In vitro and in vivo inhibition of complement activity by a single-chain Fv fragment recognizing human C5.
Complement activation has been implicated in the pathogenesis of several human diseases. Recently, a monoclonal antibody, (N19-8) that recognizes the human complement protein C5 has been shown to effectively block the cleavage of C5 into C5a and C5b, thereby blocking terminal complement activation. In this study, a recombinant N19-8 scFv antibody fragment was constructed from the N19-8 variable regions, and produced in both mammalian and bacterial cells. The N19-8 scFv bound human C5 and was as potent as the N19-8 monoclonal antibody at inhibiting human C5b-9-mediated hemolysis of chicken erythrocytes. In contrast, the N19-8 scFv only partially retained the ability of the N19-8 monoclonal antibody to inhibit C5a generation. To investigate the ability of the N19-8 scFv to inhibit complement-mediated tissue damage, complement-dependent myocardial injury was induced in isolated mouse hearts by perfusion with Krebs-Henseleit buffer containing 6% human plasma. The perfused hearts sustained extensive deposition of human C3 and C5b-9, resulting in increased coronary artery perfusion pressure, end-diastolic pressure, and a decrease in heart rate until the hearts ceased beating approximately 10 min after addition of plasma. Hearts treated with human plasma supplemented with either the N19-8 monoclonal antibody or the N19-8 scFv did not show any detectable changes in cardiac performance for at least 1 hr following the addition of plasma. Hearts treated with human plasma alone showed extensive deposition of C3 and C5b-9, while hearts treated with human plasma containing N19-8 scFv showed extensive deposition of C3, but no detectable deposition of C5b-9. Administration of a 100 mg bolus dose of N19-8 scFv to rhesus monkeys inhibited the serum hemolytic activity by at least 50% for up to 2 hr. Pharmacokinetic analysis of N19-8 scFv serum levels suggested a two-compartment model with a T1/2 alpha of 27 min. Together, these data suggest the recombinant N19-8 scFv is a potent inhibitor of the terminal complement cascade and may have potential in vivo applications where short duration inhibition of terminal complement activity is desirable.
Evans
M J
MJ
Department of Molecular Development, Alexion Pharmaceuticals, New Haven, CT 06511, USA.
Rollins
S A
SA
Wolff
D W
DW
Rother
R P
RP
Norin
A J
AJ
Therrien
D M
DM
Grijalva
G A
GA
Mueller
J P
JP
Nye
S H
SH
Squinto
S P
SP
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification* / Q000494:pharmacology; D001483:Base Sequence; D001794:Blood Pressure / Q000187:drug effects; D003167:Complement Activation / Q000187:drug effects; D003182:Complement C5 / Q000276:immunology* / Q000378:metabolism; D006339:Heart Rate / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology*; D008253:Macaca mulatta; D051379:Mice; D008969:Molecular Sequence Data; D009206:Myocardium / Q000276:immunology* / Q000473:pathology; D010477:Perfusion; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D003182:Complement C5; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins