A Kras G12D-specific T-cell receptor

Abstract

This invention relates to the field of tumor immunotherapy and discloses a Kras G12D-specific T-cell receptor comprising an α chain and a β chain. The α chain includes amino acid sequences CDR1 to CDR3 as shown in SEQ ID NO:2 to SEQ ID NO:4, and the β chain includes amino acid sequences CDR1 to CDR3 as shown in SEQ ID NO:5 to SEQ ID NO:7. Peripheral blood mononuclear cells are isolated and seeded, stimulated with anti-CD3 and anti-CD28 magnetic beads, and then transduced using a lentivirus containing the nucleic acid encoding the receptor to obtain engineered primary T cells. This invention, by including the corresponding amino acid sequences CDR1 to CDR3 in the α and β chains of the receptor, respectively, yields a Kras G12D-specific T-cell receptor with targeted recognition capabilities, providing a material basis for subsequently obtaining the nucleic acid encoding the receptor and expression plasmids or lentiviruses.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy, specifically to a Kras G12D specific T-cell receptor. Background Technology

[0002] T-cell receptor-engineered T-cell therapy plays a role in the field of tumor immunotherapy. Kras gene mutations exist in various solid tumors, with the Kras G12D mutation being a common subtype and a target antigen for tumor-targeted therapy. Utilizing T-cell receptors to recognize mutant peptides presented by corresponding molecules and mediate cell killing provides a research direction for the treatment of related tumors.

[0003] In the development of receptors targeting the Kras G12D mutant antigen, existing T-cell receptor molecules have limitations in the design of their complementarity-determining region (CDR) sequences. These receptor molecules exhibit poor affinity for the mutant antigen and are prone to cross-reaction with wild-type proteins, resulting in insufficient targeted recognition capabilities. The lack of T-cell receptors with corresponding amino acid sequences targeting this target in current technologies hinders the preparation of corresponding nucleic acids, plasmids, and lentiviral vectors based on them.

[0004] On the other hand, in the preparation process of engineered cells, existing primary T cell culture systems lack suitable magnetic bead stimulation and lentiviral infection conditions, resulting in limited in vitro transduction effects after peripheral blood separation, making it difficult to obtain engineered primary T cells expressing the corresponding receptor. Simultaneously, in the establishment of in vitro cell models, conventional methods struggle to effectively match the mixed transduction ratio of cell lines carrying reporter genes and viruses, making it impossible to obtain engineered Jurkat cell lines that meet the requirements for subsequent concentration testing. Therefore, providing a method for preparing the Kras G12D specific T cell receptor and its related engineered cells is a problem that needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a Kras G12D-specific T-cell receptor, which solves the problems of insufficient targeting and recognition ability of existing T-cell receptors for Kras G12D mutant antigens and the limitations in the preparation of corresponding engineered cells.

[0006] To address the above problems, the present invention provides the following technical solution: A Kras G12D-specific T-cell receptor, the Kras G12D-specific T-cell receptor comprising an α chain and a β chain, the α chain comprising amino acid sequences CDR1 to CDR3 as shown in SEQ ID NO:2 to SEQ ID NO:4, and the β chain comprising amino acid sequences CDR1 to CDR3 as shown in SEQ ID NO:5 to SEQ ID NO:7.

[0007] By employing the above technical solution, specific complementary determinant regions (CDRs) with amino acid sequences as shown in SEQ ID NO:2 to SEQ ID NO:7 are used. These CDRs constitute the recognition interface of the TCR molecule's antigen-binding groove, enabling them to form spatial fit and electrostatic complementarity with the Kras G12D mutant peptide presented by the HLA molecule. Therefore, the effect of having affinity and specific recognition ability for the Kras G12D mutant antigen is achieved, avoiding the off-target toxicity risk caused by cross-reaction with wild-type Kras antigen.

[0008] Preferably, the α chain includes an α chain variable region, the β chain includes a β chain variable region, the amino acid sequence of the α chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the β chain variable region is shown in SEQ ID NO:9.

[0009] By adopting the above technical solution, and by using the variable regions with amino acid sequences as shown in SEQ ID NO:8 and 9 respectively, the spatial conformation of CDR1 to CDR3 is supported and fixed by the amino acid sequences of specific framework regions. Therefore, the effect of maintaining the folding stability of TCR molecules is achieved, ensuring the structural integrity of the receptor when it is displayed on the cell membrane surface, and improving the stability of antigen binding.

