JP2005508637A - Isolated hepatocyte growth factor peptide and variants thereof, methods of preparation, and therapeutic use as anti-angiogenic agents - Google Patents

Abstract

  The present invention relates to a peptide substance selected from the group consisting of: (i) a peptide N derived from the HGF α chain and having the amino acid sequence represented by SEQ ID NO: 2, and (ii) from the peptide N sequence, Peptide variants derived by deletion, substitution or addition of one or several amino acids. The peptide N has anti-angiogenic activity by a mechanism different from the competitive binding of HGF to the HGF-R receptor. The present invention also relates to therapeutic uses and methods of preparation of the peptide substances, and (i) DNA fragments that express it, plasmids containing the DNA, and bacteria carrying the plasmid.

Description

FIELD OF THE INVENTION The present invention relates to an isolated peptide of hepatocyte growth factor (HGF) (referred to herein as N), and variants thereof, as novel industrial products. The present invention also relates to methods of preparing them, and their use in therapy as anti-angiogenic agents that intervene as inhibitors of angiogenesis.

  All abbreviations and acronyms used herein are defined later.

  HGF is now recognized as a multifaceted factor with a fairly broad specificity for many cellular targets. In particular, HGF plays an important role as a mediator in mesenchymal-epithelial / endothelial interactions involved in complex biological processes such as embryogenesis, tissue regeneration and tumorigenesis (K. Matsumoto et al., Ciba Found Symp., 1997; 212: 198-211). Although HGF is also known to be a potent activator of angiogenesis (F. Bussolino et al., J. Cell Biol., 1992; 119: 624-641), its in vivo efficacy is It is considered larger (E. van Belle et al., Circulation, 1998; 97: 381-390).

Structurally speaking, HGF is a peptide related to enzymes of the blood coagulation cascade. Mature HGF is a peptide consisting of two chains (ie subunits): an α and a β chain. In native HGF (see FIG. 1 below), the amino acid sequence portion (aa 1-478) that gives the α chain comprises the following between its N-terminus and C-terminus:
(i) Peptide L comprising 31 amino acid residues (aa 1-31), where this peptide L is (i) probably not interesting from a pharmacological point of view, but (ii) is involved as a signal peptide ,
(ii) a domain N containing 96 hair residues (aa 32-127) and containing 4 Cys residues and containing a “hairpin” loop (this peptide N is a peptide useful according to the invention) And)
(iii) 4 kringle domains (referred to as K1, K2, K3 and K4) each comprising 6 or 7 Cys residues, where K1 comprises 79 amino acid residues (aa 128-206) K2 comprises 78 amino acid residues (aa 211-288), K3 comprises 79 amino acid residues (aa 305-383) and K4 comprises 79 amino acid residues (aa 391-469).

  The alpha chain of HGF is the primary field of interaction between HGF and its receptor, which is expressed by the c-Met proto-oncogene and (after HGF binds to that receptor) tyrosine Has kinase enzyme activity. In the text below, this receptor is referred to as HGF-R.

  The β chain, which is homologous to the catalytic domain of serine protease, does not appear to bind to the HGF-R receptor, but is essential for the autophosphorylation of the receptor and eventually results in specific factors (“ Involved in intracellular signal transduction for interaction with effectors ").

  A peptide consisting of an α chain, lacking a β chain, binds to the HGF-R receptor, which is an HGF antagonist (K. Date et al., FEBS Letters, 1997; 420: 1-6) and a competitive mechanism. Acts as an inhibitor of the biological response of HGF, and more particularly the angiogenic response of this factor (K. Kuba et al., Cancer Res., 2000; 60: 6737-6743).

It is well known from WO-A-99 / 55361 in particular that the HGF α chain has been sequenced. More precisely, the peptide named NK4 starts with a pyroglutamate group and comprises the amino acid of the α chain (ie from aa Pyrglu ³² to Val ^{478 of} native HGF), which has an antitumor effect ( EP-A-0890361) and has an anti-angiogenic effect, which is evident from inhibition of angiogenesis (literature and application WO-A-99 / 55361 by K. Kuba et al., Supra).

