CA2465714A1 - Isolated peptide of the hepatocyte growth factor and its variants, preparation method and therapeutic use as anti-angiogenic agents - Google Patents

Abstract

The invention concerns a peptide substance selected from the group consisting of: (i) peptide N derived from the HGF .alpha. chain and having an amino acid sequence represented by SEQ ID No. 2, and (ii) its peptide variants derived from said peptide N sequence by suppression, substitution or addition of one or several amino acids. Said peptide N has an anti-angiogenic activity in accordance with a mechanism different from competitive fixation with HGF on the HGF-R receptor. The invention also concerns the therapeutic use and the preparation method of said peptide substance, as well as (i) the DNA fragment which expresses it, the plasmid containing said DNA and the bacteria containing said plasmid.

Description

Peptide isolates hepatocyte growth factor and its variants, preparation method and use in therapeutic in taut that antiangiogenic agents Field of invention The present invention relates, as industrial products new, has a peptide (denoted here N) isolated from growth factor hepatocyte (HGF) and its variants. It also relates to their process of preparation as well as their use in therapy as agents antiangiogenic agents acting as inhibitors of fangiogenesis.
The abbreviations and acronyms used here are defined in a way complete further.
Prior art HGF is currently recognized as a pleiotropic factor having a fairly broad specificity vis-à-vis many targets cell. In particular, he plays an important role as mediator in mesenchymal-epithelial / endothelial interactions that contribute to complex biological processes, such as fembryogenesis, regeneration tissue and tumorogenesis [see K. Matsumoto et al., Ciba Found. Symp., 1997; 212: 198-211]. We also know that HGF is a powerful activator of fangiogenese [see F. Bussolino et al., .J. Cell Biol., 1992; 119: 624-641 ]
whose efficacy in vivo may be greater than that of VEGF [see E.
van Belle et al., Circulation, 1998; 97: 381-390].
Regarding its structure, HGF is a peptide apparent to enzymes of the blood clotting cascade. Mature HGF is a peptide consists of two strings (or subunits): the strings a, and [i. In the HGF
native (see Figure 1 below), the portion of the amino acid sequence (aa 1-478), which supplies the chain a, has since its N-terminus to its C-terminate end (i) a peptide L comprising 31 amino acid residues (aa 1-31) which (i) does not probably have no interest in the plan pharmacological and (ii) acts as a signal peptide, (ii) an N domain containing a so-called hairpin loop, having 96 amino acid residues (aa 32-127) and comprising four Cys residues (peptide N is that which is useful according to the present invention), and

2 (iii) four kringle domains (named K1, K2, K3 and K4) comprising each 6 to 7 Cys residues, K1 comprising 79 amino acid residues (aa 128-206), K2 including 78 amino acid residues (aa 211-288), K3 comprising 79 amino acid residues (aa 305-383) and K4 comprising 79 amino acid residues (aa 391-469).
The HGF chain a represents the main HGF interaction site with its receptor which is expressed by the proto-oncogene c-Met and which has (after fixing HGF on its receptor) an enzymatic activity of tyrosine kinase. In what follows this receptor is called HGF-R.
The Vii chain, which has homology with the catalytic domain serine proteases, does not appear to bind to the HGF-R receptor but is essential to the phosphophorylation of said receptor and consequently to its interaction with certain agents (the "effectors") involved in the intracellular signaling.
Devoid of the chain ~ 3, the peptide constitutes by the chain a, which is linked to the HGF-R receptor, is an HGF antagonist [see K. Date et al., FEBS Letters, 1997; 420: 1-6] and acts as an inhibitor of biological responses of HGF, including the angiogenic response of this factor [see K. Kuba et al., Cancer Res., 2000; 60: 6737-6743], according to a competition mechanism.
It is known in particular from WO-A-99/55361 that the chain a of HGF a was sequenced. More precisely, we know that the peptide called NK4, which begins with a pyroglutamate group and includes the amino acids of said chain a (ie as PyrGlu32 to Va1478 of native HGF), has effects anti-tumor [see EP-A-0890361] and antiangiogenic effects which manifest by inhibition of neovascularization [see publication by K. Kuba et al. and WO-A-99/55361 cited above].
In short, the mechanisms known to date are the following (A) by cleavage of the peptide L, native HGF gives (or is "active" in) HGF

