TCR with improved activity and application thereof

Abstract

The invention discloses a TCR with improved activity and application thereof. The TCR with improved activity comprises an alpha chain variable region containing CDR1 alpha, CDR2 alpha and CDR3 alpha and a beta chain variable region containing CDR1 beta, CDR2 beta and CDR3 beta, wherein the TCR with the improved activity has T30H mutation relative to CDR1 alpha of the parent TCR, or the TCR with the improved activity has S102H or G99H mutation relative to CDR3 alpha of the parent TCR. The TCR with improved activity provided by the invention is obtained by mutation on the basis of a parent TCR, has high-sensitivity target antigen peptide binding capacity, remarkably improves target cell recognition capacity, can mediate the specific killing effect of effector cells on antigen positive target cells, and does not have allogeneic reaction on different types of HLA (human leukocyte antigen). The compound can be used for treating various cancers positive by KRAS G12D.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a TCR with enhanced activity and uses thereof. Background Art

[0002] Kirsten Rat Sarcoma Viral Oncogene (KRAS) is a common tumor driver gene, and the mutation frequency varies depending on the tumor, 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 mainly single-base missense mutations, of which 98% are located at positions 12, 13, or 61. Among all mutations, G12D mutation is one of the most common mutations (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 antigen 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 may be applicable to a wide range of patient populations.

[0003] Leveraging the TCR's superior specificity for tumor antigens, T cell receptor engineered T cells (TCR-T) therapy uses genetic engineering to transfer a TCR capable of specifically binding to a target antigen into T cells derived from the patient's peripheral blood, enabling them to specifically recognize and kill tumor cells expressing the antigen. 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 specifically targeting KRAS_G12D-HLA-A*11:01 discovered by Poole A et al. (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) is a natural TCR screened from patients. It has strong specificity but too weak affinity (KD = 63 μM), resulting in its insufficient ability to induce T cells to kill tumors. The 3D affinity of the affinity-matured mutant JDI-a41b1 TCR is significantly improved (KD = 0.743 pM), but it has obvious nonspecific killing when used in TCR-T therapy, limiting its application in clinical treatment. Therefore, it is urgent to optimize the function of JDI TCR to obtain TCR molecules with excellent activity and specificity.

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

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a TCR with enhanced activity and its use.

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

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

[0009] The parent TCR comprises 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 nomenclature of the International Immunogenetics Information System (IMGT).

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

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

[0013] NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPMGLRLI

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

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

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

[0017] The present invention further provides a nucleotide sequence encoding the TCR with enhanced 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 separated on two gene expression vectors.

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

[0021] The present invention also provides use of the activity-enhanced TCR, the nucleotide sequence, the gene expression vector, or the transgenic expression cell in preparing a product for detecting, diagnosing, preventing, alleviating or treating a KRAS G12D-positive disease or condition.

[0022] Preferably, the KRAS G12D-positive disease or disorder is pancreatic adenocarcinoma (PAAD), colorectal cancer (CRC) or lung adenocarcinoma (LUAD).

[0023] The enhanced TCR provided by the present invention is derived by mutation of the parental TCR. It possesses the ability to bind target antigen peptides with high sensitivity, significantly improving target cell recognition, mediating the specific killing of antigen-positive target cells by effector cells, and exhibiting no alloreactivity to different HLA types. It can be used to treat various cancers that are KRAS G12D-positive. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of flow cytometry sorting.

[0025] Figure 2 Screening results for functionally optimized TCRs, where Figure 2 A in the figure indicates the α-positive cell population; Figure 2 B in the figure is the α-negative cell population; Figure 2 C in the figure is the β-positive cell population; Figure 2 D in the figure represents the β-negative cell population.

[0026] Figure 3 Flow cytometry was used to detect the expression rate of TCR structure.

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

[0028] Figure 5 Figure 2 shows the results of TCR function after screening based on model cells.

[0029] Figure 6 To optimize the cytokine secretion function of TCR to antigen target cells.

