U.S. patent application number 14/710896 was filed with the patent office on 2015-08-27 for neuritogenic peptides.
The applicant listed for this patent is Neoloch ApS. Invention is credited to Vladimir Berezin, Elisabeth Bock.
Application Number | 20150238556 14/710896 |
Document ID | / |
Family ID | 36917323 |
Filed Date | 2015-08-27 |
United States Patent
Application |
20150238556 |
Kind Code |
A1 |
Berezin; Vladimir ; et
al. |
August 27, 2015 |
NEURITOGENIC PEPTIDES
Abstract
The present invention relates to peptide compounds that are
capable of stimulating neuronal differentiation, neurite outgrowth
and survival of neural cells, and enhancing synaptic plasticity,
learning and memory, methods of treating diseases and conditions of
nervous system by administration of compositions comprising said
compounds. The compounds and compositions of the invention include
peptide sequences that are derived from the sequence of human
erythropoietin or proteins that are homologous of human
erythropoietin.
Inventors: |
Berezin; Vladimir;
(Copenhagen, DK) ; Bock; Elisabeth;
(Charlottenlund, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Neoloch ApS |
Charlottenlund |
|
DK |
|
|
Family ID: |
36917323 |
Appl. No.: |
14/710896 |
Filed: |
May 13, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13680715 |
Nov 19, 2012 |
9044428 |
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14710896 |
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11913954 |
Jul 25, 2008 |
8329652 |
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PCT/DK06/00246 |
May 8, 2006 |
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13680715 |
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Current U.S.
Class: |
514/17.8 ;
514/17.7 |
Current CPC
Class: |
A61P 25/16 20180101;
A61K 38/08 20130101; A61P 25/28 20180101; A61K 38/10 20130101; A61K
38/00 20130101; A61K 38/07 20130101; C07K 14/505 20130101 |
International
Class: |
A61K 38/07 20060101
A61K038/07; A61K 38/10 20060101 A61K038/10 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 5, 2005 |
DK |
DK 2005 00671 |
Claims
1. A method for treating a condition of the central or peripheral
nervous system, said method comprising administering to a subject
in need thereof an effective amount of a composition comprising (i)
a peptide consisting of 6 to 20 consecutive amino acids of SEQ ID
NO:3; or (ii) a peptide having a length of not more than 20 amino
acids and including 6 to 20 consecutive amino acids of SEQ ID NO:3,
wherein said peptide has more than 80% homology to SEQ ID NO:3; or
(iii) a peptide having a length of not more than 20 amino acids and
including a variant of a fragment of SEQ ID NO:3, said fragment
having 6 to 20 consecutive amino acids of SEQ ID NO:3 and said
variant having more than 80% homology to said fragment; wherein
said peptide is capable of stimulating neurite outgrowth, and/or
promoting survival of neural cells, and/or stimulating neural cell
proliferation.
2. The method of claim 1, wherein said condition of the central or
peripheral nervous system is a neurodegenerative disease.
3. The method of claim 2, wherein said neurodegenerative disease is
Alzheimer's disease, Parkinson's disease or Huntington's
disease.
4. The method of claim 1, wherein said condition of the central or
peripheral nervous system is cerebral ischemia.
5. The method of claim 1, wherein said condition of the central or
peripheral nervous system is postoperative nerve damage, traumatic
nerve damage or traumatic brain injury.
6. The method of claim 1, wherein said condition of the central or
peripheral nervous system is selected from the group consisting of
epilepsy, dementia, sclerosis, schizophrenia, impaired myelination
of nerve fibers, nerve degeneration associated with diabetes
mellitus, disorders affecting the circadian clock or neuromuscular
transmission or mood disorders.
7. The method of claim 1, wherein the subject is human.
8. The method of claim 1, wherein the composition comprises a
monomer of (i) a peptide consisting of 6 to 20 consecutive amino
acids of SEQ ID NO:3, or (ii) a peptide having a length of not more
than 20 amino acids, including 6 to 20 consecutive amino acids of
SEQ ID NO:3, and having more than 80% homology to SEQ ID NO:3, or
(iii) a peptide having a length of not more than 20 amino acids and
including a variant of a fragment of SEQ ID NO:3, said fragment
having 6 to 20 consecutive amino acids of SEQ ID NO:3 and said
variant having more than 80% homology to said fragment.
9. The method of claim 1, wherein the composition comprises two or
more peptides, the peptides either (i) consisting of 6 to 20
consecutive amino acids of SEQ ID NO:3, or (ii) having a length of
not more than 20 amino acids, including 6 to 20 amino acids of SEQ
ID NO:3, and having more than 80% homology to SEQ ID NO:3, or (iii)
having a length of not more than 20 amino acids and including a
variant of a fragment of SEQ ID NO:3, said fragment having 6 to 20
consecutive amino acids of SEQ ID NO:3 and said variant having more
than 80% homology to said fragment; wherein said two or more
peptides are linked via one or more linker groups.
10. The method of claim 9, wherein said linker group is selected
from the group consisting of peptide bonds forming a polymer of the
two or more polypeptides, dendrimeric polymers, Ligand Presenting
Assembly (LPA)-type polymers, lysine dendrimers, bovine serum
albumin (BSA), lipophilic dendrimers, micelle-like carriers,
starburst carbon chain polymer conjugates, and ligand presenting
assembly polymers based on derivatives of diethylaminomethane, with
the proviso that the amino acid sequence of each copy of the
peptide is not identical to the amino acid sequence of naturally
occurring erythropoietin
11. The method of claim 9, wherein the composition comprises a
dimer or a tetramer of said peptides.
12. The method of claim 1, wherein the composition comprises a
peptide having a length of not more than 20 amino acids and
including (i) a fragment of SEQ ID NO:3 having 10 to 15 consecutive
amino acids of SEQ ID NO:3, or (ii) a variant of said fragment,
said variant having more than 80% homology to said fragment.
13. The method of claim 1, wherein the composition comprises a
peptide having an amino acid sequence consisting of from 10 to 15
amino acids of SEQ ID NO:3.
14. The method of claim 1, wherein the composition comprises a
peptide consisting of the amino acid sequence of SEQ ID NO:3.
15. The method of claim 1, wherein the arginine amino acid residue
at position 12 of SEQ ID NO:3 is arginine or conservatively
substituted with a histidine or a lysine amino acid residue in the
corresponding position within said peptide.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compounds that are capable
of stimulating neuronal differentiation, neurite outgrowth and
survival of neural cells, and enhancing synaptic plasticity,
learning and memory, methods of treating diseases and conditions of
nervous system by administration of compositions comprising said
compounds. The compounds and compositions of the invention include
peptides and/or peptide multimers that derived from human
erythropoietin or proteins that are homologous of human
erythropoietin.
BACKGROUND OF THE INVENTION
[0002] Erythropoietin (EPO) is a glycoprotein hormone produced by
the kidney in response to tissue hypoxia that stimulates red blood
cell production in the bone marrow. The gene for erythropoietin has
been cloned and expressed in Chinese hamster ovary (CHO) cells as
described in U.S. Pat. No. 4,703,008. Recombinant human
erythropoietin (r-HuEPO or Epoetin alfa) has an amino acid sequence
identical to that of human urinary erythropoietin, and the two are
indistinguishable in chemical, physical and immunological tests.
Recombinant human erythropoietin acts by increasing the number of
cells capable of differentiating into mature erythrocytes,
triggering their differentiation and augmenting hemoglobin
synthesis in developing erythroblasts (Krantz S B. Blood (1991) 77:
419-434, Beckman B S, Mason-Garcia M. The Faseb Journal (1991) 5:
2958-2964).
[0003] Epoetin alfa has been well tolerated in studies conducted to
date. Hypertensive encephalopathy and seizures have occasionally
been described in dialysis patients treated with Epoetin alfa,
particularly during the early phase of therapy when hematocrit is
rising. (Eschbach J W, Egrie J C, Downing M R, Browne J K, Adamson
J W. New Engl J Med (1987) 316: 73-78, Winearls C G, Oliver D O,
Pippard M J, et al. Lancet (1986) 2 (8517): 1175-1177). Such
reports became more rare as experience of use of the compound
developed. Occasionally, cancer patients treated with Epoetin alfa
have experienced an increase in blood pressure associated with a
significant increase in hematocrit. The risk, however, appears
substantially lower than in chronic renal failure patients.
[0004] No antibody titers against Epoetin alfa could be
demonstrated and confirmed in subjects treated with Epoetin alfa
for up to 2 years, indicating the absence of immunological
sensitivity to Epoetin alfa. Skin rashes and urticaria have been
observed rarely and when reported have been mild and transient in
nature, but these events suggest allergic hypersensitivity to some
components of the Epoetin alfa formulation.
[0005] Epoetin alfa is approved for sale in many countries for the
treatment of anemia in chronic renal failure (dialysis and
predialysis), anemia in zidovudine treated HIV positive patients
(US), anemia in cancer patients receiving platinum-based
chemotherapy, as a facilitator of autologous blood pre-donation,
and as a pert-surgical adjuvant to reduce the likelihood of
requiring allogeneic blood transfusions in patients undergoing
orthopedic surgery.
[0006] EPO influences neuronal stem cells, likely during embryonic
development, and possibly during in vitro experiments of
differentiation. (Juul et al Pediatr Dev Pathol (1999) 2(2)
148-158. Juul et al Pediatr Res (1998) 43(1) 40-49.) Further,
neonates and infants that suffer CNS injury via hypoxia,
meningitis, and intraventricular hemorrhage, show an EPO induced
neuroprotective effect (Juul et al Ped Res (1999) 46(5)
543-547.)
[0007] EPO helps prevent apoptosis of neural tissue in cases of
injury that create hypoxia. This may be the result of EPO produced
locally by astrocytes (Morishita et al Neuroscience (1996) 76(1)
105-116). Neuroprotection has been demonstrated in gerbil
hippocampal and rat cerebrocortical tissue (Sakanaka et al PNAS
(1998) 95(8) 4635-4640. Sadamoto et al Biochem Biophys Res Commun
(1998) 253(1) 26-32).
[0008] EPO induces biological effects of PC12 cells, including
changes in Ca<2+>, changes in membrane potential, and
promotion of neuronal survival. This has been interpreted that EPO
can stimulate neural function and viability (Koshimura et al J.
Neurochem (1999) 72(6) 2565-2572. Tabria et al Int J Dev Neurosci
(1995) 13(3/4) 241-252.).
[0009] A number of studies were attempted to associated diverse
biological activities of EPO with the particular structural areas
of the protein (Grodberg et al. Eur J Biochem (1993)
218(2):597-601; Wen et al. J Biol Chem (1994) 269(36):22839-22846;
Elliott et al. Blood (1997) 89(2):493-502; Cheetham et al. Nat Str
Biol (1998) 5(10):861-866; Syed et al. Nature (1998) 395:516)
Campana et al proposed that a 17 amino acid peptide sequence of EPO
can act through the EPO-R (Erythropoietin receptor) to induce
biological activity in NS20Y, S-K-N-MC, and PC12 cells, which
includes sprouting, differentiation and neuroprotection. Curiously
this peptide does not promote proliferation of hematologic cells,
thus it appears inactive in cell lines well understood for their
sensitivity to EPO activity (Campana et al Int J Mol Med (1998)
1(1) 235-241).
[0010] Short peptide fragments of Epo or other peptide sequences
that have the full range of biological activity of human
erythropoietin or only certain biological activities of
erythropoietin are of great interest as drug candidates and a
number of patent applications has already described or contemplated
the uses of such biologically active peptide fragments as
neurotrofic or neuroprotective drugs (U.S. Pat. No. 5,700,909, U.S.
Pat. No. 6,703,480, U.S. Pat. No. 6,642,363, U.S. Pat. No.
5,106,954, US2003130197). The present application provides new
peptide fragments derived from Epo or Epo functional homologues
that may be advantageously used in therapeutic treatment of
diseases or conditions of neural system.
SUMMARY OF INVENTION
[0011] The present invention identifies a new group of peptide
sequences which are potent stimulators of neurite outgrowth.
Surprisingly, the sequences are also capable to protect neuronal
cells from death and promote survival of said cells, and moreover,
are capable of stimulating cell proliferation. The inventors
identified the structural motif that present in all disclosed
herein peptide sequences and correlated the presence of this motif
with biological activity of the sequences.
[0012] Accordingly, in the first aspect the invention relates to a
compound comprising at least one isolated peptide sequence of 6 to
25 amino acid residues comprising the amino acid sequence motif of
the formula
x.sup.1-x.sup.2-x.sup.3-x.sup.4-x.sup.5-x.sup.6 (SEQ ID N0:43),
[0013] wherein [0014] x.sup.1 is a charged amino acid residue,
[0015] x.sup.6 is a hydrophobic amino acid residue or A, [0016] and
[0017] x.sup.2, x.sup.3, x.sup.4 and x.sup.5 is any amino acid
residue
[0018] The invention discloses a group of particular sequences
comprising the above motif, wherein said sequences are either short
peptide fragments of human erythropoietin or short fragments of
proteins having a structural homology to human erythropoietin. The
disclosed sequences possess neuritogenic activity, neuronal cell
survival promoting activity, synaptic plasticity stimulating
activity, and/or learning and memory stimulating activity and cell
proliferation stimulating activity.
[0019] In another aspect the invention relates to use of a peptide
sequence comprising the structural motif of above and/or a compound
comprising such sequence for [0020] the stimulating neurite
outgrowth end/or promoting survival of neural cells, [0021] the
manufacture of a medicament for treatment of conditions of the
central and peripheral nervous system, [0022] the manufacture of a
medicament for the stimulation of the ability to learn and/or the
short and/or long-term memory [0023] the production of an antibody
capable of recognizing an epitope comprising the sequence; [0024]
the treatment an individual in need.
