Preparation method and application of TCR-T cells

Abstract

The invention discloses a preparation method and application of TCR-T cells. The method for preparing the TCR-T cell disclosed by the invention comprises the following steps: connecting an intracellular structural domain of 41BB, a CD3 zeta signal domain and TCR protein to obtain a recombinant TCR; introducing the coding gene of the recombinant TCR into a T cell, and expressing the coding gene to obtain a TCR-T cell; wherein the recombinant TCR is as shown in SEQ ID No. 2. The TCR-T cell with higher killing ability is prepared by transforming the structure of the TCR, and the method for preparing the TCR-T cell and the transformed TCR show huge application potential in tumor treatment and have good application prospects.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of TCR-T cells in the field of biomedicine. Background Art

[0002] Currently, cancer treatment faces a series of obstacles. Many tumors may not show obvious symptoms in the early stages. Although the vast majority of patients receive standard treatment with existing clinical methods after diagnosis, including surgery, chemotherapy, radiotherapy, targeted therapy, and immune checkpoint inhibitors, the prognosis for most advanced tumors remains poor, with extremely low five-year survival rates. This is due to the fact that, on the one hand, tumor cells may develop resistance to chemotherapy, radiotherapy, and targeted therapy, making long-term successful treatment difficult. In addition, many cancer treatments can cause significant side effects and reduce the patient's immune function. On the other hand, there are significant differences between different types of tumors and even individual tumors within the same type, which increases the difficulty of customizing widely applicable and effective treatment plans.

[0003] Immune cell therapy technology is the fastest-growing sector in the biopharmaceutical field. In 2013, the journal Science Translational Medicine predicted that cell therapy would become the "third pillar of future medicine." Among these, T-cell immunotherapy is considered the most promising treatment for curing tumors. T-cell immunotherapy involves collecting T cells from the patient (autologous) or a donor (allogeneic), culturing them in vitro for a short period of time to enhance immune function, and then infusing these active anti-tumor T cells into the patient's body. This direct or indirect stimulation of the immune system kills tumor cells. The U.S. Food and Drug Administration (FDA) approved the first CAR-T therapy product in 2017, officially announcing the industrialization of T-cell immunotherapy and ushering the field into an era of rapid development.

[0004] T cell immunotherapy is currently divided into two main categories: chimeric antigen receptor (CAR) T cells (CAR-T) and T cell receptor (TCR) T cells (TCR-T). CAR-T therapy has achieved promising results in the clinical treatment of hematologic malignancies. T cells in peripheral blood are genetically engineered to express CARs, enabling rapid proliferation and specific recognition of target antigens, exerting their anti-tumor effects. However, adverse reactions and post-treatment relapses of CAR-T therapy limit its applicability. Furthermore, CAR-T cells can only recognize target cell membrane antigens. Therefore, most CAR-Ts target tumor-associated antigens, which often leads to the possibility of "off-target" effects and significantly limits the development of CAR-T technology. Unlike CAR-T therapy, TCR-T therapy offers many advantages, including a wider target range. Tumor-associated antigens can be presented as peptides on the major histocompatibility complex (MHC) on the cell surface. Therefore, all antigens that can be presented by the MHC can be recognized by TCR-T cells. In addition, the affinity required for TCR-T activation is lower than that of CAR-T, and therefore it is less dependent on antigen expression levels.

[0005] At present, TCR-T cell immunotherapy has undergone multiple updates and iterations. The first generation of TCR-T therapy is a T cell subset directly isolated from the patient, which can specifically recognize tumor antigens and then be re-infused into the patient for treatment after in vitro expansion. However, due to the extremely small number of such T cell clones and large individual differences, this method is difficult to industrialize. The second generation of TCR-T cells is obtained by obtaining TCR gene sequences that specifically recognize tumor-associated antigens and then introducing them into the patient's peripheral T cells. Therefore, this method makes the industrialization of TCR-T therapy possible. The third generation of TCR-T therapy optimizes the affinity of TCR, enabling it to better recognize tumor antigens, and then introduce them into the patient's T cells, thereby improving the overall drug capacity of TCR-T therapy.

[0006] More and more studies have shown that after specific T cells are infused back into the patient's body, their ability to expand and persist in solid tumors is crucial. Therefore, improving the expansion and survival of specific T cells in the tumor microenvironment is the key to improving the overall drug efficacy of TCR-T therapy. Currently, the vast majority of research on TCR-T therapy is limited to enhancing TCR-T specificity. How to enhance the function and persistence of TCR-T in solid tumors is crucial to improving the efficacy of TCR-T. Summary of the Invention

[0007] The technical problem to be solved by the present invention is how to improve the activity of TCR-T cells.

