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Recent Patents on Anti-Cancer Drug Discovery, 2012, 7, 4-13
Expanding Targets for a Metabolic Therapy of Cancer: L-Asparaginase
Daniele Covinia, Saverio Tarditob, Ovidio Bussolatib, Laurent R. Chiarellic, Maria V. Pasquettoa,
Rita Digiliod, Giovanna Valentinic and Claudia Scotti*,a
a
Department of Experimental Medicine, Section of General Pathology, University of Pavia, Via Ferrata 1, 27100 Pavia,
Italy; bDepartment of Experimental Medicine, Unit of General and Clinical Pathology, University of Parma, Via Volturno 39, 43125 Parma, Italy; cDepartment of Biochemistry, “A. Castellani”, University of Pavia, Via Taramelli 3/b,
27100 Pavia, Italy; d Centre for Innovation and Technology Transfer, University of Pavia, Corso Strada Nuova 65,
27100 Pavia, Italy
Received: March 31, 2011; Accepted: April 15, 2011; Revised: April 28, 2011
Abstract: The antitumour enzyme L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1, ASNase), which catalyses
the deamidation of L-asparagine (Asn) to L-aspartic acid and ammonia, has been used for many years in the treatment of
acute lymphoblastic leukaemia. Also NK tumours, subtypes of myeloid leukaemias and T-cell lymphomas respond to
ASNase, and ovarian carcinomas and other solid tumours have been proposed as additional targets for ASNase, with a potential role for its glutaminase activity. The increasing attention devoted to the antitumour activity of ASNase prompted us
to analyse recent patents specifically concerning this enzyme. Here, we first give an overview of metabolic pathways affected by Asn and Gln depletion and, hence, potential targets of ASNase. We then discuss recent published patents concerning ASNases. In particular, we pay attention to novel ASNases, such as the recently characterised ASNase produced
by Helicobacter pylori, and those presenting amino acid substitutions aimed at improving enzymatic activity of the classical Escherichia coli enzyme. We detail modifications, such as natural glycosylation or synthetic conjugation with other
molecules, for therapeutic purposes. Finally, we analyse patents concerning biotechnological protocols and strategies applied to production of ASNase as well as to its administration and delivery in organisms.
Keywords: Acute lymphoblastic leukaemia, amino acid metabolism, asparagine, cancer, glutaminase, glutamine, L-asparaginase.
INTRODUCTION
Initial studies concerning L-asparaginase (ASNase) date
back to the 1900s, when the enzyme was isolated from different animal sources [1] and the key biological role of Lasparagine (Asn) in metabolism was established. Only in the
1950s, the search for immune components able to prevent
and/or halt tumour development lead to the discovery of the
antibody- and complement-independent chemotherapeutic
effect of guinea pig serum against murine Gardner lymphosarcoma [2, 3] and ASNase was then identified as the agent
responsible for this effect [4-6]. The enzyme, isolated from
many bacterial organisms, including Escherichia coli (E.
coli) [7] and Erwinia carotovora (E. carotovora) [8], was
then thoroughly characterised and produced in convenient
recombinant systems, and it is nowadays a part of several
standard chemotherapeutic protocols thanks to its confirmed
anticancer activity.
In nature, Asn is synthesised by asparagine synthetase
(ASNsynt, EC 6.3.5.4) from aspartic acid and L-glutamine
(Gln), and it is the substrate of ASNase (EC 3.5.1.1), which
catalyses its deamidation giving L-aspartic acid and ammonia as reaction products. ASNase may also act on Gln
thereby obtaining L-glutamic acid and ammonia. Bacterial
ASNases are the best characterised members of this enzyme
*Address correspondence to this author at the Department of Experimental
Medicine, Section of General Pathology, University of Pavia, Via Ferrata, 1,
27100 Pavia, Italy; Tel: +39 0382 986335; Fax: +39 0382 986893;
E-mail [email protected]
2212-3970/12 $100.00+.00
family. Type I ASNases are cytoplasmic, display high Km
values vs. Asn and are also active towards Gln. Type II
ASNases are periplasmic, exhibit low Km values vs. Asn, and
have low-to-negligible activity towards Gln. These enzymes
exhibit hyperbolic response to Asn [9, 10], though ASNase I
from E. coli displays positive cooperativity towards Asn and
is allosterically regulated by the substrate itself [11]. A minority of ASNases, also referred to as glutaminasesasparaginases (EC 3.5.1.38), transform either Asn or Gln
into their corresponding acids with an activity against Gln 10
times lower than that exhibited against Asn [12].
Bacterial ASNases are 140-150kDa tetramers, more accurately described as dimers of intimate dimers [13], built up
by identical subunits of 300-350 amino acid residues. Four
independent catalytic sites are located at the intersubunit
interface of the intimate dimers [13, 14]. The enzymatic activity is likely to depend on the classic two-step ping-pong
mechanism of serine proteases and on two catalytic triads
consisting, in E. coli ASNase, of Thr12-Tyr25-Glu283 and
Thr89-Asp90-Lys162, respectively [10, 15].
Most of the information about this enzyme class derives
from studies on E. coli ASNase: it works in a pH range between 5 and 10 [16], it has optimal ASNase activity between
pH 8.0 and 9.0 and optimal glutaminase activity between pH
5.5 and 7.5 [17]. Its affinity constants (Km) for Asn and Gln
are, respectively, 1.15x10-5 e 6.25x10-3 M; its isoelectric
point is 5.2. The enzyme is not linked to either carbohydrates
or phospholipids [18].
© 2012 Bentham Science Publishers
Therapeutic L-Asparaginases
Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1
METABOLIC PATHWAYS TRIGGERED BY ASPARAGINASES
Since ASNases catalyse the hydrolysis of Asn and Gln,
the nutritional stress caused by depletion of these amino acids is reflected by the metabolic responses of treated cells.
Both amino acids work as vehicles in nitrogen transport but
their relative importance is widely different in different organisms, with Gln appearing much more important than Asn
in mammals. Gln is the most abundant amino acid in human
plasma, with concentrations ranging from 0.6 to 0.9mM,
while Asn concentration is ten-fifteen times lower [19].
Moreover, although high variability is presumed among the
various tissues, Gln is also the most abundant amino acid in
the intracellular compartment, with tissue concentrations as
high as 20mM [20]. While several widely used cell culture
media are nominally Asn-free, it has been known since the
early years of cell cultures that Gln is not dispensable for
in vitro growth of mammalian cells [21]. However, the basis
for this requirement has been never completely elucidated
[22-24], since Gln is not considered an essential amino acid
and it can be produced by most cultured cells.
