Tcr-t cells with killing and tumor inhibiting efficacy and uses thereof

Abstract

The application discloses a TCR-T cell with killing and tumor inhibiting effects and application thereof, and the TCR-T cell is a T cell expressing a tumor antigen TCR, the tumor antigen is one or more of MAGE-C2, MAGE-A3, MAGE-A4, NY-ESO-1 or TP53, the TCR-T cell stably expresses PD-L1, and the PD-1 gene in the TCR-T cell is knocked out or inhibited. The TCR-T cell targeting MAGE-C2, stably expressing PD-L1 and knocking out the self PD-1 has greater killing and tumor inhibiting effects in vitro and in animal experiments. The TCR-T cell can be used in the preparation of a drug for treating tumors.

Description

Technical fields:

[0001] This invention relates to the field of immunotherapy technology, and in particular to a TCR-T cell with killing and tumor-suppressing effects and its applications. Background technology:

[0002] TCR-T cell therapy (engineered T cell receptor T cell therapy) is a promising tumor immunotherapy. The engineered TCR complex of TCR-T cells is structurally and functionally identical to the natural TCR, recognizing specific tumor antigens presented by MHC (major histocompatibility complex) molecules. Antigen selection is crucial for the development of TCR-T cell therapy; ideally, the antigen should be tumor-specific, widely expressed in tumor cells, and presentable by MHC molecules.

[0003] MAGE-C2 (MC2), also known as CT10, belongs to the MAGE family (melanoma-associated antigen family). MAGE-C2 exhibits excellent tumor specificity and is associated with various cancer types, including lung cancer, melanoma, breast cancer, prostate cancer, liver cancer, and multiple myeloma, making it a potential candidate TCR-T antigen target. Previous studies have obtained TCR sequences targeting the MC2 antigen from melanoma patients who showed good clinical responses to tumor peptide vaccine therapy. One of the antigen targets is HLA-A02-restricted MAGE-C2. 336–344 (ALKDVEERV) exhibits antitumor activity after transduction into T cells. However, it remains unclear whether this specific antigen has the potential to be developed as a TCR-T antigen target, how TCR-T cells recognizing this antigen specifically perform in antitumor activity, and whether there is the possibility of engineered integration of other genes to improve and enhance its function.

[0004] PD-1 is a receptor on T cells. The therapeutic strategy of using immune checkpoint inhibitors (ICIs) to block the PD-1 signaling pathway and enhance T cell activity has been widely recognized and applied. However, ICIs are generally administered systemically and lack specificity in their effects on cells, which may be related to some of the side effects they cause. PD-1 is widely expressed in various cancer cells, and studies have shown that the PD-1 signaling pathway acts as a tumor suppressor in certain types of tumor cells. However, therapeutic strategies that specifically activate PD-1 signaling in malignant cells have not yet been explored. This application explores the possibility of utilizing the targeting ability of TCR-T cells to tumor antigens to actively activate the PD-1 signaling pathway in tumor cells under specific conditions (when TCR-T cells' own PD-1 is inhibited), while simultaneously combining this with the cytotoxic effects of TCR-T cells to produce an anti-cancer effect. Summary of the Invention:

[0005] The first objective of this invention is to provide a TCR-T cell with killing and tumor-suppressing effects.

[0006] The second objective of this invention is to provide an application of TCR-T cells with killing and tumor-suppressing effects.

[0007] The first objective of this invention is achieved by the following technical solution: TCR-T cells with killing and tumor-suppressing effects, wherein the TCR-T cells are T cells expressing TCRs that recognize tumor antigens, wherein the tumor antigens are one or more of MAGE-C2, MAGE-A3, MAGE-A4, NY-ESO-1, or TP53; the TCR-T cells stably express PD-L1; the function of the TCR carrying one or more of the tumor antigens MAGE-C2, MAGE-A3, MAGE-A4, NY-ESO-1, or TP53 is: on the one hand, it can target and recognize tumor cells, and on the other hand, it has a significant killing effect on tumor cells; the purpose of the TCR-T cells stably expressing PD-L1 is: when it can target and recognize tumor cells, the PD-L1 expressed by the TCR-T cells can target and activate the PD-1 signaling pathway in tumor cells, thereby achieving the killing or suppression effect on tumor cells, while inhibiting abnormal cells.

[0008] Furthermore, the TCR sequence is derived from one or more CD4 T cell clones, one or more CD8 T cell clones, or multiple CD4 T and CD8 T cell clones in the body of a tumor patient.

[0009] Furthermore, the PD-1 gene in the TCR-T cells is knocked out or suppressed, with the aim of preventing PD-L1 expressed in TCR-T cells from activating the PD-1 signaling pathway in TCR-T cells and thus avoiding the inhibitory effect on TCR-T cells.

[0010] TCR-T cells with killing and tumor-suppressing effects, wherein the TCR-T cells stably express PD-L1, and the PD-1 gene in the TCR-T cells is knocked out or suppressed.

[0011] The knockout of the PD-1 gene in TCR-T cells can be achieved using existing technologies, such as: CRISPR-Cas9; RNA interference (RNAi), including short interfering RNA (siRNA) and short hairpin interfering RNA (shRNA); TALENs (Transcription Activator-Like Effector Nucleases) gene editing; zinc finger nucleases (ZFNs); or homologous recombination, etc.

[0012] Methods for inhibiting the PD-1 gene in TCR-T cells can be implemented using existing technologies, such as: RNA interference (RNAi), including short interfering RNA (siRNA) and short hairpin interfering RNA (shRNA); antisense oligonucleotides (ASOs); CRISPR interference (CRISPRi); small molecule inhibitors; transcriptional repressors; dominant-negative mutants; epigenetic modifications; or inhibitory antibodies.

[0013] Furthermore, the PD-L1 is either endogenous (expressed by the cell's own gene) PD-L1 or exogenous (expressed by an introduced gene) PD-L1.

[0014] Methods for stable expression of endogenous PD-L1 in TCR-T cells can be achieved using existing technologies, such as engineered promoters, CRISPR activation, gene copy number amplification, histone modification, DNA demethylation, small activating RNAs (saRNAs), antisense oligonucleotides, cell signaling pathway regulation, and chemical drug induction.

[0015] Methods for stable expression of exogenous PD-L1 in TCR-T cells: Existing technologies can be used, such as: transfection of the PD-L1 gene with lentiviruses, adenoviruses, retroviruses and other viruses, electrotransfection of the PD-L1 gene, transfection of the PD-L1 gene with nanoparticles, chemical transfection of the PD-L1 gene, transfection with PD-L1 expression plasmids, mRNA transfection, and integration of the PD-L1 gene with CRISPR / Cas9, etc.

[0016] The second objective of this invention is achieved by the following technical solution: the application of TCR-T cells with killing and tumor-suppressing effects as described in the first objective of this invention in the preparation of drugs for treating tumors.

[0017] Furthermore, the tumor is any one or more of the following: lung cancer, lymphoma, leukemia, melanoma, colorectal cancer, liver cancer, stomach cancer, breast cancer, cervical cancer, ovarian cancer, kidney cancer, bladder cancer, or esophageal cancer.

[0018] PD-L1 stands for Programmed death-ligand 1 (PD-L1); PD-1 stands for Programmed cell death protein 1 (PD-1).

[0019] Symbol Explanation

[0020]

[0021]

[0022] Advantages of this invention:

[0023] (1) TCR-T cells that target MAGE-C2, express PD-L1 and knock out their own PD-1 have greater killing and tumor-suppressing effects in vitro and in animal experiments compared with cell subtypes that target MAGE-C2 and knock out their own PD-1 but do not express PD-L1, cell subtypes that target MAGE-C2 but do not knock out PD-1, or cell subtypes that target MAGE-C2 but do not express PD-L1 and do not knock out PD-1.

[0024] (2) Exogenous PD-L1 targets and activates PD-1 in tumor cells, enhancing the tumor-suppressive effect of TCR-T cells. By actively and specifically activating PD-1 signaling in tumor cells using TCR-T cells, the inhibition of tumor cells is synergistically enhanced. Silencing PD-1 through gene editing technology can effectively enhance the activity of TCR-T cells while avoiding self-suppression.

[0025] (3) This invention provides new strategies and references for optimizing, combining and enriching TCR-T therapy and immune checkpoint inhibitor therapy, and has potential clinical application prospects in the field of tumor immunotherapy. Attached image description:

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 A schematic diagram of the gene structure of the MAGE-C2 specific TCR;

[0028] Figure 2 The recombinant cloning vector pUC-SP-MC2-TCR HM With the recombinant expression vector pHR_LV-NS1-MC2-TCR HM Structural diagram;

[0029] Figure 3 Schematic diagram of a recombinant vector for tumor target cell lines;

[0030] Figure 4 A schematic diagram of MHC Tetramer;

[0031] Figure 5 The recombinant cloning vector pUC-SP-MC2-TCR HM Agarose gel electrophoresis image of the double enzyme digestion products;

[0032] Figure 6 To validate the recombinant expression vector and pHR_LV-NS1-MC2-TCR HM Plasmid mapping;

[0033] Figure 7 For MC2-TCR HM Figure 1 shows the flow cytometry results of MAGE-C2-specific TCR expression efficiency of -T.

[0034] Figure 8 For MC2-TCRHM -T cells in T2 MC2 A flow cytometry result showing that the cells produce IFN-γ more effectively upon stimulation.

[0035] Figure 9 For MC2-TCR HM -T and T2 MC2 The image shows the results of detecting higher TNF-α secretion levels after cell co-culture;

[0036] Figure 10 To prepare the pHR_LV-NS1-A02 agarose gel electrophoresis image

[0037] Figure 11 To prepare the agarose gel electrophoresis image of pHR_LV-NS1-MC2-A02

[0038] Figure 12 A diagram validating the successful expression of antigenic epitopes in tumor target cell lines;

[0039] Figure 13 For MC2-TCR HM The flow cytometry results of -T cells that produce IFN-γ after being stimulated by tumor target cells are shown.

[0040] Figure 14 For MC2-TCR HM - TNF-α ELISA kit results show that -T cells have a stronger ability to secrete TNF-α after being stimulated by tumor target cells;

[0041] Figure 15 For MC2-TCR HM Flow cytometry results of the killing efficiency of -T on target cells;

[0042] Figure 16 The recombinant cloning vector pUC-SP-T2A-PDL1 and the recombinant expression vector pHR_LV-NS1-PDL1-MC2-TCR HM Structural diagram;

[0043] Figure 17 A schematic diagram illustrating the packaging process and function of LNP-mRNA (Cas9-mRNA / PD-1-sgRNA);

[0044] Figure 18 This is a schematic diagram of the recombinant expression vector pHR_LV-NS1-PD-1;

[0045] Figure 19 To construct the recombinant expression vector pHR_LV-NS1-PDL1-MC2-TCR HM Agarose gel electrophoresis images and plasmid maps;

[0046] Figure 20 Flow cytometry results showing that exogenous PD-L1 restricts the expression of engineered TCRs of PDL1-MC2-TCR-T;

[0047] Figure 21 The results of flow cytometry analysis showed that PDL1-MC2-TCR-T cells produced low levels of IFN-γ and secreted TNF-α after being stimulated by antigenic peptides.

[0048] Figure 22 PDL1-MC2-TCR-Jurkat cells can express TCR normally. HM The validation results for PD-L1 are shown in the figure.

[0049] Figure 23 The image shows the quality control test results for (Cas9-mRNA+PD-1-sgRNA)-LNP.

[0050] Figure 24 For PDL1-MC2-TCR-T PD1- Flow cytometry results showing significantly reduced PD-1 expression in cells;

[0051] Figure 25 For PDL1-MC2-TCR-T PD1- TCR of cells HM Flow cytometry results of expression restoration;

[0052] Figure 26 For PDL1-MC2-TCR-T PD1- Cellular recovery of TCR HM Flow cytometry results of PD-L1 expression levels;

[0053] Figure 27 For PDL1-MC2-TCR-T PD1- Cells in T2 MC2 The results of flow cytometry showed increased levels of IFN-γ release and TNF-α secretion under stimulation.

[0054] Figure 28 Agarose gel electrophoresis image and plasmid map of pHR_LV-NS1-PD-1;

[0055] Figure 29 MC2-A02-K562 PD1+ Verification diagram of cell expression of pMHC complex and PD-1;

[0056] Figure 30 For PDL1-MC2-TCR-T PD1-With MC2-A02-K562 PD1+ Flow cytometry results showed that co-culture resulted in higher levels of intracellular IFN-γ.

[0057] Figure 31 For MC2-A02-K562 PD1+ Co-culture of PDL1-MC2-TCR-T PD1 The ELISA results showed higher TNF-α secretion levels.

[0058] Figure 32 PDL1-MC2-TCR-Jurkat inhibits MC2-A02-K562 PD1+ Verification diagram of the proliferation effect;

[0059] Figure 33 A comparison of the killing efficiency of TCR-T cells against tumor target cells;

[0060] Figure 34 For PDL1-MC2-TCR-T PD1- For MC2-A02-K562 PD1+ A comparison chart showing the increase in lethality over time;

[0061] Figure 35 A flowchart for animal experiments;

[0062] Figure 36 In vivo imaging of NCG mice after TCR-T reinfusion with different structures and doses;

[0063] Figure 37 A comparison of changes in total tumor fluorescence intensity in NCG-bearing mice after infusion of different TCR-T therapies;

[0064] Figure 38 This is a comparison of the changes in tumor volume and weight in NCG mice after TCR-T infusion. Detailed implementation method:

[0065] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0066] The experimental materials used in the following examples:

[0067] 1. Cells such as PBMCs

[0068] All human PBMC cells used in the following examples were purchased from Shanghai Heyousheng Biotechnology Co., Ltd. K562 cells (chronic myelogenous leukemia cell line) and T2 cells were preserved in our laboratory.

[0069] 2. Lentiviral

[0070] The lentiviral expression vector pHR_LV-NS1 used in the following examples was purchased from Shaanxi Infiltrate Biotechnology Co., Ltd. The lentiviral packaging plasmids psPAX2 and pMD2.G were products of Addgene.

[0071] 3. Experimental reagents and consumables

[0072] The main reagents and consumables used in the following examples are shown in Table 1.

[0073] Table 1: Main Reagents and Consumables

[0074]

[0075]

[0076]

[0077]

[0078] 4. Experimental apparatus

[0079] The main instruments and equipment used in the following embodiments are shown in Table 2.

[0080] Table 2: Experimental Instruments

[0081]

[0082]

[0083] Example 1: Construction and in vitro experiments of MAGE-C2 specific TCR-T cells

[0084] This embodiment includes experimental methods and results. The experimental methods section includes:

[0085] 1. Origin and design of TCR sequences targeting MAGE-C2(HLA-A02)pMHC;

[0086] 2. Recombinant vector targeting MAGE-C2(MC2)TCR and its gene sequence;

[0087] 3. Recombinant vectors of tumor target cell lines and their gene sequences;

[0088] 4. Cell culture methods;

[0089] 5. Preparation method of lentiviral recombinant plasmids;

[0090] 6. Methods for establishing the MC2-TCR-T cell line;

[0091] 7. Synthesis method of MC2 HLA-A02 Tetramer-PE;

[0092] 8. Methods for detecting the expression efficiency of MC2-specific TCRs;

[0093] 9. A method for detecting the intracellular release level of IFN-γ in MC2-TCR-T cells stimulated by peptide-loaded T2 cells using flow cytometry combined with intracellular cytokine staining.

[0094] 10. A method for detecting TNF-α secretion in MC2-TCR-T cells stimulated by peptide-loaded T2 cells;

[0095] 11. Methods for preparing tumor target cell lines;

[0096] 12. Purification method for the A02-K562 cell line;

[0097] 13. Sorting and purification methods for the MC2-A02-K562 cell line;

[0098] 14. Methods for detecting pMHC expression efficiency in tumor target cell lines;

[0099] 15. A method for detecting the intracellular release level of IFN-γ in MC2-TCR-T cells under the stimulation of tumor target cells;

[0100] 16. A method for detecting TNF-α secretion by MC2-TCR-T cells under the stimulation of tumor target cells;

[0101] 17. Detection of MC2-TCR HM Methods to improve the killing efficiency of T cells against tumor target cells;

[0102] The second part of the experimental results includes:

[0103] 1. Construction and validation of recombinant expression plasmids;

[0104] 2. MC2-TCR expressing optimized mouse-derived sequences HM -T has higher TCR expression efficiency and stronger pMHC affinity;

[0105] 3. Mouse-based optimized MC2-TCR HM -T cells are more effective at producing IFN-γ when stimulated with MC2 peptide-loaded T2 cells;

[0106] 4. MC2-TCR expressing optimized mouse-derived sequences HM -T cells stimulated with MC2 peptide-loaded T2 cells showed higher levels of TNF-α secretion;

[0107] 5. Construction and validation of tumor target cells;

[0108] 6. MC2-TCR stimulated by tumor target cells HM -T can produce higher IFN-γ release;

[0109] 7. MC2-TCR stimulated by tumor target cells HM -T can produce higher specific secretion of TNF-α;

[0110] 8. MC2-TCR HM -T cells have a killing effect on tumor target cells MC2-A02-K562;

[0111] The details are as follows:

[0112] I. Experimental Methods:

[0113] 1. TCR sequence origin and design targeting MAGE-C2(HLA-A02)pMHC

[0114] (1) Source of MAGE-C2 specific sequence

[0115] The tumor-specific TCR sequence used in this experiment was derived from the HLA-A02-restricted MAGE-C2 sequence from patients with tumor regression. 336-344 The (ALKDVEERV) antigen targets CD8+ T cell clones. This example uses this sequence as the basis for constructing an engineered TCR. Gene sequence information was obtained from relevant literature and the GenBank database. The α chain is named TCR-Vα3 (GenBank: EU427374.1), and the β chain is named TCR-Vβ28 (GenBank: EU427375.1). MAGE-C2 will be abbreviated as MC2 in the following text.

[0116] (2) Two designs for MC2-TCR recombinant genes

[0117] Human TCR double strands contain positively charged residues in their transmembrane region, resulting in poor pairing stability of the exogenous TCR double strands introduced into cells. This easily leads to the formation of mismatched mixed dimers between endogenous and transduced exogenous TCR strands, reducing the expression abundance of the introduced engineered TCR on the cell surface and thus affecting the activity of engineered T cells and their ability to recognize tumor cells. However, replacing the conserved C region of the human α and β chain sequences with a mouse-derived C region significantly enhances TCR expression levels and stability. This embodiment employs this modification strategy, modifying the conserved C region of the original human α and β chain sequences with a mouse-derived C region to improve TCR expression on the cell surface, binding to target cells, and killing function. In the design of the engineered TCR gene, this embodiment names the target gene containing the aforementioned unmodified original TCR-Vα3 and TCR-Vβ28 sequences as MC2-TCR. H (The specific TCR expressed is called TCR) H Its α and β chains are referred to hereafter as TCR H -α and TCR H -β); To optimize TCR expression and function, a method was designed to replace the constant regions (C regions) of TCR-Vα3 and TCR-Vβ28 with the target gene MC2-TCR from the mouse C region. HM (The corresponding specific TCR is called TCR) HM Its α and β chains are referred to hereafter as TCR HM -α and TCR HM -β). See the gene structure diagram. Figure 1 MC2-TCR H For unmodified original human MAGE-C2 336-344 (ALKDVEERV) / HLA-A*02:01 specific TCR gene sequences TCR-Vα3 and TCR-Vβ28, expressed as TCR H MC2-TCR HM MAGE-C2, which replaces the C region of the mouse TCR gene sequence. 336-344 (ALKDVEERV) / HLA-A*02:01 specific TCR gene sequences TCR-Vα3 and TCR-Vβ28, expressed as TCR HM .

[0118] 2. Recombinant vector targeting MAGE-C2(MC2) TCR and its gene sequence

[0119] (1) Obtaining the recombinant cloning vector

[0120] The target gene fragment designed in this experiment is a tandem arrangement of the α and β chains of the MC2-TCR gene sequence, linked by a self-cleaving P2A sequence. The target gene fragment is approximately 1800 bp in length, with NotI and SalI restriction enzyme sites at both ends. This is to accurately obtain the recognition mechanism for MC2-TCR. 336-344 The TCR of (ALKDVEERV) / HLA-A*02:01 was chemically synthesized and sequenced by Sangon Biotech (Shanghai) Co., Ltd. The cloning vector pUC-SP was modified from pUC57, utilizing synonymous mutations to eliminate commonly used sticky-terminal restriction sites in the pUC57 plasmid multiple cloning restriction sites, retaining only commonly used blunt-terminal sites. This ensures that the target gene has unique restriction sites at both ends, allowing for double digestion to obtain the target gene fragment. The MC2-TCR carrying the target gene... H The recombinant cloning vector was named pUC-SP-MC2-TCR H ; Carrying the target gene MC2-TCR HM The recombinant cloning vector (optimized from mouse sources) is named pUC-SP-MC2-TCR. HM The two vectors have the same structure, with pUC-SP-MC2-TCR as the carrier. HM For example, the structure is as follows Figure 2 As shown in Figure A. Target gene MC2-TCR H The gene sequence is shown in SEQ ID NO:01; the target gene is MC2-TCR. HM The gene sequence is shown in SEQ ID NO:02.

[0121] (2) Recombinant lentiviral expression plasmid

[0122] The lentiviral expression plasmid used for cell transfection was pHR_LV-NS1, derived from pLVX-Puro, with the CMV promoter replaced by the SFFV promoter. The target gene fragment MC2-TCR was inserted. H The recombinant plasmid is called pHR_LV-NS1-MC2-TCR H Insert the target gene fragment MC2-TCR HM The recombinant plasmid is called pHR_LV-NS1-MC2-TCR HM The two have the same structure. (The last part, "pHR_LV-NS1-MC2-TCR", appears to be a separate, unrelated instruction.) HM For example, the structure is as follows Figure 2 As shown in B.

[0123] Figure 2 In this study, the target gene of the inserted vector is MAGE-C2. 336-344The mouse-derived optimized TCRα and β chain gene sequences of (ALKDVEERV) / HLA-A*02:01 were linked by a self-cleaving polypeptide P2A sequence. The restriction enzyme sites used were NotI and SalI. Figure 2 A represents the recombinant cloning vector pUC-SP-MC2-TCR. HM . Figure 2 B is the recombinant expression vector pHR_LV-NS1-MC2-TCR HM After transfection, it is expressed as TCR. HM .

[0124] 3. Recombinant vectors for tumor target cell lines and their gene sequences

[0125] Two tumor target cell lines were constructed using K562 cells: A02-K562 cells expressing only HLA-A*02:01 and MAGE-C2 cells expressing HLA-A*02:01-restricted cells. 336–344 MC2-A02-K562 cells with (ALKDVEERV epitope).

[0126] (1) Recombinant cloning vector

[0127] A02-K562 cells express only HLA-A*02:01. The recombinant cloning vector used was a plasmid already preserved in our laboratory, with pcDNA3.1(+) as the vector. The target gene was inserted between the NotI and SalI restriction sites. This target gene fragment is called A02, and its gene sequence is shown in SEQ ID NO:03. It consists of the HLA-A*02:01 (GenBank: AJ575565.1) gene, CD34 (GenBank: AF523361.1) gene, and polomycin pLeo1209 (GenBank: MN811119.1) gene arranged in tandem, linked by a P2A sequence. This recombinant cloning vector is called pcDNA3.1(+)-A02( Figure 3 A). It should be noted that the recombinant cloning plasmid pcDNA3.1(+)-A02 is an existing plasmid in the laboratory. The CD34 in its target gene was not used in this experiment and is irrelevant to the experiment.

[0128] MC2-A02-K562 cells need to express MAGE-C2 336–344Based on the (ALKDVEERV) fragment at positions 336–344 of the MAGE-C2 [Homo sapiens] (AAF07211.1) protein amino acid sequence and the CDS of the Homo sapiens MAGE-C2 (MAGEC2) gene (AF196483.1), the gene sequence of the MC2 polypeptide fragment was determined. The recombinant cloning vector was pUC-57-simple, and the target gene fragment was designated MC2-A02, whose gene sequence is shown in SEQ ID NO:04. The sequence was determined by combining HLA-A*02:01 (GenBank: AJ575565.1) and MAGE-C2... 336–344 The gene sequence consists of two parts connected by a P2A sequence, with NotI and SalI restriction sites at both ends. The recombinant cloning vector is called pUC-57-MC2-A02. Figure 3 C) The synthesis was commissioned to Beijing Qingke Biotechnology Co., Ltd.

