U.S. patent application number 12/773320 was filed with the patent office on 2010-10-28 for recombinant host for producing l-asparaginase ii.
This patent application is currently assigned to DEFIANTE FARMACEUTICA, S.A.. Invention is credited to David Ray FILPULA, Maoliang WANG.
Application Number | 20100273236 12/773320 |
Document ID | / |
Family ID | 38957454 |
Filed Date | 2010-10-28 |
United States Patent
Application |
20100273236 |
Kind Code |
A1 |
FILPULA; David Ray ; et
al. |
October 28, 2010 |
RECOMBINANT HOST FOR PRODUCING L-ASPARAGINASE II
Abstract
The invention provides a recombinant Escherichia coli host cell
for producing an Escherichia coli L-asparaginase II enzyme. The
host cell includes an Escherichia coli chromosome and at least one
copy of a recombinant extrachromosomal vector, wherein the
recombinant extrachromosomal vector encodes the L-asparaginase II
enzyme, wherein the host cell chromosome also encodes the same
L-asparaginase II enzyme, and wherein the host chromosome does not
encode any other isoform of L-asparaginase II.
Inventors: |
FILPULA; David Ray;
(Piscataway, NJ) ; WANG; Maoliang; (East
Brunswick, NJ) |
Correspondence
Address: |
LUCAS & MERCANTI, LLP
475 PARK AVENUE SOUTH, 15TH FLOOR
NEW YORK
NY
10016
US
|
Assignee: |
DEFIANTE FARMACEUTICA, S.A.
Funchal-Madeira
PT
|
Family ID: |
38957454 |
Appl. No.: |
12/773320 |
Filed: |
May 4, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
11759988 |
Jun 8, 2007 |
|
|
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12773320 |
|
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60817817 |
Jun 30, 2006 |
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Current U.S.
Class: |
435/229 ;
435/252.33 |
Current CPC
Class: |
Y02A 50/30 20180101;
Y02A 50/473 20180101; A61P 35/00 20180101; C12N 9/82 20130101; A61K
38/00 20130101; C12N 9/96 20130101 |
Class at
Publication: |
435/229 ;
435/252.33 |
International
Class: |
C12N 9/82 20060101
C12N009/82; C12N 1/21 20060101 C12N001/21 |
Claims
1. A recombinant Escherichia coli host cell for producing an
Escherichia coli L-asparaginase II enzyme, comprising an
Escherichia coli host cell chromosome and at least one copy of a
recombinant extrachromosomal vector, wherein the recombinant
extrachromosomal vector encodes a subunit of the L-asparaginase II
enzyme, wherein the host cell chromosome also encodes the same
subunit of the L-asparaginase II enzyme, and wherein the host cell
chromosome does not encode any other isoform of L-asparaginase II,
wherein the encoded L-asparaginase II subunit comprises SEQ ID NO:
1.
2. The recombinant Escherichia coli host cell of claim 1, wherein
the extrachromosomal vector is a plasmid.
3. The recombinant Escherichia coli host cell of claim 1 wherein
the recombinant extrachromosomal vector comprises a DNA molecule
encoding the L-asparaginase protein, that is operatively connected
to a suitable promoter.
4. The recombinant Escherichia coli host cell of claim 3 wherein
the promoter is selected from the group consisting of T7, araB,
P.sub.R/P.sub.L, phoA, trc, and trp promoters.
5. The recombinant Escherichia coli host cell of claim 3 wherein
the recombinant extrachromosomal vector further comprises an
operator, ribosome binding site, signal sequence, transcriptional
terminator, antibiotic selection marker, origin of replication, and
a regulated copy of the repressor.
6. A method of producing a recombinant L-asparaginase II enzyme
substantially free of other L-asparaginase II isomers, comprising
culturing the host cell of claim 1, and isolating the produced
L-asparaginase II enzyme.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of U.S. patent
application Ser. No. 11/759,988 filed on Jun. 8, 2007, which claims
the benefit of priority from U.S. Provisional Patent Application
Ser. No. 60/817,817, filed on Jun. 30, 2006, the contents of each
of which are incorporated by reference herein in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to novel vectors, host cells
and methods of producing a specific recombinant E. coli
L-asparaginase II enzyme of uniform purity.
DESCRIPTION OF THE RELATED ART
[0003] L-asparaginase is an enzyme that hydrolyzes the amino acid
L-asparagine to L-aspartate and ammonia, i.e., it is a deaminating
enzyme. E. coli contain two asparaginase isoenzymes: L-asparaginase
I and L-asparaginase II. L-asparaginase I is located in the cytosol
and has a low affinity for asparagine. L-asparaginase II is located
in the periplasm and has a high affinity for L-asparagine.
[0004] L-asparaginase II is useful in treating tumors or cancers
that are dependent upon L-asparagine for protein synthesis by
removing extracellular asparagine. It is particularly useful in
treating leukemias, such as acute lymphoblastic leukemia.
L-asparaginase is typically used in combination with other
anti-tumor or anticancer therapies, although it can be employed
alone in certain clinical situations. L-asparaginase was originally
purified from several organisms, including Escherichia coli ("E.
coli") and Erwinia carotovora. Among mammals, L-asparaginase II is
found in more than trace amounts only in Guinea pigs (superfamily
Cavioidea) and in certain New World monkeys.
[0005] E. coli L-asparaginase II is a tetramer of identical
subunits exhibiting excellent k.sub.cat and K.sub.m. E. coli
L-asparaginase II (also art-known as L-asparagine amidohydrolase,
type EC-2, EC 3.5.1.1) is commercially available as Elspar.RTM.
(Merck & Co., Inc.) and is also available from Kyowa Hakko
Kogyo Co., Ltd.
[0006] L-asparaginase II, by itself, suffers from the usual
disadvantages of protein therapeutics, such as the high rate of
clearance of a protein foreign to the patient, and the potential
for inducing an immune response in a patient treated with this
enzyme. In order to address these shortcomings, a polyethylene
glycol-conjugated derivative of L-asparaginase II has been
developed and is marketed as pegaspargase or Oncaspar.RTM. by Enzon
Pharmaceuticals, Inc. Pegaspargase is produced using L-asparaginase
II extracted from E. coli, as supplied by Merck. Pegaspargase (also
known as monomethoxy polyethylene glycol succinimidyl
L-asparaginase) has the advantages of being substantially
non-antigenic, and of exhibiting a reduced rate of clearance from
the circulation.
[0007] However, despite these successes, it would be still more
efficient and economical if E. coli L-asparaginase II protein could
be produced by a recombinant host cell employing a suitable
extrachromosomal expression vector, e.g., such as a plasmid. Such
expression vectors can be engineered for more efficient production
of the protein than is available with production from a native E.
coli strain. Despite the potential advantages of such recombinant
production, it is believed that heretofore there has been no
accurate published polypeptide sequence for the commercial
L-asparaginase II enzyme, and no published nucleic acid sequence
for polynucleotides encoding that enzyme. For example, an
L-asparaginase II peptide sequence was previously reported by Maita
et al. 1980, Hoppe Seyler's Z. Physiol. Chem. 361(2), 105-117, and
Maita et al., 1974, J. Biochem. 76, 1351-1354 [Tokyo]. However, as
discussed hereinbelow, this early work suffered from numerous
sequencing errors.
[0008] Another potential obstacle to plasmid expression of the
L-asparaginase II enzyme subunit is the presence of the gene
encoding an L-asparaginase II subunit that is native to the
chromosome of potential E. coli strains that might be employed as
host cells. Thus, there is a concern that L-asparaginase II
harvested from an E. coli host cell carrying an extrachromosomal
expression vector could include subunits representing more than one
isoform of L-asparaginase. Given the need to have a well
characterized enzyme product, for both clinical and regulatory
purposes, this possibility has heretofore represented a serious
technical challenge to improving on the efficiency of the
production of E. coli L-asparaginase II protein.
SUMMARY OF THE INVENTION
[0009] The present invention fills the above-mentioned need for E.
coli L-asparaginase II that is produced efficiently and
economically in recombinant form, while providing an enzyme product
having the same peptide structure as E. coli L-asparaginase II
protein, marketed as Oncaspar.RTM., that is also free of detectable
amounts of alternative L-asparaginase II isoforms.
[0010] Thus, the invention provides an E. coli host cell comprising
an E. coli chromosome and at least one copy of a recombinant
extrachromosomal vector, wherein the extrachromosomal vector
encodes a subunit of the L-asparaginase II protein, wherein the E.
coli host cell chromosome encodes the same subunit of the
L-asparaginase protein, and wherein the E. coli host chromosome
does not encode any other isoform of L-asparaginase II. The
extrachromosomal vector is preferably a plasmid suitable for
replication and expression in E. coli.
[0011] Preferably, the expressed L-asparaginase protein comprises
four subunits that have a polypeptide sequence according to SEQ ID
NO:1, that corresponds to the sequence of the subunits of the
L-asparaginas II enzyme used in manufacturing Oncaspar.RTM., and
the plasmid vector comprises a nucleic acid molecule encoding a
subunit of the L-asparaginase protein, that is operatively
connected to a suitable promoter. The promoter is any suitable
promoter, but is optionally selected from the group consisting of
T7, araB, trp, tac, lac, .lamda.P.sub.L, .lamda.T.sub.R, aroH and
phoA promoters. The plasmid vector optionally includes additional
vector elements, as may be needed for efficient expression and/or
product purification, that are operably connected to the
L-asparaginase open reading frame and/or the promoter. These vector
elements include, for example, a compatible operator sequence,
ribosome binding site, transcriptional terminator, signal sequence,
drug resistance marker, and origin of replication. A plasmid borne
copy of the relevant repressor gene, e.g., lad, may also be
present.
