Application of TBL1XR1 gene and its gRNA in the preparation of drugs to enhance T cell function

Abstract

This invention provides the application of the TBL1XR1 gene and its gRNA in the preparation of drugs to enhance T cell function. By designing exon 4 sequences of the coding sequence, three gRNA sequences were detected that guide Cas9 protein to specifically cleave the TBL1XR1 coding sequence, inhibiting TBL1XR1 expression. RNA sequencing revealed significant differences in TBL1XR1 gene expression in T cells with different levels of exhaustion, and TBL1XR1 expression was continuously upregulated with increasing T cell exhaustion. TBL1XR1 expression levels showed a high correlation with TIM-3 and a moderate correlation with PD-1 expression. By knocking out TBL1XR1, this invention downregulated the expression of PD-1, TIM-3, and LAG-3 inhibitory receptors on the surface of T cells, thereby enhancing the killing ability of T cells.

Description

Technical Field

[0001] This invention relates to the field of T cell molecular biology, specifically to the application of the TBL1XR1 gene and its gRNA in the preparation of drugs that enhance T cell function. Background Technology

[0002] Primary liver cancer is one of the most common malignant tumors in my country. About 90% of primary liver cancers are hepatocellular carcinoma (HCC) (Villanueva, A., Hepatocellular Carcinoma. N Engl J Med, 2019. 380(15): p. 1450-1462.). It not only ranks sixth in global incidence, but is also the third leading cause of cancer death worldwide (Forner, A, et al. Hepatocellular carcinoma. Lancet, 2018. 391(10127): p. 1301-1314.). Currently, only 10-25% of early-stage HCC patients can be treated with surgical resection, ablation, and liver transplantation. The vast majority of HCC patients are diagnosed at an advanced stage and lack effective treatment options (Yang, JD, et al., A global view of hepatocellular carcinoma: trends, risk, prevention and management. Nat Rev Gastroenterol Hepatol, 2019.16(10):p.589-604.).Despite the availability of multiple kinase inhibitors (such as sorafenib (Bruix, J, et al., Adjuvant sorafenib for hepatocellular carcinoma after resection or ablation (STORM): a phase 3, randomized, double-blind, placebo-controlled trial. Lancet Oncol, 2015. 16(13): p. 1344-54.), lenvatinib (Kudo, M., et al., Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomized phase 3 non-inferiority trial. Lancet, 2018. 391(10126): p. 1163-1173.), regorafenib (Bruix, J., et al., Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a...), ... Randomized, double-blind, placebo-controlled, phase 3 trial. Lancet, 2017, 389(10064): p.56-66.) and VEGFR2 antagonists (ramucirumab) (Zhu, AX, et al., Ramucirumab after sorafenib in patients with advanced hepatocellular carcinoma and increased alpha-fetoprotein concentrations (REACH-2): a randomized, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol, 2019, 20(2): p.282-296.) can be used as alternative treatments, but their effectiveness is very limited.Recently, chimeric antigen receptor-T (CAR-T) cell immunotherapy based on T cells has shown promising efficacy in clinical trials (Shi, D., et al., Chimeric Antigen Receptor-Glypican-3 T-Cell Therapy for Advanced Hepatocellular Carcinoma: Results of Phase I Trials. Clin Cancer Res, 2020. 26(15): p. 3979-3989.). This suggests that HCC is a tumor sensitive to immunotherapy, and therefore immunotherapy is expected to become the most promising means of treating intermediate and advanced HCC.

[0003] However, in the tumor microenvironment, T cells gradually undergo dysfunction, a phenomenon known as T cell exhaustion (Blank, CU, et al., Defining 'T cell exhaustion'. Nat Rev Immunol, 2019. 19(11): p. 665-674.). Exhausted T cells express a series of inhibitory receptors on their surface, such as PD-1 (programmed cell death-1), TIM-3 (T cell immunoglobulin and mucin-domain-containing molecule-3), and LAG-3 (lymphocyte-activation gene-3), which in turn leads to a decrease in the T cell's ability to kill tumor cells (Crespo, J., et al., T cell anergy, exhaustion, senescence, and stemness in the tumor microenvironment. Curr Opin Immunol, 2013. 25(2): p. 214-21.).

