031] The CRISPR system is used to integrate TCRs into selected loci in a cell's genome. In some implementations, the CRISPR system uses a DNA donor template to guide homologous directed repair at a specific genetic locus (e.g., the TRAC locus). The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system is a genome editing tool found in prokaryotic cells. When used for genome editing, this system includes Cas9 (a protein capable of modifying DNA using crRNA as a guide), CRISPR RNA (crRNA, containing the RNA used by Cas9 to guide it to the correct portion of the host DNA and a region that binds tracrRNA (usually in the form of a hairpin loop) to form an active complex with Cas9), trans-activating crRNA (tracrRNA, which binds crRNA and forms an active complex with Cas9), and an optional DNA repair template portion (DNA that guides the cellular repair process that allows the insertion of a specific DNA sequence). CRISPR / Cas9 is often transfected into target cells using plasmids. In some implementations, CRISPR / Cas9 is a recombinant ribonucleoprotein complex transfected into target cells.

[0032] Ribonucleoprotein (RNP) is a complex formed by the binding of Cas9 protein and guide RNA (gRNA). Compared with other delivery methods (such as plasmids or viral vectors), RNP has advantages such as rapid action, reduced off-target effects, and avoidance of genome integration.

[0033] Methotrexate (MTX), as a dihydrofolate reductase (DHFR) inhibitor, can effectively eliminate abnormal T cell clones caused by chromosome loss through selective pressure enrichment, while retaining cells with normal chromosome copies, thereby improving the genomic stability of the final product.

[0034] The donor template refers to a transgenic cassette or gene-editing sequence with homologous regions on both sides for recombination with the host locus, and which replaces the DNA mutation with the correct sequence via HDR / SSTR. The donor template can be ssDNA, dsDNA, or a plasmid / vector, and can be covalently chemically conjugated to guide RNA or Cas protein.

[0035] Guide RNA and gRNA are used interchangeably in this article. For example, for Cas9, gRNA typically consists of crRNA and tracrRNA molecules that partially complement each other to form a complex, wherein the crRNA contains a sequence that is sufficiently complementary to the target sequence to hybridize with the target sequence and guide the CRISPR complex (Cas9+crRNA+tracrRNA) to bind specifically to the target sequence.

[0036] This invention first provides a method for preparing TCR-T cells mediated by the CRISPR / Cas9 system, comprising the following steps:

[0037] a) The dsDNA homologous template and the RNP complex are delivered into T cells via electroporation for homologous targeted repair to obtain electroporated T cells. The RNP complex contains Cas9 protein and gRNA, the gRNA targets the TRAC gene, and the homologous template contains the target TCR gene and the mutant DHFR gene.

[0038] b) Culture electroporated T cells and use methotrexate for enrichment and screening during cell culture to obtain screened TCR-T cells.

[0039] In this application, the dsDNA homologous template is complexed with the RNP complex to form an RNP-homologous template complex, which is then delivered into T cells via electroporation.

[0040] In some embodiments, the gRNA targets the first exon of the TRAC gene. Preferably, the gRNA sequence is as shown in SEQ ID NO.1; SEQ ID NO.1: 5'-CAGGGTTCTGGATATCTGT-3'.

[0041] Furthermore, the gRNA includes one or more modifications selected from 3'-thiophosphate, 2'-O-methyl ester, 2'-O-methyl, 2'-ribose 3'-thiophosphate, deoxy, and / or 5'-phosphate modifications. Further, the modification is selected from 3'-thiophosphate and 2'-O-methyl modifications. Even further, the above gRNA is chemically modified with 2'-O-methyl phosphorothioate, which significantly improves its stability by enhancing its resistance to nuclease degradation, optimizes gene editing efficiency, increases cleavage efficiency, and reduces off-target effects.

[0042] In some implementations, the dsDNA homologous template and RNP complex are delivered into T cells via electroporation for homologous targeted repair. After transfection, the RNP complex first cleaves the first exon of the TRAC gene in the genomic DNA, creating a broken double-stranded DNA nick. At this point, the dsDNA homologous template, acting as donor DNA, undergoes homologous recombination with the broken nick DNA, and the DNA successfully integrates into the target genomic TRAC gene site. In a preferred embodiment, this application inserts the dsDNA homologous template into the first exon of the TRAC gene, driving expression via an endogenous promoter.

[0043] In a preferred embodiment, the N-terminus and C-terminus of the Cas9 protein are respectively fused with SV40 nuclear localization signals. Preferably, the Cas9 protein is derived from a commercially available product.

[0044] In a preferred embodiment, the mutant DHFR (dihydrofolate reductase) gene described above is selected from the DHFR gene with a double missense mutation at L22F and F31S, namely DHFR-FS. DHFR-FS is a methotrexate resistance gene, containing a double missense mutation at L22F and F31S to further enhance resistance to methotrexate. Specifically, the amino acid sequence of the mutant DHFR is shown in SEQ ID NO.2, and its corresponding nucleotide sequence is shown in SEQ ID NO.10, specifically as follows:

[0045] SEQ ID NO.2:

[0046] MVGSLNCIVAVSQNMGIGKNGDFPWPPLRNESRYFQRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND*

[0047] SEQ ID NO.10:

[0048] ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAAAGTAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA。

[0049] In this application, the dsDNA homologous template sequentially includes a left homologous arm, an exogenous target TCR gene, a mutant DHFR gene, and a right homologous arm from the 5' end to the 3' end.

[0050] In one embodiment, the left and right homologous arms enable the exogenous target TCR gene to be integrated into the TRAC locus via homology-directed repair (HDR). The lengths of the left and right homologous arms (LHR) can be the same or different. In some embodiments, the sequence lengths of the left and right homologous arms are ≥10bp, ≥20bp, ≥30bp, ≥40bp, ≥50bp, ≥60bp, ≥70bp, ≥80bp, ≥90bp, ≥100bp, ≥150bp, ≥200bp, ≥250bp, ≥300bp, ≥400bp, ≥500bp, ≥1kb, ≥2kb, or ≥3kb. In some embodiments, the homologous arm sequence length is ≤20bp, ≤30bp, ≤40bp, ≤50bp, ≤60bp, ≤70bp, ≤80bp, ≤90bp, ≤100bp, ≤150bp, ≤200bp, ≤250bp, ≤300bp, ≤400bp, ≤500bp, ≤1kb, ≤2kb, or ≤3kb. In some embodiments, the left and right homologous arms each have a fragment length of 10 to 2000bp, preferably 300bp, 600bp, or 1000bp, and more preferably 300bp. In one specific embodiment, the nucleotide sequence of the left homologous arm is shown in SEQ ID NO.3, and the nucleotide sequence of the right homologous arm is shown in SEQ ID NO.4.