[0025] The invention also related to a pharmaceutical composition
comprising a compound and/or peptide sequence of the invention.
[0026] In further aspect the invention relates to an antibody
capable of recognizing the epitope comprising a sequence comprising
the motif of the invention.
FIGURE LEGENDS
[0027] FIG. 1 Effect of recombinant human erythropoietin (Epo) on
neurite outgrowth from CGN
[0028] FIG. 2 Effect of Epo1 (Epo-peptide 1) (SEQ ID NO: 1) on
neurite outgrowth from CGN
[0029] FIG. 3 Effect of Epo2 (Epo-peptide 2) (SEQ ID NO: 2) on
neurite outgrowth from CGN in vitro.
[0030] FIG. 4 Effect of Epo3 (Epo-peptide 3) (SEQ ID NO: 3) on
neurite outgrowth from CGN in vitro.
[0031] FIG. 5 Effect of Epo4 (Epo-peptide 4) (SEQ ID NO: 4) on
neurite outgrowth from CGN in vitro.
[0032] FIG. 6 Effect of recombinant human erythropoietin (Epo) on
survival of CGN in vitro.
[0033] FIG. 7 Effect of Epo1 (Epo-peptide 1) (SEQ ID NO: 1) on
survival of CGN in vitro.
[0034] FIG. 8 Effect of Epo2 (Epo-peptide 2) (SEQ ID NO: 2) on
survival of CGN in vitro.
[0035] FIG. 9 Effect of Epo3 (Epo-peptide 3) (SEQ ID NO: 3) on
survival of CGN in vitro.
[0036] FIG. 10 Effect of Epo4 (Epo-peptide 4) (SEQ ID NO: 4) on
survival of CGN in vitro.
[0037] FIG. 11 Effect of Epo1 (Ep1) (SEQ ID NO: 1) on proliferation
of human erythroleukemia cells TF-1 in vitro. Statistical
significance was calculated in comparison with control.
[0038] FIG. 12 Effect of Epo2 (Ep2) (SEQ ID NO: 2) on proliferation
of human erythroleukemia cells TF-1 in vitro. Statistical
significance was calculated in comparison with control.
[0039] FIG. 13 Effect of Epo3 (Ep3) (SEQ ID NO:3) on proliferation
of human erythroleukemia cells TF-1 in vitro. Statistical
significance was calculated in comparison with control.
[0040] FIG. 14 Effect of Epo4 (Ep4) (SEQ ID NO: 4) on proliferation
of human erythroleukemia cells TF-1. Statistical significance was
calculated in comparison with control.
[0041] FIG. 15 Effect of recombinant human erythropoietin (EPO) and
EPO mimetic peptides Epo2, Epo3 and Epo 4 (EP2, EP3 and EP4) (SEQ
ID NO: 2, 3 and 4) on the hematocrit in rat brain after the brain
lesion. The hematocrit (%) is significantly increased after
treatment of the rats with EPO (63.7+-1.9 EPO vs 48.7+-3.35
control, p<0.02). The peptide mimetics have no statistically
significant effect on the hematocrit (Ep2, Ep3, Ep4 vs control
p>0.02). Control--treatment with water.
[0042] FIG. 16 Binding of recombinant erythropoietin receptor
(EPOR) to the immobilized Epo3 peptide (SEQ ID NO:3). The binding
was studied by SPR analysis. Three independent experiments were
performed. The results show that EPOR binds to Epo3 with relatively
middle association rate (k.sub.a=2.1*10.sup.4+-1.4*10.sup.4) and
slow dissociation rate (k.sub.d=4.33*10.sup.-5+-1.62*10.sup.-5).
The EPOR binds with Epo3 with overall binding affinity 4.48 nM.
[0043] A--binding of soluble EPOR to Epo3 ionic bound to the chip
[0044] B--binding of soluble EPOR to Epo3 covalently bound to the
chip.
DETAILED DESCRIPTION OF THE INVENTION
[0045] Molecules with the potential to promote neurite outgrowth as
well as stimulate survival, regeneration of neuronal cells, such as
certain endogenous trophic factors, are prime targets in the search
for compounds that facilitate for example neuronal regeneration and
other forms of neuronal plasticity. Short peptide sequences of 6-25
amino acid residues have proved to be good candidate compounds
useful as in research as in medical applications.
Peptide Sequences
[0046] Thus, in one aspect the invention relates to a compound,
which comprises, essentially comprises or consists of at least one
isolated peptide sequence comprising the amino acid sequence motif
of the formula
x.sup.1-x.sup.2-x.sup.3-x.sup.4-x.sup.5-x.sup.6 (SEQ ID N0:43),
[0047] wherein [0048] x.sup.1 is a charged amino acid residue,
[0049] x.sup.6 is a hydrophobic amino acid residue or A, [0050] and
[0051] x.sup.2, x.sup.3, x.sup.4 and x.sup.5 is any amino acid
residue
[0052] Beyond the presence of two most important amino acid
residues in the structural motif of the invention, which are a
charged residue in position 1 (x.sup.1) of the motif, preferably
the negatively charged residue, and a hydrophobic amino acid
residues in position 6 (x.sup.6), preferably L, V or Y, the
invention further favours the sequences wherein the motif further
comprises i) the S residue in position 2 (x.sup.2) and/or ii) a
hydrophobic residue in position 2 (x.sup.2) and/or hydrophobic
residue in position 3 (x.sup.3) of the motif. Examples of such
preferred motifs may be the sequences (i)
R-S-x.sup.3-x.sup.4-x.sup.5-L (SEQ ID NO:44), and (ii)
R-V-x.sup.3-x.sup.4-x.sup.5-A (SEQ ID NO:45),
R-V-L-x.sup.4-x.sup.5-Y (SEQ ID NO:46), K-A-V-x.sup.4-x.sup.5-L
(SEQ ID NO:47), R-x.sup.2-L-x.sup.4-x.sup.5-L (SEQ ID NO:48), or
R-S-L-x.sup.4-x.sup.5-L (SEQ ID NO:49). Yet, the residue S or T is
in some cases preferred in position 4 (x.sup.4) independently of
the presence of a hydrophobic residue in position x.sup.2 and/or
x.sup.3.
[0053] In the present application the standard one-letter code for
amino acid residues as well as the standard three-letter code are
applied. Abbreviations for amino acids are in accordance with the
recommendations in the IUPAC-IUB Joint Commission on Biochemical
Nomenclature Eur. J. Biochem, 1984, vol. 184, pp 9-37. Throughout
the description and claims either the three letter code or the one
letter code for natural amino acids are used. Where the L or D form
has not been specified it is to be understood that the amino acid
in question has the natural L form, cf. Pure & Appl. Chem. Vol.
(56(5) pp 595-624 (1984) or the D form, so that the peptides formed
may be constituted of amino acids of L form, D form, or a sequence
of mixed L forms and D forms.
[0054] Where nothing is specified it is to be understood that the
C-terminal amino acid of a peptide of the invention exists as the
free carboxylic acid, this may also be specified as "--OH".
However, the C-terminal amino acid of a compound of the invention
may be the amidated derivative, which is indicated as "--NH.sub.2".
Where nothing else is stated the N-terminal amino acid of a
polypeptide comprise a free amino-group, this may also be specified
as "H--".
[0055] Where nothing else is specified 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, Val,
Leu, Ile, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gln,
Asp, Glu, Lys, Arg, His Aib, Nal, Sar, Orn, Lysine analogues, DAP,
DAPA and 4Hyp.
[0056] Also, according to the invention modifications of the
compounds/peptides may be performed, such as for example
glycosylation and/or acetylation of the amino acids.
[0057] Basic amino acid residues are according to invention
represented by the residues of amino acids Arg, Lys, and His,
acidic amino acid residues--by the residues of amino acids Glu and
Asp. Basic and amino acid residues constitute a group of charged
amino acid residues. The group of hydrophobic amino acid residues
is represented by the residues of amino acids Leu, Ile, Val, Phe,
Trp, Tyr, and Met.
[0058] In one embodiment variants may be understood as exhibiting
amino acid sequences gradually differing from the preferred
predetermined sequence, as the number and scope of insertions,
deletions and substitutions including conservative substitutions
increase. This difference is measured as a reduction in homology
between the predetermined sequence and the variant.
[0059] The invention relates to naturally occurring,
synthetically/recombinant 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 the polypeptide
chain, said sequences being the existing separated/isolated
individual chemical units/compounds.
[0060] The present invention identifies herein a group of short
individual peptide fragments which comprise the amino acid sequence
motif of above and correlates the presence of the motif in the
sequence with the capability of said sequence to i) induce neurite
outgrowth, and/or ii) stimulate survival of neural cells, and/or
iii) stimulate synaptic plasticity, and/or iv) stimulate learning,
and/or v) stimulate memory and vi) stimulate cell proliferation.
This group of peptide fragments consists of the following
sequences:
TABLE-US-00001 (SEQ ID NO: 1) DSRVLERYLLEAKE (SEQ ID NO: 2)
NENITVPDTKVNFYAWKR (SEQ ID NO: 3) QLHVDKAVSGLRSLTTLLRA (SEQ ID NO:
4) RVYSNFLRGKLKLYTGEA (SEQ ID NO: 5) DLRVLSKLLRDSHV (SEQ ID NO: 6)
PSTQPWEHVNAIQEARR (SEQ ID NO: 7) CSRSIWLARKIRSD (SEQ ID NO: 8)
SERIDKQIRYILDGIS (SEQ ID NO: 9) SCNMIDEIITHLKQ (SEQ ID NO: 10)
SSCLMDRHDFGFPQEEFDGNQ (SEQ ID NO: 11) MSYNLLGFLQRSSNFQCQKLLWQLN
(SEQ ID NO: 12) CYCQDPYVKEAENLKKYFNA (SEQ ID NO: 13)
PTPVLLPAVDFSLGEWKTQM (SEQ ID NO: 14) NETVEVISE (SEQ ID NO: 15)
NKNINLDSADGMPVASTD (SEQ ID NO: 16) AENNLNLPKMAEKD (SEQ ID NO: 17)
ENNLRRPNLEAFNRAVKS (SEQ ID NO: 18) QQIFNLFTTKDSSAAWDE (SEQ ID NO:
19) DRMNFDIPEEIKQLQQFQK (SEQ ID NO: 20) ADNGTLFLGILKNWKEESDR (SEQ
ID NO: 21) TAHKDPNAIFLSFQHLLRGKVRFL (SEQ ID NO: 22)
QTRLELYKQGLRGSLTKLKGPLTM (SEQ ID NO: 23) LLQVAAFAYQIEELMILLEYK (SEQ
ID NO: 24) EEQARAVQMSTKVLIQ (SEQ ID NO: 25) HIKDGDWNEFRRKLTFYLKT
(SEQ ID NO: 26) LMNADSILAVKKYFRRITLY (SEQ ID NO: 27)
KLEKEDFTRGKLMSSLHLKR (SEQ ID NO: 28) NSNKKKRDDFEKLTNYSVTD (SEQ ID
NO: 29) PNRTSGLLETNFTAS (SEQ ID NO: 30) KDFLLVIPFDCWEPVQE (SEQ ID
NO: 31) ELSQWTVRSIHDLRFISS (SEQ ID NO: 32) RSFKEFLQSSLR (SEQ ID NO:
33) FINRLTGYLRN (SEQ ID NO: 34) ELSPAAKTGKR (SEQ ID NO: 35)
SLIIGFAAGALYWKKRQPSL (SEQ ID NO: 36) DELINIIDGVLRDDDKNND (SEQ ID
NO: 37) RNRVTNNVKDVTKLV (SEQ ID NO: 38) DKLVNIVDDLVECVKE (SERQ ID
NO: 39) GLDKNTVHDQEHIMEHLEGV (SEQ ID NO: 40) SETSDCVVSSTLSPEKDSRV
(SEQ ID NO: 41) QLHYFKMHDYDGNNLL.
[0061] The identified above peptides according to the application
may used for different applications, for example for production of
different antibodies or production of different medicaments.
Therefore in different embodiments the peptide sequence may be
preferably selected.
[0062] Thus, in one preferred embodiment, the peptide fragment may
be preferably selected from the group consisting of the peptide
sequences
TABLE-US-00002 (SEQ ID NO: 1) DSRVLERYLLEAKE (SEQ ID NO: 2)
NENITVPDTKVNFYAWKR (SEQ ID NO: 3) QLHVDKAVSGLRSLTTLLRA (SEQ ID NO:
4) RVYSNFLRGKLKLYTGEA
[0063] The above group of sequences is designated herein as Group
1. The sequences of Group 1 are derived from human erythropoietin
(Swissprot Ass. No. P01588).
[0064] In another preferred embodiment, the peptide fragment may be
selected from the group consisting of the peptide sequences
TABLE-US-00003 (SEQ ID NO: 5) DLRVLSKLLRDSHV (SEQ ID NO: 13)
PTPVLLPAVDFSLGEWKTQM (SEQ ID NO: 21) TAHKDPNAIFLSFQHLLRGKVRFL (SEQ
ID NO: 29) PNRTSGLLETNFTAS.
[0065] The above group of sequences is designated herein as Group
2. The sequences of Group 2 are derived from human thrombopoietin
(Swissprot Ass. No. P40225).
[0066] In still another preferred embodiment, the peptide fragment
may be selected from the group consisting of peptide sequences
TABLE-US-00004 (SEQ ID NO: 6) PSTQPWEHVNAIQEARR (SEQ ID NO: 14)
NETVEVISE (SEQ ID NO: 22) QTRLELYKQGLRGSLTKLKGPLTM (SEQ ID NO: 30)
KDFLLVIPFDCWEPVQE.
