MULTIMERIC COMPOUNDS OF A KRINGLE DOMAIN FROM THE HEPATOCYTE GROWTH FACTOR / SCATTER FACTOR (HGF/SF)

Abstract

Disclosed are multimeric compounds of K1 domains from the Hepatocyte Growth Factor/Scatter Factor (HGF/SF) being able to induce activation of the tyrosine kinase receptor MET and their uses.

Claims

1. Multimeric compound comprising at least two K1 peptide domains (Kringle 1) of the Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and being represented by the formula (I): ##STR00008## wherein: m=0 or 1, n=0 or 1, K1.sub.a, K1.sub.b, and, if present, K1.sub.c and K1.sub.d are polypeptides, K1.sub.a and K1.sub.b and, if present, K1.sub.c and K1.sub.d contain a K1 peptide domain, said K1 peptide domain consisting of an amino acid sequence SEQ ID NO: 1 or of an amino acid sequence with at least 80%, preferably 90% identity to SEQ ID NO: 1, Biot represents one molecule of biotin, and Strept represents one molecule chosen among the group consisting of: streptavidin, avidin, neutravidin and any synthetic or recombinant derivatives thereof, K1.sub.a and K1.sub.b and, if present, K1.sub.c and K1.sub.d are C-terminally linked to a Biot by a covalent bond, and each Biot is linked to Strept by a non-covalent bond, said multimeric compound being able to induce activation of the tyrosine kinase receptor MET.

2. Multimeric compound according to claim 1, wherein Strept represents one molecule of streptavidin.

3. Multimeric compound according to claim 1, which is a K1 dimer represented by the formula (II): ##STR00009## wherein: K1.sub.a and K1.sub.b are polypeptides, K1.sub.a and K1.sub.b contain a K1 peptide domain, said K1 peptide domain consisting of an amino acid sequence SEQ ID NO: 1 or of an amino acid sequence with at least 80%, preferably 90% identity to SEQ ID NO: 1, Biot represents one molecule of biotin, and Strept represents one molecule of streptavidin, K1.sub.a and K1.sub.b are C-terminally linked to a Biot by a covalent bond, and each Biot is linked to Strept by a non-covalent bond.

4. Multimeric compound according to claim 1, which is a K1 trimer represented by the formula (III): ##STR00010## wherein: K1.sub.a, K1.sub.b and K1.sub.c are polypeptides, K1.sub.a, K1.sub.b and K1.sub.c contain a K1 peptide domain, said K1 peptide domain consisting of an amino acid sequence SEQ ID NO: 1 or of an amino acid sequence with at least 80%, preferably 90% identity to SEQ ID NO: 1, Biot represents one molecule of biotin, and Strept represents one molecule of streptavidin, K1.sub.a, K1.sub.b and K1.sub.c are C-terminally linked to a Biot by a covalent bond, and each Biot is linked to the Strept by a non-covalent bond.

5. Multimeric compound according to claim 1, which is a K1 tetramer represented by the formula (IV): ##STR00011## wherein: K1.sub.a, K1.sub.b, K1.sub.c and K1.sub.d are polypeptides, K1.sub.a, K1.sub.b, K1.sub.c and K1.sub.d contain a K1 peptide domain, said K1 peptide domain consisting of an amino acid sequence SEQ ID NO: 1 or of an amino acid sequence with at least 80%, preferably 90% identity to SEQ ID NO: 1, Biot represents one molecule of biotin, and Strept represents one molecule of streptavidin, K1.sub.a, K1.sub.b, K1.sub.c and K1.sub.d are C-terminally linked to a Biot by a covalent bond, and each Biot is linked to Strept by a non-covalent bond.

6. Multimeric compound according to claim 1, wherein K1.sub.a and K1.sub.b, and if present K1.sub.c and K1.sub.d, are identical.

7. Multimeric compound according to claim 1, wherein said multimeric compound is able to bind the tyrosine kinase receptor MET with a dissociation constant K.sub.D≦200 nM, preferably ≦100 nM, more preferably ≦10 nM.

8. Composition comprising a multimeric compound as defined in claim 1.

9. Composition according to claim 8, wherein said multimeric compound is in the form of a mix of: a K1 dimer represented by the formula (II), ##STR00012## wherein: K1.sub.a and K1.sub.b are polypeptides, K1.sub.a and K1.sub.b contain a K1 peptide domain, said K1 peptide domain consisting of an amino acid sequence SEQ ID NO: 1 or of an amino acid sequence with at least 80%, preferably 90% identity to SEQ ID NO: 1, Biot represents one molecule of biotin, and Strept represents one molecule of streptavidin, K1.sub.a and K1.sub.b are C-terminally linked to Biot by a covalent bond, and each Biot is linked to Strept by a non-covalent bond, a K1 trimer represented by the formula (III), ##STR00013## wherein: K1.sub.a, K1.sub.b and K1.sub.c are polypeptides, K1.sub.a, K1.sub.b and K1.sub.c contain a K1 peptide domain, said K1 peptide domain consisting of an amino acid sequence SEQ ID NO: 1 or of an amino acid sequence with at least 80%, preferably 90% identity to SEQ ID NO: 1, Biot represents one molecule of biotin, and Strept represents one molecule of streptavidin, K1.sub.a, K1.sub.b and K1.sub.c are C-terminally linked to Biot by a covalent bond, and each Biot is linked to Strept by a non-covalent bond, and, a K1 tetramer represented by the formula (IV), ##STR00014## wherein: K1.sub.a, K1.sub.b, K1.sub.c and K1.sub.d are polypeptides, K1.sub.a, K1.sub.b, K1.sub.c and K1.sub.d contain a K1 peptide domain, said K1 peptide domain consisting of an amino acid sequence SEQ ID NO: 1 or of an amino acid sequence with at least 80%, preferably 90% identity to SEQ ID NO: 1, Biot represents one molecule of biotin, and Strept represents one molecule of streptavidin, K1.sub.a, K1.sub.b, K1.sub.c and K1.sub.d are C-terminally linked to a Biot by a covalent bond, and each Biot is linked to Biot by a non-covalent bond.