Thus, it not surprising that, so far, Gln depletion has been
studied much more than Asn depletion in a variety of tissues
and conditions. However, Asn depletion is per se able to
induce ASNS, the gene for ASNsynt, and to prolong the shelf
life of its mRNA [25], although these effects are not exclusive of this condition but are observed also upon the depletion of Gln as well as of any single essential amino acid [25]
Fig. (1). Thus, although Asn and Gln are not essential, their
depletion seems able to induce at least part of the complex
transcriptional response elicited by complete amino acid
deprivation [26]. Under such conditions, cells activate a
kinase named General Control Nonderepressible 2 (GCN2),
which phosphorylates the -subunit of the eukaryotic Initiation Factor 2 (eIF2, Fig. (1)). This phosphorylation represents a fundamental metabolic switch that lowers the rate of
global protein synthesis, dependent on formation of the classical ternary activation complex, and leads to energy conservation needed for cell survival. At the same time, translation
of a sub-population of mRNAs, endowed with Internal Ribosome Entry sites (IRES), is favoured. These mRNAs encode
cell-defence proteins or proteins involved in apoptosis. For
example, one of these proteins is transcription factor ATF4
Fig. (1), which, once translated, leads to expression of several genes involved in cell survival or, depending on environmental conditions, apoptosis, such as GADD153/CHOP.
The direct role of GCN2 kinase in the response to ASNase
has been recently demonstrated in vivo by Anthony’s group
[27, 28]. Previous contributions of the same group had already demonstrated that treatment of mice with ASNase
leads to enhanced eIF2 phosphorylation in liver, but not in
pancreas, suggesting a tissue-specificity of the metabolic
effects of the enzyme [29]. In the same report, using
ASNases endowed with different glutaminase activities, it
was also demonstrated that eIF2 phosphorylation was dependent on Gln, rather than Asn depletion, although both
conditions are able to induce ASNS and CHOP [29].
Many of the responses elicited by ASNase treatment are
directly referable to attempts of adaptation to Asn and/or Gln
5
L-Asparaginase
Gln depletion
Asn depletion
GCN2
eIF2
GLNsynt
p-mTOR
mTOR
p-eIF2
ATF4
ASNsynt
SNAT2
ASCT1
Others
Protein synthesis
Cell Growth
CHOP
Others
Apoptosis
Fig. (1). A simplified scheme of the metabolic pathways triggered
by ASNase. See text for details.
shortage. For instance, in MOLT-4 leukaemia cells, E. coli
ASNase promotes not only induction of ASNS, but also synthesis of ASCT1 and SNAT2, two sodium-dependent transporters for Asn and Gln [30], Fig. (1). Moreover, also the
activity of Glutamine Synthetase (GLNsynt) is increased
upon ASNase treatment, although, in this case, the site of
regulation is post-transcriptional [30]. The role of GLNsynt
in the metabolic adaptation to ASNase has been directly addressed in sarcoma cells, demonstrating that increase in
GLNsynt activity parallels enhanced abundance of the enzyme, likely stabilised by intracellular depletion of Gln [31].
This adaptation is important for cell survival, since a
GLNsynt inhibitor powerfully synergises the cytotoxic effects of ASNase [32, 33]. The relationships between effects
of ASNase treatment and pathways triggered by amino acid
starvation have also been strengthened by a genome-wide
study of the response to ASNase (likely derived from E. coli,
although this detail was not given) of human ALL cells [34].
In that study, Fine et al. found that more than 800 genes have
their expression modified after exposure to the antitumour
enzyme [34]. Among these, besides ASNS, genes for tRNA
synthetases, amino acid transporters, ATF and CCAAT/enhancer-binding protein (C/EBP) families of transcription
factors were found induced, while expression of genes associated with proliferation was suppressed [34]. Interestingly,
ATF4 and members of the C/EBP family synergistically interact to promote induction of amino acid-responsive genes
by binding to composite C/EBP-ATF response elements
(CARE) [35].
Protein synthesis slowdown in ASNase-treated cells is
not due only to enhanced eIF2 phosphorylation. Another
important metabolic regulatory switch affected by the antitumour enzyme is the mTOR pathway, which controls cell
growth and division, coupling permissive environmental
conditions and protein synthesis rate. This pathway is activated on the basis of an integration of signals deriving from
growth factors, energy status, nutrient availability, and the
presence of stress of various nature (see [36] for a recent
6 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1
review). In the same report cited above [29], Reinert et al.
demonstrate that mTOR signalling is repressed in liver and
pancreas of ASNase-treated animal. Importantly, this effect
was observed upon treatment with E. coli ASNase (endowed
with glutaminolytic activity) but not with the enzyme from
Wolinella succinogenes, which is nearly devoid of glutaminase activity. This result suggests that mTOR inhibition
is a specific effect of ASNase-induced Gln depletion. Consistently, it is well known that mTOR activity is influenced
by the availability of selected amino acids and that, in particular, Gln is needed for mTOR-dependent signalling [37].
The importance of Gln in tumour metabolism has
boosted the interest towards the search for inhibitors of
GLNsynt and glutaminase, and has renewed the attention to
the therapeutic potential of glutaminases.
MECHANISMS OF ANTITUMOUR EFFECTS
The anti-tumour action of bacterial ASNases is primarily
attributed to their ability to reduce Asn blood concentration
causing a selective inhibition of growth of sensitive malignant cells [38, 39], Fig. (1). Despite the long standing experience with these drugs, however, the precise metabolic and
molecular bases of ASNase antitumoural effect have not yet
been fully elucidated. For example, cell sensitivity has been
related to low or absent expression of ASNsynt [40, 41], and,
in vitro, resistance to ASNase has been associated with upregulation of ASNsynt mRNA expression. Nevertheless,
ASNsynt up-regulation does not correlate with early poor
response to ASNase in children with ALL [42, 43]. Moreover, TEL/AML1 positive leukaemic cells are unable to progress into the S phase of cell cycle under nutrition stress
caused by ASNase, despite their ability to upregulate
ASNsynt [44]. The contribution of the glutaminolytic activity of ASNases to the biological effects of the enzyme is also
a matter of discussion. The prevailing opinion is that Gln
hydrolysis contributes to the toxic effects of ASNases [29,
45, 46]. However, Gln hydrolysis is also needed for an effective and sustained Asn depletion [47] and may contribute
significantly to the antineoplastic activities of the enzyme
[48].
CLINICAL APPLICATIONS
Today, ASNase finds its main use in medicine, with
some of the available commercial names being Elspar®
(E. coli ASNase), Erwinase® (Erwinia chrysanthemi ASNase), and Oncaspar® (pegylated E. coli ASNase). At the
moment of writing, the website of the National Institute for
Health [49] lists 132 open clinical trials involving ASNase.
Since the discovery of ASNase anticancer effect, bacteria
have been the best source of enzyme. Among all bacteria
analysed in the early years, E. coli and E. carotovora have
shown the greater production of enzymes with good antitumour activity. Unluckily, the enzymes have also shown
their toxicity, so that research regarding ASNase is still of
great interest both for the clinics and for biotechnology.
For over thirty years now, the enzyme has been part of
established anticancer protocols for the treatment of acute
lymphoblastic leukaemia (ALL) [50], but it has been used
Covini et al.
also for other types of haematological malignancies like
myeloblastic leukaemia [51], Hodgkin and non-Hodgkin
lymphomas, myelosarcoma and multiple myeloma [52].
