AU633377B2 - Novel polypeptides having growth factor activity and nucleic acid sequences encoding the polypeptides - Google Patents

Description

4 i S F Ref: 48687/8501 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 337 COMPLETE SPECIFICATI N

(ORIGINAL)

FOR OFFICE USE: Class Int Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: Name and Address of Applicant: Address for Service: Oncogen 3005 First Avenue Seattle Washington 98121 UNITED STATES OF AMERICA Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia

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7:A Complete Specification for the invention entitled: Novel Polypeptides \-v\ac o)- 0 ct1 \v Fe C C%-s.

The foilowing statement is a full description of this invention, including the best method of performing it known to me/us r.

P "S5845/3

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%T .Q V.K r i 14 The fragments will generally be of from about 15 to 50, usually 15 to

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TECHNICAL FIELD A significant number of polypeptides secreted by mammalian cells are I found to have growth factor activity. These compounds have been found to i be substantially conserved over a wide range of mammals. Much of the i interest in these compounds is stimulated by their acsociation with i oncogenicity. There is also interest in how the production of the growth factors is regulated and how they in turn regulate cellular activity.

It has also been noted that infection of mammalian cells by viruses ji results in proliferation of the growth of the cells. For the purpose of :this invention, of particular interest are members of the poxvirus family, i such as variola, vaccinia and, such viruses associated with particular diseases such as Shope fibroma virus, Yaba tumor virus, and Molluscum contagiosum virus (MCV).

SIt would be of interest to determine whether viruses causing cellular proliferation upon infection, produced polypeptides involved with growth -i factors, either acting as the growth factor or the growth factor receptor.

These compounds could then be used in the studies of viral actions, in i; assays for the presence of the virus, in nutrient media, as mitogens, and in the development of therapeutic agents for treating viral infection. It is also of interest to develop compounds which may be agonists or antagonists of growth factors for use in vitro in growing cell cultures, in I investigating mitotic processes, and in therapy.

i TMS/1416R 2 BACKGROUND ART Venkatesan et al., J. Virol. (1982) 44:637-646 describes the DNA sequencing of structural genes encoding vaccinia virus proteins. Cooper et al., ibid (1981) 37:284-294, report the translation of mRNA's encoded within the inverted terminal repetition of the vaccinia virus genome. Proliferative diseases for members of the poxvirus family have been reported for Shope fibroma virus (Shope, J. Exp. Med. (1932) 56:793-822; Yaba tumor virus (Niven et al., J. Path.

Bacteriol. (1961) 81:1-14) and Molluscum contagiosum virus (MCV) (Postlethwaite, Arch. Environ. Health (1970) 21:432-452). Descriptions of epidermal growth factor (EGF) may be found in Scott et al., Science (1983) 221:236-240 and Gray et al., Nature (1983) 303:722-725. The presence of three disulfide bridges in EGF and transforming growth factor (TGF) is reported by Savage et al., J. Biol. Chem. (1973) 248:7669-7692.

See also Doolittle et al., Nature (1984) 307:558-560.

The receptor binding region of the EGF molecule has been suggested as lying in the loop between the third and fourth cysteine residues. (Komoriya et al., Proc.

Natl. Acad. Sci. USA (1983) 81:1351-1355.) New descriptions of vaccinia virus growth factor (VGF) may be found in Brown et al., Nature (1985) 313:491-492; Reisner, Nature (1985) 313:801-803 and Blomquist et al., Proc. Natl. Acad. Sci. USA (1984) 81:7263-7367. The disclosure of these references is incorporated herein by reference.

Expression of foreign peptides employing a baculovirus vector in insect cells is described by Maeda et al., Nature (1985) 315:592-594 and Carbonell et al., J. of Virology (1985) 56:153-160. These disclosures are also incorporated herein by reference.

I: -e lil-Ijii~- i ii 3 DISCLOSURE OF THE INVENTION Polypeptide compositions are described finding analogy in fragments of viral proteins, which compositions act as mitogens and can ue used in nutrient media, as reagents for the detection of growth factor receptors or the presence of growth factors, and as competitors for transforming growth factor and epidermal growth factor. The compositions may be synthesized.

The peptides of the composition may be formed as oligopeptides or fused proteins employing recombinant techniques in a-wide variety of hosts. The polypeptides of the invention are generally immunogens or can be rendered immunogenic. The polypeptide of the invention finds therapeutic use, for example, to promote epithelialization and healing of burns and wounds.

According to a first embodiment of this invention there is provided a mature VGF polypeptide of a purity of at least 90ng equivalents of EGF per microgram of protein.

According to a second embodiment of this invention there is provided a method for inducing an immunogenic response in a mammal, comprising administering to said mammal an immunogenically effective amount of a polypeptide according to the first embodiment.

According to a third embodiment of this invention there is provided a method for enhancing cellular proliferation in a mammal, comprising administering to said mammal an effective amount of a polypeptide as defined in the first embodiment.

According to a fourth embodiment of this invention there is provided a method for promoting wound healing in or on a mammal requiring such healing, comprising administering to the mammal and/or applying to the wound on the mammal, an effective amount of a polypeptide as defined in the first embodiment.

S: BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an amino acid sequence comparison of the vaccinia virus protein with known growth factors, where the initial amino acid of the VVF is aspartic acid at position 20; and Figure 2 is a fragment of the vaccinia virus protein, beginning at amino acid 44 and terminating at amino acid 90, with residues which are identical in the polypeptide fragments, mEGF, hEGF and rTGF being crosshatched; Figure 3 is a comparison of the EGF receptor binding inhibition curves for mouse EGF and N-gene/TGF/TGF/VGF after 7 days of refolding.

TMS/1416R -4 BEST MODES FOR CARRYING OUT THE INVENTION Compositions which can act as agonists or antagonists of epidermal growth factor (EGF) or transforming growth factor (TGF) are provided, which provide for a wide variety of applications in cell culture, diagnostics, in vivo therapy, and in combination with other peptides in the formation of hybrid polypeptides as immunogens for production of antibodies.

Polynucleotide sequences may be isolated or prepared which may be introduced into an expression vector for expression of the peptides of the compositions.

The compositions find analogy in fragments of viral proteins, particularly poxvirus proteins. The poxvirus proteins are found to have regions which have structures analogous to surface membrane proteins. The structural gene coding for the polypeptide has regions analogous to regions functioning as a leader sequence and processing signal and as a transmembrane integrator sequence, with the growth factor fragment sequence intermediate the leader and integrator sequence.

The polypeptides are characterized by having at least one loop or circle, usually three loops or circles, as a result of cysteines which form bridges, where the physiologically active portion of the molecule providing a growth factor related activity will be from about 12 to 65 amino acids, usually 15 to 60 amino acids. Of the three loops, two of the loops are of from 12 to 15 amino acids (14-17 annular amino acids), exclusive of the cysteine bridge, more particularly one is of 12 or 13 amino acids (14 or annular amino acids) and the other of 15 amino acids (17 annular amino acids), where the N-terminal proximal loop will usually be of from 12 to 13 amino acids, more usually of 12 amino acids, and the middle loop will be of amino acids.

The third loop or C-proximal loop will be of 8 amino acids annular amino acids) and including flanking amino acids will have the following formula: 3 25 27 29 30 PP (aa -C-aa C-aa -aa -G-Y 4 40 39 38 35 1 PP -(aa -aa -aa R- aa- G-A1 wherein: the one letter symbols for amino acids have their conventional meaning, wherein C is cysteine, G is glycine, Y is tyrosine, and R is arginine; TMS/1416R V| Al intends a neutral amino acid, which will i be described in more detail below, particularly, i wherein Al is an aliphatic amino acid of from 2 to 6, more usually of from 3 to 6 carbon atoms having from 0 to 1 hydroxyl group, more particularly serine, threonine, valine, leucine or isoleucine, wherein the hydroxy substituted amino acids have from 3 to 4 carbon atoms, and the unsubstituted or alkyl substituted glycine amino acids have from 5 to 6 carbon atoms; aa 2 is.a neutral amino acid which may be I aliphatic, particularly of from 3 to 4 carbon atoms, or aromatic, particularly of 9 carbon atoms, and having i from 0 to 1 oxy substituents, alanine, serine, threonine or tyrosine; 27 aa may be neutral or basic, being particularly of from 4 to 6 carbon atoms, and when neutral having from 0 to 1 carboxamide group, e.g., arginine, valine, leucine, isoleucine, asparagine or I glutamine; 29 aa is a neutral amino acid, being aliphatic i or aromatic, wherein aromatic is exemplified by histidine and aliphatic is of from 2 to 6, more usually of from 3 to 6 carbon atoms, having from 0 to 1 hydroxyl group, serine, threonine, leucine, valine or isoleucine; 30 aa is a neutral amino acid, being aliphatic or aromatic, wherein aromatic is exemplified by histidine and aliphatic is of from 3 to 6 carbon atoms, having a chain of other than hydrogen or from 5 to 6 atoms, and having from 0 to 1 hydroxyl group, particularly serine, isoleucine and valine, and wherein 29 30 aa and aa will usually be different, particularly one being histidine and the other being serine; aa is a neutral or acidic amino acid, of from 4 to 6 carbon atoms', where neutral is exemplified by valine, leucine and isoleucine and where acidic is exemplified by aspartic and glutamic acids; I I 7 i

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iit i 6 11, aa 38 is an aliphatic neutral substituted amino acid or acidic amino acid, wherein the substituent is carboxamide and is of from 4 to 5 carbon atoms, asparagine, glutamine, aspartic acid or glutamic acid; aa 39 is an aromatic amino acid or a neutral allphatic amino acid of from 3 to 4 carbon atoms having an hydroxyl substituent, preferably aromatic, histidine, tyrosine, serine or threonine; aa 40 is neutral unsubstltuted aliphatic or basic amino acid, wherein the neutral amino acids are of from 3 to 5 carbon atoms, e.g., alanine, valine, lysine or arginine; m and n are 0 or 1;

PP

3 and PP 4 are either hydrogens, indicating the termination of the polypeptide or may be polypeptide chains of not greater than a total of 1000 amino acids, usually of not greater than a total of about 500 amino acids, preferably where at least 90% of the amino acids, more preferably at least about 95% of the amino acids present are in one of the two polypeptide chains; in some instances, the chain may be of only one amino acid and not more than 100 amino acids, frequently of not more than about amino acids, depending upon the use of the polypeptide and the role of the extended chain; the polypeptide chains may be related to the naturally occurring polypeptide chains of the present invention associated with naturally occurring growth factors and pox virus proteins or may be other than the naturally occurring chains or fragments thereof associated with the polypeptide chain specifically set forth in the formula, usually unrelated.

(By unsubstituted is intended no other heterosubstituents than the carboxy and amino group present in glycine. All the amino acids are the natural L-stereomer.) The definitions of the amino acids are set forth below.

Neutral (Ne) aliphatic (Al) unsubstituted G, A, V, L, I, P substituted oxy S, T thio C, M amido N, Q ::f I-t 1 t ii TMS/1416R 7 aromatic (Ar) unsubstituted F substituted Y heterocyclic H, N Charged basic (Ba) K, R acidic (Ac) D, E The abbreviations in the parentheses refer to the particular amino acid groups. The amino acids are the naturally occurring L-amino acids.

SThe loop between the cysteines in the above formula will have from 0 to 1 acidic amino acid, preferably no acidic amino acids, and the amino acids immediately flanking the cysteines outside the loop will include at least one charged amino acid, preferably a basic amino acid, more preferably, having in addition an amido substituted aliphatic amino acid.

Of the amino acids in the loop from 4 to 6, usually 5 amino acids, will be neutral aliphatic amino acids and not more than 2, usually 2, will be aromatic amino acids.

Of interest are compounds, where the polypeptide is of fewer than 130 amino acids, more particularly of fewer than 50 amino acids, and at least amino acids, preferably at least about 42 amino acids. Of interest are polypeptides including amino acids 44 to 86 as indicated in Figure 1 of VVP.

Preferably aa 29 is a substituted or unsubstituted aliphatic amino acid of from 3 to 6 carbon atoms having from 0 to 1 hydroxyl group, particularly serine; aa 3 is preferably histidine, serine or an unsubstituted aliphatic amino acid of from 5 to 6 carbon atoms, particularly where one of aa 29 S and aa 30 is histidine and the other is serine; S. Al is preferably a substituted aliphatic amino acid having one hydroxyl group and of from 3 to 4 carbon atoms; Saa 35 is preferably an unsubstituted aliphatic amino acid of from to 6 caron atoms; aa 38 is preferably a substituted aliphatic amino acid of from 4 to carbon atoms, wherein the substitution is carboxamide; 39 aa 3 is preferably aromatic, more preferably histidine.

