U.S. patent application number 16/399971 was filed with the patent office on 2019-08-15 for novel feline erythropoietin receptor agonists.
This patent application is currently assigned to AskGene Pharma Inc.. The applicant listed for this patent is AskGene Pharma Inc.. Invention is credited to Donggou He, Jian-Feng Lu, Yuefeng Lu, Kurt Shanebeck, Aijun Wang, Chen Yao.
Application Number | 20190248858 16/399971 |
Document ID | / |
Family ID | 55525142 |
Filed Date | 2019-08-15 |
United States Patent
Application |
20190248858 |
Kind Code |
A1 |
Lu; Jian-Feng ; et
al. |
August 15, 2019 |
NOVEL FELINE ERYTHROPOIETIN RECEPTOR AGONISTS
Abstract
The present specification discloses erythropoietin receptor
agonists, compositions and medicaments comprising such
erythropoietin receptor agonists, methods and uses for such
erythropoietin receptor agonists and compositions and medicaments,
and methods and uses for erythropoietin receptor agonists and
compositions and medicaments for treating an anemia.
Inventors: |
Lu; Jian-Feng; (Oak Park,
CA) ; Lu; Yuefeng; (Newbury Park, CA) ; Wang;
Aijun; (Sammamish, WA) ; He; Donggou;
(Camarillo, CA) ; Shanebeck; Kurt; (Camarillo,
CA) ; Yao; Chen; (Moorpark, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AskGene Pharma Inc. |
Camarillo |
CA |
US |
|
|
Assignee: |
AskGene Pharma Inc.
Camarillo
CA
|
Family ID: |
55525142 |
Appl. No.: |
16/399971 |
Filed: |
April 30, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14857799 |
Sep 17, 2015 |
10287336 |
|
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16399971 |
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62052407 |
Sep 18, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/30 20130101;
A61K 38/00 20130101; C07K 14/505 20130101 |
International
Class: |
C07K 14/505 20060101
C07K014/505 |
Claims
1. A modified non-human mammalian erythropoietin comprising a
non-human mammalian erythropoietin modified to have and at least
one added and/or at least one relocated glycosylation site.
2. The modified non-human mammalian erythropoietin according to
claim 1, wherein the a non-human mammalian erythropoietin comprises
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO: 11, a
sequence having at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 5
or SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO: 11, wherein Xaa at
position 18 of SEQ ID NO: 5 or SEQ ID NO: 6 is selected from E or
G; and Xaa at position 116 of SEQ ID NO: 5 or SEQ ID NO: 6 is K or
absent.
3. The modified non-human mammalian erythropoietin according to
claim 2, wherein an asparagine residue is substituted for the amino
acid residue at any one or more of positions 30, 51, 57, 69, 88,
89, 137, or 139 of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 10;
or wherein an asparagine residue is substituted for the amino acid
residue at any one or more of positions 56, 77, 83, 95, 114, 115,
163, or 165 of SEQ ID NO: 11.
4. The modified non-human mammalian erythropoietin according to
claim 2, wherein in SEQ ID NO: 5 or SEQ ID NO: 6, or SEQ ID NO: 10
either a serine or a threonine residue is substituted for the amino
acid residue at position 126 or wherein in SEQ ID NO: 11 either a
serine or threonine residue is substituted for the amino acid
residue at position 152.
5. The modified non-human mammalian erythropoietin according to
claim 2, wherein in SEQ ID NO: 5 or SEQ ID NO: 6, or SEQ ID NO: 10
one or more of the following amino acid substitution sets is
present: Asn.sup.30 and Thr.sup.32; Asn.sup.51 and Thr.sup.53;
Asn.sup.57, Thr.sup.59 and Asn.sup.69; Asn.sup.69 and Thr.sup.71;
Ser.sup.68, Asn.sup.69 and Thr.sup.71; Val.sup.87 and Asn.sup.88;
Ser.sup.87 and Asn.sup.88; Ser.sup.87, Asn.sup.88 and Gly.sup.89;
Ser.sup.87, Asn.sup.88 and Thr.sup.92; Ser.sup.87, Asn.sup.88 and
Ala.sup.163; Asn.sup.69, Thr.sup.71, Ser.sup.87 and Asn.sup.88;
Asn.sup.30, Thr.sup.32, Val.sup.87 and Asn.sup.88; Asn.sup.89,
Ile.sup.90 and Thr.sup.91; Ser.sup.87, Asn.sup.89, Ile.sup.90 and
Thr.sup.91; Asn.sup.137 and Thr.sup.139; Asn.sup.139 and
Thr.sup.141; Thr.sup.126; Pro.sup.125 and Thr.sup.126; or any
combination thereof; and wherein in SEQ ID NO: 11 one or more of
the following amino acid substitution sets is present: Asn.sup.56
and Thr.sup.58; Asn.sup.77 and Thr.sup.79; Asn.sup.83 and
Thr.sup.85; Asn.sup.69; Asn.sup.95 and Thr.sup.97; Ser.sup.94,
Asn.sup.95 and Thr.sup.97; Val.sup.113 and Asn.sup.114; Ser.sup.113
and Asn.sup.114; Ser.sup.113, Asn.sup.114 and Gly.sup.15;
Ser.sup.113, Asn.sup.114 and Thr.sup.118, Ser.sup.113, Asn.sup.114
and Ala.sup.189; Asn.sup.95, Thr.sup.97, Ser.sup.113 and
Asn.sup.114; Asn.sup.56, Thr.sup.58, Val.sup.113 and Asn.sup.114;
Asn.sup.115, Ile.sup.116 and Thr.sup.117; Ser.sup.113, Asn.sup.115,
Ile.sup.116 and Thr.sup.117; Asn.sup.163 and Thr.sup.165;
Asn.sup.165 and Thr.sup.167; Thr.sup.152; and/or Pro.sup.151 and
Thr.sup.152 or any combination thereof.
6. The modified non-human mammalian erythropoietin according to
claim 5, wherein in SEQ ID NO: 5 or SEQ ID NO: 6, or SEQ ID NO: 10
one or more of the following substitution sets is present:
Ser.sup.87 and Asn.sup.88; and/or Asn.sup.30, Thr.sup.32,
Val.sup.87 and Asn.sup.88; and wherein in SEQ ID NO: 11 one or more
of the following substitution sets is present: Ser.sup.113 and
Asn.sup.114 and/or Asn.sup.56, Thr.sup.58, Val.sup.113 and
Asn.sup.114.
7. The modified non-human mammalian erythropoietin according to
claim 5, wherein in SEQ ID NO: 5 or SEQ ID NO: 6, or SEQ ID NO: 10
one of the following amino acid substitution sets is present:
Gln.sup.24, Ser.sup.87 and Asn.sup.88; Gln.sup.38, Ser.sup.87 and
Asn.sup.88; or Gln.sup.83, Ser.sup.87 and Asn.sup.88; and wherein
in SEQ ID NO: 11 one or more of the following substitution sets is
present: Gln.sup.50, Ser.sup.113 and Asn.sup.114; Gln.sup.64,
Ser.sup.113 and Asn.sup.114; or Gln.sup.109, Ser.sup.113 and
Asn.sup.114.
8. The modified non-human mammalian erythropoietin according to
claim 1, wherein SEQ ID NO: 5 or SEQ ID NO: 6, SEQ ID NO: 10 or SEQ
ID NO: 11 is modified by deleting one or more of the glycosylation
sites that attach to N linked carbohydrate chains.
7. The modified non-human mammalian erythropoietin according to
claim 1, wherein the at least one added and/or at least one
relocated glycosylation site is a site for the attachment of an
N-linked carbohydrate chain or a site for the attachment of an
O-linked carbohydrate chain.
8. The modified non-human mammalian erythropoietin according to
claim 1, wherein the glycosylation site further comprises a
fragment of human chorionic gonadotropin.
9. The modified non-human mammalian erythropoietin according to
claim 8, wherein the fragment of human chorionic gonadotropin is
SEQ ID NO: 8 or an amino acid sequence having at least 90%, at
least 95%, at least 96%, at least 97%, at least 98% or at least 99%
sequence identity to SEQ ID NO: 8, or an amino acid sequence having
1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5
amino acid additions, deletions or substitutions to SEQ ID NO:
8.
10. A nucleic acid sequence encoding a modified non-human mammalian
erythropoietin as defined in claim 1.
11. A vector comprising a) a nucleic acid sequence encoding the
amino acid sequence for a modified non-human mammalian
erythropoietin as defined in claim 1, and b) a promoter operatively
linked to the nucleic acid sequence.
12. A host cell comprising the vector as defined in claim 11.
13. A composition comprising a) a modified non-human mammalian
erythropoietin as defined in claim 1, and b) a pharmaceutically
acceptable diluent, adjuvant, or carrier.
14. A method of treating non-regenerative anemia (NRA) in a mammal
comprising administering to a subject in need thereof an effective
amount of a modified non-human mammalian erythropoietin as defined
claim 1.
15. A fusion protein, comprising a peptide, a linker and an Fc
fragment, wherein: a) the peptide is SEQ ID NO: 1, an amino acid
sequence having at least 90%, at least 95%, at least 96%, at least
97%, at least 98% or at least 99% sequence identity to SEQ ID NO:
1, a feline EPO with the amino acid sequence SEQ ID NO: 5 or SEQ ID
NO: 6, wherein Xaa at position 18 is selected from E or G; and Xaa
at position 116 is K or absent, a sequence having at least 90%, at
least 95%, at least 96%, at least 97%, at least 98% or at least 99%
sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6, wherein Xaa at
position 18 is selected from E or G; and Xaa at position 116 is K
or absent, a canine EPO with the amino acid sequence SEQ ID NO: 10
or SEQ ID NO: 11, or a sequence having at least 90%, at least 95%,
at least 96%, at least 97%, at least 98% or at least 99% sequence
identity to SEQ ID NO: 10 or SEQ ID NO: 11; and b) the Fc fragment
has an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID
NO: 4, or a sequence having at least 90%, at least 95%, at least
96%, at least 97%, at least 98% or at least 99% sequence identity
to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; wherein the peptide
is fused, through linker, to the Fc fragment.
16. The fusion protein according to claim 15, wherein the C
terminus of the peptide is fused through the linker to the N
terminus of the Fc fragment or wherein the N terminus of the
peptide is fused through the linker to the C terminus of the Fc
fragment.
17. The fusion protein according to claim 15, modified to have and
at least one added and/or at least one relocated glycosylation
site.
18. A nucleic acid sequence encoding the fusion protein as defined
in claim 15.
19. A composition comprising: a) a fusion protein as defined in
claim 15, and b) a pharmaceutically acceptable diluent, adjuvant,
or carrier.
20. A method of treating non-regenerative anemia (NRA) in a mammal
comprising administering to a subject in need thereof an effective
amount of a fusion protein as defined in claim 15.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This patent application is a divisional of U.S. patent
application Ser. No. 14/857,799, filed Sep. 17, 2015 which claims
the right of priority pursuant to 35 U.S.C. .sctn. 119(e) and is
entitled to the benefit of the filing date of U.S. Provisional
Patent Application 62/052,407, filed on Sep. 18, 2014, the content
of which is hereby expressly incorporated by reference in its
entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The entire content of the following electronic submission of
the sequence listing via the USPTO EFS-WEB server, as authorized
and set forth in MPEP .sctn. 1730 II.B.2(a)(C), is incorporated
herein by reference in its entirety for all purposes. The sequence
listing is identified on the electronically filed text file as
follows: File Name: SEQLIST-3IPAG3-0003US; Date of Creation: Sep.
15, 2014; Size (bytes): 17 KB.
INTRODUCTION
[0003] Non-regenerative anemia (NRA) is a serious disease in cats,
dogs and other mammals and there are no adequate therapies.
Erythropoietin (EPO) is a glycoprotein hormone and is the most
important hormone regulating erythropoiesis (red blood cell
production). Recombinant human EPO products such as the
non-glycosylated EPOGEN.RTM. and the glycosylated ARANESP.RTM.
(Darbepoetin) have been commercially available to treat NRA in
human. Human EPO is a heavily glycosylated protein with a molecular
mass of 30.4 kD. The 5 exons of the EPO gene encode 193 amino
acids, 27 of which are later cleaved off to produce a 166 amino
acid long peptide, although the circulating peptide contains 165
amino acids. Its mature, circulating, structure includes a
165-amino acid backbone with three N-linked carbohydrates attached
to asparagines at amino acid positions 24, 38, and 83 and one
O-linked carbohydrate attached to Ser.sup.126. Sixty percent of the
EPO molecular mass is contributed by the 165 amino acids and 40% is
contributed by the carbohydrate. The carbohydrates probably cover
much of the surface of the molecule since they have extended and
flexible structure.
[0004] The carbohydrate residues allow for many possible isoforms
and contribute significantly to the serum half-life and biological
activity of the hormone in vivo. The isoforms with increased sialic
acid content have longer serum half-life and reduced receptor
binding affinity. For EPO, the serum half-life is the primary
determinant of in vivo activity. Darbepoetin was created through
site directed mutation of two amino acid residues, allowing for two
additional N-linked carbohydrate chains. The relative serum
half-life, receptor binding affinity and biological activity are
illustrated in FIG. 1.
[0005] It is estimated that there are over 0.5-1 million of
domestic cats with NRA in US. Based on a veterinary survey, a
number of conditions are associated with and/or can lead to NRA in
cats including chronic renal failure (51%), cancer (16%),
retroviral disease (11%), hyperthyroidism (5%), inflammatory bowel
disease (4%), as well as other miscellaneous chronic conditions
(6%) or multiple miscellaneous conditions (6%). Similar to human,
chronic renal diseases and cancers are two main causes of NRA.
[0006] There is currently no adequate treatment for NRA in cats.
Internal surveys have indicated that currently available treatments
for NRA include EPOGEN.RTM. or other non-glycosylated human
recombinant EPO (39%), the glycosylated recombinant EPO
ARANESP.RTM. (5.7%), corticosteroids (32.5%), blood transfusions
(6.7%), anabolic steroids (1.4%), as well as other miscellaneous
treatments (5.3%). However, antibodies often developed in some of
the cats against those human proteins, which resulted in serious
conditions including pure red-cell aplasia (PRCA) in 25-30% of
cats. ARANESP.RTM. may have low occurrence of PRCA in cats,
estimated to be less than 10%, which is still significant. The
second most frequently used option is steroids, which often result
in serious side effects and dubious therapeutic efficacy. Blood
transfusion is inconvenient and expensive. Thus, there is currently
a serious unmet veterinary need for cats.
[0007] No species-specific EPO is commercially available for cat as
of today. Feline EPO is only about 83% similar to that of the human
EPO. Recombinant feline EPO was produced and tested in cats.
Unfortunately, development of red cell aplasia was observed at a
similar rate as that of recombinant human EPO. Thus, the addition
of unmodified recombinant feline EPO would not address this unmet
veterinary need.
[0008] Peptide-based erythropoietin receptor (EpoR) agonists have
also been developed, such as disclosed in U.S. Pat. No. 6,703,480.
In a small clinical trial, a peptide-based EpoR agonist was also
shown to be effective for red cell aplasia in human. It is believed
that the peptide-based EpoR agonist would not induce red cell
aplasia as it does not share any sequence similarities to that of
EPO. However, no studies have been reported using the peptide-based
EpoR agonist in veterinary applications. In addition,
hypersensitivity was also reported in a small number of patients
using the pegylated peptide-based EpoR.
[0009] Present specification discloses novel EpoR agonists for
cats, dogs and other mammals. In one embodiment, modifications are
carried out on feline EPO to add one, two, three and/or more
N-linked glycosylation sites, which allow for lower immunogenicity
and higher serum half-life comparing to that of the feline EPO or
human EPO. In another embodiment, peptide EpoR agonists are fused
to feline IgG Fc to form fusion proteins, which are safe and
effective in treating cat NRA.
SUMMARY
[0010] Aspects of the present specification disclose a modified
non-human mammalian erythropoietin. The disclosed modified
non-human mammalian erythropoietin is modified by adding or
relocating at least one glycosylation site. The disclosed modified
non-human mammalian erythropoietin may be a feline erythropoietin
or a canine erythropoietin or a variant thereof. The added or
relocated glycosylation site may be located at the carboxy terminal
region of the mammalian erythropoietin or inserted between amino
acids located in the mammalian erythropoietin sequence. A disclosed
modified non-human mammalian erythropoietin may further comprise a
fragment of human chorionic gonadotropin and/or at least one
additional carbohydrate chain attached to a disclosed modified
non-human mammalian erythropoietin.
[0011] Other aspects of the present specification disclose a
nucleic acid sequence encoding a disclosed modified non-human
mammalian erythropoietin as well as vectors comprising such nucleic
acid sequences and hosts containing such vectors.
[0012] Other aspects of the present specification disclose a
composition comprising a disclosed modified non-human mammalian
erythropoietin. Such compositions further comprise one or more
pharmaceutically acceptable diluents, adjuvants, or carriers.
[0013] Other aspects of the present specification disclose a use of
a disclosed modified non-human mammalian erythropoietin in the
manufacture of a medicament for the treatment of non-regenerative
anemia (NRA).
[0014] Other aspects of the present specification disclose a method
of treating non regenerative anemia (NRA) in a mammal. A method of
treating disclosed herein comprising administering to a subject in
need thereof an effective amount of a disclosed modified non-human
mammalian erythropoietin. The treated mammal may be a cat, a dog, a
mouse, a rat, a hamster, a rabbit, a guinea pig, a ruminant, a
ferret, a non-human primate, or a pig.
[0015] Other aspects of the present specification disclose a use of
a disclosed modified non-human mammalian erythropoietin for the
treatment of non-regenerative anemia (NRA).
[0016] Other aspects of the present specification disclose a fusion
protein, comprising a peptide, a linker and an Fc fragment where
the peptide is fused through the linker to the Fc fragment. A
disclosed peptide may be one having an amino acid sequence having
at least 70% sequence identity to SEQ ID NO: 1, a feline EPO with
the amino acid sequence SEQ ID NO: 5, a sequence having at least
70%, sequence identity to SEQ ID NO: 5, a modified feline EPO with
the amino acid sequence SEQ ID NO: 6, or at least 70% sequence
identity to SEQ ID NO: 6. A disclosed Fc fragment has an amino acid
sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a
sequence having at least 70% sequence identity to SEQ ID NO: 2, SEQ
ID NO: 3 or SEQ ID NO: 4. The C terminus of the peptide may be
fused through the linker to the N terminus of the Fc fragment or
the N terminus of the peptide may be fused through the linker to
the C terminus of the Fc fragment.
[0017] Other aspects of the present specification disclose a
nucleic acid sequence encoding a disclosed fusion protein as well
as vectors comprising such nucleic acid sequences and hosts
containing such vectors.
[0018] Other aspects of the present specification disclose a
composition comprising a disclosed fusion protein. Such
compositions further comprise one or more pharmaceutically
acceptable diluents, adjuvants, or carriers.
[0019] Other aspects of the present specification disclose a use of
a disclosed fusion protein in the manufacture of a medicament for
the treatment of non-regenerative anemia (NRA).
[0020] Other aspects of the present specification disclose a method
of treating non-regenerative anemia (NRA) in a mammal. A method of
treating disclosed herein comprising administering to a subject in
need thereof an effective amount of a disclosed fusion protein. The
treated mammal may be a cat, a dog, a mouse, a rat, a hamster, a
rabbit, a guinea pig, a ruminant, a ferret, a non-human primate, or
a pig.
[0021] Other aspects of the present specification disclose a use of
a disclosed fusion protein for the treatment of non-regenerative
anemia (NRA).
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows biochemical and biological properties of
recombinant modified EPO (rHuEPO) containing four and five N-linked
carbohydrate chains.
[0023] FIG. 2A shows an embodiment disclosed herein where an EpoR
agonist peptide is fused, optionally through a peptide linker, to
the N-terminals of the Fc domain; FIG. 2B shows an embodiment
disclosed herein where an EpoR agonist peptide is fused, optionally
through a peptide linker, to the C-terminals of the Fc domain.
[0024] a peptide disclosed herein is fused to the N-terminals or
the C-terminals of the Fc domain
[0025] FIG. 3 shows results of a batch culture of clones.
[0026] FIG. 4A shows a viable cell density graph over time; FIG. 4B
shows a percent viability graph over time; and FIG. 4C shows titer
levels over time.
[0027] FIG. 5 shows the results of a TF1 EPO bioassay.
DETAILED DESCRIPTION
[0028] Disclosed herein are EpoR protein agonists which are
modified non-human mammalian EPOs comprising a non-human mammalian
erythropoietin sequence having at least one added and/or at least
one relocated glycosylation site. The disclosed EpoR protein
agonists have reduced probability for inducing red cell aplasia and
having a longer half-lives in serum, which allow for weekly dosing.
