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Cancer Immunol Immunother(199l) 34:9 - 16 034070049100089E

ancer mmunology mmunotherapy

© Springer-Verlag1991

Human recombinant interleukin-6 enhances antibody-dependent cellular cytotoxicity of human tumor cells mediated by human peripheral blood mononuclear cells K. Y. Tsang, M. D. Fineh, F. J. Primus, and J. Sehlom Laboratoryof TumorImmunologyand Biology,NationalCancer Institute, NIH, Bethesda, MD 20 892, USA Received 16 January 1991/Accepted2 May 1991

Summary. The effects of human recombinant interleukin-6 (hrIL-6) on antihody-dependent cellular cytotoxicity (ADCC) activity mediated by human peripheral blood mononuclear cells (PMNC) were investigated. Human PMNC were preincubated for 24 h with various concentrations of hrIL-6 and were used as effector cells in a 4-h 51Cr-release assay. The ability of hrIL-6 to augment ADCC was measured using anti-colorectal carcinoma mAbs D612, 17.lA and 31.1 (each directed against a distinct tumor antigen) and using three human colorectal carcinoma cell lines, LS-174T, WiDr and HT-29, as targets. A significant increase in ADCC activity was observed after PMNC were preincubated in 100-400 U/ml but not in lower concentrations of hrIL-6. Variations in activities of PMNC among donors were observed. Non-specific mAb showed no effect in augmenting ADCC activity, hrIL-6 treatment did not augment non-specific (non-mAb-mediated) cytotoxicity. The enhancement of ADCC activity was blocked by the addition of an antibody against hrIL-6 but not by an antibody to the IL-2 receptor (capable of blocking the induction of lymphokine-activated killer cell cytotoxicity by IL-2), suggesting that hrIL-6 augmentation of ADCC activity may not be mediated through IL-2. These results demonstrate that hrIL-6 augments ADCC activity of human PMNC using mAbs to human tumor antigens and human tumor cells as targets, suggesting a potential role for IL-6 in combination with anti-cancer antibodies for cancer immunotherapy. Key words: Human rIL-6 - Antibody-dependent cellular

Introduction Interleukin-6 (IL-6), originally identified as a B cell differentiation factor, is a pleiotropic cytokine that influences the functional activity of a wide range of target cells. Recent studies revealed that IL-6 has significant effects on the differentiation of cells of immune and non-immune systems, as well as on the expression of differentiated functions [6, 9, 10, 11, 16, 22, 27, 29]. IL-6 inhibits the growth of some breast carcinoma cell lines and myelomonocytic M1 cells [4, 30], but it augments the proliferation of a number of murine hybridomas and plasmacytomas [35, 36] as well as human lymphoblastoid cells infected with Epstein-Barr virus [33]. It has been shown that IL-6 functions as a killer helper factor in the in vitro induction of human cytotoxic T cells [26, 37]. In addition, hrIL-6 has been demonstrated to significantly augment delayed-type hypersensitivity responses in vivo [32]. Furthermore, IL-6 can greatly increase the lytic activity of human lymphokine-activated killer (LAK) cells [8] and can augment human natural killer (NK) cell activity [21]. Antibody-dependent cellular cytotoxicity (ADCC) has been suggested to be an important host defense mechanism for tumor cell destruction mediated by antibody [14, 20]. This report describes the effects of human rIL-6 (hrIL-6) on ADCC activity of human peripheral blood mononuclear cells (PMNC) using three distinct anti-colorectal carcinoma monoclonal antibodies (mAbs) D612 [25], 17.lA [15] and 31.1 [34] to mediate ADCC activity. Results indicate that hrIL-6 can augment ADCC activity mediated by PMNC.

cytotoxicity - Peripheral blood - Mononuclear cells

Materials and methods Human effector cells. PMNC were obtainedfrom heparinizedblood from

healthy adult donors using a lymphocyte separation medium (Organon Teknika Co., Durham, N. C.) gradient as previouslydescribed [2]. Monoclonal antibodies. D612 [25] is a murine IgG2a-~cmAb that reacts Offprint requests to: J. Schlom, Building 10, Room 8B07, NCI, NIH,

9000 RockvillePike, Bethesda,MD 20892, USA

to a 48-kDa glycoprotein[7a] found on the membraneof malignant and benign human gastrointestinal epitheliumbut not other adult normal or malignant human tissues. It has been shown to react with LS-174Tcolon

10 10 ~tg/6 × 106 cells or control mAb TEPC 183 (IgM) at the same concentration. After treatment with mah, cells were resuspended in 1.0 ml 3 to 4 week old rabbit complement (Fel-Freeae, Brown Deer, Wis.). The number of CD 16-positive cells was determined by flow cytometry.

