EP1940873A2 - Neue fgf-rezeptor-bindende verbindungen, die peptidfragmente des neuralen zelladhäsionsmoleküls l1 enthalten - Google Patents

Neue fgf-rezeptor-bindende verbindungen, die peptidfragmente des neuralen zelladhäsionsmoleküls l1 enthalten

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Publication number
EP1940873A2
EP1940873A2 EP06791468A EP06791468A EP1940873A2 EP 1940873 A2 EP1940873 A2 EP 1940873A2 EP 06791468 A EP06791468 A EP 06791468A EP 06791468 A EP06791468 A EP 06791468A EP 1940873 A2 EP1940873 A2 EP 1940873A2
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Prior art keywords
amino acid
compound
seq
compound according
fragment
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French (fr)
Inventor
Vladimir Berezin
Elisabeth Bock
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Enkam Pharmaceuticals AS
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Enkam Pharmaceuticals AS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

  • Novel FGF receptor binding compounds comprising peptide fragments of neural cell adhesion molecule L1
  • novel peptide compounds capable of binding to fibroblast growth factor receptor (FGFR) and activating said receptor.
  • Novel compounds of the invention comprise peptide fragments comprising FGFR binding motif x " -(x) n -x p -(x) n -x " , wherein x " is a basic amino acid residue, x p is a hydrophobic amino acid residue and (x) n is a sequence of any amino acid residues wherein n is a integer from 0 to 3.
  • a compound of the invention comprises a fragment of neural cell adhesion molecule L1 which is capable of binding to FGFR and activating the receptor.
  • the invention discloses the amino acid sequences of the FGFR binding, fragments of L1 and features pharmaceutical compositions comprising thereof.
  • Invention also relates to uses of the compounds and pharmaceutical compositions for treatment of different pathological conditions, wherein FGFR and/or L1 play a role in pathology and/or recovery from the disease.
  • Antibodies which are capable of binding to epitopes comprising peptide sequences of the invention are also concerned.
  • CAMs cell adhesion molecules
  • Candidate molecules include the amyloid precursor protein, which shares many properties of a classical CAM and beta- amyloid, which can masquerade as a pseudo CAM.
  • Beta-Amyloid serves as a nidus for the formation of senile plaques in Alzheimer's disease and like CAMs provides an environment for organising neurotrophic factors and other CAMs. Inflammatory responses evolve in this environment and can initiate a vicious cycle of perpetuated neuronal damage that is medicated by microglia, complement and other factors (Cotman et al. (1998) Prog Neurobiol. 55:659-69).
  • CAMs Neural cell adhesion molecules of the immunoglobulin superfamily nucleate and maintain groups of cells at key sites during early development and in the adult.
  • CAMs homophylic and heterophylic interactions can affect intracellular signalling. Their ability to influence developmental events, including cell migration, proliferation, and differentiation may therefore result from both their adhesive and signalling properties.
  • L1 plays a pivotal role during development of the nervous system, mediating binding between neuronal cells and stimulating axonal outgrowth and fasciculation.
  • L1 consists of six immunoglobulin (Ig) modules in the amino-terminal region, followed by five fibronectin type III (F3) modules, a trans- membrane domain, and a highly conserved cytoplasmic tail (Nybroe and Bock, 1990).
  • Ig immunoglobulin
  • F3 fibronectin type III
  • L1 is known to mediate cell-cell interactions by a homophilic binding, i.e. L1 on one cell binds to L1 on an adjacent cell (Grumet and Edelman, 1984; Lemmon V et al., 1989, Doherty et al., 1995).
  • NCAM neural cell adhesion molecule
  • TAG-1/axonin- 1 the axon-associated cell adhesion molecule TAG-1/axonin- 1
  • HAS/CD24 glycosyl-phosphatidylinositol- anchored molecule HAS/CD24
  • chondroitin sulphate proteoglycans neurocan and phosphacan Margolis et al., 1996)
  • Fibroblast growth factor receptors are a family of at least four closely related receptor protein tyrosine kinases, FGFR 1 , FGFR 2, FGFR 3 and FGFR 4, consisting extracellularly of three Ig-like modules and intracellular ⁇ of a split tyro- sine-kinase module (Powers et al. (2000) Endocr Relat Cancer 7:165-97).
  • the receptors are known to be key regulators of morphogenesis, development, angio- genesis, and wound healing.
  • FGFR activation and signalling are dependent on dimerization of the receptor which is induced by high affinity binding of FGFR natural ligand, fibroblast growth factor (FGF), and it also requires participation of cell surface heparin or heparan sulphate proteoglycans.
  • FGFs fibroblast growth factors
  • FGFs Fibroblast growth factors
  • their receptors constitute an elaborate signaling system that participates in many developmental and repair processes of virtually all mammalian tissues, in particular, they play a prominent role in functioning of the peripheral and central neural system.
  • NCAM neural cell adhesion molecules
  • l_1 and N-cadherin neural cell adhesion molecules
  • NCAM has recently been regarded as a member of a new class of putative alternative ligands of FGFR, low affinity ligands (Kiselyov et al. (2005) J Neurochem 94:1169- 1179).
  • NCAM has been obtained evidence for a direct interaction between NCAM and the receptor (Kiselyov et al. (2003) Structure (Camb) 11 :691-701).
  • NCAM fragment having the sequence EVYWAENQQGKSKA (FGL peptide) involved in the direct interaction between NCAM and FGFR has been suggested as a new candidate drug for the treatment of a variety of pathologic conditions where the activity of FGFR may play a key role (WO 03/016351).
  • WO 03/016351 describes some biological effects of the FGL peptide due to binding and activating FGFR.
  • L1 has been broadly studied as a neurite outgrowth stimulating and cell survival proting cell adhesion molecule (Haspel et al. (2000) J Neurobiol 15:287-302; Roonprapurt et al. (2003) J Neurotrauma 20:871-882; Wiencken-Barger et al. Cereb Cortex (2004) 14:121-131; Loers er al. (2005) J Neurochem 92:1463-1476). It has also been regarded important for neural cell precursor proliferation, differentiation and transmitter phenotype subtype generation (Dihne et al. (2003) J Neurosci 23:6638-6650), and it is known to play a role in synaptic plasticity (Saghatelyan et al.
  • Soluble molecules of L1 have been suggested for use as neurite outgrowth and survival promoting compounds in theurapetic applications (US 6,576,607).
  • the usage of such molecules for medical applications is limited as the L1 ectodomain polypeptide is very long (constituting 1100 amino acids (Swissprot P32004)).
  • L1 polypeptide lidentification of functionally important regions of L1 polypeptide and production of these regions as individual peptide fragments which are capable of mimicking the biological function of the whole L1 ectodomain polypeptide, seems might solve the problems rising when a medical application of L1 is concerned.
  • a beneficial input may also give the identification of binding sites in L1 which are involved in interaction with different L1 binding molecules. It would advantageously provide new effective drugs for the treatment of diseases where the molecules interacting with L1 are concerned, e. g. FGFR receptor.
  • information about structural requirements for the functional capabilities of L1 molecule is insufficiently disclosed in the prior art, if available at al.
  • the present invention relates to identificating FGFR binding sites in L1 and providing compounds comprising peptide fragments derived from these binding sites.
  • Novel compounds of the invention are FGFR binding compounds which are capable of modilating FGFR activity.
  • Compounds of the invention comprise peptide sequences which comprise FGFR binding motif x " -(x) n -x p -(x) n -x " , wherein x " is a basic amino acid residue, x p is a hydrophobic amino acid residue and (x) n is a sequence of any amino acid residues wherein n is a integer from 0 to 3.
  • compounds of the invention are at most 105 amino acids long and comprise a fragment of neural cell adhesion molecule L1 which is capable of binding FGFR and activating the receptor.
  • the invention discloses amino acid sequences of the FGFR binding fragments of L1 and features pharmaceutical compositions comprising thereof.
  • Invention also relates to uses of the compounds and pharmaceutical compositions comprising thereof for the treatment of different pathological conditions, wherein FGFR and/or L1 play a role in pathology and/or recovery from the disease, for example for a) treatment of conditions of the central and peripheral nervous system associated with postoperative nerve damage, traumatic nerve damage, impaired myelina- tion of nerve fibers, postischaemic damage, e.g.
  • Parkinson's disease Alzheimer's disease, Huntington's disease, dementias such as multiinfarct dementia, sclerosis, nerve degeneration associated with diabetes mellitus, disorders affecting the circadian clock or neuro-muscular transmission, and schizophrenia, mood disorders, such as manic depression;
  • promotion of wound-healing e.g., wound-healing
  • Antibodies which are capable of binding to epitopes comprising peptide sequences of the invention, are also concerned by the invention.
  • Fig. 1 Binding of L1 F3 modules I-V to FGFR1 Ig modules H-III. The binding was studied by means of SPR analysis. Approx. 3000 resonance units (RU) of the FGFR1 modules were immobilized on the sensor chip. Combined L1 F3 modules I-V or combined NCAM Ig modules l-ll (ctrl) were injected into the sensor chip at the specified concentrations. The binding is given as a response difference between the binding to the sensor chip with the immobilized FGFR1 modules and a blank sensor chip (unspecific binding). Only a very low unspecific binding was detected for the above mentioned proteins at all concentrations used in this study. Four-five independent experiments were performed using different preparations of all proteins.
  • Fig. 2 Demonstration of an ATP-induced increase of binding between FGFR1 Ig modules 11— Il I and L1 F3 modules I-V. The binding was studied by SPR analysis.
  • PCP, AMP or GTP were performed.
  • Fig. 3 Demonstration of an effect of ATP, AMP-PCP, GTP, AMP on the binding between FGFR1 Ig modules H-III and L1 F3 modules I-V.
  • Fig. 4 Effect of the L1 F3 modules I-V on phosphorylation of FGFR1 and immunoprecipitation of L1 by FGFR1.
  • Fig. 5 Effect of L1 F3 modules I-V on neur ⁇ te outgrowth from cerebellar neurons.