[0012] Preferably, the plasmid DNA molecule encoding the subunit of
the L-asparaginase II protein comprises SEQ ID NO: 2, and the
chromosomal DNA molecule encoding the L-asparaginase II protein
comprises SEQ ID NO: 3.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 illustrates a map of the pEN537 plasmid vector.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Accordingly, in order to provide the desired improvements in
the production of the L-asparaginase II corresponding to
Oncaspar.RTM. and Kyowa Hapkko L-asparaginase, it is necessary to
obtain a vector encoding the enzyme, and also to provide a host
cell that will only express a single isoform of L-asparaginase II.
Thus, L-asparaginase II enzyme from Merck & Co., Inc., as well
as L-asparaginase II enzyme obtained from Kyowa Hakko Kogyo Co.,
Ltd. were sequenced, and the resulting sequences were compared to
that of the L-asparaginase II enzyme obtained from E. coli K-12, as
reported by Jennings et al., 1990 J Bacteriol 172: 1491-1498,
incorporated by reference herein. The K12 L-asparaginase II enzyme
is encoded by the ansB gene (GeneBank No. M34277, incorporated by
reference herein).
[0015] As noted above, the artisan will appreciate that
L-asparaginase II enzyme comprises four identical subunits. Thus,
reference to a gene or DNA molecule encoding the enzyme, and the
enzyme protein sequence, refers to the gene encoding one of these
identical subunits.
[0016] The peptide sequencing was conducted by art-standard
methods, as summarized by Example 1, hereinbelow. The protein
sequences of subunits of both the Merck & Co., Inc., and the
Kyowa Hakko Kogyo Co., Ltd. were surprisingly found to be identical
(see SEQ ID NO: 1). With this data, it can now be appreciated that
earlier reports of the sequence of the Merck L-asparaginase by
Maita et al. 1980 Hoppe Seyler's Z. Physiol. Chem. 361(2), 105-117,
and Maita et al., 1974, J. Biochem. 76, 1351-1354 [Tokyo] actually
contained numerous errors.
[0017] The obtained sequences were also compared to the subunit
structure of the K12 L-asparaginase II enzyme. It was found that
the K12 L-asparaginase II enzyme subunit differs from the Merck
& Co., Inc. L-asparaginase II enzyme subunit at four specific
residue positions. Relative to the Merck L-asparaginase II enzyme,
the K12 enzyme subunit has Val.sub.27 in place of Ala.sub.27,
Asn.sub.64 in place of Asp.sub.64, Ser.sub.252 in place of
Thr.sub.252 and Thr.sub.263 in place of Asn.sub.263.
[0018] As noted supra, it is preferred that the chromosome of the
E. coli host cell does not express a different isoform of
L-asparaginase II than is expressed by the extrachromosomal vector,
i.e., by a plasmid. This desirable result can be achieved by one of
several alternative strategies. For example, any L-asparaginase II
gene present on the E. coli host chromosome could be fully or
partially deleted or knocked out. Alternatively, the expression of
any alternative L-asparaginase II gene present on the host
chromosome could be suppressed by intrinsic regulatory properties
of the natural promoter with one that fails to allow expression
under the same culture conditions that favor the expression of the
isoform of L-asparaginase II encoded by the extrachromosomal
vector. However, it is preferable to have the chromosomal and
extrachromosomal L-asparaginase II genes express the same isoform
of the L-asparaginase II enzyme.
[0019] To this end, the subunits of the L-asparaginase II enzyme
produced by several available E. coli strains were sequenced and
compared to the commercial enzyme products. It was unexpectedly
discovered that the E. coli BLR (DE3) strain [obtained from Novagen
Corporation; Cat. No. 69208-3] produces a chromosomally encoded
L-asparaginase II enzyme identical in structure to the commercially
available enzymes, whereas the E. coli GX1210 and E. coli GX6712
strains that were also tested were found to produce different
isoforms of L-asparaginase II enzyme.
[0020] With the identification of a preferred E. coli host, an
extrachromosomal expression vector, i.e., a vector which exists as
an extrachromosomal entity, the replication of which is independent
of chromosomal replication, can be constructed. Extrachromosomal
vectors suitable for use in E. coli include, for example, pUC or
pBR322 derived plasmids. These include plasmids such as pET and
pBAD, as well as a variety of plasmids having expression elements
from T7, araBAD, phoA, trc, O.sub.L, O.sub.R, P.sub.L, P.sub.R.
[0021] In the vector, the nucleic acid sequence encoding the
L-asparaginase II enzyme subunit is operably connected to a
suitable promoter sequence. Suitable promoters include, e.g., the
T7, araBAD, phoA, trc, O.sub.L, O.sub.R, P.sub.L and P.sub.R
promoters. Preferably, the promoter is a T7 viral promoter.
[0022] Suitable inducer elements include, for example, arabinose,
lactose, or heat induction, phosphate limitation, tryptophan
limitation, to name but a few. Preferably, the inducer element is a
Lac operon, which is inducible by isopropyl thiogalactoside
("IPTG").
[0023] A suitable signal sequence (signal peptide) may be derived
from pelB, fd pIII, or ompA. Preferably the signal peptide is
derived from ansB.
[0024] Suitable antibiotic selection markers are well known to the
art and include, for example, those that confer ampicillin,
kanamycin, chloramphenicol, rifampicin, or tetracycline resistance,
among others.
[0025] Suitable origin of replication sequences include those found
in the following plasmids: pUC19, pACYC177, pUB110, pE194, pAMB1,
pIJ702, pBR322, pBR327, and pSC101.
[0026] Suitable termination sequences include, for example, phage
fd major terminator, T.PHI., and rrnB.
[0027] Generally plasmids are preferred for use in E. coli.
Conventional plasmid vectors are double-stranded circular DNA
molecules preferably engineered with enzyme recognition sites
suitable for inserting exogenous DNA sequences, an antibiotic
selectable gene, an origin of replication for autonomous
propagation in the host cell, and a gene for the discrimination or
selection of clones that contain recombinant insert DNA. Available
plasmid vectors include, for example, pET3, pET9, pET11 and the
extended pET series (cataloged by Novagen Corporation), pBAD, trc,
phoA, trp, and O.sub.L/R/P.sub.L/R plasmids
[0028] As exemplified hereinbelow, a plasmid of the pET expression
system, such as pET 27b+ is preferred. In order to provide
efficient and controlled expression of the enzyme, the expression
vector also includes a promoter, an operator, ribosome binding
site, signal sequence, transcriptional terminator, origin of
replication, a regulated copy of the repressor gene (e.g.,
lacI)
[0029] The host E. coli strain will have compatible regulatory
elements in its chromosome. For example, the gene for T7 RNA
polymerase under the control of the lacUV5 promoter is present in
BLR (DE3) cells. This strain is a lysogen of bacteriophage DE3.
Addition of IPTG to the culture of BLR (DE3) induces T7 RNA
polymerase, which in turn transcribes the target gene on the pET
plasmid. BLR(DE3) is also recA.sup.- which may provide further
stability of genes on extrachromosomal plasmids.
[0030] In order to obtain a nucleic acid molecule encoding the
Merck and Kyowa Hakko Kogyo Co., Ltd. enzyme, an available
L-asparaginase II can be modified by suitable methods. The 326
mature amino acid sequence L-asparaginase II subunit of E. coli
K-12 ansB is encoded in a 978 base pair segment as reported by
Jennings M P and Beacham I R (1990 J Bacteriol 172: 1491-1498;
GeneBank No. M34277). The ansB gene, which includes a 22 amino acid
signal peptide preceding the mature protein, was cloned from
another E. coli K-12 strain (GX1210; obtained from Genex
Corporation) by conventional polymerase chain reaction (PCR)
methods. The ansB gene encoding E. coli K-12 ansB L-asparaginase II
subunit was adapted by site-directed mutagenesis (e.g., with the
Amersham Sculptor method) to express L-asparaginase II with the
residue substitutions discussed supra, to make the following base
substitutions. T to C at base 530; A to G at base 640; T to A at
base 1205 and C to A at base 1239. Numbering is based on that given
by GeneBank No. M34277, incorporated by reference herein. The
resulting codon changes [GTG to GCG; AAT to GAT; TCT to ACT and ACC
to AAC at the corresponding positions] converted the ansB gene to a
modified gene (hereinafter ansB*; SEQ ID NO: 2) that expresses an
L-asparaginase II enzyme subunit identical to that obtained from
Merck & Co., Inc. and Kyowa Hakko Kogyo Co., Ltd.
[0031] The ansB* gene can be inserted into any extrachromosomal
vector suitable for efficient protein expression in E. coli, as
discussed above. In particular, the ansB* gene was inserted into
plasmid pET-27b+ (Novagen Corporation) and introduced into E. coli
strain BLR (DE3) by electroporation, as described in detail by the
examples provided hereinbelow, to provide an E. coli carrying the
ansB* plasmid and expressing L-asparagenase II subunit as a uniform
isoform matching the Merck L-asparaginase II.