Such, EpoR protein agonists initiate, facilitate or enhance a
biological activity. For example, an EpoR protein agonist can be a
biologically active ligand which binds to its complementary
biologically active receptor and activates the latter either to
cause a biological response in the receptor, or to enhance
preexisting biological activity of the receptor.
[0029] Non-regenerative anemia (NRA) refers to a decrease in
erythrocyte production. Anemia, the condition of having lower than
normal numbers of red blood cells called erythrocytes, can have
many clinical manifestations. Affected animals often suffer from
decreased energy levels, pale or yellowed gums, an increased heart
rate, and an intolerance to exercise. NRA in cats may be caused by
chronic kidney diseases, feline leukemia virus, feline
immunodeficiency virus, and cancer.
[0030] Pure red cell aplasia (PRCA or RCA) or erythroblastopenia
refers to a type of anemia affecting the precursors to red blood
cells but not to white blood cells. In PRCA, the bone marrow ceases
to produce red blood cells. In cats, prior treatment with, for
example, recombinant human EPO may lead to generation of antibodies
against both the external human EPO and endogenous feline EPO,
which can result in RCA in cats.
[0031] An EpoR protein agonist disclosed herein is a modified
non-human mammalian erythropoietin having at least one additional
site for glycosylation, and/or at least one relocation of at least
one site for glycosylation. The added sites for glycosylation may
result in a greater number of carbohydrate chains, and higher
sialic acid content, than the native erythropoietin. Modified
non-human mammalian erythropoietin comprising amino acid sequences
which include the rearrangement of at least one site for
glycosylation are also provided. Modified non-human mammalian
erythropoietin comprising an addition of one or more amino acids to
the carboxy terminal end of erythropoietin wherein the addition
provides at least one glycosylation site are also included.
[0032] The present specification encompasses modified non-human
mammalian EPO. A modified non-human erythropoietin is one having
sufficient homology to a native mammalian EPO or a fragment of a
native mammalian EPO such that the modified EPO has agonist
activity for the mammalian EpoR. In one aspect, the modified EPO
has a non-naturally occurring amino acid sequence and/or a
non-naturally occurring glycosylation pattern. The mammalian EPO
may be from a mammal, including feline, canine, mouse, rat,
hamster, rabbit, guinea pig, ruminant, ferret, non-human primate,
pig, Siberian tiger, giant panda, or pacific walrus.
[0033] The modifications described herein may be based on sequence
differences at the nucleotide or amino acid level. A modified
nucleic acid and/or amino acid sequence is a sequence that is
different from the native sequence due to a deletion, an insertion,
a non-conservative or conservative substitution or combinations
thereof of one or more amino acid residues. In one embodiment, the
modification is a point mutation. The modifications herein may also
include the addition of glycosylation not occurring in the native
host organism, a truncated protein (i.e., a protein fragment)
and/or binding to or otherwise attaching to additional amino acids,
nucleotide(s) or nucleotide sequences, aptamers, labels, drugs,
antibodies, etc.
[0034] In one embodiment, a modified non-human mammalian
erythropoietin disclosed herein may have 1-17 amino acid additions,
deletions, or substitutions. In one aspect, the modified non-human
mammalian erythropoietin has at least 1, at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, at least 8, at least
9, at least 10, at least 11, at least 12, at least 13, at least 14,
at least 15, at least 16, at least 17 amino acid additions,
substitutions, or deletions. Substitutions may be conservative or
non-conservative. In another aspect, the modified non-human
mammalian erythropoietin may have at most 17, at most 16, at most
15, at most 14, at most 13, at most 12, at most 11, at most 10, at
most 9, at most 10, at most 11, at most 12, at most 13, at most 14,
at most 15, at most 16, or at most 17 amino acid additions,
substitutions, or deletions. In yet another aspect, the modified
non-human mammalian erythropoietin may have 1-17, 1-16, 1-15, 1-14,
1-13, 1-12, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-17,
2-16, 2-15, 2-4, 2-13, 2-12, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4,
2-3, 3-17, 3-16, 3-15, 3-4, 3-13, 3-12, 3-10, 3-9, 3-8, 3-7, 3-6,
3-5, 3-4, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-10, 4-9, 4-8, 4-7,
4-6, 4-5, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-10, 5-9, 5-8, 5-7,
5-6, 5-5, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-10, 6-9, 6-8, 6-7,
7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-10, 7-9, 7-8, 8-17, 8-16,
8-15, 8-14, 8-13, 8-12, 8-10, 8-9, 9-17, 9-16, 9-15, 9-14, 9-13,
9-12, 9-10, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 11-17, 11-16,
11-15, 11-14, 11-13, 11-12, 12-17, 12-16, 12-15, 12-14, 12-13,
13-17, 13-16, 13-15, 13-14, 14-17, 14-16, 14-15, 15-17, 15-16, or
16-17 amino acid additions, substitutions or deletions. These
additions, substitutions, or deletions may or may not add a
glycosylation site.
[0035] In one embodiment, the present modified non-human mammalian
EPO includes a set of substitutions denoted by the notation:
Xaa.sup.number, where Xaa is the three letter code for the amino
acid to be substituted, and the number is the position into which
the amino acid is substituted based on the sequence of the
mammalian EPO. In aspects of this embodiment, the mammalian
erythropoietin is feline erythropoietin where an asparagine residue
is substituted for the amino acid residue at any one or more of
positions 30, 51, 57, 69, 88, 89, 137, or 139. In other aspects of
this embodiment, the mammalian erythropoietin is feline
erythropoietin where either a serine or threonine residue is
substituted for the amino acid residue at position 126.
[0036] In one embodiment, the mammalian erythropoietin is feline.
In an aspect of this embodiment, a feline EPO is one having the
sequence shown in SEQ ID NO: 5, wherein Xaa at position 18 is
selected from E or G; and Xaa at position 116 is K or absent, a
fragment thereof, or a variant thereof. In other aspects of this
embodiment, a feline EPO disclosed herein may have, e.g., at least
70%, at least 75%, at least 80%, at least 85%, at least 87%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or 100% sequence identity with SEQ ID NO: 5, wherein Xaa at
position 18 is selected from E or G; and Xaa at position 116 is K
or absent. In yet other aspect of this embodiment, a feline EPO
disclosed herein may have, e.g., at least 1, at least 2, at least
3, at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, at least 10, at least 11, at least 12, at least 13, at
least 14, at least 15, at least 16, at least 17 amino acid
additions, substitutions, or deletions to SEQ ID NO: 5, wherein Xaa
at position 18 is selected from E or G; and Xaa at position 116 is
K or absent. In still other aspect of this embodiment, a feline EPO
disclosed herein may have, e.g., at most 17, at most 16, at most
15, at most 14, at most 13, at most 12, at most 11, at most 10, at
most 9, at most 10, at most 11, at most 12, at most 13, at most 14,
at most 15, at most 16, or at most 17 amino acid additions,
substitutions, or deletions to SEQ ID NO: 5, wherein Xaa at
position 18 is selected from E or G; and Xaa at position 116 is K
or absent. In other aspect of this embodiment, a feline EPO
disclosed herein may have, e.g., 1-17, 1-16, 1-15, 1-14, 1-13,
1-12, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-17, 2-16,
2-15, 2-4, 2-13, 2-12, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3,
3-17, 3-16, 3-15, 3-4, 3-13, 3-12, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5,
3-4, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-10, 4-9, 4-8, 4-7, 4-6,
4-5, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-10, 5-9, 5-8, 5-7, 5-6,
5-5, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-10, 6-9, 6-8, 6-7, 7-17,
7-16, 7-15, 7-14, 7-13, 7-12, 7-10, 7-9, 7-8, 8-17, 8-16, 8-15,
8-14, 8-13, 8- 12, 8-10, 8-9, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12,
9-10, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 11-17, 11-16,
11-15, 11-14, 11-13, 11-12, 12-17, 12-16, 12-15, 12-14, 12-13,
13-17, 13-16, 13-15, 13-14, 14-17, 14-16, 14-15, 15-17, 15-16, or
16-17 amino acid additions, substitutions or deletions to SEQ ID
NO: 5, wherein Xaa at position 18 is selected from E or G; and Xaa
at position 116 is K or absent. These additions, substitutions, or
deletions may or may not add a glycosylation site. Substitutions
may be conservative or non-conservative.
[0037] In an aspect of this embodiment, a feline EPO is one having
the sequence shown in SEQ ID NO: 6, wherein Xaa at position 18 is
selected from E or G; and Xaa at position 116 is K or absent, a
fragment thereof, or a variant thereof. In other aspects of this
embodiment, a feline EPO disclosed herein is one having, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 87%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94% at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% or 100% sequence identity with SEQ ID NO: 6, wherein Xaa
at position 18 is selected from E or G; and Xaa at position 116 is
K or absent. In yet other aspect of this embodiment, a feline EPO
disclosed herein may have, e.g., at least 1, at least 2, at least
3, at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, at least 10, at least 11, at least 12, at least 13, at
least 14, at least 15, at least 16, at least 17 amino acid
additions, substitutions, or deletions to SEQ ID NO: 6, wherein Xaa
at position 18 is selected from E or G; and Xaa at position 116 is
K or absent. In still other aspect of this embodiment, a feline EPO
disclosed herein may have, e.g., at most 17, at most 16, at most
15, at most 14, at most 13, at most 12, at most 11, at most 10, at
most 9, at most 10, at most 11, at most 12, at most 13, at most 14,
at most 15, at most 16, or at most 17 amino acid additions,
substitutions, or deletions to SEQ ID NO: 6, wherein Xaa at
position 18 is selected from E or G; and Xaa at position 116 is K
or absent. In other aspect of this embodiment, a feline EPO
disclosed herein may have, e.g., 1-17, 1-16, 1-15, 1-14, 1-13,
1-12, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-17, 2-16,
2-15, 2-4, 2-13, 2-12, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3,
3-17, 3-16, 3-15, 3-4, 3-13, 3-12, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5,
3-4, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-10, 4-9, 4-8, 4-7, 4-6,
4-5, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-10, 5-9, 5-8, 5-7, 5-6,
5-5, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-10, 6-9, 6-8, 6-7, 7-17,
7-16, 7-15, 7-14, 7-13, 7-12, 7-10, 7-9, 7-8, 8-17, 8-16, 8-15,
8-14, 8-13, 8-12, 8-10, 8-9, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12,
9-10, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 11-17, 11-16,
11-15, 11-14, 11-13, 11-12, 12-17, 12-16, 12-15, 12-14, 12-13,
13-17, 13-16, 13-15, 13-14, 14-17, 14-16, 14-15, 15-17, 15-16, or
16-17 amino acid additions, substitutions or deletions to SEQ ID
NO: 6, wherein Xaa at position 18 is selected from E or G; and Xaa
at position 116 is K or absent. These additions, substitutions, or
deletions may or may not add a glycosylation site. Substitutions
may be conservative or non-conservative.
[0038] In another embodiment, the modified mammalian erythropoietin
is canine. In an aspect of this embodiment, a canine EPO is one
having the sequence shown in SEQ ID NO: 10, a fragment thereof, or
a variant thereof. In aspects of this embodiment, a modified canine
EPO disclosed herein may have, e.g., at least 70%, at least 75%, at
least 80%, at least 85%, at least 87%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94% at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or 100% sequence
identity with SEQ ID NO: 10. In yet other aspect of this
embodiment, a modified canine EPO disclosed herein may have, e.g.,
at least 1, at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least
16, at least 17 amino acid additions, substitutions, or deletions
to SEQ ID NO: 10. In still other aspect of this embodiment, a
modified canine EPO disclosed herein may have, e.g., at most 17, at
most 16, at most 15, at most 14, at most 13, at most 12, at most
11, at most 10, at most 9, at most 10, at most 11, at most 12, at
most 13, at most 14, at most 15, at most 16, or at most 17 amino
acid additions, substitutions, or deletions to SEQ ID NO: 10. In
other aspect of this embodiment, a modified canine EPO disclosed
herein may have, e.g., 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-10,
1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-17, 2-16, 2-15, 2-4,
2-13, 2-12, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-17, 3-16,
3-15, 3-4, 3-13, 3-12, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-17,
4-16, 4-15, 4-14, 4-13, 4-12, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-17,
5-16, 5-15, 5-14, 5-13, 5-12, 5-10, 5-9, 5-8, 5-7, 5-6, 5-5, 6-17,
6-16, 6-15, 6-14, 6-13, 6-12, 6-10, 6-9, 6-8, 6-7, 7-17, 7-16,
7-15, 7-14, 7-13, 7-12, 7-10, 7-9, 7-8, 8-17, 8-16, 8-15, 8-14,
8-13, 8-12, 8-10, 8-9, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-10,
10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 11-17, 11-16, 11-15,
11-14, 11-13, 11-12, 12-17, 12-16, 12-15, 12-14, 12-13, 13-17,
13-16, 13-15, 13-14, 14-17, 14-16, 14-15, 15-17, 15-16, or 16-17
amino acid additions, substitutions or deletions to SEQ ID NO: 10.
These additions, substitutions, or deletions may or may not add a
glycosylation site. Substitutions may be conservative or
non-conservative.
[0039] In another embodiment, a modified mammalian erythropoietin
is canine. In an aspect of this embodiment, a modified canine EPO
is one having the sequence shown in SEQ ID NO: 11, a fragment
thereof, or a variant thereof. In aspects of this embodiment, a
modified canine EPO disclosed herein may have, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 87%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94% at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
or 100% sequence identity with SEQ ID NO: 11. In yet other aspect
of this embodiment, a modified canine EPO disclosed herein may
have, e.g., at least 1, at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 11, at least 12, at least 13, at least 14, at least
15, at least 16, at least 17 amino acid additions, substitutions,
or deletions to SEQ ID NO: 11. In still other aspect of this
embodiment, a modified canine EPO disclosed herein may have, e.g.,
at most 17, at most 16, at most 15, at most 14, at most 13, at most
12, at most 11, at most 10, at most 9, at most 10, at most 11, at
most 12, at most 13, at most 14, at most 15, at most 16, or at most
17 amino acid additions, substitutions, or deletions to SEQ ID NO:
11. In other aspect of this embodiment, a canine EPO disclosed
herein may have, e.g., 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-10,
1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-17, 2-16, 2-15, 2-4,
2-13, 2-12, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-17, 3-16,
3-15, 3-4, 3-13, 3-12, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-17,
4-16, 4-15, 4-14, 4-13, 4-12, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-17,
5-16, 5-15, 5-14, 5-13, 5-12, 5-10, 5-9, 5-8, 5-7, 5-6, 5-5, 6-17,
6-16, 6-15, 6-14, 6-13, 6-12, 6-10, 6-9, 6-8, 6-7, 7-17, 7-16,
7-15, 7-14, 7-13, 7-12, 7-10, 7-9, 7-8, 8-17, 8-16, 8-15, 8-14,
8-13, 8-12, 8-10, 8-9, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-10,
10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 11-17, 11-16, 11-15,
11-14, 11-13, 11-12, 12-17, 12-16, 12-15, 12-14, 12-13, 13-17,
13-16, 13-15, 13-14, 14-17, 14-16, 14-15, 15-17, 15-16, or 16-17
amino acid additions, substitutions or deletions to SEQ ID NO: 11.
These additions, substitutions, or deletions may or may not add a
glycosylation site. Substitutions may be conservative or
non-conservative.
[0040] A modified non-human mammalian erythropoietin disclosed
herein may be glycosylated or unglycosylated. That is, one or more
sugar residues or carbohydrate chains may be attached to the
glycosylation sites of the modified non-human mammalian
erythropoietin. This glycosylation can be performed by enzymatic
glycosylation during or after translation and assembly of the
modified non-human mammalian erythropoietin. Typically the
structure of the glycosylation site is directed to the attachment
of a carbohydrate residue or polymer. The "polymer" is a molecule
formed by covalent linkage of two or more monomers, wherein none of
the monomers is an amino acid residue. The term is intended to
cover carbohydrate molecules or chains attached by in vitro
glycosylation, i.e., a synthetic glycosylation performed in vitro
normally involving covalently linking a carbohydrate molecule to an
attachment group of the polypeptide, optionally using a
cross-linking agent.
[0041] In one embodiment the attachment of the carbohydrate is
either O-linked or N-linked. O-linked glycosidic typically attach
at specific amino acid residues, such as serine, threonine,
hydroxyproline, or hydroxylysine. In one embodiment, the addition
of or relocation of a serine or threonine may add a glycosylation
site to the mammalian erythropoietin. N-linked glycosidic chains
typically attach at specific amino acid sequences, such as
Asn-Xaa-Ser/Thr/Cystein. Xaa as used in this context may be any
amino acid.
[0042] A modified non-human mammalian EPO disclosed herein includes
at least one additional and/or at least one relocated glycosylation
site. In one embodiment, a glycosylation site is added to a
mammalian erythropoietin, resulting in a modified non-human
mammalian erythropoietin disclosed herein. This addition may be
completed by adding amino acid residue(s) to either end of the
mammalian erythropoietin, by adding amino acid residue(s) between
internal amino acids of the mammalian erythropoietin or by
replacing one or more amino acid residues in the mammalian
erythropoietin with the amino acid(s) which may attach a glycosidic
chain.
[0043] In one embodiment, a glycosylation site is relocated within
a mammalian erythropoietin, resulting in a modified non-human
mammalian erythropoietin disclosed herein. That is, the amino
acid(s) of a glycosylation site in the native protein are replaced
with amino acids which cannot be glycosylated with carbohydrate
chains, and a glycosylation site is added elsewhere to obtain a
modified non-human mammalian erythropoietin sequence.
[0044] In an embodiment, a feline EpoR protein agonist is modified
feline EPO, which include at least one additional site for
glycosylation, and/or a relocation of at least one site for
glycosylation. In aspects of this embodiment, an EpoR protein
agonist is a feline EPO or a modified feline EPO disclosed herein
having one of the following sets of substituted amino acids:
Asn.sup.30Thr.sup.32 EPO; Asn.sup.51Thr.sup.53 EPO;
Asn.sup.57Thr.sup.59 EPO; Asn.sup.69EPO; Asn.sup.69Thr.sup.71 EPO;
Ser.sup.68Asn.sup.69Thr.sup.71 EPO; Val.sup.87Asn.sup.88 EPO;
Ser.sup.87Asn.sup.88 EPO; Ser.sup.87Asn.sup.88Gly.sup.89 EPO;
Ser.sup.87Asn.sup.88Thr.sup.92 EPO; Ser.sup.87Asn.sup.88Ala.sup.163
EPO; Asn.sup.69Thr.sup.71Ser.sup.87Asn.sup.88 EPO;
Asn.sup.30Thr.sup.32Val.sup.87Asn.sup.88 EPO;
Asn.sup.89Ile.sup.90Thr.sup.91 EPO; Ser.sup.87Asn.sup.89
Ile.sup.90Thr.sup.91 EPO; Asn.sup.137Thr.sup.139 EPO;
Asn.sup.139Thr.sup.141 EPO; Thr.sup.126 EPO; or
Pro.sup.125Thr.sup.126 EPO. In other aspects of this embodiment, an
EpoR protein agonist is a feline EPO or a modified feline EPO
disclosed herein having one or more of the following substitution
sets is present: Ser.sup.87Asn.sup.88 and/or
Asn.sup.30Thr.sup.32Val.sup.87Asn.sup.88.
[0045] In aspects of this embodiment, an EpoR protein agonist is a
feline EPO of SEQ ID NO: 5, wherein Xaa at position 18 is selected
from E or G; and Xaa at position 116 is K or absent, having one of
the following sets of substituted amino acids: Asn.sup.30Thr.sup.32
EPO; Asn.sup.51Thr.sup.53 EPO; Asn.sup.57Thr.sup.59 EPO;
Asn.sup.69EPO; Asn.sup.69Thr.sup.71 EPO;
Ser.sup.68Asn.sup.69Thr.sup.71 EPO; Val.sup.87Asn.sup.88 EPO;
Ser.sup.87Asn.sup.88 EPO; Ser.sup.87Asn.sup.88Gly.sup.89 EPO;
Ser.sup.87Asn.sup.88Thr.sup.92 EPO; Ser.sup.87Asn.sup.88Ala.sup.163
EPO; Asn.sup.69Thr.sup.71Ser.sup.87Asn.sup.88 EPO;
Asn.sup.30Thr.sup.32Val.sup.87Asn.sup.88 EPO;
Asn.sup.89Ile.sup.90Thr.sup.91 EPO; Ser.sup.87Asn.sup.89
Ile.sup.90Thr.sup.91 EPO; Asn.sup.137Thr.sup.139 EPO;
Asn.sup.139Thr.sup.141 EPO; Thr.sup.126 EPO; or
Pro.sup.125Thr.sup.126 EPO. In other aspects of this embodiment, an
EpoR protein agonist is a feline EPO of SEQ ID NO: 5, wherein Xaa
at position 18 is selected from E or G; and Xaa at position 116 is
K or absent, having one or more of the following substitution sets
is present: Ser.sup.87Asn.sup.88 and/or
Asn.sup.30Thr.sup.32Val.sup.87Asn.sup.88.
