METHOD OF TREATING A MAMMAL, INCLUDING HUMAN, AGAINST CANCER USING METHIONINE AND ASPARAGINE DEPLETION

Abstract

The invention is related to a new method for treating liquid and solid cancers, in a mammal, including human, wherein methioninase is administered before asparaginase. The invention also encompasses the use of a dietary methionine deprivation, possibly combined with methioninase administration, in advance of asparaginase treatment. Methioninase and asparaginase may be used in particular under free form, pegylated form or encapsulated into erythrocytes.

Claims

1-25. (canceled)

26. A pharmaceutical composition or kit for use in treating cancer in a mammal comprising an asparagine-reducing means and a methionine-reducing means, wherein the methionine-reducing means is administered before the asparagine-reducing means and wherein there is a delay between the administration of the methionine-reducing means and the asparagine-reducing means.

27. The composition or kit of claim 26, wherein when the methionine-reducing means comprises or consists essentially of a methioninase and/or when the asparagine-reducing means comprises or consists essentially of an asparaginase, the methioninase and/or the asparaginase are under free form, pegylated form or encapsulated inside erythrocytes.

28. The composition or kit of claim 27, wherein the methioninase is under free form or is pegylated and the delay between the end of the methioninase administration and the initiation of the asparaginase administration is between about 1 h and about 7 days, between about 3 h and about 6 days, or between about 1 day and about 5 days.

29. The composition or kit of claim 27, wherein the methioninase is encapsulated into erythrocytes and the delay between the end of methioninase administration and the initiation of the asparaginase administration is between about 1 h and about 30 days, between about 1 day and about 20 days, or between about 1 day and about 10 days.

30. The composition or kit claim 29, wherein the methioninase is administered once or more in an amount of between about 100 and about 100,000 IU, between about 500 and about 50,000 IU, or between about 500 and about 5,000 IU; and wherein the asparaginase is administered once or more in an amount of between about 500 and about 100,000 IU, between about 1,000 and about 50,000 IU, or between about 5,000 and about 30,000 IU.

31. The composition or kit of claim 30, wherein (a) the methioninase is under free form or pegylated form and the delay between the methioninase and the asparaginase administration is between about 1 h and about 7 days, between about 3 h and about 6 days, or between about 1 day and about 5 days; or (b) the methioninase is encapsulated inside erythrocytes and the delay between the end of the methioninase administration and the initiation of the asparaginase administration is between about 1 h and about 30 days, between about 1 day and about 20 days, or between about 1 day and about 10 days; wherein the methioninase is administered once or more in an amount of between about 100 and about 100,000 IU, between about 500 and about 50,000 IU, or between about 500 and about 5,000 IU; and wherein the asparaginase is administered once or more in an amount of between about 500 and about 100,000 IU, between about 1,000 and about 50,000 IU, or between about 5,000 and about 30,000 IU.

32. The composition or kit of claim 31, further comprising PLP or a PLP precursor for simultaneous, separate or sequential administration with the methioninase.

33. The composition or kit of claim 32, comprising methioninase encapsulated inside erythrocytes and a non-phosphate precursor of PLP for separate or sequential administration.

34. The composition or kit of claim 31, wherein the methioninase encapsulated inside erythrocytes is administered at least once or twice before the asparaginase encapsulated inside erythrocytes is administered, and each methioninase administration is followed by administration of a solution of non-phosphate precursor of PLP before asparaginase is administered.

35. The composition or kit of claim 26, wherein the methionine-reducing means comprises or consists essentially of a methionine-restricted diet.

36. The composition or kit of claim 26, wherein the asparagine-reducing means comprises or consists essentially of an asparagine-restricted diet.

37. A method for treating a cancer in a mammal in need thereof, the method comprising administering to the mammal in need thereof, a composition comprising a methionine-reducing means, delaying for a period of time, and then administering a composition comprising an asparagine-reducing means, thereby treating the cancer.

38. The method of claim 37, wherein the methionine-reducing means comprises or consists essentially of a methioninase and the asparagine-reducing means comprises or consists essentially of an asparaginase.

39. The method of claim 37, wherein the delay comprises between about 1 h and about 30 days.

40. The method of claim 37, wherein the methionine-reducing means comprises or consists essentially of a methionine-restricted diet.

41. The method of claim 37, wherein the asparagine-reducing means comprises or consists essentially of an asparagine-restricted diet.

42. The method of claim 38, wherein the methioninase is encapsulated inside erythrocytes and is administered at least once or twice before the asparaginase, which is also encapsulated inside erythrocytes, and each methioninase administration is accompanied by administration of PLP or a precursor of PLP before the asparaginase is administered.

43. The method of claim 38, wherein a non-phosphate precursor of PLP is administered once or more after each administration of methioninase.

44. The method of claim 43, wherein the non-phosphate precursor of PLP is administered once a day, or twice or more per day, during the time of methioninase administration.

45. The method of claim 37, wherein the cancer is leukemia or gastric cancer.

Description

BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0180] FIG. 1 is a graph showing % cell viability under different conditions of treatment.

[0181] FIGS. 2 and 3 are graphs showing % cell viability under different conditions of treatment.

[0182] FIG. 4 is a graph showing individual tumor volume with median in function of time.

EXAMPLE 1

I. Abbreviations

[0183] CCK-8: Cell counting kit-8

DPBS: Dulbecco's Phosphate-Buffered Saline

IMDM: Iscove's Modified Dulbecco's

[0184] MGL: Methionine-γ-lyase
v/v: Volume to volume

II. Operating Conditions

II.1 Test Item

II.1.1. L-Asparaginase

Description: Medac® (Germany), E. Coli L-asparaginase 10 000 IU

[0185] One concentration of L-asparaginase (2.53 IU/mL) was prepared by serial dilutions in Dulbecco Phosphate Buffered Saline (DPBS) 1×. Concentration of L-asparaginase was diluted 11-fold to obtain final concentration of 0.23 IU/mL (IC50).

