Tumor shared cell membrane antigen epitope peptide and screening method thereof
By screening differentially highly expressed proteins in induced pluripotent stem cell membranes and human tumor cell membranes, and combining them with amino acid sequences strongly bound to the major histocompatibility complex, a nanovaccine platform was used to detect candidate epitope peptides with high population coverage. This solved the problem of difficulty in screening tumor-shared antigens in existing technologies, and achieved efficient screening and significant anti-tumor effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-29
- Publication Date
- 2026-03-31
AI Technical Summary
Existing technologies are insufficient for efficiently screening tumor-shared antigens, which limits the development of tumor preventive vaccines, and the success rate of existing methods is extremely low.
By screening differentially highly expressed proteins in induced pluripotent stem cell membranes and human tumor cell membranes, amino acid sequences that strongly bind to major histocompatibility complexes were identified. A nanovaccine platform was used to detect candidate epitope peptides with high population coverage, and tumor-shared cell membrane antigen epitope peptides were screened.
Six tumor-shared cell membrane antigen epitope peptides were successfully screened, all of which showed significant anti-tumor effects in vivo, significantly improving the screening ratio, saving time and costs, and inducing effective anti-tumor responses in mouse and human immune cells.
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Figure CN121762841A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of tumor antigen peptide identification technology, and relates to a tumor shared cell membrane antigen epitope peptide and its screening method. Background Technology
[0002] Antigens are the most crucial component of tumor vaccines, acting as the commanders in training the immune system to generate adaptive immunity. Current research on tumor antigens focuses on using exome sequencing, transcriptome sequencing, and ribosomal imprinting sequencing combined with bioinformatics analysis to predict tumor antigen epitopes. However, this prediction method is only suitable for predicting personalized antigens and not for identifying shared tumor antigens. Furthermore, large-scale data mining and screening are time-consuming and costly. The predicted antigenic peptide candidates still require further identification and confirmation through subsequent T-cell immune response testing and TCR (T-cell antigen receptor) sequencing. Experiments have shown that this method successfully predicts a very low proportion of antigenic peptides. Induced pluripotent stem cells (iPSCs) have been reported to have the potential for broad-spectrum anti-tumor immune responses, but currently, no research reports the immunological basis for this broad-spectrum anti-tumor effect of iPSCs.
[0003] Most non-viral cancers are considered preventable. Tumor prophylactic vaccines are the preferred method for cancer prevention, but the discovery of tumor-shared antigens has hampered their development. Current technologies identify shared antigens for tumor types with microsatellite instability, using exome sequencing and transcriptome sequencing combined with bioinformatics analysis to predict tumor mutational antigens and related epitopes. However, this predictive method is only suitable for predicting tumor mutational antigens carrying microsatellite instability and is not suitable for identifying a broad spectrum of tumor-shared antigens. Furthermore, the predicted antigenic peptide candidates are difficult to detect for anti-tumor efficacy in animal experiments, and the proportion of successfully predicted antigenic peptides using this method is extremely low.
[0004] Therefore, there is an urgent need to provide a method for efficiently and accurately screening tumor-shared cell membrane antigen epitope peptides. Summary of the Invention
[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a method for identifying tumor shared cell membrane antigenic epitopes and its application, filling a gap in tumor shared antigen screening. The method of this invention can successfully screen tumor shared cell membrane antigenic epitope peptides, and all six screened tumor shared cell membrane antigenic epitope peptides have significant anti-tumor effects in vivo.
[0006] To achieve this objective, the present invention adopts the following technical solution:
[0007] In a first aspect, the present invention provides a method for screening tumor-shared cell membrane antigen epitope peptides, the method comprising the following steps:
[0008] (1) Proteins that are differentially highly expressed compared to the surface of normal somatic cells were screened in induced pluripotent stem cell membranes and human tumor cell membranes;
[0009] (2) Screening the amino acid sequences of differentially expressed proteins to obtain amino acid sequences that strongly bind to the major histocompatibility complex of humans or mice, and obtaining candidate epitope peptides.
