Enhancement of Polymorphonuclear Cell ... - Cancer Research

[CANCER RESEARCH 61, 4055– 4060, May 15, 2001]

Enhancement of Polymorphonuclear Cell-mediated Tumor Cell Killing on Simultaneous Engagement of Fc␥RI (CD64) and Fc␣RI (CD89)1 Marjolein van Egmond, Annemiek B. van Spriel, Hans Vermeulen, Gerwin Huls, Evert van Garderen, and Jan G. J. van de Winkel2 Departments of Cell Biology/Immunology and Surgical Oncology, Vrije Universiteit, Amsterdam, the Netherlands [M. v. E.]; Department of Immunology [A. B. v. S., H. V., G. H., J. G. J. v. d. W.], Medarex Europe [A. B. v. S.], and Genmab [J. G. J. v. d. W.], University Medical Center Utrecht, Utrecht, the Netherlands; and Department of Pathology, Faculty of Veterinary Medicine, Utrecht, the Netherlands [E. v. G.]

ABSTRACT Antibodies can efficiently induce antitumor responses via recruitment of Fc receptor-bearing cytotoxic cells. Polymorphonuclear (PMN) cells represent attractive effector cells for antibody-directed immunotherapy. This, because activated PMN cells coexpress the class I receptors for IgG (Fc␥RI, CD64) and IgA (Fc␣RI, CD89), which are potent cytotoxic trigger molecules. Both receptors, however, require the FcR ␥ chain for signaling. In this study, we show that Fc␥RI and Fc␣RI can trigger function independently of one another and do not cross-compete for the FcR ␥ chain. Fc␣RI proved more efficient in initiating early signaling events and effector functions, such as redirected tumor cell killing and generation of superoxide. In addition, simultaneous engagement of Fc␥RI and Fc␣RI resulted in enhanced tumor cell lysis. These data support the development of concepts in which both Fc␥RI and Fc␣RI on PMN cells are targeted for tumor therapy.

INTRODUCTION Immunotherapeutic approaches that harness the cytotoxic ability of immune cells to reject tumor cells receive increasing levels of attention. Both T cells and myeloid cells are considered suitable effector cells and have documented ability to kill tumor cells (1). PMN3 cells represent an attractive effector cell population, and their numbers can be easily increased in vivo by G-CSF (2). To elicit cytotoxic responses, these cells require activation via trigger molecules that can be linked to target cells via mAb, and several mAb targeting to tumor cells have recently been approved for cancer therapy (3). The prototypic antitumor Ab rituximab, a chimeric anti-CD20 mAb, was shown to elicit prominent antitumor effects in patients with non-Hodgkin’s B-cell lymphoma (4, 5), whereas Herceptin, an anti-HER-2/neu mAb, induced promising results in breast cancer patients (3, 6). Because interaction with Fc receptors was reported to be crucial for therapeutic responses induced by antitumor mAb (7), the development of BsAb targeting to select Fc receptors may represent a way to further improve therapeutic activity (1, 8, 9). Both the class I receptors for IgG (Fc␥RI, CD64) and IgA (Fc␣RI, CD89) have been identified as candidate therapeutic targets (9 –11). These receptors exhibit a myeloid-restricted cell distribution and potently trigger effector functions like phagocytosis and tumor cell lysis (12, 13). PMN cells constitutively express Fc␣RI and can be induced to express Fc␥RI upon treatment with IFN-␥ or G-CSF (2, 14). We thus posed the question whether it would be feasible to use both receptors simultaneously as trigger molecules for immunotherapy. Received 2/24/00; accepted 3/14/01. 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. 1 Supported by National Organization for Scientific Research Grant 901-12-214. 2 To whom requests for reprints should be addressed, at Department of Immunology, University Medical Center Utrecht, KC.02-085.2, Lundlaan 6, 3584 EA Utrecht, the Netherlands. E-mail: [email protected]. 3 The abbreviations used are: PMN, polymorphonuclear; G-CSF, granulocyte colonystimulating factor; Ab, antibody; mAb, monoclonal Ab; BsAb, bispecific Ab; Tg, transgenic; dTg, double Tg; FcR, Fc receptor; GM-CSF, granulocyte macrophage colonystimulating factor; sTg, single Tg; TNF-␣, tumor necrosis factor ␣; PE, phycoerythrin.

