A tcr targeting a krass g12d mutation and uses thereof

Abstract

The application discloses a TCR targeting KRAS G12D mutation and a use thereof. The TCR with improved activity provided by the application is obtained by mutation on the basis of a parent TCR10, has the ability of high sensitivity to bind a target antigen peptide, significantly improves target cell recognition ability, can mediate specific killing of antigen-positive target cells by effector cells, and has no allogeneic reaction to different HLA types. The TCR can be used for treating various cancers positive to KRAS G12D.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a TCR that targets the KRAS G12D mutation and its applications. Background Technology

[0002] KRAS (Kirsten rat sarcoma virus oncogene), a common tumor driver gene, dynamically switches between an inactive state bound to guanosine diphosphate (GDP) and an active state bound to guanosine triphosphate (GTP), thereby participating in the regulation of key signal transduction for cell proliferation and survival. Mutations in this gene are widespread in various human cancers, including approximately 90% of pancreatic cancers, 30-40% of colon cancers, 15-20% of lung cancers (especially non-small cell lung cancers), and 17% of endometrial cancers, thus being considered one of the most common mutated genes in human cancers.

[0003] Of these, the G12D mutation (the most common one at codon 12) accounts for approximately 20-50% of all KRAS mutations. G12D The mutated abnormal peptides can be presented to the cell surface by human leukocyte antigen (HLA) molecules and specifically recognized by T cells. Due to its high specificity in tumors, it is a typical neoantigen of tumors and has become an ideal therapeutic target. Based on targeting KRAS... G12D T-cell receptor-engineered T-cell (TCR-T) therapy has achieved objective regression of metastases in pancreatic cancer patients carrying this mutation, demonstrating strong therapeutic potential. However, the application of this therapy is limited by the patient's HLA alleles, meaning that only patients carrying a specific HLA subtype can benefit.

[0004] Previous studies have confirmed that targeting HLA-C*08:02-KRAS... G12D The TCR10 gene has shown significant anti-tumor effects in both in vitro and in vivo experiments. However, the applicability of this TCR-T cell therapy is limited to patients carrying the HLA-C*08:02 allele (Leidner Rom, Nelson Sanjuan Silva, Huayu Huang, et alNeoantigen T-CellReceptor Gene Therapy in Pancreatic Cancer[J]. The New England Journal of Medicine, 2022, 386(22): 2112-2119). This allele has a global distribution frequency of only 3-4%, and is even lower in Asian populations, at only about 0.37%.

[0005] In contrast, the HLA-C*08:01 allele, which differs from HLA-C*08:02 by only three amino acids, has a global frequency of approximately 5-6%, exceeding 8.0% in Asian populations, particularly in China. Therefore, if a KRAS receptor agonist can be identified that recognizes HLA-C*08:01, it would be highly valuable. G12D The T-cell receptor (TCR) of the peptide could more than double the potential patient population, especially in the Chinese population, where it could expand to 22% (Zhou Fusheng, Hongzhi Cao, Xianbo Zuo). et al ., Deep Sequencing of the MHC Region in the Chinese Population Contributes to Studies of Complex Disease[J]. Nature Genetics, 2016, 48(7): 740-746). However, there is currently no treatment for HLA-C*08:01-KRAS. G12D TCR report of this tumor neoantigen. Summary of the Invention

[0006] To address the aforementioned shortcomings in the prior art, this invention provides a TCR targeting the KRAS G12D mutation and its applications.

[0007] This invention first provides a TCR targeting the KRAS G12D mutation, comprising an α-chain variable region containing CDR1α, CDR2α, and CDR3α, and a β-chain variable region containing CDR1β, CDR2β, and CDR3β. The key feature is that the amino acid sequences of CDR1α, CDR2α, and CDR3α in the pre-mutant parental TCR are shown in SEQ ID NO: 1-3, respectively, and the amino acid sequences of CDR1β, CDR2β, and CDR3β are shown in SEQ ID NO: 4-6, respectively. The TCR targeting the KRAS G12D mutation has a G3A mutation or a D1F mutation relative to the parental TCR's CDR1α, or the TCR targeting the KRAS G12D mutation has an A3V mutation relative to the parental TCR's CDR3α.

[0008] Preferably, the amino acid sequence of the α-chain variable region of the parental TCR is shown in SEQ ID NO: 7, and the amino acid sequence of the β-chain variable region is shown in SEQ ID NO: 8.

