Application of immunogenic peptide in preparation of medicine for activating anti-tumor immune response

Abstract

The invention discloses an application of an immunogenic peptide in preparation of a medicine for activating an anti-tumor immune response. The invention proves that after the endoplasmic reticulum targeted photosensitizer ER-Cy-poNO2 is activated by laser, an immune peptide group of 4T-1 tumor cells can be remarkably remodeled, and the immunogenicity of the 4T-1 tumor cells is enhanced. Transcriptome analysis shows that antigen processing and presentation pathways are up-regulated, and at the same time, immunopeptide group mass spectrometry systematically identifies a variety of major histocompatibility complex class I (MHC-I) ligands with high affinity. Functional studies show that the immunogenic peptide IF4G3986-994 can promote the maturation of dendritic cells and the activation of T lymphocytes in vitro. In vivo, the compound is combined with poly (inosinic-cytidylic acid) for use, so that a strong anti-tumor immune response can be initiated. Through the discovery, a mechanism relation is established among endoplasmic reticulum stress-driven immunogen cell death, immune peptide group remodeling and adaptive immune activation, and the potential of endoplasmic reticulum targeted phototherapy as an induction for generating immunogenic peptides and promoting a peptide-based tumor vaccine research and development platform is highlighted.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of an immunogenic peptide in the preparation of a drug for activating an anti-tumor immune response. Background Art

[0002] Optical tumor therapy (hereinafter referred to as phototherapy), including photodynamic therapy (PDT) and photothermal therapy (PTT), has the characteristics of spatio-temporal selectivity, almost no drug resistance and no cumulative toxicity, which makes it a promising non-invasive tumor treatment method. In photodynamic therapy, a photosensitizer absorbs light to generate reactive oxygen species (ROS), triggering oxidative stress damage and then leading to the death of tumor cells; while in photothermal therapy, near-infrared light is converted into heat energy to damage tumor cells under light irradiation. Recent studies have emphasized that phototherapy can not only directly eliminate tumor cells, but also induce immunogenic cell death (ICD). During this process, dying tumor cells release damage-associated molecular patterns (DAMPs) in a spatio-temporal specific manner, such as calreticulin (CRT), high-mobility group protein B1 (HMGB1), adenosine triphosphate (ATP), and heat shock proteins (HSPs), etc., thereby activating the immune system. Among them, the immunogenicity of phototherapy-induced dying tumor cells plays a key role in mediating anti-tumor immune responses. However, some key challenges, including insufficient tumor selectivity, poor tissue penetration, and complex tumor microenvironment, severely limit the clinical translation of phototherapy and affect its therapeutic effect, especially the ability to trigger effective anti-tumor immune responses. Therefore, developing innovative strategies to overcome these fundamental obstacles can improve the effect of phototherapy-induced immunogenic cell death, enhance the immunogenicity of dying tumor cells, and improve the therapeutic effect.

[0003] Recent studies have shown that selectively delivering photosensitizers to specific sub - organelles can significantly affect the immunotherapeutic effect induced by phototherapy. The endoplasmic reticulum (ER), as the largest organelle in eukaryotic cells, plays a central role in maintaining intracellular signal transduction, calcium homeostasis, and protein synthesis and processing. It has been reported that protein misfolding induced by reactive oxygen species (ROS) exacerbates ER dysfunction, leading to the unfolded protein response (UPR) and subsequent ER stress. Severe and persistent ER stress exceeds the adaptability of tumor cells, induces immunogenic cell death (ICD), and thus promotes the anti - tumor immune response. Therefore, the immunogenicity of tumor cells related to immunogenic cell death depends to a large extent on the duration and intensity of ER stress. Existing studies have confirmed that ER - targeted phototherapy can significantly amplify ER stress, enhance immunogenic cell death, and promote the release of damage - associated molecular patterns (DAMPs) in tumor cells, thereby triggering a strong anti - tumor immune response and effectively inhibiting the growth of primary and distant metastatic tumors. However, the exact mechanism by which ER - targeted phototherapy enhances immunogenic cell death and the immunogenicity of dying tumor cells remains to be elucidated.

[0004] Recent studies have shown that the immunogenicity of phototherapy - mediated immunogenic cell death (ICD) is mainly determined by two aspects: one is the adjuvanticity driven by the release of damage - associated molecular patterns (DAMPs), and the other is the antigenicity closely related to tumor antigens derived from dying tumor cells. The adjuvanticity of the signature DAMPs induced by phototherapy in promoting the anti - tumor immune response has been widely studied, and its release mechanism and role in enhancing the maturation and activation of dendritic cells (DCs) have also been fully elucidated. However, the effect and mechanism of phototherapy on the antigenicity of dying tumor cells have rarely been studied. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems in the related art to some extent. For this reason, the main object of the present invention is to provide the use of immunogenic peptides in the preparation of drugs for activating the anti - tumor immune response.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] Use of immunogenic peptides in the preparation of drugs for activating the anti - tumor immune response.

[0008] In some specific embodiments, the immunogenic peptides can enhance the antigenicity of immunogenic - death tumor cells and activate the anti - tumor immune response.

[0009] In certain specific embodiments, the immunogenic peptide comprises at least one peptide selected from the group consisting of immunogenic peptide IF4G3, immunogenic peptide RPB2, immunogenic peptide B4GT4, or immunogenic peptide NA-1, or immunogenic SAE2 peptide, or immunogenic peptide NA-2, or immunogenic peptide CARTF, or immunogenic peptide WDR12, or immunogenic peptide YIPF3, or immunogenic peptide HTR5B, or immunogenic peptide TM35B, or immunogenic peptide RENT1, or immunogenic peptide UBP30, or immunogenic peptide DNMT1, or immunogenic peptide SOS1, and the at least one peptide is bound or not bound to a carrier protein.

[0010] Further, the immunogenic peptide comprises immunogenic peptide IF4G3.

[0011] In certain specific embodiments, the immunogenic peptide IF4G3 is the amino acids at positions 986-994 of the IF4G3 protein, and its sequence is as shown in SEQ ID NO.1.

[0012] In certain specific embodiments, the immunogenic peptide RPB2 is the amino acids at positions 1043 - 1051 of the RPB2 protein, and its sequence is as shown in SEQ ID NO.2; the immunogenic peptide B4GT4 is the amino acids at positions 230 - 238 of the B4GT4 protein, and its sequence is as shown in SEQ ID NO.3; the amino acid sequence of the immunogenic peptide NA-1 is SEQ ID NO.4; the immunogenic peptide SAE2 is the amino acids at positions 143 - 151 of the SAE2 protein, and its sequence is as shown in SEQ ID NO.5; the amino acid sequence of the immunogenic peptide NA-2 is SEQ ID NO.6; the immunogenic peptide CARTF is the amino acids at positions 438 - 446 of the CARTF protein, and its sequence is as shown in SEQ ID NO.7; the immunogenic peptide WDR12 is the amino acids at positions 413 - 421 of the WDR12 protein, and its sequence is as shown in SEQ ID NO.8; the immunogenic peptide YIPF3 is the amino acids at positions 266 - 274 of the YIPF3 protein, and its sequence is as shown in SEQ ID NO.9; the immunogenic peptide HTR5B is the amino acids at positions 2012 - 2020 of the HTR5B protein, and its sequence is as shown in SEQ ID NO.10; the immunogenic peptide TM35B is the amino acids at positions 77 - 85 of the TM35B protein, and its sequence is as shown in SEQ ID NO.11; the immunogenic peptide RENT1 is the amino acids at positions 964 - 972 of the RENT1 protein, and its sequence is as shown in SEQ ID NO.12; the immunogenic peptide UBP30 is the amino acids at positions 240 - 248 of the UBP30 protein, and its sequence is as shown in SEQ ID NO.13; the immunogenic peptide DNMT1 is the amino acids at positions 1362 - 1370 of the DNMT1 protein, and its sequence is as shown in SEQ ID NO.14; the immunogenic peptide SOS1 is the amino acids at positions 746 - 754 of the SOS1 protein, and its sequence is as shown in SEQ ID NO.15. Note: The above NA indicates that the peptide sequence has not been matched to a known protein.

