American Association for Cancer Research
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Supplementary Figure S6 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

Fig. S6. huAR9.6 reduces tumor burden in models of PDAC. (A) Schematic of subcutaneous PDAC model using T3M4 cells implanted in athymic nude mice (n=5). On day 9 post implantation, mice were randomized to two treatment groups: vehicle (PBS) and huAR9.6 (500 µg/25 g bodyweight, i.p. every 72 h) for 4 doses. At the experimental endpoint (day 19), mice were euthanized. (B) Hematoxylin and eosin (H&E) staining, Ki67 staining and CD31 staining of formalin-fixed paraffin embedded (FFPE) subcutaneous tumor tissues (n=5) treated with vehicle and huAR9.6. Scale bar 100 µm. (C) Schematic of orthotopic PDAC model using T3M4 cells implanted in athymic nude mice. On day 18 post implantation, mice were randomized to three treatment groups (n=7 per group): vehicle (PBS), isotype control huIgG and huAR9.6 (500 µg/25 g bodyweight, i.p.) for 4 doses. At the experimental endpoint (day 28), mice were euthanized. (D) Differences in tumor size from the three treatment groups. (E) Schematic of PDAC patient-derived organoid (PDO) development and treatment (n=3) with huAR9.6 (40 µg/mL for 24 h) and isotype control huIgG and subjected to further studies with cell proliferation and signaling. (F) Immunofluorescent staining for MUC16 with huAR9.6 on primary human PDAC tissue and matched PDO. Scale bar 50 µm.


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Otis Glebe Foundation

American Cancer Society (ACS)



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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