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.

The Role of Antigen Presentation in Tumor-Associated Macrophages.

Macrophages are cells of the myeloid lineage with important roles not only in immune regulation and tissue repair, but also in pathological states such as autoimmune disease and cancer. A plethora of macrophage subtypes exist with distinct phenotypes and functions, not least within the tumor microenvironment (TME) of solid tumors. The abundant macrophages located within the TME are often referred to as tumor-associated macrophages (TAMs). TAMs may be pro-inflammatory with antitumor properties, or may have pro-tumor functions such as angiogenesis. Typically, TAMs are endowed with pro-tumor phenotypes, which has led to strategies to deplete or reprogram TAMs within the TME. Although historically recognized as professional antigen presenting cells (APCs), macrophages are often considered inferior in their abilities to process and present antigens in comparison with dendritic cells (DCs). Notwithstanding, this review gives an overview of the potential accessory role that macrophages might have in antigen processing and presentation to T cells within the TME, with implications for the design of novel immunotherapies.

Shaving Is an Epiphenomenon of Type I and II Anti-CD20-Mediated Phagocytosis, whereas Antigenic Modulation Limits Type I Monoclonal Antibody Efficacy.

Rituximab is an anti-CD20 mAb used in the treatment of B cell malignancies. Loss of surface CD20 Ag from the surface of target cells is thought to be one mechanism governing resistance to rituximab, but how this occurs is not completely understood. Two explanations for this have been proposed: antigenic modulation whereby mAb:CD20 complexes are internalized in a B cell intrinsic process and shaving, in which mAb:CD20 complexes undergo trogocytic removal by effector cells, such as macrophages. However, there is conflicting evidence as to which predominates in clinical scenarios and hence the best strategies to overcome resistance. In this study, we investigated the relative importance of modulation and shaving in the downregulation of surface mAb:CD20. We used both murine and human systems and treated ex vivo macrophages with varying concentrations of non-FcγR-interacting beads to achieve differential macrophage saturation states, hence controllably suppressing further phagocytosis of target cells. We then monitored the level and localization of mAb:CD20 using a quenching assay. Suppression of phagocytosis with bead treatment decreased shaving and increased modulation, suggesting that the two compete for surface rituximab:CD20. Under all conditions tested, modulation predominated in rituximab loss, whereas shaving represented an epiphenomenon to phagocytosis. We also demonstrate that the nonmodulating, glycoengineered, type II mAb obinutuzumab caused a modest but significant increase in shaving compared with type II BHH2 human IgG1 wild-type mAb. Therefore, shaving may represent an important mechanism of resistance when modulation is curtailed, and glycoengineering mAb to increase affinity for FcγR may enhance resistance because of shaving.

New revelations from an old receptor: Immunoregulatory functions of the inhibitory Fc gamma receptor, FcγRIIB (CD32B).

The Fc gamma receptor IIB (FcγRIIB/CD32B) was generated million years ago during evolution. It is the sole inhibitory receptor for IgG, and has long been associated with the regulation of humoral immunity and innate immune homeostasis. However, new and surprising functions of FcγRIIB are emerging. In particular, FcγRIIB has been shown to perform unexpected activatory roles in both immune-signaling and monoclonal antibody (mAb) immunotherapy. Furthermore, although ITIM signaling is an integral part of FcγRIIB regulatory activity, it is now clear that inhibition/activation of immune responses can occur independently of the ITIM. In light of these new findings, we present an overview of the established and noncanonical functions of FcγRIIB and discuss how this knowledge might be exploited therapeutically.

Detection of Experimental and Clinical Immune Complexes by Measuring SHIP-1 Recruitment to the Inhibitory FcγRIIB.

Fc γ receptors (FcγR) are involved in multiple aspects of immune cell regulation, are central to the success of mAb therapeutics, and underpin the pathology of several autoimmune diseases. However, reliable assays capable of accurately measuring FcγR interactions with their physiological ligands, IgG immune complexes (IC), are limited. A method to study and detect IC interactions with FcγRs was therefore developed. This method, designed to model the signaling pathway of the inhibitory FcγRIIB (CD32B), used NanoLuc Binary Interaction Technology to measure recruitment of the Src homology 2 domain-containing inositol phosphatase 1 to the ITIM of this receptor. Such recruitment required prior cross-linking of an ITAM-containing activatory receptor, and evoked luciferase activity in discrete clusters at the cell surface, recapitulating the known biology of CD32B signaling. The assay detected varying forms of experimental IC, including heat-aggregated IgG, rituximab-anti-idiotype complexes, and anti-trinitrophenol-trinitrophenol complexes in a sensitive manner (≤1 µg/ml), and discriminated between complexes of varying size and isotype. Proof-of-concept for the detection of circulating ICs in autoimmune disease was provided, as responses to sera from patients with systemic lupus erythematosus and rheumatoid arthritis were detected in small pilot studies. Finally, the method was translated to a stable cell line system. In conclusion, a rapid and robust method for the detection of IC was developed, which has numerous potential applications including the monitoring of IC in autoimmune diseases and the study of underlying FcγR biology.

Therapeutic Antibodies: What Have We Learnt from Targeting CD20 and Where Are We Going?

Therapeutic monoclonal antibodies (mAbs) have become one of the fastest growing classes of drugs in recent years and are approved for the treatment of a wide range of indications, from cancer to autoimmune disease. Perhaps the best studied target is the pan B-cell marker CD20. Indeed, the first mAb to receive approval by the Food and Drug Administration for use in cancer treatment was the CD20-targeting mAb rituximab (Rituxan®). Since its approval for relapsed/refractory non-Hodgkin's lymphoma in 1997, rituximab has been licensed for use in the treatment of numerous other B-cell malignancies, as well as autoimmune conditions, including rheumatoid arthritis. Despite having a significant impact on the treatment of these patients, the exact mechanisms of action of rituximab remain incompletely understood. Nevertheless, numerous second- and third-generation anti-CD20 mAbs have since been developed using various strategies to enhance specific effector functions thought to be key for efficacy. A plethora of knowledge has been gained during the development and testing of these mAbs, and this knowledge can now be applied to the design of novel mAbs directed to targets beyond CD20. As we enter the "post-rituximab" era, this review will focus on the lessons learned thus far through investigation of anti-CD20 mAb. Also discussed are current and future developments relating to enhanced effector function, such as the ability to form multimers on the target cell surface. These strategies have potential applications not only in oncology but also in the improved treatment of autoimmune disorders and infectious diseases. Finally, potential approaches to overcoming mechanisms of resistance to anti-CD20 therapy are discussed, chiefly involving the combination of anti-CD20 mAbs with various other agents to resensitize patients to treatment.

Regulation of Monoclonal Antibody Immunotherapy by FcγRIIB.

Monoclonal antibodies (mAb) are revolutionising the treatment of many different diseases. Given their differing mode of action compared to most conventional chemotherapeutics and small molecule inhibitors, they possess the potential to be independent of common modes of treatment resistance and can typically be combined readily with existing treatments without dose-limiting toxicity. However, treatments with mAb rarely result in cure and so a full understanding of how these reagents work and can be optimised is key for their subsequent improvement. Here we review how an understanding of the biology of the inhibitory Fc receptor, FcγRIIB (CD32B), is leading to the development of improved mAb treatments.