Both FcR classes, however, associate with the promiscuous FcR ␥ chain signaling subunit and are dependent on this FcR ␥ chain for stable surface expression (15–19). Consequently, it is conceivable that Fc␥RI and Fc␣RI “cross-compete” for the FcR ␥ chain, which would hinder the possibility of using both receptors in immunotherapy. The aim of this study was, therefore, to determine whether both receptors can function independently, irrespective of (limiting amounts of) the FcR ␥ chain. Furthermore, we analyzed whether simultaneous engagement of Fc␣RI and Fc␥RI on PMN cells facilitates destruction of malignant cells.

MATERIALS AND METHODS Antibodies. Surface expression of Fc␣RI was detected with FITC-conjugated F(ab⬘)2 fragments of CD89 mAb A77 (Medarex, Annandale, NJ) or PE-labeled CD89 mAb A59 (PharMingen, San Diego, CA). FITC-conjugated CD64 mAb 22 (Medarex) was used to determine Fc␥RI expression. Mouse PMN cells were defined with PE-conjugated Gr-1 (PharMingen). Fully human IgA1 antibodies targeting Ep-CAM were obtained by using phage display and engineering as described previously (20). BsAb Fc␥RIxHER-2/neu (22x520C9; MDXH210), Fc␣RIxHER-2/neu (A77x520C9), Fc␥RIxHLA-II (22xF3.3), and Fc␣RIxCD20 (A77x1F-5) were prepared as described in Ref. 21. mAb 520C9 (Medarex) recognizes HER-2/neu, a proto-oncogene product overexpressed on human carcinoma cells. mAb 1F5 and mAb F3.3 are directed against CD20, and MHC class II antigens, respectively, and were a kind gift from Dr. M. Glennie (Tenovus Research Laboratory, Southampton, United Kingdom). Flow Cytometry. Whole blood of mice was incubated with mAb (10 ␮g/ml) for 15 min at room temperature and subjected to fluorescence-activated cell sorting lysing solution (Becton Dickinson, San Jose, CA). Human PMN cells (2 ⫻ 105), either freshly isolated or cultured overnight, were incubated with 22-FITC and A59-PE for 30 min at 4°C. Cells were analyzed on a FACScan (Becton Dickinson). Tg Mice. Generation of Fc␥RI (22) and Fc␣RI (19) Tg mice was described earlier. Briefly, Fc␥RI Tg mice were generated by injection of an 18-kb human genomic DNA fragment into FVB/N oocytes. A 41-kb cosmid clone served as a Tg construct to create Fc␣RI Tg mice. Both Tg mice expressed the human receptor solely on myeloid cells, which parallels the human situation. Fc␥RI Tg males were crossed with Fc␣RI Tg females to generate (Fc␣RI ⫻ Fc␥RI) double Tg (dTg) mice. Expression of transgenes was determined by flow cytometry of peripheral blood cells using anti-Fc␥RI mAb 22-FITC and anti-Fc␣RI mAb A77-FITC. To induce Fc␥RI expression on PMN cells and to increase PMN cell counts in blood, mice were injected s.c. with 1.6 ␮g/mouse/day murine G-CSF for 4 days (23). Cell Culture. Peripheral blood (heparin anticoagulated) from healthy volunteers was collected and PMN cells were isolated by Ficoll-Histopaque discontinuous gradient centrifugation. PMN cells were collected at the interface between Ficoll and Histopaque and the remaining erythrocytes were removed by hypotonic shock. Both purity of PMN cells and viability checked with trypan blue exceeded 95%. The breast carcinoma cell line SK-BR-3, overexpressing HER-2/neu and the malignant B-cell line ARH-77 were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FCS and antibiotics. SK-BR-3 cells were harvested using trypsin-EDTA (Life Technologies, Inc., Paisley, United Kingdom).

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ENGAGEMENT OF Fc␥RI AND Fc␣RI ENHANCES TUMOR CELL LYSIS

Fig. 1. Effect of IFN-␥ on PMN Fc␣RI and Fc␥RI expression. A, human PMN cells were stained for expression of Fc␣RI and Fc␥RI following isolation (left panel) and after overnight culture with IFN-␥ (right panel). B, histogram overlay of Fc␣RI expression on day 0 (dotted line) and after culture with IFN-␥ (black line). This experiment was repeated three times, yielding similar results.