[0067] The above group of sequences is designated herein as Group
3. The sequences of Group 3 are derived from human
granulocyte-macrophage colony-stimulating factor (GM-CSF, Swissprot
Ass. No. P04141).
[0068] In yet another preferred embodiment, the peptide fragment
may be selected from for the group consisting of the peptide
sequences
TABLE-US-00005 (SEQ ID NO: 7) CSRSIWLARKIRSD (SEQ ID NO: 15)
NKNINLDSADGMPVASTD (SEQ ID NO: 23) LLQVAAFAYQIEELMILLEYK (SEQ ID
NO: 31) ELSQWTVRSIHDLRFISS.
[0069] The above group of sequences is designated herein as Group
4. The sequences of Group 4 are derived from human ciliary
neurotrophic factor (CNFT, Swissprot Ass. No. P26441).
[0070] In still yet another preferred embodiment, the peptide
fragment may be selected from the group consisting of the peptide
sequences
TABLE-US-00006 (SEQ ID NO: 8) SERIDKQIRYILDGIS (SEQ ID NO: 16)
AENNLNLPKMAEKD (SEQ ID NO: 24) EEQARAVQMSTKVLIQ (SEQ ID NO: 32)
RSFKEFLQSSLR.
[0071] The above group of sequences is designated herein as Group
5. The sequences of Group 5 are derived from human interleukin-6
(IL-6, Swissprot Ass. No. P05231).
[0072] In other preferred embodiments the sequence may be selected
either from the sequences of the group of
i) peptide sequences derived from human interleukin-3 (IL-3,
Swissprot Ass. No. P08700) (Group 6):
TABLE-US-00007 (SEQ ID NO: 9) SCNMIDEIITHLKQ (SEQ ID NO: 17)
ENNLRRPNLEAFNRAVKS or (SEQ ID NO: 25) HIKDGDWNEFRRKLTFYLKT;
or ii) peptide sequences derived from human interferon alpha-1
(Swissprot Ass. No. P05231) (Group 7):
TABLE-US-00008 (SEQ ID NO: 10) SSCLMDRHDFGFPQEEFDGNQ (SEQ ID NO:
18) QQIFNLFTTKDSSAAWDE (SEQ ID NO: 26) LMNADSILAVKKYFRRITLY;
or iii) peptide sequences derived from human interferon beta
(Swissprot Ass. No. P01574) (Group 8):
TABLE-US-00009 (SEQ ID NO: 11) MSYNLLGFLQRSSNFQCQKLLWQLN (SEQ ID
NO: 19) DRMNFDIPEEIKQLQQFQK (SEQ ID NO: 27) KLEKEDFTRGKLMSSLHLKR
(SEQ ID NO: 33) FINRLIGYLRN;
or iv) peptide sequences derived from human interferon gamma
(Swissprot Ass. No. P05231) (Group 9):
TABLE-US-00010 (SEQ ID NO: 12) CYCQDPYVKEAENLKKYFNA (SEQ ID NO: 20)
ADNGTLFLGILKNWKEESDR (SEQ ID NO: 28) NSNKKKRDDFEKLTNYSVTD (SEQ ID
NO: 34) ELSPAAKTGKR;
or v) peptide sequences derived from human cell stem factor
(Swissprot Ass. No. P21583) (Group 10):
TABLE-US-00011 (SEQ ID NO: 37) RNRVTNNVKDVTKLV (SEQ ID NO: 38)
DKLVNIVDDLVECVKE (SEQ ID NO: 40) SETSDCVVSSTLSPEKDSRV (SEQ ID NO:
35) SLIIGFAAGALYWKKRQPSL;
or vi) peptide sequences derived from human multiple coagulation
factor deficiency protein 2 (neural stem cell derived neuronal
survival protein/MCD2 (Swissprot Ass. No. P21583Q8NI22) (Group
11):
TABLE-US-00012 (SERQ ID NO: 39) GLDKNTVHDQEHIMEHLEGV (SEQ ID NO:
41) QLHYFKMHDYDGNNLL (SEQ ID NO: 36) DELINIIDGVLRDDDKNND,
[0073] However, any amino acid sequence other then the above
mentioned sequences, which contains the motif of the invention and
is capable of at least one biological activity selected from
stimulating neuronal differentiation, stimulating neurite
outgrowth, stimulating survival of neural cells, enhancing synaptic
plasticity, stimulating learning and memory is in the scope of
protection of the invention.
[0074] Compounds, which comprise or consist of variants of the
above sequences, are also in the scope of the invention.
[0075] "Variant of a peptide sequence" means that the peptides may
be modified, for example by substitution of one or more of the
amino acid residues. Both L-amino acids and D-amino acids may be
used. Other modification may comprise derivatives such as esters,
sugars, etc. Examples are methyl and acetyl esters.
[0076] Variants of the peptide fragments according to the invention
may comprise, within the same variant, or fragments thereof or
among different variants, or fragments thereof, at least one
substitution, such as a plurality of substitutions introduced
independently of one another. Variants of the complex, or fragments
thereof may thus comprise conservative substitutions independently
of one another, wherein at least one glycine (Gly) of said variant,
or fragments thereof is substituted with an amino acid selected
from the group of amino acids consisting of Ala, Val, Leu, and Ile,
and independently thereof, variants, or fragments thereof, wherein
at least one alanine (Ala) of said variants, or fragments thereof
is substituted with an amino acid selected from the group of amino
acids consisting of Gly, Val, Leu, and Ile, and independently
thereof, variants, or fragments thereof, wherein at least one
valine (Val) of said variant, or fragments thereof is substituted
with an amino acid selected from the group of amino acids
consisting of Gly, Ala, Leu, and Ile, and independently thereof,
variants, or fragments thereof, wherein at least one leucine (Leu)
of said variant, or fragments thereof is substituted with an amino
acid selected from the group of amino acids consisting of Gly, Ala,
Val, and Ile, and independently thereof, variants, or fragments
thereof, wherein at least one isoleucine (Ile) of said variants, or
fragments thereof is substituted with an amino acid selected from
the group of amino acids consisting of Gly, Ala, Val and Leu, and
independently thereof, variants, or fragments thereof wherein at
least one aspartic acids (Asp) of said variant, or fragments
thereof is substituted with an amino acid selected from the group
of amino acids consisting of Glu, Asn, and Gln, and independently
thereof, variants, or fragments thereof, wherein at least one
aspargine (Asn) of said variants, or fragments thereof is
substituted with an amino acid selected from the group of amino
acids consisting of Asp, Glu, and Gln, and independently thereof,
variants, or fragments thereof, wherein at least one glutamine
(Gln) of said variants, or fragments thereof is substituted with an
amino acid selected from the group of amino acids consisting of
Asp, Glu, and Asn, and wherein at least one phenylalanine (Phe) of
said variants, or fragments thereof is substituted with an amino
acid selected from the group of amino acids consisting of Tyr, Trp,
His, Pro, and preferably selected from the group of amino adds
consisting of Tyr and Trp, and independently thereof, variants, or
fragments thereof, wherein at least one tyrosine (Tyr) of said
variants, or fragments thereof is substituted with an amino acid
selected from the group of amino acids consisting of Phe, Trp, His,
Pro, preferably an amino acid selected from the group of amino
acids consisting of Phe and Trp, and independently thereof,
variants, or fragments thereof, wherein at least one arginine (Arg)
of said fragment is substituted with an amino acid selected from
the group of amino acids consisting of Lys and His, and
independently thereof, variants, or fragments thereof, wherein at
least one lysine (Lys) of said variants, or fragments thereof is
substituted with an amino acid selected from the group of amino
acids consisting of Arg and His, and independently thereof,
variants, or fragments thereof, and independently thereof,
variants, or fragments thereof, and wherein at least one proline
(Pro) of said variants, or fragments thereof is substituted with an
amino acid selected from the group of amino acids consisting of
Phe, Tyr, Trp, and His, and independently thereof, variants, or
fragments thereof, wherein at least one cysteine (Cys) of said
variants, or fragments thereof is substituted with an amino acid
selected from the group of amino acids consisting of Asp, Glu, Lys,
Arg, His, Asn, Gln, Ser, Thr, and Tyr.
[0077] It thus follows from the above that the same functional
equivalent of a peptide fragment, or fragment of said functional
equivalent may comprise more than one conservative amino acid
substitution from more than one group of conservative amino acids
as defined herein above. The term "conservative amino acid
substitution" is used synonymously herein with the term "homologous
amino acid substitution".
[0078] The groups of conservative amino acids are as the
following:
P, A, G (neutral, weakly hydrophobic), S, T (neutral, hydrophilic)
Q, N (hydrophilic, acid amine) E, D (hydrophilic, acidic) H, K, R
(hydrophilic, basic) A, L, I, V, M, F, Y, W (hydrophobic, aromatic)
C (cross-link forming)
[0079] Conservative substitutions may be introduced in any position
of a preferred predetermined peptide of the invention or fragment
thereof. It may however also be desirable to introduce
non-conservative substitutions, particularly, but not limited to, a
non-conservative substitution in any one or more positions.
[0080] A non-conservative substitution leading to the formation of
a functionally equivalent fragment of the peptide of the invention
would for example differ substantially in polarity, for example a
residue with a non-polar side chain (Ala, Leu, Pro, Trp, Val, Ile,
Leu, Phe or Met) substituted for a residue with a polar side chain
such as Gly, Ser, Thr, Cys, Tyr, Asn, or Gln or a charged amino
acid such as Asp, Glu, 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 Gly by another residue; and/or iii) differ
substantially in electric charge, for example substitution of a
negatively charged residue such as Glu or Asp for a positively
charged residue such as Lys, His or Arg (and vice versa); and/or
iv) differ substantially in steric bulk, for example substitution
of a bulky residue such as His, Trp, Phe or Tyr for one having a
minor side chain, e.g. Ale, Gly or Ser (and vice versa).
[0081] Substitution of amino acids may in one embodiment be made
based upon their hydrophobicity and hydrophilicity values and the
relative similarity of the amino acid side-chain substituents,
including charge, size, and the like. Exemplary amino acid
substitutions which take various of the foregoing characteristics
into consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine.
[0082] The addition or deletion of an amino acid may be an addition
or deletion of from 2 to to 6 amino acids, such as from 2 to 4
amino acids. However, additions of more than 6 amino acids, such as
additions from 2 to 10 amino acids, are also comprised within the
present invention. In the multimeric forms additions/deletions may
be made individually in each monomer of the multimer.
[0083] As mentioned above, a compound of the invention may
comprise, essentially comprise or consist of at least one peptide
sequence containing the motif of the invention. Accordingly, a
peptide sequence of the compound may have different length.
[0084] Thus, an isolated individual peptide sequence of the
compound may consist of 6 or more amino acid residues. The
essential amino acid motif of the invention consists of 6 amino
acid residues therefore the minimal length of a peptide sequence is
6 amino acid residues. The upper limit for the number of amino acid
residues in an isolated contiguous peptide sequence of the compound
may vary from 7 to 50 and some cases may extend beyond 50 amino
acid residues to 100 amino acid residues. However, sequences of
25-35 amino acid residues in length or less, such as 20-25, 15-20,
or 10-15 amino acid residues, are within preferred embodiments of
the invention. Peptide sequences comprising the motif of the
invention and having the length from 7 to 15 amino acid residues
are referred herein as the sequences essentially comprising the
motif of the invention.
[0085] Thus, the invention preferably features compounds consisting
of or comprising at least one contiguous peptide sequence of 6-25
amino acid residues. Thus in one embodiment, a peptide sequence may
be from 10 to 25, such as for example from 14 to 25, in another
embodiment a sequence may have the length from 14 to 20, for
example from 14 to 18 amino acid residues. In some embodiments, the
peptide sequence of the invention may comprise more then 25 amino
acid residues, such as from 26 to 50 amino acid residues, for
example 28-30, 31-35, 36-40, 41-45 or 46-49 amino acid residues,
and in some cases the sequence may extends beyond 50 amino acid
residues, for example such as a sequence having the length of
51-55, 56-60, 61-65, 66-71 or 72-75 amino acid residues, or 75-85
amino acid residues.
[0086] A compound of the invention may comprise more then one of
the above peptide sequences. Thus, the sequences of above may be
formulated as monomers, which mean that they may be represented by
a single copy of an individual peptide sequence. A compound may
also comprise more then one copy of the same sequence. Thus, the
invention also relates to polymers of individual peptide sequences
of the above. A polymer of a peptide sequence may be formulated as
a contiguous peptide chain wherein an individual peptide sequence
is repeated/copied two or more times, or it may a molecule, wherein
the copies of an individual peptide sequence are connected to each
other then the peptide bond, for example via any kind of linker
grouping. An example of such polymers may be dendromeric polymers
wherein the individual copies of a peptide sequence are attached to
a core molecule, such as for example a lysine residue. Another
example of a polymer may be the LPA (Ligand Presenting Assembly)
type polymer. Polymerisation such as repetitive sequences or
attachment to various carriers are well-known in the art, e.g.
lysine backbones, such as lysine dendrimers carrying 4 peptides, 8
peptides, 16 peptides, or 32 peptides. Other carriers may be
protein moieties, such as bovine serum albumin (BSA), or lipophilic
dendrimers, or micelle-like carriers formed by lipophilic
derivatives, or starburst (star-like) carbon chain polymer
conjugates, or ligand presenting assembly (LPA) based on
derivatives of diethylaminomethane.
[0087] A compound of the invention may comprise or consists of two
or more individual peptide fragments having different amino acid
sequences presented as isolated peptide sequence.
Proteins and Peptide Fragments Thereof
[0088] According to the invention an isolated peptide sequence
which comprises, essentially comprises or consists of the motif
described above is one embodiment derived from human erythropoietin
(EPO). The invention relates to human erythropoietin having the
sequence identified in the Swissprot database under ID No. P01588.
