Note: Descriptions are shown in the official language in which they were submitted.
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PEGYLATED GROWTH HORMONE ANTAGONISTS
REFERENCE TO A SEQUENCE LISTING
[0001] A sequence listing in computer readable form (CRF) is on file. The
sequence listing
is in an ASCII text (.txt) file entitled SEQIDNOS 1 24 ST25.txt created on
December 10, 2018
and is 33 KB in size. The sequence listing is incorporated by reference as if
fully recited herein.
BACKGROUND OF THE INVENTION
[0002] The described invention relates in general to compositions for use
as receptor
antagonists, and more specifically to novel human growth hormone antagonists
that have the
potential to be highly effective therapeutics.
[0003] Human growth hormone, also known as somatotropin or somatropin, is
a peptide
hormone that stimulates growth, cell reproduction, and regeneration in humans
and other animals.
Growth hormone is a type of mitogen that is specific only to certain kinds of
cells and is a 191-
amino acid, single-chain polypeptide that is synthesized, stored, and secreted
by somatotropic cells
within the lateral wings of the anterior pituitary gland. Acromegaly is a
syndrome that results when
the anterior pituitary gland produces excess growth hormone (hGH) after
epiphyseal plate closure
at puberty. If hGH is produced in excess prior to epiphyseal plate closure,
the result is gigantism
(or giantism). A number of disorders may increase the pituitary's hGH output,
although most
commonly it involves a tumor called pituitary adenoma, derived from a distinct
type of cell
(somatotrophs). Acromegaly most commonly affects adults in middle age and can
result in severe
disfigurement, complicating conditions, and premature death if untreated.
Because of its
pathogenesis and slow progression, the disease is hard to diagnose in the
early stages and is
frequently missed for years until changes in external features, especially of
the face, become
noticeable.
[0004] A receptor is a protein molecule usually found embedded within the
plasma
membrane surface of a cell that receives chemical signals from outside the
cell. When such
chemical signals bind to a receptor, they cause some form of cellular/tissue
response such as, for
example, a change in the electrical activity of the cell. In this sense, a
receptor is a protein molecule
that recognizes and responds to endogenous chemical signals. An agonist, such
as human growth
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hormone, is a chemical composition that binds to a receptor and activates the
receptor to produce
a biological response. Whereas an agonist causes an action, an antagonist
blocks the action of the
agonist and an inverse agonist causes an action opposite to that of the
agonist. A receptor
antagonist is a type of receptor ligand or drug that blocks or dampens agonist-
mediated responses
rather than provoking a biological response itself upon binding to a receptor.
These compositions
are sometimes called blockers and examples include alpha blockers, beta
blockers, and calcium
channel blockers. In pharmacology, antagonists have affinity but no efficacy
for their cognate
receptors, and binding will disrupt the interaction and inhibit the function
of an agonist or inverse
agonist at receptors. Antagonists mediate their effects by binding to the
active (orthosteric) site or
to other (allosteric) sites on receptors, or they may interact at unique
binding sites not normally
involved in the biological regulation of the receptor's activity. Antagonist
activity may be
reversible or irreversible depending on the longevity of the
antagonist¨receptor complex, which,
in turn, depends on the nature of antagonist¨receptor binding. The majority of
drug antagonists
achieve their potency by competing with endogenous ligands or substrates at
structurally defined
binding sites on receptors. By definition, antagonists display no efficacy to
activate the receptors
they bind and antagonists do not maintain the ability to activate a receptor.
Once bound, however,
antagonists inhibit the function of agonists, inverse agonists, and partial
agonists.
[0005] Growth hormone receptor antagonists such as the product
pegvisomant (sold under
the trademark SOMAVERT ) are used in the treatment of acromegaly. Such
compositions are
used if the tumor of the pituitary gland causing the acromegaly cannot be
controlled with surgery
or radiation and the use of somatostatin analogues is unsuccessful.
Pegvisomant is typically
delivered as a powder that is mixed with water and injected under the skin.
[0006] PEGylation is the process of both covalent and non-covalent
amalgamation of
polyethylene glycol (PEG) polymer chains to molecules and macrostructures,
such as drugs,
peptides, antibody fragments, or therapeutic proteins. PEGylation is routinely
achieved by
incubation of a reactive derivative of PEG with the target molecule and
produces alterations in
physiochemical properties, including changes in molecular size and molecular
charge. These
physical and chemical changes increase systemic retention of the therapeutic
agent and can
influence the binding affinity of the therapeutic moiety to the cell receptors
and can alter the
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absorption and distribution patterns. The covalent attachment of PEG to a drug
or therapeutic
protein can also "mask" the agent from the host's immune system (i.e.,
reducing immunogenicity
and antigenicity), and increase the hydrodynamic size (i.e., size in solution)
of the agent which
prolongs its circulatory time by reducing renal clearance. PEGylation can also
provide water
solubility to hydrophobic drugs and proteins.
[0007] PEGylation, by increasing the molecular weight of a molecule, can
impart several
significant pharmacological advantages over the unmodified form of the
molecule, such as: (i)
improved drug solubility; (ii) reduced dosage frequency, without diminished
efficacy and with
potentially reduced toxicity; (iii) extended circulating life; (iv) increased
drug stability; and (v)
enhanced protection from proteolytic degradation. PEGylated drugs also include
the following
commercial advantages: (i) opportunities for new delivery formats and dosing
regimens; and (ii)
extended patent life of previously approved drugs. PEG is a particularly
attractive polymer for
conjugation and the specific characteristics of PEG moieties relevant to
pharmaceutical
applications include: (i) water solubility; (ii) high mobility in solution;
(iii) lack of toxicity and
low immunogenicity; and (v) altered distribution in the body.
