Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
Compositions and Methods for Treating Cancer with Arginine Depletion and
Inununo Oncology
Agents.
BACKGROUND OF THE INVENTION
[0001] It has been recognized for over 50 years that certain tumor cells
have a high
demand for amino acids, such as L-arginine and are killed under conditions of
L-arginine
depletion (Wheatley and Campbell, 2002). In human cells L-arginine is
synthesized in three
steps; first L-citrulline is synthesized from L-omithine and carbamoyl
phosphate by the enzyme
omithine transcarbamylase (OTC), argininosuccinate synthetase (ASS) converts L-
citrulline and
aspartate to argininosuccinate, followed by conversion of argininosuccinate to
L-arginine and
fwnarate by argininosuccinate lyase (ASL). A large number of hepatocellular
carcinomas
(HCC), melanomas and, renal cell carcinomas (Ensor et al., 2002; Feun et al.,
2007; Yoon et al.,
2007) do not express ASS and thus are sensitive to L-arginine depletion. The
molecular basis for
the lack of ASS expression appears to be diverse and includes aberrant gene
regulation. Whereas
non-malignant cells enter into quiescence (Go) when depleted of L-arginine and
thus remain
viable for several weeks, tumor cells have cell cycle defects that lead to the
re-initiation of DNA
synthesis even though protein synthesis is inhibited, in turn resulting in
major imbalances and
rapid cell death (Shen et al., 2006; Scott et al., 2000). The selective
toxicity of L-arginine
depletion for HCC, melanoma and other ASS-deficient cancer cells has been
extensively
demonstrated in vitro, in xenograft animal models and in clinical trials
(Ensor et al., 2002; Feun
et al., 2007; Shen et al., 2006; Izzo et al., 2004). Recently Cheng et al.
(2007) demonstrated that
many HCC cells are also deficient in omithine transcarbamylase expression and
thus, they are
also susceptible to enzymatic L-arginine depletion.
100021 There is interest in the use of L-arginine hydrolytic enzymes for
cancer therapy,
especially the treatment of cancers such as hepatocarcinomas, melanomas and
renal cell
carcinomas, for example, which are common forms of cancer associated with high
morbidity.
Two L-arginine degrading enzymes have been used for cancer therapy: bacterial
arginine
deiminase and human arginases. Unfortunately, both of these enzymes display
significant
shortcomings that present major impediments to clinical use (immunogenicity,
and low catalytic
activity with very poor stability in serum, respectively). Thus, the
therapeutic success of L-
arginine depletion therapy will rely on addressing these shortcomings.
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100031 Another challenge in the treatment of many cancers is the ability
of some cancers
to evade the immune system. Some tumors, for example, do this through the
immune checkpoint
pathways, which are inhibitory pathways in the immune system that maintain
self-tolerance by
modulating immune response. These pathways can be dysregulated by tumors
resulting in
immune resistance. Some of these pathways, both agonists of prostimulatory
receptors or
antagonists of inhibitory signals, both of which result in amplification of
antigen-specific T-cell
responses, have become targets for cancer immunotherapy. Some exemplary
receptors and
ligands include cytotoxic T-lymphocyte-associated antigen 4 (CTLA4),
programmed cell death 1
(PD1), programmed cell death ligand 1 (PDL1), lymphocyte activation gene 3
(LAG3), B7-H3,
B-7-H4, and T cell membrane protein 3 (TIM3) among others. (Pardo11, 2012).
SUMMARY OF THE INVENTION
[0004] An aspect of the present disclosure generally relates to
compositions and methods
for the treatment of cancer with enzymes that deplete L-arginine in serum. In
some
embodiments, the cancer is one that does not express, or is otherwise
deficient in,
argininosuccinate synthetase (ASS), ornithine transcarbamylase (OTC), or
argininosuccinate
lyase (ASL).
[0005] In some aspects, the present invention also contemplates the use
of arginase
proteins wherein the natural metal cofactor (Mn2+) is replaced with another
metal. In particular
embodiments, the arginase protein comprises an amino acid sequence of human
Arginase I or an
amino acid sequence of human Arginase II and a non-native metal cofactor. In
some
embodiments, the metal is cobalt (Co2+). Human Arginase I and II proteins of
the present
invention have two Mn (II) sites; either or both sites can be substituted so
as to generate a
modified Arginase I or II protein with a non-native metal cofactor. In some
embodiments, the
protein displays a lccat/Km greater than 400 m1\44 s-1 at pH 7.4. In a
particular embodiment, the
protein displays a kcat/Km between 400 mM-1 s and 4,000 mM-I s at pH 7.4. In
another
embodiment, the protein displays a kcat/Km between 400 mM-1 s-1 and 2,500 niM4
s-1 at pH 7.4 at
37 C. In a particular embodiment, the present invention contemplates a
protein comprising an
amino acid sequence of human Arginase I or II and a non-native metal cofactor,
wherein said
protein exhibits a kcat/Km greater than 400 mM-1 s-1 at 37 C., pH 7.4.
[0006] Yet another aspect of the present disclosure is methods of
treating cancer or
tumors by arginine depletion in conjunction with an immunotherapeutic
treatment targeting an
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immune checkpoint pathway, for example. arginine depletion can be accomplished
with
administration of a human Arginase I or Arginase II enzyme, including
engineered or derivatized
arginase enzymes as well as arginase or other arginine depleting enzymes from
other species that
exhibit at least an additive or synergistic effect when administered with an
immune checkpoint
targeted therapy.
[0007] The present disclosure can be described in certain embodiments,
therefore, as a
method of inhibiting tumor growth in a subject, comprising administering a
pharmaceutical
composition including a therapeutic amount of a human Arginase I enzyme
comprising a cobalt
cofactor and an immuno-oncology agent. The tumor can be of various types that
respond to
arginine depletion therapy and in certain embodiments is an arginine
auxotrophic tumor, or
includes arginine dependent or auxotrophic cells. In certain embodiments the
auxotrophic cells
exhibit a reduced or inhibited expression of one or more of ASS, OTC, ASL, or
a combination
thereof, thus requiring the tumor cell to utilize arginine from the serum.
[0008] In certain embodiments the human Arginase I or other enzyme is
stabilized by
association with a stabilizing agent in order to increase the half-life of the
enzyme in the serum
of a patient. As used herein "association" can include any of a number of
types of association
including, but not limited to covalent or non-covalent bonds, and can also
include a protein
fusion expressed from an engineered nucleic acid construct, from a hydrogen
bonding or
hydrophobic interaction and others known to those of skill in the art.
Stabilizing agents for use in
the disclosed methods can include but are not limited to polyethylene glycol,
often referred to as
pegylation, conjugation to one or more homogenous synthetic protein polymers,
referred to as
extenylation and commercially available under the trade name Xten ,
conjugation to one or more
Fe fragments or to a serum protein like albumin, for example. All such
stabilized enzymes and
others that would occur to those of skill in this art are contemplated by the
present disclosure.
[0009] The disclosed methods are applicable to both human and non-human
animal
subjects including but not limited to veterinary, agricultural, domestic or
research animals. It is
an aspect of the disclosure that the immuno-oncology agent enhances the
subject's immune
response. In certain embodiments enhancing an immune system includes
increasing activity of a
patient's T-cell response to the presence of a tumor. In certain embodiments,
therefore, the
immuno-oncology agent inhibits an immune suppressor, which is sometimes a cell
surface
receptor referred to as a checkpoint inhibitor, or a ligand for such a
receptor. Examples include,
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but are not limited to PD-1 pathway inhibitors such as an anti-PD-1 antibody
or an anti-PD-Li
antibody, 0X40 (CD134) pathway inhibitors such as anti-0X40 or anti-OX4OL
(CD252), anti-4-
1BB or other anti B7 family ligands such as anti-B7-H1 and anti-B7.1 for
example. Exemplary
antibodies include but are not limited to pembrolizumab, ipilimumab,
atezolizumab or
nivolumab.
[00010] The methods of the disclosure are contemplated for the treatment
of any
responsive cancer or tumor, including, but not limited to hepatocellular
carcinoma, renal cell
carcinoma, breast cancer, melanoma, prostate cancer, pancreatic cancer,
bladder cancer, colon
carcinoma, colorectal cancer, triple negative breast cancer, Hodgkin's
lymphoma, gastric cancer,
glioblastoma, Merkel cell carcinoma, lung carcinoma, small cell lung cancers
or non-small cell
lung cancers. The administration of a combination of the human Arginase I
enzyme and the anti-
PD-1 antibody or anti-PDL-1 antibody or other immune checkpoint or TNF
receptor inhibitors
can exhibit an additive effect on tumor growth inhibition compared to the
tumor growth
inhibition exhibited by administering a therapeutic dose of the anti-PD-1
antibody alone or the
anti-PD-Li antibody alone, or the human Arginase I enzyme alone, or in certain
embodiments
exhibits a greater than additive, or synergistic effect on the tumor growth or
cancer. The two
treatment regimens can be administered concurrently or they can be
administered sequentially as
needed.
[00011] The current disclosure can also be described in certain
embodiments as a method
of treating cancer in a cancer patient comprising administering to said
patient a therapeutic
amount of a pharmaceutical composition comprising a pegylated human Arginase I
enzyme
comprising a cobalt cofactor and an immune system modulating therapy
comprising
administering a pharmaceutical composition comprising an immuno-oncology
agent.
[00012] In certain embodiments a therapeutic amount of the pegylated human
Arginase I
enzyme comprising a cobalt cofactor is from about 0.01 mg/kg to about 7.5
mg/kg, about 0.05
mg/kg to about 5 mg/kg, or about 0.1 mg/kg to about 5 mg/kg, or any amount
derivable from or
contained within the preceding ranges.
[00013] The pharmaceutical composition including a pegylated human
Arginase I enzyme
comprising a cobalt cofactor can be administered parenterally, or it can be
delivered by various
routes known in the art, including but not limited to topically,
subcutaneously, intravenously,
intradermally, intraarterially, intraperitoneally, intralesionally,
intracranially, intraarticularly,
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intraprostatically, intrapleurally, intratracheally, intraocularly,
intranasally, intravitreally,
intravaginally, intrarectally, intramuscularly,
subcutaneously, subconjunctivally,
intravesicularlly, mucosally, intrapericardially, intraumbilically, orally, by
inhalation, by
injection, by infusion, by continuous infusion, by localized perfusion bathing
target cells directly,
via a catheter, or via a lavage. In certain embodiments the pharmaceutical
composition is
administered intravenously or subcutaneously.