II.1.2. Methionine-γ-Lyase (MGL)

[0186] Description: P. Putida methionine-γ-lyase (MGL) produced in E Coli.
One concentration of MGL (2.09 IU/mL) was prepared by serial dilutions in Dulbecco Phosphate Buffered Saline (DPBS) 1×. Concentration of MGL was diluted 11-fold to obtain final concentration of 0.19 IU/mL (IC50).

II.2 Cell Lines

II.2.1. Description

[0187] Name: HL-60 cell line
Description: Human promyelocytic leukemia cell line (suspension)
Supplier and reference number: ATCC, CCL-240

II.2.2. Culture Conditions

[0188] Cells were cultivated in a IMDM with L-glutamine medium and supplemented with 20% (v/v) of foetal bovine serum, 100 IU/mL of penicillin and 100 μg/mL of streptomycin. Subculturing was performed according to PO-CELL-002 and PO-CELL-005.

II.2.3. Colorimetric Kit

Name: Cell Counting Kit-8 (CCK-8)

[0189] Supplier and reference number: Fluke 96992
Principle: the CCK-8 reagent contains a highly water-soluble tetrazolium salt WST-8. WST-8 is reduced by dehydrogenases in cells to give a yellow colored product (formazan) which is soluble in the tissue culture medium. The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells.

[0190] The colorimetric test was performed according to PO-CELL-004.

III. Cytotoxicity Assay

III.1 Method

[0191] Fifteen thousand cells in 100 μL/well were dispensed in five 96-well flat bottom plates. In addition, 2 wells were filled with culture medium for blank control on each plate. All empty wells were filled with culture medium in order to minimize evaporation and condensation. On day 0 (D0), 10 μL of IC50 concentrations of L-asparaginase or MGL was added to the corresponding wells. Controls (blank wells and control plate) received 10 μL of DPBS 1×. On day 3 (D3), medium was removed from wells and replaced by fresh medium and 10 μL of DPBS 1× or 10 μL of IC50 concentrations of L-asparaginase (for cells previously incubated with MGL) or MGL (for cells previously incubated with L-asparaginase) added to the corresponding wells. Controls (blank and positive control) received 10 μL of DPBS 1×. Then, plates were incubated for 3 more days in the incubator. At the end of the incubation period (D6), 10 μL of CCK-8 solution were added to each well according to PO-CELL-004 and plates incubated for 2 hours in the incubator. Optical density (OD) was then determined at 450 nm using a microplate reader.

III.2 Internal Controls

[0192] Controls were performed in duplicate.

III.2.1. Blank Wells

[0193] Slight spontaneous absorbance around 460 nm occurs in culture medium with CCK-8. This background absorbance depends on the culture medium, pH, incubation time and length of exposure to light. Therefore blank wells were performed containing 100 μL of culture medium and 10 μL of L-asparaginase or MGL diluent, DPBS 1×. The average absorbance of these control wells was subtracted to the others wells containing cells.

III.2.2. Viability Control (Positive Control)

[0194] As positive control for the HL-60 cell line (100% cell viability), cells were cultivated in the culture medium (100 μL) without L-asparaginase nor MGL, but with 10 μL of the diluent (DPBS 1×).

III.3 Determination of Cell Viability

[0195] Culture medium without cells constituted blank controls (OD Blank). Cells without L-asparaginase nor MGL constituted positive controls (viability control).

[0196] Percentage of living cells was calculated as shown below:

[00001]$\frac{O{D}_{L-\mathrm{aspa}+\mathrm{MGL}*}-{\mathrm{OD}}_{\mathrm{Blank}}}{{\mathrm{OD}}_{\mathrm{viability}-\mathrm{control}**}-O{D}_{\mathrm{Blank}}}\times 100$

*: cells with L-asparaginase and MGL treatment
**: cells without L-asparaginase and MGL treatment [0197] Calculations were automatically performed via the Gen 5 software that pilots the microplate reader. The mean optical density (OD) of the 2 blank wells was automatically subtracted from all optical densities. Calculations of cell viability were realized for sequential treatment.

IV. Results

IV.1 Internal Control

[0198] Internal controls were acceptable when it was not specified in raw data.

IV.2 IC50 Calculations with L-Asparaginase or MGL Alone

[0199] Percentages of cell viability with drug alone (MGL or L-asparaginase) were controlled in each experiment of drugs combination

IV.2.1. Sequential Addition of L-Asparaginase and MGL

[0200] The experiment with sequential treatment of L-asparaginase and MGL was done once with duplicate data. All quality controls (blank and positive control) were accepted in experiments. Details of % of cell viability calculations and graphical representation are presented below in table 1 and FIG. 1.

TABLE-US-00001 TABLE 1 % of cell viability for controls and enzyme association % cell viability at D6 Mean SD Cells alone 100 25 Cells + IC50 L-aspa D0 34 0 Cells + IC50 MGL D0 27 8 Cells + IC50 L-aspa D0 + IC50 32 15 MGL D3 Cells + IC50 MGL D0 + IC50 L- 8 2 aspa D3

[0201] Results indicated that enzyme association with MGL added at IC50 dose before the addition of L-asparaginase at IC50 dose (in red on FIG. 1) permitted to reduce cell viability of: [0202] 76% compared to IC50 L-asparaginase (IC50 control for L-asparaginase), [0203] 70% compared to MGL (IC50 control for MGL), [0204] 75% compared to enzyme association with L-asparaginase added in first at IC50 dose.

[0205] Yet, the reverse order of enzyme association did not give such results, with no benefits of the association on cell viability compared to enzymes alone (controls).