[0008] The addition of high molecular weight polyethylene glycols (PEGs)
to proteins has
been previously shown to increase the in-vivo half-lives of these proteins by
a size dependent
decrease in elimination by the kidneys. The addition of PEGs also lowers the
immunogenicity of
the proteins and decreases aggregation and protease cleavage (Pasut and
Vronese, 2012; and
Parveen and Sahoo, 2006). Multiple known PEGylated proteins have been approved
by the
USFDA for therapeutic use, including hormones, cytokines, antibody fragments,
and enzymes
(Pasut, and Veronese, 2012; Alconcel et al., 2011; and Kling, 2013). Thus,
there is an ongoing
need for the further development of PEGylated therapeutics, particularly for
use in the treatment
of diseases that are responsive to the use of human growth hormone (hGH)
receptor antagonists or
other receptor antagonists.
SUMMARY OF THE INVENTION
[0009] The following provides a summary of certain exemplary embodiments
of the
present invention. This summary is not an extensive overview and is not
intended to identify key
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or critical aspects or elements of the present invention or to delineate its
scope. However, it is to
be understood that the use of indefinite articles in the language used to
describe and claim the
present invention is not intended in any way to limit the described system.
Rather the use of "a"
or "an" should be interpreted to mean "at least one" or "one or more". As will
be appreciated by
one skilled in the art, the single letter amino acid abbreviations used herein
follow the IUPAC
format.
[0010] In accordance with one aspect of the present invention, a first
composition or
compound that functions as a human growth hormone receptor antagonist is
provided. This human
growth hormone receptor antagonist includes human growth hormone receptor
antagonist G120K,
wherein one amino acid of human growth hormone receptor antagonist G120K has
been mutated
to cysteine or wherein two amino acids of human growth hormone receptor
antagonist G120K
have been mutated to cysteine, and wherein the one amino acid mutated to
cysteine is selected
from the group consisting of N99, T142, and H151, and wherein the two amino
acids mutated to
cysteine are selected from the group consisting of N99/T142, N99/H151, and
T142/H151; and a
polyethylene glycol molecule conjugated to each substituted cysteine in the
human growth
hormone receptor antagonist G120K mutant.
[0011] In accordance with another aspect of the present invention, a
second composition
or compound that functions as a human growth hormone receptor antagonist is
provided. This
human growth hormone receptor antagonist includes human growth hormone
receptor antagonist
G120K, wherein one amino acid of human growth hormone receptor antagonist
G120K has been
mutated to cysteine or wherein two amino acids of human growth hormone
receptor antagonist
G120K have been mutated to cysteine, and wherein the one amino acid mutated to
cysteine is
selected from the group consisting of N99, T142, and H151, and wherein the two
amino acids
mutated to cysteine are selected from the group consisting of N99/T142,
N99/H151, and
T142/H151; and a polyethylene glycol molecule conjugated to each substituted
cysteine in the
human growth hormone receptor antagonist G120K mutant, wherein the
polyethylene glycol
molecule conjugated to the one amino acid mutated to cysteine is a
polydispersed 40 kDa branched
polyethylene glycol molecule; and wherein the polyethylene glycol molecules
conjugated to the
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two amino acids mutated to cysteine are either two 40 kDa branched
polyethylene glycol
molecules or two 4.5 kDa branched polyethylene glycols each containing three
carboxylate anions.
[0012] In yet another aspect of this invention, a third composition or
compound that
functions as a human growth hormone receptor antagonist is provided. This
human growth
hormone receptor antagonist includes human growth hormone receptor antagonist
G120K,
wherein two amino acids of human growth hormone receptor antagonist G120K have
been
mutated to cysteine, and wherein the two amino acids mutated to cysteine are
selected from the
group consisting of N99/T142, N99/H151, and T142/H151; and a polyethylene
glycol molecule
conjugated to each substituted cysteine in the human growth hormone receptor
antagonist G120K
mutant.
[0013] Additional features and aspects of the present invention will
become apparent to
those of ordinary skill in the art upon reading and understanding the
following detailed description
of the exemplary embodiments. As will be appreciated by the skilled artisan,
further embodiments
of the invention are possible without departing from the scope and spirit of
the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Exemplary embodiments of the present invention are described
below. Although
the following detailed description contains many specifics for purposes of
illustration, a person of
ordinary skill in the art will appreciate that many variations and alterations
to the following details
are within the scope of the invention. Accordingly, the following embodiments
of the invention
are set forth without any loss of generality to, and without imposing
limitations upon, the claimed
invention.
[0015] The present invention provides novel human growth hormone (hGH)
antagonists
for use primarily as therapeutics. The hGH antagonists of this invention are
typically made by
mutating one or more selected amino acids of hGH G120K, a known hGH
antagonist, to cysteines
and then conjugating the cysteines to chemically activated polyethylene glycol
molecules. The
positions of the substituted cysteines have been selected for minimal loss in
receptor binding
activity after conjugation with polyethylene glycol. The specific type and
number of polyethylene
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glycol modifiers of this invention have been selected to produce antagonists
with increased in-vivo
half-lives.
[0016] Two important variables in the preparation of PEGylated proteins
in accordance
with this invention are: (i) the amino acid position used for PEG attachment;
and (ii) the size and
type of the conjugated PEG. Initial research with similar compositions was
done using random
attachment of relatively small PEGs (e.g., about 5 kDa) to multiple lysines on
the surfaces of
proteins. This procedure successfully increased the in vivo half-lives of the
proteins, but resulted
in large decreases in the affinity of the proteins for their receptors. More
recent experimental
approaches have added PEG molecules to specific amino acid sites on proteins.
