Activity-improved variants of human asparaginase 1

Abstract

The present invention relates to variants (mutants) of human asparaginase 1 (hASN1 variants) with improved asparaginase activity and related matter, including polynucleotides encoding said hASN1 variants, expression vectors and host cells comprising said polynucleotides, methods for preparing said hASN1 variants as well as pharmaceutical compositions and medical uses of said hASN1 variants.

Description

[0001] ACTIVITY-IMPROVED VARIANTS OF HUMAN ASPARAGINASE 1

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to variants (mutants) of human asparaginase 1 (hASNl variants) with improved asparaginase activity and related matter, including polynucleotides encoding said hASNl variants, expression vectors and host cells comprising said polynucleotides, methods for preparing said hASNl variants as well as pharmaceutical compositions and medical uses of said hASNl variants.

[0004] BACKGROUND OF THE INVENTION

[0005] Asparaginases catalyze the hydrolysis of asparagine (herein, unless indicated otherwise: "asparagine") to aspartate and ammonia (EC 3.5.1.1, KEGG R00485). They are being used in the treatment of acute lymphoblastic leukemia (ALL), the most ubiquitous cancer in children, and also other hematological malignancies like myeloid leukemia (Perel et al., 2002). Targeting the substrate asparagine is also being considered for treatment of other cancers including prostate cancer (Linares et al., 2017, Sircar et al., 2012), epithelial breast cancer and breast cancer metastasis (Pavlova et al., 2018; Knott et al., 2018; Awada et al., 2019), ovarian cancer (Lorenzi et al., 2006), lymphoblastic lymphoma (LBL; Pieters et al., 2011; Mondelaers et al., 2017), colon cancer (Hinze et al., 2020) and pancreatic cancer (Hammel et al., 2020; Dufour et al., 2012) . The respective tumor cells lack the ability to replenish intracellular asparagine, and instead rely on supply of asparagine from the blood. Exogenously administered asparaginases deplete the serum levels of asparagine, ultimately killing tumor cells, but not asparagine-prototrophic, healthy cells.

[0006] The asparaginases derived from Escherichia cu / / (Elspar) and Dickeya dadantii (formerly Erwinia chrysanthemi (Samson et al., 2005), Erwinaze) are approved and tested drugs which have been used in chemotherapeutic applications. Compared to the human asparaginases, these bacterial asparaginases display a significantly higher activity at serum concentrations of asparagine. However, these bacterial asparaginases also exhibit glutaminase activity, which is undesired since said glutaminase activity is associated with many of the side-effects observed during asparaginase treatment. Additionally, non-human asparaginases, such as bacterial asparaginases, are prone to be immunogenic, i.e. trigger an immune reaction in patients, which can be a safety issue and reduce therapeutic efficacy in particular efficiency in case of repeated injections. Efforts to reduce immunogenicity and increase stability resulted in the development of pegylated E. cu / / asparaginase (Oncaspar). PEGylation of therapeutic enzymes is a common procedure to increase the molecular weight and hydrophilicity of the enzyme which in turn can improve solubility, stability and can hinder proteolytic degradation leading to a longer half-life of the therapeutic enzymes in the blood (Dinndorf et al., 2007, Radadiya et al., 2020).

[0007] In humans, three asparaginases have been described, the aspartylglucosaminidase AGA, the isoaspartyl peptidase / L-asparaginase ASRGL1 and the 60 kDa lysophospho- lipase ASPG. ASPG (UniProtKB Q86U10) is also called "human asparaginase 1" (abbreviated as "hASNl" herein) due to its N-terminal asparaginase domain.

[0008] Of said three human asparaginases, only hASNl accepts free asparagine as native substrate, albeit with a very low substrate affinity (S0.5 of 6 mM) owing to a pronounced positive cooperativity, ruling out therapeutic application of wildtype hASNl. Molecular dynamics simulations of human ASPG exploring potential structural determinants of its enzymatic activity are described in Guimaraes et al., 2021.

[0009] In contrast to hASNl, the asparaginase of Cavia porcellus (guinea pig; UniProtKB H0W0T5) is constitutively active. It does not exhibit cooperative behavior and shows good activity at low asparagine concentrations. C-terminally truncated and humanized variants of the guinea pig ASPG-homologue and their intended use for treating cancer are described in WO 2019 / 032952 and Rigouin et al., 2017.

[0010] However, there is still a need for alternative asparaginase preparations for treatment of cancer, in particular asparaginase preparations which have the potential to further reduce the risk of adverse effects observed with known non-human, particularly bacterial, asparaginases, such as immunogenicity and effects linked to glutaminase activity.

[0011] SUMMARY OF THE INVENTION

[0012] It has now surprisingly been found that the enzymatic activity of human asparaginase 1 (hASNl) can be significantly increased by substitution of the histidine (H) at position 298 by glutamine acid (Q). Using H298Q single mutants of full-length and truncated hASNl as well as other hASNl variants wherein loop residues 297-318 and / or 186-195 are replaced by the corresponding residues of guinea pig asparaginase (gpASN), the inventors demonstrated in vitro and in vivo that variants of hASNl can be generated which show with good enzymatic activity, also at low (physiological) asparagine concentrations, and are thus useful as alternative asparaginases for treating cancer. Additionally, being variants of a human protein, the hASNl variants described herein are expected to have much less potential for immunogenicity that non-human asparaginases. The amino acid residue asparagine (N) is isofunctional to the amino acid residue Q (only shorter). Like their amids N and Q, the amino acid residues glutamic acid (E) and aspartic acid (D) can act as H-bond donors and acceptors. Like H298Q, substitution of H298 of hASNl by N, D or E is therefore expected to also increase the enzymatic (asparaginase) activity of hASNl.

[0013] The amino acid residues T19, T116, D117, K188 and Y308 of hASNl are known to be catalytically relevant residues, and T19, T116, D117 and K188 are conserved among asparaginases. It is known that the substitution of Y308 by E (Y308E) does not impair the enzymatic activity of the therapeutically used Dickeya dadantii asparaginase. In the hASNl variants described herein, the T19, T116, D117 and K188 therefore remain unchanged as in wildtype hANSl, and Y308 is either unchanged or substituted by E (Y308E).

[0014] The crystal structure of hASNl and findings on the effect of certain amino acid variations on the activity of hASNl are described in the dissertation "Mechanistic and structural characterization of human L-asparaginases" by Nora Katharina Eulig. This dissertation was submitted at the Georg-August-University Gottingen, Germany on 17 February 2022 and was published on 4 April 2024. The oral examination ("defense") of Nora Katharina Eulig on 5 April 2022 took place completely in camera and in nonpublic form. The participation of the examiners was bound to the condition of confidentiality.

[0015] The present invention relates to a hASNl variant which comprises an amino acid sequence having at least 85% identity to the sequence set forth in SEQ ID NO:2; wherein the amino acid at the position of the hASNl variant corresponding to position 298 of the sequence set forth in SEQ ID NO:2 is selected from Q, N, E and D, preferably is Q or N, and most preferably is Q; wherein each of the amino acids at the positions of the hASNl variant corresponding to positions 19 and 116 of the sequence set forth in SEQ ID NO: 2 is T; wherein the amino acid at the position of the hASNl variant corresponding to position 117 of the sequence set forth in SEQ ID NO:2 is D; wherein the amino acid at the position of the hASNl variant corresponding to position 188 of the sequence set forth in SEQ ID NO:2 is K; and wherein the amino acid at the position of the hASNl variant corresponding to position 308 of the sequence set forth in SEQ ID NO: 2 is Y or E, and preferably is Y. optionally, wherein the amino acid at the position of the hASNl variant corresponding to position 297 of the sequence set forth in SEQ ID NO:2 is T or S.

[0016] Further aspects of the present invention relate to a polynucleotide comprising a sequence encoding such hASNl variant, to an expression vector comprising such polynucleotide, and to a host cell comprising such polynucleotide. The host cell may be capable of, and thus can be used for, expressing the hASNl variant.

[0017] The present invention also relates to a method for preparing a hASNl variant of the invention. The method comprises a step of culturing a host cell, which is capable of expressing said hASNl variant, under conditions where said hASNl variant is produced by said host cell. The host cell comprises a polynucleotide comprising sequence encoding said hASNl variant. The method can further comprise a step of purifying the produced hASNl variant.

[0018] The hASNl variants provided by the present invention have good asparaginase activity and are therefore useful for therapeutic applications. A further aspect of the present invention therefore relates to a pharmaceutical composition comprising a hASNl variant of the invention and a carrier.

[0019] Further aspects of the present invention relate to a hASNl variant or a pharmaceutical composition of the invention for use in a method of treatment of the human or animal body by therapy, and to a method for treating cancer in a subject in need of such treatment. Said method of treatment comprises administering to the subject an effective amount of a hASNl variant or a pharmaceutical composition of the invention. In particular, the treatment is a treatment of cancer.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] Unless otherwise defined herein, the singular article 'a' or 'an' means 'one or more than one', i.e. is not meant as a limitation to 'one'. Unless otherwise defined herein, 'at least one' means 'one or more than one'.

[0022] A main aspect of the present invention relates an hASNl as defined in the claims.

[0023] The term 'hASNl' used herein is the abbreviation of 'human asparaginase 1' which is the wild-type human 60 kDa lysophospholipase (UniPROT Accession No. Q86U10) and has the amino acid sequence set forth in SEQ ID NO:1 herein.

[0024] The term 'hASNltr' used herein is the abbreviation of 'truncated human asparaginase 1' and refers to a truncated version of hASNl which lacks amino acids 370-573 of hASNl and has amino acid sequence set forth in SEQ ID NO:2 herein.

[0025] The term 'hASNl variant' is used to refer to a variant of hASNl or hASNltr, in particular a variant as defined in the claims. Unless specified otherwise, the term 'hASNl' used herein is meant to include both full-length and truncated hASNl.

[0026] The hASNl variant of the present invention has at least 85% identity to the sequence set forth in SEQ ID NO:2, and may have - with increasing preference - at least 90%, at least 91%, at least 93%, at least 95% or at least 99% identity to the sequence set forth in SEQ ID NO:2.

[0027] Preferably, an amino acid sequence has "at least x % identity" to another amino acid sequence, e.g. SEQ ID NO: 2 as indicated herein, when the sequence identity between those two amino acid sequences is at least x % over the full length of said other amino acid sequence, e.g. SEQ ID NO: 2.

[0028] As used herein, "sequence identity" or "identity" in the context of two amino acid sequences refers to a specified percentage of residues in the two sequences which residues are the same when the sequences are aligned for maximum correspondence. The percentage of sequence identity between two amino acid sequences can be determined by sequence comparison algorithms (which are typically part of sequence alignment software), or by visual inspection and calculation of the percentage of aligned identical amino acid residues. See, for example, alignment of hASNl and gpASN in Figure 1. Thus, amino acid sequence identity can generally be determined by methods of amino acid sequence alignment and calculation of the percentage of aligned identical amino acid residues. Such methods are routine to the person skilled in the art, and software for performing amino acid sequence alignments and calculating the percentage of sequence identity is well known in the art and readily available. For example, such alignments and determination of percent identity can be performed using publicly available computer homology programs such as the "EMBOSS" program provided at the EMBL homepage at www.ebi.ac.uk / Tools / psa / emboss_needle / , using the default settings provided therein. Examples of mathematical algorithms for determining the percent identity between any two sequences include, e.g., the algorithm of Myers et al., 1988; the local homology algorithm of Smith et al, 1981; the search-for-similarity-method of Pearson et al., 1988; the algorithm of Karlin et al., 1990; modified as in Karlin et al., 1993; and the homology alignment algorithm of Needleman et al., 1970; wherein the latter is preferred herein.