Mature;
(B) mature HGF binds, by its chain a, to its HGF-R receptor;
(C) thanks to the binding of mature HGF to HGF-R, the activity enzy-HGF-R material, which is of the tyrosine kinase type, manifests; he this is the "activation" by which the enzyme contained in the intracellular domain of the HGF-R protein becomes able to exercise its tyrosine kinase-like effect;
(D) when used, the peptide NK4, qua binds to HGF-R without this exerts its enzymatic activate of the tyrosine kinase type, intervenes as a competitive HGF inhibitor.
Origin of the invention As the domains K1, K2, K3 and K4 of the a chain of HGF are quite similar to the antiangiogenic kringles of some other proteins (such as fangiostatin and the second prothrombin kringle), we asks during the genesis of the present invention if (1 °) the kringles K1, K2, K3 and / or K4 of the HGF chain a have an activate antiangiogenic and if (2 °) the mechanism of antiangiogenic activity possibly one or more of these kringles resides in the competitive fixation of K1, K2, K3 and / or K4 on the HGF-R receiver.
After isolating and testing peptides N, K1, K2, K3 and K4 from the a chain of HGF, we just found surprisingly that the peptide N, that was substantially included in the sequence of NK4 [N differs from the sequence of NK4 by the nature of the first amino acid, Glnl replaces ~ ant PyrGlul (ie in position 32 of native HGF)], has properties antiangiogenic drugs that manifest themselves in a mechanism different from that NK4 kringles.
In short, as illustrated below, it was observed according to the invention than ~ N acts as an HGF inhibitor according to a mechanism different from competitive binding with HGF on the HGF- receptor R; according to the results of the Applicant's tests, N does not settle substantially on HGF-R, unlike NK4; we have everything to think that N acts as an HGF inhibitor since it intervenes at the level of HGF interaction with GAGS in inhibiting HGF / GAG binding; and, ~ K1, unlike K2, K3 and K4, is in the current state of Applicant's knowledge the only HGF kringle to agar actually by competition with HGF at the level of the fixing on the HGF-R receptor.

4 Alternatively, peptide N, which has a hairpin of NK4 and comes from the HGF chain a, contains within its sequence the amino acids 2-96 of NK4 (ie Arg33-Asnlz7 of native HGF) but .differentiation of NK4 by the first amino acid (ie G1n32 instead of the group pyroglutamate PyrGlu32 from native HGF according to EP-A-0890361 and WO-A-99/55361 above).
Purpose of the invention According to the invention it is proposed to provide (a) a new substance which has antiangiogenic properties, (b) a process for its preparation and (c) therapeutic use of this substance.
Object of invefitioh We therefore recommend according to (invention a new peptide substance dique characterized in that it is chosen from (set constituted by (i) the peptide N originating from the a chain of HGF and having the sequence amino acid represented by SEQ ID No.: 2, and (ii) its peptide variants derived from the sequence of said peptide N
by deletion, substitution or addition of one or more amino acids, said peptide N and its variants having antiangiogenic activity according to a mechanism different from competitive binding with HGF on the receiver HGF-R.
According to another aspect of (invention, a composition is recommended therapeutic which is characterized in that it contains, in association with a pharmaceutically acceptable excipient, a quantity therapeutically effective of a peptide substance selected from (en-seems constituted by (i) the peptide N originating from the a chain of HGF and having the sequence amino acid represented by SEQ ID No.: 2, and (ii) its peptide variants derived from the sequence of said peptide N
by deletion, substitution or addition of one or more amino acids, to combat the disorders associated with fangiogenesis.
We also recommend the use of said peptide substance antiangiogenic vis-à-vis said disorders associated with angiogenesis.

WO 03/03809

5 PCT / FR02 / 03739 Finally we advocate, as new industrial products, the DNA fragment coding for peptide N or its aforementioned variants, the plasmid containing said DNA fragment and the bacteria containing said plasmid.
5 Brief description of the drawings In the accompanying drawings, Figure 1 shows schematically the structure of native HGF with the five main domains of chain a (N, K1, K2, K3 and K4), the PD fragment representing the chain [i;
- Figures 2a and 2b show the level of purification of the peptides isolates N, Kl, K2, K3 and K4 according to coomassie blue staining (Fig. 2a) and Western blotting with an anti-HGF antibody (Fig. 2b);
- Figure 3 represents, in graphic form, the results obtained at during trials related to stimulating cell proliferation HUVEC by bFGF (10ng / ml) in the presence of increasing doses (from 0 to 50 ~, g / ml) of peptide N, K1, K2, K3 or K4;
- Figure 4 is a graph illustrating the inhibition of the proliferation of HUVEC cells with peptide N in the presence of a GF such as bFGF, HGF, VEGFI6s and VEGFIZi;
- Figure 5 is a graph illustrating the effect of HGF, HGF + N, HGF
+ K1, HGF + K2, HGF + K3 and HGF + K4 on cell migration HUVEC through a porous membrane;
- Figure 6 includes six photographs relating to (inhibition of structure formation of the "capillary tube" type by HUVEC in a gel of three-dimensional collagen [comparisons of bFGF with bFGF + N, of VEGF with VEGF + N (or VEGF is VEGFI ~ s) and HGF with HGF + N];
- Figure 7 is a graph showing that the peptide N according to (invention differs from K1, K2, K3 and K4 by the intense fixation of Hep;
and, - Figures 8A-8D and 9A-9B relate to complementary results.
silences obtained with said peptide N.
abbreviations For convenience, here is the list of abbreviations and acronyms techniques which have been used in the present description.

