Structural Basis for Multivalent MUC16 Recognition and Robust Anti-Pancreatic Cancer Activity of Humanized Antibody AR9.6.

Mucin-16 (MUC16) is a target for antibody-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC) among other malignancies. The MUC16-specific monoclonal antibody AR9.6 has shown promise for PDAC immunotherapy and imaging. Here, we report the structural and biological characterization of the humanized AR9.6 antibody (huAR9.6). The structure of huAR9.6 was determined in complex with a MUC16 SEA (Sea urchin sperm, Enterokinase, Agrin) domain. Binding of huAR9.6 to recombinant, shed, and cell-surface MUC16 was characterized, and anti-PDAC activity was evaluated in vitro and in vivo. HuAR9.6 bound a discontinuous, SEA domain epitope with an overall affinity of 88 nmol/L. Binding affinity depended on the specific SEA domain(s) present, and glycosylation modestly enhanced affinity driven by favorable entropy and enthalpy and via distinct transition state thermodynamic pathways. Treatment with huAR9.6 reduced the in vitro growth, migration, invasion, and clonogenicity of MUC16-positive PDAC cells and patient-derived organoids (PDO). HuAR9.6 blocked MUC16-mediated ErbB and AKT activation in PDAC cells, PDOs, and patient-derived xenografts and induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. More importantly, huAR9.6 treatment caused substantial PDAC regression in subcutaneous and orthotopic tumor models. The mechanism of action of huAR9.6 may depend on dense avid binding to homologous SEA domains on MUC16. The results of this study validate the translational therapeutic potential of huAR9.6 against MUC16-positive PDACs.

Preclinical Evaluation of a Humanized, Near-Infrared Fluorescent Antibody for Fluorescence-Guided Surgery of MUC16-Expressing Pancreatic Cancer.

Surgery remains the only potentially curative treatment option for pancreatic cancer, but resections are made more difficult by infiltrative disease, proximity of critical vasculature, peritumoral inflammation, and dense stroma. Surgeons are limited to tactile and visual cues to differentiate cancerous tissue from normal tissue. Furthermore, translating preoperative images to the intraoperative setting poses additional challenges for tumor detection, and can result in undetected and unresected lesions. Thus, pancreatic ductal adenocarcinoma (PDAC) has high rates of incomplete resections, and subsequently, disease recurrence. Fluorescence-guided surgery (FGS) has emerged as a method to improve intraoperative detection of cancer and ultimately improve surgical outcomes. Initial clinical trials have demonstrated feasibility of FGS for PDAC, but there are limited targeted probes under investigation for this disease, highlighting the need for development of additional novel biomarkers to reflect the PDAC heterogeneity. MUCIN16 (MUC16) is a glycoprotein that is overexpressed in 60-80% of PDAC. In our previous work, we developed a MUC16-targeted murine antibody near-infrared conjugate, termed AR9.6-IRDye800, that showed efficacy in detecting pancreatic cancer. To build on the translational potential of this imaging probe, a humanized variant of the AR9.6 fluorescent conjugate was developed and investigated herein. This conjugate, termed huAR9.6-IRDye800, showed equivalent binding properties to its murine counterpart. Using an optimized dye:protein ratio of 1:1, in vivo studies demonstrated high tumor to background ratios in MUC16-expressing tumor models, and delineation of tumors in a patient-derived xenograft model. Safety, biodistribution, and toxicity studies were conducted. These studies demonstrated that huAR9.6-IRDye800 was safe, did not yield evidence of histological toxicity, and was well tolerated in vivo. The results from this work suggest that AR9.6-IRDye800 is an efficacious and safe imaging agent for identifying pancreatic cancer intraoperatively through fluorescence-guided surgery.