Fc Engineering by Monoclonal Mammalian Cell Display for Improved Affinity and Selectivity Towards FcγRs.

Fc optimization can significantly enhance therapeutic efficacy of monoclonal antibodies. However, existing Fc engineering approaches are sub-optimal with noted limitations, such as inappropriate glycosylation, polyclonal libraries, and utilizing fragment but not full-length IgG display. Applying cell cycle arrested recombinase-mediated cassette exchange, this study constructed high-quality monoclonal Fc libraries in CHO cells, displayed full-length IgG on cell surface, and preformed ratiometric fluorescence activated cell sorting (FACS) with the antigen and individual Fc gamma Rs. Identified Fc variants were quantitatively evaluated by flow cytometry, ELISA, kinetic and steady-state binding affinity measurements, and cytotoxicity assays. An error-prone Fc library focusing on the hinge-CH2 region was constructed in CHO cells with a functional diversity of 7.5 x 106. Panels of novel Fc variants with enhanced affinity and selectivity for Fc gamma Rs were isolated. Particularly, clone 2a-10 (G236E/K288R/K290W/K320M) showed increased binding strength towards Fc gamma RIIa-131R and 131H allotypes with kinetic dissociation constants (KD-K) of 140 nM and 220 nM, respectively, while reduced binding strength towards Fc gamma RIIb compared to WT Fc; clone 2b-1 (K222I/V302E/L328F/K334E) had KD-K of 180 nM towards Fc gamma RIIb; clone 3a-2 (P247L/K248E/K334I) exhibited KD-K of 190 nM and 100 nM towards Fc gamma RIIIa-176F and 176 V allotypes, respectively, and improved potency of 2.0 ng/ml in ADCC assays. Key mutation hotspots were identified, including P247 for Fc gamma RIIIa, K290 for Fc gamma RIIa, and K334 for Fc gamma RIIb bindings. Discovery of Fc variants with enhanced affinity and selectivity towards individual Fc gamma R and the identification of novel mutation hotspots provide valuable insights for further Fc optimization and serve as a foundation for advancing antibody therapeutics development. Statement of Significance: Fc optimization is critical in improving therapeutic efficacy, but current engineering methods have limitations. By constructing large monoclonal mutagenesis libraries and displaying full-length IgG on CHO cells, novel Fc variants with enhanced affinity and selectivity for individual Fc gamma Rs were isolated and validated by multiple measurements and cytotoxicity assays.