BR102018076595A2 - polypeptide with asparaginase activity, polynucleotide, expression cassette, expression vector, host cell, pharmaceutical composition, methods for producing a polypeptide with asparaginase activity and for preventing or treating neoplasms, and, use of a polypeptide - Google Patents

Abstract

The present invention relates to modified E. coli L-asparaginase in order to decrease its interaction with different amino acids other than asparagine, more preferably, glutamine. Also described herein are polynucleotides encoding the polypeptides of the invention, expression cassettes comprising said polynucleotides, expression vectors, host cells, pharmaceutical compositions, uses of the polypeptide of the invention in the manufacture of a cancer prevention or treatment drug and methods for producing the polypeptide of the invention and to prevent or treat neoplasms.

Description

POLYPEPTIDE WITH ASPARAGINASE ACTIVITY, POLYNUCLEOTIDE, EXPRESSION CASSETTE, EXPRESSION VECTOR, HOSTING CELL, PHARMACEUTICAL COMPOSITION, METHODS FOR PRODUCING A POLYPEPTIDE WITH ASPARAGINASE ACTIVITY AND FOR PREVENTING, TREATING AND TREATING FIELD OF THE INVENTION

[001] The present invention relates to the field of oncology and biotechnology. More specifically, the present invention relates to polypeptides with asparaginase activity useful in the prevention and treatment of neoplasms.

BACKGROUND OF THE INVENTION

[002] Leukemia is a disease that affects the hematopoietic organs (bone marrow, lymph nodes, spleen and liver) and is characterized by a predominance of immature lymphoid or myeloid precursors, called blasts. These immature cells replace the normal bone marrow and migrate to other tissues. Blasts undergo a neoplastic transformation, which makes these cells highly replicative, but without going into a differentiation process. These neoplastic clones are independent of the action of stimulating and inhibiting factors for normal hematopoiesis and remain crystallized in a maturation phase. As a result of this overproduction, there is a spread through the body of abnormal white blood cells, which causes an interference with the vital functions of the body.

[003] Depending on the origin of the neoplastic cell, leukemias can be divided into acute myeloid leukemia (AML - myeloid cells) or acute lymphoid leukemia (ALL - lymphoid cells) (LORENZI et al., 2006). Acute leukemias are more rapid to progress and chronic leukemias to progress more slowly.

[004] In the case of ALL, the bone marrow, instead of producing cells that would differentiate into lymphocytes, produces abnormal cells that cannot fight infections (PUI & EVANS, 2006). The symptoms of the disease are mainly due to the accumulation of abnormal cells in the bone marrow (ZANICHELLI et al, 2010). Other clinical manifestations are secondary to the proliferation of leukemic cells, which invade other tissues of the body, such as tonsils, lymph nodes, skin, spleen, kidneys, central nervous system and testicles. The most frequent signs and symptoms of the disease are fever, adenomegaly, hemorrhagic manifestations, pallor, hepatomegaly, splenomegaly, fatigue and bone pain (NEHMY et al, 2011).

[005] ALL incidence corresponds to about 3/4 of all newly diagnosed childhood leukemia cases and approximately 1/4 of all childhood malignancy cases (United States Environmental Protection Agency, 2015) . In addition, there is a second peak incidence at 50 years of age, which corresponds to the incidence rate in children. (PUI & EVANS, 2006). It is estimated that, in the United States alone, approximately 6,000 new cases of ALL appear in both adults and children (out of 10 cases, 6 are diagnosed in children), and approximately 1500 die from the disease (out of 5 deaths, 4 are from adults) (North American Cancer Society, 2015).

[006] The most common treatment for leukemia is chemotherapy, consisting of the following phases: induction of remission, consolidation and maintenance phase, comprising polychemotherapy schemes, in order to prevent the development of resistance. The different stages of treatment have varying intensities, depending on the patient's risk group, with more intense regimes in high-risk cases. The chemotherapeutics most used in the treatment phase are glucocorticoids, anthracyclines, vincristine and L-asparaginase (L-ASNase).

[007] L-ASNase is the enzyme responsible for the hydrolysis of asparagine in aspartate and ammonia. Asparagine is a critical amino acid for protein synthesis in leukemic cells, since they do not have the ability to synthesize asparagine again due to the low level or absence of expression of the enzyme asparagine synthase (enzyme that synthesizes asparagine from aspartate) (LEE et al, 1989). Thus, tumor cells require a high and unusual level of asparagine, and because they have low levels of asparagine synthase, they are dependent on serum levels of the amino acid for their proliferation and survival (KIRIYAMA et al, 1989). Normal cells, on the other hand, are able to synthesize asparagine from aspartate, using the enzyme asparagine synthetase (RICHARDS & SCHUSTER, 1998), therefore, the antineoplastic activity of L-ASNase is selective (UREN et al., 1977). The antitumor effect of L-ASNase is due to the depletion of asparagine in the bloodstream, which leads to the inhibition of DNA and protein synthesis in leukemic cells, consequently preventing tumor growth (PIETERS et al., 2011). For L-ASNase to be effective in the treatment against ALL, the level of L-asparagine in the medium must be reduced to at least 10-5 M for the protein synthesis of tumor cells to be impaired (CEDAR & SCHWARTZ, 1967 ).

[008] Clinical studies indicate that the use of L-ASNase in phases of induction of remission and consolidation are critical for the treatment of childhood ALL. The prolonged use of high doses of the enzyme has been reported as crucial for the reduction of relapse and complete remission, including in high-risk patients (SILVERMAN et al., 2001; AMYLON et al., 1999). In addition, combinations between L-ASNase and other medications (corticosteroids or other chemotherapeutic agents) can enhance the activity of L-ASNase and, consequently, improve the patient's prognosis (ASSELIN et al., 1999; ABSHIRE et al., 2000) . During the treatment of patients classified into basic, medium or high risk groups, it is possible to observe the presence of L-ASNase also in the stages of induction of remission and consolidation, with variations in dosage, depending on the treatment phase and the group of patients. patient risk (INCA, 2001; INCA, 2002).

[009] Currently, there are four L-ASNase licensed for therapy, three of which are widely marketed. E. coli native L-ASNase (Ec-A) was approved by the US regulatory agency Food and Drug Administration (FDA) for ALL treatment in 1978 and was the only commercial product available for several years. In 1994, the FDA approved PEG-asparaginase (PEG-Ec-A), a conjugate of a portion of polyethylene glycol (PEG) with E. coli L-ASNase. In 2011, the FDA approved the use of L-ASNase from Erwinia chrysanthemi (Ew-A) and in 2012, Alizé Pharma licenses Asparec®, a pegylated derivative of E. chrysanthemi asparaginase for the treatment of ALL for EUSA Pharma.

[0010] The successful use of L-ASNases in the treatment against ALL has its harmful side, making its use limited and constantly reassessed due to the serious side effects generated mainly by its toxicity. Immediate reactions to the use of L-ASNase occur in 71% of cases, including nausea, vomiting and fever. Due to the decrease in the protein synthesis of the cells, due to the depletion of the level of asparagine in the blood, hyperglycemia, thrombosis and hemorrhages occur. In Go of the cases, the organs suffer dysfunctions, as in the central nervous system, resulting in headache, disorientation, coma, convulsions and depression. Approximately 97% of cases affect the liver, 68% affect the kidneys and 15% affect the pancreas, causing pancreatitis (GUILLERME et al, 2013; HENRIKSEN et al, 2015). Usually, children are more tolerant of the side effects induced by L-ASNase, while adolescents and adults are more sensitive and can often develop significant morbidity (ALBERTSEN et al., 2002; ALVAREZ & ZIMMERMANN, 2000; KEARNEY et al., 2009 ; BARRY et al., 2007).

[0011] Side effects are mainly associated with a second specificity of L-ASNase, which can lead to the depletion of the amino acid glutamine, due to its structural similarity to asparagine and the production of glutamate that is toxic to cells in concentrations greater than 140 μM / L (MULLER & BOSS, 1998; NARTA et al, 2007). Side effects caused by the glutaminase activity of L-ASNase are pancreatitis, hemostasis abnormalities, thrombosis and neurological complications. In addition, clinical hypersensitivity reactions and inactivation by antibodies against Ec-A are also observed, which occurs in about 60% of cases, almost exclusively in post-induction regimens.

[0012] E. carotovora L-ASNase (ECAR-LANS) is much more specific to asparagine than to glutamine and such reduced glutaminase activity would be advantageous in the use of these enzymes for the treatment of neoplasms. However, this enzyme has a shorter half-life in the bloodstream (KRASOTKINA et al, 2004), a characteristic that prevents the ECAR-LANS enzyme from being efficient as a biopharmaceutical.

[0013] So, despite the known side effects of Ec-A, PEG-Ec-A and Ew-A, these are still the commercially available enzymes. However, developments are still necessary for the construction of L-ASNases that cause fewer side effects when administered to treat neoplasms.

[0014] Some attempts to reduce the glutaminase activity of the enzyme L-ASNase are found in the prior art. However, all available studies refer to specific amino acid substitutions and in none of them has a portion of more than 5 residues been replaced.

[0015] It is known in the state of the art that the replacement of just one amino acid residue in the primary structure of an enzyme can lead to its complete inactivation. Not only the number of substituted residues, but their positions in the primary polypeptide structure and the nature of the substitute residues directly influence the enzyme's catalytic activity.

