Tumor neoantigen polypeptide as well as generation method and application thereof

Abstract

The invention discloses a tumor neoantigen polypeptide as well as a generation method and application thereof. The amino acid sequence of the tumor neoantigen polypeptide is as shown in SEQ ID NO. 12-33. According to the invention, the RNA binding motif protein 39 (RNA Binding Motif Protein 39, RBM39) is knocked down in a targeted manner through CasRx driven by a bladder cancer specific promoter, and the RNA splicing process is effectively interfered, so that the tumor specific neoantigen is successfully induced to be generated in bladder cancer cells, the technical bottlenecks of high antigen heterogeneity, low generation efficiency and the like in the traditional tumor neoantigen therapy are overcome, and the tumor specific neoantigen is obtained. Efficient generation of the tumor neoantigen is realized, a new treatment strategy and a technical path are provided for immunotherapy of bladder cancer, and the tumor neoantigen has important clinical transformation prospect and application potential.

Description

Technical Field

[0001] The present invention relates to the technical field of antigen peptides, and particularly relates to a novel tumor antigen polypeptide, a method for generating the same, and an application thereof. Background Art

[0002] Novel tumor antigens are proteins or polypeptides expressed in tumor cells that are different from normal tissues. They have strong immunogenicity and are key targets for tumor immunotherapy. By inducing or enhancing the generation of novel tumor antigens, the immune system of patients can be activated to recognize and eliminate tumor cells. Traditional methods for discovering novel tumor antigens mostly rely on techniques such as whole-genome sequencing, transcriptome analysis, or proteomics screening. However, these methods are usually limited by factors such as tumor tissue sample heterogeneity, data processing complexity, and high analysis process costs, resulting in limited effects in clinical applications.

[0003] CRISPR-CasRx (or Cas13d) is a breakthrough RNA editing technology in the type II CRISPR-Cas system. Compared with traditional CRISPR systems that target DNA, the Cas13 family (subtypes a, b, c, d), including CasRx, is specifically used for RNA regulation and can avoid permanent changes to the genome. Among the Cas13 family, CasRx has multiple unique advantages: not only does it have extremely high targeting accuracy and extremely low off-target effects, its small size is convenient for viral vector packaging, and more importantly, it is not restricted by the protospacer adjacent motif (PAM). These characteristics make it an ideal choice for precise RNA editing in mammalian cells. Although CasRx has the collateral effect of degrading RNA transcripts around the target, compared with traditional RNA interference techniques and other Cas13 subtypes, it still shows higher knockdown efficiency and unprecedented precision in RNA regulation, and can also achieve precise temporal control of gene expression. More importantly, CasRx can autonomously process the required guide RNA from longer transcripts, which makes it possible to regulate multiple RNA targets simultaneously. Based on its programmability, researchers can use CasRx to achieve precise RNA editing including site-specific modification, thereby precisely regulating the processing and function of RNA. Combining these characteristics, together with the good safety shown in mammalian models, CasRx shows great application potential in the fields of basic research and clinical treatment (especially tumor treatment).

[0004] Bladder cancer (BC) is one of the most common malignant tumors in the urinary system, with approximately 570,000 new cases worldwide each year, especially prominent in men. According to clinical and pathological characteristics, bladder cancer is usually divided into two types: non-muscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Among them, NMIBC is more common, accounting for 70-80% of bladder cancer. Although NMIBC can be treated by transurethral resection of bladder tumor (TURBT), the recurrence rate within 5 years after surgery is as high as 50-70%, and about 15% of patients may progress to muscle-invasive lesions. MIBC has significant invasiveness and metastatic potential. Approximately 25% of patients already have locally advanced or metastatic lesions at the time of initial diagnosis. Even with comprehensive treatment regimens such as radical cystectomy combined with chemotherapy, the 5-year overall survival rate is still less than 50%, and the clinical prognosis is poor. In recent years, certain progress has been made in the field of immunotherapy for bladder cancer. Immune checkpoint inhibitors (such as PD-1 / PD-L1 inhibitors) have been approved for the treatment of bladder cancer and have shown good clinical efficacy. Although immunotherapy brings new hope for the treatment of bladder cancer, its clinical benefits still have obvious limitations. The complex immune escape mechanisms in the tumor microenvironment seriously affect the effect of immunotherapy. Therefore, only less than 30% of bladder cancer patients respond to immunotherapy. Therefore, finding new targets or neoantigens and developing more precise immunotherapy strategies have become an important research direction in the treatment of bladder cancer. Summary of the Invention

[0005] Aiming at the defects in the prior art, the present invention provides a tumor neoantigen polypeptide, a method for generating the same, and an application thereof.

