Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
Use of Asparaginase and Glutaminase to Treat Autoimmune
Disease and Graft Versus Host Disease
Related Applications
This application claims priority to U.S. Patent
Application Serial No. 09/094,435, by Donald L. Durden,
entitled "Utilization of Wolinella succinogenes asparaginase
in the treatment of human hematologic and autoimmune
disease" (Lyon & Lyon Docket No. 234/274), filed June 9,
1998, which claims priority to TJ.S. provisional patent
application 60/049,085, fi)_ed June 9, 1997.
Field Of Invention
The present invention relates to methods for the
utilization of recombinant microbial enzymes, including
asparaginases and glutaminases, in the treatment of
autoimmune diseases and Graft versus Host disease.
Background Of Invention
The references cited below are not admitted to be prior
art to the inventions described herein.
Juvenile rheumatoid arthritis (JRA) is the most common
rheumatic condition of childhood. Recent long-term follow-
up studies have shown that JRA is not benign and the
proportion of patients with a favorable outcome is less than
initially thought (Wallace, 1991; Levinson, 1992).
Approximately one-third of all patients achieve adequate
control of their disease with nonsteroidal anti-inflammatory
drugs (NSAIDs), but the remainder of patients are candidates
for more aggressive therapy with second-line agents.
Placebo-controlled trials and long-term prospective
studies in children with JRA showed a lack of efficacy among
agents such as penicillamine, hydroxychloroquine, oral gold,
and intravenous immune globulin. Brewer, 1986; Giannini,
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1993; Silverman, 1993. Secondary treatment failures even
with new standard medications such as methotrexate are
common, creating a high demand for new safe and effective
agents in these refractory diseases.
Asparaginases are used as front-line therapy in the
treatment of acute leukemia. Enzymes that deplete
asparagine or glutamine possess immunosuppressive effects
and have been shown to have anti-inflammatory properties.
However, the mode of action and the final lethal route of
susceptible cells deprived of L-asparagine or L-glutamine is
still undetermined.
The clinically utilized forms of L-Asparaginase are
immunogenic proteins derived either from E. coli (EC),
Erwinia carotovora, or Wolinella succinogenes (WS). E coli
possesses two asparaginase enzymes, one constitutive and
another induced by anaerobic conditions. The asparaginase
induced by anaerobic conditions is known to have a tumor
inhibitory effect. Interestingly, L-Asparaginase from E.
coli has cytotoxic, but also immunosuppressive, properties
due to its glutamine depleting effect. In fact, the
immunosuppressive effect of L-Asparaginase has been
attributed to this glutaminase property of this enzyme. The
EC asparaginase has recently been covalently modified using
polyethylene glycol (PEG) conjugation, to form PEG
asparaginase, to reduce antigenicity and extend the half-
life of the EC enzyme.
Unlike other anti-tumor agents (cyclophosphamide,
etoposide, etc.), asparaginases from E. coli (EC and EC-PEG)
are not mutagenic, and not associated with second
malignancy. At the same time, EC and EC-PEG enzymes are not
myelosuppressive. Hence, patients treated with asparaginase
are not at risk for development of sepsis or other severe
life threatening conditions, for example, infections.
EC and EC-PEG have potent antileukemic activity and
cause minimal toxicity in children. The limited toxicity of
these enzymes is restricted to rare coagulation
abnormalities in less than to of patients, which can be
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managed easily. Mild allergic reactions have also been
described.
The immunosuppressive effects of EC are restricted to
its effects on the lymphoid system. L-Asparaginase derived
from E. coli suppresses the humoral or cell-mediated
immunological response to T cell-dependent immunogens on
sheep red blood cells. The EC enzyme inhibits T-cell
immunity to the antigen, SRBC, as measured by antibody
titer, ADCC, and immunoglobulin producing cells in the
spleen (80% reduction). The effects of E. coli asparaginase
treatment on spleen histology and lymphocyte populations are
known to include a marked reduction in the size and
reactivity of the germinal centers, which correlates with a
marked reduction in the cytoplasmic immunoglobulin-
containing cells (B-cell immunoblasts).
These data support the hypothesis that depletion of
glutamine, or asparagine together with glutamine, after
treatment with E. coli asparaginase results in marked immune
suppression. In contrast, asparagine deprivation alone,
caused by the administration of the glutaminase-free
asparaginase from WS, does not affect spleen histology or
lymphocyte marker distribution and is not immunosuppressive.
Definition Of Terms
Unless otherwise expressly defined, the terms used
herein will be understood according to their ordinary
meaning in the art, although the following terms will be
understood to have the following meanings, unless otherwise
indicated.
An "analog" of a protein, e.g., asparaginase or
glutaminase, refers to a polypeptide that differs in some
way from its forms) found naturally. For example, in
certain embodiments, an analog of asparaginase or
glutaminase will refer to an enzyme wherein one or more
amino acids has been deleted from the naturally occurring
amino acid sequence. Alternatively, one or more amino acid
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residues may be substituted with a different amino acid.
Other analogs include those wherein additional amino acids
have been added to the native sequence. For example, one or
more amino acids may be added to the amino terminus and/or
carboxy-terminus of the enzyme, or be inserted between
internal amino acid residues. Such analogs can be prepared
by any suitable technique, although modifying a recombinant
gene to encode the desired changes) will typically be
employed. Other analogs include those wherein one or more
amino acid residues are derivatized, e.g., glycosylated,
pegylated, acylated, or otherwise bound covalently to a
molecule not attached to native forms) of the protein. Of
course, analogs according to the invention include those
where an amino acid residue is added to or substituted in
the native amino acid sequence, and this new residue is
itself later modified, for example, by a covalent
modification performed after the enzyme has been at least
partially purified or isolated. Moreover, as used herein,
an asparaginase or glutaminase analog includes those that
have been modified and exhibit altered biochemical or
physiological properties, e.g., different substrate
specificity and/or affinity, altered quarternary structure,
etc. After generating analogs, e.g., by a rational design
strategy, random mutagenesis, etc., the proteins can be
screened for biological activity, as described elsewhere
herein. When large numbers of analogs are generated, high
throughput screening methods are preferred in order to
identify analogs having the desired characteristics. Those
analogs found to exhibit the desired activity in vitro may
then be tested in vivo for activity and pharmacokinetic
properties.
A "unique contiguous amino acid sequence" means an
amino acid sequence not found in a naturally occurring
protein or polypeptide. Thus, a "unique contiguous amino
acid sequence of Wolinella succinogenes", for example,
refers to a sequence which contains one or more amino acid
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substitutions, insertions, or deletions, as compared to
corresponding region of the native enzyme.
A "disease which responds to asparagine or glutamine
depletion" refers to a disorder wherein the cells
5 responsible for or otherwise correlates with the disease
state either lack or have a reduced ability to synthesize,
uptake, or otherwise utilize asparagine or glutamine.
Depletion or deprivation of asparagine to such cells can be
partial or substantially complete, so long as the desired
therapeutic benefit is achieved. In certain embodiments,
more than about 50% of asparagine or glutamine in the serum
is depleted, preferably greater than about 750, with
depletion of more than 95o being most preferably achieved.
Representative examples of diseases that respond to
asparagine or glutamine depletion or deprivation include
certain non-hematologic diseases. Non-hematologic diseases
associated with asparagine or glutamine dependence include
autoimmune diseases, for example rheumatoid arthritis,
systemic Lupus erythematosus (SLE), autoimmunity, collagen
vascular diseases, AIDS, etc. Other autoimmune diseases
that may be treated according to the instant methods
include, without limitation, osteo-arthritis, Issac's
syndrome, psoriasis, insulin dependent diabetes mellitus,
multiple sclerosis, sclerosing panencephalitis, systemic
lupus erythematosus, rheumatic fever, inflammatory bowel
disease (e. g., ulcerative colitis and Crohn's disease),
primary billiary cirrhosis, chronic active hepatitis,
glomerulonephritis, myasthenia gravis, pemphigus vulgaris,
and Graves' disease. Notwithstanding the foregoing, any
disease the cells responsible for which respond, e.g., cease
proliferating, become senescent, undergo apotosis, die,
etc., to asparagine or glutamine depletion may be treated in
accordance with the instant methods. As those in the art
will appreciate, cells suspected of causing disease can be
tested for asparagine or glutamine dependence in any
suitable in vitro or in vivo assay, e.g., an in vitro assay
wherein the growth medium lacks asparagine or glutamine.
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A "patient" refers to an animal afflicted with a
disease that responds to asparagine or glutamine depletion.
Typically, patients treated in accordance with the instant
methods are mammals, e.g., bovine, canine, equine, feline,
ovine, porcine, and primate animals, particularly humans.
An "expression vector" refers to a nucleic acid,
typically a plasmid, into which heterologous genes of
interest may be cloned and subsequently expressed. For
expression, such vectors are generally introduced into a
suitable host cell or population of host cells. The
expression vector can be introduced by any appropriate
technique. Preferred techniques include transformation,
electroporation, transfection, and ballistic (e. g., "gene
gun") introduction. Depending upon the vector employed,
suitable host cells for expression of the desired
heterologous genes) include prokaryotic and eukaryotic
cells. Preferred prokaryotic cells are transformation-
competent bacterial cells such as E. coli strain and DHSa
and JM 109. Preferred eukaryotic host cells include yeast
and mammalian cell lines. As those in the art will
appreciate, the particular expression vector/host cell
system selected for expression of the desired heterologous
gene depends on many factors, and is left to the skilled
artisan to determine in the particular circumstances.
Similarly, the conditions required for expression of the
desired gene from an expression vector carrying the same
depends on many factors, including the host cell type, the
promoters) and other transcription regulation elements
employed, the media (or medium) used, etc. Again, the
selection made in a given circumstance is at the discretion
of the artisan involved, and the particular employed is
readily within the skill of such a person given the
disclosure herein.
A protein that is "biologically active" is one that has
at least one of the biological activities of the
corresponding native protein, although the activity
exhibited may differ in degree from that of the native
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protein. For example, an analog of W. succinogenes
asparaginase according to the invention may have a greater
specific activity, longer serum half-life, etc. than the
native form of the protein.
A protein that has an "epitope-tag" refers to a protein
having one or more, preferably two or more, additional amino
acids covalently attached thereto or incorporated therein.
The tag has a distinct epitope that can be recognized by
another protein, e.g., an antibody that binds that epitope,
preferably with high affinity; or a protease that cleaves in
or around a specific amino acid sequence (e. g., DAPI,
cathepsin-C), etc. For example, as used herein an "N-
terminal epitope tag" can refer to a peptide attached to the
N-terminus of a protein, where the peptide has a
conformation recognized by a particular antibody. Such a
peptide and its corresponding antibody(ies) can be used to
rapidly purify the polypeptide to which the peptide is
attached by standard affinity chromatography techniques.
Such antibodies, and any others used in the practice of this
invention (e.g., for targeting gene delivery vehicles), can
be prepared used techniques widely known in the art. For
example, see Harlow and Lane in Antibodies, a Laboratory
Manual, Cold Spring Harbor Laboratory, 1988. Epitope tags
may also be included at the C-terminus of the protein, and
in internal regions where insertion of such a tag does not
substantially and adversely affect the biological activity
or pharmacokinetic properties of the enzyme.