Of interest is the presence of two loops, with the C-proximal loop joined to the second loop, where the amino acids of the second loop may be widely varied. The second loop is preferably of from about 14 to 16 amino TMS/1416R I :I

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ii -8-

B

acids exclusive of the cystelne bridge, more preferably of about 15 amino acids. Of the amino acids, from 6 to 9, preferably 7 to 9, more preferably about 8 are aliphatic amino acids either substituted or unsubstituted, preferably not more than about 3 of the amino acids being substituted, more preferably from 1 to 2 amino acids being substituted; there will be from 2 to 4 aromatic amino acids, more particularly 3 aromatic amino acids, desirably histidine and tyrosine; there will be from 2 to 4 acidic amino acids, preferably 3 acidic amino acids, more particularly aspartic acid; and there will be from 0 to 2, more usually 1 basic amino acid, particularly arginine. Desirably, the cysteine forming the subject loop closest to the cysteine of the other loop, will be separated by from 0 to 2, more usually from 0 to 1 amino acids, particularly arginine.

Of interest for some applications for the compounds are compounds of the following formula: i:

~A

b

I

~1"

PP

1 -aa -C-aa 3 -aa -aa 5 -aa -aa -aa* -Y-C- 11* 12 12 a 14 16 2 19 aa -aa (aa -G-aa -C-aa -Ar -A 1 -aa -aa aa 2 1 Ac-aa 23 aa 24 -(aa 2 5 C-aa 2 7 C-aa 2 9 aa 30 G-Y-A l-G-aa 3 5 -R-C-aa 38 *-aa 3 9 aa 4 1 -L-aa 4 3 -aa 4 4

PP

2 wherein: the symbols between the parentheses (except for aa 2a) in the formula have already been described; PP1 and PP 2 are the same or different and may be hydrogens, indicating the terminal portion of the indicated polypeptide or may be polypeptides having a total of up to about 1000 amino acids, more usually of up to about 500 amino acids and may have a total of as few as 1 amino acid, or may individually or separately be polypeptides of from 1 to 100 amino acids, more usually from about 1 to 75 amino acids, more particularly from about 5 to 50 amino acids; these polypeptides will have specific applications in modifying the specifically described sequence for a predetermined purpose; PP 1 and PP 2 may be the same or different from the natural pox virus polypeptide, of the present invention usually different; TMS/1416R -9the indicates th conservation of the amino acid of the vaccinia virus peptide and mouse and human epidermal growth factor, where a space is introduced between amino acids 55 and 56 of the vaccinia virus peptide in order to provide for the alignment of cystelnes defining the loops, as well as other amino acids; aa may be any amino acid, more particularly an aliphatic amino acid, basic amino acid or acidic amino acid, preferably an unsubstituted aliphatic amino acid of from 2 to 6 carbon atoms, more particularly glycine and leucine; aa is a neutral amino acid, particularly of from 2 to 4 carbon atoms, more particularly glycine and proline; aa 4 may be any amino acid, particularly of from 2 to 6 carbon atoms, which may be neutral, acidic or basic, more particularly of from 3 to 6 carbon atoms, including proline, aspartic acid, serine and arginine; aa 5 is an acidic or a neutral substituted aliphatic amino acid, particularly glutamic acid or an hydroxy substituted amino acid of from 3 to 4 carbon atoms, more particularly serine; aa is an unsubstituted aliphatic amino acid of from 2 to 6, usually 2 to 3, carbon atoms or an aromatic amino acid, particularly glycine, histidine or tyrosine; aa 7 is an acidic or neutral substituted allphatic amino acid, particularly of from 3 to 5, more particularly 4 carbon atoms, such as aspartic acid and threonine; aa is a neutral unsubstituted aliphatic amino acid of from 2 to usually 2 to 3, carbon atoms, particularly glycine; or a carboxylate substituted amino acid of from 4 to 5 carbon atoms, particularly glutamine; aa 11 is a neutral amino acid, either aliphatic or aromatic, more S particularly aliphatic, particularly of from 5 to 6 carbon atoms, more particularly leucine or phenylalanlne; aa 12 is an aromatic amino acid or a carboxamlde substituted aliphatic amino acid of from 4 to 5 carbon atoms, particularly histidine or asparagine; aa 12 a is a neutral aliphatic amino acid or acidic amino acid, more particularly glycine or aspartic acid; p is 0 or 1; aa 14 is an acidic amino acid or neutral aliphatic amino acid substituted or unsubstituted of from 4 to 5 carbon atoms, having from 0 to 1 hydroxyl group, particularly aspartic acid, threonine or valine; TMS/1416R 10 aa 16 is a neutral or basic aliphatir amino acid, either substituted or unsubstituted, of from 4 to 6 carbon atoms, particularly isoleucine, arginine or methionine; Ar intends an aromatic amino acid, which may have a carbocyclic or heterocyclic ring, and includes histidine, phenylalanine or tyrosine; Al 2 is a neutral aliphatic amino acid of from 2 to 6, preferably of from 3 to 6 carbon atoms, particularly alanine, leucine and isoleucine; aa 9 may be any amino acid, particularly aliphatic acidic or basic amino acids, more particularly of from 4 to 6 carbon atoms, more preferably of from 5 to 6 carbon atoms, particularly arginine, valine and glutamic acid; aa 20 is an acidic amino acid or neutral aliphatic amino acid, either substituted or unsubstituted of from 3 to 6, usually 3 to 5, carbon atoms, having from 0 to 1 hydroxyl group, and includes aspartic acid, glutamic, serine or alanine; 21 aa is a neutral unsubstituted aliphatic amino acid or acidic amino acid of from 5 to 6 carbon atoms; Ac is an acidic amino acid which is aspartic acid or glutamic acid; aa 23 is a neutral aliphatic or basic amino acid, of from 2 to 6 carbon atoms, when neutral, of from 2 to 4 carbon atoms having from 0 to 1 hydroxyl substituent, glycine and serine; and when basic, being lysine or arginine, particularly lysine; 24 aa 24 s an aliphatic amino acid, proline or an aromatic amino acid, particularly a thiosubstituted aliphatic amino acid, more particularly methionine, or tyrosine; aa 41 is a neutral unsubstituted aliphatic or acidic amino acid of from 4 to 6 carbon atoms, particularly valine or aspartic acid; aa 43 is a neutral aliphatic or basic amino acid, particularly a neutral aliphatic amino acid which may be substituted or unsubstituted of S from 4 to 6 carbon atoms, either unsubstituted or carboxamide substituted J and includes valine, leucine, arginine and lysine; and aa 44 may be any amino acid, particularly other than a basic amino acid, and may be acidic, neutral or aromatic, when other than aromatic, being of 3 to 4 carbon atoms, particularly aspartic acid, alanine, or tryptophan.

Of interest is when PP has the following sequence: TMS/1416R 11 1 -aa-27 aa-26 aa-25aa-24aa-23-aa22aa-21-aa- 2 0 aa-19 aa-8 -aa-17 aa-16 aa-15 aa-14 aa-13 aa-12-aa11 aa -aa -aa -aa -aa -aa -aa -aa -aa -aa- 8 -aa-7_aa-6_aa-5-aa-4-aa-3-aa- 2 -aa" wherein:

PP

1 in combination with the subsequent amino acid symbols in the 1 1 above sequence is the equivalent of PP 1 The above sequence comes within the definition of P. PP may be hydrogen or an amino acid sequence.

One or more of the amino acids, symbolized by aa x (X is any number), may be a bond, so as to serve to reduce the number of amino acids in the N-terminal chain. Therefore, all or a portion of the amino acid sequence ft TMS/1416R 12- I 1 2

Y

A

A

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indicated may be present. When a portion of the sequence is present, preferably the sequence will involve contiguous amino acids, that is, amino acids in their numerical order without deletions. Usually, there will be at least one amino acid, more usually at least three, more usually at least five, where the remaining upstream amino acids may be absent, so that 1' -1 -5 PP may be joined to aa aa or the like; aa may be any amino acid, particularly aliphatic, of from. about 3 to 6 carbon atoms preferably neutral or basic, more particularly neutral or basic of from about 5 to 6 carbon atoms; -2 aa2 may be any amino acid, either aliphatic or aromatic, particularly aliphatic, more particularly 15 of from about 4 to 6 carbon atoms; -3 aa may be ali atic or aromatic, particu.'arly aliphatic, either polar or non-polar, of from 2 to 6, more usually of from 2 to 5 carbon atoms, generally having from 0 to 1 hydroxyl substituent; -4 aa may be any aliphatic amino acid, particularly neutral, generally of from 3 to 6 carbon atoms, usually of from 3 to 5 carbon atoms, and may be polar or non-polar, particularly proline and asparagine; aa may be any aliphatic amino acid, particularly a neutral or basic amino acid, more particularly of from about 4 to 6 carbon atoms, preferably an aliphatic neutral amino acid; -6 aa is an aliphatic amino acid of from about 3 to 6, more usually of from about 4 to 6 carbon atoms, particularly acidic or aliphatic, more particularly acidic; 4 4

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BI

-11, -15, -16, -17, -23 and -27 aa are aliphatic amino acids of from 3 to 5 carbon atoms, either polar or non-polar, particularly polar having from 0 to 1 hydroxyl substituent; I 13 9 -20 -23 -25 aa, and are non-polar aliphatic amino acids of from 2 to 6, more usually of from 2 to 3 carbon atoms; |a 0 and -21 are polar aliphatic amino acids of from 4 to carbon atoms, particularly having a carboxamido substituent; aa and are aliphatic amino acids of from 2 to 6 carbon atoms, more particularly of from 4 to 6 carbon atoms; S13 -18 -24 Saa- and are aliphatic acidic amino acids; and aa-26 is an aromatic amino acid, particularly phenylalanine.

Of particular interest is an N-terminal fragement of from aa to iaa 2 4 and aa to aa 7 more particularly aa to aa PP Smay conveniently be an unrelated amino acid sequence which may serve as a ;fusion protein, particularly to provide VGF of the present invention as an immunogen for production of antibodies to VGF and its congeners, such as EGF and TGF-c.

The primary aspects of the compositions are the sequence in the parentheses, particularly between the cysteine at position 28 and the cysteine at position 37 and the loops generated by the cysteines at positions 2 and 10 and the cysteines at positions 15 and 26. Thus, i desirably, the compositions have the loop created by the cysteines at positions 28 and 37 in conjunction with the loop created by the cysteines at positions 15 and 26.

Varying combinations of polypeptide sequences may be prepared by employing as one fragment the C-terminal portion of one sequence with the N-terminal portion of another sequence as a second fragment, whereby polypeptides of from about 40 to 65 amino acids, usually about 50 to amino acids are provided. Desirably, the juncture will be made at some 20 27 20 point between aa 20 and aa particularly aa and aa25 Preferably the fragments are joined at a site not more than 5 amino acids from the sequence C-X-C, where X is any amino acid. In each case, the framework structure of the cysteines is retained with the cystine bridges defining loops of the sizes described previously. Thus, a fragment may be employed from any EGF, TGF, VGF (portion of the vaccinia virus having substantial homology with other growth factors and depicted in Fig. or other growth factor having the same framework structure of the subject compositions and similar physiologic activity. This will be regardless of the mammalian source, such as primate, human, rodent, rat and mouse, bovine, avian, porcine, etc.

TMS/1416R i i 1 I 14 The fragments will generally be of from about 15 to 50, usually 15 to amino acids, since aa 20 does not Intend the twentieth amino acid of the compound, but only of the specific sequence which has been specifically defined.

Of particular Interest is where VGF of the present invention is modified where VGF has an alanine substituted for a tyrosine which introduces an SphI restriction site. This provides for a convenient site for linking one fragment from the VGF gene to a fragment from another growth factor. Of course, the entire sequence could be synthesized without any amino acid changes. Thus the polypeptides may be synthesized in accordance with conventional polypeptide synthesizing techniques, using a commercially available polypeptide synthesizer.

The compositions may be unglycosylated or glycosylated. Depending upon the particular ,mino acid sequence, one or more glycosylation sites may be present, usually not more than six, more usually not more than three, glycosylation sites. The glycosylation sites may or may not be glycosylated. The saccharides will usually add not more than 6kDal (kilodaltons), usually not more than about 5kDal, to the total molecular weight of the active polypeptide. Sugars involved will be mannose, glucose, N-acetylglucosamine, galactose, glucuronic acid, galacturonic acid, sialic acid, etc.

The compositions find a wide variety of applications in vitro and in vivo as agonists or antagonists for growth factors, such as epidermal growth factor (EGF), and transforming growth factor (TGF), particularly

TGF-I.

