CA2055961A1 - Active fragments of fibroblast growth factor - Google Patents

Active fragments of fibroblast growth factor

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Publication number
CA2055961A1
CA2055961A1 CA002055961A CA2055961A CA2055961A1 CA 2055961 A1 CA2055961 A1 CA 2055961A1 CA 002055961 A CA002055961 A CA 002055961A CA 2055961 A CA2055961 A CA 2055961A CA 2055961 A1 CA2055961 A1 CA 2055961A1
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Prior art keywords
dna sequence
growth factor
fibroblast growth
bfgf
fragment
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CA002055961A
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French (fr)
Inventor
Andrew P. Seddon
Peter Bohlen
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Wyeth Holdings LLC
Original Assignee
Andrew P. Seddon
Peter Bohlen
American Cyanamid Company
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Application filed by Andrew P. Seddon, Peter Bohlen, American Cyanamid Company filed Critical Andrew P. Seddon
Publication of CA2055961A1 publication Critical patent/CA2055961A1/en
Abandoned legal-status Critical Current

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • C07K14/50Fibroblast growth factors [FGF]
    • C07K14/503Fibroblast growth factors [FGF] basic FGF [bFGF]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Abstract

31,309-00 FIBROBLAST GROWTH FACTOR FRAGMENTS
ABSTRACT OF THE INVENTION
The present invention relates to novel fragments of basic fibroblast growth factor (bFGF). The mitogenic potency of one of these bFGF fragments, identified as HBF-2 is about 25-50 fold less than that of native bFGF
but at least 103-104 fold more active than that of previously reported synthetic fragments of bFGF.
Therefore, the present invention provide the shortest fragment of bFGF that retains substantial biologic activity.

