U.S. patent application number 17/478628 was filed with the patent office on 2022-01-06 for dual function proteins comprising fgf21 mutant protein and pharmaceutical composition comprising same.
This patent application is currently assigned to YUHAN CORPORATION. The applicant listed for this patent is YUHAN CORPORATION. Invention is credited to Hyun Ho CHOI, Mi Kyeong JU, Dohoon KIM, Jong Gyun KIM, Jun Hwan KIM, Seul Gi KIM, Sangmyoun LIM, Seyoung LIM, Su Youn NAM, Ju-Young PARK, Minji SEO.
Application Number | 20220002368 17/478628 |
Document ID | / |
Family ID | 1000005839593 |
Filed Date | 2022-01-06 |
United States Patent
Application |
20220002368 |
Kind Code |
A1 |
KIM; Jun Hwan ; et
al. |
January 6, 2022 |
DUAL FUNCTION PROTEINS COMPRISING FGF21 MUTANT PROTEIN AND
PHARMACEUTICAL COMPOSITION COMPRISING SAME
Abstract
A dual function protein is disclosed. The dual function protein
may be prepared by linking a biologically active protein and an FGF
mutant protein to an Fc region of an immunoglobulin. The dual
function protein has improved pharmacological efficacy, in vivo
duration and protein stability. The dual function protein exhibits
improved pharmacological efficacy, in vivo duration and protein
stability. A pharmaceutical composition containing the dual
function protein as an active ingredient may be effectively used as
a therapeutic agent for diabetes, obesity, dyslipidemia, metabolic
syndrome, non-alcoholic fatty liver diseases, non-alcoholic
steatohepatitis or cardiovascular diseases.
Inventors: |
KIM; Jun Hwan; (Seoul,
KR) ; LIM; Seyoung; (Yongin-si, KR) ; SEO;
Minji; (Seoul, KR) ; CHOI; Hyun Ho; (Suwon-si,
KR) ; KIM; Dohoon; (Yongin-si, KR) ; JU; Mi
Kyeong; (Suwon-si, KR) ; PARK; Ju-Young;
(Seoul, KR) ; KIM; Seul Gi; (Suwon-si, KR)
; LIM; Sangmyoun; (Seoul, KR) ; KIM; Jong
Gyun; (Anyang-si, KR) ; NAM; Su Youn; (Seoul,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
YUHAN CORPORATION |
Seoul |
|
KR |
|
|
Assignee: |
YUHAN CORPORATION
Seoul
KR
|
Family ID: |
1000005839593 |
Appl. No.: |
17/478628 |
Filed: |
September 17, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15768865 |
Apr 17, 2018 |
11136364 |
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PCT/KR2016/012300 |
Oct 28, 2016 |
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17478628 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/575 20130101;
C07K 14/605 20130101; C07K 2319/30 20130101; C07K 14/50
20130101 |
International
Class: |
C07K 14/50 20060101
C07K014/50; C07K 14/575 20060101 C07K014/575; C07K 14/605 20060101
C07K014/605 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 28, 2015 |
KR |
10-2015-0150576 |
Claims
1. A dual function protein comprising a fibroblast growth factor 21
(FGF21) mutant protein; a biologically active protein, or a mutant
or fragment thereof; and an Fc region of an immunoglobulin, wherein
the FGF21 mutant protein comprises the following mutation: (i) a
substitution of the amino acid at position 180 from the N-terminus
of a wild-type FGF21 protein with the amino acid E.
2. The dual function protein of claim 1, wherein the FGF21 mutant
protein further comprises the following mutation: (ii) a
substitution of the amino acids at positions 170 to 174 from the
N-terminus of a wild-type FGF21 protein with the amino acid
sequence of TGLEAN (SEQ ID NO: 44).
3. The dual function protein of claim 1, wherein the FGF21 mutant
protein further comprises the following mutation: (iii) a
substitution of the amino acid at position 170 from the N-terminus
of a wild-type FGF21 protein with the amino acid N.
4. The dual function protein of claim 1, wherein the FGF21 mutant
protein further comprises the following mutation: (iv) a
substitution of the amino acids at positions 98 to 101 from the
N-terminus of a wild-type FGF21 protein with the amino acid
sequence of EIRP (SEQ ID NO: 42); (v) a substitution of the amino
acids at positions 170 to 174 from the N-terminus of a wild-type
FGF21 protein with the amino acid sequence of TGLEAV (SEQ ID NO:
43); (vi) a substitution of the amino acid at position 174 from the
N-terminus of a wild-type FGF21 protein with the amino acid N; or
(vii) a combination of the substitution (iv) and the substitution
(v), or a combination of the substitution (iv) and the substitution
(v).
5. The dual function protein of claim 1, wherein the amino acid
residue N of the FGF21 mutant protein introduced by a mutation is
glycosylated.
6. The dual function protein of claim 1, wherein the biologically
active protein is one selected from the group consisting of
insulin, C-peptide, leptin, glucagon, gastrin, gastric inhibitory
polypeptide (GIP), amylin, calcitonin, cholecystokinin, peptide YY,
neuropeptide Y, bone morphogenetic protein-6 (BMP-6), bone
morphogenetic protein-9 (BMP-9), oxyntomodulin, oxytocin,
glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2),
irisin, fibronectin type III domain-containing protein 5 (FNDC5),
apelin, adiponectin, C1q and tumor necrosis factor related protein
(CTRP family), resistin, visfatin, omentin, retinol binding
protein-4 (RBP-4), glicentin, angiopoietin, interleukin-22 (IL-22),
exendin-4, growth hormone, and a combination thereof.
7. The dual function protein of claim 6, wherein the biologically
active protein is one selected from GLP-1, a mutant thereof, and
exendin-4.
8. The dual function protein of claim 7, wherein the mutant of
GLP-1 comprises an amino acid sequence selected from the group
consisting SEQ ID NOs: 43 to 46.
9. The dual function protein of claim 1, wherein the wild-type
FGF21 protein comprises the amino acid sequence represented by SEQ
ID NO: 1.
10. The dual function protein of claim 1, wherein the FGF21 mutant
protein comprises an amino acid sequence selected from the group
consisting of SEQ ID NOs: 6 to 23.
11. The dual function protein of claim 1, wherein the dual function
protein further comprises a linker.
12. The dual function protein of claim 11, wherein the linker
connects the FGF21 mutant protein to the Fc region of the
immunoglobulin.
13. The dual function protein of claim 12, wherein the linker is
connected to the C-terminus of the Fc region of the immunoglobulin
and the N-terminus of the FGF21 mutant protein.
14. The dual function protein of claim 12, wherein the linker is a
peptide consisting of 10 to 30 amino acid residues.
13. The dual function protein of claim 14, wherein the linker
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOs: 2 to 5.
14. The dual function protein of claim 1, wherein the Fc region of
the immunoglobulin is any one of the Fc region of IgG1, IgG2, IgG3,
IgG4 and IgD, or a hybrid Fc containing a combination thereof.
15. The dual function protein of claim 14, wherein the hybrid Fc
comprises an IgG4 region and an IgD region.
16. The dual function protein of claim 1, wherein the dual function
protein comprises the biologically active protein, the Fc region of
the immunoglobulin and the FGF21 mutant protein, connected in this
order from the N-terminus to the C-terminus.
17. The dual function protein of claim 16, wherein a linker is
additionally connected between the Fc region of the immunoglobulin
and the FGF21 mutant protein.
18. The dual function protein of claim 17, wherein the linker is
connected to the C-terminus of the Fc region of the immunoglobulin
and the N-terminus of the FGF21 mutant protein.
19. The dual function protein of claim 17, wherein the linker is a
peptide consisting of 10 to 30 amino acid residues.
20. The dual function protein of claim 17, wherein the linker
comprise an amino acid sequence selected from the group consisting
of SEQ ID NOs: 2 to 5.
21. The dual function protein of claim 1, wherein the dual function
protein comprise the amino acid sequence of SEQ ID NO: 65.
22. The dual function protein of claim 1, wherein the dual function
protein comprises the amino acid sequence of SEQ ID NO: 66.
23. The dual function protein of claim 1, wherein the dual function
protein has an amino acid sequence represented by SEQ ID NO:
67.
24. A pharmaceutical composition comprising the dual function
protein according to claim 1 and a pharmaceutically acceptable
carrier.
25. An isolated nucleic acid molecule encoding the fusion protein
according to claim 1.
26. An expression vector comprising the nucleic acid molecule of
claim 25.
27. A host cell comprising the nucleic acid molecule of claim
25.
28. A method for treating diabetes, obesity, dyslipidemia,
metabolic syndrome, non-alcoholic fatty liver disease,
non-alcoholic steatohepatitis or cardiovascular diseases in a
subject in need thereof, comprising administering the
pharmaceutical composition of claim 24 to the subject.
29. A method selected from the group consisting of: reducing blood
glucose level in a subject; reducing body weight in a subject;
reducing triglyceride or low-density lipoprotein levels in a
subject; and improving insulin sensitivity in a subject wherein the
method comprising administering the pharmaceutical composition of
claim 24 to the subject.
30. The method of claim 29, wherein the subject is obese.
31. The method of claim 30, wherein the subject has diabetes.
32. A method for inhibiting cAMP production in a subject in need
thereof, comprising administering the composition of claim 24 to
the subject.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is Divisional of U.S. application Ser. No.
15/768,865 filed Apr. 17, 2018, which is a National Stage of
International Application No. PCT/KR2016/012300 filed Oct. 28,
2016, claiming priority based on Korean Patent Application No.
10-2015-0150576 filed Oct. 28, 2015.
SEQUENCE LISTING
[0002] The content of the electronically submitted sequence
listing, file name: Sequence Listing.txt; size: 173,688 bytes; and
date of creation: Aug. 6, 2020, filed herewith, is incorporated
herein by reference in its entirety.
TECHNICAL FIELD
[0003] The present invention relates to a dual function protein
including a biologically active protein and a fibroblast growth
factor 21 (FGF21) mutant protein, and a pharmaceutical composition
containing same.
BACKGROUND ART
[0004] Glucagon-like peptide-1 (GLP-1) is an incretin hormone
consisting of 31 amino acids, which is secreted by L cells in the
intestinal tract when stimulated by food, etc. Its biological
effects arise via intracellular signaling through the GLP-1
receptor, a G protein-coupled receptor which is expressed in target
tissues such as .beta.-cells in the pancreas, brain, etc. GLP-1
secreted in the blood has a very short half-life of less than 2
minutes, which is caused by a loss of activity due to the cleavage
of amino acids at the N-terminus by the enzyme dipeptidyl
peptidase-4 (DPP-4). Since GLP-1 stimulates the secretion of
insulin in .beta.-cells in the pancreas based on blood glucose
level, it has a strong effect on lowering blood glucose without
inducing hypoglycemia. Further, the administration of GLP-1 results
in loss of body weight in various animal models and humans, which
is known to be caused by reduced food intake due to its effect on
appetite suppression. GLP-1 induces proliferation of .beta.-cells
and enhances the viability of .beta.-cells by inhibiting cell death
caused by glycolipid toxicity through GLP-1 receptor expressed in
.beta.-cells in the pancreas. Excessive secretion of glucagon
increases blood glucose, which is known to be one of the causes of
hyperglycemia in diabetics. In addition, it is known that GLP-1
acts on .alpha.-cells in the pancreas to inhibit fasting blood
glucose elevation by inhibiting secretion of protein kinase A (PKA)
protein-specific glucagon.
[0005] Exendin-4 is a clinically important GLP-1 receptor agonist.
Exendin-4 is a polypeptide with 39 amino acid residues, and is
normally produced in the salivary glands of the Gila Monster
lizard. It is known that exendin-4 an amino acid sequence homology
of 52% with GLP-1, and interacts with the GLP-1 receptor in mammals
(Thorens et al. (1993) Diabetes 42:1678-1682). Exendin-4 has been
shown to stimulate the secretion of insulin by insulin-producing
cells in vitro, and the induction of insulin release by
insulin-producing cells is stronger than GLP-1 under equimolar
conditions. While exendin-4 strongly stimulates the secretion of
insulin to decrease blood glucose levels in both rodents and humans
with a duration of action longer than that of GLP-1, exendin-4 has
exhibits antigenicity in mammals devoid of GLP-1 as it has
unfamiliar epitopes in such animals.
[0006] The ability of GLP-1 and exendin-4 analogs (e.g.,
liraglutide and byetta) to improve glucose control in humans has
been clinically confirmed. It has been reported that GLP-1
increases .beta.-cell mass through the inhibition of apoptosis and
induced proliferation. Furthermore, it has been also reported that
GLP-1 acts as an intestinal hormone inhibiting gastric acid
secretion and gastric emptying while enhancing satiety signals,
thereby reducing appetite. Such effects of GLP-1 can explain the
weight loss observed when GLP-1 analogs are administered to
patients with type 2 diabetes. In addition, GLP-1 exhibits
cardioprotective effects following ischemia in rodents.
[0007] Various attempts have been made to develop long-acting GLP-1
analogs. Clinically confirmed long-acting GLP-1 analogs include
dulaglutide (WO 2005/000892) and albiglutide (WO 2003/059934).
Dulaglutide is an Fc-fused GLP-1 analog, and albiglutide is an
albumin-fused GLP-1 analog, both of which have pharmacokinetic
profiles allowing for once weekly administration. Both drugs have
excellent effects on lowering blood glucose and reducing body
weight with once weekly administration, and also provide greatly
improved convenience in terms of treatment when compared to byetta
and liraglutide.
[0008] Meanwhile, fibroblast growth factor 21 (FGF21), synthesized
in the liver, is a hormone known to play an important role in
glucose and lipid homeostasis. FGF21 exhibits pharmacological
actions in the liver, adipocytes, .beta. cells of the pancreas,
hypothalamus in the brain, and muscle tissues, where both an
FGF21-specific receptor, i.e., FGF receptor, and .beta.-klotho
complex are expressed. It has been reported that in non-human
primate and murine models of various diabetic and metabolic
diseases, FGF21 can lower blood glucose levels in an
insulin-independent manner, reduce body weight, and lower
triglyceride and low-density lipoprotein (LDL) concentrations in
the blood. Additionally, due to its effect of improving insulin
sensitivity, FGF21 has potential for development as a novel
therapeutic agent for diabetes and obesity (see WO2003/011213).
[0009] Accordingly, in order to develop a novel anti-diabetic drug
based on FGF21, attempts have been made to improve its biological
activity and in vivo stability by constructing FGF21 mutants based
on the wild-type FGF21 sequence via substitution, insertion, and
deletion of some amino acids (see WO2010/065439). However, as FGF21
has a very short half-life, it has proven problematic if used
directly as a biotherapeutic agent (Kharitonenkov, A. et al. (2005)
Journal of Clinical Investigation 115:1627-1635). The in vivo
half-life of FGF21 is 1 to 2 hours in mice, and 2.5 to 3 hours in
monkeys. Therefore, for FGF21 to be used in its current form as a
therapeutic agent for diabetes, daily administration is
required.
[0010] Various approaches have been reported in attempting to
increase the in vivo half-life of FGF21 recombinant proteins. One
such example is to link polyethylene glycol (PEG), i.e., a polymer
material, to FGF21 to increase its molecular weight, thereby
inhibiting renal excretion and increasing in vivo retention time
(see WO2012/066075). Another approach attempts to improve the
half-life by fusing it with a fatty acid, which binds to human
albumin (see WO2012/010553). An additional example attempts to
increase the half-life while maintaining pharmacological activity
equivalent to that of wild-type FGF21 through the generation of an
agonist antibody, which specifically binds to the human FGF
receptor alone or as a complex with .beta.-klotho (see
WO2012/170438). In another example, the half-life was improved by
preparing long-acting fusion proteins, in which an Fc region of IgG
binds to an FGF21 molecule (see WO2013/188181).
[0011] Among the various technologies available to create
long-acting drugs, Fc fusion technology is widely used because it
has less of the disadvantages seen with other approaches, such as
inducing an immune response or toxicity while increasing in vivo
half-life. For the development of an Fc-fused FGF21 protein as a
long-acting therapeutic drug, the following conditions should be
satisfied.
[0012] First, the decrease of in vitro activity caused by fusion
should be minimized. Both the N-terminus and C-terminus of FGF21
are involved in FGF21's activity. In this regard, it is known that
the activities of FGF21 fusion proteins greatly vary depending on
the location of the fusion. Accordingly, the activities of Fc-fused
FGF21 fusion proteins, in which mutations are introduced into
FGF21, may be altered depending on the presence/absence or location
of the fusion. Second, a pharmacokinetic profile enabling
administration at an interval of once per week in humans should be
realized by the increase of in vivo half-life by the fusion. Third,
considering that immunogenicity may be expected in most patients
after administration of biopharmaceuticals, the immunogenicity risk
due to a fusion linker or mutation should be minimized. Fourth,
there should be no stability issues arising from the position of
the fusion or the introduction of the mutation. Fifth, since
undesired immune responses may occur depending on the isotypes of
fused immunoglobulin, a solution to prevent such responses is
necessary.
[0013] An attempt to develop a long-acting fusion protein by
linking the Fc region of an immunoglobulin G (IgG) to an FGF21
molecule has already been reported (see WO 2013/188181). In the
case of one Fc-FGF21 structure, where the Fc is fused to the
N-terminus of the wild-type FGF21, while there is no distinct
difference in in vitro activity as compared to that of the
wild-type FGF21, the half-life is known to be very short due to in
vivo degradation of the protein. To address this issue, there has
been an attempt to improve the in vivo half-life by introducing
several mutations at specific site locations of FGF21 to resist
protein degradation. However, immunogenicity risk may increase with
the introduction of multiple mutations. In contrast, in the case of
an FGF21-Fc structure, where the Fc is fused to the C-terminus of
the FGF21 molecule, it is known that there is a significant
decrease in activity caused by fusion at this site when compared to
the Fc-FGF21 structure.
[0014] Combined administration of GLP-1 and FGF21 may have a
synergistic effect as compared with single administration depending
on the action mechanisms and target tissues in the body, and
potentially outstanding anti-diabetic efficacy and additional
advantages are expected. The effects of combined administration of
GLP-1 and FGF21 or a GLP-1/FGF21 dual function protein have been
already investigated and reported (see WO 2010/142665 and WO
2011/020319).
[0015] Various problems must be solved in order to develop a dual
function protein comprising GLP-1 and FGF21. Since wild-type GLP-1
and wild-type FGF21 have a very short in vivo half-life, they are
required to be administered at least once daily, even if developed
as therapeutic agents. Accordingly, long-acting technologies such
as an Fc fusion are required in order to develop a long-acting dual
function protein to improve convenience for patients. In a dual
function drug for the two targets of GLP-1 and FGF21, the
introduction of mutation(s) is essential to maintain the activity
and in vivo stability of each drug, and problems associated with
changes in activity, structure or stability caused by each mutation
should be addressed. Medicinal effects for the two targets of GLP-1
and FGF21 should be well-balanced, and drug designs considering in
vitro activities, pharmacokinetic profiles, pharmacological
efficacy in animal models as well as clinical evaluation of
efficacy in humans are required for this purpose. A dual function
protein has a structure that cannot exist in a human body, and is
structurally complex as compared with a fusion protein for a single
target. In addition, since mutation or linker engineering is
required to balance the two targets, the possibility of forming
aggregate complexes may increase, and further protein engineering
to prevent this may be required. Furthermore, potential
immunogenicity may increase due to novel mutation sequences or
complex structures, which should be addressed or avoided.
[0016] The present inventors have endeavored to improve the
stability, pharmacokinetic profiles and pharmacological efficacy of
dual function proteins including GLP-1 mutant proteins and FGF21
mutant proteins, and discovered that the stability, pharmacokinetic
profiles and pharmacological efficacy of dual function proteins may
be improved when a GLP-1 mutant protein is fused to an Fc region of
an immunoglobulin and a novel FGF21 mutant protein is fused
thereto, thereby accomplishing the present invention.
DISCLOSURE OF INVENTION
Technical Problem
[0017] An object of the present invention is to provide a dual
function protein including a biologically active protein and an
FGF21 mutant protein with improved pharmacokinetic parameters, high
stability, low possibility of forming aggregation complexes, and
reduced potential immunogenicity.
[0018] Another object of the present invention is to provide a
pharmaceutical composition including the dual function protein for
preventing or treating FGF21-associated disorders.
[0019] A further object of the present invention is to provide an
isolated nucleic acid molecule encoding the dual function protein,
an expression vector including the nucleic acid molecule, and a
host cell including the expression vector.
Solution to Problem
[0020] The present invention provides a dual function protein
comprising an FGF21 mutant protein; a biologically active protein,
or a mutant or fragment thereof; and an Fc region of an
immunoglobulin, wherein the FGF21 mutant protein comprises at least
one mutation selected from the group consisting of the following
mutations (1) to (7):
[0021] (1) a substitution of amino acids at positions 98 to 101
from the N-terminus of a wild-type FGF21 protein with an amino acid
sequence of EIRP (SEQ ID NO: 68);
[0022] (2) a substitution of amino acids at positions 170 to 174
from the N-terminus of a wild-type FGF21 protein with an amino acid
sequence of TGLEAV (SEQ ID NO: 69);
[0023] (3) a substitution of amino acids at positions 170 to 174
from the N-terminus of a wild-type FGF21 protein with an amino acid
sequence of TGLEAN (SEQ ID NO: 70);
[0024] (4) a substitution of an amino acid at position 170 from the
N-terminus of a wild-type FGF21 protein with an amino acid N;
[0025] (5) a substitution of an amino acid at position 174 from the
N-terminus of a wild-type FGF21 protein with an amino acid N;
[0026] (6) a substitution of an amino acid at position 180 from the
N-terminus of a wild-type FGF21 protein with an amino acid E, along
with one or more mutations (1) to (5) above; and
[0027] (7) a mutation of 1 to 10 amino acids for reducing
immunogenicity of a wild-type FGF21 protein.
[0028] In addition, the present invention provides a pharmaceutical
composition comprising the dual function protein for treating
diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic
fatty liver disease, non-alcoholic steatohepatitis or
cardiovascular diseases.
[0029] Further, the present invention provides an isolated nucleic
acid molecule encoding the dual function protein, an expression
vector comprising the nucleic acid molecule, and a host cell
comprising the expression vector.
Advantageous Effects of Invention
[0030] A dual function protein of the present invention, prepared
by linking a biologically active protein and an FGF mutant protein
to an Fc region of an immunoglobulin, has improved pharmacological
efficacy, in vivo duration and protein stability. In addition, a
pharmaceutical composition including the dual function protein as
an active ingredient can be used as a therapeutic agent for
diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic
fatty liver diseases, non-alcoholic steatohepatitis or
cardiovascular diseases.
BRIEF DESCRIPTION OF DRAWINGS
[0031] FIGS. 1A to 1C are graphs showing the in vitro activities of
fusion proteins including FGF21 mutant proteins (hereinafter,
"FGF21 mutant fusion protein") using a HEK293 cell line in which
human .beta.-klotho is overexpressed. No FGF21 mutant fusion
proteins exhibited a significant decrease in activity due to the
introduction of a mutation.
[0032] FIGS. 2A and 2B are graphs showing the in vitro activities
of FGF21 mutant fusion proteins with various linkers connecting the
N-terminus of FGF21 to an Fc region, using a HEK293 cell line in
which human .beta.-klotho is overexpressed. No FGF21 mutant 23
fusion protein exhibited a significant decrease in activity,
although a slight difference was shown in activity depending on the
linker sequence.
[0033] FIG. 3 is a graph showing the in vitro activities of RGE
(Amgen), Fc-FGF21 (Lilly) and DFD1 using a HEK293 cell line in
which human .beta.-klotho is overexpressed. DFD1 and RGE (Amgen)
had similar activities, while Fc-FGF21 (Lilly) had in vitro
activity two times higher than the other proteins.
