Fc-Receptor Interactions Regulate Both Cytotoxic and Immunomodulatory Therapeutic Antibody Effector Functions David J

Home , FCGR1A, FCGR2A, FCGR2B, Fc receptor

Masters of Immunology Cancer Immunology Research Fc-Receptor Interactions Regulate Both Cytotoxic and Immunomodulatory Therapeutic Antibody Effector Functions David J. DiLillo and Jeffrey V. Ravetch

Abstract

Antibodies are now recognized as key therapeutic tools to through their engagement of Fc receptor (FcR) family members. It combat most forms of malignancy. Although the first wave of is now clear that multiple variables, including the nature of the therapeutic antibodies that emerged over two decades ago directly target molecules, the local presence of effector cells, and the target tumor cells for killing, a new class of antibody therapies expression patterns of FcRs, will dictate whether and how an targeting immunoregulatory pathways to boost antitumor antibody will necessitate interactions with FcRs to mediate opti- immune responses by activating the immune system is poised mal therapeutic effects. Thus, through careful in vivo mechanistic for clinical success. A notable common characteristic of both analyses of individual therapeutic antibodies, Fc domains engi- classes of therapeutic antibodies is the importance of the IgG Fc neered for optimal engagement of the appropriate cellular domain, which connects the fine specificity of an antibody with FcRs must be designed to maximize clinical success. Cancer Immunol immune cells that mediate antibody-triggered effector functions Res; 3(7); 704–13. 2015 AACR.

Disclosure of Potential Conﬂicts of Interest No potential conﬂicts of interest were disclosed.

Editor's Disclosures The following editor(s) reported relevant ﬁnancial relationships. R. Vonderheide—None.

CME Staff Planners' Disclosures The members of the planning committee have no real or apparent conﬂicts of interest to disclose.

Learning Objectives Antibodies have been used successfully to target either tumor cells for direct killing or immunoregulatory pathways to boost antitumor immune responses. The immunoglobulin constant region, termed the Fc domain, connects the ﬁne-speciﬁcity of the antibody with immune cells that mediate antibody-triggered effector functions. The nature of the target molecules, the local presence of immune effector cells, and the expression patterns of Fc-receptor (FcR) family members dictate whether and how an antibody will interact with an FcR to mediate optimal therapeutic effects. Upon completion of this activity, the participant should gain a basic knowledge of FcR biology and the various classes of antibodies being developed for cancer immunotherapy.

Acknowledgment of Financial or Other Support This activity does not receive commercial support.

Introduction often-overlooked characteristic of antibodies is their bifunctional nature. The variable Fab region of an antibody mediates specificity Antibodies have become a major therapeutic tool for the and dictates to what antigen and with what affinity the antibody treatment of all classes of malignancy. A well-defined but will bind its target. However, antibodies also contain a constant region, termed the Fc domain, which engages a diversity of cellular receptors, thereby triggering antibody-mediated effector func- The Laboratory of Molecular Genetics and Immunology, The Rock- tions. The Fc domain acts as a bridge between the specificity efeller University, New York, New York. dictated by the Fab region and cells of the innate and adaptive Corresponding Author: Jeffrey V. Ravetch, Rockefeller University, 1230 York immune system. Avenue, New York, NY 10065. Phone: 212-327-7321; Fax: 212-327-7319; E-mail: The first therapeutic antibodies designed almost three decades [email protected] ago targeted tumor antigens, and were developed based on their doi: 10.1158/2326-6066.CIR-15-0120 intrinsic activity to mediate tumoricidal effects by Fab-mediated 2015 American Association for Cancer Research. cross-linking of target molecules to trigger proapoptotic signaling

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A Type I FcgRs in mice

FcgRI FcgRIIB FcgRIII FcgRIV (Fcgr1) (Fcgr2b) (Fcgr3) (Fcgr4)

ITIM ITAM ITAM ITAM γ Expression: FcR -chain Mono/Mac + + + + NK cell – – + – Neutrophil + + + + Dendritic cell + + + + Figure 1. B cell – + – – Mouse (A) and human (B) type I FcgR family members and expression patterns. , inducible upon cytokine B Type I FcgRs in humans stimulation; , inducible upon cytokine stimulation in immature DCs; FcgRI FcgRIIA FcgRIIB FcgRIIC FcgRIIIA FcgRIIIB , functional FcgRIIC is expressed by (FCGR1A) (FCGR2A) (FCGR2B) (FCGR2C) (FCGR3A) (FCGR3B) 10% of individuals.