Description

31,309-~0 X~9~

ACTIVE FRAGMENTS OF FIBROBLAsT GROWTH FACTOR

BACKGRpUND QF TEE INVENTION
The present invention relates to novel active fragments of basic fibroblast growth factors (bFGF). The fibrobla t growth factors (FGF) are multifuntional polypeptide mitogens which exhibit broad target-cell specificity (1). In the course of study of these factors, a number have baen identified on the basis of the ability of extrac~s from various tissues, such as brain, pituitary and hypothalamus, to stimulate the mitosis of cultured cells. Numerous shorthand names have been applied to active factors in these extracts, including epidermal growth factor, platelet-derived growth factor, nerve growth factor, hematopoiPtic growth factor and fibroblast growth factor.
Fibroblast growth factor (FGF) was first described by Gospodarowicz in 1974 (2) as derived Prom bovine brain or pitui~ary tissue which wa~ mitogenic for ibroblasts and endothelial cells. It was later noted that the primary mitogen from brain was different from that isolated from pituitary. These two ~actors were named acidic and basic FGF, respectively, because they had similar if not identical biological activities but differed in their isolectric polnts. Acidic and basic fibroblast growth factors (recently reviewed by Bu.rgess, W.H. and Maciag (3~ appear to be normal members of a family of heparin-binding growth factors ~hat in~luence the general prolifera~ion capacity of a majority of mesoderm and neuroectoderm-derived cells (4), including endothelial cells, smooth muscle cells, adrenal cortex cells, prostatic and retina epithelial cells, oligodendrocytes, astrocytes, chrondocytes, myoblasts and osteoblasts tBurgess and Maciag, cited above at Page 584) t3)- Although human melanocytes respond to the mitogenic influences o~ basic fibroblast growth ~actor but not acidic FGF, most avian and mammalian cell types respond to both polypeptides (ibid.) (3).
In addition to eliciting a mitogenic response that stimulates cell growth, fibroblast growth factors can stimulate a large number of cell types to respond in a non-mitogenic manner. These activities include promotion of cell migration into wound areas (chemotaxis~, initiation of new blood vessel formulation (angiogenesis), modulation o~ nerve re~eneration (neurotropism~, and stimulation or suppression of specific cellular protein expression, extracellular matrix production and cell survival important in the healing proces~ (Burgess and Maciag, cited above, pages 584 to 588) (3).
These properties, together with cell growth promoting action, provide a basis for using fibroblast growth factors in therapeutic approaches to accelerate wound healing and in pre~ention and therapeutic applications for thrombosis, artheriosclerosis, and the like. ~hus, ~ibroblast growth factors have been suggested to promote the healing of tissue subjected to trauma (5), to minlmize myocardium damage in ~eart disease and surgery (6 and 7), and to increase neuronal survival and neurite extension (8).
Complementary DNA clones Pncoding human acidic and - human and bovine basic fibroblast growth factors have been isolated and sequenced, and the predicated amino acid sequences derived from the complementary DNAs agree with th~ structures determined ~y protein sequence analysis ~summarized by Burgess and ~aciag, cited abow e, at pages 580-581) (3). The data predict acidic fibroblast growth factor (hereafter referred to as aFGF) to have 155 amino acids (ibid) (3). The gene for basic fibroblast growth factor (hereafter referred to as bFGF~
also codes ~or a 155 residue protein. For both aFGF and bFGF N-terminally truncated forms exist that exhibit full biologic acitivity, including the 146-amino acid bFGF
orginally isolated and sequenced (9) and a 131~amino acid form. Analysis of the structuree demonstrates a 55%
identity between aFGF and bF~F (Burgess and Maciag, cited above at page 581) (3).
Basic fibroblast growth ~actor may be extract~d from mammalian tissue, bu~ this requires several steps even when heparin-linked affini~y chromatography is employed (U.S. Pat. Nos. 4,785,079 and 4,902,782 to Gospodarowicz, et al.~ (10 and 11), and the 146-amino acid species is generally obtained i~ extraction is done in the absence of protease inhibitors (ibid~, column 9, lines 29 to 32).
Bovine and human basic fibroblast growth factor cDNA have been expressed in E. ~Ql~ (12 and 13) and S. cervisiae (36). However, reporked yields o~ product are low (15), and recombinant factors exhibit a marked tendency to undergo thiol-disulfide interchanges promoted by ~ree thiol groups in the protein ~ha~ result in the formation of disulfide scrambled species (12).
A number of basic fibroblast growth ~actor analogues have been suggested. Muteins of bFGF having amino or carboxyl terminal amino acids deleted, amino acids added, cysteine substituted with a n~utral amino acid such as serine, or aspartic acid, arginine, glycine, serine, or valine substituted with other acids have been suggested to have enhanced stability (16). ~he muteins comprise two ~~~ 61109-7916 or three additions, deletions or substitutions, with substitution of serine for cysteine the most pref~rred substitution ~16). Arakawa and Fox (17) suggested replacing at least one, and more preferably two, o~ the cysteines found in natural bFGF with a different amino acid residue to yield a more stable analogue (page 4, lines 44 to 47): serine was illustrated in the Examples (page 13, lines 22 to 23). Similarly, recombinant aFGFs having extraneous bond-forming cysteine replaced with serine, and oxidation-prone cysteine, me~hionine and trypotophan replaced with alanine, valine, leucine or isoleucine, to yield factors having enhanced or improved biological activity have also been suggested (18).
A bFGF mutein lacking 7 to 46 amino acids from the lS carboxyl terminus and, optionally, having amino acid replacements was suggested to have improved stability while retaining activity in Eur. Pat. Ap. Pub. No.
326,907 to Seno, et al. (page 2, line 50 to page 3, line 4) (19). Fiddes, et al, (Eur. Pat. Ap. Pub. No. 298723) (20) suggested replacing basic or positively charged residues in the heparin binding domain encompassing residues 128 to 138 with neutral or negatively charged amino acids to produce forms of FGF having reduced haparin binding ability and enhanced potency (page 5, line 45, and page 5, line 54 to page 6, line 16).
Bergonzoni, et al.(21), suggested six analogues: 1) Ml-bFGF, lacking residues 27 to 32: M2-bFGF, lacking residues 54 to 58; M3-bFGF, lacking residues 70 to 75;
M4-bFGF, lacking residues 78 to 83; M5-bFGF, lacking residues 110 to ~20 M5a-bFGF, having the position 128 lysine and the position 129 arginine replaced with glutamine residues; and M6b-bFGF, having the positions 119 and 128 lysines and the positions 118 and 129 arginines replaced by glutamine residues.
: 35 2~SS~
Fortunately, the affinity for heparin also provides a selective method for isolation and purification of the two forms of FGF, acidic and basic (22). FGFs are structurally labile but can be protected from inactivation by heat or low pH by association wi~h heparin (23). Heparin-FGF complexes also are highly resistant to proteolytic degradation (24). Heparin, through a mechanismr most probably related to enhanced stability of the fibroblast growth factor can potentiate the biologic properties of both acidic and basic FGF (23 and 25). FGFs lack a classical signal peptide sequence which can direct secretion of the FGF into extracellular space (26~. However, considerable quantities of bFGF
have been detected in and isolated from th~ extracellular matrix (ECM) both n vitrQ (27) and ia viYo (28). This, therefore, raises the question of how secretion of FGF
occurs and suggests the po~sible use of a carrier protein or proteoglycan. ECM-associated bFGF is bound to heparin sulfate (HS3 prot~oglycans (29 and 31) and is most likely released in a controlled manner a as FGF-HS complex (31).
Thus, a current hypothesis (1) regarding the mechanism for regulation of the mitogenic activity of FGF is that FGF is sequestered in the ECM as a biologically inactive FGF-heparin sulfate proteoqlycan compIex. Then, ECM-bound FGF may be mobilized by specific cellular signals that activate, for example, matrix-degrading enzyme systems such as the plasminogen activation cascade (31) and heparin sulfate-specific endo-beta-D-glucuronidasQs (32 and ~3).
As such, it -seem~ that locating the functional domains on bFGF should involve reviewing this structure function interaction with heparin and- the receptor for FGF. In fact, structure function studies with synthetic peptide fragments of bFGF suggest the existence of two functional domains, corresponding to residues 33-77 and , 2Q5~9~.
112-lS5 (The numbering for bFGF adopted here is for the 155 amino acid form as described in (26), and refers to the methionine initiation codon as position 1. Using this system the N terminal prolyl resldue of thP
sequenced 14~ amino acid tissue derived ~orm of bFGF
corresponds to codon position 10 and is ~hus identified as bFGF (10-155)). Heparin and receptor-binding domains were identified by the ability of synthetic peptides related to FGF sequence~ to compæte with bFGF for its receptor, to bind to radiolabeled heparin and to modulate the mitogenic response of FGF (34)~ From one of these regions containing a functional domain, 112-155, an active core decapeptide (115-124) was produced. However, the peptide (115-124) had decreased affinity for heparin and ~iologic potency by 10-and 100-fold, respectively in comparison to peptide 1120155 (34).
Seno et al (35) provided another series of fragments to study th~ mitogenic and heparin-binding properties of a series of C-terminally truncated bFGF's (10-155) produced in E. ~ . As the degree of C-terminal truncation exceeded 6 residues, affinity for heparin was markedly decreased . In the same skudy, two N-terminally truncated form~ of b~GF, 23-155 and 50-155, were also studied. When compared to the native b~GF (10-lSS), bFGF
(23-155) and (5Q 155) showed no changes in affinity for heparin but exhibited about a 2-and 50-fold decrease in mitogenic activity, respectively.
~he present invention takes advantage of the specific significant interaction between heparin and bFGF
to more closely define t~e structural domains of the growth factor ~hat interact with heparin and the receptor for bFGF to yield a mitogenic response. The human bFGF
mutant glu3'5,ser7~'96bFGF is used for these studies for the following reasons: (a) bFGF and the mutants are equipotent; tb) the mutations o~ residues 3 and 5 afford 2q~
considerable higher expression than the parent protein in the expression system of this invention; and (c) the mutations of cysteines at residues 7~ and 96 to serines eliminates disulfide scrambling-related stability problems associated with recombinant preparations of the parent bFGF, particularly when expressed in high yield.
These bFGF analogues are described in more detail in copending application for limited sta~es letters Patent Serial No. _ , filed concurrently.
Proteolytic digestion of heparin sepharose-bound glu3'5, ser78'96 hbFGF(1-155), gives two peptide fra~ments that are elut~d from heparin sepharose under the same conditions used to elute intact bFGF. Thus, these bFGF peptide products are heparin-binding ~ragments (HBF), since they are protected from proteolytic degradation by virtue of their specific interaction with heparin. The 2 peptide fragments are labeled HBF-1 (bFGF
27-69) and HBF-2 (ser78'96 bFGF(79-155)). On heparin affinity ~PLC the mixture o~ HBF-1 and HBF-2 is not resolved, and the two fragments coelute with a retention time identical to that of intact bFGF. It is assumed, therefore, that both fragments possess equal affinities for heparin; although the possibility that they are covalently linked or non-covalently associa ed to form a complex, also ~xists. Intere-~tingly, HBF-l and HBF-2 each contain a single cysteine residue corresponding to positions 34 and 101 in glu3'5, ser78'96 hbFGF(1-155) which are thought to be disulfide linXed in the native structure (35). RBF-l and HBF-2 are, however, efficiently resolved by RPLC in the absence of a thiol reducing agent. The fact that N-terminal sequence analysis of RPLC-purified HBFs indicates no secondary sequences argues against HBF-1 and HBF-2 being covalently linked.