10. Multimeric compound as defined in claim 1, for use in an in vivo diagnostic method, in particular in an in vivo diagnostic method of a pathology chosen among: cancers, diseases of epithelial organs including acute and chronic liver diseases, acute and chronic kidney diseases, chronic lung diseases and chronic skin wounds, diseases of the central nervous system including neuron diseases and sclerosis, ischemic heart diseases, peripheral vascular diseases, diabetes and associated complications such as peripheral neuropathies.

11. Multimeric compound as defined in claim 1, for use in medical imaging.

12. A method for performing in vitro diagnosis, comprising providing the multimeric compound as defined in claim 1, and using the compound to perform an in vitro diagnostic of a pathology chosen among: cancers, diseases of epithelial organs including acute and chronic liver diseases, acute and chronic kidney diseases, chronic lung diseases and chronic skin wounds, diseases of the central nervous system including neuron diseases and sclerosis, ischemic heart diseases, peripheral vascular diseases, diabetes and associated complications such as peripheral neuropathies.

13. Multimeric compound as defined in claim 1, for use as a medicament.

14. Process to obtain a composition comprising a multimeric compound comprising at least two K1 peptide domains as defined in claim 1, comprising the steps of: synthesizing a molecule containing a K1 peptide domain linked to a biotin to obtain a biotinylated K1 molecule, said biotin being linked to the C-terminus of the K1 molecule, mixing said biotinylated K1 molecule with a streptavidin homotetramer to obtain a composition of a multimeric compound comprising at least 2 K1 peptide domains, said biotinylated K1 molecule and said streptavidin homotetramer being preferably mixed in a 2:1 molar ratio to obtain dimeric compounds of K1 domains, a 3:1 molar ratio to obtain trimeric compounds of K1 domains, or a 4:1 molar ratio to obtain tetrameric compounds of K1 domains.

15. Process to obtain a multimeric compound comprising at least two K1 peptide domains as defined in claim 1, comprising the steps of: synthesizing a molecule containing a K1 peptide domain linked to a biotin to obtain a biotinylated K1 molecule, said biotin being linked to the C-terminus of the K1 molecule, mixing said biotinylated K1 molecule with a streptavidin homotetramer to obtain a composition of a multimeric compound comprising at least 2 K1 peptide domains, purifying and separating multimeric compounds to obtain dimeric compounds of K1 domains, trimeric compounds of K1 domains, and tetrameric compounds of K1 domains.

16. Multimeric compound according to claim 2, which is a K1 dimer represented by the formula (II): ##STR00015## wherein: K1.sub.a and K1.sub.b are polypeptides, K1.sub.a and K1.sub.b contain a K1 peptide domain, said K1 peptide domain consisting of an amino acid sequence SEQ ID NO: 1 or of an amino acid sequence with at least 80%, preferably 90% identity to SEQ ID NO: 1, Biot represents one molecule of biotin, and Strept represents one molecule of streptavidin, K1.sub.a and K1.sub.b are C-terminally linked to a Biot by a covalent bond, and each Biot is linked to Strept by a non-covalent bond.

17. Multimeric compound according to claim 2, which is a K1 trimer represented by the formula (III): ##STR00016## wherein: K1.sub.a, K1.sub.b and K1.sub.c are polypeptides, K1.sub.a, K1.sub.b and K1.sub.c contain a K1 peptide domain, said K1 peptide domain consisting of an amino acid sequence SEQ ID NO: 1 or of an amino acid sequence with at least 80%, preferably 90% identity to SEQ ID NO: 1, Biot represents one molecule of biotin, and Strept represents one molecule of streptavidin, K1.sub.a, K1.sub.b and K1.sub.c are C-terminally linked to a Biot by a covalent bond, and each Biot is linked to the Strept by a non-covalent bond.

18. Multimeric compound according to claim 2, which is a K1 tetramer represented by the formula (IV): ##STR00017## wherein: K1.sub.a, K1.sub.b, K1.sub.c and K1.sub.d are polypeptides, K1.sub.a, K1.sub.b, K1.sub.c and K1.sub.d contain a K1 peptide domain, said K1 peptide domain consisting of an amino acid sequence SEQ ID NO: 1 or of an amino acid sequence with at least 80%, preferably 90% identity to SEQ ID NO: 1, Biot represents one molecule of biotin, and Strept represents one molecule of streptavidin, K1.sub.a, K1.sub.b, K1.sub.c and K1.sub.d are C-terminally linked to a Biot by a covalent bond, and each Biot is linked to Strept by a non-covalent bond.

19. Multimeric compound according to claim 2, wherein K1.sub.a and K1.sub.b, and if present K1.sub.c and K1.sub.d, are identical.

20. Multimeric compound according to claim 3, wherein K1.sub.a and K1.sub.b, and if present K1.sub.c and K1.sub.d, are identical.

Description

LEGENDS TO THE FIGURES

[0166] FIG. 1. K1B total chemical synthesis. (a) Structure of the K1 domain of HGF/SF (residues 125-209, extracted from PDB 1BHT). The annotation was done according to UniProt database (entry P14210) with the 3 internal cysteine bridges and C-term biotin. (b) Scheme of one-pot assembly and folding of K1B. (c) RP-HPLC characterization of the crude linear K1B domain (left), the purified K1B domain (center) and MS analysis of folded K1B domain (right).

[0167] FIG. 2. HeLa cells were treated for 7 min with 100 pM or 500 pM HGF/SF (HGF), 100 nM or 1 μM K1 and 100 nM or 1 μM K1B. Cell lysates were then analyzed by specific total MET, Akt and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot.

[0168] FIG. 3. Cell scattering assay. MDCK isolated cell islets were incubated for 18 h in presence of culture media (Ctrl) with 1 μM K1 or 1 μM K1B. Cells were then stained and observed under microscope (40×).

[0169] FIG. 4. K1B and NB MET binding properties. (a) Structure of NK1 dimer (center, PDB 1BHT) and spatial relative orientation of each N (left) and K1 (right) monomers within the dimer. Dashed arrows indicate distances between subdomain C-termini. (b) NB, K1B and MET-Fc binding assay. Increasing concentrations of NB or K1B were mixed with extracellular MET domain fused with human IgG1-Fc (MET-Fc), and incubated with streptavidin AlphaScreen donor beads and Protein A acceptor beads. Error bars correspond to standard error (+/−SD) of triplicates. (c) Endogenous MET capture. Streptavidin coated beads loaded with NB or K1B were incubated with HeLa or Capan-1 total cell lysates. Input, flow-through and elution fractions from NB or K1 loaded beads were analyzed by specific total MET western blot.