A "therapeutically effective amount" of a protein
(e. g., an asparaginase, a glutaminase, or an analog thereof)
means that amount required to produce the desired
therapeutic effect. Of course, the actual amount required
depends on many factors, such as the disease to be treated,
the progression of the disease, and the age, size, and
physical condition of the patient, as discussed in more
detail below.
By "altering a pharmacokinetic property of a protein"
is meant that a property of a drug as it acts in the body
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over a period of time, e.g., serum half-life, clearance
rate, biodistribution, immunogenicity, etc., is changed.
Such alteration can be either an increase or decrease in the
property being examined.
Summary Of Invention
One aspect of the present invention is directed to
methods for the therapeutic utilization of native and/or
recombinant forms of asparaginases and glutaminases in the
treatment of diseases which respond to asparagine and/or
glutamine depletion, including various autoimmune diseases
which respond to asparagine and/or glutamine depletion. In
preferred embodiments, these methods involve administering
to a patient a therapeutically effective amount of a W.
succinogenes asparaginase or glutaminase, an analog of
either, or an acylated asparaginase or glutaminase derived
from an organism other than W. succinogenes. Other
asparaginases or glutaminases specifically envisioned
include those from other fungal and bacterial sources, and
include, but are not limited to, both recombinant and native
asparaginases from Wolinella succinogenes, and recombinant
and native asparaginases/glutaminases from E. coli,
Acinetobacter, and Erwinia, for example.
Representative diseases that can be treated in
accordance with the instant invention include autoimmune
diseases, for example, arthritis (e. g., rheumatoid
arthritis), systemic lupus erythematosus (SLE), diabetes,
and AIDS. The methods of the invention may also be used to
treat Graft versus Host Disease, for example. Typically,
the instant methods will be applied to humans afflicted with
a disease which responds to asparagine and/or glutamine
depletion, although other patient classes, particularly
mammals (e. g., bovine, canine, equine, feline, ovine,
porcine, and primate animals) suffering from a disease which
responds to asparagine and/or glutamine depletion can be
similarly treated.
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Methods for isolating native W. succinogenes
asparaginase, producing recombinant W. succinogenes
asparaginase in vitro or in vivo, making derivatives,
analogs, and covalent modifications thereof, and making
pharmaceutical formulations therefrom were described
previously in U.S. Patent application Serial No. 09/094,435,
by Donald L. Durden, entitled "Utilization of Wolinella
succinogenes asparaginase in the treatment of human
hematologic and autoimmune disease" (Lyon & Lyon Docket No.
234/274), filed June 9, 1998, incorporated by reference
herein in its entirety including any drawings; tables, or
figures. These methods can be applied analogously to
asparaginases and glutaminases from other organisms,
including those from other bacterial and fungal sources,
including, but not limited to, recombinant and native
asparaginases/glutaminases from E. coli, Acinetobacter, and
Erwinia .
The invention has been described broadly and
generically herein. Each of the narrower species and
subgeneric groupings falling within the generic disclosure
also form part of the invention. This includes the generic
description of the invention with a proviso or negative
limitation removing any subject matter from the genus,
regardless of whether or not the excised material is
specifically recited herein. For example, in the methods of
the invention, patients can be mammals, but in some
embodiments this may not include mice or rats. Similarly,
although all asparaginases and glutaminases are envisioned
in the methods of the invention, in some embodiments this
may not include native E. coli asparaginase.
Other features and advantages of the invention will be
apparent from the following figures, detailed description,
examples, and claims.
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Brief Description Of The Drawings
The present invention may be better-understood and its
advantages appreciated by those individuals skilled in the
relevant art by referring to the accompanying drawings
5 wherein:
Figure 1: Illustrates the nucleotide sequences of the
forward [SEQ ID NO. 1] and reverse [SEQ ID
NO. 2] PCR primers used in the amplification
10 of the genomic L-asparaginase sequences of
W. succinogenes.
Figure 2: Agarose gel electrophoresis of propidium
iodine-stained W. succinogenes genomic DNA
(lanes 1 and 2) and a 1.0 kb DNA fragment
derived from PCR amplification. Lanes 3 and
4 are DNA molecular weight markers. Lane 5
is the 1.0 kb W. succinogenes-specific PCR
fragment amplified using the two PCR primers
shown in Figure 1. Lane 6 contains a X174
DNA molecular weight marker.
Figure 3: Restriction enzyme analysis of 4 colonies
which were isolated following the ligation of
the 1.0 kb W. succinogenes-specific PCR
fragment into the PCR II vector. The 1.0 kb
DNA was digested with BamHl (lanes 2-5);
EcoRl (lanes 6-9); and BamHl and EcoRl (lanes
10-13). Lane 14 represents a DNA molecular
weight ladder. The 1.0 kb W. succinogenes-
specific DNA fragment is denoted by an arrow.
Figure 4: Agarose gel electrophoresis of the DNA
fragments amplified from the selected,
"positive" clones utilizing W. succinogenes
asparaginase-specific primers. Lanes 1 and 7
are molecular weight markers. Lanes 2 and 4
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represent DNA extracted from bacterial
colonies #1 and #3 from lanes 2 and 4 of
Figure 3. Lane 6 represents a sample of the
W. succinogenes asparaginase PCR
amplification product (amplified from W.
succinogenes genomic DNA from Figure 2, lane
5) used in the initial ligation reaction. It
should be noted that the fragment cloned into
the PCR II vector was shown to be exactly the
same size (i.e., 1.0 b) as the initial PCR
amplification product.
Figure 5: Illustrates the results of a determination of
the anti-tumor activity of W. succinogenes
(WS) , E. coli (EC) and E. carotovora (Erw)
asparaginases against tumors generated by the
subcutaneous injection of 6C3HED Gardner
lymphosarcoma cells in C3H mice. Anti-tumor
activity was measured as a function of
caliper-measured tumor volume (cm3). The
negative control consisted of injections of
0.01 M phosphate buffer (pH 7.0) into C3H
mice using the same injection schedule as for
the asparaginases.
Figure 6: Illustrates the DNA sequence [SEQ ID NO. 3]
of the modified W. succinogenes asparaginase-
specific DNA insert. This sequence contains
not only the coding sequence of the native W.
succinogenes asparaginase (beginning with
codon 40 of Figure 6 and not including the
final 23 3' - terminal nucleotides of Figure
6), but also 39 codons for the N-terminal
epitope "tag" shown in Figure 6.
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Figure 7: Is a schematic representation of a chemical
modification for a protein, for example W.
succinogenes asparaginase.
Figure 8: Illustrates the lack of cross-reactivity
between different dilutions of a patient's
plasma known to contain high-titer
neutralizing antibodies against E. coli
asparaginase and the W. succinogenes enzyme.
Figure 9: Illustrates the lack of cross-reactivity
between different dilutions of polyclonal
high-titer neutralizing antibodies against E.
coli asparaginase and asparaginase derived
from W. succinogenes.
Figure 10: Demonstrates that E. coli asparaginase
reverses established arthritis in CIA model.
Digital image of mouse extremity before and
after treatment with E. coli asparaginase.
Mice were injected with bovine collagen type
II in complete Freund's adjuvant on day 0 and
boosted with same antigen on day +21.
Arthritis developed on day +35 following
immunization (Panel A) graded as 3+ arthritic
involvement. Mouse treated with 50 IU of E.
coli asparaginase daily for 1 week showed
dramatic reversal of arthritic involvement
from score of +3 to 0 on day + 42 as depicted
in Panel B.
Figure 11: Demonstrates the effects of E. coli
asparaginase on established arthritis in CIA
mouse model. CIA was induced in DBA/1 mice
as described above. On day +35 mice that
developed detectable arthritis were separated
into equivalent groups. One group received
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E. coli asparaginase 50 IU/injection daily
for 8 weeks the other group received PBS
injections. Arthritic scores were compiled
in blinded manner over the next 8 weeks of
evaluation as depicted in bar graphs for two
experimental groups. The data were analyzed
for statistic significance. The difference
between E. coli asparaginase and control PBS
treated groups on months 1 and 2 was
significant (p < 0.05).
Figure 12: Demonstrates the effects of E. coli
asparaginase on established arthritis induced
LPS/CIA model. CIA was induced in DBA/1 mice
as described above. On day +21 mice were
boosted with 100 ug collagen in Freuds
adjuvant. On day +49 and +54 we administered
LPS (40 ~g/mouse IP). Mice developed LPS/CIA
on day +61 and were separated into equal
groups based on the arthritic scores. One
group was treated with E. coli asparaginase
50 IU daily injections IP on Monday,
Wednesday and Friday and other group was
treated with PBS. Treatment was extended to
4 weeks. The bars represent the mean
arthritic score over time. The data were
evaluated by Student t-test and the
differences observed between the E. coli
asparaginase-treated mice on weeks 1-4 were
statistically significant as compared to
controls at (p< 0.01).
Detailed Description Of The Invention
Asparaginases and glutaminases can be used in the
treatment of autoimmune diseases and Graft versus Host
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Disease, and alter the natural course of autoimmunity.
There is a dramatic clinical response to L-asparaginase in
cancer treatment, although host toxicity and immuno-
suppression also arise. The advantages to using L-
asparaginase treatment for auto-immune and Graft versus Host
diseases include the fact that immuno-suppression is a
desired effect, and that lower and less frequent doses are
likely to be required, limiting toxicity to the host.
Described herein are exemplary methodologies for the
isolation of "native" asparaginases and glutaminases, as
well as for the production (using recombinant expression
vectors) of recombinant asparaginases and glutaminases and
analogs thereof, e.g., those which have been acylated and
those which have been modified to include additional or
alternate amino acids that have been acylated or otherwise
modified (e. g., by pegylation).
The following sections elaborate upon some of the
various biochemical and physiological effects of clinical
utilization of asparaginase or glutaminase therapy in the
treatment of diseases associated with asparagine or
glutamine dependence.
I. Review of the Clinical use of Asparaginase and
Glutaminase
Asparaginases are enzymes which catalyze the
deamidation of L-asparagine (asparaginase activity) and L-
glutamine (glutaminase activity). See Cantor, P. S. &
Schimmell, M. R., Enzyme Catalysis, 2nd ed., (T. Pettersonn
& Y. Tacashi, eds.) Sanders Scientific Press, New York pp.
219-23. (1990). L-glutamine serves as the amide donor in
purine biosynthesis, as well as other transamination
reactions, and hence plays a role in DNA and cyclic
nucleotide metabolism.
In vivo biochemical activity of asparaginase was first
documented to be present in guinea pig serum in 1922 (see
Clementi, A., La desamidation enzmatique de 1'asparagine
chez les differentes especes-animals et la signification
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physiologique de sa presence dass 1'organisme, 19 Arch.
Intern. Physiol. 369 (1922)). The subsequent discovery that
asparaginase isolated from guinea pig serum was the active
agent which inhibited the in vivo growth of certain
5 asparagine-dependent mammalian tumors without concomitant
deleterious effects on normal tissue (see Broome, J. D.,
Evidence that the asparaginase activity of guinea pig serum
is responsible for its anti-lymphoma effects, 191 Nature
1114 (1961)) suggested that this enzyme could be utilized as
10 an anti-neoplastic agent.
