TCR-T cell with killing and tumor inhibition effects and application thereof

Abstract

The invention discloses a TCR-T cell with killing and tumor inhibition effects and application thereof, the TCR-T cell with killing and tumor inhibition effects is a T cell expressing a tumor recognition antigen TCR, and the tumor antigen is one or more of MAGE-C2, MAGE-A3, MAGE-A4, NY-ESO-1 or TP53; the TCR-T cell is used for stably expressing the PD-L1; the PD-1 gene in the TCR-T cell is knocked out or inhibited. The TCR-T cells which are targeted to MAGE-C2, stably express PD-L1 and knock out own PD-1 have greater killing and tumor inhibition effects in in-vitro and animal experiments. The compound can be used for preparing medicines for treating tumors.

Description

Technical field:

[0001] The present invention relates to the technical field of immunotherapy, and in particular to a TCR-T cell with killing and tumor inhibition effects and an application thereof. 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 identical to the natural TCR in structure and function, and can recognize specific tumor antigens presented by MHC (major histocompatibility complex) molecules. Antigen selection is the key to the development of TCR-T cell therapy. The ideal antigen should be tumor-specific, widely expressed in tumor cells, and can be presented by MHC molecules.

[0003] MAGE-C2 (MC2), also known as CT10, belongs to the MAGE family (melanoma-associated antigen family). MAGE-C2 has good tumor specificity and is associated with many types of cancers such as lung cancer, melanoma, breast cancer, prostate cancer, liver cancer, and multiple myeloma. It is a potential candidate target for TCR-T antigens. Previous studies have obtained TCR sequences targeting MC2 antigens from melanoma patients who have a good clinical response to tumor peptide vaccine treatment. One of the antigen targets: HLA-A02 restricted MAGE-C2 336–344 (ALKDVEERV) has anti-tumor activity after transducing T cells. However, it is still unclear whether this specific antigen has the potential to be developed as a TCR-T antigen target, how the TCR-T cells that recognize this antigen perform in anti-tumor, and whether it is possible to improve and enhance the function by integrating other genes in engineering design.

[0004] PD-1 is a receptor in T cells. The treatment strategy of blocking the PD-1 signaling pathway with immune checkpoint inhibitors (ICIs) and enhancing T cell activity has been widely recognized and applied. However, ICIs are generally systemically administered at present, and their effects on cells are not specific. Some side effects caused by ICIs may be related to this. PD-1 is widely expressed in a variety of cancer cells. Studies have shown that the PD-1 signaling pathway plays the role of a tumor suppressor in certain types of tumor cells, but the treatment strategy of specifically activating the PD-1 signal in malignant cells has not yet been explored. Whether it is possible to use the targeting of TCR-T to tumor antigens, target and actively activate the PD-1 signaling pathway of tumor cells under specific conditions (the PD-1 of the TCR-T cell itself is inhibited), and combine the killing effect of TCR-T to produce a tumor suppressor effect, is the exploration direction of this application. Summary of the invention:

[0005] The first object of the present invention is to provide a TCR-T cell with killing and tumor suppression effects.

[0006] The second object of the present invention is to provide an application of TCR-T cells with killing and tumor suppression effects.

[0007] The first object of the present invention is implemented by the following technical scheme: a TCR-T cell with killing and tumor suppression efficacy, wherein the TCR-T cell is a T cell expressing a TCR that recognizes a tumor antigen, and 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; the TCR carries one or more of the tumor antigens MAGE-C2, MAGE-A3, MAGE-A4, NY-ESO-1 or TP53 to recognize the effect of: on the one hand, it can target and identify tumor cells, and on the other hand, it has a significant killing effect on tumor cells; the purpose of the TCR-T cell stably expressing PD-L1 is: when it is able to target and identify tumor cells, the PD-L1 expressed by the TCR-T cell can target and activate the PD-1 signaling pathway in tumor cells, thereby achieving the killing or inhibition effect on tumor cells, but not normal cells.

[0008] Furthermore, the TCR sequence is derived from one or more CD4 T cell clones; or one or more CD8 T cell clones; or multiple CD4T and CD8 T cell clones in a tumor patient.

[0009] Furthermore, the PD-1 gene in the TCR-T cells is knocked out or inhibited, the purpose of which is to prevent PD-L1 expressed in TCR-T cells from activating the PD-1 signaling pathway in TCR-T cells, thereby avoiding inhibitory effects on TCR-T cells.

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

[0011] The method for knocking out the PD-1 gene in the TCR-T cell can be achieved by using existing technologies, such as: CRISPR-Cas9; RNA interference (RNA Interference, RNAi) including short interfering RNA (Short interfering RNA, siRNA) and small hairpin interfering RNA (Short hairpin RNA, shRNA); TALENs (Transcription Activator-Like Effector Nucleases) gene editing; zinc finger nucleases (Zinc-Finger Nucleases, ZFNs); or homologous recombination (Homologous Recombination), etc.

[0012] The method of inhibiting the PD-1 gene in the TCR-T cell can be achieved by existing technologies, such as: RNA interference (RNA Interference, RNAi) including short interfering RNA (Short interfering RNA, siRNA) and short hairpin interfering RNA (Short hairpin RNA, shRNA); Antisense Oligonucleotides (Antisense Oligonucleotides, ASOs); CRISPR Interference (CRISPR Interference, CRISPRi); small molecule inhibitors; Transcriptional Repressors; Dominant-Negative Mutants; Epigenetic Modifications; or inhibitory antibodies.

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

[0014] The method of stably expressing endogenous PD-L1 by TCR-T cells can be achieved by 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, chemical drug induction, etc.

[0015] Methods for stably expressing exogenous PD-L1 in TCR-T cells: This can be achieved using existing technologies, such as: transfection of PD-L1 gene with lentivirus, adenovirus, retrovirus, and other viruses, electrotransfection of PD-L1 gene, nanoparticle transfection of PD-L1 gene, chemical transfection of PD-L1 gene, PD-L1 expression plasmid transfection, mRNA transfection, CRISPR / Cas9 integration of PD-L1 gene, etc.

[0016] The second object of the present invention is implemented by the following technical solution: use of the TCR-T cells with killing and tumor suppressor effects as described in the first object of the present invention in the preparation of drugs for treating tumors.

[0017] Furthermore, the tumor is any one or more of lung cancer, lymphoma, leukemia, melanoma, intestinal cancer, liver cancer, gastric 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] Explanation of symbols

[0020]

[0021]

[0022] Advantages of the present 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 suppression efficacy in vitro and 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 anti-tumor effect of TCR-T. Using TCR-T cells, the PD-1 signal of tumor cells is actively and targetedly activated, which has a synergistic effect on the inhibition of tumor cells. Silencing PD-1 through gene editing technology can effectively enhance the activity of TCR-T cells while avoiding self-inhibition.

[0025] (3) The present invention provides new strategies and reference bases 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. Description of the drawings:

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0028] Figure 2 pUC-SP-MC2-TCR is a recombinant cloning vector HM and recombinant expression vector pHR_LV-NS1-MC2-TCR HM Structural diagram;

[0029] Figure 3 This is a schematic diagram of the recombinant vector of the tumor target cell line;

[0030] Figure 4 This is a schematic diagram of MHC Tetramer;

[0031] Figure 5 pUC-SP-MC2-TCR is a recombinant cloning vector HM and agarose gel electrophoresis of double enzyme digestion products;

[0032] Figure 6 Verification of recombinant expression vector and pHR_LV-NS1-MC2-TCR HM Plasmid map;

[0033] Figure 7 For MC2-TCR HM -T's MAGE-C2-specific TCR expression efficiency is higher, flow cytometry detection results;

[0034] Figure 8 For MC2-TCRHM -T cells in T2 MC2 Flow cytometry test results of cells that produce IFN-γ more effectively under stimulation;

[0035] Fig. 9 For MC2-TCR HM -T and T2 MC2 The test result graph shows that the secretion level of TNF-α is higher after cell co-culture;

[0036] Fig.10 Agarose gel electrophoresis for the preparation of pHR_LV-NS1-A02

[0037] Fig.11 Agarose gel electrophoresis for the preparation of pHR_LV-NS1-MC2-A02

[0038] Fig.12 This is a validation diagram of the successful expression of antigen epitopes in tumor target cell lines;

[0039] Fig.13 For MC2-TCR HM -Flow cytometry test results showing that T cells produce IFN-γ more effectively after being stimulated by tumor target cells;

[0040] Fig.14 For MC2-TCR HM -The test results of TNF-αElisa kit show that the secretion of TNF-α by T cells is more powerful after being stimulated by tumor target cells;

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

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

[0043] Fig.17 Schematic diagram of the packaging process and function of LNP-mRNA (Cas9-mRNA / PD-1-sgRNA);

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

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

[0046] Fig. 20 Flow cytometry results showing that exogenous PD-L1 restricted the engineered TCR expression of PDL1-MC2-TCR-T;

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

[0048] Fig. 22 PDL1-MC2-TCR-Jurkat cells can normally express TCR HM and PD-L1 validation results diagram;

[0049] Fig.23 This is the quality control test result diagram of (Cas9-mRNA+PD-1-sgRNA)-LNP;

[0050] Fig.24 For PDL1-MC2-TCR-T PD1- Flow cytometry results showed that the PD-1 expression of cells was significantly reduced;

[0051] Fig.25 For PDL1-MC2-TCR-T PD1- TCR HM Flow cytometry results of expression recovery;

[0052] Fig.26 For PDL1-MC2-TCR-T PD1- Cells restore TCR HM and flow cytometry results of PD-L1 expression levels;

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

[0054] Fig.28 Agarose gel electrophoresis and plasmid map for the preparation of pHR_LV-NS1-PD-1;

[0055] Fig.29 For MC2-A02-K562 PD1+ Validation diagram of cells expressing pMHC complex and PD-1;

[0056] Fig.30 For PDL1-MC2-TCR-T PD1-With MC2-A02-K562 PD1+ Flow cytometry results showed that the level of intracellular IFN-γ production was higher after co-culture;

[0057] Fig.31 For use with MC2-A02-K562 PD1+ Co-culture of PDL1-MC2-TCR-T PD1 The ELISA test results showed that the secretion level of TNF-α was higher;

[0058] Fig.32 MC2-A02-K562 for PDL1-MC2-TCR-Jurkat inhibition PD1+ The effect verification diagram of proliferation;

[0059] Fig.33 This is a comparison chart of the killing efficiency of TCR-T cells on tumor target cells;

[0060] Fig.34 For PDL1-MC2-TCR-T PD1- For MC2-A02-K562 PD1+ A comparison chart of the effect of the killing efficiency increasing over time;

[0061] Fig.35 Flow chart for animal experiments;

[0062] Fig.36 Live imaging of NCG mice after TCR-T transfusion with different structures and doses;

[0063] Fig.37 This is a comparison of the changes in total fluorescence intensity of tumors in NCG tumor-bearing mice after the infusion of different TCR-T cells;

[0064] Fig.38 This is a comparison chart of the changes in tumor volume and weight in NCG mice after TCR-T transfusion. Specific implementation method:

[0065] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0067] 1. PBMC and other cells

[0068] The 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 this laboratory.

[0069] 2. Lentivirus

[0070] The lentiviral expression vector pHR_LV-NS1 used in the following examples was purchased from Shaanxi Infill 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 instruments

[0079] The main instruments and equipment used in the following examples 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 experimental results. The experimental method part includes:

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

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

[0087] 3. Tumor target cell line recombinant vector and its gene sequence;

[0088] 4. Cell culture methods;

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

[0090] 6. Method for establishing 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 TCR;

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

[0094] 10. Method for detecting the secretion of TNF-α by MC2-TCR-T cells under the stimulation of peptide-loaded T2 cells;

[0095] 11. Method for preparing tumor target cell line;

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

[0097] 13. Sorting and purification method of MC2-A02-K562 cell line;

[0098] 14. Method for detecting the pMHC expression efficiency of tumor target cell lines;

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

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

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

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

[0103] 1. Construction and verification of recombinant expression plasmid;

[0104] 2. Expression of MC2-TCR with optimized mouse sequence HM -T has higher TCR expression efficiency and stronger pMHC affinity;

[0105] 3. Mouse-optimized MC2-TCR HM -T has a stronger ability to produce IFN-γ under the stimulation of T2 cells loaded with MC2 peptide;

[0106] 4. Expression of MC2-TCR with optimized mouse sequence HM -T secretion of TNF-α was higher when stimulated by T2 cells loaded with MC2 peptide;

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

[0108] 6. MC2-TCR under tumor target cell stimulation HM -T can produce higher IFN-γ release;

[0109] 7. MC2-TCR under tumor target cell stimulation 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 specific contents are as follows:

[0112] 1. Experimental methods:

[0113] 1. Source and design of TCR sequences targeting MAGE-C2 (HLA-A02) pMHC

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

[0115] The tumor-specific TCR sequences in this experiment were derived from HLA-A02-restricted MAGE-C2 from a patient with tumor regression. 336-344 (ALKDVEERV) antigen has a targeted CD8+ T cell clone. This example selects this sequence as the basis for constructing an engineered TCR. The gene sequence information is obtained through relevant literature and GenBank database. The name of its α chain is TCR-Vα3 (GenBank: EU427374.1), and the name of its β chain is TCR-Vβ28 (Gen Bank: EU427375.1). MAGE-C2 may be referred to as MC2 in the following text.

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

[0117] The human TCR double chain has positively charged residues in its transmembrane region, which causes the exogenous TCR double chain pairing and binding stability of the transduced cells to be poor, and it is easy to form mismatched mixed dimers between the endogenous and transduced exogenous TCR chains, which reduces the expression abundance of the introduced engineered TCR on the cell surface, thereby affecting the activity of the engineered T cells and the ability to recognize tumor cells. Replacing the conserved region C region of the human α and β chain sequences with a mouse C region can significantly enhance the expression level and stability of the TCR. This embodiment adopts this modification strategy to perform a mouse replacement modification on the conserved region C region of the original α and β chain human sequences to improve the expression of the TCR on the cell surface, the binding to the target cells and the killing function. In the process of designing the engineered TCR gene, this embodiment named the target gene containing the above-mentioned unmodified original TCR-Vα3 and TCR-Vβ28 sequences as MC2-TCR H (The specific TCR expressed is called TCR H , whose α and β chains are hereinafter referred to as TCR H -α and TCR H -β); In order to optimize the expression and function of TCR, the target gene MC2-TCR was designed to replace the constant region (C region) of TCR-Vα3 and TCR-Vβ28 with the mouse C region HM (The corresponding specific TCR is called TCR HM , whose α and β chains are hereinafter referred to as TCR HM -α and TCR HM -β). See the schematic diagram of gene structure for Figure 1 , MC2-TCR H 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 to replace 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 and gene sequence of TCR molecule targeting MAGE-C2 (MC2)

[0119] (1) Obtaining the recombinant cloning vector

[0120] The target gene fragment structure designed in this experiment is a tandem arrangement of the α and β chain gene sequences of MC2-TCR, which are connected by a self-cleaving P2A sequence. The target gene fragment is about 1800 bp long, with restriction sites NotI and SalI at both ends. 336-344 (ALKDVEERV) / HLA-A*02:01 TCR, entrusted Sangon Biotech (Shanghai) Co., Ltd. to carry out chemical synthesis and sequencing of the target gene cloning vector. The cloning vector pUC-SP is modified from pUC57, and the commonly used sticky end sites in the pUC57 plasmid multi-cloning restriction site are eliminated by synonymous mutation, leaving only the commonly used blunt end sites, so that the target gene has only one restriction site at both ends, and the target gene fragment can be obtained by double restriction digestion. MC2-TCR with target gene H The recombinant cloning vector was named pUC-SP-MC2-TCR H ; With target gene MC2-TCR HM The recombinant cloning vector (mouse optimized) was named pUC-SP-MC2-TCR HM The two vectors have the same structure, with pUC-SP-MC 2-TCR HM For example, the structure is Figure 2 As shown in A. Target gene MC2-TCR H The gene sequence is shown in SEQ ID NO: 01; the target gene 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 transfecting cells is pHR_LV-NS1, which is modified from pLVX-Puro, and the CMV promoter of pLVX-Puro is replaced with SFFV promoter. Insert the target gene fragment MC2-TCR 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. HM For example, the structure is Figure 2 As shown in B.

[0123] Figure 2 The target gene inserted into the vector is MAGE-C2 336-344The mouse optimized TCRα and β chain gene sequences of (ALKDVEERV) / HLA-A*02:01 are connected by the self-cleaving peptide P2A sequence. The restriction sites used are NotI and SalI. Figure 2 A is the recombinant cloning vector pUC-SP-MC2-TCR HM . Figure 2 B is the recombinant expression vector pHR_LV-NS1-MC2-TCR HM , which is expressed as TCR after transfection into cells HM .

[0124] 3. Tumor target cell line recombinant vector and its gene sequence

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

[0126] (1) Recombinant cloning vector

[0127] A02-K562 cells only express HLA-A*02:01. The target gene recombinant cloning vector used is a stored plasmid in our laboratory. pcDNA3.1(+) is used as the vector. The target gene is inserted between the restriction sites NotI and SalI. This target gene fragment is called A02. Its gene sequence is shown in SEQ ID NO:03. It consists of HLA-A*02:01 (GenBank: AJ57556 5.1) gene, CD34 (GenBank: AF523361.1) gene and puromycin pLeo1209 (GenBank: MN811119.1) gene arranged in series, and each gene is connected by a P2A sequence. The 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 original plasmid in the laboratory, and its target gene CD34 has no use in this experiment and is irrelevant to the experiment.

[0128] MC2-A02-K562 cells need to express MAGE-C2 336–344(ALKDVEERV) / HLA-A*02:01, based on the 336–344 position (ALKDVEERV) fragment of the amino acid sequence of the MAGE-C2 [Homo sapiens] (AAF07211.1) protein and the Homo sapiens MAGE-C2 (MAGEC2) gene (AF196483.1) CDS, the gene sequence of the MC2 polypeptide fragment was determined. The recombinant cloning vector used pUC-57-simple as the vector, and the target gene fragment was called MC2-A02. Its gene sequence is shown in SEQ ID NO:04, which is composed of HLA-A*02:01 (GenBank: AJ575565.1) and MAGE-C2 336–344 The two are 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) was commissioned to Beijing Qingke Biotechnology Co., Ltd. for synthesis.

[0129] (2) Recombinant lentiviral expression vector

[0130] The lentiviral expression vector used for transfecting cells is pHR_LV-NS1. The one with target gene A02 inserted is called pHR_LV-NS1-A02, and the one with target gene MC2-A02 inserted is called pHR_LV-NS1-MC2-A02. The two have the same structure. The structure of the recombinant expression vector 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 series and connected by P2A sequences, and the restriction sites used are NotI and SalI. Figure 3 A is the recombinant cloning vector pcDNA3.1(+)-A02: it contains the target gene fragment A02, which is composed of HLA-A*02:01, puromycin pLeo1209 and CD34 gene sequence. The recombinant cloning vector pcDNA3.1(+)-A02 is an original plasmid in the laboratory. The CD34 in its target gene is 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: the target gene fragment A02 is inserted. Figure 3 C is the recombinant cloning vector pUC-57-MC2-A02: containing the target gene fragment MC2-A02, which consists of HLA-A*02:01 and MAGE-C2 336–344 of coding gene sequences. Figure 3 D is the recombinant expression vector pHR_LV-NS1-MC2-A02: insert the target gene fragment MC2-A02.