In culture experiments, human or mouse cells were cultured with human or mouse cytokines, respectively. Human PMN cells were cultured overnight at 37°C with IFN-␥ (300 units/ml; Boehringer Mannheim, Mannheim, Germany) to induce Fc␥RI expression. Mouse bone marrow cells were cultured in DMEM medium, supplemented with 4.5 g/liter glucose, 10% FCS, and antibiotics, with or without granulocyte macrophage colony-stimulating factor (GM-CSF, 50 ng/ml) and tumor necrosis factor ␣ (TNF-␣, 50 ng/ml), or IFN-␥ (250 units/ml; Amgen, Thousand Oaks, CA). After 24 h, nonadherent cells were harvested and stained with A77-FITC or 22-FITC. Gr-1-PE was used to define mouse PMN. Dr. J. Andresen (Amgen) generously provided GM-CSF and G-CSF. TNF-␣ was kindly donated by Dr. W. Buurman (University Maastricht, the Netherlands). Cytotoxicity Experiments. 51Cr release assays were used to evaluate the capacity of effector cells to lyse tumor cells (24). Either 1 ⫻ 106 SK-BR-3 or 1 ⫻ 106 ARH-77 tumor cells were incubated with 150 ␮Ci of 51Cr (Amersham, Little Chalfont, United Kingdom) for 2 h at 37°C and washed three times. In “cold target inhibition experiments,” 2.5 ⫻ 103 51Cr-labeled SK-BR-3 cells were plated with 2.5 ⫻ 103 unlabeled ARH-77 cells (or vice versa) in 96-well round-bottomed microtiter plates. Fifty microliters of whole blood of G-CSF-treated dTg mice were added (E:T ratio, 120:1). Alternatively, 50 ␮l of RPMI 1640 medium containing 3 ⫻ 105 (E:T ratio, 60:1), 6 ⫻ 105 (E:T ratio, 120:1), or 9 ⫻ 105 (E:T ratio,180:1) IFN-␥-treated human PMN cells were added. BsAb-mediated tumor cell lysis of 51Cr-labeled targets was compared with lysis of the same cells in the presence of a BsAb directed against competing unlabeled cells. In an additional set of experiments, 51Cr-labeled SK-BR-3 or 51Cr-labeled ARH-77 cells (5 ⫻ 103/well) were incubated with effector cells and increasing amounts of anti-Fc␣RI BsAb, anti-Fc␥RI BsAb, or both types of BsAb. Cells were incubated at 37°C for 6 h, after which 51Cr release in supernatants was measured. Respiratory Burst Experiments. Polystyrene tubes were coated with 100 ␮g/ml human serum IgA (Cappel, Aurora, OH), 100 ␮g/ml human IgG (CLB, Amsterdam, the Netherlands), or PBS for 3 h at 37°C. After washing three times with PBS, all tubes were blocked with HEPES complete [20 mM HEPES (pH 7.4), 132 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1.2 mM NaH2PO4, 1 mM CaCl2, 5.5 mM glucose, 0.5% BSA, and 1.5 mM MgCl2] for 1 h at 37°C. The luminol-enhanced chemiluminescence method was used for determination of

real-time respiratory burst activity (25). Human PMN cells (2 ⫻ 105/0.2 ml HEPES) were gently centrifuged (400 rpm, 5 min, 4°C) and placed in a 953 LB Biolumat (Berthold, Wildbad, Germany). Luminol (150 mM) was injected in all tubes, and light emission was recorded continuously for 30 min at 37°C. Statistics. Results were analyzed by means of the unpaired two-tailed Student’s t test (comparison experiments described in Fig. 6) or ANOVA tests (combination experiments; Fig. 5). Differences in competition experiments were analyzed by Wilcoxon rank sum tests (Fig. 4). Results are expressed as mean ⫾ SE, and significance was accepted at P ⬍ 0.05.

