Note: Descriptions are shown in the official language in which they were submitted.
DUAL-FUNCTION PROTEIN FOR LIPID AND BLOOD GLUCOSE
REGULATION
The present disclosure relates to a GLP-1-FGF21 dual-function protein and
related
pharmaceutical combination, and also relates to the use of said dual-function
protein for
preparing a medicament for treating type 2 diabetes, obesity, hyperlipidaemia,
fatty liver
disease and/or metabolic syndrome, and treatment of these diseases using the
medicament.
Glucagon-like peptide-1 (GLP-1) is an endocrine peptide consisting of 36 amino
acids
secreted by mammalian intestinal L cells, and stimulates insulin secretion
from pancreatic
beta cells in a glucose dependent manner by binding to and activating GLP-1
receptor (GLP-
1R), inhibits glucagon release from pancreatic alpha cells to maintain normal
glucose level.
In addition, it inhibits gastrointestinal movement and suppresses appetite
(Knudsen LB, J
Med Chem, 2004, 4128-4134). Native human GLP-1 is easily inactivated in vivo
by
dipeptidyl peptidase IV (DDP-IV), and has a short circulating half-life.
Exendin-4 is isolated
from the saliva of toxic lizards from South Africa and has 39 amino acids with
53%
homology to human GLP-1 and exerts a similar biological activity. Compared
with the
human GLP-1, Gly replaces Ala at the second position of N-telininus of Exendin-
4 which
enhances resistance of the peptide to the protcolytic degradation induced by
DDP-IV and
extends the circulating half-life in vivo. Exendin-4 has a special Trap-cage
structure at the
C-terminus, such that its binding affinity with GLP-1 receptor is
significantly higher than
that of human GLP-1 (Neidigh JW et al., Biochemistry, 2001, 40:13188-13200).
At the
equimolar concentration, Exendin-4 exhibits a stronger effect on promoting
insulin release
from pancreatic beta cells. Although there is slight difference in structure
and amino
sequence, both the blood glucose metabolic regulators have been marketed,
wherein most
predominant ones are Liraglutide of Novo Nordisk and Exenatide ofAstraZeneca.
Treatment
with Liraglutide or Exenatide 2-3 times daily can effectively control the
blood glucose level
1
Date Recue/Date Received 2020-07-02
in type 2 diabetes patients. However, the high injection frequency results in
high cost and
poor clinical compliance for patients. In order to prolong the in vivo half-
life and
bioavailability of GLP-1 and Exendin-4 analogs, Fc or HSA fusion technologies
have been
used for developing long-acting drugs. At present, marketed products are
Dulaglutide of Eli
Lilly and Albiglutide of GlaxoSmithKline. The most extensively used one in
clinic is
Dulaglutide, a GLP-1 hIgG4 Fc fusion protein (Dulaglutide), wherein its
average half-life is
up to 90 hours (Chinese patent CN 1802386 B), its clinical indication is type
2 diabetes, and
the recommended dose regimen is subcutaneous injection once a week. Clinical
study
showed that Dulaglutide could effectively control postprandial blood glucose
and
glycosylated hemoglobin of diabetic patients, and lower the body weight of
obese patients
by inhibiting appetite. However, varying degrees of gastrointestinal adverse
effects were
observed. Epidemiological investigation showed that the large percentage of
patients with
type 2 diabetes accompanied with nonalcoholic fatty liver disease and lipid
metabolism
disorder (Radaelli MG et al., J Endocrinol Invest, 2017, s40618). However, no
clinical study
demonstrated that human GLP-1 or Exendin-4 analogs have the effect of treating
fatty liver
and hyperlipidaemia independent of weight loss (Petit JIM, Diabetes Metab,
2017, 43, 2S28-
2S33). Therefore, GLP-1 products cannot completely satisfy all clinical needs
for patients
with type 2 diabetes.
The family of fibroblast growth factors (FGFs) has 22 members and 7
subfamilies,
wherein the FGF19 subfamily exerts physiological activity in an endocrine
manner, involves
the regulation and control of energy and cholic acid homeostasis, glucose and
lipid
metabolism, and phosphate and vitamin D homeostasis (Moore DD et al., Science,
2007,
316:1436-1438 and Beenken et al., Nature Reviews Drug Discover, 2009, 8:235).
FGF21 is
a member of FGF19 subfamily, and has 181 amino acids. The C-terminus of FGF21
binds
first to a co-factor 13-Klotho transmembrane protein, induces FGFR binding to
the N-
2
Date Recue/Date Received 2020-07-02
terminus of FGF21, then forms a stable FGF21/13-Klotho/FGFR complex, which
trigger
subsequent signaling pathway in vivo (Yie J et al., FEBS Lett, 2009, 583(1):19-
24 and
Micanovic R et al., J Cell Physiol, 2009, 219(2): 227-234). Under
physiological conditions,
FGF21 is to promote glucose utilization independent of insulin (Kharitonenkov
A et al., J
Clin Invest, 2005, 115(6): 1627-1635), to enhance insulin sensitization
(Duthchak PA et al.,
Cell, 2012, 148, 387-393), to inhibit de novo lipogenesis and promote the
fatty acid (3-
oxidation in liver, to decrease serum triglyceride level (Xu J et al.,
Diabetes, 2009, 58, 250-
259). In addition, FGF21 could decrease total cholesterol and low density
lipoprotein-
cholesterol contents in serum by inhibiting liver SREBP-2 synthesis to relieve
hypercholesteremia (Lin Z et al., Circulation, 2015, 131, 1861-1871).
In conclusion, FGF21 exerts multiple regulatory functions on metabolic
diseases, such
as obesity, type 2 diabetes, nonalcoholic fatty liver and hyperlipidaemia.
Meanwhile, FGF21
is the only discovered member without any mitogenic effect in this
superfamily, which
greatly reduces potential carcinogenicity risk in clinical applications (Wu X
et al., Proc Natl
Acad Sci USA, 2010, 170: 14158-14163). However, due to its unstable
physicochemical
property, native FGF21 does not possess druggability so far due to the
following reasons: (1)
native FGF21 protein has pool stability and is easily degraded by proteases in
vivo; (2)
FGF21 conformation is unstable with ease of aggregation, which increases
difficulty in
scale-up production; (3) native FGF21 has short circulating half-life, about
0.5-1 h in mice
and 2-3 h in Cynomolgus monkeys (Kharitonenkov A et al., J Clin Invest, 2005,
115: 1627-
1635). Various long-acting protein-engineering technologies are commonly used
for
prolonging the in vivo half-life of recombinant FGF21. For example,
conjugation of FGF21
and PEG molecule increases the molecular weight, lowers the glomerular
filtration rate, and
prolongs the in vivo retention time (see WO 2005/091944, WO 2006/050247, WO
2008/121563 and WO 2012/066075); FGF21 fuses to long chain fatty acid (which
can binds
3
Date Recue/Date Received 2020-07-02
to serum albumin) (see WO 2010/084169 and WO 2012/010553); preparation of an
agonist
antibody which specifically binds to FGFR or FGFR/13-klotho complex to mimic
the
mechanism of FGF21, and to activate FGF/FGFR signaling pathway (see WO
2011/071783,
WO 2011/130417, WO 2012/158704 and WO 2012/170438); FGF21 fuses to Fc fragment
to improve half-life (see WO 2004/110472, WO 2005/113606, WO 2009/149171, WO
2010/042747, WO 2010/129503, WO 2010/129600, WO 2013/049247, WO 2013/188181
and WO 2016/114633). At present, there is no marketed drug of long-acting
FGF21 protein,
but there are three long-acting FGF21 candidates in clinic trials, LY2405319
of Eli Lilly, PF-
05231023 of Pfizer and BMS986036 of Bristol-Myers Squibb. In the clinical
trials, for
patients with type 2 diabetes, LY2405319 and PF-05231023 had weight loss
effect and
decreased serum TG level, but had no positive therapeutic effect on blood
glucose (Gaich G
et al., Cell Metab, 2013, 18:333-340 and Dong JQ et al., Br J Clin Pharmacol,
2015, 80-
1051-1063). BM5986036 exhibited a good therapeutic effect on nonalcoholic
fatty liver, but
there was no experimental study on blood glucose control for patients with
type 2 diabetes.
Above-mentioned results showed that although the use of long-acting FGF21
protein alone
can exert many pharmacodynamic activities such as in body weight, nonalcoholic
fatty liver
and hyperlipidaemia. However, it cannot satisfy the blood glucose control
requirement
which is crucial in the treatment of patients with type 2 diabetes.
Recently, some studies reported that the combination of GLP-1 and FGF21 has a
synergistic effect on blood glucose control. For example, CN 102802657 A
disclosed that
the combination of GLP-1 and FGF21 can synergistically lower the blood glucose
level in
db/db mice. However, the combined usage of drugs not only increases the
administration
frequency for patients and reduces the patient compliance, but also greatly
increases
treatment costs. In addition, a dual-function protein prepared by fusing GLP-1
and FGF21
was also reported. In order to solve the issue that FGF21 is easily degraded
in vivo, scientists
4
Date Recue/Date Received 2020-07-02
generally introduce point mutations in native FGF21 molecule, but this
inevitably increases
the potential immunogenicity of the dual-function protein (WO 2017/074123 and
CN
104024273 B). Furthermore, the reported synergistic effect of FGF21 and GLP-1
generally
exhibited in terms of blood glucose control, but their synergistic effects in
terms of other
metabolic diseases, such as obesity, nonalcoholic fatty liver and lipid
metabolism disorder
are not investigated by comparing with marketed long-acting GLP-1 analogs. The
reasons
for lack of above-mentioned investigations may comprise the following: (1)
neither native
GLP-1 nor FGF21 is stable in vivo, and defection of structural integrity and
stability in any
molecule will eliminate the synergistic effect; (2) in the process of fusion
of GLP-1 and
FGF21 into a single protein, their respective three-dimensional conformation
needs to be
maintained to the maximum extent for preventing mutual interference, such that
the
functional synergy will be achieved, and this should be carefully considered
when designing
the molecule; (3) the functions of both GLP-1 and FGF21 depend on binding to
their
respective receptors, and it needs to be confirmed by a number of in vitro and
in vivo
experiments to clarify the conditions under which the dynamic equilibrium can
be achieved
among them. So far, there is no such report in published patents or other non-
patent
documents.