[0132] 4. Cell culture

[0133] The cells involved in the experiments in this example are all suspended cells, and the culture conditions are CO 2 5% concentration and a 37°C constant temperature cell culture incubator. The cell culture in this embodiment is a conventional culture method, including: preparing the culture medium of the cells involved in the experiment in advance, cell recovery, cell passaging, cell freezing, cell counting, cell mycoplasma detection and other operations.

[0134] 5. Preparation of Lentivirus Recombinant Plasmid

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

[0136] Before the experiment begins, prepare all reagents. Buffers and other reagents need to be taken out from -20℃ and rewarmed to room temperature to melt, vortexed and mixed, and then centrifuged slightly before use. Enzyme reagents need to be taken out from -20℃ and centrifuged (less than 4000rpm) and placed in an ice bath for later use. Mark the PCR tubes and add the corresponding ingredients in Table 3 and Table 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 them to concentrate the solution at the bottom of the tube, place them in a 37°C incubator for enzyme digestion for 3 hours. After the enzyme digestion is completed, keep them at 65°C for 5-10 minutes to terminate the enzyme digestion and prevent DNA end annealing, then place them on ice to quickly cool down for use.

[0143] (2) Agarose gel electrophoresis purification

[0144] Make 1% agarose gel, cool to 60℃, pour into the installed horizontal electrophoresis tank plate, let it stand and wait for solidification, then slowly add electrophoresis buffer (1×TAE) until it covers the horizontal plate, and pull out the comb vertically. When adding samples, electrophoresis the lentiviral vectors required for subsequent transfection of cells together. The loading order is lentiviral vector, target gene plasmid enzyme cleavage product, marker, and use a micropipette to add the samples to the sample slots of the gel plate. After adding each sample, replace a sample head to prevent contamination. Samples should be added gently to avoid damaging the gel surface around the sample hole. After adding the sample, plug in the power supply immediately, adjust the voltage to start the electrophoresis program (100V, about 30min), and the sample begins to move from the negative pole (black) to the positive pole (red). When bromophenol blue moves to about 1cm from the bottom edge of the gel plate, stop electrophoresis. Take out the gel plate, stain it in ethidium bromide staining solution for 30min, put it in the multifunctional gel imager under the UV light to start cutting the gel, MC2-TCR H and MC2-TCR HM Cut the band at 1.8k; cut the band at 2.8kb for pcDNA3.1(+)-A02; cut the band at 1.4kb for pUC-57-MC2-A02; cut the band at 8.9k for lentivirus pHR_LV-NS1. After gel cutting, recover the corresponding target fragment according to the steps of gel recovery kit.

[0145] (3) DNA purification and recovery

[0146] The gel recovery kit was restored to room temperature in advance to ensure that all relevant reagents were clear and free of precipitation. The adsorption column was placed in a collection tube, 500 μL of equilibration solution was added, and the mixture was centrifuged at 12,000 rpm for 1 min. The waste liquid was discarded and the adsorption column was reassembled for use; the target DNA band was weighed, and PN solution was added to each gel band at a ratio of 0.1 g and 100 μL was added, and the gel block was completely dissolved in a 50°C water bath. The above solution was restored to room temperature, and added to the equilibrated adsorption column with a pipette and marked to prevent confusion. It was allowed to stand at room temperature for 2 min, and centrifuged at 12,000 rpm for 1 min, and the waste liquid was discarded. 600 μL of rinsing solution was added, and the waste liquid was discarded after centrifugation at 12,000 rpm for 1 min. After repeating the rinse, the waste liquid was discarded after centrifugation at 12,000 rpm for 2 min, and the adsorption column was removed and allowed to stand at room temperature and dried thoroughly. The adsorption column was placed in a sterile enzyme-free centrifuge tube, and 50 μL of elution solution was dripped onto the adsorption membrane, and the mixture was allowed to stand at room temperature for 2 min. The target fragment solution was collected by centrifugation at 12,000 rpm for 2 min. The concentration and purity of the recovered solution were checked 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. The band position is observed under ultraviolet light to see if it is correct.

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

[0150] Connect the purified lentiviral vector pHR_LV-NS1 (vector) and the target gene fragment (insert). Prepare the corresponding reagents and add them to the labeled PCR tubes according to the system in Table 5 for ligation reaction at 25°C for 1 hour. Insert MC2-TCR H and MC2-TCR HM The lentiviral plasmids of the target gene fragments were named pHR_LV-NS1-MC2-TCR H and pHR_LV-NS1-MC2-TCR HM The target cell lentiviral expression vectors after connection 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 out the purchased E. coli DH5α from the -80℃ refrigerator and put it on ice to melt, inoculate 20μl of E. coli DH5α glycerol into 2ml of pre-prepared LB liquid medium, and shake culture at 37℃ overnight. The next day, centrifuge the above bacterial solution at 4℃, 5000rpm for 2min to recover the bacteria; carefully remove the supernatant with a pipette in the clean bench, and then resuspend the bacteria in 400μl pre-cooled 0.1M CaCl 2 The solution was gently pipetted evenly, and then an appropriate amount of sterile glycerol was added. After mixing, it was placed in an ice bath for 20 minutes. A portion of the prepared competent state was stored at -80°C, and an appropriate amount was kept on ice for later use.

[0155] ②Transformation and culture: Take 100 μL of competent cells (DH5α) and add them to the marked centrifuge tube, flick to mix, and place on ice for 30 minutes to rewarm; add 10 μL of the ligation product obtained in the previous step, place on ice for 30 minutes, heat shock at 42°C for 1 minute, and then place on ice for 2 minutes; add 400 μL of LB liquid culture medium without antibiotics to the tube, and then put it in a shaker, set it to oscillate at 32°C at a speed of 250 rpm for 1 hour; take 100 μL of bacterial solution and add it to Amp solid culture medium, spread it with a sterile coating rod and culture it, culture it at 32°C overnight, observe the grown single colonies the next day, pick 8 single colonies for each sample, and inoculate each single colony into a tube containing 1 mL of LB-Amp liquid culture medium. After vortexing and mixing, put it in a shaker, set it to oscillate at 32°C at a speed of 250 rpm for 2 hours.

[0156] ③PCR amplification, based on partial sequence information of pHR_LV-NS1 lentiviral vector and MC2-TCR H 、MC2-TCR HM Sequence information, in pHR_LV-NS1-MC2-TCR H A fragment of about 683 bp was selected and cloned into pHR_LV-NS1-MC2-TCR HM A fragment of about 526 bp was selected and the primers were designed as follows: pHR_LV-NS1-MC2-TCR H -F gene sequence is shown in SEQ ID NO: 05; pHR_LV-NS1-MC2-TCR H -R gene sequence is shown in SEQ ID NO:06; pHR_LV-NS1-MC2-TCR HM -F gene sequence 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] ④Using the partial sequence of pHR_LV-NS1 lentiviral vector to the partial sequence of the target gene fragment A02 and MC2-A02 as templates, a gene fragment of about 1327bp was selected on pHR_LV-NS1-A02, and a fragment of about 1300bp was selected on pHR_LV-NS1-MC2-A02. The primers were designed as follows:

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

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

[0160] Table 6: PCR reaction program

[0161]

[0162] After shaking and mixing, place in PCR instrument and carry out the reaction program in Table 7.

[0163] Table 7: PCR reaction system

[0164]

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

[0166] ⑦ Lentiviral plasmid amplification: Select the corresponding bacterial solution cloned correctly at the band position in step ⑥, take 1 mL and inoculate it into a T75 culture bottle containing 100 mL LB-Amp liquid culture medium, and incubate it at 250 rpm at 32°C overnight. The total shaking time should be controlled within 15 hours.

[0167] (7) Extraction of plasmid

[0168] Prepare the kit, add the balance solution to the adsorption column, centrifuge and discard the waste liquid for later use; take 100mL of overnight cultured bacterial solution and add it to a centrifuge tube, centrifuge at 8000rpm for 3min at room temperature, remove the supernatant as much as possible and reduce the liquid residue; add 8mL of solution P1 to the precipitate in the centrifuge tube and shake it thoroughly; add 8mL of solution P2, gently turn the centrifuge tube upside down several times to promote the full lysis of the bacteria, and let it stand at room temperature for 5min; add 8mL of solution P4 and then immediately turn the centrifuge tube upside down several times to mix thoroughly until a white precipitate appears, and let it stand at room temperature for 10min; centrifuge at 8000rpm for 10min to concentrate the white precipitate at the bottom of the tube; pour all the supernatant solution into the CS1 filter and slowly push the filter handle to collect the filtrate in a clean centrifuge tube; observe the filtrate volume scale, add 0.3 times the volume of isopropanol to the filtrate, mix it upside down, and transfer it to the adsorption column CP6 twice according to the volume, centrifuge it at 8000rpm for 2min and discard the waste liquid; add 10mL of rinse liquid PW, centrifuge it at 8000rpm for 2min and discard the waste liquid, repeat once; add 3mL of anhydrous ethanol to the adsorption column, centrifuge it at 8000rpm for 2min and discard the waste liquid, centrifuge it at 8000rpm for 5min again to completely remove the anhydrous ethanol; put the adsorption column into a clean centrifuge tube, add 1mL of elution liquid TB to the center of the adsorption membrane in mid-air, let it stand at room temperature for 5min, then centrifuge it at 8000rpm for 2min to obtain a plasmid solution, transfer all the solution to a clean EP tube, which can be stored at -20℃.

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

[0170] Table 8: Recombinant vector restriction enzyme digestion reaction system

[0171]

[0172] (9) The recombinant expression vectors are summarized in the table below.

[0173] Table 9: Recombinant plasmids used for transfection of cells

[0174]

[0175] 6. Establishment of 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 the cells and count them. 6Inoculate cells / well into a 6-well plate and culture them normally for about 6 hours until the cells are completely attached to the wall. Then replace the medium without dual antibodies and continue culturing until the next day for use;

[0178] ②Take a 1.5mL EP tube and mark it. Place 3.3μg of the main plasmid (pHR_LV-NS1-MC2-TCR 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 by pipetting and allowed to stand;

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

[0180] ④ Transfer the plasmid mixture in the EP tube to the corresponding 15mL centrifuge tube, mix well by pipetting, vortex and shake, and then place 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, remove the medium, slowly add the above mixture along the wall of the well plate, put it back into the CO2 incubator, and observe it from time to time;

[0182] ⑥After 6 hours, change the medium, discard the original solution, and rinse once with 2 mL of DMEM high-glucose medium without serum added along the wall. Be gentle to avoid blowing up the cells, then add 2 mL of DMEM high-glucose complete medium along the wall, CO 2 Continue culturing in the incubator for 48 h.

[0183] ⑦ Aspirate the supernatant in the above 6-well plate into the corresponding labeled 15mL centrifuge tube, centrifuge at 3000rpm for 5min at 25℃ to remove cell residues, then filter with a 0.45μm filter membrane, and divide the collected viruses into portions for use or freeze at -80℃ for storage.

[0184] (2) T cell activation

[0185] PBMC cells isolated and frozen from whole blood need to be activated before transfection, that is, T Cell TansAct and human interleukin 2 (IL-2) are added to RPMI-1640 complete medium, the ratio is 10μL T Cell TansAct and 6μL IL-2 (IL-2 storage solution concentration 100U / μL, final concentration 600U / mL) in 1mL complete medium. Resuscitate PBMC, remove the freezing solution by centrifugation, and count about 2×10 6Each PBMC was resuspended in 2 mL of complete medium containing TansAct and IL-2, and transferred into a 24-well plate and cultured for 48 h to activate T cells.

[0186] (3) Transfection of target cells

[0187] The day before transduction, a 24-well plate was coated with RetroNectin and placed at 4°C overnight. The next day, the 24-well plate coated with RetroNectin was recovered, 2 mL of packaged virus was added to each well, and target cells (PBMCs needed to be activated before transduction) were added dropwise. PBMCs were 0.5×10 6 Each group was assigned a negative control well, which contained the target cells supplemented with the corresponding culture medium. After mixing, the 24-well plate was sealed with a film and centrifuged at 2000 rpm for 90 min at room temperature. The medium speed was set to increase the speed and the slow speed was set to decrease the speed to allow the cells to settle and increase the virus transfection efficiency. After the centrifugation, the film was removed and placed in a CO 2 Culture in an incubator, carefully remove 1 mL of supernatant after 24 hours, add 1 mL of the corresponding complete culture medium, observe the cell status in real time, expand the culture according to the situation, use part of it for subsequent experiments, and store the remaining cells in a cryopreservation procedure. The cells obtained after PBMC cell transduction are MC2-specific TCR-T cells, and the cells in the negative control group are named Vector, which express the original human TCR H The TCR-T cells with the sequence were named MC2-TCR H -T, expressing C region mouse 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 H -T, MC2-TCR HM -T) should express MAGE-C2 targeting HLA-A*02:01 restriction 336–344(ALKDVEERV)-specific TCR. After obtaining the target cells, the expression of MC2-TCR must be tested, that is, the ability of TCR-T cells to bind to the corresponding pMHC must be tested. In this step, the corresponding TCR will be labeled with MHC tetramers (MHC Tetramer), and 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 connected to streptavidin through the lysine residue ends. Streptavidin carries a fluorescent marker. After Tetramer is co-incubated with specific T cells, Tetramer can specifically bind to multiple corresponding TCRs, such as Figure 4 As shown, the recombinant proteins MHC class I heavy chain and β2-microglobulin (β2m) and antigen polypeptide fragments are folded to form a soluble MHC class I / polypeptide complex monomer. The lysine residue on the C-terminus of the MHC class I heavy chain in the complex monomer is biotinylated using the BirA enzyme. The biotinylated complex monomer is purified by column chromatography. The purified biotinylated monomer is combined with streptavidin labeled with a fluorescent dye to form a tetramer Tetramer, thereby completing the preparation of T-Select MHC Tetramer. When the Tetramer is incubated with T cells, it will stably bind to multiple TCR specificities, and the proportion or number of the labeled T cell population will be quantitatively detected by flow cytometry. This combination has higher stability, and the proportion or number of specific T cells is subsequently quantitatively detected by flow cytometry.

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

[0191] pHR_LV-NS1-MC2-TCR H and pHR_LV-NS1-MC2-TCR HM Two types of TCR-T cells were obtained after transduction of PBMC cells: MC2-TCR H -T and MC2-TCR HM-T, PBMCs transduced with pHR_LV-NS1 empty vector were used as control group Vector. On the 5th day 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 (ALKDVEERV) antigen’s percentage of CD8+T cell population.

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

[0193] (1) Peptide loading of T2 cells

[0194] T2 cells are tap-deficient human lymphoblastoid cell lines that express HLA-A02 but lack antigen peptide transporters and are unable to present endogenous HLA. MC2 Cells can faithfully present the target antigen peptide prototype on the cell surface. The process of stimulating T2 with exogenous target antigen peptide is called peptide loading.

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

[0195] Prepare a round-bottom 96-well plate, take the previously cultured T2 cells, centrifuge at 25°C, 100 rpm for 5 min, resuspend in complete medium and adjust the cell concentration to 0.4×10 per well. 6 100 μL of T2 cell suspension was added to each well with 2 μL of control peptide or synthetic MC2 peptide (peptide storage solution concentration 1 mM) and placed in CO 2 The cells were incubated in the incubator for 2 h and peptide loading was performed on 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 HMOn the 9th day after PBMC transduction, the intracellular gamma interferon staining (ICS) was used to detect the proportion of cells that secreted IFN-γ intracellularly after TCR-T cells were stimulated by peptide-loaded T2 cells. The intracellular factor staining method blocks the secretion of cytokines to the extracellular space, and the produced cytokines accumulate in the cells. After the membrane is broken, the antibodies bind to specific intracellular factors, making the cytokine fluorescence signal stronger and more accurate. Combined with cell surface staining, it can analyze the proportion of cells that can release cytokines in a certain T cell population.

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

[0199] (1) TCR-T cells and peptide-loaded T2 cells were counted at 0.4×10 6 / well, the effector cells and target cells (E:T) were fully mixed at a ratio of 1:1 and returned to the incubator for co-culture for 24 hours;

[0200] (2) Before use, restore the Elisa kit to room temperature, prepare the required reagents and the gradient dilution of the standard, set up duplicate wells for the standard and sample, use 6,000pg / ml standard solution for 2-fold gradient dilution, set up 12 gradients, and use 1X sample diluent B as the zero standard (0pg / ml). According to the experiment, disassemble the corresponding 96-well plate kit, mark it, remove the remaining strips, put them back into the aluminum foil bag and seal it for next use.

[0201] (3) Add 200 μL of 1X washing solution Item B to the experimental plate and rinse for 30 seconds. After discarding the liquid, turn it upside down on filter paper and pat it dry. The experimental process should be as continuous and fast as possible to prevent the plate from drying out.

[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 replicate wells, and incubate with gentle shaking at room temperature for 2.5 h.

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

[0204] (6) Gently mix Item F (detection antibody), add 100 μl 1X Item E to make 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 make 1X Item F, add 100 μL 1X Item F to each well, and incubate at room temperature on a shaker at low speed for 1 hour.

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

[0206] (8) Gently rotate Item G (HRP-streptavidin concentrate bottle) to mix, take an appropriate amount of Item G, use 1X Item E to dilute Item G 600 times and mix well for later use, add 100 μL of diluted Item G to each detection well, and incubate at room temperature with gentle shaking for 45 minutes.

[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 with gentle shaking at room temperature for 30 min in the dark.

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

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

[0211] 11. Preparation of tumor target cell lines

[0212] The steps of lentiviral packaging and transfection were the same as described above. 2 mL of packaged virus was added to each well of the 24-well plate coated with RetroNectin, and 0.4×10 K562 cells were added to the corresponding well. 6After 24 hours, 1 mL of supernatant was removed and 1 mL of the corresponding complete medium was added to observe the cell status in real time. After expanding the culture according to the situation, some of the cells were used for subsequent experiments, and the remaining cells were stored by freezing procedures. The cells in the negative control group 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 A02-K562 cell line

[0214] Prepare A02-K562 cells expressing the puromycin resistance gene pLeo1209, 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 the transfected A02-K562 cell suspension, add 20 μL of the 1 mg / mL puromycin solution obtained in the previous step, mix well, and place in an incubator for continued culture.

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

[0218] (4) After culturing for 48 hours, the A02-K562 cells containing the resistance gene still 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 MC2-A02-K562 cell line

[0220] (1) Take 1.0×10 7 Add 6 mL of sorting buffer to each cell in a 15 mL centrifuge tube, mix thoroughly, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

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

[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, and shake and mix every 5 min during the incubation period.

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

[0225] (6) Discard the supernatant of the centrifuged cells and use 3 mL of sorting buffer to make a single cell suspension, then add it to the rinsed LS sorting column. When the cell suspension has flowed out, add 5 mL of sorting buffer to rinse, and repeat twice;

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

[0227] (8) After centrifugation, resuspend the cell pellet in 10 mL of RPMI-1640 medium and aspirate all of it and place it in 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 the HLA-A02 expression was detected by flow cytometry. The control group Vector was K562 cells transfected with an empty vector.

[0230] (1) Pipette 500 μL of the corresponding cell suspension from the culture flask into EP tubes and mark 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. Prepare HLA-A2-PE antibody during centrifugation, according to the ratio of HLA-A2-PE: FACS Buffer = 1:9. Each reaction requires 50 μL of the above solution to prepare a sufficient amount of antibody solution.

