Abstract 4117: Molecular insights into resistance mechanisms to therapy with T cell bispecific antibodies
Authors: Sam Johannes , Servera Llucia Alberti , Nassiri Sina , Sethi Atul , Ribeiro Alison , Leclercq-Cohen Gabrielle , Schwalie Petra , Hüsser Tamara , Yangueez Emilio , Herter Sylvia , Klein Christian , Umana Pablo , Bacac Marina 
Published: April 2023

T Cell Bispecific Antibodies (TCBs) are potent molecules that induce durable complete remissions with a favorable safety profile in Relapsed Refractory Non Hodgkin Lymphoma patients. Despite the promising clinical efficacy, a proportion of patients escape under treatment due to the occurrence of primary and acquired resistance mechanisms that may involve the modulation of target antigen expression, cancer cell intrinsic mechanisms, T cell functionality or treatment-induced changes in the tumor microenvironment (TME). Therefore, a better understanding of TCB-induced resistance mechanisms is of great importance to enable the design of novel therapeutic approaches and/or combination strategies to overcome resistance. We present the results of exploratory preclinical analysis using an aggressive lymphoma model in humanized mice and a syngeneic melanoma model in immunocompetent mice, focusing on the assessment of T cell functionality, TME reprogramming, and cancer cell intrinsic changes upon longitudinal TCB treatments, encompassing tumor regression and escape. Flow cytometry, IHC, scRNAseq and ex vivo functional analysis of T cell cytotoxicity and cytokine release demonstrate that intra-tumor T cell functionality is preserved over serial TCB treatments, including tumor escape. On the other hand, TCB treatment induced a reprogramming of the TME consisting of rapid myeloid cell infiltration, followed by the expansion of intra-tumor T cells at a later time point (favoring higher CD8:CD4 ratio). Cumulative evidence from in vitro and in vivo scRNAseq experiments provided insights into macrophage reaction to TCB-mediated T cell inflammation, consisting of a rapid (as early as 2 h) activation and upregulation of several pro-inflammatory molecules (IL-1b, CXCL8, CXCL9, CXCL10, CXCL1, CCL2, CCL3, CCL4, CCL5, IFN-g-induced genes: GBP2 -8 and BATF2). This was followed by a shift towards a more suppressive phenotype at later time points (20-96 h) as evidenced by the expression of PD-L1, INHBA, IDO, FLT1 genes. The analysis of cancer cells showed upregulation of Wnt/beta catenin, p53 and Hedgehog signaling pathways at tumor escape. In contrast, proliferation signatures including MYC, E2F target genes, MTORC signaling, and G2M checkpoint, along with metabolic pathways of oxidative phosphorylation and glycolysis were enriched in cancer cells at regression. Taken together, our data show that T cell cytotoxicity and functional states are preserved during tumor regression and tumor escape phases, suggesting that T cell dysfunctionality is unlikely to be the sole cause of TCB resistance. TME reprogramming, consisting of a rapid myeloid cell infiltration and their acquisition of a suppressive phenotype over time, along with a selection of cancer cell clones that escape the TCB-mediated cytotoxicity and upregulate Wnt, p53 and Hedgehog pathways could contribute to TCB-mediated resistance, leading to tumor escape.

Citation Format: Johannes Sam, Llucia Alberti Servera, Sina Nassiri, Atul Sethi, Alison Ribeiro, Gabrielle Leclercq-Cohen, Petra Schwalie, Tamara Hüsser, Emilio Yangueez, Sylvia Herter, Christian Klein, Pablo Umana, Marina Bacac. Molecular insights into resistance mechanisms to therapy with T cell bispecific antibodies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4117.