POLYPEPTIDES WITH FIBRONECTIN ACTIVITY

Abstract: A composition which can bind heparin and promote cellular adhesion and neurite outgrowth is provided which consists essentially of a polypeptide of the formula: tyr-glu-lys-pro-gly-ser-pro-pro-arg-glu-val-val-pro-arg-pro-arg-pro-gly-val, lys-asn-asn-gln-lys-ser-glu-pro-leu-ile-gly-arg-lys-lys-thr-asp-glu-leu, lys-asn-asn-gln-lys-ser-glu-pro-leu-ile-gly-arg-lys-lys-thr, leu-ile-gly-arg-lys-lys-thr, tyr-arg-val-arg-val-thr-pro-lys-glu-lys-thr-gly-pro-met-lys-glu, ser-pro-pro-arg-arg-ala-arg-val-thr, trp-gln-pro-pro-arg-ala-arg-ile, or mixtures thereof. Medical devices such as prosthetic implants, percutaneous devices and cell culture substrates coated with the polypeptide composition are also provided.

Description: POLYPEPTIDES WITH FIBRONECTIN ACTIVITY Cross Reference to Related Application This application is a continuation-in-part of 5 U.S. patent application Serial No. 89,073, filed August 25, 1987. Grant Information The present invention was made with the 10 support of Grant No. CA21463 from the National Institutes of Health. The Government has certain rights in the invention. Background of the Invention 15 The adhesion of mammalian cells to the extra¬ cellular matrix is of fundamental importance in regu¬ lating growth, adhesion, motility and the development of proper cellular phenotype. This has implications for normal development, wound healing, chronic inflam- 20 matory diseases, and tumor metastasis. Evidence accu¬ mulated over the last several years suggests that the molecular basis for the adhesion of both normal and transformed cells is complex and probably involves several distinct cell surface molecules. Extracellular 25. matrices consist of three types of macro oleculest collagens, proteoglycans and noncollagenous glycopro- teins. The extracellular matrix molecule which has been most intensively studied with regard to cell adhe¬ sion is the noncollagenous cell adhesion glycoprotein, 30 fibronectin, which is present in plasma, cell matrices, basal lamina and on cell surfaces. The plasma form of fibronectin consists of a disulfide-bonded dimer having a molecular weight of 450,000 daltons. The two subunit chains ("A" and "B"), each of about 220,000 daltons, 35 are observed under reducing conditions. This form of fibronectin will be referred to as "fibronectin" hereinafter. Fibronectin, as with other components of the extracellular matrix, has the ability to bind to itself, to other matrix constituents, and to the sur¬ face of cells, via discrete domains on the molecule. For example, fibronectin promotes the attachment of suspended cells to collagen. (See Li T. Furcht, Modern Cell Biology, B. Satir, ed., Alan R. Liss, Inc., N.Y., Vol. I (1983) at pages 53-117). The primary structure of one adhesion sequence within fibronectin was " origin¬ ally deduced by M. D. Pierschbacher et al. using mono¬ clonal antibody data and direct sequence analysis. This sequence was found to be a tetrapeptide consisting of arginyl-glycyl-aspartyl-serine (RGDS) (M. D. Pierschbacher and E. Ruoslahti, PNAS USA, 81, 5985 (1984)). Peptides containing .the' RGDS sequence are capable of directly promoting the adhesion of certain cell types, and high levels of soluble RGDS will par- tially disrupt cell adhesion to intact fibronectin. Cell adhesion to the RGDS sequence in fibronectin is believed to occur by the interaction of this sequence with a cell surface glycoprotein complex termed "integrin". Despite the importance of the RGDS/integrin complex in fibronectin mediated cell adhesion, several lines of evidence point to the involvement of addi¬ tional cellular receptors and different fibronectin determinants in this process. Many cell types form focal adhesions on intact fibronectin. These struc¬ tures ' represent regions of close apposition between the plasma membrane and the substratum. These sites also represent insertion points.for actin-rich stress fibers, and have been shown to contain several actin- associated cytos.keletal proteins. Focal adhesion sites also contain several classes of cell surface molecules implicated in cell adhesion, including integrin, heparan sulfate, chondroitin sulfate, or other proteo- glycans and gangliosides. The action of multiple receptors for "fibro¬ nectin has been implicated in adhesion plaque forma¬ tion. Cells adherent to either RGDS-containing frag- - ments or heparin-binding, adhesion promoting ligands (e.g., platelet factor 4 or heparin binding fragments of fibronectin) form only close contacts. In contrast, cells adherent on both RGDS-containing fragments and heparin-binding ligands display fully developed focal adhesions. Additionally, antibodies against heparin binding fragments of fibronectin inhibit focal adhesion formation, without drastically inhibiting the level of cell adhesion on intact fibronectin. Collectively, these results support a role of heparin-binding domain(s) of fibronectin in promoting normal and malignant cell adhesion, and in regulating phenotypic expression of cells. J. B. McCarthy et al., in 3. Cell Biol., 102, 179 (1986) recently published results identifying a 33 kD carboxyl terminal heparin-binding fragment of fibro¬ nectin which promotes the adhesion and spreading of metastatic melanoma cells by an RGDS independent mech¬ anism. This fragment originates from the A chain of the fibronectin molecule. It binds heparan sulfate proteόglycan and also promotes the adhesion of neurons and the extension of neurites by these cells. Therefore, a need exists to isolate and characterize the subset of peptides within this fragment which are responsible for its wide range of biological activities. Such lower molecular weight oligopeptides would be expected to be more readily obtainable and to exhibit a narrower profile of biolo¬ gical activity than the 33 kD fragment, thus increasing their potential usefulness as therapeutic or diagnostic agents. Brief Description of the Invention The present invention provides biologically- active polypεptides which represent fragments of the 33 kD carboxyl terminal, heparin-binding region located on the A chain of fibronectin. Two of these polypeptides, which can be prepared in substantially pure form by conventional solid phase peptide synthesis, have the formulasi tyr-glu-lys-pro-gly-ser-pro-pro-arg-glu-val-val- pro-arg-pro-arg-pro-gly-val (I) and lys-asn-asn-gln-lys-ser-glu-pro-leu-ile-gly-arg- lys-lys-thr-asp-glu-leu (II) Polypeptide I formally represents isolated fibronectin residues 1906-1924, while polypeptide II formally represents isolated fibronectin residues 1946-1963. The single letter a ino acid codes for these polypep- tides are YEKPGSPPREVVPRPRPGV and