Abstract 1979: Dissecting the cellular and molecular players of T Cell Bispecific (TCB) antibody-mediated CRS
Authors: Leclercq Gabrielle , Steinhoff Nathalie , Alberti-Serverra LLucia , Yángüez Emilio , Nassiri Sina , Giusti Anna-Maria , Schneider Anneliese , Klein Christian , Haegel Hélène , Umaña Pablo , Sam Johannes , Bacac Marina 
Published: June 2022

Abstract
T-cell bispecific antibodies (TCBs) are a promising class of immunotherapies that activate T cells by engaging the CD3ε chain of the T-cell receptor and simultaneously binding to the tumor-associated antigens on target cells. They enable the formation of a cytotoxic synapse where the release of perforin and granzyme B induces target cell killing. Nevertheless, on-target activation of T-cells is associated with an intrinsic risk of cytokine release syndrome (CRS), one of the safety risks associated with the treatment with T cell engaging therapies. In this work, we focused on dissecting the cellular and molecular players of TCB-mediated cytokine release using single cell RNA sequencing of whole blood treated with CD20-TCB, targeting CD20-expressing B cells, as model system. In particular, we aimed at dissecting the kinetics of events along with identifying the main players that “trigger” with others that “amplify” the cytokine release. In accordance with previous reports, we show that activated T-cells initiate the cytokine cascade by releasing cytokines and chemokines stimulating monocytes and neutrophils that consequently amplify TCB-induced cytokine release. Additionally, we revealed inflammatory gene signatures and pathways evolving over treatment time in T cells, monocytes and neutrophils, highlighting potential novel biomarkers of CRS. Furthermore, using an in vitro co-culture of peripheral blood mononuclear cells (PBMCs) with tumor cells or a whole blood assay in the presence of CD20-TCB and neutralizing antibodies against the identified molecular targets, we confirmed their role in triggering or amplifying cytokine release. We further corroborated their role by depleting and/or blocking different cellular and molecular players in tumor-bearing humanized NSG mice treated with CD20-TCB. Last, we assessed the effects of dexamethasone, adalimumab (anti-TNF-a), tocilizumab (anti-IL-6R) and anakinra (anti-IL-1R) on cytokine release and anti-tumor efficacy of CD20-TCB in the same in vivo tumor model. Among the considered mitigation strategies, dexamethasone and adalimumab reduced the level of cytokines and chemokines, correlating with milder CRS signs upon TCB treatment. Importantly, none of the mitigations strategies interfered with anti-tumor efficacy, except adalimumab, which partially impacted the tumor growth inhibition and affected early T cell infiltration in the tumor.In summary, our findings reveal novel molecular and cellular biomarkers of TCB-induced cytokine release, and in addition provide insights on the potential mitigation strategies of the same. Our work sheds new light on the kinetics of mechanisms of inflammation driven by TCBs.
Citation Format: Gabrielle Leclercq, Nathalie Steinhoff, LLucia Alberti-Serverra, Emilio Yángüez, Sina Nassiri, Anna-Maria Giusti, Anneliese Schneider, Christian Klein, Hélène Haegel, Pablo Umaña, Johannes Sam, Marina Bacac. Dissecting the cellular and molecular players of T Cell Bispecific (TCB) antibody-mediated CRS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1979.