U.S. patent application number 16/226499 was filed with the patent office on 2019-06-06 for modified l-asparaginase.
This patent application is currently assigned to XL-Protein GmbH. The applicant listed for this patent is Lars Friedrich, Anne O'Donnell. Invention is credited to Lars Friedrich, Anne O'Donnell.
Application Number | 20190169589 16/226499 |
Document ID | / |
Family ID | 64691458 |
Filed Date | 2019-06-06 |
View All Diagrams
United States Patent
Application |
20190169589 |
Kind Code |
A1 |
Friedrich; Lars ; et
al. |
June 6, 2019 |
Modified L-Asparaginase
Abstract
The disclosure provides a modified protein that is a combination
of (i) an L-asparaginase and (ii) one or more (poly)peptide(s),
wherein the (poly)peptide consists solely of proline and alanine
amino acid residues, and methods of preparation and use
thereof.
Inventors: |
Friedrich; Lars; (Munich,
DE) ; O'Donnell; Anne; (Dublin, IE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Friedrich; Lars
O'Donnell; Anne |
Munich
Dublin |
|
DE
IE |
|
|
Assignee: |
XL-Protein GmbH
Freisling
DE
Jazz Pharmaceutical Ireland Limited
Dublin
IE
|
Family ID: |
64691458 |
Appl. No.: |
16/226499 |
Filed: |
December 19, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15671086 |
Aug 7, 2017 |
10174302 |
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16226499 |
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62523061 |
Jun 21, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/82 20130101; C12Y
305/01001 20130101; A61K 38/48 20130101; C07K 2319/00 20130101;
C07K 14/00 20130101; A61K 38/02 20130101 |
International
Class: |
C12N 9/82 20060101
C12N009/82; A61K 38/48 20060101 A61K038/48; A61K 38/02 20060101
A61K038/02 |
Claims
1. A modified protein comprising (i) an L-asparaginase and (ii) one
or more polypeptides, wherein the one or more polypeptides consists
solely of proline and alanine amino acid residues.
2. The modified protein according to claim 1, wherein the
L-asparaginase has at least 85% sequence identity to the amino acid
sequence of SEQ ID NO: 1.
3. The modified protein according to claim 1, wherein the
L-asparaginase has at least 90% sequence identity to the amino acid
sequence of SEQ ID NO: 1.
4. The modified protein according to claim 1, wherein the
L-asparaginase has at least 95% sequence identity to the amino acid
sequence of SEQ ID NO: 1.
5. The modified protein according to claim 1, wherein the
L-asparaginase comprises the amino acid sequence of SEQ ID NO:
1.
6. The modified protein according to claim 1, wherein the
L-asparaginase consists of the amino acid sequence of SEQ ID NO:
1.
7. The modified protein according to claim 1, wherein the modified
protein is a fusion protein of the L-asparaginase and the
polypeptide.
8. The modified protein according to claim 7, wherein polypeptide
consists of 100 to 600 proline and alanine amino acid residues.
9. The modified protein according to claim 7, wherein the
polypeptide consists of 200 to 400 proline and alanine amino acid
residues, and the proline amino acid residues constitute more than
10% and less than 70% of the polypeptide.
10. The modified protein according to claim 7, wherein the
polypeptide comprises the amino acid sequence of SEQ ID NO: 5.
11. The modified protein according to claim 7, wherein the
polypeptide comprises the amino acid sequence of SEQ ID NO: 7.
12. The modified protein according to claim 7, wherein the
polypeptide comprises the amino acid sequence of SEQ ID NO: 9.
13. The modified protein according to claim 7, wherein the
polypeptide comprises the amino acid sequence of SEQ ID NO: 11.
14. The modified protein according to claim 7, wherein the
polypeptide comprises the amino acid sequence of SEQ ID NO: 13.
15. The modified protein according to claim 7, wherein the
L-asparaginase comprises the amino acid sequence of SEQ ID NO: 1
and the polypeptide comprises the amino acid sequence of SEQ ID NO:
5, 7, 9, 11 or 13.
16. The modified protein according to claim 1, wherein each of the
one or more polypeptides is independently a peptide
R.sup.N-(P/A)-R.sup.c, wherein (P/A) is an amino acid sequence
consisting solely of proline and alanine amino acid residues,
R.sup.N is a protecting group attached to the N-terminal amino
group of the amino acid sequence, and R.sup.C is an amino acid
residue bound via its amino group to the C-terminal carboxy group
of the amino acid sequence, the each of the one or more
polypeptides is conjugated to the L-asparaginase via an amide
linkage formed from the carboxy group of the C-terminal amino acid
residue R.sup.c of the peptide and a free amino group of the
L-asparaginase, and at least one of the free amino groups, which
the one or more polypeptide(s) are conjugated to, is not an
N-terminal .alpha.-amino group of the L-asparaginase.
17. The modified protein according to claim 16, wherein the each of
the one or more polypeptides independently consists of 15 to 45
proline and alanine amino acid residues.
18. The modified protein according to claim 16, wherein the each of
the one or more polypeptides independently consists of 20 to 40
proline and alanine amino acid residues, and the proline amino acid
residues constitute more than 10% and less than 70% of the amino
acid sequence.
19. The modified protein according to claim 16, wherein at least
one of the one or more polypeptides comprises the amino acid
sequence of SEQ ID NO: 5.
20. The modified protein according to claim 16, wherein at least
one of the one or more polypeptides comprises the amino acid
sequence of SEQ ID NO: 15.
21. The modified protein according to claim 16, wherein R.sup.N is
pyroglutamoyl or acetyl, and/or R.sup.C is .epsilon.-aminohexanoic
acid.
22. The modified protein according to claim 16, wherein (i) at
least one of the free amino groups, which the one or more
polypeptides are conjugated to, is an .epsilon.-amino group of a
lysine residue of the L-asparaginase; or (ii) the free amino
groups, which the one or more polypeptides are conjugated to, are
selected from the group consisting of the .epsilon.-amino group(s)
of any lysine residue(s) of the L-asparaginase and the N-terminal
.alpha.-amino group(s) of the L-asparaginase.
23. The modified protein according to claim 16, wherein the
L-asparaginase is composed of four subunits, and 9 to 13
polypeptides are conjugated to each subunit of the
L-asparaginase.
24. The modified protein according to claim 16, wherein the
L-asparaginase comprises the amino acid sequence of SEQ ID NO: 1
and the one or more polypeptides comprise the amino acid sequence
of SEQ ID NO: 5 or 15.
25. A pharmaceutical composition comprising the modified protein
according to claim 1 and one or more pharmaceutical acceptable
carriers or excipients.
26. The pharmaceutical composition of claim 25, wherein the one or
more polypeptides is present in an amount sufficient to mediate a
decreased immunogenicity of the modified protein following
administration to a human subject.
27. The pharmaceutical composition of claim 25, wherein the one or
more polypeptides is present in an amount sufficient to increase
the plasma half-life of the L-asparaginase following administration
to a human subject.
Description
[0001] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing (filename:
sequencelisting; date recorded: Jun.28, 2017; file size: 34
KB).
BACKGROUND
[0002] Proteins with L-asparagine aminohydrolase activity, commonly
known as L-asparaginases, have successfully been used for the
treatment of Acute Lymphoblastic Leukemia (ALL) in children for
many years. ALL is the most common childhood malignant cancers
(Avramis (2005) Clin. Pharmacokinet. 44, 367-393).
[0003] L-asparaginase has also been used to treat Hodgkin's
disease, acute myelocytic Leukemia, acute myelomonocytic Leukemia,
chronic lymphocytic Leukemia, lymphosarcoma, reticulosarcoma, and
melanosarcoma (Kotzia (2007) J. Biotechnol. 127, 657-669). The
anti-tumor activity of L-asparaginase is believed to be due to the
inability or reduced ability of certain malignant cells to
synthesize L-asparagine (Id). These malignant cells rely on an
extracellular supply of L-asparagine. However, the L-asparaginase
enzyme catalyzes the hydrolysis of L-asparagine to aspartic acid
and ammonia, thereby depleting circulating pools of L-asparagine
and killing tumor cells that cannot perform protein synthesis
without L-asparagine (Id).
[0004] L-asparaginase from E. coli was the first enzyme drug used
in ALL therapy and has been marketed as Elspar.RTM. in the United
States or as KIDROLASE and L-asparaginase MEDAC in Europe.
L-asparaginases have also been isolated from other microorganisms,
e.g., an L-asparaginase protein from Erwinia chrysanthemi, named
crisantaspase, that has been marketed as ERWINASE (Wriston (1985)
Meth. Enzymol. 113, 608-618; Goward (1992) Bioseparation 2,
335-341). L-asparaginases from other species of Erwinia have also
been identified, including, for example, Erwinia chrysanthemi 3937
(Genbank Accession No. AAS67028), Erwinia chrysanthemi NCPPB 1125
(Genbank Accession No. CAA31239), Erwinia carotovora (Genbank
Accession No. AAP92666), and Erwinia carotovora subsp. artroseptica
(Genbank Accession No. AAS67027). These Erwinia chrysanthemi
L-asparaginases have about 91-98% amino acid sequence identity with
each other, while the Erwinia carotovora L-asparaginases have
approximately 75-77% amino acid sequence identity with the Erwinia
chrysanthemi L-asparaginases (Kotzia (2007) J. Biotechnol. 127,
657-669).
[0005] L-asparaginases of bacterial origin have a high immunogenic
and antigenic potential and frequently provoke adverse reactions
ranging from mild allergic reaction to anaphylactic shock in
sensitized patients (Wang (2003) Leukemia 17, 1583-1588). E. coli
L-asparaginase is particularly immunogenic, with reports of the
presence of anti-asparaginase antibodies to E. coli L-asparaginase
following intravenous or intramuscular administration reaching as
high as 78% in adults and 70% in children (Id).
[0006] L-asparaginases from Escherichia Coli and Erwinia
chrysanthemi differ in their pharmacokinetic properties and have
distinct immunogenic profiles, respectively (Klug Albertsen (2001)
Brit. J. Haematol. 115, 983-990). Furthermore, it has been shown
that antibodies that developed after a treatment with
L-asparaginase from E. coli do not cross react with L-Asparaginase
from Erwinia (Wang (2003) Leukemia 17, 1583-1588). Thus,
L-asparaginase from Erwinia (crisantaspase) has been used as a
second line treatment of ALL in patients that react to E. coli
L-asparaginase (Duval (2002) Blood 15, 2734-2739; Avramis (2005)
Clin. Pharmacokinet. 44, 367-393).
[0007] In another attempt to reduce immunogenicity associated with
administration of microbial L-asparaginases, an E. coli
L-asparaginase has been developed that is modified with
methoxy-polyethyleneglycol (mPEG) This so-called
mPEG-L-asparaginase, or pegaspargase, marketed as ONCASPAR (Enzon
Inc.), was first approved in the U.S. for second line treatment of
ALL in 1994, and has been approved for first-line therapy of ALL in
children and adults since 2006.
[0008] ONCASPAR is an E. coli L-asparaginase that has been modified
at multiple lysine residues using 5 kDa mPEG-succinimidyl succinate
(SS-PEG) (U.S. Pat. No. 4,179,337). SS-PEG is a PEG reagent of the
first generation that contains an unstable ester linkage that is
sensitive to hydrolysis by enzymes or at slightly alkaline pH
values (U.S. Pat. No. 4,670,417). These properties decrease both in
vitro and in vivo stability and can impair drug safety.
[0009] Furthermore, it has been demonstrated that antibodies
developed against L-asparaginase from E. coli will cross react with
ONCASPAR (Wang (2003) Leukemia 17, 1583-1588). Even though these
antibodies were not neutralizing, this finding clearly demonstrated
the high potential for cross-hypersensitivity or cross-inactivation
in vivo. Indeed, in one report 30-41% of children who received
pegaspargase had an allergic reaction (Id).
[0010] In addition to outward allergic reactions, the problem of
"silent hypersensitivity" was recently reported, whereby patients
develop anti-asparaginase antibodies without showing any clinical
evidence of a hypersensitivity reaction (Wang (2003) Leukemia 17,
1583-1588). This reaction can result in the formation of
neutralizing antibodies to E. coli L-asparaginase and pegaspargase;
however, these patients are not switched to Erwinia L-asparaginase
because there are not outward signs of hypersensitivity, and
therefore they receive a shorter duration of effective treatment
(Holcenberg (2004) J. Pediatr. Hematol. Oncol. 26, 273-274).
[0011] Erwinia chrysanthemi L-asparaginase treatment is often used
in the event of hypersensitivity to E. coli-derived
L-asparaginases. However, it has been observed that as many as
30-50% of patients receiving Erwinia L-asparaginase are
antibody-positive (Avramis (2005), Clin. Pharmacokinet. 44,
367-393). Moreover, because Erwinia chrysanthemi L-asparaginase has
a shorter elimination half-life than the E. coli L-asparaginases,
it must be administered more frequently (Id). In a study by Avramis
et. al, Erwinia asparaginase was associated with inferior
pharmacokinetic profiles (Avramis (2007), J. Pediatr. Hematol.
Oncol. 29, 239-247). E. coli L-asparaginase and pegaspargase
therefore have been the preferred first-line therapies for ALL over
Erwinia L-asparaginase.
[0012] Numerous biopharmaceuticals have successfully been PEGylated
and marketed for many years. However, in many cases, PEGylated
biopharmaceuticals show significantly reduced activity compared to
the unmodified biopharmaceutical. In the case of L-asparaginase
from Erwinia carotovora, it has been observed that PEGylation
reduced its in vitro activity to approximately 57% (Kuchumova
(2007) Biochemistry (Moscow) Supplement Series B: Biomedical
Chemistry, 1, 230-232). The L-asparaginase from Erwinia carotovora
has only about 75% homology to the Erwinia chrysanthemi
L-asparaginase (crisantaspace). For ONCASPAR it is also known that
its in vitro activity is approximately 50% compared to the
unmodified E. coli L-asparaginase.
[0013] Thus, the technical problem underlying the present invention
is the provision of means and methods for treating cancer, such as
leukemia or non-Hodgkin's lymphoma, that avoids the limitations and
disadvantages of prior art therapies, particularly of some
PEGylated asparaginases.
[0014] The technical problem is solved by provision of the
embodiments characterized in the claims.
SUMMARY OF THE INVENTION
[0015] The present invention relates to a modified protein that is
a combination of (i) an L-asparaginase and (ii) one or more
(poly)peptide(s), wherein the (poly)peptide consists solely of
proline and alanine amino acid residues. The modified protein can
be formed in a number of ways, including chemical conjugation
between the L-asparaginase and the (poly)peptides or by expressing
the modified protein as a fusion protein. Also provided herein are
nucleic acids encoding the modified protein, vectors and/or host
cells comprising same, as well as processes for their production.
Compositions comprising the modified protein and their use in
medicine, particularly in the treatment of cancer, are disclosed.
In another aspect of the invention, the L-asparaginase can be
derived from Erwinia and/or it has at least 85% identity to the
amino acid sequence of SEQ ID NO: 1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read
in conjunction with the appended drawings.
[0017] FIG. 1: Chemistry of the Conjugation of Crisantaspase with
N-Terminally Protected P/A Peptides via Amino Groups.
[0018] (A) and (B) depict the chemical structures of P/A peptides
(SEQ ID NO: 16 and 17, amino acid sequence shown in SEQ ID NO: 5
and 15) containing either 20 or 40 Pro/Ala residues (respectively),
which were obtained by solid-phase peptide synthesis. In order to
avoid polymerization of the peptides upon chemical activation of
the C-terminus, the N-terminus was protected with pyroglutamyl
(Pga) residue Aminohexanoic acid (Ahx) was incorporated at the
C-terminus of the peptides to serve as linker. (C) In the presence
of the non-nucleophilic base N,N-diisopropylethylamine (DIPEA,
Hunig's base), and with DMSO as solvent, the N-terminally protected
P/A peptide is activated at its C-terminus with the benzotriazol
derivative O-(benzotriazol-1-yl)-N,N,N'N'-tetramethyluronium
tetrafluoroborate (TBTU). The hydroxybenzotriazol (HOBt) active
ester of the peptide is subsequently used to derivatize the amino
groups (.epsilon.-amino groups of lysine residues or .alpha.-amino
group of N-terminus) of Crisantaspase with the P/A peptide through
formation of peptide or isopeptide bonds, while free HOBt is
released. This coupling step is performed in aqueous solution (e.g.
PBS buffer) with a content of organic solvent .ltoreq.30%. The
P/A-Crisantaspase modified protein may be purified from residual
P/A peptide/coupling reagent by dialysis and/or chromatography
(e.g. ion exchange chromatography).
[0019] FIG. 2: Optimization of Crisantaspase/Pga-P/A(20)-Ahx
Coupling Ratio.
[0020] Recombinant Crisantaspase produced in E. coli was conjugated
with the Pga-P/A#1(20)-Ahx peptide (Part A of FIG. 1)(SEQ ID NO:
16, amino acid sequence shown in SEQ ID NO: 5) as described in
Example 1. The peptide-to-protein ratio was varied between 3.5 mg
and 10 mg P/A peptide per 1 mg Crisantaspase. The gel was loaded
with 7 .mu.g of Crisantaspase from each coupling reaction.
Additionally, a mix of coupling reactions with ratios of 0.3 to 10
mg peptide per mg protein was applied as size standard ("Std"). The
number of coupled P/A peptides can be determined by counting the
bands in that ladder starting from the unconjugated Crisantaspase
as marked on the right. Lane "kDa": PIERCE Unstained Protein MW
Marker (Thermo Fisher Scientific).
[0021] FIG. 3: Purification of Crisantaspase/Pga-P/A(40)-Ahx
Peptide Coupling Product via Anion Exchange Chromatography
[0022] Recombinant Crisantaspase produced in E. coli was conjugated
with the Pga-P/A(40)-Ahx peptide (Part B of FIG. 1)(SEQ ID NO: 17,
amino acid sequence shown in SEQ ID NO: 15) as described in Example
2. After dialysis against AIX running buffer (25 mM boric acid/NaOH
pH 9.0, 1 mM EDTA) anion exchange chromatography was performed on
an 85 mL SOURCE 15 Q column (A). By applying an NaCl concentration
gradient, the enzyme modified protein eluted in a single sharp
peak, as revealed by the UV trace at 280 nm. Separation of
remaining uncoupled peptide and other non-proteinous byproducts of
the chemical conjugation devoid of UV absorption at 280 nm was
monitored by the 225 nm UV trace. (B) SDS-PAGE analysis of the
Crisantaspase/Pga-P/A(40)-Ahx modified protein after purification
by anion exchange chromatography (lane 1). A mix of coupling
reactions with ratios of 0.3 to 10 mg peptide per mg protein was
applied to lane 2 to allow determination of the number of coupled
P/A peptides per Crisantaspase monomer. PAGERULER Plus Prestained
marker (Thermo Fisher Scientific) was applied to lane "M".
[0023] FIG. 4: Purification of Crisantaspase/Pga-P/A(20)-Ahx
Peptide Coupling Product via Anion Exchange Chromatography
[0024] Recombinant Crisantaspase produced in E. coli was conjugated
with the Pga-P/A(20)-Ahx peptide (Part A of FIG. 1)(SEQ ID NO: 16,
amino acid sequence shown in SEQ ID NO: 5) as described in Example
3. After dialysis against AIX running buffer (25 mM boric acid/NaOH
pH 9.0, 1 mM EDTA) anion exchange chromatography was performed on
an 85 mL SOURCE 15 Q column (A). By applying an NaCl concentration
gradient the enzyme modified protein eluted in a single sharp peak,
as revealed by the UV trace at 280 nm. Separation of remaining
uncoupled peptide and other non-proteinous byproducts of the
chemical conjugation devoid of UV absorption at 280 nm was revealed
by the 225 nm UV trace. (B) SDS-PAGE analysis of the
Crisantaspase/Pga-P/A(20)-Ahx modified protein after purification
by anion exchange chromatography (lane 1). A mix of coupling
reactions with ratios of 0.3 to 10 mg peptide per mg protein was
applied to lane 2 to allow determination of the number of coupled
P/A peptides per Crisantaspase monomer. PAGERULER Plus Prestained
marker (Thermo Fisher Scientific) was applied to lane "M".
[0025] FIG. 5: Cloning of the Expression Vectors for the Production
of PASylated Crisantaspase in E. coli
[0026] (A) Plasmid map of pASK75-SapI-Crisantaspase (SEQ ID NO: 4)
and (B) of its derivative pASK75-PA400-Crisantaspase (SEQ ID NO:
14) after seamless insertion of a PA#1c/1b(400) (SEQ ID NO: 10)
gene cassette via the two inversely oriented SapI restriction
sites. The structural gene for the biologically/pharmacologically
active (pre)protein PA#1(400)-Crisantaspase (SEQ ID NO: 13)
comprising the low repetitive nucleotide sequence encoding a PA#1
polypeptide with 401 amino acid residues and the structural gene
for Crisantaspase as well as coding region for the bacterial Enx
signal sequence (SP.sup.Enx) is cloned under transcriptional
control of the tet promoter/operator)(tet.sup.p/o). The plasmid
backbone outside the expression cassette flanked by the XbaI and
HindIII restriction sites is identical with that of the generic
expression vector pASK75 (Skerra (1994) Gene 151:131-135). A
plasmid for the expression of Crisantaspase fused to PA#1(200) (SEQ
ID NO: 11) was cloned in the same way using the PA#1b(200) gene
cassette (SEQ ID NO: 12).
[0027] FIG. 6: SDS-PGE Analysis of Recombinant Crisantaspase
Genetically Fused with PA200 or PA400
[0028] (A) Analysis of the mature PA#1(400)-Crisantaspase fusion
protein (SEQ ID NO: 13) after periplasmic extraction (PPE),
ammonium sulfate precipitation (ASP) and anion exchange
chromatography (AEX) by 10% SDS-PAGE. (B) The gel shows 5 .mu.g
samples of purified mature PA#1(200)-Crisantaspase (lane 1) (SEQ ID
NO: 11) or PA#1(400)-Crisantaspase (lane 2) (SEQ ID NO: 13). Sizes
of the marker proteins (M) are indicated on the left. The
PA#1(200)-Crisantaspase and the PA#1(400)-Crisantaspase fusion
protein appear as single homogeneous bands with an apparent
molecular size of about 105 kDa (lane 1) or 200 kDa (lane 2),
respectively. Due to poor SDS binding, PA fusion proteins generally
show significantly larger sizes (Schlapschy (2013) Protein Eng Des
Sel. 26:489-501) than, e.g., the calculated mass of 51 kDa for the
PA#1(200)-Crisantaspase monomer or 67 kDa for the
PA#1(400)-Crisantaspase monomer.
[0029] FIG. 7: Size Exclusion Chromatography of PASylated
Crisantaspase Variants
[0030] (A) Overlay of elution profiles for unmodified
Crisantaspase, as well as for Crisantaspase chemically conjugated
either to Pga-P/A(20)-Ahx or Pga-P/A(40)-Ahx peptides (described in
Examples 3 and 2, respectively) and the recombinant Crisantaspase
genetically fused with either PA#1(200) (SEQ ID NO: 7) or PA#1(400)
(SEQ ID NO: 9) polypeptides (described in Example 5). 150 .mu.L of
the purified protein at a concentration of 1 mg/ml was applied to a
SUPERDEX S200 10/300 GL column equilibrated with PBS buffer.
Absorption at 280 nm was monitored and the peak of each
chromatography run was normalized to 100%.
[0031] (B) Calibration curve for the chromatograms from (A) using a
SUPERDEX S200 10/300 GL column. The logarithm of the molecular
weight of marker proteins (ovalbumin, 43.0 kDa; bovine serum
albumin, 66.3 kDa; alcohol dehydrogenase, 150 kDa,
.quadrature.-amylase, 200 kDa, apo-ferritin, 440 kDa) was plotted
vs. their elution volumes (black circles) and fitted by a straight
line. From the observed elution volumes of the tetrameric
Crisantaspase, its PA#1 peptide modified proteins and its
recombinant PA#1 fusion proteins (black squares) their apparent
molecular sizes were determined as follows. Crisantaspase, 105 kDa
(true mass 140 kDa); Crisantaspase/Pga-P/A(20)-Ahx modified
protein, 531 kDa (true mass 228 kDa); Crisantaspase/Pga-P/A(40)-Ahx
modified protein, 820 kDa (true mass 284 kDa); PA200-Crisantaspase,
595 kDa (true mass 205 kDa); PA400-Crisantaspase, 1087 kDa (true
mass 269 kDa). These data show that both the chemically conjugated
P/A peptides and the genetic fusion with the PA#1 polypeptide
confer a much enlarged hydrodynamic volume.
