CA1340976C - Fibroblast growth factor antagonists - Google Patents

Abstract

Antagonists to basic fibroblast growth factor, a 146 amino acid residue polypeptide, are produced. These antagonists are generally between 10 and 41 residues in length and are characterized by their ability to interact with the FGF receptor and/or inhibit and therefore modulate endothelial and other cell growth. These antagonists include the sequence of bovine basic FGF(106-115;1, namely Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr; however, there may be one or more substitutions made in such sequence to create biologically potent analogs thereof. These peptides are also antagonistic to acidic FGF and other members of the family of FGF peptides. They are effective to combat FGF-promoted mitosis in melanomas and the like.

Description

FIBROBLAST GROWTH FACTOR ANTAGONISTS
They present invention is directed to fibroblast growth factor (FGF) and more particularly to FGF
antagonists produced by synthetic methods, which can be used to reduce then effects of mammalian FGF in certain instances.
BACKGROUND OF THE INVENTION
Both the brain and the pituitary gland have been known to contain rnitogenic factors for cultured cells;
however, until 19',14, it was unclear what their relationship was with classical pituitary hormones, such as TSH, LH, FSH, GH and ACTH. In 1974, the purification of a bovine growth factor called basic fibroblast growth factor (bFGF') was reported which was shown to be distinct from pituitary hormones, Gospodarowicz, D. Nature, 249, 123-127 (1974). ~.Chis growth factor is now known to have a MW of 16,4.15, is basic (a pI of 9.6), and is a potent mitogen for either normal diploid fibroblasts or established cell :Lines. Purification of another distinct growth factor, acidic brain fibroblast growth factor (aFGF) is describE:d in U.S. Patent No. 4,444,760 (Apr.
24, 1984). ComplEate characterization of bovine aFGF was reported by Esch eat al., Biochemical and Biophysical Research Communications, 133, 554-562 (1985).
Later studies confirmed that, in addition to fibroblasts, FGF .is also mitogenic for a wide variety of normal diploid mesoderm-derived and neural crest-derived cells, including granulosa cells, adrenal cortical cells, chondrocyte:>, myoblasts, corneal and vascular endothelial cells from either bovine or human origin, vascular smooth muscle cells:, and lens epithelial cells. FGF has also been shown t:o sub:atitute for platelet-derived growth factor in ita abi:Lity to support the proliferation of fibroblasts exposf~d to plasma-supplemented medium.
Consistent with ii;.s ability to stimulate the proliferation of bovine and human vascular endothelial cells, FGF teas a similar activity in vivo upon capillary endothelial cells;; therefore, FGF is considered an angiogenic factor..
SUMMARY OF THE INVENTION
They present invention provides FGF antagonists which may be produced by synthetic methods and which substantially counteract the biological effect of mammalian FGF in certain instances.
They presEant invention provides antagonists to basic and acidic i:ibroblast growth factor which may be s nthesized usin recombinant DNA techni y g ques or other suitable tec:hniquEa, such as classical or solid phase synthesis. Basic FGF is a 146 amino acid residue polypeptide having the sequence set forth hereinafter.
It appears nnost likely that, in the native bovine FGF
molecule, none of the cysteine residues are disulfide-bonded t:o each other, but that there may be bonding of ene or more of the cysteine residues to free cysteine moleculesc. In any case, the present invention provides biologic2~lly active peptides that suppress the biolo ical activit, of FGF and are g 'y generally between about 10 and 41 residues in length. They can be synthesized by a recombinant DNA technique or by standard chain elongation procedures involving stepwise addition of amino acid residues, such as solid-phase synthesis upon a solid resin support.
Pharmaceutical compositions in accordance with invention include FGF antagonists or nontoxic salts thereof dispersed in a pharmaceutically acceptable liquid or solid carrier. Such pharamaceutical compositions can be used in clinical medicine, both human and veterinary, and in acute or chronic administration for diagnostic or therapeutic purposes. They are useful both in vivo and in vitro in modulating the growth of endothelial and other related cell types.
In one particular aspect, the invention provides a peptide having the following formula:
H-Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-1 3 40 9? 6 Val-Ala-Leu-Lys-A.rg-Y', wherein 8106 is Tyr or D-Tyr, 8107 is Arg or D-Arg, 8108 is Ser or D-Ser, 8109 is Arg or D-Arg, 8110 is Lys or D-Lys, 8111 is Tyr or D-Tyr, 8112 is Ser, Thr or D-Ser, 8113 is Ser, Ala or D-Ser, 8114 is Trp or Met, 8115 is Tyr or Phe, and Y' is OH, NH2, or Thr-Gly-Gln-Tyr-Lys-Leu-Gly-8128-Lys-Thr-Gly-Pro-Gly-Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser-Y, with 8128 being Pro or Ser and Y being OH or NH2, .or a biologically active fragment which functions as an FGF antagonist or binds with the FGF
receptor, provided however that at least one of 8106 to 8113 is a D~-isomer and/or 8112 is Thr and/or 8113 is Ala and/or 8114 is Met and/or 8115 is Phe and/or Y' is other than OH or NH2.
In another particular aspect, the invention provides a method of producing an FGF antagonist comprising: obtaining a DNA chain that encodes a polypeptide containing the following sequence:
H-Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-T;yr-8112 8113 8114-8115-Val-Ala-Leu-Lys-Arg-Thr-Gly-Gln-Tyr-Lys-Leu-Gly-8128-Lys-Thr-Gly-Pro-Gly--Gln-L;ys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser, wherein :8112 is Ser or Thr, 8113 is Ser or Ala, 8114 1~' Trp or Met, 8115 is Tyr or Phe and 8128 is Pro or Se:r, or a biologically active fragment thereof coni=aining a sequence at least 29-residues in length which functions as an FGF antagonist or binds with the FGF receptor, inserting said DNA chain into a cloning vector in proper :relationship to DNA sequences which promote expression of said encoded polypeptide, transforming an organism or cell line with said cloning vector having said inserted DNA chain, culturing said transformed organism or cell line, and obtaining said polypeptide produced thereby.

1 3 40 9? 6 DETAILED DESCF;IPTION OF CERTAIN PREFERRED EMBODIMENTS
The invention provides antagonists to mammalian FGF, particularly to bovine basic FGF, but also to acidic FGF, which can be: readily synthesized. The nomenclature used to define th.e peptides is that specified by Schroder & Lubke, "The Peptides", Academic Press (1965), wherein in accordance with conventional representation the residue having the free alpha-amino group at the N-terminus appears to left and the residue having the alpha-carboxyl group at the C-terminus to the right.
Where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented. Bovine basic FGF has been found to be a peptide having the following sequence:

Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly-Ala-Phe-Pro-Pro-Gly-His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly-Phe-20 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-Ala-Glu-Glu-Arg-Gly-Val-Val--Ser-Ile-Lys-Gly-Val-Cys-Ala-Asn-Arg-Tyr-Leu-Ala-'g0 85 90 Met-Lys-Glu--Asp-G:ly-Arg-Leu-Leu-Ala-Ser-Lys-Cys-Val-Thr-Asp-!~5 100 105 Glu-Cys-Phe--Phe-Plze-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-1:10 115 120 Tyr-Arg-Ser--Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-Thr-Gly-Gln--Tyr-L!~s-Leu-Gly-Pro-Lys-Thr-Gly-Pro-Gly-Gln-Lys-Ala-Ile-Leu--Phe-L<au-Pro-Met-Ser-Ala-Lys-Ser.
The C-terminus of the native molecule is free acid.