KNNQKSEPLIGRKKTDEL, respectively. Peptide I exists in all isoforms of fibronec¬ tin. In contrast, the first 15 residues of peptide II exist in all isoforms, whereas the last three residues in peptide II are a part of a region of structural heterogeneity which occurs only in certain isoforms in fibronectin (termed A chains). In the case of plasma fibronectin, this region of structural heterogeneity, termed type III cs, can be up to 89 residues long. The 33 kD heparin-binding fragment arises from A chains and includes part of this type III cs sequence. Recently, Humphries et al., J . Biol. Chem., 262, 6886-6892 (1987), reported that the type III cs connecting segment of fibronectin has cell adhesion- promoting activity. Humphries et al. constructed over- lapping peptides which represented the entire type III cs sequence and tested these peptides for the ability to promote the adhesion and spreading of fibroblasts and melanoma cells. The results of these studies indi¬ cated that the first 24 residues of this type III cs connecting sequence promoted melanoma cell adhesion and spreading. The sequence of the biologically active peptide, termed CS I, was asp-glu-leu-pro-gln-leu-val- thr-leu-pro-his-pro-asn-leu-his-gly-pro-glu-ile-leu- asp-val-pro-ser-thr (DELPQLVTLPHPNLHGPEILDVPST) , and corresponds to residues 1961-1985. Thus, an overlap exists between the last three residues of peptide II and the first three residues of the CS I.peprtide reported by Humphries et al. However, the peptide reported by Humphries et al. differs chemically from either peptides I or II. CS I is more hydrophobic, and totally lacks lysine or arginine residues. The signif¬ icant chemical properties of each peptide are summar- ized on Table I, below: Table I Peptide Residue Nos. Hydropathy Index Net Charge I 1906 - 1924 -24.3 +2 II 1946 - 1963 -32.5 +2 CS I 1961 - 1985 -9. 9 -4 The "hydropathy index" is calculated according to the method of Kyte and Doolittle, 3. Mol. Biol., 157, 105-132 (1982). According to this method, the more (-) a value is, the more hydrophilic it is. Thus, the CS I peptide is much more hydrophobic than peptides I and II. The net charge of each peptide is calculated by assigning a (+1) charge to each lysine (K) and argi- nine (R) residue, and a (-1) charge to each glutamic acid (E) and aspartic acid. (D) residue. Additional residues are assumed to be uncharged. These charges would be expected under the conditions of heparin bind¬ ing and cell adhesion assays, which are performed near neutrality (pH 6.8 to 7.4). The only other residue which could contribute significantly to total charge is histidine (H), which occurs twice in the CS I peptide but would b-e uncharged under the pH conditions of the assays used. Despite the difference in chemical properties between CS I and peptide II, experiments were carried out to determine whether or not the biological activity of peptide .II was related to the three-residue overlap with peptide CS I (residues +1961-1963, DEL). Thus, a polypeptide was synthesized which contained the first 15 amino acid residues of polypeptide II. The formula of this polypeptide, Ha, is shown belowt lys-asn-asn-gln-lys-ser-glu-pro-leu-ile-gly- arg-lys-lys-thr (Ha) The single letter amino acid code for this polypeptide is ; .KNNQKSEPLIGRKKT, which corresponds to residues 1946-1960. This peptide differs from peptide I in that a.) the total net charge is (+4) and the total hydropathy index is -29.3. It was found that while Ila bound heparin in a concentration-dependent fashion, peptide CS I failed to bind to heparin at any concentration that was tested. Therefore, the biological activity of each peptide is due to distinct and unique structural determinants. The present invention is also directed to the biologically active peptide fragments of polypeptides I, II and Ila. For example, the present invention also provides three polypeptides which correspond to the amino terminal third (Ila-A), the central third (Ila-B), and the carboxyl terminal third (Ila-C), sequences of peptide II. Specifically, these peptides have the sequences shown on Table II, below. Table II Name Primary Sequence Net Charge IΙ(amino) KNNQKSEP +1 (Ila-A) (lys-asn-asn-gln- lys-ser-glu-pro) IΙ(central) KSEPLIGR +1 (Ila-B) (lys-ser-glu-pro- leu-ile-gly-arg) IΙ(carb.oxyl) LIGRKKT +3 (Ila-C) (leu-ile-gly-arg- lys-lys-thr) The present invention also provides three additional bioactive polypeptides, which also represent fragments of the 33 kD carboxyl terminal heparin- binding region located on the A chain of fibronectin. These polypeptides, III, IV and V, are structurally distinct from each other and from peptides I and Ila, however, they do share certain chemical properties which are summarized on Table III, belowj Table III Hydro- Primary pathy Net Peptide Structure Residue Nos. Index Charge III YRVRVTPKEKTGPMKE 1721-1736 -23.7 +3 (tyr-arg-val-arg- val-thr-pro-lys- glu-lys-thr-gly- pro-met-lys-glu) IV SPPRRARVT 1784-1792 -12.2 +3 (ser-pro-pro-arg- arg-ala-arg-val- thr) V WQPPRARI 1892-1899 -10.8 +2 (trp-gln-pro-pro- arg-ala-arg-ile) Each peptide has an identical net charge, although each peptide derives its charge from different basic residues. Peptide III contains two arginine (R) residues and three lysine (K) residues. Peptides IV and V contain only arginine residues. Furthermore, the charges are clustered in peptides IV and V, whereas the positive charges in peptide III are more dispersed. The dispersal of charge within peptide III is different from that in the other four heparin-binding peptides identified from this region to date. Peptide III is more hydrophilic than either peptide IV or V, but less hydrophilic than peptides I or II. These synthetic polypeptides were assayed for bioactivity and found to exhibit at least one of the following properties! they (a) promote neurite exten¬ sion, (b) promote the adhesion and spreading of endo- thelial cells (c) promote the adhesion and spreading of -melanoma cells, and/or (d) promote the binding of heparin to a synthetic substratum. Therefore, it is believed that these polypeptides may be useful to (a) assist in nerve regeneration, (b) promote wound healing and implant acceptance, (c) promote cellular attachment to culture substrata, (d) inhibit the metastasis of ' malignant cells, and/or (e) bind excess heparin,- a con- dition which can occur in vivo during heparin therapy. Due to the difference in the spectra of biological activities exhibited by the present polypeptides, mix¬ tures thereof are also within the scope of the inven¬ tion. Furthermore, since it is expected that further digestion/hydrolysis of polypeptides I, II, Ila and III-V, in_ vitro or _in_ vivo, will yield fragments of substantially