[0006] The present invention provides a tumor neoantigen polypeptide, and the amino acid sequence of the tumor neoantigen polypeptide is shown as SEQ ID NO. 12-33.

[0007] The present invention further provides a nucleic acid molecule encoding the tumor neoantigen polypeptide.

[0008] The present invention further provides an expression cassette or vector comprising the nucleic acid molecule.

[0009] The present invention further provides a cell comprising or expressing the antigen peptide, or comprising the nucleic acid molecule, or the expression cassette or vector.

[0010] The present invention also provides a pharmaceutical composition comprising any one of the antigenic peptides, the nucleic acid molecules, the expression cassette or vector, and the cells described above.

[0011] The present invention also provides a vaccine comprising the antigenic peptide or the nucleic acid molecule described above.

[0012] The present invention also provides a method for inducing the generation of the tumor neoantigen polypeptide, comprising the following steps:

[0013] S1: Design specific sgRNA according to the mRNA sequence of the RBM39 gene;

[0014] S2: Construct a CasRx protein expression vector driven by the mouse SPP1 gene promoter and an sgRNA expression vector driven by the mouse U6 promoter;

[0015] S3: Transfect or infect MB49 cells with the CasRx protein and the sgRNA expression vector described in S2 to interfere with RNA splicing and induce the generation of tumor neoantigen polypeptides.

[0016] The nucleotide sequence of the mouse SPP1 gene promoter is as shown in SEQ ID NO.3, and the nucleotide sequence of the mouse U6 promoter is as shown in SEQ ID NO.1.

[0017] In the present application, by replacing the original promoters of CasRx and sgRNA with the SPP1 gene promoter mSPP1 and the U6 promoter, the working efficiency of the CRISPR / CasRx system in bladder cancer cells is improved and abnormal epitopes are avoided in non-tumor cells.

[0018] In some embodiments, the sgRNA sequences are as shown in SEQ ID NOs. 5-11; preferably, the combination of SEQ ID NO.6 and SEQ ID NO.9.

[0019] The present invention also provides the application of the tumor neoantigen polypeptide in the preparation of anti-bladder cancer drugs.

[0020] The present invention also provides the application of the tumor neoantigen polypeptide in the preparation of diagnostic reagents for bladder cancer.

[0021] In summary, compared with the prior art, the present invention has achieved the following technical effects:

[0022] 1. The present invention discloses a new method for inducing bladder cancer neoantigens based on the CRISPR / CasRx system. By targeting and knocking down RBM39 with CasRx driven by a bladder cancer-specific promoter to interfere with RNA splicing, the generation of tumor neoantigens in bladder cancer cells is successfully induced.

[0023] 2. The present invention optimizes the design of CRISPR / CasRx vectors, combines the precise design of specific sgRNAs, and improves the expression level of CasRx protein in MB49 cells, thereby significantly enhancing the RNA splicing interference efficiency and inducing the generation of tumor neoantigens.

[0024] 3. By designing specific sgRNAs and through systematic screening, the present invention discovers that a combination of two specific sgRNAs can exhibit the best targeted interference effect in in vitro experiments. By using these sgRNAs in combination, the CRISPR / CasRx system can effectively interfere with the expression of RBM39, achieve precise interference of the target gene RBM39 in bladder cancer MB49 cells, and significantly improve the generation efficiency of tumor neoantigens.

[0025] 4. The present invention overcomes the technical bottlenecks such as high neoantigen heterogeneity and low generation efficiency faced by traditional tumor neoantigen therapies, realizes the efficient generation of tumor neoantigens, provides a new treatment strategy and technical path for the immunotherapy of bladder cancer, and has important clinical transformation prospects and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative efforts.