[0010] (3) Based on the proportion of each subtype of human major tissue compatibility complex that strongly binds to the candidate epitope peptide in the population, candidate epitope peptides with high population coverage are screened.
[0011] (4) Using a nanovaccine platform to detect candidate epitope peptides with high population coverage, those that can elicit an immune response are tumor-shared cell membrane antigen epitope peptides.
[0012] The method of this invention can successfully screen tumor-shared cell membrane antigen epitope peptides, and all six screened tumor-shared cell membrane antigen epitope peptides have significant anti-tumor effects in vivo.
[0013] Preferably, the number of types of human tumor cell membranes in step (1) is greater than 20.
[0014] Preferably, the differentially highly expressed protein is a protein whose result is greater than 1.5 when sorted by log2 protein abundance ratio.
[0015] Preferably, the screening method in step (2) includes: searching for potential T-cell epitopes on differentially highly expressed proteins and sorting them according to the binding scores of potential T-cell epitopes to MHC (HLA in humans) alleles and representative HLA alleles. Proteins with binding scores of potential T-cell epitopes to both MHC alleles and representative HLA alleles greater than 0.3 are candidate epitope peptides.
[0016] Preferably, the subtypes of human major tissue compatibility complexes include HLA-A 02:01, HLA-A 02:06, HLA-A 02:03, HLA-A 26:01, HLA-B 51:01, HLA-A 01:01, HLA-B 15:01, HLA-A 30:01, HLA-B 07:02, and HLA-B 58:01.
[0017] Preferably, the high population coverage rate is the sum of the population coverage rates of each subtype of strong-binding human HLA greater than 5%.
[0018] Preferably, the nanovaccine platform comprises: an induced pluripotent cell membrane and an adjuvant.
[0019] Preferably, the adjuvant comprises a Toll-like receptor (TLR) agonist.
[0020] Preferably, the mass ratio of the induced pluripotent cell membrane to the adjuvant is (1-10):1.
[0021] The specific point values in 1-10 above can be selected as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.
[0022] In a second aspect, the present invention provides a tumor-shared cell membrane antigenic epitope peptide, which is obtained by screening the tumor-shared cell membrane antigenic epitope peptide by the method described in the first aspect.
[0023] Preferably, the amino acid sequence of the tumor-shared cell membrane antigen epitope peptide includes the sequences shown in SEQ ID NO.1-SEQ ID NO.6.
[0024] SEQ ID NO.1: SLLPVMQHL.
[0025] SEQ ID NO.2: DIYAYRQL.
[0026] SEQ ID NO.3: YVKDIYAYL.
[0027] SEQ ID NO.4: KVVERLLSL.
[0028] SEQ ID NO.5: LLKDFLRNL.
[0029] SEQ ID NO.6: SSILLLQQM.
[0030] Thirdly, the present invention provides the application of the tumor-shared cell membrane antigen epitope peptide described in the second aspect in the preparation of tumor prophylactic vaccines.
[0031] Compared with the prior art, the present invention has the following beneficial effects:
[0032] (1) The screening method of the present invention can successfully screen tumor shared cell membrane antigen epitope peptides. All six tumor shared cell membrane antigen epitope peptides screened have significant anti-tumor effects in vivo.
[0033] (2) The method of the present invention screens 6 / 7 of the candidate peptides that can effectively induce antigen epitope-specific T cell responses. This screening ratio is much higher than the screening ratio of tumor antigen peptides from genome sequencing data or transcriptome sequencing data, which can save a lot of time. At the same time, the screened tumor shared cell membrane antigen epitope peptides can also induce effective anti-tumor responses in mice and can induce epitope peptide-specific T cell responses in human immune cells. Attached Figure Description
[0034] Figure 1 The graph shows the results of proteins that are differentially highly expressed in various human cancers;
[0035] Figure 2 This is a graph showing the epitope scores of peptide binding to MHC alleles and representative HLA alleles.
[0036] Figure 3 Figure showing the results of ELISpot assays to determine the specific immune response elicited by epitope candidate peptides.
