Reciprocal Regulation of Activating and Inhibitory Fcγ Receptors by TLR7/8 Activation: Implications for Tumor Immunotherapy

Abstract

Purpose: Activation of Toll-like receptors (TLR) 7 and 8 by engineered agonists has been shown to aid in combating viruses and tumors. Here, we wished to test the effect of TLR7/8 activation on monocyte Fcγ receptor (FcγR) function, as they are critical mediators of antibody therapy.Experimental Design: The effect of the TLR7/8 agonist R-848 on cytokine production and antibodydependent cellular cytotoxicity by human peripheral blood monocytes was tested. Affymetrix microarrays were done to examine genomewide transcriptional responses of monocytes to R-848 and Western blots were done to measure protein levels of FcγR. Murine bone marrow-derived macrophages from WT and knockout mice were examined to determine the downstream pathway involved with regulating FcγR expression. The efficacy of R-848 as an adjuvant for antibody therapy was tested using a CT26-HER2/neu solid tumor model.Results: Overnight incubation with R-848 increased FcγR-mediated cytokine production and antibodydependent cellular cytotoxicity in human peripheral blood monocytes. Expression of FcγRI, FcγRIIa, and the common γ-subunit was increased. Surprisingly, expression of the inhibitory FcγRIIb was almost completely abolished. In bone marrow-derived macrophage, this required TLR7 and MyD88, as R-848 did not increase expression of the γ-subunit in TLR7 −/− nor MyD88 Monocyte Fcγ receptors (FcγR) mediate clearance of IgG-immune complexes and IgG-coated tumor targets. Binding of IgG complexes to FcγR results in receptor clustering, which activates downstream events such as phagocytosis (1), release of reactive oxygen species (2), and cytokine production (3).The strength of FcγR response is largely determined by the ratio of activating (FcγRI, FcγRIIa, FcγRIII, and the γ-subunit) to inhibitory (FcγRIIb) receptors, as mice genetically deleted for FcγRIIb show markedly enhanced antibody-mediated tumor clearance in vivo (4). Conversely, mice lacking the common γ-subunit show very poor antibody-dependent cytotoxicity as mice do not express the γ-subunit-independent FcγRIIa (5). It has also been shown that Toll-like receptor (TLR) activation can enhance FcγR expression and function. For example, the TLR4 ligand lipopolysaccharide has been shown to increase FcγR-mediated phagocytosis (6) and tumor cell lysis (7). Unmethylated DNA (CpG oligonucleotides), which activates TLR9, has also proven effective, enhancing antibody-dependent cellular cytotoxicity (ADCC) against tumors (8).Agonists of TLR7 and TLR8 have come to light as an effective means of enhancing immune responses. The TLR7 agonist imiquimod has been shown in vivo to reduce the growth of MC-26 tumor cells (9), an effect abolished by blocking IFN-α. Both TLR7 and TLR7/8 agonists show antitumor (10) and antiviral (11) activities. Their major mode of action seems to be induction of cytokine production, leading to stronger proinflammatory responses (12).Here, we have studied the effects of the TLR7/8 agonist R-848 on human monocytes within the context of FcγR expression and function. Results sho...

VTX-2337 Is a Novel TLR8 Agonist That Activates NK Cells and Augments ADCC

“…This suggests the opportunity of using VTX-2337 in combination with approved mAbs, where ADCC contributes to the clinical efficacy (17). The potential for TLR agonists to enhance NK cell function and increase ADCC have been documented in previous publications (21,(38)(39)(40). For example, CpG ODN has been reported to increase IFNg production of NK cells and enhance trastuzumab-mediated lysis of breast cancer cells and rituximab-mediated lysis of lymphoma cells (21,39).…”

“…For example, CpG ODN has been reported to increase IFNg production of NK cells and enhance trastuzumab-mediated lysis of breast cancer cells and rituximab-mediated lysis of lymphoma cells (21,39). The TLR7/8 agonist resiquimod has also been shown to enhance FcgR function and ADCC and enhance the antitumor effect of HER2-targeted mAb therapy in a mouse model (40). Interestingly, we did not observe IFNg induction (Table 1) or the enhancement of ADCC by CpG ODN2006 (Supplementary Fig.…”

Toll-like Receptor 2 Ligands Regulate Monocyte Fcγ Receptor Expression and Function

“…Pam2CSK4 Enhances Antibody Therapy in Vivo-We next tested whether Pam2CSK4 could enhance antibody-mediated antitumor responses using a murine model of antibody therapy (22)(23)(24). Here, syngeneic CT26 colon adenocarcinoma cells engineered to express human Her2/neu were injected into the flanks of Balb/cJ mice.…”

“…Cells were centrifuged and resuspended at 10 ϫ 10 6 cells/ml in RPMI 1640. The murine tumor model (22) was used as described previously (23,24). Briefly, 5-week-old female Balb/cJ mice (The Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously with 1 ϫ 10 6 of syngeneic CT26-HER2/neu cells.…”

Systemic Cancer Therapy with a Small Molecule Agonist of Toll-like Receptor 7 Can Be Improved by Circumventing TLR Tolerance

“…In addition, Butchar and colleagues showed that TLR7 ligands improve the outcome of treatment with monoclonal antibodies by increasing Fc-g receptor expression and thus enhancing antibody-mediated cellular cytotoxicity (43). We have shown earlier that application of TLR7-activating RNA oligonucleotides can prevent growth of NK cell-sensitive tumors by activation of NK cells via TLR7-expressing DCs (44).…”