[0129] (2) Recombinant lentiviral expression vector

[0130] The lentiviral expression vector used for transfecting cells is pHR_LV-NS1. The vector with the target gene A02 inserted is called pHR_LV-NS1-A02, and the vector with the target gene MC2-A02 inserted is called pHR_LV-NS1-MC2-A02. Both have the same structure. The recombinant expression vector structure is as follows: Figure 3 B and Figure 3 As shown in D.

[0131] Figure 3 In the vector, the target genes are arranged in tandem and linked by the P2A sequence. The restriction enzyme sites used are NotI and SalI. Figure 3 A represents the recombinant cloning vector pcDNA3.1(+)-A02, which contains the target gene fragment A02, composed of the HLA-A*02:01, pleuromycin pLeo1209, and CD34 gene sequences. The recombinant cloning vector pcDNA3.1(+)-A02 is a pre-existing plasmid in the laboratory. The CD34 gene in this vector was not used in this experiment and is therefore not shown in the figure. Figure 3 B is the recombinant expression vector pHR_LV-NS1-A02: with the target gene fragment A02 inserted. Figure 3 C represents the recombinant cloning vector pUC-57-MC2-A02: containing the target gene fragment MC2-A02, composed of HLA-A*02:01 and MAGE-C2. 336–344 The coding gene sequence is composed of. Figure 3 D represents the recombinant expression vector pHR_LV-NS1-MC2-A02, which inserts the target gene fragment MC2-A02.

[0132] 4. Cell Culture

[0133] The cells involved in this experiment were all suspension cells, cultured in a 37°C constant temperature cell culture incubator with 5% CO2. The cell culture in this example used conventional methods, including procedures such as: pre-preparing the culture medium for the cells, cell resuscitation, cell passage, cell cryopreservation, cell counting, and mycoplasma detection.

[0134] 5. Preparation of lentiviral recombinant plasmids

[0135] (1) Enzyme digestion of the target gene plasmid

[0136] Before starting the experiment, prepare all reagents. Buffers, etc., need to be taken out of -20℃, warmed to room temperature to thaw, vortexed to mix, and then briefly centrifuged before use. Enzyme reagents should be taken out of -20℃, briefly centrifuged (less than 4000 rpm), and placed in an ice bath for later use. Label the PCR tubes and add the corresponding components as shown in Tables 3 and 4:

[0137] Table 3: Enzyme digestion reaction system of pUC-57 and pHR_LV-NS1

[0138]

[0139]

[0140] Table 4: pcDNA3.1(+)-A02 enzyme digestion reaction system

[0141]

[0142] After mixing the above solutions, use a microcentrifuge to shake the solution to concentrate it at the bottom of the tube. Place the tube in a 37°C incubator for 3 hours for enzyme digestion. After the enzyme digestion is completed, incubate at 65°C for 5-10 minutes to terminate the enzyme digestion and prevent DNA end annealing. Then place the tube on ice to cool down rapidly for later use.

[0143] (2) Purification by agarose gel electrophoresis

[0144] Prepare a 1% agarose gel, cool to 60°C, pour into a prepared horizontal electrophoresis tank, and allow to solidify. Slowly add electrophoresis buffer (1×TAE) until the gel is completely submerged. Remove the comb vertically. During sample loading, add the lentiviral vector required for subsequent cell transfection along with the gel. The loading order is: lentiviral vector, target gene plasmid digestion product, marker. Use a micropipette to add samples to the sample wells of the gel. Replace the pipette tip after each sample to prevent contamination. Load samples gently to avoid damaging the gel surface around the sample wells. Immediately after loading, plug in the power and adjust the voltage to start the electrophoresis program (100V, approximately 30 minutes). The samples will begin to move from the negative electrode (black) to the positive electrode (red). Stop electrophoresis when the bromophenol blue reaches approximately 1 cm from the bottom edge of the gel. Remove the gel plate, stain with ethidium bromide solution for 30 minutes, and place it under UV light in a multi-functional gel imaging system to begin gel cutting. MC2-TCR H and MC2-TCR HM The band at 1.8kb was excised; the band at 2.8kb was excised with pcDNA3.1(+)-A02; the band at 1.4kb was excised with pUC-57-MC2-A02; and the band at 8.9kb was excised with lentivirus pHR_LV-NS1. After gel excision, the corresponding target fragments were recovered according to the gel recovery kit procedure.

[0145] (3) DNA purification and recovery

[0146] The gel extraction kit was brought to room temperature beforehand, ensuring all reagents were clear and free of precipitation. The adsorption column was placed in a collection tube, 500 μL of equilibration buffer was added, and the column was centrifuged at 12,000 rpm for 1 min. The waste liquid was discarded, and the adsorption column was reassembled for later use. The target DNA band was weighed, and PN solution was added to each gel band at a ratio of 0.1 g to 100 μL. The gel was incubated at 50°C until the gel block was completely dissolved. The solution was brought to room temperature, and the solution was added to the equilibrated adsorption column using a pipette, clearly labeled to prevent confusion. The column was allowed to stand at room temperature for 2 min, centrifuged at 12,000 rpm for 1 min, and the waste liquid was discarded. 600 μL of wash buffer was added, and the column was centrifuged at 12,000 rpm for 1 min, and the waste liquid was discarded. This washing process was repeated once, followed by centrifugation at 12,000 rpm for 2 min, and the waste liquid was discarded. The adsorption column was then removed and allowed to air dry completely at room temperature. The adsorption column was placed in a sterile, enzyme-free centrifuge tube, and 50 μL of elution buffer was added to the adsorption membrane. The column was allowed to stand at room temperature for 2 min, and then centrifuged at 12,000 rpm for 2 min to collect the target fragment solution. The concentration and purity of the recovered solution were determined using a spectrophotometer.

[0147] (4) Detection of the purity of the target gene fragment by agarose gel electrophoresis.

[0148] The agarose gel electrophoresis method is the same as above. The loading order is 1kb marker, lentiviral vector, and target gene recovery product. Observe the band positions under UV light to see if they are correct.

[0149] (5) Ligation reaction of target gene and lentiviral vector

[0150] The purified lentiviral vector pHR_LV-NS1 (vector) and the target gene fragment (insert) were ligated. Reagents were prepared and added to labeled PCR tubes according to the system in Table 5 for the ligation reaction, which was carried out at 25°C for 1 hour. MC2-TCR insertion was then performed. H and MC2-TCR HM The lentiviral plasmids containing the target gene fragment were named pHR_LV-NS1-MC2-TCR. H and pHR_LV-NS1-MC2-TCR HM The lentiviral expression vectors for target cells after ligation were named pHR_LV-NS1-A02 and pHR_LV-NS1-MC2-A02, respectively.

[0151] Table 5: Target Gene Ligation Reaction System

[0152]

[0153] (6) Transformation and identification

[0154] ① Preparation of competent cells: Take the purchased Escherichia coli DH5α from the -80℃ freezer and thaw it on ice. Inoculate 20 μl of Escherichia coli DH5α glycerol bacteria into 2 ml of pre-prepared LB liquid medium and incubate overnight at 37℃ with shaking. The next day, centrifuge the above bacterial suspension at 4℃, 5000 rpm for 2 min and recover the bacterial cells. Carefully remove the supernatant with a pipette in a clean bench, then resuspend the bacterial cells in 400 μl of pre-cooled 0.1M CaCl2 solution, gently pipette to mix, add an appropriate amount of sterile glycerol, mix well, and incubate on ice for 20 min. Aliquot a portion of the prepared competent cells and store them at -80℃, reserving an appropriate amount to keep on ice for later use.

[0155] ② Transformation and culture: Add 100 μL of competent cells (DH5α) to a labeled centrifuge tube, gently tap to mix, and incubate on ice for 30 min to warm up; add 10 μL of the ligation product obtained in the previous step, incubate on ice for 30 min, heat shock at 42℃ for 1 min, and then incubate on ice for 2 min; add 400 μL of LB liquid medium without antibiotics to the tube, and then place it on a shaker at 250 rpm and 32℃ for 1 h; add 100 μL of bacterial culture to Amp solid medium, spread evenly with a sterile spreader, and culture overnight at 32℃. Observe the single colonies that have grown the next day. Pick 8 single colonies from each sample, and inoculate each single colony into a tube containing 1 mL of LB-Amp liquid medium. Vortex mix and then place on a shaker at 250 rpm and 32℃ for 2 h.

[0156] ③PCR amplification, based on partial sequence information of the pHR_LV-NS1 lentiviral vector and MC2-TCR H MC2-TCR HM Sequence information, in pHR_LV-NS1-MC2-TCR H A fragment of approximately 683 bp was selected and analyzed in pHR_LV-NS1-MC2-TCR. HM A fragment of approximately 526 bp was selected, and primers were designed as follows: pHR_LV-NS1-MC2-TCR H The gene sequence of -F is shown in SEQ ID NO:05; pHR_LV-NS1-MC2-TCR H The gene sequence of -R is shown in SEQ ID NO:06; pHR_LV-NS1-MC2-TCR HM The gene sequence of -F is shown in SEQ ID NO:07; pHR_LV-NS1-MC2-TCR HM The gene sequence of -R is shown in SEQ ID NO:08.

[0157] ④ Using partial sequences from the pHR_LV-NS1 lentiviral vector into the target gene fragments A02 and MC2-A02 as templates, a gene fragment of approximately 1327 bp was selected from pHR_LV-NS1-A02, and a fragment of approximately 1300 bp was selected from pHR_LV-NS1-MC2-A02. Primers were designed as follows:

[0158] The gene sequences of pHR_LV-NS1-A02-F are shown in SEQ ID NO:09; the gene sequences of pHR_LV-NS1-A02-R are shown in SEQ ID NO:10; the gene sequences of pHR_LV-NS1-MC2-A02-F are shown in SEQ ID NO:11; and the gene sequences of pHR_LV-NS1-MC2-A02-R are shown in SEQ ID NO:12.

[0159] ⑤ According to Table 6, take the bacterial culture of all 8 samples in each group and perform PCR. Mark the tube wall and add PCR reaction reagent on ice.

[0160] Table 6: PCR reaction procedure

[0161]

[0162] After vortexing and mixing, place the mixture in a PCR instrument and proceed with the reaction program in Table 7.

[0163] Table 7: PCR reaction system

[0164]

[0165] ⑥ Screening correct clones by agarose electrophoresis: Take 6 μL from each sample tube in step ⑤ and add it to the well of the agarose gel sample. Follow the electrophoresis procedure above. After electrophoresis, observe whether the band position of each clone sample meets the expectations.

[0166] ⑦ Lentiviral plasmid amplification: Select the corresponding bacterial culture that was cloned with the correct band position in step ⑥, take 1 mL and inoculate it into a T75 culture flask containing 100 mL LB-Amp liquid medium, incubate overnight at 32°C at 250 rpm, and control the total shaking time within 15 h.

[0167] (7) Plasmid extraction

[0168] Prepare the reagent kit. Add equilibration buffer to the adsorption column, centrifuge, and discard the waste liquid. Add 100 mL of overnight culture to a centrifuge tube and centrifuge at 8000 rpm for 3 minutes at room temperature. Aspirate as much supernatant as possible to minimize liquid residue. Add 8 mL of solution P1 to the precipitate in the centrifuge tube and vortex thoroughly to mix. Add 8 mL of solution P2 and gently invert the centrifuge tube several times to promote complete cell lysis. Let stand at room temperature for 5 minutes. Add 8 mL of solution P4 and immediately gently invert the centrifuge tube several times to mix thoroughly until a white precipitate appears. Let stand at room temperature for 10 minutes. Centrifuge at 8000 rpm for 10 minutes to allow the white precipitate to settle at the bottom of the tube. Pour all the supernatant into a CS1 filter and slowly push the filter handle to collect the filtrate in a clean container. In a centrifuge tube, observe the filtrate volume markings. Add 0.3 times the volume of isopropanol to the filtrate, mix thoroughly by inverting the tube, and transfer it to the adsorption column CP6 in two portions according to volume. Centrifuge at 8000 rpm for 2 minutes and discard the waste liquid. Add 10 mL of wash buffer PW, centrifuge at 8000 rpm for 2 minutes and discard the waste liquid. Repeat once. Add 3 mL of anhydrous ethanol to the adsorption column, centrifuge at 8000 rpm for 2 minutes and discard the waste liquid. Centrifuge again at 8000 rpm for 5 minutes to completely remove the anhydrous ethanol. Place the adsorption column in a clean centrifuge tube, add 1 mL of elution buffer TB dropwise to the center of the adsorption membrane, let stand at room temperature for 5 minutes, and then centrifuge at 8000 rpm for 2 minutes to obtain the plasmid-containing solution. Transfer all the solution to a clean EP tube and store at -20℃.

[0169] (8) Recombinant plasmid enzyme digestion verification: Prepare the reagents according to Table 8, shake and centrifuge briefly to mix, place in a 37℃ water bath for 2 hours for enzyme digestion reaction, and perform agarose electrophoresis on the digestion product (steps as before) to observe whether the recombinant plasmid meets the expected position to determine whether the construction is successful.

[0170] Table 8: Enzymatic digestion system of recombinant vector

[0171]

[0172] (9) A summary of recombinant expression vectors is shown in the table below.

[0173] Table 9: Recombinant plasmids used for cell transfection

[0174]

[0175] 6. Establishment of the MC2-TCR-T cell line

[0176] (1) Lentiviral packaging

[0177] ① Prepare 293T cells. When the cells are in good logarithmic growth phase, digest and centrifuge them to collect and count them, according to 0.6 × 10⁻⁶ cells / year. 6Seeds were planted into 6-well plates and cultured for about 6 hours until the cells were fully attached. Then, the medium was replaced with a medium without antibiotics and the cells were cultured until the next day.

[0178] ② Take a 1.5 mL EP tube, label it, and put 3.3 μg of main plasmid (pHR_LV-NS1-MC2-TCR) into it. H or pHR_LV-NS1-MC2-TCR HM 1.6 μg of auxiliary plasmid psPAX2 and 1.6 μg of auxiliary plasmid pMD2.G were mixed evenly by pipetting and allowed to stand.

[0179] ③ Take 15mL centrifuge tubes and label them. Add 100μL of Opti-MEM and 13μg of PEI to each tube (the ratio of plasmid to PEI is 1:2). Mix well by pipetting and let stand.

[0180] ④ Transfer the plasmid mixture in the EP tube to the corresponding 15mL centrifuge tube, mix by pipetting and vortexing, and then let it stand at room temperature for 20 minutes.

[0181] ⑤ After standing, add 2 mL of DMEM high-glucose complete medium containing 10% FBS (heat-inactivated) to the mixture in the 15 mL centrifuge tube; take out the 6-well plate with 293T cells in step a, mark it accordingly, aspirate the medium, slowly add the above mixture along the well wall, put it back into the CO2 incubator, and observe it from time to time.

[0182] ⑥ After 6 hours, change the medium, discard the original medium, add 2 mL of DMEM high glucose medium without serum along the wall to rinse once, being careful not to blow the cells. Then add 2 mL of DMEM high glucose complete medium along the wall and continue culturing in a CO2 incubator for 48 hours.

[0183] ⑦ Aspirate the supernatant from the above 6-well plate into the corresponding 15mL centrifuge tubes marked with the label, centrifuge at 3000rpm for 5min at 25℃ to remove cell residue, then filter with a 0.45μm filter membrane, and use the collected virus aliquots or freeze them at -80℃ for storage.

[0184] (2) Activation of T cells

[0185] PBMCs isolated from whole blood require activation before transfection. This is achieved by adding T Cell TansAct and human interleukin-2 (IL-2) to RPMI-1640 complete medium at a ratio of 10 μL T Cell TansAct and 6 μL IL-2 (IL-2 stock solution concentration 100 U / μL, final concentration 600 U / mL). After resuscitation, the PBMCs are centrifuged to remove the cryopreservation solution, and the cell count is approximately 2 × 10⁻⁶. 6Each PBMC was resuspended in 2 mL of complete culture medium containing TansAct and IL-2 and transferred into a 24-well plate for 48 h to activate T cells.

[0186] (3) Transfection of target cells

[0187] The day before transduction, 24-well plates were coated with RetroNectin and incubated overnight at 4°C. The following day, the RetroNectin-coated 24-well plates were retrieved, and 2 mL of pre-packaged virus was added to each well. Target cells (PBMCs need to be activated before transduction) were then added, with 0.5 × 10⁶ PBMCs per well. 6 Each well contains 500 μL of target cells and the corresponding culture medium. After mixing, the 24-well plates are sealed with a protective film and centrifuged at 2000 rpm for 90 min at room temperature. A medium-speed ramp-up and slow-speed deceleration are used to allow cell sedimentation and increase virus transfection efficiency. After centrifugation, the sealing film is removed, and the plates are placed in a CO2 incubator. After 24 hours, 1 mL of supernatant is carefully aspirated, and 1 mL of the corresponding complete culture medium is added. Cell status is monitored in real time. Depending on the results, the culture is expanded, with some cells used for subsequent experiments and the remaining cells cryopreserved. Cells obtained after PBMC transduction are MC2-specific TCR-T cells. The negative control group cells are named Vector, expressing original human TCR. H The TCR-T cells with the sequence were named MC2-TCR. H -T indicates C-region mouse-derived optimized TCR HM The TCR-T cells with the sequence were named MC2-TCR. HM -T.

[0188] 7. Synthesis of MC2 HLA-A02 Tetramer-PE

[0189] The TCR-T cells (MC2-TCR) obtained by the above transfection H -T, MC2-TCR HM -T) should express the HLA-A*02:01-restricted MAGE-C2 target. 336–344 (ALKDVEERV)-specific TCR. After obtaining the target cells, MC2-TCR expression must be detected, i.e., the ability of TCR-T cells to bind the corresponding pMHC. This step will use MHC tetramer (MHC Tetramer) to label the corresponding TCR, and the TCR-T cells will be detected at the single-cell level by flow cytometry. MBL was commissioned to synthesize T-Select MHC I Tetramer for labeling MAGE-C2-specific TCR-T cells, the full name of which is T-SelectHLA-A*02:01Tetramer-MAGE-C2.336–344 -ALKDVEERV-PE, hereinafter referred to as MAGE-C2-Tetramer-PE. Tetramer is composed of four MHC / antigen complex monomers linked to streptavidin via lysine residues. Streptavidin carries a fluorescent label. After co-incubation with specific T cells, Tetramer can specifically bind to multiple corresponding TCRs, such as... Figure 4 As shown, the recombinant protein MHC class I heavy chain, β2-microglobulin (β2m), and antigenic polypeptide fragment are folded to form a soluble MHC class I / polypeptide complex monomer. The lysine residues at the C-terminus of the MHC class I heavy chain in the complex monomer are biotinylated using BirA enzyme. The biotinylated complex monomer is purified by column chromatography. The purified biotinylated monomer binds to streptavidin labeled with a fluorescent dye to form a tetramer, thus completing the preparation of T-Select MHC Tetramer. When co-incubated with T cells, the Tetramer specifically and stably binds to multiple TCRs, and the proportion or number of labeled T cells can be quantitatively detected by flow cytometry. This binding exhibits higher stability, allowing for subsequent quantitative detection of the specific proportion or number of T cells by flow cytometry.

[0190] 8. Detection of the expression efficiency of MC2-specific TCRs

[0191] pHR_LV-NS1-MC2-TCR H and pHR_LV-NS1-MC2-TCR HM Two types of TCR-T cells were obtained after transducing PBMC cells: MC2-TCR H -T and MC2-TCR HM -T, PBMCs transduced with the pHR_LV-NS1 empty vector served as the control vector. On day 5 after transduction, cells in each group were double-stained with MAGE-C2-Tetramer-PE and CD8-APC, and flow cytometry was used to detect the ability of TCR-T cells to bind MC2. 336–344 The percentage of CD8+ T cells with (ALKDVEERV) antigen.

[0192] 9. Flow cytometry combined with intracellular cytokine staining was used to detect the intracellular release level of IFN-γ in MC2-TCR-T cells stimulated with peptide-loaded T2 cells.

[0193] (1) Peptide loading on T2 cells

[0194] T2 cells are a tap-deficient human lymphoblastic cell line that expresses HLA-AO2 but lacks antigen peptide transporters, thus failing to present endogenous HLA. T2 cells stimulated by target antigen peptides... MC2 Cells can faithfully present the original form of a target antigen peptide to the cell surface. The process by which an exogenous target antigen peptide stimulates T2 cells is called peptide loading.

[228] In this study, T2 cells loaded with the control peptide were hereinafter referred to as T2 cells. C Cells loaded with chemically synthesized MAGE-C2 336–344 –ALKDVEERV peptide (hereinafter referred to as MC2 peptide) T2 cells are hereinafter referred to as T2 MC2 cell.

[0195] Prepare a 96-well round-bottom plate. Take pre-cultured T2 cells, centrifuge at 100 rpm for 5 min at 25°C, resuspend in complete culture medium to adjust cell concentration, and divide into 0.4 × 10⁶ cells per well. 6 Seed cells at 100 μL / 100 μL. Add 2 μL of control peptide or synthetic MC2 peptide (peptide storage solution concentration 1 mM) to each well of 100 μL T2 cell suspension and incubate in a CO2 incubator for 2 h to perform peptide loading of T2 cells.

[0196] (2) Cell co-culture and flow cytometry detection

[0197] pHR_LV-NS1 empty vector, pHR_LV-NS1-MC2-TCR H or pHR_LV-NS1-MC2-TCR HM On day 9 of PBMC transduction, the proportion of TCR-T cells that produced intracellular IFN-γ secretion after stimulation with peptide-loaded T2 cells was detected using intracellular gamma interferon staining (ICS). ICS works by blocking the extracellular secretion of cytokines, causing them to accumulate intracellularly. After membrane permeation, antibodies bind to specific intracellular factors, resulting in stronger and more accurate cytokine fluorescence signals. Combined with cell surface staining, this allows analysis of the percentage of cells capable of releasing cytokines within a given T cell population.

[0198] 10. Detect the secretion of TNF-α by MC2-TCR-T cells under the stimulation of peptide-loaded T2 cells.

[0199] (1) TCR-T cells and peptide-loaded T2 cells were counted separately, each at a value of 0.4 × 10⁻⁶. 6 Each cell / well was thoroughly mixed with effector cells and target cells (E:T) at a ratio of 1:1 and then placed back into the incubator for co-culture for 24 hours.

[0200] (2) Before use, bring the ELISA kit to room temperature, prepare the necessary reagents and serial dilutions of standards, and set up replicates for both standards and samples. Use 6,000 pg / ml standard solution for 2-fold serial dilutions, setting up 12 gradients. Use 1X sample dilution B as the zero standard (0 pg / ml). According to the experiment, disassemble the corresponding 96-well plate kit, mark it, and remove the remaining strips and put them back in the aluminum foil bag for future use.

[0201] (3) Add 200 μL of 1X washing solution Item B to the well plate for 30 seconds, discard the solution, and then invert it onto the filter paper and pat it dry. The experiment should be conducted as continuously and quickly as possible to avoid drying out the well plate.

[0202] (4) Add 100 μL of each gradient standard and 100 μL of cell supernatant sample to a 96-well plate according to the number of replicates, and incubate gently at room temperature for 2.5 h.

[0203] (5) After incubation, aspirate the solution from the wells and wash four times with 200 μL 1X Item B washing solution, patting thoroughly dry to remove as much liquid as possible. After the last wash, use a pipette to completely remove any remaining washing solution, invert the plate over clean filter paper, and drain the liquid.

[0204] (6) Gently mix Item F (detection antibody), add 100 μl of 1X Item E to prepare a detection antibody concentrate and mix well (the concentrate can be stored at 4°C for 5 days). Before use, dilute the detection antibody concentrate 80 times with 1X (Item E) to prepare 1X Item F, add 100 μL of 1X Item F to each well, and incubate on a shaker at low speed for 1 h at room temperature.

[0205] (7) After incubation, discard the solution in the well and repeat the washing steps in (4).