[0029] The hASNl variants of the present invention have improved asparaginase activity compared to wildtype hASNl, in particular at physiologically relevant asparagine concentrations such as 50 pM, which is used herein as a representative "low micromolar" concentration of asparagine. The term 'asparaginase activity' refers to the enzymatic activity of an asparaginase in hydrolyzing its substrate L-asparagine. Said enzymatically catalyzed hydrolysis of L-asparagine forms aspartic acid and ammonia (NH3). The rate of this reaction can therefore be determined, for example, by an asparaginase activity assay, wherein the NH3 formation is (indirectly) monitored via a second reaction which uses the formed NH3 as a substrate and involves a conversion of NADH to NAD+ which can be monitored by measuring absorbance at 340 nm. Such second reaction can be the reaction of NH3 and o- ketoglutarate to glutamate catalyzed by the enzyme glutamate dehydrogenase (GDH), wherein simultaneously NADH is converted into NAD+. Such an asparaginase activity assay is described in Fernandez et al., 2013, and in chapter A.5 of the Examples section below.

[0030] Specifically, the asparaginase activity of a hASNl variant of the invention may be - with increasing preference - at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, or at least 13-fold higher than the asparaginase activity of wildtype hASNl (SEQ ID NO:1). For example, the asparaginase activity is determinable using the results of an asparaginase activity assay. Such an assay may comprise the steps:

[0031] (i) preparing a solution comprising the asparaginase substrate L-asparagine, glutamate dehydrogenase (GDH), the GDH substrates o-ketoglutarate and NADH, Tris buffer, at pH 7.5 and 37°C, and

[0032] (ii) measuring absorbance at 340 nm.

[0033] Asparaginase activity hydrolyses the L-asparagine forming ammonia, which ammonia reacts with the o-ketoglutarate in a GDH-catalyzed reaction, wherein the GDH also converts the NADH to NAD+, which conversion is detectable as a change in absorbance at 340 nm.

[0034] This reaction is performed with different L-asparagine concentrations. The So.s value is thereby the L-asparagine concentration at which the reaction rate is half-maximal. Accordingly, the hASNl variant may have a So.s value which is at least 2-fold lower than the So.s value of wildtype hASNl (SEQ ID NO:1).

[0035] More specifically, the So.s values of the hASNl variant and wildtype hASNl (SEQ ID NO:1) can be determined by a method comprising the steps:

[0036] (i) preparing a solution comprising a concentration of L-asparagine in the range of 0.025-70 mM, 15 mM c, 0.3 mM NADH, 30 U / mL glutamate dehydrogenase (GDH), 50 mM Tris buffer, pH 7.5;

[0037] (ii) incubating the solution for 10 min at 25°C;

[0038] (iii) then, placing the solution into the cuvette of a photometer at 37°C for 3 min and measuring absorbance at 340 nm;

[0039] (iv) then, adding the hASNl variant or wildtype hASNl to the solution in the cuvette and continuing the absorbance measurement for 10 min so as to obtain absorbance curves;

[0040] (v) determining the change in absorbance per minute from the interval of the obtained absorbance curves having the steepest slope;

[0041] (vi) repeating steps (i)-(v) at least three times using different L-asparagine concentrations within the range of 0.025-70 mM;

[0042] (vii) calculating the apparent reaction rate kappusing the formula wherein MWASN is the molecular weight of the hASNl or variant thereof, ENADH is 6220 M’1cm1, CASN is the weight concentration of the hASNl or variant thereof, and d is the width of the cuvette;

[0043] (vii) fitting the obtained absorbance values for the different L-asparagine concentrations with the Hill equation so as to determine S0.5: wherein V is the reaction velocity, kcat is the turnover number, [Asn] is the L- asparagine concentration, and n is the Hill coefficient.

[0044] The hASNl variants of the invention typically do not show cooperativity among their ligand binding sites. Their Hill coefficient (n) is therefore close to 1; and their S0.5 thus corresponds to the Michaelis constant KM which can be determined, e.g. using the above-described method, by fitting the kapp(apparent reaction rate) values determined for the different L-asparagine concentrations with the Michaelis-Menten equation: y _ kcat ' [ASTI]

[0045] KM+ [4sn] wherein V is the reaction velocity, kcat is the turnover number, [Asn] is the L- asparagine concentration, and KM is the Michaelis constant.

[0046] Methods and software for fitting data with a given equation, such as the Hill equation or the Michaelis-Menten equation, are very well known in the art. Various computational tools for data fitting to the Hill equation are known and available. One of the best-known data analysis software packages for such analysis is SigmaPlot which was originally developed by Jandel Corporation, later versions by SPSS Inc., SYSTAT Software and Grafiti LLC.

[0047] A specific example for an asparaginase activity assay which can be used to determine the So.5 values is described in chapter A.5 of the Examples section below.

[0048] The hASNl variant of the invention comprises at least the following amino acids, which are believed to be important for its asparaginase (enzymatic) activity:

[0049] - Q, N, E or D at the position of the hASNl variant corresponding to position 298 of the sequence set forth in SEQ ID NO:2;

[0050] - T at each of the positions of the hASNl variant corresponding to positions 19 and 116 of the sequence set forth in SEQ ID NO:2;

[0051] - D at the position of the hASNl variant corresponding to position 117 of the sequence set forth in SEQ ID NO:2;

[0052] - K at the position of the hASNl variant corresponding to position 188 of the sequence set forth in SEQ ID NO: 2; and

[0053] - Y or E at the position of the hASNl variant corresponding to position 308 of the sequence set forth in SEQ ID NO:2.

[0054] Additionally, the hASNl variant of the invention may have one or more of the following characteristics:

[0055] - T or S at the amino acid at the position of the hASNl variant corresponding to position 297 of the sequence set forth in SEQ ID NO:2;

[0056] - the amino acid sequence of positions 297-316 of the sequence set forth in SEQ ID NO: 14 at the amino acid sequence portion of the hASNl variant corresponding to positions 297-318 of the sequence set forth in SEQ ID NO:2;

[0057] - the amino acid sequence of positions 186-195 of the sequence set forth in SEQ ID NO: 14 at the amino acid sequence portion of the hASNl variant corresponding to positions 186-195 of the sequence set forth in SEQ ID NO:2; or, alternatively, the amino acid sequence of positions 186-195 of the sequence set forth in SEQ ID NO: 2 at the corresponding amino acid sequence portion of the hASNl variant;

[0058] - the amino acid sequences of positions 2-185, 196-296 and 319-369 of the sequence set forth in SEQ ID NO:2 at the corresponding amino acid sequence portions of the hASNl variant.

[0059] In particular embodiments, the hASNl variant comprises the sequence set forth in SEQ ID NO:1 or, preferably, SEQ ID NO:2, wherein the amino acid at position 298 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO: 2 has been substituted by Q, N, E or D, preferably by Q, in the amino acid sequence of the hASNl variant; and wherein, optionally, one or more of the amino acids at positions 186-195, 297, and 299-318 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has / have been altered.

[0060] In further particular embodiments, the hASNl variant comprises the sequence set forth in SEQ ID NO:1 or, preferably, SEQ ID NO:2, wherein the amino acid at position 298 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO: 2 has been substituted by Q, N, E or D, preferably by Q, in the amino acid sequence of the hASNl variant; and wherein the amino acid at position 1 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 is not present in the amino acid sequence of the hASNl variant; and wherein, optionally, one or more of the amino acids at positions 186-195, 297, and 299-318 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has / have been altered.

[0061] In further particular embodiments, the hASNl variant consists of the sequence set forth in SEQ ID NO:1 or, preferably, SEQ ID NO:2, wherein the amino acid at position 298 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has been substituted by Q, N, E or D, preferably by Q, in the amino acid sequence of the hASNl variant; and wherein, optionally, one or more of the amino acids at positions 186-195, 297, and 299-318 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has / have been altered.

[0062] In further particular embodiments, the hASNl variant consists of the sequence set forth in SEQ ID NO:1 or, preferably, SEQ ID NO:2, wherein the amino acid at position 298 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has been substituted by Q, N, E or D, preferably by Q, in the amino acid sequence of the hASNl variant; and wherein the amino acid at position 1 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 is not present in the amino acid sequence of the hASNl variant; and wherein, optionally, one or more of the amino acids at positions 186-195, 297, and 299-318 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has / have been altered.

[0063] In embodiments, wherein the amino acid at position 308 has been altered, said amino acid is E.

[0064] In embodiments, wherein one or more of the amino acids at positions 186-195, 297, and 299-318 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has / have been altered, said alteration(s) may be independently selection from an amino acid substitution or an amino acid deletion. In particular, said one or more of the amino acids at positions 186-195, 297, and 299-318 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 may have been altered in the amino acid sequence of the hASNl variant as follows:

[0065] - the amino acid at position 297 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been substituted by S,

[0066] - the amino acid at position 301 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been substituted by R,

[0067] - the amino acid at position 303 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been substituted by S,

[0068] - the amino acid at position 306 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by P,

[0069] - the amino acid at position 307 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been substituted by G,

[0070] - the amino acid at position 310 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been substituted by T,

[0071] - the amino acid at position 311 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been substituted by S,

[0072] - the amino acid at position 312 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been deleted,

[0073] - the amino acid at position 313 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been deleted,

[0074] - the amino acid at position 314 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been substituted by L, - the amino acid at position 318 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been substituted by N.

[0075] Particular examples of hASNl variants of the invention include hASNAl variants having an amino acid sequence which comprises, or consists of, an amino acid sequence selected from the sequences as set forth in SEQ ID NOs: 3-12, wherein the amino acid at position 1 of the sequences set forth in SEQ ID NOs: 3-12, is present or is not present in the amino acid sequence of said hASNl variants.

[0076] In particularly preferred embodiments, the amino acid at the position of the hASNl variant corresponding to position 298 of the sequence set forth in SEQ ID NO1 or SEQ ID NO:2 is Q. Specific examples of such hASNl variants include hASNAl variants having an amino acid sequence which comprises, or consists of, an amino acid sequence selected from the sequences as set forth in SEQ ID NOs: 3-10, wherein the amino acid at position 1 of the sequences set forth in SEQ ID NOs: 3-10, is present or is not present in the amino acid sequence of said hASNl variants.

[0077] Generally, in the variants of hASNl described herein, the N-terminal methionine (initiator methionine) present in any of SEQ ID NOs: 1-13 may be missing.

[0078] The hASNl variant may further comprise at least one tag.

[0079] The term 'tag' as used herein refers to any additional amino acids, protein domains or full-length proteins which are fused N- and / or C-terminally to the asparaginase sequence (i.e., the sequence of amino acid at the positions corresponding to positions in SEQ ID NO:1 or SEQ ID NO:2), optionally via a linker or spacer peptide. Accordingly, the hASNl variant may be a fusion protein.

[0080] Tags are typically used to provide additional desired properties and functionalities. For example, tags may facilitate detectability and purification of the tagged (fusion) protein (e.g., His tag, GST tag, MBP tag), may improve solubility and / or stability of the tagged protein (e.g., MBP tag, GST tag), may provide a cleavage site where more distal tags can be site-specifically cleaved off from the asparaginase sequence by a protease (e.g., SUMO tag), may increase serum circulation time and biodistribution of the tagged (fusion) protein (e.g., albumin, transferrin, XTEN), or may facilitate cellular targeting (e.g., antibody against CD7 or CD19).

[0081] The hASNl variant may therefore comprise at least one tag, independently selected from a His tag, a SUMO tag, an MPB tag and a GST tag, and / or may be a fusion protein comprising (besides its asparaginase sequence) at least one of an albumin, a transferrin, an XTEN, and an antibody against CD7 or CD19. The term 'His tag' as used herein refers to a tag sequence (i.e., a sequence outside the aspartate sequence of the hASNl variant) containing 6-14 histidines spaced over a sequence of 6-30 amino acids, possibly containing glycines, serines and / or threonines as spacer sequences. In particular, the His tag may consist of a sequence of 6-12 consecutive histidines, more particularly of 6 consecutive histidines. A His-tag may facilitate purification using nickel or cobalt containing resins (e.g., Ni-NTA resin).