[0004] T cell exhaustion can be classified into reversible early exhaustion and irreversible late exhaustion based on the expression level of PD-1 on the T cell surface (Wherry, EJand M.Kurachi, Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol, 2015.15(8):p.486-99.). Currently, the function of early exhausted T cells can be restored by blocking inhibitory receptors on the surface of T cells with blocking antibodies (Chong, EA, et al., Pembrolizumab for B-cell lymphomas relapsing after or refractory to CD19-directed CAR T-cell therapy. Blood, 2022.139(7):p.1026-1038.). However, many cancer patients are not sensitive to blocking antibodies, which is likely because this method cannot reverse the function of late exhausted T cells in the patient's body. Therefore, developing anti-tumor drugs that inhibit the development of T cells into a late exhaustion state is an urgent problem to be solved in anti-tumor immunotherapy.

[0005] Currently, research on the mechanisms of T cell exhaustion has revealed that several molecules (such as Blimp-1, NFAT, and TOX) can regulate the exhaustion state of T cells (Mackay, LK, et al., Hobit and Blimp1 instruct universal transcriptional program of tissue residency in lymphocytes. Science, 2016. 352(6284): p. 459-63. Martinez, GJ, et al., The transcription factor NFAT promotes exhaustion of activated CD8(+) T cells. Immunity, 2015. 42(2): p. 265-278. Khan, O., et al., TOX transcriptionally and epigenetically programs CD8(+) T cell exhaustion. Nature, 2019. 571(7764): p. 211-218.). This suggests that these molecules could serve as therapeutic targets to reverse T cell exhaustion. However, research into the mechanisms of T cell exhaustion is still in its early stages, and developing anti-tumor immunotherapy methods by reversing T cell exhaustion is fraught with challenges and uncertainties.

[0006] The TBL1XR1 gene is located on human chromosome 3 at q26.32, with a full length of 7959 bp, encoding 514 amino acids. The protein is expressed in cells, and its NCBI Gene ID number is 79718. The TBL1XR1 gene maintains high similarity across all species and plays an important role in transcriptional repression mediated by nuclear receptors (NRs) and other transcription factors (TFs) (Yoon, HG, et al., Purification and functional characterization of the human N-CoR complex: the roles of HDAC3, TBL1 and TBLR1. EMBO J, 2003. 22(6): p. 1336-46. Perissi, V., et al., A corepressor / coactivator exchange complex required for transcriptional activation by nuclear receptors and other regulated transcription factors. Cell, 2004. 116(4): p. 511-26.).Studies have reported that TBL1XR1 is involved in primary lung squamous cell carcinoma (Liu, Y., et al., Identification of genes differentially expressed in human primary lung squamous cell carcinoma. Lung Cancer, 2007, 56(3): p. 307-17.), cervical cancer (Wang, J., et al., TBLR1 is a novel prognostic marker and promotes epithelial-mesenchymal transition in cervical cancer. Br J Cancer, 2014, 111(1): p. 112-24.), nasopharyngeal carcinoma (Chen, SP, et al., Transducin beta-like 1X-linked receptor 1 suppresses cisplatin sensitivity in nasopharyngeal carcinoma via activation of NF-kappaB pathway. Mol Cancer, 2014, 13: p. 195.), and esophageal squamous cell carcinoma (Liu, L., et al.). TBL1XR1 promotes lymphangiogenesis and lymphatic metastasis in esophageal squamous cellcarcinoma. Gut, 2015, 64(1):p.26-36. It is also aberrantly expressed in B-cell leukemia (Venturutti, L., et al., TBL1XR1 Mutations Drive Extranodal Lymphoma by Inducing a Pro-tumorigenic Memory Fate. Cell, 2020, 182(2):p.297-316e27.), such as through gene translocation, deletion, and mutation. These abnormalities are closely related to the stage, metastasis, and prognosis of advanced tumors. However, the relationship between the TBL1XR1 gene and the exhaustion of infiltrating T cells in hepatocellular carcinoma (HCC) tissue, as well as its application methods, have not yet been reported. Therefore, there is an urgent need in this field to utilize the TBL1XR1 gene as a target to develop T-cell-based immunotherapy methods, thereby reversing T-cell exhaustion and enhancing T-cell function for the treatment of HCC. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies by isolating T cells from HCC (hepatocellular carcinoma) tissue samples. Transcriptome expression profiling analysis revealed that the TBL1XR1 gene plays a crucial role in regulating T cell exhaustion. Furthermore, gRNA targeting this gene was designed for drug application to reverse T cell exhaustion. To achieve the above objectives, this invention employs the following technical solution:

[0008] The TBL1XR1 gene and its gRNA are used to prepare drugs that enhance T cell function.

[0009] To optimize the above technical solution, the specific measures also include:

[0010] Furthermore, we designed the exon 4 sequence of the coding sequence and detected three gRNA sequences that guide the Cas9 protein to specifically cleave the TBL1XR1 coding sequence and inhibit TBL1XR1 expression.

