EXTRACELLULAR FORM OF THE HUMAN FIBROBLAST GROWTH FACTOR RECEPTOR

Abstract: A polypeptide is disclosed which is capable of binding specifically human fibroblast growth factor (bFGF) and human acidic fibroblast growth factor (aFGF) and which has: (a) the sequence shown or a part of the said sequence or (b) a said sequence (a) modified by one or more amino acid substitutions, insertions and/or deletions and/or by an extension at either or each end. A process for preparing the polypeptide, pharmaceutical compositions containing it and a DNA sequence which encodes it are also disclosed.

Description: EXTRACELLULAR FORM OF THE HUMAN FIBROBLAST GROWTH FACTOR RECEPTOR ' The present invention relates to human growth factor receptors. The formation of blood capillaries occurs in a number of important biological processes, either physiological, such as organ development and wound healing, or pathological, such as tumor growth. While the sequence of events leading to neovascularization has been characterized morphologically, the molecular mechanisms by which this process occurs are still poorly understood. The control of growth in the capillary endothelium appears to be very tightly controlled, since these cells normally form a static monolayer whose proliferation is triggered in the angiogenic process. The normally quiescent nature of the endothelial cells may be explained in' part by the apparent lack of endothelial cell growth factors to plasma. The major endothelial cell mitogenε in fact are not found in plasma, although they are present in extracts of almost all tissues t ~ ' - ■ studied and in many normal and tumor cell lines as' well. Therefore, the localized induction of rapid endothelial cell proliferation may involve the release of endothelial cell mitogens from cells in response to environmental cues. The best characterized of the endothelial cell mitogens are a family of polypeptide growth factors, including basic fibroblast growth factor (bFGF), also known as heparin-binding growth factors for their high affinity to heparin. Basic FGF has been purified ' from most mesoderm- or neuroectoderm-derived tissues or cells. Structural studies have shown that bFGF is a single chain polypeptide made of 146 amino acids, which can also exist in NH 2 -terminally truncated forms missing the first 10-20 amino acids. The truncated forms of bFGF are as potent as native bFGF, as demonstrated by radioreceptor binding and biological assays. In addition, modifications of the purification protocols by substitution of neutral for acidic extraction from homogeneized tissue and inclusion of protease inhibitors have yielded a longer 154-residue form. The observed microheterogeneity of FGFs seems to be due, at least in part, to partial proteolysiε near the amino termini that occurs either in vivo or during purification. However, because the various forms appear to be equally active, the microheterogeneity is probably physiologically, irrelevant. Basic FGF seems to have been extremely well conserved through evolution. For example, bovine and human bFGF differ in only two of their 146 amino acids, giving an - overall amino acid sequence ho ology of 98.7%. Related to ' bFGF is acidic FGF (aFGF), which shares a 55% total sequence homology with bFGF. Acidic FGF is a 140-amino acid polypeptide that can also exist in a NH 2 -terminally truncated form hissing the first 6 amino acids. As expected from their high degree of homology, both basic and acidic FGF seem to interact with the same cell-surface receptors. This explains their common range of target cells and spectrum of biological activity. Recently, Lee et &1 (Science, 245, 57-60, 1989) have described the purification, from chicken embryos, of a new membrane protein able to bind specifically basic FGF. On the base of its biochemical characteristics and on homology with other known receptors, this new protein is thought to be a basic FGF receptor. In the same article the authors describe the isolation of a chicken full-length cDNA clone coding for the described protein. The nucleotide sequence of this clone is however not disclosed. Lee e_t a_l noted that the chicken bFGF receptor shows a considerable amino acid "similarity to a previously identified human polypeptide sequence which is the product of the fig gene (Ruta e_t a_l, Oncogene, 3 ^ , 9-15, 1988). Like the chicken bFGF receptor, the fig molecule seems to be a tyrosine kinase, shows a typical hydrophobic transmembrane region and consequently it is likely that the described human sequence could be part of the human bFGF receptor. The amino acid sequence of the fig molecule was ■ deduced by Ruta e_t al_ by the translation of an open reading frame of a partial cDNA clone. The fig cDNA sequence, according to Rutal e_t a_l was obtained by low-stringency screening of a human endothelial cell' cDNA library using as probe a DNA fragment coding for a tyrosine kinase oncogene. Nevertheless, the fig cDNA clone, described to date, is