  • E, F Neurons stimulated with 1 ⁇ M F3 modules I-V in the presence of various concentrations of ATP (E) or AMP-PCP (F).
  • * and ** stand for statistical significance of p ⁇ 0.05 and p ⁇ 0.01 , respectively, compared to Ctr. +, ++ and +++ stand for statistical significance of p ⁇ 0.05, p ⁇ 0.01 and p ⁇ 0.001 , respectively, compared to the highest response.
  • Fig. 6 Sequence of peptides representing loop regions of L1 F3 domains I-V. 30 peptides representing all loops of the F3 I-V modules were synthesized and used to map binding sites between L1 and FGFR1. Shown are the loop areas of F3 I-V modules with parts of the adjacent beta strands.
  • Fig. 7. Demonstration of binding between the FGFR1 and peptides derived from L1 F3 modules I-V. The binding was studied by SPR analysis. Approx. 3000 resonance units (RU) of the FGFR1 modules were immobilized on the sensor chip. Three independent experiments were performed. A) Peptides bound to FGFR1 modules H-III (F1CDL- (8 ⁇ M), F2BCL- (8 ⁇ M), F3ABL- (40 ⁇ M), F3CDL- (2 ⁇ M), F5BCL- (80 ⁇ M), and F5FGL- (80 ⁇ M) peptides). B) peptides bound to FGFR1 module Il (F1CDL- (16 ⁇ M), F2BCL- (16 ⁇ M), F3CDL- (8 ⁇ M), and F5BCL- (130 ⁇ M) peptides).
  • Fig. 8 Effect of the peptides bound to FGFR1 on phosphorylation of FGFR1.
  • Fig. 9 Effect of the peptides bound to FGFR1 on neurite outgrowth from cerebellar neurons. Neurite length versus the concentration of the peptides which bound to FGFR1. Four independent experiments were performed. Error bar represents one standard error of the mean. * and ** stand for statistical significance of p ⁇ 0.05 and p ⁇ 0.01, respectively, compared to Ctr (PBS).
  • Fig. 10 A model of binding between L1 and FGFR1.
  • Possible compact conformation of L1 F3 modules was proposed where marked regions correspond to peptides, which bound to FGFR1, stimulated neurite outgrowth and phosphorylation of the receptor.
  • Conformation of the dimer of FGFR1 is taken from the crystal structure of FGFR1 (Pellegrini et al., 2000).
  • FIG. 11 Gel-filtration experiments demonstrating possibility of a compact conformation of L1 F3 domains I-V.
  • the C-terminal amino acid of a peptide of the invention exists as the free carboxylic acid, this may also be specified as "-OH".
  • the C-terminal amino acid of a compound of the invention may be the amidated derivative, which is indicated as "-NH 2 ".
  • the N-terminal amino acid of a polypeptide comprise a free amino-group, this may also be specified as "H-”.
  • amino acid can be selected from any amino acid, whether naturally occurring or not, such as alfa amino acids, beta amino acids, and/or gamma amino acids. Accordingly, the group comprises but are not limited to: A, V, L, I, P, F, W, M, G, S, T, C, Y, N, Q, D, E, K, R, H Aib, NaI, Sar, Orn, Lysine analogues, DAP, DAPA and 4Hyp.
  • modifications of the compounds/peptides may be performed, such as for example glycosylation and/or acetylation of the amino acids.
  • Basic amino acid residues are according to invention represented by the residues of amino acids H, K and R; acidic amino acid residues - by the residues of amino acids E and D; hydrophobic amino acid residues by the residues of amino acids A, L, I, V, M, F, Y and W; neutral, weakly hydrophobic - by P, A and G; neutral hydrophilic - by amino acid residues Q, N, S and T; cross-link forming by amino acid resudue C.
  • the present invention relates to a compound capable of interacting with Fibroblast Growth Factor Receptor (FGFR) comprising an amino acid sequence which comprises an amino acid motif of the formula wherein x " is a basic amino acid residue, x p is a hydrophobic amino acid residue and
  • FGFR Fibroblast Growth Factor Receptor
  • (X) n is a sequence of any amino acid residues, wherein n is an integer from 0 to 3.
  • x " may be any basic amino acid residue independently selected from K, R or H
  • x p may be any hydrophobic amino acid residue independ- ently selected from A, V, L, I, P, F, W, M or Y
  • (x) n is a sequence of any amino acid residues, wherein n is an integer from 0 to 3.
  • preffered motifs are, those wherein the residues x " and x p and the length of the sequence (x) are as in any of the following motifs: K-(X) 3 -L-(X) 1 -K, R-(X) 3 -L-(X) 1 -K, K-(X) 1 -L-(X) 3 -K, H-(X) 1 -L-(X) 2 -K, R-(X) 2 -W-(X) 0 -R, K-(X) 2 -L-(X) 0 -R, R-(X) 1 -V-(X) 2 -H, K-(X) 1 -F-(X) 3 -K, R-(X) 0 -Y-(X) 2 -K or R-(X) 2 -I-(X) 2 -K
  • At least one of the residues of the sequence (x) is a basic amino acid residue, hydrophilic amino acid residue or G.
  • it may be a basic residue selected from K, R or H, wherein H is more preferred.
  • it may be a hydrophilic residue selected from either charged or uncharged hydrophylic residues, such as D, E, H, K, N, Q, R, S or T, wherein H is more preferred among the charged amino acid residues, and Q or S are more preferred among the uncharged residues.
  • the residue may be G.
  • the amino acid motif as defined above is a FGFR binding amino acid motif.
  • the present invention provides the following non-limited examples of such FGFR binding amino acid motifs: KWFSLGK (SEQ ID NO: 8) RYQWR (SEQ ID NO: 9) KGHLR (SEQ ID NO: 10) RHVHSH (SEQ ID NO: 11) RFHILFK (SEQ ID NO: 12) KALPEGK (SEQ ID NO: 13) HHLAVK (SEQ ID NO: 14).
  • any of the above listed amino acid sequences is capable of binding FGFR and this capability is conferred to a compound comprising said sequence. Accordingly, a compound comprising any of the above sequences is concerned as a promising compound for the purpose of activating FGFR.
  • promising compounds of the invention are preferred peptide sequences which comprise at most 105 amino acid residues.
  • one of the preferred compounds is a compound that comprises an isolated peptide sequence which comprises a FGFR binding motif selected from the motifs identified above (SEQ ID NOs: 8-14), wherein the isolateed peptide sequence, preferably, comprises an amino acid sequence selected from the following amino acid sequences:
  • a compound that essentially comprises an amino acid sequence selected from SEQ ID NOs: 1-6 More preferred a compound that essentially comprises an amino acid sequence selected from SEQ ID NOs: 1-6.
  • the term "essentially comprising” means in the present context that a sequence selected from SEQ ID NOs: 1-6 constitutes at least
  • a compound consisting any of the sequences selected form SEQ ID NOs: 1-6 are also among preferred compounds of the invention.
  • a preferred compound which consists of a sequence selected from the sequences of SEQ ID Nos:1-6, is a compound consisting of SEQ ID NO: 1.
  • a preferred compound may consist of SEQ ID NO: 2.
  • a preferred compound may consist of SEQ ID NO: 3.
  • a preferred compound may consist of SEQ ID NO: 4.
  • the compound may consist of SEQ ID NO: 5 or SEQ ID NO: 6.
  • SEQ ID NOs:1-6 are meant to be isolated peptide sequences.
  • An isolated peptide sequence is according to the invention an amino acid sequence of a desirable length which is produced by use of any recombinant technology methods or chemical synthesis, or it was separated from a longer polypeptide or protein by a method of enzymatic or chemical cleavage
  • an isolated peptide sequence may be a fragment of a protein being separated from the other parts of the protein, e.g. other fragments of the pro- tein polypeptide.
  • the present invention relates to isolated peptide fragments of the proteins which comprise a FGFR motif described above.
  • an isolated peptide fragment of the invention may derive from any protein, however, a preferred isolated protein fragment of the invention is derived from neural cell adhesion molecule L1.
  • a compound of the invention may comprise or consist of a peptide fragment of L1.
  • the invention relates to any mammal L1 protien, such as for example as the identified in Swiss-Prot database polypeptides P32003, P11627, Q05695.
  • L1 is human which has the sequence identified in Swiss-Prot database as P32004.
  • the invention in particular relates to peptide fragments of L1 which are derived from a part of L1 polypeptide which corresponds to Fibronectin type-3 modules 1 to 5 (F3, 1-5).
  • a peptide fragment of L1 of the invention consists of the F3, 1 module of L1 and has the sequence identified as SEQ ID NO: 15.
  • the L1 -derived peptide ftadment consists of the F3, 2 module of L1 and has the sequence identified as SEQ ID NO: 16.
  • the fragment is the F3, 3 module of L1 and has the sequence identified as SEQ ID NO: 17.
  • the fragment is the F3, 4 module of L1 and has the sequence identified as SEQ ID NO: 18.
  • the fragment may consist of the F3, 5 module of L1 and has the sequence identified as SEQ ID NO: 19.
  • the invention preferably relates to L1 F3,1-F3,5 modules having the following sequences: F3.1: PGPVPRLVLSDLHLLTQSQVRVSWSPAEDHNAPIEKYDIEFEDKEMAPEKWYSLG KVPGNQTSTTLKLSPYVHYTFRVTAINKYGPGEPSPVSETV
  • F3,2 PEKNPVDVKGEGNETTNMVITWKPLRWMDWNAPQVQYRVQWRPQGTR GPWQEQIVSD PFLWSNTSTFVPYEIKVQA VNSQGKGPEP QVTIGYS
  • the invention concern fragments of the F3 modules of L1, such as fragments of the F3,1 , F3,2, F3,3, F3, 4 or F3, 5 modules. It is preferred that the fragments of the latter modules comprise at least one of the sequences selected from SEQ ID NOs: 1-6 or fragments of said sequences, such as fragments having the length of 5-7 amino acid residues and comprising the FGFR binding motif discussed above, for example a sequence selected from SEQ ID NOs: 8-14.