[0027] Figure 1 Construction and application of a CRISPR / CasRx-mediated RBM39 gene regulation system in bladder cancer cells in Example 1 of the present invention; A Detection of the RNA expression levels of the differentially expressed genes obtained by screening in MB49 cells, subcutaneous tumors, bladder epithelium (EP), and whole bladder tissue (BL) using qRT-PCR, n = 4; the data were normalized using the EP group as the standard. B RNA expression levels of different promoter genes in MB49 cells and various tissues, n = 3; the data were normalized using the MB49 group as the standard; C Detection of the promoter efficiency of each candidate promoter in MB49 cells by dual-luciferase reporter gene assay, n = 3; D Detection of the promoter efficiency of human and mouse U6 promoters in MB49 cells by dual-luciferase reporter gene assay, n = 3; E Evaluation of the efficiency of sgRNAs targeting RBM39 by qRT-PCR, n = 3; F Schematic diagram of the CasRx-sgRNA expression system; the data are expressed as mean ± standard deviation; statistical analysis was performed using two-tailed Student's t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0028] Figure 2 This is the result of Example 2 of the present invention that the downregulation of RBM39 promotes anti-tumor effects through immune activation; A is a schematic diagram of the function of the AAV9-delivered CasRx-RBM39 system in a subcutaneous tumor model of C57BL / 6 mice; B is the RNA expression level of RBM39 in subcutaneous tumors after intratumoral injection of AAV-NC or AAV-RBM39, n = 8; C is the protein expression level of RBM39 in subcutaneous tumors after intratumoral injection of AAV-NC or AAV-RBM39, n = 4; D is the expression level of CasRx in tumors and normal tissues after intratumoral injection of AAV-RBM39 detected by qRT-PCR, n = 8; E is the change in the tumorigenic volume of MB49 cells in immunocompetent C57BL / 6 mice after intratumoral injection of AAV-NC or AAV-RBM39, n = 8; F is the change in the tumorigenic volume of MB49 cells in immunodeficient BALB / c nude mice, n = 8; G is the Western blot result of RBM39 after siRNA transfection for 48 hours, n = 3; H is the RBM39 protein level at different time points after MB49 cells were transfected with a mixture of siRNAs three times, n = 3; I is the change in tumor volume in C57BL / 6 mice after subcutaneous inoculation of RBM39-knockdown MB49 cells, n = 10; J is the change in tumor volume of RBM39-knockdown MB49 cells in BALB / c nude mice, n = 10; Data are presented as mean ± standard deviation. Statistical analysis was performed using a two-tailed Student's t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0029] Figure 3 This is Example 3 of the present invention that the downregulation of RBM39 in bladder cancer cells remodels the pro-inflammatory tumor microenvironment; Figures A - F show the analysis of infiltrating lymphocytes CD45 in bladder tumors by flow cytometry after intratumoral injection of AAV + ) and dendritic cells (DCs, CD11c + ) and activated DCs (CD11c + CD86 + MHCII + ) and M1 macrophages (CD11b + CD206 - CD86 + ) and CD8 + T cells and cytotoxic CD8 + T cells (GrB + CD8 +) ratio, n = 7 - 9; Figure G performed enrichment analysis on differentially expressed genes of TILs in tumors treated with AAV-NC and AAV-RBM39, n = 3. NC: AAV-NC group; KD: AAV-RBM39 group.

[0030] Figure 4 This is for Example 4 of the present invention, where RBM39 knockdown triggers abnormal RNA splicing and generates neoantigens; A Flow chart of neoantigen screening based on RNA sequencing; B Filtering process of neoantigen screening; C Representative abnormal AS events caused by RBM39 knockdown; D Upregulated genes related to abnormal AS events; E MHC I molecular haplotypes responsible for presenting screened peptide segments; F Phagocytosis ability of DCs after treatment with different cell lysates; G IFN-γ ELISpot detection results of splenocytes after treatment with different cell lysates; H Comparison of TCR diversity indices between the NC group and the siRNA group; I Representative map of CDR3 peptide length distribution; J Map of differentially expressed VJ genes between the NC group and the KD group; K Differentially expressed CDR3 genes in TRB between the NC group and the KD group.