[0016] DERST et al. (2000), for example, tests the effect on the specificity of the enzyme to glutamine by substituting amino acid residues at positions 11, 27, 57 and 88 with supportive amino acids. The work shows that the substitution of a glycine at position 11 and 88 practically completely abolished the catalysis, while at position 57 it had little effect on the substrate specificity.

[0017] In WO2015038639, a substitution in position 58 or 59 is cited, while in CN103060299 mutations are specifically in positions 48, 49, 152 and / or 283.

[0018] WO2014170811 discloses E. coli mutant L-ASNases in which the mutation induces less immunogenicity and greater stability of the enzyme, which comprises less than five amino acid substitutions.

[0019] WO2017151707 discloses E. chrysanthemi mutant L-ASNases with substitutions in only one or more of positions 31, 63 and 254 in order to reduce L-glutaminase activity and increase enzymatic stability.

[0020] WO2013055699 refers to mutant L-ASNases of W. succinogenes with little or no glutaminase activity, which have only one mutation at position 121.

[0021] The advantages of the invention will be evident in the description of the invention provided in this document.

SUMMARY OF THE INVENTION

[0022] The present invention aims to provide a polypeptide with asparaginase activity that solves the main problems of the prior art listed above.

[0023] In a first aspect, the present invention provides a polypeptide with asparaginase activity that comprises substitutions in the region of the amino acid sequence of the peptide with Escherichia coli asparaginase activity (EC-A) by corresponding amino acid residues in the sequence amino acids of the peptide with asparaginase activity of Erwinia carotovora (ECAR-LANS), in which the polypeptide of the present invention (EC-A_mut) has less affinity for different amino acids than non-asparagine when compared to the peptide with asparaginase activity of parental E. coli.

[0024] In a second aspect, the present invention provides a polynucleotide that encodes a polypeptide with asparaginase activity as defined above.

[0025] In a third aspect, the present invention provides an expression cassette comprising a polynucleotide as defined above operatively linked to a promoter and a transcription terminator.

[0026] In a fourth aspect, an expression vector comprising a polynucleotide or an expression cassette as defined above is also provided.

[0027] In a fifth aspect, the present invention provides a host cell comprising an expression cassette or an expression vector as defined above.

[0028] In a sixth aspect, a pharmaceutical composition is provided that comprises a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient.

[0029] In a seventh aspect, the use of the polypeptide of the invention in the manufacture of a medicament for the prevention or treatment of neoplasms is provided.

[0030] In an eighth aspect, the present invention provides a method for producing a polypeptide with asparaginase activity, comprising the steps of: (a) providing a transformed host cell; (b) cultivating said cell under conditions conducive to the production of the polypeptide; and (c) isolating said polypeptide from said cell or culture medium surrounding said cell.

[0031] In a ninth aspect, a method for preventing or treating neoplasms is provided, comprising administering a therapeutically effective amount of the polypeptide with asparaginase activity to an individual in need of said prevention or treatment.

BRIEF DESCRIPTION OF THE FIGURES

[0032] Figure 1 shows the structural analysis for the generation of Ec-A_mut.

[0033] Figure 2 shows gel filtration chromatogram of Ac-A (Figure 2A) and ECAR-LANS (Figure 2B).
Figure 3A shows a chromatogram of the filtration gel column calibration curve. Peak elution volume: 1 (Ferritin, 440 kDa) - 45.99 mL; 2 (Aldolase, 158 kDa) - 51.22 ml; 3 (Conalbumin, 75 kDa) - 86.47 ml; 4 (Ovalbumin, 44 kDa) -102.98 mL. Figure 3B shows a related graph relating the Kav value and log MW of the column calibration curve used for the filtration gel tests.

[0034] Figure 4 shows the result of the gel filtration chromatography of Ec-A_mut.

[0035] Figure 5 shows a double reciprocal graph of the activity of ECAR-LANS (Figure 5A) and Ec-A (Figure 5B).

[0036] Figure 6 shows the circular dichroism spectrum of Ec-A and ECAR-LANS, with the analysis of the folding of Ec-A (red) and ECAR-LANS (black).

[0037] Figure 7 shows the circular dichroism spectrum of the enzymes Ec-A, ECAR-LANS and Ec-A_mut.

[0038] Figure 8 shows a structural analysis of Ec-A_mut.

[0039] Figure 9 shows the circular dichroism spectrum observed at 222 nm wavelength during temperature gradient, varying between 20 ° and 90 ° of the native and mutated enzymes of E. coli and native of E. carotovora.

DEFINITIONS

[0040] To ensure a better understanding of the scope of the invention, without being a limiting factor, the technical terms of the related technology areas, as used in the present invention, are defined below.

[0041] "Understanding" or variations such as "understand", "understand" or "understood from" are used throughout the specification and claims in an inclusive sense, that is, to specify the presence of the determined resources, but not to exclude the presence or addition of additional features that may materially improve the operation or usefulness of any of the modalities of the invention, unless the context otherwise requires due to the language of expression or necessary implication.

[0042] “Consists essentially of” and variations such as “consist essentially of” or “consisting essentially of”, as used throughout the specification and claims, indicate the inclusion of any cited elements or group of elements and the optional inclusion other elements, of a similar or different nature to the mentioned elements, which do not materially alter the basic or innovative properties of the claimed material.

[0043] The terms "nucleic acid" and polynucleotide "are used interchangeably, and refer to RNA and DNA. Polynucleotides can be single or double stranded. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA, siRNA, miRNA, complementary DNA, genomic DNA, synthetic DNA, recombinant DNA, cassettes, vectors, probes and primers. The term "recombinant DNA" refers to any artificial nucleotide sequence that results from the combination of DNA sequences from different sources.

[0044] The term "degenerate nucleotide sequence" denotes a nucleotide sequence that includes one or more degenerate codons when compared to a reference nucleic acid molecule that encodes a given polypeptide. Degenerate codons contain different nucleotide triplets, but they encode the same amino acid residue (eg, GAU and GAC both encode Asp).

[0045] The term "therapeutically effective amount" refers to an amount of proteins or polypeptides that provides activity against cancer, when administered according to the appropriate dose and route of administration.

[0046] The term "pharmaceutically acceptable carriers or excipients" refers to ingredients compatible with other ingredients contained in pharmaceutical preparations and which have no therapeutic effect and are not harmful to humans or animals.

[0047] The term "individual" refers to humans and animals. Preferably the individual is a human being.

[0048] The term "identity" is defined as the degree of equality between DNA or amino acid sequences when compared nucleotide by nucleotide or amino acid by amino acid with a reference sequence.

[0049] The term "percentage of sequence identity" refers to comparisons between polynucleotides or polypeptides and is determined by two sequences ideally aligned, under certain comparison parameters. This alignment can comprise gaps (spaces), generating intervals when compared to the reference sequence, which facilitate an adequate comparison of them. In general, the calculation of the percentage of identity considers the number of positions where the same nucleotide or amino acid occurs in the sequences compared to the reference sequence, being carried out through several sequence comparison algorithms and programs known in the state of the art. Such algorithms and programs include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, CLUSTALW, FASTDB.

[0050] The term "Polymerase Chain Reaction" or the English acronym, PCR, refers to a method in which a nucleic acid fragment is amplified as described in US patent 4,683,195. Generally, the information contained at the 5 'and 3' ends of the sequence of interest is used for the design of the primers or primers that they span, around 8 synthetic nucleotides. These primers have sequences complementary to the sequence to be amplified. PCR can be used to amplify RNA, DNA or cDNA sequences.

[0051] An "expression cassette" refers to a nucleic acid construct comprising a coding region and a regulatory region, operably linked that, when introduced into a host cell, results in the transcription and / or translation of an RNA or polypeptide, respectively. Generally, an expression cassette is constituted or comprised of a promoter that allows the initiation of transcription, a nucleic acid according to the invention, and a transcription terminator. The expression "operably linked" indicates that the elements are combined so that the expression of the coding sequence is under the control of the transcriptional promoter and / or signal peptide. Typically, the promoter sequence is placed upstream of the gene of interest, at a distance compatible with expression control. Likewise, the signal peptide sequence is generally fused upstream of the gene sequence of interest, and in phase with it, and downstream of any promoter. Spacing sequences may be present between regulatory elements and the gene, since they do not prevent expression and / or screening. In one embodiment, said expression cassette comprises at least one "enhancer" activator sequence operably linked to the promoter.

[0052] The term "vector" refers to nucleic acid molecules designed to transport, transfer and / or store genetic material, as well as expressing and / or integrating the genetic material into the host cell's chromosomal DNA, such as, for example, plasmids, cosmids, artificial chromosomes, bacteriophages and other viruses. The vector generally consists of at least three basic units, the origin of replication, a selection marker and the multiple cloning site.

[0053] The vectors used in this invention preferably present at least one "selection marker", which is a genetic element that allows the selection of genetically modified organisms / cells. Such markers include antibiotic resistance genes such as, but not limited to, ampicillin, chloramphenicol, tetracycline, kanamycin, hygromycin, bleomycin, phleomycin, puromycin, and / or phenotype complementing genes, such as, but not limited to methotrexate, dihydrofolate reductase , ampicillin, neomycin, mycophenolic acid, glutamine synthetase.

