Generation of anti-complement "prodrugs": cleavable reagents for specific delivery of complement regulators to disease sites.
Expression of biologically active molecules as fusion proteins with antibody Fc can substantially extend the plasma half-life of the active agent but may also influence function. We have previously generated a number of fusion proteins comprising a complement regulator coupled to Fc and shown that the hybrid molecule has a long plasma half-life and retains biological activity. However, several of the fusion proteins generated had substantially reduced biological activity when compared with the native regulator or regulator released from the Fc following papain cleavage. We have taken advantage of this finding to engineer a prodrug with low complement regulatory activity that is cleaved at sites of inflammation to release active regulator. Two model prodrugs, comprising, respectively, the four short consensus repeats of human decay accelerating factor (CD55) linked to IgG4 Fc and the three NH2-terminal short consensus repeats of human decay accelerating factor linked to IgG2 Fc have been developed. In each, specific cleavage sites for matrix metalloproteinases and/or aggrecanases have been incorporated between the complement regulator and the Fc. These prodrugs have markedly decreased complement inhibitory activity when compared with the parent regulator in vitro. Exposure of the prodrugs to the relevant enzymes, either purified, or in supernatants of cytokine-stimulated chondrocytes or in synovial fluid, efficiently cleaved the prodrug, releasing active regulator. Such agents, having negligible systemic effects but active at sites of inflammation, represent a paradigm for the next generation of anti-C therapeutics.
Harris
Claire L
CL
Department of Medical Biochemistry and Immunology, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, United Kingdom. HarrisCL@cardiff.ac.uk
Hughes
Clare E
CE
Williams
Anwen S
AS
Goodfellow
Ian
I
Evans
David J
DJ
Caterson
Bruce
B
Morgan
B Paul
BP
D051722:ADAM Proteins; D000071121:ADAMTS4 Protein; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D018958:CD55 Antigens / Q000096:biosynthesis; D016466:CHO Cells; D019902:Chondrocytes / Q000378:metabolism; D003165:Complement System Proteins / Q000378:metabolism; D006224:Cricetinae; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry; D005822:Genetic Vectors; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D011348:Procollagen N-Endopeptidase; D011355:Prodrugs / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection
D018958:CD55 Antigens; D016207:Cytokines; D007074:Immunoglobulin G; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D003165:Complement System Proteins; D010450:Endopeptidases; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C083641:aggrecanase