Because L-asparagine is a non-essential amino acid,
asparaginase was initially thought to represent a unique
prototype of selective chemotherapy in which treatment could
be directed specifically and selectively against asparagine-
15 dependent cells. However, the low levels of asparaginase in
guinea pig serum necessitated the development of a more
practical source of this enzyme.
Subsequently, microbial asparaginase isolated from
Escherichia coli and Erwinia carotovora were shown to act as
potent anti-leukemic agents (see Howard, J. B. & Carpenter,
F.H., L-asparaginase from Erwinia carotovora: substrate
specificity and enzymatic properties, 247 J. Biol. Chem.
1020 (1972); Campbell, H. A., et al., Two asparaginases from
Escherichia coli B: their separation, purification, and
anti-tumor activity, 6 Biochemistry 721 (1967)), and when
one of these enzymes was utilized in combination with the
chemotherapeutic agent vincristine and the corticosteroid
prednisone for the treatment of acute lymphoblastic or acute
undifferentiated human leukemia, an overall remission rate
of 93o was reported (see Ortega, J.A., et al., L-
asparaginase, vincristine, and prednisone for the induction
of first remission in acute lymphocytic leukemia, 37 Cancer
Res. 535 (1977)).-
While these asparaginases possess potent anti-leukemic
activity, clinical utilization of the aforementioned
microbial asparaginases resulted in a wide range of host
toxicity (e. g., hepatic, renal, splenic, pancreatic
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dysfunction and blood coagulation) and pronounced
immunosuppression (see Ohno, R. & Hersh, E. M.,
Immunosuppressive effects of L-asparaginase, 30 Cancer Res.
1605 (1970)), unlike asparaginase isolated from guinea pig
serum (see Cooney, D.A., et al., L-asparaginase and L-
asparagine metabolism, 10 Ann. Rev. Pharmacol. 421 (1970)).
Examination of the effects of E. coli asparaginase
treatment on spleen histology and lymphocyte populations
revealed a marked reduction in both the size and reactivity
of the splenic germinal centers which was concomitantly
associated with a marked reduction in the cytoplasmic
immunoglobulin-containing cells (B-cell immunoblasts; see
Distasio, J.A., et al., Alteration in spleen lymphoid
populations associated with specific amino acid depletion
during L-asparaginase treatment, 42 Cancer Res. 252 (1982)).
Additionally, examination of the lymphocyte sub-population
within the spleen revealed that there was a 40% reduction in
the percentage of surface immunoglobulin-expressing cells
(B-cells) accompanied by an increase in the ratio of Thy-
1.2-expressing cells (T-cells), whereas the ratio of Lyt-2
to Lyt-1 cells remained unchanged in comparison to the
control animal group. These results supported the
hypothesis that glutamine, or glutamine combined with
asparagine depletion initially resulting from administration
of E. coli asparaginase, caused a marked decrease in spleen
lymphocytic cells of the B-cell lineage.
Another important adverse clinical effect associated
with traditional microbial asparaginase treatment is hepatic
dysfunction (see Schein, P.S., et al., The toxicity of E.
coli asparaginase, 29 Cancer Res. 426 (1969)). Patients
treated with E. coli asparaginase generally exhibit
decreased plasma levels of albumin, antithrombin III,
cholesterol, phospholipids, and triglycerides. Other
indications of asparaginase-induced hepatic dysfunction and
pathology include fatty degenerative changes, delayed
bromosulfophthalein clearance, and increased levels of serum
glutamic-oxaloacetic transaminase and alkaline phosphatase.
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Although some investigators have reported that low dosages
of E. coli asparaginase result in limited hepatotoxic
complications, sensitive indicators of hepatic function in
some patients receiving low dosages, however, still reveals
significant hepatic disease which may result in life-
threatening coagulopathy (see Crowther, D., Asparaginase and
human malignant disease, 229 Nature 168 (1971)).
The hepatotoxic effects of microbial asparaginases may
be a result of their capability to hydrolyze both asparagine
and glutamine. One biochemical difference between E. coli
and E. carotovora asparaginases and the enzyme derived from
guinea pig is the non-specific amidohydrolase activity
associated with the microbial enzymes (see Howard, J.B. &
Carpenter, F.H., (1972) supra; Campbell, H.A., et al.,
(1967) supra). For example, E. coli asparaginase has been
shown to possess a 130-fold greater level of glutaminase
activity as compared to the activity of Wolinella
succinogenes (previously classified as Vibrio succinogenes)
asparaginase. As a result, patients treated with the
conventional microbial asparaginases show a marked reduction
in serum levels of both glutamine and asparagine (see
Schrek, R., et al., Effect of L-glutaminase on
transformation and DNA synthesis of normal lymphocytes, 48
Acta Haematol. 12 (1972)), which may demonstrate a possible
correlation between glutamine deprivation and asparaginase-
induced clinical toxicity (see Spiers, A.D.S., et al., L-
glutaminase/L-asparaginase: human pharmacology, toxicology,
and activity in acute leukemia, 63 Cancer Treat. Rep. 1019
(1979) ) .
The relative importance of L-glutamine in mammalian
intermediary metabolism served to stimulate further research
into the possible role of glutamine deprivation in
asparaginase-induced immunosuppression. Lymphoid tissue has
been shown to have relatively low levels of glutamine
synthetase activity (see E1-Asmar, F.A. & Greenberg, D.H.,
Studies on the mechanism of inhibition of tumor growth by
glutaminase, 26 Cancer Res. 116 (1966); Hersh, E.M., L-
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glutaminase: suppression of lymphocyte blastogenic responses
in vitro, 172 Science 139 (1971)), suggesting that these
tissues may be particularly sensitive to the depletion of
exogenous glutamine. In contrast, some investigators have
proposed that asparagine depletion alone may be responsible
for asparagine-induced immunosuppression (see Baechtel, F.
S., et al., The influence of glutamine, its decomposition
products, and glutaminase on the transformation of human
lymphocytes, 421 Biochem. Biophys. Acta 33 (1976)).
While the immunosuppressive effect of E. coli and E.
~arotovora asparaginases are well-documented (see Crowther,
D., (1971) supra; Schwartz, R.S., Immunosuppression by L-
asparaginase, 224 Nature 276 (1969)), the molecular
biological basis of these functions have not yet been fully
elucidated. The inhibition of lymphocyte blastogenesis by
various L-glutamine antagonists (see Hersh, E.M. & Brown,
B.W., Inhibition of immune response by glutamine antagonism:
effect of azotomycin on lymphocyte blastogenesis, 31 Cancer
Res. 834 (1980)) and glutaminase from Escherichia coli (see
Hersh, E.M., (1971) supra) tends to be illustrative of a
possible role for glutamine depletion in immunosuppression.
It has also been demonstrated that inhibition of the
lymphoid blastogenic response to phytohemagglutinin (PHA) by
E. coli asparaginase can be reversed by the addition of L-
glutamine, but not by the addition of L-asparagine. See
Simberkoff, M.S. & Thomas, L., Reversal by L-glutamine of
the inhibition of lymphocyte mitosis caused by E. coli
asparaginase, 133 Proc. Soc. Exp. Biol. (N. Y.) 642 (1970).
Additionally, a correlation between immunosuppressicn and
the relative amount of glutaminase activity has been
suggested by the observation that E. carotova asparaginase
is more effective than E. coli asparaginase in suppressing
the response of rabbit leukocytes to PHA (see Ashworth,
L.A.E. & MacLennan, A.P., Comparison of L-asparaginases from
Escherichia coli and Erwinia carotovora as
immunosuppressant, 34 Cancer Res. 1353 (1974)). However,
the significance of these in vitro studies is limited
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19
because the in vivo fates of asparaginases and the
homeostatic control of asparagine and glutamine may result
in a modification of the immunosuppressive effects of anti-
neoplastic asparaginases.
Another significant problem associated with the use of
microbial asparaginases is that patients treated with E.
coli and E. carotovora asparaginases frequently develop
neutralizing antibodies of the IgG and IgM immunoglobulin
class (see, e.g., Cheung, N. & Chau, K., Antibody response
to Escherichia coli L-asparaginase: Prognostic significance
and clinical utility of antibody measurement, 8 Am. J.
Pediatric Hematol. Oncol. 99 (1986) Howard, J.B. &
Carpenter, F.H. (1972) supra), which allows an immediate
rebound of serum levels of asparagine and glutamine. In an
attempt to mitigate both the toxic effects and
immunosensitivity associated with the therapeutic
utilization of E. coli and E. carotovora asparaginase, a
covalently-modified E. coli asparaginase (PEG-asparaginase)
was initially developed for use in patients who have
developed a delayed-type hypersensitivity to preparations
"native" of E. coli asparaginase (see Gao, S. & Zhao, G.,
Chemical modification of enzyme molecules to improve their
characteristics, 613 Ann. NY Acad. Sci. 460 (1990)).
However, subsequent studies established that the initial
development of an immune response against E. coli
asparaginase resulted in an 80% cross-reactivity against the
PEG-asparaginase with concomitant adverse pharmacokinetic
effects-neutralization of PEG-asparaginase activity and
normalization of the plasma levels of L-asparagine and L-
glutamine (see Avramis, V. & Periclou, I., Pharmodynamic
studies of PEG-asparaginase (PEG-ASNase) in pediatric ALL
leukemia patients, Seventh International Congress on Anti-
cancer Treatment, Paris, France (1997)). The development of
antibodies directed against E. coli (EC) asparaginase and
the modified PEG-asparaginase in patients is associated with
neutralization of the enzymatic activity of both the EC and
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PEG-asparaginases in vivo, thus potentially resulting in an
adverse clinical prognosis.
II. Effects of Asparaginase Treatment on Spleen and Thymus
5 Histology and Lymphocyte Population.
Examination of the effects of E. coli asparaginase
treatment on spleen histology and lymphocyte populations
shows a marked reduction in both the size and reactivity of
the splenic germinal centers, and a concomitant marked
10 reduction in the cytoplasmic immunoglobulin-containing cells
(B-cell immunoblasts; see Distasio, J. A., et al. (1982),
supra). Additionally, spleen lymphocyte sub-populations
show up to a 40% reduction in the percentage of surface
immunoglobulin-expressing cells (B-cells) accompanied by an
15 increase in the ratio of Thy-1.2-expressing cells (T-cells),
whereas the ratio of Lyt-2 to Lyt-1 cells remains unchanged.
In contrast, asparagine deprivation alone, caused by the
administration of W. succinogenes asparaginase, has no
demonstrable effect on spleen histology or lymphocyte marker
20 distribution.
Similarly, histological examination of the thymus
following E. coli asparaginase administration revealed a
pronounced depletion of cortical thymocytes, whereas no
changes in thymus histology or cellularity were found after
W. succinogenes asparaginase administration. Therefore, a
comparison of the effects of long-term administration on
spleen and thymus histology, cellularity, and weight
indicated that E. coli asparaginase treatment was associated
with a pronounced, sustained reduction in these parameters
in both the spleen and thymus.