[0206] (8) Gently rotate Item G (HRP-streptavidin concentrate bottle) to mix it well. Take an appropriate amount of Item G and dilute it 600 times with 1X Item E and mix well for later use. Add 100μL of diluted Item G to each detection well and gently shake and incubate at room temperature for 45min.

[0207] (9) Discard the solution and repeat the washing steps in (4).

[0208] (10) After washing, add 100 μL of Item H (TMB one-step substrate reagent) to each well and incubate gently with shaking at room temperature in the dark for 30 min.

[0209] (11) After incubation, add 50 μl of stop solution to each well and immediately read the value at 450 nm in the microplate reader.

[0210] (12) Calculation of ELISA results: Calculate the average OD value of each set of replicate standards, control standards and samples. Subtract the average OD value of the zero concentration standard from the average OD value. In Excel, the horizontal axis is the standard concentration and the vertical axis is the OD value. Use the Logit-log linear regression method to draw the best fit line to obtain the calculation formula and calculate the concentration value corresponding to each OD value.

[0211] 11. Preparation of tumor target cell lines

[0212] The lentivirus packaging and transfection procedures are the same as described above. In a 24-well plate coated with RetroNectin, add 2 mL of the packaged virus to each well, and then add 0.4 × 10⁴ K562 cells to the corresponding well. 6 After 24 hours, 1 mL of supernatant was aspirated, and 1 mL of the corresponding complete culture medium was added. Cell status was observed in real time. After scaling up the culture as needed, some cells were used for subsequent experiments, and the remaining cells were cryopreserved. The negative control group cells were named Vector (transfected with pHR_LV-NS1 empty vector); A02-K562 (transduced with pHR_LV-NS1-A02) were control target cells expressing only HLA; MC2-A02-K562 (transduced with pHR_LV-NS1-MC2-A02) were tumor target cells expressing HLA-A*02:01-restricted MAGE-C2. 336–344 (ALKDVEERV).

[0213] 12. Purification of the A02-K562 cell line

[0214] The pLeo1209 puromycin resistance gene was prepared on the surface of A02-K562 cells, so puromycin can be used to screen and purify the transfected cells.

[0215] (1) Weigh 10 mg of puromycin powder and dissolve it in 10 mL of PBS. Mix well to obtain a 1 mg / mL puromycin solution.

[0216] (2) Take 20 mL of transfected A02-K562 cell suspension, add 20 μL of the 1 mg / mL puromycin solution obtained in the previous step, mix well and put it in an incubator for further culture.

[0217] (3) After culturing for 48 hours, observe the cell status. Cells without the puromycin resistance gene will die, while cells transduced with the puromycin resistance plasmid will grow normally. After centrifugation, discard the supernatant and resuspend the cells in complete culture medium containing Puromycin reagent to continue purifying the cells.

[0218] (4) After culturing for another 48 hours, the A02-K562 cells containing the resistance gene continued to proliferate normally. At this time, the cells can be resuspended in normal complete culture medium to obtain purified A02-K562 cells.

[0219] 13. Sorting and purification of the MC2-A02-K562 cell line

[0220] (1) Take 1.0 × 10 7 Add 6 mL of sorting buffer to a 15 mL centrifuge tube, mix well by pipetting, centrifuge at 1000 rpm for 5 min and discard the supernatant;

[0221] (2) Resuspend the cell pellet in 180 μL sorting buffer to form a single cell suspension, add 20 μL HLA-A2-PE antibody and mix by pipetting, incubate at 4°C for 20 min, and shake to mix once every 5 min during incubation.

[0222] (3) After incubation, add 6 mL of sorting buffer, centrifuge at 1000 rpm for 5 min, and discard the supernatant;

[0223] (4) Add 80 μL of sorting buffer to resuspend the cell pellet, then add 20 μL of Anti-PE Microbeads and mix by pipetting. Incubate at 4°C for 20 min, shaking to mix every 5 min during incubation.

[0224] (5) After incubation, add 6 mL of sorting buffer to the cell suspension and mix well. Centrifuge at 1000 rpm for 5 min. At the same time, install the LS sorting column into the magnetic rack and rinse the LS sorting column once with 3 mL of sorting buffer.

[0225] (6) Discard the supernatant of the centrifuged cells and pipette them into a single-cell suspension with 3 mL of sorting buffer. Then add the suspension to the rinsed LS sorting column. When the cell suspension has finished flowing, add 5 mL of sorting buffer to rinse. Repeat twice.

[0226] (7) Remove the rinsed LS sorting column and place it into a clean 15mL centrifuge tube. Add 5mL sorting buffer to the column, push the cells down, add 5mL RPMI-1640 and mix by pipetting. Centrifuge at 1000rpm for 5min.

[0227] (8) After centrifugation, resuspend the cell pellet in 10 mL of RPMI-1640 medium and aspirate it into a culture flask for culture or use in experiments.

[0228] 14. Detection of pMHC expression efficiency in tumor target cell lines

[0229] The purified A02-K562 and MC2-A02-K562 cell suspensions were incubated with HLA-A2-PE antibody and HLA-A02 expression was detected by flow cytometry. The control vector was K562 cells transfected with the empty vector.

[0230] (1) Take 500 μL of the corresponding cell suspension from each culture flask and label them as Vector, A02-K562, and MC2-A02-K562 cells, respectively. Add 1 mL of FACS Buffer to each EP tube and centrifuge at 3000 rpm for 5 min at room temperature. During centrifugation, prepare HLA-A2-PE antibody according to the ratio of HLA-A2-PE:FACS Buffer = 1:9. Each reaction requires 50 μL of the above solution to prepare sufficient antibody solution.

[0231] (2) After centrifugation, discard the supernatant, add 50 μL of the HLA-A2-PE mixture prepared above to each reaction, mix by pipetting, incubate at 4°C for 30 min, then add 1 mL of FACS Buffer and centrifuge at 3000 rpm for 5 min.

[0232] (3) After centrifugation, discard the supernatant, resuspend in 300 μL FACS Buffer, and perform flow cytometry. Use HLA-A2-PE as the x-axis for single-parameter analysis of stained cells.

[0233] 15. Detect the intracellular release level of IFN-γ in MC2-TCR-T cells under the stimulation of tumor target cells.

[0234] After the target cells were transduced and purified, they were cultured to a suitable quantity, and then the two types of TCR-T cells (MC2-TCR) were detected. H -T and MC2-TCR HM Intracellular release of IFN-γ after TCR-T cells were cross-incubated with A02-K562 and MC2-A02-K562 cells, respectively. K562 cells transfected with the empty vector (Vector) served as the control group. The effector-target ratio of TCR-T cells to target cells was 1:1. Cells were grown at 0.4 × 10⁻⁶ cells per well. 6100 μL of cells were seeded into 96-well round-bottom plates, gently mixed, and 3 μL of blocking buffer was added to each well. The plates were then incubated in a CO2 incubator for 24 h to allow the two cell lines to interact. The remaining steps were the same as in section 2.2.2.9 of this chapter. Flow cytometry results were plotted with CD8-APC on the x-axis and IFN-γ-FITC on the y-axis to analyze the staining of TCR-T cells.

[0235] 16. Detection of TNF-α secretion by MC2-TCR-T cells under the stimulation of tumor target cells.

[0236] TCR-T cells and tumor target cells were counted separately, and after being thoroughly mixed at a 1:1 ratio, they were incubated together in an incubator for 24 hours. Before the experiment, the ELISA kit was brought to room temperature, and the required reagents and serial dilutions of the standards were prepared. The experimental steps were the same as in Example 10, detecting the secretion of TNF-α by MC2-TCR-T cells under the stimulation of peptide-loaded T2 cells.

[0237] 17. Detection of MC2-TCR HM - T cell killing efficiency against tumor target cells

[0238] CFSE dye was used to label the target cells used in the reaction to distinguish between target cells and effector cells. CFSE-labeled target cells were co-cultured with different TCR-T cells for a certain period of time, and the number of CFSE-labeled target cells after co-culture was detected by flow cytometry.

[0239] (1) CFSE target cell labeling: Prepare CFSE stock solution in advance according to the instructions. A02-K562 cells and MC2-A02-K562 cells were resuspended, counted, and their cell density adjusted to 1×10⁶ cells per cell. 6 1 mL of target cells per mL were introduced into a labeled centrifuge tube. 0.4 μL of 2.5 mM CFSE stock solution was added to each of the target cell centrifuge tubes. The final CFSE concentration was 1 μM. The cells were mixed by pipetting and incubated at 37°C in the dark for 20 min, vortexing every 5 min to mix. After incubation, 5 volumes of RPMI 1640 medium containing 10% FBS were added to each tube, and the cells were incubated on ice for 5 min to terminate staining. This termination step was repeated twice to ensure that the CFSE dye bound to the protein in the supernatant was removed. After the final termination step, the cells were centrifuged to remove the supernatant, and then resuspended in 1 mL of complete RPMI 1640 medium containing 10% FBS. The CFSE-labeled target cells were prepared as follows: A02-K562-CFSE and MC2-A02-K562-CFSE.

[0240] (2) Take 100 μL of the above-mentioned A02-K562-CFSE or MC2-A02-K562-CFSE cells and inoculate them into labeled 96-well round-bottom plates, which is equivalent to 0.1 × 10⁶ target cells per well. 6 indivual.

[0241] (3) Prepare MC2-TCR HM -T cells, MC2-TCR were added to the corresponding wells at an effector-target ratio of 1:1. HM -T cells (or other TCR-T cells) 100 μL (0.1 × 10⁻⁶ cells per well) 6 After gently mixing, the cells were placed in a cell culture incubator and incubated for 24 hours. The number of CFSE-labeled cells and the percentage of the cell population were then detected by flow cytometry.

[0242] (4) The derivation process of the lethality formula:

[0243] At the start of the experiment, the number of effector cells and target cells was equal in each group, therefore the following equation can be derived:

[0244]

[0245] Assuming no killing effect, after 14 hours of cell-cell interaction, x = (a × b) ÷ c. However, if MC2-A02-K562 cells interact with MC2-TCR... HM If T cells exhibit specific killing activity, then the number of MC2-A02-K562 target cells lost through killing should be xd (where d is the number of surviving MC2-A02-K562 cells after treatment). Therefore, the killing rate can be calculated using the following formula:

[0246]

[0247] The density of CFSE-stained cells after co-culturing effective and target cells was obtained by flow cytometry. The corresponding values ​​were then substituted into the calculation formula to obtain the MC2-TCR. HM -T cell killing rate against target cells MC2-A02-K562.

[0248] Statistical methods

[0249] Statistical analysis was performed using Prism version 8.0 (Graph Pad). For data comparing between groups, t-tests were used. One-way ANOVA was used to detect the significance of differences between groups. *p<0.05 indicates a statistically significant difference; **p<0.01 indicates a statistically significant difference; ***p<0.001 indicates an extremely statistically significant difference.

[0250] II. Experimental Results:

[0251] 1. Construction and validation of recombinant expression plasmids

[0252] The two target gene sequences for expressing engineered TCRs are MC2-TCR. H and MC2-TCR HM MC2-TCR H Contains unmodified TCR-Vα3 and TCR-Vβ28 sequences, MC2-TCR HM It contains mouse-optimized TCR-Vα3 and TCR-Vβ28 sequences. Both target HLA-A02-restricted MAGE-C2. 336-344 (ALKDVEERV) antigen. Insert the target gene fragment MC2-TCR H The pUC-SP recombinant cloning vector is called pUC-SP-MC2-TCR. H Loading the C-region mouse-derived replacement sequence MC2-TCR HM The recombinant cloning vector is called pUC-SP-MC2-TCR HM The two recombinant cloning vectors have identical structures, and their plasmid maps are pUC-SP-MC2-TCR. HM For example, Figure 5 As shown in C, the recombinant cloning vector pUC-SP-MC2-TCR HM The target gene carried is MC2-TCR HM It contains the α-chain sequence (TCRα3) and β-chain sequence (TCRβ28) that replace the mouse gene sequence in region C. The α and β chains are linked by the self-cleaving polypeptide P2A sequence. The red boxes indicate the restriction sites NotI and SalI used in this experiment.

[0253] For the recombinant cloning vector pUC-SP-MC2-TCR H pUC-SP-MC2-TCR HM The target gene fragment MC2-TCR was obtained by double digestion of the NotI and SalI restriction sites of the lentiviral expression vector pHR_LV-NS1. H MC2-TCR HM And the linear enzyme digestion product fragment of the lentiviral expression vector pHR_LV-NS1, and the target gene fragment MC2-TCR H The total length is 1806bp, MC2-TCR HM The full-length pHR_LV-NS1 linear fragment is 1821 bp, and its full-length length is 8900 bp. After agarose gel electrophoresis, the enzyme digestion products yielded two expected bands near the 2.0 kb and 8.0 kb positions, as shown below. Figure 5As shown in Figure A, lane M contains a 1Kb DNA Marker; lane 1 contains pUC-SP-MC2-TCR. H Double enzyme digestion product; lane 2 contains pUC-SP-MC2-TCR HM Double digestion product; lane 3 contains pHR_LV-NS1 double digestion product.

[0254] After gel extraction, the target band was purified by gel electrophoresis. The purified product was then subjected to agarose gel electrophoresis again to observe the band position. The linear restriction enzyme fragment of pHR_LV-NS1 was approximately 8.9 kb, located around 8.0 kb. The target gene fragment MC2-TCR was also observed. H and MC2-TCR HM Around 2.0Kb, the bands were confirmed to be in line with expectations. Figure 5 As shown in Figure B, lane M contains a 1 kb DNA marker; lane 1 contains a pHR_LV-NS1 double-digested linear fragment (approximately 8.9 kb); and lane 2 contains the target gene fragment pUC-SP-MC2-TCR. H Double enzyme digestion product (approximately 1.8 Kb); lane 3 contains the target gene fragment pUC-SP-MC2-TCR. HM Double enzyme digestion product (approximately 1.8 kb).

[0255] The above-mentioned target gene MC2-TCR was recovered and purified. H and MC2-TCR HM The recombinant lentiviral expression vector pHR_LV-NS1-MC2-TCR was obtained by ligation reaction with the linear fragment digested by the lentiviral vector pHR_LV-NS1. H and pHR_LV-NS1-MC2-TCR HM After transformation of the above vectors, bacterial culture was selected and amplified. The bacterial culture was then validated by PCR. A gene sequence containing the boundary between the expression vector and the inserted target gene from the recombinant lentiviral expression vector was selected as a template and analyzed using pHR_LV-NS1-MC2-TCR. H The medium length is 526bp, in pHR_LV-NS1-MC2-TCR HM The sequence length is 683 bp. Primers were designed based on the above sequence for PCR amplification. All eight amplification products in each group yielded the expected bands after agarose gel electrophoresis. Figure 6 A and Figure 6 B, Figure 6 Lane M in A is the DNA Marker D2000, and lanes 1 to 8 are the PCR amplification products of positive colonies, approximately 526 bp. Figure 6 In section B, lane M is for DNA Marker D2000, and lanes 1 to 8 are respectively for pHR_LV-NS1-MC2-TCR. HMThe PCR amplification products of the eight selected single-positive colonies were approximately 683 bp. Two recombinant lentiviral expression vector plasmids were amplified and cultured separately. After plasmid uptake, the extracted plasmids were subjected to double enzyme digestion for identification, and gel electrophoresis yielded bands consistent with expectations, such as... Figure 6 As shown in C, lane M1 contains a 1Kb DNA Marker, lane M2 contains a 500Kb DNA Marker, and lane 1 contains pHR_LV-NS1-MC2-TCR. H The double enzyme digestion product, lane 2 contains the recombinant plasmid pHR_LV-NS1-MC2-TCR HM Double enzyme digestion product. Target gene fragment MC2-TCR H and MC2-TCR HM The expression vector fragment is located around 1.8 kb, and the recombinant lentiviral expression vector pHR_LV-NS1-MC2-TCR is around 8.9 kb. H and pHR_LV-NS1-MC2-TCR HM The plasmid was successfully constructed and can be used for subsequent lentivirus packaging and transfection of cells. After aliquoting, it was stored at -80°C. The plasmid map is represented by pHR_LV-NS1-MC2-TCR. HM For example, Figure 6 As shown in D, the promoter of the lentiviral expression vector pHR_LV-NS1 is the SFFV promoter. The target gene is MC2-TCR. HM The lentiviral expression vector pHR_LV-NS1 is inserted between the NotI and SalI restriction sites in pHR_LV-NS1, with a full length of 10721 bp. The lentiviral expression vector used in this experiment is pHR_LV-NS1, which is a modified version of the pLVX-Puro vector. The core design involves replacing the CMV promoter in the original pLVX-Puro vector with the SFFV promoter.

[0256] 2. MC2-TCR expressing optimized mouse-derived sequences HM -T exhibits higher TCR expression efficiency and stronger pMHC affinity.

[0257] The recombinant expression vector pHR_LV-NS1-MC2-TCR was detected. H and pHR_LV-NS1-MC2-TCR HM Lentiviral particles were co-transfected into HEK-293T cells with packaging plasmids psPAX2 and pMD2.G, respectively, and incubated in an incubator for approximately 54 hours before centrifugation to collect the lentiviral particles. The collected viral particles were then used to transfect PBMC cells activated with TansAct and IL-2 to obtain MAGE-C2-specific TCR-T cells (MC2-TCR-T). pHR_LV-NS1-MC2-TCR... HThe cell containing the viral particles was named MC2-TCR H -T, this TCR-T cell line expresses primitive human TCR targeting MAGE-C2. H ; Transfection with pHR_LV-NS1-MC2-TCR HM The cell containing the viral particles was named MC2-TCR HM -T, this TCR-T cell line expresses a TCR targeting MAGE-C2 that undergoes murine sequence substitution in the C region. HM， like Figure 7 A, TCR in the figure H For the original human TCR targeting MC2; TCR HM This is a human-mouse hybrid TCR targeting the mouse-derived C region of MC2; Cαm represents the mouse-derived α-chain C region; Cβm represents the mouse-derived β-chain C region; the α and β chain sequences of the target gene are linked by a self-cleaving polypeptide P2A sequence. During translational expression, the self-cleaving polypeptide P2A breaks, and the α and β chains are expressed and assembled independently. Vector cells were used as control cells transfected with the empty vector. This study aimed to clarify the expression of the TCR by transfecting CD8+ T cell populations. H or TCR HM The efficiency of Vector and MC2-TCR H -T and MC2-TCR HM T cells were incubated with MAGE-C2-Tetramer-PE and CD8-APC antibodies, respectively, and the staining status of the cells was analyzed by flow cytometry. The unique structure of the Tetramer tetramer allows it to bind with high affinity and high stability to multiple specific TCRs on the surface of T cells. The percentage of cells stained with MAGE-C2-Tetramer-PE / CD8-APC was used to assess the TCR status. H or TCR HM The expression efficiency can also be used to verify the affinity of MC2-TCR-T for targeting and binding to antigen epitopes.

[0258] Flow cytometry results as follows Figure 7 As shown in Figure C, the horizontal axis represents MAGE-C2-Tetramer-PE, and the vertical axis represents CD8-APC; the Vector in the figure is the PBMC transfected empty vector pHR_LV-NS1; MC2-TCR H -T indicates PBMC transfection with pHR_LV-NS1-MC2-TCR H MC2-TCR HM -T indicates PBMC transfection with pHR_LV-NS1-MC2-TCR HM Compared to Vector, MC2-TCR H -T and MC2-TCR HMIn the -T phase, transduction of the recombinant expression vectors all produced a population of CD8+ T cells capable of binding MAGE-C2-Tetramer-PE. The percentage of cells in the MAGE-C2-Tetramer-PE and CD8-APC double-stained populations was [not specified in the original text]. H -T is 8.01%, MC2-TCR HM -T was 23.47%. Results from three independent, repeated transfection and flow cytometry assays, as shown... Figure 7 B displays MC2-TCR HM The proportion of MAGE-C2-Tetramer-PE and CD8-APC double-stained cells in T cells was significantly higher than that in MC2-TCR cells. H -T.

[0259] The results indicate that MAGE-C2-specific TCR-T cells (MC2-TCR-T) were successfully constructed using the recombinant lentiviral expression vector pHR_LV-NS1-MC2-TCR. H and pHR_LV-NS1-MC2-TCR HM Both methods can effectively transfect PBMCs to express MC2-specific TCR molecules; mouse sequence substitutions in the C regions of the α and β chains can further enhance the expression efficiency of exogenous TCRs, resulting in MC2-TCR. HM -T exhibits higher TCR expression efficiency and stronger affinity for pMHC.

[0260] After the recombinant expression vector was transduced into normal donor PBMCs for 54 hours, the cells were incubated with various antibodies, and the antibody staining was detected by flow cytometry. Figure 7 A is TCR H and TCR HM Structural diagram: TCR H For the original human TCR targeting MC2; TCR HM This is a human-mouse hybrid TCR targeting the murine C region of MC2. Cαm represents the murine α-chain C region; Cβm represents the murine β-chain C region. The α and β chain sequences of the target gene are linked by a self-cleaving polypeptide P2A sequence. During translational expression, the self-cleaving polypeptide P2A breaks, and the α and β chains are expressed and assembled independently. Figure 7 B represents the TCR expression efficiency detected in three independent replicate experiments (i.e., the proportion of cells in the MAGE-C2-Tetramer-PE and CD8-APC double-stained cell populations as detected by flow cytometry). Figure 7 C represents the flow cytometry results of MAGE-C2-Tetramer-PE and CD8-APC staining. Vector represents PBMCs transfected with the empty vector pHR_LV-NS1; MC2-TCR. H -T indicates PBMC transfection with pHR_LV-NS1-MC2-TCRH MC2-TCR HM -T indicates PBMC transfection with pHR_LV-NS1-MC2-TCR HM .

[0261] 3. Mouse-based optimized MC2-TCR HM -T cells have a stronger ability to produce IFN-γ when stimulated by T2 cells loaded with MC2 peptide.

[0262] To examine the function of the constructed TCR-T cells in producing and expressing IFN-γ upon stimulation by specific antigen peptides, MC2-TCR... H -T and MC2-TCR HM -T and T2 respectively C Cells (T2 cells loaded with control peptide) and T2 MC2 After co-incubation of cells (T2 cells loaded with MEGE-C2 peptide) for 24 h, intracellular cytokine staining (ICS) with IFN-γ-FITC was performed first, followed by incubation with CD8-APCs. Vector cells transfected with an empty vector were used as a control. The proportion of cells double-stained with IFN-γ-FITC and CD8-APC (i.e., the level of IFN-γ produced by the CD8+ cell population) was detected by flow cytometry. Results are as follows: Figure 8 A shows that Vector via T2 C Or T2 MC2 After cell stimulation, the proportions of cells double-stained with IFN-γ-FITC / CD8-APC were 0.83% and 0.72%, respectively; MC2-TCR expressing the original α and β chain sequences... H -T and MC2-TCR expressing murine C-region sequences HM -T, in relation to T2 C After 24 hours of co-incubation, almost no intracellular IFN-γ expression was induced, and IFN-γ signal levels were consistently low in MC2-TCR. H -T is 0.44%, MC2-TCR HM -T is 0.66%; in T2 MC2 After co-incubation for 24 hours, IFN-γ-FITC and CD8-APC double-stained cell populations were detected in both TCR-T cells, and MC2-TCR cells were also found to be positive. H -T is 9.47%, MC2-TCR HM -T is 21.3%. Statistical results show that, as Figure 8 B, Vector group and T2 C Cells and T2 MC2After the cell reaction, there was no significant difference in the proportion of cells stained with IFN-γ-FITC and CD8-APC double staining; MC2-TCR H -T and MC2-TCR HM -T passes through T2 MC2 After cell stimulation, the proportion of double-stained cells was significantly higher than that of T2 cells. C Cellular response group; MC2-TCR HM -T is affected by T2 MC2 Following cell stimulation, the proportion of cells double-stained with IFN-γ-FITC and CD8-APC was significantly higher than that of MC2-TCR cells. H -T group.

