IE83775B1 - Recombinant fibroblast growth factors - Google Patents

Abstract

ABSTRACT DNA sequences encoding human basic fibroblast growth factor (FGF) can be recombinantly expressed to obtain practical amounts of protein useful in effecting wound healing and related tissue repair.

Description

PATENTS ACT, 1992 950301 RECOMBINANT FIBROBLAST GROWTH FACTORS SCIOS INC.

The invention relates to DNA encoding human basic FGF which is a growth factor important for constructing vascular systems in healing tissues. The genes encoding bovine and human basic and acidic fibroblast growth factors (FGF) are cloned and expressed.

The process of healing when tissue is subjected to trauma. such as wounding or burns, is an extremely complex one. but it is known to be mediated by a number of protein factors. These factors are essential to the growth and differentiation of the cells which serve to replace the tissue destroyed. A number of candidate factors have been identified on the basis of the ability of extracts from various tissues. such as brain. pituitary, and hypothalamus. to stimulate the mitosis of cultured cell lines. Numerous shorthand names have been applied to the active factors in these extracts. including platelet—derived growth factor (PDGF). macrophage-derived growth factor (MGF). epidermal growth factor‘(EGF). tumor angiogenesis factor (TAP), endothelial cell growth factor (ECGF). fibroblast growth factor (FGF). hypothalamus—derived growth factor (HDGF){ retina-derived growth factor (RDGF). and heparin—binding growth factor (HGF). (See. Hunt, T.K.. ; Trauma (1984) ;5:s39-349; Lobb. R.R.. et al.

Biochemistry (1984) g;:629S-6299).

Folkman. J., et al. Science (1983) ;;;:7l9-725, reported that one of the processes involved in wound for example. healing. the formation of blood vessels. is profoundly affected in tumors by heparin. From this and other or not to bind to heparin is one measure of et al. differentiation between the activities in the various EGF and PDGF do not bind in fact, EGF does not bind to The other factors above do show strong extracts. For example. strongly to heparin: heparin at all. heparin binding. However. it is believed that acidic brain FGF. ECGF. RDGF. factor.

FGF. protein. and HGF-a are in fact the same Similarly. it is also believed that pituitary cationic brain FGF, TAP.

(Lobb. R.R.. comparison of thirteen endothelial growth factors which Gospodarowicz and Bohlen et al also reported the isolation of a basic FGF from human brain and a partial characterisation including a partial amino—terminal amino acid sequence (FEBS Letters, 1985, vol 185, pp 177-185).

The complete sequence for basic FGF derived from bovine pituitary has been determined (Esch, F., et al, ELOC gag; Agad Sci (USA) (1985) '_8_2_: 56507-6511) .

Homogeneous preparations were obtained and showed potent mitogenic activity in in vitro assays with endothelial cells (basic FGF has an ED" of 60 pg/ml) — see also WO—A— 86/07595 (Salk Institute) published after the date of filing of the European patent application of which the present application is a divisional.

Acidic FGF has an ED" of about 6,000 pg/ml. An N- terminal sequence for acidic FGF derived from bovine brain tissue was determined by Bohlen P.,et al, EMBQ Q (1985) g: 1951-1956. Gimenez-Gallego, G, et al, determined the N- terminal sequences for both acidic and basic FGF prepared from human brain, and compared them to the bovine sequences ( (1986) L35:541—548).

Their results are consistent with those disclosed herein.

Also, the complete amino acid sequence of bovine brain- derived acidic FGF was determined from the isolated protein (Gimenez-Galleco, G, et al, Sgigngfi (1985) ;3Q:l38S-1388; Esch F, et al, Biochem Bionhvs Res Comm (1985) ;;;:554-562). These two determinations are in agreement with the exception of a single amino acid.

Subsequent to much of the work herein, the complete amino acid sequence of human acidic FGF was deduced from the et al, et al, extension in et al. suggesting Proc degenerative neurological disorders, such as Alzheimer's disease and Parkinson's disease.

It would. therefore. be desirable to insure the availability of these FGF proteins in large quantities and in a form free from any toxic or infectious impurities. The human form of the protein is preferred, and perhaps required. for therapeutic use. Clearly practical availability cannot be achieved from natural human sources. as obtaining a pure preparation involves an approximately 35.000-fold purification. Even if human cadavers were otherwise a practical source. »complete purification would be crucial due to the possibility of transmitting AIDS, hepatitis. or other The recent experience with Creutzfeld-Jacob Lancet (1985) ii:244-246) precludes the use of human pituitaries as a disease.

Syndrome (Powell-Jackson et al, recombinant techniques are source. Therefore. particularly suitable to apply to the production of ‘ these proteins. The invention herein provides the means whereby basic human FGF can be obtained in practical quantities and in pure, uncontaminated form.

The invention provides the tools for synthesis and manipulation of human basic fibroblast growth factor useful in effecting accelerated healing of wounds, bone fractures, burn tissue, damaged myocardial tissue, degenerated neurological tissue, or other trauma. Cloning and expression of the genes encoding these factors are provided by the methods and materials of the invention.

In one aspect, the invention relates to an isolated cloned recombinant or synthetic DNA sequence encoding a basic human FGF protein comprising the amino acid sequence numbered l6~146 in Figure 4 (SEQ ID No 20). In particular, this includes the human genomic sequence. In other aspects, the invention relates to recombinant vectors bearing these DNA sequences and to host cells transformed with such vectors and harbouring these DNA sequences.

Figures 1-4 show the DNA sequences encoding, and the deduced amino acid sequences of, acidic bFGF, acidic hFGF, basic bFGF, and basic hFGF. Figure la shows the partial sequence for the acidic bovine FGF (SEQ. ID. No. 6 and 7).

Figure 1b shows the complete amino acid sequence of this protein (SEQ. ID. No. 8). Figures 2a (SEQ. ID. No. 9, ), 2b (SEQ. ID. No. 11, 12) and 2c (SEQ. ID. No. 13,14) show the nucleotide sequence and deduced amino acid sequence corresponding to the three exons of the human acidic FGF gene contained in X phage XHAG-9.1, XHG-3, and KHAG-3, respectively. Figure 2d (SEQ.ID.No.15, 16) shows the complete amino acid sequence and CDNA sequence encoding human acidic FGF as disclosed by Jaye et al.

Figure 3 shows the nucleotide and amino acid sequence of basic bovine FGF (SEQ.ID.No.17, 18).

Figure 4 shows the nucleotide and amino acid sequence of basic human FGF (SEQ.ID.No.19, 20).

Figure 5 shows the oligonucleotide probes 889/890 (SEQ.ID.No.27), 891 (SEQ.ID.No.23) and 853-856 (SEQ.ID.No.24) designed from the acidic bFGF N-terminal sequence (SEQ.ID.No. 21, 22, 25, 26).

Figure 6 gives restriction maps of the inserts for genomic acidic bFGF clones KBA2 and lBA3.

Figure 7 shows the DNA sequence of the bovine acidic FGF genomic probe 250/Alul (SEQ.Ib.No.28, 29).

Figure 8 shows basic FGF probes 1097/1098 (SEQ.ID.No.35, 34) and part of the amino acid and nucleotide sequence of bovine acidic FGF (SEQ.ID.No. , 31) and bovine basic FGF (SEQ ID.No. 32, 33).

Figure 9 is a restriction map of the basic bFGF CDNA clone ABB2.

Figure 10 shows the results of transient expression of basic hFGF in CV—l cells.

Figure 11 shows a map of the human basic FGF encoding gene.

A. The Fibroblast Growth Factors Two different bovine (and analogous human) fibroblast growth factors have been purified to homogeneity by others and partially or completely sequenced. Both factors are capable of mitogenic activity in ig_gig;g assays using cultured cells. such as bovine brain and adrenal cortex-derived capillary endothelial cells, human umbilical vein endothelial cells, bovine adrenal cortex cells. granulosa cells. and vascular smooth muscle cells. In vitro assays employing these cell cultures have been described by Gospodarowicz. D.. et al. J Cell Physiol (1985) J Cell Biol More recently, alternative in Gospodarowicz. D.. et al. J Cell Physiol (1986) lglz l2l—l36. angiogenic in vivo in a chicken chorioallantoic membrane Purified basic bFGF has been shown to be in Hormonal Proteins and :::___ assay. (Gospodarowicz. D.

