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

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.     

Alternative Splicing of the Angiogenesis Associated Extra-Domain B of Fibronectin Regulates the Accessibility of the B-C Loop of the Type III Repeat 8

Background

Fibronectin (FN) is a multi-domain molecule involved in many cellular processes, including tissue repair, embryogenesis, blood clotting, and cell migration/adhesion. The biological activities of FN are mediated by exposed loops located mainly at the interdomain interfaces that interact with various molecules such as, but not only, integrins. Different FN isoforms arise from the alternative splicing of the pre-mRNA. In malignancies, the splicing pattern of FN pre-mRNA is altered; in particular, the FN isoform containing the extra-domain B (ED-B), a complete FN type III repeat constituted by 91 residues, is undetectable in normal adult tissues, but exhibits a much greater expression in fetal and tumor tissues, and is accumulated around neovasculature during angiogenic processes, thus making ED-B one of the best markers and targets of angiogenesis. The functions of ED-B are still unclear; however, it has been postulated that the insertion of an extra-domain such as ED-B modifies the domain-domain interface and may unmask loops that are otherwise cryptic, thus giving FN new potential activities.

Methodology

We used the mAb C6, which reacts with ED-B containing FN, but not with ED-B-free FN and various recombinant FN fragments containing mutations, to precisely localize the epitopes recognized by the mAb C6.

Conclusion

We formally demonstrated that the inclusion of the alternatively spliced angiogenesis-associated ED-B leads to the unmasking of the FNIII 8 B-C loop that is cryptic in FN molecules lacking ED-B. Thus, the mAb C6, in addition to providing a new reagent for angiogenesis targeting, represents a new tool for the study of the potential biological functions of the B-C loop of the repeat FNIII 8 that is unmasked during angiogenic processes.     

Selective targeting and photocoagulation of ocular angiogenesis mediated by a phage-derived human antibody fragment.

Molecules that selectively target and occlude new blood vessels would be useful for diagnosis and treatment of pathologies associated with angiogenesis. We show that a phage-derived human antibody fragment (L19) with high affinity for the ED-B domain of fibronectin, a marker of angiogenesis, selectively localizes to newly formed blood vessels in a rabbit model of ocular angiogenesis. The L19 antibody, chemically coupled to a photosensitizer and irradiated with red light, mediates complete and selective occlusion of ocular neovasculature and promotes apoptosis of the corresponding endothelial cells. These results demonstrate that new ocular blood vessels can be distinguished immunochemically from preexisting ones and suggest that the targeted delivery of photosensitizers may be effective in treating angiogenesis-related pathologies.     

Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening

We describe here a method, based on iterative colony filter screening, for the rapid isolation of binding specificities from a large synthetic repertoire of human antibody fragments in single-chain Fv configuration. Escherichia coli cells, expressing the library of antibody fragments, are grown on a porous master filter, in contact with a second filter coated with the antigen, onto which antibodies secreted by the bacteria are able to diffuse. Detection of antigen binding on the second filter allows the recovery of a number of E.coli cells, including those expressing the binding specificity of interest, which can be submitted to a second round of screening for the isolation of specific monoclonal antibodies. We tested the methodology using as antigen the ED-B domain of fibronectin, a marker of angiogenesis. From an antibody library of 7 × 10⁸ clones, we recovered a number of specifically-binding antibodies of different aminoacid sequence. The antibody clone showing the strongest enzyme-linked immunosorbent assay signal (ME4C) was further characterised. Its epitope on the ED-B domain was mapped using the SPOT synthesis method, which uses a set of decapeptides spanning the antigen sequence synthesised and anchored on cellulose. ME4C binds to the ED-B domain with a dissociation constant K_d = 1 × 10^–7 M and specifically stains tumour blood vessels, as shown by immunohistochemical analysis on tumour sections of human and murine origin.     