In conclusion, if a GLP-1-FGF21 dual-function protein drug that has enhanced
stability,
prolonged half-life and low immunogenicity can be developed in the art, then
the multiple
requirements of patients with type 2 diabetes for reducing blood glucose,
relieving hepatic
steatosis, reducing body weight and improving metabolic disorders of
circulating lipids can
be met.
Summary of the Disclosure
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Date Recue/Date Received 2020-07-02
The present disclosure provides a dual-function protein having a synergistic
effect in
terms of blood glucose and lipid regulations and comprising human GLP-1 analog
and
human FGF21, the preparation method therefor and the use thereof. The present
disclosure
solves the issues such as the defects relating to unstable structure and short
in vivo half-life
of native GLP-1 or FGF21, retains the strong hypoglycemic effect of GLP-1 and
the
physiological effects of FGF21 on insulin sensitization, weight loss, fatty
liver and
hypercholesteremia treatment, and relieves gastrointestinal adverse effects
caused by GLP-
1 to some extent.
In one embodiment of the present disclosure, a dual-function protein can
synergistically
regulate blood glucose and lipids, wherein said dual-function protein
comprises human
glucagon-like peptide-1 analog (abbreviated as GLP-1 analog hereafter), linker
peptide 1
(abbreviated as L1 hereafter), human fibroblast growth factor 21 (abbreviated
as FGF21
hereafter), linker peptide 2 (abbreviated as L2 hereafter) and human
immunoglobulin Fc
fragment (abbreviated as Fc fragment hereafter) sequentially from the N-
terminus to the C-
terminus; wherein the linker peptide 1 comprises a flexible peptide; the
linker peptide 2
comprises a flexible peptide and rigid peptide, the rigid peptide comprises at
least 1 rigid
unit, and the rigid unit comprises carboxyl terminal peptide of human
chorionic
gonadotropin 13-subunit or a truncated sequence thereof
In one embodiment of the present disclosure, a dual-function protein can
synergistically
regulate blood glucose and lipids, wherein said dual-function protein consists
of sequentially
human glucagon-like peptide-1 analog (abbreviated as GLP-1 analog hereafter),
linker
peptide 1 (abbreviated as Li hereafter), human fibroblast growth factor 21
(abbreviated as
FGF21 hereafter), linker peptide 2 (abbreviated as L2 hereafter) and human
immunoglobulin
Fc fragment (abbreviated as Fc fragment hereafter) from the N-terminus to the
C-terminus;
wherein the linker peptide 1 consists of a flexible peptide; the linker
peptide 2 consists of a
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Date Recue/Date Received 2020-07-02
flexible peptide and rigid peptide, the rigid peptide consists of at least 1
rigid unit, and the
rigid unit comprises carboxyl terminal peptide of human chorionic gonadotropin
13-subunit
or a truncated sequence thereof.
In the present disclosure, said "human GLP-1 analog" refers to an analog,
fusion
peptide, or derivative which are obtained by substituting, deleting or adding
one or more
amino acid residues on the amino acid sequence of human GLP-1 (as shown in SEQ
ID NO:
1) and maintains human GLP-1 activity. For example, said GLP-1 analog
comprises but is
not limited to the amino acid sequences as shown in SEQ ID NO: 2, 3, 4 or 5 in
the sequence
listing. In at least one embodiment of the present disclosure, said GLP-1
analog is shown in
SEQ ID NO: 2, and in another embodiment, said GLP-1 analog is shown in SEQ ID
NO: 5.
In the present disclosure, said "linker peptide 1 (L1)" is a short peptide
between GLP-
1 analog and FGF21 and has connecting function. In at least one embodiment,
said linker
peptide 1 is non-immunogenic, and generates enough distal distance between GLP-
1 analog
and FGF21, such that minimal steric hindrance effect is present, which does
not affect or not
affect severely correct folding and spatial conformation of GLP-1 analog and
FGF21. A
person skilled in the art can design linker peptides according to conventional
methods in the
art. In at least one embodiment, a flexible peptide comprising 2 or more amino
acids is used,
and the amino acids are selected from the following amino acids: Gly(G),
Ser(S), Ala(A)
and Thr(T); in at least one embodiment, said linker peptide 1 comprises G and
S residues.
The length of the linker peptide is very important for the activity of the
dual-function protein,
and in at least one embodiment, the linker peptide consists of 5-30 amino
acids. In a at least
one embodiment of the present disclosure, the amino acid sequence of said
linker peptide 1
is GGGGGGGSGGGGSGGGGS.
7
Date recue / Date received 202 1-1 1-25
In the present disclosure, said "FGF21" comprises the sequence as shown in SEQ
ID
NO: 6 in which the secreting leader signal of amino acid position 1-28 is
deleted; or
comprises the isoform sequence of SEQ ID NO: 6 in which the secreting leader
signal of
amino acid position 1-28 is deleted and which has G141S or L174P substitution.
In a at least
one embodiment of the present disclosure, said FGF21 comprises the amino acid
sequence
as shown in SEQ ID NO: 6 in which the secreting leader signal of amino acid
position 1-28
is deleted and has L174P substitution.
In the present disclosure, said "linker peptide 2 (L2)" is a short peptide
between FGF21
and Fc fragment and having connect function. Said linker peptide consists of a
flexible
peptide and a rigid peptide, wherein said flexible peptide comprises 2 or more
amino acid
residues which are selected from Gly(G), Ser(S), Ala(A) and Thr(T). In at
least one
embodiment, said flexible peptide comprises G and S residues. With regard to
the present
disclosure, preferably, the general structural formula of the amino acid
composition of said
flexible peptide is (GS)a(GGS)b(GGGS)c(GGGGS)d, wherein a, b, c and d are
integers
greater than or equal to 0, and a+b+c+d> 1.
In some embodiments of the present disclosure, said flexible peptide comprised
in said
L2 is selected from the following sequences:
(i) GGGGS;
(ii) GSGGGSGGGGSGGGGS;
(iii) GSGGGGSGGGGSGGGGSGGGGSGGGGS;
(iv) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS;
(v) GGGSGGGSGGGSGGGSGGGS;
(vi) GGSGGSGGSGGS.
In the present disclosure, said rigid peptide constituting said linker peptide
2 (L2)
consists of one or more rigid units, and said rigid units are selected from a
full-length or
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Date Recue/Date Received 2020-07-02
truncated sequence consisting of carboxyl terminal amino acids 113 to 145 of
human
chorionic gonadotropin I3-subunit (known as CTP rigid unit hereafter);
specifically, said CTP
rigid unit comprises the amino acid sequence as shown in SEQ ID NO: 7 or the
truncated
sequences thereof.
In at least one embodiment, said CTP rigid unit comprises at least 2
glycosylation sites;
for example, in one at least one embodiment of the present disclosure, said
CTP rigid unit
comprises 2 glycosylation sites, for example, said CTP rigid unit comprises 10
amino acids
of N-terminus of SEQ ID NO: 7, i.e. SSSS*KAPPPS*; or said CTP rigid unit
comprises 14
amino acids of C-terminus of SEQ ID NO: 7, i.e. S*RLPGPS*DTPILPQ; for another
example, in another embodiment, said CTP rigid unit comprises 3 glycosylation
sites, for
example, said CTP rigid unit comprises 16 amino acids of N-terminus of SEQ ID
NO: 7, i.e.
SSSS*KAPPPS*LPSPS*R; for another example, in another embodiment, said CTP
rigid
unit comprises 4 glycosylation sites, for example, said CTP rigid unit
comprises 28, 29, 30,
31,32 or 33 amino acids and starts at positions 113, 114, 115, 116, 117 or 118
and terminates
at position 145 of human chorionic gonadotropin 13-subunit. Specifically, said
CTP rigid unit
comprises 28 amino acids of N-terminus of SEQ ID NO: 7, i.e.
SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ. In the present disclosure, * represents
glycosylation sites. All possibilities represent independent embodiments of
the present
disclosure.
In some embodiments, the CTP rigid unit comprised in L2 of the present
disclosure can
preferably comprise one of the following sequences:
(i) CTP': SSSSKAPPPSLPSPSRLPGPSDTPILPQ;
(ii) CTP2: PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ;
(iii) CTP3: SSSSKAPPPS;
(iv) CTP4: SRLPGPSDTPILPQ;
9
Date Recue/Date Received 2020-07-02
(v) CTP5: SSSSKAPPPSLPSPSR.
In some embodiments, the CTP rigid unit provided in the present disclosure has
at least
70% homology to the native CTP amino acid sequence; In some embodiments, the
CTP rigid
unit provided in the present disclosure has at least 80% homology to the
native CTP amino
acid sequence; In some embodiments, the CTP rigid unit provided in the present
disclosure
has at least 90% homology to the native CTP amino acid sequence; In some
embodiments,
the CTP rigid unit provided in the present disclosure has at least 95%
homology to the native
CTP amino acid sequence.