[0082] The term 'MBP' is an abbreviation of 'maltose binding protein'. The MBP tag is a common protein expression tag which is known to enhance the solubility of many proteins, and is one of the most well-known and accomplished means of tagging proteins expressed in microbes. Fusion of a protein (Target protein', e.g., a hASNl variant) to MBP permits its one-step purification using amylose resin. The MBP-fusion protein will bind to amylose resin while other proteins flow through. The MBP-protein fusion can then be eluted from the resin with maltose. The purified fusion protein can then be treated with a specific protease to cleave the M BP-tag from the target protein and the target protein can be separated from the MBP tag by affinity chromatography.

[0083] The term 'GST' is an abbreviation of 'glutathione S-transferase'. Because GST folds rapidly into a stable and highly soluble protein upon translation, inclusion of the GST tag often promotes greater expression and solubility of recombinant proteins than expression without the tag. In addition, GST-tagged fusion proteins can be purified or detected based on the ability of GST (an enzyme) to bind its substrate, glutathione (GSH).

[0084] The small ubiquitin-related modifier (SUMO) is an amino acid sequence which can be specifically cleaved by certain proteases. For example, the SUMO from Brachypodium distachyon (bdSUMO) can be specifically cleaved by the SUMO-specific protease from B. distachyon (bdSENPl) and enzymatically active variants thereof, as described in Frey et al., 2014. By including a SUMO tag in a hASNl variant which is fusion protein that contains the SUMO tag between the asparaginase sequence and one or more other tags, said tags can be specifically cleaved from the asparaginase sequence by a SUMO-specific protease.

[0085] Albumins are proteins found in the blood plasma, where they serve as transport proteins. Transferrins are iron-binding proteins located in the serum. Fusion of albumin or transferrin to a protein (e.g. a hASNl variant) can increase serum circulation time and biodistribution of the fusion protein. The albumin or transferrin used in fusion proteins such as the hASNl variants described herein are preferably of the same species origin as the patient to be treated with said fusion protein to reduce or avoid immunogenicity, but might be derived from close relatives in other instances. For example, a hASNl variant which is a fusion protein may comprise human albumin or human transferrin to reduce or avoid immunogenicity when used in human patients.

[0086] XTENs are artificial, extended disordered proteins shown to increase circulating times when used as part of fusion proteins (Schellenberger et al., 2009).

[0087] An antibody against the CD7 surface protein of T-lymphoblasts or the CD19 surface protein of B-lymphoblasts (which are the aberrant cell types in ALL) may be fused to the hASNl variant to facilitate cellular targeting in the patient, i.e. enrichment of such fusion proteins at locations within the patient where depletion of asparagine by the activity of the hASNl variant may be particularly beneficial. Antibodies with other antigen-specificities may also be useful. Expediently, an antibody is chosen based on the antigen profile of the cancer to be treated.

[0088] The hASNl variant may be PEGylated.

[0089] The term 'PEGylated' as used herein refers to a protein (such as a hASNl variant) being conjugated with polyethylene glycol polymers (PEGs). Typically, the number of monomers (-CH2CH2O- units) in such polyethylene glycol polymer is in the range of 5-100. The term PEGylated is meant to include end-capped PEG, e.g. monomethyl- capped PEG (mmPEG). PEG may be attached to cysteine residues of the (thus PEGylated) protein, for example by Michael addition, or to lysine residues of the (thus PEGylated) protein, for example by nucleophilic substitution. Such methods for attaching PEG to cysteine or lysine residues of a protein are well known in the art.

[0090] As mentioned, PEGylation of therapeutic enzymes is a common procedure to increase the molecular weight and hydrophilicity of the enzyme which in turn can improve solubility, stability and can hinder proteolytic degradation leading to a longer half-life of the therapeutic enzymes in the blood (Dinndorf et al., 2007, Radadiya et al., 2020).

[0091] Also disclosed herein is a polynucleotide comprising a sequence encoding a hASNl variant of the invention.

[0092] The term 'polynucleotide' as used herein includes DNA and RNA, and preferably means DNA. Accordingly, the term 'nucleotide sequence' as used herein includes DNA and RNA sequences, and preferably means a DNA sequence. The polynucleotide disclosed herein is not naturally occurring, and may be a nucleic acid construct, e.g., prepared by molecular cloning. Methods for generating polynucleotides (or particularly nucleic acid constructs) comprising a nucleotide sequence coding for (i.e., encoding) a protein of interest have been well known in the art since several decades. A polynucleotide comprising a nucleotide sequence encoding a hASNl variant disclosed herein can therefore be generated by a person skilled in the art in a routine manner.

[0093] The sequence of the polynucleotide can be modified for optimal expression, for example by adding suitable regulatory sequences and / or targeting sequences and / or by matching of the coding DNA sequence to the preferred codon usage of the chosen host cell (i.e., the cell chosen for expression of the hASNl variant encoded by the polynucleotide). The targeting sequence may encode a C-terminal extension targeting the hASNl variant to a particular site or compartment within the host cell, or a sequence facilitating the secretion of the hASNl variant into the medium surrounding the host cell. The sequence of the polynucleotide acid may be combined with further elements, e.g., a promoter and a transcription start and stop signal and a translation start and stop signal and a polyadenylation signal in order to provide for expression of the hASNl variant of the present disclosure. The promoter can be inducible or constitutive, general or cell specific promoter. Selection of promoters, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.

[0094] Also disclosed herein is an expression vector comprising a polynucleotide as disclosed herein, i.e. a polynucleotide comprising a nucleotide sequence encoding a hASNl variant.

[0095] Various types of expression vectors suitable for various types of host cells are well- known in the art. Examples of expression vectors include, but are not limited to, plasmids and viruses.

[0096] The polynucleotide or the expression vector disclosed herein, or at least the nucleotide sequence thereof encoding the hASNl variant, may be introduced into the host cells, for example using a virus as a carrier or by transfection. Useful methods for transfection are commonly known in the art and include, e.g., transfection using chemical transfectants (such as, e.g., Lipofectamine or Fugene), electroporation, calcium phosphate co-precipitation and direct diffusion of DNA. Transfection with DNA yields stable cells or cell lines, if the transfected DNA is integrated into the genome, or unstable (transient) cells or cell lines, wherein the transfected DNA exists in an extrachromosomal form. Furthermore, stable cell lines can be obtained by using episomal replicating plasmids, which means that the inheritance of the extrachromosomal plasmid is controlled by control elements that are integrated into the cell genome. In general, the selection of a suitable vector or plasmid depends on the intended host cell.

[0097] A host cell comprising a polynucleotide as disclosed herein, i.e. a polynucleotide comprising a sequence encoding a hASNl variant is thus also disclosed herein.

[0098] As demonstrated by the inventors, the hASNl variants, including those which are fusion proteins and comprise one or more tags, can be readily generated by methods for recombinant protein purification and expression known in the arts. To this end, host cells (preferentially, but not limited to, E. coli strains, e.g. NEBexpress) which comprise a recombinant polynucleotide having a nucleotide sequence that encodes the hASNl variant (e.g., introduced into the host cell by transfection) are cultured under suitable conditions (e.g., autoinduction ZYM-5052, Studier, 2005). The expressed hASNl variant can be isolated and purified. The purified hASNl variant can then be used as is for therapy, or preferentially be introduced in a pharmaceutical composition.

[0099] Thus, a method of preparing a hASNl variant as disclosed herein is provided, which method of preparing a hASNl variant comprises the step of culturing a host cell under conditions where said hASNl variant is produced by said host cell. Said host cell comprises a polynucleotide comprising a nucleotide sequence encoding a hASNl variant capable disclosed herein; and said host cell is capable of (recombinantly) expressing said hASNl variant. The method of preparing a hASNl variant may further comprise a step of purifying the expressed hASNl variant. Methods for the purification of a protein which has been recombinantly expressed by a host cell are well-known in the art. Expressing the protein (here: the hASNl variant) as a fusion with a tag may facilitate effective tag-based purification of the fusion as described in the context of particular tags for such purpose nucleotide sequence encoding a hASNl variant herein above. An exemplary method for the expression and purification of a hASNl variant expressed in E. coli\s provided in the examples section below (see chapters A.3 and A.4). A SUMO tag in between the asparagine sequence of the hASNl variant and the tag(s) used for purification may facilitate easy removal of the tags after purification by cleavage with a SUMO-specific protease, e.g., bdSENPl for cleaving bdSUMO.

[0100] Suitable host cells for expression of a hASNl variant as disclosed herein include, but are not limited to, bacterial cells (e.g., E. coli), yeast cells, mammalian cells, insect cells and plant cells. The mammalian cells and insect cells used for expression of the hASNl variant are typically cells of a mammalian cell line or an insect cell line, i.e. expression of the hASNl variant occurs ex vivo. The hASNl variants provided by the present invention have good asparaginase activity and are therefore useful for therapeutic applications. A further aspect of the present invention therefore relates to a pharmaceutical composition comprising a hASNl variant of the invention and a carrier. The pharmaceutical composition may comprise further excipients and / or further pharmaceutically active agents. The formulation of the hASNl variant in a pharmaceutical composition may increasing shelf-life and / or reduce administration-related side-effects for the patients. As hASNl exhibits optimal stability in buffers carrying negatively charged moieties, e.g. HEPES or phosphate buffer, pharmaceutical compositions of hASNl and variants thereof as described herein are preferentially based on such buffers.

[0101] Expediently, the carrier is a pharmaceutically acceptable carrier. The term 'pharmaceutically acceptable' is used herein to refer to those compounds or compositions (e.g., carriers) which are within the full range of medical judgment suitable for being administered to a human or non-human animal by the chosen route of administration, and thereby meet reasonable benefit I risk ratios without undue toxicity, irritation or other adverse events.

[0102] The pharmaceutical composition can be formulated in any way that is compatible with its therapeutic application, including intended route of administration, delivery format and desired dosage. Typically, for application in the treatment of cancer, the pharmaceutical composition is formulated for parenteral administration, for example by injection. For example, the hASNl variant may be encapsulated in an erythrocyte. Techniques for the encapsulation of L-asparaginase in intact erythrocytes for therapeutic application are known in the art (e.g., described in Kwon et al., 2009).

[0103] A further aspect of the present invention therefore relates to a hASNl variant as disclosed herein, or a pharmaceutical composition comprising such hASNl variant and a carrier, for use in a method of treatment of the human or animal body by therapy. In particular, the treatment may be a treatment of cancer.

[0104] A further aspect relates to a method for treating cancer in a subject in need of such treatment, wherein the method comprises administering to the subject an effective amount of the hASNl variant or the pharmaceutical composition disclosed herein.

[0105] The term 'effective amount' as used herein in the context of a medical use or method of treating cancer refers to the amount of the hASNl variant, or the pharmaceutical composition comprising same, that is capable of eliciting the beneficial biological or medical response in a subject that is sought by the treating individual, i.e. medical doctor or other clinician, in particular an inhibitory effect against the treated cancer which prevents or reduces the growth of the cancer (particularly the proliferation of cancer cells), and / or reduces the number of viable cancer cell in the treated subject.

[0106] The optimal dose of the hASNl variant administered will, of course, depend on the subject to be treated, particularly on the subject's weight, the subject's age, the manner of administration, and the judgement of the prescribing physician.

[0107] The hASNl variant, or the pharmaceutical composition comprising same, is typically administered to the subject by parenteral administration, for example by injection.

[0108] Unless specified otherwise herein, the terms 'subject' and 'patient' are used herein synonymously and refer to a human or a non-human animal, for example a companion animal. Non-limiting examples of subjects or patients which can be treated with an NPY / Y5R inhibitor as described herein are mammals such as humans, cats, dogs, and horses. Preferably, the subject or patient is a human. Typically, the subject has a cancer and needs treatment against this cancer.

[0109] The term 'cancer' as used herein refers to malignant diseases characterized by the abnormal cell growth with the potential to spread and / or to invade to other parts of the body (metastasis). Cancers include solid tumors as well as non-solid cancers (e.g., leukemia).

[0110] Expediently, the cancers treated according to the present invention are L-asparagine auxotrophic cancers, i.e. cancers which obtain all or at least the majority of the asparagine they need from their environment and thus rely on supply of asparagine from the blood.