[0011] A gRNA, wherein the nucleotide sequence of the gRNA is selected from:

[0012] 1) Any nucleotide sequence as shown in SEQ ID NO.3 to SEQ ID NO.5;

[0013] 2) The nucleotides in 1) are subjected to substitution and / or deletion of one or more bases and, or addition of a nucleotide sequence that has the same function as the nucleotide sequence in 1).

[0014] The above gRNA is used to knock out the TBL1XR1 gene.

[0015] A method for targeting and specifically knocking out TBL1XR1 in T cells using CRISPR / Cas9 gene editing technology includes the following steps:

[0016] 1) Design of TBL1XR1-gRNA: Based on the TBL1XR1 gene sequence on NCBI Gene and combined with the CRISPR / Cas9 gene editing principle, three gRNA sequences were designed by selecting three sites located in exon 4 of the TBL1XR1 gene.

[0017] 2) Preparation of RNP: CRISPR-Cas9 protein and 3 gRNAs were co-incubated to prepare the RNP complex;

[0018] 3) Electroporation: T cells were electroporated using the Lonza 4D-Nuclefector electroporator, and the RNP complex was electroporated into T cells using the P3 Primary cell 4D-Nuclefector X Kit.

[0019] 4) Electroporation mode: activated human T cell.

[0020] The beneficial effects of this invention are as follows: Compared with the prior art, this invention has the following advantages:

[0021] 1) This invention is the first to discover that the TBL1XR1 gene plays a key role in regulating T cell exhaustion;

[0022] 2) This invention is the first to design the gRNA sequence of TBL1XR1 and successfully knock out the TBL1XR1 gene in T cells, obtaining exhausted T cells with specific knockout of TBL1XR1.

[0023] 3) This invention demonstrates for the first time that TBL1XR1 gene knockout enhances the killing ability of T cells by downregulating the expression of inhibitory receptors PD-1, TIM-3 and LAG-3 on the surface of T cells. Attached Figure Description

[0024] Figure 1 A flowchart illustrating the process of identifying genes related to T depletion;

[0025] Figure 2 A schematic diagram illustrating the expression of the TBL1XR1 gene in T cells at different levels of exhaustion;

[0026] Figure 3 A schematic diagram illustrating the correlation between the TBL1XR1 gene and inhibitory receptors on T cells;

[0027] Figure 4 Schematic diagram of TBL1XR1-gRNA design;

[0028] Figure 5 A schematic diagram showing the protein expression of the TBL1XR1 gene before and after knockout in T cells;

[0029] Figure 6 A schematic diagram illustrating the expression of the inhibitory receptor in TBL1XR1 gene before and after knockout in T cells;

[0030] Figure 7 A schematic diagram illustrating the tumor-killing ability of the TBL1XR1 gene before and after knockout in T cells. Detailed Implementation

[0031] Example 1: Screening of the TBL1XR1 gene

[0032] 1.1 Obtaining infiltrative T cells

[0033] Tumor tissue from HCC patients was collected, collagenase was added, and the tissue was homogenized into single cells using a homogenizer. Then, Precoll solution was added for density gradient centrifugation, and the white haze layer was collected and washed once with PBS to isolate infiltrating T lymphocytes from the tissue. Figure 1 Using flow cytometry, non-exhausted T cells (PD-1 and TIM-3) were sorted based on their expression levels. - TIM-3 - 3 cases, early exhaustion T cells (PD-1) int TIM-3 + 4 cases and late-stage exhaustion cells (PD-1) hi TIM-3 + )3 cases.

[0034] 1.2 Identifying highly expressed genes in exhausted T cells

[0035] High-throughput RNA sequencing analysis revealed significant differences in TBL1XR1 gene expression in T cells with varying degrees of exhaustion, and that TBL1XR1 expression was continuously upregulated with increasing T cell exhaustion. Figure 2 ).

[0036] 1.3 Analysis of the relationship between the TBL1XR1 gene and T cell exhaustion-related inhibitory receptors

[0037] Correlation analysis was performed on the expression abundance of TBL1XR1 in T cells of four HCC patients in each group and the expression abundance of various T cell exhaustion-related factors. Pearson correlation coefficient calculation results showed that the expression level of TBL1XR1 was highly correlated with TIM-3, while it was moderately correlated with PD-1 expression. Figure 3 ).