[000141
The disclosure can also be described as a method of treating cancer in a
cancer
patient comprising administering to said patient an arginine depleting agent
and a checkpoint
pathway inhibitor or other immune system modulator that inhibits or reduces
cancer growth or
proliferation. The methods further include treatment of cancers in which the
therapeutic effect of
treatment with the arginine depleting agent and a checkpoint pathway inhibitor
is additive as
compared to treatment the arginine depleting agent alone or said checkpoint
pathway inhibitor
alone, or in which the therapeutic effect of treatment with said arginine
depleting agent and a
checkpoint pathway inhibitor is synergistic as compared to treatment the
arginine depleting agent
alone or said checkpoint pathway inhibitor alone. In certain embodiments the
treatment can
result in from 50% to 99%, or from 90% to 99% reduction in serum arginine in
the patient, or
reduction of serum arginine in a patient to an undetectable level.
[00015]
Enzymes useful in the practice of the methods can include arginase enzymes,
arginine deiminase enzymes or a combination thereof. The enzymes can be human
enzymes,
recombinant human enzymes, engineered human enzymes or enzymes from other
species, either
mammalian or bacterial, for example, including but not limited to mycoplasma.
[00016]
In some embodiments, the native arginase is modified only by the substitution
of
the metal cofactor. In other embodiments, the arginase is modified by
substitution of the metal
cofactor in addition to other modifications, such as substitutions, deletions,
truncations, or
stabilization by conjugation to a stabilizing protein or polymer, such as by
pegylation. In a
particular embodiment, the invention provides a protein comprising a native
amino acid
sequence of human Arginase I or II and a non-native metal cofactor, wherein
the amino acid
sequence is lacking part of the native sequence. In particular embodiments,
the non-native metal
cofactor is cobalt. In some embodiments, the arginase lacks a portion of the
wild-type sequence.
In other embodiments, the amino acid sequence comprises a truncated Arginase I
or Arginase II
sequence. In a particular embodiment, the arginase is Arginase II and lacks
the first 21 amino
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acids of the wild-type sequence. In another embodiment, the native arginases
lacks an N-terminal
methionine.
100017] In another aspect, the present invention contemplates an arginase
protein
comprising at least one amino acid substitution, wherein the protein displays
an increased
catalytic activity under physiological conditions and especially at the pH of
human serum (pH
7.4) when compared with native human Arginase I or II protein. In some
embodiments, the
arginase protein is a human Arginase I protein or human Arginase II protein.
In some
embodiments, the protein further comprises a non-native metal cofactor. In
particular
embodiments, the non-native metal cofactor is Co+2. Substitution of the Mn+2
cofactor with Co+2
results in marked increase in catalytic activity and a drastic reduction in Km
at physiological pH.
In some aspects, the present invention also contemplates fusion proteins
comprising an arginase
linked to a non-arginase amino acid sequence. In one embodiment, the non-
arginase sequence
comprises at least a portion of the Fc region of an immunoglobulin, e.g., to
increase the half-life
of the arginase in serum when administered to a patient. The Fc region or
portion thereof may be
any suitable Fc region. In one embodiment, the Fc region or portion thereof is
an IgG Fc region.
In some embodiments, the amino acid sequence having arginase activity is
selected from the
group consisting of a native or mutated amino acid sequence of human Arginase
I and a native or
mutated amino acid sequence of human Arginase II or other arginine depleting
enzymes known
in the art. In certain embodiments, a dimeric Fc-arginase fusion protein,
albumin, or a synthetic
protein conjugation is contemplated.
[00018] The arginase in the fusion protein may be native, mutated, and/or
otherwise
modified, e.g., metal cofactor modified. In some embodiments, the arginase may
contain
deletions, substitutions, truncations or a combination thereof. In a
particular embodiment, the
present invention contemplates an Fc-arginase containing fusion protein,
wherein the arginase is
an Arginase I. In one embodiment, the arginase lacks a portion of the wild-
type sequence. In
another embodiment, the arginase is Arginase I lacking an N-terminal
methionine. In yet another
embodiment, the arginase is Arginase II, wherein the Arginase II lacks the
first 21 amino acids of
the wild-type Arginase II sequence. In some embodiments, the arginase further
comprises a non-
native metal cofactor. In these embodiments, either or both sites can be
substituted to generate a
fusion protein comprising an amino acid sequence of human Arginase I or II and
a non-native
metal cofactor. In some embodiments, the non-native metal cofactor is cobalt.
In some
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embodiments, the arginase contains a substitution. Exemplary arginase enzymes
for use in the
present disclosure are more fully described in U.S. Patent No. 8,440,184,
incorporated herein in
its entirety by reference.
[00019] The present invention also contemplates methods of treatment by
the
administration of the arginase proteins of the present invention, and in
particular methods of
treating subjects with cancer. In some embodiments, the cancer is one that
does not express, or is
otherwise deficient in, ASS, OTC, or ASL. In particular embodiments, the human
cancer is an
arginine auxotrophic cancer. As discussed above, the arginase protein may be
native, mutated,
and/or otherwise modified, e.g., metal cofactor modified. In one embodiment,
the present
invention contemplates a method of treating a human cancer patient comprising
administering a
formulation comprising a fusion protein, the fusion protein comprising an
amino acid sequence
having arginase activity and at least a portion of the Fc region of a human
immunoglobulin to the
patient. In some embodiments, the administration occurs under conditions such
that at least a
portion of the cancer cells of the cancer are killed. In another embodiment,
the formulation
comprises an amino acid sequence having human arginase activity higher than
that displayed by
the authentic human arginases at physiological conditions and further
comprising one or more
attached polyethylene glycol chain(s). In some embodiment, the formulation is
a pharmaceutical
formulation comprising any of the above discussed arginase proteins and a
pharmaceutically
acceptable excipients. Such pharmaceutically acceptable excipients are well
known to those
having skill in the art. All of the above arginase variants are contemplated
as useful for human
therapy.
[00020] The cancer may be any type of cancer or tumor type. In some
embodiments, the
cancer is hepatocellular carcinoma, renal cell carcinoma, melanoma, prostate
cancer, or
pancreatic cancer. In some embodiments, the formulation is administered
topically,
intravenously, intradermally, intraarterially, intraperitoneally,
intralesionally, intracranially,
intraarticularly, intraprostaticaly, intrapleurally, intratracheally,
intraocularly, intranasally,
intravitreally, intravaginally, intrarectally, intramuscularly,
subcutaneously, subconjunctival,
intravesicularlly, mucosally, intrapericardially, intraumbilically, orally, by
inhalation, by
injection, by infusion, by continuous infusion, by localized perfusion bathing
target cells directly,
via a catheter, or via a lavage.
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[00021] All of the above mentioned arginases, variants and the like are
contemplated in a
preferred embodiment as purified or isolated proteins, and preferably
monomeric proteins.
[00022] The embodiments in the Example section are understood to be
embodiments of
the invention that are applicable to all aspects of the invention.
[00023] The use of the term "or" in the claims is used to mean "and/or"
unless explicitly
indicated to refer to alternatives only or the alternatives are mutually
exclusive, although the
disclosure supports a definition that refers to only alternatives and
"and/or."
[00024] Throughout this application, the term "about" is used to indicate
that a value
includes the standard deviation of error for the composition, device or method
being employed to
determine the value.
[00025] Following long-standing patent law, the words "a" and "an," when
used in
conjunction with the word "comprising" in the claims or specification, denotes
one or more,
unless specifically noted.
[00026] The term "therapeutically effective" as used herein refers to an
amount of an
active agent and/or therapeutic composition (such as a therapeutic
polynucleotide and/or
therapeutic polypeptide) that is employed in methods of the present invention
to achieve a
therapeutic effect, such as wherein at least one symptom of a condition being
treated is at least
ameliorated, and/or to the analysis of the processes or materials used in
conjunction with these
cells.
[00027] Other objects, features and advantages of the present invention
will become
apparent from the following detailed description. It should be understood,
however, that the
detailed description and the specific examples, while indicating specific
embodiments of the
invention, are given by way of illustration only, since various changes and
modifications within
the spirit and scope of the invention will become apparent to those skilled in
the art from this
detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[00028] The following drawings form part of the present specification and
are included to
further demonstrate certain aspects of the present invention. The invention
may be better
understood by reference to one or more of these drawings in combination with
the detailed
description of specific embodiments presented herein.
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[00029] FIG. 1 is a graph showing serum L-arginine depletion in the mouse
model. Serum
L-Arg concentrations of Balb/c mice treated with a single IP dose of Co-hArgI
are kept < to 3-4
uM for over 3 days.
[00030] FIG. 2 is a graph showing HCC tumor xenograft reduction when
treated with Co-
hArgI as compared to controls. Nude mice bearing a Hep3b tumor xenografts were
treated twice
by IP injection with either PBS (o) or Co-hArgI (*) at day 9 and at day 12.
Tumor shrinkage was
observed in the mice treated with Co-hArgI whereas PBS treated tumors grew
unchecked.
[00031] FIG. 3 is a graph showing the effect of cobalt loading on the
catalytic activity of
human Arginase I.
[00032] FIG. 4 is a graph showing colon carcinoma tumor growth inhibition
in CT26
mouse model with Co-hArgI, anti PD-Li and the combination of Co-hArgI and anti
PD-Li. As
seen in the data, the combination of the 2 agents has a greater than additive
effect on inhibition
of tumor growth.
[00033] FIG. 5 is a graph showing colon carcinoma tumor growth inhibition
in an MC38
mouse model with Co-hArgI, anti 0X40 antibodies, and the combination of Co-
hArgI and anti
0X40 antibodies. As seen in the data, the combination of the 2 agents has a
greater than additive
effect on inhibition of tumor growth.