A discussion of growth factors, used by themselves or in combination with other compositions, particularly polypeptide compositions, for regulating the growth of cells, and other activities, may be found, for example, in Handbook of Experimental Pharmacology. Tissue Growth Factors, ed. Baseraga, Vol. 57, Springer-Verlag, Berlin, 1981, Chapter 3, particularly pages 98-109; and Carpenter, Ann. Rev. Biochem. (1979) 48:193-216.

hEGF appears to be identical to human urogastrone. EGF has been found to exert a variety of effects on prenatal and neonatal tissue growth. Among the effects are precocious eye-opening, wound healing, incisor eruption, and acceleration maturation of the lung. EGF receptors are found in a wide variety of adult tissues. EGF is found to stimulate TMS/1416R 28 Partial purification of VGF. The activity competing with EGF found in W-infected BSC-1 cells was n:i-ar+i a4 1 1 \u ni -r f i P e9 f-rnm a -iI -e -j: 15 phosphorylation of Its own receptor. EGF is also found to be related to increased bone resorption.

Transforming growth factor, particularly TGF-I or TGF-o, has many analogous activities to that of EGF. TGF binds to the EGF receptor leading to phosphorylatlon of the receptor, enhancement of its tyrosine-specific kinase activity and to stimulation of cell growth. Cohen, in: Biological Response Mediators and Modulators (ed. August, Academic, New York, 1983, pp. 7-12; Tam it Nature (1984) 309:376-378; Ibbotsom et al., Science (1983) 221:1292-1294.

The mature VGF polypeptide may have particular application as drugs as agonists for EGF, and for wound TMS/1416R 16 healing, such as epithelialization of wounds, such as burns, eye wounds, surgical incisions, and the like.

The active ingredient may be employed in a convenient vehicle, Silvadene, in amounts ranging from about 0.01 to 0.5, usually from about 0.075 to 0.2ug/ml. The formulation is spread over the wound, so as to provide a complete coating of the wound with the formulation.

Treatments may be as frequent as four times a day or as infrequent as every other day or less, depending upon the nature of the wound, its response to the treatment, the concentration of the active ingredient, and the like.

The subject compositions can be used as reagents in diagnostic assays or for the preparation of reagents, such as polyclonal or monoclonal antibodies.

As reagents, they may be used for the detection of analogous growth factors or for the detection of antibodies to the growth factors in physiological fluids, such as blood.

Depending upon the particular protocol and the purpose of the reagent, the polypeptide may be labeled or unlabeled. A wide variety of labels have been used which provide for, directly or indirectly, a detectable signal. These labels include radionuclides, enzymes, fluorescers, particles, chemiluminescers, S. enzyme substrates or cofactors, enzyme inhibitors, magnetic particles, etc. See for example, U.S. Patent Nos. 3,654,090, 3,817,837, 3,935,074, 3,996,345, 4,277,437, 4,374,925, and 4,366,241.

A wide variety of methods exist for linking the labels to the polypeptides, which may involve use of the N-terminal amino group for functionalization to form a pyrazolone, while other free amino groups are protected, where the pyrazolone may then be contacted with various reagents, amino groups, to link to the detectable signal generating moiety. By protecting the arginine amino acids associated with the third loop -17 or proximal thereto, other arginines may be functionalized for conjugation to amino groups or thio groups in accordance with known ways.

Alternatively, the polypeptide may be contacted with an active agent, e.g., an activated carboxylic acid and randomly substituted, where biologically active material may be separated from biologically inactivated material as a result of the random substitution. Finally, depending upon the method of synthesis, the polypeptide may be modified to provide for the desired functionality as part of the synthetic procedure.

The subject polypeptide can also be used for monitoring EGF Sreceptors. The subject polypeptide can also be used for monitoring cellular response to EGF and/or TGF by providing or competition between these naturally occurring materials and a composition according to the subject invention. In this way, changes in the receptor conformation can be monitored.

Depending upon the particular composition of this invention which is employed and the purpose for the additive, for in vitro use, the concentration of the additive will vary widely, due to fluctuations in activity, varied purpose, and variations in receptors.

The subject polypeptide can also be used for various therapeutic purposes involving growth stimulation or control of bone formation. These compounds may be administered in appropriate physiological carriers intraperitoneally, subcutaneously, intravenously, intraarterially, or by application to the site of interest. In addition, the subject compositions can be introduced into liposomes, which may or may not involve the use of antibodies for site direction. Various carriers include phosphate buffered saline, saline, water, or the like. The concentration of the additive will vary widely, depending upon its ultimate use and activity.

Other additives may also be included in the formulations, such as EGF, TGF, other growth factors, bacteriocides, antibiotics, bacteriostats, buffers, etc.

For preparing antibodies, the subject polypeptides where PP -4 are hydrogen or short oligopeptide chains (fewer than five amino acids), may be joined to antigenic polypeptides or proteins, for injection into mammalian hosts. The antigenic protein will have at least about 60 amino acids and will usually be not more than 10 kilodaltons (kDal). Numerous techniques exist for joining to polypeptides, either at a specific site or randomly, using bifunctional reagents, a-maleimidobenzoic acid, glutaraldehyde, TMS/1416R UI~p III~IICI I i irm~- 18 -,p'-benzidine, etc. Common antigenic proteins include bovine serum albumin, keyhole limpet hemocyanin, tetanus toxoid, etc. The subject polyptntides are joined to the antigenic protein in sufficient number to provide the desired immunogenic response. Usually there will be two or more booster injections after the initial injection. For antisera, blood is removed from the immunized host and the immunoglobulin fraction isolated. For monoclonal antibodies, the spleen is isolated and splenocytes fused with an appropriate fusion partner in accordance with conventional ways. The resulting hybridomas are then screened for j antibodies binding to the epitopic sites of the subject polypeptide. These antibodies may be used for a variety of purposes, such as diagnostic reagents, therapy, etc. The antibodies when used as reagents may be labeled or unlabeled, as described for the polypeptides.

i The synthetic compositions can be prepared in a variety of ways depending on the size of the composition. Particularly below about more particularly below about 60 amino acids, the composition can be prepared by synthesis in accordance with conventional ways. See, for example, Merrifield, Solid-Phase Peptide Synthesis, "The Peptides Analysis, Synthesis, Biology," Special Methods in Peptide Synthesis, Part A, Vol. 2, Gross and Meinhofer eds., Academic Press, NY, 1980, pp. 1-284. See also, U.S. Patent No. 4,127,526.

Alternatively, the use of hybride DNA technology can be employed, where DNA sequences can be used which code for the synthetic polypeptide or precursor thereof.

DNA sequences can be synthesized employing conventional techniques such as overlapping single strands which may be ligated together to define .7 the desired coding sequence. The termini can be designed to provide restriction sites or one or both termini may be blunt-ended for ligation to complementary ends of an expression vector. For expression of the sequence an initial methionine is provided. Expression vectors are generally available and are amply described in the literature.

Instead of synthesizing the structural gene, a poxvirus may be isolated and by various techniques, the mRNA isolated, which codes for the polypeptide including the growth factor, the mRNA reverse transcribed, the resulting single-stranded (ss) DNA used as a template to prepare doublestranded (ds) DNA and the ds DNA gene isolated. This gene may then be manipulated in a variety of ways to remove undesired untranslated region or TMS/1416R kL 7 19 undesired codons, for example, employing primer repair, in vitro mutagenesis to introduce a restriction site or different amino acid at one or more appropriate sites, or introduction into a vector, followed by restriction and exonuclease digestion to remove the terminal bases. In this way, the gene may be reduced to the desired number of codons.

Where a convenient restriction site is internal to the coding region, the coding region may be restricted and the lost nucleotides replaced by employing an adaptor for joining the coding region to the desired flanking region, such as another coding region which codes for a foreign polypeptide to be joined to the synthesized polypeptides to provide a fused protein.

The DNA coding for the growth factor sequence may be excised from the VV genome by cleavage with Alul and HpaII to give a 190bp fragment. This may then be manipulated as described above.

Expression vectors are characterized by having transcriptional and translational regulatory initiation and termination signal regions, where a DNA sequence having an open reading frame may be inserted between them and will be under the transcriptional and translational control of the signals. In addition, the expression vector may have one or more markers which allow for selection of the host having the expression vector and maintenance of the expression vector in the host.

The transcriptional initiation may be subject to inducible control, by temperature change, chemicals, or the like. In this manner, one can grow the host cells to high density, prior to initiation of the produciion of the desired product.

In addition, there may be one or more replication systems. For extrachromosomal maintenance, it will be necessary to have a replication I system which is functional in thn host to be used for expression. Where the host is other than a bacterium, it will frequently be desirable to have a second replication system which allows for cloning in a bacterium, to enhance the availability of the plasmid and allow for purification and characterization.

A wide variety of prokaryotic or eukaryotic hosts may be employed, including unicellular microorganisms, both prokaryotic and eukaryotic, cold-blooded and warm-blooded eukaryotes, such as insects and mammals, or the like. By employing different constructs for expression of the gene, one can introduce the constructs into different hosts. The host may provide for different products by providing for glycosylation to varying TMS/1416R 20 degrees with the same or different sugars or providing for an unglycosylated product. Usually, the glycosylatlon may occur at one or more, usually not more than two glycosylatlon sites present in the VGF seqence, where for glycosylatlon at least one sugar will be added and usually not more than about 60kDal of sugars, usually not more than about 4kDal of sugars, frequently not more than about 2kDal of sugars.

i Desirably, one can use the leader sequence naturally present with the polypeptide of the subject invention to provide for secretion in a K eukaryotic host, particularly a mammalian host, plus other maturation processes, glycosylatlon. Thus, by employing the DNA sequence encoding for the subject oligopeptlde joined to the naturally occurring secretory leader and processing signal, with the subject DNA sequence by itself or fused to a DNA sequence coding for a foreign polypeptide not naturally joined to the subject oligopeptide, one can provide for secretion of the subject polypeptides with concommitant removal of the secretory leader processing signal in an appropriate eukaryotic host. Alternatively, where the leader and processing signal are not functional in the intended expression host, the leader and processing signal may be substituted with a leader and processing signal recognized by the intended expression host.

ee TMS/1416R 21- Where integration into the host genome is desired, a stable replication system is not required.

Normally, the sequences of interest will be flanked by sequences homologous with a sequence present in the host genome to enhance the probability for i recombination. Desirably, a marker is included, Sparticularly one which allows for amplification, such as genes expressing metallothioneins, dihydrofolate reductase or thymidine kinase, so that the insertion sequence is not only maintained but amplified.

The subject polypeptides may be used with Svarious hosts for inducing an immunogenic response, enhancing cellular proliferation, wound healing or the like. For wounds, epithelialization and vascularization is observed, as well as rapid restoration of the strength of the wound, that is, resistance to separation or tearing. Hosts include mammals, such as rodents, domestic animals, primates and humans.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL 1 The Dayhoff protein sequence library of 2676 sequences was obtained on magnetic tape from the protein identification resource (Georgetown, Washington, and searched for sequences related to rat TGF (rTGF), where the three most closely related sequences were mouse EGF (mEGF) (Scott et al., 1983, supra; Gray et al., 1983, supra) and human EGF (hEGF) (Gregory et al., Int. J. Pept. Protein Res. (1977) 9:107-118), which are known to be homologous to human, mouse and rat TGF (Marquardt et al., Proc. Natl. Acad.

Sci. USA (1983) 80:4684-4688) and residues 45 to 85 of a 140 residue polypeptide encoded by the vaccinia virus genome (Venkatesan et al., 1982, supra). The alignment of the VV polypeptide, rTGF, mEGF, and hEGF is shown in 22 Figure 1. The observed homology has a probability due to chance of,less than 0.00301 by Fisher's exact test.

The V peptide has uncharged and hydrophobic residues near the N-terminus between residues 5 and and near the C-terminus between residues 100 and 124.

These residues may be considered by analogy to integral membrane glycoproteins as having a N-terminal hydrophobic signal sequence, which is removed proteolytically during or immediately after translation, and a C-terminal transmembranous sequence, Swhich serves to anchor the mature protein in the membrane. Cleavage of the viral polypeptide at arg-43 Iand arg-90 would lead to release of a soluble polypeptide. The 140 residue VV polypeptide may be assumed to give rise first to a membrane-associated protein of approximately 120 residues after removal of a signal peptide and then to a soluble growth factor peptide of about 47 residues.

The following DNA sequences were designed for synthesis and expression in an E. coli host. The sequences were designed to employ primarily bacterial preferred codons. The sequences contain several useful restriction sites, a few being indicated. The VGF sequence differs from the natural sequence in being DGMACRC rather than DGMYCRC. The subsequent chimeric sequences are prepared by synthesizing the two fragments bounded by BssHII and SphI sites and SohI and BamHI sites and ligating these fragments into the plasmid vector containing the modified TGF or VGF structural gene.