[0037] Figure 4 The figure shows the inhibitory effect of candidate epitope peptides on the MC38 colon cancer model.
[0038] Figure 5 The graph shows the effect of candidate epitope peptide vaccines on mouse body weight.
[0039] Figure 6 The figure shows the effects of the candidate epitope peptide vaccine on the major organs of mice.
[0040] Figure 7 The image shows the results of a specific immune response to shared antigenic epitopes in induced pluripotent cell membranes stimulated by a nanovaccine platform in peripheral blood mononuclear cells (PBMCs) from healthy individuals. Detailed Implementation
[0041] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.
[0042] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0043] Example 1
[0044] This embodiment uses the method of the present invention to screen tumor-shared cell membrane antigen epitope peptides.
[0045] Mass spectrometry was used to screen 161 differentially expressed membrane proteins shared by induced pluripotent stem cell membranes and human tumor cell membranes. These proteins were then ranked according to their significantly high expression levels in human tumor samples from the GEPIA2 database, identifying three tumor-associated proteins: RACGAP1, cyclin B1, and MELK, which are highly expressed in more than 20 human cancer types. NetMHCpan 4.1 was then used to search for potential T-cell epitopes on RACGAP1, Ccnb1, and Melk. Figure 1and Figure 2 The T-cell epitope sequencing strategy is as follows: First, an MHC / HLA binding score for the epitope is introduced, which is the score of the epitope's binding to both the MHC allele and the representative HLA allele. An MHC and HLA score greater than 0.3 indicates a strong binding with higher confidence. Orange dots represent the epitope's binding to the MHC allele (H-2K) expressed in C57BL6N mice. b and H-2D b Strong binding was observed, with the same epitope sequence also strongly binding to representative human HLA alleles (i.e., the frequency of a single HLA allele in the population is greater than 2%). This parameter indicates that epitopes present on induced pluripotent stem cell (iPM) membranes are universal across species. Finally, all HLA alleles predicted to bind to these epitopes were grouped to facilitate the selection of multi-allelic epitopes, covering a different group of alleles in the population (HLA population coverage greater than 5%). These selection parameters were applied to differentially expressed tumor-shared membrane proteins on induced pluripotent stem cell membranes, and the results of NetMHCpan 4.1 predictions of epitopes sharing antigens between induced pluripotent stem cell membranes and tumor cell membranes are shown in Table 1. The screening criteria were based on MHC-I (H2K)... b H2 D b It has high affinity for HLA-A, B, and C (the sum of the population coverage of high-binding HLA is >5%) and HLA-B, B, and C (rank < 0.5%).
[0046] Table 1
[0047] serial number protein Epitope peptide Peptide population coverage Ccnb1-1 CyclinB1 SLLPVMQHL 22.32％ Ccnb1-2 CyclinB1 DIYAYLRQL 9.42％ Ccnb1-3 CyclinB1 YVKDIYAYL 24.52％ Ccnb1-4 CyclinB1 LQLVGVTAM 4.78％ Racgap1-1 RacGAP1 KVVERLLSL 33.9％ Racgap 1-2 RacGAP1 LLKDFLRNL 16.4％ Melk-1 Melk VTMQFELEV 17.32％ Melk-2 Melk SSILLLQQM 10.16％
[0048] This invention identified multiple allelic epitopes in C57BL6N mice and humans, with epitope-associated HLA population coverage ranging from 9.42% to 33.9%, indicating that induced pluripotent stem cell membrane antigens have broad prospects for transformation.
[0049] Example 2
[0050] This embodiment demonstrates an antigen-specific T-cell immune response.