[0231] (2) After centrifugation, discard the supernatant, add 50 μL of the above-prepared HLA-A2-PE mixture to each reaction, mix well 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 analysis. Use HLA-A2-PE as the horizontal axis and analyze the stained cells with a single parameter.

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

[0234] After target cells were transduced and purified, they were cultured to an appropriate number and two TCR-T cells (MC2-TCR H -T and MC2-TCR HM The intracellular release of IFN-γ after incubation with A02-K562 and MC2-A02-K562 cells. K562 cells transfected with empty vector (Vector) were used as the control group, and the effector-target ratio of TCR-T cells to target cells was 1:1. 0.4×10 6 100 μL of the cells were inoculated into a round-bottom 96-well plate, gently mixed, and 3 μL of blocking solution was added to each well and incubated in a CO2 incubator for 24 hours to allow the two cells to interact with each other. The remaining steps were the same as in 2.2.2.9 of this chapter. The flow cytometry results were analyzed with CD8-APC as the horizontal axis and IFN-γ-FITC as the vertical 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, fully mixed in a 1:1 ratio, and returned to the incubator for incubation for 24 hours; before use in the experiment, the Elisa kit was restored to room temperature, and the required reagents and gradient dilutions of the standard 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 of tumor target cells

[0238] CFSE dye was used to label the target cells used in the reaction to distinguish target cells from 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) Target cell labeling with CFSE: Prepare CFSE stock solution in advance according to the instructions. Resuspend A02-K562 cells and MC2-A02-K562 cells, count and adjust the cell density. The cell density of each cell is 1×10 6 1 mL of target cells per mL was inoculated into a labeled centrifuge tube; 0.4 μL of 2.5 mM CFSE stock solution was added to each of the above target cell centrifuge tubes, at which time the final concentration of CFSE was 1 μM, and the mixture was mixed by pipetting, and incubated in a 37°C incubator in the dark for 20 min, and vortexed and mixed every 5 min; after the incubation, 5 times the volume of RPMI 1640 culture medium containing 10% FBS was added to each tube, and the tube was incubated on ice for 5 min to terminate the staining, and the staining termination step was repeated twice to ensure that the CFSE dye bound to the protein in the supernatant was removed; after the last termination step, the supernatant was removed by centrifugation, and then resuspended with 1 mL of RPMI 1640 complete culture medium containing 10% FBS to prepare the target cells labeled with CFSE: A02-K562-CFSE and MC2-A02-K562-CFSE.

[0240] (2) Take 100 μL of the above A02-K562-CFSE or MC2-A02-K562-CFSE cells and inoculate them into a marked round-bottom 96-well plate, which is equivalent to 0.1×10 target cells per well. 6 indivual.

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

[0242] (4) Calculation process of the killing efficiency formula:

[0243] At the beginning of the experiment, the number of effector cells and target cells in each group is equal, so the following equation can be obtained:

[0244]

[0245] Then, assuming that there is no killing effect, x = (a × b) ÷ c after 14 hours of cell interaction, and if MC2-A02-K562 cells interact with MC2-TCR HM -If T cells have a specific killing effect, the number of target cells MC2-A02-K562 lost by killing should be xd (d is the number of surviving cells of MC2-A02-K562 after the effect), and the killing rate calculation formula is:

[0246]

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

[0248] Statistical methods

[0249] Statistical analysis was performed using Prism version 8.0 (Graph Pad). For data comparisons 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 significant statistical difference; ***p<0.001 indicates an extremely significant statistical difference.

[0250] 2. Experimental results:

[0251] 1. Construction and verification of recombinant expression plasmid

[0252] The two target gene sequences for expressing engineered TCR are MC2-TCR H and MC2-TCR HM , MC2-TCR H Contains unmodified TCR-Vα3 and TCR-Vβ28 sequences, MC2-TCR HMContains mouse optimized TCR-Vα3 and TCR-Vβ28 sequences. Both target the 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 C region mouse source replacement sequence MC2-TCR HM The recombinant cloning vector is called pUC-SP-MC2-TCR HM The two recombinant cloning vectors have the same structure, and the plasmid map is 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 , including the α chain sequence (TCRα3) and β sequence (TCRβ28) with the mouse gene sequence replaced in the C region. The α and β chain sequences are connected by the self-cleaving polypeptide P2A sequence. The red boxes indicate the restriction sites NotI and SalI used in this experiment.

[0253] Recombinant cloning vector pUC-SP-MC2-TCR H , pUC-SP-MC2-TCR HM Double digestion was performed with NotI and SalI restriction sites of lentiviral expression vector pHR_LV-NS1 to obtain the target gene fragment MC2-TCR H 、MC2-TCR HM As well as the linear restriction product fragment of the lentiviral expression vector pHR_LV-NS1, the target gene fragment MC2-TCR H The total length is 1806 bp, MC2-TCR HM The total length is 1821 bp, and the total length of the pHR_LV-NS1 linear fragment is 8900 bp. After agarose gel electrophoresis of the digested product, two bands at 2.0 Kb and one at 8.0 Kb were obtained, as expected. Figure 5 As shown in A, lane M is DNAMarker 1Kb; lane 1 is pUC-SP-MC2-TCR H Double enzyme digestion products; Lane 2 is pUC-SP-MC2-TCR HM Lane 3 is the double-enzyme digestion product of pHR_LV-NS1.

[0254] After the target band was cut, gel recovery and purification were performed, and the purified product was subjected to agarose gel electrophoresis again to observe the band position. The pHR_LV-NS1 linear enzyme fragment was about 8.9Kb near 8.0Kb, and the target gene fragment MC2-TCR H and MC2-TCR HM At around 2.0Kb, the confirmation bands are all in line with expectations, such as Figure 5 As shown in B, lane M is DNA Marker 1Kb; lane 1 is pHR_LV-NS1 double restriction enzyme digested linear fragment (about 8.9Kb); lane 2 is the target gene fragment pUC-SP-MC2-TCR H Double enzyme digestion product (about 1.8Kb); Lane 3 is the target gene fragment pUC-SP-MC2-TCR HM Double enzyme digestion product (about 1.8Kb).

[0255] The above recovered and purified target gene MC2-TCR H and MC2-TCR HM The recombinant lentiviral expression vector pHR_LV-NS1-MC2-TCR was obtained by ligating the linear fragments of the lentiviral vector pHR_LV-NS1. H and pHR_LV-NS1-MC2-TCR HM After the above vectors were transformed and the bacteria were propagated, PCR verification was performed on the bacterial solution: a gene sequence containing the junction of the expression vector and the inserted target gene on the recombinant lentiviral expression vector was selected as a template, and the pHR_LV-NS1-MC2-TCR H The length is 526 bp, in pHR_LV-NS1-MC2-TCR HM The length of the medium is 683 bp. Primers were designed according to the above sequence and PCR amplification was performed. After agarose gel electrophoresis, each group of 8 amplified products obtained expected bands such as Figure 6 A and Figure 6 B, Figure 6 Lane M in A is DNA Marker D2000, and lanes 1 to 8 are the PCR amplification products of positive colonies, about 526 bp; Figure 6 In B, lane M is DNA Marker D2000, and lanes 1 to 8 are pHR_LV-NS1-MC2-TCR HM The PCR amplification product of the 8 single positive colonies picked was about 683bp. The two recombinant lentiviral expression vector plasmids were amplified and shaken separately. After the plasmids were extracted, the extracted plasmids were double-digested for identification, and the expected bands were obtained by running the gel, such as Figure 6As shown in C, lane M1 is DNA Marker 1Kb, lane M2 is DNA Marker 500Kb, and lane 1 is pHR_LV-NS1-MC2-TCR H Double enzyme digestion products, lane 2 is the recombinant plasmid pHR_LV-NS1-MC2-TCR HM Double enzyme digestion product. Target gene fragment MC2-TCR H and MC2-TCR HM Around 1.8Kb, the expression vector fragment is around 8.9Kb, and the recombinant lentiviral expression vector pHR_LV-NS1-MC2-TCR H and pHR_LV-NS1-MC2-TCR HM The construction was successful and can be used for subsequent lentiviral packaging and transfection of cells. After aliquoting, store at -80°C. The plasmid map is 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 SFFV promoter. HM Inserted between the NotI and SalI restriction sites of pHR_LV-NS1, with a total length of 10721bp. The lentiviral expression vector used in this experiment is pHR_LV-NS1, which is transformed from the pLVX-Puro vector. The core design is to modify the CMV promoter in the original pLVX-Puro to the SFFV promoter.

[0256] 2. Expression of MC2-TCR with optimized mouse sequence HM -T has higher TCR expression efficiency and stronger pMHC affinity

[0257] The recombinant expression vector pHR_LV-NS1-MC2-TCR tested H and pHR_LV-NS1-MC2-TCR HM , co-transfected with packaging plasmids psPAX2 and pMD2.G into HEK-293T cells, incubated in the incubator for about 54 hours, and then centrifuged to collect lentiviral particles. The collected viral particles were used to transfect PBMC cells activated by TansAct and IL-2 to obtain MAGE-C2-specific TCR-T cells (abbreviated as MC2-TCR-T). Transfection of pHR_LV-NS1-MC2-TCR H The cells that produced the virus particles were named MC2-TCR H -T, this strain of TCR-T cells expresses the original human TCR targeting MAGE-C2 H ; Transfection with pHR_LV-NS1-MC2-TCRHM The cells that produced the virus particles were named MC2-TCR HM -T, this strain of TCR-T cells expresses a TCR targeting MAGE-C2 with a mouse-derived sequence replaced in the C region HM， like Figure 7 A, TCR in the figure H Original human TCR targeting MC2; TCR HM To target MC 2 The C region of the mouse-derived human-mouse hybrid TCR is murinized; Cαm is the mouse-derived α chain C region; Cβm is the mouse-derived β chain C region; the α and β chain sequences of the target gene are connected by the self-cleaving polypeptide P2A sequence. During translation and expression, the self-cleaving polypeptide P2A breaks, and the α and β chains are expressed and assembled independently. The control cells transfected with the empty vector are Vector. In order to clarify the CD8+T cell population transfected with TCR H or TCR HM efficiency, Vector, MC2-TCR H -T and MC2-TCR HM -T cells were incubated with MAGE-C2-Tetramer-PE and CD8-APC antibodies, and the stained cells were detected by flow cytometry. The special structure of the tetramer can bind to multiple specific TCRs on the surface of T cells with high affinity and high stability. The proportion of MAGE-C2-Tetramer-PE / CD8-APC double-stained cells was used to evaluate TCR H or TCR HM The expression efficiency can also be verified, and the affinity of MC2-TCR-T targeting binding antigen epitope can be verified.

[0258] Flow cytometry results Figure 7 As shown in C, the horizontal axis is MAGE-C2-Tetramer-PE, and the vertical axis is CD8-APC; in the figure, Vector is PBMC transfected with empty vector pHR_LV-NS1; MC2-TCR H -T is PBMC transfected with pHR_LV-NS1-MC2-TCR H ;MC2-TCR HM -T is PBMC transfected with pHR_LV-NS1-MC2-TCR HM Compared with Vector, MC2-TCR H -T and MC2-TCR HM -T, transduction of the recombinant expression vector produced a CD8+T cell population that could bind to MAGE-C2-Tetramer-PE, and the proportion of MAGE-C2-Tetramer-PE and CD8-APC double-stained cell populations in MC2-TCRH -8.01% in T, MC2-TCR HM -T was 23.47%. The results of three independent transfection and flow cytometry experiments were as follows: Figure 7 B shows MC2-TCR HM The proportion of MAGE-C2-Tetramer-PE and CD8-APC double-stained cell populations in MC2-TCR H -T.

[0259] The results showed that MAGE-C2-specific TCR-T cells MC2-TCR-T were successfully constructed, and the recombinant lentiviral expression vector pHR_LV-NS1-MC2-TCR was used. H and pHR_LV-NS1-MC2-TCR HM Both can effectively transfect PBMC to express MC2-specific TCR molecules; replacement of mouse sequences in the C region of the α and β chains can make the expression efficiency of exogenous TCR higher, and MC2-TCR HM -T has higher TCR expression efficiency and stronger affinity for pMHC.

[0260] 54 h after normal donor PBMCs were transduced with the recombinant expression vector, they were incubated with various antibodies, and antibody staining was detected by flow cytometry. Figure 7 A is TCR H and TCR HM Structural diagram: TCR H Original human TCR targeting MC2; TCR HM To target MC 2 The C region of the mouse-derived human-mouse hybrid TCR is murinized. Cαm is the mouse-derived α chain C region; Cβm is the mouse-derived β chain C region. The α and β chain sequences of the target gene are connected by a self-cleaving polypeptide P2A sequence. During translation and expression, the self-cleaving polypeptide P2A breaks, and the α and β chains are expressed and assembled independently. Figure 7 B is the TCR expression efficiency detected by three independent repeated experiments (i.e., the proportion of MAGE-C2-Tetramer-PE and CD8-APC double-stained cell population detected by flow cytometry). Figure 7 C is the flow cytometry result of MAGE-C2-Tetramer-PE and CD8-APC staining. Vector is PBMC transfected with empty vector pHR_LV-NS1; MC2-TCR H -T is PBMC transfected with pHR_LV-NS1-MC2-TCR H ;MC2-TCR HM -T is PBMC transfected with pHR_LV-NS1-MC2-TCR HM .

[0261] 3. Mouse-optimized MC2-TCR HM -T2 cells loaded with MC2 peptide have stronger ability to produce IFN-γ

[0262] In order to detect the function of the constructed TCR-T cells in producing and expressing IFN-γ under the stimulation of specific antigen peptides, MC2-TCR H -T and MC2-TCR HM -T and T2 C Cells (T2 cells loaded with control peptide) and T2 MC2 After the cells (T2 cells loaded with MEGE-C2 peptide) were co-incubated for 24 hours, intracellular gamma interferon staining (ICS) of IFN-γ-FITC was performed first, and then CD8-APC was incubated. Using the vector transfected with an empty vector 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. The results are shown in Figure 2. Figure 8 A shows that Vector is T2 C or T2 MC2 After cell stimulation, the proportions of IFN-γ-FITC / CD8-APC double-stained cell populations were 0.83% and 0.72%, respectively; MC2-TCR expressing the original α and β chain sequences H -T and MC2-TCR expressing murinized C region sequence HM -T, in with T2 C After 24 hours of co-incubation, there was almost no expression of intracellular IFN-γ, and the IFN-γ signal was low. H -0.44% in T, MC2-TCR HM -T is 0.66%; in comparison with T2 MC2 After 24 hours of co-incubation, IFN-γ-FITC and CD8-APC double-stained cell populations were detected in both TCR-T cells, and MC2-TCR H -9.47% in T, MC2-TCR HM -T is 21.3%. The statistical results show that Figure 8 B, Vector group and T2 C Cells and T2 MC2 After the cell response, there was no significant difference in the percentage of cells double-stained with IFN-γ-FITC and CD8-APC; MC2-TCR H -T and MC2-TCR HM -T passes through T2 MC2After stimulation of cells, the proportion of double-stained cells was significantly higher than that of T2 C Cellular response group; MC2-TCR HM -T is affected by T2 MC2 After cell stimulation, the proportion of cells with IFN-γ-FITC and CD8-APC double staining was significantly higher than that of MC2-TCR H -T group.

[0263] Figure 8 In, Vector, MC2-TCR H -T and MC2-TCR HM -T and negative control T2 C Cells (T2 cells loaded with control peptide) and experimental group T2 MC2 After co-culture of cells (T2 cells loaded with MEGE-C2 peptide), the CD8+ cell population producing IFN-γ in the cells was labeled with antibodies, first incubated with CD8-APC antibody, then incubated with IFN-γ-FITC antibody by intracellular factor staining method, and the stained cells were detected by flow cytometry. CD8-APC and IFN-γ-FITC double-stained cells represent the functional level of CD8+ cell population producing IFN-γ. Figure 8 A is the flow cytometry results of IFN-γ-FITC and CD8-APC staining. Figure 8 B is the percentage of CD8+ cells producing IFN-γ-FITC (percentage of cells double-stained with IFN-γ-FITC and CD8-APC) detected in three independent repeated experiments. Vector is PBMC transfected with empty vector pHR_LV-NS1; MC2-TCR H -T is PBMC transfected with pHR_LV-NS1-MC2-TCR H ;MC2-TCR HM -T is PBMC transfected with pHR_LV-NS1-MC2-TCR HM . T2 C The cells were T2 cells and incubated with control peptide (10 μM) for 2 h; T2 MC2 The cells were T2 cells co-cultured with MAGE-C2 peptide (10 μM) for 2 h. The number of TCR-T and T2 cells was 4×10 6 Pieces / hole.

[0264] The results showed that the Vector group cells that did not express MC2-specific TCR could not respond to MC2 antigen peptides, while both TCR-T cells could respond to the stimulation of MC2 antigen peptides and had the function of specifically producing and expressing IFN-γ; MC2-TCR HM -T due to mouse replacement of α and β chain C regions, TCR HMThe affinity for specific antigen peptide epitopes is stronger and the function of producing IFN-γ is stronger, while the MC2-TCR expressing the original sequence H -T has a weaker specific reaction ability.

[0265] 4. Expression of MC2-TCR with optimized mouse sequence HM -T2 cells loaded with MC2 peptide secreted more TNF-α

[0266] In order to detect the specific secretion of TNF-α by the constructed MC2-TCR-T cells under the stimulation of MC2 antigen peptide, in this example, the vector transfected with empty vector and MC2-TC R H -T and MC2-TCR HM -T and T2 C Cells and T2 MC2 After the cells were incubated with each other for 24 h, the TNF-α content in the supernatant of the co-cultured cells was measured using an Elisa kit. Fig. 9 As shown, both Vector cells and T2 C Cell or T2 MC2 After co-culture, the secretion of TNF-α was about 1pg / mL, and there was no significant difference between the two. 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 -T is the same; compared to MC2-TCR H -T, MC2-TCR HM -T and T2 MC2 After cell co-culture, the secretion of TNF-α increased significantly, and the average value of three repeated experiments was about 200pg / mL.

[0267] Fig. 9 Vector and two types of TCR-T cells were inoculated into 96-well plates and respectively C Cells and T2 MC2 The cells 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 an 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 and incubated with control peptide (10 μM) for 2 h; T2 MC2The cells were T2 cells co-cultured with MAGE-C2 peptide (10 μM) for 2 h. The number of TCR-T and T2 cells was 4×10 6 Pieces / hole.

[0268] The results showed that T cells could not respond to MC2 antigen stimulation without expressing MC2-specific TCR; MC2-TCR-T cells expressing two different TCR sequences could respond to specific antigen stimulation and produce significant TNF-α secretion; compared with the original sequence of MC2-specific TCR, MC2-TCR HM Murine TCR of T HM It has a stronger affinity for specific antigen peptide epitopes and a more sensitive response, and the specific secretion of TNF-α is higher after being stimulated by MC2 antigen.

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

[0270] In order to verify the responsiveness of MC2-TCR-T to tumor cells in subsequent tests, two tumor target cells need to be constructed. One is the control target cell A02-K562 expressing only HLA-A, and the other is the target cell expressing HLA-A02 restricted MAGE-C2 336-344 (ALKDVEERV) tumor target cells.