Ths~ pres~ant invention provides two families of FGF antagon:Lsts which are each based upon a central fragment from the native hormone bFGF. The core of the first is those re:aidues appearing at positions 36-39, and the core of the second appears to be those residues appearing at: positions 106-115. In other words, relatively :short peptides containing the four residues of the first family, as well as the tetrapeptide itself, show some suppres:aion of endothelial cell growth, when growing under nonstimulated conditions (serum alone) and also when serum i;~ supplemented by the addition of FGF to in vitro ce7.l cull:ures. The FGF antagonism of the first family is found to be very substantially increased by the inclusion of N-terminal and/or C-terminal extensions to the tetrapeF>tide. These extensions may comprise the residue sequences normally found at these locations in the native r~ormone:, e.g., bFGF(30-50) and are preferably, but not neceasari7Ly, amidated at the C-terminus.
Preferably, the extended fragment includes bFGF(24-68) which exhibits good FGF antagonism. Some substitutions may be made in thE~ sequence at selected locations, as discussed he:reinai:ter.
The: basi:~ for the antagonistic action exhibited by these peptides is an interaction with the FGF
receptor. F~eptide~s that show antagonism to mitogenisis in vitro (including all FGF target cell types) also prevent FGF from binding to its receptor, and it appears the minimum length peptide should contain either the core sequence of bFGF (36-39) or bFGF (106-115).
In the pe~ptidic fragments of the second family, which are ge.nerall.y within the sequence of bFGF (93120) and which are also antagonistic, there is also a distinct heparin-binding site, i.e., a sequence contained within the peptide fragms:nt binds radioactive heparin as well as the receptor. Because heparin is an important element in certain FGF action, peptides that inhibit binding between FGF and heparin may well also exhibit the important 134897 fi capacity to inhibit the biological action of FGF, which may be an inhibition of the binding of FGF to its receptor re::ultinc~ from this interaction between FGF and heparin. However,, it appears that it may be possible to design FGF antagonists that will bind strongly to the receptor and not bind strongly to heparin; for example, by replacing certain of the residues that account for binding to heparin, e.g., those in positions 107-110, analogs should result which will not bind heparin without substantially det~__~acting from binding to the FGF
receptor. The specificity of fragments related to bFGF(24-68) and b1?GF(93-120) is best illustrated by a) their effecta on all three parameters of FGF action (i.e., mitoc~enesi:a, heparin-binding and receptor interaction) and b) the observation that other FGF
peptide fragments which do not contain either of the afore-mentioned core sequences fail to exhibit similar activity.
The' firsi: family of FGF antagonist peptides provided by the invention may be expressed by the following formula (which is based upon the naturally occurring sequencEa of bovine bFGF): Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-LEau-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-R42-Val-Arg-Glu-Lys-R47-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-~Glu-G:Lu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-Y
wherein Y i~> eithE~r OH or NH2. R42 may be Gly or Ala or Sar, and R47 may be Ser or Ala or Thr. Sar is the abbreviation for sarcosine. Peptides having this entire length, i.e., 45 residues, function as FGF antagonists and not as ~>artial agonists. As such, they suppress endothelial cell growth both in the presence of basal FGF
as well as in the presence of added FGF. 45 residues is not considered to be a maximum limit for a peptide that will function as an FGF antagonist, a main function of such an antagonist: being simply to block the receptor on the endothe7.ia1 cE~lls without causing activation. As a result, additiona:L residues may be added to either or 1 3 40 97 fi _, _ both termini so long as the presence of these additional residues does not either (a) turn the peptide into a partial FGF agonist or (b) detract from the binding of the peptide to the receptor so as to lessen its biological .activity as an FGF antagonist.
The second family of FGF antagonist peptides provided by the invention may be expressed by the following formula (which is based on the naturally occurring s~~quence of bovine bFGF): Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-8112 8113 8:114 8115-Val-Ala-Leu-Lys-Arg-Y, wherein one or more of the residues in the sequence: Tyr-Arg-Ser-Arg-Lys-Tyr can be substituted by its D-isomer, 8112 is Ser, Thr or D-Ser, 8113 is Ser, Ala or D-Ser, 8114 is Trp or Met, 8115 is Tyr or Phe, and Y is either OH or NH2. These peptides function as FGF antagonists, suppressing endothelial cell growth (and growth in other FGF target cells) in the presence or absence of FGF, and peptides shorter in length which include the core sequence bF(~F(106~-115) are also effective to act as antagonists to FG:F. Although various of these peptides may also exhibit ;some agonist activity, if residues are be added to either or both termini of this 28-residue sequence, such changes may result in some receptor activation, creating competitive antagonists having still greater agonist activity.
It may bc_ preferable to synthesize peptides which are about 4!5 amino acids or greater in length by using recombinant DNA methods. On the other hand, it may be preferab7.e to :synthesize peptides of about 30 residues or less in 7_ength using the well-known chain elongation techniques, such as solid-phase synthesis, as on a Merrifield resin or the like.
To synthesize a bFGF peptide containing only naturally occurring amino acid residues by recombinant DNA, a double-stranded DNA chain which encodes the desired amino acid sequence is synthetically 134~9~ 6 _8_ constructed. The degeneracy of the genetic code permits a wide variety of codon combinations to be used to form the DNA chain that encodes the product polypeptide.
Certain particular codons are more efficient for polypeptide expression in certain types of organisms, and the selection of codons preferably is made according to those codon:~ which are most efficient for expression in the type of organism which is to serve as the host for the recombinant vector. However, any correct set of codons should encode the desired product, even if slightly le:~s efficiently. Codon selection may also depend upon vector construction considerations; for example, it may b~e necessary to avoid creating a particular restriction site in the DNA chain if, subsequent i~o insertion of the synthetic DNA chain, the vector is to be manipulated using a restriction enzyme that cleaver at such a site. Also, it is necessary to avoid placing restriction sites in the DNA chain if the host organi:~m which is to be transformed with the recombinant vector containing the DNA chain is known to produce a rcsstriction enzyme that would cleave at such a site within the DIHA chain. For example, polypeptides containing i:he following sequence:
H-Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-T:yr-8112 8113 8114 8115-Val-Ala-Leu-Lys-Arg-Thr-G:ly-Gln-Tyr-Lys-Leu-Gly-8128-Lys-Thr-Gly-Pro-Gly-Gln-L:ys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser, whcarein :8112 is Ser or Thr, 8113 is Ser or Ala, 8114 1~a Trp ~or Met, 8115 is Tyr or Phe and 8128 is Pro or Se:r, or biologically active fragments thereof coni:aining a sequence at least 29-residues in length which function as an FGF antagonist and/or bind with the FGl? receptor may be produced in this manner.
In addition to the bFGF antagonist-encoding sequences, i:he DN;A chain that is synthesized may contain additional sequences, depending upon vector construction considerations. 'typically, a DNA chain is synthesized ~ 3 40 9~ 6 _g_ with linkers at its ends to facilitate insertion into restriction sites within a cloning vector. The DNA chain may be constructed so as to encode the desired sequence as a portion of a fusion polypeptide; and if so, it will generally contain terminal sequences that encode amino acid residue sequences that serve as proteolytic processing sites, whereby the desired polypeptide may be proteolytic~ally cleaved from the remainder of the fusion polypeptide. The terminal portions of the synthetic DNA
chain may also contain appropriate start and stop signals.
To assemble the desired DNA chain, oligonucleotides are constructed by conventional methods, such as procedures described in T. Manatis et al., Cold Spring Harbnr Laboratory Manual, Cold Spring Harbor, New York (1982)(hereinafter, CSH). Sense and antisense oligonucleotide chains, up to about 70 nucleotide residues long, are synthesized, preferably on automated synthesizers, sudh as the Applied Biosystem Inc. model 380A DNA synthesizer. The oligonucleotide chains are constructed so that portions of the sense and antisense oligonucleoltides Overlap, associating with each other through hydrogen :bonding between complementary base pairs and thereby forming double stranded chains, in most cases with gaps in the .strands. Subsequently, the gaps in the strands are filled in and oligonucleotides of each strand are joined sand to end with nucleotide triphosphates in the presence of appropriate DNA polymerases and/or with ligases.
As an alternative to construction of a synthetic DNA chain through oligonucleotide synthesis, when a peptide is desired that is a segment of the naturally occurring molecule, cDNA corresponding to the desired bFGF fragment may be prepared. A cDNA library or an expression :Librar:y is produced in a conventional manner by reverse i:ransc:ription from messenger RNA (mRNA) from a bFGF-producing cell line. To select clones containing bFGF sequences, hybridization probes (preferably mixed probes to accommodate the degeneracy of the genetic code) corresponding to portions of the bFGF protein are produced and used to identify clones containing such sequences. Screening of the expression library with bFGF
antibodies :may also be used, alone or in conjunction with hybridization probing, to identify or confirm the presence of bFGF-encoding DNA sequences in DNA library clones. Such techniques are taught, for example in CSH, l0 supra.
The double-stranded bFGF-encoding DNA chain is shortened appropriately to the desired length to create the peptide of interest and then modified as necessary to permit its insertion into a particular appropriate cloning vector in mind. The cloning vector that is to be recombined to incorporate the DNA chain is selected appropriate to its viability and expression in a host organism or cell line, and the manner of insertion of the DNA chain depends upon factors particular to the host.
For example, if the DNA chain is to be inserted into a vector for :insertion into a prokaryotic cell, such as E.
Coli, the D1~1A chain will be inserted 3' of a promoter sequence, a Shine-Delgarno sequence (or ribosome binding site) that :is within a 5' non-translated portion and an ATG start codon. The ATG start codon is appropriately spaced from the S',hine-Delgarno sequence, and the encoding sequence is placed in correct reading frame with the ATG
start codon. The cloning vector also provides a 3' non-translai:ed region and a translation termination site. For :insertion into a eukaryotic cell, such as a yeast cell or a cell line obtained from a higher animal, the FGF fragment-.encoding oligonucleotide sequence is appropriate:Ly spaced from a capping site and in correct reading frame witlh an ATG start signal. The cloning vector also provides a 3' non-translated region and a translation termination site.