RESULTS Since both Fc␥RI and Fc␣RI crucially depend on the FcR ␥ signaling chain for stable surface expression, we investigated whether these receptors “cross-compete” for the FcR ␥ chain. We first checked expression of Fc␥RI and Fc␣RI under various conditions. Freshly isolated human PMN expressed Fc␣RI, but not Fc␥RI (Fig. 1A, left panel). To induce surface Fc␥RI, PMN cells were incubated with IFN-␥. Fc␥RI expression was up-regulated after 24 h (Fig. 1A, right panel), whereas Fc␣RI levels were unaffected (Fig. 1B). Because Fc␣RI expression on human PMN cells is difficult to manipulate experimentally, we created mice Tg for human Fc␥RI or human Fc␣RI. It was previously shown that human Fc␥RI and Fc␣RI physically interact with the murine FcR ␥ chain (17, 19) and these Tg models enabled us to modulate receptor expression separately and in vivo. As described before (19, 22), cell distribution patterns in Tg mice closely parallels the human situation; Fc␥RI is constitutively expressed on monocytes and macrophages and induced on PMN cells by G-CSF or IFN-␥ treatment (22). Fc␣RI Tg mice constitutively express Fc␣RI on PMN, whereas expression can be induced on macrophages by culture with GM-CSF and TNF-␣ (19, 26). Culturing bone marrow-derived PMN cells of (Fc␣RI ⫻ Fc␥RI) dTg mice with GM-CSF and TNF-␣ enhanced Fc␣RI expression levels (Fig. 2A, top panels), whereas IFN-␥ selectively induced Fc␥RI

Fig. 2. Differential cytokine regulation of Fc␣RI and Fc␥RI expression in (Fc␣RI ⫻ Fc␥RI) dTg mice. A, bone marrow-derived PMN cells from dTg mice were cultured overnight with GM-CSF and TNF-␣ (top panels) or with IFN-␥ (bottom panels) and stained for Fc␣RI (left panels) or Fc␥RI (right panels). Cells cultured in the presence of cytokines (black lines) were compared with cells cultured in medium alone (dotted lines). B, effect of cytokines on FcR expression of sTg versus dTg animals. After overnight culture with GM-CSF/TNF-␣ or IFN-␥, Fc␣RI and Fc␥RI expression levels on PMN cells of dTg mice (black lines) were compared with expression on sTg mice (dotted lines). Gr-1-PE was used to define mouse PMN cells. One representative experiment of four is shown.

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Fig. 3. Effect of in vivo G-CSF treatment on Fc␣RI and Fc␥RI expression. A, whole blood of dTg mice was stained for expression of Fc␣RI (left panel) or Fc␥RI (right panel). Expression on PMN cells at day 0 (dotted lines) was compared with expression after 4 days of G-CSF treatment (black lines). B, PMN cell surface expression of dTg mice (black lines) versus expression of Fc␣RI (left panel, dotted line) or Fc␥RI sTg (right panel, dotted line) mice after G-CSF treatment. Gr-1-PE served to identify PMN cells. This experiment was repeated three times, yielding essentially identical results.

expression (Fig. 2A, bottom panels). Up-regulation of either receptor class, however, did not influence expression levels of the other class. No difference in Fc␥RI expression was found between Fc␥RI sTg and dTg PMN cells upon culture with GM-CSF/TNF-␣, although the latter PMN cells expressed increased levels of Fc␣RI (Fig. 2B, top right panel). Similarly, increased levels of Fc␥RI after culture with IFN-␥ did not affect Fc␣RI expression levels (Fig. 2B, bottom left panel). When mice were injected with G-CSF, Fc␥RI was up-regulated on dTg PMN, whereas expression of Fc␣RI was unaltered (Fig. 3A). Again, enhanced Fc␥RI expression levels did not affect levels of Fc␣RI (Fig. 3B, left panel). In addition, PMN cells from dTg and Fc␥RI sTg mice showed identical Fc␥RI expression levels, indicating that Fc␣RI does not affect expression of Fc␥RI either (Fig. 3B, right panel). In summary, Fc␣RI and Fc␥RI expression levels on resting and cytokine-stimulated PMN cells are comparable between sTg and dTg mice. Since both Fc␣RI and Fc␥RI require the FcR ␥ chain for proper function (15–19), we investigated whether the FcR ␥ chain preferentially interacts with either one of these receptors. For this purpose, we set up a cold target inhibition assay. Effector cells were incubated with a mixture of 51Cr-labeled target cells X and unlabeled target cells Y