[0263] Figure 8 In the middle, Vector, MC2-TCR H -T and MC2-TCR HM -T was compared with the negative control T2. C Cells (T2 cells loaded with control peptide) and experimental T2 cells MC2 After co-culturing T2 cells (loaded with MEGE-C2 peptide), the CD8+ cell population producing IFN-γ was labeled with antibodies. First, CD8-APC antibodies were incubated, followed by IFN-γ-FITC antibody incubation using an intracellular factor staining method. The stained cells were then analyzed by flow cytometry. Double-stained cells (CD8-APC and IFN-γ-FITC) represent the functional level of IFN-γ production in the CD8+ cell population. Figure 8 A represents the flow cytometry results of IFN-γ-FITC and CD8-APC staining. Figure 8 B represents the percentage of CD8+ cells producing IFN-γ-FITC, as determined in three independent replicate experiments (percentage of cells double-stained with IFN-γ-FITC and CD8-APC). Vector represents PBMCs transfected with the empty vector pHR_LV-NS1;MC2-TCR. H -T indicates PBMC transfection with pHR_LV-NS1-MC2-TCR H MC2-TCR HM -T indicates PBMC transfection with pHR_LV-NS1-MC2-TCR HM T2 C The cells were T2 cells co-incubated with a control peptide (10 μM) for 2 h; T2 MC2 T2 cells were co-cultured with MAGE-C2 peptide (10 μM) for 2 h. The number of both TCR-T and T2 cells was 4 × 10⁻⁶. 6 One hole / hole.

[0264] The results indicate that Vector cells, which do not express MC2-specific TCRs, cannot respond to MC2 antigen peptides, while both types of TCR-T cells can respond to stimulation by MC2 antigen peptides and have the function of specifically producing and expressing IFN-γ; MC2-TCR HM -T due to murine substitution of the C region of the α and β chains, TCR HM It has a stronger affinity for specific antigenic peptide epitopes and a stronger function in producing IFN-γ, while MC2-TCR expressing the original sequence has a stronger affinity for specific antigenic peptide epitopes and a stronger function in producing IFN-γ. H -T has a weaker specific reactivity.

[0265] 4. MC2-TCR expressing optimized mouse-derived sequences HM -T cells stimulated with MC2 peptide-loaded T2 cells showed higher TNF-α secretion.

[0266] To examine the specific secretion of TNF-α by the constructed MC2-TCR-T cells in response to MC2 antigen peptide stimulation, this embodiment transfected Vector with an empty vector and MC2-TCR-T cells. H -T and MC2-TCR HM -T and T2 respectively C Cells and T2 MC2 After 24 hours of co-culture, the TNF-α content in the supernatant of the co-cultured cells was measured using an ELISA kit. The results are as follows: Figure 9 As shown, Vector cells, regardless of T2 C Cells or T2 MC2 After co-culture, the secretion of TNF-α was approximately 1 pg / mL in both groups, with no significant difference between them; MC2-TC R HM -T and T2 MC2 In the cell group, the secretion of TNF-α was significantly higher than that of T2. C Cell incubation group, MC2-TCR H The -T case is the same; compared to MC2-TCR H -T, MC2-TCR HM -T and T2 MC2 After co-culture of cells, the secretion of TNF-α was significantly increased, with the mean value of three repeated experiments being approximately 200 pg / mL.

[0267] Figure 9 In this study, Vector and two types of TCR-T cells were seeded into 96-well plates and then seeded with T2 cells. C Cells and T2 MC2Cells were co-cultured for 24 h, and the TNF-α content in the cell supernatant was detected using a TNF-α ELISA kit. The experiment was repeated three times. Vector was PBMC transfected with the empty vector pHR_LV-NS1; MC2-TCRH-T was PBMC transfected with pHR_LV-NS1-MC2-TCRH; MC2-TCRHM-T was PBMC transfected with pHR_LV-NS1-MC2-TCRHM. T2 C The cells were T2 cells co-incubated with a control peptide (10 μM) for 2 h; T2 MC2 T2 cells were co-cultured with MAGE-C2 peptide (10 μM) for 2 h. The number of both TCR-T and T2 cells was 4 × 10⁻⁶. 6 One hole / hole.

[0268] The results indicate that T cells without expressing MC2-specific TCRs cannot respond to MC2 antigen stimulation; MC2-TCR-T cells expressing two different TCR sequences can both respond to specific antigen stimulation, producing significant TNF-α secretion; compared with the original MC2-specific TCR sequence, MC2-TCR... HM -T-derived mouse-derived TCR HM It has a stronger affinity for specific antigenic peptide epitopes, is more sensitive to reactions, and has a higher specific secretion of TNF-α after stimulation by MC2 antigen.

[0269] 5. Construction and validation of tumor target cells

[0270] To further verify the responsiveness of MC2-TCR-T cells to tumor cells, two types of tumor target cells need to be constructed: one is the control target cell A02-K562 expressing only HLA-A, and the other is the HLA-A02-restricted MAGE-C2 cell. 336-344 (ALKDVEERV) tumor target cells.

[0271] 5.1 Preparation of recombinant plasmid pHR_LV-NS1-A02 for constructing A02-K562

[0272] To construct the control target cell line A02-K562, the recombinant expression plasmid pHR_LV-NS1-A02 carried the HLA-A gene and the PuroR gene for cell selection and purification. First, the NotI and SalI restriction sites of the recombinant cloning plasmid pcDNA3.1(+)-A02 and the lentiviral expression vector pHR_LV-NS1 were double-digested to obtain the target gene fragment A02, with a full length of 2.8 kb, and the linear digestion product fragment of the lentiviral expression vector pHR_LV-NS1, with a full length of approximately 8.9 kb. Agarose gel electrophoresis of both digestion products yielded the expected bands, as shown below. Figure 10As shown in Figure A, the agarose gel electrophoresis of the NotI / SalI double digestion products is shown. Lane M is the DNA Marker 1 kb; lane 1 is the pcDNA3.1(+)-A02 double digestion product, with the target gene A02 being approximately 2.8 kb. After excising the bands at these two locations, the gel was recovered, and the recovered products were purified and subjected to agarose gel electrophoresis. The band positions were observed, such as... Figure 10 As shown in Figure B, the recovered target band was observed by agarose gel electrophoresis. Lane M represents the DNA Marker (1 kb); Lane 1 represents the pHR_LV-NS1 double-digested linear fragment (approximately 8.9 kb); Lane 2 represents the target gene fragment A02 (approximately 2.8 kb). The target gene fragment A02 is between 2.0 and 3.0 kb, and the pHR_LV-NS1 linear digested fragment is between 10.0 and 8.0 kb, which is in line with expectations.

[0273] The validated double-digested target gene fragment A02 was ligated with the pHR_LV-NS1 digestion fragment to obtain the recombinant lentiviral expression vector pHR_LV-NS1-A02. This vector was transformed, and eight single colonies were selected for further amplification and shaking. PCR verification was then performed on the eight bacterial cultures. A gene sequence of approximately 1.3 kb containing the boundary between the expression vector and the inserted target gene was selected as a template, primers were designed, and PCR amplification was performed. All eight amplification products showed the expected 1.3 kb band after agarose gel electrophoresis. Figure 10 Figure C shows the electrophoresis diagram of the pHR_LV-NS1-A02 PCR product. Lane M is the DNA Marker D2000, and lanes 1 to 8 are the PCR amplification products of the stained colonies, approximately 1.3 kb each. One group of recombinant lentiviral expression vector plasmids was amplified from bacterial culture and then extracted. The extracted plasmids were identified by double enzyme digestion, and gel electrophoresis yielded bands consistent with expectations, as shown in Figure C. Figure 10 As shown in Figure D, the recombinant expression vector plasmid is verified by double enzyme digestion. Lane M in the figure is the DNA Marker 1Kb, and lane 1 is the double enzyme digestion product of pHR_LV-NS1-A02. The target gene fragment A02 is around 3.0Kb, and the expression vector fragment is around 8.9Kb. The recombinant lentiviral expression vector plasmid pHR_LV-NS1-A02 has been successfully constructed and can be used for subsequent lentiviral packaging and transfection of K562 cells. After labeling and aliquoting, it should be stored at -80℃.

[0274] 5.2 Preparation of recombinant plasmid pHR_LV-NS1-MC2-A02 for constructing MC2-A02-K562

[0275] Target cells MC2-A02-K562 need to express MAGE-C2 336–344The HLA-A02 complex was first digested with NotI / SalI to obtain the target gene fragment MC2-A02 (1.4 kb) and the linear digestion product of the lentiviral expression vector pHR_LV-NS1. The digestion products were then subjected to agarose gel electrophoresis, yielding the expected bands, as shown below. Figure 11 As shown in Figure A, the agarose gel electrophoresis results of NotI / SalI double digestion products are as follows: Lane M contains a 1 kb DNA marker; Lane 1 contains the pUC-57-MC2-A02 double digestion product, with the target gene A02 being approximately 1.4 kb. After excising the bands at these two locations, the gel was recovered, and the recovered products were purified and subjected to agarose gel electrophoresis. The band positions were observed, such as... Figure 11 As shown in B, the recovered target band was observed by agarose gel electrophoresis: Lane M is the DNA Marker 1Kb; Lane 1 is the pHR_LV-NS1 double-digested linear fragment (approximately 8.9Kb); Lane 2 is the target gene fragment MC2-A02 (approximately 1.4Kb); the target gene fragment A02 is between 1.0 and 2.0Kb, and the pHR_LV-NS1 linear digested fragment is between 10.0 and 8.0Kb, which is in line with expectations.

[0276] The restriction enzyme fragment MC2-A02 was ligated with pHR_LV-NS1 to obtain the recombinant lentiviral expression plasmid pHR_LV-NS1-MC2-A02. This plasmid was transformed, and eight single colonies were selected for amplification and culture. PCR verification was then performed on the bacterial culture. A gene sequence of approximately 1.327 kb, containing the boundary between the expression vector and the inserted target gene, was selected from the recombinant lentiviral expression vector as a template, and primers were designed for PCR amplification. All eight PCR amplification products yielded the expected 1.327 kb band after agarose gel electrophoresis. Figure 11 As shown in Figure C, this is an electrophoresis diagram of the pHR_LV-NS1-MC2-A02 PCR product: lane M is the DNA Marker D2000, and lanes 1 to 8 represent the PCR amplification products of positive colonies, approximately 1.3 kb each. One group of recombinant plasmid bacterial cultures was amplified, and the plasmid was extracted and identified by NotI / SalI double digestion. Gel chromatography yielded bands consistent with expectations, as shown below. Figure 11As shown in D, the recombinant expression vector plasmid was verified by double enzyme digestion: lane M is the DNA Marker 1Kb, and lane 1 is the double enzyme digestion product of pHR_LV-NS1-MC2-A0; the target gene fragment MC2-A02 is between 1.0Kb and 2.0Kb, and the pHR_LV-NS1 expression vector fragment is around 8.9Kb. The recombinant lentiviral expression vector plasmid pHR_LV-NS1-MC2-A02 was successfully constructed and can be used for subsequent lentiviral packaging and transfection of K562 cells. After labeling and aliquoting, it was stored at -80℃.

[0277] 5.3 HLA-A02-restricted MAGE-C2 in tumor target cells 336–344 antigen expression efficiency

[0278] The ability of MC2-TCR-T cells to recognize tumor cells is based on the restriction of MAGE-C2 on the surface of tumor cells. 336-344 Affinity of the (ALKDVEERV) / HLA-A02 complex. The affinity of HLA-A02 for the antigenic peptide MAG E-C2 in recombinant plasmid pHR_LV-NS1-MC2-A02. 336–344 Distributed in a tandem manner, after expression on K562, HLA-A02 interacts with the antigenic peptide MAGE-C2. 336–344 It can exist in the form of MHC complexes, such as Figure 12 Figure A shows the schematic diagram of the HLA complex structure of A02-K562 and MC2-A02-K562: A02-K562 expresses only HLA-A02 and polomycin pLeo1209 used for screening and purification; MC2-A02-K562 expresses HLA-A02-restricted MAGE-C2. 336-344 Antigen peptide complex. The target gene consists of multiple genes arranged in tandem, linked by a self-cleaving polypeptide P2A sequence. During translation, the self-cleaving polypeptide P2A breaks down, and each gene sequence is expressed independently. Therefore, flow cytometry can be used to detect HLA-A2-PE antibody staining to assess HLA-A02 and MAGE-C2 complexes on MC2-A02-K562 cells. 336–344 Overall expression of antigenic peptides.

[0279] In this embodiment, the tumor target cell line MC2-A02-K562 was constructed by transfecting K562 cells with the recombinant plasmid pHR_LV-NS1-MC2-A02, and the tumor control target cell line A02-K562 was constructed by transfecting K562 cells with the recombinant plasmid pHR_LV-NS1-A02. Vector cells transfected with an empty vector served as the control group. The three cell lines were co-incubated with HLA-A2-PE and then analyzed by flow cytometry. Figure 12C, with the x-axis representing HLA-A2-PE, shows that the percentage of cells stained with HLA-A2-PE in the Vector group transfected with the empty vector was 0.08%; the percentage of A02-K562 cells expressing HLA-A02 stained with HLA-A2-PE was 98.47%; and the percentage of cells expressing HLA-A02 / MAGE-C2 was... 336–344 The proportion of HLA-A2-PE stained cells in the MC2-A02-K562 complex was 99.12%. Results of three independent replicate transfections and flow cytometry assays are as follows: Figure 12 B showed that the proportion of HLA-A2-PE stained cells in both MC2-A02-K562 and A02-K562 was over 98%, with no significant difference between the two.

[0280] Figure 12 In this study, K562 cells were transduced with the recombinant expression vector for 55 hours and then incubated with HLA-A2-PE staining. The cells were then analyzed by flow cytometry. Figure 12 A represents the structural diagram of the HLA complexes of A02-K562 and MC2-A02-K562: A02-K562 expresses only HLA-A02 and uses pleomycin pLeo1209 for screening and purification; MC2-A02-K562 expresses HLA-A02-restricted MAGE-C2. 336-344 Antigen peptide complex. Multiple genes within the target gene are arranged in tandem, linked by a self-cleaving polypeptide P2A sequence. During translation and expression, the self-cleaving polypeptide P2A breaks, and each gene sequence is expressed independently. Figure 12 B represents HLA-A2-PE staining detected in three independent replicate experiments. Figure 12 C represents the HLA-A2-PE stained cells as detected by flow cytometry. Vectors are: K562 transfected empty vector pHR_LV-NS1; A02-K562 transfected recombinant vector pHR_LV-NS1-A02; MC2-A02-K562 transfected recombinant vector pHR_LV-NS1-A02-MC2.

[0281] The results indicate that both the lentiviral expression vectors pHR_LV-NS1-MC2-A02 and pHR_LV-NS1-A02 can effectively transduce K562 cells, and the target cells MC2-A02-K562 express MAGE-C2. 336-344 The (ALKDVEERV epitope) / HLA-A02 complex was successfully constructed, and the control target cells A02-K562 expressed HLA-A02, thus successfully establishing the tumor target cell line.

[0282] 6. MC2-TCR stimulated by tumor target cells HM -T can produce higher IFN-γ release.

[0283] To clarify the production of IFN-γ by MC2-TCR-T cells under specific stimulation of tumor target cells, this example used K562 cells transfected with an empty vector as the control group, and A02-K562 cells expressing only HLA-A02 and MAGE-C2 cells expressing complete HLA-A02 as the control group. 336–344 The antigen peptide complex MC2-A02-K562 was used as the experimental group, and was used with two types of MC2-TCR-T cells (MC2-TCR...). H -T and MC2-TCR HM After co-culturing with CD8+ cells for 24 hours, CD8-APC cells were incubated and IFN-γ-FITC intracellular factor staining (ICS) was performed. The proportion of cells double-stained with IFN-γ-FITC and CD8-APC was detected by flow cytometry, indicating the level of intracellular IFN-γ production in CD8+ cells. A single flow cytometry analysis is shown below. Figure 13 A showed that after Vector was co-cultured with A02-K562 and MC2-A02-K562 cells, the proportion of IFN-γ-FITC / CD8-APC double-stained cells was very low, at 0.28% and 0.14%, respectively; MC2-TCR H After co-culturing -T cells with A02-K562, the percentage of the double-stained cell population was 0.22%, in MC2-TCR HM -T is 0.16%; MC2-TCR H After co-incubation of T cells with MC2-A02-K562, the percentage of the double-stained cell population was 0.92%, while that of MC2-TCR cells was... HM After incubation of -T cells with MC2-A02-K562 cells, the percentage of the double-stained cell population was 2.82%. Results from three independent replicate experiments are shown below. Figure 13 B shows that K562 cells transfected with the empty vector, regardless of whether they are associated with MC2-TCR H -T or MC2-TCR HM After co-incubation with -T, the proportion of CD8+ cells producing IFN-γ was low in both groups, with no significant difference between the two; A02-K562 cells and MC2-TCR H -T or MC2-TCR HM After co-incubation with -T, the proportion of CD8+ cells producing IFN-γ was low in both groups, with no significant difference between the two; MC2-TCR HM The proportion of IFN-γ-FITC-labeled CD8+ cells in the T cell and MC2-A02-K562 incubation groups was significantly higher than that in the MC2-TCR groups. H -T / A02-K562 incubation group.

[0284] Figure 13 In China, MC2-TCR H-T and MC2-TCR HM -T cells were co-incubated with Vector, A02-K562, and MC2-A02-K56 at a 1:1 effector-target ratio for 24 h. The CD8+ cell population producing IFN-γ was then labeled with antibodies. First, CD8-APC antibody was incubated, followed by IFN-γ-FITC antibody incubation using an intracellular factor staining method. Flow cytometry was used to detect the IFN-γ-FITC and CD8-APC staining status of cells. Double staining of CD8-APC and IFN-γ-FITC cells represents the functional level of IFN-γ production in the CD8+ cell population. Figure 13 A represents the flow cytometry results of IFN-γ-FITC and CD8-APC staining. Figure 13 B represents the percentage of CD8+ cells producing IFN-γ-FITC, as determined in three independent replicate experiments (percentage of cells double-stained with IFN-γ-FITC and CD8-APC). MC2-TCR H -T indicates PBMC transfection with pHR_LV-NS1-MC2-TCR H MC2-TCR HM -T indicates PBMC transfection with pHR_LV-NS1-MC2-TCR HM Vector K562 was used to transfect the empty vector pHR_LV-NS1; A02-K562 was used to transfect the recombinant vector pHR_LV-NS1-A02; and MC2-A02-K562 was used to transfect the recombinant vector pHR_LV-NS1-A02-MC2. The effective target cell count was 0.4 × 10⁻⁶ cells in each vector. 6 Each hole.

[0285] The results indicate that only HLA-A02-restricted MAGE-C2 expression... 336–344 The MC2-A02-K562 target cells of the complex can stimulate MC2-TCR H -T and MC2-TCR HM -T-specific production of IFN-γ; MC2-TCR-T cells against unpresented MAGE-C2 336–344 The antigenic peptide did not elicit a specific HLA-A02 response; compared to MC2-TCR H -T, mouse-optimized MC2-TCR HM -T is restricted by HLA-A02 on MC2-A02-K562 MAGE-C2 336–344 Upon stimulation by the complex, the ability to specifically produce IFN-γ is enhanced.

[0286] 7. MC2-TCR stimulated by tumor target cells HM -T can produce higher specific secretion of TNF-α.

[0287] To test the function of the constructed tumor target cells in stimulating MC2-TCR-T cells to specifically secrete TNF-α, this embodiment uses MC2-TCR... H -T and MC2-TCR HM -T cells were co-incubated with A02-K562, MC2-A02-K562, and Vector (K562) transfected with empty vector for 24 h. The TNF-α content in the cell supernatant was then measured using an ELISA kit. Results are as follows: Figure 14 As shown, Vector and MC2-TCR H -T and MC2-TCR HM The levels of TNF-α in the supernatant were very low after -T interaction, and there was no significant difference between the two; A02-K562 and MC2-TCR HM After co-incubation with T, the TNF-α content was slightly higher than that of A02-K562 and MC2-TCR. H -T incubation group, but there was no significant difference between the two; MC2-A02-K562 and MC2-TC R HM After co-incubation with T, the TNF-α content was significantly higher than that of A02-K562 / MC2-TCR. H -T incubation group, also higher than MC2-TCR HM -T / A02-K562 incubation group and MC2-TCR HM In the -T / Vector incubation group, the mean value of three independent experiments was approximately 21 pg / mL, and the differences were statistically significant.

[0288] Figure 14 Vector cells and two types of TCR-T cells were seeded into 96-well plates and co-cultured with tumor target cells A02-K562 and MC2-A02-K562 for 24 h, respectively. After incubation, the TNF-α content in the cell supernatant was detected using a TNF-α ELISA kit. The experiment was repeated three times. MC2-TCR H -T indicates PBMC transfection with pHR_LV-NS1-MC2-TCR H MC2-TCR HM -T indicates PBMC transfection with pHR_LV-NS1-MC2-TCR HM Vector K562 was used to transfect the empty vector pHR_LV-NS1; A02-K562 was used to transfect the recombinant vector pHR_LV-NS1-A02; and MC2-A02-K562 was used to transfect the recombinant vector pHR_LV-NS1-A02-MC2. The effective target cell count was 0.4 × 10⁻⁶ cells in each vector. 6 Each hole.

[0289] The results indicate that A02-K562 cells that did not present the MC2 antigen peptide were essentially unable to stimulate MC2-TCR-T cells to specifically secrete TNF-α; both types of MC2-TCR-T cells could recognize and express fully HLA-A02-restricted MAGE-C2. 336–344 The complex targets MC2-A02-K562 cells and induces specific secretion of TNF-α; it also interacts with MC2-TCR cells expressing the original MC2-specific TCR sequence. H Compared to -T, MC2-TCR with mouse-source optimization HM -T cells exhibit a stronger specific response to MC2-specific antigenic peptides, resulting in higher specific TNF-α secretion upon antigen stimulation. Therefore, subsequent experiments will utilize MC2-TCR. HM -T cells were used as effector cells in target cell killing experiments.

[0290] 8. MC2-TCR HM -T cells have a killing effect on tumor target cells MC2-A02-K562.

[0291] To detect MC2-TCR HM In in vitro experiments, the -T cells demonstrated the ability to recognize and kill tumor target cells. In this embodiment, CFSE staining was used to label tumor target cells MC2-A02-K562 and control target cells A02-K562 to distinguish effector cells from target cells. Effector cells and target cells were mixed at a 1:1 ratio and incubated in 96-well plates for 14 hours. Then, flow cytometry was used to count the absolute volume of CFSE-stained cell particles in each well, obtaining the CFSE-stained cell particle density for each well. The killing rate of effector cells against target cells within a certain time period was calculated based on the cell particle density. Figure 15 As shown in Figure A, four reaction groups were set up: A02-K562 culture group, MC2-A02-K562 culture group, and MC2-TCR group. HM -T / A02-K562 co-incubation group and MC2-TCR HM -T / MC2-A02-K562 co-incubation group. After incubation, the cell density (unit: cell / μL) of the remaining target cells labeled with CFSE in each group was detected by flow cytometry. (A) Results of a single flow cytometry analysis. (B) Scatter plot of results from three independent replicate experiments.

[0292] The flow cytometry results of the separate culture groups of tumor target cells MC2-A02-K562 and control target cells A02-K562 under normal culture conditions were analyzed. MC2-TCR HMFlow cytometry analysis of the -T and A02-K562 incubation group showed that A02-K562 cells expressing only HLA and MC2-TCR HM Following the interaction of -T cells, the cell density of surviving A02-K562 cells; MC2-TCR HM Flow cytometry results from the incubation group of -T and MC2-A02-K562 showed expression of HLA-A02-restricted MAGE-C2. 336–344 MC2-A02-K562 and MC2-TCR of the antigen peptide complex HM The cell density of surviving MC2-A02-K562 cells after T cell interaction.

[0293] As the previous results confirmed, A02-K562 cells expressing only HLA-A could not induce MC2-TCR. HM -T cells specifically secrete IFN-γ and TNF-α, therefore, in this embodiment, MC2-TCR is considered to be... HM -T cells did not show significant specific killing effect against A02-K562, but on the other hand, MC2-TCR HM -T cells are derived from normal human PBMCs, which contain natural killer cells and some untransfected normal T cells. These cells have a certain non-specific killing effect on target cells. Therefore, this embodiment uses MC2-TCRs. HM The number of CFSE-stained cells in the group incubated with -T and A02-K562 was used to calculate MC2-TCR. HM -T is used to correct the kill rate value by representing the background kill rate of MC2-A02-K562. In addition, the proliferation rate of MC2-A02-K562 and the control target cell A02-K562 differs under the absence of external interference, so separate culture groups are set up to correct the kill rate calculation.