[0170] FIG. 5. Structure of a streptavidin homotetramer with 4 bound biotins (left, PDB 1SWE) and distances between binding sites (right).

[0171] FIG. 6. AlphaScreen competition assay. Increasing concentrations of K1B/S complex (ratio 2:1) were added to pre-mixed K1B (20 nM)/MET-Fc (2 nM)/Alpha beads. IC50 of Alpha signal was measured. Graph is representative of experiments reproduced at least 3 times with 2 different lots of K1B. Error bars correspond to standard error (+/−SD) of triplicates.

[0172] FIG. 7. Analysis of K1B/S complexes. Increasing ratio of K1B and streptavidin (from 0:1 to 8:1) were analyzed in non-denaturing condition by SDS-PAGE on a 10% NuPage® gel in MES buffer. Gel was fixed and stained with Coomassie Brilliant Blue. K1B:S ratio for each complex composition is indicated with corresponding A, B, C and D relative biotin binding sites positioned as proposed in FIG. 6.

[0173] FIG. 8. (a) Mass spectrum of K1B under native conditions. (b) Titration of streptavidin with K1B. Upon addition of K1B, new species corresponding to the binding of 1 to 4 molecules of K1B to the streptavidin are clearly visible. (c) Relative intensity of each species depending on the K1B:S ratio.

[0174] FIG. 9. Determination of optimal K1B:S ratio. HeLa cells were treated for 7 min with 50 nM streptavidin (S), 500 pM mature HGF/SF (HGF), 400 nM K1B and an increasing ratio of K1B/S mixture (from 1:1 to 8:1) with 50 nM streptavidin. Cell lysates were then analyzed by specific total MET, Akt and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot.

[0175] FIG. 10. Structure of human IgG: distance between two paratopes is 13.7 nm (PDB 1IGt).

[0176] FIG. 11. MET signaling analysis upon K1B/S stimulation. (a) HeLa cells were treated for 7 min with 50 nM streptavidin (S), 50 nM anti-biotin antibody (Ab), 500 pM mature HGF/SF (HGF), 100 nM and 1 μM K1B, 100 nM K1B/S, 100 nM K1B/Ab and 100 nM NK1. Cell lysates were then analyzed by specific total MET, Akt and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot. Ctrl: vehicle, MW: molecular weight. (b) HeLa cells were treated with increasing concentrations of mature HGF/SF, K1B/S, NK1 and K1B/Ab for 7 min. Activation levels of ERK and Akt were measured using HTRF technology, and plotted as the 665/620 nm HTRF signal ratio. (c) K1B/S and NK1, K1B/Ab kinetic analysis. HeLa cells were treated with 100 nM K1B/S or NK1, for 1, 5, 10, 20, 30, 40 or 90 min. Cell lysates were then analyzed by specific total MET, Akt and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot. (d) HGF/SF, K1B/S, NK1 and K1B/Ab kinetic analysis. HeLa cells were treated with optimal concentration of 100 pM HGF/SF, 50 nM K1B/S, 50 nM NK1 or 400 nM K1B/Ab for 1, 3, 5, 7, 10, 15, 20, 30, 60 or 90 min. Activation levels of ERK and Akt were measured using HTRF technology and plotted as the 665/620 nm HTRF signal ratio.

[0177] FIG. 12. Analysis of MET tyrosine phosphorylation profile. HeLa cells were treated for 7 min with 50 nM streptavidin (S), 50 nM anti-biotin antibody (Ab), 500 pM mature HGF/SF (HGF), 10 or 100 nM K1B, 100 nM K1B/S, 100 nM K1B/Ab or 100 nM NK1. Cell lysates were then analyzed by western blot with total MET and phospho-specific MET Y1234-1235 and Y1349-1356 residues.

[0178] FIG. 13. HGF/SF, K1B/Ab kinetic analysis. HeLa cells were treated with 500 pM HGF/SF or 100 nM K1B/Ab, for 1, 5, 10, 20, 30, 40 or 90 min. Cell lysates were then analyzed by specific total MET, Akt and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot.

[0179] FIG. 14. HeLa cells were treated for 7 min with 100 pM HGF/SF (HGF), 1 μM NB and 1 μM NB/S (2:1 ratio), and 500 nM Streptavidin (S). Ctrl: vehicle. Cell lysates were then analyzed by specific total MET, Akt and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot.

[0180] FIG. 15. Cell scattering assay. MDCK isolated cell islets were incubated for 18 h in culture media (Ctrl), 500 pM HGF/SF (HGF) 500 nM streptavidin (S), 1 μM NB or 1 μM NB/S. Cells were then stained and observed under microscope (40×).

[0181] FIG. 16. Cellular phenotypes induced by K1B/S. (a) Cell scattering assay. MDCK isolated cell islets were incubated for 18 h in culture media with 50 nM streptavidin (S), 50 nM anti-biotin antibody (Ab), 500 pM mature HGF/SF (HGF), 100 nM K1B, 100 nM K1B/S, 100 nM NK1 and 100 nM K1B/Ab. Cells were then stained and observed under microscope (40×). (b) Matrigel morphogenesis assay. MDCK cells were seeded onto a layer of Matrigel and treated for 18 h with 50 nM streptavidin (S), 50 nM antibiotin antibody (Ab), 500 pM mature HGF/SF (HGF), 100 nM K1B, 100 nM K1B/S, 100 nM NK1 and 100 nM K1B/Ab. Cells were then observed under microscope (40×). (c) MTT Assay. MDCK cells were cultured overnight (15 h) in medium containing 0.1% FBS with or without anisomycin (0.7 μM) and in the presence of 500 pM mature HGF/SF (HGF), 100 nM K1B, 100 nM K1B/S, 100 nM NK1 and 100 nM K1B/Ab. An MTT assay was then performed to evaluate cell survival. Results are expressed as the percentage of untreated control. An ANOVA test was performed to compare the 3 means, with a P-value <0.05 considered statistically significant. (d) Angiogenesis. Mice were injected with a mixture of Matrigel and 1 nM HGF/SF (HGF), 10 nM VEGF, 100 nM NK1, 100 nM K1B/S, 100 nM K1B or 50 nM S. Hemoglobin absorbance was measured and concentration was determined using a rate hemoglobin standard curve and plug weight. ANOVA tests were performed to compare all the means, and a P-value<0.001 was considered to indicate a statistically significant difference.