[0010] A nucleic acid that encodes the Kras G12D-specific T-cell receptor.

[0011] By adopting the above technical solution, since the nucleic acid sequence encoding the Kras G12D specific T cell receptor is used as the genetic material basis, the Kras G12D specific T cell receptor can be endogenously synthesized in the host cell through the transcription and translation system, thus providing a genetic information template for the subsequent construction of genetically engineered cells.

[0012] Preferably, the Kras G12D specific T cell receptor includes an α-chain variable region and a β-chain variable region; the nucleic acid includes a nucleotide sequence encoding the α-chain variable region and a nucleotide sequence encoding the β-chain variable region, wherein the nucleotide sequence encoding the α-chain variable region is shown in SEQ ID NO:10 and the nucleotide sequence encoding the β-chain variable region is shown in SEQ ID NO:11.

[0013] By adopting the above technical solution, and using nucleotide sequences as shown in SEQ ID NO:10 and 11 respectively, which are adapted to the human cell expression system in terms of codon usage frequency, the transcription and translation efficiency of the nucleic acid in T cells is improved, thus ensuring the expression level of the Kras G12D specific T cell receptor on the surface of the host cell.

[0014] An expression plasmid or lentivirus containing the nucleic acid.

[0015] By adopting the above technical solution, since the expression plasmid or lentivirus is used as a gene delivery vector to carry the nucleic acid, the effect of delivering the nucleic acid into the host cell is achieved; when the lentiviral vector is used, the nucleic acid can be integrated into the host cell genome, ensuring the stable inheritance and expression of the Kras G12D specific T cell receptor gene.

[0016] An engineered cell that expresses the Kras G12D-specific T-cell receptor.

[0017] By adopting the above technical solution, since the Kras G12D specific T cell receptor is expressed on the cell surface, the host cell targeting ability is redirected, thus giving cells that originally lacked tumor-specific recognition ability the ability to recognize and kill tumor cells carrying the Kras G12D mutation.

[0018] Preferably, the engineered cells are engineered Jurkat cell lines or engineered primary T cells.

[0019] By adopting the above technical solution, and using the Jurkat cell line or primary T cells as the host, effects suitable for different application scenarios are obtained: the engineered Jurkat cell line proliferates rapidly and has good uniformity, making it suitable for in vitro TCR function identification and pharmacodynamic model evaluation; the engineered primary T cells retain immune killing activity and proliferative potential, making them suitable for adoptive cell reinfusion therapy.

[0020] A method for preparing the engineered primary T cells includes the following steps: Peripheral blood mononuclear cells were isolated by density gradient centrifugation; The peripheral blood mononuclear cells were divided into 5.0 × 10⁻⁶ cells. 5 Cells were seeded at a density of cells per well in 24-well cell culture plates to obtain seeded cells; The seeded cells were stimulated with anti-CD3 and anti-CD28 magnetic beads and cultured in a 37°C, 5% CO2 incubator for 24 h to obtain cells to be transduced. The cells to be transduced were transduced using lentiviruses to obtain the engineered primary T cells; The lentivirus contains nucleic acid encoding the Kras G12D-specific T-cell receptor.

[0021] By adopting the above technical solution, and by using a pretreatment method that combines peripheral blood density gradient centrifugation with stimulation by anti-CD3 and anti-CD28 magnetic beads, the primary T cells were induced to enter the proliferation cycle, and the expression level of viral receptors on the cell surface was increased, thereby significantly improving the subsequent infection efficiency of the primary T cells and genome integration efficiency of the lentivirus.

[0022] Preferably, the transduction of the cells to be transduced using lentivirus to obtain the engineered primary T cells specifically includes: The lentivirus was added to the cells to be transduced at a multiplicity of infection of 2 to obtain transduced cells. The transduced cells were placed in RPMI 1640 medium containing 200 IU / mL IL-2 and 10% FBS and cultured for 3 to 4 days to obtain expanded cells. The concentration of the expanded cultured cells was adjusted to 5.0 × 10⁻⁶. 4 The engineered primary T cells were obtained by collecting 1 positive cell / mL.

[0023] By employing the above technical solution, and by controlling the multiplicity of infection of the lentivirus to 2 and amplifying it under conditions containing IL-2, the effect of ensuring nucleic acid transfer while avoiding cytotoxicity and immune exhaustion caused by high-dose virus was achieved; the final cell concentration after expansion culture was adjusted to 5.0 × 10⁻⁶. 4 The positive cell density per mL ensures a stable and controllable positive cell density in the final product per unit volume, guaranteeing the uniformity of biological function.