[0051] SEQ ID NO.3:CCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAG TTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTG CCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCTTACAGATGGTTCCGGA.

[0052] SEQ ID NO.4: ATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAA TCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAG AAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCA.

[0053] In this application, a truncated Cas9 target sequence is attached to the 5' end of the left homologous arm and / or the 3' end of the right homologous arm. The truncated Cas9 target sequence can promote the transfer of dsDNA homologous templates to the cell nucleus, thereby improving gene editing efficiency.

[0054] Specifically, the truncated Cas9 target sequence (tCTS) is selected from a 15bp truncated Cas9 target sequence near the PAM region of the gRNA, which improves transfection efficiency by binding to the Cas9 protein containing the nuclear localization signal. The nucleotide sequence of the truncated Cas9 target sequence is shown in SEQ ID NO.5, specifically: GTTCTGGATATCTGTGGG.

[0055] Preferably, the exogenous target TCR gene and the mutant DHFR are separated by a cleavage peptide. The cleavage peptide can be P2A or T2A, etc. In some embodiments, the cleavage peptide is P2A, whose nucleotide sequence is shown in SEQ ID NO:6. In some embodiments, the cleavage peptide is T2A, whose nucleotide sequence is shown in SEQ ID NO:7.

[0056] SEQ ID NO:6:

[0057] GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCC CC.

[0058] SEQ ID NO:7:

[0059] GAGGGCAGAGGCAGTCTGCTGACATGCGGTGACGTGGAAGAGAATCCCGGCCCT.

[0060] In another preferred embodiment of this application, the dsNDA homologous template further includes a polyA terminator. Preferably, the polyA can be any polyA gene, preferably bGHpApolyA. In some embodiments, the nucleotide sequence of bGHpApolyA is shown in SEQ ID NO:8:

[0061] GGGGATCAATTCTCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCT GAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGACTAGAGCTTGCGGAACCCTT.

[0062] Furthermore, the exogenous target TCR gene is composed of an α-chain variable domain and a β-chain variable domain linked by a peptide linker sequence. Even further, the length of the aforementioned exogenous target TCR gene sequence can be 100bp-20kb, 100bp-500bp, 500bp-1kb, 1kb-5kb, 5kb-10kb, 10kb-15kb, or 15kb-20kb.

[0063] Specifically, the TCR gene is selected from genes targeting the pp65 antigen. More specifically, the nucleotide sequence of the aforementioned gene targeting the pp65 antigen is shown in SEQ ID NO.9, and is as follows:

[0064]

[0065] In this invention, a non-viral vector is used as a targeting vector to deliver a site-specific integration dsDNA homologous template. This process is simpler than methods using lentiviruses, requiring only one electroporation cycle to complete all gene editing steps in the entire preparation process. The non-viral vector is designed and prepared as follows:

[0066] The template DNA is ligated into a plasmid vector: The template DNA includes a left homologous arm, the exogenous target TCR gene, the mutant DHFR, and a right homologous arm, wherein the left homologous arm is homologous to the 5' end sequence of the DNA nick, and the right homologous arm is homologous to the 3' end sequence of the DNA nick. In a preferred embodiment, the 3' end DNA sequence of the left homologous arm is consistent with the 5' end sequence 0 to 500 bases from the nick DNA, with a fragment length of 10 to 5000 bp; the 5' end DNA sequence of the right homologous arm is consistent with the 3' end sequence 0 to 500 bases from the nick DNA, with a fragment length of 100 to 5000 bp. After cloning the dsDNA homologous template, it is ligated into a plasmid vector. The plasmid vector can be any type, such as pUC57, pCDNA3.1, pCMV, etc., and the ligation position can be flexible. Alternatively, it can proceed directly to subsequent steps without ligation into a plasmid vector.

[0067] After PCR amplification, the modified template DNA can be purified and concentrated using the following methods, either individually or in combination, to obtain dsDNA homologous templates: purification using DNA purification kits from various reagent companies, purification using various DNA-binding magnetic beads, gel chromatography, ion chromatography, affinity chromatography, ultrafiltration tube ultrafiltration, dialysis membrane dialysis, etc.

[0068] In this application, electroporation is performed after T cell activation. Preferably, electroporation is performed 24–72 hours after T cell activation, more preferably 48 hours after activation. More preferably, optimal cell numbers are harvested after T cell activation to improve transfection efficiency.

[0069] In this application, 1.0 × 10 6 Based on the cell electroporation reaction system, the amount of the above-mentioned dsDNA homologous template is 1-3 μg, preferably 1-2 μg.

[0070] In one specific embodiment, the electroporation also requires the use of an electroporation buffer, which can be any buffer known in the art suitable for electroporation therein. The electroporation buffer is selected from B1 mix electroporation buffer, LONZA P3 electroporation buffer, and Celetrix electroporation buffer, preferably B1 mix electroporation buffer. All of the above electroporation buffers are commercially available.

[0071] In one specific embodiment, the electroporation selection optimization program EO115 or EO138 is used to perform the electroporation operation. More preferably, B1 mix electroporation buffer is used in conjunction with the EO138 program to perform the electroporation operation. The B1 mix electroporation buffer combined with the EO138 electroporation program exhibits higher gene knock-in efficiency, successfully integrating exogenous gene fragments into the TRAC site while maintaining high cell viability.

[0072] In this application, in step b), electroporated T cells are first cultured in a medium containing an HDR enhancer, and then the medium is replaced with one without the HDR enhancer. The HDR enhancer is used to improve the efficiency of HDR (homological recombination repair). Preferably, the HDR enhancer is selected from HDR enhancerv2, which, as an enhancer of the HDR pathway, effectively promotes homologous recombination. More preferably, based on the total volume of the medium containing the HDR enhancer, the concentration of the HDR small molecule enhancer is less than 2 μM, preferably 0.5 μM.

[0073] In one specific embodiment, the culture medium containing the HDR enhancer is cultured at 37°C and 5% CO2 for 24 hours before the HDR enhancer is removed to avoid the toxicity of the HDR enhancer due to long-term culture.

[0074] In a preferred embodiment, when using methotrexate for enrichment screening, the concentration of methotrexate (MTX) is below 1000 nM, preferably 50–1000 nM, based on the total volume of the culture medium. By adding methotrexate, selective pressure enrichment effectively eliminates abnormal T cell clones caused by chromosome loss, while retaining cells with normal chromosome copies.

[0075] The present invention also provides a method for reducing the level of chromosome loss in CRISPR / Cas9 system-mediated TCR-T cell products, wherein the TCR-T cell products are prepared using the above-described TCR-T cell preparation method, so that the level of chromosome 14 loss in the TCR-T cell products is not significantly different from the level of chromosome 14 loss in unedited T cells.

[0076] The present invention also provides a TCR-T cell prepared using the above method.