III. Covalent Modification of Asparaqinases and Glutaminases
Many proteins currently used to treat human diseases
have extremely short circulating half-lives which limit
their efficacy. In addition, the administration of many
foreign proteins (including certain recombinant proteins) is
associated with allergic hypersensitivity responses which
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21
can also lead to the production of neutralizing antibodies
which hasten the rapid elimination of these therapeutic
proteins from plasma. To overcome these and other problems,
the invention provides a covalent modification procedure to
chemically modify proteins, including asparaginases and
glutaminases, in order to extend their half-lives, reduce
their immunogenicity, and increase their efficacy. This
chemical modification regimen involves the systematic
alteration of protein structures by conjugating an aliphatic
hydrocarbon chain (saturated, partially saturated, or
unsaturated, a straight chain, a branched chain, and/or a
chain of aromatic) of an acylating agent to polar groups
within the protein structure (see Figure 7). While this
process is generally applicable to any protein to be
introduced into a patient, below conditions are described
for covalently modifying E. coli and W. succinogenes
asparaginase using an acid chloride.
IV. Compositions, Formulation, and Administration
As described above, asparaginases and glutaminases (and
analogs and derivatives thereof) can be used to treat
diseases which respond to asparagine or glutamine depletion.
These compounds may also be used to treat such diseases
prophylactically, or to treat those patients previously
diagnosed with and treated for such a disease. For example,
a patient previously diagnosed and successfully treated
whose disease is otherwise in remission, may experience a
relapse. Such patients may also be treated in accordance
with the claimed invention.
Asparaginases and glutaminases, and their biologically
active analogs and derivatives, can be administered to a
patient using standard techniques. Techniques and
formulations generally may be found in Remington's
Pharmaceutical Sciences, 18th ed., Mack Publishing Co.,
Easton, PA, 1990 (hereby incorporated by reference).
Suitable dosage forms, in part, depend upon the use or
the route of entry, for example, oral, transdermal, trans-
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mucosal, or by injection (parenteral). Such dosage forms
should allow the therapeutic agent to reach a target cell or
otherwise have the desired therapeutic effect. For example,
pharmaceutical compositions injected into the blood stream
preferably are soluble.
Pharmaceutical compositions according to the invention
can be formulated as pharmaceutically acceptable salts and
complexes thereof. Pharmaceutically acceptable salts are
non-toxic salts present in the amounts and concentrations at
which they are administered. The preparation of such salts
can facilitate pharmaceutical use by altering the physical
characteristics of the compound without preventing it from
exerting its physiological effect. Useful alterations in
physical properties include lowering the melting point to
facilitate transmucosal administration and increasing
solubility to facilitate administering higher concentrations
of the drug. The pharmaceutically acceptable salt of an
asparaginase or glutaminase may be present as a complex, as
those in the art will appreciate.
Pharmaceutically acceptable salts include acid addition
salts such as those containing sulfate, hydrochloride,
fumarate, maleate, phosphate, sulfamate, acetate, citrate,
lactate, tartrate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate,
and quinate. Pharmaceutically acceptable salts can be
obtained from acids, including hydrochloric acid, malefic
acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic
acid, citric acid, lactic acid, tartaric acid, malonic acid,
methanesulfonic acid, ethanesulfonic acid, benzenesulfonic
acid, p-toluenesulfonic acid, cyclohexylsulfamic acid,
fumaric acid, and quinic acid.
Pharmaceutically acceptable salts also include basic
addition salts such as those containing benzathine,
chloroprocaine, choline, diethanolamine, ethylenediamine,
meglumine, procaine, aluminum, calcium, lithium, magnesium,
potassium, sodium, ammonium, alkylamine, and zinc, when
acidic functional groups, such as carboxylic acid or phenol
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are present. For example, see Remington's Pharmaceutical
Sciences, supra. Such salts can be prepared using the
appropriate corresponding bases.
Pharmaceutically acceptable carriers and/or excipients
can also be incorporated into a pharmaceutical composition
according to the invention to facilitate administration of
the particular asparaginase or glutaminase. Examples of
carriers suitable for use in the practice of the invention
include calcium carbonate, calcium phosphate, various sugars
such as lactose, glucose, or sucrose, or types of starch,
cellulose derivatives, gelatin, vegetable oils, polyethylene
glycols, and physiologically compatible solvents. Examples
of physiologically compatible solvents include sterile
solutions of water for injection (WFI), saline solution and
dextrose.
Pharmaceutical compositions according to the invention
can be administered by different routes, including
intravenous, intraperitoneal, subcutaneous, intramuscular,
oral, topical (transdermal), or transmucosal administration.
For systemic administration, oral administration is
preferred. For oral administration, for example, the
compounds can be formulated into conventional oral dosage
forms such as capsules, tablets, and liquid preparations
such as syrups, elixirs, and concentrated drops.
Alternatively, injection (parenteral administration)
may be used, e.g., intramuscular, intravenous,
intraperitoneal, and subcutaneous injection. For injection,
pharmaceutical compositions are formulated in liquid
solutions, preferably in physiologically compatible buffers
or solutions, such as saline solution, Hank's solution, or
Ringer's solution. In addition, the compounds may be
formulated in solid form and redissolved or suspended
immediately prior to use. For example, lyophilized forms of
the asparaginase and glutaminase can be produced.
Systemic administration can also be accomplished by
transmucosal or transdermal means. For transmucosal or
transdermal administration, penetrants appropriate to the
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24
barrier to be permeated are used in the formulation. Such
penetrants are well known in the art, and include, for
example, for transmucosal administration, bile salts, and
fusidic acid derivatives. In addition, detergents may be
used to facilitate permeation. Transmucosal administration,
for example, may be through nasal sprays, inhalers (for
pulmonary delivery), rectal suppositories, or vaginal
suppositories. For topical administration, compounds can be
formulated into ointments, salves, gels, or creams, as is
well known in the art.
The amounts of the active therapeutic agent to be
delivered will depend on many factors, including the
particular therapeutic agent and the agent' s ICSO, the ECSO.
the biological half-life of the compound, as well as the
age, size, weight, and physical condition of the patient,
and the disease or disorder to be treated. The importance
of these and other factors to be considered are well known
to those of ordinary skill in the art. Generally, the
amount of asparaginase or glutaminase to be administered
will range from about 10 International Units per square
meter of the surface area of the patient's body (IU/M2) to
50,000 IU/M2, with a dosage range of about 1,000 IU/MZ to
about 15,000 IU/M2 being preferred, and a range of about
6,000 IU/M2 to about 10,000 IU/MZ being particularly
preferred to treat an auto-immune disease or Graft versus
Host Disease. Typically, these dosages are administered via
intramuscular or intravenous injection three times per week,
e.g. Monday, Wednesday, and Friday, during the course of
therapy. Of course, other dosages and/or treatment regimens
may be employed, as determined by the attending physician.
In addition to administering an asparaginase or
glutaminase to treat a disease which responds to asparagine
or glutamine depletion, other embodiments of the invention
concern administration of a nucleic acid construct encoding
the enzyme or an analog thereof. As those in the art will
appreciate, a variety of different gene delivery vehicles
(GDVs) may be employed for this purpose. GDVs include viral
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and non-viral delivery systems. Representative viral
delivery systems include recombinant retroviral vectors
which provide for stable, long term, and generally low level
expression of one or more heterologous genes via integration
5 in the genome of cells transfected by the virus. Here,
retroviral GDVs will encode an asparaginase or glutaminase
or an analog thereof, and may also include one or more other
heterologous genes, for example, a gene encoding a
conditionally lethal gene (e. g., thymidine kinase, which
10 converts the pro-drug gancyclovir to its cytotoxic form) to
eliminate the transfected cells, if desired.
Other viral delivery systems include those based on
adeno-associated virus (AAV) and various alpha viruses,
e.g., Sindbis and Venezuelan equine encephalitis virus.
15 These other viral GDVs may provide for higher level
expression, or expression for different duration, of the
desired heterologous gene(s). As those in the art will
appreciate, the host range for the particular virus employed
may be altered by techniques well known in the art.
20 Non-viral GDVs useful in the practice of these
embodiments of the invention include, among others, so-
called "naked DNA" systems which provide the desired
heterologous genes) in functional association with an
appropriate promoter (which in certain embodiments may be an
25 inducible or tissue-specific promoter) encoded by the
nucleic acid construct. Other regulatory elements may also
be included, for example, enhancers and other activators of
gene expression. Preferably, such non-viral systems are
incorporated into liposomes or are associated with
polycationic reagents to facilitate introduction of the
nucleic acid construct into cells of the patient. Of
course, other components can also be included in such GDVs,
e.g., molecules to target one or more particular cell types,
fusogenic peptides to facilitate endocytotic vesicle escape,
etc. Construction of these and other GDVs useful in the
practice of this invention are within the skill of those in
the art.
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Detailed Description Of The Preferred Embodiments
The following examples will serve to further illustrate
various aspects of the present invention and are not
intended to act in any manner as limitations on the claimed
invention. In addition, methodologies are provided which
will permit one of ordinary skill within the relevant arts
to determine whether a derivative asparaginase or
glutaminase is appropriate for utilization in the clinical
therapeutic treatment of humans. For a discussion of
molecular biology techniques which can be used in the
practice of this invention, in addition to those described
below, see Molecular Cloning, A Laboratory Manual, 2d ed.,
ed. Sambrook, et al., Cold Spring Harbor Laboratory Press,
1989, and Current Protocols In Molecular Biology, ed.
Ausubel, et al., John Wiley & Sons, Inc., 1995.
Example 1: In Vitro Culture of W. succinogenes
W. succinogenes was grown in 10-15 liters of liquid
culture media containing 0.4o yeast extract, 100 mM ammonium
formate, and 120 mM sodium fumerate. The medium was
adjusted to pH 7.2 prior to autoclaving. After autoclaving,
a 0.2 ~.m filter-sterilized solution of thioglycolate was
added to the room temperature culture medium to give a final
concentration of 0.050. The cultures were incubated with
continuous agitation on a shaking platform in a 37°C warm-
room. For large scale culture, a 500 mL pre-culture was
utilized to inoculate 10-15 liters of complete culture
medium.
The bacteria were collected after the cultures had
reached a optical density of approximately 1.1 at a 650 nm
wavelength, by centrifugation using a Sorvall high-speed
continuous flow rotor. Following centrifugation, the cells
were washed in a buffer containing 0.15 M sodium chloride,
0.1 M magnesium chloride, and 0.01 M mercaptoethanol. The
cells were then resuspended in 0.1 M borate buffer (pH 9.0)
at a final concentration of 0.5 g wet cell weight/mL borate
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buffer and stored frozen until subsequent processing for
enzyme purification.
Example 2: Animals and Cell Lines
The murine model animals utilized in these experiments
were Balb/C or C3H mice of 9 to 12 weeks in age (Jackson
Laboratories, Bar Harbor, ME).
