Tumor neoantigen polypeptide for inhibiting microsatellite stable colorectal cancer effect
By identifying and using neoantigens peptide sequences for microsatellite-stable colorectal cancer, tumor vaccines were solved, and the problems of great side effects and poor treatment effects were achieved, and significant tumor growth inhibition and treatment effect were improved.
Patent Information
- Application Number
- CN202411876799.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art has problems of strong toxic side effects and poor treatment effect in the treatment of microsatellite stable colorectal cancer, especially inadequate for advanced patients.
Tumor vaccines were prepared by identifying and utilizing neoantigens peptide sequences against microsatellite-stabilized colorectal cancer, and immunotherapy was performed in an animal model of humanized PDX colorectal cancer, which significantly inhibited tumor growth.
This method has high specificity and safety through personalized tumor immunotherapy, which significantly inhibits the growth of microsatellite-stable colorectal cancer and improves the effectiveness of the treatment.
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Figure CN119708139A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of medical biotechnology, and specifically relates to a new tumor antigen polypeptide that inhibits microsatellite stable colorectal cancer. Background Art
[0002] In clinical medicine, tumors are divided into two categories: solid tumors and non-solid tumors. Solid tumors are tangible masses that can be detected by X-rays, CT scans, B-ultrasound examinations or physical palpation, such as certain types of cancer. In contrast, non-solid tumors, such as blood diseases such as leukemia, cannot be directly observed by the above methods. Solid tumors are often accompanied by problems such as immune escape, recurrence and metastasis after treatment, which in turn increases the patient's cancer risk and mortality rate after treatment. Currently, traditional treatments such as surgery, radiotherapy and chemotherapy are only effective for some cancers. Immunotherapy, as an advanced cancer treatment method, activates the human immune system to identify and eliminate cancer cells. Specific methods include checkpoint inhibitors, cellular immunotherapy, oncolytic viruses and cancer vaccines.
[0003] Colorectal cancer places a heavy mental and economic burden on patients and their families. The current traditional treatment methods, which mainly include surgery, radiotherapy and chemotherapy, have many disadvantages, such as strong toxic side effects, great harm to patients, and unsatisfactory treatment effects (especially for advanced patients).
[0004] The goal of the present invention is to identify a group of new antigenic peptide sequences targeting microsatellite-stable colorectal cancer. The tumor vaccines prepared using these sequences show significant tumor growth inhibition in the humanized PDX (patient-derived tumor xenograft) colorectal cancer animal model. Summary of the invention
[0005] The purpose of the present invention is to provide a tumor neoantigen polypeptide that inhibits microsatellite stable colorectal cancer to solve the problems raised in the above background technology.
[0006] In order to achieve the above-mentioned purpose, the present invention provides the following technical solutions: a tumor neoantigen polypeptide that inhibits microsatellite-stable colorectal cancer, the tumor neoantigen polypeptide is: ZNF169-A275S and CDH4-V456M and their extended sequences; The ZNF169-A275S related mutant peptide sequence: CGRRFSQKS CGRRFSQKSS CGRRFSQKSSL ECGRRFSQKS ECGRRFSQKSS FSQKSSLS FSQKSSLSI FSQKSSLSIH FSQKSSLSIHQ GRRFSQKS GRRFSQKSS GRRFSQKSSL GRRFSQKSSLS KSSLSIHQ KSSLSIHQ KSSLSIHQRK KSSLSIHQRK PECGRRFSQKS QUR QKSSLS QKSSLSIHQ QKSSLSIHQRK RFSQKSSL RFSQKSSLS RFSQKSSLSI RFSQKSSLSIH RRFSQKSS RRFSQKSSL RRFSQKSSLS RRFSQKSSLSI SQ SQ SQKSSLSIHQ SQKSSLSIHQR SSLSIHQ SSLSIHQRK SSLSIHQRKH SSLSIHQRKHS; The CDH4-V456M-related mutant peptide sequence: DPVTNEGMVTM EGMVTMVK EGMVTMVKA EGMVTMVKAV EGMVTMVKAVD GMVTVKA GMVTMVKAV GMVTMVKAVD GMVTMVKAVDY MVKAVDYE MVKAVDYEL MVKAVDYELN MVKAVDYELNR MVTMVKAV MVTMVKAVD MVTMVKAVDY MVTMVKAVDYE NEGMVTMV NEGMVTMVK NEGMVTMVKA NEGMVTMVKAV PVTNEGMVTM PVTNEGMVTMV TMVKAVDY TMVKAVDYEL TMVKAVDYELN TNEGMVTM TNEGMVTMV TNEGMVTMVK TNEGMVTMVKA VTMVKAVD VTMVKAVDY VTMVKAVDYE VTMVKAVDYEL VTNEGMVTM VTNEGMVTMV VTNEGMVTMVK.