[0077] The present invention also provides the application of the above-mentioned TCR-T cells in the preparation of antitumor drugs.

[0078] In one embodiment, the tumor is a solid tumor or a hematologic tumor; preferably, the solid tumor is selected from: esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, and sarcoma; the hematologic tumor is selected from: leukemia, lymphoma, and myeloma.

[0079] In one embodiment, a pharmaceutical composition for treating tumors is provided, the pharmaceutical composition comprising the above-described TCR-T cells, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0080] In one embodiment, a method for inhibiting tumor cells in vitro is provided, comprising contacting tumor cells with the aforementioned TCR-T cells or the aforementioned pharmaceutical composition, thereby inhibiting the tumor cells.

[0081] This invention introduces the TCR gene and mutant DHFR into a plasmid vector using site-directed integration technology. By introducing mutant DHFR, the edited cells are conferred methotrexate (MTX) resistance, achieving efficient enrichment of CRISPR-edited cells. Methotrexate treatment can increase the editing efficiency from 13% to 70% while reducing the chromosomal abnormality rate. MTX selectively eliminates chromosomally abnormal cells by inhibiting endogenous DHFR activity, reducing the proportion of T cells lost due to abnormal chromosome 14. In vivo and in vitro experiments have verified the complete effector function and good safety of TCR-T cells, laying the foundation for subsequent clinical translation.

[0082] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0083] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0084] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0085] Example 1 - Plasmid Construction and Homologous Recombination Template Design

[0086] All exogenous gene sequences were optimized for human codons using the ExpOptimizer online codon optimization platform (Novoprotein). The homologous recombination template vector was constructed using the pUC57mini plasmid vector, with the following general structure: left homologous region (LHR) - gene trap - Furin-P2A - target gene - bGHpoly(A) - right homologous region (RHR). Wherein:

[0087] (1) Construction of electroporation plasmid pUC57mini-EGFP-HDR: Insertion of optimized enhanced green fluorescent protein (EGFP) sequence;

[0088] (2) Construction of pUC57mini-TCR-HDR: Insert the CMV-A*02:01-pp65-TCR gene cassette, the sequence of which is shown in SEQ ID NO.10 and is consistent with the elements used in the construction of the lentiviral vector;

[0089] (3) Construction of pUC57mini-TCR-DHFR-FS-HDR: A directed mutagenesis-induced dihydrofolate reductase (DHFR) mutant was introduced downstream of the CMV-A*02:01-pp65-TCR gene cassette. Its amino acid sequence is shown in SEQ ID NO.2, and the corresponding nucleotide sequence is shown in SEQ ID NO.10, separated by a P2A self-cleaving peptide. This mutant enhances methotrexate resistance through L22F, F31S double missense mutations. All modular elements were assembled into a linearized vector via blunt-end ligation.

[0090] SEQ ID NO.2:

[0091] MVGSLNCIVAVSQNMGIGKNGDFPWPPLRNESRYFQRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND*

[0092] SEQ ID NO.10:

[0093]

[0094] 1. Isolation of CD3+ T cells

[0095] In this embodiment, an appropriate amount of anti-human CD3 biotin antibody and streptavidin magnetic beads were added to a peripheral blood mononuclear cell suspension, and the target CD3+ T cells were sorted using a magnetic adsorption column. The obtained T cells were used for downstream experimental applications. The CD3+ T cell sorting experimental procedure is as follows:

[0096] ① Prepare the sorting buffer required for the experiment: phosphate-buffered saline (PBS) at pH 7.2, 2% fetal bovine serum (FBS) and 2mM EDTA. After preparation, place in a 4°C freezer for pre-cooling.

[0097] ② Wash the freshly isolated or cryopreserved samples with sorting buffer, then centrifuge at 800 rpm for 5 min, increasing speed 5 and decreasing speed 3.

[0098] ③ Gently discard the supernatant, then gently resuspend the cells and count them, and adjust the cell suspension density to 1E8 cells / mL using sorting buffer.

[0099] ④ Add 100 μL of CD3 biotin antibody to each 1 mL of cell suspension, mix well, and incubate at 4°C for 15 min. During this time, gently tap the bottom of the test tube to remix the cell suspension once.

[0100] ⑤ Wash the cells to be sorted with 5 ml of sorting buffer, centrifuge at 1000 rpm, gently discard the supernatant, and then resuspend the cells in 1 mL of sorting buffer for every 1E8 cells.

[0101] ⑥ Add 100uL of streptavidin magnetic beads to each 1mL of cell suspension, mix well, and incubate at 4℃ for 15min, gently tapping the bottom of the test tube once during the incubation period to remix the cell suspension.

[0102] ⑦ Wash the cells again, centrifuge at 1000 rpm and discard the supernatant, then resuspend in 1 mL of sorting buffer per 1E8 cells and separate by magnetic column.

[0103] ⑧ The magnetic column separation procedure is as follows: Place the sorting column on the magnetic sorting rack, rinse the sorting column with 2 ml of sorting buffer, and after the liquid has completely flowed out, add the cells from the previous step into the sorting column, collect the effluent, and then wash the sorting column with 3 ml of sorting buffer. Finally, remove the magnetic column from the magnetic field and place it in a new collection tube, add 2 ml of sorting buffer, and push the buffer down using the matching stopcock. The resulting cell suspension is CD3+ T cells.

[0104] 2. Culture of CD3+ T cells

[0105] Unless otherwise specified, primary CD3+ T cells should be cultured under the following conditions: Freshly isolated PBMCs are sorted with CD3 magnetic beads to obtain CD3+ T cells with a purity >95%, and seeded in X-VIVO 15 serum-free medium supplemented with 5% FBS and 1% penicillin-streptomycin dual antibiotic solution. During the culture activation phase (within 48 hours of activation), CD3 / CD28 Dynabeads are added at a cell:magnetic bead ratio of 1:1, and the culture medium is supplemented with human recombinant cytokines IL-2 200 IU / mL + IL-7 10 ng / mL + IL-15 10 ng / mL. The cells are then placed in suspension culture at 37°C in a 5% CO2 incubator, maintaining a cell density of 1-2 × 10⁻⁶ cells / mL. 6 cells / mL. During the T cell expansion phase (48 hours to endpoint), the activating magnetic beads were removed by magnetic adsorption at room temperature for 3 minutes. Subsequently, the IL-2 concentration gradient in the culture medium was increased to 300 IU / mL, and cell density was monitored daily, maintaining a cell concentration of 0.5-1.5 × 10⁻⁶ cells / mL through fluid replenishment. 6 cells / mL, until the observation endpoint.