In short, the mechanisms known to date are as follows:
(A) Upon cleavage of peptide L, native HGF becomes mature HGF (ie, activated);
(B) Mature HGF binds to the HGF-R receptor via its α chain;
(C) As a result of binding of mature HGF to HGF-R, the enzyme activity of HGF-R appears, which is a tyrosine kinase type activity; this means “activation”, but this activation Enables enzymes contained in the intracellular domain of the HGF-R protein to exert their tyrosine kinase potency;
(D) When peptide NK4 is used, NK4 functions as a competitive inhibitor of HGF because NK4 binds to this receptor without HGF-R exerting its own tyrosine kinase enzyme activity.

Origin of the invention
The K1, K2, K3, and K4 domains of the HGF alpha chain are very similar to the anti-angiogenic kringles of certain other proteins (eg, angiostatin, and the second kringle of prothrombin) and are therefore ongoing (1) whether the kringles K1, K2, K3 and / or K4 of the HGF α chain have anti-angiogenic activity, and (2) the expected anti-angiogenic activity of one or more of the kringles The question arises whether this mechanism is the competitive binding of K1, K2, K3 and / or K4 to the HGF-R receptor.

After isolating and testing the peptides N, K1, K2, K3 and K4 of the HGF α chain, surprisingly, peptide N is almost encompassed by the sequence of NK4, where N is the first amino acid sequence of NK4. The amino acid is different and Gln ¹ is replaced by Pyrglu ¹ (ie, position 32 of native HGF). This peptide N has anti-angiogenic properties expressed by a mechanism different from NK4 kringle. Became clear.

Briefly, as explained below, the following was observed from the present invention:
N acts as an inhibitor of HGF by a different mechanism than competitively binding HGF to the HGF-R receptor; according to the applicant's assay results, N is different from NK4 There is good evidence that N does not substantially bind to HGF-R; it is believed that N acts as an inhibitor of HGF from the point of time that interferes with the interaction between HGF and GAG by inhibiting HGF / GAG binding;
-K1, unlike K2, K3 and K4, to the best of Applicants' present knowledge, is the only Kringle of HGF that works effectively by competing with HGF for binding to the HGF-R receptor .

Supplementally, peptide N comprises a NK4 hapten and is derived from the HGF α chain, but this peptide N contains amino acids 2-96 of NK4 in its sequence (ie, Arg ^{33 of} native HGF). -Asn ¹²⁷ ), but the first amino acid is different from NK4 (ie, Gln32 instead of pyroglutamate group PyrGlu32 of native HGF according to EP-A-0890361 and WO-A-99 / 55361 above) .

Objects of the Invention It is presented according to the present invention to provide (a) a novel substance with anti-angiogenic properties, (b) a method of preparing said, and (c) a therapeutic use of said substance.

The subject of the invention, a novel peptide substance is presented, which is characterized in that it is selected from the group consisting of:
(i) peptide N derived from the HGF α chain and having the amino acid sequence represented by SEQ ID NO: 2, and
(ii) a variant of the peptide derived from the peptide N sequence by deletion, substitution or addition of one or more amino acids,
Peptide N and its variants exhibit anti-angiogenic activity by a mechanism that is different from the competitive binding of HGF to the HGF-R receptor.

According to another aspect of the present invention, a therapeutic composition is proposed, which composition, together with a pharmaceutically acceptable excipient for the purpose of treating angiogenesis related diseases, is a therapeutic selected from the following group: Containing an effective amount of peptide substance:
(i) peptide N derived from the HGF α chain and having the amino acid sequence represented by SEQ ID NO: 2, and
(ii) A variant of the peptide derived from the peptide N sequence by deletion, substitution or addition of one or more amino acids.

