WO2009100724A1 - Peptide inédit dérivé de la ncam (bcl) - Google Patents

Peptide inédit dérivé de la ncam (bcl) Download PDF

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Publication number
WO2009100724A1
WO2009100724A1 PCT/DK2009/050041 DK2009050041W WO2009100724A1 WO 2009100724 A1 WO2009100724 A1 WO 2009100724A1 DK 2009050041 W DK2009050041 W DK 2009050041W WO 2009100724 A1 WO2009100724 A1 WO 2009100724A1
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Prior art keywords
peptide
seq
medicament
antibody
amino acid
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PCT/DK2009/050041
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English (en)
Inventor
Vladimir Berezin
Elisabeth Bock
Jacob Jacobsen
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Kobenhavns Universitet
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Publication of WO2009100724A1 publication Critical patent/WO2009100724A1/fr

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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

Definitions

  • the present invention relates to a novel peptide, NLIKQDDGGSPIRHY, and variants, and fragments thereof, which are capable of binding to, and modulating the activity of FGFR.
  • the peptide is derived from the second fibronectin module type III of the neural cell adhesion molecule NCAM.
  • the invention further relates to use of said peptides for the manufacturing of a medicament for the treatment of different pathological conditions, wherein NCAM and/or FGFRs play a prominent role.
  • NCAM is a cell surface glycoprotein belonging to the Ig superfamily of CAMs (for review see Kiselyov et al., 2005). NCAM can be expressed as three major isoforms (A, B and C) with differences in the cytoplasmic domain. The extracellular part of NCAM is identical for the three isoforms and consists of five Ig-like and two fibronectin type III (F3) modules.
  • NCAM is widely expressed during embryonic development, whereas in the adult organism it is mainly found in tissues of neural origin. It induces cell adhesion by homophilic binding mechanisms, mediates neuronal migration, axon growth and guidance, and modulates synaptic plasticity associated with learning and memory (R ⁇ nn et al., 2000; Walmod et al., 2004; Kiselyov et al., 2005).
  • NCAM binding results in activation of a number of intracellular signaling pathways including activation of the fibroblast growth factor receptor (FGFR), the nonreceptor kinases Fyn and focal adhesion kinase (Ditlevsen et al., 2007).
  • FGFR fibroblast growth factor receptor
  • Fyn the nonreceptor kinases
  • Fyn focal adhesion kinase
  • NCAM binding site for FGFR has been shown by nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analyses to localize to a sequence motif in the second fibronectin type III (F3) module, the FGL motif, encompassing the F and G ⁇ strands and the interconnecting loop (Kiselyov et al., 2003).
  • NMR nuclear magnetic resonance
  • SPR surface plasmon resonance
  • FGL A synthetic peptide, FGL, has been shown to bind and activate FGFR, thereby inducing neurite outgrowth and promoting survival of primary neurons (Neiiendam et al., 2004). FGL has also been shown to enhance presynaptic function and promote synapse formation in vitro (Cambon et al., 2004). In vivo, FGL enhances spatial memory and contextual fear conditioning, promotes early postnatal sensory motor development, and enhances social memory retention.
  • NCAM The mechanism of FGFR activation by NCAM is not well understood. It is thought that most of the FGFR molecules are involved in a transient interaction with NCAM (Kiselyov et al., 2003). When NCAM is not involved in cell-cell adhesion, the NCAM molecules are supposed to be uniformly spread on the cell surface. However, when NCAM is involved in cell-cell adhesion, via homophilic binding, NCAM molecules may arrange themselves into the so-called 'zipper'-formations (Soroka et al., 2003). This leads to clustering of the NCAM molecules, resulting in a subsequent clustering of FGFR molecules (Kiselyov et al., 2005).
  • the present invention concerns a peptide consisting of at most 20 contiguous amino acids comprising the sequence NLIKQDDGGSPIRHY or a variant or fragment thereof.
  • a compound of the invention is capable of inducing neurite outgrowth and/or differentiation, modulating proliferation, stimulate regeneration, and/or neuronal plasticity.
  • Another aspect of the invention relates to use of a peptide of the invention as medicaments and for the preparation of medicaments for treatment of a condition or disease wherein FGFR and/or NCAM play a role in pathology or recovery from the disease.
  • the present invention describes a pharmaceutical composition comprising at least one peptide or compound of the invention.
  • a peptide of the invention may be used for the production of an antibody.