[0032] Preferably, the clone identified by the examples as strain
EN538 (deposited as ATCC Number PTA 7490) is employed and cultured
employing any art-known method suitable for E. coli. Suitable
culture systems include batch, fed batch and continuous culture
methods. Culture medium are selected from art-known medium
optimized for E. coli. Once the culture reaches a sufficient
density, ranging from about 20 OD.sub.660 to about 200 OD.sub.660,
an appropriate inducer, such as IPTG, is added to the culture
medium. After a sufficient period of time, ranging from about 0.5
hours to about 20 hours, the produced L-asparaginase II is purified
by standard methods from the culture medium and/or from cell mass
harvested from the culture.
[0033] The cell mass is harvested by centrifugation and/or
filtration, and lysed by any art-known method. Lysis of the cell
bodies can be accomplished by methods including enzymatic cell wall
lysis followed by osmotic lysis, freeze thaw, sonication,
mechanical disruption (e.g., microfluidization), use of lysing
agents and the like, followed by filtration and/or centrifugation
to separate the disrupted cell mass from the soluble protein
contents. Several cycles of lysis, washing and separation can be
employed to optimize recovery.
[0034] The enzyme can then be recovered and purified from
supernatant and/or culture medium by well-known purification
methods including ammonium sulfate precipitation, acid extraction,
chromatofocusing, anion or cationic exchange chromatography,
phosphocellulose chromatography, hydrophobic-interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography, FPLC.RTM. (fast protein liquid chromatography),
high performance liquid chromatography, and the like.
[0035] Several parameters of the fermentation process may be
adjusted to optimize the asparaginase expression or to control the
extent of leakage of the protein from the periplasm into the growth
medium. These variables include the medium constituents (e.g.,
carbon and nitrogen source and added amino acids or other
nutrients), temperature, pH, inducer concentration, and duration of
expression. The total E. coli genetic lineage (genotype) may also
affect expression and product leakage. It may be desirable to
harvest the asparaginase product from cells (periplasm) only, or
from medium only, or from the total fermenter contents depending on
the outcome of the protein expression and leakage from the host
cells.
Polymer-L-Asparaginase Conjugates
[0036] A preferred utility for the L-asparaginase II enzyme
prepared according to the invention is in the form of a polymer
conjugated enzyme. The L-asparaginase-polymer conjugates of the
present invention generally correspond to formula (I):
(R).sub.z--NH-(ASN) (I)
[0037] wherein
[0038] (ASN) represents the L-asparaginase or a derivative or
fragment thereof;
[0039] NH-- is an amino group of an amino acid found on the ASN,
derivative or fragment thereof for attachment to the polymer;
[0040] z is a positive integer, preferably from about 1 to about
80; and
[0041] R is a substantially non-antigenic polymer residue that is
attached to the ASN in a releasable or non-releasable form.
[0042] The non-antigenic polymer residue portion of the conjugate
(R) can be selected from among a non-limiting list of polymer based
systems such as:
##STR00001## ##STR00002##
[0043] wherein:
[0044] R.sub.1-2, R.sub.10-11, and R.sub.22-23 may be the same or
different and are independently selected non-antigenic polymer
residues;
[0045] R.sub.3-9, R.sub.12-21 and R.sub.24 (see below) are the same
or different and are each independently selected from among
hydrogen, C.sub.1-6 alkyls, C.sub.3-12 branched alkyls, C.sub.3-8
cycloalkyls, C.sub.1-6 substituted alkyls, C.sub.3-8 substituted
cycloalkyls, aryls, substituted aryls, aralkyls, C.sub.1-6
heteroalkyls, substituted C.sub.1-6 heteroalkyls, C.sub.1-6 alkoxy,
phenoxy and C.sub.1-6 heteroalkoxys;
[0046] Ar is an aromatic moiety which forms a multi-substituted
aromatic hydrocarbon or a multi-substituted heteroaromatic
group;
[0047] Y.sub.1-11 and Y.sub.13 may be the same or different and are
independently selected from O, S and NR.sub.24;
[0048] A is selected from among hydrogen, alkyl groups, targeting
moieties, leaving groups, functional groups, diagnostic agents, and
biologically active moieties;
[0049] X is O, NQ, S, SO or SO.sub.2; where Q is H, C.sub.1-8
alkyl, C.sub.1-8 branched alkyl, C.sub.1-8 substituted alkyl, aryl
or aralkyl;
[0050] Z is selected from among moieties actively transported into
a target cell, hydrophobic moieties, bifunctional linking moieties
and combinations thereof;
[0051] L.sub.1-6 and L.sub.8 may be the same or different and are
independently selected bifunctional linker groups;
[0052] a, c, d, f, g, i, j, j', k, l, n, o, p, q and t may be the
same or different and are independently 0 or a positive integer,
preferably, in most aspects;
[0053] b, e, r, r', s, h, h' and m may be the same or different and
are independently 0 or 1;
[0054] mPEG is H.sub.3CO(--CH.sub.2CH.sub.2O).sub.u-- and
[0055] u is a positive integer, preferably from about 10 to about
2,300, and more preferably from about 200 to about 1000.
[0056] Within the above, it is preferred that Y.sub.1-11 and
Y.sub.13 are O; R.sub.3-8, R.sub.12-21 and R.sub.24 are each
independently either hydrogen or C.sub.1-6 alkyls, with methyl and
ethyl being the most preferred alkyls and R.sub.9 is preferably
CH.sub.3.
[0057] In a further aspect of the invention, the polymer portion of
the conjugate can be one which affords multiple points of
attachment for the L-asparaginase. A non-limiting list of such
systems include:
##STR00003##
wherein all variables are the same as that set forth above.
[0058] The activated polymers which can be employed to make the
L-asparaginase conjugates will naturally correspond directly with
the polymer portions described above. The chief difference is the
presence of a leaving or activating group, which facilitates the
releasable attachment of the polymer system to an amine group found
on the L-asparaginase. Thus, compounds (i)-(xiii) include a leaving
or activating group such as: p-nitrophenoxy, thiazolidinyl thione,
N-hydroxysuccinimidyl
##STR00004##
or other suitable leaving or activating groups such as,
N-hydroxybenzotriazolyl, halogen, N-hydroxyphthalimidyl,
imidazolyl, O-acyl ureas, pentafluorophenol or
2,4,6-tri-chlorophenol or other suitable leaving groups apparent to
those of ordinary skill, found in the place where the
L-asparaginase attaches after the conjugation reaction.
[0059] Some preferred activated PEGs include those disclosed in
commonly assigned U.S. Pat. Nos. 5,122,614, 5,324,844, 5,612,460
and 5,808,096, the contents of which are incorporated herein by
reference. As will be appreciated by those of ordinary skill such
conjugation reactions typically are carried out in a suitable
buffer using a several-fold molar excess of activated PEG. Some
preferred conjugates made with linear PEGs like the above mentioned
SC-PEG can contain, on average, from about 20 to about 80 PEG
strands per enzyme. Consequently, for these, molar excesses of
several hundred fold, e.g., 200-1000.times. can be employed. The
molar excess used for branched polymers and polymers attached to
the enzyme will be lower and can be determined using the techniques
described in the patents and patent applications describing the
same that are mentioned hereinbelow.
[0060] For purposes of the present invention, leaving groups are to
be understood as those groups which are capable of reacting with an
amine group (nucleophile) found on an L-asparaginase, e.g. on a
Lys.
[0061] For purposes of the present invention, the foregoing is also
referred to as activated polymer linkers. The polymer residues are
preferably polyalkylene oxide-based and more preferably
polyethylene glycol (PEG) based wherein the PEG is either linear or
branched.
[0062] Referring now to the activated polymers described above, it
can be seen that the Ar is a moiety which forms a multi-substituted
aromatic hydrocarbon or a multi-substituted heteroaromatic group. A
key feature is that the Ar moiety is aromatic in nature. Generally,
to be aromatic, the .pi. (pi) electrons must be shared within a
"cloud" both above and below the plane of a cyclic molecule.
Furthermore, the number of n electrons must satisfy the Huckle rule
(4n+2). Those of ordinary skill will realize that a myriad of
moieties will satisfy the aromatic requirement of the moiety and
thus are suitable for use herein with halogen(s) and/or side chains
as those terms are commonly understood in the art.
[0063] In some preferred aspects of the invention, the activated
polymer linkers are prepared in accordance with commonly-assigned
U.S. Pat. Nos. 6,180,095, 6,720,306, 5,965,119, 6624,142 and
6,303,569, the contents of which are incorporated herein by
reference. Within this context, the following activated polymer
linkers are preferred:
##STR00005## ##STR00006##
[0064] In one alternative aspect of the invention, the
L-asparaginase polymer conjugates are made using certain branched
or bicine polymer residues such as those described in commonly
assigned U.S. Pat. Nos. 7,122,189 and 7,087,229 and U.S. patent
application Ser. Nos. 10/557,522, 11/502,108, and 11/011,818. The
disclosure of each such patent application is incorporated herein
by reference. A few of the preferred activated polymers
include:
##STR00007##
It should also be understood that the leaving group shown above is
only one of the suitable groups and the others mentioned herein can
also be used without undue experimentation.
