Arginine deiminase encapsulated inside erythrocytes and their use in treating cancer and arginase-1 deficiency

Abstract

The present invention is related to arginine deiminase encapsulated into erythrocytes for use in therapy. It is in particular related to the use thereof in treating arginase-1 deficiency. Also, it relates to novel pharmaceutical compositions comprising arginine deiminase from M. arginini encapsulated into erythrocytes and the use thereof in treating diseases that may benefit from arginine depletion, such as arginine dependent cancers, in particular arginine-auxotrophic cancers, and arginase-1 deficiency.

1 .- 14 . (canceled) 
     
     
         15 . A pharmaceutically acceptable suspension of erythrocytes encapsulating a therapeutically effective amount of a magnesium-independent arginine deiminase (ADI), wherein when administered to a patient in need thereof, the suspension of erythrocytes delivers to said patient a sustained reduction in blood arginine levels for at least 10 days post administration. 
     
     
         16 . The suspension of  claim 15 , further comprising a preservative solution for erythrocytes. 
     
     
         17 . The suspension of  claim 15 , wherein the ADI is a full length ADI, or a variant or fragment thereof retaining substantially the same enzymatic activity and magnesium independence as the ADI having the amino acid sequence as set forth in SEQ ID NO: 1. 
     
     
         18 . The suspension of  claim 15 , wherein the ADI comprises an amino acid sequence that is at least 80% identical to the amino acid sequence as set forth in sequence SEQ ID NO: 1. 
     
     
         19 . The suspension of  claim 18 , wherein the ADI comprises the amino acid sequence as set forth in SEQ ID NO: 1. 
     
     
         20 . The suspension of  claim 15 , wherein the concentration of the ADI enzymatic activity comprises about 1 to about 400 U/ml, about 5 to about 400 U/ml, or about 5 to about 350 U/ml, and/or wherein the amount of ADI comprises about 0.5 to about 6.5 mg/ml, about 1 to 6 mg/ml, about 1 to about 5 mg/ml, about 1 to about 4 mg/ml, about 1 to about 3 mg/ml, about 1.2 to about 2.8 mg/ml, about 1.4 to about 2.6 mg/ml, or about 1.6 to 2.4 about mg/ml. 
     
     
         21 . The suspension of  claim 15 , wherein extracellular hemoglobin levels do not exceed 0.2 g/dl for at least 72 hours when the suspension is maintained between 2 and 8° C. 
     
     
         22 . The suspension of  claim 15 , wherein less than 1% hemolysis occurs in 72 hours when the suspension is maintained between 2 and 8° C. 
     
     
         23 . The suspension of  claim 15 , having an osmolarity of between about 270 and about 350 mOsm/l. 
     
     
         24 . The suspension of  claim 15 , wherein the erythrocytes are human erythrocytes or are generated from human stem cells capable of becoming erythrocytes. 
     
     
         25 . The suspension of  claim 24 , wherein the stem cells have been transformed to express the magnesium-independent ADI. 
     
     
         26 . The suspension of  claim 15 , wherein the reduction in blood arginine levels provides a clinical benefit in the treatment and/or prevention of any one or all of the following: arginase-1 deficiency (A1D), arginine-dependent cancers, septic shock, and angiogenesis-related diseases. 
     
     
         27 . The suspension of  claim 15 , packaged as a single dose in a container suitable for blood transfusion, said dose having a volume of between about 150 ml to about 350 ml. 
     
     
         28 . The suspension of  claim 27 , wherein the dose comprises between about 50 to about 3500 units (U) of ADI enzymatic activity per kilogram of patient body weight. 
     
     
         29 . The suspension of  claim 15 , prepared according to a method comprising the following steps: 1) suspending erythrocytes in an isotonic solution; 2) exposing the suspension to a cooled hypotonic solution between +1 and +8° C., wherein the hypotonic solution and the suspension are separated by a dialysis membrane; 3) encapsulating the ADI at a temperature between +1 and +8° C.; and 4) resealing the erythrocytes in an isotonic or hypertonic solution at a temperature between about +30 and +42° C. 
     
     
         30 . The suspension of  claim 15 , prepared according to a method comprising the following steps: 1) suspending a pellet of erythrocytes in an isotonic solution at a hematocrit level equal to or greater than 65%, between +1 and +8° C.; 2) lysing the erythrocytes at a temperature between +1 and +8° C. by passing the suspension into a dialysis device and exposing the erythrocytes to hypotonic conditions; 3) encapsulating the erythrocytes by adding the ADI to the suspension before and/or during the lysing step, at a temperature between +1 and +8° C.; and 4) resealing the erythrocytes under isotonic or hypertonic conditions at a temperature between +30 and +42° C. 
     
     
         31 . The suspension of  claim 15 , prepared according to a method comprising the following steps: 1) encapsulating the ADI inside the erythrocytes by placing the erythrocytes into contact with a hypotonic medium, contacting the erythrocytes with the ADI, and resealing the erythrocytes; 2) preparing a suspension comprising the resealed erythrocytes of step 1 having an osmolality of between about 290 and about 330 mOsmol/kg; 3) incubating the suspension of step 2 at an osmolality of between about 290 and about 330 mOsmol/kg, for a period greater than or equal to 30 minutes; 4) removing the liquid medium of the incubated suspension of step 3; 5) suspending the erythrocytes obtained under step 4 into a solution suitable for administration of the suspension to a patient. 
     
     
         32 . The suspension of  claim 15 , wherein the suspension is a stabilized erythrocyte suspension. 
     
     
         33 . The suspension of  claim 15 , wherein the suspension is a ready-to-use stabilized erythrocyte suspension. 
     
     
         34 . The suspension of  claim 15 , wherein the ADI is co-factor independent.