Two common
methods used for site specific PEGylation are: (i) addition of PEG to the N-
terminal amine of
proteins by way of low pH reductive amination; and (ii) addition of PEG to the
thiol groups of
cysteines that are either native to the protein or engineered into specific
positions. Other methods
include PEG addition to unnatural amino acids; PEG addition to proteins C-
termini by way of
intein fusion proteins; and PEG addition to accessible glutamines by way of
transaminase catalysis
(Pasut and Veronese, 2012).
[0017] Two or more different types of polyethylene glycol (PEG) molecules
are utilized
with the present invention. A first type of PEG is prepared by polymerization
and is by nature
polydispersed, in that there is a distribution of molecular weight products
around the average
molecular weight. A second class of polyethylene glycols are discrete PEGs
(dPEG s; Quanta
BioDesign). Such dPEG s are single PEG molecules that are prepared by step-
wise, organic
chemistry so that each dPEG species is a pure single compound with a specific
structure and
molecular weight (Povosky et al., 2013). The different types of PEGs have been
produced as both
linear and branched structures. For large polydispersed PEGs, the addition of
a branched PEG to
a protein may cause less of a decrease in binding affinity and a greater
increase in half-life than
addition of a linear PEG of the same molecular weight (Zhang et al., 2012).
Branched dPEG s
have been shown to increase protein half-lives and a negatively charged dPEG
has been shown
to be particularly efficacious (Ding et al., 2013).
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[0018] Pegylated Growth Hormone Antagonists
[0019] The conversion of hGH from a growth agonist to a growth antagonist
requires only
a single amino acid change at hGH position 120 from the native glycine to any
amino acid except
alanine (Chen et al., 1994). This molecule, however, cannot be used as a
therapeutic for conditions
of excess growth (e.g., acromegaly) due to its short in vivo half-life.
Researchers have addressed
this problem by the addition of polyethylene glycol molecules to the hGH
antagonist hGH G120K
to decrease the clearance of the molecule through the kidneys. SOMAVERT , an
FDA approved
treatment for acromegaly, contains 4-6 linear PEG molecules with molecular
weights of 5000
Daltons each. The addition of the PEGs, which are attached randomly to surface
lysines (van der
Lely and Kopchick, 2006), increases the in vivo half-life of the antagonist
from less than an hour
to approximately 72 hours (Finn, 2009). The affinity of the pegylated
antagonist for the membrane
bound receptor, however, is reduced approximately 30 fold compared with the
unpegylated
molecule (Ross et al., 2001). Despite the decrease in receptor affinity,
SOMAVERT is an
effective treatment for acromegaly, although a large daily dose of 5-30 mg is
typically prescribed.
[0020] The loss of receptor binding that occurs after the addition of
multiple low molecular
weight PEGs (about 5 kDa) to random lysines on a protein was common with early
protein-PEG
conjugates (Parveen and Sahoo, 2006). More recently, however, researchers have
made conjugates
with higher receptor binding activity by adding a single higher molecular
weight PEG to specific
amino acid positions on the protein target (Pasut and Veronese, 2012). In the
case of growth
hormone antagonists, either a 20 kDa or a 40 kDa linear PEG was added to the N-
terminus of the
unpegylated precursor to SOMAVERT (B2036) using reductive alkylation at a low
pH (Wu et
al., 2013). The addition of the 20 kDa PEG and the 40 kDa PEG reduced the
affinity of the
antagonist for the soluble hGH receptor by about 50% and about 95%,
respectively. The ability of
these molecules to inhibit the production of insulin growth factor-1 (IGF-1)
in rats was tested.
While the 40 kDa PEG conjugate was inactive, the 20 kDa conjugate reduced the
IGF-1 production
by 30 to 40%.
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[0021] Pegylated Growth Hormone
[0022] Insights regarding the predicted effects of different PEGylation
strategies on
growth hormone antagonist activity and half-life can be obtained by analysis
of the results from
PEGylation of human growth hormone (hGH) (Finn, 2009). Cox and coworkers
(2007) mutated
the threonine at position 3 of human growth hormone to cysteine (T3C hGH) and
then conjugated
the cysteine to a 20 kDa linear PEG. The activity of hGH, measured by a
proliferation assay,
decreased by about 4 fold and the half-life increased by about 8 fold. Similar
increases in half-life
occurred after the enzyme catalyzed addition of a 20 kDa PEG to Gln141 and the
chemical addition
of a 20 kDa PEG to the N-terminus (Freitas et al., 2013). The addition of a 30
kDa linear PEG to
different amino acid positions, which were mutated to chemically active non-
native amino acids,
resulted, in the optimal cases, in a roughly 10 fold loss of proliferation
activity and a roughly 10
fold increase in half-life (Cho et al., 2011). The addition of a linear 43 kDa
PEG to position 141
(Gln mutated to cysteine) was reported to increase the half-life ¨30 fold and
the addition of a
branched 40 kDa PEG to the N-terminus of hGH was reported to increase the half-
life about 20
fold (Rasmussen et al., 2010).
[0023] PEGylated Cytokines
[0024] The site-specific addition of PEGs to cytokines, which have
molecular weights
similar to that of hGH, indicates that a single PEG can cause a significant
increase in half-life.
