U.S. patent application number 17/067929 was filed with the patent office on 2021-06-03 for use of cd24 proteins for treating leptin-deficient conditions.
This patent application is currently assigned to ONCOIMMUNE, INC.. The applicant listed for this patent is ONCOIMMUNE, INC.. Invention is credited to Martin DEVENPORT, Yang LIU, Pan ZHENG.
Application Number | 20210162006 17/067929 |
Document ID | / |
Family ID | 1000005397718 |
Filed Date | 2021-06-03 |
United States Patent
Application |
20210162006 |
Kind Code |
A1 |
LIU; Yang ; et al. |
June 3, 2021 |
Use of CD24 Proteins for Treating Leptin-Deficient Conditions
Abstract
This invention relates to the use of a CD24 protein for treating
leptin-deficient conditions, such as lipodystrophy, by increasing
the level of circulating leptin.
Inventors: |
LIU; Yang; (Baltimore,
MD) ; ZHENG; Pan; (Baltimore, MD) ; DEVENPORT;
Martin; (Gaithersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ONCOIMMUNE, INC. |
Rockville |
MD |
US |
|
|
Assignee: |
ONCOIMMUNE, INC.
Rockville
MD
|
Family ID: |
1000005397718 |
Appl. No.: |
17/067929 |
Filed: |
October 12, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16074726 |
Aug 1, 2018 |
10799558 |
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PCT/US2017/016120 |
Feb 2, 2017 |
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17067929 |
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62290202 |
Feb 2, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/1774 20130101;
A61K 47/6801 20170801; A61P 5/00 20180101; A61K 38/2264
20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 47/68 20060101 A61K047/68; A61K 38/22 20060101
A61K038/22; A61P 5/00 20060101 A61P005/00 |
Claims
1. A method for increasing circulating leptin levels in the
bloodstream of a subject, comprising administering a CD24 protein
to a subject in need thereof.
2. The method of claim 1, wherein the subject has reduced
leptin.
3. The method of claim 2, wherein the subject has
lipodystrophy.
4. The method of claim 2, wherein the subject has HIV.
5. The method of claim 2, wherein the subject has received
anti-viral therapy.
6. The method of claim 1, wherein the CD24 protein comprises the
sequence of mature human CD24 or a variant thereof.
7. The method of claim 6, wherein the mature human CD24 comprises a
sequence selected from the group consisting of the sequence of SEQ
ID NO: 1 and 2.
8. The method of claim 6, wherein the CD24 protein comprises an
extracellular domain of human CD24.
9. The method of claim 7, wherein the CD24 protein further
comprises a protein tag, wherein the protein tag is fused at the
N-terminus or C-terminus of the CD24 protein.
10. The method of claim 9, wherein the protein tag comprises a
portion of a mammalian immunoglobulin (Ig).
11. The method of claim 10, wherein the Ig portion is the Fc
portion of a human immunoglobulin.
12. The method of claim 11, wherein the Fc portion comprises the
hinge region and CH2 and CH3 domains of the human Ig protein, and
wherein the Ig is selected from the group consisting of IgG1, IgG1,
IgG3, IgG4, and IgA.
13. The method of claim 11, wherein the Fc portion comprises the
hinge region and CH3 and CH4 domains of IgM.
14. The method of claim 11, wherein the CD24 protein comprises the
sequence of SEQ ID NO: 6.
15.-18. (canceled)
19. The method of claim 1, wherein the CD24 protein is soluble
and/or glycosylated.
20. (canceled)
21. A method for treating or preventing lipodystrophy syndrome in a
subject, comprising administering a CD24 protein to a subject in
need thereof.
22.-23. (canceled)
24. A method for increasing circulating leptin levels in the
bloodstream of a subject, comprising administering a CD24 protein
of SEQ ID NO: 6 to a subject in need thereof.
25. A method for treating or preventing lipodystrophy syndrome in a
subject, comprising administering a CD24 protein of SEQ ID NO: 6 to
a subject in need thereof.
26.-27. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to CD24 proteins for use in
treating leptin-deficient conditions.
BACKGROUND
[0002] Leptin-deficient conditions such as leptin deficiency due to
mutations in the leptin gene, hypothalamic amenorrhea, and
lipodystrophy syndromes (LS), are characterized by partial or
complete absence of adipose tissue and hormones derived from
adipose tissue, most importantly leptin (Rodriguez et al 2015). In
those disorders, the subcutaneous adipose tissue is the most
affected and fat accumulates in non-adipose tissues. Lipoatrophy,
is a more specific term used when describing the loss of fat from
one area (usually the face). Patients deficient in leptin exhibit a
number of severe metabolic abnormalities such as hyperglycemia,
hypertriglyceridemia, and hepatic steatosis, which can progress to
diabetes mellitus, acute pancreatitis, and hepatic cirrhosis,
respectively. Lipodystrophy may also lead to osteosclerosis.
[0003] Generalized lipodystrophy is the most striking form of
lipodystrophy and can be either acquired or congenital. Acquired
lipodystrophy may develop from the use of highly active retroviral
therapy (HAART) or underlying HIV infection (the most common form
of lipodystrophy) or autoimmune conditions. The pathogenesis of
congenital forms of lipodystrophy is determined by molecular
deficiencies in several genes that orchestrate adipocyte
differentiation, lipid droplet morphology, and lipid
metabolism.
[0004] The clinical manifestations of LS are essentially determined
by the partial or complete lack of white adipose tissue, leading to
low or undetectable levels of the adipose-derived cytokine leptin.
Leptin is previously known as an adipocytokine that regulates
several metabolic processes including glucose homeostasis, insulin
sensitivity, and fatty acid oxidation. The state of leptin
deficiency seen in lipodystrophy leads to the development of
several metabolic abnormalities and approximately 80% of the
patients with lipodystrophy fulfill the diagnostic criteria for
metabolic syndrome.