V. Conclusion

[0206] Sequential enzyme association demonstrated that cell mortality could be increased with an addition of MGL at IC50 dose followed 3 days later by the addition of L-asparaginase at IC50 dose. Yet, the reverse design of enzyme addition did not permit to obtain such results.

[0207] We can hypothesize that Met deprivation induced by MGL enzyme activity makes HL-60 leukemia cells more sensitive to L-asparaginase activity. Moreover, the roles of L-asparaginase and MGL have to be discussed considering their known respective effect. Indeed, L-asparaginase is known to trigger apoptosis in leukaemia cells (Ueno et al., 1997), therefore, it could probably plays a role of cytotoxic agent. MGL being known for blocking cell division in S or G2 phase of the cell cycle probably acts more as a cytostatic agent.

EXAMPLE 2: METHOD FOR ENCAPSULATION OF L-ASPARAGINASE IN MURINE ERYTHROCYTES

[0208] The L-asparaginase (Medac® E. Coli L-asparaginase) is encapsulated in murine erythrocytes (OF1 mice) by the method of hypotonic dialysis in a dialysis bag. The blood is centrifuged beforehand to remove the plasma, and then washed three times with 0.9% NaCl. The haematocrit is adjusted to 70% in the presence of the asparaginase, added to a final concentration of 400 IU/ml of erythrocytes or red blood cells (RBC) before starting the dialysis. The dialysis lasts 50 minutes at 4° C. against a lysis buffer of low osmolarity. The murine erythrocytes are then resealed through the addition of a high osmolarity solution and incubating 30 minutes at 37° C. After two washings with 0.9% NaCl and one washing with Sag-mannitol supplemented with bovine serum albumin BSA (6%), the erythrocytes are adjusted to haematocrit 50%. The erythrocytes encapsulating the L-asparaginase are called L-Aspa RBC. The encapsulation generates L-Aspa RBC at a concentration of 40 IU of asparaginase/ml of RC at 50% haematocrit.

[0209] During the encapsulation procedure, the whole blood, the washed RBC, the RBC mixed with the L-asparaginase (before dialysis) and the RBC loaded with L-asparaginase (after dialysis) are tested for: [0210] haematocrit (Ht) [0211] average corpuscular volume (ACV) [0212] average corpuscular haemoglobin concentration (ACHC) [0213] total haemoglobin concentration and [0214] cell count.

[0215] Aliquots of the cell suspensions are withdrawn before and after the hypotonic dialysis for measurement of the L-asparaginase enzyme activity. The estimation of the L-asparaginase was performed according to the protocol published in: Orsonneau et al., Ann Biol Clin, 62: 568-572.

EXAMPLE 3: ENCAPSULATION OF L-ASPARAGINASE IN HUMAN ERYTHROCYTES

[0216] The method described in WO-A-2006/016247 is used to produce a batch of erythrocytes encapsulating L-asparaginase. In accordance with the teaching of WO-A-2006/016247, the osmotic fragility is considered and the lysis parameters are adjusted accordingly (flow rate of the erythrocyte suspension in the dialysis cartridge is adjusted). The method is further performed in conformity with the physician prescription, which takes into account the weight of the patient and the dose of L-asparaginase to be administered. The specifications of the end product are as follows: [0217] mean corpuscular volume (MCV): 70-95 fL [0218] mean corpuscular haemoglobin concentration (MCHC): 23-35 g/dL [0219] extracellular haemoglobin≤0.2 g/dL of suspension [0220] osmotic fragility≤6 g/L of NaCl [0221] mean corpuscular L-asparaginase concentration: 78-146 IU/mL [0222] extracellular L-asparaginase≤2% of the total enzyme activity.

[0223] The suspension of erythrocytes so obtained is called GRASPA® and is mentioned in the literature.

EXAMPLE 4. METHOD FOR OBTAINING AND CHARACTERIZING METHIONINE GAMMA LYASE (MGL)

[0224] Production of the strain and isolation of a hyper-producing clone: the natural sequence of MGL of Pseudomonas putida (GenBank: D88554.1) was optimized by modifying rare codons (in order to adapt the sequence stemming from P. putida to the production strain Escherichia coli). Other changes have been made to improve the context of translation initiation. Finally, silent mutations were performed to remove three elements that are part of a putative bacterial promoter in the coding sequence (box −35, box −10 and a binding site of a transcription factor in position 56). The production strain E. coli HMS174 (DE3) was transformed with the expression vector pGTPc502_MGL (promoter T7) containing the optimized sequence and a producing clone was selected. The producing clone is pre-cultivated in a GY medium+0.5% glucose+kanamycin for 6-8 h (pre-culture 1) and 16 h (pre-culture 2) at 37° C.

[0225] Fermentation: the production is then achieved in a fermenter with GY medium, with stirring, controlled pressure and pH from the pre-culture 2 at an optical density of 0.02. The growth phase (at 37° C.) takes place until an optical density of 10 is obtained and the expression induction is achieved at 28° C. by adding 1 mM IPTG into the culture medium. the cell sediment is harvested 20 h after induction in two phases: the cell broth is concentrated 5-10 times after passing over a 500 kDa hollow fiber and then cell pellet is recovered by centrifugation at 15900×g and then stored at −20° C.