In some embodiments of the present disclosure, L2 comprises 2, 3, 4 or 5 above-
mentioned CTP rigid units. In some embodiments of the present disclosure, L2
of said dual-
function protein comprises 2 CTP3 rigid unit: SSSSKAPPPSSSSSKAPPPS (CTP3-CTP3,
or
represented as (CTP3)2).
In the present disclosure, said "Fe fragment" is selected from the Fc
fragments of human
immunoglobulins IgG, IgM, IgA and variants thereof; in at least one
embodiment, is selected
from the Fc fragments of human IgGl, IgG2, IgG3 or IgG4 and variants thereof,
wherein
said human IgG Fc fragment (represented as vFc) comprises at least one amino
acid
modification located in wild type human IgG Fc, and the Fc variants have non-
lytic
characteristics, and show an extremely minimal Fc-mediated effector functions
(ADCC and
CDC functions) and/or enhanced binding affinity with FcRn receptor; most
preferably,
human IgG Fc variant is selected from the group of:
(i) vFcyl: hinge. CH2 and CH3 regions of human IgG1 containing Leu234Va1,
Leu235Ala and Pro331Ser mutations (the amino acid sequence as shown in SEQ ID
NO: 8);
(ii) vFcy2-1: hinge. CH2 and CH3 regions of human IgG2 containing Pro331Ser
mutation (the amino acid sequence as shown in SEQ ID NO: 9);
Date Recue/Date Received 2020-07-02
(iii) vFcy2-2: hinge, CH2 and CH3 regions of human IgG2 containing Thr250Gln
and
Met428Leu mutations (the amino acid sequence as shown in SEQ ID NO: 10);
(iv) vFcy2-3: hinge, CH2 and CH3 regions of human IgG2 containing Pro331Ser,
Thr250Gln and Met428Leu mutations (the amino acid sequence as shown in SEQ ID
NO:
11).
(v) vFcy4: hinge, CH2 and CH3 regions of human IgG4 containing Ser228Pro and
Leu235Ala mutations (the amino acid sequence as shown in SEQ ID NO: 12).
The Fc variants provided by the present disclosure comprises, but is not
limited to above
5 variants of (i) to (v), and also can be the combination or overlap of
functional variants
among same IgG subtypes, for example, the variant of above-mentioned (iv) is a
new
combination variant of IgG2 Fc obtained by overlapping the mutation sites in
(ii) and (iii).
The Fc variant (vFc) in the dual-function protein of the present disclosure
contains
human IgG, such as the hinge region and CH2 and CH3 regions of human IgGl,
IgG2 and
IgG4. Such CH2 region contains amino acid mutations at positions 228, 234, 235
and 331
(determined by EU numbering system). It is believed that these amino acid
mutations can
reduce Fc effector function. Human IgG2 does not bind to FcyR, but shows a
very weak
complement activity. The complement activity of Fcy2 variant having Pro331Ser
mutation
should be lower than that of native Fcy2, and is still an FcyR non-binder.
IgG4 Fc has some
defects in activation of complement cascade, and its binding affinity with
FcyR is lower than
that of IgG1 by about one order of magnitude. Compared with native Fcy4, the
Fcy4 variant
having Ser228Pro and Leu235Ala mutations should show the minimal effector
function.
Compared with native Fcyl, Fcyl having Leu234Val, Leu235Ala and Pro331Ser
mutations
also shows a reduced effector function. These Fc variants are more suitable
for preparing a
dual-function protein of FGF21 and analogs thereof than native human IgG Fc.
However,
positions 250 and 428 (positions determined by EU numbering system) contain
amino acid
11
Date Recue/Date Received 2020-07-02
substitutions, such that the binding affinity of Fe region with neonate
receptor FcRn is
increased, thus further prolonging the half-life (Paul R et al., J Biol Chem,
2004, 279:6213-
6216); the combination or overlap of above-mentioned two types of functional
variants
obtains new variants which have reduced effector function and prolonged half-
life. The Fc
variants of the present disclosure comprises, but is not limited to above-
mentioned mutations;
the substitutions at other sites can also be introduced, such that Fc has a
reduced effector
function and/or enhanced affinity with FcRn receptor, at the same time,
without causing
reduced functions/activities of Fc variants or adverse conformational changes,
and see
Shields RL et al., J Biol Chem, 2001, 276(9):6591-604 for common mutation
sites.
In one at least one embodiment of the present disclosure, the amino acid
sequence of
said dual-function protein is shown in SEQ ID NO: 13. In another at least one
embodiment
of the present disclosure, the amino acid sequence of said dual-function
protein is shown in
SEQ ID NO: 15.
The dual-function protein of the present disclosure is glycosylated;
preferably, said
dual-function protein is glycosylated by being expressed in mammalian cells;
in at least one
embodiment, said dual-function protein is glycosylated by being expressed in
Chinese
hamster ovary cells.
According to another embodiment of the present disclosure, provided is a DNA
encoding the above-mentioned dual function protein. In one at least one
embodiment of the
present disclosure, the DNA sequence encoding said dual-function protein is
shown in SEQ
ID NO: 14.
According to still another embodiment of the present disclosure, provided is a
vector.
The vector comprises the above-mentioned DNA.
12
Date Recue/Date Received 2020-07-02
According to still another embodiment of the present disclosure, provided is a
host cell.
The host cell comprises the above-mentioned vector, or is transfected with the
above-
mentioned vector.
In a particular embodiment of the present disclosure, the host cell is a CHO-
derived cell
strain DXB-11.
According to still another embodiment of the present disclosure, provided is a
pharmaceutical composition. The pharmaceutical composition comprises a
pharmaceutically
acceptable carrier, excipient or diluent, and an effective amount of the above-
mentioned
synergistic dual function protein.
According to another embodiment of the present disclosure, provided is a
method for
preparing or producing said dual-function protein from mammalian cell lines
(such as a
CHO-derived cell line), comprising following steps:
(a) introducing a DNA encoding the above-mentioned dual-function protein into
a
mammalian cell;
(b) screening a high-yield cell strain expressing more than 20 jig/b6 cells
within a
period of every 24 hours in the growth medium thereof from step (a);
(c) culturing the screened cell strain in step (b);
(d) harvesting the fermentation broth obtained from step (c), and purifying
the dual
function protein.
In at least one embodiment, said mammalian cell in step (a) is CHO cell; in at
least one
embodiment, said mammalian cell in step (a) is CHO-derived cell line DXB-11.
According to still another embodiment of the present disclosure, provided is
the use of
said dual-function protein in the preparation of a drug for treating FGF21
related diseases
and GLP-1 related diseases, and other metabolic, endocrinic and cardiovascular
diseases,
comprising obesity, types 1 and 2 diabetes, pancreatitis, dyslipidemia,
nonalcoholic fatty
13
Date Recue/Date Received 2020-07-02
liver disease, nonalcoholic steatohepatitis, insulin tolerance,
hyperinsulinemia, glucose
intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction,
high blood
pressure, cardiovascular disease, atherosclerosis, peripheral arterial
disease, stroke, cardiac
failure, coronary heart disease, nephropathy, diabetic complication,
neuropathy,
gastroparesis, and conditions associated with the severe inactivation
mutations of insulin
receptor; preferably, said diseases comprise obesity, types 1 and 2 diabetes,
dyslipidemia,
nonalcoholic fatty liver disease, nonalcoholic steatohepatitis and metabolic
syndrome.
Compared with existing products, the dual-function protein of the present
disclosure
has many advantages, which are demonstrated in detail by using, e.g., dual-
function protein
FP4I-2 of the present disclosure:
1. The half-life in vivo is prolonged, and blood glucose-lowering activity
in vivo is
maintained for a longer period of time. The glucose tolerance test performed
on C57BL/6
mice shows that at 144 h after a single dose of FP4I-2, FP4I-2 still exhibits
a good ability
for promoting glucose utilization, which is better than the commercially
available GLP-1
analog Liraglutide and Exenatide and native FGF21. Compared with GLP-1-Fc
fusion
protein Dulaglutide, FP4I-2 exhibits a more excellent blood glucose control
effect in the type
2 diabetes mice.
2. Improved safety and tolerability. Dulaglutide induces severe
gastrointestinal
adverse effects after the first administration. In db/db mice, the initial 24
hour food intake of
the mice after first administration of FP4I-2 is significantly elevated
relative to that of mice
administrated with Dulaglutide, which shows that the dual-function protein
FP4I-2 can
effectively relieve appetite inhibition induced by gastrointestinal adverse
effects.
3. Improved therapeutic effects of dual-function protein on fatty liver.
Compared
with Dulaglutide, FP4I-2 can significantly reduce the liver mass of db/db
mice, improve
liver function, and the mechanism does not completely depend on the appetite
inhibition
14
Date Recue/Date Received 2020-07-02
related to GLP-lanalog, demonstrating the physiological activity of FGF21.
Relative to
native FGF21, the in vivo half-life of FP4I-2 is significantly prolonged. In
the animal model,
the dose frequency of FP4I-2 is twice per week, which will improve the
clinical feasibility.
4. The
C-terminal sequence of FGF21 is crucial to its activity, most dual-proteins
reported in the prior art uses FGF21 N-terminal fusion, and free C-terminus is
beneficial to
maintain activity; however, the C-terminus of FGF21 also contains various
protease
hydrolysis sites, and is very easily degraded; exposed intact C-terminus is
more easily
attacked by protease and degraded. In order to overcome this problem, the
prior art avoids
using native FGF21, but introducing corresponding mutations to improve its
stability,
however, this inevitably increases the potential immunogenicity of dual-
function proteins.
