WO2014170811A2

WO2014170811A2 - Novel mutant l-asparaginases

Info

Publication number: WO2014170811A2
Application number: PCT/IB2014/060697
Authority: WO
Inventors: Avinash SONAWANE; Ranjit Kumar MEHTA
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-04-16
Filing date: 2014-04-14
Publication date: 2014-10-23
Anticipated expiration: 2015-10-16
Also published as: WO2014170811A3

Abstract

The present invention relates to non-naturally occurring L-Asparaginases mutants particularly Type II Escherichia coli L-asparaginase (Ec A) mutants comprising at least one mutation in the amino-acid sequence of wild-type Ec A, wherein such mutation causes the mutants to induce significantly lesser immunogenicity, higher stability and higher cytotoxicity against leukemic cells as compared to wild-type Ec A. The present invention also provides use of the Ec A mutants as anti-leukemic agents for treatment of Acute Lymphoblastic Leukemia (ALL) in a subject in need thereof.

Description

TITLE: NOVEL MUTANT L-ASPARAGINASES FIELD OF THE INVENTION

[0001] The present invention relates to non-naturally occurring L-Asparaginases mutants particularly Type II Escherichia coli L-asparaginase (EcA) mutants. The EcA mutants of the present invention include mutations which result in reduced immunogenicity, high stability and more cytotoxicity against leukemic cells as compared to wild-type EcA. The present invention also relates to use of the EcA mutants as anti-leukemic agents for treatment of Acute Lymphoblastic Leukemia (ALL) in a subject in need thereof.

BACKGROUND OF THE INVENTION

[0002] Asparaginases are widely distributed enzymes in nature from bacteria to mammals and play a central role in amino acid metabolism and utilization. L-asparaginase enzyme specifically catalyzes L-asparagine to L-aspartate and ammonia and play important roles both in the metabolism of all living organisms as well as in pharmacology. L-Asparaginase enzyme maintains nitrogen balance and the level of amino acids within cells. Escherichia coli (E. coli) is a source of two types of L- asparaginases. Type I E. coli L-asparaginase is located in the cystol. Whereas, type II E. coli (E. coli asparaginase-II) is located in the preplasmic region of the bacteria between plasma membrane and the cell envelope, possessing higher affinity for amino acid asparagine than type I E. coli L-asparaginase. Only the type II L-asparaginases present tumor inhibitory activity and, for this reason, have been extensively studied. Since the early 1970s, E. coli asparaginase-II (EcA) has been used as a drug in the treatment of acute lymphoblastic leukemia (ALL).

[0003] The current protocols available in market for the treatment of ALL use combination of 10 or more drugs, including EcA as an integral part of the chemotherapy. There is evidence that EcA potentiates the antileukemic effect of drugs and markedly improves overall survival in childhood ALL.

[0004] To date, there are three main types of asparaginases that are used for the treatment of ALL: 1. Native asparaginase isolated from E. coli marketed as Kidrolase® (EUSA Pharma), Elspar® (Ovation Pharmaceuticals), Crasnitin™ (Bayer), Leunase® (sanofi- aventis), Asparaginase medac™ (Kyowa Hakko)

2. A pegylated form of the native E. co/z-asparaginase (polyethylene glycol [PEG]- asparaginase marketed as Oncaspar™ (Enzon Pharmaceuticals Inc)

3. Asparaginase isolated from Erwinia chrysanthemi marketed as Erwinase® (EUSA Pharma).

[0005] However, the above anti-leukemic agents have several limitations such as they are immunogenic in patients that lead to production of antibodies against asparaginase causing neutralization of asparaginase. Due to immunogenicity patients suffer from hypersensitivity reactions. Furthermore, native form of asparaginase has short half-life due to low stability under in-vivo condition.

[0006] Thus there is a need in the art to address these key limitations of known asparaginases used as anti-leukemic agents. There is a need to develop new asparaginase as antileukemic agents that have reduced immunogenicity, high stability as well as more efficient cytotoxicity against leukemic cells as compared to native form.

[0007] In view of the foregoing, the present invention satisfies these needs, as well as others, and efficiently overcomes the deficiencies found in the background art.

