Research Of The Function And Mechanism Of Antigen Peptide Disturbance Mediated By Proteasome Inhibitor In Synergy With PD-1 Monoclonal Antibody

Background:In recent years,immunotherapy,especially immune checkpoint inhibitors targeting PD-1,CTLA-4,and other checkpoints molecules,has shown promising clinical outcomes in various cancer types,marking a new breakthrough in cancer treatment.Known for its wide range of application,long-lasting therapeutic effect and minimal side effects,immunotherapy has even led to clinical cures in some patients.However,the overall response rate falls short of expectations.In metastatic colorectal cancer（m CRC）,only about5%of patients with Microsatellite Instability-High（MSI-H）can benefit from immune checkpoint therapy,while the vast majority of Microsatellite Stable（MSS）type find it hard to gain benefits from such treatments.Enhancing the sensitivity of MSS-type tumor to immunotherapy has emerged as a focal area of research.MSI-H type tumor is predominantly characterized by an immune-inflamed subtype,representing a typical“hot”tumor microenvironment.In contrast,MSS tumor commonly exhibits immune-excluded and immune-desert subtypes,falling into the category of“cold”tumors.Currently,it is believed that this microenvironmental difference may be due to the low immunogenicity caused by the lack of mutations in MSS tumors.Therefore,strategies such as combining immune checkpoint inhibitors with chemotherapy,targeted therapy,and radiation have been developed to improve the efficacy against such tumors.While these combined immunotherapeutic strategies may emphasize various mechanistic explanations,they mostly involve changes in tumor antigenicity,essentially converting“cold tumors”into“hot tumors”.Hence,we hypothesize that intervening in the tumor antigen processing process may help increase the neoantigen load in MSS tumors,thereby promoting the transition of the immune microenvironment from“cold”to“hot”.Bortezomib is the first proteasome inhibitor approved by the FDA for cancer treatment,demonstrating good safety and efficacy in long-term clinical applications.By selectively modulating the activity of the20S proteasome subunit,it can potentially impact the processing of MHC-I peptides in tumor cells,leading to the generation of more neo-peptide segments.This intervention is expected to enhance the infiltration and cytotoxicity of immune cells against“cold”tumors,thereby synergizing with immune checkpoint inhibitors and further promoting their efficacy in treating p MMR/MSS type cancer patients.Objective:Investigate the preclinical efficacy and mechanisms of proteasome inhibitor bortezomib combined with immune checkpoint therapy,providing theoretical basis and experimental foundation for clinical application.1.Clarify the in vitro effects of the proteasome inhibitor bortezomib on tumor cells;2.Elucidate the regulation of tumor microenvironment by the proteasome inhibitor bortezomib;3.Investigate the mechanism of proteasome pretreatment at low doses in regulating the immune microenvironment;4.Explore the impact of bortezomib-induced proteasome inhibition on the peptide repertoire and neoantigen processing in tumors;5.Evaluate the efficacy of bortezomib in combination with PD-1 checkpoint inhibition in syngeneic tumor models.Method:1.Cellular experiment to detect the in vitro effects of bortezomib on tumorsCCK-8 assay was used to measure the viability of tumor cells treated with different concentrations of bortezomib after 48 hours,and a dose-response curve was plotted;flow cytometry was employed to detect Annexin V/PI staining in bortezomib-treated tumor cells to calculate cell apoptosis;PI staining was utilized for DNA staining of bortezomib-treated tumor cells,and flow cytometry analysis was carried out to determine the proportion of cells in the G1 phase,S phase,and G2/M phase;Western blot was conducted to assess the level of protein ubiquitination in tumor cells after treatment with bortezomib;a proteasome activity fluorescent reporter assay was performed to evaluate the proteasomal subunit activity in MC38 cell line after bortezomib treatment.2.Evaluation of the immune microenvironment changes in syngeneic tumor model after bortezomib treatmentSyngeneic tumor model to assess the impact of proteasome inhibitors on the tumor microenvironment;calipers used for continuous monitoring of tumor growth in vivo;flow cytometry employed to detect changes in major immune cell subpopulations infiltrating in the tumor;mice were treated with FTY720,and the effect of peripheral lymphocyte migration on bortezomib inhibition was evaluated.3.Wash-out model to explore the immune cell subpopulations and mechanisms through which bortezomib regulates the immune microenvironmentBortezomib washout model to investigate the effects of sublethal doses of bortezomib on tumor growth in vitro and in vivo;subcutaneous tumor engraftment in Rag2^-/-gene mice to determine