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Pretreatment of effector cells with human rlL-6 (hrlL-6). Effector cells 40

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Fig. 1. Effect of various concentrations of hrIL-6 on antibody-dependent cellular cytotoxicity (ADCC) activity mediated by human peripheral blood mononuclear cells (PMNC). Human PMNC were cultured with various concentrations of human recombinant interleukin-6 (hrlL-6) for 24 h. D612 mAb and myeloma protein UPC-10 were used at 1 btg/ml. The effector : target ratio was 50 : 1 and 25 : 1. D612 (50 : 1) ( • ) ; Dó12 (25: 1) (D); UPC-10 (50: 1) (A); UPC-10 (25: 1) (A). Significance level for D612 (paired t-test): 0 U/ml vs 50, 100, 200, and 400 U/ml: P 0.05

carcinoma cell line and tumor cells [25]. 31.1 [34] is a murine IgGl-~: MAb that reacts with a 72-kDa glycoprotein antigen displayed on colorectal carcinoma cell lines and tissues but not with normal and other malignant tumor tissues. 17.1A [ 15] is a murine IgG2a-~; mAb, originally produced at the Wistar Institute, that reacts with a 37-kDa protein antigen displayed prominently on adenocarcinoma cells of gastrointestinal origin (17.1A mAb was generously supplied by Dr. Peter Daddona, Centocor, Malvern, Pa.).

Antibody-dependent cellular cytotoxicity assay (ADCC). A 4-h 51Cr-release assay was used to measure ADCC activity. The target cell was the colorectal carcinoma cell line LS-174T obtained from the American Type Culture Collection (ATCC, Rockville, Md.). Target cells were labeled with 200 ~tCi sodium [» 1Cr]chromate (250-500 mCi/mg, Amersham, Arlington, Ill.) in 0.2 ml fetal calf serum for 1 h. Other colorectal carcinoma cell lines used in this study were HT-29 and WiDr (ATCC, Rockville, Md.). Target cells (1 x 104) in 50 ~tl were added to assay plates containing 96 U-bottom wells (Costar, Cambridge, Mass.) Effector-cell to target-cell ratios of 100, 50, 25, 12.5, 6.25 to 1 were assayed in the presence of mAbs (1.0 ~tg/well). The plates were incubated for 4 h at 37 ° C in a humidified atmosphere containing 5% CO2. Supernatants were harvested for gamma counting using Skatron Harvester Frames (Sterling, Va.). Experiments were carried out in triplicate. Specific lysis was calculated using the formula: observed release (cpm - spontaneous release (cpm) × 100 total release (cpm) - spontaneous release (cpm) Spontaneous release was determined by measuring the radioactivity released from target cells incubated in medium alone. Total releasable radioactivity was obtained after treatment with 2.5% Triton X-100. Results are also expressed as lytic units/106 effector cells. One lytic unit was defined as the number of effector cells required to cause 30% lysis of 104 target cells. For CD16 depletion, effector cells were treated with mAb Leul lb (Becton, Dickinson, Mountain View, Calif.) at a concentration of lysis (%) =

