Overcoming CAR-mediated CD19 downmodulation and leukemia relapse with T lymphocytes secreting anti-CD19 T-cell engagers

B Blanco(1,2,3) A Ramírez-Fernández(1,2) C Bueno(3,4,5) L Argemí-Muntadas(6) P Fuentes(7) O Aguilar-Sopeña(1,8) F Gutiérrez-Agüera(3,4) S R Zanetti(4) A Tapia-Galisteo(9) L Díez-Alonso(1,2) A Segura-Tudela(1,2) M Castellà(10) B Marzal(10) S Betriu(10) S L Harwood(6) M Compte(9) S Lykkemark(6) A Erce-Llamazares(1,2) L Rubio-Pérez(1,2,11) A Jiménez-Reinoso(1,2) C Domínguez-Alonso(1,2) M Neves(7) P Morales(2) E Paz-Artal(2,8) S Guedan(10) L Sanz(9) M L Toribio(7) P Roda-Navarro(1,8) M Juan(10,12,13,14) P Menéndez(3,4,5,14,15) L Álvarez-Vallina(1,2,3,6)

1:Instituto de Investigación Hospital 12 de Octubre (i+12); 2:Hospital Universitario 12 de Octubre; 3:Red Española de Terapias Avanzadas (TERAV), Instituto de Salud Carlos III; 4:Josep Carreras Leukemia Research Institute; 5:Centro de Investigación Biomédica en Red-Oncología (CIBERONC), Instituto de Salud Carlos III; 6:Aarhus University; 7:Centro de Biología Molecular Severo Ochoa CSIC-UAM; 8:School of Medicine, Universidad Complutense de Madrid; 9:Hospital Universitario Puerta de Hierro; 10:Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS); 11:Universidad Francisco de Vitoria; 12:Hospital Clínic de Barcelona; 13:Hospital Sant Joan de Déu; 14:Universitat de Barcelona,; 15:Institució Catalana de Recerca i Estudis Avançats (ICREA)

Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of CD19- positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission in patients with B cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. We have compared the antitumor efficacy of engineered T cells either expressing a second-generation anti-CD19 CAR (CAR-T19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19, that is coupled with lysosome-mediated degradation, 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 prevent the CD19 downmodulation observed in anti-CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 down-modulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies.