Cell type-directed Tim-3 targeting in melanoma

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PROJECT SUMMARY Immune checkpoint blockade has elicited unprecedented clinical responses in patients with metastatic melanoma and other cancers. A promising new checkpoint under investigation in cancer therapeutic trials is T- cell immunoglobulin and mucin domain 3 (Tim-3). Tim-3 blockade reverses T-cell impairment, thereby reinvigorating antitumor T-cell immunity. However, we found that Tim-3 inhibitors, including those in clinical trials, not only target T-cell-Tim-3, but also have varying affinity for Tim-3 on dendritic cells (DCs), macrophages (MΦs), NK and melanoma cells. Clinical benefit might thus not exclusively rely on antagonism of T-cell-Tim-3, but also on inhibition of these additional Tim-3-expressing cell types. In support, blockade of T- cell-Tim-3 suppressed, while melanoma-directed Tim-3 inhibition enhanced tumor growth in murine melanoma models, thereby counteracting desired efficacy of Tim-3 therapy. Consistently, enforced expression of Tim-3 on melanoma cells suppressed tumorigenesis, metastasis formation, and proliferative pathway activity. Our preliminary studies thus identify melanoma cell-intrinsic, DC-, MΦ-, and NK-cell-Tim-3 as unexpected variables and/or potential confounders of treatment outcome. They further highlight the need to define therapeutic consequences of Tim-3 antibody (Ab) responses at the level of specific cell types. The Tim-3 protein bears multiple N- and O-glycostructures that differ dramatically in composition, size, and charge between cell lineages and which might explain the marked variations in Tim-3 Ab clone reactivity we found between cell types. For example, the clinical Tim-3 trial candidate, TSR-022, avidly bound T-cell- and melanoma-, but not NK-, DC-, or MΦ-Tim-3, while other Tim-3 Abs showed high affinity for Tim-3 on T-cells, MΦs, DCs, and/or NK, but not melanoma cells. Notably, glycan-modifying regimens shifted inhibitor binding towards desired T-cell- Tim-3 recognition and reduced melanoma-Tim-3 reactivity. Our preliminary data highlights the critical need for dissecting immune- vs. melanoma cell-Tim-3 glyco-epitopes, Ab affinity, signaling, and immunobiology. Results will help optimize Tim-3 therapeutic efficacy by validating regimens that preferentially target immune cell-Tim-3 glycans, while avoiding unwanted blockade of melanoma cell-Tim-3. Our aims are to 1) define cell type- associated Tim-3 glycan moieties, ligands, Ab affinities, and signaling networks, 2) examine immune cell- vs. melanoma-intrinsic effects of existing Tim-3 antagonists and their relevance to interpreting therapeutic benefit, and 3) identify new Tim-3 targeting strategies that accentuate immune cell-Tim-3 inhibition. We will use state- of-the-art gain and loss of Tim-3 function and glycan-modifying strategies, Tim-3 inhibitors with variable tissue- associated affinities, and immune and melanoma model systems to define cell type-specific Tim-3 functions and glycomolecular targets. Our initiative also implements clinical tumor biospecimens from patients receiving immune checkpoint inhibitors. Together, these studies will pave the way for next generation biomarkers and treatment modalities that discriminate immune- from cancer cell-Tim-3 for optimized immunotherapy outcomes.