Peptides xII:20S-230 (Academic Press). Purified acidic bFGF has been shown to be angiogenic in vivo in the same assay (Thomas. K.A.. et al. Proc Natl Acad Sci (supra)).

’ Bovine pituitary basic FGF has been completely sequenced and is shown in Figure 3: the human sequence determined herein from genomic and CDNA is shown in Figure 4. The primary sequences contain 146 amino acids, beginning with the proline residues numbered "1" in the figures. and are in agreement with the sequence reported for the N—terminus of the native bovine protein by Giminez—Gallego et al. Biochem Biophys Res Comm . gland. reported from the human hepatoma cell line. additional form of each protein containing the amino acids upstream of the proline shown as residue 1 in Figures 3 and 4 is also produced. There are 9 upstream including the ATG. certain that the methionine encoded by the ATG will be codons in the DNAs, It is reasonably processed when the gene is expressed in eucaryotic systems. Such processing may or may not occur when the gene is expressed recombinantly in bacterial systems.

Thus. the long form of the protein contains an additional eight amino acid pro-sequence. or a total of 154 amino acids. It has also been shown that this extended FGF as isolated from SK—HEP—1 cells is blocked at the N-terminus (Klagsbrun. M.. et al. (supra)).

Proteins having FGF activity in the above-mentioned in vitro assays and sharing a similar putative N-terminal sequence with the bovine pituitary basic FGF shown in Figure 3 (the 146 amino acid form) have also been isolated from bovine brain. adrenal corpus luteum, retina, kidney. and from human placenta. The native protein obtained from certain of these tissues is heterogeneous -- a second form missing the putative fifteen N-terminal amino acids retains Meth En: (1986) in that bovine and activity. (Gospodarowicz. D.. press.) It is considered. tnerefore. human basic FGFs exist in three forms--those indicated as mature forms in Figures 3 and 4, longer forms containing eight additional amino acids at the N-terminus, and shorter forms lacking fifteen amino Thus. there is believed to be natively produced Wlong" basic acids of the putative mature sequences shown.

FGF containing 154 amino acids. "primary" basic FGF containing 146 amino acids. and "short" basic FGF containing 131 amino acids. These FGFS are designated "basic" FGF. because they contain a high number of basic amino acid residues (lysine. arginine. histidine) and are therefore cations at neutral pH.

A protein is defined herein as basic FGF if it binds to is a cation at neutral pH. and reacts shows FGF activity in the foregoing assays. heparin. immunologically with antibodies prepared using a synthetic analog of the amino terminal sequence [tyrlo] FGF (1-10) conjugated to bovine serum albumin (if appropriate) or to other antibodies raised against bovine (or human) FGF or synthetic or native peptides thereof. See Baird. A.. et al. Regulatory Peptides (1985) lQ:309-317.

Acidic FGF has been isolated from bovine brain by others, and the first 34 amino acid residues determined. The cloning herein of the genes for bovine and human acidic FGF has permitted amino acid sequences additional to 1-34 for acidic bFGF. to be deduced as shown in Figure la, and a partial sequence for acidic hFGF has been obtained, as shown in Figure 2a.

Subsequent to much of the work described below. the complete amino acid sequence for acidic bFGF was disclosed by Esch. et al. Biochem Biophys Res Comm 1 et al.

Also, the complete coding sequence (supra) and by Gimenez-Gallego. G.. science (supra), as shown in Figure lb. subsequent to most of the present work, for acidic hFGF was determined by the Maciag group. as shown in Figure 2b.

The acidic protein also has two known active forms, one having the 140 amino acid sequence beginning at the phenylalanine residue numbered "1" in the figures. and a second shorter form corresponding to amino acids 7-140. Both the bovine and human proteins may also occur in N-terminal extended forms.

Translation of DNA upstream of the codon for the amino acid numbered "1" in the figures (back to the ATG start codon at -15, shown in parentheses) represents the As is the the N-terminal methionine is almost additional sequence of the extended protein. case for basic FGF. certainly processed off in eucaryotic expression hosts, although it may not be if the gene is expressed in like the basic FGF described above. the native acidic protein may exist in three "short." acidic PG? containing 134 amino acids: one N—terminal extended. bacteria. Therefore. active forms: one truncated, 1.e.. i.e., "long" form containing 154 amino acids: and the other "primary" acidic FGF containing 140 amino acids beginning at the residue numbered "1" in the figures.

It has been shown by Burgess. W.H.. et al. (in press) that the bovine brain long form is blocked by an acetyl residue. These proteins contain a disproportionate number of acidic amino acid residues. i.e.. glutamic and aspartic acids and the proteins are therefore anions at neutral pH.

A protein is defined herein as acidic FGF if it shows FGF activity in in vitro assays. binds to heparin. is an anion at neutral pH, and is immunologically reactive with antibodies prepared against human or bovine acidic FGF or against synthetic or native peptides thereof.

Acidic FGF and basic FGF are thus used herein to designate the foregoing proteins or proteins having amino acid sequences represented by those shown in Figures 1-4. of course. these definitions are not restricted to the specific sequences shown. but include proteins which contain accidentally or deliberately induced alterations, such as deletions. exchanges of amino acid residues. additions. or so long as the biological activity. as measured by the foregoing in vitro and immunological assays. and respective anionic or cationic character at neutral pH does not change. Of course. modified forms may have slightly altered quantitative activity and specificity.

"Purified" or "pure" refers to material which is free from substances which normally accompany,it as found in its native state. Thus acidic hFGF, example. refers to acidic hFGF which does not contain "pure" for materials normally associated with its in situ environment in human brain or pituitary. "operably linked" refers to a juxtaposition wherein the components are configured so as to perform Thus. promoters operably linked to a coding sequence are capafile of effecting the expression of the coding their usual function. control sequences or sequence.

’ "Control sequence" refers to a DNA sequence or sequences which are capable. when properly ligated to a desired coding sequence. of effecting its expression in hosts compatible with such sequences. Such control sequences include at least promoters in both procaryotic and eucaryotic hosts. and optionally. transcription termination signals. Additional factors necessary or helpful in effecting expression may also be As used herein. identified. "control sequences" simply refers to whatever DNA sequence may be required to effect expression in the particular host used.

"Cells" or "cell cultures" or "host cells" "recombinant host cells" or are often used interchangeably as will be clear from the context. These terms include the immediate subject cell. and. of course. the progeny It is understood that not all progeny are exactly identical to the parental cell. thereof. due to chance mutations or differences in environment.

However. such altered progeny are included in these terms. so long as the progeny retain the characteristics relevant to those conferred on the originally transformed cell. In the such a characteristic might be the ability to produce recombinant FGF. present case. for example, B. General Description Utility and Administration The invention provides DNAS encoding human basic fibroblast growth factor protein which is useful in encour- aging the healing of wounds and which further may be supplied in sufficiently pure amounts to permit the design of inhibitors specific to them. The purified growth factors are generally applied topically to the traumatized tissue in order to stimulate vascularization and healing. Appropriate substrates are burns. wounds. bone fractures. surgical abrasions such as those of plastic surgery. Because they or others requiring repair. application of these factors accelerates healing. also reduce the risk of infection.

Indications wherein FGF is of value in" encouraging neovascularization include musculo—ske1etal conditions such as bone fractures. ligament and tendon repair. tendonitis. and bursitis: skin conditions such as burns, cuts. lacerations. bed sores. and slow-healing ulcers such as those seen in diabetics: and in tissue repair during ischaemia and myocardial infarction.

Formulations of the recombinantly produced growth factor using available excipients and carriers are prepared according to standard methods known to those in the art. The protein can be formulated as lotions, gels. as part of a controlled release system. or ointments with additional active ingredients. such as antibiotics. if desired.

For topical administration. which is the most appropriate with regard to superficial lesions. standard topical formulations are employed using. for example.

