Pre-neutralization of C5a-mediated effects by the monoclonal antibody 137-26 reacting with the C5a moiety of native C5 without preventing C5 cleavage

Abstract

Complement C5a is aetiologically linked to inflammatory tissue damage in conditions like septicaemia, immune complex diseases and ischaemia-reperfusion injury. We here describe a monoclonal antibody (mAb), 137-26, that binds to the C5a moiety of human C5 and neutralizes the effects of C5a without interfering with C5 cleavage and the subsequent formation of lytic C5b-9 complex. Mouse anti-human C5 mAbs were generated and the reactivity with C5 and C5a was detected by ELISA and surface plasmon resonance. The inhibition of C5a binding to C5a receptor was studied using a radioligand binding assay. The effects of the antibody on C5a functions were examined using isolated neutrophils and a novel human whole blood model of inflammation. Haemolytic assays were used to study the effect on complement-mediated lysis. mAb 137-26 reacted with both solid- and solution-phase C5 and C5a in a dose-dependent manner with high affinity. The antibody competed C5a binding to C5a receptor and inhibited C5a-mediated chemotaxis of neutrophils. Furthermore, the antibody effectively abrogated complement-dependent E. coli-induced CD11b up-regulation and oxidative burst in neutrophils of human whole blood. mAb 137-26 was more potent than a C5a receptor antagonist and a previously described anti-C5a antibody. mAb 137-26 did not inhibit complement-mediated lysis, nor did it activate complement itself. Together, mAb 137-26 binds both the C5a moiety of native C5 and free C5a, thereby effectively neutralizing the biological effects of C5a. The antibody may have therapeutic potential in inflammatory diseases where C5a inhibition combined with an operative lytic pathway of C5b-9 is particularly desired.

Fig. 1

Reactivity of mAb 137–26 with (a) human C5 and (b) human C5a in ELISA. mAb 137–26 (•) reacts with both C5 and C5a, whereas the C5 β-chain specific antibody, mAb 137–76 (○), reacts with C5 only. Isotype-matched control mAb G3-519 (□) does not react with C5 and C5a.

Fig. 2

Effects of mAb 137–26 on complement-mediated haemolysis (a) via the classical pathway in sensitized chicken red blood cells and (b) the alternative pathway in unsensitized rabbit red blood cells. mAb 137–26 (•) does not inhibit haemolysis via either the classical or the alternative complement pathway. In contrast, the anti-C2 mAb 175–62 (□, panel a) inhibits haemolysis via the classical pathway and the anti-factor D mAb 166–32 (□, panel b) inhibits haemolysis via the alternative pathway. Isotype-matched control mAb G3-519 (○) has no effect.

Fig. 3

Inhibition of C5a binding to C5aR on human neutrophils by mAb 137–26. ¹²⁵I-human C5a (0·4 nm) were incubated with purified human neutrophils, in the presence or absence of varying concentrations of the inhibitors, mAb 137–26 (•) or recombinant human C5a (□). The concentration for 50% inhibition (IC₅₀) for mAb 137–26 and C5a are 0·45 and 30 nm, respectively. Isotype-matched control mAb G3-519 (○) has no effect.

Fig. 4

Inhibition of C5a-induced chemotaxis of human neutrophils by mAb 137–26. C5a at 1 nm was used to induce chemotaxis of human neutrophils. Varying concentrations of mAb 137–26 were added for blocking of C5a. The degree of neutrophil migration was represented by the optical density of the formazan product as described in the text.

Fig. 5

Relative potency of inhibition by mAb 137–26 and C5aR antagonist AcF-[OPdChaWR] in C5a-induced chemotaxis of human neutrophils. C5a at 1 nm was used to induce chemotaxis of human neutrophils. Chemotaxis of neutrophils in the presence (▵) or absence of C5a (○) was used as control. Varying concentrations of mAb 137–26 (•) and AcF-[OPdChaWR] (▪) were added for blocking C5a. The degree of neutrophil migration was represented by the optical density of the formazan product described in the text.

Fig. 6

Inhibition of (a) CD11b expression and (b) oxidative burst by anti-C5 mAbs in human whole blood. E. coli was incubated in lepirudin-anticoagulated whole human blood at 1 × 10⁷ bacteria/ml for CD11b expression and 1 × 10⁸ bacteria/ml for oxidative burst. Neutrophils and monocytes were separately gated and examined and the neutrophil results presented. mAb 137–26 (•) abrogated very efficiently CD11b expression with a substantial higher potency than the anti-C5 mAb 137–30 (▴) and the anti-C5a mAb 561 (▪), whereas the isotype-matched control mAb G3-519 (▾) had no effect. The effect of mAb 137–26 on oxidative burst was intermediate between mAbs 137–30 and 561. T-0 = baseline sample (□). T-10 = whole blood incubated only with PBS (○). Pos. ctr. = whole blood incubated with E. coli (open triangle ▵). The experiment was repeated four times with identical results.

Fig. 7

Inhibition of (a) CD11b expression and (b) oxidative burst by mAb 137–26 and the C5aR antagonist AcF-[OPdChaWR] in human whole blood. The experiment was performed as described in Fig. 6. Both mAb 137–26 (•) and the C5aR antagonist (▪) inhibited neutrophil CD11b expression and oxidative burst efficiently, but mAb 137–26 was substantially more potent, particularly in inhibition of CD11b expression. Irrelevant control peptide (▾) has no effect. Other symbols are as described in Fig. 6.

Fig. 8

Complement activation potential of mAb 137–26. A possible complement activation capacity of mAb 137–26 (•) was investigated by incubating with human serum at 37°C for 1 h. The isotype-matched control mAb G3-519 (▾) was used as a negative control and heat-aggregated immunoglobulins HAIGG (▪) as a positive control. Detection of the complement activation products (a) C1rs-C1Inhibitor complexes, (b) C4bc, (c) C3bc and (d) TCC revealed a dose-dependent classical complement activation by HAIGG, whereas mAb 137–26 and mAb G3-519 did not activate complement. T-0, baseline serum sample (□); T-60, serum incubated with PBS for 60 min (○). The experiment was repeated once with identical results.