[0007] Preferably, the experiment based on the above tumor neoantigen polypeptides is divided into four parts, namely: enzyme-linked immunosorbent spot ELISpot experiment, T cell receptor TCR sequencing experiment and neoantigen-specific TCR clone analysis experiment and mouse tumor transplantation and neoantigen vaccination experiment.
[0008] Preferably, the specific steps of the ELISpot assay are: A1. Dendritic cell DCs culture: Dendritic cells were cultured according to established methods.
[0009] A2, T cell isolation and stimulation: A21, T cells isolated from peripheral blood mononuclear cells (PBMCs); A22, T cells were co-stimulated with autologous dendritic cells (DCs) loaded with individual neoantigen peptides (10 μg / mL) and IL-7 (10 ng / mL; PeproTech) in 24-well cell culture plates; A23, on the third day, IL-2 (5 μg / mL, PeproTech) was added; A24, half of the culture medium was replaced every three days and the corresponding cytokines were added; A3, ELISpot experiment: A31, ten days later, ELISpot assay was performed using the human IFN-γ ELISpot kit MabTech to test the response of pre-stimulated T cells to the new antigens; A32, wash Elispot plate five times with PBS; A33, pre-stimulated T cells and DCs loaded with neoantigen peptides were added to separate wells of the plate and incubated at 37°C in an atmosphere containing 5% CO2 for 18-24 h; A34, the plate was washed again five times with PBS; A35, incubated with 100 μL / well of anti-human IFN-γ (7-B6-ALP) at 37°C for 2 h; A36, the plate was washed again five times with PBS; A37, incubated with BCIP / NBT-plus substrate at room temperature; A38, scan the plate using ELISPOTCTL reader Cellular Technology Inc.; A39, Results of ELISPOT analysis using AID software; spots more than twice the size of the no-peptide control (medium only) were considered positive T cell responses.
[0010] Preferably, the T cell receptor TCR sequencing experimental steps are: B1, Cell collection and RNA extraction: B11, co-incubation of the treated patient's peripheral blood with the antigenic peptide that was positive in the ELISpot test of the patient; B12, after two weeks of incubation, cells were collected for RNA extraction; B2, TCR library construction and sequencing: B21, construction of TCR library using cDNA reverse transcribed from each RNA sample; B22, perform high-throughput sequencing.
[0011] Preferably, the neoantigen-specific TCR clonal analysis experimental steps include clonal diversity, average clonal frequency, and TCR convergence analysis of CDR3 in the TCR-β chain.
[0012] Preferably, the mouse tumor transplantation and neoantigen vaccination experimental steps include: C1, PDX model establishment: C11, tumor sample collection: tumor fragments weighing 20–30 mg were obtained from the patient; C12, primary implantation into NSG mice: Tumor fragments were implanted subcutaneously into the right inguinal region of 8-week-old male NSG mice using a puncture needle; C13, primary tumor growth and collection: wait for the primary tumor to grow to a sufficient size in the mouse; Collect tumor tissue and prepare for the next step of digestion; C14, tumor tissue digestion and PDX cell preparation: the collected tumor tissue was digested using collagenase / hyaluronidase solution; the digested cells were used for subsequent PDX model establishment; C15, secondary implantation of PDX cells into NSG mice: Digested PDX cells were implanted subcutaneously into NSG mice using Matrigel as a carrier; the number of implanted cells was 1-5×10^6, and the carrier volume was 100 μL; C2, follow-up experiments of PDX models: C21, observation of tumor growth: monitoring the growth of tumors in mice; C22, autologous PBMCs intravenous injection: Once tumor growth is observed, 1-5×10^6 autologous peripheral blood mononuclear cells (PBMCs) are injected intravenously into mice.