O.l—l0% solutions. Such solutions would be applied 3-6 times a day to the affected area. The concentration of the ointment or other formulation depends. of course. on the severity of the wound and nature of the subject. In most protocols. the dose is lowered with time to lessen likelihood of scarring. For example. the most severe wounds, such as third degree burns. are typically treated with a 10: composition. but as healing begins. the dose is progressively dropped to approximately 0.1% or lower. as the wound heals. A topical formulation for EGF using BSA as carrier was disclosed by Franklin.

J.D.y et al. Elastic and Reconstruc Surq (1979) 53:766-770.

For bone and tissue repair. administration is preferred locally. but by means of subcutaneous implant or slow release formulation implanted directly proximal the target. surgery may be required for such conditions as bone injuries. thus making implantation directly practical. Slow—release forms can be formulated in other sustained- preferred in the present invention. is suggested by Buckley, A., Proc Natl Acad Sci USA (1985) g;:7340— 7344.

As with topical administration. for sustained- release delivery. the concentration of FGF in the formulation depends on a number of factors. including the severity of the condition and the rate of FGF release from the polymer. In general. the formulations are constructed so as to achieve a constant local concentration of about 100 times the serum level of hormone or 10 times the tissue concentration. as described by Buckley et al (Proc Natl Acad sci USA (supra)). Based on an FGF concentration in tissue of -50 ng/g wet weight (comparable to EGF at 60 ng/g wet weight). release of 50-5000 ng FGF per hour is acceptable. The initial concentration. of course. depends on the severity of the wound.

It is expected that FGF may act in concert. and even synergistically. with other growth factors such as epidermal growth factor (EGF), the transforming growth factors (TGF-a or TGF-B). insulin-like growth factors (IGF-l and IGF-2). and/or platelet—derived growth factor (PDGF). may act in synergy with antagonists of parathyroid In addition. specifically for bone repair. it hormone. since parathyroid hormone promotes bone resorption. Therefore. also included within the compositions and administration protocols of the invention are embodiments wherein the FGF of the invention is administered in the same composition with, or in the same protocol with. one or more of the foregoing factors, thus more effectively to achieve the desired tissue repair.

Since FGF is effective in promoting neurite outgrowth. nerve regeneration, and neuronal survival. it may be useful for treatment of certain neurological disorders such as Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis. and general aging of the nervous system. as well as traumatic injury to the spinal cord and peripheral nerves.

Administration of the drug for these indications is preferably by implant in formulations similar to those set forth above in connection with wound healing. The drug may also be delivered by means of implants of cell cultures as in transplant therapy by treating the cultures prior to transplantation with the the FGF may be injected directly into the spinal fluid. or may FGF preparations of the invention. In addition, be applied systemically. Systemic formulations are generally as are known in the art and include formulation in buffer or physiological saline. or other appropriate excipient. Dosage levels are approximately those of wound healing: however. for tissue culture or explant maintenance. it may be supplied at 0.1-lo nq/ml of serum or culture medium.

FGF proteins are particularly useful, also. in aiding the reformation and repair of tissues traumatized For this use. during surgery. it may be helpful to ‘embed the FGF proteins in polymers used as surgical staples. The proteins are thus able to supplement biologically the mechanical suturing effected by the— staples. and to augment and abet the "natural" healing processes in the repaired tissues. invention are also useful in treatment of conditions (Gross, the FGF proteins of the which respond to these enzymes. While it may be necessary in acute situations (such as the presence of a blood clot associated with stroke or heart attack) directly to administer large doses of tPA to dissolve the clot, for treatment of chronic propensity to form embolisms. administration of FGF to maintain a suitable level of tPA in the blood stream may be desirable.

Therefore. for this indication. systemic administration of the drug, using conventional means such as intramuscular or intravenous injection, is preferred.

The invention provides practical quantities of pure human basic FGF growth factors for use in connection with the foregoing indications. Basic FGF is considered to occur in long, primary, and short forms, as described herein. It is considered that the N- terminal methionine of the long form is processed off when the protein is produced in eucaryotic systems, and that the subsequent amino acid residue is derivatized. probably by acetylation, post—translation.

While FGF in its various forms does not have a recognized signal sequence, it must somehow be secreted. since it acts outside the cells producing it at a while it is membrane—bound receptor. Therefore. probably not secreted by the recognized constitutive _]_'7_ secretion pathway. its secretion is accomplished by other means, such as by cell lysis or by exocytosis.

For most tissues from which FGF is naturally derived, and fior many mammalian expression systems. such release may be achieved by securing exocytosis with a calcium ionophore, such as the commonly employed A2318? (CalBiochem), which. is added to the culture medium at 1-10 uu in the presence of 1 mM CaCl2. For expression systems derived from macrophages or monocytes, other activation methods have been shown to be effective. in in vitro conditions, triglycerides.

Gene Retrieval The general strategy whereby the illustrated human basic FGF—encoding sequences were obtained herein is as follows. The known N—terminal sequence of bovine acidic EGF was used to design a series of probes for use with a bovine genomic library ligated into phage. Phage recombinants which hybridized to the probes were isolated from the library and digested into smaller fragments suitable for cloning into M13 cloning vectors in order to obtain a "natural" probe. This resulted in an M13 probe containing a 250 bp sequence corresponding _l8_ to a portion of the bovine acidic protein; this probe is central to obtaining the genes for the basic forms in these species.

Briefly, the fragments obtained by Alul digestion of a selected acidic bFGF gene fragment cloned into phage were shotgun cloned into M13 and a 250 bp fragment which hybridized to appropriate probe DNA selected and sequenced.

The above, designated /Alul, was transferred into pBR322 and was used to design probes for the basic form, taking advantage of the available amino acid sequence information to alter the DNA to correspond to the basic rather than acidic form. The modified probe, thus designed on the basis of a comparison of the acidic bFGF N—terminal coding sequence and the basic bFGF amino acid sequence, was used to probe a bovine pituitary CDNA library for the basic bFGF cDNA. The recovered bovine clone was then used to probe human genomic and CDNA libraries to recover the genomic sequence encoding human basic FGF—encoding DNA. Alternatively, the bovine cDNA clone ABB2 was mutagenized to convert the DNA sequence to one encoding the human form of the basic FGF protein. The CDNA and genomic clones described hereinbelow are useful in probing DNA libraries prepared from various species to obtain the analogous coding sequences from these mammalian libraries for basic FGF; in addition, the genomic clones are capable of expression in mammalian systems and may give better results than the corresponding cDNAs. cDNA libraries prepared from various tissues such as pituitary. brain. hypothalamus. or kidney can also be screened in this C .

Expression of FGF Genes The cloned genomic or CDNA sequences can be expressed in appropriate expression systems. "Of course. for the DNAs disclosed herein. the foregoing protocol but conventional chemical synthesis methods can suitably be This permits adjustment of the DNA to obtain for retrieving them need not be repeated. employed. any desired form of the protein. cDNA sequences can be provided with appropriate controls suitable for any host, including bacteria, yeast, or eucaryotic cells.

Genomic sequences containing In particular, complete DNA encoding full length human basic FGF can be constructed, for example, using a combination of recombinant and synthetic methods to obtain any of the long, primary or short forms of human basic FGF. Heterologous signal sequences may also be fused to these, and advantage taken of the known relationship of the signal sequence to cleavage site to obtain the protein in the desired form. Intracellularly produced forms of the proteins can be obtained by cell lysis. or their release from the cells can be stimulated as described above. Particularly preferred are expression systems for either the cell-associated or putatively secreted (fused to signal sequence) forms which utilize control systems compatible with mammalian such as CHO cells. which can be used for stable or cells. Also preferred are vaccinia-based systems. transient expression in susceptible cells.

The recombinant human basic FGF protein thus produced is then purified in a manner similar to that utilized for purification of FGF from natural sources, but purification is considerably simpler, as the proteins form a much larger proportion of the starting material.

Polymorphism _ It has also been shown that human genomic DNA exhibits a polymorphism in the region of the second exon of the gene. Existence of the polymorphism is'a predictor of the tendency to solid tumors, as FGF is secreted by them. and probably is necessary for their survival. as it promotes blood vessel growth that keeps nutrients flowing to the tumor.

