We examined the role of FcγR in antibody therapy of metastatic melanoma in wild-type and different FcγR knock-out mice. Treatment of B16F10-challenged wild-type mice with TA99 antibody specific for the gp75 tumor antigen resulted in a marked decrease in numbers of lung metastases. Treatment of individual FcγR knock-out mice revealed the high-affinity IgG receptor, FcγRI (CD64), to represent the central FcγR for TA99-induced antitumor effects. The potential of immune-modulating agents to further enhance the protective effect induced by monoclonal antibody (mAb) TA99 was examined in combination treatments consisting of mAb TA99 and a TLR-4 agonist, monophosphoryl lipid A (MPL). MPL did potently boost TA99 antibody-induced effects, and combination therapy was, again, found to be dependent on the presence of FcγRI. (Cancer Res 2006; 66(3): 1261-4)

Antibodies represent promising therapeutic candidates due to their high specificity and low toxicity profiles. The exact variables and mechanisms contributing to the therapeutic potential of anticancer antibodies remain unclear. Antibodies can initiate various effects, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement dependent cytotoxicity, and induction of apoptosis (1). A better understanding of the working mechanisms of therapeutic antibodies is essential to further enhance the efficacy of antibody therapy. In this study, we address the role of IgG receptors (FcγR) in antibody-induced antitumor activity. Four classes of murine leukocyte FcγR are currently distinguished [FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and the recently described FcγRIV], which differ in cell distribution and function. FcγRI, FcγRIII, and FcγRIV are activatory receptors, whereas FcγRII can mediate inhibitory effects (2, 3). The B16F10 lung metastasis model represents a validated model to study antibody therapy using TA99, a mouse IgG2a antibody recognizing the gp75 tumor antigen (4). Treatment of wild-type mice with TA99 after tumor challenge has been shown to markedly reduce numbers of lung metastases (5). A role for FcγR in TA99 antibody-mediated effects has been documented with the use of FcR γ-chain knock-out mice, which lack all activatory leukocyte FcγR (6, 7). The relative contribution of individual FcγR classes, however, has not been previously assessed. Antibody therapy is often combined with other treatment regimens, such as antiangiogenic or cytostatic drugs, to further enhance therapeutic efficacy. Agonists of Toll-like receptors (TLR) can effectively boost antibody-induced effects (8). Monophosphoryl lipid A (MPL), a TLR-4 agonist, is a chemically modified lipopolysaccharide (LPS) derived from Salmonella minnesota (9). As adjuvant effects of MPL in vivo have been documented (10, 11), we tested the influence of MPL on the outcome of treatment when administered in combination with TA99 antibody. In this report, we observed FcγRI to be essential for TA99 antibody-induced antitumor effects in C57Bl/6 mice. MPL further enhanced TA99-mediated antitumor effects.

Mice. C57Bl/6 wild-type mice were obtained from Janvier (Le Genest Saint Isle, France). FcγRI (CD64) knock-out mice (12), FcγRIII (CD16) knock-out mice (13), and FcR γ-chain knock-out mice (7), all in the C57Bl/6 background, were bred and maintained in the Central Animal Laboratory, Utrecht University. Experiments were done with 7- to 12-week-old mice and were all approved by the Utrecht University animal ethics committee.

Cell lines and TA99 antibody. The B16F10 mouse melanoma cell line was obtained from the National Cancer Institute (Frederick, MD). Cells were cultured in RPMI 1640 (Life Technologies, Paisley, United Kingdom) supplemented with 10% fetal bovine serum (Integro, Dieren, the Netherlands), 50 units/mL penicillin (Life Technologies), and 50 μg/mL streptomycin (Life Technologies). Hybridoma HB-8704 (American Type Culture Collection, Manassas, VA), which produces TA99 antibody, was cultured under serum-free conditions with HyQ ADCF-monoclonal antibody (mAb) medium (Hyclone, Logan, UT). Monoclonal antibody TA99 (mouse IgG2a), directed against the gp75 antigen present on B16F10 melanoma cells, was purified from hybridoma supernatants by protein A-Sepharose chromatography.

MPL. MPL, a derivative of lipid A from Salmonella minnesota, was obtained from Corixa (Seattle, WA).