[0008] To solve the above technical problems, the present invention first provides a method for preparing TCR-T cells, which comprises: connecting the intracellular domain of 41BB, the CD3ζ signaling domain and the TCR protein to obtain a recombinant TCR; introducing the encoding gene of the recombinant TCR into T cells and expressing the encoding gene to obtain TCR-T cells.

[0009] The recombinant TCR can be obtained by connecting the intracellular domain of 41BB, the CD3ζ signaling domain and the two chains of the TCR protein. As long as the functions of each part are not affected, the parts can be connected by a connecting peptide (such as P2A) or directly connected.

[0010] The order of connection of the parts can be determined according to the actual situation without affecting the functions of the parts in the recombinant TCR. In one embodiment of the present invention, the recombinant TCR is obtained by sequentially connecting the TCR-α chain, the connecting peptide, the TCR-β chain, the intracellular domain of 41BB, and the CD3ζ signaling domain.

[0011] The connection refers to connecting the last amino acid of the corresponding amino acid sequence with the first amino acid of the subsequent amino acid sequence through a peptide bond.

[0012] The T cells are isolated T cells.

[0013] In the above method, the intracellular domain of 41BB may be the following A1), A2) or A3):

[0014] A1) the amino acid sequence is the protein shown in positions 596-637 of SEQ ID No. 2;

[0015] A2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 2 in the sequence listing, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0016] A3) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2);

[0017] The CD3ζ signaling domain may be as follows B1), B2) or B3):

[0018] B1) the amino acid sequence of the protein shown in positions 638-749 of SEQ ID No. 2;

[0019] B2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 2 in the sequence listing, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0020] B3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of B1) or B2).

[0021] The protein in A2) is a protein that has 75% or greater identity to the amino acid sequence of the protein represented by positions 596-637 of SEQ ID No. 2 and has the same function. The protein in B2) is a protein that has 75% or greater identity to the amino acid sequence of the protein represented by positions 638-749 of SEQ ID No. 2 and has the same function. Identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, a search can be performed to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained. The said 75% or more identity is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0022] The tag described in A3) and B3) can be a polypeptide or protein fused with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag can be a Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, MBP tag, HA tag, GST tag, and / or SUMO tag, etc.

[0023] In the gene encoding the recombinant TCR described in the above method, the gene encoding the intracellular domain of 41BB may be the following a11) or a12) or a13):

[0024] a11) the DNA molecule shown in positions 1786 to 1911 of SEQ ID No. 1 in the sequence listing;

[0025] a12) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in a11) and encodes the intracellular domain of 41BB;

[0026] a13) a DNA molecule that hybridizes under stringent conditions with the nucleotide sequence defined in a11) or a12) and encodes the intracellular domain of 41BB;

[0027] The gene encoding the CD3ζ signaling domain may be the following b11) or b12) or b13):

[0028] b11) the DNA molecule shown in positions 1912-2247 of SEQ ID No. 1 in the sequence listing;

[0029] b12) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in b11) and encodes the CD3ζ signaling domain;

[0030] b13) A DNA molecule that hybridizes under stringent conditions with the nucleotide sequence defined in b11) or b12) and encodes the CD3ζ signaling domain.

[0031] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0032] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the intracellular domain of 41BB and the CD3ζ signaling domain of the present invention. Identity can be evaluated with the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0033] In the above method, the stringent conditions may be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 and 1mM EDTA, and washing at 50°C in 2×SSC, 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and washing at 50°C in 1×SSC, 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and washing at 50°C in 0.5×SSC, 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and washing at 50°C in 0.1×SSC, 0.1% The membrane can be rinsed in SDS; alternatively, hybridization can be performed at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and then rinsed in 0.1×SSC, 0.1% SDS at 65°C; alternatively, hybridization can be performed at 65°C in a solution of 6×SSC, 0.5% SDS, followed by washing the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS; alternatively, hybridization and washing the membrane twice at 68°C in a solution of 2×SSC, 0.1% SDS, for 5 minutes each, and then hybridization and washing the membrane twice at 68°C in a solution of 0.5×SSC, 0.1% SDS, for 15 minutes each; alternatively, hybridization and washing the membrane in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS, at 65°C.

[0034] The aforementioned 75% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0035] In the above method, the encoding gene of the recombinant TCR can be introduced into T cells via an expression vector.

[0036] The expression vector may be a plasmid or a virus. The virus may be a lentivirus.