  • Figure 1 A model of the first and second NCAM F3 modules based on the experimental structures obtained by NMR (Kilselyov et al., 2003) and X-ray crystallography (Mendiratta et al., 2006). Six strand-loop-strand regions in the second F3 module are marked according to the corresponding ⁇ strands they connect.
  • Results are given as percentage ⁇ SEM.
  • n 4. ** p ⁇ 0.01 compared to the control (untreated cells) set at 100%. +p ⁇ 0.05 compared to the cultures treated with FGF2 in the absence of BCL.
  • FIG. 6 Effect of peptide modification on neurite outgrowth
  • B Effect of glutamine substitution (Q ⁇ A) on neuritogenic activity of BCL. ** p ⁇ 0.01 compared to the control set at 100%. ++p ⁇ 0.01 compared to the cultures treated with non-mutated BCL.
  • FIG. 7 Effect of BCL on survival of CGNs induced to undergo apoptosis Apoptosis of CGNs grown for 7 days was induced by changing a high-potassium (40 mM) medium to a low-potassium (5 mM) medium. Survival was estimated 48 h later.
  • A Effect of 5 mM KCI (negative control) and IGF1 (positive control) on survival of CGNs. Results are given as percentage ⁇ SEM. *** p ⁇ 0.001 compared to the control (40 mM KCI) set at 100%. +++p ⁇ 0.001 compared to the cultures treated with 5 mM KCI.
  • the present invention discloses a peptide consisting of at most 20 contiguous amino acids comprising the sequence NLIKQDDGGSPIRHY (SEQ ID NO:1 ) or a variant or a fragment thereof, with the proviso that when said fragment consists of 9 contiguous amino acids the amino acid sequence is not QDDGGSPIR.
  • the peptide according to the invention comprises the sequence NLIKQDDGGSPIRHY (SEQ ID NO:1 ).
  • the peptide according to the invention comprises a fragment of NLIKQDDGGSPIRHY (SEQ ID NO:1 ).
  • the peptide according to the invention comprises a variant of NLIKQDDGGSPIRHY (SEQ ID NO:1 ).
  • the C-terminal amino acid of a peptide for use according to the invention exists as the free carboxylic acid, this may also be specified as "-OH".
  • the C-terminal amino acid of a peptide for use according to the invention may be the amidated derivative, which is indicated as "-NH 2 ".
  • the N-terminal amino acid of a polypeptide comprises a free amino-group, this may also be specified as "H-”.
  • a peptide, fragment, or variant thereof according to the invention can also comprise one or several unnatural amino acids.
  • a preferred peptide according to the invention comprises the sequence NLIKQDDGGSPIRHY or a variant or fragment thereof, with the proviso that when said fragment consists of 9 contiguous amino acids the amino acid sequence is not QDDGGSPIR.
  • a variant according to the invention of an amino acid sequence may be i) an amino acid sequence having at least 60% identity with the selected sequence, such as 61 -70% identity, for example 71 -80% identity, such as 81 -90% identity, for example 91 -95% identity, such as 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.
  • the identity between amino acid sequences may be calculated using well known algorithms such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLOSUM 90;
  • an amino acid sequence which has at least 60% positive amino acid matches with a selected sequence such as 61 -70% positive amino acid matches, for example 71 -80% positive amino acid matches, such as 81 -90% positive amino acid matches, for example 91 -95% positive amino acid matches, such as 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 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
  • variant of a peptide sequence also 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., for example methyl and acetyl esters, as well as polyethylene glycol modifications.
  • an amine group of the peptide may be converted to amides, wherein the acid part of the amide is a fatty acid.
  • variants of the amino acid sequences 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 lie
  • the same variant of a peptide fragment, or fragment of said variant 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:
  • Conservative substitutions may be introduced in any position of a preferred predetermined peptide for use according to 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 variant fragment of the peptide for use according to 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
  • Substitution of amino acids may in one embodiment be made based upon their hydrophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like.
  • a fragment is to be understood as being any part of a peptide of the invention capable of interacting with an FGF-receptor and through said binding stimulate neurite outgrowth, modulate proliferation, and/or induce differentiation, and/or stimulate regeneration, neuronal plasticity and/or survival of cells.
  • a fragment may be defined as
  • the term “functional equivalent” of an amino acid sequence is in the present context meant a molecule which meets the criteria for a variant or a fragment of said amino acid sequence described above and which is capable of one or more functional activities of said sequence or a compound comprising said sequence.