[0030] Figure 7 The results of cytokine release assay. DETAILED DESCRIPTION

[0031] Example 1: Screening of functionally optimized TCRs

[0032] Using the human JDI TCR sequence (disclosed 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 (the α chain amino acid sequence is shown in SEQ ID NO: 19, the β chain amino acid sequence is shown in SEQ ID NO: 20, and the gene sequence is obtained by artificial synthesis), NheI restriction sites were introduced upstream and downstream of the α chain (the gene sequence is shown in SEQ ID NO: 7), and MluI and EcoRI restriction sites were introduced upstream and downstream of the β chain (the gene sequence is shown in SEQ ID NO: 8), respectively. Through the 2A self-cleavage peptide (the gene sequence is shown in SEQ ID NO: 9), the α chain and β chain of TCR were constructed in series in the order of α-P2A-β (wherein α represents the α chain, P2A represents the 2A self-cleavage peptide, and β represents the β chain) into the lentiviral expression vector pSIN.

[0033] Single amino acid substitutions were made in the complementarity-determining regions (CDRs) of the JDI TCR using NNK random mutagenesis. Plasmid libraries containing single-point mutations of the α chain were pooled together to form the JDI-α initial library; plasmid libraries containing single-point mutations of the β chain were pooled together to form the JDI-β initial library. Both plasmid libraries were packaged into lentivirus and used to infect Jurkat-CD8-ZsGreen reporter cells at a 20% infection rate, constructing the JDI-α and JDI-β initial cell libraries, respectively.

[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. The cell line was then cloned and verified by single-cell flow cytometry (FACS), cell expansion, and Sanger sequencing. Finally, a cell line that stably expressed human CD8 and NFAT-ZsGreen reporter genes and did not express endogenous TCR was obtained.

[0035] The JDI-α initial cell library and the JDI-β initial cell library were respectively cross-linked with the HLA-A*11:01 expressing cells (Uniprot ID: A0A583ZB34, amino acid sequence: GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:24)) K562 cells (named K562-A11 cells) were co-incubated and incubated with KRAS_G12D VVVGA D GVGK (SEQ ID NO: 25) antigen peptide. The cell numbers of the cell library and K562-A11 were 5×10 6 and 1×10 7 , the antigen peptide concentration was 10μg / mL. After 12 hours, the positive cell population that was successfully activated and the negative cell population that was not activated were sorted by flow cytometry. The results are as follows Figure 1 shown.

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

[0037] After deep sequencing of these PCR gene fragments, the corresponding sequence maps were drawn. The results are as follows Figure 2 As shown in the figure. By comparing the amino acid enrichment of sites in the 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 sites 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 function of the screened TCRs is improved, this example expresses 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 surface of reporter cells, the constant regions of the α and β chains of the TCRs are replaced with the constant regions of mouse TCRs. Specifically, the α chain gene sequence includes the variable region and the constant region as shown in SEQ ID NO: 10; the β chain gene sequence includes the variable region and the constant region as shown in SEQ ID NO: 11.

[0040] The highly enriched mutants selected in this example were R28L, T30H, G99H, S102D, S102H, and L105M (the α chain amino acid sequence is shown in SEQ ID NOs: 12-17, and the β chain amino acid sequence is shown in SEQ ID NO: 21), and the mutant T106W with lower enrichment (the α chain amino acid sequence is shown in SEQ ID NO: 18, and the β chain amino acid sequence is shown in SEQ ID NO: 21) was selected as a negative control.

[0041] After the plasmid DNA of each single amino acid mutant was packaged into lentivirus, the lentivirus of each single point mutant TCR was transfected into Jurkat-CD8-ZsGreen reporter cells. After transfection, the expression rate of each TCR was detected by flow cytometry. The results are shown in Figure 2. Figure 3 As shown, the expression rates of each single-point mutant TCR and wild-type JDI TCR were stable between 88.2% and 95.7%.