[0054] The term "expression vector" refers to any vector that is capable of transporting, transferring and / or storing genetic material, and that, once in the host cell, is used as a source of genetic information for the production of a or more gene products (gene expression).

[0055] In addition, the expression vectors of this invention can include one or more regulatory nucleotide sequences to control gene replication, transfer, transport, storage and expression of genetic material, such as origin of replication, selection marker, site multiple cloning, promoter (for example, T7 pol, pL and pR phage lambda, SV40, CMV, HSV tk, pgk, T4 pol, or EF-1 alpha and its derivatives), ribosome binding site, splice site RNA, polyadenylation site, signal peptide for secretion and gene transcription termination sequence. However, the expression vectors of this invention are not limited to them. The technique of incorporating control sequences into a vector is well characterized in the prior art.

[0056] The expression vector used in this invention may also have "enhancer" sequences, also called "cis" elements that can positively or negatively influence the promoter-dependent gene expression.

[0057] A "coding sequence" refers to a nucleotide sequence that is transcribed into mRNA (messenger RNA) and translated into a polypeptide when it is under the control of appropriate regulatory sequences. The limits of the coding sequence are determined by a translation initiation codon at the 5 'end of the DNA sense strand and a translation termination codon at the 3' end of the DNA sense strand. As a result of the degeneration of the genetic code, different DNA sequences can encode the same polypeptide sequence. Therefore, it is considered that such degenerate substitutions in the coding region are inserted in the sequences described in this invention.

[0058] The term "promoter" is a minimum sequence of DNA sufficient to direct gene transcription, that is, a sequence that directs the binding of the RNA polymerase enzyme thus promoting the synthesis of messenger RNA. Promoters can be specific to the cell type, tissue type and species, in addition to being modulated, in certain cases, by regulatory elements in response to some external physical or chemical agent called an inducer.

[0059] The terms "transformation" and "transfection" refer to the act of inserting a vector or other carrier vehicle of exogenous genetic material in a host cell, prokaryotic or eukaryotic, for transport, transfer, storage and / or gene expression of the genetic material of interest.

[0060] The term "recombinant expression" refers to the expression of the recombinant polypeptide in host cells.

[0061] The term "host cell" refers to the cell that will receive genetic material through a vector and / or cells that have already received genetic material through a vector (transformed or transfected cells). These host cells can be either of prokaryotic origin (prokaryotic microorganisms) or eukaryotic (cells or eukaryotic microorganisms).

[0062] In the present application, the terms "peptide", "polypeptide" or "protein" can be used interchangeably, and refer to a polymer of amino acids connected by peptide bonds, regardless of the number of amino acid residues that make up this chain. Polypeptides, as used herein, include "variants" or "derivatives" thereof, which refer to a polypeptide that includes variations or modifications, for example, substitution, deletion, addition or chemical modifications in its amino acid sequence in relation to the polypeptide reference, provided that the derived polypeptide has immunosuppressive activity, stability, half-life, pharmacokinetic characteristics and / or physicochemical characteristics equal to or greater than that initially observed for the original polypeptide. Examples of chemical modifications are glycosylation, PEGylation, PEG alkylation, alkylation, phosphorylation, acetylation, amidation, etc. The amino acids of the polypeptides of the invention, depending on the orientation of the alpha group of the alpha carbon atom may belong to the L or D series. The polypeptide can be produced artificially from cloned nucleotide sequences using the recombinant DNA technique ("recombinant polypeptide") or it can be prepared by a known chemical synthesis reaction ("synthetic polypeptide").

[0063] The term "amino acid substitutions" refers to the replacement of at least one amino acid residue of polypeptides for the production of derivatives with asparaginase activity. Substituting amino acids can be natural, modified or unusual.

[0064] In this respect, the term "conservative amino acid substitution" refers to the replacement of amino acids in a polypeptide by those with similar side chains and, therefore, with very close physical-chemical properties. For example, the exchange of an alanine for a valine or leucine or isoleucine is considered conservative, since the amino acids involved have a common characteristic of an aliphatic side chain. The group containing a basic side chain is composed of lysine, arginine and histidine. The sulfur-containing group in the side chain comprises the amino acids cysteine and methionine. The amino acids phenylalanine, tyrosine and tryptophan contain an aromatic side chain. Asparagine and glutamine are part of the amino acids with side chain containing amide, while serine and threonine contain a hydroxyl linked to its aliphatic side chain. Other examples of conservative substitution include the substitution of a support or hydrophobic amino acid such as isoleucine, valine, leucine or methionine for another also support. Likewise, the invention described here contemplates the replacement of polar or hydrophilic amino acids such as arginine with lysine, glutamine with asparagine and threonine with serine. In addition, substitution between basic amino acids such as lysine, arginine or histidine or substitution between acidic amino acids such as aspartic acid or glutamic acid is also contemplated. Examples of conservative substitution of amino acids are as follows: valine for leucine or isoleucine, phenylalanine for tyrosine, lysine for arginine, alanine for valine and asparagine for glutamine.

[0065] In addition, illustrative examples of modified or unusual amino acids include 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2 -aminoisobutyric, 3-aminoisobutyric acid, 2-aminoheptanoic acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylparagine, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, aloisoleucine, N-methyl glycine, N-methyl isoleucine, 6-N-methyl-lysine, N-methyl valine, norvaline, norleucine, omitine, etc.

[0066] The objects of the present invention will be better understood from the detailed description of the invention and the attached claims.

DETAILED DESCRIPTION OF THE INVENTION

[0067] Unless they are defined differently, all technical and scientific terms used here have the same meaning understood by a technician in the subject to which the invention belongs. The terminology used in describing the invention is intended to describe particular embodiments only, and is not intended to limit the scope of the teachings. Unless otherwise indicated, all numbers expressing quantities, percentages and proportions, and other numerical values used in the specification and in the claims, should be understood as being modified, in all cases, by the term "about" . Thus, unless otherwise indicated, the numerical parameters shown in the specification and in the claims are approximations that may vary, depending on the properties to be obtained.

[0068] The present inventors have solved the problem of the state of the art by providing polypeptides with asparaginase activity that have reduced affinity for amino acids other than asparagine, particularly glutamine. The polypeptides of the present invention with reduced glutaminase activity were obtained by replacing amino acid residues located in the region of the E. coli L-asparaginase (EC-A) active site by corresponding amino acid residues in E. carotovora L-asparaginase ( ECAR-LANS).

[0069] The precise mechanism of the action of L-asparaginase is still unknown, although it is known that hydrolysis occurs in two stages through a beta-acyl-enzyme intermediate. In the first step, the nucleophilic threonine attacks the carbonyl radical of the substrate to generate an acyl-enzyme intermediate. An ammonia molecule is released. In the second step, a water molecule attacks the acyl-enzyme intermediate to produce L-aspartate. Results available in the state of the art propose a conserved catalytic triad involving T residues (position 89 of E. coli L-ASNase), K (position 162 E. coli L-ASNase) and D (position 90 of E L-ASNase coli). Threonine acts as a primary nucleophile that attacks the substrate and the proximal lysine residue, stabilized by aspartate, acts as a base to increase the nucleophilicity of the catalytic threonine residue.

[0070] Residues responsible for binding to the substrate form the rigid part of the active site. The flexible part of the active site (residues 14-33, from E. coli) controls access to the Ugation site. This region is often disordered in the crystals of the L-ASNases, indicating high mobility of this loop. The state of the art further demonstrates that the bonding of the substrate induces rapid closure of the flexible part, whereas in the absence of binders, the flexible part of the active site remains in the open conformation predominantly.

[0071] Evaluation of contacts in the structure of E. carotovora asparaginase shows that conformational changes or different flexibility in the loop formed by residues G61-E63 can affect the glutaminase activity of an L-ASNase, as well as a more bulky residue can further reduce the area of the active site, possibly leading to a further reduction in glutaminase activity.

[0072] To decrease the toxic glutaminase activity of E. coli L-ASNase, the inventors proposed to replace residues from the region of the active site of L-ASNase of that organism with the corresponding residues in E. carotovora L-ASNase, which include the loop suggested as a possible target of the specificity of this enzyme.

[0073] In one embodiment, the mutated E. coli L-ASNase sequence is the amino acid sequence of SEQ ID NO: 1 with substitutions for corresponding residues of E. carotovora L-ASNase of SEQ ID NO: 2.

[0074] In one embodiment, modifications made to the amino acid sequence of E. coli L-ASNase comprise substitutions at residues 53-75 of SEQ ID NO: 1.

[0075] In one embodiment, substitutions are made on amino acid residues in at least one of positions 54, 55, 59, 60, 62, 63, 64, 66, 68, 70, 72, 73 and 75 of the amino acid sequence of SEQ ID NO: 1.

[0076] In particular, the substitutions comprise at least one of the following substitutions in the amino acid sequence of SEQ ID NO: 1: V54A, N55S, Q59E, D60N, N62T, D63S, N64D, W66L, T68K, A70S, K72R, I73V and T75E .

[0077] In a particular embodiment, the polypeptide with asparaginase activity claimed herein comprises the amino acid sequence of SEQ ID NO: 3.

[0078] In a particular embodiment, the polypeptide with asparaginase activity claimed herein consists of the amino acid sequence of SEQ ID NO: 3.

[0079] Also included in the scope of protection of the present invention are amino acid sequences containing the modifications as set out in SEQ ID NO: 3 and other conservative substitutions in different amino acid residues.