  • the functional equivalent of an amino acid sequence according to the invention is capable of binding and modulating activity of FGFRs.
  • the invention relates both to isolated peptides according to the invention and fusion proteins comprising peptides according to the invention.
  • the peptide according to the invention is an isolated peptide.
  • isolated peptide is meant that the peptide according to the invention is an individual compound and not a part of another compound.
  • the isolated peptide may be produced by use of any recombinant technology methods or chemical synthesis and separated from other compounds, or it may be separated from a longer polypeptide or protein by a method of enzymatic or chemical cleavage and further separated from other protein fragments.
  • 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.
  • the 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, 1981 , Tetrahedron Lett. 22:1859-1869, or the method described by Matthes et al., 1984, EMBO J. 3:801 -805.
  • 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
  • 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
  • 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
  • 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 extrachromosomal 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 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 (Vasuvedan et al., 1992, FEBS Lett. 31 1 :7-1 1 ).
  • 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.
  • 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 TPM (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, enterokinase, with the amino acid sequence DDDDK, thrombin, with the amino acid sequence LVPR/GS, or Acharombacter 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 eukaryotic cell, such as invertebrate (insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells.
  • a eukaryotic cell such as invertebrate (insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells.
  • suitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCC
  • fungal cells may be used as host cells.
  • suitable yeast cells include cells of Saccharomyces spp. or Schizosaccharomyces 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
  • Peptides may be synthesised either batchwise in a polyethylene vessel equipped with a polypropylene filter for filtration or in the continuous-flow version of the polyamide 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.
  • An isolated peptide sequence of the invention may be connected to another isolated peptide sequence by a chemical bond in a fusion protein or the amino acid sequences may be connected to each other through a linker group.
  • a peptide sequence of the invention may be formulated as an oligomer (multimer) of monomers, wherein each monomer is as a peptide sequence defined above.
  • multimeric peptides such as dendrimers may form conformational determinants or clusters due to the presence of multiple flexible peptide monomers.
  • the compound is a dimer, in another embodiment the compound is a trimer or a tetramer.
  • the compound is a dendrimer, such as four peptides linked to a lysine backbone, or coupled to a polymer carrier, for example a protein carrier, such as BSA.
  • a polymer carrier for example a protein carrier, such as BSA.
  • Polymerisation such as repetitive sequences or attachment to various carriers are well-known in the art, e.g. lysine backbones, such as lysine dendrimers carrying 4 peptides, 8 peptides, 16 peptides, or 32 peptides.
  • Other carriers may be lipophilic dendrimers, or micelle-like carriers formed by lipophilic derivatives, or starburst (star-like) carbon chain polymer conjugates.
  • a multimeric compound may be a polymer comprising two or more identical or different peptide sequences of the invention, wherein in a preferred embodiment, at least one of the two or more amino acid sequences is selected from a sequence of a peptide according to this invention.
  • the compound may comprise two identical amino acid sequences or the compound may comprise four identical copies of an amino acid 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 the peptide sequences shown herein, or fragments or variants thereof. More preferably two or more sequences are selected from the sequences shown herein.
  • the invention relates to the use of one or more of the peptides comprising a sequence corresponding to NCAM or a fragment thereof or a variant for the manufacture of a medicament.
  • the medicament of the invention comprises at least one of the amino acid sequences set forth in SEQ ID NOS: 1 -18 or fragments or variants of said sequences.
  • the medicament of the invention comprises an antibody capable of binding to an epitope comprising NCAM or a fragment or variant thereof or a fragment or variant of said antibody.
  • the medicament of the invention comprises an effective amount of one or more of the compounds as defined above, or a composition comprising compound as defined above, in combination with pharmaceutically acceptable additives.
  • Such medicament may suitably be formulated for oral, percutaneous, intramuscular, intravenous, intracranial, intrathecal, intracerebroventricular, 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, microcapsules, 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%.
  • formulations 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 (for example formed with the free amino groups of the peptide compound) and which are formed with inorganic acids such as, for example, hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like, or such organic acids as formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzene
  • 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 isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • compositions include the pharmaceutically acceptable salts listed in J. Pharm. Sci. 1977, 66, 2, which is incorporated herein by reference.
  • metal salts include lithium, sodium, potassium, magnesium salts and the like.
  • ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts 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.
  • the preparation further comprises pharmaceutically acceptable additives and/or carriers.
  • 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.