[0111] Particular non-limiting examples of cancers which can be treated with hASNl according to the invention include acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), prostate cancer, ovarian cancer, colon cancer, pancreatic cancer and breast cancer (including epithelial breast cancer and breast cancer metastasis).

[0112] The terms 'treatment' and 'treating' as used herein with regard to a medical treatment and particularly treatment of cancer refer to an intervention to prevent or reduce the growth of the cancer (particularly the proliferation of cancer cells) in the treated subject, and / or reduce the number of viable cancer cell in the treated subject by administering a hASNl variant described herein to the subject.

[0113] The treatment of cancer by administering a hASNl variant as described herein to the subject can be combined with other anti-cancer treatments. Strategies for using asparaginases in the treatment of cancer are known, e.g. from the bacterial asparaginases which have already been approved for such application. Said known strategies can provide valuable guidance for the use of a hASNl variant disclosed herein in a treatment of cancer.

[0114] Particularly, the present invention includes, but is not limited to, the following embodiments E1-E27:

[0115] El. A human asparaginase 1 (hASNl) variant which comprises an amino acid sequence having at least 85% identity to the sequence set forth in SEQ ID NO:2; wherein the amino acid at the position of the hASNl variant corresponding to position 298 of the sequence set forth in SEQ ID NO:2 is selected from Q, N, E and D; wherein each of the amino acids at the positions of the hASNl variant corresponding to positions 19 and 116 of the sequence set forth in SEQ ID NO:2 is T; wherein the amino acid at the position of the hASNl variant corresponding to position 117 of the sequence set forth in SEQ ID NO:2 is D; wherein the amino acid at the position of the hASNl variant corresponding to position 188 of the sequence set forth in SEQ ID NO:2 is K; and wherein the amino acid at the position of the hASNl variant corresponding to position 308 of the sequence set forth in SEQ ID NO:2 is Y or E.

[0116] E2. The hASNl variant of El, wherein the hASNl variant has a S0.5 value which is at least 2-fold lower than the S0.5 value of wildtype hASNl having the amino acid sequence set forth in SEQ ID NO:1, wherein S0.5 is determinable using the results of an asparaginase activity assay which assay comprises:

[0117] (i) preparing a solution comprising the asparaginase substrate L- asparagine, glutamate dehydrogenase (GDH), the GDH substrates o- ketoglutarate and NADH, Tris buffer, at pH 7.5 and 37°C, and

[0118] (ii) measuring absorbance at 340 nm, wherein asparaginase activity hydrolyses the L-asparagine and thereby forms ammonia, which ammonia reacts with the o-ketoglutarate in a GDH- catalyzed reaction, wherein the GDH also converts the NADH to NAD+, which conversion is detectable as a change in absorbance at 340 nm, and wherein S0.5 is the L-asparagine concentration at which the reaction rate is half-maximal. E3. The hASNl variant of El or E2, wherein (l.a) the amino acids at the positions of the hASNl variant corresponding to positions 2-185, 196-296 and 319-369 of the sequence set forth in SEQ ID NO:2 are identical to (l.b) the amino acids at corresponding positions 2-185, 196-296 and 319-369 of the sequence set forth in SEQ ID NO:2; optionally, wherein (2.a) the amino acids at the positions of the hASNl variant corresponding to positions 186-195 of the sequence set forth in SEQ ID NO:2 are identical to (2.b) the amino acids at positions 186-195 of the sequence set forth in SEQ ID NO:2.

[0119] E4. The hASNl variant of any of E1-E3, wherein the amino acid at the amino acid position of the hASNl variant corresponding to position 297 of the sequence set forth in SEQ ID NO:2 is T or S.

[0120] E5. The hASNl variant of any of E1-E4, wherein the amino acid sequence of the hASNl variant corresponding to positions 297-318 of the sequence set forth in SEQ ID NO:2 is the amino acid sequence of positions 297-316 of the sequence set forth in SEQ ID NO: 14.

[0121] E6. The hASNl variant of any of E1-E5, wherein the amino acid sequence of the hASNl variant corresponding to positions 186-195 of the sequence set forth in SEQ ID NO:2 is the amino acid sequence of positions 186-195 of the sequence set forth in SEQ ID NO: 14.

[0122] E7. The hASNl variant of any of E1-E6, wherein the amino acid sequence of the hASNl variant comprises the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, wherein the amino acid at position 298 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has been substituted by Q, N, E or D in the amino acid sequence of the hASNl variant; and wherein the amino acid at position 1 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO: 2 is present or is not present in the amino acid sequence of the hASNl variant; optionally, wherein one or more of the amino acids at positions 186-195, 297, and 299-318 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has / have been altered in the amino acid sequence of the hASNl variant, in particular has / have been substituted by another amino acid or deleted, more particularly has / have been substituted by the corresponding amino acid(s) of the sequence set forth in SEQ ID NO: 14.

[0123] E8. The hASNl variant of any of E1-E7, wherein the amino acid sequence of the hASNl variant comprises the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, wherein the amino acid at position 298 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has been substituted by Q, N, E or D in the amino acid sequence of the hASNl variant; and wherein the amino acid at position 1 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 is present or is not present in the amino acid sequence of the hASNl variant; optionally, wherein one or more of the amino acids of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has / have been altered in the amino acid sequence of the hASNl variant as follows:

[0124] - the amino acid at position 297 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been substituted by S,

[0125] - the amino acid at position 301 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by R,

[0126] - the amino acid at position 303 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been substituted by S,

[0127] - the amino acid at position 306 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by P,

[0128] - the amino acid at position 307 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by G,

[0129] - the amino acid at position 310 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by T,

[0130] - the amino acid at position 311 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by S,

[0131] - the amino acid at position 312 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been deleted,

[0132] - the amino acid at position 313 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been deleted, - the amino acid at position 314 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been substituted by L,

[0133] - the amino acid at position 318 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by N.

[0134] E9. The hASNl variant of E7 or E8, wherein the amino acid sequence of the hASNl variant consists of the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2, wherein the amino acid at position 298 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, and optionally one or more of the amino acids at positions 1, 186-195, 297, and 299-318 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has / have been altered in the amino acid sequence of the hASNl variant as defined in E7 or E8.

[0135] E10. The hASNl variant of any of E1-E9 comprising an amino acid sequence selected from the sequences as set forth in SEQ ID NOs: 3-12, and wherein the amino acid at position 1 of the sequences set forth in SEQ ID NOs: 3-12, is present or is not present in the amino acid sequence of the hASNl variant.

[0136] Ell. The hASNl variant of any of E1-E10, wherein the amino acid at the position of the hASNl variant corresponding to position 298 of the sequence set forth in SEQ ID NO1 or SEQ ID NO:2 is Q.

[0137] E12. The hASNl variant of any of El-Ell, further comprising at least one tag independently selected from a His tag, a SUMO tag, an MPB tag and a GST tag, and / or wherein the hASNl variant is PEGylated.

[0138] E13. The hASNl variant of any of E1-E12 which is a fusion protein comprising at least one of the following: an albumin, a transferrin, an XTEN, and an antibody against CD7 or CD19.

[0139] E14. A polynucleotide comprising a nucleotide sequence encoding a hASNl variant of any of E1-E13.

[0140] E15. An expression vector comprising the polynucleotide of E14.

[0141] E16. A host cell comprising the polynucleotide of E14; optionally, wherein the host cell is capable of expressing said hASNl variant.

[0142] E17. The host cell of E16, which host cell is a bacterial cell, particularly an E. coli cell. E18. A method for preparing a hASNl variant of any of E1-E13, the method comprising: culturing a host cell of E16 or E17, which is capable of expressing said hASNl variant, under conditions where said hASNl variant is produced by said host cell; and, optionally, purifying the produced hASNl variant.

[0143] E19. A pharmaceutical composition comprising a hASNl variant of any of E1-E13, and a carrier.

[0144] E20. The pharmaceutical composition of E19, wherein the hASNl variant is encapsulated in an erythrocyte.

[0145] E21. A hASNl variant of any of E1-E13 or a pharmaceutical composition of E19 or E20 for use in a method of treatment of the human or animal body by therapy.

[0146] E22. A hASNl variant of any of E1-E13 or a pharmaceutical composition of E19 or E20 for use in the treatment of cancer.

[0147] E23. The hASNl variant or pharmaceutical composition for use according to E22, wherein the cancer is an L-asparagine auxotrophic cancer.

[0148] E24. The hASNl variant or pharmaceutical composition for use according to E22 or E23, wherein the cancer is selected from acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), prostate cancer, ovarian cancer, colon cancer, pancreatic cancer and breast cancer.

[0149] E25. A method for treating cancer in a subject in need of such treatment, wherein the method comprises administering to the subject an effective amount of a hASNl variant of any of E1-E13 or a pharmaceutical composition of E19 or E20.

[0150] E26. The method of E25, wherein the cancer is an L-asparagine auxotrophic cancer.

[0151] E27. The method of E25 or E26, wherein the cancer is selected from acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), prostate cancer, ovarian cancer, colon cancer, pancreatic cancer and breast cancer. SEQUENCES

[0152] Table 1. Overview of asparaginase sequences

[0153] SEQ ID NO:1 human asparaginase 1, full-length (amino acids 1-573)

[0154] MARAVGPERRLLAVYTGGTIGMRSELGVLVPGTGLAAILRTLPMFHDEEHARARGLSEDTLV LPPASRNQRILYTVLECQPLFDSSDMTIAEWVCLAQTIKRHYEQYHGFWIHGTDTMAFAAS MLSFMLENLQKTVILTGAQVPIHALWSDGRENLLGALLMAGQYVIPEVCLFFQNQLFRGNRA

[0155] TKVDARRFAAFCSPNLLPLATVGADITINRELVRKVDGKAGLWHSSMEQDVGLLRLYPGIP

[0156] AALVRAFLQPPLKGWMETFGSGNGPTKPDLLQELRVATERGLVIVNCTHCLQGAVTTDYAA

[0157] GMAMAGAGVISGFDMTSEAALAKLSYVLGQPGLSLDVRKELLTKDLRGEMTPPSVEERRPSL

[0158] QGNTLGGGVSWLLSLSGSQEADALRNALVPSLACAAAHAGDVEALQALVELGSDLGLVDFNG

[0159] QTPLHAAARGGHTEAVTMLLQRGVDVNTRDTDGFSPLLLAVRGRHPGVIGLLREAGASLSTQ

[0160] ELEEAGTELCRLAYRADLEGLQVWWQAGADLGQPGYDGHSALHVAEAAGNLAWAFLQSLEG AVGAQAPCPEVLPGV

[0161] SEQ ID NO:2 human asparaginase 1, truncation of amino acids 370-573 (A370)

[0162] MARAVGPERRLLAVYTGGTIGMRSELGVLVPGTGLAAILRTLPMFHDEEHARARGLSEDTLV

[0163] LPPASRNQRILYTVLECQPLFDSSDMTIAEWVCLAQTIKRHYEQYHGFWIHGTDTMAFAAS

[0164] MLSFMLENLQKTVILTGAQVPIHALWSDGRENLLGALLMAGQYVIPEVCLFFQNQLFRGNRA

[0165] TKVDARRFAAFCSPNLLPLATVGADITINRELVRKVDGKAGLWHSSMEQDVGLLRLYPGIP

[0166] AALVRAFLQPPLKGWMETFGSGNGPTKPDLLQELRVATERGLVIVNCTHCLQGAVTTDYAA GMAMAGAGVISGFDMTSEAALAKLSYVLGQPGLSLDVRKELLTKDLRGEMTPPSVEERR

[0167] SEQ ID NO:3 human asparaginase 1, full-length, loop amino acids 297-318 replaced by corresponding amino acid sequence of guinea pig asparaginase (underlined)

