Method to prepare therapeutically active aldesleukin highly stable in liquid pharmaceutical compositions

Abstract

The invention relates to a liquid pharmaceutical composition of aldesleukin/SDS aggregates and its use in the treatment of auto-immune disease, inflammatory disorders and cancer. A method for preparing said composition is also described.

What is claimed: 
     
         1 . A method of producing aldesleukin suitable for the preparation of a composition comprising aldesleukin in a pharmaceutical aqueous vehicle, the method comprising:
 fermenting a bacterial cell transfected with an expression vector encoding an aldesleukin gene;   disrupting the bacterial cell;   collecting inclusion bodies containing aldesleukin;   subjecting the inclusion bodies to dissolution by a detergent followed by oxidation and a first chromatography;   diluting with an organic nitrile and subjecting to a second chromatography;   subjecting the aldesleukin to diafiltration and sterilization.   
     
     
         2 . The method of  claim 1 , wherein the bacterial cell is an  Escherichia coli  bacterial cell. 
     
     
         3 . The method of  claim 1 , wherein the detergent is sodium dodecyl sulfate (SDS). 
     
     
         4 . The method of  claim 1 , wherein the oxidation is conducted with an oxidizing compound. 
     
     
         5 . The method of  claim 1 , wherein the first chromatography is a ceramic hydroxyapatite chromatography. 
     
     
         6 . The method of  claim 1 , wherein the second chromatography is a high-performance liquid chromatography (HPLC). 
     
     
         7 . The method of  claim 6 , wherein the HPLC is C4 column HPLC chromatography. 
     
     
         8 . The method of  claim 1 , wherein the aldesleukin remains stable in the pharmaceutical liquid composition for at least a year as measured by an in vitro biological activity assay. 
     
     
         9 . The method of  claim 1 , wherein the aldesleukin has a stable biological activity, at least for a year in the liquid composition, as measured in HT-2 cell culture. 
     
     
         10 . The method of  claim 1 , wherein the aldesleukin is biologically active to be used in any cell therapy procedure involving cells expressing IL-2 receptors in their membranes. 
     
     
         11 . The method of  claim 1 , wherein the aldesleukin is biologically active to be used in cell therapies involving NK and T cells in cell culture and any modified NK and T cells expressing IL-2 receptors in their membranes. 
     
     
         12 . A pharmaceutical composition containing the aldesleukin obtained by the method of  claim 1  in which the aldesleukin remains stable in the pharmaceutical liquid composition for at least a year as measured by an in vitro biological activity assay. 
     
     
         13 . The pharmaceutical composition of  claim 12 , wherein the aldesleukin has a stable aldesleukin/SDS aggregate distribution in the liquid composition at least for a year in the range of 4-18 nm range with a peak at about 8 nm as measure by dynamic light scattering. 
     
     
         14 . The pharmaceutical composition of  claim 12 , wherein the aldesleukin has a stable therapeutic activity, at least for a year in the liquid composition, as measured in an animal model of human cancer. 
     
     
         15 . The pharmaceutical composition of  claim 12 , wherein the aldesleukin is active for use in cell therapies involving cells expressing IL-2 receptor in their membrane. 
     
     
         16 . The pharmaceutical composition of  claim 15 , wherein the aldesleukin amplifies human regulatory T cells, CAR-T cells, CAR-NK cells, cytotoxic T cells, cells comprising IL-2 receptors in a cell culture. 
     
     
         17 . A pharmaceutical composition comprising: SEQ ID NO: 1, in a range from about 0.001 mg/ml to about 3 mg/ml, sodium dodecyl sulfate (SDS) in a range from about 0.05 mg/ml to about 20 mg/ml, osmolytes, a pH 7-8 phosphate buffer or combinations thereof. 
     
     
         18 . The pharmaceutical composition of  claim 17 , wherein the osmolytes comprise sodium chloride, mannitol, sorbitol, xylitol or combinations thereof. 
     
     
         19 . A pharmaceutical composition consisting of SEQ ID NO: 1, in a range from about 0.001 mg/ml to about 3 mg/ml, sodium dodecyl sulfate (SDS) in a range from about 0.05 mg/ml to about 20 mg/ml, osmolytes and a pH 7-8 phosphate buffer. 
     
     
         20 . The pharmaceutical composition of  claim 19 , wherein the osmolytes comprise sodium chloride, mannitol, sorbitol, xylitol or combinations thereof.