An activity-enhanced TCR and its applications

Abstract

This invention discloses an enhanced TCR and its uses. The enhanced TCR includes an α-chain variable region containing CDR1α, CDR2α, and CDR3α, and a β-chain variable region containing CDR1β, CDR2β, and CDR3β; wherein the enhanced TCR has a T30H mutation relative to the parental TCR's CDR1α, or the enhanced TCR has an S102H or G99H mutation relative to the parental TCR's CDR3α. The enhanced TCR provided by this invention is obtained by mutation based on the parental TCR, possessing a highly sensitive ability to bind target antigen peptides, significantly improving target cell recognition ability, and mediating the specific killing effect of effector cells on antigen-positive target cells, and exhibiting no allogeneic reaction to different HLA subtypes. It can be used to treat various cancers caused by KRAS G12D positivity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an activity-enhancing TCR and its applications. Background Technology

[0002] Kirsten Rat Sarcoma Viral Oncogene (KRAS) is a common tumor driver gene, with mutation frequencies varying depending on the tumor type, ranging from 90% in pancreatic cancer to 50% in colorectal cancer, 32% in lung adenocarcinoma, and less than 5% in many rare tumors. KRAS mutations are predominantly single-base missense mutations, with 98% located at positions 12, 13, or 61. Among all mutations, G12D mutation is one of the most common (Huang L, Guo Z, Wang F, et al. KRAS mutation: from undruggable to druggable in cancer[J]. Signal Transduction and Targeted Therapy, 2021, 6(1), 386: 1-16.). The antigenic peptide VVVGADGVGK is a key epitope fragment of the KRAS_G12D protein and can be presented by HLA-A*11:01. Therefore, immunotherapy targeting KRAS_G12D-HLA-A*11:01 can be applied to a wide range of patient populations.

[0003] Based on the specificity of TCRs for tumor antigens, T cell receptor-engineered T cell therapy (TCR-T) uses genetic engineering to transfer TCRs capable of specifically binding to target antigens into peripheral blood-derived T cells, enabling them to specifically recognize and kill tumor cells expressing the antigens. Neoantigen targets such as KRAS_G12D-HLA-A*11:01 are ideal targets for TCR-T therapy due to their high tumor specificity.

[0004] The JDI TCR, which specifically targets KRAS_G12D-HLA-A*11:01 and was discovered by Poole A et al. (Poole A, Karuppiah V, Hartt A, et al. Therapeutic highaffinity T cell receptor targeting a KRAS_G12D cancer neoantigen[J]. Nature Communications, 2022, 13(1), 5333:1-13), is a natural TCR screened from patients. While it exhibits high specificity, its affinity is too weak (KD = 63 μM), resulting in insufficient ability to induce T cells to kill tumors. The affinity-matured mutant JDI-a41b1 TCR shows a significantly enhanced 3D affinity (KD = 0.743 pM), but exhibits significant non-specific killing when used in TCR-T therapy, limiting its clinical application. Therefore, functional optimization of the JDI TCR is urgently needed to obtain a TCR molecule with both excellent activity and specificity.

[0005] Currently, phage and yeast display systems have been successfully used for TCR optimization, but they cannot fully reflect human post-translational modifications (such as glycosylation). This may adversely affect the optimization process of TCRs composed of natural glycoproteins. Mammalian cell surface display technology can effectively solve these problems. The displayed TCRs are closest to the natural TCRs in terms of molecular structure, physicochemical properties, and biological function, and are stably and highly expressed in mammalian cells. Therefore, mammalian cell display technology has potential advantages that other technologies do not possess. Summary of the Invention

[0006] To address the aforementioned shortcomings in the prior art, this invention provides a TCR with enhanced activity and its applications.

[0007] The present invention first provides an activity-enhancing TCR, comprising an α-chain variable region containing CDR1α, CDR2α and CDR3α and a β-chain variable region containing CDR1β, CDR2β and CDR3β;

[0008] Wherein, the TCR with enhanced activity has a T30H mutation relative to the CDR1α of the parental TCR, or the TCR with enhanced activity has an S102H or G99H mutation relative to the CDR3α of the parental TCR;

[0009] The parental TCR contains the amino acid sequences CDR1α as shown in SEQ ID NO:1, CDR2α as shown in SEQ ID NO:2, CDR3α as shown in SEQ ID NO:3, CDR1β as shown in SEQ ID NO:4, CDR2β as shown in SEQ ID NO:5, and CDR3β as shown in SEQ ID NO:6.

