WO2008022645A2

WO2008022645A2 - Ncam fibronectin type 3 binding peptides

Info

Publication number: WO2008022645A2
Application number: PCT/DK2007/050112
Authority: WO
Inventors: Vladimir Berezin; Elisabeth Bock
Original assignee: Enkam Pharmaceuticals A/S
Priority date: 2006-08-21
Filing date: 2007-08-21
Publication date: 2008-02-28
Also published as: WO2008022645A3

Abstract

The present invention discloses the discovery of a new class of NCAM binding peptides, which has a beneficial mode of modulating NCAM function which differs from previously identified NCAM binding compounds. One aspect of the present invention is to provide a compound which binds to a fibronectin type 3 module of NCAM, wherein said compound is capable of modulating NCAM signaling. The invention also relates to pharmaceutical compositions comprising the peptide sequences and uses thereof for treatment of conditions wherein NCAM play a prominent role.

Description

Title

NCAM fibronectin type 3 binding peptides

Field of invention

The present invention discloses the discovery of a new class of NCAM binding peptides, which has a beneficial mode of modulating NCAM function which differs from previously identified NCAM binding compounds.

Background of invention

The neural cell adhesion molecule (NCAM) plays an important role in formation of neuronal connections in the developing nervous system. In addition considerable evidence suggests that in the adult nervous system, NCAM is involved in learning and regeneration.

NCAM is expressed as three major isoforms in the nervous system of which two, NCAM-180 (NCAM-A) and NCAM-140 (NCAM-B) are transmembrane, while the third, NCAM-120 (NCAM-C) is linked to the membrane via a GPI-anchor (see Figure 1 for general schematic of NCAM). In addition soluble forms of NCAM may be generated by truncation and shedding.

NCAM mediates cell-cell adhesion primarily through a homophilic (NCAM-NCAM) mechanism. In addition, NCAM binds heterophilically to other cell surface receptors and extracellular matrix components, including heparan sulphate proteoglycans and the cell adhesion molecules L1 and TAG-1/axonin-1 .

The extracellular part of NCAM is composed of five immunoglobulin-like homology modules (Ig) and two fibronectin type III like modules (F3) (See Figure 1 ). NCAM homophilic binding is believed to depend on the first three Ig modules. The two membrane-proximal F3 modules of NCAM appear not to be involved in hemophilic NCAM interactions but have have been shown to be involved in fibroblast growth factor receptor (FGFR) binding (Kiselyov et al. 2003). Homophilic NCAM binding initiates a signalling cascade involving activation of a receptor tyrosine kinase: the fibroblast growth factor receptor (FGFR), and a nonreceptor tyrosine kinase: Fyn, and the signaling appear to converge on the Ras-MAP- kinase signaling pathway. Studies in primary neurons as well as in the neuronal-like cell line PC12, have provided evidence that activation of both the FGFR and Fyn appear to be required for NCAM homophilic binding to lead to neurite outgrowth (for review see Hinsby et al. 2004).

A signaling cascade initiated by NCAM homophilic binding has also been shown to implicate other signaling proteins and second messengers such as: phospholipase C (PLC), Frs2, Grb2, SOS, Ras, Raf, She, focal adhesion kinase (FAK), MAP kinase kinase (MEK), MAP kinase (Erk1 and Erk2), Protein kinase A (PKA), protein kinase C (PKC), GAP43, DAG Lipase, CREB, c-FOS, CaMK, Ca²⁺, cAMP, IP₃.

Previous efforts have described the identification of NCAM binding peptides and antibodies, which target the homophilic binding site of NCAM. WO00/18801 and WO03/020749 disclose peptides capable of binding to the NCAM Ig domains, which peptides initiate NCAM signaling through the FGFR but abrogates NCAM homophilic binding. US5,667,978 describes an antibody directed at Ig3 domain of NCAM, which also induces neurite outgrowth.

The present invention concerns compounds capable of binding the NCAM fibronectin type 3 modules, and modulating NCAM functions, such as cell aggregation, neurite outgrowth, and regulation of intracellular calcium.

Summary of invention

NCAM is an important molecule of the central and peripheral nervous system. It is highly expressed on the surface of neuronal and glia cells, and plays a role in the formation of neuronal connections during development. Additionally, evidence is accumulating for a role of NCAM in learning and memory events.

The present invention discloses the discovery of a new class of NCAM binding peptides, which has a beneficial mode of modulating NCAM function which differs from previously identified NCAM binding compounds. Firstly, these compounds do not mimic NCAM homophilic binding, but are directed at the Fibronectin type 3 (F3) modules 1 and 2, F3.1 and F3.2 respectively. In a preferred embodiment, the compounds and peptides of the invention do not have a direct effect on NCAM homophilic binding, such as by physically blocking NCAM's homophilic binding site. For example, said compounds and peptides do not sterically obstruct homophilic binding. Preferably, said compounds and peptides do not abrogate NCAM homophilic binding. Without being bound by theory, this is a consequence of the targeted modules (F3.1 and F3.2) not being involved in homophilic binding events, and accordingly the peptides identified within the present invention do not appear to abrogate NCAM homophilic binding. Surprisingly the peptides are capable of inducing activation of NCAM signaling events.

NCAM has been shown to signal through the FGFR and Fyn tyrosine kinases, and the F3 modules of NCAM has been shown capable of interacting with and activating the FGFR. It is another surprising discovery that the peptides of the present invention appear to activate NCAM signaling through a pathway which is independent of the FGFR and Fyn tyrosine kinases. The signaling pathway appear to involve a novel G protein dependent pathway.

It is therefore a primary aspect of the present invention to provide: A compound which binds to a fibronectin type 3 module of NCAM, wherein said compound is capable of modulating NCAM signaling.

It is preferred that said compound contains, or in some embodiments consists of, a peptide sequence, a peptide derivative or a peptide-mimicking compound.

It is also preferred that said compound is capable of activating NCAM mediated cell signaling. In a preferred embodiment said signaling includes activation of G-protein coupled pathways.

It is furthermore preferred that said compound is capable of promoting neurite outgrowth from NCAM presenting cells. Preferably, said NCAM presenting cells are selected from primary cells and cell lines wherein NCAM activation, such as induced by homophilic interaction in trans, is known to promote neurite outgrowth. For example, said cells include cerebellar granule neurons and PC12 cells. The present invention provides peptides that are capable of binding one of, or both, the Fibronectin type 3 domains of NCAM and activating NCAM signaling.

Accordingly, the present invention concerns a compound, wherein said compound comprise an 1 1 residue peptide sequence, which is defined by the motif:

Ala - Xaa{h} - Aro - Xaa{i} - Aro - Xaa{j} - Aro - Xaa{k},

wherein Ala is alanine,

Aro is any amino acid containing an aromatic side chain

Xaa is any amino acid,

Xaa{h}, Xaa{i}, Xaa{j}, Xaa{k} represent h, i, j, k independent amino acid residues, respectively, wherein h, i, j, k are integers from 0 to 4

In a preferred embodiment this 1 1 residue peptide is: Ala-Arg-Trp-Ser-Lys-Gly-Phe-Asp-Gln-Trp-Met (SEQ ID NO 1 ), or a functional homologue hereof, wherein said homologue is at least 75% identical to SEQ ID NO 1 and is capable of binding NCAM and modulating NCAM signaling.

or

Ala-Phe-Tyr-Arg-Thr-lle-Gln-Trp-Thr-Met-Glu (SEQ ID NO 2), or a functional homologue hereof, wherein said homologue is at least 75% identical to SEQ ID NO 2 and is capable of binding NCAM and modulating NCAM signaling. or

Ala-Phe-Tyr-Arg-Leu-Val-Phe-Asn-Gln-Asp-Thr (SEQ ID NO 3), or a functional homologue hereof, wherein said homologue is at least 75% identical to SEQ ID NO 3 and is capable of binding NCAM and modulating NCAM signaling. or

Ala-Gly-Gln-Ala-Gly-Arg-Ala-Phe-lle-Phe-Phe (SEQ ID NO 4), or a functional homologue hereof, wherein said homologue is at least 75% identical to SEQ ID NO 4 and is capable of binding NCAM and modulating NCAM signaling.

It is an aspect of the invention to provide said compounds and/or said peptides as a pharmaceutical composition.

Furthermore the invention concerns a use of said compounds and/or said peptides for the manufacture of a medicament for the treatment of diseases or conditions of the central or peripheral nervous system, or of the muscles or of various organs. Preferably, a condition or disease wherein stimulating neural cell differentiation, neural cell survival, neurogenesis, stem cell proliferation, stem cell differentiation, learning and memory, and/or modulating activity of NCAM is beneficial for said treatment.

Description of Drawings

Figure 1 : Schematic of the NCAM protein. NCAM-120, -140, -180 are identical on the extracellular side. However NCAM-120 does not have a transmembrane domain, but are attached to the membrane through a GPI anchor.

Figure 2: Binding kinetics of ENFIN2 and ENFIN 11.

Figure 3: a-d. Neurite outgrowth in single-cell cultures of CGNs. a-c: Images of representative cells grown in the absence of peptides (a) or in the presence of 39.3 μM ENFIN2 (b) or 4.47 μM ENFIN1 1 (c). The images show neurons collected from several separate micrographs. Scale bar: 10 μm. d: Quantitative concentration-response curves showing the effects of ENFIN2 (black) and ENFIN11 (grey) on the neurite outgrowth of CGNs. Data have been normalized to the values of the respective control cultures. The individual data points indicate mean and SEM calculated on the basis of 4-6 independent experiments. ^*, ^** and ^*** indicate statistically significant differences relative to the corresponding control values. Figure 4: Concentration dependent effect on neurite outgrowth. Concentration dependent effect of ENFIN3 (LibF3N3) and ENFIN5 (LibF3N5) on neurite outgrowth in CGNs. P2 is a positive control, a potent inducer of NCAM dependent neurite outgrowth.