[0013] C23, vaccination: On the third day after intravenous injection of PBMCs, mice were vaccinated subcutaneously; the vaccine included 0.3 mg of ZNF169-A275S or CDH4-V456M and 0.5 mg of polyI:C; injections were given twice a week for 3 weeks; C24, tumor volume measurement: The tumor volume in mice was measured three times a week to evaluate the tumor growth; C25, mouse sacrifice and sample collection: On the 80th day after tumor inoculation, mice were sacrificed and relevant samples were collected for subsequent experimental analysis.
[0014] Preferably, the dendritic cells are derived from a patient and stimulate PBMCs to produce NRTs in vitro.
[0015] Preferably, the mouse tumor transplantation and neoantigen vaccination experiments use mutant polypeptides ZNF169-A275S and CDH4-V456M that have been shown to be highly immunogenic in vitro to immunize humanized mice.
[0016] The beneficial effects of the present invention are as follows: The present invention uses personalized tumor immunotherapy based on tumor neoantigens, which is highly specific and safe. Clinical data show that the therapy has excellent effects in a variety of tumors. This project uses molecular biology, immunology and pharmaceutical technology to screen lung cancer neoantigens, and found that the target neoantigens can inhibit the growth of colorectal cancer by enhancing the activity of T cell response, especially the neoantigen peptides ZNF169-A275S and CDH4-V456M show potential for the treatment of colorectal cancer. Through sequencing and tumor neoantigen prediction analysis of tissues from a patient with microsatellite-stable colorectal cancer, the enzyme-linked spot assay verified that the top ten antigens with high scores can induce significant T cell responses in vitro, and the immune response of mutant antigen peptides is more significant. Vaccination with two mutant antigen peptides ZNF169-A275S and CDH4-V456M can effectively inhibit tumor growth in mouse models, indicating that personalized tumor neoantigen vaccines are a promising strategy for the treatment of solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 Immunogenicity profiles of neoantigens in colorectal cancer patients evaluated for the present invention; Figure 2 is a graph of immune response of colorectal cancer patients to personalized new antigens of the present invention; Figure 3 This is a diagram of vaccination in the neoantigen autologous humanized mouse model of the present invention; Figure 4 This is a sequence table of the new antigen of the present invention. DETAILED DESCRIPTION
[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0019] like Figures 1 to 4 As shown, the embodiment of the present invention provides a tumor neoantigen polypeptide that inhibits microsatellite stable colorectal cancer, and the tumor neoantigen polypeptide is: ZNF169-A275S and CDH4-V456M and their extended sequences; The ZNF169-A275S related mutant peptide sequence: CGRRFSQKS CGRRFSQKSS CGRRFSQKSSL ECGRRFSQKS ECGRRFSQKSS FSQKSSLS FSQKSSLSI FSQKSSLSIH FSQKSSLSIHQ GRRFSQKS GRRFSQKSS GRRFSQKSSL GRRFSQKSSLS KSSLSIHQ KSSLSIHQ KSSLSIHQRK KSSLSIHQRK PECGRRFSQKS QUR QKSSLS QKSSLSIHQ QKSSLSIHQRK RFSQKSSL RFSQKSSLS RFSQKSSLSI RFSQKSSLSIH RRFSQKSS RRFSQKSSL RRFSQKSSLS RRFSQKSSLSI SQ SQ SQKSSLSIHQ SQKSSLSIHQR SSLSIHQ SSLSIHQRK SSLSIHQRKH SSLSIHQRKHS; The CDH4-V456M-related mutant peptide sequence: DPVTNEGMVTM EGMVTMVK EGMVTMVKA EGMVTMVKAV EGMVTMVKAVD GMVTVKA GMVTMVKAV GMVTMVKAVD GMVTMVKAVDY MVKAVDYE MVKAVDYEL MVKAVDYELN MVKAVDYELNR MVTMVKAV MVTMVKAVD MVTMVKAVDY MVTMVKAVDYE NEGMVTMV NEGMVTMVK NEGMVTMVKA NEGMVTMVKAV PVTNEGMVTM PVTNEGMVTMV TMVKAVDY TMVKAVDYEL TMVKAVDYELN TNEGMVTM TNEGMVTMV TNEGMVTMVK TNEGMVTMVKA VTMVKAVD VTMVKAVDY VTMVKAVDYE VTMVKAVDYEL VTNEGMVTM VTNEGMVTMV VTNEGMVTMVK.
[0020] Among them, the experiments based on the above tumor neoantigen peptides are divided into four parts, namely: enzyme-linked immunosorbent spot (ELISpot) experiment, T cell receptor (TCR) sequencing experiment, neoantigen-specific TCR clone analysis experiment, mouse tumor transplantation and neoantigen vaccination experiment.
[0021] Using bioinformatics analysis technology, the following new antigen sequences were screened: Figure 4 As shown, chemically synthesized neoantigen peptides were then used to experimentally evaluate the immunogenicity of the neoantigens, the immunogenicity of the neoantigens (mutant peptides) and wild-type peptides, and the immune effects of the immunohumanized colorectal cancer animal model on tumors. It was finally found that ZNF169-A275S and CDH4-V456M produced a significant inhibitory effect on tumor growth.
[0022] The specific steps of the ELISpot assay are as follows: A1. Dendritic cell DCs culture: Dendritic cells were cultured according to established methods.
[0023] A2, T cell isolation and stimulation: A21, T cells isolated from peripheral blood mononuclear cells (PBMCs); A22, T cells were co-stimulated with autologous dendritic cells (DCs) loaded with individual neoantigen peptides (10 μg / mL) and IL-7 (10 ng / mL; PeproTech) in 24-well cell culture plates; A23, on the third day, IL-2 (5 μg / mL, PeproTech) was added; A24, half of the culture medium was replaced every three days and the corresponding cytokines were added; A3, ELISpot experiment: A31, ten days later, ELISpot assay was performed using the human IFN-γ ELISpot kit MabTech to test the response of pre-stimulated T cells to the new antigens; A32, wash Elispot plate five times with PBS; A33, pre-stimulated T cells and DCs loaded with neoantigen peptides were added to separate wells of the plate and incubated at 37°C in an atmosphere containing 5% CO2 for 18-24 h; A34, the plate was washed again five times with PBS; A35, incubated with 100 μL / well of anti-human IFN-γ (7-B6-ALP) at 37°C for 2 h; A36, the plate was washed again five times with PBS; A37, incubated with BCIP / NBT-plus substrate at room temperature; A38, scan the plate using ELISPOTCTL reader Cellular Technology Inc.; A39, Results of ELISPOT analysis using AID software; spots more than twice the size of the no-peptide control (medium only) were considered positive T cell responses.