Rosendahl et al. (2005) reported that addition of a 10 kDa PEG, a 20 kDa PEG,
and a 40 kDa PEG
to position 5 of Interferon a-2, after mutation of this position to cysteine,
reduced the in vitro
bioactivity by a factor of 2-3. In contrast, the half-life increased for the
10 kDa, 20 kDa PEG, and
40 kDa PEG by factors of 14, 23 and 40 respectively. A similar result was
found for the addition
of different molecular weight PEGs to human granulocyte-macrophage colony-
stimulating factor
(Doherty et al., 2005). Bell et al. (2008) found that the addition of either a
20 kDa or a 40 kDa
PEG to position 111 (M111C) of interferon a led to a three-fold decrease in
receptor binding
activity. The half-lives for the 20 kDa and the 40 kDa substituted interferons
increased by 25 fold
and 39 fold respectively. In contrast, the site specific addition of a 40 kDa
PEG to interferon 13-lb
only increased the half-life of by about three-fold (Lee et al., 2013).
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[0025] Qiu et al. (2013) substituted position 22 of human thyroid
stimulating hormone
with a linear 40 kDa PEG, a two branched 40 kDa PEG, and a three branched 40
kDa PEG and
found that the receptor binding activity decreased by 5-fold, 14 fold, and 11
fold respectively. A
comparison of the half-lives of these different conjugates was not reported.
Fam et al. (2014)
studied the effect of the addition of 10 kDa, 20 kDa, and 40 kDa PEGs to
position103 of interferon
y and found that the PEGs did not significantly affect the cytokine's in vitro
activity, but in all
cases the half-life was increased by about 30 fold.
[0026] Antibody Fragments
[0027] Extensive work has been done on the addition of PEGs to antibody
fragments to
increase their residence time in the body. Lee et al. (1999) conjugated a
single chain antibody
fragment (scFv MAb; 26 kDa) with six different PEG polymers with MWs ranging
from 2 to 20
kDa. These conjugates showed longer half-lives compared to their nonPEGylated
parent.
Increasing PEG polymer length was found to be more effective for half-life
extension than
increasing total PEG mass. Li et. al. (2010) showed that the addition of two
discrete linear PEG
units conjugated to random lysines on a diabody resulted in longer blood
retention times than
unpegylated or polydisperse pegylated products. Chapman et al. (2002)
demonstrated that the half-
lives of antibody Fab' fragments (about 50 kDa) are directly related to the
size and numbers of
site-directed PEGs.
[0028] Lee et al. (2013) found that an increase in linear, randomly
conjugated PEG mass
(4-20 kDa) in scFv-PEG conjugates effectively increased half-lives roughly
linearly with mass.
These workers found that a single 20 kDa PEG was more effective than four
5,000 PEGs,
concluding that PEG length was more important than PEG mass. In a study of the
roughly 50 kDa
Fab' antibody fragment, the site specific addition of a 4.4 kDa branched
discrete PEG (dPEG)
increased the half-life by a factor of about two over unconjugated Fab' (Ding
et al., 2013).
[0029] Selection of Amino Acid Positions of hGH G120K to Mutate to
Cysteine
[0030] In various embodiments of the present invention, PEGylated
versions of the
antagonist hGH G120K were prepared by attaching PEGs to cysteine residues that
have been
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incorporated into the antagonist sequence through genetic engineering. The
antagonist positions
selected for mutation to cysteine were selected using the X-ray structure of
the complex of hGH
with an hGH receptor dimer (hGHR2; Sundstrom et al., 1996). The structure of
hGH when bound
to hGHR2 is almost identical to the structure of the hGH antagonist hGH G12OR
when the
antagonist is bound to the same receptor (Sundstrom et al., 1996).
[0031] Two criteria, based on the hGH-hGHR2 crystal structure, were used
to select amino
acids for cysteine mutation: (i) accessibility of the amino acid to solvent;
and (ii) making the
substitution of cysteine for the selected amino acid needs close to an
energetically neutral process.
The solvent accessibility of each amino acid in hGH-hGHR2 was determined by
way of the
modeling programs Swiss PDB Viewer (Guex and Peitsch, 1997) and PoPMuSIC
(Dehouck et al.,
2009; Dehouck et al., 2011). The energetic cost of substituting cysteine for
each amino acid
position was determined using the Prediction of Protein Mutant Stability
Changes (PoPMuSIC)
program (http://babylone.ulb.ac.be/popmusic/).
[0032] The amino acids selected by solvent (e.g., water) accessibility
and mutation energy
considerations are listed below in Table 1 under "All Selected Positions" in
seven spatially
separate domains. However, being accessible to water is not necessarily a sole
criteria for selection;
the amino acids need to be accessible to the much larger PEG molecules in
order for the PEGylated
antagonists to bind to a target receptor. The X-ray structure of hGH-hGHR2 was
inspected to
determine if the side chains of the selected amino acids are directed towards
solvent or towards
the hGH-hGHR2 protein complex. Amino acid positions whose side chains point
into the solvent
are the most desirable candidates for PEG substitution and are listed below in
Table 1 under "Final
Selection". An additional position, H151, was also selected for cysteine
mutation. This position is
part of a section of Loop 3 that was not apparent in the crystal structure.
The general location of
the missing segment is pointing away from the hGH-hGHR2 protein complex.
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[0033] TABLE 1. Final Selection of Amino Acid Positions
All Selected Positions Final Selection
(amino acids whose side chains point away
from the protein structure)
Domain 1 (N-Terminus): Fl, T3 T3
Domain 2 (Loop 1): E39 E39
Domain 3 (Loop 1): P48 P48
Domain 4 (Loop 1): Q69 Q69
Domain 5 (Loop 2): N99, L101 N99, L101
Domain 6 (Loop 3): T142, D147, D154 T142
Domain 7 (C-Terminus) G190 G190
[0034] Selection of
PEGs for Conjugation to hGH G120K Mutants
[0035] Two different classes of polyethylene glycol (PEG) molecules are
utilized with the
present invention. The first class of PEGs was prepared by polymerization and
has been used to
modify proteins in order to increase their in vivo half-lives (Kling, 2013).