Figure 5: Neurite outgrowth in co-cultures of CGNs. CGNs were plated on top of a confluent layer of fibroblasts not expressing or expressing NCAM (denoted LVN and LBN, respectively). Cells were grown for 24 h in the absence (white columns) or presence of 4.37 μM ENFIN2 (grey columns) or 4.47 μM ENFIN1 1 (black columns). Data have been normalized to the values of control cultures grown in LVN cells, and are given as mean and SEM on the basis of 5 independent experiments. ^* indicate a statistically significant difference relative to the corresponding control values.

Figure 6: Neurite outgrowth in single-cell cultures of CGNs. Cells were grown in the absence or presence ENFIN2 and ENFIN1 1 in combination with protein overexpression or in the presence of various enzyme inhibitors, a: Cells grown in the absence or presence of 40 μg mL^'1 SU5402, an inhibitor of FGFR-mediated tyrosine auto-phosphorylation. b: Cells transiently transfected with vectors encoding wild type (wt) or dominant negative (dn) FGFR. c: Cells grown in the absence or presence of 5 μM PP2, and inhibitor of Fyn and related Src family kinases, d: Cells grown in the absence or presence of 1 μg mL^'1 PTX, and inhibitor of Gi proteins. Cells in "a", "b" and "c" were grown in the absence or presence of 39.3 μM ENFIN2 or 4.47 μM ENFIN1 1 . Cells in "d" were grown in the absence or presence of 4.37 μM ENFIN2 or 4.47 μM ENFIN1 1 . Data have been normalized to the values of the respective control cultures, and the individual data points indicate mean and SEM calculated on the basis of 4-5 independent experiments. With the exception of cells grown in the presence of ENFIN2 in combination with PP2, all average results for peptide treatments are statistically significantly higher than the corresponding controls untreated with peptide. ^* and ^*** indicate statistically significant differences relative to the corresponding control values.

Figure 7: Effect of ENFIN2 (N2) and ENFIN11(N11) on neurite outgrowth in PC12 cells. White bars: PC12 - E2 cells (with NCAM expression). Dark bars : PC12 -E2 cell (NCAM knock-down by siRNA). Detailed description of the invention

The present invention concerns a new class of NCAM binding compounds, which has a beneficial mode of modulating NCAM function which differs from previously identified NCAM binding compounds.

Accordingly, it is primary feature of the invention to provide a compound which binds to a fibronectin type 3 module of NCAM, wherein said compound is capable of modulating NCAM signaling.

Amino acids

Throughout the description and claims either the three letter code or the one letter code for natural amino acids are used. The abbreviations for natural amino acids are specified in the table below:

However, it is also within the scope of the invention that the peptide may contain other amino acids, such as, but not limited to: Aib - aminoisobutyric acid, NaI - 2- naphthylalanine, Sar - Sarcosine, Orn - ornithine, DAP - diaminopimelic acid, DAPA - Diaminopimelic acid, HYP - hydroxyproline. Furthermore, amino acids of the present invention include analogs of natural amino acids, such as lysine analogs, as well as amino acids which have been post-translationally modified such as, but not limited to, by acetylation, oxidation, phosphorylation, methylation, deamidation, glycosylation, glycation, or modified by lipid modification.

Where the L or D form has not been specified it is to be understood that the amino acid in question has the natural L form or the D form, so that the peptides formed may be constituted of amino acids of L form, D form, or a sequence of mixed L forms and D forms.

Where nothing else is specified Xaa 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: Ala, VaI, Leu, lie, Pro, Phe, Trp, Met, GIy, Ser, Thr, Cys, Tyr, Asn, GIn, Asp, GIu, Lys, Arg, His, Aib, NaI, Sar, Orn, Lysine analogues, DAP, DAPA and 4Hyp.

Compounds An NCAM binding compound according to the present invention binds to a fibronectin type 3 (F3) module of NCAM. In embodiments of the invention the compound binds the first (F3.1 ) and/or second (F3.2) F3 module. It is disclosed herein, in Examples 1 to 3, how such a compound can be identified. The methods described herein are not meant to limit the scope of the invention as other means of identifying binding partners are well known to a person skilled in the art.

An NCAM binding compound according to the present invention can be any organic or inorganic chemical entity which binds to a fibronectin type 3 (F3) module of NCAM. It is preferred that these compounds modulate NCAM signaling. In preferred embodiments said compound contains a peptide sequence, a peptide derivative or a peptide- mimicking compound. In more preferred embodiments said compound consists of a peptide sequence, a peptide derivative or a peptide-mimicking compound, such as, but not limited to, SEQ ID NO 1 to 4 and functional homologs and fragments thereof.

A peptide derivative according to the invention is meant to include any peptide that has been modified in order to increase biological effect, the biological availability, the efficiency of uptake and/or stability of the compound, such as the stability of a pharmaceutical composition comprising said compound. A peptide derivative according to the invention also includes peptides that have been modified for decreasing the cost of manufacturing or the cost of a treatment scheme. E.g. a peptide derivative includes a peptide which has been modified, e.g. by covalent bonding to another peptide for example with or without a linker moiety, in order to obtain multimeric compounds such as, but not limited to, those disclosed in the sections below.

A peptide-mimicking compound according to the invention can be any compound mimicking a NCAM binding peptide sequence, such as but not limited to a compound which binds to an identical or similar part of NCAM as an NCAM binding peptide, and/or a compound which modulates NCAM function in an identical or similar manner. Such compounds may include, but are not limited to, small organic compounds and antibodies.

In a preferred embodiment said compound consists of, comprises and/or contains a peptide sequence. In preferred embodiments the compound is a peptide and/or a peptide derivative comprising several copies of a peptide covalently coupled to each other. In another embodiment the compound can be a peptide derivative comprising several different peptides covalently coupled to each other. The covalent coupling may be direct or through one or more linker molecules.

In a preferred embodiment of the invention the compound contains one or multiple copies of a peptide. It is within the scope of the invention that said peptide is formulated as a multimeric compound comprising 2 or more copies of the peptide, such as 3, 4, 5, 6, 7, 8 or 9 copies of a peptide. The peptide sequences of a multimeric compound may be linked to each other by a peptide bond, or connected through a linker or a grouping. It is also within the scope of the invention to provide peptide multimers, wherein different peptides of the invention are linked or bonded, such as by the method described in the section below.

In embodiments where the peptide is formulated as a multimeric compound, said formulation may involve modification of the peptide, such as covalent linkage to a backbone structure or another peptide. In preferred embodiments the compounds are formulated as dendrimers. A dendrimer is built from a monomer, with new branches added in steps until a tree-like structure is created (dendrimer comes from the Greek dendra, meaning tree). A dendrimer is thus technically a type of polymer. Depending on the dendrimer core (central part), the dendrimer can start with 3 to 8 (or more) branches, with 3 and 4 being the most common numbers observed. In a preferred embodiment of the invention a dendrimer contains four peptides linked to a lysine backbone, or coupled to a protein carrier such as BSA (bovine serum albumin). In a preferred embodiment the peptide is formulated as a dendrimer comprising 4 copies of the peptide linked to a backbone structure of lysine residues, preferably a backbone of 3 lysine residues.

In a preferred embodiment of the invention the compound contains one or multiple copies of a peptide linked as LPA-dimers.

In a first aspect the invention concerns a compound, wherein said compound comprise an 1 1 residue peptide sequence, which is defined by Motif 1 :

Ala - Xaa{h} - Aro - Xaa{i} - Aro - Xaa{j} - Aro - Xaa{k} (Motif 1 ), wherein Ala is alanine,

Aro is any amino acid containing an aromatic side chain Xaa is any amino acid, Xaa{h}, Xaa{i}, Xaa{j}, Xaa{k} represent h, i, j, k independent amino acid residues, respectively, wherein h, i, j, k are integers from O to 4

Aro may be selected from any natural as well as non-natural amino acid, which contains an aromatic side chain. In preferred embodiments Aro is selected from the group consisting of Phe, Tyr and Trp. In one embodiment of the invention, the peptide contains at least 2 Trp residues. In another embodiment of the invention, the peptide contains at least 2 residues selected from Trp and Phe residues.

It is understood that the integer value discloses a number of amino acids in a sequence, so for example an integer value of 3, for example Xaa{h=3}, defines a stretch of exactly 3 amino acids which are selected independently of each other. Accordingly, as a non-limiting example, a motif where Xaa{h=3} defines the motif: Ala - AA1 - AA2 - AA3 - Aro - Xaa{i} - Aro - Xaa{j} - Aro - Xaa{k} wherein AA1 , AA2, and AA3 are amino acids selected independently of each other.

It is also understood that h, i, j and k are independently selected, however it is preferred that: h + i + j + k = 7.

In preferred embodiments of the invention the integer h = 0 or 1. Preferably, integer h = 0. In another preferred embodiment integer h = 1 .

In another preferred embodiment the integer h = 0 and i = 0. It is even more preferred that integer h = 0, integer i = 0, and integer j = 3 or 4. It is preferred that integer h = 0, integer i = 0, and integer j = 4. It is preferred that integer h = 0, integer i = 0, and integer j = 3.