only partial and lacks the nucleotide sequence coding for the extracellular portion of the human bFGF receptor. In summary, the only amino acid or nucleotide sequences available prior to the present invention were those corresponding to the complete chicken bFGF receptor and to a portion of the human basic FGF receptor. The complete extracellular amino acid sequence of the human bFGF receptor, which is responsible for the specific binding of human basic and acidic FGFs, was unknown. We have now cloned the human bFGF receptor and have identified its extracellular portion. The receptor and its extracellular portion may be used as antagonists for bFGF or aFGF. Accordingly, the present invention provides a polypeptide which is capable of binding specifically human basic fibroblast growth factor (bFGF) and human acidic fibroblast growth factor (aFGF) and which has: (a) the sequence shown in Figure 3 or a part of the said sequence or (b) a said sequence (a) modified by one or more amino acid substitutions, insertions and/or deletions and/or by an extension at either or each end. A preferred polypeptide has: (a ) the sequence underlined in Figure 3 or a part of the said underlined sequence or (b ) a said sequence (a χ ) modified by one or more amino acid substitutions, insertions and/or deletions and/or by an extensipn at either or each end. The invention also provides a DNA sequence which encodes a polypeptide of the invention. The DNA sequence may be: (a 2 ) the sequence shown in Figure 2 or a part of the said sequence or (b 2 ) a said sequence (a 2 ) modified by one or more nucleotide substitutions, insertions and/or deletions and/or by an extension at either or each end. A suitable DNA " sequence is (a 3 ) the sequence from nucleotide 64 to nucleotide 1128 shown in Figure 2 or a part of the said sequence or (b 3 ) a said sequence (a. ) modified by one or more nucleotide substitutions, insertions and/or deletions and/or by an extension at either or each end. The invention further provides a vector which • incorporates a DNA sequence " of the invention and which is capable, when provided in k transformed host, of expressing the polypeptide of the invention encoded by the DNA sequence. A host transformed with such a vector forms part of the present invention too. A polypeptide of the invention is prepared by a • process which comprises culturing a transformed host according to the invention under such conditions that the polypeptide is expressed. The polypeptide can then be isolated. The polypeptide may be recovered in biologically pure form. In the accompanying drawings: Figure 1 is a schematic illustration of clones PL5 Figure 2 shows the nucleotide sequence of the bFGF receptor molecule; and Figure 3 shows the amino acid sequence of the bFGF receptor. The putative leader peptide is in italic characters. The previously unknown extracellular portion is underlined. The arginine (R) residue which is thought to be the first amino acid of the mature molecule is in a bold character. Figure 4 shows the two intermediate constructions (plasmids L23 and pFC42) as well as the two final expression plasmids (pFC138 and pFC164) carrying the sequence coding for the extracellular form of the FGF receptor. For details, see Example 2 below. The polypeptide of the invention having the sequence shown in Figure 3 is therefore the human bFGF receptor. A polypeptide which is composed of part of the sequence shown in Figure 3 and which is capable of binding specifically human bFGF and human aFGF also forms part of the invention. Other polypeptides of the invention are polypeptides which comprise modified versions of the sequence shown in Figure 3 or of part of the sequence of Figure 3 and which are capable of binding specifically human bFGF and human aFGF. For a suitable receptor-binding test, see Dower e_t l ^ , J. Immunol. , 4314-4320, 1989. A preferred polypeptide is the polypeptide -having the sequence from amino acid 1 to amino acid 376 shown in Figure 3. This is the sequence of the extracellular portion of the bFGF receptor with the putative leader sequence. Another preferred polypeptide is the polypeptide having the sequence underlined in Figure 3 from .amino acid 22 to amino acid 376. This is the sequence of the extracellular portion of the bFGF receptor without the putative leader sequence. The sequence, or an ' appropriate part of the sequence, shown in Figure 3 may be modified by one or more amino acid substitutions, insertions and/or deletions and/or by an extension at either or each end. A polypeptide composed of such a modified sequence must of course still be capable of binding human bFGF and aFGF. When the sequence or a part of the sequence shown in Figure 3, i.e. an unmodified sequence is modified typically there is a degree of homology of 75% or more between the modified sequence and the unmodified sequence. The degree of homology may be 85% or more or 95% or more. For example, one or more amino acid residues of the sequence or a part of the sequence shown in Figure 3 may be substituted or deleted or one or more additional amino acid residues may be inserted; provided the phyεicochemical character of the original sequence is preserved, i.e. in terms of charge density, hydrophobicity/hydrophilicity, size and configuratio- Candidate substitutions are, based on the one-letter code (Eur. J. Biochem. 