[0024] A method for preparing the engineered Jurkat cell line includes the following steps: JK8NF cells carrying CD8 and NFAT reporter genes were provided as transduction cells; The cells to be transduced were mixed with lentivirus at a ratio of 10 to obtain transduced cells; The lentivirus contains nucleic acid encoding the Kras G12D-specific T-cell receptor; The transduced cells were cultured at 37°C for 72 h and then expanded to obtain expanded cultured cells. The concentration of the expanded cultured cells was adjusted to 1.6 × 10⁻⁶. 6 The engineered Jurkat cell line was obtained by using cells / mL.

[0025] By employing the above technical solution, and using JK8NF cells carrying the NFAT reporter gene as the transduction host, along with a transduction system with a multiplicity of infection of 10, an in vitro TCR signaling evaluation system was successfully constructed. After the Kras G12D-specific T cell receptor recognizes the mutant antigen, the cell activation state can be quantified by the expression level of the NFAT reporter gene, providing a stable cell model for affinity verification and in vitro pharmacodynamic evaluation of the Kras G12D-specific T cell receptor.

[0026] This invention provides a Kras G12D-specific T cell receptor. It has the following beneficial effects: 1. This invention obtains a Kras G12D-specific T-cell receptor with targeting recognition capability by including CDR1 to CDR3 corresponding amino acid sequences in the α chain and β chain of the Kras G12D-specific T-cell receptor, respectively. This provides a material basis for obtaining nucleic acids encoding the Kras G12D-specific T-cell receptor and expression plasmids or lentiviruses.

[0027] 2. This invention separates peripheral blood mononuclear cells using density gradient centrifugation and stimulates them with anti-CD3 and anti-CD28 magnetic beads after inoculation to obtain cells to be transduced. Then, the cells to be transduced are transduced using a lentivirus containing nucleic acid encoding the Kras G12D specific T cell receptor, thereby obtaining engineered primary T cells expressing the Kras G12D specific T cell receptor.

[0028] 3. This invention provides JK8NF cells carrying CD8 and NFAT reporter genes as transduction cells, mixes the transduction cells with lentivirus containing nucleic acid encoding Kras G12D specific T cell receptor in a certain proportion for transduction, and adjusts the cell concentration after expansion culture to obtain an engineered Jurkat cell line expressing Kras G12D specific T cell receptor. Attached Figure Description

[0029] Figure 1 This is a graph showing the transfection positivity rate detection results of the engineered Jurkat cell line in this embodiment of the invention; Figure 2 This is a graph showing the activation response of engineered Jurkat cells in the test examples of this invention. Figure 3 This is a graph showing the transfection efficiency detection results of engineered primary T cells in an embodiment of the present invention; Figure 4 This is a real-time proliferation curve of target cells under the action of different effector cells in the test examples of this invention. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] The main raw materials and reagents used in the following examples, preparation examples and test examples are as follows. Unless otherwise specified, all reagents are commercially available analytical grade or higher grade products.

[0032] Kras G12D antigen short peptide: The amino acid sequence is shown in SEQ ID NO:1, and it was obtained by artificial synthesis.

[0033] 293T cells: deposited at the American Center for Type Culture Collection, catalog number CRL-1573.

[0034] T2 cells and Jurkat cells: both are deposited at the American Center for Type Culture Collection (ACC), with accession numbers CRL-1992 and TIB-152, respectively. The T2-A11 cells and JK8NF cells used in subsequent experiments were cell lines constructed based on these deposits.

[0035] PANC0813 cell line: Pancreatic cancer cells carrying the Kras oncogene mutation, derived from solid tumors. The PANC0813-A11 cell line used in subsequent experiments was a mutant cell line with the genotype HLA-A*11:01 constructed from this line.

[0036] Preparation Example 1: This preparation example provides a method for obtaining and identifying specific TCR sequence information, including the following steps: pHLA monomers refolded from biotin-labeled HLA-A*11:01 and SEQ ID NO:1 short peptide were prepared using conventional techniques in the art, and then combined with PE-labeled streptavidin to form PE-labeled tetramers.