[0226] Purification: The cell pellet is thawed and suspended in lysis buffer (7v/w). Lysis is performed at 10° C. in three steps by high pressure homogenization (one step at 1000 bars, and then two steps at 600 bars). The cell lysate then undergoes clarification at 10° C. by adding 0.2% PEI and centrifugation at 15900×g. The soluble fraction is then sterilized by 0.2 μm before precipitation with ammonium sulfate (60% saturation) at 6° C., over 20 h. Two crystallization steps are carried out on the re-solubilized sediment using solubilization buffer, the first crystallization step is realized by addition of PEG-6000 at 10% (final concentration) and ammonium sulfate at 10% saturation, and the second crystallization is then performed by addition of PEG-6000 at 12% final concentration and 0.2M NaCl (final concentration) at 30° C. The pellets containing the MGL protein are harvested at each stage after centrifugation at 15900×g. The pellet containing the MGL protein is re-suspended in a solubilization buffer and passed over a 0.45 μm filter before being subject to two anion exchange chromatographies (DEAE sepharose FF). The purified protein is then subject to a polishing step and passed over a Q membrane chromatography capsule for removing the different contaminants (endotoxins, HCP host cell protein, residual DNA). Finally, the purified MGL protein is concentrated at 40 mg/ml and diafiltered in formulation buffer using a 10 kDa cut-off tangential flow filtration cassette. Substance is then aliquoted at ˜50 mg of protein per vial, eventually freeze-dried under controlled pressure and temperature, and stored at ˜80° C.

[0227] Characterization: The specific activity of the enzyme is determined by measuring the produced NH.sub.3 as described in WO 2015/121348. The purity is determined by SDS-PAGE. The PLP level after being taken up in water was evaluated according to the method described in WO 2015/121348. The osmolarity is measured with an osmometer (Micro-Osmometer Loser Type 15).

[0228] The following table 2 summarizes the main characteristics of one produced batch of MGL:

TABLE-US-00002 MGL of P. putida Freeze-dried (amount per tube: 49.2 mg). Formulation Characteristics after being taken up in 625 pL of water: 78.7 mg/ml, ~622 μM of PLP, 50 mM of Na phosphate, pH 7.2, Osmolarity 300 mOsmol/kg. Specific activity 13.2 IU/mg Purity >98%

[0229] Discussion of the production method. The method for purifying the MGL described in in WO 2015/121348 is established on the basis of the method detailed in patent EP 0 978 560 B1 and of the associated publication (Takakura et al., Appl Microbiol Biotechnol 2006). This selection is explained by the simplicity and the robustness of the crystallization step which is described as being particularly practical and easily adaptable to large scale productions according to the authors. This step is based on the use of PEG6000 and of ammonium sulfate after heating the MGL solution obtained after the lysis/clarification and removal of impurities by adding PEG6000/ammonium sulfate steps. The other notable point of this step is the possibility of rapidly obtaining a high purity level during the step for removing the impurities by achieving centrifugation following the treatment of the MGL solution with PEG6000. The impurities are again found in the centrifugation pellet, the MGL being in majority found in solution in the supernatant. Because of this purity, the passing of the MGL solution in a single chromatography step over an anion exchanger column (DEAE), associated with a purification step by gel filtration on a sephacryl S200 HR column, gives the possibility of obtaining a purified protein.

[0230] Upon setting into place the patented method for small scale tests, it appeared that the obtained results were not able to be reproduced. According to patent EP 0 978 560 B1, at the end of the step for removing the impurities (treatment with PEG6000/ammonium sulfate and centrifugation), the MGL enzyme is in majority found in the soluble fraction, centrifugation causing removal of the impurities in the pellet. During small scale tests conducted according to the described method in EP 0 978 560 B1, the MGL protein is again in majority found (˜80%) in the centrifugation pellet. The table 3 below lists the percentage of MGL evaluated by densitometry on SDS-PAGE gel in soluble fractions.

TABLE-US-00003 MGL percentage in Purification the soluble fraction Average Test no. 1 11% 17% Test no. 2 23%

[0231] This unexpected result therefore led to optimization of the patented method by: 1) operating from the centrifugation pellet containing MGL, 2) carrying out two successive crystallization steps for improving the removal of the impurities after loading on a DEAE column, 3) optimizing chromatography on a DEAE column.

[0232] For this last step, it is found that the DEAE sepharose FF resin is finally not a sufficiently strong exchanger in the tested buffer and pH conditions. After different additional optimization tests, the selection was finally directed to 1) replacement of the phosphate buffer used in the initial method with Tris buffer pH 7.6 for improving the robustness of the method and 2) carrying out a second passage on DEAE in order to substantially improve the endotoxin level and the protein purity without any loss of MGL (0.8 EU/mg according to Takakura et al., 2006 versus 0.57 EU/mg for the modified method).

[0233] Finally, in order to obtain a method compatible with the requirements for large scale GMP production, a polishing step on a membrane Q was added in order to reduce the residual endotoxins and HCP levels. This final step of polishing avoids the implementation of the S200 gel filtration chromatography which is a difficult step to be used in production processes at an industrial scale (cost and duration of the chromatography).

[0234] Product obtained is summarized in the following table 4 using the two methods.

TABLE-US-00004 Patent Method of EP 978 560 B1 the application Amount of Yield Amount of Yield Step enzyme (g) (%) enzyme (g) (%) Solubilised pellet 125 100 70 100 before DEAE Concentrated solution.sup.$  80  64 46  65 .sup.$post sephacryl S-200 HR (EP 978 560) or post Membrane Q (method of the invention).

EXAMPLE 5. CO-ENCAPSULATION OF MGL AND PLP IN MURINE ERYTHROCYTES

[0235] Whole blood of CD1 mice (Charles River) is centrifuged at 1000×g, for 10 min, at 4° C. in order to remove the plasma and buffy coat. The RCs are washed three times with 0.9% NaCl (v:v). The freeze-dried MGL is re-suspended in water at a concentration of 78.7 mg/ml and added to the erythrocyte suspension in order to obtain a final suspension with a hematocrit of 70%, containing different concentrations of MGL and of the PLP. The suspension was then loaded on a hemodialyzer at a flow rate of 120 ml/h and dialyzed against a hypotonic solution at a flow rate of 15 ml/min as a counter-current. The suspension was then resealed with a hypertonic solution and then incubated for 30 min at 37° C. After three washes in 0.9% NaCl, 0.2% glucose, the suspension was taken up in a preservation solution SAG-Mannitol supplemented with 6% BSA. The obtained products are characterized at D0 (within the 2 h following their preparation) and at D1 (i.e. after ˜18 h-24 h of preservation at 2-8° C.). The hematologic characteristics are obtained with a veterinary automaton (Sysmex, PocH-100iV).