138, 9-37, 1984): A for G and vice versa, V by A, L or G; K by R; S by T and vice versa; E for D and vice versa; and Q by N and vice versa. As far as extensions are concerned, a short sequence of up to 50 amino acid residues may be provided at either or each terminal. The sequence may have up to 30, for example up to 20 or up to 10, amino acid residues. Alternatively, a much longer extension may be present. " Longer amino acid sequences may be fused to either or each end. A chimaeric protein may therefore be provided in which the or each extension is a heterologous amino acid sequence, i.e. a sequence not naturally linked to the sequence derived from Figure 3. Such a chimaeric protein may therefore combine the ability to bind specifically to bFGF and aFGF with another functionality. The polypeptides are prepared by recombinant DNA technology. The preparation of the polypeptides therefore depends upon the provision of a DNA sequence encoding the polypeptide. DNA having the sequence shown in Figure 2 may be obtained by probing a human placenta cDNA library,- for example a λgtll library. Such a library is available from Clontech. Suitable probes are: ATAACGGACCTTGTAGCCTCCAATTCTGTG, GGGTCTCTGTGAGGGCACTGCATGCCAGCA, and ACGGCCTAGCGGTGCAGAGTGTGGCTGTGA. A shorter sequence than that shown in Figure 2 may be obtained by use of restriction endonucleases and/σr exonucleases. A DNA sequence from nucleotide 1 to nucleotide 1128 or from nucleotide 64 to nucleotide 1128 may be provided. Modified sequences may be obtained by use of any appropriate technique, including restriction with an endonuclease, insertion of linkers, use of an exonuclease and/or a polymerase and site-directed mutagenesis. Whether - 9 - a shortened and/or modified DNA sequence encodes a polypeptide of the invention can be readily ascertained. The polypeptide encoded by the sequence can be expressed in a suitable host and tested for its ability to bind specifically human bFGF and aFGF. For expression of a polypeptide of the invention, an expression vector is constructed. An expression vector is prepared which comprises a DNA sequence encoding a polypeptide of the invention and which is capable of expressing the polypeptide when provided in a suitable host. Appropriate transcriptional and translational control elements are provided, including a promoter for the DNA sequence, a transcriptional termination site, and translational start and stop codonε. The DNA sequence is provided in the correct frame such as to enable expression of the polypeptide to occur in a host compatible with the vector. The expression vector is then provided in an appropriate host. Cells harbouring the vector are grown so as to enable expression to occur. The vector may be a - plasmid or a viral vector. Any appropriate host-Vector system may be employed. The transformed host may be a prokaryotic or eukaryotic host. A bacterial or yeast host may be employed, for example E. coli or S. cerevisiae. Insect cells can v alternatively be used, in which case a baculoviruε expression system may be appropriate. As a further alternative, cells of a mammalian cell line, such as Chinese Hamster Overy (CHO) cells may be transformed. The polypeptide of the invention can be isolated and purified. The polypeptide can be employed as a human bFGF or aFGF antagonist. It may sequester toFGF ' _in vivo, * thus preventing the biological activity of bFGF and acting as an inhibitor of bFGF activities. Antagonists of bFGF and aFGF activities could have clinical applications in several pathologies related to abnormal angiogenesis such as diabetic, retinopathy, neovascular glaucoma, rheumatoid arthritis, psoriasis, artheroεclerosis and as contraceptives. In addition, the observation that certain solid tumors require neovascularization to grow εuggests that FGF antagonists could be therapeutically developed to treat these diεeaεes. For thiε purpoεe, a polypeptide of the preεent invention may be chemically coupled to another , material to provide a conjugate. The other material may be a carrier molecule or a molecule with another biological function. The polypeptide of the invention may also be formulated- in a pharmaceutical composition. The pharmaceutical omposition also comprises a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition may contain the polypeptide of the invention in the form of a conjugate. The polypeptide of the invention may be administered to a patient by any convenient route. The choice of whether an oral route or a parenteral route, such as subcutaneous, intravenous or intramuscular