The therapeutic activity of L-asparaginases was
determined utilizing the 6C3HED Gardner's lymphosarcoma
(Gardner, W.U., Cancer Res., vol. 4: 73 (1944)) and P1798
lymphosarcoma cell lines (ATCC) which as ascites tumors in
C3H and Balb/cc mice, respectively. Alternately, the two
lymphosarcoma cell lines were cultured in RPMI 1640 medium
supplemented with 10% fetal calf serum. The 6C3HED
Gardner's lymphosarcoma originated in the thymus of C3H mice
that were initially given high doses of estradiol. The
lymphosarcoma was subsequently perpetuated by serial
transplantation in the C3H mice.
W.S. asparaginase showed potent anti-tumor activity.
Example 3: Isolation of W. succinoqenes Genomic DNA
Genomic DNA from W. succinogenes was extracted from
bacteria grown in basal medium. Typically, bacterial cells
from a 50 mL of culture were collected by centrifugation and
resuspended by gentle vortexing in 1.5 mL TE buffer (pH
7 . 0 ) . To the cell suspension was added 15 ~,L of 10% SDS to
give a final concentration of O.lo and 3 ~L of a 20 mg/mL
stock solution of proteinase K. The mixture was then
incubated at 37°C for approximately 60 minutes, followed by
several phenol/chloroform extractions. The genomic DNA was
ethanol precipitated and collected by centrifugation. The
W, succinogenes genomic DNA so isolated was sufficiently
pure to use in high stringency PCR amplification.
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Example 4: PCR Amplification of W. succinoQenes Asparaginase
Sequences
The nucleotide sequence of a 2.5 kb Hind III fragment
containing the 993 nucleotide coding region of W.
succinogenes asparaginase was published in 1995. See
GenBank accession number X89215. The elucidation of this
sequence facilitated the synthesis of primers specific for
PCR amplification of the gene coding, for the W.
succinogenes enzyme. As illustrated in Figure 1, the
forward and reverse W. succinogenes asparaginase-specific
PCR primers forward and reverse had the following sequences:
5' -TCCGGATCCAGCGCCTCTGTTTTGATGGCT-3' Forward PCR Primer
[SEQ ID NO. 1]
(BamHI] Restriction
Site Underlined)
5' -TGGGAATTCGGTGGAGAAGATCTTTTGGAT-3' Reverse PCR Primer
[SEQ ID N0. 2]
(EcoRl Site
Restriction
Underlined)
It should be noted that the genomic W. succinogenes
asparaginase coding sequence does not naturally contain
either a BamHl or EcoRl restriction site. However, PCR
amplification utilizing these aforementioned primers
introduced a BamHl and EcoRl restriction site to the 5'- and
3'-termini, respectively to facilitate directional cloning
of this amplified genomic sequence into sequencing and/or
expression vectors.
With respect to PCR amplification, W. succinogenes
genomic DNA (purified as per Example 3) was subjected to 30
cycles of PCR amplification under the following reaction
conditions: 10 ~,L PCR II reaction buffer; 6 ~.L of 25 mg/mL
magnesium chloride, 8 ~L of 10 mM stock solutions of dNTPs,
1 ~L of Taq DNA polymerase (Stratagene Corp.); 1 ~,L ( about
50 ng) each of the W. succinogenes asparaginase-specific
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forward and reverse PCR primers; 1 ~.L of W. succinogenes
genomic DNA; and nuclease-free PCR-grade water to bring the
reaction mixture to 100 ~,L total volume. Following
amplification, 2 ~,L of the PCR products were electrophoresed
through a 1o agarose gel and stained with propidium iodine
to assess both the specificity of the amplification reaction
and the molecular weight of the resulting DNA fragments.
The amplification resulted in the production of a
homogeneous, 1.0 kb W. succinogenes asparaginase-specific
DNA fragment.
Example 5: Cloning of W. succinogenes Asparaginase Sequences
The amplified W. succinogenes asparaginase-specific
amplified DNA fragment was subsequently sub-cloned into the
BamHl and EcoRl sites of the PCRII cloning vector
(Stratagene, La Jolla, CA) utilizing the following reaction
conditions: 2 ~,L of the PCR amplified reaction products, 2
~L of the PCRII cloning vector; 1 ~.L of lOX ligation buffer;
4 ~,L of T4 DNA ligase (Stratagene, La Jolla, CA); and
distilled/deionized water to bring the total reaction volume
to 10 ~L. The ligation reaction was incubated at 16°C
overnight and 2 ~,L of this reaction was utilized to
transform competent E. coli strains DH-5a and M15.
IPTG-induced colorimetric selection (medicated by
expression of (3-galactosidase in the presence of X-GAL) was
utilized to identify recombinant bacterial colonies. Three
white colonies (putative positive recombinants) and one blue
colony (putative negative recombinants) were chosen,
inoculated into a 5 mL culture of LB medium containing 100
~.g/mL ampicillin, and incubated overnight at 37°C on a
shaking platform. Plasmid DNA was isolated from these
cultures via standard DNA "mini-prep" methodology and the
DNA was dissolved in 30 ~.L TE buffer and digested with 3
different restriction endonucleases: BamHl; EcoRl; and
BamHl/EcoRl, to ensure that the isolated plasmid DNA
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contained the expected 1.0 kb W. succinogenes asparaginase-
specific insert.
The electrophoretic results, as illustrated in Figure
3, lanes 2 and 4, demonstrated that colonies #1 and #3
5 contained the expected 1.0 kb insert. To confirm that these
clones contained the W. succinogenes asparaginase gene, the
W. succinogenes asparaginase-specific PCR primers were used
to amplify the W. succinogenes asparaginase-specific
fragments isolated from the aforementioned clones (Figure 3,
10 lanes 2 and 4). These primers did not mediate amplification
of non-insert-containing bacterial DNA (Figure 3, lane 3).
Results of this second PCR amplification demonstrated that
colonies #1 and #3 contained the W. succinogenes
asparaginase-specific DNA insert within the PCRII cloning
15 vector, resulting in the generation of a 1.0 kb
amplification product (see Figure 3, lanes 2 and 4).
The W. succinogenes asparaginase-specific DNA insert in
the PCR II cloning vector was then removed by BamHl and
EcoRl digestion of 10 g of plasmid DNA derived from colony
20 #1, gel-purified via the use of Gene Clean Kit~ (Stratagene,
La Jolla, CA) . The DNA insert was eluted from the gel with
10 ~L distilled/deionized water and then ligated overnight
at 16°C into the similarly restricted pGEX-2T (Amersham
Pharmacia Biotech, Piscataway, N.J.) and pET-28a (Novagen,
25 Inc., Madison, WI) vectors under the following reaction
conditions: 3 ~L DNA insert; 3 ~,L vector DNA; 4 ~,L 5X
ligation reaction buffer; 1 ~.L T4 DNA lipase; and 9 ~L of
distilled/deionized water to give a final reaction volume of
20 ~,L. 10 ~L of each ligation reaction mixture was used to
30 transform 50 ~L of competent E. coli DH-5a cells.
Transformants were then plated onto LB agar plates
containing 100 mg/mL ampicillin. Positive transformants
(i.e., W. succinogenes asparaginase-specific DNA insert-
containing transformants, pGEX-2T-WSA and pET-28-WSA,
respectively) were obtained following approximately 18 hours
of incubation at 37°C. To confirm that the transformants
contained the W. succinogenes asparaginase-specific DNA
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insert, restriction endonuclease digestion using BamH1 and
EcoRl was performed, as well as PCR amplification and DNA
sequence analysis. Results of these analyses demonstrated
that each of the selected "positive" transformants contained
the W. succinogenes asparaginase-specific DNA insert. The
nucleotide sequence of the W. succinogenes asparaginase
specific DNA insert is shown in Figure 6 [SEQ ID NO. 3],
which sequence contains 117 nucleotides 5' to the initial
codes of the Wolinella gene and 23 nucleotides 3' to the
gene's termination codon.
Example 6: Expression of Recombinant W. succinogenes
Asparaginase Analogs
To facilitate isolation of the recombinant W.
succinogenes (rWS) asparaginase protein, several types of
epitope-labeled asparaginase analogs have been constructed.
These epitope labels included: influenza hemagglutinin (HA);
glutathione-S-transferase (GST); DYLD (FLAG) and poly-
histidine (p-His). In each instance, the label is placed on
the N-terminus of the enzyme.
The following methodologies are utilized to isolate
these various epitope labeled rWS asparaginase proteins:
(1) GST-sepharose (Pharmacia AB, Upsala, Sweden) column
chromatography is utilized to purify the GST-labeled
rWS asparaginase enzyme expressed from the pGEX-2T-WSA
vector, followed by cleavage by thrombin.
(2) Protein-G-sepharose immobilized anti-HA and anti-
FLAG antibodies (Pharmacia AB, Upsala, Sweden) is
utilized to affinity purify the HA-or FLAG-labeled rWS
asparaginase enzyme.
(3) Nickel resin (Ni-NTA [nitilo-tri-acetic acid
resin]; Novagen, Inc., Chatsworth, CA) is used to
affinity purify p-His-labeled rWS asparaginase enzyme.
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More specifically, for example, production of poly-
histidine (p-His)-labeled, glutathione-S-transferase (GST)-
rWS asparaginase requires the induction of positively
transformed E. coli with IPTG, followed by harvesting of the
bacteria (see Hochuli, E., & Dobell, N, New metal chelate
absorbents selective for protein and peptide containing
neighboring histidine residues, 411 J. Chromatography 177
(1987)). In such expression systems, vectors such as pGEX-
2T and pET-28a expression vectors may be utilized to
facilitate the expression of a non-epitope-labeled form of
the rWS asparaginase following IPTG induction. The p-His-
labeled constructs, localized in the N-terminus of the rWS
asparaginase, can then be sub-cloned into the BamHl to EcoRl
site of the pET-28a vector (Novagen, Inc., Chatsworth, CA)
for expression of the p-His-labeled rWS enzyme.
Example 7: Purification of Native Wolinella succinogenes
Asparaginase
The native, homotetrameric form of W. succinogenes
asparaginase was purified according to the following
methodology. W. succinogenes cell lysates were prepared by
subjecting bacteria cultured and frozen in accordance with
Example 1 to 3 to 4 freeze/thaw cycles with sonication,
followed by high-speed centrifugation to remove cell debris.
After centrifugation, the supernatant was brought to 0.1 M
concentration of ammonium sulfate at a temperature of 4°C.
