Abstract 2978: Characterization of novel antibodies for understanding MUC16 cleavage and its pathological implications in cancer
Authors: Aithal Abhijit , Junker Wade , Kaur Sukhwinder , Das Srustidhar , Kshirsagar Prakash , Rachagani Satyanarayana , Mallya Kavita , Jain Maneesh , Batra Surinder K. 
Published: July 2016

Abstract
Introduction: MUC16 (CA125) is a type I transmembrane mucin that exhibits differential expression in multiple malignancies including ovarian, pancreatic and breast cancers. It was initially predicted to undergo cleavage in the penultimate and/or last sea urchin, enterokinase, and agrin (SEA) domain (among a total of 56) to generate circulating CA125. However, our recent studies demonstrated the presence of a unique cleavage site that involves 12 amino acid residues proximal to transmembrane segment. Upon ectopic overexpression, this cleaved intracellular C-terminal (C-ter) fragment promotes tumor metastasis, invasion and upregulates cancer stem cell inducing genes in cancer cells. However, physiological presence of C-ter cleavage and its role in cancer remains obscure since most available antibodies recognize the N-terminal tandem repeat domain of MUC16. The present study aims to develop novel antibodies against MUC16 C-ter fragment to dissect its role in tumor development and progression.
Methods: Monoclonal antibodies (mAbs) were generated by immunizing mice with purified C-ter (114 amino acid) domain of MUC16. MUC16 reactive hybridomas were screened by indirect ELISA. Positive hybridomas were isolated, cloned and the antibodies were purified from culture supernatant. The specificity of the newly generated anti-MUC16 antibodies was determined using immunoprecipitation, immunoblot, confocal, and FACS analysis. Finally, the reactivity of novel antibodies was evaluated on mouse and human tumor tissues by immunohistochemistry.
Results: Two mAbs (3H1 and 5E6) showed specific reactivity to recombinant MUC16 C-ter fragment in ELISA. Stringent checkpoints were included such as expression analyses in MUC16 expressing and non-expressing cell lines, histological reactivity in normal and MUC16 expressing cancer tissues and MUC16 C-ter over expression constructs to delineate the highly specific nature of these antibodies. Epitope mapping studies using MUC16 C-ter constructs transfected into HEK293T cells identified the epitope to most probably lie in a 61 amino acid region just proximal to transmembrane domain. Interestingly, in immunoblot and immunoprecipitation analysis, these mAbs identified a unique 20kDa cleavage product in some but not all MUC16 expressing cancer cell lines. This suggests the existence of a cell-context dependent cleavage mechanism for MUC16. Both mAbs preferentially recognize intracellular antigen as compared to cell surface exposed epitope. Further, these exhibited strong reactivity in pancreatic and ovarian cancer tissues in the same regions that stained positive for mAb CA125.
Conclusions: The novel mAbs exhibit unique specificity towards cleaved MUC16 C-ter in cell lysates and tissues and hence can be valuable reagents for studying the mechanisms and role of MUC16 cleavage in cancer cells.
Citation Format: Abhijit Aithal, Wade Junker, Sukhwinder Kaur, Srustidhar Das, Prakash Kshirsagar, Satyanarayana Rachagani, Kavita Mallya, Maneesh Jain, Surinder K. Batra. Characterization of novel antibodies for understanding MUC16 cleavage and its pathological implications in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2978.