~ .

Thus, HBF-1 is subjected to S-pyridylethylation (37) under non-reducing conditions, ~ollowed by N-terminal sequence analysis. This procedure gives a quantitative reaction with vinylpyridine and release of phenylhydantoin S-pyridylethyl cysteine derivative on the 8th sequencer cycle, whereas a similiary treated control, somatostatin-28 (Bachem Bioscience), in which cysteines 17 and 28 are disulfide-linked, gives no detectable phenylhydantoin- cysteine deriva~ive at sequencer cycle 17. Thus, the data support the presence of a free sulfhydryl group in HBF-l, and by analogy, in HBF-2, as well. This implies that cysteines 34 and 101 may not be stably disulfide-linked in glu3'5,ser~76'96~bFGF.
Alternatively, the possibili~y exists of non-covalent interactions between the 2 pep~ides. Indirect evidence for an association between HaF-l and -2 comes from the failure of cation exchange chromatography, despite the marked differences in net charge of the peptides and lack of disulfide linkage, to resolve the 2 fragments. Also, their behavior on both heparin and cation exchange chromatographies is indistinguishable from that of bFGF.
This may imply that in the native state or when bound to heparin, regions of bFGF contained in the sequences 27-69 and 70-155 interact to yield a unique 3-dimensional structure that is required for high affinity binding to heparin. Additional support for this comes from the observation that RPLC-purified bFGF, HBF-1 and HBF-2 do not retain their a~finity for heparin, even after removal of HPLC solvents, but RPLC-purified bFGF and HBF-2 3 exhibit biologic activities.
Baird et at (34) using synthetic bFGF fragments identified 2 peptide regions, residues 33-77 and 109-129 in bFGF, that bind to heparin and exhibik weak partial agoinst antagonist activities in biological assays.
HBF-2 (ser73'96b~GF(70-155)) contains the bFGF sequence , , , ;, -9- 61109~7916 109-129. A comparison of the potencies of FGF sequences relative to intact bFGF (Table 2) shows that the activity of HBF-2 is at least 10~-104 fold greater than the most active synthetic peptide known (bFGF (112-155)) (34).
s Recently, Seno et al (35) expressed and examined the properties of a series of C-terminally truncated versions of bFGF. These studies conclude that essential elements for receptor binding are contained in the sequence 50-109 and for heparin binding in the sequence 110-150; however, other interpretations are possible. Seno et al., also described an N-terminally truncated from of bFGF (50-155) which retains full affinity for heparin and exhibits about 2~ the mitogenic activity oP bFGF (~0-155). Since bFGF (50-155) and H~F-2 (70-155) seem equipot~nt (Table 2), the FGF sequence 50-69 can thus be eliminated as contributing signi~icantly to receptor recognition and the mitogenic response.
The presen~ invention provides FGF fragments obtained upon proteolytic degradation of bFGF bound to heparin or heparin sepharose and include fragments which immobilize heparin sepharose analogues or heparin analogues, including ones with mitogenic activity. This invention also encompasse6 analogues haviny the cysteine residues at positions 78 and 96 replaced with oth r amino acids, such as, for example alanine, glycine, arginine, tryptophan, lysine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, isoleucine, leucine, valine, phenylalanine, tyrosine, methionine, serine, threonine or proline. More over, the bFGF fragments of the presen~ invention are not species specific, a~d include instance, bovine, ovine, porcine and others that share similar sequences homology with human bFGF.
The novel bFGF fragments of the present invention also may be prepared by recombinant protein synthesis involving preparation of DNA encoding thç bFGF fragments, ~Q~:59~
insertion of the DNA into a vector, expression of the vector in host cells, and isolation o~ the recombinant bFGF fragments thereby produced.
Because of the degeneracy of the gen~tic code, a variety of codon ~hange combinations can be selected to form DNA that encodes the bFGF ~ragments of the present invention, so that any nucleotide deletion(s3, addition(s), or point mutation(s~ that result in a DNA
encoding the bFGF ~ragment~ herein are encompassed by this invention. Since certain codons are more efficient for polypeptide expression in certain types of organisms, the selection of gene alterations to yield DNA material that codes for the b~GF fragments of the present invention are preferably those that yield the most efficient expression in the type o~ organism which is to servive a~ the host of the recombinant vector. Altered codon selection may also depend upon vector construction considerations.
DNA starting material which can be altered to form 2 the DNA of the present invention may be natural ~isolated from tissue), recombinant or synthetic. Thus, DNA
starting ~aterial may be isolated from tissue or tissue culture, constructed ~rom oligonucleotides using conventional methods, obtained commercially, or prepared 2S by isolating RNA coding ~or bFGF from ~ibro~lasts, using this RNA to synthesize single-stranded cDNA which can be used as a template to synthesize the corresponding double stranded DNA. As pointed out herein, proteolytic degradation of native bFGF bound to heparin or immoblized 3~ heparin can generate the fragments, as well.
Also encompassed are DNA sequences homologous or closeiy related to complementary DNA described herein, namely DNA sequences which hybridize, particularly under stringent conditions, to the cDNA described herein and RNA corresponding thereto.

.