To detect the polymorphism. human genomic DNA is obtained by conventional methods. from a blood sample, for example, and subjected to size separation on polyacrylamide gels and probed using standard Southern blot techniques. An effective probe is the 1.4 Kb EcoRI fragment obtained from the 2.1 Kb insert into XBBZ described hereinbelow. when such a probe or its equivalent is used to hybridize to gels containing Hindlll digests of the isolated human DNA. a 2.7 Kb _2]_..

In some individuals. an These fragments map to the region of the gene surrounding exon fragment is normally detected. additional 2.9 kb fragment is also found. . as shown in Figure 17.

Of three individuals tested. two exhibited only the 2.7 kb fragment: one exhibited both the 2.7 and 2.9 Kb fragments. The hybridization intensity showed that the individual with both fragments contains both alleles. which is supported by results obtained by Southern blot analysis of DNA from mouse/human hybrid cell lines. In such hybrids. wherein only one chromosome is transferred. only one of the two fragments appears in each line.

C. Standard Methods Most of the techniques which are used to transform cells. construct vectors. extract messenger RNA. prepare CDNA libraries. and the like are widely practiced in the art. and most practitioners are familiar with the standard resource materials which describe specific conditions and procedures. However. for convenience. the following paragraphs may serve as a guideline.

C.l.

Both procaryotic and eucaryotic systems may be Hosts and Control Sequences used to express the FGF encoding sequences: procaryotic hosts are. of course. the most convenient for cloning procedures. Procaryotes most frequently are represented by various strains of E. coli: however. other microbial strains may also be used. Plasmid vectors which contain replication sites. selectable markers and control sequences derived from a species compatible with the host are used: for example, E. coli is typically lambda—derived PL promoter and N-gene ribosome site (shimatake. et al. Nature (1981) 292:l28).

In addition to bacteria. eucaryotic microbes. such as yeast, may also be used as hosts. Laboratory of saccharomyces cerevisiae. Baker's yeast. are most used although a number of other strains or species are commonly available. strains Vectors employing. for example. the 2 u origin of replication of Breach. J. R.. Meth Enz (1983) 1012307. or other yeast compatible origins of replication (see. for example. Stinchcomb. et al. Nature additional advantage of transcription controlled by growth conditions and/or genetic background are the promoter regions for alcohol dehydrogenase 2. isocytochrome C. acid phosphatase. degradative enzymes associated with nitrogen metabolism. the alpha factor system and enzymes responsible for maltose and galactose utilization. It is also believed terminator sequences are desirable at the 3' end of the coding sequences.

Such terminators are found in the 3' untranslated region following the coding sequences in yeast-derived genes.

It is also, of course. possible to express genes encoding polypeptides in eucaryotic host cell cultures derived from multicellular organisms.

Axel, 4.399.216. additional advantage of the ability to splice out See. for example. et al. These systems have the introns and thus can be used directly to express genomic Useful host cell lines include VERO and HeLa (CHO) cells.

Expression vectors for such cells ordinarily include fragments. cells. and Chinese hamster ovary promoters and control sequences compatible with mammalian cells such as, for example, the commonly used early and late promoters from simian Virus 40 (SV40) (Fiers. et al. Nature (1978) ;1;:ll3). or other viral promoters such as those derived from polyoma, Adenovirus 2, bovine papilloma virus. or avian sarcoma viruses.

The controllable promoter. hMTII (Karin, M.. et al.

Nature (1982) ;ggi797-802) may also be used. General aspects of mammalian cell host system transformations have been described by Axel (supra). It now appears. also that "enhancer" regions are important in optimizing expression: these are. generally. sequences found upstream or downstream of the promoter region in noncoding DNA regions. Origins of replication may be obtained, if needed. from viral sources. However. ' understood in the art. integration into the chromosome is a common mechanism for DNA replication in eucaryotes.

C.2. Transformations Depending on the host cell used. transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium described in Maniatis. et al. Molecular Cloning: A chloride. as described by Cohen.

Transformations C.3.

Construction of suitable vectors containing the Vector Construction desired coding and control sequences employs standard ligation and restriction techniques which are well Isolated plasmids. DNA sequences. or synthesized oligonucleotides are cleaved. tailored, and religated in the form desired.

The DNA sequences which form the vectors are available from a number of sources. Backbone vectors and control systems are generally found on available "host" vectors which are used for the bulk of the sequences in construction. Typical sequences have been set forth in 1C.1 above. For the pertinent coding sequence. initial construction may be. and usually is. a matter of retrieving the appropriate sequences from CDNA or genomic DNA libraries. However, once the sequence is disclosed it is possible to synthesize the entire gene sequence in gitgg starting from the individual nucleoside derivatives. The entire gene sequence for genes of sizeable length, e g.. 500-1000 bp may be prepared by synthesizing individual overlapping complementary oligonucleotides and filling in single stranded nonoverlapping portions using DNA polymerase in the presence of the deoxyribonucleotide triphosphates.

This approach has been used successfully in the Synthetic oligonucleotides are prepared by either the phosphotriester method as described by Edge, et al. Nature (supra) and Duckworth, et al. Nucleic Acids Res (1981) 321691 or the phosphoramidite method as described by Beaucage, S.L.. and Caruthers. M.H.. get ggttg (1981) ;;:1a59 and Matteucci. M.D.. and Caruthers.

M.H., J Am Chem Soc (1981) 1Q;:318S using commercially available automated oligonucleotide and can be prepared synthesizers. Kinasing of single strands prior to annealing or for labeling is achieved using an excess, e.g.. approximately 10 units of polynucleotide kinase to l nmole substrate in the presence of 50 mM Tris. pH 7.6. mM MgCl2. 5 mM dithiothreitol. 1-2 mM ATP. 1.7 pmoles y32P-ATP (2.9 mci/mmole). 0.1 mM spermidine. .1 mM EDTA.

Once the components of the desired vectors are thus available. they can be excised and ligated using standard restriction and ligation procedures.

Site specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art. and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See. e.g.. New England Biolabs.

Product Catalog. In general. about 1 ug of plasmid or DNA sequence is cleaved by one unit of enzyme in about ul of buffer solution: in the examples herein. typically. an excess of restriction enzyme is used to insure complete digestion of the DNA substrate.

Incubation times of about one hour to two hours at about 37°C are workable. although variations can be tolerated. After each incubation. protein is removed by extraction with phenol/chloroform. and may be followed by ether extraction, and the nucleic acid recovered from aqueous fractions by precipitation with ethanol. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in ‘ polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 min at 20 to 25°C in 50 mM Tris pH 7.6. 50 mM NaC1. 6 mM MgC12. 6 mM DTT and 0.1-1.0 mM dNTPs. single-stranded overhangs but chews back protruding 3' The Klenow fragment fills in at 5' single strands. even though the four dNTPs are present. -27..

If desired. selective repair can be performed by supplying only one of the, or selected, dNTPs within the limitations dictated by the nature of the overhang.

After treatment with Klenow. the mixture is extracted with phenol/chloroform and ethanol precipitated.

Treatment under appropriate conditions with S1 nuclease or BAL-31 results in hydrolysis of any single-stranded portion.

Ligations are performed in 15-50 ul volumes under the following standard conditions and mM Tris-Cl pH 7.5, 10 mM MgC12. 10 mM DTT, 33 ug/ml BSA. 10 mM-S0 mM NaCl. and either 40 uM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C (for "sticky end" ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C (for "blunt temperatures: for example. end" ligation). Intermolecular "sticky end" liqations are usually performed at 33-100 uq/ml total DNA concentrations (5-100 nM total end concentration).

Intermolecular blunt end.ligations are performed at 1 uM total ends concentration.

In vector construction employing "vector fragments". the vector fragment is commonly treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase (CIP) in order to remove the 5' phosphate and prevent self-ligation of the vector.

Digestions are conducted at pH 8 in approximately 10 mM Tris-HCl. 1 mM EDTA using about 1 unit of BAP or CIP per ug-of vector at 60° for about one hour. In order to recover the nucleic acid fragments. the preparation is extracted with phenol/chloroform and ethanol precipitated. Alternatively. religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion and separation of the unwanted fragments.

For portions of vectors derived from cDNA or genomic DNA which require sequence modifications. site specific primer directed mutagenesis may be used (Zoller, M.J.. and Smith. M. Nucleic Acids Res (1982) Lg:6487-6500 and Adelman. J.P., et al, QQA (1983) ;:1.e3-193). oliqonucleotide complementary to a single stranded phage DNA to be mutagenized except for limited mismatching.

