Note: Descriptions are shown in the official language in which they were submitted.
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
COMPOSITIONS CONTAINING FUSION PROTEIN OF ALBUMIN AND
ANALOGS THEREOF, METHODS FOR MAKING AND USING THE SAME
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. Patent Application 15/249,346 filed
August
26, 2016, the contents of which are incorporated herein by reference.
U.S. Patent Application 15/249,346 is a continuation-in-part of
PCT/U52016/019950,
which claims the benefit of priority from U.S. Provisional Patent Application
Serial No. US
62/121,487 filed on February 26, 2015, the contents of each of which are
incorporated herein
by reference.
U.S. Patent Application 15/249,346 also claims benefit from Taiwanese Patent
Application No. 105106088 filed February 26, 2016, which also claims benefit
of priority
from U.S. Provisional Patent Application No. 62/121,487 filed on Feb 26, 2015,
the contents
of each of which are incorporated herein by reference.
FIELD OF INVENTION
The present invention relates to a fusion protein comprising a somatostatin,
or its
analogue or derivatives, a linker or spacer and an albumin, or its analogue or
variant.
The present invention also relates to recombinant fusion proteins containing a
human
.. serum albumin moiety, and a somatostatin moiety, separated by a spacer
sequence and
analogues thereof.
BACKGROUND OF THE INVENTION
Somatostatin ("SST") is a secretory product of a variety of endocrine and non-
.. endocrine tissues and is widely distributed throughout the body.
Somatostatin inhibits
pituitary, pancreatic, and gastrointestinal hormone secretion release, as well
as cytokine
production, intestinal motility and absorption, vascular contractility, and
cell proliferation.
Recent studies have found that SST has use as a treatment for cancer,
inhibiting tumor
growth, inhibiting the proliferation of endocrine tumors, and many other solid
tumors, such
.. as breast cancer, colorectal cancer, liver cancer, lung cancer, endocrine
cancer,
neuroendocrine cancers, pancreatic cancer and prostate cancer. The
somatostatin molecule
1
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
has two biologically active forms: somatostatin-14 (SST-14), the cyclic
tetradecapeptide, and
somatostatin-28 (SST-28), an N-terminally elongated form of SST-14. SST-14 is
a cyclic
peptide with a length of 14 residues, containing a disulfide linkage between
cysteines at
positions 3 and 14. SST-28 is an N-terminal extension form (28 residues) of
the same
precursor that is proteolytically cleaved to generate SST-14. Although the two
have similar
activity, their respective potency and histological characteristics vary. For
example, SST-14
displays more pronounced inhibition of glucagon and gastrin, while SST-28
displays more
pronounced inhibition of growth hormone and insulin action. Both forms of
somatostatin
exert their respective biological functions through receptors on target cells
and intracellular
pathways. Five subtypes of somatostatin receptors (SSTR 1-5) have been
recognized, with
two spliced variants for SSTR2: SSTR2A and SSTR2B, with a different carboxyl
terminus.
The beneficial effects of somatostatin in the treatment of certain
hypersecretory
endocrine disorders, and its anti-proliferation effect on tumors are well
recognized. However,
the half-life of somatostatin in vivo is only 2-3 minutes due to enzymatic
degradation and
endocytosis, limiting clinical utility of somatostatin. In the past decade,
numerous stable
somatostatin analogs have been developed. For example, octreotide and
lanreotide are used
in treatment of growth hormone (GH)-secreting adenomas and carcinoids.
However,
therapeutic limitations still exist due to altered binding affinity to SSTRs.
As a result, there
remains a need in the art for somatostatin constructs that achieve high in
vivo half-life while
maintaining a desirable binding affinity to SSTRs.
Albumin, the most abundant protein in the blood plasma, is produced in the
liver as a
monomeric protein of 67 kDa and responsible for 80% of the colloid osmotic
pressure of
plasma. Human granulocyte colony stimulating factor (G- CSF), human growth
hormone
(GH), human insulin, human interferon-a-2b (INF-2b), and interleukin-28B (IL-
28B) fused
with HSA were used effectively to construct long-acting therapeutic drug
candidates.
However, the comparative studies between HSA fusion proteins and the parent
molecules in
the biological and molecular mechanisms are less reported.
Chinese patent applications CN102391376A and CN102675467A, both hereby
incorporated by reference, disclose somatostatin-albumin fusion proteins.
However, there
remains a need for further development of somatostatin-albumin fusion
proteins.
2
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
SUMMARY OF THE INVENTION
The present invention provides somatostatin-albumin fusion proteins and
analogues
thereof and methods of producing and using the same. Constructs prepared
according to the
invention include an albumin (or an analog thereof) moiety, a somatostatin
moiety (SST-14,
SST-28), and a spacer, such as a spacer or linker peptide, separating the two
moieties.
A fusion protein according to the invention is also described as a polypeptide
herein.
The polypeptide according to the invention may optionally include, in certain
embodiments,
one or more non-naturally occurring amino acids or amino acid residues.
The somatostatin-albumin fusion proteins and analogues thereof broadly include
a
human SST peptide moiety, a linker or spacer, and a human albumen moiety. The
SST
peptide moiety can include analogues and derivatives thereofõ that actively
inhibit the
activity of human growth hormone. Optionally, the SST peptide moiety is
obtained from
natural or synthetic sources. The albumin moiety is, e.g., human albumin
and/or active
fragments or subdomains thereof. The linker or spacer is selected to enhance
the stability of
the somatostatin-albumin fusion protein. More particularly, the somatostatin-
albumin fusion
proteins and analogues thereof have a structure as follows.
The invention provides for a fusion protein comprising:
an SST;
an L; and
an ALB,
wherein,
SST is a somatostatin, its analogue or derivative;
L is a spacer or a linker; and
ALB is an albumin, its analogue or variant.
Preferably, the inventive fusion protein is isolated and purified.
Optionally, the ALB component of the inventive fusion protein is mammalian
serum
albumin. In one embodiment, the mammalian serum albumin is SEQ ID NO: 25, or a
sequence having at least 85 % sequence identity thereto.
In other particular embodiments, the inventive fusion protein is selected from
the
group consisting of:
SST-(L)xi-ALB (I);
3
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
ALB -(L)x) -SST (II);
[SST-(L)x)] yi -ALB (III);
ALB- [(L)x) -SST] yl (IV);
[ S S T- (L)x 1] yi -ALB - [(L)x2-SST] y2 (V);
[SST-(L)x)] yi -ALB -[(L)2-SST] y2-(L)3-ALB (VI);
[SST-(L)x)] yi -ALB -[(L)2-SST] y2-(L)3-ALB- [(L)4-S ST] y3 (VII);
ALB-(L)) - [ S ST-(L)x2] yi -ALB (VIII);
ALB-(L)) - [ S ST-(L)x2] yi -ALB - [(L)x3-S ST] y2-(L)x) -ALB (IX);
and
ALB-(L)xi-[SST-(L)x2]yi-ALB-RL)x3-SST]y2-(L)xi-ALB-RL)x4-S ST] y3 (X);
wherein,
x 1, x2, x3, x4, yl, y2, or y3 is independently zero or an integer selected
from 1-10, or
more particularly from 1-5, or an integer from 1-4, provided that there is at
least one
L present in the nucleotide sequence encoding an albumin-somatostatin fusion
protein.
In alternative embodiments, the inventive fusion protein comprises an SST that
is
either naturally occurring or synthetically manufactured.