Endothelins: regulators of extracellular matrix protein production in diabetes

Fibronectin (FN), a key extracellular matrix protein, is upregulated in target organs of diabetic angiopathy and in cultured cells exposed to high levels of glucose. FN has also been reported to undergo alternative splicing to produce the extra domain-B (ED-B) containing isoform, which is exclusively expressed during embryogenesis, tissue repair, and tumoral angiogenesis. The present study was aimed at elucidating the role and mechanism of endothelins (ETs) in FN and ED-B FN expression in diabetes. We investigated vitreous samples for ED-B FN expression from patients undergoing vitrectomy for proliferative diabetic retinopathy. Our results show increased FN and ED-B FN expression in the vitreous of diabetic patients in association with augmented ET-1. Using an antibody specific to the ED-B segment of FN, we show an increase in serum ED-B FN levels in patients with diabetic retinopathy and nephropathy. We further examined retinal tissues, as well as renal and cardiac tissues, from streptozotocin-induced diabetic rats. Diabetes increased FN and ED-B FN in all three organs, which was prevented by ET antagonist bosentan. To provide insight into the mechanism of glucose-induced and ET-mediated ED-B FN upregulation, we assayed endothelial cells (ECs). Inhibition of mitogen-activated protein kinase with pharmacological inhibitors and protein kinase B with dominant negative transfections prevented glucose- and ET-1-mediated FN and ED-B FN expression. Furthermore, treatment of cells exposed to high levels of glucose with ET antagonist prevented the activation of all signaling pathways studied and normalized glucose-induced ED-B FN expression. We then determined the functional significance of ED-B in ECs and show that ED-B FN is involved in vascular endothelial growth factor expression and cellular proliferation. These studies show that glucose-induced and ET-mediated FN and ED-B FN expressions involve complex interplays between signaling pathways and that ET may represent an ideal target for therapy in chronic diabetic complications.     

Expression of fibronectin variants in vascular and visceral smooth muscle cells in development

Monoclonal antibodies recognizing extra domain A (ED-A) and extra domain B (ED-B) fibronectin (FN) sequences were used to characterize FN variants expressed in human vascular smooth muscle cells (SMC) during fetal and postnatal development and to compare spectrum of FN variants produced by vascular and visceral SMC. In 8- to 12-week-old fetuses both ED-A-containing FN (A-FN) and ED-B-containing FN (B-FN) were found in all smooth muscles studied--aorta, esophagus, stomach, and jejunum. By 20-25 weeks of gestation relative amounts of both A-FN and B-FN were reduced significantly in the aortic media (fivefold for A-FN and twofold for B-FN), while in visceral SMC only B-FN content was decreased. All the adult visceral smooth muscles examined contained A-FN rather than B-FN. Therefore, the cells from adult aortic media appear to be the only SMC so far known to produce FN that contains neither ED-A nor ED-B. Moreover, the data obtained show that, unlike other cells, medial SMC are embedded in vivo in the extracellular matrix that contains FN lacking both ED-A and ED-B. SMC from the minor intimal thickenings in the human child aorta as well as those from the atherosclerotic plaques produce A-FN rather than B-FN. We conclude that (1) vascular SMC change the spectrum of produced FN variants at least twice--during prenatal development between 12 and 20 weeks of gestation, and during the postnatal period, when they are recruited into the intimal cell population; (2) the production of FN variants in visceral SMC is also developmentally regulated; (3) all visceral SMC unlike the cells from adult aortic media produce A-FN; (4) the presence of ED-A and ED-B sequences in the FN molecule is not necessary for the extracellular matrix assembly in vivo.     

Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon.

Purification and amino acid sequence analysis of a proteolytic fragment of fibronectin (FN) from transformed human cells demonstrated that a high percentage of these FN molecules contains an extra amino acid sequence which is present only in a very low percentage of FN molecules from normal fibroblasts and is undetectable in plasma FN. This new amino acid sequence introduces into the FN molecule a site very sensitive to a number of proteolytic enzymes. By analyzing the cellular mRNA and genomic clones, we have demonstrated that this sequence derives from a differential splicing pattern of the FN mRNA precursors, which leads in transformed cells to a high-level expression of an extra type III homology repeat (ED-B) coded for by a previously unobserved exon. Here we also report the complete sequence of this new exon. These results demonstrate that in malignant cells the mechanisms regulating the splicing of FN mRNA precursors are altered.     

The inclusion of the type III repeat ED-B in the fibronectin molecule generates conformational modifications that unmask a cryptic sequence.

We have previously reported an anti-fibronectin monoclonal antibody (mAb) (BC-1) which reacts with an ED-B-containing beta-galactosidase-fibronectin fusion protein but not with an identical beta-galactosidase-fibronectin fusion protein in which the ED-B sequence is omitted. In further experiments aimed at localizing more precisely the epitope recognized by this mAb, we demonstrate that 1) the mAb BC-1 is indeed specific for ED-B-containing fibronectin (FN) molecules even though the epitope recognized by this mAb is localized on the type III homology repeat 7 (the one which precedes the ED-B sequence) and 2) in fibronectin molecules lacking the ED-B sequence, this epitope is masked. We further demonstrate that, to mask the epitope recognized by the mAb BC-1, the presence of at least half of the FN type III homology repeat 9 is necessary. We also report the production of the mAb IST-6 which recognizes only FN molecules in which the ED-B sequence is lacking. These data clearly demonstrate that the presence of the ED-B sequence within FN molecules generates conformational modification in the central part of the molecules that unmasks previously cryptic sequences and masks others.     

Enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature

Interleukin-12 (IL-12) is a heterodimeric cytokine with potent immunostimulatory activity and anti-angiogenic properties. Its clinical applications are limited, however, by severe side-effects. Here we report that an IL-12 fusion protein, consisting of IL-12 fused to a human antibody fragment specific to the oncofetal ED-B domain of fibronectin, markedly enhances the antitumor activity of this cytokine, as demonstrated in a mouse lung-metastasis model and in two models of mice bearing different aggressive murine tumors. The residual small tumor masses seen in the treated mice were infiltrated with lymphocytes, macrophages, and natural killer cells and had elevated interferon gamma (IFN-gamma). These results are of therapeutic relevance as the ED-B domain of fibronectin, a naturally occurring marker of angiogenesis identical in mouse and man, is expressed in the majority of aggressive solid tumors but is not detectable in normal vessels and tissues.     

Teaching an old scaffold new tricks: monobodies constructed using alternative surfaces of the FN3 scaffold

The fibronectin type III domain (FN3) has become one of the most widely used non-antibody scaffolds for generating new binding proteins. Because of its structural homology to the immunoglobulin domain, combinatorial libraries of FN3 designed to date have primarily focused on introducing amino acid diversity into three loops that are equivalent to antibody complementarity-determining regions. Here, we report an FN3 library that utilizes alternative positions for presenting amino acid diversity. We diversified positions on a β-sheet and surface loops that together form a concave surface. The new library produced binding proteins (termed "monobodies") to multiple target proteins, generally with similar efficacy as the original, loop-focused library. The crystal structure of a monobody generated from the new library in complex with its target, the Abl SH2 domain, revealed that a concave surface of the monobody, as intended in our design, bound to a convex surface of the target with the interface area being among the largest of published structures of monobody-target complexes. This mode of interaction differs from a common binding mode for single-domain antibodies and antibody mimics in which recognition loops recognize clefts in targets. Together, this work illustrates the utilization of different surfaces of a single immunoglobulin-like scaffold to generate binding proteins with distinct characteristics.     

Production of functionalized single-chain Fv antibody fragments binding to the ED-B domain of the B-isoform of fibronectin in Pichia pastoris

The Pichia pastoris expression system was used to produce functionalized single-chain antibody fragments (scFv) directed against the ED-B domain of the B-fibronectin (B-Fn) isoform which was found to be present only in newly formed blood vessels during tumor angiogenesis. Therefore, scFv antibody fragments recognizing the ED-B domain are potential markers for angiogenesis. We constructed four functionalized scFv antibody fragments for direct labeling with radioactive molecules or toxins or for attachment to liposomes serving as carriers for cytotoxic or antiangiogenic compounds. The C-termini of the scFv antibody fragments contain 1-3 cysteine residues that are separated by a hydrophilic linker (GGSSGGSSGS) from the binding domain and are accessible for site-specific functionalization with thiol-reactive reagents. Plasmid expression, culture conditions, and purification were optimized in 1-L cultures. The scFv antibody fragments were purified by anion exchange chromatography. The yields were 5-20 mg/L culture medium. The large-scale production of one scFv antibody fragment in a 3.7-L fermenter gave a yield of 60 mg. The reactivity of the cyteines was demonstrated by labeling with the thiol-reactive fluorescent dye ABD-F. The four scFv antibody fragments bound specifically to ED-B-modified Sepharose and binding was further confirmed by immunofluorescence on cell cultures using ED-B-positive human Caco-2 tumor cells. Furthermore, we could demonstrate specific binding of scFv-modified liposomes to ED-B-positive tumor cells. Our results indicate that the P. pastoris expression system is useful for the large-scale production of cysteine-functionalized alpha-ED-B scFv antibody fragments.     

Detailed mapping of the antigenicity of the surface unit glycoprotein of equine infectious anemia virus by using synthetic peptide strategies.

We describe here a detailed analysis of the antigenic determinants of the surface unit glycoprotein (gp90) of equine infectious anemia virus (EIAV), using a comprehensive panel of synthetic peptides in enzyme-linked immunosorbent assays with immune serum from naturally and experimentally infected horses and with a panel of gp90-specific neutralizing and nonneutralizing monoclonal antibodies. The results of these studies identify immunoreactive segments throughout the conserved and variable domains of gp90 but localize immunodominant (100% reactivity) determinants to the amino and carboxyl termini of the glycoprotein molecule. Analysis of peptide reactivities with longitudinal serum samples taken from experimentally infected ponies revealed that antibody responses to conserved B-cell determinants appeared earlier and at higher titers than do antibodies specific for determinants contained in the variable domain of gp90. These observations suggest an evolution of antibody responses in EIAV-infected ponies that may correspond to the establishment of immunological control of virus replication and disease routinely observed in EIAV infections. In addition, the mapping of monoclonal antibody epitopes to peptides of 9 to 12 amino acids demonstrated that all of the neutralizing epitopes are located in the variable domain of gp90. The arrangement of neutralizing epitopes and critical structural considerations suggest that EIAV gp90 contains a principal neutralizing domain similar to the V3 loop of human immunodeficiency virus type 1. These antigenic analyses provide an important foundation for further analyzing the protective immune response generated during persistent EIAV infections and also provide potential peptide substrates for diagnostic assays and for vaccine strategies.     

