WO2008086813A2 - Peptides dérivés de protéines de la superfamille de l'insuline - Google Patents

Peptides dérivés de protéines de la superfamille de l'insuline Download PDF

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WO2008086813A2
WO2008086813A2 PCT/DK2008/050009 DK2008050009W WO2008086813A2 WO 2008086813 A2 WO2008086813 A2 WO 2008086813A2 DK 2008050009 W DK2008050009 W DK 2008050009W WO 2008086813 A2 WO2008086813 A2 WO 2008086813A2
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Prior art keywords
peptide
peptide according
amino acid
seq
insulin
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PCT/DK2008/050009
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WO2008086813A3 (fr
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Elisabeth Bock
Vladimir Berezin
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Kobenhavns Universitet
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to peptides derived from members of the insulin super- family.
  • the insulin super-family and its receptors are the insulin super-family and its receptors.
  • the insulin super-family consists of a group of small, structurally related proteins, such as insulin, insulin-like growth factors (IGFs) and relaxins. Insulin signaling molecules are not confined to vertebrates but have also been identified in invertebrates such as nematodes, mollusks and insects, and even in unicellular eukaryotes. The proteins initiate an evolutionary conserved signal transduction pathway. The overall degree of high structural conservation yields a recognizable 'finger-print' of the members of the insulin super-family.
  • the primary peptide sequence of each member of the family is characterized by three domains comprised of an N-terminal B peptide (or chain) joined to a C-terminal A peptide by an intervening C peptide.
  • a member of the insulin super-family comprises this B-C-A element.
  • the B and A chain are relatively invariant and exhibit a pattern of distinct and highly conserved cysteine motifs.
  • cysteine motifs characterize the family; specifically the motif (C-C-X 1 - S -C-X 1 - S -C) present in the A peptide has been termed the insulin signature (Kasik JW et al. 2000 Pediatric Diabetes 1 :169-177).
  • the family comprises insulin, IGF1 and -2, INSL(insulin- like protein)-3, -4, -5, -6, and -7, as well as relaxin, relaxin-1 , -2, and -3, and IGFL (insulin growth factor-like protein)-1 , -2, -3, -4, -5, and -6.
  • the archetypical receptors of the insulin super-family of proteins are the structurally similar receptor tyrosine kinases insulin receptor (IR) and IGF1 receptor (IGF1 R). In line with the high structural similarity of the ligands IGF1 , IGF2 and insulin, there is significant similarity between the IGF1 R and the IR, which results in overlapping functions of the receptors.
  • Insulin, IGF1 and IGF2 all bind to the IGF1 receptor and activate the intracellular tyrosine kinase activity. This yields a variety of responses such as proliferation, differentiation, inhibition of apoptosis and migration.
  • the IR exists in two isoforms, IR-A and IR-B, differing in the number of amino acids of the ⁇ -subunit due to alternative splicing of exon 11 of the IR gene.
  • the IR-B isoform which includes exon 1 1 , has low affinty for the IGFs and high affinity for insulin, which induces metabolic responses upon binding to the receptor.
  • the resulting IR-A isoform has high affinity for insulin as well as for IGF2, and that activation of the IR-A leads to mitogenic responses similar to those of lGFI R.
  • the IGF1 R and IR are expressed throughout the CNS. There is a great overlap in the expression of the two receptors such as in the olfactory bulbs, cerebellar cortex and hippocampal formation. However, there are also specific cell populations with selective enrichment for IR or IGF1 R expression.
  • the anterior thalamic and hypothalamic nuclei are enriched for IR, whereas the suprachiasmatic nucleus of the hypothalamus and the dorsal thalamic sensory nuclei selectively express the IGF1 R.
  • the receptors are expressed in both fetal and adult brains, and their expression pattern does not vary during development.
  • the IGF2 receptor also known as the cation-independent mannose-6- phosphate receptor, binds IGF2 and proteins containing mannose-6-phosphate such as proliferin, transforming growth factor ⁇ (TGN ⁇ ) and renin.
  • the IGF2R does not have an intrinsic signaling domain. Instead, its major function is to clear circulating IGF2 and thereby modulate the availability of the ligand to the IGF1 R and IR. This is achieved by sequestering the ligands, internalization of the ligand receptor complex and final degradation of the ligands.
  • the IGF2R is expressed in the frontal cortex, hippocampus and cerebellum of fetal and adult human brains.
  • GPCRs G-protein coupled receptors
  • LGRs Leucine-rich repeat-containing G-protein coupled receptors
  • IGF1 and IGF2 Proteins of the insulin super-family in the nervous system.
  • the IGF1 and IGF2 have growth promoting effects as well as insulin-like metabolic activities, most of which are mediated through the IGF1 R.
  • IGF1 is described as a neuronal survival factor, and it has been demonstrated that IGF1 and IGF2 play a significant role in brain development.
  • IGF1 The neurotrophic effects of IGF1 have been further characterized as promoting proliferation and differentiation of developing neuronal cells. Furthermore, in vitro and in vivo studies have shown that IGF1 promotes survival and prevents apopto- sis in neuronal cells. IGF1 is also an important factor for the survival, development and myelination of oligodendrocytes.
  • IGF2 The role of IGF2 in postnatal brain development is less understood, but it has been proposed that IGF2 plays a role in differentiation and proliferation in the CNS. In cultured neuroblastoma, sensory, sympathetic and motor neurons IGF2 has been demonstrated to support neurite growth. Similarly to IGF1 , IGF2 is associated with oligodendrocytes and myelin, which implies its role in myelination. Many neurodegenerative disorders are related to a change of IGF1 levels in serum and brain.
  • IGF1 R signaling which is most likely due to desensitization of nerve cells to IGF1 .
  • the signaling stimulated by IGF1 , IGF2 and insulin is also impaired in Alzheimer's disease (AD) and it has been suggested that this abnormality is the underlying basis of the disease.
  • AD Alzheimer's disease
  • the neurodegeneration is mediated by depletion of IGF/insulin leading to increased levels of GSK-3, yielding neuronal oxidative stress and cell death. Consequently, for patients with neurodegenerative disorders caused by malfunction in IGF1 signaling, the therapeutic rescue could be provided by IGF1 , IGF2 and insulin sensitizers and/or mimicks, to enhance the neuronal survival and reduce the oxidative stress.
  • the present invention concerns peptides derived from proteins of the insulin super- family, which preferably bind and modify the activation status of receptors of proteins of the insulin super-family.
  • the invention concerns a peptide derived from a protein of the insulin super-family, or a variant or fragment thereof, wherein said peptide is not more than 16 amino acids and capable of binding a receptor of a protein of the insulin super-family.
  • a peptide of the invention comprises a motif selected from:
  • the C-terminal amino acid of a compound of the invention may be the amidated derivative, which is indicated as “-NH 2 ". Where nothing else is stated the N- terminal amino acid of a polypeptide comprises a free amino-group, this may also be specified as "H-”.
  • an amino acid can be selected from any amino acid, whether naturally occurring or not, such as alfa amino acids, beta amino acids, and/or gamma amino acids. Accordingly, the group comprises but are not limited to: 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.
  • modifications of the compounds/peptides may be performed, such as for example glycosylation and/or acetylation of the amino acids.
  • the invention relates to naturally occurring, synthetically/recombinantly prepared peptide sequence/fragments, and/or peptide sequence/fragments prepared by means of enzymatic/chemical cleavage of a bigger polypeptide, wherein said peptide sequence/fragments are integral parts of said bigger polypeptides.
  • the invention relates to isolated individual peptide sequences.
  • the present invention relates to a peptide derived from a protein of the insulin super-family, or a variant or fragment thereof, wherein said peptide is not more than 16 amino acid residues and capable of binding a receptor of a protein of the insulin super-family.