[0046] In one embodiment the mammalian erythropoietin is a feline
EPO or a modified feline EPO disclosed herein modified by deleting
one or more of the glycosylation sites that attach to N-linked
carbohydrate chains and by adding a glycosylation site that
attaches to an N-linked carbohydrate chain at amino acid position
88. In one aspect of this embodiment, one of the following amino
acid substitution sets is present: Gln.sup.24Ser.sup.87Asn.sup.88;
Gln.sup.38Ser.sup.87Asn.sup.88; or
Gln.sup.83Ser.sup.87Asn.sup.88.
[0047] In a further embodiment, an EpoR protein agonist has an
amino acid sequence of SEQ ID No.6, whereas Xaa at position 18 is
selected from E or G; and Xaa at position 116 is K or absent. In
aspects of this embodiment, an EpoR protein agonist has at least
70%, at least 75%, at least 80%, at least 85%, at least 87%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or 100% sequence identity with SEQ ID NO: 6, whereas Xaa at
position 18 is selected from E or G; and Xaa at position 116 is K
or absent.
[0048] Additional sequences may also be added to the mammalian
erythropoietin to obtain a modified non-human mammalian EPO. For
instance, in one embodiment, the glycosylation sequence comprises a
fragment of a mammalian chorionic gonadotropin or a sequence having
at least 70% sequence homology thereto. In one aspect, fragment of
mammalian chorionic gonadotropin is a fragment from the carboxy
terminal (aka "C-terminal") region of the mammalian chorionic
gonadotropin or a sequence having at least 70% sequence homology
thereto. In one aspect, the carboxy terminal of the mammalian
chorionic gonadotropin comprises at least 50% of the protein
located at the C terminal. In one embodiment, the carboxy terminal
of the mammalian chorionic gonadotropin includes at least 40% of
the protein from the C-terminal, at least 30% of the protein from
the C-terminal, at least 25% of the protein from the C-terminal, at
least 20% of the protein from the C-terminal, or at least 10% of
the protein from the C-terminal.
[0049] Optionally, a mammalian EPO disclosed herein includes a
signal sequence. In an aspect of this embodiment, a signal sequence
disclosed herein includes a signal sequence from human EPO (SEQ ID
NO: 9), a signal sequence from feline EPO (SEQ ID NO: 12), or a
signal sequence from canine EPO (SEQ ID NO: 13) or another
conventionally recognized signal sequence. For instance, SEQ ID NO:
11 is a canine EPO including a signal sequence (amino acids
1-26).
[0050] In one embodiment, the chorionic gonadotropin is human
chorionic gonadotropin. In an aspect of this embodiment, a
chorionic gonadotrophin comprises SEQ ID NO: 8, or a sequence
having, e.g., at least 70%, at least 75%, at least 80%, at least
85%, at least 87%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94% at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or 100% sequence identity with SEQ ID
NO: 8. In an aspect of this embodiment, a chorionic gonadotrophin
comprises an amino acid sequence having 1, 2, 3, 4, 5, 1-2, 1-3,
1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 amino acid additions,
deletions or substitutions to SEQ ID NO: 8.
[0051] The present specification also discloses nucleic acid
sequences encoding modified non-human mammalian EPO disclosed
herein. The nucleic acid sequence may include introns or may be a
cDNA. The nucleic acid sequence may also encode a sequence which is
cleaved off after translation. The nucleic acids may be DNA or RNA.
The nucleic acids described herein may also include the complements
of the coding sequences.
[0052] The present specification also discloses a fusion protein,
comprising a peptide, a linker and a Fc fragment, wherein the
peptide is fused through linker to the Fc fragment. A peptide
disclosed herein may be one with the amino acid sequence of SEQ ID
NO: 1 wherein Xaa at position 13 is Trp, 1-naphthylalanine, or
2-naphthylalanine, or a sequence having, e.g., at least 70%, at
least 75%, at least 80%, at least 85%, at least 87%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94% at least
95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 1
wherein Xaa at position 13 is Trp, 1-naphthylalanine, or
2-naphthylalanine. In an aspect of this embodiment, a peptide
disclosed herein comprises an amino acid sequence having 1, 2, 3,
4, 5, 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 amino
acid additions, deletions or substitutions to SEQ ID NO: 1 wherein
Xaa at position 13 is Trp, 1-naphthylalanine, or
2-naphthylalanine.
[0053] A peptide disclosed herein may be a feline EPO as disclosed
herein. In aspects of this embodiment, a feline EPO disclosed
herein may be SEQ ID NO: 5, wherein Xaa at position 18 is selected
from E or G; and Xaa at position 116 is K or absent, or a sequence
having, e.g., at least 70%, at least 75%, at least 80%, at least
85%, at least 87%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94% at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or 100% sequence identity to the amino
acid sequence of SEQ ID NO: 5, wherein Xaa at position 18 is
selected from E or G; and Xaa at position 116 is K or absent. In
other aspect of this embodiment, a feline EPO disclosed herein is
one having, e.g., at least 1, at least 2, at least 3, at least 4,
at least 5, at least 6, at least 7, at least 8, at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17 amino acid additions,
substitutions, or deletions to SEQ ID NO: 5, wherein Xaa at
position 18 is selected from E or G; and Xaa at position 116 is K
or absent. In yet other aspect of this embodiment, a feline EPO
disclosed herein may have, e.g., at most 17, at most 16, at most
15, at most 14, at most 13, at most 12, at most 11, at most 10, at
most 9, at most 10, at most 11, at most 12, at most 13, at most 14,
at most 15, at most 16, or at most 17 amino acid additions,
substitutions, or deletions to SEQ ID NO: 5, wherein Xaa at
position 18 is selected from E or G; and Xaa at position 116 is K
or absent. In still other aspect of this embodiment, a feline EPO
disclosed herein may have, e.g., 1-17, 1-16, 1-15, 1-14, 1-13,
1-12, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-17, 2-16,
2-15, 2-4, 2-13, 2-12, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3,
3-17, 3-16, 3-15, 3-4, 3-13, 3-12, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5,
3-4, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-10, 4-9, 4-8, 4-7, 4-6,
4-5, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-10, 5-9, 5-8, 5-7, 5-6,
5-5, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-10, 6-9, 6-8, 6-7, 7-17,
7-16, 7-15, 7-14, 7-13, 7-12, 7-10, 7-9, 7-8, 8-17, 8- 16, 8-15,
8-14, 8-13, 8-12, 8-10, 8-9, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12,
9-10, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 11-17, 11-16,
11-15, 11-14, 11-13, 11-12, 12-17, 12-16, 12-15, 12-14, 12-13,
13-17, 13-16, 13-15, 13-14, 14-17, 14-16, 14-15, 15-17, 15-16, or
16-17 amino acid additions, substitutions or deletions to SEQ ID
NO: 5, wherein Xaa at position 18 is selected from E or G; and Xaa
at position 116 is K or absent. These additions, substitutions, or
deletions may or may not add a glycosylation site. Substitutions
may be conservative or non-conservative.
[0054] A peptide disclosed herein may be a modified feline EPO. A
modified feline EPO disclosed herein may be SEQ ID NO: 6, or a
sequence having, e.g., at least 70%, at least 75%, at least 80%, at
least 85%, at least 87%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94% at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 6. In other aspect of this
embodiment, a modified feline EPO disclosed herein may have, e.g.,
at least 1, at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least
16, at least 17 amino acid additions, substitutions, or deletions
to SEQ ID NO: 6. In yet other aspect of this embodiment, a modified
feline EPO disclosed herein may have, e.g., at most 17, at most 16,
at most 15, at most 14, at most 13, at most 12, at most 11, at most
10, at most 9, at most 10, at most 11, at most 12, at most 13, at
most 14, at most 15, at most 16, or at most 17 amino acid
additions, substitutions, or deletions to SEQ ID NO: 6. In still
other aspect of this embodiment, a modified feline EPO disclosed
herein may have, e.g., 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-10,
1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-17, 2-16, 2-15, 2-4,
2-13, 2-12, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-17, 3-16,
3-15, 3-4, 3-13, 3-12, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-17,
4-16, 4-15, 4-14, 4-13, 4-12, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-17,
5-16, 5-15, 5-14, 5- 13, 5-12, 5-10, 5-9, 5-8, 5-7, 5-6, 5-5, 6-17,
6-16, 6-15, 6-14, 6-13, 6-12, 6-10, 6-9, 6-8, 6-7, 7-17, 7-16,
7-15, 7-14, 7-13, 7-12, 7-10, 7-9, 7-8, 8-17, 8-16, 8-15, 8-14,
8-13, 8-12, 8-10, 8-9, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-10,
10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 11-17, 11-16, 11-15,
11-14, 11-13, 11-12, 12-17, 12-16, 12-15, 12-14, 12-13, 13-17,
13-16, 13-15, 13-14, 14-17, 14-16, 14-15, 15-17, 15-16, or 16-17
amino acid additions, substitutions or deletions to SEQ ID NO: 6.
These additions, substitutions, or deletions may or may not add a
glycosylation site. Substitutions may be conservative or
non-conservative.
[0055] A peptide disclosed herein may be a modified canine EPO. A
canine EPO disclosed herein may be SEQ ID NO: 10, a fragment
thereof, or a variant thereof. In aspects of this embodiment, a
canine EPO disclosed herein may have, e.g., at least 70%, at least
75%, at least 80%, at least 85%, at least 87%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94% at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity with SEQ ID NO: 10. In yet other aspect of this
embodiment, a canine EPO disclosed herein may have, e.g., at least
1, at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10, at least 11, at least
12, at least 13, at least 14, at least 15, at least 16, at least 17
amino acid additions, substitutions, or deletions to SEQ ID NO: 10.
In still other aspect of this embodiment, a canine EPO disclosed
herein may have, e.g., at most 17, at most 16, at most 15, at most
14, at most 13, at most 12, at most 11, at most 10, at most 9, at
most 10, at most 11, at most 12, at most 13, at most 14, at most
15, at most 16, or at most 17 amino acid additions, substitutions,
or deletions to SEQ ID NO: 10. In other aspect of this embodiment,
a canine EPO disclosed herein may have, e.g., 1-17, 1-16, 1-15,
1-14, 1-13, 1-12, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2,
2-17, 2-16, 2-15, 2-4, 2-13, 2-12, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5,
2-4, 2-3, 3-17, 3-16, 3-15, 3-4, 3-13, 3-12, 3-10, 3-9, 3-8, 3-7,
3-6, 3-5, 3-4, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-10, 4-9, 4-8,
4-7, 4-6, 4-5, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-10, 5-9, 5-8,
5-7, 5-6, 5-5, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-10, 6-9, 6-8,
6-7, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-10, 7-9, 7-8, 8-17,
8-16, 8-15, 8-14, 8-13, 8-12, 8-10, 8-9, 9-17, 9-16, 9-15, 9-14,
9-13, 9-12, 9-10, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 11-17,
11-16, 11-15, 11-14, 11-13, 11-12, 12-17, 12-16, 12-15, 12-14,
12-13, 13-17, 13-16, 13-15, 13-14, 14-17, 14-16, 14-15, 15-17,
15-16, or 16-17 amino acid additions, substitutions or deletions to
SEQ ID NO: 10. These additions, substitutions, or deletions may or
may not add a glycosylation site. Substitutions may be conservative
or non-conservative.
[0056] A peptide disclosed herein may be a modified canine EPO. A
canine EPO disclosed herein may be SEQ ID NO: 11, a fragment
thereof, or a variant thereof. In aspects of this embodiment, a
canine EPO disclosed herein may have, e.g., at least 70%, at least
75%, at least 80%, at least 85%, at least 87%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94% at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity with SEQ ID NO: 11. In yet other aspect of this
embodiment, a canine EPO disclosed herein may have, e.g., at least
1, at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10, at least 11, at least
12, at least 13, at least 14, at least 15, at least 16, at least 17
amino acid additions, substitutions, or deletions to SEQ ID NO: 11.
In still other aspect of this embodiment, a canine EPO disclosed
herein may have, e.g., at most 17, at most 16, at most 15, at most
14, at most 13, at most 12, at most 11, at most 10, at most 9, at
most 10, at most 11, at most 12, at most 13, at most 14, at most
15, at most 16, or at most 17 amino acid additions, substitutions,
or deletions to SEQ ID NO: 11. In other aspect of this embodiment,
a canine EPO disclosed herein may have, e.g., 1-17, 1-16, 1-15,
1-14, 1-13, 1-12, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2,
2-17, 2-16, 2-15, 2-4, 2-13, 2-12, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5,
2-4, 2-3, 3-17, 3-16, 3-15, 3-4, 3-13, 3-12, 3-10, 3-9, 3-8, 3-7,
3-6, 3-5, 3-4, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-10, 4-9, 4-8,
4-7, 4-6, 4-5, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-10, 5-9, 5-8,
5-7, 5-6, 5-5, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-10, 6-9, 6-8,
6-7, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-10, 7-9, 7-8, 8-17,
8-16, 8-15, 8-14, 8-13, 8-12, 8-10, 8-9, 9-17, 9-16, 9-15, 9-14,
9-13, 9-12, 9-10, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 11-17,
11-16, 11-15, 11-14, 11-13, 11-12, 12-17, 12-16, 12-15, 12-14,
12-13, 13-17, 13-16, 13-15, 13-14, 14-17, 14-16, 14-15, 15-17,
15-16, or 16-17 amino acid additions, substitutions or deletions to
SEQ ID NO: 11. These additions, substitutions, or deletions may or
may not add a glycosylation site. Substitutions may be conservative
or non-conservative.
[0057] In one embodiment, a peptides disclosed herein is selected
from the amino acid sequences disclosed in U.S. Pat. No. 6,703,480,
the entire contents of which are hereby expressly incorporated by
reference. In one embodiment, a peptide disclosed herein is SEQ ID
NO: 1, wherein Xaa at position 13 is Trp, 1-naphthylalanine, or
2-naphthylalanine.
[0058] The term "Fc fragment" or "immunoglobulin Fc region" as used
herein, refers to a protein that contains the heavy-chain constant
region 2 (CH2) and the heavy-chain constant region 3 (CH3) of an
immunoglobulin, excluding the variable regions of the heavy and
light chains, the heavy-chain constant region 1 (CH1) and the
light-chain constant region 1 (CL1) of the immunoglobulin. It may
further include a hinge region at the heavy-chain constant region.
Also, the immunoglobulin Fc region disclosed herein may contain a
part or all of the Fc region including the heavy-chain constant
region 1 (CH1) and/or the light-chain constant region 1 (CL1),
except for the variable regions of the heavy and light chains, as
long as it has a physiological function substantially similar to or
better than the native protein. Also, the immunoglobulin Fc region
may be a fragment having a deletion in a relatively long portion of
the amino acid sequence of CH2 and/or CH3. That is, the
immunoglobulin Fc region disclosed herein may comprise 1) a CH1
domain, a CH2 domain, a CH3 domain and a CH4 domain, 2) a CH1
domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2
domain and a CH3 domain, 5) a combination of one or more domains
and an immunoglobulin hinge region (or a portion of the hinge
region), and 6) a dimer of each domain of the heavy-chain constant
regions and the light-chain constant region.
[0059] The immunoglobulin Fc region disclosed herein includes a
native amino acid sequence, and a modified sequence (mutant)
thereof. A modified amino acid sequence is a sequence that is
different from the native amino acid sequence due to a deletion, an
insertion, a non-conservative or conservative substitution or
combinations thereof of one or more amino acid residues.
[0060] Also, other variants of the Fc are possible, including one
in which a region capable of forming a disulfide bond is deleted,
or certain amino acid residues are eliminated at the N-terminal end
of a native Fc form or a methionine residue is added thereto.
Further, to remove effector functions, a deletion may occur in a
complement-binding site, such as a C1q-binding site and an ADCC
(antibody dependent cell mediated cytotoxicity) site. Techniques of
preparing such sequence variants of the immunoglobulin Fc region
are disclosed in WO 97/34631 and WO 96/32478.
[0061] The aforementioned Fc variants are variants that have a
biological activity identical to the Fc region disclosed herein or
improved structural stability, for example, against heat, pH, or
the like.
[0062] In addition, these Fc regions may be obtained from native
forms isolated from cats and other animals including dogs, cows,
goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, or may
be recombinants, variants, or derivatives thereof, obtained from
transformed animal cells or microorganisms. Herein, they may be
obtained from a native immunoglobulin by isolating whole
immunoglobulins from human or animal organisms and treating them
with a proteolytic enzyme. Papain digests the native immunoglobulin
into Fab and Fc regions, and pepsin treatment results in the
production of pF'c and F(ab)2 fragments. These fragments may be
subjected, for example, to size exclusion chromatography to isolate
Fc or pF'c. Preferably, a feline-derived Fc region is a recombinant
immunoglobulin Fc region that is obtained from a microorganism.
[0063] In addition, the immunoglobulin Fc region disclosed herein
may be in the form of having native sugar chains, increased sugar
chains compared to a native form or decreased sugar chains compared
to the native form, or may be in a deglycosylated form. The
increase, decrease or removal of the immunoglobulin Fc sugar chains
may be achieved by methods common in the art, such as a chemical
method, an enzymatic method and a genetic engineering method using
a microorganism. The removal of sugar chains from an Fc region
results in a sharp decrease in binding affinity to the C1q part of
the first complement component C1 and a decrease or loss in
antibody-dependent cell-mediated cytotoxicity or
complement-dependent cytotoxicity, thereby not inducing unnecessary
immune responses in vivo. In this regard, an immunoglobulin Fc
region in a deglycosylated or aglycosylated form may be more
suitable to the object disclosed herein as a drug carrier.
[0064] As used herein, the term "deglycosylation" refers to
enzymatically removing sugar moieties from an Fc region, and the
term "aglycosylation" means that an Fc region is produced in an
unglycosylated form by a prokaryote, preferably E. coli.
[0065] In addition, the immunoglobulin Fc region may be an Fc
region that is derived from a IgG, IgA, IgD, IgE and IgM, or that
is made by combinations thereof or hybrids thereof. In one
embodiment it is derived from a mammalian IgG or IgM, which are
among the most abundant proteins in human blood, and most
preferably from IgG, which is known to enhance the half-lives of
ligand-binding proteins. In one embodiment, the immunoglobulin Fc
region is an Fc region derived from feline IgG, IgA, IgE, or
IgM.
[0066] On the other hand, the term "combination", as used herein,
means that polypeptides encoding single-chain immunoglobulin Fc
regions of the same origin are linked to a single-chain polypeptide
of a different origin to form a dimer or multimer. That is, a dimer
or multimer may be formed from two or more fragments selected from
the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc
fragments.
[0067] Meanwhile, the Fc fragment or the Fc region of the dummy
antibody may be modified to increase its affinity with neonatal Fc
receptor (FcRN) and further extend its half-life in vivo. As
mentioned above, Fc may be used as carrier to extend the in vivo
half-lives of therapeutic peptides. Fc fusion proteins have in vivo
half-lives ranging from a few days to over a week. Meanwhile, a
full antibody molecule may also be used as carrier for therapeutic
peptides. Antibody molecules can have in vivo half-lives over 3
weeks.
[0068] In addition, modifications can be made to the Fc domain to
further extend the in vivo half-life of an antibody molecule or an
Fc fusion protein. For example, introduction of mutations
T250Q/M428L to an IgG1 molecule led to a significant increase in
the serum half-life of the IgG1 molecule, also as disclosed in U.S.
Pat. No. 7,217,798. Other mutations have also been made to increase
the binding affinity of Fc to FcRN and extend the serum half-life,
such as disclosed in U.S. Pat. No. 8,394,925.
[0069] Another modification, referred as "knob-and-hole" may also
be made to the Fc region to form heterogeneous dimers. Antibody
molecules have heavy chains and light chains. It is convenient to
fuse two different peptides to an antibody molecule. Fc fusion
proteins are often homo-dimers. However, hetero-dimers can also be
constructed. For example, U.S. Pat. No. 7,642,228 disclosed that
heterodimers of antibodies and Fc-fusion proteins can be
constructed using the Knob-and-Hole concept.
