Ž . Immunopharmacology 42 1999 209–218 Neutralization of complement regulatory proteins augments lysis of breast carcinoma cells targeted with rhumAb anti-HER2 Katrin Jurianz a , Sabine Maslak a , Helena Garcia-Schuler a , Zvi Fishelson b , ¨ Michael Kirschfink a, ) a Institute of Immunology, UniÕersity of Heidelberg, Im Neuenheimer Feld 305, Heidelberg 69120, Germany b Department of Cell Biology and Histology, Sackler School of Medicine, Tel AÕiÕ UniÕersity, Tel AÕiÕ, Israel Accepted 29 December 1998 Abstract HER2 Ž . The capacity of recombinant human monoclonal anti-p185 IgG rhumAb anti-HER2 to activate human complement was investigated. Complement activation by rhumAb anti-HER2 on various human breast carcinoma cell lines resulted in deposition of complement proteins on these cells. Complement activation was also observed in a solid-phase binding assay, in which purified p185 HER2 was immobilized onto a microtiter plate. rhumAb anti-HER2 induced some complement-media- ted tumor cell lysis by rabbit complement, but not by human complement. Analysis of membrane complement regulatory Ž . proteins mCRP on breast carcinoma cells revealed a heterogenous expression of CD46, CD55 and CD59. After blocking the mCRP activity with specific antibodies, rhumAb anti-HER2 induced about 15% lysis of p185 HER2 -expressing tumor cells. Tumor cell sensitization with rabbit polyclonal anti-tumor antiserum following mCRP neutralization, augmented cell lysis from 10 to 80%. Expression of mCRP was upregulated by treatment with PMA, and correlated with increased protection of the tumor cells from complement lysis. These results suggest that humanized antibodies like rhumAb anti-HER2 promote complement activation leading to tumor cell phagocytosis and cell-mediated cytotoxicity. They further demonstrate that a successful tumor immunotherapeutical approach, based on antibody and complement treatment, requires mCRP neutralization. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Complement resistance; Membrane regulatory proteins; Breast carcinoma; HER2; Humanized antibody AbbreÕiations: ADCC, antibody-dependent cellular cytotoxic- Ž . X X X Y ity; BATDA, bis acetoxymethyl 2,2 :6 ,2 -terpyridine-6,6 - dicarboxylic acid; DMSO, dimethyl sulfoxide; FACS, fluores- cence activated cell sorter; mAb, monoclonal antibody; MAC, membrane attack complex of complement; mCRP, membrane complement regulatory proteins; NHS, normal human serum; NRS, normal rabbit serum; PMA, phorbol myristate acetate; PO, peroxidase; rhumAb, recombinant humanized antibody ) Corresponding author. Tel.: q49-6221-564076; fax: q49- 6221-565586; e-mail: k92@ix.urz.uni-heidelberg.de 1. Introduction Clinical and experimental evidence supports a role of the HER2-protooncogene, encoding a 185- Ž HER2 . kDa transmembrane protein p185 , in various human malignancies, including breast carcinoma Ž . Slamon et al., 1988; Wright et al., 1989 . A mono- HER2 Ž clonal murine antibody to p185 mmoAb anti- . HER2 4D5 was developed, and was shown to 0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0162-3109 99 00006-5