Internalization via Antennapedia protein transduction domain of an scFv antibody toward c-Myc protein.

We constructed a single-chain variable fragment miniantibody (G11-scFv) directed toward the transactivation domain of c-Myc, which is fused with the internalization domain Int of Antennapedia at its carboxyl terminus (a cargo-carrier construct). In ELISA experiments, an EC(50) for binding saturation was achieved at concentrations of G11-scFv-Int(-) of approximately 10(-8) M. Internalization of a fluoresceinated Fl-G11-scFv-Int(+) construct was observed in intact human cultured cells with confocal microscopy. After 5 h of incubation in medium containing 1 microM Fl-G11-scFv-Int(+) or Fl-G11-scFv-Int(-), fluorescence intensity was determined in individual cells, both for cytoplasmic and nuclear compartments: concentration levels of Fl-G11-scFv-Int(+), relative to the extracellular culture medium concentration, were 4-5 times higher in the cytoplasm, 7-8 times higher in the nucleus, and 10 times higher in the nucleoli. In the same experimental conditions, the Fl-G11-scFv-Int(-) construct was 3-4 times more concentrated outside of the cells than inside. Cell membranes kept their integrity after 5 h of incubation. The antiproliferative activity of our miniantibody was studied on HCT116 cells. Incubation with 4 microM G11-scFv-Int(+) for 4 days induced very significant statistical and biological growth inhibition, whereas Int alone was completely inactive. Miniantibodies capable of penetrating cell membranes dramatically broaden the potential for innovative therapeutic agents and attack of new targets.

Modulation of matrix adhesive responses of human neuroblastoma cells by neighboring sequences in the fibronectins.

Attachment and neurite extension have been measured when Platt or La-N1 human neuroblastoma cells respond to tissue culture substrata coated with a panel of complementary fragments from the individual chains of human plasma (pFN) or cellular fibronectins (cFN) purified from thermolysin digests. A 110-kD fragment (f110), which contains the Arg-Gly-Asp-Ser sequence (RGDS)-dependent cell-binding domain but no heparin-binding domains and whose sequences are shared in common by both the alpha- and beta-subunits of pFN, facilitated attachment of cells that approached the level observed with either intact pFN or the heparan sulfate-binding platelet factor-4 (PF4). This attachment on f110 was resistant to RGDS-containing peptide in the medium. Neurite outgrowth was also maximal on f110, and half of these neurites were also resistant to soluble RGDS peptide. Treatment of cells with glycosaminoglycan lyases failed to alter these responses on f110. Therefore, there is a second "cell-binding" domain in the sequences represented by f110 that is not RGDS- or heparan sulfate-dependent and that facilitates stable attachment and some neurite outgrowth; this domain appears to be conformation-dependent. Comparisons were also made between two larger fragments generated from the two subunits of pFN-f145 from the alpha-subunit and f155 from the beta-subunit--both of which contain the RGDS-dependent cell-binding domain and the COOH-terminal heparin-binding domain but which differ in the former's containing some IIICS sequence at its COOH terminus and the latter's having an additional type III homology unit. Heparin-binding fragments (with no RGDS activity) of f29 and f38, derived from f145 or f155 of pFN, respectively, and having the same differences in sequence, were also compared with f44 + 47 having the "extra domain" characteristic of cFN. Attachment on f145 was slightly sensitive to soluble RGDS peptide; attachment on f155 was much more sensitive. There were also differences in the percentage of cells with neurites on f145 vs. f155 but neurites on either fragment were completely sensitive to RGDS peptide. Mixing of f29, f38, or PF4 with f110 could not reconstitute the activities demonstrated in f145 or f155, demonstrating that covalently linked sequences are critical in modulating these responses. However, mixing of f44 + 47 from cFN with f110 from pFN increased the sensitivity to RGDS peptide.(ABSTRACT TRUNCATED AT 400 WORDS)

Fibronectin-mediated adhesion of fibroblasts: inhibition by dermatan sulfate proteoglycan and evidence for a cryptic glycosaminoglycan-binding domain.