[0070] A Fc fragment disclosed herein may be a feline IgG1a Fc, a
feline IgG1b Fc, a feline IgG2 Fc, a canine IgG1a Fc, a canine
IgG1b Fc, or a canine IgG2 Fc. A Fc fragment disclosed herein may
be a peptide with the amino acid sequence of SEQ ID NO: 2, SEQ ID
NO: 3 or SEQ ID NO: 4, or a sequence having, e.g., at least 70%, at
least 75%, at least 80%, at least 85%, at least 87%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94% at least
95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 2, SEQ
ID NO: 3 or SEQ ID NO: 4. A Fc fragment disclosed herein may be a
peptide with the amino acid sequence of SEQ ID NO: 2 wherein the
first 1 to 8 amino acids on the N-terminal end may be absent, SEQ
ID NO: 3 wherein the first 1 to 8 amino acids on the N-terminal end
may be absent, or SEQ ID NO: 4 wherein the first 1 to 8 amino acids
on the N-terminal end may be absent.
[0071] In yet other aspect of this embodiment, a Fc fragment
disclosed herein may have, e.g., at least 1, at least 2, at least
3, at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, at least 10, at least 11, at least 12, at least 13, at
least 14, at least 15, at least 16, at least 17 amino acid
additions, substitutions, or deletions to SEQ ID NO: 2, SEQ ID NO:
3 or SEQ ID NO: 4. In still other aspect of this embodiment, a Fc
fragment disclosed herein may have, e.g., at most 17, at most 16,
at most 15, at most 14, at most 13, at most 12, at most 11, at most
10, at most 9, at most 10, at most 11, at most 12, at most 13, at
most 14, at most 15, at most 16, or at most 17 amino acid
additions, substitutions, or deletions to SEQ ID NO: 2, SEQ ID NO:
3 or SEQ ID NO: 4. In other aspect of this embodiment, a Fc
fragment disclosed herein may have, e.g., 1-17, 1-16, 1-15, 1-14,
1-13, 1-12, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-17,
2-16, 2-15, 2-4, 2-13, 2-12, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4,
2-3, 3-17, 3-16, 3-15, 3-4, 3-13, 3-12, 3-10, 3-9, 3-8, 3-7, 3-6,
3-5, 3-4, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-10, 4-9, 4-8, 4-7,
4-6, 4-5, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-10, 5-9, 5-8, 5-7,
5-6, 5-5, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-10, 6-9, 6-8, 6-7,
7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-10, 7-9, 7-8, 8-17, 8-16,
8-15, 8-14, 8-13, 8-12, 8-10, 8-9, 9-17, 9-16, 9-15, 9-14, 9-13,
9-12, 9-10, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 11-17, 11-16,
11-15, 11-14, 11-13, 11-12, 12-17, 12-16, 12-15, 12-14, 12-13,
13-17, 13-16, 13-15, 13-14, 14-17, 14-16, 14-15, 15-17, 15-16, or
16-17 amino acid additions, substitutions or deletions to SEQ ID
NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. These additions,
substitutions, or deletions may or may not add a glycosylation
site. Substitutions may be conservative or non-conservative.
[0072] In yet another embodiment, the Fc fragments may be modified
to remove its glycosylation sites. In addition, the fusion proteins
described herein may also include the addition of glycosylation, a
truncated protein (i.e., a protein fragment) or a protein fused,
bound or otherwise attached to additional amino acids,
nucleotide(s) or nucleotide sequences, aptamers, labels, drugs,
antibodies, etc.
[0073] The Fc domains may be further modified to remove its
N-glycosylation site. In addition, modifications can be made to the
Fc domain to further extend half-life in vivo of an antibody
molecule or an Fc fusion protein. For example, introduction of
mutations T250Q/M428L to a human IgG1 molecule led to a significant
increase in the serum half-life of the IgG1 molecule, as disclosed
in U.S. Pat. No. 7,217,798. Other mutations have also been made to
increase the binding affinity of Fc to FcRN and extend the serum
half-life, such as disclosed in U.S. Pat. No. 8,394,925. The
present specification further encompasses DNA sequences encoding
such Fc fusion proteins, and recombinant plasmids and host cells
for expression.
[0074] In one embodiment, the Fc fragment is further modified so
that the half-life of the fusion protein in vivo is further
extended by, e.g., at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95% or at least
100%. In one aspect of this embodiment, the half-life of the fusion
protein is extended by at least 50%, when compared to a fusion
protein constructed using the sequence of an un-modified Fc
fragment. In a further embodiment, a therapeutic and its variants
have half-lives of 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14
hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours,
21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, one month,
two months, three months, four months or more.
[0075] A fusion protein disclosed herein is also optionally is
fused or linked to a Fc fragment disclosed herein using a linker.
An example amino acid linkers include GGGGS (SEQ ID NO: 7), where
(GGGGS)n, where n=1, 2, 3, and 4. In one embodiment, a peptide
disclosed herein is fused to the N-terminals or the C-terminals of
the Fc domain (FIGS. 2A & B).
[0076] In one embodiment, an EpoR protein agonist disclosed herein
is formulated, and used as a medicament, for use in the treatment
of NRA caused by conditions including chronic renal failure,
cancer, viral infections, and red blood cell aplasia due to
antibodies against recombinant human EPO.
[0077] The term "isolated proteins" refers to a protein
substantially free of cellular material or culture medium when
produced by recombinant DNA techniques, or chemical precursors or
other chemicals when chemically synthesized.
[0078] The term "sequence homology" or "sequence identity" as used
herein refers to the percentage of sequence identity between two
polypeptide sequences. In order to determine the percentage of
identity between two polypeptide sequences, the amino acid
sequences of such two sequences are aligned, preferably using the
Clustal W algorithm (Thompson, J D, Higgins D G, Gibson T J, 1994,
Nucleic Acids Res. 22 (22): 4673-4680), together with BLOSUM 62
scoring matrix (Henikoff S, and Henikoff J. G., 1992, Proc. Natl.
Acad. Sci. USA 89: 10915-10919) and a gap opening penalty of 10 and
gap extension penalty of 0.1, so that the highest order match is
obtained between two sequences wherein at least 50% of the total
length of one of the sequences is involved in the alignment. Other
methods that may be used to align sequences are the alignment
method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443), as
revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) so
that the highest order match is obtained between the two sequences
and the number of identical amino acids is determined between the
two sequences. Other methods to calculate the percentage identity
between two amino acid sequences are generally art recognized and
include, for example, those described by Carillo and Lipton (SIAM
J. Applied Math., 1988, 48:1073) and those described in
Computational Molecular Biology, Lesk, e.d. Oxford University
Press, New York, 1988, Biocomputing: Informatics and Genomics
Projects. Generally, computer programs will be employed for such
calculations. Computer programs that may be used in this regard
include, but are not limited to, GCG (Devereux et al., Nucleic
Acids Res., 1984, 12: 387) BLASTP, BLASTN and FASTA (Altschul et
al., J. Molec. Biol., 1990: 215: 403). In one aspect the present
modified non-human mammalian EPO or fusion proteins have at least
70%, at least 75%, at least 80%, at least 85%, at least 87%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or 100% sequence identity with another sequence, either on a
local or a full-length basis.
[0079] If on a local basis, the locality is determined by a region
of the non-modified or native sequence, or a specifically
identified motif of non-modified or native sequence. In one aspect
the locality is at least 8, at least 9, at least 10, at least 11,
at least 12, at least 13, at least 14, at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 25, at
least 30, at least 35, at least 40, at least 45, at least 50, at
least 55, at least 60, at least 65, at least 70, or at least 75
nucleic acids or amino acids of the non-modified or native
sequence.
[0080] The term "variant" as used herein includes modifications or
chemical equivalents of the amino acid and nucleotide sequences
disclosed herein that perform substantially the same function as
the proteins or nucleic acid molecules disclosed herein in
substantially the same way. For example, variants of proteins
disclosed herein include, without limitation, conservative amino
acid substitutions. Variants of proteins disclosed herein also
include additions and deletions to the proteins disclosed herein.
In addition, variant peptides and variant nucleotide sequences
include analogs and chemical derivatives thereof.
[0081] The present modified non-human mammalian erythropoietin or
fusion protein may have amino acid additions, deletions, or
substitutions. A modified amino acid sequence is a sequence that is
different from the native amino acid sequence due to a deletion, an
insertion, a non-conservative or conservative substitution or
combinations thereof of one or more amino acid residues. In one
embodiment, the modification is a point mutation. In one aspect,
the modified non-human mammalian erythropoietin does not have a
naturally occurring sequence. Similarly, in one aspect, the P of
the fusion protein is a non-naturally occurring amino acid
sequence.
[0082] The amino acid substitutions may be conservative or
non-conservative. A "conservative amino acid substitution", as used
herein, is one in which one amino acid residue is replaced with
another amino acid residue having a similar side chain. Families of
amino acid residues having similar side chains have been defined in
the art, including basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). The most
commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu,
Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Ala/Pro,
Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, in both
directions. Amino acid exchanges in proteins and peptides, which do
not generally alter the activity of the proteins or peptides, are
known in the art (H. Neurath, R. L. Hill, The Proteins, Academic
Press, New York, 1979).
[0083] The term "derivative of a peptide" refers to a peptide
having one or more residues chemically derivatized by reaction of a
functional side group. Such derivatized molecules include for
example, those molecules in which free amino groups have been
derivatized to form amine hydrochlorides, p-toluene sulfonyl
groups, carbobenzoxy groups, t-butyloxycarbonyl groups,
chloroacetyl groups or formyl groups. Free carboxyl groups may be
derivatized to form salts, methyl and ethyl esters or other types
of esters or hydrazides. Free hydroxyl groups may be derivatized to
form O-acyl or O-alkyl derivatives. The imidazole nitrogen of
histidine may be derivatized to form N-im-benzylhistidine. Also
included as derivatives are those peptides which contain one or
more naturally occurring amino acid derivatives of the twenty
standard amino acids. For examples: 4-hydroxyproline may be
substituted for proline; 5-hydroxylysine may be substituted for
lysine; 3-methylhistidine may be substituted for histidine;
homoserine may be substituted for serine; and ornithine may be
substituted for lysine.
[0084] The term "nucleic acid sequence" as used herein refers to a
sequence of nucleoside or nucleotide monomers consisting of
naturally occurring bases, sugars and intersugar (backbone)
linkages. The term also includes modified or substituted sequences
comprising non-naturally occurring monomers or portions thereof.
The nucleic acid sequences disclosed herein may be deoxyribonucleic
acid sequences (DNA) or ribonucleic acid sequences (RNA) and may
include naturally occurring bases including adenine, guanine,
cytosine, thymidine and uracil. The sequences may also contain
modified bases. Examples of such modified bases include aza and
deaza adenine, guanine, cytosine, thymidine and uracil; and
xanthine and hypoxanthine.
[0085] The present modified non-human mammalian EPO or fusion
proteins may made by conventional means, such as recombination. The
term "recombinant" as used herein refers to a polypeptide produced
through a biological host, selected from a mammalian expression
system, an insect cell expression system, a yeast expression
system, and a bacterial expression system.
[0086] Accordingly, the nucleic acid molecules disclosed herein may
be incorporated in a known manner into an appropriate expression
vector which ensures good expression of the proteins disclosed
herein. Possible expression vectors include but are not limited to
cosmids, plasmids, or modified viruses (e.g. replication defective
retroviruses, adenoviruses and adeno-associated viruses), so long
as the vector is compatible with the host cell used. The expression
vectors are "suitable for transformation of a host cell", which
means that the expression vectors contain a nucleic acid molecule
disclosed herein and regulatory sequences selected on the basis of
the host cells to be used for expression, which is operatively
linked to the nucleic acid molecule. Operatively linked is intended
to mean that the nucleic acid is linked to regulatory sequences in
a manner which allows expression of the nucleic acid.
[0087] The present specification therefore contemplates a
recombinant expression vector containing a nucleic acid molecule
disclosed herein, or a fragment thereof, and the necessary
regulatory sequences for the transcription and translation of the
inserted protein-sequence. Suitable regulatory sequences may be
derived from a variety of sources, including bacterial, fungal,
viral, mammalian, or insect genes (For example, see the regulatory
sequences described in Goeddel, Gene Expression Technology Methods
in Enzymology 185, Academic Press, San Diego, Calif. (1990)).
Selection of appropriate regulatory sequences is dependent on the
host cell chosen as discussed below, and may be readily
accomplished by one of ordinary skill in the art. Examples of such
regulatory sequences include: a transcriptional promoter and
enhancer or RNA polymerase binding sequence, a ribosomal binding
sequence, including a translation initiation signal. Additionally,
depending on the host cell chosen and the vector employed, other
sequences, such as an origin of replication, additional DNA
restriction sites, enhancers, and sequences conferring inducibility
of transcription may be incorporated into the expression
vector.
[0088] The recombinant expression vectors disclosed herein may also
contain a selectable marker gene which facilitates the selection of
host cells transformed or transfected with a recombinant molecule
disclosed herein. Examples of selectable marker genes are genes
encoding a protein such as G418 and hygromycin which confer
resistance to certain drugs, .beta.-galactosidase, chloramphenicol
acetyltransferase, firefly luciferase, or an immunoglobulin or
portion thereof such as the Fc portion of an immunoglobulin
preferably IgG. Transcription of the selectable marker gene is
monitored by changes in the concentration of the selectable marker
protein such as .beta.-galactosidase, chloramphenicol
acetyltransferase, or firefly luciferase. If the selectable marker
gene encodes a protein conferring antibiotic resistance such as
neomycin resistance transformant cells can be selected with G418.
Cells that have incorporated the selectable marker gene will
survive, while the other cells die. This makes it possible to
visualize and assay for expression of recombinant expression
vectors disclosed herein and in particular to determine the effect
of a mutation on expression and phenotype. It will be appreciated
that selectable markers can be introduced on a separate vector from
the nucleic acid of interest.
[0089] The recombinant expression vectors may also contain genes
which encode a fusion moiety which provides increased expression of
the recombinant protein; increased solubility of the recombinant
protein; and aid in the purification of the target recombinant
protein by acting as a ligand in affinity purification. For
example, a proteolytic cleavage site may be added to the target
recombinant protein to allow separation of the recombinant protein
from the fusion moiety subsequent to purification of the fusion
protein. Typical fusion expression vectors include pGEX (Amrad
Corp., Melbourne, Australia), pMal (New England Biolabs, Beverly,
Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the recombinant protein.
[0090] Recombinant expression vectors can be introduced into host
cells to produce a transformed host cell. The terms "transformed
with", "transfected with", "transformation" and "transfection" are
intended to encompass introduction of nucleic acid (e.g. a vector)
into a cell by one of many possible techniques known in the art.
The term "transformed host cell" as used herein is intended to also
include cells capable of glycosylation that have been transformed
with a recombinant expression vector disclosed herein. Prokaryotic
cells can be transformed with nucleic acid by, for example,
electroporation or calcium-chloride mediated transformation. For
example, nucleic acid can be introduced into mammalian cells via
conventional techniques such as calcium phosphate or calcium
chloride co-precipitation, DEAE-dextran mediated transfection,
lipofectin, electroporation or microinjection. Suitable methods for
transforming and transfecting host cells can be found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold
Spring Harbor Laboratory Press, 2001), and other laboratory
textbooks.
[0091] Suitable host cells include a wide variety of eukaryotic
host cells and prokaryotic cells. For example, the proteins
disclosed herein may be expressed in yeast cells or mammalian
cells. Other suitable host cells can be found in Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1991). In addition, the proteins disclosed
herein may be expressed in prokaryotic cells, such as Escherichia
coli (Zhang et al., Science 303 (5656): 371-3 (2004)). In addition,
a Pseudomonas based expression system such as Pseudomonas
fluorescens can be used (US Patent Application Publication No. US
2005/0186666, Schneider, Jane C et al.).
[0092] Yeast and fungi host cells suitable for carrying out the
disclosed methods include, but are not limited to Saccharomyces
cerevisiae, the genera Pichia or Kluyveromyces and various species
of the genus Aspergillus. Examples of vectors for expression in
yeast S. cerevisiae include pYepSec1(Baldari. et al., Embo J.
6:229-234 (1987)), pMFa (Kurjan and Herskowitz, Cell 30:933-943
(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2
(Invitrogen Corporation, San Diego, Calif.). Protocols for the
transformation of yeast and fungi are well known to those of
ordinary skill in the art (see Hinnen et al., Proc. Natl. Acad.
Sci. USA 75:1929 (1978); Itoh et al., J. Bacteriology 153:163
(1983), and Cullen et al. (Bio/Technology 5:369 (1987)).
[0093] Mammalian cells suitable for carrying out the disclosed
methods include, among others: COS (e.g., ATCC No. CRL 1650 or
1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa
(e.g., ATCC No. CCL 2), 293 (ATCC No. 1573) and NS-1 cells.
Suitable expression vectors for directing expression in mammalian
cells generally include a promoter (e.g., derived from viral
material such as polyoma, Adenovirus 2, cytomegalovirus and Simian
Virus 40), as well as other transcriptional and translational
control sequences. Examples of mammalian expression vectors include
pCDM8 (Seed, B., Nature 329:840 (1987)) and pMT2PC (Kaufman et al.,
EMBO J. 6:187-195 (1987)).
[0094] Given the teachings provided herein, promoters, terminators,
and methods for introducing expression vectors of an appropriate
type into plant, avian, and insect cells may also be readily
accomplished. For example, within one embodiment, the proteins
disclosed herein may be expressed from plant cells (see Sinkar et
al., J. Biosci (Bangalore) 11:47-58 (1987), which reviews the use
of Agrobacterium rhizogenes vectors; see also Zambryski et al.,
Genetic Engineering, Principles and Methods, Hollaender and Setlow
(eds.), Vol. VI, pp. 253-278, Plenum Press, New York (1984), which
describes the use of expression vectors for plant cells, including,
among others, PAPS2022, PAPS2023, and PAPS2034).
[0095] Insect cells suitable for carrying out the disclosed methods
include cells and cell lines from Bombyx, Trichoplusia or Spodotera
species. Baculovirus vectors available for expression of proteins
in cultured insect cells (SF 9 cells) include the pAc series (Smith
et al., Mol. Cell. Biol. 3:2156-2165 (1983)) and the pVL series
(Lucklow, V. A., and Summers, M. D., Virology 170:31-39 (1989)).
Some baculovirus-insect cell expression systems suitable for
expression of the recombinant proteins disclosed herein are
described in PCT/US/02442.
[0096] Alternatively, the proteins disclosed herein may also be
expressed in non-human transgenic animals such as rats, rabbits,
sheep and pigs (Hammer et al. Nature 315:680-683 (1985); Palmiter
et al. Science 222:809-814 (1983); Brinster et al. Proc. Natl.
Acad. Sci. USA 82:4438-4442 (1985); Palmiter and Brinster Cell
41:343-345 (1985) and U.S. Pat. No. 4,736,866).
[0097] The proteins disclosed herein may also be prepared by
chemical synthesis using techniques well known in the chemistry of
proteins such as solid phase synthesis (Merrifield, J. Am. Chem.
Assoc. 85:2149-2154 (1964); Frische et al., J. Pept. Sci. 2 (4):
212-22 (1996)) or synthesis in homogenous solution (Houbenweyl,
Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II,
Thieme, Stuttgart (1987)).
[0098] N-terminal or C-terminal fusion proteins comprising the
proteins disclosed herein conjugated with other molecules, such as
proteins may be prepared by fusing, through recombinant techniques.
The resultant fusion proteins contain a protein disclosed herein
fused to the selected protein or marker protein as described
herein. The recombinant protein disclosed herein may also be
conjugated to other proteins by known techniques. For example, the
proteins may be coupled using heterobifunctional thiol-containing
linkers as described in WO 90/10457,
N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5
thioacetate. Examples of proteins which may be used to prepare
fusion proteins or conjugates include cell binding proteins such as
immunoglobulins, hormones, growth factors, lectins, insulin, low
density lipoprotein, glucagon, endorphins, transferrin, bombesin,
asialoglycoprotein glutathione-S-transferase (GST), hemagglutinin
(HA), and truncated myc.
[0099] Accordingly, the present specification provides a
recombinant expression vector comprising the nucleic acid sequences
that encode the proteins disclosed herein, such as the light and
heavy chain complementarity determining regions, the light and
heavy chain variable regions, the binding proteins, such as
antibodies and antibody fragments, immunoconjugates disclosed
herein and novel isolated proteins disclosed herein. Further, the
present specification provides a host cell comprising the
recombinant expression vector disclosed herein.
