C6: A Monoclonal Antibody Specific for a Fibronectin Epitope Situated at the Interface between the Oncofoetal Extra-Domain B and the Repeat III8

RESEARCH ARTICLE C6: A Monoclonal Antibody Specific for a Fibronectin Epitope Situated at the Interface between the Oncofoetal Extra-Domain B and the Repeat III8 Elisa Ventura1¤, Cinzia Cordazzo2, Rodolfo Quarto3, Luciano Zardi2*, Camillo Rosano4 1 Laboratory of Oncology, G. Gaslini Institute, Genova, Italy, 2 Sirius-biotech, c/o Advanced Biotechnology Center, Genova, Italy, 3 Laboratory of Stem Cells, University of Genoa and Laboratory of Regenerative Medicine, IRCCS AOU San Martino-IST, Genova, Italy, 4 Laboratory of Biopolymers and Proteomics, IRCCS AOU San Martino-IST, Genova, Italy ¤ Current address: Laboratory of Molecular Neuro-Oncology, Department of Neurology, University Hospital and University of Zurich, Zurich, Switzerland * OPEN ACCESS Citation: Ventura E, Cordazzo C, Quarto R, Zardi L, Rosano C (2016) C6: A Monoclonal Antibody Specific for a Fibronectin Epitope Situated at the Interface between the Oncofoetal Extra-Domain B and the Repeat III8. PLoS ONE 11(2): e0148103. doi:10.1371/journal.pone.0148103 Editor: Sompop Bencharit, University of North Carolina at Chapel Hill, UNITED STATES Received: September 6, 2015 Accepted: January 13, 2016 Published: February 11, 2016 Copyright: © 2016 Ventura et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its supporting information files. Funding: This study was partially supported by Sirius-biotech. The funder provided support in the form of salaries for authors (L.Z. and partially to C.C.) and for research materials but did not have any additional role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The specific roles of these authors are articulated in the author contribution sections. Abstract Background Fibronectin (FN) is a large multidomain molecule that is involved in many cellular processes. Different FN isoforms arise from alternative splicing of the pre-mRNA including, most notably, the FN isoform that contains the “extra-domain-B” (ED-B). The FN isoform containing ED-B (known as B-FN) is undetectable in healthy adult tissues but is present in large amounts in neoplastic and foetal tissues as well as on the blood vessels during angiogenesis. Thus, antibodies specific for B-FN can be useful for detecting and targeting neoplastic tissues in vivo. We previously characterised C6, a new monoclonal antibody specific for human B-FN and we suggested that it reacts with the B-C loop of the type III repeat 8 which is masked in FN isoforms lacking ED-B and that the insertion of ED-B in FN molecules unmasked it. Here we have now consolidated and refined the characterization of this B-FN specific antibody demonstrating that the epitope recognized by C6 also includes loop E-F of ED-B. Methodology We built the three dimensional model of the variable regions of the mAb C6 and of the FN fragment EDB-III8 and performed protein:protein docking simulation using the web server ClusPro2.0. To confirm the data obtained by protein:protein docking we generated mutant fragments of the recombinant FN fragment EDB-III8 and tested their reactivity with C6. Conclusion The monoclonal antibody C6 reacts with an epitope formed by the B-C loop of domain III8 and the E-F loop of ED-B. Both loops are required for an immunological reaction, thus this PLOS ONE | DOI:10.1371/journal.pone.0148103 February 11, 2016 1/9 FN mAb to the EDB-III8 Interface Competing Interests: Sirius-biotech commercialised the antibody C6. This does not alter the authors' adherence to all PLOS ONE policies on sharing data and materials. monoclonal is strictly specific for B-FN but the part of the epitope on III8 confers the human specificity. Introduction Fibronectin (FN) is a multi-domain molecule present in the extracellular matrix (ECM) and in body fluids. It is a dimer of two subunits of about 220/250kDa, linked at the C-termini by two disulfide bonds and each monomer consists of three types of repeating units. FN is involved in many cellular processes and different FN isoforms arise from the alternative splicing of its premRNA [1–2]. In particular, the FN isoform containing the extra-domain B (ED-B), a complete FN type III repeat formed by 91 amino acids, is expressed only during physiological or pathological tissue remodelling such as in embryogenesis, wound healing, in uterus and ovary during the female reproductive cycle, in tumorigenesis and in degenerative chronic inflammatory diseases. The ED-B primary structure is highly conserved in different species, having 100% homology in all mammals thus far tested and 96% homology with a similar domain in chicken. The FN isoform containing ED-B (B-FN) is undetectable in healthy adult tissues but its expression levels are highly increased in tumour tissues and it accumulates around neovasculature during angiogenesis. This makes it one of the oncologist’s best markers of angiogenesis and neoplastic tissues [3–8]. The demonstration that monoclonal antibodies to B-FN can be used to selectively deliver therapeutic substances to diseased tissues [9] prompted the generation of human recombinant antibodies for preclinical and clinical diagnostic and therapeutic purposes [10–13].The biological function(s) of B-FN are still unclear, however it has been suggested that B-FN increases vascular endothelial growth factor (VEGF) expression, endothelial proliferation and tube formation [14]. More recently Kraft et al [15] reported that B-FN enhances phagocytosis more than plasma FN and that this enhancements is mediated by the integrin alphaVbeta3. On the whole the biological activities are mediated by exposed loops located mainly at the inter-domain interface, therefore the insertion of ED-B within repeat III7 and III8 modifies the domain-domain interface and would be expected to lead to changes in biological activities [16]. We have previously described C6, a monoclonal antibody specific for human B-FN [17]. Using various recombinant FN fragments containing mutations we concluded that its epitope was located within the loop B-C of III8 and we speculated that, in FN isoforms lacking ED-B, this loop is masked [18]. Here, to better understand the interaction between human B-FN and C6, we performed protein:protein docking simulation of the three dimensional models of the scFv of the mAb C6 and of the FN recombinant fragment containing the type III domains B and 8. The results confirm the interaction with the loop B-C of domain III8 but also with the loop E-F of ED-B. Further experiments using a FN fragment with mutation on ED-B confirmed that its loop E-F is part of the epitope recognized by C6. Results and Discussion In immunohistochemistry experiments on human tissue, the mAb C6 behaves exactly as an anti (...truncated)