administration, is adopted; of the dose; and of the frequency of administration depends upon a variety of factors. These factors include the purpose of the administration, * the ag and weight of. the_ patient being treated and the condition of the patient. Typically, however, the polypeptide is administered in an amount of from 1 to 1000 μq per doέe, more preferably from 10 to 100 μg per dose, for each route of administration. The following Example illustrates the invention. EXAMPLE 1 A cDNA clone coding for basic FGF receptor was isolated by screening a human placenta λgtll cDNA library. Thiε library is commercially available from Clontech. A partial cDNA clone thought to encode a portion of the human bFGF receptor wa ? s already published in the literature (Ruta et al, 19$8) . According to this article, we designed two oligonucleotide probes with the following sequenceε: 5' ATAACGGACCTTGTAGCCTCCAATTCTGTG 3' (named OAB965) and 5' GGGTCTCTGTGAGGGCACTGCATGCCAGCA 3' (named OAB984 ) . A first screening was carried out using only the probe OAB965. The oligonucleotide was kinased using (γ 32 P)ATP and purified oh a Nensorb column (Nen). Following the determination of the cDNA library titre (2 x pfu/ml), appropriate dilutions were made and plated using the E. coli strain Y1090. 0.05 ml of an overnight culture of Y1090 were mixed with 0.1 ml of sterile lambda diluent (10 mM Triε HCl pH 7.5, 10 mM MgCl 2 , 0.1 mM EDTA) containing a 4 x 10 " 3 dilution of the library and incubated at 37°C for 15 minutes to allow phage adsorption. Plating was carried but on 20 Petri ' dishes of LB containing ampicillin 100 According to published procedures (Maniatis e_t al, Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 1982) 10 6 plaques were transferred onto nitrocellulose filters and hybridized under low stringency conditionε (5X SSC, 0.1% SDS, 5x Denhardt solution, 0.1 mg/ml salmon sperm DNA), overnight at 65°C. The labelled probe was added at 10 6 cpm/ml. Washeε were performed at 65°C in 6X SSC, 0.1% SDS and in 3X SSC, 0.1% SDS. Three poεitive cloneε, named PL3, PL10, and PL16 were detected after an overnight exposure with Emersham Hyperfilm -MP. Theεe poεitive signals were confirmed in a separate screening using the same probe. A second screening was then performed on PL3, PL10 and PL16 on duplicate filters using the two probes OAB 965 and OAB 984. Intereεtingly, these clones were recognized by both probes. The three clones were plaque-purified and further analyzed. Phage DNA of PL3, PL10, and PL16 was purified using Lambdasorb phage adsorbent (Promega) and digested with EcoRI. From the different digestions, we selected two inserts' of 2.8 Kb and 0.6 Kb from PL10 and subcloned them into M13mpl9.. Nucleotide sequence analysiε waε carried out by primed DNA εyntheεiε on single-stranded DNA templates in the presence of dideoxynucleoside triphosphates (Sanger e_t al, Proc. Natl. Acad. Sci. USA 7_4, 5463-5467, 1977) by using Sequenaεe (United Stateε Biochemical Corp. Cleveland, Ohio). Both strands of the subclones were εequenced using either universal or specific primer oligonucleotides based on the generated sequence. A single open reading frame for 817 amino acids with a termination codόn could be detected. The first 196 amino acids of the mature bFGF receptor are codified by the insert of 0.6 Kb. The final sequence of thiε fragment contains an EcoRI εite, εo that the 626 amino acidε of the COOH-terminuε are codified by the 2.8 Kb i'nsert and followed by a 3' end untranslated region, aε schematically illustrated in Fig. 1. The chicken receptor' ^for bFGF, as it has been described by Lee e_t al (1989), is a protein of 819 amino acids including a leader peptide of 21 amino acidε, followed by a mature protein of 798 amino acidε. By homology to this sequence, our clone contained the complete mature bFGF • receptor molecule, preceded by 16 amino acids of : the leader peptide. In order to ' find the remaining 5 amino acids of the leader peptide, including the εtarting methionine, we εyntheεized a new oligonucleotide probe with.the following sequence, derived from the 0.6 kb insert of PL10: 5' ACGGCCTAGCGGTGCAGAGTGTGGCTGTGA 3' (named OAB 1088) By screening the library with this probe, we εelected a new clone, name PL5, which contained a 0.7 kb EcoRI fragment. Thiε fragment waε εequenced and found to contain the 0.6 kb sequence from PLlO, while it further extended at the 5' end. More precisely, the 0.7 kb sequence from PL5 codes for the 196 amino acidε of the NH 2 terminal sequence, also found in PLlO, preceded by 21 amino acids of the leader peptide, starting with a methionine. A schematic description of clones PL5 and PLlO is illustrated in Fig. 1. The full length DNA sequence for a bFGF receptor, as obtained from the two overlapping clones PL5 and PLlO, is depicted in Figure 2. The corresponding amino acid sequence is depicted in Figure 3. In thiε εequence, the putative leader peptide of 21 amino acids is in italicε. The arginine (R) reεidue which is thought to be the first amino acid of the mature molecule is in bold character. The previously unknown extracellular portion is underlined. The full length clone and the extracellular domain have been expressed in both mammalian and bacterial cells. Cross-linking experiments with labelled basic and 'acidic FGFs have shown that the recombinant complete receptor and extracellular portion are indeed able to bind specifically basic and acidic FGF. EXAMPLE 2 To obtain the expression of the extracellular portion of the human FGF receptor in E. coli, a vector was constructed using as regulatory signals the tryptophan promoter of E.coli and the ribosome binding site region of the lambda CII protein (Hendrix R.W., Roberts J.W., Stahl F.W. and Weisberg R.A.: Lambda II Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y. 1983). The 5' end of the receptor sequence was synthetically reconstructed to be cloned into the expression vector. For this purpose a HindLTI-Pstl synthetic DNA fragment coding for the CII ribosome binding site followed by the ATG codon and the first 95 nucleotides of the mature recep'tor sequence (Fig.2: nucleotides 61 to 155) was synthesized using complementary oligonucleotides. The complete sequence of such synthetic fragment is: 5 * AGCTTGGGCATACATTCAATCAATTGTTATCTAAGGAAATACTTAC ATATCKαCTAGGCCGTCCCCGACCTTGCCIOAA AGCCCCTGTGGAAGTGGAGTCCTTCCTGGTCCACCCCGGTGACCTGCT GCA 3' This Hindlll-PstI fragment was subcloned into M13mpl8 previously cut with Hindlll and EcoRI, together with a Pstl-EcoRI fragment from PL5, harbouring the receptor sequence corresponding to nucleotides 156 to 586 of fig.2. The resulting plasmid L23 carries the CII ribosome binding site followed by nucleotides 61 to 586 of the, receptor sequence (See Fig.4), corresponding to the first 175 amino acids of the mature protein. To obtain the whole extracellular portion of the FGF receptor an EcoRI-BamHI fragment was recovered by polymerase chain reaction (PCR) on PLlO. The reaction wajj performed in a Perkin Elmer DNA thermal cycler, using the Perkin Elmer Amplitaq DNA Polymerase and the following cycles: 2'30 at 94°c 30 cycles made by one step of 1'30" at.94°C and a second step of 2'3C" at 72°C and a final cycle of 7' at 72°C. The primers used for the reaction , called OAB 1192 and OAB 1195 ' , were the following: OAB 1192 : 5' GTAAAACGACGGCCAGT 3' OAB 1195 : 5' ATGAGGATCCTCACTCCAGGTACAGGGGCGAGGTCA 3' The amplified product was digested with EcoRI and BamHI to obtain a fragment of 546 bp, corresponding in fig.2 to nucleotides 587 to 1128 of the mature receptor sequence. The expression plasmid for the mature extracellular receptor protein, named pFC138, was constructed by joining the following three fragments: 1) The Hindlll-EcoRI fragment from L23. 2) The EcoRI-BamHI fragment from PLlO. 3) The Hindlll-BamHI large fragment from plasmid pFC42, previously obtained in our laboratory (Isacchi A., Sarmientos P., Lorenzetti R. and Soria M.: Mature apolipoprotein Al and its precursor proApo Al: influence of the sequence at the 5' end of the gene on the efficiency of expression in Escherichia colt Gene 8_1 p.129- 137 1989), harbouring the tryptophan promoter and the β- lactamase gene for resistance to ampicillin. An E.coli B strain ( Delbruck M.: Bacterial viruses or bacteriophages. Biol.Rev. 21. p.30-40 1946) was used for expression of the mature extracellular FGF receptor protein. Induction and analysis of protein expression were performed as described (Isacchi A. et al.). The protein was recognized in western blot by rabbit anti-human FGF receptor polyclonal antibodies (Promega). Alternatively, the recombinant extracellular domain could be synthesized as a fusion protein where additional amino acid residues are present either at the NH - end or at the C O O H- end of the mature extracellular region. Thiε approach is currently used for increasing intracellular protein stability, yield, detection and isolation. Both patent and scientific literature report many examples of different expression systems based on fusion proteins [see, for example, International Patent Application PCT/WO84/00380; European Patent Application EP 243333; Harris, T.J.R. 1983, Expression of eukaryotiσ genes in E. co i. pp. 127-185. In Williamson, R. (Ed.), Genetic Engineering Vol. 4, Academic Press, London; Hellebust, M. et al. 1989, Biotechnology Vol. 7, pp 165-168]. We have constructed a new plasmid named pFC 164, shown in figure 4, wherein the amino acids 9 to 23 of the E. coli S-galactosidase are inserted upstream of the mature extracellular domain of the FGF receptor. Plasmid pFC 164 is constructed in a way similar to pFC 138 and when introduced in the E. coli host strain B, yields higher levels of expression of the extracellular domain of the FGF receptor. The 9-galactosidase moiety is also responsible for an increased intracellular stability of the fusion protein. Plasmids pFC 138 and pFC 164 are therefore objects of the present invention.

