Immune capture and protein profiling of tumor-derived exosomes (TEX) in plasma of patients with Head and neck squamous cell carcinoma (HNSCC)

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1. Development and optimization of a method for the targeted isolation of tumor-derived exosomes (TEX) and non-TEX from plasma of patients with head and neck squamous cell carcinoma (HNSCC). The proposed study is based on extensive preclinical experiments demonstrating the feasibility of targeted isolation as the method of choice for biomarker discovery. Since no monoclonal antibody (mAb) that specifically binds to HNSCC tumor antigens is known, mAb against overexpressed tumor Ag are used for immune detection of TEX. In this project, a combination of three different capture antibodies (Abs) will be used, based on preliminary results, which were recently collected in Prof. Whiteside's working group. The combination of anti-CSPG4, anti-B7H3 and CD44v3 mAbs has previously been shown to recognize Ag, which is highly overexpressed by HNSCC cells and which is also carried in exosomes produced by these cells. Therefore, we hypothesize that the combination of anti-CSPG, anti-B7-H3, and anti-CD44v3 mAbs, all of which recognize three different Ags, overexpressed those from HNSCC cells and from plasma-derived HNSCC exosomes worn, can be used successfully to isolate HNSCC-derived exosomes from patient plasma. 2. Use of the developed methodology for the targeted isolation of TEX and non-TEX from plasma of a cohort of 25 HPV(+) and 25 HPV(-) patients with HNSCC for subsequent high-resolution mass spectrometry (HRMS) to search for protein profiles as well as viral to look for antigens, including E2 and E5, that characterize TEX 3. Analysis of the phenotypic and functional influence of the separated exosome fractions on CD8+ T cells and NK cells will also be performed.