  • a peptide according to the invention can be derived from any member of the insulin super-family, such as the proteins characterized by distinct structural features as described by (Kasik JW et al., 2000, Pediatric Diabetes 1 :169- 177) which is hereby incorporated in its entirety by reference. Without being bound by theory, it is envisioned that a member of the insulin super-family contains within the primary sequence a three-domain moiety consisting of an A, B and C chain/peptides as disclosed by (Lu C et al., 2005, Pediatric Research 57:70-73). From the N-terminal the B-chain is linked to the C-chain which is further joined to the A-chain.
  • a protein of the insulin super-family is selected from the group consisting of insulin, insulin-like growth factors (IGFs), IGF-like proteins (IGFLs), insulin-like proteins (INSLs) and relaxins.
  • IGFs insulin-like growth factors
  • IGFLs IGF-like proteins
  • INSLs insulin-like proteins
  • insulin IGF1 , IGF2, Relaxin, Relaxin 1 (RLN1 ), Relaxin 2 (RLN2), Relaxin 3 (RLN3), insulin-like protein (INSL), insulin-like protein 4 (INSL4), insulin-like protein 5 (INSL5), insulin-like protein 6 (INSL6), insulin growth factor-like protein 1 (IGFL1 ), insulin growth factor-like protein 2 (IGFL2), insulin growth factor-like protein 3 (IGFL3), insulin growth factor-like protein 4 (IGFL4), insulin growth factor-like protein 5 (IGFL5), insulin growth factor-like protein 6 (IGFL6) or homologues thereof, insulin-like 3 peptide (Leydig insulin-like peptide, INSL3, P51460), early placenta insulin-like peptide (EPIL, Q14641), insulin- like peptide (INSL5, Q9Y5Q6).
  • a peptide of the invention is derived from a protein selected from the group consisting of insulin, IGF1 , IGF2, insulin growth factor-like protein 1 (IGFL1 ), insulin growth factor-like protein 2 (IGFL2), insulin growth factor-like protein 3 (IGFL3), insulin growth factor-like protein 4 (IGFL4), insulin growth factor-like protein 6 (IGFL6), Relaxin 1 (RLN1 ), Relaxin 2 (RLN2), Relaxin 3 (RLN3). More preferably, the peptide of the invention is derived from a protein selected from the group consisting of insulin, IGF1 and IGF2.
  • a peptide according to the invention is capable of binding to a receptor of a protein selected from the group consisting of insulin, insulin-like growth factors (IGFs), IGF-like proteins (IGFLs) and relaxins.
  • insulin IGF1 , IGF2, Relaxin, Relaxin 1 (RLN1 ), Relaxin 2 (RLN2), Relaxin 3 (RLN3), insulin-like protein (INSL), insulin-like protein 4 (INSL4), insulin-like protein 5 (INSL5), insulin-like protein 6 (INSL6), insulin growth factor-like protein 1 (IGFL1 ), insulin growth factor-like protein 2 (IGFL2), insulin growth factor-like protein 3 (IGFL3), insulin growth factor-like protein 4 (IGFL4), insulin growth factor-like protein 5 (IGFL5), insulin growth factor-like protein 6 (IGFL6) or homologues thereof.
  • said receptor is selected from the group consisting of the insulin receptor, IGF receptor 1 and 2 (IGFR1 and IGFR2), GPCR135, GPCR142, LGR7 and LGR8.
  • said receptors are selected from the group consisting of the insulin receptor, IGFR1 and IGFR2.
  • said receptor is selected from the group of insulin and IGF receptors belonging to the family of receptor tyrosine kinases.
  • the ligand and receptors can be selected from any species where homologues of said proteins and receptors can be identified.
  • said species is selected from metazoans, and more preferably from mammals.
  • the proteins and receptors are selected from a species in which they are predicted to function in a manner similar to their function in humans.
  • Preferred species include human, mouse, rat, pig, monkey, ape, dog, cow and horse.
  • the proteins and receptors are of human origin.
  • a peptide according to the present invention preferably binds to a receptor of a protein of the insulin super-family. 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 receptors of IGFs/insulin and peptides by means of surface plasmon resonance (SPR). This is a real-time analysis, and the output reveals information on the dissociation constant (Kd) and association constant (Ka) of the interactions measured.
  • SPR surface plasmon resonance
  • a dissociation constant (Kd) is commonly used to describe how tightly a ligand binds to a receptor.
  • Said receptor being for example a polypeptide.
  • binding is usually non- covalent, and is usually best described as a two-state equilibrium:
  • P polypeptide/receptor
  • L ligand
  • C bound complex of L and P.
  • the corresponding dissociation constant can be defined as:
  • M mol/L
  • a peptide according to the present invention interacts with a receptor of a protein of the insulin super-family with a Kd of less than 15 mM, for example less than 10 mM, such as less than 5 mM, for example less than 1 mM, such as less than 750 ⁇ M, for example less than 500 ⁇ M, such as less than 250 ⁇ M, for example less than 150 ⁇ M, such as less than 100 ⁇ M, for example less than 50 ⁇ M, such as less than 25 ⁇ M, for example less than 15 ⁇ M, such as less than 10 ⁇ M, for example less than 5 ⁇ M, for example less than 1 ⁇ M, such as less than 100 nM.
  • said Kd is measured by SPR.
  • said Kd is measured for a dendrimer.
  • a dendrimer of a peptide according to the invention for example, a dendrimer of a peptide according to the invention.
  • the peptides of the invention are identified from the analysis method outlined above, and described in Example 2.
  • the interaction between a peptide and a receptor of a protein of the insulin super-family 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).
  • NMR nuclear magnetic resonance
  • FRET or BRET bio luminescence resonance energy transfer
  • RIA radio immunoassay
  • ELISA cross-linking
  • confocal microscopy peptide arrays, peptide pull-down, proteinchips, antibody microarrays, multiple photon detection (MPD), mass spectrometry (MS).
  • the effect of peptides of the present invention on the activation status of signaling events initiated by a receptor of a protein of the insulin super-family can be assessed by analyzing receptor activation and/or any downstream event in any cell type.
  • a receptor of a protein of the insulin super-family Preferably said cell expresses a receptor of a protein of the insulin super-family.
  • Said downstream event includes differentiation, proliferation, motility and/or survival of a neuronal, neuronal-like or non-neuronal cell.
  • a peptide of the present invention may modulate signaling at the level of one or more of the proteins and/or second messengers of a signaling cascade initiated by a receptor of a protein of the insulin super-family.
  • peptides of the present invention activate a receptor of a protein of the insulin super-family and/or the intracellular signaling initiated by said receptor.
  • a peptide according to the present invention may induce phosphorylation of the receptor to which it is bound.
  • a peptide according to the present invention functions as a partial agonist, inverse agonist, antagonist and/or inhibitor of a receptor of a protein of the insulin super-family.
  • a peptide according to the invention is capable of promoting neurite outgrowth from neuronal or neuronal-like cells.
  • said cells express a receptor of a protein of the insulin super-family.
  • a neuronal-like cell of the invention includes, but is not confined to, the PC12 cell line.
  • Primary neuronal cells include, but are not confined to, cerebellar granule neurons, hippocampal, midbrain (dopaminergic), and cortex neurons.
  • a peptide according to the invention is capable of preventing cell death of cells presenting a receptor of a protein of the insulin super-family.
  • said cell is a cell of the nervous system, such as a neuron, a glial cell and/or a precursor of a neuron or glial cell.
  • a peptide according to the invention is capable of inducing cell death.
  • a single peptide according to the invention consists of a contiguous amino acid sequence of not more than 16 amino acid residues, preferably it is 6 to 16 amino acid residues, such as 7 to 16 amino acid residues, for example 8 to 16 amino acid residues, such as 9 to 16 amino acid residues, for example 10 to 16 amino acid residues, such as 1 1 to 16 amino acid residues.
  • a peptide of 10, 11 , 12, 13, 14, 15 or 16 amino acid residues is also within the scope of the present invention.
