| Background and objectiveCancer vaccines have attracted extensive attention for their great potential for personalized therapy,prophylactic effects,and long-term antitumor effects.Cancer vaccines are designed to involve tumor antigens with adjuvants to activate DC,inducing antigen presentation for T cell priming,producing cytotoxic T lymphocytes(CTLs)for tumor infiltration.In general,cancer vaccines are designed to target tumor-associated antigens(TAA)and tumor-specific antigens(TSA),also called as neoantigens.TSA are derived from random genome mutations only in tumor cells but not expressing in normal cells,which are considered as the most effective targets for personalized cancer vaccines.However,the clinical translation is still hampered by the following challenges.Firstly,the therapeutic efficacy of cancer vaccines is largely limited by multiple biological barriers,including rapid clearance,poor lymphatic transport,insufficient cellular uptake as well as endosome entrapment.Secondly,the defects of antigen-presenting cells(APC)for antigen processing and presentation will induce modest T cell responses,and the immunosuppressive factors,including immunosuppressive cells,cytokines,and checkpoint blockades,existing in tumor microenvironment(TME)also induce attenuate therapeutic benefits.Thereby,we designed and established two kinds of nanovaccines as universal platforms for delivery of peptide-based and mRNA-based neoantigens,respectively.Meanwhile,novel adjuvants were combined with neoantigens for APC activation,potentially improving antigen-specific T cell responses and therapeutic effects in tumor-bearing mice.This study provided an inspiration for design and development of a novel cancer vaccine,expanding applications in various tumors.Contents and methodsGiven that activation of stimulator interferon genes(STING)pathway in DC could enhance antigen presentation efficiency,inducing robust tumor-specific T cell responses.For the proof of concept,we engineered an intelligent nanovaccine,called Man-PDPM@Ag,based on acid-responsive polymers for co-delivery of neoantigen peptide and STING agonist,DMXAA.The acid-responsive polymers were synthesized by reversible additionfragmentation chain transfer polymerization(RAFT)and DMXAA was conjugated onto the backbone.Man-PDPM@Ag nanovaccines were fabricated by nanoprecipitation method and then characterized.The biodistribution and lymph nodes accumulation of nanovaccines were detected by fluorescence imaging in vivo and ex vivo,while the cellular uptake of antigens was determined by flow cytometry.To determine STING pathway activation,the expression of downstream proteins,related genes and cytokines secretion were analyzed by western blot.real-time PCR and ELISA assay,respectively.The immune analysis,including DC maturation,antigen presentation,generation of tumor-specific T cells and memory T cells in spleen,as well as T cell subsets in tumors were all determined by flow cytometry.For antitumor studies,the therapeutic efficacy of nanovaccines were evaluated by tumor volume,overall survival,and biosafety in subcutaneous tumor models of B16-OVA and 4T1 tumors.In another work,we proposed a liponanoparticle(Lipo-ORG)for tailored delivery of mRNA tumor antigen and STING agonist,2’3’-cGAMP.OEI-PBA polymer was synthesized by substitution reaction under different feeding ratio of OEI to PBA.Then the ability for mRNA condensation was studied by agarose gel electrophoresis assay and transfection efficiency of mRNA was assessed by flow cytometry.Then ORG nanoparticles were formulated by complexing OEI-PBA.mRNA and 2’3’-cGAMP via electrostatic interaction.flow cytometry and confocal laser scanning microscopy were used to investigate cytosolic delivery and intracellular localization of cGAMP,respectively.Lipo-ORG was constructed by film dispersion and its morphology was observed by cryo-TEM.Other experiments with the same contents or purposes were performed as above illustrated.ResultsMan-PDPM@Ag nanovaccine displayed uniform and spherical morphology,with a hydrodynamic diameter of 69.7±5.9 nm.The nanovaccine showed the encapsulation efficiency of OVA 257-264 of～70%and over 80%of DMXAA released within 20 min in the presence of esterase.The nanovaccines have good accumulation in lymph node and facilitated DC uptake of antigens.DMXAA in the nanovaccines activated STING pathway in DC and promoted DC maturation as well as antigen presentation,eliciting tumor-specific T cell priming and intratumoral infiltration of CTLs.The antitumor immune responses significantly inhibited tumor growth,prolonged mice survival and showed good biosafety in both tumor models of B16-OVA and 4T1.Combination therapy with anti-PD-L1 antibody further improved antitumor effects and suppressed tumor metastasis.Meanwhile,we established a universal nanoplatform for delivering mRNA and other nucleic acid drugs to lymph node.The synthesized polymer OEI-PB A has a superior capability for mRNA condensation and transfection.The formulated ORG nanoparticles displayed a diameter of～40 nm and highly positive surface charge of 18.6±3.6 mV,while Lipo-ORG exhibited a diameter of～100 nm and negatively charge of-14.0 ± 0.4 mV.The cytosolic delivery of cGAMP was dramatically prompted,which lead to STING activation both in vitro and in vivo.Taking advantages of efficient lymphatic transport and DC uptake,tumor-specific T cell responses,increased tumor infiltrating of CTLs and decreased regulatory T cells were detected after vaccination in B16-OVA tumor-bearing mice,which contributed to suppressed tumor growth and extended overall survival.Herein,we presented feasible and flexible nanoplatforms for delivering peptide-based and mRNA-based neoantigens for personalized cancer immunotherapy.These nanovaccines were highly efficient for overcoming multistage biological barriers to stimulating robust and long-lasting adaptive immune responses.We also verified the mechanisms and superior efficacy for combination STING-activation with nanovaccines,which is also a promising therapeutic strategy for clinical translation. |
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