[0037] In the above method, the TCR protein may be a specific TCR protein for tumor cells or virus-infected cells.

[0038] Furthermore, the tumor cells may be human liver cancer cells; and the virus-infected cells may be human liver cells infected with hepatitis B virus.

[0039] In one embodiment of the present invention, the human liver cancer cells are HepG2.2.15 (A1101+) cells.

[0040] In the above method, the TCR protein may be a specific TCR protein for the HLA-A1101 restricted hepatitis B virus HBc141-151 epitope peptide.

[0041] Furthermore, the TCR-α chain of the TCR protein may be as shown in positions 1-268 of SEQ ID No. 2, and the TCR-β chain may be as shown in positions 291-595 of SEQ ID No. 2.

[0042] In the above method, the recombinant TCR may be as shown in SEQ ID No.2.

[0043] The TCR-T cells obtained by the method for preparing TCR-T cells also fall within the scope of protection of the present invention.

[0044] The recombinant TCR also falls within the scope of protection of the present invention.

[0045] The use of TCR-T cells obtained by the method for preparing TCR-T cells in the preparation of tumor treatment products or in the treatment of tumors also falls within the scope of protection of the present invention.

[0046] The use of TCR-T cells obtained by the method for preparing TCR-T cells in the preparation of products for treating hepatitis B, or in the treatment of hepatitis B, also falls within the scope of protection of the present invention.

[0047] The use of the recombinant TCR in preparing a product for treating tumors, or in treating tumors, or in preparing a product for enhancing the killing ability of T cells, or in enhancing the killing ability of T cells, or in preparing a product for treating hepatitis B, or in treating hepatitis B, also falls within the scope of protection of the present invention.

[0048] The present invention prepares TCR-T cells with higher killing ability by modifying the structure of TCR. The method for preparing TCR-T cells and the modified TCR of the present invention show great application potential in tumor treatment and have good application prospects.

[0049] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic diagram of the sequence design of TCR-CAR2.

[0051] Figure 2The results of flow cytometry detection of TCR-CAR2 expression are shown.

[0052] Figure 3 The following table shows the TCR downstream signaling detection results of TCR-CAR2 expressing cells. The vertical axis shows the ratio of firefly luciferase expression to Renilla luciferase expression.

[0053] Figure 4 Efficiency detection of human T cells expressing TCR-CAR2.

[0054] Figure 5 This is the killing efficiency test result of TCR-CAR2-T. DETAILED DESCRIPTION

[0055] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. The quantitative tests in the following examples were all repeated at least three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0056] Example 1. Construction and application of novel TCR-T cells

[0057] 1. Construction of a novel TCR-T gene (TCR-CAR2) and recombinant vector

[0058] The existing HBc141-151 specific TCR sequence was selected as the initial sequence. First, the ACT at position 538-540 of the TCR-α chain nucleotide sequence was mutated to TGC, so that the threonine residue at position 180 of the TCR-α chain amino acid sequence was mutated to a cysteine ​​residue. The A at position 565 of the TCR-β chain nucleotide sequence was mutated to T, so that the serine residue at position 189 of the TCR-β chain amino acid sequence was mutated to a cysteine ​​residue. The TCR-α chain and the TCR-β chain were connected with P2A to obtain TCR protein, and then the intracellular domain of the costimulatory signal 41BB and the CD3ζ signal domain were connected to the TCR protein to obtain the full-length target protein - a new TCR-T, named TCR-CAR2. Figure 1 The amino acid sequence of TCR-CAR2 is shown in SEQ ID No. 2 in the sequence listing, and its encoding gene (i.e., TCR-CAR2 gene) is shown in SEQ ID No. 1.

[0059] In SEQ ID No. 1, positions 1-804 represent the gene encoding the TCR-α chain, positions 805-870 represent the gene encoding P2A, positions 871-1785 represent the gene encoding the TCR-β chain, positions 1786-1911 represent the gene encoding the intracellular domain of 41BB, and positions 1912-2247 represent the gene encoding the CD3ζ signaling domain;

[0060] In SEQ ID No. 2, positions 1-268 represent the TCR-α chain, positions 269-290 represent P2A, positions 291-595 represent the TCR-β chain, positions 596-637 represent the intracellular domain of 41BB, and positions 638-749 represent the CD3ζ signaling domain.