(Fig. 4A). Lysis of 51Cr(X), either in the absence (Fig. 4, B–D, open columns) or presence (Fig. 4, B–D, filled columns) of a BsAb targeting the competing unlabeled cell line Y, was measured after incubation for 6 h. IFN-␥-treated human PMN cells (Fig. 4, B and C) or whole blood of G-CSF-treated dTg animals (Fig. 4D) were used as effector cells. When 51Cr-labeled ARH-77 was used as target cell line X and the unlabeled SK-BR-3 as Y, neither lysis via Fc␥RI nor that via Fc␣RI was influenced when BsAb directed against SK-BR-3 and the competing receptor were present (Fig. 4B). In the alternate case, with 51 Cr-labeled SK-BR-3 as X and ARH-77 as Y, no differences in Fc␥RI-mediated tumor cell lysis were observed, either in the absence or presence of BsAb targeting Fc␣RI and ARH-77 (Fig. 4C, left panel). In addition, at E:T ratios 120:1 or 180:1 Fc␣RI-mediated lysis of 51Cr-labeled SK-BR-3 cell was unaffected in the presence of a BsAb targeting Fc␥RI (Fig. 4C, right panel). However, at E:T ratios of 60:1 tumor cell lysis was somewhat reduced. No differences in Fc␥RI- or Fc␣RI-mediated lysis of either cell lines were observed when whole blood of dTg mice was used as effector population (Fig. 4D). In an additional set of experiments in which 51Cr release was measured after 2 or 4 h of incubation, similar results were found (data not shown, n ⫽ 2).

Fig. 4. Investigation of functional competition for FcR ␥ chain between Fc␣RI and Fc␥RI in cold target inhibition assays. A, effector cells (PMN) were incubated with a mixture of 51Cr-labeled cell line X (black cells) and unlabeled cell line Y. BsAb-mediated lysis of 51Cr(X) was determined in the absence or presence of a second BsAb targeting the competing unlabeled cell line Y. B and C, human PMN cells were incubated with 51Cr(ARH-77) cells and unlabeled SK-BR-3 cells (B) or vice versa (C). Fc␥RI (left panels)- or Fc␣RI- (right panels) mediated lysis of the 51Cr-labeled cell line in the absence of the second BsAb (䡺) was set at 100% and compared with tumor cell lysis in the presence of competing BsAb (f). D, whole blood of G-CSF-treated dTg mice was used as effector population. Lysis of 51Cr(SK-BR-3) or 51Cr(ARH-77) tumor cells is shown in the left and right panel, respectively. 51 Cr release from triplicate (human PMN) or duplicate (mouse blood) samples was measured after 6 h of incubation at 37°C. Data represent mean ⫾ SE of three separate experiments. ⴱ, P ⬍ 0.05.

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NADPH-oxidase complex, but respiratory burst activity was consistently higher with IgA-coated tubes (Fig. 6D). This was observed with a range of IgA and IgG concentrations (data not shown, n ⫽ 2).

DISCUSSION

Fig. 5. Simultaneous engagement of both Fc␣RI and Fc␥RI results in enhanced tumor cell kill. A, lysis of ARH-77 tumor cells by IFN-␥-treated human PMN cells (E:T, 120:1) in the presence of increasing amounts of Fc␥RI ⫻ anti-HLAII (䡺), Fc␣RI ⫻ CD20 (F), or both BsAb (Œ). B, lysis of SK-BR-3 tumor cells by IFN-␥-treated human PMN cells (E:T, 60:1) in the presence of increasing amounts of Fc␥RI ⫻ HER-2/neu (䡺), IgA anti-Ep-CAM (F), or both BsAb (Œ). On the X axis, the concentration of each separate BsAb/Ab is indicated. Data are expressed as mean ⫾ SE of triplicate samples. One representative experiment of three is shown. ⴱ, P ⬍ 0.01.