  It is also proposed to use the anti-angiogenic peptide substance for the angiogenesis related diseases.

  Finally, a DNA fragment encoding peptide N, or a variant thereof as described above, a plasmid containing the DNA fragment, and a bacterium containing the plasmid are also presented as new industrial products.

Detailed Description of the Invention
The hHGF factor has already been sequenced. In particular, the sequence PyrGlu ³² -Val ⁴⁷⁸ of NK4 is given in WO-A-99 / 55361. According to the prior art, NK4 functions as an anti-tumor and anti-angiogenic agent by a competitive mechanism between NK4 and HGF for the HGF receptor HGF-R.

  After synthesis (by expression based on genetic engineering techniques) and subsequent purification, peptide N of the invention (the first amino acid at the N-terminus of this peptide is Gln, not PyrGlu) and its variants were tested. Peptide N is novel to the teaching of the NK4 sequence, but this peptide N and its variants include GAG (especially Hep (high molecular weight heparin, normal and low molecular weight heparin), dermatan sulfate, heparan sulfate ], It differs from K1, K2, K3 and K4 in terms of their interaction. Considering all, it is noted that N and its variants inhibit the interaction of HGF (and other specific GFs such as VEGF and bFGF, for example) with GAG; ) N and its variants prevent GF binding to the receptor, and (ii) the activity of the GF is reduced or almost absent.

The following content is presumed, but this is one theory and the applicant is not bound to it. That is, the peptide N and its variants bind to GAG,
(a) prevents GAG adhesion to HGF or activation of HGF by GAG, and (b) prevents HGF binding to HGF-R to activate HGF-R.

  In conclusion, in the absence of the above point (b) binding, the angiogenic action of HGF is not exerted.

  In addition, the inhibition of the mechanism of (a) + (b) is not limited to HGF but also to HGF-R, which is a receptor for HGF, and to other receptors that are not capable of binding to their own specific receptors. It is noted that it also affects GF.

  The mutant of the present invention is derived from peptide N by deletion, substitution or addition of one or more amino acids.

Here, deletion or elimination does not include a Cys residue. On the other hand, this deletion or elimination can involve from 1 to 5 adjacent amino acid groups. For example, first one or more Met, Trp and / or Tyr residues are deleted, then in the sequence of N, more particularly (a) Val ³³ to Glu ³⁸ , (b) Leu ⁶⁶ to Val ⁷⁷ , Or (c) a peptide fragment of no more than 5 amino acids between Glu ⁸⁴ and Asn ⁹⁰ can be excised.

  Now, the substitution involves one or more amino acid groups consisting of 1 to 3 amino acids. For example, at least one Cys (or even all of Cys) can be replaced with Ser, and certain residues with basic side chains (especially Arg, His and Lys) can be replaced with Ala, Glu or Can be replaced with Asp.

Addition mainly in close proximity to N isolated basic amino acids, in particular Lys ¹⁶ , Lys ²¹ , Arg ⁴² , Arg ⁴⁵ , Lys ⁴⁷ , Lys ⁵⁴ , Lys ⁶⁰ , His ⁸³ , Lys ⁹¹ and Close to Arg ⁹⁵ is involved a fragment consisting of 1 to 3 amino acids with a basic side chain (preferably Arg and / or Lys). Depending on the expression vector used, it is possible to incorporate 1 to 22 amino acids upstream of Gln ¹ (ie upstream of Gln ³² of the native HGF sequence) without altering the activity of N. Thus, ^one of the following N-termini can be added upstream of Gln ¹ :
(A) M,
(A2) MASMTGGQQMGRD,
(A3) MGSSHHHHHSSGLVPRGSHM.
The variants of peptide N of the invention naturally result from a combination of deletions, substitutions and / or additions.