[0009] Preferably, the TCR targeting the KRAS G12D mutation further includes a TCR constant region or a fragment thereof, wherein the TCR constant region is of mouse or human origin.

[0010] Preferably, the amino acid sequence of the α chain of the TCR targeting the KRAS G12D mutation is as shown in any one of CDR1α-D1F (SEQ ID NO: 23), CDR1α-G3A (SEQ ID NO: 25), and CDR3α-A3V (SEQ ID NO: 27), and the amino acid sequence of the β chain is as shown in SEQ ID NO: 29.

[0011] The present invention further provides a nucleotide sequence encoding the TCR that targets the KRAS G12D mutation.

[0012] The present invention further provides a gene expression vector containing the nucleotide sequence, wherein the nucleotide sequence encoding the α chain and the nucleotide sequence encoding the β chain are on the same gene expression vector or on two separate gene expression vectors.

[0013] The present invention also provides transgenic expression cells comprising the gene expression vector.

[0014] The present invention also provides the use of the TCR targeting KRAS G12D mutation, the nucleotide sequence, the gene expression vector, or the transgenic expression cell in the preparation of products for detecting, diagnosing, preventing, alleviating, or treating KRAS G12D positive diseases or conditions.

[0015] Preferably, the patient carries the HLA-C*08:01 or HLA-C*08:02 allele.

[0016] The enhanced TCR provided by this invention is obtained by mutation of the parental TCR10, possessing a highly sensitive ability to bind to target antigen peptides, significantly improving target cell recognition, and mediating the specific killing of antigen-positive target cells by effector cells. Furthermore, it does not exhibit allogeneic reactions to different HLA subtypes. It can be used to treat various cancers caused by KRAS G12D positivity. Attached Figure Description

[0017] Figure 1 For flow cytometry detection of TCR10 and HLA-C*08:01-KRAS G12D Detection of binding affinity of tetramers.

[0018] Figure 2 To detect TCR10-transduced Jurkat-CD8-ZsGreen cells by HLA-C*08:01-KRAS in flow cytometry G12D The situation of antigen activation.

[0019] Figure 3 The results of the construction and functional screening of the TCR10 single-point mutation library are shown below. Figure 3 In the figure, A represents the viral infection positivity rate verification of two single-point mutation libraries; Figure 3 In the figure, B represents the activation rate assessment after co-incubation of two single-point mutation libraries with the target antigen.

[0020] Figure 4 The sequence fitness landscape of TCR10 α-chain single-point mutation libraries after functional screening.

[0021] Figure 5 The sequence fitness landscape of the TCR10 β-chain single-point mutation library after functional screening.

[0022] Figure 6 The activation rates of Jurkat-CD8-ZsGreen cells transduced with the target antigen were compared.

[0023] Figure 7 The results show the effects of different concentrations of antigen on Jurkat-CD8-ZsGreen cells transduced with candidate mutants.

[0024] Figure 8 To evaluate the cytotoxic activity of candidate TCR mutants after co-incubation with target cells carrying the target antigen (lactate dehydrogenase release assay).

[0025] Figure 9 The results show the release of cytokines after the candidate TCR was co-incubated with target cells carrying the target antigen. Detailed Implementation

[0026] The sequences involved in this application are: The amino acid sequence of CDR1α in human TCR10: SEQ ID NO: 1.

[0027] The amino acid sequence of CDR2α in human TCR10: SEQ ID NO: 2.

[0028] The amino acid sequence of CDR3α in human TCR10: SEQ ID NO: 3.

[0029] The amino acid sequence of CDR1β in human TCR10: SEQ ID NO: 4.

[0030] The amino acid sequence of CDR2β in human TCR10: SEQ ID NO: 5.

[0031] The amino acid sequence of CDR3β in human TCR10: SEQ ID NO: 6.

[0032] The amino acid sequence of the α-chain variable region in human TCR10: SEQ ID NO: 7.

[0033] The amino acid sequence of the β-chain variable region in human TCR10: SEQ ID NO: 8.

[0034] The amino acid sequence of the α chain of CD8: SEQ ID NO: 9.

[0035] The amino acid sequence of the β chain of CD8: SEQ ID NO: 10.

[0036] Gene sequence of self-cleaving peptide P2A: SEQ ID NO: 11.