[0100] In addition, the present polypeptide sequence may be capable
of selective binding to a solid support by including a positively
or a negatively charged amino acid sequence, a cysteine-containing
amino acid sequence, avidin, streptavidin, a functional fragment of
Staphylococcus protein A, GST, a His-tag, a FLAG-tag or Lex A. As
is described in the appended Examples, the polypeptide disclosed
herein exemplified by a single-chain antibody may also be expressed
with an N-terminal FLAG-tag and/or C-terminal His-tag that allow
for easy purification and detection. The FLAG-tag may include 8
amino acids. However, FLAG-tags comprised of shortened versions of
the FLAG used in the appended examples such as the amino acid
sequence Asp-Tyr-Lys-Asp are suitable as well. Thus, the present
peptides may be included in kits for the purification of an
analyte, or for detection of an analyte in a sample taken from a
subject. Thus, the present peptides may also be used for diagnosis
of NRA or other conditions.
[0101] The term "sample" as used herein refers to any fluid, cell
or tissue sample from a subject which can be assayed for an EpoR.
That is, any biological tissue or excreted material from an
organism. Examples of samples include blood, mucous, sperm, urine,
organ tissues, finger and toe nails, hair, skin cells, stool, milk,
tears, bile, marrow and tissue samples from organs including the
lungs, liver, kidney, heart, bladder, esophagus, stomach, breasts,
prostate, arteries, veins, lymph nodes, lymph ducts, tongue,
salivary gland, large intestine, small intestine, pituitary gland,
pineal gland, thymus, thyroid gland, parathyroid gland, adrenal
gland, ovary, oviduct, uterus, vagina, vulva, penis, testis,
spleen, brain, spinal cord, nose, pharynx, larynx, trachea, eye,
ear, and bowel.
[0102] The term "refolding" as used herein refers to the process by
which a protein structure assumes its functional shape or
conformation. It is the physical process by which a polypeptide
folds into its characteristic and functional three-dimensional
structure from random coil. It takes place at a basic pH (typically
pH 8.0-10.0, preferably pH 8.5-10, more preferably pH 8.5-9.6), a
low temperature (typically 0.0.degree. C. to 10.0.degree. C.,
preferably 2.0.degree. C. to 8.0.degree. C.), preferably with the
presence of a redox pair at suitable concentrations, and/or at the
presence of oxygen, and/or at the presence of catalyst(s) such as
copper ions at suitable concentration.
[0103] The present disclosures also relate to the treatment of a
subject with the modified non-human mammalian EPO or fusion protein
disclosed herein. The term "subject" is those suspected of having
anemia, which includes but is not limited to mammals including a
cat, a dog, a mouse, a rat, a hamster, a rabbit, a guinea pig, a
ruminant, a ferret, a non-human primate, a pig, or other livestock
having NRA or having the possibility of NRA, In one embodiment, the
subject is a cat, a dog, a mouse, a rat, a hamster, a rabbit, a
guinea pig, a ruminant, a ferret, a non-human primate, or a pig.
However, any non-human subject to be treated with the fusion
proteins or the pharmaceutical composition disclosed herein is
Included without limitation. In an aspect of this embodiment, the
subject is not a human being. In another aspect of this embodiment,
the treatment of a subject with the modified non-human mammalian
EPO or fusion protein disclosed herein is for a veterinary
treatment. The pharmaceutical composition including the novel
proteins disclosed herein is administered to a subject suspected of
anemia, thereby treating the subject effectively.
[0104] Since EPO has been shown to have a mitogenic and chemotactic
effect on vascular endothelial cells as well as an effect on
central cholinergic neurons (see, e.g., Amagnostou et al. (1990)
Proc. Natl. Acad. Sci. USA 87:5978-5982 and Konishi et al. (1993)
Brain Res. 609:29-35), the compounds disclosed herein may also find
use for the treatment of a variety of vascular disorders in
subjects, such as promoting wound healing, growth of collateral
coronary blood vessels (such as those that may occur after
myocardial infarction), trauma, and post-vascular graft treatment,
and a variety of neurological disorders, generally characterized by
low absolute levels of acetyl choline or low relative levels of
acetyl choline as compared to other neuroactive substances e.g.,
neurotransmitters.
[0105] In still another aspect, the present specification provides
a pharmaceutical composition (aka, "therapeutic") for the
prevention or treatment of NRA comprising the fusion proteins and
modified feline EPO proteins.
[0106] The term "formulation" as used herein refers to the modified
non-human mammalian EPO and fusion proteins disclosed herein and
excipients combined together which can be administered and has the
ability to bind to the corresponding receptors and initiate a
signal transduction pathway resulting in the desired activity. The
formulation can optionally comprise other agents so long as the
present modified non-human mammalian EPO or fusion protein retains
the ability to bind the corresponding receptors and ligands.
[0107] In an embodiment, a first therapeutic is administered to a
subject and at a later date, a second therapeutic is administered
to the same subject. In an embodiment, a first therapeutic is
administered to a subject at the same time as a second therapeutic
is administered to the subject.
[0108] In aspects of this embodiment, a sustained release
therapeutic delivery platform releases a therapeutic disclosed
herein with substantially zero order release kinetics over a period
of, e.g., about 7 days after administration, about 15 days after
administration, about 30 days after administration, about 45 days
after administration, about 60 days after administration, about 75
days after administration, or about 90 days after administration.
In other aspects of this embodiment, a sustained release
therapeutic delivery platform releases a therapeutic disclosed
herein with substantially zero order release kinetics over a period
of, e.g., at least 7 days after administration, at least 15 days
after administration, at least 30 days after administration, at
least 45 days after administration, at least 60 days after
administration, at least 75 days after administration, or at least
90 days after administration.
[0109] In aspects of this embodiment, a sustained release
therapeutic delivery platform releases a therapeutic disclosed
herein with substantially first order release kinetics over a
period of, e.g., about 7 days after administration, about 15 days
after administration, about 30 days after administration, about 45
days after administration, about 60 days after administration,
about 75 days after administration, or about 90 days after
administration. In other aspects of this embodiment, a sustained
release therapeutic delivery platform releases a therapeutic
disclosed herein with substantially first order release kinetics
over a period of, e.g., at least 7 days after administration, at
least 15 days after administration, at least 30 days after
administration, at least 45 days after administration, at least 60
days after administration, at least 75 days after administration,
or at least 90 days after administration.
[0110] In an embodiment, a therapeutic disclosed herein in may be
in any concentration desired. In aspects of this embodiment, the
concentration of a therapeutic disclosed herein may be, e.g., at
least 0.00001 mg/mL, at least 0.0001 mg/mL, at least 0.001 mg/mL,
at least 0.01 mg/mL, at least 0.1 mg/mL, at least 1 mg/mL, at least
10 mg/mL, at least 25 mg/mL, at least 50 mg/mL, at least 100 mg/mL,
at least 200 mg/mL, at least 500 mg/mL, at least 700 mg/mL, at
least 1,000 mg/mL, or at least 1,200 mg/mL. In other aspects of
this embodiment, the concentration of a therapeutic disclosed
herein in the solution may be, e.g., at most 1,000 mg/mL, at most
1,100 mg/mL, at most 1,200 mg/mL, at most 1,300 mg/mL, at most
1,400 mg/mL, at most 1,500 mg/mL, at most 2,000 mg/mL, at most
2,000 mg/mL, or at most 3,000 mg/mL. In other aspects of this
embodiment, the concentration of a therapeutic disclosed herein on
may be in a range of, e.g., about 0.00001 mg/mL to about 3,000
mg/mL, about 0.0001 mg/mL to about 3,000 mg/mL, about 0.01 mg/mL to
about 3,000 mg/mL, about 0.1 mg/mL to about 3,000 mg/mL, about 1
mg/mL to about 3,000 mg/mL, about 250 mg/mL to about 3,000 mg/mL,
about 500 mg/mL to about 3,000 mg/mL, about 750 mg/mL to about
3,000 mg/mL, about 1,000 mg/mL to about 3,000 mg/mL, about 100
mg/mL to about 2,000 mg/mL, about 250 mg/mL to about 2,000 mg/mL,
about 500 mg/mL to about 2,000 mg/mL, about 750 mg/mL to about
2,000 mg/mL, about 1,000 mg/mL to about 2,000 mg/mL, about 100
mg/mL to about 1,500 mg/mL, about 250 mg/mL to about 1,500 mg/mL,
about 500 mg/mL to about 1,500 mg/mL, about 750 mg/mL to about
1,500 mg/mL, about 1,000 mg/mL to about 1,500 mg/mL, about 100
mg/mL to about 1,200 mg/mL, about 250 mg/mL to about 1,200 mg/mL,
about 500 mg/mL to about 1,200 mg/mL, about 750 mg/mL to about
1,200 mg/mL, about 1,000 mg/mL to about 1,200 mg/mL, about 100
mg/mL to about 1,000 mg/mL, about 250 mg/mL to about 1,000 mg/mL,
about 500 mg/mL to about 1,000 mg/mL, about 750 mg/mL to about
1,000 mg/mL, about 100 mg/mL to about 750 mg/mL, about 250 mg/mL to
about 750 mg/mL, about 500 mg/mL to about 750 mg/mL, about 100
mg/mL to about 500 mg/mL, about 250 mg/mL to about 500 mg/mL, about
0.00001 mg/mL to about 0.0001 mg/mL, about 0.00001 mg/mL to about
0.001 mg/mL, about 0.00001 mg/mL to about 0.01 mg/mL, about 0.00001
mg/mL to about 0.1 mg/mL, about 0.00001 mg/mL to about 1 mg/mL,
about 0.001 mg/mL to about 0.01 mg/mL, about 0.001 mg/mL to about
0.1 mg/mL, about 0.001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to
about 10 mg/mL, or about 0.001 mg/mL to about 100 mg/mL. The final
concentration of a therapeutic disclosed herein in a pharmaceutical
composition disclosed herein may be of any concentration desired.
In an aspect of this embodiment, the final concentration of a
therapeutic in a pharmaceutical composition may be a
therapeutically effective amount. In other aspects of this
embodiment, the final concentration of a therapeutic in a
pharmaceutical composition may be, e.g., at least 0.00001 mg/mL, at
least 0.0001 mg/mL, at least 0.001 mg/mL, at least 0.01 mg/mL, at
least 0.1 mg/mL, at least 1 mg/mL, at least 10 mg/mL, at least 25
mg/mL, at least 50 mg/mL, at least 100 mg/mL, at least 200 mg/mL,
at least 500 mg/mL, at least 700 mg/mL, at least 1,000 mg/mL, or at
least 1,200 mg/mL. In other aspects of this embodiment, the
concentration of a cancer therapeutic disclosed herein in the
solution may be, e.g., at most 1,000 mg/mL, at most 1,100 mg/mL, at
most 1,200 mg/mL, at most 1,300 mg/mL, at most 1,400 mg/mL, at most
1,500 mg/mL, at most 2,000 mg/mL, at most 2,000 mg/mL, or at most
3,000 mg/mL. In other aspects of this embodiment, the final
concentration of a therapeutic in a pharmaceutical composition may
be in a range of, e.g., about 0.00001 mg/mL to about 3,000 mg/mL,
about 0.0001 mg/mL to about 3,000 mg/mL, about 0.01 mg/mL to about
3,000 mg/mL, about 0.1 mg/mL to about 3,000 mg/mL, about 1 mg/mL to
about 3,000 mg/mL, about 250 mg/mL to about 3,000 mg/mL, about 500
mg/mL to about 3,000 mg/mL, about 750 mg/mL to about 3,000 mg/mL,
about 1,000 mg/mL to about 3,000 mg/mL, about 100 mg/mL to about
2,000 mg/mL, about 250 mg/mL to about 2,000 mg/mL, about 500 mg/mL
to about 2,000 mg/mL, about 750 mg/mL to about 2,000 mg/mL, about
1,000 mg/mL to about 2,000 mg/mL, about 100 mg/mL to about 1,500
mg/mL, about 250 mg/mL to about 1,500 mg/mL, about 500 mg/mL to
about 1,500 mg/mL, about 750 mg/mL to about 1,500 mg/mL, about
1,000 mg/mL to about 1,500 mg/mL, about 100 mg/mL to about 1,200
mg/mL, about 250 mg/mL to about 1,200 mg/mL, about 500 mg/mL to
about 1,200 mg/mL, about 750 mg/mL to about 1,200 mg/mL, about
1,000 mg/mL to about 1,200 mg/mL, about 100 mg/mL to about 1,000
mg/mL, about 250 mg/mL to about 1,000 mg/mL, about 500 mg/mL to
about 1,000 mg/mL, about 750 mg/mL to about 1,000 mg/mL, about 100
mg/mL to about 750 mg/mL, about 250 mg/mL to about 750 mg/mL, about
500 mg/mL to about 750 mg/mL, about 100 mg/mL to about 500 mg/mL,
about 250 mg/mL to about 500 mg/mL, about 0.00001 mg/mL to about
0.0001 mg/mL, about 0.00001 mg/mL to about 0.001 mg/mL, about
0.00001 mg/mL to about 0.01 mg/mL, about 0.00001 mg/mL to about 0.1
mg/mL, about 0.00001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to
about 0.01 mg/mL, about 0.001 mg/mL to about 0.1 mg/mL, about 0.001
mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 10 mg/mL, or
about 0.001 mg/mL to about 100 mg/mL.
[0111] In an aspect, a concentration of a therapeutic disclosed
herein typically may be between about 50 mg/mL to about 1,000
mg/mL. In aspects of this embodiment, a therapeutically effective
amount of a therapeutic disclosed herein may be from, e.g., about
50 mg/mL to about 100 mg/mL, about 50 mg/mL to about 200 mg/mL,
about 50 mg/mL to about 300 mg/mL, about 50 mg/mL to about 400
mg/mL, about 50 mg/mL to about 500 mg/mL, about 50 mg/mL to about
600 mg/mL, about 50 mg/mL to about 700 mg/mL, about 50 mg/mL to
about 800 mg/mL, about 50 mg/mL to about 900 mg/mL, about 50 mg/mL
to about 1,000 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100
mg/mL to about 300 mg/mL, about 100 mg/mL to about 400 mg/mL, about
100 mg/mL to about 500 mg/mL, about 100 mg/mL to about 600 mg/mL,
about 100 mg/mL to about 700 mg/mL, about 100 mg/mL to about 800
mg/mL, about 100 mg/mL to about 900 mg/mL, about 100 mg/mL to about
1,000 mg/mL, about 200 mg/mL to about 300 mg/mL, about 200 mg/mL to
about 400 mg/mL, about 200 mg/mL to about 500 mg/mL, about 200
mg/mL to about 600 mg/mL, about 200 mg/mL to about 700 mg/mL, about
200 mg/mL to about 800 mg/mL, about 200 mg/mL to about 900 mg/mL,
about 200 mg/mL to about 1,000 mg/mL, about 300 mg/mL to about 400
mg/mL, about 300 mg/mL to about 500 mg/mL, about 300 mg/mL to about
600 mg/mL, about 300 mg/mL to about 700 mg/mL, about 300 mg/mL to
about 800 mg/mL, about 300 mg/mL to about 900 mg/mL, about 300
mg/mL to about 1,000 mg/mL, about 400 mg/mL to about 500 mg/mL,
about 400 mg/mL to about 600 mg/mL, about 400 mg/mL to about 700
mg/mL, about 400 mg/mL to about 800 mg/mL, about 400 mg/mL to about
900 mg/mL, about 400 mg/mL to about 1,000 mg/mL, about 500 mg/mL to
about 600 mg/mL, about 500 mg/mL to about 700 mg/mL, about 500
mg/mL to about 800 mg/mL, about 500 mg/mL to about 900 mg/mL, about
500 mg/mL to about 1,000 mg/mL, about 600 mg/mL to about 700 mg/mL,
about 600 mg/mL to about 800 mg/mL, about 600 mg/mL to about 900
mg/mL, or about 600 mg/mL to about 1,000 mg/mL.
[0112] In a further aspect, an amount of a therapeutic disclosed
herein typically may be between about 0.01% to about 45% by weight.
In aspects of this embodiment, an amount of a therapeutic disclosed
herein may be from, e.g., about 0.1% to about 45% by weight, about
0.1% to about 40% by weight, about 0.1% to about 35% by weight,
about 0.1% to about 30% by weight, about 0.1% to about 25% by
weight, about 0.1% to about 20% by weight, about 0.1% to about 15%
by weight, about 0.1% to about 10% by weight, about 0.1% to about
5% by weight, about 1% to about 45% by weight, about 1% to about
40% by weight, about 1% to about 35% by weight, about 1% to about
30% by weight, about 1% to about 25% by weight, about 1% to about
20% by weight, about 1% to about 15% by weight, about 1% to about
10% by weight, about 1% to about 5% by weight, about 5% to about
45% by weight, about 5% to about 40% by weight, about 5% to about
35% by weight, about 5% to about 30% by weight, about 5% to about
25% by weight, about 5% to about 20% by weight, about 5% to about
15% by weight, about 5% to about 10% by weight, about 10% to about
45% by weight, about 10% to about 40% by weight, about 10% to about
35% by weight, about 10% to about 30% by weight, about 10% to about
25% by weight, about 10% to about 20% by weight, about 10% to about
15% by weight, about 15% to about 45% by weight, about 15% to about
40% by weight, about 15% to about 35% by weight, about 15% to about
30% by weight, about 15% to about 25% by weight, about 15% to about
20% by weight, about 20% to about 45% by weight, about 20% to about
40% by weight, about 20% to about 35% by weight, about 20% to about
30% by weight, about 20% to about 25% by weight, about 25% to about
45% by weight, about 25% to about 40% by weight, about 25% to about
35% by weight, or about 25% to about 30% by weight.
[0113] As used herein, the term "prevention" means all of the
actions by which the occurrence of the disease is suppressed,
restrained or retarded. In the present specification, "prevention"
means that the occurrence of anemia is suppressed, restrained or
retarded by administration of the conjugates disclosed herein.
[0114] As used herein, the term "treatment" means all of the
actions by which one or more symptoms of the disease have been
alleviated, improved or ameliorated. In the present specification,
"treatment" means that the symptoms of NRA are alleviated, improved
or ameliorated by administration of the novel proteins disclosed
herein.
[0115] As used herein, the term "administration" means introduction
of an amount of a predetermined substance into a patient by a
certain suitable method. The composition disclosed herein may be
administered via any of the common routes, as long as it is able to
reach a desired tissue, for example, but is not limited to,
intraperitoneal, intravenous, intramuscular, subcutaneous,
intradermal, oral, topical, intranasal, intrapulmonary, or
intrarectal administration. However, since peptides are digested
upon oral administration, active ingredients of a composition for
oral administration should be coated or formulated for protection
against degradation in the stomach.
[0116] The composition disclosed herein may be formulated into a
variety of dosage forms in combination with pharmaceutically
acceptable carriers, binders, lubricants, disintegrants,
excipients, diluents, solubilizers, dispersing agents, stabilizers,
suspending agents, colorants, flavorants, buffering agents,
preserving agents, anti-oxidants, analgesics, solubilizers,
isotonic agents, and base materials.
[0117] For example, for oral administration, the pharmaceutical
composition may be formulated into tablets, troches, capsules,
elixirs, suspensions, syrups or wafers. For injectable
preparations, the pharmaceutical composition may be formulated into
an ampule as a single dosage form or a multi-dose container. The
pharmaceutical composition may also be formulated into solutions,
suspensions, tablets, pills, capsules and long-acting
preparations.
[0118] Further, the pharmaceutical composition disclosed herein may
have any formulation selected from the group consisting of tablets,
pills, powders, granules, capsules, suspensions, liquids for
internal use, emulsions, syrups, sterile aqueous solutions,
non-aqueous solvents, lyophilized formulations and
suppositories.
[0119] The pharmaceutical composition disclosed herein may further
include a pharmaceutically acceptable excipient or diluent. As used
herein, the term "pharmaceutically acceptable" means that the
composition is sufficient to achieve the therapeutic effects
without deleterious side effects, and may be readily determined
depending on the type of the diseases, the subject's species, age,
body weight, health conditions, gender, and drug sensitivity,
administration route, administration mode, administration
frequency, duration of treatment, drugs used in combination or
coincident with the composition disclosed herein, and other factors
known in medicine.
[0120] For oral administration, the pharmaceutical composition may
include, but is not limited to, a carrier, a binder, a lubricant, a
disintegrant, an excipient, a diluent, a solubilizer, a dispersing
agent, a stabilizer, a suspending agent, a colorant, and a
flavorant. For injectable preparations, the pharmaceutical
composition may include a buffering agent, a preserving agent, an
analgesic, a solubilizer, an isotonic agent, and a stabilizer. For
preparations for topical administration, the carrier may include a
base, an excipient, a lubricant, and a preserving agent.
