[CANCER RESEARCH 50. 2991-2996. May 15. 1990| Antibody-dependent, Cell-mediated Cytotoxicity by an Anti-Class II Murine Monoclonal Antibody: Effects of Recombinant Interleukin 2 on Human Effector Cell Lysis of Human B-Cell Tumors William C. Biddle,1 James Pancook, Martin Goldrosen, Tin Han, Kenneth A. Foon, and Louis Vaickus Divisions of Clinical Immunology [W. C. B., J. P., M. G., K. A. F., L. V.¡andHematological Oncology ¡T.H.], Roswell Park Cancer Institute, Buffalo, New York 14263 ABSTRACT exists as to which effector cell type is most effective at ADCC (4, 5). This is partly attributable to the wide spectrum of Lym-1 is an IgG2a murine monoclonal antibody that reacts with variant antibodies (and species of origin) and target cells used in in Class II molecules expressed on B-cell malignancies. Lym-1 was shown vitro assays. A considerable amount of our present knowledge to mediate antibody-dependent cellular cytotoxicity (ADCC) of human concerning ADCC is based on previous studies using heterolo- effector cells against a variety of malignant B-cell lines. Tumor cell lysis was Lym-1 specific because (a) the reaction was dose dependent with gous antisera, irrelevant targets (e.g., chicken red blood cells), significant ADCC detectable at Lym-1 concentrations as low as 1 MH/nil: or nonhuman effector cells, and direct extrapolation to the (b) tumor targets not expressing the Lym-1 antigen were unaffected; (c) human system may not be valid. Murine MoAbs of the IgG2a an isotype-matched irrelevant monoclonal antibody and an IgGl anti- and IgG3 subclass are felt to be the best mediators of ADCC Class II monoclonal antibody failed to mediate ADCC; and (d) addition (6-8). There is no a priori method to determine MoAb activity of Protein A (which binds avidly to Lym-1) blocked ADCC by 90 to in ADCC based on isotype alone, however. In fact, not all 100%. Peripheral blood mononuclear cells obtained from normal donors IgG2a MoAbs mediate ADCC, and their functionality may be as well as from cancer patients were able to interact with Lym-1 to elicit based on antigen density, kinetics of antigen modulation, and ADCC. Recombinant interleukin 2 (rIL-2) enhanced non-antibody-me interaction with Fc receptors, among others. Indeed, it has been diated tumor lysis and Lym-1 ADCC with an optimal concentration of recently reported that IgG2a and IgG3 antiidiotype MoAbs 100 units/ml. Pulse treatment of normal peripheral blood mononuclear cells with rIL-2 was able to augment Lym-1 ADCC but was less effective were unable to mediate ADCC against lymphoma targets with than having the rIL-2 present through the assay. Peripheral blood human effector cells (9). mononuclear cells obtained from patients being treated with high doses IL-2 has been reported to enhance effector cell function in of rIL-2 administered by continuous i.v. infusion demonstrated Lym-1 ADCC following in vivo or in vitro administration in the murine ADCC levels which were higher than normal individuals and which were system (10, 11). Similarly, in vitro exposure to IL-2 has been further augmented by in vitro incubation with rIL-2. shown to result in increased ADCC in human model systems (12). This is, however, not a universal finding. Treatment with IL-2 was shown to increase non-antibody-mediated cytotoxicity INTRODUCTION by human effector cells, but ADCC against bladder carcinoma Lym-1 is an IgG2a Mo Ab2 that recognizes variant DR mol cells was not affected (13). Thus, it is critical to evaluate each ecules present on a variety of B-cell lymphomas and leukemias. new therapeutic MoAb individually to determine whether it can Expression of the Lym-l-Ag appears to be highly restricted in mediate ADCC and to learn whether this reaction can be that Lym-1 reacts with only a limited number of normal cells augmented by recombinant cytokines. This knowledge may help and with lower avidity as compared with tumor cells. The Lym- to optimize the clinical utility of MoAbs used in their uncon l-Ag does not appear to modulate nor be shed into the circu jugated or conjugated forms. lation (1). These attributes make Lym-1 an attractive candidate MoAb for immunotherapy. Previous trials using unaltered MoAbs against B-cell malig MATERIALS AND METHODS nancies have met with limited success (reviewed in Ref. 2). In Human Cell Lines and Culture Conditions. The human Burkitt's contrast, successful treatment of xenografted human tumors in lymphoma cell lines Raji, Jijoye, and EB3 and the K562 erythroleuke- murine models has been demonstrated using unconjugated mia cell line were obtained from the American Type Culture Collection MoAbs and appeared to be largely dependent upon the anti (Rockville, MD). The