6 as amino acid (s);

c-Met fragment of proto-oncogene DNA encoding a protein, which has an enzymatic activity of tyrosine kinase and represents the receiver [here called HGF-R]
HGF;

cpm counts per minute;

DNA acids deoxyribonuclsique (from English: "deoxyrubonucleic acid ");

FBS bovine fetal serum;

FGF fibroblast growth factor (from English : "fibroblast growth factor "); among the FGFs we distinguish especially the aFGF (fibroblast growth factor acids) and bFGF

(basic fibroblast growth factor) ;

GAG glycosylaminoglycan;

GF growth factor;

Hep hsparine;

HEPES acids N-2-hydroxysthylpiperazine-N-2-sthanesulfonic, product acting as a buffer;

HGF hspatocyte growth factor (from English : "hepatocyte growth factor "); unless otherwise noted HGF designs here on human hspatocyte growth factor (hHGF);

hHGF human hspatocyte growth factor ;

HGF-R HGF receptor (the notation "HGF-R" is prsfsrse here at the confusing "c-Met" nomenclature with the gene "c-Met");
HUVEC human umbilical cord endothslial cells (of English: "human umbilical cord endothelial cells");
K1 domains kringle 1 of HGF or corresponding peptide;
K2 domains kringle 2 of HGF or corresponding peptide;
K3 domains kringle 3 of HGF or corresponding peptide;
K4 domains kringle 4 of HGF or corresponding peptide;
M 199 culture medium sold by the American socists LIFE TECHNOLOGIES (Rockville, MD);
M 1991 medium M 199 supplements with 15 mM HEPES (pH 7.4), 2 mM glutamine, 50 U / ml of penicillin, 50 ~, g / ml of streptomycin and 2.5 ~ .g / ml amphotsricin B;

7 N area of the HGF hairpin loop, the peptide corresponding or better the peptide according to the invention whose N-terminate end is Gln (instead of PyrGlu according to previous wax illustrated by WO-A-99/55361);
NK4 peptide constituted by the amino acid sequence from N to K4 in HGF (ie as 32-478 of native HGF);
RT ambient temperature;
RU resonance unit (1000 RU corresponds to a fixing of 1 ng of protein per mm2);
VEGF vascular endothelial growth factor (from English "vascular endothelial growth factor"); among the VEGFs we distinguishes in particular the factors VEGF »5 and VEGF121.
Detailed description of the invention The hHGF factor has already been sequenced. In particular, the sequence PyrGlu32-Va14 ~ 8 from NK4 is given in WO-A-99/55361). According to fart NK4 acts as an anti-tumor substance and antiangiogenic according to a mechanism of competition between NK4 and HGF
vis-à-vis HGF-R, which is the HGF receptor.
After synthesis (by expression according to a engineering technique genetics) puffs purification, peptide N according to the invention (in which the first ace of N-terminate fextremite is Gln instead of PyrGlu) and its variants were tested. Said peptide N, guff is new compared to teaching of the sequence of NK4, and its variants are distinguished from Kl, K2, K3 and K4 by their interactions with GAGS [in particular Hep (high molecular weight heparin, normal heparin and low heparin molecular weight), dermatan sulfate, heparan sulfate, etc]. overall we find that N and its variants inhibit (interaction of HGF (and certain other GFs such as VEGF and bFGF) with GAGs, therefore (i) its prevent the binding of the GF concerned on its receptor, and (ii) the activity of said GF is reduced or even deleted.
We presume, but it is the dune theory guff does not bind the Applicant, that said peptide N and its variants bind GAGs, this guff stop (a) the adhesion of GAGs to HGF or the activation of HGF by GAGs, puffs

8 (b) the binding of HGF to HGF-R to activate the latter.
The consequence is that, in the absence of the fixing of point (b) above above, HGF does not exert its angiogenic effects.
We also note that the inhibition of the mechanism (a) + (b) concerns not only HGF but also other GFs which cannot attach to HGF-R, the HGF receptor, and to specific receptors from other GFs.
The variants according to the invention are those which derive from peptide N by - deletion, substitution or addition of one or more aa.
Here the deletion or deletion does not concern the Cys residue. In however the deletion or deletion can relate to a set of 1 to 5 aces contiguous. For example, one or more of the residues Met, Trp and / or Tyr, and, on the other hand, a peptide fragment of at most 5 ace located in the sequence of N notably between (a) Va133 and G1u38, (b) Leu66 and Va177, or (c) G1u84 and Asn9 °.
The substitutions here relate to one or more sets from 1 to 3 aa. For example, at least one of the Cys (or even all of them) can be replaced by Ser, and some residues with basic side chain (in particular Arg, His and Lys) can be replaced by Ala, Glu or Asp.
Additions mainly concern fragments from 1 to 3 as a basic side chain (preferably Arg and / or Lys) in the vicinity of basic amino acids isolated from N, especially in the vicinity of Lysl6, LysZy Ar 42 Ar 45 L s47 L s54 L s6 ° His83 L s9 'and Ar 95. Without modifying g ~ g ~ Y ~ Y ~ Y>> Y g factivite of N, can also incorporate 1 to 22 as upstream of Glnl (ie upstream of G1n32 in the sequence of native HGFJ, according to the vector of expression uses. So upstream of Gln ~, we can add Tune of following N-terminal ends (Al) M, (A2) MASMTGGQQMGRD, (A3) MGSSHHHHHSSGLVPRGSHM.
The variants of peptide N according to the invention can, of course, result from a combination of deletions, substitutions and / or additions.
The preparation process which is recommended according to (invention, involves using a DNA molecule encoding the N peptide or One of its puffs variants,