[0080] The structural analysis of the asparaginase of the present invention is shown in Figure 1. In Figure 1A, the conserved active site of L-ASNases is identified, mainly residues T89, D90 and K162. In Figure 1B, the modified region in the enzyme of the present invention is identified, where the amino acid residues of the E. coli enzyme (blue regions) have been replaced by the corresponding ones in E. carotovora (green region).

[0081] In thermal denaturation tests, it was observed that the L-ASNase of E. coli is more stable than those of E. corotovora. This difference in stability is already described by other studies, where the incubation was carried out for 3 minutes at 35 ° C, which led to a 60% irreversible decrease in the activity of ECAR-LANS, while Ec-A maintained a large part of its activity after incubation under the same conditions. ECAR-LANS also showed low stability in solution with urea and the catalytic activity was totally lost after one hour of incubation with 2 M urea. In contrast, there was no observation of activity in Ec-A with up to 4 M urea (PAPAGEORGIOU et al, 2008).

[0082] Thermal denaturation analysis also showed that amino acid substitutions that differ Ec-A from Ec-A mut do not affect the thermal stability of E. coli enzymes. Both have Tm (temperature where 50% of the sample is unfolded) of 60 ° C.

[0083] These data allow us to conclude that the β-loop-α motif, formed by the 53-75 region, is not involved in the thermal stability of Ec-A, as its absence does not modify the enzyme's denaturation profile. The substitution of amino acids between residues 53-75, although altering the content of secondary structure in relation to the enzyme without modifications, produces an enzyme with folding and with a quaternary structure similar to the other L-ASNses.

[0084] The L-ASNase of the present invention was not as specific to asparagine when the L-ASNase of parental E. carotovora, which, according to the state of the art, is 12,000 times more specific for L-asparagine than for L -glutamine. However, the proposed substitutions increased the specificity of the enzyme of the present invention in relation to parental E. coli L-ASNase.

[0085] Thus, the inventors surprisingly obtained a modified E. coli enzyme, correctly folded, with conserved secondary and quaternary structures. In addition, the kinetic and enzymatic activity values shown by the recombinant L-ASNase of the present invention increased the specificity of the enzyme compared to the parental E. coli L-ASNase.

[0086] These data further indicate that the L-ASNase of the present invention has a half-life in the bloodstream similar to the L-ASNase of parental E. coli.

[0087] Since the desired properties for therapeutic L-ASNases include high enzymatic activity, low specificity for glutamine and a long half-life in the bloodstream, the enzyme of the present invention proves to be an excellent alternative to L -ASNase of the state of the art that present all the problems already discussed here.

[0088] In one embodiment, the polypeptide of the invention is for use in preventing or treating neoplasms. In a preferred embodiment, the neoplasm is leukemia, being particularly selected from acute myeloid leukemia or acute lymphoid leukemia.

[0089] In another aspect, the present invention provides polynucleotides that encode the polypeptides described herein.

[0090] In one embodiment, the polynucleotides according to the invention comprise the nucleic acid sequence of SEQ ID NO: 4 and its degenerations.

[0091] A person skilled in the art would recognize that degenerations are fully supported based on the information provided in the application and common knowledge of the state of the art. For example, the degeneration of the genetic code (that is, different codons being able to encode the same amino acids) is common knowledge in the art and the identity of the amino acid encoded by each codon is well established.

[0092] Based on information well known and established in the state of the art, the person skilled in the art is able to identify nucleotide substitutions that do not alter the resulting amino acid sequence. For example, if a nucleotide sequence contains the CTA codon that encodes a leucine, a person skilled in the art would understand that replacing “A” with any other nucleotide (i.e., T, C or G) would still result in a codon that encodes for leucine. Thus, when in possession of both the nucleotide sequence of a gene and the amino acid sequence of the encoded protein, the person skilled in the art will easily identify the degenerations that encode the same protein, with the same amino acid sequence.

[0093] The use of the preferred codons can be adapted according to the host cell in which the nucleic acid is to be transcribed. These steps can be performed according to methods well known to the person skilled in the art and some of which are described in the reference manual Sambrook et al, 2001.

[0094] In this sense, different species may exhibit a preferential “codon usage”. See Grantham et al., 1980, Haas et al., 1996, Wain-Hobson et al., 1981, Grosjean and Fiers, 1982, Holm, 1986, Ikemura, 1982, Sharp and Matassi, 1994, Kane, 1995 and Makrides 1996 As used here, the term "preferred codon usage", or "preferred codons" is a term used in the art referring to codons that are most often used in cells of certain species. For example, the amino acid threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells, ACC is the most commonly used codon. In other species, for example, different Thr codons may be preferred. Preferred codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. The introduction of preferred codon sequences into recombinant DNA can, for example, increase the production of the polypeptide by making translation more efficient in a given cell type. Thus, the polynucleotide sequences of the invention can be optimized for different species.

[0095] The polynucleotides of this invention are obtained by methods already known in the art. For example, additional strings can be identified and functionally annotated by comparing strings. Therefore, a person skilled in the art can readily identify a sequence functionally equivalent to the polynucleotides of the present invention in a suitable database, for example, GenBank, using publicly available sequence analysis programs and parameters.

[0096] In another example, polynucleotides of the invention can be obtained through a reverse transcription reaction followed by PCR amplification. Both oligo-dT and random primers can be used in the reverse transcription reaction to prepare single stranded cDNAs, from the isolated RNA of the snake L. muta, which contain the sequences of interest. RNA can be isolated by methods known as using the Trizol reagent (GIBCO-BRL / Life Technologies, Gaithersburg, Maryland).

[0097] Gobinda et al. (PCR Methods Applic. 2: 318-22, 1993), describes “restriction-site PCR” as a direct method that uses universal primers to obtain unknown sequences adjacent to a known locus. First, genomic DNA is amplified in the presence of an adapter-primer, which is homologous to an adapter sequence attached to the ends of the genomic DNA fragments, and in the presence of a specific primer for a known region. The amplified sequences are subjected to a second round of PCR with the same adapter-primer and another specific primer, internal to the first. The products from each round of PCR are transcribed with a suitable RNA polymerase and sequenced using a reverse transcriptase.

[0098] Still illustratively, the inverse PCR allows obtaining unknown sequences starting with primers based on a known region. The method uses several restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR. Divergent initiators are drawn from the known region.

[0099] In addition, it is known that sequences with reduced degrees of identity can also be obtained with the aid of degenerate primers and PCR-based methodologies.

[00100] Typically, the nucleic acid sequence of a primer useful for amplifying nucleic acid molecules by PCR can be based on the amino acid sequences of the polypeptides of the invention. In the present invention, the primers used for the amplification of the genes encoding E. coli mutated L-asparaginase, and E. coli and E. carotovora wild-type are represented by SEQ ID NOs: 7-12.

[00101] In another aspect, the present invention provides an expression cassette comprising a polynucleotide according to the invention operably linked to the sequences necessary for its expression. Typically, the coding and regulatory regions are heterologous to each other.

[00102] In another aspect of the present invention, the present invention provides an expression vector comprising a polynucleotide or an expression cassette according to the invention. This expression vector can be used to transform a host cell and allow expression of the nucleic acid according to the invention in said cell.

[00103] Advantageously, the expression vector comprises regulatory elements that allow the expression of the nucleic acid and elements that allow its selection in the host cell according to the invention. The methods for selecting these elements depending on the host cell in which the expression is desired, are well known to the person skilled in the art and widely described in the literature.

[00104] Vectors can be constructed by classical molecular biology techniques, well known to those skilled in the art. Non-limiting examples of expression vectors suitable for expression in host cells are plasmids and viral or bacterial vectors.

[00105] In another aspect of the present invention, the present invention provides a polynucleotide, expression cassette or an expression vector according to the invention for transforming or transfecting a cell. The host cell can be transformed / transfected in a transient or stable manner and the nucleic acid, cassette or vector can be contained in the cell in the form of an episome or in a chromosomal form.

[00106] The polynucleotide, expression cassette or vector is inserted into competent prokaryotic or eukaryotic host cells. The recombinant clones are selected and then subjected to analysis by restriction enzymes and DNA sequencing, enabling the confirmation of the cloned sequence, using methods, kits and equipment widely known by a technician in the subject.

[00107] Thus, the polypeptides of the invention can be prepared using recombinant DNA technology, wherein a cassette or expression vector comprising a polynucleotide sequence of the invention, for example, which encodes the polypeptide comprising or consisting of SEQ ID No: 4 or its degenerations, is operationally linked to a promoter. Host cells are cultured under appropriate conditions and the polypeptide is expressed. The host cell can be a bacterial, fungal, plant or animal cell. The polypeptide is recovered from the culture, where the recovery may include a polypeptide purification step. The obtained recombinant polypeptide is analyzed and treated in order to solubilize it, when pertinent. The solubilized polypeptide is then purified and characterized biochemically, using, for example, methods common to the field of biochemistry, such as HPLC, SDS-PAGE, Western Blotting, isoelectric focusing with pH gradient, circular dichroism. Through these methods, it is possible to determine characteristics such as, for example, the expression yield of the recombinant polypeptide; the determination of the characteristics of the secondary structures, in addition to other characteristics whose determination is important for the development of a biotechnological drug.

