A high-throughput luciferase cancer cell panel driving DLL3-targeted TCE discovery

Background: T-cell engagers (TCEs) have rapidly moved into the spotlight as a next-generation immunotherapy, lauded for their (1) “off-the-shelf” convenience, (2) dual-arm precision that re-directs, rather than re-engineers, a patient's own T cells, and (3) capacity to deliver deep, durable responses even after conventional checkpoint blockade has failed. Solid-tumor TCE development is now a major frontier, yet conventional assays-confined to few cell lines-offer limited throughput and cannot probe the full antigen expression spectrum, leaving a blind spot for candidate triage. To close this gap, we selected delta-like ligand 3 (DLL3)-an embryonic Notch inhibitor that is transcriptionally resurrected in high-grade neuroendocrine tumors yet absent from normal adult tissues-and constructed a fully integrated discovery platform anchored by a 20-member luciferase-reporter tumor panel that systematically recapitulates clinically relevant expression gradients. Methods : The platform merges four modules: (1) biophysical profiling via SPS binding kinetics and cell-based affinity screens; (2) parallel 2-D/3-D tumor co-culture assays as well as ICI combination to evaluate T-cell mediated cytotoxicity; (3) multiparametric T-cell activation readouts (CD69/CD25, proliferation); and (4) 10-plex cytokine release assays to map safety-relevant inflammatory signatures. Results Here we showcase several DLL-3 targeting TCEs using a diverse luciferase-neuroendocrine cell panel. Preclinical studies demonstrate their binding, activation, antitumor activity, and T cell expansion in DLL3+ cell lines across cancer types, juxtaposed with a diminished impact on DLL3- cell lines. Conclusions: This platform enables high-throughput evaluation of TCEs across the full antigen expression spectrum, systematically linking target density to therapeutic activity and safety outcomes. By linking target density to TCE performance, this system guides candidate selection and accelerates solid-tumour TCE development, while being readily adaptable to other antigens.