ITIM ITAM ITAM ITAM ITAM FcR γ-chain Expression: Mono/Mac + + + – + – NK cell – – – +*** + – Neutrophil –* + + – - + Dendritic cell –** + + – – – B cell – – + – – –

cascades or through inhibition of autocrine loops. Little con- to next-generation therapies with enhanced antitumor activities sideration was placed on the potential contributions from the capable of sustained responses. Fc domains and Fc receptors (FcR). However, it is now clear that the Fc domain plays an instrumental role in the effector Fc Receptor Expression and Function mechanisms elicited by multiple classes of therapeutic antibodies, whether they directly target tumor cells or alternatively Upon binding their cognate antigens, IgG antibodies mediate target the immune system, modulating either positive or neg- downstream effector functions by interacting with either type I or ative regulatory pathways. In this Masters of Immunology type II FcRs. Type I FcRs are members of the immunoglobulin (Ig) primer, we discuss FcR biology and the mechanisms of action superfamily and include the canonical FcRs for IgG (FcgR; Fig. 1; of cytotoxic antitumor antibodies as well as newer classes of ref. 1). Type II FcRs are members of the C-type lectin receptor agonistic/antagonistic immunomodulatory antibodies, with an family and currently include CD209 (also known as DC-SIGN, emphasis on how they differentially engage various members which is homologous to SIGN-R1 in mice) and CD23. Whether of the FcR family to mediate their effector functions. New data type I or type II FcRs are engaged by an antibody is determined by demonstrating that cytotoxic antitumor antibodies can stimu- the conformational state of its Fc domain, which is regulated by late long-term antitumor T-cell memory responses through an the complex, biantennary N-linked glycan attached to Asn297 FcR-dependent "vaccinal effect" are also discussed. Through a (1, 2). Thus, differences in Fc glycosylation at Asn297 can lead to thoughtful analysis of their respective mechanisms of action, modulated afﬁnities for FcRs: sialylated Fc domains adopt a more engineering Fc domains of therapeutic antibodies for optimal ﬂexible, "closed" conformation that allows engagement of type II engagement of appropriate members of the FcR family will lead FcRs and reduces binding to type I FcRs (2, 3). By contrast,