In another embodiment the peptide sequence is derived from a
variant or a homologue of human erythropoietin.
[0089] Thus, the invention relates to proteins, sequences of which
have homology to the sequence of erythropoietin (Swissprot Ass. No.
P01588). The invention defines a protein as being a homologue of
human erythropoietin when the amino acid of said protein shares at
least 35% homology with the sequence of erythropoietin (Swissprot
Ass. No. P01588). The homology of one amino acid sequence with
another amino acid sequence is defined as a percentage of identical
amino acids in the two collated sequences. The homology between
amino acid sequences may be calculated using well known algorithms
such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55,
BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80,
BLOSUM 85, or BLOSUM 90.
[0090] Preferably the invention relates to human erythropoietin
structural homologues selected from [0091] thrombopoietin
(Swissprot Ass. No. P40225), 82% homology to erythropoitin, [0092]
granulocyte-macrophage colony-stimulating factor (GM-CSF, Swissprot
Ass. No. P04141) 63% homology to erythropoitin, [0093] ciliary
neurotrophic factor (CNFT, Swissprot Ass. No. P26441) 41% of
homology to erythropoitin, [0094] interleukin-6 (IL-6, Swissprot
Ass. No. P05231) 45% homology to erythropoitin, [0095]
interleukin-3 (IL-3, Swissprot Ass. No. P08700) 36% homology to
erythropoitin, [0096] interferon alpha-1 (Swissprot Ass. No.
P05231) 57% homology to erythropoitin, [0097] interferon beta
(Swissprot Ass. No. P01574) 59% homology to erythropoitin, [0098]
interferon gamma (Swissprot Ass. No. P05231) 41% homology to
erythropoitin, [0099] cell stem factor (Swissprot Ass. No. P21583)
37% homology to erythropoitin, [0100] multiple coagulation factor
deficiency protein 2 (MCD2, Swissprot Ass. No. Q8NI22) 38% homology
to erythropoitin.
[0101] According to the invention the above proteins are both
structural and functional homologues of human erythropoietin.
[0102] An amino acid sequence, which is 35-100%, preferably
45-100%, more preferably 50-100%, such as about 55, 60, 65, 70, 75,
80% homologous to the sequence of erythropoietin of Swissprot Ass.
No. P01588, and possesses at least one biological activity of said
erythropoietin is regarded by the application as a functional
homologue of erythropoietin. Non-limited examples of functional
homologues of human erythropoietin are given above. Examples of the
biological activities of human erythropoietin relevant for the
present application include, but not limited by stimulation of
proliferation, differentiation and survival of haemopoietic cells,
stimulating neuronal cell differentiation and survival. In a
preferred embodiment the application relates to biological
activities, which are most relevant for neural cells, such as the
capability of stimulating neuronal differentiation, for example
stimulating neurite outgrowth, stimulating neuronal survival, such
as inhibiting apoptosis of neural cells, and/or enhancing synaptic
plasticity, learning and memory. These biological functions of
erythropoietin are according to the invention to be executed by an
area(s) of the protein, which essentially comprise a
sequence(s)
TABLE-US-00013 (SEQ ID NO: 1) DSRVLERYLLEAKE (SEQ ID NO: 2)
NENITVPDTKVNFYAWKR (SEQ ID NO: 3) QLHVDKAVSGLRSLTTLLRA (SEQ ID NO:
4) RVYSNFLRGKLKLYTGEA.
[0103] The invention also contemplates variants of human
erythropoietin having the sequence of Swissprot Ass. No. P01588 as
functional homologues of the protein "Variant" in the present
content means i) erythropoietin-like polypeptides of non-human
origin, such as other species origin, ii) recombinant molecules of
human erythropoietin; iii) molecules of i) and ii) which comprise
amino acid substitutions or modifications of amino acids in
relation to the sequence of Ass. No. P01588, iv) polypeptides that
share 90-99% homology with the sequence of Ass. No. P01588, v)
fragments of the sequence of Ass. No. P01588, which are of 90-99%
of the sequence length, or vi) natural or recombinant human
erythropoietin molecules which have post-translational
modifications, such as for example additional/alternative
glycosylation of the polypeptide chain when compared to naturally
occurring erythropoietin polypeptides.
[0104] The sequences derived from human erythropoietin identified
as SEQ ID NOS: 1-4 all
1) comprise the motif of the invention
x.sup.1-x.sup.2-x.sup.3-x.sup.4-x.sup.5-x.sup.6 (SEQ ID NO:43),
[0105] wherein [0106] x.sup.1 is a charged amino acid residue,
[0107] x.sup.6 is a hydrophobic amino acid residue or A, [0108]
x.sup.2, x.sup.3, x.sup.4 and x.sup.5 is any amino acid residue,
and 2) possess neuritogenic activity, 3) possess survival promoting
activity, 4) possess synaptic plasticity enhancing activity, and/or
5) possess learning and memory enhancing activity, and 6) possess
cell proliferation stimulating activity
[0109] The invention considers the presence of the motif of the
invention in a peptide sequence to be essential structural element
to enable the peptide sequence to induce neuronal differentiation,
such as stimulate neurite growth, promote survival of neuronal
cells and/or enhance synaptic plasticity, learning capability and
improve memory. Accordingly, any peptide sequence of at least 6
amino acids comprising the above motif, having about 40-99%
structural homology to SEQ ID NO: 1, 2, 3 or 4 and having at least
one activity of the SEQ ID NO: 1, 2, 3 or 4 is considered to be a
functional homologue of SEQ ID NOS: 1, 2, 3 and 4. Homology between
two peptide sequences is defined as described above.
[0110] As described above, the invention preferably relates to
short contiguous peptide sequences of 6 to 25 amino acids long
comprising the motif of above. The invention further prefers those
of the latter peptide sequences that have at least 40% homology to
any of identified above fragments of EPO (SEQ ID NO: 1, 2, 3 or 4).
More preferred the sequences having about 50-55, 55-60 or 60-65%
homology. Yet, more preferred the sequences which are more then 65%
homologous to any of the EPO peptide fragments identified herein as
SEQ ID NOs: 1-4. Homology between sequences is identified as
described above. However, in some embodiments a sequence which has
homology to a particular sequence selected from SEQ ID NOS: 1, 2, 3
or 4, may be preferred. The invention discloses herein that the
sequences of SEQ ID NOS: 1-4 very in their activity in relation to
stimulating of neurite outgrowth, neural cell survival and cell
proliferation, therefore, depending on the purpose it may be
favourable to select a particular sequence to achieve the maximal
effect of the sequence on neurite outgrowth, cell survival or cell
proliferation.
[0111] Thus, in one embodiment of the invention, such preferred
sequence may be the sequence of SEQ ID NO: 1 or a sequence
homologous to SEQ ID NO: 1, which may be selected form the group
consisting of the sequences
TABLE-US-00014 (SEQ ID NO: 5) DLRVLSKLLRDSHV (SEQ ID NO: 6)
PSTQPWEHVNAIQEARR (SEQ ID NO: 7) CSRSIWLARKIRSD (SEQ ID NO: 8)
SERIDKQIRYILDGIS (SEQ ID NO: 9) SCNMIDEIITHLKQ (SEQ ID NO: 10)
SSCLMDRHDFGFPQEEFDGNQ (SEQ ID NO: 11) MSYNLLGFLQRSSNFQCQKLLWQLN
(SEQ ID NO: 12) CYCQDPYVKEAENLKKYFNA (SEQ ID NO: 37)
RNRVTNNVKDVTKLV (SEQ ID NO: 39) GLDKNTVHDQEHIMEHLEGV
[0112] The sequences homologous to SEQ ID NO: 2 may preferably be
selected form the group consisting of the sequences:
TABLE-US-00015 (SEQ ID NO: 3) QLHVDKAVSGLRSLTTLLRA (SEQ ID NO: 4)
RVYSNFLRGKLKLYTGEA. (SEQ ID NO: 5) DLRVLSKLLRDSHV (SEQ ID NO: 13)
PTPVLLPAVDFSLGEWKTQM (SEQ ID NO: 14) NETVEVISE (SEQ ID NO: 15)
NKNINLDSADGMPVASTD (SEQ ID NO: 16) AENNLNLPKMAEKD (SEQ ID NO: 17)
ENNLRRPNLEAFNRAVKS (SEQ ID NO: 18) QQIFNLFTTKDSSAAWDE (SEQ ID NO:
19) DRMNFDIPEEIKQLQQFQK (SEQ ID NO: 20) ADNGTLFLGILKNWKEESDR.
[0113] The sequences homologous to SEQ ID NO: 3 may preferably be
selected form the group consisting of the sequences:
TABLE-US-00016 (SEQ ID NO: 21) TAHKDPNAIFLSFQHLLRGKVRFL (SEQ ID NO:
22) QTRLELYKQGLRGSLTKLKGPLTM (SEQ ID NO: 23) LLQVAAFAYQIEELMILLEYK
(SEQ ID NO: 24) EEQARAVQMSTKVLIQ (SEQ ID NO: 25)
HIKDGDWNEFRRKLTFYLKT (SEQ ID NO: 26) LMNADSILAVKKYFRRITLY (SEQ ID
NO: 27) KLEKEDFTRGKLMSSLHLKR (SEQ ID NO: 28) NSNKKKRDDFEKLTNYSVTD
(SEQ ID NO: 40) SETSDCVVSSTLSPEKDSRV (SEQ ID NO: 41)
QLHYFKMHDYDGNNLL.
[0114] A sequence homologous to SEQ ID NO: 4 may preferably be
selected form the group consisting of the sequences:
TABLE-US-00017 (SEQ ID NO: 29) PNRTSGLLETNFTAS (SEQ ID NO: 30)
KDFLLVIPFDCWEPVQE (SEQ ID NO: 31) ELSQWTVRSIHDLRFISS (SEQ ID NO:
32) RSFKEFLQSSLR (SEQ ID NO: 33) FINRLTGYLRN (SEQ ID NO: 34)
ELSPAAKTGKR (SEQ ID NO: 35) SLIIGFAAGALYWKKRQPSL (SEQ ID NO: 36)
DELINIIDGVLRDDDKNND
[0115] The sequences of SEQ ID NOs: 5-41 are derived from the amino
acid sequences of the following human EPO homologues
thrombopoietin (Swissprot Ass. No. P40225), granulocyte-macrophage
colony-stimulating factor (GM-CSF, Swissprot Ass. No. P04141),
ciliary neurotrophic factor (CNFT, Swissprot Ass. No. P26441),
interleukin-6 (IL-6, Swissprot Ass. No. P05231), interleukin-3
(IL-3, Swissprot Ass. No. P08700), interferon alpha-1 (Swissprot
Ass. No. P05231), interferon beta (Swissprot Ass. No. P01574),
interferon gamma (Swissprot Ass. No. P05231). cell stem factor
(Swissprot Ass. No. P21583) multiple coagulation factor deficiency
protein 2 (MCD2, Swissprot Ass. No. Q8NI22)
[0116] The term "derived" in relation to an individual peptide
sequence means that said peptide sequence is originally an integral
part/fragment of a naturally occurring polypeptide/protein which
has been prepared as an isolated individual chemical entity by
means of biochemical or chemical methods, for example by enzymatic
cleavage of the original polypeptide or synthetic preparation of
the peptide sequence. The present patent application relates to
peptide sequences derived from the above identified proteins as
isolated individual peptide fragments of said proteins, and does
not relate to the original proteins/polypeptides sequences of which
include these fragments.
[0117] Individual peptide sequences/fragments of the invention
possess biological activity(s) that can mimic certain biological
activity(s) of the original polypeptide. Thus, the individual
peptide sequence/fragment of the invention may be used as a
functional equivalent/homologue of the predetermined polypeptide
selected from the above identified, or a functional equivalent of
human EPO or a biological fragment thereof.
Production of Individual Peptide Sequences
[0118] The peptide sequences of the present invention may be
prepared by any conventional synthetic methods, recombinant DNA
technologies, enzymatic cleavage of full-length proteins which the
peptide sequences are derived from, or a combination of said
methods.
Recombinant Preparation
[0119] Thus, in one embodiment the peptides of the invention are
produced by use of recombinant DNA technologies.
[0120] 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. 22:1859-1889, or the method described by Matthes
et al., 1984, EMBO J. 3:801-805. According to the phosphoamidine
method, oligonucleotides are synthesised, e.g. in an automatic DNA
synthesiser, purified, annealed, ligated and cloned in suitable
vectors.
[0121] 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, N.Y.,
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, N.Y., 1989.
[0122] 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 U.S. Pat. No. 4,683,202 or
Saiki et al., 1988, Science 239:487-491.
[0123] The DNA sequence is then inserted into a recombinant
expression vector, which may be any vector, which may conveniently
be subjected to recombinant DNA procedures. The choice of vector
will often depend on the host cell into which it is to be
introduced. Thus, the vector may be an autonomously replicating
vector, i.e. a vector that exists as an extrachromosomal entity,
the replication of which is independent of chromosomal replication,
e.g. a plasmid. Alternatively, the vector may be one which, when
introduced into a host cell, is integrated into the host cell
genome and replicated together with the chromosome(s) into which it
has been integrated.
[0124] In the vector, the DNA sequence encoding a peptide or a
full-length protein should be operably connected to a suitable
promoter sequence. The promoter may be any DNA sequence, which
shows transcriptional activity in the host cell of choice and may
be derived from genes encoding proteins either homologous or
heterologous to the host cell. Examples of suitable promoters for
directing the transcription of the coding DNA sequence in mammalian
cells are the SV 40 promoter (Subramani at al., 1981, Mol. Cell
Biol. 1:854-884), 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.