[0236] Results:

[0237] In the different studies mentioned hereafter, the MGL activity in the finished products is assayed with the method described in example 5 against an external calibration range of MGL in aqueous solution. These results, combined with explanatory studies, show that MGL activity in the finished products increases with the amount of enzyme introduced into the method and that it is easily possible to encapsulate up to 32 IU of MGL per ml of finished product while maintaining good stability.

[0238] In another study, three murine finished products RC-MGL-PLP1, RC-MGL-PLP2 and RC-MGL-PLP3 were prepared according to the following methods: [0239] RC-MGL-PLP1: co-encapsulation of MGL and of PLP from a suspension containing 3 mg/ml of MGL and ˜30 μM of PLP. The final product was taken up in SAG-Mannitol, 6% BSA supplemented with final 10 μM PLP. [0240] RC-MGL-PLP2: co-encapsulation of MGL and of PLP from a suspension containing 3 mg/ml of MGL and ˜30 μM of PLP. The finished product was taken up in SAG-Mannitol 6% BSA. [0241] RC-MGL-PLP3: this product stems from a co-encapsulation of MGL and PLP from a suspension containing 3 mg/ml of MGL and ˜124 μM of PLP. The final product was taken up in SAG-Mannitol 6% BSA.

[0242] In a third study, a murine finished product RC-MGL-PLP4 was prepared from a new batch of MGL according to the following methods: [0243] RC-MGL-PLP4: co-encapsulation of MGL and the PLP from a suspension containing 5 mg/ml of MGL and ˜35 μM of PLP. The finished product was taken up in SAG-Mannitol 6% BSA.

[0244] Finally in a fourth study, a murine product RC-MGL-PLP5 was prepared from a third batch of MGL according to the following methods: [0245] RC-MGL-PLP5: co-encapsulation of MGL and PLP from a suspension containing 6 mg/ml of MGL and ˜100 μM of PLP. The finished product was taken up in SAG-Mannitol 6% BSA.

[0246] The hematologic and biochemical characteristics of the three finished products at D0 (after their preparation) are detailed in the table 5 below. The encapsulation yields are satisfactory and vary from 18.6% to 30.5%.

TABLE-US-00005 RC- RC- RC- RC- RC- MGL- MGL- MGL- MGL- MGL- PLP1 PLP2 PLP3 PLP4 PLP5 Hematolo- Hematocrit (%) 50.0 49.6 50.0 50.0 50.0 gical data Corpuscle volume (fl) 46.3 46.5 46.8 42.4 45.6 Corpuscle hemoglobin (g/dl) 24.7 24.0 24.2 27.4 25.1 RC concentration (10.sup.6/μl) 6.5 6.9 6.6 7.2 6.0 Total hemoglobin (g/dl) 14.8 15.4 15.0 16.6 13.8 Extracellular Hb (g/dl) 0.1 0.1 0.1 0.2 0.05 mgl Intra-erythrocyte concentration of 0.97 0.94 0.79 1.01 1.36 MGL (mg/ml of RC) Intra-erythrocyte activity of MGL 12.8 12.4 8.8 5.0 8.6 (IU/ml of RC)* Extracellular activity (%) 0.92% 0.97% 1.32% 1.18% 2.23% Intracellular activity (%) 99.08% 99.03% 98.68% 98.82% 97.77% Encapsulation yield of MGL (%) 18.6% 30.5% 22.6% 19.4% 22.7% PLP Intra-erythrocyte concentration of ND 13.4 71.4 10.2 ND PLP (μmol/l of RC) Intracellular PLP fraction (%) ND 99.5 98.7 98.1 ND Extracellular PLP fraction (%) ND 0.5 1.3 1.92 ND PLP encapsulation yield (%) ND 44.8 57.4 30.7 ND *Calculated from the specific activity of each batch.

EXAMPLE 6. PRODUCTION OF HUMAN RCS ENCAPSULATING METHIONINE GAMMA LYASE AND PLP ACCORDING TO THE INDUSTRIAL METHOD

[0247] A pouch of leukocyte-depleted human Red Cell RCs (provided by the “Etablissement Français du Sang”) is subject to a cycle of three washes with 0.9% NaCl (washer Cobe 2991). The freeze-dried MGL is re-suspended with 0.7% NaCl and added to the erythrocyte suspension in order to obtain a final suspension with a hematocrit of 70%, containing 3 mg/ml of MGL and ˜30 μM of PLP (stemming from the formulation of MGL). The suspension is homogenized and it is proceeded with encapsulation according to the method described in EP 1 773 452. The suspension from the resealing is then incubated for 3 h at room temperature in order to remove the most fragile RCs. The suspension is washed three times with a 0.9% NaCl, 0.2% glucose solution (washer Cobe 2991) and then re-suspended with 80 ml of preservation solution (AS-3). The encapsulated MGL level is assayed like in Example 6, see table 6 below.