[0065] In alternative aspects, the activated polymer linkers are
prepared using branched polymer residues such as those described
commonly assigned U.S. Pat. Nos. 5,643,575; 5,919,455 and 6,113,906
and 6,566,506, the disclosure of each being incorporated herein by
reference. Such activated polymers correspond to polymer systems
(v)-(ix) with the following being representative:
##STR00008##
wherein B is L-asparaginase II and all other variables are as
previously defined.
Substantially Non-Antigenic Polymers
[0066] As stated above, R.sub.1-2, R.sub.10-11, and R.sub.22-23 are
preferably each water soluble polymer residues which are preferably
substantially non-antigenic such as polyalkylene oxides (PAO's) and
more preferably polyethylene glycols such as mPEG. For purposes of
illustration and not limitation, the polyethylene glycol (PEG)
residue portion of R.sub.1-2, R.sub.10-11, and R.sub.22-23 can be
selected from among:
J-O--(CH.sub.2CH.sub.2O).sub.u--
J-O--(CH.sub.2CH.sub.2O).sub.u--CH.sub.2C(O)--O--,
J-O--(CH.sub.2CH.sub.2O).sub.u--CH.sub.2CH.sub.2NR.sub.25--,
and
J-O--(CH.sub.2CH.sub.2O).sub.u--CH.sub.2CH.sub.2SH--,
[0067] wherein:
[0068] u is the degree of polymerization, i.e. from about 10 to
about 2,300;
[0069] R.sub.25 is selected from among hydrogen, C.sub.1-6 alkyls,
C.sub.2-6 alkenyls, C.sub.2-6 alkynyls, C.sub.3-12 branched alkyls,
C.sub.3-8 cycloalkyls, C.sub.1-6 substituted alkyls, C.sub.2-6
substituted alkenyls, C.sub.2-6 substituted alkynyls, C.sub.3-8
substituted cycloalkyls, aryls substituted aryls, aralkyls,
C.sub.1-6 heteroalkyls, substituted C.sub.1-6 heteroalkyls,
C.sub.1-6 alkoxy, phenoxy and C.sub.1-6 heteroalkoxy, and
[0070] J is a capping group, i.e. a group which is found on the
terminal of the polymer and, in some aspects, can be selected from
any of NH.sub.2, OH, SH, CO.sub.2H, C.sub.1-6 alkyls, preferably
methyl, or other PEG terminal activating groups, as such groups are
understood by those of ordinary skill.
[0071] In one particularly preferred embodiment, R.sub.1-2,
R.sub.10-11, and R.sub.22-23 are selected from among,
CH.sub.3--O--(CH.sub.2CH.sub.2O).sub.u--,
CH.sub.3--O--(CH.sub.2CH.sub.2O).sub.u--CH.sub.2C(O)--O--, and
CH.sub.3--O--(CH.sub.2CH.sub.2O).sub.u--CH.sub.2CH.sub.2NH-- and
CH.sub.3--O--(CH.sub.2CH.sub.2O).sub.u--CH.sub.2CH.sub.2SH--,
[0072] where u is a positive integer, preferably selected so that
the weight average molecular weight from about 200 to about 80,000
Da. More preferably, R.sub.1-2, R.sub.10-11, and R.sub.22-23
independently have an average molecular weight of from about 2,000
Da to about 42,000 Da, with an average molecular weight of from
about 5,000 Da to about 40,000 Da being most preferred. Other
molecular weights are also contemplated so as to accommodate the
needs of the artisan.
[0073] PEG is generally represented by the structure:
##STR00009##
and R.sub.1-2, R.sub.10-11, and R.sub.22-23 preferably comprise
residues of this formula. The degree of polymerization for the
polymer represents the number of repeating units in the polymer
chain and is dependent on the molecular weight of the polymer.
[0074] Also useful are polypropylene glycols, branched PEG
derivatives such as those described in commonly-assigned U.S. Pat.
No. 5,643,575 (the '575 patent), "star-PEG's" and multi-armed PEG's
such as those described in Shearwater Corporation's 2001 catalog
"Polyethylene Glycol and Derivatives for Biomedical Application".
The disclosure of each of the foregoing is incorporated herein by
reference. The branching afforded by the '575 patent allows
secondary or tertiary branching as a way of increasing polymer
loading on a biologically active molecule from a single point of
attachment. It will be understood that the water-soluble polymer
can be functionalized for attachment to the bifunctional linkage
groups if required without undue experimentation.
[0075] For example, the conjugates of the present invention can be
made by methods which include converting the multi-arm PEG-OH or
"star-PEG" products such as those described in NOF Corp. Drug
Delivery System catalog, Ver. 8, April 2006, the disclosure of
which is incorporated herein by reference, into a suitably
activated polymer, using the activation techniques described in the
aforementioned '614 or '096 patents. Specifically, the PEG can be
of the formula:
##STR00010##
[0076] wherein:
[0077] u' is an integer from about 4 to about 455, to preferably
provide polymers having a total molecular weight of from about
5,000 to about 40,000; and up to 3 terminal portions of the residue
is/are capped with a methyl or other lower alkyl.
[0078] In some preferred embodiments, all 4 of the PEG arms are
converted to suitable leaving groups, i.e. N-hydroxysuccinimidyl
carbonate (SC), etc., for facilitating attachment to the
recombinant protein. Such compounds prior to conversion
include:
##STR00011## ##STR00012##
[0079] The polymeric substances included herein are preferably
water-soluble at room temperature. A non-limiting list of such
polymers include polyalkylene oxide homopolymers such as
polyethylene glycol (PEG) or polypropylene glycols,
polyoxyethylenated polyols, copolymers thereof and block copolymers
thereof, provided that the water solubility of the block copolymers
is maintained.
[0080] In a further embodiment, and as an alternative to PAO-based
polymers, R.sub.1-2, R.sub.10-11, and R.sub.22-23 are each
optionally selected from among one or more effectively
non-antigenic materials such as dextran, polyvinyl alcohols,
carbohydrate-based polymers, hydroxypropylmeth-acrylamide (HPMA),
polyalkylene oxides, and/or copolymers thereof See also
commonly-assigned U.S. Pat. No. 6,153,655, the contents of which
are incorporated herein by reference. It will be understood by
those of ordinary skill that the same type of activation is
employed as described herein as for PAO's such as PEG. Those of
ordinary skill in the art will further realize that the foregoing
list is merely illustrative and that all polymeric materials having
the qualities described herein are contemplated and that other
polyalkylene oxide derivatives such as the polypropylene glycols,
etc. are also contemplated.
Bifunctional Linker Groups:
[0081] In many aspects of the invention, L.sub.1-6 and L.sub.8 are
linking groups which facilitate attachment of the polymer strands,
e.g. R.sub.1-2, R.sub.10-11, and/or R.sub.22-23. The linkage
provided can be either direct or through further coupling groups
known to those of ordinary skill. In this aspect of the invention,
L.sub.1-6 and L.sub.8 may be the same or different and can be
selected from a wide variety of groups well known to those of
ordinary skill such as bifunctional and heterobifunctional
aliphatic and aromatic-aliphatic groups, amino acids, etc. Thus,
L.sub.1-6 and L.sub.8 can be the same or different and include
groups such as:
--[C(.dbd.O)].sub.v'(CR.sub.32R.sub.33).sub.t'--,
--[C(.dbd.O)].sub.v'O(CR.sub.32R.sub.33).sub.t'O--,
--[C(.dbd.O)].sub.v'O(CR.sub.32R.sub.33).sub.t'N.sup.R.sub.36--,
--[C(.dbd.O)].sub.v'O(CR.sub.32R.sub.33O).sub.t'NR.sub.36--,
--[C(.dbd.O)].sub.v'NR.sub.31(CR.sub.32R.sub.33).sub.t'--,
--[C(.dbd.O)].sub.v'NR.sub.31(CR.sub.32R.sub.33).sub.t'O--,
--[C(.dbd.O)].sub.v'NR.sub.31(CR.sub.32R.sub.33O).sub.t'--,
--[C(.dbd.O)].sub.v'NR.sub.31(CR.sub.32R.sub.33O).sub.t'(CR.sub.34R.sub.-
35).sub.y'--,
--[C(.dbd.O)].sub.v'NR.sub.31(CR.sub.32R.sub.33O).sub.t'(CR.sub.34R.sub.-
35).sub.y'O--,
--[C(.dbd.O)].sub.v'NR.sub.31(CR.sub.32R.sub.33).sub.t'(CR.sub.34CR.sub.-
35O).sub.y'--,
--[C(.dbd.O)].sub.v'NR.sub.31(CR.sub.32R.sub.33).sub.t'(CR.sub.34CR.sub.-
35O).sub.y'NR.sub.36--,
--[C(.dbd.O)].sub.v'NR.sub.31(CR.sub.32R.sub.33).sub.t'NR.sub.36--,
##STR00013##
[0082] wherein:
[0083] R.sub.31-R.sub.37 are independently selected from the group
consisting of hydrogen, amino, substituted amino, azido, carboxy,
cyano, halo, hydroxyl, nitro, silyl ether, sulfonyl, mercapto,
C.sub.1-6 alkylmercapto, arylmercapto, substituted arylmercapto,
substituted C.sub.1-6 alkylthio, C.sub.1-6 alkyls, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.3-19 branched alkyl, C.sub.3-8
cycloalkyl, C.sub.1-6 substituted alkyl, C.sub.2-6 substituted
alkenyl, C.sub.2-6 substituted alkynyl, C.sub.3-8 substituted
cycloalkyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, C.sub.1-6 heteroalkyl, substituted C.sub.1-6
heteroalkyl, C.sub.1-6 alkoxy, aryloxy, C.sub.1-6 heteroalkoxy,
heteroaryloxy, C.sub.2-6 alkanoyl, arylcarbonyl, C.sub.2-6
alkoxycarbonyl, aryloxycarbonyl, C.sub.2-6 alkanoyloxy,
arylcarbonyloxy, C.sub.2-6 substituted alkanoyl, substituted
arylcarbonyl, C.sub.2-6 substituted alkanoyloxy, substituted
aryloxycarbonyl, C.sub.2-6 substituted alkanoyloxy, substituted and
arylcarbonyloxy, [0084] wherein the substituents are selected from
the group consisting of acyl, amino, amido, amidine, araalkyl,
aryl, azido, alkylmercapto, arylmercapto, carbonyl, carboxylate,
cyano, ester, ether, formyl, halogen, heteroaryl, heterocycloalkyl,
hydroxy, imino, nitro, thiocarbonyl, thioester, thioacetate,
thioformate, alkoxy, phosphoryl, phosphonate, phosphinate, silyl,
sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamide, and
sulfonyl;
[0085] (t') and (y') are independently selected from zero or
positive integers , preferably 1 to 6; and
[0086] (v') is 0 or 1.