[0106] Example 3 - Homologous Template Amplification and Reconstruction

[0107] Using plasmids pUC57mini-EGFP-HDR, pUC57mini-TCR-HDR, or pUC57mini-TCR-DHFR-FS-HDR from Example 1 as templates (1 pg template per reaction), PCR amplification was performed in a 50 μL system using GXL high-fidelity DNA polymerase. Primer sequences: TRAC-FP: TGGCGGGACTAGTGGCTCAAG; TRAC-RP: CACC ACTTCCAGCACCTCAAG.

[0108] To purify and concentrate homologous templates, this experiment used Hieff. DNA purification magnetic beads (YeasenBiotech) are based on solid-phase reversible immobilization (SPRI) technology for purification and concentration. The specific steps are as follows:

[0109] (1) Remove the magnetic beads from the 4℃ refrigerator and allow them to equilibrate at room temperature for 30 minutes. During this time, prepare an 80% ethanol solution for later use.

[0110] (2) Vortex magnetic beads for 30s;

[0111] (3) Mix the PCR product with 80% ethanol at a volume ratio of 1:1 and let stand at room temperature for 7 min.

[0112] (4) Transfer to a magnetic rack and let stand for 5 minutes until the solution is clear, then discard the supernatant;

[0113] (5) Add freshly prepared 80% ethanol to wash the magnetic beads, let stand for 30 seconds and then discard the supernatant;

[0114] (6) Wash the magnetic beads again;

[0115] (7) Dry the magnetic beads at room temperature for 5 minutes until the magnetic beads just begin to crack. Then, add 5 μL of ultrapure water for every 100 μL of original PCR product to elute.

[0116] (8) After full resuspension, let stand at room temperature for 15 minutes, then magnetically separate and collect the eluent, which is the target product;

[0117] (9) The purified dsDNA homologous templates (EGFP-HDR homologous template, TCR-HDR homologous template, or TCR-DHFR-FS-HDR homologous template) were verified for fragment size and purity by 1% agarose gel electrophoresis, and their concentration and A260 / 280 ratio were determined by Nanodrop2000 ultra-micro spectrophotometry. All qualified templates were aliquoted and stored at -20℃ for later use.

[0118] The TCR-HDR homologous template includes, from the 5' to the 3' end, a left homologous arm, the CMV-A*02:01-pp65-TCR gene, and a right homologous arm, as shown in SEQ ID NO.11.

[0119]

[0120] The TCR-DHFR-FS-HDR homologous template includes, from the 5' to the 3' end, a left homologous arm, the CMV-A*02:01-pp65-TCR gene, the DHFR-FS gene, and a right homologous arm, as shown in SEQ ID NO.12.

[0121]

[0122] Example 4 – Preparation of Ribonucleoprotein Complex

[0123] The ribonucleoprotein complex (RNP) was prepared using a guide RNA (gRNA sequence as shown in SEQ ID NO.1: 5'-CAGGGTTCTGGATATCTGT-3') custom-synthesized by Genscript Biotech Ltd. Its backbone was chemically modified with 2'-O-methyl phosphorothioate, which significantly improved its stability by enhancing its resistance to nuclease degradation and optimized gene editing efficiency. The Cas9 nuclease was purchased from KACTUS-Cas9 Biotech (Shanghai) Co., Ltd. Its N-terminus and C-terminus are fused with the SV40 nuclear localization signal (NLS), respectively, which ensures effective protein localization in target cells by promoting nuclear transport mechanisms. For the preparation of RNP complexes in a 20 μL electroporation system, a strictly nuclease-free procedure was employed: First, 100 pmol gRNA solution (dissolved in DEPC water) was transferred to a low-adsorption PCR tube. Using a pre-chilled pipette tip, 50 pmol Cas9 protein was slowly added dropwise, gently mixing with a pipette tip. The reaction mixture was then incubated at 37°C for 15 minutes to promote the self-assembly of the RNP complex. Immediately after the reaction was terminated, the complexes were transferred to a pre-chilled ice box (≤4°C) for storage, and subsequent electroporation experiments were completed within 2 hours. RNase-free reagents and consumables were used throughout the process to maintain the functional integrity of the complexes.

[0124] Example 5 – Construction of a T cell electroporation and TCR-HDR homologous localization repair system

[0125] The specific experimental procedures in this embodiment are as follows:

[0126] (1) Pre-equilibration electroporation buffer: Electroporation was performed using a Lonza P3 Primary Cell 4D-Nucleofector X system. Activated T cells, after 48 hours of activation with magnetic beads, were counted at 1.0 × 10⁻⁶. 6 Cell / reaction was used as the baseline. Before the experiment, 1 mL of pre-filled X-VIVO 15 complete culture medium in 24-well plates was placed in a 37°C incubator for 30 min to equilibrate. For the electroporation buffer system, commercially available Lonza P3 buffer or B1 Mixbuffer was used; the buffer was gently mixed and equilibrated at room temperature before use.

[0127] (2) Preparation of RNP-homological template complex: The Cas9 / gRNARNP complex pre-assembled in Example 4 was mixed with the TCR-HDR homologous template purified in Example 3 (final concentration 2 μg / μL) in a low adsorption EP tube. After collecting the droplets by instantaneous centrifugation, the mixture was incubated at room temperature for 5 min to form the RNP-HDR ternary complex. After incubation, the mixture was placed on ice for later use.

[0128] (3) Cell pretreatment: Collect T cells activated for 48 hours, gently pipette them and place them in a magnetic rack. Let them stand at room temperature for 5 minutes to remove the magnetic beads, collect the supernatant cell suspension and centrifuge at 90×g for 10 minutes to obtain the cell pellet. Then use a pipette tip to completely remove the supernatant.

[0129] (4) Cell resuspension: 1×10 6 Resuspend cells in pre-equilibrated P3 buffer or B1 Mix buffer at a cell / 20 μL ratio.

[0130] (5) System mixing: Gently blow and aspirate the cell suspension and RNP-HDR complex together, then transfer it to a Lonza 4D electroporation strip and tap the cup wall to remove air bubbles;

[0131] (6) Electrical pulse parameters: Select the optimized program EO138 and perform the electroporation operation;

[0132] (7) Cell recovery: Immediately and gently add 90 μL of preheated X-VIVO 15 complete culture medium and let stand for 15 minutes to allow the cells to recover.

[0133] (8) Cell culture: After gently pipetting 3 times, transfer to a pre-equilibrated 24-well plate and add culture medium to a final volume of 1 mL / well.

[0134] (9) To improve HDR efficiency, some experimental groups added Alt-R at a volume ratio of 1:100 to the culture medium after electroporation. TM HDREnhancerV2 was cultured at 37°C and 5% CO2 for 24 hours, after which the enhancer was removed.

[0135] (10) After electroporation, cell morphology and proliferation status were monitored daily using an inverted microscope to obtain TCR-T cells for downstream experiments.

