Abstract B030: Identifying B7-H3 ligands that mediate tumor-immune cell interactions
Authors: Davoodbeglou Foad , Santis George , Parsons Maddy 
Published: February 2025

B7-H3 (CD276) is a member of the B7 superfamily of immune checkpoint molecules and is over-expressed in a range of tumors including non-small cell lung cancer, where high levels correlate with poor prognosis. B7-H3 expression is proposed to positively contribute to tumor invasion and metastasis, and our recent work has demonstrated a role in cancer cell metabolic flux leading to chemoresistance. B7-H3 has been proposed as both costimulatory and inhibitory in the immune compartment, but most studies support a T-cell suppressive role for this receptor. B7-H3 has also been shown to exist as a soluble, membrane cleaved form in culture and in patients. However, the ligands for B7-H3 that mediate these roles remain unclear. We hypothesized that tumor and immune cells express distinct ligands for B7-H3 that mediate roles in tumorigenesis. We aimed to define potential membrane or soluble ligands for B7-H3 and how these ligands, and presence of B7-H3 in cancer cells, controls tumor-immune cell interactions and function. Recombinant B7-H3 extracellular domain labelled with a fluorescent dye was incubated with a range of immune cell lines (T cell, monocyte, macrophage, T-lymphoblast, neutrophil) or 2 different lung cancer cell lines to identify binding to B7-H3. Purified His-tagged B7-H3 and nickel-affinity chromatography was used for proteomic identification of specific binding partners and confirmation of putative ligands AAMP and IL20RA. Identified targets were validated using pulldown assays. Tumor-immune cell interactions and cell viability were analyzed using control and B7-H3 CRISPR cancer cell co-cultures and confocal microscopy. Data demonstrates that soluble B7-H3 can bind both lung cancer and immune cells, including B7-3 CRISPR lines. Proteomic analysis of B7-H3 bound proteins from incubation with cell lysates and follow-up pulldowns did not validate AAMP and IL20RA as B7-H3 binding partners. However, Annexin A2 emerged as a potential ligand identified by proteomics analysis and subsequently validated by pulldown assays using supernatants from both cancer and immune cells. B7-H3 CRISPR lung cancer cells showed a marked increase in T cell and T-lymphoblast binding compared to parental cells. This enhanced interaction correlated with dispersed B7-H3 CRISPR cancer cell colonies. No other immune cells showed notable interactions with cancer cells or changes following B7-H3 depletion. Incubation of soluble recombinant B7-H3 with immune cells resulted in reduced viability, suggesting the membrane-cleaved form may be cytotoxic. Our findings suggest that B7-H3 may mediate distinct interactions between lung cancer and immune cells, with Annexin A2 as a potential novel ligand. Increased immune cell binding to B7-H3 CRISPR cells further support the notion that B7-H3 in tumor cells suppresses immune cell function, potentially enhanced by soluble B7-H3. These data pave the way for the identification of cell-type specific roles for B7-H3 in the tumor microenvironment and increase our understanding of this potential therapeutic target in lung cancer.

Citation Format: Foad Davoodbeglou, George Santis, Maddy Parsons. Identifying B7-H3 ligands that mediate tumor-immune cell interactions [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B030.