1:GENyO- Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia; 2:LentiStem Biotech; 3:Universidad de Granada; 4:Hospital Clínic

The efficacy of chimeric antigen receptor (CAR)-redirected T cells for treating solid tumors remains challenging. Main hurdles include heterogeneous antigen expression, inability of CAR-T cells to infiltrate into the tumor, inhibitory effect of tumor microenvironment and poor persistence of CAR-T cells . Arming CAR-T cells with immunostimulatory molecules (4th generation CARs/TRUCKs) could be determinant to achieve therapeutic efficacy against solid tumors. However, continuous expression of these potent molecules may cause unacceptable side effects on patients. In this work, we used a doxycycline (dox)-inducible, transactivator-free lentiviral vector (LV) (Lent-On-Plus) to generate clinically-relevant inducible TRUCKs. Previous publications showed that IL-18 releasing CAR-T cells present enhanced antitumor effect in complex tumor models, since IL-18 is a master regulator of the immune environment. Here, we describe first-in-class anti-CD19-CAR-T cells engineered to release IL-18 only in the presence of dox (αCD19-iTRUCK-IL-18). These iTRUCKs tightly controlled IL-18 expression upon dox addition and exhibited enhanced antitumor potency against a metastatic pancreatic ductal adenocarcinoma model expressing different levels of CD19. Interestingly, the presence of the inductor increases clinically-desirable TSCM/TCM phenotype and reduces exhaustion markers after multiple tumor re-challenges. Additionally, αCD19-iTRUCK-IL-18 were able to polarize M2 (pro-tumor) to M1 (anti-tumor) macrophages in a dox-dependent manner. Finally, the oral administration of dox to orthotopically implanted pancreatic tumor model mice and inoculated with αCD19-iTRUCK-IL-18 induced stronger antitumor responses compared with those without dox. These results showed that Lent-On-Plus LVs can generate iTRUCKs controlling functional IL-18 release, constituting a promising and safer strategy to externally control the activity and potency of TRUCKs.