[CANCER RESEARCH 58, 3051-3058. July 15, 1998] Evaluating Antibodies for Their Capacity to Induce Cell-mediated Lysis of Malignant B Cells1 Dieter Wiirflein, Michael Dechant, Bernhard Stockmeyer, Alison L. Tutt, Peisheng Hu, Roland Repp, Joachim R. Kalden, Jan G. J. van de Winkel, Alan L. Epstein, Thomas Valerius,2 Martin Glennie, and Martin Gramatzki Division of Hemalology/Oncology, Department of Medicine III, University of Erlangen-NUmherg. 91054 Erlangen. Germany ¡D.W.. M. D., B. S., K. R.. J. R. K., T. V., M. Gr.]; Department of Pathologv, University of Southern California. Los Angeles, California 90033-4526 ¡P.H., A. L. E.j: Medarex Europe and Department of Immunology. University Hospital, 3508 GA Utrecht, the Netherlands ¡J.G. J. v. d. W.J; anil Tenovus Research Laboratory, Universitv of Southampton. Southampton SOI6 6YD England {A. L T.. M. Gì.I ABSTRACT Promising results from clinical trials have led to renewed interest in effector mechanisms operating in antibody-based therapy of leukemia and lymphoma. We tested a panel of B-cell antibodies from the Sixth Human Leukocyte Differentiation Antigen workshop for their capacity to mediate antibody-dependent cellular cytotoxicity, often considered to be one of the most potent effector mechanisms in vivo. As effector cells, mononuclear cells and polymorphonuclear (PMN) cells from healthy donors were com pared with FcyRI (CD64)-expressing PMN cells from patients receiving granulocyte colony-stimulating factor (G-CSF) treatment. Of the 29 IgG workshop antibodies binding most strongly to the tested malignant human B-cell lines, only 3 consistently induced target cell lysis. These three antibodies were determined to be 11LA DR reactive. Experiments with a panel of HLA class II antibodies showed the involvement of individual Key receptors on effector cells to be strongly dependent on the antibody isotype. We then compared killing mediated by chimi1rie IgG 1 antibodies with that from FcyRI-directed bispecific antibodies, targeting classical HLA class II, or the Lym-1 and Lym-2 antigens. The latter two are variant forms of HLA class II, which are highly expressed on the surface of malignant B cells but which are found only at low levels in normal cells. With blood from G-CSF-treated donors, bispecific antibodies showed enhanced killing compared to their chimeric IgGl derivatives, because they were more effective in recruiting FcyRI-expressing PMN cells. G- CSF- and FcyRI-directed bispecific antibodies to HLA class II, therefore, seem to be an attractive combination for lymphoma therapy. INTRODUCTION Malignant lymphomas are the most common neoplasm of young adults, with increasing mortality over the last decades (1). In the Western world, most cases are of B-cell origin, and, although chemo- and radiotherapy have proven to be effective treatments, the majority of patients with disseminated low-grade lymphoma or relapses of high-grade lymphoma will ultimately die from their disease. The application of MoAbs3 has the potential to become another therapeu tic option (2). Hematological malignancies seem to be particularly promising targets for antibody therapy, because antibodies to well- defined and rather specific surface molecules are available, therapeu tic antibodies usually reach their targets, and induction of human antimouse or antichimeric antibody is less pronounced than in patients with solid tumors (3). Clinical trials with customized antibodies to patients' tumor idiotype were the first to show encouraging results in Received 2/11/98; accepted 4/30/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by Grani Va 124/1-3 from the Deutsche Forschungsge meinschaft. 2 To whom requests for reprints should be addressed, at Division of Hematology/ Oncology, Department of Medicine III, University Erlangen-Nürnberg, Kranken hausstraße12, 91054 Erlangen, Germany. Phone: 49-9131-853434; Fax: 49-9131- 854770. 1 The abbreviations used are: MoAb, monoclonal antibody: ADCC, antibody-depen dent cellular cytotoxicity; CSF, colony-stimulating factor; G-CSF, granulocyte CSF; PMN, polymorphonuclear; MNC. mononuclear cell; o-PDM. o-phenlendimaleimide; RFI, relative fluorescence intensity; NK, natural killer. lymphoma patients (4), and IDEC-C2B8 [a chimeric CD20 antibody (5)] was the first MoAb to be approved by the United States Food and Drug Administration for treatment in oncology. MoAbs mediate their antitumor effects either by directly acting on tumor cells (e.g., by blocking growth factors, inhibiting cell prolifer ation, or inducing programmed cell death or dormancy) or by recruit ing immune effector mechanisms such as cell- or complement-depen dent cytotoxicity. Studies with isotype switch variants showed a positive correlation between the capacity to induce ADCC in vitro and therapeutic efficacy in vivo, suggesting that ADCC can be an impor tant mechanism of antibody action in vivo (6). Neutrophils, the most abundant Fc receptor-expressing effector cells, showed cytolytic ac tivity against a broad spectrum of tumor cells in vitro (7) and were critically involved in the rejection of cytokine-transfected tumor cells in vivo (8). In vitro, we found cell-mediated target cell lysis by neutrophils to be a major effector mechanism for HER-2/neu-directed MoAbs (9). The contribution of neutrophils can be further enhanced by clinical application of hematopoietic growth factors, such as G- CSF or granulocyte-macrophage CSF, which dramatically raise neu- trophil numbers in vivo and at the same time stimulate important functions, such as phagocytosis, release of oxygen radicals, and ADCC (10). Cell-mediated effects of MoAbs require interaction between the Fc region of antibodies with activating Fc receptors on immune effector cells (11). Depending on their specificity for the heavy chains of IgA, IgE, or IgG, Fc receptors are grouped as Fca, Fee, or Fey receptors, respectively (12, 13). The majority of Fc receptors consist of ligand- specific a chains, which associate with shared molecules for signaling (14, 15). Neutrophils constitutively express the myeloid receptor for IgA (FcaRI, CD89) and two low-affinity IgG receptors, Fcylla (CD32) and FcylIIb (CD16; Ref. 16). IFN-y (17) or G-CSF (18) induces neutrophils to additionally express the high-affinity IgG re ceptor (FcyRI, CD64). Tumor-cytolytic activity on neutrophils has been established for FcyRI, FcyRII and, more recently, for FcaRI (19) but not for FcyRIIIb, which is glycosylphosphatidylinositol- linked on PMN cells. Monocytes/macrophages mediate tumor cell killing via molecules belonging to all three Fey receptor classes, whereas NK cells express only the cytotoxically active FcyRIIIa (11). In a previous study, comparing the capacity of B cell-directed antibodies to induce ADCC of malignant cells, we observed an unexpected antigen restriction whereby PMN cells induced high lev els of target cell lysis with antibodies to HLA class II but not with antibodies to classical B-cell antigens, such as CD 19, CD20, CD21, CD37, or CD38 (20). Here, we report on our results with an extended panel of B-cell antibodies from the Sixth Human Leukocyte Differ entiation Antigen workshop and with engineered antibody derivatives. The results extend our understanding of HLA class H-directed re agents and their ability to recruit effector cells and underscore the potential of bispecific antibodies as therapeutic reagents in the treat ment of lymphoma. 3051 Research. on February 10, 2016. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from