Prokaryotic transformation vectors, such as pBR322, pMB~~, Col E1, pCRl, RP4 and lambda-phage, are available for inserting a DNA chain of the length necessary to encode the FGF fragments of interest with substantial assurance of at least some expression of the encoded polypeptide. Typically, such vectors are constructed or modified to have a unique restriction sites) approprialtely positioned relative to a promoter, such as the lac promoter. The DNA chain may be inserted with appropriate :Linkers into such a restriction site, with substantial assurance of production of FGF in a prokaryotic cell .line transformed with the recombinant vector. To assurca the proper reading frame, linkers of various lengths may be provided at the ends of the FGF
peptide-encoding :sequence. Alternatively, cassettes, which include sequences, such as the 5' region of the lac Z gene (inc7_uding the operator, promoter, transcription start site, Shine Delgarno sequence and translation initiation signal;l, the regulatory region from the tryptophane gene (trp operator, promoter, ribosome binding sites and i~ranslation initiator) , and a fusion gene containing these two promoters, called the trp-lac or commonly called the Tac promoter, are available into which a synthetic DNA chain may be conveniently inserted before the c:asseti~e is inserted into a cloning vector of choice.
Similarl;t, eukaryotic transformation vectors, such as the cloned bovine papilloma virus genome, the cloned genomes of the murine retroviruses, and eukaryotic cassettes, :such as the pSV-2 gpt system (described by Mulligan and Berg" Nature 277, 108-114, 1979), the Okayama-Berc~ cloning system (Mol. Cell Biol. 2, 161-170, 1982) and tree exp~.~ession cloning vector recently described b~~ Generics Institute (Science 228, 810-815, 1985), are available which provide substantial assurance of at least some expression of the FGF peptide in the transformed eukar;totic cell line.

Another 'way to produce bFGF fragments of desired length is to produce the polypeptide initially as a segment of a gene-encoded fusion polypeptide. In such case, the D1JA chain is constructed so that the expressed polypeptide has enzymatic processing sites flanking the bFGF fragment seqvuence. A bFGF-fragment-encoding DNA
chain may bca inserted, for example, into the beta-galactosidas~e gene for insertion into E. Coli, in which case, the e:Kpressed fusion polypeptide is subsequentl~~ cleaved with appropriate proteolytic enzymes to release i~he bFGF fragment from beta-galactosidase peptide sequences.
An advantage of inserting the bFGF-fragment-encoding sequence so that it is expressed as a cleavable segment of a fusion polypeptide, e.g., as the bFGF-fragment sequence fused within the beta-galactosidasce peptide sequence, is that the endogenous polypeptide into which the bFGF fragment sequence is inserted is generally rendered non-functional, thereby facilitating selection for vectors encoding the fusion peptide.
The peptides can be synthesized by suitable chain elongation or coupling-type methods, such as by exclusively solid--phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings. The techniques of exclusively solid--phase synthesis are set forth in the textbook "Solid-Phase Peptide Synthesis", Stewart &
Young, Pierce Chemical Co., Rockford, Illinois, 1984, and are exemplified by the disclosure of U.S. Patent No.
4,105,603, issued August 8, 1978. The fragment condensation method of synthesis is exemplified in U.S.
Patent No. =~,972,F359 (August 3, 1976). Other available syntheses are exemplified by U.S. Patent No. 3,842,067 (October 15, 1974;1 and U.S. Patent No. 3,862,925 (January 28, 1975;1.