In a further embodiment, the SST of the inventive fusion protein comprises one
or
more tandem repeats of a sequence encoding SST-14 or SST-28, represented by
SEQ ID
NOS: 17 or 18, respectively, or a sequence having at least 85% identity to
either of these
sequences.
The SST moiety is optionally SST-14 or SST-28.
In a further embodiment, the fusion protein comprises an L that is either
flexible or
alpha helically structured polypeptide linker or spacer.
In a further embodiment, the fusion protein comprises an L that is a
polypeptide
having 2-100 amino acids. The linkers or spacers according to a further
embodiment of the
invention encompass peptides covalently linked to somatostatin on one terminal
and to
albumin on another terminal.
The terms "linker" or "spacer" are used interchangeably herein to refer to
short amino
acid sequences used to separate multiple domains in a single protein. Absence
of linkers
between two or more discrete domains in a protein may result in reduced or
improper
functionality of the protein domains e.g., a reduction in catalytic activity
or binding affinity
4
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
for a receptor/ligand, due to the steric hindrance. Linking protein domains in
the chimeric
proteins using an artificial linker can increase the space between the
domains. Preferably, the
linker or spacer is selected independently of the somatostatin and albumin.
The linker L is either a flexible or alpha helically structured polypeptide
linker or
spacer. In certain embodiments, L contains at least one GGGGS, A(EAAAK)4A,
(AP)n,
wherein n is an integer selected from 10-34, (G)8, (G)5, or any combination
thereof.
The albumin-somatostatin fusion constructs described herein may also include a
signal peptide sequence ("SP"). Signal peptides are understood to refer to
short amino acid
sequences present at the N-terminus of a polypeptide that direct the cellular
placement of a
newly-synthesized protein. For example, signal peptides may lead to a protein
being
localized to a given intracellular region (e.g., the nucleus), inserted into a
membrane (e.g., the
cell membrane or the endoplasmic reticulum) or secreted from the cell. Besides
directing
localization, signal peptides may also be incorporated into recombinant
proteins in order to
improve stability, modify expression levels, and to aid in the proper folding
of the
recombinant proteins. The signal peptide sequence of the precursor protein is
usually
removed by signal peptidase in the host cell to produce a mature protein.
The albumin-somatostatin fusion constructs described herein may also include
an
affinity or purification tag as part of the polypeptide sequence to facilitate
purification. Such
tags are used as part of affinity chromatographic methods, e.g., high
performance liquid
chromatography (HPLC) in order to purify a protein sample from a crude
biological source.
Suitable purification tags include, but are not limited to: poly-histidine
(e.g., His-6 or H6),
glutathione-S-transferase (GST), maltose-binding protein (MBP), chitin binding
protein
(CBP), FLAG-tag (FLAG octapeptide). When it is necessary to remove the
affinity tag from
the fusion protein, specific enzymatic cleavage site can be introduced in the
linker region.
Enzymes commonly used for removal of affinity tags include, but are not
limited to: factor
Xa, entrokinase, thrombin, TEV protease, and rhinovirus 3C protease.
In a further embodiment, the invention provides a nucleotide sequence encoding
a
polypeptide comprising:
an SST;
an L; and
an ALB,
5
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
wherein,
SST is a somatostatin or its analogues or derivatives;
L is a spacer or a linker; and
ALB is an albumin or its analogues or variants.
In particular embodiments the inventive nucleotide encodes a polypeptide that
is
selected from the group consisting of,
SST-(L)xi-ALB (I);
ALB-(L)xi-SST (II);
[SST-(L)Ayi-ALB (III);
ALB-[(L)xi-SST] yl (IV);
[SST-(L)xi]yi-ALB-[(L)x2-SST] y2 (V);
[S ST-(L)Ayi-ALB-[(L)x2-SST]y2-(L)o-ALB (VI);
[SST-(L)Ayi-ALB-[(L)x2-SST]y2-(L)o-ALB-[(L)x4-SST] y3 (VII);
ALB-(L)xi-[SST-(L)x2]yi-ALB (VIII);
ALB-(L)xi- [SST-(L)x2]yi-ALB-[(L)3-SST]y2-(L)xi-ALB (IX);
and
ALB-(L)xi-[SST-(L)xflyi-ALB-[(L)x3-SST]y2-(L)xi-ALB-[(L)x4-SST] y3 (X);
wherein,
each of xl, x2, x3, x4, yl, y2, or y3 is independently zero or an integer
selected from 1-10, or more particularly from 1-5 or from 1-4, provided that
there is
at least one L present in the polypeptide.
In a further embodiment, the nucleotide sequence encodes a fusion protein
wherein
the SST comprises one or more tandem repeats of a sequence encoding SST-14 or
SST-28,
represented by SEQ ID NOS: 17 or 18, respectively, or a sequence having at
least 85%
identity to either of these sequences.
The invention is further contemplated to include an expression vector, e.g., a
plasmid
construct, a host cell comprising the expression vector, that is capable of
expressing the
inventive albumin-somatostatin fusion protein. The host cell can be a suitable
bacterial host
cell, a suitable mammalian host cell, a suitable plant host cell, or a
suitable insect host cell.
The invention also provides for methods of treating a disease or disorder of
endocrine
release in a mammal, such as in a human subject, by administering an effective
amount of a
6
CA 03032995 2019-02-04
WO 2018/038803
PCT/US2017/039477
pharmaceutical composition comprising the inventive fusion protein, wherein
the disease or
disorder of endocrine release is a condition that responds to the
administration of
somatostatin.
For example, the disease or disorder is a cancer selected from the group
consisting of
breast cancer, colorectal cancer, liver cancer, endocrine cancer,
neuroendocrine cancers,
pancreatic cancer, prostate cancer, brain cancer, and lung cancer. In certain
embodiments,
the cancer expresses somatostatin receptor type 1, 2, 3, 4 or 5.
It should also be understood that singular forms such as "a," "an," and "the"
are used
throughout this application for convenience, however, except where context or
an explicit
statement indicates otherwise, the singular forms are intended to include the
plural. Further,
it should be understood that every journal article, patent, patent
application, publication, and
the like that is mentioned herein is hereby incorporated by reference in its
entirety and for all
purposes.
All numerical ranges should be understood to include each and every numerical
point
within the numerical range, and should be interpreted as reciting each and
every numerical
point individually. The endpoints of all ranges directed to the same component
or property
are inclusive, and intended to be independently combinable.
As used herein, the term "about" means within 10% of the reported numerical
value,
preferably within 5% of the reported numerical value.
The phrase "consisting essentially of" means that the composition or method
may
include additional ingredients and/or steps, but only if the additional
ingredients and/or steps
do not materially alter the basic and novel characteristics of the claimed
composition or
method.
The SST and albumin fusion proteins of present application provide advantages
over
natural SST of (a) higher in vivo stability, (b) higher binding affinity to
SST receptors, (c)
higher protein expression yield, and (d) better
pharmacokinetic/pharmacodynamics behavior.
Before the present invention is described in detail below, it is to be
understood that
this invention is not limited to the particular methodology, protocols and
reagents described
herein, as these may vary. It is also to be understood that the terminology
used herein is for
the purpose of describing particular embodiments only, and is not intended to
limit the scope
of the present invention, which will be limited only by the appended claims.
Unless defined
7
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
otherwise, all technical and scientific terms used herein have the same
meanings as
commonly understood by one of ordinary skill in the art.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the pharmacokinetic profile of SST in rat:
wherein
the diamond (*) labeled curve represents the data from Rat #6;
the triangle (A) labeled curve represents the data from Rat #7;
the square (M) labeled curve represents the data from Rat #8;
the "Y" axis is Ln(Ct/C0) that represents the natural log of the measured
plasma
concentration of SST at time;
"t" (Ct) divided by the initial measured plasma concentration (CO) of SST; and
the "X" axis is plasma sampling time ('t") in hours.