[0121] Examples of the carrier for the pharmaceutical compositions
include physiological saline, organic solvents, lactose, dextrose,
sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch,
acacia rubber, alginate, gelatin, calcium phosphate, calcium
silicate, cellulose, methylcellulose, microcrystalline cellulose,
polyvinylpyrrolidone, water, methylhydroxybenzoate,
propylhydroxybenzoate, talc, magnesium stearate and mineral oils.
In addition, the excipient or diluent for pharmaceutical
formulations may further include fillers, anti-coagulating agents,
solubilizer, anti-oxidants, lubricants, humectants, flavorants, and
antiseptics. In one embodiment, the pharmaceutical composition may
be obtained by blending with a variety of pharmaceutically
acceptable carriers. In order to increase the stability or
absorptivity, carbohydrates such as glucose, sucrose or dextrans,
antioxidants such as ascorbic acid or glutathione, chelating
agents, low molecular weight proteins or other stabilizers may be
used.
[0122] Further, the pharmaceutical composition may be formulated
into a single dosage form suitable for the subject's body. For
example, the pharmaceutical composition is formulated into a
preparation useful for peptide drugs according to the typical
method in the pharmaceutical field so as to be administered by an
oral or parenteral route such as through skin, intravenous,
intramuscular, intra-arterial, intramedullary, intramedullary,
intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal, intracolonic, topical, sublingual,
vaginal, or rectal administration, but is not limited thereto.
[0123] The administration dose and frequency of the pharmaceutical
composition disclosed herein are determined by the type of active
ingredient, together with various factors such as the disease to be
treated, administration route, subject's species, age, gender, and
body weight, and disease severity.
[0124] Timing of Administration Methods disclosed herein include
administration of the modified non-human mammalian EPO or fusion
proteins disclosed herein prior to, substantially contemporaneously
with or after the subject has been diagnosed with NRA, and
administration prior to, substantially contemporaneously with or
after a pathology or a development of one or more adverse symptoms
of NRA or pathologies caused by NRA. Methods, compositions and uses
disclosed herein also include administration of the modified
non-human mammalian EPO or fusion proteins to a subject prior to,
substantially contemporaneously with or following the
identification of an adverse symptom, disorder, illness or disease
caused by or associated with NRA, or pathology resulting from NRA.
A subject with NRA may have NRA or present symptoms of NRA over a
period of 1-5, 5-10, 10-20, 20-30, 30-50, 50-100 hours, days,
weeks, months, or years.
[0125] The compositions disclosed herein can be combined with any
compound, agent, drug, treatment or other therapeutic regimen or
protocol having a desired therapeutic, beneficial, additive,
synergistic or complementary activity or effect. Exemplary
combination compositions and treatments include second actives such
as, e.g., drugs or agents related to the treatment of bone marrow
disease, tick disease, abscesses, cancer, kidney failure, toxic
chemical exposure, radiation exposure, lead poisoning, and
inherited NRA. Such drugs, agents, treatments and therapies can be
administered or performed prior to, substantially contemporaneously
with or following any other method disclosed herein, for example, a
therapeutic method of treating a subject for an NRA thereto, or a
method of prophylactic treatment of a subject for NRA. Moreover,
the pharmaceutical composition may be administered alone or in
combination or coincident with other pharmaceutical formulations
showing prophylactic or therapeutic effects on anemia, especially
NRA.
[0126] The compositions disclosed herein can be administered as a
combination composition, or administered separately, such as
concurrently or in series or sequentially (prior to or following)
administering a second active, to an individual. The present
specification therefore provides combinations in which a method or
use is used in a combination with any compound, agent, drug,
therapeutic regimen, treatment protocol, process, remedy or
composition, such as drugs or agents related to the treatment of
bone marrow disease, tick disease, abscesses, cancer, kidney
failure, toxic chemical exposure, radiation exposure, lead
poisoning, and inherited NRA. The compound, agent, drug,
therapeutic regimen, treatment protocol, process, remedy or
composition can be administered or performed prior to,
substantially contemporaneously with or following administration of
one or more of the present modified non-human mammalian EPO or
fusion proteins or subsequences, portions or modifications thereof,
or a nucleic acid encoding all or a portion of the present modified
non-human mammalian EPO or fusion proteins, subsequence, portion or
modification thereof, to a subject. Specific non-limiting examples
of combination embodiments therefore include the foregoing or other
compound, agent, drug, therapeutic regimen, treatment protocol,
process, remedy or composition.
[0127] Single Dose/Period of Time The total effective dose of the
composition disclosed herein may be administered to a subject in a
single dose, or may be administered for a period of time in
multiple doses according to a fractionated treatment protocol. For
instance, the dose may be determined as a total dose over the
lifetime of the subject, a total dose over the expected treatment
period, a total monthly dose, a total weekly dose, or a total daily
dose.
[0128] In an embodiment, the period of administration of a modified
non-human mammalian EPO or fusion proteins disclosed herein is for
1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9
days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4
weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11
weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months,
9 months, 10 months, 11 months, 12 months, or more.
[0129] Dosing can be single dosage or cumulative (serial dosing),
and can be readily determined by one skilled in the art. For
instance, treatment of anemia may comprise a one-time
administration of a sufficient dose of a composition disclosed
herein. As a non-limiting example, a sufficient dose of a
composition disclosed herein can be administered once to a subject,
e.g., as a single injection or deposition or a single oral
administration. Alternatively, treatment may comprise a one-time
administration of a sufficient dose of a composition disclosed
herein carried out over a range of time periods, such as, e.g.,
daily, once every few days, weekly, monthly or yearly. As a
non-limiting example, an antigen or a composition disclosed herein
can be administered once or twice weekly to a subject. The timing
of administration can vary from subject to subject, depending upon
such factors as the severity of the subject's symptoms. For
example, a sufficient dose of an antigen or a composition disclosed
herein can be administered to a subject once a month for an
indefinite period of time, or until the subject no longer requires
therapy. A person of ordinary skill in the art will recognize that
the condition of the subject can be monitored throughout the course
of treatment and that the sufficient amount of a composition
disclosed herein that is administered can be adjusted
accordingly.
[0130] Total Daily Dose In one embodiment the total daily dose of
the modified non-human mammalian EPO or fusion protein disclosed
herein may be approximately 0.01 .mu.g to 5 mg per 1 kg of body
weight of a patient (aka .mu.g/kg or mg/kg). In one embodiment, the
total daily dose is at least 0.01 .mu.g/kg, at least 0.05 .mu.g/kg,
at least 0.1 .mu.g/kg, at least 0.5 .mu.g/kg, at least 1 .mu.g/kg,
at least 5 .mu.g/kg, at least 0.01 mg/kg, at least 0.05 mg/kg, at
least 0.1 mg/kg, at least 0.5 mg/kg, at least 1 mg/kg, at least 1.5
mg/kg, at least 2 mg/kg, at least 2.5 mg/kg, at least 3 mg/kg, at
least 3.5, mg/kg, at least 4 mg/kg, at least 4.5 mg/kg, or at least
5 mg/kg. In one aspect, the maximum daily dose is at most 5 mg/kg,
at most 4.5 mg/kg, at most 4.0 mg/kg, at most 3.5 mg/kg, at most 3
mg/kg, at most 2.5 mg/kg, at most 2 mg/kg, at most 1.5 mg/kg, at
most 1 mg/kg, at most 0.5 mg/kg, at most 0.1 mg/kg, at most 0.05
mg/kg, at most 10 .mu.g/kg, at most 5 .mu.g/kg, at most 1 .mu.g/kg,
at most 0.05 .mu.g/kg, or at most 0.1 .mu.g/kg. In yet another
aspect the daily dose of the modified non-human mammalian EPO or
fusion protein disclosed herein may be approximately 0.05 .mu.g/kg
to 5 mg/kg, 0.1 .mu.g/kg to 5 mg/kg, 1 .mu.g/kg to 5 mg/kg, 50
.mu.g/kg to 5 mg/kg, 0.1 mg/kg to 5 mg/kg, or 1 mg/kg to 5 mg/kg.
In an even further aspect the daily dose of the modified non-human
mammalian EPO or fusion protein may be approximately 0.01 .mu.g/kg
to 2 mg/kg, 0.05 .mu.g/kg to 2 mg/kg, 0.1 .mu.g/kg to 2 mg/kg, 1
.mu.g/kg to 2 mg/kg, 5 .mu.g/kg to 2 mg/kg, 10 .mu.g/kg to 2 mg/kg,
50 .mu.g/kg to 2 mg/kg, 100 .mu.g/kg to 2 mg/kg, 0.5 mg/kg to 2
mg/kg, or 1 mg/kg to 2 mg/kg. In still other aspects of this
embodiment, a sufficient amount of a modified non-human mammalian
EPO or fusion protein disclosed herein is the dosage sufficient to
reduce a symptom associated with anemia for, e.g., at least one
week, at least one month, at least two months, at least three
months, at least four months, at least five months, at least six
months, at least seven months, at least eight months, at least nine
months, at least ten months, at least eleven months, or at least
twelve months.
[0131] However, the effective dose of the modified non-human
mammalian EPO or fusion protein is determined considering various
factors including subject's species, age, body weight, health
conditions, gender, disease severity, diet, and secretion rate, in
addition to administration route and treatment frequency of the
pharmaceutical composition. In view of this, those skilled in the
art may easily determine an effective dose suitable for the
particular use of the pharmaceutical composition disclosed herein.
The pharmaceutical composition disclosed herein is not particularly
limited to the formulation, and administration route and mode, as
long as it shows a beneficial effects.
[0132] The pharmaceutical composition disclosed herein is expected
to have longer in vivo duration of efficacy and titer, thereby
remarkably reducing the number and frequency of administration
thereof when compared to administration of native EPO (i.e., EPO
having the full-length amino acid sequence of the naturally
occurring EPO amino acid sequence). In one embodiment, a
composition disclosed herein and its derivatives or variants have
half-lives of 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours,
8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours,
15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21
hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6
days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, one month, two
months, three months, four months or more.
[0133] In an embodiment, an individual is provided a treatment
protocol wherein a pharmaceutical composition is to be administered
to a subject on a periodic schedule, wherein the individual is
informed by electronic notification to administer the therapeutic
on a period schedule. In an aspect of this embodiment, the
electronic notification is by email, text, instant messaging or by
another electronic notification method. In an embodiment, an
individual is informed to administer the presently disclosed
therapeutic on a period schedule through receipt of a telephone
call, postal mail, overnight express (including, without
limitation, FedEx and UPS) or other method of notification.
[0134] Aspects of the present specification can also be described
as follows: [0135] 1. A modified non-human mammalian erythropoietin
comprising a mammalian erythropoietin sequence and at least one
added or relocated glycosylation site. [0136] 2. The modified
non-human mammalian erythropoietin according to embodiment 1,
wherein the mammalian erythropoietin comprises an amino acid
sequence having at least 90% sequence homology to a sequence
selected from the group consisting of: feline erythropoietin, or
canine erythropoietin. [0137] 3. The modified non-human mammalian
erythropoietin according to embodiment 1 or embodiment 2, wherein
the mammalian erythropoietin comprises an amino acid sequence
having at least 95% sequence identity to an amino acid sequence of
a feline erythropoietin or a canine erythropoietin. [0138] 4. The
modified non-human mammalian erythropoietin according to any one of
embodiments 1-3, wherein the mammalian erythropoietin comprises an
amino acid sequence having at least 90%, at least 95%, at least
96%, at least 97%, at least 98% or at least 99% sequence identity
to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO: 11,
wherein Xaa at position 18 of SEQ ID NO: 5 or SEQ ID NO: 6 is
selected from E or G; and Xaa at position 116 of SEQ ID NO: 5 or
SEQ ID NO: 6 is K or absent. [0139] 5. The modified non-human
mammalian erythropoietin according to any one of embodiments 1-4,
wherein the mammalian erythropoietin comprises from about 3 to
about 5, about 3 to about 8, about 3 to about 10, about 3 to about
12, about 3 to about 15 or about 3 to about 17 amino acid
additions, deletions, or substitutions. [0140] 6. The modified
non-human mammalian erythropoietin according to any one of
embodiments 1-5, wherein the glycosylation site has the structure
Asn-Xaa-Ser/Thr, wherein Xaa is any amino acid. [0141] 7. The
modified non-human mammalian erythropoietin according to any one of
embodiments 1-6, wherein the glycosylation site is a site for the
attachment of an N-linked carbohydrate chain. [0142] 8. The
modified non-human mammalian erythropoietin according to any one of
embodiments 1-7, wherein the glycosylation site comprises a serine,
threonine, hydroxyproline, or hydroxylysine. [0143] 9. The modified
non-human mammalian erythropoietin according to any one of
embodiments 1-8, wherein the glycosylation site is a site for the
attachment of an O-linked carbohydrate chain. [0144] 10. The
modified non-human mammalian erythropoietin according to any one of
embodiments 1-9, wherein the glycosylation site is located at the
carboxy terminal region of the mammalian erythropoietin. [0145] 11.
The modified non-human mammalian erythropoietin according to any
one of embodiments 1-9, wherein the glycosylation site is inserted
between amino acids in the mammalian erythropoietin sequence.
[0146] 12. The modified non-human mammalian erythropoietin
according to any one of embodiments 1-11, wherein the glycosylation
site further comprises a fragment of human chorionic gonadotropin.
[0147] 13. The modified non-human mammalian erythropoietin
according to embodiment 12, wherein the fragment of human chorionic
gonadotropin is SEQ ID NO: 8 or an amino acid sequence having at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%
or at least 99% sequence identity to SEQ ID NO: 8, or an amino acid
sequence having 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5,
3-4, 3-5, or 4-5 amino acid additions, deletions or substitutions
to SEQ ID NO: 8. [0148] 14. The modified non-human mammalian
erythropoietin according to any one of embodiments 1-13, further
comprising at least one additional carbohydrate chain attached
thereto. [0149] 15. The modified non-human mammalian erythropoietin
according to any one of embodiments 1-14, wherein the mammalian
erythropoietin is feline erythropoietin having the amino acid
sequence of SEQ ID NO: 5 or SEQ ID NO: 6, wherein Xaa at position
18 is selected from E or G; and Xaa at position 116 is K or absent,
or a sequence having at least 90%, at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% sequence identity to SEQ ID
NO: 5 or SEQ ID NO: 6, wherein Xaa at position 18 is selected from
E or G; and Xaa at position 116 is K or absent, and wherein in SEQ
ID NO: 5 or SEQ ID NO: 6 an asparagine residue is substituted for
the amino acid residue at any one or more of positions 30, 51, 57,
69, 88, 89, 137, or 139. [0150] 16. The modified non-human
mammalian erythropoietin according to any one of embodiments 1-15,
wherein the mammalian erythropoietin is feline erythropoietin
having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6,
wherein Xaa at position 18 is selected from E or G; and Xaa at
position 116 is K or absent, or a sequence having at least 90%, at
least 95%, at least 96%, at least 97%, at least 98% or at least 99%
sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6, wherein Xaa at
position 18 is selected from E or G; and Xaa at position 116 is K
or absent, and wherein in SEQ ID NO: 5 or SEQ ID NO: 6 either a
serine or a threonine residue is substituted for the amino acid
residue at position 126. [0151] 17. The modified non-human
mammalian erythropoietin according to any one of embodiments 1-14,
wherein the mammalian erythropoietin is feline erythropoietin
having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6,
wherein Xaa at position 18 is selected from E or G; and Xaa at
position 116 is K or absent, or a sequence having at least 90%, at
least 95%, at least 96%, at least 97%, at least 98% or at least 99%
sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6, wherein Xaa at
position 18 is selected from E or G; and Xaa at position 116 is K
or absent, and wherein in SEQ ID NO: 5 or SEQ ID NO: 6 one or more
of the following amino acid substitution sets is present:
Asn.sup.30 and Thr.sup.32; Asn.sup.51 and Thr.sup.53; Asn.sup.57,
Thr.sup.59 and Asn.sup.69; Asn.sup.69 and Thr.sup.71; Ser.sup.68,
Asn.sup.69 and Thr.sup.71; Val.sup.87 and Asn.sup.88; Ser.sup.87
and Asn.sup.88; Ser.sup.87, Asn.sup.88 and Gly.sup.89; Ser.sup.87,
Asn.sup.88 and Thr.sup.92; Ser.sup.87, Asn.sup.88 and Ala.sup.163;
Asn.sup.69, Thr.sup.71, Ser.sup.87 and Asn.sup.88; Asn.sup.30,
Thr.sup.32, Val.sup.87 and Asn.sup.88; Asn.sup.89, Ile.sup.90 and
Thr.sup.91; Ser.sup.87, Asn.sup.89, Ile.sup.90 and Thr.sup.91;
Asn.sup.137 and Thr.sup.139; Asn.sup.139 and Thr.sup.141;
Thr.sup.126; Pro.sup.125 and Thr.sup.126; or any combination
thereof. [0152] 18. The modified non-human mammalian erythropoietin
according to embodiment 17, wherein the mammalian erythropoietin is
feline erythropoietin having the amino acid sequence of SEQ ID NO:
5 or SEQ ID NO: 6, wherein Xaa at position 18 is selected from E or
G; and Xaa at position 116 is K or absent, or a sequence having at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%
or at least 99% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6,
wherein Xaa at position 18 is selected from E or G; and Xaa at
position 116 is K or absent, and wherein in SEQ ID NO: 5 or SEQ ID
NO: 6 one or more of the following substitution sets is present:
Ser.sup.87 and Asn.sup.88; and/or Asn.sup.30, Thr.sup.32,
Val.sup.87 and Asn.sup.88. [0153] 19. The modified non-human
mammalian erythropoietin according to embodiment 17, wherein the
mammalian erythropoietin is feline erythropoietin having the amino
acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, wherein Xaa at
position 18 is selected from E or G; and Xaa at position 116 is K
or absent, with one of the following amino acid substitution sets
is present: Gln.sup.24, Ser.sup.87 and Asn.sup.88; Gln.sup.38,
Ser.sup.87 and Asn.sup.88; or Gln.sup.83, Ser.sup.87 and
Asn.sup.88. [0154] 20. The modified non-human mammalian
erythropoietin according to any one of embodiments 1-14, wherein
the mammalian erythropoietin is feline erythropoietin of SEQ ID NO:
5 or SEQ ID NO: 6, wherein Xaa at position 18 is selected from E or
G; and Xaa at position 116 is K or absent, modified by deleting one
or more of the glycosylation sites that attach to N linked
carbohydrate chains and by adding a glycosylation site that
attaches to an N linked carbohydrate chain at amino acid position
88 of SEQ ID NO: 5 or SEQ ID NO: 6. [0155] 21. The modified
non-human mammalian erythropoietin according to any one of
embodiments 1-14, wherein the mammalian erythropoietin is canine
erythropoietin having the amino acid sequence of SEQ ID NO: 10 or
SEQ ID NO: 11, or a sequence having at least 90%, at least 95%, at
least 96%, at least 97%, at least 98% or at least 99% sequence
identity to SEQ ID NO: 10 or SEQ ID NO: 11, wherein in SEQ ID NO:
10 an asparagine residue is substituted for the amino acid residue
at any one or more of positions 30, 51, 57, 69, 88, 89, 137, or 139
and wherein in SEQ ID NO: 11 an asparagine residue is substituted
for the amino acid residue at any one or more of positions 56, 77,
83, 95, 114, 115, 163, or 165. [0156] 22. The modified non-human
mammalian erythropoietin according to any one of embodiments 1-14
and 21, wherein the mammalian erythropoietin is canine
erythropoietin having the amino acid sequence of SEQ ID NO: 10 or
SEQ ID NO: 11, or a sequence having at least 90%, at least 95%, at
least 96%, at least 97%, at least 98% or at least 99% sequence
identity to SEQ ID NO: 10 or SEQ ID NO: 11, wherein in SEQ ID NO:
10 either a serine or threonine residue is substituted for the
amino acid residue at position 126; and wherein in SEQ ID NO: 11
either a serine or threonine residue is substituted for the amino
acid residue at position 152. [0157] 23. The modified non-human
mammalian erythropoietin according to any one of embodiments 1-14,
wherein the mammalian erythropoietin is canine erythropoietin
having the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11,
or a sequence having at least 90%, at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% sequence identity to SEQ ID
NO: 10 or SEQ ID NO: 11, wherein in SEQ ID NO: 10 one or more of
the following amino acid substitution sets is present: Asn.sup.30
and Thr.sup.32; Asn.sup.51 and Thr.sup.53; Asn.sup.57 and
Thr.sup.59; Asn.sup.69; Asn.sup.69 and Thr.sup.71; Ser.sup.68,
Asn.sup.69 and Thr.sup.71; Val.sup.87 and Asn.sup.88; Ser.sup.87
and Asn.sup.88; Ser.sup.87, Asn.sup.88 and Gly.sup.89; Ser.sup.87,
Asn.sup.88 and Thr.sup.92; Ser.sup.87, Asn.sup.88 and Ala.sup.163;
Asn.sup.69, Thr.sup.71, Ser.sup.87 and Asn.sup.88; Asn.sup.30,
Thr.sup.32, Val.sup.87 and Asn.sup.88; Asn.sup.89, Ile.sup.90 and
Thr.sup.91; Ser.sup.87, Asn.sup.89, Ile.sup.90 and Thr.sup.91;
Asn.sup.137 and Thr.sup.139; Asn.sup.139 and Thr.sup.141;
Thr.sup.126; and/or Pro.sup.125 and Thr.sup.126; and wherein in SEQ
ID NO: 11 one or more of the following amino acid substitution sets
is present: Asn.sup.56 and Thr.sup.58; Asn.sup.77 and Thr.sup.79;
Asn.sup.83 and Thr.sup.85; Asn.sup.69; Asn.sup.95 and Thr.sup.97;
Ser.sup.94, Asn.sup.95 and Thr.sup.97; Val.sup.113 and Asn.sup.114;
Ser.sup.113 and Asn.sup.114; Ser.sup.113, Asn.sup.114 and
Gly.sup.115; Ser.sup.113, Asn.sup.114 and Thr.sup.118; Ser.sup.113,
Asn.sup.114 and Ala.sup.189; Asn.sup.95, Thr.sup.97, Ser.sup.113
and Asn.sup.114; Asn.sup.56, Thr.sup.58, Val.sup.113 and
Asn.sup.114; Asn.sup.115, Ile.sup.116 and Thr.sup.117; Ser.sup.113,
Asn.sup.115, Ile.sup.116 and Thr.sup.117; Asn.sup.163 and
Thr.sup.165; Asn.sup.165 and Thr.sup.167; Thr.sup.152; and/or
Pro.sup.151 and Thr.sup.152. [0158] 24. The modified non-human
mammalian erythropoietin according to embodiment 23, wherein the
mammalian erythropoietin is a canine erythropoietin having the
amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or a
sequence having at least 90%, at least 95%, at least 96%, at least
97%, at least 98% or at least 99% sequence identity to SEQ ID NO:
10 or SEQ ID NO: 11, wherein in SEQ ID NO: 10 one or more of the
following substitution sets is present: Ser.sup.87 and Asn.sup.88
and/or Asn.sup.30, Thr.sup.32, Val.sup.87 and Asn.sup.88; and
wherein in SEQ ID NO: 11 one or more of the following substitution
sets is present: Ser.sup.113 and Asn.sup.114 and/or Asn.sup.56,
Thr.sup.58, Val.sup.113 and Asn.sup.114. [0159] 25. The modified
non-human mammalian erythropoietin according to embodiment 23,
wherein the mammalian erythropoietin is a canine erythropoietin
having the amino acid sequence of SEQ ID NO: 10 with one of the
following amino acid substitution sets present: Gln.sup.24,
Ser.sup.87 and Asn.sup.88; Gln.sup.38, Ser.sup.87 and Asn.sup.88;
or Gln.sup.83, Ser.sup.87 and Asn.sup.88; or having the amino acid
sequence of SEQ ID NO: 11 with one of the following amino acid
substitution sets present: Gln.sup.50, Ser.sup.113 and Asn.sup.114,
Gln.sup.64, Ser.sup.113 and Asn.sup.114; or Gln.sup.109,
Ser.sup.113 and Asn.sup.114. [0160] 26. The modified non-human
mammalian erythropoietin according to any one of embodiments 1-14,
wherein the mammalian erythropoietin is canine erythropoietin of
SEQ ID NO: 10 or SEQ ID NO: 11, modified by deleting one or more of
the glycosylation sites that attach to N linked carbohydrate chains
and by adding a glycosylation site that attaches to an N linked
carbohydrate chain at amino acid position 88 of SEQ ID NO: 10 or
SEQ ID NO: 11. [0161] 27. A nucleic acid sequence encoding a
modified non-human mammalian erythropoietin comprising a mammalian
erythropoietin sequence and at least one added or relocated
glycosylation site. [0162] 27. A nucleic acid sequence encoding a
modified non-human mammalian erythropoietin as defined in any one
of embodiments 1-27. [0163] 28. A nucleic acid sequence encoding a
modified feline erythropoietin comprising the amino acid sequence
of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO: 11 or
encoding an amino acid sequence having at least 90%, at least 95%,
at least 96%, at least 97%, at least 98% or at least 99% sequence
identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO:
11. [0164] 29. A vector comprising [0165] a) a nucleic acid
sequence encoding a modified non-human mammalian erythropoietin
comprising a mammalian erythropoietin sequence and at least one
added or relocated glycosylation site, and [0166] b) a promoter
operatively linked to the nucleic acid sequence. [0167] 30. A
vector comprising [0168] a) a nucleic acid sequence encoding the
amino acid sequence for a modified non-human mammalian
erythropoietin as defined in any one of embodiments 1-26 or a
nucleic acid sequence as defined in embodiments 27-28, and [0169]
b) a promoter operatively linked to the nucleic acid sequence.