[0032] FIG. 8: ESI-MS Analysis of PASylated Crisantaspase
Variants
[0033] (A) The raw m/z spectrum obtained by Electrospray Ionisation
Mass Spectrometry (ESI-MS) of the purified
Crisantaspase/Pga-P/A(20)-Ahx modified protein prepared as
described in Example 3 was deconvoluted yielding the mass spectrum
(B). The observed mass species could unambiguously be assigned to
Crisantaspase conjugated with 9 to 14 peptides (cf. Table 3). Major
peaks, however, were observed only for protein species with 10 to
13 peptides, what corresponds to the determination of the peptide
coupling ratio by SDS-PAGE (cf. Part B of FIG. 4). (C) and (E) show
raw m/z spectra of the PA200-Crisantaspase and PA400-Crisantaspase
fusion proteins prepared in Example 5. The deconvoluted mass
spectra (D) and (F) revealed masses of 51164.75 Da and 67199.17 Da,
respectively, which correspond almost perfectly to the calculated
masses of 51163.58 Da.
[0034] FIG. 9: Mean (.+-.SD) Plasma Concentration Versus Time
Profiles Following a Single IV Bolus Dose to Male CD-1 Mice
[0035] The figures shows plasma asparaginase activity of
PA-crisantaspase conjugates following a single IV bolus dose to
male mice.
DETAILED DESCRIPTION OF THE INVENTION
[0036] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs. All patents
and publications referred to herein are incorporated by reference
in their entireties.
[0037] In one aspect, the present invention relates to a modified
protein comprising (i) an L-asparaginase and (ii) one or more
(poly)peptide(s), wherein the (poly)peptide consists solely of
proline and alanine amino acid residues. In a preferred aspect the
invention relates to a modified protein comprising (i) an
L-asparaginase having at least 85% identity to the amino acid
sequence of SEQ ID NO: 1 and (ii) one or more (poly)peptide(s),
wherein the (poly)peptide consists solely of proline and alanine
amino acid residues.
[0038] The present invention relates, inter alia, to a modified
protein comprising (i) an L-asparaginase and (ii) one or more
(poly)peptide(s), wherein the (poly)peptide consists solely of
proline and alanine amino acid residues. In some embodiments, said
L-asparaginase has at least 85% or 100% identity to the amino acid
sequence of SEQ ID NO: 1. In additional embodiments, the modified
protein has an asparaginase or glutaminase activity higher than
that of the unmodified L-asparaginase. In further embodiments, said
modified protein has an L-asparagine depletion activity at least
about 20% higher than the unmodified L-asparaginase. In yet further
embodiments, said L-asparaginase is a tetramer.
[0039] In some embodiments, the modified protein described herein
is a modified protein of said L-asparaginase and a polypeptide,
wherein the polypeptide consists solely of proline and alanine
amino acid residues. In some embodiments, said polypeptide consists
of about 100 to 600 proline and alanine amino acid residues,
particularly about 200 to about 400 proline and alanine amino acid
residues. In further embodiments, said polypeptide consists of a
total of about 200 proline and alanine amino acid residues or a
total of about 400 proline and alanine amino acid residues. In
additional embodiments, said proline residues constitute more than
about 10% and less than about 70% of the polypeptide. In yet
additional embodiments, said polypeptide comprises a plurality of
amino acid repeats, wherein said repeat consists of proline and
alanine residues and wherein no more than 6 consecutive amino acid
residues are identical. For example, said polypeptide comprises or
consists of the amino acid sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID
NO: 5) or circular permuted versions or (a) multimers(s) of the
sequences as a whole or parts of the sequence. In one aspect, said
polypeptide comprises or consists of an amino acid sequence as
shown in SEQ ID NO: 7 or 9; said polypeptide comprises or consists
of an amino acid sequence encoded by a nucleic acid having a
nucleotide sequence as shown in SEQ ID NO: 8 or 10; said modified
protein comprises or consists of an amino acid sequence as shown in
SEQ ID NO: 11 or 13; or said modified protein comprises or consists
of an amino acid sequence encoded by a nucleic acid having a
nucleotide sequence as shown in SEQ ID NO: 12 or 14. In some
embodiments, said polypeptide is a random coil polypeptide. In
further embodiments, the modified protein is a fusion protein of
the L-asparaginase and the polypeptide.
[0040] In one aspect, the modified protein is a modified protein of
L-asparaginase and one or more peptide(s), wherein each is
independently a peptide R.sup.N-(P/A)-R.sup.c, (P/A) is an amino
acid sequence consisting solely of proline and alanine amino acid
residues, wherein R.sup.N is a protecting group attached to the
N-terminal amino group of the amino acid sequence, and R.sup.c is
an amino acid residue bound via its amino group to the C-terminal
carboxy group of the amino acid sequence, each peptide is
conjugated to the L-asparaginase via an amide linkage formed from
the carboxy group of the C-terminal amino acid residue R.sup.C of
the peptide and a free amino group of the L-asparaginase, and at
least one of the free amino groups, which the peptides are
conjugated to, is not an N-terminal .alpha.-amino group of the
L-asparaginase. In some embodiments, said amino acid sequence
consists of a total of between 15 to 45 proline and alanine amino
acid residues. In additional embodiments, said amino acid sequence
consists of 20 or 40 proline and alanine amino acid residues. In
further embodiments, said proline residues constitute more than
about 10% and less than about 70% of the amino acid sequence. For
example, said amino acid sequence is AAPAAPAPAAPAAPAPAAPA (SEQ ID
NO: 5) or AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 15).
In yet further embodiments, R.sup.N is pyroglutamoyl or acetyl,
and/or R.sup.C is .epsilon.-aminohexanoic acid. In some
embodiments, the peptides comprised in said modified protein adopt
a random coil conformation. In additional embodiments, all of the
peptides comprised in said modified protein are the same. In yet
additional embodiments, at least one of the free amino groups,
which the peptides are conjugated to, is an .epsilon.-amino group
of a lysine residue of the L-asparaginase. In further embodiments,
the free amino groups, which the peptides are conjugated to, are
selected from the group comprising the .epsilon.-amino group(s) of
any lysine residue(s) of the L-asparaginase and the N-terminal
.alpha.-amino group(s) of the L-asparaginase. In yet further
embodiments, the L-asparaginase is composed of four subunits, and
wherein 9 to 13 peptides as defined in any one of items 15 to 24
are conjugated to each subunit of the L-asparaginase.
[0041] In one aspect, the polypeptide or peptide described herein
mediates a decreased immunogenicity of said modified protein.
[0042] In another aspect, the disclosure is related to a nucleic
acid encoding the modified protein described herein. In some
embodiments, said nucleic acid is selected from the group
consisting of: (a) the nucleic acid comprising the nucleotide
sequence of SEQ ID NO: 12 or 14; (b) the nucleic acid comprising
the nucleotide sequence having at least 85% identity to the
nucleotide sequence as defined in (a); and (c) the nucleic acid
being degenerate as a result of the genetic code to the nucleotide
sequence as defined in (a) or (b).
[0043] In another aspect, the disclosure is related to a vector
comprising the nucleic acid described herein. In another aspect,
the disclosure is related to a host cell comprising the nucleic
acid and/or the vector described herein. In some embodiments, said
host cell is selected from the group consisting of Pseudomonas
fluorescens and Corynebacterium glutamicum.
[0044] In another aspect, the disclosure is related to a process
for the preparation of a modified protein described herein or of a
nucleic acid described herein. In some embodiments, the mprocess
comprises culturing the host cell according to item 33 or 34 and
isolating said modified protein from the culture or from said
cell.
[0045] In another aspect, the disclosure is related to a process of
preparing a modified protein as defined in any one of items 17 to
29, the process comprising: (a) coupling an activated peptide of
the formula R.sup.N-(P/A)-R.sup.C-act, wherein R.sup.C-act is a
carboxy-activated form of R.sup.C, wherein R.sup.C and (P/A) are as
defined in the modified protein to be prepared, and wherein R.sup.N
is a protecting group which is attached to the N-terminal amino
group of (P/A), with L-asparaginase to obtain a modified protein of
the L-asparaginase and peptides in which R.sup.N is a protecting
group. In some embodiments, the activated carboxy group of the
amino acid residue R.sup.C-act in the activated peptide is an
active ester group.
[0046] In another aspect, the disclosure is related to a
composition comprising the modified protein described herein or the
modified protein prepared by the process described herein. In some
embodiments, the composition is a pharmaceutical composition,
optionally further comprising (a) pharmaceutical acceptable
carrier(s) or excipient(s).
[0047] In another aspect, the modified protein described herein,
the modified protein prepared by the process described herein, or
the composition described herein may be used as a medicament. In
another aspect, the modified protein described herein, the modified
protein prepared by the process described herein, or the
composition described herein may be used in the treatment of a
disease, e.g. a disease treatable by L-asparagine depletion in a
patient. In another aspect, the modified protein described herein,
the modified protein prepared by the process described herein, or
the composition described herein may be used in the treatment of
cancer.
[0048] In another aspect, the disclosure is related to a method of
treating a disease treatable by L-asparagine depletion in a
patient, said method comprising administering to said patient an
effective amount of the modified protein described herein, the
modified protein prepared by the process described herein, or the
composition described herein. In some embodiments, said disease
treatable by L-asparagine depletion is a cancer. In another aspect,
the disclosure is related to a method of treating cancer comprising
the administration of the modified protein described herein, the
modified protein prepared by the process described herein, or the
composition described herein, to a subject.
[0049] In some embodiments, said cancer is a non-solid cancer. In
additional embodiments, said non-solid cancer is leukemia or
non-Hodgkin's lymphoma. In yet additional embodiments, said
leukemia is acute lymphoblastic leukemia (ALL) or acute myeloid
leukemia (AML); or the method according to item 48, wherein said
leukemia is acute lymphoblastic leukemia (ALL) or acute myeloid
leukemia (AML).
[0050] In another aspect, said modified protein described herein
elicits a lower immunogenic response in said patient compared to
the unmodified L-asparaginase. In some embodiments, said modified
protein has a longer in vivo circulating half-life after a single
dose compared to the unmodified L-asparaginase. In additional
embodiments, said modified protein has a greater AUC value after a
single dose compared to the unmodified L-asparaginase. In yet
additional embodiments, said patient has had a previous
hypersensitivity to an E. coli L-asparaginase or PEGylated form
thereof. In further embodiments, said patient has had a previous
hypersensitivity to an Erwinia L-asparaginase. In yet further
embodiments, the treatment comprises intravenous administration of
said modified protein.
[0051] In one aspect, the present invention relates to a modified
protein comprising (i) a recombinant L-asparaginase having at least
85% identity to the amino acid sequence of SEQ ID NO: 1 and (ii)
one or more (poly)peptide(s), wherein the (poly)peptide consists
solely of proline and alanine amino acid residues. The explanations
and definitions given herein in relation to the terms "modified
protein", "L-asparaginase", "(poly)peptide(s)" and the like
provided herein apply mutatis mutandis. The term "recombinant
L-asparaginase" as used herein refers to a recombinant form of
L-asparaginase having at least 85% identity to the amino acid
sequence of a native Erwinia L-asparaginase. The term "recombinant"
may refer to a recombinantly produced L-asparaginase, e.g. a
L-asparaginase produced in a host cell comprising a nucleic acid
encoding the L-asparaginase.
[0052] The modified proteins further show an enhanced plasma
half-life and, thus, a prolonged duration of action as compared to
the respective unconjugated L-asparaginase. This allows for a
reduction of the dosing frequency and thus the side-effect burden.
The invention also provides processes of preparing the modified
proteins as described herein.
[0053] In certain aspects, the invention relates to a modified
protein comprising (i) an L-asparaginase having at least 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity
to the amino acid sequence of SEQ ID NO: 1 and (ii) one or more
(poly)peptide(s), wherein the (poly)peptide(s) consist(s) solely of
proline and alanine amino acid residues. It is understood that the
term "consisting solely of proline and alanine amino acid residues"
means that at least one proline residue and at least one alanine
residue are present, i.e. both at least one proline residue and at
least one alanine residue must be present. In a preferred aspect,
the invention relates to a modified protein comprising (i) a
recombinant L-asparaginase having the amino acid sequence of SEQ ID
NO: 1 and (ii) one or more (poly)peptide(s), wherein the
(poly)peptide consists solely of proline and alanine amino acid
residues. In one aspect, the L-asparaginase is a tetramer (i.e. the
L-asparaginase composed of four subunits or monomers). One
exemplary subunit or monomer has the amino acid sequence of SEQ ID
NO: 1.
[0054] In one aspect, the (poly)peptide (i.e. polypeptide or
peptide) mediates a decreased immunogenicity of the modified
protein described herein, e.g. a decreased immunogenicity of the
modified protein as compared to the unconjugated
L-asparaginase.
[0055] As shown in the appended examples, the
PA#1(200)-Crisantaspase protein had 109% and the
PA#1(400)-Crisantaspase protein had 118% of enzyme activity
compared to the unmodified Crisantaspase; see Example 5. This
demonstrates that the fusion of asparaginases as described herein
with polypeptides does not affect enzymatic activity. Surprisingly,
the activity even increased with the length of the
PA-polypeptide.
[0056] More generally, the herein provided modified proteins have
the same or substantially the same (enzymatic) activity compared to
unmodified asparaginase. The (enzymatic) activity may be assessed
by the Nessler assay. Details of the Nessler assay are provided in
the appended examples and/or are disclosed in the prior art e.g.
Mashburn (1963) Biochem. Biophys. Res. Commun. 12, 50 (incorporated
herein by reference in its entirety). Accordingly, in one aspect,
the herein provided modified proteins have the same or
substantially the same (enzymatic) activity compared to unmodified
asparaginase as assessed by a Nessler assay. The term "unmodified
asparaginase" as used herein refers to a native asparaginase, i.e.
an asparaginase that is not modified by fusion/conjugation with
(poly)peptides as defined herein.
[0057] For example, an "unmodified asparaginase" is an
L-asparaginase having at least 85% identity to the amino acid
sequence of SEQ ID NO: 1. In a preferred aspect, an "unmodified
asparaginase" is an L-asparaginase having the amino acid sequence
of SEQ ID NO: 1.
[0058] In some aspects, the herein provided modified proteins have
an (enzymatic) activity higher than that of the unmodified
L-asparaginase. The (enzymatic) activity may be assessed by the
Nessler assay for example. Details of the Nessler assay are
provided in the appended examples and/or are disclosed in the prior
art e.g. Mashburn (1963) Biochem. Biophys. Res. Commun. 12, 50
(incorporated herein by reference in its entirety). Accordingly, in
one aspect, the herein provided modified proteins have an
(enzymatic) activity higher than that of the unmodified
L-asparaginase as assessed by a Nessler assay. The term "unmodified
asparaginase" as used herein refers to a native asparaginase, i.e.
an asparaginase that is not modified by fusion/conjugation with
(poly)peptides as defined herein. For example, an "unmodified
asparaginase" is an L-asparaginase having at least 85% identity to
the amino acid sequence of SEQ ID NO: 1. In a preferred aspect, an
"unmodified asparaginase" is an L-asparaginase having the amino
acid sequence of SEQ ID NO: 1. For example, the modified proteins
have an (enzymatic) activity that can be at least 5% and/or up to
30% (e.g. at least 10%, 15%, 20%, 25% (or more)) higher than that
of the L-asparaginase, particularly higher than that of the
unmodified L-asparaginase, particularly as assessed by the Nessler
assay. The above explanations apply in particular to the herein
provided fusion proteins (e.g. modified protein of L-asparaginase
and a polypeptide, wherein the polypeptide consists solely of
proline and alanine amino acid residues), but are not limited
thereto.
[0059] In some aspects, the modified proteins have an asparaginase
activity or glutaminase activity higher than that of the unmodified
L-asparaginase. For example, the modified proteins can have an
asparaginase activity or glutaminase activity at least 5% and/or up
to 30% (e.g. at least 10%, 15%, 20%, 25% (or more)) higher than
that of the L-asparaginase, particularly higher than that of the
unmodified L-asparaginase. In some embodiments, the asparaginase
activity or glutaminase activity may be measured by Nessler assay.
The rate of hydrolysis of asparagine may be determined by measuring
released ammonia, and the amount of released ammonia from using the
modified proteins disclosed herein may be compared with that from
using the L-asparaginase or unmodified L-asparaginase. In
additional aspects, said modified proteins have an L-asparagine
depletion activity higher than that of the unmodified
L-asparaginase. In additional embodiments, the modified proteins
have an L-asparagine depletion activity at least 5% and/or up to
30% (e.g. at least 10%, 15%, 20%, 25% (or more)) higher than that
of the L-asparaginase, particularly higher than that of the
unmodified L-asparaginase, for example as assessed by the Nessler
assay. The invention also relates to a pharmaceutical composition
comprising the modified protein, and the modified protein or the
pharmaceutical composition for use in therapy, or for use as a
medicament, or for use in medicine.
[0060] Generally, a modified protein can be obtained by chemical
coupling or by genetic fusion (in the case of conjugation with
another protein or peptide). The term "fusion protein" as used
herein relates primarily to a modified protein comprising (i) an
L-asparaginase and (ii) one or more polypeptide(s), wherein the
polypeptide consists solely of proline and alanine amino acid
residues. In this context, the polypeptide can consist of about 200
to about 400 proline and alanine amino acid residues. Exemplary
amino acid sequences of such polypeptides are shown in SEQ ID NO: 7
or 9.
[0061] If the modified protein is obtained by chemical coupling, it
comprises (i) an L-asparaginase and (ii) one or more peptide(s),
wherein the peptide consists solely of proline and alanine amino
acid residues. In this context, the peptide can consist of a total
of between 10 to 100 proline and alanine amino acid residues, from
about 15 to about 60 proline and alanine amino acid residues, from
about 15 to 45 proline and alanine amino acid residues, e.g. from
about 20 to about 40, for example, 20 proline and alanine amino
acid residues or 40 proline and alanine amino acid residues.
Exemplary amino acid sequence of such peptides are
AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5) or
AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 15).
[0062] The term "modified protein" as used herein can be used
interchangeably with the term "conjugate", particularly if the term
"modified protein" refers to a modified protein obtained by
chemical coupling or as a fusion protein, i.e. primarily if it
comprises (i) an L-asparaginase and (ii) one or more
(poly)peptide(s), wherein the (poly)peptide consists solely of
proline and alanine amino acid residues. Likewise, the terms
"unmodified" and "unconjugated" can be used interchangeably
herein.
[0063] The invention also relates to a process of preparing the
modified protein, comprising (a) coupling an activated peptide of
the formula R.sup.N-(P/A)-R.sup.C-act, wherein R.sup.C-act is a
carboxy-activated form of R.sup.C, wherein R.sup.C and (P/A) are as
defined in the modified protein to be prepared, and wherein R.sup.N
is a protecting group which is attached to the N-terminal amino
group of (P/A), with L-asparaginase to obtain a modified protein of
the L-asparaginase and peptides in which R.sup.N is a protecting
group.
[0064] It has been demonstrated in the appended examples (cf.
Example 1, Table 1) that the modified protein can be prepared using
a variety of mass ratios of the activated peptide and asparaginase.
For example, mass ratios of 10:1 (activated peptide: asparaginase),
7.5:1, 5:1 or 3.5:1 can be used. It was observed that (enzymatic)
activity of the modified protein was highest, if a ratio of 5:1 or
below was used (cf. Example 1, Table 2). Thus, it may be
advantageous to use a mass ratio of activated peptide :
asparaginase of 5:1 or below, e.g. 5:1, 4:1, 3.5:1 or 3:1, in the
process described herein above. The term "mass ratio" as used
herein refers to the ratio of the molecular weight of the activated
peptide as defined herein and of the asparaginase as defined herein
(e.g. asparaginase as shown in SEQ ID NO: 1 and proteins with at
least 85% identity to SEQ ID NO: 1). The "molecular weight" is
typically indicated herein using the scientific unit Dalton (Da).
It is well known that the molecular weight unit of the asparaginase
or peptide as indicated herein in dalton (Da), is an alternative
name for the unified atomic mass unit (u). A molecular weight of,
e.g., 500 Da is thus equivalent to 500 g/mol. The term "kDa"
(kilodalton) refers to 1000 Da.
[0065] The molecular weight of asparaginase or peptide can be
determined using methods known in the art, such as, e.g., mass
spectrometry (e.g., electrospray ionization mass spectrometry,
ESI-MS, or matrix-assisted laser desorption/ionization mass
spectrometry, MALDI-MS), gel electrophoresis (e.g., polyacrylamide
gel electrophoresis using sodium dodecyl sulfate, SDS-PAGE),
hydrodynamic methods (e.g., gel filtration/size exclusion
chromatography, SEC, or gradient sedimentation), or dynamic (DLS)
or static light scattering (e.g., multi-angle light scattering,
MALS), or the molecular weight of the asparaginase or peptide can
be calculated from the known amino acid sequence (and the known
post-translational modifications, if present) of the asparaginase
or peptide. Preferably, the molecular weight of the asparaginase or
peptide is determined using mass spectrometry.
[0066] The invention also relates to a process for the preparation
of the modified protein or of a nucleic acid encoding the modified
protein. In some aspects, the process comprises producing an
L-asparaginase in a host selected from the group comprising yeasts,
such as Saccharomyces cerevisiae and Pichia Pistoris, as well as
bacteria, actinomycetes, fungi, algae, and other microorganisms,
including Escherichia coli, Bacillus sp., Pseudomonas fluorescens,
Corynebacterium glutamicum and bacterial hosts of the following
genuses, Serratia, Proteus, Acinetobacter and Alcaligenes. Other
hosts are known to those of skill in the art, including
Nocardiopsis alba, which expresses a variant of Asparaginase
lacking on glutaminase-activity (Meena et al. (2014) Bioprocess
Biosyst. Eng. October 2014 Article, which is incorporated by
reference herein in its entirety), and those disclosed in Savitri
et al. (2003) Indian Journal of Biotechnology, 2, 184-194, which is
incorporated by reference herein in its entirety.
[0067] The modified protein can be a fusion protein comprising (i)
a L-asparaginase having at least 85% identity to the amino acid
sequence of SEQ ID NO: 1 and (ii) one or more polypeptide(s),
wherein the polypeptide consists solely of proline and alanine
amino acid residues.
[0068] The proline residues in the polypeptide consisting solely of
proline and alanine amino acid residues may constitute more than
about 10% and less than about 70% of the polypeptide. Accordingly,
it is preferred that 10% to 70% of the total number of amino acid
residues in the polypeptide are proline residues; more preferably,
20% to 50% of the total number of amino acid residues comprised in
the polypeptide are proline residues; and even more preferably, 30%
to 40% (e.g., 30%, 35% or 40%) of the total number of amino acid
residues comprised in the polypeptide are proline residues.
[0069] The polypeptide may comprise a plurality of amino acid
repeats, wherein said repeat consists of proline and alanine
residues and wherein no more than 6 consecutive amino acid residues
are identical. Particularly, the polypeptide may comprise or
consist of the amino acid sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO:
5) or circular permuted versions or (a) multimers(s) of the
sequences as a whole or parts of the sequence.
[0070] Preferably, the polypeptide comprises or consists of the
amino acid sequence as shown in SEQ ID NO: 7 or 9, or the
polypeptide comprises or consists of an amino acid sequence encoded
by a nucleic acid having a nucleotide sequence as shown in SEQ ID
NO: 8 or 10. It is preferred herein that the modified protein (a)
comprises or consists of an amino acid sequence as shown in SEQ ID
NO: 11 or 13; or (b) comprises or consists of an amino acid
sequence encoded by a nucleic acid having a nucleotide sequence as
shown in SEQ ID NO: 12 or 14. In one aspect, the polypeptide is a
random coil polypeptide.
[0071] In some aspects, the modified protein, e.g. the fusion
protein, has an asparaginase or glutaminase activity higher than
that of the unconjugated L-asparaginase. For example, the modified
proteins can have an asparaginase or glutaminase activity at least
5% and/or up to 30% (e.g. at least 10%, 15%, 20%, 25% (or more))
higher than that of the unmodified L-asparaginase, particularly as
assessed by the Nessler assay. In further aspects, the
L-asparaginase in the modified protein, e.g. in the fusion protein,
is covalently linked to a terminal residue of the polypeptide
directly by an amine bond, and/or the fusion protein is
manufactured recombinantly. In preferred aspects, the modified
protein, e.g. the fusion protein, includes a linker between the
L-asparaginase and the polypeptide. An exemplary linker may be an
alanine amino acid residue. The invention also relates to a
pharmaceutical composition comprising the modified protein, e.g.
the fusion protein, or its use in therapy, or for use as a
medicament, or for use in medicine.
[0072] The invention also relates to a nucleic acid encoding the
modified protein, particularly a fusion protein as defined herein.
