PH/redox Dual Sensitive-minicelle Co-delivery Trp2/CpG Vaccine And IL-15 For Cancer Combined Immunotherapy Based On "Cascaded Cytosol Delivery" Strategy

Cancer immunotherapy is a novel clinical strategy for cancer treatment,and exploring new strategies for cancer immunotherapy have become one of the hotspots in recent years.The therapeutic target cells in cancer immunotherapy mainly include T cell,nature killer(NK)cell,macrophage,B cell and so on.Among these target cells,T cell based cancer immunotherapy,such as cancer vaccine,immune checkpoint inhibitors,chimeric antigen receptor T-Cell(CAR-T),have made great process.Among these T cell based cancer immunotherapy strategies,cancer vaccine has been entered into clinical or clinical trials due to its high specificity,low side effects and potential radical treatment.Cancer vaccine can activate tumor-associated antigens,restore and enhance anti-cancer immune response,resulting in destroying tumor cells.Cytotoxic T lymphocyte(CTL)is the terminal effect cell of cancer vaccine,and it can specifically recognize tumor antigens presented by major histocompatibility complex I(MHC I)on the surface of cancer cells,and directly kill cancer cells by secreting granzyme and perforin.At present,although cancer vaccine,such as Provenge(Sipuleucel-T),has achieved good therapeutic effect,the response rate and therapeutic effect of the cancer vaccine still can’t meet the expectations.One of the main reasons that limited the therapeutic effect of cancer vaccines is the insufficiency of CTL-mediated anticancer immune response:after be uptake by professional antigen presenting cells(APC),cancer vaccines are mainly presented to CD4+T cells by major histocompatibility complex(MHC Ⅱ),while only a few CD8+T cells are further differentiated into CTL by MHC I molecules.Therefore,how to increase the binding of antigens with MHC I,promote the antigen presentation to CD8+T cells and CD8+T cells differentiation into CTLs,and eventually increase the immunotherapeutic effect of cancer vaccine,remains a great challenge.Another main reason that limited the therapeutic effect of cancer vaccines is that cancer cells have evolved a variety of immune escape mechanisms to escape immune surveillance and killing of T cells,such as MHC I mutation or deletion.NK cells are anther main immune effector cells to kill cancer cells besides T cells,and NK cells also play an important role in cancer immunotherapy.NK cells have "natural" killing effect on cancer cells through cytotoxicity effect(secretion of perforin and granzyme B,induction of Fas/FasL)and secretion of IFN-γ(for immunoregulation).The effect of NK cells do not require the existence of MHC I and NK cells can effectively kill cancer cells without MHC I molecular,thus effectively solve the problem of immune escape caused by MHC I deficiency in T cell immunotherapy.Hence,the mechanism of T cells and NK cells is complementary.If T cells and NK cells can be activated at the same time,cancer immunotherapy effect could be further enhanced.Based on this,the project aims to meet the respective requirements of cancer vaccine,NK cell activation factor and combination cancer vaccine with NK cell activation factor by using novel formulation technologies,overcome the existing problem of cancer vaccine,and thus improve the efficacy of combined cancer immunotherapy.A novel cancer vaccine was firstly designed and constructs to improve the antigen MHC I presentation rate and efficiency of antigen cross presentation,and thus enhance the therapeutic effect of cancer vaccine.After cancer vaccines are uptake by antigen presenting cell(APC),if some measures could be taken to deliver antigens to cytoplasm,the antigens could bind with major histocompatibility complex I(MHC I)molecular and be presented to CD8+T cells.Then the CD8+ T cell could be differentiated into cytotoxic T-lymphocyte(CTL),and this process is called antigen cross-presentation.Therefore,increasing the cytosol delivery of antigens in APC is a widely accepted strategy to enhance antigen cross-presentation.In this study,"cascade cytosol delivery" strategy was proposed for the first time.Based on the design strategy,multiple cascade and pH/redox micelles to accelerate lysosomal escape were developed to enhance antigen cross-presentation and increase the anti-cancer immune response mediated by CD8+ T cells,therefore enhancing effect of cancer immunotherapy.In this study,Ovalbumin was selected as model antigen,and Ova loaded micellar cancer vaccine(Ovalbumin-loaded pH/redox dual-sensitive micellar vaccine,OLM-D)was prepared.The cascade cytosol delivery process was proved by in vitro studies,and the results of antigen presentation test,BMDC maturation test and in vivo immunity test were performed to prove