Synapse topology and downmodulation events determine the functional outcome of anti-CD19 T cell-redirecting strategies

A Ramírez-Fernández(1,2) O Aguilar-Sopeña(3,4) L Díez-Alonso(1,2) A Segura-Tudela(1,2) J Arroyo-Rodenas(1,2) C Domínguez-Alonso(1,2) P Roda-Navarro(3,4) L Álvarez-Vallina(1,2) B Blanco(1,2)

1:Cancer Immunotherapy Unit (UNICA), Department of Immunology, Hospital Universitario 12 de Octubre, Madrid; 2:Immuno-Oncology and Immunotherapy Group, Instituto de Investigación Sanitaria 12 de Octubre (imas12), Madrid; 3:Department of Immunology, Ophthalmology and ENT, School of Medicine, Universidad Complutense, Madrid; 4:Lymphocyte Immunobiology Group, Instituto de Investigación Sanitaria 12 de Octubre (imas12), Madrid

Immunotherapeutic approaches focused on the redirection of T cell activity toward tumor cells are actively being investigated. Strategies based on the endogenous secretion of T cell redirecting bispecific antibodies by engineered T lymphocytes (STAb-T) are emerging as alternative or complementary approaches to those based on chimeric antigen receptors (CAR-T). The anti-tumor efficacy of bispecific anti-CD19 x anti-CD3 (CD19xCD3) T cell engager (BiTE)-secreting STAb-T cells has been demonstrated in several mouse models of B cell acute leukemia.

Here, we have investigated the spatial topology and downstream signaling of the artificial immunological synapses (IS) that are formed by CAR-T or STAb-T cells. Upon interaction with CD19-positive target cells, STAb-T cells form IS with structure and signal transduction which more closely resemble those of physiological cognate IS, compared to IS formed by CAR-T cells expressing a second-generation CAR bearing the same CD19-single-chain variable fragment. Importantly, while CD3 is maintained at detectable levels on the surface of STAb-T cells, indicating sustained activation mediated by the secreted BiTE, the anti-CD19 CAR was rapidly downmodulated, which correlated with a more transient downstream signaling. Furthermore, CAR-T cells, but not STAb-T cells, provoked an acute loss of CD19 in target cells. Such differences might represent advantages of the STAb-T strategy over the CAR-T approach and should be carefully considered in order to develop more effective and safer treatments for hematological malignancies.