[0136] To evaluate the impact of different activation time points on the transfection efficiency and homology-directed repair efficiency of primary T cells after electroporation, T cells were activated for 24, 36, 48, 60, and 72 hours with a magnetic bead-to-cell ratio of 1:1. 1 μg of pMAX-EGFP plasmid or 1 μg of EGFP-HDR was transfected into the cells using a Lonza 4D nuclear transfection instrument. At fixed electroporation doses, the proportion of EGFP-positive cells was analyzed by flow cytometry on day four after electroporation at each time point, and the number of EGFP+ cells was quantified using trypan blue staining. Results are as follows: Figure 2 The results showed that transfection efficiency at 48 hours post-activation was significantly higher than at other time points. Furthermore, the 48-hour group also yielded the optimal number of EGFP-positive cells. Subsequently, this study co-transfected primary T cells with 50 pmol RNP complex and 1 μg EGFP homologous template at different time points to compare HDR efficiency. The results showed that the 48-hour group still exhibited the highest EGFP positivity and the largest number of EGFP-positive cells, indicating that 48 hours post-T cell activation is the optimal time for electroporation.

[0137] This embodiment also used CD3 / CD28 magnetic beads to pre-activate human primary T cells for 48 hours for electroporation experiments. 50 pmol of CRISPR-Cas9 ribonucleoprotein complex (RNP) (Cas9:sgRNA molar ratio 1:2) and 1 μg of EGFP homology-directed repair (HDR) donor template (1098 bp, containing a TRAC site homologous arm) were delivered. On day 4 after electroporation, the proportion of EGFP+ cells was analyzed by flow cytometry to quantify the targeted integration efficiency. Results are as follows: Figure 3 The results showed that, compared to LONZAP3 electroporation buffer and the officially recommended EO115 electroporation program, B1 mix electroporation buffer combined with the EO138 electroporation program exhibited higher gene knock-in efficiency. B1 mix electroporation buffer combined with the EO138 electroporation program successfully integrated a 1098 bp exogenous fragment into the TRAC site in approximately 20% of primary T cells while maintaining high cell viability, suggesting that synergistic optimization of buffer ionic components and the electroporation program can improve the efficiency of large fragment HDR.

[0138] The control group used a modular molecular cloning strategy to construct the lentiviral expression vector. LV-TCR-T cells were constructed using a codon-optimized HLA-A02:01-restricted pp65-TCR gene sequence, shown in SEQ ID NO. 10. The lentiviral vector was directionally cloned into the pHR-SFFV-P2A-puro lentiviral expression vector via BamHI / NotI double restriction sites. After constructing the recombinant plasmid, it was transfected into 293T cells (80-90% confluence) for lentiviral packaging. Viral supernatants were collected at 48h and 72h post-transfection and filtered through a 0.45μm PES membrane to remove impurities. PBMCs were isolated from healthy donor peripheral blood, and CD3+ was purified by density gradient centrifugation. + T cells were pre-activated at 37°C and 5% CO2 for 48 hours with CD3 / CD28 Dynabeads, 300 U / mL recombinant human IL-2, 10 ng / mL IL-7, and 10 ng / mL IL-15 in a 1:1 ratio of cells to magnetic beads, and then injected with 1×10⁻⁶ cells. 7 One activated T cell was added with 1× protamine sulfate (final concentration 4 μg / mL), and 300 μL of pp65-TCR lentivirus solution was added to the cell supernatant. Transduction was completed by centrifugation at 1,200×g and 32℃ for 90 minutes. After incubation for 24 hours, the medium was replaced with X-VIVO serum-free medium containing 300 U / mL IL-2 for amplification culture (half the medium was replaced every 48 hours) to obtain control LV-TCR-T cells. TCR expression efficiency, cell killing experiments and activation verification were detected by flow cytometry.

[0139] Example 6—Exploring the effects of truncated Cas9 targeting sequences and homologous template dosage on T cell electroporation

[0140] This embodiment utilizes primers carrying a truncated Cas9 targeting sequence (tCTS, sequence shown in SEQ ID NO. 5, SEQ ID NO. 5: GTTCTGGATATCTGTGGG) and primers without tCTS to produce two homologous templates (2181 bp) expressing CMVpp65-A*02:01-TCR: a homologous template containing tCTS and a homologous template without tCTS. The effects on editing efficiency and cell viability are compared. Simultaneously, since dsDNA exhibits significant cytotoxicity, dsDNA quality is also a crucial optimization parameter for electroporation; therefore, this experiment will also simultaneously explore the optimal dsDNA quality for electroporation.

[0141] In this experiment, primary T cells activated for 48 hours were electroporated with homologous templates of varying masses, some containing and some without tCTS. Four days after electroporation, the expression efficiency of exogenous TCRs was detected by flow cytometry, and cell counting was used to calculate the results. The results showed that tCTS on the homologous template significantly improved the editing efficiency of T cells after electroporation and yielded a higher total number of mTCRβ+ T cells. Figure 5 Meanwhile, the study found that tCTS increased the dose of homologous template transfected into the cell nucleus, causing a more significant cytotoxic effect. At an electroporation dose of 4 μg, the number of positive cells harvested was less than with conventional homologous template. Therefore, electroporation using 1-2 μg of homologous template containing tCTS can achieve optimal editing efficiency and yield the most target cells.

[0142] The pUC57mini-TCR-HDR plasmid and pUC57mini-TCR-DHFR-FS-HDR plasmid were constructed using the method in Example 1. The homologous arm sequences were replaced with Cas9 target sequences containing truncated sequences. The plasmids were amplified and purified using the method in Example 3 to obtain TCR-HDR homologous templates containing tCTS and TCR-DHFR-HDR homologous templates containing tCTS.

[0143] Example 7—Exploring the impact of HDR enhancer v2 on editing efficiency

[0144] HDR enhancer v2 is an enhancer of the HDR pathway, and previous studies have reported its effective promotion of homologous recombination. This embodiment focuses on the HDR enhancer v2 and systematically optimizes its effect on the targeted integration efficiency of primary T cells. Human primary T cells pre-activated with CD3 / CD28 magnetic beads for 48 hours (CD25 expression >90%) were electrotransferred with the CRISPRRNP complex and TCR-HDR homologous template donor. Cells were then cultured for 24 hours in X-VIVO 15 medium containing gradient concentrations of HDR enhancer v2 (0-2 μM), followed by drug-free medium to avoid long-term toxicity. Subsequently, mTCR was quantitatively analyzed by flow cytometry on day 4 after electroporation. + Cell percentage and viable cell count. Results showed that treatment with 0.5 μM HDR enhancer v2 for 24 hours exhibited the best editing efficiency while maintaining high cell viability, with a maximum editing efficiency of 24.2%. Figure 6 Therefore, in the targeted integration gene editing operation of primary T cells based on the HDR repair pathway, primer small molecule HDR pathway enhancers can significantly improve HDR repair efficiency.