[0061] The TCR-CAR2 gene shown in SEQ ID No. 1 was artificially synthesized, and a small DNA fragment between the restriction endonuclease EcoRI and XbaI recognition sequences of the mammalian expression vector pEF-BOS-EX (whose sequence is SEQ ID No. 5 in the sequence listing) was replaced with the TCR-CAR2 gene shown in SEQ ID No. 1 to obtain the recombinant vector pEF-BOS-EX-TCR-CAR2, which can express the TCR-CAR2 protein shown in SEQ ID No. 2.

[0062] The TCR gene DNA sequence was obtained by adding the stop codon TAA after positions 1-1785 of SEQ ID No. 1. The TCR gene was artificially synthesized by replacing a small DNA fragment between the restriction endonuclease EcoRI and XbaI recognition sequences of the mammalian expression vector pEF-BOS-EX with the TCR gene to generate the recombinant vector pEF-BOS-EX-TCR, which is capable of expressing the TCR protein shown in positions 1-595 of SEQ ID No. 2.

[0063] II. Obtaining Jukat Cells Expressing TCR-CAR2 and Identifying TCR-CAR2 Expression

[0064] 1. Culture Jurkat cells to a number of 2×10 7 The cells were collected and washed twice with antibiotic-free 1640 medium. After washing twice, they were resuspended with antibiotic-free 1640 medium to obtain a cell concentration of 5×10 7The obtained cell suspension was divided into 400 μL / portion and added to an electroporation cup (BIO-RAD, catalog number: 165-2088). At the same time, 40 μg of the pEF-BOS-EX-TCR-CAR2 recombinant vector was added and mixed. The electroporation cup was placed in an electroporator (BIO-RAD, Gene Pulser XcellTM) and electroporated at a voltage of 250 V and a capacitance of 950 μF. The recombinant vector pEF-BOS-EX-TCR-CAR2 was introduced into Jurkat cells to obtain Jurkat recombinant cells expressing TCR-CAR2, which were recorded as Jurkat / TCR-CAR2.

[0065] According to the above method, pEF-BOS-EX-TCR-CAR2 was replaced with pEF-BOS-EX-TCR, and pEF-BOS-EX empty vector was also electroporated to obtain recombinant cells Jurkat / TCR and Jurkat / pEF-BOS-EX, respectively.

[0066] 2. Use Tetramer (HLA-A1101 / HBc141-151) to stain the recombinant Jurkat cells obtained in step 1.

[0067] The recombinant cells after electroporation were stained by flow cytometry using TCRvb10 PE (BD bioscience, Cat. No. 553285) and Tetramer APC. The results of flow cytometry staining are shown in Figure 2 The results showed that the modified new TCR-T sequence, namely TCR-CAR2, can be successfully expressed on the surface of T cells, and the expression ratio increased from 77.5% of TCR to 81.2%, and the expression intensity was higher than that of TCR protein.

[0068] Tetramer APC was prepared according to the instructions of the Flex-TTMHLA-A*11:01Monomer UVX (Biolgend, catalog number: 280007) kit.

[0069] 3. Detection of TCR Downstream Signaling in Jukat Cells Expressing TCR-CAR2

[0070] 1. Culture Jurkat cells to a number of 2×10 7 The cells were collected and washed twice with antibiotic-free 1640 medium. After washing twice, they were resuspended with antibiotic-free 1640 medium to obtain a cell concentration of 5×10 7 / mL cell suspension; the resulting cell suspension was divided into 400 μL / portion and added to an electroporation cup (BIO-RAD, catalog number: 165-2088), and 30 μg of pEF-BOS-EX-TCR-CAR2 recombinant vector, 15 μg of NF-kB promoter-Luc plasmid, and 0.375 μg of pRL-TK plasmid were added at the same time, mixed, and the electroporation cup was placed in an electroporator (BIO-RAD, Gene Pulser XcellTM), and the voltage was 250 V and the capacitance was 950 μF. The above three plasmids were simultaneously introduced into Jurkat cells, and 1640 complete culture medium containing 10% FBS was added to the electroporated cells for use.

[0071] Among them, the NF-kB promoter-Luc plasmid contains the NF-kB promoter and the luciferase reporter gene, and the expression of the luciferase reporter gene can be driven by the NF-kB promoter after the NF-kB promoter is activated.

[0072] NF-kB promoter-Luc plasmid: Promega product, catalog number: E8491.

[0073] pRL-TK plasmid: Promega product (Cat. No. E2241), this plasmid can express Renilla luciferase.