[0031] Figure 5 This is for Example 5 of the present invention, verification of the immunogenicity of neoantigen epitopes induced by abnormal RNA splicing in vitro and in vivo; A List of candidate peptide segments obtained from in vitro neoantigen prediction based on RNA sequencing; B IFN-γ ELISpot detection results of co-culture of primary bone marrow-derived DCs pre-incubated with DMSO, candidate peptide segments, or OVA with naive spleen-derived CD8 + T cells, n = 3; C Statistical analysis of the spots in (B) by quantitative analysis of spot forming units (SFUs); D Flow chart of the polypeptide immunization experiment in C57BL / 6 mice; E Subcutaneous tumors formed after inoculation of MB49 cells treated with indisulam in C57BL / 6 mice immunized with polypeptides, n = 5; F Statistical results of tumor volumes in E; G Flow chart of the ELISpot experiment; H IFN-γ ELISpot detection results of co-culture of CD8 + T cells from immunized mice with homologous bone marrow DCs pulsed with polypeptides; I IFN-γ ELISpot detection results of co-culture of CD8 + T cells from immunized mice with bone marrow-derived DCs pre-incubated with cell lysates; Data are presented as mean ± standard deviation. Statistical analysis was performed using two-tailed Student's t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0032] Figure 6In Example 6 of the present invention, the combined application of the CRISPR / CasRx-AAV system targeting RBM39 and immune checkpoint blockade (ICB) can effectively improve the anti-tumor immune efficiency; A is the combined treatment protocol of the CRISPR / CasRx vector AAV and ICB in C57BL / 6 mice; B shows the changes in subcutaneous tumor volume in C57BL / 6 mice in the control group (IgG+AAV-NC), αPD-1 group, AAV-RBM39 group, and αPD-1+AAV-RBM39 combined treatment group; Antibody treatment: Intraperitoneal injection, 2 times a week for 3 weeks. AAV treatment: Intratumoral injection, once every 3 days for 3 times. n = 7-9; C shows the statistical results of tumor volume in each group in B on the 28th day after tumor inoculation; D-I are the representative images and quantitative statistical analysis results of the indicated immune cell subsets in the MB49 subcutaneous tumors of each group, n = 7-9; J shows the changes in body weight of mice in each group during and after combined treatment; K shows the concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum after combined treatment; L shows the statistical data of organ weights of mice in each group after combined treatment; M shows the representative hematoxylin and eosin (H&E) staining pictures of each organ after treatment; Data are expressed as mean ± standard deviation. Statistical analysis was performed using two-tailed Student's t-test, *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Detailed implementation manners

[0033] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0035] Cell culture:

[0036] The MB49 cell line was purchased from Shanghai Yikai Biotechnology Co., Ltd., and the NIH / 3T3 cell line was purchased from Shanghai Aisen Biotechnology Co., Ltd. Based on the MB49 cell line, stable cell lines of MB49-GFP and MB49-OVA were constructed using lentivirus technology.

[0037] MB49 and NIH / 3T3 cells were cultured in high-glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 5% carbon dioxide, 37°C constant temperature incubator.

[0038] The experimental animals were provided by Zhuhai Best Experimental Animal Co., Ltd. and maintained by Shenzhen Zhongketai Industrial Holdings Co., Ltd. All animal experiments were approved by the Experimental Animal Ethics Committee of Shenzhen Zhongketai Industrial Holdings Co., Ltd.

[0039] Construction of subcutaneous transplant tumor model: According to the specific experimental purpose, 6-8 week-old male C57BL / 6 mice or BALB / c nude mice were fixed in supine position and 5×10 5 MB49 cells.

[0040] The CasRx expression backbone plasmid was purchased from Beijing Hesheng Gene Technology Co., Ltd.

[0041] Example 1 Design and construction of RBM39-targeted CRISPR / CasRx system

[0042] In order to optimize the gene editing efficiency of the CRISPR-CasRx system, this example first needs to identify bladder tumor-specific promoters to achieve efficient expression of CasRx protein in bladder cancer cells while reducing the impact on non-tumor tissues. First, the MB49 bladder cancer cell line, mouse bladder tissue, and bladder epithelium were comprehensively analyzed using transcriptome sequencing technology. When screening for differentially expressed genes (DEGs) between MB49 cells and normal bladder tissue, in order to prevent the nonspecific expression of CasRx in non-targeted cells, genes related to inflammatory response and developmental processes were first eliminated, and the ten candidate genes with the most significant expression differences were preliminarily screened out, and their promoter sequences may be suitable for the construction of a bladder tumor-specific expression system.

[0043] The differential expression characteristics of each candidate gene in MB49 cells, MB49 cell-derived xenografts, bladder tissues and bladder epithelial samples were analyzed by qRT-PCR system. To confirm the tumor specificity of these promoters, the expression levels of the above differentially expressed genes in MB49 cells and multiple important organs (including bladder, heart, liver, spleen, lung and kidney) were further detected.

[0044] like Figure 1 As shown in A, FOSL1 and SPP1 showed the most significant up-regulation in MB49 cells and their derived tumors. Figure 1The B results showed that these genes were generally maintained at low expression levels in normal tissues, but were highly expressed in MB49 cells. Among them, FOSL1 and SPP1 showed the most significant tissue-specific differences.

[0045] Subsequently, a luciferase reporter gene system was constructed. SPP1 and FOSL1 promoter reporter plasmids were constructed using the pGL4.10 plasmid as the backbone; mU6 and hU6 promoter reporter plasmids were constructed using the pGL4.17 plasmid as the backbone. All promoter sequences were located upstream of the luciferase gene.