  • a compound of the invention comprises an amino acid sequence comprising a FGFR binding motif of at most 105 amino acid residues.
  • the compound may comprise a peptide sequence comprising 3-105 amino acid residues, for example 3-100 amino acid residues, such as 3-95 amino acid residues, for example 3-90 amino acid residues, such as 3-85 amino acid residues, for example 3-80 amino acid residues, such as 3-75 amino acid residues, for example 3-70 amino acid residues, such as 3-65 amino acid residues, for example 3-60, such as 3-55 amino acid residues, for example 3-50 amino acid residues, such as 3-45 amino acid residues, for example 3-40 amino acid residues, such as 3-35 amino acid residues, such as 3-30 amino acid residues, for example 3-25 amino acid residues, such as 3-20 amino acid residues, for example 3-15, such as 3-10 amino acid residues, for example 3-5 amino acid residues.
  • 3-105 amino acid residues for example 3-100 amino acid residues, such as 3-95 amino acid residues, for example 3-90 amino acid residues, such as 3-85 amino acid residues, for example 3-80 amino acid residues, such as 3-75 amino acid residues, for
  • a peptide sequence of the compound may have the length of between 4-100 amino acid residues, such as 4-90 amino acid residues, for example 4-80 amino acid residues, such as 4-70, for example 4-60, such as 4-50 amino acid residues, for example 4-40 amino acid residues, such as 4-30 amino acid residues, for example 4-20 amino acid residues, such as 4-15 amino acid residues or 4-10.
  • a peptide sequence may have the length between 5 and 100 amino acid residues, such as 5 to 90 amino acid residues, for example 5 to 80 amino acid residues, such as 5 to 70, for example 5 to 60, such as 5 to 50 amino acid residues, for example 5 to 40 amino acid residues, such as 5 to 30 amino acid residues, for example 5 to 20 amino acid residues, such as 5 to 15 amino acid residues, for example 5 to 10 amino acid residues.
  • sequence may be of 6-100 amino acid residues, such as 6-90 amino acid residues, for example 6-80 amino acid residues, such as 6-70, for example 6-60, such as 6-50 amino acid residues, for example 6-40 amino acid residues, such as 6-30 amino acid residues, for example 6-20 amino acid residues, such as 6-15 amino acid residues, for example 6-10 amino acid residues.
  • the invention also relates to amino acid sequences, which have the length of between 7-100 amino acid residues, such as 7-90 amino acid residues, 7-80 amino acid residues, such as 7-70, for example 7-60, such as 7-50 amino acid residues, for example 7-40 amino acid residues, such as 7-30 amino acid residues, for example 7-20 amino acid residues, such as 7-15 amino acid residues, for example 8, 9, 10, 11, 12, 13, or 14 amino acid residues.
  • the length of the isolated sequences may also be of 8-80 amino acid residues, such as 8-70, for example 8-60, such as 8-50 amino acid residues, for example 8-40 amino acid residues, such as 8-30 amino acid residues, for example 8-20 amino acid residues.
  • it may be of 9-80 amino acid residues, such as 9-70 amino acid residues, for example 9-60 amino acid residues, such as 9-50 amino acid residues, for example 9-40 amino acid residues, such as 9-30 amino acid residues, for example 9-20 amino acid residues.
  • a compound which comprises an amino acid sequence which is of 10-80 amino acid residues, such as 10-70 amino acid residues, for example 10-60 amino acid residues, such as 10-50 amino acid residues, for example 10-40 amino acid residues, such as 10-30 amino acid residues, for example 10-20 amino acid residues is also within the scope of the invention.
  • the minimal length of an amino acid sequences of the compound be of 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 amino acid residues, and the maximal is 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues.
  • a compound comprising an amino acid sequence the length of which is in the range of 25 - 36 amino acid residues is also preferred.
  • more preferred compounds are those that comprise isolated peptide sequences the length of which is at most 20 amino acid residues.
  • the compounds comprising individual peptide fragments having the length of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 or 18 amino acid residues are among most preferred compounds. It is understood that all the above discussed peptide sequences comprise either an FGFR binding motif of the invention or a peptide fragment of L1 described above, and are capable of binding to FGFR.
  • An isolated peptide sequence of the invention may be connected to another isolated peptide sequence by a chemical bond in a fusion protein with the proviso that the fusion protein is not L1 protein.
  • Two or more isolated sequences of the invention may also be connected to each other through a linker grouping making other types of multimeric presentation of peptide sequences of the invention. Both the latter fusion proteins and multimers are concerned as compounds of the invention.
  • a peptide sequence of the invention may in some embodiments be formulated as a polymer comprising several copies of said peptide sequence.
  • the polymers comprising two sequences are termed herein as dimers.
  • the polymers comprising four peptide sequences are termed herein as tetramers.
  • a multimeric compound may be a polymer of two or more identical or different peptide sequences of the invention, wherein in a preferred embodiment, one of the two or more amino acid sequences is selected from SEQ ID NOs: 1-6, or fragments or variants of said sequences.
  • the compound may comprise two identical amino acid se- quences selected from SEQ ID NOs:1-6, or two identical fragments or variants of the selected sequence, wherein said amino acid sequences, fragments or variants.
  • the compound may comprise four identical copies of an amino acid sequence selected from SEQ ID NOs:1-6, or four identical fragments or variants of the selected sequence.
  • the compound may comprise two or more different amino acid sequences, wherein at least one of the two amino acid sequences is a sequence selected from SEQ ID NOs: 1-6, or fragments or variants thereof.
  • the compound may comprise two or more different amino acid sequences, wherein said two or more amino acid sequences are selected from SEQ ID NO: 1-6, or fragments or variants thereof.
  • a preferred multimeric compound of the invention is a compound wherein the amino acid sequences are connected to each other through a linker or a linker grouping.
  • a linker is according to the invention may be any molecule or chemical moiety capable of cross-linking two or more peptide sequences, for example it may be an achiral di-, tri- or tetracarboxylic acid of the general formula
  • n and m independently are an integer of from 1 to 20,
  • X is HN, H 2 N(CR 2 )PCR, RHN(CR 2 )pCR, HO(CR 2 )pCR, HS(CR 2 )pCR, halogen- (CR 2 )pCR, HOOC(CR 2 )pCR, ROOC(CR 2 )pCR, HCO(CR 2 )pCR, RCO(CR 2 )pCR, [HOOC(A)n][HOOC(B)m]CR(CR 2 )pCR, H 2 N(CR 2 )p, RHN(CR 2 )P, HO(CR 2 )p, HS(CR 2 )P, halogen-(CR 2 )p, HOOC(CR 2 )p, ROOC(CR 2 )P, HCO(CR 2 )P, RCO(CR 2 )p, or [HOOC(A)n][HOOC(B)m](CR 2 )p , wherein
  • Ci_ 10 alkyl is meant straight or branched chain alkyl groups having 1- 10 carbon atoms, e.g. methyl, ethyl, isopropyl, butyl, and tertbutyl.
  • C 2-10 alkenyl is meant straight or branched chain alkenyl groups hav- ing 2-10 carbon atoms, e.g. ethynyl, propenyl, isopropenyl, butenyl, and tert-butenyl.
  • cyclic moiety is meant cyclohexan, and cyclopentane.
  • aromatic moiety is meant phenyl.
  • a and B forms a cyclic, heterocyclic or aromatic moiety denotes cyclohexan, piperidine, benzene, and pyridine.
  • a multimeric compound of the invention comprises the linker of above
  • the compound is preferably obtained by the LPA method (a ligand presentation assembly method) as described in WO0018791 and WO2005014623.
  • a preferred linker of the invention may be amino acid lysine.
  • Individual peptide sequences may be attached to a core molecule such as lysine forming thereby a dendritic multimer (dendrimer) of an individual peptide sequence(s).
  • dendrimers Production of dendrimers is also well known in the art (PCT/US90/02039, Lu et al., (1991) MoI Immunol. 28:623-630; Defoort et al., (1992) lnt J Pept Prot Res. 40:214-221 ; Drijfhout et al. (1991) lnt J Pept Prot Res. 37:27- 32), and dedrimers are at present widely used in research and in medical applications.
  • amino acid cystein may be preferred a linker molecule.
  • One of the referred embodiments of the invention concernes a compound comprising four individual amino acid sequences attached to the lysine core molecule, a dendritic tetramer/dendrimer of a peptide sequence of the invention.
  • Multimeric compounds of the invention such as LPA-dimers or dendrimers, are most preferred compounds of the invention.
  • other types of multimeric compounds comprising two or more individual sequences of the invention are also in the scope of the invention. These compounds may be produced using thechnologies known in the art.
  • the peptide sequences may be covalently bound to the linker through their amino- or carboxy-groups, preferably through the N- or C terminal amino- or carboxy- groups.
  • a compound may comprise a variant or a fragment of an FGFR binding sequences of the invention, preferably a fragment or variant of a sequence selected from SEQ ID NOs:1-6.
  • a variant of an amino acid sequence selected from the sequences SEQ ID NOs: 1-6 may be i) an amino acid sequence which has at least 60% identity with a selected sequence, such as 61-65% identity, for example 66-70% identity, such as 71-75% identity, for example 76-80% identity, such as 81-85 % identity, for example 86-90% identity, such as 91-95% identity, for example 96- 99% identity, wherein the identity is defined as a percentage of identical amino acids in said sequence when it is collated with the selected sequence.
  • amino acid sequences may be calculated using well known algorithms such as BLOSUM 30, BLOSUM 40, BLO- SUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLO- SUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLO-
  • positive amino acid matches for example 76-80% positive amino acid matches, such as 81-85 % positive amino acid matches, for example 86-90% positive amino acid matches, such as 91-95% positive amino acid matches, for example 96-99% positive amino acid matches, wherein the positive amino acid match is defined as the presence at the same position in two compared sequences of amino acid residues which has similar of physical and/or chemical properties.