Common to coupling-type syntheses is the protection of the labile side-chain groups of the various amino acid moieticas with suitable protecting groups which will prevent: a chemical reaction from occurring at that site until t:he group is ultimately removed. Usually also common is tree proi~ection of an alpha-amino group on an amino acid or a fragment while that entity reacts at the carboxyl group, followed by the selective removal of the alpha-amino protecaing group to allow subsequent reaction to take place at t:hat location. Accordingly, it is common that, as a step in the synthesis, an intermediate compound is produced which includes each of the amino acid residues located in its desired sequence in the peptide chain with side-chain protecting groups linked to the appropriate rE~sidues.
Such an intermediate for the first family may have the formula:
X1-Tyr(X2)-C'ys(X4)-Lys(X~)-Asn(X8)-Gly-Gly-Phe-Phe-Leu-Arg(X6)-Ile-His(X~~)-Pro-Asp(X3)-Gly-Arg(X6)-Val-Asp(X3)-R42-Val-Arg(X6)-G7.u(X3)-Lys(X~)-R4~(X5)-Asp(X3)-Pro-His(X9)-Ile-~Lys(X'~)-Leu-Gln(X8)-Leu-Gln(X$)-Ala-Glu(X3)-Glu(X3)-Arg(X6)-Gly-Val-Val-Ser(X5)-Ile-Lys(X~)-Gly-Val-X10.
Such an intermediate for the second family may have the formula:
X1-Phe-Phe-F~he-Glu(X3)-Arg(X6)-Leu-Glu(X3)-Ser(X5)-Asn(X8)-Asn(X8)-Tyr(X2)-Asn(X8)-Thr(X5)-Tyr(X2)-Arg(X6)-Ser(X5)-Arg(X6)-Lys(X~)-Tyr(X2)-Ser(X5)-Ser(X5)-Trp-Tyr(X'~)-Val-Ala-Leu-Lys(X~)-Arg(X6)-X10.
In. theses formulae: X1 is either hydrogen or an alpha-amino protecting group. The alpha-amino protecting groups contemplated by X1 are those known to be useful in the art of step-wise synthesis of polypeptides. Among the classes of alpha-amino protecting groups covered by X1 are (1) acyl-type protecting groups, such as formyl, trifluoroacetyl, phthalyl, toluene~;ulfonyl(Tos), benzensulfonyl, nitrophenylsulfenyl, tritylsulfenyl, o-nitrophenoxyacetyl, chloroacetyl, acetyl, and -chlorobutyryl; (2) aromatic urethan-type protecting groups, such as benzyloxycarbonyl(Z) and substituted Z, such as p-c:hlorobenzyloxycarbonyl, p-nitrobenz:yloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybe:nzyloxycarbonyl; (3) aliphatic urethan protecting groups, such as t-butyloxycarbonyl (BOC), diisopropylmethyloxycarbonyl, isopropyloxycarbonyl, ethox carbon 1 all lox carbon 1;
Y Y . Y y y (4) cycloalkyl urethan-type protecting groups, such as cyclopentyloxycar:bonyl, adamantyloxycarbonyl,and cyclohexylo:~cycarb~onyl; (5) thiourethan-type protecting groups, such as phenylthiocarbonyl; (6) alkyl-type protecting groups, such as triphenylmethyl (trityl), benzyl;(7) i:rialkylsilane groups, such as trimethylsi:Lane. The preferred alpha-amino protecting group i s BOC~ .
X2 is a protecting group for the phenolic h drox 1 rou of T r selected from the Y Y g P Y group consisting of tetrahydropyranyl, tert-butyl, trityl, Bzl, CBZ, 4Br-CBZ and 2,6-d:ichlorobenzyl. The preferred protecting group is 2,E>-dich:lorobenzyl. X2 can be hydrogen which means that there :is no protecting group on the hydroxyl group.
X3 is hydrogen or an ester-forming protecting group for the carboxyl group of Asp or Glu and is selected from the group consisting of Bzl, cyclohexyl, cycloheptal, 2,6-dichlorobenzyl, methyl and ethyl.
X4 is a ~rotectin g group for Cys selected from the group consisting of p-methoxy-benzyl(MeOBzl), p-methylbenz;yl, ac:etamidomethyl, trityl and Bzl. The most preferred protecting group is p-methoxybenzyl. X6 can also be hydrogen, meaning that there is no protecting group on the: sulfhydryl.
X5 is a protecting group for the hydroxyl group of Thr and ~ter and is selected from the group consisting of acetyl, benzoyl, tert-butyl, trityl, tetrahydrop5~ranyl,, Bzl, 2,6-dichlorobenzyl and CBZ. The preferred protecting group is Bzl. X5 can be hydrogen, which means there is no protecting group on the hydroxyl group.
X6 is a protecting group for the guanido group of Arg selecaed from the group consisting of nitro, Tos, CBZ, adamant:yloxyc:arbonyl, and BOC, or is hydrogen.
X~ is hydrogen or a protecting group for the side-chain amino substituent of Lys. Illustrative of suitable side-chain amino protecting groups are 2-chloroben2;yloxyc:arbonyl(2-C1-Z), Tos, CBZ, t-amyloxycarbonyl and BOC.
The selecaion of a side-chain amino protecting group is not: critical except that it must be one which is not removed during deprotection of the alpha-amino groups during the synthesis. Hence, the alpha-amino protecting group and tree sidEa-chain amino protecting group cannot be the same.
X$ is a protecting group for the side-chain amido group of Gln and/or Asn and is preferably xanthyl (Xan). Optionally X8 can be hydrogen.
X9 is a protecting group for the imidazole nitrogen of His, :such as Tos or dinitrophenyl, or may be hydrogen.
X1~~ is selected from the class consisting of OH, OCH3, esters, amides, hydrazides, -O-CH2-resin support and -NH-resin support, with the groups other than OH and amidea being broadly considered as protecting groups.
In the formula for the intermediate, at least one of X1, x:2, X3, X4, X5, X6, X~, X8, X9 and X10 is a protecting' group.
In selecting a particular side-chain protecting group to be used in the synthesis of the peptides, the following rules are followed: (a) the protecting group should be stable t:o the reagent and under the reaction conditions :selected for removing the alpha-amino protecting croup <~t each step of the synthesis, (b) the protecting croup :should retain its protecting properties and not be :split off under coupling conditions, and (c) the side-chain protecting group should be removable, upon the completion of the synthesis containing the desired amino acid sequence, under reaction conditions that will not alter tree pepi=ide chain.
The pept:Ldes are preferably prepared using solid phase synthesis, such as that described by Merrifield, J.
Am. Chem. Soc., 8~i, p 2149 (1963), although other equivalent chemical syntheses known in the art can also be used as previously mentioned. Solid-phase synthesis is commenced from the C-terminal end of the peptide by coupling a ~~rotect:ed alpha-amino acid to a suitable resin. Such a starting material can be prepared by attaching alpha-annino-protected Val by an ester linkage to a chloromethylated resin or a hydroxymethyl resin, or by an amide bond t:o a BHA resin or MBHA resin. The preparation of the: hydroxymethyl resin is described by Bodansky et al., C:hem. Ind. (London) 38, 1597-98 (1966).
Chloromethyl.ated resins are commercially available from Bio Rad Laboratories, Richmond, California and from Lab.
Systems, Inc. Then preparation of such a resin is described by Stewart et al., "Solid Phase Peptide Synthesis" (Freeman & Co., San Francisco 1969), Chapter 1, pp 1-6. BHA and MBHA resin supports are commercially available and are generally used only when the desired polypeptide being synthesized has an alpha-carboxamide at the C-terminal.
For examx>le, a peptide of the first family can be prepared by coupling Val, protected by BOC, to a chloromethylated resin according to the procedure of Monahan and Gilon, Biopolymer 12, pp 2513-19, 1973 when, for example, it is. desired to synthesize such a peptide with free carboxy terminus. Following the coupling of BOC-Val, the alpha-amino protecting group is removed, as by using tr:ifluoroacetic acid(TFA) in methylene chloride, TFA alone o~~ HC1 in dioxane. The deprotection is carried out at a temperature between about 0°C. and room temperature.. Other standard cleaving reagents and conditions ~Eor removal of specific alpha-amino protecting groups may be used as described in Schroder & Lubke, "The Peptides", .L pp 72-75 (Academic Press 1965).
Afi~er removal of the alpha-amino protecting group of va:l, the remaining alpha-amino- and side-chain-protected amino acids are coupled stepwise in the desired order to obtain an intermediate compound as defined here~inbefore. As an alternative to adding each amino acid :separately in the synthesis, some of them may be coupled i.o one another prior to their addition to the solid phase react~~r. The selection of an appropriate coupling reagent :is within the skill of the art;
particularly suitable as a coupling reagent is N,N'-dicyclohexyl carbodiimide (DCCI).
Activating reagents used in solid phase synthesis oi' the peptides are well known in the peptide synthesis ant. Ex<~mples of suitable activating reagents are: (1) carbodiimides, such as N,N'-diisopropyl carbodiimide~, N-N'-dicyclohexylcarbodiimide(DCCI); (2) cyanamides :such a:a N,N'-dibenzylcyanamide; (3) keteimines; (4) i:~oxazolium salts, such as N-ethyl-5-phenyl :isoxazolium-3'-sulfonate; (5) monocyclic nitrogen-containing heterocyclic amides of aromatic character containing one through four nitrogens in the ring, such as imidazolides, pyrazolides, and 1,2,4-triazolides. Specific heterocyclic amides that are useful include N,N'-carbonyl diimidazole, N,N'-carbon~~l-di-:1,2,4-triazole; (6) alkoxylated acetylene, ~~uch as ethoxyacetylene; (7) reagents which form a mixed anhydride with the carboxyl moiety of the amino acid, such as ethylchloroformate and isobutylchloroforrnate and (8) reagents which form an active ester- with the carboxyl moiety of the amino acid, such as nitrogen-containing heterocyclic compounds having a hydroxy group on one ring nitrogen, e.g.
N-hydroxyphthalimide, N-hydroxysuccinimide and 1-hydroxybenzotriazole(HOBT). Other activating reagents and their use in peptide coupling are described by Schroder & Lubke supra, in Chapter III and by Kapoor, J.
Phar. Sci., 59, pp 1-27 (1970).
Ea~~h protected amino acid or amino acid sequence is introduced into the solid phase reactor in about a twofold or more excess, and the coupling may be carried out in a medium of dimethylformamide(DMF):CH2C12 (1:1) or in DMF o:r CH2C12 alone. In cases where incomplete coupling occurs, the coupling procedure is repeated before remo~~al of the alpha-amino protecting group prior to the cou :Lin of the next amino acid. If p g performed manually, the success of the coupling reaction at each stage of the synthesis is monitored by the ninhydrin reaction, a:~ described by E. Kaiser et al., Anal.
Biochem. 34, 595 (1970).
Afl;.er the desired amino acid sequence has been completed, l~he intermediate peptide is removed from the resin suppo~:~t by 'treatment with a reagent, such as liquid hydrogen fluoride, which not only cleaves the peptide from the resin but also cleaves all remaining side-chain protecting groups X2, X3, X4, X5, X6, X7, X8 and X9 and the alpha-amino protecting group X1 to obtain the peptide.
As an alternative route, the intermediate peptide may be separated from the resin support by alcoholysis after which the recovered C-terminal alkyl ester is convertec9 to the acid by hydrolysis. Any side-chain protecting groups may then be cleaved as previously described or by other known procedures, such as catalytic: reduction (e. g. Pd on BaSO4). When using h dro en fluoride for cleavin y g g, anisole and methylethyl sulfide are included in the reaction vessel for scavenging.

~ ~~4976 ThEa following Examples set forth preferred methods for synthesizing FGF antagonists by the solid-phase technique. It will of course be appreciated that the synthesis of a correspondingly shorter peptide fragment is effected in the same manner by merely eliminating the requisite number of amino acids at either end of the chain.
EXAMPLE I
The synthesis of bFGF(24-68)-amide having the formula: H--Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-G:Ly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro--His-I:Le-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val--Val-Ser-Ile-Lys-Gly-Val-NH2 is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer and an MBHA resin. Coupling of BOC-Val to the resin is performed by the general procedure set forth in U.S.
Patent No. ~t,292,:313, and it results in the substitution of about 0.2-0.6 mmol Val per gram of resin depending on the substitution of the MHBA resin used.
After deprotection and neutralization, the peptide chap.n is built step-by-step on the resin.
Deprotection, neui~ralization and addition of each amino acid is peri:ormed in general accordance with the procedure sE~t fori~h in detail in Guillemin et al. U.S.
patent No. ;1,904,!594. The couplings are specifically carried out as sei~ out in the following schedule.

134097fi SCHEDULE
MIX TIMES

STEP :REAGENTS AND OPERATIONS MIN.

