The Construction And Antitumor Immunological Activity Of Adjuvant-free Peptide Vaccine And Biomimetic Nanovaccine By Targeting Clec9a~+ Dendritic Cell

Recently,tumor immunotherapy has become an important treatment strategy in clinical tumor treatment.A large number of checkpoint inhibitors(ICIs)have been used for clinically and demonstrated good therapeutic effects.However,the efficacy of ICIs varies widely,with less than 20%of cancer patients can benefit from ICIs.Hence,there is an urgent need to identify novel tumor immunotherapy strategies or drugs.Tumor vaccines are able to overcome resistance to ICIs and has good curative effect in cold tumors.Therefore,tumor vaccines represent an attractive tumor immunotherapy strategy.Dendritic cells(DCs)are a pivotal part of the immune system and widely explored for tumor immunotherapies,especially for tumor vaccine studies.The key of generating effective tumor vaccines is the ability to stimulate antigen specific cytotoxic T lymphocyte(CTL)responses by DCs.cDC1s excel at antigen cross-presentation,and thereby stimulate robust antigen specific CTL responses.The C-type lectin domain family 9 member A(Clec9a)is an endocytic receptor and selectively expressed on cDC1s.Antigens targeting Clec9a is a promising strategy for tumor vaccine.Although antigen targeting cDC1s by Clec9a antibody is feasible,antibodies relatively large molecular masses and complex structures,it is difficult to conjugate or fuse antibodies with antigens.Clinically,tumor vaccine requires adjuvant in T cell-mediated antitumor immune responses,however,adjuvants can randomly activate innate immune cells and induce inflammatory side effects.To obtain peptides with high affinity and selectivity toward mClec9a,computeraided in silico method was applied for peptide mutation.The binding and MST experiments showed that a 12-mer Clec9a binding peptide(CBP-12)with high affinity for mClec9a protein.Through in vitro and in vivo phagocytosis experiments,the results showed that CBP-12 was effectively taken up by cDC1s.Antigen cross-presentation experiments showed that CBP-12-OVA was more effective in stimulating the proliferation of antigen specific CD8+ T cells and IFN-γ secretion.In naive mice,CBP12-OVA induced strong antigen specific CTL responses without adjuvant.We further validated the antitumor activity of adjuvant-free CBP-12 peptide vaccine,the results showed that adjuvant-free CBP-12 peptide vaccine significantly inhibited tumor growth,increased the proportion of tumor-infiltrating T cells and stimulated antigen specific CD8+T cell activation in B 16-OVA and B16 tumor models.In terms of the underlying mechanisms,the results found that CBP-12 may be stimulated cDC1s to release IL-21 through the activation of Clec9a-Syk signal pathway.IL-21 further enhanced antigen cross-presentation and CD8+ T cells activation.Finally,we observed synergistic effects of adjuvant-free CBP-12 peptide vaccine in combination with radiotherapy.The cyclic guanosine monophosphate-adenosine monophosphate synthase(cGAS)and stimulator of interferon gene(STING)pathway belongs to innate immunity and plays an important role in antitumor immunity.cGAS-STING pathway is found in various cells,for tumor cells,macrophage and DCs.The cGAS-STING pathway is more efficiently activated in DCs compared to tumor cells.Activation of STING pathway can trigger the release of type Ⅰ interferon(IFN),which leads to DCs activation and tumor antigen cross-presentation to enhance cytotoxicity of CTL.Therefore,tumor immunotherapy based on STING agonists is a feasible treatment method,especially for tumor vaccine.Most of STING agonists can only be injected intratumorally due to its side effect,thus,limiting the clinical application of STING agonists.Although noncyclic dinucleotide(CDN)STING agonists can systemic administration,significant toxicity is observed in mice.Drug delivery system can enhance cytosolic delivery of STING agonists and increase its biological potency.Nevertheless,these STING agonists delivery systems all fail to achieve optimal STING pathway activation cell subsets.This study found that,compared to tumor cells,cGAS-STING pathway was easier to activate in Clec9a+DCs.What’s more,cGAS-STING pathway activation enhanced CD8+T-cell proliferation and IFN-y secretion.Thus,this study employed a strategy of nanovaccine to target Clec9a+DCs and delivery STING agonist.CBP-12 was originally engineered to be expressed on biomimetic cancer cell membrane(CCM)to coat nanoparticles(PLGA/STING@EPBM).Tumor antigens and 2’,3’-cGAMP of PLGA/STING@EPBM were delivered to Clec9a+ DCs by CBP-12 engineered biomimetic CCM.The vector was constructed consisting of the base sequences of signal peptide,CBP-12,transmembrane segment,intracellular domain and His tag.B 16-OVA,4T1 and TC-1 cells stably expressing CBP-12 were established by viral infection.The hydrodynamic diameter and zeta potential of PLGA/STING@EPBM were 157.5±1.9 nm and-21.1±2.0 mV,respectively.PLGA/STING@EPBM was spherical in shape,exhibited a clear core-shell structure and stable in PBS 7.2 for 4 days.The phagocytosis assays found that PLGA/STING@EPBM exhibited significant uptake by Clec9a+DCs in vitro and in vivo.The effects of PLGA/STING@EPBM on cytosolic delivery and cross-presentation of antigens were then investigated.PLGA/STING@EPBM illustriously promoted interferon stimulating genes(ISGs)activation and antigen crosspresentation of Clec9a+DCs,which further promoted the proliferation of naive T cells.Blood samples,liver injury and organ histopathology were analyzed to evaluate the potential toxicity of nanoparticles(NPs).The results showed that nanovaccines have no obvious toxicity under the treatment dose and frequency.The antitumor effects of PLGA/STING@EPBM nanovaccine was evaluated in the B 16-OVA melanoma model.PLGA/STIENG@EPBM nano vaccine inhibited tumor growth and induced strong antigen specific CD8+T cell responses.Moreover,PLGA/STING@EPBM nanovaccine significantly inhibited the growth of 4T1 breast tumors and lung metastases.PLGA/STING@EPBM nanovaccine therapy remarkably increased the ratio of infiltrating T lymphocyte cells in tumor and stimulated killer CD8+T cell activation.Similarly,the combination of PLGA/STING@EPBM nanovaccine with IR showed synergy effects.In summary,adjutant-free CBP-12 peptide vaccine demonstrated excellent antitumor performance,and synergistic effects with radiotherapy.Mechanistically,this study found that adjuvant-free CBP-12 peptide vaccine enhanced antitumor immune response through Syk/IL-21 axis.In addition,this study found that cGAS-STING pathway was easier activated in Clec9a+DCs.Thus,STING agonist was delivered to Clec9a+DCs by CBP-12-expressed CCM.PLGA/STING@EPBM nanovaccine was systemic administration without obvious toxicity.PLGA/STING@EPBM nanovaccine targeting Clec9a+DCs remarkably reduced the dosage and enhanced the tumor immunotherapy effects of STING agonist.