Note that at certain time points, the detection of SST plasma concentration
was below limits
of quantitation
FIG. 2 illustrates bi-phasic pharmacokinetic profile of SST Fusion Protein in
rat
(Black dotted line distinguishes between a-Phase (0-0.5 hour) and 13-Phase
(0.75-4 hours):
wherein,
the diamond (*) labeled curve represents the data from Rat #1;
the triangle (A) labeled curve represents the data from Rat #2;
the star (*) labeled curve represents the data from Rat #3;
the square (M) labeled curve represents the data from Rat #4;
the "x" ( x ) labeled curve represents the data from Rat #5;
the "Y" axis is Ln(Ct/C0) that represents the natural log of the measured
plasma
concentration of SST fusion protein at time;
"t" (Ct) divided by the initial measured plasma concentration (CO) of SST
fusion
protein; and
the "X" axis is plasma sampling time ('t") in hours.
8
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
DETAILED DESCRIPTION OF THE INVENTION
The present invention encompasses somatostatin-albumin fusion proteins and
analogues thereof and methods of producing and using the same. Constructs
prepared
according to the invention include an albumin (or an analog thereof) moiety, a
somatostatin
moiety (e.g., SST-14, SST-28), and a spacer separating the two moieties.
The somatostatin-albumin fusion proteins of the certain embodiment of the
invention
include variants of albumin including human serum albumin and / or derivatives
of
somatostatin. The spacers of another embodiment of the invention encompass
peptides
covalently linked to somatostatin on one terminal and albumin on another
terminal. The
spacers in other embodiments of the invention include peptide sequences having
2-100 amino
acids.
In one embodiment, the present invention provides a fusion protein comprising:
an SST;
an L; and
an ALB,
wherein,
SST is a somatostatin or its analogues or derivatives;
L is a spacer or a linker;
ALB is an albumin or its analogues or variants.
In certain embodiments, the fusion protein of the present invention is
selected from
among formulas I-X, as follows.
SST-(L)xi-ALB (I);
ALB-(L)xi-SST (II);
[SST-(L)Ayi-ALB (III);
ALB-[(L)xi-SST] yl (IV);
[SST-(L)xi]yi-ALB-[(L)x2-SST] y2 (V);
[ S ST-(L)xi]yi-ALB-[(L)x2-SST]y2-(L)o-ALB (VI);
[SST-(L)xi]yi-ALB-[(L)x2-SST]y2-(L)o-ALB-[(L)x4-SST] y3 (VII);
ALB-(L)xi-[SST-(L)x2]yi-ALB (VIII);
ALB-(L)xi- [SST-(L)x2]yi-ALB-[(L)3-SST]y2-(L)xi-ALB (IX); and
9
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
ALB-(L)) - [ S S T-(L)x2] yi -ALB - [(L)x3-SS T] y2-(L)x 1 -ALB - [(L)x4-S S
T] y3 (X);
wherein,
each xl, x2, x3, x4, yl, y2, or y3 is independently zero or an integer
selected
from 1-10,
provided that there is at least one L present in the fusion protein.
In yet another embodiment, the present invention provides a nucleotide
sequence
encoding an albumin-somatostatin fusion protein comprising:
an SST;
an L; and
an ALB,
wherein,
SST is a somatostatin or its analogues or derivatives;
L is a spacer or a linker;
ALB is an albumin or its analogues or variants.
In certain embodiments, the nucleotide sequence of the present invention is
selected
to encode an albumin-somatostatin fusion protein from among,
SST-(L)xi-ALB (I);
ALB-(L)xi-SST (11);
[SST-(L)Ayi-ALB (III);
ALB-[(L)xi-SST] yl (IV);
[SST-(L)xi ] yi -ALB - [(L)x2.-SS 1] y2 (V);
[SST-(L)x)] yi -ALB - [(L)x2.-SS 1] y2-(L)3-ALB (VI);
[SST-(L)x)] yi -ALB -[(L)2-SST] y2-(L)3-ALB- [(L)4-S S T] y3 (VII);
ALB-(L)) - [ S S T-(L)x2] yi -ALB (VIII);
ALB-(L)) - [ S S T-(L)x2] yi -ALB - [(L)x3-S S 1] y2-(L)x) -ALB (IX); and
ALB-(L)xi-[SST-(L)xflyi-ALB-[(L)x3-SST]y2-(L)xi-ALB-[(L)x4-SST] y3 (X);
wherein,
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
each xl, x2, x3, x4, yl, y2, or y3 is independently zero or an integer
selected
from 1-10, 1-5 or 1-4, provided that there is at least one L present in the
nucleotide
sequence encoding an albumin-somatostatin fusion protein.
Another embodiment of the present invention provides a nucleotide sequence
.. encoding an albumin-somatostatin fusion protein, wherein the spacer
sequence consists of the
sequence encoding the amino acid sequence represented by SEQ ID NO: 31 or
¨GGGGS-.
Another certain embodiment of the present invention provides a nucleotide
sequence
encoding an albumin-somatostatin fusion protein, wherein the second region (b)
encodes a
polypeptide having at least 85% sequence identity to SEQ ID NO: 19, albumin or
a fragment
.. thereof.
One embodiment of the present invention provides a nucleotide sequence
encoding an
albumin-somatostatin fusion protein, wherein the first region (a) encodes a
polypeptide
having at least 85% sequence identity to either SEQ ID NOS: 17 or 18, SST-14,
SST-28, or a
fragment thereof.
The present invention also provides a nucleotide sequence encoding an albumin-
somatostatin fusion protein comprising:
(a) a first region comprising a nucleotide sequence containing one or more
adjacent
repeats of a sequence encoding a human somatostatin peptide;
(b) a second region comprising a nucleotide sequence encoding human serum
albumin, or a fragment thereof;
(c) a spacer region comprising a nucleotide sequence encoding a polypeptide of
2-100
residues in length;
wherein the spacer region is present between the first region and the second
region, or
or between the first region and another first region;
wherein one or more adjacent repeats of a sequence encoding a human
somatostatin
peptide encodes either SST-14 or SST-28, as represented by SEQ ID NOS:17 and
18,
respectively, or a sequence having at least 85% identity to either of these
two sequences; or
wherein the spacer sequence consists of the sequence encoding the amino acid
sequence represented by SEQ ID NO: 31 or GGGGS or by SEQ ID NO: 30 A(EAAAK)4A;
or
11
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
wherein the region (a) consists of one or more adjacent repeats of either SST-
14 or of
SST-28, as represented by SEQ ID NOS: 23 and 24, respectively, or a sequence
having at
least 85% identity to either of these two sequences.
Furthermore, the present invention provides a polypeptide sequence an albumin-
somatostatin fusion protein comprising:
(a) a first region comprising a polypeptide sequence of a somatostatin peptide
(which
may be a human somatostatin peptide);
(b) a second region comprising a polypeptide sequence of serum albumin (which
may
be a human serum albumin), or a fragment thereof;
(c) a spacer region comprising a polypeptide of 2-100 residues in length.
The spacer region (c) may be present between region (a) and region (b) or
between region (a)
and region (a). In addition, the region (a) may comprise one or more tandem
repeats of a
sequence encoding SST-14 or SST-28, represented by SEQ ID NOS: 17 or 18,
respectively,
or sequence having 85% identity to either of these sequences.