23 Synthetic human TGF gene BssHI.

CGCGCCATCGTTGTTTCTCACTTTA.ACOACTCCCOGACTCTCATACTCAGTTT

M V VS H F NDC P D S H TQ F Sh T GCTTT CATGGTAC CTGC C TTTT CTG GTT CACO AAGAAAAAC CGO CATCG TT C F HG TC RF L VQ ED K PA C V BamHI TGC CATT C TGCTAC OTTGC CCAC OTTGCGCAACAC GCT GAC CT CCTOCCTTAAGGATC C C H S G Y V G A R C E H A D L L A Ter Synthetic human TGF/VGF gene B s sHI I COCGCCCATGGTTGTTTCTCACTTTAkACGACTOCCCGOACTCTCATACTCAOTTT M VV S H FND CFP D S H TQ F TO CTTT CAT OCTAC CT GC CGTTTT CTG GTTCACGAAOAAAAACCCO CATGC TT C F H G TC R F L VQ E D K PAC V

TOF

BamHI

TGCTCTCATOOCTACACTGOAATTCOTTGCCAOCA'TGTTOTTCTGOTCCACTACCAGCGTTAAOCATCC

C S H G Y T G I R C Q IH V V L V D Y Q R Ter

VGF

Synthetic VGF/human TGF gene 4 BssHII CCGOCCATGCTGTGCGCGC CC GCAAGOCGACGCTAG M LC G P EOGD G Y S~h I TOO CTCATG GC CACTGCATC CATGCCACCT CACAT CGAO GGCATGGCATC TT C L HGD C I HA RD I D G MA C V VGF TGF BamHI TGC CATT CT GOCTAC GTTGGC OCAC GTTGC AACAGCT GAG CTOCT00 CTTA.AG C H S C Y V C A R C E H A D L L A Ter Synthetic

TGCCTGCATGGCG

CLHG D 24 VGF gene BssHII

CGCCCATGCTGTCCGGCCCGGAAGGCGACGGCTAC

MLCGPEGDGY

SphI

ACTGCATCCATGCACGTGACATCCACGGCATGGCATGCCGT

CIHARDIDGMACR

BamHI

TGCTCTCATGGCTACACTGGAATTCCTTGCCAGCATGTTCTTCTGCTCGACTACCACCGTTAACCATCC

C S H G Y T G I R C Q H V V L V D Y Q R Ter A synthetic VGF gene is prepared from the following fragments: SohI EcoRI VGFs1 VGFs2

VCSHGYTG

CGTTTGCTCTCATGGCTACACTGG

GTACCCAAACGAGACTACCGATGTGACCTTAA

EcoRI HincII BamHI

IRCQHVVLVDYQR*

-AATTCCTTGCCACCATGTTGTTCTGGTCGACTACCAGCGTTAAG

GCAACGGTCGTACAACAAGACCAGCTGATGGTCGCAATTCCTAG

VGFs3 VGFs4 d' The VGF gene may be assembled according to conventional procedures and used for insertion into an appropriate expression vector, joined to other fragments to extend the N- and/or C-terminus, or the like.

Isolation of Naturally Occurring VGF Cell culture and virus. Cercopithecus monkey kidney (BSC-1) cell monolayers were maintained in Eagle's basal medium supplemented with 10% fetal calf serum. VV (strain WR) w-s grown in Hela cells and purified by sucrose densi Z gradient sedimentation (Moss, (1981) in Gene Amplification and Analysis, eds. Chirickjian, J.G. and Papis, T.S.

(Elsevier/North--Holland, NY), Vol. 2, pp. 253-266).

I

BSC-1 cell monolayers were infected with plaque-forming units (pfu) per cell of purified virus, and incubated at 37 0 C with approximately 1ml of Eagle's basal medium supplemented with 2% fetal calf serum per 2x106 cells. Mock-infected cells were treated in an identical manna;. Cell culture supernatants were clarified by low speed centrifugation and lyophilized.

The residue was then resuspended in IM acetic acid and dialyzed extensively against 0.2M acetic acid.

Insoluble material was removed by centrifugation and the supernatant was lyophilized and resuspended in 1/100th of the original volume of 1M acetic acid and stored at Chromatography. Gel filtration was performed on columns of Bio-Gel P-10 (Bio-Rad) equilibrated in 1M acetic acid. Sizing by HPLC utilized two Bio-Sil TSK-250 columns (Bio-Rad) in series.

125 Radioreceptor Assay. The binding of !2I-labeled EGF (1I-EGF) to its receptor on monolayers of A431 cells was modified from the procedure described by Cohen and Carpenter, Proc. Natl. Acad. Sci. USA (1975) 72:1317-1321. Cells (lxl03 per well) were fixed on 24-well plates (Linbro, Flow Laboratories) with formalin in phosphate-buffered saline prior to assay.

Formalin-fixed cells do not sloughoff plates as easily as do unfixed cells, and replicate values were thus more consistent. Under these assay conditions, 12SI-EGF (1x10 10 cpm/nmol) saturates the binding assay at 3nM; assays were performed at 10% of the saturation value. TGF and VGF concentrations are expressed as ng equivalents of EGF per the amount required to 125 produce an inhibition of 125I-EGF binding equivalent to that produced by a known amount of EGF.

26 Radioimmunoassay. Each 50l reaction contained the following: 20mM sodium phosphate at pH 7.4, 200nM NaC1, 40mM dithiothreitol, 0.1% bovine serum albumin, 0.1% NaN, 125I-labeled peptide (2x104cpm) corresponding to the 17 carboxyl-terminal residues of TGF-a (Linsley et al., Proc. Natl. Acad. Sci. USA (1985) 82:356-369), antiserum at a final dilution of 1:5000, and other additions as specified. The reaction was initiated by the addition of antiserum and was continued at 23 0 C for 90min. An equal volume of formalin-fixed S. aureus (Pansorbin, Calbiochem) was then added, and incubation was continued for an additional 30min at 23OC. The immunoadsorbant was removed by sedimentation, and the amount of bound 125 I-labeled peptide was measured. The amount of bound peptide as corrected for nonspecific binding measured in the absence of antibody (less than 5% of the total) and expressed as a percentage of maximal binding.

Cellular DNA synthesis assay. Diploid human fibroblasts obtained from explant of newborn foreskin 4 were seeded at a density of 3x10 cells per well (96-weil plates, Nunclon, Roskilde, Denmark) and were grown to confluency in Dulbecco's modified Eagle's medium (GIBCO)/10% newborn calf serum. Cultures were then placed in medium containing 0.2% newborn calf serum, and two days later EGF (10ng/ml) or VGF equivalents of EGF per ml) was added. After 8hr, 125 cultures were labeled with I]iodo-2'-deoxyuridine (Amersham, 10pCi/ml, 5Ci/mg; ICi 37GBq) and the amount of isotope incorporated into trichlo,'oacetateinsoluble material was determined as described (Van Zoelen et al., Proc. Natl. Acad. Sci. USA. (1984) 81:4085-4089).

r C Results i n 27 Ii

I

The medium derived from BSC-1 cells 24hr after infection with W was tested for the presence of 125 material that could compete with 125 I-EGF for binding to EGF receptor-rich human epidermoid carcinoma cells (A431). VV-infected cells released a potent activity that competed with EGF, which activity is designated VGF. Control medium volume from mock-infected BSC-1 control cultures contained minimal activity in competition with EGF.

Monitoring VGF production, at the earliest time examined, 2hr after infection, enhanced levels of VGF were observed in the culture medium. By 12hr, maximum amounts of this activity were found in culture supernatant with only a slight increase noted at 24hr.

The level of EGF production was found to be a function of the multiplicity of virus infection as demonstrated by the following Table 1.

TABLE 1: Effect of Multiplicity of Infection on VGF Release Virus multiplicity, VGF released, pfu per cell ng eq. of EGF per ml

:I

'V.

10.1 Cultures of BSC-1 cells were infected at the pfu-to-cell ratio indicated, and the supernatants (approx. 1ml/2x10 6 cells) were harvested at 24hr after infection. Samples of each were acidified, lyophilized, and tested in duplicate radioreceptor assays for EGF as described (Delarco and Todaro, Proc. Natl Acad. Sci.

,c USA (1978) 75:401-405). ng eq., nanogram equivalents.

i n C I 28 Partial purification cf VGF. The activity competing with EGF found in VV-infected BSC-1 cells was partially purified from acid-extracted culture supernatants at 24hr after infection as described above. Acid-solubilized polypeptides (10.5mg) from the supernatants were applied to a Bio-Gel P10 column equilibrated with 1M acetic acid and samples of each fraction tested for activity competing with EGF. The major active peak (fraction 42) eluted slightly after the Mr 29,000 carbonic anhydrase marker with an apparent M of 25,000. The molecular weight was confirmed by utilizing tandemly linked Bio-Sil TSK 250 HPLC sizing columns, with all of the activity eluting as a major peak in the region of the M r 25,000 protein marker. One microgram of partially purified VGF was equivalent to 90ng of EGF in the radioreceptor competition assay.

Immunological comparison of VGF and TGF. In the radioimmunoassay described above, a 50% displacement of antigen from antibody to the carboxyl-terminal 17 amino acids of rat TGF-a molecule is observed at an antigen concentration of approximately 0.2-0.3ng equivalents of 125 EGF, where I-labeled TGF-a competes with TGF-a.

When VGF was tested at equivalent concentrations, no competition was observed. In a competitive radioimmunoassay for native EGF, VGF preparations, even when tested at 50ng of EGF equivalents/ml, exhibited a 1 125 minimal displacement of 1I-EGF from a polyclonal antibody to native EGF.

Biological activity of VGF. A comparison of levels of VGF produced by BSC-1 cells infected with of VV per cell with that of TGF-a produced by retrovirus-transformed cells is shown in the following Table 2.

29 TABLE 2: Comparison of the biological activity of VGF with TGF-a and EGF Stimulation of EGF receptor Induction of anchoragebinding, 2 DNA synthesis, 3 independent ng eq. of 125 1]IdU cell growth, 4 Growth EGF/ml of incorporated Soft-agar factor 1 medium (cpm/dish) colonies/plate None 1,779 VGF 2.3 3,760 108 TGF-a 0.2 NT 294 EGF 8,482 346 NT, not tested.

1 VGF [90ng equivalents (eq) of EGF per pg of protein] was purified by gel filtration followed by elution from a C pBondapak (Waters Associates) column. TGF-a was purified from Snyder-Theilen feline sarcoma virus-transformed Fisher rat embryo cells as described in Marquardt et al., Proc. Natl, Acad. Sci. USA (1983) 80:4684-4688; EGF was purified from mouse submaxillary gland (Cohen et al., J. Biol. Chem. (1980) 255:41834-41842).

2 Quantitation of EGF equivalents was based on a standard 125

I-EGF

binding competition curve as described.

3 Mitogenesis assays were as described; quiescentccultures of diploid human fibroblasts received 10ng of purified EGF/ml or the same number of EGF equivalent of VGF/ml. Values for [1 25 1]iododeoxyuridine 125 1]IdU) incorporated represent the average of triplicate determinations.

4 The number of soft-agar colonies represent the average number of colonies containing a minimum of 20 NRK cells per six random lowpower fields 10 days after seeding (1.5xl0 4 cell/ml) with purified EGF (5ng/ml) or the same number of EGF equivalents of VGF/ml and 2.0ng of TGF-B/ml purified from human platelets as described in Assoian et al., ibid. (1983) 258:7155-7160. Plates of NRK cells treated with TGF-B alone above did not form colonies.

Further purification of VGF. Pooled fractions from the gel filtration column (25-35) were concentrated by vacuum centrifugation, resuspended in 0.05% trifluoroacetic acid (TFA), clarified and injected into a 3.9mm x 30cm pBondapak C 18 column 180

T

30 (Waters, Milford, MA). Peptides were eluted with a linear 20-60% gradient of acetonitrile in 0.05% TEA at a flow rate of 1.0ml/min at 22 0 C. Aliquots of each fraction were assayed in a radioreceptor assay for EGF-competing activity. The peptide corresponding to the peak activity was collected and diluted with 0.05% TFA and reinjected into a uBondapak column and eluted utilizing isocratic conditions. The acetonitrile concentration was about 22 to Amino-Terminal Sequence of VGF VGF (18pmol) purified, as described, from vaccinia virus-infected monkey cells, was subjected to automated repetitive Edman degradation in the Model 470A protein sequences (Applied Biosystems). The phenylthiohydantoin amino acids were analyzed by rpHPLC.

The amino-terminal amino acid sequence is as follows:

D-S-G-N-A-I-E-X-X-X-P-E-I-X-N-A

unidentified residues) r, r A comparison of these data with those deduced from the vaccinia virus DNA sequence shows that VGF begins with aspartic acid at residue 20 of the primary translation product (Fig. 1).

Preparation of VGF as TrpE Fusion Protein pVG3, a plasmid containing part of the early region of Vaccinia virus is available from B. Moss (NIH). A 260bp Sau3AI-HpaII fragment was inserted into the TrpE expression pl.asmid pJH14, using the BamHI and HindIII sites. The recombinant plasmid was introduced into E. coli by transfection, and expression of the recombinant protein was induced by indoleacetic acid.