TABLE-US-00006 J0 J1 J7 Hematocrit (%) 52.0 51.6 52.7 Corpuscle volume (fl) 91.0 92.0 88.0 Corpuscle hemoglobin (g/dl) 30.3 29.8 31.6 RC concentration (10.sup.6/μl) 6.00 5.92 5.98 Total hemoglobin (g/dl) 16.4 16.2 16.6 Extracellular Hb (g/dl) 0.119 0.197 0.280 Osmotic fragility (g/l) 1.17 Hemolysis (%) 0.7% 1.2% 1.7% Total MGL concentration (mg/ml) 0.36 0.35 MGL supernatant concentration 0.01 0.01 (mg/ml) MGL intra-erythrocyte concentra- 0.68 0.67 tion (mg/ml, 100% Ht) Extracellular activity (%) 1.3% 1.4% Intracellular activity (%) 98.7% 98.6% Encapsulation yield (%) 19.7%

EXAMPLE 7

Additional Abbreviations

[0248] RPMI: Le Roswell park memorial institute medium

I. Operating Conditions

I.1 Test Item

I.1.1. L-Asparaginase

[0249] Description: Medac® (Germany), E. Coli L-asparaginase 10 000 IU.

[0250] One concentration of L-asparaginase (2.2 IU/mL) was prepared by serial dilutions in Dulbecco Phosphate Buffered Saline (DPBS) 1×. Concentration of L-asparaginase was diluted 11-fold to obtain final concentration of 0.20 IU/mL (IC50).

I.1.2. Methionine-γ-Lyase (MGL)

[0251] Description: P. Putida methionine-γ-lyase (MGL) produced in E. Coli.

[0252] One concentration of MGL (3.85 IU/mL) was prepared by serial dilutions in Dulbecco Phosphate Buffered Saline (DPBS) 1×. Concentration of MGL was diluted 11-fold to obtain final concentration of 0.35 IU/mL (IC50).

I.2 Cell Lines

I.2.1. Description

[0253] Name: NCI-N87 cell line
Description: Human gastric carcinoma cell line (adherent)
Supplier and reference number: ATCC, CRL-5822

I.2.2. Culture Conditions

[0254] Cells were cultivated in a RPMI media supplemented with 10% (v/v) of foetal bovine serum, 100 IU/mL of penicillin and 100 μg/mL of streptomycin. Subculturing was performed according to PO-CELL-002 and PO-CELL-005.

I.2.3. Colorimetric Kit

Name: Cell Counting Kit-8 (CCK-8)

[0255] Supplier and reference number: Fluka 96992

[0256] Principle: the CCK-8 reagent contains a highly water-soluble tetrazolium salt WST-8. WST-8 is reduced by dehydrogenases in cells to give a yellow colored product (formazan) which is soluble in the tissue culture medium. The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. The colorimetric test was performed according to PO-CELL-004.

II. Cytotoxicity Assay

II.1 Method

[0257] Two thousand five hundred cells in 100 μL/well were dispensed in 96-well flat bottom plates (cf. number of plates in raw data). In addition, two wells were filled with culture medium for blank control on each plate. All empty wells were filled with culture medium in order to minimize evaporation and condensation. On day 0 (D0), 10 μL of IC50 concentrations of L-asparaginase or MGL were added to the corresponding wells. Controls (blank wells and control plate) received 10 μL of DPBS 1×. On day 4 (D4), medium was removed from wells and replaced by fresh medium and 10 μL of DPBS 1× or 10 μL of IC50 concentrations of L-asparaginase (for cells previously incubated with MGL) or MGL (for cells previously incubated with L-asparaginase) added to the corresponding wells. Controls (blank and positive control) received 10 μL of DPBS 1×. Then, plates were incubated for 4 more days in the incubator. At the end of the incubation period (D8), 10 μL of CCK-8 solution were added to each well according to PO-CELL-004 and plates incubated for 4 hours. Optical density (OD) was then determined at 450 nm using a microplate reader.

II.2 Internal Controls

[0258] Controls were performed in duplicate.

II.2.1. Blank Wells

[0259] As above in Example 1.

II.2.2. Viability Control (Positive Control)

[0260] As positive control for the NCI-N87 cell line (100% cell viability), cells were cultivated in the culture medium (100 μL) without L-asparaginase nor MGL, but with 10 μL of the diluent (DPBS 1×).

II.3 Determination of Cell Viability

[0261] As above in Example 1.

III. Results

III.1 Internal Control

[0262] Internal controls were acceptable when it was not specified in raw data.

III.2 IC50 Calculations with L-Asparaginase or MGL Alone

[0263] Percentages of cell viability with drug alone (MGL or L-asparaginase) were controlled in each experiment of drugs combination. Fifty percent of cell viability are expected at half of the test (D4) because IC50 value used here for enzymes were previously validated in single treatment at D4.

III.2.1. Sequential Addition of L-Asparaginase and MGL

[0264] The experiment with sequential treatment of L-asparaginase and MGL was done twice with duplicate data. All quality controls (blank and positive control) were accepted in experiments.

[0265] Details of % of cell viability calculations and graphical representation are presented below in table 7 and FIG. 2.

TABLE-US-00007 TABLE 7 % of cell viability for controls and enzyme association % cell viability at D8 Mean SD Cells alone 100 0 Cells + IC50 L-aspa D0 56 8 Cells + IC50 MGL D0 45 4 Cells + IC50 L-aspa D0 + IC50 MGL D3 44 0 Cells + IC50 MGL D0 + IC50 L-aspa D3 25 6

[0266] Results indicated that enzyme association with MGL added at IC50 dose before the addition of L-asparaginase at IC50 dose (cf. FIG. 2) permitted to reduce cell viability of: [0267] 55% compared to IC50 L-asparaginase (IC50 control for L-asparaginase), [0268] 44% compared to MGL (IC50 control for MGL), [0269] 43% compared to enzyme association with L-asparaginase added in first at IC50 dose.

IV. Conclusion

[0270] Sequential enzyme association demonstrated that cell mortality could be increased with an addition of MGL at IC50 dose followed 4 days later by the addition of L-asparaginase at IC50 dose.

[0271] We can hypothesize that Met deprivation induced by MGL enzyme activity makes NCI-N87 gastric cells more sensitive to L-asparaginase activity. Moreover, the roles of L-asparaginase and MGL have to be discussed considering their known respective effect.