Melanoma model. Wild-type mice, FcR γ-chain knock-out, FcγRI knock-out, or FcγRIII knock-out mice, were injected i.v. with 1.5 × 105 B16F10 tumor cells (in 100 μL saline) on day 0. For treatments with an antibody dosage of 200 μg (6), mice were injected i.p. with TA99 antibody (or PBS as control) on days 0, 2, 4, 7, 9, and 11. For combination treatments with suboptimal antibody and MPL concentrations, mice were injected with MPL (0.5 μg in 100 μL PBS) or 100 μL PBS s.c. on days −1, 4, and 8. A suboptimal dose of TA99 antibody (10 μg in 100 μL PBS) or 100 μL PBS (as control) were injected i.p. at days 0, 2, 4, 7, 9, and 11. At day 21, mice were sacrificed, and lungs were scored for numbers of metastases and tumor load. Tumor load was defined as the sum of the following scores: metastases <1 mm were scored as 1; metastases between 1 and 2 mm were scored as 3; and metastases >2 mm were scored as 10, as detailed in ref. (14).

Statistical analyses. ANOVA analyses were done using SPSS software (Chicago, IL). All experiments were done a minimum of two times. Ps < 0.05 were considered significant.

To dissect the role of individual FcγR in antibody therapy of melanoma, we injected wild-type and FcγR knock-out mice i.v. with B16F10 tumor cells and studied the effect of antibody treatment. The TA99 antibody, specific for the gp75 tumor antigen, induced a profound protective effect in wild-type mice, which was abrogated in FcR γ-chain−/− mice (Fig. 1). These results confirmed an earlier report, exemplifying the importance of activating FcγR in antibody-mediated antitumor effects (6). We then did experiments in FcγRI and FcγRIII knockout mice. FcγRI represents the sole FcγR class capable of binding monomeric IgG with high affinity (2) and can potently initiate various immune cell functions, including antibody-dependent, cell-mediated cytotoxicity; antigen uptake; and induction of antigen presentation (12). FcγRI−/− mice exhibit various defects, such as an impaired phagocytosis of IgG2a-immune complexes, impaired ADCC, and antigen presentation (12). FcγRIII plays a role in anaphylactic and inflammatory responses (13). Antibody TA99 induced a profound antitumor effect in FcγRIII knockout mice (Fig. 1). Expression of FcγRI proved essential for mAb TA99-mediated effects, as antibody treatment in FcγRI−/− mice induced no therapeutic effect (Fig. 1). These data indicated FcγRI to be instrumental for the TA99-induced effects.

Figure 1.
Figure 1. FcγRI is essential for antitumor effects induced by TA99 antibody. Wild-type, FcR γ-chain−/−, FcγRI−/−, or FcγRIII−/− mice were challenged i.v. with 1.5 × 105 B16F10 tumor cells and injected i.p. with 200 μg mAb TA99 or PBS (control) on days 0, 2, 4, 7, 9, and 11. After 21 days, mice were sacrificed, lungs were excised, and tumor loads were scored as detailed in Materials and Methods. A, lungs of FcγRI−/− and FcγRIII−/− mice treated with PBS or mAb TA99; black nodules represent metastases. B, tumor load scores in wild-type, FcR γ-chain−/−, FcγRI−/−, and FcγRIII−/− mice. Tumor loads in PBS-treated mice were set at 100%. Columns, average; bars, SE. Representative for two experiments, each with six mice per group.
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FcγRI is essential for antitumor effects induced by TA99 antibody. Wild-type, FcR γ-chain−/−, FcγRI−/−, or FcγRIII−/− mice were challenged i.v. with 1.5 × 105 B16F10 tumor cells and injected i.p. with 200 μg mAb TA99 or PBS (control) on days 0, 2, 4, 7, 9, and 11. After 21 days, mice were sacrificed, lungs were excised, and tumor loads were scored as detailed in Materials and Methods. A, lungs of FcγRI−/− and FcγRIII−/− mice treated with PBS or mAb TA99; black nodules represent metastases. B, tumor load scores in wild-type, FcR γ-chain−/−, FcγRI−/−, and FcγRIII−/− mice. Tumor loads in PBS-treated mice were set at 100%. Columns, average; bars, SE. Representative for two experiments, each with six mice per group.