[0013] As a concept of the same invention, the present invention also discloses the application of an immunogenic peptide combined with polyinosinic-polycytidylic acid (poly IC) in the preparation of a drug for activating an anti-tumor immune response.

[0014] Regarding the expression of the drug:

[0015] Preferably, the drug further comprises a pharmaceutically acceptable carrier, and such carriers include (but are not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulants, excipients, fillers, binders, sprays, transdermal absorbents, wetting agents, disintegrants, absorption promoters, surfactants, colorants, flavoring agents or adsorption carriers.

[0016] The medicament of the present invention can be prepared into various dosage forms as needed, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories.

[0017] The administration route of the medicament of the present invention is not restricted as long as it can achieve the desired therapeutic or prophylactic effect.

[0018] The medicament of the present invention can also be used in combination with other medicaments that activate the anti-tumor immune response. The combined use of multiple medicaments can greatly improve the success rate of treatment.

[0019] Compared with the prior art, the present invention has at least the following advantages:

[0020] 1) The application of the immunogenic peptide provided by the present invention in the preparation of a medicament for activating the anti-tumor immune response has for the first time discovered and confirmed that endoplasmic reticulum-targeted phototherapy significantly enhances the immunogenicity of 4T-1 tumor cells undergoing immunogenic cell death. In tumor prophylactic and therapeutic models, these cells effectively activate the maturation of dendritic cells (DCs), promote the activation of CD8 + T lymphocytes, and inhibit the infiltration of regulatory T cells (Tregs), thereby triggering a strong anti-tumor immune response and inhibiting tumor growth.

[0021] 2) Transcriptome analysis shows that ER-Cy-poNO 2 -mediated phototherapy upregulates genes involved in antigen processing and immune signaling pathways, while downregulating pro-tumor pathways, providing a mechanistic basis for the enhancement of tumor cell immunogenicity. Immunopeptidome mass spectrometry analysis shows that ER-Cy-poNO 2 -mediated phototherapy remodels the immunopeptidome of 4T-1 cells, generating high-affinity major histocompatibility complex class I (MHC-I) ligands. Among these ligands, the IF4G3 986-994 peptide exhibits excellent immunogenicity, can effectively activate dendritic cells and T lymphocytes in vitro, and when combined with polyinosinic:polycytidylic acid (poly IC) as an adjuvant, triggers a strong anti-tumor immunity in vivo. The present invention establishes a mechanistic link between endoplasmic reticulum stress-driven immunogenic cell death, immunopeptidome remodeling, and adaptive immune activation, providing important insights into the potential mechanism by which endoplasmic reticulum-targeted phototherapy enhances tumor cell immunogenicity and induces a potent anti-tumor immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention, the following will briefly introduce the drawings required for the specific embodiments or the description of the prior art.

[0023] Figure 1Schematic diagram of the potential mechanism of endoplasmic reticulum-targeted phototherapy in remodeling the tumor immunopeptidome to enhance the immunogenicity of immunogenic cell death tumor cells and adaptive anti-tumor immunity in the present invention;

[0024] Figure 2 In the present invention, endoplasmic reticulum-targeted phototherapy enhanced the immunogenicity of 4T-1 tumor cells that underwent immunogenic cell death (ICD); among them, (a) representative enzyme-linked immunospot (ELISPOT) images showing the interferon-γ (IFN-γ) spots generated after co-incubation of 4T-1 cells with splenocytes under different treatment conditions. (b) Summary of the experimental data in (a), showing the number of IFN-γ spots generated per 2×10 5 average splenocytes. (n = 3, mean ± standard deviation). (c) Workflow of the tumor vaccination experiment. (d) Representative ELISPOT images showing the IFN-γ spots generated by splenocytes of mice vaccinated with 4T-1 cells treated differently. (e) Summary of the experimental data in (d), showing the number of IFN-γ spots generated per 2×10 5 average splenocytes. (n = 3, mean ± standard deviation). (f) Tumor volume curve during the observation period (n = 5, mean ± standard deviation). (g) Representative images of flow cytometry analysis of the maturation of dendritic cells (DCs) (CD80 + CD86 + ) in local lymph nodes. (h) Quantitative analysis of the proportion of CD80 + CD86 + DCs (n = 3, mean ± standard deviation). (i) Representative images of flow cytometry analysis of T lymphocytes (CD3 + CD8 + ) in the spleen. (j) Quantitative analysis of the proportion of CD3 + CD8 + T lymphocytes (n = 3, mean ± standard deviation). (k) Representative images of flow cytometry analysis of T lymphocytes (CD8 + IFN-γ + ) in the spleen. (l) Quantitative analysis of the proportion of CD8 + IFN-γ + T lymphocytes (n = 3, mean ± standard deviation). (m) Representative images of flow cytometry analysis of T lymphocytes (CD3 + CD8 + ) in the tumor. (n) Quantitative analysis of the proportion of CD3 + CD8 + T lymphocytes (n = 3, mean ± standard deviation). (o) Flow cytometry analysis of regulatory T cells (Tregs, CD4 +CD25 + FoxP3 + ) Representative images for flow cytometry analysis. (p) Quantitative analysis of the proportion of CD4 + CD25 + FoxP3 + Tregs in (o) (n = 3, mean ± standard deviation). Statistical analysis was performed using one-way ANOVA followed by Tukey's test. When significant differences between groups were observed, a t-test was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0025] Figure 3 In this invention, tumor cells undergoing immunogenic cell death (ICD) induced by endoplasmic reticulum-targeted phototherapy triggered a strong anti-tumor immune response in 4T-1 tumor-bearing mice; among them, (a) Workflow of the tumor treatment experiment. (b) Tumor volume curve during treatment (n = 5, mean ± standard deviation). (c) Images of the tumors (n = 5). (d) Representative images for flow cytometry analysis of the maturation of dendritic cells (DCs) in local lymph nodes (CD80 + CD86 + ). (e) Quantitative analysis of the proportion of CD80 + CD86 + DCs in (d) (n = 3, mean ± standard deviation). (f) Representative images for flow cytometry analysis of the maturation of dendritic cells (DCs) in distant lymph nodes (CD80 + CD86 + ). (g) Quantitative analysis of the proportion of CD80 + CD86 + DCs in (f) (n = 3, mean ± standard deviation). (h) Representative images for flow cytometry analysis of T lymphocytes (CD3 + CD8 + ) in tumors. (i) Quantitative analysis of the proportion of CD3 + CD8 + T lymphocytes in (h) (n = 3, mean ± standard deviation). (j) Representative images for flow cytometry analysis of regulatory T cells (Tregs, CD4 + CD25 + FoxP3 + ) in tumors. (k) Quantitative analysis of the proportion of CD4 + CD25 + FoxP3 +Quantitative analysis of the proportion of Tregs (n = 3, mean ± standard deviation). (l) Quantitative analysis of the concentration of interferon-γ (IFN-γ) in serum (n = 3, mean ± standard deviation). When significant differences between groups were observed, a t-test was used. *p < 0.05, **p < 0.01, ***p < 0.001.