cell lines designated MO 1011, MO 1018, MO body's ability to mediate ADCC (3). Improvements in the 1046, and MO 2007 were established from tumor cells obtained from clinical utility of MoAbs may be attained by further understand patients with B-cell CLL and PLL, respectively. These cell lines were ing this antitumor mechanism. Although much is known about generated by Epstein-Barr virus transformation and retain many of the ADCC interactions, many areas are still unresolved. phenotypic, molecular, and cytogenetic characteristics of the original tumor cell (14). They were kindly provided by Dr. Tin Han, Roswell In general, the ability to lyse tumor cells depends on the Park Cancer Institute, Buffalo, NY. Cell lines were cultured in CM MoAb subclass, type of effector cell, degree of antigen expres consisting of RPMI 1640 medium, 10% heat-inactivated fetal bovine sion, and susceptibility of the target cell to lysis. Controversy serum, and standard supplements. Cell lines were maintained at 37°C Received 9/20/89; revised 1/10/90. in a humidified 95% air, 5% CO2 environment. All tissue culture The costs of publication of this article were defrayed in part by the payment reagents were obtained from GIBCO, Grand Island, NY. of page charges. This article must therefore be hereby marked advertisement in Monoclonal Antibodies. The Lym-1 monoclonal antibody was sup accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Clinical Immunology, plied by Dr. F. Dürr,Lederle Laboratory, Division of American Cy- Roswell Park Cancer Institute. Elm and Carlton Streets, Buffalo, NY 14263. anamid Corporation, Pearl River, NY. MoAb L227 (IgGl) is reactive 1The abbreviations used are: MoAb. monoclonal antibody; ADCC, antibody- with nonpolymorphic DR, DP, and DQ molecules, whereas MoAb dependent cellular cytotoxicity; CM, complete medium; FcR, Fc receptor; FITC, L243 (IgG2a) recognizes nonpolymorphic DR (15). fluorescein ¡sothiocyanate:LU. lytic unit; LAK. lymphokine-activated killer cell; An isotype matched (IgG2a) control MoAb (UPC-10) was obtained Lym-l-Ag. Lym-1 antigen; PBMC. peripheral blood mononuclear cells; rIL-2. recombinant interleukin 2; SpA, Staphylococcus aureus Protein A; IL-2, interleu from Sigma Chemical Corp., St. Louis, MO. All antibodies were used kin 2; PLL, prolymphocytic leukemia; CLL. chronic lymphocytic leukemia; E:T, as purified immunoglobulin in phosphate-buffered saline. MoAb purity effectortarget. was determined to be greater than 90% by sodium dodecyl sulfate- 2991 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. EFFECTS OF rlI.-2 ON HUMAN EFFECTOR CELL LYSIS OF B-CELL TUMOR polyacrylamide gel electrophoresis. Antibodies were sterilized by filtra effectors required to cause 33% target lysis was extrapolated (1 lytic tion through 0.2-/JIT1filters prior to use. unit). Results are expressed as lytic units (LU33) per IO7effector cells Biological Response Modifier Treatment Protocols. The 1L-2/LAK (17). protocols consisted of continuous i.v. infusion of rIL-2, RO 23-6019 Protein A Inhibition of Lym-1 ADCC. SpA and FITC-SpA were (Hoffmann-LaRoche, Nutley, NJ), 6 x IO6 units/m2 daily for 5 days purchased as lyophilized powders (Sigma Corp.). Lym-1 (5 Mg) was (Days 1 to 5), then daily leukapheresis for 3 days (Days 7 to 9), and used to saturate the Lym-1-Ag binding sites on 2.5 x IO5 Raji cells by infusion of IL-2 and LAK cells for 3 days followed by IL-2 alone for 2 incubation on ice for 20 min. After washing, varying amounts of FITC- days (Days 10 to 14). Cells from Patient 1 were obtained on Day 13 of SpA were added for an additional 20 min. Binding of FITC-SpA to Cycle 1; Patient 4 on Day 7 of Cycle 1; Patient 5 on Day I of Cycle 3; Lym-1 was determined by flow cytometry based on mean log fluores and Patient 7 on Day 14 of Cycle 1. The IL-2/a-interferon protocol cence and the percentage of positive cells as compared with control consisted of concomitantly administered rIL-2 at 3 x IO6units/m2/day MoAbs. This method was used to determine a saturating concentration continuous i.v. infusion with a-interferon (Roferon-A, RO 22-8I8I/ of SpA. A 4-fold higher concentration of unlabeled SpA (20 ^g) was 002; Hoffman-LaRoche) at 5 x IO6units/m2/day by i.m. or s.c. injec added in the ADCC assays to determine whether binding of SpA to the tion, both for 4 days. Cells from Patient 2 were obtained after 3 Fc portion of Lym-1 could inhibit Lym-1 ADCC. complete cycles; Patient 3 after 2 cycles. The IL-2/cyclophosphamide Statistical Analysis. Healthy controls and cancer patients were com protocol consisted of cyclophosphamide (400 mg/m2) administered by pared using a Mann-Whitney test. Levels for various combinations of bolus i.v.