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nonsialylated Fc domains assume an "open" conformation, there- tially engages the inhibitory msFcgRIIB receptor (11). An activating: by allowing binding to type I FcRs and preventing engagement of inhibitory (A:I) ratio can be assigned to each antibody isotype by type II FcRs. Detailed biochemical mechanisms regulating type I creating a ratio of the Fc's affinity for the relevant activating FcgR and type II FcR engagement, as well as the anti-inflammatory versus the Fc's affinity for the inhibitory FcgR. Thus, msIgG2a downstream consequences of type II FcR engagement, have antibodies have an A:I ratio of approximately 70, whereas msIgG1 been reviewed recently (1, 4, 5). Engagement of type II FcRs by antibodies show an A:I ratio of 0.1. The in vivo activities of msIgG2a sialylated IgG typically results in suppression of antibody- and T and msIgG1 antibodies tightly correlate with their A:I ratios, as cell–mediated inflammation (6), as exemplified by the use of antitumor antibodies of the msIgG2a subclass potently activate intravenous immunoglobulin (IVIG) in patients suffering from effector cells to kill msIgG2a antibody-opsonized target cells, various autoimmune diseases. By contrast, engagement of type I whereas msIgG1 antibodies demonstrate little tumoricidal activity FcRs results in antibody-dependent cellular cytotoxicity and in vivo (11). Human IgG isotypes also demonstrate preferential phagocytosis (ADCC and ADCP), demonstrating the ability of engagement of FcgRs, but to a lesser degree than the murine FcgR an IgG to bridge target cells/pathogens and FcgR-expressing system, and without selective engagement of either activating or effector cells to mediate cytotoxicity or phagocytosis (7). Type I inhibitory receptors. Human (hu)IgG2 and IgG4 interact poorly FcRs also engage antigen–antibody immune complexes (IC) and with huFcgRs, whereas huIgG1 and huIgG3 interact more strongly mediate their downstream immunomodulatory effects on anti- (12). Further, allelic variants of huFcgRs exist in the human pop- gen-presenting cells (APC) and B cells. Immunomodulatory ulation that can considerably affect binding affinities to the huIgG1 effects mediated by ICs are observed on dendritic cells (DC), and huIgG3 subclasses. For example, the FcgRIIIA158V allele has a where ICs can enhance antigen uptake and regulate DC matura- higher affinity for huIgG1 than the FcgRIIIA158F allele, and the tion in an FcgR-dependent fashion, thereby shaping T-cell FcgRIIA131H allele demonstrates increased binding to huIgG1 than responses (8). ICs can also regulate affinity maturation of B FcgRIIA131R (13). The in vivo consequences of these allelic variants cells in the germinal center, thus setting thresholds for B-cell are clearly evident, as patients with cancer carrying the FcgRIIIA158V activation (1, 9). and FcgRIIA131H alleles show greater responses to antitumor mono- Type I FcR family members comprise the canonical FcgRs, clonal antibody (mAb) therapies (14, 15). This is discussed in more which can be classified into two functionally defined sub- detail below. classes: activating and inhibitory FcgRs (Fig. 1). The activating FcgRs, which include murine FcgRI, FcgRIII, and FcgRIV, as well Monoclonal Antibody Therapy for as human FcgRI, FcgRIIA, and FcgRIIIA, initiate cellular activa- Malignancies tion through their intracellular immunoreceptor tyrosine– based activation motif (ITAM), while inhibitory signaling path- Antibodies have become an important tool in the battle against ways are triggered by the intracellular immunoreceptor tyro- all forms of malignancy, and the number of therapeutic antibo- sine–based inhibitory motifs (ITIM) of the mouse and human dies in clinical use or under investigation has dramatically inhibitory FcgR, FcgRIIB (10). Both activating and inhibitory increased in recent years. There are several classes of therapeutic FcgRs are expressed by cells of the innate immune system. antibodies, each with the ultimate effect of priming either the Mouse natural killer (NK) cells exclusively express FcgRIII, and innate or the adaptive arm of the immune system to target tumor human NK cells primarily express FcgRIIIA; B cells of both cells for destruction: (i) antitumor antibodies that directly engage species exclusively express the inhibitory FcgRIIB. Murine DCs tumor antigens and target the tumor for destruction by the innate express the full array of activating and inhibitory FcgRs, while immune system; (ii) agonistic immunomodulatory antibodies human DCs only express a single activating FcgR, huFcgRIIA, that target immune system costimulatory molecules to potentiate and the inhibitory huFcgRIIB (Fig. 1; ref. 7). With the exception antitumor immune responses; and (iii) antagonistic immuno- of FcgRI, type I FcRs are of intermediate or low affinity and thus modulatory antibodies that target immune system regulatory will not bind monomeric IgG at normal physiologic concen- molecules to "release the brakes" on antitumor immune trations. These receptors have evolved to engage immune responses (Fig. 2). complexes or IgG-coated targets, resulting in receptor cross- linking and triggering of cellular responses. The cellular out- Antitumor antibodies come of IgG interactions with FcgRs is governed by the affinity The fine specificity demonstrated by mAbs has enabled the of the Fc for the specificFcgR and the expression pattern of targeting of tumor-specific or tumor-associated antigens (TAA) those receptors on the effector cells. restricted to malignant cells, thereby allowing for precise targeting Because most effector cells coexpress activation and inhibitory of tumor cells. Although radionucleotide-conjugated or toxin- FcgRs, it is the relative ratio of the binding affinities of a specific conjugated antibodies have demonstrated therapeutic activities, IgG Fc to these receptors that will determine the outcome of the "naked" Fc-engineered antibodies have the potential to recruit IgG–FcgRinteraction.Thesebindingaffinities are determined by immune effector pathways that may amplify and extend the the amino acid sequences of the different IgG Fc subclasses and the activity of antitumor therapies already in clinical use. Several N-linked glycan patterns of the IgG Fc domains. Thus, the IgG Fc potential mechanisms had been proposed by which antitumor composition can dramatically influence the in vivo outcome of an mAbs mediate their tumoricidal effects in vivo, including cross- antibody–antigen complex engaging FcgRs on an innate cell, by linking–mediated activation of signaling cascades that lead to cell directing the cell into either a proinflammatory or an anti-inflam- death, blockade of ligands required for tumor cell survival, þ matory state. This is best exemplified by isotype-specific activity in recruitment of the complement cascade, or recruitment of FcgR the murine FcgR system. Mouse (ms)IgG2a demonstrates a 2-log cytotoxic or phagocytic effector cells (16). Rituximab, an anti- greater affinity for the activating msFcgRIV receptor as compared CD20 antibody, was the first mAb to be approved to fight cancer in with the inhibitory msFcgRIIB receptor, while msIgG1 preferen- lymphoma patients, and its mechanism of action has been