[00108] Polypeptides can be expressed "fused" to a tag. The term "tag" or the English term "tag" refers to coding sequences incorporated near the multiple cloning site of an expression vector, enabling its translation concurrently and adjacent to the sequence of the cloned recombinant polypeptide. Thus, the tag is expressed fused to the recombinant polypeptide. Such tags are well known in the art and include compounds and peptides such as polyhistidine, polyarginine, FLAG, glutathione-S-transferase, maltose-binding protein (MBP), cellulose-binding domain (CBD), Beta-Gal , OMNI, thioredoxin, NusA, mistin, chitin-binding domain, cutinase, fluorescent compounds (such as GFP, YFP, FITC, rhodamine, lanthanides), enzymes (such as peroxidase, luciferase, alkaline phosphatase), chemiluminescent compounds, biotinyl groups, recognized epitopes by antibodies such as leucine zipper, c-myc, metal-binding domains and binding sites for secondary antibodies.

[00109] Polypeptides can also be obtained synthetically using methods known in the art. Direct synthesis of the polypeptides of the invention can be carried out using solid phase synthesis, solution synthesis or other conventional means, generally using protecting groups of α-aminogroup, α-carboxyl and / or functional groups of amino acid side chains. For example, in solid phase synthesis, an appropriately protected amino acid residue is attached through its carboxyl group to an insoluble polymeric support, such as a cross-linked polystyrene or polyamide resin. Solid-phase synthesis methods include both BOC and FMOC methods, which use tert-butyloxycarbonyl, and 9-fluorenylmethyloxycarbonyl as α-amino protecting groups, respectively, both well known to those skilled in the art (Sambrook et al., 1995).

[00110] The following protecting groups can be examples used for the synthesis of the polypeptides of the invention: 9-fluorenylmethyloxycarboyl (Fmoc), tert-butyloxycarbonyl (Boc), carbobenzyloxy (Cbz), 2-chloro-3-indenylmethoxycarbonyl (Climoc), benz (f) indene-3-yl-methoxycarbonyl (Bimoc), 1,1 dioxobenzo [b] thiophene-2-yl-methoxycarbonyl (Bsmoc), 2,2,2-trichloroethoxycarbonyl (Troc), 2- (trimethylsilyl) ethoxycarbonyl ( Teoc), homobenzyloxycarbonyl (hZ), 1,1-dimethyl-2,2,2-trichloroetoxycarbonyl (TCBoc), 1-methyl-1 (4-biphenyl) ethoxycarbonyl (Bpoc), 1- (3,5-di-t -butylphenyl) -1-methylethoxycarbonyl (t-Bumeoc), 2- (2'- or 4'-pyridyl) ethoxycarbonyl (Pyoc), vinyloxycarbonyl (Voc), l-isopropylalyloxycarbonyl (Ipaoc), 3- (pyridyl) allyloxycarbonyl (Paloc), p-methoxybenzyloxycarbonyl (Moz), p-nitrocarbamate (PNZ), 4-azidobenzyloxycarbonyl (AZBZ), Benzyl (Bn) MeO, BnO, Methoxymethyl (Mom), methylthiomethyl (MTM), phenyldimethylihlmetoxymethyl (SMOM) butyldimethylsihl (TBDMS), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), nitrobenzyloxymethyl (NBOM), p-anisiloxymethyl (p-AOM), pBuOCH20-, 4-pentenyloxymethyl (POM), 2-methoxymethoxymethyl (MEM), 2- (trimethylsihl) ethoxymethyl (SEM), menthoxy (MM), tetrahidropiran , - OCOCOph, Acetyl, C1CH2C02-, -C02CH2CC13, 2- (Trimethylsilyl) ethyl (TMSE), 2 (p-toluenesulfonyl) ethyl (Tse). (Greene T.W. Wuts P.G.M., 1999).

[00111] After the chemical reaction, the polypeptides can be separated and purified by a known purification method. An example of such purification methods may include a combination of solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like.

[00112] In one embodiment, the purification of the polypeptide of the present invention has three stages: affinity, ion exchange and filtration gel, the latter being responsible for separating from the total extract those forms of the enzyme that are not in the active tetrameric form.

[00113] In another aspect, a pharmaceutical composition is provided herein comprising a polypeptide with asparaginase activity according to the invention and at least one pharmaceutically acceptable carrier or excipient.

[00114] Pharmaceutically acceptable carriers or excipients are selected according to the final presentation of the composition of the present invention which can be in the form of capsules, tablets or solution for administration for injection for intramuscular or intravenous administration.

[00115] Pharmaceutically acceptable excipients, carriers or stabilizers do not present toxicity to the recipient organism at the dosages and concentrations used and include buffers such as phosphate, citrate and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens such as methyl- and propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; proteins such as albumin, gelatin or immunoglobulins; amino acids, monosaccharides, disaccharides and other carbohydrates such as glucose, mannose, sucrose, mannitol or sorbitol; polymeric excipients such as polyvinylpyrrolidones, Ficoll®, dextrins and polyethylene glycols; flavoring agents; sweeteners; anti-static agents; chelating agents such as EDTA or EGTA; ion-releasing salts such as sodium; metal complexes; nonionic surfactants such as polysorbates 20 and 80; lipids like phospholipids, fatty acids and steroids like cholesterol. Methods for preparing various pharmaceutical compositions are well known, or will be apparent in light of the present invention, by the skilled artisan in pharmaceutical technology.

[00116] In addition, the compositions may comprise additives in order to increase the ease of administration, the capacity to be stored, resistance to degradation, bioavailability, half-life, to provide isotonic preparations, etc. Usual additives for the preparation of pharmaceutical compositions are well known in the art.

[00117] The composition according to the present invention may comprise at least one additional chemotherapeutic agent selected from alkylating agents, antimetabolites, kinase inhibitors, antifuse poison plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, antiestrogens and selective estrogen receptor modulators (SERMs), antiprogesterones, downward estrogen receptor regulators (ERDs), estrogen receptor antagonists, luteinizing hormone releasing hormone agonists, antiandrogens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors , antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth. Chemotherapeutic agents useful in the treatment methods of the present invention include cytostatic and / or cytotoxic agents.

[00118] The pharmaceutical compositions of the present invention must comprise a therapeutically effective amount of the polypeptide. For any compound, the therapeutically effective dose can be estimated initially, either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs or pigs. The animal model can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

[00119] The pharmaceutical composition according to the present invention comprises from 0.1% to 99% w / w, preferably 1% to 60% w / w, particularly 10% to 50% w / w of the polypeptides of the present invention. In a particular embodiment, the pharmaceutical composition according to the present invention comprises 10,000 IU of the polypeptides of the present invention.

[00120] According to the present invention, the administration of said pharmaceutical compositions can be carried out by intravenous and intramuscular administration routes. In a preferred embodiment, the composition of the present invention is for intravenous administration.

[00121] In another aspect, the present invention provides for the use of the polypeptides of the invention in the manufacture of a medicament for the prevention or treatment of neoplasms. In a preferred embodiment, the neoplasm is leukemia. In particular, leukemia is acute myeloid leukemia or acute lymphoid leukemia.

[00122] The present invention further relates to a method for producing polypeptide according to the invention with asparaginase activity comprising inserting a polynucleotide, cassette or expression vector according to the invention into an in vivo expression system and the collection of the polypeptide produced by said system. Numerous in vivo expression systems, including the use of suitable host cells, are commercially available and the use of these systems is well known to the person skilled in the art.

Particularly suitable expression systems include microorganisms, such as bacteria transformed with bacteriophage, plasmid or cosmid recombinant DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (eg, baculovirus); plant cell systems transformed with virus expression vectors (eg, cauliflower mosaic virus, CaMV [cauliflower mosaic virus]; tobacco mosaic virus, TMV [tobacco mosaic virus]) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems. It is also possible to employ cell-free translation systems to produce the polypeptides of the invention.

[00124] The introduction of polynucleotides that encode a polypeptide of the present invention into host cells can be performed using methods described in many standard laboratory manuals, such as Davis et al., 1986) and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989).

The transformed or transfected host cell described above is then cultured in a suitable nutrient medium under conditions conducive to the expression of the immunosuppressive polypeptides of the invention. The medium used to grow the cells can be any conventional medium suitable for developing the host cells, such as minimal or complex medium containing appropriate supplements. Suitable media are available from commercial suppliers or can be prepared according to published recipes (for example, in the catalogs of the American Type Culture Collection). The polypeptides of the invention produced by the cells can then be recovered from the cell or the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the aqueous protein components from the supernatant or filtered through a salt, for example. example, ammonium sulfate, purification by a variety of chromatographic procedures, for example ion exchange chromatography, exclusion chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography or the like, depending on the type of polypeptide in question.

[00126] In accordance with another aspect of the invention there is provided a method for producing a polypeptide with asparaginase activity according to the invention comprising:
(a) providing a host cell comprising an expression cassette or expression vector comprising a polynucleotide encoding a polypeptide with asparaginase activity according to the present invention;
(b) culturing the transformed or transfected host cell to obtain a cell culture;
(c) expressing the polynucleotide of the present invention in a host cell transformed or transfected to produce a polypeptide; and
(e) isolating the polypeptide of the present invention from the cell or cell culture.

[00127] In a particular aspect of the invention, the host cell is a prokaryotic microorganism or a eukaryotic cell or microorganism. In a further aspect of the invention, said polypeptide is provided with a "tag".