[0024] To detect interferon-γ (IFN-γ) in neoantigen-specific T cells, we performed ELISpot assays using the IFN-γ Elispot Kit from MabTech. Dendritic cells (DCs) were cultured as described previously. T cells isolated from peripheral blood mononuclear cells (PBMCs) were co-stimulated with autologous DCs loaded with individual neoantigen peptides (10 μg / mL) and IL-7 (10 ng / mL; PeproTech) in 24-well cell culture plates. On day 3, IL-2 (5 μg / mL, PeproTech) was added. Half of the medium was changed every three days, and cytokines were added as described previously. Ten days later, the responses of prestimulated T cells to neoantigens were tested by Elispot assay using the Human IFN-γ Elispot Kit (MabTech). Elispot plates were washed five times with PBS. Prestimulated T cells and DCs loaded with neoantigen peptides were added to separate wells of the plate and incubated at 37°C in an atmosphere containing 5% CO2 for 18–24 h. Afterwards, the plates were washed five times with PBS and incubated with 100 μL / well of anti-human IFN-γ (7-B6-ALP) at 37°C for 2 hours. Then, the plates were washed five times again with PBS and incubated with BCIP / NBT-plus substrate at room temperature. Next, the plates were scanned using an ELISPOT CTL reader (Cellular Technology Inc.). The results were analyzed using ELISPOT software (AID). Spots that were more than twice the size of the no-peptide control (medium only) were considered positive T cell responses.
[0025] Wherein, the T cell receptor TCR sequencing experimental steps are: B1, Cell collection and RNA extraction: B11, co-incubation of the treated patient's peripheral blood with the antigenic peptide that was positive in the ELISpot test of the patient; B12, after two weeks of incubation, cells were collected for RNA extraction; B2, TCR library construction and sequencing: B21, construction of TCR library using cDNA reverse transcribed from each RNA sample; B22, perform high-throughput sequencing.
[0026] Among them, the experimental steps of the new antigen-specific TCR clonal analysis include clonal diversity, average clonal frequency, and TCR convergence analysis of CDR3 in the TCR-β chain.
[0027] The treated patient's peripheral blood was co-incubated with the antigenic peptide that was positive in the patient's ELISpot test. After two weeks of incubation, the cells were collected for RNA extraction. The cDNA obtained by reverse transcription from each RNA sample was used to construct a TCR library and perform high-throughput sequencing. The CDR3 in the TCR-β chain was then analyzed for clonal diversity, average clonal frequency, and TCR convergence.
[0028] Experimental results: The immunogenicity of the new antigenic peptides was evaluated using the patient's PBMCs. The results of the ELISPOT experiment showed that the new antigen candidates SOX9-V144M, ZNF169-A275S, CDH4-V456M, NIM1K-T66M and MAP3K9-R1008Q from the patient induced peptide-specific T cell responses compared with medium (no peptide) or an irrelevant peptide VSV-NP43-69 (STKVALNDLRAYVYQGIKSGNPSILHI) ( Figure 1 In particular, the new antigenic peptides SOX9-V144M, ZNF169-A275S, CDH4-V456M, and MAP3K9-R1008Q showed significant peptide-specific T cell responses ( Figure 1 ). To evaluate the ability of NRTs to respond to mutant and wild-type peptides, we used autologous DCs from patients loaded with SOX9-V144M, ZNF169-A275S, CDH4-V456M, NIM1K-T66M, or MAP3K9-R1008Q to stimulate PBMCs to produce NRTs in vitro. Specific NRTs to the mutant peptides were generated in vitro, and their immune responses were compared with those induced by the WT peptide. ELISPOT analysis showed that NRTs secreted higher levels of IFN-γ when stimulated with the mutant peptides than with the corresponding WT peptides ( Figure 2 A). In addition, a significant decrease in T cell receptor (TCR) clonal diversity, an increase in the average frequency of clones, and enhanced TCR convergence were observed after incubation with mutant peptides, indicating the influence of new antigens on TCR ( Figure 2 BD).
[0029] The mouse tumor transplantation and neoantigen vaccination experimental steps include: C1, PDX model establishment: C11, tumor sample collection: tumor fragments weighing 20–30 mg were obtained from the patient; C12, primary implantation into NSG mice: Tumor fragments were implanted subcutaneously into the right inguinal region of 8-week-old male NSG mice using a puncture needle; C13, primary tumor growth and collection: wait for the primary tumor to grow to a sufficient size in the mouse; Collect tumor tissue and prepare for the next step of digestion; C14, tumor tissue digestion and PDX cell preparation: the collected tumor tissue was digested using collagenase / hyaluronidase solution; the digested cells were used for subsequent PDX model establishment; C15, secondary implantation of PDX cells into NSG mice: Digested PDX cells were implanted subcutaneously into NSG mice using Matrigel as a carrier; the number of implanted cells was 1-5×10^6, and the carrier volume was 100 μL; C2, follow-up experiments of PDX models: C21, observation of tumor growth: monitoring the growth of tumors in mice; C22, autologous PBMCs intravenous injection: Once tumor growth is observed, 1-5×10^6 autologous peripheral blood mononuclear cells (PBMCs) are injected intravenously into mice.