The preparation method proposed by the present invention is to use a DNA molecule encoding peptide N or one of its variants, and subsequently carry out the following sections:
(1) introducing a DNA fragment encoding the first restriction enzyme cleavage site at the 5 ′ end;
(2) introducing a DNA fragment encoding a second restriction enzyme cleavage site at the 3 ′ end, steps 1 and 2 are performed in any order, then
(3) inserting the peptide resulting from steps 1 and 2 into a bacterial plasmid, in particular a plasmid of Escherichia coli,
(4) culturing the strain having the plasmid modified as described above at a temperature of 30-37 ° C.,
(5) To express peptide N or one of its variants at a temperature of 25-30 ° C.

  The peptide thus obtained is purified by methods known per se, in particular by chromatography.

Examples 1 to 4 Example 1 (Ex1) of the present invention is composed of peptide N, which is the amino acid sequence given by SEQ ID NO: 2. A DNA fragment expressing Ex1 is shown in SEQ ID NO: 1.

  Example 2 (Ex2) of the present invention is a peptide consisting of A3-N. This comprises peptide A3 at its N-terminus, upstream of peptide N. The amino acid sequence of Ex2 is shown in SEQ ID NO: 4. A DNA fragment expressing Ex2 is given in SEQ ID NO: 3.

Example 3 (Ex3) of the present invention results from deletion of a 4-amino acid fragment from Ex2 (amino acids Met ⁹⁴ to Gly ⁹⁷ ) (ie, Met ⁷³ to Gly ⁷⁶ of peptide N). The sequence of Ex3 and the DNA sequence encoding it are given in SEQ ID NO: 5 and SEQ ID NO: 7, respectively.

In Example 4 (Ex4) of the present invention, from Ex2, firstly, Lys ¹¹² (ie, Lys ^{91 of} N) was inserted followed by 2 amino acids (Lys-Arg), and then the second and third Cys residues [ It is obtained by replacing Ex2 Cys ⁶⁴ and Cys ⁷⁴ (ie, N Cys ⁴³ and Cys ⁵³ )] with Ser. The sequence of Ex4 and the DNA sequence encoding it are given in SEQ ID NO: 6 and SEQ ID NO: 8, respectively.

  Some of the results of the assays performed are summarized below. In the assay below, HUVEC cells were used as the primary model for analyzing angiogenesis ex vivo. HUVEC cells do not produce HGF; on the other hand, the cells provide a c-Met proto-oncogene that expresses the HGF receptor HGF-R, whose levels can be regulated by culture conditions. Confluent, quiescent cells give a small amount of HGF-R. This expression increases when cells enter the logarithmic growth phase or when they form capillary-like structures (or capillaries) in a three-dimensional collagen gel.

Assay 1
HFGFEC cell growth stimulation by bFGF was evaluated without and with increasing amounts of N, K1, K2, K3 and K4 peptides. HUVEC cell proliferation was measured with [ ³ H] thymidine.

When HUVEC cells reach confluence, the cells are collected and placed in a 24 well plate at a concentration of 2 × 10 ⁴ cells / well in 0.5 ml of M199i supplemented with 2.5% FBS. Cells are allowed to attach for 4 hours at 37 ° C., and 10 ng / ml bFGF alone is added, or the amount of purified peptides N, K1, K2, K3 and K4 is increased (0.1-50 μg / ml). ml) In combination, induce proliferation. After an additional 24 hours of incubation, 3.7 × 10 ⁴ Bq (1 μCi) of [ ³ H] thymidine (a product sold by Amersham Pharmacia Biotech) is added to each well during the last 18 hours of incubation. The cells are washed three times with PBS and then treated with a 10% (w / v) trichloroacetic acid solution cooled in an ice bath for 0.5 hour. The resulting precipitate is solubilized with 0.3 N NaOH (0.5 ml / well) for 0.5 h at room temperature. The solution is then neutralized with 1N HCl. Radioactivity [ ³ H] is measured using a liquid scintillation counter (Beckman LS-6500 multipurpose liquid scintillation counter). Results are expressed as the mean [count / min (± standard error)] of 3 cultures per test product.

