Cancer Letters, 62 (1992) 179 - 183 Elsevier Scientific Publishers

Ireland Ltd.

Antibody dependent cell mediated cytotoxicity on human cervical carcinoma cell line, ME-HO, with human monoclonal antibody K. Osumia, Ohnod ‘Central

J. Nagaob,



H. Yuasab,


H. Fujimoto”,




H. Hagiwarab,



Y. Kodera”






*Hagiwara Institute for Health, Asazuma-cho Maruyama, Kasaishi, Hyogo 679-01, ‘Nagoya First Red Cross Hospital, Michisita-cho. Nakamura-ku, and dFirst Department of Internal Medicine, Nagoya University School of Medicine, Turumai-cho, Syowa-ku, Nagoya 466 (Japan) (Received 28 November 1991) (Revision received 9 October 1991) (Accepted 13 November 1991)

Summary A human monoclonal antibody CLN-lgG, showed cytotoxic effect

(MCA), in


line, MEby antibody dependent cell-mediated cytotoxicity (ADCC). To determine which fractions of cells in peripheral blood lymphocyte (PBL) mediate ADCC, PBL were separated with nylon wool column and sheep red blood cells (SRBC). Both adherent celts (monocyte) and non-T, non-B cells showed cytotoxicity by ADCC. Human non-T, non-B cells showed higher cytotoxic activity against ME-180 cells

against 180,







cells were less effective in effector cells with human

antibody; cytotoxiciimmunity;




ADCC than human MCA.

Correspondence to: K. Osumi, Research Laboratory, Mitsubishi Shimura, Itabashi-ku, Tokyo 174, Japan. Abbreuiations: ADCC, antibody dependent cell-mediated cytotoxicity; MCA, monoclonal antibody; E:T, effector to target cell ratio; D’MEM, Dulbecco’s Modified Eagle Medium; F-12, Ham’s F-12 medium; FBS, foetal bovine serum; anti-A +B

Yuka BCL, 3-30-1,

serum, human anti-human blood type A and B serum; SRBC, sheep red blood cells; PBL, peripheral blood lymphocyte; MNC, mononuclear cells; PEC, peritoneal exudated macrophage; Kcell, killer cell

0304.3835/92/$05.00 Printed and Published

Keywords: human monoclonal antibody dependent cell-mediated ty; anti-cancer antibody; tumor cytotoxicity; effector cell

0 1992 Elsevier Scientific Publishers in Ireland

Introduction Since the methodology of MCA production procedure was reported [ 11, a large number of MCA including human and chimeric MCA were produced against tumor cells. Some monoclonal antibodies showed cytotoxicity against tumor cells [2 - 81. Herlyn et al. reported that mouse peritoneal macrophage showed ADCC activity with mouse MCA [9] and Chassoux et al. showed killer cell (K-cell)mediated cytotoxicity with rat MCA [lo]. Hagiwara and Sato reported the production of human x human hybridoma, CLNHll, by fusing a 6-thioguanine resistant human B lymphoblastoid cell line, UC729-6, with lymphocytes obtained from a patient with adenosquamous cell carcinoma of the cervix [ 111. In this report using this human IgG type MCA (IgGl, K), we demonstrate the non-T, non-B cells are the main effector cells in ADCC and Ireland Ltd


human effector cells have higher cytotoxicity than murine effector cells. Materials and methods Cell culture ME-180 cells were cultured in DF medium (Dulbecco’s Modified Eagle Medium, D’MEM: F-12 = 1: 1) supplemented with 10% heat inactivated foetal bovine serum FBS. All cell cultures were maintained in an incubator with 50% CO2 at 37OC. Reagents CLN-IgG was purified from IgG fraction of CLNHll spent medium using ammonium sulfate precipitation and protein A affinity chromatography [ 111. Trypsin-EDTA and SRBC were purchased from Immuno-Biological Laboratory Co. (Gunma, Japan). Nylon wool was purchased from Wako Pure Chemicals (Osaka, Japan). Human anti-human blood type A and B (anti-A + B) serum was purchased from Cooper Biochemical (Malverin, PA, U.S.A.). Preparations of effector cells PBL samples were obtained from normal healthy adult donors. Mononuclear cells (MNC) were separated by centrifugation on a Ficoll-Conray gradient. Non-adherent cells were obtained by depletion of monocytes from MNC according to the method of Fischer et al. [12], and adherent cells were collected by treatment with PBS containing 0.02% EDTA (monocyte fraction). For preparation of nonadherent and non-B cells (T cell rich fraction), MNC were passed monocytes-depleted through a nylon wool column [13]. Non-T and non-B cells were obtained by the depletion of T cells from T cell rich fraction with SRBC [ 141. Mouse non-adherent cells were obtained from pooled heparinized blood of ten Balb/c and Balb/c nu/nu mice. ADCC assay Monolayer cells which grew in logarithmic phase were dispersed with 0.25% trvpsin in

Ca*+- and Mg”+-free PBS containing 0.02% EDTA. A 2 x lo6 quantity of dispersed tumor cells were suspended in 0.3 ml of DF medium supplemented with 10% heat inactivated FBS and labeled for 1.5 h with 50 ~1 of 51Cr (sp. act. 37 MBq/ml; Amersham, U.K.). After washing the cells, 50 ~1 of cells (2 x 106/ml) were seeded in a 96-well microtiter plate (Corning no. 25860) in quadruplicate and then incubated at 37OC. After 2 h, 50 ~1 of antibody and 100 ~1 of effector cells were added to each well and then incubated at 37OC with 5% CO2 for 8 h. The supernatant was collected with supernatant collection system (Skatron SX-7072 and 7073). Spontaneous and maximal release of radioactivity by the target cells were determined as described by Mantovani et al. [15]. The percentage of specific lysis was calculated as follows: % specific lysis =


