# Cell-Based Immunization Combined with Single-Round Cell Panning Enables Discovery of PSMA-Targeting Nanobodies from Phage Display Libraries

## TL;DR

This study shows that using tumor cells and cell panning can successfully identify nanobodies that target PSMA, a cancer-related protein, for potential use in imaging and therapy.

## Contribution

The study demonstrates a novel non-targeted immunization and panning strategy to discover PSMA-targeting nanobodies.

## Key findings

- Nanobodies specific to PSMA were successfully isolated using non-targeted immunization and panning.
- Two distinct epitopes on PSMA were identified and confirmed through competition assays.
- The approach proved effective for discovering cancer-targeting nanobodies without prior antigen knowledge.

## Abstract

There is a strong need for nanobodies that target novel cancer-associated antigens to advance radioligand imaging and antibody-based therapeutics. In this study, we investigated whether non-targeted llama immunization using tumor cells, combined with non-targeted phage-display panning of human cell lines, could yield nanobodies specific to Prostate-Specific Membrane Antigen (PSMA). Nanobody selection using both classical three-round PSMA negative–positive panning and single-round panning of cell lines (positive or negative) for PSMA showed clear enrichment for PSMA binders in both strategies. Using shRNA knockdown, flow cytometry, cell-ELISA, immunohistochemistry and structural modeling and docking, we confirmed the PSMA-targeting of selected nanobodies. Two distinct epitopes were predicted to be bound by nanobodies PSMANb9 and A7 (JVZ-007), and this was corroborated by epitope competition assays. These findings support the feasibility of non-targeted immunization and panning strategies for isolating antigen-targeting cancer nanobodies.