[0005] Current approved methods of treatment for leptin-deficient
conditions like lipodystrophy involve leptin replacement therapy
(LRT), such as with the leptin analog metreleptin (MYALEPT).
However, LRT is associated with risks related to the development of
neutralizing anti-drug antibodies and lymphoma, and it is currently
approved only for cases of generalized non-HIV lipodystrophy.
Furthermore, due to its low half-life, metreleptin must be
administered once or twice daily (usually at the same time of day)
to mimic the natural leptin circadian cycle. Therefore, there is a
need for alternative methods of treating LS and
leptin-deficiencies, particularly for patients with HIV.
SUMMARY OF THE INVENTION
[0006] This invention describes a novel method for treating
leptin-deficient conditions in a subject by administering a CD24
protein to a subject in need thereof. Also provided is a method for
treating or preventing treating lipodystrophy in patients with HIV
by administering the CD24 protein to a subject in need thereof.
[0007] The CD24 protein may comprise the sequence of mature human
CD24 or a variant thereof. The mature human CD24 may comprise the
sequence of SEQ ID NO: 1 or 2. The CD24 protein may comprise any or
all of the extracellular domain of human CD24. The CD24 protein may
comprise the signal sequence of SEQ ID NO: 4 to allow secretion
from a cell expressing the protein. The signal peptide sequence may
be one that is found on other transmembrane or secreted proteins,
or one modified from the existing signal peptides known in the art.
The CD24 protein may be soluble and/or may be glycosylated. The
CD24 protein may be produced using a eukaryotic protein expression
system, which may comprise a vector contained in a Chinese Hamster
Ovary cell line or a replication-defective retroviral vector. The
replication defective retroviral vector may be stably integrated
into the genome of a eukaryotic cell.
[0008] The CD24 protein may comprise a protein tag, which may be
fused at the N- or C-terminus of the CD24 protein. The protein may
comprise a portion of a mammalian immunoglobulin (Ig), which may be
the Fc portion of a human Ig protein. The human Ig protein may
comprise the hinge region and CH2 and CH3 domains of the human Ig
protein, and the human Ig protein may be IgG1, IgG2, IgG3, IgG4, or
IgA. The Fc portion may also comprise the hinge region and CH3 and
CH4 domains of IgM. The CD24 protein may comprise the sequence of
SEQ ID NO: 5, 6, 8 or 9.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1A shows the amino acid composition of the full length
CD24 fusion protein, CD24IgG1Fc (also referred to herein as CD24Fc)
(SEQ ID NO: 5). The underlined 26 amino acids are the signal
peptide of CD24 (SEQ ID NO: 4), which are cleaved off during
secretion from a cell expressing the protein and thus missing from
the mature version of the protein (SEQ ID NO: 6). The bold portion
of the sequence is the extracellular domain of the mature CD24
protein used in the fusion protein (SEQ ID NO: 2). The last amino
acid (A or V) that is ordinarily present in the mature CD24 protein
has been deleted from the construct to avoid immunogenicity. The
non-underlined, non-bold letters are the sequence of IgG1 Fc,
including the hinge region and CH1 and CH2 domains (SEQ ID NO: 7).
FIG. 1B shows the sequence of CD24.sup.vFc (SEQ ID NO: 8), in which
the mature human CD24 protein (bold) is the valine polymorphic
variant of SEQ ID NO: 1. FIG. 1C shows the sequence of CD24.sup.AFc
(SEQ ID NO: 9), in which the mature human CD24 protein (bold) is
the alanine polymorphic variant of SEQ ID NO: 1. The various parts
of the fusion protein in FIGS. 1B and 1C are marked as in FIG. 1A
and the variant valine/alanine amino acid is double underlined.
[0010] FIG. 2 shows the ratio of leptin in the serum of healthy
human subjects at day 3 post CD24Fc treatment compared to day-1
pre-treatment. The drug was administered on day 0. The data is
represented by CD24Fc dosing cohort; the 0 mg/kg group represents
the placebo control group.
DETAILED DESCRIPTION
[0011] Here we describe the surprising discovery that the
administration of a CD24 protein to human subjects causes an
increase in the circulating levels of leptin within the blood. This
is useful for the treatment of leptin-deficient conditions, such as
lipodystrophy, by increasing the level of circulating leptin. In a
specific embodiment the invention relates to methods of treating
lipodystrophy in patients with HIV.
[0012] As described in more detail herein, CD24 is a small
glycosyl-phosphatidyl-inositol (GPI)-anchored glycoprotein with
widespread expression among both hematopoietic and
non-hematopoietic cells, which is encoded by a coding sequence of
240 base pairs. Of the 80 amino acids, the first 26 constitute the
signal peptide, while the last 23 serve as a signal for cleavage to
allow for the attachment of the GPI tail. As a result, the mature
human CD24 molecule has only 31 amino acids. Of the 31 amino acids
in the mature CD24 protein, the last (C-terminal) amino acid is
polymorphic among the human population. A C to T transition at
nucleotide 226 results in the substitution of alanine (a) with
valine (v). Since this residue is immediately N-terminal to the
cleavage site, and since the replacement is non-conservative, these
two alleles may be expressed at different efficiencies on the cell
surface.