[00128] In a particular aspect of the invention, the host cell is a bacterium. In a particular aspect of the invention, the bacterium is of the genus Escherichia. In a particular aspect of the invention, the host cell is E. coli.

[00129] In another aspect of the invention, a method of preventing or treating neoplasms is provided, characterized in that it comprises administering to a subject in need of said prevention or treatment, a therapeutically effective amount of a polypeptide according to the invention .

[00130] The effective amount needed for a human individual will depend on the severity of the disease state, the general health of the individual, the age, weight, and sex of the subject, the diet, the time and frequency of administration, the combination / combinations of drugs, reaction sensitivities, and tolerance / response to therapy. Thus, doses to be delivered depend on a number of factors that cannot be measured before clinical trial studies are done. The technician in the subject, however, knows how to reach adequate doses for different treatments.

[00131] The examples cited below are merely illustrative, and should be used only for a better understanding of the developments contained in the present invention, however, they should not be used in order to limit the objects described.

EXAMPLES EXAMPLE 1: Cloning of wild and modified L-ASNases

[00132] E. coli derived enzyme constructs were amplified by polymerase chain reaction (PCR), using E. coli strain C43 genomic DNA (amplification region: 430-1044pb - fragment 3) and DNA synthetic with the modifications inserted produced by the company IDT (amplification region: 66-429pb - fragment 2). The amplification products were cloned into pGEM T-easy (Promega) and analyzed by sequencing.

[00133] The sequencing result was compared to the sequence deposited in the database used as a template sequence. In the E. coli nucleotide sequence used in the present invention there is a substitution (C321T). This is a silent mutation for the creation of an Ncol restriction site that will be used to join Ec-A_frag1 and Ec-A_frag2 to the vector.

[00134] After confirming the correct sequence, they were subcloned into expression vectors pET28a-TEV (CARNEIRO et al., 2006) to transform E. coli bacteria from different strains for expression tests.

[00135] The coding sequences of constructs derived from E. carotovora were optimized for expression in E. coli, using the OptimumGene ™ algorithm, regarding the use of codons, removal of unwanted restriction sites, adequacy of CG content, removal of ORFs (Open Reading Frames) and secondary mRNA structures that may interfere with translation efficiency. The optimization process consisted of modifying 80 nucleotides, which did not alter the native amino acid sequence.

[00136] The E. carotovora constructions were synthesized and acquired commercially from the company GenScript (New Jersey, United States) in vector pUC57. In the optimization stage, restriction sites for NdeI and Xhol enzymes were inserted at the ends of the E. carotovora genes. The ECAR-LANS-pUC57 vector was synthesized containing the signal peptide sequence for periplasmic secretion, so that primers were designed for amplification of ECAR-LANS without signal peptide (Table 3) and subcloning in pET28a-TEV.

[00137] The methodology used to obtain the clones followed the following order:
Oligonucleotide design;
Gene amplification by PCR;
Agarose gel electrophoresis;
DNA extraction from agarose gel;
Linking the genes of interest in pGEM T-easy;
Transformation into E. coli Dh5α;
Selection of transformants and sequencing;
Plasmid DNA extraction;
Plasmid DNA digestion;
Digestion and binding in vector pET28a_TEV;
Transformation into E. coli Dh5α;
Selection of transformants and sequencing;
Plasmid DNA extraction; and
Transformation into expression strain.

1.1. Design of primer oligonucleotides

[00138] For the cloning of the Ec-A construct, the initiator oligonucleotides (Table 1) were designed based on the gene sequence deposited in the database (www.nbci.nlm.nih.gov). Site for restriction enzyme NdeI (BioLabs) was added in the 5 'region of each oligonucleotide, allowing subcloning in vector pET28a-TEV.

Tabela 2: Oligonucleotídeos utilizados para clonagem de L-ASNases derivadas de E. carotovora. Os sítios para enzimas de restrição estão sublinhados.

[00139] The same pair of primer oligonucleotides was used to amplify the gene encoding ECAR-LANS with and without modifications (Table 2), which differed in the amplification reaction was the template DNA, which was ECAR-LANS_pUC57 without modifications for amplification of the wild gene and ECAR-LANS_pUC57 with modifications for amplification of the gene with modifications.
Table 1: Oligonucleotides used for cloning L-ASNases derived from E. coli. The restriction enzyme sites are underlined.

Table 2: Oligonucleotides used for cloning L-ASNases derived from E. carotovora. The restriction enzyme sites are underlined.

1.2. Gene amplification by PCR

[00140] For amplification, the enzyme Taq DNA Polymerase (Invitrogen) was used. The final concentration of the primers, specific for each sequence, was 5 pmol. The travel temperature was 55 ° C to amplify Ec-A_fragl and Ec-A_frag2, 58 ° C for ECAR-LANS and 64 ° C for Ec-A_frag3. The amplification was performed in 30 cycles, with 30 seconds of extension for Ec-A_fragl and Ec-A_frag2 and one minute of extension for Ec-A_frag3 and ECAR-LANS.

1.3. DNA extraction in agarose gel

[00141] The amplification products were submitted to electrophoresis on 1% agarose gel. 1 kb Plus DNA Ladder (Invitrogen) was used as a molecular weight marker and MassRuler Loading Dye Solution 6X (Fermentas) supplemented with GelRed ™ (Biotium) 1: 500 as sample buffer. Electrophoresis was performed at 100 V for 35 minutes.

[00142] After agarose gel electrophoresis, the bands referring to the desired nucleotide sequences were extracted and purified using the QIAquick Gel Extraction Kit Protocol (QIAGEN).

1.4. Cloning vector link

[00143] The insertion of the pGEM T-easy vector insert (Promega) was done using 25 ng of purified gel sample, 50 ng of vector and 3 U of T4 DNA ligase enzyme (Promega). The reaction was maintained at 16 ° C for 18 hours and then transformed into a competent Escherichiaslklk coli Dh5α cell, as described below.

1.5. Bacteria preparation and transformation by thermal shock

[00144] An isolated colony of E. coli Dh5α was inoculated in Luria-Bertani (LB) culture medium under agitation of 200 rpm for approximately 16 hours at 37 ° C. After this period, a 1: 100 dilution was performed in LB medium. The culture was incubated at 37 ° C until OD600 of 0.6 - 0.8. After 10 minutes at 4 ° C, cells were collected by centrifugation at 4000 g for 10 minutes at 4 ° C. The pellet was resuspended in 10 mL of 0.1 M CaCl2 solution and 10% glycerol. After 15 minutes at 4 ° C, the cells were centrifuged at 4000 g for 15 minutes at 4 ° C. Aliquots in 1 mL of 0.1 M CaCl2 solution and 10% glycerol were stored at -80 ° C.

[00145] Transformation was performed by adding approximately 100 ng of the plasmid. After 30 minutes of incubation at 4 ° C, thermal shock was performed by incubating the cells for 2 minutes at 42 ° C, followed by 2 minutes at 4 ° C. 1 ml of LB medium was added to the cells. After 60 minutes, at 37 ° C and 200 rpm, the cells were centrifuged for 2 minutes at 5000 g and the pellet resuspended in approximately 70 μL of LB medium. 2 were then plated in solid LB medium containing the appropriate antibiotic. The plates were incubated for 16 hours in an oven at 37 ° C.

1.6. Selection of transformants

[00146] The selection of transformants was carried out by gene amplification by PCR or plasmid digestion. For amplification, 1 isolated bacterial colony was diluted in 30 μL of water and heated to 94 ° C for 10 minutes. 2 μL of this suspension was used as a DNA template.

[00147] For plasmid digestion selection, the purified vectors were incubated for 1 hour at 37 ° C with the restriction enzymes flanking the region of interest (Table 2) and the fragment release evaluated by agarose gel electrophoresis.

[00148] The selected plasmids were sequenced by the sequencing service of the Carlos Chagas Institute - FIOCRUZ / PR. The sequencing used is the Single Extension. After sample preparation carried out by the company Macrogen (Korea), the sample is precipitated with ethanol and sequenced using Automatic Sequencer 3730x1.

1.7. Small scale plasmid DNA extraction

[00149] The extraction step was performed using the QIAprep Spin Miniprep Kit (QIAGEN) kit, where the DNA present in the bacterial lysate adsorbed on the silica membrane available in the kit. After washing steps to separate DNA from contaminants in bacterial culture, the DNA adsorbed on silica is eluted in water or buffer.

1.8. Subcloning in expression vector

[00150] For the expression of E. carotovora enzymes, the purified plasmid DNA was used for subcloning in the expression vector pET28a (Qiagen). For this, the plasmid insert pGEM-T easy was cleaved with the enzymes NdeI and XhoI. 2 U of T4 DNA ligase (Fermentas), and plasmid: insert in a 1: 3 ratio were used. The final volume was 10 μL and the reaction incubated for 16 hours at 16 ° C. The pET28a vector had previously been digested with the same enzymes. After the insertion of the insert in the vector, transformation into the DH5α strain was performed, following the transformation protocol by thermal shock.

[00151] The nucleotide sequence of wild ECAR-LANS is as shown in SEQ ID NO: 6.

The sequence encoding the E. coli L-ASNase was cloned in pGEM T-easy into two separate parts: nucleotide sequence 1-267 without (Ec-A_frag1) or with modifications (Ec-A_frag2) and the sequence of nucleotides 268-978 (Ec-A_frag3).