[0037] ZsGreen reporter gene sequence: SEQ ID NO: 12.

[0038] The α-chain amino acid sequence of human TCR10: SEQ ID NO: 13.

[0039] The β-chain amino acid sequence of human TCR10: SEQ ID NO: 14.

[0040] The α-chain gene sequence of human TCR10 (including the variable and constant regions): SEQ ID NO: 15.

[0041] Human TCR10 β-chain gene sequence (including variable and constant regions): SEQ ID NO: 16.

[0042] The amino acid sequence of HLA-C*08:01: SEQ ID NO: 17.

[0043] The amino acid sequence of human β2m: SEQ ID NO: 18.

[0044] The amino acid sequence of KRAS mutant antigen peptide 9: SEQ ID NO: 19.

[0045] The amino acid sequence of KRAS mutant antigen peptide 10: SEQ ID NO: 20.

[0046] The amino acid sequence of the α-chain constant region of the mouse TCR: SEQ ID NO: 21.

[0047] The amino acid sequence of the constant region of the β chain of a mouse TCR: SEQ ID NO: 22.

[0048] The α-chain amino acid sequence of mutant CDR1α-D1F: SEQ ID NO: 23.

[0049] The α-chain amino acid sequence of the mutant CDR1α-R2P: SEQ ID NO: 24.

[0050] The α-chain amino acid sequence of mutant CDR1α-G3A: SEQ ID NO: 25.

[0051] The α-chain amino acid sequence of mutant CDR1α-S4I: SEQ ID NO: 26.

[0052] The α-chain amino acid sequence of the mutant CDR3α-A3V: SEQ ID NO: 27.

[0053] The α-chain amino acid sequence of the mutant CDR3α-M5C: SEQ ID NO: 28.

[0054] β-chain amino acid sequences of each mutant: SEQ ID NO: 29.

[0055] The α-chain amino acid sequence of the mutant CDR3α-D6M: SEQ ID NO: 30.

[0056] Example 1: Candidate TCR targeting HLA-C*08:01-KRAS G12D Confirmation Given that HLA-C*08:01 and HLA-C*08:02 both belong to the C*08 subtype and differ only in a few amino acids, we first evaluated whether TCR10, which originally targets HLA-C*08:02, could also recognize KRAS presented by HLA-C*08:01. G12D Mutant peptide.

[0057] Jurkat-CD8-ZsGreen reporter cell construction was based on the reference Vazquez-Lombardi R, Jung JS, Schlatter FS, et al., High-throughput T cell receptor engineering by functional screening identifies candidates with enhanced potency and specificity. Immunity. 2022, 55(10):1953-1966.e10. In short, the endogenous TCR gene of Jurkat cells (human leukemia T lymphocytes) was knocked out using CRISPR-Cas9 gene editing technology, and the co-receptor CD8 (UniProt ID: P01732 for the α chain, amino acid sequence as shown in SEQ ID NO: 9; UniProt ID: P10966 for the β chain, amino acid sequence as shown in SEQ ID NO: 10, both chains were obtained artificially; the self-cleaving peptide P2A (gene sequence as shown in SEQ ID NO: 10) was used. (As shown in SEQ ID NO: 11) The α and β chains were tandemly constructed into the lentiviral expression vector pLVX-puro and the ZsGreen reporter gene (SEQ ID NO: 12). Subsequently, the cell line was cloned and verified by single-cell flow cytometry (FACS), cell expansion and Sanger sequencing. Finally, a cell line that stably expressed human CD8 and ZsGreen reporter gene and did not express endogenous TCR was obtained.

[0058] The human TCR10 gene sequence was artificially synthesized (disclosed in the literature Sim Malcolm JW, Jinghua Lu, Matthew Spencer, et al., High-Affinity Oligoclonal TCRs Define Effective Adoptive T Cell Therapy Targeting Mutant KRAS-G12D[J]. Proceedings of the National Academy of Sciences, 2020, 117(23): 12826-12835), the α-chain amino acid sequence is shown in SEQ ID NO: 13, and the β-chain amino acid sequence is shown in SEQ ID NO: 14. The gene sequence was obtained artificially; the α-chain gene sequence includes variable and constant regions as shown in SEQ ID NO: 15; the β-chain gene sequence includes variable and constant regions as shown in SEQ ID NO: 16. NheI restriction sites (gene sequence 5'-GCTAGC-3') were introduced upstream and downstream of the α chain, and EcoRI restriction sites (gene sequence 5'-GAATTC-3') were introduced upstream and downstream of the β chain, respectively. The α and β chains of TCR were then tandemly constructed into the lentiviral expression vector pLVX-puro via P2A self-cleaving peptide in the order α-P2A-β (where α represents the α chain, P2A represents the 2A self-cleaving peptide, and β represents the β chain).