~ 61109-7916 DNA encoding the bFGF fragments of the present invention, or RNA corresponding thereto, can then be inserted into a vector, e.g., a pBR, pUC, pUB or pET
series plasmid, and the recombinant vector used to transform a microbial host organisms. Host organisms may be bacterial (e.g., E. coli or B subtilis, y~ast (e.g., S. cervisiae) or mammalian ~e.g., mouse fibroblast).
Culture of host organisms stably transformed or transfected with such vectors under conditions Pacilitative or large scale expression of the exogenous, vector-borne DNA or RNA sequences and isolation of the desired polypeptides from the growth medium, cellular lysates, or cellular membrane fractions yields the desired products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1: RPLC e~E~ication of HB~-l and HBF-2 A portion (3~g) of the 3~ NaCl eluent from heparin sepharose is subject to reverse-pha~ed HPLC on a Vydac C4 column as described.
FIGURE 2: The e~fec~_of the ~ixture of ~BF-l and HBF-2 eluted f~om he~rin se~aros~ on the ~roliferation - of bovine aortic arch endothelial cells Prot~in in the 3~ NaC1 eluate from heparin-Sepharose is determined by amino acids analysis and aliquots, after appropriats dilution in DM~M/~SA, are added to the cells for assay of mitogenic activity Cell growth is assessed by cell counting, as dPscribed.
FIGURE 3: The effect of RPLC purified HE3F-l. HBE-~
a qlu3~5 ~e~ 78~96 bFGF on the Proli~eration of bovine aorti~ axch endothellal cells Samples of RPLC-purified mutant bFGF(-~ -) HBF-l ; (-o-), HBF-2(-~-), appropriately diluted (in DMEM/BSA), ~59~
are added to cells, and growth is determined using the acid phosphatase assay.

SUMMARY~OF THE INVENTION
The present invention relates to novel fragments of bFGF. The bFGF fragments are obtained by proteolytic degradation of the native bFGF or analogues thereof and result in fragments that are bound to heparin or heparin sepharose alone or in combination with other fragments of bFGF which bind to heparin or heparin sepharose. More specifically, the fragments of the present invention contain about amino acids 27 to 69 and about amino acids 70 to 155 of the native bFGF or ~nalogues thereof which bind to heparin or which immobilize heparin or heparin sepharose. It has been unexpectedly found that the polypeptides of the pr~esent invention, those with about amino acids 70 to 155, exhibit a mitoyenic activity much greater than pep~ide~ previously ~enerate~ (34 and 35).
As such, it is an objective o~ the present invention to provide biologically active bFGF fragments, which bind, alone or in combination with other FGF fragments to heparin. Further, the ~ragment 70-155 also exhibits mitogenic activity. It is a further object of the invention to provide a method for generating said peptide fragments. Also, these fragments are useful in treating animals and patients to more quickly regenerate damaged and/or destroyed tissues as previously discussed. These and other objects of the present invention will become more apparent by the more detailed description of the invention provided hereinbelow.

~:' 9~.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is exemplified by the following examples, which are illustrative and not limitative thereof.

ConstructiQn o~_Ex~pression Plasmid A synthetic gene encoding the 155 amino acid form of human basic FGF (26) cloned into pUC 18 was purchased from British Bio-technology, Oxford, UX. To facilitate manipulations, the internal Ncol restriction site, which includes the N-terminal methionine codo~ of the bFGF
cDNA, is destroyed and replaced with a unique Ndel site.
This is accomplished by excision of the nucleotide seguence (-12 ~o 36) with HindIII and BspMII and cloning of a synthetic ~ra~ment containing an internal Ndel site into pUC 18. The cDNA encoding bFGF is then excised from pUC 18 with Ndel and Ba~ H1 and cloned into the Ndel and Bam Hl sites of the expression vector pT7 Kan 5, a 2~ derivative of pET-3a containing the T7 promoter ~rom RNA
polymerase (38)~

EX~MP~E 2 Cons ~uction of a Synthetic Gene 50rresPondina to the Human bFGF analo~ alu3'5,ser78'96bFGF
In a first step, glu3'5-bFGF a chimeric ~GF is constxucted using a protocol that is identical to that described above for the introduction of the Ndel restriction site excep~ ~hat the codons for alanine and serine at positions 3 and 5, respectively, (a) are changed to encode glutamic acid (b);

5' AGCTTCAT~TGGCAGCCGGGAGCATCACCACGCTGCCCGCCCTT 3' (a) 5' AGCTTCATATGGCTQ~GGG~aATCACCACGCTGCCCGCCCTT 3' (b) , -14- Z~ 9~

Only the sense strands are shown for the original (a) and modified (b) fragments, respectively. The codons underlined indicate those changed to encode glutamic acid at positions 3 and 5.
The expression plasmid, pT7 glu3'5-hbFGF, is then used as a template for oligonucleotide site-directed mutagenesis. Two mutagenic oligonucleotide primers are designed to change codons for cysteine at positions 78 and 96 to serine codons. The primer for serine at position 96 is to the sense strand (60-mer; 238-297) whereas that for serine at position 96 is to the antisense strand (30-mer; 251-222). In addition to these mutagenic primers, primers to the T7 promoter (nucleotide -12 to ~13~ and terminator regions (nucleotide -75 to -51) are designed (38). Mutation of the modified FGF
gene is accomplished by use of the Polymerase Chain Reaction (PCR). Two reac~ion mixtures containing HindIII
cut plasmid DNA are prepared; (i) T7 sense plus Ser 78 antisense primers to yield an expected 319 bp product, and (ii) T7 antis~nse plus Ser 96 sense primers to produce an expected 294 bp product. PCR mixtures are prepared according to standard instructions. PCR is performed using Taq polymerase for 30 amplification cycles each of g2C for 1 min, 50C for 5 sec, 72C for 1 min and the products analyzed by agarose gel 2S electrophoresis. Excess primers are separated from the amplified DNA fragments by 3 successi~e rounds of concentration and dialysis using 30,000 MW cut of microconcentrators (Millipore). Portions of the retentates are combined and amplified using the PCR as described above except that the primers used correspond to the T7 promoter (sense) and T7 terminator (antisense) regions. The 599 bp PCR product is then treated with Ndel and BamHl and purified by agarose gel electrophoresis.

59'~
The purified fragment is then cloned into the T7 expression vector, pET-3a(M-13), a derivative of pET-3a.