[0145] Furthermore, using the method for constructing TRAC-TCR-T cells shown in Example 5, TRAC-TCR-T cells were obtained by constructing 1-2 μg of TCR-HDR homologous template containing tCTS as shown in Example 6.

[0146] Example 8 - Cell Killing Assay and Activation Detection

[0147] To quantitatively evaluate the TRAC-TCR-T cell-specific killing efficacy described in Example 7, a co-culture experiment was conducted using the K562-A*02:01-pp65 target cell system (HLA-A*02:01-restricted pp65 antigen presentation model). First, target cells were labeled with CellTrace Violet cell tracer dye: target cells were gently resuspended in DPBS solution containing CellTrace Violet dye and incubated at room temperature in the dark for 20 min. Then, 10 ml of DPBS containing 2% FBS was added, followed by centrifugation at 300×g for 5 min to remove free dye. Cells were then resuspended in pre-warmed RPMI 1640 complete medium (containing 10% FBS) to a final concentration of 2.5×10⁻⁶ cells / mL. 5 Effector cells (A*02:01-pp65-TCR-T cells) and target cells were seeded at effector-to-target ratios (E:T) of 1:1, 5:1, and 10:1 in 96-well round-bottom plates, with a final volume of 200 μL per well. The cells were co-cultured at 37°C and 5% CO2 for 24 hours. After co-culture, a 1:10000 dilution of LIVE / DEAD Fixable Near-IR staining reagent was added, and the cells were incubated in the dark for 30 min. The proportion of dead target cells was detected by flow cytometry. Data analysis was performed using FlowJo v10.8 software and CellTraceViolet. + / Near-IR + The specific killing efficiency was calculated based on the proportion of double-positive cells. At the same time, an untreated T cell group (control group) and a non-effect cell well were set up as negative controls to exclude interference from spontaneous apoptosis.

[0148] To systematically evaluate the expression of activated surface molecular markers of TRAC-TCR-T cells obtained in Example 7, an in vitro antigen-specific stimulation model was established: K562-A*02:01-pp65 target cells and A*02:01-pp65-TCR-T cells were seeded in a flat-bottomed 96-well plate at an effector-to-target ratio (E:T) of 5:1, with a final volume of 200 μL per well (containing 2.5 × 10⁻⁶ cells). 5 Effector cells and 5×10 4Target cells (labeled with CellTrace Violet cell tracer dye) were co-cultured at 37°C and 5% CO2 for 24 hours. After stimulation, the co-cultured cells were collected, washed with PBS containing 2% fetal bovine serum, and then subjected to surface labeling: anti-human CD69 and CD137 were added, and the cells were incubated at 4°C in the dark for 30 min. After staining, the cells were washed twice with FACS buffer, and the expression of activation markers was then detected by flow cytometry.

[0149] The primary evaluation criteria for assessing the normality of TRAC-TCR-T cell function are the in vitro killing function of T cells and the ability to effectively activate them. Therefore, this experiment utilized T cell-tumor cell co-culture, labeling K562-A*02:01-pp65 target cells with celltrace violet and co-culturing them for 24 hours at different effector-to-target ratios. After culture, dead cells were labeled with LIVE / DEAD Fixable Near-IR staining, and the percentage of cell killing was subsequently detected by flow cytometry.

[0150] Experimental results showed that both LV-TCR-T and TRAC-TCR-T effectively killed target cells at different effector-to-target ratios, and both exhibited comparable killing levels with no significant statistical differences. Figure 7 Subsequently, to further investigate cell activation levels, the above model was repeated. Cells from the co-culture system were collected after 24 hours of culture, and cell populations expressing exogenous TCRs were identified using CD8 and mTCRβ antibodies. These cell populations were then analyzed using CD69 and CD137 antibodies. The experimental results are as follows: Figure 8 Figures B and D show that both LV-TCR-T and TRAC-TCR-T can be effectively activated in this co-culture system, and the ratio of CD69+ cells to CD137+ cells is not significantly different between the two types of TCR-T cells.

[0151] Example 9 - Detection of Cytokine Release Function

[0152] To evaluate the cytokine secretion levels of TRAC-TCR-T cells obtained in Example 7, target cells and T lymphocytes were co-cultured in 96-well plates with an effector-to-target ratio (E:Tratio) of 5:1. After maintaining the co-culture system at 37°C and 5% CO2 for 12 hours, the protein transport inhibitor BD GolgiStop (BD Biosciences, USA) was added at the recommended working concentration, and incubation continued for 4 hours to enrich intracellular cytokines. After the experiment was terminated, cell surface molecular marker staining was performed, followed by cell fixation and permeabilization. Intracellular staining was completed using specific fluorescently labeled antibodies, and flow cytometry was used to quantitatively analyze the protein expression levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and granzyme B (GRZB).

[0153] Cytokine release function evaluation is also an important part of T cell function assessment. After recognizing target cells, TCR-T cells directly kill target cells through cytokines or further amplify their sensitivity to target cells through related signaling pathways, thereby indirectly improving cell killing efficiency. This experiment relied on the above-mentioned classic co-culture model, and under the condition of an effector-to-target ratio of 5 / 1, two types of TCR-T cells were co-cultured with target cells for 12 hours. Then, a Golgi transport inhibitor was added to the system to inhibit the transfer of cytokines from the intracellular to the extracellular space, and co-culture was continued for another 4 hours. After the experiment, the proportion of cytokine-positive cells was detected by flow cytometry. The LV-TCR-T cells of Example 8 were used to detect cytokine release function. The results showed that LV-TCR-T cells had significantly higher expression of granzyme B (GZMB) than TRAC-TCR-T cells, while TRAC-TCR-T cells had a stronger response in the expression levels of IFN-γ and TNF-α. Figure 9 This suggests that the TCR-T cells produced by the two construction methods have different cytokine response patterns.

[0154] Example 10 - Fluorescence in situ hybridization

[0155] Fluorescence in situ hybridization (FISH) was performed using standardized procedures from Kanglu Biotechnology Co., Ltd. to detect chromosomal abnormalities in peripheral blood-derived T lymphocytes. A specific two-color break probe (TRA / TRD apart probe) was used to target the T cell receptor alpha / delta locus (TRA / TRD) gene in the q11.2 region of chromosome 14 to detect V(D)J recombination-related chromosomal translocation events. The specific experimental steps were as follows: After pretreatment with cell fixation medium (methanol: glacial acetic acid = 3:1), the samples were denatured at 80℃ for 5 min to unwind the DNA double strands. Subsequently, the samples were hybridized with the probe system (10 μL / sample) in a humidified chamber at 37℃ in the dark for 16 h. After rigorous elution (2×SSC / 0.3% NP-40, 72℃) to remove non-specific bindings, the samples were mounted with an anti-quenching agent. Digital images were acquired using the Metafer 5.10.5 fully automated scanning and analysis system (MetaSystems, Germany). The criteria for interpreting positive signals were set as follows: the distance between red and green fluorescent signals ≥ 3 times the diameter of the signal point (signal separation threshold = 1.5 μm), and ≥ 200 intact cell nuclei were counted per sample.

