GÉTICA 2021

51 [ F I T C á n c e r - 7 ] and cell cytotoxicity against CD19+ targets were analyzed by luciferase assays, flow cy- tometry and impedance-based real-time cytotoxicity assays. IFN- γ secretion secretion was studied by ELISA. Synapse studies were performed using immunofluorescence and confocal microscopy. In vivo B-ALL xenograft models: nine-week-old NOD.C γ -Prkdcscid-IL- 2rgtm1Wjl/SzJ (NSG) mice were infused intra- venously with the B-ALL cell line NALM6 or pri- mary human B-ALL samples and 2 days after received CAR-T19 or STAb-T19 cells. Results: In this study we demonstrated that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoi- ding leukemia escape in vitro , and preventing relapse in vivo. We observed that leukemia escape is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degrada- tion, leading to the emergence of transiently CD19-negative- leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immuno- logical synapses and prevent the CD19 down- modulation observed in anti-CD19 CAR-me- diated interactions. While both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient derived xenograft-derived mouse model, where STAb-T19 cells efficient- ly eradicated leukemia cells, whereas leuke- mia relapsed after CAR-T19 therapy (Figs. 2-5). Conclusions: Our findings suggest that the absence of CD19 down-modulation in the STAb-T19 strategy, coupled with the conti- nued antibody secretion, allows an efficient recruitment of the endogenous T cell pool, re- sulting in fast and effective elimination of can- cer cells that may prevent relapses, frequently associated with CAR-T19 therapies. Funding: we thank CRIS Cancer Foundation, CERCA/Generalitat de Catalunya and Funda- ció Josep Carreras-Obra Social la Caixa for core support. Financial support for this work was obtained from the European Research Council (CoG-2014-646903, PoC-2018-811220 to PM); the Spanish Ministry of Science and Inno- vation (PID2019-105623RB-I00 to MLT, SAF2016- 75656-P and RTC-2017-5944-1 to PR-N, SAF- 2019-108160-R to PM, and SAF2017-89437-P to LA-V), partially supported by the European Regional Development Fund (ERDF); the Car- los III Health Institute (ISCIII, PI20/01030 to BB; PI20/00822 to CB; PI16/00357 and PI19/00132 to LS; PICI14/122, PI13/676, PIE13/33, and PI18/775 to MJ; DTS20/00089 to LA-V), partia- lly supported by the ERDF; the Obra Social La Caixa. (LCF/PR/HR19/52160011 to PM), CatSa- lut, Fundació La Caixa (CP042702 to MJ); the Spanish Association Against Cancer (AECC CICPF18030TORI to MLT, AECC 19084 to LA-V); Fundación Uno Entre Cien Mil, and Fundación Ramón Areces to MLT; and the CRIS Cancer Foundation (FCRIS-IFI-2018 to LA-V). SG has received funding from the Spanish Ministry of Science and Innovation under a Ramon y Cajal grant (RYC2018-024442-I). AT-G was supported by predoctoral fellowship from Comunidad Autónoma de Madrid (PEJD-2018- PRE/BMD- 8314). LD-A was supported by a Rio Hortega fe- llowship from the ISCIII (CM20/00004). LR-P was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck. CD-A was supported by a pre- doctoral fellowship from the Spanish Ministry of Science and Innovation (PRE2018-083445). MN was supported by a grant from Portuguese Foundation for Science and Technology (SFRH/ BD/136574/2018).