whether the inhibitory effect of bortezomib in vivo is mediated by innate or adaptive immunity;co-culturing bone marrow-derived dendritic cells（BMDC）with Di O-stained tumor cells and using flow cytometry to assess changes in antigen-presenting cell function;depleting corresponding cell populations in C57BL/6 mice using CD8,CD4,CD19,and NK1.1 m Abs;detecting immune cell depleting efficiency through flow cytometry;monitoring tumor growth in the drug washout model after immune cell depleted to identify the key immune cell subpopulations involved;constructing B2m knockout MC38cell lines using CRISPR-Cas9;conducting in vivo and in vitro experiments using MHC-I deficient MC38 cells to validate the role of MHC-I molecules in the drug washout model;establishing MC38-OVA-Luc cell lines stably expressing ovalbumin,co-culturing with OT-I CD8⁺T cells to detect antigen-specific killing function,and conducting fluorescence reporter assays to measure MC38-OVA viability;using flow cytometry to assess changes in membrane H-2 and H-2Kb^SIINFEKL expression in bortezomib-treated MC38-OVA cells.4.Multi-omics analysis of changes in the proteomics,peptidomes,and immunopeptidomes induced by bortezomibUsing proteomics to detect differential expressed proteins after bortezomib treatment;LMWP mass spectrometry for non-enzyme-treated samples,analyzing peptidomes using the Peaks algorithm;immunoprecipitation to enrich MHC-I antigen peptides,mass spectrometry to analyze the immunopeptidomes;employing bioinformatics methods to screen for neo-antigenic peptides;conducting immunogenicity analysis on peptides using the IEDB database and Net MHCpan-4.1 algorithm.5.Evaluation of the therapeutic efficacy and side effects of bortezomib in combination with PD-1 monoclonal antibody in a syngeneic modelEvaluating of the efficacy of bortezomib in combination with the PD-1 monoclonal antibody in syngeneic mouse models;histopathological changes in major relevant organs assessed through tissue HE staining;blood biochemistry,and complete blood counts to evaluate liver and kidney target-organ toxicity as well as hematologic toxicity;immunohistochemistry staining to detect the number of CD8-positive cells within the tumor post-treatment;flow cytometry to measure changes in the quantity and functionality of infiltrating lymphocytes within the tumor endured combination therapy.Results:1.In vitro effects of proteasome inhibitor bortezomib on tumor cellsThe IC50 values of bortezomib for the cell lines HCT116,HT29,4T1,MC38,CT26,and B16 are 51.59 n M,56.57 n M,42.89 n M,34.83 n M,27.58 n M,and 26.85 n M,respectively.After treating tumor cells with different concentrations of bortezomib for 48hours,there was a significant increase in apoptosis and an elevation in the proportion of G2/M phase cells when the concentration reached 30～60 n M.Western blot analysis showed an increase in ubiquitination levels after bortezomib treatment,with the most prominent changes observed in K48 ubiquitination.Following incubation of tumor cells with bortezomib,the relative fluorescence intensity of Suc-Leu-Leu-Val-Tyr-AMC and Z-LEU-LEU-GLU-AMC substrates was significantly lower compared to the control group,while no significant difference was observed in the relative fluorescence intensity of Bz-Val-Gly-Arg-AMC between the two groups.2.The regulatory effects of bortezomib on the tumor microenvironmentIn the CT26 and B16 mouse syngeneic tumor models,the tumor growth curve of the bortezomib treatment group was significantly inhibited compared to the control group.The bortezomib treatment group showed a significant increase in the number of CD3⁺T cells,CD8⁺T cells,CD4⁺T cells,NK cells,and TCR-γδ⁺T cells within the tumors compared to the control group.In the bortezomib treatment group,there was a significant increase in the number of monocytes,macrophages,and dendritic cells in the myeloid immune cells compared to the control group,with a significant increase in the ratio of M1/M2macrophages as well.In the FTY720 combined with bortezomib treatment group and the control group,no significant difference was observed in tumor growth in vivo,while tumor growth was inhibited in the bortezomib treatment group.3.The mechanisms by which bortezomib regulates the tumor microenvironmentIn drug washout model,after treating MC38 cells with a low concentration of bortezomib,there was no observable difference in the in vitro growth between the treated group and the control group.However,a significant difference in tumor growth in vivo was observed when comparing the bortezomib-treated group to the control group.Rag2^-/-mice implanted with MC38 cell lines pre-treated with bortezomib or DMSO showed no differences in tumor growth between the treated group and the control group.Co-culturing dendritic cells induced ex vivo from bone marrow with Di O-stained bortezomib-pretreated tumor cells did not show any differences in the proportion of Di O⁺CD11c⁺cells at