Abstract 1836: Structural basis for recognition of MUC16 expressed in various cancers

Abstract Background: Overexpressed Mucin 16 (MUC16) is highly related with cancer progression, metastasis, and therapy resistance in multiple malignancies. The proteolytic cleavages of MUC16 form independent bimodular fragments, a circulating shed form known as an ovarian cancer biomarker (CA 125) and a C-terminal tail-connected juxtamembrane region as an unshed form which is critical in determining the unique mechanistic roles of MUC16 and therapeutic interventions. Effective neutralizing antibody for MUC16-positive solid cancers is urgently needed for earlier diagnosis and immunotherapeutic approaches. Methods: The novel 4H11-scFv (single chain antibody) targeting MUC16-juxtamembrane was optimized for mammalian cell usage and Liter-scale cultures of HEK293T were infected with high-titer lentiviral stocks expressing the 4H11-scFv and the recombinant proteins were purified by a series of purification process and prepared for further characterization and crystallization. The sequence corresponding to human MUC16-juxtamembrane was cloned with maltose binding protein (MBP) and expressed by the bacterial system. The purified MBP-fused-MUC16 was prepared for co-crystallization with 4H11-scFv. A series of biochemical studies are performed: Thermal stability measurement of 4H11-scFv, MBP-pull down, Isothermal titration calorimetry (ITC) for interaction analysis, bioactivity of Alexa-labeled 4H11-scFv, analytical size-exclusion chromatography, multi-mutagenesis studies at the interface of 4H11-scFv-MUC16 complex. Crystallizations were performed using high-throughput crystallization screen kits, followed by extensive manual optimization. X-ray diffraction data were collected at the Advanced Photon Source (APS) and processed with HKL2000, PHENIX, CCP4 suite package and COOT. Results: We determined the crystal structures of 4H11-scFv at 2.36 Å and 4H11-scFv-MUC16 at 2.47 Å resolutions, respectively. Structural analysis of a 4H11-scFv demonstrated that all six complementarity-determining regions (CDRs) are composed of 5 loop-like coiled coil regions and 1 short-helix in VH (Heavy chain), while juxtamembrane portion showing flexible loop regions with β-Turns. The complex was stabilized through extensive hydrogen bond-mediated interaction, salt bridge, and cation-π interaction. We identified some specific residues of MUC16, critical to form the complex, from mutagenesis studies. Biochemical analysis demonstrated the 4H11-scFv potently neutralizes MUC16 and stable at the cancer microenvironmental and endosomal pH with a possibility of pre-clinical diagnosis/treatment in various cancers. Conclusion: We investigate the structures and the neutralizing mechanisms for 4H11-scFv targeting the MUC16. Our study offer insight into antibody to neutralize the non-cleaved cell surface retained portion of MUC16 and advance our ability to design anti-MUC16 antibody with improved therapeutic properties. Citation Format: Kwangkook Lee, Kay Perry, Oladapo O. Yeku, David R. Spriggs. Structural basis for recognition of MUC16 expressed in various cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1836.