Briefly. the synthetic oligonucleotide is used as a primer to direct This is conducted using a primer synthetic representing the desired mutation. synthesis of a strand complementary to the phage. and the resulting partially or fully double-stranded DNA is transformed into a phage-supporting host bacterium.

Cultures of the transformed bacteria are plated in top agar. permitting plaque formation from single cells A which harbor the phage.

Theoretically. 50% of the new plaques will contain the phage having. the 50% will have the original sequence. The resulting plaques are washed after hybridization with kinased synthetic primer at a wash temperature which as a single strand. mutated form: permits binding of an exact match. but at which the mismatches with the original strand are sufficient to prevent binding. Plaques which hybridize with the probe are then picked. cultured. and the DNA recovered.

C.4.

In the constructions set forth below.

Verification of Construction by ampicillin, tetracycline or other antibiotic Holmes. Anal Biochem nucleotide method of Sanger. F..

C.5. Hosts Exemplified Host strains used in cloning and procaryotic expression herein are as follows: For cloning and sequencing. and for expression of construction under control of most bacterial promoters. E. coli strains such as MCl06l. DHI, RRL.

C600hfl, K803. HBl01. JA221, and JMl0l were used.

D. Illustrative Procedure The following examples are intended to The DNA encoding the illustrated FGF sequences is obtained illustrate but not to limit the invention. initially by screening a bovine genomic library and obtaining a pivotal probe. additional DNA. followed by retrieval of However, it would not be necessary to repeat this procedure, as the sequence of the pivotal probe is now known and could thus be constructed chemically in vitro. In addition, bacteriophage harboring the four illustrated sequences are deposited at the American Type Culture Collection.

Example 1 Construction of the 250/AluI Probe: Preparation of Acidic bFGF Genomic DNA A 250 bp Alul bovine genomic fragment was used to probe both human and bovine libraries in order to obtain complete coding sequences for the illustrated acidic FGF proteins. This probe. designated 250/Alul. was obtained as follows.

The N-terminal amino acid sequence for residues 1-34 of bovine acidic FGF is known. Three long probes were prepared. based on codon choice (Anderson. 8.. et al. Proc Natl Acad Sci (USA) (1983) gg:6838—6842: Jaye.

M.. et al. Nucleic Acids Res (1983) l1:232S-2335) using an automatic synthesizer and a phosphoramidite coupling reagent. The sequences of these nucleotide probes are shown in Figure 5. Probe 891 is a 48—mer corresponding to amino acids 1-16: probes 889 and 890 are Sl-mers corresponding to amino acids 18-34 and are used as a 50-50 mixture of the two oligonucleotides which are identical except for the codon for arginine at position 24. The probes were used to screen a bovine genomic library obtained from Dr. Fritz Rottman. Case Western Reserve. which had been prepared as a partial Mbol digest and was cloned into BamHI treated phage vector Charon 28 (Woychik. R.F.. et al. Nucleic Acids Res (1982) lQ:7197-7210).

Hybridization was conducted on denatured DNA replicated onto filters using a modification of the method described by Ullrich. A.. et a1. EMBO J (1984) 3:361-364: and the washing conditions were those of Wood. W.I., et al. Nature (1984) 3l2:330—337.

Prehybridization/hybridization buffer contained 20% 5x Denhardt‘s solution (lO0x Denhardt's equals 2% bovine serum albumin. 2% polyvinyl pyrollidone: 2% Ficoll); 6x SSC (20x SSC equals 3 M Nacl, 0.3 M Na citrate); pH 6.8; formamide. mM sodium phosphate. 100 ug/ml herring sperm DNA; hybridization buffer further included 10% dextran sulfate and about 105-106 Prehybridization and hybridization were at 42°C for 1 hr cpm/ml kinased probes 891 or 889/890.

The filters were then washed 2x 0.1% SDS at 22°C. followed by l ten 0.1% SDS at 55°C. the filters were exposed for 1 day using intensifying and l6 hr respectively. min with 1x SSC. minute wash in 1x SSC. After washing, screens.

The screened bovine genomic library contained phage out of 106 both probes. recombinants which hybridized to These 50 phage were further screened with mixtures of probes 853-856. In this screen. prehybridization/hybridization buffer contained 6x SSC. lx Denhardt's. 0.1% SDS. 0.05% Na pyrophosphate, and 100 uq/ml salmon sperm DNA; hybridization buffer further contained 105-106 Probes 853-856 are pools of 16 sequences each of the 64 (total) 17-mers cpm/ml probe. corresponding to amino acids 7-12. synthesized using the phosphotriester method. However. 46 of the 50 clones further hybridized to the shorter probes. This hybridization was performed at between 65°C to 35°C for 16 hr and the base composition-independent washing method using tetramethyl ammonium chloride at 50°C was used (Wood. W.I.. Proc Natl Acad Sci (USA) (1985) ggzises-isee).

Two positively hybridizing phage were selected (XBA2 and XBA3) and the phage inserts were restriction mapped, as shown in Figure 6. and partially sequenced as shown in Figure la. Comparison of the deduced amino acid sequence with that published for the N—termina1 34 residues of the bovine acidic FGF native protein confirmed that these clones are correct. From the nature of the coding sequence it is apparent that amino acid residues l-41 (as shown in Figure la) are encoded in these clones: immediately subsequent The length but it is nucleotides appear to represent an intron. of this intron is. at present. uncertain, possible that the complete acidic bFGF encoding sequence resides on these LBAZ and XBAS DNAs. However any additional DNA required to obtain the complete coding sequences for this protein can be obtained from the same gene library using the XBAZ or XBA3 in "walking" techniques. The codons preceding the N-terminal residue are believed to encode the indicated fifteen amino acid prosequence. or. as discussed above. the vlong" form of the native protein extended by fifteen amino acids at the N-terminus (or by fourteen if the N-terminal methionine is cleaved) as compared to isolated "primary" form.

To prepare the 250/Alul probe. XBAZ was partially digested with Alul and shotgun cloned into M13 et al. 92$ (1982) l9:269-276). The M13 plaques were hybridized in duplicate with 853-856 and 889/890. sequenced.

(Messing. J..

Phage hybridizing to both probes were The resulting 250 bp AluI probe is shown in Figure 7 along with the corresponding deduced amino acid sequence: its location on the XBA2 and kBA3 inserts of Figure 6 corresponds to the site of probes 889/890 and 891.

N—terminal portion of the acidic bFGF protein.

The 250/Alul probe corresponds to the _amino acid sequence was the same.

Example 2 Retrieval of Basic bFGF Genomic and cDNA Clones The 250/A1uI probe was used to design appropriate probes to obtain the corresponding basic bFGF sequences. Advantage was taken of the finding of Esch. F.. et al (supra) that amino acids 4-29 of acidic bFGF align with amino acids 13-38 of the basic bFGF sequence. Probes were designed based on the basic bFGF residues 18-36 and acidic bFGF residues 9-27. which regions are homologous at 14 of the 19 amino acids.

Probes 1097 and 1098. 40-mers designed to encode this region. were prepared using the The probes are shown in Figure 8: they overlap in the amino acid 23-31 region of the basic sequence. phosphoramidite method on an automatic synthesizer.

In designing the probes. the 250/Alul sequence was used where the and where different. minimum nucleotide differences in order to effect the required change in encoded sequence were incorporated.

The bovine pituitary cDNA library obtained by the method of Huynh. V.T.. . was screened with 1098. as set forth in Example 2.

Correct conditions for hybridization were determined using genomic DNA (Example 1) for Southern blot as follows: A bovine pituitary CDNA library was obtained from pituitary mRNA using the Xgt 10 vector of Huynh V.T [DNA cloning techniques )] A Practical Approach (IRL Press, Oxford, and was screened with 1098. Correct conditions for hybridization were determined using genomic DNA (Example 1) for Southern blot as follows: It was. of course. expected that the 1097 and 1098 probes would cross-hybridize with acidic FGF encoding DNA under low stringency conditions. Southern blot analysis showed that genomic sequences known to encode acidic bFGF which hybridized to 1097 and 1098 at 55°C wash temperatures failed to hybridize at 65°C.