In contrast, the dual-function protein constructed in the present disclosure
uses native FGF21,
and has an Fc fragment connected at its C-terminus. The dual-function protein
provided by
the present disclosure not only has a significantly prolonged in vivo half-
life in circulation,
but also has a synergistic effect in terms of blood glucose and lipid
regulations, which
suggests that the constructed dual-function protein well maintains the
properties of this two
active molecules, and has good stability. This benefits result from the new
type of linker
peptide among FGF21 and Fc variants; the linker peptide consists of a flexible
peptide and
a rigid peptide, and the CTP rigid unit contains multiple 0-carbohydrate side
chains, can
forms a relatively stable three-dimensional conformation, which can
effectively separate
FGF21 and Fc, thereby lowers the steric hindrance effect caused by Fc fragment
to the
utmost extent and keep relatively good FGF21 biological activity. In addition,
carbohydrate
side chain of CTP rigid unit can mask the enzymolysis site of FGF21. The
protective effect
lowers the sensibility of enzymolysis to proteases and achieves the purpose
for protein
stability.
Date Recue/Date Received 2020-07-02
5. Mutations on Fc only retains the long half-life property in
circulation of Fc,
reducing or eliminating ADCC and CDC effects (such as P33 1S), thus increases
the safety
of drug use. In addition, Fc variants (such as T250Q/M428L) having an enhanced
binding
affinity with neonate receptor (FcRn) can further prolong the half-life of
dual function
protein.
DETAILED DESCRIPTION OF THE DISCLOSURE
Human GLP-1 analog
The term "human GLP-1 analog" used herein refers to an analog, fusion peptide,
and
derivative which are obtained by substituting, deleting or adding one or more
amino acid
residues on the amino acid sequence of human GLP-1 (as shown in SEQ ID NO: 1)
and
maintain human GLP-1 activity. For example, said human GLP-1 analog comprises
but are
not limited to the amino acid sequences as shown in SEQ ID NO: 2, 3, 4 or 5 in
the sequence
listing.
Human FGF21
The term "human FGF21" used herein refers to a wild type human FGF21
polypeptide.
The sequence of the wild type FGF21 protein can be obtained from UNIPROT
database,
and the accession number is Q9NSA1. The precursor protein consists of 209
amino acids,
comprising a signal peptide (amino acids 1-28) and a mature protein (amino
acids 29-209).
US 2001012628 Al teaches the isoform or allelic form of the wild type FGF21
having
the substitution from Leu to Pro in the mature protein (in the present
disclosure, as shown in
positions 47-227 of SEQ ID NO: 13); another isoform of wild type FGF21 having
the
substitution from Gly to Ser (Gly at the position 141 of SEQ ID NO: 6 is
substituted or
replaced by Ser).
16
Date Recue/Date Received 2020-07-02
WO 2003/011213 teaches another isoform (see SEQ ID NO: 2 disclosed in WO
2003/011213, which has a signal peptide of 27 amino acid residues) having a
relatively short
signal peptide (in the present disclosure, Leu at position 23 of SEQ ID NO: 6
deleted).
In the present disclosure, the wild type FGF21 comprises SEQ ID NO: 6 and the
sequence of the mature protein portion (amino acids 29-209) of the isoform
having L 174P
or G141S substitutions after removing the leader peptide; in addition, also
comprised is the
full-length sequence of the precursor protein with the above-mentioned 27 or
28 amino acid
signal peptide added before those above sequences.
hCG-I3 carboxyl terminal peptide (CTP)
CTP is a short carboxyl terminal peptide of human chorionic gonadotropin (hCG)
(3-
subunit. Four reproduction-related polypeptide hormones follicle-stimulating
hormone
(FSH), luteinizing hotinone (LH), thyroid stimulating hormone (TSH) and
chorionic
gonadotropin (hCG) contain the same alpha subunit and the different specific
beta subunit
from each other. The in vivo half-life of hCG, compared with other three
hormones, is
prolonged, which is mainly due to the specific carboxyl terminal peptide (CTP)
of its beta
subunit (Fares FA et al., Proc Natl Acad Sci USA, 1992, 89: 4304-4308). Native
CTP
contains 37 amino acid residues, has 4 0-glycosylation sites, and has sialic
acid residue at
the terminus. Negative charged, highly sialylated CTP can resist the clearance
of kidneys on
same, thus prolong the in vivo half-life of same. However, the inventors
herein creatively
connects a rigid peptide including at least one CTP rigid unit with a flexible
peptide with a
suitable length, collectively as linker peptide 2 for connecting FGF21 and Fc
fragment.
N-terminal and C-terminal sequences of FGF21 are crucial to the functions of
FGF21.
The spatial conformation of FGF21 is complex and fragile, such that FGF21 has
a poor
stability, is easily degraded and aggregated; if FGF21 is fused to a ligand,
the steric hindrance
effect will interfere with the correct folding of FGF21, making the activity
of FGF21
17
Date Recue/Date Received 2020-07-02
significantly lowered or even lost, or more easily to generate a polymer.
Adding CTP rigid
units between FGF21 and Fc variants is equivalent to adding a section of rigid
linker peptide.
In one embodiment, it ensures that the FGF21 fused at N-terminus will not
affect the binding
site of Fc variants and FcRn, thereby not affecting the half-life; in
addition, the Protein A
binding site of Fc is very important for the purification step in the
preparation process, and
connecting CTP rigid units ensures that N-terminus fused FGF21 also will not
"mask" its
binding site with protein A. In another embodiment, the addition of CTP rigid
units also
makes Fc fragments with about 25 KD not interfere with the correct folding of
N-terminus
fused FGF21, and not cause the biological activity/functions of FGF21 lowered
or lost. This
may be interpreted that a CTP rigid polypeptide with multiple carbohydrate
side chains,
which, relative to the random coil of (GGGGS)n of flexible linker peptides,
can form a stable
three-dimensional conformation, and this "barrier- effect promotes FGF21 and
Fc fragment
to fold and form a correct three-dimensional conformation independently, thus
not affecting
their respective biological activity. In another embodiment, the protective
effect of
carbohydrate side chain of CTP can lower the sensibility of linker peptides to
proteases, such
that the dual-function protein is not easily degraded in the linker region. In
addition, CTP is
derived from native human hCG, has no immunogenicity, therefore, relative to
non-native
encoded amino acid sequence, is more suitable for being used as a linker
peptide.
IgG Fc variant
Non-lytic Fc variants
Fc elements are derived from the constant region Fc of immunoglobulin IgG, and
have
an important effect in immune defense for eliminating pathogens. The effector
function of
IgG mediated by Fc is exerted via two mechanisms: (1) binding to the Fc
receptors (FcyRs)
on the cell surface, digesting pathogens by phagocytosis or lysis, or by
killer cells through
antibody-dependent cytotoxic (ADCC) pathway, or (2) binding to Clq of the
first
18
Date Recue/Date Received 2020-07-02
complement component Cl, triggering the complement dependent cytotoxic (CDC)
pathway,
thereby lysing pathogens. Among four human IgG subtypes, IgG1 and IgG3 can
effectively
bind to FeyRs, the binding affinity of IgG4 with FcyRs is relatively low, and
the binding of
IgG2 with FeyRs is too low to be determined, and therefore, human IgG2 almost
has no
ADCC effect. In addition, human IgG1 and IgG3 also can effectively bind to C 1
q, thereby
activating the complement cascade. The binding of human IgG2 with Clq is
relatively weak,
and IgG4 does not bind with C 1 q (Jefferis R et al., Immunol Rev, 1998, 163:
59-76), and
therefore, the CDC effect of human IgG2 is also weak. There is no native IgG
subtype which
is very suitable for constructing GLP-1-FGF21 dual function protein. In order
to obtain a
non-lytic Fc without effector function, the most effective method is
performing mutation
modification on the complement and receptor binding domain of Fc fragment,
regulating the
binding affinity of Fc with related receptors, reducing or eliminating ADCC
and CDC effects,
only retaining the long half-life property of Fc in circulation, and not
generating cytotoxicity.
For additional mutation sites comprised in non-lytic Fc variants, one can
refer to RL et al., J
Biol Chem, 2001, 276(9):6591-604 or Chinese invention patent CN
201280031137.2.
Fc variants having an enhanced binding affinity with neonate receptor (FcRn)
The plasma half-life of IgG depends on its binding with FcRn, and generally,
they bind
at pH 6.0, and dissociate at pH 7.4 (plasma pH). By the study of the binding
site of the two,
the binding site on IgG with FcRn is modified, such that the binding ability
thereof is
increased at pH 6Ø It is proven that the mutations of some residues in human
Fey domain
which is important for binding with FcRn can increase the serum half-life. It
has been
reported that the mutations of T250, M252, S254, T256, V308, E380, M428 and
N434 can
increase or reduce FcRn binding affinity (Roopenian et al., Nat Rview
Immunology, 2007,
7:715-725). South Korea patent no. KR 10-1027427 discloses Trastuzumab
(Herceptin,
Genentech) variants with an increased FcRn binding affinity, and these
variants comprise
19
Date Recue/Date Received 2020-07-02
one or more amino acid modifications selected from 257C, 257M, 257L, 257N,
257Y, 279Q,
279Y, 308F and 308Y. South Korea patent no. KR 2010-0099179 provides
bevacizumab
(avastin, Genentech) variants, and these variants comprise amino acid
modifications of
N4345, M252Y/M428L, M252Y/N4345 and M428L/N4345, and show an increased half-
life in vivo. In addition, Hinton et al. also find that 2 mutants of T250Q and
M428L can
increase the binding with FcRn by 3 and 7 times respectively. Mutating the 2
sites at the
same time, the binding will be increased by 28 times. In rhesus monkeys, the
mutants of
M428L or T250QM/428L show the plasma half-life in vivo is increased by 2 times
(Paul R.