9 (1 °) introduce, on the side of fextremite 5 ′, a fragment of DNA coding for a first restriction site, (2 °) introduce on the side of the fextremite 3 ′ a DNA fragment coding for a second restriction site, steps 1 ° and 2 ° being carried out in any order, puffs, (3 °) insert the peptide resulting from all of the steps 1 ° and 2 ° in a bacterial plasmid, in particular an Escherichia plasmid coli, (4 °) cultivating the strain containing the plasmid thus modified to a temperature of 30-37 ° C, (5 °) express the N or fun peptide of its variants at a temperature from 25-30 ° C.
The peptide thus obtained is purified according to a method known per se, in particular by chromatography.
Examples 1 d 4 Example 1 of (invention is constituted by peptide N whose sequence of aces is given in SEQ ID No.: 2. The DNA fragment expressing Ex 1 is described in SEQ ID No.: 1.
Example 2 of (invention is the peptide constituted by A3-N. It has the A3 peptide at its upstream N-terminus of peptide N. The sequence of the aces of Ex 2 is given in SEQ ID No.: 4.
The DNA fragment expressing Ex 2 is provided in SEQ ID No.: 3.
Example 3 of the invention results from the deletion of a fragment of 4-amino acids (aa Met94 to G1y97) of Ex 2 (ie Met73 to G1y76 of peptide N).
The sequence of Ex 3 and that of the DNA which codes it are given in SEQ
ID No.: 5 and, respectively, SEQ ID No.: 7.
Example 4 of (invention results from Ex 2 by insertion of 2 aces (Lys-Arg) after Lyst ~ 2 (ie Lys91 of N), on the one hand, and replacement of the second-me and third residues Cys [Cys64 and Cys74 from Ex 2 (ie Cys43 and Cys53 from N)] by Ser, on the other hand. The sequence of Ex 4 and that of the DNA which codes it are given in SEQ ID No.: 6 and respectively, SEQ ID No.: 8.
Some of the results of the tests which have been carried out are summarized below.
undertaken. In these tests, HUVEC cells were used as main model of ex vivo angiogenesis analysis. HUVEC cells do not do not produce HGF, however they provide the proto-oncogene c-Met which expresses the HGF-R receptor of HGF and whose level is modular according to the culture conditions. Quiescent cells, at confluence, provide a small amount of HGF-R. This expression increases when the cells enter the exponential growth phase, or 5 when they form structures similar to capillaries (or tubes capillaries) in a three-dimensional collagen gel.
Trial 1 We learned how to stimulate the proliferation of HUVEC cells by bFGF in the absence and in the presence of increasing amounts of peptides

10 N, Kl, K2, K3 and K4, the proliferation of HUVEC cells being measured at [3 H] thymidine means.
The HUVEC cells are collected when they have reached the fetus of confluence and placed on 24-well plates at a concentration of 2 x 104 cells / well in 0.5 ml of M199i supplements with 2.5% FBS.
The cells are allowed to attach for 4 ha 37 ° C. and are induced the proliferation using 10 ng / ml of bFGF added alone or in combination with increasing quantities (0.1-50 ~ g / ml) of purified peptide N, K1, K2, K3 or K4. After additional 24 h incubation, 3.7 x 104 Bq are added (1 ~ .Ci) of [3H] thymidine (product sold by the company Amersham Pharmacia Biotech) at each well during the last 18 h of incubation.
The cells are washed three times with PBS puffs treated for 0.5 h with a 10% (w / v) solution of trichloroacetic acid cooled in the ice cream. The resulting precipitates are solubilized with NaOH 0.3 N (0.5 ml / well) for 0.5 ha RT. Then neutralized with 1N HCl. We measures radioactivity [3H] using a liquid scintillation counter (Beckman LS-6500 multipurpose scintillation counter). The results are expressed as average [count / minute (~ error standard to average)] of three cultures per dose of product to be tested.
The results recorded in Figure 3 show that none of the kringles K1 to K4 does not have an effect on the proliferation of HUVEC cells induced by bFGF since the counting of [3H] thymidine does not vary in a way statistically significant. In contrast, peptide N inhibits the bFGF-induced HWEC cell proliferation in one mode depending on the concentration, half of the maximum effect a dose of peptide N of approximately 7 ~ .M.

11 Trial 2 We appreciated the proliferation of cells. HWEC according to modalities described above by inducing the proliferation of these cells by addition of 10 ng / ml of HGF, or 10 ng / ml of each of the isoforms of VEGF (VEGFI6s and VEGFIZi) with bFGF in the punches, in (absence or in presence of 4 ~, M of peptide N. The results recorded in FIG. 4 (which are expressed in cpm) show that the peptide N inhibits in a way remarkable the proliferations of HUVEC cells induced by fun any of the FGs.
Trial 3 Chemotactic migration of HUVEC cells was evaluated according to a technique derived from previous wax (modified test with Boyden). Cell culture chamber inserts ("Transwells") paristale marketed by the company Corning Costar Corporation (Cambridge, MA) and provided with porous polycarbonate filters (pore dimensions: 8 Vim) were coated with 0.1% gelatin. of the HUVEC cells (4 x 104) in M199i medium supplements with 2.5% of FBS have been added to Transwell devices. The inserts are placed above above chambers containing 10 ng / ml of HGF and one of the purified peptides N, K1, K2, .K3 or K4, each has a dose of 50 ~ g / ml. We incubate them rooms for 5 ha 37 ° C. The filters are then rinsed with PBS, fixed and stained using hematoxylin. The upper surface of the filters is scraped to remove cells that have not migrated. We record the number of cells in a high power field (x 400) is due to 10 fields / filter in triplicate.
The results recorded in Figure 5 highlight that the peptide N completely inhibits the induced migration of HWEC cells a through the porous membrane. We also observe that some kringles (K3 and K4) have a weak inhibitory effect, this effect however not statistically significant.
Trial 4 Peptide N inhibition of tube formation has been studied capillaries of HUVEC cells in a three-dimensional collagen gel.
Type 1 rat tail collagen was obtained from the BD company Biosciences (Bedford, MA) and gels were formed according to