[00034] FIG. 6 is a graph showing CD45+ Tcells present in CT26 mouse model
with Co-
hArgI, anti PD-L1 and the combination of Co-hArgI and anti PD-Li.
[00035] FIG. 7 is a graph showing percent CD8+ cells present in CD45+
Tcells as shown
in FIG. 6.
DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS
[00036] The invention generally relates to compositions and methods for
the treatment of
cancer with enzymes that deplete L-arginine in serum. In some embodiments, the
cancer is one
that does not express, or is otherwise deficient in, argininosuccinate
synthetase (ASS), ornithine
transcarbamylase (OTC), or argininosuccinate lyase (ASL), or other enzymes
required for
arginine biosynthesis. Both native and mutated enzymes are contemplated, as
well as enzymes
with modified metal cofactors, enzymes fused to other polypeptides as well as
enzymes
conjugated to polymers that increase serum persistence, e.g., high molecular
weight polyethylene
glycol
I. Arginase
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[00037] Arginase is a manganese-containing enzyme. It is the final enzyme
of the urea
cycle. Arginase is the fifth and final step in the urea cycle, a series of
biophysical reactions in
mammals during which the body disposes of harmful ammonia. Specifically,
arginases convert
L-arginine into L-ornithine and urea.
[00038] L-arginine is the nitrogen donating substrate for nitric oxide
synthase (NOS),
producing L-citrulline and NO. Although the Km of arginase (2-5 mM) has been
reported to be
much higher than that of NOS for L-arginine (2-20 p,M), arginase may also play
a role in
regulating NOS activity. Under certain conditions Arginase I is Cys-S-
nitrosylated, resulting in
higher affinity for L-arginine and reduced availability of substrate for NOS.
[00039] Arginase is a homo-trimeric enzyme with an a/P fold of a parallel
eight-stranded
I3sheet surrounded by several helices. The enzyme contains a di-nuclear metal
cluster that is
integral to generating a hydroxide for nucleophilic attack on the guanidinium
carbon of L-
arginine. The native metal for arginase is Mn2+. These Mn2+ ions coordinate
water, orientating
and stabilizing the molecule and allowing water to act as a nucleophile and
attack L-arginine,
hydrolyzing it into ornithine and urea.
[00040] Mammals have two arginase isozymes (EC 3.5.3.1) that catalyze the
hydrolysis of
L-arginine to urea and L-ornithine. The Arginase I gene is located on
chromosome 6 (6q.23), is
highly expressed in the cytosol of hepatocytes, and functions in nitrogen
removal as the final step
of the urea cycle. The Arginase II gene is found on chromosome 14 (14q.24.1).
Arginase II is
mitochondrially located in tissues such as kidney, brain, and skeletal muscle
where it is thought
to provide a supply of L-ornithine for proline and polyamine biosynthesis
(Lopez et al., 2005).
[00041] Arginases have been investigated for nearly 50 years as a method
for degrading
extracellular L-arginine (Dillon et al., 2002). Some promising clinical
results have been achieved
by introducing arginase by transhepatic arterial embolisation; following
which, several patients
experienced partial remission of HCC (Cheng et al., 2005). However, since
arginase has a high
Km (-2-5 mM) and exhibits very low activity at physiological pH values, high
dosing is required
for chemotherapeutic purposes (Dillon et al., 2002). While native arginase is
cleared from
circulation within minutes (Savoca et al., 1984), a single injection of PEG-
arginase MW5000 in
rats was sufficient to achieve near complete arginine depletion for ¨3 days
(Cheng et al., 2007).
[00042] Cheng et al. made the surprising observation that many human HCC
cells lines do
not express OTC (in addition to ASS) and thus they are susceptible to PEG-
arginase (Cheng et
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al., 2007). In mice implanted with Hep3b hepatocarcinoma cells weekly
administration of PEG-
arginase resulted in tumor growth retardation which was accentuated by co-
administration of 5-
fluorouracil (5-FU). However, PEG-arginase was used at the very high doses
that are impractical
for human therapy, reflecting its lower physiological activity.
[00043] To address these issues a bacterial arginine hydrolyzing enzyme,
arginine
deiminase or ADI which displays good kinetics and stability has been tested in
vitro and
clinically. Unfortunately ADI is a bacterial enzyme and therefore it induces
strong immune
responses and adverse effects in most patients. However, for those patients
who do not develop
significant adverse responses, an impressive percentage exhibit stable disease
or remission.
[00044] For clinical use, it is essential that the arginase is engineered
to allow it to persist
for long times (e.g., days) in circulation. In the absence of any
modification, human arginase has
a half-life of only a few minutes in circulation primarily because its size is
not sufficiently large
to avoid filtration though the kidneys. Unmodified human arginase is very
susceptible to
deactivation in serum and it is degraded with a half-life of only four hours.
Therefore, the present
invention developed novel and improved forms of arginase for clinical research
and potential
therapeutic use with improved circulation persistence.
II. Arginase Variants
[00045] Mammals have two arginase isozymes (EC 3.5.3.1) that catalyze the
hydrolysis of
L-arginine to urea and L-ornithine. The Arginase I gene is located on
chromosome 6 (6q.23), is
highly expressed in the cytosol of hepatocytes, and functions in nitrogen
removal as the final step
of the urea cycle. The Arginase II gene is found on chromosome 14 (14q.24.1).
Arginase II is
mitochondrially located in tissues such as kidney, brain, and skeletal muscle
where it is thought
to provide a supply of L-omithine for proline and polyamine biosynthesis
(Lopez et al., 2005).
L-arginine is the sole substrate for nitric oxide synthase (NOS), producing L-
citrulline and NO.
Although the Km of arginase (2-5 mM) has been reported to be much higher than
that of NOS for
L-arginine (2-20 M), arginase may also play a role in regulating NOS activity
(Durante et al.,
2007). Under certain conditions Arginase I is Cys-S-nitrosylated, resulting in
higher affinity for
L-arginine and reduced availability of substrate for NOS (Santhanam et al.,
2007). Arginase is a
homo-trimeric enzyme with an a/13 fold of a parallel eight-stranded n-sheet
surrounded by
several helices. The enzyme contains a di-nuclear metal cluster that is
integral to generating a
hydroxide for nucleophilic attack on the guanidinium carbon of L-arginine
(Cama et al., 2003;
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Dowling et al., 2008). The native metal for arginase is Mn2t arginase with the
native metal (i.e.
Mn2+) exhibits a pH optimum of 9. At physiological pH the enzyme exhibits more
than a 10-
fold lower kcat / Km. in the hydrolysis of L-arginine. The low catalytic
activity displayed by the
authentic human arginase with the native Mn2+ enzyme presents a problem for
human therapy
since it means that impractical doses of the enzyme may have to be used to
achieve a
therapeutically relevant reduction in L-arginine plasma levels.
1000401 In some aspects, the present invention contemplates mutant
arginases wherein the
natural metal cofactor (Mn2+) is replaced with another metal. It has been
found that substitution
of the metal cofactor in human arginase exerts a beneficial effect on the rate
of hydrolysis of L-
Arginine and stability under physiological conditions when compared to native
human arginase
with the natural metal cofactor. The substitution of the native metal (Mn2+)
with other divalent
cations can be exploited to shift the pH optimum of the enzyme to a lower
values and thus
achieve high rates of L-arginine hydrolysis under physiological conditions.
Human Arginase I
and II proteins of the present invention have two Mn (II) sites; therefore,
either or both sites can
be substituted so as to generate a mutated Arginase I or II protein with a non-
native metal
cofactor.
[00047.1 In some embodiments, the metal is cobalt (Co2 ). Incorporation of
Co2+ in the
place of Mn2+ in human Arginase I or human Arginase II results in dramatically
higher activity at
physiological pH. It was found that a human Arginase I enzyme containing Co2+
("Co-hArgI")
displayed a 10 fold increase in kcat / Km in vitro at pH 7.4, which in turn
translated into a 15 fold
increase in HCC cytotoxicity and a 13-fold increase in melanoma cytotoxity as
compared to the
human Arginase I which contains Mn2+ ("Mn-hArgI"). It was also found that a
pharmacological
preparation of Co-hArgI could clear serum L-Arg for over 3 days in mice with a
single injection.
Furthermore, it was found that a pharmacological preparation of Co-hArgI could
shrink HCC
tumor xenografts in nude mice whereas Mn-hArgI only slowed tumor growth (Ensor
et al.,
2002).
[00048.1 In certain aspects of the invention, methods and compositions
related to pegylated
arginase are disclosed. Specifically, pegylation of arginase at an engineered
cysteine residue
(e.g., substituting the third residue of the N-terminal) may be used to
produce a homogenous
pegylated arginase composition. Methods for isolation of pegylated arginase
based on temporary
disruption of polymerization are also disclosed.
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[00049] Pegylation is the process of covalent attachment of poly(ethylene
glycol) polymer
chains to another molecule, normally a drug or therapeutic protein. Pegylation
is routinely
achieved by incubation of a reactive derivative of PEG with the target
macromolecule. The
covalent attachment of PEG to a drug or therapeutic protein can "mask" the
agent from the host's
immune system (reduced immunogenicity and antigenicity), increase the
hydrodynamic size
(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.
[00050] The first step in pegylation is the suitable functionalization of
the PEG polymer at
one or both terminals. PEGs that are activated at each terminus with the same
reactive moiety are
known as "homobifunctional", whereas if the functional groups present are
different, then the
PEG derivative is referred as "heterobifunctional" or "heterofunctional." The
chemically active
or activated derivatives of the PEG polymer are prepared to attach the PEG to
the desired
molecule.
[00051] The choice of the suitable functional group for the PEG derivative
is based on the
type of available reactive group on the molecule that will be coupled to the
PEG. For proteins,
typical reactive amino acids include lysine, cysteine, histidine, arginine,
aspartic acid, glutamic
acid, serine, threonine, tyrosine. The N-terminal amino group and the C-
terminal carboxylic acid
can also be used.
[00052] The techniques used to form first generation PEG derivatives are
generally
reacting the PEG polymer with a group that is reactive with hydroxyl groups,
typically
anhydrides, acid chlorides, chloroforrnates and carbonates. In the second
generation pegylation
chemistry more efficient functional groups such as aldehyde, esters, amides
etc. made available
for conjugation.