[00153] Ec-A_frag3 was digested with the enzymes NcoI and XhoI and linked to the vector pET28a previously digested with the same enzymes. After selection of positive clones, new digestion was performed with the enzymes NdeI and NcoI. Then, Ec-A_frag2, previously selected and digested with the enzymes NcoI and NdeI, was linked to the vector pET28a-TEV + Ec-A_frag3. The combination of Ec-A_frag1 and Ec-A_frag3 in pET28a-TEV resulted in the Ec-A vector (unmodified gene -SEQ ID NO: 5). The combination Ec-A_frag2 and Ec-A_frag3 in pET28a-TEV resulted in the vector Ec-A_mut (gene with the modifications - SEQ ID NO: 4).

[00154] The synthetic DNAs used as templates in the present invention were synthesized by Genscript.

EXAMPLE 2: L-ASNases expression test

[00155] The expression of the enzymes Ec-A, Ec-A_mut, ECAR-LANS was evaluated at different induction temperatures in the following strains of E.coli: BL21 (DE3) -Star, Rosetta-Gami 2, Tuner and pLysS. For induction, isolated colonies were inoculated in 5 mL of LB medium with appropriate antibiotic and incubated for 16 hours, at 37 ° C and 200 rpm. Then, a 1:40 dilution was performed and the cells cultured at 37 ° C until OD600nm of 0.6. For expression induction, 0.1 mM of IPTG was added to the inoculants, except in the control cultures, and the incubation was continued at 20 ° C for 16 hours, 30 ° C and 37 ° C for 4 hours.

[00156] After the induction period, the cultures were centrifuged for 15 minutes, at 5000 g and 4 ° C. The precipitate was resuspended in 1 ml of buffer A (20 mM phosphate, 500 mM NaCl, pH 7.5) supplemented with 1 mM PMSF and 5 mM benzamidine. After a 30-minute incubation with lysozyme (10 μg / mL), cell lysis was performed by sonication (8 pulses, 20% potency). The samples were centrifuged at 20,000 g for 15 minutes. The supernatants were separated (soluble fraction) and the pellets were resuspended in 1 mL of buffer A (insoluble fraction).

[00157] The analysis of the solubility was performed by electrophoresis in polyacrylamide gel. Separation gels 13% and stacking 5% were prepared according to a pre-established protocol (LAEMMLI, 1970). In 15 μL of aliquot taken at each pass (pellet and supernatant), 5 μL of sample buffer (240 mM Tris-HCl, 6% SDS, 30% glycerol, 16% β-mercaptoethanol, 0.6 mg / mL was added bromophenol blue, pH 6.8), and were incubated at 95 ° C for 5 minutes for later application to the gels. 10 μL of prepared sample were applied to each well of the gel. The molecular weight markers used were Unstained / Prestained Protein Molecular Weight Marker (Ferments) or BenchMark ™ Protein Ladder (Thermo Fisher Scientific). The gel was stained with Coomassie blue R-250 or G-250, depending on the case, and discolored with an appropriate solution (45% methanol and 10% acetic acid).

[00158] After analyzing the best expression condition for each protein (Table 4), the protein expression was performed on a larger scale, maintaining the proportions of volume and induction conditions. Thus, soluble fractions of each protein were obtained for further purification. Table 4: Condition of expression of L-ASNases. E. coli strains used in the expression of each of the enzymes under study, with the best induction temperature and growth time for E. coli and Erwinia carotovora L-ASNases.

EXAMPLE 3: Purification of recombinant proteins 3.1. Affinity chromatography

[00159] The expression vector pET28a-TEV allows the recombinant protein to be expressed fused to a histidine tail, enabling the use of chromatographic columns containing nickel immobilized in solid phase. Therefore, the soluble fractions obtained from each of the enzymes were purified by a first stage of affinity chromatography with the HisTrap HP 1 mL column (GE Healthcare). The buffers used were buffer A (20 mM sodium phosphate, 500 mM NaCl, pH 7.5) and buffer B (20 mM sodium phosphate, 500 mM NaCl, 1 M imidazole, pH 7.5). The purification column and Äkta system (models Äkta Pure M25 or Äkta Purifier UPC 100) were washed first with 5 column volumes (CV) of milliQ water and then with 5 CV of buffer A. Then the sample was injected with flow varying between 0.3 - 1 mL / min, depending on the system pressure. After sample injection, the column was washed with 2 CV of buffer A to remove proteins not bound to the column and then elution started with a segmented gradient of 0 - 100% buffer B in 20 CV.

[00160] The elution of Ec-A, ECAR-LANS and Ec-A_mut was performed with 160 mM, 220 mM and 180 mM imidazole, respectively. The elution was collected in fractions of 1.5 mL. After the end of the chromatographic run, the purity of the fractions obtained was analyzed by polyacrylamide gel electrophoresis. The asparaginases of E. coli and E. carotovora, as well as of the mutated asparaginase as described in the present invention were purified in the presence of contaminants and, therefore, a second chromatography was performed.

3.1. Ion exchange chromatography

[00161] Affinity chromatography fractions that contained the protein of interest were dialyzed against buffer C (20 mM Tris-HCl pH 8) to remove the salt. The column used in this purification step was HiTrap Q 1 mL (GE Healthcare). Elution occurred by segmented gradient of 0 -100% buffer D (20 mM Tris-HCl, 1 M NaCl, pH 8). After the end of the chromatographic run, the purity of the fractions obtained was analyzed by polyacrylamide gel electrophoresis.

[00162] In the case of Ec-A_mut protein, the charge balance of the protein is negative (pI Ec-A_mut = 4.95). The column was then equilibrated with buffer C. The sample was injected into the column and the elution occurred due to the gradual increase in NaCl concentration, reaching 1M. Elution was performed at 300 mM.

3.2. Gel chromatography filtration

[00163] Samples from ion exchange chromatography that contained the protein of interest were concentrated through filtration. The solution was placed in an Amicon Ultra MWCO 10,000 filter (Milipore) and centrifuged at 4,000 rpm at 4 ° C until the sample volume was equal to 500 μL.

[00164] The experiment was performed in Äkta Pure M25 system (GE Healthcare), using HiLoad 16/60 Superdex 200 pg column (GE Healthcare), in 20 mM Tris-HCl pH 8, 150 mM NaCl buffer.

[00165] Fractions from chromatography were analyzed using polyacrylamide gel electrophoresis. With this assay, it is possible to separate the proteins contained in the sample according to the molecular size. The column used in the chromatography is composed of a matrix with pores of different diameters, therefore, the smaller the particle, the greater the path taken and the greater the elution volume. In addition, the technique also allows estimating the size of the eluted molecules, based on the hydrodynamic radius and elution volume, compared to a standard curve made with samples of known sizes.

[00166] The degree of purity of the samples necessary for further enzymatic and structural characterization was achieved after gel chromatography filtration. The yield of each of the proteins was 15 mg and 8 mg per liter of culture of E. coli BL21 (DE3) -Star producing the native E. coli and E. carotovora L-ASNase, respectively.

[00167] By way of comparison, the commercial L-ASNase Ew-A is expressed for 16 hours, at 23 ° C with induction made by IPTG. The purification of this enzyme is carried out by cation exchange chromatography and gel filtration on a Superdex 200 pg column (Alize Pharma II, 2011). The expression of commercial Ec-A is done in vector pET27b in a strain of E. coli EN538, to allow control of protein production. The culture in LB medium at 37 ° C until reaching OD600nm equal to 0.8 is induced with 1 mM of IPTG and maintained at 37 ° C for another 4 hours. Purification is performed by a cationic exchange chromatography step and then by anionic purification.

[00168] The elution volume of Ec-A, and ECAR-LANS was 68.95 and 70.29 mL, respectively (Figure 2). Through a calibration curve (Figure 3) that relates the elution volume and molecular mass of standard proteins, it is possible to verify that the quaternary structure of the recombinant proteins E. coli and E. carotovora is composed of 4 monomers.

[00169] By the result of gel filtration chromatography (Figure 4), with an elution volume of approximately 79 ml, it is possible to confirm that the enzymes Ec-A-mut was produced in tetrameric form in solution, as well as Ec-A and ECAR -LANS.

EXAMPLE 4: Enzymatic assays and kinetic analysis

[00170] The enzyme activity tests were performed by detecting ammonia by the Nessler reagent. Firstly, it was necessary to perform a standard curve to relate OD436nm and ammonia concentration generated in the reaction. For that, different concentrations of ammonium sulfate solution (0.03 - 0.075 - 0.15 - 0.3 - 0.45 - 0.6 - 1.05 - 1.5 mM) in 50 mM Tris-HCl pH 8.6 were incubated for 30 minutes at 37 ° C. Then 60 mM TCA was added to each of the tubes. Centrifugation was performed for 2 minutes at 5,000 rpm and 0.2 mL of the supernatant was collected and added to tubes with 4.3 mL of milliQ water. Then, 500 μL of Nessler's reagent (Sigma) was added. The volume of 200 μL of each tube was transferred to 96-well ELISA plates for reading at a wavelength of 436 nm.

[00171] To evaluate enzymatic kinetics with the calculation of the maximum velocity (Vmax) and the Michaelis-Menten coefficient (Km), the asparaginase activity assays were performed by incubating 1.5 μM of each of the proteins with different concentrations (0.1 ; 0.25; 0.5; 1 and 2 mM) asparagine or glutamine in 50 mM Tris-HCl pH 8.6. Activity measures were measured for 30 minutes every 2 minutes.