[0294] Based on the results of a single flow cytometry analysis, such as Figure 15 A. Substituting into the kill rate calculation formula, the kill rate is calculated as follows:

[0295]

[0296] Three independent replicate experiments were conducted on MC2-TCR. HM -T cells showed a killing efficiency of approximately 50% against target cells MC2-A02-K562. Figure 15 B.

[0297] Figure 15 In this study, CFSE staining was used to label tumor target cells MC2-A02-K562 and control target cells A02-K562 to differentiate effector cells. Effector cells MC2-TCR... HM-T cells and target cells A02-K562 and MC2-A02-K562, at an effector-target ratio of 1:1, were seeded into 96-well plates and incubated for 14 hours. Four reaction groups were established: A02-K562 culture alone, MC2-A02-K562 culture alone, and MC2-TCR... HM -T / A02-K562 co-incubation group and MC2-TCR HM -T / MC2-A02-K562 co-incubation group. After incubation, the cell density (unit: cell / μL) of the remaining target cells labeled with CFSE in each group was detected by flow cytometry. Figure 15 A represents the result of a single flow cytometry analysis. Figure 15 B is a scatter plot of the results from three independent repeated experiments.

[0298] The results indicate that, in in vitro experiments, with an effector-to-target ratio of 1:1, MC2-TCR HM -T is used for the expression of HLA-A02-restricted MAGE-C2. 336–344 The complex exhibits stable and specific killing activity against the target cell MC2-A02-K562.

[0299] Example 2: PDL1-MC2-TCR-T cells and PDL1-MC2-TCR-T PD1- Cell construction and in vitro experiments

[0300] This embodiment includes experimental methods and results. The experimental methods section includes:

[0301] 1. Method for constructing PDL1-MC2-TCR-T cells;

[0302] 2. Methods for preparing MC2-TCR-Jurkat and PDL1-MC2-TCR-Jurkat cells;

[0303] 3. Construct PDL1-MC2-TCR-T PD1- Cellular methods;

[0304] 4. Flow cytometry method for detecting MC2-TCR expression efficiency;

[0305] 5. Flow cytometry method for detecting PD-L1 expression efficiency;

[0306] 6. Target cells MC2-A02-K562 PD1+ The construction method;

[0307] 7. A method for detecting intracellular expression of IFN-γ in TCR-T cells using flow cytometry (ICS);

[0308] 8. The ELISA method for detecting TNF-α secretion;

[0309] 9. Flow cytometry method for detecting inhibition of target cell proliferation;

[0310] 10. Flow cytometry method for detecting the killing efficiency of target cells;

[0311] The experimental results section includes:

[0312] 1. Successfully constructed with PD-L1 and TCR HM Recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR containing gene sequence HM ;

[0313] 2. The expression of exogenous PD-L1 has an inhibitory effect on TCR-T cell function;

[0314] 3. LNP packaging of Cas9-mRNA / PD-1-sgRNA effectively knocks out PD-1 in PDL1-MC2-TCR-T cells;

[0315] 4. PDL1-MC2-TCR-T knockout of PD-1 PD1- Cells enhanced TCR HM and PD-L1 expression;

[0316] 5. PDL1-MC2-TCR-T knockout of PD-1 PD1- Cells were T2 MC2 Cell activation enhances cytotoxicity.

[0317] 6. Successfully constructed simultaneous expression of MAGE-C2 336–344 MC2-A02-K562, a tumor target cell containing HLA-A02 (pMHC) and PD-1. PD-1+ ;

[0318] 7. PDL1-MC2-TCR-T PD1- It has higher cytotoxicity against PD-1 positive tumor target cells;

[0319] 8. PD-L1 co-expressed on TCR-T cells has an inhibitory effect on the proliferation of PD-1 positive tumor target cells;

[0320] 9. PDL1-MC2-TCR-T PD1- It has a higher in vitro killing efficiency against PD-1 positive target cells;

[0321] 10. PDL1-MC2-TCR-T PD1- The killing efficiency against PD-1 positive tumor target cells increased with the extension of co-culture time;

[0322] The details are as follows:

[0323] I. Experimental Methods:

[0324] 1. Construction of PDL1-MC2-TCR-T cells

[0325] (1) Design of recombinant expression vector for MC2-TCR-T cells co-expressing PD-L1

[0326] PDL1-MC2-TCR-T cells simultaneously express the C-region murine substitution sequence MC2-TCR described in Example 1. HM and PD-L1, MC2-TCR HM It contains α and β strand sequences representing mouse gene sequence substitutions in region C. The construction process of its recombinant expression plasmid is as follows: Figure 16 As shown: A self-cleaving polypeptide T2A sequence was added to the front end of the designed PD-L1 gene sequence. The cloning plasmid pUC-SP-T2A-PDL1 was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and then double-digested with RsrII and MluI to obtain the target gene T2A-PDL1. Simultaneously, the recombinant expression vector pHR_LV-NS1-MC2-TCR described in Example 1 was... HM The product was also subjected to RsrII / MluI double digestion, and after purification and recovery, it was ligated to obtain the recombinant expression vector pHR_LV-NS1-PDL1-MC2-TCR co-expressing PD-L1. HM Its target gene is PDL1-MC2-TCR HM This is a tandem arrangement of the α and β chain gene sequences linked by T2A in PD-L1 and P2A. Target gene: PDL1-MC2-TCR HM The gene sequence is shown in SEQ ID NO:SEQ ID NO:13.

[0327] (2) Lentiviral recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR HM Preparation

[0328] ① Enzyme digestion of the T2A-PDL1 target gene plasmid

[0329] The experimental conditions and procedures are the same as in Part 5, "Preparation of Lentiviral Recombinant Plasmids," of Example 1, specifically (1) enzyme digestion of the target gene plasmid. The enzyme digestion reaction system is shown in Tables 10 and 11:

[0330] Table 10: pUC-SP-T2A-PDL1 double digestion (RsrII / MluI) reaction system

[0331]

[0332] Table 11: pHR_LV-NS1-MC2-TCRHM double digestion (RsrII / MluI) reaction system

[0333]

[0334] (3) Purification and detection of the target gene fragment

[0335] The experimental conditions and steps are the same as in Part 5 of Example 1, "Preparation of Lentiviral Recombinant Plasmids": (2) agarose gel electrophoresis purification; (3) DNA purification and recovery; (4) agarose gel electrophoresis detection of the purity of the target gene fragment; the difference is that when cutting the gel, the T2A-PDL1 fragment is cut at 880bp, and the pHR_LV-NS1-MC2-TCR band is cut. HM A band at 10.7 kb was extracted from the fragment. The target fragment was then recovered according to the gel extraction kit instructions, and the concentration and purity of the recovery solution were determined using a spectrophotometer. Agarose gel electrophoresis was then performed again to assess the purity of the target gene fragment. The loading order was: D2000 marker, T2A-PD-L1 fragment recovery product, pHR_LV-NS1-MC2-TCR. HM The linear fragment recovery product and 1kb marker were examined under a UV lamp to check if the band positions were correct.

[0336] (4) Connection reaction

[0337] The purified T2A-PDL1 fragment (insert) and pHR_LV-NS1-MC2-TCR were then processed. HM (vector) Ligation is performed. Prepare the appropriate reagents and add them to the labeled PCR tubes according to the system in Table 12 for the ligation reaction. The recombinant expression plasmid after ligation is pHR_LV-NS1-PDL1-MC2-TCR. HM The specific steps are the same as those in Part 5 of Example 1, “Preparation of Lentiviral Recombinant Plasmid”, specifically (5) the ligation reaction of the target gene and the lentiviral vector.

[0338] Table 12: Connection Reaction System

[0339]

[0340] (5) Identification of recombinant expression vectors

[0341] Transform competent cells into culture medium, and perform PCR amplification and identification of the bacterial culture. The results are then determined based on pHR_LV-NS1-PDL1-MC2-TCR. HM The target gene-vector junction sequence is approximately 523 bp in length. Primers were designed as follows:

[0342] pHR_LV-NS1-PDL1-MC2-TCRHM The gene sequence of -F is shown in SEQ ID NO:14; pHR_LV-NS1-PDL1-MC2-TCR HM The gene sequence of -R is shown in SEQ ID NO:15.

[0343] According to Table 13, take sample bacterial cultures and perform PCR separately, label them, and add PCR reaction reagents on ice.

[0344] Table 13: PCR reaction procedure

[0345]

[0346] After vortexing and mixing, place the mixture in a PCR instrument and proceed with the reaction program in Table 14.

[0347] Table 14: PCR reaction system

[0348]

[0349]

[0350] For each reaction, 6 μL of PCR product was added to the agarose gel sample wells. After electrophoresis, the band position of each clone sample was observed to see if it met the expectation (523 bp). The corresponding bacterial culture of the clone with the correct band position was selected, and the culture was shaken overnight before plasmid extraction. Reagents were added according to Table 15, and enzyme digestion was performed. The digestion products were then subjected to agarose gel electrophoresis (steps as above) to observe whether the recombinant plasmid met the expected position to determine whether the construction was successful.

[0351] Table 15: Recombinant plasmid enzymatic digestion reaction system

[0352]

[0353] (6) Preparation of PDL1-MC2-TCR-T cells

[0354] Before transfection, T cells need to be activated. The activation and transfection steps are the same as in Example 1. pHR_LV-NS1-PDL1-MC2-TCR HM The PDL1-MC2-TCR-T cells obtained after transfecting PBMC cells are MC2-specific TCR-T cells that co-express PD-L1, and the negative control cells transfected with empty vector are Vector cells.

[0355] 2. Preparation of MC2-TCR-Jurkat and PDL1-MC2-TCR-Jurkat cells

[0356] Jurkat cells were cultured in 90% RPMI-1640 medium with 10% fetal bovine serum. MC2-TCR-Jurkat cells used pHR_LV-NS1-MC2-TCR. HM Jurkat cells were transfected with pHR_LV-NS1-PDL1-MC2-TCR Jurkat. HM The steps of viral transduction, T cell activation, and cell culture are the same as in Example 1.

[0357] 3. Construct PDL1-MC2-TCR-T PD1- cell

[0358] This experiment used lipid nanoparticles (LNPs) to package Cas9 mRNA and PD-1-sgRNA and precisely delivered them into the nucleus of constructed PDL1-MC2-TCR-T cells to specifically knock out the endogenous PD-1 gene. Figure 17 Cas9-mRNA and PD-1-sgRNA were dissolved in citrate buffer to prepare an aqueous phase containing mRNA. SM-102, DSPC, cholesterol, and DMG-PEG-2000 were dissolved in anhydrous ethanol to prepare an ethanol phase, which was then connected to the two inlets of the microfluidic chip. The aqueous and ethanol phases were mixed in the microfluidic instrument at a flow rate ratio of 3:1. The LNP product was transferred into a MWCO dialysis tube and dialyzed with PBS buffer at 4°C for 4 hours. Finally, it was concentrated through an ultrafiltration tube to obtain the LNP-mRNA (Cas9-mRNA / PD-1-sgRNA) product for later use. LNP-mRNA (Cas9-mRNA / PD-1-sgRNA) was transfected into cells. Cas9-mRNA was transiently expressed in the cells, and all components entered the cell nucleus. PD-1-sgRNA could target the target sequence near PAM through base complementarity pairing. Under the guidance of PD-1-sgRNA, the Cas9 protein caused DNA double-strand breaks upstream and downstream of the gene. DNA damage repair mechanisms connect the upstream and downstream sequences of a break, thereby knocking out the target gene PD-1.

[0359] (1) Cas9 mRNA

[0360] The Cas9 mRNA used in this embodiment has a Cap1 structure and a 100-poly A tail, and the entire Cas9 mRNA sequence is modified with m1Ψ or 5-MOU. The product was synthesized by Genscript Biotech Co., Ltd.

[0361] (2) PD-1-sgRNA

[0362] The sgRNA (small guide RNA) used in the experiment was in single-stranded form. crRNA (CRISPRRNA) and tracrRNA (trans-activating crRNA) were linked together by a linker to fuse them into a single-stranded molecule without the need for annealing.

[0363] The PD-1-sgRNA sequence is shown in SEQ ID NO:16.

[0364] (3) Preparation and packaging of lipid nanoparticles for Cas9-mRNA / PD-1-sgRNA using microfluidic mixing method

[0365] The stock solution was diluted with ultrapure water to prepare a 50 mM citrate buffer (pH = 4). Cas9-mRNA:PD-1-sgRNA was dissolved in the citrate buffer at a molar ratio of 1:5 to a final concentration of 100 μg / mL to prepare an aqueous phase containing mRNA. At a fixed molar ratio, SM-102 (50%), DSPC (10%), cholesterol (38.5%), and DMG-PEG-2000 (1.5%) were dissolved in anhydrous ethanol to prepare an ethanol phase. The microfluidic instrument was preheated, and the microfluidic... With the Luer tube facing upwards, the flow control chip is inserted into the adapter. After assembling the chip, the entire module is placed into the microfluidic reaction chamber. The chip is pre-filled with citrate buffer and anhydrous ethanol. The injection adapter is inserted into the reaction chamber. Two syringes are used to draw ethanol and buffer solution respectively, and air bubbles are removed. Two 15ml centrifuge tubes are installed as product and waste collection tubes, respectively. The software parameters are set so that the aqueous phase and ethanol phase are mixed in the microfluidic device at a flow rate ratio of 3:1. The mixed product is placed in an MWCO dialysis tube at 4°C, and 14ml of PBS (1X) is added. Dialysis is performed on a shaker, with the medium changed every 2 hours. This process is repeated until dialysis is completed after 4 hours. The solution is then filtered through a 0.22µM filter membrane, and the filtered product is concentrated into LNP-mRNA (Cas9-mRNA / PD-1-sgRNA) using a 100kD ultrafiltration tube. The product is then stored at 4°C or -20°C.

[0366] LNP-mRNA (Cas9-mRNA / PD-1-sgRNA) transfection into cells results in transient expression of Cas9-mRNA within the cells. The components then enter the nucleus. PD-1-sgRNA, through base complementarity, targets the sequence near PAM. Guided by PD-1-sgRNA, the Cas9 protein causes DNA double-strand breaks upstream and downstream of the gene. DNA damage repair mechanisms then reconnect the sequences at both ends of the breaks, thereby achieving the knockout of the target gene PD-1.

[0367] (4) Determine the encapsulation efficiency of LNP

[0368] The average particle size, PDI (polymer dispersion index), and zeta potential of LNP-mRNA were measured using a nanoparticle size analyzer (Zetasizer Nano ZS / ZEN 3600).

[0369] (5) Preparation of PDL1-MC2-TCR-T by transfecting PDL1-MC2-TCR-T with LNP-mRNA (Cas9-mRNA / PD-1-sgRNA). PD1- cell

[0370] Stably transfected PDL1-MC2-TCR-T cells were obtained according to the schedule, and the cells were counted and seeded into 24-well plates. Each well contained 1 ml of 10% FBS DMEM medium with 5 × 10⁶ cells / well. 4 100 cells; add 500 ng of encapsulated LNP-mRNA (Cas9-mRNA / PD-1-sgRNA) to each well, continue culturing for 24 h, change the medium, and proceed with subsequent experiments. MC2-TCR-T PD1- The transfection methods are the same.

[0371] 4. Flow cytometry detection of MC2-TCR expression efficiency

[0372] After obtaining the corresponding TCR-T cells through transfection, cells were cultured for 5 days. PBMCs transduced with the pHR_LV-NS1 empty vector served as the control vector. Cells from each group were subjected to double staining with HLA-A02-restricted MAGE-C2-Tetramer-PE and CD8-APC. Flow cytometry was used to detect the ability of TCR-T cells to bind MC2. 336–344 The percentage of CD8+ T cells with (ALKDVEERV) antigen.

[0373] 5. Flow cytometry detection of PD-L1 expression efficiency

[0374] After transfection to obtain the corresponding TCR-T cells, the cells were cultured for 5 days. Each group of cells was then subjected to double staining with HLA-A02-restricted MAGE-C2-Tetramer-PE and PDL1-APC. Flow cytometry was used to detect the ability of TCR-T cells to bind HLA-A02 / MC2. 336–344 The cell population percentages of the ALKDVEERV pMHC complex and PD-L1 antibody were measured. The FACS buffer was PBS containing 0.5% FBS. Each reaction required an antibody mixture of 2 μL PDL1-APC, 2 μL Tetramer-PE, and 46 μL FACS buffer.

[0375] 6. Target cells MC2-A02-K562 PD1+ Construction

[0376] (1) Structure of the recombinant lentiviral expression vector

[0377] The target gene fragment was synthesized by Sangon Biotech (Shanghai) Co., Ltd., with NotI and SalI restriction enzyme sites at both ends of the PD-1 gene sequence. The lentiviral expression vector used for cell transfection was pHR_LV-NS1. The enzyme digestion, ligation, and transformation steps were as described above. After inserting the PD-1 target gene fragment, it was named pHR_LV-NS1-PD-1, as shown below. Figure 18 As shown, the target gene PD-1 sequence was inserted into the lentiviral expression vector pHR_LV-NS1 to obtain the recombinant expression vector pHR_LV-NS1-PD-1. The target gene PD-1 sequence is shown in SEQ ID NO:17.

[0378] (2) Obtain MC2-A02-K562 PD1+ cell

[0379] MC2-A02-K562 cells were obtained by transfecting the recombinant expression vector pHR_LV-NS1-PD-1 into the successfully constructed MC2-A02-K562 cells. PD1+ Cells, cell culture, viral transfection, and target cell purification are detailed in Example 1.

[0380] 7. Detection of intracellular IFN-γ expression in TCR-T cells by flow cytometry (ICS).

[0381] The mixing ratio of TCR-T cells and target cells (or peptide-loaded T2 cells) was 1:1. After co-incubation for 24 hours, ICS (intracytoplasmic cytokine staining) was performed, and the cell population that could be labeled with Anti-IFN-γ / FITC was detected by flow cytometry. The specific experimental procedures were the same as in Example 1.

[0382] 8. Detection of TNF-α secretion by ELISA

[0383] TCR-T cells and tumor target cells (or peptide-loaded T2 cells) in each group were counted. Following a 1:1 ratio of effector cells to target cells, the cells were thoroughly mixed and incubated together in the incubator for 24 hours. After incubation, the cell supernatant was collected and the experiment was performed according to the ELISA kit requirements, following the same steps as in Example 1.

[0384] 9. Flow cytometry detection of inhibition of target cell proliferation

[0385] MC2-TCR-Jurkat, PDL1-MC2-TCR-Jurkat, and target cells MC2-A02-K562 were prepared. PD1+Afterwards, cells were counted and seeded. The FACS buffer was PBS containing 0.5% FBS. 50 μL of HLA-A2-PE antibody working solution (5 μL HLA-A2-PE antibody stock solution plus 45 μL FACS buffer) was added to each reaction; other experimental procedures were the same as in Example 1. At 0 h after cell mixing and at 24 h and 48 h after co-incubation, target cells were labeled with HLA-A2-PE antibody, and then analyzed by flow cytometry to obtain the number and percentage of target cells labeled with HLA-A2-PE antibody at each time point. The inhibition rate of effector cells against target cells at the corresponding time point was then calculated using the inhibition rate calculation formula. The inhibition rate calculation formula is (based on 24 h effector cells MC2-TCR-Jurkat and target cells MC2-A02-K562). PD1+ (Taking the calculation of inhibition rate of co-incubation group as an example):

[0386]

[0387] 10. Flow cytometry to detect the killing efficiency against target cells

[0388] Target cells were labeled with CFSE dye. TCR-T cells and target cells were co-incubated at effector-to-target ratios of 1:1 and 1:2, respectively. A separate target cell group was also established. The number of CFSE-labeled target cells after co-incubation was detected by flow cytometry at 24h, 48h, and 72h. The killing rate of TCR-T cells against target cells in each group was calculated using a formula. The CFSE labeling and flow cytometry experimental procedures were the same as in Example 1. The formula for calculating the killing rate at a certain time point is:

[0389]

[0390] Statistical methods:

[0391] Statistical analysis was performed using Prism version 8.0 (Graph Pad). For data comparing between groups, t-tests were used. One-way ANOVA was used to detect the significance of differences between groups. *p<0.05 indicates a statistically significant difference; **p<0.01 indicates a statistically significant difference; ***p<0.001 indicates an extremely statistically significant difference.

[0392] II. Experimental Results:

[0393] 1. Successfully constructed with PD-L1 and TCR HM Recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR containing gene sequence HM

[0394] To investigate whether exogenous PD-L1, under the targeting guidance of MC2-TCR-T, can activate PD-1 signaling in malignant T cells and synergistically enhance the tumor-killing and inhibitory effects of MC2-TCR-T, this embodiment constructed the recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR. HM Integrating the PD-L1 gene with TCR HM The sequence was transfected into activated T cells to form engineered PDL1-MC2-TCR-T cells.

[0395] First, the recombinant plasmid pUC-SP-T2A-PDL1 (containing the T2A self-cleaved polypeptide sequence and the PD-L1 gene) and the recombinant plasmid pHR_LV-NS1-MC2-TCR were digested with double enzymes. HM The restriction endonuclease sites RsrII and MluI were used. After digestion, the enzyme products were subjected to agarose gel electrophoresis to obtain the target gene fragment T2A-PDL1 of approximately 880 bp and the recombinant plasmid pHR_LV-NS1-MC2-TCR of approximately 10.7 kb, respectively. HM The linear enzyme digestion product fragments were recovered, and the two bands were collected again by agarose gel electrophoresis. The T2A-PDL1 band was found between 750 bp and 1.0 kb, and the pHR_LV-NS1-MC2-TCR band was found above 10.0 kb. HM The strips all met expectations, such as Figure 19 A represents pUC-SP-T2A-PDL1 and pHR_LV-NS1-MC2-TCR. HM Double enzyme digestion agarose gel electrophoresis: Lane M1 is the standard DNA molecular weight DNA Marker D2000, lane 1 is the T2A-PDL1 fragment (880bp), and lane 2 is the pHR_LV-NS1-MC2-TCR fragment. HM Linear fragment (10.7Kb), lane M2 is the standard DNA molecular weight DNA marker 1Kb.

[0396] The validated double-enzyme digestion target gene fragment T2A-PDL1 and pHR_LV-NS1-MC2-TCR were then digested. HM Enzyme digestion fragments were ligated to obtain the recombinant lentiviral expression vector pHR_LV-NS1-PDL1-MC2-TCR HM The target gene is PDL1-MC2-TCR. HM After transforming the vector into selected bacteria and amplifying them again, all eight groups of bacterial cultures were subjected to PCR verification. All eight amplification products, after agarose gel electrophoresis, yielded the expected 523bp band. Figure 19B is the electrophoresis diagram of PCR amplification products: lane M is DNA Marker D2000, lanes 1-8 are pHR_LV-NS1-PDL1-MC2-TCR. HM PCR amplification product (523bp). One group of bacterial cultures was used for amplification and culture followed by plasmid extraction. The plasmid was double-digested with RsrII and MluI. The digestion products were run on a gel electrophoresis gel, yielding the expected 880bp and 10.7kb bands. Figure 19 C is pHR_LV-NS1-PDL1-MC2-TCR HM Double enzyme digestion verification: Lane M1 is DNA Marker D2000, and lane 1 is pHR_LV-NS1-PDL1-MC2-TCR. HM Double enzyme digestion results showed that lane M2 contained DNA Marker 1 kb. The recombinant lentiviral expression vector plasmid pHR_LV-NS1-MC2-TCR... HM Successfully built, as follows Figure 19 D, for pHR_LV-NS1-PDL1-MC2-TCR HM Plasmid mapping: The target genome is named TCRα3-P2A-TCRβ28-T2A-PDL1, where TCRα3-P2A-TCRβ28 is the mouse-derived optimized TCR from Example 1. HM Sequence MC2-TCR HM It can be used for subsequent lentiviral packaging and transfection of PBMC cells.