  • a variant in the present context is defined as a contiguous amino acid sequence of not more than 16 amino acid residues, which has at least 50% sequence identity with a sequence selected from the sequences of SEQ ID NOs:1 to 34, preferably, more then 50% sequence identity with a sequence selected from sequences of SEQ ID NOs:1 to 34, such as for example from 51 % to 60%, preferably more then 60%, for example between 61 % and 70%, more preferably more than 70% sequence identity, such as from 71 % to 80%, more preferred a sequence identity of more than 80%, such as from 85% to 90%, such as about 90%, even more preferred sequence identity of 95% or higher, such as about 99% and most preferred more than 99% identical to a sequence selected from the sequences of SEQ ID NOs:1 to 34.
  • a variant of a sequence selected from SEQ ID NOs: 1 to 34 may be a sequence of SEQ ID NOs:1 to 34 which comprises modifications of amino acid residues discussed below.
  • Other variants of peptide sequences of the invention concerned are also discussed below. Sequence identity is calculated relative to the full sequence length of the original peptide, i.e. the relevant sequence from SEQ ID NOs 1 to 34.
  • Sequence identity and similarity may be calculated using well known tools such as BLAST, LALIGN and FASTA, by the application of any suitable substitution matrices such as a BLOSUM, PAM or identity matrix scoring sequences against a sequence of the invention, such as SEQ ID NO 1 to 34.
  • Sequence identity calculations should include alignment of putative low complexity regions. Gaps can be allowed, using a gap opening penalty score of 8 or more, such as 9 or higher, such as 10, for example 1 1 , such as 12, for example 14, such as 15, for example 16 or higher.
  • a gap opening penalty score 8 or more, such as 9 or higher, such as 10, for example 1 1 , such as 12, for example 14, such as 15, for example 16 or higher.
  • an alignment does not contain a gap of more than 2 residues, more preferably an alignment does not contain a gap of more than 1 residue, most preferably an alignments does not contain a gap.
  • a variant in the present context also encompass a contiguous amino acid sequence of not more than 16 amino acid residues, which has at least 50% sequence similarity with a sequence selected from the sequences of SEQ ID NOs:1 to 34, preferably, more then 50% sequence similarity with a sequence selected from sequences of SEQ ID NOs:1 to 34, such as for example from 51% to 60%, preferably more then 60%, for example between 61% and 70%, more preferably more than 70% sequence similarity, such as from 71% to 80%, more preferred sequence similarity of more than 80%, such as from 85% to 90%, preferably 90% or higher, and even more preferred sequence similarity of 95% or higher, such as about 99% similar to a sequence selected from the group consisting of SEQ ID NOs:1 to 34.
  • Similar amino acid residues are amino acid residues derived from the same group of conservative amino acid residues.
  • a group of similar amino acids is thus a selection of amino acids exhibiting similarity of at least one physico-chemical feature, such as, but not limited to, size, charge, polarity, aromaticity, hydrophobicity, or hydrophilicity.
  • a Venn diagram which represents common knowledge to a person skilled in the art, defines non-exclusive groups of shared physico-chemical properties of the 20 naturally occurring amino acids.
  • a substitution of one amino acid to another similar amino acid constitutes a 'conservative substitution'. For example, it is within the scope of the present invention that substitutions within the groupings of the Wenn diagram can be considered conservative substitutions.
  • Figure 1 and Table 2 depicts groups of similar amino acids and preferred conservative amino acid substitutions, respectively.
  • Figure 1 shows a Wenn diagram with groups of amino acids with shared physico-chemical properties.
  • Substitution of amino acids may in one embodiment be made based upon their hydrophobicity, hydropathicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like.
  • conservative substitutions are not based on physico-chemical properties per se, but on 'natural selection', such as that disclosed by the PAM or BLOSUM substitution matrices.
  • the substition matrices contain values for the propensity of particular amino acid substitutions in closely related biological amino acid sequences.
  • a conservative substition can, in one embodiment, be assigned to substitutions in a BLOSUM matrix with a score of 0 or higher, preferably a score higher than 0
  • BLOSUM62 or BLOSUM80 is used for said purpose
  • the BLOSUM62 matrix is depicted in Table 3
  • Conservative substitutions may be introduced at any position of a preferred predetermined peptide of the invention or fragment thereof It may, however, also be desirable to introduce non-conservative substitutions, particularly, but not limited to, a non-conservative substitution at any one or more positions
  • a non-conservative substitution leading to the formation of a functionally equivalent fragment of the peptide of the invention would for example i) differ substantially in polarity, for example a residue with a non-polar side chain, such as Ala, Leu, Pro, Trp, VaI, lie, Leu, Phe or Met, substituted for a residue with a polar side chain such as GIy, Ser, Thr, Cys, Tyr, Asn, or GIn or a charged amino acid such as Asp, GIu, Arg, or Lys; or substituting a charged or a polar residue for a non-polar one; and/or ii) differ substantially in its effect on peptide backbone orientation such as substitution of or for Pro or GIy by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as GIu or Asp for a positively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ substantially in ster
  • PTM post-translational modifications
  • PTMs are known to a person skilled in the art. Common PTMs include, but are not limited to, phosphorylation (primarily on residues S 1 T or Y), lipid modifications, prenylation, farnesylation, palmitoylation, acetylation, glycation, glycosylates, sulfation, SUMOylation, biotinylation, myristoylation, GPI-addition a.s.o.
  • the present invention also concerns fragments of the peptide sequences described above and below.
  • the following fragments and variants are preferred by the invention.
  • a fragment contains at least 6 amino acid residues.
  • the fragment comprises a motif such as disclosed herein below.
  • the preferred fragments and variants of the invention are functional equivalents of sequences identified as SEQ ID NOs:1 to 34, which means that a fragment or variant shares a function with the original peptide sequence from which they are derived.
  • a fragment or variant is capable of binding the same receptor protein as the original peptide sequence from which it is derived. It is also preferred that a fragment or variant of the invention is capable of modifying the activation status of said receptor to a similar or higher level as the peptide from which it is derived. It is also preferred that a fragment or variant of the invention is capable of promoting neurite outgrowth. In another preferred embodiment a fragment or variant of the invention is capable of preventing cell death.
  • a fragment or variant of the invention has the capability of stimulating neural plasticity, such as associated with neural cell differentiation and/or such as associated with memory and learning. 4 Peptide sequences
  • a peptide according to the invention comprises an amino acid motif of the formula: X1 -X2-X3 m -X4-X5-X6-C-G-X7-X8-X9-X10-X1 1 (Motif: M1 ),
  • the invention concerns a peptide comprising motif M1 , wherein said peptide does not comprise more than 13 amino acid residues.
  • the invention concerns a peptide comprising motif M1 , wherein X2 is selected from the group of amino acids consisting of P, V, N, A and G. More preferably, X2 is P.
  • the invention concerns a peptide comprising motif M1 , wherein X5 is selected from the group of amino acids consisting of T, H, K, R, and P. More preferably, X5 is T.
  • the invention concerns a peptide comprising motif M1 , wherein X8 is selected from the group of amino acids consisting of E, H, Y, K and W. More preferably, X8 is E.
  • the invention concerns a peptide comprising motif M1 , wherein X9 is selected from the group of amino acids consisting of L, I, F and T. More preferably, X9 is L or I.
  • the invention concerns a peptide comprising motif M1 , wherein X10 is selected from the group of amino acids consisting of V, I and Y. More preferably, X10 is V. In another preferred embodiment, the invention concerns a peptide comprising motif M1 , wherein X1 1 is an acidic or basic amino acid. More preferably, X1 1 is D or E.
  • the invention concerns a peptide comprising motif M1 , wherein the peptide contains or consists of a sequence selected from SEQ ID NOs 1 to 1 1 or variants or fragments hereof.
  • the invention concerns a peptide comprising motif M1 , wherein the peptide contains or consists of a sequence selected from SEQ ID NOs 1 to 1 1 .
  • the invention concerns a peptide comprising motif M1 , wherein the peptide contains or consists of the sequence: R-P-S-E-T-L-C-G-G-E-L-V-D (SEQ ID NO 1 ), or a variant, fragment or homologue thereof.
  • the invention concerns a peptide comprising motif M1 , wherein the peptide sequence is R-P-S-E-T-L-C-G-G-E-L-V-D (SEQ ID NO 1 ).