1 CH2C12 wash (2 times) 0.5 . 2 45% t:rifluoroacetic acid (TFA) 0.5 - + 5~ 1,2-ethanedithiol CH2C12 (1 time) in 3 45% t:rifluoroacetic acid (TFA) 20.0 + 5~ 1,2-ethanedithiol CH2C12 (1 time) in 4 CH2C1,2 wash (3 times) 0.5 5 CH30H wash (2 times) 0.5 6 10s t:riethylamine (Et3N) in CH2C12 0.5 neutralization (2 times) 7 CH30H wash (2 times) 0.5 8 10~ t:riethylamine (Et3N) in CH2C12 0.5 neutralization (2 times) 9 CH30H wash (2 times) 0.5 10 CH2C1.~ wash (2 times) 0.5 11 *Boc-amino a~~id (1 mmole/gresin) plus Equivalent amount 120 of dicyc:Lohexylcarbodiimide (DCC) in CH2C1;~

12 CH2C1;~ wash ( 1 time) 0. 5 13 50% dimethy:lformamide CH2C12 0.5 in wash (2 times) 14 10$ triethy:lamine (Et3N) in CH2C12 0.5 wash (1 time) 15 CH30H wash (2 times) 0.5 16 CH2C1~~ wash (2 times) 0.5 17 25$ acetic anhydride in 20.0 (2 ml/g resin) 18 CH2C1~, wash (2 times) 0.5 19 CH30H wash (2 times) 0.5 i~vr Lnc: coupling of Asn ana c~ln, ari 1. 136 molar excess of 1--hydro:~cybenzotriazole (HOBt) is included in this step.

~~40976 Brp.efly, for the coupling reaction, one mmol. of BOC-protected amino acid in methylene chloride is used per gram of resin,, plus one equivalent of 0.5 molar DCCI
in methylenea chlo~__~ide or 30% DMF in methylene chloride, for two hours. When Arg is being coupled, a mixture of 10% DMF and methylene chloride is used. Bzl is used as the hydroxyl. side--chain protecting group for Ser and Thr. 2-chloro-benzyloxycarbonyl (2C1-Z) is used as the protecting c_~roup for the Lys side chain. Tos is used to protect the guanidino group of Arg, and the Glu or Asp carboxyl group is protected as the Bzl ester. The phenolic hydroxyl group of Tyr is protected with 2,6-dichlorobenzy:L. Asn and Gln are left unprotected.
At the end of the synthesis, the following composition is obtained. X T r .~ -C s X -L s X -Asn-G1 -G1 -Phe-Phe ~ ( ~-) Y (' 2) Y ( 4) Y ( ~) Y Y
Leu-Arg(X6)-~Ile-H_Ls(X9)-Pro-Asp(X3)-Gly-Arg(X6)-Val-Asp(X3)-Gly-~Val-Arg(X6)-Glu(X3)-Lys(X~)-Ser(X5)-Asp(X3)-Pro-His(X9)-~Ile-L~rs(X~)-Leu-Gln-Leu-Gln-Ala-Glu(X3)-Glu(X3)-Arg(X6)-Gly-~Val-Val-Ser(X5)-Ile-Lys(X~)-Gly-Val-X10 wherein Xl i.s BOC,, X2 is 2,6-dichlorobenzyl, X3 is benyzl ester, X4 is MeOBzl, X5 is Bzl, X6 is Tos, X~ is 2C1-Z, X9 is Tos and X10 is -NH-MBHA resin support.
After the' final Tyr residue has been coupled to the resin, t:he BOC: group is removed with 45% TFA in CH2C12. In order to cleave and deprotect the remaining protected peptide-resin, it is treated with 1.5 ml. anis;ole, 0.25 ml. methylethylsulfide and 10 ml.
hydrogen fluoride (HF) per gram of peptide-resin, at -20°C. for one-ha7.f hour and at 0°C. for one-half hour.
After elimination of the HF under high vacuum, the resin-peptidle remainder is washed alternately with dry diethyl ether and chloroform, and the peptide is then extracted with dec_Lassed 2N aqueous acetic acid.
Lyophilizati.on of the acetic acid extract provides a white fluffy material.

~340g76 The cleaved and deprotected peptide is then dissolved i:n 30~ acetic acid and subjected to Sephadex ~

fine gel filtration.

The peptide is then further purified by CM-32~

:; carboxymeth;yl cellulose (Whatman) cation-exchange chromatogra;phy(1.8x 18 cm., V - 50 ml.) using a bed concave gradient generated by dropping 1 L. of 0.4 M

NH40Ac, pH ~6.5 into a mixing flask containing 400 ml.

0.01 M. NH40Ac, pH 4.5. Final purification is carried out using preparative HPLC on a Vydec C4~ column using a 0.1~ TFA and acetonitrile solvent system. Purification details are generally set forth in Ling et al. Biochem.

Biophys. Re;s. Com:mun. 95, 945 (1980). The chromatogra~~hic fractions are carefully monitored by TLC, and only the fractions showing substantial purity are pooled.

The synthesis is repeated using a chloromethy:Lated resin to produce the same peptide having a free acid C-terminus, generally following the procedure described in BlOp~olvmers, 12, 2513-19 (1973) to link Val to the chlooometh;ylated resin.
EXAMPLE II
To determine the effectiveness of the bFGF
fragment peptide to inhibit the growth endothelial cells, the peptide is to;sted under conditions to measure its ability to modulate both basal cell growth and bFGF-simulai:ed cell proliferation. A bioassay was employed of the t:~pe set forth in detail in Gospodarowicz et al., J. c=ell B.iol., 122, 323-333 (1985), using BARE
cells.
For each test, an initial cell density of between about 0.3~-0.5 x 104 cells per well was established in 24~-miniwell plates. After 6-8 hours, the cells in each well were treated with a challenge dose of bFGF in the absence, or presence to a varying concentration, of a synthetic FGF antagonist. The precise tre~itment was repeated 48 hours later. On the d n a cJ W Yr~ ca r W

fifth day, the cells were digested with trypsin, and the total number of cells in each well was determined using a Coulter particle counter. Testing of the peptide bFGF(24-68)-NH2 shows full antagonist activity to both basal cell growth and to bFGF-stimulated cell growth, with cell population being reduced by about 84% and about 92%, respectively, at a concentration of about 100 ~,g/ml. Like results are obtained from the testing of bFGF(24-68)-OH, with both peptides exhibiting an ID50 of about 5 micromoles.

Testing is then carried out to determine the effect of the fragments of bFGF on the binding of 1125-bFGF to BHK cells, in order to determine the interaction with the receptors of FGF target cells, and is also carried out to determine the binding of the fragments to [3H]-heparin. bFGF(24-68)-NH2, at a concentration of 100 ~.g/ml., reduces the amount of radioactive bFGF :bound to the cells by about 54% and shows strong affinity to bind heparin.

EXAMPLE III

ThEa syntlhesis of [ Tyr50 ] -bFGF ( 3 0-50 ) -NH2 having the jEormula: H-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg--Val-A;sp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-Tyr-NH2 is conducted in a stepwise manner using a Beckman 990~~synthesizer and an MBHA resin in the manner described in Example I. The peptide is judged to be substantial7~~y pure using TLC and HPLC. Testing in the manner set i:orth :in Example II shows that the peptide has full antagonist activity to both basal and bFGF-stimulated endothelial cell growth, reducing cell population by about 19% and about 16%, respectively.

