Novel Strategy of Antibody Affinity Maturation and Enhancement of Nucleolin‐Mediated Antibody‐Dependent Cellular Cytotoxicity Against Triple‐Negative Breast Cancer

ABSTRACT Triple‐negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer that remains an unmet medical need. Because TNBC cells do not express the most common markers of breast cancers, there is an active search for novel molecular targets in triple‐negative tumors. Additionally, this subtype of breast cancer presents strong immunogenic characteristics which have been encouraging the development of immunotherapeutic approaches against the disease. In this context, nucleolin arises as a promising target for immunotherapy against TNBC. Our group has previously developed an anti‐nucleolin VHH‐Fc antibody capable of eliciting antibody‐dependent cellular cytotoxicity (ADCC). Moreover, we constructed and characterized an antibody library, that was screened against nucleolin‐overexpressing cells, originating an enriched anti‐nucleolin antibody pool. In this work, a strategy to select individual clones from the pool was designed, combining NGS data with 3D modeling. Two antibodies demonstrated a significant 4.4‐ and 6.1‐fold increase in binding to nucleolin‐overexpressing and TNBC cells, and an improvement in affinity to the sub‐micromolar range (0.19 µM and 83.69 nM). Additionally, an increment in 4.6‐ and 3.1‐fold in ADCC activity against respective cell lines was observed for the M2 antibody clone. Herein, the affinity maturation strategy developed was validated and corroborated a positive, but not proportional, correlation between antibody binding, affinity, and ADCC.