The fusion protein was purified from the bacteria, and TMS/1416R 31 the VGF sequence was excised by digestion with the enzyme Lys C. The digest was shown to contain an activity that competed with EGF in the radioreceptor assay. Rabbit antiserum to vaccinia virus precipitates polypeptides of 10kDal and 25kDal from lysates of these cells. The EGF receptor binding activity was found to be about llOng eq./mg protein. By comparison TGF-a expressed in yeast is 115ng eq./mg protein.

Preparation of VGF in Silkworm using Baculovirus Vector An additional expression system which is used to express the VGF recombinant protein is an insect system. See Maeda et al. and Carbonell et al., supra.

In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodootera frugiperda cells. The envelope gene can be cloned into non-essential regions (for example, the polyhedrin gene) of the virus and is placed under control of an AcNPV promoter (for example, the polyhedrin promoter).

Successful insertion of the VGF gene construct will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the I inserted gene is expressed.

Construction. The VGF gene of interest is placed under the control of a suitable promoter for an insect cell system. Plasmids pAc610 and pAc611 contain the polyhedrin gene cloned into a plasmid vector possessing an ampicillin resistance marker. Polylinkers are inserted into this gene which are 50 bases downstream from the transcriptional start site of the polyhedrin gene and 7 bases before the first ATG. The VGF gene is cloned into a convenient polylinker site, so that it is TMS/1416R 32 under the control of the polyhedrin promoter. The ATG initiation methionine codon and the translational termination codons are those of the VGF gene; the transcriptional start sites and polyadenylation signals are those of the polyhedrin gene.

Transformation. The host cell for AcNPV is Spodootera frugiperda In order to produce a recombinant virus stock, the VGF containing plasmid is mixed with AcNPV DNA and transfected into SF cells 1 using the calcium phosphate technique. Recombinant viruses are isolated from the medium and plaque purified on SF cells. Recombinant plaques are identified by hybridization using radiolabeled VGF DNA as probe.

Expression. Once recombinant virus is identified, it is then expahded on SF cells. The recombinant virus stock is then used to infect SF cells, the cells lysed, and supernatants screened for production of VGF protein, which is purified as described above for vaccinia virus-infected mammalian cell.

The predicted sequence of VGF produced in insect cells is:

CHO

1 DSGNAIETTSPEITNATTDIPAIRLCGPECDGYCLHGDCIKARDIDGMYCRCSHGYTGIR

CQHVVLVDYQRSENPNTTTSYIPSPGIMLVLVGIIIITCCLLSVYRFTRRTKLPIQDMVVP

This 121-residue sequence starts with the N-terminal sequence that has been determined directly for VGF purified from VV-infected monkey cells (that is, at residue 20 of the VGF open reading frame) and continues to the last residue of the open reading frame. It thus lacks the signal peptide (residues 1-19 33 of the open reading frame) but includes the transmembranous sequence (YIPSPGIMLVLVGIIIITCCLLSVY).

The predicted MW of the 121-residue peptide is 13,304, not counting carbohydrate. The apparent MW of the baculovirus-produced VGF is 17,000, I significantly smaller than that obtained from VV-infected cells, indicating that the two forms have probably been processed differently. The baculovirus-produced VGF may lack an additional portion of the N-terminal .sequence, or a portion of the C-terminal sequence, or both, and/or could differ in the type and extent of glycosylation.

I~

Burn Treatment with Natural VGF Three female piglets of approximately pounds were anesthetized with Ketamine and Rompum, their backs shaved and the hair totally removed with a commercial depilatory cream. A brass template (3x3cm, 147gm) was equilibrated in a 70°C water bath and then placed in firm contact with the skin for exactly l0sec.

Five wounds were placed on each side of the spine and were separated from each other by approximately one inch. The top of the resulting blister was totally removed and treated twice a day with vehicle (Silvadene) alone, vehicle containing the factor, or untreated. The pigs ate and drank at will. After 9 or days of treatment, the pigs were anesthetized again and the eschar was removed as much as possible. All burns were photographed and a punch biopsy was taken in each burn in an area judged to be epithelialized.

The following Table 3 represents the approximate percentage of each burn epithelialized as judged visually.

4.

I..

I

C. C C TABLE 3 E p ithlie1a Ii z ed Right Side Left Side Untreated Silvadene Growth Factor i'g /ml human EGF Untreated Silvadene Growth Factor 11g/ml natural VGF Pig 1 9 days Post-Burn Pig 2 days Post-Burn 75 30,60,70 0.1 15 70,65 rVGF* rVGF* 50 60 75 50 human TGF rat TGF Pig 3 9 days Post-Burn 20 90 0.1 rVGF VGF prepared in insect cells described above I 35 It is evident from the above results, that the VGF is a potent epithelializing agent comparing satisfactorily with other growth factors which have been previously tested and demonstrated to have mitogenic activity.

EXPERIMENTAL 2 I. PREPARATION OF SYNTHETIC TGF AND VGF OLIGONUCLEOTIDES TGF and VGF synthetic growth factor genes were designed which use bacterial codons optimized for high levels of expression. In addition, several convenient restriction sites were designed into the synthetic genes. Nhen possible, the new restriction sites left the amino acid sequence of the growth factor gene unaltered, however, in some cases incorporation of the new restriction site yielded an altered amino acid sequence. These sites roughly divide the synthetic genes into thirds yielding N-terminal, middle and C-terminal domains. The natural VGF gene product contains an extreme N-terminal domain which has no counterpart in mature TGF. VGF fragments lacking this domain are referred to as truncated. The restriction sites were used for initial construction of the final genes from partial synthetic oligonucleotide fragments extending from one restriction site to another. The oligonucleotides were synthesized on S" an Applied Biosystems oligonucleotide synthesizer and were purified on an S acrylamide gel. The oligonucleotides were phosphorylated at the 5' end using T 4 polynucleotide kinase and each oligonucleotide was then annealed to its complement.

A. TGF Synthetic Oligonucleotides 1. Human TGF N-terminal domain: TGF BssHIINcol

K-

IV V S H F 1N D C P D S H T Q F C F H G T COCOCCATGGTTCTTTCTCACTTTACACTTCCCG ACTCTCAACTCAGTTCTCATCGTC-. 3 TCFHC:- 3' G 3 TACCAACAAAGTCAAATTGCTGACGGCCTGAGA GTATGAGTCAAAACGAAAGTAC 5' TGF IC TMS/1416R 1 36- 2. Modified human TGF middle domain with the human sequence QEDK being altered to QEEK. the sequence found in rat TGF: KpnI -SphI C R FL VQ EE K P A C CTGCCCTTTTCTCGTTCA GGAACAAAAACCGGCATC 3CATCGACCGCAAAAC ACCAACTCCTTCTTTTTGGCC 3' TGF1 01 5' TGF102 3. Human TGF C-terminal domain: S:)h IB m H 1.

V C H S C Y V G A R C E H A D L L, A Te r CCT*TTC/-CA TTCTCGCTACCTTCCA CCTTCQ.GAACACCC TCA CCTCClTCGC TTA A C GT ACCXAACGTA.PCACCATGI-AACCCCCTGCAACCTTCTCACTOGACGP -CCAATTCCT.G 3' TCEF205 5' TCF206 B. VGF Synthetic Oligonucleotides 1. VGF extreme N-terminal domain: Ti :-C..ATCAACTAC'riC'!'CUzAAATCfkCUCAC-TA,--, ACT 3 VCPOS 2. Modified VGF N-terminal domain Including Asp-Pro cleavage site, with the sequence HGT replacing the natural sequence HGD: BarMH T v 4a KpnI I D PM D P A iR LCCGPE GD CY C HOT '3AT.'CrOATCCCArTCACA ATCCCGC!--ATCCG'CTTCTCCCCCCCCO,'AAGCACCGCTACTGCCTCCATCCIGTAC CTACGCTA CCTCT*IAGCCCCATAGGC AGACACCCCCGCCTTFCCGCTQ(-CGATCA COACCTA C VCF103a 3. Modified VGF middle-dQmain having the sequence GYAC replacing the natural sequence GMYC: KpiiI Sph I Tr C I H A R D I D G Y- A C CTCATCCATGCACGTCACATCGACGOCTACG 3' VGO01a 3' CATGGACCTACGTACCTGCACTCTACCTCCCCATCGC-TAC 5' VGF'102a TMS/ 1 416R 37 4. VGF C-terminal domain, 5' end: Sph IEcoRi CR CS H CYT G CCCTTCCTCTCATGCCTACACTGG 3' VOFi A 3' GTACCCCA-ACCACACTACCCATCYCOACCTFTA.A 5 VCF2A Modified VGF C-terminal domain. 5' end, with the sequence VC-S replacing the natural sequence RCS: Spliw EcoRi V' C S H C Y T C CCTTGCTCCATGCCTACA CTGC 3 'VCE'1 GTA.CCAACCACACTACCCATGTGtICCTTAA 5'1 VOF2 6. VGF C-terminal domain, 3' fragment. ending at YOR instead of PNT. the deduced C-terminus of natural secreted VGF: EcoRI BamHT R C Q H k V1 L V DY Q Ri Ter II. DESCRIPTION OF CLONING AND EXPRESSION PLASMIDS A. Plasmid pLEBam was used to clone synthetic oligonucleotide fragments because of its convenient BssHII and BamHI restriction sites. A plasmid with NcoI and BamHI restriction sites such as pBMll or pBMll/NDP (described below) can be used for cloning the synthetic nucleotide fragments.

B. Plasmid.-PBMII, described in EP 0 314 184 by Liu, Bruce, Rose and Franceschini, allows cloning of a foreign gene downstream of the DNA sequences coding for TMS/ I416R 38 the 33 N-terminal amino acids of the bacteriophage X N-gene at a BamHI restriction site. Upon induction of the X PL promoter by inactivation of the C1857 temperature-sensitive repressor at 42*C, the foreign gene product is expressed as the C-terminal part of a fusion protein whose N-terminal sequence is that of the N-gene.

C. Plasmid. pBM11M4, described in EP 0 314 184 by Llu, Bruce, Rose and Franceschini, is derived from pBM11 and allows a foreign gene to be cloned at a BamHI restriction site directly after the initiating methlon'ne of the N-gene.

D. Plasmid pBM11MS, described in EP 0 314 184 by Liu, Bruce, Rose and Franceschini, is derived from pBM1l in which an Ncol site present in the neomycin resistance gene has been removed by site-direction mutagenesis.

E. Plasmid pBM11/NDP, described in EP 0 314 184 by Liu, Bruce, Rose and Franceschini, is derived from pBM11 and has DNA sequences coding for an acid labile aspartic acld-proline dipeptide Inserted between the sequences coding for the N-gene and the foreign gene.

.*goo F. Plasmid pBMll/PAD, described in EP 0 314 184 by Liu, Bruce, Rose and Franceschini, is derived from plasmid pBM11M4 and allows a foreign gene to be cloned at a HindIII, Smal or BamHI downstream from a modified alkaline phosphatase signal sequence.

TMS/1416R 39 G. Plasmid plac/cro-lacZ was constructed by ligating a 0.96 kb PsI-BlII fragment from pTR213 (Roberts t aIl., Proc. Natl. Acad. Sc. USA (1979) 76:760-764) and a 5.54 kb PstI-BjTHI fragment from pLG400 (Guarante t Al., Cell (1980) 2Q:543-553) in the presence of oligodeoxynucleotide linkers. Nine different linkers were used. These linkers served the following purposes: to regenerate the BgllII and BamHI sites from the parental plasmids; to provide additional sites for the insertion of foreign DNA; and to allow the inserted DNA to be in the correct translational reading frames with respect to the Cro and W-galactosidase coding sequences. Fusion proteins expressed by this vector consist of the N-terminus of bacteriophage X protein Cro, the amino acid sequence encoded by the inserted DNA, and the C-terminus of 6-galactosidase. The controlling elements of this vector consist of the operator-promoter region of E. coli lactose (lac) operon, as well as the RBSes of lac and cro.

H. Plasmid ptac/cro-lacZ, allows a foreign gene to be cloned downstream of the N-terminal 21 amino acids of the bactertal Cro protein. It was constructed by inserting a 0.87 kb RsaI fragment of the plac/cro-lacZ plasmid into pDR540 at the BamHI site, which was previously converted to blunt ends by the action of PolIk enzyme. The orientation of the inserted DNA was such that the RBS and the coding sequence of Cro were located downstream from the RBS of lac. The resulting plasmid, ptac/cro, contained both RBSes of lac and cro, and the N-terminal coding sequences of Cro. The second step in the construction of ptac/cro-lacZ was achieved by ligating the 1.16 kb and the 5.54 kb PitI-BamHI fragments from ptac/cro and pLG400 plasmids, respectively. Expression vector ptac/cro-lacZ is thus similar to plac/cro-lacZ, with the exception that the promoter of ptac/cro-lacZ TMS/1416R 40 consists o" the -35 region from the promoter of the tryptophan operon and the Pribnow box of lac operon. This hybrid promoter allows a higher level Sof expression than plac/cro-lacZ.