[0034] FIG. 4 shows the stability of DFD4 and that of DFD13 in
order to confirm the effect of the EIRP (SEQ ID NO: 68) mutation of
FGF21 on the stability of fusion protein. It was confirmed that
DFD13 was associated with a lower rate of high molecular weight
aggregates (HMW %) at the initial stage and at a time-point of more
than 2 weeks later as compared with DFD4, indicating that the
introduction of the EIRP (SEQ ID NO: 68) mutation improves the
stability of the FGF21 mutant fusion protein, thereby reducing HMW
% significantly.
[0035] FIG. 5 shows the concentration of each protein in the blood
over time for % hours after subcutaneous administration of FGF21
mutant fusion proteins. Data are indicated as mean values and
standard deviation.
[0036] FIG. 6 shows blood glucose levels in an ob/ob mouse model
after single subcutaneous injection of DFD18, DFD72, DFD74 or
Fc-FGF21 (Lilly). DFD18, DFD72 and DFD74 all had an effect of
lowering blood glucose level continuously. Data are indicated as
mean values and standard error of the mean (S.E.M.).
[0037] FIG. 7 shows graphs indicating the changes in body weights
in the ob/ob mouse model from the day of administration to the
14.sup.th day after single subcutaneous injection of DFD18, DFD72,
DFD74 or Fc-FGF21 (Lilly). DFD18, DFD72 and DFD74 all had an effect
of reducing body weight as compared with the PBS-treated group.
Data are indicated as mean values and standard error of the
mean.
[0038] FIG. 8 shows graphs indicating the changes in glycated
hemoglobin levels in the ob/ob mouse model at the day of
administration (1.sup.st day) and the 16.sup.th day after single
subcutaneous injection of DFD18, DFD72, DFD74 or Fc-FGF21 (Lilly).
DFD18, DFD72 and DFD74 all reduced glycated hemoglobin levels at
the 16.sup.th day as compared with those at the day of
administration. Data are indicated as mean values and standard
error of the mean.
[0039] FIG. 9 shows blood glucose levels in an HFD/STZ mouse model
after single subcutaneous injection of DFD72 or DFD74. Both DFD72
and DFD74 had the effect of lowering blood glucose level
continuously. Data are indicated as mean values and standard error
of the mean.
[0040] FIG. 10 shows the changes in animal body weights in the
HFD/STZ mouse model from the day of administration to the 14.sup.th
day after single subcutaneous injection of DFD72 or DFD74. Both
DFD72 and DFD74 had the effect of reducing body weight as compared
with the PBS-treated group. Data are indicated as mean values and
standard error of the mean.
[0041] FIG. 11 shows graphs indicating the changes in glycated
hemoglobin levels in the HFD/STZ mouse model at the 1.sup.st day
and the 13.sup.th day after single subcutaneous injection of DFD72
or DFD74. It was observed that both DFD72 and DFD74 resulted in
greater reduction of glycated hemoglobin levels as compared with
the PBS-treated group. Data are indicated as mean values and
standard error of the mean.
[0042] FIG. 12 shows the changes in body weights measured in the
diet-induced obesity mouse model from the day of administration to
the 14.sup.th day after single administration of DFD18. DFD18 had a
significant effect on body weight reduction. Data are indicated as
mean values and standard error of the mean.
[0043] FIG. 13 is a graph showing the in vitro GLP-1 activities of
dual function proteins depending on the hinges which link the
C-terminus of GLP-1 mutants and GLP-1 to the Fc region using a CHO
cell line in which human GLP-1 receptor is overexpressed.
Generally, the dual function protein including a GLP-1 (A2G)
sequence (DFD23) exhibited 2 to 3 times lower activity than those
of other dual function proteins including other GLP-1 mutant
sequences. No significant difference in GLP-1 activities was shown
between the dual function proteins including mutant sequences
except the GLP-1 (A2G) sequence.
[0044] FIG. 14 shows graphs indicating the GLP-1 activities of
DFD59, DFD69, DFD112 and DFD114 and the FGF21 activities of DFD69,
DFD112 and DFD114. In vitro GLP-1 activities of three dual function
proteins (DFD69, DFD112 and DFD114) and Fc-fused GLP-1 mutant
including no FGF21 (DFD59) were measured using a CHO cell line in
which human GLP-1 receptor is overexpressed. The three dual
function proteins showed similar EC.sub.50 values, and the Fc-fused
GLP-1 mutant (DFD59) showed about 2 times higher activity than
those of dual function proteins. In vitro activities of dual
function proteins depending on FGF21 mutants were measured using a
HEK293 cell line in which human pi-klotho is overexpressed. It was
confirmed that the in vitro activities of the FGF21 portion were
similar in the three dual function proteins.
[0045] FIG. 15 shows the concentrations of proteins in the blood
versus time for 240 hours after subcutaneous administration of dual
function proteins. Data are indicated as mean values and standard
deviation.
[0046] FIG. 16 shows the blood glucose levels in a db/db mouse
model after single subcutaneous injection of DFD114 or DFD59 and
single subcutaneous injection of combination of DFD59 and DFD74.
The groups treated with dual function proteins showed stronger
effects on lowering blood glucose levels than those treated with
single function proteins. Data are indicated as mean values and
standard error of the mean (S.E.M.).
[0047] FIG. 17 shows graphs indicating the changes in body weights
in db/db mouse model from the day of administration to the
14.sup.th day after single subcutaneous injection of DFD114 or
DFD59 and single subcutaneous injection of combination of DFD59 and
DFD74. The groups treated with dual function proteins showed
stronger effects on reducing body weight than those treated with
single function proteins. Data are indicated as mean values and
standard error of the mean (S.E.M.).
[0048] FIG. 18 shows graphs indicating the changes in glycated
hemoglobin levels in a db/db mouse model at the day of
administration (1.sup.st day) and the 16.sup.th day after single
subcutaneous injection of DFD114 or DFD59 and single subcutaneous
injection of a combination of DFD59 and DFD74. The groups treated
with dual function proteins showed stronger effects on reducing
glycated hemoglobin levels than those treated with single function
proteins or a combination thereof. Data are indicated as mean
values and standard error of the mean.
[0049] FIG. 19 shows the blood glucose levels in an HFD/STZ mouse
model after single subcutaneous injection of DFD114, DFD59, DFD74
or DFD72 and single subcutaneous injection of combination of DFD59
and DFD74. The groups treated with dual function proteins showed
stronger effects on lowering blood glucose levels than those
treated with single function proteins. Data are indicated as mean
values and standard error of the mean (S.E.M.).
[0050] FIG. 20 shows the changes in body weights in the HFD/STZ
mouse model from the day of administration to the 14.sup.th day
after single subcutaneous injection of DFD59, DFD72, DFD74 or
DFD114 and single subcutaneous injection of combination of DFD59
and DFD74. The groups treated with dual function proteins showed
stronger effects on reducing body weight than those treated with
single function proteins. Data are indicated as mean values and
standard error of the mean (S.E.M.).
[0051] FIG. 21 shows the changes in glycated hemoglobin levels in
the HFD/STZ mouse model at the day of administration (1.sup.st day)
and the 16.sup.th day after single subcutaneous injection of DFD59,
DFD72, DFD74 or DFD114 and single subcutaneous injection of
combination of DFD59 and DFD74. The groups treated with dual
function proteins showed stronger effects on reducing glycated
hemoglobin levels than those treated with single function proteins
or a combination thereof. Data are indicated as mean values and
standard error of the mean.
BEST MODE FOR CARRYING OUT THE INVENTION
[0052] Hereinafter, the present invention will be described in more
detail.
[0053] In an aspect, the present invention provides a dual function
protein comprising a fibroblast growth factor 21 (FGF21) mutant
protein; a biologically active protein, or a mutant or fragment
thereof; and an Fc region of an immunoglobulin, wherein the FGF21
mutant protein comprises at least one mutation selected from the
group consisting of the following mutations (1) to (7).
[0054] (1) a substitution of amino acids at positions 98 to 101
from the N-terminus of a wild-type FGF21 protein with an amino acid
sequence of EIRP (SEQ ID NO: 68) (hereinafter, "EIRP");
[0055] (2) a substitution of amino acids at positions 170 to 174
from the N-terminus of a wild-type FGF21 protein with an amino acid
sequence of TGLEAV (SEQ ID NO: 69) (hereinafter, "TGLEAV");
[0056] (3) a substitution of amino acids at positions 170 to 174
from the N-terminus of a wild-type FGF21 protein with an amino acid
sequence of TGLEAN (SEQ ID NO: 70) (hereinafter, "TGLEAN");
[0057] (4) a substitution of an amino acid at position 170 from the
N-terminus of a wild-type FGF21 protein with an amino acid N;
[0058] (5) a substitution of an amino acid at position 174 from the
N-terminus of a wild-type FGF21 protein with an amino acid N;
[0059] (6) a substitution of an amino acid at position 180 from the
N-terminus of a wild-type FGF21 protein with an amino acid E, along
with one or more mutations (1) to (5) above; and
[0060] (7) a mutation of 1 to 10 amino acids for reducing
immunogenicity of a wild-type FGF21 protein.
[0061] The wild-type FGF21 protein, a hormone known to play an
important role in glucose and lipid homeostasis, may be one derived
from mammals such as humans, mice, pigs, monkeys, etc., preferably
from humans. More preferably, the wild-type FGF21 protein may be
the wild-type human FGF21 protein having an amino acid sequence
represented by SEQ ID NO: 1.
[0062] The mutation included in the FGF21 mutant proteins may be,
preferably, any one of the mutations of EIRP (SEQ ID NO: 68),
TGLEAV (SEQ ID NO: 69), TGLEAN (SEQ ID NO: 70), G170N and G174N; a
combination of any one of the mutations of TGLEAV (SEQ ID NO: 69),
TGLEAN (SEQ ID NO: 70), G170N and G174N and the mutation of EIRP
(SEQ ID NO: 68); a combination of any one of the mutations of EIRP
(SEQ ID NO: 68), TGLEAV (SEQ ID NO: 69), TGLEAN (SEQ ID NO: 70),
G170N and G174N and the mutation of A180E; or a combination of any
one of the mutations of TGLEAV (SEQ ID NO: 69), TGLEAN (SEQ ID NO:
70), G170N and G174N, the mutation of EIRP (SEQ ID NO: 68) and the
mutation of A180E. Furthermore, the FGF21 mutant proteins may have
a conformation, in which 1 to 10 amino acids at the N-terminus or
C-terminus is (are) deleted as compared to the wild-type FGF21
protein. More preferably, the FGF21 mutant proteins may include an
amino acid sequence represented by any one of SEQ ID NO: 6 to 23.
Still more preferably, the FGF21 mutant proteins may include an
amino acid sequence represented by any one of SEQ ID NO. 6 to 23
and further have a conformation, in which 1 to 10 amino acids at
the N-terminus or C-terminus is (are) deleted as compared to the
wild-type FGF21 protein.
[0063] In the dual function protein, an amino acid residue N of
FGF21 mutant protein introduced by a mutation may be
glycosylated.
[0064] The biologically active protein may be one selected from the
group consisting of insulin, C-peptide, leptin, glucagon, gastrin,
gastric inhibitory polypeptide (GIP), amylin, calcitonin,
cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic
protein-6 (BMP-6), bone morphogenetic protein-9 (BMP-9),
oxyntomodulin, oxytocin, glucagon-like peptide-1 (GLP-1),
glucagon-like peptide-2 (GLP-2), irisin, fibronectin type III
domain-containing protein 5 (FNDC5), apelin, adiponectin, C1q and
tumor necrosis factor related protein (CTRP family), resistin,
visfatin, omentin, retinol binding protein-4 (RBP-4), glicentin,
angiopoietin, interleukin-22 (IL-22), exendin-4 and growth hormone.
Preferably, the biologically active protein may be one selected
from GLP-1, a mutant thereof and exendin-4.
[0065] The GLP-1 protein is an incretin hormone consisting of 31
amino acids, which is secreted by L cells in the intestinal tract
stimulated by food, etc. For example, the GLP-1 protein may be
represented by the amino acid sequence of SEQ ID NO: 42.
[0066] A mutant of GLP-1 may be represented, for example, by the
amino acid sequence of any one of SEQ ID NO: 43 to 46.
[0067] As used herein, the term "Fc region," "Fc fragment," or "Fc"
refers to a protein, which includes a heavy chain constant region 1
(CH1), a heavy chain constant region 2 (CH2) and a heavy chain
constant region 3 (CH3) of an immunoglobulin, but does not include
variable regions of the heavy and light chains and a light chain
constant region 1 (CL1) of an immunoglobulin. Additionally, as used
herein, the term "Fc region mutant" refers to one prepared by
substituting part of amino acid(s) of an Fc region or by combining
Fc regions of different types.
[0068] The Fc region of immunoglobulin may be an entire Fc region
constituting an antibody, a fragment thereof, or an Fc region
mutant. Additionally, the Fc region includes a molecule in the form
of a monomer or multimer, and may further include a hinge region of
the heavy chain constant region. The Fc region mutant may be
modified to prevent cleavage at the hinge region. Furthermore, the
hinge sequence of the Fc may have a substitution in some amino acid
sequences to reduce antibody-dependent cell-mediated cytotoxicity
(ADCC) or complement-dependent cytotoxicity (CDC). In addition,
part of the amino acid sequence of the Fc hinge sequence may be
substituted to inhibit the rearrangement of the Fab region. A
lysine residue at the C-terminus of the Fc may be removed.
[0069] Preferably, the Fc region of immunoglobulin may be any one
of IgG1, IgG2, IgG3, IgG4 and IgD Fc regions; or a hybrid Fc, which
is a combination thereof. Further, the hybrid Fc may include an
IgG4 region and an IgD region. Further, the hybrid Fc region may
include part of the hinge sequence and CH2 of an IgD Fc, and CH2
and CH3 sequences of IgG4 Fc.
[0070] In addition, the Fc fragment of the present invention may be
in the form of wild-type glycosylated chain, more glycosylated
chain than the wild-type, less glycosylated chain than the
wild-type, or deglycosylated chain. The increase, decrease, or
removal of glycosylated chain may be performed by a conventional
method known in the art, such as a chemical method, an enzymatic
method, and a genetic engineering method using microorganisms.
[0071] Preferably, the immunoglobulin Fc region may be represented
by an amino acid sequence selected from SEQ ID NO: 24 to 26, 47 and
48.
[0072] The dual function protein may include a biologically active
protein, an Fc region of an immunoglobulin and an FGF21 mutant
protein, linked in this order from the N-terminus to the
C-terminus. Further, the dual function protein may include an FGF21
mutant protein, an Fc region of an immunoglobulin and a
biologically active protein, linked in this order from the
N-terminus to the C-terminus. Preferably, the dual function protein
may include a biologically active protein, an Fc region of an
immunoglobulin and an FGF21 mutant protein, linked in this order
from the N-terminus to the C-terminus.
[0073] Furthermore, the dual function protein may include a GLP-1
mutant protein, an Fc region of an immunoglobulin and an FGF21
mutant protein, linked in this order from the N-terminus to the
C-terminus. Further, the dual function protein may include an FGF21
mutant protein, an Fc region of an immunoglobulin and a GLP-1
mutant protein, linked in this order from the N-terminus to the
C-terminus. Preferably, the dual function protein may include a
GLP-1 mutant protein, an Fc region of an immunoglobulin and an
FGF21 mutant protein, linked in this order from the N-terminus to
the C-terminus.
[0074] Additionally, the dual function protein may further include
a linker. The dual function protein may be in the form, in which
the FGF21 mutant protein is directly connected to the N-terminus or
C-terminus of the immunoglobulin Fc region, or the FGF21 mutant
protein is connected to the immunoglobulin Fc region via a
linker.
[0075] In such case, the linker may be connected to the N-terminus,
C-terminus, or a free radical of the Fc fragment, and also, may be
connected to the N-terminus, C-terminus, or a free radical of the
FGF21 mutant protein. When the linker is a peptide linker, the
connection may occur in any region. For example, the linker may be
connected to the C-terminus of the immunoglobulin Fc region and the
N-terminus of the FGF21 mutant protein to form a fusion protein of
the immunoglobulin Fc region and the FGF21 mutant protein.
[0076] Furthermore, the dual function protein of the present
invention may be in the form, in which a biologically active
protein is linked to the N-terminus of the Fc region of
immunoglobulin of the fusion protein.
[0077] When the linker and Fc are separately expressed and then
connected, the linker may be a crosslinking agent known in the art.
Examples of the crosslinking agent may include
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, imidoesters
including N-hydroxysuccinimide ester such as 4-azidosalicylic acid
and disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides
such as bis-N-maleimido-1,8-octane, but are not limited
thereto.
[0078] Further, the linker may be a peptide. Preferably, the linker
may be a peptide consisting of 10 to 30 amino acid residues.
[0079] Furthermore, alanine may additionally be attached to the end
of linker. Preferably, the linker may be a peptide having an amino
acid sequence represented by any one of SEQ ID NO: 2 to 5.
[0080] The dual function protein may be in a form in which a dimer
or multimer of FGF21 mutant proteins, in which one or more FGF21
mutant proteins linked together, is connected to an immunoglobulin
Fc region. Additionally, the dual function protein may be in a form
of a dimer or multimer in which two or more immunoglobulin Fc
regions are linked, wherein the immunoglobulin Fc regions have the
FGF21 mutant protein connected thereto.
[0081] Additionally, the dual function protein may be a peptide
which preferably has an amino acid sequence represented by any one
of SEQ ID NO: 58 to 67. More preferably, the dual function protein
may be a peptide which has an amino acid sequence represented by
SEQ ID NO: 65, 66 or 67.
[0082] The FGF21 mutant protein may further include a mutation of 1
to 10 amino acids for reducing immunogenicity of the wild-type
FGF21 protein. The immunogenicity may be predicted by a
conventional method known in the art. For example, the potential
immunogenicity of a protein may be screened by using, e.g.,
ITOPE.TM. and TCED.TM. methods.
[0083] Further, the mutation for minimizing the immunogenicity may
be designed by a conventional method known in the art. For example,
when immunogenicity is observed by performing an EPISCREEN.TM.
analysis to evaluate potential immunogenicity, the amino acid
sequences inducing the immunogenicity may be identified through
T-cell epitope mapping, and the mutants with minimized
immunogenicity may be designed via in silico prediction.
[0084] In another aspect, the present invention provides a
pharmaceutical composition containing the dual function protein for
treating FGF21-associated disorders.
[0085] As used herein, the term "FGF21-associated disorder" may
include obesity, type I- and type II diabetes, pancreatitis,
dyslipidemia, non-alcoholic fatty liver disease (NAFLD),
non-alcoholic steatohepatitis (NASH), insulin resistance,
hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic
syndrome, acute myocardial infarction, hypertension, cardiovascular
diseases, atherosclerosis, peripheral arterial disease, apoplexy,
heart failure, coronary artery heart disease, renal disease,
diabetic complications, neuropathy, gastroparesis, disorder
associated with a serious inactivation mutation in insulin
receptor, and other metabolic disorders. Preferably, the
FGF21-associated disorder may be diabetes, obesity, dyslipidemia,
metabolic syndrome, non-alcoholic fatty liver disease,
non-alcoholic steatohepatitis or cardiovascular diseases.
[0086] Further, the pharmaceutical composition may further include
a pharmaceutical carrier. The pharmaceutical carrier may be any
carrier as long as it is a non-toxic material suitable for
delivering antibodies to patients. For example, distilled water,
alcohol, fats, waxes and inactive solids may be included as a
carrier. Pharmaceutically acceptable adjuvants (buffering agents,
dispersants) may also be included in the pharmaceutical
composition. In these formulations, the concentration of the dual
function protein may vary greatly.
[0087] Specifically, the pharmaceutical composition may contain a
formulation material for altering, maintaining, or conserving the
pH, osmolarity, viscosity, transparency, color, isotonicity, odor,
sterility, stability, dissolution or release rate, adsorption, or
permeability of the composition. Examples of the suitable
formulating material may include amino acids (e.g., glycine,
glutamine, asparagine, arginine or lysine), anti-microorganism
agents, anti-oxidants (e.g., ascorbic acid, sodium sulfite or
sodium bisulfite), buffering agents (e.g., borate, bicarbonates,
Tris-HCl, citrate, phosphate or other organic acids), bulking
agents (e.g., mannitol or glycine), chelating agents (e.g.,
ethyelenediaminetetraacetic acid (EDTA)), complexing agents (e.g.,
caffeine, polyvinylpyrrolidione, .beta.-cyclodextrin or
hydroxypropyl-.beta.-cyclodextrin), fillers, monosaccharides,
disaccharides and other carbohydrates (e.g., glucose, mannose or
dextrin), proteins (e.g., serum albumin, gelatin or
immunoglobulin), coloring agents, flavoring agents, diluents,
emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidione),
low molecular weight polypeptides, salt-forming counterions (e.g.,
sodium), preservatives (e.g., benzalkonium chloride, benzoic acid,
salicylic acid, thimerosal, phenethyl alcohol, methylparaben,
propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide),
solvents (e.g., glycerin, propylene glycol or polyethylene glycol),
sugar alcohols (e.g., mannitol or sorbitol), suspending agents,
surfactants or humectants (e.g., pluronics; PEG; sorbitan ester;
polysorbate, e.g., polysorbate 20 or polysorbate 80; triton;
tromethamine; lecithin; cholesterol or tyloxapol), stability
improvers (e.g., sucrose or sorbitol), growth improvers (e.g.,
alkali metal halides, preferably, sodium chloride or potassium
chloride; or mannitol, sorbitol), delivery vehicles, diluents,
excipients and/or pharmaceutical adjuvants, but are not limited
thereto.
[0088] In another aspect, the present invention provides a method
for preventing or treating FGF21-associated disorders including
administering the dual function protein to a subject in need of
such prevention or treatment. This method includes, in particular,
administering an effective amount of the dual function protein of
the present invention to a mammal having a symptom of diabetes,
obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty
liver disease, non-alcoholic steatohepatitis or cardiovascular
diseases which are FGF21-associated disorders.
[0089] The pharmaceutical composition of the present invention may
be administered via any route. The composition of the present
invention may be provided to an animal directly (e.g., topically,
by administering into tissue areas by injection, transplantation,
or by topical administration) or systemically (e.g., by oral- or
parenteral administration) via any appropriate means. When the
composition of the present invention is parenterally provided via
intravenous-, subcutaneous-, ophthalmic-, intraperitoneal-,
intramuscular-, oral-, rectal-, intraorbital-, intracerebral-,
intracranial-, intraspinal-, intraventricular-, intrathecal-,
intracistenal-, intracapsular-, intranasal-, or aerosol
administration, the composition is preferably aqueous or may
include a portion of a physiologically applicable body liquid
suspension or solution. Accordingly, the carrier or vehicle may be
added to the composition and be delivered to a patient since it is
physiologically applicable. Therefore, a
physiologically-appropriate saline solution may generally be
included as a carrier like a body fluid for formulations.
[0090] Further, the administration frequency may vary depending on
the pharmacokinetic parameters of the dual function protein in the
formulations to be used. Typically, physicians would administer the
composition until an administration dose to achieve a desired
effect is reached. Accordingly, the composition may be administered
as a unit dose, at least two doses with time intervals (may or may
not contain the same amount of a target dual function protein) or
administered by a continuous injection via a transplantation device
or catheter. The precision of addition of an appropriate
administration dose may be routinely performed by those skilled in
the art, and corresponds to the scope of work being routinely
performed by them.
[0091] Additionally, the preferable unit dose of the dual function
protein in humans may be in a range from 0.01 .mu.g/kg to 100 mg/kg
of body weight, and more preferably from 1 .mu.g/kg to 10 mg/kg of
body weight. Although this is the optimal amount, the unit dose may
vary depending on the disease to be treated or the presence/absence
of adverse effects. Nevertheless, the optimal administration dose
may be determined by performing a conventional experiment. The
administration of the dual function protein may be performed by a
periodic bolus injection, an external reservoir (e.g., an
intravenous bag), or a continuous intravenous-, subcutaneous-, or
intraperitoneal administration from the internal source (e.g., a
bioerodible implant).