More recently, ASNase has been used for extranodal NK/T
cell lymphoma, nasal type, a highly aggressive neoplasm
relatively rare in Europe and North America but more common in Asia and South America [53]. Both ASNases from E.
coli and E. carotovora seem effective [54]. A recent phase II
study has confirmed that ASNase has an excellent activity on
this tumour and, in particular, has suggested that enzymebased treatments are indicated for salvage therapy in patients
with disseminated disease, thus far characterised by unfavourable prognosis [55].
Given the presumed relationship between sensitivity to
ASNase and low expression of ASNsynt, efforts have been
performed to identify other possible targets of antitumour
activity. This approach led to the identification of a subset of
ovarian cancer cell lines endowed with low ASNsynt mRNA
expression and a sensitivity to ASNase greatly enhanced by
ASNsynt silencing [56]. More recently, the same group, using a larger panel of ovarian cancer cell lines, has found a
stronger correlation between sensitivity to ASNase and expression of ASNsynt at the protein level [57].
In vitro sensitivity to ASNases of subsets of several other
solid human neoplasms, such as soft tissue sarcoma [33],
hepatocellular carcinoma (Tardito, unpublished results), and
gastric carcinoma [58, 59] has also been demonstrated, but
the translational relevance of these studies awaits confirmation. In general, access of the tetrameric active enzyme to the
tumour microenvironment is expected to be limited, thus
preventing a sustained local depletion of Asn and Gln.
Hence, modifications of subunit aggregation status to enhance accessibility of the active enzyme to the tumour microenvironment have been recently proposed [48].
PROBLEMS
Clinical applications of ASNases are severely restricted
by their side effects. First of all, administration of ASNase in
childhood ALL may cause coagulative disorders (haemorrhage, disseminated intravascular coagulation (DIC), or
thrombotic events), alterations of the gastrointestinal system
(loss of appetite, nausea, vomiting), central nervous system
symptoms (agitation, depression, hallucinations, disorientation, convulsions, and somnolence or even coma), a mild
increase of body temperature, changes in endocrine and exocrine pancreas with acute pancreatitis, and impaired liver
function [60]. A number of these drawbacks are probably
linked to the glutaminase activity of ASNase [61].
Drug resistance and hypersensitivity, at least in part associated with appearance of neutralising antibodies, can
emerge during treatment. Besides causing immune-mediated
reactions, which range from mild allergy to anaphylaxis,
these antibodies lead to increased clearance of the enzyme by
the reticuloendothelial system and to a consequent fast decrease of ASNase activity in plasma [62]. However, correlation between hypersensitivity, duration of remission, and
development of specific antibodies in treated patients is still
controversial [29, 45, 63, 64].
Therapeutic L-Asparaginases
Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1
SOLUTIONS
In general, most studies in the field have aimed at modulating the catalytic activity of ASNase, improving pharmacokinetics, and achieving greater tolerance by structural
modifications [65, 66]. Significant enhancements have also
been proposed for production, administration, and detection
methods, Fig. (2). These data will be reviewed below.
Novel Asparaginases
It is expected that ASNases with different biochemical
characteristics, derived from new or known microorganisms,
may be the solution to enzyme toxicity, so that novel, more
effective ASNases with less toxic effects are more and more
investigated.
Recently, a new ASNase was discovered in Helicobacter
pylori (H. pylori) by our group [58]. The protein was produced in recombinant form, isolated, purified and characterised. The sensitivity of HL60 leukaemic, cells supports the
hypothesis that this enzyme might represent a candidate
drug, also considering that its IC50 value is significantly
lower than that of E. coli enzyme. The higher cytotoxic effects displayed by H. pylori ASNase compared to the E. coli
enzyme are difficult to explain, but this fact underlines the
potential of the former as a chemotherapeutic drug for its
minimal glutaminase activity, high thermal stability, and
maximum activity at physiological pH [67].
Recombinant ASNase derived from W. succinogenes [68]
has a very low activity towards Gln and this characteristic
7
may lead to a drastic reduction of side effects. Besides potentially becoming a first line therapy, it could be used to treat
patients who have developed hypersensitisation to other microbial ASNases thanks to its low cross-reactivity [14, 69,
70]. Thus, Wolinella ASNase is currently under development
through the NIH/NCI-RAID Developmental Therapeutics
program, although it has not yet been administered to patients [14].
Particular attention has been given to GLNase-ASNase
from Pseudomonas 7A [71], capable of depleting Gln and
Asn for prolonged periods. Its considerable antineoplastic
activity has been related to the synergic depletion of both
amino acids. Pseudomonas 7A GLNase-ASNase appears to
be suited for therapeutic use because of its low Km for Gln
(micromolar range), good stability and activity in a physiological milieu, and long plasma half-life in tumour-bearing
hosts [72, 73].
The widespread problem of neutralising antibodies
against non-human protein drugs has led to a great effort in
the development of human molecules with therapeutic purposes. Thus, a promising solution to the problem of antiASNase antibodies would be to use recombinant human glycosylasparaginase (also named aspartylglucosaminidase, EC
3.5.1.26) [74]. This enzyme, previously found defective in
aspartylglycosaminuria, hydrolyzes the N-glycosidic carbohydrate-to-protein linkage region, aspartylglucosamine, to Laspartic acid and L-amino-N-acetylglucosamine through a
reaction mechanism similar to ASNase [75]. Noronkoski and
Kelo [76, 77] have demonstrated that the enzyme also has an
NOVEL ASNases
Pseudomonas A7
H. pylori
W. succinogenes
Human glycosil-ASNase
Clini
cal
ap
pl
MODIFICATIONS
at
ic
ASNase for
cancer therapy:
s
ion
pH range of activity
Endopeptidase resistance
PEG conjugation
Acylation
Bi
Innovations
DELIVERY
ot
Reduction of
immunoreactivity
Erythrocytes
Liposomes
Human albumin
conjugation
Increase of activity
Rosmarinic acid
ec
hn
ol
og
y
DRUG PRODUCTION
Modified hosts
ansB- hosts
Novel hosts
ASSAY
Prediction of
neutralizing factors
Fig. (2). Recent innovations in ASNase usage for cancer therapy: strategies adopted in biotechnology and clinical applications are illustrated.
8 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1
Covini et al.
ASNase activity similar to the Erwinia enzyme but that, unlike this, glycosylasparaginase lacks glutaminase activity.
The characteristics mentioned above make the enzyme a
potential anticancer agent.
Table 1 summarises the biochemical features of some
representative ASNases [40, 58, 69, 72, 76, 78-83], where
Km is the substrate concentration at which the reaction rate is
half maximal, and kcat or turnover number is the number of
catalytic events per second per active site. The former indicates the binding affinity of the enzyme for substrate, the
latter represents the maximal rate of conversion obtained at
saturating concentration of substrate. In principles, enzymes
with a high efficiency have high kcat and low Km and, consequently, a high kcat / Km ratio. Kinetic data can allow some
predictions about the performance of ASNase in cancer
treatment. Enzymes best suited as drugs would be those with
a high activity (high kcat), a high affinity (low Km) for Asn,
and a strong preference for Asn over Gln [83]. Among the
enzymes used today in the clinics, none is endowed with all
these ideal features.