311:7-11). Suitable promoters for use in yeast host cells include
promoters from yeast glycolytic genes (Hitzeman et al., 1980, J.
Biol. Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. Mol.
Appl. Gen. 1: 419-434) or alcohol dehydrogenase genes (Young et
al., 1982, in Genetic Engineering of Microorganisms for Chemicals,
Hollaender et al, eds., Plenum Press, New York), or the TPI1 (U.S.
Pat. No. 4,599,311) or ADH2-4c (Russell et al., 1983, Nature
304:652-654) promoters. Suitable promoters for use in filamentous
fungus host cells are, for instance, the ADH3 promoter (McKnight et
al., 1985, EMBO J. 4:2093-2099) or the tpiA promoter.
[0125] 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 TPI1 (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).
[0126] The recombinant expression vector may further comprise a DNA
sequence enabling the vector to replicate in the host cell in
question. An example of such a sequence (when the host cell is a
mammalian cell) is the SV 40 origin of replication. The vector may
also comprise a selectable marker, e.g. a gene the product of which
complements a defect in the host cell, such as the gene coding for
dihydrofolate reductase (DHFR) or one which confers resistance to a
drug, e.g. neomycin, hydromycin or methotrexate.
[0127] The procedures used to ligate the DNA sequences coding the
peptides or full-length proteins, the promoter and the terminator,
respectively, and to insert them into suitable vectors containing
the information necessary for replication, are well known to
persons skilled in the art (cf., for instance, Sambrook et al.,
op.cit.).
[0128] To obtain recombinant peptides of the invention the coding
DNA sequences may be usefully fused with a second peptide coding
sequence and a protease cleavage site coding sequence, giving a DNA
construct encoding the fusion protein, wherein the protease
cleavage site coding sequence positioned between the HBP fragment
and second peptide coding DNA, inserted into a recombinant
expression vector, and expressed in recombinant host cells. In one
embodiment, said second peptide selected from, but not limited by
the group comprising glutathion-S-reductase, calf thymosin,
bacterial thioredoxin or human ubiquitin natural or synthetic
variants, or peptides thereof. In another embodiment, a peptide
sequence comprising a protease cleavage site may be the Factor Xa,
with the amino acid sequence IEGR, enterokinase, with the amino
acid sequence DDDDK, thrombin, with the amino acid sequence
LVPR/GS, or Acharombacter lyticus, with the amino acid sequence
XKX, cleavage site.
[0129] The host cell into which the expression vector is introduced
may be any cell which is capable of expression of the peptides or
full-length proteins, and is preferably a eukaryotic cell, such as
invertebrate (insect) cells or vertebrate cells, e.g. Xenopus
laevis oocytes or mammalian cells, in particular insect and
mammalian cells. Examples of suitable mammalian cell lines are the
HEK293 (ATCC CRL-1573), COS (ATCC CRL-1650), BHK (ATCC CRL-1632,
ATCC CCL-10) or CHO (ATCC CCL-61) cell lines. Methods of
transfecting mammalian cells and expressing DNA sequences
introduced in the cells are described in e.g. Kaufman and Sharp, J.
Mol. Biol. 159, 1982, pp. 601-621; Southern and Berg, 1982, J. Mol.
Appl. Genet. 1:327-341; Loyter et al., 1982, Proc. Natl. Acad. Sci.
USA 79: 422-426; Wigler et al., 1978, Cell 14:725; Corsaro and
Pearson, 1981, in Somatic Cell Genetics 7, p. 603; Graham and van
der Eb, 1973, Virol. 52:456; and Neumann et al., 1982, EMBO J.
1:841-845.
[0130] Alternatively, fungal cells (including yeast cells) may be
used as host cells. Examples of suitable yeast cells include cells
of Saccharomyces spp. or Schizosaccharomyces spp., in particular
strains of Saccharomyces cerevisiae. Examples of other fungal cells
are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora
spp., in particular strains of Aspergillus oryzae or Aspergillus
niger. The use of Aspergillus spp. for the expression of proteins
is described in, e.g., EP 238 023.
[0131] 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).
[0132] The peptides or full-length proteins recombinantly produced
by the cells may then be recovered from the culture medium by
conventional procedures including separating the host cells from
the medium by centrifugation or filtration, precipitating the
proteinaceous components of the supernatant or filtrate by means of
a salt, e.g. ammonium sulphate, purification by a variety of
chromatographic procedures, e.g. HPLC, ion exchange chromatography,
affinity chromatography, or the like.
Synthetic Preparation
[0133] The methods for synthetic production of peptides are well
known in the art. Detailed descriptions as well as practical advice
for producing synthetic peptides may be found in Synthetic
Peptides: A User's Guide (Advances in Molecular Biology), Grant G.
A. ed., Oxford University Press, 2002, or in: Pharmaceutical
Formulation: Development of Peptides and Proteins, Frokjaer and
Hovgaard eds., Taylor and Francis, 1999.
[0134] Peptides may for example be synthesised by using Fmoc
chemistry and with Acm-protected cysteins. After purification by
reversed phase HPLC, peptides may be further processed to obtain
for example cyclic or C- or N-terminal modified isoforms.
[0135] The methods for cyclization and terminal modification are
well-known in the art and described in detail in the above-cited
manuals.
[0136] In a preferred embodiment the peptide sequences of the
invention are produced synthetically, in particular, by the
Sequence Assisted Peptide Synthesis (SAPS) method.
[0137] By 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.
[0138] When synthesised, 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.
Antibody
[0139] It is an objective of the present invention to provide an
antibody, antigen binding fragment or recombinant protein thereof
capable of recognizing and selectively binding to an epitope on
human erythropoietin, thrombopoietin, granulocyte-macrophage
colony-stimulating factor (GM-CSF), ciliary neurotrophic factor
(CNFT), interleukin-.beta. (IL-.beta.), interleukin-3 (IL-3),
interferon alpha-1, interferon beta, interferon gamma, cell stem
factor, multiple coagulation factor deficiency protein 2 (MCD2),
said epitope comprising at least one of the sequences selected from
SEQ ID NOs:1-41, or a fragment of said sequence.
[0140] By the term "epitope" is meant the specific group of atoms
(on an antigen molecule) that is recognized by (that antigen's)
antibodies (thereby causing an immune response). The term "epitope"
is the equivalent to the term "antigenic determinant". The epitope
may comprise 3 or more amino acid residues, such as for example 4,
5, 6, 7, 8 amino acid residues, located in close proximity, such as
within a contiguous amino acid sequence, or located in distant
parts or the amino acid sequence of an antigen, but due to protein
folding have been approached to each other.
[0141] 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.
[0142] Native antibodies and immunoglobulins are usually
heterotetrameric glycoproteins of about 150,000 daltons, composed
of two identical light (L) chains and two identical heavy (H)
chains. Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies
between the heavy chains of different immunoglobulin isotypes. Each
heavy and light chain also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at one end a variable
domain (VH) followed by a number of constant domains. Each light
chain has a variable domain at one end (VL) and a constant domain
at its other end. The constant domain of the light chain is aligned
with the first constant domain of the heavy chain, and the light
chain variable domain is aligned with the variable domain of the
heavy chain. Particular amino acid residues are believed to form an
interface between the light and heavy chain variable domains
(Novotny J, & Haber E. Proc Natl Acad Sci USA. 82(14):4592-6,
1985).
[0143] Depending on the amino acid sequences of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are at least five (5) major classes of
immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these
may be further divided into subclasses (isotypes), e.g. IgG-1,
IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. The heavy chains constant
domains that correspond to the different classes of immunoglobulins
are called alpha (.alpha.), delta (.delta.), epsilon (.epsilon.),
gamma (.gamma.) and mu (.mu.), respectively. The light chains of
antibodies can be assigned to one of two clearly distinct types,
called kappa (.kappa.) and lambda (.lamda.), based on the amino
sequences of their constant domain. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
[0144] The term "variable" in the context of variable domain of
antibodies, refers to the fact that certain portions of the
variable domains differ extensively in sequence among antibodies.
The variable domains are for binding and determine the specificity
of each particular antibody for its particular antigen. However,
the variability is not evenly distributed through the variable
domains of antibodies. It is concentrated in three segments called
complementarity determining regions (CDRs) also known as
hypervariable regions both in the light chain and the heavy chain
variable domains.
[0145] The more highly conserved portions of variable domains are
called the framework (FR). The variable domains of native heavy and
light chains each comprise four FR regions, largely a adopting a
.beta.-sheet configuration, connected by three CDRs, which form
loops connecting, and in some cases forming part of, the
.beta.-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.
[0146] 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 preferred embodiments, in the context of both
the therapeutic and screening methods described below, an antibody
or fragment thereof is used that is immunospecific for an antigen
or epitope of the invention.
[0147] The term "antibody fragment" refers to a portion of a
full-length antibody, generally the antigen binding or variable
region. Examples of antibody fragments include Fab, Fab',
F(ab').sub.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').sub.2 fragment that has two
antigen binding fragments that are capable of cross-linking
antigen, and a residual other fragment (which is termed pFc').
Additional fragments can include diabodies, linear antibodies,
single-chain antibody molecules, and multispecific antibodies
formed from antibody fragments. As used herein, "functional
fragment" with respect to antibodies, refers to Fv, F(ab) and
F(ab').sub.2 fragments.
[0148] The term "antibody fragment" is used herein interchangeably
with the term "antigen binding fragment".
[0149] Antibody fragments may be as small as about 4 amino adds, 5
amino acids, 6 amino acids, 7 amino acids, 9 amino acids, about 12
amino acids, about 15 amino acids, about 17 amino acids, about 18
amino acids, about 20 amino acids, about 25 amino acids, about 30
amino acids or more. In general, an antibody fragment of the
invention can have any upper size limit so long as it is has
similar or immunological properties relative to antibody that binds
with specificity to an epitope comprising a peptide sequence
selected from any of the sequences identified herein as SEQ ID NOs:
1-41, or a fragment of said sequences. Thus, in context of the
present invention the term "antibody fragment" is identical to term
"antigen binding fragment".
[0150] Antibody fragments retain some ability to selectively bind
with its antigen or receptor. Some types of antibody fragments are
defined as follows: [0151] (1) Fab is the fragment that contains a
monovalent antigen-binding fragment of an antibody molecule. A Fab
fragment can be produced by digestion of whole antibody with the
enzyme papain to yield an intact light chain and a portion of one
heavy chain. [0152] (2) Fab' is the fragment of an antibody
molecule can be obtained by treating whole antibody with pepsin,
followed by reduction, to yield an intact light chain and a portion
of the heavy chain. Two Fab' fragments are obtained per antibody
molecule.
[0153] 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. [0154] (3) (Fab').sub.2 is the fragment of an antibody that
can be obtained by treating whole antibody with the enzyme pepsin
without subsequent reduction. [0155] (4) F(ab').sub.2 is a dimer of
two Fab' fragments held together by two disulfide bonds.
[0156] 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.sub.H-V.sub.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.sub.H-V.sub.L dimer. Collectively, the six CDRs confer antigen
binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding site.
[0157] (5) Single chain antibody ("SCA"), defined as a genetically
engineered molecule containing the variable region of the light
chain, the variable region of the heavy chain, linked by a suitable
polypeptide linker as a genetically fused single chain molecule.
Such single chain antibodies are also referred to as "single-chain
Fv" or "sFv" antibody fragments. Generally, the Fv polypeptide
further comprises a polypeptide linker between the VH and VL
domains that enables the sFv to form the desired structure for
antigen binding. For a review of sFv see Pluckthun in The
Pharmacology of Monoclonal Antibodies 113: 269-315 Rosenburg and
Moore eds. Springer-Verlag, NY, 1994.
[0158] The term "diabodies" refers to a small antibody fragments
with two antigen-binding sites, which fragments comprise a heavy
chain variable domain (VH) connected to a light chain variable
domain (VL) in the same polypeptide chain (VH-VL). By using a
linker that is too short to allow pairing between the two domains
on the same chain, the domains are forced to pair with the
complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161, and Hollinger et al., Proc. Natl.
Acad Sci. USA 90: 6444-6448 (1993).
[0159] 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.
[0160] 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.
[0161] The preparation of monoclonal antibodies likewise is
conventional. See, for example, Kohler & Milstein, Nature,
256:495-7 (1975); Coligan, et al., sections 2.5.1-2.6.7; and
Harlow, et al., in: Antibodies: A Laboratory Manual, page 726, Cold
Spring Harbor Pub. (1988), Monoclonal antibodies can be isolated
and purified from hybridoma cultures by a variety of
well-established techniques. Such isolation techniques include
affinity chromatography with Protein-A Sepharose, size-exclusion
chromatography, and ion-exchange chromatography. See, e.g.,
Coligan, et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3;
Barnes, et al., Purification of Immunoglobulin G (IgG), in: Methods
in Molecular Biology, 1992, 10:79-104, Humana Press, NY.
[0162] Methods of in vitro and in vivo manipulation of monoclonal
antibodies are well known to those skilled in the art. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler and Milstein, 1975, Nature 256, 495-7, or may be made by
recombinant methods, e.g., as described in U.S. Pat. No. 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 Mol Biol 222: 581-597. Another method
involves humanizing a monoclonal antibody by recombinant means to
generate antibodies containing human specific and recognizable
sequences. See, for review, Holmes, et al., 1997, J Immunol
158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma
& Immunol 81:105-115.
[0163] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies. i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional polyclonal
antibody preparations that typically include different antibodies
directed against different determinants (epitopes), each monoclonal
antibody is directed against a single determinant on the antigen.
In additional to their specificity, the monoclonal antibodies are
advantageous in that they are synthesized by the hybridoma culture,
uncontaminated by other immunoglobulins. The modifier "monoclonal"
indicates the character of the antibody 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.