[0038] Example 2: Knockout of the TBL1XR1 gene

[0039] 2.1 Isolation of PBMCs from peripheral blood

[0040] Peripheral blood was collected from healthy individuals and mixed with an equal volume of PBS. A 50 mL centrifuge tube was prepared, and Ficoll lymphocyte separation medium was added. The blood and PBS mixture was then slowly added to the tube, taking care not to disrupt the surface of the Ficoll lymphocyte mixture. The tube was centrifuged at 2000 rpm for 10 minutes, with an ascending speed of 9 and a descending speed of 1. After centrifugation, the white layer in the middle was carefully aspirated; this was PBMCs. 20 mL of PBS buffer was added, and the tube was centrifuged again at 300 × g for 8 minutes. The supernatant was discarded, and the pellet was resuspended in PBS buffer. The pellet was centrifuged again at 300 × g for 8 minutes. The supernatant was discarded, and the pellet was resuspended in PBS buffer. 10 μL of 0.1% trypan blue and 10 μL of the cell suspension were mixed thoroughly, and the cells were counted.

[0041] 2.2 Transfection of AFP TCR cells

[0042] HEK293T cells were seeded in DMEM + 10% FBS medium. When the density was about 70%, the viral vector containing the AFP TCR gene and the helper plasmid were added to the cells according to the instructions of Lipofectamine 3000 Transfection Reagent. The medium was changed after 6 hours, and the viral fluid was collected after 48 hours for later use.

[0043] The isolated PBMCs and magnetic beads (Human T-activation anti-CD3 / 28 dynabeads) were stimulated at a 1:1 ratio in RPMI-1640 + 50 IU / mL IL-2 + 10% FBS medium for 72 h. Then, the AFP TCR viral solution was added to T cells at an MOI of 5. After 48 h, the infected T cells were stained with anti-mouse beta chain and anti-CD3 antibodies, and the TCR positivity rate was detected by flow cytometry.

[0044] Experimental results are as follows Figure 5 As shown in B: In Figure 5 In section B, isotype is used as a blank control in the analysis. The CD3 positivity rate of both uninfected and infected T cells was over 90%, indicating that the vast majority of stimulated PBMCs were T cells. Simultaneously, using anti-mouse TCR beta chain antibodies, only AFP TCR-infected T cells showed a positivity rate of approximately 30%, indicating successful T cell transfection.

[0045] 2.3 Preparation of Cas9 protein: gRNA ribonucleoprotein (RNP)

[0046] Based on the TBL1XR1 gene sequence on NCBI gene and combined with the CRISPR / Cas9 gene editing principle, three sites were selected from exon 4 of TBL1XR1, and three gRNA sequences were designed.

[0047] The gRNA was dissolved in NEB buffer 3.1 and then incubated with Cas9 recombinant protein at room temperature for 20 minutes to form an RNP complex for subsequent experiments. The molar ratio of Cas9 protein to gRNA was 1:1.5, and the molar ratio of the three gRNAs was also 1:1:1.

[0048] like Figure 4 As shown, the RNP complex formed by gRNA-1 and Cas9 binds to the antisense strand of the TBL1XR1 gene, the RNP complex formed by gRNA-2 and Cas9 binds to the antisense strand of the TBL1XR1 gene, and the RNP complex formed by gRNA-3 and Cas9 binds to the sense strand of the TBL1XR1 gene. This indicates that the RNPs of the Cas9 recombinant protein and the three gRNAs bind to the sense and antisense strands of the TBL1XR1 gene, respectively, thereby cleaving it.

[0049] 2.4 Electrical knockout

[0050] Take 1.2 × 10 6 Infected T cells were placed in 1.5 mL centrifuge tubes and centrifuged at 300×g for 8 minutes. They were then resuspended in PBS and centrifuged again at 300×g for 8 minutes. The T cells were then resuspended in Lonza electroporation buffer, and 120 pmol of Cas9 protein (control) and RNP complex (experimental group) were added to the T cell suspension, respectively. After repeated pipetting and mixing, the mixture was transferred to an electroporation cuvette and electroporated using a Lonza electroporator. Preheated culture medium (RPMI-1640 + 50 IU / mL IL-2 + 10% FBS) was then added, and the cells were cultured further to maintain a T cell density of 1.2 × 10⁶ cells / mL. 6 / mL.