Another embodiment of the present invention provides a plasmid construct
expressing
an albumin-somatostatin fusion protein with any of the fusion protein or
polypeptide
sequences described above.
Yet another embodiment of the present invention includes a bacterial cell
transformed
with the plasmid construct described above.
A further embodiment of the present invention includes an isolated and
purified
albumin-somatostatin fusion protein having the polypeptide sequence described
above (e.g.,
a polypeptide sequence of an albumin-somatostatin fusion protein or the
plasmid construct
expressing such protein).
Table 1. A non-exclusive list of polypeptide sequences
SEQ ID NO: Description
SEQ ID NO: 1 55T14-A(EAAAK)4A-HSA-A(EAAAK)4A-55T14
SEQ ID NO: 2 HSA-A(EAAAK)4A-55T14
12
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
SEQ ID NO: 3 His6-GGS-HSA-GGGGS-55T14-HSA
SEQ ID NO: 4 His6-GGS-HSA-GGGGS-(55T14-GGGGS)2-HSA
SEQ ID NO: 5 HSA-GGGGS-(55T14-GGGGS)2- HSA
SEQ ID NO: 6 Linker GGGGGGGG
SEQ lD NO: 7 SST14-(GGGGS)3-HSA
SEQ lD NO: 8 SST14-A(EAAAK)4A-HSA
SEQ lD NO: 9 His6-GGS-HSA-GGGGS-55T14
SEQ lD NO: 10 55T14-GGGGS-HSA-GGS-His6
SEQ lD NO: 11 HSA-GGGGS-55T14
SEQ lD NO: 12 55T14-GGGGS-HSA
SEQ lD NO: 13 (55T14-GGGGS)2- HSA
SEQ lD NO: 14 (55T14-GGGGS)4- HSA
SEQ lD NO: 15 HSA-(GGGGS)3-55T14
SEQ lD NO: 16 HSA-(GGGGS)6-55T14
SEQ ID NO: 17 Somatostatin-14 (SST-14)
SEQ ID NO: 18 Somatostatin-28 (SST-28)
SEQ ID NO: 19 Human Serum Albumin (HSA)
SEQ lD NO: 20 MDMRVPAQLLGLLLLWLRGARC (Signal Peptide)
SEQ lD NO: 21 Linker APAPAPAPAPAPAPAPAPAP
SEQ lD NO: 22 Linker APAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAP
SEQ lD NO: 30 A(EAAAK)4A peptide
SEQ lD NO: 31 GGGGS peptide
SEQ ID NO: 32 Linker GGGGSLVPRGSGGGGS
SEQ lD NO: 33 Linker GSGSGS
SEQ lD NO: 34 Linker GGGGSLVPRGSGGGG
13
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
SEQ ID NO: 35 Linker GGGGSLVPRGSGGGGS
SEQ ID NO: 36 Linker GGSGGHMGSGG
SEQ ID NO: 37 Linker GGSGGSGGSGG
SEQ ID NO: 38 Linker GGSGGHMGSGG
SEQ ID NO: 39 Linker GGSGG
SEQ ID NO: 40 Linker GGGGSLVPRGSGGGGS
SEQ ID NO: 41 Linker GGSGGGGG
SEQ ID NO: 42 Linker GSGSGSGS
SEQ ID NO: 43 Linker GGGSEGGGSEGGGSEGGG
SEQ ID NO: 44 Linker AAGAATAA
SEQ ID NO: 45 Linker GGGGG
SEQ ID NO: 46 Linker GGSSG
SEQ ID NO: 47 Linker GSGGGTGGGSG
SEQ ID NO: 48 Linker GSGSGSGSGGSGGSGGSGGSGGSGGS
For the fusion proteins, e.g., SEQ ID NOs: 1-5, 7-10 and 13-16, it should be
noted that these
are encoded as pro-proteins with a 22 residue signal peptide (SEQ ID NO: 20).
Somatostatin-Albumin Fusion Proteins
The invention encompasses polypeptide constructs wherein the somatostatin
moiety
is encoded by a nucleotide having at least 85% sequence identity to the
nucleotide sequence
of endogenous human SST-14 or SST-28 (SEQ ID Nos: 23 and 24, respectively).
The invention also encompasses polypeptide constructs wherein the human serum
albumin moiety is encoded by a nucleotide having at least 85% sequence
identity to the
nucleotide sequence of endogenous human serum albumin (SEQ ID NO: 25). The
invention
further encompasses polypeptide constructs wherein the human serum albumin
moiety is a
fragment of the endogenous human serum albumin protein, e.g., where it is
encoded by a
nucleotide consisting of a subsequence of SEQ ID NO: 25. For example, the
human serum
14
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
albumin fragment optionally includes one or more of the three human serum
albumin globular
domains, each of which contains two subdomains, denominated subdomain IA, TB,
IIA, JIB,
IIIA, and IIIB (Dockal, 1999, The Journal Of Biological Chemistry, 274(41):
29303-29310).
The invention also encompasses polypeptide constructs wherein the somatostatin
moiety has a polypeptide sequence at least 85% sequence identity, preferably
at least 90% to
the polypeptide sequence of endogenous SST-14 or SST-28 (SEQ ID NOs:17 and 18,
respectively).
The invention also encompasses polypeptide constructs wherein the human serum
albumin moiety has a polypeptide sequence at least 85% sequence identity to
the polypeptide
sequence of mature human serum albumin (SEQ ID NO: 19).
The invention also encompasses a fusion protein comprising a signal peptide, a
purification tag (His-6), a first linker, a human serum albumin moiety, a
second linker and a
somatostatin moiety. In one embodiment, the fusion protein is a polypeptide is
represented
by SEQ ID NO: 9 or a sequence having 85% sequence identity to the same.
The invention also encompasses a fusion protein comprising a somatostatin
moiety, a
first linker, a human serum albumin moiety, a second linker, a somatostatin
moiety and a
purification tag (His-6). In one embodiment, the fusion protein is a
polypeptide is represented
by SEQ ID NO: 10 or a sequence having 85% sequence identity to the same.
The invention also encompasses a nucleotide sequence (SEQ ID NO: 11) encoding
a
fusion protein comprising an N-terminal human serum albumin moiety and a C-
terminal
somatostatin moiety separated by a peptide spacer. The invention further
encompasses
nucleotide sequences encoding an albumin-somatostatin fusion construct which
have 85%
sequence identity to SEQ ID NO: 11.
The invention also encompasses a nucleotide sequence (SEQ ID NO: 12) encoding
a
fusion protein comprising an N-terminal somatostatin moiety and a C-terminal
human serum
albumin moiety separated by a peptide spacer. The invention further
encompasses nucleotide
sequences encoding an albumin-somatostatin fusion construct which have 85%
sequence
identity to SEQ ID NO: 12.
The invention also encompasses polypeptide constructs wherein the somatostatin
moiety comprises two or more copies of the SST-14 or SST-28 sequence arranged
in tandem,
i.e., "(SST-14)2" or "(SST-14)3"or "(SST-28)2" or "(SST-28)3", respectively.
Optionally, a
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
linker sequence is included between the two or more tandem somatostatin
moieties, and/or a
signal peptide sequence is included at the N-terminus of the fusion protein.
The invention also encompasses polypeptide constructs wherein the somatostatin
moiety comprises two or more copies of the SST-14 sequence arranged in a way
that at least
one copy of the SST14 is linked on both sides of albumin, respectively.