[0164] 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 (U.S. Pat. No. 4,816,567); Morrison et
al., 1984, Proc Natl Acad Sci 81: 6851-6855.
[0165] Methods of making antibody fragments are also known in the
art (see for example, Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, NY, 1988, incorporated
herein by reference). Antibody fragments of the present invention
can be prepared by proteolytic hydrolysis of the antibody or by
expression in E. coli of DNA encoding the fragment. Antibody
fragments can be obtained by pepsin or papain digestion of whole
antibodies conventional methods. For example, antibody fragments
can be produced by enzymatic cleavage of antibodies with pepsin to
provide a 5S fragment denoted F(ab').sub.2. This fragment can be
further cleaved using a thiol reducing agent, and optionally a
blocking group for the sulfhydryl groups resulting from cleavage of
disulfide linkages, to produce 3.5S Fab' monovalent fragments.
Alternatively, an enzymatic cleavage using pepsin produces two
monovalent Fab' fragments and an Fc fragment directly. These
methods are described, for example, in U.S. Pat. No. 4,036,945 and
U.S. Pat. No. 4,331,647, and references contained therein. These
patents are hereby incorporated in their entireties by
reference.
[0166] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody. For
example, Fv fragments comprise an association of V.sub.H and
V.sub.L chains. This association may be noncovalent or the variable
chains can be linked by an intermolecular disulfide bond or
cross-linked by chemicals such as glutaraldehyde. Preferably, the
Fv fragments comprise V.sub.H and V.sub.L chains connected by a
peptide linker. These single-chain antigen binding proteins (sFv)
are prepared by constructing a structural gene comprising DNA
sequences encoding the V.sub.H and V.sub.L domains connected by an
oligonucleotide. The structural gene is inserted into an expression
vector, which is subsequently introduced into a host cell such as
E. coli. The recombinant host cells synthesize a single polypeptide
chain with a linker peptide bridging the two V domains. Methods for
producing sFvs are described, for example, by Whitlow, et al.,
1991, in: Methods: A Companion to Methods in Enzymology, 2:97; Bird
et al., 1988, Science 242:423-426; U.S. Pat. No. 4,946,778; and
Pack, et al., 1993, BioTechnology 11:1271-77.
[0167] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") are often involved in antigen
recognition and binding. CDR peptides can be obtained by cloning or
constructing genes encoding the CDR of an antibody of interest.
Such genes are prepared, for example, by using the polymerase chain
reaction to synthesize the variable region from RNA of
antibody-producing cells. See, for example, Larrick, et al.,
Methods: a Companion to Methods in Enzymology, Vol. 2, page 106
(1991).
[0168] The invention contemplates human and humanized forms of
non-human (e.g. murine) antibodies. Such humanized antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) that contain a minimal
sequence derived from non-human immunoglobulin, such as the epitope
recognising sequence. For the most part, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a nonhuman species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. Humanized antibody(es)
containing a minimal sequence(s) of antibody(es) of the invention,
such as a sequence(s) recognising the epitope(s) described herein,
is one of the preferred embodiments of the invention.
[0169] In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are
found neither in the recipient antibody nor in the imported CDR or
framework sequences. These modifications are made to further refine
and optimize antibody performance. In general, humanized antibodies
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details, see: Jones et al., 1986, Nature 321, 522-525; Relchmann et
al., 1988. Nature 332, 323-329; Presta, 1992, Curr Op Struct Biol
2:593-596; Holmes et al., 1997, J Immunol 158:2192-2201 and
Vaswani, et al., 1998, Annals Allergy, Asthma & Immunol
81:105-115.
[0170] The generation of antibodies may be achieved by any standard
methods in the art for producing polyclonal and monoclonal
antibodies using natural or recombinant fragments of human
erythropoietin, thrombopoietin, granulocyte-macrophage
colony-stimulating factor (GM-CSF), ciliary neurotrophic factor
(CNFT), interleukin-6 (IL-6), interleukin-3 (IL-3), interferon
alpha-1, interferon beta, interferon gamma, cell stem factor,
multiple coagulation factor deficiency protein 2 (MCD2) comprising
a sequence selected from any of the sequences identified as SEQ ID
NO: 1-41 as an antigen. Such antibodies may be also generated using
variants or fragments of peptide fragments of SEQ ID NOs:1-41, said
fragments being immunogenic fragments which meet at least two of
the following criteria: [0171] (i) being a contiguous amino acid
sequence of at least 6 amino acids; [0172] (ii) comprising an amino
acid sequence derived from the sequence of human human
erythropoietin, thrombopoietin, granulocyte-macrophage
colony-stimulating factor (GM-CSF), ciliary neurotrophic factor
(CNFT), interleukin-6 (IL-6), interleukin-3 (IL-3), interferon
alpha-1, interferon beta, interferon gamma, cell stem factor or
multiple coagulation factor deficiency protein 2 (MCD2).
[0173] 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.
[0174] 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.
[0175] The invention relates both to an antibody, which is capable
of modulating, such as enhancing or attenuating, biological
function of human erythropoietin, thrombopoietin,
granulocyte-macrophage colony-stimulating factor (GM-CSF), ciliary
neurotrophic factor (CNFT), interleukin-6 (IL-6), interleukin-3
(IL-3), interferon alpha-1, interferon beta, interferon gamma, cell
stem factor or multiple coagulation factor deficiency protein 2
(MCD2), in particular a function related to neural cell growth,
differentiation, survival and/or plasticity, and to an antibody,
which can recognise and specifically bind the latter proteins
without modulating biological activity thereof.
[0176] The invention relates to use of the above antibodies for 1)
therapeutic applications when the modulation of activity of human
erythropoietin, thrombopoietin, granulocyte-macrophage
colony-stimulating factor (GM-CSF), ciliary neurotrophic factor
(CNFT), interleukin-6 (IL-6), interleukin-3 (IL-3), interferon
alpha-1, interferon beta, interferon gamma, cell stem factor or
multiple coagulation factor deficiency protein 2 (MCD2) is needed,
2) detecting and/or monitoring the latter proteins in vitro and/or
in vivo for diagnostic purposes, 3) research purposes.
Pharmaceutical Composition
[0177] The invention also relates to a pharmaceutical composition
comprising one or more of the compounds defined above, wherein the
compound is capable of stimulating neurite outgrowth and/or neural
cell differentiation, survival of neural cells and/or stimulating
learning and/or memory. Thus, the invention concerns a
pharmaceutical composition capable of stimulating differentiation
of neuronal cells and/or stimulating regeneration of neuronal
cells, and/or stimulating neuronal plasticity in connection with
learning and memory, and/or stimulating survival of neural
cells.
[0178] In the present context the term "pharmaceutical composition"
is used synonymously with the term "medicament".
[0179] In a composition the peptide sequences may be formulated as
comprising isolated individual peptide fragments or multimers or
dimers thereof as discussed above.
[0180] The pharmaceutical composition may have the described above
effects on cells in vitro or in vivo, wherein the composition is
administered to a subject.
[0181] The medicament of the invention comprises an effective
amount of one or more of the compounds as defined above, or a
composition as defined above in combination with the
pharmaceutically acceptable additives. Such medicament may suitably
be formulated for oral, percutaneous, intramuscular, intravenous,
intracranial, intrathecal, intracerebroventricular, intranasal or
pulmonal administration.
[0182] 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.
[0183] Injectables are usually prepared either as liquid solutions
or suspensions, solid forms suitable for solution in, or suspension
in, liquid prior to injection. The preparation may also be
emulsified. The active ingredient is often mixed with excipients
which are pharmaceutically acceptable and compatible with the
active ingredient. Suitable excipients are, for example, water,
saline, dextrose, glycerol, ethanol or the like, and combinations
thereof. In addition, if desired, the preparation may contain minor
amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, or which enhance the effectiveness or
transportation of the preparation.
[0184] 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.
[0185] The preparation may suitably be administered by injection,
optionally at the site, where the active ingredient is to exert its
effect. Additional formulations which are suitable for other modes
of administration include suppositories, nasal, pulmonal and, in
some cases, oral formulations. For suppositories, traditional
binders and carriers include polyalkylene glycols or triglycerides.
Such suppositories may be formed from mixtures containing the
active ingredient(s) in the range of from 0.5% to 10%, preferably
1-2%. Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, and the like. These compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders and generally contain 10-95% of the active
ingredient(s), preferably 25-70%.
[0186] Other formulations are such suitable for nasal and pulmonal
administration, e.g. inhalators and aerosols.
[0187] 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.
[0188] The preparations are administered in a manner compatible
with the dosage formulation, and in such amount as will be
therapeutically effective. The quantity to be administered depends
on the subject to be treated, including, e.g. the weight and age of
the subject, the disease to be treated and the stage of disease.
Suitable dosage ranges are per kilo body weight normally of the
order of several hundred .mu.g active ingredient per administration
with a preferred range of from about 0.1 .mu.g to 5000 .mu.g per
kilo body weight. Using monomeric forms of the compounds, the
suitable dosages are often in the range of from 0.1 .mu.g to 5000
.mu.g per kilo body weight, such as in the range of from about 0.1
.mu.g to 3000 .mu.g per kilo body weight, and especially in the
range of from about 0.1 .mu.g to 1000 .mu.g per kilo body weight.
Using multimeric forms of the compounds, the suitable dosages are
often in the range of from 0.1 .mu.g to 1000 .mu.g per kilo body
weight, such as in the range of from about 0.1 .mu.g to 750 .mu.g
per kilo body weight, and especially in the range of from about 0.1
.mu.g to 500 .mu.g per kilo body weight such as in the range of
from about 0.1 .mu.g to 250 .mu.g per kilo body weight. In
particular when administering nasally smaller dosages are used than
when administering by other routes. Administration may be performed
once or may be followed by subsequent administrations. The dosage
will also depend on the route of administration and will vary with
the age and weight of the subject to be treated. A preferred dosage
of multimeric forms would be in the interval 1 mg to 70 mg per 70
kg body weight.
[0189] For some indications a localised or substantially localised
application is preferred.
[0190] Some of the compounds of the present invention are
sufficiently active, but for some of the others, the effect will be
enhanced if the preparation further comprises pharmaceutically
acceptable additives and/or carriers. Such additives and carriers
will be known in the art. In some cases, it will be advantageous to
include a compound, which promotes delivery of the active substance
to its target.
[0191] In many instances, it will be necessary to administrate the
formulation multiple times. Administration may be a continuous
infusion, such as intraventricular infusion or administration in
more doses such as more times a day, daily, more times a week,
weekly, etc. It is preferred that administration of the medicament
is initiated before or shortly after the individual has been
subjected to the factor(s) that may lead to cell death. Preferably
the medicament is administered within 8 hours from the factor
onset, such as within 6 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.
[0192] In connection with the use in nerve guides, the
administration may be continuous or in small portions based upon
controlled release of the active compound(s). Furthermore,
precursors may be used to control the rate of release and/or site
of release. Other kinds of implants and well as oral administration
may similarly be based upon controlled release and/or the use of
precursors.
[0193] As discussed above, the present invention relates to
treatment of individuals for inducing differentiation, stimulating
regeneration, plasticity and survival of neural cells in vitro or
in vivo, said treatment involving administering an effective amount
of one or more compounds as defined above.
[0194] Another strategy for administration is to implant or inject
cells capable of expressing and secreting the compound in question.
Thereby the compound may be produced at the location where it is
going to act.
Treatment
[0195] In a further aspect, the present invention relates to said
peptides, fragments, or variants thereof for use in the induction
of differentiation and/or stimulation of regeneration, plasticity
and/or survival of neural cells. The use is for the treatment for
preventing diseases and conditions of the central and peripheral
nervous system, and of the muscles or of various organs.
[0196] Treatment by the use of the compounds/compositions according
to the invention is in one embodiment useful for inducing
differentiation, modulating proliferation, stimulate regeneration,
neuronal plasticity and survival of cells being implanted or
transplanted. This is particularly useful when using compounds
having a long term effect.
[0197] Thus, the treatment comprises treatment and/or prophylaxis
of cell death in relation to diseases or conditions of the central
and peripheral nervous system, such as postoperative nerve damage,
traumatic nerve damage, e.g. resulting from spinal cord injury,
impaired myelination of nerve fibers, postischaemic damage, e.g.
resulting from a stroke, multiinfarct dementia, multiple sclerosis,
nerve degeneration associated with diabetes mellitus,
neuro-muscular degeneration, schizophrenia, Alzheimer's disease,
Parkinson's disease, or Huntington's disease.
[0198] Also, in relation to diseases or conditions of the muscles
including conditions with impaired function of neuro-muscular
connections, such as genetic or traumatic atrophic muscle
disorders; or for the treatment of diseases or conditions of
various organs, such as degenerative conditions of the gonads, of
the pancreas, such as diabetes mellitus type I and II, of the
kidney, such as nephrosis the compounds according to the invention
may be used for inducing differentiation, modulating proliferation,
stimulate regeneration, neuronal plasticity and survival, i.e.
stimulating survival.
[0199] In yet a further embodiment the use of the compound and/or
pharmaceutical composition is for the stimulation of the ability to
learn and/or of the short and/or long term memory.