Next, tumor cell kill upon simultaneous engagement of Fc␣RI and Fc␥RI was assessed. Maximal lysis of ARH-77 cell was observed with either 1 ␮g/ml BsAb Fc␥RIxHLAII or Fc␣RIxCD20, which was not increased in the presence of higher BsAb concentrations. Tumor cell lysis was, however, enhanced upon incubation with two targeting BsAb, relative to either one of them separately (Fig. 5A). Comparable data were obtained with whole blood of G-CSF-treated dTg mice (data not shown, n ⫽ 3). We, furthermore, investigated whether the observed reduction in Fc␣RI-mediated tumor cell lysis upon engagement of Fc␥RI at E:T ratios of 60:1 would abrogate this enhancement in tumor cell lysis. Since BsAb Fc␣RIxCD20 did not induce tumor cell lysis at the E:T ratio 60:1 (data not shown), SK-BR-3 cells expressing both HER-2/neu and Ep-CAM were used. Because an IgA Ab targeting Ep-CAM was available (20), BsAb Fc␥RIxHER-2/neu and IgA anti-Ep-CAM Ab were used in these experiments. Maximal lysis of SK-BR-3 tumor cells was observed in the presence of 0.4 ␮g/ml Fc␥RIxHER-2/neu or 2.0 ␮g/ml IgA anti-Ep-CAM, and again enhanced tumor cell lysis was observed upon addition of two (bispecific) Abs, relative to addition of only one (Fig. 5B). Notably, Fc␣RI proved more efficient in triggering tumor cell kill than Fc␥RI (Fig. 6, A and B). Therefore, the capacity of Fc␣RI and Fc␥RI to initiate PMN cell signaling was investigated. Cross-linking of Fc␣RI resulted in a more rapid induction of rises in intracellular free calcium levels than cross-linking of Fc␥RI (Fig. 6C). Initiation of respiratory burst activity was tested as a more distal signaling event using a sensitive chemiluminescence method. No PMN oxygen radical production was evoked when PBS/HEPES-coated tubes were used. Tubes coated with either IgA or IgG activated the PMN

In this study, we aimed to evaluate whether simultaneous targeting of tumor cells to two types of trigger molecules on PMN cells results in enhanced tumor cell destruction. As trigger molecules, we chose Fc␥RI and Fc␣RI, because both are selectively expressed on myeloid effector cells which can easily be mobilized in vivo and potently trigger tumor cell lysis in vitro (2, 9, 10, 27). Moreover, treatment with a combination of G-CSF and BsAb, targeting Fc␥RI and idiotype, led to effective antitumor responses in lymphoma-bearing Fc␥RI Tg mice, which were not observed in control mice (including treated nontransgenic litter mates) (28). Similarly, treatment of Fc␣RI Tg mice with an anti-Fc␣RI BsAb resulted in prolonged survival, compared with control mice (29). A possible drawback of therapies engaging both Fc␣RI and Fc␥RI, however, may be the (limiting) amount of FcR ␥ chain in effector cells restricting simultaneous triggering via two FcR ␥ chain-dependent receptors (15–19). In the present work, however, no evidence was found that the FcR ␥ chain limits expression of Fc␣RI or Fc␥RI, neither in vitro nor in vivo. Up-regulation of Fc␥RI did not result in decreased expression of Fc␣RI and vice versa (Figs. 1–3). Furthermore, cold target inhibition experiments showed that in most circumstances both receptors function independently of each other without any cross-competition for the FcR ␥ chain. Since BsAb targeting either HLA class II or HER-2/neu induced efficient tumor cell lysis, it is feasible that these BsAb induce maximal occupancy of receptors and associating FcR ␥ chain. In one situation, where Fc␣RIxHER-2/ neu and Fc␥RIxHLAII BsAb were used at an E:T ratio 60:1, Fc␣RImediated tumor cell lysis was somewhat reduced upon engagement of

Fig. 6. Fc␣RI cross-linking results in more efficient tumor cell killing and signaling. A, ARH-77 cells were incubated with IFN-␥-treated human PMN cells in the presence of increasing amounts of Fc␥RI ⫻ anti-HLAII (䡺) or Fc␣RI ⫻ anti-HLAII (F) BsAb. B, SK-BR-3 cells were incubated with IFN-␥-treated human PMN cells in the presence of increasing amounts of Fc␥RI ⫻ anti-HER-2/neu (䡺) or Fc␣RI ⫻ anti- HER-2/neu (F) BsAb. After incubation for 6 h at 37°C, 51Cr release from triplicate samples was measured. Data represent mean ⫾ SE. ⴱ, P ⬍ 0.001. C, calcium release assays using white blood cells of G-CSF-treated dTg mice showed a rapid rise in intracellular free calcium after Fc␣RI-cross-linking (F) and a delayed response after Fc␥RI (䡺) cross-linking. D, tubes were coated with PBS/HEPES (⽧), IgG (䡺), or IgA (F), after which oxygen radical production by human PMN cells was measured. Experiments were repeated three times, yielding similar results.