The results recorded in Figure 3 show that none of Kringle K1 through K4 has an effect on bFGF-induced HUVEC cell proliferation because [ ³ H] thymidine counts do not change statistically significantly. Indicates. In contrast, peptide N inhibits bFGF-induced HUVEC cell proliferation in a concentration-dependent manner, but half of its maximum effect occurs when using approximately 7 μM peptide N.

Assay 2
Proliferation of HUVEC cells was assessed according to the above-described manner, with 10 ng / ml HGF or 10 ng / ml VEGF isoform (VEGF ₁₆₅ and The proliferation of the cells was induced by the addition of VEGF ₁₂₁ ), respectively. The results shown in FIG. 4 (expressed as cpm) clearly show that peptide N inhibits proliferation of HUVEC cells induced by any GF.

Assay 3
Migration due to chemotaxis of HUVEC cells was evaluated by a technique derived from the prior art (a modification of the Boyden chamber method). Wall cell culture chamber (“Transwell”) inserts, sold by Corning Costar Corporation (Cambridge, MA) and equipped with a porous polycarbonate filter (pore size: 8 μm), were coated with 0.1% gelatin. HUVEC cells (4 × 10 ⁴ ) in M199i medium supplemented with 2.5% FBS were added to the transwell apparatus. The insert is mounted on a chamber containing 10 ng / ml HGF and one of each of the purified peptides N, K1, K2, K3 or K4 in an amount of 50 μg / ml each. Incubate the chamber at 37 ° C. for 5 hours. The filter is then rinsed with PBS, fixed with hematoxylin and stained. Scrape the top surface of the filter to remove cells that did not migrate. The number of cells in the high migration area (× 400) is recorded in triplicate at a rate of 10 areas / filter. The results shown in FIG. 5 show that peptide N completely inhibits the induced migration of HUVEC cells that cross the porous membrane. Some kringles (K3 and K4) were also observed to show weak inhibitory effects, however, this effect is not statistically significant.

Assay 4
We investigated whether peptide N inhibits the formation of HUVEC cell capillaries in 3D collagen gel. Rat tail type 1 collagen was obtained from BD Biosciences (Bedford, MA) and the gel was made according to the manufacturer's instructions. Briefly, ice-cold collagen solution is neutralized by addition of 1N NaOH and mixed with PBS at a 9/1 v / v ratio. HUVEC cells are individually separated by trypsin-EDTA treatment and suspended in M199i medium supplemented with 2.5% FBS, and this is added to the neutralized collagen solution to a final concentration of 1 mg / ml collagen. Mix. Pour 0.4 ml of this suspension (5 × 10 ⁵ cells / ml) into a well of a 24-well plate and incubate at 37 ° C. for 40 minutes to form a gel. The gel is covered with 0.5 ml of M199i medium supplemented with 2.5% FBS and 10 ng / ml of one of three GF: bFGF, VEGF ₁₆₅ or HGF. Purified peptide N (1 μM) is added to the wells simultaneously with the growth factor. Cultures are incubated at 37 ° C. for 48 hours in a humidified environment containing 5% CO2. Examine the gel using a microscope (Zeiss Axiovert 25) and record images using Archimed Pro 2.1 software.

  When HUVEC cells grow in collagen gels, the cells rapidly undergo morphogenesis to give a capillary network.

These capillary formations occur spontaneously or are caused by growth factors. The results shown in FIG. 6 show that bFGF, VEGF ₁₆₅ and HGF induce the organization of most HUVEC cells into capillaries under the experimental conditions used. Such capillary formation is completely inhibited when peptide N of the invention is added simultaneously with any one of the growth factors. Furthermore, no alteration in morphogenesis of HUVEC cells is observed when capillary formation is induced in the presence of one of kringles K1, K2, K3 or K4 (this result is not evident in FIG. 6).

Assay 5
In order to show that peptide N of the present invention constitutes a major site involved in the interaction between HGF and GAG (eg, Hep), the affinity of peptides N, K1, K2, K3 and K4 for heparin , And analyzed by a surface plasmon resonance apparatus using a BIAcore 2000 apparatus (available from BIAcore, Uppsala, Sweden). High molecular weight heparin is biotinylated using the product EZ-Link Biotin-LC-Hydrazide (Pierce, Rockford, IL) so that it can be captured on the surface of a detector (sensorship) coated with streptavidin. used. The purified peptide was diluted with normal buffer for BIAcore instrument (PBS containing 0.005% Tween-20) and injected over immobilized heparin at a rate of 20 ml / min. Experiments were performed within the peptide concentration range. The results obtained were automatically analyzed by the BIAcore BIA evaluation program. The obtained results are shown in FIG. 7, where it can be seen that peptide N has a much higher affinity for heparin than the other four kringles K1, K2, K3 and K4. These five peptides have the ability to bind heparin, but the binding is different. The largest reaction is that obtained for 0.7 μM peptide N binding, but this concentration is about 20 times lower than the concentration of kringle (600 K for K3 versus peptide N About 2000RU). Thus, at physiological concentrations, the HGF / GSG interaction is primarily provided by peptide N.

Assay 6
Affinity of soluble HGF-R for HGF or N [a chimeric product (HGF-R / IgG), a fusion protein comprising the HGF-binding portion of the extracellular domain of HGF-R and a portion of IgG]], To assess the affinity of normal molecular weight heparin (HEP) and the affinity of resolved low molecular weight heparin (LMWH), assay 5 above was reproduced. Each of these was immobilized on the surface of the detector (“sensorship”).

  Of note is that Hep has a higher affinity (25 times higher) than LMWH for HGF and N.

  Regarding the binding of soluble HGF-R and Hep to HGF and N, the results obtained are shown in FIGS. 8A, 8B, 8C and 8D for three different experiments, which show selective binding of Hep to N, And the lack of binding of soluble HGF-R to N.

Assay 7
The binding and displacement of the radiolabeled ligand was examined. Use Iodo-gen Pre-Coated Iodination Tubes according to the manufacturer's instructions and add HGF (5 μg), VEGF ₁₆₅ (10 μg) and N (25 μg) (or K1 (25 μg) where appropriate) Iodination with 1 mCi (ie 3.7 × 10 ⁷ Bq) of [ ¹²⁵ I] Na. The separated protein is purified on a column equilibrated with 25 mM Tris-HC, pH 7.4 containing 0.4 M NaCl, 5 mM EDTA and 0.25% AlbuMAX (supplied by INVITROGEN) and concentrated by centrifugation did. The specific activity measured by precipitation with trichloroacetic acid is:
For [ ¹²⁵ I] HGF, 18 × 10 ⁶ cpm / pmol,
^[125 I] For ^{_{VEGF 165, 8 × 10 6 cpm}} / pmol,
For [ ¹²⁵ I] N, 9 × 10 ⁵ cpm / pmol
It is.

Binding and displacement studies with iodinated proteins were performed on 24-well plates at 4 ° C. in confluent HUVEC cells. Cells were washed with PBS and preincubated with 0.5 ml binding medium (normal medium containing 0.1% AlbuMIX) for 0.5 hours. To determine binding parameters for N, increasing amounts of radiolabeled N were added to HUVEC cells with or without a molar excess of 100 unlabeled N and incubated for 2 hours . For displacement experiments, constant amounts of HGF (1 nM) or VEGF ₁₆₅ (0.2 nM) were added to HUVEC cells for 4 hours and 2 hours, respectively, in increasing concentrations in the presence of unlabeled N. At the end of the incubation, the cells were washed 3 times with binding medium and then solubilized with 0.3 M NaOH. Radioactivity associated with HUVEC cells was measured by a suitable counter.

The results obtained indicate that N inhibits the binding of HGF and VEGF ₁₆₅ to HUVEC and that K1 does not function as an inhibitor in this case. Inhibition by N depends on the concentration of N.

With regard to substitution, it is observed that the kinetics correspond to a bimolecular simple reaction model. The Kd value calculated using an appropriate program is
20.40 ± 5.11 nM for HGF, and
56.17 ± 17.18 nM for VEGF ₁₆₅ .

  See Figures 9A and 9B.

Conclusion In light of the results given above in assay 1-7 and shown in FIGS. 3-9, it is clear that peptide N functions as an HGF inhibitor. Unlike the known peptide NK4, the peptide N of the invention hardly interferes with competitive binding with HGF for HGF-R. On the other hand, considering assays 5-6 and FIGS. 7-8, peptide N inhibits the attachment of HGF to GGF, such as Hep, by preventing Hep from attaching to HGF or by activating HGF by Hep. It more reliably mediates the interaction; therefore, HGF can no longer act on HGF-R, nor can it activate HGF-R so that HGF-R exerts tyrosine kinase action.

The peptide of Example 2-4 shows the same mode of action as peptide N. As anti-angiogenic agents, N and its variants are useful for the treatment of diseases associated with angiogenesis, particularly those caused by excessive angiogenesis. These are especially as angiogenesis inhibitors and HGF antagonists (especially with regard to rheumatoid arthritis, diabetic retinopathy, age-related macular degeneration, and excessive scar formation in the wound healing process)
As a regulator of abnormal cell growth (especially with respect to psoriasis, Osler-Weber syndrome, hemangioma, and benign or malignant tumors), and as an inhibitor of endothelial cell hyperstimulation
Needed.

  Briefly, peptide N and its variants are particularly useful as anti-angiogenic, anti-rheumatic, anti-arthritic, anti-inflammatory, anti-psoriatic, anti-proliferative, anti-tumor and / or cytostatic agents. Useful.

In the accompanying drawings,
Shows schematically the structure of native HGF with the five major domains of the α chain (N, K1, K2, K3 and K4), and the fragment PD represents the β chain; Figures 2a and 2b show the purity of isolated N, K1, K2, K3 and K4 peptides by Coomassie blue staining (Figure 2a) and Western blotting with anti-HGF antibody (Figure 2b) ; Results from assays for stimulating HUVEC cell proliferation using bFGF (10 ng / ml) in the presence of peptide N, K1, K2, K3 or K4 (increased from 0 to 50 μg / ml) Is represented as a graph; Is a graph demonstrating inhibition by peptide N of HUVEC cell proliferation in the presence of GF such as bFGF, HGF, VEGF ₁₆₅ and VEGF ₁₂₁ ; Figure 2 is a graph that reveals the effects of HGF, HGF + N, HGF + K1, HGF + K2, HGF + K3 and HGF + K4 on HUVEC cell migration across the porous membrane; Comprises 6 photographs of the inhibition of “capillary-like tube” structure formation by HUVEC in 3D collagen gel [comparison of bFGF and bFGF + N, VEGF and VEGF + N (in this case VEGF is VEGF ₁₆₅ ) and HGF and HGF + N]]; Is a graph showing that peptide N of the present invention differs from K1, K2, K3 and K4 in terms of strong binding of Hep; Figures 8a-8d and 9A-9B relate to the complementary results obtained for the peptide N. Figures 8a-8d and 9A-9B relate to the complementary results obtained for the peptide N.

