PURPOSE: Targeting intracellular oncogenic mutations with T-cell engagers has the potential to expand precision immunotherapy beyond surface antigens. Here, we describe the preclinical characterization of CLSP-1025, a mutation-specific T-cell engager that targets the p53R175H neoantigen presented by HLA-A*02:01. EXPERIMENTAL DESIGN: CLSP-1025 binding, selectivity, and functional activity were evaluated using biochemical assays, T-cell activation and cytotoxicity assays, primary human cells, patient-derived tumor organoids, and humanized mouse models. Off-target activity was assessed using broad HLA cross-reactivity screens, primary tissue panels, and cytokine release assays. Pharmacokinetic and pharmacodynamic properties were characterized in rats and human CD3 transgenic mice. RESULTS: CLSP-1025 bound with nM affinity to both CD3 and the HLA-A*02:01-p53R175H complex, with structural modeling suggesting spacing consistent with a physiologic immune synapse. The molecule mediated potent and mutation-dependent cytotoxicity across tumor cell lines and patient-derived organoids expressing p53R175H, with minimal activity against p53 wild-type targets. Extensive cross-reactivity screening demonstrated a narrow HLA recognition profile and limited off-target T-cell activation. In humanized mouse models, CLSP-1025 induced robust tumor growth inhibition, accompanied by increased intratumoral CD8⁺ and CD4⁺ T-cell infiltration. Pharmacokinetic analyses showed dose-proportional exposure with an IgG-like half-life, and CD3 receptor occupancy correlated with systemic exposure. CONCLUSIONS: These data establish CLSP-1025 as a highly selective, mutation-specific T-cell engager and support the feasibility of targeting shared intracellular neoantigens with pHLA-directed immunotherapies. This favorable preclinical activity and specificity support ongoing clinical investigation of CLSP-1025 in HLA-A*02:01+ patients with cancers harboring p53R175H and outline a framework for developing next-generation TCEs against additional driver mutations.
Maloney et al. (Fri,) studied this question.