[0200] In particular the compound and/or pharmaceutical composition
of the invention may be used in the treatment of clinical
conditions, such as psychoses, such as senile and presenile organic
psychotic conditions, alcoholic psychoses, drug psychoses,
transient organic psychotic conditions, Alzheimer's disease,
cerebral lipidoses, epilepsy, general paresis [syphilis],
hepatolenticular degeneration, Huntington's chorea,
Jakob-Creutzfeldt disease, multiple sclerosis, Pick's disease of
the brain, syphilis, Schizophrenic disorders, affective psychoses,
neurotic disorders, personality disorders, including character
neurosis, nonpsychotic personality disorder associated with organic
brain syndromes, paranoid personality disorder, fanatic
personality, paranoid personality (disorder), paranoid traits,
sexual deviations and disorders, mental retardation, disease in the
nerve system and sense organs, cognitive anomalies, inflammatory
disease of the central nervous system, such as meningitis,
encephalitis, Cerebral degenerations such as Alzheimer's disease,
Pick's disease, senile degeneration of brain, communicating
hydrocephalus, obstructive hydrocephalus, Parkinson's disease
including other extra pyramidal disease and abnormal movement
disorders, spinocerebellar disease, cerebellar ataxia, Marie's,
Sanger-Brown, Dyssynergia cerebellar is myoclonica, primary
cerebellar degeneration, such as spinal muscular atrophy, familial,
juvenile, adult spinal muscular atrophy, motor neuron disease,
amyotrophic lateral sclerosis, motor neuron disease, progressive
bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, other
anterior horn cell diseases, anterior horn cell disease,
unspecified, other diseases of spinal cord, syringomyelia and
syringobulbia, vascular myelopathies, acute infarction of spinal
cord (embolic) (nonembolic), arterial thrombosis of spinal cord,
edema of spinal cord, subacute necrotic myelopathy, subacute
combined degeneration of spinal cord in diseases classified
elsewhere, myelopathy, drug-induced, radiation-induced myelitis,
disorders of the autonomic nervous system, disorders of peripheral
autonomic, sympathetic, parasympathetic, or vegetative system,
familial dysautonomla [Riley-Day syndrome], idiopathic peripheral
autonomic neuropathy, carotid sinus syncope or syndrome, cervical
sympathetic dystrophy or paralysis; peripheral autonomic neuropathy
in disorders classified elsewhere, amyloidosis, diseases of the
peripheral nerve system, brachial plexus lesions, cervical rib
syndrome, costoclavicular syndrome, scalenus anterior syndrome,
thoracic outlet syndrome, brachial neuritis or radiculitis,
including in newborn. Inflammatory and toxic neuropathy, including
acute infective polyneuritis, Guillain-Barre syndrome,
Postinfectious polyneuritis, polyneuropathy in collagen vascular
disease, disorders affecting multiple structures of eye, purulent
endophthalmitis, diseases of the ear and mastoid process,
abnormality of organs and soft tissues in newborn, including in the
nerve system, complications of the administration of anesthetic or
other sedation in labor and delivery, diseases in the skin
including infection, insufficient circulation problem, injuries,
including after surgery, crushing injury, burns. Injuries to nerves
and spinal cord, including division of nerve, lesion in continuity
(with or without open wound), traumatic neuroma (with or without
open wound), traumatic transient paralysis (with or without open
wound), accidental puncture or laceration during medical procedure,
injury to optic nerve and pathways, optic nerve injury, second
cranial nerve, injury to optic chiasm, injury to optic pathways,
injury to visual cortex, unspecified blindness, injury to other
cranial nerve(s), injury to other and unspecified nerves. Poisoning
by drugs, medicinal and biological substances, genetic or traumatic
atrophic muscle disorders; or for the treatment of diseases or
conditions of various organs, such as degenerative conditions of
the gonads, of the pancreas, such as diabetes mellitus type I and
II, of the kidney, such as nephrosis.
[0201] A further aspect of the invention is a process of producing
a pharmaceutical composition, comprising mixing an effective amount
of one or more of the compounds of the invention, or a
pharmaceutical composition according to the invention with one or
more pharmaceutically acceptable additives or carriers, and
administer an effective amount of at least one of said compound, or
said pharmaceutical composition to a subject.
[0202] In one embodiment of the process as mentioned above, the
compounds are used in combination with a prosthetic device, wherein
the device is a prosthetic nerve guide. Thus, in a further aspect,
the present invention relates to a prosthetic nerve guide,
characterised in that it comprises one or more of the compounds or
the pharmaceutical composition as defined above. Nerve guides are
known in the art.
[0203] Another aspect of the invention relates to the use of a
compound as defined above. In particular the use of a compound
according to the invention is for the production of a
pharmaceutical composition. The pharmaceutical composition is
preferably for the treatment or prophylaxis of any of the diseases
and conditions mentioned above.
[0204] In yet a further aspect the invention relates to a method of
treating a disease or condition as discussed above by administering
a compound as defined herein.
EXAMPLES
Example 1
Stimulation of Neurite Outgrowth In Vitro
Methods:
[0205] Cerebellar granule neuron (CGN) cultures were obtained from
3-4-day-old Wistar rats (Charles River, Sulzfeld, Germany, or
Moellegaard, Denmark) as previously described by Schousboe et al.
(1989). Briefly, the cerebella were dissected, cleared of meninges
and blood vessels, chopped, and trypsinized. The neurons were
washed in the presence or DNAse 1 and soybean trypsin inhibitor
(Sigma), and cellular debris was pelleted by centrifugation; the
cells resuspended and then plated on poly-L-lysine (PLL;
Sigma)-coated or uncoated microtiter plates in Neurobasal medium
supplemented with 4% (w/v) bovine serum albumin (BSA), 2% (v/v)
B27, 1% (v/v) glutamax, 100 U/ml penicillin, 100 g/ml streptomycin,
4.5 g D-glucose/L (Sigma) 0.25% (v/v) sodium pyruvate, and 2% (v/v)
1 M HEPES (Gibco BRL).
Results and Conclusions
[0206] Three out of four Epo fragments (SEQ ID NO: 2, 3, and 4)
strongly induce neurite outgrowth from CGN in a dose dependent
manner, whereas the recombinant Epo protein (rhEPO) and the peptide
having the sequence of SEQ ID NO: 1 do not have any neuritogenic
activity. The effect of 24 h treatment of CGN cultures with the
peptides and rhEPO is demonstrated in FIGS. 1-5.
Example 2
Stimulation of Survival of Neural Cells In Vitro
Methods:
[0207] Primary cultures of CGN were plated at a density of 100 000
cells/cm.sup.2 on poly-L-lysine coated 8-well permanox slides in
Neurobasal-A medium (Gibco BRL) supplemented with 2% (v/v) B27,
0.5%(v/v) glutamax, 100 units/mL penicillin, 100 .mu.g/mL
streptomycin and KCl, making the final concentration of KCl in the
medium 40 mm. Twenty-four hours after plating, Ara-C(Sigma-Aldrich)
was added to a final concentration of 10 .mu.m to avoid
proliferation of glial cells, after which the neurons were allowed
to differentiate for a further 6 days at 37.degree. C. Apoptotic
cell death was induced by washing twice and changing the medium to
Basal Medium Eagle (BME; Gibco BRL) supplemented with 1% (v/v)
glutamine, 100 U/mL penicillin and 100 .mu.g/mL streptomycin, 3.5 g
d-glucose/L and 1% (v/v) sodium pyruvate (Gibco BRL) together with
various concentrations of peptide. Thereby the concentration of
potassium in the cultures was reduced to 5 mm KCl (D'Mello et al.
1993). Two days after induction of apoptosis, the cells were fixed
with 4% (v/v) formaldehyde and stained with Hoechst 33258 as
described for the survival assay employing hippocampal neurons.
Results and Conclusions:
[0208] All Epo-derived peptides and the recombinant Epo protein are
capable of protection of CGN induced to undergo apoptosis. The
effect is dose-dependent The Epo-derived peptides 2, 3 and 4 (SEQ
ID NOs: 2-4) have the strongest neuroprotective effect, which is
comparable to the effect of insulin growth factor-1 (IGF-1), a
known strong promoter of cell survival The effect of the treatment
of CGN cultures with the peptides is demonstrated in FIGS.
6-10.
Example 3
Stimulation of Proliferation of Haemopoietic Cells
Methods:
[0209] Evaluation of the effect of four EPO-derived peptides on
cell proliferation was performed using a TF-1 cell line. The TF-1
cell line was established from sample from patient with severe
pancytopenia (human erythroleukemia). The growth of these cells is
dependent of the presence of the granulocyte-macrophage
colony-stimulating factor (GM-SCF). The TF-1 cells were grown in
Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v)
heat inactivated foetal calf serum (FCS), 2 mM GlutaMAX, penicillin
(100 U/ml) and streptomycin (100 .mu.g/ml), 10 mM HEPES, 1 mM
sodium pyruvate, 1% fungizone. The growth medium was supplemented
with 2 ng/ml GM-CSF. Cell proliferation was determined using a
Biotrak ELISA system, version 2 (Amersham Biosciences, UK). The
procedure is based on measurement of the levels of
5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis
in proliferating cells. Briefly, passaged cells were washed once
with starving medium (culturing medium without GM-CSF) then plated
in 96-well Nuclon culture microplates (Nunc, Denmark) at a density
of 5000 cells/well and grown in starving medium containing 0.008,
0.04, 0.2, 1.0, 5.0, 25 and 50 .mu.g/ml of the tested compounds. As
a positive control the human recombinant erythropoietin (EPO)
(Calbiochem, Denmark) was used in a concentration of 2.0 ng/ml.
TF-1 cells were allowed to grow in the presence of peptides for 24
h and then to all wells (except negative controls) BrdU was added
to a final concentration of 10 .mu.M and cultures were further
incubated for 24 h, fixed and processed according to the procedure
recommended by manufacturer.
Results/Conclusions:
[0210] The recombinant Epo protein in a concentration of 2 ng/ml
stimulates proliferation of TF-1 cells. The Epo-derived peptides,
Epo3 and Epo4, (SEQ ID NOs:3 and 4) stimulate proliferation of TF-1
cells in a concentration of 50 .mu.g/ml, but not in concentration
25 .mu.g/ml or below. The Epo-derived peptide 2, Epo2, (SEQ ID
NO:2) stimulates proliferation of TF-1 cells in concentrations of
25 and 50 .mu.g/ml, but not in concentration below 25 .mu.g/ml. The
Epo-derived peptide 1, Epo1, stimulates proliferation of TF-1 cells
in a concentration of 50 .mu.g/ml and probably had a stimulatory
effect in a concentration of 0.2 .mu.g/ml.
General Conclusion
[0211] All four tested Epo peptides Epo1, 2, 3 and 4 (SEQ ID
NOs:1-4) possess biological activity. However, biological activity
of Epo fragments and the full-length recombinant Epo protein is
different. Thus, peptides Epo2, 3 and 4 are capable of stimulating
neurite outgrowth, whereas the full-length recombinant Epo protein
does not possess neurite stimulatory activity, nor does the Epo1
peptide. However, all peptides and the Epo protein as well are
potent in stimulating neuronal cells survival. All peptides and the
Epo protein are also capable of stimulating haemopoietic cell
proliferation, most potent among them being the Epo peptides 3 and
4. Interestingly, biological activity of different Epo peptides
depends on their concentration in cell medium. Thus, the
neuritogenic activity of both Epo peptides 2, 3 and 4 is higher
when the peptides are present in low concentrations, whereas
stimulation of cell proliferation demands the presence of
significantly larger amounts of the peptides.
Example 4
Therapeutic Effects of Erythropoietin Mimetic Peptides Epo 2, 3 and
4 in Rat Brain In Vivo
Experimental Setup:
[0212] Male rats (wistar) (tree groups, 5 rats/group) were injected
s.c with different Epo peptides (each group individually with Epo
2, Epo 3 or Epo 4) three times according to the scheme: [0213] 1.
the day before the lesion: a single injection of a peptide per
animal (1 mg/100 g animal weight); [0214] 2. the 1.sup.st day after
the lesion: a single injection of a peptide per animal (1 mg/100 g
animal weight); [0215] 3. the 2.sup.nd day after the lesion: a
single injection of a peptide per animal (1 mg/100 g animal
weight); [0216] 4. the 3.sup.rd day after the lesion: animals are
sacrificed.
[0217] Control groups were a group (4 rats) injected with the
vehicle (water) and a group (4 rats), injected with human
recombinant EPO (hrEPO) in dose 50 .mu.g/100 g rat according to the
above scheme.
Methods
1. Traumatic Brain Injury
[0218] A traumatic brain injury (TBI) was induced by applying dry
ice (-78 C) to the extracranial surface of the skull for 60 sec,
which induces a focal injury in the right fronto-parietal cortex.
Three days after the lesion (3 dpl) rats were deeply anesthetized
with Brietal (10 mg/100 gr) and fixed by transcardial perfusion
with 0.9% NaCl and 0.3% heparin, followed by Zamboni's fixative.
The brains were removed, dehydrated according to the standard
protocol, embedded in paraffin, and cut in 6-.mu.m-thick frontal
sections for further evaluation by immunohistochemistry and
TUNEL.
2. Immunocytochemistry
[0219] For immunohistochemistry the sections of the brains were
incubated overnight with polyclonal rabbit anti-cow GFAP, GAP-43,
.beta.-oxoguanine, or PSA-NCAM antibodies 1:250 (Dakopalts). The
primary antibodies were detected in the sections with biotinylated
secondary antibodies followed by incubation with
streptavidin-biotin-peroxidase (30 min). Afterwards, the sections
were incubated with biotinylated tyramide and
streptavidin-peroxidase complex (NEN, Life Science Products, USA,
code NEL700A) prepared following the manufacturer recommendations,
and visualized with 0.015% H.sub.2O.sub.2 in DAB/TBS for 10
min.
3. TUNEL (In Situ Detection of DNA Fragmentation):
[0220] Terminal deoxynucleotidyl transferase (TdT)-mediated
biotin-linked deoxynucleotide nick end labeling (TUNEL) was
performed using the Fragment End Labeling (FragEL.TM.) Detection
Kit (Calbiochem, USA, code QIA33). The FragEL kit contains all the
materials used below and each step was performed according to the
manufacturer recommendations. The tissue was processed and
rehydrated as mentioned above, and sections were incubated with 20
.mu.g/ml proteinase K for 20 min to strip off nuclear proteins.