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

[0272] To construct the control target cell A02-K562, the recombinant expression plasmid pHR_LV-NS1-A02 carries the HLA-A gene and the PuroR gene for screening and purifying cells. 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 total length of 2.8Kb, and the linear restriction product fragment of the lentiviral expression vector pHR_LV-NS1, with a total length of about 8.9Kb. After agarose gel electrophoresis, the two restriction products obtained the expected bands, as shown in Figure 2. Fig.10 As shown in A, it is the agarose gel electrophoresis of NotI / SalI double digestion products. Lane M in the figure is DNA Marker 1Kb; Lane 1 is the pcDNA3.1(+)-A02 double digestion product, and the target gene A02 is about 2.8Kb. After cutting the gel, the bands at the above two positions are gel-recovered, and the recovered products are purified by agarose gel electrophoresis to observe the band positions, such as Fig.10As shown in B, it is the agarose gel electrophoresis of the recovered target bands, in which lane M is DNA Marker 1Kb; lane 1 is the pHR_LV-NS1 double-enzyme-cut linear fragment (about 8.9Kb); lane 2 is the target gene fragment A02 (about 2.8Kb); the target gene fragment A02 is between 2.0 and 3.0Kb, and the pHR_LV-NS1 linear enzyme-cut fragment is between 10.0 and 8.0Kb, which is in line with expectations.

[0273] The double-digested target gene fragment A02 and the pHR_LV-NS1 digested fragment were ligated to obtain the recombinant lentiviral expression vector pHR_LV-NS1-A02. The vector was transformed and 8 single colonies were selected for amplification and shaking. Then, PCR verification was performed on the 8 groups of bacterial liquids: a gene sequence of about 1.3Kb at the junction of the expression vector and the inserted target gene on the recombinant lentiviral expression vector was selected as a template, primers were designed, and PCR amplification was performed. After agarose gel electrophoresis, the 8 groups of amplified products all obtained the expected 1.3Kb band, as shown in Figure 2. Fig.10 C shows the electrophoresis of pHR_LV-NS1-A02 PCR products. Lane M in the figure is DNAMarker D2000, and lanes 1 to 8 are PCR amplification products of stained colonies, about 1.3Kb. One group of recombinant lentiviral expression vector plasmid bacterial solution was amplified and shaken to extract plasmids. The extracted plasmids were double-digested for identification, and the expected bands were obtained by running the gel, as shown in Figure 1. Fig.10 As shown in D, it is the double enzyme digestion verification of the recombinant expression vector plasmid. In the figure, lane M is DNAMarker 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 is successfully constructed and can be used for subsequent lentiviral packaging and transfection of K562 cells. After labeling and packaging, it is stored at -80℃.

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

[0275] Target cell MC2-A02-K562 needs to express MAGE-C2 336–344 / HLA-A02 complex. First, the synthesized recombinant cloning plasmid pUC-57-MC2-A02 and the lentiviral expression vector pHR_LV-NS1 were double-digested with NotI / SalI to obtain the target gene fragment MC2-A02, with a total length of 1.4Kb, and the linear digestion product fragment of the lentiviral expression vector pHR_LV-NS1, with a total length of about 8.9Kb. After agarose gel electrophoresis of the digestion product, the expected band was obtained, such as Fig.11As shown in A, it is the agarose gel electrophoresis of NotI / SalI double digestion products: Lane M is DNA Marker 1Kb; Lane 1 is the pUC-57-MC2-A02 double digestion product, and the target gene A02 is about 1.4Kb. After cutting the gel, the bands at the above two positions are recovered by gel recovery, and the recovered products are purified by agarose gel electrophoresis to observe the band positions, such as Fig.11 As shown in B, it is the agarose gel electrophoresis of the recovered target bands: Lane M is DNA Marker 1Kb; Lane 1 is the pHR_LV-NS1 double-enzyme-digested linear fragment (about 8.9Kb); Lane 2 is the target gene fragment MC2-A02 (about 1.4Kb); the target gene fragment A02 is between 1.0 and 2.0Kb, and the pHR_LV-NS1 linear enzyme-digested fragment is between 10.0 and 8.0Kb, which is in line with expectations.

[0276] The enzyme-cut fragment MC2-A02 was ligated with pHR_LV-NS1 to obtain the recombinant lentiviral expression plasmid pHR_LV-NS1-MC2-A02. The plasmid was transformed and 8 single colonies were selected for amplification and shaken. The bacterial solution was then verified by PCR: a gene sequence of about 1.327Kb at the junction of the expression vector and the inserted target gene on the recombinant lentiviral expression vector was selected as a template, and primers were designed for PCR amplification. After agarose gel electrophoresis, the 8 groups of PCR amplification products all obtained the expected 1.327Kb band, as shown in Figure 2. Fig.11 C shows the electrophoresis of pHR_LV-NS1-MC2-A02 PCR products: Lane M is DNAMarker D2000, and lanes 1 to 8 are the PCR amplification products of positive colonies, about 1.3 Kb. After amplification of one group of recombinant plasmid bacterial solution, the plasmid was extracted, and the extracted plasmid was identified by NotI / SalI double enzyme digestion. The expected bands were obtained by running the gel, as shown in Figure 1. Fig.11 As shown in D, it is the double enzyme digestion verification of the recombinant expression vector plasmid: Lane M is 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 is successfully constructed and can be used for subsequent lentiviral packaging and transfection of K562 cells. After labeling and packaging, it is 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 (ALKDVEERV) / HLA-A02 complex. HLA-A02 and antigen peptide MAG E-C2 in recombinant plasmid pHR_LV-NS1-MC2-A02 336–344 It is distributed in series. After expression on K562, HLA-A02 and antigen peptide MAGE-C2 336–344 It will exist in the form of MHC complexes, such as Fig.12 As shown in A, the schematic diagram of the HLA complex structure of A02-K562 and MC2-A02-K562: A02-K562 only expresses HLA-A02 and puromycin pLeo1209 used for screening and purification; MC2-A02-K562 expresses HLA-A02-restricted MAGE-C2 336-344 Antigen peptide complex. Multiple genes are arranged in series inside the target gene and connected by the self-cleavage polypeptide P2A sequence. When translated and expressed, the self-cleavage polypeptide P2A breaks and each gene sequence is expressed independently. Therefore, flow cytometry can be used to detect HLA-A2-PE antibody staining to evaluate the interaction between HLA-A02 and MAGE-C2 on MC2-A02-K562 cells. 336–344 Overall expression of antigenic peptides.

[0279] In this example, the recombinant plasmid pHR_LV-NS1-MC2-A02 was used to transfect K562 cells to construct the tumor target cell line MC2-A02-K562, and the recombinant plasmid pHR_LV-NS1-A02 was used to transfect K562 cells to construct the tumor control target cell line A02-K562. The vector transfected with an empty vector was used as the control group. The above three cell lines were co-incubated with HLA-A2-PE and then subjected to flow cytometry detection. Fig.12 C, the horizontal axis is HLA-A2-PE, it can be seen that the proportion of HLA-A2-PE stained cells in the Vector group transfected with empty vector is 0.08%; the proportion of HLA-A2-PE stained cells in A02-K562 cells expressing HLA-A02 is 98.47%; the proportion of HLA-A02 / MAGE-C2 336–344 The percentage of HLA-A2-PE stained cells in MC2-A02-K562 of the complex was 99.12%. The results of the transfection and flow cytometry experiments were repeated three times independently. Fig.12 B shows that the proportion of HLA-A2-PE stained cells in MC2-A02-K562 and A02-K562 was above 98%, with no significant difference between the two.

[0280] Fig.12 In the experiment, the recombinant expression vector was transduced into the conventionally cultured K562 cells for 55 h, and then the HLA-A2-PE staining was performed and incubated, and the HLA-A2-PE staining was performed and detected by flow cytometry. Fig.12A is a schematic diagram of the HLA complex structure of A02-K562 and MC2-A02-K562: A02-K562 only expresses HLA-A02 and puromycin pLeo1209 used for screening and purification; MC2-A02-K562 expresses HLA-A02-restricted MAGE-C2 336-344 Antigenic peptide complex. Multiple genes are arranged in series inside the target gene and connected by the self-cleavage polypeptide P2A sequence. During translation and expression, the self-cleavage polypeptide P2A breaks and each gene sequence is expressed independently. Fig.12 B shows HLA-A2-PE staining detected by three independent repeated experiments. Fig.12 C is the result of flow cytometry detection of HLA-A2-PE stained cells. Vector is K562 transfected with empty vector pHR_LV-NS1; A02-K562 is transfected with recombinant vector pHR_LV-NS1-A02; MC2-A02-K562 is transfected with recombinant vector pHR_LV-NS1-A02-MC2.

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

[0282] 6. MC2-TCR under tumor target cell stimulation HM -T can produce higher IFN-γ release

[0283] In order to clarify the production of IFN-γ by MC2-TCR-T cells under the specific stimulation of tumor target cells, this example used K562 cells transfected with empty vector as the control group, A02-K562 cells expressing only HLA-A02 and A02-K562 cells expressing complete HLA-A02 restricted MAGE-C2 336–344 The antigen peptide complex MC2-A02-K562 was used as the experimental group and was combined with two MC2-TCR-T cells (MC2-TCR H -T and MC2-TCR HM After 24 h of co-culture, CD8-APC incubation and IFN-γ-FITC intracellular factor staining (ICS) were performed, and the proportion of IFN-γ-FITC and CD8-APC double-stained cells was detected by flow cytometry, that is, the level of intracellular IFN-γ production in CD8+ cells. Fig.13A shows that after Vector was co-cultured with A02-K562 and MC2-A02-K562, the percentage of IFN-γ-FITC / CD8-APC double-stained cell population was very low, 0.28% and 0.14%, respectively; MC2-TCR H After co-culture of MC2-T and A02-K562, the proportion of double-stained cell population was 0.22%. HM -0.16% in T; MC2-TCR H After co-incubation of T cells with MC2-A02-K562, the percentage of double-stained cell population was 0.92%, while MC2-TCR HM After incubation of -T with MC2-A02-K562 cells, the percentage of double-stained cell population was 2.82%. The results of three independent repeated experiments were as follows Fig.13 B shows that K562 cells transfected with empty vector, regardless of MC2-TCR H -T or MC2-TCR HM After co-incubation with MC2-TCR, the proportion of CD8+ cells producing IFN-γ in the cells was low, and there was no significant difference between the two. H -T or MC2-TCR HM After co-incubation with MC2-TCR, the proportion of CD8+ cells producing IFN-γ in the cells was low, and there was no significant difference between the two. HM The proportion of CD8+ cells labeled with IFN-γ-FITC in the group incubated with MC2-A02-K562 was significantly higher than that in the group incubated with MC2-TCR H -T / A02-K562 incubation group.

[0284] Fig.13 In MC2-TCR H -T and MC2-TCR HM -T was co-incubated with Vector, A02-K562 and MC2-A02-K56 at a 1:1 effector-target ratio for 24 hours, and the CD8+ cell population producing IFN-γ in the cells was labeled with antibodies. CD8-APC antibody was first incubated, and then IFN-γ-FITC antibody was incubated by intracellular factor staining method, and IFN-γ-FITC and CD8-APC stained cells were detected by flow cytometry. CD8-APC and IFN-γ-FITC double-stained cells represent the functional level of CD8+ cell population producing IFN-γ. Fig.13 A is the flow cytometry results of IFN-γ-FITC and CD8-APC staining. Fig.13B is the percentage of CD8+ cells producing IFN-γ-FITC (percentage of cells double-stained with IFN-γ-FITC and CD8-APC) detected in three independent repeated experiments. MC2-TCR H -T is PBMC transfected with pHR_LV-NS1-MC2-TCR H ;MC2-TCR HM -T is PBMC transfected with pHR_LV-NS1-MC2-TCR HM The vector was K562 transfected with empty vector pHR_LV-NS1; A02-K562 was transfected with recombinant vector pHR_LV-NS1-A02; MC2-A02-K562 was transfected with recombinant vector pHR_LV-NS1-A02-MC2. The number of effective target cells was 0.4×10 6 Each hole.

[0285] The results showed that only the expression of HLA-A02 restricted MAGE-C2 336–344 The complex of MC2-A02-K562 target cells can stimulate MC2-TCR H -T and MC2-TCR HM -T-specific IFN-γ production; MC2-TCR-T cells do not present MAGE-C2 336–344 There is no specific response to HLA-A02 antigen peptide; compared to MC2-TCR H -T, mouse optimized MC2-TCR HM -T is restricted by MAGE-C2 on HLA-A02 on MC2-A02-K562 336–344 After stimulation by the complex, the function of specifically producing IFN-γ is stronger.

[0286] 7. MC2-TCR under tumor target cell stimulation HM -T can produce higher specific secretion of TNF-α

[0287] In order to detect the function of the constructed tumor target cells to stimulate MC2-TCR-T cells to specifically secrete TNF-α, this example used MC2-TCR H -T and MC2-TCR HM -T was incubated with A02-K562, MC2-A02-K562 and Vector (K562) transfected with empty vector for 24 hours, and the TNF-α content in the supernatant of the incubated cells was measured using an Elisa kit. Fig.14 As shown, Vector and MC2-TCR H -T and MC2-TCR HM-T interaction, the content of TNF-α in the supernatant was very low, and there was no significant difference between the two; A02-K562 and MC2-TCR HM -T after co-incubation, 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 A02-K562 / MC2-TCR, the content of TNF-α was significantly higher than that of A02-K562 / MC2-TCR H -T incubation group, and also higher than MC2-TCR HM -T / A02-K562 incubation group and MC2-TCR HM -T / Vector incubation group, the mean of three independent experiments was approximately 21 pg / mL, and the differences were statistically significant.

[0288] Fig.14 Vector and two TCR-T cells were inoculated into 96-well plates and co-cultured with tumor target cells A02-K562 and MC2-A02-K562 for 24 hours. After incubation, the TNF-α Elisa kit was used to detect the TNF-α content in the supernatant of the incubated cells. The experiment was repeated three times. MC2-TCR H -T is PBMC transfected with pHR_LV-NS1-MC2-TCR H ;MC2-TCR HM -T is PBMC transfected with pHR_LV-NS1-MC2-TCR HM The vector was K562 transfected with empty vector pHR_LV-NS1; A02-K562 was transfected with recombinant vector pHR_LV-NS1-A02; MC2-A02-K562 was transfected with recombinant vector pHR_LV-NS1-A02-MC 2. The number of effective target cells was 0.4×10 6 Each hole.

[0289] The results showed that A02-K562 cells without MC2 antigen peptides could not stimulate MC2-TCR-T cells to specifically secrete TNF-α; both MC2-TCR-T cells could recognize MAGE-C2 expressing complete HLA-A02 restriction. 336–344 The complex was expressed in MC2-A02-K562 target cells and caused specific secretion of TNF-α; MC2-TCR expressing the original sequence of MC2-specific TCR H -T, the mouse optimized MC2-TCR HM-T has a stronger specific response to MC2-specific antigen peptides, and the specific TNF-α secretion is higher after antigen stimulation, so the subsequent experiments will use MC2-TCR HM -T was used as effector cell to carry out target cell killing experiment.

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

[0291] To detect MC2-TCR HM -T in vitro experiments for the recognition and killing ability of tumor target cells, this example uses CFSE staining to mark tumor target cells MC2-A02-K562 and control target cells A02-K562 to distinguish effector cells from target cells, the effector cells and target cells are mixed in a 1:1 ratio in a 96-well plate and incubated for 14 hours, and then the CFSE-stained cell particles in each well are counted by absolute volume using a flow cytometer to obtain the density of CFSE-stained cell particles in each well, and the cell particle density is used to calculate the killing rate of effector cells to target cells within a certain period of time. Fig.15 As shown in A, a total of four reaction groups were set up: A02-K562 single culture group, MC2-A02-K562 single culture group, MC2-TCR HM -T / A02-K562 co-incubation group and MC2-TCR HM -T / MC2-A02-K562 co-incubation group. After the incubation, the cell density of the remaining target cells labeled with CFSE in each group was detected by flow cytometry (unit: cell / μL). (A) Flow cytometry results. (B) Scatter plot of the results of three independent repeated experiments.

[0292] The flow cytometry results of the separate culture groups of tumor target cells MC2-A02-K562 and control target cells A02-K562 are the cell densities of the two tumor target cells under normal culture conditions; MC2-TCR HM The results of flow cytometry in the incubation group of A02-K562 and A02-K562 cells showed that only HLA-expressing A02-K562 cells and MC2-TCR HM -T interaction, the cell density of surviving A02-K562; MC2-TCR HM The results of flow cytometry analysis of the incubation group of -T and MC2-A02-K562 showed that the expression of HLA-A02 restricted MAGE-C2 336–344 Antigen-peptide complex of MC2-A02-K562 and MC2-TCR HM - 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 cannot trigger MC2-TCR HM -T cell-specific secretion produces IFN-γ and TNF-α, so this example believes that MC2-TCR HM -T cells had no significant specific killing effect on A02-K562, but on the other hand, MC2-TCR HM -T cells are derived from normal human PBMC, which contain natural killer cells and some untransfected normal T cells. These cells have a certain non-specific killing effect on target cells. Therefore, in this example, MC2-TCR HM -T The number of CFSE-stained cells in the group incubated with A02-K562 was used to calculate MC2-TCR HM -T to MC2-A02-K562 killing rate background to correct the killing rate value. In addition, without external interference, the proliferation rate of MC2-A02-K562 and the control target cell A02-K562 itself is also different, so separate culture groups are set up to correct the calculation of the killing rate.

[0294] According to the results of a flow cytometric test, Fig.15 A, substitute into the kill rate calculation formula, calculate the kill rate:

[0295]

[0296] The experiment was repeated three times independently. HM -T has a killing efficiency of about 50% for target cells MC2-A02-K562. Fig.15 B.

[0297] Fig.15 In the experiment, CFSE staining was used to mark tumor target cells MC2-A02-K562 and control target cells A02-K562 to distinguish effector target cells. HM -T and target cells A02-K562 and MC2-A02-K562 were inoculated into 96-well plates at a 1:1 effector-target ratio and incubated for 14 hours. HM -T / A02-K562 co-incubation group and MC2-TCR HM -T / MC2-A02-K562 co-incubation group. After the incubation, the cell density of the remaining target cells labeled with CFSE in each group was detected by flow cytometry (unit: cell / μL). Fig.15 A is the result of a flow cytometry test. Fig.15B is a scatter plot of the results of three independent repeated experiments.