Dermatan sulfate proteoglycans (DS-PGs) isolated from bovine articular cartilage have been examined for their effects on the adhesive responses of BALB/c 3T3 cells and bovine dermal fibroblasts on plasma fibronectin (pFN) and/or type I collagen matrices, and compared to the effects of the chondroitin sulfate/keratan sulfate proteoglycan monomers (CS/KS-PGs) from cartilage. DS-PGs inhibited the attachment and spreading of 3T3 cells on pFN-coated tissue culture substrata much more effectively than the cartilage CS/KS-PGs reported previously; in contrast, dermal fibroblasts were much less sensitive to either proteoglycan class unless they were pretreated with cycloheximide. Both cell types failed to adhere to substrata coated only with the proteoglycans; binding of the proteoglycans to various substrata has also been quantitated. While a strong inhibitory effect was obtained with the native intact DS-PGs, little inhibitory effect was obtained with isolated DS chains (liberated by alkaline-borohydride cleavage) or with core protein preparations (liberated by chondroitinase ABC digestion). In marked contrast, DS-PGs did not inhibit attachment or spreading responses of either 3T3 or dermal fibroblasts on type I collagen-coated substrata when the collagen was absorbed with pFN alone, DS-PGs alone, or the two in combination. These results support evidence for (a) collagen-dependent, fibronectin-independent mechanisms of adhesion of fibroblasts, and (b) different sites on the collagen fibrils where DS-PGs bind and where cell surface "receptors" for collagen bind. Experiments were developed to determine the mechanism(s) of inhibition. All evidence indicated that the mechanism using the intact pFN molecule involved the binding of the DS-PGs to the glycosaminoglycan (GAG)-binding sites of substratum-bound pFN, thereby inhibiting the interaction of the fibronectin with receptors on the cell surface. This was supported by affinity chromatography studies demonstrating that DS-PGs bind completely and effectively to pFN-Sepharose columns whereas only a subset of the cartilage CS/KS-PG binds weakly to these columns. In contrast, when a 120-kD chymotrypsin-generated cell-binding fragment of pFN (CBF which has no detectable GAG-binding activity as a soluble ligand) was tested in adhesion assays, DS-PGs inhibited 3T3 adherence on CBF more effectively than on intact pFN. A variety of experiments indicated that the mechanism of this inhibition also involved the binding of DS-PGs to only substratum-bound CBF due to the presence of a cryptic GAG-binding domain not observed in the soluble CBF.(ABSTRACT TRUNCATED AT 400 WORDS)

Transformed human cells release different fibronectin variants than do normal cells.

Fibronectin molecules are dimers composed of subunits whose primary structures may differ. This is due to alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA. Using two monoclonal antibodies specific for two different epitopes of domain 5 (high affinity for heparin), we have quantitatively analyzed the expression of the IIICS sequence in human fibronectins from different sources. The results demonstrated that the percentage of fibronectin subunits containing the IIICS is higher in fibronectins from tumor-derived or simian virus 40-transformed human cells than in fibronectins from human plasma or normal human fibroblasts. Furthermore, we observed that 45-65% of fibronectin subunits from transformed cells or normal embryonic fibroblasts are sialylated on the heparin-binding domain 5, whereas this occurs in only 24-28% of fibronectin subunits from normal adult fibroblasts. On the contrary, no sialylation was observed on domain 5 in fibronectin from human plasma.

A tumor-associated fibronectin isoform generated by alternative splicing of messenger RNA precursors.