After immersion in equilibration buffer for 20 min, sections were
incubated with TdT and biotin-labeled deoxynucleotides
(dNTP-biotin) in a humified chamber at 37.degree. C. for 1.5 hr.
This was followed by washing with the buffer and stop solution for
5 min at room temperature to stop the reaction. After washing in
TBS and incubation in blocking buffer for 10 min, the sections were
incubated with Peroxidase-Streptavidin for 30 min and afterwards,
DAB was used as chromogen. The sections were counterstained with
methyl-green.
[0221] 1. The EPO mimetics dearly increase astrocytosis in the rat
brain after TBI as judged by GFAP IHC. The most pronounced increase
in reactive astrogllosis is obtained after treating the rats with
the Epo 2 peptide; and then with the Epo 3 peptide. The Epo 4
peptide has the least effect. Treatment with the EPO protein
results in slightly increased astrocytosis in comparison to control
rats (treated with water), which does increases astrocytosis very
slightly, if any.
[0222] 2. The Epo peptides inhibit programmed cell death in the
areas surrounding the necrotic cavity after TBI. All groups show
TUNEL-positive apoptotic cells after TBI, but the numbers of dying
cells is the highest in control group (treated with water) compared
to the rats receiving the EPO protein and Epo 2 peptide. The lowest
level of apoptosis is observed in Epo 3 and Epo 4 treated groups of
rats.
[0223] 3. Immunostaining with a marker of oxidative DNA damage,
8-oxoguanine, demonstrates a higher number of the 8-oxoguanine
positive cells in the control group. The EPO and Epo 4 treated rats
have a significantly reduced oxidative DNA damage relative to
control, and the lowest number of the 8-oxoguanine positive cells
is observed in Epo 2 and Epo 3 treated rats.
[0224] 4. EPO mimetic peptides stimulate neuroplasticity and
neurite outgrowth in vivo after TBI of rat brain as judged by
PSA-NCAM and GAP-43 immunostaining of rat brain tissue sections.
The Epo 3 and Epo 4 treatment significantly increases the level of
PSA-NCAM staining both in the area adjusted to the lesion, of
Neural Stem Cells (NSC) and in the subventricular zone (SVZ),
whereas only a few cells in the lesion area of control rats are
PSA-NCAM positive. Treatment with Epo 2 and Epo 3 also stimulates
neurite outgrowth in the area of cortical lesion and in the choroid
plexus, hippocampus, and hypothalamus.
[0225] 4. The hematocrit (%) significantly increases after EPO
treatment (63.7+-1.9 EPO vs 48.7+-3.35 control, p<0.02). All
peptide mimetics Epo 2, Epo 3 and Epo 4 have no significant effect
on the hematocrit (Ep2, Ep3, Ep4 vs control p>0.02). The results
are demonstrated in FIG. 15.
Example 5
Binding of the Epo 3 Peptide to Recombinant Erythropoietin Receptor
(EPOR-Fc)
[0226] The binding of Epo 3 to EPOR-Fc was studied by SPR analysis.
The Epo 3 peptide was immobilized on a chip and the chip was
incubated with soluble recombinant EPOR-Fc under standard
conditions. Three independent experiments were performed. The
results show that EPOR binds to the immobilized Epo 3 (ionic bonded
with the chip) with association rate
k.sub.a=2.1*10.sup.4+/-1.4*10.sup.4 and dissociation rate
k.sub.d4.33*10.sup.-5+/-1.62*10.sup.-5. The affinity of the binding
of EPOR to Ep3 is 4.48 nM. Epo 3 covalently bonded to the chip does
not bind to EPOR. The results of binding are shown on FIG. 16.
Sequence CWU 1
1
52114PRTArtificial sequenceDerived from human erythropoietin, Swiss
Prot P01588 1Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys
Glu 1 5 10218PRTArtificial sequenceDerived from human
erythropoietin, Swiss Prot P01588 2Asn Glu Asn Ile Thr Val Pro Asp
Thr Lys Val Asn Phe Tyr Ala Trp 1 5 10 15 Lys Arg320PRTArtificial
sequenceDerived from human erythropoietin, Swiss Prot P01588 3Gln
Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr 1 5 10
15 Leu Leu Arg Ala 20418PRTArtificial sequenceDerived from human
erythropoietin, Swiss Prot P01588 4Arg Val Tyr Ser Asn Phe Leu Arg
Gly Lys Leu Lys Leu Tyr Thr Gly 1 5 10 15 Glu Ala514PRTArtificial
sequenceDerived from human thrombopoietin, Swiss Prot P40225. 5Asp
Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val 1 5
10617PRTArtificial sequenceDerived from human GM-CSF, Swiss Prot
P04141 6Pro Ser Thr Gln Pro Trp Glu His Val Asn Ala Ile Gln Glu Ala
Arg 1 5 10 15 Arg714PRTArtificial sequenceDerived from human CNFT,
Swiss Prot P26441 7Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg
Ser Asp 1 5 10816PRTArtificial sequenceDerived from human
interleukin-6, Swiss Prot P05231 8Ser Glu Arg Ile Asp Lys Gln Ile
Arg Tyr Ile Leu Asp Gly Ile Ser 1 5 10 15914PRTArtificial
sequenceDerived from human interleukin-3, Swiss Prot P08700 9Ser
Cys Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln 1 5
101021PRTArtificial sequenceDerived from human interferon alpha-1,
Swiss Prot P05231 10Ser Ser Cys Leu Met Asp Arg His Asp Phe Gly Phe
Pro Gln Glu Glu 1 5 10 15 Phe Asp Gly Asn Gln 201124PRTArtificial
sequenceDerived from human interferon beta, Swiss Prot P01574 11Met
Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10
15 Cys Gln Lys Leu Leu Trp Gln Leu 201220PRTArtificial
sequenceDerived from human interferon gamma, Swiss Prot P05231
12Cys Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys 1
5 10 15 Tyr Phe Asn Ala 201320PRTArtificial sequenceDerived from
human thrombopoietin, Swiss Prot P40225 13Pro Thr Pro Val Leu Leu
Pro Ala Val Asp Phe Ser Leu Gly Glu Trp 1 5 10 15 Lys Thr Gln Met
20149PRTArtificial sequenceDerived from human GM-CSF, Swiss Prot
P04141 14Asn Glu Thr Val Glu Val Ile Ser Glu 1 51518PRTArtificial
sequenceDerived from human CNFT, Swiss Prot P26441 15Asn Lys Asn
Ile Asn Leu Asp Ser Ala Asp Gly Met Pro Val Ala Ser 1 5 10 15 Thr
Asp1614PRTArtificial sequenceDerived from human interleukin-6,
Swiss Prot P05231 16Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Ala Glu
Lys Asp 1 5 101718PRTArtificial sequenceDerived from human
interleukin-3, Swiss Prot P08700 17Glu Asn Asn Leu Arg Arg Pro Asn
Leu Glu Ala Phe Asn Arg Ala Val 1 5 10 15 Lys Ser1818PRTArtificial
sequenceDerived from human interferon alpha-1, Swiss Prot P05231
18Gln Gln Ile Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala Trp 1
5 10 15 Asp Glu1919PRTArtificial sequenceDerived from human
interferon beta, Swiss Prot P01574 19Asp Arg Met Asn Phe Asp Ile
Pro Glu Glu Ile Lys Gln Leu Gln Gln 1 5 10 15 Phe Gln
Lys2020PRTArtificial sequenceDerived from human interferon gamma,
Swiss Prot P05231 20Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys
Asn Trp Lys Glu 1 5 10 15 Glu Ser Asp Arg 202124PRTArtificial
sequenceDerived from human thrombopoietin, Swiss Prot P40225 21Thr
Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu 1 5 10
15 Leu Arg Gly Lys Val Arg Phe Leu 202224PRTArtificial
sequenceDerived from human GM-CSF, Swiss Prot P04141 22Gln Thr Arg
Leu Glu Leu Tyr Lys Gln Gly Leu Arg Gly Ser Leu Thr 1 5 10 15 Lys
Leu Lys Gly Pro Leu Thr Met 202321PRTArtificial sequenceDerived
from human CNFT, Swiss Prot P26441 23Leu Leu Gln Val Ala Ala Phe
Ala Tyr Gln Ile Glu Glu Leu Met Ile 1 5 10 15 Leu Leu Glu Tyr Lys
202416PRTArtificial sequenceDerived from human interleukin-6, Swiss
Prot P05231 24Glu Glu Gln Ala Arg Ala Val Gln Met Ser Thr Lys Val
Leu Ile Gln 1 5 10 152520PRTArtificial sequenceDerived from human
interleukin-3, Swiss Prot P08700 25His Ile Lys Asp Gly Asp Trp Asn
Glu Phe Arg Arg Lys Leu Thr Phe 1 5 10 15 Tyr Leu Lys Thr
202620PRTArtificial sequenceDerived from human interferon alpha-1,
Swiss Prot P05231 26Leu Met Asn Ala Asp Ser Ile Leu Ala Val Lys Lys
Tyr Phe Arg Arg 1 5 10 15 Ile Thr Leu Tyr 202720PRTArtificial
sequenceDerived from human interferon beta, Swiss Prot P01574 27Lys
Leu Glu Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser Leu 1 5 10
15 His Leu Lys Arg 202820PRTArtificial sequenceDerived from human
interferon gamma, Swiss Prot P05231. 28Asn Ser Asn Lys Lys Lys Arg
Asp Asp Phe Glu Lys Leu Thr Asn Tyr 1 5 10 15 Ser Val Thr Asp
202915PRTArtificial sequenceDerived from human thrombopoietin,
Swiss Prot P40225 29Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe
Thr Ala Ser 1 5 10 153017PRTArtificial sequenceDerived from human
GM-CSF, Swiss Prot P04141 30Lys Asp Phe Leu Leu Val Ile Pro Phe Asp
Cys Trp Glu Pro Val Gln 1 5 10 15 Glu3118PRTArtificial
sequenceDerived from human CNFT, Swiss Prot P26441 31Glu Leu Ser
Gln Trp Thr Val Arg Ser Ile His Asp Leu Arg Phe Ile 1 5 10 15 Ser
Ser3212PRTArtificial sequenceDerived from human interleukin-6,
Swiss Prot P05231 32Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg
1 5 103311PRTArtificial sequenceDerived from human interferon beta,
Swiss Prot P01574 33Phe Ile Asn Arg Leu Thr Gly Tyr Leu Arg Asn 1 5
103411PRTArtificial sequenceDerived from human interferon gamma,
Swiss Prot P05231 34Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg 1 5
103520PRTArtificial sequenceDerived from human cell stem factor,
Swiss Prot P21583 35Ser Leu Ile Ile Gly Phe Ala Ala Gly Ala Leu Tyr
Trp Lys Lys Arg 1 5 10 15 Gln Pro Ser Leu 203619PRTArtificial
sequenceDerived from human multiple coagulation factor deficiency
protein 2 (neural stem cell derived neuronal survival
protein/MCD2), Swiss Prot P21583Q8NI22 36Asp Glu Leu Ile Asn Ile
Ile Asp Gly Val Leu Arg Asp Asp Asp Lys 1 5 10 15 Asn Asn
Asp3715PRTArtificial sequenceDerived from human cell stem factor,
Swiss Prot P21583 37Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr
Lys Leu Val 1 5 10 153816PRTArtificial sequenceDerived from human
cell stem factor, Swiss Prot P21583 38Asp Lys Leu Val Asn Ile Val
Asp Asp Leu Val Glu Cys Val Lys Glu 1 5 10 153920PRTArtificial
sequenceDerived from human multiple coagulation factor deficiency
protein 2 (neural stem cell derived neuronal survival
protein/MCD2), Swiss Prot P21583Q8NI22 39Gly Leu Asp Lys Asn Thr
Val His Asp Gln Glu His Ile Met Glu His 1 5 10 15 Leu Glu Gly Val
204020PRTArtificial sequenceDerived from human cell stem factor,
Swiss Prot P21583 40Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu
Ser Pro Glu Lys 1 5 10 15 Asp Ser Arg Val 204116PRTArtificial
sequenceDerived from human multiple coagulation factor deficiency
protein 2 (neural stem cell derived neuronal survival
protein/MCD2), Swiss Prot P21583Q8NI22 41Gln Leu His Tyr Phe Lys
Met His Asp Tyr Asp Gly Asn Asn Leu Leu 1 5 10 1542193PRTHomo
sapiens 42Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu
Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala
Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr
Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys
Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp
Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Gly
Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser
Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110
Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115
120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys
Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu
Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg
Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr
Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg 436PRTArtificial
sequenceMotif sequence 43Xaa Xaa Xaa Xaa Xaa Xaa 1
5446PRTArtificial sequenceMotif sequence 44Arg Ser Xaa Xaa Xaa Leu
1 5456PRTArtificial sequenceMotif sequence 45Arg Val Xaa Xaa Xaa
Ala 1 5466PRTArtificial sequenceMotif sequence 46Arg Val Leu Xaa
Xaa Tyr 1 5476PRTArtificial sequenceMotif sequence 47Lys Ala Val
Xaa Xaa Leu 1 5486PRTArtificial sequenceMotif sequence 48Arg Xaa
Leu Xaa Xaa Leu 1 5496PRTArtificial sequenceMotif sequence 49Arg
Ser Leu Xaa Xaa Leu 1 5506PRTArtificial sequenceMotif sequence
50Arg Val Xaa Xaa Xaa Tyr 1 5516PRTArtificial sequenceMotif
sequence 51Lys Val Xaa Xaa Xaa Ala 1 5526PRTArtificial
sequenceMotif sequence 52Lys Ala Xaa Xaa Xaa Leu 1 5
* * * * *