[0298] The results show that in in vitro experiments, when the effector-target ratio is 1:1, MC2-TCR HM -T for expression of HLA-A02 restricted MAGE-C2 336–344 The complex has a stable and specific killing effect on 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 experimental results. The experimental method part includes:

[0301] 1. Construction method of PDL1-MC2-TCR-T cells;

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

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

[0304] 4. Method for detecting the expression efficiency of MC2-TCR by flow cytometry;

[0305] 5. Method for detecting PD-L1 expression efficiency by flow cytometry;

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

[0307] 7. Method for detecting the intracellular expression of IFN-γ in TCR-T cells by flow cytometry ICS method;

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

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

[0310] 10. Method for detecting the killing efficiency of target cells by flow cytometry;

[0311] The experimental results section includes:

[0312] 1. Successfully constructed a gene with PD-L1 and TCR HM Gene sequence of the recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR HM ;

[0313] 2. The expression of exogenous PD-L1 inhibits TCR-T cell function;

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

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

[0316] 5. PDL1-MC2-TCR-T with PD-1 knockout PD1- Cells were T2 MC2 Cytotoxic function is enhanced after cell activation;

[0317] 6. Successful construction and simultaneous expression of MAGE-C2 336–344 / HLA-A02 (pMHC) and PD-1 tumor target cell MC2-A02-K562 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 inhibits 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 of PD-1-positive tumor target cells increased with the extension of co-culture time;

[0322] The specific contents are as follows:

[0323] 1. Experimental methods:

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

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

[0326] PDL1-MC2-TCR-T cells simultaneously express the C region mouse replacement sequence MC2-TCR described in Example 1 HM and PD-L1, MC2-TCR HMThe α and β chain sequences of the C region are replaced by mouse gene sequences. The construction process of the recombinant expression plasmid is as follows Fig.16 As shown: the self-cleaving polypeptide T2A sequence was added to the front end of the PD-L1 gene sequence, and the cloning plasmid pUC-SP-T2A-PDL1 was synthesized by entrusting Sangon Biotechnology (Shanghai) Co., Ltd., and then the target gene T2A-PDL1 was obtained by double restriction digestion with RsrII and MluI. At the same time, the recombinant expression vector pHR_LV-NS1-MC2-TCR described in Example 1 was HM RsrII / MluI double digestion was also performed, and the digestion products were purified and recovered, and then connected to obtain the recombinant expression vector pHR_LV-NS1-PDL1-MC2-TCR co-expressing PD-L1 HM , whose target gene is PDL1-MC2-TCR HM The α and β chain gene sequences of PD-L1 with T2A and P2A are arranged in series. 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 T2A-PDL1 target gene plasmid

[0329] The experimental conditions and steps are the same as those in (1) digestion of the target gene plasmid in Part 5 "Preparation of Lentivirus Recombinant Plasmid" of Example 1. The digestion reaction system is shown in Tables 10 and 11:

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

[0331]

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

[0333]

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

[0335] The experimental conditions and steps were the same as those in Part 5 “Preparation of Lentivirus Recombinant Plasmid” of Example 1, including (2) agarose gel electrophoresis purification; (3) DNA purification and recovery; and (4) agarose gel electrophoresis to detect the purity of the target gene fragment. The differences were that: when cutting the gel, the T2A-PDL1 fragment was cut at the 880 bp band, and pHR_LV-NS1-MC2-TCR HM The fragment was cut at the 10.7 kb band, and then the corresponding target fragment was recovered according to the gel recovery kit steps and the concentration and purity of the recovered solution were detected by spectrophotometer. Then agarose gel electrophoresis was performed again to detect 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 Recover the linear fragment product and 1kb marker, and observe whether the band position is correct under UV light.

[0336] (4) Ligation reaction

[0337] The T2A-PDL1 fragment (insert) obtained after the above purification and pHR_LV-NS1-MC2-TCR HM Prepare the corresponding reagents and add them into the marked PCR tubes according to the system in Table 12 for ligation reaction. The recombinant expression plasmid after ligation is pHR_LV-NS1-PDL1-MC2-TCR HM The specific steps are the same as (5) the ligation reaction between the target gene and the lentiviral vector in Part 5 “Preparation of lentiviral recombinant plasmid” of Example 1.

[0338] Table 12: Ligation reaction system

[0339]

[0340] (5) Identification of recombinant expression vector

[0341] Transform competent bacteria, perform PCR amplification and identification on the bacterial solution, and according to pHR_LV-NS1-PDL1-MC2-TCR HM The sequence at the junction of the target gene and the vector is about 523bp in length. The primers are designed as follows:

[0342] pHR_LV-NS1-PDL1-MC2-TCR HM -F gene sequence 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, sample bacterial liquids were taken for PCR respectively, marked, and PCR reaction reagents were added on ice.

[0344] Table 13: PCR reaction program

[0345]

[0346] After shaking and mixing, place it in a PCR instrument and perform the reaction program in Table 14.

[0347] Table 14: PCR reaction system

[0348]

[0349]

[0350] Take 6 μL of PCR product from each reaction and add it to the sample well of agarose gel for electrophoresis to observe whether the band position of each clone sample meets the expectation (523 bp). Select the corresponding bacterial solution with the correct band position, shake the bacteria overnight, and then extract the plasmid. Add reagents according to Table 15, perform enzyme digestion, and perform agarose electrophoresis on the enzyme digestion product (same steps as above) to observe whether the recombinant plasmid meets the expected position to determine whether the construction is successful.

[0351] Table 15: Recombinant plasmid restriction enzyme digestion reaction system

[0352]

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

[0354] Before transfection, T cells need to be activated, and 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 transfection of PBMC cells are MC2-specific TCR-T cells co-expressing PD-L1, and the negative control group cells transfected with empty vector are Vector.

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

[0356] Jurkat cell culture medium is 90% RPMI-1640 medium plus 10% fetal bovine serum. MC2-TCR-Jurkat uses pHR_LV-NS1-MC2-TCR HM Jurkat cells were transfected with pHR_LV-NS1-PDL1-MC2-TCR for PDL1-MC2-TCR-Jurkat. HMThe steps of virus transduction, T cell activation and cell culture are the same as those in Example 1.

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

[0358] In this experiment, lipid nanoparticles (LNPs) were used to package Cas9 mRNA and PD-1-sgRNA and precisely deliver them into the nucleus of the constructed PDL1-MC2-TCR-T cells to specifically knock out the endogenous PD-1 gene. Fig.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 connected to the two liquid inlets of the microfluidic chip, and the aqueous phase and the ethanol phase were mixed in the microfluidic instrument at a flow rate ratio of 3:1. The LNP product was transferred to a MWCO dialysis tube, dialyzed with PBS buffer for 4 hours at 4°C, and finally concentrated into a LNP-mRNA (Cas9-mRNA / PD-1-sgRNA) product through an ultrafiltration tube for standby use. LNP-mRNA (Cas9-mRNA / PD-1-sgRNA) was transfected into cells, Cas9-mRNA was transiently expressed in cells, and each component entered the cell nucleus. PD-1-sgRNA could target the target sequence near PAM through base complementary pairing, and Cas9 protein caused DNA double-strand breaks upstream and downstream of the gene under the guidance of PD-1-sgRNA. The DNA damage repair mechanism connects the sequences at the upstream and downstream ends of the break, thereby achieving the knockout of the target gene PD-1.

[0359] (1) Cas9 mRNA

[0360] The Cas9 mRNA used in this example 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 commissioned to be synthesized by GenScript Biotech Co., Ltd.

[0361] (2) PD-1-sgRNA

[0362] The sgRNA (small guide RNA) used in the experiment is in single-stranded form. The crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) are connected by a linker and fused 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 of lipid nanoparticles for packaging Cas9-mRNA / PD-1-sgRNA by microfluidic mixing

[0365] The stock solution was diluted with ultrapure water to 50 mM citrate buffer (pH = 4), and Cas9-mRNA:PD-1-sgRNA was dissolved in citrate buffer at a molar ratio of 1:5 to a final concentration of 100 μg / mL to prepare an mRNA-containing aqueous phase; SM-102 (50%), DSPC (10%), cholesterol (38.5%), and DMG-PEG-2000 (1.5%) were dissolved in anhydrous ethanol at a fixed molar ratio to prepare an ethanol phase; the microfluidic instrument was preheated and the microfluidic The Luer port of the fluidic chip faces upwards and is installed in the adapter. The chip is assembled and the entire module is installed in the microfluidic reaction chamber; the chip is pre-filled with citrate buffer and anhydrous ethanol; the injection adapter is installed in the reaction chamber, and ethanol and buffer are respectively drawn with two syringes to exhaust air bubbles, and two 15ml centrifuge tubes are installed as product and waste liquid collection tubes respectively; the software parameters are set, and 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 a MWCO dialysis tube at 4℃ and dialyzed on a shaker with 14ml PBS (1X), and the liquid is changed every 2 hours, and the operation is repeated until the dialysis is completed after 4 hours. Then filter with a 0.22uM filter membrane, and after filtration, use a 100kD ultrafiltration tube to ultrafilter and concentrate into the LNP-mRNA (Cas9-mRNA / PD-1-sgRNA) finished product, which is frozen at 4℃ or -20℃.

[0366] LNP-mRNA (Cas9-mRNA / PD-1-sgRNA) transfects cells, Cas9-mRNA is transiently expressed in cells, and each component enters the cell nucleus. PD-1-sgRNA can target the target sequence near PAM through base complementary pairing. Under the guidance of PD-1-sgRNA, Cas9 protein breaks the DNA double strands upstream and downstream of the gene. The DNA damage repair mechanism connects the sequences at both ends of the break, thereby achieving the knockout of the target gene PD-1.

[0367] (4) Determination of LNP encapsulation efficiency

[0368] Nanoparticle size analyzer (Zetasizer Nano ZS / ZEN 3600) was used to measure the average particle size, PDI (polymer dispersity index) and zeta potential of LNP-mRNA.

[0369] (5) LNP-mRNA (Cas9-mRNA / PD-1-sgRNA) transfection of PDL1-MC2-TCR-T to prepare PDL1-MC2-TCR-T PD1- cell

[0370] Obtain stably transfected PDL1-MC2-TCR-T cells according to the schedule, count the cells, and inoculate 24-well plates for plating. Each well contains 1 ml of 10% FBS DMEM medium containing 5×10 4 cells; add 500ng of encapsulated LNP-mRNA (Cas9-mRNA / PD-1-sgRNA) to each well, continue to culture normally for 24h, and change the medium for subsequent experiments. PD1- The transfection method was the same.

[0371] 4. Detection of MC2-TCR expression efficiency by flow cytometry

[0372] After transfection, the corresponding TCR-T cells were adapted to culture for 5 days, and the PBMCs transduced with the pHR_LV-NS1 empty vector were used as the control group. The cells in each group were double-stained with HLA-A02 restricted 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 (ALKDVEERV) antigen’s percentage of CD8+T cell population.

[0373] 5. Detection of PD-L1 expression efficiency by flow cytometry

[0374] After transfection, the corresponding TCR-T cells were adapted to culture for 5 days, and the cells in each group were double-stained with HLA-A02-restricted MAGE-C2-Tetramer-PE and PDL1-APC. Flow cytometry was used to detect the TCR-T cells that could bind to HLA-A02 / MC2. 336–344 -ALKDVEERV pMHC complex and PD-L1 antibody cell population ratio. FACS Buffer is PBS containing 0.5% FBS, and each reaction requires 2μL PDL1-APC, 2μL Tetramer-PE and 46μL FACS Buffer antibody mixture.

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

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

[0377] The target gene fragment was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the structure was that the restriction sites NotI and SalI were at both ends of the PD-1 gene sequence. The lentiviral expression vector used for transfection of cells was pHR_LV-NS1. The restriction, ligation and transformation steps were the same as described above. After the PD-1 target gene fragment was inserted, it was called pHR_LV-NS1-PD-1. Fig.18As shown in SEQ ID NO: 17, the target gene PD-1 sequence is 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] The recombinant expression vector pHR_LV-NS1-PD-1 was used to transfect the successfully constructed MC2-A02-K562 cells to obtain MC2-A02-K562 PD1+ Cells, cell culture, virus transfection and target cell purification, the specific steps are shown in Example 1.

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

[0381] The mixing ratio of TCR-T cells and target cells (or peptide-loaded T2 cells) was 1:1, and after 24 hours of co-incubation, ICS (intracellular 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 steps were the same as those in Example 1.

[0382] 8. Elisa method to detect TNF-α secretion

[0383] Each group of TCR-T cells and tumor target cells (or peptide-loaded T2 cells) were counted, and the effector cells and target cells were still in a 1:1 ratio, fully mixed, and returned to the incubator for incubation for 24 hours. After the incubation was completed, the cell supernatant was taken and the experiment was carried out according to the requirements of the Elisa kit, and the specific steps were the same as in Example 1.

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

[0385] Preparation of MC2-TCR-Jurkat, PDL1-MC2-TCR-Jurkat and target cells MC2-A02-K562 PD1+Afterwards, count the inoculated cells, the FACS Buffer is PBS containing 0.5% FBS, and 50 μL HLA-A2-PE antibody working solution (5 μL HLA-A2-PE antibody stock solution plus 45 μL FACS Buffer) is added to each reaction; the other experimental steps are the same as in Example 1. After the cells are mixed, that is, 0h and at 24h and 48h of co-incubation, the target cells are labeled with the HLA-A2-PE antibody, and then detected by flow cytometry to obtain the number and proportion of target cells labeled with the HLA-A2-PE antibody at this time point, and then the inhibition rate calculation formula is used to calculate the inhibition rate of effector cells on target cells at the corresponding time point. The inhibition rate calculation formula is (based on 24h effector cells MC2-TCR-Jurkat and target cells MC2-A02-K562 PD1+ Take the inhibition rate calculation of the co-incubation group as an example):

[0386]

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

[0388] The target cells were labeled with CFSE dye, and each group of TCR-T cells was mixed and co-incubated with target cells at two ratios of 1:1 and 1:2, and a separate target cell group was set up. The number of CFSE-labeled target cells after co-incubation was detected by flow cytometry at 24h, 48h and 72h, and the killing rate of each group of TCR-T on target cells was calculated according to the formula. The experimental steps of CFSE labeling and flow cytometry are the same as those in Example 1. The killing rate calculation formula at a certain time point is:

[0389]

[0390] Statistical methods:

[0391] Statistical analysis was performed using Prism version 8.0 (Graph Pad). For data comparisons 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 significant statistical difference; ***p<0.001 indicates an extremely significant statistical difference.

[0392] 2. Experimental results:

[0393] 1. Successfully constructed a gene with PD-L1 and TCR HM Gene sequence of the recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR HM

[0394] In order to explore whether exogenous PD-L1 can activate the PD-1 signal of malignant T cells under the targeted guidance of MC2-TCR-T, and synergistically enhance the tumor killing inhibitory effect of MC2-TCR-T, this example constructed the recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR HM , PD-L1 gene and TCR HM The sequences were transfected into activated T cells to form engineered PDL1-MC2-TCR-T cells.

[0395] First, the recombinant plasmid pUC-SP-T2A-PDL1 (with T2A self-cleavage polypeptide sequence and PD-L1 gene) and the recombinant plasmid pHR_LV-NS1-MC2-TCR were double-digested HM The restriction endonuclease sites RsrII and MluI were added, and the digested products were subjected to agarose gel electrophoresis to obtain the target gene fragment T2A-PDL1 of about 880 bp and the recombinant plasmid pHR_LV-NS1-MC2-TCR of about 10.7 kb. HM The linear enzyme digestion product fragments were recovered and the above two bands were recovered. After agarose gel electrophoresis again, the T2A-PDL1 band located between 750 bp and 1.0 Kb was obtained, and the pHR_LV-NS1-MC2-TCR above 10.0 Kb was obtained. HM The strips are in line with expectations, such as Fig.19 A, pUC-SP-T2A-PDL1 and pHR_LV-NS1-MC2-TCR HM Double enzyme digestion agarose gel electrophoresis: M 1 The lanes are standard DNA molecular weight DNA Marker D2000, lane 1 is T2A-PDL1 fragment (880bp), lane 2 is pHR_LV-NS1-MC2-TCR HM Linear fragment (10.7Kb), M 2 The lane is the standard DNA molecular weight DNA Marker 1Kb.

[0396] The double-enzyme digested target gene fragment T2A-PDL1 was combined with pHR_LV-NS1-MC2-TCR HM The enzyme-cut fragments were ligated to obtain the recombinant lentiviral expression vector pHR_LV-NS1-PDL1-MC2-TCR HM , whose target gene is PDL1-MC2-TCR HM After the vector was transformed into bacteria and amplified again, all 8 groups of bacterial solutions were verified by PCR. After agarose gel electrophoresis, the 8 groups of amplified products all obtained the expected 523bp band. Fig.19B, is the electrophoresis 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). Take one of the bacterial solutions for amplification and shake the bacteria for plasmid extraction. RsrII and MluI double-digest the plasmid. The digestion product is run on the gel to obtain the expected 880bp and 10.7kb bands. Fig.19 C, pHR_LV-NS1-PDL1-MC2-TCR HM Double enzyme digestion verification: Lane M 1 is DNA Marker D2000, lane 1 is pHR_LV-NS1-PDL1-MC2-TCR HM Double enzyme digestion results, lane M 2 DNA Marker 1Kb. Recombinant lentiviral expression vector plasmid pHR_LV-NS1-MC2-TCR HM The build is successful, such as Fig.19 D, pHR_LV-NS1-PDL1-MC2-TCR HM Plasmid map: The target gene is composed of TCRα3-P2A-TCRβ28-T2A-PDL1, where TCRα3-P2A-TCRβ28 is the mouse optimized TCR in 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] 2.1PDL1-MC2-TCR-T cells can express TCR simultaneously 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 and MC2-TCR-T, PDL1-MC2-TCR-T and control group Vector cells were obtained by transfection. MAGE-C2-Tetramer-PE and CD8-APC double staining of the above cells were detected by flow cytometry to observe the expression of TCR HM The proportion of CD8+ cell population; TCR was observed by double staining of 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 HM PDL1-MC2-TCR-T cells obtained after transfection of PBMCs express C-region mouse-optimized TCR on their cell surface HM and PD-L1 protein, such as Fig. 20 A. TCR HM The results of a flow cytometric assay of expression were as follows Fig. 20 D, the horizontal axis is MAGE-C2-Tetramer-PE, the vertical axis is CD8-APC. In both MC2-TCR-T and PDL1-MC2-TCR-T, there are cell populations with MAGE-C2-Tetramer-PE and CD8-APC double staining, accounting for 38.7% in MC2-TCR-T, 1.69% in PDL1-MC2-TCR-T, and 0.29% in the Vector group. The results of three independent repeated experiments showed that Fig. 20 B, The proportion of MAGE-C2-Tetramer-PE and CD8-APC double-stained cell population in PDL1-MC2-TCR-T was significantly higher than that in Vector, but significantly lower than that in MC2-TCR-T. HM The results of a flow cytometric analysis of cells co-expressing PD-L1 were as follows: Fig. 20 E, the horizontal axis is MAGE-C2-Tetramer-PE, the vertical axis is PDL1-APC, the percentage of double-stained cell populations in Vector and MC2-TCR-T is 0.04% and 0.25%, respectively, and in PDL1-MC2-TCR-T is 3.09%. The results of three independent repeated experiments showed that Fig. 20 C, The proportion of MAGE-C2-Tetramer-PE and PDL1-APC double-stained cell population in PDL1-MC2-TCR-T was significantly higher than that in Vector and MC2-TCR-T.

[0401] The results show that PDL1-MC2-TCR-T cells can simultaneously express the C region mouse optimized TCR HM and PD-L1, but TCR HM The expression of PD-L1 or the function of binding to MAGE-C2 (MC2) antigen epitope is significantly reduced compared with MC2-TCR-T. This example speculates that the engineered expression of PD-L1 in PDL1-MC2-TCR-T cells interferes with TCR HM expression or ability to bind to antigenic epitopes.

[0402] For the sake of simplicity, the MC2-TCR-T cells mentioned in this example are the MC2-TCR constructed in Example 1. HM-T cells, composed of the recombinant plasmid pHR_LV-NS1-MC2-TCR HM Transfected PBMCs expressing TCR targeting MAGE-C2 with mouse-derived sequence replacement in the C region HM Similarly, transfect the recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR HM The TCR-T cells were named PDL1-MC2-TCR-T, which is MC2-TCR co-expressing PD-L1. HM -T cells. MAGE-C2 will be referred to as MC2 in the future.