Claims: CLAIMS 1. A polypeptide which is capable of binding specifically human fibroblast growth factor (bFGF) and human acidic fibroblast growth factor (aFGF) and which has: (a) the sequence shown in Figure 3 or a part of the said sequence or (b) a said sequence (a) modified by one or more amino acid subεtitutionε, inεertionε and/or deletionε and/or by an extension at either or each end. 2. A polypeptide according to claim 1, which has: (a χ ) the εequence underlined in Figure 3 or a part of the said underlined sequence or (b 1 ) a said εequence (a 1 ) modified by one or more amino acid εubεtitutions, insertionε and/or deletionε and/or by an extenεion at either or each end. 3. A DNA sequence which encodes a polypeptide as defined in claim 1 or 2. 4. A DNA sequence according to claim 3, which is: (a 2 ) the sequence shown in Figure 2 or a part of the said sequence or (b 2 ) a said sequence (a 2 ) modified by one or more nucleotide substitutions,, insertions and/or deletions and/or by an extension at either or each end. 5. A DNA sequence according to claim 4, which is (a 3 ) the εequence from nucleotide 64 to nucleotide 1128 shown in Figure 2 or a part of the said sequence or -19 - (b 3 ) a said sequence (a 3 ) modified by one or more nucleotide εubstitutions, inεertions and/or deletionε and/or by an extenεion at either or each end. 6. A vector which incorporateε a DNA sequence as claimed in any one of claims 3 to 5 anu which is capable, when provided in a suitable host, of expressing the said polypeptide. 7. A vector according to claim 6, which iε a plasmid. 8. The plasmid pFC 138. 9. The plasmid pFC 164. 10 . A host transformed with a vector or a plasmid as claimed in claims 6 to 9. 11 . A host according to claim 10 which iε a mammalian cell line. 12 . A host according to claim ιo which iε a . bacterium. 13 . A process for the preparation of a polypeptide as defined in claim 1 or 2, which process comprises culturing a transformed host as claimed in any one of claims 10 to 12 under such conditions that the said polypeptide is expresεed. 14. A fusion protein comprising a polypeptide as claimed in claim 1 or 2 coupled to a carrier protein or polypeptide. 15. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as an active principle, a polypeptide as claimed in claim 1 or 2. 16. A composition according to claim 15, wherein the said polypeptide is in the form of a conjugate as claimed in claim 14. 17. A polypeptide as defined ' in claim 1 or 2 for se as an antagonist for human bFGF or human aFGF.

Inventors: BERGONZONI LAURA, ISACCHI ANTONELLA, MAZUE GUY, RONCUCCI ROMEO, SARMIENTOS PAOLO, BERGONZONI, LAURA, ISACCHI, ANTONELLA, MAZUE, GUY, RONCUCCI, ROMEO, SARMIENTOS, PAOLO
Applicants: ERBA CARLO SPA, FARMITALIA CARLO ERBA S.R.L.
Application year: 1991
Main classification: C12N15
IPC codes: A61K38, A61K47, A61P9, A61P17, A61P27, A61P29, A61P35, C07K14, C07K19, C12N1, C12N5, C12N15, C12P21, C12R1