[0010] The amino acid positions are numbered according to the naming rules of the International Immunogenetic Information System (IMGT).

[0011] Preferably, the amino acid sequence of the variable region of the α chain of the parental TCR is AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFL IRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSGPSGAGSYQLTFGKGTKLSVIPN (as shown in SEQ ID NO:22);

[0012] The amino acid sequence of the β-chain variable region of the parental TCR

[0013] NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPMGLRLI

[0014] HYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYGPGQHNSPLHFGNGTRLTVT (shown in SEQ ID NO: 23).

[0015] More preferably, the activity-enhanced TCR further comprises a TCR constant region or a fragment thereof, wherein the TCR constant region is a mouse constant region or a human constant region.

[0016] More preferably, the amino acid sequence of the α chain of the activity-enhancing TCR is as shown in any one of SEQ ID NO:12 to 17; and the amino acid sequence of the β chain is as shown in SEQ ID NO:21.

[0017] The present invention further provides a nucleotide sequence encoding the TCR that enhances the activity.

[0018] The present invention also provides a gene expression vector comprising the nucleotide sequence.

[0019] Preferably, 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.

[0020] The present invention also provides a transgenic expression cell, wherein the transgenic expression cell comprises the nucleotide sequence or the gene expression vector.

[0021] The present invention also provides the use of the enhanced TCR, the nucleotide sequence, the gene expression vector, or the transgenic expression cell in the preparation of products for the detection, diagnosis, prevention, relief, or treatment of KRAS G12D positive diseases or conditions.

[0022] Preferably, KRAS G12D positive diseases or conditions are pancreatic adenocarcinoma (PAAD), colorectal cancer (CRC), or lung adenocarcinoma (LUAD).

[0023] The enhanced TCR provided by this invention is obtained by mutation of the parental TCR, 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

[0024] Figure 1 This is the result of flow sorting.

[0025] Figure 2 The selection results for TCR optimization include, among which, Figure 2 In this context, A represents the α-positive cell population; Figure 2 B in the diagram represents the α-negative cell population. Figure 2 C in the text represents the β-positive cell population; Figure 2 D in the diagram represents the β-negative cell population.

[0026] Figure 3 To detect the expression rate and structure of TCR by flow cytometry.

[0027] Figure 4 The activation rate of each reporter cell.

[0028] Figure 5 This demonstrates the results of TCR function after pattern cell validation screening.

[0029] Figure 6 To optimize the function of TCR in the secretion of cytokines by antigen target cells.

[0030] Figure 7 The results are from the cytokine release assay. Detailed Implementation

[0031] Example 1: Screening for Functionally Optimized TCRs

[0032] Using the human JDI TCR sequence (published in the literature Poole A, Karuppiah V, Hartt A, et al. Therapeutic high affinity T cell receptor targeting a KRAS_G12D cancer neoantigen[J]. Nature Communications, 2022, 13(1), 5333:1-13) as a template (α chain amino acid sequence as shown in SEQ ID NO:19, β chain amino acid sequence as shown in SEQ ID NO:20, gene sequence obtained by artificial synthesis), NheI restriction sites were introduced upstream and downstream of the α chain (gene sequence as shown in SEQ ID NO:7), and MluI and EcoRI restriction sites were introduced upstream and downstream of the β chain (gene sequence as shown in SEQ ID NO:8). The α chain and β chain of the TCR were tandemly constructed into the lentiviral expression vector pSIN through a 2A self-cleaving peptide (gene sequence as shown in SEQ ID NO:9) 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).

[0033] Single amino acid substitutions were performed on the complementarity-determining region (CDR) of the JDI TCR using NNK random mutations. The plasmid libraries containing single-point mutations in the α chain were pooled together to form the JDI-α initial library; the plasmid libraries containing single-point mutations in the β chain were pooled together to form the JDI-β initial library. After packaging the two plasmid libraries into lentiviruses, Jurkat-CD8-ZsGreen reporter cells were infected at a 20% infection rate to construct the JDI-α and JDI-β initial cell libraries.