[0046] As Figure 1 shown in C, the experiments confirmed that these promoters had good transcriptional activity, especially the SPP1 promoter showed the strongest transcriptional driving ability in MB49 cells. Based on the above experimental results, the SPP1 promoter was selected as the best promoter to drive CasRx expression.

[0047] During the optimization of the sgRNA expression system, the RNA polymerase III-dependent U6 promoter was selected. By comparing the performance of mouse and human U6 promoters in mammalian cells, as Figure 1 shown in D, it was found that the mouse U6 promoter (mU6) had higher transcriptional activity in MB49 cells, so it was used to drive the expression of sgRNA.

[0048] The list of screened promoter sequences is shown in Table 1:

[0049] Table 1 Promoter Sequences

[0050] Promoter Sequence mU6 SEQ ID NO.1 hU6 SEQ ID NO.2 mSPP1 SEQ ID NO.3 mFOSL1 SEQ ID NO.4

[0051] Using an online Cas13d gRNA design tool, seven high-score sgRNA sequences were designed and screened, as shown in Table 2:

[0052] Table 2 sgRNA List

[0053] Name Sequence sgRNA1 SEQ ID NO.5 sgRNA2 SEQ ID NO.6 sgRNA3 SEQ ID NO.7 sgRNA4 SEQ ID NO.8 sgRNA5 SEQ ID NO.9 sgRNA6 SEQ ID NO.10 sgRNA7 SEQ ID NO.11

[0054] These sequences were integrated into the optimized CRISPR / CasRx system, that is, the SPP1 promoter was used to drive CasRx expression, and the mU6 promoter regulated sgRNA transcription. When performing functional verification in NIH3T3 cells, as Figure 1 shown in the qRT-PCR analysis in E, gRNA2 and gRNA5 had the best target gene knockdown efficiency. To further improve the knockdown efficiency, by combining gRNA2 and gRNA5, a more significant gene knockdown effect was successfully obtained compared to using either sgRNA alone.

[0055] Example 2: Knockdown of RBM39 Mediated by CRISPR / CasRx System Enhances Anti-Tumor Immune Response in Bladder Cancer

[0056] In this example, the optimized CRISPR / CasRx system was used to knockdown RBM39 in a mouse model of bladder cancer to evaluate its anti-tumor effect in vivo.

[0057] To evaluate the gene knockdown efficiency in vivo, the AAV9 vector with good solid tumor penetration ability was selected to deliver the system, as shown in Figure 2 A. The CRISPR / CasRx-RBM39 system was constructed on the pcDNA3.1 plasmid backbone. In this plasmid, the mU6 promoter drives the expression of a single sgRNA or two sgRNAs in a sandwich structure (each sgRNA flanked by a DR30 sequence on both sides), and the SPP1 promoter drives the expression of CasRx, resulting in the CRISPR / CasRx-RBM39 plasmid.

[0058] Lentivirus packaging and infection:

[0059] HEK-293T cells were seeded in a 10-cm culture dish until the cell confluence reached 70 - 80%. According to the instructions, Lipofectamine TM 3000 transfection reagent (Invitrogen) was used to transfect pMD2.G, psPAX2, and the target plasmid into HEK-293T cells and continue culturing. The culture supernatants were collected at 48 hours and 72 hours after transfection respectively. After removing cell debris, the supernatant was mixed with a universal virus concentration kit (Beyotime) and centrifuged at 4000 g for 40 minutes at 4°C. The obtained lentivirus precipitate was resuspended in PBS solution and stored at -80°C. When infecting with the virus, MB49 cells were seeded in a 6-well plate, and the resuspended lentivirus was added to infect for 12 hours, followed by replacing with fresh medium. Two days after infection, specific antibiotics were used for screening, and after maintaining for at least 1 week, it was used for subsequent experiments.

[0060] AAV infection: The packaging, purification, and titer determination of AAV9 virus particles were completed by Shanghai GeneChem Co., Ltd. In the in vivo infection experiment, when the tumor volume reached 50 - 100 cubic millimeters, 5×10 11 viral genomes (vg) of AAV9 virus particles were injected into the tumor for the first time.

[0061] Administered by intratumoral injection, the results are shown in Figure 2 B - C. AAV-mediated knockdown of RBM39 significantly reduced the expression of RBM39 at both the RNA and protein levels, confirming the strong gene silencing ability of this system in vivo. In addition, Figure 2The D results showed that the expression of CasRx was limited to tumor tissues, and almost no leakage expression was observed in normal tissues, verifying the tumor specificity of the SPP1 promoter in vivo.

