Preparation Of Self-assembled Short Peptide-based Hydrogels Anti-tumor Vaccine And Its Immunotherapy Efficacy

Tumor immunotherapy is one of the most promising therapy against cancer,and tumor vaccine is an effective approach to eradicate tumor cells.Vaccine is a form of active immunotherapeutic option.It can either stimulate or mobilize the host immune system,and enhance antitumor immunity against the self-formed inhibitory microenvironment,including the release of inhibitory cytokines and metabolites and the presence of immune checkpoint receptors on the effector T-cells.As a result,tumor vaccine has become an ideal option to control and eliminate tumor.The strong cytotoxic T lymphocyte(CTL)response is needed to be activated against the rapid growth of tumor cells,however,currently the efficacy of vaccine is limited by weak cellular immune responses and short duration in vivo.The development of vaccine delivery system based on nanomaterial was substantially enhanced the immunogenicity and antitumor response of conventional vaccine.In particular,the application of self-assembled short peptide-based hydrogels has emerged as a new research hotspot as tumor vaccine.Aims:Dendritic cells(DCs)are antigen presenting cells that are crucial to the efficacy of vaccine stimulation.Immunotherapy based on the injection of vaccine suffers from low cell viability upon injection,short duration in vivo and poor cell homing efficiency.The short peptide-based hydrogel formed by self-assembly technique could function as a scaffold and effective carrier of DCs.On the other hand,the immunogenicity of epitope is weak due to its low molecular weight and simple chemical structure.In addition,epitopes are easily degraded in vivo,which leads to weak immune responses and unsatisfactory tumor inhibition efficiency.In order to improve the immunogenicity and antigen-specific cellular immune response of epitopes,chemical modification and self-assembly technology have been implemented to design and develop novel epitope vaccine.In the present study,tumor vaccine will be obtained by covalently conjugating epitope to self-assembling small molecular peptides.We hope that our research could advance the development of personalized tumor vaccine and cancer immunotherapy.Methods:For DCs vaccine,the short peptide-based hydrogel RADA16 was constructed by self-assembly technique.The effect of hydrogel for DCs vitality and function was analysis by live and dead staining and flow cytometry.For in vivo distribution experiment,the GFP+DCs were used.Specifically,C57BL/6 mice were immunized with GFP+DCs and antigen loaded hydrogel by subcutaneous injection at the back flank.The GFP+DCs in the hydrogel and draining lymph nodes(dLNs)were recovered at scheduled time points post-injection.In vivo DCs duration and homing efficiency were analysed by flow cytometry and immunostaining.Both prophylactic and therapeutic tumor models were used in the study to measure the antitumor effect of combination therapy of DCs vaccine and anti-PD-1 antibody.Regarding the antigenic epitope vaccine,it was obtained by disulfide bond conjugated between the CD8+T epitope TRP2180-188(SVYDFFVWL)to self-assembling small molecular peptides KKFKFEFEF(KKEF).The conjugated epitope vaccine was characterized by TEM,CD spectrum and rheology.The effects of epitope vaccine,such as DCs vitality and function were analysed by live and dead staining and flow cytometry respectively.Splenocyte proliferation and serum cytokine levels were analysed by flow cytometer and ELISA using cocultured sensitized DCs and spleen cells.The antitumor effects and mechanisms were studied in vivo using combination therapy of epitope vaccine and anti-PD-1 antibody.Results:The short peptide hydrogel RADA16 was synthesized and the purity was proved by HPLC and MS.The peptide forms a typical ?-sheet conformation when it is exposed to aqueous solution.DCs were encapsulated into the nanofiber hydrogel by gently mixing the peptide with cell suspension,and rheological analysis demonstrates that the encapsulation of DCs did not significantly change the hydrogel modulus.Meanwhile,the hydrogel efficiently maintained the viability and the biological activity of encapsulated DCs,including antigen uptake and maturation properties.Peptide hydrogel encapsulated with GFP+DCs and antigen was subcutaneously injected into C57BL/6 mice,and DCs in the hydrogels and dLNs were recovered at scheduled time points post-injection.These data indicate that the self-assembled nanofibrous hydrogel can harbor plentiful exogenous DCs,maintain their viability,prolong their duration time period at the injection site,and recruit vast autologous DCs,which could sustain the immune response induced by the exogenous DCs vaccines.In addition,it also effectively promoted the homing of exogenous DCs to lymph nodes,which would provide a better chance to stimulate T cell response.Proliferation and infiltration of intratumoral activated CD8+ T cells and inhibition of intratumoral immunosuppression were achieved by the injection of such a vaccine nodule in combination with anti-PD-1 antibody,resulting in superior protective and therapeutic antitumor effects.KKEF-TRP2 could self-assemble into hydrogel at a concentration of 20 mg/mL with the addition of several drops of salt such as sodium chloride or phosphate.TEM revealed that networks of entangled nano fibers were found in the supramolecular hydrogel.The KKEF-TRP2 vaccine can effectively activate DCs in vitro and stimulate the expression of CD86 and MHCI molecules,likely due to the fact that the amphiphilic supramolecular assembly changed the single molecular state of TRP2 epitope in culture medium or its water-insoluble nature and the resulting nanofibers improved the cellular uptake of epitopes by DCs.The enhanced maturation of DCs by KKEF-TRP2 vaccine may contribute to the initiation of vaccine-specific T-cell immunity.The supramolecular KKEF-TRP2 hydrogel vaccine could elicit powerful therapeutic antitumor T-cell responses and boost infiltration of activated CD8+ T cells into tumors,which is more efficacious than the vaccine formulation used in clinic.Meanwhile,the combination of PD-1 blockade further improved the therapeutic antitumor T-cell immunity by inhibiting the PD-1/PD-L1 binding or converting anergic T cells into functional effector CD8 T cells.Conclusion:The short peptide hydrogel RADA16 loading DCs and antigen formed vaccine nodules,which maintained exogenous DCs viability and recruited vast autologous DCs.Proliferation and infiltration of intratumoral activated CD8 T cells were achieved by the injection of such a vaccine nodule,resulting in superior protective and therapeutic antitumor effects.The KKEF-TRP2 could self-assemble into nanofibers hydrogel and can effectively activate DCs in vitro and stimulate the expression of surface markers,becase the amphiphilic supramolecular assembly changed the single molecular state of TRP2 and the resulting nanofibers improved the cellular uptake of epitopes by DCs.The supramolecular KKEF-TRP2 hydrogel vaccine could elicit a powerful therapeutic antitumor T-cell response and boost the infiltration of activated CD8+ T cells into tumors,which is more efficacious than the currently used clinical vaccine formulation.