[0168] MARAVGPERRLLAVYTGGTIGMRSELGVLVPGTGLAAILRTLPMFHDEEHARARGLSEDTLV

[0169] LPPASRNQRILYTVLECQPLFDSSDMTIAEWVCLAQTIKRHYEQYHGFWIHGTDTMAFAAS

[0170] MLSFMLENLQKTVILTGAQVPIHALWSDGRENLLGALLMAGQYVIPEVCLFFQNQLFRGNRA

[0171] TKVDARRFAAFCSPNLLPLATVGADITINRELVRKVDGKAGLWHSSMEQDVGLLRLYPGIP

[0172] AALVRAFLQPPLKGWMETFGSGNGPTKPDLLQELRVATERGLVIVNCSQCLRGSVTPGYAT

[0173] SLAGANVISGFDMTSEAALAKLSYVLGQPGLSLDVRKELLTKDLRGEMTPPSVEERRPSLQG

[0174] NTLGGGVSWLLSLSGSQEADALRNALVPSLACAAAHAGDVEALQALVELGSDLGLVDFNGQT

[0175] PLHAAARGGHTEAVTMLLQRGVDVNTRDTDGFSPLLLAVRGRHPGVIGLLREAGASLSTQEL EEAGTELCRLAYRADLEGLQVWWQAGADLGQPGYDGHSALHVAEAAGNLAWAFLQSLEGAV GAQAPCPEVLPGV

[0176] SEQ ID NO:4 human asparaginase 1, truncation of amino acids 370-573 (A370), loop amino acids 297-318 replaced by corresponding amino acid sequence of guinea pig asparaginase (underlined)

[0177] MARAVGPERRLLAVYTGGTIGMRSELGVLVPGTGLAAILRTLPMFHDEEHARARGLSEDTLV

[0178] LPPASRNQRILYTVLECQPLFDSSDMTIAEWVCLAQTIKRHYEQYHGFWIHGTDTMAFAAS

[0179] MLSFMLENLQKTVILTGAQVPIHALWSDGRENLLGALLMAGQYVIPEVCLFFQNQLFRGNRA

[0180] TKVDARRFAAFCSPNLLPLATVGADITINRELVRKVDGKAGLWHSSMEQDVGLLRLYPGIP

[0181] AALVRAFLQPPLKGWMETFGSGNGPTKPDLLQELRVATERGLVIVNCSQCLRGSVTPGYAT SLAGANVISGFDMTSEAALAKLSYVLGQPGLSLDVRKELLTKDLRGEMTPPSVEERR SEQ ID NO:5 human asparaginase 1, full-length, loop amino acids 186-195 and loop amino acids 297-318 replaced by corresponding amino acid sequences of guinea pig asparaginase (underlined)

[0182] MARAVGPERRLLAVYTGGTIGMRSELGVLVPGTGLAAILRTLPMFHDEEHARARGLSEDTLV LPPASRNQRILYTVLECQPLFDSSDMTIAEWVCLAQTIKRHYEQYHGFWIHGTDTMAFAAS MLSFMLENLQKTVILTGAQVPIHALWSDGRENLLGALLMAGQYVIPEVCLFFQNQLFRGNRy

[0183] TKypSQKFEAFCSPNLLPLATVGADITINRELVRKVDGKAGLWHSSMEQDVGLLRLYPGIP AALVRAFLQPPLKGWMETFGSGNGPTKPDLLQELRVATERGLVIVNCSQCLRGSVTPGYAT SLAGANVISGFDMTSEAALAKLSYVLGQPGLSLDVRKELLTKDLRGEMTPPSVEERRPSLQG

[0184] NTLGGGVSWLLSLSGSQEADALRNALVPSLACAAAHAGDVEALQALVELGSDLGLVDFNGQT PLHAAARGGHTEAVTMLLQRGVDVNTRDTDGFSPLLLAVRGRHPGVIGLLREAGASLSTQEL EEAGTELCRLAYRADLEGLQVWWQAGADLGQPGYDGHSALHVAEAAGNLAWAFLQSLEGAV GAQAPCPEVLPGV

[0185] SEQ ID NO:6 human asparaginase 1, truncation of amino acids 370-573 (A370), loop amino acids 186-195 and loop amino acids 297-318 replaced by corresponding amino acid sequences of guinea pig asparaginase (underlined)

[0186] MARAVGPERRLLAVYTGGTIGMRSELGVLVPGTGLAAILRTLPMFHDEEHARARGLSEDTLV LPPASRNQRILYTVLECQPLFDSSDMTIAEWVCLAQTIKRHYEQYHGFWIHGTDTMAFAAS MLSFMLENLQKTVILTGAQVPIHALWSDGRENLLGALLMAGQYVIPEVCLFFQNQLFRGNRy

[0187] TKypSQKFEAFCSPNLLPLATVGADITINRELVRKVDGKAGLWHSSMEQDVGLLRLYPGIP AALVRAFLQPPLKGWMETFGSGNGPTKPDLLQELRVATERGLVIVNCSQCLRGSVTPGYAT SLAGANVISGFDMTSEAALAKLSYVLGQPGLSLDVRKELLTKDLRGEMTPPSVEERR

[0188] SEQ ID NO:7 human asparaginase 1, full-length (amino acids 1-573), amino acid substitution H298Q (underlined)

[0189] MARAVGPERRLLAVYTGGTIGMRSELGVLVPGTGLAAILRTLPMFHDEEHARARGLSEDTLV LPPASRNQRILYTVLECQPLFDSSDMTIAEWVCLAQTIKRHYEQYHGFWIHGTDTMAFAAS MLSFMLENLQKTVILTGAQVPIHALWSDGRENLLGALLMAGQYVIPEVCLFFQNQLFRGNRA TKVDARRFAAFCSPNLLPLATVGADITINRELVRKVDGKAGLWHSSMEQDVGLLRLYPGIP

[0190] AALVRAFLQPPLKGWMETFGSGNGPTKPDLLQELRVATERGLVIVNCTQCLQGAVTTDYAA GMAMAGAGVISGFDMTSEAALAKLSYVLGQPGLSLDVRKELLTKDLRGEMTPPSVEERRPSL QGNTLGGGVSWLLSLSGSQEADALRNALVPSLACAAAHAGDVEALQALVELGSDLGLVDFNG QTPLHAAARGGHTEAVTMLLQRGVDVNTRDTDGFSPLLLAVRGRHPGVIGLLREAGASLSTQ ELEEAGTELCRLAYRADLEGLQVWWQAGADLGQPGYDGHSALHVAEAAGNLAWAFLQSLEG

[0191] AVGAQAPCPEVLPGV

[0192] SEQ ID NO:8 human asparaginase 1, truncation of amino acids 370-573 (A370), amino acid substitution H298Q (underlined)

[0193] MARAVGPERRLLAVYTGGTIGMRSELGVLVPGTGLAAILRTLPMFHDEEHARARGLSEDTLV LPPASRNQRILYTVLECQPLFDSSDMTIAEWVCLAQTIKRHYEQYHGFWIHGTDTMAFAAS MLSFMLENLQKTVILTGAQVPIHALWSDGRENLLGALLMAGQYVIPEVCLFFQNQLFRGNRA TKVDARRFAAFCSPNLLPLATVGADITINRELVRKVDGKAGLWHSSMEQDVGLLRLYPGIP AALVRAFLQPPLKGWMETFGSGNGPTKPDLLQELRVATERGLVIVNCTQCLQGAVTTDYAA GMAMAGAGVISGFDMTSEAALAKLSYVLGQPGLSLDVRKELLTKDLRGEMTPPSVEERR

[0194] SEQ ID NO:9 human asparaginase 1, full-length (amino acids 1-573), amino acid substitutions T297S and H298Q (underlined)

[0195] MARAVGPERRLLAVYTGGTIGMRSELGVLVPGTGLAAILRTLPMFHDEEHARARGLSEDTLV LPPASRNQRILYTVLECQPLFDSSDMTIAEWVCLAQTIKRHYEQYHGFWIHGTDTMAFAAS MLSFMLENLQKTVILTGAQVPIHALWSDGRENLLGALLMAGQYVIPEVCLFFQNQLFRGNRA TKVDARRFAAFCSPNLLPLATVGADITINRELVRKVDGKAGLWHSSMEQDVGLLRLYPGIP AALVRAFLQPPLKGWMETFGSGNGPTKPDLLQELRVATERGLVIVNCSQCLQGAVTTDYAA GMAMAGAGVISGFDMTSEAALAKLSYVLGQPGLSLDVRKELLTKDLRGEMTPPSVEERRPSL QGNTLGGGVSWLLSLSGSQEADALRNALVPSLACAAAHAGDVEALQALVELGSDLGLVDFNG QTPLHAAARGGHTEAVTMLLQRGVDVNTRDTDGFSPLLLAVRGRHPGVIGLLREAGASLSTQ ELEEAGTELCRLAYRADLEGLQVWWQAGADLGQPGYDGHSALHVAEAAGNLAWAFLQSLEG AVGAQAPCPEVLPGV

[0196] SEQ ID NO: 10 human asparaginase 1, truncation of amino acids 370-573 (A370), amino acid substitution T297S and H298Q (underlined)

[0197] MARAVGPERRLLAVYTGGTIGMRSELGVLVPGTGLAAILRTLPMFHDEEHARARGLSEDTLV LPPASRNQRILYTVLECQPLFDSSDMTIAEWVCLAQTIKRHYEQYHGFWIHGTDTMAFAAS MLSFMLENLQKTVILTGAQVPIHALWSDGRENLLGALLMAGQYVIPEVCLFFQNQLFRGNRA TKVDARRFAAFCSPNLLPLATVGADITINRELVRKVDGKAGLWHSSMEQDVGLLRLYPGIP AALVRAFLQPPLKGWMETFGSGNGPTKPDLLQELRVATERGLVIVNCSQCLQGAVTTDYAA GMAMAGAGVISGFDMTSEAALAKLSYVLGQPGLSLDVRKELLTKDLRGEMTPPSVEERR

[0198] SEQ ID NO: 11 human asparaginase 1, full-length (amino acids 1-573), amino acid substitution H298X (underlined), wherein X is selected from Q, N, E and D

[0199] MARAVGPERRLLAVYTGGTIGMRSELGVLVPGTGLAAILRTLPMFHDEEHARARGLSEDTLV LPPASRNQRILYTVLECQPLFDSSDMTIAEWVCLAQTIKRHYEQYHGFWIHGTDTMAFAAS MLSFMLENLQKTVILTGAQVPIHALWSDGRENLLGALLMAGQYVIPEVCLFFQNQLFRGNRA TKVDARRFAAFCSPNLLPLATVGADITINRELVRKVDGKAGLWHSSMEQDVGLLRLYPGIP AALVRAFLQPPLKGWMETFGSGNGPTKPDLLQELRVATERGLVIVNCTXCLQGAVTTDYAA GMAMAGAGVISGFDMTSEAALAKLSYVLGQPGLSLDVRKELLTKDLRGEMTPPSVEERRPSL QGNTLGGGVSWLLSLSGSQEADALRNALVPSLACAAAHAGDVEALQALVELGSDLGLVDFNG QTPLHAAARGGHTEAVTMLLQRGVDVNTRDTDGFSPLLLAVRGRHPGVIGLLREAGASLSTQ ELEEAGTELCRLAYRADLEGLQVWWQAGADLGQPGYDGHSALHVAEAAGNLAWAFLQSLEG AVGAQAPCPEVLPGV SEQ ID NO: 12 human asparaginase 1, truncation of amino acids 370-573 (A370), amino acid substitution H298X (underlined), wherein X is selected from Q, N, E and D

[0200] MARAVGPERRLLAVYTGGTIGMRSELGVLVPGTGLAAILRTLPMFHDEEHARARGLSEDTLV LPPASRNQRILYTVLECQPLFDSSDMTIAEWVCLAQTIKRHYEQYHGFWIHGTDTMAFAAS MLSFMLENLQKTVILTGAQVPIHALWSDGRENLLGALLMAGQYVIPEVCLFFQNQLFRGNRA TKVDARRFAAFCSPNLLPLATVGADITINRELVRKVDGKAGLWHSSMEQDVGLLRLYPGIP AALVRAFLQPPLKGWMETFGSGNGPTKPDLLQELRVATERGLVIVNCTXCLQGAVTTDYAA GMAMAGAGVISGFDMTSEAALAKLSYVLGQPGLSLDVRKELLTKDLRGEMTPPSVEERR