Optionally, a linker
sequence is included between the two or more tandem somatostatin moieties and
between
somatostatin and albumin, and/or a signal peptide sequence is included at the
N-terminus of
the fusion protein. For example, the polypeptide construct may include a
signal peptide, two
SST-14 moieties separated by a spacer, a second spacer, and an HSA moiety as
represented.
Optionally, the construct omits the N-terminal signal peptide.
The invention also encompasses polypeptide constructs wherein the somatostatin
moiety comprises two or three copies of the SST-28 sequence arranged in
tandem, i.e.,
"(SST-28)2" or "(SST-28)3", respectively. Optionally, a linker sequence is
included between
the two or more tandem somatostatin moieties.
The invention also encompasses polypeptide constructs comprising any of the
albumin-somatostatin fusion proteins described in the preceding paragraphs,
where the
albumin-somatostatin fusion protein has an in vivo half-life longer than the
endogenous SST-
14 or SST-28 peptides.
The invention also encompasses polypeptide constructs comprising any of the
albumin-somatostatin fusion proteins described in the preceding paragraphs,
wherein the
albumin-somatostatin fusion protein has an approximately equal or a greater
binding affinity
for a somatostatin receptor compared to endogenous SST-14 or SST-28.
The invention also encompasses albumin-somatostatin fusion proteins comprising
an
N-terminal albumin moiety as represented by SEQ ID NO: 15, SEQ ID NO: 16, and
SEQ ID
NO: 2, an internal SST moiety and a C-terminal Albumin moiety as represented
by SEQ ID
NO: 7 and SEQ ID NO: 8. Optionally, the N-terminus may further include a
signal peptide.
Optionally, one or more of the albumin and SST domains may each be separated
by an
independently selected linker sequence as represented by SEQ ID NO: 1.
In some embodiments, the SST moiety may comprise a pair or plurality of tandem
SST sequences, e.g., (SST-14)2 or (SST-28)3, with or without intervening
spacing sequences
between the two or more tandem SST repeats. Optionally, one or more
purification tag
16
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
sequences may be included in the sequence between two moieties or at the N or
C-terminus
in order to assist with purification of the fusion protein. An alternative
embodiment includes
a pair of SST-14 moieties separated by a spacer, as represented by SEQ ID NO:
4. A further
embodiment may omit the purification tag (e.g., His6) as shown by the
polypeptide sequence
represented by SEQ ID NO: 5.
Somatostatin
The somatostatin for use with the present invention may be any somatostatin,
its
analogue or derivative. It may be a human somatostatin, any other isolated or
naturally
occurring somatostatin. The SST moiety can be an analogue such as octreotide,
lanreotide,
pasireotide, seglitide, or vapreotide.
The invention also encompasses polypeptide constructs wherein the somatostatin
moiety comprises a somatostatin analog. Preferably, such an analog is suitable
for
expression, as part of a fusion protein, in a recombinant host cell. It is
understood that a
suitable somatostatin analog sequence may be used in place of the SST-14 or
SST-28
sequences included in any of the examples disclosed herein.
The invention also encompasses polypeptide constructs wherein the somatostatin
moiety comprises two or more tandem repeats of a somatostatin polypeptide
sequence e.g.,
SST-14 or SST-28; SEQ ID NOS: 17 and 18, respectively. Each of the repeated
somatostatin
polypeptide sequences may be a polypeptide sequence having at least 85%
sequence identity
to SST-14 or SST-28. These repeated variant sequences are independently
selected, i.e., in
some embodiments the repeats are identical, whereas in other embodiments they
are unique.
Albumin
The albumin for use with the present invention may be any albumin, its
analogue or
variant. The albumin may be human serum albumin, or any other isolated or
naturally
occurring albumin.
The invention also encompasses polypeptide constructs wherein the human serum
albumin moiety comprises a polypeptide sequence variant with alternative
arrangement or
number of disulfide bonds due to the presence of additional or fewer cysteine
residues than
the natural form (SEQ ID NO: 25).
17
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
Spacer or Linker
As described earlier, a spacer or linker can be used with the present
invention. The
spacer or linker may be independent of the somatostatin or albumin.
The invention also encompasses polypeptide constructs wherein the peptide
spacer of
alternatively referred to as a linker, consists of a polypeptide sequence of
from about 2 to
about 100 amino acid residues in length. The invention further encompasses
polypeptide
constructs wherein the peptide spacer is from about 2 to about 50 amino acid
residues in
length, preferably from about 2 to about from 30, or more preferably from
about 3 to about
20 amino acid residues in length.
The invention also encompasses polypeptide constructs wherein the peptide
spacer
(alternatively referred to as a linker) has the polypeptide sequence "GGGGS"
(SEQ ID NO:
31). Polypeptides rich in Gly, Ser or Thr offer special advantages include,
but not limited to:
(i) rotational freedom of the polypeptide backbone, so that the adjacent
domains are free to
move; (ii) enhanced solubility; (iii) resistance to proteolysis. In addition,
many natural linkers
exhibited alpha-helical structures. The alpha-helical structure is more rigid
and stable than
Gly rich linker. An empirical rigid linker with the sequence of A(EAAAK)4A
(SEQ ID NO:
30) can be used to separate functional domains. In addition to the role of
linking protein
domains together, artificial linkers may offer other advantages to the
production of fusion
proteins, such as improving biological activity, increasing protein
expression, and achieving
desirable pharmacokinetic profiles.
Table 2. A non-exhaustive list of linker sequences that may be used in the
fusion protein constructs of the present invention.
GGGGSLVPRGSGGGGS (SEQ ID NO: 32)
GSGSGS (SEQ ID NO: 33)
GGGGSLVPRGSGGGG (thrombin proteolytic site is underlined)
_
(SEQ ID NO: 34)
GGGGSLVPRGSGGGGS (thrombin proteolytic site is underlined)
_
(SEQ ID NO: 35)
GGSGGHMGSGG (SEQ ID NO: 36)
18
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
GGSGGSGGSGG (SEQ ID NO: 37)
GGSGGHMGSGG (SEQ ID NO: 38)
GGSGG (SEQ ID NO: 39)
GGGGSLVPRGSGGGGS (thrombin proteolytic site is underlined)
(SEQ ID NO: 40)
GGSGGGGG (SEQ ID NO: 41)
GSGSGSGS (SEQ ID NO: 42)
GGGSEGGGSEGGGSEGGG (SEQ ID NO: 43)
AAGAATAA (SEQ ID NO: 44)
GGGGG (SEQ ID NO: 45)
GGSSG (SEQ ID NO: 46)
GSGGGTGGGSG (SEQ ID NO: 47)
GT
GSGSGSGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 48)
GGS
GGGGGGGG (SEQ ID NO: 6)
A(EAAAK)4A (SEQ ID NO: 20)
APAPAPAPAPAPAPAPAPAP (SEQ ID NO: 21)
APAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAP
(SEQ ID NO: 22)
Preparation of Somatostatin-Albumin fusion protein
An embodiment of the invention provides a method for preparation of the
somatostatin-albumin fusion protein. In one embodiment, the somatostatin-
albumin fusion
protein of the invention is prepared by expressing a vector containing the
encoding gene and
introducing the vector into a suitable host cell. For example, the fusion
protein is obtained by
expression of a suitable vector in a host such as yeast. In one embodiment,
Pichia pastoris
GS115 may be used as a suitable expression host, and the vector is pPIC9K. In
particular,
mammalian cell lines such as CHO or HEK293 can be used as an expression host.