OBJECTS OF THE INVENTION

[0008] It is an object of the present invention to provide novel Type II Escherichia coli L- asparaginase-II (EcA) mutants as anti-leukemic agents.

[0009] It is a further object of the present invention to provide novel EcA mutants wherein the mutants have significantly lesser immunogenicity as compared to wild-type EcA.

[0010] It is a further object of the present invention to provide novel EcA mutants wherein the mutants have higher stability as compared to wild-type EcA. [0011] It is yet another object of the present invention to provide novel EcA mutants wherein the mutants have higher cytotoxicity as compared to wild-type EcA without affecting survival of normal lymphocytes.

[0012] It is yet another object of the present invention to provide novel EcA mutants wherein the mutants are used in the treatment of Acute Lymphoblastic Leukemia (ALL) in a subject in need thereof

[0013] Various objects, features, aspects and advantages of the present invention will become more apparent from the detailed description of the invention herein below along with the accompanying drawing figures in which like numerals represent like components.

SUMMARY OF THE INVENTION

[0014] The present invention provides novel, non-naturally occurring Type II Escherichia coli L-asparaginase (EcA) mutants comprising the amino acid sequence of wild-type EcA, wherein the sequence includes at least a mutation which results in significantly lesser immunogenicity against leukemic cells than the wild-type EcA.

[0015] The EcA mutants of the present invention further have at least a mutation in the amino acid sequence of wild-type EcA wherein the sequence includes at least a mutation which results in higher stability against leukemic cells than wild-type EcA.

[0016] The EcA mutants of the present invention further have at least a mutation in the amino acid sequence of wild-type EcA wherein the sequence includes at least a mutation which results in greater cytotoxicity against leukemic cells than wild-type EcA.

[0017] In one embodiment, the immunogenicity of the mutants of the present invention is more than 7-fold less than wild-type EcA.

[0018] In another embodiment, the mutants of the present invention induce 3-fold more killing of leukemic cells than wild-type EcA, without affecting survival of normal lymphocytes.

[0019] The EcA mutants of the present invention differ from the wild-type EcA by at least one amino-acid substitution. Preferably, the EcA mutants of the invention have less than five amino-acid substitutions. [0020] In an embodiment, EcA mutants of the present invention can be derived by replacement of at least one amino acid residue of wild-type EcA of SEQ ID 03 wherein the at least amino acid residues are selected from the group comprising Lysine at position 288 (K288), Tyrosine at position 176 (Y176) and Tyrosine at position 66 (W66).

[0021] In one embodiment, a new amino-acid residue can be substituted for K288 wherein the new amino-acid residue can be Serine (S) or Histidine (H).

[0022] In another embodiment, a second new amino-acid residue can be substituted for Y 176 wherein the second new amino-acid residue can be Phenylalanine (F).

[0023] In a yet another embodiment, a third new amino acid can be substituted for W66 wherein the third amino acid residue can be Tyrosine (Y).

[0024] The EcA mutants of the present invention can be single substitutions at specific locations such as a mutant where one amino-acid acid residue selected from K288, Y 176 or W66 is replaced by a new amino-acid residue.

[0025] Some of the preferred EcA mutants of the present invention are specific combinations of substitutions such as a mutant where two or more amino-acid residues selected from K288, Y176 and/or W66 are replaced by new amino acid residues. For example, one of the preferred mutants of the invention can include two substitutions wherein K288, Y176 are replaced by Serine (S) and Phenylalanine (F) respectively and one of the other preferred mutants of the invention can include three substitutions wherein K288, Y176 and W66 are replaced by Histidine (H), Phenylalanine (F) and Tyrosine (Y) respectively.

[0026] In an embodiment, the EcA mutants of the present invention can be used as antileukemic agents for the treatment of Acute Lymphoblastic Leukemia (ALL) in a subject in need thereof.

[0027] The present invention further provides a method of treatment of Acute Lymphoblastic Leukemia (ALL) comprising administering a therapeutically effective amount of EcA mutant of the invention to a subject in need thereof. [0028] The present invention further provides methods of production of EcA mutants of the invention.