[0397] 2. The expression of exogenous PD-L1 inhibits TCR-T cell function.

[0398] 2.1 PDL1-MC2-TCR-T cells can simultaneously express TCR HM and PD-L1 but TCR HM Low expression

[0399] To clarify the specific TCR on PDL1-MC2-TCR-T cells HM The expression of PD-L1 was investigated, and MC2-TCR-T, PDL1-MC2-TCR-T, and control Vector cells were obtained by transfection. MAGE-C2-Tetramer-PE and CD8-APC double staining of these cells were analyzed by flow cytometry to observe TCR expression. HM The percentage of CD8+ cells; TCR was observed using double staining with MAGE-C2-Tetramer-PE and PDL1-APC. HM The percentage of cells co-expressing PD-L1.

[0400] Recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR HMPDL1-MC2-TCR-T cells obtained after transfection with PBMCs simultaneously express mouse-derived optimized TCRs in the C region on their cell surface. HM And PD-L1 protein, such as Figure 20 A. TCR HM The results of a single flow cytometry analysis of the expression are as follows: Figure 20 D, with the x-axis representing MAGE-C2-Tetramer-PE and the y-axis representing CD8-APC, showed that both MC2-TCR-T and PDL1-MC2-TCR-T cells exhibited double staining for both MAGE-C2-Tetramer-PE and CD8-APC. The percentage of cells in MC2-TCR-T was 38.7%, in PDL1-MC2-TCR-T it was 1.69%, and in the Vector group it was 0.29%. Three independent replicate experiments showed that... Figure 20 B, the proportion of MAGE-C2-Tetramer-PE and CD8-APC double-stained cells in PDL1-MC2-TCR-T was significantly higher than that in Vector, but significantly lower than that in MC2-TCR-T. TCR HM The results of a single flow cytometry analysis of cells co-expressing PD-L1 are as follows: Figure 20 E, with the x-axis representing MAGE-C2-Tetramer-PE and the y-axis representing PDL1-APC, showed that the percentages of double-stained cells in Vector and MC2-TCR-T were 0.04% and 0.25%, respectively, while in PDL1-MC2-TCR-T it was 3.09%. Results from three independent replicate experiments showed that, as Figure 20 In C, the proportion of MAGE-C2-Tetramer-PE and PDL1-APC double-stained cell populations in PDL1-MC2-TCR-T was significantly higher than that in Vector and MC2-TCR-T.

[0401] The results indicate that PDL1-MC2-TCR-T cells can simultaneously express C-region mouse-derived optimized TCR. HM And PD-L1, but TCR HM The expression of PD-L1, or rather the function of binding to the MAGE-C2 (MC2) antigenic epitope, was significantly reduced compared to MC2-TCR-T. This embodiment suggests that engineered expression of PD-L1 in PDL1-MC2-TCR-T cells interferes with TCR. HM The ability of it to express or bind to antigenic epitopes.

[0402] For the sake of brevity, the MC2-TCR-T cells mentioned in this embodiment are the MC2-TCR cells constructed in Example 1. HM -T cells, generated from recombinant plasmid pHR_LV-NS1-MC2-TCR HMDerived from PBMCs, expressing a TCR targeting MAGE-C2 that performs mouse-derived sequence substitution in the C region. HM Similarly, transfecting the recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR... HM The TCR-T cells were named PDL1-MC2-TCR-T, which are MC2-TCR cells co-expressing PD-L1. HM -T cells. MAGE-C2 will be abbreviated as MC2 from now on.

[0403] Figure 20 In China, flow cytometry was used to detect TCR. HM Expression (MAGE-C2-Tetramer-PE and CD8-APC double staining), and TCR HM Co-expression of PD-L1 and TCR (double staining with MAGE-C2-Tetramer-PE and PDL1-APC). (A) Schematic diagram of TCR and PD-L1 on the surface of PDL1-MC2-TCR-T cells: PDL1-MC2-TCR-T cells express murine TCR targeting the C region of MC2. HM , and co-express PD-L1. The expression vector target gene is composed of α, β chains and PD-L1 gene sequences tandemly, with the genes linked by self-cleaved polypeptide sequences. (B) TCR expression efficiency detected in three independent replicate experiments (i.e., the proportion of MAGE-C2-Tetramer-PE and CD8-APC double-stained cell populations detected by flow cytometry). (C) PD-L1 expression efficiency detected in three independent replicate experiments (i.e., the proportion of MAGE-C2-Tetramer-PE and PDL1-APC double-stained cell populations detected by flow cytometry). (D) Flow cytometry results of MAGE-C2-Tetramer-PE and CD8-APC staining. (E) Flow cytometry results of MAGE-C2-Tetramer-PE and PDL1-APC staining. Vector: PBMC transfected with empty vector pHR_LV-NS1; MC2-TCR HM -T: PBMC transfection with pHR_LV-NS1-MC2-TCR HM ;PDL1-MC2-TCR-T: PBMC transfection with pHR_LV-NS1-PDL1-MC2-TCR HM After transducing normal donor PBMCs with the recombinant expression vector for 54 hours, the cells were incubated with various antibodies, and the antibody staining was detected by flow cytometry.

[0404] 2.2 The ability of PDL1-MC2-TCR-T cells to produce IFN-γ and secrete TNF-α was significantly reduced after activation.

[0405] PDL1-MC2-TCR-T cells express TCR HM If the level of the MC2 antigen epitope is low, how effective is the ability of PDL1-MC2-TCR-T cells to produce cytokines after stimulation with the MC2 antigen peptide? To investigate this question, this example uses MC2-TCR-T cells, which have been demonstrated to have normal function in previous examples, as a positive control. MC2-TCR-T and PDL1-MC2-TCR-T cells were obtained by transfecting cells and then compared with T2 cells loaded with the control peptide. C and T2 loaded with MC2 antigen peptide MC2 After co-culture, the production and secretion levels of IFN-γ and TNF-α were detected.

[0406] IFN-γ was detected using flow cytometry (ICS). Cells were incubated with CD8-APC and IFN-γ-FITC to label the intracellular IFN-γ-producing CD8+ cell population. The percentage of the double-stained cell population was then analyzed by flow cytometry. A single flow cytometry analysis is shown below. Figure 21 Display A shows that the horizontal axis is CD8-APC and the vertical axis is IFN-γ-FITC, with values ​​at MC2-TCR-T and PDL1-MC2-TCR-T and T. 2C Under co-culture conditions, the proportions of CD8-APC / IFN-γ-FITC double-stained cell populations were both very low, at 0.32% and 0.17%, respectively; compared with T2... MC2 During co-incubation, the percentages of cells double-stained with CD8-APC and IFN-γ-FITC were 4.61% and 2.01%, respectively. Results from three independent replicate experiments showed that... Figure 21 B: PDL1-MC2-TCR-T and T2 MC2 The proportion of double-stained cell populations in the co-culture group was significantly lower than that in the MC2-TCR-T group, but significantly higher than that in the PDL1-MC2-TCR-T / T2 group. C Group.

[0407] TNF-α secretion was detected using an ELISA kit. MC2-TCR-T and PDL1-MC2-TCR-T were respectively compared with T... 2C and T2 MC2 The cell supernatant after co-culture was incubated for color development, and the results were as follows: Figure 21 As shown in C, PDL1-MC2-TCR-T / T2 MC2 TNF-α secretion in the group was significantly lower than that in the MC2-TCR-T / T2 group. MC2 The group, but significantly higher than PDL1-MC2-TCR-T / T2 C Group.

[0408] Figure 21In the middle, MC2-TCR-T and PDL1-MC2-TCR-T are respectively with T2 C Cells (T2 cells loaded with control peptide) and T2 MC2 After co-culturing cells (T2 cells loaded with MC2 peptide), flow cytometry was used to detect CD8-APC and IFN-γ-FITC staining. Double-stained cells (CD8-APC and IFN-γ-FITC) represent the functional level of IFN-γ production in the CD8+ cell population. The TNF-α content in the supernatant of the co-cultured cells was detected using an ELISA kit. Figure 21 A represents the results of flow cytometry staining with CD8-APC and IFN-γ-FITC. Figure 21 B represents the percentage of CD8+ cells producing IFN-γ-FITC (i.e., the percentage of cells double-stained with IFN-γ-FITC and CD8-APC) as detected in three independent replicate experiments. Figure 21 C represents the TNF-α secretion data from three independent, repeated assays. In the figure, MC2-TCR... HM -T indicates PBMC transfection with pHR_LV-NS1-MC2-TCR HM PDL1-MC2-TCR-T is PBMC transfection of pHR_LV-NS1-PDL1-MC2-TCR HM T 2C The cells were T2 cells co-incubated with a control peptide (10 μM) for 2 h; T2 MC2 The cells were T2 cells and co-incubated with MEGE-C2 peptide (10 μM) for 2 h.

[0409] The results indicate that PDL1-MC2-TCR-T cells co-expressing PDL1 can specifically produce IFN-γ and secrete TNF-α upon stimulation by the MC2 antigen peptide, but the production and secretion levels are much lower than those of MC2-TCR-T cells. Therefore, it is speculated that the PD-L1 co-expressed in PDL1-MC2-TCR-T cells affects the activation of TCR and the function of cytokine secretion after activation through some mechanism.

[0410] 2.3 PDL1-MC2-TCR-Jurkat cells, which do not express PD-1 themselves, can express TCR at high levels. HM and PD-L1

[0411] The detailed mechanism by which PD-1 inhibits T cell activation is still unclear, but PD-1 signaling activation inhibits the binding of the TCR to the pMHC complex. This embodiment hypothesizes whether the TCR function of PDL1-MC2-TCR-T cells is inhibited due to the activation of their own PD-1 by co-expressed PD-L1. Therefore, to investigate the role of T cell-specific PD-1 in TCR function inhibition, this embodiment performed pHR_LV-NS1-PDL1-MC2-TCR on Jurkat (human acute T-lymphoblastic leukemia cells) and normal PBMCs that do not express PD-1. HM PDL1-MC2-TCR-Jurkat and PDL1-MC2-TCR-T cells were transfected, and then subjected to MAGE-C2-Tetramer-PE and PDL1-APC double staining before being analyzed by flow cytometry.

[0412] RNA was extracted from Jurkat cells, and PD-1 expression was detected by RT-PCR. The results are as follows: Figure 22 As shown in Figure C, with β-actin as the internal control, no PD-1 mRNA was expressed in Jurkat. Flow cytometry results are as follows. Figure 22 A shows that the x-axis represents MAGE-C2-Tetramer-PE, and the y-axis represents PDL1-APC. The percentage of double-stained cells in both Vector groups transfected with the empty vector was very low, at 0.019%; the percentage of double-stained cells in PDL1-MC2-TCR-T was 3.83%, and in PDL1-MC2-TCR-Jurkat it was 52.2%. Statistical analysis of three independent replicate experiments showed that... Figure 22 B: The proportion of MAGE-C2-Tetramer-PE / PDL1-APC double-stained cells in PDL1-MC2-TCR-T was significantly higher than that in Vector; the proportion of double-stained cell population in PDL1-MC2-TCR-Jurkat was significantly higher than that in Vector and PDL1-MC2-TCR-T.

[0413] like Figure 22 Vector (Jurkat), PDL1-MC2-TCR-Jurkat, and Vector (Tcell), PDL1-MC2-TCR-T cells were prepared, and MAGE-C2-Tetramer-PE and PDL1-APC double staining was performed and the cells were detected by flow cytometry. Figure 22 A shows the flow cytometry results of MAGE-C2-Tetramer-PE and PDL1-APC staining. Figure 22 B represents the percentage of MAGE-C2-Tetramer-PE and PDL1-APC double-stained cell populations as determined in three independent replicate experiments. Figure 22 C represents the RT-PCR detection of PD-1 expression in normally cultured Jurkat cells: Lane M is the standard DNA molecular weight marker D2000; lanes 1-3 are the internal control β-actin (540bp); lanes 4-6 show no PD-1 expression (512bp). The red boxes indicate the locations of the internal control and PD-1 bands. In the figure, Vector (Jurkat) is Jurkat transfected with the empty vector pHR_LV-NS1; PDL1-MC2-TCR-Jurkat is Jurkat transfected with the recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR. HM Vector (T cell) is an empty vector transfected with PBMCs; PDL1-MC2-TCR-T is a recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR transfected with PBMCs. HM .

[0414] The results indicate that, compared with primary T cells, Jurkat cell lines that do not express PD-1 showed better performance after transfection with the same recombinant vector pHR_LV-NS1-PDL1-MC2-TCR. HM Post-TCR HM Both PD-1 and PD-L1 are expressed at higher levels. PDL1-MC2-TCR-T cells are derived from primary T cells expressing PD-1, and after transfection with a recombinant vector, TCR... HM With low PD-L1 expression levels, the T cell's own PD-1 signaling pathway may be activated by co-expressed PD-L1. Therefore, it is preliminarily concluded that T cell-expressed PD-1 is a cause of TCR. HM Limited expression or function and low expression of PD-L1 are key reasons.

[0415] 3. LNP packaging of Cas9-mRNA / PD-1-sgRNA effectively knocks out PD-1 in PDL1-MC2-TCR-T cells.

[0416] 3.1 LNP packaging of Cas9-mRNA / PD-1-sgRNA

[0417] To verify the effect of T cell-derived PD-1 on the expression or function of PDL1-MC2-TCR-T cells, this study attempted to knock out the PD-1 gene in PDL1-MC2-TCR-T cells to observe changes in TCR function and PD-L1 expression after PD-1 knockout. Lipid nanoparticles (LNPs) can directly encapsulate nucleic acids and deliver them into the cell to release the target nucleic acid. PDL1-MC2-TCR-T cells were prepared by packaging and delivering Cas9-mRNA / PD-1-sgRNA to knock out the PDCD1 gene in the genome of PDL1-MC2-TCR-T cells using LNPs.PD1- This method offers low cytotoxicity and high knockout efficiency in cells. First, nucleic acid nanoparticles are prepared using microfluidic mixing technology. Cas9-mRNA / PD-1-sgRNA in a citric acid aqueous solution and a lipid ethanol solution are respectively connected to the inlets of two channels in a microfluidic chip. The two phases are rapidly mixed using a microfluidic injector to produce lipid-nucleic acid nanoparticles with a specific particle size range. After dialysis and ultrafiltration concentration, these nanoparticles are ready for cell transfection. Dynamic light scattering analysis data shows that... Figure 23 A. The average particle size of the (Cas9-mRNA+PD-1-sgRNA)-LNP particles prepared in this embodiment is 69.4 nm, the lipid content in the 40-90 nm range is 91.7%, and the polydispersity index is 0.142 (<0.3). The nucleic acid LNPs were visualized and detected using cryo-electron microscopy, such as... Figure 23 B. As can be seen, most LNPs are bilayered, spherical with no obvious internal defects, and the particle size distribution is uniform. The above characterization and detection results indicate that the (Cas9-mRNA+PD-1-sgRNA)-LNPs prepared in this embodiment have high encapsulation efficiency and uniform particle size distribution.

[0418] Preparation of (Cas9-mRNA+PD-1-sgRNA)-LNP using a microfluidic mixer. Figure 23 A is a graph showing the particle size data of (Cas9-mRNA+PD-1-sgRNA)-LNP detected by dynamic light scattering. Figure 23 Image B shows the overall morphology of (Cas9-mRNA+PD-1-sgRNA)-LNP encapsulation efficiency, roundness, layered distribution, and particle size distribution as revealed by cryo-electron microscopy. Scale bar: 200 nm.

[0419] 3.2 (Cas9-mRNA+PD-1-sgRNA)-LNP knockout of PDL1-MC2-TCR-T cells PD-1

[0420] The customized Cas9-mRNA and PD-1-sgRNA in this embodiment have the necessary gene modifications, low cytotoxicity, and stable knockout efficiency. (Cas9-mRNA+PD-1-sgRNA)-LNP, after transfection into PDL1-MC2-TCR-T cells, results in genomic PD-1 knockout, obtaining PDL1-MC2-TCR-T cells. PD1- Cells. MC2-TCR-T cells were simultaneously transfected to prepare MC2-TCR-T cells. PD1- As an experimental control, the percentage of PD-1-PE stained cells was detected using flow cytometry. The flow cytometry results showed that... Figure 24 A, MC2-TCR-T knocked out PD-1 PD1- and PDL1-MC2-TCR-T PD1-The percentages of cells labeled with PD-1-PE antibody were 17.4% and 13.3% in the cells, respectively. In Vector and MC2-TCR-T cells that did not undergo PD-1 knockout, the percentages of cells stained with PD-1-PE antibody were 26.3% and 25.2%, respectively. (PDL1-MC2-TCR-T...) PD1- Cells and MC2-TCR-T PD1- The proportion of PD-1-PE stained cells was significantly lower in PDL1-MC2-TCR-T than in Vector and MC2-TCR-T. PD1- Cells and MC2-TCR-T PD1- There was no significant difference in the proportion of PD-1-PE stained cells, such as Figure 24 B.

[0421] The results indicate that (Cas9-mRNA+PD-1-sgRNA)-LNP can safely and effectively knock out the PD-1 gene in TCR-T cells, significantly reducing the PDL1-MC2-TCR-T... PD1- Expression of PD-1 in cells.

[0422] PDL1-MC2-TCR-T cells and MC2-TCR-T cells were co-incubated with (Cas9-mRNA+PD-1-sgRNA)-LNP solution for 24 h to knock out PD-1 and obtain PDL1-MC2-TCR-T cells. PD1- and MC2-TCR-T PD1- Cells. Each group was incubated with PD-1-PE, and the percentage of PD-1-PE stained cells was detected by flow cytometry. Figure 24 A represents the results of flow cytometry staining with PD-1-PE. Figure 24 B represents the PD-1 staining results detected in three independent replicate experiments. Vector represents PBMC transfection with pHR_LV-NS1; MC2-TCR. HM -T is PBMC transfected with pHR_LV-NS1-MC2-TCR HM MC2-TCR HM -T PD1- Transfection of pHR_LV-NS1-MC2-TCR with PBMC HM LNP knockout of PD-1; PDL1-MC2-TCR-T PD1- Transfection of pHR_LV-NS1-PDL1-MC2-TCR into PBMC HM PD-1 was then knocked out by LNP.

[0423] 4. PDL1-MC2-TCR-T knockout of PD-1 PD1- Cells enhanced TCR HMand PD-L1 expression

[0424] 4.1PDL1-MC2-TCR-T PD1- Cells express TCR HM And the level of pMHC binding is higher than that of MC2-TCR-T.

[0425] To determine the PD-1 knockout post-PDL1-MC2-TCR-T PD1- Cellular specific TCR HM To investigate any changes in the expression of [a specific substance] or its binding ability to antigenic epitopes, and after preparing TCR-T cells for each group, the proportion of CD8-APC and MAGE-C2-Tetramer-PE double-stained cell populations was detected using flow cytometry. Results are as follows: Figure 25 A and Figure 25 B. The x-axis of the flow cytometry plot is MAGE-C2-Tetramer-PE, and the y-axis is CD8-APC, PDL1-MC2-TCR-T. PD1- The proportion of double-stained cells was higher in the middle group, reaching 36.8% in a single flow cytometry analysis. In the other three groups, Vector accounted for 0.022%, MC2-TCR-T for 31.8%, and MC2-TCR-T for [the remaining percentage is missing from the original text]. PD1 It is 34.2%. PDL1-MC2-TCR-T PD1- The proportion of double-stained cell populations was significantly higher in the MC2-TCR-T group than in the other two TCR-T and Vector groups. PD1 The proportion of positive double-positive cells was slightly higher than that of MC2-TCR-T, but the difference was not statistically significant.

[0426] The results indicate that PD-1 knockout restores and enhances PDL1-MC2-TCR-T. PD1- TCR on cells HM The expression or binding ability of PD-1 to the MC2 antigen epitope is higher than that of MC2-TCR-T; for MC2-TCR-T cells that do not co-express PD-L1, PD-1 gene knockout treatment did not affect TCR. HM Its expression or function has even been enhanced.

[0427] TCR-T cells were prepared and incubated with antibodies. MAGE-C2-Tetramer-PE and CD8-APC staining was performed on the cells using flow cytometry. Figure 25 A represents the flow cytometry results of MAGE-C2-Tetramer-PE and CD8-APC staining. Figure 25B represents the TCR expression efficiency (i.e., the percentage of cells double-stained with MAGE-C2-Tetramer-PE and CD8-APC as detected by flow cytometry) as determined in three independent replicate experiments. Vector is PBMC transfected with the empty vector pHR_LV-NS1; MC2-TCR-T is PBMC transfected with pHR_LV-NS1-PDL1-MC2-TCR. HM MC2-TCR-T PD1- Transfection of pHR_LV-NS1-MC2-TCR with PBMC HM LNP knockout of PD-1; PDL1-MC2-TCR-T PD1 Transfection of pHR_LV-NS1-PDL1-MC2-TCR into PBMC HM PD-1 was then knocked out by LNP.

[0428] 4.2PDL1-MC2-TCR-T PD1- Cells enhanced the co-expression of exogenous PD-L1.

[0429] The next step in this embodiment was to examine the co-expression of PD-L1 to clarify the effect of PD-1 knockout on PD-L1 co-expression. After preparing each group of cells, MAGE-C2-Tetramer-PE and PDL1-APC were used for labeling, and flow cytometry was used to analyze the proportion of double-stained cell populations.

[0430] See results Figure 26 A and Figure 26 B, PDL1-MC2-TCR-T PD1- The proportion of double-stained cells in the Vector group was significantly higher than in other groups. A single flow cytometry analysis showed that the proportion of double-stained cells (MAGE-C2-Tetramer-PE / PDL1-APC) was 0.021% in the Vector group and 0.068% in the MC2-TCR-T group. PD1 It is 0.21%, PDL1-MC2-TCR-T PD1- The percentage was 45.7%. Furthermore, MC2-TCR-T and MC2-TCR-T... PD1- The lower right quadrant of the flow cytometry image shows a population of cells stained with MAGE-C2-Tetramer-PE, each accounting for approximately 50% of the total. PDL1-MC2-TCR-T cells are visible. PD1- With MC2-TCR-T (or MC2-TCR-T) PD1 They have similar TCRs HM Expression levels, and relatively good PD-L1 expression.

[0431] The results indicate that PD-1 knockout PDL1-MC2-TCR-T PD1-Cells can simultaneously express TCR at high levels HM And PD-L1.

[0432] TCR-T cells were prepared and incubated with antibodies. Cells were then detected by flow cytometry using MAGE-C2-Tetramer-PE and PDL1-APC staining. Figure 26 A shows the flow cytometry results of MAGE-C2-Tetramer-PE and PDL1-APC staining. Figure 26 B represents the PD-L1 expression efficiency (i.e., the percentage of cells double-stained with MAGE-C2-Tetramer-PE and PDL1-APC as detected by flow cytometry) as determined in three independent replicate experiments. Vector represents PBMCs transfected with the empty vector pHR_LV-NS1; MC2-TCR-T represents PBMCs transfected with pHR_LV-NS1-PDL1-MC2-TCR. HM MC2-TCR-T PD1- Transfection of pHR_LV-NS1-MC2-TCR with PBMC HM LNP knockout of PD-1; PDL1-MC2-TCR-T PD1- Transfection of pHR_LV-NS1-PDL1-MC2-TCR into PBMC HM PD-1 was then knocked out by LNP.

[0433] 5. PDL1-MC2-TCR-T knockout of PD-1 PD1- Cells were T2 MC2 Cell cytotoxicity is enhanced after cell activation.