[0051] Example 3: Effects of TBL1XR1 knockout on T cell function

[0052] 3.1 TBL1XR1 knockout efficiency test

[0053] Collect the same number of T cells from the control and experimental groups after electroporation, and centrifuge at 300×g for 8 minutes. Wash once with PBS, centrifuge again at 300×g for 8 minutes. Discard the PBS, resuspend in cell lysis buffer, place on ice for 10 minutes, then centrifuge at 14000×g for 10 minutes to collect the cell lysis supernatant. Add loading buffer to the supernatant, incubate in boiling water for 5 minutes, and then perform 12% SDS-PAGE gel electrophoresis. After electrophoresis, transfer to a membrane, and incubate overnight with primary antibodies against anti-TBL1XR1 and anti-βactin. The next day, incubate with secondary antibody for 1 hour, and then develop the color using a chromogenic solution to compare the expression of TBL1XR1 in wild-type and knockout T cells.

[0054] like Figure 5 As shown in Figure A, T cells were infected with AFP-TCR virus, and then the TBL1XR1 gene was knocked out by electroporation. The knockout effect was verified by Western blotting. Figure 5 As shown in Figure C, NC represents the wild-type control group, and KO represents the experimental group with TBL1XR1 gene knockout. The results indicate that βactin expression in T cells of wild type and TBL1XR1 gene knockout is comparable, suggesting that the overall cell number and total protein content are not significantly different. However, the expression of TBL1XR1 protein in T cells after TBL1XR1 gene knockout is significantly lower than that in wild type, indicating that the TBL1XR1 gene in T cells was successfully knocked out using RNP complex electroporation.

[0055] 3.2 Detection of inhibitory receptor expression on T cell surface after TBL1XR1 gene knockout

[0056] T cells from the control and experimental groups were collected separately after electroporation and centrifuged at 300×g for 8 minutes. They were then washed once with PBS and centrifuged again at 300×g for 8 minutes. Anti-CD3, anti-PD-1, anti-TIM-3, and LAG-3 were added, and the cells were incubated at 4°C in the dark for 20 minutes. After washing twice with PBS, the expression of PD-1, TIM-3, and LAG-3 on the surface of TBL1XR1 gene-knockout T cells before and after the knockout was detected by flow cytometry.

[0057] like Figure 6 As shown, the expression of inhibitory receptors on the surface of T cells knocked out by the TBL1XR1 gene was reduced compared to the wild-type group. This indicates that the TBL1XR1 gene regulates the expression of PD-1, TIM-3, and LAG-3 inhibitory receptors, thereby affecting the occurrence and development of T cell exhaustion.

[0058] 3.3 Effects of TBL1XR1 gene knockout on T cell killing

[0059] Lactate dehydrogenase (LDH) cannot cross the cell membrane in living cells, but when tumor cells are killed by effector cells, the permeability of their cell membrane changes, causing LDH to be released into the culture supernatant. Therefore, by detecting LDH in the culture supernatant, the killing effect function of T cells on target cells can be obtained.

[0060] The target cell density was adjusted to 2 × 10⁶ cells / day using RPMI-1640 medium. 5 / mL, effector T cell density adjusted to 2×10 5 / mL and 4×10 5 / mL. Target cells and effector cells were added to 96-well round-bottom plates at effector-to-target ratios of 1:1 and 2:1. Target cell spontaneous wells, effector cell spontaneous wells, and target cell maximum release wells were set up as control wells. After 24 hours, the supernatant was collected, and the OD value at 490 nm was measured using an ELISA reader according to the LDH kit instructions. The T cell killing function was calculated using the following formula: Killing rate = (Experimental wells - Effector cell spontaneous wells - Target cell spontaneous wells) / (Target cell maximum lysis wells - Target cell spontaneous wells).

[0061] like Figure 7 As shown, T cells knocked out of the TBL1XR1 gene exhibited stronger cytotoxicity than wild-type T cells in the presence of target cells. This indicates that TBL1XR1 gene knockout enhances the cytotoxic ability of T cells by downregulating the expression of inhibitory receptors PD-1, TIM-3, and LAG-3 on the surface of T cells.

[0062] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Application of three gRNAs that inhibit TBL1XR1 gene expression in the preparation of drugs to enhance T cell function; wherein the gRNAs are the three gRNAs shown in SEQ ID NO.3-SEQ ID NO.5, and the molar ratio of the three gRNAs is 1:1:1; the drugs to enhance T cell function are drugs used to restore T cell exhaustion and enhance T cell killing power.

2. The application according to claim 1, characterized in that, We designed exon 4 sequences to detect three gRNA sequences that guide Cas9 protein to specifically cleave the TBL1XR1 coding sequence and inhibit TBL1XR1 expression.

3. A gRNA for reducing TBL1XR1 protein expression, characterized in that, The gRNA consists of three gRNAs with nucleotide sequences as shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively; the molar ratio of the three gRNAs is 1:1:1.

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