EXAMPLE IV

The' synthesis of bFGF(30-49)-NH2 having the formula: H--Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-~Arg-G:Lu-Lys-Ser-Asp-Pro-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example I except 134(Ig76 that cyclohe:xyl instead of Bzl is used to protect Asp and Glu. The peptide is judged to be substantially pure using TLC and HPLC. Testing in the manner set forth in Example II shows that the peptide has full antagonist activity to both basal and FGF-stimulated endothelial cell growth.
EXAMPLE IV A
The: synthesis of bFGF(25-37)-NH2 having the formula: H-~Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-NH2 is conducted in a stepwise manner using a Beckman 990 synthEasizer and an MBHA resin in the manner described in Example I. The peptide is judged to be substantially pure' using TLC and HPLC. Testing in the manner set forth in Example II shows that the peptide has some antagonist ac;tivity to basal endothelial cell growth and has a fairly ~>trong binding affinity for heparin and a fair affinity for BHK cells.
EXAMPLE V
The: synthesis of [Tyr25]-bFGF(25-68)-NH2 having the formula: H-Tyr-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-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-NH2 is conducted in. a stE:pwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example I. The peptide is judged to be substantially pure using Z'LC and HPLC. Testing in the manner set forth in Example II shows that the peptide has full antagonist activity to both basal and bFGF-stimulated endothelial cell growth, reducing cell population by about 86% and about 95%, respectively, and that it has a very strong binding affinity for BHK cells and heparin.
EXAMPLE VI
The synthesis of [Tyr30,50~-bFGF(30-50)-OH
having the formula: H-Tyr-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gl.y-Val-Arg-Glu-Lys-Ser-Asp-Pro-Tyr-OH is conducted in a stepwise manner using a Beckman 990 synthesizer and a chloromethylated resin in the manner described hereinbefore. The peptide is judged to be substantially pure using TLC and HPLC. Testing in the manner set forth in Example II shows that the peptide has full antagonist activity to both basal and bFGF-stimulated endothelial cell growth.
' EXAMPLE VII
The synthesis of bFGF(32-53)-NH2 having the i formula: H-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-His-Ile-Lys-Leu-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example I. The peptide is judged to be substantially pure using 'rLC and HPLC. Testing in the manner set forth in Example II shows that the peptide has weak antagonist activity to both basal and bFGF-stimulated endothelial cell growth.
EXAMPLE VIII
The synthesis of bFGF(32-39)-NH2 having the formula: H-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example I. The peptide is judged to be substantially pure using TLC and HPLC. Testing in the manner set forth in Example :1:I shows that the peptide has full antagonist activity to both :basal and bFGF-stimulated endothelial cell growth, reducing cell population by about 37~ and about 11~, :respectively.
EXAMPLE IX
Th~~ synt:hesis of bFGF(24-63)-NH2 having the formula: H-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-Ala-Glu-Glu-Arg-Gly-Val~-Val-N~H2 is conducted in a stepwise manner using a BecJcman 9'90 synthesizer and an MBHA resin in the manner described in Example I. The peptide is judged to be substantially pure using TLC and HPLC. Testing in the 134p976 manner set forth in Example II shows that the peptide has full antagonist activity to both basal and bFGF-stimul;eted endothelial cell growth.
EXAMPLE X
Th~~ synthesis of [Ala4~]-bFGF(24-63)-NH2 having the formula: H-Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ala-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu~-Arg-Gly-Val-Val-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin :in the manner described in Example I. The peptide is :judged to be substantially pure using TLC and HPLC. Testing in the manner set forth in Example II
shows that i~he peptide has full antagonist activity to both basal and bFGF-stimulated endothelial cell growth.
EXAMPLE XI
The. synthesis of [Sar42]-bFGF(36-68)-NH2 having the :Pormul.a: H-Pro-Asp-Gly-Arg-Val-Asp-Sar-Val-Arg-Glu--Lys-S~er-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu--Glu-A:rg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example I. The pa_ptide is judged to be substantially pure using ':PLC and HPLC. Testing in the manner set forth in Example :CI shows that the peptide has full antagonist activity to both basal and bFGF-stimulated endothelial cell growth..
EXAMPLE XII
Thsa synt)zesis of [A1a42 ] -bFGF ( 3 6-68 ) -NH2 having the i'ormula: H-Pro-Asp-Gly-Arg-Val-Asp-Ala-Val-Arg-Glu--Lys-Ser-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu--Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example I. The peptide is judged to be substantially pure using TLC and HPLC. Testing in the manner set forth in Example 7:I shows that the peptide has full antagonist 134097fi activity to both basal and bFGF-stimulated endothelial cell growth.
EXAMPLE XIII
The synthesis of bFGF(35-50)-NH2 having the formula: H~-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-His-NH2 is conducted in a stepwise manner usin<~ a Beckman 990 synthesizer and an MBHA resin in the manncar described in Example I. The peptide is judged to be. substantially pure using TLC and HPLC.
l0 Testing in i:.he manner set forth in Example II shows that the peptide has full antagonist activity to both basal and bFGF-st:imulat~ed endothelial cell growth.
EXAMPLE XIV
Thca syntlhesis of [A1a42, Thr47]-bFGF(35-50)-NH2 having the :Eormul;a: H-His-Pro-Asp-Gly-Arg-Val-Asp-Ala-Val-Arg-Glu-Lys-Tlhr-Asp-Pro-His-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin :in the manner described in Example I. The peptide is =judged to be substantially pure using TLC and HPLC. Testing in the manner set forth in Example II
shows that i~he peptide has full antagonist activity to both basal and bFGF-stimulated endothelial cell growth.
EXAMPLE XV
ThE~ synthesis of bFGF(36-39)-NH2 having the formula: H--Pro-Aap-Gly-Arg-NH2 is conducted in a stepwise manner u:~ing a Beckman 990 synthesizer and an MBHA resin :Ln the manner described in Example I. The tetrapeptidE~ is judged to be substantially pure using TLC
and HPLC. ~~esting in the manner set forth in Example II
shows that l:he peptide has full antagonist activity to both basal and bFGF-stimulated endothelial cell growth, reducing cel.1 population by about 37% and about 54%, respectivel~r. It has biological potency less than that of bFGF(24-E>8), exhibiting an ID50 at between about 30 and 50 micromoles.
EXAMPLE XVI
The: synthesis of bFGF(93-120)-NH2 having the formula: H--Phe-Plae-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-NH2 is conducted in a stepwise manner using a Beckman 9!30 synthesizer and an MBHA resin in the manner described in Example I. The peptide is judged to be substantially pure using TLC and HPLC. Testing in the manner set :forth in Example II shows that the peptide has full antagonist a~~tivity to both basal and bFGF-stimul<~ted endothelial cell growth, that it binds to heparin, an<i that it inhibits the binding of bFGF to BHK
cells.
EXAMPLE XVII
Thca syntlhesis of bFGF(106-118)-NH2 having the formula: H-Tyr-A:rg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example I. The peptide is judged to be substantial:Ly pure using TLC and HPLC. Testing in the manner set Forth .in Example II shows that the peptide has partial antagonist activity in mitogenic assays and inhibits binding of bFGF to its receptor in BHK cells.
EXAMPLE XVIII
Then synthesis of bFGF(103-146)-NH2 having the formula: H--Tyr-Aan-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val--Ala-Leu-Lys-Arg-Thr-Gly-Gln-Tyr-Lys-Leu-Gly-Pro-Lys-Thr--Gly-P:ro-Gly-Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala--Lys-Ser-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the mannear described in Example I. The peptide is judged to bea substantially pure using TLC and HPLC.
Testing in t:he manner set forth in Example II shows that the peptide very :strongly inhibits FGF binding to BHK
cells and to heparin.
EXAMPLE XVIII A
Ths~ synthesis of bFGF(97-120)-NH2 having the formula: H--Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys--Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and a:n MBHA resin in the manner described in Example I. The peptide is judged to be substantially pure using ~~LC and HPLC. Testing is carried out using a culture of serum-;starved 3T3 cells which are incubated for 24 hours with the bFGF peptide fragment and a challenge dose of bFGF and then incubated for 5 hours with radioacaive [3H]-thymidine to determine whether the fragmeni~ will inhibit the incorporation of [3H]-DNA
in the cell line which will be indicative of its inhibiting cell growth. It is shown that the peptide exhibits very good inhibition of bFGF-induced mitosis, and further testing shows that it very strongly inhibits bFGF binding to BI~K cells and that it binds itself to heparin.
EXAMPLE XVIII B
Then synthesis of bFGF(100-120)-NH2 having the formula: H--Ser-A:an-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser--Trp-Tyr-Val-Ala-Leu-Lys-Arg-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example I. The peptide is judged to be substantially pure using 'TLC and HPLC. Testing is carried out using a culture of :serum-:starved 3T3 cells which are incubated for 24 hour: with the bFGF peptide fragment and a challenge dose of bFGF and then incubated for 5 hours with radioacaive [3H]-thymidine to determine whether the fragment: will inhibit the incorporation of [3H]-DNA
in the cell line which will be indicative of its inhibiting cell growth. It is shown that the peptide exhibits very good inhibition of bFGF-induced mitosis, and further testing shows that it very strongly inhibits bFGF binding to BHK cells and that it binds itself to heparin.
EXAMPLE XVIII C
The: synthesis of bFGF ( 103-120) -NH2 having the formula: H-Tyr-A:an-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leau-Lys-Arg-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example I. The peptide is judged to be substantially pure using TLC and HPLC. Testing is carried out using a culture of serum-starved 3T3 cells which are incubated for 24 hours with the bF~~F peptide fragment and a challenge dose of bFGF and then incubated for 5 hours with radioactive [3H]-thymidine to determine whether the fragment will inhibit the incorporation of [3H]-DNA in the cell line which will lae indicative of its inhibiting cell growth.
It is shown that the peptide exhibits very good inhibition of bFGF-induced cell mitosis; further testing shows that :it very strongly inhibits binding of bFGF to BHK cells and that it binds to heparin.
EXAMPLE XVIII D
The: synt:hesis of bFGF(106-120)-NH2 having the formula: H~-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-N:H2 is conducted in a stepwise manner using a Beckman 9'90 synthesizer and an MBHA resin in the manner descoibed in Example I. The peptide is judged to be substantially pure using TLC and HPLC. Testing is carried out using a culture of serum-starved 3T3 cells which are incubated for 24 hours with the bFGF peptide fragment and a challenge dose of bFGF and then incubated for 5 hours with :radioactive [3H]-thymidine to determine whether the fragment will inhibit the incorporation of [3H]-DNA in the cell line which will be indicative of :its inhibiting cell growth. It is shown that the peptide ~axhibits very good inhibition of bFGF-induced cell mitosis; further testing shows that it very strong7Ly inhabits binding of bFGF to BHK cells and that it binds to heparin.
EXAMPLE XVIII E
The synthesis of [Met114]-bFGF(106-120)-NH2 having the formula: H-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Met-Tyr-Val--Ala-L<su-Lys-Arg-NH2 is conducted in a stepwise manner u:aing a Beckman 990 synthesizer and an 134097fi MBHA resin in the manner described in Example I. The peptide is :judged to be substantially pure using TLC and HPLC. Test_Lng is carried out as described in Example XVIIID. ThE~ peptide exhibits very good inhibition of bFGF-induced cell mitosis and very strongly inhibits binding of bFGF to BHK cells.
EXAMPLE XVIII F
ThE: synthesis of [Phe115]-bFGF(106-120)-NH2 having the i'ormul<~: H-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Phe-Val-Ala-Lcau-Lys-Arg-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin :Ln the manner described in Example I. The peptide is _judged to be substantially pure using TLC and HPLC. Test:Lng is carried out as described in Example XVIIID. The. peptide exhibits very good inhibition of bFGF-induced cell mitosis and very strongly inhibits binding of bFGF to BHK cells.
EXAMPLE XVIII G
ThE~ synthesis of bFGF(106-115)-NH2 having the formula: H--Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example I. The p<eptide is judged to be substantially pure using ~'LC and HPLC. Testing is carried out using a culture of :serum-:starved 3T3 cells which are incubated for 24 hour:a with the bFGF peptide fragment and a challenge dose of bFGF and then incubated for 5 hours with radioacaive [3H]-thymidine to determine whether the fragment: will inhibit the incorporation of [3H]-DNA
in the cell line which will be indicative of its inhibiting cell growth. It is shown that the peptide exhibits very good inhibition of bFGF-induced cell mitosis; further nesting shows that it very strongly inhibits binding of bFGF to BHK cells and that it binds to heparin.

EXAMPLE XVIII H
Th~~ syntheses of the following compounds are conducted in a stepwise manner using a Beckman 990 synthesizer and a:n MBHA resin in the manner described in Example I:
bFGF(1~D6-125)-NH2, " ( 1~D6-130) - " , " (1~D6-135)- " , (1~D6-140)- " , (106-146)- " .
These peptides aria each judged to be substantially pure using TLC and HPLC. Testing in the manner set forth in Example II :shows 'that all the peptides exhibit strong inhibition of bFGIF binding to the receptor and inhibition of bFGF induced mitosis.
EXAMPLE XVIII J
ThEa syntheses of the following compounds are conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example I:
[ I)-Tyrl ~) 6 ] -bFGF ( 10 6-12 0 ) -NH2 , [ I)-Arg l l) 7 ] - n n [ I)-Serll)8 ] _ n n [ I)-Arg 1 c) 9 ] _ .. n 2 5 [ I)-Lys 1:L 0 ] _ [ I)-Tyr l:l l ] _ .. n [ I)-Serlv2 ] - " " and [ D-Serl:l3 ] _ n n .
These peptides arEa each judged to be substantially pure using TLC and HPLC. Testing in the manner set forth in Example II shows .inhibition of bFGF binding to the FGF
receptors of BHK cells nearly as strong as does bFGF(106-120)-NH2. Moreover, D-Ser113 showed inhibition t:o bFGF-induced mitosis substantially as strong as bP'GF ( 10(i-120) -NH2 .

-33- 934097fi EXAMPLE XVIII K
The synthesis of [A1a113~-bFGF(103-146)-NH2 having the formula: H-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ala~-Trp-Tyr-Val-Ala-Leu-Lys-Arg-Thr-Gly-Gln-Tyr-Lys-Leu-Gly~-Pro-Lys-Thr-Gly-Pro-Gly-Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser-NH2 is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin .in the manner described in Example I. The peptide is :judged to be substantially pure using TLC and l0 HPLC. Testing in the manner set forth in Example II
shows that 'the peptide very strongly inhibits FGF binding to BHK cello.
EXAMPLE XIX
Using conventional methods, described in CSH, supra., a s!~nthetic bFGF-fragment gene is constructed having the :Following formula:
5' AATTCATG~t'ATTGT,~1AAAACGGGGGGTTC
3' GTACi~TAACA'rTTTTGCCCCCCAAG
'.~TCCTA~CGAATCCACCCAGATGGGCGAGTAGATGGGGTACGAGAA
i~AGGATiJCTTAGGTGGGTCTACCCGCTCATCTACCCCATGCTCTT
i~AATCCGATCCACACATCAAACTACAACTACAAGCCGAAGAACGA
~~TTAGGCTAGGTGTGTAGTTTGATGTTGATGTTCGGCTTCTTGCT
(~GGGTAGTATCCATCAAAGGGGTATAAG 3' (:CCCATCATAGGTAGTTTCCCCATATTCAGCT 5' :~yntheais of such a bFGF-fragment-encoding DNA
chain is acc:ompliahed by synthesizing oligonucleotides on an Applied Biosystems automatic synthesizer with overlapping complementary sequences.
~Che ovc=rlapping oligonucleotides are fused to form a doub7Le-stranded DNA chain, gaps being filled in with DNA po7Lymerase and with T4 ligase. Immediately 5' of the bFGF--fragment-encoding sequence in the sense strand is provided an ATG start signal, which results in an extraneous metlzionine being added to the N-terminus of the expressead poll~peptide. Immediately 3' of the bFGF-fragment-encoding sequence is a stop signal. At the 5' end is a Eco R:C overhang and at the 3' end is a Sal I

1 3 4~ 97 6 overhang, whereby the synthetic DNA strand is directly insertable in the Eco RI and Sal I site of the plasmid pUC8, described by Vieira et al. Gene 14, 259-268 (1982). Then DNA :strand is annealed into the pUC8 plasmid where it is under the control of the beta galactosidase promoter with the ATG start signal and the Shine Delgarno sequence regained in their natural orientation and association with the promoter.
~'he rec:ombinant vector, designated bFGF(24-68), is transformed into the DH-1 strain of E.
Coli by the calcium chloride procedure, CSH, supra.
~'he tr<~nsformed E. Coli is cultured in L
broth, and ~~mpicillan-resistant strains are selected.
Because the DNA chain was inserted into the plasmid in an orientation which could be expected to lead to expression of protein product= of the DNA chain, the ampicillan-resistant colonies are screened for reactivity with antiserum raised against bFGF. These colonies are screened by the immunological method of Healfman et al., Proc. Natl. Acad. Sci. USA 80, 31-35 (1983), and colonies reacting positively with bFGF antibody are further characterized. The cells, following separation from their culture media, are lysed, and their supernatent obtained. ~~upernatent from these transformed cells is determined by RIA to be reactive with antibodies raised against bFGF.
7.00 ml., of cell supernatant is obtained, and the desired bFGF(:?4-68) fragment is purified as described above. Approximately 0.01 mg. of bFGF(24-68), purified to upwards of 98% by weight of total protein, is produced.
~'he biological activity of the synthetic bFGF
fragment, which contains the extraneous N-terminal methionine residuEa, is tested for biological activity with respect: to ability to inhibit the growth of adult bovine aortic arch endothelial cells in culture, using an assay similar to i:hat described in J. Cell Biol. 97, 1677-1685 (:L983). Briefly, cells (at passage 3-10) are seeded at a dens ity of 2 x 103 cells/dish on plastic tissue culture dishes and exposed to Dulbecco's modified Eagle's med_Lum (DMEM) supplemented with 10% calf serum.
Test samples, at a dilution ranging from 10-1 to 10-3, are added on day 0 and day 2 to the dishes. On day 4, trip7Licate dishes are trypsinized and counted in a Coulter counter. Background levels are ordinarily 105 cells/dish, while those exposed to specified varying concentrations of the FGF antagonist contain as few as 104 cells/dp.sh. hor a potency assay, a log response curve is established. For this purpose, 10 microliter-aliquoia of a dilution (ranging from 10-1 to 10-5) of the: original solution made in 0.5% bovine serum albumin (BSA)/DMEM are added in triplicate.
'fhe superfluous N-terminal residue is removable b~~ part_Lal chemical digestion with cyanogen bromide or phenyl isothiocyanate followed by treatment with a strong anhydrous acid, such as trifluoroacetic acid. After subjEaction to such cyanogen bromide treatment, t:he bFCiF fragment continues to substantially reduce the total number of cells present per dish.
EXAMPLE XX
p, plasmid, following amplification in one of the bFGF-fragment producing E. Coli clones of Example XIX, is isolated and cleaved with Eco RI and Sal I. This digested pla,smid is electrophoresed on an agarose gel allowing for the reparation and recovery of the amplified bFGF fragmerut inseart. The insert is inserted into the plasmid pYE~~, a shuttle vector which can be used to transform both E- Coli and Saccharomyces cerevisiae yeast. Insertion of the synthetic DNA chain at this point assurea that: the DNA sequence is under the control of a promoter, in proper reading frame from an ATG signal and properly spaced relative to a cap site. The shuttle vector is used to transform URA3, a strain of S.
cerevisiae yeast from which the oratate monophosphate decarboxylase gene: is deleted.

'.the transformed yeast is grown in medium to attain log <~rowth. The yeast is separated from its culture medium, and cell lysates are prepared. Pooled cell lysatea are determined by RIA to be reactive with antibody raised against bFGF, demonstrating that a peptide coni~aining bFGF peptide segments is expressed within the ~teast cells.
~Che invention provides polypeptides which are biologicall~t active antagonists of both basic FGF and acidic FGF, because both have been shown to act upon the same receptors, and should be available for biological and therapeutic u;se. The production of longer bFGF
fragments can be carried out in both prokaryotic and eukaryotic cell lines. While such synthesis is easily demonstrated using either bacteria or yeast cell lines, the synthetic genes should be insertable for expression in cells of higher animals, such as mammalian tumor cells. Such mammalian cells may be grown, for example, as peritoneal tumors in host animals, and the desired bFGF fragments suitably harvested therefrom. The shorter bFGF fragments can simply be made by solid-phase or other coupling-type synthesis.
~~lthough the above examples demonstrate that bFGF-fragments can be synthesized through recombinant DNA
techniques, the e:Kamples do not purport to have maximized production. It i;s expected that subsequent selection of more efficiESnt cloning vectors and host cell lines will increase thsa yield of bFGF fragments. Known gene amplification techniques for both eukaryotic and prokaryotic cells may be used to increase production.
Secretion off' the gene-encoded polypeptide from the host cell line into thca culture medium is also considered to be an impori~ant factor in obtaining synthetic bFGF
fragments in largce quantities.
~3rain and pituitary basic FGF preparations, as reported earlier, are mitogenic for a wide variety of normal diploid cu:Ltured cells derived from tissue 134097fi originating from 'the primary or secondary mesenchyme, as well as from neuroectoderm. These include rabbit chondrocytes, bovine granulosa and adrenal cortex cells, bovine cornEaal endothelial cells, capillary endothelial cells derivead from bovine adrenal cortex and human umbilical endothelial cells. FGF antagonists are useful biological materials for regulating in vitro growth of cultured ce:Ll limes and are expected to also function in this manner when administered in vivo locally and otherwise. Accordingly, FGF antagonist peptides have many potent:lal therapeutic applications such as the treatment oi: vasoproliferative diseases of the eye, e.g.
diabetic ret~inopaithies, of proliferative diseases of the kidney, e.g., glomerulonephritis, of certain tumors, e.g.
chondrosarcoma, and adrenal vascularization, as well as inhibiting neovascularization of solid tumors in formation, and of other similar infirmities.
F3ecause it appears that the growth of human melanomas and other melanocytes is promoted by bFGF, the FGF antagon~Lsts should be effective to combat the growth of these cells and the growth promotion of certain related oncogenes, such as hst/KS3. More specifically, it is found that bFGF antagonists, in the presence of heparin, inhibit the response of melanocytes to the transforming oncogene protein KS3. It is expected that these peptides will also be antagonists to other of the FGF family of pepi~ides, such as FGF-5.
:~ynthei;.ic FGF antagonists or the nontoxic salts thereof, combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition, may be administered to mammals, including humans, either intravenously, subcutaneously, intramuscularly or orally. The' required dosage will vary with the particular c:ondit:ion being treated, with the severity of the condition and with the duration of desired treatment.
Such peptides are often administered in the form of phai°maceui~ically acceptable nontoxic salts, such 1 3 4Q 97 fi as acid addition salts or metal complexes, e.g., with zinc, iron ~~r the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active .ingredient is to be administered in tablet form, the tablet may contain a binder, such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.
'rhe peptides should be administered under the guidance of a physician, and pharmaceutical compositions will usuall~~ contain the peptide in conjunction with a conventional, pharmaceutically-acceptable carrier.
i~lthou~gh the invention has been described with regard to ii~s preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood i=hat various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which i;s set forth in the claims appended hereto. Fo~~ example, Thr can be substituted for Ser in what would be position 112 of the bFGF molecule, and Ser can be subsi=ituted for Pro in position 128, as these differences appear in the highly homologous human bFGF
molecule. ~~s also indicated hereinbefore, Met can be substituted for T:rp in the 114-position, Phe can be substituted for Tyr in the 115-position, and Ala can be substituted for Sa_r in the 113-position. Other similar substitutions as would be obvious to one skilled in peptide chemistry may be made to provide equivalent FGF
antagonists without departing from the scope of the invention. Extensions which do not change the FGF

antagonist peptide into an FGF partial agonist having substantial agonist activity can be added to either or both termini., so 7Long as they do not significantly lessen its biological potency as an FGF antagonist, and such polypeptide~~ are considered to be equivalents of those disclosed. For example, the residue Tyr can be added at either terminus o1. a synthetic FGF antagonist without substantially affescting the biological potency of that particular antagonist. Inasmuch as the function of the peptide is primarily one of binding, it is the sequence that is most: important, and the C-terminus can be free acid, amide or Borne equivalent moiety.
~~pecif_ic features of the invention are emphasized i.n the claims which follow.

Claims (15)

1. A peptide having the following formula:
H-Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-R106 -R107-R108-R109-R110-R111-R112-R113-R114-R115-Val-Ala-Leu-Lys-Arg-Y', wherein R106 is Tyr or D-Tyr, R107, is Arg or D-Arg, R108 is Ser or D-Ser, R109 is Arg or D-Arg, R110 is Lys or D-Lys, R111 is Tyr or D-Tyr, R112 is Ser, Thr or D-Ser, R113 is Ser, Ala or D-Ser, R114 is Trp or Met, R115 is Tyr or Phe, and Y' is (a) OH, (b) NH2, or (c)Thr-Gly-Gln-Tyr-Lys-Leu-Gly-R128-Lys-Thr-Gly-Pro-Gly-Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser-Y, with R128 being Pro or Ser and Y being OH or NH2, or a biologically active fragment thereof which contains the sequence R106 to R115 and which functions as an FGF antagonist or binds with the FGF receptor, provided however that at least one of the following is present:
R106 to R113 is a D-isomer: R112 is Thr: R113 is Ala; R114 is Met; R115 is Phe; and Y' is other than OH or NH2.

2. A peptide according to Claim 1 wherein R113 is D-Ser.

3. A peptide according to Claim 1 wherein R112 is D-Ser.

4. A peptide according to Claim 1 wherein R111 is D-Tyr.

5. A peptide according to Claim 1 wherein R110 is D-Lys.

6. A peptide according to Claim 1 wherein R109 is D-Arg.

7. A peptide according to Claim 1 wherein R108 is D-Ser.

8. A peptide according to Claim 1 wherein R107 is D-Arg.

9. A peptide according to Claim 1 wherein R106 is D-Tyr.

10. A peptide according to Claim 1 wherein R112 is Thr.

11. A peptide according to Claim 1 wherein R113 is Ala.

12. A peptide according to Claim 1 wherein R114 is Met.

13. A peptide according to Claim 1 wherein R115 is Phe.

14. A peptide .according to Claim 1 wherein Y' is Thr-Gly-Gln-Tyr-Lys-Leu-Gly-R128-Lys-Thr-Gly-Pro-Gly-Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser-Y.

15. A method of producing an FGF antagonist comprising: obtaining a DNA chain that encodes a polypeptide containing the following sequence:
H-Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-R112-R113-R114-R115-Val-Ala-Leu-Lys-Arg-Thr-Gly-Gln-Tyr-Lys-Leu-Gly-R128-Lys-Thr-Gly-Pro-Gly-Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser, wherein R112 is Ser or Thr, R113 is Ser or Ala, R114 is Trp or Met, R115 is Tyr or Phe and R128 is Pro or Ser, or a biologically active fragment thereof containing a sequence at least 29-residues in length which includes the decapeptide Tyr106-R115 and which functions as an FGF
antagonist or binds with the FGF receptor, inserting said DNA chain into a cloning vector in proper relationship to DNA sequences which promote expression of said encoded polypeptide, transforming an organism or cell line with said cloning vector having said inserted DNA chain, culturing said transformed organism or cell line, and obtaining said polypeptide produced thereby.