[0434] To understand the impact of removing PD-1, PDL1-MC2-TCR-T PD1- The ability of cells to produce cytokines when stimulated by the MC2 antigen peptide; this embodiment sets up PDL1-MC2-TCR-T and PDL1-MC2-TCR-T. PD1- The three groups of TCR-T cells, MC2-TCR-T, were compared with T2 cells loaded with control peptides. C and T2 loaded with MC2 peptide MC2 Co-culture was performed to analyze and compare the intracellular release of IFN-γ and the secretion levels of TNF-α. First, CD8-APC antibody was incubated, then intracellular IFN-γ was labeled using an intracellular factor staining method. The proportion of CD8-APC and IFN-γ-FITC double-stained cells was detected by flow cytometry. Flow cytometry results are shown below. Figure 27 A and Figure 27 B shows that the horizontal axis represents CD8-APC, and the vertical axis represents IFN-γ-FITC, indicating the relationship between the three groups of TCR-T cells and T2. CDuring co-incubation, the proportions of CD8-APC / IFN-γ-FITC double-stained cell populations were low, at 0.3%, 0.71%, and 0.98%, respectively; compared with T2... MC2 After co-incubation, PDL1-MC2-TCR-T PD1- The proportion of double-stained cells was 10.3% in the middle chromatid population, 1.19% in PDL1-MC2-TCR-T, and 24% in MC2-TCR-T. (PDL1-MC2-TCR-T) PD1- T2 MC2 Following stimulation with the antigenic peptide, the proportion of double-stained cell populations was significantly higher than that of PDL1-MC2-TCR-T cells. PD1- / T2 C Co-culture group and PDL1-MC2-TCR-T / T2 MC2 Group, but significantly lower than MC2-TCR-T / T2 MC2 Co-culture group.

[0435] The detection of TNF-α secretion was also performed using the same three groups of TCR-T, i.e., compared with T2. C and T2 MC2 After co-culturing, the cell supernatant was used for ELISA incubation and color development. The results are as follows: Figure 27 As shown in C, PDL1-MC2-TCR-T PD1- At T2 MC2 Following antigen stimulation, the secretion level of TNF-α was significantly lower than that of MC2-TCR-T / T2. MC2 The group, but significantly higher than PDL1-MC2-TCR-T / T2 C Group.

[0436] The results show that, compared with PDL1-MC2-TC-T, PDL1-MC2-TCR-T PD1- It significantly enhanced the specific release of IFN-γ and the specific secretion of TNF-α in response to MC2 antigen peptide stimulation. PD-1 knockout disrupted the PDL1-MC2-TCR-T... PD1- The ability to secrete cytokines was significantly enhanced. Although PDL1-MC2-TCR-T PD1- Cytokine secretion levels remained significantly lower than MC2-TCR-T, but this embodiment suggests that the response to peptide-loaded T2 cells at this time only represents PDL1-MC2-TCR-T. PD1- Regarding the specific reactivity of the MC2 antigen, PDL1-MC2-TCR-T PD1- The biological function of co-expressed PD-L1 needs further validation through cell experiments.

[0437] Three types of TCR-T cells were compared with the negative control T2. CCells (T2 cells loaded with control peptide) and experimental T2 cells MC2 After incubation with T2 cells loaded with MC2 peptide, the CD8+ cell population producing IFN-γ was labeled with antibodies. First, CD8-APC antibody was incubated, followed by IFN-γ-FITC staining using an intracellular factor staining method. Flow cytometry was used to detect the staining status of CD8-APC and IFN-γ-FITC cells. Double staining of CD8-APC and IFN-γ-FITC cells represents the functional level of IFN-γ production in the CD8+ cell population. TNF-α levels in the supernatant of co-cultured cells were detected using an ELISA assay. Figure 27 A represents the results of flow cytometry staining with CD8-APC and IFN-γ-FITC. Figure 27 B represents the percentage of CD8+ cells producing IFN-γ-FITC (i.e., the percentage of cells double-stained with IFN-γ-FITC and CD8-APC) as detected in three independent replicate experiments. Figure 27 C represents TNF-α secretion as measured in three independent replicate experiments. MC2-TCR-T represents PBMC transfection with pHR_LV-NS1-PDL1-MC2-TCR. HM MC2-TCR-T PD1- Transfection of pHR_LV-NS1-MC2-TCR with PBMC HM LNP knockout of PD-1; PDL1-MC2-TCR-T PD1- Transfection of pHR_LV-NS1-PDL1-MC2-TCR into PBMC HM PD-1 was subsequently knocked out by LNP. 2C Cells: T2 cells were co-incubated with a control peptide (10 μM) for 2 h; T2 MC2 Cells: T2 cells were co-incubated with MC2 peptide (10 μM) for 2 h.

[0438] 6. Successfully constructed simultaneous expression of MAGE-C2 336–344 MC2-A02-K562, a tumor target cell containing HLA-A02 (pMHC) and PD-1. PD1+

[0439] 6.1 Preparation of pHR_LV-NS1-PD-1 recombinant plasmid

[0440] To verify PDL1-MC2-TCR-T PD1- In vitro function was demonstrated in this embodiment by transfecting the pre-constructed MC2-A02-K562 cell line, enabling it to express HLA-A02-restricted MAGE-C2. 336–344PD-1 was further expressed based on the antigenic peptide. The artificially synthesized PD-1 gene sequence with NotI / SalI restriction sites and the expression vector pHR_LV-NS1 were subjected to NotI / SalI double digestion to obtain the target gene fragment PD-1 (888 bp) and the linear digested fragment of pHR_LV-NS1 (8.9 kb). After gel purification, agarose gel electrophoresis was performed, as shown below. Figure 28 A and Figure 28 B. The band positions all met expectations. The two were ligated to obtain the recombinant expression plasmid pHR_LV-NS1-PD-1. After transformation, eight single colonies were selected for further amplification and shaking. PCR verification was then performed on the eight bacterial cultures. All eight PCR amplification products, after agarose gel electrophoresis, showed the expected 438bp band. Figure 28 C. Select one group of plasmid bacterial cultures for amplification and shaken plasmid extraction, followed by double enzyme digestion and identification. The desired bands were obtained, such as... Figure 28 D. The target gene fragment PD-1 is between 750bp and 1000bp, and the pHR_LV-NS1 fragment is between 8000bp and 10000bp. The recombinant lentiviral expression vector plasmid pHR_LV-NS1-PD-1 was successfully constructed. The pHR_LV-NS1-PD-1 plasmid map can be found here. Figure 28 E.

[0441] like Figure 28 Lane A represents the NotI / SalI double digestion product on an agarose gel electrophoresis gel; lane M is the DNA Marker D200; lane 1 contains the PD-1 insert double digestion product, with the target gene PD-1 being approximately 880 bp. Figure 28 Lane B is the agarose gel electrophoresis of the pHR_LV-NS1 NotI / SalI double digestion product: Lane M is the DNA Marker 1Kb; Lane 1 is the pHR_LV-NS1 double digestion linear fragment (approximately 8.9Kb). Figure 28 Electrophoresis diagram of pHR_LV-NS1-PD-1 PCR products (C): Lane M is DNA Marker D2000; lanes 1 to 8 contain PCR amplification products, approximately 438 bp. Figure 28 D represents the NotI / SalI double digestion verification of the recombinant expression vector: lane M1 contains the DNA Marker 1Kb; lane 1 contains the pHR_LV-NS1-PD-1 double digestion product; lane M2 contains the DNA Marker D2000. For example... Figure 28 D represents the pHR_LV-NS1-PD-1 plasmid map.

[0442] 6.2MC2-A02-K562 PD1+ Cellular pMHC and PD-1 expression efficiency is high.

[0443] MC2-A02-K562 was obtained by transfecting MC2-A02-K562 with recombinant plasmid pHR_LV-NS1-PD-1. PD1+ Cells were analyzed using flow cytometry to detect HLA-A-PE and PD-1-PE staining, respectively, to clarify their expression levels. Figure 29 As shown, regarding MC2 antigen expression, MC2-A02-K562 and MC2-A02-K562 PD1+ All cell lines significantly expressed HLA-A02-restricted MAGE-C2 336–344 The proportion of HLA-A-PE stained cells was above 99%, which was statistically significant compared to Vector-K562. Regarding PD-1 expression, MC2-A02-K562... PD1+ The proportion of PD-1-PE stained cells in the cell line reached 99%, which was significantly different from that in Vector-K562 and MC2-A02-K562. However, the proportion of PD-1-PE stained cells in Vector-K562 and MC2-A02-K562 was lower, and there was no statistical difference between the two.

[0444] The results show that MC2-A02-K562 PD1+ Cells can simultaneously express HLA-A02-restricted MAGE-C2 at high levels 336–344 Antigen and PD-1, PD-1 positive tumor target cells were successfully constructed.

[0445] Flow cytometry was used to detect HLA-A-PE and PD-1-PE staining in each group of cells. Figure 29 A represents the results of HLA-A-PE staining flow cytometry. Figure 29 B represents the results of flow cytometry analysis of PD-1-PE stained cells. Figure 29 C represents the HLA-A and PD-1 expression levels as determined in three independent replicate experiments. Vector represents K562 transfection of the empty vector pHR_LV-NS1; MC2-A02-K562 represents K562 transfection of pHR_LV-NS1-MC2-A02; MC2-A02-K562. PD1+ Transfect pHR_LV-NS1-PD-1 with MC2-A02-K562.

[0446] 7. PDL1-MC2-TCR-T PD1- It has higher cytotoxicity against PD-1 positive tumor target cells.

[0447] 7.1 With tumor target cells MC2-A02-K562 PD1+ During co-incubation, PDL1-MC2-TCR-T PD1-A higher percentage of cells produce IFN-γ intracellularly.

[0448] As mentioned above, with T2 MC2 After the reaction, PDL1-MC2-TCR-T PD1- There is a CD8+ cell population that specifically produces IFN-γ at a much higher rate than that of PDL1-MC2-TCR-T, then PDL1-MC2-TCR-T... PD1- With tumor target cells MC2-A02-K562 PD1+ What is the percentage of CD8+ cells producing IFN-γ after co-culture? This example uses CT, MC2-TCR-T, and MC2-TCR-T cells. PD1- , PDL1-MC2-TCR-T and PDL1-MC2-TCR-T PD1- Five groups, respectively with MC2-A02-K562 and MC2-A02-K562 PD1+ After co-culturing for 24 hours, flow cytometry was used to detect the staining of CD8-APC and IFN-γ-FITC (ICS). The results are as follows: Figure 30 The flow cytometry plot uses CD8-APC as the x-axis and IFN-γ-FITC as the y-axis. Within each group, PDL1-MC2-TCR-T... PD1- With tumor target cells MC2-A02-K562 PD1+ The co-culture group showed the highest proportion of CD8+ cells producing IFN-γ (i.e., CD8-APC and IFN-γ-FITC double-stained cells), at 14.1% in a single flow cytometry test. This was significantly higher than the co-culture group with MC2-A02-K562, and also significantly higher than other TCR-T cells co-cultured with MC2-A02-K562. PD1+ Co-culture group; PDL1-MC2-TCR-T regardless of whether it is with MC2-A02-K562 or MC2-A02-K562 PD1+ Co-culture did not produce a significant population of CD8+ cells generating IFN-γ, and the proportion of double-stained cells was very low, showing no significant difference compared to the CT group; compared to MC2-TCR-T, MC2-TCR-T... PD1- and target cells MC2-A02-K562 PD1+ After co-culture, the proportion of I double-stained cells increased significantly and was higher than that of the co-culture group with MC2-A02-K562; after co-culturing MC2-TCR-T with the two target cells, there was no significant difference in the proportion of double-stained cells.

[0449] The results showed that, compared with other TCR-T groups, PDL1-MC2-TCR-T PD1- In PD-1-expressing tumor target cells MC2-A02-K562 PD1+Under stimulation, the highest proportion of IFN-γ-stained CD8+ cells were produced, meaning that more TCR-T cells produced the cytokine IFN-γ after co-expression of PD-L1 and PD-1 knockout. Furthermore, the primary T cell CT group could not specifically respond to tumor target cells; the PDL1-MC2-TCR-T cells without PD-1 knockout could not be stimulated by tumor target cells to produce the cytokine IFN-γ; compared to MC2-TCR-T, MC2-TCR-T... PD1- The knockout of its own PD-1 improves its ability to generate IFN-γ to some extent.

[0450] Set up CT, MC2-TCR-T, MC2-TCR-TPD-1-, PDL1-MC2-TCR-T and PDL1-MC2-TCR-T PD1- Five groups of TCR-T cells interacted with tumor target cells MC2-A02-K562 and MC2-A02-K562, respectively. PD1+ After co-culturing for 24 hours, the cells were first incubated with CD8-APC antibody, followed by incubation with IFN-γ-FITC antibody using intracellular factor staining. Flow cytometry was used to detect the percentage of CD8-APC and IFN-γ-FITC double-stained cell populations, representing the functional level of IFN-γ production in the CD8+ cell population. Figure 30 A represents the results of flow cytometry staining with CD8-APC and IFN-γ-FITC. Figure 30 B represents the percentage of CD8-APC and IFN-γ-FITC double-stained cell populations as detected in three independent replicates. CT represents PBMC culture medium with added transfection reagent; MC2-TCR-T represents PBMC transfected with pHR_LV-NS1-MC2-TCR. HM MC2-TCR-T PD1- Transfection of pHR_LV-NS1-MC2-TCR with PBMC HM LNP knockout of PD-1; PDL1-MC2-TCR-T is PBMC transfection of pHR_LV-NS1-PDL1-MC2-TCR HM ;PDL1-MC2-TCR-T PD1- Transfection of pHR_LV-NS1-PDL1-MC2-TCR into PBMC HM PD-1 was then knocked out by LNP. MC2-A02-K562 was generated by K562 transfection of pHR_LV-NS1-MC2-A02; MC2-A02-K562 PD1+ Transfect pHR_LV-NS1-PD-1 with MC2-A02-K562. n = 3; *p < 0.05; **p < 0.01; ***p < 0.001.

[0451] 7.2 With tumor target cells MC2-A02-K562 PD1+ Co-incubation of PDL1-MC2-TCR-T PD1- Higher TNF-α secretion in cells

[0452] To detect tumor target cell stimulation of PDL1-MC2-TCR-T PD1- The secretion of TNF-α in cells was also monitored in this embodiment, with CT, MC2-TCR-T, and MC2-TCR-T also included. PD1- , PDL1-MC2-TCR-T and PDL1-MC2-TCR-T PD1- Five groups, respectively with MC2-A02-K562 and MC2-A02-K562 PD1+ Cells were co-cultured at a 1:1 effector-to-target ratio for 24 hours, and the TNF-α ELISA kit was used to detect the cell supernatant. Results are as follows: Figure 31 PDL1-MC2-TCR-T PD1- With tumor target cells MC2-A02-K562 PD1+ The co-culture group had significantly higher levels of TNF-α secretion than other TCR-T and MC2-A02-K562 groups. PD1+ The co-culture group also showed significantly higher levels than the PDL1-MC2-TCR-T group. PD1- Co-culture group with MC2-A02-K562; PDL1-MC2-TCR-T PD1- The TNF-α secretion level in the co-culture group with MC2-A02-K562 was also significantly higher than that in other incubation groups; PDL1-MC2-TCR-T with MC2-A02-K562 or MC2-A02-K562 PD1+ After co-culture, the secretion of TNF-α was very low in both groups, only significantly higher than in the CT group; compared with MC2-TCR-T, MC2-TCR-T... PD1- and target cells MC2-A02-K562 PD1+ The secretion of TNF-α was significantly increased after co-culturing with MC2-A02-K562; there was no significant difference in the secretion of TNF-α after co-culturing MC2-TCR-T with the two target cells; the secretion of TNF-α in the CT group was significantly lower than that in the other groups.

[0453] The results showed that, compared with other TCR-Ts, PDL1-MC2-TCR-T... PD1- In PD-1 positive tumor target cells MC2-A02-K562 PD1+ Under stimulation, TNF-α secretion is highest, and it also has higher levels than MC2-TCR-T and MC2-TCR-T in non-PD-1 target cells. PD1-The TNF-α secretion function is affected by PD-L1 co-expression, therefore PD-L1 does not affect the TNF-α secretion function of TCR-T, while PD-1 knockout disrupts the PDL1-MC2-TCR-T function. PD1- They exhibit higher TNF-α secretion. PDL1-MC2-TCR-T cells without PD-1 knockout have poorer TNF-α secretion function when stimulated by tumor target cells; compared to MC2-TCR-T, MC2-TCR-T cells show higher TNF-α secretion. PD1- The knockout of PD-1 itself also improved its ability to secrete TNF-α to some extent.

[0454] Figure 31 In the middle, set CT, MC2-TCR-T, MC2-TCR-T PD1- , PDL1-MC2-TCR-T and PDL1-MC2-TCR-T PD1- Five groups of TCR-T cells interacted with tumor target cells MC2-A02-K562 and MC2-A02-K562, respectively. PD1+ After co-culturing for 24 hours, the TNF-α content in the cell supernatant was detected by ELISA. The experiment was repeated three times. CT consisted of PBMC medium with added transfection reagent; MC2-TCR-T consisted of PBMC transfected with pHR_LV-NS1-MC2-TCR. HM MC2-TCR-T PD1- Transfection of pHR_LV-NS1-MC2-TCR with PBMC HM LNP knockout of PD-1; PDL1-MC2-TCR-T is PBMC transfection of pHR_LV-NS1-PDL1-MC2-TCR HM ;PDL1-MC2-TCR-T PD1- Transfection of pHR_LV-NS1-PDL1-MC2-TCR into PBMC HM PD-1 was then knocked out by LNP. MC2-A02-K562 was generated by K562 transfection of pHR_LV-NS1-MC2-A02; MC2-A02-K562 PD1+ Transfect pHR_LV-NS1-PD-1 with MC2-A02-K562.

[0455] 8. PD-L1 co-expressed on TCR-T cells inhibits the proliferation of PD-1-positive tumor target cells.

[0456] The binding of PD-1 and PD-L1 can negatively regulate T cell proliferation. Therefore, can PD-L1 co-expressed on TCR-T cells also produce the same inhibitory effect on PD-1-positive tumor cells? To verify this hypothesis, this embodiment uses human acute T-lymphoblastic leukemia cells (Jurkat) to construct a TCR-T cell line: PDL1-MC2-TCR-Jurkat. Previous embodiments have verified that Jurkat cells do not express PD-1 under normal conditions, and the engineered co-expression of PD-L1 and MC2-TCR in Jurkat cells should not trigger PD-1 activation-related TCR inhibition, making it a viable model of natural PD-1-negative T cells. Therefore, this embodiment uses Jurkat cells as a parallel TCR-T model to demonstrate the enhancing effect of exogenous PD-L1 on inhibiting target cells. Using MC2-TCR-Jurkat cells that do not express PD-L1 as a control, and comparing them with tumor target cells MC2-A02-K562... PD1+ After co-culturing for 0h, 24h, and 48h, HLA-A2-PE was used to target MC2-A02-K562 cells. PD1+ HLA-A2-PE labeled target cells were identified, and the number of target cells was detected by flow cytometry. The inhibitory rate of PDL1-MC2-TCR-Jurkat on target cells was calculated. Flow cytometry results are as follows: Figure 32 As shown in Figure A, at the initial 0h, MC2-TCR-Jurkat / MC2-A02-K562 PD1+ Co-culture group and PDL1-MC2-TCR-Jurkat / MC2-A02-K562 PD1+ In the co-culture groups, the percentages of target cells stained with HLA-A2-PE were 47.3% and 47.0%, respectively; after 24 hours, the percentages decreased to 37.4% and 32.7%, respectively; and after 48 hours, the percentages continued to decrease to 29.7% and 25.3%, respectively. Using the number of HLA-A2-PE-labeled target cells from the above single flow cytometry analysis, the target cell inhibition rate of each co-culture group at each time point was calculated. The inhibition rate of MC2-TCR-Jurkat on target cells MC2-A02-K562 was calculated. PD1+ The inhibition rate was 25.6% at 24 h and 49.7% at 48 h; while PDL1-MC2-TCR-Jurkat inhibited MC2-A02-K562. PD1+ The inhibition rate was 30.3% at 24 h and 61.4% at 48 h. The above inhibition rates of MC2-TCR-Jurkat on target cells include the background inhibition rates of all cellular components in the mixed cells against the target cells. Using this as a control group for statistical analysis of the inhibition rates of PDL1-MC2-TCR-Jurkat on target cells is more scientific and reliable. Statistical results are as follows: Figure 32B. At both 24 and 48 hours, PDL1-MC2-TCR-Jurkat showed significantly higher inhibition rates against target cells than MC2-TCR-Jurkat. Simultaneously, PDL1-MC2-TCR-Jurkat also inhibited the inhibition of MC2-A02-K562 cells. PD1+ The inhibition rate at 48 hours was significantly higher than that at 24 hours.

[0457] The results showed that, in addition to the baseline killing or inhibitory effect of MC2-TCR-Jurkat on target cells, PDL1-MC2-TCR-Jurkat had an additional inhibitory effect on the proliferation of target cells, and the inhibitory effect became more significant with the extension of the treatment time.

[0458] MC2-TCR-Jurkat and PDL1-MC2-TCR-Jurkat respectively interacted with tumor target cells MC2-A02-K562. PD1+ After co-culturing for 0h, 24h and 48h, target cells were labeled with HLA-A2-PE, and the number and percentage of HLA-A2-PE labeled target cells were detected by flow cytometry. Figure 32 Figure A shows the flow cytometry results of HLA-A2-PE staining of tumor target cells at different time points. Figure 32 B represents the effects of MC2-TCR-Jurkat and PDL1-MC2-TCR-Jurka on tumor target cells MC2-A02-K562, as detected in three independent replicate experiments. PD1+ The inhibition rate. MC2-TCR-Jurkat is Jurkat transfected pHR_LV-NS1-MC2-TCR. HM ;PDL1-MC2-TCR-Jurkat is Jurkat transfection of pHR_LV-NS1-PDL1-MC2-TCR HM .MC2-A02-K562 PD1+ Transfect pHR_LV-NS1-PD-1 with MC2-A02-K562.

[0459] 9. PDL1-MC2-TCR-T PD1- It has higher in vitro killing efficiency against PD-1 positive target cells.

[0460] To further verify PDL1-MC2-TCR-T PD1- In this embodiment, three types of TCR-T cells were obtained to demonstrate their in vitro killing and inhibitory effects on tumor target cells, including MC2-TCR-T, MC2-TCR-T, and MC2-TCR-T. PD1- and PDL1-MC2-TCR-T PD1-PBMCs treated with the transfection reagent served as the control group (CT), and were compared with CFSE-labeled tumor target cells MC2-A02-K562 and PD-1 positive tumor target cells MC2-A02-K562. PD1+ The cells were co-cultured at different effector-to-target ratios (E:T), and the number of remaining target cells after different co-culture times was detected. MC2-A02-K562 and MC2-A02-K562 were used as examples. PD1+ Individual culture groups, results are expressed as lethality.

[0461] The results are as follows Figure 33 As shown in Figure A, after 72 hours of co-culture, with an effector-to-target ratio of 2:1, PDL1-MC2-TCR-T PD1- For MC2-A02-K562 or MC2-A02-K562 PD1+ The kill efficiency of PDL1-MC2-TCR-T was the highest compared to other groups. PD1- For MC2-A02-K562 PD1+ The kill rate of MC2-TCR-T was significantly higher than that of MC2-A02-K562; at the same time, MC2-TCR-T PD1- For MC2-A02-K562 or MC2-A02-K562 PD1+ The killing efficiency of PDL1-MC2-TCR-T was significantly higher than that of the MC2-TCR-T group. At 72 hours and a target-effect ratio of 1:1, the PDL1-MC2-TCR-T group showed significantly higher killing efficiency. PD1- The killing efficiency showed a trend consistent with that at an effector-to-target ratio of 2:1, both being significantly higher than other co-culture groups, particularly against MC2-A02-K562. PD1+ The kill rate was also significantly higher than that against MC2-A02-K562; at this time, MC2-TCR-T PD1- For MC2-A02-K562 or MC2-A02-K562 PD1+ The lethality of the PDL1-MC2-TCR-T group was also significantly higher than that of the MC2-TCR-T group. PD1- At an effectiveness-to-target ratio of 2:1, the killing efficiency was significantly higher than that at an effectiveness-to-target ratio of 1:1. PD1- The same applies to the other groups. The CT group showed a significantly lower killing rate than other TCR-T groups after different effector-to-target ratios and co-culture with two types of tumor target cells, but there was no significant difference between the CT groups under different conditions.