Fibronectin (FN) represents the mixture of a number of structurally different molecules (isoforms) whose make-up varies depending on the FN sources. FN from cultured transformed human cells has a very different isoform composition with respect to its normal counterpart. In fact, SV-40-transformed WI-38VAI3 human fibroblasts produce high levels of a FN isoform (B-FN) which is very poorly expressed in their normal, WI-38, counterpart. We have recently demonstrated that the B-FN isoform derives from a differential splicing pattern of the FN primary transcript which leads, in transformed cells, to a high level expression of the exon ED-B (Zardi, L., B. Carnemolla, A. Siri, T. E. Petersen, G. Paolella, G. Sebastio, and F. E. Baralle. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:2337-2342). Here we report on the production and characterization of a monoclonal antibody (BC-1) which recognizes an epitope within the protein sequence coded for by the ED-B exon. This monoclonal antibody makes it possible to carry out immunohistochemical analysis of the distribution of the ED-B-containing FN isoform (B-FN) in human tissues. The results show that while in normal, adult, human tissues total FN has a widespread distribution, the B-FN isoform is restricted only to synovial cells, to some vessels and areas of the interstitium of the ovary, and to the myometrium. On the contrary, the B-FN isoform has a much greater expression in fetal and tumor tissues. These results demonstrate that, in vivo, different FN isoforms have a differential distribution and indicate that the B-FN isoform may play a role in ontogenesis and oncogenetic processes.

The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1.

Transforming growth factor-beta1 (TGFbeta1), a major promoter of myofibroblast differentiation, induces alpha-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes alpha-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFbeta1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of alpha-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se alpha-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFbeta1-triggered enhancement of alpha-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A-containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFbeta1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.

Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells.

Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing have offered an explanation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using these monoclonals, we have quantitatively analyzed the expression of the ED sequence in human fibronectin from different sources. The results demonstrated that, at the protein level, the ED segment is not expressed in plasma fibronectin and that, in fibronectin from the tissue culture medium of tumor-derived or simian virus-40-transformed human cells, the percentage of fibronectin molecules containing the ED segment is about 10 times higher than in fibronectin from normal human fibroblasts. These results suggest that in malignant cells the mechanisms that regulate the splicing of mRNA precursors are altered.

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.

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.

Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform.

The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.

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 x 10(8) 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 x 10(-7) M and specifically stains tumour blood vessels, as shown by immunohistochemical analysis on tumour sections of human and murine origin.

Targeted delivery of tissue factor to the ED-B domain of fibronectin, a marker of angiogenesis, mediates the infarction of solid tumors in mice.

The selective thrombosis of tumor blood vessels, leading to the starvation and subsequent death of tumor cells, is an attractive anticancer strategy. Here we report that a fusion protein, consisting of an antibody fragment specific for the oncofoetal ED-B domain of fibronectin fused to the extracellular domain of tissue factor, selectively targets tumor blood vessels in vivo. Furthermore, this fusion protein mediates the complete and selective infarction of three different types of solid tumors in mice. At the highest doses administered, complete tumor eradication was observed in 30% of the mice treated without apparent side effects. These results are of therapeutic relevance because 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 undetectable in normal vessels and tissues.

Concentration of fibronectin in plasma of tumor-bearing mice and synthesis by Ehrlich ascites tumor cells.

In the present paper we have studied: (a) the concentration of fibronectin (FN) in plasma and in ascitic fluid of mice at different times after inoculation of Ehrlich ascites tumor cells; (b) the ability of Ehrlich ascites cells to synthesize and release FN; and (c) the localization of FN in Ehrlich ascites cells by immunofluorescence microscopy. It was found that (a) 4 to 5 days after inoculation of the tumor, the plasma concentration of FN was significantly higher [1.7 +/- 0.07% (S.E.) of total plasma protein] than that in the normal control mice (0.8 +/- 0.035); (b) FN is present in the ascitic fluid in all phases of tumor growth; (c) Ehrlich ascites cells cultured in vitro synthesize and release large amounts of FN in the culture medium; and (d) only about 1 to 2% of the tumor cells show a very small amount of FN, and this is mostly in the area of cell-cell contact.

Increased binding affinity and valence of recombinant antibody fragments lead to improved targeting of tumoral angiogenesis.