[0092] In addition, the dual function protein of the present
invention may be administered to a subject recipient along with
other biologically active molecules. The optimal combination of the
dual function protein and other molecule(s), dosage forms, and
optimal doses may be determined by a conventional experiment well
known in the art.
[0093] In still another aspect, the present invention provides an
isolated nucleic acid molecule encoding the dual function
protein.
[0094] As used herein, the term "isolated nucleic acid molecule"
refers to a nucleic acid molecule of the present invention, which
is isolated from about at least 50% of proteins, lipids,
carbohydrates, or other materials, discovered in nature when total
nucleic acids are isolated from a source cell; which is operatively
linked to a polynucleotide which is not linked in nature; or which
is a part of a larger polynucleotide sequence and does not occur in
nature. Preferably, in the isolated nucleic acid molecules of the
present invention, there are not substantially present any other
contaminated nucleic acids, or other contaminants which are
discovered in the natural environment and inhibit uses of the
nucleic acids in the production of polypeptides, or treatment,
diagnosis, prevention, or research.
[0095] In such case, the isolated nucleic acid molecules encoding
the dual function protein may have different sequences with each
other due to codon redundancy. Furthermore, as long as the isolated
nucleic acid can produce the dual function protein, the isolated
nucleic acid may be appropriately modified, or a nucleotide may be
added to the N-terminus or C-terminus of the isolated nucleic acid
according to desired purposes.
[0096] The isolated nucleic acid may include, for example, a
nucleotide sequence represented by any one of SEQ ID NO: 71 to
80.
[0097] In still another aspect, the present invention provides an
expression vector comprising the isolated nucleic acid
molecule.
[0098] As used herein, the term "expression vector" refers to a
vector containing a nucleic acid sequence, which is suitable for
the transformation of a host cell and directs or controls the
expression of an inserted heterogenous nucleic acid sequence. The
expression vector includes a linear nucleic acid, a plasmid, a
phagemid, a cosmid, an RNA vector, a viral vector, and analogs
thereof. Examples of the viral vector include a retrovirus, an
adenovirus and an adeno-associated virus, but are not limited
thereto.
[0099] As used herein, the term "expression of a heterogeneous
nucleic acid sequence" or "expression" of a target protein refers
to transcription of an inserted DNA sequence, translation of an
mRNA transcript, and production of an Fc fusion protein product, an
antibody or an antibody fragment.
[0100] A useful expression vector may be RcCMV (Invitrogen,
Carlsbad) or a mutant thereof. The useful expression vector may
include a human cytomegalovirus (CMV) promoter for promoting a
continuous transcription of a target gene in a mammalian cell, and
a bovine growth hormone polyadenylation signal sequence for
enhancing the level of post-transcriptional RNA stability. In an
exemplary embodiment of the present invention, the expression
vector is pAD15, which is a modified vector of RcCMV.
[0101] In still another aspect, the present invention provides a
host cell comprising the expression vector.
[0102] As used herein, the term "host cell" refers to a prokaryotic
cell or eukaryotic cell into which a recombinant expression vector
may be introduced. As used herein, the term "transformed" or
"transfected" refers to introduction of a nucleic acid (e.g., a
vector) into a cell by various technologies known in the art.
[0103] An appropriate host cell may be transformed or transfected
with a DNA sequence of the present invention and may be used for
the expression and/or secretion of the target protein. Examples of
the appropriate host cell that may be used in the present invention
include immortal hybridoma cells, NS/0 myeloma cells, 293 cells,
Chinese hamster ovary (CHO) cells, HeLa cells, CAP cells (human
amniotic fluid-derived cells), and COS cells.
[0104] Hereinafter, exemplary embodiments of the present invention
will be described in detail with reference to the examples.
However, these examples according to the present invention can be
modified in many different forms and the scope of the present
invention should not be construed as limited to the examples set
forth herein.
MODE FOR THE INVENTION
Preparation Example 1. Preparation and Purification of Fusion
Protein Containing FGF21 Mutant Protein
Preparation Example 1-1. Preparation of Expression Vectors for
Expression of FGF21 Mutant Proteins
[0105] In order to improve the stability, activity and
pharmacokinetic profiles of the FGF21 in an Fc-FGF21 structure,
mutation studies of FGF21 were performed.
[0106] Specifically, mutant proteins were designed for the LLLE
(SEQ ID NO: 81) region (the amino acids at positions 98 to 101 from
the N-terminus of the FGF21 protein) and GPSQG (SEQ ID NO: 82)
region (the amino acids at positions 170 to 174 from the N-terminus
of the FGF21 protein), and A180 site, which were expected to
significantly affect protein activities based on 3-dimensional
structure analysis of the FGF21 proteins.
[0107] The position, sequence information, target and expected
effect of each mutation introduced into the FGF21 protein are
listed in Table 1 below (in Table 1, N refers to glycosylated
asparagine (N)). Further, FGF21 mutant proteins including the
mutations described in Table 1 are listed in Table 2 below.
TABLE-US-00001 [TABLE 1] Se- Posi- Original Mutated quence tion
sequence sequence Target Expected effect EIRP 98-101 LLLE EIRP
Substitution Improvement of (SEQ ID (SEQ ID (SEQ ID with FGF19
stability and NO: 68) NO: 81) NO: 68) sequence pharmacokinetics
TGLEAV 170-174 GPSQG TGLEAV Substitution Improvement of (SEQ ID
(SEQ ID (SEQ ID with FGF19 pharmacokinetics NO: 69) NO: 82) NO: 69)
sequence TGLEAN 170-174 GPSQG TGLEAN Substitution Improvement of
(SEQ ID (SEQ ID (SEQ ID with FGF19 pharmacokinetics NO: 70) NO: 82)
NO: 70) sequence, and addition of N-glycosylation G170N 170 G N
Point Improvement of mutation, pharmacokinetics and addition of
N-glycosylation G174N 174 G N Point mutation, Improvement of and
addition pharmacokinetics of N-glycosylation A180E 180 A E Point
mutation Improvement of pharmacokinetics
TABLE-US-00002 SEQ ID NO Sequence of FGF21 mutant protein 6 FGF21
(EIRP (SEQ ID NO: 68)) 7 FGF21 (TGLEAV (SEQ ID NO: 69)) 8 FGF21
(TGLEAN (SEQ ID NO: 70)) 9 FGF21 (G170N) 10 FGF21 (G174N) 11 FGF21
(EIRP (SEQ ID NO: 68), TGLEAV (SEQ ID NO: 69)) 12 FGF21 (EIRP (SEQ
ID NO: 68), TGLEAN (SEQ ID NO: 70)) 13 FGF21 (EIRP (SEQ ID NO: 68),
G170N) 14 FGF21 (EIRP (SEQ ID NO: 68), G174N) 15 FGF21 (EIRP (SEQ
ID NO: 68), A180E) 16 FGF21 (TGLEAV (SEQ ID NO: 69), A180E) 17
FGF21 (TGLEAN (SEQ ID NO: 70), A180E) 18 FGF21 (G170N, A180E) 19
FGF21 (G174N, A180E) 20 FGF21 (EIRP (SEQ ID NO: 68), TGLEAV (SEQ ID
NO: 69), A180E) 21 FGF21 (EIRP (SEQ ID NO: 68), TGLEAN (SEQ ID NO:
70), A180E) 22 FGF21 (EIRP (SEQ ID NO: 68), G170N, A180E) 23 FGF21
(EIRP (SEQ ID NO: 68), G174N, A180E)
[0108] Expression vectors were prepared to express the amino acids
of the three components: fusion carrier, linker and FGF21 mutant in
this order from the N-terminus to C-terminus. The material code of
each FGF21 mutant fusion protein, sequence of mutation introduced
into FGF21, sequence of fusion carrier and linker sequence are
listed in Table 3 below (in Table 3, N refers to glycosylated
asparagine (N)).
TABLE-US-00003 [TABLE 3] Sequence SEQ of ID Material FGF21 Fusion
Linker NO code mutation carrier sequence 27 DFD1 EIRP hyFc C (SEQ
ID (SEQ ID (SEQ ID NO: 68), NO: 26) NO: 2) TGLEAV (SEQ ID NO: 69)
28 DFD3 TGLEAV hyFc AKA (SEQ ID (SEQ ID (SEQ ID NO: 69) NO: 26) NO:
3) 29 DFD4 TGLEAV hyFc GS3 (SEQ ID (SEQ ID (SEQ ID NO: 69) NO: 26)
NO: 4) 30 DFD5 TGLEAN hyFc GS3 (SEQ ID (SEQ ID (SEQ ID NO: 70) NO:
26) NO: 4) 31 DFD6 G170N hyFc GS3 (SEQ ID (SEQ ID NO: 26) NO: 4) 32
DFD6 G170N hyFc GS3 (E. coli) (SEQ ID (SEQ ID NO: 26) NO: 4) 33
DFD7 G174N hyFc GS3 (SEQ ID (SEQ ID NO: 26) NO: 4) 34 DFD9 none
hyFc GS3 (SEQ ID (SEQ ID NO: 26) NO: 4) 35 DFD13 EIRP hyFc GS3 (SEQ
ID (SEQ ID (SEQ ID NO: 68), NO: 26) NO: 4) TGLEAV (SEQ ID NO: 69)
36 DFD18 EIRP hyFc GS3 (SEQ ID (SEQ ID (SEQ ID NO: 68), NO: 26) NO:
4) TGLEAV (SEQ ID NO: 69), A180E 37 DFD72 EIRP hyFc GS3 (SEQ ID
(SEQ ID (SEQ ID NO: 68), NO: 26) NO: 4) TGLEAN (SEQ ID NO: 70),
A180E 38 DFD73 EIRP hyFc GS3 (SEQ ID (SEQ ID (SEQ ID NO: 68), NO:
26) NO: 4) G170N 39 DFD74 EIRP hyFc GS3 (SEQ ID (SEQ ID (SEQ ID NO:
68), NO: 26) NO: 4) G170N, A180E 40 RGE L98R, IgG1Fc GS3 (Amgen)
P171G, mutant (SEQ ID A180E NO: 4) 41 Fc-FGF21 X IgG4Fc GS3A
(Lilly) mutant (SEQ ID (SEQ ID NO: 5) NO: 25)
[0109] In order to produce the FGF21 mutant fusion proteins, the
nucleotide sequences encoding each of the FGF21 mutant proteins
were synthesized by consulting with Bioneer Corporation (Korea)
based on the amino acid sequence of each protein. NheI and NotI
restriction enzyme sequences were added to the 5' terminus and 3'
terminus of the nucleotide sequences encoding each of the FGF21
mutant proteins and an initiation codon for protein translation and
a leader sequence (MDAMLRGLCCVLLLCGAVFVSPSHA) (SEQ ID NO: 83)
capable of secreting the expressed protein to the outside of a cell
were inserted next to the restriction enzyme sequence at the 5'
terminus. A termination codon was inserted next to the nucleotide
sequence, which encodes each of the FGF21 mutant fusion proteins.
The nucleotide sequence encoding each of the FGF21 mutant fusion
proteins was cloned into a pTrans-empty expression vector by using
the two restriction enzymes of NheI and NotI. The pTrans-empty
expression vector, which has a simple structure including a CMV
promoter, a pUC-derived replication origin, an SV40-derived
replication origin and an ampicillin-resistant gene, was purchased
from CEVEC Pharmaceuticals (Germany).
[0110] In the case of the fusion proteins of DFD6 (E. coli) and RGE
(Amgen), the nucleotide sequence encoding each fusion protein was
inserted into a pET30a expression vector for expression in E.
coli.
Preparation Example 1-2. Construction of Plasmid DNA for Expression
of FGF21 Mutant Fusion Proteins
[0111] E. coli was transformed with each of the expression vectors
constructed in Preparation Example 1-1 to obtain a large amount of
plasmid DNA to be used for expression. E. coli cells, whose cell
walls were weakened, were transformed with each expression vector
through heat shock, and the transformants were plated out on LB
plates to obtain colonies. The colonies thus obtained were
inoculated into LB media, cultured at 37.degree. C. for 16 hours,
and each E. coli culture containing each expression vector was
obtained in a volume of 100 mL. The E. coli thus obtained was
centrifuged to remove the culture medium, and then P1, P2, P3
solutions (QIAGEN, Cat No.:12963) were added to break the cell
walls, thereby obtaining a DNA suspension in which proteins and
DNAs were separated. Plasmid DNA was purified from the DNA
suspension thus obtained by using a Qiagen DNA purification column.
The eluted plasmid DNA was identified through an agarose gel
electrophoresis, and concentrations and purities were measured by
using a NANODROP.TM. device (Thermo scientific, NANODROP.TM. Lite).
The DNA thus obtained was used for expression.
Preparation Example 1-3. Expression of Fusion Proteins in CAP-T
Cells
[0112] Human cell lines were transfected with each plasmid DNA type
obtained in Preparation Example 1-2. Each plasmid DNA type was
transduced into CAP-T cells (CEVEC), which had been cultured in PEM
medium (Life technologies), by using PEI solution (Polyplus, Cat.
No.:101-10N). The mixed solution of DNA and the PEI solution was
mixed with the cell suspension by using a FREESTYLE.TM. 293
expression medium (Invitrogen), cultured at 37.degree. C. for 5
hours, and PEM medium was added. After culturing at 37.degree. C.
for 5-7 days, the culture was centrifuged to remove cells and a
supernatant including FGF21 mutant fusion proteins was
obtained.
Preparation Example 1-4. Expression and Purification of FGF21
Mutant Fusion Proteins in E. coli
[0113] E. coli strain BL21 (DE3) was transformed with each plasmid
DNA expressing DFD6 (E. coli) and RGE (Amgen) fusion proteins. The
transformed E. coli expressing each fusion protein was inoculated
into 20 mL of LB media, cultured at 37.degree. C. for 15 hours with
shaking, and then a portion of the culture media was inoculated
into 100 mL of LB media, and cultured at 37.degree. C. for 16 hours
with shaking. Upon completion of culturing, the culture was
centrifuged to obtain E. coli pellets, and then cells were
disrupted using a high pressure cell disruptor to obtain inclusion
bodies.
[0114] The obtained inclusion bodies were purified by washing and
elution, followed by a protein refolding process. Specifically, the
obtained inclusion bodies were washed 2-3 times with a buffer
solution (pH 8.0) containing 0.5% Triton X-100, 50 mM Tris, 1 mM
EDTA and 0.1 M NaCl to remove bacterial protein, and then
resuspended in 8 M urea buffer containing 8 M urea, 50 mM Tris and
1 mM DTT. Since the proteins in 8 M urea buffer were completely
denatured, a protein refolding process was performed as
follows.
[0115] To begin, 8 M urea buffer was gradually diluted with 20 mM
glycine buffer solution (pH 9.0) to remove urea, and from the
concentration of 2 M, CuSO.sub.4 was added to the concentration of
80 .mu.M to induce stable protein folding. The protein completing
the refolding process was suspended in PBS buffer solution (pH
7.4), and the suspension was filtered with a 0.22 .mu.m filter to
remove impurities, and then loaded into a Protein A affinity
chromatography column. The column was washed with 1.times.PBS
buffer solution (pH 7.4) and then the proteins were eluted using
100 mM glycine buffer solution (pH 3.0) to prepare DFD6 (E. coli)
fusion protein.
[0116] In the case of RGE (Amgen) fusion protein, the protein
completing the refolding process was suspended in 50 mM Tris buffer
solution (pH 8.0), the suspension was filtered with a 0.22 .mu.m
filter to remove impurities, and then loaded into an anion exchange
resin column (POROS.RTM. HQ 50 m, Thermo Fisher Scientific). The
column was washed with 50 mM Tris buffer solution (pH 8.0), and
then 50 mM Tris buffer solution (pH 8.0) was administered along the
concentration gradient to elute RGE (Amgen) fusion protein. The RGE
(Amgen) fusion protein obtained by the anion exchange resin was
mixed with ammonium sulfate to the concentration of 1 M, and then
purified using a hydrophobic interaction chromatography column
(Phenyl sepharose FF, GE Healthcare). Specifically, the column was
washed with 50 mM Tris buffer solution (pH 8.0) containing 1 M
ammonium sulfate, 50 mM Tris buffer solution (pH 8.0) was
administered along the concentration gradient, and the eluted
fractions were analyzed through 10% Tris-glycine gel
electrophoresis. The gel was dyed with coomassie brilliant blue R
with mild shaking, and the fractions containing FGF21 mutant fusion
protein with high purity were collected and then dialyzed overnight
at 4.degree. C. using a final buffer solution (1.times.PBS, 1 mM
EDTA, pH 7.4). Upon completion of the dialysis, the obtained
protein stock solution was concentrated at 3,000 rpm by using a
30,000 MW cut-off centrifugation filter at 4.degree. C. The
concentration of FGF21 mutant fusion protein was measured via BCA
quantitative analysis.
Preparation Example 1-5. Purification of FGF21 Mutant Fusion
Proteins
[0117] Protein A affinity chromatography column (GE Healthcare) was
equilibrated with 1.times.PBS buffer solution (pH 7.4). The culture
supernatant including each FGF21 mutant fusion protein obtained in
Preparation Example 1-3 was filtered with a 0.2 .mu.m filter, and
then loaded into a Protein A affinity chromatography column. The
column was washed with 1.times.PBS buffer solution (pH 7.4) and
then proteins were eluted using 100 mM glycine buffer solution (pH
3.0). The fusion proteins obtained by affinity chromatography were
purified using an anion exchange resin column (POROS.RTM. HQ 50
.mu.m, Thermo Fisher Scientific). The anion exchange resin column
was equilibrated with 50 mM Tris buffer solution (pH 8.0), before
the FGF21 mutant fusion proteins were eluted from the column.
Specifically, after washing the column with 50 mM Tris buffer
solution (pH 8.0), 50 mM Tris buffer solution (pH 8.0) was
dispensed along the concentration gradient and the eluted fractions
were analyzed. Each eluted fraction was analyzed using size
exclusion chromatography (SEC-HPLC), and the fractions including
FGF21 mutant fusion proteins with high purity were collected. The
concentration and quantitative analysis were performed in
accordance with the methods described in Preparation Example
1-4.
Experimental Example 1. In Vitro Activities of Fusion Proteins
Experimental Example 1-1. Effect of FGF21 Mutations on Protein
Activity
[0118] The in vitro activities of fusion proteins DFD4, DFD5, DFD6,
DFD6 (E. coli), DFD7, DFD9, DFD13, DFD18, DFD72, DFD73 and DFD74
prepared in Preparation Example 1 were measured.
[0119] Specifically, the in vitro FGF21 activities of the fusion
proteins were evaluated using a HEK293 cell line (Yuhan
Corporation, Korea) which was modified to overexpress human
.beta.-klotho, a coreceptor of FGF21. For the evaluation of
activity, the concentrates containing the fusion proteins prepared
in Preparation Examples 1-4 and 1-were subjected to a 3-fold serial
dilution at a concentration of 3 .mu.M. After having been cultured
in a serum-deficient state for 5 hours, the cell line
overexpressing human .beta.-klotho was treated with the diluted
fusion proteins for 20 minutes, and then was lysed by adding
cytolysis buffer (Cisbio/Cat #64ERKPEG) with stirring at 60 rpm for
30 minutes at room temperature. The cell lysate solution was mixed
with antibodies (Cisbio/Cat #64ERKPEG), which can detect
extracellular signal-regulated kinase (ERK) and phosphorylated ERK,
and the mixture was maintained at room temperature for 2 hours.
Fluorescence was detected using a fluorometric detector
(TECAN/GENiosPro). The activities of the fusion proteins were
measured by comparing their EC.sub.50 values.
[0120] As shown in FIGS. 1A to 1C, it was confirmed that the in
vitro activities of the fusion proteins prepared by introducing
mutation sequences into the wild-type FGF21 protein were not
inhibited, and the activities of each fusion protein were similar
to each other. It was also confirmed that through the DFD6 (E.
coli) sample expressed in E. coli and the DFD6 sample expressed in
animal cells, the in vitro activities of the fusion proteins
prepared by introducing N-glycosylation mutation into the wild-type
FGF21 protein were not inhibited.
Experimental Example 1-2. Effect of Linker Sequence on Protein
Activity
[0121] The in vitro activities of fusion proteins DFD1, DFD3, DFD4
and DFD13 prepared in Preparation Example 1 were measured.
[0122] Specifically, the FGF21 activities of the fusion proteins
were measured by using the concentrates containing the fusion
proteins prepared in Preparation Example 1-5 in accordance with the
methods described in Experimental Example 1-1. The results are
shown in FIGS. 2A and 2B.
[0123] It was confirmed that no FGF21 mutant fusion protein showed
a significant decrease in the activity, although a slight
difference was shown in the activity depending on the linker
sequence, as shown in FIGS. 2A and 2B.
Experimental Example 1-3. Experimental Results for DFD1, RGE
(Amgen) and Fc-FGF21 (Lilly)
[0124] The in vitro activities of fusion protein DFD1 prepared in
Preparation Example 1 and control proteins RGE (Amgen) and Fc-FGF21
(Lilly) were measured.
[0125] Specifically, the FGF21 activities of the fusion proteins
were measured by using the concentrates containing the fusion
proteins prepared in Preparation Example 1-5 and the control
proteins in accordance with the methods described in Experimental
Example 1-1. The results are shown in FIG. 3.
[0126] It was confirmed that DFD1 and RGE (Amgen) had similar in
vitro activity, while Fc-FGF21 (Lilly) had in vitro activity two
times higher than those of the other proteins, as shown in FIG.
3.
Experimental Example 2. Evaluation of Stability of Fusion
Proteins
Experimental Example 2-1. Experimental Method for Evaluating
Stability
[0127] In order to measure the quantity of protein aggregates at
the initial stage of the sample preparation, high molecular weight
aggregates (% HMW) were quantified using a size-exclusion
chromatography (SEC-HPLC) method. The results are shown in FIG.
4.
[0128] Specifically, a TOSOHAAS.TM. model TSK-GEL.RTM.
G3000SW.sub.XL column was used for the SEC-HPLC method. The column
was equilibrated by flowing a buffer solution (1.times.PBS, 1 mM
EDTA, pH 7.4) at a flow rate of 1 mL/min. The DFD4 and DFD13
protein stock solutions prepared in Preparation Examples 1-5 were
concentrated to a target concentration of 20 mg/mL or higher at
3,000 rpm using a 30,000 MW cut-off centrifugation filter at
4.degree. C. After the measurement of the concentration of each
sample by BCA quantitative analysis, the samples were diluted with
a buffer solution (1.times.PBS, 1 mM EDTA, pH 7.4) to a final
concentration of 20 mg/mL. In order to measure the initial % HMW of
DFD4 and DFD13, 20 mg/mL of the samples were diluted with the
buffer solution (1.times.PBS, 1 mM EDTA, pH 7.4) to a final
concentration of 1 mg/mL, and each sample in a volume of 100 .mu.L
was analyzed by SEC-HPLC column.
[0129] For the stability evaluation of each sample, % HMW of the
samples was measured using the SEC-HPLC method on the 4.sup.th, the
8.sup.th and the 14.sup.th days while storing them at 5.degree. C.,
25.degree. C. and 37.degree. C. for two weeks.
[0130] As shown in FIG. 4, it was confirmed that DFD13 had a lower
quantity of high molecular weight aggregates (HMW %) at the initial
stage and up to the point of 2 weeks as compared with DFD4,
indicating that the introduction of the EIRP (SEQ ID NO: 68)
mutation improves the stability of the FGF21 mutant fusion protein,
thereby reducing HMW % significantly.