[0029] Additional objects and embodiments of the invention will be set forth in part in the description, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] These and other features, aspects and advantages of the invention will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein:

[0031] Figure 1 illustrates immunogenicity of EcA wild-type (WT) and K288S/Y176F mutant in mice model wherein EcA WT and the mutant were administered in BALB/c mice and IgG (A), IgGl (B), IgM (C) antibody titers were determined 29 days after immunization. D depicts Indirect ELISA of wild-type EcA WT and K288S/Y176F in MV4: 11 cells.

[0032] Figure 2 illustrates thermal stability of EcA WT and the mutants K288H/Y176F/W66Y and K288S/Y176F.

[0033] Figure 3 illustrates cytotoxicity assay in Acute Lymphoblastic Leukemia (ALL) cell line with 0.6 and 0.8U/ml of the EcA WT and the mutants K288H/Y176F/W66Y and K288S/Y176F after 96 h treatment.

[0034] Figure 4 illustrates an apotosis analysis with 0.8U/ml of the EcA WT and the mutants K288H/Y176F/W66Y and K288S/Y176F after 96 h treatment in ALL cell line (MV4: 11).

Figure 5 illustrates a cell cycle analysis with 0.8U/ml of the EcA WT and the mutants K288H/Y176F/W66Y and K288S/Y176F after 96 h treatment in ALL cell line (MV4: 11).

[0035] Figure 6 depicts three main advantages of the mutants of the present invention including: A. Reduced immunogenicity, B. High Stability, C. Better Cytotoxicity, as compared to wide-type form.

[0036] Figure 7 depicts the amino-acid sequence for K288S/Y176F (SEQ ID 01) [0037] Figure 8 depicts the amino-acid sequence for K288H/Y176F/W66Y (SEQ ID 02)

[0038] Figure 9 depicts the amino-acid sequence for wild-type EcA (SEQ ID 03) DETAILED DESCRIPTION OF THE INVENTION

[0039] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying figures and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

[0040] The term "therapeutically effective amount" or "effective amount", as used herein means that amount of active ingredient that elicits the biological or medicinal response in a mammal which includes at least partial alleviation of the symptoms of the disease being treated.

[0041] The term "subject', as used herein refers to a mammal including but not limited to, for example, human, dog, monkey, horse, mouse, rabbit etc.

[0042] A substitution at a position in a polypeptide is indicated with [designation for original amino acid] [position number] [designation for replacing (new) amino acid]. For example, substitution of Lysine at position 288 with Histidine will be indicated by K288H.

[0043] In one exemplary embodiment of the present invention, EcA mutant has two mutations including substitutions of two new amino acids Serine (S) and Phenylalanine (F) for K288 and Y176 respectively, as shown in SEQ ID 02 in Figure 7. This mutant has been referred to as K288S/Y176F in the Examples section hereinafter.

[0044] In another exemplary embodiment of the present invention, EcA mutant has three mutations including substitutions of three new amino acids Histidine (S), Phenylalanine (F) and Tyrosine (Y) for K288, Y176 and W66 respectively, as shown in SEQ ID 03 in Figure 8. This mutant has been referred to as K288H/Y176F/W66Y in the Examples section hereinafter.

[0045] The EcA mutants of the present invention can also be used in combination therapy along with other anti-leukemic agents for the treatment of Acute Lymphoblastic Leukemia (ALL) in a subject in need thereof.

EXAMPLES

I. Construction of EcA mutants by site directed mutagenesis

[0046] Escherichia coli asparaginase (EcA) is a homotetramer enzyme. A few amino acid residues that are present at the dimer interfaces and B-cell epitopes of EcA were selected. These amino acid residues were mutated by rational protein engineering approach using site directed mutagenesis method. More than 10,000 mutants were obtained after mutagenesis and these mutants were checked individually for their antigenicity, stability and cytotoxicity properties. Out of 10,000 mutants, the inventors have surprisingly identified two new mutants K288S/Y176F and K288H/Y176F/W66Y that showed low immunogenicity, high stability and increased cytotoxicity against leukemic cells as described below.