  • Preferred positive amino acid matches of the present invention are K to R, E to D, L to M, Q to E, I to V, I to L, A to S, Y to W, K to Q, S to T, N to S and Q to R;
  • 95% positive amino acid matches for example 96-99% positive amino acid matches and comprises other chemical moieties, e. g. phosphoryl, sulphur, acetyl, glycosyl moieties.
  • variant of a peptide sequence means that the peptide sequence may be modified, for example by substitution of one or more of the amino acid residues. Both L-amino acids and D-amino acids may be used. Other modification may comprise derivatives such as esters, sugars, etc. Examples are methyl and acetyl esters.
  • variants of the peptide fragments according to the invention may comprise, within the same variant, or fragments thereof or among different variants, or fragments thereof, at least one substitution, such as a plurality of substitutions introduced independently of one another.
  • Variants of the complex, or fragments thereof may thus comprise conservative substitutions independently of one another, wherein at least one glycine (GIy) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Ala, VaI, Leu, and lie, and independently thereof, variants, or fragments thereof, wherein at least one alanine (Ala) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, VaI, Leu, and lie, and independently thereof, variants, or fragments thereof, wherein at least one valine (VaI) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, Ala, Leu, and
  • the same functional equivalent of a peptide frag- ment, or fragment of said functional equivalent may comprise more than one conservative amino acid substitution from more than one group of conservative amino acids as defined herein above.
  • conservative amino acid substitution is used synonymously herein with the term “homologous amino acid substitution”.
  • the groups of conservative amino acids are as the following: A, G (neutral, weakly hydrophobic), Q, N, S, T (hydrophilic, non-charged) E, D (hydrophilic, acidic) H, K, R (hydrophilic, basic) L, P, I 1 V, M, F, Y, W (hydrophobic, aromatic) C (cross-link forming)
  • Conservative substitutions may be introduced in any position of a preferred predetermined peptide of the invention or fragment thereof. It may however also be desirable to introduce non-conservative substitutions, particularly, but not limited to, a non-conservative substitution in any one or more positions.
  • a non-conservative substitution leading to the formation of a functionally equivalent fragment of the peptide of the invention would for example differ substantially in polarity, for example a residue with a non-polar side chain (Ala, Leu, Pro, Trp, VaI, lie, Leu, Phe or Met) substituted for a residue with a polar side chain such as GIy, Ser, Thr, Cys, Tyr, Asn, or GIn or a charged amino acid such as Asp, GIu, Arg, or Lys, or substituting a charged or a polar residue for a non-polar one; and/or ii) differ substantially in its effect on peptide backbone orientation such as substitution of or for Pro or GIy by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as GIu or Asp for a positively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ substantially in steric bulk, for example
  • Substitution of amino acids may in one embodiment be made based upon their hy- drophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like
  • a fragment of a selected sequence of the invention may be an amino acid sequence, which has about 25 - 99 % of the length of the selected amino acid sequence.
  • Both fragments and variants of selected sequences of the invention are according to the invention functional homologues of said selected sequences.
  • functional homologue of a selected amino acid sequence is in the present context meant a molecule which is capable of one or more biological functions of the selected sequence or a compound comprisinf said selected sequence, in a preferred embodiment biological functions, which are executed through the mecha- nism of binding and activating FGFR.
  • FGFR Fibroblast Growth Factor Receptor 1
  • FGFR2 Fibroblast Growth Factor Receptor 2
  • FGFR3 Fibroblast Growth Factor Receptor 3
  • FGFR4 Fibroblast Growth Factor Receptor 4
  • FGFR5 Fibroblast Growth Factor Receptor 5
  • the above compound according to the invention is capable of binding to any of the latter FGFRs at a binding site located in the extracellular part of the receptor, such as a binding site located in the Ig1, Ig2 and/or Ig3 domains of FGFR, preferably in the Ig2 and/or Ig3 domains of FGFR.
  • the latter binding site is different from the other described binding sites of FGFR (e.g. see: Plotnikov AN, Hubbard SR, Schlessinger J, Mohammadi M..Cell. 2000 May 12;101 (4):413-24).
  • the invention relates to a functional cell-surface receptor.
  • functional cell- surface receptor is meant that a receptor is located in the outer plasma membrane of the cell and has an identifiable group of extracellular ligands. Binding of these ligands to the receptor induces intracellular signal transduction, which results in a physiological response of the cell.
  • the physiological response such as for example the ligand binding induced change in cell metabolism, induction of cell differentiation, termination or induction of cell proliferation, survival or death of the cell, change in motile bihavior of the cell, depends on the nature of receptor ligand, the receptor cellular and extracellular environments and/or particularity of the ligand-receptor interaction, e.g. affinity and/or duration of interaction.
  • Ligand binding to a functional receptor normally results in a change in the activation status of the receptor, such as the receptor becoming capable of initiating a cascade of biochemical reactions in- side the cell resulting in one of the above mentioned physiological responses of the cell collectively termed "receptor signalling" or “signal transduction”. Binding of a ligand may also result in inhibiting the receptor activity which means that the receptor is becoming “silent” or “inactive” and is not any more capable of initiating a cascade of biochemical reactions which it normally does due to the ligand binding.
  • the invention relates to both compounds which are capable of activating FGFR and compounds which are capable of inhibiting FGFR.
  • the invention relates to compounds that are capable of activating FGFR, in another preferred embodiment the invention relates to compounds that are capable of inhibiting FGFR. Preferably, the invention relates to compounds which are capable of activat- ing FGFR 1 and stimulating FGFR 1 signalling.
  • the invention relates to a compound that is capable of dimerising of FGFR.
  • Compounds of the invention may be capable of attenuating binding of other ligands to FGFR despite of occupying an alternative binding site(s).
  • the compounds of the invention may modulate receptor signalling dependent on another ligand binding. It is known that a cellular response to the activation of a receptor depends on the strength of receptor stimulation, which may, for example, be characterised by the value of affinity of interaction of a ligand with the receptor, and/or by the duration of such interaction. Thus, both affinity and duration of interaction of FGF and the receptor may be affected by a compound of the invention. Accordingly, it is another embodiment of the invention to provide a compound, which is capable of modulating the receptor signalling induced by another receptor ligand, for example FGF or FGL- peptide of WO 03/016351.
  • the term "interacting" is used interchangeably with the term “binding” and refers to a direct or indirect contact between a compound of the invention and the FGF receptor, preferably a direct interaction.
  • the term “direct interaction” means that the compound in question binds directly to the receptor.
  • the binding affinity of the compound according to the invention preferably has Kd value in the range of 10 "3 to 10 '10 M, such as preferably in the range of 10 "4 to 10 "8 M.
  • the binding affinity may be determined by any available assayes suitable for this purpose, such as for example surface plasmon resonance (SPR) analysis or nuclear magnetic resonance (NMR) spectroscopy.
  • the binding affinity of the compound to FGFR according to the invention can be modulated, such as enhanced or attenuated, at the presence of a nucleotide compound.
  • the affinity of the interaction may be enhanced.
  • the affinity of the interaction may be attenuated.
  • the affinity of the interaction is enhanced at the presence of a trinucleoti- dephosphate, such as for example ATP, GTP or UTP, and the affinity is attenuated at the presence of a mononucleotidephospate, such as for example AMP, GMP, or at the presence of dinucleotidephosphate, for example ADP or GDP.
  • the compound of the invention may be used combination with a nucleotide for the purpose of optimal activating or inhibiting FGFR.
  • Binding of the compound of the invention to FGFR leads to a series of cellular re- sponces mediated by FGFR.
  • the compound which is capable of binding to FGFR and activating/inhibiting FGFR is also capable of inducing differentiation of FGFR presenting cells, modulating of proliferation of FGFR presenting cells, stimulating survival of FGFR presenting cells, and /or stimulating morphological plasticity of FGFR presenting cells, inducing angiogenesis, anti-oxidative stress and antiinflammatory activity.
  • cells presenting FGFR cells expressing FGFR on the external membrane of the cells, these cells are for example neurons, glial cells, all types of muscle cells, neuroendocrine cells, gonadal cells and kidney cells, endothelial cells fibroblasts, osteoblasts, cancer, stem and embryonic cells.
  • the compound is capable of stimulating neurite outgrowth.
  • the compound is capable of stimulating cell sur- vival. In another preferred embodiment the compound is capable of stimulating synaptic plasticity. Accordingly, the compound is capable of stimulating empering and memory as well.
  • the compound is capable of stimulating differentiation of stem cells.
  • Compounds of the present invention are shown to promote neurite outgrowth and are therefore considered to be good promoters of regeneration of neuronal connec- tions, and thereby of functional recovery after damages as well as promoters of neuronal function in other conditions where such effect is required. Furthermore, compounds of the present invention are capable of stimulating neuronal progenitor cell differention into marture neurons. Compounds of the present invention are also potent stimulators of morphological plasticity of neurons associated with learning and memory.
  • differentiation is related both to the processes of initiation of differentiation of neuronal precursor cells, maturation of immature neurons, such as neurite outgrowth which take place after the last cell division of said neurons, and morphological plasticity of mature neurons, such as takes place in the brain in connection with learning and memory.
  • the compounds of the present invention may be capable of stopping neural precursor and immature neural cell division and initiating maturation said cells, such as initiating extension of neurites.
  • “differentiation” is related to initiation of the process of genetic, biochemical, morphological and physiological transformation of neuronal progenitor cells, immature neural cells or embryonic stem cells leading to formation of cells having functional characteristics of normal neuronal cell as such characteristics are defined in the art.
  • the invention defines "immature neural cell” as a cell that has at least one feature of neural cell accepted in the art as a feature characteristic for the neural cell.
  • a compound comprising at least one of the above peptide sequences is capable of stimulating neurite outgrowth.