[0062] Tumorigenesis experiments were conducted in immunocompetent and immunodeficient mice, and the changes in tumor volume were statistically analyzed. As Figure 2 shown in E - F, in both models, the tumors treated with AAV - RBM39 had smaller volumes than those in the AAV - NC treatment group, and this difference was more significant in immunocompetent mice, indicating that the immune system enhanced the anti - tumor effect of RBM39 knockdown. Similar results were also observed in the experiment of treating MB49 cells with RBM39 - targeted siRNA. As Figure 2 shown in I - J, in which tumor growth was significantly inhibited in immunocompetent mice. Although a certain degree of tumor suppression was also observed in immunodeficient mice, this may be due to the anti - proliferative effect caused by RBM39 deletion or the residual immune function of C57BL / 6 mice. The stronger inhibitory effect observed in immunocompetent mice indicates that the tumor suppression caused by RBM39 deletion is related to the immune system response.

[0063] Example 3 Remodeling of the pro - inflammatory tumor microenvironment by CRISPR / CasRx - mediated RBM39 gene knockdown

[0064] To explore the contribution of the immune system to the anti - tumor effect induced by RBM39 deletion, this example analyzed tumor - infiltrating lymphocytes (TILs) in the AAV - NC and AAV - RBM39 treatment groups in C57BL / 6 mice. Flow cytometry analysis was as Figure 3 shown in A - F. The number of TIL (CD45 + ) in the AAV - RBM39 group increased significantly, and the phagocyte population was significantly elevated. In particular, activated DCs (aDCs; CD11c + CD86 + MHC II + ), which play a key role in antigen presentation and T - cell activation, were significantly enriched in RBM39 - knockdown tumors. In addition, M1 - type macrophages (CD11b + CD206 - CD86 + ), which have anti - tumor properties, were also highly enriched in the tumor microenvironment of the AAV - RBM39 group. The cytotoxic CD8 +The infiltration of T cells and the expression levels of Granzyme B (GrB) and Interferon gamma (IFN-γ) were both significantly increased. Similar immune activation was also observed in the tumors treated with indisulam, and RBM39 degradation also induced the formation of a pro-inflammatory tumor microenvironment in C57BL / 6 mice.

[0065] As Figure 3 shown in G, RNA sequencing analysis of TILs further revealed significant differences in gene expression profiles between the AAV-NC group and the AAV-RBM39 group. The differentially expressed genes were mainly enriched in pathways related to cytotoxic T cell activation and the functions of antigen presenting cells (APCs). These findings indicate that CRISPR / CasRx-mediated knockdown of the RBM39 gene in bladder cancer cells can induce a potent inflammatory immune response, thereby enhancing the anti-tumor immune response in vivo.

[0066] Example 4 CRISPR / CasRx System-Mediated RBM39 Gene Silencing Triggers RNA Splicing Regulation and Neoantigen Generation

[0067] The flowchart for neoantigen screening based on RNA sequencing is as Figure 4 shown in A.

[0068] To verify whether the immune editing effect produced by CRISPR / CasRx-mediated RBM39 knockdown in MB49 cells is driven by abnormal RNA splicing and subsequent neoantigen generation, this example performed RNA sequencing analysis on cells treated with lentivirus-delivered CasRx-NC and CasRx-RBM39.

[0069] Subsequently, alternative splicing (AS) events were screened according to indicators such as read coverage, P value, false discovery rate (FDR), and splicing change (ΔPSI). The rMATS-turbo software (version 4.3.0) was used for quantitative analysis of AS events in the BAM files obtained from RNA sequencing analysis. The screening criteria were: the number of covered reads greater than 20, p value less than 0.01, FDR less than 0.01, and ΔPSI greater than 0.1. As Figure 4 shown in B–C, differential AS events unique to CasRx-RBM39 knockdown cells were identified, and the corresponding mRNA sequences were translated into peptide sequences. Subsequently, these peptides were input into the NetMHCpan 4.1 system to evaluate their affinity with MHC I molecules (H-2D b or H-2K b alleles), and those with an affinity for H-2Db or H-2K b peptides that bind and have a percentile score less than 0.5, such as Figure 4 shown in E. Peptides that were significantly upregulated and had strong MHC I affinity in the CasRx-RBM39 group were classified as neoantigens. CRISPR / CasRx-mediated knockdown of RBM39 significantly affected AS, especially exon skipping, and ultimately 15 abnormal AS events were identified in 9 upregulated genes, most of which involved exon skipping. These findings suggest that RBM39 knockdown drives the generation of neoantigens.

