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.

Sequence specific peptidomimetic molecules inhibitors of a protein-protein interaction at the helix 1 level of c-Myc.

Our work is focused in the broad area of strategies and efforts to inhibit protein-protein interactions. The possible strategies in this field are definitely much more varied than in the case of ATP-pocket inhibitors. In our previous work (10), we reported that a retro-inverso (RI) form of Helix1 (H1) of c-Myc, linked to an RI-internalization sequence arising from the third alpha-helix of Antennapedia (Int) was endowed with an antiproliferative and proapoptotic activity toward the cancer cell lines MCF-7 and HCT-116. The activity apparently was dependent upon the presence of the Myc motif. In this work, by ala-scan mapping of the H1 portion of our molecules with D-aa, we found two amino acids necessary for antiproliferative activity: D-Lys in 4 and D-Arg in 5 (numbers refer to L-forms). In the natural hetero-dimer, these two side chains project to the outside of the four alpha-helix bundle. Moreover, we were able to obtain three peptides more active than the original lead. They strongly reduced cell proliferation and survival (RI-Int-VV-H1-E2A,S6A,F8A; RI-Int-VV-H1-S6A,F8A,R11A; RI-Int-VV-H1-S6A,F8A,Q13A): after 8 days at 10 muM total cell number was approximately 1% of the number of cells initially seeded. In these more potent molecules, the ablated side chains project to the inside in the corresponding natural four alpha-helix bundle. In the present work, we also investigated the behavior of our molecules at the biochemical level. Using both a circular dichroism (CD) and a fluorescence anisotropy approach, we noted that side chains projecting at the interior of the four alpha-helix bundle are needed for inducing the partial unfolding of Myc-H2, without an opening of the leucine zipper. Side chains projecting at the outside are not required for this biochemical effect. However, antiproliferative activity had the opposite requirements: side chains projecting at the outside of the bundle were essential, and, on the contrary, ablation of one side chain at a time projecting at the inside increased rather than decreased biological activity. We conclude that our active molecules probably interfere at the level of a protein-protein interaction between Myc-Max and a third protein of the transcription complex. Finally, CD and nuclear magnetic resonance (NMR) data, plus dynamic simulations, suggest a prevalent random coil conformation of the H1 portion of our molecules, at least in diluted solutions. The introduction of a kink (substitution with proline in positions 5 or 7) led to an important reduction of biological activity. We have also synthesized a longer peptido-mimetic molecule (RI-Int-H1-S6A,F8A-loop-H2) with the intent of obtaining a wider zone of interaction and a stronger interference at the level of the higher-order structure (enhanceosome). RI-Int-H1-S6A,F8A-loop-H2 was less active rather than more active in respect to RI-Int-VV-H1-S6A,F8A, apparently because it has a clear bent to form a beta-sheet (CD and NMR data).

Identification of a glioblastoma-associated tenascin-C isoform by a high affinity recombinant antibody.

Tenascin-C exists in several polymorphic isoforms due to alternative splicing of nine fibronectin-like type III repeats. Large Tenascin-C isoforms are present in almost all normal adult tissues but are upregulated in fetal, regenerating, and neoplastic tissues. Here, we report a human antibody fragment, TN11, derived from a phage library with high affinity for the spliced repeat C and demonstrate that this repeat is undetectable in normal adult tissues, barely detectable or undetectable in breast, lung and gastric carcinomas, meningioma, and low grade astrocytoma, but extremely abundant in high grade astrocytoma (grade III and glioblastoma), especially around vascular structures and proliferating cells. The antibody appears to have potential for development of a therapeutic agent for patients with high grade astrocytoma.

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.

Fibronectin type III5 repeat contains a novel cell adhesion sequence, KLDAPT, which binds activated alpha4beta1 and alpha4beta7 integrins.

The region of fibronectin encompassing type III repeats 4-6 contains a low affinity heparin binding domain, but its physiological significance is not clear. We have studied whether this domain is able to interact with cells as already shown for other heparin binding domains of fibronectin. A computer search based on homologies with known active sites in fibronectin revealed the sequence KLDAPT located in FN-III5. A synthetic peptide containing this sequence induced lymphoid cell adhesion upon treatment with the activating anti-beta1 monoclonal antibody (mAb) TS2/16 or with Mn2+, indicating that KLDAPT was binding to an integrin. A recombinant fragment containing repeat III5 (FN-III5) also mediated adhesion of TS2/16/Mn2+-treated cells while the FN-III6 fragment did not. Soluble KLDAPT peptide inhibited cell adhesion to FN-III5 as well as to a 38-kDa fibronectin fragment and VCAM-1, two previously known ligands for alpha4beta1 integrin. KLDAPT also competed with the binding of soluble alkaline phosphatase-coupled VCAM-Ig to Mn2+-treated alpha4beta1. Furthermore, mAbs anti-alpha4 and anti-alpha4beta7, but not mAbs to other integrins, inhibited cell adhesion to FN-III5 and KLDAPT. These results therefore establish a cell adhesive function for the FN-III5 repeat and show that KLDAPT is a novel fibronectin ligand for activated alpha4 integrins.

Phage antibodies with pan-species recognition of the oncofoetal angiogenesis marker fibronectin ED-B domain.

Fibronectin (FN) exists in several polymorphic forms due to alternative splicing. The B-FN isoform (with ED-B domain inserted by splicing) is present in the stroma of foetal and neoplastic tissues and in adult and neoplastic blood vessels during angiogenesis but is undetectable in mature vessels. This isoform, therefore, represents a promising marker for angiogenesis, as already shown using the mouse monoclonal antibody (MAb) BC-1 directed against an epitope on human B-FN. However, this MAb does not directly recognise the human ED-B domain nor does it recognise B-FN of other species; therefore, it cannot be used as a marker of angiogenesis in animal models. In principle, antibodies directed against the human ED-B domain should provide pan-species markers for angiogenesis as the sequence of this domain is highly conserved in different species (and identical in humans and mice). As it has proved difficult to obtain such antibodies by hybridoma technology, we used phage display technology. Here, we describe the isolation of human antibody fragments against the human ED-B domain that bind to human, mouse and chicken B-FN. As shown by immunohistochemistry, the antibody fragments stain human neoplastic tissues and the human, mouse and chicken neovasculature.

Human tenascin-R. Complete primary structure, pre-mRNA alternative splicing and gene localization on chromosome 1q23-q24.

We have established the primary structure of human tenascin-R (TN-R), a component of the extracellular matrix of the central nervous system, by sequencing cDNA clones which cover its complete coding region. The deduced amino acid sequence of human TN-R (1358 amino acids) showed a homology to chicken and rat TN-R of 75 and 93%, respectively. By reverse transcriptase-polymerase chain reaction we have studied the existence of TN-R isoforms generated by pre-mRNA alternative splicing in various human astrocytomas and meningiomas. Our findings demonstrate the existence of a human isoform in which one fibronectin-like repeat is omitted. Northern blot analysis of the poly(A)-rich RNA from different tissues showed two mRNAs having sizes of about 10 and 11 kilobases. Using DNA from a panel of human-hamster and human-mouse somatic cell hybrids and by fluorescence in situ hybridization, we have assigned the gene for human TN-R to the region 1q23-q24. The mouse mutation loop-tail (Lp), which has been proposed as a model for human neural tube defects, maps to region of mouse chromosome 1 syntenic with human 1q23-q24.

Extracellular matrix proteins in colorectal carcinomas. Expression of tenascin and fibronectin isoforms.