  • the invention concerns the neurite outgrowth improvement/stimulation such as about 75% improvement/stimulation above the value of neurite outgrowth of control/non- stimulated cells, for example 50%, such as about 150%, for example 100%, such as about 250, for example 200%, such as about 350 %, for example 300%, such as about 450%, for example 400%, such as about 500%.
  • Estimation of capability of a candidate compound to stimulate neurite outgrowth may be done by using any known method or assay for estimation of neurite outgrowth, such as for example as the described in Examples.
  • a compound has neuritogenic activity both as an insoluble immobile component of cell growth substrate and as a soluble component of cell growth media.
  • immobile means that the compound is bound/attached to a substance which is insoluble in water or a water solution and thereby it becomes insoluble in such solution as well.
  • insoluble and soluble compounds are considered by the application, however soluble compounds are preferred.
  • soluble compound is understood a compound, which is soluble in water or a water solution.
  • an antibody, antigen binding fragment or recombinant protein thereof capable of recognizing and selectively binding to an epitope comprising an FGFR motif of the invention, such as epitope comprising a sequence selected from SEQ ID NOs: 8-14 or a sequence selected from SEQ ID NOs:1-6, or a fragment of said sequence, preferably en epitope in L1 poly- peptide.
  • epitope is meant the specific group of atoms (on an antigen molecule) that is recognized by (that antigen's) antibodies (thereby causing an immune response).
  • epitopope is the equivalent to the term “antigenic determinant”.
  • the epitope may comprise 3 or more amino acid residues, such as for example 4, 5, 6, 7, 8 amino acid residues, located in close proximity, such as within a contiguous amino acid sequence, or located in distant parts of the amino acid sequence of an antigen, but due to protein folding have been approached to each other.
  • Antibody molecules belong to a family of plasma proteins called immunoglobulins, whose basic building block, the immunoglobulin fold or domain, is used in various forms in many molecules of the immune system and other biological recognition systems.
  • a typical immunoglobulin has four polypeptide chains, containing an antigen binding region known as a variable region and a non-varying region known as the constant region.
  • Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disul- fide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end.
  • VH variable domain
  • VL variable domain at one end
  • the con- stant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Novotny J, & Haber E. Proc Natl Acad Sci U S A. 82(14):4592-6, 1985).
  • immunoglobulins can be assigned to different classes. There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. lgG-1, lgG-2, lgG-3 and lgG-4; lgA-1 and lgA-2.
  • the heavy chains constant domains that correspond to the different classes of immunoglobulins are called alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) and mu ( ⁇ ), respectively.
  • the light chains of antibodies can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino sequences of their constant domain.
  • K kappa
  • ⁇ lambda
  • the subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known.
  • variable in the context of variable domain of antibodies, refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies.
  • the variable domains are for binding and determine the specificity of each particular antibody for its particular antigen.
  • variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) also known as hypervariable regions both in the light chain and the heavy chain variable domains.
  • CDRs complementarity determining regions
  • variable domains The more highly conserved portions of variable domains are called the framework (FR).
  • the variable domains of native heavy and light chains each comprise four FR regions, largely a adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen- binding site of antibodies.
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • an antibody that is contemplated for use in the present invention thus can be in any of a variety of forms, including a whole immunoglobulin, an antibody fragment such as Fv, Fab, and similar fragments, a single chain antibody which includes the variable domain complementarity determining regions (CDR), and the like forms, all of which fall under the broad term "antibody”, as used herein.
  • the present invention contemplates the use of any specificity of an antibody, polyclonal or monoclonal, and is not limited to antibodies that recognize and immunoreact with a specific antigen.
  • an antibody or fragment thereof is used that is immuno- specific for an antigen or epitope of the invention.
  • antibody fragment refers to a portion of a full-length antibody, generally the antigen binding or variable region.
  • antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments.
  • Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen binding fragments that are capable of cross-linking antigen, and a residual other fragment (which is termed pFc').
  • Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from anti- body fragments.
  • “functional fragment” with respect to antibodies refers to Fv, F(ab) and F(ab') 2 fragments.
  • antibody fragment is used herein interchangeably with the term “antigen binding fragment”.
  • Antibody fragments may be as small as about 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 9 amino acids, about 12 amino acids, about 15 amino acids, about 17 amino acids, about 18 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids or more.
  • an antibody fragment of the invention can have any upper size limit so long as it is has similar or immunological properties relative to antibody that binds with specificity to an epitope comprising a peptide sequence selected from any of the sequences identified herein as SEQ ID NOs: 1-14, or a fragment of said sequences.
  • the term "antibodv fragment' is identical to term "antigen binding fragment".
  • Antibody fragments retain some ability to selectively bind with its antigen or receptor. Some types of antibody fragments are defined as follows:
  • Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule.
  • a Fab fragment can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain.
  • Fab' is the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per anti- body molecule. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH 1 domain including one or more cysteines from the antibody hinge region.
  • (Fab') 2 is the fragment of an antibody that can be obtained by treat- ing whole antibody with the enzyme pepsin without subsequent reduction.
  • F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds.
  • Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V H -V L dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the V H -V L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single chain antibody defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
  • Such single chain antibodies are also referred to as "single-chain Fv” or “sFv” antibody fragments.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.
  • diabodies refers to a small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161 , and Hollinger et at., Proc. Natl. Acad Sci. USA 90: 6444-6448 (1993).
  • the invention contemplate both polyclonal and monoclonal antibody, antigen binding fragments and recombinant proteins thereof which are capable of binding an epiyope according to the invention.
  • polyclonal antibodies The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green et al. 1992. Production of Polyclonal Antisera, in: Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press); Coligan, et al., Production of Polyclonal Antisera in Rabbits, Rats Mice and Hamsters, in: Current Protocols in Immunology, section 2.4.1, which are hereby incorporated by reference.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256, 495-7, or may be made by recombinant methods, e.g., as described in US 4,816,567.
  • the monoclonal antibodies for use with the present invention may also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991, Nature 352: 624- 628, as well as in Marks et al., 1991, J MoI Biol 222: 581-597.
  • Another method in- volves humanizing a monoclonal antibody by recombinant means to generate antibodies containing human specific and recognizable sequences See, for review, Holmes, et al., 1997, J Immunol 158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma & Immunol 81 :105-115.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly spe- cific, being directed against a single antigenic site.
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal" indicates the character of the antibody indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US 4,816,567); Morrison et al., 1984, Proc Natl Acad Sci 81 : 6851-6855.
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody
  • Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monova- lent fragments.
  • a thiol reducing agent and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monova- lent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent or the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde.
  • the Fv fragments comprise V H and V L chains connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene compris- ing DNA sequences encoding the V H and V L domains connected by an oligonucleotide.
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • CDR peptides (“minimal recognition units") are often involved in antigen recognition and binding.
  • CDR peptides can be obtained by cloning or constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick, et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 106 (1991).
  • the invention contemplates human and humanized forms of non-human (e.g. murine) antibodies.
  • humanized antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other anti- gen-binding subsequences of antibodies) that contain a minimal sequence derived from non-human immunoglobulin, such as the eitope recognising sequence.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a nonhuman species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody nonhuman species
  • humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • antibodies may be achieved by any standard method in the art for producing polyclonal and monoclonal antibodies using natural or recombinant fragments of L1 , said fragment comprising a sequence selected from SEQ ID NO: 1-14, as an antigen.
  • Such antibodies may be also generated using variants, homologues or fragments of peptide sequences of SEQ ID NOs:1-14, said variants, homologues and fragments are immunogenic peptide sequences which meet the following criteria:
  • the antibodies may also be produced in vivo by the individual to be treated, for example, by administering an immunogenic fragment according to the invention to said individual. Accordingly, the present invention further relates to a vaccine comprising an immunogenic fragment described above.
  • the application also relates to a method for producing an antibody of the invention said method comprising a step of providing of an immunogenic fragment described above.
  • the invention relates both to antibodies, which are capable of modulating, such as enhancing or attenuating, biological function of L1 , in particular a function related to neural cell differentiation, survival and/or plasticity, and to an antibody, which can recognise and specifically bind the latter proteins without modulating biological activity thereof.
  • the invention relates to use of the above antibodies for 1 ) therapeutic applications when the modulation of activity of L1 or FGFR is needed, 2) detecting and/or monitoring l_1 or a protein comprising the above described epotope in vitro and/or in vivo for diagnostic purposes, 3) research purposes.
  • the peptide sequences of the present invention may be prepared by any conventional synthetic methods, recombinant DNA technologies, enzymatic cleavage of full-length proteins which the peptide sequences are derived from, or a combination of said methods.
  • the peptides of the invention are produced by use of recombinant DNA technologies.
  • DNA sequence encoding a peptide or the corresponding full-length protein the peptide originates from may be prepared synthetically by established standard methods, e.g. the phosphoamidine method described by Beaucage and Caruthers,
  • oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in suitable vectors.
  • the DNA sequence encoding a peptide may also be prepared by fragmentation of the DNA sequences encoding the corresponding full-length protein of peptide origin, using DNAase I according to a standard protocol (Sambrook et al., Molecular cloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor, NY, 1989).
  • the present invention relates to full-length proteins selected from the groups of proteins identified above.
  • the DNA encoding the full-length proteins of the invention may alternatively be fragmented using specific restriction endonucleases.
  • the fragments of DNA are further purified using standard procedures described in Sambrook et al., Molecular cloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor, NY, 1989.
  • the DNA sequence encoding a full-length protein may also be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the full-length protein by hybridisation using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).
  • the DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., 1988, Science 239:487-491.
  • a recombinant expression vector which may be any vector, which may conveniently be subjected to recombinant DNA procedures.
  • the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector that exists as an extrachromosoma! entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the DNA sequence encoding a peptide or a full-length protein should be operably connected to a suitable promoter sequence.
  • the promoter may be any promoter sequence.
  • DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the coding DNA sequence in mammalian cells are the SV 40 promoter (Subramani et al., 1981 , MoI. Cell Biol. 1 :854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., 1983, Science 222: 809-814) or the adenovirus 2 major late promoter.