  • a peptide according to the invention comprises an amino acid motif of the formula:
  • X1 m or X5 n is not present in the peptide.
  • the invention concerns a peptide comprising motif M2, wherein said motif is additionally characterized according to the formula:
  • X1 1 to X15 can independently be any amino acid, and wherein X12 0 is optional; integer o is 0 or 1 , when integer o is 1 X12 0 can be any amino acid, when integer o is 0 X12 is not present in the peptide, wherein X4 is amino acid G or C.
  • the invention concerns a peptide comprising motif M3, wherein X7 is selected from the group of amino acids consisting of G, S, D and A. More preferably, X7 is G.
  • the invention concerns a peptide comprising motif M3, wherein X9 is a hydrophobic or aromatic amino acid. More preferably, X9 is selected from the group of amino acids consisting of F, W and Y.
  • the invention concerns a peptide comprising motif M3, wherein X8, X9 and X10 are all aromatic amino acids independently selected from the group consisting of F, W and Y.
  • the invention concerns a peptide comprising motif M3, wherein one or more of X13, X14 and X15 is S or T.
  • the invention concerns a peptide comprising motif M3, wherein X3 is V, X5 n is acidic, X6 is basic, X7 is G, X8 is F, X9 and X10 are aromatic and X13 is P.
  • the invention concerns a peptide comprising motif M3, wherein the peptide contains or consists of a sequence selected from SEQ ID NOs 12 to 22 or fragments or variants hereof.
  • the invention concerns a peptide comprising motif M3, wherein the peptide contains or consists of a sequence selected from SEQ ID NOs 12 to 22.
  • the invention concerns a peptide comprising motif M3, wherein the peptide contains or consists of the sequence:
  • the invention concerns a peptide comprising motif M3, wherein the peptide sequence is
  • the present invention concerns a peptide comprising motif M2, wherein m is 1 and n is 1 , and wherein X1 is acidic, X3 is aromatic and X4 is A.
  • X1 is E and X3 is Y.
  • said peptide comprises the sequence: A-K-S.
  • the present invention concerns a peptide comprising motif
  • the present invention concerns a peptide comprising motif M2, wherein the peptide contains or consists of a sequence selected from SEQ ID NOs 33 to 34.
  • the present invention concerns a peptide comprising motif M2, wherein the peptide contains or consists of the sequence:
  • E-T-Y-C-A-T-R-A-K-S-E (SEQ ID NO 33) or a variant, fragment or homologue thereof.
  • the present invention concerns a peptide comprising motif M2, wherein the peptide sequence is
  • the present invention concerns a peptide comprising motif M2, wherein m is 1 and n is 1 , and wherein X8 is a hydrophobic amino acid.
  • X8 is L or I. More preferably, X8 is L or I, and X5 is L or K. Even more preferably, X8 is L or l, X5 is L or K and X9 is E.
  • the present invention concerns a peptide comprising motif M2, wherein the peptide contains or consists of a sequence selected from SEQ ID NOs 26 to 32 or variants or fragments hereof.
  • the present invention concerns a peptide comprising motif M2, wherein the peptide contains or consists of a sequence selected from SEQ ID NOs 26 to 32.
  • the present invention concerns a peptide comprising motif M2, wherein the peptide contains or consists of the sequence:
  • T-S-I-C-S-L-Y-Q-L-E-N-Y-C-N SEQ ID NO 28
  • SEQ ID NO 28 or a variant, fragment or homologue thereof.
  • the present invention concerns a peptide comprising motif M2, wherein the peptide sequence is
  • a peptide according to the invention comprises an amino acid motif of the formula:
  • X4 m or X5 n is not present in the peptide, and wherein X a is an acidic amino acid.
  • the present invention concerns a peptide comprising motif
  • the present invention concerns a peptide comprising motif M4, wherein m is 1 and n is 1 .
  • the present invention concerns a peptide comprising motif M4, wherein the peptide contains or consists of a sequence selected from SEQ ID NOs 23 to 25 or variants or fragments hereof.
  • the present invention concerns a peptide comprising motif M4, wherein the peptide contains or consists of a sequence selected from SEQ ID NOs 23 to 25.
  • the invention also relates to a pharmaceutical composition containing a compound comprising one or more of the peptides and/or fragments and/or variants as defined above.
  • a compound of the invention is preferably formulated as a dimer or multimer such as disclosed herein below.
  • 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.
  • a compound may contain a single copy of an individual peptide of the invention selected from any of the peptides and peptide sequences described herein above, or it may contain two or more copies of such a peptide.
  • a compound of the invention may be formulated as a monomer of said peptide sequence, such as containing a single individual peptide sequence, or it may be formulated as a multimer of a peptide sequence, i.e containing two or more individual peptide sequences.
  • a compound may be formulated as a multimer comprising two or more different peptides according to the invention, such as two or more of the peptide sequences SEQ ID NO 1 to 34.
  • a multimer may also comprise a combination of the full-length sequence and one or more fragments thereof.
  • a compound may contain two or more amino acid sequences, such compound is defined herein as a multimer.
  • a compound may be dimer and contain two amino acid sequences.
  • a compound may contain two different amino acid sequences, and is thus defined herein as a heterodimer.
  • a compound may contain more then two amino acid sequences, such for example three, four or more sequences.
  • said multimers are heteromultimers, such as comprising two or more different sequences of the invention.
  • the compounds may be formulated as dimers or multimers comprising more than two copies of individual peptide fragments which may have the identical amino acid sequences or different amino acid sequences.
  • One example of such compound may be a heterodimeric compound containing SEQ ID NO 3 and SEQ ID NO 28 or SEQ ID NO 25 and SEQ ID NO 14 or SEQ ID 25 and SEQ ID NO 3 or SEQ ID NO 28 and SEQ ID NO 14 or SEQ ID NO 2 and SEQ ID NO 26 or SEQ ID NO 23 and SEQ ID NO 13 or SEQ ID NO 23 and SEQ ID NO 2 or SEQ ID NO 26 and SEQ ID NO 2.
  • sequences of the invention may be connected to each other via peptide bond, or connected to each other through a linker molecule or grouping.
  • a compound of the invention may contain two or more copies of a single sequence, such as for example two copies of any of the sequences selected from SEQ ID NOs: 1 -34, wherein said two sequences may be connected to each other via a linker molecule or grouping.
  • a compound wherein the sequences are connected via a linker grouping is preferred.
  • One example of such linking grouping may be an achiral di-, tri- or tetracarboxylic acid. Suitable achiral di-, tri- or tetracarboxylic acids and a method of production such a compound (a ligand presentation assembly method (LPA)) are described in WO0018791 and WO2005014623.
  • Another example of a possible linker may be the amino acid lysine.
  • Individual peptide sequences may be attached to a core molecule such as lysine forming thereby a dendritic multimer (dendrimer) of an individual peptide sequence(s).
  • dendrimer dendritic multimer
  • Production of dendrimers is also well known in the art (PCT/US90/02039, Lu et al., (1991 ) MoI Immunol. 28:623-630; Defoort et al., (1992) lnt J Pept Prot Res. 40:214-221 ; Drijfhout et al. (1991 ) lnt J Pept Prot Res.
  • a dendrimeric compound comprising four individual amino acid sequences attached to the lysine core molecule. It is also preferred that at least one of the four individual amino acid sequences comprises an amino acid sequence comprising a motif as defined above, preferably said sequence is a peptide selected from SEQ ID NO 1 to 34 or a fragment or variant hereof. It is even more preferred that two or more, more preferably all four, individual peptide sequences of a dendrimeric compound are peptides according to the present invention.
  • Multimeric compounds of the invention such as LPA-dimers or Lysin-dendrimers, are preferred compounds of the invention.
  • other types of multimeric compounds comprising two or more individual sequences of the invention may be preferred depending on the embodiments.
  • a medicament of the invention may suitably be formulated for oral, percutaneous, parenteral, intramuscular, intravenous, intracranial, intrathecal, intracerebroventricular, intranasal or pulmonal administration to a subject in need hereof.