T t

x 100 (%)

where E is the experimental release, S is the spontaneous release, and M is the maximum release [ 161. Results




human PBL

CLN-IgG showed the cytotoxicity against ME-180 cells with human adherent cells (monocyte fraction) and non-adherent cells (T, B and non-T, non-B cells) of PBL. Cytotoxici-



20 40 80 EFFECTOR:TAlt6ET

Fig.1. Induction


10 20 RATIO

40 10

of ADCC against ME-180 ceils by CLN-IgG. ADCC was performed in the absence (A - A) or in the presence (0 - 0) of CLN-IgG at 12.5~g/mI. Adherent cells (monocyte fraction) (a) or monocyte depleted mononuclear cells (b) were used as effector cells.


Table I.


of human

effector cells involved

in ADCC against ME-180 cells.

E:T Ratio

Effector cells

2.5 0.1 -0.2 1.0 -1.5 16.1

Mononuclear cells Non-adherent cells Adherent cells T cell rich fraction Non-T, non-B cells ‘Results were mean bNot done.



7.0 7.3 8.0 7.0

f 3.3” f 0.5 f 4.5 f 0.8 f 3.8

S.D. for quadruplicate

f 1.0 f 2.2 + 1.8 f 0.4 NDb


CLN-IgG” Anti-A + B serumb None “ADCC was performed bADCC was performed ‘Results were mean f

15.0 f 0.3 13.0 +x 0.6 15.2 +I 1.1 13.3 f 0.8 ND

16.0 + 2.3 16.1 ztz 1.1 17.5 f 1.8 15.5 f 3.1 ND

Comparison of ADCC actioity of human and murine effector cells ADCC activities against ME-180 cells were compared with human and mouse effector cells at E:T = 40. Mouse effector cells obtained from Balb/c (nu/nu) and Balb/c mice were 2 -4-times less effective than human effector cells with CLN-IgG. To elucidate whether this phenomenon is restricted in CLN-IgG or not, we examined the ADCC activity of other human antibody, such as anti-A + B serum, with human and murine effector cells. AntiA + B serum showed 3-5-times lower cytotoxicities with murine effector cells than human effector cells against ME-180 cells, which expressed blood type A antigen on their cell surfaces (Table II).

Comparison of ADCC activity of fractionated human effector cells Between 16% and 17% specific cytotoxic activity of CLN-IgG was observed with MNC, adherent cells (monocyte fraction), monocyte depleted MNC (T, B, and non-T, non-B cells fraction), and monocyte and B cell depleted cells (T cell rich fraction) against ME-180 cells at E:T = 40. Non-T, non-B cells showed 16% specific lysis at E:T = 2.5, whereas no ADCC activity by CLN-IgG was detected with the cells in other fractions at the same E:T ratio (Table I).




ty increased with an increase in the effector to target cell ratio (E:T) and reached a maximum at E:T = 40 (Fig. 1).

Table II.


of ADCC activity of human

and murine effector cells against ME- 180 cells

Effector cells obtained





19.8 + 4.2’ 32.5 f 1.5 5.1 f 1.0

8.0 ztz 2.5 6.0 f 1.1 5.2 f 2.9

5.2 ztz 0.2 10.0 f 1.5 3.0 f 1.1

by CLN-lgG (12.5 pg/ml) at E:T = 40. by anti-A + B serum (1:lOO dilution) at E:T = 40. S.D. for quadruplicate samples.





In this study, we demonstrate the ADCC activity of human MCA (CLN-IgG) against monolayer tumor cells with various effector cells. Tumor target cells, ME-180, were lysed by CLN-IgG with monocyte and non-T, non-B cells. Herlyn et al. reported mouse peritoneal exudated macrophage (PEC) showed ADCC activity [9] and Chassoux et al. reported with human K cells [lo]. Our data showed both monocyte and non-T, non-B cells had ADCC activity and higher ADCC activity was shown by non-T, non-B cells than monocytes. Herlyn et al. reported that murine PEC had shown the higher ADCC activity than human monocyte with murine MCA [9]. They discussed how murine PEC had higher binding affinity to the Fc portion of murine MCA than human antibody. Human effector cells showed 32.5% lysis by anti-A + B serum and 20% lysis by CLN-IgG; on the other hand murine effector cells showed 6 - 10% lysis by anti-A + B serum and 5 - 8% by CLN-IgG, respectively. Therefore human effector cells might have higher binding affinity to human antibody than that of murine effector cells. Although some papers reported the efficacy of murine MCA in immunotherapy [7,17,X4], most of murine MCA have failed to induce patients into complete remission [ 191. The most probable reasons for low efficiency of anticancer treatment by murine MCA seem to be the escape of tumor cells from the immune system by the antigenic modulation and the inactivation of murine MCA by human antimouse antibody. Furthermore, the low efficacy of murine MCA might be due to the difference between the affinity of human and murine immunoglobulin to the human effector cells. From these investigations of ADCC with CLN-IgG, human MCA including chimeric MCA have more advantage than murine MCA in immunotherapy against human cancer.










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Antibody dependent cell mediated cytotoxicity on human cervical carcinoma cell line, ME-180, with human monoclonal antibody.

A human monoclonal antibody (MCA), CLN-IgG, showed cytotoxic effect in vitro against the cervical carcinoma cell line, ME-180, by antibody dependent c...
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