Hinton et al., J Immunol, 2006, 176:346-356). For additional mutation sites
comprised in Fc
variants having an enhanced binding affinity with neonate receptor (FcRn), one
can refer to
Chinese invention patent CN 201280066663.2. In addition, in some studies
performing
T250Q/M428L mutations on the Fc fragment of five humanized antibodies, the
interaction
between Fc and FcRn is improved, and in the subsequent in vivo pharmacokinetic
test, it
finds that using subcutaneous injection administration, the pharmacokinetic
parameters of
Fc mutation antibodies are improved compared with wild type antibodies, such
as an
increased in vivo exposure, lowered clearance rate and improved subcutaneous
bioavailability (Datta-Mannan A et al., MAbs. Taylor & Francis, 2012, 4(2):
267-273).
Terms "FGF21-related conditions" and "GLP-1-related conditions" comprise
obesity,
types 1 and 2 diabetes, pancreatitis, dyslipidemia, nonalcoholic fatty liver
disease,
nonalcoholic steatohepatitis, insulin tolerance, hyperinsulinemia, glucose
intolerance,
hyperglycemia, metabolic syndrome, acute myocardial infarction, high blood
pressure,
cardiovascular disease, atherosclerosis, peripheral arterial disease, stroke,
cardiac failure,
coronary heart disease, nephropathy, diabetic complication, neuropathy,
gastroparesis, and
conditions associated with the severe inactivation mutations of insulin
receptor.
Date Recue/Date Received 2020-07-02
"Conditions associated with the severe inactivation mutations of insulin
receptor"
describe the conditions of subjects with insulin receptor (or a direct
downstream possible
protein thereof) mutation, wherein said mutation results in a severe insulin
tolerance, but
generally no obesity which is common in type 2 diabetes. In many embodiments,
subjects
with these conditions exhibit the symptom complex of types 1 and 2 diabetes.
Therefore, the
involved subjects are divided into several types according to the severity,
comprising: type
A diabetes resistance, type C insulin resistance (AKA HAIR-AN syndrome),
Rabson-
Mendenhall syndrome, Donohue's syndrome or Leprechaunism. These conditions are
associated with a very high endogenous insulin level, and results in an
elevated blood
glucose level. Therefore, in the involved subjects, there are many clinical
features associated
with "insulin toxicity", wherein the clinical features comprise androgen
excess, polycystic
ovarian syndrome (PCOS), hirsutism and acanthosis nigricans (overgrowth of
wrinkly skin
and pigmentation).
"Diabetic complications" is the dysfunction of other tissue/organs of the body
induced
by chronic hyperglycemia, such as diabetic nephropathy, diabetic neuropathy,
diabetic feet
(foot ulcers and low blood circulation) and eye lesions (retinopathy).
Diabetes also increases
the risks of heart disease and osteoarticular diseases. The other long-term
complications of
diabetes comprise skin, digestive, sexual function, teeth and gums disease.
"Metabolic syndrome (MS)" is the morbidness caused by abnormal metabolic
parameters, comprising: (1) abdominal obesity or overweight; (2)
atherosclerosis and
dyslipidemia, such as hypertriglyceride and reduction in high density
lipoprotein cholesterol
(HDL-C); (3) hypertension; (4) insulin resistance and/or abnormal glucose
tolerance. In
some criteria, microalbuminuria, hyperuricemia, pro-inflammatory state (C-
reactive protein)
and pro-thrombogenesis state (increase in Fibrinogen and Plasminogen inhibitor-
1) are also
comprised.
21
Date Recue/Date Received 2020-07-02
"Dyslipidemia" is a lipoprotein metabolic disorder, comprising the
oversynthesis or
defect of lipoprotein. Dyslipidemia can exhibit as the elevated concentration
of total
cholesterol, low density lipoprotein (LDL) cholesterol and triglycerides, and
the reduced
concentration of high density lipoprotein (HDL) cholesterol.
"Nonalcoholic fatty liver disease (NAFLD)" is a liver disease which is not
associated
with abused alcohol consumption and is characterized in hepatocellular
steatosis.
"Nonalcoholic steatohepatitis (NASH)" is a liver disease which is not
associated with
abused alcohol consumption and is characterized in hepatocellular steatosis
accompanied by
lobular inflammation and fibrosis.
"Atherosclerosis" is an angiopathy and is characterized in lipid deposits
irregularly
distributed on endangium of large and medium-sized arteries, which results in
hemadostenosis, and eventually develops into fibrosis and calcification.
Brief Description of the Drawinks
Fig. 1. shows the nucleotide sequence of dual-function protein FP4I-2 of
SpeI/EcoRI
fragment in PCDNA3.1 expression vector according to the embodiments of the
present
disclosure and the deduced amino acid sequence, consisting of alpha 1
microglobulin leader
peptide (1-19), GLP-1 analog (20-47), Li (48-65), FGF21 mature protein (66-
246), L2 (247-
301) and IgG2 Fc (302-524).
Fig. 2a. Reduced SDS-PAGE electrophoretogram of GLP-1-FGF21 dual-function
protein FP41-2.
Fig. 2b. SEC-HPLC spectrogram of GLP-1-FGF21 dual-function protein FP4I-2.
Fig. 3a. Glucose tolerance test curve of GLP-1-FGF21 dual-function proteins
FP4I-1
and FP4I-2 16 h after a single injection (means SEM, n = 8).
22
Date Recue/Date Received 2020-07-02
Fig. 3b. Glucose tolerance test iAUC of GLP-1-FGF21 dual-function proteins
FP4I-1
and FP4I-2 16 h after a single injection (means SEM, n = 8); statistical
difference symbols:
compared with the control group, *P < 0.05, and **P < 0.01.
Fig. 4a. Glucose tolerance test curve of GLP-1-FGF21 dual-function proteins
FP4I-1
and FP4I-2 96 h after a single injection (means SEM, n = 8).
Fig. 4b. Glucose tolerance test iAUC of GLP-1-FGF21 dual-function proteins
FP4I-1
and FP4I-2 96 h after a single injection (means SEM, n = 8); statistical
difference symbols:
compared with the control group, *P < 0.05, and **P < 0.01.
Fig. 5a. Glucose tolerance test curve of GLP-1-FGF21 dual-function proteins
FP4I-1
and FP4I-2 144 h after a single injection (means SEM, n = 8).
Fig. 5b. Glucose tolerance test iAUC of GLP-1-FGF21 dual-function proteins
FP4I-1
and FP41-2 144 h after a single injection (means SEM, n = 8); statistical
difference symbols:
compared with the control group, *P < 0.05, and **P < 0.01.
Fig. 6a. Glucose tolerance test curve of Exendin4-FGF21 dual-function proteins
FP4I-
3 16 h after a single injection (means SEM, n = 8).
Fig. 6b. Glucose tolerance test iAUC of Exendin4-FGF21 dual-function proteins
FP4I-
3 16 h after a single injection (means SEM, n = 8); statistical difference
symbols: compared
with the control group, *P < 0.05, and **P < 0.01; compared with the
Dulaglutide group, #P
<0.05, and "P < 0.01.
Fig. 7a. Glucose tolerance test curve of Exendin4-FGF21 dual-function proteins
FP4I-
3 96 h after a single injection (means SEM, n = 8).
Fig. 7b. Glucose tolerance test iAUC of Exendin4-FGF21 dual-function proteins
FP4I-
3 96 h after a single injection (means SEM, n = 8); statistical difference
symbols: compared
with the control group, *P < 0.05, and **P < 0.01; compared with the
Dulaglutide group, #P
<0.05, and "P < 0.01.
23
Date Recue/Date Received 2020-07-02
Fig. 8a. Glucose tolerance test curve of Exendin4-FGF21 dual-function proteins
FP4I-
3 144 h after a single injection (means SEM, n = 8).
Fig. 8b. Glucose tolerance test iAUC of Exendin4-FGF21 dual-function proteins
FP4I-
3 144 h after a single injection (means SEM, n = 8); statistical difference
symbols:
compared with the control group, *P < 0.05, and **P < 0.01; compared with the
Dulaglutide
group, #P < 0.05, and 44P < 0.01.
Fig. 9. Effect of GLP-1-FGF21 dual-function proteins FP4I-1 and FP4I-2 on 24 h
food
intake in db/db mice after first administration (means SEM, n = 6);
statistical difference
symbols: compared with the control group, *P < 0.05, and **P < 0.01; compared
with the
Dulaglutide group, #P < 0.05, and "P < 0.01.
Fig. 10. Effect of multiple administrations of GLP-1-FGF21 dual-function
proteins
FP4I-1 and FP4I-2 on glycated hemoglobin in db/db mice (means SEM, n = 6);
statistical
difference symbols: compared with the control group, *P < 0.05, and **P <
0.01; compared
with the Dulaglutide group, #P < 0.05, and "P < 0.01.
Fig. 11. Effect of multiple administrations of GLP-1-FGF21 dual-function
proteins
FP4I-1 and FP4I-2 on accumulative food intake in db/db mice (means SEM, n =
6);
statistical difference symbols: compared with the control group, *P < 0.05,
and **P < 0.01;
compared with the Dulaglutide group, #P < 0.05, and 44P < 0.01.