[0013] CD24 is known to be a genetic modifier for multiple
sclerosis (MS), rheumatoid arthritis (RA), and systemic lupus
erythematosus (SLE). At the population level, the CD24.sup.v/v
genotype is more than twice as frequent as it is in the normal
population. Among multiplex MS families, the CD24.sup.v allele is
preferentially transmitted to the MS patients in comparison to
healthy controls. Furthermore, among the MS patients who have a
more severe form of the disease (Expanded Disability Status Scale
[EDSS] at or exceeding 6.0, when the patients lose the ability to
walk independently), CD24.sup.v/v individuals took, on average, 7
years to reach EDSS 6.0 from the first clinical symptom, yet the
CD24.sup.a/v or CD24.sup.a/a individuals reached EDSS 6.0 in 13 to
15 years. Conversely, a dinucleotide deletion in the 3'
untranslated region of CD24 messenger ribonucleic acid (mRNA),
which reduces CD24 mRNA stability and thus reduces CD24 expression,
protects humans against MS and other autoimmune diseases. To date,
CD24 has not been shown to affect lipid levels.
1. Definitions
[0014] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting. As
used in the specification and the appended claims, the singular
forms "a," "an" and "the" include plural referents unless the
context clearly dictates otherwise.
[0015] For recitation of numeric ranges herein, each intervening
number there between with the same degree of precision is
explicitly contemplated. For example, for the range of 6-9, the
numbers 7 and 8 are contemplated in addition to 6 and 9, and for
the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
[0016] A "peptide" or "polypeptide" is a linked sequence of amino
acids and may be natural, synthetic, or a modification or
combination of natural and synthetic.
[0017] "Substantially identical" may mean that a first and second
amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,
230, 240, 250, 260, 270, 280, 290, or 300 amino acids.
[0018] "Treatment" or "treating," when referring to protection of a
human or an animal from a disease, means preventing, suppressing,
repressing, or completely eliminating the disease. Preventing the
disease involves administering a composition of the present
invention to a human or an animal prior to onset of the disease.
Suppressing the disease involves administering a composition of the
present invention to a human or an animal after induction of the
disease but before its clinical appearance. Repressing the disease
involves administering a composition of the present invention to a
human or an animal after clinical appearance of the disease.
[0019] A "variant" may mean a peptide or polypeptide that differs
in amino acid sequence by the insertion, deletion, or conservative
substitution of amino acids, but retain some biological activity.
Representative examples of "biological activity" for CD24 include
the ability to bind to a lectin, in particular, Siglecs (Sialic
acid-binding immunoglobulin-type lectins), and to be bound by a
specific antibody. Variant may also mean a protein with an amino
acid sequence that is substantially identical to a referenced
protein with an amino acid sequence that retains at least one
biological activity. A conservative substitution of an amino acid,
i.e., replacing an amino acid with a different amino acid of
similar properties (e.g., hydrophilicity, degree and distribution
of charged regions) is recognized in the art as typically involving
a minor change. These minor changes can be identified, in part, by
considering the hydropathic index of amino acids, as understood in
the art. The hydropathic index of an amino acid is based on a
consideration of its hydrophobicity and charge. It is known in the
art that amino acids of similar hydropathic indexes can be
substituted and still retain protein function. In one aspect, amino
acids having hydropathic indexes of .+-.2 are substituted. The
hydrophilicity of amino acids can also be used to reveal
substitutions that would result in proteins retaining biological
function. A consideration of the hydrophilicity of amino acids in
the context of a peptide permits calculation of the greatest local
average hydrophilicity of that peptide, a useful measure that has
been reported to correlate well with antigenicity and
immunogenicity. U.S. Pat. No. 4,554,101, incorporated fully herein
by reference. Substitution of amino acids having similar
hydrophilicity values can result in peptides retaining biological
activity, for example immunogenicity, as is understood in the art.
Substitutions may be performed with amino acids having
hydrophilicity values within .+-.2 of each other. Both the
hyrophobicity index and the hydrophilicity value of amino acids are
influenced by the particular side chain of that amino acid.
Consistent with that observation, amino acid substitutions that are
compatible with biological function are understood to depend on the
relative similarity of the amino acids, and particularly the side
chains of those amino acids, as revealed by the hydrophobicity,
hydrophilicity, charge, size, and other properties.
2. CD24
[0020] Provided herein is a CD24 protein, which may comprise the
amino acid sequence of mature human CD24 or those from other
mammals, which corresponds to the extracellular domain (ECD), or a
variant thereof. As described above, the sequence of the mature
human CD24 protein is 31 amino acids long with a variable alanine
(a) with valine (v) residue at its C-terminal end:
TABLE-US-00001 (SEQ ID NO: 1)
SETTTGTSSNSSQSTSNSGLAPNPTNATTK(V/A)
[0021] The C-terminal valine or alanine may be immunogenic and may
be omitted from the CD24 protein to reduce its immunogenicity.
Therefore, in another embodiment, the CD24 protein may comprise the
amino acid sequence or mature human CD24 lacking the C-terminal
amino acid:
TABLE-US-00002 (SEQ ID NO: 2) SETTTGTSSNSSQSTSNSGLAPNPTNATTK
[0022] Despite considerable sequence variations in the amino acid
sequence of the mature CD24 proteins from mouse and human, they are
functionally equivalent as human CD24Fc has been shown to be active
in the mouse. The amino acid sequence of the human CD24 ECD shows
some sequence conservation with the mouse protein (39% identity;
Genbank accession number NP_033976). However, it is not that
surprising that the percent identity is not higher as the CD24 ECD
is only 27-31 amino acids in length depending on the species, and
binding to some of its receptor(s), such as Siglec 10/G, is
mediated by its sialic acid and/or galactose sugars of the
glycoprotein. The amino acid sequence identity between the
extracellular domains of the human Siglec-10 (GenBank accession
number AF310233) and its murine homolog Siglec-G (GenBank accession
number NP_766488) receptor proteins is 63%. As a result of sequence
conservation between mouse and human CD24 primarily in the
C-terminus and in the abundance of glycosylation sites, significant
variations in the mature CD24 proteins may be tolerated in using
the CD24 protein, especially if those variations do not affect the
conserved residues in the C-terminus or do not affect the
glycosylation sites from either mouse or human CD24. Therefore, the
CD24 protein may comprise the amino acid sequence of mature murine
CD24:
TABLE-US-00003 (SEQ ID NO: 3) NQTSVAPFPGNQNISASPNPTNATTRG.