[0037] CD14-positive cells from the peripheral blood of healthy volunteers with genotype HLA-A*11:01 were isolated and used to induce mature dendritic cells. The mature dendritic cells were loaded with the aforementioned short peptide at a final concentration of 20 μg / ml to stimulate naïve CD8-positive T cells isolated from peripheral blood mononuclear cells. After three rounds of stimulation, flow cytometry was used to separate tetramer- and CD8-double-positive antigen-specific single cells.

[0038] The single-cell TCR α and β chains obtained above through a one-step RT-PCR kit were amplified and sequenced. The sequencing results were compared with the IMGT database to obtain the Kras G12D-specific TCR, named the TCR107 sequence. The CDR1 to CDR3 amino acid sequences of its α chain (TCR107-α) and β chain (TCR107-β) are shown in SEQ ID NO:2 to SEQ ID NO:4 and SEQ ID NO:5 to SEQ ID NO:7, respectively; the variable region amino acid sequences of the α and β chains are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively; and the nucleotide sequences of the α and β chains are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0039] Examples 1-3: Example 1: This embodiment provides a Kras G12D-specific T cell receptor TCR107 lentiviral vector and a method for preparing lentivirus, including the following steps: S1. Using a multi-fragment recombination cloning method, the α and β chain variable region sequences of the Kras G12D specific T cell receptor obtained in Preparation Example 1, whose nucleotide sequences are shown in SEQ ID NO:10 and SEQ ID NO:11 respectively, together with the mouse α and β chain constant region sequences, were cloned into a pLKO-based expression plasmid. S2. The above recombinant expression plasmid was transformed into competent Escherichia coli Stbl3, and single clones were picked and cultured in a medium containing ampicillin. S3. Extract and purify the plasmid. After verifying the sequence is correct by sequencing, obtain the expression plasmid expressing the Kras G12D specific T cell receptor. S4. Seed 293T cells in a culture dish. When the cell confluence is appropriate, mix the virus packaging plasmid and the expression plasmid that has been verified above at a mass ratio of 1:1, with a total mass of 10 μg. Add polyethyleneimine and serum-free DMEM medium to prepare a mixture, and then add it to 293T cells for transfection incubation. S5. After culturing at 37℃ for 72 hours, the cell culture supernatant was collected and concentrated using a 100kd ultrafiltration tube to obtain a lentiviral vector carrying the Kras G12D specific T cell receptor gene.

[0040] Example 2: This embodiment provides a method for constructing an engineered Jurkat cell line expressing the above-mentioned Kras G12D specific T cell receptor, including the following steps: S1. JK8NF cells carrying CD8 and NFAT reporter genes, i.e. Jurkat-CD8-NFAT cells, are mixed with lentiviruses carrying the Kras G12D specific T cell receptor gene prepared in Example 1 at a ratio of 10 for transduction. S2. After culturing the above cells at 37°C for 72 hours, the transfection positivity rate was detected by flow cytometry. The results are as follows: Figure 1 As shown, this indicates that the receptor gene has been successfully transferred and expressed; S3. The successfully transduced cells were cultured in a large-scale manner, and then the cells were collected and the cell concentration was adjusted to 1.6 × 10⁻⁶. 6 We obtained engineered Jurkat cell lines expressing the Kras G12D-specific T cell receptor at a density of 1 cell / ml, which were then used as effector cells for subsequent testing.

[0041] Example 3: This embodiment provides a method for constructing engineered primary T cells expressing the above-mentioned Kras G12D specific T cell receptor, including the following steps: S1. Peripheral blood mononuclear cells were separated by density gradient centrifugation at 5.0 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell per well in 24-well cell culture plates; S2, Add anti-CD3 and anti-CD28 magnetic beads to stimulate cells, and incubate in a 37℃, 5% CO2 incubator for 24h; S3. At a multiplicity of infection ratio of 2, add the lentivirus carrying the Kras G12D specific T cell receptor gene prepared in Example 1 to the well plate for transduction. S4. The transduced cells were placed in RPMI 1640 medium containing 200 IU / ml IL-2 and 10% FBS and cultured for 3 to 4 days for further expansion. S5. Flow cytometry was used to detect cell transfection efficiency. The results are as follows: Figure 3 As shown, the cell concentration after expansion culture was then adjusted to 5.0 × 10⁻⁶. 4We obtained engineered primary T cells expressing the Kras G12D specific T cell receptor by 1 positive cell / ml, which were then used as effector cells for subsequent testing.