[0403] Fig. 20 Flow cytometry detection of TCR HM Expression (MAGE-C2-Tetramer-PE and CD8-APC double staining), and TCR HM Co-expression of TCR and PD-L1 on the surface of PDL1-MC2-TCR-T cells: PDL1-MC2-TCR-T cells express targeted MC 2 Murine TCR HM , and co-express PD-L1. The target gene of the expression vector is composed of α, β chain and PD-L1 gene sequences in series, and the genes are connected by self-cleaving polypeptide sequences. (B) TCR expression efficiency detected by three independent repeated experiments (i.e., the proportion of double-stained cell populations detected by flow cytometry for MAGE-C2-Tetramer-PE and CD8-APC). (C) PD-L1 expression efficiency detected by three independent repeated experiments (i.e., the proportion of double-stained cell populations detected by flow cytometry for MAGE-C2-Tetramer-PE and PDL1-APC). (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 transfected with pHR_LV-NS1-MC2-TCR HM ; PDL1-MC2-TCR-T: PBMC transfected with pHR_LV-NS1-PDL1-MC2-TCR HM 54 hours after normal donor PBMCs were transduced with the recombinant expression vector, they were incubated with various antibodies, and antibody staining was detected by flow cytometry.

[0404] 2.2 The function 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 Or the level of binding to MC2 antigen epitope is low, then what is the ability of PDL1-MC2-TCR-T cells to produce cytokines after being stimulated by MC2 antigen peptide? In order to explore this issue, this example uses the MC2-TCR-T with normal function that has been demonstrated in the example as a positive control, and transfects cells to obtain MC2-TCR-T and PDL1-MC2-TCR-T, respectively, and then interacts with T2 loaded with 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 the cytokine flow cytometry method (ICS). Cells were incubated with CD8-APC and IFN-γ-FITC to mark the CD8+ cell population that produced IFN-γ in the cells, and the proportion of double-stained cell populations was detected by flow cytometry. Fig.21 A shows that the horizontal axis is CD8-APC and the vertical axis is IFN-γ-FITC. 2C In the case of co-culture, the proportions of CD8-APC / IFN-γ-FITC double-stained cell populations were very low, 0.32% and 0.17%, respectively; MC2 When co-incubated, the cell populations with CD8-APC / IFN-γ-FITC double staining accounted for 4.61% and 2.01%, respectively. The results of three independent repeated experiments showed that Fig.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 Elisa kit. MC2-TCR-T and PDL1-MC2-TCR-T were respectively 2C and T2 MC2 The cell supernatant after co-culture was incubated for color development, and the results were as follows Fig.21 C, PDL1-MC2-TCR-T / T2 MC2 The secretion of TNF-α in the MC2-TCR-T / T2 group was significantly lower than that in the MC2-TCR-T / T2 group. MC2group, but significantly higher than PDL1-MC2-TCR-T / T2 C Group.

[0408] Fig.21 In the study, MC2-TCR-T and PDL1-MC2-TCR-T were respectively C Cells (T2 cells loaded with control peptide) and T2 MC2 After co-culture of cells (T2 cells loaded with MC2 peptide), flow cytometry was used to detect CD8-APC and IFN-γ-FITC staining. CD8-APC and IFN-γ-FITC double-stained cells represent the functional level of IFN-γ produced by CD8+ cell populations. Elisa kit was used to detect the content of TNF-α in the supernatant of co-cultured cells. Fig.21 A is the flow cytometry results of CD8-APC and IFN-γ-FITC staining. Fig.21 B is the percentage of CD8+ cells producing IFN-γ-FITC (i.e., the percentage of cells doubly stained with IFN-γ-FITC and CD8-APC) detected in three independent repeated experiments. Fig.21 C shows the secretion of TNF-α detected by three independent repetitions. HM -T is PBMC transfected with pHR_LV-NS1-MC2-TCR HM , PDL1-MC2-TCR-T is PBMC transfected with pHR_LV-NS1-PDL1-MC2-TCR HM . T 2C The cells were T2 cells and incubated with control peptide (10 μM) for 2 h; T2 MC2 The cells were T2 cells and incubated with MEGE-C2 peptide (10 μM) for 2 h.

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

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

[0411] The details of the mechanism by which PD-1 inhibits T cell activation are still unclear, but activation of PD-1 signals inhibits the binding of TCR to pMHC complexes. This example speculates whether the function of TCR in PDL1-MC2-TCR-T cells is inhibited because the co-expressed PD-L1 activates its own PD-1. In order to explore the role of T cell's own PD-1 in causing TCR function obstruction, this example performs pHR_LV-NS1-PDL1-MC2-TCR on Jurkat (human acute T lymphoblastic leukemia cells) and normal PBMCs that do not express PD-1. HM After transfection, PDL1-MC2-TCR-Jurkat and PDL1-MC2-TCR-T cells were obtained and double-stained with MAGE-C2-Tetramer-PE and PDL1-APC before detection by flow cytometry.

[0412] Jurkat cell RNA was extracted and RT-PCR was used to detect PD-1 expression. Fig. 22 As shown in C, with β-actin as the internal reference, there is no PD-1 mRNA expression in Jurkat. Fig. 22 A shows: the horizontal axis is MAGE-C2-Tetramer-PE, the vertical axis is PDL1-APC, the percentage of double-stained cells in the two groups of Vector cells transfected with empty vector is very low, both 0.019%; the percentage of double-stained cells in PDL1-MC2-TCR-T is 3.83%, and the percentage of double-stained cells in PDL1-MC2-TCR-Jurkat is 52.2%. The statistical results of three independent repeated experiments show that Fig. 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 populations in PDL1-MC2-TCR-Jurkat was significantly higher than that in Vector and PDL1-MC2-TCR-T.

[0413] like Fig. 22 Vector (Jurkat), PDL1-MC2-TCR-Jurkat and Vector (Tcell), PDL1-MC2-TCR-T cells were prepared, double-stained with MAGE-C2-Tetramer-PE and PDL1-APC and detected by flow cytometry. Fig. 22 A is the flow cytometry results of MAGE-C2-Tetramer-PE and PDL1-APC staining. Fig. 22 B shows the percentage of MAGE-C2-Tetramer-PE and PDL1-APC double-stained cell populations detected by three independent repeated experiments. Fig. 22 C is RT-PCR detection of PD-1 expression in normal cultured Jurkat cells: Lane M is the standard DNA molecular weight Marker D2000; Lanes 1-3 are the internal reference β-actin (540bp); Lanes 4-6 are PD-1 (512bp) without expression. The red frame is the location of the internal reference and PD-1 bands. In the figure, Vector (Jurkat) is the empty vector pHR_LV-NS1 transfected by Jurkat; PDL1-MC2-TCR-Jurkat is the recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR transfected by Jurkat HM ; Vector (T cell) is the empty vector transfected with PBMC; PDL1-MC2-TCR-T is the recombinant plasmid pHR_LV-NS1-PDL1-MC2-TCR transfected with PBMC HM .

[0414] The results showed that compared with primary T cells, Jurkat cells that do not express PD-1 showed a higher expression of PD-1 in the Jurkat cells transfected with the same recombinant vector pHR_LV-NS1-PDL1-MC2-TCR. HM Post-TCR HM PDL1-MC2-TCR-T cells are derived from primary T cells expressing PD-1. After transfection with the recombinant vector, TCR HM The expression levels of PD-L1 and PD-1 are low, and the PD-1 signaling pathway of T cells itself may be activated by the co-expressed PD-L1. Therefore, it is preliminarily concluded that the PD-1 expressed by T cells itself is the cause of TCR HM The key reasons for limited expression or function and low expression of PD-L1.

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

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

[0417] In order to verify the effect of T cell's own PD-1 on PDL1-MC2-TCR-T expression or function, this example attempts to knock out the PD-1 gene of PDL1-MC2-TCR-T cells to observe the changes in TCR function and PD-L1 expression after knocking out PD-1. Lipid nanoparticles LNP can directly encapsulate nucleic acids and deliver them to cells to release target nucleic acids. LNP (lipid nanoparticles) are used to package and deliver Cas9-mRNA / PD-1-sgRNA to knock out the PDCD1 gene in the genome of PDL1-MC2-TCR-T cells to prepare PDL1-MC2-TCR-TPD1- Cells, this method has low cytotoxicity and high knockout efficiency. First, nucleic acid nanoparticles are prepared using microfluidic mixing technology. The Cas9-mRNA / PD-1-sgRNA citric acid aqueous solution and lipid ethanol solution are connected to the two channel entrances of the microfluidic chip respectively. The two phases are quickly mixed by a microfluidic injector to prepare lipid nucleic acid nanoparticles with a certain particle size range. After dialysis and ultrafiltration concentration, they can be used for transfection of cells. Dynamic light scattering instrument detection data show that Fig.23 A. The average particle size of the (Cas9-mRNA+PD-1-sgRNA)-LNP particles prepared in this example is 69.4 nm, the lipid content of 40-90 nm is 91.7%, and the polydispersity coefficient is 0.142 (<0.3). The nucleic acid LNPs were visualized using cryo-electron microscopy, as shown in Figure 2. Fig.23 B, it can be seen that most of the LNPs are double-layered, spherical with no obvious internal defects, and the particle size distribution is uniform. The above characterization tests show that the (Cas9-mRNA+PD-1-sgRNA)-LNP prepared in this example has high encapsulation efficiency and uniform particle size distribution.

[0418] Microfluidic mixer to prepare (Cas9-mRNA+PD-1-sgRNA)-LNP. Fig.23 A is the data graph of (Cas9-mRNA+PD-1-sgRNA)-LNP particle size detected by dynamic light scattering instrument. Fig.23 B is the overall appearance of (Cas9-mRNA+PD-1-sgRNA)-LNP encapsulation efficiency, circularity, lamellar distribution and particle size distribution shown by cryo-electron microscopy. Scale bar: 200 nm.

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

[0420] The Cas9-mRNA and PD-1-sgRNA customized in this example have the necessary gene modifications, low cytotoxicity, and stable knockout efficiency. After transfection of (Cas9-mRNA+PD-1-sgRNA)-LNP into PDL1-MC2-TCR-T cells, genomic PD-1 was knocked out, and PDL1-MC2-TCR-T cells were obtained. PD1- Cells. Simultaneously transfected with MC2-TCR-T cells to prepare MC2-TCR-T cells. PD1- As an experimental control, flow cytometry was used to detect the percentage of PD-1-PE stained cells. The flow cytometry results showed that Fig.24 A, MC2-TCR-T cells with PD-1 knocked out PD1- and PDL1-MC2-TCR-T PD1-The proportion of PD-1-PE antibody-labeled cells in the cells was 17.4% and 13.3%, respectively. The proportion of PD-1-PE-stained cells in Vector and MC2-TCR-T cells without PD-1 knockout was 26.3% and 25.2%, respectively. PD1- Cells and MC2-TCR-T PD1- The proportion of PD-1-PE stained cells in the PDL1-MC2-TCR-T cells was significantly lower than that in the Vector and MC2-TCR-T cells. PD1- Cells and MC2-TCR-T PD1- There was no significant difference in the proportion of PD-1-PE stained cell populations. Fig.24 B.

[0421] The results showed that (Cas9-mRNA+PD-1-sgRNA)-LNP can safely and effectively knock out the PD-1 gene of TCR-T cells themselves, 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 PD1- and MC2-TCR-T PD1- Each group was incubated with PD-1-PE, and the proportion of PD-1-PE stained cells was detected by flow cytometry. Fig.24 A is the flow cytometry result of PD-1-PE staining. Fig.24 B is the PD-1 staining detected by three independent repeated experiments. Vector is PBMC transfected 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 PBMC with pHR_LV-NS1-MC2-TCR HM LNP knockout PD-1; PDL1-MC2-TCR-T PD1- Transfection of PBMC with pHR_LV-NS1-PDL1-MC2-TCR HM Then LNP knocks out PD-1.

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

[0424] 4.1PDL1-MC2-TCR-T PD1- Cells expressing TCR HM and binding to pMHC at higher levels than MC2-TCR-T

[0425] To investigate the relationship between PDL1-MC2-TCR-T PD1- Specific TCR on cells HM After preparing each group of TCR-T cells, flow cytometry was used to detect the proportion of CD8-APC and MAGE-C2-Tetramer-PE double-stained cell populations. Fig.25 A and Fig.25 B, flow cytometry graph with MAGE-C2-Tetramer-PE on the horizontal axis and CD8-APC, PDL1-MC2-TCR-T on the vertical axis PD1- The proportion of double-stained cell populations was higher in the two groups, with a flow cytometry result of 36.8%. In the other three groups, Vector was 0.022%, MC2-TCR-T was 31.8%, and MC2-TCR-T was 31.8%. PD1 34.2%. PDL1-MC2-TCR-T PD1- The proportion of double-stained cell populations was significantly higher than that of the other two groups of TCR-T cells and Vector group cells, while MC2-TCR-T PD1 The proportion of double positive cell population was slightly higher than that of MC2-TCR-T, but it was not significant.

[0426] The results showed that PD-1 knockout restored and improved the expression of PDL1-MC2-TCR-T PD1- TCR on cells HM The expression of MC2 antigen epitopes or binding ability was higher than that of MC2-TCR-T. For MC2-TCR-T without co-expression of PD-L1, PD-1 gene knockout did not affect TCR HM expression or function, or even improved.

[0427] TCR-T cells were prepared and incubated with antibodies, and MAGE-C2-Tetramer-PE and CD8-APC stained cells were detected by flow cytometry. Fig.25 A is the flow cytometry results of MAGE-C2-Tetramer-PE and CD8-APC staining. Fig.25B is the TCR expression efficiency detected by three independent repeated experiments (i.e., the proportion of MAGE-C2-Tetramer-PE and CD8-APC double-stained cell population detected by flow cytometry). Vector is PBMC transfected with 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 PBMC with pHR_LV-NS1-MC2-TCR HM LNP knockout PD-1; PDL1-MC2-TCR-T PD1 Transfection of PBMC with pHR_LV-NS1-PDL1-MC2-TCR HM Then LNP knocks out PD-1.

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

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

[0430] Results Fig.26 A and Fig.26 B, PDL1-MC2-TCR-T PD1- The percentage of double-stained cell populations in the Vector group was significantly higher than that in the other groups. The results of a flow cytometric assay showed that the percentage of MAGE-C2-Tetramer-PE / PDL1-APC double-stained cell populations was 0.021% in the Vector group, 0.068% in the MC2-TCR-T group, and 0.1% in the MC2-TCR-T group. PD1 0.21%, PDL1-MC2-TCR-T PD1- In addition, MC2-TCR-T and MC2-TCR-T PD1- The lower right quadrant of the flow cytometer graph is the MAGE-C2-Tetramer-PE stained cell population, which accounts for about 50%. PD1- With MC2-TCR-T (or MC2-TCR-T PD1 ) have similar TCR HM Expression level, and better PD-L1 expression.

[0431] The results showed that PDL1-MC2-TCR-T cells with PD-1 knockout PD1-Cells can simultaneously express high levels of TCR HM and PD-L1.

[0432] TCR-T cells were prepared and incubated with antibodies, and MAGE-C2-Tetramer-PE and PDL1-APC stained cells were detected by flow cytometry. Fig.26 A is the flow cytometry results of MAGE-C2-Tetramer-PE and PDL1-APC staining. Fig.26 B is the PD-L1 expression efficiency detected by three independent repeated experiments (i.e., the proportion of MAGE-C2-Tetramer-PE and PDL1-APC double-stained cell population detected by flow cytometry). Vector is PBMC transfected with 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 PBMC with pHR_LV-NS1-MC2-TCR HM LNP knockout PD-1; PDL1-MC2-TCR-T PD1- Transfection of PBMC with pHR_LV-NS1-PDL1-MC2-TCR HM Then LNP knocks out PD-1.

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

[0434] To understand the impact of PD-1 removal, PDL1-MC2-TCR-T PD1- The ability of cells to produce cytokines when stimulated by MC2 antigen peptides. In this example, PDL1-MC2-TCR-T, PDL1-MC2-TCR-T PD1- The three groups of TCR-T cells, MC2-TCR-T and T2 loaded with control peptide, were respectively C and T2 loaded with MC2 peptide MC2 Co-culture, analyze and compare the intracellular release of IFN-γ and the secretion level of TNF-α. First, incubate with CD8-APC antibody, then use intracellular factor staining method to mark intracellular IFN-γ, and detect the proportion of CD8-APC and IFN-γ-FITC double-stained cells by flow cytometry. The flow cytometry test results are as follows Fig. 27 A and Fig. 27 B shows that the horizontal axis is CD8-APC, the vertical axis is IFN-γ-FITC, and the three groups of TCR-T cells and T2 CWhen co-incubated, the proportions of CD8-APC / IFN-γ-FITC double-stained cell populations were low, at 0.3%, 0.71%, and 0.98%, respectively; MC2 After co-incubation, PDL1-MC2-TCR-T PD1- The proportion of double-stained cell populations was 10.3% in PDL1-MC2-TCR-T, 1.19% in PDL1-MC2-TCR-T, and 24% in MC2-TCR-T. PD1- Affected by T2 MC2 After stimulation with antigenic peptide, the proportion of double-stained cell population was significantly higher than that of PDL1-MC2-TCR-T 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 set up with the above three groups of TCR-T, namely, T2 C and T2 MC2 After co-culture, the cell supernatant was taken for Elisa incubation and color development. Fig. 27 C, PDL1-MC2-TCR-T PD1- At T2 MC2 After antigen stimulation, the secretion level of TNF-α was significantly lower than that of MC2-TCR-T / T2 MC2 group, but significantly higher than PDL1-MC2-TCR-T / T2 C Group.

[0436] The results showed that compared with PDL1-MC2-TC-T, PDL1-MC2-TCR-T PD1- The specific release of IFN-γ and secretion of TNF-α under the stimulation of MC2 antigen peptide were significantly improved. PD1- The ability to secrete cytokines was greatly enhanced. PD1- The cytokine secretion level is still significantly lower than that of MC2-TCR-T, but this example speculates that the reaction with peptide-loaded T2 cells at this time can only represent the PDL1-MC2-TCR-T PD1- For the specific response ability of MC2 antigen, PDL1-MC2-TCR-T PD1- The biological function of co-expressed PD-L1 needs further verification by cell experiments.

[0437] The three TCR-T cells were compared with the negative control T2 CCells (T2 cells loaded with control peptide) and experimental group T2 MC2 After incubation with cells (T2 cells loaded with MC2 peptide), the CD8+ cell population that produces IFN-γ in the cells is labeled with antibodies. First, incubate with CD8-APC antibody, and then incubate with IFN-γ-FITC by intracellular factor staining method. Flow cytometry was used to detect the CD8-APC and IFN-γ-FITC stained cells. CD8-APC and IFN-γ-FITC double-stained cells represent the functional level of IFN-γ produced by the CD8+ cell population. Elisa method was used to detect the content of TNF-α in the supernatant of co-cultured cells. Fig. 27 A is the flow cytometry results of CD8-APC and IFN-γ-FITC staining. Fig. 27 B is the percentage of CD8+ cells producing IFN-γ-FITC (i.e., the percentage of cells doubly stained with IFN-γ-FITC and CD8-APC) detected in three independent repeated experiments. Fig. 27 C is the secretion of TNF-α detected by three independent repeated experiments. MC2-TCR-T is PBMC transfected with pHR_LV-NS1-PDL1-MC2-TCR HM ;MC2-TCR-T PD1- Transfection of PBMC with pHR_LV-NS1-MC2-TCR HM LNP knockout PD-1; PDL1-MC2-TCR-T PD1- Transfection of PBMC with pHR_LV-NS1-PDL1-MC2-TCR HM LNP knocks out PD-1. 2C Cells: T2 cells were incubated with control peptide (10 μM) for 2 h; T2 MC2 Cells: T2 cells were incubated with MC2 peptide (10 μM) for 2 h.