[0026] Figure 4 In this invention, endoplasmic reticulum-targeted phototherapy upregulated the antigen processing and immune signaling pathways in 4T-1 cells; among which (a) The volcano plot shows the differentially expressed genes (DEGs) upregulated and downregulated in the ER-Cy-poNO 2 + laser group compared with the phosphate buffer saline (PBS) group. Red indicates high expression, and blue indicates low expression (log2|fold change| > 1, adjusted P value < 0.05). (b) The heatmap shows the differentially expressed genes upregulated and downregulated. Red indicates high expression, and blue indicates low expression. (c) Gene Ontology (GO) enrichment analysis of the upregulated differentially expressed genes in the ER-Cy-poNO 2 + laser group compared with the PBS group. (d) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes in the ER-Cy-poNO 2 + laser group compared with the PBS group. (e) Gene Set Enrichment Analysis (GSEA) of antigen processing and presentation (MMU04612) in the ER-Cy-poNO 2 + laser group compared with the PBS group. (f) Expression differences of key genes in the antigen processing and presentation (MMU04612) pathway.

[0027] Figure 5 In this invention, endoplasmic reticulum-targeted phototherapy reshaped the immunopeptidome of 4T-1 cells, generating high-affinity major histocompatibility complex class I (MHC-I) ligands; among which (a) Workflow for identifying the immunopeptidome (MIP) derived from 4T-1 cells. (b) Length distribution of MIP in the phosphate buffer saline (PBS) group, ER-Cy-poNO 2 + laser group, and overall (7 - 16 amino acids). (c) Overlap of MIP between the PBS group and the ER-Cy-poNO 2 + laser group. (d) The volcano plot shows that among the common immunopeptides, compared with the PBS group, ER-Cy-poNO 2+ Upregulated and downregulated differentially expressed MIPs in the laser group, with red indicating high expression and blue indicating low expression (log2|fold change| > 1, P value < 0.05). (e) Peptide affinity predicted by NetMHCpan-4.1, SB: strong-binding peptide (percentile rank < 0.5); WB: weak-binding peptide (percentile rank: 0.5 - 2). (f) Binding motifs of nonamer peptides identified from MIPs derived from 4T-1 cells under different treatment conditions. The x-axis represents the residue positions in the nonamer peptide sequence. The y-axis represents the size of each amino acid symbol proportional to its frequency of occurrence.

[0028] Figure 6 IF4G3 in the present invention 986-994 The peptide shows significant immunogenic advantages compared to other tested candidate peptides; among them, (a) the figure shows the screening process of immunopeptides for immunogenicity evaluation. (b) The figure shows the amino acid sequences, protein sources, and MHC-I haplotypes bound to the 15 synthesized candidate immunopeptides. (c) Workflow of the enzyme-linked immunospot (ELISPOT) assay for interferon-γ (IFN-γ). (d) Representative ELISPOT images show IFN-γ produced by splenocytes from BALB / c mice pre-stimulated with specific peptides and re-stimulated with either bone marrow-derived dendritic cells (BMDCs) alone or BMDCs loaded with specific peptides (BMDCs + specific peptide). (e) Summary of the experimental data in (d), showing the average number of IFN-γ spots produced per 1×10 5 splenocytes (n = 3, mean ± standard deviation). (f) Quantitative analysis of the proportion of CD80 + CD86 + BMDCs (n = 3, mean ± standard deviation).

[0029] Figure 7 The highly immunogenic peptide IF4G3 in the present invention 986–994 can effectively activate the anti-tumor immune response in mice and inhibit tumor growth. (a) Workflow of the in vivo tumor vaccine experiment with IF4G3. (b) Representative enzyme-linked immunospot (ELISPOT) images showing interferon-γ (IFN-γ) spots produced by splenocytes from mice receiving different treatments. (c) Summary of the experimental data in (b), showing the average number of IFN-γ spots produced per 2×10 986-994 splenocytes. (n = 3, mean ± standard deviation). (d) Tumor volume curve during the tumor vaccine experiment (n = 4, mean ± standard deviation). (e) Representative images of tumors (n = 4). (f) Tumor weights on the 21st day after different treatments (n = 4, mean ± standard deviation). (g) Flow cytometry analysis of the maturation of dendritic cells (DCs) in lymph nodes (CD80 5 ​+ CD86 + ) representative images. (h) Quantitative analysis of the proportion of CD80 in (g) + CD86 + Quantitative analysis of the proportion of DCs (n = 3, mean ± standard deviation). (i) Flow cytometry analysis of T lymphocytes (CD3 + CD8 + ) representative images. (j) Quantitative analysis of the proportion of CD3 + CD8 + T lymphocytes (n = 3, mean ± standard deviation). (k) Flow cytometry analysis of T lymphocytes (CD8 + IFN-γ + ) representative images. (l) Quantitative analysis of the proportion of CD8 + IFN-γ + T lymphocytes (n = 3, mean ± standard deviation). (m) Flow cytometry analysis of regulatory T cells (Tregs, CD4 + CD25 + FoxP3 + ) representative images. (n) Quantitative analysis of the proportion of CD4 + CD25 + FoxP3 + Tregs (n = 3, mean ± standard deviation). Statistical analysis was performed using one-way ANOVA followed by Tukey's test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Detailed implementation manners

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are only descriptive and not restrictive, and the protection scope of the present invention cannot be limited thereby.

[0031] When a quantity, concentration, or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper and lower limit of a value, it should be understood that it is equivalent to specifically disclosing any range obtained by combining any pair of upper limits of the range or preferred values with any lower limit of the range or preferred values, regardless of whether the range is specifically disclosed. Unless otherwise indicated, the numerical range values listed herein include the endpoints of the range and all integers and fractions within the range.

[0032] Unless otherwise specified, all percentages, parts, ratios, etc. herein are by weight.

[0033] The materials, methods, and embodiments herein are exemplary and should not be construed as restrictive unless otherwise specified.