[0170] 31. A host cell comprising the vector of embodiment 29 or
embodiment 30. [0171] 32. A composition comprising [0172] a) a
modified non-human mammalian erythropoietin comprising a mammalian
erythropoietin sequence and at least one added or relocated
glycosylation site, and [0173] b) a pharmaceutically acceptable
diluent, adjuvant, or carrier. [0174] 33. A composition comprising
[0175] a) a modified non-human mammalian erythropoietin as defined
in any one of embodiments 1-26 or a vector as defined in embodiment
29 or embodiment 30, and
[0176] b) a pharmaceutically acceptable diluent, adjuvant, or
carrier. [0177] 34. Use of a modified non-human mammalian
erythropoietin comprising a mammalian erythropoietin sequence and
at least one added or relocated glycosylation site in the
manufacture of a medicament for the treatment of non-regenerative
anemia (NRA). [0178] 35. Use of a modified non-human mammalian
erythropoietin as defined in any one of embodiments 1-26 or a
vector as defined in embodiment 29 or embodiment 30 in the
manufacture of a medicament for the treatment of non-regenerative
anemia (NRA). [0179] 36. A method of treating non regenerative
anemia (NRA) in a mammal comprising administering to a subject in
need thereof an effective amount of a modified non-human mammalian
erythropoietin comprising a mammalian erythropoietin sequence and
at least one added or relocated glycosylation site or a composition
comprising a modified non-human mammalian erythropoietin comprising
a mammalian erythropoietin sequence and at least one added or
relocated glycosylation site. [0180] 37. A method of treating non
regenerative anemia (NRA) in a mammal comprising administering to a
subject in need thereof an effective amount of a modified non-human
mammalian erythropoietin as defined in any one of embodiments 1-26
or a composition as defined in embodiment 32 or embodiment 33.
[0181] 38. Use of a modified non-human mammalian erythropoietin
comprising a mammalian erythropoietin sequence and at least one
added or relocated glycosylation site for the treatment of
non-regenerative anemia (NRA). [0182] 39. Use of a modified
non-human mammalian erythropoietin as defined in any one of
embodiments 1-26 or a composition as defined in embodiment 32 or
embodiment 33 for the treatment of non-regenerative anemia (NRA).
[0183] 40. The method of embodiment 34 or embodiment 35 or the use
of embodiment 38 or embodiment 39, wherein the mammal is a cat, a
dog, a mouse, a rat, a hamster, a rabbit, a guinea pig, a ruminant,
a ferret, a non-human primate, or a pig. [0184] 41. A fusion
protein, comprising a peptide, a linker and an Fc fragment,
wherein: [0185] a) the peptide is SEQ ID NO: 1, an amino acid
sequence having at least 90%, at least 95%, at least 96%, at least
97%, at least 98% or at least 99% sequence identity to SEQ ID NO:
1, a feline EPO with the amino acid sequence SEQ ID NO: 5 or SEQ ID
NO: 6, wherein Xaa at position 18 is selected from E or G; and Xaa
at position 116 is K or absent, a sequence having at least 90%, at
least 95%, at least 96%, at least 97%, at least 98% or at least 99%
sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6, wherein Xaa at
position 18 is selected from E or G; and Xaa at position 116 is K
or absent, a canine EPO with the amino acid sequence SEQ ID NO: 10
or SEQ ID NO: 11, or a sequence having at least 90%, at least 95%,
at least 96%, at least 97%, at least 98% or at least 99% sequence
identity to SEQ ID NO: 10 or SEQ ID NO: 11; and [0186] b) the Fc
fragment has an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3
or SEQ ID NO: 4, or a sequence having at least 90%, at least 95%,
at least 96%, at least 97%, at least 98% or at least 99% sequence
identity to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; [0187]
wherein the peptide is fused, through linker, to the Fc fragment.
[0188] 42. The fusion protein according to embodiment 41, wherein
the C terminus of the peptide is fused through the linker to the N
terminus of the Fc fragment. [0189] 43. The fusion protein
according to embodiment 42, wherein the N terminus of the peptide
is fused through the linker to the C terminus of the Fc fragment.
[0190] 44. A nucleic acid sequence encoding the fusion protein of
any one of embodiments 40-42. [0191] 45. A vector comprising the
nucleic acid sequence according to embodiment 44 and a promoter
operatively linked to the nucleic acid sequence. [0192] 46. A host
cell comprising the vector of embodiment 45. [0193] 47. A
composition comprising: [0194] a) a fusion protein as defined in
any one of embodiments 40-42 or a vector as defined in embodiment
45, and [0195] b) a pharmaceutically acceptable diluent, adjuvant,
or carrier. [0196] 48. Use of a fusion protein as defined in any
one of embodiments 40-3429 or a vector as defined in embodiment 45
in the manufacture of a medicament for the treatment of
non-regenerative anemia (NRA). [0197] 49. A method of treating non
regenerative anemia (NRA) in a mammal comprising administering to a
subject in need thereof an effective amount of a fusion protein as
defined in any one of embodiments 37-39 or a composition as defined
in embodiment 47. [0198] 50. Use of a fusion protein as defined in
any one of embodiments 40-3429 or a composition as defined in
embodiment 47 for the treatment of non-regenerative anemia (NRA).
[0199] 51. The method of embodiment 49 or the use of embodiment 50,
wherein the mammal is a cat, a dog, a mouse, a rat, a hamster, a
rabbit, a guinea pig, a ruminant, a ferret, a non-human primate, or
a pig.
EXAMPLES
[0200] The following non-limiting examples are provided for
illustrative purposes only in order to facilitate a more complete
understanding of representative embodiments now contemplated. These
examples should not be construed to limit any of the embodiments
described in the present specification, including those pertaining
to the compounds, pharmaceutical compositions, or methods or uses
of treating a disorder disclosed herein.
Example 1--Production of Modified Feline EPO
[0201] DNA for modified feline EPO having the modifications
Val.sup.87Asn.sup.88 was synthesized (SEQ ID NO: 14) and cloned
into a bacterial expression vector. The complete expression
construct comprising the modified feline EPO DNA gene (named as
ASKBH01) was confirmed by DNA sequencing. The expression construct
was amplified by transforming into DH10B E. coli and culturing the
cells overnight. DNA for the expression construct was prepared and
purified by endo-free plasmid kit (from QIAGEN.RTM.).
[0202] Cell lines stably expressing a modified Feline EPO protein
were obtained by transfecting the expression construct into
GS.sup.-/- Chinese hamster ovarian cells (CHO) by electroporation
and screening for transfected CHO cells using a selective culture
medium without glutamine (EX-CELL.RTM. CD CHO Fusion Growth
Medium). In this manner 32 stable minipools were established and
the leading mini-pool was selected based on expression level in
batch and fed-batch cultures. FIG. 3 shows ELISA titer results of
the batch cell culture during the 1st round of batch culture clone
selection. Single cloning was performed by limited dilution and
using clone media, two leading single clones out of 132 positive
clones (in total 960 wells) were selected based on productivity and
cell growth in batch and fed-batch culture. The lead clones were
expanded and seeded at 0.5.times.10.sup.6 cells/mL, total 300 mL in
2 L shake flasks, and the cells were cultured at 37.degree. C., 5%
CO.sub.2, 70% HMR conditions and shaking at 120 rpm. The cultures
were fed by using 5% Acti CHO.RTM. Feed A+0.5% Feed B (from GE
Health) on Day 3, 6, 7, 8 and 9. The cell viability, viable cell
density were monitored every other day, the cultures were harvested
on Day 11. The cell growth profile, viability and titer are shown
in FIG. 4.
[0203] Modified Feline EPO protein was harvested by clarifying
approximately 600 mL of the cultured cell medium through
centrifugation at 2000 rpm for 10 minutes followed by filtration.
The clarified supernant was concentrated to approximately 100 mL
and buffer exchanged into 10 mM Tris (pH 7.1). A Pellicone II UF
membrane with 30 kDa molecular weight cut-off was used for this
UFDF step. The UFDF pool was aliquoted and frozen at -80.degree. C.
until further purification.
[0204] The UFDF pool containing the modified feline EPO was thawed
and loaded to a column packed with Q sepahrose FF resin at neutral
pH. The column has a diameter of 2 cm and a bed height of
approximately 1.5 cm. The column was washed with 10 mM Tris, pH
7.1, followed by a 2.sup.nd wash with 2 mM acetic acid, 1 mM
glycine, 6 M urea, 20 micoM CuSO4, pH 4.8 to remove host cell
impurities and feline EPO isoforms with approximately 7 sialic
acids or less. The column was further washed with 40 mM acetic
acid, 1 mM glycine, 6 M urea, 20 micoM CuSO4, pH 4.0. This step
removed additional cell impurities and feline EPO isoforms
containing approximately 8 to approximately 12 sialic acids. The
column was then eluted with 10 mM Tris, 140 mM NaCl, 20 micoM
CuSO4, pH 7.1 to elute the modified feline EPO isoforms containing
more than 12 sialic acids. Chromatography and SDS-PAGE analysis
showed that isoforms containing more than 12 sialic acids were
expressed at dominant level comparing to the fractions presumably
containing the modified feline EPO with lower levels of sialic
acids.
[0205] The elution pool containing 12 or more sialic acids is
further purified using reverse phase chromatography, a 2.sup.nd
anion exchange chromatography, size exclusion chromatography,
and/or hydroxyapatite chromatography to achieve high purity,
wherein the level of aggregates is lower than 0.5%, preferably
<0.2%, and further preferably <0.1%.
Example 2--Production of EpoR Agonist Peptide-Fc Fusion Protein
[0206] DNA for a GGTYSCHFGPLTBVCRPQGG-Linker-feline IgG1a Fc fusion
protein is synthesized and cloned into a bacterial expression
vector. The complete expression construct comprising the
GGTYSCHFGPLTBVCRPQGG-Linker-feline IgG1a Fc fusion protein gene is
confirmed by DNA sequencing. The expression construct was amplified
by transforming into DH10B E. coli and culturing the cells
overnight. DNA for the expression construct was prepared and
purified by endo-free plasmid kit (from QIAGEN.RTM.).
Alternatively, the fusion protein is recovered in the insoluble
inclusion body (IB). The IB is solubilized and the fusion protein
is refolded at pH between 8.0-10.0, preferably 8.5-9.5, at a
temperature between 0-8.degree. C.
[0207] The expression construct is transfected into GS.sup.-/-
Chinese hamster ovarian cells (CHO) and stable cell pools are
obtained as discussed in Example 1. The
GGTYSCHFGPLTBVCRPQGG-Linker-feline IgG1a Fc fusion protein is
expressed, harvested as discussed in Example 1. The harvested
GGTYSCHFGPLTBVCRPQGG-Linker-feline IgG1a Fc fusion protein is
purified by chromatography steps including Protein A affinity, ion
exchange, hydroxyapatite and/or hydrophobic interaction. The
purified bulk is formulated and lyophilized.
Example 3--Analysis of Sialic Acid Content in the Modified Feline
EPO
[0208] Sialic acid content of recombinant modified feline EPO was
determined using the OPD-labeling method as described previously,
see, e.g., Anumula, K. R., Rapid quantitative determination of
sialic acids in glycoproteins by High-Performance Liquid
Chromatography with a sensitive fluorescence detection. Anal.
Biochem. 230 (1): 24-30 (1995). In brief, sialic acid was detached
from purified rhEPO in 0.5 M NaHSO.sub.4 for 20 minutes at
80.degree. C., and derivatized with OPD (o-phenylenediamine-2HCl;
Sigma) for 40 minutes at 80.degree. C. OPD-labeled sialic acid was
separated on a C18 reversed-phase column (Shim-pack CLC-ODS;
Shimadzu, Kyoto, Japan) and detected by a fluorescence detector
(Model 474; Waters) with wavelengths 230 nm emission and 425 nm
excitation.
Example 4--Analysis of Sialylation Profile of N-Linked Glycans from
Recombinant Modified Feline EPO
[0209] To release N-linked glycans, purified modified feline EPO
(50 .mu.g) was resuspended in N-glycanase reaction buffer
comprising 20 mM sodium phosphate, 0.02% sodium azide, (pH 7.5)
containing 0.1% SDS, and 50 mM .beta.-mercaptoethanol. rhEPO was
denatured by boiling for 5 minutes, and added with 0.75% NP-40. The
mixture was incubated with 2 units N-glycosidase F (Roche)
overnight at 37.degree. C., to release N-linked glycans from rhEPO.
Peptides and detergents were then removed using GLYCOCLEAN.TM. R
cartridge, and salts were removed using GLYCOCLEAN.TM. H cartridge
(GLYKO.RTM.; PROZYME.RTM., Hayward, Calif.). Purified N-linked
glycans were derivatized with 2-AB (2-aminobenzamide) using
SIGNAL.TM. labeling kit (GLYKO.RTM.), and nonreacted 2-AB reagents
were removed using GLYCOCLEAN.TM. S cartridge. The above procedures
were performed following manufacturer's instructions. 2-AB
derivatized N-linked glycans from rhEPO were separated according to
number of sialic acids using an anion-exchange column (TSKgel
DEAE-SPW, 7.5 mm.times.75 mm; Tosoh, Tokyo, Japan). Eluents and
gradient conditions were as described previously (Llop et al.
2007). Samples were eluted over a 35 min linear gradient from 0%
(v/v) to 100% (v/v) solvent A (solvent A: 50% 500 mM ammonium
formate, pH 4.5. 30% water, and 20% acetonitrile (ACN); solvent B:
80% water, 20% ACN) at a flow rate of 0.4 mL/min at 30.degree. C.,
and detected by a fluorescence detector (Model 474; Waters) at
wavelengths 330 nm emission and 420 nm excitation. Number of sialic
acid in each peak was determined by comparison with peaks in
standard 2-AB bovine fetuin N-linked glycan library
(GLYKO.RTM.).
Example 5--Cell-Based Activity Assay: TF-1 Proliferation Assay
[0210] Cultured TF-1 cells were maintained in complete medium
consisting of RPMI 1640 mediume supplemented with 2 mM L-glutamine,
1% penicillin-streptomycin, n 5% FBS, and recombinant human
granulocyte-macrophage colony-stimulating factor at 2 ng/mL. Cells
were cultured in a flask and incubated at 37.degree. C. in a
humidified incubator with 5% CO.sub.2. Diluted recombinant modified
feline EPO samples to 2.times. (200 ng/mL) into 200 mL assay medium
in the first well of 96 well plate. Performed serial 2.times.
dilutions for 12 wells (carry 100 .mu.L from first well into 100
.mu.L medium in second well etc.). Assay samples in at least
triplicate. Harvested TF-1 cells. Centrifuged at 1200 rpm for 7
minute. Re-suspend cell pellet in 10 mL assay medium. Repeated
centrifugation and re-suspension 2 more times to remove growth
factor. Diluted cells to 100,000 cells/mL in assay medium. Added
100 .mu.L/well (10,000 cells/well). Cultured for 3 days (37.degree.
C., 5% CO.sub.2). Add 20 mL/well Alamar Blue. Incubate 6 hours
(37.degree. C., 5% CO.sub.2). Using microplate reader, measure
fluorescence (545 nm excitation, 590 nm emission). FIG. 5 shows the
cell-based activity assay data for the Elution Fraction from the
Initial Purification of Example 1. A recombinant canine
erythropoietin (cEPO) sample from R&D Systems was used as
reference for this analysis. As discussed above, this elution pool
was expected to contain feline EPO with higher levels (12 or more)
of sialic acids. This elution pool showed higher EC.sub.50 as
compared to the reference material dog EPO. This is consistent with
prior report, where human EPO analogs with higher levels of
glycosylation showed higher EC50 in vitro binding and cell-based
activity, see, e.g., Angus M Sinclair: "Erythropoiesis stimulating
agents: approaches to modulate activity". Biologics. 2013; 7:
161-174.