[0201] SEQ ID NO:13 human asparaginase 1, full-length (amino acids 1-573), amino acid substitution T297S (underlined)

[0202] MARAVGPERRLLAVYTGGTIGMRSELGVLVPGTGLAAILRTLPMFHDEEHARARGLSEDTLV LPPASRNQRILYTVLECQPLFDSSDMTIAEWVCLAQTIKRHYEQYHGFWIHGTDTMAFAAS MLSFMLENLQKTVILTGAQVPIHALWSDGRENLLGALLMAGQYVIPEVCLFFQNQLFRGNRA TKVDARRFAAFCSPNLLPLATVGADITINRELVRKVDGKAGLWHSSMEQDVGLLRLYPGIP AALVRAFLQPPLKGWMETFGSGNGPTKPDLLQELRVATERGLVIVNCSHCLQGAVTTDYAA GMAMAGAGVISGFDMTSEAALAKLSYVLGQPGLSLDVRKELLTKDLRGEMTPPSVEERRPSL QGNTLGGGVSWLLSLSGSQEADALRNALVPSLACAAAHAGDVEALQALVELGSDLGLVDFNG QTPLHAAARGGHTEAVTMLLQRGVDVNTRDTDGFSPLLLAVRGRHPGVIGLLREAGASLSTQ ELEEAGTELCRLAYRADLEGLQVWWQAGADLGQPGYDGHSALHVAEAAGNLAWAFLQSLEG AVGAQAPCPEVLPGV

[0203] SEQ ID NO:14 guinea pig asparaginase, full-length (amino acids 1-565)

[0204] MARASGSERHLLLI YTGGTLGMQSKGGVLVPGPGLVTLLRTLPMFHDKEFAQAQGLPDHALA LPPASHGPRVLYTVLECQPLLDSSDMTIDDWIRIAKI IERHYEQYQGFWIHGTDTMASGAS MLSFMLENLHKPVILTGAQVPIRVLWNDARENLLGALLVAGQYI IPEVCLFMNSQLFRGNRV TKVDSQKFEAFCSPNLSPLATVGADVTIAWDLVRKVKWKDPLWHSNMEHDVALLRLYPGIP ASLVRAFLQPPLKGWLETFGSGNGPSKPDLLQELRAAAQRGLIMVNCSQCLRGSVTPGYAT SLAGANIVSGLDMTSEAALAKLSYVLGLPELSLERRQELLAKDLRGEMTLPTADLHQSSPPG STLGQGVARLFSLFGCQEEDSVQDAVMPSLALALAHAGELEALQALMELGSDLRLKDSNGQT LLHVAARNGRDGWTMLLHRGMDVNARDRDGLSPLLLAVQGRHRECIRLLRKAGACLSPQDL KDAGTELCRLASRADMEGLQAWGQAGADLQQPGYDGRSALCVAEAAGNQEVLALLRNLALVG PEVPPAI

[0205] In the variants of hASNl described herein, the N-terminal methionine (initiator methionine) present in SEQ ID NOs:l-13 may be missing.

[0206] DESCRIPTION OF THE FIGURES

[0207] Figure 1. Pairwise amino acid sequence alignment of human asparaginase 1 (hASNl, SEQ ID NO:1) and guinea pig asparaginase (gpASN, SEQ ID NO: 12). The amino acids at positions 19, 116, 117, 188, 308 of hASNl are underlined. The amino acid at position 298 of hASNl is in bold font, and marked by an arrow. The loop amino acid sequences at position 186-195 and 297-318 of hASNl and the corresponding aligned amino acid positions in gpASN are marked by frames. Due to gaps in the aligned gpASN, amino acids 297-318 of hASNl align with amino acids 297-316 of gpASN. The hASNl and variants of hASNl described herein may be truncated. The truncation site (after position 269 of hASNl) is marked by a vertical dashed line.

[0208] Figures 2A-2B. Crystal structure of truncated hASNl (hASNltr). (A) Functional tetramer of hASNltr indicating chains A, B, C and D. (B) Allosteric center of hASNltr showing residues 296-319 and 186-195 in black. The structure of the closely related non-allosteric asparaginase from guinea pig (gpASN) is shown in white. The important allosteric residue His298 in hASNltr and the corresponding Gln298 in gpASN are highlighted.

[0209] Figure 3. Enzymatic activity of full-length hASNl (hASNlfl), hASNlfl with amino acids 297-318 replaced by amino acids 297-316 of gpASN (hASNlfl-loop), hASNlfl with amino acid substitution H298Q (hASNlfl-H298Q), hASNlfl with amino acids 186- 195 and 297-318 replaced by amino acids 186-195 and 297-316 of gpASN (hASNlfl- loop-conl). The calculated apparent reaction rate constant (kObs in s1; = kaPP) was plotted against the corresponding concentration of the substrate L-asparagine (in mM). The data points were fitted a Michaelis-Menten equation.

[0210] Figure 4. Enzymatic activity of truncated hASNl (hASNltr), hASNltr with amino acids 297-318 replaced by amino acids 297-316 of gpASN (hASNltr-loop), hASNltr with amino acid substitution H298Q (hASNltr-H298Q), hASNltr with amino acids 186-195 and 297-318 replaced by amino acids 186-195 and 297-316 of gpASN (hASNltr-loop-conl). The calculated apparent reaction rate constant (kObs in s1;= kaPP) was plotted against the corresponding concentration of the substrate L- asparagine (in mM). The data points were fitted a Michaelis-Menten equation.

[0211] Figures 5A-5C. Jurkat T-ALL cells were lentivirally transduced with shRNA against GSK3o, and knockdown efficiency was assessed by qRT-PCR analysis. Jurkat T-ALL cells lentivirally transduced with shRNA against luciferase were used as a negative control. Subsequently, cells were treated for 8 days at the indicated doses of (A) bacterial asparaginase (Oncaspar), (B) hASNlfl with amino acids 297-318 replaced by amino acids 297-316 of gpASN (hASNlfl-loop), or (C) hASNlfl with amino acid substitution H298Q (hASNlfl-H298Q). Note that Jurkat T-ALL cells are intrinsically resistant to asparaginase, and knockdown of GSK3o is known to highly sensitize these cells to asparagine shortage.

[0212] Figure 6. Asparagine levels in C57BL / 6J mice treated either with 1000 U / kg hASNlfl-loop ("hASNase"), bacterial asparaginase (Oncaspar; "bASNase") or without asparaginase (control; "PBS") at day 0 (left) and day 7 (right) of the treatment. The amino acid profile was determined by LC / MS-MS. Statistical significance was assessed using a one-way ANOVA with Dunnett's adjustment for multiple comparisons. All error bars represent standard error of the mean (SEM). **** p < 0.0001, * p < 0.05, ns (not significant) p > 0.05.

[0213] Figure 7. For treatment of patient-derived xenograft (PDX) cells, ALL clinical specimens collected from children enrolled on AIEOP-BFM ALL protocols were used with informed consent and institutional review board approval in accordance with the Declaration of Helsinki. PDXs were generated by engraftment of viably frozen leukemic cells into immunodeficient mice followed by harvesting and viably freezing. PDX cells were thawed and subsequently resuspended in alpha-MEM medium supplemented with 10% human serum, 10% FBS, 1% L-glutamine, 1% penicillin / streptomycin, 50 ng / ml human SCF, 20 ng / ml FLT3, and 10 ng / ml human IL7. After thawing, cells were cultured for 24 h at 37°C, 5% CO2. Subsequently, PDX cells were seeded in complete alpha-MEM growth medium and treated with vehicle ("PBS") or 100 U / L pegaspargase (Oncaspar, Shire Pharmaceuticals, Lexington, MA; "bASNase") or 100 U / L hASNlfl-loop ("hASNase"), in biological duplicates. Relative viability was assessed by counting viable cells using trypan blue exclusion dye. PDXs were classified as asparaginase sensitive (3418(S) and M5424(S)) and asparaginase resistant (1572(R) and M4119(R)) based on their treatment response to bacterial asparaginase (Oncaspar). The viability cutoff to distinguish sensitive from resistant cases was set at 50%. Statistical significance was assessed using a one-way ANOVA with Dunnett's adjustment for multiple comparisons. All error bars represent SEM. **** P < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05.

[0214] EXAMPLES

[0215] A Experimental Procedures

[0216] A.l Generation of new hASNl constructs

[0217] Codon-optimized DNA sequences encoding wildtype hASNl and hASNl variants were ordered from GeneArt Gene Synthesis (Thermo Fisher Scientific, Waltham, MA, USA). The DNA sequences encoding full-length wildtype hASNl (hASNlfl) and hASNlfl variants were cloned in the vector G031 (hereafter "H14-bdSUMO") using Gibson assembly method. The H14-bdSUMO vector contained an N-terminal discontinuous polyhistidine tag with 14 histidines in total as well as a sequence for the cleavage tag of the highly specific SUMO-protease from the bacterium Brachypodium distachyon (bdSENPl; Frey et al., 2014).

[0218] The construct for the truncated hASNl (hASNltr) lacking the C-terminal ankyrin domain of mammalian asparaginases was obtained by applying site-directed mutagenesis by inverse PCR (Ho et al., 1989). The used primers were designed to hybridize to the regions on either side of the area to be deleted oriented in the reverse direction. This results in the amplification of the entire circular plasmid except for the desired sequence region. After analysis of PCR products via agarose gel electrophoresis, the samples with the desired PCR product were digested with the restriction enzyme Dpnl to remove methylated template DNA. The additional 5'- phosphorylation of the used primers allows ligation and thereby recircularization of the linearized amplification product after PCR. Digestion and subsequent ligation were done according to the respective manufacturer's protocol (Thermo Fisher Scientific, Waltham, MA, USA).

[0219] After inactivation of the ligase enzyme, the reaction product was directly used and introduced into chemically competent E. coli cells and plated onto LB-agar plates with the respective antibiotic for selection. Grown colonies were cultivated in liquid cultures, plasmids purified and the sequence validated by Sanger sequencing.

[0220] A.2 Polymerase chain reaction

[0221] To amplify specific DNA sequences as well as to introduce mutations and generate human asparaginase variants, the polymerase chain reaction (PCR) was applied. The preparation of DNA mixture and the PCR program were adapted according to the manufacturer's manual for the Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) following a 3-step protocol. Thus, the annealing temperature was adapted to primer melting temperatures and the extension time chosen according to construct length. To find suitable PCR conditions, a temperature gradient was applied for annealing and both buffers as well as different concentrations of DMSO and template were tested for each amplification individually. Designed DNA oligonucleotide primers were ordered from Sigma-Aldrich (St. Louis, MO, USA) in desalted quality grade. After the run, the samples were either stored at -20°C or analyzed via agarose gel electrophoresis.

[0222] A.3 Protein expression of asparaginase variants

[0223] For the overexpression of hASNlfl, hASNltr and variants thereof, E. coli NEBExpress cells were transformed with corresponding expression plasmids. An auto-induction protocol (Studier, 2005) was applied. Precultures of 200 mL LB medium were prepared in 1 L cell culture flasks with chicanes by adding the respective antibiotic (Kanamycin) for selection and four colonies of the transformed cells. The precultures were incubated overnight at 37°C and 200 rpm. The OD at 600 nm was determined and the large-scale cultures of 1 L were prepared in 2 L non-baffled cell culture flasks. The cultures were incubated for 1 h at 37°C and 200 rpm and then incubated for three days (72 h) at 16°C and 200 rpm.

