Shortened ex vivo manufacturing time of EGFRvIII-specific chimeric antigen receptor (CAR) T cells reduces immune exhaustion and enhances antiglioma therapeutic function.

BACKGROUND: Non-viral manufacturing of CAR T cells via the Sleeping Beauty transposon is cost effective and reduces the risk of insertional mutagenesis from viral transduction. However, the current gold standard methodology requires ex vivo numerical expansion of these cells on artificial antigen-presenting cells (AaPCs) for 4 weeks to generate CAR T cells of presumed sufficient quantity and function for clinical applications. METHOD: We engineered EGFRvIII-specific CAR T cells and monitored phenotypic changes throughout their ex vivo manufacturing. To reduce the culture time required to generate the CAR T-cell population, we selected for T cells in peripheral blood mononuclear cells prior to CAR modification (to eliminate the competing NK cell population). RESULTS: While we found increased expression of exhaustion markers (such as PD-1, PD-L1, TIM-3, and LAG-3) after 2 weeks in culture, whose levels continued to rise over time, we were able to generate a CAR+ T-cell population with comparable CAR expression and cell numbers in 2 weeks, thereby reducing manufacturing time by 50%, with lower expression of immune exhaustion markers. The CAR T cells manufactured at 2 weeks showed superior therapeutic efficacy in mice bearing established orthotopic EGFRvIII+ U87 gliomas. CONCLUSION: These findings demonstrate a novel, rapid method to generate CAR T cells by non-viral modification that results in CAR T cells superior in phenotype and function and further emphasizes that careful monitoring of CAR T-cell phenotype prior to infusion is critical for generating an optimal CAR T-cell product with full antitumor potential.

Localized Treatment with Oncolytic Adenovirus Delta-24-RGDOX Induces Systemic Immunity against Disseminated Subcutaneous and Intracranial Melanomas.

PURPOSE: Intratumoral injection of oncolytic adenovirus Delta-24-RGDOX induces efficacious antiglioma immunity in syngeneic glioma mouse models. We hypothesized that localized treatment with the virus is effective against disseminated melanomas. EXPERIMENTAL DESIGN: We tested the therapeutic effect of injecting Delta-24-RGDOX into primary subcutaneous (s.c.) B16-Red-FLuc tumors in s.c./s.c. and s.c./intracranial (i.c.) melanoma models in C57BL/6 mice. Tumor growth and in vivo luciferase-expressing ovalbumin-specific (OT-I/Luc) T cells were monitored with bioluminescence imaging. Cells were profiled for surface markers with flow cytometry. RESULTS: In both s.c./s.c. and s.c./i.c. models, 3 injections of Delta-24-RGDOX significantly inhibited the growth of both the virus-injected s.c. tumor and untreated distant s.c. and i.c. tumors, thereby prolonging survival. The surviving mice were protected from rechallenging with the same tumor cells. The virus treatment increased the presence of T cells and the frequency of effector T cells in the virus-injected tumor and mediated the same changes in T cells from peripheral blood, spleen, and brain hemispheres with untreated tumor. Moreover, Delta-24-RGDOX decreased the numbers of exhausted T cells and regulatory T cells in the virus-injected and untreated tumors. Consequently, the virus promoted the in situ expansion of tumor-specific T cells and their migration to tumors expressing the target antigen. CONCLUSIONS: Localized intratumoral injection of Delta-24-RGDOX induces an in situ antovaccination of the treated melanoma, the effect of which changes the immune landscape of the treated mice, resulting in systemic immunity against disseminated s.c. and i.c. tumors.