equivalent bioactivity, such lower molec¬ ular weight polypeptides are considered to be within the scope of the present invention. Brief Description of the Drawings Figure 1 is a schematic depiction of plasma fibronectin, indicating the relative location of RGDS and CS-I on the protein with respect to the heparin- binding peptides I-V of the present invention, which are located on the 33 kD carboxyl terminal heparin binding fragment of the A .chain. Figure 2 is a graph depicting the heparin binding activity of peptides I and II of the invention (nitrocellulose binding assay). Figure 3 is a graph depicting the heparin binding activity of fibronectin (fn) (nitrocellulose binding assay). Figure 4 is a graph depicting the relative heparin-binding activity of peptides Ila and CS I (Immulon C binding assay). Figure 5 is a graph depicting the heparin- binding activities of peptides Ila-A, Ila-B, and Ila-C (Immulon C binding assay). Figure 6 is a graph depicting the relative heparin-binding activity of peptides I, Ila and III-V (Immulon C binding assay). Figure 7 is a graph depicting the neurite extension promoted by peptide Ila. Figure 8 is a graph depicting the neurite extension promoted by peptide CS I. Figure 9 is a graph depicting the neurite extension promoted by peptide III. Figure 10 is a graph depicting the relative melanoma cell adhesion promoted by peptides Ila and III. Figure 11 is a schematic representation of the amino acid sequence of the portion of the plasma fibro¬ nectin molecule from which these peptides are formally derived. Detailed Description of the Invention Structure of Fibronectin. Referring to Figure 1, the two types of chains (A and B) of plasma fibronectin are shown as a disulfide (-S-S-) bonded heterodimer. The six domains (I-VI) of fibronectin are labeled according to previous nomenclature (L. T. Furcht in Modern Cell Biology, B. Satir, ed., Alan R. Liss, Inc., NY (1983) at pages 53-117. ) Biological activities within each domain include: (1) weak heparin binding, (II) noncovalent collagen binding, (III) DNA binding, (IV) RGDS-mediated cell adhesion, shown as box (0), (V) heparin binding, RGDS independent cell adhesion, and (VI) free sulfhydryl. The molecular weight estimates of pro- teolytic fragments containing each domain are based on a previously described digestion and purification scheme. (J. B. McCarthy, J. Cell Biol., 102, 179 (1986)). Proteolytic cleavage sites (X) are shown for trypsin (T) and Cathepsin D (C). By these schemes, domains V and VI isolated from digests of the B chain are located on a 66 kD fragment. In contrast, the A chain digests contain a 33 kD fragment (domain V) and a 31 kD fragment (domain VI). The difference is a result of a trypsin sensitive site in the A-chain specific type IIIcs insert, shown as a bold line. Amino Terminal Sequence of the Tryptic/Catheptic 66 kD and 33 kD Heoarin-Bindinα Fragments and Carboxyl Terminal Tryptic 31 kD Free-Sulfhydryl Containing Fragment. The entire primary structure of fibronectin has either been determined directly (T. E. Peterson et al., PNAS USA, 80, 137 (1983)) or has been predicted from reco binant DNA technology. (J. E. Schwarzbauer et al., Cell, 135, 421 (1983)). The amino terminal sequences of tryptic/catheptic (t/c) 33 kP, t/c66 kD, and tryptic (t) 31 kD fragments were established by direct amino acid sequencing on an Applied Biosyste s gas phase sequenator (Model 470A), in order to deter¬ mine the exact location of these fragments with respect to the known human sequence. The first 21 amino acids which were determined for the t/c66 heparin binding fragment (Figure 11, underlined residues which begin in line 1 and continue in line 2). This fragment starts with the amino acid alanine which corresponds to residue 1583 on intact plasma fibronectin (A. R. Kornblihtt et al., EMBO 3. , 4-, 1755 (1985)). The presence of tyrosine to the amino terminal side of this alanine in intact fibronectin is consistent with a preference of Cathepsin D for peptide bonds involving aromatic residues. The sequence of the t/c66 fragment does not contain the EDIII insert, since the sequence proceeds" from a threonine at residue number 1599 (double asterisks followed by a slash at the end of line 1) to an alanine at residue 1690 (first residue, line 2). This lack of the EDIII region is a characteristic feature which distinguishes plasma- or liver-derived fibronectin from cellular, or fibroblast derived fibronectin. The t/c33 fragment also shares a common amino terminal sequence with the t/c66 fragment (Figure 11, line 1), beginning with alanine at position 1583, and it also lacks the EDIII domain. These results illu- stratε that the amino terminal sequences of these fragments are identical, and support the contention that the size heterogeneity of the t/c33 and t/c66 heparin binding fragments results from the action of trypsin within the type IIIcs insert of the A chains of plasma fibronectin. Localization of the 33 kD heparin binding fragment within the A chain of plasma fibronectin was established by determining the amino terminal sequence of the first 21 amino acids of a tryptic 31 kD fragment. This fragment, which is produced during the purification of 33 and 66 kD heparin binding fragments, contains a free sulfhydryl and orginates from the car¬ boxyl terminal end of the A chain of plasma fibronec¬ tin. See P. E. Smith and L. T. Furcht, 3. Biol. Chem. , 257, 6518 (1982). Furthermore, the 31 kD fragment also originates from a subset of fibronectin molecules which give rise to the 33 kD heparin binding fragment of fibronectin. The amino terminal end of the t31 fragment begins at histidine residue 2040, underlined, line 5 of Figure 11. This is consistent with the known specific¬ ities of trypsin, since the residue to the amino ter¬ minal side of this histidine is an arginine. This sequence is present in the type IIIcs insert which occurs in a subset of fibronectin molecules. This fragment contains 9 additional amino acids from the type IIIcs insert, skips the last 31 amino acids of this insert (Figure 11, line 5, parentheses), then con¬ tinues as a type III homology (Figure 11, line 6, underlined) until the tyrosine at residue 2062 where the current sequence information ends. These results demonstrate that the t31 fragment contains a portion (the first 89 amino acids) of the maximum possible 120 residue type IIIcs inserted sequence, in agreement with previously established sequence data for this " region of plasma fibronectin. The sequence information indicates the maximum possible carboxyl terminal limit of the t/c33 heparin binding fragment at arginine residue 2039, within the type IIIcs insert (Figure 11, line 5). Synthesis of Polypeptides