[0074] 2. After 12 hours of electroporation, spleen cells of HLA-I transgenic mice were used as antigen presenting cells. The spleen cells of HLA-I transgenic mice were resuspended in 1640 complete medium containing 10% FBS. 1×10 cells and spleen cells of HLA-I transgenic mice were taken from the electroporated cells in step 1 and 1×10 cells were taken from the spleen cells of HLA-I transgenic mice. 6 Transfer cells to the same well of a 24-well plate, add 1640 complete medium to a volume of 500 μL, then add the synthetic HBc141-151 polypeptide to a final concentration of 10 μg / mL, and incubate in an incubator for 5 h.

[0075] The sequence of the HBc141-151 polypeptide is shown as SEQ ID No. 3 in the sequence listing.

[0076] HLA-I transgenic mouse spleen cells: HLA-I transgenic mice were sacrificed by cervical dislocation. The spleen was removed and placed in a 6 cm dish. 5 mL of 1640 complete medium was added and the spleen was ground using a sterile frosted glass slide. The sample was collected in a 15 mL centrifuge tube and centrifuged at 2000 rpm for 3 min. The supernatant was then removed and the red blood cells were lysed with erythrocyte lysis buffer (Gibco, A1049201). After 1 min, the lysis reaction was terminated with sufficient 1640 complete medium. The supernatant was removed. The cells were resuspended in 1 mL of 1640 complete medium and impurities and spleen tissue were filtered using a sterile 200 mesh nylon membrane to obtain HLA-I transgenic mouse spleen cells for later use. Among them, HLA-I transgenic mice are described in a Chinese patent (patent number: ZL201510136784.5; publication (announcement) number: CN106148405B). The HLA-I transgenic mice used in the present invention are the HLA-A1101 transgenic mice in this patent, and the HLA-A1101 transgenic mice are C57BL / 6J×BALB / c background mice into which the HLA-A1101 gene is transferred.

[0077] 3. After incubation, use a dual fluorescence reporter system assay kit (Promega, Cat. No. E1910) to detect the expression levels of firefly luciferase and Renilla luciferase in the cells. Calculate the ratio of firefly luciferase expression to Renilla luciferase expression, which represents the level of TCR downstream signaling.

[0078] According to the method of steps 1-3 above, the HBc141-151 polypeptide was replaced with the NP366 polypeptide (as a negative control), and the other steps remained unchanged. The level of TCR-CAR2 downstream signal transduction under the action of NP366 polypeptide was detected.

[0079] The sequence of the NP366 polypeptide is shown as SEQ ID No. 4 in the sequence listing.

[0080] The above steps 1-3 were followed, but HBc141-151 was not added and other steps remained unchanged, as a control.

[0081] Following steps 1-3 above, the pEF-BOS-EX-TCR-CAR2 recombinant vector was replaced with pEF-BOS-EX-TCR, while other steps remained unchanged. The downstream signal transduction level of the TCR protein under the action of the TCR-CAR2 peptide was detected.

[0082] Following steps 1-3 above, the pEF-BOS-EX-TCR-CAR2 recombinant vector was replaced with pEF-BOS-EX-TCR, and the HBc141-151 polypeptide was replaced with the NP366 polypeptide. Other steps remained unchanged, and the downstream signaling level of the TCR protein under the action of the NP366 polypeptide was detected.

[0083] Following the above steps 1-3, the pEF-BOS-EX-TCR-CAR2 recombinant vector was replaced with pEF-BOS-EX-TCR, without adding the HBc141-151 polypeptide, and the other steps remained unchanged, as a control.

[0084] Test results are shown in Figure 3 After stimulating TCR and TCR-CAR2 with a specific peptide (HBc141-151), both TCR and TCR-CAR2 can be activated. TCR and TCR-CAR2 can further activate the NF-kB promoter to drive the expression of firefly luciferase. However, the relative expression of firefly luciferase after TCR-CAR2 treatment is significantly higher than that of TCR, indicating that TCR signaling mediated by TCR-CAR2 is significantly higher than that of TCR protein. This shows that TCR-CAR2 can significantly enhance the conduction of TCR downstream signals, that is, TCR-CAR2 can more effectively activate downstream pathways than TCR alone, further promoting T cell function.

[0085] 4. Preparation of Human T Cells Expressing TCR-CAR2

[0086] In order to prove that TCR-CAR2-expressing T cells (TCR-CAR2-T) have stronger effector function than TCR-T cells, human T cells expressing TCR-CAR2 or TCR must first be prepared. The specific steps are as follows:

[0087] 1. Preparation and Concentration of Lentivirus

[0088] (1) EcoRI and BamHI were used to connect the TCR gene and TCR-CAR2 gene into the multiple cloning site of the lentiviral backbone plasmid pCDH-MSCV-MCS-IRES-GFP (System Biosciences, No.: CD731B-1) to obtain recombinant plasmids pCDH-MSCV-TCR-GFP and pCDH-MSCV-TCR-CAR2-GFP. pCDH-MSCV-TCR-GFP can express the fusion protein formed by the TCR protein shown in positions 1-595 of SEQ ID No. 2 and GFP, and pCDH-MSCV-TCR-CAR2-GFP can express the fusion protein formed by the TCR-CAR2 protein shown in SEQ ID No. 2 and GFP.