[0182] FIG. 17. In vivo MET activation assays. (a) FVB mice were injected intravenously with PBS (ctrl), 25 pmol K1B (250 ng), 25 pmol K1B/S complex (250 ng K1/700 ng S), 25 pmol NK1 (500 ng) or 2.5 pmol mature HGF/SF (250 ng) per g of body weight. After 10 min, livers were extracted, snap frozen and crushed. MET, Akt and ERK phosphorylation status in cell lysates was analyzed by western blot. Data obtained from 2 mice are representative of 3 independent experiments. (b) FVB mice were injected intravenously with 125 ng anti-Fas monoclonal antibody (aFas) mixed with 25 pmol K1B (250 ng), 25 pmol K1B/S complex (250 ng/700 ng), 25 pmol NK1 (500 ng) or 2.5 pmol mature HGF/SF (250 ng) per g of bodyweight, or PBS. A second injection without anti-Fas was performed 90 min later.

[0183] Livers were extracted and fixed in formalin after 3 additional hours. (c) Frozen liver sections were stained with hematoxylin-eosin for histological observation (40×). (d) Frozen liver sections were treated with Apoptag® Kit for apoptotic nuclei labelling (green) and counterstained with DAPI for total nuclei labelling (blue) (100×, insert: 200× on apoptotic cells).

[0184] FIG. 18. Mice were injected with an increased concentration of K1B/S complex (0.5, 2.5 or 25 pmol/g, corresponding to 5 ng K1B/14 ng S, 25 ng K1B/70 ng S and 250 ng K1B/700 ng S), 25 pmol K1B/g (250 ng/g) or 25 pmol/g NK1 (500 ng/g). After 10 min, livers were extracted, snap frozen and crushed. Cell lysates were analyzed by specific total MET, Akt and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot.

[0185] FIG. 19. In vivo MET activation kinetics. Mice were injected with 25 pmol K1B/S (250 ng/700 ng) per g of body weight, and livers were extracted after 0, 10, 20 or 30 min, snap frozen and crushed. Cell lysates were analyzed by specific total MET, Akt and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot.

[0186] FIG. 20. Fas-induced fulminant hepatitis. FVB mice were injected intravenously with 125 ng anti-Fas monoclonal antibody (aFas) mixed with 25 pmol K1B, 25 pmol K1B/S complex, 25 pmol NK1, 12.5 pmol Streptavidin (S) or 2.5 pmol mature HGF/SF per g of body weight, or PBS. A second injection without anti-Fas was performed 90 min later. Livers were extracted, snap frozen and crushed. Proteins were analyzed by specific total MET, PARP 1/2, Caspase 3, cleaved Caspase 3 and total ERK western blot.

EXAMPLES

Example 1. Total Chemical Synthesis of Biotinylated K1 and N Domains

[0187] The K1 domain (HGF/SF 125-209) is composed of 85 amino acid residues, and its tertiary structure is stabilized by three disulfide bonds (FIG. 1A). In K1B, the K1 primary structure was extended at the C-terminus by addition of two glycine residues and a lysine residue modified on its side chain with a biotin group. The chemical synthesis of K1B was performed using solid phase peptide synthesis (SPPS) in a one-pot sequential three peptide segments assembly process, which required the preparation of HGF/SF segments 125-148 (segment 1), 149-176 (segment 2) and 177-209 (segment 3), the latter with the GGK (biotin) extension (FIG. 1B). A thioester and bis(2-sulfanylethyl)amido cyclic disulfide (SEAoff) group were introduced on the C-terminus of peptide segments 1 and 2 respectively. Assembly of K1B linear polypeptide started by joining thioester segment 1 with segment 2 using the Native Chemical Ligation reaction. The reaction led to the successful formation of segment 1-2 featuring a blocked C-terminal SEAoff group. Activation of the SEAoff group by reduction with tris(2-carboxyethyl)phosphine (TCEP) and addition of biotinylated segment 3 triggered the SEA native peptide ligation step and the successful formation of linear K1B HGF/SF domain as shown by the LC-MS of the crude reaction mixture (FIG. 1C, left). Linear K1B was purified by HPLC to give 3.6 mg (40% overall) of homogeneous material (FIG. 1C, center) and folded subsequently using the glutathione-glutathione disulfide redox system. Proteomic analysis of the folded K1B domain demonstrated the formation of the native disulfide bond pattern (FIG. 1C, right).

[0188] Interestingly, a MET phosphorylation assay using HeLa cells (FIG. 2) and cell scattering assays using MDCK cells (FIG. 3) showed that K1B activity was indistinguishable from unmodified synthetic K1 domain and behaved as a micromolar MET agonist, as it is known for recombinant K1 domain. Consequently, introduction of the biotin group had no detectable influence on the biological activity of K1B at this stage.

Example 2. Design of K1 Multivalent Complexes

[0189] Analysis of the relative positions of N and K1 domains in the NK1 homodimer crystal structure reveals that the C-termini of the two N domains and the C-termini of the two K1 domains are separated by only ˜1.3-2 nm (FIG. 4A). Interestingly, the individual biotin binding sites within a streptavidin homotetramer (S) are separated by distances of ˜2.0-3.5 nm (FIG. 5). Therefore, it was anticipated that the formation of K1B/S or NB/S complexes might recapitulate the relative distances and positions of N and K1 domains found in NK1 dimer independently of each other.