The polypeptides of the invention were synthesized using the Merrifield solid phase method. This is the method most commonly used for peptide synthesis, and it is extensively described by 3. M. Stewart and J. D. Young in Solid Phase Peptide Synthesis, Pierce Chemical Company, pub., Rockford, IL (2d ed., 1984), the disclosure of which is incorpor¬ ated by reference herein. The ' Merrifield system of peptide synthesis uses a 1% crosslinked polystyrene resin functionalized -14- with benzyl chloride groups. The halogens, when reacted with the salt of a protected amino acid will form an ester, linking it covalently to the resin. The benzyloxy-carbonyl (BOC) group is used to protect the free amino group of the amino acid. This protecting group is removed with 25% trifluoroacetic acid (TCA) in dichloromethane (DCM). The newly exposed amino group is converted to the free base by 10% triethylamine (TEA) in DCM. The next BOC-protected amino acid is then coupled to the free amine of the previous amino acid by the use of dicyclohexylcarbodiimide (DCC). Side chain functional groups of the amino acids are protected during synthesis by TFA stable benzyl deriva¬ tives. All of these repetitive reactions can be auto- mated, and the peptides of the present invention were synthesized at the University of Minnesota Microchemi- cal facility by the use of a Beckman System 990 Peptide synthesizer. Following synthesis of a blocked polypeptide 0 on the resin, the polypeptide resin is treated with anhydrous hy.drofluoric acid (HF) to cleave the benzyl ester linkage to the resin and thus to release the free polypeptide. The benzyl-derived side chain protecting groups are also removed by the HF treatment. The poly- 5 peptide is then extracted from the resin, using 1.0 M acetic acid, followed by lyophilization of the extract. Lyophilized crude polypeptides are purified by preparative high performance liquid chromatography (HPL-C) by reverse phase technique on a C-18 column. A 0 typical elution gradient is 0% to 60% acetonitrile with 0.1% TFA in H2O. Absorbance of the eluant is monitored at 220 nm, and fractions are collected and lyophilized. Characterization of the purified polypeptides is by amino acid analysis. The polypeptides are first 5 hydrolyzed anaerobically for 24 hours at 110°C in 6 M HC1 (constant boiling) or in 4 N methanesulfonic acid, when cysteine or tryptophane are present. The hydro- lyzed amino acids are separated by ion exchange chroma- tography using a Beck an System 6300 amino acid analyzer, using citrate buffers supplied by Beckman. Quantitation is by absorbance at 440 and 570 nm, and comparison with standard curves. The polypeptides may be. further characterized by sequence determination. This approach is especially useful for longer polypep- tides, where amino acid composition data are inherently less informative. Sequence determination is carried out by sequential Edman degradation from the amino ter¬ minus, automated on a Model 470A gas-phase sequenator (Applied Biosystems, Inc.), by the methodology of R. M. Hewick et al., 3. Biol. Chem., 256, 7990 (1981). The invention will be further described by reference to the following detailed examples. Example 1. Heparin-Bindinq Assay The assay for heparin binding utilizes nitro¬ cellulose sheets as substrata to bind peptides or pro¬ teins to be tested -for heparin binding activity. Peptides I and II or intact fibronectin (fn) were solu- bilized in 50 mM (NH 4 )2C03 and diluted to the concen- trations indicated in Figures 2 and 3. Nitrocellulose sheets which had been presoaked in 50 mM NH4CO3 were placed in a 96 well dot blot apparatus (Bethesda Research Laboratories, Bethesda, MD), and 250 μl of various concentrations of each peptide were aspirated through the wells. Each well was then washed three times with binding buffer (10 mM Tris-HCl, pH 8.0, 0.15 M NaCl), and the filters were removed and allowed to air dry overnight. The filters were then equilibriated for 5 minutes at room temperature in binding buffer which contained 10 mM CaCl2- 3 H-heparin was then diluted to a concentration of 50,000 cpm/ml in binding buffer (with Ca ), and nitrocellulose sheets were incubated in the presence of this mixture for 2 hours. The filters were then washed four times with binding buffer, and air dried. The individual spots of samples were cut out of the nitrocellulose and bound heparin was quantitated with a liquid scintillation counter. A. Polypeptides I and II The results show that peptide II bound 3 H-heparin in a concentration dependent manner (Figure 2). In con¬ trast, 3 H-heparin bound poorly to peptide I at any con¬ centration tested. The lowest concentration of peptide II which promoted 3 H-hεparin binding was 0.25 x 10-4 M with a saturation of binding observed at higher coating concentrations (0.25 - 0.5 x 10~ 2 M). Similarly, fibronectin also bound 3 H-heparin in a concentration dependent manner, with maximum binding observed at 10-6 M fibronectin (Figure 3). B. Polypeptides Ila and CS I An additional lysine residue was added at the carboxyl terminus of CS I in order to facilitate coupling of this peptide to the substrata used in this assay. Both peptides Ila and CS I were then compared for relative heparin-binding activity. Plastic Immulon C plates (Oynatech, Alexandria, VA) were adsorbed with 100 μl (in triplicate) of the indicated levels of peptides Ila, CS I or BSA as described. The ability of the 33 kD fragment to bind heparin was also determined, for comparison, although due to the rela¬ tive size of this fragment compared with the peptides, different coating concentrations were used. The actual coating levels of the 33 kD fragment were 4, 20, 100 and 500 μg/ l. Following the blocking of nonspecific binding sites with BSA, the ability of these various substrata to bind 3 H-heparin was determined by the addition of approximately 4,000 disintegrations per minute (dpm) of this ligand. All conditions of the assay were as described in U.S. patent application Serial No. 89,073, the disclosure of which is incorpor¬ ated by reference herein. As shown in Figure 4, peptide Ila retained the ability to bind 3 H-heparin in a concentration dependent fashion. In contrast, peptide CS I failed to bind heparin at any concentration tested, indicating that the heparin-binding activity ascribed to peptide II does not involve the area of structural overlap with CS I. C. Polypeptides Ila-A, Ila-B and Ila-C The three peptides derived from peptide Ila and intact peptide Ila, were adsorbed to Immulon C plates at the indicated concentrations (100 μl/well) in triplicate and tested for the ability to bind 3 H- heparin as described hereinabove. The results of this study are summarized