This type of PEG is by
nature polydispersed, meaning that there is a distribution of molecular weight
products around the
average molecular weight. The PEGs include a 20 kDa linear PEG (Layson Bio,
MPEG-MAL-
20,000), a 40 kDa branched PEG (NOF, Sunbright GL2-400MA), and a linear 40 kDa
PEG (NOF,
Sunbright ME-400MA).These PEGS each contain a maleimide group for conjugation
to the free
sulfhydryl groups of the mutant proteins. The second class of polyethylene
glycols are "discrete"
PEGs (dPEG s; Quanta BioDesign). These dPEG s are pure single PEG molecules
that are
prepared using step-wise, organic chemistry so that each dPEG species is a
pure single compound
with a specific structure and molecular weight (Povosky et al., 2013). The
dPEGs used in this
invention, which typically contain a maleimide group for coupling to free
thiols, include the
following: a tri-branched molecule with a molecular weight of 4473 Daltons and
a carboxylate
anion at the terminus of each branch (Quanta BioDesign #10451, MAL-dPEGA); a
neutral tri-
branched molecule with a molecular weight of 4299 Daltons (Quanta BioDesign
#4229, MAL-
dPEGB); a neutral 9-branched molecule with a molecular weight of 8324 (Quanta
Biodesign
#10484; MAL-dPEGE); and a neutral 9-branched molecule with a molecular weight
of 15,592
(Quanta Biodesign #11487; MAL-dPEGF). The tri-branched 4473 Da molecule has
been
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conjugated to an antibody fragment and its effect on blood clearance in mice
has been investigated
(Ding et al., 2013). While the added dPEG increased the molecular weight of a
50 kDa protein
molecular weight by only about 8%, the "area under the curve" (AUC) for blood
clearance
increased by a factor of about 2.5 over the AUC for the unPEGylated protein.
[0036] Purification and Pegylation of hGH G120K Mutants
[0037] Cell Disruption
[0038] A cell pellet obtained from centrifugation of 250 mL of growth
medium containing
the expressed mutant was suspended in 10 mL PBS and combined with 0.05 mL of a
protease
inhibitor cocktail without EDTA (Sigma P8849). The solution was cooled in an
ice water mixture
and sonicated for four minutes in 30 second bursts. After each sonication, the
sample was cooled
in the ice-water mixture until the temperature was below 4 C. The sonicated
suspension was then
centrifuged at 4 C and 25,000 X g for 30 minutes and the supernatant was
collected and kept on
ice.
[0039] Affinity Purification
[0040] The sonicated supernatant was adjusted to 0.3 M sodium chloride
and made 5 mM
imidazole by addition of a pH 7 solution of 150 mM imidazole. The sample was
then applied to a
gravity flow column having a stoppered outlet packed with 5 mL of TALON
(Clontech)
immobilized metal affinity resin (IMAC). The resin was equilibrated in 0.05 M
sodium phosphate
buffer, pH 7.0 containing 5 mM imidazole and 0.3 M sodium chloride prior to
addition of the
supernatant. The top of the column was then also stoppered and the column
mixed end-over-end
at room temperature for 30 minutes. The column was then allowed to drain and
washed with at
least five 5 mL aliquots of equilibration buffer. Washing was continued until
the A(280) nm of the
eluent no longer decreased. The column was then eluted with pH 7 equilibration
buffer containing
150 mM imidazole and the product containing fractions were made 5 mM EDTA by
addition of a
100 mM solution of disodium EDTA adjusted to pH 7.
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[0041] TEV Protease Cleavage of His-Tag
[0042] The IMAC purified mutant was concentrated by molecular filtration
to 2 mg/mL
and 0.5 mL of the solution was combined with 0.05 mL of a solution containing
15 mM reduced
glutathione + 1.5 mM oxidized glutathione. An aliquot of 0.04 mg TEV Protease
(TurboTev,
Accelagen) was then added and the solution incubated for two hours at room
temperature followed
by overnight incubation at 4 C. The imidazole containing buffer was then
exchanged on a spin
column for a buffer containing 0.05 M sodium phosphate, pH 7.0 and 0.3 M
sodium chloride.
[0043] Pegylation and Purification
[0044] The desalted mutant was PEGylated by making the solution 0.5 mM
maleimide-
PEG and incubating the reaction for two hours at room temperature followed by
overnight
incubation at 4 C. The PEGylated mutant was then applied to a gravity flow
IMAC column
containing 1 mL TALON resin equilibrated in the spin column buffer and the
column was washed
with 5 CVs of the same buffer. The TALON flow through and wash contained the
product, which
was then concentrated by a centrifugal concentrator to 0.3 mL and purified by
size exclusion
chromatography on a Superdex 200 Increase 10/300 GL column (GE Healthcare)
equilibrated in
0.05 M Tris Buffer, pH 8, containing 0.15 M sodium chloride and 10% glycerol.
The product
fractions were combined and analyzed for protein concentration by absorption
at A(280) nm and
for purity by SDS-PAGE. The addition of a single dPEGB to the single cysteine
mutants and two
dPEGB s to the double cysteine mutants was confirmed by MALDI mass
spectrometry.