that OLM-D could improve the binding rate of MHC I molecule,antigen cross-presentation and enhance anti-tumor immune response.Tyrosinase related protein-2(Trp2)is a melanoma related epitope peptide,which can be presented in human and mouse melanoma cells,and is an ideal antigen for immunotherapy of melanoma.In this project,Trp2 was selected as tumor antigen to prepare Trp2 loaded pH/redox double-sensitive micelle vaccine for proving enhanced cancer vaccine therapeutic efect of "cascade cytosol delivery" strategy.The combination of immune adjuvant and tumor antigen is one of the effective ways to improve the immune response of cancer vaccine.Immune adjuvant is a kind of non-specific substance that can enhance the immune response to antigen or change the type of immune response.CpG is an agonist of TLR9 receptor and an oligonucleotide containing non-methylated CpG motif.CpG activates TLR9 receptors in DC and macrophages,which produces pro-inflammatory cytokines associated with Th1 cells,promotes macrophage polarization,and promotes CD8+T cells to differentiate into CTL.CpG is a new type of immune adjuvant or immune therapeutic agent.In this project,CpG was selected as immune adjuvant to prepare Trp2/CpG co-loaded pH/redox double-sensitive micelle vaccine(TCCM).Since,the particle size of micellar vaccine has effect on lymph node aggregation and APC uptake rate.Hence,Trp2/CpG co-loaded vaccines at about 70 nm and 150 nm were prepared.The size of the vaccine was screened by lymph node distribution and cell uptake experiments,and the ability of the vaccine to co-deliver Trp2 and CpG was evaluated.In order to further enhance the therapeutic effect of cancer vaccine and block the immune escape of cancer cells based on the MHC I deficiency,this project explored the feasibility of combining cancer vaccine with NK cell activator for combined immunotherapy.IL-15 is a decisive factor for the development of NK cells in vitro and in vivo hematopoietic precursor cells,which can promote NK cell proliferation,regulate the cytotoxic activity of NK,and induce cytokines production in NK cells.Hence,IL-15 was selected as NK activation factor and IL-15 loaded pH/redox dual sensitive micelle(ILM)were both prepared to enhance in vivo delivery efficiency of IL-15.TCCM and ILM had similar particle sizes,and can synchronously distribute to lymph nodes.TCCM plays the role of cancer vaccine,and ILM activates NK cells,and thus enhance the cancer immune response.Hence,both adaptive and innate immune responses can be activated,resulting in enhanced anti-cancer immune response.In vivo and in vitro experiments verified that TCCM and ILM could respectively promote DC maturation and activate NK cell killing.In vivo tumor inhibition and immune experiments proved that the combination TCCM with ILM can simultaneously enhance the anti-cancer immune response mediated by CTL and NK cells,and exert the synergistic anti-tumor effect,which is a feasible combination of cancer immunotherapy.The main contents and results of this study are listed as follows:Chapter Ⅰ:Study on using pH/redox dual sensitive micelle based on"cascade cytosol delivery" strategy as novel cancer vaccine carrier to improve cancer immunotherapy effect1.Preparation and characterization of OVA loaded multiple cascade micelles:to develop cancer vaccine capable of achieving cascade cytosol delivery,multifunctional materials PLH-PEG-NH2 and PLH-PEG-SPDP were designed and synthesized.The structures of two materials were verified by 1H-NMR.PLH-PEG-SPDP had low critical micelle concentration,which can self-assemble into polymeric micelle.The pKb value of PLH was pH 6.2,which has the effect of disassembly and proton pumping in acid lysosome.Then,OVA loaded micelles through disulfide bond(OLM-D,124.0±4.780 nm,-9.98±1.50 mV)and amido bond(OLM-A,123.5±1.422 nm,-6.80±0.38mV)with similar particle size were both prepared.2.Evaluation of cascade cytosol delivery ability of OLM-D:Using OLM-A as control,the antigen cascade cytosol delivery process was verifiedr:①and the redox sensitive release behavior of OLM-D was characterized by SDS-PAGE and particle size distribution;②The disassembly of OLM-D in lysosome environment(pH 6.0 PBS+10 mM GSH)was confirmed by acid-base titration,particle size distribution,Zeta potential and TEM images;③the disassembly of OLM-D can further accelerate lysosomal escape.The results of in vitro cross-presentation of BMDC antigen showed that the cross-expression of OLM-D antigen was increased compared with that of OLM-A(p<0.05).And the ability of OLM-D to promote maturation of BMDC was stronger than that of OLM-A(p<0.05).The ex vivo NIFR imaging results showed that the accumulation capacity of DiR loaded OLM-D was significantly increased in lymph nodes compared with free