[0190] The binding of K1B/S complexes to MET was examined using AlphaScreen technology. K1B was loaded on streptavidin-coated donor beads and incubated with recombinant extracellular MET-Fc chimera loaded on Protein A-coated acceptor beads. If K1B/S donor beads interact with MET-Fc/Protein A acceptor beads, a chemical energy transfer is possible between the beads, leading to fluorescence emission upon laser excitation. K1B induced strong signal intensities with an apparent dissociation constant KD (˜16 nM) about 100-fold lower than the KD reported for monomeric K1 protein-MET interaction (FIG. 4B). Since the bead-based AlphaScreen assay can generate avidity and thus introduce a bias in the estimation of the apparent KD in saturation experiments, it was performed the reciprocal competition assay by adding increasing concentrations of preformed K1B/S complex (2:1 molar ratio) into the K1B/MET-Fc/AlphaScreen bead mixture (FIG. 6). With this competition assay, an IC50 (˜14 nM) was determined in perfect agreement with the apparent K1B/MET-Fc KD from the saturation assay. This study was completed by examining the binding of K1B/S complexes to endogenous MET from a whole cell lysate (FIG. 4C). Streptavidin-coated agarose beads were incubated with K1B to form immobilized complexes, which were subsequently incubated with whole lysate from HeLa or Capan-1 cells. Western blot analysis of the eluted material showed that K1B/S complexes were able to capture MET from cell lysates. Collectively, these data show that the semisynthetic K1B/S complex interacts with MET at low nanomolar concentration, and indicate the importance of multivalency in the K1-MET interaction system.

Example 3. Semisynthetic K1B/S Complex is a Potent MET Agonist

[0191] These results set the stage for evaluating the K1B/S complex agonistic activity using in vitro cell assays in the human HeLa cell line. For this, the stoichiometry for K1B/S complex formation was fixed to 2:1, which generates several species varying in the number of K1B proteins bound per streptavidin tetramer. With this molar ratio, and by assuming that each biotin binding unit is independent, the probability of having 0, 1, 2, 3 or 4 K1B proteins bound per streptavidin should correspond to 6%, 25%, 38%, 25% and 6% respectively, meaning 69% of K1B/S multimers in theory. These K1B/S multimers were indeed identified by SDS-PAGE analysis (FIG. 7) and by native mass spectrometry analysis (FIG. 8). Using the latter technique, it was estimated that the 2:1 K1B:S molar ratio resulted in 75% of the K1 domain presented at least as pairs within K1B/S multimers. In practice, it was noticed that a 2:1 K1B:S molar ratio was sufficient to achieve a maximum cellular response, since a higher proportion of K1B in the mixture from 3:1 up to 8:1 led to no improvement in potency (FIG. 9).

[0192] Another complex produced by mixing K1B with an anti-biotin antibody (Ab) in a 2:1 molar ratio was also designed. The antibody is expected to produce consistent K1B dimers, albeit with a distance of ˜13-20 nm between each K1B protein, which is significantly greater than those found in NK1 crystal structure or K1B/S complexes (FIG. 10).

[0193] MET activation and downstream signaling in HeLa cells upon HGF/SF, K1B, K1B/S, K1B/Ab or recombinant NK1 incubation was analyzed by western blot and quantified by HTRF approaches (FIGS. 11A & B). Typically, HGF/SF triggered maximal ERK and Akt activation down to pM concentrations. Impressively, K1B/S complexes were able to trigger ERK and Akt phosphorylation levels down to a low nM range, and thus displayed an agonist activity similar to NK1 protein. Moreover, K1B/S but not K1B induced a strong MET phosphorylation at 100 nM. The fact that activation of MET by K1B was detected only for μM concentrations, as reported in the literature for recombinant K1, highlights the critical role of multivalency for achieving strong receptor activation. A similar multivalent process was evident for the K1B/Ab complex, which unlike K1B, also induced a significant MET phosphorylation at 100 nM.

[0194] However, K1B/Ab was significantly less active than K1B/S since it was unable to trigger significant ERK and Akt downstream signaling (FIG. 11A). The MET phosphorylation pattern was analyzed at the tyrosine level. Indeed, auto-phosphorylation of tyrosines 1234 and 1235 is the first event leading MET activation, and is crucial for unlocking and maintaining sustained kinase activity. Subsequently, phosphorylation of C-terminal tyrosines 1349 and 1356 is required to provide recognition sites for scaffolding partners that propagate, amplify and diversify MET signaling. Both K1B/Ab and K1B/S activated MET auto-phosphorylation onto tyrosines 1234 and 1235. However, unlike K1B/S, K1B/Ab failed to trigger phosphorylation of tyrosines 1349 and 1356 (FIG. 12), and thus failed to trigger the downstream signaling cascade. This fact might be due to the large distance between K1B domains in the antibody complex and thus to the suboptimal stabilization of MET dimers.

[0195] It was also determined the MET and downstream signaling activation kinetics (0-90 min) using western Blot (FIG. 11C) and HTRF (FIG. 11D). Typically, HGF/SF induced a maximum of MET autophosphorylation between 5 and 10 min (FIG. 13), followed by a maximum of Akt and ERK phosphorylation at around 10-15 min, which slowly decreased over time. In comparison, MET phosphorylation proceeded much faster with K1B/S and NK1, i.e. within the very first minute, and then decreased below HGF/SF levels. Accordingly, maximum ERK and Akt activation was observed earlier, after only 3-7 min. In contrast, K1B/Ab complex induced weak MET activation (FIG. 13), and downstream signaling faded faster than for HGF/SF, NK1 or K1B/S.

[0196] Finally, and as expected from binding experiments, NB/S complex showed no agonistic activity (FIG. 14), and did not promote any cellular phenotypes (FIG. 15).

[0197] Together these results indicate that K1B/S complex recapitulates NK1 agonist activity, and demonstrate that K1 is the minimal HGF/SF functional domain required for MET activation. Moreover, these data show that the distance and/or orientation which separates the two K1 domains within a dimeric structure (natural or synthetic) is important to induce full MET activation.

Example 4. K1B/S Promotes Cell Scattering, Morphogenesis, Survival and Angiogenic Phenotypes

[0198] The ability of MET agonists to induce cell scattering in MDCK cells (the reference cell line for this phenotypic assay) was evaluated (FIG. 16A). In the presence of HGF/SF (100 pM) for 18-24h, MDCK cells acquired a mesenchymal-like phenotype and scatter.