I. Plasmid pRSV allows expression in eukaryotic cells with the r3SV LTR promoter, Gorman et Proc. Natl. Acad. Sci. USA (1982) 79:6777-6781.

J. Plasmid pAc610, Smith t al., Molec. Cell. Biol. (1983) 12:2156-2165, allows expression of a foreign gene in insect cells, (see specification, page 33, line 10 through page 34, line 1 III. PREPARATION OF RECOMBINANT GRONTH FACTOR GENES A. Modified Synthetic TGF 1 1. Preparation of pBMIl/N/TGF: A modified human TGF gene was prepared essentially as described on page 24, lines 19-30 and page 25, lines 1-10 by cloning into the expression plasmid pBM11. The modified human TGF is expressed in this system as part of a fusion with the 33 N-terminal amino acids of the N-gene and has the sequence QEEK replacing the human sequence QEDK.

Preparation of a 780 bp SphI-PvuI fragment of pBM11/N/TTV.

S Plasmid pBM11/N/TTV (described below), was digested with ShI and P~Il and the 780 bp S£hI-PvuI fragment was gel purified.

1 For the sequence of naturally occurring mature hTGF, see Appendix A, incorporated herein by reference.

TMS/1416R -41 This fragment contains part of the pBM11 plasmid at the PvuI end and at the Spl end, the N-gene and N-terminal two-thirds of the human TGF gene.

Preparation of the 5 kb BamHI-PvuI fragment of pBM11/N/TTV.

Plasmid pBM11/N/TTV, as described below, was digested with BamHI and Pvul and the 5 kb BamHI-PvuI fragment was gel purified.

Ligation and isolation of pBM11/N/TGF. Oligonucleotides TGF 205 and 206, the 780 bp ShI-PvuI fragment and the 5 kb BamHI-PvuI fragment of pBM11/N/TTV were ligated together and used to transform competent HB101.

The transformants were selected on neomycin and were screened by restriction analysis using EcoRI and nucleotide sequencing following the Sanger-dideoxy method. A correct construction was isolated and denoted pBM11/N/TGF.

B. Modified Synthetic VGF and Fragments Thereof 2 Synthetic VGF genes were prepared essentially as described on page 24, lines 19-30 and page 26, lines 1-28 of the specification.

1. Preparation of pBM11/NDP/VGFA: The N-terminal sequence of the synthetic VGFA gene is a truncated version of the natural VGF sequence and begins with the sequence DIPAIR. In this plasmid the VGFA fragment is located downstream of 32 amino acids of the X N-protein and the dipeptide aspartic acid-proline.

S In order to preserve the KpnI cloning site, the synthetic sequence was altered to code for CLHCGTC instead of the natural VGF sequence CLHGDC and terminates with the sequence YQR upstream of the natural sequence PNT. In addition, the VGFA gene codes for the sequence GYACVC which replaces the natural sequence GMYCRC.

2 For the sequence of naturally occurring mature VGF, see Appendix A, incorporated herein by reference.

TMS/1416R .i 42 Preparation of a KpnI-BamHI 80bp C-terminal fragment of the synthetic VGF gene. Plasmid pLEBam/TVV, described below, was digested with KpnI and BamHI and the 80bp KpnI-BamHI fragment was gel purified. This fragment contains the C-terminal two-thirds of the synthetic VGF gene with the KpnI site at the 5' end.

Preparation of BamHI digested dephosphorvlated pBM11. Plasmid pBM11/N/TTV was digested with BamHI and the 5' phosphates were removed by treatment with calf intestinal alkaline phosphatase. The 5.6kp BamHI plasmid fragment was gel purified.

Ligation and isolation of pBMI1/NDP/VGFA. 01igonucleotides VGF 103a, 104a, the 5.6kb BamHI fragment of pBM11 and the 80bp KpnI-BamHI fragment of pLEBam/TVV were ligated together using DNA ligase and then used to transform competent HB101. The transformants were selected on neomycin and were screened by restriction analysis using ClaI and nucleotide sequencing following the Sanger-dideoxy technique. A correct construction was isolated and denoted pBM11/NDP/VGFA. This construction has the sequences GTC and GYACVC instead of the authentic VGF sequences GDC and

GMYCRC.

2. Preparation of pBM11/NDP/VGFa: The N-terminal sequence of VGFa is a truncated version of the natural VGF sequence and starts with the sequence DIPAIR. In addition, the S VGFa sequence contains the altered sequences GTC and GYACRC instead of the natural VGF sequences GDC and GMYCRC. In this plasmid the VGFa gene is located downstream of 32 amino acids of the A N-protein and the dipeptide aspartir acid-proline. Treatment of the purified fusion protein with formic acid results in cleavage at the acid labile aspartic acid-proline peptide bond allowing separation of the VGFa protein from the N-protcin TMS/1416R ~sill 43 amino-terminus. Cleavage is such that the VGFa protein is left with the proline residue at the amino terminus.

Preparation of SphI digested, dephosphorylated pBM11/DP/VGFA.

j Plasmid pBM11/DP/VGFA (10 gg) was digested with 30 units of SphI and the phosphates were removed by treatment with calf intestinal alkaline phosphatase. The 5kb plasmid fragment was recovered after electrophoresis on an agarose gel.

Preparation of an EcoRI-SphI 70bp fragment of pBMll/DP/VGFA.

Plasmid pBM11/DP/VGFA (10 gg) was digested with 30 units of EcoRI and then 30 units of iphl. The 70bp fragment was recovered after electrophoresis on an agarose gel.

Ligation and isolation of pBM11/NDP/VGFa. The 24bp fragment containing oligonucleotides VGF 1A and 2A, the 5kb SphI fragment and the EcoRI-Spl fragment of pBM11/DP/VGFA were 11gated together and the mixture was used to transform competent E. coli HB101 cells. The transformants were screened by nucleotide sequencing using the Sangerdideoxy nucleotide method. A correct clone was isolated and denoted pBM11/NDP/VGFa.

3. Preparation of pBM11/PAD/nVGFa: Synthetic oligonucleotides were designed to link the VGFa S synthetic gene with an alkaline phosphatase modified signal sequence to provide for an optimal signal sequence cleavage site by coding for the additional N-terminal residues occurring immediately downstream of the signal sequence cleavage site in the natural VGF, denoted extreme N-terminus above. The nVGFa sequence contains the altered sequences GTC and GYACRC instead of the natural VGF sequences GDC and GMYCRC and terminates with the sequence YQR upstream of the natural sequence PNT. In TMS/1416R 44 this expression system, the signal sequence remains attached to the nVGFa forming a fusion protein with nVGFa at the C-terminus.

Preparation of 0.5kb HindIII-PvuI digested pBM11/PAD. Plasmid pBM11/PAD was digested with HindIII and Pvul and the 0.5kb fragment was gel purified. The HindIII site is located at the C-terminus of the modified alkaline phosphatase signal sequence.

Preparation of the 5.2kb PvuI-BamHI pBM11 plasmid fragment.

Plasmid pBM11/NDP/VGFa was digested with PvuI and BamHI and the 5.2kb plasmid fragment was gel purified.

Preparation of the 170bp NcoI(blunt)-BamHI synthetic VGFa gene.

Plasmid pBMII/NDP/VGFa was digested with Ncol and the 5' overhangs were removed by treatment with Sl-nuclease. This created a blunt end at the first codon of the VGFa truncated synthetic gene. The DNA was then digested with BamHI and the 170bp icoI(blunt)-BamHI fragment was gel purified.

Ligation and isolation of pBM11/nVGFa. Oligonucleotides VGF105 and 106, the 0.5kb HindIII-yPuI fragment of pBM11/PAD, the 5.2kb PvuI-BamHI pBM11 fragment and the 170bp NQI(blunt)-BamHI synthetic VGFa gene were ligated together using DNA ligase and the resulting mixture was used to transform competent HB101. The transformants were selected on neomycin and screened by restriction analysis and nucleotide sequencing using the Sanger-dideoxy technique. A correct construct was isolated containing the modified alkaline phosphatase signal sequence in frame with the nVGFa gene.

C. Modified Synthetic Chimeric TGF/VGF Genes Chimeric TGF/VGF genes were prepared essentially as described on page 24, lines 19-20 and page 25, lines 13-38 of the specification, by TMS/1416R 45 exchanging restriction fragments coding for one growth factor for restriction fragments coding for another.

1. Preparation of Plasmid pLEBam/TTV: The synthetic chimeric growth factor, denoted TTV or (TGF/TGF/VGF) was assembled in the cloning vector pLEBam. This hybrid growth factor contained the amino acid sequence of human TGF in the amino terminal two-thirds of the gene with the exception of the sequence QEEK which was altered from the natural human sequence QEDK. The carboxy terminus was derived from the amino acid sequence of VGF and terminated with the sequence YQR upstream of the natural sequence PNT.

Preparation of BssHII-BamHI pLEBam. Plasmid pLEBam was digested with BssHII and BamHI.

Ligation and isolation of pLEBam/TTV. BsiHII-BamHI pLEBam was ligated to oligonucleotides TGF101, 102, 103, and 104, and VGF1, 2, 3 and 4 using DNA ligase and used to transform competent HB101. The transformants were selected on ampicillin and screened by restriction analysis using EcoRI, NcoI and BamHI and by nucleotide sequencing using the Maxam-Gilbert protocol. A correct construction was isolated and denoted pLEBam/TTV.

2. Preparation of Plasmid ptac/TTV: The synthetic chimeric growth factor, denoted TTV or S (TGF/TGF/VGF) was expressed in this system as a fusion protein with the N-terminal 21 amino acids of the X Cro protein. This hybrid growth factor contained the amino acid sequence of human TGF in the amino terminal two-thirds of the gene with the exception of the sequence QEEK which was altered from the natural human sequence QEDK. The carboxy terminus was derived from the amino acid sequence of VGF and terminated with the sequence YQR upstream of the natural sequence PNT.

TMS/1416R P- 46 Preparation of BamHI plasmid fragment. Plasmid pLEBam/TTV was cleaved with BssHII, filled in with Klenow, cleaved with BamHI and the TTV encoding fragment was purified by agarose gel electrophoresis.

Ligation and isolation of ptac/TTV. The BamHI TVV fragment was ligated to ptac/cro-lacZ which had been cleaved with StuI BamHI and used to transform E. coli NF1829. Colonies harboring the correct plasmids were identified by restriction digestion of mini prep DNA.

3. Preparation of Plasmid pBMII/N/TTV: In this construct, the synthetic modified TTV chimeric gene was expressed as the C-terminal portion of a fusion protein having the first 33 amino acids of the N-gene at the N-terminus. This hybrid growth factor contained the amino acid sequence of human TGF in the amino terminal two-thirds of the gene with the exception of the sequence QEEK which was altered from the natural human sequence QEDK. The carboxy terminus was derived from the amino acid sequence of VGF and terminated with the sequence YQR upstream of the natural sequence PNT.

Preparation of NcoI(blunt)-BamHI TTV synthetic gene. Plasmid pLEBam/TTV was digested with NcoI and the ends were made blunt by filling in the overhangs using the Klenow fragment of DNA polymerase. The DNA was then digested with BamHI and the 170bp NcoI(blunt)-BamHI TTV fragment was gel purified.

Preparation of BamHI digested pBM11. Plasmid pBM11 was digested with BamHI.

Ligation and isolation of pBM11/TTV. BamHI digested pBM11, the NcoI(blunt)-BamHI TTV fragment, and BamHI linkers (5'GATCCG3') were ligated together using DNA ligase and the resulting mixture was used to transform competent HB101. The transformants were selected on neomycin and were TMS/1416R 47 screened using restriction analysis and nucleotide sequencing using the Sanger-dideoxy method. A correct construct was isolated and denoted pBM11/N/TTV.

4. Preparation of Plasmid pBMII/NDP/TTV: In this construct, the synthetic modified TTV chimeric gene is expressed as the C-terminal portion of a fusion protein having the first 32 amino acids of the N-gene at the N-terminus. An acid labile aspartic acid-proline dipeptide separates the two parts of the fusion. The hybrid Igrowth factor contains the amino acid sequence of human TGF in the amino Iterminal two-thirds of the gene with the exception of the sequence QEEK which was altered from the natural human sequence QEDK. The carboxy terminus was derived from the amino acid sequence of VGF and terminated with the sequence YQR upstream of the natural sequence PNT.

Preparation of 5kb NcoI pBM1 plasmid fragment. Plasmid pBM11/NDP/VGFA was digested with NcoI and the 5kb NcoI plasmid fragment was gel purified. This fragment has one NgQI overhang at the aspartic acidproline cleavage site downstream of the sequences coding for the first 32 amino acids of the N-gene. The other NcoI site is in the neomycin resistance gene.