DiR(p<0.05),indicating that OLM-D had better lymph node retention ability.The in vivo immune effect results showed that OLM-D could promote the secretion of cytokines IL-2 and IFN-y(p<0.05).Moreover,the proportion of CD3+CD8+T cells and OVA specific CD3+CD8+T cells in spleen of OLM-D sc,OLM-D ip and OLM-D LPS groups was significantly higher than that of free OVA(p<0.05).These results showed that OLM-D had better ability to enhance cancer immunotherapy effect.The safety of OLM-D was preliminarily verified by body weight changes of mice and H&E staining.These results indicate that OLM-D vaccine with cascade cytosol delivery capability can accelerate lysosome escape,enhance the ability of antigen cross-presentation,and "cascade cytosol delivery"strategy can improve the immunotherapeutic effect of cancer vaccine.Chapter Ⅱ:Studies on using Trp2/CpG co-loaded multiple cascade pH/redox dual sensitive micelle as novel cancer vaccine1.Preparation and particle size screening of OVA/CpG co-loaded multiple cascade micelles:Then the pH sensitive micelles with two sizes of Trp2/CpG were prepared.The size of large Trp2/CpG co-loaded pH/redox dual sensitive micelle(L-TCCM)was 152.5±13.37 nm,and the zeta potential of L-TCCM was-2.87±1.12 mV.The size of small Trp2/CpG co-loaded pH/redox dual sensitive micelle(S-TCCM)was 68.02±2.32 nm and the zeta potential was-3.65±0.80 mV.The results of cellular uptake test showed that S-TCCM had higher cellular uptake rate in DC2.4 cells within 2 h than L-TCCM.Meanwhile,the fluorescence intensity of DiR loaded S-TCCM was higher than that of DiR loaded L-TCCM after 24 h,which confirmed that S-TCCM had better lymph node accumulation ability.Therefore,S-TCCM was selected for subsequent experiments.2.Study on immunotherapeutic effect of Trp2/CpG co-loaded multi-cascade micellar vaccine:the results of in vitro and in vivo co-delivery test proved that the co-delivery ability of Trp2 and CpG in S-TCCM was higher than the mix groups(the mixture of Trp2 loaded micelle and CpG loaded micelle).The rapid lysosome escape of S-TCCM was observed by CLSM.The results showed that C6 loaded S-TCCM had faster lysosome escape rate than free C6,which indicated that S-TCCM could accelerate antigen cytosol delivery.Then the ability of S-TCCM to activate dendritic cells was verified by in vitro BMDC maturation test.Compared with free Trp2,free CpG and STLM,S-TCCM can enhance the mature ability of BMDC(p<0.05).The results of the Polarization of M2-type TAMs test showed that S-TCMM has better abaility to enhance polarization of tumor associated macrophage(TAM).These results indicate that S-TCCM can co-deliver Trp2 and CpG,and CpG as adjuvant can enhance the ability of S-TLM to stimulate maturation of BMDC and polarization of macrophage,which proves that S-TCCM with multiple cascade ability was a type of ideal new cancer vaccine.Chapter Ⅲ:Using multiple cascade pH/redox dual sensitive micelles for co-delivery of Trp2/CpG vaccine and IL-15 for cancer combined immunotherapyIL-15 loaded pH/redox dual sensitive micelles(ILM)were prepared,the particle size and zeta potential of ILM was 69.67±7.08 nm and 1.40±0.35 mV,respectively.The positive rate of CD3-CD49+NK cell extracted was 96.1%,which could be used directly in the follow-up experiment.The NK cells activated by ILM had equal cytotoxicity compared with IL-2 and IL-15 at different effect-target ratios.Finally,the in vivo antitumor test was investigated on the B16-F10 melanoma bearing mice.The results showed that compared with Trp2、Trp2+CpG、BM、S-TLM,S-TCCM had better antitumor effect(p<0.05).Compared with S-TCCM and IL-15+S-TCCM,ILM+S-TCCM had enhanced antitumor effect(p<0.01).In addition,the types of CD3+CD4+T,CD3+CD8+T,IFN-γ+CD3+CD8+T,CD3-CD49+NK cells in tumor tissue and secretion of IFN-y in serum were quantitatively studied.The results showed that compared with Trp2 and S-TCCM,the proportion of CD3+CD4+T,CD3+CD8+T,IFN-γ+CD3+CD8+T and IFN-γ secretion were enhanced(p<0.05).The proportion of CD3+CD4+T、CD3+CD8+T、IFN-γ+CD3+CD8+T cell in IL-15+S-TCCM,ILM+S-TCCM and S-TCCM groups had no significant difference(p<0.05).And the CD3-CD49+NK cell proportion and IFN-γ secretion of IL-15+S-TCCM,ILM+S-TCCM increased compared with S-TCCM(p<0.05).These results indicated that S-TCCM can realize the synergistic therapy of Trp2 and CpG.ILM+S-TCCM can realize the synergistic therapy of S-TCCM and IL-15.The results of body weight change in mice and the results of H&E staining of main organs(heart,liver,spleen,lung and kidney)showed that ILM+S-TCCM was safe for in vivo test.These results preliminarily showed that co-delivery of Trp2/CpG vaccine and IL-15 by multiple cascade pH/redox double-sensitive micelles(IL-15+S-TCCM)was a promising new method for combined cancer immunotherapy.