[0023] The amino acid sequence of the human CD24 ECD shows more
sequence conservation with the cynomolgus monkey protein (52%
identity; UniProt accession number UniProtKB-I7GKK1) than with
mouse. Again, this is not surprising given that the percent
identity is not higher as the ECD is only 29-31 amino acids in
length in these species, and the role of sugar residues in binding
to its receptor(s). The amino acid sequence of cynomolgous
Siglec-10 receptor has not been determined but the amino acid
sequence identity between the human and rhesus monkey Siglec-10
(GenBank accession number XP_001116352) proteins is 89%. Therefore,
the CD24 protein may also comprise the amino acid sequence of
mature cynomolgous (or rhesus) monkey CD24:
TABLE-US-00004 (SEQ ID NO: 10) TVTTSAPLSSNSPQNTSTTPNPANTTTKA
[0024] The CD24 protein may be soluble. The CD24 protein may
further comprise an N-terminal signal peptide, to allow secretion
from a cell expressing the protein. The signal peptide sequence may
comprise the amino acid sequence MGRAMVARLGLGLLLLALLLPTQIYS (SEQ ID
NO: 4), which is from the endogenous human CD24 signal peptide.
Alternatively, the signal sequence may be any of those that are
found on other transmembrane or secreted proteins, or those
modified from the existing signal peptides known in the art.
[0025] a. Fusion
[0026] The CD24 protein may be fused at its N- or C-terminal end to
a protein tag, which may comprise a portion of a mammalian Ig
protein, which may be human or mouse. The portion may comprise an
Fc region of the Ig protein. The Fc region may comprise the hinge
region and CH2 and CH3 domains of the Ig protein. The Ig protein
may be human IgG1, IgG2, IgG3, IgG4, IgM, or IgA. The Fc portion
may comprise the human immunoglobulin G1 (IgG1) isotype SEQ ID NO:
7. The Ig protein may also be IgM, and the Fc portion may comprise
the hinge region and CH3 and CH4 domains of IgM. The protein tag
may also comprise GST, His, or FLAG. Methods for making fusion
proteins and purifying fusion proteins are well known in the
art.
[0027] Based on our preclinical research, for the construction of
the fusion protein CD24Fc identified in the examples, we have
chosen to use the truncated form of native CD24 molecule of 30
amino acids which lacks the final polymorphic amino acid before the
GPI signal cleavage site (SEQ ID NO: 2). This protein is fused to a
human IgG1 Fc domain (SEQ ID NO: 7). The full length CD24Fc fusion
protein is provided in SEQ ID NO: 5 (FIG. 1) and the mature version
of CD24Fc fusion protein that is secreted from the cell (i.e.
lacking the signal sequence which is cleaved off) is provided in
SEQ ID NO: 6. Processed polymorphic variants of mature CD24 (that
is, mature CD24 protein having SEQ ID NO: 1) fused to IgG1 Fc may
comprise SEQ ID NO: 11 or 12.
[0028] b. Production
[0029] The CD24 proteins described herein may be heavily
glycosylated, and the sugar moieties may be involved in the
biological activity of CD24, such as immune cell costimulation,
interaction with Siglecs (Sialic acid-binding immunoglobulin-type
lectins) and interaction with a damage-associated molecular pattern
molecule (DAMP). The CD24 protein may be prepared using a
eukaryotic expression system. The expression system may entail
expression from a vector in mammalian cells, such as Chinese
Hamster Ovary (CHO) cells. The system may also be a viral vector,
such as a replication defective retroviral vector that may be used
to infect eukaryotic cells. The CD24 protein may also be produced
from a stable cell line that expresses the CD24 protein from a
vector or a portion of a vector that has been integrated into the
cellular genome. The stable cell line may express the CD24 protein
from an integrated replication-defective retroviral vector. The
expression system may be GPEx.TM..
[0030] c. Pharmaceutical composition
[0031] The CD24 proteins described herein may be contained in a
pharmaceutical composition, which may comprise at least one
pharmaceutically acceptable excipient. The pharmaceutical
composition may comprise a solvent, which may keep the CD24 protein
stable over an extended period. The solvent may be PBS, which may
keep the CD24 protein stable for at least 66 months at about
-20.degree. C. (-15 to -25.degree. C.). The solvent may be capable
of accommodating the CD24 protein in combination with another
drug.
[0032] d. Dosage
[0033] The dose of the CD24 protein may ultimately be determined
through a clinical trial to determine a dose with acceptable
toxicity and clinical efficacy. The initial clinical dose may be
estimated through pharmacokinetics and toxicity studies in rodents
and non-human primates. The dose of the CD24 protein may be 0.01
mg/kg to 1000 mg/Kg, and may be 1 to 500 mg/kg, depending on the
desired amount of leptin increase and the route of administration.
The CD24 protein may be administered by intravenous infusion or
subcutaneous or intramural injection, and the dose may be
10-100,000 mg, 10-10,000 mg, 10-1000 mg, 10-500 mg, 10-240 mg,
10-120 mg, or 10, 30, 60, 120, or 240 mg, where the subject is a
human.
3. Methods of Treatment
[0034] The CD24 protein may be administered to a subject in need of
increasing leptin levels, such as those with a leptin-deficient
conditions such as leptin deficiency due to hypomorphic mutations
or epigenetic silencing in the leptin gene, hypothalamic
amenorrhea, and lipodystrophy syndromes (LS), including
lipoatrophy. Furthermore, the subject may have generalized
lipodystrophy that is either acquired or congenital. Acquired
lipodystrophy includes those subjects using highly active
retroviral therapy (HAART) or those with an underlying HIV
infection (the most common form of lipodystrophy) or autoimmune
conditions. Congenital lipodystrophy includes those subjects with a
molecular deficiency in any of several genes that orchestrate
adipocyte differentiation, lipid droplet morphology, and lipid
metabolism. The subject may be a mammal such as a human.