Preferably, the nucleic acid is selected from the group consisting
of: (a) the nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO: 12 or 14; (b) the nucleic acid molecule
comprising the nucleotide sequence having at least 85% identity to
the nucleotide sequence as defined in (a); and (c) the nucleic acid
molecule being degenerate as a result of the genetic code to the
nucleotide sequence as defined in (a).
[0073] One aspect of the invention further relates to a process for
the preparation of a modified protein as defined herein or of a
nucleic acid as defined herein. The process may comprise culturing
a host cell as defined herein and isolating said modified protein
from the culture or from said cell. The process of preparing the
modified protein as defined herein, particularly the fusion
protein, can comprise culturing a host cell transformed with or
comprising a vector comprising a nucleic acid encoding the modified
protein, particularly the fusion protein, under conditions causing
expression of the modified protein, particularly of the fusion
protein. In some aspects, the host cell is selected from the group
recited above.
[0074] The invention further relates to a method of treating a
disease treatable by L-asparagine depletion in a patient, said
method comprising administering to said patient an effective amount
of the modified protein as defined herein, e.g. the fusion protein.
The disease treatable by L-asparagine depletion may be a cancer.
The modified protein as defined herein may elicit a lower
immunogenic response in the patient compared to unconjugated
L-asparaginase, may have a longer in vivo circulating half-life
after a single dose compared to the unconjugated L-asparaginase,
and/or may have a greater AUC value after a single dose compared to
the L-asparaginase (particularly the unconjugated
L-asparaginase).
[0075] The problem to be solved by the invention can be seen to be
the provision of an L-asparaginase preparation with: high in vitro
bioactivity; a stable protein-modifier linkage; prolonged in vivo
half-life; significantly reduced immunogenicity, as evidenced, for
example, by the reduction or elimination of an antibody response
against the L-asparaginase preparation following repeated
administrations; and/or usefulness as a second-line therapy for
patients who have developed sensitivity to first-line therapies
using, e g , non-E. coli-derived L-asparaginases.
[0076] This problem is solved according to the present invention by
the embodiments characterized in the claims, in particular by
providing a modified protein comprising an L-asparaginase and a
modifier, i.e. (ii) one or more (poly)peptide(s), wherein the
(poly)peptide consists solely of proline and alanine amino acid
residues, and by providing methods for preparing and using the
same.
[0077] In one aspect, described herein is a modified L-asparaginase
with improved pharmacological properties as compared with the
unmodified L-asparaginase protein.
[0078] The term "modified L-asparaginase" as used herein refers to
"a modified protein comprising (i) L-asparaginase and (ii) one or
more (poly)peptide(s), wherein the (poly)peptide consists solely of
proline and alanine amino acid residues" as defined and described
herein. In one aspect of the invention the L-asparaginase is
derived from Erwinia having at least 85% identity to the amino acid
of SEQ ID NO: 1.
[0079] The modified L-asparaginase described herein, e.g.,
L-asparaginase conjugated or fused to one or more (poly)peptide(s),
wherein the (poly)peptide consists solely of proline and alanine
amino acid residues, serves as a therapeutic agent particularly for
use in patients who show hypersensitivity (e.g., an allergic
reaction or silent hypersensitivity) to treatment with
L-asparaginase or PEGylated L-asparaginase from Erwinia and/or E.
coli, or unmodified L-asparaginase from Erwinia. The modified
L-asparaginase described herein is also useful as a therapeutic
agent for use in patients who have had a disease relapse, e.g., a
relapse of ALL, and have been previously treated with another form
of asparaginase.
[0080] Erwinia chrysanthemi (also known as Pectobacterium
chrysanthemi) has been renamed Dickeya chrysanthemi. Thus, the
terms Erwinia chrysanthemi, Pectobacterium chrysanthemi and Dickeya
chrysanthemi are used interchangeably herein.
[0081] Unless otherwise expressly defined, the terms used herein
will be understood according to their ordinary meaning in the
art.
[0082] As used herein, the term "including" means "including,
without limitation," and terms used in the singular shall include
the plural, and vice versa, unless the context dictates
otherwise.
[0083] As used herein, the terms "comprising", "including",
"having" or grammatical variants thereof are to be taken as
specifying the stated features, integers, steps or components but
do not preclude the addition of one or more additional features,
integers, steps, components or groups thereof. The terms
"comprising"/"including"/"having" encompass the terms "consisting
of" and "consisting essentially of". Thus, whenever the terms
"comprising"/"including"/"having" are used herein, they can be
replaced by "consisting essentially of" or, preferably, by
"consisting of".
[0084] The terms "comprising"/"including"/"having" mean that any
further component (or likewise features, integers, steps and the
like) can be present.
[0085] The term "consisting of" means that no further component (or
likewise features, integers, steps and the like) can be
present.
[0086] The term "consisting essentially of" or grammatical variants
thereof when used herein are to be taken as specifying the stated
features, integers, steps or components but do not preclude the
addition of one or more additional features, integers, steps,
components or groups thereof but only if the additional features,
integers, steps, components or groups thereof do not materially
alter the basic and novel characteristics of the claimed product,
composition, device or method and the like.
[0087] Thus, the term "consisting essentially of" means that
specific further components (or likewise features, integers, steps
and the like) can be present, namely those not materially affecting
the essential characteristics of the product, composition, device
or method. In other words, the term "consisting essentially of"
(which can be interchangeably used herein with the term "comprising
substantially"), allows the presence of other components in the
product, composition, device or method in addition to the mandatory
components (or likewise features, integers, steps and the like),
provided that the essential characteristics of the product,
composition, device or method are not materially affected by the
presence of other components.
[0088] As used herein, the term "about" refers to .+-.10%, unless
indicated otherwise herein.
[0089] As used herein, "a" or "an" may mean one or more.
[0090] As used herein, the term "disease treatable by depletion of
asparagine" refers to a condition or disorder wherein the cells
involved in or responsible for the condition or disorder either
lack or have a reduced ability to synthesize L-asparagine.
Depletion or deprivation of L-asparagine can be partial or
substantially complete (e.g., to levels that are undetectable using
methods and apparatus that are known in the art).
[0091] As used herein, the term "therapeutically effective amount"
refers to the amount of a protein (e.g., asparaginase or modified
protein thereof), required to produce a desired therapeutic
effect.
[0092] As used herein, the term, "L-asparaginase" is an enzyme with
L-asparagine aminohydrolase activity. L-asparaginase's enzymatic
activity may include not only deamidation of asparagine to aspartic
acid and ammonia, but also deamidation of glutamine to glutamic
acid and ammonia. Asparaginases are typically composed of four
monomers (although some have been reported with five or six). Each
monomer can be about 32,000 to about 36,000 daltons.
[0093] Many L-asparaginase proteins have been identified in the
art, isolated by known methods from microorganisms. (See, e.g.,
Savitri and Azmi, Indian J. Biotechnol 2 (2003) 184-194,
incorporated herein by reference in its entirety). The most widely
used and commercially available L-asparaginases are derived from E.
coli or from Erwinia chrysanthemi, both of which share 50% or less
structural homology.
[0094] The following relates to "L-asparaginase" to be used in
accordance with the invention. Within the Erwinia species,
typically 75-77% sequence identity was reported between Erwinia
chrysanthemi and Erwinia carotovora-derived enzymes, and
approximately 90% sequence identity was found between different
subspecies of Erwinia chrysanthemi (Kotzia (2007), Journal of
Biotechnology 127, 657-669, incorporated herein by reference in its
entirety). Some representative Erwinia L-asparaginases include, for
example, those provided in Table 1 below which discloses percent
sequence identity to Erwinia Chrysanthemi NCPPB 1066:
TABLE-US-00001 TABLE 1 Species Accession No. % Identity Erwinia
chrysanthemi 3937 AAS67028 91% Erwinia chrysanthemi NCPPB 1125
CAA31239 98% Erwinia carotovora subsp. atroseptica AAS67027 75%
Erwinia carotovora AAP92666 77%
[0095] The sequences of the Erwinia L-asparaginases and the GenBank
entries of Table 1 are herein incorporated by reference. Exemplary
L-asparaginases used in therapy are L-asparaginase isolated from E.
coli and from Erwinia, specifically, Erwinia chrysanthemi.
[0096] The L-asparaginases may be native enzymes isolated from the
microorganisms. They can also be produced by recombinant enzyme
technologies in producing microorganisms such as E. coli. As
examples, the protein used in the modified protein of the invention
can be a recombinant protein produced in an E. coli strain,
preferably a protein from an Erwinia species, particularly Erwinia
chrysanthemi, produced in a recombinant E. coli strain.
[0097] Enzymes can be identified by their specific activities. This
definition thus includes all polypeptides that have the defined
specific activity also present in other organisms, more
particularly in other microorganisms. Often enzymes with similar
activities can be identified by their grouping to certain families
defined as PFAM or COG. PFAM (protein family database of alignments
and hidden Markov models; pfam.xfam.org) represents a large
collection of protein sequence alignments. Each PFAM makes it
possible to visualize multiple alignments, see protein domains,
evaluate distribution among organisms, gain access to other
databases, and visualize known protein structures. COGs (Clusters
of Orthologous Groups of proteins; ncbi.nlm.nih.gov/COG/) are
obtained by comparing protein sequences from 43 fully sequenced
genomes representing 30 major phylogenetic lines. Each COG is
defined from at least three lines, which permits the identification
of former conserved domains.
[0098] The means of identifying percentage sequence identity are
well known to those skilled in the art, and include in particular
the BLAST programs, which can be used from the website
blast.ncbi.olo.nih.gov/Blast.cgi with the default parameters
indicated on that website. The sequences obtained can then be
exploited (e.g., aligned) using, for example, the program CLUSTALW
(ebi.ac.uk/Tools/clustalw2/index.html) with the default parameters.
Using the references given on GenBank for known genes, those
skilled in the art are able to determine the equivalent genes in
other organisms, bacterial strains, yeasts, fungi, mammals, plants,
etc. This routine work is advantageously done using consensus
sequences that can be determined by carrying out sequence
alignments with genes derived from other microorganisms, and
designing degenerate probes to clone the corresponding gene in
another organism.
[0099] The person skilled in the art will understand how to select
and design proteins retaining substantially their L-asparaginase
activity. One approach for the measuring L-asparaginase activity is
a Nessler assay as described by Mashburn (1963) Biochem. Biophys.
Res. Commun. 12, 50 (incorporated herein by reference in its
entirety).
[0100] In a particular aspect of the modified protein of the
invention, the L-asparaginase has at least about 85% homology or
sequence identity to the amino acid sequence of SEQ ID NO: 1, more
specifically at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence
identity to the amino acid sequence of SEQ ID NO:1 as set forth in
the attached sequence listing. The terms "homology" and "sequence
identity" are used interchangeably herein.
[0101] The term "comprising the sequence of SEQ ID NO:1" (e.g. if
the L-asparaginase has 100% homology or sequence identity to the
amino acid sequence of SEQ ID NO: 1) means that the amino-acid
sequence of the asparaginase may not be strictly limited to SEQ ID
NO:1 but may contain one, two, three, four, five, six, seven,
eight, nine, ten or more additional amino-acids. In other words, if
the L-asparaginase to be used herein has 100% homology or sequence
identity to the amino acid sequence of SEQ ID NO: 1, the
L-asparaginase can comprise or consist of the amino acid sequence
of SEQ ID NO: 1. The term "comprising" means in this context that
the amino acid sequence of the L-asparaginase of SEQ ID NO: 1 may
contain one, two, three, four, five, six, seven, eight, nine, ten
or more additional amino-acids.
[0102] In a particular aspect, the protein is the L-asparaginase of
Erwinia chrysanthemi comprising or consisting of the sequence of
SEQ ID NO: 1. In another aspect, the L-asparaginase is from Erwinia
chrysanthemi NCPPB 1066 (Genbank Accession No. CAA32884,
incorporated herein by reference in its entirety), either with or
without signal peptides and/or leader sequences.
[0103] Fragments of the L-asparaginase, preferably the
L-asparaginase of SEQ ID NO:1, are also comprised within the
definition of the L-asparaginase used in the modified protein of
the invention. The term "a fragment of asparaginase" (e.g. a
fragment of the asparaginase of SEQ ID NO: 1) means that the
sequence of the asparaginase may include less amino-acid than in
the asparaginases exemplified herein (e.g. the asparaginase of SEQ
ID NO: 1) but still enough amino-acids to confer L-aminohydrolase
activity. For example, the "fragment of asparaginase" is a fragment
that is/consists of at least about 150 or 200 contiguous amino
acids of one of the asparaginases exemplified herein (e.g. the
asparaginase of SEQ ID NO: 1) (e.g. about 150, 160, 170, 180, 190,
200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,
321, 322, 323, 324, 325, 326 contiguous amino acids) and/or wherein
said fragment has up to 50 amino acids deleted from the N-terminus
of said asparaginase exemplified herein (e.g. the asparaginase of
SEQ ID NO: 1) (e.g. up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 35, 40, 45, or 50) and/or has up to up to 75 or 100 amino
acids deleted from the C-terminus of said asparaginase exemplified
herein (e.g. the asparaginase of SEQ ID NO: 1) (e.g. up to 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70,
75, 80, 85, 90, 95 or 100) and/or has deleted amino acids at both
the N-terminus and the C-terminus of said asparaginase exemplified
herein (e.g. the asparaginase of SEQ ID NO: 1), wherein the total
number of amino acids deleted can be up to 125 or 150 amino
acids.
[0104] It is well known in the art that a polypeptide can be
modified by substitution, insertion, deletion and/or addition of
one or more amino-acids while retaining its enzymatic activity. The
term "one or more amino acids" in this context can refer to one,
two, three, four, five, six, seven, eight, nine, ten or more amino
acids. For example, substitution of one amino-acid at a given
position by a chemically equivalent amino-acid that does not affect
the functional properties of a protein is common Substitutions may
be defined as exchanges within one of the following groups: [0105]
Small aliphatic, non-polar or slightly polar residues: Ala, Ser,
Thr, Pro, Gly [0106] Polar, negatively charged residues and their
amides: Asp, Asn, Glu, Gln [0107] Polar, positively charged
residues: His, Arg, Lys [0108] Large aliphatic, non-polar residues:
Met, Leu, Ile, Val, Cys [0109] Large aromatic residues: Phe, Tyr,
Trp.
[0110] Thus, changes that result in the substitution of one
negatively charged residue for another (such as glutamic acid for
aspartic acid) or one positively charged residue for another (such
as lysine for arginine) can be expected to produce a functionally
equivalent product.
[0111] The positions where the amino-acids are modified and the
number of amino-acids subject to modification in the amino-acid
sequence are not particularly limited. The skilled artisan is able
to recognize the modifications that can be introduced without
affecting the activity of the protein. For example, modifications
in the N- or C-terminal portion of a protein may be expected not to
alter the activity of a protein under certain circumstances. With
respect to asparaginases, in particular, much characterization has
been done, particularly with respect to the sequences, structures,
and the residues forming the active catalytic site. This provides
guidance with respect to residues that can be modified without
affecting the activity of the enzyme. All known L-asparaginases
from bacterial sources have common structural features. All are
homotetramers with four active sites between the N- and C-terminal
domains of two adjacent monomers (Aghaipour (2001) Biochemistry 40,
5655-5664, incorporated herein by reference in its entirety). All
have a high degree of similarity in their tertiary and quaternary
structures (Papageorgiou (2008) FEBS J. 275, 4306-4316,
incorporated herein by reference in its entirety). The sequences of
the catalytic sites of L-asparaginases are highly conserved between
Erwinia chrysanthemi, Erwinia carotovora, and E. coli
L-asparaginase II (Id). The active site flexible loop contains
amino acid residues 14-33, and structural analysis show that Thr15,
Thr95, Ser62, Glu63, Asp96, and Ala120 contact the ligand (Id).
Aghaipour et al. have conducted a detailed analysis of the four
active sites of Erwinia chrysanthemi L- asparaginase by examining
high resolution crystal structures of the enzyme complexed with its
substrates (Aghaipour (2001) Biochemistry 40, 5655-5664). Kotzia et
al. provide sequences for L-asparaginases from several species and
subspecies of Erwinia and, even though the proteins have only about
75-77% identity between Erwinia chrysanthemi and Erwinia
carotovora, they each still have L-asparaginase activity (Kotzia
(2007) J. Biotechnol. 127, 657-669). Moola et al performed epitope
mapping studies of Erwinia chrysanthemi 3937 L-asparaginase and
were able to retain enzyme activity even after mutating various
antigenic sequences in an attempt to reduce immunogenicity of the
asparaginase (Moola (1994) Biochem. J. 302, 921-927). In view of
the extensive characterization that has been performed on
L-asparaginases, one of skill in the art could determine how to
make fragments and/or sequence substitutions while still retaining
enzyme activity.
[0112] As used herein, the term "about" modifying, for example, the
dimensions, volumes, quantity of an ingredient in a composition,
concentrations, process temperature, process time, yields, flow
rates, pressures, and like values, and ranges thereof, refers to
variation in the numerical quantity that can occur, for example,
through typical measuring and handling procedures used for making
compounds, compositions, concentrates or use formulations; through
inadvertent error in these procedures; through differences in the
manufacture, source, or purity of starting materials or ingredients
used to carry out the methods; and like considerations. The term
"about" also encompasses amounts that differ due to aging of, for
example, a composition, formulation, or cell culture with a
particular initial concentration or mixture, and amounts that
differ due to mixing or processing a composition or formulation
with a particular initial concentration or mixture. Whether
modified by the term "about" the claims appended hereto include
equivalents to these quantities. The term "about" further may refer
to a range of values that are similar to the stated reference
value. In certain embodiments, the term "about" refers to a range
of values that fall within 10, 9, 8,7, 6, 5,4, 3, 2, 1 percent or
less of the stated reference value.
[0113] In the context of the present invention, it has surprisingly
been found that the chemical conjugation of one or more peptides
consisting solely of proline and alanine amino acid residues via a
specific C-terminal amino acid residue (R.sup.C) to L-asparaginase
allows to provide an L-asparaginase modified protein having a
particularly high coupling ratio of said peptides per molecule of
asparaginase and, thus, a considerably reduced immunogenicity and
enhanced plasma half-life. It has further been found that this
novel technique can also be applied to L-asparaginase without
impairing its catalytic activity, which greatly enhances the
therapeutic value of the corresponding modified proteins described
herein.
[0114] In one aspect, described herein is a modified protein
comprising (i) an L-asparaginase having at least 85, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99 or 100% identity to the amino acid
sequence of SEQ ID NO: 1 and (ii) one or more peptide(s), wherein
the peptide consists solely of proline and alanine amino acid
residues.
[0115] In a preferred aspect, the modified protein is a modified
protein of L-asparaginase and one or more peptide(s), wherein each
is independently a peptide R.sup.N-(P/A)-R.sup.C, wherein (P/A) is
an amino acid sequence consisting solely of proline and alanine
amino acid residues, wherein R.sup.N is a protecting group attached
to the N-terminal amino group of the amino acid sequence, and
wherein R.sup.C is an amino acid residue bound via its amino group
to the C-terminal carboxy group of the amino acid sequence, wherein
each peptide is conjugated to the L-asparaginase via an amide
linkage formed from the carboxy group of the C-terminal amino acid
residue R.sup.C of the peptide and a free amino group of the
L-asparaginase, and wherein at least one of the free amino groups,
which the peptides are conjugated to, is not an N-terminal a-amino
group of the L-asparaginase.
[0116] In some aspect, the monomer of the modified protein has from
about 350, 400, 450, 500, amino acids to about 550, 600, 650, 700,
or 750 amino acids after modification. In additional aspects, the
modified protein has from about 350 to about 750 amino acids, or
about 500 to about 750 amino acids.
[0117] Each peptide that is comprised in the modified protein as
described herein is independently a peptide R.sup.N-(P/A)-R.sup.C.
Accordingly, for each of the peptides comprised in a modified
protein described herein, the N-terminal protecting group R.sup.N,
the amino acid sequence (P/A), and the C-terminal amino acid
residue R.sup.C are each independently selected from their
respective meanings. The two or more peptides comprised in the
modified protein may thus be the same, or they may be different
from one another. In one aspect, all of the peptides comprised in
the modified protein are the same.
[0118] Furthermore, the peptides comprised in the modified protein
preferably adopt a random coil conformation, particularly when the
modified protein is present in an aqueous environment (e.g., an
aqueous solution or an aqueous buffer). The presence of a random
coil conformation can be determined using methods known in the art,
in particular by means of spectroscopic techniques, such as
circular dichroism (CD) spectroscopy.
[0119] The moiety (P/A) in the chemically conjugated modified
protein, which is comprised in the peptide R.sup.N-(P/A)-R.sup.C,
is an amino acid sequence that can consist of a total of between 10
to 100 or more proline and alanine amino acid residues, a total of
15 to 60 proline and alanine amino acid residues, a total of 15 to
45 proline and alanine amino acid residues, e.g. a total of 20 to
about 40 proline and alanine amino acid residues, e.g. 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 proline and alanine
amino acid residues. In a preferred aspect, said amino acid
sequence consists of about 20 proline and alanine amino acid
residues. In another preferred aspect, said amino acid sequence
consists of about 40 proline and alanine amino acid residues. In
the peptide R.sup.N-(P/A)-R.sup.C, the ratio of the number of
proline residues comprised in the moiety (P/A) to the total number
of amino acid residues comprised in (P/A) is preferably .gtoreq.10%
and .ltoreq.70%, more preferably .gtoreq.20% and .ltoreq.50%, and
even more preferably .gtoreq.25% and .ltoreq.40%. Accordingly, it
is preferred that 10% to 70% of the total number of amino acid
residues in (P/A) are proline residues; more preferably, 20% to 50%
of the total number of amino acid residues comprised in (P/A) are
proline residues; and even more preferably, 25% to 40% (e.g., 25%,
30%, 35% or 40%) of the total number of amino acid residues
comprised in (P/A) are proline residues. Moreover, it is preferred
that (P/A) does not contain any consecutive proline residues (i.e.,
that it does not contain any partial sequence PP). In a preferred
aspect, (P/A) is the amino acid sequence AAPAAPAPAAPAAPAPAAPA (SEQ
ID NO: 5). In another preferred aspect, (P/A) is the amino acid
sequence AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA (SEQ ID NO:
15).
[0120] The group R.sup.N in the peptide R.sup.N-(P/A)-R.sup.C may
be a protecting group which is attached to the N-terminal amino
group, particularly the N-terminal .alpha.-amino group, of the
amino acid sequence (P/A). It is preferred that R.sup.N is
pyroglutamoyl or acetyl.
[0121] The group R.sup.C in the peptide R.sup.N-(P/A)-R.sup.C is an
amino acid residue which is bound via its amino group to the
C-terminal carboxy group of (P/A), and which comprises at least two
carbon atoms between its amino group and its carboxy group. It will
be understood that the at least two carbon atoms between the amino
group and the carboxy group of R.sup.C may provide a distance of at
least two carbon atoms between the amino group and the carboxy
group of R.sup.C (which is the case if, e.g., R.sup.C is an
.omega.-amino-C.sub.3-15 alkanoic acid, such as
.epsilon.-aminohexanoic acid). It is preferred that R.sup.C is
.epsilon.-aminohexanoic acid.
[0122] In one embodiment, the peptide is
Pga-AAPAAPAPAAPAAPAPAAPA-Ahx-COOH (SEQ ID NO: 16) or
Pga-AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA-Ahx-COOH (SEQ ID NO:
17). The term "Pga" is an abbreviation of "pyroglutamoyl" or
"pyroglutamic acid". The term "Ahx" is an abbreviation of
".epsilon.-aminohexanoic acid".
[0123] As also demonstrated in the appended examples, it has
surprisingly been found that the use of a C-terminal amino acid
residue R.sup.C as defined herein, including in particular
.epsilon.-aminohexanoic acid, allows to provide modified proteins
with an advantageously high coupling ratio of peptides consisting
solely of proline and alanine amino acid residues per molecule of
asparaginase and, thus, an advantageously reduced immunogenicity
and an advantageously enhanced plasma half-life.
[0124] In the modified proteins as described herein, each peptide
R.sup.N-(P/A)-R.sup.C, can be conjugated to the L-asparaginase via
an amide linkage formed from the carboxy group of the C-terminal
amino acid residue R.sup.C of the peptide and a free amino group of
the L-asparaginase. A free amino group of the L-asparaginase may
be, e.g., an N-terminal .alpha.-amino group or a side-chain amino
group of the L-asparaginase (e.g., an .epsilon.-amino group of a
lysine residue comprised in the L-asparaginase). If the
L-asparaginase is composed of multiple subunits, e.g. if the
L-asparaginase is a tetramer, there may be multiple N-terminal
.alpha.-amino groups (i.e., one on each subunit). In one aspect, 9
to 13 peptides as defined herein (e.g. 9, 11, 12, or 13 peptides)
can be chemically conjugated to the L-asparaginase (e.g. to each
subunit/monomer of the L-asparaginase).