on Figure 5. As demonstrated by these data, peptide Ila-A. exhibits extremely poor heparin-binding activity, peptide Ila-B exhibits slightly higher heparin activity at high coating con¬ centrations, whereas peptide Ila-C exhibits extremely high heparin-binding activity, even when used at very low coating concentrations. In fact, peptide Ila-C binds heparin much better than does the parent peptide (peptide Ila), despite the fact that the net charge on peptide Ila-C is (+3) whereas the net charge on peptide Ila is (+4). This demonstrates that net charge per se is not the only primary consideration for the heparin- binding activities observed in synthetic peptides -18- derived from fibronectin. Rather, a specific primary structure is crucial for this activity. In the case of peptide Ila, (KNNQKSEPLIGRKKT) the heparin-binding activity can be localized to the carboxyl terminal third of this peptide (corresponding to the sequence LIGRKKT, peptide Ila-C). Furthermore, at least one of the two lysine residues in this peptide is important for heparin-binding activity, since peptide II- (central), which contains an arginine, binds heparin much more weakly than peptide IΙ(carboxyl), which con¬ tains the same arginine and two additional lysines. D. Polypeptides III-V Peptides I, Ila and III-V were adsorbed at the indicated concentrations (100 μl/well), in triplicate, to Immulon C plates as described hereinabove. Peptides III-V bind 3 H-heparin ' substantially in the solid phase heparin-binding assay, as shown in Figure 6, and pep¬ tides III and IV bind heparin more strongly than does peptide V. 'Importantly, peptide I, which did not bind heparin well in the nitrocellulose-binding assay, bound heparin well and specifically in the Immulon C binding assay. The specificity of each peptide for heparin binding is indicated by the fact that the 50% inhibi¬ tion point of 3 H-heparin binding caused by dextran sulfate or chondroitin/dermatan sulfate is at a concen¬ tration which is 1 to 3 orders of magnitude higher than that exhibited by heparin. These results are similar to those observed for intact fibronectin or for pep¬ tides I and II. - 19- Example 2. Neurite Outgrowth Assay A_. Preparation of Plates Peptides I, II, Ila, III, CSI or intact fibro¬ nectin were diluted in Voller's buffer (0.05 M Carbon- ate buffer, pH 9. 6 ) and 100 μl of each concentration was dipensed into 96 well tissue culture plates in triplicate. The plates were then placed in a sterile hood overnight to evaporate the buffer and to dry the peptides onto the plate. The following morning, 200 μl of phosphate-buffered saline (PBS) containing 5 mg/ml bovine serum albumin (PBS/BSA) were added to each well and the plates were incubated for an additional 3 hours. At that point, the PBS/BSA was aspirated and cells in the appropriate media were added to each well. B. Isolation of Neurons and Assay for Neurite Out- rowth Embryonic CNS nerve cell cultures were pre¬ pared by the method of Rogers et al., Devel. Biol . , 98, 212-220 (1983).- Briefly, spinal cords from 6-day chick embryos were, isolated and their dorsal halves removed and placed in Ca -Mg free (CMF) Hank's balanced salt solution for 10 minutes at 37°C. Only the ventral por¬ tions, containing predominantly motor neurons, were prepared for culture. The cords were then dissociated in 0.25% trypsin (Bactotrypsin, Difco) in CMF Hanks for 25 minutes at 37°C. The trypsin containing medium was replaced with Ham's F12, buffered with HEPES and supplemented with 10% . fetal calf serum, and the cells repeatedly pipetted to complete dissociation. The single-cell suspension was pelleted by centrifugation, rinsed with Ham's F12-HEPES plus serum, centrifuged, and resuspended in Ham's F12 supplemented with sodium bicarbonate and gluta ine (2 M) , penicillin (100 U/ml), streptomycin (100 U/ml) and plated into wells -20- which had been prepared as described below in the pre¬ sence and absence of the indicated concentrations of heparin. Cultures were incubated for 24 hours at 37°C in a humidified incubator in 5% CO2 and then fixed in glutaraldehyde. The number of neurons with neurites was quantitated by randomly sampling 10 fields with the aid of a dissecting microscope. C. Polypeptides I and II The results of this assay are summarized on Tables IV and V, below. Table IV. Comparison of Neurite Extension by CNS Neurons on Peptide I and Peptide II Coating Number of Neurons with Neurites Cone* Without Heparin With 10 μq/ml Heparin " I 12;3** 3 II 220;194** 75 Control (BSA) 2 2 * 500 μq/ml ** Data represented ' as duplicate values. Table V. Dose Response of Peptide II Coating Concentration of Peptide II ' 2 mg/ml 1 mg/ml 500 μg/ml 250 μg/ml Background * Data represented as duplicate values -21- These results indicate that peptide II is much more effective than peptide I at promoting neurite out¬ growth, and that the neurite promoting activity of pep¬ tide II is apparently related to the heparin-binding activity of this peptide. Thus, peptide II may be use¬ ful in providing a synthetic substratum to promote nerve growth in situations where nerve regeneration is desirable (e.g., in crush injuries). D. Polypeptides Ila and CS I Further evidence for the- distinctive nature of peptides Ila and CS I was obtained by examining the growth of neurites exhibited by central and peripheral system embryonic chicken neurons when plated onto sub- strata coated with these peptides. Substrata were coated with 100 μl of a 500 μg/ml solution of the indicated ' peptides of with 5 μg/ml of the 33 kD heparin-binding fragment of fibro¬ nectin as described hereinabove. Following the block- ing of nonspecific sites with BSA, suspensions of either central nervous system (CNS) neurons isolated from the spinal cords of empryonic chickens, or peri¬ pheral nervous system (PNS) neurons isolated from the dorsal root ganglia of embryonic chickens, were plated onto the various coated substrata. Data represent the average number of neurons expressing neurites in these culture, and represent the mean of triplicate cultures. As shown in Figures 7 and 8, both peptides were able to promote neurite extension by embryonic neurons. However, CNS neurons appeared to be prefer¬ ably stimulated by peptide CS I, while peptide Ila appeared most effective at stimulating neurite exten¬ sion by peripheral neurons. Thus, we conclude that, despite the three residue overlap between peptide II and peptide CS I, the biological activity of each pep¬ tide is due to distinct and unique structural deter¬ minants. E. Peptide III Peptide III was examined for the ability to promote neurite outgrowth by embryonic chicken neurons in vitro. As shown in Figure 9, peptides Ila and III showed