Modified ASNases
ASNase finds a further application in food industry processes, for example during the preparation of French fries,
whose Asn, exposed at high-temperatures, generates acrylamide. For this application, amino acid substitutions of the
enzyme sequence have been designed to change the pH
range of peak activity [84]. However, the power of this approach is certainly not limited to industrial applications.
Structural modifications can be introduced at the amino acid
level by site-directed mutagenesis, like in [84], either structure or sequence-based, or by random mutagenesis followed
by phenotypic selection [85]. Both can have extraordinary
effects on the properties of the wild type protein and are,
therefore, a powerful system to modulate both ASNase and
Table 1.
GLNase activities for therapeutic purposes at will. In this
respect, active site transplant by loop grafting is also an alternative, once an appropriate human framework is found.
In order to tackle a further mechanism of resistance in
clinical sets, a modified E. coli ASNase enzyme has been
produced, which is resistant to cleavage by human asparaginyl endopeptidase (legumain), a lysosomal cysteine protease that can cleave native E. coli ASNase [48, 86]. It is
expected that the modified enzyme will have a longer halflife in vivo and it will be less allergenic than the native enzyme [87].
Important results have been achieved with pegylated
(PEG)-ASNase, a form of E. coli ASNase covalently linked
to polyethylene glycol, that decreases immunogenicity of the
enzyme and prolongs its half-life [88-90]. Beyond advantages, PEG conjugation has some adverse effects, such as the
formation of anti-PEG antibodies, which has led to the development of strategies and methods to select patients who
are specifically sensitive to the PEG-conjugated drug and to
pursue the selective elimination of anti-PEG antibodies [91,
92]. As an alternative to decrease immunogenicity and avoid
acute toxicity, native ASNase has been also acylated, binding palmitoyl residues to epsilon-NH2 groups of lysines [93,
94].
Methods of Administration and/or Delivery
Among new administration approaches, an erythrocytecarried ASNase (Graspa®) shows the absence of hypersensitivity reactions. The enzyme can be encapsulated into homologous erythrocytes with two methods: the most widely
used requires an incubation under hypotonic conditions or in
the presence of membrane-translocating, low-molecularweight protamine; the second method consists in the attachment of the enzyme to the erythrocyte membrane [95].
Biochemical Properties of Representative ASNases.
Asparagine
Glutamine
References
Organism
kcat (s-1)
Km (mM)
0.04-0.07
60
0.04-0.07
60
[78]
Erwinia chrysanthemi*
0.058-0.080
397-440
1.7-6.7
65-72
[40, 79]
Erwinia carotovora*
0.085-0.098
524-1033
3.0-6.8
2.9-7.6
[80, 81]
Arabidopsis thaliana
>4
0.23
n.d.
n.d.
[82]
Escherichia coli
0.015
24.0
3.5
0.33
[83]
Homo sapiens
0.656
1.09
n.d.
n.d.
[76]
Pseudomonas 7A
0.0046
93.1
0.0044
93.1
[72]
Helicobacter pylori
0.290
19.2
46.4
22.1
[58]
Wolinella succinogenes
0.0478
166.6
n.d.
n.d.
[69]
Km (mM)
Acinetobacter glutaminasificans
†
†
†
kcat (s-1 )
In some cases the kcat was calculated from specific activity values reported in the literature.
* Taxonomy of the genus Erwinia is in continuous progress, thanks to the introduction of ever new biochemical and genomic methods. Though several Authors report, the two names
E. carotovora and E. chrystanthemi as synonyms, this is taxonomically incorrect and the respective ASNases have only a limited amino acid sequence identity (77%, UNIPROT
codes: C6DB03 and P06608, respectively).
Therapeutic L-Asparaginases
Moreover, the improvement in the pharmacokinetics associated with the use of erythrocytes makes it possible to use
much lower quantities of enzyme compared to those needed
with the free form or with the PEG-conjugated form, thus
reducing toxicity risks [96].
Palmitoyl-conjugated ASNase can also be encapsulated
in large liposomes (simplified Dehydration-Rehydration
Vesicles, sDRV; median diameter 1.249 nm) [97, 98] and in
small liposomes (extruded vesicles, VET: median diameter
158-180nm). Both devices mitigate anaphylactic reactions
and enhance antitumour activity, with VET also prolonging
the circulation time of the enclosed enzyme [97, 98].
Poznansky and his group have described the advantage of
ASNase cross-linked to homologous albumin in a polymeric
form. This modification leads to an enzyme much more resistant to proteolytic degradation and less immunogenic than
the free enzyme, due to the masking of antigenic determinants by albumin. Moreover, immobilisation of ASNase with
homologous albumin reduces the dose needed for antileukaemia activity [99, 100].
The co-administration of the natural polyphenol rosmarinic acid with E. coli ASNase can significantly improve, at
least in vitro, the activity of ASNase when the concentration
of rosmarinic acid is between 0.375 x 10-4 and 3.0 x 10-4g/L
[101].
Drug Production
A group of recent patents concerns biotechnological
techniques for production of recombinant ASNases, with
particular attention to the improvement of methods and expression vectors. The presence of the gene encoding an
ASNase II subunit, native to the chromosome of E. coli host
strains, could have been a potential obstacle and several
methods have been implemented to circumvent this problem.
In one, the host cell chromosome has been modified to encode the same ASNase II cloned in the plasmid for recombinant protein expression [102]; in another, the E. coli genome
has been modified to delete native ASNase genes [103, 104].
An alternative method to produce E. coli ASNase exploits
the construction of the encoding pNAN5 plasmid and its
transformation in Bacillus cereus 1676 cells. This process
allows improvement of industrial production and purification
of the enzyme [105].
Assays
Methods to monitor activity of serum ASNase to tailor
the therapeutic dose to the single patient are currently used in
research settings, but have not been validated for patient
care. Inactivation of ASNase by neutralising factors present
in patients' serum is not necessarily accompanied by clinical
signs and, therefore, can remain undetected. For this reason,
a quick and easy-to-use test for predicting the presence of
ASNase neutralising factors, mainly antibodies, in patient
serum has been proposed so as to adjust the dose of administered enzyme or to replace the ASNase used with another
enzyme not sensitive, or less sensitive, to the neutralising
factors [106].
Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1
9
CURRENT & FUTURE DEVELOPMENTS
Thanks to more than one century of studies, it is now
clear that Asn and Gln are pivotal in normal metabolism. On
the other hand, recent work on amino acid metabolism in
cancer has made ASNase and related enzymes potentially
key therapeutic tools in previously untested tumours.
Moreover, the search for natural variants and the production
of novel modified forms of ASNase by protein engineering
appear as the most powerful approaches to improve the
therapeutic potential of the enzyme. Recent studies on human L-ASNases [74, 77, 107, 108] would suggest that these
molecules will be the most likely to be exploited in future
clinical applications. However, their distribution, limited to
immunologically privileged tissues (brain and testis), along
with the discovery that ASNase is an autoantigen in rats
[108] indicate that this route should be undertaken with caution. Alternatively, novel sources of ASNases, such as
plants, have not yet been fully considered. In this respect, a
bioinformatic analysis for immunogenicity of representative
ASNases from different species was performed by Ellipro
[109] and CBTOPE [110], Table 2. Ellipro [109] predicts
linear and discontinuous antibody epitopes based on a
protein antigen's 3D structure or a model generated from a
protein sequence [111, 112]. CBTOPE [110] discriminates
the antibody epitope residues and non-epitope residues for a
given protein sequence by using the amino acid composition
generated from the query sequence(s). Our result suggests
that the number of predicted epitopes for several plant enzymes is similar to that of bacterial enzymes, Table 2.