[0042] Comparative Examples 1-3: Comparative Example 1: This comparative example provides a wild-type JK8NF control cell. The difference from Example 2 is that the lentivirus prepared in Example 1 was not added for transduction of the JK8NF cells; all other parameters and procedures are the same.

[0043] Comparative Example 2: This comparative example provides a primary T control cell line that has not been transduced. The difference from Example 3 is that peripheral blood mononuclear cells from the same volunteer were stimulated with anti-CD3 and anti-CD28 magnetic beads, but the lentivirus prepared in Example 1 was not added for transduction. All other parameters and steps are the same.

[0044] Comparative Example 3: This comparative example provides an engineered primary T control cell expressing an irrelevant TCR. The difference from Example 3 is that the added lentivirus carries an irrelevant TCR gene targeting a non-Kras G12D site; all other parameters and procedures are the same.

[0045] Test Example 1-2: Test Example 1: Experimental description: The specific activation response of engineered Jurkat cells prepared in Example 2 to target cells loaded with a mutant antigen peptide was verified using an activated T-cell nuclear factor reporter gene system. This test focused on examining whether the cells possess antigen-specific recognition capabilities and concentration-dependent activation function.

[0046] Experimental steps: In this test, experimental and control groups were set up in parallel. In a 96-well plate, 50 μl of T2-A11 target cell suspension in the logarithmic growth phase was first added to each well, so that the number of target cells in a single well reached 80,000. Next, using RPMI 1640 basal medium, the short peptide shown in SEQ ID NO:1 was prepared into short peptide dilutions with different concentration gradients. 50 μl of the short peptide dilution was added to each well to bring the final concentration of the short peptide in the well to 10. -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M and 10 -10 M; Subsequently, 50 μl of effector cells were added to each well. The experimental group was added with engineered Jurkat cells prepared in Example 2, and the control group was added with control cells from Comparative Example 1. The amount of each type of effector cell added was 80,000 per well to ensure an effector-to-target ratio of 1:1. Finally, add 50 μl of RPMI 1640 basal medium to each well to make up the volume of the test system; The plated 96-well plates were placed in a cell culture incubator at 37°C and 5% CO2 and incubated for 12 hours. After incubation, flow cytometry was used to detect the expression of nuclear factor reporter genes of activated T cells. The expression level was used as the readout value for the cells to produce an antigen-specific activation response and the data was analyzed.

[0047] Experimental data: Table 1. Cell activation response under stimulation with different concentrations of antigenic short peptides.

[0048] Experimental conclusion: Combination Figure 2 As shown in Table 1, wild-type cells in the control group did not show significant activation responses at any of the antigen concentration gradients, indicating that unmodified cells do not have the ability to recognize this target.

[0049] like Figure 2 The activation response curve is shown in the figure. The horizontal axis represents the logarithm of the molar concentration of the antigen short peptide, and the vertical axis represents the positive rate of activated T cell nuclear factor. The engineered cells in the experimental group showed a significant activation response to the target cells loaded with Kras G12D short peptide. The curve trend shows that when the logarithm of the antigen molar concentration was in the range of -10 to -8, the positive rate of activated T cell nuclear factor was at a low level; when the logarithm increased from -8 to -6, the positive rate showed a significant upward trend; when the concentration continued to increase to -4, the positive rate gradually leveled off and remained at a high level. This indicates that the intensity of the activation response is positively correlated with the molar concentration of the antigen short peptide, exhibiting an S-shaped dose-response curve.

[0050] The above experimental results indicate that the Kras G12D specific T cell receptor can recognize the corresponding antigens presented on the surface of target cells, thereby enabling engineered Jurkat cells to produce an antigen-specific activation response.

[0051] Test Example 2: Experimental description: A real-time label-free cell analysis system was used to monitor the cell index derived from microelectrode impedance to reflect the proliferation and survival status of adherent target cells in real time, thereby verifying the in vitro cell killing ability and targeting specificity of the engineered primary T cells prepared in Example 3 against pancreatic cancer solid tumor cell lines that endogenously express Kras G12D mutant antigen and HLA-A*11:01.