Ex~ession of qlu3'5 ser78,96bFG~
Following sequsnce verification ~39), the gene encoding the bFGF mutant is transformed into competent BL21 plys S cells. E. coli (E. coli skrain that contains the lysozyme "s" plasmid) cells harboring the plasmid are grown in Luria broth containing ampicillin (100 ~g/ml) and chloroamphenicol (34 ~g/ml) at 37C to about 0.6 absorbance units at 600 nm, and bF~F synthesis is induced by addition of isopropyl-beta-thiogalactopyranoside (IPTG, 1 mM). The cells are then harvested 2 hours post induction by centrifugation at 4C.
EXA~PLE 4 Puri~icatio~ of al 3~5 ser78~96bFGF
Cell pellets from 1 liter-cultures are resuspended in 50 ~M Tris, 0.1 mM EDTA bu~fer (pH 7.6; 30ml) and lysed by 3 rapid freeze/thaw cycles~ The lysate is then treatsd with DNase 1 (20 ~g/ml) in the presence of 5 mM
MgCl2 for 20 min at 4C and centrifu~ed to remove cell debris ~10,000 x g; 20 min). bFGF is purified from the supernatant solution by heparin sepharose af~iniby chromatography essentially as described (22) using a linear salt gradient from 0.6-3.OM NaCl. Fractions containing growth factor are pooled, diluted with Tris buffer (10 mM; pH 7.6) to give a final NaCl concentration of about 0.6 M and loaded onto a TSK Heparin 5PW column (0.75 X 7.5 cm; TosoHaas, Philadelphia, PA) equilibrated with lO mM Tris, 0.6M NaCl (pH 7.6). Elution of bound material is monitored at 280 nm and is accomplished using a linear salt gradient (0.6-3.0 M NaCl in 60 min) at a flow rate o~ 0.7 ml/min.

.
' .

;2~S5~
Growth factor purified in this manner is analyzRd for homogeneity by using reverse phase HPLC, monitoring elution at 210 nm, (C4, Vydac; The Separations Group, Hesperia, CA) in an acetonitrile gradient (0-28% CH3CN in 15 min; 28-60% CH3CN in 90 min) in 0~1% trifluoroacetic acid at a flow rate of 0.7 ml/min, N-terminal sequence analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10-15% gradient and 20%
homogeneous gels using a silver-s~ain detection system (Phastgel System, Pharmacia/LKR).

Proteolytic Diaestion of qlu3l5~-ser78~96bFGF
A solution containing glu3'5,ser78'96bFGF (712 ~g in 0.475 ml) is added to a drained slurry (0.2 ml) of heparin-Sepharose (Pharmacia/LKB, Uppsala, Sweden), previously equilibrated with Tris-H~l buffer (50 mM; pH
7.6), mixed and incuba~ed at ~C. After 60 min the supernatant solution is removed and the gel washed with 20 ` 50 mM Tris-RCl pH 7.6 ~0.4 ml). The g~l slurry is resuspended in Tris-HCl buffer (0.2 ml), and pronase (Sigma Chemical Co.; Type XXV) is added to giv~ a ratio of bFGF to pronase of 0.75:1.0 (w/w). The mixture is then incubated at 37C with agitation. After 24 hours the gel mixture is centrifuged, the supernatant solution ramovad and the drained gel washed with 10 mM Tris buffer (pH 7.6;3 x 0.4 ml~ and Tris bu~fer containing 0.6 M NaCl (2 X 0.4 ml) to ramove non-specifically bound proteolytic products. Elution o~ bound material is efected by washing the heparin-Sepharose gel twice with Tris buffer containing 3M NaCl.

-17- 2~9~

Analysis of Proteolytic Fraoments of qlu ' ser ' bFGF
Heparin-binding proteolytic fragments of bFGF are analyzed under reducing conditions on 20% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) using a silver-stain detection system (Phastgel System, Pharmacia/LXB). Peptide fragments are resolved by reverse phase HPLC (C4, Vydac, Hesperia, CA) using an acetonitrile gradient in 0.1% trifluoroacetic acid at a flow rate of 0.7 ml/min. Elution of bound material is monitored at 210 nm. N-terminal sequence analyses of reverse phase purified peptides are perfonmed on a model 477A pulsed liguid phased sequence (Applied Biosystems, CA) equipped with an on-line PTH-derivative analyses (Model 120A, Applied Biosystems). Amino acid compositions are determined after HCl gas phase hydrolysis (5.7 M HCl 10.1% phenol; 24 hours at 110 C) using a model 420A phenylisothiocyanate-derivatizer equipped with an on-line model 130A separa~ion system (Applied Biosystems). Unless otherwise stated, procedures are performed according to the manufacturer's protocols.
Analysis by SDS-PAGE of heparin binding proteolytic fragments of hbFGF shows complete digestion of the 18kD
band corresponding to glu3'5,ser78'96-bFGF and the generation o 2 lower molecular weight peptides that migrate as llkD and 9kD species. RPLC of the growth factor digest eluted ~rom heparin-Sepharo~e with 3M NaCl reveals 2 ma~or peaks, termed heparin-binding fragment-1 and -2 (HBF-l and HBF-2~ (Figure 1). The early eluting fragment, HBF-l, is identified by SDS-PAGE as the 9 kD
species present in material eluted from heparin sepeharose, whereas, the more retarded fragment HBF-2 migrates in a position identical to that of the 11 kD
fragmen~. N-~erminal sequence analysis of the 2 RPLC-purified fragments gives single sequences consistent with peptide regions that begin at residue 27 and residue 70 of glu3'5,ser78'96bFGF for HBF-l and HBF-2, respectively. The amino acid compositions of HBF-l and HBF-2 (Table 1) correspond, within experimental error, to glu3'5ser78'96-bFGF sequence regions of residues 27-69 and 70-155 respectively.
.

.
, .. , --19--5gS~, :
`. TABLE 1 MOL OF AMINO ACID/MOL OF PEPTIDE
. , _ ` HBF- 1 HBF 2 `
, . .
: 5 Amino acid Found Theory Found Theory Asx 4.87 5 6.04 6 Glx 5.02 5 5.76 6 - Ser 1.23 1 7.56 1o lo Gly 4.44 4 7.64 7 His 2.15 2 0.00 0 Arg 5.01 5 6.79 6 Thr o.oo 0 5.71 5 Ala 0.99 1 6.16 6 P~o 2.99 3 2.3~ 2 Tyr 1.00 1 5.82 6 Val 2.07 2 4.84 5 Met o.oo o 1.86 2 Cys N.D. 1 N.D.
Ile 1.87 2 2.43 2 L4u 3-93 4 8.3~ 8 P~e 2.28 2 4.30 4 Trp N.D. 0 N.D.
Lys 4O73 5 8.32 9 _..................... .. _. . . ~ _ Total 43 86 AminQ acid çom~os tions of. HBF=1 and~ F-2 Amino acid analyses of HBF-l (4spmol) and HBF-2(25pmol) are performed in triplicate. Relative standard deviations are less than 10%. (N.D., not determined).