[00053] As applications of pegylation have become more and more advanced
and
sophisticated, there has been an increase in need for heterobifunctional PEGs
for conjugation.
These heterobifunctional PEGs are very useful in linking two entities, where a
hydrophilic,
flexible and biocompatible spacer is needed. Preferred end groups for
heterobifunctional PEGs
are maleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids and
NHS esters.
[00054] The most common modification agents, or linkers, are based on
methoxy PEG
(mPEG) molecules. Their activity depends on adding a protein-modifying group
to the alcohol
end. In some instances polyethylene glycol (PEG diol) is used as the precursor
molecule. The
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diol is subsequently modified at both ends in order to make a hetero- or homo-
dimeric PEG-
linked molecule (as shown in the example with PEG bis-vinylsulfone).
[00055] Proteins are generally PEGylated at nucleophilic sites such as
unprotonated thiols
(cysteinyl residues) or amino groups. Examples of cysteinyl-specific
modification reagents
include PEG maleimide, PEG iodoacetate, PEG thiols, and PEG vinylsulfone. All
four are
strongly cysteinyl-specific under mild conditions and neutral to slightly
alkaline pH but each has
some drawbacks. The amide formed with the maleimides can be somewhat unstable
under
alkaline conditions so there may be some limitation to formulation options
with this linker. The
amide linkage formed with iodo PEGs is more stable, but free iodine can modify
tyrosine
residues under some conditions. PEG thiols form disulfide bonds with protein
thiols, but this
linkage can also be unstable under alkaline conditions. PEG-vinylsulfone
reactivity is relatively
slow compared to maleimide and iodo PEG; however, the thioether linkage formed
is quite
stable. Its slower reaction rate also can make the PEG-vinylsulfone reaction
easier to control.
[00056] Site-specific pegylation at native cysteinyl residues is seldom
carried out, since
these residues are usually in the form of disulfide bonds or are required for
biological activity.
On the other hand, site-directed mutagenesis can be used to incorporate
cysteinyl pegylation sites
for thiol-specific linkers. The cysteine mutation must be designed such that
it is accessible to the
pegylation reagent and is still biologically active after pegylation.
[00057] Amine-specific modification agents include PEG NHS ester, PEG
tresylate, PEG
aldehyde, PEG isothiocyanate, and several others. All react under mild
conditions and are very
specific for amino groups. The PEG NHS ester is probably one of the more
reactive agents;
however, its high reactivity can make the pegylation reaction difficult to
control at large scale.
PEG aldehyde forms an imine with the amino group, which is then reduced to a
secondary amine
with sodium cyanoborohydride. Unlike sodium borohydride, sodium
cyanoborohydride will not
reduce disulfide bonds. However; this chemical is highly toxic and must be
handled cautiously,
particularly at lower pH where it becomes volatile.
[00058] Due to the multiple lysine residues on most proteins, site-
specific pegylation can
be a challenge. Fortunately, because these reagents react with unprotonated
amino groups, it is
possible to direct the pegylation to lower-pK amino groups by performing the
reaction at a lower
pH. Generally the pK of the a-amino group is 1-2 pH units lower than the
epsilon-amino group
of lysine residues. By PEGylating the molecule at pH 7 or below, high
selectivity for the N-
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terminus frequently can be attained. However; this is only feasible if the N-
terminal portion of
the protein is not required for biological activity. Still, the
pharmacokinetic benefits from
pegylation frequently outweigh a significant loss of in vitro bioactivity,
resulting in a product
with much greater in vivo bioactivity regardless of pegylation chemistry.
[000591 There are several parameters to consider when developing a
pegylation procedure.
Fortunately, there are usually no more than four or five key parameters. The
"design of
experiments" approach to optimization of pegylation conditions can be very
useful. For thiol-
specific pegylation reactions, parameters to consider include: protein
concentration, PEG-to-
protein ratio (on a molar basis), temperature, pH, reaction time, and in some
instances, the
exclusion of oxygen. Oxygen can contribute to intermolecular disulfide
formation by the protein,
which will reduce the yield of the PEGylated product. The same factors should
be considered
(with the exception of oxygen) for amine-specific modification except that pH
may be even more
critical, particularly when targeting the N-terminal amino group.
[00060] For both amine- and thiol-specific modifications, the reaction
conditions may
affect the stability of the protein. This may limit the temperature, protein
concentration, and pH.
In addition, the reactivity of the PEG linker should be known before starting
the pegylation
reaction. For example, if the pegylation agent is only 70% active, the amount
of PEG used
should ensure that only active PEG molecules are counted in the protein-to-PEG
reaction
stoichiometry. How to determine PEG reactivity and quality will be described
later.
IV. Proteins and Peptides
[00061] In certain embodiments, the present invention concerns novel
compositions
comprising at least one protein or peptide, such as stabilized arginase
multimers. These peptides
may be comprised in a fusion protein or conjugated to an agent as described
supra.
A. Proteins and Peptides
[00062] As used herein, a protein or peptide generally refers, but is not
limited to, a
protein of greater than about 200 amino acids, up to a full length sequence
translated from a
gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of
from about 3 to
about 100 amino acids. For convenience, the terms "protein," "polypeptide" and
"peptide" are
used interchangeably herein.
[00063] In certain embodiments the size of at least one protein or peptide
may comprise,
but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23,
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24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, 100,
about 110, about 120, about 130, about 140, about 150, about 160, about 170,
about 180, about
190, about 200, about 210, about 220, about 230, about 240, about 250, about
275, about 300,
about 325, about 350, about 375, about 400, about 425, about 450, about 475,
about 500, about
525, about 550, about 575, about 600, about 625, about 650, about 675, about
700, about 725,
about 750, about 775, about 800, about 825, about 850, about 875, about 900,
about 925, about
950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400,
about 1500, about
1750, about 2000, about 2250, about 2500 or greater amino acid residues.
[000641 As used herein, an "amino acid residue" refers to any naturally
occurring amino
acid, any amino acid derivative or any amino acid mimic known in the art. In
certain
embodiments, the residues of the protein or peptide are sequential, without
any non-amino acid
interrupting the sequence of amino acid residues. In other embodiments, the
sequence may
comprise one or more non-amino acid moieties. In particular embodiments, the
sequence of
residues of the protein or peptide may be interrupted by one or more non-amino
acid moieties.
Accordingly, the term "protein or peptide" encompasses amino acid sequences
comprising at
least one of the 20 common amino acids found in naturally occurring proteins,
or at least one
modified or unusual amino acid, including but not limited to those shown on
Table 1 below.
TABLE 1
Modified and Unusual Amino Acids
Abbr. Amino Acid
Aad 2-Aminoadipic acid
Baad 3-Aminoadipic acid
Bala 0-alanine, P-Amino-propionic
acid
Abu 2-Aminobutyric acid
4Abu 4-Aminobutyric acid,
piperidinic acid
Acp 6-Aminocaproic acid
Ahe 2-Aminoheptanoic acid
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Aib 2-Aminoisobutyric acid
Baib 3-Aminoisobutyric acid
Apm 2-Aminopimelic acid
Dbu 2,4-Diaminobutyric acid
Des Desmosine
Dpm -Diaminopimelic acid
Dpr 2,3-Diaminopropionic acid
EtGly N-Ethylglycine
EtAsn N-Ethylasparagine
Hyl Hydroxylysine
AHyl allo-Hydroxylysine
3Hyp 3 -Hydroxyproline
4Hyp 4-Hydroxyproline
Ide Isodesmosine
Alle allo-Isoleucine
MeGly N-Methylglycine, sarcosine
MeIle N-Methylisoleucine
MeLys 6-N-Methyllysine
MeVal N-Methylvaline
Nva Norvaline
Nle Norleucine
Om Ornithine
[000651 Proteins or peptides may be made by any technique known to those
of skill in the
art, including the expression of proteins, polypeptides or peptides through
standard molecular
biological techniques, the isolation of proteins or peptides from natural
sources, or the chemical
synthesis of proteins or peptides. The nucleotide and protein, polypeptide and
peptide sequences
corresponding to various genes have been previously disclosed, and may be
found at
computerized databases known to those of ordinary skill in the art. One such
database is the
National Center for Biotechnology Information's Genbank and GenPept databases
(available on
the world wide web at ncbi.nlm.nih.gov/). The coding regions for known genes
may be amplified
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and/or expressed using the techniques disclosed herein or as would be know to
those of ordinary
skill in the art. Alternatively, various commercial preparations of proteins,
polypeptides and
peptides are known to those of skill in the art.
B. Nucleic Acids and Vectors
[00066] In certain aspects of the invention, nucleic acid sequences
encoding a fusion
protein as a stabilized multimeric arginase may be disclosed. Depending on
which expression
system to be used, nucleic acid sequences can be selected based on
conventional methods. For
example, human arginase I and II contain multiple codons that are rarely
utilized in E. coli that
may interfere with expression, therefore the respective genes or variants
thereof may be codon
optimized for E. coli expression. Various vectors may be also used to express
the protein of
interest, such as a fusion multimeric arginase or a cysteine-substituted
arginase. Exemplary
vectors include, but are not limited, plasmid vectors, viral vectors,
transposon or liposome-based
vectors.
C. Host cells
[00067] Host cells, preferably eukaryotic cells, useful in the present
invention are any that
may be transformed to allow the expression and secretion of arginase and
fusion multimers
thereof. The host cells may be bacteria, mammalian cells, yeast, or
filamentous fungi. Various
bacteria include Escherichia and Bacillus. Yeasts belonging to the genera
Saccharomyces,
Kluyveromyces, Hansenula, or Pichia would find use as an appropriate host
cell. Various species
of filamentous fungi may be used as expression hosts including the following
genera:
Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya,
Podospora,
Endothia, Mucor, Cochliobolus and Pyricularia.