[0052] Experimental steps: 100 μl of PANC0813-A11 pancreatic cancer target cell suspension in the logarithmic growth phase was added to the E-plate wells of the real-time label-free cell analysis system to make the number of target cells per well reach 5000. Next, 100 μl of effector cells were added to each of the above well plates. The experimental group was filled with engineered primary T cells expressing the Kras G12D specific T cell receptor prepared in Example 3. The two control groups were filled with untransduced primary T cells from Comparative Example 2 and engineered primary T control cells expressing irrelevant TCRs from Comparative Example 3, respectively. The number of effector cells of each type was 5000 per well to ensure that the effector-to-target ratio was 1:1. The E-plate containing the above co-culture system was mounted onto the detection stage of the real-time label-free cell analyzer. The instrument was placed in an incubator at 37℃ and 5% CO2 and continuously statically cultured for 72 hours. The instrument automatically recorded and output the real-time proliferation curve data of the target cells.

[0053] Experimental data: Table 2. Trends in target cell proliferation index among different groups

[0054] Experimental conclusion: Combination Figure 4 As shown in Table 2, the experimental data indicates that in the real-time label-free cell analysis system, the cell index derived from microelectrode impedance is positively correlated with the number of adherent target cells. Figure 4 As shown in the figure, the horizontal axis represents time in hours, and the vertical axis represents the cell index. The figure illustrates the proliferation status of target cells under the influence of different effector cells. The negative control group, which added untransduced primary T cells, corresponds to the TCRneg curve in the figure. Its target cells can continuously proliferate, and the cell index shows a significant upward trend over time. The specific control group, which added cells expressing irrelevant TCRs, corresponds to the irrelevant TCR curve in the figure. Its cell index generally shows an initial increase followed by a flattening out, indicating that neither of these two control groups produced a significant killing effect on the pancreatic cancer target cells.

[0055] In the experimental group incorporating engineered primary T cells from Example 3, the proliferation of target cells was significantly inhibited, as shown by the TCR107 curve in the figure. From the initial stage of culture, the cell index in this group remained close to the baseline level of 0 and did not show an upward trend over time, reflecting that the target cells in the wells underwent lysis and death.

[0056] The above experimental results indicate that engineered primary T cells expressing the Kras G12D specific T cell receptor have significant specific lysis and killing functions against tumor cell lines endogenously expressing HLA-A*11:01 and Kras G12D mutations, and this killing effect depends on the specific recognition of antigens.

[0057] Appendix: Kras G12D amino acid sequence: SEQ ID NO:1: VVVGADGVGK.

[0058] TCR107-α complementarity-determining region CDR1 amino acid sequence: SEQ ID NO:2: TSGFNG.

[0059] TCR107-α complementarity-determining region CDR2 amino acid sequence: SEQ ID NO:3: NVLDGL.

[0060] TCR107-α complementarity-determining region CDR3 amino acid sequence: SEQ ID NO:4: CAVRGAGNMLTF.

[0061] TCR107-β complementarity-determining region CDR1 amino acid sequence: SEQ ID NO:5: SQVTM.

[0062] TCR107-β complementarity-determining region CDR2 amino acid sequence: SEQ ID NO:6: ANQGSEA.

[0063] TCR107-β complementarity-determining region CDR3 amino acid sequence: SEQ ID NO:7: CSVLAGYGEQYF.

[0064] TCR107-α variable region (Vα) amino acid sequence: SEQ ID NO:8: GQNIDQPTEMTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQLKDSASYLCAVRGAGNMLTFGGGTRLMVKPH。

[0065] Amino acid sequence of the variable region (Vβ) of TCR107-β: SEQ ID NO:9: SAVISQKPRYDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLAGYGEQYFGPGTRLTVT。

[0066] Nucleotide sequence of the variable region of TCR107-α: SEQ ID NO:10: ATGTGGGGAGTTTTCCTTCTTTATGTTTCCATGAAGATGGGAGGCACTACAGGACAAAACATTGACCAGCCCACTGAGATGACAGCTACGGAAGGTGCCATTGTCCAGATCAACTGCACGTACCAGACATCTGGGTTCAACGGGCTGTTCTGGTACCAGCAACATGCTGGCGAAGCACCCACATTTCTGTCTTACAATGTTCTGGATGGTTTGGAGGAGAAAGGTCGTTTTTCTTCATTCCTTAGTCGGTCTAAAGGGTACAGTTACCTCCTTTTGAAGGAGCTCCAGATGAAAGACTCTGCCTCTTACCTCTGTGCTGTGAGGGGTGCAGGCAACATGCTCACCTTTGGAGGGGGAACAAGGTTAATGGTCAAACCCCAT。