Preferably, L.sub.1-6 and L.sub.8 are selected from among:
--C(O)CH.sub.2OCH.sub.2C(O)--;
--C(O)CH.sub.2NHCH.sub.2C(O)--;
--C(O)CH.sub.2SCH.sub.2C(O)--;
--C(O)CH.sub.2CH.sub.2CH.sub.2C(O)--, and
--C(O)CH.sub.2CH.sub.2C(O)--.
[0087] Alternatively, suitable amino acid residues can be selected
from any of the known naturally-occurring L-amino acids is, e.g.,
alanine, valine, leucine, etc. and/or a combination thereof, to
name but a few. L.sub.1-6 and L.sub.8 can also include a peptide
which ranges in size, for instance, from about 2 to about 10 amino
acid residues.
[0088] Derivatives and analogs of the naturally occurring amino
acids, as well as various art-known non-naturally occurring amino
acids (D or L), hydrophobic or non-hydrophobic, are also
contemplated to be within the scope of the invention.
A Moieties
[0089] 1. Leaving or Activating Groups
[0090] In those aspects where A is an activating group, suitable
moieties include, without limitation, groups such as
N-hydroxybenzotriazolyl, halogen, N-hydroxyphthalimidyl,
p-nitrophenoxyl, imidazolyl, N-hydroxysuccinimidyl; thiazolidinyl
thione, O-acyl ureas, pentafluorophenoxyl, 2,4,6-trichlorophenoxyl
or other suitable leaving groups that will be apparent to those of
ordinary skill.
[0091] For purposes of the present invention, leaving groups are to
be understood as those groups which are capable of reacting with a
nucleophile found on the desired target, i.e. a biologically active
moiety, a diagnostic agent, a targeting moiety, a bifunctional
spacer, intermediate, etc. The targets thus contain a group for
displacement, such as NH.sub.2 groups found on proteins, peptides,
enzymes, naturally or chemically synthesized therapeutic molecules
such as doxorubicin, spacers such as mono-protected diamines. It is
to be understood that those moieties selected for A can also react
with other moieties besides biologically active nucleophiles.
[0092] 2. Functional Groups
[0093] A can also be a functional group. Non-limiting examples of
such functional groups include maleimidyl, vinyl, residues of
sulfone, hydroxy, amino, carboxy, mercapto, hydrazide, carbazate
and the like which can be attached to the bicine portion through an
amine-containing spacer. Once attached to the bicine portion, the
functional group, (e.g. maleimide), can be used to attach the
bicine-polymer to a target such as the cysteine residue of a
polypeptide, amino acid or peptide spacer, etc.
[0094] 3. Alkyl Groups
[0095] In those aspects of formula (I) where A is an alkyl group, a
non-limiting list of suitable groups consists of C.sub.1-6 alkyls,
C.sub.2-6 alkenyls, C.sub.2-6 alkynyls, C.sub.3-19 branched alkyls,
C.sub.3-8 cycloalkyls, C.sub.1-6 substituted alkyls, C.sub.2-6
substituted alkenyls, C.sub.2-6 substituted alkynyls, C.sub.3-8
substituted cycloalkyls, aralkyls, C.sub.1-6 heteroalkyls, and
substituted C.sub.1-6 heteroalkyls.
Z Moieties and Their Function
[0096] In one aspect of the invention Z is L.sub.7-C(.dbd.Y.sub.12)
wherein L.sub.7 is a bifunctional linker selected from among the
group which defines L.sub.1-6, and Y.sub.12 is selected from among
the same groups as that which defines Y.sub.1. In this aspect of
the invention, the Z group serves as the linkage between the
L-asparaginase and the remainder of the polymer delivery system. In
other aspects of the invention, Z is a moiety that is actively
transported into a target cell, a hydrophobic moiety, and
combinations thereof. The Z' when present can serve as a
bifunctional linker, a moiety that is actively transported into a
target cell, a hydrophobic moiety, and combinations thereof.
[0097] In this aspect of the invention, the releasable polymer
systems are prepared so that in vivo hydrolysis cleaves the polymer
from the L-asparaginase and releases the enzyme into the
extracellular fluid, while still linked to the Z moiety. For
example, one potential Z--B combination is
leucine-L-asparaginase
Preparation of L-Asparaginase Conjugates
[0098] For purposes of illustration, suitable conjugation reactions
include reacting L-asparaginase with a suitably activated polymer
system described herein. The reaction is preferably carried out
using conditions well known to those of ordinary skill for protein
modification, including the use of a PBS buffered system, etc. with
the pH in the range of about 6.5-8.5. It is contemplated that in
most instances, an excess of the activated polymer will be reacted
with the L-asparaginase.
[0099] Reactions of this sort will often result in the formation of
conjugates containing one or more polymers attached to the
L-asparaginase. As will be appreciated, it will often be desirable
to isolate the various fractions and to provide a more homogenous
product. In most aspects of the invention, the reaction mixture is
collected, loaded onto a suitable column resin and the desired
fractions are sequentially eluted off with increasing levels of
buffer. Fractions are analyzed by suitable analytical tools to
determine the purity of the conjugated protein before being
processed further. Regardless of the synthesis route and activated
polymer selected, the conjugates will conform to Formula (I) as
defined herein. Some of the preferred compounds which result from
the synthetic techniques described herein include:
##STR00014##
wherein B is L-asparaginase.
[0100] Still further conjugates made in accordance with the present
invention include:
##STR00015##
wherein all variables are the same as that set forth above. For
example, some of embodiments included in the conjugates are
selected from the group consisting of:
##STR00016## ##STR00017##
wherein B is L-asparaginase.
[0101] Further conjugates include:
##STR00018##
wherein B is L-asparaginase. A non-limiting list employed in the
conjugates are among
##STR00019##
wherein B is L-asparaginase.
[0102] A particularly preferred conjugate is:
##STR00020##
wherein the molecular weight of the mPEG is from about 10,000 to
about 40,000.
[0103] When the bicine-based polymer systems are used, two
preferred conjugates are:
##STR00021##
wherein the molecular weights of the mPEG are the same as
above.
[0104] It is noted that PEGylation of L-asparaginase will be
empirically optimized for total PEG attachments per protein, PEG
polymer size, and PEG linker design. Key characteristics of the
PEGylated L-asparaginase for evaluation of PEGylation optimization
include both in vitro assays (e.g., enzyme activity and stability)
and in vivo assays (e.g., pharmacokinetics and
pharmacodynamics).
Methods of Treatment
[0105] The L-asparaginase produced by the DNA, vectors and host
cells described herein is useful for all of the methods and
indications already art-known for Elspar.RTM. (Merck & Co.,
Inc.) and Oncaspar.RTM. (Enzon Pharmaceuticals, Inc.). Thus, the
inventive L-asparaginase II enzyme, whether polyalkylene oxide
conjugated, or as an unconjugated protein is administered to a
patient in need thereof in an amount that is effective to treat a
disease or disorder or other condition that is responsive to such
treatment. The artisan will appreciate suitable amounts, routes of
administration and dosing schedules extrapolated from the known
properties of Elspar.RTM. and Oncaspar.RTM..
Examples
[0106] The following non-limiting examples set forth hereinbelow
illustrate certain aspects of the invention.
Example 1
Sequencing of L-Asparagine Amidohydrolase, Type EC-2, EC 3.5.1.1;
E. coli L-Asparaginase II Protein
[0107] In order to obtain the amino acid sequences of the
L-asparaginase II enzymes commercially available from Merck &
Co. and Kyowa Hakko Kogyo Co., respectively, these proteins were
subject to protein sequence analysis and compared to the sequence
of the published E. coli K-12 ansB gene (GenBank Accession Number
M34277).