BACKGROUND: Interactions of tumor cells and extracellular matrix (ECM) components are crucial determinants of tumor cell spreading and metastatic activity. Particularly tenascin (TN) as a member of the adhesion modulating family of ECM and its alternatively spliced isoforms became the matter of interest in ECM changes associated with malignancy. EXPERIMENTAL DESIGN: We analyzed the composition of the stromal- and basement membrane-associated ECM of colorectal adenomas and carcinomas using indirect immunofluorescence. Tenascin was investigated by immunoblot of snap frozen tumor specimens. RESULTS: Fibronectin (FN), TN, and chondroitin sulfate proteoglycan were the major components of the tumor stroma. Normal basement membrane components like laminin (LM), collagen type IV, and heparan sulfate proteoglycan were down-regulated. In the center of the tumor, tumor glands were surrounded by discontinuous basement membranes. At the tumor-host interface and in solid, poorly differentiated tumors, no immunoreactivity with normal basement membrane components was found. However, in cases with pericellular anti-LM staining, LM immunoreactivity was also found at the tumor-host interface. An alternatively spliced isoform of TN with a molecular weight of 330 kDa was found in seven of 15 carcinomas. In four of these cases, an alternatively spliced isoform of FN containing the ED-B segment was present. CONCLUSIONS: The coexpression of alternative splicing of FN and TN suggests that there may be common regulation mechanisms. The matrix composition found in the present study resembles that of healing wounds and probably favors the invasive spread of tumor cells.

Cell-cycle dependent alternative splicing of the tenascin primary transcript.

Functionally different tenascin (TN) isoforms may be generated by alternative splicing of the TN primary transcript. In fact, it has been demonstrated that only the larger TN isoform containing the alternatively spliced region induces loss of focal adhesion in cultured cells and seems able to facilitate cell migration. Recent studies have shown that the higher molecular mass TN isoform is a marker of stromal cell proliferation in hyperplastic and neoplastic breast tissues. This finding prompted us to study the pattern of TN alternative splicing in proliferating and non-proliferating cultured fibroblasts. Here, we show that the mitogenic stimulation of fibroblasts with serum or cytokines leads to an early and striking modification in the steady-state levels of the two major TN mRNAs. We also show that de novo protein synthesis is not necessary for this modification, indicating that it is a "primary response" event. Similarly, mitogenic stimulation induces changes both in synthesis and accumulation of the different TN isoforms.

Expression of different tenascin isoforms in normal, hyperplastic and neoplastic human breast tissues.

Functionally different tenascin (TN) isoforms, containing varying numbers of a 91 amino-acid motif resembling the fibronectin type-III homology repeat, may be generated by alternative splicing of the TN primary transcript. In fact, only the TN isoform containing the alternatively spliced region can induce loss of focal adhesion in cultured cells and seems to be able to facilitate cell migration. We examined the patterns of alternative splicing of the TN primary transcript in normal, hyperplastic and neoplastic breast tissues, and found that, in all the invasive breast carcinomas analyzed, the relative amount of TN mRNA in which the alternatively spliced region was included was about 10 times higher than in RNA from normal breast tissues. A similar result was observed in phyllodes tumors and in those fibroadenomas which showed very high stromal cellularity. Western-blot analysis using different monoclonal antibodies showed the same pattern as that seen in Northern blotting. The data reported here suggest that, in the breast, expression of the high-molecular-mass TN isoform is a marker of stromal element proliferation and that, in invasive breast carcinomas, this TN isoform could play a role in generating a permissive environment for proliferation, invasion and metastasis of neoplastic epithelial cells.

Comparison of human tenascin expression in normal, simian-virus-40-transformed and tumor-derived cell lines.

Tenascin is a polymorphic high-molecular-mass extracellular-matrix glycoprotein composed of six similar subunits. Using two-domain-specific anti-tenascin monoclonal antibodies, we have studied the expression and distribution of tenascin in four cultured normal human fibroblasts, two simian-virus-40-(SV40)-transformed and three tumor-derived (melanoma, rhabdomyosarcoma and fibrosarcoma) cell lines. We found that (a) cultured normal human fibroblasts accumulate considerable amounts of tenascin and retain 60-90% in the extracellular matrix, while they release the remainder into the tissue-culture medium; (b) of the two SV40-transformed counterparts we have tested, the AG-280 cell line accumulates no detectable amounts of tenascin and the WI-38-VA cell line accumulates about 10-times less tenascin than its normal counterpart and releases about 90% of it into the culture medium; (c) some tumor-derived cell lines accumulate considerable amounts of tenascin, but in these cases, more than 90% is released into the culture media; (d) in normal human fibroblasts, two major tenascin isoforms, generated by alternative splicing of the mRNA precursor, are detectable (280 kDa and 190 kDa, respectively) and the lower-molecular-mass tenascin isoform is accumulated preferentially in the extracellular matrix; (e) in SV40-transformed or tumor-derived cell lines, only the higher-molecular-mass isoform is detectable and it is more sialylated than the tenascin produced by the normal human fibroblast cell lines.

