493 Identification of different classes of antagonist monoclonal antibodies targeting the myeloid checkpoint CLEC-1 and their associated anti-tumoralin vivoefficacies in humanized preclinical models

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Background The c-type lectin receptor CLEC-1 is a pattern recognition receptor 1 expressed by endothelial and myeloid cells in mice, non-human primates, and humans. While genetic deletion of CLEC-1 in mice does not lead to any developmental defect, CLEC-1 deletion or CLEC-1 targeting using monoclonal antibodies increases damaged or necrotic cell antigen cross-presentation by cDC1 dendritic cells, as well as subsequent T-cell activation and anti-tumor response.2 However, the identification of CLEC-1 endogenous ligands and their relative involvement in the immune checkpoint activity of CLEC-1 remained to be fully investigated.Methods Endogenous CLEC-1 ligand candidates were identified by affinity capture assays followed by LC/MS analysis. Ligand candidates were validated through protein-protein binding assays and Biacore affinity measurements. Through an immunization campaign, a library of anti-CLEC-1 monoclonal antibodies was generated and screened for CLEC-1 protein binding. Monoclonal antibodies were also assessed for their abilities to inhibit the binding of CLEC-1 to its newly identified endogenous ligands. Different classes of antagonist anti-CLEC-1 antibodies were thereby identified and subsequently evaluated for their anti-tumor efficacies in hepatocellular carcinoma (Hepa1.6) and colorectal cancer (MC38) preclinical models, using human CLEC-1 knock-in mice.Results While we confirm CLEC-1 specific binding to the E3 ubiquitin ligase TRIM21 2 and to the secreted histidine rich glycoprotein (HRG),3 we also identify several novel intra-cellular and cell surface CLEC-1 ligands. We show that the binding of CLEC-1 to these newly identified ligands is protein-specific, as deglycosylation does not impair CLEC-1 binding to its ligands. Finally, we document the antitumoral activities of several classes of antagonist anti-CLEC-1 monoclonal antibodies: while blocking of CLEC-1 binding to its secreted ligand HRG moderately increases anti-tumor responses, inhibition of CLEC-1 binding to its cytoplasmic membrane ligands significantly impairs MC38 tumor growth (n=12, p=0.04) and increases the overall survival of Hepa1.6-bearing mice (n=12, p=0.002), as compared to corresponding isotype control treatment.Conclusions Altogether, by shedding new light on the role of CLEC-1/CLEC-1 ligand interactions, our results further dissect the mechanism of action of the myeloid checkpoint CLEC-1 in its ability to impair anti-tumor immunity and support its use as a novel and highly promising target for cancer immunotherapy.References Drouin M, Saenz J, Chiffoleau E. C-Type Lectin-Like Receptors: Head or Tail in Cell Death Immunity. Front Immunol. 2020;11:251.Drouin M, Saenz J, Gauttier V, et al. CLEC-1 is a death sensor that limits antigen cross-presentation by dendritic cells and represents a target for cancer immunotherapy. Sci Adv. 2022;8(46):eabo7621.Gao S, Wake H, Sakaguchi M, et al. Histidine-Rich Glycoprotein Inhibits High-Mobility Group Box-1-Mediated Pathways in Vascular Endothelial Cells through CLEC-1A. iScience. 2020;23(6):101180.

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