  • a suitable promoter for use in insect cells is the polyhedrin promoter (Vasu- vedan et al., 1992, FEBS Lett. 311:7-11).
  • Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., 1980, J. Biol. Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. MoI. Appl. Gen. 1: 419-434) or alcohol dehydrogenase genes (Young et al., 1982, in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al, eds., Plenum Press, New York), or the TPM (US 4,599,311) or ADH2-4c (Russell et al., 1983, Nature 304:652-654) promoters.
  • Suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., 1985, EMBO J. 4:2093-2099) or the tpiA promoter.
  • the coding DNA sequence may also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) the TPH (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) promoters.
  • the vector may further comprise elements such as polyadenylation signals (e.g. from SV 40 or the adenovirus 5 EIb region), transcriptional enhancer sequences (e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).
  • the recombinant expression vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question.
  • An example of such a sequence is the SV 40 origin of replication.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomycin, hydromycin or methotrexate.
  • DHFR dihydrofolate reductase
  • the coding DNA sequences may be usefully fused with a second peptide coding sequence and a protease cleavage site coding sequence, giving a DNA construct encoding the fusion protein, wherein the protease cleavage site coding sequence positioned between the HBP fragment and second peptide coding DNA, inserted into a recombinant expression vector, and expressed in recombinant host cells.
  • said second peptide selected from, but not limited by the group comprising glutathion-S-reductase, calf thymosin, bacterial thioredoxin or human ubiquitin natural or synthetic variants, or peptides thereof.
  • a peptide sequence comprising a protease cleavage site may be the Factor Xa, with the amino acid sequence IEGR, en- terokinase, with the amino acid sequence DDDDK, thrombin, with the amino acid sequence LVPR/GS, or Acharomhacter lyticus, with the amino acid sequence XKX, cleavage site.
  • the host cell into which the expression vector is introduced may be any cell which is capable of expression of the peptides or full-length proteins, and is preferably a eu- karyotic cell, such as invertebrate (insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells.
  • a eu- karyotic cell such as invertebrate (insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells.
  • Exam- pies of suitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCC CRL-1650), BHK (ATCC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL- 61) cell lines.
  • fungal cells may be used as host cells.
  • suitable yeast cells include cells of Saccharomyces spp. or Schizosaccharo- myces spp., in particular strains of Saccharomyces cerevisiae.
  • Other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora spp., in particular strains of Aspergillus oryzae or Aspergillus niger.
  • Aspergillus spp. for the expression of proteins is described in, e.g., EP 238 023.
  • the medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements, or a suitable medium for growing insect, yeast or fungal cells. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the peptides or full-length proteins recombinantly produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. HPLC, ion exchange chromatography, affinity chromatography, or the like.
  • a salt e.g. ammonium sulphate
  • Peptides may for example be synthesised by using Fmoc chemistry and with Acm- protected cysteins. After purification by reversed phase HPLC, peptides may be further processed to obtain for example cyclic or C- or N-terminal modified isoforms.
  • the methods for cyclization and terminal modification are well-known in the art and described in detail in the above-cited manuals.
  • the peptide sequences of the invention are produced synthetically, in particular, by the Sequence Assisted Peptide Synthesis (SAPS) method.
  • SAPS Sequence Assisted Peptide Synthesis
  • Sequence Assisted Peptide Synthesis may be synthesised either batchwise in a polyethylene vessel equipped with a polypropylene filter for filtration or in the continuous-flow version of the poly- amide solid-phase method (Dryland, A. and Sheppard, R.C., (1986) J.Chem. Soc. Perkin Trans. I, 125 - 137.) on a fully automated peptide synthesiser using 9- fluorenylmethyloxycarbonyl (Fmoc) or tert. -Butyloxycarbonyl, (Boc) as N-a-amino protecting group and suitable common protection groups for side-chain functionality's.
  • SAPS Sequence Assisted Peptide Synthesis
  • FGFR and L1 are known to be involved in a number of body processes in normal conditions and in disease, in particular in the neural system. These proceses include differentiation, proliferation, survival, plasticity and motility of cells.
  • FGFRs and L1 have been shown to be important determinants of neuronal survival both during development and during adulthood (Haspel et al. (2000) J Neurobiol 15:287-302; Roonprapurt et al. (2003) J Neurotrauma 20:871-882; Wiencken-Barger et al. Cereb Cortex (2004) 14:121-131 ; Loers er al.
  • the invention features compounds that promote survival of neural cells and can be used as medicaments for the treatment of conditions involving neural cell death.
  • a compound of the invention may also be used as a medicament for promotion of survival of another type of cells, e.g. different type of muscle cells, or, alternatively, for promotion of cell death of still another. type of cells, e.g.
  • the present invention provides compounds capable of modulation of the activity of FGFRs. Consequently, said compounds are concerned by the invention as medica- ment for the treatment of diseases, wherein modulation of the activity of FGFRs may be considered as an essential condition for the curing.
  • the medicament of the invention is in one embodiment for prevention and/or treatment of 1 ) diseases and conditions of the central and peripheral nervous system, or of the muscles or of various organs, and/or
  • diseases or conditions of the central and peripheral nervous system such as postoperative nerve damage, traumatic nerve damage, impaired myelination of nerve fibers, postischaemic damage, e.g. resulting from a stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, dementias such as multiin- farct dementia, sclerosis, nerve degeneration associated with diabetes mellitus, disorders affecting the circadian clock or neuro-muscular transmission, and schizophrenia, mood disorders, such as manic depression;
  • diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, such as after organ transplan- tation, or such as genetic or traumatic atrophic muscle disorders; or for treatment of diseases or conditions of various organs, such as degenerative conditions of the gonads, of the pancreas such as diabetes mellitus type I and II, of the kidney such as nephrosis and of the heart, liver and bowel, and/or 4) cancer disease, and/or 5) prion diseases.
  • diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, such as after organ transplan- tation, or such as genetic or traumatic atrophic muscle disorders; or for treatment of diseases or conditions of various organs, such as degenerative conditions of the gonads, of the pancreas such as diabetes mellitus type I and II, of the kidney such as nephrosis and of the heart, liver and bowel, and/or 4) cancer disease, and/or 5) prion diseases.
  • the invention concerns cancer being any type of solid tumors requiring neoangio- genesis.
  • the invention concerns prion diseases selected from the group consisting of scrapie, Creutzfeldt- Jakob disease. It has been shown that FGFRs plays a distinct role in prion diseases (Castelnau et al. (1994) Exp Neurobiol. 130:407-10; Ye and Carp (2002) J MoI Neurosci. 18:179-88).
  • L1 has been broadly studied as a neurite outgrowth stimulator. It has also been regarded important for neural cell precursor proliferation, differentiation and transmitter phenotype subtype generation (Dihne et al. (2003) J Neurosci 23:6638-6650), and it is known to play a role in synaptic plasticity (Saghatelyan et al. (2004) MoI Cell Neurosci 26:191-203). FGFRs and their ligands play important roles in CNS, in particular they are involved in the processes associated with memory and learning (Reuss and von Bohlen und Halbach (2003) Cell Tissue Res. 313:139-57). Thus, in still another embodiment a compound of the invention may be used for stimulation of the ability to learn and/or of the short and/or long-term memory. This embodiment is one of the preferred embodiments of the invention.
  • the invention concerns normal, degenerated or damaged L1 presenting cells.
  • Diseases such as X linked hydrocephalus and MASA syndrome corpus callosum hypoplasia, mental retardation, adducted thumbs, spasticity and hydrocephalus (CRASH) syndrome are of a particular interest of the present invention.
  • the medicament of the invention comprises an effective amount of one or more compounds as defined above, or a pharmaceutical composition comprising one or more compounds and pharmaceutically acceptable additives.
  • the invention in another aspect also concerns a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound of the invention.
  • a further aspect of the invention is a process of producing a pharmaceutical composition, comprising mixing an effective amount of one or more of the compounds of the invention, or a pharmaceutical composition according to the invention with one or more pharmaceutically acceptable additives or carriers.
  • the compounds are used in combination with a prosthetic device, wherein the device is a prosthetic nerve guide.
  • the present invention relates to a prosthetic nerve guide, characterised in that it comprises one or more of the compounds or the pharmaceutical composition as defined above. Nerve guides are known in the art.
  • the invention relates to use of a medicament and/or pharmaceutical composition comprising the compound of invention for the treatment or prophylaxis of any of the diseases and conditions mentioned below.
  • Such medicament and/or pharmaceutical composition may suitably be formulated for oral, percutaneous, intramuscular, intravenous, intracranial, intrathecal, in- tracerebroventricular, intranasal or pulmonal administration.
  • Injectables are usually prepared either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection.
  • the preparation may also be emulsified.
  • the active ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof.
  • excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof.
  • the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH- buffering agents, or which enhance the effectiveness or transportation of the preparation.
  • Formulations of the compounds of the invention can be prepared by techniques known to the person skilled in the art.
  • the formulations may contain pharmaceutically acceptable carriers and excipients including microspheres, liposomes, micro- capsules, nanoparticles or the like.
  • the preparation may suitably be administered by injection, optionally at the site, where the active ingredient is to exert its effect.
  • Additional formulations which are suitable for other modes of administration include suppositories, nasal, pulmonal and, in some cases, oral formulations.
  • traditional binders and carriers include polyalkylene glycols or triglycerides.
  • Such suppositories may be formed from mixtures containing the active ingredient(s) in the range of from 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and generally contain 10-95% of the active ingredient(s), preferably 25-70%.
  • compositions are such suitable for nasal and pulmonal administration, e.g. inhalators and aerosols.
  • the active compound may be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide compound) and which are formed with inorganic acids such as, for ex- ample, hydrochloric or phosphoric acids, or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like.
  • Salts formed with the free carboxyl group may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as iso- propylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides
  • organic bases as iso- propylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the preparations are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective.
