Screening, Optimization And Application Of Targeted Therapies For Human Non-small Cell Lung Cancer And AIDS (HIV-1 Infection)

In the current study,we have separately selected,developed,and optimized targeting therapeutics for non-small cell lung cancer(NSCLC)and HIV-1-infected patients.Lung cancer is the leading cause of cancer-related mortality in both China and the rest of the world.A majority(85?90%)of lung malignancies diagnosed are NSCLC.Aptamers are single-stranded oligonucleotides(DNA or RNA)folded into particular secondary or tertiary structures that bind to molecular targets with high specificity and affinity.Due to these properties,aptamers are widely applied in in vitro diagnosis,in vivo imaging,and drug delivery,thus playing important roles in biomedical research,biomarker discovery,drug development,disease diagnosis and treatments.Since the emergence of AIDS epidemic,more than 70 million people have been infected with HIV and about half of them have succumbed to the disease.Although combined antiretroviral therapy(cART)has achieved great success in reducing the viral load the virus remains in patient body.In the past decade,chimeric antigen receptors(CARs)have offered great potential toward a complete cure of AIDS through the elimination of HIV.We optimized CD4-based chimeric antigen receptor(CAR)that was previously used without toxicity in clinical trials.Methods to NSCLC targeting bio-product selection are as follows:1)We established an in vivo SELEX selection platform.NSCLC(NCI-H460)tumor bearing mice were used as a model system and PEGylated RNA pools were used as the library to select specific PEGylated RNA aptamers against NCI-H460;2)Cell binding assay,flow cytometry-based dissociation constant determination and IC50 detection were applied to determine specificity and affinity of selected RNA aptamer(RA16)in vitro;3)In vivo imaging,quantitative RT-PCR,and tumor growth assessment were applied to further examine targeting specificity and efficacy of RA16 in vivo;4)Same experiments were done on syn-RA 16 to assess its targeting and inhibitory effects;5)RA16-EPI adduct was then formed to investigate the potential of RA16 as drug delivery vector both in vitro and in vivo.Methods to anti-HIV-1 CD4? CAR optimization:CD4? CAR with or without costimulatory signaling domains were transduced into Pan T or naive T cells.CAR-T cells expression levels,T cell phenotypes and CAR-T cell specific cytotoxicity were evaluated in vitro.Results of NSCLC targeting bio-product selection:A 2'-F RNA aptamer(RA16)that specifically binds to non-small-cell lung cancer cells NCI-H460 was evolved from an in vivo SELEX in mice using a PEGylated RNA library,with an affinity(Kd)of 9 ± 2 nM.Interestingly,RA16 potently inhibited cancer cell proliferation in a dose-dependent manner with an IC50 of 116.7 nM.When tested in vivo in xenografted mice,RA16 showed gradual migration toward tumor and accumulation at tumor site over time.An in vivo anti-cancer study showed that the average inhibition rate for mouse tumors in the RA16-treated group was 54.26 ± 5.87%on day 16 versus the control group.Besides,we conducted sequential studies of the synthesized RNA aptamer(syn-RA16)and compared it with trans-RNA in terms of their specific targeting and direct inhibitory activity towards NCI-H460 cells.We demonstrated aptamer binding to NCI-H460 cells with Kd values of 24.75 ± 2.28 nM and 12.14 ± 1.46 nM for syn-RA16 and trans-RA16,respectively.Similar to trans-RA 16,syn-RA16 was capable of inhibiting NCI-H460 cell viability in a dose-dependent manner.IC50 values were 118.4 nM(n = 4)for syn-RA16 and 105.7 nM(n = 4)for trans-RA 16.Moreover,in vivo imaging demonstrated the gradual accumulation of both syn-RA16 and trans-RA 16 at the tumor site,and qRT-PCR showed the high retention of syn-RA16 in tumor tissues.In addition,a truncated fragment of RA16(S3)was identified and exhibited binding affinity for NCI-H460 cells with a Kd value of 63.20 ± 0.91 nM.Moreover,the aptamer RA16 adducted with epirubicin(RA16-epirubicin)showed significantly higher toxicity against targeted NCI-H460 cells and low toxicity against non-targeted tumor cells.Furthermore,RA16-epirubicin adduct exhibited in vivo anticancer efficacy,with an inhibition rate of 64.38 ± 7.92%when administrated in 1460 xenograft mouse model.Results of anti-HIV-1 CD4? CAR optimization:Introduction of costimulatory signaling domains have no negative impact on expression of CD4? CAR,CAR-T cell growth and anti-HIV-1-specific cytotoxicity in vitro.Moreover,1)Engineered CAR-Tscm cells were enriched in the Tscm culture condition;2)Costimulatory signaling domains have minimal impact on T cell phenotypes;3)Costimulatory signaling domains enhance anti-HIV-1 cytotoxicity in vitro;4)CD28 signaling domain impairs the stability of CD4? CAR-T cells in vitro.CD4? CAR with 41BB signaling domain was optimized for further development.Conclusions:A specific bi-functional RNA aptamer RA16 has been developed that specifically targets and inhibits growth of NCI-H460 cells both in vitro and in vivo.Syn-RA16 and its truncated aptamer can help the development of a diagnostic and/or treatment agent for NSCLC in clinical settings.Besides,anti-HIV-1 CD4? CAR with 41BB signaling domain possesses high stability and enhanced cytotoxicity,which may facilitate further development of CAR-T based therapies for anti-HIV-1.