Targeting the myeloid immune checkpoint ILT3 (LILRB4) with small molecules enables reprogramming of suppressive tumor...

Cancer immunotherapy has transformed cancer treatment; however, durable responses remain limited by suppressive myeloid populations within the tumor microenvironment. Leukocyte immunoglobulin-like receptor B4 (LILRB4/ILT3) is an emerging myeloid immune checkpoint implicated in immune evasion and resistance to immunotherapy, yet small molecule targeting of ILT3 remains largely unexplored. Here, we report the discovery of small molecule ILT3 modulators identified using a Dianthus-based temperature-related intensity change (TRIC) screening platform. Screening of an 8961-member Enamine Protein Mimetic Library identified multiple direct ILT3 binders, with lead compound ICB-7 demonstrating high-affinity binding to recombinant human ILT3 by microscale thermophoresis (K_D = 156 nM) and robust cellular target engagement in CETSA assays. Molecular docking and molecular dynamics simulations revealed a stable hydrophobic binding pocket within the D2 domain of ILT3. Functionally, ICB-7 disrupted the ILT3-SCG2 interaction and inhibited downstream SHP1, SHP2, and STAT3 signaling. In patient-derived colorectal cancer and acute myeloid leukemia co-culture models, ICB-7 restored IFN-γ and IL-2 secretion, enhanced cytotoxic T-cell activity, and reduced tumor-cell viability. The compound also demonstrated favorable pharmacokinetic and safety properties together with significant anti-tumor efficacy in the CT26 syngeneic colorectal carcinoma model. Collectively, these findings establish ILT3 as a tractable target for small-molecule immunomodulation and support pharmacological targeting of suppressive myeloid checkpoints as a promising cancer immunotherapy strategy.