[0199] This marked phenotype was also induced by NK1 protein and K1B/S complex, whereas scattering with K1B and K1B/Ab was weak. Notably, the ability of the agonists to induce a scattering phenotype seemed to be strongly correlated with their capacity to induce sustained phosphorylation of MET, ERK and Akt kinases.

[0200] Further cell assays were performed using lumina basal like matrix (Matrigel) as a mimic of basement extracellular matrix. In these conditions and without treatment, MDCK cells spontaneously form tight spherical clusters on Matrigel within 24 h. In contrast, when stimulated with HGF/SF, MDCK cells self-organize into branched and connected structures. Notably, NK1 and K1B/S widely promoted the formation of such structures (FIG. 16B), while K1B and K1B/Ab were unable to do so.

[0201] The capacity of the agonists to promote the survival of cells after apoptotic stress was examined. This phenotype is a hallmark of HGF/SF, which can protect many cell types against death induced by serum depletion, ultra-violet radiation, ischemia or some chemical substances. MDCK cells were stressed using anisomycin, a DNA and protein synthesis inhibitor which induces apoptosis. Anisomycin treatment induced ˜90% of cell death after 16 h, but only 50% of cell death when pretreated with HGF/SF (FIG. 16C). K1B/S or NK1 displayed similar survival rates, whereas K1B or K1B/Ab complex failed to protect the cells to a significant extent.

[0202] Clearly, these results show that in vitro K1B/S fully mimics the properties of NK1 as a potent MET agonist. To extend this observation in vivo, the different agonists were injected subcutaneously with Matrigel plugs into immunodeficient SCID mice to induce angiogenesis. Indeed, HGF/SF is a potent angiogenic factor that stimulates endothelial cell proliferation and migration. The plugs were extracted after 11 days to determine the quantity of hemoglobin infiltrated into the plug as a measure of angiogenesis induced (FIG. 16D). As expected, VEGF or HGF/SF showed potent angiogenic properties compared to control plugs. K1B/S induced the formation of vessels with a hemoglobin content comparable to that of VEGF and significantly higher than those induced by NK1 or K1B. Thus, while NK1 and K1B/S displayed similar potencies in in vitro cell assays, their angiogenic properties were significantly different in vivo.

Example 5. The K1B/S Complex Activates MET in the Liver and Impairs FAS-Induced Fulminant Hepatitis

[0203] In this last assay it was examined whether the K1B/S complex could act in vivo on distant tissues when injected systemically, and thus could constitute a basis for designing potent MET agonists of potential therapeutic interest. In a first approach, the different agonists were injected intravenously to see if they could activate MET and downstream pathways in the liver, an organ well known to strongly express MET receptor. After 10 min, livers were extracted and MET, ERK and Akt phosphorylation status was determined by western Blot (FIG. 17A). K1B/S, NK1 and HGF/SF injection induced a clear MET phosphorylation associated with a strong Akt and ERK activation in the liver. Importantly, activation by K1B/S was detectable at doses as low as 2.5 pmol (250 ng) per mg of body weight (FIG. 18) and even up to 30 min post-injection (FIG. 19). In contrast, K1B and streptavidin control led to no detectable signal.

[0204] Considering the fact that K1B/S complex is able to diffuse into the liver through the blood circulation and induce MET activation, it was examined whether the complex could promote hepatocyte survival when an apoptotic stress was induced in the liver. Indeed, injection of an anti-FAS antibody (anti-CD95) in mice quickly induces a massive hepatocellular apoptosis leading to fulminant hepatitis and death of the animals. Previous studies showed that HGF/SF was able to abrogate FAS induced fulminant hepatitis, but required prohibitive amounts to show significant effects (usually 1 nmol, i.e. ˜100 μg per mouse). In the present assay, anti-FAS antibody was mixed with 25 pmol of K1B, K1B/S or NK1, or 2.5 pmol of mature HGF/SF per mg of body weight. These concentrations were sufficient to promote strong MET signaling for at least 30 min. After 90 min, a second injection of each protein was performed to sustain signaling. Livers were extracted after 3 additional hours for histological and molecular analysis. Macroscopically, mice treated with anti-FAS antibody and K1B, NK1 or mature HGF/SF presented an altered liver, retaining a deep brown color even after PBS perfusion and elimination of vascular blood content (FIG. 17B). Remarkably, mice treated with K1B/S maintained a clear liver, almost intact. Histological analysis demonstrated that this dark color was mostly induced by a vascular congestion attributable to a massive hepatocyte loss and subsequent blood infiltration. Controls and HGF/SF treated mice showed totally disorganized livers with significant blood infiltration. In contrast, K1B/S mice kept well organized structures, although some blood infiltration could be visualized. NK1 treated mice presented an intermediate phenotype, retaining some organized areas but with massive blood infiltration. Further analysis confirmed that these disorganized regions corresponded to large clusters of apoptotic hepatocytes (FIG. 17D). Interestingly, all the mice challenged with anti-Fas antibody showed the early molecular markers characteristic for apoptosis such as cleaved caspase 3 and PARP1/2, even for the animals which were protected by K1B/S complex (FIG. 20). These results show that K1B/S does not act on the initial steps following FAS receptor activation but rather on downstream intracellular apoptotic signaling.

[0205] These histological and molecular analyses demonstrated that K1B/S complex acts systematically, efficiently activates MET signaling in the liver and is a potent survival factor even in extreme apoptotic stress conditions. The fact that K1B/S was more potent than NK1 highlights the significance of these findings for future MET agonist design.

METHODS

[0206] Chemical Protein Synthesis

[0207] Total chemical synthesis of K1 C-terminal biotin (K1B) was performed using 3 fragments in a one-pot protocol process, as described for the synthesis of biologically active K1 domain of HGF-SF (Ollivier et al., A one-pot three-segment ligation strategy for protein chemical synthesis. Angew Chem Int Ed 51, 209-213, 2012). Final purification of the full length synthetic 88 residues polypeptide and folding with concomitant formation of the 3 disulfide bridges gave synthetic biologically active K1B. The protein was aliquoted and stored at −80° C.