12 manufacturer's recommendations. In short, a cooled collagen solution with ice is neutralized by addition of 1N NaOH and mixed with PBS
in the 9/1 v / v ratio. HUVEC cells, isolated by treatment with using trypsin-EDTA and suspended in M199i medium supplements with 2.5% FBS, are mixed with the neutralized solution of collagen to a final concentration of 1 mg / ml of collagen. We pour 0.4 ml of this suspension (5 x 10 cells / ml) into the well of a 24-well plate and the gels are allowed to form by incubation at 37 ° C
for 40 minutes. The gels are coated with 0.5 ml of M199i medium supplements with 2.5% FBS and 10 ng / ml of one of the three FGs: bFGF, VEGF ~ 6s or HGF. The purified peptide N (1 ~ .M) is added to the wells.
same time as the croissant factor. Cultures are incubated for 48 ha 37 ° C in a humid atmosphere containing 5% C02. We examine the gels using a microscope ("Zeiss Axiovert 25") and record images using "Archimed Pro 2.1" software.
When the HUVEC cells are grown in a gel of collagen, they quickly give rise to a morphogenesis to sleep a net of capillary tubes.
The formation of these tubes occurs spontaneously or is induced by increasing factors. The results recorded in Figure 6 show that bFGF, VEGFI6s and HGF induce in experimental conditions used (organization of the majority of HUVEC cells in tubes capillaries. The formation of these tubes is totally inhibited by the peptide N according to the invention when added at the same time as any fun said increasing factors. No changes are also observed of HUVEC cell morphogenesis when tube formation capillaries is induced in the presence of one of the kringles K1, K2, K3 or K4 (these results are not shown in figure 6).
Trial 5 To demonstrate that the peptide N according to the invention constitutes the main site involves in the HGF interaction with a GAG such as Hep, we analyzed the affinities of peptides N, K1, K2, K3 and K4 for hsparine by surface plasmon resonance (in English: "surface plasmon resonance ") using a" BIAcore 2000 "instrument (sold by the company BIAcore, Uppsala, Sweden). High-weight fsparin

13 molecule has been biotinylated with the product EZ-Link Biotin-LC-Hydrazide (manufactured by Pierce, Rockford, IL) and was used to be captured on the surface of a detector ("sensor ship") coated with streptavidin.
The purified peptides were diluted in the usual buffer of (instrument S BIAcore (PBS containing 0.005% tween-20) and injected at a rate of 20 ml / minute on immobilized heparin. The experiments were performed in a peptide concentration range. The results obtained were automatically analyzed using software BIAevaluation of the company BIAcore. The results obtained were instructions in Figure 7 where Fon sees that peptide N has a very large life-to-life affinity of fheparin with respect to the four kringles Kl, K2, K3 and K4. All five peptides are capable of fheparin-like but they have differences in connection. The most answer important is that obtained for the binding of 0.7 ~ M of peptide N, that is a say at a concentration approximately 20 times lower than those kringles (approximately 2,000 RU for peptide N instead of 600 RU for K3). At physiological concentrations (HGF / GAG interaction is therefore mainly provided by peptide N.
Trial 6 Test 5 above has been reproduced to assess the affinity of the fixation of soluble HGF-R [chemical product which is a protein of fusion (HGF-R / IgG) comprising the portion of the extracellular domain of HGF-R fixing HGF and a portion of IgG], weight heparin normal molecular (Hep) and low-weight fractionated heparin molecular (LMWH), on HGF or N each immobilized on the surface of the detector ("sensor ship").
We find that Hep has a greater affinity (25 times more) than LMWH vis-à-vis HGF and N.
Regarding the binding of soluble HGF-R and Hep to HGF and N, the results obtained, which are recorded in FIGS. 8A, 8B, 8C and 8D for three different doses, highlight the selective fixation of Hep on N and the absence of binding of soluble HGF-R on N.
Trial 7 The fixation and displacement of radiolabeled ligands have been studied.
HGF (5 fig), VEGF ~ 65 (10 fig) and N (25 fig) [or if applicable K1 (25 fig)]

