Abstract 5163: A colon cancer survivor-derived antibody recognizes a previously unidentified truncated, O-mannosylated 70kDa variant of E-cadherin
Authors: Kedde Martijn , Beaumont Tim , Merat Sabrina J. , Kwakkenbos Mark J. , Bartels Lina , Berg Dorien van de , Wagner Koen , Bakker Arjen Q. , Maijoor Kelly , Böhne Martino , Bru Camille , Kattler Veronika , Eenennaam Hans van , Roos Victorine H. , Kallenberg Frank G.J. , Medema Jan Paul , Hensbergen Paul J. , van Helden Pauline , Dekker Evelien , Spits Hergen 
Published: September 2020

Abstract
INTRODUCTION Colorectal cancer (CRC) associated with Lynch syndrome is characterized by an abundance of infiltrating lymphocytes. To study whether tumor-specific antibodies with therapeutic potential can be isolated from these patients, the B-cell repertoire from a patient with Lynch syndrome who recovered from a stage IV colon carcinoma was screened. Here we describe a novel human antibody, AT1636 that recognizes a previously unidentified O-mannosylated 70kDa form of E-cadherin. The intercellular interactions by E-cadherin on tumor cells have for long been recognized as protective in cancer metastasis, and deregulation of E-cadherin is a hallmark for epithelial-mesenchymal transition (EMT).
METHODS AIMM's BCL6 and Bcl-xL immortalization method[1] was used to interrogate the human antibody repertoire against targets on colon cancer cells. From a carrier of a pathogenic gene variant in the MSH6 gene diagnosed with stage IV CRC and liver metastasis that had been treated with avastin, capecitabine and oxaliplatin, peripheral-blood memory B cells were obtained 9 years after last treatment. Antibodies-containing supernatant of cultured B-cells were screened for binding to 3 different CRC cell lines (DLD1, LS174T and COLO205) and absence of binding to fibroblast by flow cytometry. High-affinity variants of AT1636 (AT1636IYN) were sorted from the AID-expressing immortalized B-cells clone[2].
RESULTS Patient derived antibodies that demonstrated differential binding to CRC cells were further characterized. Targets recognized by such antibodies were identified using immunoprecipitation and mass-spectrometry. AT1636 binds to a previously unidentified single O-mannosylated 70kDa E-cadherin variant (ECV). Although the 70 kDa ECV is found in all cells that express full length E-cadherin, tumor-specific binding of AT1636 is dependent on the single O-mannosylation pattern in the antibody epitope on ECV. Using shRNA knock-down AT1636 binding was shown to depend on the transmembrane O-mannosyltransferase targeting cadherins 3 (TMTC3)[3]. In accordance, coexpression of TMTC3 and E-cadherin in tumor cells is predictive for AT1636 binding. In addition, we observed that (over)expression of ECV results in a strong de-adhesive, EMT-like phenotype. Although AT1636 by itself is not able to induce ADCC, the CD3-bispecific antibody (single-chain UCHT1) AT1636 format specifically killed CRC cell lines.
CONCLUSION The AT1636 antibody retrieved from a patient with Lynch syndrome binds a previous unidentified cancer-specific O-mannosylated 70kDa form of E-cadherin. This variant might play a role in tumor-cell invasion and metastasis. More importantly, we provide a rationale to advance AT1636 based therapeutics for treatment of CRC.
references
1) Kwakkenbos et al. Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming. Nature Medicine 2010
2) Wagner et al. Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity. PNAS 2014
3) Larsen et al. Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins. PNAS 2017
Citation Format: Martijn Kedde, Tim Beaumont, Sabrina J. Merat, Mark J. Kwakkenbos, Lina Bartels, Dorien van de Berg, Koen Wagner, Arjen Q. Bakker, Kelly Maijoor, Martino Böhne, Camille Bru, Veronika Kattler, Hans van Eenennaam, Victorine H. Roos, Frank G.J. Kallenberg, Jan Paul Medema, Paul J. Hensbergen, Pauline van Helden, Evelien Dekker, Hergen Spits. A colon cancer survivor-derived antibody recognizes a previously unidentified truncated, O-mannosylated 70kDa variant of E-cadherin [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5163.