[0034] In the following examples, the dyes and antibodies used were: viable cell fixable dyes 780 (#565388, BD Biosciences, New Jersey, USA), anti-CD11c antibody conjugated with phycoerythrin (PE) (#12-0114-82, eBioscience, California, USA), anti-CD80 antibody conjugated with allophycocyanin (APC) (#17-0801-82, eBioscience, California, USA), anti-CD86 antibody conjugated with PE-Cy7 (#25-0862-82, eBioscience, California, USA), anti-CD3 antibody conjugated with fluorescein isothiocyanate (FITC) (#11-0031-82, eBioscience, California, USA), anti-CD45 antibody conjugated with APC (#17-0451-82, eBioscience, California, USA), anti-CD4 antibody conjugated with PE-Cy7 (#25-0041-82, eBioscience, California, USA), anti-CD8 antibody conjugated with peridinin-chlorophyll protein-cyanine 5.5 (PerCP-Cy5.5) (#45-0081-82, BioLegend, California, USA), anti-forkhead box protein P3 (FoxP3) antibody conjugated with PE (#320008, BioLegend, California, USA), anti-CD25 antibody conjugated with BV421 (#102043, BioLegend, California, USA), anti-interferon-γ (IFN-γ) antibody conjugated with PE (#505807, BioLegend, California, USA). Cytokines and kits: interleukin-2 (IL-2, #212-12, Peprotech, New Jersey, USA), granulocyte-macrophage colony-stimulating factor (GM-CSF, #315-03, Peprotech, New Jersey, USA), interleukin-4 (IL-4, #214-14, Peprotech, New Jersey, USA), mouse IFN-γ enzyme-linked immunosorbent spot (ELISPOT) kit (#2210005, Dakewe Biotech Co., Ltd., Shenzhen, China) and mouse IFN-γ enzyme-linked immunosorbent assay (ELISA) kit (SMK2918A, Enzyme Science Co., Ltd., Jiangsu, China).

[0035] The photosensitizer ER-Cy-poNO described in the following examples 2 has the molecular structural formula as follows:

[0036]

[0037] Among them, n = 1 - 10; R1, R2, R3, R4, R5 and R6 are any one of hydrogen, halogen, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl, methoxy, ethoxy, propoxy, hydroxyl, amino, nitro, and cyano; X− is any one of iodide ion, chloride ion, and bromide ion. The photosensitizer ER-Cy-poNO 2 can be prepared by the preparation method disclosed in the previous patent number CN115010643B of our team, which is a preparation method and use patent of a benzosulfonamide-modified heptamethine indocyanine small molecule; in the following examples, the photosensitizer with the molecular formula is used as an example for experiments:

[0038]

[0039] In the following examples, the tumor cells that undergo immunogenic cell death (ICD) are taken as 4T-1 cells. The culture and treatment method of 4T-1 cells is as follows:

[0040] 4T-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in complete Dulbecco's modified Eagle's medium (DMEM, Gibco, New York, USA) containing 10% fetal bovine serum (FBS, Hyclone, Utah, USA) and 1% penicillin / streptomycin (Gibco, New York, USA); incubation was carried out in an environment of 37 °C and 5% carbon dioxide.

[0041] To induce immunogenic cell death (ICD), 5×10 4 4T-1 cells per well were seeded in a 6-well plate and allowed to adhere overnight; the next day, in the ER-Cy-poNO 2 + laser group, 4 μM ER-Cy-poNO 2 was added to each well and incubated overnight, and then the cells were irradiated with an 808 nm laser at a power of 1 W·cm 2 for 5 min per well (denoted as ICD 4T-1 cells). For the phosphate buffer solution (PBS, Hyclone, Utah, USA) group, only an equal volume of sterile PBS solution was added. After the above experimental steps were completed, the 4T-1 cells were cultured for another 24 hours; the 4T-1 cells in each group were collected and the cell number was adjusted to more than 2×10 7 for subsequent experiments.

[0042] Example 1: Excellent immunogenicity

[0043] The endoplasmic reticulum-targeted photosensitizer ER-Cy-poNO 2 (hereinafter referred to as ER-Cy-poNO 2 ) in the present invention can improve the immunogenicity of tumor cells that undergo immunogenic cell death (ICD). Specifically:

[0044] Materials and testing methods:

[0045] The immunogenicity of 4T-1 cells treated with ER-Cy-poNO 2 combined with laser irradiation was analyzed by interferon-γ enzyme-linked immunospot (ELISPOT) assay.

[0046] Results and discussion: The results are shown in Figure 2 a and Figure 2 b. As can be seen from the figure, the number of interferon-γ spots in the ER-Cy-poNO 2 + laser group increased significantly (more than 500), even higher than that of the positive control group (phorbol myristate acetate (PMA) and ionomycin). This indicates that 4T-1 cells receiving endoplasmic reticulum-targeted phototherapy mediated by ER-Cy-poNO 2 showed strong immunogenicity.

[0047] Example 2: Potential to activate anti-tumor immune responses with excellent efficacy

[0048] To further evaluate the potential of 4T-1 cells that underwent immunogenic cell death to activate anti-tumor immune responses, we conducted a tumor vaccination experiment in mice. Specifically:

[0049] Materials and methods: Six-week-old female BALB / c mice were randomly divided into two groups. The PBS group received 100 μL of sterile PBS, while the treatment group (ICD 4T-1 cell group) received 1×10 6 ICD 4T-1 cells suspended in 100 μL of sterile PBS, and the injection was performed subcutaneously in the right hind limb. The vaccine was administered once every 3 days for a total of 3 doses ( Figure 2 c). One week after the last vaccination, 3 mice from each group were sacrificed, and dendritic cells were isolated from the lymph nodes for further flow cytometry analysis. Meanwhile, lymphocytes were isolated from the spleen for ELISPOT detection. The remaining mice in each group were then inoculated with 1×10 6 normal 4T-1 cells, suspended in 100 μL of sterile PBS, and subcutaneously injected into the left posterior side. The body weight and tumor volume of the mice were measured every 3 days. When the average tumor volume of the PBS group reached 600 cubic millimeters, the mice were euthanized, and lymphocytes were isolated from the spleen and tumor tissues for flow cytometry analysis.

[0050] Results and discussion:

[0051] Compared with the phosphate-buffered saline (PBS) group, immunization with 4T-1 cells that underwent immunogenic cell death (ICD 4T-1 cell group) significantly activated mouse splenocytes, resulting in a significant difference in the presence of ELISPOT spots (Figure 2 d, e). Compared with the PBS group, tumor growth in the ICD 4T-1 cell group was significantly inhibited ( Figure 2 f).

[0052] Among them, the results of the maturation of dendritic cells in the lymph nodes on the injection side where 4T-1 cells that underwent immunogenic cell death were injected showed that the proportion of mature dendritic cells (CD80 + CD86 + ) in the lymph nodes of the ICD 4T-1 cell group increased to approximately 15%, while it was only 5% in the PBS group ( Figure 2 g, h).

[0053] CD8 + The activation of T lymphocytes was as Figure 2 i, j. As can be seen from the figure, the proportions of CD3 + CD8 + T lymphocytes in the spleen of the ICD 4T-1 cell group were significantly higher than those in the PBS group, reaching 40% and 30% respectively. The proportion of CD8 + IFN-γ + cells in the ICD 4T-1 cell group increased significantly to approximately 12%, while the CD8 + T lymphocytes producing interferon-γ in the PBS group were only approximately 3% ( Figure 2 k, l).

[0054] CD8 + The results of the infiltration of T lymphocytes into the tumor tissue showed that the proportion of CD3 + CD8 + T lymphocytes in the tumor of the PBS group was approximately 20%, while it increased significantly in the ICD 4T-1 cell group, approximately 35% ( Figure 2 m, n).

