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 sever al human diseases. Recently, a monoclonal antibody (N19-8) that recogn izes the human complement protein C5 has been shown to effectively blo ck the cleavage of C5 into C5a and C5b, thereby blocking terminal comp lement activation. In this study, a recombinant N19-8 scFv antibody fr agment was constructed from the N19-8 variable regions, and produced i n both mammalian and bacterial cells. The N19-8 scFv bound human C5 an d was as potent as the N19-8 monoclonal antibody at inhibiting human C 5b-9-mediated hemolysis of chicken erythrocytes. In contrast, the N19- 8 scFv only partially retained the ability of the N19-8 monoclonal ant ibody to inhibit C5a generation. To investigate the ability of the N19 -8 scFv to inhibit complement-mediated tissue damage, complement-depen dent myocardial injury was induced in isolated mouse hearts by perfusi on with Krebs-Henseleit buffer containing 6% human plasma. The perfuse d hearts sustained extensive deposition of human C3 and C5b-9, resulti ng in increased coronary artery perfusion pressure, end-diastolic pres sure, and a decrease in heart rate until the hearts ceased beating app roximately 10 min after the addition of plasma. Hearts treated with hu man plasma supplemented with either the N19-8 monoclonal antibody or t he N19-8 scFv did not show any detectable changes in cardiac performan ce for at least 1 hr following the addition of plasma. Hearts treated with human plasma alone showed extensive deposition of C3 and C5b-9, w hile hearts treated with human plasma containing the N19-8 scFv showed extensive deposition of C3, but no detectable deposition of C5b-9. Ad ministration of a 100 mg bolus dose of N19-8 scFv to rhesus monkeys in hibited the serum hemolytic activity by at least 50% for up to 2 hr. P harmacokinetic analysis of N19-8 scFv serum levels suggested a two-com partment model with a T(1/2)alpha of 27 min. Together, these data sugg est the recombinant N19-8 scFv is a potent inhibitor of the terminal c omplement cascade and may have potential in vivo applications where sh ort duration inhibition of terminal complement activity is desirable.