Design of K1B/S Complex NK1 (entry 1BHT) and streptavidin (entry 1SWE) structures were obtained from the PDB database. Extraction of K1 domain portion, visualization and distance measurements were performed on PyMOL v1.7 software.

[0208] Binding and Competition Assay

[0209] Competition assays for binding of K1B to recombinant MET-Fc protein were performed in 384-well microtiter plates (OptiPlate™-384, PerkinElmer, CA, USA, 50 μL of final reaction volume). Final concentrations were 0-300 nM for K1B, 2.5 nM for MET-Fc, 10 μg/mL for streptavidin coated donor beads and protein A-conjugated acceptor beads. The buffer used for preparing all protein solutions and the bead suspensions was: PBS, 5 mM HEPES pH 7.4, 0.1% BSA.

[0210] For K1B and MET-Fc binding assay, K1B (10 μL, 0-1.5 μM) was mixed with solutions of hMET-Fc (10 μL, 10 nM). The mixture was incubated for 10 min (final volume 15 μL). Protein A-conjugated acceptor beads (10 μL, 50 μg/mL) were then added to the vials. The plate was incubated at 23° C. for 30 min in a dark box. Finally, streptavidin coated donor beads (10 μL, 50 μg/mL) were added and the plate was further incubated at 23° C. for 30 min in a dark box. The emitted signal intensity was measured using standard Alpha settings on an EnSpire® Multimode Plate Reader (PerkinElmer). For the competition assay: increasing concentrations of K1B/S complex (ratio 2:1) were added to pre-mixed K1B (20 nM)/MET-Fc (2 nM)/ALPHA bead (10 μg/mL) complex.

[0211] Endogenous MET Capture

[0212] Streptavidin coated beads loaded with NB or K1B were incubated with HeLa or Capan-1 total cell lysates. Input, flow-through and elution fractions from NB or K1 loaded beads were analyzed by specific total MET western blot.

[0213] Cell Culture and Drug Treatment

[0214] Madin Darby Canine Kidney (MDCK) and Human cervical cancer HeLa cells, purchased from ATCC® (American Type Culture Collection, Rockville, Md., USA), were cultured in DMEM medium (Dulbecco's Modified Eagle's Medium, Gibco, Karlsruhe, Germany), supplemented with 10% FBS (Fetal Bovine Serum, Gibco®, Life technologies, Grand Island, N.Y., USA) and 5 mL of ZellShield™ (Minerva Biolabs GmbH, Germany). Twenty-four hours before drug treatment, the medium was exchanged with DMEM containing 0.1% FBS, and cells were then treated for different times with different compounds.

[0215] Akt and ERK Phosphorylation Assay by HTRF Method

[0216] The assay was performed according to the manufacturer's protocol mentioned in HTRF® (Cisbio bioassays, Bedford, Mass., USA). Briefly, cells were plated, stimulated with different agonists (HGF/SF, NK1, K1B/S and K1B/Ab), and then lysed in the same 96-well culture plate. Lysates (16 μL) were transferred to 384-well microplates for the detection of phosphorylated Akt (Ser473) and ERK (Thr202/Tyr204) by HTRF® reagents via a sandwich assay format using 2 different specific monoclonal antibodies: an antibody labelled with d2 (acceptor) and an antibody labelled with Eu3+-cryptate (donor). Antibodies were pre-mixed (2 μL of each antibody) and added in a single dispensing step. When the dyes are in close proximity, the excitation of the donor with a light source (laser) triggers a Fluorescence Resonance Energy Transfer (FRET) towards the acceptor, which in turn fluoresces at a specific wavelength (665 nm). Upon laser excitation, energy transfer between d2 and Eu3+-cryptate molecules occurs and fluorescence is detected at 620 and 665 nm on an EnVision® Multilabel reader (PerkinElmer). Data are presented as a 620/665 nm ratio for signal normalization.

[0217] Angiogenesis

[0218] Immunodeficient SCID mice weighing 19-21 g were used for this experiment. Mice were housed in a facility with a 12 h light/dark cycle at 22° C. and had free access to food and water. Mature HGF/SF, VEGF-A, NK1, K1B, Streptavidin and K1/S complexes were added to growth factor reduced Matrigel™ (BD Biosciences, Becton Dickinson, Belgium). Mice (n=6) were injected subcutaneously in the flank with 400 μL of Matrigel. After 11 days, mice were sacrificed, Matrigel plugs were removed and weighed, and 300 μL of water was added to induce hypotonic red blood cell lysis and hemoglobin release. Hemoglobin absorbance (405 nm) was measured, and concentration was determined against a hemoglobin standard curve and plug weight.

[0219] All experimental procedures were conducted with the approval of the Ethics Committee for Animal Experimentation of the Nord Pas de Calais Region (CEEA 75).

[0220] Fas Induced Fulminant Hepatitis

[0221] FVB mice weighing 19-21 g (Charles River) were used for this experiment. After anesthesia with isoflurane (Aerrane, Baxter, USA), mice (n=3) were given intravenous injections of 125 ng/g body weight of anti-Fas antibody (Clone Jo-2, CD95, Pharmingen, BD Biosciences) mixed with different agonists (HGF/SF, NK1, and K1/S) in PBS. The mice were injected a second time with each agonist 90 min after the first injection. The mice were sacrificed after 3 additional hours, and their livers perfused with PBS supplemented with protease and phosphatase inhibitors.

[0222] In parallel, to visualize MET activation in the liver, mice were given intravenous injections of each agonist for 10 min.

[0223] For histological analysis, liver tissue was collected, fixed overnight in 4% paraformaldehyde, and snap frozen in isopentane, submerged in liquid nitrogen, and embedded in OCT (Tissue-Tek®, VWR, PA, USA). Frozen liver sections (5 μm) were stained with hematoxylin and eosin (HE) for general morphology. TUNEL staining for apoptosis was also performed on liver sections according to the manufacturer's instructions (Apoptag® Fluorescein Direct In Situ kit, Merck Millipore, Billerica, Mass., USA). For molecular analysis, extracted liver tissue was immediately frozen in liquid nitrogen. Livers were crushed in lysis buffer supplemented with freshly added protease and phosphatase inhibitors.