[0030] C23, vaccination: On the third day after intravenous injection of PBMCs, mice were vaccinated subcutaneously; the vaccine included 0.3 mg of ZNF169-A275S or CDH4-V456M and 0.5 mg of polyI:C (polyinosinic acid: polycytidylic acid mixture); injections were given twice a week for 3 weeks; C24, tumor volume measurement: The tumor volume in mice was measured three times a week to evaluate the tumor growth; C25, mouse sacrifice and sample collection: On the 80th day after tumor inoculation, mice were sacrificed and relevant samples were collected for subsequent experimental analysis.
[0031] Patient-derived xenograft (PDX) establishment process: Patient tumor fragments (20-30 mg) were implanted subcutaneously (sc) into the right inguinal region of 8-week-old male NSG mice using a puncture needle. After the primary tumor grew, the tumor tissue was collected, digested with collagenase / hyaluronidase solution, and then injected sc into NSG mice with Matrigel as a carrier. For tumor growth experiments, 1-5×106 digested PDX cells were implanted sc into NSG mice with 100 μl of Matrigel as a carrier. Once tumor growth was observed, 1-5×106 autologous PBMCs were injected intravenously into the mice. Three days later, the mice were vaccinated sc twice a week (0.3 mg ZNF169-A275S or CDH4-V456M mixed with 0.5 mg polyI:C (polyinosinic acid:polycytidylic acid) for 3 weeks. Tumor volume was measured three times a week, and the mice were killed on day 80 after tumor inoculation.
[0032] Experimental results: To determine whether the new antigens could be used as effective vaccines to limit tumor growth in vivo, we selected mutant peptides ZNF169-A275S and CDH4-V456M, which were shown to be highly immunogenic in vitro, to immunize humanized mice. Mice vaccinated with mutant peptides ZNF169-A275S or CDH4-V456M showed significantly delayed tumor growth ( Figure 3 A). In addition, 8 out of 10 mice vaccinated with the ZNF169-A275S vaccine and 7 out of 10 mice vaccinated with the CDH4-V456M vaccine had significantly longer survival times (more than 80 days) after tumor inoculation ( Figure 3 B). All mice in the control group died between days 20 and 40. Therefore, these results indicate that immunization with the new antigenic peptides ZNF169-A275S and CDH4-V456M induced effective anti-tumor responses in the experimental animals. No adverse reactions were observed in the mice throughout the experiment.
[0033] The dendritic cells are from patients and stimulate PBMCs to produce NRTs in vitro.
[0034] Among them, the mouse tumor transplantation and neoantigen vaccination experiments used mutant polypeptides ZNF169-A275S and CDH4-V456M, which were proven to be highly immunogenic in vitro, to immunize humanized mice.
[0035] Figure 1 Autologous PBMCs were stimulated every 3 days with candidate mutant peptides in the presence of IL-2. On day 10, T cell responses to each antigen were measured by IFN-γ ELISPOT assay. PBMCs were obtained from patients with CRC. No peptide (medium only) or VSV-NP43-69 (STKVALNDLRAYVYQGIKSGNPSILHI) stimulation was used as control. Data are presented as mean ± SD of three independent experiments. **p<0.01 and *p<0.05 compared to IFN-γ production by PBMCs not stimulated with peptide or stimulated with VSV-NP43-69.