  • the present invention relates to treatment of individuals for inducing differentiation, modulating proliferation, stimulate regeneration, neuronal plasticity and survival of cells in vitro or in vivo, the treatment involving administering an effective amount of one or more compounds as defined above.
  • Another strategy for administration is to implant or inject cells capable of expressing and secreting the compound in question. Thereby the compound may be produced at the location where it is going to act.
  • the present invention relates to said peptides, fragments, or variants thereof for use in the modulation of proliferation and/or induction of differentiation and/or stimulation of regeneration and/or neuronal plasticity.
  • the use is for the treatment for preventing diseases and conditions of the central and peripheral nervous system, and/or of the muscles and/or of various organs.
  • Treatment according to the invention is in one embodiment useful for inducing differentiation, modulating proliferation, stimulating regeneration, and/or neuronal plasticity of cells.
  • 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 surgical 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 surgical treatments and/or diagnostic methods that may cause formation of free radicals or otherwise have cytotoxic effects, such as X-rays and chemotherapy.
  • chemotherapy peptides according to the invention are useful in cancer treatment.
  • the treatment may comprises treatment and/or prophylaxis 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 associated 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 associated 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, and/or neuronal plasticity.
  • the treatment may be for preventing cell death of heart muscle cells, such as after acute myocardial infarction, in order to induce angiogenesis.
  • the treatment is for the stimulation of the survival of heart muscle cells, such as survival after acute myocardial infarction.
  • the treatment is for revascularisation, such as after injuries.
  • the present peptides are capable of stimulating angiogenesis and thereby they can promote the wound healing process.
  • the invention further discloses a use of peptides in the treatment of cancer. Regulation of activation of receptor tyrosine kinases is important for tumor agiogenesis, proliferation and spreading.
  • a use of the peptides 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 peptide for use according to 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 peptide is still another embodiment of the invention.
  • a peptide may be for the treatment of clinical conditions, such as neoplasms such as malignant neoplasms, benign neoplasms, carcinoma in situ and neoplasms of uncertain behavior, cancer in breast, thyroidal, pancreas, brain, lung, kidney, prostate, liver, heart, skin, blood organ, muscles (sarcoma), 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
  • 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 infarction of spinal
  • Inflammatory and toxic neuropathy including acute infective polyneuritis, Guillain-Barre syndrome, 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 continuity (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, poisoning by drugs, medicinal and biological substances, genetic or traumatic atrophic muscle disorders; or for the 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.
  • 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 nephros
  • Scrapie Creutzfeldt-Jakob disease, Gerstmann-Straussler- Sheinker (GSS) disease; 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.
  • 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.
  • 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 invention relates to any antibody capable of selectively binding to an epitope comprising a contiguous amino acid sequence derived from NCAM or a fragment, or a variant therof, selected from any of the sequences set forth in SEQ ID NOS: 1 -18, or a fragment or variant of said sequence.
  • epitope is meant the specific group of atoms (on an antigen molecule) that is recognized by (that antigen's) antibodies.
  • epitope 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 disulfide 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 constant 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.
  • 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
  • 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 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. In the context of both the therapeutic and screening methods described below, preferred embodiments are the use of an antibody or fragment thereof that is immunospecific 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 antibody 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 -18, or a fragment of said sequences.
  • the term "antibody 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:
  • (1 ) 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 antibody 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 treating 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.
  • V H -V L dimer tight, non-covalent association
  • 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 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
  • VH-VL polypeptide chain
  • the invention also contemplates multivalent antibodies having at least two binding domains.
  • the binding domains may have specificity for the same ligand or for different ligands.
  • the multispecific molecule is a bispecific antibody (BsAb), which carries at least two different binding domains, at least one of which is of antibody origin.
  • BsAb bispecific antibody
  • Multivalent antibodies may be produced by a number of methods. Various methods for preparing bi- or multivalent antibodies are for example described in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881 ,175; 5,132,405; 5,091 ,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.
  • the invention contemplate both polyclonal and monoclonal antibody, antigen binding fragments and recombinant proteins thereof which are capable of binding an epitope 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. See, e.g., Coligan, et al., sections 2.7.1 -2.7.12 and sections 2.9.1 -2.9.3; Barnes, et al., Purification of Immunoglobulin G (IgG). In: Methods in Molecular Biology, 1992, 10:79-104, Humana Press, NY.
  • 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 involves 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 :105-1 15.