[0034] Construction of Jurkat-CD8-ZsGreen reporter cells: 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 was inserted. 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 expresses human CD8 and NFAT-ZsGreen reporter genes and does not express endogenous TCR was obtained.

[0035] The JDI-α and JDI-β initial cell libraries were respectively compared with cells expressing HLA-A*11:01 (Uniprot ID: A0A583ZB34, amino acid sequence: GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID) K562 cells (named K562-A11 cells) were co-incubated with NO:24) and then ex-incubated with KRAS_G12D VVVGA. D GVGK (SEQ ID NO:25) antigenic peptide. The cell library and K562-A11 cell numbers were 5 × 10⁻⁶. 6 and 1×10 7 The antigen peptide concentration was 10 μg / mL. After 12 hours, the successfully activated positive cell population and the unactivated negative cell population were separated by flow cytometry. The results are as follows: Figure 1 As shown.

[0036] Once the cells had expanded to fill a small dish, they were collected, and RNA was extracted from each cell and reverse transcribed into a cDNA library. Using the cDNA as a template, gene fragments of the α-chain CDR1, CDR2, and CDR3 regions from α-positive and α-negative cell populations, and gene fragments of the β-chain CDR1, CDR2, and CDR3 regions from β-positive and β-negative cell populations were obtained by PCR. The band lengths of all fragments were approximately 250 bp.

[0037] After deep sequencing of these PCR gene fragments, corresponding sequence maps were drawn, and the results are as follows: Figure 2 As shown in the figure, by comparing the site-specific amino acid enrichment of positive and negative cell populations, it was found that the CDR region of the β chain was relatively conserved, with no sites showing high enrichment, and some amino acids enriched in the positive cell population were also enriched in the negative cell population. In contrast, the CDR region of the α chain showed high enrichment at some sites, such as the R28L and T30H mutations in the CDR1 region, the S53H mutation in the CDR2 region, and the G99H, S102D, S102H, and L105M mutations in the CDR3 region.

[0038] Example 2: Validation of TCR function after screening based on model cells

[0039] To preliminarily verify whether the screened TCRs showed improved function, this embodiment expressed highly enriched TCRs on Jurkat-CD8-ZsGreen reporter cells to verify their 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 TCRs were replaced with the constant regions of mouse TCRs. Specifically, the α chain gene sequence includes variable and constant regions as shown in SEQ ID NO:10; the β chain gene sequence includes variable and constant regions as shown in SEQ ID NO:11.

[0040] In this embodiment, the high-enrichment mutants selected were R28L, T30H, G99H, S102D, S102H, and L105M (α-chain amino acid sequences as shown in SEQ ID NO:12-17, β-chain amino acid sequences as shown in SEQ ID NO:21), and the low-enrichment mutant T106W (α-chain amino acid sequence as shown in SEQ ID NO:18, β-chain amino acid sequence as shown in SEQ ID NO:21) was selected as a negative control.

[0041] After packaging plasmid DNA of each single-amino acid mutant into lentiviruses, these lentiviruses were transfected into Jurkat-CD8-ZsGreen reporter cells with each single-point mutant TCR. Following transfection, the expression rate of each TCR was detected by flow cytometry. Results are as follows: Figure 3 As shown, the expression rates of each single-point mutant TCR and wild-type JDI TCR remained stable between 88.2% and 95.7%.

[0042] JDI and each single-point mutant reporter cell were co-incubated with K562-A11 cells, and then ex-incubated with KRAS_G12D VVVGA cells. D GVGK antigen peptide. The number of reporter cells and K562-A11 cells were 1×10⁻⁶. 5 and 2×10 5 The antigen peptide concentration was 10 μg / mL. The activation rate of each reporter cell was measured after 12 hours. The results are as follows: Figure 4 As shown, the activation rate of the negative control mutant αCDR3-T106W was significantly reduced, while the activation rates of each positive mutant were on par with JDI. Furthermore, the activation rates of αCDR1-T30H, αCDR3-G99H, and αCDR3-S102H were slightly higher than those of JDI.