Experimental Example 2-2. Stability Results
[0131] In order to investigate the effects of the EIRP (SEQ ID NO:
68) mutation introduced into the original sequence LLLE (SEQ ID NO:
81) (amino acid residues at 98-101 of SEQ ID NO: 1) of FGF21 on
stability, the stability of DFD4 (SEQ ID NO: 29) and DFD13 (SEQ ID
NO: 35) was measured in accordance with the methods described in
Experimental Example 2-1. The analysis results for the zero-hour
sample (initial stage; Day 0) and 4-, 8-, and 14 day-stored samples
of DFD4 and DFD13 are summarized in Table 4 below (in Table 4, N.D.
means "not detected").
TABLE-US-00004 TABLE 4 Stability of DFD4 and DFD13 for 2 weeks at a
concentration of 20 mg/mL (% HMW) DFD4 DFD13 Day 5.degree. C.
25.degree. C. 37.degree. C. 5.degree. C. 25.degree. C. 37.degree.
C. 0 0.91 0.56 4 4.25 11.64 5.12 0.36 0.34 0.84 8 6.16 9.99 4.87
N.D. N.D. N.D. 14 8.15 8.83 4.71 N.D. N.D. 0.32
[0132] As shown in Table 4, the quantity of % HMW at the initial
stage (Day 0) was 0.91% for DFD4, and 0.56% for DFD13. After 2
weeks, the amount of % HMW increased to 8.83% for DFD4, but it was
not observed in DFD13, under the condition of storage at 25.degree.
C. DFD13 was shown to have a lower % HMW rate at the initial stage
and 2 weeks, as compared with DFD4, which indicates that the % HMW
rate of FGF21 mutant fusion protein decreased significantly due to
the introduction of the EIRP (SEQ ID NO: 68) mutation.
Experimental Example 3. Pharmacokinetic Assessment of Fusion
Proteins
Experimental Example 3-1. Experimental Method for Pharmacokinetic
Assessment
[0133] Six-week old male ICR mice purchased from Orient BIO (Korea)
were partitioned into groups (n=3/blood sampling time) in order to
have similar mean values for body weight one day before drug
treatment, and subcutaneously administered once with a respective
sample at 1 mg/kg (2 mg/kg for RGE). Blood samples were then
collected at 1, 4, 8, 12, 24, 48, 72, and 96 hours after the
injection, respectively. The concentration of intact full length
FGF21 protein in the blood was measured using a intact human FGF21
ELISA Kit (F1231-K01, Eagle Biosciences, USA), which has
immunoreactivity to the N-terminus and C-terminus of FGF21 protein.
The concentrations of the samples in the blood collected until 96
hours after the subcutaneous injection of each fusion protein into
the mice were measured, and pharmacokinetic parameters of each
sample were calculated.
Experimental Example 3-2. Assessment of Pharmacokinetic
Activity
[0134] Based on the graph showing the concentrations of each
protein in the blood versus time after the subcutaneous
administration of fusion proteins in mice (FIG. 5), the
pharmacokinetic parameters were calculated. The data are shown in
Table 5 below.
TABLE-US-00005 TABLE 5 DFD6 Parameters DFD4 DFD5 DFD6 DFD7 DFD9
DFD13 DFD18 DFD72 DFD73 DFD74 (E.coli) RGE* T.sub.max (hour) 12 12
12 4 4 12 12 8 8 8 8 12 C.sub.max (ng/mL) 1288 1732 2868 696 384
1070 3428 2962 3296 3996 1399 9921 AUC.sub.last 25856 40706 100107
14118 4656 28785 104230 115977 123511 206634 37269 325747 (ng
hr/mL) Half-life (hour) 5.5 8.0 14.9 19.7 17.4 7.1 11.0 14.4 16.6
26.0 9.1 12.9
[0135] The pharmacokinetic profile of each fusion protein was
compared and evaluated based on the value of the area under the
curve (AUC) indicating the degree of drug exposure.
[0136] As shown in Table 5, upon comparing DFD4 with DFD13, and
DFD6 with DFD73, it was determined that the introduction of the
EIRP (SEQ ID NO: 68) sequence resulted in an approximate 10 to 20%
increase in AUC value. Comparing DFD9 with DFD4, the introduction
of TGLEAV (SEQ ID NO: 69) resulted in an approximate 6-fold
increase in AUC value.
[0137] Furthermore, the mutations of TGLEAN (SEQ ID NO: 70), G170N
and G174N are designed to extend the half-life by introducing
N-glycosylation into the C-terminus of FGF21, which is known to be
proteolyzed in vivo. The increase in AUC due to the introduction of
N-glycosylation was confirmed by comparing the mutants with each
control material. In order to confirm the effect of improvement in
AUC due to the introduction of N-glycosylation, the AUC value for
DFD6 (E. coli) produced by E. coli which has no glycosylation was
compared with that in DFD6 produced by a human cell line. DFD6
produced by the human cell line showed a 3-fold or higher increase
in the AUC value as compared with DFD6 (E. coli) produced by E.
coli, which demonstrated an improvement of pharmacokinetic profile
due to glycosylation.
[0138] The A180E is a mutation disclosed in WO 2009/149171 owned by
Amgen Inc. When the mutation of A180E was further introduced into
the mutant DFD13 or DFD73 including the mutation of TGLEAV (SEQ ID
NO: 69) or G170N, respectively, the resulting mutant DFD18 or
DFD74, respectively, showed an approximate 2- to 3-fold additional
increase in AUC value.
[0139] In summary, it was confirmed that the pharmacokinetic
parameters were improved by the introduction of various mutations
and combinations thereof, as compared with DFD9, the wild-type
FGF21 fusion protein. The fusion protein showing the most improved
AUC value was DFD74 containing the mutations of EIRP (SEQ ID NO:
68), G170N and A180E, which showed an approximate 45-fold
improvement in AUC value as compared with DFD9. Furthermore,
considering RGE (Amgen) at the dose of 2 mg/kg of body weight,
DFD74 may have a higher degree of drug exposure as compared with
RGE. The overall effects of improvement in pharmacokinetics due to
the mutations are summarized in Table 6 below.
TABLE-US-00006 [TABLE 6] Control Material Position vs Assessment of
Mutation of improved pharmacokinetic sequence mutation material
parameters EIRP 98-101 DFD4 vs Improvement (SEQ ID DFD13 of AUC NO:
68) DFD6 vs DFD73 TGLEAV 170-174 DFD9 vs Improvement (SEQ ID DFD4
of AUC NO: 69) TGLEAN 170-174 DFD9 vs Improvement (SEQ ID DFD5 of
AUC NO: 70) G170N 170 DFD9 vs Improvement DFD6 of AUC DFD6
Improvement (E. coli) of AUC vs DFD6 G174N 174 DFD9 vs Improvement
DFD7 of AUC AI80E 180 DFD13 vs Improvement DFD18 of AUC DFD73 vs
Improvement DFD74 of AUC
Experimental Example 4. Activity Evaluation of Fusion Proteins in
Ob/Ob Mice
Experimental Example 4-1. Experimental Method for Evaluating
Activity in Ob/Ob Mice
[0140] The ob/ob mice, characterized as exhibiting hyperglycemia,
insulin resistance, hyperphagia, fatty liver and obesity due to a
genetic deficiency in leptin, are widely used for the study of type
2 diabetes. Male ob/ob mice (Harlan, USA) were purchased from
Raonbio (Korea). These mice were 5 to 6 weeks old at the time of
arrival, and 8 to 9 weeks old at the time of drug treatment after 3
weeks of adaptation. The mice were partitioned into groups
(n=8/group) in order to have similar mean values for body weight
and caudal blood glucose levels one day before the drug treatment
(Day 0), and the samples were subcutaneously administered once
according to each of their respective dosages. Dulbecco's phosphate
buffered saline (DPBS, Gibco, USA) was administered as the vehicle
treatment, and the glucose concentration in the blood was measured
using a glucose meter, GLUCODR.TM. (All Medicus, Korea). The
non-fasting glucose levels and body weights were measured every day
until the 14.sup.th day after administration. Glycated hemoglobin
levels were also measured in each group before the administration
and after the test. The glycated hemoglobin levels were calculated
using a DCA.TM. 2000 HbA1c kit (Siemens, 5035C).
Experimental Example 4-2. Evaluation of Activity in Ob/Ob Mice
[0141] The changes in non-fasting blood glucose levels and body
weights in male ob/ob mice were observed after single subcutaneous
injection of 30 or 100 nmol/kg of DFD18 and DFD72, or 10, 30 or 100
nmol/kg of DFD74.
[0142] It was confirmed that DFD18, DFD72 and DFD74 all had the
effect of lowering blood glucose level in a dose-dependent manner.
Comparing the three agents at the high dose of 100 nmol/kg, DFD72
and DFD74 showed an improved effect on lowering blood glucose level
than DFD18 (FIG. 6). In addition, Fc-FGF21 (Lilly) which was used
as a control material in the test, was less effective in lowing
blood glucose level as compared with DFD18, DFD72 and DFD74 at the
same dose level (30 nmol/kg).
[0143] As for the effect on body weight reduction, comparing the
three agents at the high dose of 100 nmol/kg, DFD72 was the most
effective in ob/ob mice resulting in an approximate 6% reduction in
body weight, and DFD18 was the next most effective, followed by
DFD74 (FIG. 7).
[0144] After the termination of the test, the glycated hemoglobin
levels indicative of the mean values of blood glucose were measured
and the changes in mean blood glucose were analyzed in each test
group. All of the treated groups except the control group treated
with control protein Fc-FGF21 (Lilly) showed negative values in the
differences between before administration and after the test, which
confirmed the effectiveness of the test proteins as compared with
the control material in lowering blood glucose (FIG. 8).
Experimental Example 5. Activity Evaluation of Fusion Proteins in
HFD/STZ Mice
Experimental Example 5-1. Experimental Method for Evaluating
Activity in HFD/STZ Mice
[0145] The effects of the FGF21 mutant fusion proteins on lowering
blood glucose and body weight were compared and evaluated in
another diabetic model, the HFD/STZ mouse model. Conventional
dietary-induced obesity mouse models (induced by feeding 60 kcal %
high fat diet to C57BL/6 mice for eight weeks or longer) have weak
hyperglycemic and diabetic features, although they invoke insulin
resistance. The HFD/STZ mice, which may compensate for defects in
the conventional dietary-induced obesity mouse models, are capable
of producing dysfunctional .beta. cells in the pancreas and
decreased secretion of insulin as a result of a high fat diet (HFD)
and administration of low level streptozotocin (STZ), and are
therefore useful for pharmacological studies of type 2
diabetes.
[0146] Specifically, in order to induce the HFD/STZ mouse model,
C57BL/6 mice (Japan SLC) were fed on a 60 kcal % high fat diet for
four weeks, and then 50 mg/kg of STZ (Sigma, 85882) was
administered intraperitoneally every day for 3 days to induce
dysfunction in the 0 cells of the pancreas. After feeding on the
high fat diet for an additional 2 weeks, the mice with non-fasting
blood glucose levels of 200 mg/dL or higher were used for the test.
The mice were partitioned into groups (n=6/group) in order to have
similar mean values of body weight and caudal blood glucose levels
one day before the drug treatment (Day 0), and the samples were
subcutaneously administered once according to each of their
respective dosages. Dulbecco's phosphate buffered saline (DPBS,
Gibco, USA) was administered as the vehicle treatment, and the
glucose concentration in the blood was measured using a glucose
meter, GLUCODR.TM. (All Medicus, Korea). The non-fasting glucose
levels and body weights were measured every day until the 14.sup.th
day after administration. Glycated hemoglobin levels were also
measured in each group before the administration and after the
test. The glycated hemoglobin levels were calculated using a
DCA.TM. 2000 HbA1c kit (Siemens, 5035C).
Experimental Example 5-2. Activity Evaluation in HFD/STZ Mice
[0147] The changes in non-fasting blood glucose levels and body
weights over time in male HFD/STZ mice were observed after single
subcutaneous injection of 10 nmol/kg of DFD72 or DFD74.
[0148] Regarding the changes in non-fasting blood glucose levels,
it was confirmed that DFD72 and DFD74 had similar effects on
lowering blood glucose levels, and the blood glucose lowering
effect was maintained until the 10.sup.th day after administration
and then lost with metabolism of the drugs after the 10.sup.th day
(FIG. 9). DFD72 showed a more prolonged effect than DFD74 in terms
of changes in non-fasting blood glucose levels after the 10.sup.th
day after administration.
[0149] In terms of the effect on body weight reduction due to the
administration of FGF21 mutant proteins, it was confirmed that both
DFD72 and DFD74 had similar effects on reducing body weight by
approximately 5%, and the effect disappeared after the 10.sup.th
day after administration (FIG. 10).
[0150] After the termination of the test, the glycated hemoglobin
levels indicative of the mean value of blood glucose were measured
and the changes in mean blood glucose were analyzed in each test
group. While the vehicle group had an increase of 0.25 in glycated
hemoglobin levels, the group treated with DFD74 had an increase of
0.1 and the group treated with DFD72 had an decrease of 0.27 (FIG.
11).
Experimental Example 6. Activity of Fusion Proteins in Diet-Induced
Obese Mice
Experimental Example 6-1. Experimental Method for Evaluating
Activities in Diet-Induced Obese Mice
[0151] The body weight-reduction effect of DFD18, an FGF21 mutant
fusion protein, was evaluated in diet-induced obese mice. For the
diet-induced obesity model, C57BL/6J mice were purchased from
Central Lab. Animal Inc. and fed on a high-fat diet containing 60
kcal % fat (Research diet) for 8 to 12 weeks. The mice were
partitioned into groups (n=8/group) in order to have a similar mean
value of body weight one day before the drug treatment (Day 0), and
then 30 nmol/kg of samples were subcutaneously administered once.
The changes in body weights were compared with the group treated
with vehicle (PBS).
Experimental Example 6-2. Protein Activity in Diet-Induced Obese
Mice
[0152] For changes in body weight over time in the diet-induced
obesity mouse model following single administration of 30 nmol/kg
DFD18, it was confirmed that the weight-reducing effect was
continuing by the 10.sup.th day after the administration, and the
maximum weight reduction (about 18%) was at the 1.sup.th day after
the administration, which was maintained by the 14.sup.th day (FIG.
12).
Preparation Example 2. Preparation and Purification of Dual
Function Proteins
Preparation Example 2-1. Preparation of Expression Vectors for
Expression of Dual Function Proteins
[0153] In order to identify the effects of the sequence of the
GLP-1 mutant protein and the sequence of the Fc hinge fused thereto
on the in vitro activity, pharmacokinetic profiles and
pharmacological efficacy, various sequences for the Fc-fused GLP-1
mutant proteins were designed. The sequences of the GLP-1 mutant
proteins are listed in Table 7 below, and the sequences of Fc-fused
GLP-1 mutants are listed in Table 8.
TABLE-US-00007 TABLE 7 SEQ ID NO: Sequence of GLP-1 mutant protein
43 GLP-1(A2G) 44 GLP-1(GE) 45 GLP-1(GG) 46 GLP-1(GEG)
TABLE-US-00008 TABLE 8 SEQ ID NO: Fc-fused GLP-1 mutant protein 49
DFD52: GLP1(A2G)-HyFc5 50 DFD53: GLP1(A2G)-HyFc40 51 DFD54:
GLP1(GE)-HyFc5 52 DFD55: GLP1(GE)-HyFc40 53 DFD56: GLP1(GG)-HyFc5
54 DFD57: GLP1(GG)-HyFc40 55 DFD58: GLP1(GEG)-HyFc5 56 DFD59:
GLP1(GEG)-HyFc40
[0154] In Table 8, HyFc5 refers to SEQ ID NO: 47, and HyFc40 refers
to SEQ ID NO: 48.
[0155] In order to investigate the effects of the sequences of the
GLP-1 mutant proteins and FGF21 mutant proteins, the sequence of
the Fc hinge fused to the GLP-1 mutants, the sequence of the linker
connected between the FGF21 mutant proteins and Fc on the in vitro
activity, pharmacokinetic profiles and pharmacological efficacy,
various sequences for the dual function proteins were designed. The
sequences of the dual function proteins including the GLP-1 mutant
proteins and FGF21 mutant proteins are listed in Table 9 below.
Each dual function protein contains a GLP-1 mutant protein, an Fc
region of an immunoglobulin, a linker and an FGF21 mutant protein
connected in this order from the N-terminus to C-terminus.
TABLE-US-00009 [TABLE 9] Sequence of Changes SEQ GLP-1 In ID
Material mutant Fusion Linker FGF21 NO: code protein carrier
sequence sequence 58 DFD23 GLP-1 hyFc40 GS3 FGF21 (A2G) (SEQ ID
(SEQ ID (EIRP NO: NO: 4) (SEQ ID 48) NO: 68), TGLEAV (SEQ ID NO:
69)) 59 DFD24 GLP-1 hyFc5 GS3 FGF21 (GE) (SEQ ID (SEQ ID (EIRP NO:
47) NO: 4) (SEQ ID NO: 68), TGLEAV (SEQ ID NO: 69)) 60 DFD25 GLP-1
hyFc40 GS3 FGF21 (GE) (SEQ ID (SEQ ID (EIRP NO: 48) NO: 4) (SEQ ID
NO: 68), TGLEAV (SEQ ID NO: 69)) 61 DFD26 GLP-1 hyFc5 GS3 FGF21
(GG) (SEQ ID (SEQ ID (EIRP NO: 47) NO: 4) (SEQ ID NO: 68), TGLEAV
(SEQ ID NO: 69)) 62 DFD27 GLP-1 hyFc40 GS3 FGF21 (GG) (SEQ ID (SEQ
ID (EIRP NO: 48) NO: 4) (SEQ ID NO: 68), TGLEAV (SEQ ID NO: 69)) 63
DFD28 GLP-1 hyFc5 GS3 FGF21 (GEG) (SEQ ID (SEQ ID (EIRP NO: 47) NO:
4) (SEQ ID NO: 68), TGLEAV (SEQ ID NO: 69)) 64 DFD29 GLP-1 hyFc40
GS3 FGF21 (GEG) (SEQ ID (SEQ ID (EIRP, NO: 48) NO: 4) TGLEAV) 65
DFD69 GLP-1 hyFc40 GS3 FGF21 (GEG) (SEQ ID (SEQ ID (EIRP NO: 48)
NO: 4) (SEQ ID NO: 68), TGLEAV (SEQ ID NO: 69)) AI80E) 66 DFD112
GLP-1 HyFc40 GS3 FGF21 (GEG) (SEQ ID (SEQ ID (EIRP NO: 48) NO: 4)
(SEQ ID NO: 68, TGLEAN (SEQ ID NO: 70), A180E) 67 DFD114 GLP-1
hyFc40 GS3 FGF2I (GEG) (SEQ ID (SEQ ID (EIRP NO: 48) NO: 4) (SEQ ID
NO: 68), G170N, A180E)
[0156] Specifically, the nucleotide sequences encoding each of the
dual function proteins were synthesized after consulting with
Bioneer Corporation (Korea) based on the amino acid sequence of
each protein. NheI and NotI restriction enzyme sequences were added
to the 5' terminus and 3' terminus of the nucleotide sequences
encoding each of the dual function proteins and an initiation codon
for protein translation and a leader sequence
(MDANLRGLCCVLLLCGAVFVSPSHA) (SEQ ID NO: 83) enabling secretion of
the expressed protein to the outside of a cell were inserted next
to the restriction enzyme sequence at the 5' terminus. A
termination codon was inserted next to the nucleotide sequence,
which encodes each of the dual function proteins. The nucleotide
sequence encoding each of the dual function proteins was cloned
into a pTrans-empty expression vector by using the two restriction
enzymes NheI and Noll. The pTrans-empty expression vector, which
has a simple structure including a CMV promoter, a pUC-derived
replication origin, an SV40-derived replication origin and an
ampicillin-resistance gene, was purchased from CEVEC
Pharmaceuticals (Germany).
Preparation Example 2-2. Construction of Plasmid DNA for Expression
of Fc-Fused GLP-1 Mutant and Dual Function Proteins
[0157] E. coli was transformed with each of the expression vectors
constructed in Preparation Example 2-1 to obtain a large quantity
of plasmid DNA to be used for expression. E. coli cells, with cell
walls weakened through heat shock, were transformed with each
expression vector, and the transformants were plated out on an LB
plate to obtain colonies. The colonies thus obtained were
inoculated into LB media, cultured at 37.degree. C. for 16 hours,
and each E. coli culture containing each expression vector was
obtained in a volume of 100 mL. The E. coli thereafter obtained was
centrifuged to remove the culture medium, and then P1, P2, P3
solutions (QIAGEN, Cat No.:12963) were added to break the cell
walls, thereby obtaining a DNA suspension in which proteins and DNA
were separated. Plasmid DNA was purified from the DNA suspension
thus obtained by using a QIAGEN.TM. DNA purification column. The
eluted plasmid DNA was identified by agarose gel electrophoresis,
and the concentrations and purities were measured using a
NANODROP.TM. device (Thermo Scientific, NANODROP.TM. Lite). The DNA
thus obtained was used for expression.
Preparation Example 2-3. Expression of Fc-Fused GLP-1 Mutants and
Dual Function Proteins in CAP-T Cells
[0158] Human cell lines were transformed with each plasmid DNA
obtained in Preparation Example 2-2. Each plasmid DNA type was
transduced into CAP-T cells (CEVEC), which had been cultured in PEM
medium (Life Technologies), by using a PEI solution (Polyplus, Cat.
No.:101-10N). The mixed solution of DNA and the PEI solution was
mixed with the cell suspension using FREESTYLE.TM. 293 expression
medium (Invitrogen), cultured at 37.degree. C. for 5 hours, and PEM
medium was added. After culturing at 37.degree. C. for 5-7 days,
the culture was centrifuged to remove cells and supernatant
containing each protein was obtained.
Preparation Example 2-4. Purification of Fc-Fused GLP-1 Mutants and
Dual Function Proteins
[0159] Protein A affinity chromatography column (GE Healthcare) was
equilibrated with 1.times.PBS buffer solution (pH 7.4). The culture
supernatant including each of the Fc-fused GLP-1 mutants and dual
function proteins obtained in Preparation Example 2-3 was filtered
with a 0.2 .mu.m filter, and then loaded into a Protein A affinity
chromatography column. The column was washed with 1.times.PBS
buffer solution (pH 7.4) and then the proteins were eluted using
100 mM glycine buffer solution (pH 3.0). The proteins obtained by
affinity chromatography were purified using an anion exchange resin
column (POROS.RTM. HQ 50 .mu.m, Thermo Fisher Scientific). The
anion exchange resin column was equilibrated with 50 mM Tris buffer
solution (pH 8.0), before the proteins eluted from the affinity
chromatography were loaded thereto.
[0160] After washing the column with 50 mM Tris buffer solution (pH
8.0), 50 mM Tris buffer solution (pH 8.0) was dispensed along the
concentration gradient and the eluted fractions were analyzed. Each
eluted fraction was analyzed by using size exclusion chromatography
(SEC-HPLC), and the fractions including the Fc-fused GLP-1 mutants
and dual function proteins with high purity were collected and
dialyzed overnight at 4.degree. C. using a final buffer solution
(1.times.PBS, 1 mM EDTA, pH 7.4). Upon completion of the dialysis,
the obtained protein stock solution was concentrated at 3,000 rpm
using a 30,000 MW cut-off centrifugation filter at 4.degree. C. The
concentration of each protein was measured via BCA quantitative
analysis.
Experimental Example 7. In Vitro Activity of Dual Function
Proteins
Experimental Example 7-1. Activity of DFD23, DFD24, DFD25, DFD26,
DFD27, DFD28 and DFD29
[0161] The in vitro GLP-1 activities of the dual function proteins
DFD23, DFD24, DFD25, DFD26, DFD27, DFD28 and DFD29 were measured.
