Mentored Scientist Award

VRC07-523LS, a modified potent variant of bnAb VRC01 targeting the CD4 binding site of the HIV-1 envelope protein, is currently undergoing rigorous clinical evaluation to assess its potential as a novel antiretroviral therapeutic for both preventing and treating HIV-1 infections. However, accurate measurement of this anti-HIV bnAb in plasma, PBMC, and tissue samples poses challenges due to limitations in specificity and sensitivity with ligand binding assays. This can result in discrepancies between laboratory results and impact patient treatment. To address this issue, we introduce an LC-MS /MS method capable of precisely quantifying the bnAb in human tissue samples (VRC07-523LS, ~72 kDa) without interference across the clinically relevant concentration range of 0.5 to 50 ng/mL. Our novel approach involves rapid enzymatic digestion followed by microdroplet mass spectrometry quantification of a surrogate peptide for the bnAb, as well as 2D-LC preparation utilizing size exclusion chromatography for tissue bnAb separation, reduction of heavy and light chains to subunit level. Additionally, the use of internal standards consisting of custom peptides will ensure absolute quantitation with improved accuracy. Validation of the method according to regulatory guidance will demonstrate excellent precision and recovery. The method's consistent performance across various clinical sample types and conditions will be compared with immunoassay methods (for example ELISA) to avoid discrepancies and support ongoing pharmacokinetic studies aimed at determining durable responses following treatment.