Preparation of 0.6kb NcoI-BamHI pBM11 plasmid fragment. Plasmid S pBM11/N/TTV was digested with Ncol and BamHI and the 0.6kb NcoI-BamHI plasmid fragment was gel purified. This fragment has the Ncol overhang in the neomycin resistance gene.

Preparation of the 170bp synthetic TGF/TGF/VGF fragment.

Plasmid pLEBam/TTV was digested with NcoI and BamHI and the NcoI-BamHI 170bp fragment containing the TGF/TGF/VGF synthetic gene was gel purified.

This fragment has the NcoI overhang at the 5' end of the gene and the BamHI TMS/1416R 48 overhang at the 3' end of the gene.

Ligation and isolation of pBM11/NDP/TTV. The 5kb Ncol and the 0.6kb NcoI-BamHI plasmid fragments were ligated with the 170bp NLoI-BamHI TTV gene using DNA ligase and the resulting mixture was used to transform competent HB101. The transformants were selected on neomycin such that only colonies with correctly reconstructed neomycin resistance genes would survive. Transformants were screened using restriction analysis with Ncol and nucleotide sequencing using the Sanger-dideoxy technique. A correct construction was isolated and denoted pBM11/NDP/TTV.

Preparation of Plasmid pBM11/NDP/VTV: In this construct, the synthetic modified VTV chimeric gene was expressed as the C-terminal portion of a fusion protein having the first 32 amino acids of the N-gene at the N-terminus. An acid labile aspartic acid-proline dipeptide separates the two parts of the fusion. The hybrid growth factor contained the amino acid sequence of human TGF in the middle domain with the amino acid sequence QEEK replacing the natural sequence QEDK. The N-terminal and C-terminal domains were derived from the truncated VGF sequence and begin with the sequence DIPAIR and end with the sequence YQR which is upstream of the natural sequence PNT.

Preparation of a 5kb BamHI-NcoI fragment of pBMl. Plasmid pBM11/N/TTV was digested with BamHI and NclI and the 5kb BamHI-NcDI fragment was gel purified. This fragment contains a BamHI overhang at the 3' end of the sequences coding for the first 32 amino acids of the N-gene and a NcoI site in the neomycin resistance gene.

Preparation of a 700 bp KpnI-Ncol fragment of pBM11/N/TTV.

Plasmid pBMI1/N/TTV was digested with KpnI and N IQa and the 700 bp KlI-NcoI fragment was gel purified. This fragment is made up of part of TMS/1416R 49 plasmid pBMll containing part of the neomycin resistance gene at the NcoI overhang, and the C-terminal VGF domain of the TTV synthetic gene at the Kpnl overhang.

Ligation and isolation of pBM11/NDP/VTV. Oligonucleotides VGF 103a and 104a, the 5kb BamHI-NcolI fragment of pBM11 and the 700 bp KpnI-Nc__gI fragment of pBM11/N/TTV were ligated together using DNA ligase and then used to transform competent HB101. The transformants were selected on neomycin and were screened by restriction analysis using CiI and nucleotide sequencing following the Sanger-dideoxy technique.

6. Preparation of Plasmid pLEBam/TVV: The synthetic chimeric growth factor, denoted TVV or (TGF/VGF/VGF) was assembled in the cloning vector pLEBam. This hybrid growth factor contained the amino acid sequence of human TGF in the N-terminal domain of the gene. The middle and C-terminal domains are derived from the truncated VGF sequence and end with the sequence YQR upstream of the natural sequence PNT. In addition, the synthetic gene has the modification, GYACVC for GMYCRC.

Preparation of a 4.3kb KpnI-SphI fragment of pLEBam/TTV.

Plasmid pLEBam/TTV was digested with KpnI and SgpI and the 4.3kb KPnI-pbhI fragment was gel purified. This digestion removes the middle of TGF domain from the synthetic gene TTV in the cloning plasmid pLEBam.

Ligation and isolation of pLEBam/TVV. Ollgonucleotides VGFlOla and 102a were ligated to the 4.3kb KpaI-!pl fragment of pLEBam/TTV using DNA ligase and the resulting mixture was used to transform competent HBIOl. The transformants were selected on ampicillin and were screened by nucleotide sequencing using the Sanger-dideoxy method. A correct construct was isolated and denoted pLEBam/TVV.

TMS/1416R 1 i

I

50 7. Preparation of Plasmid pBM16/NDP/TVV: In this construct, the synthetic modified TVV chimeric gene was expressed as the C-terminal portion of a fusion protein having the first 32 amino acids of the N-gene at the N-terminus. An acid labile aspartic acid-proline dipeptide separates the two parts of the fusion. The hybrid growth factor contained the amino acid sequence of human TGF in the N-terminal domain. The middle and C-terminal domains were derived from the truncated VGF sequence and end with the sequence YQR. In addition, the synthetic gene has the modification GYACVC for GMYCRC.

Preparation of 4.3kb NcoI-BglII fragment of pBM11. Plasmid pBM11/NDP was digested with NcoI and BgiII and the 4.3kb fragment was gel purified. The NcoI overhang Is positioned at the aspartic acid-proline cleavage site just downstream of the first 32 amino acids of the N-gene.

Preparation of the 1.2kb BamHI-BalII fragment of Plasmid pBM11M5 was digested with BamHI and BgiII and the 1.2kb fragment was gel purified. This fragment differs from the normal pBMll fragment in that the NcoI site in the neomycin resistance gene has been removed.

Preparation of the 170bp NcoI-BamHI TVV synthetic gene. Plasmid pLEBam/TVV was digested with NcoI and BamHI and the 170 bp NcoI-BamHI fragment was gel purified. This synthetic gene fragment has the NcoI site at the 5' end and the BamHI site at the 3' end.

Ligation and isolation of pBM16/NDP/TVV. The 4.3kb NcoI-BgII fragment of pBM11, and 1.2kb BamHI-BgilII fragment of pBM11M5, and the 170 bp N-I-BamHI TVV synthetic gene fragment were ligated together using DNA ligase and the resulting mixture was used to transform competent HB101.

The transformants were selected on neomycin and screened by restriction analysis and nucleotide sequencing using the Sanger-dideoxy technique. The r e

I

I

rr TMS/1416R 51 plasmid is denoted pBM16 to indicate the loss of the Ncol restriction site in the neomycin resistance gene.

8. Preparation of Plasmid pRSV/VGF: Transfection of this plasmid into Chinese hamster ovary cells resulted in the production of natural VGF.

Preparation of HindIII(blunt)-BglII(blunt) pRSV. Plasmid pRSV was digested with HindIII and BglII and the 4kb fragment was gel purified.

The protruding ends were made blunt with the Klenow fragment of DNA polymerase and then the 5' phosphates were removed with calf alkaline intestinal phosphatase.

Preparation of a DdeI fragment containing the VGF gene. A subcloned genomic fragment of vaccinia DNA was digested with DdeI, the protruding ends were made blunt using the Klenow fragment, and a 550 bp DdeI fragment was gel purified.

Ligation and isolation of pRSV/VGF. The 4kb HindIII (blunt)-BglII(blunt) fragment of pRSV and the 550 bp )_dI(blunt) fragment of vaccinia DNA were ligated together using DNA ligase and the transformants were selected with ampicillin and screened by restriction analysis. A correct construction was isolated and denoted pRSV/VGF.

Transfection of CHO cells by pRSV/VGF. Plasmid pRSV/VGF was cotransfected with a plasmid pRSV by calcium phosphate precipitation into Chinese hamster ovary (CHO) cells. Transfectants were screened jy Southern dot blot hybridization and a positive clone designated pRSV/VGF52 was isolated.

9. Preparation of Plasmid pAC/VGF: Transfection of this plasmid into insect cells, Spodoptera frugiperda, allows VGF to be expressed under the control of the baculovirus 1 frugierda. allows VGF to be expressed under the control of the baculovirus TMS/1416R 52 polyhedrin gene regulators as described in the specification (see page 33, line 10 through page 35, line 13).

Preparation of SmaI-BamHI pAc610. Plasmid pAc610 was digested with SmaI and BamHI.

Preparation of HindIII(blunt)-BglII fragment of the VGF gene.

Plasmid pRSV/VGF was digested with HindIII and the protruding ends were made blunt using the Klenow fragment. The DNA was the digested with Bglll and the 560 bp fragment was gel purified.

Ligation and isolation of pAc/VGF. The SmaI-BamHI fragment of pAc610 and the 560 bp fragment of pRSV/VGF containing the VGF gene were ligated together using DNA ligase and the transformants were screened by restriction analysis. A correct construct was isolated and termed pAc/VGF.

Transfection of Sf9 cells with pAc/VGF. Plasmid pAc/VGF was cotransfected with AcNPV wild-type baculovirus DNA into Sf9 insect cells (Spodoptera frugiperda). The transfectants were screened for occlusion negative phenotype in plaque assays. One occlusion negative plaque was isolated and after five rounds of successive plaque purification, a high titer recombinant virus stock was prepared.

IV. PREPARATION OF POLYPEPTIDES A. Solid-phase Synthesis of VGF and TGF Synthetic VGF fragment. The sequence corresponding to truncated VGF, beginning with the sequence DIPAIR and ending with the sequence LVDY was assembled on an Applied Biosystems Model 430A Peptide Synthesizer using the standard protocol. Treatment of the final peptide resin with liquid HF under standard "low-high" conditions gave the crude deprotected peptide.

The peptide was subjected to chromatofocusing on PBE94 <Pharmacia). The TMS/1416R 53 partially purified peptide was eluted at pH 6.5. Brief treatment with 0.2 N sodium hydroxide removed the cysteine protecting groups (ethylcarbamoyl) and the peptide was oxidized in the presence of oxidized and reduced glutathione. Purification by gel filtration and HPLC gave highly purified

VGF.

2. Synthetic human and rat TGF-c. Human and rat TGF-a were chemically synthesized, essentially as described above for VGF, and were provided by Penninsula Labs.

B. Isolation of VGF Produced by Eukaryotic Expression of the Natural VGF Gene 1. Natural VGF was produced by infection of monkey kidnc.' cells with vaccinia virus and purified as described in the specification (see page 26, line 30 through page 27, line 13).

2. Natural VGF was also produced by transfection of the plaamid pRSV/VGF containing the VGF gene fragment Into Chinese hamster ovary cells. The transfected cells were expanded and the media and cell pellet were assayed for the presence of VGF by immunoprecipitation using an antisera against an N-terminal VGF pept'le. Low levels of VGF were detected. The media and cell pellet were tested without purification.

3. Natural VGF was produced under a baculovirus promoter in insect S cells as described in the specification (see page 33, line 10 through page 35, line 12). Recombinant virus stock was used to infect Sf9 cells and the media and cell pellets were assayed for the preser of VGF by immunoprecipitation using an antisera against an N-terminal VGF peptide.

High levels of VGF were detected. VGF from the medium of infected cells was purified by size exclusion and reversed phase HPLC to greater than purity.

fMS/1416R 54 C. Isolation of Growth Factors Expressed in Recombinant Bacteria Recombinant bacteria isolated as described in the individual preparations above, were induced by appropriate means to produce the recombinant growth factor. These recombinant growth factors were expressed at high levels as fusion proteins in the bacteria carrying a plasmid r-i i designed to express the respective synthetic growth factor gene. The growth factors were purified from an aggregate preparation essentially as follows: after pelleting and one freeze/thaw treatment, bacteria were resuspended in mM Tris, pH 7.9, 0.2 M NaCl, 2 mM EDTA, 2 mM 2-mercaptoethanol and incubated with 200 g/ml lysozyme for 20 min on ice. Triton X-100 was added to 1% and incubation continued for 20 min. Zwittergent 3-14 (Calbiochem) was added to 0.5% and after a further incubation for 15 min, the mixture was sonicated, layered onto 40% sucrose in 0.01 M Tris, pH 7.2, S0.001 M EDTA, 0.15 M NaCI and centrifuged for 20 min at 5°C in an SN28 Beckman rotor at 13,000 rpm. The pellets were resuspended in 0.01 M Tris, pH 7.2, 0.15 M NaCl and a sample was analyzed by SDS-PAGE. The purity of the recombinant factors :las greater than The purified growth factors underwent a refolding step to 's an i active protein. The growth factor fusion protein was thei either assayed as such or was separated from the bacterial leader sequer.e by chemical treatment and subsequently further purified by size exclusion and HPLC as indicated for individual preparations.

1. TGF and Modified TGF TGF: Recombinant human TGF was purified and supplied by Triton Biosciences.

N/TGF: Recombinant modified human TGF was produced from plasmid TMS/1416R 55 pBM11/N/TGF and contained 33 amino acids of the N-gene at the N-terminus and the sequence modification QEEK instead of the natural human sequence

QEDK.