Specifically, a CHO cell line (Eurofins, HTS163C2), overexpressing
the human GLP-1 receptor was purchased and used to evaluate the
GLP-1 activities of the dual function proteins. For the evaluation
of activity, samples containing the fusion proteins (protein stock
solutions prepared in Preparation Example 2-4, hereinafter,
"sample") were subjected to a 4-fold serial dilution at a
concentration of 25 nM. After the human GLP-1
receptor-overexpressing CHO cell line was treated for 30 minutes,
the intracellular cAMP produced was measured (Cisbio, 62AM4PEB).
The activity of each protein was evaluated by comparing the
EC.sub.50 values.
[0162] As shown in FIG. 13, the dual function protein containing
the GLP-1 (A2G) sequence showed activity approximately 2-3 times
lower than that for the dual function proteins containing other
GLP-1 mutant sequences. No significant difference in GLP-1
activities was observed between the dual function proteins
containing the mutation sequences except the GLP-1 (A2G)
sequence.
Experimental Example 7-2. Activities of DFD59, DFD69, DFD112 and
DFD114
[0163] The in vitro GLP-1 activities of the dual function proteins
DFD69, DFD112 and DFD114 prepared in Preparation Example 2 and
DFD59 (an Fc-fused GLP-1 mutant) were measured. Specifically, a CHO
cell line (Eurofins, HTS163C2) overexpressing the human GLP-1
receptor was purchased and used to evaluate the GLP-1 activities of
the dual function proteins. For the evaluation of activity, the
sample containing each of the fusion proteins was subjected to a
4-fold serial dilution at a concentration of 25 nM. After the human
GLP-1 receptor-overexpressing CHO cell line was treated for 30
minutes, the intracellular cAMP produced was measured (Cisbio,
62AM4PEB).
[0164] As shown in FIG. 14, the activity of each protein was
evaluated by comparing the EC.sub.50 value. The three dual function
proteins showed similar EC.sub.50 values, and DFD59 (containing no
FGF21 mutant) showed activity approximately 2 times higher than
that of the dual function proteins.
[0165] Next, the in vitro activities of the FGF21 portion in DFD69,
DFD112 and DFD114 were measured. Specifically, the in vitro
activities of the FGF21 portion in the dual function proteins were
evaluated using a HEK293 cell line overexpressing human
.beta.-klotho (a co-receptor of FGF21). For the evaluation of
activity, samples containing each of the dual function proteins
were subjected to a 3-fold serial dilution at a concentration of 3
.mu.M. After having been cultured in a serum-deficient state for 5
hours, the human .beta.-klotho-overexpressing HEK293 cell line was
treated for 20 minutes, before the cells were lysed by adding
cytolysis buffer (Cisbio/Cat #64ERKPEG) with stirring at 60 rpm for
30 minutes at room temperature. The cell lysate solution was mixed
with antibodies which can detect ERK and phosphorylated ERK, and
the mixture was maintained at room temperature for 2 hours.
Fluorescence was detected using a fluorometric detector
(TECAN/GENiosPro). The activities were measured by comparing their
EC.sub.50 values.
[0166] It was confirmed that the in vitro activities of the FGF21
portion of the dual function proteins DFD69, DFD112 and DFD114 were
similar, as shown in FIG. 14.
Experimental Example 8. Pharmacokinetic Assessment of Dual Function
Proteins
Experimental Example 8-1. Experimental Method for Pharmacokinetic
Assessment
[0167] Six-week old male ICR mice purchased from Orient BIO (Korea)
were partitioned into groups (n=3/blood sampling time) in order to
have a similar mean value of body weight one day before drug
treatment, and subcutaneously administered once with a respective
sample in a volume of 1 mg/kg. The blood samples were collected at
1, 4, 8, 12, 24, 48, 72, 96, 144, 192 and 240 hours after the
injection, respectively. The concentration of each dual function
protein in the blood was measured based on the FGF21 portion and
the GLP-1-Fc portion separately. The concentration of the intact
full length FGF21 portion of the dual function protein in the blood
was measured using an Intact human FGF21 ELISA Kit (F1231-K01,
Eagle Biosciences, USA), which has immunoreactivity to the
N-terminus and C-terminus of FGF21 protein. Further, the
concentration of the active GLP-1-Fc portion of the dual function
protein in the blood was measured using an antibody, which has
immunoreactivity to the N-terminus of GLP-1 and Fc, as determined
through ELISA analysis. The concentrations of the FGF21 and
GLP-1-Fc portions of each protein in the blood samples collected
until 240 hours after single subcutaneous injection of each protein
into the mice were measured, and the pharmacokinetic parameters of
each protein was calculated.
Experimental Example 8-2. Pharmacokinetic Activity Results
[0168] Based on the concentration of each active substance in the
blood over time after single subcutaneous administration of each
protein in mice (FIG. 15), pharmacokinetic parameters for the FGF21
and GLP-1-Fc portions of the dual function proteins were
calculated. The data are shown in Table 10 below.
TABLE-US-00010 TABLE 10 FGF21 detection GLP-1-Fc detection
Parameter DFD69 DFD112 DFD114 DFD59 DFD69 DFD112 DFD114 T.sub.max
(hour) 8 8 24 4 4 8 4 C.sub.max (ng/mL) 2715 3619 3711 5202.1 3234
4454 3616 AUC.sub.last 100907 144395 222504 182852 149083 189338
171687 (ng hr/mL) Half-life (hour) 13.4 14.2 39.9 20.7 23.3 24.7
27.2
[0169] The pharmacokinetic profiles of each dual function protein
were compared and evaluated based on the value of the area under
the curve (AUC), indicating the degree of drug exposure.
[0170] As shown in Table 10, for the pharmacokinetic parameters of
the FGF21 portion, DFD114 showed the highest degree of drug
exposure (AUC) and half-life, and DFD112 showed the next highest
AUC value, followed by DFD69. DFD114 exhibited an approximate
2-fold or higher increase in AUC value as compared with DFD69. For
the pharmacokinetics of the GLP-1-Fc portion, the four proteins
(DFD59, DFD69, DFD112 and DFD114) containing the same GLP-1 mutant
sequence showed similar AUC values.
Experimental Example 9. Activity Evaluation in Db/Db Mice
Experimental Example 9-1. Method for Evaluating Activities in Db/Db
Mice
[0171] The db/db mice, characterized as having hyperglycemia,
insulin resistance, hyperphagia, fatty liver and obesity due to a
genetic deficiency for the leptin receptor and exhibiting more
serious hyperglycemia and obesity than ob/ob mice, are widely used
for the study of type 2 diabetes. Male db/db mice (Harlan, USA)
were purchased from Raonbio (Korea). These mice were 5 to 6 weeks
old at the time of arrival, and 8 to 9 weeks old at the time of
drug treatment, after 3 weeks of adaptation. The mice were
partitioned into groups (n=6/group) in order to have a similar mean
value of body weight and caudal blood glucose levels one day before
the drug treatment (Day 0), and the samples were subcutaneously
administered once according to each of their respective dosages.
Dulbecco's phosphate buffered saline (DPBS, Gibco, USA) was
administered as the vehicle treatment, and the glucose
concentration in the blood was measured using a glucose meter,
GLUCODR.TM. (All Medicus, Korea). The non-fasting glucose levels
and body weights were measured every day until the 14.sup.th day
after administration. Glycated hemoglobin levels were also measured
in each group before the administration and after the test. The
glycated hemoglobin levels were calculated using a DCA.TM. 2000
HbA1c kit (Siemens, 5035C).
Experimental Example 9-2. Evaluation of Activity in Db/Db Mice
[0172] The changes in non-fasting blood glucose levels and body
weights in male db/db mice were observed after single subcutaneous
injection of 10 or 30 nmol/kg of dual function protein DFD114,
single subcutaneous injection of 30 nmol/kg of long-acting GLP-1-Fc
single function protein DFD59, and combined administration of 30
nmol/kg of DFD59 and DFD74 (which are GLP-1-Fc and Fc-FGF21 single
function proteins, respectively) to compare the effect of the dual
function protein DFD114 with combined administration of Fc-FGF21
and GLP-1-Fc single function proteins.
[0173] The long-acting GLP-1-Fc protein DFD59 caused a sharp
reduction in blood glucose levels by the 1.sup.st day after
administration, but the reduction in blood glucose decreased after
the 2.sup.nd day and the blood glucose level was similar to that of
the vehicle-treated group after the 4.sup.th day. Meanwhile, the
group treated with DFD114 showed excellent effects on blood glucose
reduction by the 3.sup.rd day after administration, and the effects
on lowering blood glucose level disappeared more rapidly after the
40 day from the administration at the dose of 10 nmol/kg than for
30 nmol/kg, indicating dose-dependent differences in the duration
of the blood glucose lowering effect. The groups treated with
combined administration of each protein showed the most sustained
effects for lowering blood glucose levels as compared with those of
the other groups, indicating that the combination of GLP-1 and
FGF21 had an excellent effect on controlling blood glucose level
(FIG. 16).
[0174] As for the effect on body weight reduction, the groups
treated with a combination of DFD59 and DFD74 showed the greatest
effects on reducing body weight, and the group treated with 30
nmol/kg of DFD114 also showed an outstanding effect on reducing
body weight (FIG. 17).
[0175] After the termination of the tests, the glycated hemoglobin
levels indicative of the mean value of blood glucose were measured
and the changes in mean blood glucose were analyzed in each test
group. As shown in FIG. 18, the group treated with vehicle showed
increased glycated hemoglobin levels after the termination of the
tests as compared with the group before the administration, and the
group treated with DFD59 showed a similar increase. The group
treated with 30 nmol/kg of DFD114 showed the greatest decrease in
glycated hemoglobin levels, and the group receiving combined
administration showed the next highest effectiveness, followed by
the group treated with 10 nmol/kg of DFD114. When evaluating the
proteins by comparing them based on the decrease in glycated
hemoglobin levels in each group treated, it was confirmed that the
dual function protein DFD114 showed a stronger effect on lowering
blood glucose level than GLP-1-Fc or Fc-FGF21 single function
protein alone.
Experimental Example 10. Activity of Fusion Proteins in HFD/STZ
Mice
Experimental Example 10-1. Experimental Method for Evaluating
Activities in HFD/STZ Mice
[0176] The effects of the dual function proteins on lowering blood
glucose and body weight were compared and evaluated in another
diabetic model, the HFD/STZ mouse model.
[0177] The conventional dietary-induced obesity mouse model
(induced by feeding 60 kcal % high fat diet to C57BL/6 mice for
eight weeks or longer) has weak hyperglycemic and diabetic
features, although invokes insulin resistance. The HFD/STZ mice,
which may compensate for the deficiencies of the conventional
dietary-induced obesity mouse model, are capable of generating
dysfunctional .beta. cells of the pancreas and decreased secretion
of insulin following a high fat diet (HFD) and administration of
low level streptozotocin (STZ), and are used for pharmacological
studies of type 2 diabetes. In order to induce the HFD/STZ mouse
model, C57BL/6 mice were fed on a 60 kcal % high fat diet for four
weeks, and then 50 mg/kg of STZ (Sigma, 85882) was administered
intraperitoneally every day for 3 days to induce dysfunction of the
.beta. cells of the pancreas. After feeding on the high fat diet
for an additional 2 weeks, the mice with non-fasting blood glucose
levels of 200 mg/dL or higher were selected for the test. The mice
were partitioned into groups (n=6/group) in order to have a similar
mean value of body weight and caudal blood glucose levels one day
before the drug treatment (Day 0), and the samples were
subcutaneously administered once according to each of their
respective dosages. Dulbecco's phosphate buffered saline (DPBS,
Gibco, USA) was administered as the vehicle treatment, and the
glucose concentration in the blood was measured using a glucose
meter, GLUCODR.TM. (All Medicus, Korea). The non-fasting glucose
levels and body weights were measured every day until the 14.sup.th
day after administration. Glycated hemoglobin levels were also
measured in each group before the administration and after the
test. The glycated hemoglobin levels were calculated using a
DCA.TM. 2000 HbA1c kit (Siemens, 5035C).
Experimental Example 10-2. Activity in HFD/STZ Mice
[0178] The changes in non-fasting blood glucose levels and body
weights over time in male HFD/STZ mice were observed after single
subcutaneous injection of 3 nmol/kg or nmol/kg of dual function
protein DFD114, 10 nmol/kg of Fc-fused GLP-1 mutant DFD59, or 10
nmol/kg of each of the Fc-fused FGF21 mutants DFD72 and DFD74.
DFD59 and DFD74 were also subcutaneously injected once at 10
nmol/kg each in order to compare the effect of combined
administration of the single function proteins with that of the
dual function protein.
[0179] As shown in FIG. 19, regarding the changes in blood glucose
levels until the 4.sup.th day, DFD72 and DFD74 (long-acting FGF21
single function proteins) administration resulted in slower
reductions of blood glucose, while DFD114 (long-acting protein
including GLP-1), DFD59 and combined administration of DFD59 and
DFD74 showed a more rapid reduction of blood glucose from the 1a
day of administration. Similar to the results in db/db mice, DFD59
showed a sharp reduction in blood glucose at an early stage, but
the reduction of blood glucose disappeared slowly after the
4.sup.th day. DFD114 showed a similar pattern at the low dose of 3
nmol/kg. In the groups treated with 10 nmol/kg of DFD114, DFD72,
DFD74 and combined administration, similar non-fasting blood
glucose profiles were observed.
[0180] As for the effect on body weight reduction, the group
treated with combined administration of DFD59 and DFD74 showed the
greatest effect on body weight reduction (7 to 8%), and the group
treated with 10 nmol/kg of DFD114 also showed an outstanding effect
on reducing body weight (approximately 6%) (FIG. 20). The group
treated with DFD59 exhibited a reduction in body weight by 5% at
the 1.sup.st day after administration, but the effect disappeared
after the 2.sup.nd day and became similar to that of the vehicle
group after the 7.sup.th day. The group treated with each of the
long-acting FGF21 single function proteins DFD72 and DFD74 showed a
slower reduction in body weight by 4 to 5% until the 7.sup.th day
after the administration, and the effect disappeared after the
10.sup.th day.
[0181] After the termination of the tests, the glycated hemoglobin
levels indicative of the mean value of blood glucose were measured
and the changes in mean blood glucose were analyzed in each test
group (FIG. 21). The vehicle group had an increase in glycated
hemoglobin levels after the termination of the test as compared
with before administration, and the group treated with DFD59 showed
a similar increase. In contrast, the group treated with DFD114
showed reductions in glycated hemoglobin levels in a dose-dependent
manner, and the group treated with 10 nmol/kg of DFD114 had the
greatest effect in terms of reduced glycated hemoglobin levels
(-0.42%). The group treated with combined administration of DFD59
and DFD74 showed reduced glycated hemoglobin levels (-0.38%)
similar to that of DFD114. For the long-acting FGF21 single
function proteins, it was observed that DFD72 was superior to
DFD74. Comparing the proteins based on the reduced levels of
glycated hemoglobin in each group, it was confirmed that the dual
function protein DFD114 was superior to both GLP-1-Fc and Fc-FGF21
single function proteins.
Experimental Example 11. Prediction and Evaluation of
Immunogenicity
Experimental Example 11-1. Prediction Method for Immunogenicity and
Results
[0182] In order to predict the potential immunogenicity of dual
function proteins, in silico analysis of immunogenicity was
performed for each protein.
[0183] Specifically, the potential immunogenicity of dual function
proteins was rapidly screened by using ITOPE.TM. and TCED.TM.
methods (Prediction of immunogenicity of therapeutic proteins:
validity of computational tools, BioDrugs, 2010). According to the
two methods, the T-cell epitope may be more accurately predicted as
compared with the in silico analytical method which depends on MHC
class II binding analysis only.
Experimental Example 11-2. Ex Vivo Evaluation Method for
Immunogenicity and Results
[0184] In order to evaluate the potential immunogenicity of dual
function proteins, EPISCREEN.TM. analysis (Increased brain
bio-distribution and chemical stability and decreased
immunogenicity of an engineered variant of GDNF, Exp Neurol, 2015)
was performed. When immunogenicity is detected, the amino acid
sequences inducing immunogenicity may be identified through T-cell
epitope mapping, and deimmunized mutants with minimized
immunogenicity may be designed and prepared via in silico
prediction to reevaluate immunogenicity.
Sequence CWU 1
1
831181PRTHomo sapienshuman FGF21 1His Pro Ile Pro Asp Ser Ser Pro
Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg Tyr Leu Tyr Thr
Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30Leu Glu Ile Arg Glu Asp
Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro Glu Ser Leu Leu
Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55 60Ile Leu Gly Val
Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly65 70 75 80Ala Leu
Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95Glu
Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105
110Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro
115 120 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu
Pro Pro 130 135 140Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln
Pro Pro Asp Val145 150 155 160Gly Ser Ser Asp Pro Leu Ser Met Val
Gly Pro Ser Gln Gly Arg Ser 165 170 175Pro Ser Tyr Ala Ser
180230PRTArtificial Sequencelinker 2Ala Lys Ala Thr Thr Ala Pro Ala
Thr Thr Arg Asn Thr Gly Arg Gly1 5 10 15Gly Glu Glu Lys Lys Lys Glu
Lys Glu Lys Glu Glu Gln Glu 20 25 30317PRTArtificial Sequencelinker
3Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly1 5
10 15Gly415PRTArtificial Sequencelinker 4Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15516PRTArtificial
Sequencelinker 5Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Ala1 5 10 156181PRTArtificial SequenceFGF21 variant 6His
Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10
15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
20 25 30Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln
Ser 35 40 45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val
Ile Gln 50 55 60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg
Pro Asp Gly65 70 75 80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu
Ala Cys Ser Phe Arg 85 90 95Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val
Tyr Gln Ser Glu Ala His 100 105 110Gly Leu Pro Leu His Leu Pro Gly
Asn Lys Ser Pro His Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro Ala
Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro Glu
Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val145 150 155 160Gly
Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser 165 170
175Pro Ser Tyr Ala Ser 1807182PRTArtificial SequenceFGF21 variant
7His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5
10 15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala
His 20 25 30Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp
Gln Ser 35 40 45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly
Val Ile Gln 50 55 60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln
Arg Pro Asp Gly65 70 75 80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro
Glu Ala Cys Ser Phe Arg 85 90 95Glu Leu Leu Leu Glu Asp Gly Tyr Asn
Val Tyr Gln Ser Glu Ala His 100 105 110Gly Leu Pro Leu His Leu Pro
Gly Asn Lys Ser Pro His Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro
Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro
Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val145 150 155
160Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Val Arg
165 170 175Ser Pro Ser Tyr Ala Ser 1808182PRTArtificial
SequenceFGF21 variant 8His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln
Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala
Gln Gln Thr Glu Ala His 20 25 30Leu Glu Ile Arg Glu Asp Gly Thr Val
Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro Glu Ser Leu Leu Gln Leu Lys
Ala Leu Lys Pro Gly Val Ile Gln 50 55 60Ile Leu Gly Val Lys Thr Ser
Arg Phe Leu Cys Gln Arg Pro Asp Gly65 70 75 80Ala Leu Tyr Gly Ser
Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95Glu Leu Leu Leu
Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110Gly Leu
Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro 115 120
125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
130 135 140Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro
Asp Val145 150 155 160Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly
Leu Glu Ala Asn Arg 165 170 175Ser Pro Ser Tyr Ala Ser
1809181PRTArtificial SequenceFGF21 variant 9His Pro Ile Pro Asp Ser
Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg Tyr Leu
Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30Leu Glu Ile Arg
Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro Glu Ser
Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55 60Ile Leu
Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly65 70 75
80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg
85 90 95Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala
His 100 105 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His
Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu
Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro Glu Pro Pro Gly Ile Leu
Ala Pro Gln Pro Pro Asp Val145 150 155 160Gly Ser Ser Asp Pro Leu
Ser Met Val Asn Pro Ser Gln Gly Arg Ser 165 170 175Pro Ser Tyr Ala
Ser 18010181PRTArtificial SequenceFGF21 variant 10His Pro Ile Pro
Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg
Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30Leu Glu
Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro
Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55
60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly65
70 75 80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe
Arg 85 90 95Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu
Ala His 100 105 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro
His Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro
Leu Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro Glu Pro Pro Gly Ile
Leu Ala Pro Gln Pro Pro Asp Val145 150 155 160Gly Ser Ser Asp Pro
Leu Ser Met Val Gly Pro Ser Gln Asn Arg Ser 165 170 175Pro Ser Tyr
Ala Ser 18011182PRTArtificial SequenceFGF21 variant 11His Pro Ile
Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln
Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30Leu
Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40
45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
50 55 60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp
Gly65 70 75 80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys
Ser Phe Arg 85 90 95Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln
Ser Glu Ala His 100 105 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys
Ser Pro His Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro Ala Arg Phe
Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro Glu Pro Pro
Gly Ile Leu Ala Pro Gln Pro Pro Asp Val145 150 155 160Gly Ser Ser
Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Val Arg 165 170 175Ser
Pro Ser Tyr Ala Ser 18012182PRTArtificial SequenceFGF21 variant
12His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1
5 10 15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala
His 20 25 30Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp
Gln Ser 35 40 45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly
Val Ile Gln 50 55 60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln
Arg Pro Asp Gly65 70 75 80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro
Glu Ala Cys Ser Phe Arg 85 90 95Glu Glu Ile Arg Pro Asp Gly Tyr Asn
Val Tyr Gln Ser Glu Ala His 100 105 110Gly Leu Pro Leu His Leu Pro
Gly Asn Lys Ser Pro His Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro
Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro
Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val145 150 155
160Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Asn Arg
165 170 175Ser Pro Ser Tyr Ala Ser 18013181PRTArtificial
SequenceFGF21 variant 13His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln
Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala
Gln Gln Thr Glu Ala His 20 25 30Leu Glu Ile Arg Glu Asp Gly Thr Val
Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro Glu Ser Leu Leu Gln Leu Lys
Ala Leu Lys Pro Gly Val Ile Gln 50 55 60Ile Leu Gly Val Lys Thr Ser
Arg Phe Leu Cys Gln Arg Pro Asp Gly65 70 75 80Ala Leu Tyr Gly Ser
Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95Glu Glu Ile Arg
Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110Gly Leu
Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro 115 120
125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
130 135 140Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro
Asp Val145 150 155 160Gly Ser Ser Asp Pro Leu Ser Met Val Asn Pro
Ser Gln Gly Arg Ser 165 170 175Pro Ser Tyr Ala Ser
18014181PRTArtificial SequenceFGF21 variant 14His Pro Ile Pro Asp
Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg Tyr
Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30Leu Glu Ile
Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro Glu
Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55 60Ile
Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly65 70 75
80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg
85 90 95Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala
His 100 105 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His
Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu
Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro Glu Pro Pro Gly Ile Leu
Ala Pro Gln Pro Pro Asp Val145 150 155 160Gly Ser Ser Asp Pro Leu
Ser Met Val Gly Pro Ser Gln Asn Arg Ser 165 170 175Pro Ser Tyr Ala
Ser 18015181PRTArtificial SequenceFGF21 variant 15His Pro Ile Pro
Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg
Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30Leu Glu
Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro
Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55
60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly65
70 75 80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe
Arg 85 90 95Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu
Ala His 100 105 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro
His Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro
Leu Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro Glu Pro Pro Gly Ile
Leu Ala Pro Gln Pro Pro Asp Val145 150 155 160Gly Ser Ser Asp Pro
Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser 165 170 175Pro Ser Tyr
Glu Ser 18016182PRTArtificial SequenceFGF21 variant 16His Pro Ile
Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln
Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30Leu
Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40
45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
50 55 60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp
Gly65 70 75 80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys
Ser Phe Arg 85 90 95Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln
Ser Glu Ala His 100 105 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys
Ser Pro His Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro Ala Arg Phe
Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro Glu Pro Pro
Gly Ile Leu Ala Pro Gln Pro Pro Asp Val145 150 155 160Gly Ser Ser
Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Val Arg 165 170 175Ser
Pro Ser Tyr Glu Ser 18017182PRTArtificial SequenceFGF21 variant
17His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1
5 10 15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala
His 20 25 30Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp
Gln Ser 35 40 45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly
Val Ile Gln 50 55 60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln
Arg Pro Asp Gly65 70 75 80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro
Glu Ala Cys Ser Phe Arg 85 90 95Glu Leu Leu Leu Glu Asp Gly Tyr Asn
Val Tyr Gln Ser Glu Ala His 100 105 110Gly Leu Pro Leu His Leu Pro
Gly Asn Lys Ser Pro His Arg Asp Pro 115
120 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro
Pro 130 135 140Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro
Pro Asp Val145 150 155 160Gly Ser Ser Asp Pro Leu Ser Met Val Thr
Gly Leu Glu Ala Asn Arg 165 170 175Ser Pro Ser Tyr Glu Ser