[0070] To evaluate the antigen presentation effect, bone marrow-derived DCs were co-cultured with lysates of MB49 cells treated with indisulam or DMSO, as Figure 4 shown in F. DCs that contacted the lysates of indisulam-treated cells showed enhanced phagocytic activity and induced CD8 + T cells in spleen lymphocytes to secrete more IFN-γ, as Figure 4 shown in G. Bladder cancer cells with RBM39 knockdown were able to stimulate an immune response.

[0071] To study the changes in T cell receptor (TCR) diversity caused by bladder cancer cells after RBM39 knockdown, TCR sequencing was performed on splenocytes stimulated with lysates of MB49 cells treated with CasRx-RBM39 or CasRx-NC. As Figure 4 shown in H, RBM39 knockdown led to an increase in TCR diversity, which was reflected in higher Simpson's reciprocal index, D50 value, and CDR3 peptide length distribution ( Figure 4 shown in I), as well as lower clonality. Although some indicators did not reach the significant level due to individual differences, the overall trend of these indicators revealed that CRISPR / CasRx-mediated RBM39 knockdown increased TCR diversity. In addition, as Figure 4 shown in J-K, RBM39 knockdown led to significant changes in the expression profiles of V / J segments and CDR3 genes. These evidences support the role of CRISPR / CasRx-driven RBM39 knockdown in promoting TCR diversity and immune response in bladder cancer.

[0072] Example 5 Neoantigens generated by abnormal splicing are immunogenic

[0073] A total of 22 potential neoantigens were identified through the screening method in Example 4, as Figure 5 shown in A and Table 3:

[0074] Table 3 List of candidate peptides obtained from in vitro neoantigen prediction based on RNA sequencing

[0075]

[0076]

[0077] Subsequently, the immunogenicity of these peptides was evaluated by in vitro and in vivo experiments. First, the synthesized peptides were co-incubated with DCs respectively, and then the pre-incubated DCs were co-cultured with naïve CD8 + T cells from the spleen. The IFN-γ produced by CD8 + T cells was measured by ELISpot. To extend these findings to the in vivo environment, peptides with ELISpot SFUs exceeding 300 were selected to verify their immunogenicity in mice. The results of the in vitro experiments are shown in Figure 5 B–C. Multiple peptides showed significant immunogenicity, even higher than ovalbumin (OVA).

[0078] Subsequently, three intramuscular injections of each peptide were performed in the hind limbs of C57BL / 6 mice. The flow chart is shown in Figure 5 D. The synthesized polypeptide (10 mg / mL, dissolved in DMSO) was diluted to 1 mg / mL with PBS. Subsequently, an equal volume of complete Freund's adjuvant (Sigma-Aldrich) was added to the aqueous polypeptide solution, and the mixture was vigorously vortexed to form an oil-in-water emulsion. The emulsified polypeptide was injected into the hind limb muscles at 20 μL / dose. Subsequently, RBM39-knockdown MB49 cells were transplanted. As shown in Figure 5 E–F, consistent with the in vitro results, most of the candidate neoantigens showed obvious in vivo antitumor effects, manifested as a significant reduction in tumor volume.

[0079] To confirm the immunogenicity of these peptides in vivo, CD8 + T cells were isolated from the inguinal lymph nodes after inoculation with the candidate peptides and co-cultured with bone marrow-derived DCs pre-incubated with each peptide. The ELISpot detection process is shown in Figure 5 G. The results are shown in Figure 5 F. Approximately half of the peptides showed significant immunogenicity by triggering antigen presentation-dependent CD8 + T cell responses.

[0080] To confirm that the candidate peptides were indeed presented by CRISPR / CasRx-mediated RBM39-knockdown bladder cancer cells, the cross-reactivity between peptide-immunized CD8 + T cells from the inguinal lymph nodes and DCs pre-incubated with CasRx-RBM39 or CasRx-NC MB49 cell lysates was tested. Figure 5ELISpot assays in I confirmed that RBM39 knockdown cells could cross-react with DCs immunized with some of the candidate peptides, indicating that these epitopes were indeed presented by RBM39 knockdown cells. The above results indicate that immunogenic neoantigens induced by CRISPR / CasRx-mediated RBM39 knockdown can trigger potent anti-tumor responses.

