Abstract 3289: Melanoma patients with high and low target expression can benefit from TCR-CD3 bispecifics through direct and indirect mechanisms of tumor control

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Abstract Background: ImmTAC molecules are TCR-CD3 bispecific fusion proteins that can redirect and activate polyclonal T cells to kill tumors. Tebentafusp, which targets gp100, is the first ImmTAC to demonstrate an overall survival benefit and is approved for the treatment of HLA-A*02:01 adult patients with unresectable or metastatic uveal melanoma1. Clinical benefit was observed in patients with both high and low gp100 expression2, suggesting direct and indirect mechanisms of tumor control, which we explored using in vitro models. Method: Uveal and cutaneous melanoma cell lines were pulsed with 0-1 µM peptide concentrations or were treated with supernatants from ImmTAC-activated PBMC, with or without blocking antibodies against IFNβ, IFNγ, TNFα, and IL6. Peptide-HLA (pHLA) molecules on tumor cell surface were quantified by microscopy. ImmTAC-redirected T cell activation and killing were measured by ELISPOT, MSD, and xCelligence assays. Antigen presentation machinery (APM) gene expression was measured by qPCR. Linear regression models were used to assess the influence of variables on ImmTAC-mediated T cell activation. Results: Peptide-pulsing of tumor cells showed a dose response relationship between surface pHLA number and ImmTAC-mediated T cell killing (R2= 0.85), which was confirmed by ImmTAC redirected killing assays using tumor lines with differential endogenous expression of pHLA targets. Higher effector: target (E: T) and higher pHLA number are the key factors, by regression analyses, influencing ImmTAC mediated T cell activation. These account for 53% and 29% variability in IFNγ production (p<0.01), respectively. To assess the indirect impact of ImmTAC-activated T cells on tumor cells, cell lines were pre-treated with supernatants. ImmTAC-activated PBMC supernatants reduced tumor growth (2 to 25-fold) and increased tumor death (20 - 80%) in 5 of 8 cell lines tested. Blocking IFNγ restored tumor growth by over 40%, which was further enhanced by additional blocking of IFNβ, TNFα, and IL6. All cell lines significantly upregulated APM genes and became more susceptible to direct ImmTAC-mediated T cell killing (1.4 to 2-fold increase). Conclusions: In vitro, ImmTAC bispecifics kill cancer cells by both direct and indirect mechanisms. Redirection of polyclonal T cells to kill melanoma cells is greatly influenced by surface pHLA number and E:T ratio. ImmTAC-induced cytokines inhibited tumor growth, increased tumor apoptosis and resulted in higher antigen presentation demonstrating the indirect effect of ImmTAC. These observations highlight how ImmTAC-treated melanoma patients could benefit through direct and indirect mechanisms of tumor control, and in part help to explain how patients with low gp100 expression can derive benefit from tebentafusp.