Fig. 12. Effect of GLP-1-FGF21 dual-function proteins FP4I-1 and FP4I-2 on
body
weight in obese mice induced by high-fat diet (means SEM, n = 7);
statistical difference
symbols: compared with the obesity control group, *P < 0.05, and **P < 0.01;
compared
with the Dulaglutide group, #P < 0.05, and 44/' < 0.0/; compared with the FP4I-
1 group, &P
<0.05, and "P < 0.01.
Fig. 13. Effect of GLP-1-FGF21 dual-function proteins FP4I-1 and FP4I-2 on
liver
mass in obese mice induced by high-fat diet (means SEM, n = 7); statistical
difference
24
Date Recue/Date Received 2020-07-02
symbols: compared with the obesity control group, *P < 0.05, and **P < 0.01;
compared
with the Dulaglutide group, #P < 0.05, and "P < 0.01.
Fig. 14. Effect of GLP-1-FGF21 dual-function proteins FP4I-1 and FP4I-2 on
liver
triglyceride content in obese mice induced by high-fat diet (means SEM, n =
7); statistical
difference symbols: compared with the obesity control group, *P < 0.05, and
**P < 0.01;
compared with the Dulaglutide group, #P < 0.05, and 44P < 0.01.
Fig. 15. Effect of GLP-1-FGF21 dual-function proteins FP4I-1 and FP4I-2 on
serum
triglyceride in obese mice induced by high-fat diet (means SEM, n = 7);
statistical
difference symbols: compared with the obesity control group, *P < 0.05, and
**P < 0.01;
compared with the Dulaglutide group, #P < 0.05, and "P < 0.01; compared with
the FP4I-1
group, &P < 0.05, and "P < 0.01.
Fig. 16. Effect of GLP-1-FGF21 dual-function proteins FP4I-1 and FP4I-2 on
serum
total cholesterol content in obese mice induced by high-fat diet (means SEM,
n = 7);
statistical difference symbols: compared with the obesity control group, *P <
0.05, and **P
<0.01; compared with the Dulaglutide group, #P < 0.05, and "P < 0.01.
Fig. 17. Effect of GLP-1-FGF21 dual-function proteins FP4I-1 and FP4I-2 on
serum
low density lipoprotein cholesterol content in obese mice induced by high-fat
diet (means
SEM, n = 7); statistical difference symbols: compared with the obesity control
group, *P <
0.05, and **P < 0.01; compared with the Dulaglutide group, 4/' < 0.05, and "P
< 0.01.
The present disclosure is further described below in combination with specific
embodiments. It is to be understood that these embodiments serve only to
illustrate the
present disclosure and are not limiting the scope of the present disclosure.
In the following
embodiments, experimental methods without specifying specific conditions are
generally
performed under conventional conditions, for example, those described in
Sambrook et al.,
Date Recue/Date Received 2020-07-02
Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor
Laboratory Press,
1989), or the conditions recommended by the manufacturer.
Generally, the dual-function protein of the present disclosure is prepared
synthetically.
The nucleotide sequence according to the present disclosure, a person skilled
in the art can
conveniently use various known methods to prepare the encoding nucleic acid of
the present
disclosure. These methods are for example but not limited to: PCR, and DNA
artificial
synthesis etc., the specific methods can refer to J. Sambrook, "Molecular
Cloning: A
Laboratory Manual". As an embodiment of the present disclosure, a method
comprising
fragment synthesis of nucleotide sequences, followed by overlap extension of
PCR can be
used for constructing the encoding nucleic acid sequence of the present
disclosure.
Also provided in the present disclosure is an expression vector comprising a
sequence
encoding the dual-function protein of the present disclosure and a regulatory
element
transcriptionally linked thereto. Said "transcriptionally linked" or
"transcriptionally linked
to" refer to such a condition that some parts of a linear DNA sequence can
regulate or control
the activity of other parts in the same linear DNA sequence. For example, if a
promoter
controls the transcription of a sequence, then the promoter is
transcriptionally linked to the
encoding sequence.
The expression vector can use commercially available ones, for example but not
limited
to: vectors pcDNA3, pIRES, pDR and pUC18 which can be used for expression in
an
eukaryotic system. A person skilled in the art can select a suitable
expression vector
according to the host cell.
According to the restriction map of the known expression vector, a person
skilled in the
art can insert the sequence encoding the dual-function protein of the present
disclosure into
a suitable restriction site to prepare the recombinant expression vector of
the present
disclosure following conventional methods via restriction digestion and
ligation.
26
Date Recue/Date Received 2020-07-02
Also provided in the present disclosure is a host cell expressing the dual-
function
protein of the present disclosure, wherein said host cell comprises the
sequence encoding the
dual-function protein of the present disclosure. In at least one embodiment,
said host cell is
a eukaryotic cell, for example but not limited to CHO, a COS cell, a 293 cell
and a RSF cell
etc. As a at least one embodiment of the present disclosure, said cell is a
CHO cell, which
can well express the dual-function protein of the present disclosure, and the
dual-function
protein with a good binding activity and stability can be obtained.
Also provided in the present disclosure is a method for preparing the dual-
function
protein of the present disclosure using recombinant DNA, the steps thereof
comprise:
1) Providing a nucleic acid sequence encoding the synergistic dual function
protein;
2) Inserting the nucleic acid sequence of 1) into a suitable expression
vector, and
obtaining a recombinant expression plasmid;
3) Introducing the recombinant expression plasmid of 2) into a suitable host
cell;
4) Culturing the transformed host cell under a condition suitable for
expression;
5) Collecting the supernatant, and purifying the dual-function protein
product.
To introduce said encoding sequence into the host cell one can use multiple
known
technologies in the art, for example but not limited to: calcium phosphate
precipitation,
protoplast fusion, liposome transfection, electroporation, microinjection,
reverse
transcription method, phage transduction method, and alkali metal ion method.
With respect to the culture of and expression in the host cell can refer to
Olander RM
Dev Biol Stand, 1996, 86:338. Cells and debris in the suspension can be
removed by
centrifugation, and the supernatant is collected. Agarose gel electrophoresis
technique can
be used for identification.
The dual-function protein prepared as described herein can be purified to have
a
substantially homogeneous property, such as has a single band on SDS-PAGE
27
Date Recue/Date Received 2020-07-02
electrophoresis. For example, when the recombinant protein is expressed for
secretion, a
commercially available ultrafiltration membrane (such as products of Millipore
and Pellicon
etc.) can be used to separate said protein, wherein firstly, the expression
supernatant is
concentrated. The concentrate can be purified by the method of gel
chromatography, or by
the method of ion exchange chromatography, for example, by anion exchange
chromatography (DEAE etc.) or cation exchange chromatography. The gel matrix
can be
common matrices for protein purification, such as agarose, glucan, and
polyamide etc. Q- or
SP-groups is a relatively ideal ion exchange group. Finally, the above-
mentioned purified
product can be further refined and purified by the methods of hydroxyapatite
adsorption
chromatography, metal chelate chromatography, hydrophobic interaction
chromatography
and reversed high performance liquid chromatography (RP-HPLC). All the above-
mentioned purification steps can be used in different combination in order to
make the
protein purity substantially homogeneous.
The expressed dual-function protein can be purified using an affinity column
containing
a specific antibody, receptor or ligand of said dual function protein.
According to the
properties of the affinity column, conventional methods, such as high salt
buffer and
changing pH etc. can used to elute the fusion polypeptide binding to the
affinity column.
Optionally, at the amino terminus or carboxyl terminus of said dual function
protein, one or
more polypeptide fragments also can be contained as protein tags. Any suitable
tags can be
used in the present disclosure. For example, said tags can be FLAG, HA, HAL c-
Myc, 6-
His or 8-His etc. These tags can be used for purifying the dual function
protein.
Non-limitipe Exemplary Embodiments
28
Date Recue/Date Received 2020-07-02
EXAMPLES
Example 1: Construction of an expression plasmid of the synergistic dual
function protein
All gene sequences encoding alpha 1 microglobulin secretion leader signal, GLP-
1
analog, Li, FGF21 mature protein, L2 (comprising a flexible linker unit and
rigid linker unit)
and human IgG Fc variants were optimized using CHO preferred codons and the
full-length
gene sequences were synthesized. There are a SpeI at the 5' and a EcoRI at the
3' for
subcloning the target gene encoding the fusion protein into the expression
vector PXY1A1
modified from PCDNA3.1 (Fig. 1 exemplarily set forth the nucleotide sequence
of the dual-
function protein FP4I-2 and the translated amino acid sequence). The
expression plasmid
contained the early promoter of cytomegalovirus, leading to high expression of
exogenous
genes in mammalian cells. The plasmid also contained a selective marker
conferring
kanamycin resistance in bacteria, and G418 resistance in mammalian cells.
Furthermore, the
host cell carrying DHFR- mutant, PXY1A1 expression vector contained the gene
of mouse
dihydrofolate reductase (DHFR) could amplify the fusion gene and DHFR gene in
the
absence of methotrexate (MTX) (see U.S. Patent No. 4,399,216).
Various dual-function proteins comprising GLP-1 and FGF21 were constructed.
Here,
three are exemplified: FP4I-1, FP4I-2 and FP4I-3. The amino acid composition
is shown in
Table 1 (L1 and L2 were underlined, and mutated amino acids in Fc variants
were boxed).