  • 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 defined above in combination with pharmaceutically acceptable additives and optionally one or more carriers.
  • compositions comprising one or more of the compounds described above administered in vitro or in vivo in an effective amount.
  • 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.
  • the administration may be continuous or in small portions based upon controlled release of the active compound(s).
  • precursors may be used to control the rate of release and/or site of release.
  • Other kinds of implants as 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.
  • 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.
  • 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.
  • virus reduction is a reduction of any virus contaminants.
  • the preparation further comprises pharmaceutically acceptable additives and/or carriers.
  • additives and carriers will be known in the art.
  • Formulations of the compounds of the invention can be prepared by techniques known to the person skilled in the art.
  • the formulations may contain pharmaceutically acceptable carriers and excipients including microspheres, liposomes, microcapsules, nanoparticles or the like.
  • the preparation may suitably be administered by injection, optionally at the site, where the active ingredient is to exert its effect.
  • Additional formulations which are suitable for other modes of administration include suppositories, nasal, pulmonal and, in some cases, oral formulations.
  • traditional binders and carriers include polyalkylene glycols or triglycerides.
  • Such suppositories may be formed from mixtures containing the active ingredient(s) in the range of from 0.5% to 10%, preferably 1 -2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and generally contain 10-95% of the active ingredient(s), preferably 25-70%.
  • formulations are such suitable for nasal and pulmonal administration, e.g. inhalators and aerosols.
  • the active compound may be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include acid addition salts (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 ⁇ g active ingredient per administration with a preferred range of from about 0.1 ⁇ g to 100 mg, such as in the range of from about 1 ⁇ g to 100 mg, and especially in the range of from about 10 ⁇ g 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 according to paramenters such as 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.
  • 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.
  • 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.
  • the medicament is administered within 8 hours from the factor onset, such as within 5 hours from the factor onset.
  • Many of the compounds exhibit a long term effect whereby administration of the compounds may be conducted with long intervals, such as 1 week or 2 weeks.
  • the administration 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.
  • an acute injury such as an acute stroke
  • the administration according to the invention is not time dependent, i.e. it may be administered at any time.
  • the compound(s) according to the invention may be advantageous to administer with other substances to obtain a synergistic effect.
  • 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 compounds may be synthesised and secreted from implanted or injected gene manipulated cells.
  • the present invention also relates to a pharmaceutical composition capable of promoting cell differentiation and/or modulating neuronal plasticity and/or proliferation of neural cells, and/or modulating survival and/or regeneration of cells expressing a receptor of a protein of the insulin super-family.
  • 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.
  • peptides, compounds and compositions may be used to treat conditions affecting the peripheral and/or the central nervous system and/or muscles and other tissues.
  • said tissue expresses receptors of one or more proteins of the insulin super-family, such as defined herein above.
  • a compound of the invention may be for the manufacture of a medicament for treatment of normal, degenerated or damaged cells which express receptors of one or more proteins of the insulin super-family, such as defined herein above.
  • compounds according to the invention are useful for the stimulation of regeneration of cells which are degenerated or are at risk of dying due to a variety of factors, such as traumas and injuries, acute diseases, chronic diseases and/or disorders.
  • compounds of the present invention may be useful for the treatment of degenerative diseases normally leading to cell death, other external factors, such as medical and/or surgical treatments and/or diagnostic methods that may cause formation of free radicals or otherwise have cytotoxic effects, such as X- rays and chemotherapy.
  • chemotherapy 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.
  • said compounds can be used to stimulate survival of neurons, glial cells, or precursors of neurons and glial cells.
  • 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.
  • the treatment comprises the use of said compound for diseases or conditions of the central and peripheral nervous system, such as neurological and psychiatric disorders. Such conditions include postoperative nerve damage, traumatic nerve damage, impaired myelination of nerve fibers, postischaemic damage, e.g.
  • Parkinson's disease including other extra pyramidal disease and abnormal movement disorders, spinocerebellar disease, cerebellar ataxia, Marie's Sanger- Brown, Dyssynergia cerebellaris myoclonica, 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, Cerebral degenerations such as Alzheimer's disease, Pick's disease, senile degeneration of brain, senility NOS, communicating hydrocephalus, Obstructive hydrocephalus.
  • the conditions furthermore include 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.
  • 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
  • the invention concerns treatment of diseases in the nervous system and sense organs, such as affecting sight, hearing, smell, felling, tasting.
  • Diseases of the ear and mastoid process and inflammatory disease of the central nervous system, such as meningitis and encephalitis.
  • Further conditions to be treated include 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, and peripheral autonomic neuropathy in disorders classified elsewhere.
  • conditions to be treated include diseases of the peripheral nerve system, brachial plexus lesions, cervical rib syndrome, costoclavicular syndrome, scalenus anticus syndrome, thoracic outlet syndrome, brachial neuritis or radiculitis NOS, including in newborn; and inflammatory and toxic neuropathy, including acute infective polyneuritis, Guillain-Barre syndrome, Postinfectious polyneuritis, polyneuropathy in collagen vascular disease.
  • Further conditions to be treated by compounds or compositions of the invention include cognitive anomalies after disease, injury (e.g.
  • pain syndrome such as non- opioid pain, neuropatic pain, or in pain related to other disorders, (e.g. diabetes or HIV), encephalitis, drug/alcohol abuse, anxiety, perioperative ischemia.
  • psychiatric disorders such as mental retardation, inherited or in relation with disease or trauma, psychoses, such as senile and presenile organic psychotic conditions, alcoholic psychoses, drug psychoses, transient organic psychotic conditions, depression and other mood disorders including manic and bipolar disorders, 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, sleep disorders, and sexual deviations and disorders or dysfunctions (including reduced sexual motivation or capability for what ever reason) may also be treated with compounds and compositions of the present invention.
  • psychoses such as senile and presenile organic psychotic conditions, alcoholic psychoses, drug psychoses, transient organic psychotic conditions, depression and other mood disorders including manic and bipolar disorders, schizophrenic disorders, affective psychoses, neurotic disorders, personality disorders, including character neurosis, nonpsychotic personality disorder associated with organic brain syndromes
  • the present compound may be used in relation to infections disorders such as inflammatory disorders with tissue damage, either by affecting the infections agent or protecting the tissue, disorders of the globe, including disorders affecting multiple structures of eye, such as purulent endophtalmitis.
  • infections disorders such as inflammatory disorders with tissue damage, either by affecting the infections agent or protecting the tissue, disorders of the globe, including disorders affecting multiple structures of eye, such as purulent endophtalmitis.
  • 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, and of the kidneys, such as nephrosis.
  • 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.
  • the compound and/or pharmaceutical composition is for the stimulation of the survival of heart muscle cells, such as survival after acute myocardial infarction.
  • the compound and/or pharmaceutical composition is for revascularisation, such as after injuries.
  • cardiovascular diseases in the pulmonary system, respiratory system, sensoring e.g. oxygen, and asthma.
  • conditions including complications of the administration of anesthetic or other sedation in labor and delivery, and acute myocardial infarction, and other related disorders or sequel from AMI.
  • 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 a receptor of a protein of the insulin super-family is beneficial for treatment of said condition or disease.
  • the compound and/or pharmaceutical composition is used to stimulate neurogenesis, stem cell proliferation and/or stem cell differentiation, maturation and protection in vivo or in vitro.
  • 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.
  • a compound of the invention may be useful for the treatment of autoimmune diseases, such as such as rheumatoid arthritis, SLE, ALS and MS, anti-inflammatory effects, Amyloidosis, Chronic rheumatic heart disease, ischaemic heart disease, arrhythmia, asthma and other allergic reactions.
  • autoimmune diseases such as such as rheumatoid arthritis, SLE, ALS and MS
  • anti-inflammatory effects such as Amyloidosis, Chronic rheumatic heart disease, ischaemic heart disease, arrhythmia, asthma and other allergic reactions.