[0125] In accordance with the above, in one aspect at least one of
the free amino groups, which the peptides are chemically conjugated
to, is not (i.e., is different from) an N-terminal .alpha.-amino
group of the L-asparaginase. Accordingly, it is preferred that at
least one of the free amino groups, which the peptides are
conjugated to, is a side-chain amino group of the L-asparaginase,
and it is particularly preferred that at least one of the free
amino groups, which the peptides are conjugated to, is an
.epsilon.-amino group of a lysine residue of the
L-asparaginase.
[0126] Moreover, it is preferred that the free amino groups, which
the peptides are conjugated to, are selected from the
.epsilon.-amino group(s) of any lysine residue(s) of the
L-asparaginase, the N-terminal .alpha.-amino group(s) of the
L-asparaginase or of any subunit(s) of the L-asparaginase, and any
combination thereof. It is particularly preferred that one of the
free amino groups, which the peptides are conjugated to, is an
N-terminal .alpha.-amino group, while the other one(s) of the free
amino groups, which the peptides are conjugated to, is/are each an
.epsilon.-amino group of a lysine residue of the L-asparaginase.
Alternatively, it is preferred that each of the free amino groups,
which the peptides are conjugated to, is an .epsilon.-amino group
of a lysine residue of the L-asparaginase.
[0127] The modified proteins as described herein are composed of
L-asparaginase and one or more peptides as defined herein. A
corresponding modified protein may, e.g., consist of one
L-asparaginase and one, two, three, four, five, six, seven, eight,
nine, ten, 15, 20, 25, 30, 35, 40, 45, 50, 55 (or more) peptides
which are each conjugated to the L-asparaginase. The L-asparaginase
may be, e.g., a monomeric protein or a protein composed of multiple
subunits, e.g. a tetramer. If the L-asparaginase is a monomeric
protein, a corresponding modified protein may, e.g., consist of one
monomeric L-asparaginase and nine to thirteen (or more) (e.g. 9,
10, 11, 12, or 13), peptides which are each conjugated to the
monomeric L-asparaginase. An exemplary amino acid sequence of a
monomeric L-asparaginase is shown in SEQ ID NO: 1. If the
L-asparaginase is a protein composed of multiple subunits, e.g. of
four subunits (i.e. if said L-asparaginase is a tetramer), a
corresponding modified protein may, e.g., consist of four
L-asparaginase subunits and nine to thirteen (or more) (e.g. 9, 10,
11, 12, or 13), peptides as defined herein which are each
conjugated to each subunit of the L-asparaginase. An exemplary
amino acid sequence of a subunit of L-asparaginase is shown in SEQ
ID NO. 1. Likewise, if the L-asparaginase is a protein composed of
multiple subunits, e.g. of four subunits (i.e. if said
L-asparaginase is a tetramer), a corresponding modified protein
may, e.g., consist of four L-asparaginase subunits and 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55 (or more) peptides which are each conjugated
to the L-asparaginase tetramer. In one aspect the invention relates
to a modified protein having an L-asparaginase and multiple
chemically attached peptide sequences. In a further aspect the
length of the peptide sequences are from about 10 to about 100,
from about 15 to about 60 or from about 20 to about 40.
[0128] The peptide consisting solely of proline and alanine amino
acid residues may be covalently linked to one or more amino acids
of said L-asparaginase, such as lysine residues and/or N-terminal
residue, and/or the peptide consisting solely of proline and
alanine amino acid residues may be covalently linked to at least
from about 40, 50, 60, 70, 80 or 90% to about 60, 70, 80, 90 or
100% of the accessible amino groups including amino groups of
lysine residues and/or N-terminal residue on the surface of the
L-asparaginase. For example, there may be about 11 to 12 lysine
residues accessible per L-asparaginase, and about 9 to 12 lysines
would be conjugated to the peptide consisting solely of proline and
alanine amino acid residues. In further aspects, the peptide
consisting solely of proline and alanine amino acid residues is
covalently linked to from about 20, 30, 40, 50, or 60% to about 30,
40, 50, 60, 70, 80 or 90% of total lysine residues of said
L-asparaginase. In further embodiments, the peptide consisting
solely of proline and alanine amino acid residues is covalently
linked to the L-asparaginase via a linker. Exemplary linkers
include linkers disclosed in U.S. Patent Application Publication
No. 2015/0037359, which is herein incorporated by reference in its
entirety.
[0129] In addition, said modified protein may have a half-life of
at least about 5, 10, 12, 15, 24, 36, 48, 60, 72, 84 or 96 hours at
a dose of about 25 .mu.g protein/kg, and/or a longer in vivo
circulating half-life compared to the unmodified L-asparaginase.
Moreover, said modified protein may have a greater area under the
plasma drug concentration-time curve (AUC) compared to the
L-asparaginase.
[0130] The modified protein according to the present invention can
be prepared using methods known in the art. In particular, it can
be prepared using the process described in the following, and/or in
accordance with or in analogy to the procedures described in the
examples.
[0131] The invention further relates to a process of preparing a
modified protein as defined herein, the process comprising: (a)
coupling an activated peptide of the formula
R.sup.N-(P/A)-R.sup.C-act, wherein R.sup.C-act is a
carboxy-activated form of R.sup.C, wherein R.sup.C and (P/A) are as
defined in the modified protein to be prepared, and wherein R.sup.N
is a protecting group which is attached to the N-terminal amino
group of (P/A), with L-asparaginase to obtain a modified protein of
the L-asparaginase and peptides in which R.sup.N is a protecting
group.
[0132] The carboxy-activated C-terminal amino acid residue
R.sup.C-act, which is comprised in activated peptide, may be any
amino acid residue R.sup.C, as described and defined herein with
respect to the peptide, wherein the carboxy group of R.sup.C is in
the form of an activated carboxy group. Preferably, the activated
carboxy group of the amino acid residue R.sup.C-act in the
activated peptide is an active ester group.
[0133] If the activated carboxy group of R.sup.C-act is an active
ester group, it is preferably selected from any one the following
active ester groups:
##STR00001##
[0134] A particularly preferred active ester group is a
1-hydroxybenzotriazole (HOBt) active ester group. Accordingly, it
is particularly preferred that the activated carboxy group of
R.sup.C-act is a group of the following formula:
##STR00002##
[0135] The process may additionally comprise, before step (a), a
further step of converting a peptide of the formula
R.sup.N-(P/A)-R.sup.C, wherein R.sup.C and (P/A) are as defined in
the modified protein to be prepared, and wherein R.sup.N is a
protecting group which is attached to the N-terminal amino group of
(P/A), into the activated P/A peptide.
[0136] For example, in order to obtain an activated peptide having
a 1-hydroxybenzotriazole active ester group as the activated
carboxy group of R.sup.C-act, the step of converting the peptide
into the activated peptide can be conducted by reacting the peptide
with a salt of a phosphonium, uronium or immonium ester of
1-hydroxybenzotriazole (HOBt) in the presence of a base. The salt
of the phosphonium, uronium or immonium derivative of HOBt is
preferably O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU).
[0137] The coupling step (a) and the preceding optional step of
converting a peptide into an activated peptide can be conducted,
e.g., using any of the peptide coupling or amide bond formation
procedures described in the literature, e.g., in any of: El-Faham
et al., (2011) Chem Rev. 111(11), 6557-6602; Montalbetti et al.,
(2005) Tetrahedron, 61(46), 10827-10852; Klose et al. (1999) Chem.
Commun. 18, 1847-1848; Carpino et al. (1995) J. Am. Chem. Soc.
117(19), 5401-5402); Valeur et al., (2009) Chem. Soc. Rev., 38(2),
606-631; or Hermanson, (2013) Bioconjugate techniques. Third
edition. Academic press. Suitable reagents and reaction conditions
for such procedures are further described in the aforementioned
literature and in the further references cited therein. Additional
descriptions are found in U.S. Pat. No. 8,563,521; 9,260,494; and
9,221,882, all of which are incorporated by references herein in
their entirety.
[0138] Procedures for removing the protecting groups R.sup.N, as
required in the optional step (b), are well-known in the art and
are described, e.g., in Wuts et al., (2012) Greene's Protective
Groups in Organic Synthesis. Fourth Edition. John Wiley & Sons,
and/or in Isidro-Llobet et al., (2009) Chem. Rev. 109(6),
2455-2504. The optional step (b) can thus be conducted, e.g., as
described for the corresponding protecting group R.sup.N in any of
the aforementioned references.
[0139] In some aspects, the invention relates to a modified protein
comprising (i) an L-asparaginase having at least 85, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99 or 100% identity to the amino acid
sequence of SEQ ID NO: 1 and (ii) and a polypeptide, wherein the
polypeptide consists solely of proline and alanine amino acid
residues. In one aspect, the modified protein is a fusion protein.
The polypeptide consisting solely of proline and alanine amino acid
residues may have a length of about 200 to about 400 proline and
alanine amino acid residues. In other words the polypeptide may
consist of about 200 to about 400 proline and alanine amino acid
residues. In a preferred aspect, the polypeptide consists of a
total of about 200 (e.g. 201) proline and alanine amino acid
residues (i.e. has a length of about 200 (e.g. 201) proline and
alanine amino acid residues) or the polypeptide consists of a total
of about 400 (e.g. 401) proline and alanine amino acid residues
(i.e. has a length of about 400 (e.g. 401) proline and alanine
amino acid residues). In some preferred embodiments, the
polypeptide comprises or consists of an amino acid sequence as
shown in SEQ ID NO: 7 or 9; or the polypeptide comprises or
consists of an amino acid sequence encoded by a nucleic acid having
a nucleotide sequence as shown in SEQ ID NO: 8 or 10. In some
aspects, the modified protein, preferably wherein the modified
protein is a fusion protein, and each monomer has from about 350,
400, 450, 500, amino acids to about 550, 600, 650, 700, 750 or
1,000 amino acids including the monomer and the P/A amino acid
sequence. In additional aspects, the modified protein has from
about 350 to about 800 amino acids or about 500 to about 750 amino
acids.
[0140] For example, the polypeptide includes the peptides prepared
in U.S. Pat. No. 9,221,882.
[0141] In a preferred aspect, the modified protein (a) comprises or
consists of an amino acid sequence as shown in SEQ ID NO: 11 or 13;
or (b) comprises or consists of an amino acid sequence encoded by a
nucleic acid having a nucleotide sequence as shown in SEQ ID NO: 12
or 14. It is contemplated herein that the modified protein
comprises (a) a protein having an amino acid sequence as shown in
SEQ ID NO: 11 or 13; (b) a protein as defined in (a) wherein one to
65 amino acids are deleted, inserted, added or substituted in the
asparaginase; (c) a protein encoded by a nucleic acid having a
nucleotide sequence as shown in SEQ ID NO: 12 or 14; (d) a protein
having an amino acid sequence encoded by a nucleic acid hybridizing
under stringent conditions to the complementary strand of nucleic
acid molecules as defined in (c); (e) a protein having at least 85%
identity to the protein of any one of (a) to (d); and (f) a protein
having an amino acid sequence encoded by a nucleic acid being
degenerate as a result of the genetic code to the nucleotide
sequence of a nucleic acid as defined in (c) or (d).
[0142] The modified protein as defined herein may be composed of
four subunits, wherein the subunits are selected from the group
consisting of (a) a protein having an amino acid sequence as shown
in SEQ ID NO: 1; (b) a protein as defined in (a) wherein one to 65
amino acids are deleted, inserted, added or substituted in the
asparaginase; (c) a protein encoded by a nucleic acid molecule
having a nucleotide sequence as shown in SEQ ID NO: 2; (d) a
protein having an amino acid sequence encoded by a nucleic acid
hybridizing under stringent conditions to the complementary strand
of nucleic acid molecules as defined in (c); (e) a protein having
at least 85% identity to the protein of any one of (a) to (d); and
(f) a protein having an amino acid sequence encoded by a nucleic
acid being degenerate as a result of the genetic code to the
nucleotide sequence of a nucleic acid as defined in (c) or (d).
[0143] The invention relates to a nucleic acid encoding the
modified protein as defined herein, specifically if the modified
protein is a modified protein of the L-asparaginase and a
polypeptide, wherein the polypeptide consists solely of proline and
alanine amino acid residues. In a preferred aspect, the modified
protein is a fusion protein. In a preferred aspect, the nucleic
acid is selected from the group consisting of: (a) the nucleic acid
comprising the nucleotide sequence of SEQ ID NO: 12 or 14; (b) the
nucleic acid comprising the nucleotide sequence having at least 85%
identity to the nucleotide sequence as defined in (a); and (c) the
nucleic acid being degenerate as a result of the genetic code to
the nucleotide sequence as defined in (a).
[0144] In a further aspect, the invention relates to a nucleotide
sequence encoding the fusion protein, including a nucleotide
sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99
or 100% identity to the nucleotide sequence selected from the group
consisting of SEQ ID NO: 12 or 14. While the encoded polypeptide
comprises a repetitive amino acid sequence that may form a random
coil, the encoding nucleic acid comprises preferably a low
repetitive nucleotide sequences. In other words, the nucleic acid
can comprise a nucleotide sequence encoding a PA-rich polypeptide,
wherein said coding nucleotide sequence comprises nucleotide
repeats having a maximum length of 14, 15, 16, 17, about 20, about
25, about 30, about 35, about 40, about 45, about 50 or about 55
nucleotides. The low repetitive nucleic acid as disclosed herein
can be advantageous compared to highly repetitive nucleic acid
molecules. In particular, the genetic stability of the low
repetitive nucleic acid molecules to be used herein can be
improved.
[0145] In some aspects, the nucleotide sequence is a sequence
encoding any of the modified proteins comprising the L-asparaginase
and a polypeptide, wherein the polypeptide consists solely of
proline and alanine amino acid residues, preferably wherein the
modified protein is a fusion protein, described herein, except that
one or more amino acid is added, deleted, inserted or substituted,
with the proviso that the fusion protein having this amino acid
sequence has L-asparaginase activity.
[0146] In additional aspects, the invention relates to a
(recombinant) vector comprising the nucleotide sequence encoding
the modified protein comprising the L-asparaginase and a
polypeptide, wherein the polypeptide consists solely of proline and
alanine amino acid residues, preferably wherein the modified
protein is a fusion protein, as described herein, wherein the
vector can express the modified protein (e.g. fusion protein). In
further aspects, the invention also relates to a host comprising
the (recombinant) vector described herein. The host may be yeasts,
such as Saccharomyces cerevisiae and Pichia Pistoris, bacteria,
actinomycetes, fungi, algae, and other microorganisms, including
Escherichia coli, Bacillus sp., Pseudomonas fluorescens,
Corynebacterium glutamicum and bacterial hosts of the following
genuses, Serratia, Proteus, Acinetobacter and Alcaligenes. Other
hosts are known to those of skill in the art, including
Nocardiopsis alba, which expresses a variant of Asparaginase
lacking on glutaminase-activity (Meena et al. (2014) Bioprocess
Biosyst. Eng. October 2014 Article, which is incorporated by
reference herein in its entirety), and those disclosed in Savitri
et al. (2003) Indian Journal of Biotechnology, 2, 184-194, which is
incorporated by reference herein in its entirety.
[0147] The present invention relates to a vector comprising the
nucleic acid as described herein above, i.e. a nucleic acid
encoding the modified protein as defined herein, particularly a
modified protein of the L-asparaginase and a polypeptide, wherein
the polypeptide consists solely of proline and alanine amino acid
residue, such as a fusion protein. In a preferred aspect, the
nucleic acid is selected from the group consisting of: (a) the
nucleic acid comprising the nucleotide sequence of SEQ ID NO: 12 or
14; (b) the nucleic acid comprising the nucleotide sequence having
at least 85% identity to the nucleotide sequence as defined in (a);
and (c) the nucleic acid being degenerate as a result of the
genetic code to the nucleotide sequence as defined in (a).
[0148] The invention relates to a host cell comprising the nucleic
acid as defined herein or comprising the vector as defined herein.
Example hosts are listed above.
[0149] The invention further relates to a process of preparing the
modified protein as described herein, preferably the fusion
protein, or of the nucleic acid encoding same. The process can
comprise culturing a host cell as defined herein and isolating said
modified protein from the culture or from said cell. The process
can comprise culturing a host cell (e.g. a host cell transformed
with or a host cell comprising the nucleic acid and/or the vector
comprising a nucleotide sequence encoding the modified protein
(preferably the fusion protein) under a condition causing
expression of the modified protein (preferably the fusion protein).
Example hosts are listed above.
[0150] Many suitable vectors are known to those skilled in
molecular biology. The choice of a suitable vector depends on the
function desired, including plasmids, cosmids, viruses,
bacteriophages and other vectors used conventionally in genetic
engineering.
[0151] Methods which are well known to those skilled in the art can
be used to construct various plasmids; see, for example, the
techniques described in Sambrook (2012) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press. Typical
plasmid vectors include, e.g., pQE-12, the pUCseries of plasmids,
pBluescript (Stratagene), the pET series of expression vectors
(Novagen) or pCRTOPO (Invitrogen), lambda gt11, pJOE, the pBBR1-MCS
series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1. Typical vectors
compatible with expression in mammalian cells include E-027 pCAG
Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4
(Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5
(Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo,
pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vector pcDV1
(Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1,
pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech),
pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro
(Novagen) and pCINeo (Promega). Non-limiting examples for plasmid
vectors suitable for Pichia pastoris comprise e.g. the plasmids
pAO815, pPIC9K and pPIC3.5 K (all Invitrogen).
[0152] Generally, vectors can contain one or more origins of
replication (ori) and inheritance systems for cloning or
expression, one or more markers for selection in the host, e.g.,
antibiotic resistance, and one or more expression cassettes.
Examples of suitable origins of replication include, for example,
the full length ColE1, its truncated versions such as those present
on the pUC plasmids, the SV40 viral and the M13 phage origins of
replication. Non-limiting examples of selectable markers include
ampicillin, chloramphenicol, tetracycline, kanamycin, dhfr, gpt,
neomycin, hygromycin, blasticidin or geneticin. Further, said
vector comprises a regulatory sequence that is operably linked to
said nucleotide sequence or the nucleic acid molecule defined
herein.
[0153] The coding sequence(s), e.g., said nucleotide sequence
encoding the polypeptide, comprised in the vector can be linked to
(a) transcriptional regulatory element(s) and/or to other amino
acid encoding sequences using established methods. Such regulatory
sequences are well known to those skilled in the art and include,
without being limiting, regulatory sequences ensuring the
initiation of transcription, internal ribosomal entry sites (IRES)
and, optionally, regulatory elements ensuring termination of
transcription and stabilization of the transcript. Non-limiting
examples for such regulatory sequences ensuring the initiation of
transcription comprise promoters, a translation initiation codon,
enhancers, insulators and/or regulatory elements ensuring
transcription termination. Further examples include Kozak sequences
and intervening sequences flanked by donor and acceptor sites for
RNA splicing, nucleic acid sequences encoding secretion signals or,
depending on the expression system used, signal sequences capable
of directing the expressed protein to a cellular compartment or to
the culture medium.
[0154] Examples of suitable promoters include, without being
limiting, the cytomegalovirus (CMV) promoter, SV40 promoter, RSV
(Rous sarcome virus) promoter, the lacZ promoter, chicken
.beta.-actin promoter, CAG promoter (a combination of chicken
.beta.-actin promoter and cytomegalovirus immediate-early
enhancer), human elongation factor 1.alpha. promoter, AOX1
promoter, GAL1 promoter, CaM-kinase promoter, the lac, trp or tac
promoter, the lacUV5 promoter, the T7 or T5 promoter, the
Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)
polyhedral promoter or a globin intron in mammalian and other
animal cells. One example of an enhancer is, e.g., the SV40
enhancer. Non-limiting additional examples for regulatory
elements/sequences ensuring transcription termination include the
SV40 poly-A site, the tk poly-A site or the AcMNPV polyhedral
polyadenylation signals.
[0155] Furthermore, depending on the expression system, leader
sequences capable of directing the polypeptide to a cellular
compartment or secreting it into the medium may be added to the
coding sequence of the nucleic acid provided herein. The leader
sequence(s) is (are) assembled in frame with translation,
initiation and termination sequences, and preferably, a leader
sequence is capable of directing secretion of translated protein,
or a portion thereof, into the periplasmic space or into the
extracellular medium. Suitable leader sequences are, for example,
the signal sequences of BAP (bacterial alkaline phosphatase), CTB
(cholera toxin subunit B), DsbA, ENX, OmpA, PhoA, stII, OmpT, PelB,
Tat (Twin-arginine translocation) in E. coli, and the signal
sequences of bovine growth hormone, human chymotrypsinogen, human
factor VIII, human ig-kappa, human insulin, human interleukin-2,
luciferase from Metrida or Vargula, human trypsinogen-2, inulinase
from Kluyveromyces marxianus, mating factor alpha-1 from
Saccharomyces cerevisiae, mellitin, human azurocidin and the like
in eukaryotic cells.
[0156] The vectors may also contain an additional expressible
nucleic acid sequence coding for one or more chaperones to
facilitate correct protein folding.
[0157] In some aspects, the vector of the present invention is an
expression vector. An expression vector is capable of directing the
replication and the expression of the nucleic acid molecule of the
invention, e.g., the nucleic acid comprising the nucleotide
sequence encoding the polypeptide and the nucleotide sequence
encoding asparaginase.
[0158] The nucleic acid molecules and/or vectors as described
herein above may be designed for introduction into cells by, e.g.,
non-chemical methods (electroporation, sonoporation, optical
transfection, gene electrotransfer, hydrodynamic delivery or
naturally occurring transformation upon contacting cells with the
nucleic acid molecule of the invention), chemical-based methods
(calcium phosphate, DMSO, PEG, liposomes, DEAE-dextrane,
polyethylenimine, nucleofection etc.), particle-based methods (gene
gun, magnetofection, impalefection), phage or phagemid vector-based
methods and viral methods. For example, expression vectors derived
from viruses such as retroviruses, vaccinia virus, adeno-associated
virus, herpes viruses, Semliki Forest Virus or bovine papilloma
virus, may be used for delivery of the nucleic acid molecules into
a targeted cell population.
[0159] The present invention also relates to a host cell or a
non-human host transformed with a vector or the nucleic acid
described herein. It will be appreciated that the term "host cell
or a non-human host transformed with the vector" relates to a host
cell or a non-human host that comprises the vector or the nucleic
acid as described herein. Host cells for the expression of
polypeptides are well known in the art and comprise prokaryotic
cells as well as eukaryotic cells. Appropriate culture media and
conditions for the above described host cells are known in the
art.
[0160] "Culturing the host or host cell" includes expression of the
modified protein, including as a fusion protein, as defined herein
and/or the polypeptide as defined herein and/or of the asparaginase
in the host or host cell.
[0161] Methods for the isolation of the modified protein and/or the
polypeptide as defined herein and/or of the asparaginase comprise,
without limitation, purification steps such as affinity
chromatography (preferably using a fusion tag such as the Strep-tag
II or the His.sub.6-tag), gel filtration (size exclusion
chromatography), anion exchange chromatography, cation exchange
chromatography, hydrophobic interaction chromatography, high
pressure liquid chromatography (HPLC), reversed phase HPLC,
ammonium sulfate precipitation or immunoprecipitation. These
methods are well known in the art and have been generally
described, e.g., in Scopes (1994) Protein Purification--Principles
and Practice, Springer. Such methods provide substantially pure
polypeptides. Said pure polypeptides have a homogeneity of,
preferably, at least about 90 to 95% (on the protein level), more
preferably, at least about 98 to 99%. Most preferably, these pure
polypeptides are suitable for pharmaceutical use/applications.
[0162] It is envisaged herein that, a modified protein comprising
L-asparaginase and the polypeptide can be prepared by expressing
the nucleic acid molecule comprising the nucleotide sequence
encoding the polypeptide and the nucleic acid sequence encoding the
asparaginase. The expressed modified protein can be isolated.
Alternatively, the modified protein can be prepared by
culturing/raising the host comprising the nucleotide sequence or
the nucleic acid molecule encoding said polypeptide consisting
solely of proline and alanine. Thus, the nucleic acid is expressed
in the host. The produced polypeptide can be isolated. The produced
polypeptide can be conjugated to the asparaginase, e.g., via a
peptide bond or a non-peptide bond.
[0163] The modified proteins described herein can be used in the
treatment of a disease treatable by depletion of asparagine. The
disease treatable by depletion of asparagines is preferably cancer,
such as non-solid cancer. Preferably, the non-solid cancer is
leukemia or non-Hodgkin's lymphoma. The leukemia preferably is
acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).