low concentrations.It was observed that after removing CD19⁺,NK1.1⁺,and CD4⁺cells,tumor growth inhibition was present in the bortezomib-treated group in the drug washout model while the inhibitory effect disappeared when CD8⁺cells was removed.Constructing MHC-I molecule knock-out cells and pretreating them with low concentrations of bortezomib did not show any differences in tumor growth both in vivo and in vitro.Co-culturing OT-I CD8⁺T cells with bortezomib-treated MC38-OVA cells resulted in increased tumor cell viability in the bortezomib-treated group,indicating inhibition of the antigen-specific cytotoxicity of OT-I T cells.After treating MC38-OVA cells with bortezomib,there were no observed changes in the membrane expression of H-2 molecules on tumor cells,while H-2Kb^SIINFEKLexpression decreased.4.Impact of bortezomib-induced proteasome inhibition on peptides repertoire and neoantigen processingMass spectrometry revealed 407 differential proteins and GO/KEGG functional enrichment showed that these differential proteins are related to the protein degradation process.Peptidomes identified 5739 peptide segments,with an increase in peptide diversity after proteasome inhibition,particularly evident in peptides with arginine residues at the C-terminal.The efficiency of MHC-I antigen peptide complex immunoprecipitation（IP）and mass spectrometry detection met quality control requirements.Antigen peptide mass spectrometry detected 6607 immune peptides,with lengths ranging from 7 to 16 amino acids.Treatment with bortezomib led to a significant decrease in the proportion of hydrophobic and neutral amino acids.Integrating results from the protein group,peptidome,and immunopeptidome,it was found that Vimentin-derived peptides exhibited the most significant changes in both the peptidome and immunopeptidome,with increased abundance and variety after bortezomib treatment.Predictions from the IEDB database and Net MHCpan-4.1 algorithm indicated enhanced immunogenicity of Vimentin-derived peptides induced by bortezomib.5.Preliminary observations on the efficacy and adverse effects of the combination of Bortezomib with PD-1 m AbsAfter treatment with the combination of Bortezomib and PD-1 m Abs in mouse syngeneic tumor models,the tumor growth in the combination therapy group was significantly inhibited,and tumor regression rate was markedly higher compared to the single group.HE staining results did not show any significant pathological changes in major organs of all treatment groups,including the combination therapy group.Hematological toxicity and liver-kidney toxicity-related indicators did not show significant changes in all treatment groups.Immunohistochemistry（IHC）results demonstrated that both bortezomib monotherapy and combination therapy significantly increased the number of CD8⁺cells compared to the control group and PD-1 inhibitor monotherapy group.Flow cytometry analysis detected a significant increase in the proportion of CD8⁺T cells in tumors in the combination therapy group,along with a notable increase in the proportions of IFN-γ⁺and GZMB⁺CD8⁺T cells.Conclusion:1.Bortezomib promotes apoptosis in a concentration-dependent manner,induces G2/M cell cycle arrest,and facilitates the accumulation of K48-linked ubiquitinated proteins in vitro,thereby inhibiting the proliferative capacity of tumor cells.In vitro,Bortezomib selectively and efficiently inhibits the activity of the 20S proteasome subunitsβ5 andβ1.2.Bortezomib treatment enhances the infiltration and function of anti-cancer immune cells in“cold”tumors,promoting the conversion towards a“hot”microenvironment.The therapeutic efficacy of Bortezomib in vivo relies on the migration of T cells from lymphoid tissues to the tumor site.3.Bortezomib can exert in vivo anti-tumor effects after low-dose pretreatment,a mechanism dependent on the recognition of CD8⁺T cells in adaptive immunity with tumor p MHC.Bortezomib can alter the presentation of specific antigen peptides without affecting total MHC-I molecules expression.4.Bortezomib influences the composition of peptide repertoires within tumor cells and the antigen peptide processing process,promoting the generation of neo epitopes,thereby enhancing the immunogenicity of tumoral MHC-I peptides.5.The combination therapy of Bortezomib with PD-1 treatment significantly inhibits tumor growth in syngeneic models and shows no apparent adverse effects.This combined treatment enhances the infiltration of CD8⁺T cells within tumors and their anti-tumor activity.In conclusion,bortezomib can promote neoepitopes by intervening in the proteasomal cleavage process,thereby driving an anti-tumor immune response in“cold”tumors.Bortezomib is expected to offer a novel combined strategy for immunologically“cold”tumors of the microsatellite stable（MSS）type.This involves enhancing tumor immunogenicity through antigen peptide perturbation to improve responses to immune checkpoint therapies.