  • 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
  • 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 disorders affecting the circadian clock or neuro-muscular transmission
  • schizophrenia mood disorders, such as manic depression.
  • the invention further discloses the use of the compound and/or pharmaceutical composition in the treatment of neoplasms, such as benign and malignant neoplasms or tumors.
  • a compound of the invention can be used for the treatment of cancer, such as, but not limited to, carcinoma in situ and neoplasms of uncertain behavior, hematological malignancy, sarcoma or glioma. More specifically cancer in breast, thyroidal, pancreas, brain, lung, kidney, prostate, liver, heart, skin, blood organ (including but not limited to CML and AML), muscles (sarcoma).
  • 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.
  • 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 and colon cancer.
  • 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.
  • 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.
  • a virus induced tumor such as HPV (human papilloma virus) and CMV (cytomegalovirus) induced cancers.
  • 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 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 ALL, such as acute myelogenous leukemia
  • 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.
  • a compound of the invention is used for treatment of Metabolic and endocrine disorders, such as obscenity lipid disorders (e.g. hyper cholestorolamia, artherosclerosis), diseases of endocrine glands, such as diabetes mellitus I and II, pituitary gland tumor; and disorders of amino-acid transport and metabolism, disorders of purine and pyrimidine metabolism and gout.
  • Further disorders include myelopathy, drug-induced, radiation-induced, myelitis, bone disorders, such as fracture osteoporosis, osteo arthritis (OA), and obesity.
  • a compound of the invention is used for treatment of conditions of the skin, such as diseases in the skin including infection, insufficient circulation problem, burn injury and other mechanic and or physical injuries, atopic dermatitis, psoriasis, and infection cased disorders.
  • 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.
  • 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 cells expressing a receptor of a protein of the insulin super-family.
  • the invention also discloses the use, wherein said compound is for the manufacture of a medicament for the treatment comprising the stimulation of differentiation and/or survival of cells expressing a receptor of a protein of the insulin super-family.
  • 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.
  • 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.
  • 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 contains peptide sequences of the invention, or a combination of said methods. It is within the scope of the invention to concern nucleotides encoding peptides of the invention.
  • the invention concerns nucleotides encoding SEQ ID NO 1 to 34 or variants and fragments hereof. More preferably, the invention concerns nucleotides encoding peptides SEQ ID NO 1 to 34 of the invention, preferably SEQ ID NO 1 , 12 and 33.
  • the peptides of the invention are produced by use of recombinant DNA technologies.
  • the DNA sequence encoding a peptide or the corresponding full-length protein the peptide originates from may be prepared synthetically by established standard methods, e.g. the phosphoamidine method described by Beaucage and Caruthers, 1981 , Tetrahedron Lett. 22:1859-1869, or the method described by Matthes et al., 1984, EMBO J. 3:801 -805.
  • oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in suitable vectors.
  • the DNA sequence encoding a peptide may also be prepared by fragmentation of the DNA sequences encoding the corresponding full-length protein of peptide origin, using 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.
  • a recombinant expression vector which may be any vector, which may conveniently be subjected to recombinant DNA procedures.
  • vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the DNA sequence encoding a peptide or a full-length protein should be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the coding DNA sequence in mammalian cells are the SV 40 promoter (Subramani et al., 1981 , MoI. Cell Biol. 1 :854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., 1983, Science 222: 809-814) or the adenovirus 2 major late promoter.
  • a suitable promoter for use in insect cells is the polyhedrin promoter (Vasuvedan et al., 1992, FEBS Lett. 31 1 :7-1 1 ).
  • Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., 1980, J. Biol. Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. MoI. Appl. Gen.
  • Suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., 1985, EMBO J. 4:2093-2099) or the tpiA promoter.
  • the coding DNA sequence may also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) the TPM (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) promoters.
  • the vector may further comprise elements such as polyadenylation signals (e.g. from SV 40 or the adenovirus 5 EIb region), transcriptional enhancer sequences (e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).
  • the recombinant expression vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question.
  • An example of such a sequence is the SV 40 origin of replication.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomycin, hydromycin or methotrexate.
  • DHFR dihydrofolate reductase
  • the coding DNA sequences may be usefully fused with a second peptide coding sequence and a protease cleavage site coding sequence, giving a DNA construct encoding the fusion protein, wherein the protease cleavage site coding sequence positioned between the HBP fragment and second peptide coding DNA, inserted into a recombinant expression vector, and expressed in recombinant host cells.
  • said second peptide selected from, but not limited by the group comprising glutathion-S-reductase, calf thymosin, bacterial thioredoxin or human ubiquitin natural or synthetic variants, or peptides thereof.
  • a peptide sequence comprising a protease cleavage site may be the Factor Xa, with the amino acid sequence IEGR, enterokinase, with the amino acid sequence DDDDK, thrombin, with the amino acid sequence LVPR/GS, or Acharombacter lyticus, with the amino acid sequence XKX, cleavage site.
  • the host cell into which the expression vector is introduced may be any cell which is capable of expression of the peptides or full-length proteins, and is preferably a eukaryotic cell, such as invertebrate (insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells.
  • a eukaryotic cell such as invertebrate (insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells.
  • suitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCC
  • fungal cells may be used as host cells.
  • suitable yeast cells include cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae.
  • Other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora spp., in particular strains of Aspergillus oryzae or Aspergillus niger.
  • Aspergillus spp. for the expression of proteins is described in, e.g., EP 238 023.
  • the medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements, or a suitable medium for growing insect, yeast or fungal cells. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the peptides or full-length proteins recombinantly produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. HPLC, ion exchange chromatography, affinity chromatography, or the like.
  • a salt e.g. ammonium sulphate
  • the peptide sequences of the invention are produced synthetically, in particular, by the Sequence Assisted Peptide Synthesis (SAPS) method.
  • SAPS Sequence Assisted Peptide Synthesis
  • SAPS 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.
  • individual peptide sequences may then be formulated as multimers using well-known in the art techniques, for examples dimers of the sequences may be obtained by the LPA method described in WO 00/18791 , dendrimeric polymers by the MAP synthesis described in PCT/US90/02039.
  • the antibody is an antibody that recognizes and binds to an epitope on insulin, insulin-like growth factors (IGFs), IGF-like proteins (IGFLs), insulin-like proteins (INSLs) or relaxins said epitope comprising at least one of the sequences selected from SEQ ID NOs: 1 - 34, or a fragment or variant of said sequence.
  • IGFs insulin-like growth factors
  • IGFLs IGF-like proteins
  • INSLs insulin-like proteins
  • epitope is meant the specific group of atoms (on an antigen molecule) that is recognized by (that antigen's) antibodies.
  • epitope is the equivalent to the term “antigenic determinant”.
  • the epitope may comprise 3 or more amino acid residues, such as for example 4, 5, 6, 7, 8 amino acid residues, located in close proximity, such as within a contiguous amino acid sequence, or located in distant parts of the amino acid sequence of an antigen, but due to protein folding have been approached to each other.
  • Antibody molecules belong to a family of plasma proteins called immunoglobulins, whose basic building block, the immunoglobulin fold or domain, is used in various forms in many molecules of the immune system and other biological recognition systems.
  • a typical immunoglobulin has four polypeptide chains, containing an antigen binding region known as a variable region and a non-varying region known as the constant region.
  • Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end.
  • VH variable domain
  • VL variable domain at one end
  • the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Novotny J, & Haber E. Proc Natl Acad Sci U S
  • immunoglobulins can be assigned to different classes. There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. lgG-1 , lgG-2, lgG-3 and lgG-4; lgA-1 and lgA-2.
  • the heavy chains constant domains that correspond to the different classes of immunoglobulins are called alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) and mu ( ⁇ ), respectively.
  • the light chains of antibodies can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino sequences of their constant domain.
  • K kappa
  • ⁇ lambda
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • variable in the context of variable domain of antibodies, refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies.
  • the variable domains are for binding and determine the specificity of each particular antibody for its particular antigen.
  • variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) also known as hypervariable regions both in the light chain and the heavy chain variable domains.
  • CDRs complementarity determining regions
  • variable domains The more highly conserved portions of variable domains are called the framework (FR).
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies.
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • an antibody that is contemplated for use in the present invention thus can be in any of a variety of forms, including a whole immunoglobulin, an antibody fragment such as Fv, Fab, and similar fragments, a single chain antibody which includes the variable domain complementarity determining regions (CDR), and the like forms, all of which fall under the broad term "antibody”, as used herein.
  • the present invention contemplates the use of any specificity of an antibody, polyclonal or monoclonal, and is not limited to antibodies that recognize and immunoreact with a specific antigen. In the context of both the therapeutic and screening methods described below, preferred embodiments are the use of an antibody or fragment thereof that is immunospecific for an antigen or epitope of the invention.
  • antibody fragment refers to a portion of a full-length antibody, generally the antigen binding or variable region.
  • antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments.
  • Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen binding fragments that are capable of cross-linking antigen, and a residual other fragment (which is termed pFc').
  • Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • “functional fragment” with respect to antibodies refers to Fv, F(ab) and F(ab') 2 fragments.
  • antibody fragment is used herein interchangeably with the term “antigen binding fragment”.
  • Antibody fragments may be as small as about 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 9 amino acids, about 12 amino acids, about 15 amino acids, about 17 amino acids, about 18 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids or more.
  • an antibody fragment of the invention can have any upper size limit so long as it is has similar or immunological properties relative to antibody that binds with specificity to an epitope comprising a peptide sequence selected from any of the sequences identified herein as SEQ ID NOs: 1 -34, or a fragment of said sequences.
  • the term "antibody fragment” is identical to term "antigen binding fragment".
  • Antibody fragments retain some ability to selectively bind with its antigen or receptor.
  • Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule.
  • a Fab fragment can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain.
  • Fab' is the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per antibody molecule.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • (Fab') 2 is the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction.
  • F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds.
  • Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V H -V L dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the V H -V L dimer.
  • V H -V L dimer tight, non-covalent association
  • Single chain antibody defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
  • Such single chain antibodies are also referred to as "single-chain Fv” or “sFv” antibody fragments.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH-VL polypeptide chain
  • the invention also contemplates multivalent antibodies having at least two binding domains.
  • the binding domains may have specificity for the same ligand or for different ligands.
  • Multivalent antibodies may be produced by a number of methods. Various methods for preparing bi- or multivalent antibodies are for example described in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881 ,175; 5,132,405; 5,091 ,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.
  • the invention contemplate both polyclonal and monoclonal antibody, antigen binding fragments and recombinant proteins thereof which are capable of binding an epitope according to the invention.
  • polyclonal antibodies The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green et al. 1992. Production of Polyclonal Antisera, in: Immunochemical Protocols (Manson, ed.), pages 1 -5 (Humana Press); Coligan, et al., Production of Polyclonal Antisera in Rabbits, Rats Mice and Hamsters, in: Current Protocols in Immunology, section 2.4.1 , which are hereby incorporated by reference.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion- exchange chromatography. See, e.g., Coligan, et al., sections 2.7.1 -2.7.12 and sections 2.9.1 -2.9.3; Barnes, et al., Purification of Immunoglobulin G (IgG). In: Methods in Molecular Biology, 1992, 10:79-104, Humana Press, NY.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256, 495-7, or may be made by recombinant methods, e.g., as described in US 4,816,567.
  • the monoclonal antibodies for use with the present invention may also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991 , Nature 352: 624-628, as well as in Marks et al., 1991 , J MoI Biol 222: 581 -597.
  • Another method involves humanizing a monoclonal antibody by recombinant means to generate antibodies containing human specific and recognizable sequences. See, for review, Holmes, et al., 1997, J
  • 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.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US 4,816,567); Morrison et al., 1984, Proc Natl Acad Sci 81 : 6851 -6855.
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another
  • Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • Fv fragments comprise an association of V H and V L chains. This association may be noncovalent or the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde.
  • the Fv fragments comprise V H and V L chains connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains connected by an oligonucleotide.
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • CDR peptides (“minimal recognition units") are often involved in antigen recognition and binding.
  • CDR peptides can be obtained by cloning or constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick, et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 106 (1991 ).
  • humanized antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) that contain a minimal sequence derived from non-human immunoglobulin, such as the eitope recognising sequence.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a nonhuman species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Humanized antibody(es) containing a minimal sequence(s) of antibody(es) of the invention, such as a sequence(s) recognising an epitope(s) described herein is one of the preferred embodiments of the invention.
  • humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • antibodies may be achieved by any standard methods in the art for producing polyclonal and monoclonal antibodies using natural or recombinant fragments of a protein of the insulin super family comprising a sequence selected from any of the sequences identified as SEQ ID NOs: 1 -34, as an antigen.
  • Such antibodies may be also generated using variants or fragments of SEQ ID NOs: 1 -34, said fragments being immunogenic fragments which meet the following criteria: (i) being a contiguous amino acid sequence of at least 6 amino acids, and (ii) comprising an amino acid sequence derived from the sequence of a protein of the insulin super family.
  • 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 a protein of the insulin super family in particular a function related to neural cell growth and survival, and to an antibody, which can recognise and specifically bind to the latter proteins without modulating biological activity thereof.
  • the invention relates to use of the above antibodies for 1 ) therapeutic applications involving the modulation of activity of a protein of the insulin super family 2) detecting and/or monitoring the latter proteins in vitro and/or in vivo for diagnostic purposes, e.g. determine levels of insulin, IGF-1 and IGF-2 in biological fluids and/or tissue samples. 3) research purposes.
  • the invention relates to a pharmaceutical composition comprising an antibody described above.
  • the invention relates to use of the above pharmaceutical composition for treatment of cancers whose growth depend upon and can be promoted by IGF-1 and/or IGF-2 (e.g. non-small lung cancer, see Han et. al., Lung Cancer 2006, 54, 227-234)
  • Example 1 Neuritogenic effect of peptides derived from proteins of the insulin super-family.
  • the IGF1 - and IGF2-derived peptides as well as the positive control P2d were purchased from Schafer-N (Copenhagen, DK), were synthesised as tetrameric dendrimers, composed of four monomers coupled to a lysine backbone.
  • the IGF1 - and IGF2-derived peptides were purified by gel-filtration utilizing Sephadex G-10 (Amersham). Peptide concentrations were determined using spectrophotometric measurements at 205 nm.
  • CGNs cerebellar granule neurons
  • the experiments were performed essentially as described in Schousbo et al. Neurochemical Research (1989).
  • the cerebellum was removed and placed in a solution of Krebs buffer with 0.3 % (w/v) BSA, 0.03 % (v/v) MgSO4 and 2OmM HEPES (solution 1 ).
  • solution 1 Using a stereomicroscope the cerebellum was cleared from meninges and blood vessels and homogenised by chopping with a scalpel blade.
  • the neurons were dissociated by mild trypsinization (12 min at 37 °C) in solution 1 supplemented with 0.2 mg/ml trypsin.
  • solution 1 consists of solution 1 supplemented with 0.08 mg/ml DNAase 1 , 0.52 mg/ml soybeen trypsin inhibitor and 1 .5 mM MgSO4.
  • centrifugation (1500 rpm for 2 minutes), the supernatant was discarded and the cells were resuspended in solution 3.
  • the cell solution was centrifuged (100 rpm for 15 seconds) to let tissue pellet, and supernatant was transferred to washing solution (132 ⁇ M CaCI2 and 120 ⁇ M MgSO4 in solution 1 ).
  • the cells were pelleted by centrifugation and resuspended in Neurobasal medium supplemented with 0.4% (w/v) BSA, 2% (v/v) B27, 0.5 % (v/v) glutamax, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
  • the cell concentration was determined using a Burker-Turk counting chamber and the cells were seeded in eight- well LabTek Permanox Chamber slides (Nunc, Roskilde, Denmark) to a density of 10 000 cells per well. Peptides or growth factors were added to a final volume of 300 ⁇ l/well, and the cells were grown at 37°C, 5 % CO2 for 24 hours. Medium was added to the untreated controls and P2d peptide was added to the positive controls. P2d (GRILARGEINFK) has previously been shown to efficiently induce neurite outgrowth at the concentration of 2 ⁇ g/ml.