[0036] Figure 2 (A) IFN-γ secretion by neoantigen-reactive T cell lines upon stimulation with mutant and WT peptides. IFN-γ-positive SFCs / 105NRTs detected by cytokine-specific ELISPOT. TCR diversity (B), average clonal frequency (C), and TCR convergence (D) were examined after treatment with WT or mutant peptides. (**P<0.01, ***P<0.001).
[0037] Figure 3The control group received poly(I:C) only. The untreated group did not receive any treatment. (A) Tumor growth was observed every 3 days and recorded as the average tumor size (mm3). (B) Survival of mice after tumor inoculation was monitored. (10 mice per group, **P<0.01).
[0038] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.
[0039] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A tumor neoantigen polypeptide that inhibits microsatellite-stable colorectal cancer, characterized in that: The tumor neoantigen peptides are: ZNF169-A275S and CDH4-V456M; The ZNF169-A275S related mutant peptide sequence: CGRRFSQKS CGRRFSQKSS CGRRFSQKSSL ECGRRFSQKS ECGRRFSQKSS FSQKSSLS FSQKSSLSI FSQKSSLSIH FSQKSSLSIHQ GRRFSQKS GRRFSQKSS GRRFSQKSSL GRRFSQKSSLS KSSLSIHQ KSSLSIHQ KSSLSIHQRK KSSLSIHQRK PECGRRFSQKS QUR QKSSLS QKSSLSIHQ QKSSLSIHQRK RFSQKSSL RFSQKSSLS RFSQKSSLSI RFSQKSSLSIH RRFSQKSS RRFSQKSSL RRFSQKSSLS RRFSQKSSLSI SQ SQ SQKSSLSIHQ SQKSSLSIHQR SSLSIHQ SSLSIHQRK SSLSIHQRKH SSLSIHQRKHS; The CDH4-V456M-related mutant peptide sequence: DPVTNEGMVTM EGMVTMVK EGMVTMVKA EGMVTMVKAV EGMVTMVKAVD GMVTVKA GMVTMVKAV GMVTMVKAVD GMVTMVKAVDY MVKAVDYE MVKAVDYEL MVKAVDYELN MVKAVDYELNR MVTMVKAV MVTMVKAVD MVTMVKAVDY MVTMVKAVDYE NEGMVTMV NEGMVTMVK NEGMVTMVKA NEGMVTMVKAV PVTNEGMVTM PVTNEGMVTMV TMVKAVDY TMVKAVDYEL TMVKAVDYELN TNEGMVTM TNEGMVTMV TNEGMVTMVK TNEGMVTMVKA VTMVKAVD VTMVKAVDY VTMVKAVDYE VTMVKAVDYEL VTNEGMVTM VTNEGMVTMV VTNEGMVTMVK.
2. The tumor neoantigen polypeptide for inhibiting microsatellite-stable colorectal cancer according to claim 1, characterized in that: The experiments based on the above tumor neoantigen peptides are divided into four parts: enzyme-linked immunosorbent spot (ELISpot) experiment, T cell receptor (TCR) sequencing experiment, neoantigen-specific TCR clone analysis experiment, and mouse tumor transplantation and neoantigen vaccination experiment.
3. The tumor neoantigen polypeptide for inhibiting microsatellite-stable colorectal cancer according to claim 2, characterized in that: The specific steps of the ELISpot assay are as follows: A1, Dendritic cell culture: Dendritic cells were cultured according to the established method; A2, T cell isolation and stimulation: A21, T cells isolated from peripheral blood mononuclear cells (PBMCs); A22, T cells were co-stimulated with autologous dendritic cells (DCs) loaded with individual neoantigen peptides (10 μg / mL) and IL-7 (10 ng / mL; PeproTech) in 24-well cell culture plates; A23, on the third day, IL-2 (5 μg / mL, PeproTech) was added; A24, half of the culture medium was replaced every three days and the corresponding cytokines were added; A3, ELISpot experiment: A31, ten days later, ELISpot assay was performed using the human IFN-γ ELISpot kit MabTech to test the response of pre-stimulated T cells to the new antigens; A32, wash Elispot plate five times with PBS; A33, pre-stimulated T cells and DCs loaded with neoantigen peptides were added to separate wells of the plate and incubated at 37°C in an atmosphere containing 5% CO2 for 18-24 h; A34, the plate was washed again five times with PBS; A35, incubated with 100 μL / well of anti-human IFN-γ (7-B6-ALP) at 37°C for 2 h; A36, the plate was washed again five times with PBS; A37, incubated with BCIP / NBT-plus substrate at room temperature; A38, scan the plate using ELISPOTCTL reader Cellular Technology Inc.; A39, Results of ELISPOT analysis using AID software; spots more than twice the size of the no-peptide control (medium only) were considered positive T cell responses.