Table 1. Amino acid composition of each synergistic dual function protein
FP4I- 1 HGEGT FT S DVS SYLEGQAAKEF IAWLVKGRGGGGSGGGGSGGGGSH
PI PDS S PLLQFGGQVRQRYLYT DDAQQTEAHLE I REDGTVGGAADQ
S PE SLLQLKALKPGVIQ I LGVKT SRFLCQRPDGALYGSLHFDPEAC
SFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPL
PGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRS PSYASGGGG
SGGGGSGGGGSGGGGSGGGGSSSSSKAPPPSSSSSKAPPPSESKYG
PPC P PC PAPE FAGGP SVFLF PPKPKDTLMI SRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKGLPSS I EKT I SKAKGQPREPQVYTLPPSQEEMTK
NQVSLTCLVKGFY PSDIAVEWE SNGQPENNYKTT PPVLDSDGS EEL
YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
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Date Recue/Date Received 2020-07-02
HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGS
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAAD
QSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEA
CSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLP
LPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASGSG
FP4I-2 GGGSGGGGSGGGGSGGGGSGGGGSSSSSKAPPPSLPSPSRLPGPSD
TPILPOVECPPCPAPPVAGPSVFLFPPKPKDQLMISRTPEVICVV
VDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLIVV
HQDWLNGKEYKCKVSNKGLPAIIIEKTISKTKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVIIHEALHNHYTQKSLSLSP
GK
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGGG
SGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEI
REDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGA
LYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRD
PAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQ
GRSPSYASGSGGGGSGGGGSGGGGSGGGGSGGGGSSSSSKAPPPSL
FP4I-3
PSPSRLPGPSDTPILPQVECPPCPAPPVAGPSVFLFPPKPKDQLM
ISRTPEVICVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPADEKTISKTKGQP
REPQVYTLPPSREEMTKNQVSLICLVKGFYPSDIAVEWESNGQPEN
NYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNH
YTQKSLSLSPGKVDKSRWQQGNVFSCSVIIHEALHNHYTQKSLSLS
PGK
Example 2: Expression of the dual-function protein in a transfected cell line
A recombinant expression vector plasmid was transfected into a mammalian host
cell
line to express the synergistic dual function protein. In order to stabilize
the high expression,
a preferred host cell line was DHFR defective CHO-cell (U.S. Patent No.
4,818,679). In the
present example, the host cell was selected from CHO-derived cell line DXB11.
A preferred
transfection method was electroporation, but other methods such as calcium
phosphate and
liposome-induced transfection also can be used. A Gene PulserTM
electroporation apparatus
(Bio-Rad Laboratories, Hercules, CA) setting at 300 V of electric field and
1500 pF'd of
capacitance was used in the present experiment and 50 pg pure expression
plasmid was
mixed with 5 x 107 CHO cells in the cuvette. Two days after the transfection,
a selection
Date recue / Date received 202 1-1 1-25
medium containing 0.6 mg/mL G418 was used. Quantitative ELISA using anti-human
IgG
Fc was applied to screen the transfectants with the resistance to G418. Anti-
human FGF21
or anti-human GLP-1 by ELISA was used to quantify expression of the dual-
function protein.
A 96 well culture plate was subjected to the limiting dilution, the well
generating a high level
of the dual-function protein was subcloned.
In order to achieve a relatively high expression of the dual-function protein,
it was
appropriate to use the DHFR gene inhibited by MTX for co-amplification. In
another
selection medium containing incremental concentrations of MTX, the gene of the
dual-
function protein was co-amplified with the DHFR gene. The subclone with a
positive DHFR
expression was subjected to the limiting dilution, the selection pressure was
gradually
increased and the transfectant which can grow in a medium with up to 6 p,M MTX
was
selected. The secreting rate of transfectant was determined and the cell line
with a high
expression of exogenous protein was screened out. The cell lines with
secretory rate higher
than about 10 (preferably about 20) g/106 (i.e. a million) cells/24 hours was
subjected to an
adaptive suspension culture in serum-free medium, then the dual-function
protein was
purified by a specified medium.
Example 3: Purification and qualification of the dual-function protein
This example describes the exemplary purification and qualification methods of
FP4I-
2. The cell culture supernatant was subjected to clarifying treatments, such
as high speed
refrigerated centrifugation and 0.22 pm sterile filtration etc., then purified
by three
chromatograph steps including protein A, anion exchange and hydrophobic
chromatography,
the specific method was as follows: In the first step, protein A was used for
capture, wherein
the equilibrium solution was PBS buffer, the eluant was a citrate buffer at pH
3.5, then the
eluted protein was neutralized by 1 M Tris solution. In the intermediate
purification process,
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Date Recue/Date Received 2020-07-02
high resolution anion exchange packing material Q SepharoseTM HP (GE company)
was
selected to remove residual impurity proteins. A combined mode was used, that
is, 20 mM
Tris-HC1, 0.2 M NaCl, pH 7.5 solution was used for rinsing, and 20 mM Tris-
HC1, 0.3 M
NaCl, pH 7.5 solution was used for elution. In the fine purification step,
Butyl Sepharose FF
(GE) was selected to remove polymers; due to different hydrophobic properties
of FP4I-2
monomer and polymer, the monomer with weak hydrophobic property flowed through
directly, but the polymer with high hydrophobic property bound to the medium;
hydrophobic
chromatography was selected as flow through mode, and the equilibrium solution
was PBS
buffer.
The qualitative analysis result is shown in Figs. 2a and 2b. The theoretical
molecular
weight of single stranded FP4I-2 was about 53 KD, due to the absence of
glycosylation sites,
under the reducing condition, SDS-PAGE electrophoresis showed that the actual
mass of the
single stranded FP4I-2 molecule was about 70 KB. FP4I-1 and -3 were prepared
by the same
method.
Example 4: Effect of a single injection of the dual-function protein on
glucose utilization
in C57BL/6 mice
8 weeks aged male C57BL/6J mice at SPF grade (purchased from Beijing HFK
Bioscience Ltd.) were selected. Housing conditions: temperature 22-25 C,
relative humidity
45-65%, and 12h-light/dark cycle. After acclimation for 1 week, mice were
randomly
divided into control group, Dulaglutide 120 nmol/kg group, FP4I-2 120 nmol/kg
group and
FP4I-1 120 nmol/kg group (n = 7) according to body weight. The mice in the
treatment
groups were injected subcutaneously with corresponding drug solutions, while
the mice in
the control group were injected subcutaneously with PBS buffer. After the
injection, mice in
each group were fasted for 16 h, and then glucose tolerance test was
performed. The fasting
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blood glucose values of the mice were determined followed by an
intraperitoneal injection
of a 2g/kg glucose solution, the blood glucose values were determined at 15
min, 30 min, 60
min, 90 min and 120 min after glucose injection, and the increased area below
the curve and
above the baseline (iAUC) was calculated by the trapezoidal method. The
glucose tolerance
test was further performed on the mice of each group at 96 h and 144 h after
the
administration, and the method was the same as above. The data were
represented as means
SEM, and analyzed using SPSS18. 0 statistical software. For the Gaussian
distribution data,
statistical comparison of the means among the groups was performed using one-
way
ANOVA, followed by LSD test for the homogeneity of variance or Dunnet T3 test
for the
heterogeneity of variance; non-parametric test was used for the Non-Gaussian
distribution
data. P < 0.05 represented a significant statistical difference.
As shown in Figs.3a and 3b, FP4I-1 and FP41-2 significantly improved glucose
utilization in the mice at 16 h after administration when compared with the
control group (P
<0.01). It can be known from Figs.4a and 4b that FP4I-1 and FP4I-2 also can
significantly
improve the glucose utilization level in mice at 96 h after administration as
well (P < 0.01).
It can be known from Figs.5a and 5b that FP4I-1 and FP4I-2 significantly
improved the
glucose utilization level in mice even at 144 h after administration (P <
0.05). The results
showed that in the case of a suddenly increased glucose level in vivo, the GLP-
1-FGF21
dual-function protein had a rapid response to the glucose level and normalized
it to the
physiological level by promoting release and secretion of insulin with a long-
acting activity,
and therefore it could be used for treating diabetes and the complications
induced by the
absolute or relative deficiency of insulin. When the mice were fasted for 16 h
after FP4I-1
or FP4I-2 administration, no shock or death due to hypoglycemia were noted in
any mouse,
indicating that the dual-function protein would not result in hypoglycemic
symptoms as
insulin.
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Date Recue/Date Received 2020-07-02
In addition, the activity of Exendin4-FGF21 dual-function protein FP4I-3 on
the
glucose utilization was determined by above-mentioned method as well. C57BL/6
mice were
divided into the control group, Dulaglutide group and FP4I-3 group.
Corresponding drug
solutions (120nmol/kg) were administrated subcutaneously to the mice in
Dulaglutide group
and FP4I-3 group, respectively, and PBS buffer was administrated to the mice
in the control
group. The glucose tolerance test was performed at 16 h, 96 h, and 144 h after
the injection.
As shown in Figs. 6a and 6b, 16 h after the administration, compared with the
control group,
FP4I-3 significantly improved the glucose utilization (P < 0.01), but the
activity was
significantly weaker than that of Dulaglutide (P < 0.01). As shown in Figs. 7a
and 7b, 96 h
after the administration, FP4I-3 significantly improved the glucose
utilization in mice (P <
0.01) though the efficacy was significantly lower than Dulaglutide as well (P
< 0.01). As
shown in Figs. 8a and 8b, 144 h after the administration, the ability of FP41-
3 to improve
glucose utilization in mice was not observed (P> 0.05).
