Construction of proteolysis resistant human interleukin-2 by fusion to its protective single chain antibody.
Interleukin-2 (IL-2) is a 15 kDa cytokine secreted by T-cells. Consequence to its natural function as locally secreted short-term messenger, its half-life in circulation is short and provided mainly by fast renal clearance due to its low molecular weight and its proteolytic susceptibility. These are common characteristics for most cytokines, resulting in low clinical utility. In this study we report the construction of an IL-2 fusion-protein comprising a protective anti-hIL-2 single chain antibody that was selected from a phage display library and the hIL-2. This IL-2 fusion-protein is fully human and resistant to inactivation by the ubiquitous lysosomal protease-cathepsin D, which is implicated in the in vivo inactivation of IL-2. In contrast, the native IL-2 lost practically all of its activity under these conditions. This resistance is due to the interaction of the single chain domain with its epitope on IL-2 thus masking possible cleavage sites. We suggest that this 45 kDa proteolysis resistant IL-2 fusion-protein upon further increase of its molecular weight by common fusion techniques to at least 75 kDa will exhibit significantly longer half-life in vivo and a higher clinical utility than either the native IL-2 or any of its reported long T/2 derivatives.
Klimka
Alexander
A
Graduate Program in Biology, York University, Ontario, Toronto, Canada.
Yu
Nelson
N
Shami
Ezekiel Y
EY
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002402:Cathepsin D / Q000378:metabolism*; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D007376:Interleukin-2 / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
D000906:Antibodies; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D002402:Cathepsin D