The formation of new blood vessels (angiogenesis) is an important step in tumor progression. Molecules capable of selectively targeting markers of angiogenesis may offer opportunities for the in vivo imaging of aggressive tumors and for the delivery of toxic agents to the tumoral vasculature. Using antibody phage display libraries and combinatorial mutagenesis, we isolated single-chain Fv antibody fragments, which recognize with different affinities the same epitope of the ED-B domain of fibronectin, a marker of angiogenesis. Two single-chain Fv fragments, E1 and L19, with dissociation constants of 41 nM and 0.054 nM, respectively, were investigated for their ability to target F9 murine teratocarcinoma grafted s.c. in nude mice when injected i.v. in either monomeric or homodimeric form (Mr 27,000 and 54,000, respectively). Biodistribution studies, performed at two time points (4 h and 24 h) with radiolabeled samples, showed that the higher affinity antibody targets the tumor significantly better than the lower affinity one, in terms both of tumor:organ ratios and of the amounts of antibody delivered to the tumor. In particular, more than 20% of the injected dose of dimeric L19 accumulated per gram of tumor at 4 h; the tumor:organ ratios at 4 h and 24 h were in the (2.1-8.6):1 and (10.3-29.4):1 range, respectively. This study demonstrates that, although vasculature represents only a small fraction of the total tumor mass, anti-ED-B antibodies can selectively target tumors in vivo and that this process is particularly efficient if very high-affinity binders are used.

Adhesion of Kirsten-ras+ tumor-progressing and Kirsten-ras- revertant 3T3 cells on fibronectin proteolytic fragments.

Adhesion has been evaluated for tumor cell populations derived from Kirsten murine sarcoma virus (KiMSV)-transformed BALB/c 3T3 cells responding to substrata coated with intact plasma fibronectin (pFN), a family of related proteolytic fragments from pFN or cellular fibronectins (FNs), and the heparan sulfate-binding platelet factor-4 (PF4). Both early-passage KiMSV cells, harboring the viral Kirsten ras oncogene (v-Ki-ras+), and late-passage KiMSV cells, in which most cells have lost the oncogene (v-Ki-ras-), are compared with primary tumor and lung metastatic tumor cells after three routes of injection into nude mice; nontumorigenic v-Ki-ras- revertant cells have been cloned from the late-passage KiMSV population. Attachment of early-passage KiMSV, primary tumor, and lung metastatic tumor cells was optimal and resistant to soluble RGDS peptide in the medium on intact pFN, on fragment F-155 from pFN containing the RGDS cell-binding domain and the heparinII domain, and on PF4 but decreased for metastatic cells on F110 containing only the RGDS domain (and sensitive to RGDS peptide). Cytoplasmic spreading of early-passage KiMSV and all tumor cells was good to excellent in polygonal patterns on pFN and on F155, while most cells remained round on F110. Responses for KiMSV and tumor cells varied on different heparin-binding proteins; cells remained rounded or detached on F38 derived from pFN or on PF4 but spread effectively with long linear process extension on cellular FN-derived fragments F44 + 47 harboring the extra domaina sequence. That F44 + 47 may contain a new cell-binding site for v-Ki-ras+ cells was also indicated by resistance to bacterial heparitanase in cell responses on F44 + 47 but not on PF4 and extensive catabolism of proteoglycans in the substratum-attached material of these cells. v-Ki-ras- revertant cells, nontumorigenic in nude mice, have reacquired 3T3-like responses to proteolytic fragments, including much more effective spreading on PF4 or on F38 substrata, and have reverted in generating microfilament stress fibers on pFN, a competence lacking in all v-Ki-ras+ cells. These results indicate that (a) v-Ki-ras+ primary and metastatic tumor cells respond similarly to most proteolytic fragments of FNs harboring known binding domains, with a few exceptions; (b) v-Ki-ras gene expression correlates with a new cell surface receptor activity recognized by extra domaina-containing fragments from cellular FNs; and (c) loss of the viral oncogene to generate v-Ki-ras- revertant cells reverts their FN-mediated adhesion responses.