In yet another preferred embodiment integer h = 1 and integer i = 3. Preferably, integer h = 1 , integer i = 3 and integer j =2.

In yet another preferred embodiment integer h is 6. In a preferred embodiment said 1 1 residue peptide sequence is: Ala-Arg-Trp-Ser-Lys-Gly-Phe-Asp-Gln-Trp-Met (SEQ ID NO 1 ), or a functional homologue hereof, wherein said homologue is at least 75% identical to SEQ ID NO 1 and is capable of binding NCAM and modulating NCAM signaling.

In another preferred embodiment said 1 1 residue peptide sequence is: Ala-Phe-Tyr-Arg-Thr-lle-Gln-Trp-Thr-Met-Glu (SEQ ID NO 2), or a functional homologue hereof, wherein said homologue is at least 75% identical to SEQ ID NO 2 and is capable of binding NCAM and modulating NCAM signaling.

In another preferred embodiment said 1 1 residue peptide sequence is: Ala-Phe-Tyr-Arg-Leu-Val-Phe-Asn-Gln-Asp-Thr (SEQ ID NO 3), or a functional homologue hereof, wherein said homologue is at least 75% identical to SEQ ID NO 3 and is capable of binding NCAM and modulating NCAM signaling.

In another preferred embodiment said 1 1 residue peptide sequence is:

In a preferred embodiment said 1 1 residue peptide sequence is:

Ala-Arg-Trp-Ser-Lys-Gly-Phe-Asp-Gln-Trp-Met (SEQ ID NO 1 ).

In another preferred embodiment said 1 1 residue peptide sequence is: Ala-Phe-Tyr-Arg-Thr-lle-Gln-Trp-Thr-Met-Glu (SEQ ID NO 2).

In another preferred embodiment said 1 1 residue peptide sequence is: Ala-Phe-Tyr-Arg-Leu-Val-Phe-Asn-Gln-Asp-Thr (SEQ ID NO 3).

In another preferred embodiment said 1 1 residue peptide sequence is: Ala-Gly-Gln-Ala-Gly-Arg-Ala-Phe-lle-Phe-Phe (SEQ ID NO 4).

In a preferred a embodiment the compound of the invention comprises or consists of a peptide of at least 1 1 amino acid residues, such as a peptide of length 1 1 to 100 amino acid residues, such as 1 1 to 50 amino acid residues, for example 1 1 to 25 amino acid residues, such 1 1 to 18 amino acid residues, for example 1 1 to 15 amino acid residues, such as 1 1 to 13 residues, for example exactly 1 1 amino acid residues.

For example, in a preferred embodiment the compound comprises or consists of a peptide of at least 12 amino acid residues, such as a peptide of length 12 to 100 amino acid residues, such as 12 to 50 amino acid residues, for example 12 to 25 amino acid residues, such 12 to 18 amino acid residues, for example 12 to 15 amino acid residues, such as 12 to 13 residues, for example exactly 12 amino acid residues.

For example, in a preferred embodiment the compound comprises or consists of a peptide of at least 13 amino acid residues, such as a peptide of length 13 to 100 amino acid residues, such as 13 to 50 amino acid residues, for example 13 to 25 amino acid residues, such 13 to 18 amino acid residues, for example 13 to 15 amino acid residues, for example exactly 13 amino acid residues.

Moreover, it is within the scope of the invention that a compound according to the invention comprises, or consists of, fragments of Motif 1 , which are capable of binding NCAM and modulating NCAM signaling. Preferably, said fragments are fragments of peptides SEQ ID NO 2, SEQ ID NO 1 , SEQ ID NO 3 or SEQ ID NO 4, which are capable of binding NCAM and modulating NCAM signaling. Said fragments are preferably at least 5 amino acid residues, such as at least 6 amino acid residues, for example at least 7 amino acid residues, such as at least 8 amino acid residues, for example at least 9 amino acid residues, such as 10 amino acid residues. In a preferred embodiment said peptide fragment comprises at least one aromatic amino acid residue, such as at least 2 aromatic amino acid residues, for example at least 3 aromatic amino acid residues. In one embodiment of the invention, said fragment contains at least 2 Trp residues. In another embodiment of the invention, the fragment contains at least 2 residues selected from Trp and Phe residues. In a preferred embodiment a compound of the present invention does not comprise or consist of a peptide sequence derived from an FGFR. For example, the said compound does not comprise or consist of a protein sequence corresponding to an FGFR protein. In another preferred embodiment the compound of the present invention does not comprise or consist of an FGFR protein or any peptide fragment hereof. An FGFR is selected from the family of FGFRs, such as FGFR1 -4.

A compound according to the present invention preferably binds to a fibronectin type 3 (F3) module of NCAM. Said binding may be determined by any method of determining protein interactions known to a person skilled in the art. For example, one method disclosed in the present invention detects interaction between the NCAM F3 modules and peptides by means of incubating a combinatorial library of peptides linked to polystyrene beads with recombinant polypeptide containing the F3 modules of NCAM (see Example 1 ). This procedure is similar to the screening procedure described in Rønn et al. 2002, which is hereby incorporated by reference.

A method of determining biomolecular interactions which also yields quantitative information is surface plasmon resonance (SPR). This is a real-time analysis, and the output reveals information on the dissociation constant (Kd) and of the association constant (Ka) of the interactions measured.

A dissociation constant (Kd) is commonly used to describe how tightly a ligand binds to a receptor. Said receptor being for example a polypeptide. Such binding is usually non- covalent, and is usually best described as a two-state equilibrium:

P + L <-> C

wherein P = polypeptide/receptor, L = ligand, C = bound complex of L and P.

The corresponding dissociation constant is defined as:

Kd = [P]x[L]/[C]

where [P], [L] and [C] represent the concentrations of the protein, ligand and bound complex, respectively. The dissociation constant has the units of molar (M = mol/L), and corresponds to the concentration of ligand [L] at which the binding site on the protein is half occupied, i.e., when the concentration of protein with ligand bound [C] equals the concentration of protein with no ligand bound [P]. The smaller the dissociation constant, the more tightly bound the ligand is; for example, a ligand with a nanomolar (nM) dissociation constant binds more tightly than a ligand with a micromolar (μM) dissociation constant.

It is preferred that an NCAM binding compound according to the present invention interacts with F3.1 and/or F3.2 of NCAM with a Kd of less than 10 mM, such as less than 5 mM, for example less than 1 mM, such as less than 750 μM, for example less than 500 μM, such as less than 250 μM, for example less than 150 μM, such as less than 100 μM, for example less than 50 μM, such as less than 25 μM, for example less than 15 μM, such as less than 10 μM, for example less than 5 μM, for example less than 1 μM, such as less than 100 nM.

In a preferred embodiment said Kd is measured by SPR.

In another preferred embodiment said Kd is measured for a dendrimer. For example a dendrimer of a peptide according to the invention.

In embodiments of the invention, the NCAM binding compounds are identified from the analysis method outlined above, and described in Examples 1 and 2. Both these methods are known to a person skilled in the arts, and he/she will also know how to calculate the Kd from the output of a SPR analysis.

However, in other embodiments of the invention, the interaction between a compound and a fibronectin type 3 (F3) module of NCAM is detected and/or quantified by other techniques. Such techniques include, but are not limited to, phage display, nuclear magnetic resonance (NMR), co-immunoprecipitation, fluorescence or bio luminescence resonance energy transfer (FRET or BRET) analysis, radio immunoassay (RIA), ELISA, cross-linking, confocal microscopy, peptide arrays, peptide pull-down, proteinchips, antibody microarrays, multiple photon detection (MPD), mass spectrometry (MS). NCAM signaling can be activated by NCAM homophilic or heterophilic interactions. Analysis of NCAM signaling can be performed by a variety of techniques. One means of analysing the activation and level, i.e. amplitude, of the NCAM signaling process is by observing specific end-points of NCAM signaling. These end-points include influence on events such as differentiation, cell motility, cell survival and proliferation of NCAM expressing cells. Neurite outgrowth in NCAM expressing neuronal or neuronal- like cells is one end-point which is particularly useful in the analysis of NCAM signaling. In particular, neurite outgrowth as a consequence of NCAM signaling can be observed in primary NCAM expressing neurons such as but not limited to cerebellar granule neurons or neuronal like cell lines such as the PC12 cell line. Functional abrogation and/or partial or full knock out of these components, such as downstream signaling proteins, of the NCAM signaling pathway can be surveyed by analysis of the above- mentioned end-points. Other means of analysing NCAM signaling include direct assessment of activation and/or expression status of signaling or reporter proteins of the NCAM signaling pathway. Particularly, the activation status of a protein can be determined at the protein level by detection of the phosphorylation status of particular residues in said protein. In another example, the enzymatic activation status, e.g. kinase activity, can be assessed to determine NCAM signaling activity. Another common analysis tool include measuring the level, e.g. concentration, of particular second messengers such as Ca²⁺, cAMP or lipid metabolites (e.g. IP₃ and DAG). Finally, the activation status and level of NCAM can be assessed by detecting particular protein-protein interactions. A review on protein components of NCAM signaling is hereby incorporated by reference (Hinsby et al. 2004). It is within the scope of the present invention that analysis of each of these components can be used to measure the activation status and level of NCAM signaling. The proteins in NCAM signaling include, but are not limited to, NCAM (-120, -140, -180), the FGFRs (-1 , -2, -3 and/or -4), Fyn tyrosine kinase, Erk1 and Erk2 kinases, GFRalpha, Phospholipase Cgamma, GAP43, CREB, CaMKII, She, FAK, DAG lipase, Protein kinase C, Protein kinase A, GRB2, N- and L-type calcium channels, CB1 cannabinoid receptor. Moreover, second messengers such as Ca²⁺, cAMP, IP₃ and DAG can also be analysed to measure the activation status and level of NCAM signaling.