[0156] This embodiment collected control T (untreated group), LV-TCR-T (lentivirus production group), and TRAC-TCR-T (non-viral gene-edited group) cells from D10 (cultured to D12) after gene editing. The cells were then fixed with methanol-glacial acetic acid fixative, and a standardized procedure was used to perform fluorescence in situ hybridization (FISH) experiments to detect chromosome breakage. Experimental data are as follows: Figure 10 B showed that LV-TCR-T did not significantly increase chromosome loss, suggesting that lentiviruses did not significantly cause changes at the chromosome level. However, compared to the control group, the chromosome loss rate of TRAC-TCR-T increased from 3.7% to 9.1%, and Fisher's exact test showed a statistically significant difference. Figure 10 (B) This indicates that CRISPR-induced DNA double-strand breaks can significantly cause changes at the chromosome level. Therefore, further optimization of the system is still needed to reduce the level of chromosome loss.

[0157] Example 11 -- RNA-Seq Sequencing

[0158] To further evaluate the functional phenotype of T cells at the transcriptome level, this experiment separated the CD8+TCR+ (mTCR+) population from LV-TCR-T cells (day 12 post-activation) and TRAC-TCR-T cells (from Example 7) using flow cytometry sorting (FACS). RNA was then extracted and sequenced. LV-TCR-T and TRAC-TCR-T cells that were not co-incubated with K562 target cells were collected on day 10 post-gene editing (day 12 post-activation). After collection, the cells were washed twice with DPBS, and 1 mL of TRIZOL reagent was added. The cells were thoroughly lysed and stored at -80°C. Subsequent samples were sent to Gedio BioSequencing for sequencing analysis, and online analysis was performed using Gedio Bio's online analysis platform (http: / / www.omicsmart.com), including differential gene analysis and heatmap generation, differential gene pathway enrichment, and GSEA pathway enrichment.

[0159] Differential Gene Fever Figure 11 A showed that TRAC-TCR-T cells exhibited significantly differential transcriptomic characteristics, with significantly lower expression levels of exhaustion-related markers (such as LAG3 and TOX) compared to LV-TCR-T cells. Subsequent pathway enrichment of differentially expressed genes (DEGs) was performed. Figure 11 B showed that the differentially expressed genes were mainly enriched in immune and cytokine-related pathways, such as signal transduction and cytokine signaling in the immune system.

[0160] Example 12—Obtaining TRAC-DHFR-TCR-T cells and exploring the concentration and treatment time of methotrexate.

[0161] To reduce the probability of chromosomal abnormalities in non-viral site-directed integration TCR-T cells constructed using CRISPR technology, such as... Figure 12As shown, the methotrexate resistance gene DHFR-FS was introduced into the pUC57mini-TCR-HDR homologous template. Using 1-2 μg of the TCR-DHFR-FS-HDR homologous template containing tCTS (as shown in Example 6) and the RNP complex from Example 3, electroporated T cells were used to explore the treatment concentration and time of methotrexate. It is hypothesized that chromosome loss occurs in cells that have not successfully undergone homologous recombination. Methotrexate (MTX), as a dihydrofolate reductase (DHFR) inhibitor, can effectively eliminate abnormal T cell clones caused by chromosome loss through selective pressure enrichment, while retaining cells with normal chromosome copies, thereby improving the genomic stability of the final product. Simultaneously, this strategy can further enrich T cells with exogenous gene integration, thereby improving the purity of the final product, achieving a "two birds with one stone" effect.

[0162] Using the method for preparing TCR-T cells as described in Example 5, T cells were obtained by electroporating T cells with 1-2 μg of TCR-DHFR-FS-HDR homologous template containing tCTS as shown in Example 6 and the RNP complex of Example 3. The electroporated T cells were cultured with HDR enhancer v2 as shown in Example 7 for 24 hours and then subjected to MTX treatment. TRAC-DHFR-TCR-T cells were obtained by screening.

[0163] To determine the optimal concentration and treatment duration of MTX, we tested concentration gradients of 0, 1, 10, 50, 100, 500, and 1000 nM. The results showed that the half-maximal inhibitory concentration (IC50) of MTX was 27.94 nM, and a concentration of 100 nM effectively eliminated most T cells that had not undergone homologous recombination. Figure 13 C). Further investigation into the timeliness of MTX screening revealed that with 100 nM MTX treatment, a 6-day screening cycle was sufficient to efficiently enrich TRAC-DHFR-TCR-T cells, increasing the editing efficiency from ~13% to ~70%. Figure 13 D).

[0164] Example 13—Methotrexate enrichment strategy reduces chromosome loss level in TCR-T cells

[0165] To systematically evaluate the regulatory effect of methotrexate enrichment strategy on the genomic stability of gene-edited T cells, this study performed fluorescence in situ hybridization (FISH) analysis on three groups of D12 cultured cells based on TRAC-DHFR-TCR-T cells obtained in Example 12: the Control group (unedited T cells), the TRAC-TCR-T group obtained in Example 7 (TRAC sites integrating TCR but not selected by MTX), and the TRAC-DHFR-TCR-T group obtained in Example 12 (integrating TCR and DHFR-FS reporter genes and continuously selected by 100 nM MTX for 5 days). Subsequently, the cells were fixed and subjected to fluorescence in situ hybridization (FISH) experiments to detect chromosome breakage using a standardized procedure. Experimental data are as follows: Figure 14 The results showed that the TRAC-TCR-T group exhibited significantly increased chromosomal instability (mean cellular abnormal events, TRAC-TCR-T group ~10% vs Control group ~5%, p<0.001), suggesting that random integration or CRISPR off-target effects may lead to genomic structural variations; the incidence of abnormal events in the TRAC-DHFR-TCR-T group returned to baseline levels (TRAC-DHFR-TCR-T group 6% vs Control group ~5%, ns), confirming that MTX pressure can effectively clear the cell subset that has experienced abnormal loss of chromosome 14. Figure 14 ).