  • 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 specific, being directed against a single antigenic site. Furthermore, in contrast to conventional polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In additional to their specificity, 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 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 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' monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • Fv fragments comprise an association of V H and V L 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 comprising 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 ).
  • humanized antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-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.
  • Humanized antibody(es) containing a minimal sequence(s) of antibody(es) of the invention, such as a sequence(s) recognising an epitope(s) described herein is one of the preferred embodiments of the invention.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported
  • 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 methods in the art for producing polyclonal and monoclonal antibodies using natural or recombinant fragments of a sequence selected from any of the sequences identified as SEQ ID NOs: 1 -18, as an antigen. Such antibodies may be also generated using variants or fragments of SEQ ID NOs: 1 -18.
  • 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 an antibody, which is capable of modulating, such as enhancing or attenuating, biological function of NCAM in particular a function related to neural cell growth, and to an antibody, which can recognise and specifically bind to NCAM without modulating biological activity thereof.
  • the invention relates to use of the above antibodies for therapeutic applications involving the modulation of activity of NCAM.
  • the invention relates to the use of a pharmaceutical composition comprising an antibody described above.
  • the BCL peptide derived from the U-terminus of the folded second NCAM fibronectin module binds to FGFR
  • peptides derived from five regions of the second F3 module of rat NCAM (SwissProt P13596) (ABL: 618-EGQMGEDGNSIK-629; BCL: 631 - NLIKQDDGGSPIRHY-645; CDL: 648-KYRALASEWKPEIR-661 ; DEL: 661 - RLPSGSDHVM-670; EFL: 672-KSLDWNAEYE-681 ) were synthesized as tetrameric dendrimers and tested for their ability to bind to FGFR by means of SPR analysis.
  • FGFR1 Ig modules 2 and 3 were immobilized on a sensor chip, and peptide binding to FGFR was measured after injecting the peptides at various concentrations. Peptide association with and dissociation from immobilized FGFR lg2-3 are shown in Fig. 2A. Only the BCL peptide bound to FGFR1 with any measurable affinity. The ABL peptide had a very low binding capacity, whereas CDL, DEL, and EFL did not bind at all to immobilized FGFR1. To evaluate the equilibrium dissociation constant (K 0 ), BCL binding at various concentrations to FGFR1 was studied (Fig. 2B). BCL bound FGFR1 with an apparent K 0 value of 2.4 ⁇ 0.3 ⁇ M.
  • TREX293 cells (Invitrogen A/S, Taastrup, Denmark) stably transfected with human FGFR1 (splice variant INc) with a C-terminal Strep Il tag (IBA Biotech, G ⁇ ttingen, Germany) were used for analysis as previously described (Kiselyov et al., 2003).
  • DMEM Dulbecco's Modified Eagle's Medium
  • the cells were treated with 300 ⁇ l lysis buffer containing 1% (v/v) NP-40 (Sigma-Aldridge A/S, Copenhagen, Denmark), complete protease inhibitors (Roche, Mannheim, Germany; 1 :50), and phosphatase inhibitors (Calbiochem inhibitor cocktail III; 1 :100) in phosphate buffered saline (PBS). Protein concentrations were determined using the bicinchoninic acid assay (Pierce, Rockford, IL, USA).
  • the BCL peptide induces FGFR1 phosphorylation
  • TREX-293 cells stably transfected with FGFR containing a C-terminal Strep Il tag were treated for 20 min either with BCL at various concentrations or 0.58 nM FGF2. After stimulation, the amount of phosphorylated FGFR1 was determined. From Fig. 3A it appears that BCL induced FGFR phosphorylation within a broad range of peptide concentrations. FGF2 had a much higher receptor activation potency and efficacy than BCL.
  • the TREX-292 cells were treated with a low concentration, 0.029 nM, of FGF2 in the presence of increasing, high concentrations of BCL. From Fig. 3B it appears that FGF2-induced receptor phosphorylation was partially inhibited in the presence of high BCL concentrations, indicating that BCL was capable of competing with FGF2 for receptor binding and activation. This suggests that BCL is an FGFR partial agonist.
  • CGNs cerebellar granule neurons
  • CGNs were prepared from postnatal day 7 Wistar rats (Charles River, Sultzhild, Germany) essentially as described by Schousboe and Pasantes-Morales (1989).