For example, the modified proteins are useful in the treatment or
the manufacture of a medicament for use in the treatment of acute
lymphoblastic Leukemia (ALL) in both adults and children or acute
myeloid leukemia (AML) in both adults and children. The use of the
modified proteins described herein in the treatment of other
conditions where asparagine depletion is expected to have a useful
effect is contemplated. Such conditions include, but are not
limited to the following: malignancies, or cancers, including but
not limited to hematalogic malignancies, NK lymphoma, pancreatic
cancer, Hodgkin's disease, acute myelocytic Leukemia, acute
myelomonocytic Leukemia, chronic lymphocytic Leukemia,
lymphosarcoma, reticulosarcoma, melanosarcoma, and diffuse large
B-cell lymphoma (DLBCL). The cancer may be a solid cancer, e.g.
lung cancer or breast cancer. Representative non-malignant
hematologic diseases which respond to asparagine depletion include
immune system-mediated Blood diseases, e.g., infectious diseases
such as those caused by HIV infection (i.e., AIDS). Non-hematologic
diseases associated with asparagine dependence include autoimmune
diseases, for example rheumatoid arthritis, SLE, autoimmune,
collagen vascular diseases, etc. Other autoimmune diseases include
osteo-arthritis, Issac's syndrome, psoriasis, insulin dependent
diabetes mellitus, multiple sclerosis, sclerosing panencephalitis,
systemic lupus erythematosus, rheumatic fever, inflammatory bowel
disease (e.g., ulcerative colitis and Crohn's disease), primary
billiary cirrhosis, chronic active hepatitis, glomerulonephritis,
myasthenia gravis, pemphigus vulgaris, and Graves' disease. Cells
suspected of causing disease can be tested for asparagine
dependence in any suitable in vitro or in vivo assay, e.g., an in
vitro assay wherein the growth medium lacks asparagine.
[0164] The invention further relates to a method of treating a
disease treatable by L-asparagine depletion in a patient, said
method comprising administering to said patient an effective amount
of the modified protein. In some preferred aspects, said disease
treatable by L-asparagine depletion is Acute Lymphoblastic Leukemia
(ALL), acute myeloid leukemia (AML) or non-Hodgkin's lymphoma. In
some aspects, said disease treatable by L-asparagine depletion is a
cancer including, but not limited to NK lymphoma, and pancreatic
cancer. In additional embodiments, the modified protein described
herein elicits a lower immunogenic response in said patient
compared to the L-asparaginase of said modified protein.
[0165] In some aspects, the modified protein described above has a
longer in vivo circulating half-life after a single dose compared
to the unmodified L-asparaginase of said modified protein. The
modified protein described herein can reduce plasma L-asparagine
levels for a time period of at least about 12, 24, 48, 72, 96, or
120 hours when administered at a dose of 5 U/kg body weight (bw) or
10 .mu.g/kg (protein content basis). The modified protein described
herein can reduce plasma L-asparagine levels to undetectable levels
for a time period of at least about 12, 24, 48, 72, 96, 120, or 144
hours when administered at a dose of 25 U/kg bw or 50 .mu.g/kg
(protein content basis). The modified protein described herein can
reduce plasma L-asparagine levels for a time period of at least
about 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hours
when administered at a dose of 50 U/kg bw or 100 .mu.g/kg (protein
content basis). The modified protein described herein can reduce
plasma L-asparagine levels to undetectable levels for a time period
of at least about 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, or
240 hours when administered at a dose ranging from about 10,000 to
about 15,000 IU/m.sup.2 (about 20-30 mg protein/m.sup.2).
[0166] The modified protein described herein can result in a
similar level of L-asparagine depletion over a period of time
(e.g., 24, 48, or 72 hours) after a single dose.
[0167] The modified protein described herein can have a longer
t.sub.1/2 than the unmodified L-asparaginase administered at an
equivalent protein dose. The modified protein described above can
have a greater AUC value (e.g. at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9
or 10 times) after a single dose compared to the L-asparaginase of
said unmodified protein.
[0168] In some aspects the modified protein described herein does
not raise any significant antibody response for a particular period
of time after administration of a single dose, e.g., greater than
about 1 week, 2 weeks, 3 weeks, 4, weeks, 5 weeks, 6 weeks, 7
weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, etc. For
example, the modified protein does not raise any significant
antibody response for at least 8 weeks. In one example, "does not
raise any significant antibody response" means that the subject
receiving the modified protein is identified within art-recognized
parameters as antibody-negative. Antibody levels can be determined
by methods known in the art, for example ELISA or surface plasmon
resonance assays (Zalewska-Szewczyk (2009) Clin. Exp. Med. 9,
113-116; Avramis (2009) Anticancer Research 29, 299-302, each of
which is incorporated herein by reference in its entirety). The
modified protein may have any combination of these properties.
[0169] In some aspects, treatment with the modified protein
described herein will be administered as a first line therapy. In
another aspect, treatment with the modified protein will be
administered as a second line therapy in patients, particularly
patients with ALL, where objective signs of allergy or
hypersensitivity, including "silent hypersensitivity," have
developed to other asparaginase preparations, in particular, the
native Escherichia coli-derived L-asparaginase or its PEGylated
variant (pegaspargase). Non-limiting examples of objective signs of
allergy or hypersensitivity include testing "antibody positive" for
an asparaginase enzyme. In a specific aspect, the modified protein
is used in second line therapy after treatment with pegaspargase.
The patient may have had a previous hypersensitivity to an E. coli
L-asparaginase, and/or may have had a previous hypersensitivity to
an Erwinia L-asparaginase. The hypersensitivity may be selected
from the group consisting of allergic reaction, anaphylactic shock,
and silent hypersensitivity.
[0170] The incidence of relapse in ALL patients following treatment
with L-asparaginase remains high, with approximately 10-25% of
pediatric ALL patients having early relapse (e.g., some during
maintenance phase at 30-36 months post-induction) (Avramis (2005)
Clin. Pharmacokinet. 44, 367-393). If a patient treated with E.
coli-derived L-asparaginase has a relapse, subsequent treatment
with E. coli preparations could lead to a "vaccination" effect,
whereby the E. coli preparation has increased immunogenicity during
the subsequent administrations. The modified protein described
herein may be used in a method of treating patients with relapsed
ALL who were previously treated with other asparaginase
preparations, in particular those who were previously treated with
E. coli-derived asparaginases. The disease relapse may occur after
treatment with an E. coli L- asparaginase or PEGylated form
thereof.
[0171] In another aspect, the invention is directed to a method for
treating acute lymphoblastic Leukemia comprising administering to a
patient in need of the treatment a therapeutically effective amount
of the modified protein described above. In a specific aspect,
treatment will be administered at a dose ranging from about 1500
IU/m.sup.2 to about 15,000 IU/m.sup.2, typically about 10,000 to
about 15,000 IU/m.sup.2 (about 20-30 mg protein/m.sup.2), at a
schedule ranging from about twice a week to about once a month,
typically once per week or once every other week. The modified
protein described above may be administered as a single agent
(e.g., monotherapy) or as a part of a combination of chemotherapy
drugs, including, but not limited to glucocorticoids,
corticosteroids, anticancer compounds or other agents, including,
but not limited to methotrexate, dexamethasone, prednisone,
prednisolone, vincristine, cyclophosphamide, and anthracycline. As
an example, patients with ALL will be administered the modified
protein described above as a component of multi-agent chemotherapy
during 3 chemotherapy phases including induction, consolidation or
intensification, and maintenance. In a specific example, the
modified protein described above is not administered with an
asparagine synthetase inhibitor (e.g., such as set forth in WO
2007/103290, which is herein incorporated by reference in its
entirety). In another specific example, the modified protein
described above is not administered with an asparagine synthetase
inhibitor, but is administered with other chemotherapy drugs. The
modified protein described above can be administered before, after,
or simultaneously with other compounds as part of a multi-agent
chemotherapy regimen.
[0172] In a specific embodiment, the method comprises administering
the modified protein described above at an amount of about 1 U/kg
to about 25 U/kg (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 U/kg) or
an equivalent amount thereof 20 (e.g., on a protein content basis).
The amounts of the modified protein to be delivered will depend on
many factors, for example, the IC.sub.50, EC.sub.50, the biological
half-life of the compound, the age, size, weight, and physical
condition of the patient, and the disease or disorder to be
treated. The importance of these and other factors to be considered
are well known to those of ordinary skill in the art. In certain
embodiments, the amount of modified protein to be administered may
range from about 10 International Units per square meter of the
surface area of the patient's body (IU/m.sup.2) to 50,000
IU/m.sup.2. In additional aspects, the modified protein is
administered at an amount selected from the group consisting of
about 5, about 10, and about 25 U/kg. In another specific aspect,
the modified protein is administered at a dose ranging from about
1,000 IU/m2 to about 20,000 IU/m 2 (e.g., 1,000 IU/m.sup.2, 2,000
IU/m.sup.2, 3,000 IU/m.sup.2, 4,000 IU/m.sup.2, 5,000 IU/m.sup.2,
6,000 IU/m.sup.2, 7,000 IU/m.sup.2, 8,000 IU/m.sup.2, 9,000
IU/m.sup.2, 10,000 IU/m.sup.2, 11,000 IU/m.sup.2, 25 12,000
IU/m.sup.2, 13,000 IU/m.sup.2, 14,000 IU/m.sup.2, 15,000
IU/m.sup.2, 16,000 IU/m.sup.2, 17,000 IU/m.sup.2, 18,000
IU/m.sup.2, 19,000 IU/m.sup.2, or 20,000 IU/m.sup.2). In another
specific aspect, the modified protein described above is
administered at a dose that depletes L-asparagine to undetectable
levels using methods and apparatus known in the art for a period of
about 3 days to about 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10
days) for a single dose.
[0173] The modified protein may be administered in a dose that
depletes L-asparagine to undetectable levels for a period of about
3 days to about 10 days, about 5 days to 20 days, about 1 day to 15
days, or about 2 day to 30 days. The modified protein may be
administered in a dose that depletes L-asparagine to undetectable
levels for a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days
to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or
20 days. The modified protein may be administered intravenously or
intramuscularly. In additional embodiments, said modified protein
may be administered once or twice per week, less than once per
week, or as monotherapy.
[0174] The present invention relates to a composition comprising
the modified protein as defined herein or the modified protein
prepared by the process as described herein. The composition may be
a pharmaceutical composition, optionally further comprising (a)
pharmaceutical acceptable carrier(s) or excipient(s).
[0175] The invention also relates to a pharmaceutical composition
comprising the modified protein described above. In a specific
aspect, the pharmaceutical composition is contained in a vial as a
lyophilized powder to be reconstituted with a solvent, such as
currently available native L-asparaginases, whatever the bacterial
source used for its production (e.g. KIDROLASE, ELSPAR, ERWINASE).
In another aspect, the pharmaceutical composition is a solution,
such as pegaspargase (ONCASPAR) enabling, further to an appropriate
handling, an administration through, e.g., intramuscular,
intravenous (infusion and/or bolus), intra-cerebro-ventricular
(icv), sub-cutaneous routes.
[0176] The modified protein, including compositions comprising the
same (e.g., a pharmaceutical composition) can be administered to a
patient using standard techniques. Techniques and formulations
generally may be found in Remington's Pharmaceutical Sciences, 22nd
ed., Pharmaceutical Press, (2012). Suitable dosage forms, in part,
depend upon the use or the route of entry, for example, oral,
transdermal, transmucosal, or by injection (parenteral). Such
dosage forms should allow the therapeutic agent to reach a target
cell or otherwise have the desired therapeutic effect. For example,
pharmaceutical compositions injected into the blood stream
preferably are soluble. The pharmaceutical compositions according
to the invention can be formulated as pharmaceutically acceptable
salts and complexes thereof Pharmaceutically acceptable salts are
non-toxic salts present in the amounts and concentrations at which
they are administered. The preparation of such salts can facilitate
pharmaceutical use by altering the physical characteristics of the
compound without preventing it from exerting its physiological
effect. Useful alterations in physical properties include lowering
the melting point to facilitate transmucosal administration and
increasing solubility to facilitate administering higher
concentrations of the drug. The pharmaceutically acceptable salt of
a modified protein as described herein may be present as a complex,
as those in the art will appreciate. Pharmaceutically acceptable
salts include acid addition salts such as those containing sulfate,
hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate,
citrate, lactate, tartrate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, and
quinate. Pharmaceutically acceptable salts can be obtained from
acids, including hydrochloric acid, maleic acid, sulfuric acid,
phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic
acid, tartaric acid, malonic acid, methanesulfonic acid,
ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,
cyclohexylsulfamic acid, fumaric acid, and quinic acid.
Pharmaceutically acceptable salts also include basic addition salts
such as those containing benzathine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine, procaine, aluminum,
calcium, lithium, magnesium, potassium, sodium, ammonium,
alkylamine, and zinc, when acidic functional groups, such as
carboxylic acid or phenol are present. For example, see Remington's
Pharmaceutical Sciences, supra. Such salts can be prepared using
the appropriate corresponding bases. Pharmaceutically acceptable
carriers and/or excipients can also be incorporated into a
pharmaceutical composition according to the invention to facilitate
administration of the particular asparaginase. Examples of carriers
suitable for use in the practice of the invention include calcium
carbonate, calcium phosphate, various sugars such as lactose,
glucose, or sucrose, or types of starch, cellulose derivatives,
gelatin, vegetable oils, polyethylene glycols, and physiologically
compatible solvents. Examples of physiologically compatible
solvents include sterile solutions of water for injection (WFI),
saline solution and dextrose. Pharmaceutical compositions according
to the invention can be administered by different routes, including
intravenous, intraperitoneal, subcutaneous, intramuscular, oral,
topical (transdermal), or transmucosal administration. For systemic
administration, oral administration is preferred. For oral
administration, for example, the compounds can be formulated into
conventional oral dosage forms such as capsules, tablets, and
liquid preparations such as syrups, elixirs, and concentrated
drops. Alternatively, injection (parenteral administration) may be
used, e.g., intramuscular, intravenous, intraperitoneal, and
subcutaneous injection. For injection, pharmaceutical compositions
are formulated in liquid solutions, preferably in physiologically
compatible buffers or solutions, such as saline solution, Hank's
solution, or Ringer's solution. In addition, the compounds may be
formulated in solid form and redissolved or suspended immediately
prior to use. For example, lyophilized forms of the modified
protein can be produced. In a specific aspect, the modified protein
is administered intramuscularly. In preferred specific aspect, the
modified protein is administered intravenously.
[0177] Systemic administration can also be accomplished by
transmucosal or transdermal means. For transmucosal or transdermal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are well
known in the art, and include, for example, for transmucosal
administration, bile salts, and fusidic acid derivatives. In
addition, detergents may be used to facilitate permeation.
Transmucosal administration, for example, may be through nasal
sprays, inhalers (for pulmonary delivery), rectal suppositories, or
vaginal suppositories. For topical administration, compounds can be
formulated into ointments, salves, gels, or creams, as is well
known in the art.
[0178] In one aspect, the invention also relates to the use of the
modified protein as described herein in therapy. The use may be for
treating a disease treatable by L-asparagine depletion described
above as a method of treating a disease treatable by L-asparagine
depletion. In one aspect, the invention relates to the modified
protein as described herein or the modified protein prepared by the
process as described herein, or the composition comprising the
modified protein as described herein, for use as a medicament/for
use in therapy/for use in medicine.
[0179] In one aspect, the invention relates to the modified protein
as described herein or the modified protein prepared by the process
as described herein, or the composition comprising the modified
protein as described herein, for use in the treatment of a disease
treatable by L-asparagine depletion in a patient. The present
invention also relates to the use of the modified protein as
described herein or of the modified protein prepared by the process
as described herein, or of the composition comprising the modified
protein as described herein in the preparation of a medicament for
treating a disease treatable by L-asparagine depletion in a
patient, The present invention also relates to a method of treating
a disease treatable by L-asparagine depletion in a patient, said
method comprising administering to said patient an effective amount
of the modified protein as described herein, the modified protein
prepared by the process as described herein, or composition as
described herein. Preferably, the disease treatable by L-asparagine
depletion is a cancer.
[0180] In a preferred aspect, the invention relates to the modified
protein as described herein or the modified protein prepared by the
process as described herein, or the composition comprising the
modified protein as described herein for use in the treatment of
cancer. The present invention also relates to the use of the
modified protein as described herein or of the modified protein
prepared by the process as described herein, or of the composition
comprising the modified protein as described herein in the
preparation of a medicament for treating cancer. The present
invention also relates to a method for treating cancer comprising
the administration of the modified protein described herein, the
modified protein prepared by the process described herein, or the
composition described herein, to a subject.
[0181] It is preferred herein that the subject to be treated is a
mammal, particularly a human
[0182] The cancer may be a non-solid cancer, e.g. is leukemia or
non-Hodgkin's lymphoma. Preferably, said leukemia is acute
lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).
[0183] The modified protein may elicit a lower immunogenic response
in the patient compared to the unconjugated L-asparaginase. The
modified protein may have a longer in vivo circulating half-life
after a single dose compared to the unconjugated L-asparaginase.
The modified protein can have a greater AUC value after a single
dose compared to the unconjugated L-asparaginase. The patient may
have had a previous hypersensitivity to an E. coli L-asparaginase
or PEGylated form thereof.
[0184] The following examples illustrate exemplary embodiments of
the invention:
Example 1
[0185] Optimization of Coupling Ratio for the Preparation of
Pyroglutamoyl-P/A(20)-aminohexanoyl-Crisantaspase
[0186] 4.38 mg Pga-P/A#1(20)-Ahx peptide (Part A of FIG. 1; TFA
salt, purity 98%; PSL Peptide Specialty Laboratories, Heidelberg,
Germany) (SEQ ID NO: 16, amino acid sequence shown in SEQ ID NO: 5)
was dissolved in 66.3 .mu.l DMSO. The chemical activation of the
P/A peptide via its terminal carboxylate group was started by
addition of 23.7 .mu.L of a solution of 500 mM TBTU
(CAS#125700-67-6; Iris Biotech, Marktredwitz, Germany) in DMSO and
2.7 .mu.L DIPEA to the peptide solution and vortexing (cf. Part C
of FIG. 1). In this setup, the concentration of the peptide was
25.8 mM and the molar ratio between DIPEA, TBTU and
Pga-P/A#1(20)-Ahx was 5:5:1. After 10 min incubation at 25.degree.
C. the mixture was diluted in Eppendorf tubes according to Table
1.
[0187] A solution of Dickeya chrysanthemi L-Asparaginase
(Crisantaspase, SEQ ID NO: 1, recombinant, produced in E. coli (lot
RE-LAP-P57D) with a concentration of 2 mg/mL was prepared in
phosphate-buffered saline (PBS: 115 mM NaCl, 4 mM KH.sub.2PO.sub.4
and 16 mM Na.sub.2HPO.sub.4, pH 7.4) and pipetted into each
Eppendorf tube according to the volumes stated in table 1. After
mixing by repeated pipetting and vortexing, the coupling reaction
was allowed to take place at 25.degree. C. for 30 min. The reaction
was quenched by addition of glycine (pH 8.0 adjusted with Tris
base) to a final concentration of 250 mM.
TABLE-US-00002 TABLE 1 Dilution series of activated P/A peptide for
coupling with Asparaginase Peptide stock solution DMSO Asparaginase
Mass ratio [.mu.L] [.mu.L] [.mu.L] 10x 21 0 50 7.5x 21 7 66.7 5x 21
21 100 3.5x 21 39 143
[0188] SDS-PAGE analysis of the modified proteins is shown in FIG.
2. The individual bands correspond to protein modified proteins
varying by one coupled P/A peptide each. The additional application
of a mix of coupling reactions with ratios of 0.3 to 10 mg peptide
per mg protein allowed counting of the bands in a successive ladder
starting from the unconjugated protein and thus the number of
coupled P/A peptides could be precisely determined. The band
intensities were quantified densitometrically using the Quant v12.2
software (TotalLab, Newcastle upon Tyne, UK) and arithmetic mean
values of the number of coupled peptides per Crisantaspase monomer
weighted for their band intensities were calculated (cf. Table 2).
3.5 mg P/A peptide per mg Crisantaspase resulted in a coupling
ratio in the range of 9 to 12 P/A peptides per Crisantaspase
monomer (mean value: 10.4). Increasing the applied mass ratio to 10
mg P/A peptide per mg Crisantaspase led only to a slight increase
of the resulting coupling ratio of 10 to 13 P/A peptides per
Crisantaspase (mean value 12.0), indicating a saturation of
accessible amino groups.
[0189] The modified proteins were purified by anion exchange
chromatography (AEX) on a MonoQ HR5/5 column (GE Healthcare) using
25 mM Na-borate pH 9.0, 1 mM EDTA as running buffer and a NaCl
concentration gradient from 0 to 1 M to elute the proteins.
L-asparaginase aminohydrolase activity of each Crisantaspase
modified protein was determined by reaction of ammonia that is
liberated via L-asparagine enzymatic activity with the Nessler
reagent. Briefly, 50 .mu.L of enzyme solution was mixed with 20 mM
of L-asparagine in a 100 mM sodium borate buffer pH 8.6 containing
0.015% (w/v) bovine serum albumin and incubated for 15 min at
37.degree. C. The reaction was stopped by addition of 200 .mu.L of
Nessler reagent (Sigma-Aldrich). Absorbance of this solution was
measured at 450 nm. The activity was calculated from a calibration
curve that was obtained from ammonium sulphate as reference. The
results are summarized in Table 2.
TABLE-US-00003 TABLE 2 Enzymatic activities of Crisantaspase
conjugated with Pga-P/A(20)-Ahx peptide in different amounts mg PA
peptide/ mol PA peptide/ Specific activity Rel. activity mg
Crisantaspase mol monomer [U/mg] [%] 0 -- 540 .+-. 32 100 3.5 10.4
508 .+-. 20 94.1 5 11.2 436 .+-. 22 80.7 7.5 11.7 401 .+-. 21 74.3
10 12.0 256 .+-. 20 47.4
Example 2
[0190] Preparation of
Pyroglutamoyl-P/A(40)-aminohexanoyl-Crisantaspase
[0191] 28 mg of the Pyroglutamoyl-P/A#1(40)-Ahx peptide (SEQ ID NO.
17, amino acid sequence shown in SEQ ID NO: 15), Part B of FIG. 1,
TFA salt, purity 98%; Almac Group, Craigavon, UK) was dissolved in
1324 .mu.L of anhydrous DMSO (99.9%; Sigma-Aldrich, Taufkirchen,
Germany). To achieve chemical activation of the P/A peptide via its
terminal carboxylate group, 162 .mu.L of a solution of 500 mM TBTU
(CAS# 125700-67-6; Iris Biotech, Marktredwitz, Germany) in DMSO
and, after mixing, 14 .mu.L DIPEA (99.5%, biotech. Grade,
Sigma-Aldrich) were added. The whole mixture was vortexed briefly
and incubated for 20 min at 25.degree. C. (cf. Part C of FIG. 1).
In this setup, the peptide concentration was 5.41 mM and the molar
ratio between DIPEA, TBTU and Pga-P/A#1(40)-Ahx was 10:10:1.
[0192] 3.5 mL of an ice-cold Crisantaspase solution (SEQ ID NO:
1)(2 mg/mL in PBS) was mixed with the activated peptide solution
(1.5 mL), resulting in a mass ratio between Pga-P/A#1(40)-Ahx and
Crisantaspase of 5:1, and incubated at room temperature for 30 min
to allow coupling. Using a regenerated cellulose membrane dialysis
tube (MWCO 50 kDa, Spectrum Laboratories, Los Angeles, Calif.), the
solution was dialyzed against 5 L AEX running buffer (25 mM
Na-borate pH 9.0, 1 mM EDTA) and subjected to anion exchange
chromatography on a HISCALE 16/40 column packed with SOURCE 15 Q
resin (GE Healthcare). The column was equilibrated with AEX running
buffer and the protein modified protein was eluted using a
segmented NaCl concentration gradient from 0 to 150 mM in 1 column
volume and from 150 to 1000 mM in 0.25 column volumes (Part A of
FIG. 3).
[0193] Applying the eluate to SDS-PAGE alongside a ladder obtained
from a mix of coupling reactions with ratios of 0.3 to 10 mg
peptide per mg protein allowed determination of the coupling ratio
of 9-11 PA peptides per Crisantaspase monomer (mean value: 10.0)
(Part B of FIG. 3). Enzyme activity of the Crisantaspase/PA(40)
modified protein determined using the Nessler assay described in
example 1 was 78.2% of the activity of the equally assayed
non-modified Crisantaspase.