[0059] The preparation of the pMHC complex followed the previously reported experimental procedure (Zhao Xiang, Elizabeth MKolawole, Waipan Chan, et al., Tuning T Cell Receptor Sensitivity through Catch Bond Engineering[J]. Science, 2022, 376(6589): eabl5282.). In short, HLA-C*08:01 (Uniprot ID: P10321, SEQ ID NO: 17) and human β2m (Uniprot ID: P61769, SEQ ID NO: 18) were expressed and purified as inclusion bodies in *E. coli*. HLA-C*08:01 inclusion bodies, human β2m inclusion bodies, and KRAS mutant antigen peptides (9-peptide, SEQ ID NO: 19 or 10-peptide, SEQ ID NO: 20) were added to refolding buffer, and the refolding reaction was maintained at 4 °C with low-speed magnetic stirring for 32 h. Subsequently, the refolding buffer containing the target product was collected, concentrated by ultrafiltration, and then biotinylated. The biotinylated protein was purified by size exclusion chromatography to obtain the pHLA complex. The pHLA complex was co-incubated with APC-labeled streptavidin (BioLegend Cat #740452) at 4°C to obtain the pHLA tetramer for flow cytometry experiments.

[0060] The artificially synthesized TCR10 gene was introduced into Jurkat-CD8-ZsGreen cells via lentiviral infection to obtain Jurkat-CD8-ZsGreen cells expressing TCR10. HLA-C*08:01-KRAS was then used to analyze the TCR10 expression. G12D The binding affinity of the tetramer was detected by flow cytometry. Results are as follows: Figure 1 As shown, TCR10 can react with two tetramers, including HLA-C*08:01-GA. D GVGKSA (9-peptide, SEQ ID NO: 19) and HLA-C*08:01-GA D It binds to GVGKSAL (10-peptide, SEQ ID NO: 20) and has a stronger affinity for binding to the 10-peptide.

[0061] K562 cells expressing HLA-C*08:01 (named K562-C*08:01 cells) were co-incubated with 10 μg / mL of 9-peptide or 10-peptide, respectively. Subsequently, Jurkat-CD8-ZsGreen cells expressing TCR10 were co-incubated with K562-C*08:01 cells loaded with 9-peptide or 10-peptide, respectively, for 12 h. The activation status of Jurkat-CD8-ZsGreen cells was detected by flow cytometry. Results are shown below. Figure 2 K562-HLA-C*08:01 cells were able to activate TCR10-transduced Jurkat-CD8-ZsGreen cells only when loaded with 10 peptides.

[0062] Figure 1 and Figure 2 The results showed that TCR10 can recognize the KRAS mutant antigen peptide GA presented by HLA-C*08:01. D GVGKSAL has potential, but may be difficult to activate effectively under low antigen density conditions.

[0063] Example 2: Functionally Optimized Targeted HLA-C*08:01-KRAS G12D TCR screening Using the human TCR10 sequence as a template, single amino acid substitutions were performed on the complementarity-determining regions (CDR1 and CDR3) of TCR10 using the NNK random mutation method. The plasmid libraries containing single-point mutations in the α chain were pooled together to form the TCR10-α initial library; the plasmid libraries containing single-point mutations in the β chain were pooled together to form the TCR10-β initial library. After packaging the two plasmid libraries into lentiviruses, they were used to infect Jurkat-CD8-ZsGreen reporter cells, respectively, to construct the TCR10-α and TCR10-β initial cell libraries. The infection efficiency was assessed by detecting CD3-positive cell populations using flow cytometry. Results are as follows: Figure 3 As shown in A, the infection efficiencies of the TCR10 α and β single-point mutant libraries were 26.4% and 26.2%, respectively.