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Therapeutic antibodies with FcγR-dependent and FcγR-independent mechanisms of action

Fcg RIIb B NK CD40 anti- Fcg R f DC CD40 mAb M Fcg R ↑MHC ↑ Lymphoma Costimulation ↑Ag presentation Mf Immunomodulation: Anti–CTLA-4, -GITR, -OX40: Direct ADCC/ADCP Agonistic mAbs Fc-dependent Treg depletion Fc-dependent & anti-CD20 mAb Fc-dependent Fc-independent blockade

Anti–CTLA-4 TREG CTLA-4 mAb Figure 2. Immunomodulation: Schematic diagram of therapeutic Antagonistic mAbs antibodies with FcgR-dependent and Signaling: FcgR-independent mechanisms of Anti–PD-1 PD-1 Anti–PD-1: Induction of apoptosis action for treating malignancies. mAb TEFF Fc-independent Fc-independent blockade

g Anti–PD-L1 Fcg R Fc R PD-L1 DC mAb Anti–PD-L1: Mf Fc-dependent & Fc-independent Teff MHC TCR T CD28 Signaling inhibition: eff Immune-complex delivery ligand blockade Teff T & maturation of APCs Fc-independent (antitumor vaccinal effect) Teff Fc-dependent

studied extensively. Although in vitro studies indicated that ritux- (20, 21). Specifically, huFcgRIIIA expressed by monocytes/macro- imab is capable of activating complement and inducing apopto- phages is both necessary and sufficient for huIgG1 mAb-mediated sis, in vivo studies, both in animal models and patients undergoing clearance of lymphoma cells (21). treatment, have clearly demonstrated that engagement of FcgRs The requirement for Fc–FcgR interactions during the cyto- on innate cell populations is crucial for this mAb to mediate its toxic clearance of tumor cells by antitumor mAbs in vivo has antitumor cytotoxic effects (1, 17, 18). been confirmed for other antitumor mAbs. The anti-Her2 mAb Experiments in immunodeficient mice with xenografts of trastuzumab requires Fc–FcgR interactions to mediate cytotox- þ þ huCD20 human tumor cells were the first in vivo studies to icityofxenograftsofHer2 tumor cells in mice (17). Accord- demonstrate that expression of murine activating FcgRs is ingly, functional polymorphisms in huFcgRIIIA also correlate required for the clearance of lymphoma cells by rituximab with efficacy in breast cancer patients treated with trastuzumab (17). Subsequent studies in immunocompetent mice treated with (22). In addition to blocking downstream epidermal growth anti-msCD20 mAbs further demonstrated that macrophages factor receptor (EGFR) signaling, the efficacy of the anti-EGFR expressing activating FcgRs mediated clearance (ADCC/ADCP) of mAb cetuximab also correlates with huFcgRIIA and FcgRIIIA þ CD20 B cells or syngeneic lymphoma cells (18, 19). No con- polymorphisms (23). Thus, multiple antitumor mAbs mediate þ tributions by FcgR NK cells, complement pathways, or the their cytotoxic antitumor effects through engaging FcgRs induction of apoptotic signaling pathways could be detected expressed by innate cell populations, including monocytes and in vivo. These results are consistent with results from studies in macrophages. humans demonstrating that functional polymorphisms in huFcgRIIIA correlate with response rates in lymphoma patients Fc-optimization of cytotoxic antitumor antibodies treated with rituximab (14, 15). Investigators have begun to Because both activating and inhibitory FcgRs are coexpressed perform therapeutic studies utilizing FcgR-humanized mice, in by the majority of innate immune cells that mediate antitumor which the full array of huFcgRs is expressed in fully immuno- ADCC/ADCP upon engagement of cell-bound mAbs or immune competent mice that lack all murine FcgRs (20). These mice fully complexes, a threshold for immune cell activation exists. Thereby, recapitulate huFcgR expression patterns and function, and dem- modulation of an antibody's relative engagement of activating onstrate that huIgG1 mAb-mediated ADCC of tumor cells or other versus inhibitory FcgRs (the A:I ratio) represents a key approach cellular populations is mediated by Fc–FcgR interactions in vivo for augmenting the in vivo activity of a therapeutic mAb. This