Moreover, immunodominant epitopes can be tackled by
structural modifications [40]. As an example, in Fig. (3a,b,c)
epitopes predicted by Ellipro [109] are mapped in dark grey
on the molecular surface of E. coli ASNase (Protein Database ID: 3ECA). Linear epitopes demonstrated to be biologically relevant according to [111] (§) and [112] (*) are shown
in black. Biologically relevant epitope 115-124 was also
predicted by Ellipro. In panel D, discontinuous epitopes predicted by Ellipro [109] are represented.
New modifications are also under development. For example, Zhang and his group conjugated E. coli ASNase to
silk fibroin and silk sericin from Bombyx mori, using a crosslinker [113]. The modified enzyme has enhanced thermal
and storage stability, resistance to trypsin digestion, a
lengthened circulatory half-life in vitro, and reduced immunogenicity. Only recently, E. chrysanthemi L-asparaginase
has been pegylated [114], while the capacity of ASNase to
deplete asparagine has been combined with the inhibitory
activity of a small interfering RNA (siRNA) towards
ASNsynt [115].
It is likely that the combination of different approaches
mentioned above will sort out the issues related to the clinical usage of ASNase reasonably soon.
ACKNOWLEDGEMENTS
Financial support for all the authors derive from the Italian Ministry for University and Research. ST is a Rina Fallini scholar of the Medical Faculty of the University of
10 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1
Table 2.
Covini et al.
Number of Epitopes Predicted for L-Asparaginase from Different Species.
ElliPro [109]
Uniprot code
CBTOPE [110]
Epitope Prediction
Organism
Epitope Prediction
Linear
Discontinous
P20933
Homo sapiens
3
4
19
Q7L266
Homo sapiens
2
2
22
P06608
Erwinia chrysanthemi
5
4
29
CSDB03
Erwinia carotovora
13
8
23
P00805
Escherichia coli K12
4
5
21
B6ZCD8
Helicobacter pylori CCUG
4
14
24
O34245
Wolinella succinogenes
17
3
Q8GXG1
Arabidopsis thaliana
3
12
19
Q9ZSD6
Lupinus luteus
4 chain A
12 chain A
15
4 chain B
14 chain B
4
14
(Genbank) CAE11777.1
Cavia porcellus
A
44
B
304-313 [*]
244-265
115-124 [*]
14-52
75-81
55-58 [§]
311-318
C
D
260-261
264-265
198-211
1-2 [§]
19-20-120
137-144
181-195
15-18
14-30-31
Fig. (3). Epitopes mapped on the atomic surface of E. coli ASNase (Protein Database ID: 3ECA). Panels A, B and C: linear epitopes mapped
on subunit B (white) of the tetramer. Dark grey islands: epitopes predicted by Ellipro [109]. Black islands: linear epitopes demonstrated to be
biologically relevant according to [111] (§) and [112] (*). Epitope 115-124 of panel B was also predicted by Ellipro. Panel D: discontinuous
epitopes predicted by Ellipro [109]. §: [111], *: [112].
Therapeutic L-Asparaginases
Parma. The authors would like to thank Leonardo Barozzi
(Artware Solutions S.r.l.) for his precious support with
hardware and software and Luca Vecchia for figures production and text revision.
CONFLICT OF INTEREST
The authors declare that they have no conflicts of inter-
Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1
[20]
[21]
[22]
[23]
est.
[24]
REFERENCES
[25]
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
Clementi A. La desamidation enzmatique de I'asparagine chez les
differentes especes-animals et la signification physiologique de sa
presence dans l'organisme. Arch Intern Physiol 1922; 19: 369-98.
Kidd J. Regression of transplanted lymphomas induced in vivo by
means of normal guinea pig serum. I. Course of transplanted
cancers of various kinds in mice and rats given guinea pig serum,
horse serum, or rabbit serum. J Exp Med 1953; 98: 565-82.
Kidd J. Regression of transplanted lymphomas induced in vivo by
means of normal guinea pig serum. II. Studies on the nature of the
active serum constituent: Histological mechanism of the regression:
tests for effects of guinea pig serum on lymphoma cells in vitro:
Discussion. J Exp Med 1953; 98: 583-606.
Broome J. Evidence that the L-asparaginase activity of guinea pig
serum is responsible for its antilymphoma effects. Nature 1961;
191: 1114-5.
Broome J. Evidence that the L-asparaginasc of guinea pig serum is
responsible for its antilymphoma effects. I. Properties of the Lasparaginase of guinea pig serum in relation to those of the
antilymphoma substance. J Exp Med 1963; 118: 99-120.
Broome J. Evidence that the L-asparaginase of guinea pig serum is
responsible for its antilymphoma effects. II. Lymphoma 6C3HED
cells cultured in a medium devoid of L-asparagine lose their
susceptibility to the effects of guinea pig serum in vivo. J Exp Med
1963; 118: 121-47.
Tsuji Y. Studies on the amidase. IV. Supplemental studies on the
amidase action of the bacteria. Japan Arch Internal Med 1957; 4:
222-4.
Wade HE, Elsworth R, Herbert D, Keppie J, Sargeant K. A new Lasparaginase with antitumour activity? Lancet 1968; 2: 776-7.
Michalska K, Jaskolski M. Structural aspects of L-asparaginases,
their friends and relations. Acta Biochim Pol 2006; 53: 627-40.
Sanches M, S. K, Polikarpov I. Structure, Substrate Complexation
and reaction Mechanism of Bacterial Asparaginases. Curr Chem
Biol 2007; 1: 75-86.
Yun MK, Nourse A, White SW, Rock CO, Heath RJ. Crystal
structure and allosteric regulation of the cytoplasmic Escherichia
coli L-asparaginase I. J Mol Biol 2007; 369: 794-811.
Aghaiypour K, Wlodawer A, Lubkowski J. Structural basis for the
activity and substrate specificity of Erwinia chrysanthemi Lasparaginase. Biochemistry 2001; 40: 5655-64.
Swain AL, Jaskolski M, Housset D, Rao JK, Wlodawer A. Crystal
structure of Escherichia coli L-asparaginase, an enzyme used in
cancer therapy. Proc Natl Acad Sci USA 1993; 90: 1474-8.
Lubkowski J, Palm GJ, Gilliland GL, Derst C, Rohm KH,
Wlodawer A. Crystal structure and amino acid sequence of
Wolinella succinogenes L-asparaginase. Eur J Biochem 1996; 241:
201-7.
Dodson G, Wlodawer A. Catalytic triads and their relatives. Trends
Biochem Sci 1998; 23: 347-52.