Figure 1.
Figure 1. FcγRI is essential for antitumor effects induced by TA99 antibody. Wild-type, FcR γ-chain−/−, FcγRI−/−, or FcγRIII−/− mice were challenged i.v. with 1.5 × 105 B16F10 tumor cells and injected i.p. with 200 μg mAb TA99 or PBS (control) on days 0, 2, 4, 7, 9, and 11. After 21 days, mice were sacrificed, lungs were excised, and tumor loads were scored as detailed in Materials and Methods. A, lungs of FcγRI−/− and FcγRIII−/− mice treated with PBS or mAb TA99; black nodules represent metastases. B, tumor load scores in wild-type, FcR γ-chain−/−, FcγRI−/−, and FcγRIII−/− mice. Tumor loads in PBS-treated mice were set at 100%. Columns, average; bars, SE. Representative for two experiments, each with six mice per group.
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FcγRI is essential for antitumor effects induced by TA99 antibody. Wild-type, FcR γ-chain−/−, FcγRI−/−, or FcγRIII−/− mice were challenged i.v. with 1.5 × 105 B16F10 tumor cells and injected i.p. with 200 μg mAb TA99 or PBS (control) on days 0, 2, 4, 7, 9, and 11. After 21 days, mice were sacrificed, lungs were excised, and tumor loads were scored as detailed in Materials and Methods. A, lungs of FcγRI−/− and FcγRIII−/− mice treated with PBS or mAb TA99; black nodules represent metastases. B, tumor load scores in wild-type, FcR γ-chain−/−, FcγRI−/−, and FcγRIII−/− mice. Tumor loads in PBS-treated mice were set at 100%. Columns, average; bars, SE. Representative for two experiments, each with six mice per group.

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Choice of antibody isotype can influence binding to Fcγ receptors. Mouse IgG2a antibodies can bind FcγRI and FcγRIII, albeit with far higher affinity to FcγRI (12). However, in the case of the absence of FcγRI, the TA99 antibody should still be able to bind to FcγRIII, thus not explaining the dramatic effect on antitumor response in FcγRI−/− mice. If an antibody of another subclass would be employed (e.g., IgG1), different results might be expected, although this antibody would still be able to bind to FcγRI. Very recently, a new class of murine FcγR, FcγRIV, has been characterized as an IgG2a and IgG2b receptor (3). Because the effect of TA99 antibody was observed to be absent in FcγRI−/− mice, FcγRIV may play only a minor role, if any, in TA99 antibody-induced effects.

We next evaluated the influence of MPL on TA99-induced antitumor effects. MPL is a TLR-4 agonist, which has similar adjuvant properties as LPS, without inducing toxicity (9). With suboptimal amounts of TA99 or MPL used as monotherapies, mice developed metastases (Fig. 2). Combination of TA99 antibody and MPL, however, consistently led to lower numbers of metastases (Fig. 2). The FcγR dependency of TA99 effects in the presence of MPL was analyzed in FcR γ-chain−/− mice. These animals were challenged with tumor cells and injected with antibody or MPL alone or in combination. The protective effect of the combination was abrogated in FcR γ-chain−/− mice (Fig. 3A). We evaluated the contribution of FcγRI by challenging FcγRI−/− mice with B16F10 tumor cells followed by treatment with antibody, MPL, or antibody plus MPL. FcγRI was, again, found essential for induction of a therapeutic effect with the combination therapy (Fig. 3B).

Figure 2.
Figure 2. Effect of MPL on therapeutic efficacy of TA99 antibody. Wild-type mice were challenged i.v. with tumor cells and treated with PBS, a suboptimal dose of mAb TA99 (10 μg), a suboptimal dose of MPL (0.5 μg), or with TA99 plus MPL. After 21 days, mice were sacrificed, lungs were excised, and tumor loads scored as detailed in Materials and Methods. A, lungs of wild-type mice; black nodules represent metastases. B, tumor loads scored in wild-type mice. Representative for two experiments, each with six mice per group.
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Effect of MPL on therapeutic efficacy of TA99 antibody. Wild-type mice were challenged i.v. with tumor cells and treated with PBS, a suboptimal dose of mAb TA99 (10 μg), a suboptimal dose of MPL (0.5 μg), or with TA99 plus MPL. After 21 days, mice were sacrificed, lungs were excised, and tumor loads scored as detailed in Materials and Methods. A, lungs of wild-type mice; black nodules represent metastases. B, tumor loads scored in wild-type mice. Representative for two experiments, each with six mice per group.

Figure 2.
Figure 2. Effect of MPL on therapeutic efficacy of TA99 antibody. Wild-type mice were challenged i.v. with tumor cells and treated with PBS, a suboptimal dose of mAb TA99 (10 μg), a suboptimal dose of MPL (0.5 μg), or with TA99 plus MPL. After 21 days, mice were sacrificed, lungs were excised, and tumor loads scored as detailed in Materials and Methods. A, lungs of wild-type mice; black nodules represent metastases. B, tumor loads scored in wild-type mice. Representative for two experiments, each with six mice per group.
View largeDownload slide

Effect of MPL on therapeutic efficacy of TA99 antibody. Wild-type mice were challenged i.v. with tumor cells and treated with PBS, a suboptimal dose of mAb TA99 (10 μg), a suboptimal dose of MPL (0.5 μg), or with TA99 plus MPL. After 21 days, mice were sacrificed, lungs were excised, and tumor loads scored as detailed in Materials and Methods. A, lungs of wild-type mice; black nodules represent metastases. B, tumor loads scored in wild-type mice. Representative for two experiments, each with six mice per group.