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Table 1. Next-generation antitumor antibodies optimized for Fc-mediated effector function Name Target Indication Company Fc optimization strategy MGA271 B7-H3 B7-H3þ carcinomas (solid MacroGenics Fc domain point mutants tumors) MGAH22 (margetuximab) HER2 Breast cancer Macrogenics Fc domain point mutants XmAb-2513 CD30 Hodgkin lymphoma Xencor Fc domain point mutants XmAb-5574 CD19 Leukemia/lymphoma (CLL) Xencor Fc domain point mutants AME-133v (ocaratuzumab, LY2469298) CD20 Leukemia/lymphoma (NHL: Mentrik/Eli Lilly Fc domain point mutants CLL, FL) Obinutuzumab (GA101) CD20 Leukemia/lymphoma (NHL: Roche Afucosylated Fc domain CLL, FL) GA201 (RG7160) EGFR Various carcinomas Roche Afucosylated Fc domain MEDI-551 CD19 Leukemia/lymphoma (CLL) Medimmune Afucosylated Fc domain KW2871 GD3 ganglioside Melanoma Life Science Afucosylated Fc domain Pharmaceuticals ARGX-110 CD70 CD70þ tumors Argen-x Afucosylated Fc domain ARGX-111 c-Met cMetþ tumors Argen-x Afucosylated Fc domain Quilizumab M1 of IgE IgEþ B cells Genentech Afucosylated Fc domain OBT357 (MEN1112) Bst1 (CD157) Acute myeloid leukemia Menarini Group/Oxford Afucosylated Fc domain BioTherapeutics BIW-8692 Ganglioside GM2 Lung cancer Kyowa Hakko Kirin Afucosylated Fc domain Mogamulizumab (KW-0761) CCR4 Peripheral and cutaneous Kyowa Hakko Kirin Afucosylated Fc domain T-cell lymphomas CetuGEX EGFR Various carcinomas Glycotope Afucosylated Fc domain Pankomab-GEX MUC1 Ovarian cancer Glycotope Afucosylated Fc domain Tras-GEX HER2 Breast cancer Glycotope Afucosylated Fc domain KB004 EphA3 Hematologic and solid KaloBios Pharaceuticals Afucosylated Fc domain tumors Abbreviations: CLL, chronic lymphocytic leukemia; FL, follicular lymphoma; NHL, non-Hodgkin lymphoma.

rationale is demonstrated by studies in which genetic deletion of of IgE, CD157, CCR4, or EphA3, will demonstrate significant the inhibitory FcgR, FcgRIIB, results in augmented mAb-mediated efficacy against the tumors they target (Table 1). cytotoxicity (17, 19). Alternatively, two routes for engineering the Afucosylation of mAbs offers a limited approach to Fc-engi- huIgG Fc region for selectively enhanced engagement of activating neering, because this class of mAb shows augmented engagement FcgRs are under investigation in both preclinical and clinical of only huFcgRIIIA. By contrast, the introduction of Fc domain studies: (i) modification of Fc–FcgR interactions through manip- point mutations may be used to manipulate interactions with ulating the Fc glycan at Asn297; and (ii) modification of Fc–FcgR any and/or multiple FcgR family members. For example, known interactions through the introduction of Fc domain point mutations are capable of allowing the Fc domain to selectively mutants. engage huFcgRIIA, huFcgRIIB, huFcgRIIIA, or both huFcgRIIA/IIIA Although N-linked glycosylation of the IgG Fc is absolutely simultaneously (20, 21). Animal studies using FcgR-humanized required for FcR binding (1), modification of specific carbohy- mice have demonstrated that appropriate Fc point mutations can drates, such as fucose and branching GNAc, can modulate the Fc's dramatically modulate the in vivo efficacy of anti-melanoma and affinity for FcgR. For example, afucosylated IgG demonstrates anti-CD20 mAbs (20, 21), as well as antibodies that neutralize enhanced affinity specifically for huFcgRIIIA, thereby increasing pathogens (29–31). Preclinical studies have also demonstrated the A:I ratio and resulting in increased ADCC/ADCP effector that an anti-CD19 mAb Fc-optimized using point mutations function both in vitro and in vivo (24). Thus, the next-generation showed augmented antitumor activity in vivo (32). Thus, the next antitumor mAb therapeutics lacking fucose residues for enhanced generation of point-mutated, Fc-optimized antitumor mAbs now effector functions are exploiting this technique (25, 26). A remark- in clinical testing, including mAbs reactive with B7-H3, HER2, able example of the in vivo consequences of selectively engaging CD30, CD19, and CD20, are likely to show superior efficacy in activating FcgRs and increasing the A:I ratio of a mAb's Fc is the patients (Table 1). clinical success of the FDA-approved anti-CD20 mAb obinutuzumab (GA101). Obinutuzumab's N-linked Fc glycan has been engineered to contain a bisecting N-acetylglucosamine (GlcNAc), Immunomodulatory Antibodies which precludes the addition of fucose residues. Thus, Fc-opti- Tumors have the unique ability to shield themselves from mized, afucosylated obinutuzumab plus chemotherapy extended attack from the immune system through a variety of immuno- progression-free survival by almost a full year and showed marked- suppressive mechanisms that occur in the tumor microenviron- ly higher rates of complete responses in patients with chronic ment (33). Thus, a major focus in cancer research is to understand lymphocytic leukemia compared with treatment with unmodified the underlying mechanisms of this immunosuppression and anti-CD20 rituximab plus chemotherapy (27). Preclinical studies design therapies to "release the brakes" on antitumor immune with an afucosylated anti-EGFR antibody also demonstrated responses. Therapeutic success has been demonstrated in both increased efficacy compared with unmodified cetuximab (28). preclinical and clinical studies of antibodies targeting not the Therefore, it is likely that many of the next-generation Fc-opti- tumors but the immunoregulatory pathways mediated by mole- mized, afucosylated antitumor antibodies now under examination cules expressed by cells of both the innate and adaptive immune in clinical trials, including mAbs targeting CD70, c-Met, EGFR, M1 system. Initially, it was assumed that using agonistic mAbs to