distinct differences in the ability to promote neurite outgrowth PNS and CNS neurons. Both peptides Ila and III demonstrate neurite promoting activity in both populations of neurons, however, peptide III is far more capable of promoting neurite extension by CNS neurons, whereas peptide Ila appears more effective at promoting neurite extension by PNS neurons. Example 3. Adhesion of Endothelial Cells A. Isolation of Bovine Aortic Endothelial Cells Bovine aortic endothelial cells were isolated according to the following protocol. Aortas were obtained from a local slaughterhouse, washed in cold phosphate buffered saline (PBS) (136 mM NaCl, 2.6 mM KC1, 15.2 mM Na 2 HP04, pH 7.2) and processed within 2 hours. Crude collagenase- (CLS III, 125-145 units per mg dry weight, Cooper Biomedical) was used at 2 mg/ml in Dulbecco's modified Eagle's medium (DMEM) (GIBCO). The vessel was clamped at the distal end, filled with the collagenase-PBS solution and digestion was carried out for 10 minutes. The lumenal contents were har- vested, followed by the addition of fresh collagenase for two additional 10-minute periods. The enzyme-cell suspensions were added to an equal volume of DMEM con¬ taining 10% fetal bovine serum (FBS) to inhibit the enzyme and spun in a centrifuge at 400 x g for 10 minutes. The resulting cell pellet was resuspended in •23- DMEM containing 10% FBS, 100 units/ml of penicillin G, 100 μg/ml of streptomycin and 100 μg/ml of crude fibroblast growth factor. Cells are cultured in 75 cm 2 flasks in a humidified 5% CO2 atmosphere at 37°C. Cultures were fed twice a week with the same medium and cells were used in assays when approximately 75% con¬ fluent. Cells were identified as endothelial in nature by characteristic cobblestone morphology, contact inhi¬ bition of growth upon reaching confluency, and positive immunofluorescent staining for factor VIIItRAg (Miles Laboratories) [s. Schwartz, In Vitro, 14, 966 (1978)]. Only endothelial cells, megakaryocytes and platelets are known to contain the factor VIIIiRAg. This method routinely gives a high yield of endothelial cells with little contamination (less than 5%) by smooth muscle cells, pericytes or fibroblasts as judged by phase contrast microscopy as well as by immuπostaiπiπg.- B. Aortic Endothelial Cell Adhesion Assay Adhesion was measured using 96 well microtiter plates adsorbed with fibronectin or peptides I and II. Cultures of cells which were 60-80% confluent were metabolically labeled overnight with the addition of 10 μCi/ml of 3 H«amino acids. On the day of the assay, the cells were harvested by trypsinization, the trypsin was inhibited by the addition of serum, and the cells were washed free of this mixture and resuspended in DMEM buffered with HEPES at pH 7.2. The adhesion medium also contained 5 mg/ml BSA. The cells were adjusted to a concentration of 3-4 x lOVml, and 100 μl of this cell suspension was added to the wells. The assay mixture was then incubated at 37°C for 90 minu¬ tes. At the end of the incubation, the wells were washed with warm PBS containing 10 mM Ca , and the adherent population was solubilized with 0.5 N NaOH -24- containing 1% sodium dodecyl sulfate. The solubilized cells were then quantitated using a liquid scintilla¬ tion counter. Each determination was done in tripli¬ cate. The results of this study are summarized in Table VI, below. Table VI. Adherent Cells Coating Concentration (Counts Per Minute) Background 403 Peptide I 40 μg/ml 1024 400 μg/ml 1107 4000 μg/ml 981 Peptidε II • 40 μg/ml 901 400 μg/ml 1734 40 ' 00 μg/ml 14,199 Fibronεctin 5 μg/ml 13,714 Thεsε rεsults indicate that peptide II is much more effective than peptide I at promoting endothelial cell adhesion, in agreemεnt with thε rεsults obsεrvεd for neurons. Thus, peptide II may be useful to promote endothelial cell adhesion to artificial or natural substrata. Example 4. Adhesion of Cancer Cells A. Isolation of Metastatic Melanoma Cells Highly metastatic melanoma cells, K1735M4, were originally provided by Dr. I. 3. Fidler of Houston, TX. When the cells were received, a large -25- nu bεr of early passage cells were propagated and frozen in liquid nitrogen. The tumor cells are usually cultured in_ vitro for no longer than six weeks. Following this period, the cells are discarded and nεw cεlls withdrawn from storage for use in further in vitro or _in_ vivo expεrimεπts. This prεcaution is taken to minimizε phεnotypic drift that can occur as a result of continuous in vitro passage. The cells werε cul- turεd in Dulbecco' s- Modifiεd Eaglε's Mεdiu containing 5% heat inactivatεd fεtal calf sεrum. The culturεs wεrε grown -in 37°C incubators with a humidified atmosphεrε containing 5% CO2. Cells were subculturεd twice weεkly by relεasing cells gently from the flask, using 0.05% trypsin and 1 mM EDTA. The melanoma cells were pulsεd in the samε fashion as thε εndothεlial cεlls dεscribεd hereiπabovε, except tha ' t 2 μCi/ml' 3 HTd(tritiated thymidiπe) was added to each culture instead of amino acids. The labelεd cεlls wεre harvested as described for the -endothelial cells. The cell adhesion assay was iden¬ tical to that describεd hεrεinabove for the bovine aortic endothelial cell assay. 1. Polypeptides I and II. The results of this assay are summarized on Table VII, below. Tablε VII. Tumor Cεll Adhεsion* Adhεrεnt Cεlls Coating Concentration (Counts Pεr Minutε) Background 1400 Pεptidε I 40 μg/ml 3900 200 μg/ml 3500 400 μg/ml 3000 2000 μg/ml 4000 Pεptidε II 40 μg/ml 4600 200 μg/ml 4700 400 μg/ml 4300 2000 μg/ml 3900 Fibronεctin 1 μg/ml 4700 10 μg/ml 7900 50 μg/ml 11,000 100 μq/ml 9700 * Mεasurεd onε hour following thε start of thε assay. In contrast to thε rεsults obtainεd abovε using nεurons and εndothεlial cεlls, pεptidεs I and II arε both capable of promoting the adhesion of melanoma cells. This may suggest cell specific differεncεs in the " adhe¬ sion of differεnt cεll typεs to this rεgion of fibro- nectin. 