[0166] Example 14 – Construction of a mouse metastatic tumor model and in vivo imaging

[0167] This embodiment evaluates the tumor-clearing efficacy of methotrexate-optimized TRAC-DHFR-TCR-T cells obtained in Example 12 using a cell-derived xenograft model. Six- to eight-week-old female NCG mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were randomly assigned to groups. On day -3 of the experiment, 2 × 10⁻⁶ NCG cells were injected via the tail vein. 6 K562-A*02:01-pp65-luciferase fluorescently labeled tumor cells (resuspended in 200 μL sterile PBS). 72 hours later, three groups of T-cell therapy agents (non-genetically edited T cells, LV-TCR-T cells, and TRAC-DHFR-TCR-T cells, 1×10⁻⁶) were infused. 7Cells / mouse, resuspended in 200 μL PBS. To maintain the viability of adoptive T cells, recombinant human IL-2 (2,500 IU / mouse, PeproTech) was administered daily via intraperitoneal injection for 14 days after cell infusion. In vivo imaging was performed as follows: D-fluorescein potassium contrast agent (150 μL / mouse, 15 mg / mL, Aladdin Biochemical Technology) was injected intraperitoneally at designated time points. After equilibration in the dark for 15 min, signal acquisition was performed using the Tanon ABL small animal in vivo imaging system (Shanghai Tianneng Technology Co., Ltd.). Using Tianneng ImageLab 5.2 professional software, based on a standardized region of interest (ROI) template, the total photon flux (unit: photons / sec / cm²) was quantitatively analyzed. 2 / sr).

[0168] Experimental results are as follows Figure 15 The results showed that unedited T cells (Mock group) could not effectively control tumor progression, and their bioluminescent signal increased exponentially. Figure 15 C); while both the LV-TCR-T group and the MTX-enriched TRAC-DHFR-TCR-T group showed significant tumor clearance capabilities. Figure 15 D and E) showed comparable tumor burden suppression efficiency and kinetic characteristics. Survival analysis further confirmed that the MTX enrichment strategy did not affect the therapeutic efficacy of TCR-T cells; the median survival of mice in the TRAC-DHFR-TCR-T group and the LV-TCR-T group was significantly longer than that in the Mock group. Figure 15 G).

[0169] This application utilizes the pUC57 mini-HDR vector system to introduce the TCR expression cassette and L22F / F31S double mutant DHFR into TCR-T cells via site-directed integration technology. By introducing the L22F / F31S double mutant DHFR, the edited cells are resistant to methotrexate (MTX), achieving efficient enrichment of CRISPR-edited cells. Methotrexate treatment can increase the editing efficiency from 13% to 70% while reducing the chromosomal abnormality rate. MTX selectively eliminates chromosomally abnormal cells by inhibiting endogenous DHFR activity, reducing the proportion of T cells with abnormal loss of chromosome 14. In vivo and in vitro experiments have verified the complete effector function and good safety of TCR-T cells, laying the foundation for subsequent clinical translation.

[0170] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A method for preparing TCR-T cells mediated by a CRISPR / Cas9 system, comprising the following steps: a) The dsDNA homologous template and the RNP complex are delivered into T cells via electroporation for homologous targeted repair to obtain electroporated T cells. The RNP complex contains Cas9 protein and gRNA, the gRNA targets the TRAC gene, and the homologous template contains the target TCR gene and the mutant DHFR gene. b) Culture electroporated T cells and use methotrexate for enrichment and screening during cell culture to obtain screened TCR-T cells.

2. The method for preparing TCR-T cells as described in claim 1, characterized in that, In step a): The dsDNA homologous template complexes with the RNP complex to form the RNP-homologous template complex, which is then delivered into T cells via electroporation. And / or, the gRNA targets the first exon of the TRAC gene; And / or, the gRNA includes one or more modifications selected from one or more of 3'-thiophosphate, 2'-O-methyl ester, 2'-O-methyl, 2'-ribose 3'-thiophosphate, deoxy and / or 5' phosphate modifications, preferably 2'-O-methyl thiophosphate modification; And / or, the N-terminus and C-terminus of the Cas9 protein are respectively fused with SV40 nuclear localization signals.

3. The method for preparing TCR-T cells as described in claim 1, characterized in that, In step a), the mutant DHFR gene is selected from the DHFR gene with a double missense mutation at L22F and F31S. Preferably, the amino acid sequence of the mutant DHFR is shown in SEQ ID NO.

2.

4. The method for preparing TCR-T cells as described in claim 1, characterized in that, The dsDNA homologous template includes, from the 5' to the 3' end, a left homologous arm, an exogenous target TCR gene, a mutant DHFR gene, and a right homologous arm; preferably, the exogenous target TCR gene and the mutant DHFR are separated by a cleavage peptide.

5. The method for preparing TCR-T cells as described in claim 4, characterized in that, The 5' end of the left homologous arm and / or the 3' end of the right homologous arm are connected to a truncated Cas9 target sequence. More preferably, the truncated Cas9 target sequence is selected from a truncated Cas9 target sequence of 15 bp near the PAM region of the gRNA. More preferably, the amino acid sequence of the truncated Cas9 target sequence is as shown in SEQ ID NO.

5. More preferably, the sequence of the left homologous arm is as shown in SEQ ID NO.3, and the sequence of the right homologous arm is as shown in SEQ ID NO.

4.

6. The method for preparing TCR-T cells as described in claim 1, characterized in that, The method also includes one or more of the following features: A: In step a), T cells are activated and electroporation is performed 24 to 72 hours after activation, preferably 48 hours after activation; B: In step a), the amount containing 1.0 × 10 6 Based on the cell electroporation reaction system, the amount of the dsDNA homologous template used is 1-3 μg, preferably 1-2 μg; C: In step b), the electroporated T cells are first cultured in a medium containing an HDR enhancer, and then replaced with a medium without an HDR enhancer. Preferably, the HDR enhancer is HDR enhancer v2. More preferably, based on the total volume of the medium containing the HDR enhancer, the concentration of the HDR small molecule enhancer is below 2 μM, preferably 0.5 μM. D: In step b), when using methotrexate enrichment screening, the concentration of methotrexate is below 1000 nM, preferably 100 to 1000 nM, based on the total volume of the culture medium.

7. A method for reducing the level of chromosome loss in CRISPR / Cas9 system-mediated TCR-T cell products, wherein the TCR-T cell products are prepared using the TCR-T cell preparation method according to any one of claims 1 to 6, so that the level of chromosome 14 loss in the TCR-T cell products is not significantly different from the level of chromosome 14 loss in unedited T cells.

8. A TCR-T cell, said TCR-T cell being prepared using the method described in any one of claims 1 to 6.

9. The use of TCR-T cells as described in claim 8 in the preparation of antitumor drugs.

10. The application as described in claim 9, characterized in that, The tumor is a solid tumor or a hematologic tumor. Preferably, the solid tumor is selected from: esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, and sarcoma; the hematologic tumor is selected from: leukemia, lymphoma, and myeloma.

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