[0108] L-asparaginase II was sequenced as follows. An aliquot of 2
mL of L-asparaginase II (80 mg/mL; Merck) was diluted in reagent
grade water to yield a diluted solution with a protein
concentration of 5.0 mg/mL. The diluted solution was filtered
through a 0.22 .mu.m filter into vials in order to reduce bioburden
before conducting the protein sequence analysis. Similarly, 100 mg
of L-asparaginase II (Kyowa Hakko Kogyo) was dissolved in 20 mL of
reagent grade water to yield a diluted solution of 5.6 mg/mL and
sterile filtered. Quantitative amino acid analyses, N-terminal
sequence determinations, peptide mapping, and mass spectrometry
were used to determine the complete sequences of the two proteins.
Tryptic digest, chymotryptic digest, Lys-C digest and cyanogen
bromide (CnBr) fragments were prepared and separated by high
pressure liquid chromatography ("HPLC"), and mass spectrometry and
amino acid sequencing were performed on the isolated peptides. The
completed analyses demonstrated an apparent sequence identity
between the two commercial L-asparaginase II enzymes. However, four
amino acid positions differed from the gene sequence derived
asparaginase from E. coli K-12. These four differing positions are
shown by Table 1, below.
TABLE-US-00001 TABLE 1 Residue Position 27 64 252 263 Merck and Ala
Asp Thr Asn KH K12 AnsB Val Asn Ser Thr
Example 2
Construction of E. coli Strain EN538 Expressing Recombinant
L-Asparaginase II
[0109] The gene encoding E. coli K-12 ansB L-asparaginase II was
adapted to express L-asparaginase II with the residue substitutions
illustrated by Table 1 of Example 1, as follows. The 326 mature
amino acid sequence L-asparaginase II of E. coli K-12 ansB is
encoded in a 978 base pair segment as reported by Jennings M P and
Beacham I R (1990 J Bacteriol 172: 1491-1498; GeneBank No. M34277).
The ansB gene, which includes a 22 amino acid signal peptide
preceding then mature protein, was cloned from another E. coli K-12
strain (GX1210; obtained from Genex Corporation) by conventional
polymerase chain reaction (PCR) methods. Specifically, the
oligonucleotides 5'-TACTGAATTCATGGAGTTTTTCAAAAAGACGGCA-3' (SEQ ID
NO: 4) and 5'-ACAGTAAGCTTAGTACTGATTGAAGATCTGCTG-3' (SEQ ID NO: 5)
were employed as primers using a Perkin Elmer Gene Amp 9600
thermocycler, Taq polymerase, and standard reagents with these
cycling parameters: 30 sec 94.degree. C., 30 sec 40.degree. C., 1
min 72.degree. C., for 25 cycles.
[0110] The amplified .about.1 kb band was purified on TBE agarose
gel electrophoresis, digested with Eco RI and Hind III, and cloned
into the bacteriophage vector M13mp8. The DNA sequence of the ansB
gene [Genebank No. M34277] was confirmed by manual DNA dideoxy
sequencing methods. The cloned ansB gene was used next in
site-directed mutagenesis to change four codons of ansB gene [GTG
to GCG at base 530; AAT to GAT at base 640; TCT to ACT at base 1205
and ACC to AAC at base 1239] to encode the alternate amino acids
(Val27Ala; Asn64Asp; Ser252Thr; and Thr263Asn) using the Amersham
RPN 1523 version 2 mutagenesis kit as described by Whitlow and
Filpula [Single Chain Fvs, In Tumour Immunology. A Practical
Approach, Ed. G. Gallagher, R. C. Rees, and C. W. Reynolds, 1993,
Oxford University Press, pp 279-291].
[0111] Specifically, mutagenic oligonucleotides employed were
5'-CAACTTTACCCGCTGTGTAGTTAG-3' (SEQ ID NO: 6) for Val27Ala change;
5'-CAGCCAGACATCATCGTTCATGTC-3' (SEQ ID NO: 7) for Asn64Asp change;
5'-GTCGAACACAGTTTTATACAGGTTGC-3' (SEQ ID NO: 8) for Ser252Thr
change; 5'-CTGCAGTACCGTTTTTCGCGGCGG-3' (SEQ ID NO: 9) for Thr263Asn
change. All four changes were made in a single batch and DNA
sequencing confirmed the modified ansB gene sequence [designated
herein as the ansB* gene (SEQ ID NO: 2)].
[0112] Cloning of the ansB* gene into plasmid pET-27b+ (Novagen
Corporation) was accomplished by introducing the flanking
restriction sites, NdeI and BamHI, at the 5' and 3' termini of the
gene, respectively, by PCR amplification. Following digestion of
the synthetic DNA with the restriction enzymes NdeI and BamHI, the
1 kilobase gene was ligated via T4 DNA ligase into the plasmid
vector pET-27b(+) plasmid which had also been digested with these
two enzymes. The recombinant plasmid was introduced into E. coli
strain BLR (DE3) by electroporation using a BTX Electro Cell
Manipulator 600 according to the manufacturer's instructions.
[0113] The pET vector construction places the ansB* gene behind a
T7 promoter which is inducible as a consequence of IPTG addition.
IPTG induces expression of the chromosomal T7 RNA polymerase gene
under the control of a lacUV5 promoter and the T7 RNA polymerase
then transcribes the ansB* gene yielding high level expression of
the ansB* protein product.
[0114] The transformation mixture was plated on LB agar plates
containing kanamycin (15 .mu.g/ml) to allow for selection of
colonies containing the plasmid pET-27b(+)/ansB*. This is
designated as plasmid pEN537, as illustrated by FIG. 1. Isolated
colonies were further purified by plating and analyzed for IPTG
inducible gene expression by standard methods such as those
described in Novagen pET System Manual Ninth Edition. The gene
sequences were verified using an Applied Biosystems Prism310
Genetic Analyzer.
Example 3
Expression of Recombinant L-Asparaginase II and Partial
Characterization of the Enzyme
[0115] Strain EN538 was cultured in LB medium at 37.degree. C. with
kanamycin (15 .mu.g/ml). At OD.sub.600 of about 0.8, IPTG (1 mM)
was added to the culture and induction of gene expression was
allowed to progress for either 2, 3, or 4 hr. SDS-PAGE analysis of
the culture confirmed high level expression of the 34.6 kDa ansB*
polypeptide. Western blotting using anti-E. coli asparaginase II
rabbit polyclonal antibody confirmed that the major induced protein
band on SDS-PAGE was L-asparaginase II.
[0116] Since L-asparaginase II is normally secreted into the
periplasmic space following signal peptide removal, additional
experiments were conducted to examine location of the asparaginase
in the cells or medium. The culture was centrifuged and the
pelleted cells were resuspended in a lysozyme solution to disrupt
the cell walls before examining the soluble and insoluble cell
associated proteins, plus the proteins released into the growth
medium during culture, by SDS-PAGE.
[0117] These analyses demonstrated that either a 3 or 4 hr
induction at 37.degree. C. provides near maximal ansB* expression
of about 30% of total cell proteins. At least 70% of the
asparaginase can be solubilized from the cell pellet by lysozyme
treatment. The amount of asparaginase released into the growth
medium during culture is about 25% of the total asparaginase
expressed.
[0118] The solubilized asparaginase released from the periplasm by
lysozyme treatment was further examined for enzyme activity using
an RP-HPLC assay that measures aspartic acid, the product of the
asparaginase reaction from the substrate, asparagine. Enzyme
activity in crude extracts from the IPTG induced samples was about
60 IU/mg, while only about 2 IU/mg in samples prepared from
uninduced cultures. Since the protein is only about 20% pure at
this stage, this compares well to the reported specific activity of
pure asparaginase II (.about.250-300 IU/mg). N-terminal sequence
analysis of this asparaginase preparation was also achieved using
an Applied BioSystems PROCISE protein sequencer. The N-terminal
sequence LPNITILATGGTIAGGGDSA (SEQ ID NO: 10) matches exactly the
predicted N-terminal protein sequence of mature, correctly
processed, asparaginase. LC-MS analysis (Jupiter C-18 revered-phase
column) was also performed on this sample. The principal protein
species demonstrated a mass of 34,592 which exactly matches the
predicted mass as mature ansB* asparaginase. No evidence of a
protein species bearing norleucine substitutions was observed.
Example 4
Protein Coding Sequences of L-Asparaginase II (ansB & ansB*
Genes) from pEN537 Plasmid and E. coli BLR Chromosome
[0119] Chromosomal DNA was prepared from E. coli BLR (DE3)
[obtained from Novagen Corporation; Cat. No. 69208-3]. A 2 ml
culture of BLR grown in LB medium with kanamycin (15 .mu.g/ml) at
37.degree. C. was centrifuged for 2 min in a microfuge and cell
pellet was resuspended in 0.5 ml of STET buffer. Phenol/chloroform
(0.5 ml) was added and the mixture was vortexed and centrifuged for
5 min at room temperature. The supernatant was collected and mixed
with 50 .mu.l of 3 M sodium acetate and 1 ml of ethanol. After
incubating on ice for 10 min, the DNA was pelleted by
centrifugation and resuspended in 100 .mu.l of water. PCR was
conducted on the sample to isolate the chromosomal ansB gene. The
PCR reaction mixture contained 5 .mu.l of 10.times. High Fidelity
PCR buffer, 5 .mu.l of 10 mM dNTP mixture, 1 .mu.l of 50 mM
MgSO.sub.4, 0.5 .mu.l (50 pmol) of oligonucleotide
TABLE-US-00002 (SEQ ID NO: 11)
5'-GATCCATATGGAGTTTTTCAAAAAGACGGCAC-3',
0.5 .mu.l (50 pmol) of oligonucleotide
TABLE-US-00003 (SEQ ID NO: 12)
5'-GTACGGATCCTCATTAGTACTGATTGAAGATC-3',
1 .mu.l of BLR DNA, 36 .mu.l of distilled water, and 1 .mu.l of
Platinum Taq High Fidelity polymerase. The PCR product was cloned
using the commercial TOPO cloning system obtained from Invitrogen
Corporation and conducted as described by the manufacturer.