II. K288S/Y176F and K288H/Y176F/W66Y mutants are less immunogenic as compared to wild-type EcA

[0047] The immunogenicity of K288S/Y176F and wild-type EcA was studied in mouse model. For this, wild-type EcA and K288S/Y176F EcA mutant were administered in BALB/c mice (5 mice in each group) (Day 1). On day 21, booster dose of wild-type EcA and K288S/Y176F EcA mutant was given to the mice. Then the mice were bleed on day 29 and serum was prepared from the blood samples obtained from all mice. The serum was used for determining the titer of total IgG, IgGl and IgM antibodies. Before administration of Wild-type EcA and K288S/Y176F EcA mutant, mice were bleed (pre-bleed) and serum was prepared. This prebleed serum was used as a control. As shown in Figure 1A-C, administration of K288S/Y176F EcA mutant showed approximately 7-fold less production of IgG, IgGl and IgM antibodies in mice as compared to wild-type EcA administered mice. These results indicate the K288S/Y176F EcA mutant is significantly less immunogenic as compared to wild- type EcA.

[0048] The antigenicity of wild-type EcA, K288S/Y176F and K288H/Y176F/W66Y mutants was estimated by indirect ELISA method. Wells of microtiter plates were coated overnight with 100 μΐ of EcA solution (2-5 μg/ml) in 50 mM carbonate/bicarbonate buffer, pH 9.5. Then the plates were blocked for 90 min with 300μ1 0.1 M phosphate- buffered saline (PBS), pH 7.2, containing 0.1% bovine serum albumin (BSA) and 0.05% Tween-20. Then ΙΟΟμΙ per well of 1 :8,000 diluted primary antibody (anti-EcA IgG, Abeam, USA) was added. After 1 h incubation, the plates were washed and incubated with ΙΟΟμΙ HRP -conjugated polyclonal goat anti-human IgG (1 : 10,000 v/v; Thermo Scientific, Rockford, IL) for 1 h. Then the wells were incubated with ΙΟΟμΙ of freshly prepared ABTS (2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid, 0.5 mg/ml) in 0.03% H₂0₂, 0.1 M Na₂HP0₄ and 0.08M citric acid, pH 4.0 for 30 min in the dark. Finally the absorption was measured at 405 nm using a microplate reader (Epoch, Biotek). As shown in Figure ID, both K288S/Y176F and K288H/Y176F/W66Y are significantly less antigenic as compared to wild-type EcA. Among both mutants, K288H/Y176F/W66Y showed significantly less antigenicity.

III. K288S/Y176F and K288H/Y176F/W66Y mutants are more stable than wild-type EcA

[0049] The asparaginase activity of EcA was measured using the synthetic substrate L- aspartic β-hydroxamate (AHA). The enzyme solution was incubated at different temperatures indicated in Figure 2. One unit (U) of asparaginase activity is defined as the amount of enzyme that liberates 1.0 μπιοΐ NH₂OH per min from AHA at 25°C. As shown in Figure 2, K288H/Y176F/W66Y mutant is stable up to 75 °C, whereas K288S/Y176F showed significantly higher activity up to 70 °C when compared with wild-type EcA (Figure 2). These results indicate that both mutants are significantly more stable as compared to wild-type EcA.

IV. K288S/Y176F and K288H/Y176F/W66Y mutants exhibit higher cytotoxicity against leukemic cell lines

[0050] A MTT viability assay was performed to estimate the cytotoxic activity of EcA variants against MV4: 11 ALL cell line. MV4: 11 cells were grown in RPMI medium for 24 h and then cells were treated with different concentrations of purified wild-type EcA and mutants for 96 h. MTT dye was added to each well and incubated for 1 h at 37°C, 5% CO₂ in dark. The formazan crystals formed as a result of cellular reduction of MTT were dissolved in dissolving buffer (1 lg SDS in 50 ml 0.02M HC1 and 50 ml isopropanol), and incubated for 1 h at 37°C, then the absorption was read at 570 nm in an ELISA reader. The percentage of viable cells was calculated as cell viability (%) = average O.D. of wells/average O.D. of control wells. Treatment with 0.6 and 0.8 U/ml of wild-type EcA led to a 78% and 80% reduction in cell viability, respectively (Figure 3). Interestingly, MV4: 11 cells treated with 0.6 and 0.8U/ml of K288S/Y176F and K288H/Y176F/W66Y showed a much greater reduction in viability as compared to EcA wild-type (Figure 3). K288S/Y176F showed 87% and 93% killing of MV4: 11 cells at 0.6 and 0.8 U/ml concentrations, respectively; While K288H/Y176F/W66Y showed 87% and 95% leukemic cell killing at 0.6 and 0.8U/ml concentration, respectively. These results indicate that both mutants show more cytotoxicity effect against leukemic cells as compared to wild-type EcA.

