HIF activation enhances FcγRIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy.

BACKGROUND: Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb. METHODS: We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. RESULTS: We report that TAMs are FcγRIIbbright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes. CONCLUSION: Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.

Fc-Engineering for Modulated Effector Functions-Improving Antibodies for Cancer Treatment.

The majority of monoclonal antibody (mAb) therapeutics possess the ability to engage innate immune effectors through interactions mediated by their fragment crystallizable (Fc) domain. By delivering Fc-Fc gamma receptor (FcγR) and Fc-C1q interactions, mAb are able to link exquisite specificity to powerful cellular and complement-mediated effector functions. Fc interactions can also facilitate enhanced target clustering to evoke potent receptor signaling. These observations have driven decades-long research to delineate the properties within the Fc that elicit these various activities, identifying key amino acid residues and elucidating the important role of glycosylation. They have also fostered a growing interest in Fc-engineering whereby this knowledge is exploited to modulate Fc effector function to suit specific mechanisms of action and therapeutic purposes. In this review, we document the insight that has been generated through the study of the Fc domain; revealing the underpinning structure-function relationships and how the Fc has been engineered to produce an increasing number of antibodies that are appearing in the clinic with augmented abilities to treat cancer.

STING Activation Reverses Lymphoma-Mediated Resistance to Antibody Immunotherapy.

Tumors routinely attract and co-opt macrophages to promote their growth, angiogenesis, and metastasis. Macrophages are also the key effector cell for mAb therapies. Here we report that the tumor microenvironment creates an immunosuppressive signature on tumor-associated macrophages (TAM), which favors expression of inhibitory rather than activating Fcγ receptors (FcγR), thereby limiting the efficacy of mAb immunotherapy. We assessed a panel of TLR and STING agonists (a) for their ability to reprogram macrophages to a state optimal for mAb immunotherapy. Both STINGa and TLRa induced cytokine release, modulated FcγR expression, and augmented mAb-mediated tumor cell phagocytosis in vitro However, only STINGa reversed the suppressive FcγR profile in vivo, providing strong adjuvant effects to anti-CD20 mAb in murine models of lymphoma. Potent adjuvants like STINGa, which can improve FcγR activatory:inhibitory (A:I) ratios on TAM, are appealing candidates to reprogram TAM and curb tumor-mediated immunosuppression, thereby empowering mAb efficacy. Cancer Res; 77(13); 3619-31. ©2017 AACR.