Example 3
[0194] Preparation of
Pyroglutamoyl-P/A(20)-aminohexanoyl-Crisantaspase
[0195] 21 mg of the Pyroglutamoyl-P/A#1(20)-Ahx peptide (SEQ ID NO:
5, Part A of FIG. 1; TFA salt, purity 98%; PSL Peptide Specialty
Laboratories, Heidelberg, Germany) was dissolved in 1376 .mu.L of
anhydrous DMSO (99.9%; Sigma-Aldrich, Taufkirchen, Germany). To
achieve chemical activation of the P/A peptide via its terminal
carboxylate group, 114 .mu.L of a solution of 500 mM TBTU (CAS#
125700-67-6; purchased from Iris Biotech, Marktredwitz, Germany) in
DMSO and, after mixing, 10 .mu.L DIPEA (99.5%, biotech. Grade,
Sigma-Aldrich) were added. The whole mixture was vortexed briefly
and incubated for 20 min at 25.degree. C. (Part C of FIG. 1). In
this setup, the peptide concentration was 7.58 mM and the molar
ratio between DIPEA, TBTU and Pga-P/A#1(20)-Ahx was 5:5:1.
[0196] 3.5 mL of an ice-cold Crisantaspase solution (SEQ ID NO:
1)(2 mg/mL in PBS) was mixed with the activated peptide solution
(1.5 mL), resulting in a mass ratio between Pga-P/A#1(40)-Ahx and
Crisantaspase of 5:1, and incubated at room temperature for 30 min
to allow coupling. Using a regenerated cellulose membrane dialysis
tube (MWCO 50 kDa, Spectrum Laboratories, Los Angeles, Calif.), the
solution was dialyzed against 5 L AEX running buffer (25 mM
Na-borate pH 9.0, 1 mM EDTA) and subjected to anion exchange
chromatography on a HISCALE 16/40 column packed with SOURCE 15 Q
resin (GE Healthcare). The column was equilibrated with AEX running
buffer and the protein modified protein was eluted using a
segmented NaCl concentration gradient from 0 to 150 mM in 1 column
volume and from 150 to 1000 mM in 0.25 column volumes (Part A of
FIG. 4).
[0197] Applying the eluate to SDS-PAGE alongside a ladder obtained
from a mix of coupling reactions with ratios of 0.3 to 10 mg
peptide per mg protein, allowed determination of the coupling ratio
of 10-13 PA peptides per Crisantaspase monomer (mean value 11.9)
(Part B of FIG. 4). Enzyme activity of the Crisantaspase/PA(20)
modified protein determined using the Nessler assay described in
Example 1 was 91.2% of the activity of the equally assayed
non-modified Crisantaspase.
Example 4
Cloning of Expression Plasmids for the Periplasmic Production of
Crisantaspase N-Terminally Fused to P/A Sequences of Varying
Length
[0198] A synthetic DNA fragment encoding the mature amino acid
sequence of Dickeya chrysanthemi L-asparaginase (UniProt ID P06608)
was obtained from a gene synthesis provider (Thermo Fisher
Scientific, Regensburg, Germany). This gene fragment (SEQ ID NO: 4)
comprised an XbaI restriction site, followed by a ribosomal binding
site, the nucleotide sequence encoding the Enx signal peptide,
followed by a GCC alanine codon, a first SapI recognition sequence
GCTCTTC on the non-coding strand, an 11-nucleotide spacer, and a
second SapI restriction sequence in reverse complementary
orientation, with its recognition sequence GCTCTTC on the coding
strand, followed by a GCC alanine codon directly linked to the
coding sequence for mature L-asparaginase, which was finally
followed by a HindIII restriction site.
[0199] This gene fragment was cloned on pASk75 via the flanking
restriction sites XbaI and HindIII according to standard procedures
(Sambrook (2012) Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press). The resulting plasmid (Part A of
FIG. 5) was digested with SapI, which led to the liberation of a
small (30 bp) DNA insert containing both SapI recognition sites and
a cleaved vector backbone with compatible 5'-GCC/5'-GGC sticky ends
at the position directly in front of the encoded mature N-terminus
of L-asparaginase, which is ideally suited for insertion of the low
repetitive nucleic acid molecule encoding the proline/alanine-rich
amino acid repeat sequence. After isolation of the vector fragment
using the Promega Wizard gel extraction kit (Promega, Mannheim,
Germany) and dephosphorylation with the thermosensitive alkaline
phosphatase FastAP (Thermo Fisher Scientific, Waltham, Mass.), both
according to the manufacturer's instructions, it was ligated with
the PA#1b(200) gene cassette excised from pXL2-PA#1b(200) (SEQ ID
NO: 8) or PA#1c/1b(400) gene cassette excised from
pXL2-PA#1c/1b(400) (SEQ ID NO: 10) via EarI restriction digest. The
resulting plasmids (SEQ ID NO: 12 and SEQ ID NO: 14)(Part B of FIG.
5) allow the bacterial expression of fusion proteins (SEQ ID NO: 11
and SEQ ID NO: 13) consisting of a proline/alanine-rich amino acid
repeat sequence fused with the biologically active protein
Crisantaspase (after in vivo processing of the Enx signal peptide
upon periplasmic secretion in E. coli).
Example 5
Bacterial Production and Purification of Fusion Proteins Between
Either the PA#1(200) or the PA#1(400) Sequence and
Crisantaspase
[0200] Both, the PA#1(200)-Crisantaspase and the
PA#1(400)-Crisantaspase fusion protein (calculated mass: 51 kDa and
67 kDa, respectively) were produced at 25.degree. C. in E. coli
W3110 harboring the expression plasmid pASK75-PA200-Crisantaspase
or pASK75-PA400-Crisantaspase (Part B of FIG. 5) from Example 4
using an 8 L bench top fermenter with a synthetic glucose mineral
medium supplemented with 100 mg/L ampicillin according to a
published procedure (Schiweck (1995) Proteins 23: 561-565).
Recombinant gene expression was induced by addition of 500 .mu.g/L
anhydrotetracycline (Skerra (1994) loc. cit.) as soon as the
culture reached OD.sub.550=40. After an induction period of 2.5 h,
cells were harvested by centrifugation and resuspended during 10
min in ice-cold periplasmic fractionation buffer (500 mM sucrose, 1
mM EDTA, 200 mM boric acid/NaOH pH 8.0; 2 ml per L and OD.sub.550).
After adding 15 mM EDTA and 250 .mu.g/mL lysozyme, the cell
suspension was incubated for 20 min on ice, centrifuged several
times, and the cleared supernatant containing the recombinant
protein was recovered.
[0201] The periplasmic extracts were dialyzed twice at 4.degree. C.
against 15 L PBS containing 1 mM EDTA for at least 6 h,
respectively, filtered using a 0.2 gm cellulose nitrate membrane
(GE Healthcare) and precipitated by addition of ammonium sulfate
(Ph.Eur. grade; Applichem, Darmstadt, Germany) to a saturation of
25% at 25.degree. C. After centrifugation, the supernatant was
removed and the sediment was resuspended in AEX running buffer (25
mM Na-borate pH 9.0, 1 mM EDTA) and dialyzed at 4.degree. C.
against 5L AEX running buffer for at least 6 h. The dialyzed
protein solution was cleared from remaining insoluble matter by
centrifugation and subjected to subtractive anion exchange
chromatography using a 85 ml HISCALE column (GE Healthcare,
Freiburg, Germany) packed with
[0202] Source15 Q resin, connected to an AKTA purifier system (GE
Healthcare, Freiburg, Germany), equilibrated in AEX running buffer.
The column flow-through containing the pure protein (cf. Part A of
FIG. 6 and Part B of FIG. 6) was dialyzed twice against 5 L
PBS.
[0203] Homogeneous protein preparations without signs of
aggregation were obtained with a final yield of 128 mg for
PA#1(200)-Crisantaspase and 48 mg for PA#1(400)-Crisantaspase from
one 8 L fermenter, respectively. Protein concentrations were
determined by measuring the absorption at 280 nm using a calculated
extinction coefficient (Gill (1989) Anal. Biochem. 182: 319-326) of
19370 M.sup.-1 cm.sup.-1. Enzyme activities of the fusion proteins
were determined using the Nessler assay described in example 1. In
this setup, the PA#1(200)-Crisantaspase fusion protein had 109% and
the PA#1(400)-Crisantaspase had 118% of enzyme activity compared to
the equally assayed non-modified Crisantaspase. This demonstrates
that the N-terminal fusion of Crisantaspase with P/A polypeptides
to the length of at least 401 amino acids does not affect enzymatic
activity.
Example 6
Measurement of the Hydrodynamic Volume for Both Genetically and
Chemically PASylated Crisantaspase by Analytical Gel Filtration
[0204] Size exclusion chromatography (SEC) was carried out on a
SUPERDEX S200 increase 10/300 GL column (GE Healthcare Europe,
Freiburg, Germany) at a flow rate of 0.5 mL/min using an AKTA
Purifier 10 system (GE Healthcare) with PBS (115 mM NaCl, 4 mM
KH.sub.2PO.sub.4, 16 mM Na.sub.2HPO.sub.4; pH 7.4) as running
buffer. Using regenerated cellulose disposable ultrafiltration
devices (MWCO 10 kDa; Merck-Millipore, Darmstadt, Germany)
recombinant Crisantaspase genetically fused with PA#1(200) or
PA#1(400) polypeptides (described in Example 5) and Crisantaspase
chemically conjugated with either the Pga-P/A(40)-Ahx peptide
(described in Example 2) or with the Pga-P/A(20)-Ahx peptide
(described in Example 3) were adjusted to a concentration of 1
mg/mL in PBS. 150 .mu.L samples of the concentrated PASylated
enzymes and of non-PASylated Crisantaspase were individually
applied to the column and the chromatography traces were overlaid
(Part A of FIG. 7). All five proteins eluted in single homogenous
peaks.
[0205] For column calibration (Part B of FIG. 7) 150 .mu.L of an
appropriate mixture of the following globular proteins (Sigma,
Deisenhofen, Germany) was applied in PBS at protein concentrations
between 0.5 mg/ml and 1.0 mg/ml: cytochrome c, 12.4 kDa; ovalbumin,
43.0 kDa; bovine serum albumin, 66.3 kDa; alcohol dehydrogenase,
150 kDa; .beta.-amylase, 200 kDa; apo-ferritin, 440 kDa;
thyroglobulin, 660 kDa.
[0206] As result, both the recombinant PA fusion proteins and the
chemically conjugated enzyme preparations exhibited a significantly
larger size than corresponding globular proteins with the same
molecular weight. With increasing size of the P/A (poly-)peptide
moiety this mol. weight/hydrodynamic volume disproportion increased
further. The apparent size increase for PA(200)-Crisantaspase was
5.1-fold compared with the unfused Crisantaspase whereas the true
mass was only larger by 1.5-fold. The apparent size increase for
PA(400)-Crisantaspase compared with the unfused Crisantaspase was
10.4-fold whereas the true mass was only larger by 1.9-fold. This
observation clearly indicates a much increased hydrodynamic volume
conferred to the biologically active Crisantaspase enzyme by the
Pro/Ala polypeptide segment according to this invention.
Example 7
ESI-MS Analysis of Chemically or Genetically PASylated
Crisantaspase
[0207] 250 .mu.l of the purified chemical modified protein of
Crisantaspase with Pga-P/A(20)-Ahx from Example 3 and of the
recombinant PA200- and PA400-fusion proteins from Example 5, all at
a concentration of 1 mg/mL, were applied to a 1 mL Resource.TM. RPC
column (GE Healthcare, Freiburg, Germany) connected to an AKTA
purifier system using 2% v/v acetonitrile, 1% v/v formic acid as
running buffer. The proteins were eluted using an acetonitrile
gradient from 2% v/v acetonitrile, 1% v/v formic acid to 80% v/v
acetonitrile, 0.1% v/v formic acid over 20 column volumes. The
eluted proteins were directly analyzed via ESI mass spectrometry on
a MAXIS micrOTOF instrument (Bruker Daltonik, Bremen, Germany)
using the positive ion mode. The raw m/z spectrum of the
Crisantaspase/Pga-P/A(20)-Ahx chemical modified protein is shown in
Part A of FIG. 8. The masses revealed by the deconvoluted mass
spectrum (Part B of FIG. 8) are given in Table 3. The distribution
of masses matches the coupling ratios determined by SDS-PAGE
analysis described in Example 2.
[0208] The raw m/z spectrum of the recombinant
PA#1(200)-Crisantaspase (SEQ ID NO: 11) fusion protein is shown in
Part C of FIG. 8. The deconvoluted mass spectrum revealed a mass of
51164.75 Da (Part D of FIG. 8), which essentially coincides with
the calculated mass of this protein (51163.58 Da). The raw m/z
spectrum of the recombinant PA#1(400)-Crisantaspase fusion protein
(SEQ ID NO: 13) is shown in Part E of FIG. 8. The deconvoluted
spectrum (Part F of FIG. 8) revealed a mass of 67199.17 Da, which
essentially coincides with the calculated mass of this protein
(67201.99 Da). This clearly demonstrates that intact Crisantaspase
enzyme genetically fused to either PA200 or PA400 can be produced
in E. coli in a highly homogeneous form.
TABLE-US-00004 TABLE 3 Comparison of calculated and measured masses
detected in the preparation of the Crisantaspase/Pga- P/A(20)-Ahx
chemical modified protein Coupling ratio Calculated mass Measured
mass 9x 51506.1 51503.9 10x 53334.1 53333.2 11x 55162.1 55161.7 12x
56990.1 56990.1 13x 58818.1 58819.4 14x 60646.1 60645.8
Example 8
Asparaginase Activity
[0209] PASylated L-asparaginase enzyme activity was determined by
catalysis of the conversion of L-asparagine into L-aspartic acid.
This reaction liberates one mole of ammonia per mole of converted
L-asparagine. The released ammonia is detected using Nessler's
reagent. In the presence of Nessler's reagent the ammonia will form
a water-soluble yellow complex that can be quantified by absorbance
measurement at 450 nm (Mashburn et al. (1963) Biochem. Biophys.
Res. Commun. 12, 50). One unit of L- asparaginase enzyme activity
(International Unit or IU) is defined as the amount of enzyme that
catalyzes the conversion of one .mu.mol of L-asparagine per minute.
The specific activity of the samples (IU/mg) is determined by
dividing the value of L-asparaginase activity expressed in IU/mL by
the protein concentration expressed in mg/mL. The mass of the
protein monomer with the PASylated sequence was measured.
[0210] The measurement of L-asparaginase activity is based on an
endpoint assay in which the sample is diluted to a series of final
enzyme concentrations which are then incubated at 37.degree. C.
under saturating L-asparagine concentration for 15 minutes. The
reaction is stopped by addition of Nessler's reagent and the amount
of ammonia produced by the reaction is extrapolated from a
calibration curve constructed from known quantities of ammonium
sulfate used as standard. A plot of enzyme concentration versus
ammonia is then created for each sample and the slope of the curve
divided by the reaction time to obtain the specific activity in
IU/mg. Specific activity is reported as IU/mg and is reported to
the nearest whole number.
[0211] The initial testing results are displayed in the table below
for each of the modified proteins or fusion proteins.
TABLE-US-00005 Crisantaspase Modified Nessler Nessler Nessler
Expression protein Plate 1 Plate 2 Plate 3 Nessler System Type
(IU/mg) (IU/mg) (IU/mg) (Average) E. coli PA200- 626 666 556 616
Crisantaspase E. coli Crisantaspase- 694 732 602 676 P/A(20)n E.
coli PA400- 790 748 699 746 Crisantaspase E. coli Crisantaspase-
567 528 490 528 P/A(40)n
Example 9
Pharmacokinetics
[0212] The pharmacokinetic profile of E. coli expressed recombinant
crisantaspase as a PASylated fusion protein (PA-200) or chemically
conjugated to PA-peptides (PA-20) was characterized following
administration of a single intravenous bolus dose to CD-1 mice. The
CD-1 mice is a model for a healthy mouse.
[0213] All animals received a single intravenous (IV) bolus via the
lateral tail vein (10 mL/kg) based on the body weight taken prior
to dosing. Individual doses were calculated based upon the most
recent individual body weights to provide the proper dose. The
first day of dosing was based on study day 0 body weights. All
animals were observed for mortality, abnormalities, and signs of
pain and distress twice daily, once in the morning and once in the
afternoon.
[0214] PASylated asparaginase was administered as a single IV dose
of 25 IU/kg body weight to mice. Groups of mice were dosed at 25
IU/kg body weight and plasma samples were collected at scheduled
times for up to 10 days (240 h) following dosing. Asparaginase
activity in mouse plasma was measured using a qualified biochemical
assay as described in the previous examples. Mean plasma
asparaginase activity (n=4) versus time data are plotted (FIG. 1)
and pharmacokinetic analyses were conducted.
[0215] Blood samples were taken prior to dosing and at
approximately 6, 24 (Day 1), 48 (Day 2), 51 (Day 2), 54 (Day 2), 60
(Day 2), 96 (Day 4), 168 (Day 7), and 240 (Day 10) hours post dose.
Tail-snip (cut end of tail) blood collection procedure was
employed. Approximately 1 to 2 mm was cut off the distal end of the
tail for the first blood collection, all sequential blood
collections were collected from the same site by removing the scab
and facilitating blood flow by stroking the tail. Approximately 100
.mu.L blood per time point was collected into chilled K.sub.3EDTA
(Minivette) sampling tubes. Blood was transferred into tubes
appropriate for centrifugation. For plasma isolation, all samples
were centrifuged within approximately 20 minutes of sampling at
3,000.times.g in a refrigerated centrifuge set to maintain
approximately 4.degree. C. for approximately 10 minutes. Following
centrifugation, the maximum amount of plasma was recovered
(targeting 30 .mu.L) and placed into plastic vials. The plastic
vials were stored at -65.degree. C. to -85.degree. C. until
testing.
[0216] Asparaginase activity was measured as the concentration of
asparaginase in the plasma samples as previously described (Allas
et al. (2009) Blood, 114, 2033). Parameters dependent on sufficient
characterization of the terminal phase of the concentration versus
time profile (t1/2, CL, and V.sub.SS) were only reported if R.sup.2
(the square of the correlation coefficient for linear regression
used to estimate the terminal elimination rate constant, .lamda.z)
was greater than 0.8. The pharmacokinetic data was imported into
Phoenix WinNonlin v6.4 (Certara/Pharsight) for analysis. The plasma
asparaginase activity versus time data were analyzed using
non-compartmental methods with sparse sampling in an IV bolus
administration model. Activity values below the limit of
quantitation of the assay (10 U/L) were set to zero in the
calculation of group means. Nominal dose levels and sample
collection times were used for the calculations. The estimated t1/2
values were 50.2 h for PA-20 crisantaspase and 17.9 h for PA-200
crisantaspase.
[0217] The present invention refers to the following nucleotide and
amino acid sequences:
[0218] Some sequences provided herein are available in the NCBI
database and can be retrieved from
ncbi.nlm.nih.gov/sites/entrez?db=gene; Theses sequences also relate
to annotated and modified sequences. The present invention also
provides techniques and methods wherein homologous sequences, and
variants of the concise sequences provided herein are used.
Preferably, such "variants" are genetic variants.
SEQ ID NO: 1:
Amino Acid Sequence of Dickeya Chrysanthemi L-Asparaginase.
TABLE-US-00006 [0219]
ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLA
NVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEE
SAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGR
GVMVVLNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRID
KLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGM
GAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNP
AHARILLMLALTRTSDPKVIQEYFHTY
SEQ ID NO: 2:
Nucleotide Sequence Encoding Dickeya Chrysanthemi
L-Asparaginase
TABLE-US-00007 [0220]
GCAGATAAACTGCCGAATATTGTTATTCTGGCAACCGGTGGCACCATTGC
AGGTAGCGCAGCAACCGGCACCCAAACCACAGGTTATAAAGCCGGTGCAC
TGGGTGTTGATACCCTGATTAATGCAGTTCCGGAAGTTAAAAAACTGGCC
AATGTGAAAGGTGAACAGTTTAGCAATATGGCCAGCGAAAATATGACCGG
TGATGTTGTTCTGAAACTGAGCCAGCGTGTTAATGAACTGCTGGCACGTG
ATGATGTTGATGGTGTGGTTATTACCCATGGCACCGATACCGTTGAAGAA
AGCGCCTATTTTCTGCATCTGACCGTGAAAAGCGATAAACCGGTTGTTTT
TGTTGCAGCAATGCGTCCGGCAACCGCAATTAGCGCAGATGGTCCGATGA
ATCTGCTGGAAGCAGTTCGTGTTGCCGGTGATAAACAGAGCCGTGGTCGT
GGTGTTATGGTTGTTCTGAATGATCGTATTGGTAGCGCACGCTATATTAC
CAAAACCAATGCAAGCACCCTGGATACCTTTAAAGCCAATGAAGAAGGTT
ATCTGGGCGTTATTATTGGCAATCGCATTTATTATCAGAATCGCATTGAT
AAACTGCATACCACCCGTAGCGTTTTTGATGTTCGTGGTCTGACCAGCCT
GCCGAAAGTTGATATTCTGTATGGCTATCAGGATGATCCGGAATATCTGT
ATGATGCAGCCATTCAGCATGGTGTTAAAGGTATTGTGTATGCAGGTATG
GGTGCAGGTAGCGTTAGCGTTCGTGGTATTGCAGGTATGCGTAAAGCAAT
GGAAAAAGGCGTTGTTGTTATTCGTAGCACCCGTACCGGTAATGGTATTG
TTCCGCCGGATGAAGAACTGCCGGGTCTGGTTAGCGATAGCCTGAATCCG
GCACATGCACGTATTCTGCTGATGCTGGCACTGACCCGTACCAGCGATCC
GAAAGTGATTCAGGAATATTTTCATACCTAT
SEQ ID NO: 3:
Amino Acid Sequence of Dickeya Chrysanthemi L-Asparaginase
[0221] Signal peptide: 1-28; removed during cloning: 29-39; 40-366
asparaginase
TABLE-US-00008 MFKFKKNFLVGLSAALMSISLFSATASAARRAIVGRSSAADKLPNIVILA
TGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMA
SENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKS
DKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIG
SARYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDV
RGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVSVRGIA
GMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLAL
TRTSDPKVIQEYFHTY
SEQ ID NO: 4
Nucleotide Sequence (Synthetic) Encoding Dickeya Chrysanthemi
L-Asparaginase
[0222] mature asparaginase coded from base 160-1140 (bold letters).
Thus, a nucleotide sequence encoding L-Asparaginase ranges from
nucleotides at position 160 to 1140.
