Th148 - Rapid Preclinical Development of Immtac Bispecifics in Mammalian Protein Expression Systems

Abstract Text: ImmTACs are a novel class of fusion proteins combining a CD8-associated TCR domain with the immune-activating properties of an anti-CD3ζ single-chain variable fragment (scFv). These molecules trigger anti-tumor responses by targeting peptide-loaded HLA complexes presented by cancer cells. Tebentafusp, a gp100(280-288)/A2-specific ImmTAC, has shown superior overall survival in uveal melanoma compared to anti-PD1 therapy. Tebentafusp is produced using recombinant DNA technology in E. coli, a system that limits the proper folding and activity of complex eukaryotic proteins.
Here, we describe the use of a serum-free mammalian system for ImmTAC production and the functional characterization of Tebentafusp and IMDA-001, a modified version with a proprietary anti-CD3ζ scFv. Chinese Hamster Ovary cells (CHO-S) transduced with lentiviral vectors encoding the ImmTAC cDNA were cultured in fed-batch conditions for protein production for up to 14 days. ImmTACs were purified from culture supernatants with affinity columns and, after final formulation, yielded 168 mg/L for Tebentafusp and 126 mg/L for IMDA-001. Functionality was validated by cytotoxicity assays, where both proteins exhibited potent T cell-mediated killing of gp100(280-288)/A2+ target cells at picomolar concentrations. IMDA-001 demonstrated enhanced cytotoxicity, higher proinflammatory cytokine secretion, and preferential activation of CD4+ T cells compared to Tebentafusp. This study provides a robust and scalable method for bi-specific protein production in CHO-S cells and accelerated ImmTAC pre-clinical development from conceptualization to functional testing within only two months. These findings support the use of mammalian systems for the production of next-generation ImmTACs and underscore the potential for further optimization, as exemplified by IMDA-001.