[0272] Indeed, L-asparaginase is known to trigger apoptosis in leukaemia cells (Ueno et al., 1997), therefore, it could probably plays a role of cytotoxic agent. MGL being known for blocking cell division in S or G2 phase of the cell cycle probably acts more as a cytostatic agent.

EXAMPLE 8

I. Additional Abbreviations

[0273] F12K: Kaighn's modification of ham's F-12

II. Operating Conditions

II.1 Test Item

II.1.1. L-Asparaginase

[0274] Description: Medac® (Germany), E. Coli L-asparaginase 10 000 IU.

[0275] One concentration of L-asparaginase (2.97 IU/mL) was prepared by serial dilutions in Dulbecco Phosphate Buffered Saline (DPBS) 1×. Concentration of L-asparaginase was diluted 11-fold to obtain final concentration of 0.27 IU/mL (IC50).

II.1.2. Methionine-γ-Lyase (MGL)

[0276] Description: P. Putida methionine-γ-lyase (MGL) produced in E. Coli.

[0277] One concentration of MGL (1.43 IU/mL) was prepared by serial dilutions in Dulbecco Phosphate Buffered Saline (DPBS) 1×. Concentration of MGL was diluted 11-fold to obtain final concentration of 0.13 IU/mL (IC50).

II.2 Cell Lines

II.2.1. Description

[0278] Name: AGS cell line
Description: Human gastric adenocarcinoma cell line (adherent)
Supplier and reference number: ATCC, CRL-1739

II.2.2. Culture Conditions

[0279] Cells were cultivated in a F12K media with L-glutamine supplemented with 10% (v/v) of foetal bovine serum, 100 IU/mL of penicillin and 100 μg/mL of streptomycin. Subculturing was performed according to PO-CELL-002 and PO-CELL-005.

II.2.3. Colorimetric Kit

Name: Cell Counting Kit-8 (CCK-8)

[0280] Supplier and reference number: Fluka 96992

[0281] Principle: the CCK-8 reagent contains a highly water-soluble tetrazolium salt WST-8. WST-8 is reduced by dehydrogenases in cells to give a yellow colored product (formazan) which is soluble in the tissue culture medium. The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. The colorimetric test was performed according to PO-CELL-004.

III. Cytotoxicity Assay

III.1 Method

[0282] One thousand cells in 100 μL/well were dispensed in 96-well flat bottom plates (cf. number of plates in raw data). In addition, two wells were filled with culture medium for blank control on each plate. All empty wells were filled with culture medium in order to minimize evaporation and condensation. On day 0 (D0), 10 μL of IC50 concentrations of L-asparaginase or MGL were added to the corresponding wells. Controls (blank wells and control plate) received 10 μL of DPBS 1×. On day 4 (D4), medium was removed from wells and replaced by fresh medium and 10 μL of DPBS 1× or 10 μL of IC50 concentrations of L-asparaginase (for cells previously incubated with MGL) or MGL (for cells previously incubated with L-asparaginase) added to the corresponding wells. Controls (blank and positive control) received 10 μL of DPBS 1×. Then, plates were incubated for 4 more days in the incubator. At the end of the incubation period (D8), 10 μL of CCK-8 solution were added to each well according to PO-CELL-004 and plates incubated for 4 hours. Optical density (OD) was then determined at 450 nm using a microplate reader.

III.2 Internal Controls

[0283] Controls were performed in duplicate.

III.2.1. Blank Wells

[0284] As above in Example 1.

III.2.2. Viability Control (Positive Control)

[0285] As positive control for the AGS cell line (100% cell viability), cells were cultivated in the culture medium (100 μL) without L-asparaginase nor MGL, but with 10 μL of the diluent (DPBS 1×).

III.3 Determination of Cell Viability

[0286] As above in Example 1.

IV. Results

IV.1 Internal Control

[0287] Internal controls were acceptable when it was not specified in raw data.

IV.2 IC50 Calculations with L-Asparaginase or MGL Alone

[0288] Percentages of cell viability with drug alone (MGL or L-asparaginase) were controlled in each experiment of drugs combination. Fifty percent of cell viability are expected at half of the test (D4) because IC50 value used here for enzymes were previously validated in single treatment at D4.

IV.2.1. Sequential Addition of L-Asparaginase and MGL

[0289] The experiment with sequential treatment of L-asparaginase and MGL was done twice with duplicate data. All quality controls (blank and positive control) were accepted in experiments.

[0290] Details of % of cell viability calculations and graphical representation are presented below in table 8 and FIG. 3.

TABLE-US-00008 TABLE 8 % of cell viability for controls and enzyme association % cell viability at D8 Mean SD Cells alone 100 4 Cells + IC50 L-aspa D0 101 1 Cells + IC50 MGL D0 106 1 Cells + IC50 L-aspa D0 + IC50 MGL D3 88 2 Cells + IC50 MGL D0 + IC50 L-aspa D3 79 6

[0291] Results indicated that enzyme association with MGL added at IC50 dose before the addition of L-asparaginase at IC50 dose (cf. FIG. 3) permitted to reduce cell viability of: [0292] 22% compared to IC50 L-asparaginase (IC50 control for L-asparaginase), [0293] 26% compared to MGL (IC50 control for MGL), [0294] 10% compared to enzyme association with L-asparaginase added in first at IC50 dose.

[0295] Moreover, for precision here, IC50 control for L-asparaginase or MGL (used and validated initially at D4) returned to 100% of cell viability after 8 days of culture with renewal of media at D4/half of the test. Indeed, remaining viable cells at D4 could re-growth with addition of “fresh” nutrients. Results were conform for IC50 controls (enzyme alone) reaching 50% of cell viability at D4.

V. Conclusion

[0296] Sequential enzyme association demonstrated that cell mortality could be increased with an addition of MGL at IC50 dose followed 4 days later by the addition of L-asparaginase at IC50 dose.

