Hepatitis C virus (HCV) is world-wide a major cause of liver related morbidity and mortality. No vaccine is available to prevent HCV infection. To design an effective vaccine, understanding immunity against HCV is necessary. The memory B cell repertoire was characterized from an intravenous drug user who spontaneously cleared HCV infection 25 years ago. CD27+IgG+ memory B cells were immortalized using BCL6 and Bcl-xL. These immortalized B cells were used to study antibody-mediated immunity against the HCV E1E2 glycoproteins. Five E1E2 broadly reactive antibodies were isolated: 3 antibodies showed potent neutralization of genotype 1 to 4 using HCV pseudotyped particles, whereas the other 2 antibodies neutralized genotype 1, 2 and 3 or 1 and 2 only. All antibodies recognized non-linear epitopes on E2. Finally, except for antibody AT12-011, which recognized an epitope consisting of antigenic domain C /AR2 and AR5, all other four antibodies recognized epitope II and domain B. These data show that a subject, who spontaneously cleared HCV infection 25 years ago, still has circulating memory B cells that are able to secrete broadly neutralizing antibodies. Presence of such memory B cells strengthens the argument for undertaking the development of an HCV vaccine.

This study was supported by the Virgo consortium, funded by the Dutch government, project number FES0908 and by the Netherlands Genomics Initiative (NGI) project number 050-060-452. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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BackgroundColorectal 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 an 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).MethodsThe study protocol was approved by the Medical Ethical Committee of the Academic Medical Centre, Amsterdam, The Netherlands (NL42718.018.12). AIMM’s BCL6 and Bcl-xL immortalization method1 was used to interrogate the human antibody repertoire. 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. A high-affinity variant of AT1636 (AT1636IYN) was sorted from the original AT1636, AID-expressing B-cell clone.2ResultsAntibodies that demonstrated differential binding to CRC cells were characterized and targets recognized by such antibodies were identified using immunoprecipitation and mass-spectrometry. One of the antibodies, AT1636, recognized a previously unidentified O-mannosylated 70kDa E-cadherin variant (ECV). Although the 70kDa ECV is found in all full-length E-cadherin expressing cells, tumor-specific binding of AT1636 is dependent on the 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.ConclusionsThe 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.Ethics ApprovalThe study protocol was approved by the Medical Ethical Committee of the Academic Medical Centre, Amsterdam, The Netherlands (NL42718.018.12)ReferencesM.J. Kwakkenbos, et al. Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming. Nature Medicine 2010;16:123–128.K. Wagner, et al. Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity. Proc Natl Acad Sci USA 2014; 111: 16820–16825.I.S.B. Larsen, et al. Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins. Proc Natl Acad Sci USA 2017;114:11163–11168.