[0081] Example 6 CRISPR / CasRx-AAV targeting RBM39 combined with immune checkpoint blockade produces a synergistic anti-tumor immune effect

[0082] ICB therapy relieves T cell immunosuppression by overcoming inhibitory signals between tumor cells and T cells. In this example, the effect of using CRISPR / CasRx-AAV targeting RBM39 in combination with ICB therapy (anti-PD-1) was studied in a mouse model of bladder cancer. The treatment protocol was as Figure 6 shown in A. Male C57BL / 6 mice, 6-8 weeks old, were randomly divided into 4 groups (control group; aPD-1 group; AAV-RBM39 group; aPD-1 combined with AAV-RBM39 group), with 10 mice in each group. Treatment began one week after inoculating MB49 cells on the left side of the abdomen. PD-1 blockade was performed by intraperitoneal injection of anti-PD-1 antibody (10 mg / kg dissolved in 100 μL PBS). AAV virus treatment was carried out by intratumoral injection of 5×10 11 viral genomes (vg) of AAV9 virus, once every 3 days, for a total of 3 injections.

[0083] According to Figure 6 the results in B - C, single therapies (CRISPR / CasRx and aPD-1) both significantly inhibited tumor growth, and the combination therapy showed better tumor control.

[0084] To verify the pro-inflammatory effect of the combination therapy, the immune microenvironment of subcutaneous bladder tumors in each group was analyzed. Figure 6 Analysis of TILs in D - I showed that AAV-CasRx-RBM39 combined with aPD-1 significantly increased the infiltration of total CD8 + T cells, especially IFN-γ + and GrB + cytotoxic CD8 + T cells. In addition, the number of APCs (including activated DCs and M1 macrophages) also increased in the AAV-CasRx-RBM39 group. This inflammatory microenvironment demonstrated that in vivo administration of the CRISPR / CasRx system could reshape the immune environment and produce a synergistic anti-tumor effect with PD-1 blockade.

[0085] To evaluate the safety of in vivo CRISPR / CasRx therapy delivered by AAV and its combination therapy with aPD-1, we continuously monitored the physiological status of all groups throughout the treatment period. The body weights of mice receiving AAV and / or antibody treatment remained stable ( Figure 6 J). Liver function tests showed no significant liver damage ( Figure 6 K). At the end point, the weights of major organs such as the heart, liver, spleen, lungs, and kidneys were normal ( Figure 6 L). ( Figure 6 M shown in Figure 6 demonstrated that neither anti-PD-1 nor AAV-RBM39 damaged the histological structure of the organs. On the contrary, the combination of AAV-RBM39 and anti-PD-1 could alleviate the accumulation of eosinophilic substances and cell necrosis in glomeruli, as well as the dilation of the renal capsule. Therefore, the in vivo RBM39-targeted CRISPR / CasRx therapy delivered by AAV is safe as a single therapy or in combination with ICB. The CRISPR / CasRx therapy targeting RBM39 provides a safe and effective strategy for bladder cancer treatment by regulating RNA splicing and inducing neoantigen production, especially when combined with ICB therapy.

[0086] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

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Claims

1. A novel tumor antigen polypeptide, characterized in that, The amino acid sequences of the tumor neoantigen polypeptides are shown in SEQ ID NOs. 12 to 33.

2. A nucleic acid molecule, characterized in that, Encoding the tumor neoantigen polypeptide according to claim 1.

3. An expression cassette or vector, characterized in that, Comprising the nucleic acid molecule according to claim 2.

4. A cell, characterized in that, Comprising or expressing the antigen peptide according to claim 1, or comprising the nucleic acid molecule according to claim 2 or the expression cassette or vector according to claim 3.

5. A pharmaceutical composition, characterized in that, It comprises any one of the antigen peptide according to claim 1, the nucleic acid molecule according to claim 2, the expression cassette or vector according to claim 3, and the cell according to claim 4.

6. A vaccine, characterized in that, Comprising the antigen peptide according to claim 1 or the nucleic acid molecule according to claim 2.

7. A method for inducing the generation of the tumor neoantigen polypeptide according to claim 1, characterized in that, Comprising the following steps: S1: Designing a specific sgRNA according to the mRNA sequence of the RBM39 gene; S2: Constructing a CasRx protein expression vector driven by the mouse SPP1 gene promoter and an sgRNA expression vector driven by the mouse U6 promoter; S3: Transfecting or infecting MB49 cells with the CasRx protein and the sgRNA expression vector described in S2, interfering with RNA splicing, and inducing the generation of tumor neoantigen polypeptides.

8. The induction method according to claim 7, characterized in that The sgRNA sequences are shown in SEQ ID NOs. 5 to 11.

9. Use of the tumor neoantigen polypeptide according to claim 1 in the preparation of a drug for treating bladder cancer.

10. Use of the tumor neoantigen polypeptide according to claim 1 in the preparation of a diagnostic reagent for bladder cancer.

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