(Prehybridization/hybridization buffer and conditions were as for 889/890 and 891 probes in Example 1.) Therefore. a wash temperature of 65°C was chosen. At this temperature. a 10 kb fragment in an EcoRI digest and a 3.4 kb fragment in a Pstl digest hybridized to probes 1097 and 1098. when the CDNA library was probed as above using a 65°C wash temperature, a single clone designated X882. representing a 2.1 kb cDNA with EcoRI linkers. was recovered. A restriction map of this phage is shown Subfiragments of the insert in XBB2 were transferred to M13 for sequencing and a 1.4 kb in Figure 9.

EcoRI—digested subfragment was shown to encode amino acids 1-146 (the complete "primary" sequence) of bovine basic FGF. The sequence upstream from the N¥terminal codon is believed to encode either a nine amino acid prosequence or an N-terminal extended "long" form of the native protein which retains activity. The N—terminal extension may contain only eight residues. of course. depending on whether the methionine is cleaved during post-translational processing. The portion of this subfragment encoding basic bFGF is shown in Figure 3: amino acid numbering starting at position 1 corresponds to the N-terminus of the isolated "primary" protein.

The upstream nine codons are translated in parentheses.

The possibility that this extension represents an integral part of the native active protein is suggested by the higher MW form of the human basic FGF prepared _35_ from hepatoma cells by Klagsbrun, et al, Proc Natl Acad ggi (supra).

The 1.4 kb subfragment is then nick translated and used to screen a bovine genomic library constructed in a manner similar to that of Example 1 for the basic bFGF genomic sequences.

The 1.4 Kb basic bFGF—encoding cDNA fragment is also used to probe alternate mammalian cDNA libraries. such as those from rat. pig, feline, equine or murine sources to obtain the basic PG? or bovine. canine. encoding sequences from these species.

Example 3 Preparation of Human Basic FGF Genomic and CDNA Clones A kgtlo cDNA library prepared from human kidney mRNA was also probed using the l.4 kb bovine basic subfragment. Prehybridization/hybridization buffer contained 40% formamide. 5.mM Na phosphate. pH 6.5. Sx Denhardt's. 5x SSC. and 50 ug/ml herring sperm DNA: hybridization buffer also included 10% dextran I sulfate and lO4-105 isolated; Three clones were This clone. designated XKB7. contained an approximately 1.4 cpm/ml probe. and one selected for characterization. kb EcoRI fragment which was partially sequenced to yield the data shown in Figure 4. along with the deduced amino acid sequence. The sequenced coding region permits deduction of amino acids l-50 shown in the Figure: the continuing sequence immediately downstream appears to represent the cDNA copy of an unspliced mRNA. indicating that an intron occurs in the basic FGF gene in a homologous position to the intron after amino acid 41 in The XKB7 clone also provides upstream DNA encoding the nine amino acid the bovine and human acidic FGF genes.

N—terminal extension of the long form shown. _36_ Additional genomic and cDNA libraries were screened 1Jsing the same 1.4 Kb basic bFGF-encoding fragment. under precisely the same hybridization comfitions as those employed for the human kidney xgtlo library above. obtained.

Four additional clones were which between them encode the entire 146 amino acid protein corresponding to the isolated basic bFGF. as Mwwn in Figure 4. Nine upstream codons included in XKB7 above translate into a sequence having complete homology’ with the translated upstream codons in the bovine basic FGF clone. although there is a silent nucleotide substitution in codon -8. This translated extension is shown in parentheses in Figure above, may represent a prosequence or the amino acids of an N—terminal extended active N-terminal 4: and. as additiotial protein.

In more detail. two positively hybridizing clones from a human genomic library in X Charon 4A. prepared as described by Lawn. R.M.. et al (supra) were designated XMG4 and XMGIO. acids (-9)-S1: XMGIO encodes amino acids 86-146. representing the third of three exons contained in the mature protein-encoding region of the gene. (The XMG4 encodes amino ‘location of exon/intron boundaries was determined by homology to the bovine sequence.) genomic library in X Charon 28.

A slightly different obtained from E.

Fritsch. yielded XHTl which contains the second mature protein exon. encoding amino acids Sl-85. Finally.

XHFLI. a CDNA clone obtained from a human fetal liver library prepared in Xgtlo as described above, encodes amino acids 56-146. confirming the location of the relevant intron/exon junction.

There are only two amino acid differences between basic bFGF and hFGF, at position ll2, where the _37_ bovine protein has set and the human protein has Thr. and at position 128, where the bovine protein has Pro and the human has Ser. These differences are the result of a single nucleotide difference in each case; therefore boyine cDNA may conveniently be modified by site directed mutagenesis as described below to encode the human protein. and. indeed. standard site-specific mutagenesis.techniques were used to alter these codons.

The ABB2 clone of Example 2 was digested with ECORI and the 1.4 Kb region spanning the bFGF protein-encoding portion was ligated into the EcoRI site of Ml3mp8. The ig_g;££g mutagenesis was carried out in the presence of a l7—mer: three oligonucleotides: the "universal" primer, the mutagenic l6—mer 5'~GAAATACACCAGTTGG-3' (SEQ ID NO. which alters the coding sequence at codon 112, and the mutagenic l7—mer 5'—ACTTGGATCCAAAACAG—3' (SEQ ID NO. 4), which alters the sequence at codon 128. The mutagenized phage was also subjected to a second round of in vitro primer—directed mutagenesis to create a HindIII site 34 bp downstream from the translation termination codon using the mutagenic 25—mer, 5'~TTTTACATGAAGCTTTATATTTCAG—3’ (SEQ.lD.NO.5) The resultant mutated DNA was sequenced by dideoxy sequencing to confirm that the desired mutagenesis had occurred, and the approximately 630 bp fragment spanning the FGF coding region was excised with Hindlll and ligated into pUC13 to obtain the intermediate plasmid pJJ15—l.

Of course. modified forms of the coding sequence to encode any of the three known N-terminal modifications of basic FGF may also be prepared by using standard synthesis techniques.

Example 4 Construction of Expression Vectors and Stable Expression of FGF in Mammalian Cells The cDNA clones encoding FGF are most conveniently used to produce the recombinant proteins in a variety of hosts. as set forth in 1C.l above.

Emwever, expression in mammalian systems is favored as the host is capable of post translational processing analogous to that experienced by the natively produced protein, and either CDNA or genomic sequences may be used, as the host is also capable of processing introns.

Thus. a fullelength cDNA or genomic FGF encoding clone is prepared for insertion into a host vector. illustrated by. but not limited to. those described below.

To construct the vectors, the cloned FGF—encodinq insert is excised with EcoRI (by partial digestion if the insert itself contains EcoRI sites). or other appropriate enzyme. provided with EcoRI or other appropriate linkers if necessary. and then inserted into an appropriate host vector such as pHSl or its derivatives as described below. construction of Host vectors The plasmid pHSl is suitable for expression of inserted DNA in mammalian hosts. It contains 840 bP Of the hMT-II sequence from p84H (Karin. M.. et a1. (1982) gggz 297-802) which spans from the HindIII site at position -765 of the hMT-II gene to the BamHI cleavage site at base + 70.

Nature To construct pHSl. plasmid p84H was digested to completion with BamHI. treated with exonuclease BAL—31 to remove terminal nucleotides, and then digested with HindIII. The desired 840 bp fragment was ligated into pUC8 (Vieira. J.. et al. gene (1982) 13: 259-268) which had been opened with HindIII and HincII digestion. The ligation mixture was used to transform E. coli HB10l to AmpR, and one candidate plasmid. designated pHSl, was isolated and sequenced by dideoxy sequencing. pHSl contains the hMT-II control sequences upstream of a polylinker containing convenient restriction sites.

The workable host plasmid pHS1 can be further modified to contain additional control elements besides the metallothionein promoter. In particular. enhancer elements of viral systems. such as SV40. can be included, as well as termination signals associated with the 3' untranslated regions of other proteins such as hGH.

