Eur. J. Immunol. 1987.17: 303-304 Effect of monoclonal antibodies to Bb on C3b.Bb 303

Short paper

Zvi Fishelson' and Hans J. Muller-Eberhard'

Department of Chemical Immunology, The Weizmann Institute of Science', Rehovot and Division of Molecular Immunology, Research Institute of Scripps Clinic', La Jolla

1 Introduction

C3 convertase of the human alternative pathway of complement: modulation of enzyme activity and stability by mouse monoclonal antibodies to Bb*

To further characterize functional sites on C3b,Bb, the C3 convertase of the alterna- tive pathway of complement, we examined the effect of four monoclonal antibodies on its activity and stability. These antibodies recognized antigenic epitopes on human Bb which were not fully exposed in intact factor B. Three of the monoclonal anti- bodies inhibited lysis of rabbit erythrocytes by normal human serum in the presence of Mg2+ and EGTA. Two of these antibodies markedly inhibited the activity of purified C3b,Bb deposited on rabbit erythrocytes. However, all four monoclonal antibodies increased the half-life of the C3 convertase. Thus, these results demon- strate that binding of antibodies to Bb may concomitantly stabilize C3b,Bb and abate its activity. It is likely that such antibodies induce in Bb conformational changes which increase the C3b,Bb complex stability but may also hinder its catalytic site.

The C3 convertase of the alternative pathway of complement, C3b ,Bb, is a Mg2+-dependent labile protein complex (reviewed in [l]). This enzyme has a central role in pathway activation and in deposition of the membrane attack complex on target cells [I]. Employing mouse monoclonal antibodies (mAb) to C3b [2-41 contributed to understanding of the interaction between C3b and other complement proteins. In this study, we examined the effects of four mouse mAb raised against purified Bb on activity and stability of C3b,Bb. As demonstrated below, three of these antibodies inhibited C3b,Bb activity and reduced activation of the human alterna- tive pathway by rabbit erythrocytes (ER), while stabilizing the C3b,Bb complex, suggesting that they exert an allosteric effect on Bb in the complex.

2 Materials and methods

The mouse mAb designated 1ODll-11, 10Dll-F12, 10Dll- 1F12 and 15-6-19-1 were generated as previously described [5] and the IgG fractions were isolated using a protein A-Sepha- rose column [3]. Antibody specificity was tested by enzyme- linked immunosorbent assay on purified factor B or Bb [6] or on normal human plasma containing 10 mM EDTA. The plasma-EDTA was diluted to give the same concentration of factor B assuming a concentration of 0.2 mg/ml factor B in plasma. The two mAb 10Dll-F12 and 15-6-19-1 bind more strongly to Bb than to B (20 times and 7 times, respectively, after correction for the difference in molecular weights of Bb and B; not shown). A preferential binding to Bb over factor B was also observed with antibodies 10Dll-11 and 10Dll-1F12. The antibody titers of 1ODll-11, 10Dll-F12 and 10Dll-1F12 were similar and the titer of 15-6-19-1 fourfold lower.

[I 58861 ~~~~~ ~ ~~

* These results were presented in part at the 11th International Com-

Correspondence: Zvi Fishelson, Department of Chemical Immu- nology, The Weizmann Institute of Science, Rehovot 76100, Israel

Abbreviations: ER: Rabbit erythrocytes GVB: Veronal-buffered saline containing 0.1% gelatin GVBE: GVB containing 10 mM EDTA mAb: Monoclonal antibody(ies)

plement Workshop, November 1985, Miami, FL.

3 Results

The effect of the mAb on alternative pathway activation was first tested.ER were incubated with normal human serum con- taining 2.5 mM Mg2+ and 10 mM EGTA together with increas- ing mouse mAb. The three antibodies 1ODll-11, 10Dll-F12 and 10Dll-1F12 inhibited complement activation and lysis of E R (Fig. l), though with different efficiencies. The fourth anti- body, 15-6-19-1, enhanced lysis of E R under the same condi- tions (not shown). The reason for this enhancement is not known. Inhibition of alternative pathway activation may have resulted from a reduced C3b,Bb activity effected by the anti- bodies, as is shown in Fig. 2. ER carrying C3b,Bb was pre- pared and enzyme activity was measured using guinea pig serum-EDTA [7]. The mAb were added to the preformed C3b,Bb. Two antibodies, 10Dll-11 and 10Dll-F12, markedly inhibited the activity of the preformed enzyme (Fig. 2). 10Dll-1F12 induced a partial inhibition and 15-6-19-1 at low concentration slightly enhanced C3b,Bb activity. To deter- mine whether the antibodies effected the stability of C3b,Bb the following experiment was performed. Factor B was radiolabeled with 1251 (Amersham Corp., Arlington Heights, IL) by the Iodogen technique [8]. C3b,'251-Bb(Mg2+) was

80 !-

0 ' 1 I I I I I

0 10 20 30 40 50 60

Figure 1. Effect of mAb to Bb on lysis of ER by normal human serum- Mg2+ EGTA. Ten million ER were incubated with normal human serum diluted 1 : 5 in 0.1 ml GVB (see Abbreviations) containing 2.5 mM Mg2+, 10 mM EGTA and different concentrations of mAb. After 20 min at 37 "C, 1 ml GVBE was added and the percent cell lysis was measured [7].