[0035] The subject may have been previously treated with a leptin
replacing drug, such as leptin replacement therapy (LRT), or a drug
that increases leptin levels in the blood. A specific example of an
LRT therapy is the leptin analog metreleptin (Myalept, Aegerion
Pharmaceuticals, Inc., Cambridge, Mass., USA).
[0036] a. Administration
[0037] The route of administration of the pharmaceutical
composition may be parenteral.
[0038] Parenteral administration includes, but is not limited to,
intravenous, intraarterial, intraperitoneal, subcutaneous,
intramuscular, intrathecal, intraarticular and direct injection
into affected joints. For veterinary use, the agent may be
administered as a suitably acceptable formulation in accordance
with normal veterinary practice. The veterinarian can readily
determine the dosing regimen and route of administration that is
most appropriate for a particular animal. The pharmaceutical
composition may be administered to a human patient, cat, dog, large
animal, or an avian.
[0039] The CD24 protein may be administered simultaneously or
metronomically with other treatments. The term "simultaneous" or
"simultaneously" as used herein, means that the CD24 protein and
other treatment be administered within 48 hours, preferably 24
hours, more preferably 12 hours, yet more preferably 6 hours, and
most preferably 3 hours or less, of each other. The term
"metronomically" as used herein means the administration of the
agent at times different from the other treatment and at a certain
frequency relative to repeat administration.
[0040] The CD24 protein may be administered at any point prior to
another treatment including about 120 hr, 118 hr, 116 hr, 114 hr,
112 hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96
hr, 94 hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr,
76 hr, 74 hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58
hr, 56 hr, 54 hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr,
38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20
hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2
hr, 1 hr, 55 mins., 50 mins., 45 mins., 40 mins., 35 mins., 30
mins., 25 mins., 20 mins., 15 mins, 10 mins, 9 mins, 8 mins, 7
mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins. The
CD24 protein may be administered at any point prior to a second
treatment of the CD24 protein including about 120 hr, 118 hr, 116
hr, 114 hr, 112 hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr,
98 hr, 96 hr, 94 hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80
hr, 78 hr, 76 hr, 74 hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr,
60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42
hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28 hr, 26 hr, 24 hr,
22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr,
3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40 mins., 35 mins.,
30 mins., 25 mins., 20 mins., 15 mins., 10 mins., 9 mins., 8 mins.,
7 mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins.
[0041] The CD24 protein may be administered at any point after
another treatment including about 1 min, 2 mins., 3 mins., 4 mins.,
5 mins., 6 mins., 7 mins., 8 mins., 9 mins., 10 mins., 15 mins., 20
mins., 25 mins., 30 mins., 35 mins., 40 mins., 45 mins., 50 mins.,
55 mins., 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr,
16 hr, 18 hr, 20 hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34
hr, 36 hr, 38 hr, 40 hr, 42 hr, 44 hr, 46 hr, 48 hr, 50 hr, 52 hr,
54 hr, 56 hr, 58 hr, 60 hr, 62 hr, 64 hr, 66 hr, 68 hr, 70 hr, 72
hr, 74 hr, 76 hr, 78 hr, 80 hr, 82 hr, 84 hr, 86 hr, 88 hr, 90 hr,
92 hr, 94 hr, 96 hr, 98 hr, 100 hr, 102 hr, 104 hr, 106 hr, 108 hr,
110 hr, 112 hr, 114 hr, 116 hr, 118 hr, and 120 hr. The CD24
protein may be administered at any point prior after a previous
CD24 treatment including about 120 hr, 118 hr, 116 hr, 114 hr, 112
hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr,
94 hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76
hr, 74 hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr,
56 hr, 54 hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38
hr, 36 hr, 34 hr, 32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr,
18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1
hr, 55 mins., 50 mins., 45 mins., 40 mins., 35 mins., 30 mins., 25
mins., 20 mins., 15 mins., 10 mins., 9 mins., 8 mins., 7 mins., 6
mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins.
[0042] b. Combination Treatment
[0043] The CD24 protein may be combined with another drug or
treatment regimen, such as leptin replacement therapy (LRT), or a
drug that increases leptin levels in the blood. A specific example
of an LRT therapy is the leptin analog metreleptin (Myalept,
Aegerion Pharmaceuticals, Inc., Cambridge, Mass., USA). The CD24
protein and the other drug may be administrated together or
sequentially.
[0044] The present invention has multiple aspects, illustrated by
the following non-limiting examples.
EXAMPLES
Example 1
[0045] This example demonstrates that CD24Fc increases circulating
leptin levels. Changes of leptin in plasma from baseline were
analyzed in a clinical study which is described in more detail
below (see the Methods section of this example).
[0046] Using a Luminex bead-based immunoassay, plasma leptin levels
were determined in 80 samples obtained on Day-1 pre-treatment and
Day 3-post treatment from 40 healthy subjects receiving CD24Fc or
placebo. The data are summarized in Table 1.