2. Polypeptidεs II, Ila and CS I Polypεptidεs II, Ila and CS I wεrε tεsted for thε ability to promotε thε adhεsion of mεlanoma cεlls. A comparison of thε mεlanoma adhεsion promoting activi- tiεs is shown in Tablε VIII, bεlow. Table VIII Melanoma Cell Adhεsion to Polypeptides Concentration (μg/ml) Percent Adhesion Peptide 11(80) 13.7 Pεptidε 11(400) 11.3 Pεptidε IIa(80) 6.4 Pεptide IIa(400) 18.1 Peptide CS 1(80) 71.7 Peptide CS 1(400) 71.0 Bovine Serum Albumin 3.5 " As demonstrated by the data on Table VIII, peptides II, Ila and CS I promoted the adhesion of melanoma cells in culture. Importantly, the deletion of the DEL sequence from peptide II did not eliminate the ability of this peptidε to pro otε cεll adhesion. Furthermore, the comparatively high lεvel of melanoma adhεsion-pro oting activity in CS I indicatεs that thε failurε of thε pεptidε to bind 3 H-heparin was not due to a lack of peptidε on thε substratum (since identical coating protocols were used for both the cell adhεsion and hεparin-binding assays). It is clear that pεptides Ila and CS I bind melanoma cells through distinct mechanisms. The heparin-binding activity of Ila strongly suggests that adhesion of melanoma cells to this peptidε is related to the heparin-binding properties of the peptide. These results are consistent with an ability of peptidε Ila to intεract with cell surfacε proteoglycans- glycosaminoglycans on melanoma cells (which have heparin-like qualitiεs). In contrast, pεptide CS I apparently promotes adhεsion of tumor cεlls by a heparin iπdεpεndεnt mεchanism. Thus, whilε melanoma cεlls adhεrε to both CS I and pεptidε Ila, thε biologi¬ cal activity of εach pεptidε is distinctive. 3. Peptidε III Pεptide III was examinεd for thε ability to promotε mεlanoma cεll adhεsion and sprεadlng, as des¬ cribed herεinabovε. As shown in Figurε 10, pεptidε III is activε at promoting mεlanoma cεll adhεsion in a concεntration dεpendent fashion. .In fact, pεptidε III is twicε as active as pεptidε Ila at thε highεst concεntration tεstεd, suggεsting that it could havε a highεr affinity than pεptide Ila for the surface of melanoma cεlls. This rεsult is -consistent with the.greater ability of low concentrations of peptide III to bind 3 H-hεparin whεn co parεd to pεptide Ila. A number of practical applications for these polypeptides can be envisioned. Such applications include the promotion of thε healing of wounds causεd by thε placεmεnt of natural or synthεtic substrata within the body. Such synthetic substrata can include artificial vessεls, intraocular contact lεnsεs, hip rεplacεmεnt implants, nεrvε guides and the like, where cell adhesion is an important factor in the acceptance of the synthetic implant by normal host tissue. As described in U.S. patent No. 4,578,079, medical devices can be designed making use of these polypeptidεs to attract cεlls to thε surfacε _in_ vivo or εvεn to promotε thε growing of a desired cell type on a particular surface prior to grafting. An example of such an approach is the induction of endothεlial cεll growth on a prosthetic device such as a blood vessεl or vascular graft, which is generally woven or knitted from a synthetic resin such as nitrocellulosε, εxpandεd polytεtrafluoroεthylεnε or polyεstεr fibεr, particular¬ ly Dacron™ (polyεthylεne terεphthalate) fiber. Hydrogels such as polymεthylolmεthacrylamidε (PMMA) can also be usεd for implants in the body or for objects to bε usεd in contact with mucous mεmbranes such as con¬ tact lenses. See U.S. Patent No. 3,966,902. Devicεs intεndεd for cardiac insεrtion includε temporary left ventricular assist devices, heart valves, intraortic balloon pumps and artificial hearts. Such devicεs arε prεfεrably formεd from synthεtic resins such as polyether-type polyurethane elastomers (Cardiothane TM, Koπtron) or from vulcanized polyolefin rubbers (Hexsyn , Goodyear). Most types of cells are attracted to fibroπec- tin and to the prεsεnt polypεptidεs, but εndothεlial cells, epithelial cells and fibroblastic cells in par¬ ticular arε attracted to the presεnt polypeptides. The latter point indicates the potential usefulness of thesε defined polypeptidεs in coating a patch graft or thε likε for aiding wound closurε and hεaling following an accident or surgεry. Thε coating and implantation of synthεtic polymεrs may also assist in the regenera¬ tion of nervεs following crush traumaε, e.g., spinal cord injuries. In such cases, it may be advantageous to couple or bind the peptide to a biological moleculε, such as collagεn, a glycosaminoglycan or a protεogly- can. Collagεns, protεoglycans and glycosaminoglycans arε major componεnts of connective tissues and basement membranes. In some cases, prosthetic devices formed entirely or in part from naturally-occurring mammalian -30- tissues instead of synthetic polymers are used. One example is the use of porcine heart valves to replacε defective human heart valves. Such artificial valves can also comprisε human dura matter or bovine pericar- dium. Another example is the use of bovine arteriεs as vascular grafts. It may be useful to coat surfaces of these biological substrata with the present polypeptidεs, in order to modify the cellular responsε, n_ vivo, thus improving the therapeutic outcome. This can be achieved by a variety of methods known to the art, e.g., by direct binding of the polypeptidεs to thε target surfaces based on the affinities describεd hεre- inabovε, or by thε covalεntly bonding thε polypεptidεs to the substrate using various crosslinking rεactions or reagents. For a reviεw of thε usε of synthεtic resins and biomaterials in prosthεtic dεvicεs, sεε Chεm. & Enq. Nεws (April 14, 1986) at pagεs 30-48, thε disclosurε of which is incorporatεd by rεfεrεncε hεrεin. It is also indicative of their value in coat¬ ing surfaces of a prosthetic devicε which is intendεd to sεrvε as a tεmporary or sεmipεrmanεnt εntry into thε body, ε.g., into a blood vεssel or into thε pεritonεal cavity, somεtimεs rεfεrrεd to as a pεrcutaneous devicε. Such dεvicεs includε catheters, and controlled drug . delivery reservoirs or infusion pumps. Also, the polypεptidεs, ε.g., I and II, can bε usεd to promotε εndothεlial, fibrob-last or εplthεlial cεll adhεsion to naturally occurring or artificial substrata intεndεd for usε _in_ vitro. For example, a culture substratum such as the wells of a microtiter platε or thε mεdium contacting surfacε of microporous fibεrs or beads, can be coated with the cell-attachmεnt polypεptidεs. This can obviatε thε usε of fibronεctin -31- in thε mεdium, thus providing bεtter definεd conditions for the culturε as well as bεttεr rεproducibility. As onε εxample of commercial use of cell- attachment surfaces, Cytodex particles, manufactured by Pharmacia, are coated with gelatin, making it possible to grow the samε numbεr of adhεrεnt cells in a much smaller volume of mεdium than would be possiblε in dishes. The activity of- these beads is gεnerally depεndent upon the use of fibronectin in the growth medium and the present polypeptidεs arε expected to provide an improved, chemically-defined coating for such purposes. Other surfaces or materials may be coated to enhancε attachment, such as glass, agarosε, synthεtic rεsins or long-chain polysaccharidεs. The invention has bεen describεd with rεferencε to various spεcific and prεfεrrεd εmbodimεnts and tεchniques. However, it should be understood that many variations and modifications may be made while rεmaining within the spirit and scopε of the invention.