[0438] 6. Successful construction and simultaneous expression of MAGE-C2 336–344 / HLA-A02 (pMHC) and PD-1 tumor target cell MC2-A02-K562 PD1+

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

[0440] To validate the PDL1-MC2-TCR-T PD1- In this example, the constructed MC2-A02-K562 cell line was transfected to express the HLA-A02 restricted MAGE-C2 336–344PD-1 was further expressed on the basis of the antigen peptide. The artificially synthesized PD-1 gene sequence with NotI / SalI restriction sites and the expression vector pHR_LV-NS1 were double-digested with NotI / SalI to obtain the target gene fragment PD-1 (888 bp) and the pHR_LV-NS1 linear restriction fragment (8.9 Kb). After gel recovery and purification, agarose gel electrophoresis was performed. Fig.28 A and Fig.28 B, the band positions are consistent with expectations. After the above two were ligated, the recombinant expression plasmid pHR_LV-NS1-PD-1 was obtained. After transformation, 8 single colonies were selected for amplification and shaking. Then, PCR was performed on the 8 groups of bacterial liquids. After agarose gel electrophoresis, the 8 groups of PCR amplification products all obtained the expected 438bp band, as shown in Figure 1. Fig.28 C. Select one of the plasmid bacterial solutions for amplification and plasmid extraction and double enzyme digestion identification to obtain the expected bands, such as Fig.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 is shown in Fig.28 E.

[0441] like Fig.28 A is the agarose gel electrophoresis of NotI / SalI double digestion products: Lane M is DNAMarker D200; Lane 1 is the PD-1insert double digestion product, the target gene PD-1 is about 880bp. Fig.28 B is the agarose gel electrophoresis of the double digestion product of pHR_LV-NS1 NotI / SalI: Lane M is DNA Marker 1Kb; Lane 1 is the double digestion linear fragment of pHR_LV-NS1 (about 8.9Kb). Fig.28 C is the electrophoresis of pHR_LV-NS1-PD-1 PCR product: Lane M is DNAMarker D2000, and lanes 1 to 8 are PCR amplification products, about 438 bp. Fig.28 D is the verification of the double restriction enzyme digestion of the recombinant expression vector NotI / SalI: Lane M1 is DNA Marker 1Kb; Lane 1 is the double restriction enzyme digestion product of pHR_LV-NS1-PD-1; Lane M2 is DNA Marker D2000. Fig.28 D is the pHR_LV-NS1-PD-1 plasmid map.

[0442] 6.2MC2-A02-K562 PD1+ High expression efficiency of cell pMHC and PD-1

[0443] MC2-A02-K562 was obtained by transfecting MC2-A02-K562 with the recombinant plasmid pHR_LV-NS1-PD-1. PD1+ Cells were stained with HLA-A-PE and PD-1-PE by flow cytometry to clarify their expression. Fig.29 As shown, in terms of MC2 antigen expression, MC2-A02-K562 and MC2-A02-K562 PD1+ All cell lines significantly expressed HLA-A02 restricted MAGE-C2 336–344 Antigen, HLA-A-PE staining cell population ratio is more than 99%, compared with Vector-K562, the difference is statistically significant. PD1+ The proportion of PD-1-PE stained cell population in the cell line can reach 99%, which is significantly different from Vector-K562 and MC2-A02-K562. However, the proportion of PD-1-PE stained cell population in Vector-K562 and MC2-A02-K562 is lower, and there is no statistical difference between the two.

[0444] The results show that MC2-A02-K562 PD1+ Cells can simultaneously express high levels of HLA-A02-restricted MAGE-C2 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 of cells in each group. Fig.29 A is the result of HLA-A-PE staining flow cytometry. Fig.29 B is the flow cytometry result of PD-1-PE stained cells. Fig.29 C is the expression of HLA-A and PD-1 detected by three independent repeated experiments. Vector is K562 transfected with empty vector pHR_LV-NS1; MC2-A02-K562 is K562 transfected with pHR_LV-NS1-MC2-A02; MC2-A02-K562 PD1+ MC2-A02-K562 was transfected with pHR_LV-NS1-PD-1.

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

[0447] 7.1 and tumor target cells MC2-A02-K562 PD1+ When co-incubated with PDL1-MC2-TCR-T PD1-The proportion of cells producing IFN-γ is higher

[0448] As mentioned above, with T2 MC2 After the reaction, PDL1-MC2-TCR-T PD1- If there is a CD8+ cell population with much higher specificity of producing IFN-γ than PDL1-MC2-TCR-T, then PDL1-MC2-TCR-T PD1- Target tumor cells MC2-A02-K562 PD1+ After co-culture, what is the proportion of CD8+ cells producing IFN-γ? In this example, CT, MC2-TCR-T, MC2-TCR-T PD1- , PDL1-MC2-TCR-T and PDL1-MC2-TCR-T PD1- Five groups, respectively with MC2-A02-K562 and MC2-A02-K562 PD1+ After 24 h of co-culture, flow cytometry was used to detect the staining of CD8-APC and IFN-γ-FITC (ICS). Fig.30 The horizontal axis of the flow cytometry graph is CD8-APC, and the vertical axis is IFN-γ-FITC. PD1- Target tumor cells MC2-A02-K562 PD1+ The proportion of CD8+ cells producing IFN-γ in the co-culture group (i.e., CD8-APC and IFN-γ-FITC double-stained cells) was the highest, with a flow cytometry result of 14.1%, which was significantly higher than that in the co-culture group with MC2-A02-K562 and significantly higher than that in the co-culture group with other TCR-T cells and MC2-A02-K562 cells. PD1+ Co-culture group: PDL1-MC2-TCR-T cells with either MC2-A02-K562 or MC2-A02-K562 PD1+ There was no obvious IFN-γ-producing CD8+ cell population in co-culture, and the proportion of double-stained cell population was very low, with no significant difference compared with the CT group; compared with MC2-TCR-T, MC2-TCR-T PD1- and target cells MC2-A02-K562 PD1+ After co-culture, the proportion of I double-stained cell population increased significantly and was higher than that of the co-culture group with MC2-A02-K562; after MC2-TCR-T was co-cultured with the two target cells, there was no significant difference in the proportion of double-stained cell population.

[0449] The results showed that PDL1-MC2-TCR-T PD1- In the tumor target cell MC2-A02-K562 expressing PD-1 PD1+Under the stimulation of , the highest proportion of IFN-γ stained CD8+ cell population was produced, which means that after co-expression of PD-L1 and knockout of PD-1, more TCR-T cell individuals produced the release of cytokine IFN-γ. In addition, the primary T cell CT group could not respond specifically to tumor target cells; PDL1-MC2-TCR-T without knockout of PD-1 could not be stimulated by tumor target cells to produce cytokine IFN-γ; compared with MC2-TCR-T, MC2-TCR-T PD1- Knockout of PD-1 itself improved its ability to produce IFN-γ to a certain extent.

[0450] Set CT, MC2-TCR-T, MC2-TCR-TPD-1-, PDL1-MC2-TCR-T and PDL1-MC2-TCR-T PD1- Five groups of TCR-T, respectively interacting with tumor target cells MC2-A02-K562 and MC2-A02-K562 PD1+ After 24 hours of co-culture, the cells were first incubated with CD8-APC antibody, and then with IFN-γ-FITC antibody by intracellular factor staining. Flow cytometry was used to detect the percentage of cells with double staining of CD8-APC and IFN-γ-FITC, which represented the functional level of IFN-γ produced by CD8+ cell population. Fig.30 A is the flow cytometry results of CD8-APC and IFN-γ-FITC staining. Fig.30 B is the percentage of CD8-APC and IFN-γ-FITC double-stained cell populations detected by three independent repeated experiments. CT is the addition of transfection reagent to PBMC culture medium; MC2-TCR-T is PBMC transfected with pHR_LV-NS1-MC2-TCR HM ;MC2-TCR-T PD1- Transfection of PBMC with pHR_LV-NS1-MC2-TCR HM LNP knocks out PD-1; PDL1-MC2-TCR-T is PBMC transfected with pHR_LV-NS1-PDL1-MC2-TCR HM ;PDL1-MC2-TCR-T PD1- Transfection of PBMC with pHR_LV-NS1-PDL1-MC2-TCR HM LNP knocks out PD-1. MC2-A02-K562 is K562 transfected with pHR_LV-NS1-MC2-A02; MC2-A02-K562 PD1+ MC2-A02-K562 cells were transfected with pHR_LV-NS1-PD-1. n=3; *p<0.05; **p<0.01; ***p<0.001.

[0451] 7.2 Tumor Target Cells MC2-A02-K562 PD1+ Co-incubation of PDL1-MC2-TCR-T PD1- Higher secretion of TNF-α

[0452] To detect tumor target cells stimulate PDL1-MC2-TCR-T PD1- The secretion of TNF-α in cells, this example also sets CT, MC2-TCR-T, MC2-TCR-T PD1- , PDL1-MC2-TCR-T and PDL1-MC2-TCR-T PD1- Five groups, respectively with MC2-A02-K562 and MC2-A02-K562 PD1+ The cells were co-cultured for 24 h at a 1:1 effector-target ratio, and the cell supernatant was detected by TNF-α Elisa kit. Fig.31 ,PDL1-MC2-TCR-T PD1- Target tumor cells MC2-A02-K562 PD1+ The secretion of TNF-α in the co-culture group was significantly higher than that of other TCR-T cells and MC2-A02-K562 cells. PD1+ The co-culture group was also significantly higher than the PDL1-MC2-TCR-T PD1- Co-cultured with MC2-A02-K562; PDL1-MC2-TCR-T PD1- The secretion of TNF-α in the co-culture group with MC2-A02-K562 was also significantly higher than that in the other incubation groups; PD1+ After co-culture, the secretion of TNF-α was very low, only significantly higher than that of the CT group. Compared with MC2-TCR-T, MC2-TCR-T PD1- and target cells MC2-A02-K562 PD1+ After co-culture with MC2-A02-K562, the secretion of TNF-α was significantly increased; there was no significant difference in the secretion of TNF-α after co-culture of 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 PDL1-MC2-TCR-T PD1- In PD-1 positive tumor target cells MC2-A02-K562 PD1+ Under the stimulation of , the secretion of TNF-α is the highest, and it has a higher effect on target cells without PD-1 than MC2-TCR-T and MC2-TCR-T PD1-Therefore, the co-expressed PD-L1 did not affect the TNF-α secretion function of TCR-T, while the knockout of PD-1 caused the PDL1-MC2-TCR-T PD1- There is a higher secretion of TNF-α. PDL1-MC2-TCR-T cells without PD-1 knockout have poorer function of secreting TNF-α when stimulated by tumor target cells; compared with MC2-TCR-T cells, MC2-TCR-T cells PD1- The knockout of PD-1 itself also improved its ability to secrete TNF-α to a certain extent.

[0454] Fig.31 In, set CT, MC2-TCR-T, MC2-TCR-T PD1- , PDL1-MC2-TCR-T and PDL1-MC2-TCR-T PD1- Five groups of TCR-T, respectively interacting with tumor target cells MC2-A02-K562 and MC2-A02-K562 PD1+ After 24 hours of co-culture, the TNF-α content in the cell supernatant was detected by Elisa. The experiment was repeated three times. CT is PBMC culture medium with transfection reagent added; MC2-TCR-T is PBMC transfected with pHR_LV-NS1-MC2-TCR HM ;MC2-TCR-T PD1- Transfection of PBMC with pHR_LV-NS1-MC2-TCR HM LNP knocks out PD-1; PDL1-MC2-TCR-T is PBMC transfected with pHR_LV-NS1-PDL1-MC2-TCR HM ;PDL1-MC2-TCR-T PD1- Transfection of PBMC with pHR_LV-NS1-PDL1-MC2-TCR HM LNP knocks out PD-1. MC2-A02-K562 is K562 transfected with pHR_LV-NS1-MC2-A02; MC2-A02-K562 PD1+ MC2-A02-K562 was transfected with pHR_LV-NS1-PD-1.

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

[0456] The combination of PD-1 and PD-L1 can negatively regulate T cell proliferation. So, can the PD-L1 co-expressed on TCR-T also have the same inhibitory effect on PD-1-positive tumor cells? In order to verify this hypothesis, this example uses human acute T lymphoblastic leukemia cells Jurkat to construct TCR-T: PDL1-MC2-TCR-Jurkat. The previous text example verified that Jurkat does not express PD-1 in the normal state, and the engineered co-expression of PD-L1 and MC2-TCR in Jurkat cells should not trigger TCR inhibition associated with PD-1 activation, and can be used as a natural PD-1 negative T cell model. Therefore, this example uses Jurkat cells as a parallel TCR-T model to confirm the enhancing effect of exogenous PD-L1 on the inhibition of target cells. Using MC2-TCR-Jurkat that does not express PD-L1 as a control, it was co-expressed with tumor target cells MC2-A02-K562, respectively. PD1+ After 0h, 24h, and 48h of co-culture, HLA-A2-PE was used to target MC2-A02-K562 cells. PD1+ The number of HLA-A2-PE labeled target cells was detected by flow cytometry, and the inhibition rate of PDL1-MC2-TCR-Jurkat on target cells was calculated. Fig.32 As shown in 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 group, the percentage of target cells stained with HLA-A2-PE was 47.3% and 47.0%, respectively; after 24 hours, the percentage of target cells decreased to 37.4% and 32.7%, respectively; after 48 hours, the percentage of target cells continued to decrease to 29.7% and 25.3%, respectively. Using the number of target cells labeled with HLA-A2-PE in the above flow cytometry test data, the target cell inhibition rate of each co-culture group at each time period was calculated. The inhibition rate of MC2-TCR-Jurkat on target cells MC2-A02-K562 was calculated. PD1+ The inhibition rate of PDL1-MC2-TCR-Jurkat on MC2-A02-K562 was 25.6% at 24h and 49.7% at 48h. PD1+ The inhibition rate of MC2-TCR-Jurkat on target cells was 30.3% at 24h and 61.4% at 48h. The above inhibition rate of MC2-TCR-Jurkat on target cells includes the background inhibition rate of all cell components in mixed cells on target cells. It is more scientific and reliable to use it as a control group to conduct statistical analysis on the inhibition rate of PDL1-MC2-TCR-Jurkat on target cells. The statistical results are as follows Fig.32B, The inhibitory rate of PDL1-MC2-TCR-Jurkat on target cells at 24 or 48 hours was significantly higher than that of MC2-TCR-Jurkat. PD1+ The inhibition rate at 48 hours was significantly higher than that at 24 hours.

[0457] The results showed that in addition to the background 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 as the duration of action increased.

[0458] MC2-TCR-Jurkat and PDL1-MC2-TCR-Jurkat were respectively expressed in tumor target cells MC2-A02-K562 PD1+ After 0 h, 24 h and 48 h of co-culture, the target cells were labeled with HLA-A2-PE, and the number of HLA-A2-PE labeled target cells and the proportion of the cell population were detected by flow cytometry. Fig.32 A is the flow cytometry results of HLA-A2-PE staining of tumor target cells at each time point. Fig.32 B shows the effects of MC2-TCR-Jurkat and PDL1-MC2-TCR-Jurka on tumor target cells MC2-A02-K562 detected by three independent repeated experiments. PD1+ MC2-TCR-Jurkat is Jurkat transfected with pHR_LV-NS1-MC2-TCR HM ; PDL1-MC2-TCR-Jurkat is Jurkat transfected with pHR_LV-NS1-PDL1-MC2-TCR HM .MC2-A02-K562 PD1+ MC2-A02-K562 was transfected with pHR_LV-NS1-PD-1.

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

[0460] To further validate the PDL1-MC2-TCR-T PD1- In vitro, three types of TCR-T cells were obtained, including MC2-TCR-T, MC2-TCR-T PD1- and PDL1-MC2-TCR-T PD1-PBMC cells treated with transfection reagent were used as control group CT, and CFSE-labeled tumor target cells MC2-A02-K562 and PD-1 positive tumor target cells MC2-A02-K562 were used as control group CT. PD1+ , co-cultured with different effector-to-target ratios (E:T), and detected the number of remaining target cells after different co-culture times. PD1+ For the single culture group, the results were expressed as killing efficiency.

[0461] The results are as follows Fig.33 As shown in A, after 72 hours of co-culture, when the effector-target ratio was 2:1, PDL1-MC2-TCR-T PD1- For MC2-A02-K562 or MC2-A02-K562 PD1+ The killing efficiency of PDL1-MC2-TCR-T PD1- For MC2-A02-K562 PD1+ The killing rate of MC2-TCR-T was higher than that of MC2-A02-K562, and the difference was significant. PD1- For MC2-A02-K562 or MC2-A02-K562 PD1+ The killing efficiency of PDL1-MC2-TCR-T was significantly higher than that of MC2-TCR-T group. PD1- The killing efficiency of MC2-A02-K562 was consistent with the trend when the effector-target ratio was 2:1, and was significantly higher than that of other co-culture groups. PD1+ The killing rate of MC2-TCR-T was also significantly higher than that of MC2-A02-K562. PD1- For MC2-A02-K562 or MC2-A02-K562 PD1+ The killing efficiency of PDL1-MC2-TCR-T was also significantly higher than that of MC2-TCR-T group. PD1- When the effector-target ratio was 2:1, the killing efficiency was significantly higher than that when the effector-target ratio was 1:1. PD1- The same is true for the CT group. After co-culture with different effector-target ratios and two tumor target cells, the killing rate produced by the CT group was significantly lower than that of other TCR-T groups, but there was no significant difference between the CT groups in different situations.

[0462] In order to have a more comprehensive understanding of PDL1-MC2-TCR-T PD1-In order to determine the in vitro killing efficiency and characteristics, this embodiment also sets two other time points: 48h and 24h. The killing efficiency results at 48h are as follows: Fig.33 As shown in B, when the effector-target ratio was 2:1, PDL1-MC2-TCR-T PD1- The killing efficiency of two target cells is similar to that of MC2-TCR-T PD1- No significant difference, MC2-TCR-T PD1- The killing efficiency of 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 of PDL1-MC2-TCR-T was higher than that of MC2-A02-K562. PD1- For MC2-A02-K562 PD1+ The killing efficiency of MC2-TCR-T PD1- , but PDL1-MC2-TCR-T PD1- The killing efficiency of MC2-A02-K562 is similar to that of MC2-TCR-T PD1- No significant difference, MC2-TCR-T PD1- The killing efficiency of both tumor cells was significantly higher than that of MC2-TCR-T. PD1- and PDL1-MC2-TCR-T PD1- The killing efficiency at an effect-target ratio of 2:1 was significantly higher than that at an effect-target ratio of 1:1.