TABLE-US-00005 TABLE 1 Leptin levels in subject plasma. Replicate
1: Replicate 2: Average: Sample Leptin, Leptin, Leptin, Subject #
Cohort Day pg/ml pg/ml pg/ml 002 Placebo Day -1 2057.5 2151.1
2104.3 002 Placebo Day 3 3101.0 2603.7 2852.4 003 10 mg Day -1
8764.2 7524.7 8144.4 003 10 mg Day 3 10738.8 9318.1 10028.4 006 10
mg Day -1 3205.3 3461.2 3333.2 006 10 mg Day 3 4919.7 5651.1 5285.4
009 10 mg Day -1 26019.6 33582.9 29801.2 009 10 mg Day 3 25430.8
26998.1 26214.4 010 10 mg Day -1 3657.9 3961.1 3809.5 010 10 mg Day
3 4705.2 5613.2 5159.2 008 10 mg Day -1 4055.9 4856.4 4456.2 008 10
mg Day 3 11582.4 14660.8 13121.6 012 Placebo Day -1 12345.2 14724.4
13534.8 012 Placebo Day 3 14293.5 17111.0 15702.3 016 10 mg Day -1
5281.3 6345.6 5813.4 016 10 mg Day 3 5562.1 5491.4 5526.7 033 30 mg
Day -1 7906.4 8295.2 8100.8 033 30 mg Day 3 15080.8 15884.3 15482.5
042 Placebo Day -1 3795.8 4013.6 3904.7 042 Placebo Day 3 4153.9
4767.0 4460.4 047 30 mg Day -1 11751.1 13536.1 12643.6 047 30 mg
Day 3 14161.9 16374.2 15268.1 052 Placebo Day -1 4022.3 4668.0
4345.2 052 Placebo Day 3 5699.2 6002.9 5851.0 060 30 mg Day -1
13672.4 16908.8 15290.6 060 30 mg Day 3 18703.5 19928.7 19316.1 063
30 mg Day -1 6375.7 7636.9 7006.3 063 30 mg Day 3 8173.2 9556.2
8864.7 066 30 mg Day -1 15790.3 17753.7 16772.0 066 30 mg Day 3
24460.6 27606.1 26033.3 067 30 mg Day -1 2141.7 1618.8 1880.2 067
30 mg Day 3 1908.2 1721.9 1815.0 088 Placebo Day -1 2389.5 1932.3
2160.9 088 Placebo Day 3 2273.8 2305.3 2289.6 090 60 mg Day -1
4883.8 4147.8 4515.8 090 60 mg Day 3 4884.7 4864.1 4874.4 096 60 mg
Day -1 6991.8 6135.6 6563.7 096 60 mg Day 3 9448.9 8672.7 9060.8
105 60 mg Day -1 21867.1 20502.8 21185.0 105 60 mg Day 3 27647.6
28394.9 28021.2 124 Placebo Day -1 787.0 811.4 799.2 124 Placebo
Day 3 1038.7 1140.8 1089.8 129 60 mg Day -1 10978.2 12103.2 11540.7
129 60 mg Day 3 12475.3 15487.5 13981.4 130 60 mg Day -1 10948.9
13845.4 12397.2 130 60 mg Day 3 14237.8 18069.0 16153.4 131 60 mg
Day -1 7087.5 9026.8 8057.1 131 60 mg Day 3 7537.5 9046.6 8292.1
134 120 mg Day -1 27893.0 31498.7 29695.9 134 120 mg Day 3 38629.1
43787.4 41208.2 149 Placebo Day -1 494.4 386.1 440.3 149 Placebo
Day 3 740.8 650.8 695.8 143 Placebo Day -1 11114.0 9806.6 10460.3
143 Placebo Day 3 13506.2 10982.0 12244.1 147 120 mg Day -1 7143.4
5834.3 6488.8 147 120 mg Day 3 10558.2 8223.6 9390.9 148 120 mg Day
-1 1830.8 1432.5 1631.7 148 120 mg Day 3 2193.1 1930.4 2061.7 157
120 mg Day -1 884.3 781.7 833.0 157 120 mg Day 3 1224.7 1087.3
1156.0 161 120 mg Day -1 6576.9 5863.6 6220.3 161 120 mg Day 3
8689.1 7117.7 7903.4 171 120 mg Day -1 2899.6 2606.4 2753.0 171 120
mg Day 3 2872.4 2592.9 2732.6 181 Placebo Day -1 2433.6 2206.1
2319.8 181 Placebo Day 3 2384.8 2171.0 2277.9 212 Placebo Day -1
6780.4 6600.6 6690.5 212 Placebo Day 3 10183.2 9953.6 10068.4 179
240 mg Day -1 19575.6 20203.2 19889.4 179 240 mg Day 3 29443.7
30264.3 29854.0 213 240 mg Day -1 19.4 19.9 19.7 213 240 mg Day 3
31.0 31.6 31.3 216 240 mg Day -1 321.2 326.1 323.7 216 240 mg Day 3
448.1 453.2 450.6 211 240 mg Day -1 811.7 817.6 814.7 211 240 mg
Day 3 1883.2 1889.4 1886.3 214 240 mg Day -1 888.5 887.8 888.1 214
240 mg Day 3 981.2 976.5 978.9 218 240 mg Day -1 900.6 892.7 896.6
218 240 mg Day 3 1831.2 1808.1 1819.7
[0047] The analytical sensitivity or limit of detection (LOD) was
determined as the value calculated from the standard curve at the
point lying 2 standard deviations above the mean background (twenty
zero standard replicates). The lower limit of quantification (LLOQ)
was determined using a 2-fold dilution series of the standards in
standard diluent assayed in triplicate over three different rounds,
and is defined as the point at which the coefficient of variation
(CV) for the measurement was 30%. The CV was calculated and plotted
against concentration, and LLOQ was interpolated from the plot. The
assay performance characteristics are as follow in Table 2.