The mixture was then brought to a final volume of 1200 by
the addition of a 2o protamine solution, followed by
centrifugation for 30 min. at 21,000 x g. The supernatants
were recovered, pooled, and brought to a 50o ammonium
sulfate saturation and equilibrated for 30 minutes on ice
with continuous stirring. The resulting solution was then
dialyzed against 0.01 M potassium phosphate buffer (pH 8.0)
and applied to a 3 cm x 20 cm hydroxyapatite column
(prepared by: Pharmacia, Inc.) equilibrated with 0.1 M
potassium phosphate buffer pH 8Ø
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The W. succinogenes asparaginase was eluted from the
hydroxyapatite column utilizing step-wise concentrations of
phosphate buffer (i.e., 0.10, 0.20, 0.25, 0.30, 0.35 M
phosphate buffer, pH 8.0). The eluted fractions (10
mL/fraction) were collected, assayed for asparaginase
enzymatic activity, and pooled. The enzymatically-active
fractions were dialyzed against 0.1 M sodium borate buffer
(pH 7 . 0 ) and applied to a 3 cm x 20 cm DEAF-Sephadex column
(prepared by Pharmacia, Inc.) equilibrated in 0.1 M sodium
borate buffer, pH 7Ø The enzyme was eluted by use of a
linear gradient of sodium chloride (0 to 1.O.M) in O.1 M
sodium borate buffer (pH 7.0). 60 mL asparaginase-containing
fractions were retained. W. succinogenes L-asparaginase
prepared utilizing this methodology has been shown to be
homogeneous by SDS-PAGE electrophoresis and silver staining.
E. coli EC-2 asparaginase (Merck, Sharp & Dohme, West
Point, PA) was further purified by gel filtration on
Ultragel~ AcA-44 (LKB Instruments, Inc., Rockville, NM).
Erwinia carotovora asparaginase (Microbiological Research
Establishment, Salisbury, England) was provided by
Pharmaceutical Resources Branch of the National Cancer
Institute.
Example 8: Determination of the Biochemical Characteristics
of Asparaginase
The X-ray crystallographic structures of several
microbial asparaginases have been elucidated (see Lubkowski,
J. & Palm, N. (1996), supra). Recombinant W. succinogenes
asparaginase which possesses acceptable clinical properties
has the following characteristics: (1) catalytic activity in
vitro, (2) preferably a native-protein-like homotetrameric
structure required for functional enzymatic catalysis, and
(3) with respect to the recombinant form of W. succinogenes
asparaginase, similar to that of the native, homotetrameric
form of W. succinogenes asparaginase, greater substrate
specificity for L-asparagine and not catalyzing the
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34
deamidation of L-glutamine to any physiologically
significant degree.
In order to quantitate the biochemical characteristics
of both the native, homotetrameric and recombinant
asparaginase enzymes, Km and VmaX enzyme kinetics, substrate
specificity, pH optimum, and temperature optimum can be
determined. In addition, SDS-PAGE under both reducing and
non-reducing conditions, followed by silver and Coomassie
Blue staining of the gels, can be utilized to establish
enzyme homogeneity, evaluate subunit composition, and
determine enzyme molecular weight (see Park, R. & Liu, K., A
role for Shc, grb2 and raf-1 in FcR1 signal relay, 271. J.
Biol. Chem. 13342 (1996).
The enzymatic activity of L-asparaginase can be
quantitatively determined by the amount of ammonia produced
upon the hydrolysis of 0.08 M L-asparagine using 0.01 M
sodium phosphate buffer (pH 7.0) as the reaction buffer (see
Durden, D. L. & Distasio, J. A. (1980), supra). The assay
mixture can consist of 10 to 40 IU of a homogeneous solution
of L-asparaginase enzyme diluted to 2.0 mL with 0.01 M
sodium phosphate buffer (pH 7.0). Briefly, this assay
system measures the deamidation of L-asparagine indirectly
by quantitating the release of NH3 as colormetrically-
detected by Nessler's Reagent. A standard curve of NH90H may
be prepared to initially derive an extinction coefficient
for NH3, based upon absorbance at 420 nm. The enzyme
reaction may be initiated by the addition of the L-
asparagine substrate (0.04 M). For the determination of Km
and Vmax enzyme kinetics, a more sensitive NADPH-dependent L-
asparaginase assay system can utilized (see Distasio, J. A.
& Niederman, T. (1976), supra).
Example 9: Therapeutic Administration of Asparaginase in
Murine Animal Models
The recombinant and native forms of W. succinogenes
asparaginase may be titrated between 5 and 50 IU per
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injection and the mice can receive up to 3 daily
intraperitoneal (I. P.) injections at each dose.
Toxicological and pharmacological studies for the native and
recombinant enzymes can be performed by the determination of
5 serum enzyme activity (i.e., serum enzyme half-life) as
described in Example 8.
Example 10: Determination of Asparaginase Enzymatic Activity
(Serum Half-Life)
Serum half-life determinations can be performed on
10 Balb/c mice intraperitoneally-injected with 5 or 10 IU of
native (WS) or recombinant (rWS) Wolinelia succinogenes
asparaginase. Enzyme half-life measurements can be
performed by a slight modification of a previously published
procedure (see burden, D. L., et al., Kinetic analysis of
15 hepatotoxicity associated with anti-neoplastic
asparaginases, 43 Cancer Res. 1602 (1983)). Specifically,
enzyme half-life measurements can be performed by obtaining
a 5 ~.L blood sample from the tall vein of the Balb/c mice at
specific intervals following the I.P. injection of the WS or
20 rWS asparaginase. The blood samples are then kept on ice
until all samples had been collected. Once sampling was
completed, each 5 ~L blood sample can then be immediately
pipetted into 0.5 mL of cold 1.190 sodium chloride in 0.1 M
sodium phosphate buffer (pH 7.0) and mixed by vigorous
25 vortexing.
To determine serum asparaginase activity (and hence
serum half-life), two 0.2 mL aliquots from each time point
can be equilibrated in a 37°C water bath. The enzymatic
reaction is subsequently initiated by the addition of 0.03
30 mL of 0.04 M L-asparagine, pre-equilibrated to 37°C prior to
addition, into one of the 0 . 2 mL samples . The other 0 . 2 mL
aliquot receives only 0.3 mL of distilled water and will
serve as a control "blank." The substrate-containing
reaction tube may be incubated at 37°C for 1 hour after which
35 the reaction is stopped by the addition of 0.2 mL of 5% TCA.
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36
In addition, a 0.2 mL aliquot of 5o TCA is also added to the
control "blank." The tubes are then centrifuged at 5000 x g
to remove the resulting TCA-produced precipitate. Enzymatic
activity may be colormetrically-determined by the addition
of a 0.2 mL aliquot of the substrate-containing sample to
0.2 mL of distilled water and 0.2 mL a freshly-prepared
Nessler's Reagent and the absorbance at 420 nm is read using
a spectrophotometer (Gilford Instrument Laboratories,
Oberlin, OH).
Example 11: Determination of the Anti-Neoplastic Activity of
Asparaginase
The anti-neoplastic (anti-lymphoma) activity of
homogeneous preparation of both native (WS) and recombinant
(rWS) W. succinogenes asparaginase, as well as that of
native E. coli (EC) and E. carotovora (Erw) asparaginases,
can be determined utilizing the 6C3HED Gardner lymphosarcoma
cell line implanted in C3H mice. This lymphoid tumor
originated in the thymus of C3H mice given high doses of
estradiol and was perpetuated by serial transplantation in
the C3H mice. In these studies, the tumor is maintained as
an ascites tumor through I. P injection of 2 x 108 viable
lymphosarcoma cells in 0.1 mL of PBS (pH 7.0).
To determine asparaginase anti-tumor activity, 2.5 x 106
viable 6C3HED lymphosarcoma cells from an ascites tumor is
injected in a volume of 0.05 mL of PBS (pH 7.0)
subcutaneously in the left ventral groin of 9 to 12 week-old
C3H mice. Similarly, in another series of experiments, 2.5
x 106 viable P1798 lymphosarcoma cells from an ascites tumor
is injected in a volume of 0.05 mL of PBS (pH 7.0)
subcutaneously in the left ventral groin of 9 to 12 week-old
Balb/c mice (see Jack, G. W., et al., The effect of
histidine ammonia-lyase on some murine tumors, 7 Leukemia
Res. 421 (1983)). Palpable solid tumor growth generally
occurred within 4 to 7 days after injection of the
lymphosarcoma cells. Changes in solid tumor volume are then
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37
subsequently measured by daily caliper-based measurement of
tumor dimensions along three axes. When the average tumor
volume reaches 1 cm3, intraperitoneal injection of
asparaginase can be performed. A total dosage of 3 or 6 IU
of asparaginase may be administered in a total of six I. P
injections of 0.5 or 1.0 IU asparaginase/injection,
respectively. Injections may be administered twice daily
for three consecutive days.
The negative control animal group receives I.P.
injections of 0.01 M phosphate buffer (pH 7.0) utilizing a
similar injection schedule. E. coli and E. carotovora
asparaginases serve as positive controls for comparison of
anti-tumor activity in this series of experiments.
Student's t-test will be utilized for all statistical
analysis of data.
Example 12: Immune Cross-Reactivity W. succinoqenes
Asparaginase
This example describes how it was determined if
antibodies in patients known to neutralize E. coli
asparaginase react with W. succinogenes. Specifically, an
ELISA assay was performed to make this determination, as
described below.
The ELISA assay was performed on two 96 well microtiter
plates, as follows: asparaginase (EC on one plate, WS on the
other) was diluted in carbonate buffer (prepared by
dissolving 1.59 g Na2C03, 2.93 g NaHCo3, and 0.2 g NaN3 1
in L
of purified water; pH was adjusted to 9.0 - 9.5 using 1N HCl
or 1N NaOH; the buffer was stored at 4C for no more than two
weeks before use) to a final concentration of 0.10 IU/mL.
54 wells on each plate were coated with 100 uL of the
respective diluted asparaginase solution and incubated
overnight at 4C after being wrapped in aluminum foil to
allow the enzyme to become associated with the plates.
The following morning the plates were removed, and the
solution from each of the wells was removed. These wells
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38
were then blocked with 300 uL of a 1 mg/mL solution of BSA-
PBS blocking buffer, pH 7.0 (prepared fresh by adding the
appropriate amount of bovine serum albumin to PBS buffer,
0.010 M sodium phosphate, pH 7.0 - 7.2, 0.9% saline). The
plates were then incubated for 1 hour at room temperature.
Thereafter, the plates were washed with 300 mL of saline-
Tween buffer (0.145 M NaCl, 0.05% Tween 20) per well using a
Dynatech Ultrawash plate washer.
The antibodies used to screen the two plates were
diluted as follows: 1:100, 1:1,000; 1:2,000; 1:4,000;
1:8,000; 1:16,000; and 1:32,000. As a control, serum from a
norma l human patient was used. Patient serum and rabbit
anti-EC asparaginase serum and normal human serum were
diluted in PBS-Tween (PBS containing 0.050 Tween 20) and
100uL of each dilution was placed on each plate in
triplicate according to the following grid:
CONTROL HUM AN PATIENT RABBIT
ANTIBODIES
1 2 3 1 2 3 1 2 3
2 0 1:1,00 1:1,0 1:1,001:1,00 1:1,00 1:1,00 1:1,001:1,00 1:1,00
0 00 0 0 0 0 0 0 0
1:2,00 1:2,0 1:2,001:2,00 1:2,00 1:2,00 1:2,001:2,00 1:2,00
0 00 0 0 0 0 0 0 0
1:3,00 1:3,0 1:3,001:3,00 1:3,00 1:3,00 1:3,001:3,00 1:3,00
0 00 0 0 0 0 0 0 0
1:4,00 1:4,0 1:4,001:4,00 1:4,00 1:4,00 1:4,001:4,00 1:4,00
0 00 0 0 0 0 0 0 0
1:8,00 1:8,0 1:8,001:8,00 1:8,00 1:8,00 1:8,001:8,00 1:8,00
0 00 0 0 0 0 0 0 0
1:16,0 1:16, 1:16,01:16,0 1:16,0 1:16,0 1:16,01:16,0 1:16,0
00 000 00 00 00 00 00 00 00
1:32,0 1:32, 1:32,01:32,0 1:32,0 1:32,0 1:32,01:32,0 1:32,0
00 000 00 00 00 00 00 00 00
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39
After adding the above dilutions, the plates were
incubated for at least 1.5 hour at room temperature,
followed by washing each plate three times with saline-Tween
as described above. A 1:1,000 dilution of Horse radish
peroxidase-conjugated goat anti-human immunoglobulin
(BioSource International) was then prepared in PBS-Tween.