[0055] Regulatory T cells are a key component of the immunosuppressive tumor microenvironment. The results of analyzing the proportion of regulatory T cells (Tregs) in each group found that, compared with the PBS group, the proportion of CD4 + CD25 + FoxP3 + regulatory T cells in the tumor tissue of the ICD 4T-1 cell group was significantly reduced ( Figure 2 o, p). The above results indicate that 4T-1 cells that underwent immunogenic cell death exhibit strong immunogenicity, enabling them to promote the maturation of dendritic cells, promote the activation and proliferation of CD8 + T lymphocytes, and inhibit the infiltration of regulatory T cells.

[0056] Example 3: Can activate the immune system and inhibit tumor growth

[0057] To further evaluate the potential of 4T-1 cells undergoing immunogenic cell death to activate antitumor immune responses, we conducted a tumor therapeutic immunization experiment in tumor-bearing mice ( Figure 3 a), specifically as follows:

[0058] Materials and methods:

[0059] Six-week-old female BALB / c mice were randomly divided into two groups. 100 μL of 1×10 6 4T-1 cells in sterile PBS were subcutaneously injected into the right hind limb of each mouse to establish a 4T-1 xenograft tumor model. Three days later, the PBS group received 100 μL of sterile PBS, while the treatment group (ICD 4T-1 cell group) received 1×10 6 ICD 4T-1 cells suspended in 100 μL of sterile PBS, and the injection was subcutaneously into the left hind limb. Treatments were administered once every 3 days for a total of 3 doses. The body weight and tumor volume of the mice were measured every 3 days. When the average tumor volume in the PBS group reached 600 mm

[0060] Results and discussion:

[0061] Tumor growth curves and excised tumor images showed that tumor progression in the ICD 4T-1 cell group was significantly inhibited compared with the phosphate-buffered saline (PBS) group ( Figure 3 b, c).

[0062] Further analysis of the lymph nodes showed that the proportion of mature dendritic cells (CD80 + CD86 + ) in the local and distant lymph nodes was increased in the ICD 4T-1 cell group compared with the PBS group ( Figure 3 d-g). In addition, flow cytometry analysis of the tumor microenvironment showed that the infiltration of CD3 + CD8 + T lymphocytes in the 4T-1 tumors was increased and the number of immunosuppressive regulatory T cells (Tregs) was decreased in the ICD 4T-1 cell group compared with the PBS group ( Figure 3 h-k).

[0063] In addition, the serum γ-interferon (IFN-γ) level in the ICD 4T-1 cell group further confirmed a strong antitumor immune response compared with the PBS group.Figure 3 l). In summary, ER-Cy-poNO 2 4T-1 cells that undergo immunogenic cell death mediated by phototherapy can significantly activate the immune system and inhibit tumor growth.

[0064] Example 4: Transcriptome sequencing analysis

[0065] The present invention performs transcriptome sequencing experiments and bioinformatics analysis on 4T-1 cells that undergo immunogenic cell death (ICD) induced by the endoplasmic reticulum-targeted photosensitizer ER-Cy-poNO 2 (hereinafter referred to as ER-Cy-poNO 2 ). Specifically:

[0066] Materials and methods:

[0067] 4T-1 cells were divided into a phosphate-buffered saline (PBS) control group and a treatment group (ER-Cy-poNO 2 + laser). After 24 hours of treatment, 4T-1 cells from each group were collected, and the cell number was adjusted to more than 2×10 7 . Then the cell samples were frozen in liquid nitrogen for 20 minutes and subsequently stored at -80°C. The transcriptome sequencing experiments and bioinformatics analysis were performed by Shanghai Bap Biotechnology Co., Ltd. The identification criteria for differentially expressed genes (DEGs) were Log 2 (fold change) > 1 and adjusted P value < 0.05 (calculated using the Benjamini-Hochberg method). For gene ontology (GO) enrichment analysis, the top 20 upregulated pathways were ranked in ascending order according to the false discovery rate (FDR) for all categories. For Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, pathways with an FDR threshold < 0.05 were screened, and pathways related to tumors were highlighted.

[0068] Results and discussion:

[0069] The results are shown in the volcano plot in Figure 4 a. Compared with the phosphate-buffered saline (PBS) group, there were 2,694 differentially expressed genes (DEGs) (1,459 upregulated and 1,235 downregulated) in the ER-Cy-poNO 2 + laser treatment group, indicating that endoplasmic reticulum-targeted phototherapy induced significant transcriptional changes. The heatmap shows these differentially expressed genes, further highlighting the different gene expression profiles between the PBS group and the ER-Cy-poNO 2 + laser treatment group ( Figure 4 b).

[0070] To reveal the functional categories of differentially expressed genes, the present application conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The Gene Ontology analysis showed that ER-Cy-poNO 2 -mediated phototherapy significantly upregulated the gene expressions related to multiple biological processes, including cellular stress response and immune response ( Figure 4 c). The KEGG enrichment analysis further indicated that ER-Cy-poNO 2 + laser treatment led to the upregulation of enriched genes in immune-related signaling pathways, including interleukin-17 (IL-17) signaling pathway, cytokine-cytokine receptor interaction, tumor necrosis factor (TNF) signaling pathway, calcium signaling pathway, and antigen processing and presentation. Conversely, after ER-Cy-poNO 2 -mediated phototherapy, some pro-tumor pathways, such as mammalian target of rapamycin (mTOR) signaling pathway, phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt) signaling pathway, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance pathway, transforming growth factor-β (TGF-β) signaling pathway, and Ras signaling pathway, etc., were significantly downregulated ( Figure 4 d).

[0071] Meanwhile, gene set enrichment analysis (GSEA) showed that ER-Cy-poNO 2 + laser treatment significantly upregulated the gene expressions involved in antigen processing and presentation (normalized enrichment score NES = 1.7396, false discovery rate FDR q = 0.006), indicating enhanced activation of the immune system ( Figure 4 e). In addition, ER-Cy-poNO 2 -mediated phototherapy significantly upregulated the expressions of multiple key genes involved in antigen processing and presentation, including Hspa1b, Hspa1a, HSpa8, HSp90aa1, Hsp90ab1, H2-M10.1, H2-T23, and Psme1 ( Figure 4 f). In summary, these results suggest that ER-Cy-poNO 2 -mediated phototherapy upregulates the gene expressions involved in antigen processing and presentation, cytokine signaling, and immune effector functions, which may be driven by immunogenic cell death of 4T-1 cells, thereby triggering a strong anti-tumor immune response.

[0072] Example 5: Immunopeptidomics analysis, peptide-MHC-I binding affinity, and immunogenicity prediction

[0073] This example provides an endoplasmic reticulum-targeted photosensitizer ER-Cy-poNO 2 (hereinafter referred to as ER-Cy-poNO2 ) Immunopeptidomic mass spectrometry and its bioinformatics analysis of 4T-1 cells undergoing immunogenic cell death (ICD), specifically:

[0074] Materials and testing methods:

[0075] The 4T-1 cells were also divided into a PBS control group and a treatment group (ER-Cy-poNO 2 + laser). 2×10 7 4T-1 cells from each treatment group were collected. The collected cells were placed on ice and resuspended in an equal volume of PBS. An equal volume of cell lysis buffer was added, and after gentle mixing, the mixture was incubated on ice for 60 minutes. Then the lysate was sonicated for 5 minutes and centrifuged (4°C, 2000g, 20 minutes) to remove cell debris. The resulting supernatant was transferred to a new tube and further centrifuged (4°C, 20000g, 20 minutes). Then, the NEO Discovery MHC-I Peptide Enrichment Kit from Biozhen Biotechnology Co., Ltd. was used to enrich and elute the clarified supernatant containing mouse MHC peptide complexes. The extracted MHC-I-peptide complexes were separated using a reverse-phase column to obtain the immunopeptide fraction, which was subsequently analyzed using a high-performance liquid chromatograph (HPLC, nanoElute, Bruker, Massachusetts, USA) in combination with a tandem mass spectrometry system (LC-MS / MS, timsTOF Pro2, Bruker, Massachusetts, USA). The PEAKSDeepNovo peptidomics software (Bioinformatics Solutions Inc.) was used to process and analyze the raw mass spectrometry data files generated during the LC-MS / MS analysis.