[0042] JDI and each single point mutation reporter cell were co-incubated with K562-A11 cells, and KRAS_G12D VVVGA was incubated D GVGK antigen peptide. The number of reporter cells and K562-A11 cells were 1×10 5 and 2×10 5 The antigen peptide concentration was 10 μg / mL. The activation rate of each reporter cell was detected after 12 hours. Figure 4 As shown, the activation rate of the negative control mutant αCDR3-T106W was greatly reduced, the activation rates of each positive mutant were equal to that of JDI, and the activation rates of αCDR1-T30H, αCDR3-G99H and αCDR3-S102H were slightly higher than that of JDI.

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

[0044] Example 3: TCR function after TCR-T cell validation screening

[0045] It has been verified that the αCDR1-T30H and αCDR3-S102H mutants have significantly improved activation efficiency compared to the wild type. Therefore, this example further studies whether these two mutants improve the therapeutic effect when used in 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 the influence of expression level on activity evaluation, the transduction positive rate of each TCR-T cell was controlled to be consistent (between 50% and 56%, such as Figure 6 As shown), PBMCs that were not transduced with TCR were expanded and cultured in parallel as a control for 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 performed with TCR-T cells: target cells = 20W:10W. Two groups of target cells were set up, one group was K562-KRAS_G12D-A11 cells, and the other group was K562-KRAS_G12D-A11 cells and incubated with peptide (10 μg / mL). ELISA experiments were then performed to detect the release levels of IL-2 and TNF-α after co-culture to evaluate the activity differences.

[0047] The results are as follows Figure 7 As shown, in the absence of peptide incubation, no cytokine release was detected by any TCR-T cell. After peptide incubation, the TCR-T cell's cytotoxicity increased significantly, and the cytotoxicity of JDI-αCDR1-T30H TCR-T and JDI-αCDR3-S102H TCR-T cells was significantly enhanced compared to JDI-wt TCR-T cells. This suggests that these two mutant TCRs have significantly improved their functionality compared to wild-type cells, but are still somewhat limited in the presence of low-density tumor antigens in clinical settings.

Claims

1. A TCR with enhanced activity, characterized in that: including an α chain variable region comprising CDR1α, CDR2α, and CDR3α and a β chain variable region comprising CDR1β, CDR2β, and CDR3β; wherein the affinity-enhanced TCR has a T30H mutation relative to CDR1α of the parental TCR, or the affinity-enhanced TCR has an S102H or G99H mutation relative to CDR3α of the parental TCR; The parent TCR comprises 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.

2. The TCR with enhanced activity according to claim 1, characterized in that The amino acid sequence of the α chain variable region of the parent TCR is shown in SEQ ID NO: 22; the amino acid sequence of the β chain variable region of the parent TCR is shown in SEQ ID NO:

23.

3. The TCR with enhanced activity according to claim 2, characterized in that The TCR with enhanced activity 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.

4. The TCR with enhanced activity according to claim 3, characterized in that The amino acid sequence of the α chain of the TCR with enhanced activity is shown in any one of SEQ ID NOs: 12 to 17; The amino acid sequence of the β chain is shown in SEQ ID NO:

21.

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

6. A gene expression vector, characterized in that: Comprising the nucleotide sequence of claim 5.

7. The gene expression vector according to claim 6, characterized in that The nucleotide sequence encoding the α chain and the nucleotide sequence encoding the β chain are on the same gene expression vector or separated on two gene expression vectors.

8. A transgene-expressing cell, characterized in that: The transgenic expression cell comprises the nucleotide sequence of claim 5 or the gene expression vector of claim 6 or 7.

9. Use of the TCR with enhanced activity according to any one of claims 1 to 4, the nucleotide sequence according to claim 5, the gene expression vector according to claim 6 or 7, or the transgenic expression cell according to claim 8 in the preparation of a product for detecting, diagnosing, preventing, alleviating or treating a KRAS G12D-positive disease or condition.

10. The use according to claim 9, characterized in that The KRAS G12D-positive disease or condition is pancreatic cancer, colorectal cancer, or lung adenocarcinoma.

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