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Figure 3.
Figure 3. FcγRI is essential for combination therapy. FcR γ-chain−/− (A) or FcγRI−/− mice (B) were challenged i.v. with tumor cells and treated with PBS, a suboptimal dose of mAb TA99 (10 μg), a suboptimal dose of MPL (0.5 μg), or with TA99 combined with MPL. After 21 days, mice were sacrificed, lungs were excised, and tumor loads scored. Representative for two experiments, each with six mice per group.
View largeDownload slide

FcγRI is essential for combination therapy. FcR γ-chain−/− (A) or FcγRI−/− mice (B) were challenged i.v. with tumor cells and treated with PBS, a suboptimal dose of mAb TA99 (10 μg), a suboptimal dose of MPL (0.5 μg), or with TA99 combined with MPL. After 21 days, mice were sacrificed, lungs were excised, and tumor loads scored. Representative for two experiments, each with six mice per group.

Figure 3.
Figure 3. FcγRI is essential for combination therapy. FcR γ-chain−/− (A) or FcγRI−/− mice (B) were challenged i.v. with tumor cells and treated with PBS, a suboptimal dose of mAb TA99 (10 μg), a suboptimal dose of MPL (0.5 μg), or with TA99 combined with MPL. After 21 days, mice were sacrificed, lungs were excised, and tumor loads scored. Representative for two experiments, each with six mice per group.
View largeDownload slide

FcγRI is essential for combination therapy. FcR γ-chain−/− (A) or FcγRI−/− mice (B) were challenged i.v. with tumor cells and treated with PBS, a suboptimal dose of mAb TA99 (10 μg), a suboptimal dose of MPL (0.5 μg), or with TA99 combined with MPL. After 21 days, mice were sacrificed, lungs were excised, and tumor loads scored. Representative for two experiments, each with six mice per group.

Close modal

MPL is known to activate macrophages and dendritic cells, and adjuvant studies in vivo documented MPL to induce antigen-specific CTL and to skew T-helper balance toward a Th1 phenotype (11, 15). MPL improve the capacity of B cells and macrophages to prime T and B cells and induce the maturation of splenic dendritic cells in situ. Furthermore, MPL triggers cytokine production, which can have an effect on the development of both humoral and cellular immune responses (16), indicating MPL to be a suitable candidate adjuvant for antibody therapy.

The effector cells, which can be involved in antibody-mediated antitumor effects, are natural killer (NK) cells, polymorphonuclear (PMN), monocytes, and macrophages. Earlier studies examining effector cells in the B16 model during antibody treatment excluded a role for NK cells and B and T cells (17). In these studies, microscopic analyses of lung tissues documented abundant infiltration of macrophages (5), supporting a role for macrophages during antibody therapy. Murine FcγRI is expressed on monocytes, macrophages, and dendritic cells but not on PMN (12). As we observed FcγRI to be central for antibody-mediated antitumor effects, we hypothesize FcγRI-expressing monocytes/macrophages to be of importance for the TA99-induced antitumor effects and not NK cells or PMN. We are currently performing studies (e.g., monocytes/macrophage depletion with use of clodronate liposomes) to further address the role of the effector cells involved in this model.

The importance of human Fcγ receptors for tumor therapy has been documented, where FcγR polymorphisms were shown to affect the outcome of antibody treatments in cancer patients (18–20). A better understanding of the role of individual FcγR in antibody therapies is important to further optimize antibody therapeutic approaches in man.

Note: J.G.J. van de Winkel and J.H.W. Leusen contributed equally to this work.

Grant support: Dutch Cancer Society “K.W.F.” grant UU 2001-2496.

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.

We thank Anja van der Sar, Toon Hesp, Wendy Kaspers, Sabine Versteeg, Agnes Goderie, and Gerard Geelen for excellent animal care; Esther Rudolph and Patrick Luijk for help with digital photography; and Soeniel Jhakrie, Edwin van Voskuilen, Marcel Brandhorst, and Judy Bos-de Ruijter for culturing and purification of TA99 antibody.

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2006