The glucose tolerance test of FP4I-3 in animals demonstrated that Exendin-4
did not
display a synergistic effect with FGF21. The hypoglycemic effect of FP4I-3 was
significantly weaker than that of Dulaglutide indicated that the circulating
half-life of FP4I-
3 was shorter than Dulaglutide. In contrast, the preferred GLP-1-FGF21 dual-
function
proteins FP4I-2 and FP4I-1 had a relatively strong stability in vivo, and were
not easily
degraded and inactivated, and maintained a longer in vivo pharmacodynamic
activity relative
to Exendin4-FGF21 dual-function protein FP4I-3. Above results indicated that
the
combination modes of three functional components, GLP-1 analogs, FGF21 and Fc
fragment
in the dual-function protein were not random and arbitrarily, wherein the
selection of GLP-
1 analogs, the structure of linker peptide, the fusion sequence, even the
difference of
glycosylation pattern would affect accuracy and stability of the dual-function
protein
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Date Recue/Date Received 2020-07-02
conformation to varying degrees, and it determined whether the active
molecules were
functionally synergetic and the half-life was prolonged or not.
Example 5: Hypoglycemic effect of dual-function protein in db/db mice
8 weeks aged male db/db mice were purchased from Shanghai SLAC Laboratory
Animal Ltd. Housing conditions: temperature 22-25 C, relative humidity 45-65%,
and 12 h-
light/dark cycle. After housed individually for 1 week as acclimation, the
mice were divided
into 4 groups according to body weight, blood glucose and food intake: control
group,
Dulaglutide group, FP4I-1 group and FP4I-2 group (n = 7). Mice in the control
group were
injected subcutaneously with PBS buffer, and mice in other groups were
injected
subcutaneously with 120 nmol/kg corresponding drug solutions (twice per week,
totally 8
times). Daily food intake of each mouse was recorded. At the end of the dosing
period, mice
were fasted for 16 hours, 5 pL whole blood sample was collected from the eye
socket to
measure glycosylated hemoglobin. The data were represented as means standard
error (
s), and were analyzed using SPSS 18.0 statistical software. For the data
follow Gaussian
distribution, one-way analysis of variance was used for comparing mean
difference among
groups, followed by LSD test for the homogeneity of variance or Dunnet T3 test
for the
heterogeneity of variance; non-parametric test was used for the data follow
non-normal
distribution. P < 0.05 represented a significant statistical difference.
As shown in Fig. 9, compared with Dulaglutide group, after the first
administration, the
food intakes within 24 hour of the mice in FP4I-1 and FP41-2 groups were
significantly
elevated (P < 0.01). The results showed that GLP-1-FGF21 dual-function protein
could
significantly relieve symptoms of severe gastrointestinal adverse effects
induced by the first
administration of long-acting GLP-1 receptor agonist drugs. As shown in Fig.
10, FP4I-1,
FP4I-2, Dulaglutide group can significantly lower the glycosylated hemoglobin
values of
Date Recue/Date Received 2020-07-02
db/db mice (P < 0.01), and the glycosylated hemoglobin values of mice in FP4I-
1 and FP4I-
2 groups were significantly lower than that in Dulaglutide group (P < 0.05).
db/db mouse is
a spontaneously hyperglycemic animal model with a severe insulin tolerance.
The GLP-1-
FGF21 dual-function protein exhibited a better property than Dulaglutide in
the long-term
glycemic control. Based on the data in Example 4, the insulinotropic activity
of GLP-1-
FGF21 dual-function protein was not significantly better than Dulaglutide.
Wild type FGF21
exhibited a good insulin sensitization effect in the hypeinsulinemic-
euglycemic clamp test
(Xu J et al., Diabetes, 2009, 58:250-259), but there was no direct evidence
showing that
Dulaglutide had an insulin sensitization effect in vivo. In conclusion, the
superiority in blood
glucose control exhibited by GLP-1-FGF21 dual-function protein should be the
result of
synergistic effect of GLP-1 analog promoting release and secretion of insulin
and FGF21
enhancing insulin sensitivity. As shown in Fig. 11, in the experimental
period, the cumulative
food intake of mice in FP4I-1 and FP4I-2 groups was significantly higher than
that in
Dulaglutide group (P < 0.05), the results showed that in the condition of
excluding factors
intervening food intake, the blood glucose control activity of FP4I-1 and FP4I-
2 groups on
type 2 diabetes should be higher than that of Dulaglutide.
Example 6. Therapeutic effects of the dual-function protein on weight loss,
hepatic
steatosis and lipid metabolism disorder in obese mice induced by high-fat diet
8 weeks aged C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal
Ltd. Housing conditions: temperature 22-25 C, relative humidity 45-65%, and
lighting time
12 h/d. After acclimation for 1 week, 7 mice were selected and fed with low-
fat diet
(D12450B, Research Diets), and other mice were fed with high-fat diet (D12451,
Research
Diets). 40 weeks later, obese mice were subjected to adaptive feeding with
single
animal/cage for 1 week, then the obese mice were divided into five groups
according to body
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Date Recue/Date Received 2020-07-02
weight and weekly food intake: obese control group, Dulaglutide group, high
fat diet pair-
fed group, FP4I-1 group and FP4I-2 group (n = 7). In the experiment, the
amounts of daily
diet per mouse in high fat diet pair-fed, FP4I-land FP4I-2 groups were
consistent with daily
food intake per mouse in Dulaglutide group. Mice in the obese control group
and high fat
diet pair-fed group were injected subcutaneously with PBS buffer solution, and
mice in other
groups were injected subcutaneously with 120 nmol/kg corresponding drug
solutions, once
every 6 days, and totally 2 times. The body weight of each mouse was recorded
before and
after the dosing period. At the end of the dosing period, mice in each group
were fasted for
16 hours, whole blood was collected from the eye socket, and centrifuged at
2000 xg for 15
min to obtain serum. Serum lipid profiles were determined by an automatic
biochemical
analyzer. Liver tissue was excised, washed with normal saline, then removed
residual liquid
with filter paper and weighed. About 50 mg liver tissue at the same part of
each live was
taken, and the triglyceride content was determined using the Folch method. The
results were
represented in the form of triglyceride content per mg liver tissue. The data
were represented
as means SEM, and analyzed using SPSS18.0 statistical software. For the
Gaussian
distribution data, statistical comparison of the means among the groups was
performed using
one-way ANOVA, followed by LSD test for the homogeneity of variance or Dunnet
T3 test
for the heterogeneity of variance; non-parametric test was used for the Non-
Gaussian
distribution data. P < 0.05 represented a significant statistical difference.
As shown in Figs. 12 to 17, after administrated with Dulaglutide, body weight,
liver
mass, liver triglyceride, triglycerides, total cholesterol and low density
lipoprotein-
cholesterol contents in serum were significantly lowered (P<0.01) in the obese
mice induced
by high-fat diet. Dulaglutide could cause severe gastrointestinal adverse
effects and
suppressed appetite by regulating central nervous system, resulted in
reduction in food intake.
In this example, daily supplied the same amount of diet to the mice in the
high-fat diet pair-
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Date Recue/Date Received 2020-07-02
fed group as the corresponding mice in Dulaglutide group, despite the
parameters mentioned
above were significantly lowered when compared with that in the obese control
group, there
was no significant statistical difference from that of Dulaglutide group (P >
0.05). The
results showed that the effects of Dulaglutide on weight loss, hepatic
steatosis and lipid
metabolic disorder substantially depended on inhibition of appetite without
any other
mechanisms. The obese mice in FP4I-1 group and FP4I-2 group were given the
same amount
of diet as the corresponding mice in Dulaglutide group, compared with
Dulaglutide group,
body weight and serum triglyceride level of the mice in FP4I-2 group were
significantly
decreased (P < 0.01), which demonstrated that FP4I-2 had additional functions
of reducing
fatty acid synthesis and promoting fatty acid metabolism and utilization in
vivo. The results
indicated that FP4I-2 could be used for treating obesity and obesity-induced
metabolic
syndrome.
Compared with Dulaglutide group, liver mass and liver triglyceride content of
mice in
FP4I-2 group were significantly decreased (P < 0.01 or P < 0.05), which
demonstrated that
FP4I-2 effectively reduced the excessive accumulation of triglyceride in
liver, improved
liver function. The results indicated that FP4I-2 could be used for treating
various liver
diseases induced by hepatic steatosis, such as nonalcoholic fatty liver,
nonalcoholic
steatohepatitis, liver fibrosis and liver cirrhosis.
Compared with Dulaglutide group, both total serum cholesterol and low density
lipoprotein-cholesterol content of mice in FP4I-2 group were significantly
reduced (P < 0.01
or P < 0.05), indicating that FP4I-2 can be used for treating
hypercholesteremia and relevant
cardiovascular and cerebrovascular diseases, such as hypertension, coronary
heart disease,
chronic heart failure, cerebral infarction and atherosclerosis. Compared with
Dulaglutide
group, the body weight, liver mass, liver triglyceride content, serum
triglycerides, total
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Date Recue/Date Received 2020-07-02
cholesterol and low density lipoprotein cholesterol levels in FP4I-1 group
were mildly
decreased but no significant differences were observed.
The present study demonstrated that FP4I-1 and FP4I-2 could treat obesity,
fatty liver
disease and lipid metabolic disorder via the physiological activity of FGF21,
and was not
completely dependent on the food intake regulation effect of GLP-1 analogs;
the therapeutic
effect of FP4I-2 in the obese mice was superior to Dulaglutide, which
indicated that it could
compensate for the deficiency of Dulaglutide in the clinic. In conclusion, the
therapeutic
mechanisms of FP4I-2 are more abundant than that of Dulaglutide, which is more
suitable
for the requirement of diversified clinical therapy.
This disclosure provides merely exemplary embodiments of the disclosure. One
skilled in the art will readily recognize from the disclosure and claims, that
various changes,
modifications and variations can be made therein without departing from the
spirit and scope
of the disclosure as defined in the following claims.
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