2~g~

BIOASSAY
- Reverse phase ~PLC-purified samples are immediately diluted (1:5) in Dulbecco~s modified Eagle's medium (DMEM~ containing 1~ bovine serum albumin (BSA).
Mitogenic activities of bFGF and bFGF peptide fxagments are determined using bovine vascular endothelial cells derived Prom adult aortic arch as described (22).
Briefly, cells are seeded at an initial density of 0.8 x 104 cells per 24 well plate in 0.5 ml DMEM/10% calf serum (Hyclone, Logan, UT), penicillin ~lOo units/ml), streptomycin (100 ~g/ml), and L-glu~amine (2 mM). Two hours after plating, 20~1 aliquo~s of appropriate dilutions of bFGF in DMEM containing 0.5~ BSA are added.
After 5 day~ in culture, duplicate plates are trysinized and cell densities determined by cell counting in a Coulter counter. Al~ernatively, growth curves in ~he presence and absence of bFGF ar~ determined by measuring acid phosphatase levels after cell lysis (40). Cells are seeded at an initial cell density of 1000-1200 cells per well (0.32 cm2 lat bottom 96-well plates) in 0.25 ml DMEM/10% calf serum containing antibiotics and L-glutamine (see above). After plating of cells, 10 ~l-aliquots of appropriate dilutions of growth factor and peptide fragments in DMEM/0.5% BSA are added. A~ter 4-5 days, cell growth is assessed in each well by measuring acid phosphatase levels after cell lysis using p-nitrophenyl phosphate as substrate (40). The absorbance at 405 nm for each sample is determined using W max kinetic microplate reader (Molecular Devices).
Determinations are made in triplicate. No significant differences are observed in the shape o~ dose response curves or in concentrations of bFGF re~uired for half maximal and maximal stimulation of cell growth when c211 growth is determined by either meth~od.

. . : .

. .
.

. -2~-59~

The 3M NaC1 eluate from heparin-Sepharsse containing an approximately equimolar mixture of HBF-1 and HBF-2 is examined for mitogenic activity. The mixture induces a dose-dependent proliferation of bovine endothelial cells, with the dose for hal-maximal growth stimulation being approximately 10 fold higher as compared to intact bFGF
(Figure 2). In order to ~etermine the mitogenic properties of each component, an attempt to re~olve the 2 mutant FGF fragments is made. Unexpectedly, the chromatographic behaviors of ~BF-l and HBF~2, on Mono-S
cation exchange and TSK heparin HPhC under a variety of conditions, are identical to those of intact bFGF and give no resolution of HBF-~ and HBF-2. However, since RPLC affords effective separation of the 2 components ~Figure 1) the mitogenic properties of RPLC-purified HBF-l and HBF-2 are compared to those of RPLC-purified bFGF. These conditions are known to reduce the mitogenic activity of bFGF by 10-~0 fold, presumably as a consequence of protein denaturation. ~n the present experiment RPLC-purified bFGF is l/lOth as active as intact bFGF. RPLC-purified HBF-1 does not appear to affect cell growth, whereas HBF-2 exhibits a dose-dependent stimulatory response (Figure 3) with a potency that is 25-50 fold lower than that of RPLC-purified glu3'5,ser-78'96-bFGF (ED50of RPLC-purified HBF-2 and glu3'5,ser78'95-bFGF are about 3 and 0.1 pmol/ml, respectively). Assuming that the mitogenic activity of HBF-2 is reduced ~y the same degree as bFGF
under RPLC-conditions, then an ED50 of about 150-300 fmol/ml may be predicted for native HBF 2. This estimate is consistent with an ED50 f about 150 fmol/ml obtained from the data presented in Figure 2 for the mixture containing HBF-l and HBF-2 eluted from heparin-Sepharose.
In our assay, the potency of intact bF~F or - . . ' , . .

2~ 9~
glu3 ' 5, ser78 ' 96-bFGF is approximately 17 fmol/ml Thus HBF-2 with an intact conformation is about 10 ~old less potent than intact bFGF.

.

., ', x~
T~BL~ 2 PEPTIDE MASS MITOGENIC ACTIVITY REF

-bFGF(2-155) 17.2 1.00 (14) bFGF(10-155) 16.4 1.00 (35) bFGF(23-155) 15.2 0.68 (35) bFGF(50-155) 12.1 0.02 (35) bFGF(70-155) 9.5 0.04-0.02HBF-2 bFGF(112-155) 5.0 10 -10 (34) bFGF(33-77) 5.0 10 5-10 6(34) bFGF(109-129) 2.7 10 5 10-~(34) Relati~e mito~g~iC activities of bFGF ~ptide_fra~ments.
Mitogenic activities for each peptide are estimated from published data as indicated and are expres~ed relative to the ED50 values for bFGF(2-155) or bFGF~10-155).
Relative mitogenic potencies quoted from (34) are est~mated from incomplPte data. Values reported for HBF-2 are relative to the ED50 values determined for RPLC
purified bFGF. Similar values may be obtained by comparison o~ the ED50 value for bFGF to that of the mixture containing HBF-l and HBF-2 eluted from heparin sepharose (see text).

. -24-Z~'~5g5~.
The DNA se~uences, plasmids and/or microorganisms deposited in connection with the present patent application, except where specified to the contrary, are deposited in American Cyanamid Company's culture collection maintained in Pearl River, New york and are available to the public when legally appropriate to do so. Further, the following are deposited additionally with the American ~ype Culture Collection (ATCC) in Rockville, ~aryland 20952, U.S.A. on th dake indicated with the ATCC accession numbers indicated:
BL21 lysS/pET glu3'5ser78'96 deposited on November 13, 1990 with ATCC No. 68478.

BL21 lys-S/pET glu3~5hbFGF deposited on November 13, 1990 with ATCC No. 68477.

The above two contain the DNA of glu3'5ser78'96 hbFGF and glu3'5hbFGF as described herein.

, ~C5~
BI BLI OÇ:R~APHY

1. Baird, A. and Bohlen, P. tl990) in "Peptide Growth Factors and their Receptors" (Sporn, M. and Roberts, A., Eds) Handbook of Exp. Pharmacol. 95~1), pp369-418, 5pringer.

2. Gospodarowicz, D., Nature 249:123-127 ~1974).

3. Burgess, W.H. and Maciag, T., Ann Rev. Biochem 8:575-606 (1989).
4. Gospodarowicz D., et al., Nat. Cancer Insti. Mon.
48:109-130 (1978).
5. Davidson, J.M., et al., J. Cell Bio.100: 1219-1227 (1985).
6. U. Franco, W. P., U. S. Pat. No. 4,296,100 (1981).