14 were iodized with 1 mCi (ie 3.7 x 10 'Bq) of [l2sl] Na using the "Iodo-geri Pre-Coated Iodination Tubes" material as indicated in the maker. The isolated proteins were purified on balanced colonies at 25 mM Tris-HCl pH 7.4 containing 0.4 M NaCl, 5 mM
EDTA and 0.25% of AIbuMAX (supplied by the company called INVITROGEN), and concentrated by centrifugation. The specific activities determined by precipitation with trichloroacetic acid are 18 x 106 cpm / pmol for (lasl] HGF, 8 x 106 cpm / pmol for (l2sl] VEGFI6s, and 9 x 10 cpm / pmol for (l2sl] N.
Fixation and displacement studies with iodine proteins have was carried out on HUVEC cells at confluence, at 4 ° C., on plates at 24 wells. Cells were washed with PBS and preincubated for 0.5 h with 0.5 ml of fixing medium (normal medium containing 0.1 of AIbuMAX). To determine the fixation parameters with regard to for N, increasing amounts of N radiolabels have been added to HUVEC cells, with or without molar excess of 100 in N unmarked, and incubated for 2 h. For displacement experiments, quantities constants of HGF (1 nM) or VEGF ~ 65 (0.2 nM) were added to HUVEC cells in the presence of an increasing concentration of N non mark, for 4h and 2h, respectively. At the end of the incubation the cells were washed three times with puffs fixation medium solubilized with 0.3 M NaOH. The radioactivity associated with HUVEC cells has been determined by means of an appropriate counter.
The results obtained show that N inhibits the binding of HGF and VEGFI6s on HUVEC, and that K1 does not intervene here as an inhibitor.
The inhibition by N is a function of the concentration of said N.
In this guff concerns the displacement, we observe that the kinetics corresponds to a simple reaction model of the bimolecular type. The Kd values calculated using appropriate software are 20.40 ~ 5.11 nM for HGF, and 56.17 t 17.18 nM for VEGF ~ 6s.
See Figure 9A and 9B.

conclusions With regard to the results given above in tests 1-7 and instructions in Figures 3-9, it is clear that peptide N acts as as an HGF inhibitor. Unlike the already known NK4 peptide, the 5 peptide N according to the invention does not substantially affect the fixation competitive with HGF vis-à-vis the HGF-R receptor. However, with regard to tests 5-6 and FIGS. 7-8, the peptide N is more involved precisely at the level of the interaction of HGF with a GAG such as Hep in preventing Hep adhesion to HGF or Hep activation of HGF;
10 consequently HGF can no longer act on or activate HGF-R so that it exerts its tyrosine kinase effect.
The peptides of Examples 2-4 exhibit the same mode of action.
than peptide N. As antiangiogenic substances, N and its variants are useful in the treatment of disorders associated with

15 angiogenesis and in particular induced by hyperangiogenesis. They are particularly indicated ~ as an inhibitor of neovascularization and HGF antagonists (especially with regard to rheumatoid arthritis, diabetic retinopathy, degeneration of the macula related to Page and excessive scar formation during scarring injury), ~ as modulators of abnormal cell proliferation (especially vis-à-vis psoriasis, Osler-Webber syndrome, angiomas and benign or malignant tumors), and ~ as inhibitors of cell hyperstimulation Endothelial.
In short, peptide N and its variants are particularly interesting as anti-neovascularization, anti-rheumatism agents, antiarthritis, anti-inflammatory, anti-sporiasis, anti-proliferation, antitumor and / or cytostatic.

SEQUENCE LISTING

<110> INSTITUTE AND U
BLOOD VESSELS

<120> Peptide growth factor hepatocytaire isolated from fa from a t its variants, of eparation use in proce d pr and therapeutic iangioge niques in what that <130> 16224SC

1 ~

<140>

<141>

<160> 8 <170> PatentIn Worm. 2.1 <210> 1 <211> 288 <212> DNA

<213> HUMAN

<220>

<221> CDS

2S <222> (1) .. (288) <400> 1 caa agg aaa agaaat acaattcatgaa ttcaaaaaa tcagcaaag 48 aga G1n Arg Lys ArgAsn ThrIleHisGlu PheLysLys SerAlaLys Arg 3 ~ 1 5. 10 15 act acc cta aaaata gatccagcactg aagataaaa accaaaaaa 96 atc Thr Thr Leu LysIle AspProA1aLeu LysIleLys ThrLysLys Isle gtg aat act gaccaa tgtgetaataga tgtactagg aataaagga 144 gca Val Asn Thr AspGln CysAlaAsnArg CysThrArg AsnLysGly To the 35 40 ~ 95 4 ~ ctt cca ttc tgcaag gettttgttttt gataaagca agaaaa.caa 192 act Leu Pro Phe .CysLys AlaPheValPhe AspLysAla ArgLysGln Thr tgc ctc tgg cccttc aatagcatgtca agtggagtg aaaaaagaa 240 ttc 45 Cys Leu Trp ProPhe AsnSerMetSer SerGlyVal LysLysGlu Phe ttt ggc cat tttgac ctctatgaaaac aaagactac attagaaac 288 gaa Phe Gly His PheAsp LeuTyrGlu ~ -1snLysAspTyr IleArgAsn Glue $ ~ 85 90 95 <210> 2 <211> 96 <212> PRT
$$ <213> HUMAN

<400> 2 Gln Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr Thr LeuI7.eLysIleAspPro AlaLeuLys IleLysThr LysT;, ys Va1 Asn ThrAla AspG1nCysAla AsnArgCys ThrArgPan LysGly, Leu Pro PheThr CysLysAlaPhe ValPheAsp LysAlaArg LysG1n Cys Leu TrpPhe ProPheAsnSer MetSerSer G1yValLys LysGlu Phe Gly HisGlu PheAspLeuTyr GluAsnLys AspTyrIle ArgAsn 20 <210>

<211>

<212>
DNA

<213>
SYNTHETIC

25 <220>

<221>
CDS

<222> (351) (1) ..