[0043] Therefore, in this embodiment, an incubation peptide concentration gradient activation experiment was conducted to further evaluate the three positive mutants αCDR1-T30H, αCDR3-G99H, and αCDR3-S102H, and the half-maximal effect concentration (EC50) was measured.50 The changes are as follows. Figure 5 As shown, the EC values ​​of JDI-wt, αCDR1-T30H, αCDR3-G99H, and αCDR3-S102H are respectively... 50 The optimal αCDR1-T30H and αCDR3-S102H mutations were 1.374 μM, 0.1117 μM, 0.4332 μM, and 0.08030 μM, respectively. The optimal αCDR1-T30H and αCDR3-S102H mutations increased antigen sensitivity by more than 10 times.

[0044] Example 3: Validation of TCR function after screening based on TCR-T cells

[0045] It was verified that the αCDR1-T30H and αCDR3-S102H mutants showed a significant improvement in activation efficiency compared to the wild type. Therefore, this embodiment further investigated whether these two mutants improved the therapeutic effect when applied to TCR-T therapy.

[0046] TCR-T cells expressing JDI-αCDR1-T30H TCR, JDI-αCDR3-S102H TCR, or JDI TCR were used as effector cells. To avoid infection affecting activity assessment, the transduction positivity rate of each TCR-T cell type was kept consistent (between 50% and 56%). Figure 6 As shown in the figure, PBMCs without TCR transduction were expanded and cultured in parallel as effector cells; K562-KRAS_G12D-A11 cells (KRAS G12D positive, HLA-A*11:01 positive) were used as HLA-antigen peptide-matched positive target cells. Co-culture experiments were conducted with a TCR-T cell:target cell ratio of 20 weeks:10 weeks. Two groups of target cells were established: one group consisted of K562-KRAS_G12D-A11 cells, and the other group consisted of K562-KRAS_G12D-A11 cells incubated with a peptide (10 μg / mL). ELISA was then performed to detect the release levels of IL-2 and TNF-α after co-culture, thereby evaluating the differences in activity.

[0047] The results are as follows Figure 7 As shown, no cytokine release was detected in any TCR-T cells without exotropic peptides. After exotropic peptide exotropic peptides, the killing effect of TCR-T cells significantly increased, and the killing effects of JDI-αCDR1-T30H TCR-T and JDI-αCDR3-S102H TCR-T cells were significantly enhanced compared to JDI-wt TCR-T cells. This indicates that these two mutant TCRs have significantly improved function compared to wild-type cells, but they are still slightly insufficient when facing low-density tumor antigens in clinical settings.

Claims

1. An active boosting TCR, characterized in that, The alpha chain variable region comprises a CDR1a, a CDR2a and a CDR3a, and the beta chain variable region comprises a CDR1b, a CDR2b and a CDR3b; The activity-improved TCR is obtained by mutating the CDR1a of a parent TCR, and the mutation is a T30H mutation of the CDR1a. The amino acid sequence of the CDR1a of the parent TCR is shown as SEQ ID NO: 1, the amino acid sequence of the CDR2a is shown as SEQ ID NO: 2, the amino acid sequence of the CDR3a is shown as SEQ ID NO: 3, the amino acid sequence of the CDR1b is shown as SEQ ID NO: 4, the amino acid sequence of the CDR2b is shown as SEQ ID NO: 5, and the amino acid sequence of the CDR3b is shown as SEQ ID NO:

6.

2. The potency-enhanced TCR of claim 1, wherein, The amino acid sequence of the alpha chain variable region of the parent TCR is shown as SEQ ID NO: 22, and the amino acid sequence of the beta chain variable region of the parent TCR is shown as SEQ ID NO:

23.

3. The potency-enhanced TCR of claim 2, wherein, The activity-improved TCR further comprises a TCR constant region, which is a murine constant region or a human constant region.

4. The potency-enhanced TCR of claim 3, wherein, The amino acid sequence of the alpha chain of the activity-improved TCR is shown as SEQ ID NO:

13. The amino acid sequence of the beta chain is shown as SEQ ID NO:

21.

5. A nucleotide sequence encoding the activity-improved TCR according to any one of claims 1-4.

6. A gene expression vector, characterized by, The nucleotide sequence according to claim 5.

7. The gene expression vector of claim 6, wherein, The nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are on the same gene expression vector or are separately on two gene expression vectors.

8. A transgenic expression cell, wherein, The transgenic expression cell comprises the nucleotide sequence according to claim 5 or the gene expression vector according to claim 6 or 7.

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