[0045] Competition ELISA Assay of Relative Affinity for the hGH Receptor
[0046] A Competition ELISA required the preparation of biotinylated hGH,
which was
prepared by standard methods (Hermanson, 2008) using Biotin-dPEG12-NHS (Quanta
BioDesign). Microtiter plates (Corning 96 well plates, half-area, polystyrene)
were coated with
0.05 mL of 0.125 vg/mL solutions of the hGH receptor (R&D Systems, 1210-GR-50;
cloned as a
chimira with an antibody Fc region) in a 0.05 M sodium carbonate buffer at pH
9.6 and incubated
either at 37 C for one hour or overnight at 4 C. After washing the plate three
times, with three
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minute incubations between washes, with of 0.125 mL PBS, 0.05% Tween 20 (Wash
Buffer), the
plates were blocked for one hour by incubation with 2% BSA in PBS.
[0047] Preliminary ELISA assays were performed to determine the
concentration of
biotin-hGH to use in the completion ELISA. Plates coated with the hGH receptor
and blocked
were incubated for one hour at RT with different concentrations of
biotinylated hGH dissolved in
PBS, 0.1% BSA, 0.05% Tween 20 (Dilution Buffer). The plates were then washed
3X with Wash
Buffer and incubated for 1 hour at room temperature with 0.5 ug/mL
Streptavidin-HRP (Pierce,
21130) in Dilution Buffer. The plates were again washed 3 times and developed
by the addition of
0.05 mL TMB (KPL). After incubation for 2-20 minutes at room temperature, the
plates were then
quenched by the addition of 0.1 mL 1 M HC1 and read on a plate reader at 450
nm.
[0048] A concentration of biotin-hGH was selected such that the assay
response was in the
linear range of the plot of biotin-hGH versus A(450) nm (approximately 1 OD
unit). Competition
assays were performed by the preparation of solutions in polypropylene 96 well
plates that
contained the selected concentration of biotin-hGH and varying concentrations
of hGH, hGH
G120K mutant, or pegylated hGH G120K mutant. Ninety-six well immunoassay
plates were
coated with the hHG receptor, blocked as described above, and then incubated
for 1 hour at RT
with the solutions containing the selected concentration of biotin-hGH and
different concentrations
of the inhibitors. The plates were then washed and treated with Streptavidin-
HRP and TMB as
described above.
[0049] The concentration of recombinant hGH that gave a 50% inhibition of
the assay
response (IC50) was used as the standard to determine the relative affinities
of the mutants for the
hGH receptor. Each assay plate contained a series of concentrations of both
the hGH standard and
the mutants to be tested and the relative IC50 values were determined. Two
polydispersed PEGs
(ME400MA and GL2-400MA) were conjugated to the free thiols of hGH G120K-H151C
and hGH
G120K-N99C. G120K-H151C-ME400MA and G120K-H151C-GL2-400MA had, respectively,
20% and 50% of the inhibitory activity of G120K. N99C-ME400MA and N99C-GL2-
400MA had
20% and 2% of the inhibitory activity of G120K.
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[0050]
The pegylated hGH antagonist hGH G120K-T142C-GL2-400MA was been
prepared and purified using the procedures described herein. GL2-400MA is a 40
kDa two-
branched PEG containing a maleimide group that was reacted with the inserted
cysteine of hGH
G120K-T142C. This molecule, which is expected to have a long serum half-life
(see Zhang et al.,
2012), retained 50% of the hGH receptor binding activity of unmodified hGH.
The molecule hGH
G120K-H151C-GL2-400MA, which is also disclosed herein, was shown to also
retain 50% of the
hGH receptor binding activity of unmodified hGH.
[0051]
The binding affinities of the different dPEG conjugated mutants of the
present
invention relative to that of hGH are shown in Table 2, below. Seven single
mutants and three
double mutants were conjugated to a single tri-branched molecule dPEG with a
molecular weight
of 4473 Daltons (dPEGA) and the molecule was purified as described. As shown
in Table 2, certain
of these single mutants were also conjugated to three other dPEGs. Three
double cysteine mutants
were also prepared and conjugated to different dPEGs, as shown in Table 2.
[0052] TABLE 2.
Receptor Binding Activity of hGH 120K Mutants
hGH Mutant Percent Receptor Binding Activities
Relative to that of hGH
-All Mutants Contain the G120K Mutation Determined from the Concentration
of Each Sample that Yields
50% Inhibition (I50)
dPEG Substitution dPEGA2 dPEGB2 dPEGE2
dPEGF2
G120K-T3C-dPEGX 70 70 NT
NT
G120K-E39C-dPEGX 20 NT NT
NT
G120K-P48C-dPEGX 20 NT NT
NT
G120K-Q69C-dPEGX 20 NT NT
NT
G120K-N99C-dPEGX 90 70 40 4
G120K-T142C-dPEGX 50 90 50
20
G120K-11151C-dPEGX 100 60 40 4
G120K-N99C-dPEGX-11151C-dPEGX 20 40 20
G120K-T142C-dPEGX-N99C-dPEGX 50 80 30
G120K-T142C-dPEGX-11151C-dPEGX 50 40 10
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1 The receptor binding activities were determined using a competitive ELISA
where the
recombinant receptor was bound to a plate and the concentration of each sample
needed to
inhibit the binding of biotin-hGH to the coated plate by 50% (I50) was
determined. The
Table entries show the 1505 relative to that of hGH, which is defined as 100%,
and are
rounded to a single significant figure. Only a single competitive ELISA was
run for most
of the mutants and the estimated relative standard deviation is 25%. Entries
marked NT
were not tested in this assay.