Human tenascin: primary structure, pre-mRNA splicing patterns and localization of the epitopes recognized by two monoclonal antibodies.

By sequencing cDNA clones which cover the complete coding region of human tenascin (TN), we have established its primary structure. This confirms that, as in the case of chicken, TN is mainly made up of three groups of sequences with a high homology to Epidermal Growth Factor (EGF) fibronectin (FN) type III repeat and fibrinogen. Furthermore, we have determined the amino-terminal sequence of the mature peptide directly on purified TN. The main differences with respect to the chicken TN molecule are that in the human there are 14 and half EGF-like repeats compared to 13 and half in the chicken and that, as previously reported, there are 15 FN-like repeats compared to 11 in the chicken. By Polymerase Chain Reaction (PCR) amplification we have also studied the different splicing patterns of the TN pre-mRNA in cultured cells. The results show the presence of at least four different isoforms containing different numbers of FN-like type III repeats. Using purified human TN as immunogen, we have obtained numerous monoclonal antibodies (Mabs) to TN. By screening a human melanoma cDNA library in the expression vector lambda gt11 with these Mabs and subsequently sequencing the insert of the positive clones, we have been able to localize, within the TN molecule, the epitopes recognized by two of these Mabs: BC-4, which recognizes an epitope within the EGF-like sequence and BC-2 which recognizes an epitope within the FN like type III repeats whose expression is regulated by alternative splicing of the TN pre-mRNA. Thus, while the Mab BC-4 may be useful in studies on TN distribution (since it recognizes all different TN isoforms) BC-2 may be useful in the study of the expression of particular TN isoforms generated by the alternative splicing of the TN primary transcript.

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.

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)

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.

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.

DNA-binding domains of human plasma fibronectin. pH and calcium ion modulation of fibronectin binding to DNA and heparin.

We have studied the binding of fibronectin and its thermolysin fragments to DNA and heparin. Elution of polypeptides bound to DNA-cellulose and heparin-Sepharose affinity chromatography columns was performed by NaCl linear gradients in buffers at different pH and in the presence and absence of calcium ions. The NaCl concentration required to elute fibronectin from both types of column increased as the pH decreased. Fibronectin was not retained on DNA-cellulose or heparin-Sepharose affinity chromatography columns using a buffer containing physiological concentrations of Ca2+, Mg2+ and NaCl, at pH 7.4. On the other hand at pH 6.4 in conditions of physiological ionic strength, fibronectin was retained by both columns, eluting from the DNA-cellulose at 280 mM NaCl and from the heparin-Sepharose column at 210 mM. Furthermore, we have studied the interaction of thermolysin-digested fibronectin both with DNA-cellulose and heparin-Sepharose using the above procedure. The results demonstrate that there are four distinct domains, which interact both with DNA and heparin. We also report here the modulation by pH and Ca2+ ions of the interaction with DNA and heparin of these different domains.

Fibronectin concentrations in pleural effusions of patients with malignant and non-malignant diseases.

We have determined the amount of fibronectin in pleural fluids and plasma of 131 patients suffering from pleurisy of malignant and non-malignant etiology. We observed a significantly higher concentration of fibronectin in pleural fluids of a few patients with mesothelioma as compared with that found in pleural fluids of patients with pleurisies of different etiology. In these patients the pleural fluid fibronectin concentration was higher than that measured in plasma, suggesting local synthesis of fibronectin.

Increased plasma fibronectin concentrations in tumor bearing mice.

Fibronectin concentration was studied in plasma of mice at different times after inoculation of 3 different experimental tumors, Colon 26 adenocarcinoma, mFS6 fibrosarcoma and B16 melanoma. A significant increase in fibronectin concentrations 10-30 days after inoculation of all tumors was found. Highest and earliest fibronectin increases were observed in BALB/c mice inoculated with Colon 26 adenocarcinoma cells. Control animals inoculated with previously irradiated cells (5000 rad) showed neither tumor growth nor significant variations in plasma fibronectin levels.