V. Treatment with EcA K288S/Y176F induces apoptosis in leukemic cells

[0051] To further understand the mechanism of enhanced cytotoxicity of EcA variants, the inventors examined K288S/Y176F effects on apoptosis in MV4: 11 cells. MV4: 11 cells were treated with 0.8 U/ml of wild-type EcA and K288S/Y176F for 96 h and then analyzed for apoptosis by annexinV/FITC and propidium iodide (PI) staining (Figure 4). After treatment with K288S/Y176F, 3-fold more cells were positive for apoptosis (annexin V-FITC⁺; ΡΓ) as compared to wild-type EcA.

VI. Treatment with EcA K288S/Y176F arrests the cells at the G0/G1 control point

[0052] The growth behavior of MV4: 11 cells after treatment with wild-type EcA and K288S/Y176F mutant by flow cytometry was also studied. The inventors observed cell cycle arrest at the G0/G1 transition. The number of arrested cells was significantly higher after treatment with K288S/Y176F (59 %) as compared to wild- type EcA treated cells(24%, Figure 5). It appears that K288S/Y176F block protein synthesis at the early phase of the cell cycle. These data demonstrate that K288S/Y176F is more efficient in inducing cell cycle arrest at the G0/G1 control point. Blocking the leukemic cell cycle progression at an early phase may prove crucial for ALL therapy.

[0053] Various modifications of these embodiments will readily apparent to those skilled in the art in view of present disclosure, and generic method defined herein may be applied to other embodiments.

[0054] All structural and functional equivalents to the elements of the various embodiments of the invention described throughout the disclosure that are known or later come to be known to those ordinary skills in the art are expressly incorporated herein by reference and intended to be encompassed by the invention.

[0055] The above description and drawings are only illustrative of preferred embodiments which achieve the objects, features and advantages of the present invention, and it is not intended that present invention be limited thereto. Any modification of the present invention which comes within the spirit and scope of following claims is considered part of the present invention. Furthermore, to extent that the term "include", "have" or "like" is used in the description or the claims, such term is intended to be inclusive in manner similar to the term "comprise" is interpreted when employed as a transitional word in claim.

ADVANTAGES OF THE INVENTION

[0056] The present invention provides novel Type II Escherichia coli L-asparaginase (EcA) mutants as anti-leukemic agents.

[0057] The present invention provides novel EcA mutants wherein the mutants have significantly lesser immunogenicity as compared to wild-type EcA.

[0058] The present invention provides novel EcA mutants wherein the mutants have higher stability as compared to wild-type EcA.

[0059] The present invention provides novel EcA mutants wherein the mutants have higher cytotoxicity as compared to wild-type EcA without affecting the survival of normal lymphocytes. [0060] The present invention provides novel EcA mutants wherein the mutants are used in the treatment of Acute Lymphoblastic Leukemia (ALL) in a subject in need thereof.

SEQUENCE LISTING

Number of SEQ ID Nos : 3

SEQUENCE DESCRIPTION: SEQ ID No. 01:

LENGTH: 330

TYPE: amlno-acld

40 5_0

LPNITILATG GTIAGGGDSA TKSNYTVGKV GVENLVNAVP QLKDIANVKG EQWNIGSQD

7^ 8^ 10^ 110

NDNVWLTLA KKINTDCDKT DGFVITHGTD T EETAYFLD LTVKCDKPW VGAMRPSTS

13_0 14_0 15_0 16_0 17_0 18_0

SADGPFNLY NAWTAADKA SANRGVLWM NDTVLDGRDV TKTNTTDVAT FKSVNFGPLG

19_0 20_0 21_0 22_0 23_0 24_0

YIHNGKIDYQ RTPARKHTSD TPFDVSKLNE LPKVGIVYNY ANASDLPAKA LVDAGYDGIV

25_0 26_0 27_0 28_0 29_0 30_0

SAGVGNGNLY KSVFDTLATA AKTGTAWRS SRVPTGATTQ DAEVDDASYG FVASGTLNPQ

31_0 32_0 33_0

KARVLLQLAL TQTKDPQQIQ QIFNQYSTOP

SEQUENCE DESCRIPTION: SEQ ID No. 02:

LENGTH: 330

TYPE: amlno-acld

l^

LPNITILATG GTIAGGGDSA TKSNYTVGKV GVENLVNAVP QLKDIANVKG EQWNIGSQD

MNDNVYLTLA KKINTDCDKT DGFVITHGTD TMEETAYFLD LTVKCDKPW MVGAMRPSTS

13^ 140 16^ 180

MSADGPFNLY NAWTAADKA SANRGVLWM NDTVLDGRDV TKTNTTDVAT FKSVNFGPLG

19_0 20_0 21_0 22_0 23_0 24_0

YIHNGKIDYQ RTPARKHTSD TPFDVSKLNE LPKVGIVYNY ANASDLPAKA LVDAGYDGIV

25^ 28^ 30^

SAGVGNGNLY KSVFDTLATA AKTGTAWRS SRVPTGATTQ DAEVDDAHYG FVASGTLNPQ

31_0 32_0 33_0

KARVLLQLAL TQTKDPQQIQ QIFNQYSTOP

SEQUENCE DESCRIPTION: SEQ ID No. 03:

LENGTH: 330

TYPE: amlno-acld

1

LPNITILATG GTIAGGGDSA TKSNYTVGKV GVENLVNAVP QLKDIANVKG EQWNIGSQD

7 8_0 9_0 10_0 110 12_0

MNDNVWLTLA KKINTDCDKT DGFVITHGTD TMEETAYFLD LTVKCDKPW MVGAMRPSTS

13_0 14_0 15_0 16_0 17_0 18_0

MSADGPFNLY NAWTAADKA SANRGVLWM NDTVLDGRDV TKTNTTDVAT FKSVNYGPLG

19_0 20_0 21_0 22_0 23_0 24_0

YIHNGKIDYQ RTPARKHTSD TPFDVSKLNE LPKVGIVYNY ANASDLPAKA LVDAGYDGIV

SAGVGNGNLY KSVFDTLATA AKTGTAWRS SRVPTGATTQ DAEVDDAKYG FVASGTLNPQ 310 32^ 330

KARVLLQLAL TQTKDPQQIQ QIFNQYSTOP

Claims

CLAIMS We Claim:

1. A non-naturally occurring EcA mutant wherein at least one amino acid residue of wild- type EcA is replaced by a new amino acid residue wherein the at least one amino acid residue is selected from the group comprising K288, Y176 and W66.

2. The non-naturally occurring EcA mutant of claim 1, wherein said K288 is replaced by said new amino acid residue wherein said new amino acid residue is selected from Serine or Histidine.

3. The non-naturally occurring EcA mutant of claim 1, wherein said Y176 is replaced by said new amino acid residue wherein said new amino acid residue is Phenylalanine.

4. The non-naturally occurring EcA mutant of claim 1, wherein said W66 is replaced by said new amino acid residue wherein said new amino acid residue is Tyrosine.

5. The non-naturally occurring EcA mutant of claim 1, wherein said at least one amino acid residue of wild-type EcA is replaced by said new amino acid residue, as set forth in SEQ ID 01.

6. The non-naturally occurring EcA mutant of claim 1 , wherein said at least one amino acid residue of wild-type EcA is replaced by said new amino acid residue, as set forth in SEQ ID 02.

7. The non-naturally occurring EcA mutant of claim 1, wherein said mutant results in more than 7-fold lesser immunogenicity than the wild-type EcA.

8. The non-naturally occurring EcA mutant of claim 1, wherein said mutant results in 3-fold greater cytotoxicity than the wild-type EcA.

9. The non-naturally occurring EcA mutant of claim 1, wherein said mutant results in greater stability than the wild-type EcA.

10. Use of the non-naturally occurring EcA mutant of claim 1 as an anti-leukemic agent treating acute lymphoblastic leukemia in a subject in need thereof.

11. A method of treating acute lymphoblastic leukemia comprising administering therapeutically effective amount of the EcA mutant of claim 1 to a subject in need thereof.