<400>

30atg ggc agcagc catcatcatcat catcacagc agcggcctg gtgccg 48 Met Gly SerSer HisHisHisHis HisHisSer SerGly, Leu ValPro cgc ggc agccat atgcaaagg ~ aaa agaagaaat acaattcat gaattc 96 35Arg Gly SerHis MetGlnArgLys ArgArgAsn ThrIleHis GluPhe aaa aaa tca ~ gca aagactacccta ~ atcaaaata gatccagca ctgaag 144 Lys Lys SerAla LysThrThrLeu IleLysIle AspProAla LeuLys ata aaa accaaa aaagtgaatact gcagaccaa tgtgetaat agatgt 192 Ile Lys ThrLys LysValAsnThr AlaAspGln CysAlaAsn ArgCys act agg aataaa ggacttccattc acttgcaag gettttgtt tttgat 240 Thr Arg AsnLys GlyLeuProPhe ThrCysLys AlaPheVal Phe.
Asp S ~ aaa gca agaaaa caatgcctctgg ttccccttc aatagcatg tcaagt 288 Lys Ala ArgLys GlnCysLeuTrp PheProPhe AsnSerMet SerSer gga gtg aaaaaa gaatttggccat gaatttgac ctctatgaa aacaaa 336 55GIy Val LysLys GluPheGlyHis GluPheAsp LeuTyrGlu AsnLys gac tac att aga aac 351 Asp Tyr Ile Arg Asn <210> 4 <211> 117 <212> PRT

IO <213> SYNTHETIC

<400> 4 Met Gly Ser HisHisHisHis HisHisSer SerGlyLeu Pro Ser Val Arg G2y His MetGlnArgLys ArgArgAsn ThrIleHis GluPhe Ser 2p 25 30 Lys Lys A1a LysThrThrLeu IleLysIle AspProAla LeuLys Ser 20 ~ 35 4 4 Ile Lys Lys LysVa1AsnThr AlaAspGln CysAlaAsn ArgCys Thr 50 55 6.0 25Thr Arg Lys GlyLeuProPhe ThrCysLys AlaPheVal PheAsp Asn Lys Ala Lys GlnCysLeuTrp PheProPhe AsnSerMet SerSer Arg Gly Val Lys GluPheGlyHis GluPheAsp LeuTyrGlu AsnLys Lys Asp Tyr Ile Arg Asn $ 3 115 <210> 5 <211> 113 40 <212> PRT
<213> SYNTHETIC
<90Q>

45 Met GlySerSer HisHisHis HisHisHis SerSerGlyLeu ValPro Arg GlySerHis MetG1nArg LysArgArg AsnThrIleHis GluPhe 2p 25 30 Lys LysSerAla LysThrThr LeuIleLys IleAspProAla LeuLys Ile LysThrLys LysValAsn ThrAlaAsp GlnCysAlaAsn ArgCys Thr Arg Asn Gly LeuPro Phe CysLysAla Phe PheA
Lys Thr Val Sp 65 70 75 gp Lys Ala Arg Gln CysLeu Trp ProPheAsn Ser LysL
Lys Phe Val ys $ g5 90 95 Glu Phe Gly GlizPheAsp Leu GluAsnLys Asp IleArg His Tyr Tyr Asn <210> 6 <211> 119 <212> PRT
<213> SYNTHETIC
2 ~ <900>

Met GlySerSer HisHisHisHis HisHis SerSerGlyLeu ValPro Arg G1ySerHis MetGlnArgLys ArgArg AsnThrIleHis GluPhe Lys LysSerAla LysThrThrLeu IleLys IleAspProAla LeuLys 3 ~ Ile LysThrLys LysValAsnThr AlaAsp G1nCysAlaAsn ArgSer Thr ArgAsnLys GlyLeuProPhe ThrSer LysAlaPheVal PheAsp Lys AlaArgLys GlnCysLeuTrp PhePro PheAsnSerMet SerSer Gly ValLysLys GluPheGlyHis G1uPhe AspLeuTyrG1u AsnLys loo l05 110 Lys ArgAspTyr IleArgAsn $ 4 <210> 7 <211> 339 <212> DNA
<2i3> SYNTHETIC
<220>
<221> CDS
<222> (1) .. (339) $$