[0076] As described in the published literature (Reynisson, B. et al., 2020), the state-of-the-art MhcVizPipe program and the well-established IEDB analysis resource NetMHCpan-4.1 were used to evaluate the potential binding ability of the identified peptides to the mouse MHC-I alleles H2-Kd, H2-Dd, and H2-Ld. This comprehensive evaluation provided valuable insights into the MHC-I binding ability of the detected immunopeptides. In addition, the DeepImmu TM neoantigen discovery platform (Bioinformatics Solutions Inc.) was used to predict the immunogenicity of the identified immunopeptides.

[0077] Results and discussion:

[0078] Given that the repertoire of immune peptides presented on the surface of tumor cells by MHC-I is a key determinant of the anti-tumor immune response, we further performed a comprehensive immunopeptidomics analysis to characterize the changes in the surface immunopeptidome of 4T-1 cells between the phosphate-buffered saline (PBS) group and the ER-Cy-poNO 2 + laser treatment group ( Figure 5 a).

[0079] A total of 6,671 peptides were identified, and the length distribution of MHC-I peptides in each group was plotted ( Figure 5 b). The Venn diagram showed that 720 peptides were unique to the PBS group, 2,694 peptides were unique to the ER-Cy-poNO 2 + laser group, and 3,257 peptides were common to both groups, indicating that phototherapy significantly altered the peptidome of 4T-1 tumor cells ( Figure 5 c).

[0080] Among these common peptides, 307 peptides showed significant differential expression. After ER-Cy-poNO 2 + laser treatment, 127 of these peptides were upregulated and 180 peptides were significantly downregulated ( Figure 5 d). The NetMHCpan tool (version 4.1) was used to predict the affinity of the significantly upregulated peptides for MHC-I (including H2-Kd, H2-Dd, and H2-Ld). Strong-binding peptides (SBs) were defined as having a percentile rank of the binding affinity score < 0.5, and weak-binding peptides (WBs) had a percentile rank of 0.5 - 2.

[0081] Among the upregulated peptides, 71.5% of the strong-binding peptides bound to H2-Dd, 39.2% bound to H2-Kd, and 31.5% bound to H2-Ld ( Figure 5 e). Analysis of the corresponding sequence motifs revealed different patterns for each haplotype, indicating that phototherapy-induced changes in the immunopeptidome involve the selective presentation of specific peptide sequences by MHC-I ( Figure 5 f).

[0082] Collectively, these findings indicate that ER-Cy-poNO 2 -mediated endoplasmic reticulum-targeted phototherapy induced significant changes in the immunopeptidome of 4T-1 tumor cells, generating multiple immunopeptides that were upregulated at multiple levels and had high affinity binding to MHC-I.

[0083] Example 6: Screening of candidate peptides with high immunogenicity

[0084] In this example, based on the transcriptome sequencing in Example 4 and the immunopeptidomics analysis results in Example 5, we further systematically screened and evaluated candidate peptides with high immunogenicity (Figure 6 a). First, 4,892 peptide segments with lengths ranging from 8 to 11 amino acids were selected. Second, 108 significantly up-regulated peptide segments were identified. Third, among these up-regulated peptide segments, 82 were predicted to have a strong binding affinity with MHC-I. Finally, based on the predicted immunogenicity scores, the top 15 immunogenic peptide segments were selected, and their sequences, protein sources, and predicted MHC-I haplotypes to which they bind were summarized. These 15 immunogenic peptides are selected from at least one peptide selected from immunogenic peptide IF4G3, immunogenic peptide RPB2, immunogenic peptide B4GT4, or immunogenic peptide NA, or immunogenic peptide SAE2, or immunogenic peptide ACTG, or immunogenic peptide CARTF, or immunogenic peptide WDR12, or immunogenic peptide YIPF3, or immunogenic peptide HTR5B, or immunogenic peptide TM35B, or immunogenic peptide RENT1, or immunogenic peptide UBP30, or immunogenic peptide DNMT1, or immunogenic peptide SOS1;

[0085] Among them, the immunogenic peptide IF4G3 is the amino acids at positions 986 - 994 of the IF4G3 protein, and its sequence is QGPKTIEQI (SEQ ID NO.1); the immunogenic peptide RPB2 is the amino acids at positions 1043 - 1051 of the RPB2 protein, and its sequence is IGPTYYQRL (SEQ ID NO.2); the immunogenic peptide B4GT4 is the amino acids at positions 230 - 238 of the B4GT4 protein, and its sequence is KYFGGVTAL (SEQ ID NO.3); the amino acid sequence of the immunogenic peptide NA - 1 is TYKPTTNGL (SEQ ID NO.4); the immunogenic peptide SAE2 is the amino acids at positions 143 - 151 of the SAE2 protein, and its sequence is GYLGQVTTI (SEQ ID NO.5); the amino acid sequence of the immunogenic peptide NA - 2 is ESGPSIVHRL (SEQ ID NO.6); the immunogenic peptide CARTF is the amino acids at positions 438 - 446 of the CARTF protein, and its sequence is SYFPTVNDI (SEQ ID NO.7); the immunogenic peptide WDR12 is the amino acids at positions 413 - 421 of the WDR12 protein, and its sequence is SYSPTASHV (SEQ ID NO.8); the immunogenic peptide YIPF3 is the amino acids at positions 266 - 274 of the YIPF3 protein, and its sequence is VGPTQRLLL (SEQ ID NO.9); the immunogenic peptide HTR5B is the amino acids at positions 2012 - 2020 of the HTR5B protein, and its sequence is IGPLYPHAF (SEQ ID NO.10); the immunogenic peptide TM35B is the amino acids at positions 77 - 85 of the TM35B protein, and its sequence is VGPPVLQEI (SEQ ID NO.11); the immunogenic peptide RENT1 is the amino acids at positions 964 - 972 of the RENT1 protein, and its sequence is MYFQTHDQI (SEQ ID NO.12); the immunogenic peptide UBP30 is the amino acids at positions 240 - 248 of the UBP30 protein, and its sequence is QSPVRFDTF (SEQ ID NO.13); the immunogenic peptide DNMT1 is the amino acids at positions 1362 - 1370 of the DNMT1 protein, and its sequence is SGPFRTITV (SEQ ID NO.14); the immunogenic peptide SOS1 is the amino acids at positions 746 - 754 of the SOS1 protein, and its sequence is NGPGHNITF (SEQ ID NO.15), ( Figure 6 b).

