Background MSLN is a membrane protein overexpressed by many cancers with limited expression in normal tissues. One challenge in developing MSLN-targeted therapies has been the presence of a shed MSLN sink found in both blood and the tumor microenvironment. LNK101 is a fully humanized bispecific TCE that employs an IgG-scFv architecture with an effector-silenced IgG1 backbone, full cross-reactivity to cynomolgus monkey (cyno), and a relatively low affinity but high avidity for membrane-bound MSLN, minimizing the impact of the shed sink. LNK101 is being developed as a therapy for advanced cancers associated with MSLN expression.

Methods Biolayer interferometry (BLI) and flow cytometry evaluated binding affinities, avidity, and species cross-reactivity of LNK101. In vitro T-cell killing (TCK), T-cell activation and cytokine production were assessed across multiple donor T cells and target cell lines. In vivo antitumor activity was evaluated in immune-compromised mice with intraperitoneally implanted tumor and reconstituted with human T cells. Tissue specificity was examined in a frozen human tissue cross reactivity (TCR) panel. LNK101 was further evaluated in a one-month Good Laboratory Practice (GLP) repeat-dose cyno toxicity study.

Results LNK101 bound to human and cyno MSLN and CD3 with comparable binding affinities, with no binding to murine targets. BLI experiments showed differential binding pattern for soluble versus immobilized MSLN. LNK101 bound immobilized MSLN with no off rate, consistent with high avidity and preferential binding to the membrane-bound form. Avoidance of soluble MSLN was confirmed in TCK experiments. In vitro, LNK101 has high potency, upregulates CD25-expression on T cells and stimulates cytokine production in the presence of cells with high MSLN expression but does not activate T cells in the absence of MSLN. In vivo, significant tumor regression and tumor growth inhibition were observed. In a human TCR study, LNK101 bound only to CD3- or MSLN-expressing tissues. In the GLP study, LNK101 demonstrated dose-related pharmacologic activity (T-cell margination and activation and cytokine release) and safety at exposures above predicted human efficacious doses.

Conclusions In vitro results indicate that LNK101 binds specifically to MSLN-expressing cells and CD3-expressing T cells, is minimally impacted by soluble MSLN, activates T cells only in the presence of target, and has potent antitumor activity. In cynomolgus monkeys, LNK101 is tolerated and shows pharmacologic activity. LNK101 is a promising novel treatment for MSLN-expressing solid tumors.

Ethics Approval In vivo mice studies and the GLP toxicity studies in cynomolgus monkeys were approved by Ethics Board of the respective contract research organizations (CROs).