The effect of NCAM binding compounds and/or peptides of the present invention on the activation status of NCAM mediated signaling can be assessed by analysing any downstream event in any cell type. Preferably said cell expresses NCAM. Said downstream event includes differentiation, proliferation, motility and/or survival of a neuronal, neuronal-like or non-neuronal cell. In a non-limiting example NCAM activation can be measured by analysing proliferation rate of glial cells, preferably said cells express NCAM, such as astrocytes.

A compound and/or peptide of the present invention may modulate NCAM signaling at the level of one or more of the proteins and/or second messengers of the NCAM signaling cascade, such as but not limited to the components mentioned herein above. In preferred embodiments compounds of the present invention activate intracellular signaling by NCAM. Preferably, a compound of the present invention can promote neurite outgrowth in a in an NCAM expressing primary neuron or neuronal-like cell. For example, the NCAM expressing cell is a cerebellar granule neuron or a PC12 cell. In another preferred embodiment, NCAM signaling initiated by said compound involves activation of G-protein coupled signaling pathways. In another preferred embodiment said activation leads to signaling through the phospholipase C pathway including the activation of second messengers such as DAG, IP3 (inositol triphosphate) and/or Ca²⁺. Furthermore, said activation may proceed through a cAMP driven pathway involving activation of adenylate cyclase and/or PKA). In another embodiment said signaling does not depend on activation of an FGFR. Preferably, it does not depend on the FGFR-1. In yet another embodiment said signaling does not depend on the Fyn tyrosine kinase. In yet another embodiment said signaling does not depend on the combined effects of the FGFR and Fyn kinases.

Examples 4 and 5 describe measurements of NCAM signaling activity, and the reliance on particular proteins, such as the FGFR, Fyn and G-protein. These non-limiting examples show how NCAM activation can be measured and how the role of signaling proteins can be assessed.

Functional homologues and fragments

It is preferred that a functional homologue according to the invention is related by primary structure, i.e. the sequence of amino acids, to a parent peptide of the invention. In preferred embodiments a parent peptide of the invention is selected from SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4. Preferably a functional homologue is 50%, such as 60%, for example 75% identical to, such as 80% identical to, for example 90% identical to, such 95% identical to, for example 99% identical to, the parent peptide.

It is preferred that substitutions in parent peptides of the invention are conservative amino acid substitutions. A person skilled in the art knows how to make and assess conservative amino acid substitutions, by which one amino acid is substituted for another with one or more shared chemical and/or physical characteristics. Conservative amino acid substitutions are less likely to affect the functionality of the peptide. Amino acids may be grouped according to shared characteristics. A conservative amino acid substitution is a substitution of one amino acid within a predetermined group of amino acids for another amino acid within the same group, wherein the amino acids within a predetermined groups exhibit similar or substantially similar characteristics. Within the meaning of the term "conservative amino acid substitution" as applied herein, one amino acid may be substituted for another within groups of amino acids characterised by having

i) polar side chains (Asp, GIu, Lys, Arg, His, Asn, GIn, Ser, Thr, Tyr, and Cys,)

ii) non-polar side chains (GIy, Ala, VaI, Leu, lie, Phe, Trp, Pro, and Met)

iii) aliphatic side chains (GIy, Ala VaI, Leu, lie)

iv) cyclic side chains (Phe, Tyr, Trp, His, Pro)

v) aromatic side chains (Phe, Tyr, Trp)

vi) acidic side chains (Asp, GIu)

vii) basic side chains (Lys, Arg, His)

viii) amide side chains (Asn, GIn)

ix) hydroxy side chains (Ser, Thr)

x) sulphor-containing side chains (Cys, Met), and xi) amino acids being monoamino-dicarboxylic acids or monoamino- monocarboxylic-monoamidocarboxylic acids (Asp, GIu, Asn, GIn).

It is preferred that a functional homologue and/or a fragment according to the invention is capable of binding NCAM, preferably it binds to the first (F3.1 ) and/or second (F3.2) F3 module of NCAM. In a most preferred embodiment a functional homologue and/or a fragment binds to the first (F3.1 ) and/or second (F3.2) F3 module in a manner similar to the parent peptide to which it is related by primary structure.

It is also preferred that a functional homologue and/or a fragment according to the invention is capable of modulating NCAM signaling. Preferably, a functional homologue and/or a fragment can activate signaling by NCAM. More preferably, a functional homologue and/or a fragment can activate signaling by NCAM in a manner similar to the parent peptide to which it is related by primary structure.

The functionality and binding capacity of the substituted peptide can assessed by the methods described herein above and in Examples 1 to 5.

Production of peptide sequences

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.

Recombinant preparation

Thus, in one embodiment 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. According to the phosphoamidine method, 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 .

The DNA sequence is then inserted into 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. Thus, 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. Alternatively, 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.

In the vector, 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. Examples of 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. 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 4,599,31 1 ) 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 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. An example of such a sequence (when the host cell is a mammalian cell) 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.

The procedures used to ligate the DNA sequences coding the peptides or full-length proteins, the promoter and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op.cit.). To obtain recombinant peptides of the invention 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. In one embodiment, 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. In another embodiment, 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. Examples 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. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. MoI. Biol. 159, 1982, pp. 601 -

621 ; Southern and Berg, 1982, J. MoI. Appl. Genet. 1 :327-341 ; Loyter et al., 1982,

Proc. Natl. Acad. Sci. USA 79: 422-426; Wigler et al., 1978, Cell 14:725; Corsaro and

Pearson, 1981 , in Somatic Cell Genetics 7, p. 603; Graham and van der Eb, 1973, Virol. 52:456; and Neumann et al., 1982, EMBO J. 1 :841 -845.

Alternatively, fungal cells (including yeast cells) may be used as host cells. Examples of suitable yeast cells include cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae. Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora spp., in particular strains of Aspergillus oryzae or Aspergillus niger. The use of 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.

Synthetic preparation

The methods for synthetic production of peptides are well known in the art. Detailed descriptions as well as practical advice for producing synthetic peptides may be found in Synthetic Peptides: A User's Guide (Advances in Molecular Biology), Grant G. A. ed., Oxford University Press, 2002, or in: Pharmaceutical Formulation: Development of Peptides and Proteins, Frokjaer and Hovgaard eds., Taylor and Francis, 1999.

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.

In a preferred embodiment the peptide sequences of the invention are produced synthetically, in particular, by the Sequence Assisted Peptide Synthesis (SAPS) method.

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. Pharmaceutical composition and administration

The invention also relates to a pharmaceutical composition comprising one or more of the compounds and/or peptides and/or fragments as defined above. A compound of the invention is preferably formulated as a dimer or multimer such as discussed above.

In the present context the term pharmaceutical composition is used synonymously with the term medicament, and relates to a composition comprising a compound according to the present invention plus pharmaceutically acceptable additives and optionally carriers.

Such medicament may suitably be formulated for oral, percutaneous, parenteral, intramuscular, intravenous, intracranial, intrathecal, intracerebroventricular, intranasal or pulmonal administration to a subject in need hereof.

The invention further relates to a pharmaceutical composition capable of promoting cell differentiation and/or modulating neuronal plasticity and/or proliferation of neural cells, and stimulation of survival and/or regeneration of NCAM presenting cells. The medicament of the invention comprises an effective amount of one or more of the compounds in combination with pharmaceutically acceptable additives and optionally one or more carriers.

The invention relates to compositions comprising one or more of the compounds described above administered in vitro or in vivo in an effective amount.

The present invention further concerns a medicament for the treatment of diseases and conditions of the central and peripheral nervous system, of the muscles or of various organs, wherein said medicament comprises an effective amount of one or more of the compounds as defined above in combination with pharmaceutically acceptable additives and optionally one or more carriers. Such medicament may suitably be formulated for oral, percutaneous, parenteral, intramuscular, intravenous, intracranial, intrathecal, intracerebroventricular, intranasal or pulmonal administration.

For most indications a localised or substantially localised application is preferred. The compounds are in particular used in combination with a prosthetic device such as a prosthetic nerve guide. Thus, in a further aspect, the present invention relates to a prosthetic nerve guide, characterized in that it comprises one or more of the compounds defined above. Nerve guides are known in the art.

In connection with the use in nerve guides, the administration may be continuous or in small portions based upon controlled release of the active compound(s). Furthermore, 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.

Strategies in formulation development of medicaments and compositions based on the compounds of the present invention generally correspond to formulation strategies for any other protein-based drug product. Potential problems and the guidance required to overcome these problems are dealt with in several textbooks, e.g. "Therapeutic Peptides and Protein Formulation. Processing and Delivery Systems", Ed. A. K. Banga, Technomic Publishing AG, Basel, 1995.

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. In addition, if desired, the preparation may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, which enhance the effectiveness or transportation of the preparation, and/or a stabilizer.

In general, for injection and infusion the pharmaceutical composition should be a sterile liquid, and it is also within the scope of the present invention to provide a pharmaceutical composition that has been subjected to a virus reduction step, i.e. virus filtration and/or acidic treatment. The purpose of virus filtration is a reduction of any virus contaminants.

Some of the compounds of the present invention are sufficiently active, but for 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. In some cases, it will be advantageous to include a compound, which promotes delivery of the active substance to its target.

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. For suppositories, 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%.