(1 x 106 cells/ml) in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 U/tal penicillin, 100 ~tg/ml streptomycin, and 10% heat-inactivated fetal calf serum (Gibco, Grand Island, N. Y.) (complete medium) containing various concentrations of hrIL-6 (Gibco, BRL, Gaithersburg, Md.) were incubated for 24 h, 48 h, 72 h and 96 h at 37°C in humidified air with 5% CO2. Control effectors were incubated with complete medium only. After incubation, the nonadherent cells were removed and adherent cells were harvested from flasks with scraping. Both adherent and nonadherent cells were washed three times in complete medium. Human recombinant IL-2 was kindly supplied by the Cetus Corp (Emeryville, Calif.). Various concentrations of hrIL-2 were used in combination with hrIL-6 for pretreatment of effector cells in synergistic experiments. Antibody to IL-2 receptor (IL-2R), anti-Tac, was kindly supplied by Dr. T. Waldmann (NCI, NIH). Anti-Tac at a concentration of 1.0 ~tg/ml or 0.1 [tg/ml was added to the effector cell cultures for 24 h prior to ADCC assay. Rabbit antiserum against hrIL-6 was obtained from Amgen (Thousand Oaks, Calif.). Anti-IL-6 was added to the effector cell cultures at 0.5, 0.25 and 0.125 ~tg/ml for 24 h prior to ADCC assay.

Flow «ytometry. PMNC were cultured for 24 h with or without hrIL-6 at 100 U/ml. The cells were then washed three times in cold phosphatebuffered saline (PBS) and 1 x 106 cells were stained with either 20 ~tl of fluorescein-isothiocyanate(FITC)-labeled L e u l l a antibody (Becton, Dickinson, Mountain View, Calif.) or control antibody (MOPC-21). Cells were incubated for 45 min and then washed twice in cold PBS. For depletion experiments, samples were stained with FITC-labeled antiLeu4, anti-LeuM3, anti-Leulla, or phycoerythrin-labeled anti-Leul2. FACS analysis was performed on a FACSCAN (Becton, Dickinson, Mountain View, Calif.).

Statistical analysis. Statistical analysis of differences between means was done by a paired t-test.

Results Dose-dependent effect o f hrIL-6 on induction o f A D C C Initial e x p e r i m e n t s u s e d the D 6 1 2 m A b (1 p g / w e l l ) to det e r m i n e i f the A D C C m e d i a t e d b y P M N C c o u l d be enh a n c e d by h r I L - 6 u s i n g L S - 1 7 4 T as target. E f f e c t o r cells w e r e i n c u b a t e d w i t h h r I L - 6 at c o n c e n t r a t i o n s o f 0, 5, 25, 50, 100, 200, and 400 U / t a l for 24 h p r i o r to A D C C assay. O p t i m a l a c t i v i t y w a s o b s e r v e d at a c o n c e n t r a t i o n o f 100 U / m l h r I L - 6 and, therefore, this c o n c e n t r a t i o n w a s c h o s e n for the r e m a i n i n g e x p e r i m e n t s (Fig. 1). In the abs e n c e o f hrIL-6, P M N C l y s e d 13% a n d 2 4 % o f L S - 1 7 4 T t a r g e t cells in the p r e s e n c e o f D 6 1 2 m A b at e f f e c t o r : target ratios o f 25 : 1 a n d 5 0 : 1 r e s p e c t i v e l y . T h e c o r r e s p o n d i n g lysis in the p r e s e n c e o f c o n t r o l m A b U P C - 1 0 ( w h i c h d o e s not r e a c t w i t h L S - 1 7 4 T targets) w a s 5 % and 7 % for the 25 : 1 and 5 0 : 1 e f f e c t o r : target ratios r e s p e c t i v e l y . U p o n e x p o s u r e to h r I L - 6 , an i n c r e a s e in A D C C b e g a n at 50 U / t a l and w a s m a x i m a l at 100 U / t a l , r e m a i n i n g c o n s t a n t at l e v e l s a b o v e 100 U / r e l . T h e r e w a s a slight i n c r e a s e in n o n - s p e c i f ic c y t o t o x i c i t y f o l l o w i n g e x p o s u r e o f P M N C to hrIL-6, w h i c h f o l l o w e d the s a m e pattern o f i n c r e a s e as A D C C .

11 50

Table 1. Effect of hrIL-6 on ADCC activity mediated by human PMNC from various donors Donors

hrIL-6

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Incubation time (h)

Fig. 2. Effect of incubation time on ADCC activity mediated by human PMNC. PMNC were cultured for various times with 0 or 100 U/ml hrIL-6. The effector : target ratio was 50 : 1. Significance level (paired t-test) D612 (0 U/tal vs 100 U/tal): P