journal contribution
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. S9. Evaluation of huAR9.6’s potential toxicity. Liver enzyme profile showing (A) ALT and (B) AST measured in vehicle control (PBS), huIgG and huAR9.6 (500 µg/25 g bodyweight, i.p. for 4 doses) treated orthotopic PDAC tumor-bearing mice (n=5). (C) Representative H&E staining of liver, lung, spleen, and kidney collected from the vehicle control (PBS), huIgG and huAR9.6-treated orthotopic PDAC tumor-bearing mice (n=7) on day 28. (D) Schematic of a single-dose toxicity model in athymic nude mice (n=6). Blood collection was done on day 0 (pre-treatment), after which mice were treated with a single dose of vehicle (PBS) or huAR9.6 (1 mg/25 g bodyweight, i.p.), followed by blood collection on days 3 and 14. Body weight measurements were done daily. At the experimental endpoint (day 14), mice were euthanized. (E) Body weight measurements in grams (g) for mice treated with single-dose vehicle (PBS) or huAR9.6 (1 mg/mL). Day 0 serves as the pre-treatment timepoint. Liver enzyme profile showing (F) ALT and (G) AST measured in serum at pre-treated, 3- and 14-days post treatment timepoints. (H) Representative H&E staining of pancreas, liver, lung, spleen, and kidney collected from mice 14 days after single-dose treatment with vehicle or huAR9.6 (1 mg/mL).
Funding
National Cancer Institute (NCI)
United States Department of Health and Human Services
Find out more...Otis Glebe Foundation
American Cancer Society (ACS)
History
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.