Other 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 (formed with the free amino groups of the peptide compound) and which are formed with inorganic acids such as, for example, 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 isopropylamine, 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 of the order of several hundred μg active ingredient per administration with a preferred range of from about 0.1 μg to 100 mg, such as in the range of from about 1 μg to 100 mg, and especially in the range of from about 10 μg to 50 mg. 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 dosis would be in the interval 0.5 mg to 50 mg per 70 kg body weight.

The pharmaceutical compositions according to the present invention may be administered once or more than once, for example they may be administered in the range of 2 to 5 times, such as 5 to 10 times, for example 10 to 20 times, such as 20 to 50 times, for example 50 to 100 times, such as more than 100 times.

In many instances, it will be necessary to administer the formulation multiple times. The 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, or weekly.

In particular embodiments, 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.

In one embodiment of the invention the administration of the present compound may be immediately after an acute injury, such as an acute stroke, or at the most 8 hours after said stroke in order for the present compound to have a stimulatory effect on cell survival. Further, in cases concerning proliferation and/or differentiation the administration according to the invention is not time dependent, i.e. it may be administered at any time.

In another embodiment it may be advantageous to administer the compound(s) according to the invention with other substances to obtain a synergistic effect. Examples of such other substances may be a growth factor, which can induce differentiation, or a hormone, or a transplant of cells, including a transplant of stem cells, or gene therapy, or immunotherapy.

The mentioned compounds and compositions may be used to treat conditions affecting the peripheral and/or the central nervous system and/or muscles and other tissues expressing NCAM as well as other conditions in which a stimulation of NCAM function is beneficial.

Furthermore, a compound of the invention and/or a fragment thereof may be for the manufacture of a medicament for treatment of normal, degenerated or damaged NCAM and/or NCAM ligand presenting cells.

In one aspect of the invention treatment by the use of the compounds according to the invention is useful for the stimulation of regenerating cells which are degenerating or 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. In relation to chemotherapy the NCAM binding compounds according to the invention are useful in cancer treatment.

The compounds according to the invention may be used for preventing cell death, i.e. stimulating survival.

Furthermore, the compounds according to the invention may be used for preventing cell death of cells being implanted or transplanted. This is particularly useful when using compounds having a long term effect.

In another aspect of the invention the compounds may be synthesised and secreted from implanted or injected gene manipulated cells.

The treatment comprises the use of said compound for 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 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 and bipolar disorders; for treatment of diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, such as after organ transplantation, 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.

Also, the present compound may be used in relation to diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, such as 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.

Furthermore, the compound and/or pharmaceutical composition may be administered to prevent cell death of heart muscle cells, such as after acute myocardial infarction, or after angiogenesis. Furthermore, in one embodiment the compound and/or pharmaceutical composition is for the stimulation of the survival of heart muscle cells, such as survival after acute myocardial infarction. In another aspect the compound and/or pharmaceutical composition is for revascularisation, such as after injuries.

In another aspect the compound and/or pharmaceutical composition is used to treat a condition or disease wherein stimulating neural cell differentiation, neural cell survival, neurogenesis, stem cell proliferation, stem cell differentiation, learning and memory, and/or modulating activity of NCAM is beneficial for treatment of said condition or disease.

In a preferred embodiment the compound and/or pharmaceutical composition is used to stimulate neurogenesis, stem cell proliferation and/or stem cell differentiation. In another aspect the compound and/or pharmaceutical composition is used for the stimulation of the ability to learn and/or of the short and/or long term memory.

In particular the compound and/or pharmaceutical composition of the invention may be used in the treatment of clinical conditions, such as diseases of endocrine glands, such as diabetes mellitus, 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, Jakob-Creutzfeldt disease, multiple sclerosis, Pick's disease of the brain, polyarteriti nodosa, syphilis, Schizophrenic disorders, affective psychoses, neurotic disorders, personality disorders, including character neurosis, nonpsychotic personality disorder associated with organic brain syndromes, paranoid personality disorder, fanatic personality, paranoid personality (disorder), paranoid traits, sexual deviations and disorders or dysfunctions including reduced sexual motivation or capability for what ever reason, sleep disorders, mental retardation, inherited or in relation with disease or trauma, disease in the nervesystem and sense organs such as affecting sight, hearing, smell, feeling, tasting, cognitive anomalies, pain syndrome, 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 cord (embolic) (nonembolic), arterial thrombosis of spinal cord, edema of spinal cord, hematomyelia, subacute necrotic myelopathy, subacute combined degeneration of spinal cord in diseases classified elsewhere, myelopathy, drug-induced, radiation- induced myelitis, disorders of the autonomic nervous system, disorders of peripheral autonomic, sympathetic, parasympathetic, or vegetative system, familial dysautonomia [Riley-Day syndrome], idiopathic peripheral autonomic neuropathy, carotid sinus syncope or syndrome, cervical sympathetic dystrophy or paralysis, peripheral autonomic neuropathy in disorders classified elsewhere, amyloidosis, diseases of the peripheral nerve system, brachial plexus lesions, cervical rib syndrome, costoclavicular syndrome, scalenus anterior syndrome, thoracic outlet syndrome, brachial neuritis or radiculitis NOS, including in newborn. 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, purulent endophthalmitis, inflammatory disorders with tissue damage, either by affecting the infection agent or protecting the tissue, HIV, hepatitis, and following symptoms, diseases of the ear and mastoid process, chronic rheumatic heart disease, ischaemic heart disease, arrhythmia, autoimmune disorders, such as rheumatoid arthritis, SLE, ALS, and MS, anti-inflammatory effects, asthma and other allergic reactions, diseases in the pulmonary system, respiratory system, sensoring e.g. oxyhene, asthma, acute myocardial infarction, and other related disorders or sequel from AMI, 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, atrophic dermatitis, psoriasis, infection caused disorders, 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, both acute dysfunction and chronic dysfunction e.g. deficit in cognition, mood, social functioning after injury, genetic or traumatic atrophic muscle disorders; or for the treatment of diseases or conditions of various organs, such as metabolic disorders, such as obscenity lipid disorders, such as disorders of amino-acid transport and metabolism, disorders of purine and pyrimidine metabolism and gout, such as degenerative conditions of the gonads, of the pancreas, such as diabetes mellitus type I and II, of the kidney, such as nephrosis, such as bone disorders such as fracture, osteoporosis, osteoarthritis.

Particularly, for the treatment of 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 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; for treatment of diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, such as after organ transplantation, 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 and bowel, and for the treatment of postoperative nerve damage, traumatic nerve damage, impaired myelination of nerve fibers, postischaemic, e.g. resulting from a stroke, Parkinson's disease, Alzheimer'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.

It is also within the scope of the invention to use the compound and/or pharmaceutical composition for the promotion of wound-healing.

NCAM regulates motility, inhibits cancer cells from spreading and has been found to be expressed in numerous types of cancer and tumors. The invention further discloses the use of the compound and/or pharmaceutical composition in the treatment of neoplasia, such as benign and malignant neoplasms or tumors. In preferred embodiments a compound of the invention can be used for the treatment of cancer, such as, but not limited to, carcinoma, hematological malignancy, sarcoma or glioma.

In preferred embodiments a compound of the invention is to be used for treatment of carcinomas, such as adenocarcinomas, such as squamous cell carcinoma, such as small cell carcinoma, for example large cell undifferentiated carcinomas, such as lung cancer. Said use also includes treatment of breast, prostate, liver, heart, skin, and colon cancer. In a preferred embodiment said use is for the treatment of endocrine tumors, preferably neuroendocrine tumors, for example Merkel cell carcinoma. Examples of use include conditions selected from the group, but are not limited to, pituitary tumors, thyroid tumors, pancreatic neoplasms, mammary small cell carcinoma (SmCC) and Ewing's sarcoma family.

In one embodiment a compound of the invention is to be used for treatment of a virus induced tumor, such as HPV (human papilloma virus) and CMV (cytomegalovirus) induced cancers.

In other embodiments a compound of the invention is used for treatment of hematological malignancies, such as leukemia and lymphoma, such as T-cell and B- cell lymphomas, for example acute myelomonocytic leukaemia, such as multiple myeloma, for example acute lymphoblastic leukemia (ALL), such as acute myelogenous leukemia (AML), for example acute lymphoblastic natural killer-cell lymphoma, for example chronic lymphocytic leukemia (CLL), such as chronic myelogenous leukemia (CML), for example non-Hodgkin's lymphoma, such as Hodgkin's lymphoma.

In further embodiments a compound of the invention is used for treatment of tumors and/or cancers of the brain or the peripheral nervous system, preferably gliomas such as oligodendroglioma, for example glioblastoma, such as astrocytoma. In further embodiments a compound of the invention is used for treatment of neuroblastoma.

In another aspect the invention relates to a process of producing a pharmaceutical composition, comprising mixing an effective amount of one or more of the compounds of the invention.

A further aspect of the present invention relates to the use of compounds and/or compositions of the invention. In one embodiment of the invention the use of a compound and/or pharmaceutical composition is for the manufacture of a medicament. Such use may be of any of the compounds of the invention.

The use of said compound, in one embodiment, is for the manufacture of a medicament for the treatment of conditions and diseases as those disclosed herein above in an individual in need thereof, for example conditions and diseases involving normal, degenerated or damaged NCAM presenting cells. The invention also discloses the use, wherein said compound is for the manufacture of a medicament for the treatment comprising the stimulation of differentiation of NCAM presenting cells and/or survival thereof.

For example said use is for the manufacture of a medicament comprising treatment of diseases and conditions of the central and peripheral nervous system, or of the muscles or of various organs as discussed above.