[0086] Among these peptide segments, the 15th peptide segment IF4G3 986-994(QGPKTIEQI) elicited the strongest γ-interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) response, even exceeding that of the positive control (phorbol myristate acetate (PMA) plus ionomycin), which is known to strongly activate T lymphocytes. Figure 6 c-e). This indicates that among the tested candidate peptides, the IF4G3 986-994 peptide may be the most effective stimulator for activating the mouse immune system.

[0087] Example 7: In vitro immunogenicity assessment of candidate peptides

[0088] In this example, the in vitro immunogenicity of the candidate peptides was tested as follows:

[0089] Materials and testing methods:

[0090] 1) Synthesis and in vitro immunogenicity assessment of candidate peptides

[0091] The candidate peptides were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. with a purity greater than 95%. The immunogenicity of the candidate peptides was detected by a γ-interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay involving BALB / c mouse splenocytes. Briefly, freshly isolated splenocytes were incubated with each candidate peptide at a concentration of 50 μg / ml, and interleukin-2 (IL-2) at a concentration of 50 U / ml was added during this process. Bone marrow-derived dendritic cells (BMDCs) were cultured from six-week-old female BALB / c mice according to a previously reported protocol. BMDCs were cultured in complete RPMI-1640 medium (Gibco, New York, USA) containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, granulocyte-macrophage colony-stimulating factor (GM-CSF, 25 ng / ml), and interleukin-4 (IL-4, 10 ng / ml). On the sixth day, immature DCs were incubated overnight with each candidate peptide at a concentration of 50 μg / ml. The next day, peptide-prestimulated splenocytes (2 × 10 5 cells per well) were co-cultured with peptide-loaded BMDCs (1 × 10 4 cells per well) in a 96-well ELISPOT plate pre-coated with IFN-γ-specific capture antibody. Then this co-culture was placed in an incubator at 37°C and 5% carbon dioxide for 20 hours. After the incubation period, the cells were removed from the plate, and then the plate was processed according to the guidelines provided by the manufacturer of the IFN-γ ELISPOT kit. Finally, the number of spots was counted using a spot reading system.

[0092] 2) Six-week-old female BALB / c mice were randomly divided into 3 groups. The negative control group was injected with 100 μl of sterile PBS. The polyinosinic:polycytidylic acid (poly IC) control group received 10 μg of poly IC dissolved in 100 μl of sterile PBS. The treatment group received 10 μg of the candidate peptide IF4G3 986-994 in a mixture with 10 μg of poly IC. All injections were subcutaneous, administered once every three days for a total of 3 doses. Seven days after the last vaccination, the spleens of 3 mice from each group were collected for interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assays. Then, 1 × 10 6 normal 4T-1 cells were subcutaneously injected into the right hind limb of the remaining mice in each group. The body weight and tumor volume of the mice were measured every 3 days, and a complete blood count was performed once a week. When the average tumor volume in the PBS group and the poly IC control group reached 600 cubic millimeters, the mice were euthanized, and serum samples were collected for subsequent enzyme-linked immunosorbent assay (ELISA) analysis. Dendritic cells (DCs) and lymphocytes were collected for subsequent flow cytometry analysis, and tumor tissues were collected for weighing and photography.

[0093] Among them, the flow cytometry analysis was specifically as follows:

[0094] To analyze the maturation of dendritic cells (DCs), cells isolated from lymph nodes were stained with the fixable viability dye 780, phycoerythrin (PE)-conjugated anti-CD11c antibody, allophycocyanin (APC)-conjugated anti-CD80 antibody, and PE-Cy7-conjugated anti-CD86 antibody. The staining was performed for 20 minutes at 4°C in the dark, and then the percentage of mature DCs (CD11c + CD80 + CD86 + ) was quantified using a CytoFlex LX flow cytometer (Beckman Coulter, Inc., California, USA). The data were analyzed using FlowJo V10 software (Beckman Coulter, Inc., California, USA).

[0095] For the analysis of T lymphocytes, first, staining was performed with the fixable viability dye 780. Subsequently, staining was performed using fluorescein isothiocyanate (FITC)-conjugated anti-CD3 antibody, APC-conjugated anti-CD45 antibody, PE-Cy7-conjugated anti-CD4 antibody, and peridinin chlorophyll protein-cyanine 5.5 (PerCP-Cy5.5)-conjugated anti-CD8 antibody.

[0096] To analyze regulatory T cells (Tregs), the cells were first washed to remove residues, then fixed with a fixative and permeabilized with a permeabilization buffer to label nuclear proteins. After fixation and permeabilization were completed, the cells were stained with a PE-conjugated anti-forkhead box protein P3 (FoxP3) antibody and a BV421-conjugated anti-CD25 antibody.

[0097] For IFN-γ + For the analysis of T lymphocytes, the cells were also washed, fixed, and permeabilized. Subsequently, a PE-conjugated anti-IFN-γ antibody was added to the permeabilization buffer and incubated at 4 °C for 20 minutes to complete the staining. After staining, all samples were washed again to remove unbound antibodies, and then detected using a CytoFlex LX flow cytometer, and the data were analyzed using FlowJo V10 software.

[0098] The in vitro BMDCs maturation experiment was as follows:

[0099] BMDCs were cultured from six-week-old female BALB / c mice according to a previously reported protocol. Briefly, BMDCs were cultured in RPMI-1640 (Gibco, New York, USA) medium supplemented with 1% penicillin / streptomycin, 10% FBS, GM-CSF (25 ng / mL), and IL-4 (10 ng / mL). On day 6, immature BMDCs were co-cultured overnight with the candidate immunopeptides or the positive stimulant lipopolysaccharide (LPS, 1 μg / mL, Sigma, Missouri, USA). To evaluate the purity and maturity of the cultured BMDCs, the cells harvested on day 7 were stained. In the dark at 4 °C, the cells were stained with a fixable viability dye 780, a phycoerythrin (PE)-conjugated anti-mouse CD11c antibody, a PE-Cy7-conjugated anti-mouse CD86 antibody, and an allophycocyanin (APC)-conjugated anti-mouse CD80 antibody for 20 minutes. After washing the cells, the percentage of mature BMDCs (CD11c + CD80 + CD86 + ) was quantified using a CytoFlex LX flow cytometer. Subsequently, the obtained data were analyzed using FlowJo V10 software.

[0100] Results and discussion:

[0101] To further verify the immunogenicity of these candidate peptides, their ability to stimulate the maturation of mouse bone marrow-derived dendritic cells (BMDCs) was tested in vitro ( Figure 6f). The maturation of BMDCs was evaluated by upregulation of surface markers such as CD80 and CD86. Consistent with the IFN-γ ELISPOT results, among the tested peptides, peptide IF4G3 986-994 showed the strongest ability to promote BMDCs maturation ( Figure 6 f). In summary, the comprehensive analysis of IFN-γ ELISPOT and BMDCs maturation experiments showed that among the 15 candidate peptides evaluated, peptide IF4G3 986-994 exhibited the strongest immunogenicity and the ability to activate the immune response.

