Interrogating tumor infiltrating B cell populations for discovery of novel tumor-reactive antibodies

In order to discover novel tumor-reactive antibodies, single cell BCR-sequencing of enriched plasma cells isolated from the tumor microenvironment of a non-small cell lung cancer patient treated with pembrolizamab. One of the antibodies derived from this process was used to probe a HuProt Human Protein Microarray to identify potential target antigens. The HuProt array was used for the High-Spec antibody cross-reactivity assay of the antibody. The array was incubated with blocking buffer (1xTBS / 0.1% Tween 20 / 5% BSA) at room temperature for 1 hr. with gentle shaking. Then, the antibody was diluted to 1 μg/mL in blocking buffer and probed on the array at room temperature for 1 hour with gentle shaking. After probing, the array was washed with TBST (1xTBS / 0.1% Tween 20) for 10 min X 3, and then probed with Alexa647-anti-human IgG Fc secondary antibody (0.25 ug/mL) at room temperature for 1 hour in a light-proof box with gentle shaking, followed by 3 washes with TBST for 10 min each and 3 rinses with ddH2O. The array was then dried with an air duster and scanned using a GenePix 4000B scanner for data collection. The raw data was obtained from the GenePix software and processed data from CDI’s proprietary data analysis software (Z-Score analysis).