Regulated Immune Checkpoint By Drug Delivery System For Combinatorial Cancer Therapy

Objectives:As an immune checkpoint expressed on tumor cells,programmed death-ligand 1(PD-L1)can bind to programmed cell death 1(PD-1)on T cells to cause T cell exhaustion and disability,which inhibits anti-tumor immune response.Blocking PD-L1 with monoclonal antibody has achieved revolutionary clinical outcomes.However,there are some challenges such as systemic immune side effect,limited response efficiency and high treatment costs for further application.Generally,the clinical benefit of immunotherapy is mainly restricted by tumor immunogenicity and immunosuppressive microenvironment.Recently,combination chemotherapy and immunotherapy has received extensive attention to synergistically regulate immune microenvironment.Herein,we reported two drug delivery systems co-loaded PD-L1blockade and chemotherapeutic agent to achieve effective synergistic anti-tumor therapy.Methods:1).A synthetic PEI-PLGA cationic polymer gene carrier was constructed.Gene drug(CRISPR/Cas9)and chemotherapy drug(paclitaxel,PTX)were co-loaded into the polymer by nanoprecipitation method to form core nanoparticles.Subsequently,PEG was modified to the surface of the core nanoparticles via a p H-sensitive linker to enhance the stability and long circulation characteristics of nanoparticles,which obtained a drug-loaded nanosystem with good biocompatibility(PCNP_PTX+Cdk5).Afterwards,the physicochemical properties,stability,biological safety,p H response characteristics,and in vitro gene transfection and PD-L1 blocking efficiency of nanosystem were evaluated.Moreover,the mouse B16F10 melanoma and CT26 colon cancer models was established to investigate the anti-tumor ability of PCNP_PTX+Cdk5 in vivo.Furthermore,the potential anti-tumor immune mechanisms mediated by PCNP_PTX+Cdk5were also analyzed via flow cytometry and immunofluorescence.2).PD-L1 small molecule inhibitor(BMS-202)and histone deacetylase inhibitor(chidamide,CHI)were chosen as the research objects to explore the synergistic anti-tumor effect of PD-L1 blocking therapy combined with epigenetic regulation.Firstly,the effect of CHI on apoptosis and tumor immunogenicity of 4T1 cells were investigated.Besides,the PD-1/PD-L1 blockade ability of BMS-202 was also evaluated.Subsequently,a pluronic hybrid liposome was constructed to co-deliver BMS-202 and CHI.Due to the poor hydrophilicity and hydrophobicity of chidamide,the drug loading capacity of chidamide in liposome was low through conventional liposome preparation methods.To solve this problem,CHI was firstly formed a complex(CHI-F127)with pluronic F127 to enhance the water solubility.Afterwards,BMS-202 and CHI-F127 were co-loaded into the different drug-loading areas of liposome via reverse phase evaporation to obtain drug-loaded liposome(CHI/BMS-202@lip F).Finally,mouse 4T1 orthotopic breast cancer model and lung metastasis model were established to evaluate the anti-tumor and anti-tumor metastasis ability of CHI/BMS-202@lip F in vivo.Moreover,the potential anti-tumor immune mechanism was also investigated.Results:1).We constructed a tumor microenvironment-responsive nanoparticle to encapsulate CRISPR/Cas9 and paclitaxel(PTX).The encapsulated CRISPR/Cas9could specifically knock out cyclin-dependent kinase 5(Cdk5)gene to significantly attenuate the expression of PD-L1 on tumor cells.PTX could enhance tumor immunogenicity and anti-tumor function of immune cells in tumor microenvironment.The proposed nanoparticle(PCNP_PTX+Cdk5)consisted of cationic drug-loaded core and p H-sensitive PEG shell.The PEG layer enhanced the stability of nanoparticle during blood circulation,while it could be rapidly deshielded under tumor weakly acidic microenvironment to expose the core with high positive charge for enhancing cellular uptake and penetration of nanoparticle at tumor site.The nanoparticles exhibited excellent encapsulation capacity for PTX(encapsulation efficiency was above 90%)and CRISPR/Cas9(minimum mass ratio was 1:1).PCNP_PTX+Cdk5 showed a diameter of about 100 nm and near neutral surface charge.Meanwhile,PCNP_PTX+Cdk5 also exhibited well stability and biocompatibility,p H-dependent surface charge transition and drug release behavior.In vitro results showed that