In yet a further aspect the invention relates to a method of treating an individual suffering from one or more of the diseases discussed above by administering the said individual a compound as described herein or a pharmaceutical composition comprising said compound.

Antibody

It is an objective of the present invention to provide the use of an antibody, antigen binding fragment or recombinant protein thereof capable of selectively binding to an epitope comprising a contiguous amino acid sequence derived from a fibronectin type 3 module of NCAM or a fragment, homologue or variant thereof. The invention relates to any antibody capable of selectively binding to an epitope comprising a contiguous amino acid sequence derived from fibronectin type 3 module of NCAM, selected from any of the sequences set forth in SEQ ID NOS: 1 -4, or a fragment or variant of said sequence.

By the term "epitope" is meant the specific group of atoms (on an antigen molecule) that is recognized by (that antigen's) antibodies. The term "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. 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).

Depending on the amino acid sequences of the constant domain of their heavy chains, 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 (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), respectively. The light chains of antibodies can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino sequences of their constant domain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

The term "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. However, the 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.

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 β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-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.

The term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen binding or variable region. Examples of 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. As used herein, "functional fragment" with respect to antibodies, refers to Fv, F(ab) and F(ab')₂ fragments. The term "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. In general, 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 -4, or a fragment of said sequences. Thus, in context of the present invention 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.

(2) 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 CH1 domain including one or more cysteines from the antibody hinge region.

(3) (Fab')₂ is the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction. (4) 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. (5) Single chain antibody ("SCA"), 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. Generally, 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. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies 1 13: 269-315 Rosenburg and Moore eds. Springer- Verlag, NY, 1994.

The term "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). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/1 1 161 , and Hollinger et al., Proc. Natl. Acad Sci. USA 90: 6444-

6448 (1993).

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. In one embodiment 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. 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. 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.

The preparation of monoclonal antibodies likewise is conventional. See, for example, Kohler & Milstein, Nature, 256:495-7 (1975); Coligan, et al., sections 2.5.1 -2.6.7; and Harlow, et al., in: Antibodies: A Laboratory Manual, page 726 ,CoId Spring Harbor Pub. (1988), 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.

Methods of in vitro and in vivo manipulation of monoclonal antibodies are well known to those skilled in the art. For example, 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.

Methods of making antibody fragments are also known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1988, incorporated herein by reference). 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. For example, 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. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, in US 4,036,945 and US 4,331 ,647, and references contained therein. These patents are hereby incorporated in their entireties by reference.

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. For example, 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. Preferably, the Fv fragments comprise V_H and V_L chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) 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. Methods for producing sFvs are described, for example, by Whitlow, et al., 1991 , In: Methods: A Companion to Methods in Enzymology, 2:97; Bird et al., 1988, Science 242:423-426; US 4,946,778; and Pack, et al., 1993, BioTechnology 1 1 :1271 -77.

Another form of an antibody fragment is a peptide coding for a single complementarity- determining region (CDR). 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. Such 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. For the most part, 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. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, 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. In general, 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. For further details, see: Jones et al., 1986, Nature 321 , 522- 525; Reichmann et al., 1988, Nature 332, 323-329; Presta, 1992, Curr Op Struct Biol 2:593-596; Holmes et al., 1997, J Immunol 158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma & Immunol 81 :105-115.

The generation of 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 -4, as an antigen. Such antibodies may be also generated using variants or fragments of SEQ ID NOs: 1 -4.

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 survival, 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

In one aspect the invention relates to the use of a pharmaceutical composition comprising an antibody described above.

References

Hinsby et al., Front Biosci. 2004 9:2227-44

Kasper et al. 1996., J Neurosci Res. 1996 46(2): 173-86.

Kiselyov et al., Structure. 2003 1 1 (6):691 -701

Rønn et al., Nat Biotechnol. 1999 17(10):1000-5 Rønn et al., Eur J Neurosci. 2002 16(9):1720-30

Examples

Example 1. Identification of NCAM-Binding Peptides by Combinatorial Chemistry

In order to design ligands for the two fibronectin type 3 homology modules of NCAM (denoted F3.1 and F3.2), combinatorial libraries of undecapeptides linked to polystyrene beads were synthesised and incubated with a biotinylated, unglycosylated recombinant construct of NCAM F3.1 -F3.2. The recombinant NCAM F3.1 -F3.2 modules produced in Escherichia coli and purified by FPLC as previously described (Kasper et al. 1996).

Synthesis of a resin-bound, one-bead one-peptide library was performed essentially as described previously (Rønn et al. 2002). Briefly, in each synthesis step the resin was divided into 19 portions, one for each of the protein L-amino acids except for cysteine. All the obtained undecameric peptides had an N-terminal alanine followed by 10 random amino acids. The subsequent screening for NCAM F3.1 -F3.2 binding peptides was carried out by incubating 2 ml_ resin, equivalent to ~10⁶ beads, each carrying a unique primary sequence, with biotinylated recombinant NCAM F3.1 -F3.2 modules. Binding events was identified by an alkaline phosphatase-streptavidin-based staining reaction as a dark stain.

Bead-linked peptides binding to the NCAM construct were detected by means of a enzyme-based staining reaction. Subsequently, the beads were isolated and the peptides microsequenced. The screening resulted in the isolation of ten peptides with different sequences, denoted ENFIN1 , 2, 3, 5, 6, 7, 9, 10, 1 1 , and 12 see Table 1 below.

Name Sequence

ENFIN1 ANYRWDTWTDR

ENFIN2 (SEQ ID NO 2) AFYRT IQWTME

ENFIN3 (SEQ ID NO 3) AFYRLVFNQDT

ENFIN5 (SEQ ID NO 4) AGQAGRAFIFF

ENFIN6 AGQPYWIMI IW

ENFIN7 ASFLFYGGDEA

ENFIN9 AGILFRLSSYM

ENFIN10 AFKRYPSDQWD

ENFIN1 1 (SEQ ID NO 1 ) ARWSKGFDQWM

ENFIN12 AGMDPYWNRE I

Table 1 .

Example 2.

Characterization of Binding Kinetics by Surface Plasmon Resonance (SPR)

ENFIN2 (SEQ ID NO 2) and ENFIN1 1 (SEQ ID NO 1 ) identified from the assay outlined in Example 1 were synthesised as dendrimers on a lysine backbone (Rønn et al. 1999).

Peptide dendrimers in solution (at a concentration of 6 μM) were tested for binding to immobilized recombinant NCAM F3.1 -F3.2 double modules using SPR.

To ensure that the identified interactions between ENFIN2 (SEQ ID NO 2) and ENFIN1 1 (SEQ ID NO 1 ) were specific, both peptides were checked for binding to another protein, metallothionein-ll. As expected, neither of the peptides bound to this protein (data not shown).

SPR experiments were conducted using a Biacore 2000 (Biacore International AB, Uppsala, Sweden) and CM-4 sensor chips with two chambers. The NCAM F3 modules were immobilized on the carboxymethylated dextran surface CM-4 chips using amine coupling according to the manufacturers recommendation. Briefly, recombinant NCAM protein (32 μl_, 100 μg mL^'1 in 10 mM sodium phosphate buffer, pH 7 or 10 mM acetate buffer, pH 5) was applied to the activated chip surface. The chip was blocked with 35 μl_ 1 M ethanolamine hydrochloride, pH 8.5.

Data of the SPR experiments are shown in Figure 2 and summarized in Table 2.

Table 2.

Example 3.

Characterization of Neuritogenic Activity Induced by ENFIN Peptides In Vitro

NCAM is well known for its ability to promote neuritogenesis. Hence, the NCAM- binding peptides, ENFIN2 (SEQ ID NO 2), ENFIN3 (SEQ ID NO 3), ENFIN5 (SEQ ID NO 4) and ENFIN1 1 (SEQ ID NO 1 ), were evaluated for their ability to modulate neurite outgrowth in vitro. First, in order to evaluate the general effects on neurite outgrowth, the peptides were tested in a single-cell culture system. Second, in order to evaluate the effects on NCAM-mediated neurite outgrowth, the peptides were tested in a co- culture system.

For studies with single-cell cultures, dissociated cultures of cerebellar granule neurons (CGNs) grown on plastic were incubated in the presence or absence of the individual peptides for 24 h, after which the degree of neurite outgrowth was quantified by stereology. As shown in Figure 3a-c ENFIN2 (SEQ ID NO 2) and ENFIN11 (SEQ ID NO 1 ) were able to induce neurite outgrowth. Quantitative dose-response curves for the effects of the peptides exhibited bell-shaped relationships demonstrating statistically significant effects on neurite outgrowth (Kruskal-Wallis test; ENFIN2 (SEQ ID NO 2), P<0.001 ; ENFIN1 1 (SEQ ID NO 1 ), P<0.002), ENFIN2 (SEQ ID NO 2) causing a maximal -640% increase in neurite outgrowth around 39 μM, and ENFIN1 1 (SEQ ID NO 1 ) causing a maximal -475% increase around 4.5 μM (Figure 3d). One scrambled control peptide, ENFIN2-scr, did not induce statistically significant changes in neurite outgrowth at any tested concentration, whereas the scrambled control peptide for ENFIN1 1 (SEQ ID NO 1 ), ENFIN1 1 -scr, did cause a statistically significant increase in neurite outgrowth to a maximum of -200% of control at concentrations at 40 μM (the highest tested concentration; data not shown). Hence, the strong stimulatory effects of ENFIN2 (SEQ ID NO 2) and ENFIN1 1 (SEQ ID NO 1 ) on neurite outgrowth were not the result of the overall amino acid composition of the peptides, but of their specific amino acid sequences.

