Abstract 3829: Generation and characterization of novel antibodies against the β subunit of MUC4

Abstract

Abstract Mucins are high molecular weight glycoproteins that are primarily expressed by the apical surfaces of respiratory, gastrointestinal and reproductive tracts. They are involved in protection, growth and repair of epithelial surfaces. Apart from its protective function, mucins also regulate diverse functions in normal cells and have a crucial role in oncogenesis. MUC4 is a type-1 transmembrane mucin, which is overexpressed in several cancers and has been implicated in oncogenic transformation, proliferation, invasion, inhibition of apoptosis, and chemoresistance properties associated with these cancer cells. It is a 930 kD glycoprotein, consisting of a large high molecular weight subunit, MUC4α, containing the identical 16 amino acid tandem repeat region and MUC4β, a membrane-bound subunit, containing three EGF (Epidermal Growth Factor)-like domains. The latter subunit is responsible for the oncogenic functions of this molecule and can potentially be targeted. The monoclonal antibody 8G7 has been used extensively to study MUC4 expression. Since 8G7 binds an epitope in the tandem repeat (TR) region on the MUC4α subunit it cannot detect the cleaved cell surface associated form of MUC4 or isoforms that lack the TR region or heavily glycosylated TR regions. The MUC4β subunit however, is attached to the surface of the cell making it an ideal candidate for studying and targeting MUC4. The aim of this study is to generate and characterize monoclonal antibodies (mAbs) against the β subunit of MUC4. Thirty-two mAbs against MUC4β subunit were initially isolated using ELISA screening. Flow cytometry was used to test high affinity binders in MUC4 transfected cells. It was then tested whether these mAbs were able to immunoprecipitate transfected MUC4 from detergent extracted lysates. Finally, four high affinity mAbs (E9 (IgG2b), 6E8 (IgG2b), 1C7 (IgG1), and 3B4 (IgA)) were selected from these analyses. All the four mAbs immunoprecipitated full length MUC4 from native cancer cell lines. All the four mAbs bound to native human MUC4 expressed on cancer cell surface of different cell lines albeit with varying affinities. 3B4 recognized a conformational epitope and exhibited cell surface binding. The mAb 3B4 was internalized into MUC4 expressing cells in a concentration time-dependent manner. 6E8 showed partial internalization while E9 mAb did not show any internalization. Both 3B4 and 8G7 were able to cap MUC4 at the cell surface of cancer cell lines. Our results suggested that these antibodies can potentially block MUC4 function. The mAbs, 3B4, 1C7 and 6E8 inhibited the growth of MUC4 expressing cells in MTT and colony forming assays. Studies are currently underway to evaluate the utility of these novel MUC4β-domain specific mAbs to target MUC4-expressing tumors in vivo. Overall these mAbs can be used as reagents to study MUC4 cleavage and can also be developed into MUC4 specific diagnostic and therapeutic agents. Citation Format: Catherine Orzechowski, Abhijit Aithal, Wade Junker, Prakash Kshirsagar, Surinder Batra, Maneesh Jain. Generation and characterization of novel antibodies against the β subunit of MUC4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3829.