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

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 monoclonal is strictly specific for B-FN but the part of the epitope on III8 confers the human specificity.

Fig 1. Immunohistochemistry experiments using the mAb C6 on cryostat sections.

A) Normal human lung; B) human lung adenocarcinoma; C) Normal human brain; D) Human mesothelioma; E) Human glioblastoma; F) Murine teratocarcinoma. Immunohistochemistry experiments using the recombinant antibody CGS1 on cryostat sections of human glioblastoma (G) and murine teratocarcinoma (H). CGS1 reacts directly with the ED-B (10) and thus, contrary of C6, it also reacts with murine tumours. C6 reacts, in an identical manner of CGS1, with normal and cancer human tissues but not with murine tumours (Modified from ref.[17] under CC-BY license, with permission f from John Wiley and Sons, original copyright 2009.).

Fig 2. Structures of FN and of the scFv C6.

A) Model of the domain structure of a FN subunit. The three different types of repeats, the three sites of alternative splicing (ED-A, ED-B, IIICS) and the specificity of the mAb C6 for the interface between type III repeats B and 8 are shown. B) Sequence of the scFv C6; the linker between the VL and the VH is in red; the CDRs are framed. Amino acids involved in the interaction with C6 are in bold. C) Sequence of the FN type III repeats numbers 7 (blue), B (black) and 8 (red); the various loops between the beta sheets structures are framed. The amino acids involved in the interaction with C6 are in bold. Sequence from http://www.ncbi.nlm.gov.nuccore/47132556.

Fig 3. Interaction between C6 and the FN recombinant fragment formed by the type III repeats B and 8.

A) Schematic representation of the main interactions between the two proteins. The type III domains B and 8 are drawn as green ribbons, the scFv C6 is orange. Residues involved in the binding are reported as sticks, hydrogen bonds between amino acids are shown as dotted black lines. B) Residues of the scFv and of the type III repeats B and 8 that interact. The distances are displayed. C) Representation of the interaction between the type III domains B-8 (green surface) and the scFv C6 (orange surface).

Fig 4. Reactivity of the mAb C6 with FN recombinant fragments.

A) Different FN recombinant fragments tested with the mAb C6. B) Reactivity in ELISA of various concentrations of the mAb C6 with the FN recombinant fragment containing the type III repeats B and 8; it fragment B-8 with the mutation Glu1329Ala and fragment B-8 with the mutation Asp1385Glu.