TABLE-US-00006 TABLE 2 Analyte Leptin Unit pg/ml LOD 8.1 LLOQ 18.2
Standard Curve Range 7.7-600,000
[0048] FIG. 2 displays the ratio of leptin on day3/day-1 for
patients grouped by dosing cohort. As the figure shows, there is a
upward trend in the relative amount circulating leptin following
CD24Fc treatment and between the 0, 60, 120 and 240 mg cohorts this
increase is statistically significant (P=0.009397, dose-dependent
general linear model regression), demonstrating a dose dependent
increase above 60 mg. Furthermore, there is a statistically
significant increase in the level of leptin following CD24Fc
administration in the 240 mg cohort compared to placebo (0 mg)
(P=0.05 as determined by Student's T test), indicating that CD24Fc
is effective for increasing leptin in human patients.
Methods
[0049] This was a Phase I, randomized, double-blind,
placebo-controlled, single ascending dose study to assess the
safety, tolerability, and PK of CD24Fc in healthy male and female
adult subjects. A total of 40 subjects in 5 cohorts of 8 subjects
each were enrolled in this study. Six of the 8 subjects in each
cohort received study drug and 2 subjects received placebo (0.9%
sodium chloride, saline). The first cohort was dosed with 10 mg and
succeeding cohorts received 30 mg, 60 mg, 120 mg, and 240 mg of
CD24Fc or matching placebo. Dosing was based on a fixed amount of
CD24Fc and not based weight or BSA. Subjects were dosed at least 3
weeks apart to allow for review of safety and tolerability data for
each prior cohort. Administration of the next higher dose to a new
cohort of subjects was permitted only if adequate safety and
tolerability had been demonstrated.
[0050] In each cohort, the initial 2 subjects were 1 study drug
recipient and 1 placebo recipient on Day 1. The 3rd to 5th and 6th
to 8th subjects were dosed after Day 7 (a minimum of 24 hours apart
between the subgroups). Each subject was dosed at least 1 hour
apart in the same subgroup. If necessary, dosing of the rest of
subjects was delayed pending review of any significant safety
issues that may have arisen during the post-dose period involving
the first or second subgroups in that cohort. The subsequent cohort
was dosed at least 3 weeks after the prior cohort.
[0051] Screening Period: The Screening Visit (Visit 1) occurred up
to 21 days prior to the beginning of the active treatment period.
After providing informed consent, subjects underwent screening
procedures for eligibility.
[0052] Treatment Period: Subjects were admitted to the Clinical
Pharmacology Unit (CPU) on Day -1 (Visit 2), and the randomized
treatment period began on Day 1 following a 10-hour minimum
overnight fast. Subjects were randomly assigned to treatment with
CD24Fc or placebo as a single dose. Subjects remained confined
until the morning of Day 4.
[0053] Follow-up: All subjects returned to the CPU on Day 7, Day
14, Day 21, Day 28, and Day 42 (.+-.1 day) for follow-up visits
(Visit 3, Visit 4, Visit 5, Visit 6, and Visit 7). Visit 7 was the
final visit for all subjects.
Duration of Treatment:
[0054] The total study duration for each subject was up to 63
days.
[0055] Single-dose administration occurred on Day 1.
[0056] Number of Subjects: [0057] Planned: 40 subjects [0058]
Screened: 224 subjects [0059] Randomized: 40 subjects [0060]
Completed: 39 subjects [0061] Discontinued: 1 subject
[0062] Diagnosis and Main Criteria for Inclusion: The population
for this study was healthy males and females between the ages of 18
and 55 years, inclusive, with a body mass index between 18
kg/m.sup.2 and 30 kg/m.sup.2, inclusive.
[0063] Investigational Product and Comparator Information: CD24Fc:
single dose of 10 mg, 30 mg, 60 mg, 120 mg, or 240 mg administered
via IV infusion; lot number: 09MM-036. CD24Fc is a fully humanized
fusion protein consisting of the extracellular domain of human CD24
and the fragment crystallizable region of human immunoglobulin G1
(IgG1Fc). CD24Fc was supplied as a sterile, clear, colorless,
preservative-free, aqueous solution for IV administration. CD24Fc
was formulated as single dose injection solution, at a
concentration of 10 mg/mL and a pH of 7.2. Each CD24Fc vial
contained 160 mg of CD24Fc, 5.3 mg of sodium chloride, 32.6 mg of
sodium phosphate dibasic heptahydrate, and 140 mg of sodium
phosphate monobasic monohydrate in 16 mL.+-.0.2 mL of CD24Fc.
CD24Fc was supplied in clear borosilicate glass vials with
chlorobutyl rubber stoppers and aluminum flip-off seals.
[0064] Matching placebo (0.9% sodium chloride, saline) administered
via IV infusion; lot numbers: P296855, P311852, P300715,
P315952.
[0065] The intent-to-treat (ITT) Population consisted of all
subjects who received at least 1 dose of the study drug. The ITT
Population was the primary analysis population for subject
information and safety evaluation.
[0066] Plasma samples were collected at Day 1 pre-dose, at Day
dosing, and at Day 2, Day 3, Day 4, Day 7, Day 14, Day 21 (.+-.1),
Day 28 (.+-.1), and Day 42 (.+-.1). [0067] Reference cited:
Rodriguez A J, Mastronardi C A, Paz-Filho G J. New advances in the
treatment of generalized lipodystrophy: role of metreleptin.
Therapeutics and Clinical Risk Management. 2015; 11:1391-1400.
doi:10.2147/TCRM.S66521.