[00068] Examples of usable host organisms include bacteria, e.g.,
Escherichia coli
MC1061, derivatives of Bacillus subtilis BRB1 (Sibakov et al., 1984),
Staphylococcus aureus
SAI123 (Lordanescu, 1975) or Streptococcus lividans (Hopwood et al., 1985);
yeasts, e.g.,
Saccharomyces cerevisiae AH 22 (Mellor et al., 1983) and Schizosaccharomyces
pombe;
filamentous fungi, e.g., Aspergillus nidulans, Aspergillus awamori (Ward,
1989), Trichoderma
reesei (Penttila et al., 1987; Harkki et al, 1989).
[00069] Examples of mammalian host cells include Chinese hamster ovary
cells (CHO-
Kl; ATCC CCL61), rat pituitary cells (Gill; ATCC CCL82), HeLa S3 cells (ATCC
CCL2.2), rat
hepatoma cells (H-4-II-E; ATCCCRL 1548) SV40-transformed monkey kidney cells
(COS-1;
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ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658). The
foregoing
being illustrative but not limitative of the many possible host organisms
known in the art. In
principle, all hosts capable of secretion can be used whether prokaryotic or
eukaryotic.
[00070] Mammalian host cells expressing the arginase and/or their fusion
multimers are
cultured under conditions typically employed to culture the parental cell
line. Generally, cells are
cultured in a standard medium containing physiological salts and nutrients,
such as standard
RPM!, MEM, IMEM or DMEM, typically supplemented with 5-10% serum, such as
fetal bovine
serum. Culture conditions are also standard, e.g., cultures are incubated at
37 C. in stationary or
roller cultures until desired levels of the proteins are achieved.
D. Protein Purification
[00071] Protein purification techniques are well known to those of skill
in the art. These
techniques involve, at one level, the homogenization and crude fractionation
of the cells, tissue
or organ to polypeptide and non-polypeptide fractions. The protein or
polypeptide of interest
may be further purified using chromatographic and electrophoretic techniques
to achieve partial
or complete purification (or purification to homogeneity) unless otherwise
specified. Analytical
methods particularly suited to the preparation of a pure peptide are ion-
exchange
chromatography, gel exclusion chromatography, polyacrylamide gel
electrophoresis, affinity
chromatography, immunoaffinity chromatography and isoelectric focusing. A
particularly
efficient method of purifying peptides is fast performance liquid
chromatography (FPLC) or
even high performance liquid chromatography (HPLC).
[00072] A purified protein or peptide is intended to refer to a
composition, isolatable from
other components, wherein the protein or peptide is purified to any degree
relative to its
naturally-obtainable state. An isolated or purified protein or peptide,
therefore, also refers to a
protein or peptide free from the environment in which it may naturally occur.
Generally,
"purified" will refer to a protein or peptide composition that has been
subjected to fractionation
to remove various other components, and which composition substantially
retains its expressed
biological activity. Where the term "substantially purified" is used, this
designation will refer to
a composition in which the protein or peptide forms the major component of the
composition,
such as constituting about 50%, about 60%, about 70%, about 80%, about 90%,
about 95%, or
more of the proteins in the composition.
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[00073] Various techniques suitable for use in protein purification are
well known to those
of skill in the art. These include, for example, precipitation with ammonium
sulphate, PEG,
antibodies and the like, or by heat denaturation, followed by: centrifugation;
chromatography
steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and
affinity
chromatography; isoelectric focusing; gel electrophoresis; and combinations of
these and other
techniques. As is generally known in the art, it is believed that the order of
conducting the
various purification steps may be changed, or that certain steps may be
omitted, and still result in
a suitable method for the preparation of a substantially purified protein or
peptide.
[00074] Various methods for quantifying the degree of purification of the
protein or
peptide are known to those of skill in the art in light of the present
disclosure. These include, for
example, determining the specific activity of an active fraction, or assessing
the amount of
polypeptides within a fraction by SDS/PAGE analysis. A preferred method for
assessing the
purity of a fraction is to calculate the specific activity of the fraction, to
compare it to the specific
activity of the initial extract, and to thus calculate the degree of purity
therein, assessed by a "-
fold purification number." The actual units used to represent the amount of
activity will, of
course, be dependent upon the particular assay technique chosen to follow the
purification, and
whether or not the expressed protein or peptide exhibits a detectable
activity.
[00075] There is no general requirement that the protein or peptide always
be provided in
their most purified state. Indeed, it is contemplated that less substantially
purified products may
have utility in certain embodiments. Partial purification may be accomplished
by using fewer
purification steps in combination, or by utilizing different forms of the same
general purification
scheme. For example, it is appreciated that a cation-exchange column
chromatography
performed utilizing an HPLC apparatus will generally result in a greater "-
fold" purification than
the same technique utilizing a low pressure chromatography system. Methods
exhibiting a lower
degree of relative purification may have advantages in total recovery of
protein product, or in
maintaining the activity of an expressed protein.
[00076] In certain embodiments a protein or peptide may be isolated or
purified, for
example, a stabilized arginase multimeric fusion protein, or an arginase prior
or post pegylation.
For example, a His tag or an affinity epitope may be comprised in such a
arginase variant to
facilitate purification. Affinity chromatography is a chromatographic
procedure that relies on the
specific affinity between a substance to be isolated and a molecule to which
it can specifically
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bind. This is a receptor-ligand type of interaction. The column material is
synthesized by
covalently coupling one of the binding partners to an insoluble matrix. The
column material is
then able to specifically adsorb the substance from the solution. Elution
occurs by changing the
conditions to those in which binding will not occur (e.g., altered pH, ionic
strength, temperature,
etc.). The matrix should be a substance that itself does not adsorb molecules
to any significant
extent and that has a broad range of chemical, physical and thermal stability.
The ligand should
be coupled in such a way as to not affect its binding properties. The ligand
should also provide
relatively tight binding. And it should be possible to elute the substance
without destroying the
sample or the ligand.
[000771 Size exclusion chromatography (SEC) is a chromatographic method in
which
molecules in solution are separated based on their size, or in more technical
terms, their
hydrodynamic volume. It is usually applied to large molecules or
macromolecular complexes
such as proteins and industrial polymers. Typically, when an aqueous solution
is used to
transport the sample through the column, the technique is known as gel
filtration
chromatography, versus the name gel permeation chromatography which is used
when an
organic solvent is used as a mobile phase.
[00078] The underlying principle of SEC is that particles of different
sizes will elute
(filter) through a stationary phase at different rates. This results in the
separation of a solution of
particles based on size. Provided that all the particles are loaded
simultaneously or near
simultaneously, particles of the same size should elute together. Each size
exclusion column has
a range of molecular weights that can be separated. The exclusion limit
defines the molecular
weight at the upper end of this range and is where molecules are too large to
be trapped in the
stationary phase. The permeation limit defines the molecular weight at the
lower end of the range
of separation and is where molecules of a small enough size can penetrate into
the pores of the
stationary phase completely and all molecules below this molecular mass are so
small that they
elute as a single band.
[00079] High-performance liquid chromatography (or High pressure liquid
chromatography, HPLC) is a form of column chromatography used frequently in
biochemistry
and analytical chemistry to separate, identify, and quantify compounds. HPLC
utilizes a column
that holds chromatographic packing material (stationary phase), a pump that
moves the mobile
phase(s) through the column, and a detector that shows the retention times of
the molecules.
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Retention time varies depending on the interactions between the stationary
phase, the molecules
being analyzed, and the solvent(s) used.
V. Pharmaceutical Compositions
[000801 It is contemplated that the novel arginases of the -present
invention can be
administered systemically or locally to inhibit tumor cell growth and, most
preferably, to kill
cancer cells in cancer patients with locally advanced or metastatic cancers.
They can be
administered intravenously, intrathecally, and/or intraperitoneally. They can
be administered
alone or in combination with anti-proliferative drugs. In one embodiment, they
are administered
to reduce the cancer load in the patient prior to surgery or other procedures.
Alternatively, they
can be administered after surgery to ensure that any remaining cancer (e.g.
cancer that the
surgery failed to eliminate) does not survive.
[00081] It is not intended that the present invention be limited by the
particular nature of
the therapeutic preparation. For example, such compositions can be provided in
formulations
together with physiologically tolerable liquid, gel or solid carriers,
diluents, and excipients.
These therapeutic preparations can be administered to mammals for veterinary
use, such as with
domestic animals, and clinical use in humans in a manner similar to other
therapeutic agents. In
general, the dosage required for therapeutic efficacy will vary according to
the type of use and
mode of administration, as well as the particularized requirements of
individual subjects.
[00082] Such compositions are typically prepared as liquid solutions or
suspensions, as
injectables. Suitable diluents and excipients are, for example, water, saline,
dextrose, glycerol, or
the like, and combinations thereof. In addition, if desired the compositions
may contain minor
amounts of auxiliary substances such as wetting or emulsifying agents,
stabilizing or pH
buffering agents.
[000831 Where clinical applications are contemplated, it may be necessary
to prepare
pharmaceutical compositions--expression vectors, virus stocks, proteins,
antibodies and drugs¨in
a form appropriate for the intended application. Generally, pharmaceutical
compositions of the
present invention comprise an effective amount of one or more arginase
variants or additional
agent dissolved or dispersed in a pharmaceutically acceptable carrier. The
phrases
"pharmaceutical or pharmacologically acceptable" refers to molecular entities
and compositions
that do not produce an adverse, allergic or other untoward reaction when
administered to an
animal, such as, for example, a human, as appropriate. The preparation of an
pharmaceutical
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composition that contains at least one arginase variant, such as a stabilized
multimeric arginase
or a pegylated arginase isolated by the method disclosed herein, or additional
active ingredient
will be known to those of skill in the art in light of the present disclosure,
as exemplified by
Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by
reference.
Moreover, for animal (e.g., human) administration, it will be understood that
preparations should
meet sterility, pyrogenicity, general safety and purity standards as required
by FDA Office of
Biological Standards.
[00084] As used herein, "pharmaceutically acceptable carrier" includes any
and all
solvents, dispersion media, coatings, surfactants, antioxidants, preservatives
(e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying agents,
salts, preservatives, drugs,
drug stabilizers, gels, binders, excipients, disintegration agents,
lubricants, sweetening agents,
flavoring agents, dyes, such like materials and combinations thereof, as would
be known to one
of ordinary skill in the art (see, for example, Remington's Pharmaceutical
Sciences, 18th Ed.,
1990, incorporated herein by reference). Except insofar as any conventional
carrier is
incompatible with the active ingredient, its use in the pharmaceutical
compositions is
contemplated.