Viral Enhancer 0 A pair of host expression vectors containing the SV40 enhancer in operable linkage to the MT-II promoter was constructed by inserting an 1120 bp sv4o DNA fragment into the HindIII site preceding the MT-II promoter sequences in pHSl. The SV40 DNA fragment spans the sv4o origin of replication and includes nucleotide 5171 through nucleotide 5243 (at the origin). the duplicated 72 bp repeat from nucleotide 107-250. and continues through nucleotide 1046 on the side of the origin containing the 5' end of late viral mRNAs. This HindIII 1120 bp fragment is obtained from a HindIII digest of SV40 DNA (Buchman. A.R.. et al. DNA Tumor viruses. 2d ed (J. Too2e;‘ed.). Cold Spring Harbor Laboratory. New York (1981), pp. 799-841), into pBR322 for amplification. The cloning vector was cut with HindIII, and the 1120 bp SV4O DNA fragment isolated by gel electrophoresis and ligated into HindIII—digested. CIP—treated. pHSl. and cloned The resulting _4Q_ vectors, designated pHsl-SV(9) and pHsl—SV(l0), contain the fragment in opposite orientations preceding the MT-II promoter. In pHSl-SV(9), the enhancer is about l600 bp from the 5' mRNA start site; in the opposite orientation it is approximately 980 bp from the 5‘ mRNA start site. Both orientations are operable. but the orientation wherein the enhancer sequences are proximal .to the start site provides higher levels of expression.

It is believed that deletions which place the enhancer 250-400 bp upstream of the transcription start are optimal.

Additional vectors were constructed which place the svqo enhancer 3‘ 250 bp. and 360 bp respectively upstream from the 5‘ end of the MT promoter TATA of the upstream regulatory regions of the human ME terminus l90 bp.

These vectors, designated pHS'-SVl90. pHS'-SVZSO. and pHs‘-SV360 are prepared as follows: all constructions are identical except for the length of sequence containing the metallothionein promoter and upstream region which is supplied as a fragment excised mm pl-Isl.

For pHs'—sv19o. pHSl is digested with SacIL blunted, and ligated to Kpnl linkers. The DNA is then digested with EcoRI and Kpnl to liberate the appropriate portion of the MT—II control sequences. similarly. for pHS'—Sv2S0. pHsl is digested with Hgal. blunted. ligated to KpnI linkers and digested with EcoRI and Kpnl: for pHS'—SV360. Ddel is used in the initial digestion.

' An intermediate vector containing the SV40 is prepared by inserting the HindIII/Kpnl of sv4o (which extends from position 5171 to enhancer fragment position 294 and which contains the enhancer element 50 bp from the Kpnl site) into Kpnl/HindIII digested pUCl9 to obtain pUC—sV. (pUCl9 contains three convenient restriction sites in the polylinker region. in order.

Hindlll. KpnI, and EcoRI.) obtained by inserting the Kpnl/EcoRI fragments prepared as described above into Kpnl/EcoRI digested pUC—SV.

The finished vectors are All of the foregoing modified vectors. thus. take advantage of the SV40 enhancer element. Other viral enhancers could, be used in an of course. analogous manner.

Transcription Termination Sequences To provide transcription termination control sequences. DNA representing the coding sequence and 3' untranslated sequence of human growth hormone was ligated into pHSl. The intermediate vector can provide the hGH 3' untranslated sequence to coding sequences ~subsequently ligated into the vector in place of the hGH coding sequence.

The genomic sequences encoding hGH were isolated from p2.6-3 (DeNoto. Nucleic Acids Res (1981) ;g:3719) by digestion with BamHI. which cuts at the 5' and EcoRI. of the functional gene. followed by polyacrylamide gel et al. end of the first exon. which cuts 3' purification. The isolated fragment was ligated into Bamfll/EcoRl digested pflsl and the ligation mixture transformed into E. coli MCl06l to AmpR. Successful transformants were screened by restriction analysis. and a strain containing the desired plasmid, pMT—hGHq. was further propagated to prepare quantities of plasmid DNA.

In a manner similar to that described above for constructing pHSl—SV(9) or pHSl-SV(l0), but substituting for pHSl. pMT-hGHg. a pair of vectors containing the hGH gene under the control of the MT promoter, and operably linked to sv4o enhancer. and designated, respectively. phGHq-SV(9) and phGHg—SV(l0). were obtained. The I ligation mixtures were used to transform E. coli 1061 to AmpR, and the correct constructions verified.

Construction of Expression Vectors phGHg-SV(l0) was then used as a host vector to accommodate Syn—acidic hFGF. phGHg-Sv(1o) was digested with BamHI and Smal. blunted with Klenow, and treated with CIP to excise the hGH coding sequence. This opened vector was ligated to the NcoI(blunt)/EcoRI(blunt) Syn-acidic hFGF fragment to obtain the desired expression vector pahFGF—SV(10). in which the NcoI site of the syn-acidic hFGF fragment is recreated.

Similarly. the remaining FGF-encoding fragments described above are ligated into phGHg-sV(l0) to prepare analogous vectors containing these coding sequences under control of the viral enhancer. MT—II promoter and the hGH 3' untranslated regions. For example. the ~S00 bp NcoI (blunt)/HindIII (blunt) fragment from pJJl5—l of Example 6 is conveniently inserted into BamHI (blunt)/SmaI—digested phGH-SV(l0) to obtain pJJl6—2.

In addition. other host vectors may be used to obtain expression of these sequences. including pHSl and pHSl modified to contain the various configurations of SV enhancer as above described. Insertion is by analogous means, using BamHI/EcoRI digestion of the host vector. Also. DNA modified to encode any of the "long". .to 10-4 _43... "primary" or "short" forms of the acidic or basic FGF may be employed.

These vectors are generically designated pMT—FGF for the purposes of the discussion below.

Production of FGF by Mammalian Recombinants A Chinese hamster ovary (CHO)—Kl cells were grown on medium composed of a 1:1 mixture of F12 medium and DME medium with 12% fetal calf serum. The competent cells were co-transformed with pMT-FGF and pSV2:NEo (Southern. P.. et al, J Mol Appl Genet (1982) 1: 327-341). pSV2:NEO contains a functional gene conferring resistance to the neomycin analog G418. In the transformation. 500 ng of pSV2—NEO and 5 ug of pMT—FGF were applied to a 16 mm dish of cells in a calcium phosphate-DNA co-precipitate according to the protocol of Wigler. M}. et al. C_e_l__l_ (1979) l_6_: 777-735, with the inclusion of a two minute "shock" with 15% glycerol after four hours of exposure to the DNA. later, the cells were subjected to 1 mg/ml G418 to provide a pool of G418-resistant colonies. which were assayed for FGF production and then can be cloned out.

Successful transformants. also having a stable inheritance of pMT-FGF. are plated at low density for purification of clonal isolates.

A day Small amounts of these isolates are grown in multi-well plates after exposure M zinc chloride for convenient assay of FGF production. FGF determinations are made by standard ELISA or radio—immunoassays against the antisera prepared against the appropriate FGF protein using standard methods. Clonal isolates which produce large amounts of the desired FGF are selected.

The cells, which have been shown to produce FGF under suitable conditions, are seeded at 1/10 confluency in basal medium supplemented with 10% fetal calf serum. incubated overnight. and then induced for FGF production by addition of zinc chloride in the concentration range of 1 x 10"‘ M to 3 x 10" M. 7-10 days. under optimal inducing conditions. 2 x 10- M ZnCl2.

FGF levels rise for 4 In a particular experiment. CHO cells were transformed using pMT—FGF containing the approximately S00-bp Ncol(blunt)/HindIII(blunt) fragment encoding human basic FGF derived from pJJlS—l of Example 6. This fragment was inserted into BamHI(blunt)/SmaI—digested phGH—SV(l0). particular form of pMT-FGF (designated pJJl6—2, as described above. to obtain this hereinabove). The cells were cotransformed with this and pHSl—MT. After G418 selection. the pooled resistant colonies produced approximately 500 pg vector. pSV-neo. of human basic FGF per 106 cells.

The amount of FGF produced was determined by affinity-purifying the basic FGF from lysed cells using heparin-Sepharose, followed by assay for growth The heparin affinity purification is performed by standard The foregoing pools. producing at a level of S00 pg/106 cells. were then selected for cadmium resistance by growing them in the presence of 10 um Cdcl with 100 uM ZnCl2 as inducer. resistant clones were then assayed. as described above, Pools of Production levels of 5.6 ng/106 cells were found in one assay.