Sequence CWU 1
1
12131PRTHomo sapiensVARIANT(31)...(31)Valine or Alanine 1Ser Glu
Thr Thr Thr Gly Thr Ser Ser Asn Ser Ser Gln Ser Thr Ser1 5 10 15Asn
Ser Gly Leu Ala Pro Asn Pro Thr Asn Ala Thr Thr Lys Xaa 20 25
30230PRTHomo sapiens 2Ser Glu Thr Thr Thr Gly Thr Ser Ser Asn Ser
Ser Gln Ser Thr Ser1 5 10 15Asn Ser Gly Leu Ala Pro Asn Pro Thr Asn
Ala Thr Thr Lys 20 25 30327PRTMus musculus 3Asn Gln Thr Ser Val Ala
Pro Phe Pro Gly Asn Gln Asn Ile Ser Ala1 5 10 15Ser Pro Asn Pro Thr
Asn Ala Thr Thr Arg Gly 20 25426PRTHomo sapiens 4Met Gly Arg Ala
Met Val Ala Arg Leu Gly Leu Gly Leu Leu Leu Leu1 5 10 15Ala Leu Leu
Leu Pro Thr Gln Ile Tyr Ser 20 255287PRTArtificialFusion protein
5Met Gly Arg Ala Met Val Ala Arg Leu Gly Leu Gly Leu Leu Leu Leu1 5
10 15Ala Leu Leu Leu Pro Thr Gln Ile Tyr Ser Ser Glu Thr Thr Thr
Gly 20 25 30Thr Ser Ser Asn Ser Ser Gln Ser Thr Ser Asn Ser Gly Leu
Ala Pro 35 40 45Asn Pro Thr Asn Ala Thr Thr Lys Pro Lys Ser Cys Asp
Lys Thr His 50 55 60Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val65 70 75 80Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr 85 90 95Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 100 105 110Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 115 120 125Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 130 135 140Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys145 150 155
160Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
165 170 175Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 180 185 190Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu 195 200 205Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 210 215 220Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser225 230 235 240Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 245 250 255Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 260 265 270His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 275 280
2856261PRTArtificialFusion protein 6Ser Glu Thr Thr Thr Gly Thr Ser
Ser Asn Ser Ser Gln Ser Thr Ser1 5 10 15Asn Ser Gly Leu Ala Pro Asn
Pro Thr Asn Ala Thr Thr Lys Pro Lys 20 25 30Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 35 40 45Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 50 55 60Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val65 70 75 80Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 85 90 95Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 100 105
110Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
115 120 125Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala 130 135 140Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro145 150 155 160Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln 165 170 175Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala 180 185 190Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 195 200 205Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 210 215 220Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser225 230
235 240Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser 245 250 255Leu Ser Pro Gly Lys 2607231PRTHomo sapiens 7Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro1 5 10 15Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25
30Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
35 40 45Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp 50 55 60Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr65 70 75 80Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp 85 90 95Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu 100 105 110Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 115 120 125Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys 130 135 140Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp145 150 155 160Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170
175Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
180 185 190Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 195 200 205Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser 210 215 220Leu Ser Leu Ser Pro Gly Lys225
2308288PRTArtificialFusion protein 8Met Gly Arg Ala Met Val Ala Arg
Leu Gly Leu Gly Leu Leu Leu Leu1 5 10 15Ala Leu Leu Leu Pro Thr Gln
Ile Tyr Ser Ser Glu Thr Thr Thr Gly 20 25 30Thr Ser Ser Asn Ser Ser
Gln Ser Thr Ser Asn Ser Gly Leu Ala Pro 35 40 45Asn Pro Thr Asn Ala
Thr Thr Lys Val Pro Lys Ser Cys Asp Lys Thr 50 55 60His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser65 70 75 80Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 85 90 95Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 100 105
110Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
115 120 125Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val 130 135 140Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr145 150 155 160Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr 165 170 175Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu 180 185 190Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 195 200 205Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 210 215 220Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp225 230
235 240Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser 245 250 255Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala 260 265 270Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 275 280 2859288PRTArtificialFusion protein 9Met
Gly Arg Ala Met Val Ala Arg Leu Gly Leu Gly Leu Leu Leu Leu1 5 10
15Ala Leu Leu Leu Pro Thr Gln Ile Tyr Ser Ser Glu Thr Thr Thr Gly
20 25 30Thr Ser Ser Asn Ser Ser Gln Ser Thr Ser Asn Ser Gly Leu Ala
Pro 35 40 45Asn Pro Thr Asn Ala Thr Thr Lys Ala Pro Lys Ser Cys Asp
Lys Thr 50 55 60His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser65 70 75 80Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 85 90 95Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 100 105 110Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala 115 120 125Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 130 135 140Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr145 150 155 160Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 165 170
175Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
180 185 190Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys 195 200 205Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 210 215 220Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp225 230 235 240Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 245 250 255Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 260 265 270Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 275 280
2851029PRTMacaca fascicularis 10Thr Val Thr Thr Ser Ala Pro Leu Ser
Ser Asn Ser Pro Gln Asn Thr1 5 10 15Ser Thr Thr Pro Asn Pro Ala Asn
Thr Thr Thr Lys Ala 20 2511262PRTArtificialFusion protein 11Ser Glu
Thr Thr Thr Gly Thr Ser Ser Asn Ser Ser Gln Ser Thr Ser1 5 10 15Asn
Ser Gly Leu Ala Pro Asn Pro Thr Asn Ala Thr Thr Lys Val Pro 20 25
30Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
35 40 45Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp 50 55 60Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp65 70 75 80Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly 85 90 95Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn 100 105 110Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp 115 120 125Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro 130 135 140Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu145 150 155 160Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 165 170
175Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
180 185 190Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr 195 200 205Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys 210 215 220Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys225 230 235 240Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu 245 250 255Ser Leu Ser Pro Gly
Lys 26012262PRTArtificialFusion protein 12Ser Glu Thr Thr Thr Gly
Thr Ser Ser Asn Ser Ser Gln Ser Thr Ser1 5 10 15Asn Ser Gly Leu Ala
Pro Asn Pro Thr Asn Ala Thr Thr Lys Ala Pro 20 25 30Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 35 40 45Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 50 55 60Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp65 70 75
80Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
85 90 95Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn 100 105 110Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp 115 120 125Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro 130 135 140Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu145 150 155 160Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 165 170 175Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 180 185 190Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 195 200
205Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
210 215 220Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys225 230 235 240Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu 245 250 255Ser Leu Ser Pro Gly Lys 260
* * * * *