  • CGN cells were plated on uncoated eight-well Lab-Tek chamber slides (Nunc A/S, Roskilde, Denmark) in Neurobasal A medium (Gibco BRL) supplemented with 0.4% (w/v) bovine serum albumin (BSA), 2% (v/v) B27 supplement (Gibco, BRL), 0.5% (v/v) glutamax, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin at a density of 10 5 cells/well. Peptides at various concentrations were included in to the medium immediately after plating, and cells were maintained at 37 0 C and 5% CO 2 for 24 h.
  • BCL induces neurite outgrowth, and this effect is abrogated by an FGFR inhibitor
  • One of the functions of NCAM is to promote neurite outgrowth from primary neurons through FGFR activation (Ditlevsen et al., 2007).
  • CGNs were plated at a low density and grown for 24 h in the presence or absence of BCL at various concentrations.
  • Fig. 4 shows examples of images of control CGNs (A) and CGNs treated with 3.72 ⁇ M (B) and 33.5 ⁇ M BCL (C). Quantification of the effect of
  • BCL concentrations resulted in a continual increase of peptide efficacy.
  • the neuritogenic effect of BCL was totally abrogated by concomitant treatment of cultures with a FGFR pharmacological inhibitor, SU5402 (Fig. 5B), indicating that the effect of the peptide depended on receptor activation.
  • the glutamine residue in the fifth position is critical for neuritogenic activity of BCL
  • Fig. 6A it appears that effect of the truncated peptides on neurite outgrowth was reduced as compared to the intact peptide.
  • CGNs were plated at a density of 10 6 cells/cm 2 on poly-L-lysine-coated chamber slides in modified Neurobasal A medium supplemented with 2% (v/v) B27 supplement, 0.5% (v/v) glutamax, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 40 mM KCI. Twenty- four hours after plating, cytosine- ⁇ -D-arabinofuranoside (Sigma-Aldrich A/S) was added at a concentration of 10 ⁇ M to avoid glial cell proliferation.
  • the cultures were grown for another 6 days, and apoptosis was induced by changing the medium to Basal Medium Eagle (BME) containing 5 mM KCI, followed by 48 h of incubation in the same medium in the presence of peptides, FGF2, or recombinant human insulin-like growth factor 1 (IGF-1 ; Gibco BRL).
  • BME Basal Medium Eagle
  • IGF-1 recombinant human insulin-like growth factor 1
  • Cell viability was estimated by staining with Hoechst 33258 (Molecular Probes) as previously described (K ⁇ hler el al., 2003). Survival was estimated by comparing the number of live neurons with the total number of neurons.
  • Peptides All peptides were synthesized as dendrimers composed of four monomers coupled to a lysine backbone by Schafer-N (Copenhagen, Denmark). The peptides were purified by gel filtration using SephadexTM G-10 (Amersham BioScience, Uppsala, Sweden) and dissolved in sterile distilled water. Concentration was determined by spectrophotometry at 205 nm.
  • a peptide from the first fibronectin domain of NCAM acts as an inverse agonist and stimulates FGF receptor activation, neurite outgrowth and survival. J. Neurochem. 95, 570-583.
  • a synthetic neural cell adhesion molecule mimetic peptide promotes synaptogenesis, enhances presynaptic function, and facilitates memory consolidation. J. Neurosci. 24, 4197-4204.
  • a neural cell adhesion molecule-derived peptide reduces neuropathological signs and cognitive impairment induced by A ⁇ 25 - 35 . Neuroscience 145, 209-224.
  • An NCAM-derived FGF- receptor agonist, the FGL-peptide induces neurite outgrowth and neuronal survival in primary rat neurons. J. Neurochem. 91 , 920-935.
  • a neural cell adhesion molecule- derived fibroblast growth factor receptor agonist promotes early postnatal sensorimotor development and enhances social memory retention. Neuroscience 141 , 1289-1299.

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Abstract

La présente invention concerne un peptide inédit, NLIKQDDGGSPIRHY, ainsi que des variantes et fragments de celui-ci, capables de se lier au FGFR et d'en moduler l'activité. Ce peptide est dérivé du second module de la fibronectine de type III de la molécule d'adhésion cellulaire neuronale (NCAM, de l'anglais neural cell adhesion molecule). L'invention concerne, en outre, l'utilisation desdits peptides en vue de la fabrication d'un médicament destiné au traitement de différents états pathologiques, dans lesquels la NCAM et/ou les FGFR jouent un rôle important.
PCT/DK2009/050041 2008-02-11 2009-02-10 Peptide inédit dérivé de la ncam (bcl) WO2009100724A1 (fr)

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