  • the quantity to be administered depends on the subject to be treated, including, e.g. the weight and age of the subject, the disease to be treated and the stage of disease. Suitable dosage ranges are per kilo body weight normally of the order of several hundred ⁇ g active ingredient per administration with a preferred range of from about 0.1 ⁇ g to 5000 ⁇ g per kilo body weight.
  • the suitable dosages are often in the range of from 0.1 ⁇ g to 5000 ⁇ g per kilo body weight, such as in the range of from about 0.1 ⁇ g to 3000 ⁇ g per kilo body weight, and especially in the range of from about 0.1 ⁇ g to 1000 ⁇ g per kilo body weight.
  • the suitable dosages are often in the range of from 0.1 ⁇ g to 1000 ⁇ g per kilo body weight, such as in the range of from about 0.1 ⁇ g to 750 ⁇ g per kilo body weight, and especially in the range of from about 0.1 ⁇ g to 500 ⁇ g per kilo body weight such as in the range of from about 0.1 ⁇ g to 250 ⁇ g per kilo body weight.
  • Administration may be performed once or may be followed by subsequent administrations.
  • the dosage will also depend on the route of administration and will vary with the age and weight of the subject to be treated.
  • a preferred dosage of multimeric forms would be in the interval 1 mg to 70 mg per 70 kg body weight.
  • a localised or substantially localised application is preferred.
  • intranasal application is preferred.
  • Some of the compounds of the present invention are sufficiently active, but for some of the others, the effect will be enhanced if the preparation further comprises pharmaceutically acceptable additives and/or carriers.
  • Such additives and carriers will be known in the art.
  • Administration may be a continuous infusion, such as intraventricular infusion or administration in more doses such as more times a day, daily, more times a week, weekly, etc. It is preferred that administration of the medicament is initiated before or shortly after the individual has been subjected to the factor(s) that may lead to cell death. Preferably the medicament is administered within 8 hours from the factor onset, such as within 5 hours from the factor onset. Many of the compounds exhibit a long term effect whereby administration of the compounds may be conducted with long intervals, such as 1 week or 2 weeks.
  • the administration may be continuous or in small portions based upon controlled release of the active compound(s).
  • precursors may be used to control the rate of release and/or site of release.
  • Other kinds of implants and well as oral administration may similarly be based upon controlled release and/or the use of precursors.
  • Treatment by the use of the compounds/compositions according to the invention is in one embodiment useful for inducing differentiation, modulating proliferation, stimulate regeneration, neuronal plasticity and survival of cells, for example cells being implanted or transplanted. This is particularly useful when using compounds having a long term effect.
  • the treatment may be for stimulation of survival of cells which are at risk of dying due to a variety of factors, such as traumas and injuries, acute diseases, chronic diseases and/or disorders, in particular degenerative diseases normally leading to cell death, other external factors, such as medical and/or surgi- cal treatments and/or diagnostic methods that may cause formation of free radicals or otherwise have cytotoxic effects, such as X-rays and chemotherapy.
  • factors such as traumas and injuries, acute diseases, chronic diseases and/or disorders, in particular degenerative diseases normally leading to cell death
  • other external factors such as medical and/or surgi- cal treatments and/or diagnostic methods that may cause formation of free radicals or otherwise have cytotoxic effects, such as X-rays and chemotherapy.
  • FGFR binding compounds according to the invention are useful in cancer treatment.
  • the treatment comprises treatment and/or prophylaxis of cell death in relation to diseases or conditions of the central and peripheral nervous system, such as postoperative nerve damage, traumatic nerve damage, e.g. resulting from spinal cord injury, impaired myelination of nerve fibers, postischaemic damage, e.g. resulting from a stroke, multiinfarct dementia, multiple sclerosis, nerve degeneration as- sociated with diabetes mellitus, neuro-muscular degeneration, schizophrenia, Alzheimer's disease, Parkinson's disease, or Huntington's disease.
  • diseases or conditions of the central and peripheral nervous system such as postoperative nerve damage, traumatic nerve damage, e.g. resulting from spinal cord injury, impaired myelination of nerve fibers, postischaemic damage, e.g. resulting from a stroke, multiinfarct dementia, multiple sclerosis, nerve degeneration as- sociated with diabetes mellitus, neuro-muscular degeneration, schizophrenia, Alzheimer's disease, Parkinson's disease, or Huntington's disease
  • the compounds according to the invention may be used for inducing differentiation, modulating proliferation, stimulate regeneration, neuronal plasticity and survival , i.e. stimulating survival.
  • the compound and/or pharmaceutical composition may be for preventing cell death of heart muscle cells, such as after acute myocardial infarction, in order to induce angiogenesis.
  • the compound and/or pharmaceutical composition is for the stimulation of the survival of heart muscle cells, such as survival after acute myocardial infarction.
  • the compound and/or pharmaceutical composition is for revascularisation, such as after injuries.
  • the present compounds are capable of stimulating angiogenesis and thereby they can promote the wound healing process.
  • the invention further discloses a use of the compound and/or pharmaceutical composition in the treatment of cancer. Regulation of activation of FGFR is important for tumor agiogenesis, proliferation and spreading.
  • a use of the compound and/or pharmaceutical composition is for the stimulation of the ability to learn and/or of the short and/or long term memory, as FGFR activity is important for differentiation of neural cells.
  • a compound and/or pharmaceutical composition of the invention is for the treatment of body damages due to alcohol consumption. Developmental malformations of foetuses, long-term neurobehavioral alterations, alcoholic liver disease are particularly concerned.
  • Therapeutic treatment of prion diseases including using a compound and/or phar- maceutical composition is still another embodiment of the invention.
  • Neoplasms such as malignant neoplasms, benign neoplasms, carcinoma in situ and neoplasms of uncertain behavior
  • cancers with dysfunction and/or over- or under-expression of specific receptors and/or expression of mutated receptors or associated with soluble receptors such as but not limited to Erb-receptors and FGF- receptors
  • diseases of endocrine glands such as diabetes mellitus I and II, pituitary gland tumor
  • psychoses such as senile and presenile organic psychotic conditions
  • alcoholic psychoses drug psychoses, transient organic psychotic conditions
  • Alzheimer's disease cerebral lipidoses
  • epilepsy general paresis [syphilis], hepatolenticular degeneration, Huntington's chorea, Ja
  • inflammatory disease of the central nervous system such as meningitis, encephalitis, Cerebral degenerations such as Alzheimer's disease, Pick's disease, senile degeneration of brain, senility NOS, communicating hydrocephalus, obstructive hydrocephalus, Parkinson's disease including other extra pyramidal disease and abnormal movement disorders, spinocerebellar disease, cerebellar ataxia, Marie's Sanger-Brown, Dyssynergia cerebellaris myoclonica, primary cerebellar degeneration, such as spinal muscular atrophy, familial, juvenile, adult spinal muscular atrophy, motor neuron disease, amyotrophic lateral sclerosis, motor neuron disease, progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, other anterior horn cell diseases, anterior horn cell disease, unspecified, other diseases of spinal cord, syringomyelia and syringobulbia, vascular myelopathies, acute infar
  • Inflammatory and toxic neuropathy including acute infective polyneuritis, Guillain-Barre syn- drome, Postinfectious polyneuritis, polyneuropathy in collagen vascular disease, disorders of the globe including disorders affecting multiple structures of eye, such as purulent endophthalmitis, diseases of the ear and mastoid process, chronic rheumatic heart disease, ischaemic heart disease, arrhythmia, diseases in the pulmonary system, respiratory system, sensoring e.g. oxygene, astma, abnormality of organs and soft tissues in newborn, including in the nerve system, complications of the administration of anesthetic or other sedation in labor and delivery, diseases in the skin including infection, insufficient circulation problem, burn injury and other mechanic and/or physical injuries.
  • Injuries including after surgery, crushing injury, burns. Injuries to nerves and spinal cord, including division of nerve, lesion in conti- nuity (with or without open wound), traumatic neuroma (with or without open wound), traumatic transient paralysis (with or without open wound), accidental puncture or laceration during medical procedure, injury to optic nerve and pathways, optic nerve injury, second cranial nerve, injury to optic chiasm, injury to optic pathways, injury to visual cortex, unspecified blindness, injury to other cranial nerve(s), injury to other and unspecified nerves.
  • Scrapie Creutzfeldt-Jakob disease, Gerstmann-Straussler-Sheinker (GSS) disease; X linked hydrocephalus and MASA syndrome corpus callosum hypoplasia, mental retardation, adducted thumbs, spasticity and hydrocephalus (CRASH) syndrome, pain syndrome, encephalitis, drug/alcohol abuse, anxiety, postoperative nerve damage, peri-operative ischemia, inflammatory disorders with tissue damage, either by affecting the infections agent or protecting the tissue, HIV, hepatitis, and following symptoms, autoimmune disorders, such as rheumatoid arthritis, SLE, ALS, and MS.
  • GSS Gerstmann-Straussler-Sheinker
  • CRASH spasticity and hydrocephalus
  • obscenity lipid disorders e.g. hyper cholestorolamia, artheslerosis, disorders of amino-acid transport and metabolism, disorders of purine and pyrimidine metabolism and gout
  • bone disorders such as fracture, osteoporosis, osteo arthritis (OA), Atrophic dermatitis, psoriasis, infection cased disorders, stem cell protection or maturation in vivo or in vitro.
  • the treatment and/or prevention of the above conditions and symptoms comprises a step of administering an effective amount of a compound and/or pharmaceutical composition to an individual in need.
  • the L1 F3 modules I-V were produced by RT-PCR using rat brain total RNA.
  • the FGFR1 Ig modules H-III were produced using mouse FGFR1 (IIIC isoform) cDNA (kindly provided by Dr. Patrick Doherty, King's College, London).
  • the L1 F3 modules consist of RSPWPG, amino acids 608-1109 of L1 (swissprot Q05695) and HHHHHH.