[0089] (2) 293T cells with a confluence greater than 90% were digested and resuspended in DMEM complete medium at a rate of 5 × 10 6 Plate the cells in 10 cm dishes, shake well, and place in an incubator for overnight culture.

[0090] (3) When the confluence of the 293T cells plated in step (2) reaches 70%-80%, the cell culture medium is replaced with 10 mL of fresh DMEM complete culture medium to obtain a 293T cell suspension.

[0091] (4) Prepare the transfection DNA-calcium phosphate mixture, taking the transfection of a 10 cm dish as an example. Take a sterile 1.5 mL EP tube, add 12 μg pCDH-MSCV-TCR-GFP plasmid, 9 μg psPAX2 (Hanheng Biotechnology, catalog number: HH20220721ZJ-PC02) and 6 μg pMD2.G (Hanheng Biotechnology, catalog number: HH20220722ZJ-PC01), then add 96 μL 2M CaCl2 aqueous solution, and use sterile ddH2O to make up the volume to 750 μL to obtain a DNA and CaCl2 mixture. Take another 6-well plate, add 2xHBS to the 6-well plate, and then add an equal volume of DNA and CaCl2 mixture dropwise to the 2x HBS, gently shaking while adding to mix. After the addition is complete, a DNA-calcium phosphate mixture is obtained, and let it stand for 5 minutes.

[0092] (5) After step (4) is completed, take the 293T cell suspension from step (3) and add the DNA-calcium phosphate mixture from step (4) dropwise thereto. After the addition is complete, gently shake to mix and place in an incubator for culture.

[0093] (6) After culturing for 4-6 h, the cell culture medium was replaced with 15 mL of fresh DMEM complete medium and the cells were placed in an incubator for further culture.

[0094] (7) The virus supernatant was collected at 48 h and 72 h of culture, and the virus was concentrated after the 72 h collection. First, the virus supernatant was filtered using a 0.45 μm PES filter to remove cell debris and culture medium impurities. The resulting filtrate was the virus stock solution. The virus stock solution was added to a sterile ultracentrifuge tube, balanced, and centrifuged at 70,000 g for 2 h at 4 °C.

[0095] (8) After centrifugation, discard the supernatant and carefully aspirate the remaining liquid with a pipette. The resulting precipitate is the pCDH-MSCV-TCR-GFP recombinant lentivirus. Immediately add 200 μL of fresh 1640 complete culture medium to the precipitate, place the centrifuge tube in a 4°C refrigerator and let it stand for 2-4 hours. After the standing period, repeatedly pipette and aspirate the liquid in the centrifuge tube to fully resuspend the virus in the culture medium to obtain the pCDH-MSCV-TCR-GFP recombinant lentivirus concentrate. Then collect the virus concentrate into a 1.5 mL EP tube and store at -80°C for later use.

[0096] According to the method of the above steps (1)-(8), the pCDH-MSCV-TCR-GFP plasmid in (4) was replaced with the pCDH-MSCV-TCR-CAR2-GFP plasmid, and the other steps remained unchanged to obtain the pCDH-MSCV-TCR-CAR2-GFP recombinant lentivirus and its concentrate.

[0097] According to the method of the above steps (1)-(8), the pCDH-MSCV-TCR-GFP plasmid in (4) was replaced with the pCDH-MSCV-MCS-IRES-GFP plasmid, and the other steps remained unchanged, thereby obtaining the pCDH-MSCV-MCS-IRES-GFP recombinant lentivirus and its concentrate.

[0098] 2. Activation of human T cells and lentiviral infection

[0099] (1) Take frozen human PBMC (Shanghai Aoneng Biotechnology Co., Ltd.), thaw in a 37°C water bath, and add the cells to a 15 mL centrifuge tube. Add 10 mL of 1640 complete medium to the centrifuge tube and centrifuge at 400 g for 10 minutes. After centrifugation, discard the supernatant and add 1640 complete medium to adjust the cell concentration to 2×10 6 PBMC cell suspension was obtained.

