Transcription factors ThPOK and Runx3 regulate the differentiation of “helper” CD4+ and “cytotoxic” CD8+ T cell lineages respectively, inducing single positive (SP) T cells that enter the periphery with the expression of either the CD4 or CD8 co-receptor. Despite the expectation that these cell fates are mutually exclusive and that mature CD4+CD8+ double positive (DP) T cells are present in healthy individuals and augmented in the context of disease, yet their molecular features and pathophysiologic role are disputed. Here, we show DP T cells in murine and human tumors as a heterogenous population originating from SP T cells which re-express the opposite co-receptor and acquire features of the opposite cell type’s phenotype and function following TCR stimulation. We identified distinct clonally expanded DP T cells in human melanoma and lung cancer by scRNA sequencing and demonstrated their tumor reactivity in cytotoxicity assays. Our findings indicate that antigen stimulation induces SP T cells to differentiate into DP T cell subsets gaining in polyfunctional characteristics.

Purpose: Targeting intracellular oncogenic mutations with T-cell engagers (TCE) has the potential to expand precision immunotherapy beyond surface antigens. In this study, we describe the preclinical characterization of CLSP-1025, a mutation-specific TCE 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 human leukocyte antigen (HLA) cross-reactivity screens, primary tissue panels, and cytokine release assays. Pharmacokinetic (PK) 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. PK 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 TCE and support the feasibility of targeting shared intracellular neoantigens with peptide–HLA–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.

Purpose:Targeting intracellular oncogenic mutations with T-cell engagers (TCE) has the potential to expand precision immunotherapy beyond surface antigens. In this study, we describe the preclinical characterization of CLSP-1025, a mutation-specific TCE 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 human leukocyte antigen (HLA) cross-reactivity screens, primary tissue panels, and cytokine release assays. Pharmacokinetic (PK) 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. PK 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 TCE and support the feasibility of targeting shared intracellular neoantigens with peptide–HLA–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.