2 dPEGA is a tri-branched molecule with a molecular weight of 4473 Daltons and
a
carboxylate anion at the terminus of each branch; dPEGB is a neutral tri-
branched molecule
with a molecular weight of 4299 Daltons, dPEGE is a neutral 9-branched
molecule with a
molecular weight of 8324; and dPEGF is a neutral 9-branched molecule with a
molecular
weight of 15,592.
3 These reactions did not proceed to the double PEGylated product.
[0053] Western Blot Assay for the Ability of PEGylated Mutants to Inhibit
the
Stimulation of STAT 5 Phosphorylation by hGH
[0054] The ability of the PEGylated mutants of the present invention to
inhibit the
stimulation of Stat5 Protein phosphorylation by hGH was measured in a cell-
based assay. IM9
cells were incubated in RPMI media for two hours. The cells were then
resuspended in fresh RPMI
media at 1 million cells per mL and treated with either hGH, pegylated hGH
mutants, or hGH +
pegylated mutants at concentrations from 0 to 5000 ng/mL. The treated cells
were then incubated
for 15 minutes at 37 C in a 5% carbon dioxide incubator. The cells were then
spun down, lysed in
a buffer containing 1% Triton X-100 and sodium orthovanadate, and loaded on an
SDS PAGE gel.
The gel was run under standard conditions and the proteins then transferred
electrophoretically to
a PVDF membrane. The membrane was blocked and then incubated overnight at 4 C
with a
mixture of rabbit anti-Stat5 Protein antibody and rabbit anti-3-actin antibody
(positive cell
control). The membrane was then washed and incubated with a HRP conjugated
goat anti-rabbit
antibody for one hour at room temperature. Finally, the bands were visualized
using Pierce
Supersignal West chemiluminescent substrate. Qualitative results for the
PEGylated mutants are
given in Table 3, below. The relative abilities of the hGH G120K pegylated
double mutants of the
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present invention to inhibit stimulation of STAT 5 phosphorylation by hGH as
measured by
Western blot assay are presented in Table 4, below.
[0055]
TABLE 3. Western Blot Assay for the Ability of the PEGylated Mutants
to Inhibit the Stimulation of STAT 5 Phosphorylation by hGH1
hGH Mutant Ability of the Pegylated Mutants to
Inhibit the Stimulation of
STAT 5 Phosphorylation by hGH
-All Mutants Contain the G120K Mutation
dPEG Substitution dPEGA2 dPEGB2 dPEGE2
dPEGF2
G120K-T3C-dPEGX NT NT NT
G120K-E39C-dPEGX NT NT NT NT
G120K-P48C-dPEGX NT NT NT NT
G120K-Q69C-dPEGX NT NT NT NT
G120K-N99C-dPEGX NT NT
G120K-T142C-dPEGX
G120K-11151C-dPEGX NT NT
G120K-N99C-dPEGX-11151C-dPEGX NT NT
G120K-T142C-dPEGX-N99C-dPEGX NT
G120K-T142C-dPEGX-11151C-dPEGX NT NT
1 The Western assay qualitatively measures the abilities of hGH
antagonists to inhibit the
hGH stimulation of STAT 5 phosphorylation. The inhibition is expressed as
relative to the
inhibition obtained with the parent antagonist hGH G120K. In all cases, the
relative
abilities of the pegylated antagonists to inhibit STAT 5 phosphorylation was
between
¨20% and ¨100% that of hGH G120K. The variation between duplicate runs was too
great
to make this a quantitative assay. Entries marked NT were not tested in this
assay.
2 dPEGA is a tri-branched molecule with a molecular weight of 4473 Daltons and
a
carboxylate anion at the terminus of each branch; dPEGB is a neutral tri-
branched molecule
with a molecular weight of 4299 Daltons, dPEGE is a neutral 9-branched
molecule with a
molecular weight of 8324; and dPEGF is a neutral 9-branched molecule with a
molecular
weight of 15,592.
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[0056] TABLE 4. Western blot assay for the Ability of the hGH G120K
PEGylated Double Mutants to Inhibit the Stimulation of STAT 5
Phosphorylation by hGH3
hGH G120K Mutant
Percent Inhibition of Stimulation of STAT 5
Phosphorylation by hGH3
-All Mutants Contain the G120K Mutation
G120K-N99C-dPEGA-H151C-dPEGM 20%
G120K-T142C-dPEGA-N99C-dPEGA4 20%
G120K-T142C-dPEGA-H151C-dPEGM 50%
3 This Western Blot assay measures the abilities of pegylated hGH
antagonists to inhibit
the hGH stimulation of STAT 5 phosphorylation. The inhibition is expressed as
relative
to the inhibition obtained with the parent antagonist hGH G120K. The
quantification was
obtained from the intensities of the phosphorylated STAT 5 band on the Western
Blots.
4 dPEGA is a tri-branched molecule with a molecular weight of 4473 Daltons
and a
carboxylate anion at the terminus of each branch.
[0057] As indicated by the disclosure above, the compositions of the
present invention
provide novel human growth hormone receptor antagonists that are useful in
therapeutic
applications. For reference purposes, SEQ ID NO: 1 provides the DNA sequence
for human
growth hormone WThGH and SEQ ID NO: 2 and provides the amino acid sequence for
human
growth hormone WThGH (mature form). Human growth hormone receptor antagonist
G120K is
the parent receptor antagonist for the compositions of the present invention,
and for reference
purposes, SEQ ID NO: 3 provides the DNA sequence for human growth hormone
receptor
antagonist G120K and SEQ ID NO: 4 provides the amino acid sequence for human
growth
hormone receptor antagonist G120K (mature form). As previously stated, the
single letter amino
acid abbreviations used herein follow the IUPAC format.