18018181PRTArtificial SequenceFGF21 variant 18His Pro Ile Pro Asp
Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg Tyr
Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30Leu Glu Ile
Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro Glu
Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55 60Ile
Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly65 70 75
80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg
85 90 95Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala
His 100 105 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His
Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu
Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro Glu Pro Pro Gly Ile Leu
Ala Pro Gln Pro Pro Asp Val145 150 155 160Gly Ser Ser Asp Pro Leu
Ser Met Val Asn Pro Ser Gln Gly Arg Ser 165 170 175Pro Ser Tyr Glu
Ser 18019181PRTArtificial SequenceFGF21 variant 19His Pro Ile Pro
Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg
Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30Leu Glu
Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro
Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55
60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly65
70 75 80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe
Arg 85 90 95Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu
Ala His 100 105 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro
His Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro
Leu Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro Glu Pro Pro Gly Ile
Leu Ala Pro Gln Pro Pro Asp Val145 150 155 160Gly Ser Ser Asp Pro
Leu Ser Met Val Gly Pro Ser Gln Asn Arg Ser 165 170 175Pro Ser Tyr
Glu Ser 18020182PRTArtificial SequenceFGF21 variant 20His Pro Ile
Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln
Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30Leu
Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40
45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
50 55 60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp
Gly65 70 75 80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys
Ser Phe Arg 85 90 95Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln
Ser Glu Ala His 100 105 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys
Ser Pro His Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro Ala Arg Phe
Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro Glu Pro Pro
Gly Ile Leu Ala Pro Gln Pro Pro Asp Val145 150 155 160Gly Ser Ser
Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Val Arg 165 170 175Ser
Pro Ser Tyr Glu Ser 18021182PRTArtificial SequenceFGF21 variant
21His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1
5 10 15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala
His 20 25 30Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp
Gln Ser 35 40 45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly
Val Ile Gln 50 55 60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln
Arg Pro Asp Gly65 70 75 80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro
Glu Ala Cys Ser Phe Arg 85 90 95Glu Glu Ile Arg Pro Asp Gly Tyr Asn
Val Tyr Gln Ser Glu Ala His 100 105 110Gly Leu Pro Leu His Leu Pro
Gly Asn Lys Ser Pro His Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro
Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro
Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val145 150 155
160Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Asn Arg
165 170 175Ser Pro Ser Tyr Glu Ser 18022181PRTArtificial
SequenceFGF21 variant 22His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln
Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala
Gln Gln Thr Glu Ala His 20 25 30Leu Glu Ile Arg Glu Asp Gly Thr Val
Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro Glu Ser Leu Leu Gln Leu Lys
Ala Leu Lys Pro Gly Val Ile Gln 50 55 60Ile Leu Gly Val Lys Thr Ser
Arg Phe Leu Cys Gln Arg Pro Asp Gly65 70 75 80Ala Leu Tyr Gly Ser
Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95Glu Glu Ile Arg
Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110Gly Leu
Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro 115 120
125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
130 135 140Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro
Asp Val145 150 155 160Gly Ser Ser Asp Pro Leu Ser Met Val Asn Pro
Ser Gln Gly Arg Ser 165 170 175Pro Ser Tyr Glu Ser
18023181PRTArtificial SequenceFGF21 variant 23His Pro Ile Pro Asp
Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg Tyr
Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30Leu Glu Ile
Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro Glu
Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55 60Ile
Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly65 70 75
80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg
85 90 95Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala
His 100 105 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His
Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu
Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro Glu Pro Pro Gly Ile Leu
Ala Pro Gln Pro Pro Asp Val145 150 155 160Gly Ser Ser Asp Pro Leu
Ser Met Val Gly Pro Ser Gln Asn Arg Ser 165 170 175Pro Ser Tyr Glu
Ser 18024229PRTArtificial SequenceHuman IgG4 Fc 24Glu Ser Lys Tyr
Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe1 5 10 15Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45Ser
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55
60Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65
70 75 80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu 85 90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser 100 105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro 115 120 125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys Asn Gln 130 135 140Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala145 150 155 160Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190Thr
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200
205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220Leu Ser Leu Gly Lys22525228PRTArtificial SequenceHuman
IgG4 Fc variant 25Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
Ala Pro Glu Ala1 5 10 15Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val 50 55 60Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125Gln
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135
140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala145 150 155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser 195 200 205Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu
Gly22526223PRTArtificial SequenceHybrid Fc variant 26Glu Thr Lys
Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val1 5 10 15Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25 30Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 35 40
45Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
50 55 60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
Ser65 70 75 80Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys 85 90 95Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile 100 105 110Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 115 120 125Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu 130 135 140Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn145 150 155 160Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 165 170 175Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 180 185
190Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
195 200 205His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys 210 215 22027435PRTArtificial Sequencemodified FGF21 variant
connected to hybrid Fc 27Glu Thr Lys Thr Pro Glu Cys Pro Ser His
Thr Gln Pro Leu Gly Val1 5 10 15Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 20 25 30Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu 35 40 45Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 50 55 60Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser65 70 75 80Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 100 105 110Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120
125Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
130 135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn145 150 155 160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser 165 170 175Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys Ser Arg 180 185 190Trp Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu 195 200 205His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala 210 215 220Lys Ala Thr
Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly225 230 235
240Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu Gln Glu His Pro Ile
245 250 255Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg
Gln Arg 260 265 270Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala
His Leu Glu Ile 275 280 285Arg Glu Asp Gly Thr Val Gly Gly Ala Ala
Asp Gln Ser Pro Glu Ser 290 295 300Leu Leu Gln Leu Lys Ala Leu Lys
Pro Gly Val Ile Gln Ile Leu Gly305 310 315 320Val Lys Thr Ser Arg
Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr 325 330 335Gly Ser Leu
His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Glu Ile 340 345 350Arg
Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro 355 360
365Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg
370 375 380Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala
Leu Pro385 390 395 400Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro
Asp Val Gly Ser Ser 405 410 415Asp Pro Leu Ser Met Val Thr Gly Leu
Glu Ala Val Arg Ser Pro Ser 420 425 430Tyr Ala Ser
43528422PRTArtificial Sequencemodified FGF21 variant connected to
hybrid Fc 28Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu
Gly Val1 5 10 15Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr 20 25 30Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
Glu Asp Pro Glu 35 40 45Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 50 55 60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser65 70 75 80Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile 100 105 110Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120 125Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 130 135
140Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn145 150
155 160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser 165 170 175Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
Lys Ser Arg 180 185 190Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 195 200 205His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu Gly Lys Ala 210 215 220Lys Ala Thr Thr Ala Pro Ala
Thr Thr Arg Asn Thr Gly Arg Gly Gly225 230 235 240His Pro Ile Pro
Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 245 250 255Arg Gln
Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 260 265
270Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser
275 280 285Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val
Ile Gln 290 295 300Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln
Arg Pro Asp Gly305 310 315 320Ala Leu Tyr Gly Ser Leu His Phe Asp
Pro Glu Ala Cys Ser Phe Arg 325 330 335Glu Leu Leu Leu Glu Asp Gly
Tyr Asn Val Tyr Gln Ser Glu Ala His 340 345 350Gly Leu Pro Leu His
Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro 355 360 365Ala Pro Arg
Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 370 375 380Ala
Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val385 390
395 400Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Val
Arg 405 410 415Ser Pro Ser Tyr Ala Ser 42029420PRTArtificial
Sequencemodified FGF21 variant connected to hybrid Fc 29Glu Thr Lys
Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val1 5 10 15Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25 30Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 35 40
45Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
50 55 60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
Ser65 70 75 80Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys 85 90 95Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile 100 105 110Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 115 120 125Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu 130 135 140Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn145 150 155 160Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 165 170 175Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 180 185
190Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
195 200 205His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys Gly 210 215 220Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser His Pro225 230 235 240Ile Pro Asp Ser Ser Pro Leu Leu Gln
Phe Gly Gly Gln Val Arg Gln 245 250 255Arg Tyr Leu Tyr Thr Asp Asp
Ala Gln Gln Thr Glu Ala His Leu Glu 260 265 270Ile Arg Glu Asp Gly
Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu 275 280 285Ser Leu Leu
Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu 290 295 300Gly
Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu305 310
315 320Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu
Leu 325 330 335Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala
His Gly Leu 340 345 350Pro Leu His Leu Pro Gly Asn Lys Ser Pro His
Arg Asp Pro Ala Pro 355 360 365Arg Gly Pro Ala Arg Phe Leu Pro Leu
Pro Gly Leu Pro Pro Ala Leu 370 375 380Pro Glu Pro Pro Gly Ile Leu
Ala Pro Gln Pro Pro Asp Val Gly Ser385 390 395 400Ser Asp Pro Leu
Ser Met Val Thr Gly Leu Glu Ala Val Arg Ser Pro 405 410 415Ser Tyr
Ala Ser 42030420PRTArtificial Sequencemodified FGF21 variant
connected to hybrid Fc 30Glu Thr Lys Thr Pro Glu Cys Pro Ser His
Thr Gln Pro Leu Gly Val1 5 10 15Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 20 25 30Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu 35 40 45Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 50 55 60Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser65 70 75 80Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 100 105 110Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120
125Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
130 135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn145 150 155 160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser 165 170 175Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys Ser Arg 180 185 190Trp Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu 195 200 205His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly 210 215 220Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Pro225 230 235
240Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln
245 250 255Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
Leu Glu 260 265 270Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp
Gln Ser Pro Glu 275 280 285Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro
Gly Val Ile Gln Ile Leu 290 295 300Gly Val Lys Thr Ser Arg Phe Leu
Cys Gln Arg Pro Asp Gly Ala Leu305 310 315 320Tyr Gly Ser Leu His
Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu 325 330 335Leu Leu Glu
Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu 340 345 350Pro
Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro 355 360
365Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu
370 375 380Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
Gly Ser385 390 395 400Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu
Ala Asn Arg Ser Pro 405 410 415Ser Tyr Ala Ser
42031419PRTArtificial Sequencemodified FGF21 variant connected to
hybrid Fc 31Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu
Gly Val1 5 10 15Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr 20 25 30Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
Glu Asp Pro Glu 35 40 45Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 50 55 60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser65 70 75 80Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile 100 105 110Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120 125Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 130 135 140Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn145 150
155 160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser 165 170 175Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
Lys Ser Arg 180 185 190Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 195 200 205His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu Gly Lys Gly 210 215 220Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser His Pro225 230 235 240Ile Pro Asp Ser
Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln 245 250 255Arg Tyr
Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu 260 265
270Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu
275 280 285Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
Ile Leu 290 295 300Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro
Asp Gly Ala Leu305 310 315 320Tyr Gly Ser Leu His Phe Asp Pro Glu
Ala Cys Ser Phe Arg Glu Leu 325 330 335Leu Leu Glu Asp Gly Tyr Asn
Val Tyr Gln Ser Glu Ala His Gly Leu 340 345 350Pro Leu His Leu Pro
Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro 355 360 365Arg Gly Pro
Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu 370 375 380Pro
Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser385 390
395 400Ser Asp Pro Leu Ser Met Val Asn Pro Ser Gln Gly Arg Ser Pro
Ser 405 410 415Tyr Ala Ser32419PRTArtificial Sequencemodified FGF21
variant connected to hybrid Fc 32Glu Thr Lys Thr Pro Glu Cys Pro
Ser His Thr Gln Pro Leu Gly Val1 5 10 15Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25 30Pro Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro Glu 35 40 45Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 50 55 60Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser65 70 75 80Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 100 105
110Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
115 120 125Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu 130 135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn145 150 155 160Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser 165 170 175Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp Lys Ser Arg 180 185 190Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu 195 200 205His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly 210 215 220Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Pro225 230
235 240Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg
Gln 245 250 255Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala
His Leu Glu 260 265 270Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala
Asp Gln Ser Pro Glu 275 280 285Ser Leu Leu Gln Leu Lys Ala Leu Lys
Pro Gly Val Ile Gln Ile Leu 290 295 300Gly Val Lys Thr Ser Arg Phe
Leu Cys Gln Arg Pro Asp Gly Ala Leu305 310 315 320Tyr Gly Ser Leu
His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu 325 330 335Leu Leu
Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu 340 345
350Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro
355 360 365Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
Ala Leu 370 375 380Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro
Asp Val Gly Ser385 390 395 400Ser Asp Pro Leu Ser Met Val Asn Pro
Ser Gln Gly Arg Ser Pro Ser 405 410 415Tyr Ala
Ser33419PRTArtificial Sequencemodified FGF21 variant connected to
hybrid Fc 33Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu
Gly Val1 5 10 15Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr 20 25 30Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
Glu Asp Pro Glu 35 40 45Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 50 55 60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser65 70 75 80Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile 100 105 110Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120 125Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 130 135 140Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn145 150
155 160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser 165 170 175Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
Lys Ser Arg 180 185 190Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 195 200 205His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu Gly Lys Gly 210 215 220Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser His Pro225 230 235 240Ile Pro Asp Ser
Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln 245 250 255Arg Tyr
Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu 260 265
270Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu
275 280 285Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
Ile Leu 290 295 300Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro
Asp Gly Ala Leu305 310 315 320Tyr Gly Ser Leu His Phe Asp Pro Glu
Ala Cys Ser Phe Arg Glu Leu 325 330 335Leu Leu Glu Asp Gly Tyr Asn
Val Tyr Gln Ser Glu Ala His Gly Leu 340 345 350Pro Leu His Leu Pro
Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro 355 360 365Arg Gly Pro
Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu 370 375 380Pro
Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser385 390
395 400Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Asn Arg Ser Pro
Ser 405 410 415Tyr Ala Ser34419PRTArtificial Sequencemodified FGF21
variant connected to hybrid Fc 34Glu Thr Lys Thr Pro Glu Cys Pro
Ser His Thr Gln Pro Leu Gly Val1 5 10 15Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25 30Pro Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro Glu
35 40 45Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys 50 55 60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
Val Ser65 70 75 80Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys 85 90 95Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile 100 105 110Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro 115 120 125Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu 130 135 140Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn145 150 155 160Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 165 170
175Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
180 185 190Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 195 200 205His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys Gly 210 215 220Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser His Pro225 230 235 240Ile Pro Asp Ser Ser Pro Leu
Leu Gln Phe Gly Gly Gln Val Arg Gln 245 250 255Arg Tyr Leu Tyr Thr
Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu 260 265 270Ile Arg Glu
Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu 275 280 285Ser
Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu 290 295
300Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala
Leu305 310 315 320Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser
Phe Arg Glu Leu 325 330 335Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln
Ser Glu Ala His Gly Leu 340 345 350Pro Leu His Leu Pro Gly Asn Lys
Ser Pro His Arg Asp Pro Ala Pro 355 360 365Arg Gly Pro Ala Arg Phe
Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu 370 375 380Pro Glu Pro Pro
Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser385 390 395 400Ser
Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser 405 410
415Tyr Ala Ser35420PRTArtificial Sequencemodified FGF21 variant
connected to hybrid Fc 35Glu Thr Lys Thr Pro Glu Cys Pro Ser His
Thr Gln Pro Leu Gly Val1 5 10 15Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 20 25 30Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu 35 40 45Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 50 55 60Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser65 70 75 80Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 100 105 110Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120
125Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
130 135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn145 150 155 160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser 165 170 175Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys Ser Arg 180 185 190Trp Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu 195 200 205His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly 210 215 220Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Pro225 230 235
240Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln
245 250 255Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
Leu Glu 260 265 270Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp
Gln Ser Pro Glu 275 280 285Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro
Gly Val Ile Gln Ile Leu 290 295 300Gly Val Lys Thr Ser Arg Phe Leu
Cys Gln Arg Pro Asp Gly Ala Leu305 310 315 320Tyr Gly Ser Leu His
Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Glu 325 330 335Ile Arg Pro
Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu 340 345 350Pro
Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro 355 360
365Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu
370 375 380Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
Gly Ser385 390 395 400Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu
Ala Val Arg Ser Pro 405 410 415Ser Tyr Ala Ser
42036420PRTArtificial Sequencemodified FGF21 variant connected to
hybrid Fc 36Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu
Gly Val1 5 10 15Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr 20 25 30Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
Glu Asp Pro Glu 35 40 45Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 50 55 60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser65 70 75 80Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile 100 105 110Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120 125Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 130 135 140Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn145 150
155 160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser 165 170 175Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
Lys Ser Arg 180 185 190Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 195 200 205His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu Gly Lys Gly 210 215 220Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser His Pro225 230 235 240Ile Pro Asp Ser
Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln 245 250 255Arg Tyr
Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu 260 265
270Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu
275 280 285Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
Ile Leu 290 295 300Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro
Asp Gly Ala Leu305 310 315 320Tyr Gly Ser Leu His Phe Asp Pro Glu
Ala Cys Ser Phe Arg Glu Glu 325 330 335Ile Arg Pro Asp Gly Tyr Asn
Val Tyr Gln Ser Glu Ala His Gly Leu 340 345 350Pro Leu His Leu Pro
Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro 355 360 365Arg Gly Pro
Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu 370 375 380Pro
Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser385 390
395 400Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Val Arg Ser
Pro 405 410 415Ser Tyr Glu Ser 42037420PRTArtificial
Sequencemodified FGF21 variant connected to hybrid Fc 37Glu Thr Lys
Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val1 5 10 15Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25 30Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 35 40
45Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
50 55 60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
Ser65 70 75 80Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys 85 90 95Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile 100 105 110Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 115 120 125Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu 130 135 140Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn145 150 155 160Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 165 170 175Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 180 185
190Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
195 200 205His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys Gly 210 215 220Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser His Pro225 230 235 240Ile Pro Asp Ser Ser Pro Leu Leu Gln
Phe Gly Gly Gln Val Arg Gln 245 250 255Arg Tyr Leu Tyr Thr Asp Asp
Ala Gln Gln Thr Glu Ala His Leu Glu 260 265 270Ile Arg Glu Asp Gly
Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu 275 280 285Ser Leu Leu
Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu 290 295 300Gly
Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu305 310
315 320Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu
Glu 325 330 335Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala
His Gly Leu 340 345 350Pro Leu His Leu Pro Gly Asn Lys Ser Pro His
Arg Asp Pro Ala Pro 355 360 365Arg Gly Pro Ala Arg Phe Leu Pro Leu
Pro Gly Leu Pro Pro Ala Leu 370 375 380Pro Glu Pro Pro Gly Ile Leu
Ala Pro Gln Pro Pro Asp Val Gly Ser385 390 395 400Ser Asp Pro Leu
Ser Met Val Thr Gly Leu Glu Ala Asn Arg Ser Pro 405 410 415Ser Tyr
Glu Ser 42038419PRTArtificial Sequencemodified FGF21 variant
connected to hybrid Fc 38Glu Thr Lys Thr Pro Glu Cys Pro Ser His
Thr Gln Pro Leu Gly Val1 5 10 15Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 20 25 30Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu 35 40 45Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 50 55 60Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser65 70 75 80Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 100 105 110Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120
125Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
130 135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn145 150 155 160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser 165 170 175Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys Ser Arg 180 185 190Trp Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu 195 200 205His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly 210 215 220Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Pro225 230 235
240Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln
245 250 255Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
Leu Glu 260 265 270Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp
Gln Ser Pro Glu 275 280 285Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro
Gly Val Ile Gln Ile Leu 290 295 300Gly Val Lys Thr Ser Arg Phe Leu
Cys Gln Arg Pro Asp Gly Ala Leu305 310 315 320Tyr Gly Ser Leu His
Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Glu 325 330 335Ile Arg Pro
Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu 340 345 350Pro
Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro 355 360
365Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu
370 375 380Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
Gly Ser385 390 395 400Ser Asp Pro Leu Ser Met Val Asn Pro Ser Gln
Gly Arg Ser Pro Ser 405 410 415Tyr Ala Ser39419PRTArtificial
Sequencemodified FGF21 variant connected to hybrid Fc 39Glu Thr Lys
Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val1 5 10 15Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25 30Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 35 40
45Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
50 55 60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
Ser65 70 75 80Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys 85 90 95Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile 100 105 110Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 115 120 125Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu 130 135 140Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn145 150 155 160Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 165 170 175Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 180 185
190Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
195 200 205His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys Gly 210 215 220Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser His Pro225 230 235 240Ile Pro Asp Ser Ser Pro Leu Leu Gln
Phe Gly Gly Gln Val Arg Gln 245 250 255Arg Tyr Leu Tyr Thr Asp Asp
Ala Gln Gln Thr Glu Ala His Leu Glu 260 265 270Ile Arg Glu Asp Gly
Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu 275 280 285Ser Leu Leu
Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu 290 295 300Gly
Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu305 310
315 320Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu
Glu 325 330 335Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala
His Gly Leu 340 345 350Pro Leu His Leu Pro Gly Asn Lys Ser Pro His
Arg Asp Pro Ala Pro 355 360 365Arg Gly Pro Ala Arg Phe Leu
Pro Leu Pro Gly Leu Pro Pro Ala Leu 370 375 380Pro Glu Pro Pro Gly
Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser385 390 395 400Ser Asp
Pro Leu Ser Met Val Asn Pro Ser Gln Gly Arg Ser Pro Ser 405 410
415Tyr Glu Ser40423PRTArtificial SequenceREG(Amgen) 40Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40
45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly225 230 235 240Gly Ser His Pro Ile Pro Asp Ser Ser
Pro Leu Leu Gln Phe Gly Gly 245 250 255Gln Val Arg Gln Arg Tyr Leu
Tyr Thr Asp Asp Ala Gln Gln Thr Glu 260 265 270Ala His Leu Glu Ile
Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp 275 280 285Gln Ser Pro
Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val 290 295 300Ile
Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro305 310
315 320Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys
Ser 325 330 335Phe Arg Glu Arg Leu Leu Glu Asp Gly Tyr Asn Val Tyr
Gln Ser Glu 340 345 350Ala His Gly Leu Pro Leu His Leu Pro Gly Asn
Lys Ser Pro His Arg 355 360 365Asp Pro Ala Pro Arg Gly Pro Ala Arg
Phe Leu Pro Leu Pro Gly Leu 370 375 380Pro Pro Ala Leu Pro Glu Pro
Pro Gly Ile Leu Ala Pro Gln Pro Pro385 390 395 400Asp Val Gly Ser
Ser Asp Pro Leu Ser Met Val Gly Gly Ser Gln Gly 405 410 415Arg Ser
Pro Ser Tyr Glu Ser 42041424PRTArtificial SequenceFGF21 connected
to Fc(lilly) 41Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro Glu Ala1 5 10 15Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val 50 55 60Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110Ser Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125Gln Val
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135
140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala145 150 155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser 195 200 205Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu Gly
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly225 230 235 240Gly
Gly Ser Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe 245 250
255Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln
260 265 270Thr Glu Cys His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly
Cys Ala 275 280 285Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys
Ala Leu Lys Pro 290 295 300Gly Val Ile Gln Ile Leu Gly Val Lys Thr
Ser Arg Phe Leu Cys Gln305 310 315 320Arg Pro Asp Gly Ala Leu Tyr
Gly Ser Leu His Phe Asp Pro Glu Ala 325 330 335Cys Ser Phe Arg Glu
Asp Leu Lys Glu Asp Gly Tyr Asn Val Tyr Gln 340 345 350Ser Glu Ala
His Gly Leu Pro Leu His Leu Pro Gly Asp Lys Ser Pro 355 360 365His
Arg Lys Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro 370 375
380Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro
Gln385 390 395 400Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Arg Leu
Val Glu Pro Ser 405 410 415Gln Leu Arg Ser Pro Ser Phe Glu
4204231PRTArtificial SequenceGLP-1 42His Ala Glu Gly Thr Phe Thr
Ser Asp Val Ser Ser Tyr Leu Glu Gly1 5 10 15Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 304331PRTArtificial
SequenceGLP-1 variant 43His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser
Ser Tyr Leu Glu Gly1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Lys Gly Arg Gly 20 25 304431PRTArtificial SequenceGLP-1 variant
44His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu1
5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 304531PRTArtificial SequenceGLP-1 variant 45His Gly Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly1 5 10 15Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly 20 25
304631PRTArtificial SequenceGLP-1 variant 46His Gly Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu1 5 10 15Gln Ala Ala Lys Glu
Phe Ile Ala Trp Leu Val Lys Gly Gly Gly 20 25 3047245PRTArtificial
Sequencehybrid Fc5 47Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys
Lys Glu Lys Glu Lys1 5 10 15Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr
Pro Glu Cys Pro Ser His 20 25 30Thr Gln Pro Leu Gly Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 35 40 45Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val 50 55 60Ser Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val65 70 75 80Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120
125Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
130 135 140Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln145 150 155 160Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala 165 170 175Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 180 185 190Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205Thr Val Asp Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser225 230 235
240Leu Ser Leu Gly Lys 24548233PRTArtificial Sequencehybrid Fc40
48Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu1
5 10 15Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro
Lys 20 25 30Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val 35 40 45Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr 50 55 60Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu65 70 75 80Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His 85 90 95Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys 100 105 110Gly Leu Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln 115 120 125Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 130 135 140Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro145 150 155
160Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu 180 185 190Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val 195 200 205Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln 210 215 220Lys Ser Leu Ser Leu Ser Leu Gly
Lys225 23049276PRTArtificial SequenceGLP-1 variant connected to
hybrid Fc5 49His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
Leu Glu Gly1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg Gly Arg 20 25 30Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys
Glu Lys Glu Lys Glu 35 40 45Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro
Glu Cys Pro Ser His Thr 50 55 60Gln Pro Leu Gly Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu65 70 75 80Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 85 90 95Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 100 105 110Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 115 120 125Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 130 135
140Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser145 150 155 160Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 165 170 175Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val 180 185 190Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val 195 200 205Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 210 215 220Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr225 230 235 240Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 245 250
255Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270Ser Leu Gly Lys 27550264PRTArtificial SequenceGLP-1
variant connected to hybrid Fc40 50His Gly Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg Gly Glu 20 25 30Lys Glu Lys Glu Glu Gln
Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys 35 40 45Pro Ser His Thr Gln
Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro 50 55 60Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val65 70 75 80Val Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 85 90 95Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 100 105
110Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
115 120 125Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly 130 135 140Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro145 150 155 160Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Gln Glu Glu Met Thr 165 170 175Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser 180 185 190Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 195 200 205Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 210 215 220Ser
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe225 230
235 240Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys 245 250 255Ser Leu Ser Leu Ser Leu Gly Lys
26051276PRTArtificial SequenceGLP-1 variant connected to hybrid Fc5
51His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu1
5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
Arg 20 25 30Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu
Lys Glu 35 40 45Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro
Ser His Thr 50 55 60Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu65 70 75 80Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser 85 90 95Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu 100 105 110Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr 115 120 125Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 130 135 140Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser145 150 155
160Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln Val 180 185 190Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 195 200 205Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro 210 215 220Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Arg Leu Thr225 230 235 240Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 245 250 255Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 260 265 270Ser
Leu Gly Lys 27552264PRTArtificial SequenceGLP-1 variant connected
to hybrid Fc40 52His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Glu1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val
Lys Gly Arg Gly Glu 20 25 30Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu
Thr Lys Thr Pro Glu Cys 35 40
45Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro
50 55 60Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val65 70 75 80Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val 85 90 95Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln 100 105 110Phe Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln 115 120 125Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly 130 135 140Leu Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro145 150 155 160Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 165 170 175Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 180 185
190Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
195 200 205Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr 210 215 220Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
Gly Asn Val Phe225 230 235 240Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys 245 250 255Ser Leu Ser Leu Ser Leu Gly
Lys 26053276PRTArtificial SequenceGLP-1 variant connected to hybrid
Fc5 53His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
Gly1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly
Gly Arg 20 25 30Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys
Glu Lys Glu 35 40 45Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys
Pro Ser His Thr 50 55 60Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu65 70 75 80Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 85 90 95Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu 100 105 110Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 115 120 125Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 130 135 140Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser145 150 155
160Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln Val 180 185 190Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 195 200 205Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro 210 215 220Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Arg Leu Thr225 230 235 240Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 245 250 255Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 260 265 270Ser
Leu Gly Lys 27554264PRTArtificial SequenceGLP-1 variant connected
to hybrid Fc40 54His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val
Lys Gly Gly Gly Glu 20 25 30Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu
Thr Lys Thr Pro Glu Cys 35 40 45Pro Ser His Thr Gln Pro Leu Gly Val
Phe Leu Phe Pro Pro Lys Pro 50 55 60Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val65 70 75 80Val Asp Val Ser Gln Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 85 90 95Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 100 105 110Phe Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 115 120 125Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 130 135
140Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro145 150 155 160Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu Met Thr 165 170 175Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser 180 185 190Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr 195 200 205Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 210 215 220Ser Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe225 230 235 240Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 245 250
255Ser Leu Ser Leu Ser Leu Gly Lys 26055276PRTArtificial
SequenceGLP-1 variant connected to hybrid Fc5 55His Gly Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu1 5 10 15Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Arg 20 25 30Asn Thr Gly
Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu 35 40 45Glu Gln
Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr 50 55 60Gln
Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu65 70 75
80Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu 100 105 110Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser Thr 115 120 125Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn 130 135 140Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser145 150 155 160Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 165 170 175Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 180 185 190Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 195 200
205Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr225 230 235 240Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val 245 250 255Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 260 265 270Ser Leu Gly Lys
27556264PRTArtificial SequenceGLP-1 variant connected to hybrid
Fc40 56His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
Glu1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly
Gly Glu 20 25 30Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr
Pro Glu Cys 35 40 45Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe
Pro Pro Lys Pro 50 55 60Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val65 70 75 80Val Asp Val Ser Gln Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val 85 90 95Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln 100 105 110Phe Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln 115 120 125Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 130 135 140Leu Pro
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro145 150 155
160Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
165 170 175Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser 180 185 190Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr 195 200 205Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr 210 215 220Ser Arg Leu Thr Val Asp Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe225 230 235 240Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 245 250 255Ser Leu Ser
Leu Ser Leu Gly Lys 26057275PRTArtificial SequenceDulaglutide 57His
Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu1 5 10
15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
Glu 35 40 45Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala 50 55 60Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu65 70 75 80Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser 85 90 95Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu 100 105 110Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr 115 120 125Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn 130 135 140Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser145 150 155 160Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 165 170
175Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val 195 200 205Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro 210 215 220Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr225 230 235 240Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val 245 250 255Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 260 265 270Ser Leu Gly
27558461PRTArtificial SequenceGLP1(A2G)-HyFc40-GS3-FGF21(EIRP,
TGLEAV) 58His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly
Arg Gly Glu 20 25 30Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys
Thr Pro Glu Cys 35 40 45Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu
Phe Pro Pro Lys Pro 50 55 60Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val65 70 75 80Val Asp Val Ser Gln Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val 85 90 95Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln 100 105 110Phe Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln 115 120 125Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 130 135 140Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro145 150
155 160Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
Thr 165 170 175Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser 180 185 190Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr 195 200 205Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr 210 215 220Ser Arg Leu Thr Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe225 230 235 240Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 245 250 255Ser Leu
Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly 260 265
270Gly Ser Gly Gly Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu
275 280 285Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr
Asp Asp 290 295 300Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu
Asp Gly Thr Val305 310 315 320Gly Gly Ala Ala Asp Gln Ser Pro Glu
Ser Leu Leu Gln Leu Lys Ala 325 330 335Leu Lys Pro Gly Val Ile Gln
Ile Leu Gly Val Lys Thr Ser Arg Phe 340 345 350Leu Cys Gln Arg Pro
Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp 355 360 365Pro Glu Ala
Cys Ser Phe Arg Glu Glu Ile Arg Pro Asp Gly Tyr Asn 370 375 380Val
Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn385 390
395 400Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe
Leu 405 410 415Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro
Gly Ile Leu 420 425 430Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp
Pro Leu Ser Met Val 435 440 445Thr Gly Leu Glu Ala Val Arg Ser Pro
Ser Tyr Ala Ser 450 455 46059473PRTArtificial
SequenceGLP1(GE)-HyFc5-GS3-FGF21(EIRP, TGLEAV) 59His Gly Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu1 5 10 15Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Arg 20 25 30Asn Thr
Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu 35 40 45Glu
Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr 50 55
60Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu65
70 75 80Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser 85 90 95Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu 100 105 110Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr 115 120 125Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn 130 135 140Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser145 150 155 160Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 165 170 175Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 180 185 190Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 195 200
205Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr225 230 235 240Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val 245 250 255Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 260 265 270Ser Leu Gly Lys Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly 275 280 285Gly Gly Ser His Pro
Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 290 295 300Gly Gln Val
Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr305 310 315
320Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala
325 330 335Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys
Pro Gly 340 345 350Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe
Leu Cys Gln Arg 355 360 365Pro Asp Gly Ala Leu Tyr Gly Ser Leu His
Phe Asp Pro Glu Ala Cys 370
375 380Ser Phe Arg Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln
Ser385 390 395 400Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn
Lys Ser Pro His 405 410 415Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg
Phe Leu Pro Leu Pro Gly 420 425 430Leu Pro Pro Ala Leu Pro Glu Pro
Pro Gly Ile Leu Ala Pro Gln Pro 435 440 445Pro Asp Val Gly Ser Ser
Asp Pro Leu Ser Met Val Thr Gly Leu Glu 450 455 460Ala Val Arg Ser
Pro Ser Tyr Ala Ser465 47060461PRTArtificial
SequenceGLP1(GE)-HyFc40-GS3-FGF21(EIRP, TGLEAV) 60His Gly Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu1 5 10 15Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Glu 20 25 30Lys Glu
Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys 35 40 45Pro
Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro 50 55
60Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val65
70 75 80Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val 85 90 95Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln 100 105 110Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln 115 120 125Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly 130 135 140Leu Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro145 150 155 160Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 165 170 175Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 180 185 190Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 195 200
205Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
210 215 220Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe225 230 235 240Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys 245 250 255Ser Leu Ser Leu Ser Leu Gly Lys Gly
Gly Gly Gly Ser Gly Gly Gly 260 265 270Gly Ser Gly Gly Gly Gly Ser
His Pro Ile Pro Asp Ser Ser Pro Leu 275 280 285Leu Gln Phe Gly Gly
Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp 290 295 300Ala Gln Gln
Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val305 310 315
320Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala
325 330 335Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser
Arg Phe 340 345 350Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser
Leu His Phe Asp 355 360 365Pro Glu Ala Cys Ser Phe Arg Glu Glu Ile
Arg Pro Asp Gly Tyr Asn 370 375 380Val Tyr Gln Ser Glu Ala His Gly
Leu Pro Leu His Leu Pro Gly Asn385 390 395 400Lys Ser Pro His Arg
Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu 405 410 415Pro Leu Pro
Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu 420 425 430Ala
Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val 435 440
445Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Tyr Ala Ser 450 455
46061473PRTArtificial SequenceGLP1(GG)-HyFc5-GS3-FGF21(EIRP,
TGLEAV) 61His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly
Gly Gly Arg 20 25 30Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu
Lys Glu Lys Glu 35 40 45Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu
Cys Pro Ser His Thr 50 55 60Gln Pro Leu Gly Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu65 70 75 80Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser 85 90 95Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu 100 105 110Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 115 120 125Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 130 135 140Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser145 150
155 160Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln 165 170 175Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val 180 185 190Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val 195 200 205Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro 210 215 220Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr225 230 235 240Val Asp Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 245 250 255Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 260 265
270Ser Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
275 280 285Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln
Phe Gly 290 295 300Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp
Ala Gln Gln Thr305 310 315 320Glu Ala His Leu Glu Ile Arg Glu Asp
Gly Thr Val Gly Gly Ala Ala 325 330 335Asp Gln Ser Pro Glu Ser Leu
Leu Gln Leu Lys Ala Leu Lys Pro Gly 340 345 350Val Ile Gln Ile Leu
Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg 355 360 365Pro Asp Gly
Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 370 375 380Ser
Phe Arg Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser385 390
395 400Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro
His 405 410 415Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro
Leu Pro Gly 420 425 430Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile
Leu Ala Pro Gln Pro 435 440 445Pro Asp Val Gly Ser Ser Asp Pro Leu
Ser Met Val Thr Gly Leu Glu 450 455 460Ala Val Arg Ser Pro Ser Tyr
Ala Ser465 47062461PRTArtificial
SequenceGLP1(GG)-HyFc40-GS3-FGF21(EIRP, TGLEAV) 62His Gly Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly1 5 10 15Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Glu 20 25 30Lys Glu
Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys 35 40 45Pro
Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro 50 55
60Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val65
70 75 80Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val 85 90 95Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln 100 105 110Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln 115 120 125Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly 130 135 140Leu Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro145 150 155 160Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 165 170 175Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 180 185 190Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 195 200
205Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
210 215 220Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe225 230 235 240Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys 245 250 255Ser Leu Ser Leu Ser Leu Gly Lys Gly