4. The tumor neoantigen polypeptide for inhibiting microsatellite-stable colorectal cancer according to claim 2, characterized in that: The T cell receptor TCR sequencing experimental steps are: B1, Cell collection and RNA extraction: B11, co-incubation of the treated patient's peripheral blood with the antigenic peptide that was positive in the ELISpot test of the patient; B12, after two weeks of incubation, cells were collected for RNA extraction; B2, TCR library construction and sequencing: B21, construction of TCR library using cDNA reverse transcribed from each RNA sample; B22, perform high-throughput sequencing.
5. The tumor neoantigen polypeptide for inhibiting microsatellite-stable colorectal cancer according to claim 3, characterized in that: The experimental steps of the new antigen-specific TCR clone analysis include clonal diversity, average clonal frequency, and TCR convergence analysis of CDR3 in the TCR-β chain.
6. The tumor neoantigen polypeptide for inhibiting microsatellite-stable colorectal cancer according to claim 1, characterized in that: The mouse tumor transplantation and neoantigen vaccination experimental steps include: C1, PDX model establishment: C11, tumor sample collection: tumor fragments weighing 20–30 mg were obtained from the patient; C12, primary implantation into NSG mice: Tumor fragments were implanted subcutaneously into the right inguinal region of 8-week-old male NSG mice using a puncture needle; C13, primary tumor growth and collection: wait for the primary tumor to grow to a sufficient size in the mouse; Collect tumor tissue and prepare for the next step of digestion; C14, tumor tissue digestion and PDX cell preparation: the collected tumor tissue was digested using collagenase / hyaluronidase solution; the digested cells were used for subsequent PDX model establishment; C15, secondary implantation of PDX cells into NSG mice: Digested PDX cells were implanted subcutaneously into NSG mice using Matrigel as a carrier; the number of implanted cells was 1-5×10^6, and the carrier volume was 100 μL; C2, follow-up experiments of PDX models: C21, observation of tumor growth: monitoring the growth of tumors in mice; C22, autologous PBMCs intravenous injection: Once tumor growth was observed, 1-5×10^6 autologous peripheral blood mononuclear cells (PBMCs) were injected intravenously into mice; C23, vaccination: On the third day after intravenous injection of PBMCs, mice were vaccinated subcutaneously; the vaccine included 0.3 mg of ZNF169-A275S or CDH4-V456M and 0.5 mg of polyI:C; injections were given twice a week for 3 weeks; C24, tumor volume measurement: The tumor volume in mice was measured three times a week to evaluate the tumor growth; C25, mouse sacrifice and sample collection: On the 80th day after tumor inoculation, mice were sacrificed and relevant samples were collected for subsequent experimental analysis.
7. The tumor neoantigen polypeptide for inhibiting microsatellite-stable colorectal cancer according to claim 3, characterized in that: The dendritic cells are derived from patients and stimulate PBMCs to produce NRTs in vitro.
8. The tumor neoantigen polypeptide for inhibiting microsatellite-stable colorectal cancer according to claim 6, characterized in that: The mouse tumor transplantation and neoantigen vaccination experiments used mutant polypeptides ZNF169-A275S and CDH4-V456M, which were confirmed to be highly immunogenic in vitro, to immunize humanized mice.