Abstract
Background:ImmTAC® (immune-mobilizing monoclonal T cell receptor against cancer) molecules are soluble bi-specific TCR molecules that redirect T lymphocytes to kill tumor cells. Tebentafusp, which targets the melanocyte lineage gp100, is the first ImmTAC molecule approved for the treatment of HLA-A*02:01 adult patients with metastatic uveal melanoma.1 While the roles of Granzyme B and Perforin in the anti-tumor activity of CD3 T cell engagers are well described, the contribution of other apoptotic pathways, e.g. pyroptosis (inflammatory apoptosis), is poorly understood. For this reason, we explored apoptosis mechanisms induced by ImmTACs in vitro and their relevance to the anti-tumor activity of tebentafusp in a Phase 2 clinical trial.
Methods: Cell lines derived from different malignancies, MP41 (uveal melanoma), MEL624 (cutaneous melanoma) and NCI-H1755 (non-small cell lung carcinoma) were co-cultured with PBMC and ImmTAC molecules for defined time periods, followed by cytolysis measurements (xCelligence) and protein expression analysis (western blotting). Relevant cell death pathways were targeted by genetic (CRISPR knockout) or pharmacological inhibition. Translational relevance of in vitro experiments was assessed using RNAseq of tumor biopsies at baseline (N=70) and matched on-treatment at 16 days post treatment [N=35] or at progression [N=14]) from a Phase 2 trial of tebentafusp in HLA-A*02:01+ mUM patients (NCT02570308). Clinical data cut-off: 17-Oct-2022.
Results: ImmTAC-mediated tumor killing in vitro occurred in a caspase-dependent manner and accompanied a profound induction of several apoptotic, necroptotic and pyroptotic proteins, such as GZMB, Caspases 8 and 7, RIPK3, MLKL and GSDMD in several cell lines. In NCI-H1755 cells, genetic knockout of GSDMD significantly diminished ImmTAC-mediated cytolysis (92% reduction), thereby confirming the involvement of pyroptosis in ImmTAC-mediated tumor killing in these cells. In agreement, RNAseq analysis of tumor biopsies from mUM patients after 3 doses of tebentafusp revealed enhanced expression of genes involved in different cell death pathways, including Caspase 7, RIPK3, MLKL and GSDMD (fold changes of 1.2-2). In addition, patients exhibiting above-median tumor expression of GSDMD at progression had longer overall survival (Hazard ratio = 0.074 95% C.I. 0.01-0.64).
Conclusions: ImmTAC redirection of T cells resulted in a caspase-dependent, pyroptotic cell death of tumor cell lines and enhanced gene expression of necroptotic and pyroptotic pathways in tumor biopsies. GSDMD, induced during ImmTAC-mediated cytolysis, also correlated with better overall survival in tebentafusp-treated patients. Induction of multiple cell death pathways, particularly pyroptosis, helps to explain the anti-tumor activity of tebentafusp.
References: 1. Nathan, P. et al. New Engl J Med 385, 1196-1206 (2021).
Citation Format: Shankar Varadarajan, Aleksandra Raczka, Sophie Khakoo, Laura Collins, Adel Benlahrech. TCR-CD3 bispecifics kill tumor cells in a caspase- and gasdermin-dependent manner by inducing multiple cell death pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4984.
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