Localization of an immunodominant domain on baculovirus-produced parvovirus B19 capsids: correlation to a major surface region on the native virus particle.

An immunodominant region on baculovirus-produced parvovirus B19 VP2 capsids was localized between amino acids 259 and 426 by mapping the binding sites of a panel of monoclonal antibodies which recognize determinants on the particles. The binding sites of three monoclonal antibodies were fine-mapped within this antigenic domain. Six VP2-specific monoclonal antibodies recognized determinants common to both the empty capsids and native parvovirus. The defined antigenic region is most probably exposed on the native B19 virion and corresponds to part of the threefold spike on the surface of canine parvovirus particles.     

Chemotaxis of 3T3 and SV3T3 cells to fibronectin is mediated through the cell-attachment site in fibronectin and a fibronectin cell surface receptor

Fibronectin (FN) is a multidomain extracellular matrix protein that induces attachment and chemotactic migration of fibroblastic cells. In this study we analyzed the molecular determinants involved in the FN-induced chemotactic migration of normal and SV40-transformed 3T3 cells. Two different monoclonal antibodies to the cell-binding site of FN blocked chemotaxis to a 140-kD FN fragment (Ca 140) containing the cell-binding domain. A monoclonal antibody to a determinant distant from the cell-binding site did not affect chemotaxis. A synthetic tetrapeptide, RGDS, which represents the major cell-attachment sequence, was able to compete with FN and the Ca 140 fragment in chemotaxis assays, but this peptide itself had no significant chemotactic activity. A larger peptide encompassing this sequence, GRGDSP, was chemotactic, while the peptide GRGESP, where a glutamic acid residue was substituted for aspartic acid, was inactive. Chemotactic migration could be prevented in a dose-dependent manner by a rabbit polyclonal antiserum to a 140-kD cell surface FN receptor. This antibody was more effective on normal than on transformed 3T3 cells. Neither the anti-FN receptor antiserum nor a monoclonal antibody to the cell-binding site of FN blocked migration induced by another potent chemoattractant, platelet-derived growth factor. These data indicate that FN-induced chemotaxis of 3T3 and SV3T3 cells is mediated via the RGDS cell-attachment site of FN and the 140-kD cell surface FN receptor. The interaction is specific and can be altered by transformation.     

Fibronectin Isoform Distribution in the Mouse II. Differential Distribution of the Alternatively Spliced EIIIB,EIIIA, and V Segments in the Adult Mouse

The alternatively spliced EIIIB, EIIIA. and V segments of fibronectin (FN) show widespread codistribution in the mouse embryo, suggesting that EIIIB+, EIIIA+, and V+ isoforms serve to facilitate morphogenesis and organogenesis (Peters, JH, and Hynes, RO, 1996, this issue). To gain further clues to functions of these segments, we have used segment-specific anti-FN antibodies to perform immunofluorescence microscopy on tissue sections obtained from mice aged 9 to 15 weeks. Staining for each of the three spliced segments, relative to that for the total FN pool, was reduced in the adult as compared with the embryo. Anti-V antibodies produced patterns which were most similar to those obtained with anti-total FN antibodies, localizing to basement membranes, connective tissues subjacent to epithelia, walls of blood vessels, and cartilage. Anti-EIIIA antibodies produced the next most widespread pattern. which included prominent staining of the walls of blood vessels of all sizes, the lung inter-stitium. and smooth muscle associated with the gastrointestinal (GI), genitourinary (GU), and respiratory tracts. Although anti-EIIIB antibodies produced the faintest and most restricted pattern of staining, EIIIB+ FN could be detected in the walls of some smaller blood vessels, smooth muscle of the GI, GU, and respiratory tracts, as well as within cartilaginous structures, and eye. There were quantitative and/or qualitative differences in the staining patterns produced by the three segment-specific antibodies in a variety of tissues, including liver, cartilage, synovium, cornea, muscle, peripheral nerve, and lymph node. These findings suggest that each of the spliced segments of the FN molecule may occupy unique physical or functional positions within the extracellular matrix of the adult mouse.