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Fc␥RI, suggesting competition for the FcR ␥ chain. No FcR ␥ chain competition was observed when Fc␥RIxHER-2/neu and Fc␣RIxCD20 BsAb were added, which is likely attributable to less efficient tumor cell lysis via BsAb targeting CD20 than HLA class II. This indicates that FcR ␥ chain cross-competition may occur in situations where both classes of receptors are maximally engaged, while numbers of effector cells are limited. However, at E:T ratios of 60:1 simultaneous triggering of both receptor classes still enhanced tumor cell killing (Fig. 5B), suggesting that even under these conditions only minimal FcR ␥ chain competition occurs. Notably, earlier data in mast cells supported strong competition between Fc⑀RI and FcR␥IIIa for the FcR ␥ chain (30, 31). It may, thus, be possible that differences exist between FcR ␥ chain levels in PMN cells versus mast cells or that expression of Fc⑀RI and Fc␥RIIIa is more strictly regulated than expression of Fc␣RI and Fc␥RI. Additionally, in mast cells, Fc␥RIIIa and Fc⑀RI exist as multisubunit complexes consisting of FcR ␤ chain and FcR ␥ signaling units (32). Rather than FcR ␥ chain, the FcR ␤ chain might, therefore, be limiting. Since Fc␣RI was capable of triggering early and late signaling events more potently than Fc␥RI (Fig. 6; Refs. 33 and 34), it is conceivable that different signaling pathways are initiated upon either Fc␣RI or Fc␥RI cross-linking. Simultaneous engagement of both pathways may, therefore, amplify effector functions. The higher capacity of Fc␣RI to initiate PMN cell activation might well be attributable to the presence of the positively charged amino acid (Arg209) in the transmembrane region of Fc␣RI (18). Because the FcR ␥ signaling chain bears a negatively charged amino acid in its transmembrane region, we hypothesize that this results in a stronger association of the FcR ␥ chain with Fc␣RI than with Fc␥RI (which lacks such a positively charged amino acid) (15). Alternatively, it is possible that the increased number of tumor antigens bound by effector cells resulted in enhanced tumor cell lysis. Whereas BsAb targeting Fc␥RI and CD20 were reported unable to initiate Ab-dependent cellular cytotoxicity, tumor cells were lysed in the presence of BsAb targeting Fc␣RI and CD20 (35). Also, in our experiments, Fc␣RI proved more efficient in initiating tumor cell killing. An attractive feature of Fc␥RI as target molecule, however, is the ability of this receptor to induce a “vaccine” effect. Fc␥RI was shown to initiate efficient antigen presentation in vitro and in vivo (22, 36), and recently a unique motif for antigen presentation has been identified in its cytoplasmic tail (37). We, therefore, speculate that targeting to Fc␥RI might induce a memory response to recirculating tumor cells. Indeed, Fc␥RI Tg mice with lymphoproliferative disease injected with BsAb (targeting Fc␥RI) were not only cured, but also protected against tumor rechallenge (28). In conclusion, this study documents the FcR ␥ chain not to be limiting for either expression or function of Fc␣RI or Fc␥RI on PMN cells. Because of this, Fc␣RI and Fc␥RI can be simultaneously engaged for induction of cell lysis, resulting in improved tumor cell killing. Importantly, it was shown that IgA1 and IgG1 anti-Ep-CAM Abs do not synergize (20). This has been attributed to binding of IgG1 anti-Ep-CAM Abs to inhibitory Fc␥RIIb (CD32) receptors on PMN cells, which would inhibit rather than enhance Fc␣RI-mediated killing. The usage of BsAb, selectively targeting to activatory PMN FcR, would overcome this problem and may thus be a prerequisite for combined treatment. Moreover, whereas Fc␣RI was shown to be more active in killing malignant cells, Fc␥RI might more potently induce a vaccine response. Immunotherapy, involving combined engagement of Fc␥RI and Fc␣RI, may, therefore, constitute an attractive option for the treatment of malignant disorders.

ACKNOWLEDGMENTS We thank Toon Hesp for excellent animal care and Dr. H. van Ojik for critical reading of this manuscript.

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