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γ Fc RIIIA-mediated ADCC/ADCP Chemotherapy/radiation-induced cell death

Fcg RIIIA Mf

Tumor (lymphoma) Tumor Tumor (lymphoma) (lymphoma)

Anti-tumor mAb

Fc-engineered TAAs Figure 3. Abs for enhanced Generation revealed Left, mechanism of antitumor vaccinal ADCC effect mediated by cytotoxic of Ab:Ag antitumor antibody. Antitumor mAb immune opsonizes tumor cells and targets complexes them for killing by FcgR-mediated (IC) antibody-dependent cellular cytotoxicity or phagocytosis (ADCC IC binding or ADCP), a process that generates γ – to Fc RIIA TAAs antibody tumor antigen immune DC Mf complexes (IC). These ICs engage on DC Fcg RIIA processed & activating FcgRs expressed by DCs, presented by which results in stimulation of DC APC maturation and presentation of tumor MHC MHC antigens to T cells, thereby leading to CD86 CD86 TCR Agonistic Abs long-term antitumor cellular memory CD28 CD28 TCR (e.g., anti-CD40) formation. Right, tumor cell death by DC maturation; T T other means may trigger the release of Ag presentation T reg TAAs, which could be captured, processed, and presented to T cells to stimulate antitumor cellular immunity. Teff Blue boxes represent steps at which T Antagonistic Abs antibody therapeutics may intervene eff and augment antibody-mediated (e.g., anti–CTLA-4, anti-GITR, Teff cytotoxicity or stimulation of Antagonistic Abs anti-OX40 Treg depletion and antitumor T-cell memory responses. blockade) (e.g., anti–PD-1 or Teff anti–PD-L1)

Generation of TAA-speciﬁc Tmem Tmem memory T cells

simply cross-link activator immune molecules (such as costimu- mAbs to mediate their proinﬂammatory, T-cell priming, and latory molecules) or using antagonistic mAbs to purely block antitumor effects (35, 36). This ﬁnding has been extended to ligand–receptor interactions for negative regulatory immune mAbs targeting other members of the TNFR superfamily, includ- molecules (such as CTLA-4) would potentiate antitumor immune ing CD95 (Fas), and death receptors (DR)4, and DR5 (37–40). responses. Unexpectedly, it has now been demonstrated that both Furthermore, it is now known that anti-TNFR agonistic mAbs agonistic and antagonistic immunomodulatory mAbs often function in trans by engaging FcgRIIB on a cell other than the require interactions with various FcgR family members for opti- antibody-bound target cell; distinct cellular populations provide mal in vivo antitumor activities (Fig. 2). FcgRIIB depending on the TNFR target molecule and the local microenvironment (41). Because FcgRIIB signaling is not required Agonistic immunomodulatory antibodies for the agonistic effect of these antibodies, engagement of FcgRIIB The costimulatory receptor CD40, a member of the tumor- provides a scaffold to mediate clustering of TNFR molecules to necrosis factor receptor (TNFR) superfamily, has been targeted initiate downstream signaling pathways (41). using agonistic mAbs due to its important role as a positive Because clustering of these molecules is absolutely required for regulator of innate and adaptive immunity (34). Animal studies the agonistic effect of CD40, Fas, DR4, and DR5 mAbs, reengi- have demonstrated an unexpected and absolute requirement for neering the Fc for augmented engagement of FcgRIIB represents an interactions with the inhibitory FcgR, FcgRIIB, for anti-CD40 approach for enhancing the agonistic effect of these mAbs.