Example 6--In Vivo Activity Study
[0211] Cats are dosed subcutaneously once per week of the fusion
protein at 0.05-0.1 mg/kg or 1 .mu.g/kg of the modified feline EPO
for up to 6 weeks. The hematocrits of all cats were determined at
baseline and twice weekly thereafter. At the conclusion of the
experiment, serum from all animals was collected and assayed for
antibodies to the injected product.
[0212] In closing, it is to be understood that although aspects of
the present specification are highlighted by referring to specific
embodiments, one skilled in the art will readily appreciate that
these disclosed embodiments are only illustrative of the principles
of the subject matter disclosed herein. Therefore, it should be
understood that the disclosed subject matter is in no way limited
to a particular compound, composition, article, apparatus,
methodology, protocol, and/or reagent, etc., described herein,
unless expressly stated as such. In addition, those of ordinary
skill in the art will recognize that certain changes,
modifications, permutations, alterations, additions, subtractions
and sub-combinations thereof can be made in accordance with the
teachings herein without departing from the spirit of the present
specification. It is therefore intended that the following appended
claims and claims hereafter introduced are interpreted to include
all such changes, modifications, permutations, alterations,
additions, subtractions and sub-combinations as are within their
true spirit and scope.
[0213] Certain embodiments of the present invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Of course, variations on these described
embodiments will become apparent to those of ordinary skill in the
art upon reading the foregoing description. The inventor expects
skilled artisans to employ such variations as appropriate, and the
inventors intend for the present invention to be practiced
otherwise than specifically described herein. Accordingly, this
invention includes all modifications and equivalents of the subject
matter recited in the claims appended hereto as permitted by
applicable law. Moreover, any combination of the above-described
embodiments in all possible variations thereof is encompassed by
the invention unless otherwise indicated herein or otherwise
clearly contradicted by context.
[0214] Groupings of alternative embodiments, elements, or steps of
the present invention are not to be construed as limitations. Each
group member may be referred to and claimed individually or in any
combination with other group members disclosed herein. It is
anticipated that one or more members of a group may be included in,
or deleted from, a group for reasons of convenience and/or
patentability. When any such inclusion or deletion occurs, the
specification is deemed to contain the group as modified thus
fulfilling the written description of all Markush groups used in
the appended claims.
[0215] Unless otherwise indicated, all numbers expressing a
characteristic, item, quantity, parameter, property, term, and so
forth used in the present specification and claims are to be
understood as being modified in all instances by the term "about."
As used herein, the term "about" means that the characteristic,
item, quantity, parameter, property, or term so qualified
encompasses a range of plus or minus ten percent above and below
the value of the stated characteristic, item, quantity, parameter,
property, or term. Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the specification and
attached claims are approximations that may vary. For instance, as
mass spectrometry instruments can vary slightly in determining the
mass of a given analyte, the term "about" in the context of the
mass of an ion or the mass/charge ratio of an ion refers to +/-0.50
atomic mass unit. At the very least, and not as an attempt to limit
the application of the doctrine of equivalents to the scope of the
claims, each numerical indication should at least be construed in
light of the number of reported significant digits and by applying
ordinary rounding techniques.
[0216] Use of the terms "may" or "can" in reference to an
embodiment or aspect of an embodiment also carries with it the
alternative meaning of "may not" or "cannot." As such, if the
present specification discloses that an embodiment or an aspect of
an embodiment may be or can be included as part of the inventive
subject matter, then the negative limitation or exclusionary
proviso is also explicitly meant, meaning that an embodiment or an
aspect of an embodiment may not be or cannot be included as part of
the inventive subject matter. In a similar manner, use of the term
"optionally" in reference to an embodiment or aspect of an
embodiment means that such embodiment or aspect of the embodiment
may be included as part of the inventive subject matter or may not
be included as part of the inventive subject matter. Whether such a
negative limitation or exclusionary proviso applies will be based
on whether the negative limitation or exclusionary proviso is
recited in the claimed subject matter.
[0217] Notwithstanding that the numerical ranges and values setting
forth the broad scope of the invention are approximations, the
numerical ranges and values set forth in the specific examples are
reported as precisely as possible. Any numerical range or value,
however, inherently contains certain errors necessarily resulting
from the standard deviation found in their respective testing
measurements. Recitation of numerical ranges of values herein is
merely intended to serve as a shorthand method of referring
individually to each separate numerical value falling within the
range. Unless otherwise indicated herein, each individual value of
a numerical range is incorporated into the present specification as
if it were individually recited herein.
[0218] The terms "a," "an," "the" and similar references used in
the context of describing the present invention (especially in the
context of the following claims) are to be construed to cover both
the singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. Further, ordinal indicators--such
as "first," "second," "third," etc.--for identified elements are
used to distinguish between the elements, and do not indicate or
imply a required or limited number of such elements, and do not
indicate a particular position or order of such elements unless
otherwise specifically stated. All methods described herein can be
performed in any suitable order unless otherwise indicated herein
or otherwise clearly contradicted by context. The use of any and
all examples, or exemplary language (e.g., "such as") provided
herein is intended merely to better illuminate the present
invention and does not pose a limitation on the scope of the
invention otherwise claimed. No language in the present
specification should be construed as indicating any non-claimed
element essential to the practice of the invention.
[0219] Specific embodiments disclosed herein may be further limited
in the claims using consisting of or consisting essentially of
language. When used in the claims, whether as filed or added per
amendment, the transition term "consisting of" excludes any
element, step, or ingredient not specified in the claims. The
transition term "consisting essentially of" limits the scope of a
claim to the specified materials or steps and those that do not
materially affect the basic and novel characteristic(s).
Embodiments of the present invention so claimed are inherently or
expressly described and enabled herein.
[0220] All patents, patent publications, and other publications
referenced and identified in the present specification are
individually and expressly incorporated herein by reference in
their entirety for the purpose of describing and disclosing, for
example, the compositions and methodologies described in such
publications that might be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0221] Lastly, the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the present invention, which is defined solely
by the claims. Accordingly, the present invention is not limited to
that precisely as shown and described.
Sequence CWU 1
1
16120PRTUnknownArtificial Sequence related to EPOR Agonist
peptides.VARIANT(13)..(13)X at position 13 may be Trp,
1-naphthylalanine, or 2-naphthylalanine. 1Gly Gly Thr Tyr Ser Cys
His Phe Gly Pro Leu Thr Xaa Val Cys Arg1 5 10 15Pro Gln Gly Gly
202237PRTFelis catusVariable(1)..(8)Amino acids 1-8 may be each be
present or absent. 2Arg Lys Thr Asp His Pro Pro Gly Pro Lys Pro Cys
Asp Cys Pro Lys1 5 10 15Cys Pro Pro Pro Glu Met Leu Gly Gly Pro Ser
Ile Phe Ile Phe Pro 20 25 30Pro Lys Pro Lys Asp Thr Leu Ser Ile Ser
Arg Thr Pro Glu Val Thr 35 40 45Cys Leu Val Val Asp Leu Gly Pro Asp
Asp Ser Asp Val Gln Ile Thr 50 55 60Trp Phe Val Asp Asn Thr Gln Val
Tyr Thr Ala Lys Thr Ser Pro Arg65 70 75 80Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Pro Ile 85 90 95Leu His Gln Asp Trp
Leu Lys Gly Lys Glu Phe Lys Cys Lys Val Asn 100 105 110Ser Lys Ser
Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Lys 115 120 125Gly
Gln Pro His Glu Pro Gln Val Tyr Val Leu Pro Pro Ala Gln Glu 130 135
140Glu Leu Ser Arg Asn Lys Val Ser Val Thr Cys Leu Ile Lys Ser
Phe145 150 155 160His Pro Pro Asp Ile Ala Val Glu Trp Glu Ile Thr
Gly Gln Pro Glu 165 170 175Pro Glu Asn Asn Tyr Arg Thr Thr Pro Pro
Gln Leu Asp Ser Asp Gly 180 185 190Thr Tyr Phe Val Tyr Ser Lys Leu
Ser Val Asp Arg Ser His Trp Gln 195 200 205Arg Gly Asn Thr Tyr Thr
Cys Ser Val Ser His Glu Ala Leu His Ser 210 215 220His His Thr Gln
Lys Ser Leu Thr Gln Ser Pro Gly Lys225 230 2353237PRTFelis
catusVariable(1)..(8)Amino Acids 1-8 may each be present or absent.
3Arg Lys Thr Asp His Pro Pro Gly Pro Lys Pro Cys Asp Cys Pro Lys1 5
10 15Cys Pro Pro Pro Glu Met Leu Gly Gly Pro Ser Ile Phe Ile Phe
Pro 20 25 30Pro Lys Pro Lys Asp Thr Leu Ser Ile Ser Arg Thr Pro Glu
Val Thr 35 40 45Cys Leu Val Val Asp Leu Gly Pro Asp Asp Ser Asp Val
Gln Ile Thr 50 55 60Trp Phe Val Asp Asn Thr Gln Val Tyr Thr Ala Lys
Thr Ser Pro Arg65 70 75 80Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Pro Ile 85 90 95Leu His Gln Asp Trp Leu Lys Gly Lys
Glu Phe Lys Cys Lys Val Asn 100 105 110Ser Lys Ser Leu Pro Ser Pro
Ile Glu Arg Thr Ile Ser Lys Asp Lys 115 120 125Gly Gln Pro His Glu
Pro Gln Val Tyr Val Leu Pro Pro Ala Gln Glu 130 135 140Glu Leu Ser
Arg Asn Lys Val Ser Val Thr Cys Leu Ile Glu Gly Phe145 150 155
160Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ile Thr Gly Gln Pro Glu
165 170 175Pro Glu Asn Asn Tyr Arg Thr Thr Pro Pro Gln Leu Asp Ser
Asp Gly 180 185 190Thr Tyr Phe Leu Tyr Ser Arg Leu Ser Val Asp Arg
Ser Arg Trp Gln 195 200 205Arg Gly Asn Thr Tyr Thr Cys Ser Val Ser
His Glu Ala Leu His Ser 210 215 220His His Thr Gln Lys Ser Leu Thr
Gln Ser Pro Gly Lys225 230 2354237PRTFelis
catusVariable(1)..(8)Amino acids 1-8 each may independently be
present or absent. 4Pro Lys Thr Ala Ser Thr Ile Glu Ser Lys Thr Gly
Glu Gly Pro Lys1 5 10 15Cys Pro Val Pro Glu Ile Pro Gly Ala Pro Ser
Val Phe Ile Phe Pro 20 25 30Pro Lys Pro Lys Asp Thr Leu Ser Ile Ser
Arg Thr Pro Glu Val Thr 35 40 45Cys Leu Val Val Asp Leu Gly Pro Asp
Asp Ser Asn Val Gln Ile Thr 50 55 60Trp Phe Val Asp Asn Thr Glu Met
His Thr Ala Lys Thr Arg Pro Arg65 70 75 80Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Pro Ile 85 90 95Leu His Gln Asp Trp
Leu Lys Gly Lys Glu Phe Lys Cys Lys Val Asn 100 105 110Ser Lys Ser
Leu Pro Ser Ala Met Glu Arg Thr Ile Ser Lys Ala Lys 115 120 125Gly
Gln Pro His Glu Pro Gln Val Tyr Val Leu Pro Pro Thr Gln Glu 130 135
140Glu Leu Ser Glu Asn Lys Val Ser Val Thr Cys Leu Ile Lys Gly
Phe145 150 155 160His Pro Pro Asp Ile Ala Val Glu Trp Glu Ile Thr
Gly Gln Pro Glu 165 170 175Pro Glu Asn Asn Tyr Gln Thr Thr Pro Pro
Gln Leu Asp Ser Asp Gly 180 185 190Thr Tyr Phe Leu Tyr Ser Arg Leu
Ser Val Asp Arg Ser His Trp Gln 195 200 205Arg Gly Asn Thr Tyr Thr
Cys Ser Val Ser His Glu Ala Leu His Ser 210 215 220His His Thr Gln
Lys Ser Leu Thr Gln Ser Pro Gly Lys225 230 2355166PRTFelis
catusVariable(18)..(18)Amino acid at position 18 may be selected
from E or G.Variable(116)..(116)Amino acid at position 116 may be K
or absent. 5Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg
Tyr Ile1 5 10 15Leu Xaa Ala Arg Glu Ala Glu Asn Val Thr Met Gly Cys
Ala Glu Gly 20 25 30Cys Ser Phe Ser Glu Asn Ile Thr Val Pro Asp Thr
Lys Val Asn Phe 35 40 45Tyr Thr Trp Lys Arg Met Asp Val Gly Gln Gln
Ala Val Glu Val Trp 50 55 60Gln Gly Leu Ala Leu Leu Ser Glu Ala Ile
Leu Arg Gly Gln Ala Leu65 70 75 80Leu Ala Asn Ser Ser Gln Pro Ser
Glu Thr Leu Gln Leu His Val Asp 85 90 95Lys Ala Val Ser Ser Leu Arg
Ser Leu Thr Ser Leu Leu Arg Ala Leu 100 105 110Gly Ala Gln Xaa Glu
Ala Thr Ser Leu Pro Glu Ala Thr Ser Ala Ala 115 120 125Pro Leu Arg
Thr Phe Thr Val Asp Thr Leu Cys Lys Leu Phe Arg Ile 130 135 140Tyr
Ser Asn Phe Leu Arg Gly Lys Leu Thr Leu Tyr Thr Gly Glu Ala145 150
155 160Cys Arg Arg Gly Asp Arg 1656166PRTFelis
catusVariable(18)..(18)The amino acid a position 18 may selected
from E or G.Variable(116)..(116)The amino acid a position 116 may
be K or absent. 6Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu
Glu Arg Tyr Ile1 5 10 15Leu Xaa Ala Arg Glu Ala Glu Asn Val Thr Met
Gly Cys Asn Glu Thr 20 25 30Cys Ser Phe Ser Glu Asn Ile Thr Val Pro
Asp Thr Lys Val Asn Phe 35 40 45Tyr Thr Trp Lys Arg Met Asp Val Gly
Gln Gln Ala Val Glu Val Trp 50 55 60Gln Gly Leu Ala Leu Leu Ser Glu
Ala Ile Leu Arg Gly Gln Ala Leu65 70 75 80Leu Ala Asn Ser Ser Gln
Val Asn Glu Thr Leu Gln Leu His Val Asp 85 90 95Lys Ala Val Ser Ser
Leu Arg Ser Leu Thr Ser Leu Leu Arg Ala Leu 100 105 110Gly Ala Gln
Xaa Glu Ala Thr Ser Leu Pro Glu Ala Thr Ser Ala Ala 115 120 125Pro
Leu Arg Thr Phe Thr Val Asp Thr Leu Cys Lys Leu Phe Arg Ile 130 135
140Tyr Ser Asn Phe Leu Arg Gly Lys Leu Thr Leu Tyr Thr Gly Glu
Ala145 150 155 160Cys Arg Arg Gly Asp Arg 16575PRTArtificial
SequenceLinker sequence.Repeat(1)..(5)The sequence may be extended
by repetition 2-4 times. 7Gly Gly Gly Gly Ser1 5828PRTArtificial
SequenceChorionic Gonadotropin fragment. 8Ser Ser Ser Ser Lys Ala
Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg1 5 10 15Leu Pro Gly Pro Ser
Asp Thr Pro Ile Leu Pro Gln 20 25927PRTHomo sapiensSIGNAL(1)..(27)
9Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu1 5
10 15Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly 20 2510166PRTCanis
familiaris 10Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu
Arg Tyr Ile1 5 10 15Leu Glu Ala Arg Glu Ala Glu Asn Val Thr Met Gly
Cys Ala Gln Gly 20 25 30Cys Ser Phe Ser Glu Asn Ile Thr Val Pro Asp
Thr Lys Val Asn Phe 35 40 45Tyr Thr Trp Lys Arg Met Asp Val Gly Gln
Gln Ala Leu Glu Val Trp 50 55 60Gln Gly Leu Ala Leu Leu Ser Glu Ala
Ile Leu Arg Gly Gln Ala Leu65 70 75 80Leu Ala Asn Ala Ser Gln Pro
Ser Glu Thr Pro Gln Leu His Val Asp 85 90 95Lys Ala Val Ser Ser Leu
Arg Ser Leu Thr Ser Leu Leu Arg Ala Leu 100 105 110Gly Ala Gln Lys
Glu Ala Met Ser Leu Pro Glu Glu Ala Ser Pro Ala 115 120 125Pro Leu
Arg Thr Phe Thr Val Asp Thr Leu Cys Lys Leu Phe Arg Ile 130 135
140Tyr Ser Asn Phe Leu Arg Gly Lys Leu Thr Leu Tyr Thr Gly Glu
Ala145 150 155 160Cys Arg Arg Gly Asp Arg 16511192PRTCanis
familiaris 11Met Gly Ala Cys Glu Cys Pro Ala Leu Phe Leu Leu Leu
Ser Leu Leu1 5 10 15Leu Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro
Pro Arg Leu Ile 20 25 30Cys Asp Ser Arg Val Leu Glu Arg Tyr Ile Leu
Glu Ala Arg Glu Ala 35 40 45Glu Asn Val Thr Met Gly Cys Ala Gln Gly
Cys Ser Phe Ser Glu Asn 50 55 60Ile Thr Val Pro Asp Thr Lys Val Asn
Phe Tyr Thr Trp Lys Arg Met65 70 75 80Asp Val Gly Gln Gln Ala Leu
Glu Val Trp Gln Gly Leu Ala Leu Leu 85 90 95Ser Glu Ala Ile Leu Arg
Gly Gln Ala Leu Leu Ala Asn Ala Ser Gln 100 105 110Pro Ser Glu Thr
Pro Gln Leu His Val Asp Lys Ala Val Ser Ser Leu 115 120 125Arg Ser
Leu Thr Ser Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala 130 135
140Met Ser Leu Pro Glu Glu Ala Ser Pro Ala Pro Leu Arg Thr Phe
Thr145 150 155 160Val Asp Thr Leu Cys Lys Leu Phe Arg Ile Tyr Ser
Asn Phe Leu Arg 165 170 175Gly Lys Leu Thr Leu Tyr Thr Gly Glu Ala
Cys Arg Arg Gly Asp Arg 180 185 1901226PRTFelis
catusSIGNAL(1)..(26) 12Met Gly Ser Cys Glu Cys Pro Ala Leu Leu Leu
Leu Leu Ser Leu Leu1 5 10 15Leu Leu Pro Leu Gly Leu Pro Val Leu Gly
20 251340PRTCanis familiarisSIGNAL(1)..(40) 13Met Cys Glu Pro Ala
Pro Pro Lys Pro Thr Gln Ser Ala Trp His Ser1 5 10 15Phe Pro Glu Cys
Pro Ala Leu Leu Leu Leu Leu Ser Leu Leu Leu Leu 20 25 30Pro Leu Gly
Leu Pro Val Leu Gly 35 4014593DNAArtificial SequenceDNA sequence
for Modified Feline EPO including 5'-Hind III and 3'-Pac I
restriction endonuclease cleavage sites 14aagcttatgg gttcctgtga
atgccctgcc ctcctcctcc tgctgtccct gttgttgctc 60cccctcggac tcccggtcct
gggcgcgccc ccaagactga tctgcgattc acgcgtgctg 120gagcggtaca
ttcttgaggc tcgggaagcc gagaacgtga ccatgggttg taacgagact
180tgctcgttct ccgaaaacat taccgtgccg gacaccaagg tcaacttcta
cacctggaaa 240cggatggacg tgggacagca agccgtggaa gtgtggcagg
ggcttgccct gctgtccgag 300gccatcctgc gcggccaggc cctgctggcc
aactcaagcc aggtcaacga gactctgcaa 360cttcacgtgg ataaggccgt
gtcgagcctg aggagcctca cctcgctcct gcgggcactg 420ggagcccaga
aggaagccac ttccctgcct gaagcaacat ccgctgcgcc gctgaggacc
480tttactgtgg acacgctgtg caagctgttc cgcatctact ccaatttcct
gcgggggaag 540ctgaccttgt ataccggaga agcgtgccgc agaggcgaca
gatagttaat taa 593154PRTArtificial sequenceshortened version of a
FLAG tag 15Asp Tyr Lys Asp11620PRTArtificial sequenceLinker-feline
IgG1a Fc fusion protein 16Gly Gly Thr Tyr Ser Cys His Phe Gly Pro
Leu Thr Asx Val Cys Arg1 5 10 15Pro Gln Gly Gly 20
* * * * *