[0058] A first human growth hormone antagonist in accordance with an
exemplary
embodiment of the present invention includes human growth hormone antagonist
G120K, wherein
amino acid T3 has been mutated to cysteine, and wherein a polyethylene glycol
molecule has been
conjugated to the cysteine mutation. SEQ ID NO: 5 provides the DNA sequence
for human growth
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hormone antagonist G120K-T3C and SEQ ID NO: 6 provides the amino acid for
sequence human
growth hormone antagonist G120K-T3C.
[0059] A second human growth hormone antagonist in accordance with an
exemplary
embodiment of the present invention includes human growth hormone antagonist
G120K, wherein
amino acid E39 has been mutated to cysteine, and wherein a polyethylene glycol
molecule has
been conjugated to the cysteine mutation. SEQ ID NO: 7 provides the DNA
sequence for human
growth hormone antagonist G120K-E39C and SEQ ID NO: 8 provides the amino acid
sequence
for human growth hormone antagonist G120K-E39C.
[0060] A third human growth hormone antagonist in accordance with an
exemplary
embodiment of the present invention includes human growth hormone antagonist
G120K, wherein
amino acid P48 has been mutated to cysteine, and wherein a polyethylene glycol
molecule has
been conjugated to the cysteine mutation. SEQ ID NO: 9 provides the DNA
sequence for human
growth hormone antagonist G120K-P48C and SEQ ID NO: 10 provides the amino acid
sequence
for human growth hormone antagonist G120K-P48C.
[0061] A fourth human growth hormone antagonist in accordance with an
exemplary
embodiment of the present invention includes human growth hormone antagonist
G120K, wherein
amino acid Q69 has been mutated to cysteine, and wherein a polyethylene glycol
molecule has
been conjugated to the cysteine mutation. SEQ ID NO: 11 provides the DNA
sequence for human
growth hormone antagonist G120K-Q69C and SEQ ID NO: 12 provides the amino acid
sequence
for human growth hormone antagonist G120K-Q69C.
[0062] A fifth human growth hormone antagonist in accordance with an
exemplary
embodiment of the present invention includes human growth hormone antagonist
G120K, wherein
amino acid N99 has been mutated to cysteine, and wherein a polyethylene glycol
molecule has
been conjugated to the cysteine mutation. SEQ ID NO: 13 provides the DNA
sequence for human
growth hormone antagonist G120K-N99C and SEQ ID NO: 14 provides the amino acid
sequence
for human growth hormone antagonist G120K-N99C.
[0063] A sixth human growth hormone antagonist in accordance with an
exemplary
embodiment of the present invention includes human growth hormone antagonist
G120K, wherein
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amino acid T142 has been mutated to cysteine, and wherein a polyethylene
glycol molecule has
been conjugated to the cysteine mutation. SEQ ID NO: 15 provides the DNA
sequence for human
growth hormone antagonist G120K-T142C and SEQ ID NO: 16 provides the amino
acid sequence
for human growth hormone antagonist G120K-T142C.
[0064] A seventh human growth hormone antagonist in accordance with an
exemplary
embodiment of the present invention includes human growth hormone antagonist
G120K, wherein
amino acid H151 has been mutated to cysteine, and wherein a polyethylene
glycol molecule has
been conjugated to the cysteine mutation. SEQ ID NO: 17 provides the DNA
sequence for human
growth hormone antagonist G120K-H151C and SEQ ID NO: 18 provides the amino
acid sequence
for human growth hormone antagonist G120K-H151C.
[0065] An eighth human growth hormone antagonist in accordance with an
exemplary
embodiment of the present invention includes human growth hormone antagonist
G120K, wherein
amino acids N99 and H151 have been mutated to cysteine, and wherein a
polyethylene glycol
molecule has been conjugated to each cysteine mutation. SEQ ID NO: 19 provides
the DNA
sequence for human growth hormone antagonist G120K-N99C-dPEGX-H151C and SEQ ID
NO:
20 provides the amino acid sequence for human growth hormone antagonist G120K-
N99C-
dPEGX-H151C.
[0066] A ninth human growth hormone antagonist in accordance with an
exemplary
embodiment of the present invention includes human growth hormone antagonist
G120K, wherein
amino acids T142 and N99 have been mutated to cysteine, and wherein a
polyethylene glycol
molecule has been conjugated to each cysteine mutation. SEQ ID NO: 21 provides
the DNA
sequence for human growth hormone antagonist G120K-T142C-dPEGX-N99C and SEQ ID
NO:
22 provides the amino acid sequence for human growth hormone antagonist G120K-
T142C-
dPEGX-N99C.
[0067] A tenth human growth hormone antagonist in accordance with an
exemplary
embodiment of the present invention includes human growth hormone antagonist
G120K, wherein
amino acids T142 and H151 have been mutated to cysteine, and wherein a
polyethylene glycol
molecule has been conjugated to each cysteine mutation. SEQ ID NO: 23 provides
the DNA
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sequence for human growth hormone antagonist G120K-T142C-dPEGX-H151C and SEQ
ID NO:
24 provides the amino acid sequence for human growth hormone antagonist G120K-
T142C-
dPEGX-H151C.
[0068] While the present invention has been illustrated by the description
of exemplary
embodiments thereof, and while the embodiments have been described in certain
detail, there is
no intention to restrict or in any way limit the scope of the appended claims
to such detail.
Additional advantages and modifications will readily appear to those skilled
in the art. Therefore,
the invention in its broader aspects is not limited to any of the specific
details, representative
devices and methods, and/or illustrative examples shown and described.
Accordingly, departures
may be made from such details without departing from the spirit or scope of
the general inventive
concept.
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