[0064] The TCR10-α and TCR10-β naïve cell libraries were co-incubated with K562-C*08:01 cells and then ex-incubated with KRAS cells. G12D GA D GVGKSAL antigen peptide. The cell library and the number of cells in K562-C*08:01 were 3×10⁻⁶. 6 and 6×10 6 The antigen peptide concentration was 5 μg / mL. After 12 h, the successfully activated positive cell population was sorted by flow cytometry, and the results are as follows. Figure 3As shown in B, the positive rates of the TCR10-α library and the TCR10-β library were 14.2% and 13.8%, respectively. The positive cells were sorted by flow cytometry and then cultured.

[0065] Once the cells had expanded to fill a small dish, the cells were collected and RNA was extracted and reverse transcribed into a cDNA library. Using the cDNA as a template, gene fragments of the α-chain CDR1, CDR2, and CDR3 regions in the α-positive cell population and gene fragments of the β-chain CDR1, CDR2, and CDR3 regions in the β-positive cell population were obtained by PCR. The band lengths of both were approximately 260 bp.

[0066] After deep sequencing of these PCR gene fragments, corresponding sequence maps were drawn, and the results are as follows: Figure 4 and Figure 5 As shown in the figure. By comparing the amino acid enrichment of each site before and after screening, it was found that the enrichment of single amino acid mutants of CDR1α was generally higher than that of other regions, indicating that this region has a stronger capacity to accommodate amino acid substitutions. Mutation in this region is expected to produce HLA-C*08:01-GA. D An activity-enhancing mutant of GVGKSAL.

[0067] Example 3: Validation of TCR function after screening based on model cells To preliminarily verify whether the screened TCR function was improved, this embodiment expressed the highly enriched TCR mutant on Jurkat-CD8-ZsGreen reporter cells and verified its function. To increase the correct pairing and expression of exogenous TCRs on the reporter cell surface, the constant regions of the α and β chains of the TCR mutant were replaced with the constant regions of mouse TCRs. Specifically, the amino acid sequence of the α chain constant region is shown in SEQ ID NO: 21; the amino acid sequence of the β chain constant region is shown in SEQ ID NO: 22.

[0068] In this embodiment, the high-enrichment mutants selected were CDR1α-D1F, CDR1α-R2P, CDR1α-G3A, CDR1α-S4I, CDR3α-A3V, and CDR3α-M5C (α-chain amino acid sequences are shown in SEQ ID NO: 23-28, and β-chain amino acid sequences are shown in SEQ ID NO: 29), and the low-enrichment mutant CDR3α-D6M (α-chain amino acid sequence is shown in SEQ ID NO: 30, and β-chain amino acid sequence is shown in SEQ ID NO: 29) was selected as a negative control.

[0069] After packaging the plasmid DNA of each single-amino acid mutant into lentiviruses, the lentiviruses of each single-point mutant TCR were transfected into Jurkat-CD8-ZsGreen reporter cells. After transfection, the expression rate of each TCR was detected by flow cytometry to ensure that the expression rate of each single-point mutant TCR and wild-type TCR10 was >90%.

[0070] TCR10 and each single-point mutant reporter cell were co-incubated with K562-C*08:01 cells, and then ex-incubated with KRAS cells. G12D GA D GVGKSAL antigen peptide. The number of reporter cells and K562-C*08:01 cells were 1×10⁻⁶. 5 and 2×10 5 The antigen peptide concentration was 5 μg / mL. The activation rate of each reporter cell was measured after 12 h. The results are as follows: Figure 6 As shown, Jurkat-CD8-ZsGreen cells transduced with the negative control mutant CDR3α-D6M could not be activated by the target antigen, while CDR1α-D1F, CDR1α-R2P, CDR1α-W1F, CDR1α-S4I, CDR3α-A3V, and CDR3α-M5C could all be successfully activated by the target antigen, and the activation rates were all higher than those of the parent plate TCR10.

[0071] Furthermore, TCR10 and various single-point mutant reporter cells were co-incubated with K562-C*08:01 cells, and then exoculated with different concentrations of KRAS. G12D GA D The GVGKSAL antigenic peptide was further evaluated against three positive mutants: CDR1α-D1F, CDR3α-A3V, and CDR1α-G3A. The number of reporter cells and K562-C*08:01 cells were 1×10⁻⁶. 5 and 2×10 5 The antigenic peptide concentrations were 0.046, 0.034, 0.023, 0.011, 0.006, 0.002, 0.001, and 0.572 × 10⁻⁶, respectively. -3 1.140×10 -4 5.721×10 -5 1.144×10 -5 5.721×10 -6 μM. Results as follows Figure 7 As shown, the three candidate mutants showed higher sensitivity to the antigen than the parent TCR10 at multiple peptide concentrations.

