Background
Tumor-targeting mAbs can be leveraged to stimulate innate anti-cancer immunity. NEO-102 (Ensituximab) is a chimeric human IgG1 mAb targets a glycosylated variant of MUC5AC with specificity to colorectal and pancreatic cancer. In a phase 2 study, patients with advanced, refractory metastatic colorectal cancer treated with NEO-102 showed promising results. Enhancing binding affinity stands as a prominent avenue within the realm of antibody engineering, offering the potential to augment the therapeutic efficacy of antibodies. The objective of this investigation is modifying the structure of NEO-102 to enhance its binding affinity to malignant tumors.
Methods
The VH and VL sequences of NEO-102 were re-engineered through Fast Screening for Expression Biophysical Properties and Affinity, with the aim to maintain the binding to its target antigen while achieving a lower KD. To this end, we constructed a library for saturation mutagenesis into all residues in the VH and VL region of the antibody. Flow cytometry analysis was used to compare the binding affinity between the NEO-102 and the affinity maturation generated clone (PB-223) to various tumor cell lines. O-glycan microarray was utilized to identify the O-glycan binding epitope of the PB-223. IHC tumor tissue and normal tissue microarrays, and antibody internalization were performed on PB-223.
Results
Clone PB-223 was selected through the affinity maturation process, The KD of NEO-102 was 5.60×10-9 M while PB-223 exhibited a significantly improved KD (1.23×10-9 M). Flow cytometry analysis revealed that the PB-223 displayed markedly enhanced binding to multiple tumor cell lines and IHC analysis indicated that PB-223 can bind to a broader range of tumor types compared to NEO-102. PB-223 does not bind to normal tissues. PB-223 binds strongly to truncated Core-2 O-glycan. PB-223 can be internalized in the PB-223 antigen positive human ovarian cancer cell line OV-90.
Conclusions
We have generated a high affinity clone PB-223 which comprises a KD of 4.5-fold less than NEO-102. Flow cytometry and IHC analysis suggest that PB-223 can bind to a wider spectrum of tumor types than NEO-102 but not to normal tissues. We identified the Core-2 O-glycan as the binding epitope of PB-223. In addition, PB-223 can be internalized in the PB-223 antigen positive cell line OV-90. This finding supports PB-223 as a potential candidate for the development of antibody-drug conjugates for treatment of various human carcinomas.