100 uL of the HP-conjugated anti-human Ig was then added to
each well. The plates were then covered and allowed to
incubate at room temperature for 1 hour.
After the 1 hour incubating each plate was again washed
three times with saline-Tween, as before. To detect
antibody binding, 100 uL of OPD (o-
phenylenediaminedihydrochloride) substrate (40 mg of OPD in
100 mL a citrate phosphate buffer (O.1M, pH 6.0, prepared by
combining a solution containing 13.4 g Na2HP04'7H20 (dibasic)
in 500 mL distilled water with an amount of a solution
containing 9.60 g citric acid (anhydrous) in 500 mL
distilled water sufficient to adjust the pH to 6.0) with 334
uL of 3 o H202 prepared immediately before use and kept at
room temperature in the dark) was added to each well and
allowed to incubate at room temperature in the dark for
approximately 40 minutes. The reaction in each well was
stopped by adding 100 uL of 1 M phosphoric acid. The
absorbance of each well was then measured at 40 nm.
As is shown in Figure 8, high titer neutralizing
antibodies against the E. coli enzyme present in patient
plasma failed to bind to the Wolinella asparaginase. This
figure shows one of 6 plasma specimens collected from
patients known to be allergic to the E. coli enzyme as well
as rabbit antisera raised against the E. coli asparaginase.
None of these anti-E. coli reactive antisera bind or
neutralize the Wolinella asparaginase activity (Figures 8
and 9). From these data it was concluded that the W.
succinogenes enzyme is immunologically distinct from E.
coli, and that the Wolinella enzyme can be used in patients
allergic to the E. coli enzyme (as exemplified by titration
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of patient plasma shown in Figure 8 and rabbit anti-E, coli
antisera shown in Figure 9).
A highly specific antisera against the W. succinogenes
enzyme which does not cross react with E. coli asparaginase
5 in Western blot analysis has also been prepared. This
reagent is useful for performing immunological
characterizations of the native, recombinant, and various
analog forms of the Wolinella enzyme. Analysis of native,
recombinant, and analog forms of W. succinogenes
10 asparaginase for this type of immunologic cross reactivity
will be useful in characterization of genetically and
chemically modified proteins. Importantly, these analyses
will be applied to analysis of clinical specimens during
phase I and II clinical trials of the different forms of the
15 W. succinogenes enzyme.
Example 13: Methodology for Protein Modification using
Acylation.
Protein acylation is accomplished by using different
acylating agents, such as acyl halides (e. g., acyl
20 chlorides), carbodiimide compounds, or acid anhydrides, each
with a different number of carbon atoms comprising a
straight or branched aliphatic chain attached to the
carbonyl, or the modified carbonyl (in the case of
carbodiimides), carbon atom. The acylating agents
25 contemplated for use in practicing this invention have the
ability to react with a polar group contained within the
peptide sequence of a protein to form an amide side chain.
The polar group is the side chain of any of the amino acids
in the primary sequence, for example, the amine group of
30 lysine or arginine, the hydroxy group of threonine, serine,
or tyrosine, or the thiol group of cysteine. Preferably,
the reaction is carried out under conditions which do not
substantially reduce (i.e., reduce by more than 900,
preferably less than 50%, and more preferably less than 25%)
35 the catalytic activity of the enzyme.
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Briefly, the chemical reaction was started at zero time
with the dropwise addition of acetyl chloride to 5,000 IU of
asparaginase, derived from either E. coli or W.
succinogenes, in a volume of 10 mL of 0.1 M borate buffer at
pH 8.5. The final concentration of each acid chloride is
0.1 M. The chemical reaction involves a nucleophilic attack
of the polar group, e.g., the free amino group, within the
peptide sequence of the protein, e.g., asparaginase (which
is maintained in an unprotonated form in the borate buffer,
pH 8.5) with the reactive acylating agent. The polar group
reacts with the acylating agent yielding an aliphatic
hydrocarbon modified amino acid side chain. If the
acylating agent is an acyl halide, an equivalent of the
respective hydrohalic acid is produced. Thus, if the
acylating agent is acyl chloride and the amino acid to be
modified is lysine, then the reaction yields an acylated
amino group and 1 equivalent of HC1 (see Figure 7). To
prevent acid conditions from destroying the structure of the
protein molecule (decreasing yield of enzyme, Table 1,
below), a 1 N solution of NaOH is added drop-wise to the
reaction mixture every 5-10 seconds. Aliquots of 2 mL were
removed at the indicated reaction times (see Table l,
below), and immediately dialyzed against 0.01 M phosphate
buffer at pH 7Ø Protein concentration is measured by
Bradford method. Enzyme activity is determined by the
amount of ammonia produced upon hydrolysis of L-asparagine
(0.08 M L-asparagine) with a Nessler's reagent (see Durden,
D.L. et al, Cancer Res. 40: 1125, (1980)). Free amino
groups are measured by the method of Habeeb (see Habeeb,
A.F.S.A., Analytical Biochemistry, 14:328, 1966).
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TABLE I
Effect of acylation with acetyl chloride on W, succinogenes
asparaginase
Reaction Specific Reduction Recovery Half-Life
timea (hr) Activityb~~of free of (hr)
(IU/mg) aminesd Activit
y
Native 0 150.0 0 100.0 i.8
enzyme
Derivatized0.5 120.0 29.0 80.0 8.0
enzyme
1.0 129.0 ~ 26.8 86.0 8.2
2.0 130.0 32.4 86.6 i.4
3.0 120.0 30.2 80.0 ?.3
4.5 90.0 31.3 60.0 6.2
a . The reaction is started at time 0 with the addition of
acetyl chloride to 5,000 IU of W. succinogenes asparaginase
in 10 mL of 0.1 M borate buffer, pH 8.5. Aliquots of 2.0 mL
are removed at the times indicated and dialyzed against 0.01
M phosphate buffer, pH 7Ø
b. Protein is measured in triplicate by method of
Bradford.
c. Enzyme activity is measured by determining the amount
of ammonia produced upon hydrolysis of L-asparagine with
Nessler's reagent.
d. Free amino groups are measured by method of Habeeb.
Acyl modification is performed with acylating agents of
different aliphatic chain lengths, e.g., a 2 carbon
aliphatic chain (C2), a ~-_ carbon aliphatic chain (C4), a 6
carbon aliphatic chain (C6), etc. Importantly, each
specific protein (e. g., asparaginase) has different numbers
of free polar groups in different positions within the
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protein molecule and hence each protein is optimally
modified with a different length acylating agent which
conjugates a different aliphatic carbon chain to the free
amino groups. These include, for example, acetyl chloride
(C2), butyryl chloride (C4), hexanoyl chloride (C6),
decanoyl chloride (C10), as well as the use of branched
chain acid chlorides including trimethyl-acetyl chloride.
Also, different acylating agents may be used for different
proteins. For example, with some proteins acetyl chloride
may be used, whereas for other proteins acetic anhydride may
be the best acylating agenst. By way of illustration, the
covalent modification of the W. succinogenes asparaginase
with the acetyl chloride is presented in Table 1.
A. Results of Modification
There are a number of problems that have been
associated with the use of enzymes for therapeutic purposes.
Many of these enzymes have extremely short half-lives which
severely limits their effectiveness in vivo. The
modification of proteins using organic modification
techniques of the present invention is a promising solution
to many of these problems. The C2 modification of W.
succinogenes asparaginase results in an enzyme which has a
half-life of 8.2 hours in mice as compared to the 1.8 hour
half-life of the native enzyme. The increase in half-life
is consistent with the time course of acetylation reaction
(resulting in 20-40° decrease in enzyme activity while the
activity of the W. succinogenes asparaginase decreases with
the increasing reaction time). An about 80o recovery of
enzyme activity after a 30 min. reaction time was observed,
a time of maximum alteration of pharmacokinetic extension of
half-life to 8.0 hours. Other modification procedures which
involve polymerization (e. g., polyethylene-glycol
modification) result in heterogenous groups of modified
reaction products which may not be suitable for
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administration in humans. The acid chloride modification
procedure is a systematic approach which does not yield such
heterogeneity in reaction products (see Figure 7). The
greater reproducibility and more restricted nature of
reaction products result in a well controlled modification
of proteins and a more reliable product with predictable
extension of half-life which decrease the immunogenicity,
and with the advantage of being able to very carefully
control the extent of modification of the polar groups
present in a specific protein molecule. Current data
modifying W. succinogenes asparaginase demonstrate that the
enzyme is modified with a C2 acylation reaction which
results in the augmentation of half-life approximately four
fold. The modification of the free amino groups and the
asparaginase molecule is responsible for extension of half-
life. It is suggested that the extension of half-life will
correlate with a decrease in the electrostatic charge,
increase in hydrophobicity and decreased immunogenicity of
the Wolinella enzyme. The extension of half-life and
decreased immunogenicity will increase the efficacy of the
W. succinogenes enzyme when this drug is used in the
treatment of acute lymphoblastic leukemia, autoimmune
disease, or AIDS, for example, in humans. Through this
modification procedure, we are able to generate foreign
proteins which have lower immunogenicity, extended half-
life, and augmented efficacy. With this systematic approach
of modification, any protein can be modified and the
modified protein can then be used in the treatment of human
disease. Essentially, any protein that has polar groups
available in its native state (essentially all known
proteins) is amenable to the modification technique of the
present invention. Hence this invention extends to all
proteins currently used in treatment of human, animal and
plant diseases.
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Example 14: Mouse Autoimmune Disease Model
Collagen induced arthritis (CIA) in DBA/1 mice is a
recognized experimental autoimmune disease model that
reflects aspects of human rheumatoid arthritis. When
immunized with human collagen type II, these mice develop
severe arthritis with inflammation and erosions of their
joints. Cellular and humoral immune mechanisms against
collagen characterized by synovial proliferation and joint
infiltration by inflammatory cells are believed to be
involved in the pathogenesis of this arthritis model.
Susceptibility to CIA is linked to HLA class II but
also requires the presence of T cells expressing variable V
beta chains of their T cell receptor. Due to the T cell
depleting effect of L-Asparaginase, the severity of CIA can
be reduced and arthritis can be prevented (or, if initiated,
the progression of the disease at least halted) by
prophylactic administration of L-Asparaginase prior to
immunization with collagen.