[0297] We can hypothesize that Met deprivation induced by MGL enzyme activity makes AGS gastric cells more sensitive to L-asparaginase activity. Moreover, the roles of L-asparaginase and MGL have to be discussed considering their known respective effect. Indeed, L-asparaginase is known to trigger apoptosis in leukaemia cells (Ueno et al., 1997), therefore, it could probably plays a role of cytotoxic agent. MGL being known for blocking cell division in S or G2 phase of the cell cycle probably acts more as a cytostatic agent.

EXAMPLE 9

I. Additional Abbreviations

[0298] A.M.: Ante meridiem
ERY-ASP: L-asparaginase encapsulated into red blood cells
ERY-MET: Methionine gamma-lyase encapsulated into red blood cells
IG: Intragastric injection (gavage)
IU: International Unit corresponding to pmol/min

IV: Intravenous

[0299] ND: Not determined

PN: Pyridoxine

[0300] TGI: Tumor growth inhibition

II. Objective of In Vivo Study

[0301] The objective of this study is to determine if combination of methioninase-loaded erythrocytes (ERY-MET) with L-asparaginase-loaded erythrocytes (ERY-ASP) can improve the antitumor activity observed with ERY-MET alone in a NCI-N87 gastric tumor subcutaneous xenograft mouse model.

III. Operating Conditions

[0302] NCI-N87 cells were cultivated at ERYTECH Pharma and prepared at 5.10.sup.7 cells/mL in DPBS 1× for injection. Four groups of 10 or 12 female NMRI nude mice (groups 1, 2, 3 and 4) were subcutaneously injected with the cell line at the fixed concentration of 5.10.sup.6/100 μL. ERY-MET and ERY-ASP injections were administrated (I.V. route) respectively at 108 IU/kg (8 mL/kg) and 200 IU/kg (4-5.4 mL/kg). Group 2 received 3 injections of ERY-MET on days 7, 14 and 21. Group 3 (ERY-ASP/ERY-MET) received 1 injection of ERY-ASP on day 7 and then, 2 injections of ERY-MET on days 21 and 28. Group 4 (ERY-MET/ERY-ASP) received 2 injections of ERY-MET on days 7 and 14 and then 1 injection of ERY-ASP on day 21. Group 1 was administered with the preservative solution of ERY-MET (SAG mannitol/plasma) at 8 mL/kg on days 7, 14 and 21.

[0303] Oral administrations (gavage) of PN co-factor was performed 6 hours after each ERY-MET injection (Day 7+6 h, Day 15+6 h, Day 21+6 h for group 2; Day 21+6 h, Day 28+6 h for group 3; Day 7+6 h, Day 15+6 h for group 4) and once a day (A.M.) for the other days (without ERY-MET administration) until Day 20 (for group 4), Day 27 (for group 2) or Day 34 (for group 3).

IV. Results

[0304] Tumor volume regression associated to ERY-MET/ERY-ASP combination appeared different to this observed for ERY-MET arm; indeed at D37, mice ERY-MET displayed a mean tumor volume of 298.3±36.2 mm.sup.3 and mice ERY-MET/ERY-ASP displayed a mean tumor volume of 189.7±29.8 mm.sup.3 corresponding to respectively 37% and 57% of mean tumor volume reduction while mice given vehicle (control) had a mean tumor volume of 441.5±56.6 mm.sup.3. Percentage of tumor growth inhibition (TGI*) were calculated for the enzyme association ERY-MET/ERY-ASP vs control (vehicle group) or vs ERY-MET group according to the following formula:

[00002]$100-\left(\frac{\mathrm{Tumor}{\mathrm{Volume}}_{\mathrm{enzyme}\mathrm{association}}\mathrm{at}\mathrm{Day}X}{\mathrm{Tumor}{\mathrm{Volume}}_{\mathrm{vehicle}\mathrm{or}\mathrm{ERY}-\mathrm{MET}\mathrm{alone}}\mathrm{at}\mathrm{Day}X}\times 100\right)$

[0305] Results are presented below in the table 9 below:

TABLE-US-00009 TABLE 9 TGI calculations for the association ERY-MET/ERY-ASP % TGI for ERY-MET/ ERY-ASP treatment vs control vs ERY-MET alone Day 7 ND** ND** Day 20 41% 33% Day 37 57% 36% **Not determined (not relevant) due to low volume measure disparity at the beginning of the study (D7 is the first time point of tumor volume measure).

[0306] In order to assess significance between groups and efficiency of ERY-MET/ERY-ASP treatment compared to ERY-MET alone on NCI-N87 gastric tumors, a two-way ANOVA test was performed with GraphPad Prism software (version 5.04) on tumor growth measures. Analysis comparing vehicle (control), ERY-MET and ERY-MET/ERY-ASP treatment indicating significance between groups at D37 with a P value inferior to 0.0001 (cf. FIG. 4) revealing efficacy of the combination ERY-MET/ERY-ASP 16 days after last injections for treatment against gastric tumors. With the reverse scheme of administration ERY-ASP/ERY-MET treatment compared to ERY-MET alone on NCI-N87 gastric tumors, two-way ANOVA test (cf. FIG. 4) revealed no significance between groups for three time points of follow-up (D7/D20/D37) with a P value>0.05.

V. Conclusion

[0307] ERY-MET was combined to ERY-ASP with 2 scheme of administrations: 1-ERY-ASP (D7)-ERY-MET (D21/D28) and 2-ERY-MET (D7/D15)-ERY-ASP (D21). Positive response compared to ERY-MET alone seems to appear when ERY-MET was administrated (twice) before ERY-ASP. This significance of result is supported by the obtaining of a P value inferior to 0.0001 at D37 on individual tumor volume measure.