Transforming growth factor beta regulates the levels of different fibronectin isoforms in normal human cultured fibroblasts.

Fibronectin (FN) polymorphism is caused by alternative splicing patterns in at least three regions of the primary transcript of a single gene. Using a monoclonal antibody (Mab) specific for an FN segment (ED-A), that can be included or omitted from the molecule depending on the pattern of splicing, we have examined whether transforming growth factor beta (TGF-beta) and dexamethasone, which are both known to increase the level of total FN, regulate the levels of different FN isoforms. We found that, while dexamethasone does not significantly change the ratio between the total FN and the ED-A containing FN, TGF-beta preferentially increases the expression of the FN isoform containing the ED-A sequence.

Human amnion epithelial cells assemble tenascins and three fibronectin isoforms in the extracellular matrix.

Monoclonal antibodies (MAb) were used to show that cultured human amnion epithelial (HuA) cells produce tenascins (Tn) and isoforms of cellular fibronectin (cFn). Tn polypeptides of M(r) 280,000 and 190,000, assembled into extracellular matrix (ECM) but not secreted into the culture medium by HuA cells, were electrophoretically similar to those produced by human fibroblasts as revealed with domain-specific MAbs. The results suggested that most Fn produced by HuA cells contained the extradomain (ED) A and an oncofetal domain but only a minor fraction EDB. In immunofluorescence Tn and Fn were seen in different cytoplasmic granules upon monensin-induced intracellular accumulation. Tn appeared to be deposited in the ECM in colocalization with Fn but distinctly slower. The present results show that cultured normal human epithelial cells synthesize Tn and three isoforms of cFn and secrete them by using different cytoplasmic pathways.

The first untranslated exon of the human tenascin-C gene plays a regulatory role in gene transcription.

The transcription of the human tenascin-C (TN-C) gene is directed by a single promoter. Here we demonstrate, in transiently transfected cells, that two distinct regions of the untranslated 179 bp-long exon 1 play antagonistic roles in transcriptional regulation: bases from 1 to 20 strongly increase the transcription of the reporter gene CAT directed by the human TN-C gene promoter, while bases from 79 to 179 significantly reduce this activation.

Binding characteristics of complementary fibronectin fragments on artificial substrata.

Various properties have been evaluated for the binding to tissue culture substrata of proteolytic fragments of human plasma or cellular fibronectins containing complementary sequences from the individual and alternatively spliced chains, since related fragments are known to yield differing adhesive responses from cells. These studies utilize ELISA methods and a polyclonal antiserum directed to human pFN for direct measurement or an occupancy test utilizing anti-albumin. Very related fragments (with or without an extra type III homology unit or extra domaina or b) have significantly different properties in substratum binding and such differences provide a partial explanation for alteration of cellular adhesive responses on such fragments.

Transforming growth factor-beta regulates the splicing pattern of fibronectin messenger RNA precursor.

Fibronectin (FN) polymorphism is caused by alternative splicing patterns in at least three regions (ED-A, ED-B and IIICS) of the primary transcript of a single gene. Using monoclonal antibodies, we previously demonstrated that transforming growth factor-beta (TGF-beta) preferentially increases the accumulation of the FN isoforms containing the ED-A sequence in cultured normal human fibroblasts [Balza et al., (1988) FEBS Lett. 228, 42-44]. To determine the basis of this effect, we have examined through S1 nuclease analysis, the levels of ED-A- and ED-B-containing mRNAs in cultured normal human skin fibroblasts before and after TGF-beta treatment. These experiments have shown that TGF-beta increases the relative amount of m-RNA for ED-A- and ED-B-containing FN isoforms. These data demonstrate that a growth factor may regulate the splicing pattern of a pre-mRNA.