[0462] To gain a more comprehensive understanding of PDL1-MC2-TCR-T PD1-In this embodiment, two additional time points, 48 ​​hours and 24 hours, were also set to measure the in vitro killing efficiency and characteristics. The killing efficiency results at 48 hours are as follows: Figure 33 As shown in B, at an effective-to-target ratio of 2:1, PDL1-MC2-TCR-T PD1- Killing efficiency against two target cells and MC2-TCR-T PD1- No significant difference, MC2-TCR-T PD1- The killing efficiency against tumor cells was significantly higher than that of MC2-TCR-T and PDL1-MC2-TCR-T. PD1- For MC2-A02-K562 PD1+ The killing efficiency is higher than that against MC2-A02-K562; with an effectiveness-to-target ratio of 1:1, PDL1-MC2-TCR-T PD1- For MC2-A02-K562 PD1+ The lethality is significantly higher than that of MC2-TCR-T PD1- But PDL1-MC2-TCR-T PD1- The lethality against MC2-A02-K562 is comparable to that against MC2-TCR-T. PD1- No significant difference, MC2-TCR-T PD1- The killing efficiency against both types of tumor cells was significantly higher than that against MC2-TCR-T. MC2-TCR-T, MC2-TCR-T PD1- and PDL1-MC2-TCR-T PD1- The kill efficiency at an effective-to-target ratio of 2:1 was significantly higher than that at an effective-to-target ratio of 1:1.

[0463] 24-hour lethality results are as follows Figure 33 C. At an effectiveness-to-target ratio of 2:1, PDL1-MC2-TCR-T PD1- The killing efficiency against both target cells was similar to that of MC2-TCR-T. PD1- No significant difference; MC2-TCR-T PD1- The killing rate of tumor cells was significantly higher than that of MC2-TCR-T; PDL1-MC2-TCR-T PD1- For MC2-A02-K562 PD1+ The killing efficiency of PDL1-MC2-TCR-T was higher than that of MC2-A02-K562, but the difference was not significant. At an effectiveness-to-target ratio of 1:1, the killing efficiency of PDL1-MC2-TCR-T was higher. PD1- For MC2-A02-K562 PD1+ The lethality of MC2-TCR-T PD1- No significant difference; MC2-TCR-T PD1- For MC2-A02-K562 PD1+The kill rate of MC2-TCR-T was significantly higher than that of MC2-TCR-T; however, after co-culturing with MC2-A02-K562, the kill rate of MC2-TCR-T and MC2-TCR-T was significantly higher. PD1- and PDL1-MC2-TCR-T PD1- There was no significant difference in lethality between the two; MC2-TCR-T PD1- and PDL1-MC2-TCR-T PD1- For MC2-A02-K562 PD1+ Its killing efficiency is slightly higher than that of MC2-TCR-T.

[0464] The results showed that PDL1-MC2-TCR-T PD1- It has a higher killing effect on PD-1 positive tumor target cells; PDL1-MC2-TCR-T PD1- While MC2-TCR targets and kills MC2-specific tumor cells, its co-expressed PD-L1 can utilize TCR-T as a carrier to precisely bind to PD-1 on tumor target cells and inhibit their proliferation, thus synergistically inhibiting the specific cell killing effect of TCR-T. Simultaneously, PDL1-MC2-TCR-T... PD1- For non-PD-1 positive tumor cells but with MC2 antigen epitopes, there are at least MC2-TCR-T... PD1- The lethality of the MC2-TCR-T; and the MC2-TCR-T PD1- Because the knockout of endogenous PD-1 results in a higher tumor cell killing rate than MC2-TCR-T, especially against PD-1-positive tumor target cells. Furthermore, the difference in killing rate caused by setting different effector-to-target ratios indicates that TCR-T can produce higher tumor-killing efficacy at an effector-to-target ratio of 2:1.

[0465] Control cell CT and three types of TCR-T cells: MC2-TCR-T, MC2-TCR-T PD1- and PDL1-MC2-TCR-T PD1- CFSE-labeled tumor target cells MC2-A02-K562 and MC2-A02-K562, respectively PD1+ At effector-to-target ratios of 1:1 and 2:1, the number of CFSE-labeled tumor target cells was detected by flow cytometry after 72 h, 48 h, and 24 h of co-culture, and the killing efficiency was calculated. The experiment was repeated three times. Figure 33 A represents the kill efficiency of each group over 72 hours; Figure 33 B represents the kill efficiency of each group over 48 hours; Figure 33 C represents the killing efficiency of each group at 24 hours. CT represents PBMC culture medium with added transfection reagent; MC2-TCR-T represents PBMC transfected with pHR_LV-NS1-MC2-TCR. HM MC2-TCR-TPD1- PBMC transfection with pHR_LV-NS1-MC2-TCRHM followed by LNP knockout of PD-1; PDL1-MC2-TCR-T PD1- Transfection of pHR_LV-NS1-PDL1-MC2-TCR into PBMC HM PD-1 was then knocked out by LNP. MC2-A02-K562 was generated by K562 transfection of pHR_LV-NS1-MC2-A02; MC2-A02-K562 PD1+ pHR_LV-NS1-PD-1 was transfected with MC2-A02-K562. The effector-to-target ratio is the ratio of effector TCR-T cells to tumor target cells.

[0466] 10. PDL1-MC2-TCR-T PD1- The killing efficiency against PD-1 positive tumor target cells increased with prolonged co-culture time.

[0467] The previous section compared the efficiency of different types of TCR-T cells in killing tumor target cells within the same incubation time. To further illustrate the effectiveness of PDL1-MC2-TCR-T... PD1- The change in tumor target cell killing efficiency with co-incubation time was investigated in this embodiment using the same method to obtain CT, MC2-TCR-T, and MC2-TCR-T cells. PD1- and PDL1-MC2-TCR-T PD1- Cells were co-cultured at a 2:1 effector-to-target ratio, which yielded the best killing efficiency, and then MC2-A02-K562 cells were labeled with CFSE. PD1+ Cells were analyzed using flow cytometry to detect the number of CFSE-labeled cells after 24, 48, and 72 hours, and the killing efficiency at different time points was calculated. Figure 34 A represents a single flow cytometry result, PDL1-MC2-TCR-T. PD1- In the incubation group, MC2-A02-K562 tagged with CFSE PD1+ The number of cells decreased significantly with prolonged incubation time, PDL1-MC2-TCR-T PD1- The cell count in the incubation group was lower than that in other incubation groups at all time points. Statistical results are as follows: Figure 34 B shows that PDL1-MC2-TCR-T PD1- For MC2-A02-K562 PD1+ The lethality of MC2-TCR-T consistently increased from 24 to 72 hours, with an average lethality of 95.52% at 72 hours, and was higher than other groups at every time point; PD1-The lethality of the group increased significantly from 24h to 48h, but decreased at 72h. However, its lethality was always higher than that of the MC2-TCR-T group. The lethality of MC2-TCR-T changed little at the three time points. It was basically the same from 24h to 48h, and increased slightly at 72h.

[0468] The results indicate that PDL1-MC2-TCR-T within 72 hours PD1- The killing efficiency against PD-1 positive tumor target cells increased with prolonged interaction time and maintained a consistently high killing level. MC2-TCR-T PD1- The killing efficiency against PD-1 positive tumor target cells is higher than that of MC2-TCR-T, and it can maintain a certain degree of growth over a certain period of time. The killing efficiency of MC2-TCR-T remains at a relatively constant level, with no significant change within 72 hours.

[0469] CT, MC2-TCR-T, MC2-TCR-T PD1- and PDL1-MC2-TCR-T PD1- CFSE-labeled tumor target cells MC2-A02-K562 PD1+ At an effector-to-target ratio of 2:1, after co-culturing for 24h, 48h, and 72h, the number of CFSE-labeled tumor target cells was detected by flow cytometry, and the killing efficiency was calculated. The yellow box indicates the tumor target cells MC2-A02-K562. PD1+ Flow cytometry results of the cultured group alone. Figure 34 Figure A shows the results of flow cytometry analysis of CFSE-labeled cells. Figure 34 B represents the CT, MC2-TCR-T, and MC2-TCR-T levels detected in three independent, repeated experiments. PD1- and PDL1-MC2-TCR-T PD1- For MC2-A02-K562 PD1 The killing efficiency. CT: PBMC medium with added transfection reagent; MC2-TCR-T: PBMC transfected with pHR_LV-NS1-MC2-TCR HM MC2-TCR-T PD1- PBMC transfection with pHR_LV-NS1-MC2-TCR HM LNP knockout of PD-1; PDL1-MC2-TCR-T PD1- PBMC transfection with pHR_LV-NS1-PDL1-MC2-TCR HM PD-1 was subsequently knocked out by LNP. MC2-A02-K562 PD1+ : MC2-A02-K562 transfected pHR_LV-NS1-PD-1.

[0470] Example 3: PDL1-MC2-TCR-T PD1- Animal experiments on antitumor effects

[0471] This embodiment includes experimental methods and results. The experimental methods section includes:

[0472] 1. Preparation of tumor-bearing material MC2-A02-K562 PD1+ -Luc cells;

[0473] 2. PDL1-MC2-TCR-T PD1- Cells were reinfused into the negative tumor MC2-A02-K562 PD1+ -Luc's NCG mouse model;

[0474] 3. In vivo imaging of tumor-bearing mice;

[0475] The second part of the experimental results includes:

[0476] 1. PDL1-MC2-TCR-T PD1- It exhibits the strongest tumor burden inhibition effect in NCG-bearing mice;

[0477] 2. PDL1-MC2-TCR-T PD1- MC2-TCR-T infusion therapy inhibited the growth of tumor cells in mice;

[0478] The details are as follows:

[0479] I. Experimental Methods:

[0480] 1. Preparation of tumor-bearing material MC2-A02-K562 PD1+ -Luc cells

[0481] Using the laboratory's existing pHR_LV-NS1-Luc recombinant vector, carrying the luciferase gene, transfected A02-K562 and MC2-A02-K562 cells. PD1+ Cells (specific experimental steps are the same as in Example 1) were used to obtain the tumor-bearing Luc stable cell line MC2-A02-K562. PD1+ -Luc.

[0482] 2. PDL1-MC2-TCR-T PD1- Cells were reinfused into the negative tumor MC2-A02-K562 PD1+ -Luc's NCG mouse model

[0483] Female NCG mice aged 5-6 weeks were randomly divided into five groups: control CT group, MC2-TCR-T (1M) group, PDL1-MC2-TCR-T group, and PDL1-MC2-TCR-T group. PD1-(1M) group, MC2-TCR-T (5M) group, PDL1-MC2-TCR-T PD1- (5M) group; each mouse was given MC2-A02-K562 on day 0. PD1+ - Luc had a subcutaneous tumor, and different TCR-T cells were infused into the mice every other day according to their group. Starting from Day 0, in vivo imaging was performed on the mice every 5 days to observe the tumor burden. At the end of the experiment, the mice were sacrificed, the subcutaneous tumors were removed, photographed, weighed, and their volume was measured. The animal experimental procedure is as follows: Figure 35 As shown:

[0484] The constructed tumor target cells and TCR-T cell lines were expanded and cultured. Before use, a homogeneous cell suspension was prepared, and 1×10⁻⁶ cells were added. 6 MC2-A02-K562 per 100μL PD1+ -Luc was subcutaneously injected into the right rib of each group of female NCG mice, marked as day 0. 24 hours later, each group was injected via tail vein with 1×10⁻⁶ therapeutic TCR-T cell suspension. 6 100μL or 5×10 6 Each mouse was examined per 100 μL. In vivo testing was performed every 5 days starting from day 0 until the last photograph on day 25. After the last photograph, the mice were euthanized, and subcutaneous tumors were removed for measurement and recording.

[0485] According to the animal experiment schedule, tumor-bearing animals were treated with MC2-A02-K562. PD1+ -Luc cells and various groups of TCR-T cells for in vivo treatment were expanded and cultured at 37°C under 5% CO2 conditions for later use.

[0486] 1) Experimental NCG female mice, 5-6 weeks old, were acclimatized in an SPF-grade animal facility for 7 days after receipt;

[0487] 2) NCG mice were randomly divided into five groups: CT group, MC2-TCR-T (1M) group, PDL1-MC2-TCR-T group, and PDL1-MC2-TCR-T group. PD1- (1M) group, MC2-TCR-T (5M) group, PDL1-MC2-TCR-T PD1- (5M) group;

[0488] 3) Use MC2-A02-K562 PD1+ To perform tumor-bearing cell manipulation, the hair at the right last rib of the mouse was shaved, and MC2-A02-K562 cells were collected. PD1+ -Luc cells were resuspended in PBS and injected subcutaneously at a dose of 1×10⁻⁶ per animal. 6 The number of tumors per 100 μL is recorded as day 0 on the day the tumor is borne.

[0489] 4) TCR-T cells from each group were reinfused via the tail vein 24 hours after tumor implantation, resuspended in PBS, with a reinfusion volume of 100 μL per animal. The CT group received 100 μL of PBS, while the MC2-TCR-T (1M) group received 1 × 10⁻⁶ PBS. 6 One MC2-TCR-T, PDL1-MC2-TCR-T PD1- (1M) group return 1×10 6 PDL1-MC2-TCR-T PD1- MC2-TCR-T(5M) group reintroduces 5×10 6 One MC2-TCR-T, PDL1-MC2-TCR-T PD1- (5M) group return 5×10 6 PDL1-MC2-TCR-T PD1- .

[0490] ① Gently mix the TCR-T solution and use a 1mL syringe to draw 200μL;

[0491] ② Fix the mouse on the syringe, display the location of the tail vein, and wipe the injection site on the mouse tail with an alcohol swab;

[0492] ③ After inserting the syringe needle into the mouse's tail vein, inject 100 μL of TCR-T solution at a uniform speed, gently twist to withdraw the needle, press a sterile cotton ball against the injection site for 5 seconds, put the mouse back into the cage, observe for about 20 minutes, and continue normal feeding.

[0493] 3. In vivo imaging of tumor-bearing mice

[0494] The first in vivo imaging was started on day 0 after tumor implantation, and was repeated every 5 days until day 25, for a total of 6 times.

[0495] 1) Turn on the live imaging device before starting the experiment and allow the instrument to warm up;

[0496] 2) Anesthetize mice with isoflurane gas. Prepare a 15 mg / mL fluorescein stock solution with sterile PBS in advance using the in vivo imaging tracer D-fluorescein potassium salt. After mixing, filter the solution through a 0.2 μm filter membrane for sterilization and use immediately. Administer intraperitoneally at a fluorescein / body weight concentration of 150 mg / kg. For example, if an NCG mouse weighs about 20g, the dosage is 3 mg per mouse.

[0497] 3) Shave the hair at the tumor site of the mouse to prepare. After injecting fluorescein into the body, the light signal reaches the strongest stable plateau period in about 10-15 minutes. Place the mouse in the imaging dark box platform small animal fixation device to expose the tumor site. Close the box door, select "Bioluminescence mode", check the parameters, and click Acquire Sequence to start shooting. Under the condition of no external light source, the luminescence of the tumor in the mouse is photographed to obtain the in vivo imaging image data of the mouse.

[0498] 4) The experimental results were analyzed and statistically processed using software such as Image and GraphPad Prism 8.0;

[0499] 5) After the final photograph, euthanize the mouse, dissect and remove the subcutaneous tumor, collect and photograph the tumor, and use calipers to measure the long axis (L) and short axis (W) of the tumor. Calculate TV using the formula: TV = 0.5 × L × W 2 Calculate tumor volume (cm) 3 Weigh and record the tumor mass.

[0500] Statistical methods

[0501] Statistical analysis was performed using Prism version 8.0 (Graph Pad). For data comparing between groups, t-tests were used. One-way ANOVA was used to detect the significance of differences between groups. *p<0.05 indicates a statistically significant difference; **p<0.01 indicates a statistically significant difference; ***p<0.001 indicates an extremely statistically significant difference.

[0502] II. Experimental Results:

[0503] 1. PDL1-MC2-TCR-T PD1- It has the strongest tumor burden inhibition effect in tumor-bearing NCG mice.

[0504] The experimental results above have demonstrated the effectiveness of MC2-TCR-T and PDL1-MC2-TCR-T. PD1 In vitro activity against tumor target cells MC2-A02-K562 PD1+ It has both killing and proliferative effects. To investigate the in vivo tumor-inhibiting effect of the above-mentioned TCR-T, a tumor-inhibiting MAGE-C2 gene expressing HLA-A*02:01 was used. 336–344 -ALKDVEERV antigenic epitope and MC2-A02-K562 overexpressing PD1 PD1+ - Establishment of a mouse model of NCG xenograft tumor using Luc cells. Injection of MC2-A02-K562. PD1+ -Luc cells were implanted subcutaneously into the right rib area of ​​NCG mice (n=4, 1×10⁻⁶). 6 (each animal), and 24 hours later, TCR-T cells of different groups and doses were reinfused via the tail vein according to the grouping. The day of subcutaneous tumor inoculation was recorded as day 0. From day 0, a fluorescent tracer was injected intraperitoneally every 5 days, and the animal was anesthetized before being photographed using a small animal in vivo imaging system. Figure 36As shown, no significant fluorescence signal appeared in the control group and any of the TCR-T infusion groups from 0-10 days; by 15 days, fluorescence signals appeared in all groups, with the highest in the MC2-TCR-T(1M) group; by 20 days, the fluorescence signal intensity increased in all groups, with the MC2-TCR-T(1M) group higher than the other groups, the CT group also showing higher intensity, and the MC2-TCR-T(5M) group and PDL1-MC2-TCR-T PD1- The fluorescence signal in the (5M) group was weaker; by the end of the 25-day experiment, the CT group showed the highest fluorescence signal intensity in the tumor burden burst, and the MC2-TCR-T (1M) group also showed higher intensity, as did the MC2-TCR-T (5M) group and the PDL1-MC2-TCR-T group. PD1- The fluorescence signals of the (1M) group were similar, while those of the PDL1-MC2-TCR-T group were different. PD1- The (5M) group has the lowest signal strength.

[0505] Figure 36 In the middle, MC2-A02-K562 PD1+ -Luc cells were administered subcutaneously at 1×10 6 One per mouse was injected subcutaneously into the right rib area of ​​NCG mice. 24 hours later, different TCR-T cells were reinfused via the tail vein: the CT group received 100 μL of PBS; the MC2-TCR-T (1M) group received 1 × 10⁻⁶ TCR-T cells. 6 One / each MC2-TCR-T cell was infused; PDL1-MC2-TCR-T PD1- Group 1×10 6 One / only PDL1-MC2-TCR-T return PD1- Cells; MC2-TCR-T (5M) group at 5×10 6 One / each MC2-TCR-T cell was infused; PDL1-MC2-TCR-T PD1- (5M) group according to 5×10 6 One / only PDL1-MC2-TCR-T return PD1- Cells. The day of xenograft inoculation is recorded as day 0. Starting from day 0, fluorescent tracers are injected intraperitoneally every 5 days at a dose of 150 mg / kg, and in vivo imaging of the animals is performed after anesthesia.

[0506] The tumor fluorescence signals in all groups of NCG-bearing mice showed no significant changes in the first 10 days. Total tumor burden was detected at day 15, but the differences between groups were small and not statistically significant. At day 20, tumor fluorescence intensity increased significantly in all groups, with the MC2-TCR-T (1M) group showing the highest intensity, followed by the CT group, and then the MC2-TCR-T (5M) and PDL1-MC2-TCR-T groups. PD1-The fluorescence intensity of the (5M) group was relatively weak; by the end of the 25-day experiment, the tumor burden fluorescence intensity of the CT group was significantly higher than that of the other groups, and the tumor fluorescence intensity of the MC2-TCR-T (1M) group was significantly higher than that of the MC2-TCR-T (5M) group and the PDL1-MC2-TCR-T group. PD1- (5M) group. Furthermore, although the MC2-TCR-T (1M) group was higher than the PDL1-MC2-TCR-T group... PD1- The tumor fluorescence intensity in the (1M) group was not significant, among which PDL1-MC2-TCR-T PD1- The (5M) group showed the lowest tumor fluorescence signal intensity, which was statistically significant. Figure 37 .

[0507] In vivo imaging results demonstrate that, in the in vivo environment, our novel TCR-T cell line, PDL1-MC2-TCR-T, exhibits [positive / positive] activity. PD1- It has the strongest inhibitory effect on the growth of subcutaneous xenografts; PDL1-MC2-TCR-T PD1- The inhibitory effect on subcutaneous tumors increased with increasing infusion dose, and the 5M group showed better tumor-suppressing effect than the 1M group; PDL1-MC2-TCR-T PD1- The tumor-suppressing effect became more pronounced with prolonged action after reinfusion. Furthermore, MC2-TCR-T also exhibited inhibitory effects on subcutaneous xenografts in mouse models, but both its final and long-term inhibitory effects were weaker than those of PDL1-MC2-TCR-T. PD1- .

[0508] 2. PDL1-MC2-TCR-T PD1- MC2-TCR-T infusion therapy inhibited the growth of tumor cells in mice.

[0509] To fully verify PDL1-MC2-TCR-T PD1- MC2-TCR-T vs MC2-A02-K562 PD1+ To investigate the inhibitory effect on tumor growth, this embodiment involved euthanizing NCG mice that had undergone the aforementioned 25-day in vivo imaging, dissecting subcutaneous tumors, measuring their volume, and calculating the tumor size. For example... Figure 38 A and Figure 38 As shown in B, compared with the CT group, PDL1-MC2-TCR-T PD1- In both the MC2-TCR-T and PD-L1-MC2-TCR-T groups, TCR-T intervention significantly reduced tumor volume; PD1- In the two dose groups of MC2-TCR-T, the high-dose group (5×10) 6 The tumor volume of the group with 1 × 10⁶ tumors / animals was significantly lower than that of the low-dose group (1 × 10⁶ tumors / animals). 6Groups (each / animal); PDL1-MC2-TCR-T PD1- The tumor volume in the (5M) group was significantly lower than that in both MC2-TCR-T groups and the PDL1-MC2-TCR-T group. PD1- (1M) group, while PDL1-MC2-TCR-T PD1- The tumor volume in the (1M) group was lower than that in the MC2-TCR-T (1M) group, but the difference was not statistically significant. Tumor quality data also showed a similar trend, such as... Figure 38 C. Tumor quality in the CT group was significantly higher than in other groups; PDL1-MC2-TCR-T PD1- (5M) group tumor quality was significantly lower than that of PDL1-MC2-TCR-T PD1- (1M) group and MC2-TCR-T (5M) group.

[0510] The above results indicate that the HLA-A*02:01-restricted MAGE-C2 is specifically targeted. 336–344 Both structures of the -ALKDVEERV antigenic epitope in TCR-T cells can inhibit the growth of transplanted tumors in vivo; the therapeutic effect of specific TCR-T cells in vivo is dose-dependent, with the high-dose group showing stronger inhibitory effects than the low-dose group; PDL1-MC2-TCR-T PD1- Based on the specific targeting and killing of MAGE-C2 tumor cells, the overexpressed PD-L1 protein can further inhibit tumor cell growth, exhibiting a more potent tumor-suppressing ability. In animal experiments, mice in both high and low dose groups did not show significant adverse reactions or death during the experiment, indicating that the PD-L1-MC2-TCR-T... PD1- And the reinfusion dose of MC2-TCR-T, at 5×10 6 It is safe within the range of one per unit.

[0511] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. TCR-T cells with killing and tumor suppression efficacy, characterized in that, The TCR-T cells are T cells expressing the tumor antigen TCR, which is MAGE-C2; the TCR-T cells stably express PD-L1; the PD-1 gene in the TCR-T cells is knocked out; and the target gene MC2-TCR is used to replace the constant C region of TCR-Vα3 and TCR-Vβ28 with the mouse C region. HM The T cell receptor was modified into a murine form.

2. The TCR-T cell having a killing and tumor suppression efficacy according to claim 1, characterized in that, The TCR sequence, in whole or in part, is derived from one or more CD4 T cell clones, one or more CD8 T cell clones, or multiple CD4 T and CD8 T cell clones in a tumor patient.

3. The TCR-T cell having a killing and tumor suppression efficacy according to claim 1 or 2, characterized in that, The PD-L1 is either endogenous or exogenous.

4. The use of TCR-T cells with killing and tumor-suppressing effects as described in any one of claims 1-3 in the preparation of a drug for treating tumors; wherein the tumor is a tumor expressing MAGE-C2 antigen and expressing or being induced to express PD-1.

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