[0072] Example 4: Validation of the antitumor activity of screened TCRs based on TCR-T cells Example 3 has demonstrated that the CDR1α-D1F, CDR3α-A3V, and CDR1α-G3A mutants can recognize the target antigen in vitro, and their activation efficiency is significantly improved compared to wild-type TCR10. Therefore, this example further investigates the tumor-killing activity of these three mutants when applied to TCR-T therapy.

[0073] TCR-T cells expressing CDR1α-D1F, CDR3α-A3V, CDR1α-G3A, or TCR10 were used as effector cells. To avoid infection from expression levels affecting activity assessment and to ensure consistent transduction positivity rates among TCR-T cells, untransduced PBMCs were expanded and cultured in parallel as effector cell controls. K562-C*08:01 cells were used as HLA-antigen peptide-matched positive target cells and were inoculated with 5 μg / mL target antigen GA. D GVGKSAL co-incubation was performed. The ratio of TCR-T cells to target cells was 30W:10W. After 72 h of co-culture, LDH release was assessed, and ELISA was used to detect the release level of the cytokine TNF-α, thereby evaluating the differences in cell activity.

[0074] The results are as follows Figure 8 As shown, the LDH release experiment indicated that both TCR10-WT and the mutant mediated LDH release, and the LDH release mediated by the three mutants was higher than that WT, indicating that the mutants CDR1α-G3A, CDR1α-D1F, and CDR3α-A3V have the ability to target and kill the antigen HLA-C*08:01-GA. D GVGKSAL's capabilities and lethality are superior to TCR10-WT.

[0075] Results of cytokine release experiments as follows Figure 9 As shown, no cytokine release was detected in any of the TCR-T cells without the exotropic peptide. After exotropic peptide ...

Claims

1. A TCR targeting the KRAS G12D mutation, comprising an α-strand variable region containing CDR1α, CDR2α, and CDR3α and a β-strand variable region containing CDR1β, CDR2β, and CDR3β, characterized in that, The amino acid sequences of CDR1α, CDR2α, and CDR3α in the parental TCR before mutation are shown in SEQ ID NO: 1-3, and the amino acid sequences of CDR1β, CDR2β, and CDR3β are shown in SEQ ID NO: 4-6, respectively. The TCR targeting the KRAS G12D mutation has a G3A mutation or a D1F mutation relative to the parental TCR's CDR1α, or the TCR targeting the KRAS G12D mutation has an A3V mutation relative to the parental TCR's CDR3α.

2. The TCR targeting the KRAS G12D mutation according to claim 1, characterized in that, The amino acid sequence of the α-chain variable region of the parental TCR is shown in SEQ ID NO: 7, and the amino acid sequence of the β-chain variable region is shown in SEQ ID NO:

8.

3. The TCR targeting the KRAS G12D mutation according to claim 1, characterized in that, The TCR targeting the KRAS G12D mutation also includes a TCR constant region or a fragment thereof, wherein the TCR constant region is of mouse or human origin.

4. The TCR targeting the KRAS G12D mutation according to claim 1, characterized in that, The amino acid sequence of the α chain of the TCR targeting the KRAS G12D mutation is shown in any one of SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 27, and the amino acid sequence of the β chain is shown in SEQ ID NO:

29.

5. A nucleotide sequence encoding the TCR targeting the KRAS G12D mutation as described in any one of claims 1 to 4.

6. A gene expression vector comprising the nucleotide sequence of claim 5, characterized in that, The nucleotide sequence encoding the α chain and the nucleotide sequence encoding the β chain are either on the same gene expression vector or on two separate gene expression vectors.

7. Transgenic expression cells comprising the gene expression vector of claim 6.

8. The use of the TCR targeting KRAS G12D mutation as described in any one of claims 1 to 4, the nucleotide sequence as described in claim 5, the gene expression vector as described in claim 6, or the transgenic expression cell as described in claim 7 in the preparation of products for the detection, diagnosis, prevention, relief, or treatment of KRAS G12D positive diseases or conditions.

9. The use according to claim 8, characterized in that, The patient carries the HLA-C*08:01 or HLA-C*08:02 allele.

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