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1987 001 4-298018710202-0303$02.5010

304 Z. Fishelson and H. J. Muller-Eberhard Eur. J. Immunol. 1987.17: 303-304

0 1 I I I I I I I I 0 2 4 6 8 10 1 2 1 4 1 6

Em32 Kl IgG

Figure 2. Effect of mAb to Bb on C3b,Bb activity. Ten million ER with cell-bound C3b,Bb were incubated in 0.1 ml GVBE with differ- ent concentrations of mAb for 30 min at 4°C. One ml guinea pig serum diluted 1 : 50 in GVBE was then added and the cells were further incubated for 15 min at 37°C. Percent cell lysis was deter- mined and enzyme activity was calculated as percent of control lysis in absence of mAb.

1 0 " ' I " I , , ,

0 5 1 0 1 5 2 0 25 30 5 10 1 5 20 25 30

Minutes at 2 4 T

Figure 3. Effect of mAb to Bb on stability of C3b,Bb. C3b,Bb was generated with 1251-labeled factor B, factor D and Mg2+ and deposited on ER carrying C3b. The cells were washed and then 2 X lo7 E, carry- ing C3b,'ZSI-labeled Bb(Mg2+) were mixed with different concentra- tions of mAb and incubated at 24°C. At different times, duplicate samples were removed and the quantity of cell-bound enzyme was determined [7]. Results are expressed as percent residual cell-bound C3b,'251-labeled Bb(Mg2+) relative to time zero.

deposited on ER and then incubated either with or without the mAb at 24°C. The percent residual '251-labeled protein on the cell was determined at different time points by centrifuging the cells through 20% sucrose as previously described [7]. Upon addition of the mAb lODll-ll,10Dll-F12,10Dll-1F12 or 15- 6-19-1, the half life of C3b,Bb at 24°C increased from 10-11 min to 23,22, 16 or 14 min, respectively (Fig. 3). Thus, by binding to C3b,Bb these antibodies stabilized the protein complex.

4 Discussion

These results support the previous suggestion [5] that the anti- bodies are directed to a conformational antigen on Bb which is partly cryptic in native factor B. They further indicate that by

binding to Bb in C3b,Bb the antibody may allosterically stabilize the complex. To date, stabilization of C3b,Bb has been observed with properdin and nephritic factor (reviewed in [l]) and with the metal ions Gd3+ [9] and NiZ+ [7]. Stabiliz- ing mAb may be most helpful for studies on the mechanism underlying the extreme lability of C3b,Bb. That these mAb also abrogated enzyme activity may indicate that the allosteric effect on Bb was accompanied by a conformational change around the proteolytic site of Bb which diminished its activity. Alternatively, the inhibitory effect of the antibodies could be due to steric hindrance.

Daha et al. [lo] described 2 IgM mAb directed against fac- tor B which stabilize. C3b,Bb and enhance C3 consumption. Unlike our IgG mAb, these IgM mAb bind to both factor B and to Bb. Hence, they probably recognize different epitopes on Bb. This may explain the contrasting results obtained when measuring their effect on C3b,Bb enzymatic activity. That IgM mAb could bind to Bb on C3b,Bb without interfering with its activity [lo] suggests that the inhibitory activity of the IgG mAb on enzyme activity (Fig. 2) is not due to simple steric hindrance. More recently, Ueda et al. [ l l ] reported an IgG mAb which inhibits the hemolytic activity of factor B and accelerates the decay of C3b,Bb. This report further under- scores the uniqueness of our mAb which are directed to neoantigens on Bb and which inhibit the convertase activity while stabilizing the C3b,Bb complex.

mAb to antigenic determinants on Bb may be useful for study- ing the interaction of the C3 convertase with the complement regulatory proteins factor H and properdin, with C5 and with cell surface receptors such as the decay-accelerating factor [12]. The mAb 10Dll-11 was used in a recent study to support the suggestion that isolated Bb has proteolytic and hemolytic activities [ 131.

Received November 21. 1986.

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