Mechanistic Studies of the Effects of Anti-factor H Antibodies on Complement-mediated Lysis

Michael J Corey,Robert J Kinders,Cristina M Poduje,Connie L Bruce,Halli Rowley,Lisha G Brown,G.Michael Hass,Robert L Vessella

doi:10.1074/jbc.275.17.12917

Abstract

We have recently reported that complement factor H, a negative regulator of complement-mediated cytotoxicity, is produced and secreted by most bladder cancers. This observation was exploited in the development of the BTA stat and BTA TRAK diagnostic assays, both of which make use of two factor H-specific monoclonal antibodies in sandwich format. Here we show that both antibodies exert interesting effects on the biochemistry of complement activation in in vitro systems. Antibody X13.2 competes with C3b for association with factor H and strongly inhibits factor H/factor I-mediated cleavage of C3b, thereby evidently inactivating a negative regulator of complement; yet, the antibody strongly inhibits complement-mediated lysis as well. Conversely, antibody X52. 1, which does not compete with C3b and has no effect on solution-phase cleavage of C3b, is capable of enhancing complement-mediated lysis of various cell types, including cancer cells, by over 10-fold. Our observations indicate that it is possible to deconvolute the biochemical roles of factor H in complement by means of appropriate inhibitors, a finding with potentially valuable implications for both basic research and cancer therapy.

Highlights

Factor H, a soluble negative regulator of the complement system, is produced and secreted by most transitional cell carcinomas of the bladder [1,2,3]
Antibody Specificity—EIAs performed in the process of screening the antibody panels showed that monoclonal antibodies (mAbs) X46.3 was specific for the C3 ␣ chain and exhibited no detectable crossreactivity with factor H (FH)
Mapping of Sites of Antibody Association—According to data from Western blots, mAb X52.1 associated with the 120-kDa and 48-kDa fragments of trypsinized FH, consistent with assignment of its primary site of association to SCR domains 15–20 of FH. mAb X13.2 associated with the same fragments, and with the 38-kDa fragment reported to contain SCRs 1–5, as well as the factor I cofactor activity of FH [28]

Summary

EXPERIMENTAL PROCEDURES

Materials—Human complement serum, factor B-depleted human serum, zymosan, sensitized sheep erythrocytes, other miscellaneous chemicals, and ATP assay reagent (50 mM Tricine, 10 mM MgSO4, 1 mM EDTA, 100 ␮M dithiothreitol, 1 mg/ml bovine serum albumin, 66 ␮g/ml luciferase, 590 ␮M luciferin as reconstituted) were purchased from Sigma. A typical APC assay contained 1/20 volume of complement serum, 6 mM EGTA, 4 mM MgCl2, and mAbs diluted in GVB to final volume before addition of erythrocytes, which were added in 0.5 volume. The lowest concentrations of coating antibodies that yielded a reliable signal were used to minimize ligand depletion Such measurements cannot be assumed to yield accurate dissociation constants for the solution-phase complexes, for several reasons: 1) the alkaline phosphatase conjugate may not bind as well as the free molecule; 2) the dissociation constants of the two antibodies are convoluted in the result, since dissociation of either the X13.2-FH or X52.1-FH complex leads to loss of signal in each case; 3) the antigen immobilized in a microtiter well and “displayed” by a coating antibody may be sterically hindered from forming the strongest possible association with the detection antibody.

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RESULTS

TABLE II Summary of hemolysis results and control procedures

Effects of mAbs

DISCUSSION