The invention also encompasses plasmid constructs capable of expressing an
albumin
19
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
somatostatin fusion protein comprising a nucleotide sequence encoding a
somatostatin
albumin fusion protein as described in any of the preceding paragraphs. For
example,
suitable plasmid constructs include, but are not limited to, the pcDNA3.1
vector represented
by SEQ ID NO: 26 with a DNA sequence encoding any of the albumin-somatostatin
fusion
proteins disclosed herein ligated into the multiple cloning site of this
vector. Other suitable
protein expression vectors known in the art may be selected based upon the
expression host
(e.g., an expression vector with a mammalian promoter system would be suitable
for
expression in a human cell line whereas a yeast or bacterial expression
plasmid would be
selected if expression in either of these organisms was desired.
The invention also encompasses a bacterial or yeast protein expression system
comprising a bacterial or yeast cell transformed with a plasmid construct
comprising a
nucleotide sequence that encodes a somatostatin albumin fusion protein, as
described in any
of the preceding paragraphs. Suitable bacterial strains include, for example,
Escherichia coli.
Suitable yeast strains include, for example, Pichia pastoris. An exemplary
plasmid construct
.. includes pPIC9K (Invitrogen) as represented by SEQ ID NO: 27, with a
nucleotide sequence
encoding any of the albumin-somatostatin fusion proteins described herein
incorporated into
the multiple cloning site of the vector.
The invention also encompasses isolated and purified fusion somatostatin
fusion
proteins having a polypeptide sequences as described in any of the preceding
paragraphs.
Table 3. A list of nucleotide sequences in certain embodiments of the
invention
Nucleotide Sequence Encodes the
SEQ ID NO: Description
following:
SEQ ID NO: 23 55T14 Somatostatin-14 (SST-
14)
SEQ ID NO: 24 55T28 Somatostatin-28 (SST-
28)
Human Serum Albumin
SEQ ID NO: 25 Human Serum Albumin mature form
(HSA)
pcDNA3.1(+) Vector
SEQ ID NO: 26 pcDNA3.1(+) Vector
mammalian expression vector
SEQ ID NO: 27 pPIC9K Vector yeast expression vector
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
SEQ ID NO: 28 GGGGS GGGGS Linker
SEQ ID NO: 29 A(EAAAK)4A alpha-helical linker
When the SST is a somatostatin analogue, an alternative method known in the
field
can be employed to prepare the conjugate.
Utility of Somatostatin-Albumin Fusion Protein
The fusion protein of the present invention can be used to treat conditions
for which
somatostatin is art-known to be employed. As such, the invention also
encompasses methods
of treating cancer in a human subject by administering an isolated and
purified albumin-
somatostatin fusion protein as described in any of the preceding paragraphs,
wherein the
cancer is any cancer known to respond to somatostatin treatment, e.g.,
selected from breast
cancer, colorectal cancer, liver cancer, lung cancer, endocrine cancer,
neuroendocrine
cancers, pancreatic cancer and prostate cancer.
The invention also encompasses methods of treating cancer in a human subject
by
administering a composition containing the fusion protein of the present
invention, such as an
isolated and purified albumin-somatostatin fusion protein as described in any
of the
preceding paragraphs. The composition can also include a suitable carrier.
Eleven 55T14-Albumin fusion protein constructs with various linker sequences
were
designed. Eight of these constructs were made into a fusion gene within a
plasmid and
produced by HEK 293 transient expression at 100 mL scale. The proteins were
collected
from the culture media, purified through albumin-based affinity purification,
and dialyzed to
a storage buffer. These fusion proteins were evaluated for their binding
affinity to SSTR2
receptor, and also for cell-based activity in inhibiting cAMP production in a
SSTR2-
overexpression CHO-Kl cell line. The results of these studies indicated that
the length and
type of linkers significantly affected the SSTR2 receptor binding affinity,
the in-vitro cell-
based functional activity, and the fusion protein production yield.
SST-Albumin fusion protein of this invention exhibited a significantly longer
serum
half-life and/or improved pharmacokinetic profile in solution or in a
pharmaceutical
composition in vitro and/or in vivo compared to the corresponding unfused,
free SST
21
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
molecules. The stability of free SST and SST fusion protein was compared in in
vitro rat
plasma. When incubated in freshly prepared rat plasma at 37 C, free SST and
SST fusion
protein exhibited degradation half-lives of 33 minutes and 5.5 hours,
respectively (Table 4).
In vivo pharmacokinetic profiles were also generated to demonstrate the
improved
stability of SST fusion protein relative to free SST. Rats administered
intravenously with
SST showed a calculated T1/2 of 3.5 minutes. On the other hand, rats
administered
intravenously with SST fusion protein exhibited a bi-phasic pharmacokinetic
profile, where
the a-phase Ti/2 was 1.01 hour and the 0-phase Ti/2 was 6.14 hour. The
calculated half-life of
SST fusion protein is significantly longer than the calculated T1/2 of free
SST in rat (3.5
.. minutes) and the reported plasma Ti/2 of free SST in rat (< 1 minute;
Reference #1) (Table
4).
Table 4. Calculated Half-life of Free SST and SST Fusion Protein for In Vitro
Plasma
Stability and In Vivo Rat Model
T1/2 In Vitro Plasma Stability In Vivo Rat
Pharmacokinetic
Free SST 33 minutes 3.5 1.0 minutes
less than 1 minutes (Ref. #1)
a phase (0-0.5 hr) 1.01
0.60 hr
SST Fusion Protein 5.5 hour
[3 phase (0.75 - 4 hr) 6.14
1.27 hr
Reference #1: Yogesh C. Patel and Thomas Wheatley. In Vivo and in Vitro Plasma
Disappearance and Metabolism of Somatostatin-28 and Somatostatin-14 in the
Rat.
Endocrinology. Vol. 112, No. 1(1992), pages 220-225.
22
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
EXAMPLES
Selected embodiments of the invention will be described in further detail with
reference to the following experimental and comparative examples. These
examples are for
illustrative purposes only and are not intended to limit the scope of the
invention.
EXAMPLE 1: EXPRESSION IN MAMMALIAN SYSTEMS
Example 1-1. Recombinant gene synthesis
Eight constructs corresponding to the fusion proteins listed in Table 5 were
prepared.
First, the gene sequence coding each fusion protein was de novo synthesized
and
subsequently inserted into the pcDNA3.1 vector.
Example 1-2. Plasmid generation
Maxi-prep or Mega-prep was used to generate ¨20 mg of each DNA
Example 1-3. Transfection and protein production
(A) Suspension cell method
FreeStyleTM 293-F Cells were seeded at 0.55-0.6x106 cells/mL in a flask. After
about
24 hours, the cells were seeded in a shake flask at 1.1-1.2x106 cells/mL. DNA
was prepared
at 500 g DNA / 80 mL in a FreeStyle medium. Polyethylenimine (PEI) was
prepared at 1.8
mL PEI per 80 mL in a FreeStyle medium. DNA was mixed in the FreeStyle medium,
and
the effective amount of PEI was added to the DNA solution, and the mixture is
vortexed
incubated for about 15 minutes at room temperature to form a DNA-PEI complex.
An 80 mL
of the incubated DNA-PEI complex is added to a cell culture. About 3 hours
later, TC
Yeastolate feed (BD) is added to have the final concentration of 4 gram /
liter of culture.
After about 7-8 days, the medium is harvested by centrifugation.
(B) Adherent cell method
About 24 hours before transfection, HEK293 cells were seeded to 50-90 %
confluency in a flask, and complete medium is added. After about 24 hours,
cells were
washed followed by adding basal medium.