the gene transfection efficiency of nanocarriers was above 60%on B16F10 and CT26 cells.Moreover,the expression of Cdk5 and PD-L1 were significantly down-regulated on tumor cells after treated with nanoparticles,which could enhance the killing sensitivity of cytotoxic T cells(CTLs)to tumor cells.In vivo animal studies indicated that nanoparticle was capable of effectively accumulating into tumor and significantly improved the pharmacokinetic properties of PTX.In the animal models of B16F10 melanoma with high expression of PD-L1 and CT26 colon cancer with low expression of PD-L1,PCNP_PTX+Cdk5 exhibited effective tumor growth inhibition.The analysis of immunological mechanism indicated that PCNP_PTX+Cdk5 could effectively convert‘cold'tumor into‘hot'tumor by activating antitumor immune cells(DCs and CTLs)and relieving immunosuppressive factors(PD-L1,Tregs,and TAMs),which significantly enhanced the anti-tumor immune effect.2).Inspired by the close relationship between epigenetic alteration and tumor immunogenicity,we speculated that the combination of epigenetic regulation and PD-L1 blockade therapy might achieve synergistic anti-tumor efficiency.Thus,we constructed a poloxamer hybrid liposome(CHI/BMS-202@lip F)that encapsulated BMS-202(a PD-L1 small molecule inhibitor)and CHI(a histone deacetylase inhibitor(HDACi)).In vitro studies have shown that BMS-202 could block the inhibition effect of recombinant PD-L1 protein on T cells function,theraby restoring the ability of proliferation and IFN-?secretion for T cells.Meanwhile,CHI could promote intracellular ROS levels in cancer cells,induce mitochondrial function disorder,and then activate the mitochondrial apoptosis pathway to induce 4T1 cell apoptosis.Further studies found that CHI could promote the exposure of calreticulin(CRT)on the surface of 4T1 cells,high mobility group protein-1(HMGB-1)release and ATP secretion.Additionally,the mice that subcutaneously inoculated with CHI-treated 4T1 apoptotic cells were able to elicit a"vaccine-like"protective function to prevent the tumors onset and progress of mice after rechanllenged with living 4T1 cell.Collectively,CHI was verified to induce immunogenic cell death with tumor cells,which was beneficial for the subsequently anti-tumor immune responses.To achieve the co-delivery of CHI and BMS-202,a poloxamer hybrid liposome was described.The CHI-F127 complex with Pluronic F127 was firstly prepared for increasing water solubility of CHI,and then encapsulated with BMS-202 into liposome with different drug-loaded spaces via reverse evaporation method(CHI/BMS-202@lip F).The liposome could effectively encapsulate CHI and BMS-202 through different drug-loading areas with encapsulation efficiency of 85%and 95%,respectively.Meanwhile,CHI/BMS-202@lip F possessed uniform size distribution and well stability.In animal studies in vivo,the liposome could effectively accumulate into tumor sites and stay for long time.In mouse 4T1 orthotopic breast cancer model,CHI/BMS-202@lip F could effectively inhibit primary tumor growth and lung tumor metastasis,thereby extended the survival time of tumor-bearing mice.Moreover,in mouse 4T1 lung metastasis model,CHI/BMS-202@lip F obviously inhibited the invasion and deposition of circulating 4T1 tumor cells in lung,which significantly reduced the number of lung tumor nodules.The nodule inhibition rate of CHI/BMS-202@lip F was as high as 96%.The analysis of immunological mechanism showed that CHI/BMS-202@lip F was capable of promoting DCs maturation,the infiltration and activation of NK cells,and T cells proliferation and infiltration within tumor,indicating that CHI/BMS-202@lip F could effectively regulate tumor immune microenvironment and enhance anti-tumor immune effect.Conclusions:In summary,we constructed two kinds of drug delivery systems to regulate the expression or function of PD-L1 on tumor,which could significantly improve the tumor immunosuppressive microenvironment and promote the conversion of immune‘cold'tumor to‘hot'tumor after combined with immunomodulatory chemotherapeutic drugs.Thereby,these combinatorial strategies were capable of effective inhibiting tumor growth and metastasis.Based on the study in this article,we expect to provide new strategies and sights for clinical cancer therapy.