[0102] Example 8: In vivo experiments to evaluate the ability of candidate peptides to activate anti-tumor immune responses

[0103] In this example, the in vivo immunogenicity of the candidate peptides was tested, specifically as follows:

[0104] Materials and testing methods:

[0105] Six-week-old female BALB / c mice were randomly divided into 3 groups. The negative control group was injected with 100 μl of sterile PBS. The polyinosinic:polycytidylic acid (poly IC) control group received 10 μg of poly IC dissolved in 100 μl of sterile PBS. The treatment group received a mixture of 10 μg of candidate peptide IF4G3 986-994 and 10 μg of poly IC dissolved in 100 μl of sterile PBS. All injections were subcutaneous, administered every three days for a total of 3 doses. Seven days after the last vaccination, the spleens of 3 mice from each group were collected for interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assays. Then, 1×10 6 normal 4T-1 cells were subcutaneously injected into the right hind limb of the remaining mice in each group. The body weight and tumor volume of the mice were measured every 3 days, and a complete blood count was performed once a week. When the average tumor volume in the PBS group and the poly IC control group reached 600 cubic millimeters, the mice were euthanized, and dendritic cells (DCs) and lymphocytes were collected for subsequent flow cytometry analysis. Meanwhile, tumor tissues were collected for weighing and photography.

[0106] Among them, the methods for flow cytometry analysis, T lymphocyte analysis, regulatory T cell analysis, and IFN-γ + T lymphocyte analysis were the same as in Example 7;

[0107] Results and discussion:

[0108] Through in vivo tumor vaccine experiments, the immunogenicity of immune peptide IF4G3 986-994 and its ability to activate anti-tumor immune responses were further evaluated ( Figure 7 a).

[0109] Compared with the phosphate-buffered saline (PBS) group and the group using the immune adjuvant poly IC alone, poly IC+IF4G3 986-994 significantly increased the activation of splenocytes, which was confirmed by the increased number of spots in the IFN-γ enzyme-linked immunospot (ELISPOT) assay ([[]] Figure 7 b, c). In addition, tumor growth in the poly IC+IF4G3 986-994 group was significantly slower ([[]] Figure 7 d). Compared with the PBS group and the poly IC group, the tumor size and weight in the poly IC+IF4G3 986-994 group were significantly reduced ([[]] Figure 7 e, 7f). The proportion of mature CD80 986-994 CD86 + dendritic cells (DCs) in the lymph nodes and the proportion of CD8 + T lymphocytes in the spleen in the poly IC+IF4G3 + group were significantly higher than those in the PBS group ([[]] Figure 7 g-j). Notably, the percentage of IFN-γ 986-994 CD8 + T lymphocytes in the spleen in the poly IC+IF4G3 + group reached nearly 30%, while it was 5% in the PBS group and 15% in the poly IC group ([[]] Figure 7 k, l). In addition, the proportion of CD4 986-994 CD25 + FoxP3 + regulatory T cells (Tregs) in the tumors of the poly IC+IF4G3 + group decreased by approximately 10%, indicating that the immunosuppressive tumor microenvironment was weakened during the strong immune activation process ([[]] Figure 7 m, n). These results indicate that the highly immunogenic IF4G3 986-994 peptide combined with poly IC can strongly activate the anti-tumor immune response and inhibit tumor growth in mice.

[0110] In summary: The schematic diagram of the potential mechanism by which endoplasmic reticulum-targeted phototherapy remodels the tumor immunopeptidome to enhance the immunogenicity of dying tumor cells and activate the adaptive anti-tumor immune response in the present invention is as Figure 1 shown. The light-triggered immunotherapy uses a tumor-endoplasmic reticulum dual-targeted photosensitizer (ER-Cy-poNO 2) is achieved by using a photosensitizer that can strongly induce endoplasmic reticulum stress-driven immunogenic cell death in tumor cells (immunogenic cell death occurring in 4T-1 cells in the present invention). As a result, a large number of tumor-derived peptides that bind to major histocompatibility complex class I (MHC-I) are produced. After multiple screenings from these peptides, IF4G3 986-994 (QGPKTIEQI) has the most excellent immunogenicity and can effectively enhance anti-tumor immunity.

[0111] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention, and they should all be covered by the scope of the claims and the description of the present invention.

Claims

1. Application of immunogenic peptides in the preparation of drugs for activating anti-tumor immune responses.

2. The use according to claim 1, characterized in that: The immunogenic peptide can enhance the antigenicity of immunogenic dead tumor cells and activate anti-tumor immune response.

3. The use according to claim 1, characterized in that: The immunogenic peptide contains at least one peptide selected from the group consisting of immunogenic peptide IF4G3, immunogenic peptide RPB2, immunogenic peptide B4GT4, or immunogenic peptide NA-1, or immunogenic peptide SAE2, or immunogenic peptide NA-2, or immunogenic peptide CARTF, or immunogenic peptide WDR12, or immunogenic peptide YIPF3, or immunogenic peptide HTR5B, or immunogenic peptide TM35B, or immunogenic peptide RENT1, or immunogenic peptide UBP30, or immunogenic peptide DNMT1, or immunogenic peptide SOS1, and the at least one peptide is bound to a carrier protein or not bound to a carrier protein.

4. The use according to claim 3, characterized in that: The immunogenic peptide contains the immunogenic peptide IF4G3.

5. The use according to claim 4, characterized in that: The immunogenic peptide IF4G3 is the amino acids 986-994 of the IF4G3 protein, and its sequence is shown in SEQ ID NO.

1.

6. The use according to claim 3, characterized in that: The immunogenic peptide RPB2 is the 1043-1051 amino acids of the RPB2 protein, and its sequence is shown in SEQ ID NO.2; the immunogenic peptide B4GT4 is the 230-238 amino acids of the B4GT4 protein, and its sequence is shown in SEQ ID NO.3; the amino acid sequence of the immunogenic peptide NA-1 is SEQ ID NO.4; the immunogenic peptide SAE2 is the 143-151 amino acids of the SAE2 protein, and its sequence is shown in SEQ ID NO.5; the amino acid sequence of the immunogenic peptide NA-2 is SEQ ID NO.6; the immunogenic peptide CARTF is the 438-446 amino acids of the CARTF protein, and its sequence is shown in SEQ ID NO.7; the immunogenic peptide WDR12 is the 413-421 amino acids of the WDR12 protein, and its sequence is shown in SEQ ID NO.8; the immunogenic peptide YIPF3 is the 266-274 amino acids of the YIPF3 protein, and its sequence is shown in SEQ ID NO.9; the immunogenic peptide HTR5B is the amino acids 2012-2020 of the HTR5B protein, and its sequence is shown in SEQ ID NO.10; the immunogenic peptide TM35B is the amino acids 77-85 of the TM35B protein, and its sequence is shown in SEQ ID NO.11; the immunogenic peptide RENT1 is the amino acids 964-972 of the RENT1 protein, and its sequence is shown in SEQ ID NO.12; the immunogenic peptide UBP30 is the amino acids 240-248 of the UBP30 protein, and its sequence is shown in SEQ ID NO.13; the immunogenic peptide DNMT1 is the amino acids 1362-1370 of the DNMT1 protein, and its sequence is shown in SEQ ID NO.14; the immunogenic peptide SOS1 is the amino acids 746-754 of the SOS1 protein, and its sequence is shown in SEQ ID NO.

15.

7. Application of immunogenic peptides combined with polyinosinic-polycytidylic acid in the preparation of drugs for activating anti-tumor immune response.

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