23
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
DNA and PEI solutions were prepared by adding DNA to a serum free medium. The
PEI solution was added to the DNA solution and incubated for 15 minutes to
form DNA-PEI
complex at room temperature.
The PEI/DNA mixture was added to cells, and the mixture incubated for about 4-
6
hours at 37 C. The medium was removed and fresh medium with Glutamine and
serum was
added, followed by incubating at 37 C for 4 days.
The medium was harvested after about 4 days, by centrifuging to collect the
supernatant. The precipitate was replenished with fresh medium with L-
Glutamine for
another 3-day incubation to repeat the harvesting process.
Example 1-4: Protein Concentration, Ni-NTA Purification and Buffer
Exchange
The collected medium was concentrated by TFF system (Millipore) to a certain
volume depending on purification methods (either continuous chromatography or
manual
batch purification).
The concentrated proteins was incubated with fresh Ni-NTA resin at about 4 C
in
binding buffer and washed with wash buffer using either chromatography or
batch system.
The protein was eluted with elute buffer and fractions were collected and
concentrated to
recover the purified protein. The protein can be further purified using size
exclusion
chromatography purification.
The buffer of the final eluate can be exchanged by dialysis to a desired
buffer.
EXAMPLE 2: YIELDS OF SEVERAL SST-ALBUMIN FUSION PROTEINS
The SST-HSA fusion proteins were all expressed in soluble form with high
yield. The
length or the nature of the linkers can affect the protein yield and
solubility of the fusion
proteins. The results indicated that the production yield slightly decreased
as the fusion
protein constructs became longer and more complex. However, all the constructs
exhibited
yield for scale up production.
24
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
Table 5. SST14-HSA fusion protein expression yield
Total
Production
Sequence ID Design amino MW (kDa) Yield
acids (g/L)
SST14-A(EAAAK)4A-HSA-
SEQ ID NO: 1 657 73.8364 0.26
A(EAAAK)4A-SST14
SEQ ID NO: 2 HSA A(EAAAK)4A-55T14 621 70.1543 0.27
SEQ ID NO: 7 55T14-(GGGGS)3-HSA 614 69.112 0.33
SEQ ID NO: 8 55T14-A(EAAAK)4A-HSA 621 70.1543 0.25
H6-GGGGS-HSA-GGGGS-
SEQ ID NO: 9 613 69.4874 0.30
SST14
55T14-GGGGS-HSA-GGGGS-
SEQ ID NO: 10 613 69.4874 0.41
H6
SEQ ID NO: 15 HSA-(GGGGS)3-55T14 614 69.112 0.28
SEQ ID NO: 16 HSA-(GGGGS)6-55T14 629 70.1119 0.29
EXAMPLE 3: BINDING AFFINITY OF SEVERAL SST-ALBUMIN FUSION
PROTEINS
This assay measures binding of [125I]Somatostatin to human somatostatin sst2
receptors. CHO-Ki cells stably transfected with a plasmid encoding the human
somatostatin
sst2 receptor are used to prepare membranes in modified HEPES pH 7.4 buffer
using
standard techniques. A 0.1 mg aliquot of membrane is incubated with 0.03 nM
[125I]Somatostatin and tested fusion proteins for 240 minutes at 25 C. Non-
specific binding
is estimated in the presence of 1 v.1\4 Somatostatin. Membranes are filtered
and washed 3
times and the filters are counted to determine [125I]Somatostatin specifically
bound.
The competitive binding study 125I-Tyr-somatostatin versus the fusion proteins
demonstrated the following results. The efficiency of the inhibition varied
depending on the
construct of the fusion proteins. The fusion protein construct (SEQ ID NO: 1)
with two
alpha-helical linker, A(EAAAK)4A, showed 100 % inhibition of the somatostatin
and its
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
receptor interaction. The SEQ ID NO: 1 construct has two somatostatin moiety
on both N
and C terminal sides of human serum albumin. The smaller construct with one
somatostatin
on the C terminal side of human serum albumin linked by the same alpha-helical
linker (SEQ
ID NO: 2) showed 96 % inhibition. The same construct with the more flexible
GGGGS
linker showed lower inhibition of 82 ¨ 85 % depending on the length. The
length of GGGGS
linkers also affected the inhibition. The construct with five amino acid GGGGS
linker (SEQ
ID NO: 9 and SEQ ID NO: 8) showed 57-59 % inhibition whereas the constructs
with 15
amino acid (SEQ ID NO: 15) or 30 amino acid GGGGS linkers (SEQ ID NO: 16)
showed
over 80 %, suggesting that longer than five amino acid GGGGS would be more
advantageous to SST function. A more rigid A(EAAAK)4A (a-helical) linker would
be more
efficient in binding than flexible GGGGS linker. A multiple SST can increase
the effective
concentration of the ligand for SST receptor binding. The position of
Histidine purification
tag may not affect the binding. Changing the orientation or position of
albumin in the fusion
protein may further increase the efficiency of the protein binding.
Table 6. Inhibition of 125I-Tyr1-somatostatin binding on SSTR2 by the fusion
proteins
Inhibition %
Sequence ID Construct ICso (nM)
at 0.1 p,M
SEQ ID NO: 1 55T14-A(EAAAK)4A-Albumin- 100 2.38
A(EAAAK)4A-55T14
SEQ ID NO: 2 Albumin-A(EAAAK)4A-55T14 96 9.41
SEQ ID NO: 7 55T14-(GGGGS)3-Albumin 70
SEQ ID NO: 8 55T14-A(EAAAK)4A-Albumin 79
SEQ ID NO: 9 His6-GGS-Albumin-GGGGS-55T14 59
SEQ ID NO: 10 55T14-GGGGS-Albumin-His6 57
SEQ ID NO: 15 Albumin-(GGGGS)3-55T14 85 33
SEQ ID NO: 16 Albumin-(GGGGS)6-55T14 82
SEQ ID NO: 17 SST-14 0.0069
26
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
EXAMPLE 4. INHIBITION OF SEVERAL SST-ALBUMIN FUSION PROTEINS TO
CAMP ACCUMULATION IN SSTR2-EXPRESSING CELLS
Human recombinant somatostatin sst2a receptors expressed in CHO-Kl cells were
used. Test compound and/or vehicle was incubated with the cells (2 x 105
cells/mL) in
incubation buffer for 20 minutes at 37 C. Test compound-induced decrease of
cAMP by 50
percent or more (50%) relative to the 10 nM Octreotide response indicated
sst2a receptor
agonist activity.
The inhibition of the accumulation of cAMP was observed in SST receptor type 2
expressing CHO-Kl cells. The value of EC50 was 260 nM. The constructs with
longer linkers
(SEQ ID NOS: 1, 15, and 2) exhibited lower EC50 values, which coincided with
the binding
assay data. The alpha-helical linker appeared to be more efficient in the
inhibition of cAMP
production, when Albumin-(GGGGS)3-SST14 and Albumin-(GGGGS)3-SST14 EC50 values
were compared.
Table 7. EC50 value for the inhibition of cAMP production
ECso values of the
Sequence ID Construct inhibition of cAMP
production (nM)
SEQ ID NO: 1 55T14-A(EAAAK)4A-Albumin- 5.14
A(EAAAK)4A-55T14
SEQ ID NO: 2 Albumin-A(EAAAK)4A-55T14 17.6
SEQ ID NO: 9 H6-GGGGS-Albumin-GGGGS-55T14 260
SEQ ID NO: 15 Albumin-(GGGGS)3-55T14 23
Octreotide 0.041
EXAMPLE 5. DETERMINATION OF STABILITY OF FREE SST AND SST
FUSION PROTEIN IN RAT PLASMA
Improved stability of SST fusion protein in rat plasma was proven using ELISA.