Gly Gly Gly Ser Gly Gly Gly 260 265 270Gly Ser Gly Gly Gly Gly Ser
His Pro Ile Pro Asp Ser Ser Pro Leu 275 280 285Leu Gln Phe Gly Gly
Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp 290 295 300Ala Gln Gln
Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val305 310 315
320Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala
325 330 335Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser
Arg Phe 340 345 350Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser
Leu His Phe Asp 355 360 365Pro Glu Ala Cys Ser Phe Arg Glu Glu Ile
Arg Pro Asp Gly Tyr Asn 370 375 380Val Tyr Gln Ser Glu Ala His Gly
Leu Pro Leu His Leu Pro Gly Asn385 390 395 400Lys Ser Pro His Arg
Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu 405 410 415Pro Leu Pro
Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu 420 425 430Ala
Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val 435 440
445Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Tyr Ala Ser 450 455
46063473PRTArtificial SequenceGLP1(GEG)-HyFc5-GS3-FGF21(EIRP,
TGLEAV) 63His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Glu1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly
Gly Gly Arg 20 25 30Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu
Lys Glu Lys Glu 35 40 45Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu
Cys Pro Ser His Thr 50 55 60Gln Pro Leu Gly Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu65 70 75 80Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser 85 90 95Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu 100 105 110Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 115 120 125Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 130 135 140Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser145 150
155 160Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln 165 170 175Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val 180 185 190Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val 195 200 205Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro 210 215 220Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr225 230 235 240Val Asp Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 245 250 255Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 260 265
270Ser Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
275 280 285Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln
Phe Gly 290 295 300Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp
Ala Gln Gln Thr305 310 315 320Glu Ala His Leu Glu Ile Arg Glu Asp
Gly Thr Val Gly Gly Ala Ala 325 330 335Asp Gln Ser Pro Glu Ser Leu
Leu Gln Leu Lys Ala Leu Lys Pro Gly 340 345 350Val Ile Gln Ile Leu
Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg 355 360 365Pro Asp Gly
Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 370 375 380Ser
Phe Arg Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser385 390
395 400Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro
His 405 410 415Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro
Leu Pro Gly 420 425 430Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile
Leu Ala Pro Gln Pro 435 440 445Pro Asp Val Gly Ser Ser Asp Pro Leu
Ser Met Val Thr Gly Leu Glu 450 455 460Ala Val Arg Ser Pro Ser Tyr
Ala Ser465 47064461PRTArtificial
SequenceGLP1(GEG)-HyFc40-GS3-FGF21(EIRP, TGLEAV) 64His Gly Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu1 5 10 15Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Glu 20 25 30Lys Glu
Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys 35 40 45Pro
Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro 50 55
60Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val65
70 75 80Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val 85 90 95Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln 100 105 110Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln 115 120 125Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly 130 135 140Leu Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro145 150 155 160Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 165 170 175Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 180 185 190Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 195 200
205Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
210 215 220Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe225 230 235 240Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys 245 250 255Ser Leu Ser Leu Ser Leu Gly Lys Gly
Gly Gly Gly Ser Gly Gly Gly 260 265 270Gly Ser Gly Gly Gly Gly Ser
His Pro Ile Pro Asp Ser Ser Pro Leu 275 280 285Leu Gln Phe Gly Gly
Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp 290 295 300Ala Gln Gln
Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val305 310 315
320Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala
325 330 335Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser
Arg Phe 340 345 350Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser
Leu His Phe Asp 355 360 365Pro Glu Ala Cys Ser Phe Arg Glu Glu Ile
Arg Pro Asp Gly Tyr Asn 370 375 380Val Tyr Gln Ser Glu Ala His Gly
Leu Pro Leu His Leu Pro Gly Asn385 390 395 400Lys Ser Pro His Arg
Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu 405 410 415Pro Leu Pro
Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu 420 425 430Ala
Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val 435 440
445Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Tyr Ala Ser 450
455 46065461PRTArtificial SequenceGLP1(GEG)-HyFc40-GS3-FGF21(EIRP,
TGLEAV, A180E) 65His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Glu1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val
Lys Gly Gly Gly Glu 20 25 30Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu
Thr Lys Thr Pro Glu Cys 35 40 45Pro Ser His Thr Gln Pro Leu Gly Val
Phe Leu Phe Pro Pro Lys Pro 50 55 60Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val65 70 75 80Val Asp Val Ser Gln Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 85 90 95Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 100 105 110Phe Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 115 120 125Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 130 135
140Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro145 150 155 160Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu Met Thr 165 170 175Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser 180 185 190Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr 195 200 205Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 210 215 220Ser Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe225 230 235 240Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 245 250
255Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly
260 265 270Gly Ser Gly Gly Gly Gly Ser His Pro Ile Pro Asp Ser Ser
Pro Leu 275 280 285Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu
Tyr Thr Asp Asp 290 295 300Ala Gln Gln Thr Glu Ala His Leu Glu Ile
Arg Glu Asp Gly Thr Val305 310 315 320Gly Gly Ala Ala Asp Gln Ser
Pro Glu Ser Leu Leu Gln Leu Lys Ala 325 330 335Leu Lys Pro Gly Val
Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe 340 345 350Leu Cys Gln
Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp 355 360 365Pro
Glu Ala Cys Ser Phe Arg Glu Glu Ile Arg Pro Asp Gly Tyr Asn 370 375
380Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly
Asn385 390 395 400Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro
Ala Arg Phe Leu 405 410 415Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro
Glu Pro Pro Gly Ile Leu 420 425 430Ala Pro Gln Pro Pro Asp Val Gly
Ser Ser Asp Pro Leu Ser Met Val 435 440 445Thr Gly Leu Glu Ala Val
Arg Ser Pro Ser Tyr Glu Ser 450 455 46066461PRTArtificial
SequenceGLP1(GEG)-HyFc40-GS3-FGF21(EIRP, TGLEAN, A180E) 66His Gly
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Glu 20 25
30Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys
35 40 45Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys
Pro 50 55 60Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val65 70 75 80Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val 85 90 95Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln 100 105 110Phe Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln 115 120 125Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly 130 135 140Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro145 150 155 160Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 165 170
175Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
180 185 190Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr 195 200 205Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr 210 215 220Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe225 230 235 240Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys 245 250 255Ser Leu Ser Leu Ser
Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly 260 265 270Gly Ser Gly
Gly Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu 275 280 285Leu
Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp 290 295
300Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr
Val305 310 315 320Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu
Gln Leu Lys Ala 325 330 335Leu Lys Pro Gly Val Ile Gln Ile Leu Gly
Val Lys Thr Ser Arg Phe 340 345 350Leu Cys Gln Arg Pro Asp Gly Ala
Leu Tyr Gly Ser Leu His Phe Asp 355 360 365Pro Glu Ala Cys Ser Phe
Arg Glu Glu Ile Arg Pro Asp Gly Tyr Asn 370 375 380Val Tyr Gln Ser
Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn385 390 395 400Lys
Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu 405 410
415Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu
420 425 430Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser
Met Val 435 440 445Thr Gly Leu Glu Ala Asn Arg Ser Pro Ser Tyr Glu
Ser 450 455 46067460PRTArtificial
SequenceGLP1(GEG)-HyFc40-GS3-FGF21(EIRP, G170N, A180E) 67His Gly
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Glu 20 25
30Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys
35 40 45Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys
Pro 50 55 60Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val65 70 75 80Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val 85 90 95Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln 100 105 110Phe Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln 115 120 125Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly 130 135 140Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro145 150 155 160Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 165 170
175Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
180 185 190Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr 195 200 205Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr 210 215 220Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe225 230 235 240Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys 245 250 255Ser Leu Ser Leu Ser
Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly 260 265 270Gly Ser Gly
Gly Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu 275 280 285Leu
Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp 290 295
300Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr
Val305 310 315 320Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu
Gln Leu Lys Ala 325 330 335Leu Lys Pro Gly Val Ile Gln Ile Leu Gly
Val Lys Thr Ser Arg Phe 340 345 350Leu Cys Gln Arg Pro Asp Gly Ala
Leu Tyr Gly Ser Leu His Phe Asp 355 360 365Pro Glu Ala Cys Ser Phe
Arg Glu Glu Ile Arg Pro Asp Gly Tyr Asn 370 375 380Val Tyr Gln Ser
Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn385 390 395 400Lys
Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu 405 410
415Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu
420 425 430Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser
Met Val 435 440 445Asn Pro Ser Gln Gly Arg Ser Pro Ser Tyr Glu Ser
450 455 460684PRTArtificial SequenceFGF21 variant 68Glu Ile Arg
Pro1696PRTArtificial SequenceFGF21 variant 69Thr Gly Leu Glu Ala
Val1 5706PRTArtificial SequenceFGF21 variant 70Thr Gly Leu Glu Ala
Asn1 5711383DNAArtificial Sequencenucleic acid molecule coding for
DFD23 71cacggcgagg ggaccttcac aagcgacgtg tcctcttatc tggaaggaca
ggccgctaag 60gagtttatcg catggctcgt caaaggcaga ggcgaaaagg agaaggaaga
gcaggaggag 120agagaaacca aaacacccga gtgtcccagt cacactcagc
ctctgggagt gtttctcttc 180ccacctaagc ccaaggatac ccttatgatt
tctaggacac ctgaggtgac ctgcgtcgtt 240gtggacgtga gtcaagagga
cccagaggtc cagtttaact ggtatgttga cggcgtggaa 300gtgcataatg
caaaaactaa accccgcgag gaacaattca attcaaccta ccgggtcgtt
360tctgtgttga cagtgctgca tcaagattgg ctgaacggga aggagtataa
gtgtaaagtc 420agtaataagg gactcccctc tagtatcgaa aaaactattt
caaaggccaa aggccagcct 480agagagccac aggtgtacac ccttcctcca
tcccaagagg agatgacaaa gaaccaggtg 540tctctgactt gtctcgtgaa
ggggttctac cctagtgaca tcgctgtcga atgggagtca 600aacggacagc
cagagaataa ttataagaca actcctcccg ttctggattc tgacggcagc
660ttctttctgt actctaggct tactgtggac aaaagtcgct ggcaagaagg
gaacgtcttt 720tcatgttctg ttatgcacga ggccttgcac aatcattata
cacagaagtc tctgagtctc 780tcactgggca aaggcggggg aggcagcggg
ggaggcgggt ccggaggcgg gggatctcat 840cccatccctg actccagtcc
tctcctgcaa ttcgggggcc aagtccggca gcggtacctc 900tacacagatg
atgctcagca gacagaagcc cacctggaga tcagggagga tgggaccgtg
960gggggcgctg ctgaccagag ccccgaaagt ctcctgcagc tgaaagcctt
gaagcctgga 1020gttattcaaa tcttgggagt caagactagt aggttcctgt
gccagcggcc agatggggcc 1080ctgtatggat ctctccattt tgaccctgag
gcctgcagct tccgggagga gatcagaccc 1140gacggataca atgtttacca
gtccgaagcc cacggcctcc ctctgcatct gcccgggaac 1200aagtctcctc
accgggaccc tgcccccaga ggacctgctc gcttcctgcc actcccaggc
1260ctgccccccg cattgcctga gccacccgga atcctggccc cccagccccc
tgatgtggga 1320tcctctgacc ctctgagcat ggtgacaggc ctggaggccg
tgagaagccc cagctacgct 1380tcc 1383721419DNAArtificial
Sequencenucleic acid molecule coding for DFD24 72cacggcgagg
ggaccttcac aagcgacgtg tcctcttacc tggaagagca ggccgctaag 60gaatttatcg
catggctcgt caaaggaaga gggaggaaca ccggacgggg cggggaagag
120aagaagaaag aaaaggagaa ggaagagcag gaggagagag aaaccaaaac
acccgagtgt 180cccagtcaca ctcagcctct gggagtgttt ctcttcccac
ctaagcccaa ggataccctt 240atgatttcta ggacacctga ggtgacctgc
gtcgttgtgg acgtgagtca agaggaccca 300gaggtccagt ttaactggta
tgttgacggc gtggaagtgc ataatgcaaa aactaaaccc 360cgcgaggaac
aattcaattc aacctaccgg gtcgtttctg tgttgacagt gctgcatcaa
420gattggctga acgggaagga gtataagtgt aaagtcagta ataagggact
cccctctagt 480atcgaaaaaa ctatttcaaa ggccaaaggc cagcctagag
agccacaggt gtacaccctt 540cctccatccc aagaggagat gacaaagaac
caggtgtctc tgacttgtct cgtgaagggg 600ttctacccta gtgacatcgc
tgtcgaatgg gagtcaaacg gacagccaga gaataattat 660aagacaactc
ctcccgttct ggattctgac ggcagcttct ttctgtactc taggcttact
720gtggacaaaa gtcgctggca agaagggaac gtcttttcat gttctgttat
gcacgaggcc 780ttgcacaatc attatacaca gaagtctctg agtctctcac
tgggcaaagg cgggggaggc 840agcgggggag gcgggtccgg aggcggggga
tctcatccca tccctgactc cagtcctctc 900ctgcaattcg ggggccaagt
ccggcagcgg tacctctaca cagatgatgc tcagcagaca 960gaagcccacc
tggagatcag ggaggatggg accgtggggg gcgctgctga ccagagcccc
1020gaaagtctcc tgcagctgaa agccttgaag cctggagtta ttcaaatctt
gggagtcaag 1080actagtaggt tcctgtgcca gcggccagat ggggccctgt
atggatctct ccattttgac 1140cctgaggcct gcagcttccg ggaggagatc
agacccgacg gatacaatgt ttaccagtcc 1200gaagcccacg gcctccctct
gcatctgccc gggaacaagt ctcctcaccg ggaccctgcc 1260cccagaggac
ctgctcgctt cctgccactc ccaggcctgc cccccgcatt gcctgagcca
1320cccggaatcc tggcccccca gccccctgat gtgggatcct ctgaccctct
gagcatggtg 1380acaggcctgg aggccgtgag aagccccagc tacgcttcc
1419731383DNAArtificial Sequencenucleic acid molecule coding for
DFD25 73cacggcgagg ggaccttcac aagcgacgtg tcctcttacc tggaagagca
ggccgctaag 60gaatttatcg catggctcgt caaaggaaga ggggaaaagg agaaggaaga
gcaggaggag 120agagaaacca aaacacccga gtgtcccagt cacactcagc
ctctgggagt gtttctcttc 180ccacctaagc ccaaggatac ccttatgatt
tctaggacac ctgaggtgac ctgcgtcgtt 240gtggacgtga gtcaagagga
cccagaggtc cagtttaact ggtatgttga cggcgtggaa 300gtgcataatg
caaaaactaa accccgcgag gaacaattca attcaaccta ccgggtcgtt
360tctgtgttga cagtgctgca tcaagattgg ctgaacggga aggagtataa
gtgtaaagtc 420agtaataagg gactcccctc tagtatcgaa aaaactattt
caaaggccaa aggccagcct 480agagagccac aggtgtacac ccttcctcca
tcccaagagg agatgacaaa gaaccaggtg 540tctctgactt gtctcgtgaa
ggggttctac cctagtgaca tcgctgtcga atgggagtca 600aacggacagc
cagagaataa ttataagaca actcctcccg ttctggattc tgacggcagc
660ttctttctgt actctaggct tactgtggac aaaagtcgct ggcaagaagg
gaacgtcttt 720tcatgttctg ttatgcacga ggccttgcac aatcattata
cacagaagtc tctgagtctc 780tcactgggca aaggcggggg aggcagcggg
ggaggcgggt ccggaggcgg gggatctcat 840cccatccctg actccagtcc
tctcctgcaa ttcgggggcc aagtccggca gcggtacctc 900tacacagatg
atgctcagca gacagaagcc cacctggaga tcagggagga tgggaccgtg
960gggggcgctg ctgaccagag ccccgaaagt ctcctgcagc tgaaagcctt
gaagcctgga 1020gttattcaaa tcttgggagt caagactagt aggttcctgt
gccagcggcc agatggggcc 1080ctgtatggat ctctccattt tgaccctgag
gcctgcagct tccgggagga gatcagaccc 1140gacggataca atgtttacca
gtccgaagcc cacggcctcc ctctgcatct gcccgggaac 1200aagtctcctc
accgggaccc tgcccccaga ggacctgctc gcttcctgcc actcccaggc
1260ctgccccccg cattgcctga gccacccgga atcctggccc cccagccccc
tgatgtggga 1320tcctctgacc ctctgagcat ggtgacaggc ctggaggccg
tgagaagccc cagctacgct 1380tcc 1383741419DNAArtificial
Sequencenucleic acid molecule coding for DFD26 74cacggcgagg
ggaccttcac aagcgacgtg tcctcttatc tggaaggaca ggccgctaag 60gagtttatcg
catggctcgt caaaggcggc ggcaggaaca ccggacgggg cggggaagag
120aagaagaaag aaaaggagaa ggaagagcag gaggagagag aaaccaaaac
acccgagtgt 180cccagtcaca ctcagcctct gggagtgttt ctcttcccac
ctaagcccaa ggataccctt 240atgatttcta ggacacctga ggtgacctgc
gtcgttgtgg acgtgagtca agaggaccca 300gaggtccagt ttaactggta
tgttgacggc gtggaagtgc ataatgcaaa aactaaaccc 360cgcgaggaac
aattcaattc aacctaccgg gtcgtttctg tgttgacagt gctgcatcaa
420gattggctga acgggaagga gtataagtgt aaagtcagta ataagggact
cccctctagt 480atcgaaaaaa ctatttcaaa ggccaaaggc cagcctagag
agccacaggt gtacaccctt 540cctccatccc aagaggagat gacaaagaac
caggtgtctc tgacttgtct cgtgaagggg 600ttctacccta gtgacatcgc
tgtcgaatgg gagtcaaacg gacagccaga gaataattat 660aagacaactc
ctcccgttct ggattctgac ggcagcttct ttctgtactc taggcttact
720gtggacaaaa gtcgctggca agaagggaac gtcttttcat gttctgttat
gcacgaggcc 780ttgcacaatc attatacaca gaagtctctg agtctctcac
tgggcaaagg cgggggaggc 840agcgggggag gcgggtccgg aggcggggga
tctcatccca tccctgactc cagtcctctc 900ctgcaattcg ggggccaagt
ccggcagcgg tacctctaca cagatgatgc tcagcagaca 960gaagcccacc
tggagatcag ggaggatggg accgtggggg gcgctgctga ccagagcccc
1020gaaagtctcc tgcagctgaa agccttgaag cctggagtta ttcaaatctt
gggagtcaag 1080actagtaggt tcctgtgcca gcggccagat ggggccctgt
atggatctct ccattttgac 1140cctgaggcct gcagcttccg ggaggagatc
agacccgacg gatacaatgt ttaccagtcc 1200gaagcccacg gcctccctct
gcatctgccc gggaacaagt ctcctcaccg ggaccctgcc 1260cccagaggac
ctgctcgctt cctgccactc ccaggcctgc cccccgcatt gcctgagcca
1320cccggaatcc tggcccccca gccccctgat gtgggatcct ctgaccctct
gagcatggtg 1380acaggcctgg aggccgtgag aagccccagc tacgcttcc
1419751383DNAArtificial Sequencenucleic acid molecule coding for
DFD27 75cacggcgagg ggaccttcac aagcgacgtg tcctcttatc tggaaggaca
ggccgctaag 60gagtttatcg catggctcgt caaaggcggc ggcgaaaagg agaaggaaga
gcaggaggag 120agagaaacca aaacacccga gtgtcccagt cacactcagc
ctctgggagt
gtttctcttc 180ccacctaagc ccaaggatac ccttatgatt tctaggacac
ctgaggtgac ctgcgtcgtt 240gtggacgtga gtcaagagga cccagaggtc
cagtttaact ggtatgttga cggcgtggaa 300gtgcataatg caaaaactaa
accccgcgag gaacaattca attcaaccta ccgggtcgtt 360tctgtgttga
cagtgctgca tcaagattgg ctgaacggga aggagtataa gtgtaaagtc
420agtaataagg gactcccctc tagtatcgaa aaaactattt caaaggccaa
aggccagcct 480agagagccac aggtgtacac ccttcctcca tcccaagagg
agatgacaaa gaaccaggtg 540tctctgactt gtctcgtgaa ggggttctac
cctagtgaca tcgctgtcga atgggagtca 600aacggacagc cagagaataa
ttataagaca actcctcccg ttctggattc tgacggcagc 660ttctttctgt
actctaggct tactgtggac aaaagtcgct ggcaagaagg gaacgtcttt
720tcatgttctg ttatgcacga ggccttgcac aatcattata cacagaagtc
tctgagtctc 780tcactgggca aaggcggggg aggcagcggg ggaggcgggt
ccggaggcgg gggatctcat 840cccatccctg actccagtcc tctcctgcaa
ttcgggggcc aagtccggca gcggtacctc 900tacacagatg atgctcagca
gacagaagcc cacctggaga tcagggagga tgggaccgtg 960gggggcgctg
ctgaccagag ccccgaaagt ctcctgcagc tgaaagcctt gaagcctgga
1020gttattcaaa tcttgggagt caagactagt aggttcctgt gccagcggcc
agatggggcc 1080ctgtatggat ctctccattt tgaccctgag gcctgcagct
tccgggagga gatcagaccc 1140gacggataca atgtttacca gtccgaagcc
cacggcctcc ctctgcatct gcccgggaac 1200aagtctcctc accgggaccc
tgcccccaga ggacctgctc gcttcctgcc actcccaggc 1260ctgccccccg
cattgcctga gccacccgga atcctggccc cccagccccc tgatgtggga
1320tcctctgacc ctctgagcat ggtgacaggc ctggaggccg tgagaagccc
cagctacgct 1380tcc 1383761419DNAArtificial Sequencenucleic acid
molecule coding for DFD28 76cacggcgagg ggaccttcac aagcgacgtg
tcctcttacc tggaagagca ggccgctaag 60gaatttatcg catggctcgt caaaggaggc
gggaggaaca ccggacgggg cggggaagag 120aagaagaaag aaaaggagaa
ggaagagcag gaggagagag aaaccaaaac acccgagtgt 180cccagtcaca
ctcagcctct gggagtgttt ctcttcccac ctaagcccaa ggataccctt
240atgatttcta ggacacctga ggtgacctgc gtcgttgtgg acgtgagtca
agaggaccca 300gaggtccagt ttaactggta tgttgacggc gtggaagtgc
ataatgcaaa aactaaaccc 360cgcgaggaac aattcaattc aacctaccgg
gtcgtttctg tgttgacagt gctgcatcaa 420gattggctga acgggaagga
gtataagtgt aaagtcagta ataagggact cccctctagt 480atcgaaaaaa
ctatttcaaa ggccaaaggc cagcctagag agccacaggt gtacaccctt
540cctccatccc aagaggagat gacaaagaac caggtgtctc tgacttgtct
cgtgaagggg 600ttctacccta gtgacatcgc tgtcgaatgg gagtcaaacg
gacagccaga gaataattat 660aagacaactc ctcccgttct ggattctgac
ggcagcttct ttctgtactc taggcttact 720gtggacaaaa gtcgctggca
agaagggaac gtcttttcat gttctgttat gcacgaggcc 780ttgcacaatc
attatacaca gaagtctctg agtctctcac tgggcaaagg cgggggaggc
840agcgggggag gcgggtccgg aggcggggga tctcatccca tccctgactc
cagtcctctc 900ctgcaattcg ggggccaagt ccggcagcgg tacctctaca
cagatgatgc tcagcagaca 960gaagcccacc tggagatcag ggaggatggg
accgtggggg gcgctgctga ccagagcccc 1020gaaagtctcc tgcagctgaa
agccttgaag cctggagtta ttcaaatctt gggagtcaag 1080actagtaggt
tcctgtgcca gcggccagat ggggccctgt atggatctct ccattttgac
1140cctgaggcct gcagcttccg ggaggagatc agacccgacg gatacaatgt
ttaccagtcc 1200gaagcccacg gcctccctct gcatctgccc gggaacaagt
ctcctcaccg ggaccctgcc 1260cccagaggac ctgctcgctt cctgccactc
ccaggcctgc cccccgcatt gcctgagcca 1320cccggaatcc tggcccccca
gccccctgat gtgggatcct ctgaccctct gagcatggtg 1380acaggcctgg
aggccgtgag aagccccagc tacgcttcc 1419771383DNAArtificial
Sequencenucleic acid molecule coding for DFD29 77cacggcgagg
ggaccttcac aagcgacgtg tcctcttacc tggaagagca ggccgctaag 60gaatttatcg
catggctcgt caaaggaggc ggggaaaagg agaaggaaga gcaggaggag
120agagaaacca aaacacccga gtgtcccagt cacactcagc ctctgggagt
gtttctcttc 180ccacctaagc ccaaggatac ccttatgatt tctaggacac
ctgaggtgac ctgcgtcgtt 240gtggacgtga gtcaagagga cccagaggtc
cagtttaact ggtatgttga cggcgtggaa 300gtgcataatg caaaaactaa
accccgcgag gaacaattca attcaaccta ccgggtcgtt 360tctgtgttga
cagtgctgca tcaagattgg ctgaacggga aggagtataa gtgtaaagtc
420agtaataagg gactcccctc tagtatcgaa aaaactattt caaaggccaa
aggccagcct 480agagagccac aggtgtacac ccttcctcca tcccaagagg
agatgacaaa gaaccaggtg 540tctctgactt gtctcgtgaa ggggttctac
cctagtgaca tcgctgtcga atgggagtca 600aacggacagc cagagaataa
ttataagaca actcctcccg ttctggattc tgacggcagc 660ttctttctgt
actctaggct tactgtggac aaaagtcgct ggcaagaagg gaacgtcttt
720tcatgttctg ttatgcacga ggccttgcac aatcattata cacagaagtc
tctgagtctc 780tcactgggca aaggcggggg aggcagcggg ggaggcgggt
ccggaggcgg gggatctcat 840cccatccctg actccagtcc tctcctgcaa
ttcgggggcc aagtccggca gcggtacctc 900tacacagatg atgctcagca
gacagaagcc cacctggaga tcagggagga tgggaccgtg 960gggggcgctg
ctgaccagag ccccgaaagt ctcctgcagc tgaaagcctt gaagcctgga
1020gttattcaaa tcttgggagt caagactagt aggttcctgt gccagcggcc
agatggggcc 1080ctgtatggat ctctccattt tgaccctgag gcctgcagct
tccgggagga gatcagaccc 1140gacggataca atgtttacca gtccgaagcc
cacggcctcc ctctgcatct gcccgggaac 1200aagtctcctc accgggaccc
tgcccccaga ggacctgctc gcttcctgcc actcccaggc 1260ctgccccccg
cattgcctga gccacccgga atcctggccc cccagccccc tgatgtggga
1320tcctctgacc ctctgagcat ggtgacaggc ctggaggccg tgagaagccc
cagctacgct 1380tcc 1383781383DNAArtificial Sequencenucleic acid
molecule coding for DFD69 78cacggcgagg ggaccttcac aagcgacgtg
tcctcttacc tggaagagca ggccgctaag 60gaatttatcg catggctcgt caaaggaggc
ggggaaaagg agaaggaaga gcaggaggag 120agagaaacca aaacacccga
gtgtcccagt cacactcagc ctctgggagt gtttctcttc 180ccacctaagc
ccaaggatac ccttatgatt tctaggacac ctgaggtgac ctgcgtcgtt
240gtggacgtga gtcaagagga cccagaggtc cagtttaact ggtatgttga
cggcgtggaa 300gtgcataatg caaaaactaa accccgcgag gaacaattca
attcaaccta ccgggtcgtt 360tctgtgttga cagtgctgca tcaagattgg
ctgaacggga aggagtataa gtgtaaagtc 420agtaataagg gactcccctc
tagtatcgaa aaaactattt caaaggccaa aggccagcct 480agagagccac
aggtgtacac ccttcctcca tcccaagagg agatgacaaa gaaccaggtg
540tctctgactt gtctcgtgaa ggggttctac cctagtgaca tcgctgtcga
atgggagtca 600aacggacagc cagagaataa ttataagaca actcctcccg
ttctggattc tgacggcagc 660ttctttctgt actctaggct tactgtggac
aaaagtcgct ggcaagaagg gaacgtcttt 720tcatgttctg ttatgcacga
ggccttgcac aatcattata cacagaagtc tctgagtctc 780tcactgggca
aaggcggggg aggcagcggg ggaggcgggt ccggaggcgg gggatctcat
840cccatccctg actccagtcc tctcctgcaa ttcgggggcc aagtccggca
gcggtacctc 900tacacagatg atgctcagca gacagaagcc cacctggaga
tcagggagga tgggaccgtg 960gggggcgctg ctgaccagag ccccgaaagt
ctcctgcagc tgaaagcctt gaagcctgga 1020gttattcaaa tcttgggagt
caagactagt aggttcctgt gccagcggcc agatggggcc 1080ctgtatggat
ctctccattt tgaccctgag gcctgcagct tccgggagga gatcagaccc
1140gacggataca atgtttacca gtccgaagcc cacggcctcc ctctgcatct
gcccgggaac 1200aagtctcctc accgggaccc tgcccccaga ggacctgctc
gcttcctgcc actcccaggc 1260ctgccccccg cattgcctga gccacccgga
atcctggccc cccagccccc tgatgtggga 1320tcctctgacc ctctgagcat
ggtgacaggc ctggaggccg tgagaagccc cagctacgag 1380tcc
1383791383DNAArtificial Sequencenucleic acid molecule coding for
DFD112 79cacggcgagg ggaccttcac aagcgacgtg tcctcttacc tggaagagca
ggccgctaag 60gaatttatcg catggctcgt caaaggaggc ggggaaaagg agaaggaaga
gcaggaggag 120agagaaacca aaacacccga gtgtcccagt cacactcagc
ctctgggagt gtttctcttc 180ccacctaagc ccaaggatac ccttatgatt
tctaggacac ctgaggtgac ctgcgtcgtt 240gtggacgtga gtcaagagga
cccagaggtc cagtttaact ggtatgttga cggcgtggaa 300gtgcataatg
caaaaactaa accccgcgag gaacaattca attcaaccta ccgggtcgtt
360tctgtgttga cagtgctgca tcaagattgg ctgaacggga aggagtataa
gtgtaaagtc 420agtaataagg gactcccctc tagtatcgaa aaaactattt
caaaggccaa aggccagcct 480agagagccac aggtgtacac ccttcctcca
tcccaagagg agatgacaaa gaaccaggtg 540tctctgactt gtctcgtgaa
ggggttctac cctagtgaca tcgctgtcga atgggagtca 600aacggacagc
cagagaataa ttataagaca actcctcccg ttctggattc tgacggcagc
660ttctttctgt actctaggct tactgtggac aaaagtcgct ggcaagaagg
gaacgtcttt 720tcatgttctg ttatgcacga ggccttgcac aatcattata
cacagaagtc tctgagtctc 780tcactgggca aaggcggggg aggcagcggg
ggaggcgggt ccggaggcgg gggatctcat 840cccatccctg actccagtcc
tctcctgcaa ttcgggggcc aagtccggca gcggtacctc 900tacacagatg
atgctcagca gacagaagcc cacctggaga tcagggagga tgggaccgtg
960gggggcgctg ctgaccagag ccccgaaagt ctcctgcagc tgaaagcctt
gaagcctgga 1020gttattcaaa tcttgggagt caagactagt aggttcctgt
gccagcggcc agatggggcc 1080ctgtatggat ctctccattt tgaccctgag
gcctgcagct tccgggagga gatcagaccc 1140gacggataca atgtttacca
gtccgaagcc cacggcctcc ctctgcatct gcccgggaac 1200aagtctcctc
accgggaccc tgcccccaga ggacctgctc gcttcctgcc actcccaggc
1260ctgccccccg cattgcctga gccacccgga atcctggccc cccagccccc
tgatgtggga 1320tcctctgacc ctctgagcat ggtgacaggc ctggaggcca
acagaagccc cagctacgag 1380tcc 1383801380DNAArtificial
Sequencenucleic acid molecule coding for DFD114 80cacggcgagg
ggaccttcac aagcgacgtg tcctcttacc tggaagagca ggccgctaag 60gaatttatcg
catggctcgt caaaggaggc ggggaaaagg agaaggaaga gcaggaggag
120agagaaacca aaacacccga gtgtcccagt cacactcagc ctctgggagt
gtttctcttc 180ccacctaagc ccaaggatac ccttatgatt tctaggacac
ctgaggtgac ctgcgtcgtt 240gtggacgtga gtcaagagga cccagaggtc
cagtttaact ggtatgttga cggcgtggaa 300gtgcataatg caaaaactaa
accccgcgag gaacaattca attcaaccta ccgggtcgtt 360tctgtgttga
cagtgctgca tcaagattgg ctgaacggga aggagtataa gtgtaaagtc
420agtaataagg gactcccctc tagtatcgaa aaaactattt caaaggccaa
aggccagcct 480agagagccac aggtgtacac ccttcctcca tcccaagagg
agatgacaaa gaaccaggtg 540tctctgactt gtctcgtgaa ggggttctac
cctagtgaca tcgctgtcga atgggagtca 600aacggacagc cagagaataa
ttataagaca actcctcccg ttctggattc tgacggcagc 660ttctttctgt
actctaggct tactgtggac aaaagtcgct ggcaagaagg gaacgtcttt
720tcatgttctg ttatgcacga ggccttgcac aatcattata cacagaagtc
tctgagtctc 780tcactgggca aaggcggggg aggcagcggg ggaggcgggt
ccggaggcgg gggatctcat 840cccatccctg actccagtcc tctcctgcaa
ttcgggggcc aagtccggca gcggtacctc 900tacacagatg atgctcagca
gacagaagcc cacctggaga tcagggagga tgggaccgtg 960gggggcgctg
ctgaccagag ccccgaaagt ctcctgcagc tgaaagcctt gaagcctgga
1020gttattcaaa tcttgggagt caagactagt aggttcctgt gccagcggcc
agatggggcc 1080ctgtatggat ctctccattt tgaccctgag gcctgcagct
tccgggagga gatcagaccc 1140gacggataca atgtttacca gtccgaagcc
cacggcctcc ctctgcatct gcccgggaac 1200aagtctcctc accgggaccc
tgcccccaga ggacctgctc gcttcctgcc actcccaggc 1260ctgccccccg
cattgcctga gccacccgga atcctggccc cccagccccc tgatgtggga
1320tcctctgacc ctctgagcat ggtgaaccct tcccagggca gaagccccag
ctacgagtcc 1380814PRTArtificial SequencePosition 98-101 of FGF21
81Leu Leu Leu Glu1825PRTArtificial SequencePosition 170-174 of
FGF21 82Gly Pro Ser Gln Gly1 58325PRTArtificial SequenceLeader
sequence 83Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu
Cys Gly1 5 10 15Ala Val Phe Val Ser Pro Ser His Ala 20 25
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