Results for concentration dependent induction of neurite outgrowth by ENFIN3 (SEQ ID NO 3) and ENFIN5 (SEQ ID NO 4) in CGNs are shown in Figure 4.

For co-culture studies, CGNs were grown for 24 h on top of confluent layers of fibroblasts not expressing or expressing NCAM (LVN and LBN cells, respectively). The difference in the neuritogenic response in the two situations represents the effects induced by frans-homophilic NCAM-interactions. Consequently, the assay can be utilized to determine whether ENFIN2 (SEQ ID NO 2) and ENFIN1 1 (SEQ ID NO 1 ) modulate neurite outgrowth induced by frans-homophilic NCAM-interactions.

Consistent with previous studies, CGNs grown on NCAM-expressing cells (LBN) demonstrated a statistically significant higher degree of neurite outgrowth than cells grown on cells not expressing NCAM (LVN) (see Figure 5, Newman-Keuls Multiple Comparison Test, LVN control vs. LBN control, p<0.05). In the presence of ENFIN2 (SEQ ID NO 2) or ENFIN1 1 (SEQ ID NO 1 ), CGNs grown on LBN cells demonstrated a neurite outgrowth not statistically significantly different from control cultures grown on LBN cells. In CGNs grown on LVN cells, ENFIN1 1 (SEQ ID NO 1 ) induced a statistically significant increase (p<0.05) in the neurite outgrowth, to a level comparable to that of CGNs grown on LBN cells, whereas ENFIN2 (SEQ ID NO 2) caused an insignificant increase in neurite outgrowth to a level between that of control cultures grown in LVN and LBN cells, respectively.

In conclusion, these results demonstrate that ENFIN2 (SEQ ID NO 2) and ENFIN1 1 (SEQ ID NO 1 ) are able to induce neurite outgrowth of CGNs in a co-culture system in the absence of frans-homophilic NCAM interactions. Furthermore, the results demonstrate that the two peptides do not appear to abrogate neurite outgrowth induced by frans-homophilic NCAM interactions.

Example 4.

The role of peptides in NCAM-mediated signalling

Since ENFIN2 (SEQ ID NO 2) and ENFIN11 (SEQ ID NO 1 ) had pronounced effects on neurogenesis, the intracellular signalling necessary for the effects of the peptides was investigated by quantification of neurite outgrowth in single-cells cultures of CGNs.

NCAM is known to be able to induce neurite outgrowth through c/s-interactions with FGFR, leading to autophosphorylation of FGFR and activation of downstream signalling pathways. To determine whether ENFIN2 (SEQ ID NO 2) and ENFIN1 1 (SEQ ID NO 1 ) stimulates neurite outgrowth by activation of FGFR, CGNs were incubated in the absence or presence of SU5402, an inhibitor of FGFR-mediated signalling. SU5402 inhibits the tyrosine kinase activity of FGFR with an IC50 around 10-20 μM. However, as shown in Figure 6a, treatment with 135 μM SU5402 had no statistically significant effect on neurite outgrowth in control cultures or cultures exposed to ENFIN2 (SEQ ID NO 2) and ENFIN1 1 (SEQ ID NO 1 ). As an alternative method of investigating the importance of FGFR-mediated signalling, the neurite outgrowth of CGNs were evaluated following transfection with vectors encoding wild type (wt) or a dominant negative version (dn) of FGFR. As shown in Figure 6b, neither overexpression of wtFGFR or dnFGFR had any effect on the neurite outgrowth induced by ENFIN2 (SEQ ID NO 2) and ENFIN1 1 (SEQ ID NO 1 ). Together, Figure 6a-b indicate that both ENFIN2 (SEQ ID NO 2) and ENFIN1 1 (SEQ ID NO 1 ) induce neurite outgrowth through FGFR-independent signalling. The Src-related non-receptor tyrosine kinase Fyn binds directly or indirectly to the cytoplasmic tail of NCAM, and NCAM-mediated activation of Fyn can induce neurite outgrowth. To determine whether ENFIN2 (SEQ ID NO 2) and ENFIN1 1 (SEQ ID NO 1 ) stimulates neurite outgrowth through activation of Fyn, CGNs were incubated in the absence or presence of PP2, an inhibitor of Src-related non-receptor tyrosine kinases, which inhibits the kinase activity of Fyn with an IC50 around 5 nM. Treatment with 20 nM (data not shown) and 5 μM (Figure 6c) had no statistically significant effects on neurite outgrowth in control cultures or cultures exposed to ENFIN2 (SEQ ID NO 2) and ENFIN1 1 (SEQ ID NO 1 ). Treatment with 15 μM PP2 had a statistically significant inhibitory effect on the neurite outgrowth induced by ENFIN2 (SEQ ID NO 2) (Newman- Keuls Multiple Comparison Test, p<0.05) but not ENFIN1 1 (SEQ ID NO 1 ) (data not shown). However, at this concentration, any effects of PP2 are likely to be unspecific. Hence, it appears that ENFIN2 (SEQ ID NO 2) and ENFIN1 1 (SEQ ID NO 1 ) do not mediate signalling through Fyn or related kinases of the Src-family.

Studies have suggested that NCAM can stimulate neurite outgrowth through a pertussis toxic-, PTX, sensitive activation of G-protein-coupled Ca²⁺-channels. To determine whether ENFIN2 (SEQ ID NO 2) and ENFIN1 1 (SEQ ID NO 1 ) stimulates neurite outgrowth by activation of PTX-sensitive G-proteins, CGNs were incubated in the absence or presence of PTX. As shown in Figure 6d the neurite outgrowth of control cultures unstimulated with ENFIN2 (SEQ ID NO 2) or ENFIN1 1 (SEQ ID NO 1 ) was insensitive to exposure to 1 μg mL^'1 PTX. In contrast, neurite outgrowth of cultures treated with ENFIN2 (SEQ ID NO 2) and ENFIN11 (SEQ ID NO 1 ) was statistically significantly inhibited by the PTX-treatment (Newman-Keuls Multiple Comparison Test, ENFIN2 (SEQ ID NO 2), p<0.05; ENFIN1 1 (SEQ ID NO 1 ), p<0.001 ). These observations suggest that both peptides induce neurite outgrowth through a PTX- sensitive G-protein-coupled pathway. However, the neurite outgrowth of cells exposed to PTX in combination with ENFIN1 1 (SEQ ID NO 1 ) were still statistically significantly higher than for control cultures untreated with PTX and ENFIN1 1 (SEQ ID NO 1 ) (Newman-Keuls Multiple Comparison Test, p<0.001 ), suggesting that PTX only partially could inhibit neurite outgrowth induced by ENFIN1 1 (SEQ ID NO 1 ).

Example 5.

To verify that the peptides do in fact function through NCAM activation, their effect was tested in the NCAM expressing neuronal-like PC12 cell line. NCAM activation is a potent inducer of neurite outgrowth in PC12 cells. The results show that the peptides ENFIN2 (SEQ ID NO 2) (N2) and ENFIN1 1 (SEQ ID NO 1 ) (N1 1 ) are both capable of stimulating neurite outgrowth in wild type PC12-E2 cells. In PC12 cells without NCAM expression (NCAM knock out) neither peptide showed a neuritogenic effect (see Figure 7). These results indicate a role for NCAM in the neuritogenic effect of these peptides.

NCAM knock out in the PC12 cells was accomplished by transfecting cells with NCAM siRNA.

Claims

1 . A compound which binds to a fibronectin type 3 module of NCAM, wherein said compound is capable of modulating NCAM signaling.

2. The compound according to claim 1 , wherein said compound contains a peptide sequence, a peptide derivative or a peptide-mimicking compound.

3. The compound according to claim 1 , which is capable of activating NCAM mediated cell signaling.

4. The compound according to claim 3, wherein said signaling includes activation of G-protein coupled pathways.

5. The compound according to claim 1 , which is capable of promoting neurite outgrowth from NCAM presenting cells.

6. The compound according to claim 5, wherein said neurite outgrowth does not depend on signaling through the receptor tyrosine kinase FGFR.

7. The compound according to claim 5, wherein said neurite outgrowth does not depend on signaling through the non-receptor tyrosine kinase Fyn.

8. The compound according to claim 1 , which binds to the first fibronectin type 3 module (F3.1 ) of NCAM.

9. The compound according to claim 1 , which binds to the second fibronectin type 3 module (F3.2) of NCAM.

10. The compound according to claim 1 , which binds to both fibronectin type 3 modules of NCAM.

1 1 . The compound according to claim 1 , wherein said compound contains a peptide sequence.

12. The compound according to claim 1 , which does not abrogate NCAM homophilic binding.

13. The compound according to claim 1 , wherein said compound comprise an 1 1 residue peptide sequence, which is defined by the motif:

Ala - Xaa{h} - Aro - Xaa{i} - Aro - Xaa{j} - Aro - Xaa{k},

wherein

Ala is alanine,

Aro is any amino acid containing an aromatic side chain

Xaa is any amino acid,

14. The compound according to claim 13, wherein h is 0 or 1.

15. The compound according to claim 14, wherein h is 0 and i is 0.

16. The compound according to claim 14, wherein h is 1 and i is 3.

17. The compound according to claim 13, wherein Aro is selected from: Trp, Phe, and Tyr.

18. The compound according to claim 13, wherein the first four residues of said 1 1 residue peptide sequence are defined by the motif:

Ala - Phe/Asn - Tyr - Arg

19. The compound according to claim 13, wherein said 1 1 residue peptide sequence comprise two Trp residues.

20. The compound according to claim 13, wherein said 1 1 residue peptide sequence is: Ala-Arg-Trp-Ser-Lys-Gly-Phe-Asp-Gln-Trp-Met (ENFIN1 1 ), or a functional homologue or fragment hereof, wherein said homologue is at least 75% identical to ENFIN1 1 and wherein said functional homologue or fragment is capable of binding NCAM and modulating NCAM signaling.