[0100] (2) T cell TransACT (Miltenyi Biotec, catalog number: 170-076-156) was added to the PBMC cell suspension in the following ratio: PBMC cell suspension: T cell TransACT = 1 mL: 20 μL. Then rhIL2 (peprotech, 200-02-500 μg) was added. The concentration of rhIL2 in the resulting system was 100 U / mL. The cells were then added to a 24-well plate at a volume of 1 mL / well and cultured for 48 h.

[0101] (3) After 48 h of culture, the cells were transferred to a 15 mL centrifuge tube and centrifuged at 400 g for 5 min. After centrifugation, the supernatant was discarded and the pCDH-MSCV-TCR-GFP recombinant lentiviral concentrate obtained in step 1 was added to the cell pellet, followed by polybrene at a final concentration of 8 μg / mL. The resulting mixture was added to a 24-well plate and then centrifuged at 1000 g at 31°C for 1.5 h.

[0102] (4) After centrifugation, the cells were placed in an incubator and cultured for 4 h. The cell culture medium was then replaced with 1 mL of 1640 complete culture medium containing 100 U / mL rhIL2. TCR-T cells were obtained after two days of culture. TCR-T cells were then flow stained using TCRvb10 PE and Tetramer APC to detect TCR expression efficiency.

[0103] According to the method of steps (1)-(4) above, the pCDH-MSCV-TCR-GFP recombinant lentiviral concentrate was replaced with the pCDH-MSCV-TCR-CAR2-GFP recombinant lentiviral concentrate, and the other steps remained unchanged to obtain TCR-CAR2-T cells, and the expression efficiency of TCR-CAR2 was detected.

[0104] According to the method of steps (1)-(4) above, the pCDH-MSCV-TCR-GFP recombinant lentiviral concentrate was replaced with the pCDH-MSCV-MCS-IRES-GFP recombinant lentiviral concentrate, and the other steps remained unchanged to obtain control T cells (denoted as Mock-T cells).

[0105] See the results Figure 4 , TCR-CAR2 can be successfully expressed on the surface of human T cells, and the expression ratio increased from 41.7% to 47.5% compared with the TCR control group, indicating that the expression intensity of TCR-CAR2 is higher than that of the TCR control group.

[0106] 5. TCR-CAR2-T killing efficiency detection

[0107] Using CytoTox The T cell killing efficiency was tested using the Non-Radioactive Cytotoxicity Assay Kit (Promega, Cat. No. G1780). The T cells used were the mock-T cells, TCR-T cells, and TCR-CAR2-T cells obtained in step 4. The specific steps are as follows:

[0108] 1. Take the pre-cultured human liver cancer cell HepG2.2.15-A1101 cells (expressing HBc141-151), digest them, and wash them twice with 10 mL of phenol red-free 1640 medium.

[0109] 2. Take the T cells to be tested and wash them once with 10 mL of phenol red-free 1640 culture medium.

[0110] 3. Add HepG2.2.15-A1101 cells to a U-bottom 96-well plate, 1×10 4 Test T cells were then added to HepG2.2.15-A1101 cells at effector-target ratios of 1:1 and 10:1 (i.e., the ratio of test T cells to HepG2.2.15-A1101 cells). The corresponding control groups were added to the 96-well plate according to the kit instructions. Incubate at 37°C for 4 hours.

[0111] 4. After incubation, centrifuge the 96-well plate at 200g for 5 minutes. Carefully pipette 50 μL of the supernatant into a new flat-bottom 96-well plate. Add 50 μL of the substrate provided with the kit to the plate. Incubate at room temperature in the dark for 30 minutes. After incubation, add 50 μL of the stop solution provided with the kit. Measure the OD value of each well at 490 nm using a microplate reader.

[0112] 5. Calculate the killing activity according to the kit instructions. The specific calculation formula is killing activity (%) = (experimental group value - T cell self-release value - target cell self-release value) / (target cell maximum release value - target cell self-release value).

[0113] Test results such as Figure 5 As shown, Mock-T cells are ordinary human T cells, so their cytotoxic activity against target cells cannot be detected. TCR-CAR2-T cells have higher cytotoxic activity than TCR-T cells regardless of whether the effector-target ratio is 1:1 or 10:1, indicating that TCR-CAR2 can enhance the cytotoxic effect of T cells on target cells.