DBA/1 (H-2q) mice were purchased from Jackson
Laboratories (Bar Harbour, ME), and males 8-12 weeks of age
were used for immunization experiments.
A. Induction of Arthritis
Sedated mice were immunized with 200 ~g of bovine
collagen type II emulsified 1:1 in complete Freud's adjuvant
(CFA) (Difco, Detroit, MI) at the base of the tail.
Arthritis typically developed 4-6 weeks after immunization
in 60-800 of the animals. All animal manipulations were
performed under ether anesthesia.
B. Assessment of Arthritis
Arthritis of fore and hind paws was assessed using a
subjective scoring system in which "0" = normal, "1" - minor
swelling or erythema, "2" - pronounced, edematous swelling,
and "3" - rigidity. Each limb was graded separately, giving
a maximal possible score of 12 per mouse.
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C. Effect of L-Asparaginase on Existing Arthritis
(therapeutic protocol)
At onset of arthritis symptoms, mice were treated with
5, 10, 25, or 50 IU, respectively, of EC asparaginase
intraperitoneally once a day for a total of 3 months and
compared to untreated controls. Additional experiments
using EC-PEG and WS asparaginases can be similarly conducted
using the same outcome parameters. WS asparaginase, which
is believed to solely deplete L-asparagine, has no known
immunosuppressive effects. Thus, the effect of L-asparagine
depletion on the severity and prevention of arthritis can be
assessed using the WS enzyme.
Arthritis was scored every other day for the first
month, every third day during the second month, and once a
week in the third month after onset and treatment of
arthritis symptoms. After 3 months, mice were sacrificed
for histopathological studies.
The data showed that E, coli asparaginase has potent
anti-arthritic activity. E. coli asparaginase treatment
resulted in the reversal of pre-existing arthritis in this
model (see Figures 10 and 11). Given the recognized
correlation between this model and human disease,
asparaginase treatment should reverse, prevent, or halt the
progression of human rheumatoid arthritis and other
autoimmune states.
Other data showed that E. coli asparaginase treatment
reversed the arthritic state induced by collagen and LPS
see Figure 12). Activity in this highly resistant form of
autoimmune arthritis confirmed the results from the mouse
model shown herein, and further supports the usefulness of
asparaginases and glutaminases in the treatment of
autoimmune diseases. The differences in arthritic scores
between E. coli treated animals and control animals were
statistically significant (p<0.001).
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D. Effect of L-Asparaginase on Arthritis Prevention
(preventive protocol)
To study the ability of L-Asparaginase to prevent
arthritis, DBA/1 mice were treated I.P. with 5, 10, 25, or
50 IU, respectively, of EC asparaginase prior to
immunization (-1) , parallel to immunization (0) , and then at
the consecutive days 5, 10, 15, and 30 thereafter. Arthritis
was scored every other day for the first month, every third
day during the second month, and once a week in the third
and fourth month after onset of arthritis symptoms.
After four months mice were sacrificed for
histopathological studies. The administration of E. coli
asparaginase concomitantly with type II collagen in the DBA
mouse model completely abrogated the development of
autoimmune CIA. These results also strongly support the
role for asparaginase and/or glutaminase in the prevention
and/or treatment of autoimmune and/or Graft Versus Host
disease in humans.
E. Assessment of Histology
Removed limbs were fixed in 10o buffered formaldehyde
for four days. After decalcification using 5o formic acid,
specimens were embedded in paraffin, cut into thin slices,
and stained for hematoxylin and eosin. Sections
were
obtained from the femoro-patellar area for the knee joints
and calcaneal area for the ankle joints. Histological
parameters included the amount of inflammatory cells in the
synovial cavity and synovial tissues, amount of proteoglycan
depletion, and the destruction of articular cartilage.
Histologic specimens were interpreted by a blinded
histopathologist.
Pathologic evaluation of involved joints in E. coli
asparaginase-treated and control mice revealed
a dramatic
difference in histopathology. Previously arthritic
joints
from E. coli asparaginase-treated mice demonstrated
persistence of some pannus formation, but no destruction
of
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joint cartilage. In contrast, joints from control mice
showed massive destruction of joint cartilage and underlying
bone, along with pronounced pannus and inflammatory
processes.
Example 15: Enzymatic and Pharmacokinetic Studies
The EC, PEG, and WS asparaginases are purified and
biochemical and pharmacological analysis are performed in
DBA/1 (H-2q) animals. The enzyme levels in animals treated
with these asparaginases are determined in order to
correlate efficacy with catalytic activity.
A. Pharmacoloaic Evaluation of EC, PEG, and WS in DBA/1
M; r.o
Pharmacologic analysis of EC, PEG, and WS asparaginases
is performed in DBA animals. Plasma L-asparagine and L-
glutamine is determined. Administration of asparaginase is
correlated with depletion of asparagine and/or glutamine.
Neutralizing antisera to EC, WS, and PEG asparaginases is
used to establish a cause and effect relationship between
immunosuppressive effects of PEG and WS. A WS asparaginase-
specific antibody is administered to mice as a negative
control for EC asparaginase experiments. The in vivo
effects of administration of neutralizing antisera to PEG
and WS is correlated with plasma amino acid levels and anti-
arthritic effects in the DBA mouse model (see above).
Enzyme half-life measurements are performed as follows:
Five ~.L of blood from the tail vein of mice is obtained at
specific time intervals after the injection of the
particular asparaginase. The 5 ~L blood specimen is
immediately pipetted into a 0.5 mL of cold 1.190 NaCl in 0.1
M sodium phosphate buffer (pH 7.0) and vigorously vortexed.
Blood samples are collected and kept at 4°C until all
specimens are collected. For the asparaginase assay, two
0.2 mL aliquots of each time point are equilibrated to 37°C
in a water bath. To start the reaction, 0.03 mL of a 0.04 M
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L-asparagine solution is pipetted into one of the tubes.
The other aliquot receives 0.03 mL of distilled H20 and serve
as a blank. The enzyme reaction is stopped after 60 minutes
incubation by pipetting 0.2 mL of 5o TCA into both the
reaction mixture and the blank. Tubes are then centrifuged
at 5000 x g, to remove precipitate. A 0.2 mL aliquot of the
supernatant is then be added to 0.2 mL of distilled H20, and
0.2 mL of a freshly prepared Nessler's solution is added.
Absorbance at 420 nm is determined using a spectrophotometer
(Gilford Instrument Laboratories, Oberlin, Ohio).
B. Purification of the WS and EC Asparaginases.
WS and EC asparaginase can be purified to homogeneity
as described by Durden, et a1. in order to characterize
these enzymes and compare their biological and enzymologic
activities. PEG asparaginase is obtained from Rhone Polec
Rorer, Inc. L-asparaginase preparations are shown to be
homogeneous by SDS PAGE and free of endotoxin contamination.
The efficacy of the PEG asparaginase preparation is also
tested in these experiments.
Biochemical analysis of the native WS, EC, and PEG
enzymes is also performed, and the Km, Vmax, and substrate
specificity of these enzymes are determined. The purity of
the enzyme preparations is established by SDS PAGE followed
by silver and Coomassie blue staining of gels.
L-asparaginase activity is determined by the amount of
ammonia produced upon hydrolysis of L-asparagine (.08 M L-
asparagine) using a 0.01 M sodium phosphate buffer (pH 7.0)
as the reaction mixture. The assay mixture consists of 10
to 40 IU of a homogeneous enzyme solution diluted to 2.0 mL
with 0.01 M sodium phosphate buffer, pH 7Ø Briefly, this
assay measures the deamidation of asparagine indirectly by
quantitating the release of NH3 as detected by the Nesslers
reagent. A standard curve of NH9S0~ is prepared in order to
derive an extinction coefficient for NH3 based on the
absorbance at 420 nm. The enzyme reaction is initiated by
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the addition of L-asparagine. For Km and Vmax enzyme
kinetics, a more sensitive NADPH dependent asparaginase
assay system is used.
C. DATA ANALYSIS
Student's t-test is utilized to evaluate the observed
differences between asparaginase-treated animals and control
DBA animals, the effects of different asparaginase
preparations, and different doses of asparaginases.
Example 16: Asparaainase for Treatment of Graft versus
Host Disease
A murine bone marrow transplant model for Graft versus
Host disease (GVHD)(B6--B6D2F1) (Hill GR, et al. J Clin
Invest 102:115, 1998) is used to determine if asparaginase
and/or glutaminases can reverse or prevent acute or chronic
form of GVHD. This involves the transfer of splenocytes and
lymph node cells isolated from C57BL/6J mice to Fl progeny
of C57BL/6J x DBA/2J mouse breeding (termed B6D2F1),
resulting in bone marrow transplantation across MHC and
minor H antigen barriers. In this model, parameters of
survival, spleen index, histopathology of liver, skin, small
intestine, lung, and spleen are measured with cr without
asparaginase/glutaminase treatment. This model has shown
predictive value in testing agents for treatment of
clinically significant GVHD (Kelemen E, et al. Int Arch
Allergy Immunol 102:309, 1993).
For these experiments, 13-16 week B6D2F1 mice are
irradiated 1300 cGy total body radiation split into two
fractions 3 hours apart !13~ Cs Source). These mice serve as
recipients of 60 x l0E' splenocytes and lymph node cells from
C57BL/6J mouse administered by tail vein injection in 0.3 mL
of HBSS on day 0 as described (Ellison CA, et al. J Immunol
155:4189, 1995; Ellison CA, et al. J Immunol 161:631, 1998).
Mice are monitored daily for toxicity, body weight, and
evidence of GVHD. Mice are treated on day +1 with Wolinella
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or E col? asparaginase (50 IU/injection, on Monday,
Wednesday, and Friday) for 4 weeks duration.
In another experimental group, mice are treated at time
of onset of GVHD with a similar regimen of asparaginase or
glutaminase. Splenomegaly associated with GVHD in these
mice is monitored in a subset of mice by monitoring total
body weight of mice and determining spleen weight. A
splenic index (SI) is determined as shown below and spleens
are submitted for histopathological analysis.
Spleen wt. Spleen wt
(experimental) (control)
SI =
Total body weight Total body weight
(experimental) (control)
Pathological analysis includes examination of H and E
stained paraffin-embedded sections of liver, spleen, skin,
kidney, lungs, and small intestine for lymphoid infiltration
and inflammatory damage to tissues. These are graded
according to a histopathological scale as described (Kelemen
E, et al. Int Arch Allergy Immunol 102:309, 1993), hereby
incorporated by reference herein, including any figures,
drawings, or tables. E. coli asparaginase can ameliorate
the severity of acute GVHD in this model.
While embodiments and applications of the present
invention have been described in some detail by way of
illustration and example for purposes of clarity and
understanding, it would be apparent to those individuals
whom are skilled within the relevant art that many
additional modifications would be possible without departing
from the inventive concepts contained herein. The
invention, therefore, is not to be restricted in any manner
except in the spirit of the appended claims.
All references cited herein are hereby incorporated in
their entirety. When used above, the term "including" means
"including, without limitation," and terms used in the
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singular shall include the plural, and vice versa, unless
the context dictates otherwise.