[00085] The present invention may comprise different types of carriers
depending on
whether it is to be administered in solid, liquid or aerosol form, and whether
it need to be sterile
for such routes of administration as injection. The present invention can be
administered
intravenously, intradermally, transdermally, intrathecally, intraarterially,
intraperitoneally,
intranasally, intravaginally, intrarectally, topically, intramuscularly,
subcutaneously, mucosally,
orally, topically, locally, inhalation (e.g., aerosol inhalation), injection,
infusion, continuous
infusion, localized perfusion bathing target cells directly, via a catheter,
via a lavage, in lipid
compositions (e.g., liposomes), or by other method or any combination of the
forgoing as would
be known to one of ordinary skill in the art (see, for example, Remington's
Pharmaceutical
Sciences, 18th Ed., 1990, incorporated herein by reference).
[00086] The arginase variants may be formulated into a composition in a
free base, neutral
or salt form. Pharmaceutically acceptable salts, include the acid addition
salts, e.g., those formed
with the free amino groups of a proteinaceous composition, or which are formed
with inorganic
acids such as for example, hydrochloric or phosphoric acids, or such organic
acids as acetic,
oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups
can also be derived
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from inorganic bases such as for example, sodium, potassium, ammonium, calcium
or ferric
hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine
or procaine.
Upon formulation, solutions will be administered in a manner compatible with
the dosage
formulation and in such amount as is therapeutically effective. The
formulations are easily
administered in a variety of dosage forms such as formulated for parenteral
administrations such
as injectable solutions, or aerosols for delivery to the lungs, or formulated
for alimentary
administrations such as drug release capsules and the like.
[00087] Further in accordance with the present invention, the composition
of the present
invention suitable for administration is provided in a pharmaceutically
acceptable carrier with or
without an inert diluent. The carrier should be assimilable and includes
liquid, semi-solid, i.e.,
pastes, or solid carriers. Except insofar as any conventional media, agent,
diluent or carrier is
detrimental to the recipient or to the therapeutic effectiveness of a
composition contained therein,
its use in administrable composition for use in practicing the methods of the
present invention is
appropriate. Examples of carriers or diluents include fats, oils, water,
saline solutions, lipids,
liposomes, resins, binders, fillers and the like, or combinations thereof. The
composition may
also comprise various antioxidants to retard oxidation of one or more
component. Additionally,
the prevention of the action of microorganisms can be brought about by
preservatives such as
various antibacterial and antifungal agents, including but not limited to
parabens (e.g.,
methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid,
thimerosal or combinations
thereof
[00088] In accordance with the present invention, the composition is
combined with the
carrier in any convenient and practical manner, i.e., by solution, suspension,
emulsification,
admixture, encapsulation, absorption and the like. Such procedures are routine
for those skilled
in the art.
[00089] In a specific embodiment of the present invention, the composition
is combined or
mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried
out in any
convenient manner such as grinding. Stabilizing agents can be also added in
the mixing process
in order to protect the composition from loss of therapeutic activity, i.e.,
denaturation in the
stomach. Examples of stabilizers for use in an the composition include
buffers, amino acids such
as glycine and lysine, carbohydrates such as dextrose, mannose, galactose,
fructose, lactose,
sucrose, maltose, sorbitol, mannitol, etc.
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1000901 In further embodiments, the present invention may concern the use
of a
pharmaceutical lipid vehicle compositions that include arginase variants, one
or more lipids, and
an aqueous solvent. As used herein, the term "lipid" will be defined to
include any of a broad
range of substances that is characteristically insoluble in water and
extractable with an organic
solvent. This broad class of compounds are well known to those of skill in the
art, and as the
term "lipid" is used herein, it is not limited to any particular structure.
Examples include
compounds which contain long-chain aliphatic hydrocarbons and their
derivatives. A lipid may
be naturally occurring or synthetic (i.e., designed or produced by man).
However, a lipid is
usually a biological substance. Biological lipids are well known in the art,
and include for
example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,
lysolipids,
glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-
linked fatty acids and
polymerizable lipids, and combinations thereof. Of course, compounds other
than those
specifically described herein that are understood by one of skill in the art
as lipids are also
encompassed by the compositions and methods of the present invention.
100091] One of ordinary skill in the art would be familiar with the range
of techniques that
can be employed for dispersing a composition in a lipid vehicle. For example,
the stabilized
multimeric or pegylated arginase may be dispersed in a solution containing a
lipid, dissolved
with a lipid, emulsified with a lipid, mixed with a lipid, combined with a
lipid, covalently bonded
to a lipid, contained as a suspension in a lipid, contained or complexed with
a micelle or
liposome, or otherwise associated with a lipid or lipid structure by any means
known to those of
ordinary skill in the art. The dispersion may or may not result in the
formation of liposomes.
[000921 The actual dosage amount of a composition of the present invention
administered
to an animal patient can be determined by physical and physiological factors
such as body
weight, severity of condition, the type of disease being treated, previous or
concurrent
therapeutic interventions, idiopathy of the patient and on the route of
administration. Depending
upon the dosage and the route of administration, the number of administrations
of a preferred
dosage and/or an effective amount may vary according to the response of the
subject. The
practitioner responsible for administration will, in any event, determine the
concentration of
active ingredient(s) in a composition and appropriate dose(s) for the
individual subject.
[000931 In certain embodiments, pharmaceutical compositions may comprise,
for
example, at least about 0.1% of an active compound. In other embodiments, the
an active
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compound may comprise between about 2% to about 75% of the weight of the unit,
or between
about 25% to about 60%, for example, and any range derivable therein.
Naturally, the amount of
active compound(s) in each therapeutically useful composition may be prepared
is such a way
that a suitable dosage will be obtained in any given unit dose of the
compound. Factors such as
solubility, bioavailability, biological half-life, route of administration,
product shelf life, as well
as other pharmacological considerations will be contemplated by one skilled in
the art of
preparing such pharmaceutical formulations, and as such, a variety of dosages
and treatment
regimens may be desirable.
1000941 In other non-limiting examples, a dose may also comprise from
about 1
microgram/kg/body weight, about 5 microgram/kg/body weight, about 10
microgram/kg/body
weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight,
about 200
microgram/kg/body weight, about 350 microgram/kg/body weight, about 500
microgram/kg/body weight, about 1 milligram/kg/body weight, about 5
milligram/kg/body
weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight,
about 100
milligram/kg/body weight, about 200 milligram/kg/body weight, about 350
milligram/kg/body
weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or
more per
administration, and any range derivable therein. In non-limiting examples of a
derivable range
from the numbers listed herein, a range of about 5 mg/kg/body weight to about
100 mg/kg/body
weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body
weight, etc., can be
administered, based on the numbers described above.
VII. Definitions
[00095] The term "aa" refers to amino acid(s). Amino acid substitutions
are indicated by
the amino acid position, e.g. 303, in the molecule using a letter code (the
letter in front of the
number indicates the amino acid being replaced, while the letter after the
number indicates the
amino acid being introduced).
[00096] As used herein the term "portion" when in reference to a protein
(as in "a portion
of a given protein") refers to fragments of that protein. The fragments may
range in size from
four amino acid residues to the entire amino acid sequence minus one amino
acid.
[00097] As used herein the terms "protein" and "polypeptide" refer' to
compounds
comprising amino acids joined via peptide bonds and are used interchangeably.
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1000981 As used herein, the term "fusion protein" refers to a chimeric
protein containing
the protein of interest (i.e., a human arginase or variant thereof) joined (or
operably linked) to an
exogenous protein fragment (the fusion partner which consists of a non-
arginase protein). The
fusion partner may enhance serum half-life, solubility, or both. It may also
provide an affinity tag
(e.g. his-tag) to allow purification of the recombinant fusion protein from
the host cell or culture
supernatant, or both.
[00099] The terms "in operable combination", "in operable order" and
"operably linked"
refer to the linkage of nucleic acid sequences in such a manner that a nucleic
acid molecule
capable of directing the transcription of a given gene and/or the synthesis of
a desired protein
molecule is produced. The term also refers to the linkage of amino acid
sequences in such a
manner so that a functional protein is produced.
[000100] The term "Km" as used herein refers to the Michaelis-Menten
constant for an
enzyme and is defined as the concentration of the specific substrate at which
a given enzyme
yields one-half its maximum velocity in an enzyme catalyzed reaction.
[000101] The term kcat as used herein refers to the turnover number or the
number of
substrate molecule each enzyme site converts to product per unit time, and in
which the enzyme
is working at maximum efficiency.
[000102] The term KcatiKm as used herein is the specificity constant which
is a measure of
how efficiently an enzyme converts a substrate into product.
[000103] The term "Mn-hArgI" refers to human Arginase I with an Mn (II)
cofactor. The
term "Co-hArgI" refers to human Arginase I (mutant or native) with a Co (II)
cofactor.
The term "IC so" is the half maximal (50%) inhibitory concentration (IC) and
thus a measure of
effectiveness.
[000104] The term "pegylated" refers to conjugation with polyethylene
glycol (PEG),
which has been widely used as a drug carrier, given its high degree of
biocompatibility and ease
of modification. (Harris et al., 2001). Attachment to various drugs, proteins,
and liposomes has
been shown to improve residence time and decrease toxicity. (Greenwald et al.,
2000; Zalipsky
et al., 1997). PEG can be coupled (e.g. covalently linked) to active agents
through the hydroxyl
groups at the ends of the chain and via other chemical methods; however, PEG
itself is limited to
at most two active agents per molecule. In a different approach, copolymers of
PEG and amino
acids have been explored as novel biomaterials which would retain the
biocompatibility
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properties of PEG, but which would have the added advantage of numerous
attachment points
per molecule (providing greater drug loading), and which can be synthetically
designed to suit a
variety of applications (Nathan et al., 1992; Nathan etal., 1993).
[0001051 The term "gene" refers to a DNA sequence that comprises control
and coding
sequences necessary for the production of a polypeptide or precursor thereof.
