Towards CAR T cell therapy optimization by in vivo electroporation with vectors co-expressing IL15/IL15RA

Resumen

Cancer immunotherapy is the use of strategies to activate and stimulate the immune system to fight against tumors. CAR T cells are chimeric antigen receptors, which are expressed in T cells after genetic modification of lymphocytes. These artificial receptors are inserted by genetic modification and they recognize antigens bound to the cell membrane of tumor cells. Nowadays, CAR T cell therapy has been approved in hematologic tumors such as B-cell lymphoma or Acute Lymphoblastic Leukemia, with high success percentages. Among others, there are some difficulties that do not allow the implementation of CAR T cell therapy to a great number of patients. Por example, the poor persistence of CARs in the patients, the safety concerns of using lentiviral vectors, cytokine release syndrome and neurotoxicity caused by the treatment. Thirdly, the cost and complexity of the process of manufacturing explained before. Therefore, we hypothesized that CAR T cells could be generated in vivo by using electroporation techniques with plasmids exploiting the Sleeping Beauty technology and IL15-IL15R fusion protein may provide an advantage to favor the in vivo persistence and antitumor efficacy of CART cells. The objectives of this project are: 1. To demonstrate that the expression of IL15/IL15Rα in CAR T cells can improve their persistence in vivo. 2. To advance in the in vivo generation of CAR T cells by in situ electroporation with plasmids as an alternative to ex vivo conventional CAR-T cell manufacture. The results showed that the expression of murine IL15-IL15Rα fusion protein provides to CD19 CAR T cells a more active and cytolytic potential in vitro, as demonstrated in the results regarding p-STAT5 activation and expression of Granzyme b and KI67. RNA seq analysis demonstrated that genes related to lymphocyte activation, glycolyisis, STAT-5 signaling, regulation of DNA replication, antiapoptic processes and cytokine production were differentially expressed in CD19-IL15 CAR T cells. The murine IL15-IL15Rα construct augments the anti-tumor efficacy of anti-CD19 CAR T cells in vivo. As opposed to conventional CD19-CAR T cells, CD19-IL15 CAR T cells can elicit their in vivo effect even at low lymphodepletion regimens (1 Gy). GPC3-IL15 CAR T cells were not able to recognize their antigen and did not show any antitumor effect in a model of hepatocellular carcinoma. This lack of efficacy might be associated to a lower packaging efficacy of the vector. CD19-IL15 CAR T cells cause a certain grade of systemic toxicity in the long term, with total body weight loss and elevation of serum transaminases. This toxicity could be related to the immunosuppression caused by the long-term B cell aplasia associated to the CAR T cell efficacy. The sleeping beauty transposon system could be a safe method to genetically modify T cells to express CARs in vivo. Expression of LacZ gene was found in murine splenocytes after in vivo electroporation with optimized conditions of 8 pulses of 0.1ms and 500 V/cm. In vivo electroporation with plasmids expressing CD19 CAR and IL15-IL15R achieved a transient depletion of CD19+ cells, suggesting that it is possible to generate CAR T cells in situ by this technique. An improvement in this technique might provide an alternative to conventional CAR T cell manufacturing.