If desired, lysed cells and purified according to the procedures set the FGF can be obtained from the forth above for the native protein. or by other standard methods known in the art.

In addition. as discussed above. secretion of the FGF proteins produced by the foregoing constructs can be achieved by exocytosis initiated by a calcium ionophore or other suitable stimulant. While it is not expected that proteins produced by CHO cells. specifically. would be released by LPS or phorbol ester stimulation. for example. by substituting for CHO cells. cell lines derived from macrophage as recombinant hosts.

Also. by altering the construction so as to provide a signal sequence. such as such secretion can be effected. that exemplified below, derived from hGH, secretion using the norma1~constitutive pathways could also be effected using CHO or other mammalian cell hosts.

Effecting secretion has some advantages. of course. since the protein purification task becomes much simpler.

Transfection with a pMT-FGF vector containing the Syn-acidic hFGF partially synthetic sequence will result in the production of "long" FGF containing the 14 amino acid pro region upstream of the 140 amino acids of the mature primary form; the processed Met residue may also be replaced with a blocking group such as acetyl.

Processing may also occur in mammalian cells to result it is established that the long form containing the leader sequence minus the in the mature form: however. initiating Met. and with the now N—terminal alanine residue acetylated. is active as a mitogen.

In any event, FGF is partially purified by passage over heparin/sepharose. and elution with 1.2 M Nacl for acidic FGF and 2 M Nacl for basic FGF. The eluate is analyzed for the presence of acidic or basic FDF by SDS-PAGE and by mitogenic activity on endothelial or 3T3 cells.

Example 5 Construction of Vaccinia vectors for Human FGF and Transient Expression in CV-1 Cells The basic hFGF—encoding sequences were provided with the 3' untranslated region from hGH by digesting phGH-SV(10) (supra) with BamHI and Smal. blunting with Klenow, and inserting the approximately 500 bp Ncol (blunt)/HindIII (blunt) fragment spanning the FGF from pJJlS-1. The resulting plasmid. pJJl6-2. can be used directly as an expression vector. as described above.

However. the Ncol/EcoRI fragment (approximately 1.1 kb) containing the basic bFGF coding region and the hGH poIyA addition signal was purified on a 5% acrylamide gel. eluted. blunted with Klenow. and ligated into SmaI—digested phosphatased pGS20 (Mackett et al. 1 giro; (1984) $g:857-864). designated pJV1-1. was amplified in E. coli MCl06l. and the plasmid DNA was isolated using a cesium chloride The resulting plasmid. gradient. were assayed for the production of basic hFGF using SDS-PAGE autoradiography. The results are shown in Lanes 1 and 2 are the media of cells transfected with pJVl—l and pGS2O respectively, lanes 3 and 4 are samples of the corresponding cell lysates. and Figure 10. lanes 5 and 6 are the same as lanes 3 and 4 except that the samples of lysate were bound to heparin sepharose in the presence of 0.6 M Nacl. washed with 10 mM phosphate, pH 7.4/1.1 M Nacl. and eluted from the column with 2 M Nacl in the same buffer. (The eluates were precipitated with TCA before loading on the gel.) 1251-labeled basic FGF in the 146 amino acid form.

The band at approximately 18 Rd in lane 5, which has a slightly higher molecular weight than the FGF standard.

Lane 7 is shows that the "long" form of the bovine sequence is formed in preference to the "primary" protein obtained from tissues.

Samples prepared as described for lanes 5 and 6 (except for the TCA precipitation) were also tested for mitogenic activity on bovine brain capillary endothelial cells. (see Example 6.) No activity was present in the pGS20 sample. but the pJvl-l sample contained activity equivalent to 20 pg FGF/ul.

Example 6 In Vitro Assay for FGF The assay was performed substantially as described by Esch et al. Proc Natl Acad Sci (USA) (1985) Briefly. bovine brain capillary endothelial cells were maintained in the presence of DMEM supplemented with 10% calf serum. Monolayers were dissociated by exposure to a solution containing 0.9% Nacl. pH 7.4. 0.05% trypsin. and 0.02% EDTA for 2-3 minutes at room temperature. .01 M sodium phosphate.

After the cells had rounded up. they were resuspended in DMEM and 10% calf serum and an aliquot of the cell The cells cells per suspension was counted in a Coulter counter. were seeded at an initial density of 2 x 104 mm dish. each dish containing a total of 2 ml DMEM plus 10% calf serum. Six to twelve hours later. a set of duplicated plates was trypsinized and cells were counted to determine the plating efficiency.

Aliquots of the sample to be tested for FGF activity were diluted 1:2. l:4. and 1:8 with DMEM plus 0.5% BSA, and 10 ul of the dilutions were added to triplicate assay plates on days 0 and 2. on day 4. triplicate plates for each sample dilution were trypsinized and the cell densities determined by Coulter COUIICEC . _49__ Exam le 7 Bacterial Expression of FGF The CDNA sequences encoding basic human FGF, which are uninterrupted by introns, are also expressible in bacterial systems. A convenient host Vector for expression is pKT52, which contains the "trc" promoter, followed by an ATG start codon.

The "trc" promoter contains the upstream portions of the trp promoter and lac operator.) The cells are then harvested. lysed by sonication or treatment with 5% trichloroacetic acid.

FGF be purified from the extracts by methods used for and the cell extracts assayed for the desired FGF. can the native protein or by other procedures known in the art.

Example 8 al. In the alternative.

J Surg Res (1982) ;;:394-401).

In the standard procedure. a total of four rats The treated with heparin received two identically treated sponges each. sponges were either not treated. sepharose beads, treated with FGF bound to heparin sepharose beads using 5 ug FGF per sponge: or treated with 5 ug FGF in solution. The sponges were removed after 6 days and examined histologically for granulation tissue. which is indicative of wound healing.

Sponges which contained FGF showed a higher amount of granulation. which was centered around the heparin sepharose beads in the case of the sponges where the FGF was supplied bound to these beads.

Similar results are observed whether the FGF is from native or recombinant sources. the downstream. operator-containing. regions of the lac pKTS2. containing this promoter was constructed by a simple manipulation of pKK233—2. promoter. which is described by Amman. 8.. et a1. Gene (1985) gg:1e3—19o: pKK233—2 was digested with EcoRI and PvuII, filled in with dATP and dTTP. the desired vector. and religated to obtain pKTS2 contains in addition to the desired trc promoter and downstream ATG start codon, Ncol. Pat! and HindIII sites. downstream For construction of expression vectors, the basic human FGF—encoding CDNA is obtained by excising with ECORI or other appropriate enzyme digestion and isolating and. if necessary. modifying the appropriate fragment. The 3' end is prepared for insertion into pKTS2 by cutting downstream of the termination codon at any convenient restriction site and supplying Pstl or HindIII linkers.

The 5' end is prepared by cutting at a site inside the coding sequence and supplying the missing codons and an Ncol site using a synthetic DNA. or by providing an appropriately located Ncol site by mutagenesis. reading frame with the ATG start codon.

For bacterial expression. the resulting expression vectors are used to transform E. coli Mcloel and the transformed cells are then grown on M9 medium containing 1 mM IPTG for 3-5 hr to an 0.D. of 0.2-0.5. (IPTG is a standard inducer for control sequences regulated by the or other appropriate host cells to AmpR.

On or before 9 September 1985, Applicant deposited with the American Type Culture Collection (ATCC), Rockville, MD, USA, the A phage ABA2, ABA3, ABB2. and AKB—7 which were assigned ATCC accession numbers 40195, 40194, 40196, and 40198, respectively.

These deposits were made under conditions as provided under ATCC's agreement for Culture Deposit for Patent conditions of Purposes. On or before 12 September 1986, deposit of ABB2 (ATCC 40196) was converted to conform to those specified under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms (Budapest Treaty). Availability of the deposited strains is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

Claims (1)

1.CLAIMS: An isolated, cloned recombinant or synthetic DNA sequence which encodes an FGF protein comprising the amino acid sequence numbered 16-146 in