The polypeptide
can be encoded by a full length coding sequence or by any portion of the
coding sequence so
long as the desired enzymatic activity is retained.
[000106] The term "subject" refers to animals, including humans.
[000107] The term "wild-type" refers to a gene or gene product which has
the
characteristics of that gene or gene product when isolated from a naturally
occurring source. A
wild-type gene is that which is most frequently observed in a population and
is thus arbitrarily
designated the "normal" or "wild-type" form of the gene. In contrast, the term
"modified" or
"variant" or "mutant" refers to a gene or gene product which displays
modifications in sequence
and or functional properties (i.e., altered characteristics) when compared to
the wild-type gene or
gene product. It is noted that naturally-occurring mutants can be isolated;
these are identified by
the fact that they have altered characteristics when compared to the wild-type
gene or gene
product.
VIII. Kits
10001081 The present invention provides kits, such as therapeutic kits. For
example, a kit
may comprise one or more pharmaceutical composition as described herein and
optionally
instructions for their use. Kits may also comprise one or more devices for
accomplishing
administration of such compositions. For example, a subject kit may comprise a
pharmaceutical
composition and catheter for accomplishing direct intravenous injection of the
composition into a
cancerous tumor. In other embodiments, a subject kit may comprise pre-filled
ampoules of a
stabilized multimeric arginase or isolated pegylated arginase, optionally
formulated as a
pharmaceutical, or lyophilized, for use with a delivery device.
[000109] Kits may comprise a container with a label. Suitable containers
include, for
example, bottles, vials, and test tubes. The containers may be formed from a
variety of materials
such as glass or plastic. The container may hold a composition which includes
an antibody that is
effective for therapeutic or non-therapeutic applications, such as described
above. The label on
the container may indicate that the composition is used for a specific therapy
or non-therapeutic
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application, and may also indicate directions for either in vivo or in vitro
use, such as those
described above. The kit of the invention will typically comprise the
container described above
and one or more other containers comprising materials desirable from a
commercial and user
standpoint, including buffers, diluents, filters, needles, syringes, and
package inserts with
instructions for use.
IX. EXAMPLES
[000/10] The following examples serve to illustrate certain preferred
embodiments and
aspects of the present invention and are not to be construed as limiting the
scope thereof. In the
experimental disclosure which follows, the following abbreviations apply: eq
(equivalents); M
(Molar); AM (micromolar); mM (millimolar); N (Normal); mol (moles); mmol
(millimoles);
iumol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); Ag
(micrograms); L (liters);
ml (milliliters); Al (microliters); cm (centimeters); mm (millimeters); gm
(micrometers); nm
(nanometers); EC (degrees Centigrade); MW (molecular weight); PBS (phophate
buffered
saline); mM (minutes).
Example 1
Incorporating and Determining Metal Content in Arginase I
[000/11] Incorporation of Mn2+ and Co2+ can be achieved by purifying
arginase, followed
by an incubation step with 10 mM metal at 50 C. for 10 minutes. In order to
determine the final
metal content and identity of the arginase preparations, protein samples of Mn-
hArgI (145 iM),
Co-hArgI (182 AM) and associated dialysis buffers (100 mM Hepes, pH 7.4) were
diluted in 2%
nitric acid and analyzed by inductively coupled plasma mass spectrometry (ICP-
MS, Department
of Geological Sciences, University of Texas at Austin) to quantify the
protein's cobalt, iron,
manganese and zinc content by subtracting the concentration of metals found in
the dialysis
buffer from the metal concentration of the final protein samples and dividing
by protein
concentration. To determine protein concentrations, an extinction coefficient
was calculated for
hArgI based on the amino acid sequence (Gill and von Hippel, 1989). All
protein concentrations
for Arginase I were calculated based upon the calculated 8280=24,180 M-1 cm-1
in a final buffer
concentration of 6 M guanidinium hydrochloride, 20 mM phosphate buffer, pH
6.5. For
comparison, arginase concentration was also calculated by BCA assay using
dilutions of BSA as
a standard. Using this method it was found that arginase samples incubated
with Co2+ contain
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2.10.5 equivalents Co and 0.4 0.1 equivalents Fe, with no detectable amounts
of Zn or Mn.
Samples incubated with Mn2+ contain 1.5 0.2 equivalents Mn and 0.4 0.1
equivalents Fe, and
no detectable amounts of Zn or Co. Thus, heat incubation is an efficient
method for
incorporation of cobalt.
[0001121 Additional studies of cobalt loading have demonstrated that a
higher proportion of
cobalt loading is achievable and results in a higher specific activity. The
results of these studies
is shown on the following table and in Fig.3.
TABLE 2
Co-Arginase I Cobalt Loading
Total Co Specific
Co Temp Time Total Mn
Identity (pg/mg
Activity
(mM) ( C) (Min)
Arginase) (pg/mg Arginase)
(U/mg)
APO-Arginase I* NA NA NA <0.025 0.008 24
APO Loading 1* 0.1 5 15 0.3 ND
117
Coh-Arg I* 10 20 60 2 0.06
410
APO Loading 2* 1 5 15 2.4 ND
395
APO Loading 3* 10 20 15 2.8 ND
493
APO Loading 4* 10 20 60 2.9 ND
489
APO Loading 5 10 37 15 2.8 ND NT
APO Loading 6 10 53 15 2.6 ND NT
Co-Argl-PEG 10 53 15 3 ND
500
Theoretical 3.4
*Graphed
[000113] The starred data are shown in Fig. 3.
Example 2
Cytotoxicty of Co-Arg and its Variants Towards Hepatocellular Carcinoma Cells
and
Metastatic Melanomas
[000114] In order to test the in vitro cytotoxicity of engineered
arginase, varying
concentrations (0-100 nM) of Mn-ArgI, Co-ArgI, or Co-hArgI variants were
incubated with
HCC (Hep 3b) cells or melanoma (A375) cells (American Type Culture Collection)
in 96-well
plates at a seeding density of 500 cells/well, in DMEM media supplemented with
fetal bovine
serum. After 24 hours of incubation at 37 C, the cells were treated with
arginase containing
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media in triplicate at various concentrations. The control solution was a
balanced salt solution in
media. The treated cells were maintained at 37 C and 5% CO2. Cells were
tested by standard
MTT assay (Sigma-Aldrich) on days 1, 3, 5, & 7 by addition of 100 L/well of
MTT (5 mg/mL),
and incubated for 4 hours with gentle agitation one to two times per hour.
Following this, the
solution was aspirated and 200 IAL of DMSO was then added to each well.
Absorbance at 570
nm was interpreted for each well using an automated plate reader to determine
the relative
number of surviving cells compared to controls. The resulting data was fit to
an exponential
equation to determine an apparent IC50 value for arginase cytotoxicity. The
IC50 values from day
were calculated, yielding an IC50 value for Mn-hArgI of 5 0.3 nM (-0.18 g/ml)
and a value
of 0.33 0.02 nM for Co-hArgI (-0.012 jig/m1). Thus, the Co-ArgI enzyme appears
to be 15 fold
more cytotoxic than the Mn substituted enzyme against HCC. Against the
metastatic melanoma
cell line (A375) Mn-hArgI resulted in an apparent IC50 of 4.1 0.1 nM (-0.15
jig/m1). Incubation
with Co-hArgI lead to a 13-fold increase in cytotoxicity with an apparent IC50
of 0.32 0.06 nM
(-0.012 jig/m1).
Example 3
Engineering an Fc-Arginase Fusion Protein for Enhanced In Vivo Half-Life
[0001151 Fusion to the IgG Fc domain has been employed extensively for
prolonging the in
vivo half-lives of therapeutic polypeptides such as the TNF-a inhibitor
etanercept (EnbrilTm).
The Fc domain binds to the FcyRn receptor, which is expressed on vascular
endothelium and
many other tissues (Roopenian and Akilesh, 2007). The affinity of FcyRn for
the IgG Fc domain
is strongly pH dependent. Binding occurs at the acidic pH of endosomal
compartments allowing
the protein to be recycled onto the cell surface and thus escape proteolytic
degradation. At the
cell surface, the Fc domain is released from FcyRn because the binding
affinity is very low at
physiological pH. Endosomal recycling via FcyRn is estimated to increase the
serum half-life of
immunoglobulins at least 4-7 fold, to about 7-14 days in humans. Fc fusions
exploit this property
to endow short lived molecules with a long half-life. However, the human
arginase is a
homotrimer and therefore if fused to the IgG Fc, which itself is a dimer, the
resulting Fc-arginase
polypeptide will likely form high molecular weight aggregates.
[000116] This problem was avoided by employing mutant forms of arginase
that disrupt
trimerization and are stable in the monomeric form. The trimerization and
subunit interface of
Arginase I have been studied in some detail (Lavulo et al., 2001). A single
amino acid
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substitution at Glu256Gln has been shown to disrupt trimerization resulting in
the formation of
monomeric Arginase I enzyme (Sabio et al., 2001). After expression and
purification of this
variant, the steady-state kinetic analysis revealed nearly identical activity
compared to Co-hArgI
with a kcat/KM of 1,320 s-1 mM-1.
10001171 This construct was then cloned into Fe expression vectors. The Fe
expression
vector is a construct based on a pTRC99a plasmid (Amersham) that contains a
DsbA leader
sequence followed by the IgG Fe coding region, an EcoRI restriction site and a
stop codon. The
monomeric arginase gene was placed in frame behind the Fe coding region by
digesting both
vector and gene with EcoRI, and was subsequently ligated and transformed into
E. coli (BL21)
for sequencing and expression. Since the IgG Fe is normally a glycosylated
protein, expression
of recombinant IgGs or of Fe fusions has so far been carried out in
recombinant mammalian cells
that, unlike bacteria, are capable of N-linked glycosylation. However, while
glycosylation at
Asn297 is critical for the binding to the activating and inhibitory Fcy
receptors (FcyRI-III in
humans) it does not have a noticeable effect on the affinity or pH dependent
binding to FcyRn
(Tao and Morrison, 1989; Simmons et al., 2002). Thus, aglycosylated IgG
antibodies expressed
in bacteria exhibit serum persistence in primates nearly indistinguishable
from that of fully
glycosylated antibodies expressed in mammalian cells (Simmons et al., 2002).