<400>

atg ggc agcagccat catcatcat catcacagcagc ggcctggtg c 48 VS

Met Gly SerSerHis HisHisHis HisHisSer5er GlyLeuVal g pro cgc ggc agccatatg caaaggaaa agaagaaataca attcatgaa t 96 tc Arg Gly SerHisMet GlnArgLys ArgArgAsnThr IleHisGlu P
Hey l ~ aaa aaa tcagcaaag actacccta atcaaaatagat ccagcactg a 144 ag Lys Lys SerAlaLys ThrThrLeu IleLysIleAsp ProAlaLeu L
ys 35 40 q5 ata aaa accaaaaaa gtgaatact gcagaccaatgt getaataga t 192 gt 15Ile Lys ThrLysLys ValAsnThr AlaAspGlnCys AlaAsnArg Cys 50 55. 60 act agg aataaagga cttccatte acttgcaagget tttgttttt gat 240 Thr Arg AsnLysGly LeuProPhe ThrCysLysAla PheValPhe Asp 2065 70 7s 80 aaa gca aga aaa caa tgc ctc tgg ttc ccc ttc aat agc gtg aaa aaa 288 Lys Ala Arg Lys Gln Cys Leu Trp Phe Pro Phe Asn Ser Val Lys I, ys gaa ttt ggc cat gaa ttt gac ctc tat gaa aac aaa gac tac att aga 336 Glu Phe G1y His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg 3 ~ aac 339 Asn <210> s <211> 357 <212> DNA
<213> SYNTHETIC
<22p>
<221> CDS
<222> (1) .. (357) <900> 8 atg ggc agc agc cat cat cat cat cat cac agc agc, ggc ctg gtg ccg 98 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val rro cgc ggc agc cat atg caa agg aaa aga aga aat aca att cat gaa ttc 96 S ~ Arg Gly 5er His Met Gln Arg Lys Arg Arg Asn Thr Ile His Glu Phe aaa aaa tca gca aag act acc cta atc aaa ata gat cca gca ctg aag 194 Lys Lys Ser Ala Lys Thr Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys 35 40 q5 ata aaa accaaaaaagtg aatactgca gaccaatgtget aatagaa 192 gc Ile Lys ThrLysLysVal AsnThrAla AspG1nCysAla AsnArgS
st act agg aataaaggactt ccattcact agcaaggetttt gtttttg 240 Has Thr Arg AsnLysGlyLeu ProPheThr SerLysAlaPhe ValPheZ ~ sp aaa gca agaaaacaatgc ctctgg.ttccccttcaatagc atgtcaa 288 gt 10Lys Ala ArgLysGlnCys LeuTrpPhe ProPheAsnSer MetSerSer g5 90 95 gga gtg aaaaaagaattt ggccatgaa tttgacctctat gaaaacaaa 336 Gly Val LysLysGluPhe GlyHisGlu PheAspLeuTyr GluAsnZ, ys aaa agg gactacattaga aac 357 Lys Arg AspTyrIleArg Asn

Claims (11)

Claims 1. Use of a peptidic substance, characterized in that one makes use of a peptidic substance chosen from the group consisting of:
(i) peptide N from the a chain of HGF and having the sequence amino acid represented by SEQ ID No.:2, and (ii) its peptide variants derived from the sequence of said peptide N by deletion, substitution or addition of one or more amino acids, said peptide N and its variants having antiangiogenic activity according to a different mechanism of competitive binding with HGF on the receptor HGF-R, said peptide N and its variants which selectively bind GAGs such as heparin, and preventing the interaction of HGF with GAGs by which HGF binds to or activates the HGF-R receptor, for the preparation of an antiangiogenesis medicament for use in therapy for disorders associated with angiogenesis. 2. Therapeutic composition characterized in that it contains, in association with a pharmaceutically acceptable excipient, an amount therapeutically effective peptide substance selected from the group seems to consist of:
(i) peptide N from the .alpha. of HGF and having the sequence amino acid represented by SEQ ID No.:2, and (ii) its peptide variants derived from the sequence of said peptide N by deletion, substitution or addition of one or more amino acids, to combat disorders associated with angiogenesis. 3. Peptide substance belonging to the group comprising:
(i) the .alpha chain peptide N. of HGF, and (ii) variants derived therefrom by deletion, substitution or addition one or more amino acids, said peptidic substance being characterized in that it is chosen from the peptides represented by SEQ ID No.:2, SEQ ID No.:4, SEQ ID No.:5 and SEQ ID No.:6. 4. DNA fragment, characterized in that it codes for the substance peptide according to claim 3. 5. Plasmid characterized in that it contains the following DNA fragment claim 4. 6. Bacterial strain containing the plasmid according to claim 5. 7. Process for preparing a peptide substance according to the claim 3, in which use is made of a molecule, of DNA encoding for peptide N or one of its variants, said method being characterized in this that it includes the steps of:
(1~) introduce on the side of the 5 'end a DNA fragment encoding for a first restriction site, (2~) introduce on the side of the 3 'end a DNA fragment encoding for a second restriction site, steps 1~ and 2~ being carried out in any order, then, (3~) insert the peptide resulting from all of steps 1~ and 2~ into a bacterial plasmid, in particular an Escherichia coli plasmid, (4~) cultivating the strain containing the plasmid thus modified at a temperature of 30-37°C, and (5~) expressing the peptide N or one of its variants at a temperature of 25-30°C. 8. Peptide substance according to reveradication 3, characterized in that its amino acid sequence is that of SEQ ID No.:4 9. Peptide substance according to claim 3, characterized in that that its amino acid sequence is that of SEQ ID No.:5. 10. Peptide substance according to claim 3, characterized in that that its amino acid sequence is that of SEQ ID No.:6. 11. Peptide substance according to claim 3, characterized in that that its amino acid sequence is cells of SEQ ID No.:2.