Illarionova N, Petrov L, Olennikova L, Roshchin S, Pasechnik V.
Study of the secondary structure of L-asparaginase over a broad
range of pH values. Mol Biol (Mosk) 1980; 14: 951-5.
Mardashev SR, Ia Nikolaev A, Kozlov EA, Potrii OP. Purification
of microbial asparaginases by the aid of affinity chromatography.
Biokhimiia 1975; 40: 78-82.
Ho PP, Milikin EB, Bobbitt JL, Grinnan EL, Burck PJ, Frank BH,
et al. Crystalline L-asparaginase from Escherichia coli B. I.
Purification and chemical characterization. J Biol Chem 1970; 245:
3708-15.
Cooney DA, Capizzi RL, Handschumacher RE. Evaluation of Lasparagine metabolism in animals and man. Cancer Res 1970; 30:
929-35.
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
11
Souba WW. Glutamine and cancer. Ann Surg 1993; 218: 715-28.
Eagle H, Oyama VI, Levy M, Horton CL, Fleischman R. The
growth response of mammalian cells in tissue culture to Lglutamine and L-glutamic acid. J Biol Chem 1956; 218: 607-16.
Salzman NP, Eagle H, Sebring ED. The utilization of glutamine,
glutamic acid, and ammonia for the biosynthesis of nucleic acid
bases in mammalian cell cultures. J Biol Chem 1958; 230: 1001-12.
Kovacevic Z, Morris HP. The role of glutamine in the oxidative
metabolism of malignant cells. Cancer Res 1972; 32: 326-33.
Reitzer LJ, Wice BM, Kennell D. Evidence that glutamine, not
sugar, is the major energy source for cultured HeLa cells. J Biol
Chem 1979; 254: 2669-76.
Gong SS, Guerrini L, Basilico C. Regulation of asparagine
synthetase gene expression by amino acid starvation. Mol Cell Biol
1991; 11: 6059-66.
Shan J, Lopez MC, Baker HV, Kilberg MS. Expression profiling
after activation of the amino acid deprivation response in HepG2
human hepatoma cells. Physiol Genomics 2010;
Bunpo P, Cundiff JK, Reinert RB, Wek RC, Aldrich CJ, Anthony
TG. The eIF2 kinase GCN2 is essential for the murine immune
system to adapt to amino acid deprivation by asparaginase. J Nutr
2010; 140: 2020-7.
Bunpo P, Dudley A, Cundiff JK, Cavener DR, Wek RC, Anthony
TG. GCN2 protein kinase is required to activate amino acid
deprivation responses in mice treated with the anti-cancer agent Lasparaginase. J Biol Chem 2009; 284: 32742-9.
Reinert RB, Oberle LM, Wek SA, Bunpo P, Wang XP, Mileva I,
et al. Role of glutamine depletion in directing tissue-specific
nutrient stress responses to L-asparaginase. J Biol Chem 2006; 281:
31222-33.
Aslanian AM, Kilberg MS. Multiple adaptive mechanisms affect
asparagine synthetase substrate availability in asparaginaseresistant MOLT-4 human leukaemia cells. Biochem J 2001; 358:
59-67.
Bussolati O, Belletti S, Uggeri J, Gatti R, Orlandini G, Dall'Asta V,
et al. Characterization of apoptotic phenomena induced by
treatment with L-asparaginase in NIH3T3 cells. Exp Cell Res 1995;
220: 283-91.
Rotoli BM, Uggeri J, Dall'Asta V, Visigalli R, Barilli A, Gatti R,
et al. Inhibition of glutamine synthetase triggers apoptosis in
asparaginase-resistant cells. Cell Physiol Biochem 2005; 15: 28192.
Tardito S, Uggeri J, Bozzetti C, Bianchi MG, Rotoli BM, FranchiGazzola R, et al. The inhibition of glutamine synthetase sensitizes
human sarcoma cells to L-asparaginase. Cancer Chemother
Pharmacol 2007; 60: 751-8.
Fine BM, Kaspers GJ, Ho M, Loonen AH, Boxer LM. A genomewide view of the in vitro response to l-asparaginase in acute
lymphoblastic leukemia. Cancer Res 2005; 65: 291-9.
Kilberg MS, Shan J, Su N. ATF4-dependent transcription mediates
signaling of amino acid limitation. Trends Endocrinol Metab 2009;
20: 436-43.
Zoncu R, Efeyan A, Sabatini DM. mTOR: From growth signal
integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol
2011; 12: 21-35.
Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler
B, et al. Bidirectional transport of amino acids regulates mTOR and
autophagy. Cell 2009; 136: 521-34.
Taylor C, Dorr R, Fanta P, Hersh E, Salmon S. A phase I and
pharmacodynamic evaluation of polyethylene glycol-conjugated Lasparaginase in patients with advanced solid tumors. Cancer
Chemother Pharmacol 2001; 47: 83-8.
Agrawal N, Bukowski R, Rybicki L, Kurtzberg J, Cohen L,
Hussein M. A Phase I-II trial of polyethylene glycol-conjugated Lasparaginase in patients with multiple myeloma. Cancer 2003; 98:
94-9.
Moola ZB, Scawen MD, Atkinson T, Nicholls DJ. Erwinia
chrysanthemi L-asparaginase: epitope mapping and production of
antigenically modified enzymes. Biochem J 1994; 302 ( Pt 3): 9217.
Keating MJ, Holmes R, Lerner S, Ho DH. L-asparaginase and PEG
asparaginase--past, present, and future. Leuk Lymphoma 1993; 10
Suppl: 153-7.
Krasotkina J, Borisova A, Gervaziev Y, Sokolov N. One-step
purification and kinetic properties of the recombinant L-
12 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
asparaginase from Erwinia carotovora. Biotechnol Appl Biochem
2004; 39: 215-21.
Borek D, Jaskólski M. Sequence analysis of enzymes with
asparaginase activity. Acta Biochim Pol 2001; 48: 893-902.
Appel IM, den Boer ML, Meijerink JP, Veerman AJ, Reniers NC,
Pieters R. Up-regulation of asparagine synthetase expression is not
linked to the clinical response L-asparaginase in pediatric acute
lymphoblastic leukemia. Blood 2006; 107: 4244-9.
Ollenschlager G, Roth E, Linkesch W, Jansen S, Simmel A,
Modder B. Asparaginase-induced derangements of glutamine
metabolism: The pathogenetic basis for some drug-related sideeffects. Eur J Clin Invest 1988; 18: 512-6.
Bunpo P, Murray B, Cundiff J, Brizius E, Aldrich CJ, Anthony TG.
Alanyl-glutamine consumption modifies the suppressive effect of
L-asparaginase on lymphocyte populations in mice. J Nutr 2008;
138: 338-43.
Panosyan EH, Grigoryan RS, Avramis IA, Seibel NL, Gaynon PS,
Siegel SE, et al. Deamination of glutamine is a prerequisite for
optimal asparagine deamination by asparaginases in vivo (CCG1961). Anticancer Res 2004; 24: 1121-5.
Offman MN, Krol M, Patel N, Krishnan S, Liu J, Saha V, et al.