  • the FGFR1 Ig module Il consist of RSHHHHHH and amino acids 253- 365 of FGFR1 (swissprot p16092).
  • the FGFR1 combined Ig modules ll-lll consist of RSHHHHHH and amino acids 141-365 of FGFR1 (swissprot p16092).
  • the FGFR1 Ig modules ll-lll and L1 F3 modules I-V were expressed in Drosophila S2 cells (Invi- trogen, USA) according to the manufacturer's instructions. All the proteins were purified by affinity chromatography using Ni 2+ -NTA resin (Qiagen, USA), ion exchange chromatography, and gel filtration (all chromatography columns used for purification were bought from Amersham Biosciences, Sweden).
  • the IgII module of FGFR1 was expressed in Pichia Pastoris KM71 cells expression system (Invitrogen, USA) according to the manufacturer's instructions.
  • the protein was purified by affinity chromatography using Ni 2+ -NTA resin (Qiagen, USA), ion exchange chromatography and gel filtration (all chromatography columns used for purification were bought from Amersham Biosciences, Sweden).
  • the combined NCAM Ig modules I and Il RV and amino acids 20-208 of rat NCAM, swissprot p13596) were produced as described (Jensen et. al., 1999).
  • Binding analysis was performed using a BIAcoreX instrument (Biosensor AB, Sweden) at 25 0 C using 10 mM sodium phosphate pH 7.4, 150 mM NaCI as running buffer. The flow-rate was 5 l/min. Data were analysed by non-linear curve-fitting using the manufacturer's software.
  • the FGFR1 Ig modules 2-3 were immobilized on the sensor chip CM5 using the amine coupling kit (Biosensor AB) as follows: 1) the two halves of the chip (designated Fd and Fc2) were activated by 20 I activation solution; 2) the protein was immobilized on Fd using 12 I 20 g/ml protein in 10 mM sodium phosphate buffer pH 6.0; 3) Fd and Fc2 were blocked by 35 I blocking solu- tion. Binding of various compounds to the immobilized FGFR1 modules was studied as follows: A compound was injected simultaneously into Fd (with the immobilized FGFR1 modules) and Fc2 (with nothing immobilized).
  • the curve representing un- specific binding of the compound to the surface of Fc2 was subtracted from the curve representing binding of the protein to the immobilized FGFR1 modules and the surface of Fd .
  • the resulting curve was used for analysis.
  • ATP competition experiments various compounds were preincubated for 10 min with ATP at a specified concentration.
  • the affinity between ATP and the L1 F3 modules was estimated from the following procedure: the initial binding rates of 14 ⁇ M F3 modules, V 0 , and of 14 ⁇ M F3 modules preincubated with ATP at a specified concentration, V A TP, were determined.
  • V A TP can be calculated from the equation:
  • V 0 jfcj where k 2 and fc, are proportionality constants between the initial binding rate and concentration at the initial moment for the ATP-F3 complex and F3 modules, respectively;
  • F is the con- centration of the F3 modules;
  • a is the concentration of ATP;
  • K D is the equilibrium dissociation constant.
  • VAT P was plotted against the ATP concentration, and K D was calculated by non-linear fitting of the theoretical curve to the experimental data.
  • Dissociated neurons from rat cerebellum (postnatal day 7-8) were grown on 8-well
  • ATP (Kiselyov et al., 2003). It was therefore of interest to see whether ATP had the same effect for the L1-FGFR1 binding. To test this we used SPR analysis. As can be seen from Fig. 2A and 3A, adding ATP to 14 ⁇ M L1 F3 modules I-V enhanced the binding of the F3 modules to FGFR1 Ig modules IMII. In order to test whether this effect was specific for ATP, AMP-PCP (a non-hydrolysable analogue of ATP) (Fig. 2B, 3A), AMP (Fig. 2C, 3A), and GTP (Fig. 2D, 3A), were tested in the same setup.
  • AMP-PCP a non-hydrolysable analogue of ATP
  • GTP and AMP-PCP also bound to the L1 F3 modules increasing the affinity of the interaction between the modules and FGFR1 , whereas AMP demonstrated insignificant inhibition of the binding (Fig. 2C, 3A).
  • ATP, GTP and AMP-PCP binding to the L1 F3 modules may change the conformation of the protein thereby increasing the affinity between L1 and FGFR1.
  • the Kd of the interaction between 14 ⁇ M L1 F3 modules and FGFR1 in the presence of 2mM ATP (GTP or AMP-PCP) decreased from 3.246 ⁇ 0.452 ⁇ M to 1.246+0.129 ⁇ M (0.944+0.041 ⁇ M or 0.861+0.032 ⁇ M, respectively) (Fig. 3A).
  • L1 F3 modules bind to FGFR1, they may be expected to induce FGFR1 activation in living cells.
  • TREX-293 cells stably transfected with FGFR1 containing a C-terminal Strepll-tag, were stimulated with the F3 modules of L1 at different concentrations. After stimulation, FGFR1 was im- munopurified using a fixed amount of anti-phosphotyrosine antibodies and then ana- lyzed by immunoblotting using antibodies against the Strepll-tag. As appears from Fig.
  • L1 F3 modules substantially increased FGFR1 phosphorylation compared to the non-stimulated cells and cells treated with the combined NCAM Ig Ml modules which did not bind to FGFR1 by SPR analysis (see Fig. 1).
  • the highest phosphorylation level was achieved at a concentration of the L1 F3 modules of 10 ⁇ M. This indicates that binding of the L1 F3 modules to FGFR1 results in the activation of the receptor.
  • FGFR1 activation by the L1 F3 modules stimulates neurite outgrowth and this stimulatory effect can be modulated by ATP Since the L1 F3 modules activate FGFR1 , we suggested that they were capable of mimicking a characteristic function of L1 : neuronal differentiation as reflected by neurite outgrowth. To test this assumption, dissociated cerebellar neurons were seeded on plastic and allowed to grow for 24 h in the presence of the below de- scribed compounds. As can be seen from Fig. 5A, at a concentration of 4 ⁇ M the L1 F3 modules substantially increased the length of neurites compared to non- stimulated neurons.
  • the effect was quantified in a dose-response study demonstrating that the F3 modules induced neurite outgrowth with a bell-shaped curve typical of growth factor induced neurite outgrowth (Hatten et al., 1988).
  • the stimulatory effect of the F3 modules could be completely abrogated by an inhibitor of FGFR1 , SU5402 (Fig. 5B), further supporting the notion that the modules interact with FGFR1.
  • ATP enhanced the L1-FGFR1 binding
  • ATP might interfere with the FGFR1 activation by the L1 F3 modules, and, consequently, affect the neurite promoting activity of the modules.
  • neurons were stimulated with the L1 F3 modules in the presence of ATP or AMP-PCP.
  • both ATP and AMP-PCP substantially reduced the neuritogenic effect induced by the F3 modules when using a concentration of the L1 F3 modules (4 ⁇ M) giving a maximal response in the absence of ATP (Fig. 5A).
  • the concentration of the F3 modules should be lower than the most effective concentration to demonstrate the enhancement of neurite outgrowth by adding ATP.
  • the secondary structure of a finbronectin type 3 module of L1 consists of two beta-sheets containing seven antiparallel beta-strands A, C, E, G and B, D, F, respectively, which fold up to form a beta-sandwich.
  • FGFR1 has an intrinsic ability to dimerise, although with a very low affinity. This leads to a dynamic equilibrium in which a small fraction of FGFR1 molecules forms transient dimers, resulting in a low background level of FGFR1 phosphorylation. FGFR1 binding of any ligand may change the dimerisation slightly, shift- ing the equilibrium to either association or dissociation. Thus, we presume that binding of the F3 modules to FGFR1 may shift the equilibrium towards association and, as a result, increase the FGFR1 phosphorylation.
  • the F3 modules were capable of mimicking a characteristic function of L1: promotion of neurite outgrowth. Stimulation of the cells by the F3 modules could be blocked by an inhibitor of L1 -stimulated neurite outgrowth, the FGFR1 inhibitor SU5402, further supporting the notion that the modules interact with FGFR1.
  • ATP enhanced the binding of the F3 modules to FGFR1 Ig modules IMII.
  • ATP modulated the stimula- tory effect of the F3 modules on outgrowth of neurites. This effect depended on the concentration of the L1 F3 modules that was used to stimulate neurite outgrowth. If the concentration of the protein corresponded to the one with the maximal effect on neurite outgrowth induction (Fig. 5) ATP acted inhibitory. On the other hand, if the concentration of the protein was lower, ATP enhanced the induction of neurite out- growth.
  • L1 is a very long molecule having 11 modules in the extracellular part.
  • synthesized peptides mimicking loop regions of L1 we demonstrated that a possible model for binding between L1 and FGFR1 requires compact conformation of F3 modules.
  • ATP is an abundant neurotransmitter in the CNS.
  • the release of ATP from the synapse may regulate the coupling between L1 and FGFR1 and, therefore, affect, e. g. inhibit axonal growth in the area of a newly formed synaptic contact, where it is no longer necessary.
  • ATP release may trigger L1-FGFR1 interactions and subsequent signaling in areas with a relatively low density of L1 molecules.
  • our results provide evidence for a direct interaction between L1 and FGFR1 and indicate that ATP is a regulator of L1 induced axonal outgrowth through regulation of the L1-FGFR1 interaction.
  • TAG-1 can mediate homophilic binding, but neurite outgrowth on TAG-1 requires an L1-like molecule and beta 1 integrins. Neuron. 12(3), 675-90.
  • the crystal structure of the ligand binding module of axonin-1/TAG-1 suggests a zipper mechanism for neural cell adhesion. Cell. 12; 101 (4), 425-33.
  • the fourth immunoglobulin-like domain of NCAM contains a carbohydrate recognition domain for oligomannosidic glycans implicated in association with L1 and neurite outgrowth. J Cell Biol. 121(6), 1409-21.
  • L1 -mediated axon outgrowth occurs via a homophilic binding mechanism. 2(6), 1597-603.

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