[0120] The cloning reaction using the PCR product and the TOPO TA
vector was conducted in 6 .mu.l at room temperature for 30 min. The
ligation product of the reaction was transformed in competent TOP10
E. coli cells and plated on LB agar plates with kanamycin
selection. DNA sequence analysis of the cloned ansB BLR chromosomal
gene and the pEN537 ansB* gene was conducted on the plasmids using
an Applied Biosystems Prism 310 Genetic Analyzer. Both strands were
sequenced. The coding sequences of the BLR ansB gene and pEN537
ansB* gene differ by 29 mismatched base assignments in the mature
protein coding sequences. However, none of these base substitutions
resulted in an alteration in the amino acid sequence due to codon
degeneracy. The encoded ansB protein from BLR and the encoded ansB*
protein from pEN537 was confirmed to be identical in amino acid
sequence. All 326 positions were shown to be identical in these two
asparaginase proteins.
Example 5
Purification from Cells and Culture Medium
[0121] The following process was adapted from Harms et al., 1991
Protein Expression and Purification 2: 144-150.
[0122] Cultures of E. coli strain EN538, as described above, are
grown in Luria broth in the presence of kanamycin (15 .mu.g/ml) at
37.degree. C., in a shaker incubator. At an OD.sub.660 of 0.8, IPTG
is added to a final concentration of 1 mM, and growth continued for
an additional 4 h. Cells are harvested by centrifugation. For
analytical purposes, 2-ml cultures are used.
[0123] To make cell extracts, the pellets are suspended in 1 ml
disruption buffer (50 mM KPO, pH 7.5, 1 mM EDTA, 0.5 mM
dithiothreitol] and cells disrupted by microfluidization. Cell
debris is removed by centrifugation and the supernatant fluid is
assayed for L-asparaginase II activity and also used to assess
enzyme production by polyacrylamide gel electrophoresis (SDS PAGE).
Osmotic shock fractionation is carried out as described by Boyd et
al., 1987, Proc. Natl. Acad. Sci. USA 84:8525-8529, incorporated by
reference herein. In brief, the pellet is suspended in 2 ml
spheroblast buffer (0.1 M Tris-HCl, pH 8.0, 0.5 M sucrose, 0.5 mM
EDTA), incubated on ice for 5 min, and centrifuged. The pellet is
warmed to room temperature, resuspended in 0.3 ml ice-cold water,
incubated on ice for 5 min, and again centrifuged. The supernatant
periplasmic fraction is used without further treatment for activity
determination and electrophoresis.
Enzyme Purification
[0124] For large-scale L-asparaginase II preparations cells are
grown in batch cultures (10 liters) and subjected to osmotic shock
as above. Per liter of culture volume 50-100 ml spheroblast buffer
and 30-40 ml water are employed. The following protocol starts with
the periplasmic extract obtained from a 2-liter culture. All steps
are performed at 5-10.degree. C.
Ammonium Sulfate Fractionation
[0125] To 100 ml of supernatant fluid 29.5 g solid ammonium sulfate
is added to give 50% saturation. After 2 hours the precipitate is
removed by centrifugation, and the pellet discarded. The
supernatant was brought to 90% saturation with ammonium sulfate
(27.2 g to 100 ml). After the pellet stood overnight it is
collected by centrifugation, dissolved in a few milliliters of 25
mM piperazine-HCl buffer, pH 5.5, and dialyzed against the same
buffer. This same process is also applied to the remaining cell
culture medium to recover secreted L-asparaginase II.
Chromatofocusing
[0126] A 1.times.30-cm column of Poly-buffer exchanger PBE 94 was
equilibrated with 200 ml of the above piperazine-HCl buffer
(starting buffer). After the sample solution (10 ml) is applied,
the column is eluted with 200 ml elution buffer (Polybuffer 74,
diluted 10-fold with H.sub.20 and adjusted to pH 4.0 with HCl) at a
flow rate of 30 ml/h. Fractions of 2 ml are collected and assayed
for L-asparaginase II activity after appropriate dilution of
20-.mu.l samples. The asparaginase-containing fractions are pooled
and dialyzed against saturated ammonium sulfate solution. The
enzyme pellet is washed with 90% ammonium sulfate and stored as a
suspension in this medium.
Example 6
Purification from Cells and Culture Medium
[0127] Cultures of E. coli strain EN538, as described above, are
grown in culture medium [e.g., as described in Filpula, D.,
McGuire, J. and Whitlow, M. (1996) Production of single-chain Fv
monomers and multimers, In Antibody Engineering: A Practical
Approach (J. McCafferty, H. Hoogenboom, and D. J. Chiswell, eds.;
Oxford University Press, Oxford, UK) pp. 253-268] in the presence
of kanamycin (15 .mu.g/ml) at 25.degree. C. to 37.degree. C., in a
fermenter. At an OD.sub.660 of 20 to 200, IPTG is added to a final
concentration of 0.1-1 mM, and growth continued for an additional
1-12 h. Cells are harvested by centrifugation and passed through a
Manton-Gaulin cell homogenizer. The cell lysate is centrifuged at
24,300 g for 30 min at 6.degree. C. and the supernatant is
collected and subjected to ultrafiltration/diafiltration, and the
conductivity is adjusted to 3 mS. The pH of the lysate is adjusted
to 4.1 with 25% acetic acid and diafiltered with buffer 5 mM sodium
acetate, 25 mM NaCl, pH 4.1.
[0128] The asparaginase is captured on S-Sepharose cation exchange
column chromatography. The bound asparaginase is eluted with 12.5
mM potassium phosphate, 25 mM NaCl, pH 6.4 (buffer NK64).
[0129] The collected asparaginase peak fractions from S-Sepharose
chromatography are pooled and 0.1% Tween80 is added and incubated
for 20 min at room temperature. One volume of buffer NK64 is added
and the sample is loaded onto a Q-Sepharose column. The Q column is
washed with Q-25 buffer (25 mM NaCl, 10 mM potassium phosphate pH
6.4) and the asparaginase is then eluted with buffer Q-135 (135 mM
NaCl in 10 mM potassium phosphate pH 6.4).
[0130] To the pooled enzyme fractions is added magnesium sulfate
powder to a final concentration of 0.25 M and is loaded onto a
phenyl hydrophobic interaction column pre-equilibrated with 0.25 M
MgSO.sub.4 in 10 mM potassium phosphate, pH 7.8. The asparaginase
is collected in the flow through fraction and diafiltered in a
Filtron unit using a 30 kDa molecular weight cut-off polysulfone
membrane with the buffer, 75 mM NaCl, 1 mM potassium phosphate, pH
7.2.
[0131] The asparaginase fraction is diluted with an equal volume of
water and loaded onto a hydroxyapatite column. Impurities are
removed with elution with buffer H15 (50 mM NaCl, 15 mM potassium
phosphate, pH 7.8). The purified asparaginase is eluted with buffer
H150 (50 mM NaCl, 150 mM potassium phosphate, pH 7.8).
Example 7
Purification from Cells and Culture Medium
[0132] Cultures of E. coli strain EN538, grown, induced, and
homogenized as described in Example 6, are diafiltered against 20
mM sodium acetate, 40 mM NaCl, pH 4.6 with 8 product volumes with a
50 kDa Microgon hollow fiber at a flow rate of 2.9 L/min, 16 psi
until the A.sub.280 is less than 0.1 and conductivity is 5 mS. The
product is filtered using a 0.22 .mu.m membrane.
[0133] Cation exchange chromatography is conducted with a Poros-HS
column. The column is equilibrated in 20 mM sodium acetate, ph 4.6,
40 mM NaCl. The diafiltered clarified media is loaded at 0.5 column
volume (CV)/min and the column is washed with 5 CV of 20 mM sodium
acetate, pH 4.6, 40 mM NaCl. The asparaginase is eluted with 20 mM
sodium acetate, pH 4.6, 135 mM NaCl.
[0134] To the above product is added 0.2 M dibasic sodium
phosphate, pH 9.2 to adjust the pH to 6.3. The sample is then
diafiltered against 10 mM sodium phosphate, pH 6.3 with a 50 kDa
Microgon hollow fiber filter at a flow rate of 0.74 L/min, 16.5
psi.
[0135] Anion exchange chromatography is conducted on TMAE
Fractogel. The column is equilibrated in 10 mM sodium acetate, pH
6.4. The diafiltered cation column eluate is loaded at 0.5 CV/min
and the column is washed with 5 CV of 10 mM sodium acetate, pH 6.4.
