P1361: sleeping beauty platform for engineering car-cik cells toward a multi-targeting strategy in b-all

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Adoptive transfer of Chimeric Antigen Receptor (CAR) T cells have revolutionized B-cell acute lymphoblastic leukemia (B-ALL) treatment but after initial impressive results, some criticalities emerged such as the occurrence of CD19 negative relapses. To overcome viral method’s limitations in T cell engineering, our group developed an anti-CD19 CAR engineered Cytokine Induced Killer (CIK) cells through a Sleeping –Beauty (SB) trasposon-based platform (CARCIK-CD19). A phase I/II with CARCIK-CD19 was performed in our centre showing encouraging results. In order to preventing and address post-CAR-T leukemia relapses, we leveraged SB technology to enable multi-targeting approach. Here, we present data about targeting the B-cell specific CD22 in combination with BAFF-R, which we have previously developed and showed to be active towards B-ALL. Aims: The aim of our project was to develop anti-CD22 and anti-BAFFR CAR to be used in patients resistant to anti-CD19 CAR T cells, in a multi-targeting strategy Methods: We generated CARCIK cells starting from healthy donor PBMCs with electroporation of SB plasmids encoding anti-CD22 and anti-BAFFR CARs accordingly to the clinical protocol previous describe. CAR expression, T-cell immunophenotype and exhaustion profile of CARCIK cells were analyzed by flow cytometry. In vitro killing activity was evaluated with single and double targeting CAR T cells. To assess safety, we evaluated the vector copy number (VCN) at the end of differentiations through qPCR. In vivo efficacy was evaluated both in acute leukemia and lymphoma mice models. Results: A second-generation CAR directed against CD22 was generated in the optimized SB pT4 plasmid, together with 2 anti-BAFFR CAR constructs, a third generation in the SB pT plasmid, and a second generation in a pT4 plasmid. Anti-CD22 and anti-BAFFR CAR T cell products had a similar kinetic of expansion after electroporation and activation in vitro to the benchmark CARCIK-CD19 (panel A). Mean CAR expression (panel B) was 50,4±5,6% for CARCIK-CD22, 35,6±8,1% for CARCIK-BAFFR3rd generation and 22,3±10,3% for CARCIK-BAFFR2rd generation. Regarding the CAR+ cells immunophenotype, we observed the typical enrichment in CD3+/CD56+ cells, and as expected, CD8+ population was prevalent (panel C). Memory profile, at the end of differentiation, showed an enrichment in central memory and effector memory subsets both in CD4 and CD8 CAR+ subpopulations (Panel D-E). Robust killing activity was observed for both anti-CD22 and anti-BAFFR CAR against the leukemic NALM6 cell line (panel F). To evaluate quality and safety of the CARCIK products, we evaluated the transposon VCN, that resulted below 5 copies/cells, which is considered the safety release threshold for the clinical-grade products (panel G). Then, we tested CARCIK-CD22 in vivo using DAUDI leukemia mouse models. Notably, CARCIK-CD22 eradicate disease, which it was not longer detectable in peripheral blood and organs at the sacrifice, and this was associated with persistence, measurable thorough cytometry in peripheral blood at day 60 (panel H). Summary/Conclusion: In this study we demonstrated the efficacy, quality and safety of donor-derived SB-transfected CARCIK-CD22 with a remarkable anti-tumor activity in vitro and in in vivo mice models. Further experiments are ongoing for evaluating the activity of BAFFR CAR T cells in vivo. Overall, we demonstrated the feasibility of CARCIK-CD22 and CARCIK-BAFFR non-viral engineering to be employed in the context of multi-targeting approach to prevent and treat CD19 negative B-ALL relapses.Keywords: Acute leukemia, ALL, Cellular therapy, CAR-T