[0463] The 24h kill rate results are as follows Fig.33 C. At an effector-target ratio of 2:1, PDL1-MC2-TCR-T PD1- The killing efficiency of 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 there was no significant difference. PD1- For MC2-A02-K562 PD1+ The killing efficiency of MC2-TCR-T PD1- No significant difference; MC2-TCR-T PD1- For MC2-A02-K562 PD1+The killing rate of MC2-TCR-T cells was significantly higher than that of MC2-TCR-T cells. However, after co-culture with MC2-A02-K562 cells, the killing rate of MC2-TCR-T cells and MC2-TCR-T cells was significantly higher than that of MC2-TCR-T cells. PD1- and PDL1-MC2-TCR-T PD1- There was no significant difference in the killing efficiency of MC2-TCR-T PD1- and PDL1-MC2-TCR-T PD1- For MC2-A02-K562 PD1+ The killing efficiency is slightly higher than that of MC2-TCR-T.

[0464] The results showed that PDL1-MC2-TCR-T PD1- The killing effect on PD-1 positive tumor target cells is higher; PDL1-MC2-TCR-T PD1- While MC2-TCR can target and kill MC2-specific tumor cells, its co-expressed PD-L1 can use TCR-T as a carrier to accurately bind to PD-1 of tumor target cells and produce proliferation inhibition, which has a synergistic effect on TCR-T's specific cell killing. PD1- For non-PD-1 positive tumor cells but with MC2 antigen epitopes, there is no less than MC2-TCR-T PD1- and MC2-TCR-T; while MC2-TCR-T PD1- Due to the knockout of endogenous PD-1, the killing rate of tumor cells is higher than that of MC2-TCR-T, especially for PD-1 positive tumor target cells. In addition, the difference in killing rate caused by setting different effector-target ratios can indicate that TCR-T can produce higher tumor killing power when the effector-target ratio is 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- , respectively, with CFSE-labeled tumor target cells MC2-A02-K562 and MC2-A02-K562 PD1+ When the effector-target ratio was 1:1 and 2:1, the number of CFSE-labeled tumor target cells was detected by flow cytometry after 72h, 48h and 24h of co-culture and the killing efficiency was calculated. The experiment was repeated three times. Fig.33 A is the killing efficiency of each group at 72 h; Fig.33 B is the killing efficiency of each group at 48 h; Fig.33 C is the killing efficiency of each group at 24 hours. CT is the PBMC culture medium with transfection reagent added; MC2-TCR-T is PBMC transfected with pHR_LV-NS1-MC2-TCR HM ;MC2-TCR-TPD1- PBMCs were transfected with pHR_LV-NS1-MC2-TCRHM and then LNP knocked out PD-1; PDL1-MC2-TCR-T PD1- Transfection of PBMC with pHR_LV-NS1-PDL1-MC2-TCR HM LNP knocks out PD-1. MC2-A02-K562 is K562 transfected with pHR_LV-NS1-MC2-A02; MC2-A02-K562 PD1+ MC2-A02-K562 was transfected with pHR_LV-NS1-PD-1. The effector-target ratio is the ratio of the number of effector cells TCR-T to tumor target cells.

[0466] 10. PDL1-MC2-TCR-T PD1- The killing efficiency of PD-1 positive tumor target cells increases with the extension of co-culture time

[0467] The previous article compared the efficiency of different types of TCR-T in killing tumor target cells within the same incubation time. PD1- The same method was used in this example to obtain the changes in the killing efficiency of tumor target cells with the co-incubation time, CT, MC2-TCR-T, MC2-TCR-T PD1- and PDL1-MC2-TCR-T PD1- Cells were co-cultured with a 2:1 effector-target ratio for optimal killing efficiency, and MC2-A02-K562 were labeled with CFSE. PD1+ Cells were analyzed and the number of CFSE-labeled cells was detected by flow cytometry after 24 hours, 48 ​​hours and 72 hours, and the killing efficiency at different times was analyzed and calculated. Fig.34 A is the result of a flow cytometry test, PDL1-MC2-TCR-T PD1- In the incubation group, MC2-A02-K562 labeled with CFSE PD1+ The number of cells decreased significantly with the extension of incubation time. PD1- The number of cells in the incubation group was less than that in other incubation groups at each time period. Fig.34 B shows that PDL1-MC2-TCR-T PD1- For MC2-A02-K562 PD1+ The killing efficiency of MC2-TCR-T was always on the rise from 24 hours to 72 hours, with an average killing efficiency of 95.52% at 72 hours, and was higher than that of other groups at each time point; PD1-The killing efficiency of the MC2-TCR-T group increased significantly from 24h to 48h, but decreased at 72h, but its killing efficiency was always higher than that of the MC2-TCR-T group; the killing efficiency of MC2-TCR-T changed slightly at the three time points, was basically the same from 24h to 48h, and increased slightly at 72h.

[0468] The results showed that within 72 hours, PDL1-MC2-TCR-T PD1- The killing efficiency of PD-1 positive tumor target cells increases with the extension of interaction time and always maintains a higher killing level. PD1- The killing efficiency of PD-1 positive tumor target cells is higher than that of MC2-TCR-T, and can maintain a certain degree of growth within a certain period of time. The killing efficiency of MC2-TCR-T basically maintains a certain level, and there is no obvious 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-target ratio of 2:1, after 24h, 48h, and 72h of co-culture, flow cytometry was used to detect the number of CFSE-labeled tumor target cells and calculate the killing efficiency. The yellow frame is the tumor target cell MC2-A02-K562 PD1+ Flow cytometry results of the single culture group. Fig.34 A is the flow cytometry results of CFSE-labeled cells. Fig.34 B is the CT, MC2-TCR-T, and MC2-TCR-T detected by three independent repeated experiments. PD1- and PDL1-MC2-TCR-T PD1- For MC2-A02-K562 PD1 CT: PBMC culture medium with transfection reagent; MC2-TCR-T: PBMC transfected with pHR_LV-NS1-MC2-TCR HM ;MC2-TCR-T PD1- PBMC transfected with pHR_LV-NS1-MC2-TCR HM LNP knockout PD-1; PDL1-MC2-TCR-T PD1- PBMC transfected with pHR_LV-NS1-PDL1-MC2-TCR HM LNP knocks out PD-1. MC2-A02-K562 PD1+ :MC2-A02-K562 was transfected with pHR_LV-NS1-PD-1.

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

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

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

[0473] 2. PDL1-MC2-TCR-T PD1- Cells were infused back into MC2-A02-K562 tumor cells PD1+ -NCG mouse model of Luc;

[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 had the strongest inhibitory effect on tumor burden in tumor-bearing NCG mice;

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

[0478] The specific contents are as follows:

[0479] 1. Experimental methods:

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

[0481] The pHR_LV-NS1-Luc recombinant vector with luciferase gene was used to transfect A02-K562 and MC2-A02-K562. PD1+ Cells (specific experimental steps are the same as in Example 1) were used to obtain the tumor-bearing Luc stably transfected cell line MC2-A02-K562 PD1+ -Luc.

[0482] 2. PDL1-MC2-TCR-T PD1- Cells were infused back into MC2-A02-K562 tumor cells PD1+ -Luc 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 PD1-(1M) group, MC2-TCR-T (5M) group, PDL1-MC2-TCR-T PD1- (5M) group: On day 0, each mouse was treated with MC2-A02-K562 PD1+ -Luc subcutaneously loaded with tumors, and different TCR-T cells were infused back into the groups every other day. Starting from Day 0, mice were imaged in vivo every 5 days to observe the tumor burden of the mice. At the end of the experiment, the mice were killed, the subcutaneous tumors were removed, photographed, weighed, and the volume was measured. Fig.35 As shown:

[0484] The constructed tumor target cells and each group of TCR-T cell lines were expanded and cultured. A uniform cell suspension was prepared for use. 1×10 6 MC2-A02-K562 / 100μL PD1+ -Luc was subcutaneously inoculated into the right rib of each group of NCG female mice, which was marked as 0 day. 24 hours later, 1×10 therapeutic TCR-T cell suspensions of each group were injected into the tail vein. 6 / 100μL or 5×10 6 / 100μL, live detection was performed every 5 days from 0 day until the last photo was taken on 25 days, then the mice were killed and the subcutaneous tumors were removed for measurement and recording.

[0485] According to the animal experiment schedule, the tumor-bearing cells were treated with MC2-A02-K562 PD1+ -Luc cells and each group of TCR-T cells for in vivo treatment were incubated at 37°C and 5% CO 2 Conditional amplification culture is used for future use.

[0486] 1) The NCG female mice used in the experiment were 5-6 weeks old and were fed adaptively in an SPF animal room 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 PD1- (1M) group, MC2-TCR-T (5M) group, PDL1-MC2-TCR-T PD1- (5M) group;

[0488] 3) Using MC2-A02-K562 PD1+ The cells were used for tumor-bearing operation. The hair of the last rib on the right side of the mouse was shaved and MC2-A02-K562 PD1+ -Luc cells were counted and resuspended in PBS, and 1 × 10 were injected subcutaneously into each mouse. 6 / 100μL, the day of tumor loading was recorded as day 0;

[0489] 4) After 24 hours of tumor loading, TCR-T cells of each group were resuspended in PBS and the infusion volume was 100 μL per mouse. 100 μL PBS was infused into the CT group and 1×10 6 MC2-TCR-T, PDL1-MC2-TCR-T PD1- (1M) Group return 1×10 6 PDL1-MC2-TCR-T PD1- MC2-TCR-T(5M) group received 5×10 6 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 draw 200 μL with a 1 mL syringe;

[0491] ② Fix the mouse on the injection instrument to display the location of the tail vein, and wipe the injection site of the mouse tail with an alcohol cotton ball;

[0492] ③ After the syringe needle is inserted into the mouse's tail vein, 100 μL of TCR-T solution is pushed in at a constant speed, the needle is gently rotated to withdraw, and a sterile cotton ball is pressed on the injection site for 5 seconds. The mouse is returned to the cage, observed for about 20 minutes, and continued to be raised normally.

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

[0494] The first live imaging was started on day 0 after tumor loading, and the images were taken every 5 days until day 25, for a total of 6 times.

[0495] 1) Turn on the in vivo imaging instrument and preheat the instrument before the experiment;

[0496] 2) Mice were anesthetized with isoflurane gas, and the in vivo imaging tracer D-luciferin potassium salt was prepared in advance with sterile PBS to prepare a 15 mg / mL luciferin stock solution, mixed evenly, filtered with a 0.2 μm filter membrane for sterilization and used immediately, and injected intraperitoneally at a concentration of 150 mg / kg luciferin / body weight. For example, NCG mice weighing about 20 g were used for each mouse;

[0497] 3) The mouse tumor-bearing part was shaved for preparation. After the fluorescent substance was injected into the body, the light signal reached the strongest stable plateau about 10-15 minutes later. The mouse was placed on the imaging dark box platform small animal fixing device in a side-lying position to expose the tumor position. The door of the box was closed and the "bioluminescence mode" was selected. Each parameter was checked and the Acquire Sequence was clicked to start shooting. The tumor-bearing luminescence in the mouse body was shot without external light source to obtain the mouse live imaging image data;

[0498] 4) The experimental results were analyzed and statistically analyzed using Image and GraphPad Prism8.0 software;

[0499] 5) After the last photo was taken, the mice were killed, the subcutaneous tumors were dissected and removed, and the tumors were collected and photographed. The long diameter (L) and short diameter (W) of the tumors were measured using a caliper and calculated according to 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 comparisons 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 significant statistical difference; ***p<0.001 indicates an extremely significant statistical difference.

[0502] 2. Experimental results:

[0503] 1. PDL1-MC2-TCR-T PD1- The strongest inhibitory effect on tumor burden in NCG tumor-bearing mice

[0504] The previous experimental results have shown that MC2-TCR-T and PDL1-MC2-TCR-T PD1 In vitro, the tumor target cell MC2-A02-K562 PD1+ To investigate the in vivo inhibitory effect of TCR-T on tumor growth, we used MAGE-C2 expressing HLA-A*02:01 restriction. 336–344 -MC2-A02-K562 with ALKDVEERV antigen epitope and overexpression of PD1 PD1+ -Luc cells were used to establish a transplanted tumor model in NCG mice. PD1+ -Luc cells were implanted subcutaneously in the right rib of NCG mice (n=4, 1×10 6 Each animal was injected with TCR-T cells of different groups and doses through the tail vein 24 hours later. The day of subcutaneous tumor inoculation was designated as day 0. From day 0 onwards, fluorescent tracers were injected intraperitoneally every 5 days and the animals were anesthetized and photographed using a small animal live imaging device. Fig.36As shown, no obvious fluorescence signal appeared in the control group and each TCR-T transfusion group at 0-10 days; at 15 days, fluorescence signals appeared in all groups, with the highest in the MC2-TCR-T (1M) group; at 20 days, the fluorescence signal intensity of each group increased, with the MC2-TCR-T (1M) group being higher than the other groups, and the CT group being higher, and the MC2-TCR-T (5M) group and the PDL1-MC2-TCR-T PD1- The fluorescence signal of the (5M) group was weaker. At the end of the 25th day of the experiment, the intensity of the tumor load burst fluorescence signal in the CT group was the highest, and that in the MC2-TCR-T(1M) group was also higher. The intensity of the tumor load burst fluorescence signal in the MC2-TCR-T(5M) group and the PDL1-MC2-TCR-T PD1- The fluorescence signals of the (1M) group were similar, while those of the PDL1-MC2-TCR-T PD1- The (5M) group has the lowest signal volume.

[0505] Fig.36 In the MC2-A02-K562 PD1+ -Luc cells were subcutaneously divided into 1×10 6 1×10 TCR-T cells were injected into the right rib of NCG mice. 24 hours later, different TCR-T cells were injected into the tail vein: 100 μL PBS was injected into the CT group; 1×10 TCR-T cells were injected into the MC2-TCR-T (1M) group 6 MC2-TCR-T cells were infused; PDL1-MC2-TCR-T PD1- Group by 1×10 6 PDL1-MC2-TCR-T PD1- Cells; MC2-TCR-T (5M) group, 5×10 6 MC2-TCR-T cells were infused; PDL1-MC2-TCR-T PD1- (5M) group by 5×10 6 PDL1-MC2-TCR-T PD1- The day of transplantation was defined as day 0, and the fluorescent tracer was injected intraperitoneally at 150 mg / kg every 5 days from day 0 and the animals were anesthetized for in vivo imaging.

[0506] The tumor fluorescence signals of the above-mentioned groups of NCG tumor-bearing mice did not change significantly in the first 10 days. The total tumor burden of each group was detected at 15 days, but the difference between the groups was small and not significant. At 20 days, the tumor fluorescence intensity of each group increased significantly, with the MC2-TCR-T (1M) group being higher than the other groups, followed by the CT group, and the MC2-TCR-T (5M) group and the PDL1-MC2-TCR-T PD1-At the end of the 25th day of the experiment, the fluorescence intensity of the tumor load in the CT group was significantly higher than that in the other groups, and the fluorescence intensity of the tumor in the MC2-TCR-T(1M) group was significantly higher than that in the MC2-TCR-T(5M) group and the PDL1-MC2-TCR-T PD1- (5M) group. In addition, although the MC2-TCR-T(1M) group was higher than the PDL1-MC2-TCR-T PD1- The tumor fluorescence intensity of the (1M) group was not significant, among which PDL1-MC2-TCR-T PD1- The fluorescence signal intensity of the tumor in the (5M) group was the lowest and statistically significant. Fig.37 .

[0507] The results of in vivo imaging showed that the novel TCR-T cell PDL1-MC2-TCR-T PD1- The inhibitory effect on subcutaneous transplanted tumor growth was the strongest; PDL1-MC2-TCR-T PD1- The inhibitory effect on subcutaneous tumors increased with the increase of the reinfusion dose, and the tumor inhibition effect of the 5M group was better than that of the 1M group; PD1- After reinfusion, the tumor inhibition effect became more obvious as the duration of action increased. In addition, MC2-TCR-T also had an inhibitory effect on subcutaneous transplanted tumors in the mouse model, but both the final inhibitory effect and the long-term inhibitory effect 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 confirm that PDL1-MC2-TCR-T PD1- and MC2-TCR-T to MC2-A02-K562 PD1+ In order to investigate the inhibitory effect of transplanted tumor growth, in this example, the NCG mice that had completed the above 25-day in vivo imaging were killed and the subcutaneous tumors were dissected to measure and calculate the volume. Fig.38 A and Fig.38 As shown in B, compared with the CT group, PDL1-MC2-TCR-T PD1- In both the PDL1-MC2-TCR-T and 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 low-dose group (1×10 6PDL1-MC2-TCR-T PD1- The tumor volume of the (5M) group was significantly lower than that of the two dose groups of MC2-TCR-T and PDL1-MC2-TCR-T PD1- (1M) group, while PDL1-MC2-TCR-T PD1- The tumor volume of the (1M) group was lower than that of the MC2-TCR-T (1M) group, but not significantly. The tumor mass data also showed the same trend, such as Fig.38 C, The tumor mass in the CT group was significantly higher than that in the other groups; PDL1-MC2-TCR-T PD1- The tumor mass in the (5M) group was significantly lower than that in the PDL1-MC2-TCR-T PD1- (1M) group and MC2-TCR-T (5M) group.

[0510] The above results show that the specific targeting of HLA-A*02:01 restricted MAGE-C2 336–344 -ALKDVEERV antigen epitope TCR-T of two structures can inhibit the growth of transplanted tumors in vivo; the effect of specific TCR-T cells in vivo treatment is dose-dependent, and the inhibitory effect of the high-dose group is stronger than that of the low-dose group; PDL1-MC2-TCR-T PD1- On the basis of specific targeted killing of MAGE-C2 tumor cells, overexpressed PD-L1 protein can further inhibit the growth of tumor cells and has a stronger ability to inhibit tumors; in animal experiments, mice in the high and low dose groups did not show obvious adverse reactions or deaths during the experiment, indicating that PDL1-MC2-TCR-T PD1- The MC2-TCR-T infusion dose was 5×10 6 It is safe within the range of one / only.

[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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. TCR-T cells with killing and tumor suppression effects, characterized in that: The TCR-T cells are T cells that express TCRs that recognize tumor antigens, and 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.

2. The TCR-T cell with killing and tumor suppression efficacy according to claim 1, characterized in that: All or part of the TCR sequence is derived from one or more CD4 T cell clones in a tumor patient; or one or more CD8 T cell clones; or multiple CD4T and CD8 T cell clones.

3. The TCR-T cell with killing and tumor suppression efficacy according to claim 1, characterized in that: The PD-1 gene in the TCR-T cells is knocked out or inhibited.

4. TCR-T cells with killing and tumor suppression effects, characterized in that: The TCR-T cells stably express PD-L1, and the PD-1 gene in the TCR-T cells is knocked out or inhibited.

5. The TCR-T cell with killing and tumor suppressor efficacy according to any one of claims 1 to 4, characterized in that: The PD-L1 is endogenous PD-L1 or exogenous PD-L1.

6. Use of the TCR-T cell with killing and tumor suppressor efficacy as claimed in any one of claims 1 to 4 in the preparation of a drug for treating tumors.

7. Use of the TCR-T cells having killing and tumor suppressing effects as claimed in claim 5 in the preparation of drugs for treating tumors.

8. The use according to claim 6, characterized in that: The tumor is any one or more of lung cancer, lymphoma, leukemia, melanoma, intestinal cancer, liver cancer, gastric cancer, breast cancer, cervical cancer, ovarian cancer, kidney cancer, bladder cancer or esophageal cancer.

9. The use according to claim 7, characterized in that: The tumor is any one or more of lung cancer, lymphoma, leukemia, melanoma, intestinal cancer, liver cancer, gastric cancer, breast cancer, cervical cancer, ovarian cancer, kidney cancer, bladder cancer or esophageal cancer.

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