[00172] The data obtained in the reading were processed to determine the values of enzymatic activity in IU / mg of protein, Km and Vmax. A mixture of all reaction components without the presence of the enzyme was used as a negative control. All experiments were carried out in triplicate.

onde o volume (vol.) é apresentado em mL, massa em mg e tempo de reação em minutos.[00173] The equations used to determine enzyme activity were as follows:

where the volume (vol.) is presented in mL, mass in mg and reaction time in minutes.

[00174] The maximum speed (Vmax) of E. coli asparaginase is 25.1 μΜ / min and the dissociation coefficient (Km), 26.5 μΜ (Figure 5B). The enzyme activity presented was 149.3 IU / mg. These data are in accordance with the value described by PHILIPS et al., 2013.

[00175] The maximum speed (Vmax) of E. carotovora asparaginase is 27.4 μΜ / min and the dissociation coefficient (Km), 75 μΜ (Figure 5A). The enzyme activity presented was 340 IU / mg. These results are in accordance with the one already described by WARANGKAR & KHOBRAGADE, 2010.

Tabela 6: Dados da análise cinética das enzimas incubadas com o substrato glutamina.

[00176] The verification of the L-glutaminase activity of Ec-A_mut in comparison with Ec-A and ECAR-LANS was carried out. The kinetic analysis data of the enzymes when incubated with asparagine and glutamine are shown in Tables 5 and 6 below:
Table 5: Data from the kinetic analysis of enzymes incubated with the substrate asparagine.

Table 6: Data from the kinetic analysis of enzymes incubated with the substrate glutamine.

EXAMPLE 5: Circular dichroism spectroscopy

onde θobs é a elipsidade molar observada em graus, MRW é o peso molecular médio dos resíduos da proteína, d é o caminho óptico da cubeta em centímetros e c é a concentração da proteína em mg/mL. O MRW foi calculado dividindo-se o peso molecular da proteína pelo número de resíduos.[00177] The circular dichroism spectrum was collected using the Jasco J-815 spectropolarimeter (Jasco Corporation, Japan). The data were collected in quartz cuvettes, with variations in the optical path (0.2 - 1 mm), depending on the need for each protein and the buffer components. The molar ellipsity in degrees was measured in the distant UV region, in the wavelength range of 190 - 260 nm, and this value can be limited depending on the voltage in the equipment detector, which should not exceed 800 V. The reading speed was 100 nm / min, with 10 readings for proteins and 6 readings for buffers, with 1 second response in continuous scan. The values obtained in milligrams were converted to molar ellipsity per residue ([θ] MRW), in milligrams.cm2 / dmol, which is defined by the equation (ADLER et al, 1974):

where θobs is the molar ellipsity observed in degrees, MRW is the average molecular weight of the protein residues, d is the optical path of the cuvette in centimeters and c is the protein concentration in mg / mL. The MRW was calculated by dividing the molecular weight of the protein by the number of residues.

[00178] The circular dichroism spectra of E. coli and E. carotovora asparaginases indicate the presence of β-tapes and α-helices (Figure 6). This can be seen by the minimum values at wavelengths 208 and 222 nm, in addition to positive values at 190 nm, a pattern resulting from the presence of a-helices in the structure. In addition, negative values at 218 nm and positive values at 196 nm are verified due to the contribution of β-tapes. The presence of β-tapes is also evidenced by the value at 208 nm being less than at 222 nm. In the case of unstructured samples, it would be possible to identify a positive band at 212 nm and a negative band at 195 nm. These data are strong indications that the recombinant proteins are correctly folded.

[00179] The circular dichroism spectrum of Ec-A_mut indicates the presence of secondary structures in the protein, a strong indication that it is folded (Figure 7). However, the overlapping of the Ec-A, ECAR-LANS and Ec-A_mut spectra indicates that the replacement of the 53-75 region of Ec-A by the region conserved in ECAR-LANS resulted in structural changes in comparison with the Ec- A and ECAR-LANS.

[00180] The main change in Ec-A_mut is observed by the decrease in signal at wavelengths smaller than 215 nm, this reflects changes mainly in α-helices (208 nm) and β-tapes (deviation of the spectrum to the right in wavelengths less than 208 nm). The structural model shows that the interactions that stabilize the helix formed by residues 64-76 (Ec-A_mut numbering) are maintained with the changes in that region. There are still interactions with residues K79, T95, A81, T26-N35, D208-L215 of the same molecule and with the region formed by G245-K251 of the adjacent molecule. Structural changes in these regions must be responsible for the differences in the circular dichroism spectra between Ec-A_mut, Ec-A and ECAR-LANS.

[00181] Figure 8 shows the region that differs from Ec-A in dark blue. The modified region can interact either with regions of the same monomer (intra-chain) or with regions of the monomer of another chain (inter-chain). Regions of the same monomer (in red) or the adjacent monomer (in orange) that interact with the modified part of Ec-A_mut are colored red and orange, respectively. The interactions between these regions are shown as dashed lines.

EXAMPLE 6: Thermal denaturation by circular dichroism spectroscopy

[00182] For the thermal denaturation tests, a temperature variation of 20 to 90 ° C was made, and the data were acquired in a Jasco J-815 spectropolarimeter (Jasco Corporation, Japan) at a wavelength of 222 nm.

[00183] According to the circular dichroism value at 222nm, the percentage of folded protein at anahed temperatures was inferred, which resulted in a graph that describes the% of folded fraction present in the sample as a function of temperature (Figure 9). For comparative analysis of the effects of the substitutions found in Ec-A_mut, the same experiment was carried out with the enzymes Ec-A and ECAR-LANS.

[00184] The results obtained in the thermal denaturation test illustrate well that the E. coli L-ASNase is more stable than those of Erwinia. At 40 ° C, approximately 50% of the ECAR-LANS sample is unfolded, while only 3.2% of Ec-A has lost structure. Pharmacokinetic studies of commercial L-ASNases Ec-A and Ew-A show that the half-life of Ec-A (2430 hours) is approximately double the half-life of Ew-A (16 hours), resulting in fewer doses throughout treatment (HemOnc Today, 2012).

Claims (19)

Polypeptide with asparaginase activity, characterized by the fact that it comprises substitutions in the region of the active site of the amino acid sequence of SEQ ID NO: 1 with corresponding amino acid residues in the amino acid sequence of SEQ ID NO: 2, in which the said polypeptide has less affinity by different amino acids other than asparagine when compared to the peptide with parental asparaginase activity. Polypeptide according to claim 1, characterized in that said region of the active site comprises amino acid residues at positions 53 to 75 of the amino acid sequence of SEQ ID NO: 1. Polypeptide according to claim 1, characterized in that the substitutions are made in amino acid residues in at least one of the positions 54, 55, 59, 60, 62, 63, 64, 66, 70, 72, 73 and 75 of amino acid sequence of SEQ ID NO: 1. Polypeptide according to claim 3, characterized in that the substitutions comprise at least one of the following substitutions in the amino acid sequence of SEQ ID NO: 1: V54A, N55S, Q59E, D60M, N62T, D63S, N64D, W66L, A70S, K72R, I73V and T75E. Polypeptide according to any one of claims 1 to 4, characterized in that it comprises the amino acid sequence of SEQ ID NO: 3. Polypeptide according to any one of claims 1 to 5, characterized in that it is additionally covalently conjugated to polyethylene glycol (PEG). Polypeptide according to any one of claims 1 to 6, characterized in that it is for use in the prevention or treatment of neoplasms. Polypeptide according to claim 7, characterized by the fact that the neoplasm is acute myeloid leukemia or acute lymphoid leukemia. Polynucleotide, characterized by the fact that it encodes the polypeptide as defined in any one of claims 1 to 6. Polynucleotide according to claim 9, characterized in that it comprises the nucleic acid sequence of SEQ ID NO: 4 and its degenerations. Expression cassette, characterized in that it comprises a polynucleotide as defined in claim 9 or 10, operably linked to a promoter and a transcription terminator. Expression vector, characterized in that it comprises a polynucleotide as defined in claim 9 or 10 or an expression cassette as defined in claim 11. Host cell, characterized by the fact that it comprises an expression cassette as defined in claim 11, or an expression vector as defined in claim 12. Pharmaceutical composition, characterized in that it comprises a polypeptide as defined in any one of claims 1 to 6. Use of a polypeptide as defined in any one of claims 1 to 6, characterized by the fact that it is in the manufacture of a medicament for the prevention or treatment of neoplasms. Use according to claim 15, characterized by the fact that the neoplasm is acute myeloid leukemia or acute lymphoid leukemia. Method for producing a polypeptide with asparaginase activity, characterized by the fact that it comprises:

• (a) providing a host cell as defined in claim 13;
• (b) cultivating said cell under conditions conducive to the production of the polypeptide; and
• (c) isolating said polypeptide from said cell or culture medium surrounding said cell.

Method according to claim 17, characterized in that said polypeptide is covalently conjugated to polyethylene glycol (PEG). Method for preventing or treating neoplasms, characterized in that it comprises administering a therapeutically effective amount of the polypeptide as defined in any one of claims 1 to 6 to an individual in need of said prevention or treatment.

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