21 . The compound according to claim 13, wherein said 1 1 residue peptide sequence is:

Ala-Phe-Tyr-Arg-Thr-lle-Gln-Trp-Thr-Met-Glu (ENFIN2), or a functional homologue or fragment hereof, wherein said homologue is at least 75% identical to ENFIN2 and wherein said functional homologue or fragment is capable of binding NCAM and modulating NCAM signaling.

22. The compound according to claim 13, wherein said 1 1 residue peptide sequence is:

Ala-Phe-Tyr-Arg-Leu-Val-Phe-Asn-Gln-Asp-Thr (ENFIN3), or a functional homologue or fragment hereof, wherein said homologue is at least 75% identical to ENFIN3 and wherein said functional homologue or fragment is capable of binding NCAM and modulating

NCAM signaling.

23. The compound according to claim 13, wherein said 1 1 residue peptide sequence is: Ala-Gly-Gln-Ala-Gly-Arg-Ala-Phe-lle-Phe-Phe (ENFIN5), or a functional homologue or fragment hereof, wherein said homologue is at least 75% identical to ENFIN5 and wherein said functional homologue or fragment is capable of binding NCAM and modulating

NCAM signaling.

24. The compound according to claim 13, wherein said 1 1 residue peptide sequence is:

Ala-Arg-Trp-Ser-Lys-Gly-Phe-Asp-Gln-Trp-Met (ENFIN1 1 ).

25. The compound according to claim 13, wherein said 1 1 residue peptide sequence is: Ala-Phe-Tyr-Arg-Thr-lle-Gln-Trp-Thr-Met-Glu (ENFIN2).

26. The compound according to claim 13, wherein said 1 1 residue peptide sequence is: Ala-Phe-Tyr-Arg-Leu-Val-Phe-Asn-Gln-Asp-Thr (ENFIN3).

27. The compound according to claim 13, wherein said 1 1 residue peptide sequence is:

Ala-Gly-Gln-Ala-Gly-Arg-Ala-Phe-lle-Phe-Phe (ENFIN5)

28. The compound according to claim 2, wherein said peptide is formulated as a multimeric compound comprising 2 or more copies of said individual peptide.

29. The compound according to claim 2, wherein said peptide derivative is a dendrimer comprising 4 copies of the peptide linked to a backbone structure of three lysine residues.

30. The compound according to any of the preceding claims, wherein said compound is capable of stimulating learning and memory.

31 . A peptide comprising at least 1 1 amino acid residues containing sequence motif of the formula: Ala - Xaa{h} - Aro - Xaa{i} - Aro - Xaa{j} - Aro - Xaa{k},

wherein Ala is alanine,

Aro is any amino acid containing an aromatic side chain Xaa is any amino acid,

32. The peptide according to claim 31 , containing the sequence: Ala-Arg-Trp-Ser-Lys-Gly-Phe-Asp-Gln-Trp-Met (ENFIN1 1 ). or a functional homologue or fragment hereof, wherein said homologue is at least 75% identical to ENFIN1 1 and wherein said functional homologue or fragment is capable of binding NCAM and modulating NCAM signaling.

33. The peptide according to claim 31 , containing the sequence: Ala-Arg-Trp-Ser-Lys-Gly-Phe-Asp-Gln-Trp-Met (ENFIN1 1 ).

34. The peptide according to claim 31 , containing the sequence:

Ala-Phe-Tyr-Arg-Thr-lle-Gln-Trp-Thr-Met-Glu (ENFIN2) or a functional homologue or fragment hereof, wherein said homologue is at least 75% identical to ENFIN2 and wherein said functional homologue or fragment is capable of binding NCAM and modulating NCAM signaling.

35. The peptide according to claim 31 , containing the sequence: Ala-Phe-Tyr-Arg-Thr-lle-Gln-Trp-Thr-Met-Glu (ENFIN2)

36. The peptide according to claim 31 , containing the sequence:

Ala-Phe-Tyr-Arg-Leu-Val-Phe-Asn-Gln-Asp-Thr (ENFIN3), or a functional homologue or fragment hereof, wherein said homologue is at least 75% identical to ENFIN3 and wherein said functional homologue or fragment is capable of binding NCAM and modulating NCAM signaling.

37. The peptide according to claim 31 , containing the sequence: Ala-Phe-Tyr-Arg-Leu-Val-Phe-Asn-Gln-Asp-Thr (ENFIN3).

38. The peptide according to claim 31 , containing the sequence:

Ala-Gly-Gln-Ala-Gly-Arg-Ala-Phe-lle-Phe-Phe (ENFIN5), or a functional homologue or fragment hereof, wherein said homologue is at least 75% identical to ENFIN5 and wherein said functional homologue or fragment is capable of binding NCAM and modulating NCAM signaling.

39. The peptide according to claim 31 , containing the sequence: Ala-Gly-Gln-Ala-Gly-Arg-Ala-Phe-lle-Phe-Phe (ENFIN5)

40. The peptide according to any of claims 31 to 39, wherein said peptide consists of 1 1 to 20 amino acid residues, preferably 1 1 to 18 amino acid residues, for example 1 1 to 15 amino acid residues.

41 . A pharmaceutical composition comprising the compound according to any of claims 1 to 30 and/or the peptides according to any of claims 31 to 40.

42. The pharmaceutical composition according to claim 41 , wherein the compounds are formulated as multimers.

43. The pharmaceutical composition according to claim 42, wherein the compounds are formulated as dendrimers, such as four peptides linked to a lysine backbone, or coupled to a protein carrier such as BSA.

44. The pharmaceutical composition according to any of the claims 41 to 43 formulated for oral, percutaneous, intramuscular, intracranial, intraventricular, intranasal or pulmonal administration.

45. The pharmaceutical composition according to any of the claims 41 to 44 wherein the pharmaceutical composition comprises an effective amount of one or more of the compounds according to any of the claims 1 to 26 and one or more pharmaceutically acceptable additives or carriers.

46. A use of the compound according to any of claims 1 to 26 and/or the peptide according to any of the claims 31 to 40 for the manufacture of a medicament for the treatment of diseases or conditions of the central or peripheral nervous system, or of the muscles or of various organs.

47. A use of the compound according to any of claims 1 to 26 for the manufacture of a medicament for the stimulation of learning and memory.

48. The use according to claim 46, wherein the medicament for treatment of a condition or disease wherein stimulating neural cell differentiation, neural cell survival, neurogenesis, stem cell proliferation, stem cell differentiation, learning and memory, and/or modulating activity of NCAM is beneficial for said treatment.

49. The use according to claim 48, comprising the stimulation of differentiation of N-CAM presenting cells.

50. The use according to claim 46, wherein said compound is for the treatment of diseases or conditions of the central and peripheral nervous system, such as postoperative nerve damage, traumatic nerve damage, impaired myelination of nerve fibers, post-ischaemic damage, e.g. resulting from a stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, dementias such as multi-infarct 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; for treatment of diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, such as after organ transplantation, 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.

51 . The use according to claim 46, comprising treatment of postoperative nerve damage, traumatic nerve damage, impaired myelination of nerve fibers, post- ischaemic, e.g. resulting from a stroke, Parkinson's disease, Alzheimer's disease, dementias such as multi-infarct 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.

52. The use according to claim 46, wherein the compound and/or pharmaceutical composition is for the promotion of wound-healing.

53. A use of the compound according to any of claims 1 to 26 and/or the peptide according to any of the claims 31 to 40 for the manufacture of a medicament for the treatment of cancer.

54. The use according to claim 46, wherein the compound and/or pharmaceutical composition is for the treatment of a neuroendocrine tumor.

55. The use according to claim 46, wherein the compound and/or pharmaceutical composition is for preventing cell death of heart muscle cells, such as after acute myocardial infarction, or after angiogenesis.

56. The use according to claim 46, wherein the compound and/or pharmaceutical composition is for revascularisation.

57. An antibody capable of binding to an epitope comprising a sequence corresponding to a binding site on a fibronectin type 3 module of NCAM or a fragment thereof or a variant.

58. An antibody capable of binding to an epitope comprising at least one of the following sequences:

Ala-Arg-Trp-Ser-Lys-Gly-Phe-Asp-Gln-Trp-Met (ENFIN1 1 ) (SEQ ID NO:1 ) Ala-Phe-Tyr-Arg-Thr-lle-Gln-Trp-Thr-Met-Glu (ENFIN2) (SEQ ID NO:2) Ala-Phe-Tyr-Arg-Leu-Val-Phe-Asn-Gln-Asp-Thr (ENFIN3) (SEQ ID NO:3) Ala-Gly-Gln-Ala-Gly-Arg-Ala-Phe-lle-Phe-Phe (ENFIN5) (SEQ ID NO:4)

59. An antibody according to claim 57, wherein said antibody are capable of modulating biological activity mediated by NCAM.

60. Use of an antibody according to claims 57-59 for the manufacture of a medicament for treatment of conditions as defined in claims 46-56.

61 . A pharmaceutical composition comprising an antibody according to any of claims 57-59. 