Affinity Maturation and Characterization of the Novel Monoclonal Antibody (mAb) PB-223 Targeting Cancer- specific Core 2 O-Glycans

Background: Enhancing the binding affinity of monoclonal antibodies (mAbs) has the potential to improve their therapeutic efficacy. In this study, we generated a novel mAb, PB-223, through affinity maturation of the parental antibody NEO-102. NEO-102 (Ensituximab) is a chimeric human IgG1 mAb that targets a cancer-specific glycosylated variant of MUC5AC expressed on colorectal and pancreatic cancers, while sparing healthy tissues. In a Phase 2 clinical study, involving heavily pretreated patients with advanced, refractory colorectal cancer, NEO-102, as a monotherapy, exhibited modest efficacy. Methods: We engineered the VH and VL regions of NEO-102 through affinity maturation to enhance antigen binding while preserving target specificity. The optimized clone, PB-223, was evaluated for improved binding by ELISA, flow cytometry, immunohistochemistry (IHC). Specific PB-223 target antigen was discovered using O-glycan array. Internalization assay evaluated the ability of PB-223 to internalize human cancer cell lines expressing its target antigen. Results: PB-223 exhibits a 4.55-fold lower dissociation constant (KD) compared with NEO-102 by ELISA. PB-223 has enhanced binding to human cancer cell lines recognized by NEO-102 by flow cytometry. O-glycan array analysis identified core 2 O-glycans, expressed by human cancer cell lines reactive with PB-233 in flow cytometry, as the specific binding epitope of PB-223. IHC analysis of human tumor tissues showed that PB-223 does not bind to healthy tissues, displays superior binding to multiple malignant tissues previously recognized by NEO-102 and binds to tumor types not recognized by NEO-102. PB-223 internalizes human cancer cell lines expressing core 2 O-glycans. Conclusions: PB-223 can potentially be used as a targeting moiety for antibody-based therapeutics, including antibody–drug conjugates (ADCs), bispecific antibodies, immune-engaging constructs, and radiopharmaceuticals, for the treatment of human cancers expressing core 2 O-glycans.