[0224] A.4 Purification of human asparaginase proteins and its variants

[0225] After expression of the target protein, the cells were harvested by centrifugation in 1 L beakers for 20 min at 4800 rpm and 4°C. The cell pellet was washed in lysis buffer (50 mM Tris-HCI, pH 7.5, 300 mM NaCI, 20 mM imidazole, 5 mM DTT), centrifuged in a 50 mL tube for 15 min at 9000 rpm and 4°C, and the supernatant was discarded. The final cell pellets were weighed, frozen in liquid nitrogen, and stored at -20°C. The cell lysis was initiated by resuspending the cell pellet in 2-4 mL of lysis buffer per 1 g of cells and adding 1 mM phenylmethyl-sulfonylfluoride (PMSF), a tip of a spatula lysozyme, 5 pg / mL DNasel and 2 mM MgCL. To completely resuspend the cell, the mixture was incubated and stirred at 6°C for 45-60 min. Cell disruption was performed using a microfluidizer under a pressure of 80 psi in five cycles. The cell lysate was clarified to separate the soluble protein by centrifugation for 60 min at 10°C (hASNl 75000 x g). The constructs of the human asparaginase proteins hASNlfl and hASNltr and their variants possess a His-tag and therefore were subjected to a Ni-affinity chromatography. The supernatant after centrifugation was loaded onto a 5 mL Ni Sepharose column equilibrated with lysis buffer and washed with 100 mL of ATP-wash buffer (50 mM Tris-HCI, pH 7.5, 300 mM NaCI, 5 mM MgCL, 5 mM DTT, 1 mM ATP) to remove chaperones. The proteins bound to N- columns were eluted using 100 % elution buffer (50 mM Tris-HCI, pH 7.5, 300 mM NaCI, 10 mM MgCL, 400 mM imidazole, 5mM DTT). The buffer was exchanged to the gel filtration buffer (50 mM Tris-HCI, pH 7.5, 300 mM NaCI, 5 mM DTT) using a desalting column. SUMO protease bdSENPl was added to the protein fraction at the final concentration of 100 nM after desalting to cleave the His-SUMO tag. The digestion was incubated for 1 hour at 6 °C while slowly shaking. Afterwards, the protein solution was again loaded onto the His-trap column, this time equilibrated with gel filtration buffer to remove the His-SUMO tag. The flow-through was concentrated to a volume of 1 mL and injected onto gel filtration column (Superdex 200 10 / 300 GL). A.5 Asparaginase activity assay

[0226] In order to assess the activity of the purified asparaginases and their variants an asparaginase activity assay was used. The implemented assay is based on the ammonia (NH3) which is formed during the enzymatic hydrolysis of the substrate L- asparagine. The NH3 is subsequently exploited by the helping enzyme glutamate dehydrogenase (GDH) to convert o-ketoglutarate into glutamate. This reaction takes place in the presence of NADH involves the simultaneous conversion of NADH into NAD+. This conversion can be observed as a decrease in absorbance at 340 nm over time. To be close to physiological conditions (which is relevant for application of asparaginases as therapeutics), the measurements were carried out at 37°C and pH 7.5. Stock solutions for the substrates o-ketoglutarate (300 mM, pH 7.5), 0-NADH (6 mM), L-asparagine (200 mM, pH 7.5) and the buffer Tris-HCI (100 mM, pH 7.5) were prepared. For each measurement of a data point the asparaginase enzyme was freshly diluted. A quartz cuvette with a width of 1 cm was used. The total reaction volume was adjusted to 1000 pL. The blank was done against air. After preparation of the sample without adding asparaginase (0.025-70 mM L-Asn, 15 mM o- ketoglutarate, 0.3 mM NADH, 30 U / mL GDH, 50 mM Tris buffer), it was first incubated at room temperature (25°C) for 10 min. Then, the sample was placed in the photometer (37°C) and incubated for approximately 3 min while detecting the absorbance at 340 nm until it was relatively stable. Directly after addition of asparaginase, the absorbance was measured over 600 seconds with a data pitch of 0.5 seconds. The measured curves were fitted in the interval with the steepest slope, selected with help of the first derivative, to determine the change in absorption per minute. From the obtained decrease in absorption per time (AAbs) the apparent reaction rate kapp(= "kobs" in Figures 3 and 4) can be calculated as follows: frapp MWASN

[0227] AAbs ■

[0228] £NADH ’CASN ’ d wherein MWASN is the molecular weight of the hASNl or variant thereof (g mol1), ENADH is the extinction coefficient of NADH (6220 M’1cm-1), CASN is the weight concentration of the enzyme (g L1) and d is the width of the cuvette (1 cm). The calculated apparent reaction rates were plotted against the corresponding asparagine concentration as scatter plot of the mean with error bars displaying the standard deviation (Sigma Plot Version 11.0) and fitted to the Hill equation: wherein V is the reaction velocity, kcat is the turnover number, [Asn] is the concentration of the substrate L-asparagine, So.s is the substrate concentration at which the rate of the reaction is half-maximal and n is the Hill coefficient. Provided that an enzyme has no cooperativity among the ligand binding sites, the Hill coefficient n is close to 1 and the kinetic parameters can also be determined with the Michaelis-Menten equation:

[0229] A.6 Cell Lines and Cell Culture

[0230] Jurkat T-ALL cell line was purchased from ATCC (Manassas, VA, USA), and cultured in RPMI-1640 (Thermo Fisher Scientific) with 10% fetal bovine serum (FBS, Sigma- Aldrich, Saint Louis, MO) and 1% penicillin / streptomycin (Thermo Fisher Scientific) at 37°C, 5% CO2. Cells with early passages were exclusively used for these studies, and mycoplasma contamination was excluded using the MycoAlert Mycoplasma Detection Kit according to the manufacturer's instructions (Lonza, Portsmouth, NH; most recently in November 2022).

[0231] A.7 Lentiviral Transduction and Transient Transfection

[0232] Lentiviruses were generated by co-transfecting the plasmid together with packaging vectors psPAX2 (a gift from Didier Trono; addgene plasmid # 12260) and VSV.G (a gift from Tannishtha Reya; addgene plasmid # 14888) using OptiMEM (Invitrogen, Carlsbad, CA) and Polyethyleneimine (PEI), as previously described (Hinze et al., 2019). For concentrated virus, the virus-containing medium was ultracentrifuged at 24.000 rpm for 2 hours at 4C (Beckman Coulter), and the obtained pellet was resuspended in RPMI. Selection with antibiotics was started 24 hours after infection with puromycin (1 pg / ml for a minimum of 48 hours; Thermo Fisher Scientific). Lentiviral infections with the concentrated virus were performed without spinoculation by directly adding the virus to the cultured cells. Antibiotic selections were performed as described above.

[0233] A.8 Assessment of Asparaginase Response in Jurkat T-ALL cells

[0234] Jurkat T-ALL cells are intrinsically resistant to asparaginase. Knockdown of GSK3o is known to highly sensitize these cells. Jurkat T-ALL cells were therefore lentivirally transduced with shRNA against GSK3o, and knockdown efficiency was assessed by qRT-PCR analysis. Jurkat T-ALL cells lentivirally transduced with shRNA against luciferase were used as a negative control. See Hinze et al., 2019 and Hinze et al., 2020 for details. The Jurkat T-ALL cells (25,000 per well) were seeded in 100 pl of complete growth medium in 96-well plates and incubated with the indicated amount of the to-be-tested asparaginase. Cells were split every 48 hours. Briefly, 20 pl of cells were mixed with 80 pl fresh culture medium, supplemented the to-be-tested asparagine at the indicated doses or vehicle (PBS only, control). The tested asparaginases were pegaspargase (Oncaspar, Shire Pharmaceuticals, Lexington, MA), an FDA-approved PEGylated form of E. coli asparaginase, and hASNl variants.

[0235] A.9 Amino Acid Quantification of cell culture medium

[0236] Jurkat T-ALL cells (400,000 per well) were seeded in 1 ml of complete growth medium in a 12-well format. The growth medium was supplemented with final concentrations of 100 U / L asparaginase. After 48 hours of treatment, the medium was collected and stored at -80°C until amino acid quantification. The entire amino acid profile was determined by means of LC / MS-MS.

[0237] A.10 Animal models

[0238] C57BL / 6J mice were generated at the Central Animal facility of Hannover Medical School (MHH). Eight to 12-week-old mice were used for experiments, and littermates were kept in individual cages. Mice were randomly assigned to experimental groups, and handled in strict accordance with good animal practice. All animal work was done at the MHH Central Animal Facility upon approval by the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES, protocol 33.12-42502-04- 21 / 3850).

[0239] A.11 Amino Acid Quantification In Vivo

[0240] Mice were treated by tail vein injection with 1000 lU / kg hASNlfl-loop or with 1000 lU / kg bacterial asparaginase Oncaspar) or without asparaginase (PBS, control). Blood was collected at indicated time points by punction of the vena facialis, and collected in 1.3 ml micro sample tubes coated with EDTA (Sarstedt). Subsequently, collected blood was centrifuged (5000 rpm x 10 min) for plasma collection, and stored at -80°C prior to amino acid quantification. The entire amino acid profile was determined by means of LC / MS-MS.

[0241] B Experiments

[0242] B.l EXAMPLE 1: Cloning, expression and purification of hASNl and hASNl variants

[0243] As a starting point for the development of asparaginase variants originating from the human enzyme, the purification of the wildtype protein needed to be established and its activity verified. The production of hASNl has been found to be challenging before as the commonly used bacterial host systems for expression tend to result in high amounts of insoluble protein leading to relatively low yields (Karamitros et al., 2014; Schalk et al., 2014). The constructs for expression of full-length and truncated hASNl were developed to contain a discontinuous N-terminal polyhistidine tag with 14 histidines in total (H14) as well as a following sequence for the cleavage tag of the highly specific SUMO-protease from the bacterium Brachypodium distachyon (bdSENPl; Frey et al., 2014). The construct is referred to as H14-bdSUMO-hASNl herein.

[0244] The fusion construct was expressed in E. coli NEBExpress, cells were harvested and lysed. Lactose-driven autoinduction of recombinant protein expression was used to achieve high cell density prior to induction. The supernatant was applied to a Ni- affinity chromatography column which binds the polyhistidine tag of the construct. An additional washing step with ATP-wash buffer over 100 mL was carried out to remove chaperone proteins. The buffer of the eluted protein solution was exchanged by desalting and the protein was subjected to cleavage by addition of the specific protease dSENPl. The construct without tag has a reduced size and can be separated from the cleaved off tag by a second Ni-affi nity chromatography. To remove remaining impurities and denatured or aggregated proteins, size exclusion chromatography was applied. Additionally, the growth parameters were adapted to lower temperatures over several days. This gentler approach slowed down growth and expression and allowed more time for synthesis and folding of a stable target protein. In addition to the full-length human asparaginase 1 (hASNlfl) containing the ankyrin domain, a truncated version (hASNltr), as well as hASNl variants (SEQ ID NOs:3-10 and 13) were expressed and purified. The truncated hASNl enzyme (hASNltr) and hASNl variants were cloned into the same vector system as the full- length enzyme (hASNlfl) and expressed and purified analogously.

[0245] B.2 EXAMPLE 2: hASNl variants showed enhanced catalytic activity compared to wild-type hASNl

[0246] Catalytic activity of hASNl variants was measured using the asparaginase activity assay described above and compared to corresponding wild-type hASNl (either full- length or truncated hASNl).

[0247] Surprisingly, compared to the catalytic activity ("KM" in table 2) of wildtype full-length hASNl (hASNlfl; 6.03 mM), a single H298Q substitution in hASNfl (hASNlfl-H298Q) resulted in a 15-fold improvement (0.40 mM), the loop substitution of residues 297- 318 of hASNlfl with corresponding residues 297-316 of gpASN (hASNlfl-loop) resulted in a 13-fold improvement (0.48 mM), the loop substitution of residues 186- 195 and 297-318 of hASNlfl with corresponding residues 186-195 and 297-316 of gpASN (hASNlfl-loop-Conl) resulted in a 15-fold improvement (0.43 mM), and hASNlfl with the two substitutions T297S and H298Q showed a 3-fold improvement (1.80 mM).