Rational engineering of L-asparaginase reveals importance of dual
activity for cancer cell toxicity. Blood 2011; 117: 1614-21.
U.S. National Institution of Health. Clinical trials. Available at:
http://clinicaltrials.gov. (accessed on March 29, 2011)
Hill J. L-asparaginase therapy for leukemia and other malignant
neoplasms. Remission in human leukemia. JAMA 1967; 202: 8828.
Duval M, Suciu S, Ferster A, Rialland X, Nelken B, Lutz P, et al.
Comparison of Escherichia coli-asparaginase with Erwiniaasparaginase in the treatment of childhood lymphoid malignancies:
results of a randomized European Organisation for Research and
Treatment of Cancer Children's Leukemia Group phase 3 trial.
Blood 2002; 99: 2734-9.
Perel Y, Auvrignon A, Leblanc T, Vannier J, Michel G, Nelken B,
et al. Impact of addition of maintenance therapy to intensive
induction and consolidation chemotherapy for childhood acute
myeloblastic leukemia: results of a prospective randomized trial,
LAME 89/91. Leucámie Aiqüe Myéloïde Enfant. J Clin Oncol
2002; 20: 2774-82.
Ishida F, Kwong YL. Diagnosis and management of natural killercell malignancies. Expert Rev Hematol 2011; 3: 593-602.
Matsumoto Y, Nomura K, Kanda-Akano Y, Fujita Y, Nakao M,
Ueda K, et al. Successful treatment with Erwinia L-asparaginase
for recurrent natural killer/T cell lymphoma. Leuk Lymphoma
2003; 44: 879-82.
Jaccard A, Gachard N, Marin B, Rogez S, Audrain M, Suarez F,
et al. Efficacy of L-asparaginase with methotrexate and
dexamethasone (AspaMetDex regimen) in patients with refractory
or relapsing extranodal NK/T-cell lymphoma, a phase 2 study.
Blood 2011; 117: 1834-9.
Lorenzi PL, Reinhold WC, Rudelius M, Gunsior M, Shankavaram
U, Bussey KJ, et al. Asparagine synthetase as a causal, predictive
biomarker for L-asparaginase activity in ovarian cancer cells. Mol
Cancer Ther 2006; 5: 2613-23.
Lorenzi PL, Llamas J, Gunsior M, Ozbun L, Reinhold WC, Varma
S, et al. Asparagine synthetase is a predictive biomarker of Lasparaginase activity in ovarian cancer cell lines. Mol Cancer Ther
2008; 7: 3123-8.
Cappelletti D, Chiarelli LR, Pasquetto MV, Stivala S, Valentini G,
Scotti C. Helicobacter pylori L-asparaginase: A promising
chemotherapeutic agent. Biochem Biophys Res Commun 2008;
377: 1222-6.
Scotti C, Sommi P, Pasquetto MV, Cappelletti D, Stivala S,
Mignosi P, et al. Cell-cycle inhibition by Helicobacter pylori Lasparaginase. PLoS One 2011; 5: e13892.
Stams WA, den Boer ML, Beverloo HB, Meijerink JP, Stigter RL,
van Wering ER, et al. Sensitivity to L-asparaginase is not
associated with expression levels of asparagine synthetase in
t(12;21)+ pediatric ALL. Blood 2003; 101: 2743-7.
Krejci O, Starkova J, Otova B, Madzo J, Kalinova M, Hrusak O,
et al. Upregulation of asparagine synthetase fails to avert cell cycle
arrest induced by L-asparaginase in TEL/AML1-positive leukaemic
cells. Leukemia 2004; 18: 434-41.
Müller H, Boos J. Use of L-asparaginase in childhood ALL.
Critical Reviews in Oncology/Hematology 1998; 28: 97-113.
Covini et al.
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
Distasio JA, Salazar AM, Nadji M, Durden DL. Glutaminase-free
asparaginase from Vibrio succinogenes: An antilymphoma enzyme
lacking hepatotoxicity. Int J Cancer 1982; 30: 343-7.
Gallagher MP, Marshall RD, Wilson R. Asparaginase as a drug for
treatment of acute lymphoblastic leukaemia. Essays Biochem 1989;
24: 1-40.
Killander D, Dohlwitz A, Engstedt L, Franzén S, Gahrton G,
Gullbring B, et al. Hypersensitivity reactions and antibody
formation during L-asparaginase treatment of children and adults
with acute leukemia. Cancer 1976; 37: 220-8.
Asselin B, Whitin J, Coppola D, Rupp I, Sallan S, Cohen H.
Comparative pharmacokinetic studies of three asparaginase
preparations. J Clin Oncol 1993; 11: 1780-6.
Scotti, C., Valentini, G., Cappelletti, D., Pasquetto, M.V., Stivala S,
Chiarelli L.R. L-asparaginase from Helicobacter pylori.
WO2010015264 (2010).
Durden, D.L. Utilization of Wolinella succinogenes asparaginase to
treat diseases associated with asparagine dependence. US6991788
(2006).
Distasio JA, Niederman RA, Kafkewitz D, Goodman D.
Purification and characterization of L-asparaginase with antilymphoma activity from Vibrio succinogenes. J Biol Chem 1976;
251: 6929-33.
Distasio JA, Niederman RA, Kafkewitz D. Antilymphoma activity
of a glutaminase-free L-asparaginase of microbial origin. Proc Soc
Exp Biol Med 1977; 155: 528-31.
Roberts, J., Macallister, T.W., Sethuraman, N., Freeman, A.G.
Genetically engineered glutaminase and its use in antiviral and
anticancer therapy. US6312939 (2001).
Roberts J. Purification and properties of a highly potent antitumor
glutaminase-asparaginase from Pseudomonas 7A. J Biol Chem
1976; 251: 2119-23.
Roberts J, Schmid FA, Rosenfeld HJ. Biologic and antineoplastic
effects of enzyme-mediated in vivo depletion of L-glutamine, Ltryptophan, and L-histidine. Cancer Treat Rep 1979; 63: 1045-54.
Mononen, I., Kelo, E. Method for treatment of cancers or
inflammatory diseases. EP2155237 (2010).
Kaartinen V, Mononen T, Laatikainen R, Mononen I. Substrate
specificity and reaction mechanism of human glycoasparaginase.
The N-glycosidic linkage of various glycoasparagines is cleaved
through a reaction mechanism similar to L-asparaginase. J Biol
Chem 1992; 267: 6855-8.
Noronkoski T, Stoineva IB, Petkov DD, Mononen I. Recombinant
human glycosylasparaginase catalyzes hydrolysis of L-asparagine.
FEBS Lett 1997; 412: 149-52.
Kelo E, Noronkoski T, Mononen I. Depletion of L-asparagine
supply and apoptosis of leukemia cells induced by human
glycosylasparaginase. Leukemia 2009; 23: 1167-71.
Steckel J, Roberts J, Philips FS, Chou TC. Kinetic properties and
inhibition of Acinetobacter glutaminase-asparaginase. Biochem
Pharmacol 1983; 32: 971-7.