[0051] Three tumor-shared tumor-associated antigens (RACGAP1, cyclin B1, and MELK) present in the iPM were selected, and seven candidate epitopes were synthesized to evaluate antigen-specific T-cell immune responses. Of the seven epitope peptides, six significantly restimulated spleen cells to secrete large amounts of IFN-γ, including three cyclin B1 epitopes: SLLPVMQHL, DIYAYRQL, and YVKDIYAYL; KVVERLLSL and LLKDFLRNL; and one Melk epitope: SSILLLQQM. In the positive control group, IFN-γ secretion also significantly increased when induced pluripotent stem cell membrane restimulated spleen cells immunized with the nanovaccine platform. This indicates that the induced pluripotent stem cell membrane nanovaccine platform immunization indeed induced a specific immune response against the induced pluripotent stem cell membrane. These experimental results suggest that the induced pluripotent stem cell membrane nanovaccine platform can induce a T-cell response specifically against tumor-associated proteins that are highly expressed in both induced pluripotent stem cell membranes and tumor cell membranes. Figure 3 ).
[0052] Example 3
[0053] This embodiment conducts a preventative trial using the MC38 tumor model.
[0054] Mice were immunized three times a week with MF59 as the adjuvant, using three epitopes of cyclin B1 (SLLPVMQHL, DIYAYRQL, and YVKDIYAYL), two epitopes of RACGAP1 (KVVERLLSL and LLKDFLRNL), and one epitope of Melk (SSILLLQQM). Mice were also immunized three times a week with a neoantigen peptide vaccine for MC38. Vaccination with combinations of epitopes from RACGAP1, cyclin B1, or Melk significantly inhibited MC38 tumors. This indicates that targeting tumor-shared antigens on the membrane of induced pluripotent stem cells can effectively prevent tumor progression. Furthermore, the positive control group inoculated with MC38 neoantigen epitopes and the tumor-shared antigens on the membrane of induced pluripotent stem cells showed similar inhibitory effects on tumor progression. Figure 4 This indicates that, in the context of cancer prevention, the immunogenic properties of targeting shared tumor antigens and neoantigens are essentially consistent, suggesting the effectiveness of shared tumor antigens in cancer prevention. Furthermore, targeting shared tumor-associated antigens did not cause any significant changes in body weight. Figure 5 ) or damage to major organs and tissues ( Figure 6 This result also demonstrates that targeted sharing of tumor-associated antigens has good safety.
[0055] Example 4
[0056] This embodiment provides an application of tumor-shared cell membrane antigen epitope peptides.
[0057] Preparation of the induced pluripotent stem cell membrane nanovaccine platform (iPM nanovax): 15 μL of FeCl2 was added to 150 μL of CpG adjuvant (a TLR agonist, 25 μM), and 50 μL of imidazoquinone R848 (1 mg / mL) was mixed in a 1.5 mL PCR tube to obtain a suspension. The resulting suspension was vortexed for 30 s and then immediately placed at 95 °C for 1.5 h to obtain the adjuvant nanoparticle core. The nanoparticles were washed twice with deionized water and centrifuged at 11000 × g for 15 min. Next, 10 μL of dimethyl sulfoxide solution (1 mg / mL) containing monophosphoryl lipid A was sonicated and mixed with the induced pluripotent stem cell membrane solution for 10 min. The mixed membrane suspension was added to an equal volume of nanoparticle core for 3 min, and then sequentially extruded through a 400 nm polycarbonate porous membrane using an Avanti liposome extruder to obtain the induced pluripotent stem cell membrane nanovaccine platform.
[0058] A blood sample from a healthy human with an unknown HLA type was randomly selected to investigate the potential immune response and protective probability induced by the induced pluripotent stem cell membrane nanovaccine platform in a human population. Epitope combinations that had been validated in animal experiments were chosen. Fresh PBMCs were isolated from the blood sample and co-incubated for four days with PBS, a mixture of epitope peptides and monophospholipid A, or the induced pluripotent stem cell membrane nanovaccine platform (iPM nanovax). After four days, PBMCs were harvested and restimulated with the mixed epitopes. Compared to the PBS group, PBMCs in the induced pluripotent stem cell membrane nanovaccine platform group and the mixed epitope + monophospholipid A group showed significant epitope-specific IFN-γ release upon re-examination with the mixed epitopes. Figure 7 This result indicates that epitopes sharing antigens on induced pluripotent stem cell (iPSC) membranes can elicit epitope-specific T-cell responses in human immune cells, suggesting that the immunogenicity of shared antigen epitopes on iPSC membranes is sufficient to induce a certain anti-tumor immune response in healthy human immune cells. Furthermore, the data also show that the iPSC membrane nanovaccine platform can be recognized and processed by human immune cells, thereby stimulating a specific immune response against the shared antigen epitopes, indicating that the iPSC membrane nanovaccine platform has the potential for large-scale tumor prevention.