[0248] Similarly, compared to the catalytic activity of truncated wildtype hASNl (hASNltr; 55.48 mM), a single H298Q substitution (hASNltr-H298Q) provided a 40-fold improvement (1.50 ± 0.07 mM), substitution of residues 297-318 with corresponding resides 297-316 of gpASN (hASNltr-loop) provided an improvement of 65 or more (0.86 mM for dimer, 0.56 mM for tetramer), the loop substitution of residues 186- 195 and 297-318 of hASNltr with corresponding residues 186-195 and 297-316 of gpASN (hASNltr-loop-Conl) resulted in a 15-fold improvement (0.43 mM), and combined substitutions T297S and H298Q (hANSltr-T297S_H298Q) provided a 15- fold improvement (3.79 mM) in catalytic activity.

[0249] Table 2. Characteristics of the catalytic activities of asparaginases. arepresentative concentration for "low micromolar" (physiological) asparagine concentrationbvalues in parentheses refer to x-fold increase or x-fold decrease relative to the respective wildtype hASNl (full-length or truncated)cEnzymes which show positive cooperativity (allosteric regulation) do not follow the Michaelis-Menten kinetics as their curves are sigmoidal (not hyperbolic). These curves were therefore fitted instead with the Hill's equation, and the S0.5 value was indicated in the column "KM" in this table.dAccording to the gel filtration analysis, wildtype hASNl and all hASNl variants, except for hASNltr-loop eluted exclusively as dimers. Only hASNltr-loop uniquely also showed a tetramer that had higher enzymatic activity than hASNltr-loop dimer.

[0250] The improvements in enzymatic activity by the H298Q single mutation, the substitution of loop residues by corresponding residues of gpASN are also apparent from the curves in Figures 3 and 4. B.3 EXAMPLE 3: hASNlfl-H298Q or hASNlfl-loop decreased viability of cancer cells

[0251] Jurkat T-ALL cells with shRNA knockdown of GSK3o (which confers sensitivity to L- asparaginases) were subjected to treatment with a bacterial asparaginase (Oncaspar), hASNlfl-loop (SEQ ID NO:3) or hASNlfl-H298Q (SEQ ID NO:7). For assessing baseline viability levels, Jurkat T-ALL cells transfected with shLuc (instead of shGSK3o) served as a control. As shown in Figure 5A, treatment with bacterial asparaginase exhibited a dose-dependent reduction in viability, with approximately an 80% decrease observed upon exposure to 1000 U / L of bacterial asparaginase. Remarkably, comparable viability reduction was observed in cells treated with hASNl having the single H298Q substitution, as shown in Figure 5C. Moreover, treatment with hASNlfl-loop also resulted in a dose-dependent viability reduction, with over 60% decrease in viability.

[0252] Taken together, this experiment showed that the single H298Q substitution in hASNl leads to L-asparagine depletion in cell culture model of leukemia (Jurkat T-ALL cells with GSK3o knockdown), resulting in a substantial reduction in viability comparable to that induced by a bacterial asparaginase currently utilized in cancer treatment (Oncaspar). Similarly, treatment with hASNl having only amino acids 297-318 replaced by corresponding amino acids 297-316 of gpASN (hASNlfl-loop) also showed significant viability reduction. The therapeutic use of such variants of hASNl having only one or few alterations compared to wildtype hASNl is expected to be advantageous over the use of less human-like and bacterial asparaginases due to lower immunogenicity.

[0253] B.4 EXAMPLE 4: hASN variants of the invention led to a decrease in L- asparagine levels in the cell medium, and did not affect L-glutamine levels

[0254] Jurkat T-ALL cells were cultured in complete growth medium in the presence of 100 U / L of human asparaginase variant hASNfl-loop or hASNfl-H298Q, and the medium was collected after 48h. The entire amino acid profile of the collected medium was determined using LC / MS-MS. Surprisingly, only the concentration of L-asparagine exhibited a noticeable reduction as a result of the treatment with hASNfl-loop or hASNfl-H298Q, while the levels of all other amino acids, including L-glutamine, remained basically unaffected. This underscores the substrate specificity of the hASNl variants which is a particular advantage over bacterial asparaginases having undesirable glutaminase activity which is linked to adverse effects observed with those bacterial asparaginases.

[0255] B.5 EXAMPLE 5: Susceptibility of tumors to asparaginase treatment tested using patient-derived xenografts

[0256] To test efficacy of hASNl variants and distinguish cancer patients who would benefit from asparaginase therapy the most, patient-derived xenografts (PDX) were established. ALL clinical specimens were collected from children enrolled on AIEOP- BFM ALL protocols, wherein viably frozen leukemic cells were later engrafted into immunodeficient mice, followed by harvesting and viably freezing. PDX cells were thawed and seeded in complete alpha-MEM growth medium and treated with vehicle or 100 U / L pegaspargase (Oncaspar, Shire Pharmaceuticals, Lexington, MA) or 100 U / L hASNlfl-loop in biological duplicates. Relative viability was assessed by counting viable cells using trypan blue exclusion dye. PDXs were classified as asparaginase sensitive (S) and Relative viability was assessed by counting viable cells using trypan blue exclusion dye. PDXs were classified as asparaginase sensitive (S) and asparaginase resistant (R) based on their treatment response to bacterial asparaginase (Oncaspar). Out of four tested PDX, two were sensitive to asparaginase treatment (3418 and M5424). Moreover, the reduction of relative viability in sensitive PDXs was comparable to the reduction previously observed in Jurkat T-ALL cells, as the reduction upon bacterial asparaginase treatment reached approximately 80%, while the reduction in viability of cells treated with hASNlfl-loop was reduced by around 60-70% (Figure 7).

[0257] This experiment not only provided a means to assess patient sensitivity to asparaginase treatment but also demonstrated the clinical relevance of employing the hASNl variants with enhanced catalytic activity in cancer therapy.

[0258] B.6 EXAMPLE 6: Significant reduction of asparagine levels in serum by in vivo treatment with hASNlfl-H298Q

[0259] Treatment of C57BL / 6J mice with PBS control, bacterial asparaginase (Oncaspar) or hASNlfl-loop with subsequent quantification of serum asparagine levels by LC-MS, showed significant reduction of asparagine as a result of treatment with either of the tested asparaginases. Compared to PBS control, at day 7 of treatment mice treated with the bacterial asparaginase showed almost complete depletion of asparagine, and mice treated with hASNlfl-loop also showed very effective (about 50%) depletion of asparagine. This was particularly remarkable considering that, in contrast to Oncaspar, the administered hASNlfl-loop preparation did not even include any half-life-extending or stability-improving modification such as PEGylation. Moreover, since hASNlfl-loop as a variant of a human protein is more likely to be immunogenic in mice, an even stronger beneficial effect of hASNlfl-loop can be expected in human patients. REFERENCES

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Claims

Claims1. A human asparaginase 1 (hASNl) variant which comprises an amino acid sequence having at least 85% identity to the sequence set forth in SEQ ID NO:2; wherein the amino acid at the position of the hASNl variant corresponding to position 298 of the sequence set forth in SEQ ID NO:2 is selected from Q, N, E and D, and in particular is Q; wherein each of the amino acids at the positions of the hASNl variant corresponding to positions 19 and 116 of the sequence set forth in SEQ ID NO:2 is T; wherein the amino acid at the position of the hASNl variant corresponding to position 117 of the sequence set forth in SEQ ID NO:2 is D; wherein the amino acid at the position of the hASNl variant corresponding to position 188 of the sequence set forth in SEQ ID NO: 2 is K; and wherein the amino acid at the position of the hASNl variant corresponding to position 308 of the sequence set forth in SEQ ID NO:2 is Y or E.

2. The hASNl variant of claim 1, wherein the hASNl variant has a So.s value which is at least 2-fold lower than the So.s value of wildtype hASNl having the amino acid sequence set forth in SEQ ID NO:1, wherein So.s is determinable using the results of an asparaginase activity assay which assay comprises:(i) preparing a solution comprising the asparaginase substrate L-asparagine, glutamate dehydrogenase (GDH), the GDH substrates o-ketoglutarate and NADH, Tris buffer, at pH 7.5 and 37°C, and(ii) measuring absorbance at 340 nm, wherein asparaginase activity hydrolyses the L-asparagine and thereby forms ammonia, which ammonia reacts with the o-ketoglutarate in a GDH- catalyzed reaction, wherein the GDH also converts the NADH to NAD+, which conversion is detectable as a change in absorbance at 340 nm, andwherein So.s is the L-asparagine concentration at which the reaction rate is half-maximal.

3. The hASNl variant of any of the preceding claims, wherein the hASNl variant is further characterized by one or more of the following features:(i) the amino acid at the amino acid position of the hASNl variant corresponding to position 297 of the sequence set forth in SEQ ID NO: 2 is T or S;(ii) the amino acid sequence of the hASNl variant corresponding to positions 297-318 of the sequence set forth in SEQ ID NO:2 is the amino acid sequence of positions 297-316 of the sequence set forth in SEQ ID NO: 14;(iii) the amino acid sequence of the hASNl variant corresponding to positions 186-195 of the sequence set forth in SEQ ID NO:2 is the amino acid sequence of positions 186-195 of the sequence set forth in SEQ ID NO: 14;(iv) the amino acids at the positions of the hASNl variant corresponding to positions 2-185, 196-296 and 319-369 of the sequence set forth in SEQ ID NO:2 are identical to the amino acids at corresponding positions 2-185, 196-296 and 319-369 of the sequence set forth in SEQ ID NO:2.

4. The hASNl variant of any of the preceding claims, wherein the amino acid sequence of the hASNl variant comprises the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, wherein the amino acid at position 298 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has been substituted by Q, N, E or D, in particular by Q, in the amino acid sequence of the hASNl variant; and wherein the amino acid at position 1 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 is present or is not present in the amino acid sequence of the hASNl variant; in particular, wherein one or more of the amino acids at positions 186-195, 297, and 299-318 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has / have been altered in the amino acid sequence of the hASNl variant. more particularly, wherein one or more of the amino acids at positions 186- 195, 297, and 299-318 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 has / have been altered in the amino acid sequence of the hASNl variant as follows:- the amino acid at position 297 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by S,- the amino acid at position 301 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by R,- the amino acid at position 303 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by S,- the amino acid at position 306 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by P,- the amino acid at position 307 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by G,- the amino acid at position 310 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by T,- the amino acid at position 311 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been substituted by S,- the amino acid at position 312 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been deleted,- the amino acid at position 313 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO: 2 has been deleted,- the amino acid at position 314 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by L,- the amino acid at position 318 of the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:2 has been substituted by N. more particularly, wherein the hASNl variant comprises an amino acid sequence selected from the sequences as set forth in SEQ ID NOs: 3-12, and wherein the amino acid at position 1 of the sequences set forth in SEQ ID NOs: 3-12, is present or is not present in the amino acid sequence of the hASNl variant.

5. The hASNl variant of any of the preceding claims which is further characterized by one or more of the following features:(i) the hASNl variant further comprises at least one tag independently selected from a His tag, a SUMO tag, an MPB tag and a GST tag;(ii) the hASNl variant is PEGylated;(iii) the hASNl variant is a fusion protein comprising at least one of the following: an albumin, a transferrin, an XTEN, and an antibody against CD7 or CD19.

6. A polynucleotide comprising a nucleotide sequence encoding a hASNl variant of any of the preceding claims.

7. A method for preparing a hASNl variant of any of claims 1-5, the method comprising: culturing a host cell comprising the polynucleotide of claim 6, which host cell is capable of expressing said hASNl variant, under conditions where said hASNl variant is produced by said host cell; and, optionally, purifying the produced hASNl variant.

8. A pharmaceutical composition comprising a hASNl variant of any of claims 1- 5, and a carrier, optionally, wherein the hASNl variant is encapsulated in an erythrocyte.

9. A hASNl variant of any of claims 1-5 or a pharmaceutical composition of claim 8 for use in a method of treatment of the human or animal body by therapy.

10. A hASNl variant of any of claims 1-5 or a pharmaceutical composition of claim 8 for use in the treatment of cancer; optionally, wherein the cancer is an L-asparagine auxotrophic cancer and / or is selected from acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), prostate cancer, ovarian cancer, colon cancer, pancreatic cancer and breast cancer.

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