Kotzia GA, Labrou NE. L-Asparaginase from Erwinia
chrysanthemi 3937: Cloning, expression and characterization. J
Biotechnol 2007; 127: 657-69.
Warangkar SC, Khobragade CN. Purification, characterization, and
effect of thiol compounds on activity of the Erwinia carotovora LAsparaginase. Enzyme Res 2010; 2010: 165878.
Kotzia GA, Labrou NE. Cloning, expression and characterisation
of Erwinia carotovora L-asparaginase. J Biotechnol 2005; 119:
309-23.
Hejazi M, Piotukh K, Mattow J, Deutzmann R, Volkmer-Engert R,
Lockau W. Isoaspartyl dipeptidase activity of plant-type
asparaginases. Biochem J 2002; 364: 129-36.
Derst C, Henseling J, Rohm KH. Engineering the substrate
specificity of Escherichia coli asparaginase. II. Selective reduction
of glutaminase activity by amino acid replacements at position 248.
Protein Sci 2000; 9: 2009-17.
van der Laan, J.M., Stor, M.C., Lange De, I., Mohrmann, L. Novel
asparaginases and uses thereof. WO2008128975 (2008).
Zaccolo M, Williams DM, Brown DM, Gherardi E. An Approach
to Random Mutagenesis of DNA Using Mixtures of Triphosphate
Derivatives of Nucleoside Analogues. J Mole Biol 1996; 255: 589603.
Patel N, Krishnan S, Offman MN, Krol M, Moss CX, Leighton C,
et al. A dyad of lymphoblastic lysosomal cysteine proteases
Therapeutic L-Asparaginases
[87]
[88]
[89]
[90]
[91]
[92]
[93]
[94]
[95]
[96]
[97]
[98]
[99]
[100]
[101]
degrades the antileukemic drug L-asparaginase. J Clin Invest 2009;
119: 1964-73.
Saha, V. Enzyme. WO2007083100 (2007).
Ebeid E, Kamel M, Ali B. Detection of anti-asparaginase
antibodies during therapy with E. coli asparaginase in children with
newly diagnosed acute lymphoblastic leukemia and lymphoma. J
Egypt Natl Canc Inst 2008; 20: 127-33.
Cheung N, Chau I, Coccia P. Antibody response to Escherichia
coli L-asparaginase. Prognostic significance and clinical utility of
antibody measurement. Am J Pediatr Hematol Oncol 1986; 8: 99104.
Feng, J., Wenjie, Z., Wei, G., Chunjia, X., Ning, Z. Polyethylene
glycol modified L-asparaginyl amine enzyme. CN101082043
(2006).
Armstrong, J.K., Fisher, T.C. Poly (ethylene glycol) anti-body
detection assays and kits for performing thereof. WO2008063663
(2008).
Armstrong, J.K., Fisher, T.C. Therapeutic methods using pegconjugated agents and devices for performing the same.
US20090191175 (2009).
Martins MB, Jorge JC, Cruz ME. Acylation of L-asparaginase with
total retention of enzymatic activity. Biochimie 1990; 72: 671-5.
Cruz, M.E.M., Jorge, J., Martins, M.B., Gaspar, M.M., Esteves, A.,
Perez-soler, R. Liposomal compositions and processes for their
production. EP0485143 (1997).
van den Berg H. Asparaginase revisited. Leuk Lymphoma 2011;
52: 168-78.
Godfrin, Y. Lysis/resealing process and device for incorporating an
active ingredient in erythrocytes. WO2006016247 (2006).
Jorge JC, Perez-Soler R, Morais JG, Cruz ME. Liposomal
palmitoyl-L-asparaginase: characterization and biological activity.
Cancer Chemother Pharmacol 1994; 34: 230-4.
Gaspar MM, Perez-Soler R, Cruz ME. Biological characterization
of L-asparaginase liposomal formulations. Cancer Chemother
Pharmacol 1996; 38: 373-7.
Poznansky MJ, Shandling M, Salkie MA, Elliott JF, Lau E.
Advantages in the use of L-asparaginase-albumin polymer as an
antitumor agent. Cancer Res 1982; 42: 1020-5.
Poznansky, M.J. Targeting conjugates of albumin and therapeutic
agents. US4749570 (1988).
Nan, S., Ronggui, L. Application of rosmarinic acid in Bacillus coli
L-asparaginase activator. CN101423827 (2008).
Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1
[102]
[103]
[104]
[105]
[106]
[107]
[108]
[109]
[110]
[111]
[112]
[113]
[114]
[115]
13
Filpula, D.R., Wang, M. Recombinant host for producing LAsparaginase II. US7807436 (2010).
Blattner, F.R., Posfai, G., Herring, C.D., Plunkett, G., Glasner,
J.D., Twose, T.M. Bacteria with reduced genome. WO03070880
(2003).
Campbell, J.W., Blattner, F.R., Plunkett, G., Posfai, G. Reduced
genome E. coli. US20090075333 (2009).
Noskov, A.N., Noskov, K.A.E., Shishkova, N.A. Plasmid pNAN5
determining synthesis of L-asparaginase EcA2, Bacillus cereus
1576-pNAN5 strain - industrial strain-producer of recombinant Lasparaginase EcA2 and method for its preparing. RU2313575
(2006).
Godfrin, Y. Test for predicting neutralization of asparaginase
activity. WO2010052315 (2010).
Cantor JR, Stone EM, Chantranupong L, Georgiou G. The human
asparaginase-like protein 1 hASRGL1 is an Ntn hydrolase with
beta-aspartyl peptidase activity. Biochemistry 2009; 48: 11026-31.
Bush LA, Herr JC, Wolkowicz M, Sherman NE, Shore A,
Flickinger CJ. A novel asparaginase-like protein is a sperm
autoantigen in rats. Mol Reprod Dev 2002; 62: 233-47.
Ponomarenko J, Bui H-H, Li W, Fusseder N, Bourne P, Sette A,
et al. ElliPro: a new structure-based tool for the prediction of
antibody epitopes. BMC Bioinformatics 2008; 9: 514.
Ansari HR, Raghava GP. Identification of conformational B-cell
Epitopes in an antigen from its primary sequence. Immunome Res
2010; 6: 6.
Werner A, Rohm KH, Muller HJ. Mapping of B-cell epitopes in
E. coli asparaginase II, an enzyme used in leukemia treatment. Biol
Chem 2005; 386: 535-40.
Cantor JR, Yoo TH, Dixit A, Iverson BL, Forsthuber TG, Georgiou
G. Therapeutic enzyme deimmunization by combinatorial T-cell
epitope removal using neutral drift. Proc Natl Acad Sci USA 2011;
108: 1272-7.
Zhang YQ, Zhou WL, Shen WD, Chen YH, Zha XM, Shirai K,
et al. Synthesis, characterization and immunogenicity of silk
fibroin-L-asparaginase bioconjugates. J Biotechnol 2005; 120: 31526.
Abribat, T. Pegylated L-asparaginase. WO2011003633 (2011).
Lorenzi, P.L., Weinstein, J.N., Caplen, N.J. Materials and methods
directed to asparagine synthetase and asparaginase therapies.
US7985548 (2011).