ABSTRACT Tumor associated macrophages (TAMs) are key components of the tumor microenvironment and are associated with immunosuppression, poor prognosis and an inadequate response to immune checkpoint therapy (ICT) in different types of tumors. Reprogramming of TAMs towards a more phagocytic and “type 1” pro-inflammatory phenotype can improve tumor control. Thus, several molecules associated with the immunosuppressive phenotype in TAMs have been targeted in both tumor models and patients, alone or in combination with ICT. Recently, we showed that the human inhibitory receptor ILT3, also known as LILRB4, is an attractive target for TAM reprogramming in tumor therapy. We found that ILT3 is highly expressed in TAMs in various types of cancer. Moreover, we discovered that ILT3 recognizes fibronectin (Fn), which is ubiquitously expressed in the extracellular matrix and enriched in various primary tumors and metastatic sites. ILT3-Fn interaction polarizes macrophages and other myeloid cells, such as dendritic cells and monocytes, towards an immunosuppressive state, which debilitates their capacity to stimulate and activate T cells. Thus, the ILT3-Fn axis is a mechanism underlying stromal-driven inhibition of anti-tumor activity that represents a promising target to promote reprogramming of TAMs and myeloid cells in general in cancer. In this grant application we propose to determine the impact of ILT3 blockade on the control of primary and metastatic tumor models in vivo with a specific monoclonal antibody, mAb1, that blocks ILT3Fn interaction. Since ILT3 is a human receptor with a distantly related mouse paralogue, studying its function in vivo has been difficult. To date, the evidence of ILT3 immunosuppressive activity is based on expression data in human samples and in vitro assays, while the in vivo impact of ILT3 on myeloid cell functions and anti-tumor activity remains to be elucidated. To overcome this issue, we generated a transgenic mouse that carries a bacterial artificial chromosome encompassing a portion of human chromosome 19 that contains the ILT3 gene, together with its putative regulatory regions. Analysis of ILT3 expression in this mouse demonstrated a pattern consistent with that in humans among different tissues. In specific aim 1, we will test the impact of ILT3 blockade on tumor growth and anti-tumor immune responses in two models of sarcoma and colorectal carcinoma in vivo. We will further test the ability of ILT3 blockade to restore an effective response to ICT in a model of ICT resistance. In specific aim 2, we will test the impact of ILT3 blockade on the composition of pre-metastatic and metastatic niches in two models of lung metastases and, ultimately, on the formation of metastatic lesions. This proposal will advance our knowledge of the mechanisms regulating the immune landscape in primary and metastatic tumors and validate the anti-human ILT3 mAb as a therapeutic agent that can be brought to the clinic, either alone or in a...