The column is further washed with 5 CV of 10 mM sodium acetate, pH
6.4, 25 mM NaCl. The asparaginase is eluted with 10 mM sodium
acetate, pH 6.4, 100 mM NaCl.
[0136] The product is diafiltered against 10 mM sodium phosphate,
pH 7.5 with a 50 kDa membrane to a concentration of 40 mg/ml and
filtered through a 0.22 .mu.m membrane.
Sequence CWU 1
1
121326PRTEscherichia coli 1Leu Pro Asn Ile Thr Ile Leu Ala Thr Gly
Gly Thr Ile Ala Gly Gly1 5 10 15Gly Asp Ser Ala Thr Lys Ser Asn Tyr
Thr Ala Gly Lys Val Gly Val 20 25 30Glu Asn Leu Val Asn Ala Val Pro
Gln Leu Lys Asp Ile Ala Asn Val 35 40 45Lys Gly Glu Gln Val Val Asn
Ile Gly Ser Gln Asp Met Asn Asp Asp 50 55 60Val Trp Leu Thr Leu Ala
Lys Lys Ile Asn Thr Asp Cys Asp Lys Thr65 70 75 80Asp Gly Phe Val
Ile Thr His Gly Thr Asp Thr Met Glu Glu Thr Ala 85 90 95Tyr Phe Leu
Asp Leu Thr Val Lys Cys Asp Lys Pro Val Val Met Val 100 105 110Gly
Ala Met Arg Pro Ser Thr Ser Met Ser Ala Asp Gly Pro Phe Asn 115 120
125Leu Tyr Asn Ala Val Val Thr Ala Ala Asp Lys Ala Ser Ala Asn Arg
130 135 140Gly Val Leu Val Val Met Asn Asp Thr Val Leu Asp Gly Arg
Asp Val145 150 155 160Thr Lys Thr Asn Thr Thr Asp Val Ala Thr Phe
Lys Ser Val Asn Tyr 165 170 175Gly Pro Leu Gly Tyr Ile His Asn Gly
Lys Ile Asp Tyr Gln Arg Thr 180 185 190Pro Ala Arg Lys His Thr Ser
Asp Thr Pro Phe Asp Val Ser Lys Leu 195 200 205Asn Glu Leu Pro Lys
Val Gly Ile Val Tyr Asn Tyr Ala Asn Ala Ser 210 215 220Asp Leu Pro
Ala Lys Ala Leu Val Asp Ala Gly Tyr Asp Gly Ile Val225 230 235
240Ser Ala Gly Val Gly Asn Gly Asn Leu Tyr Lys Thr Val Phe Asp Thr
245 250 255Leu Ala Thr Ala Ala Lys Asn Gly Thr Ala Val Val Arg Ser
Ser Arg 260 265 270Val Pro Thr Gly Ala Thr Thr Gln Asp Ala Glu Val
Asp Asp Ala Lys 275 280 285Tyr Gly Phe Val Ala Ser Gly Thr Leu Asn
Pro Gln Lys Ala Arg Val 290 295 300Leu Leu Gln Leu Ala Leu Thr Gln
Thr Lys Asp Pro Gln Gln Ile Gln305 310 315 320Gln Ile Phe Asn Gln
Tyr 32521530DNAEscherichia coli 2aaatgggcgc gaaagcggtg ctgaaaagcg
gcggtaaccc attacagaat gtgctgggaa 60gcctgggaag cctggggggg ctgcaatcct
caatccaaac cgagtggaaa aagcaggaaa 120aagatttcca gcagtttggc
aaagatgttt gtagccgcgt tgtgactctg gaagatagcc 180gcaaagccct
ggtcgggaat ttaaaataat cctctatttt aagacggcat aatacttttt
240tatgccgttt aattcttcgt tttgttacct gcctctaact ttgtagatct
ccaaaatata 300ttcacgttgt aaattgttta acgtcaaatt tcccatacag
agctaaggga taatgcgtag 360cgttcacgta actggaggaa tgaaatggag
tttttcaaaa agacggcact tgccgcactg 420gttatgggtt ttagtggtgc
agcattggca ttacccaata tcaccatttt agcaaccggc 480gggaccattg
ccggtggtgg tgactccgca accaaatcta actacacagc gggtaaagtt
540ggcgtagaaa atctggttaa tgcggtgccg caactaaaag acattgcgaa
cgttaaaggc 600gagcaggtag tgaatatcgg ctcccaggac atgaacgatg
atgtctggct gacactggcg 660aaaaaaatta acaccgactg cgataagacc
gacggcttcg tcattaccca cggtaccgac 720acgatggaag aaactgctta
cttcctcgac ctgacggtga aatgcgacaa accggtggtg 780atggtcggcg
caatgcgtcc gtccacgtct atgagcgcag acggtccatt caacctgtat
840aacgcggtag tgaccgcagc tgataaagcc tccgccaacc gtggcgtgct
ggtagtgatg 900aatgacaccg tgcttgatgg ccgtgacgtc accaaaacca
acaccaccga cgtagcgacc 960ttcaagtctg ttaactacgg tcctctgggt
tacattcaca acggtaagat tgactaccag 1020cgtaccccgg cacgtaagca
taccagcgac acgccattcg atgtctctaa gctgaatgaa 1080ctgccgaaag
tcggcattgt ttataactac gctaacgcat ccgatcttcc ggctaaagca
1140ctggtagatg cgggctatga tggcatcgtt agcgctggtg tgggtaacgg
caacctgtat 1200aaaactgtgt tcgacacgct ggcgaccgcc gcgaaaaacg
gtactgcagt cgtgcgttct 1260tcccgcgtac cgacgggcgc taccactcag
gatgccgaag tggatgatgc gaaatacggc 1320ttcgtcgcct ctggcacgct
gaacccgcaa aaagcgcgcg ttctgctgca actggctctg 1380acgcaaacca
aagatccgca gcagatccag cagatcttca atcagtacta atcgcctcgc
1440cccggtatcg tgccggggct ttttcacttc agactcacgt ccattgccaa
ttttaattac 1500cctaatgata atcaccggaa taaattattt
153031044DNAEscherichia coli 3atggagtttt tcaaaaagac ggcacttgcc
gcactggtta tgggttttag tggtgcagca 60ttggcattac ccaatatcac cattttagca
accggcggga ccattgccgg tggtggtgac 120tccgcaacca aatctaacta
cacagcgggt aaagttggcg tagaaaatct ggttaatgcg 180gtgccgcaac
tgaaggacat tgcgaacgtt aaaggcgagc aggtagtgaa tattggctcc
240caggacatga acgatgatgt ctggctgaca ctggcgaaaa aaattaacac
cgactgcgat 300aaaactgacg gcttcgtcat tacccacggt accgacacga
tggaagaaac cgcttacttc 360ctcgacctga cggtgaaatg cgacaaaccg
gtggtgatgg tcggtgcaat gcgtccgtcc 420acgtctatga gcgcagacgg
tccattcaac ctgtataacg cggtagtgac tgcagctgat 480aaagcctccg
ctaatcgtgg cgtactggta gtgatgaacg acaccgtgct tgatggccgt
540gatgtcacca aaaccaacac caccgatgta gcgaccttca agtctgttaa
ctacggtcct 600ctgggttaca ttcacaacgg taagattgac taccaacgta
ccccggcacg taagcacacc 660agcgacacgc cgttcgatgt ctctaagctg
aatgaactgc cgaaagtcgg cattgtttat 720aactacgcta acgcatccga
tcttccggct aaagcactgg tagatgcggg ctatgatggc 780atcgttagcg
ctggcgtggg taacggcaac ctgtataaaa ccgtatttga cacccttgca
840accgctgcga aaaacggcac tgcagtagtg cgttcttccc gcgtaccgac
gggcgctacc 900actcaggatg ccgaagtgga tgatgcgaaa tacggcttcg
tcgcctctgg cacgttgaac 960ccgcaaaaag cgcgcgttct gctgcaactg
gctctgacgc aaactaaaga tccgcagcag 1020atccagcaga tcttcaatca gtac
1044434DNAArtificial Sequenceoligonucleotide primer 4tactgaattc
atggagtttt tcaaaaagac ggca 34533DNAArtificial
Sequenceoligonucleotide primer 5acagtaagct tagtactgat tgaagatctg
ctg 33624DNAArtificial Sequenceoligonucleotide primer 6caactttacc
cgctgtgtag ttag 24724DNAArtificial Sequenceoligonucleotide primer
7cagccagaca tcatcgttca tgtc 24826DNAArtificial
Sequenceoligonucleotide primer 8gtcgaacaca gttttataca ggttgc
26924DNAArtificial Sequenceoligonucleotide primer 9ctgcagtacc
gtttttcgcg gcgg 241020PRTEscherichia coli 10Leu Pro Asn Ile Thr Ile
Leu Ala Thr Gly Gly Thr Ile Ala Gly Gly1 5 10 15Gly Asp Ser Ala
201132DNAArtificial Sequenceoligonucleotide primer 11gatccatatg
gagtttttca aaaagacggc ac 321232DNAArtificial
Sequenceoligonucleotide primer 12gtacggatcc tcattagtac tgattgaaga
tc 32
* * * * *