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Alternatively, clustering can be achieved through Fc-independent Antibodies Prime Antitumor Immune means, as has been reported for the human IgG2 subclass that can Responses: The Vaccinal Effect adopt a "B" form structure that enhances CD40 signaling (42). Indeed, introduction of Fc domain point mutations that selec- Although most mechanistic studies of antitumor antibodies tively enhance the affinity of the Fc for huFcgRIIB dramatically have focused on their short-term cytotoxic properties, an alternate augments the antitumor potency of agonistic anti-CD40 mAbs in concept has emerged by which antitumor antibodies may func- preclinical FcgR-humanized mouse models (35). Thus, future tion to activate the immune system to stimulate a potent, long- antibody-based agonistic therapeutics must incorporate ideal Fc term antitumor memory immune response. This phenomenon is domains or other strategies for clustering these trimeric receptors known as a vaccinal effect. for optimal efficacy. As early as 1907, Emil von Behring demonstrated that immu- nization with a mixture of diphtheria toxin and antitoxin (anti- Antagonistic immunomodulatory antibodies diphtheria antibody) produced safe and lasting immunity to Although most tumors contain some immunogenic antigens, diphtheria in animals and humans (48). We now understand antitumor immune responses are hampered by multiple immu- these mixtures to be antigen–antibody immune complexes, which nosuppressive mechanisms used by malignant cells in the enhance the immunogenicity of soluble molecules. While tumor microenvironment. Immunomodulatory antagonistic immune complexes are excellent inducers of humoral immunity mAbs (also known as checkpoint inhibitors) target regulatory (antibody responses), their ability to engage FcgRs expressed by molecules expressed by immune cells in order to disrupt their APCs of the innate immune system to prime cellular immunity (T- immunoregulatory pathways and promote antitumor immune cell responses) is most relevant in the context of tumor immunity responses (43). Examples of the regulatory pathways currently (Fig. 3; ref. 49). DCs, the most potent of the immune system's being targeted by mAbs include CTLA-4, GITR, OX40, and the APCs, primed with immune complexes are capable of processing PD-1/PD-L1 axis. As with the agonistic mAbs above, it was and presenting the relevant antigen to both CD4 (through tradi- unexpectedly found that engagement of activating FcgRs is tional antigen presentation) and CD8 T cells (through cross- required for optimal antitumor activity in preclinical models presentation; refs. 50, 51). Studies using various antigen–anti- using anti–CTLA-4 (44, 45), anti-GITR (46), and anti-OX40 body immune complexes have demonstrated that DCs primed (47) mAbs. Mechanistically, each of these immunomodulatory with immune complexes cross-present antigens and generate molecules is highly expressed by intratumoral regulatory T cells more potent CD8 T-cell responses compared with DCs primed (Treg), which, upon opsonization by mAbs, are targeted for with antigen alone. Activating FcgRs are required for this process, cytotoxic depletion by activating FcgR-expressing innate cells and modulating the balance of activating versus inhibitory FcgR (intratumoral macrophages). The removal of Tregs from the tumor engagement (A:I ratio) toward activating FcgRs augments CD8 microenvironment thereby enhances the ability of the remaining immune responses and tumor immunity in vivo (8, 52). Mecha- effector T cells to kill the tumor. By contrast, anti–PD-1 mAbs now nistically, immune-complex engagement of FcgRs leads to DC in clinical use have been engineered to abrogate interactions with maturation and upregulation of MHC molecules and costimula- FcgRs, because PD-1 is broadly expressed by T cells, including tory molecules, including CD80, CD86, and CD40 (52, 53). þ effector CD8 T cells that mediate antitumor activities; depletion Thus, immune complexes are potent inducers of cell-mediated of this T-cell subset would likely inhibit antitumor immunity and immunity. augment tumor growth (Fig. 2). Whether Fc–FcgR interactions are Passive administration of therapeutic antitumor cytotoxic anti- required for the in vivo activity of anti–PD-L1, anti-CD137, antibodies likely results in the in vivo generation of immune com- CD27, and anti-KIR mAbs now in clinical trials is the subject of plexes (Fig. 3), and recent studies have investigated whether such active investigation. antibodies initiate a vaccinal effect that is characterized by an The unexpected requirement for interactions with the inhibi- antitumor cellular immune response. Examples of cytotoxic anti- tory FcgRIIB for agonistic anti-TNFR family immunomodulatory body-induced vaccinal effects from the clinic include patients antibodies, or with activating FcgRs for some antagonistic anti- treated with anti-MUC1 mAbs or anti-HER2/Neu mAbs, who bodies, demonstrates how essential it is to understand the mech- generated MUC1-specific and HER2-specific T-cell responses, anism of action with regard to FcgR engagement of each agonistic respectively (54, 55). Because a single course of treatment with or antagonistic immunomodulatory antibody on a case-by-case anti-CD20 mAb (rituximab) can result in long-lasting, durable basis. It is likely that the expression levels of the therapeutic target responses, it has been hypothesized that treatment with this mAb on effector immune cells, regulatory immune cells, or the tumor may also induce a vaccinal effect in patients with lymphoma (56). itself will dictate whether engagement of FcgRs is necessary to In support of this, lymphoma-specific anti-idiotype T-cell augment or inhibit the effectiveness of a therapeutic antibody in responses were detected in some patients treated with anti-CD20 vivo. Expression patterns of activating and inhibitory FcgRs, as well rituximab (57, 58). Anti-CD20 mAb treatment of lymphoma cells in vitro stimulates DC maturation and CD8 T-cell activation (59), as the presence of the appropriate effector cells in the tumor þ microenvironment, will further dictate whether and which FcgRs and vaccination with huCD20 tumor cells and anti-huCD20 may be involved. Because the complex conditions of the tumor mAb treatment showed a synergistic antitumor effect in mice (60). microenvironment cannot be recreated in vitro, careful in vivo Studies using mouse models have also demonstrated that passive mechanistic analyses of each immunomodulatory antibody must administration of anti-CD20 mAbs can initiate antitumor T-cell be performed to inform the proper engineering of the Fc domain responses and vaccinal effects (21, 61). for ideal interaction with the appropriate FcgRs for optimum Recent studies have elucidated how an antitumor vaccinal effect in vivo efficacy. Thus, future generations of immunomodulatory could be generated upon treatment with a cytotoxic antitumor mAbs will undoubtedly incorporate engineered Fc domains for mAb using a murine model in which syngeneic EL4 lymphoma optimal activity in vivo. cells express huCD20 (EL4-huCD20 cells) as a tumor neoantigen