- ` 7. U. Franco, W. P., U.S. Pat. No. 4,378,347 (19833 8. Walicke, P., Et al~, Pro¢. N~t. Acac. Sci. USA 83:
3012-3016 tl98~) 9. Esch, F., et al., Proc. Nat. Acad. Sci USA 82:
6507-6511 (1985) 10. Go3podarowicz, D. et al., U. S. Pat. No. 4,785,079 (1988).
11. Gospodarowicz, D. et al, U. S. Pat. No5 4,902782 ( lggo) -.

~59~.
12. Iwane, M., et al., Biochem. Biophys. Res. Commun.
146:470-477 (1987).

. 13. Squires, C.H., et al., J. Biol. Chem. 263:
: 5 16297-16302 (1988).

14. Barr, P.J., et al., Biol. Chem. 263:16471-16478 (1988).

15. Esch., F., et al., Eur. Pat. Appl. Pub. 281,822 (198~).

16. Seno, M., et al., Eur. Pat. Appl. Pub. 281,822 (1~88).

17. Arakawa, T. and Fox, G.M., Eur. Pat. Appl. Pub.
320,148.

18. Thomas and Linemeyer, Eur. Pat. Appl. Pub. 319,052 (1989).
19. Seno, M., et al., ~ur. Pat ~ppl. Pub. 326,907 (1989).

20. Fiddes, J. C., et al., Eur. Pa~. Appl. PUb. 298,723 t1989).

21. Bergonzoni, L., e~ al., Eur. Pat Appl. Pub. 3G3,675 (1989) 22. Gospodarowicz, D., Cheng, J.~ Lui, G., Baird, Ao~
and Bohlen, P. (1984) Proc. Natl. Acad. Sci. 81, 6963-69S7.

, 27~ 55~

23. Gospodarowicz. D. and Cheng, J. (1986) J. Cell.
Physiol. 128, 475-484.
.~
24. Sommer, A. and Rifkin, D.B. (19~9) J. Cell. Physiol.
138, 215-220.

25. Damon, D.H. Lobb, R. R., D'~more, P.A., and Wagner, J.A. (1989) J. Cell. Physiol 138, 221-226.

`; 10 26. Abraham, J.A~, Whang, ~. L., Tumolo, A., Mergia, A., Friedman, J., Gospodarowicz. D., and Fiddes, J.C. (1986) EMB0 J. 5, 2523-2528.

27. Vlodavsky, I., Folkman, J., Sullivan, R., Fridman, R., Ishai-Michaeli, R., Sasse, J., and Klagsbrun, M.
(1987) Proc. Natl. Acad. Sci. 84, 2292-2296.

28. Folkman, J., Klagsbrun, M., Sasse, J., Wadzinski, M.
Ingber, D., and Vlodavsky, 1. (1988) Am. J. Pathol- 1 29. Bashkin, P., Doctrow, S., Klagsbrun, M. Svahn, C.M., Folkman, J., and Vlodavsky, I. (1989) Biochemistry 28, 1737-1743.

30. Presta, M., Maier, J.A.M. Rusnati, M., and Ragnotti, G. (1989) J. Cell. Physiol. 1~0 68-74.

31. Saksela, 0. and RifXin, D. B. (1990) J. Cell. Biol.
110~ 767-775.
32. Vlodavsky, I., Michaeli, R. I., Bar-Ner, M., Fridman, R., Horowitz. A. T. Fuks, Z. and Biran , S .
(1988) Israeli J. Med. Sci 24, 464-470.

;~C ~5~

. 33. Nakajima, M., Irimura, T. and Nicolson, G.L. (1988) J. Cell Biochem. 36, 157-167.

s 34. Baird, A., Schubart, D., Ling, N. and Guillemin, R.
(1988) Proc. Natl. Acad. Sci. 85, 2324-2328.

35. Seno, M., Sasada, R. KuroKawa, T. and Igarashi, K.
(1990) Eur. J. Biochem. 188, 239-245.
.
36. Fox G. M., Schiffer, S. G., Rohd~, M. F., Tsai, L.B.
Banks, A. L. and Arakawa, T., ~19~8) J. Biol. Chem 263, 18452-18458.

37. Andrews, P. C. and Dixon, J. E., (1987) Anal.
Biochem. 161, 525.
38. Rosenberg, A. H., Lade, B.N., Chui, D., Dunn, J. J.
~nd Studier, F. W., (1987) Gene 56, 125-135.

39. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc. Natl. ~cad. Sci. 74, 5463-5467.

~- 40. Connolly, D. T., Knight, M. B. Harakas, N. K.
Wittwer, A.J. and Feder, J., (1986) Anal. Biochem 152, 136-140.

-: 35 5~

(1) GENERAL INFORMATION:

(i) APPLICANT: Andrew P. Seddon and Peter Bohlen r (ii) TITLE OF INVENTION: Active Fra~ments of Fibroblast Growth Factor (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:

(A3 ADDRESSEE: Dr. Est~Ile J. Tsevdos, American Cyanamid Company (B) STREET: 1937 West Main Street, P. O. Box 60 (C) CITY: Stamford (D) STATE: Co~necticut .

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(A) TET.EPHONE: 203 321 2756 (B) TELEFAX: 203 321 297}

(C) TELEX: 710 474 4059 .~. . ~ . . ;
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t2) INFORMATION FOR SEQ ID NO: 1:

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(A) LENGTH: 132 base pairs (B) TYPE: nucleic acid (G) STRANDEDNES5S: single (D) TOPOLOGY: linear (ii) MOLE~ULE TYPE: DNA

(iii) HYPOTHETICAL:
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(v) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN:
(C):INDIVIDUAL ISOL~TE:

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(F) PAGES:

(G) DATE:

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(J) PUBLICATION DATE:
(K~ RELEVANT.RESIDUES:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
2~

Lys Asp Pro Lys Arg ~eu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu Gln ~O

Ala Glu Glu Arg , ~

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(2) INFORMATION FOR SEQ ID NO: 2:

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(A) LENGTH: 258 base pairs (B) TYPE: nucleic acid (C) 5TRAND~DNESSS:- single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:

(v) FRAGMENT TYPE:

(vi) ORIGIMAL SOURCE:

(A) ORGANISM:

(B) STRAIN

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STA~-E:

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i 15 (B) MAP POSITION:

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(ix) FEATURE:
(A) NAME/KEY:

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(J) PUBLICATION DATE:

(K3 RELEVANT RESIDUES:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn Arg Tyr CTG GCT ATG AAG GAA GAT GGA A ::A TTA CTG GCT TCT AAA 7 8 ~- 25 L~u Ala Plet Ly3 Glu Asp Gly Arg L~u Iæu Ala Ser Lys TGT GTT ACG GAT GAG TGT TTC TTT TTT GAA CGP~ TTG GAA 117 Cy~ Val Thr A-~p Glu cys Phe Phe Phe Glu Arg hsu Glu TCT AAT AAC TAC AAT ACT TP.C CGG TCT AGA AAA TAC ACC 15 6 Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr .:: 35 ~ . --37- 2~

Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Ph~ Leu Pro Met Ser Ala Lys Ser ~s~

. (2) INFORMATION FOR SEQ I~ NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 258 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESSS: single (D) TOPOLOGY: linear j (ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Gly Val Val Ser Ile Lys Gly Val Ser Ala Asn Arg Tyr Leu Ala m~t Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys TCT GTT ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG &AA 117 Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Gl~
lQ0 105 Ser Asn A~n Tyr Asn Thr Tyr Arg Ser Arg Ly~ Tyr Thr 21~!~5951 .

Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys CTT GGT TCC AAA ACA G(:A CCT GGG CAG AAA GCT ATA CTT 2 3 4 Leu Gly Ser Lys Thr Gly Pro Gly ~:lr Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser ,,

Claims (20)

1. A fragment of basic fibroblast growth factor which binds to heparin, heparin sepharose or analogues of each thereof, alone or in combination with other fragments of FGF.
2. A fragment of basic fibroblast growth factor according to Claim 1, wherein said fragment comprises about amino acids 27 to 69 of basic fibroblast growth factor or about amino acids 70 to 155 of basic fibroblast growth factor.
3. A fragment of basic fibroblast growth factor according to Claim 1, wherein said fragment is mitogenic.
4. A fragment of basic fibroblast growth factor according to Claim 3, wherein said fragment comprises about amino acids 70 to 155 of basic fibroblast growth factor.
5. A fragment of basic fibroblast growth factor according to Claim 1, wherein the cysteines at positions 78 and 96 are replaced with alanine, glycine, arginine, tryptophan, lysine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, isoleucine, leucine, valine, phenylalanine, tyrosine, methionine, serine, threonine or proline.
6 A fragment of basic fibroblast growth factor according to Claim 5, wherein the cysteines at position 78 and 96 are replaced with serine.
7. A fragment of basic fibroblast growth factor according to Claim 2, wherein the cysteine at positions 78 and 96 are replaced with alanine, glycine, arginine, tryptophan, lysine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, isoleucine, leucine, valine, phenylalanine, tyrosine, methionine, serine, threonine or proline.
8. A fragment of basic fibroblast growth factor according to Claim 7, wherein the cysteines at positions 78 and 96 are replaced with serine.
9. A DNA sequence that codes for a fragment of a recombinant basic fibroblast growth factor, said fragment binding to heparin, heparin sepharose or analogues of each thereof, alone or in combination with other fragments of basic fibroblasts growth factor and a DNA
sequence that will hybridize under stringent conditions to a DNA sequence that codes for a recombinant basic fibroblast growth factor, said fragment binding to heparin, heparin sepharose or analogues of each thereof.
10. A DNA sequence according to Claim 9, wherein said DNA sequence codes for a fragment of basic fibroblast growth factor having about amino acids 26 to 69, or about amino acids 70 to 155 of basic fibroblast growth factor.
11. A DNA sequence according to Claim 9, wherein said DNA seguence codes for a fragment of basic fibroblast growth factor, said fragment being mitogenic.
12. A DNA sequence according to Claim 11, wherein said DNA sequence codes for a fragment of basic fibroblast growth factor having about amino acids 70 to 155 of basic fibroblast growth factor.
13. A DNA sequence according to Claim 9, wherein said DNA equence coding for cysteines at positions 78 and 96 is replaced by a DNA sequence coding for alanine, glycine, arginine, tryptophan, lysine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, isoleucine, leucine, valine, phenylalanlne, tyrosine, methionine, serine, threonine or proline.
14. A DNA sequence according to Claim 13, wherein said DNA positions 78 and 96 is are replaced by a DNA
sequence coding for serine.
15. A DNA sequence according to Claim 10, wherein said DNA sequence coding for cysteines at positions 78 and 96 is replaced by a DNA sequence coding for alanine, glycine, arginine, tryptophan, lysine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, isoleucine, leucine, valine, phenylalanine, tyrosine, methionine, serine, threonine or proline.
16. A DNA sequence according to Claim 15, wherein said DNA sequence coding for cysteines at positions 78 and 96 is replaced by a DNA sequence coding for serine.
17. A DNA sequence according to Claim 11, wherein said DNA sequence coding for cysteines at positions 78 and 96 is replaced by a DNA sequence coding for alanine, glycine, arginine, tryptophan, lysine, aspartic acid, glutamia acid, asparagine, glutamine, histidine, isoleucine, leucine, valine, phenylalanine, tyrosine, methionine, serine, threonine or proline.
18. A DNA sequence according to Claim 17, wherein said DNA sequence coding ror cysteines at positions 78 and 96 is replaced by a DNA sequence coding for serine.
19. A DNA sequence according to Claim 12, wherein said DNA sequence coding for cysteines at positions 78 and 96 is replaced by a DNA sequence coding for alanine, glycine, arginine, tryptophan, lysine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, isoleucine, leucine, valine, phenylalanine, tyrosine, methionine, serine, threonine or proline.
20. A DNA sequence according to Claim 19, wherein said DNA seguence coding for cysteins at position 78 and 96 is replaced by a DNA sequence coding for serine.
CA002055961A 1990-11-23 1991-11-21 Active fragments of fibroblast growth factor Abandoned CA2055961A1 (en)

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AU4645993A (en) * 1992-06-22 1994-01-24 New York University Mammalian growth factor
AU7205894A (en) * 1993-06-08 1995-01-03 Neogenix, Inc. Purified natural and synthetic compounds for the treatment of osteoarthritis
US6221854B1 (en) 1996-03-05 2001-04-24 Orquest, Inc. Method of promoting bone growth with hyaluronic acid and growth factors
US20110207666A1 (en) * 1996-03-05 2011-08-25 Depuy Spine, Inc. Method of promoting bone growth with hyaluronic acid and growth factors
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US5206354A (en) 1993-04-27
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