For convenience of abbreviations, the following is a list of technical abbreviations and acronyms used in the description of this specification.

aa amino acid;
a proto-oncogene DNA fragment encoding the c-Met protein, where the protein has tyrosine kinase enzymatic activity and corresponds to the HGF receptor (referred to herein as HGF-R);
cpm count per minute;
DNA deoxyribonucleic acid;
FBS fetal bovine serum;
FGF fibroblast growth factor; among FGFs, especially aFGF (acidic fibroblast growth factor) and bFGF (basic fibroblast growth factor) are famous;
GAG glycosaminoglycan;
GF growth factor;
Hep Heparin;
HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, a product that functions as a buffer;
HGF hepatocyte growth factor; unless otherwise indicated herein, HGF means human hepatocyte growth factor (hHGF);
hHGF human hepatocyte growth factor;
HGF-R HGF receptor (in this specification the notation “HGF-R” is chosen over the term “c-Met” which leads to confusion with the “c-Met” gene);
HUVEC human umbilical vein endothelial cells;
K1 HGF kringle-1 domain or corresponding peptide;
Kringle-2 domain of K2 HGF or corresponding peptide;
Kringle-3 domain of K3 HGF or corresponding peptide;
Kringle-4 domain of K4 HGF or corresponding peptide;
M199 Medium sold by US company LIFE TECHNOLOGIES (Rockville, MD);
M199i medium supplemented with M199i 15 mM HEPES (pH 7.4), 2 mM glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin and 2.5 μg / ml amphotericin B;
N HGF hairpin loop domain, corresponding peptide or peptide of the invention, whose N-terminus is PyrGlu (instead of PyrGlu according to the prior art described by WO-A-99 / 55361) Gln It is even better if the peptide is;
A peptide consisting of the amino acid sequence from N to K4 of NK4 HGF (i.e. aa 32-478 of native HGF);
RT room temperature;
RU resonance unit (1000 RU corresponds to 1 ng protein binding per mm ² );
VEGF Vascular Endothelial Growth Factor; Among VEGF, VEGF ₁₆₅ factor and VEGF ₁₂₁ factor are particularly well known.

[Sequence Listing]

Claims (10)

(i) a peptide N derived from the HGF α chain and having the amino acid sequence represented by SEQ ID NO: 2, and
(ii) a peptide variant thereof derived from the peptide N sequence by deletion, substitution or addition of one or more amino acids,
Peptide substance characterized in that it is selected from the group consisting of the peptide N and its variants exhibit anti-angiogenic activity by a mechanism different from competitive binding of HGF to the HGF-R receptor. It has a peptide substance. (i) a peptide N derived from the HGF α chain and having the amino acid sequence represented by SEQ ID NO: 2, and
(ii) peptide variants thereof derived from the peptide N sequence by deletion, substitution or addition of one or more amino acids,
A therapeutic composition for treating angiogenesis-related diseases, comprising a therapeutically effective amount of a peptide substance selected from the group consisting of: and a pharmaceutically acceptable excipient.   Use of a peptide substance according to claim 1, characterized in that said substance is used for preparing an anti-angiogenic medicament for therapeutic use against angiogenesis-related diseases.   A DNA fragment encoding the peptide substance according to claim 1.   A plasmid comprising the DNA fragment according to claim 4.   A bacterial strain containing the plasmid according to claim 5. A method for preparing a peptide substance according to claim 1 using a DNA molecule encoding peptide N, or one of its variants, comprising:
(1) introducing a DNA fragment encoding the first restriction enzyme cleavage site at the 5 ′ end;
(2) introducing a DNA fragment encoding a second restriction enzyme cleavage site at the 3 ′ end, steps 1 and 2 are performed in any order, then
(3) inserting the peptide resulting from steps 1 and 2 into a bacterial plasmid, in particular a plasmid of Escherichia coli,
(4) culturing the strain carrying the plasmid modified as described above at a temperature of 30-37 ° C; and
(5) A method characterized by expressing peptide N or one of its variants at a temperature of 25-30 ° C.   The peptide substance according to claim 1, wherein the amino acid sequence is the sequence of SEQ ID NO: 4.   The peptide substance according to claim 1, wherein the amino acid sequence is the sequence of SEQ ID NO: 5.   The peptide substance according to claim 1, wherein the amino acid sequence is the sequence of SEQ ID NO: 6.

Images