The
test results showed that SST fusion protein (SEQ ID NO: 1) exhibited a
degradation half-life
27
CA 03032995 2019-02-04
WO 2018/038803
PCT/US2017/039477
of 5.5 hours as opposed to free SST, which showed less than 33 minute half-
life when
incubated in rat plasma at 37 C (Table 4).
Example 5-1: Preparation of Sample
500 pg/mL of free SST or with 750 ng/mL of SST fusion protein (SEQ ID NO: 1)
were incubated in pooled rat plasma for 1 minute, 2 minute, 5 minute, 20
minute, 60 minute,
80 minute, 100 minute, 120 minute, 150 minute, and 180 minute. The blood
samples at each
time point were incubated in triplicate. All the blood samples were
centrifuged at 5500 rpm
for 10 minutes to obtain plasma samples for analysis. Pooled rat plasma was
used as blank
for background measurement. All samples were analyzed in duplicate.
All plasma samples with SST fusion protein were diluted 15-fold with pooled
rat
plasma. For example, to a 10 [iL plasma sample with SST fusion protein was
added 140 [iL
pooled rat plasma.
Example 5-2: Preparation of Free SST and SST Fusion Protein Standards
(1). Preparation of Free SST standard
Standard curve was prepared by the following procedure. Duplicate standard
points
were prepared by serially diluting Free SST Stock (1 mg/mL) with diluent
buffer to produce
5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 and 0.039 ng/mL solutions.
(2). Preparation of SST fusion protein standard
Standard curve was prepared by the following procedure. Duplicate standard
points
were prepared by serially diluting SST fusion protein Stock (2.42 mg/mL) with
diluent buffer
to produce 225, 112.5, 56.2, 28.1, 14.1, 7.03, 3.51 and 1.76 ng/mL solutions.
Example 5-3: ELISA Assay Procedure
1) All kit components were maintained at room temperature (20-25 C) before
analysis.
2) 50 .t.L/well of standard, sample, or positive control solution was added to
the kit. Then, 25
i.t.L/well of primary antibody was added into each well except the Blank well.
At last, 25
.t.L/well biotinylated peptide was added into each well except the Blank well.
The
immunoplate was incubated for 2 hours at room temperature with shaking at 300-
400 rpm.
28
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
The wells were emptied and washed three times with 300 [IL washing solution.
After the last
wash, the wells were emptied by tapping the strip on an absorbent tissue.
3) The contents of the wells were discarded and each well was washed with 300
0_, of lx
ETA assay buffer, discard the buffer was, invert and blot dry plate. Repeat 4
times.
4) Add 100 0_, of SA-HRP solution into each well. Incubate the immunoplate for
1 hour at
room temperature with shaking at 300-400 rpm.
5) Wash and blot dry the immunoplate 4 times with lx ETA assay buffer as
described above
in step 3.
6) Add 100 0_, of TMB substrate solution into each well. Cover the immunoplate
to protect
from light. Incubate the immunoplate for 1 hour at room temperature with
shaking at 300-
400 rpm.
7) Add 100 0_, 2N HC1 into each well to stop the reaction. The color in the
well should
change from blue to yellow. If the color change does not appear to be uniform,
gently tap the
plate to ensure thorough mixing. Proceed to the next step within 20 minutes.
8) Load the immunoplate onto Plate Reader. Read absorbance O.D. at 450nm.
EXAMPLE 6. DETERMINATION OF IN VIVO PHARMACOKINETIC PROFILE
OF FREE SST and SST FUSION PROTEIN IN RAT
SST and SST fusion protein (SEQ ID NO: 1) were administered at 0.02 and 27.1
mg/kg, respectively, doses via tail vein injection to three and five,
respectively, male
Sprague Dawley rats to determine the pharmacokinetic profiles and parameters
of SST and
SST fusion protein, respectively (Table 8 and Table 9). The animals were
fasted overnight
with free access to water prior to injection, and no negative clinical signs
were observed
afterwards. SST exhibited rapid pharmacokinetic profile in each of the rats
administered with
SST (Figure 1), and the calculated Ti/2 was 3.5 minutes (Table 4). SST fusion
protein
exhibited a bi-phasic pharmacokinetic profile in each of the rats administered
with SST
fusion protein (Figure 2), where the average a-phase T1/2 (0-0.5 hour) and 0-
phase T1/2 (0.75-
4 hours) was calculated as 1.01 hour and 6.14 hour, respectively (Table 4).
The calculated
half-life of SST fusion protein was significantly longer than the calculated
plasma T1/2 of free
SST (3.5 minutes) and reported plasma T1/2 of free SST in rat (< 1 minute;
Reference #1)
29
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
(Table 4). This set of results indicated the SST fusion protein significantly
improves the
stability and prolongs the half-life of SST in vivo.
Example 6-1: Preparation of Sample
The rat was restrained manually at the designated time points, approximately
300 i.t.L
of blood sample was collected via jugular vein into EDTA-K2 tubes and
subsequently
centrifuged at 4 C and 1500 g for 10 min to obtain plasma samples.
Example 6-2: Preparation of SST and SST Fusion Protein Standards
(1). Preparation of Free SST Standard
Standard curve was prepared by the following procedure. Duplicate standard
points
were prepared by serially diluting Free SST Stock (1 mg/mL) with diluent
buffer to produce
5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 and 0.039 ng/mL solutions.
(2). Preparation of SST Fusion Protein Standard
Standard curve was prepared by the following procedure. Duplicate standard
points
were prepared by serially diluting SST fusion protein (2.42 mg/mL) with pooled
rat plasma
to produce 225, 112.5, 56.2, 28.1, 14.1, 7.03, 3.51 and 1.76 ng/mL solutions.
Example 6-3: ELISA Assay Procedure
ELISA assay has been performed as described in Example 5-3.
Table 8. Plasma SST and SST Fusion Protein Concentration after IV injection to
Rat
SST Dose Dose Sampling Time
Mean (ng/mL) Standard Deviation
(mg/kg) Route (min)
0.02 IV 0 0 NA
1 7.13 5.0
2 4.95 2.1
4 4.30 NA
6 1.07 0.33
8 2.95 NA
27.1 IV 0.00 0.36 NA
CA 03032995 2019-02-04
WO 2018/038803 PCT/US2017/039477
0.05 756393.18 223856.37
0.13 743515.21 233145.74
0.25 634640.70 243150.70
0.50 649841.53 252801.38
0.75 560439.29 183395.09
1.25 480207.30 105178.15
2.00 493399.21 90422.74
3.00 416740.09 98435.97
4.00 366465.56 98751.94
Table 9. Pharmacokinetic Parameters of SST and SST Fusion Protein in Rat
after Intravenous Administration
SST Fusion Protein SST
PK
Unit Mean SD Mean SD
Parameters
AUCo-t mg= h/mL 1960799 427419 29.0 16.0
AUCO-mf mg=h/mL 4730184 1698725 43.8 20.1
AUMCo-t mg-112/mL 3502133 775860 55.9 27.1
AUMCo-nif mg-112/mL 36520105 19055736 173 49.9
MRTw h 7.2 2.0 4.1 0.75
CL mL/kg = min 0.11 0.049 510 234
CL mL/ kg-h 6.53 2.9 NA NA
Vdss L/ kg 0.043 0.0058 2.19 1.3
31