Claims: WHAT IS CLAIMED IS; 1. A composition consisting εssεntially of a polypεp- tide of the formula sεlεcted from thε group con¬ sisting ofJ tyr-glu-lys-pro-gly-ser-pro-pro-arg-glu-val-val- pro-arg-pro-arg-pro-gly-val, lys-asn-asn-gln-lys-ser-glu-pro-leu-ile-gly-arg- lys-lys-thr-asp-glu-lεu, lys-asn-asn-gln-lys-ser-glu-pro-lεu-ilε-gly-arg- lys-lys-thr, lεu-ilε-gly-arg-lys-lys-thr, tyr-arg-val-arg-val-thr-pro-lys-glu-lys-thr-gly- pro-mεt-lys-glu, ser-pro-pro-arg-arg-ala-arg-val-thr, trp-gln-pro-pro-arg-ala-arg-ile, and mixtures thεrεof. 2. A prosthεtic dεvice dεslgnεd for placεment _in_ vivo, comprising a surfacε coatεd with a composition con¬ sisting εssεntially of a polypεptidε of thε form¬ ula sεlεctεd from. the group consisting of» tyr-glu-lys-pro-gly-ser-pro-pro-arg-glu-val-val- pro-arg-pro-arg-pro-gly-val, lys-asn-asn-gln-lys-ser-glu-pro-lεu-ilε-gly-arg- lys-lys-thr-.asp-glu-lεu, lys-asn-asn-gln-lys-ser-glu-pro-leu-ile-gly-arg- lys-lys-thr , leu-ile-gly-arg-lys-lys-thr , tyr-arg-val-arg-val-thr-pro-lys-glu-lys-thr-gly- pro-mεt-lys-glu, sεr-pro-pro-arg-arg-ala-arg-val-thr, trp-gln-pro-pro-arg-ala-arg-ilε, and mixturεs thεrεof. 3. Thε prosthεtic dεvicε of claim 2, whεrεin said sur¬ facε constitutεs a portion of a vascular graft. 4. Thε prosthεtic dεvicε of claim 2, whεrεin said sur¬ facε constitutεs a portion of an intraocular con¬ tact lens. 5. The prosthetic devicε of claim 2, whεrεin said sur¬ facε constitutεs a portion of a hεart valvε. 6. Thε prosthεtic dεvicε of claim 2, whεrεin said sur¬ facε constitutεs a portion of a hip rεplacεmεnt implant. 7. Thε prosthεtic dεvicε of claim 2, wherein said sur¬ face constitutes a portion of a percutanεous dεvicε. 8. A prosthetic device of claim 2 wherein said surface is made of a synthetic resin. 9. A prosthetic, device in accordance with claim 8 wherein said synthetic resin is selected from the group consisting of nitrocεllulosε, polyurεthanε, expanded polytetrafluoroethylene, polyestεr and polyolefin. 10. A prosthetic device of claim 2 whεrεin said surfacε is made of a naturally-occurring tissue. 11. A cell culture substrate having a surface coated with a composition consisting essεntially of a polypeptide of the formula* tyr-glu-lys-pro-gly-ser-pro-pro-arg-glu-val-val- pro-arg-pro-arg-pro-gly-val, lys-asn-asn-gln-lys-sεr-glu-pro-lεu-ilε-gly-arg- lys-lys-thr-asp-glu-lεu, lys-asn-asn-glπ-lys-sεr-glu-pro-lεu-ilε-gly-arg- lys-lys-thr, lεu-ilε-gly-arg-lys-lys-thr, tyr-arg-val-arg-val-thr-pro-lys-glu-lys-thr-gly- pro-mεt-lys-glu, sεr-pro-pro-arg-arg-ala-arg-val-thr, trp-gln-pro-pro-arg-ala-arg-ile, and mixturεs thereof. 12. The cell culturε substratε of claim 11 wherein said surface is made of a synthetic resin. 13. The cell ' culturε substratε of claim 11 wherein said surface constitutes a portion of a bead. 14. The cell culture medium of claim 11 wherεin said surface constitutes a portion of a microporous fiber. 15. The -cell culturε mεdium of claim 11 whεrein said surface constitutεs the wells of a microtiter plate.

Inventors: FURCHT LEO T, MCCARTHY JAMES B, FURCHT, LEO, T., MCCARTHY, JAMES, B.
Applicants: UNIV MINNESOTA, REGENTS OF THE UNIVERSITY OF MINNESOTA
Application year: 1988
Main classification: C07K7
IPC codes: A61F2, A61K38, A61L15, A61L27, A61P43, C07K7, C07K14, C12N5