TABLE-US-00009 TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTTCAA
ATTCAAAAAAAACTTCCTGGTGGGTCTGAGCGCAGCACTGATGAGCATTA
GCCTGTTTAGCGCAACCGCAAGCGCAGCCAGAAGAGCGATTGTAGGACGC
TCTTCTGCCGCAGATAAACTGCCGAATATTGTTATTCTGGCAACCGGTGG
CACCATTGCAGGTAGCGCAGCAACCGGCACCCAGACCACCGGTTATAAAG
CCGGTGCACTGGGTGTTGATACCCTGATTAATGCAGTTCCGGAAGTTAAA
AAACTGGCCAATGTTAAAGGTGAGCAGTTTAGCAATATGGCCAGCGAAAA
TATGACCGGTGATGTTGTTCTGAAACTGAGCCAGCGTGTTAATGAACTGC
TGGCACGTGATGATGTTGATGGTGTTGTTATTACCCATGGCACCGATACC
GTTGAAGAAAGCGCATATTTTCTGCATCTGACCGTGAAAAGCGATAAACC
GGTTGTTTTTGTTGCAGCAATGCGTCCGGCAACCGCCATTAGCGCAGATG
GTCCGATGAATCTGCTGGAAGCAGTTCGTGTTGCCGGTGATAAACAGAGC
CGTGGTCGTGGTGTTATGGTTGTGCTGAATGATCGTATTGGTAGCGCACG
TTATATTACCAAAACCAATGCAAGCACCCTGGATACCTTTAAAGCAAATG
AAGAAGGTTATCTGGGCGTCATTATTGGCAATCGTATCTATTATCAGAAC
CGCATCGACAAACTGCATACCACCCGTAGCGTTTTTGATGTTCGTGGTCT
GACCAGCCTGCCGAAAGTGGATATTCTGTATGGTTATCAGGATGATCCGG
AATATCTGTATGATGCAGCAATTCAGCATGGTGTGAAAGGTATTGTTTAT
GCAGGTATGGGTGCGGGTAGCGTTAGCGTTCGTGGTATTGCCGGTATGCG
TAAAGCAATGGAAAAAGGTGTTGTTGTGATTCGTAGCACCCGTACCGGTA
ATGGTATTGTTCCGCCTGATGAAGAACTGCCTGGTCTGGTTAGCGATAGC
CTGAATCCGGCACATGCACGTATTCTGCTGATGCTGGCACTGACCCGTAC
CAGCGATCCGAAAGTTATTCAAGAATATTTTCATACCTATTAAGCTT
SEQ ID NO: 5:
Amino Acid Sequence of PA(20) Peptide
TABLE-US-00010 [0223] AAPAAPAPAAPAAPAPAAPA
SEQ ID NO: 6:
Nucleotide Sequence Encoding PA(20) Peptide
TABLE-US-00011 [0224]
GCCGCGCCAGCGGCCCCGGCCCCTGCCGCGCCCGCTGCTCCCGCCCCTGC TGCCCCAGCC
SEQ ID NO: 7:
Amino Acid Sequence of PA(200)-Polypeptide
TABLE-US-00012 [0225]
AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA
PAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA
AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA
PAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA A
SEQ ID NO: 8:
Nucleotide Sequence Encoding PA(200)-Polypeptide
TABLE-US-00013 [0226]
GCCGCGCCAGCGGCCCCGGCCCCTGCCGCGCCCGCTGCTCCCGCCCCTGC
TGCCCCAGCCGCCGCTCCTGCGGCACCTGCGCCCGCCGCGCCGGCAGCGC
CGGCACCGGCAGCTCCGGCGGCCGCGCCTGCAGCTCCTGCACCGGCGGCT
CCAGCAGCCCCGGCGCCGGCCGCACCTGCGGCGGCGCCCGCGGCGCCTGC
ACCCGCAGCGCCTGCGGCACCGGCCCCAGCAGCCCCTGCCGCCGCACCGG
CTGCGCCTGCCCCAGCGGCCCCCGCTGCCCCGGCCCCGGCGGCTCCAGCC
GCAGCGCCTGCCGCCCCAGCGCCCGCAGCACCGGCGGCACCAGCTCCGGC
GGCGCCGGCGGCGGCTCCGGCAGCTCCGGCCCCTGCTGCGCCGGCTGCGC
CGGCTCCGGCGGCCCCTGCGGCGGCTCCGGCCGCACCTGCACCTGCCGCG
CCGGCTGCTCCGGCCCCGGCTGCCCCAGCAGCGGCACCAGCAGCGCCTGC
TCCTGCGGCGCCTGCAGCTCCGGCGCCGGCAGCCCCGGCCGCCGCACCCG
CGGCTCCAGCCCCCGCCGCTCCAGCAGCCCCCGCGCCAGCTGCACCTGCT GCC
SEQ ID NO: 9:
Amino Acid Sequence of PA(400)-Polypeptide
TABLE-US-00014 [0227]
AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA
PAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA
AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA
PAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA
AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA
PAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA
AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA
PAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA A
SEQ ID NO: 10:
Nucleotide Sequence Encoding PA(400)-Polypeptide
TABLE-US-00015 [0228]
GCCGCGCCAGCGGCCCCGGCCCCTGCCGCGCCCGCTGCTCCCGCCCCTGC
TGCCCCAGCCGCCGCTCCTGCGGCACCTGCGCCCGCCGCGCCGGCAGCGC
CGGCACCGGCAGCTCCGGCGGCCGCGCCTGCAGCTCCTGCACCGGCGGCT
CCAGCAGCCCCGGCGCCGGCCGCACCTGCGGCGGCGCCCGCGGCGCCTGC
ACCCGCAGCGCCTGCGGCACCGGCCCCAGCAGCCCCTGCCGCCGCACCGG
CTGCGCCTGCCCCAGCGGCCCCCGCTGCCCCGGCCCCGGCGGCTCCAGCC
GCAGCGCCTGCCGCCCCAGCGCCCGCAGCACCGGCGGCACCAGCTCCGGC
GGCGCCGGCGGCGGCTCCGGCAGCTCCGGCCCCTGCTGCGCCGGCTGCGC
CGGCTCCGGCGGCCCCTGCGGCGGCTCCGGCCGCACCTGCACCTGCCGCG
CCGGCTGCTCCGGCCCCGGCTGCCCCAGCAGCGGCACCAGCAGCGCCTGC
TCCTGCGGCGCCTGCAGCTCCGGCGCCGGCAGCCCCGGCCGCCGCACCCG
CGGCTCCAGCCCCCGCCGCTCCAGCAGCCCCCGCGCCAGCTGCACCTGCT
GCCGCTCCTGCTGCCCCTGCTCCCGCTGCCCCCGCCGCCCCCGCCCCAGC
TGCCCCCGCTGCCGCACCTGCTGCCCCAGCTCCCGCTGCCCCAGCCGCGC
CGGCCCCCGCAGCTCCAGCCGCGGCACCAGCTGCCCCAGCTCCAGCGGCG
CCTGCTGCCCCGGCCCCCGCGGCACCGGCTGCCGCGCCCGCAGCTCCAGC
GCCTGCTGCACCGGCTGCTCCGGCACCCGCCGCGCCAGCAGCTGCCCCTG
CGGCACCAGCTCCTGCTGCCCCCGCGGCACCTGCACCCGCTGCCCCGGCG
GCAGCTCCCGCCGCGCCAGCCCCTGCAGCTCCTGCTGCACCTGCTCCTGC
CGCCCCTGCTGCTGCCCCTGCTGCTCCAGCCCCTGCAGCACCGGCCGCTC
CAGCTCCTGCCGCTCCTGCCGCTGCGCCCGCTGCTCCAGCCCCAGCTGCG
CCAGCAGCTCCTGCACCTGCTGCCCCTGCCGCCGCCCCTGCGGCTCCAGC
ACCTGCTGCACCGGCCGCCCCGGCGCCCGCTGCCCCCGCAGCAGCCCCAG
CCGCACCCGCTCCAGCAGCTCCCGCAGCCCCAGCACCCGCAGCACCAGCC GCC
SEQ ID NO: 11:
Amino Acid Sequence of Asparaginase-PA(200)-Fusion Protein
TABLE-US-00016 [0229]
AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA
PAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA
AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA
PAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA
AADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKL
ANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVE
ESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRG
RGVMVVLNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRI
DKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAG
MGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLN
PAHARILLMLALTRTSDPKVIQEYFHTY
SEQ ID NO: 12:
Nucleotide Sequence Encoding Asparaginase-PA(200)-Fusion Protein
(XbaI/HindIII)
[0230] Mature fusion protein (SEQ ID NO: 11) coded from base
127-1710 (bold letters). Thus, a nucleotide sequence encoding a
fusion protein can range from nucleotides at position 127 to 1710
of SEQ ID NO: 12. Accordingly, the term "modified protein
comprising or consisting of an amino acid sequence encoded by a
nucleic acid having a nucleotide sequence as shown in SEQ ID NO:
12" as used herein can be more narrowly defined as "modified
protein comprising or consisting of an amino acid sequence encoded
by a nucleic acid having a nucleotide sequence as shown in
positions 127 to 1710 of SEQ ID NO: 12".
TABLE-US-00017 TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTTCAA
ATTCAAAAAAAACTTCCTGGTGGGTCTGAGCGCAGCACTGATGAGCATTA
GCCTGTTTAGCGCAACCGCAAGCGCAGCCGCGCCAGCGGCCCCGGCCCCT
GCCGCGCCCGCTGCTCCCGCCCCTGCTGCCCCAGCCGCCGCTCCTGCGGC
ACCTGCGCCCGCCGCGCCGGCAGCGCCGGCACCGGCAGCTCCGGCGGCCG
CGCCTGCAGCTCCTGCACCGGCGGCTCCAGCAGCCCCGGCGCCGGCCGCA
CCTGCGGCGGCGCCCGCGGCGCCTGCACCCGCAGCGCCTGCGGCACCGGC
CCCAGCAGCCCCTGCCGCCGCACCGGCTGCGCCTGCCCCAGCGGCCCCCG
CTGCCCCGGCCCCGGCGGCTCCAGCCGCAGCGCCTGCCGCCCCAGCGCCC
GCAGCACCGGCGGCACCAGCTCCGGCGGCGCCGGCGGCGGCTCCGGCAGC
TCCGGCCCCTGCTGCGCCGGCTGCGCCGGCTCCGGCGGCCCCTGCGGCGG
CTCCGGCCGCACCTGCACCTGCCGCGCCGGCTGCTCCGGCCCCGGCTGCC
CCAGCAGCGGCACCAGCAGCGCCTGCTCCTGCGGCGCCTGCAGCTCCGGC
GCCGGCAGCCCCGGCCGCCGCACCCGCGGCTCCAGCCCCCGCCGCTCCAG
CAGCCCCCGCGCCAGCTGCACCTGCTGCCGCAGATAAACTGCCGAATATT
GTTATTCTGGCAACCGGTGGCACCATTGCAGGTAGCGCAGCAACCGGCAC
CCAGACCACCGGTTATAAAGCCGGTGCACTGGGTGTTGATACCCTGATTA
ATGCAGTTCCGGAAGTTAAAAAACTGGCCAATGTTAAAGGTGAGCAGTTT
AGCAATATGGCCAGCGAAAATATGACCGGTGATGTTGTTCTGAAACTGAG
CCAGCGTGTTAATGAACTGCTGGCACGTGATGATGTTGATGGTGTTGTTA
TTACCCATGGCACCGATACCGTTGAAGAAAGCGCATATTTTCTGCATCTG
ACCGTGAAAAGCGATAAACCGGTTGTTTTTGTTGCAGCAATGCGTCCGGC
AACCGCCATTAGCGCAGATGGTCCGATGAATCTGCTGGAAGCAGTTCGTG
TTGCCGGTGATAAACAGAGCCGTGGTCGTGGTGTTATGGTTGTGCTGAAT
GATCGTATTGGTAGCGCACGTTATATTACCAAAACCAATGCAAGCACCCT
GGATACCTTTAAAGCAAATGAAGAAGGTTATCTGGGCGTCATTATTGGCA
ATCGTATCTATTATCAGAACCGCATCGACAAACTGCATACCACCCGTAGC
GTTTTTGATGTTCGTGGTCTGACCAGCCTGCCGAAAGTGGATATTCTGTA
TGGTTATCAGGATGATCCGGAATATCTGTATGATGCAGCAATTCAGCATG
GTGTGAAAGGTATTGTTTATGCAGGTATGGGTGCGGGTAGCGTTAGCGTT
CGTGGTATTGCCGGTATGCGTAAAGCAATGGAAAAAGGTGTTGTTGTGAT
TCGTAGCACCCGTACCGGTAATGGTATTGTTCCGCCTGATGAAGAACTGC
CTGGTCTGGTTAGCGATAGCCTGAATCCGGCACATGCACGTATTCTGCTG
ATGCTGGCACTGACCCGTACCAGCGATCCGAAAGTTATTCAAGAATATTT
TCATACCTATTAAGCTT
SEQ ID NO: 13:
Amino Acid Sequence of Asparaginase-PA(400)-Fusion Protein
TABLE-US-00018 [0231]
AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA
PAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA
AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA
PAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA
AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA
PAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA
AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA
PAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA
AADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKL
ANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVE
ESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRG
RGVMVVLNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRI
DKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAG
MGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLN
PAHARILLMLALTRTSDPKVIQEYFHTY
SEQ ID NO: 14:
Nucleotide Sequence Encoding Asparaginase-PA(400)-Fusion
Protein
(XbaI/HindIII)
[0232] Mature fusion protein (SEQ ID NO: 13) coded from base
127-2184 (bold letters). Thus, a nucleotide sequence encoding a
fusion protein can range from nucleotides at position 127 to 2184
of SEQ ID NO: 14. Accordingly, the term "modified protein
comprising or consisting of an amino acid sequence encoded by a
nucleic acid having a nucleotide sequence as shown in SEQ ID NO:
14" as used herein can be more narrowly defined as "modified
protein comprising or consisting of an amino acid sequence encoded
by a nucleic acid having a nucleotide sequence as shown in
positions 127 to 2184 of SEQ ID NO: 14".
TABLE-US-00019 TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTTCAA
ATTCAAAAAAAACTTCCTGGTGGGTCTGAGCGCAGCACTGATGAGCATTA
GCCTGTTTAGCGCAACCGCAAGCGCAGCCGCGCCAGCGGCCCCGGCCCCT
GCCGCGCCCGCTGCTCCCGCCCCTGCTGCCCCAGCCGCCGCTCCTGCGGC
ACCTGCGCCCGCCGCGCCGGCAGCGCCGGCACCGGCAGCTCCGGCGGCCG
CGCCTGCAGCTCCTGCACCGGCGGCTCCAGCAGCCCCGGCGCCGGCCGCA
CCTGCGGCGGCGCCCGCGGCGCCTGCACCCGCAGCGCCTGCGGCACCGGC
CCCAGCAGCCCCTGCCGCCGCACCGGCTGCGCCTGCCCCAGCGGCCCCCG
CTGCCCCGGCCCCGGCGGCTCCAGCCGCAGCGCCTGCCGCCCCAGCGCCC
GCAGCACCGGCGGCACCAGCTCCGGCGGCGCCGGCGGCGGCTCCGGCAGC
TCCGGCCCCTGCTGCGCCGGCTGCGCCGGCTCCGGCGGCCCCTGCGGCGG
CTCCGGCCGCACCTGCACCTGCCGCGCCGGCTGCTCCGGCCCCGGCTGCC
CCAGCAGCGGCACCAGCAGCGCCTGCTCCTGCGGCGCCTGCAGCTCCGGC
GCCGGCAGCCCCGGCCGCCGCACCCGCGGCTCCAGCCCCCGCCGCTCCAG
CAGCCCCCGCGCCAGCTGCACCTGCTGCCGCTCCTGCTGCCCCTGCTCCC
GCTGCCCCCGCCGCCCCCGCCCCAGCTGCCCCCGCTGCCGCACCTGCTGC
CCCAGCTCCCGCTGCCCCAGCCGCGCCGGCCCCCGCAGCTCCAGCCGCGG
CACCAGCTGCCCCAGCTCCAGCGGCGCCTGCTGCCCCGGCCCCCGCGGCA
CCGGCTGCCGCGCCCGCAGCTCCAGCGCCTGCTGCACCGGCTGCTCCGGC
ACCCGCCGCGCCAGCAGCTGCCCCTGCGGCACCAGCTCCTGCTGCCCCCG
CGGCACCTGCACCCGCTGCCCCGGCGGCAGCTCCCGCCGCGCCAGCCCCT
GCAGCTCCTGCTGCACCTGCTCCTGCCGCCCCTGCTGCTGCCCCTGCTGC
TCCAGCCCCTGCAGCACCGGCCGCTCCAGCTCCTGCCGCTCCTGCCGCTG
CGCCCGCTGCTCCAGCCCCAGCTGCGCCAGCAGCTCCTGCACCTGCTGCC
CCTGCCGCCGCCCCTGCGGCTCCAGCACCTGCTGCACCGGCCGCCCCGGC
GCCCGCTGCCCCCGCAGCAGCCCCAGCCGCACCCGCTCCAGCAGCTCCCG
CAGCCCCAGCACCCGCAGCACCAGCCGCCGCAGATAAACTGCCGAATATT
GTTATTCTGGCAACCGGTGGCACCATTGCAGGTAGCGCAGCAACCGGCAC
CCAGACCACCGGTTATAAAGCCGGTGCACTGGGTGTTGATACCCTGATTA
ATGCAGTTCCGGAAGTTAAAAAACTGGCCAATGTTAAAGGTGAGCAGTTT
AGCAATATGGCCAGCGAAAATATGACCGGTGATGTTGTTCTGAAACTGAG
CCAGCGTGTTAATGAACTGCTGGCACGTGATGATGTTGATGGTGTTGTTA
TTACCCATGGCACCGATACCGTTGAAGAAAGCGCATATTTTCTGCATCTG
ACCGTGAAAAGCGATAAACCGGTTGTTTTTGTTGCAGCAATGCGTCCGGC
AACCGCCATTAGCGCAGATGGTCCGATGAATCTGCTGGAAGCAGTTCGTG
TTGCCGGTGATAAACAGAGCCGTGGTCGTGGTGTTATGGTTGTGCTGAAT
GATCGTATTGGTAGCGCACGTTATATTACCAAAACCAATGCAAGCACCCT
GGATACCTTTAAAGCAAATGAAGAAGGTTATCTGGGCGTCATTATTGGCA
ATCGTATCTATTATCAGAACCGCATCGACAAACTGCATACCACCCGTAGC
GTTTTTGATGTTCGTGGTCTGACCAGCCTGCCGAAAGTGGATATTCTGTA
TGGTTATCAGGATGATCCGGAATATCTGTATGATGCAGCAATTCAGCATG
GTGTGAAAGGTATTGTTTATGCAGGTATGGGTGCGGGTAGCGTTAGCGTT
CGTGGTATTGCCGGTATGCGTAAAGCAATGGAAAAAGGTGTTGTTGTGAT
TCGTAGCACCCGTACCGGTAATGGTATTGTTCCGCCTGATGAAGAACTGC
CTGGTCTGGTTAGCGATAGCCTGAATCCGGCACATGCACGTATTCTGCTG
ATGCTGGCACTGACCCGTACCAGCGATCCGAAAGTTATTCAAGAATATTT
TCATACCTATTAAGCTT
SEQ ID NO: 15
Amino Acid Sequence of PA(40) Peptide
TABLE-US-00020 [0233] AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA
SEQ ID NO: 16
Modified PA(20) Peptide
TABLE-US-00021 [0234] Pga-AAPAAPAPAAPAAPAPAAPA-Ahx-COOH
SEQ ID NO: 17
Modified PA(40) Peptide
TABLE-US-00022 [0235]
Pga-AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA-Ahx- COOH
[0236] All references cited herein are fully incorporated by
reference. Having now fully described the invention, it will be
understood by a person skilled in the art that the invention may be
practiced within a wide and equivalent range of conditions,
parameters and the like, without affecting the spirit or scope of
the invention or any embodiment thereof.