[0067] Nucleotide sequence of the variable region of TCR107-β: SEQ ID NO:11: ATGCTGAGTCTTCTGCTCCTTCTCCTGGGACTAGGCTCTGTGTTCAGTGCTGTCATCTCTCAAAAGCCAAGCAGGGATATCTGTCAACGTGGAACCTCCCTGACGATCCAGTGTCAAGTCGATAGCCAAGTCACCATGATGTTCTGGTACCGTCAGCAACCTGGACAGAGCCTGACACTGATCGCAACTGCAAATCAGGGCTCTGAGGCCACATATGAGAGTGGATTTGTCATTGACAAGTTTCCCATCAGCCGCCCAAACCTAACATTCTCAACTCTGACTGTGAGCAACATGAGCCCTGAAGACAGCAGCATATATCTCTGCAGCGTTTTAGCGGGGTATGGGGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACA。

Claims

1. A Kras G12D-specific T cell receptor, characterized in that, The Kras G12D specific T cell receptor comprises an α chain and a β chain, wherein the α chain comprises amino acid sequences CDR1 to CDR3 as shown in SEQ ID NO:2 to SEQ ID NO:4, and the β chain comprises amino acid sequences CDR1 to CDR3 as shown in SEQ ID NO:5 to SEQ ID NO:

7.

2. The Kras G12D specific T cell receptor according to claim 1, characterized in that, The α chain includes an α chain variable region, the β chain includes a β chain variable region, the amino acid sequence of the α chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the β chain variable region is shown in SEQ ID NO:

9.

3. A nucleic acid, characterized in that, The nucleic acid encodes the Kras G12D specific T cell receptor as described in claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that, The Kras G12D specific T cell receptor includes an α-chain variable region and a β-chain variable region; The nucleic acid includes a nucleotide sequence encoding the α-chain variable region and a nucleotide sequence encoding the β-chain variable region, wherein the nucleotide sequence encoding the α-chain variable region is shown in SEQ ID NO:10 and the nucleotide sequence encoding the β-chain variable region is shown in SEQ ID NO:

11.

5. An expression plasmid or lentivirus, characterized in that, The expression plasmid or lentivirus comprises the nucleic acid as described in claim 3.

6. An engineered cell, characterized in that, The engineered cells express the KrasG12D-specific T-cell receptor as described in claim 1 or 2.

7. The engineered cell according to claim 6, characterized in that, The engineered cells are engineered Jurkat cell lines or engineered primary T cells.

8. A method for preparing engineered primary T cells as described in claim 7, characterized in that, Includes the following steps: Peripheral blood mononuclear cells were isolated by density gradient centrifugation; The peripheral blood mononuclear cells were divided into 5.0 × 10⁻⁶ cells. 5 Cells were seeded at a density of cells per well in 24-well cell culture plates to obtain seeded cells; The seeded cells were stimulated with anti-CD3 and anti-CD28 magnetic beads and cultured in a 37°C, 5% CO2 incubator for 24 h to obtain cells to be transduced. The cells to be transduced are transduced using lentivirus to obtain the engineered primary T cells; wherein the lentivirus contains nucleic acid encoding the Kras G12D specific T cell receptor.

9. The preparation method according to claim 8, characterized in that, The process of transducing the cells to be transduced using lentivirus to obtain the engineered primary T cells specifically includes: The lentivirus was added to the cells to be transduced at a multiplicity of infection of 2 to obtain transduced cells. The transduced cells were placed in RPMI 1640 medium containing 200 IU / mL IL-2 and 10% FBS and cultured for 3 to 4 days to obtain expanded cells. The concentration of the expanded cultured cells was adjusted to 5.0 × 10⁻⁶. 4 The engineered primary T cells were obtained by collecting 1 positive cell / mL.

10. A method for preparing an engineered Jurkat cell line as described in claim 7, characterized in that, Includes the following steps: JK8NF cells carrying CD8 and NFAT reporter genes were provided as transduction cells; The cells to be transduced were mixed with lentivirus at a ratio of 10 to obtain transduced cells; The lentivirus contains nucleic acid encoding the Kras G12D-specific T-cell receptor; The transduced cells were cultured at 37°C for 72 h and then expanded to obtain expanded cultured cells. The concentration of the expanded cultured cells was adjusted to 1.6 × 10⁻⁶. 6 The engineered Jurkat cell line was obtained by using cells / mL.

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