Reagents and Antibodies

[0224] Recombinant human HGF/SF was purchased from Invitrogen (Breda, Netherlands), recombinant VEGF-A from R&D Systems (Minneapolis, Minn., USA), Streptavidin (Streptomyces avidinii) from ProZyme (Hayward, Calif., USA) and Anisomycin (Streptomyces griseolus) from CalbioChem (Germany). Recombinant human NK1 protein (residues 28-209) was kindly provided by Prof. Ermanno Gherardi (University of Pavia (Italy). Antibodies directed against the kinase domain of MET were purchased from Invitrogen, anti-phospho-MET (Tyr1234/1235), anti-phospho-MET (Tyr1349), anti-total Akt, anti-phospho-Akt (Ser473), anti-phospho-ERK1/2 (Thr202/Tyr204) and anti-Caspase-3 from Cell Signaling (Massachusetts, USA), anti-ERK2 (C-14) and anti-PARP1/2 from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Anti-biotin monoclonal antibody and horseradish peroxidase (HRP)-conjugated antibodies directed against rabbit or mouse IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, Pa., USA).

[0225] Characterization of K1B/S Complex

[0226] K1B and streptavidin complex ratios were analyzed by SDSPAGE using 10% NuPage precast gels run in MES buffer (Life Technologies) without heating the samples. Gels were fixed in 20% methanol and 5% acetic acid for 30 min, and stained in Coomassie Brilliant Blue solution.

[0227] Native Mass Spectrometry

[0228] Streptavidin and K1B were first buffer exchanged in 200 mM ammonium acetate pH 7.4, using Zeba™ bench-top spin desalting columns (Thermo Scientific). Protein concentrations were determined by measuring the absorbance at 280 nm and using extinction coefficients of 16,500 and 165,000 M.sup.−1 cm.sup.−1 for K1B and streptavidin, respectively. Titration was performed by adding 0 to 5 molar equivalents of K1B to streptavidin. A 10 μl volume was prepared per sample, and final concentrations ranged from 1 to 20 μM. Noncovalent MS analysis was performed on a Synapt G2 HDMX (Waters, Manchester, UK) coupled to an automated chip-based nanoelectrospray device (Triversa Nanomate, Advion Biosciences, Ithaca, USA) operating in the positive ion mode.

[0229] Instrument parameters were as follows: capillary, sample cone and extraction cone voltages were set at 1.55 kV, 65 V and 5 V, respectively. The backing pressure was increased to 6 mbar to improve the transmission of high molecular weight species by collisional cooling. Calibration was performed with a 2 mg/ml cesium iodide solution and data were analyzed with MassLynx software v.4.1 (Waters, Manchester, UK).

[0230] Endogenous MET Capture

[0231] HeLa and Capan-1 cells were collected by scraping and then lysed on ice with a lysis buffer (20 mM Tris HCl, 50 mM NaCl, 5 mM EDTA and 1% Triton X-100). Lysates were clarified by centrifugation (20,000 g×15 min) and protein concentration was determined (BCA protein assay Kit, Pierce®, Thermo scientific, IL, USA). Streptavidin-Sepharose beads (GE Healthcare) were washed and equilibrated in PBS. Beads were loaded with 15 μg K1B or NB (100 μl beads in a 50:50 PBS:bead slurry) for 20 min at room temperature and immediately washed with PBS. Beads were incubated with 250 μg of protein cell lysates overnight at 4° C. under mild agitation. Beads were quickly washed with PBS and bound proteins were eluted with 200 mM glycine buffer pH 2. Elution fractions were then analyzed by western blotting.

[0232] Western Blots

[0233] Cells were collected by scraping and then lysed on ice with a lysis buffer (20 mM HEPES pH 7.4, 142 mM KCl, 5 mM MgCl2, 1 mM EDTA, 5% glycerol, 1% NP40 and 0.1% SDS) supplemented with freshly added protease and phosphatase inhibitors (#P8340 and #P5726, respectively, Sigma). Lysates were clarified by centrifugation (20,000 g×15 min) and protein concentration was determined (BCA protein assay Kit, Pierce®, Thermo scientific, IL, USA). The same protein amount of cell extracts was separated by either classical SDS-PAGE or NuPAGE (4-12% or 10% Bis-Tris precast gels) (Life technologies) and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore). Membranes were probed with indicated primary antibodies, followed by incubation with appropriate HRP conjugated secondary antibodies. Protein-antibody complexes were visualized by chemiluminescence with the SuperSignal® West Dura Extended Duration Substrate (Thermo scientific), using a LAS-3000 imaging system (Fujifilm, Tokyo, Japan) or X-ray films (CL-Xposure™ Film, Thermo scientific).

[0234] MTT Assay

[0235] Cells were washed with PBS to eliminate dead cells and then incubated in medium containing 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Invitrogen) for 1 h. After a washing step with PBS, the formazan crystals were solubilized and mixed thoroughly with 0.04 M HCl in isopropanol. For each condition, 60 μl of formazan solution was loaded in triplicate onto a 96-well plate. Absorbance was then measured with a microplate spectrophotometer at 550 nm and 620 nm, as test and reference wavelengths, respectively. The absorbance correlates with cell number.

[0236] Scattering Assay

[0237] Cells were seeded at low density (2,000 cells/well on a 12-well plate) to form compact colonies. After treatment, when colony dispersion was observed, the cells were fixed and colored by Hemacolor® stain (Merck, Darmstadt, Germany) according to the manufacturer's instructions. Representative images were snap-captured using a phase contrast microscope with 40× magnification (Nikon Eclipse TS100, Tokyo, Japan).

[0238] Morphogenesis Assay

[0239] Cells were seeded onto a layer of Growth Factor Reduced Matrigel™ (BD Biosciences) (100,000 cells/well of a 24-well plate), treated and observed under phase contrast microscope. Representative images were snap-captured with 40× magnification (Nikon Eclipse TS100).

[0240] Statistical Analysis

[0241] Data were obtained in triplicate from at least 3 independent experiments, and expressed either as mean values or percentages of control values+/−SD or SEM depending on the experiments performed. When indicated, differences between data groups were determined by ANOVA using Prism 5 (GraphPad Software, Inc., San Diego, Calif., USA), and considered to be statistically significant for P<0.05.