In contrast to
prevailing earlier notions, IgG antibodies and Fe proteins can be expressed
efficiently in E. coli
up to g/L levels in fermenters. E. coli expression is technically much simpler
and faster. In
addition, since the resulting protein is aglycosylated, it does not display
glycan heterogeneity, an
important issue in the expression of therapeutic glycoproteins (Jefferis,
2007). The fusion protein
is purified by Protein A chromatography and the yield of correctly folded,
dimeric Fc-arginase
fusion relative to polypeptides that fail to dimerize is quantified by FPLC
gel filtration
chromatography. This formulation has led to a highly active and very stable
form of human
arginase, suitable for in vivo trials.
Example 4
Pegylation of Arginase
10001181 Arginase was purified and was then made 10 mM with CoC12 and
heated at 50 C
for 10 minutes. After centrifuging to remove any precipitates, the PEG-5000
arginase was
extensively buffer exchanged (PBS with 10% glycerol) using a 100,000 MWCO
filtration device
(Amicon), and sterilized with a 0.2 micron syringe filter (VWR). All pegylated
enzyme was
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analyzed for lipopolysaccharide (LPS) content using a Limulus Amebocyte Lysate
(LAL) kit
(Cape Cod Incorporated).
0001191 Pegylated Co-hArgI was found to have nearly identical serum
stability to wild
type enzyme and displayed a kcat/Km value of 1690 290 s-1 mM-1.
Example 5
Serum Depletion of L-Arg in the Mouse Model
[000120] Balb/c mice were treated by single IP injection with 500 jig of
pharmacologically
prepared, pegylated Co-hArgI or an equal volume of PBS. Mice were sacrificed
by cardiac veni-
puncture for blood collection at the time points of 0, 48, 72, and 96 hrs.
Blood samples were
immediately mixed 50:50 (v/v) with a 400 mM sodium citrate buffer pH 4,
allowed to clot for 30
minutes and centrifuged for serum separation. The resulting serum was then
filtered on a 10,000
MWCO device (Amicon) for the removal of large proteins and precipitates and
the flow-through
was collected for analysis. L-arginine standards, control mouse serum and
experimental samples
were derivatized with OPA (Agilent) and separated on a C18 reverse phase HPLC
column
(Agilent) (5 pm, 4.6x150 mm) essentially as described by Agilent Technologies
(Publication
Number: 5980-3088) except for modification of the separation protocol slightly
by reducing the
flow rate by 1/2 and doubling the acquisition time to get better peak
separation. An L-arginine
standard curve was constructed by plotting L-Arg peak area versus
concentration in order to
quantify serum L-Arg levels. A single dose of pharmacologically prepared Co-
hArgI was
sufficient to keep L-Arg at or below detection limits for over 3 days (FIG.
1).
Example 6
HCC Tumor Xenograft Treatment with Co-hArgI
[000121] Nude mice were injected subcutaneously in the flank with ¨106 HCC
cells
collected from a 75% confluent tissue culture. After the HCC xenografted
tumors had grown to
¨0.5 cm3 in diameter (Day 9), mice were sorted into two groups. The
experimental group
received a 500 tig IP injection of pharmacologically optimized Co-hArgI at day
9 and at day 12.
The control group received IP injections of PBS at days 9 and 12. As can be
seen in FIG. 2, the
PBS treated tumors had increased 3-fold in size by day 15. In stark contrast,
Co-hArgI treated
tumors had decreased in size by day 15. Mn-hArgI treated tumors had only been
shown to be
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retarded in growth rate (Cheng et al., 2007). Co-hArgI appears to be a highly
effective
chemotherapeutic agent against HCCs both in vitro and in vivo.
Example 7
Disruption of the L-Arginine Balance in the Tumor Microenvironment with Co-
hArgI and
anti-PD-Li Ab
[000122] Human arginase I (hArgI) is a Mn"-dependent enzyme that displays
low activity
and low stability in serum. Myeloid-derived suppressor cells (MDSC) express
hArgI and nitric-
oxide synthase (NOS), which control the availability of L-arginine in the
tumor
microenvironrnent and in turn regulate the function of T-cells. Depletion of L-
arginine by MDSC
has been correlated to impairment of T-cell anti-tumor function and tumor
evasion of host
immunity. The expression of enzymes of the L-arginine biosynthetic pathway in
peripheral blood
mononuclear cells, bone marrow mononuclear cells and CD34+ cells was analyzed
revealing that
these cells express low levels of OTC and ASS, suggestive of dependence of
these cells on
exogenous/extracellular L-arginine for physiological function. Based on this
finding it is
contemplated that long term depletion of L-arginine may negatively impact the
MDSC
population and therefore enhance immune regulation of tumor growth. This
hypothesis was
tested using engineered hArgI (AEB1102), developed by replacement of the Mn"
natural
cofactor with Co" which results in significantly improved catalytic activity
and serum stability
compared to endogenous hArgI. The engineered enzyme is also pegylated as
described above.
The effects of chronic, extensive pegylated Co-hArgI-mediated depletion of L-
arginine in vivo in
the murine CT26 colon-cancer model dosed alone and in combination with anti-PD-
L1 and anti-
PD-1 monoclonal antibodies (mAbs) were tested.
[000123] Female Envigo Balb/c mice (BALB/cAnNHsd) were used in these
studies. They
were 6-7 weeks old on Day 1 of the test. Test animals were implanted
subcutaneously on Day 0
with 5.0E+05 CT26.WT cells. All mice were sorted into study groups and
treatment was started
as follows:
AEB-001-1037
- Group 1: Vehicle (PBS) IP, Q7Dx4; plus Isotype Control, 10mg/kg IP,
(Q3Dx2; 3off)x4
- Group 2: AEB1102, (3mg/kg IP, Q7Dx4)
- Group 3: anti-PD-Li Ab, 10mg/kg IP, (Q3Dx2; 3off)x4
- Group 4: AEB1102, 3mg/kg IP, Q7Dx4; plus anti-PD-Li Ab, 10mg/kg IP,
(Q3Dx2; 3off)x4;
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[000124] AEB1102 was dosed 4 times, once weekly, (Days 3, 10, 17, 24)
[0001251 Anti-PD-Li was dosed 8 times, one on two off, one on three off
each week (Days
3,6, 10, 13, 17, 20, 24, 27)
10001261 All animals were observed for clinical signs at least once daily.
Individual body
weights and tumor volumes were recorded three times weekly. Individual mice
were terminated
when tumor size reached a value of 2000 mm3.
[0001271 In vivo treatment of CT26 mice with AEB1102 (peglylated Co-hArgI)
resulted in
a therapeutic effect comparable to standard immunomodulatory antibodies that
target PD-1 and
PD-Li. Of significance, combination therapy of AEB1102 with anti-PD-1 and PD-
Li rnAbs
resulted in an apparently synergistic or at least additive anti-tumor effect
compared to AEB1102
alone and immunotherapy alone.
[0001281 The data from this study is shown graphically in Fig. 4. The data
reflect the effect
of treatment with a pegylated Co-hArgI in combination with an anti-immune
checkpoint protein
receptor (anti-PD-1) and ligand, (anti-PD-L1). In the figure the upper curve
is an isotype control
antibody, the second curve is anti-PD-Li antibody, the third curve is
pegylated Co-hArgI and the
lowest curve is the combination treatment of the pegylated Co-hArgI and anti-
PD-Li antibody.
As seen in the data, the combination of the 2 agents has a greater than
additive effect on
inhibition of tumor growth.
[0001291 The effect on lymphocyte Tcell activation was also measured in
samples taken on
day 3. The percentage of total live cells that expressed CD45+ in the four
groups as well as the
percentage of CD45+ cells that were also CD8+ are shown in Table 3. These data
are also shown
in graphical form in Figs. 6 and 7.
Groups Day 3 CD45+ cells Day 3 CD8+ cells
Vehicle 14.8 3.4 19.4 3.9
Isotype 13.8 2.2 18.7 2.1
AEB1102 26.5 3.7 18.7 7.1
anti-PD-L1 13.1 3.5 26.3 3.24
AEB1102 + anti-PD-Li 22.4 2.3 34.2 5.0
Table reports mean SEM
CA 03054150 2019-08-20
WO 2018/032020 PCT/US2017/050816
10001301 Collectively these results demonstrate that disrupting the L-
arginine physiological
balance in the tumor microenvironment inhibits tumor growth and further
sensitizes the tumor to
immunotherapy.
Example 8
Effect of treatment of Colon Carcinoma Treatment with Argil and 0X40
[000131] Suspensions of MC38 colon carcinoma cells were injected into the
flanks of
female C57B L/6 mice. When tumor volume reached 75-100 inm3 on day 0, mice
were
randomized into groups. Tumor volume was measured twice a week using calipers.
Treatments
were started on Day 0.
[000134 A first group was injected with 10 mg/kg isotype control biweekly
for 6 weeks,
second group was injected with 3 mg/kg co-Arginase I, weekly for 6 weeks, a
third group was
injected with 10 mg/kg anti-OX40ab weekly for 6 weeks, and a fourth group was
injected with 3
mg/kg co-ArgI and 10 mg/kg anti OX40ab weekly for 6 weeks. The data from this
study is
shown in Fig. 5. Although it appears that the co-ArgI was given at a
suboptimal dose, a trend is
seen in which the combination of ArgI and a0X40 has a more than additive
effect on the
reduction or inhibition of tumor volume.
[000133] All of the compositions and methods disclosed and claimed herein
can be made
and executed 'without undue experimentation in light of the present
disclosure. While the
compositions and methods of this invention have been described in terms of
preferred
embodiments, it will be apparent to those of skill in the art that variations
may be applied to the
compositions and methods, and in the steps or in the sequence of steps of the
methods described
herein without departing from the concept, spirit and scope of the invention.
More specifically, it
will be apparent that certain agents which are both chemically and
physiologically related may
be substituted for the agents described herein while the same or similar
results would be
achieved. All such similar substitutes and modifications apparent to those
skilled in the art are
deemed to be within the spirit, scope and concept of the invention as defined
by the appended
claims.
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