  • GAP-43 growth associated protein-43
  • the cells were incubated for 1 hour at room temperature with secondary Alexa Flour 568 goat anti-rabbit antibodies (diluted 1 :1000 in PBS with 1 % BSA). Finally, the cells were washed in PBS and mounted with flourescent mounting medium.
  • Images of the neurons were recorded by computer-assisted fluorescent microscopy using a Nikon diaphot inverted microscope (Nikon, Tokyo, Japan) equipped with an epiflourescent attachment and a Nikon Plan 2Ox objective. The images were taken using a CCD video camera (Grunding Electronics, Germany) and the software program "Prima” (Protein Laboratory, University of Copenhagen, Denmark). Images of approximately 200 ⁇ 20 cells were recorded randomly in order to get a representative collection of the cells in each well. Using the software package "Process length" the length of neuronal processes per cell was quantified.
  • Figure 2 Neurogenic effect of human IGF2 derived peptides on CGNs.
  • CGNs from 7-8 days old rats were treated with IGF2 derived peptides or medium alone (control) for 24 hours.
  • the average length of neurites for the controls was 16.2 ⁇ 1.3 ⁇ m (a) or 17.9 ⁇ 1.6 ⁇ m (b) or 19.1 ⁇ 2.2 ⁇ m (c) or 19.1 ⁇ 1 .5 ⁇ m (d).
  • the results are expressed as percentage ⁇ SEM, where the control is set at 100 %.
  • the figures are based on experiments with two different series of concentrations: first series being 0.3, 1 , 3, 9, 27 and 81 ⁇ g/ml and second series being 0.1 , 3, 27 and 243 ⁇ g/ml (a); or 0.1 , 1 , 9 and 243 (b) ⁇ g/ml; or 27, 81 , 243 ⁇ g/ml (c) and (d).
  • the figures are subsequently based on results from two sets of experiments for each peptide.
  • the number of independent experiments per set is eight and five (a) or seven and five (b) or four (c) or six (d) for the first and second concentration series, respectively.
  • concentrations common for the two were compared using unpaired t-test.
  • the mean neurite lengths were compared applying one-way ANOVA for repeated measurements.
  • the P2d was used as positive control (data not shown).
  • IGF2-derived peptides induced statistically significant neurite outgrowth.
  • the tested peptides were: a) IGF2-I (SEQ ID NO 1 ) b) IGF2-II (SEQ ID NO 12) c) IGF2-III (SEQ ID NO 24) d) IGF2-V (SEQ ID NO 33).
  • Figure 3 Neuritogenic effect of human IGF1 -derived peptides on CGNs.
  • Example 2 peptides derived from proteins of the insulin super-family binds to the IR, IGF1 receptor and IGF2 receptor.
  • the recombinant human carrier free IGF1 , IGF2, IGF1 R, IGF2R, IR were from R and D systems (Oxon, UK).
  • CM5 Biacore AB, Uppsala, Sweden
  • one type of receptor was immobilized per chip utilizing an amine coupling kit. More specifically, the chip was activated by 30 ⁇ l activation solution followed by protein immobilization using 30 ⁇ l and 15 ⁇ l 10 ⁇ g/ml IGF2 receptor or insulin receptor, respectively, in 10 mM sodium phosphate buffer pH 6 at 5 ⁇ l/min.
  • the IGF1 receptor was immobilized using 1 1 ⁇ l 10 ⁇ g/ml of the protein in 10 mM acetic acid buffer pH 4 at 5 ⁇ l/min.
  • the final receptor density was 4200 RU, 3200 and 3033 RU for IGF1 R, IGF2R and IR, respectively.
  • a sensor chip CM4 (Biacore AB, Uppsala, Sweden) had all three receptor types immobilized essentially as described for the BIAIite experiments.
  • the chip was activated by 35 ⁇ l activation solution and protein was immobilized accordingly: 20 ⁇ l and 10 ⁇ l 25 ⁇ g/ml IGF1 receptor and insulin receptor respectively and 20 ⁇ l 50 ⁇ g/ml IGF2 receptor. All receptors were diluted in 10 mM acetic acid buffer pH 4 and injected at 5 ⁇ l/min.
  • the final receptor density for the sensor chip CM4 was in the range 1700-4000 resonance units.
  • both sensor chips CM4 and CM5 were blocked by 35 ⁇ l blocking solution.
  • Peptides and growth factors were injected over the sensor chip at indicated concentrations.
  • the peptides were injected at 200 ⁇ g/ml over a sensor chip CM5 immobilized with the first and second fibronectin type 3 (F3) module of NCAM and run at 20 ⁇ l/min in HBS-EP buffer on the Biacore instrument.
  • Analysis of the data was performed by non-linear curve-fitting using the software BIAevaluation v.4 (Biacore, Uppsala, Sweden) and/or Origin v.6.1 software (Originlab, MA, USA). All samples were also run over an unmodified flow cell in the sensor chip, thereby enabling low unspecific binding and changes in bulk refractive index to be subtracted.
  • Affinity constants were calculated based upon the curve corresponding to the difference between binding to receptor and the reference flow cell.
  • the curves were fitted to a 1 :1 Langmuir binding model, which describes the interaction of two molecules in a 1 :1 complex. This model was chosen as the most appropriate, even though the peptides are synthesized as tetrameric dendrimers.
  • IGF1 R IGF1 R
  • IGF2R Insulin Receptor-A
  • IR-A Insulin Receptor-A
  • the binding is expressed in resonance units and corresponds to the difference in binding to the flow cell with immobilized protein and a reference flow cell.
  • Peptides were injected at concentrations 12.5, 25, 50, 100, 200 and 300 ⁇ g/ml peptide in HBS- EP buffer at flow rate 20 ⁇ l/min.
  • the apparent affinity constants were calculated by fitting the curves to a 1 :1 Langmuir binding model applying the BIAevalution software.
  • the affinity constants are expressed as means of the KD of the different concentrations ⁇ SEM, where SEM is calculated from the obtained KDs at different concentrations. Representative curves of the peptides at 200 ⁇ g/ml are displayed.
  • the tested peptides were: a) IGF2-I(SEQIDNO1) b) IGF2-II(SEQIDNO12) c) IGF2-III (SEQ ID NO 24) d) IGF2-IV(SEQIDNO27) e) IGF2-V(SEQIDNO33)
  • FIG. 5 Binding of insulin and IGF1 derived peptides to the Insulin receptor, IGF1 receptor and IGF2 receptor. The study was performed as described in the legend of Figure 4. Binding curves of the peptides at various concentrations are displayed. The figure shows binding of peptide
  • IGF1-V and Ins-I to all three tested receptors.
  • the tested peptides were: a) IGFI-V(SEQ ID NO 34) b) Ins-I (SEQ ID NO 3)

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Abstract

La présente invention concerne des peptides dérivés de membres de la superfamille de l'insuline. De préférence, ces peptides se lient aux récepteurs de protéines de la superfamille de l'insuline et modifient l'état d'activation desdits récepteurs. L'invention concerne également une composition pharmaceutique contenant un composé renfermant un ou plusieurs peptides capables de favoriser la différenciation cellulaire et/ou de moduler la plasticité neuronale et/ou la prolifération de cellules nerveuses, et/ou de moduler la survie et/ou la régénération de cellules exprimant un récepteur d'une protéine de la superfamille de l'insuline. Lesdits peptides peuvent en outre être utilisés pour traiter des pathologies affectant le système nerveux périphérique et/ou central et/ou les muscles et d'autres tissus, ou pour traiter des cellules normales, dégénérées ou endommagées exprimant les récepteurs d'une ou plusieurs protéines de la superfamille de l'insuline.
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