Sequence CWU 1
1
171327PRTDickeya chrysanthemiL-Asparaginase 1Ala Asp Lys Leu Pro
Asn Ile Val Ile Leu Ala Thr Gly Gly Thr Ile1 5 10 15Ala Gly Ser Ala
Ala Thr Gly Thr Gln Thr Thr Gly Tyr Lys Ala Gly 20 25 30Ala Leu Gly
Val Asp Thr Leu Ile Asn Ala Val Pro Glu Val Lys Lys 35 40 45Leu Ala
Asn Val Lys Gly Glu Gln Phe Ser Asn Met Ala Ser Glu Asn 50 55 60Met
Thr Gly Asp Val Val Leu Lys Leu Ser Gln Arg Val Asn Glu Leu65 70 75
80Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile Thr His Gly Thr Asp
85 90 95Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser
Asp 100 105 110Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr
Ala Ile Ser 115 120 125Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val
Arg Val Ala Gly Asp 130 135 140Lys Gln Ser Arg Gly Arg Gly Val Met
Val Val Leu Asn Asp Arg Ile145 150 155 160Gly Ser Ala Arg Tyr Ile
Thr Lys Thr Asn Ala Ser Thr Leu Asp Thr 165 170 175Phe Lys Ala Asn
Glu Glu Gly Tyr Leu Gly Val Ile Ile Gly Asn Arg 180 185 190Ile Tyr
Tyr Gln Asn Arg Ile Asp Lys Leu His Thr Thr Arg Ser Val 195 200
205Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu Tyr
210 215 220Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile
Gln His225 230 235 240Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly
Ala Gly Ser Val Ser 245 250 255Val Arg Gly Ile Ala Gly Met Arg Lys
Ala Met Glu Lys Gly Val Val 260 265 270Val Ile Arg Ser Thr Arg Thr
Gly Asn Gly Ile Val Pro Pro Asp Glu 275 280 285Glu Leu Pro Gly Leu
Val Ser Asp Ser Leu Asn Pro Ala His Ala Arg 290 295 300Ile Leu Leu
Met Leu Ala Leu Thr Arg Thr Ser Asp Pro Lys Val Ile305 310 315
320Gln Glu Tyr Phe His Thr Tyr 3252981DNADickeya
chrysanthemiL-Asparaginase 2gcagataaac tgccgaatat tgttattctg
gcaaccggtg gcaccattgc aggtagcgca 60gcaaccggca cccaaaccac aggttataaa
gccggtgcac tgggtgttga taccctgatt 120aatgcagttc cggaagttaa
aaaactggcc aatgtgaaag gtgaacagtt tagcaatatg 180gccagcgaaa
atatgaccgg tgatgttgtt ctgaaactga gccagcgtgt taatgaactg
240ctggcacgtg atgatgttga tggtgtggtt attacccatg gcaccgatac
cgttgaagaa 300agcgcctatt ttctgcatct gaccgtgaaa agcgataaac
cggttgtttt tgttgcagca 360atgcgtccgg caaccgcaat tagcgcagat
ggtccgatga atctgctgga agcagttcgt 420gttgccggtg ataaacagag
ccgtggtcgt ggtgttatgg ttgttctgaa tgatcgtatt 480ggtagcgcac
gctatattac caaaaccaat gcaagcaccc tggatacctt taaagccaat
540gaagaaggtt atctgggcgt tattattggc aatcgcattt attatcagaa
tcgcattgat 600aaactgcata ccacccgtag cgtttttgat gttcgtggtc
tgaccagcct gccgaaagtt 660gatattctgt atggctatca ggatgatccg
gaatatctgt atgatgcagc cattcagcat 720ggtgttaaag gtattgtgta
tgcaggtatg ggtgcaggta gcgttagcgt tcgtggtatt 780gcaggtatgc
gtaaagcaat ggaaaaaggc gttgttgtta ttcgtagcac ccgtaccggt
840aatggtattg ttccgccgga tgaagaactg ccgggtctgg ttagcgatag
cctgaatccg 900gcacatgcac gtattctgct gatgctggca ctgacccgta
ccagcgatcc gaaagtgatt 960caggaatatt ttcataccta t 9813366PRTDickeya
chrysanthemiL-Asparaginase with signal peptide 3Met Phe Lys Phe Lys
Lys Asn Phe Leu Val Gly Leu Ser Ala Ala Leu1 5 10 15Met Ser Ile Ser
Leu Phe Ser Ala Thr Ala Ser Ala Ala Arg Arg Ala 20 25 30Ile Val Gly
Arg Ser Ser Ala Ala Asp Lys Leu Pro Asn Ile Val Ile 35 40 45Leu Ala
Thr Gly Gly Thr Ile Ala Gly Ser Ala Ala Thr Gly Thr Gln 50 55 60Thr
Thr Gly Tyr Lys Ala Gly Ala Leu Gly Val Asp Thr Leu Ile Asn65 70 75
80Ala Val Pro Glu Val Lys Lys Leu Ala Asn Val Lys Gly Glu Gln Phe
85 90 95Ser Asn Met Ala Ser Glu Asn Met Thr Gly Asp Val Val Leu Lys
Leu 100 105 110Ser Gln Arg Val Asn Glu Leu Leu Ala Arg Asp Asp Val
Asp Gly Val 115 120 125Val Ile Thr His Gly Thr Asp Thr Val Glu Glu
Ser Ala Tyr Phe Leu 130 135 140His Leu Thr Val Lys Ser Asp Lys Pro
Val Val Phe Val Ala Ala Met145 150 155 160Arg Pro Ala Thr Ala Ile
Ser Ala Asp Gly Pro Met Asn Leu Leu Glu 165 170 175Ala Val Arg Val
Ala Gly Asp Lys Gln Ser Arg Gly Arg Gly Val Met 180 185 190Val Val
Leu Asn Asp Arg Ile Gly Ser Ala Arg Tyr Ile Thr Lys Thr 195 200
205Asn Ala Ser Thr Leu Asp Thr Phe Lys Ala Asn Glu Glu Gly Tyr Leu
210 215 220Gly Val Ile Ile Gly Asn Arg Ile Tyr Tyr Gln Asn Arg Ile
Asp Lys225 230 235 240Leu His Thr Thr Arg Ser Val Phe Asp Val Arg
Gly Leu Thr Ser Leu 245 250 255Pro Lys Val Asp Ile Leu Tyr Gly Tyr
Gln Asp Asp Pro Glu Tyr Leu 260 265 270Tyr Asp Ala Ala Ile Gln His
Gly Val Lys Gly Ile Val Tyr Ala Gly 275 280 285Met Gly Ala Gly Ser
Val Ser Val Arg Gly Ile Ala Gly Met Arg Lys 290 295 300Ala Met Glu
Lys Gly Val Val Val Ile Arg Ser Thr Arg Thr Gly Asn305 310 315
320Gly Ile Val Pro Pro Asp Glu Glu Leu Pro Gly Leu Val Ser Asp Ser
325 330 335Leu Asn Pro Ala His Ala Arg Ile Leu Leu Met Leu Ala Leu
Thr Arg 340 345 350Thr Ser Asp Pro Lys Val Ile Gln Glu Tyr Phe His
Thr Tyr 355 360 36541147DNADickeya chrysanthemiL-Asparaginase
4tctagaaata attttgttta actttaagaa ggagatatac atatgttcaa attcaaaaaa
60aacttcctgg tgggtctgag cgcagcactg atgagcatta gcctgtttag cgcaaccgca
120agcgcagcca gaagagcgat tgtaggacgc tcttctgccg cagataaact
gccgaatatt 180gttattctgg caaccggtgg caccattgca ggtagcgcag
caaccggcac ccagaccacc 240ggttataaag ccggtgcact gggtgttgat
accctgatta atgcagttcc ggaagttaaa 300aaactggcca atgttaaagg
tgagcagttt agcaatatgg ccagcgaaaa tatgaccggt 360gatgttgttc
tgaaactgag ccagcgtgtt aatgaactgc tggcacgtga tgatgttgat
420ggtgttgtta ttacccatgg caccgatacc gttgaagaaa gcgcatattt
tctgcatctg 480accgtgaaaa gcgataaacc ggttgttttt gttgcagcaa
tgcgtccggc aaccgccatt 540agcgcagatg gtccgatgaa tctgctggaa
gcagttcgtg ttgccggtga taaacagagc 600cgtggtcgtg gtgttatggt
tgtgctgaat gatcgtattg gtagcgcacg ttatattacc 660aaaaccaatg
caagcaccct ggataccttt aaagcaaatg aagaaggtta tctgggcgtc
720attattggca atcgtatcta ttatcagaac cgcatcgaca aactgcatac
cacccgtagc 780gtttttgatg ttcgtggtct gaccagcctg ccgaaagtgg
atattctgta tggttatcag 840gatgatccgg aatatctgta tgatgcagca
attcagcatg gtgtgaaagg tattgtttat 900gcaggtatgg gtgcgggtag
cgttagcgtt cgtggtattg ccggtatgcg taaagcaatg 960gaaaaaggtg
ttgttgtgat tcgtagcacc cgtaccggta atggtattgt tccgcctgat
1020gaagaactgc ctggtctggt tagcgatagc ctgaatccgg cacatgcacg
tattctgctg 1080atgctggcac tgacccgtac cagcgatccg aaagttattc
aagaatattt tcatacctat 1140taagctt 1147520PRTArtificial
SequencePA(20) peptide 5Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro
Ala Ala Pro Ala Pro1 5 10 15Ala Ala Pro Ala 20660DNAArtificial
SequenceNucleotide sequence encoding PA(20) peptide 6gccgcgccag
cggccccggc ccctgccgcg cccgctgctc ccgcccctgc tgccccagcc
607201PRTArtificial SequencePA(200)-polypeptide 7Ala Ala Pro Ala
Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro1 5 10 15Ala Ala Pro
Ala Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala 20 25 30Ala Pro
Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro 35 40 45Ala
Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala 50 55
60Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala65
70 75 80Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala
Pro 85 90 95Ala Ala Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala
Pro Ala 100 105 110Ala Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala
Ala Pro Ala Pro 115 120 125Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala
Pro Ala Ala Ala Pro Ala 130 135 140Ala Pro Ala Pro Ala Ala Pro Ala
Ala Pro Ala Pro Ala Ala Pro Ala145 150 155 160Ala Ala Pro Ala Ala
Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro 165 170 175Ala Ala Pro
Ala Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala 180 185 190Ala
Pro Ala Pro Ala Ala Pro Ala Ala 195 2008603DNAArtificial
SequenceNucleotide sequence encoding PA(200)- polypeptide
8gccgcgccag cggccccggc ccctgccgcg cccgctgctc ccgcccctgc tgccccagcc
60gccgctcctg cggcacctgc gcccgccgcg ccggcagcgc cggcaccggc agctccggcg
120gccgcgcctg cagctcctgc accggcggct ccagcagccc cggcgccggc
cgcacctgcg 180gcggcgcccg cggcgcctgc acccgcagcg cctgcggcac
cggccccagc agcccctgcc 240gccgcaccgg ctgcgcctgc cccagcggcc
cccgctgccc cggccccggc ggctccagcc 300gcagcgcctg ccgccccagc
gcccgcagca ccggcggcac cagctccggc ggcgccggcg 360gcggctccgg
cagctccggc ccctgctgcg ccggctgcgc cggctccggc ggcccctgcg
420gcggctccgg ccgcacctgc acctgccgcg ccggctgctc cggccccggc
tgccccagca 480gcggcaccag cagcgcctgc tcctgcggcg cctgcagctc
cggcgccggc agccccggcc 540gccgcacccg cggctccagc ccccgccgct
ccagcagccc ccgcgccagc tgcacctgct 600gcc 6039401PRTArtificial
SequencePA(400)-polypeptide 9Ala Ala Pro Ala Ala Pro Ala Pro Ala
Ala Pro Ala Ala Pro Ala Pro1 5 10 15Ala Ala Pro Ala Ala Ala Pro Ala
Ala Pro Ala Pro Ala Ala Pro Ala 20 25 30Ala Pro Ala Pro Ala Ala Pro
Ala Ala Ala Pro Ala Ala Pro Ala Pro 35 40 45Ala Ala Pro Ala Ala Pro
Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala 50 55 60Ala Pro Ala Pro Ala
Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala65 70 75 80Ala Ala Pro
Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro 85 90 95Ala Ala
Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala 100 105
110Ala Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro
115 120 125Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Ala
Pro Ala 130 135 140Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro
Ala Ala Pro Ala145 150 155 160Ala Ala Pro Ala Ala Pro Ala Pro Ala
Ala Pro Ala Ala Pro Ala Pro 165 170 175Ala Ala Pro Ala Ala Ala Pro
Ala Ala Pro Ala Pro Ala Ala Pro Ala 180 185 190Ala Pro Ala Pro Ala
Ala Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro 195 200 205Ala Ala Pro
Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala 210 215 220Ala
Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala225 230
235 240Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala
Pro 245 250 255Ala Ala Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro Ala
Ala Pro Ala 260 265 270Ala Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro
Ala Ala Pro Ala Pro 275 280 285Ala Ala Pro Ala Ala Pro Ala Pro Ala
Ala Pro Ala Ala Ala Pro Ala 290 295 300Ala Pro Ala Pro Ala Ala Pro
Ala Ala Pro Ala Pro Ala Ala Pro Ala305 310 315 320Ala Ala Pro Ala
Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro 325 330 335Ala Ala
Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala 340 345
350Ala Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro
355 360 365Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Ala
Pro Ala 370 375 380Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro
Ala Ala Pro Ala385 390 395 400Ala101203DNAArtificial
SequenceNucleotide sequence encoding PA(400)- polypeptide
10gccgcgccag cggccccggc ccctgccgcg cccgctgctc ccgcccctgc tgccccagcc
60gccgctcctg cggcacctgc gcccgccgcg ccggcagcgc cggcaccggc agctccggcg
120gccgcgcctg cagctcctgc accggcggct ccagcagccc cggcgccggc
cgcacctgcg 180gcggcgcccg cggcgcctgc acccgcagcg cctgcggcac
cggccccagc agcccctgcc 240gccgcaccgg ctgcgcctgc cccagcggcc
cccgctgccc cggccccggc ggctccagcc 300gcagcgcctg ccgccccagc
gcccgcagca ccggcggcac cagctccggc ggcgccggcg 360gcggctccgg
cagctccggc ccctgctgcg ccggctgcgc cggctccggc ggcccctgcg
420gcggctccgg ccgcacctgc acctgccgcg ccggctgctc cggccccggc
tgccccagca 480gcggcaccag cagcgcctgc tcctgcggcg cctgcagctc
cggcgccggc agccccggcc 540gccgcacccg cggctccagc ccccgccgct
ccagcagccc ccgcgccagc tgcacctgct 600gccgctcctg ctgcccctgc
tcccgctgcc cccgccgccc ccgccccagc tgcccccgct 660gccgcacctg
ctgccccagc tcccgctgcc ccagccgcgc cggcccccgc agctccagcc
720gcggcaccag ctgccccagc tccagcggcg cctgctgccc cggcccccgc
ggcaccggct 780gccgcgcccg cagctccagc gcctgctgca ccggctgctc
cggcacccgc cgcgccagca 840gctgcccctg cggcaccagc tcctgctgcc
cccgcggcac ctgcacccgc tgccccggcg 900gcagctcccg ccgcgccagc
ccctgcagct cctgctgcac ctgctcctgc cgcccctgct 960gctgcccctg
ctgctccagc ccctgcagca ccggccgctc cagctcctgc cgctcctgcc
1020gctgcgcccg ctgctccagc cccagctgcg ccagcagctc ctgcacctgc
tgcccctgcc 1080gccgcccctg cggctccagc acctgctgca ccggccgccc
cggcgcccgc tgcccccgca 1140gcagccccag ccgcacccgc tccagcagct
cccgcagccc cagcacccgc agcaccagcc 1200gcc 120311528PRTArtificial
SequenceAsparaginase-PA(200)-fusion protein 11Ala Ala Pro Ala Ala
Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro1 5 10 15Ala Ala Pro Ala
Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala 20 25 30Ala Pro Ala
Pro Ala Ala Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro 35 40 45Ala Ala
Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala 50 55 60Ala
Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala65 70 75
80Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro
85 90 95Ala Ala Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro
Ala 100 105 110Ala Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala Ala
Pro Ala Pro 115 120 125Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro
Ala Ala Ala Pro Ala 130 135 140Ala Pro Ala Pro Ala Ala Pro Ala Ala
Pro Ala Pro Ala Ala Pro Ala145 150 155 160Ala Ala Pro Ala Ala Pro
Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro 165 170 175Ala Ala Pro Ala
Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala 180 185 190Ala Pro
Ala Pro Ala Ala Pro Ala Ala Ala Asp Lys Leu Pro Asn Ile 195 200
205Val Ile Leu Ala Thr Gly Gly Thr Ile Ala Gly Ser Ala Ala Thr Gly
210 215 220Thr Gln Thr Thr Gly Tyr Lys Ala Gly Ala Leu Gly Val Asp
Thr Leu225 230 235 240Ile Asn Ala Val Pro Glu Val Lys Lys Leu Ala
Asn Val Lys Gly Glu 245 250 255Gln Phe Ser Asn Met Ala Ser Glu Asn
Met Thr Gly Asp Val Val Leu 260 265 270Lys Leu Ser Gln Arg Val Asn
Glu Leu Leu Ala Arg Asp Asp Val Asp 275 280 285Gly Val Val Ile Thr
His Gly Thr Asp Thr Val Glu Glu Ser Ala Tyr 290 295 300Phe Leu His
Leu Thr Val Lys Ser Asp Lys Pro Val Val Phe Val Ala305 310 315
320Ala Met Arg Pro Ala Thr Ala Ile Ser Ala Asp Gly Pro Met Asn Leu
325 330 335Leu Glu Ala Val Arg Val Ala Gly Asp Lys Gln Ser Arg Gly
Arg Gly 340 345 350Val Met Val Val Leu Asn Asp Arg Ile Gly Ser Ala
Arg Tyr Ile Thr 355 360 365Lys Thr Asn Ala Ser Thr Leu Asp Thr Phe
Lys Ala Asn Glu Glu Gly 370
375 380Tyr Leu Gly Val Ile Ile Gly Asn Arg Ile Tyr Tyr Gln Asn Arg
Ile385 390 395 400Asp Lys Leu His Thr Thr Arg Ser Val Phe Asp Val
Arg Gly Leu Thr 405 410 415Ser Leu Pro Lys Val Asp Ile Leu Tyr Gly
Tyr Gln Asp Asp Pro Glu 420 425 430Tyr Leu Tyr Asp Ala Ala Ile Gln
His Gly Val Lys Gly Ile Val Tyr 435 440 445Ala Gly Met Gly Ala Gly
Ser Val Ser Val Arg Gly Ile Ala Gly Met 450 455 460Arg Lys Ala Met
Glu Lys Gly Val Val Val Ile Arg Ser Thr Arg Thr465 470 475 480Gly
Asn Gly Ile Val Pro Pro Asp Glu Glu Leu Pro Gly Leu Val Ser 485 490
495Asp Ser Leu Asn Pro Ala His Ala Arg Ile Leu Leu Met Leu Ala Leu
500 505 510Thr Arg Thr Ser Asp Pro Lys Val Ile Gln Glu Tyr Phe His
Thr Tyr 515 520 525121717DNAArtificial SequenceNucleotide sequence
encoding Asparaginase- PA(200)-fusion protein 12tctagaaata
attttgttta actttaagaa ggagatatac atatgttcaa attcaaaaaa 60aacttcctgg
tgggtctgag cgcagcactg atgagcatta gcctgtttag cgcaaccgca
120agcgcagccg cgccagcggc cccggcccct gccgcgcccg ctgctcccgc
ccctgctgcc 180ccagccgccg ctcctgcggc acctgcgccc gccgcgccgg
cagcgccggc accggcagct 240ccggcggccg cgcctgcagc tcctgcaccg
gcggctccag cagccccggc gccggccgca 300cctgcggcgg cgcccgcggc
gcctgcaccc gcagcgcctg cggcaccggc cccagcagcc 360cctgccgccg
caccggctgc gcctgcccca gcggcccccg ctgccccggc cccggcggct
420ccagccgcag cgcctgccgc cccagcgccc gcagcaccgg cggcaccagc
tccggcggcg 480ccggcggcgg ctccggcagc tccggcccct gctgcgccgg
ctgcgccggc tccggcggcc 540cctgcggcgg ctccggccgc acctgcacct
gccgcgccgg ctgctccggc cccggctgcc 600ccagcagcgg caccagcagc
gcctgctcct gcggcgcctg cagctccggc gccggcagcc 660ccggccgccg
cacccgcggc tccagccccc gccgctccag cagcccccgc gccagctgca
720cctgctgccg cagataaact gccgaatatt gttattctgg caaccggtgg
caccattgca 780ggtagcgcag caaccggcac ccagaccacc ggttataaag
ccggtgcact gggtgttgat 840accctgatta atgcagttcc ggaagttaaa
aaactggcca atgttaaagg tgagcagttt 900agcaatatgg ccagcgaaaa
tatgaccggt gatgttgttc tgaaactgag ccagcgtgtt 960aatgaactgc
tggcacgtga tgatgttgat ggtgttgtta ttacccatgg caccgatacc
1020gttgaagaaa gcgcatattt tctgcatctg accgtgaaaa gcgataaacc
ggttgttttt 1080gttgcagcaa tgcgtccggc aaccgccatt agcgcagatg
gtccgatgaa tctgctggaa 1140gcagttcgtg ttgccggtga taaacagagc
cgtggtcgtg gtgttatggt tgtgctgaat 1200gatcgtattg gtagcgcacg
ttatattacc aaaaccaatg caagcaccct ggataccttt 1260aaagcaaatg
aagaaggtta tctgggcgtc attattggca atcgtatcta ttatcagaac
1320cgcatcgaca aactgcatac cacccgtagc gtttttgatg ttcgtggtct
gaccagcctg 1380ccgaaagtgg atattctgta tggttatcag gatgatccgg
aatatctgta tgatgcagca 1440attcagcatg gtgtgaaagg tattgtttat
gcaggtatgg gtgcgggtag cgttagcgtt 1500cgtggtattg ccggtatgcg
taaagcaatg gaaaaaggtg ttgttgtgat tcgtagcacc 1560cgtaccggta
atggtattgt tccgcctgat gaagaactgc ctggtctggt tagcgatagc
1620ctgaatccgg cacatgcacg tattctgctg atgctggcac tgacccgtac
cagcgatccg 1680aaagttattc aagaatattt tcatacctat taagctt
171713728PRTArtificial SequenceAsparaginase-PA-(400)-fusion protein
13Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro1
5 10 15Ala Ala Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro
Ala 20 25 30Ala Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala Ala Pro
Ala Pro 35 40 45Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala
Ala Pro Ala 50 55 60Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro
Ala Ala Pro Ala65 70 75 80Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala
Pro Ala Ala Pro Ala Pro 85 90 95Ala Ala Pro Ala Ala Ala Pro Ala Ala
Pro Ala Pro Ala Ala Pro Ala 100 105 110Ala Pro Ala Pro Ala Ala Pro
Ala Ala Ala Pro Ala Ala Pro Ala Pro 115 120 125Ala Ala Pro Ala Ala
Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala 130 135 140Ala Pro Ala
Pro Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala145 150 155
160Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro
165 170 175Ala Ala Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala
Pro Ala 180 185 190Ala Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala
Ala Pro Ala Pro 195 200 205Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala
Pro Ala Ala Ala Pro Ala 210 215 220Ala Pro Ala Pro Ala Ala Pro Ala
Ala Pro Ala Pro Ala Ala Pro Ala225 230 235 240Ala Ala Pro Ala Ala
Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro 245 250 255Ala Ala Pro
Ala Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala 260 265 270Ala
Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro 275 280
285Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala
290 295 300Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala
Pro Ala305 310 315 320Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro
Ala Ala Pro Ala Pro 325 330 335Ala Ala Pro Ala Ala Ala Pro Ala Ala
Pro Ala Pro Ala Ala Pro Ala 340 345 350Ala Pro Ala Pro Ala Ala Pro
Ala Ala Ala Pro Ala Ala Pro Ala Pro 355 360 365Ala Ala Pro Ala Ala
Pro Ala Pro Ala Ala Pro Ala Ala Ala Pro Ala 370 375 380Ala Pro Ala
Pro Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala385 390 395
400Ala Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala Thr Gly Gly Thr
405 410 415Ile Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr Gly Tyr
Lys Ala 420 425 430Gly Ala Leu Gly Val Asp Thr Leu Ile Asn Ala Val
Pro Glu Val Lys 435 440 445Lys Leu Ala Asn Val Lys Gly Glu Gln Phe
Ser Asn Met Ala Ser Glu 450 455 460Asn Met Thr Gly Asp Val Val Leu
Lys Leu Ser Gln Arg Val Asn Glu465 470 475 480Leu Leu Ala Arg Asp
Asp Val Asp Gly Val Val Ile Thr His Gly Thr 485 490 495Asp Thr Val
Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser 500 505 510Asp
Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr Ala Ile 515 520
525Ser Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val Arg Val Ala Gly
530 535 540Asp Lys Gln Ser Arg Gly Arg Gly Val Met Val Val Leu Asn
Asp Arg545 550 555 560Ile Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn
Ala Ser Thr Leu Asp 565 570 575Thr Phe Lys Ala Asn Glu Glu Gly Tyr
Leu Gly Val Ile Ile Gly Asn 580 585 590Arg Ile Tyr Tyr Gln Asn Arg
Ile Asp Lys Leu His Thr Thr Arg Ser 595 600 605Val Phe Asp Val Arg
Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu 610 615 620Tyr Gly Tyr
Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile Gln625 630 635
640His Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly Ala Gly Ser Val
645 650 655Ser Val Arg Gly Ile Ala Gly Met Arg Lys Ala Met Glu Lys
Gly Val 660 665 670Val Val Ile Arg Ser Thr Arg Thr Gly Asn Gly Ile
Val Pro Pro Asp 675 680 685Glu Glu Leu Pro Gly Leu Val Ser Asp Ser
Leu Asn Pro Ala His Ala 690 695 700Arg Ile Leu Leu Met Leu Ala Leu
Thr Arg Thr Ser Asp Pro Lys Val705 710 715 720Ile Gln Glu Tyr Phe
His Thr Tyr 725142317DNAArtificial SequenceNucleotide sequence
encoding Asparaginase- PA(400)-fusion protein 14tctagaaata
attttgttta actttaagaa ggagatatac atatgttcaa attcaaaaaa 60aacttcctgg
tgggtctgag cgcagcactg atgagcatta gcctgtttag cgcaaccgca
120agcgcagccg cgccagcggc cccggcccct gccgcgcccg ctgctcccgc
ccctgctgcc 180ccagccgccg ctcctgcggc acctgcgccc gccgcgccgg
cagcgccggc accggcagct 240ccggcggccg cgcctgcagc tcctgcaccg
gcggctccag cagccccggc gccggccgca 300cctgcggcgg cgcccgcggc
gcctgcaccc gcagcgcctg cggcaccggc cccagcagcc 360cctgccgccg
caccggctgc gcctgcccca gcggcccccg ctgccccggc cccggcggct
420ccagccgcag cgcctgccgc cccagcgccc gcagcaccgg cggcaccagc
tccggcggcg 480ccggcggcgg ctccggcagc tccggcccct gctgcgccgg
ctgcgccggc tccggcggcc 540cctgcggcgg ctccggccgc acctgcacct
gccgcgccgg ctgctccggc cccggctgcc 600ccagcagcgg caccagcagc
gcctgctcct gcggcgcctg cagctccggc gccggcagcc 660ccggccgccg
cacccgcggc tccagccccc gccgctccag cagcccccgc gccagctgca
720cctgctgccg ctcctgctgc ccctgctccc gctgcccccg ccgcccccgc
cccagctgcc 780cccgctgccg cacctgctgc cccagctccc gctgccccag
ccgcgccggc ccccgcagct 840ccagccgcgg caccagctgc cccagctcca
gcggcgcctg ctgccccggc ccccgcggca 900ccggctgccg cgcccgcagc
tccagcgcct gctgcaccgg ctgctccggc acccgccgcg 960ccagcagctg
cccctgcggc accagctcct gctgcccccg cggcacctgc acccgctgcc
1020ccggcggcag ctcccgccgc gccagcccct gcagctcctg ctgcacctgc
tcctgccgcc 1080cctgctgctg cccctgctgc tccagcccct gcagcaccgg
ccgctccagc tcctgccgct 1140cctgccgctg cgcccgctgc tccagcccca
gctgcgccag cagctcctgc acctgctgcc 1200cctgccgccg cccctgcggc
tccagcacct gctgcaccgg ccgccccggc gcccgctgcc 1260cccgcagcag
ccccagccgc acccgctcca gcagctcccg cagccccagc acccgcagca
1320ccagccgccg cagataaact gccgaatatt gttattctgg caaccggtgg
caccattgca 1380ggtagcgcag caaccggcac ccagaccacc ggttataaag
ccggtgcact gggtgttgat 1440accctgatta atgcagttcc ggaagttaaa
aaactggcca atgttaaagg tgagcagttt 1500agcaatatgg ccagcgaaaa
tatgaccggt gatgttgttc tgaaactgag ccagcgtgtt 1560aatgaactgc
tggcacgtga tgatgttgat ggtgttgtta ttacccatgg caccgatacc
1620gttgaagaaa gcgcatattt tctgcatctg accgtgaaaa gcgataaacc
ggttgttttt 1680gttgcagcaa tgcgtccggc aaccgccatt agcgcagatg
gtccgatgaa tctgctggaa 1740gcagttcgtg ttgccggtga taaacagagc
cgtggtcgtg gtgttatggt tgtgctgaat 1800gatcgtattg gtagcgcacg
ttatattacc aaaaccaatg caagcaccct ggataccttt 1860aaagcaaatg
aagaaggtta tctgggcgtc attattggca atcgtatcta ttatcagaac
1920cgcatcgaca aactgcatac cacccgtagc gtttttgatg ttcgtggtct
gaccagcctg 1980ccgaaagtgg atattctgta tggttatcag gatgatccgg
aatatctgta tgatgcagca 2040attcagcatg gtgtgaaagg tattgtttat
gcaggtatgg gtgcgggtag cgttagcgtt 2100cgtggtattg ccggtatgcg
taaagcaatg gaaaaaggtg ttgttgtgat tcgtagcacc 2160cgtaccggta
atggtattgt tccgcctgat gaagaactgc ctggtctggt tagcgatagc
2220ctgaatccgg cacatgcacg tattctgctg atgctggcac tgacccgtac
cagcgatccg 2280aaagttattc aagaatattt tcatacctat taagctt
23171540PRTArtificial SequencePA(40) peptide 15Ala Ala Pro Ala Ala
Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro1 5 10 15Ala Ala Pro Ala
Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala 20 25 30Ala Pro Ala
Pro Ala Ala Pro Ala 35 401622PRTArtificial Sequencemodified PA(20)
peptideVARIANT1Xaa = Pga (pyroglutamic acid)VARIANT22Xaa = Ahx
(6-aminohexanoic acid) 16Xaa Ala Ala Pro Ala Ala Pro Ala Pro Ala
Ala Pro Ala Ala Pro Ala1 5 10 15Pro Ala Ala Pro Ala Xaa
201742PRTArtificial Sequencemodified PA(40)-peptideVARIANT1Xaa =
Pga (pyroglutamic acid)VARIANT42Xaa = Ahx (6-aminohexanoic acid)
17Xaa Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala1
5 10 15Pro Ala Ala Pro Ala Ala Ala Pro Ala Ala Pro Ala Pro Ala Ala
Pro 20 25 30Ala Ala Pro Ala Pro Ala Ala Pro Ala Xaa 35 40
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