[0059] In summary, the method of the present invention can successfully screen tumor-shared cell membrane antigen epitope peptides, and all six screened tumor-shared cell membrane antigen epitope peptides have significant anti-tumor effects in vivo.
[0060] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims
1. A method of screening for tumor shared cell membrane antigen epitope peptides, characterized by, The method comprises the following steps: (1) screening proteins differentially highly expressed in induced pluripotent stem cell membranes and human tumor cell membranes compared with normal somatic cell surfaces; (2) screening amino acid sequences that strongly bind to human or murine major histocompatibility complexes from the amino acid sequences of the differentially highly expressed proteins to obtain candidate epitope peptides; (3) screening candidate epitope peptides with high population coverage according to the proportion of each subtype of human major histocompatibility complexes that strongly bind to the candidate epitope peptides in the population; (4) detecting the candidate epitope peptides with high population coverage using a nano-vaccine platform, and those that can induce an immune response are tumor-shared cell membrane antigen epitope peptides.
2. The method of claim 1, wherein the tumor shared cell membrane epitope peptide is selected from the group consisting of SEQ ID NOs: 1- 10. The number of types of the human tumor cell membranes in step (1) is greater than 20; Preferably, the differentially highly expressed proteins are proteins with a result greater than 1.5 when arranged according to log2 protein abundance ratio.
3. The method of claim 1 or 2, wherein the tumor shared cell membrane epitope peptide is selected from the group consisting of SEQ ID NOs: 1 to 20. The screening method in step (2) comprises searching for potential T cell epitopes on the differentially highly expressed proteins and ranking them according to the scores of potential T cell epitopes binding to MHC alleles and representative HLA alleles, and proteins with a score greater than 0.3 are candidate epitope peptides.
4. The method of screening for tumor shared cell membrane antigen epitope peptides according to any one of claims 1-3, wherein, The subtypes of human major histocompatibility complexes include HLA-A 02:01, HLA-A 02:06, HLA-A 02:03, HLA-A 26:01, HLA-B 51:01, HLA-A 01:01, HLA-B 15:01, HLA-A 30:01, HLA-B 07:02, and HLA-B 58:
01.
5. The method of screening for tumor shared cell membrane antigen epitope peptides according to any one of claims 1-4, wherein, The high population coverage is the sum of the population coverage of each subtype of human HLA strongly bound to greater than 5%.
6. The method of screening for tumor shared cell membrane antigen epitope peptides according to any one of claims 1-5, wherein, The nano-vaccine platform comprises an induced pluripotent stem cell membrane and an adjuvant.
7. The method of claim 6, wherein the tumor shared cell membrane epitope peptide is selected from the group consisting of SEQ ID NOs: 1- 12. The adjuvant comprises a TLR agonist. Preferably, the mass ratio of the induced pluripotent stem cell membrane to the adjuvant is (1-10):
1.
8. A tumor shared cell membrane antigen epitope peptide, characterized in that, The tumor-shared cell membrane antigen epitope peptide is screened by the method for screening tumor-shared cell membrane antigen epitope peptides according to any one of claims 1-7.
9. The tumor sharing cell membrane antigen epitope peptide according to claim 8, characterized in that, The amino acid sequence of the tumor-shared cell membrane antigen epitope peptide comprises the sequence shown in SEQ ID NO. 1-SEQ ID NO.
6.
10. Use of the tumor-shared cell membrane antigen epitope peptide of claim 8 or 9 in the preparation of a tumor preventive vaccine.