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Supplementary Figure S8 from Structural Basis for Multivalent MUC16 Recognition and Robust Anti-Pancreatic Cancer Activity of Humanized Antibody AR9.6

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posted on 2024-06-04, 07:42 authored by Eric N. Aguilar, Satish Sagar, Brandy R. Murray, Christabelle Rajesh, Eric K. Lei, Sarah A. Michaud, David R. Goodlett, Thomas C. Caffrey, Paul M. Grandgenett, Benjamin Swanson, Teresa M. Brooks, Adrian R. Black, Henk van Faassen, Greg Hussack, Kevin A. Henry, Michael A. Hollingsworth, Cory L. Brooks, Prakash Radhakrishnan

Figure S8. huAR9.6 induces ADCC and CDC in PDAC cells. (A) Flow cytometry-based assay showing ADCC against CFSE-labeled T3M4, Capan-2 and T3M4-MUC16KO cells via FVD (viability stain) CFSE double positive cells following incubation with peripheral blood mononuclear cells (PBMCs, effector cells) with or without the huAR9.6 antibody (B) Flow cytometry-based assay showing CDC against target T3M4, Capan-2 and T3M4-MUC16KO cells using Annexin-V/PI staining following incubation with human healthy donor derived serum with or without the huAR9.6 antibody.

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National Cancer Institute (NCI)

United States Department of Health and Human Services

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Otis Glebe Foundation

American Cancer Society (ACS)

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ARTICLE ABSTRACT

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.

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