[0114] To construct HLA-A1101-positive HepG2.2.15 cells (HepG2.2.15-A1101), HLA-A1101 was first introduced into the human liver cancer cell line HepG2.2.15 via a lentiviral approach. This established a liver cancer cell line (HepG2.2.15-A1101) that highly expressed the antigen-presenting complex HLA-A1101, serving as the tumor target cell for in vitro specific immune recognition and killing experiments with HLA-A1101-restricted TCR-T cells. The specific steps are as follows:

[0115] Preparation of lentivirus: Use EcoRI and BamHI to ligate the A1101 gene into the multiple cloning site of the lentiviral backbone plasmid pCDH-MSCV-MCS-IRES-GFP (System Biosciences, No. CD731B-1) to generate the recombinant plasmid pCDH-MSCV-A1101-GFP. Follow the procedures (2)-(8) of Step 1 in Step 4 to replace the pCDH-MSCV-TCR-GFP plasmid with pCDH-MSCV-A1101-GFP to obtain the target lentivirus.

[0116] The A1101 gene sequence is shown as SEQ ID No. 6 in the sequence listing.

[0117] Virus infection: Follow the method of step 2 in step 4, replace the cells with HepG2.2.15 cells, replace the lentivirus with the above lentivirus, complete the infection of HepG2.2.15 cells with the target lentivirus, and obtain HLA-A1101-positive HepG2.2.15 cells.

[0118] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for preparing TCR-T cells, comprising: The intracellular domain of 41BB, the CD3ζ signaling domain and the TCR protein are connected to obtain a recombinant TCR; the encoding gene of the recombinant TCR is introduced into T cells and the encoding gene is expressed to obtain TCR-T cells.

2. The method according to claim 1, wherein: The intracellular domain of 41BB is as follows A1), A2) or A3): A1) the amino acid sequence is the protein shown in positions 596-637 of SEQ ID No. 2; A2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 2 in the sequence listing, wherein one or more amino acid residues are substituted and / or deleted and / or added; A3) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2); The CD3ζ signaling domain is as follows B1), B2) or B3): B1) the amino acid sequence of the protein shown in positions 638-749 of SEQ ID No. 2; B2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 2 in the sequence listing, wherein one or more amino acid residues are substituted and / or deleted and / or added; B3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of B1) or B2).

3. The method according to claim 1 or 2, characterized in that: Among the genes encoding the recombinant TCR, the gene encoding the intracellular domain of 41BB is the following a11) or a12) or a13): a11) the DNA molecule shown in positions 1786 to 1911 of SEQ ID No. 1 in the sequence listing; a12) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in a11) and encodes the intracellular domain of 41BB; a13) a DNA molecule that hybridizes under stringent conditions with the nucleotide sequence defined in a11) or a12) and encodes the intracellular domain of 41BB; The coding gene of the CD3ζ signaling domain is as follows b11) or b12) or b13): b11) the DNA molecule shown in positions 1912-2247 of SEQ ID No. 1 in the sequence listing; b12) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in b11) and encodes the CD3ζ signaling domain; b13) A DNA molecule that hybridizes under stringent conditions with the nucleotide sequence defined in b11) or b12) and encodes the CD3ζ signaling domain.

4. The method according to any one of claims 1 to 3, characterized in that: The coding gene of the recombinant TCR is introduced into T cells via an expression vector.

5. The method according to any one of claims 1 to 4, characterized in that: The TCR protein is a specific TCR protein for tumor cells or virus-infected cells; Furthermore, the tumor cells are human liver cancer cells; and the virus-infected cells are human liver cells infected with hepatitis B virus.

6. The method according to claim 5, characterized in that: The TCR protein is a specific TCR protein for the HLA-A1101 restricted hepatitis B virus HBc141-151 epitope peptide; Furthermore, the TCR-α chain of the TCR protein is shown as positions 1-268 of SEQ ID No. 2, and the TCR-β chain is shown as positions 291-595 of SEQ ID No.

2.

7. The method according to claim 6, characterized in that: The recombinant TCR is shown in SEQ ID No.

2.

8. TCR-T cells obtained by the method according to any one of claims 1 to 7.

9. The recombinant TCR according to any one of claims 1 to 7.

10. Use of the TCR-T cells obtained by the method according to any one of claims 1 to 7 in the preparation of a product for treating tumors, or in the treatment of tumors; Or, use of the TCR-T cells obtained by the method according to any one of claims 1 to 7 in the preparation of a product for treating hepatitis B, or in the treatment of hepatitis B; Or, the use of the recombinant TCR described in any one of claims 1-7 in the preparation of a product for treating tumors, or in the treatment of tumors, or in the preparation of a product for enhancing the killing ability of T cells, or in the enhancement of the killing ability of T cells, or in the preparation of a product for treating hepatitis B, or in the treatment of hepatitis B.

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