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Background We have previously shown that novel bifunctional ‘STAR’ antibody-fusion molecules comprising germline b chain TCR-targeting agonist antibodies fused to costimulatory cytokines co-engage the TCR and T cell cytokine receptors, thereby promoting expansion of subsets of T cells expressing distinct germline variable b (Vb) chain TCRs. Here we describe a new class of T cell engager (TriSTAR) that utilizes a tri-specific format comprising the ‘STAR’ TCR b chain antibody agonist and IL-2 fusion module coupled to an additional Fab arm targeting tumor associated antigens. By design, TriSTAR molecules promote the selective activation and expansion of Vb T cell subsets that adopt a novel memory-like effector phenotype and are re-targeted to tumors. TriSTAR0701, a human Nectin-4 (hN4)-targeting TriSTAR molecule induced potent and durable anti-tumor activity in mice bearing refractory solid tumors expressing hN4.Methods TriSTAR0701 constructs with optimized affinities to hN4 and TCRVb were generated and tested in vitro and in vivo in relevant hN4-expressing tumor cell lines and murine transgenic tumor models expressing hN4, respectively. In mouse and cynomolgus monkey models, the pharmacology of TriSTAR0701 were assessed using flow cytometry, NanoString and other immunoassays.Results TriSTAR0701 elicited potent in vitro activation and expansion of human Vβ6/10 T cells that adopted a characteristic memory-like effector phenotype. In human in vitro T cell/cancer cell co-cultures, these constructs also induced potent cytotoxicity of hN4-expressing tumor lines across a range of hN4 expression levels. In cynomolgus monkeys and murine tumor-bearing mice TriSTAR0701 molecules were efficiently distributed and induced expected pharmacology in target compartments. In mice, TriSTAR0701 accumulated in tumors and induced potent anti-tumor activity that was superior to untargeted STAR T cell activators and hN4-targeting anti-CD3 TCEs. This superior anti-tumor activity was attributed to accumulation of CD8 +, CD25+ and Granzyme B+ T cells within expanded Vβ T cell subsets in tumors. Further immune profiling using flow cytometry and gene expression assays in tumors and blood isolated from treated mice highlighted significant remodeling of tumor-infiltrating lymphocyte (TIL) populations.Conclusions Germline b chain TCR-targeting TriSTAR molecules represent a new class of selective T cell engager with the potential to redirect Vβ T cell subsets with a memory phenotype to tumors resulting in improved anti-tumor activity compared with established anti-CD3 T cell engagers. Here we show that TriSTAR0701, a hN4-targeting TCE, drives potent anti-tumor activity in human in vitro and murine in vivo models making them a promising emerging treatment modality for hN4-expressing solid tumors.