2. Modified and Truncated VGF PAD/nVGFa: Recombinant modified VGF was produced from plasmid pBM11/PAD/nVGFa containing the extreme N-terminal sequence of VGF and the modified sequences GTC and GYACRC instead of the natural VGF sequence GDC and GMYCRC. The nVGFA fragment was expressed as a fusion protein with a modified alkaline phosphatase signal sequence at the N-te 's and was truncated at the sequence YQR at the C-terminus.

NDP/VGFa: Recombinant modified VGF was produced from plasmid pBMII/NDP/VGFa beginning at the DIPAIR sequence and ending at the YKQR sequence in VGF. It has the modified sequences GTC and GYACRC instead of the natural VGF sequence GDC and GMYCRC. The VGFa fragment is expressed as a fusion protein with 32 amino acids of the N-gene at the N-terminus and the acid labile dipeptide DP.

VGFa: The VGF fragment was prepared as described in and after cleavage from the fusion protein by acid treatment and subsequently further purified by HPLC.

NDP/VGFA: Recombinant modified VGF was produced from plasmid pBM11/NDP/VGFA beginning at the DIPAIR sequence and ending at the YKQR sequence in VGF and having the modified sequences GTC and GYACVC instead of the natural VGF sequence GDC and GMYCRC. The VGFA fragment was expressed as a fusion protein with 32 amino acids of the N-gene at the N-terminus and the acid labile dipeptide aspartic acid-proline.

3. Chimeric TGF/VGF Hybrids TTV (TGF/TGF/VGF): Recombinant modified TTV was produced from TMS/1416R 56 plasmid ptac/TGF/VGF and contained the amino acid sequence of human TGF in the amino terminal two-thirds of the gene with the exception of the sequence QEEK which was altered from the natural human sequence QEDK. The carboxy terminus was derived from the amino acid sequence of VGF and terminated with the sequence YQR upstream of the natural sequence PNT.

N/TTV: Recombinant TTV was produced from plasmid pBM1'/N/TTV and modified as described in except that the TTV fragment was expressed as a fusion protein with 33 amino acids of the N-gene at the N-terminus.

NDP/TTV: Recombinant TTV was produced from plasmid pBM11/N/TTV and modified as described in except that the TTV fragment was expressed as a fusion protein with 32 amino acids of the N-gene at the N-terminus and the acid labile dipeptide aspartic acid-proline.

NDP/VTV: Recombinant modified VTV was produced from plasmid pBM11/NDP/VTV and contained the amino acid sequence of human TGF in the middle domain with the amino acid sequence QEEK replacing the natural sequence QEDK. The N-terminal and C-terminal domains were derived from the truncated VGF sequence and begin with the sequence DIPAIR and end with the sequence YQR. The VTV fragment was expressed as a fusion protein with 32 amino acids of the N-gene at the N-terminus and the acid labile dipeptide aspartic acid-proline.

NDP/TVV: Recombinant modified VTV was produced from plasmid S pBMII/NDP/TVV and contained the amino acid sequence of human TGF in the N-terminal domain of the gene. The middle and C-terminal domains were derived from the truncated VGF sequence and end with the sequence YQR. In addition, the synthetic gene has the modification GYACVC for GMYCRC. The TVV fragment was expressed as a fusion protein with 32 amino acids of the N-gene at the N-terminus and the acid labile dipeptide aspartic acid- TMS/1416R 57 proline.

D. Natural EGF 1. Natural EGF was isolated from mouse salivary glands and was purchased from Collaborative Research.

V. ACTIVITY ASSAYS A. EGF Receptor Binding Inhibition Assay The radioreceptor assays were performed as described in the specification, page 27, lines 20-35. This assay determines the ability of a molecule to bind to the EGF receptor as measured by its ability to inhibit the binding of EGF to its receptor. All growth factors and chimeric growth factors, whether modified or truncated, isolated to date, (see Section IV above) were active in the EGF receptor binding inhibition assay. A summary of these results is shown in Table I.

m...e ,t TMS/1416R r "ili YI1-I-.._ 58 Table 1 Method of Binds to Peptide Preparation Purity EGF Receptor VGF synthetic truncated IV Al >95% Yes TGF synthetic IV A2 >95% Yes VGF natural** IV B1 >95% Yes VGF transfected CHO** IV B2 Yes VGF baculo*** IV B3 >50% Yes TGF recombinant IV Cla >95% Yes N/TGF modified fusion IV Clb >95% Yes PAD/nVGFa modified truncated fusion IV C2a >95% Yes NDP/VGFa modified truncated fusion IV C2b >95% Yes n.p. not purified Contains glycosylation sites Contains probable glycosylation sites **r *If TMS/1416R ua~ C~

I

I

I

59 Table 1 (Continued) Method of Binds to Peptide Preparation Purity EGF Receptor VGFa modified truncated IV C2c >95% Yes NDP/VGFA modified truncated fusion IV C2d >95% Yes TTV modified truncated chimeric fusion IV C3a >95% Yes N/TTV modified truncated chimeric fusion IV C3b >95% Yes NDP/TTV modified truncated chimeric fusion IV C3c >95% Yes NDP/VTV modified truncated chimeric fusion IV C3d >95% Yes NDP/TVV modified truncated chimeric fusion IV C3e >95% Yes n.p. not purified Contains glycosylation sites Contains probable glycosylation sites A comparison of the binding inhibition curves for natural mouse EGF and the bacterially expressed recombinant chimeric growth factor N/TTV (a polypeptide fusion of the 32 N-terminal amino acids of the lambda N-gene and the modified and truncated TGF/VGF hybrid, see IV.b above) suggested that there were no differences in the binding activity, Figure 3.

C

TMS/1416R B. Mitogenic Assay The mitogenesis assays were performed as follows: diploid human fibroblasts obtained from explants of newborn foreskin were seeded at a density of 3 x 10 cells/well (96-well plates, Nuclon, Roskilde, Denmark) and were grown to confluency in Dulbecco's modified Eagle's medium newborn calf serum. Cultures were then placed in medium containing 0.2% newborn calf serum, and two days later EGF (10 ng/ml) or the growth factor to be tested (10 ng equivalents of EGF/ml) was added.

After 8 hrs, cultures were labeled with 5-[ 125 Ilodo-2'deoxyuridine (Amersham, 10 RCi/ml, 5 Ci/mg; 1 Ci 37 GBq), and the amount of isotope incorporated into TCA precipitate was determined as described (Twardzik et i al., Proc. Natl. Acad. Sci. USA (1985) 182:5300-5304). The activity of several of the purified growth factors was tested and the activity determined in all cases was comparable to the effect caused by EGF. The I compounds tested are as indicated in Table II.

Table II ui Method of Mitogenic Peptide Preparation Activity* TGF synthetic IV A2 Yes TTV modified truncated chimeric IV C3a Yes N/TTV modified truncated chimeric fusion IV C3b Yes VGF natural IV Bl Yes As measured by 3H-thymidine or 125I-IdU incorporation TMS/1416R 61- C. Wound Healing Several of the growth factors were assayed for their ability to promote wound healing In viv.

1. Mid-dermal Thermal Injuries: Mid-dermal thermal injuries were made on the dorsal thorax of anesthetized female Yorkshire pigs (30 Ibs) whose backs had been shaved and depilatated with commercial hair cream remover. A brass template (3 x 3 cm, 147 gm) was equilibrated in a 70 0 °C water bath and placed in firm contact with the skin for exactly 10 seconds. The resulting blister was then removed. Five mid-dermal burns were placed on each side of the spine and were separated from each other by approximately one inch. Burns were treated twice a day with approximately 3 ml of vehicle cream (Silvadene which is a registered trade mark) alone or containing growth factor or were untreated. The growth factors were all tested at 0.1 g.g/ml. This concentration had previously been shown to be suboptimal for EGF. After 9 days of treatment with chemically synthesized TGF-a or 10 days of treatment with natural VGF, the pigs were anesthetized and eschar was removed from the burns. Biopsies were taken of each burn from re-epithelialized areas. The percent of the original burn area which had healed was measured by computer-assisted telemetry. This and similar "I experiments are summarized in Table III.

et m, TMS/1416R 62 Table III Method of Cone. Acceleration Treatment* Preparation (pg/ml) of Healing*** None Silvadene alone EGF natural IV D1 0.01 TGF synthetic IV A2 0.01 VGF natural IV B1 0.01 TGF recombinant IV Cla 0.1 VGFa modified truncated IV C2c 0.1 TTV modified truncated chimeric fusion IV C3b 0.1 Growth factors are mixed in Silvadene Concentrations of growth factors in Silvadene, given in EGF equivalents Acceleration of healing over that seen with no treatment 2. Mid-dermal Donor Graft Injuries: The modified truncated VGFa (Section IV. C2c) was assayed for its ability to accelerate wound healing in a donor graft model. A 20.5 kg micropig was anesthetized with 20 mg/kg ketamine and 2 mg/kg ketamine and 2 mg/kg Rompum. The dorsal thorax was shaved, prepped with betadine and thoroughly rinsed with saline. A series of six 5 x 5 cm donor sites were made on each side of the dorsal thorax with a Padgett S dermatome at 60/1000 inch by taking two swipes at 30/1000 inch. Topical therapy included 1 ml of saline in 20 grams of Silvadene distributed evenly between the six wounds on the left side. The right side was treated with 1 ml VGFa, 5 .g/ml, in 20 grams of Silvadene (1 Rg/ml final) divided TMS/1416R

^I

63 evenly between the six wounds. All wounds were covered with a large burn dressing, chux, ace wrap and gerkin. The animal was anesthetized as described above on post operative days 1, 2, 3, 4, 7, 8, 9, 10 and 11. The dressings were removed. The wounds were gently wiped with betadine and thoroughly rinsed with saline. The appropriate agent was applied and the wounds were recovered as described above. Photographs were taken on a daily basis. A summary of the results is provided in Table IV.

Table IV Treatment* POD POD 8 POD 9 POD Saline very open open wound mostly healed wounds with some healed healing VGFa modified, open wound, mostly healed healed truncated apparent healed (IV C2c) epithellalization Silvadene is the vehiele POD Post Operative Day a I1 jii tn or~ r r r TMS/1416R j 64 The modified truncated VGFa accelerated healing of the wound as compared to the carrier control.

In accordance with the subject invention, polypeptides of the present invention are described having a wide ranging capability in a variety of fields, such as diagnostics, in vitro and in vivo effects on cells, acting as mitogens, additives in nutrient media, use as agonists and antagonists to EGF and TGF, acting as immunogens, acting as therapeutics, enhancing wound healing, and the like. Furthermore, by providing for a small oligopeptide having binding activity, the oligopeptide can be used by itself or in combination with other polypeptides, fused to the other polypeptides to vary the properties of the other polypeptides, resulting in binding of the polypeptide to cells having growth factor receptors. Thus, one can reversibly bind various polypeptides to cells having growth factor receptors, affecting the properties of the cells in predetermined ways.

*iJ TMS/1416R 7 65

APPENDIX-A

The following are the sequences of naturally occurring mature VGF and human TGF.

VG F h TFG VG F h TG F VG F h TG F D S GNA I E T TS P ElI TN A T TDI P A V VS H P EG D G Y C L H G GD C I H A R D I D D S H T Q F C F H- G TC R F L VQ E DK I R L CG F N D C P -G MY -P A R C SHG YT GI R CQ HV V LVD Y QRS E NPN T V CH S G YVG AR C EHAD L LA S. 9? a TMS/1I41 6R 7 66 APPENDIX B Explanation of S~'mbols in Table III Schultz kt aI i, 5Saj (1987) 2,U:350-352 'Interim Report (Schultz, 04/04/87) Interim Report (Schultz, 02/05/87) 10-30% epithelialization 30-50% epithelialization 50-80% epithelialization S. iv 0S TMS,'l41 6R

L

Claims (6)

1. A mature VGF polypeptide of a purity of at least Sequivalents of EGF per microgram of protein.

2. A process of preparing a mature VGF of a purity of at least equivalents of EGF per microgram of protein, substantially as herein described with reference to the section entitled Experimental.

3. A mature VGF polypeptide of a purity of at least 90 ng equivalents of EGF per microgram of protein whenever prepared by the process according to claim 2.

4. A method for inducing an immunogenic response in a mammal, comprising administering to said mammal an immunogenically effective amount of a polypeptide according to claim 1 or claim 3. A pharmaceutical formulation comprising an immunogenically effective amount of a polypeptide according to cla 4 m 1 or claim 3 together with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

6. A method for enhancing cellular proliferation in a mammal, comprising administering to said mammal an effective amount of a polypeptide as defined in claim 1 or claim 3 or of a formulation defined in claim

7. A method for promoting wound healing in or on a mammal requiring such healing, comprising administering to the mammal and/or applying to the wound on the mammal, an effective amount of a polypeptide as defined in claim 1 or claim 3 or of a formulation as defined in claim DATED this NINTH day of NOVEMBER 1990 Oncogen i Patent Attorneys for the Applicant SPRUSON FERGUSON TMS/1416R