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(21, 61). Mice given EL4-huCD20 cells and subsequently treated huFcgRIIA expressed by human DCs induces DC maturation and with anti-huCD20 mAbs cleared their tumors in an activating upregulation of costimulatory molecules for optimal antigen FcgR-dependent manner, again demonstrating that the initial presentation and cross-presentation to stimulate long-term anti- cytotoxic effect of antitumor mAbs requires Fc–FcgR interactions tumor T-cell memory (Fig. 3, left). As discussed above, the current (21). Anti-huCD20 mAb-treated mice that survived this primary collection of next-generation antitumor antibodies are being tumor challenge were rested for 90 days and then rechallenged optimized through glycoengineering or with point mutations for without additional mAb treatment. The mAb/tumor-primed mice superior huFcgRIIIA engagement to augment their cytotoxic survived rechallenge with EL4-huCD20 cells as well as a distinct effects. However, these new antibodies have not been optimized tumor cell line expressing huCD20, but did not survive rechal- for huFcgRIIA engagement, as defucosylation does not affect lenge with wild-type EL4 cells (21). Thus, mice primed with EL4- binding to this receptor. Therefore, an ideal antitumor antibody hCD20 and mAb generate a vaccinal effect memory immune will need to be optimized for both effector functions: optimized response against the CD20 antigen and subsequently reject rechal- huFcgRIIIA engagement for augmented immediate cytotoxicity lenge with EL4-huCD20 cells, but not wild-type EL4 cells. This and optimized huFcgRIIA binding for more potent induction of process relies on both CD4 and CD8 T cells, and adoptive transfer long-term antitumor vaccinal effect T-cell responses. of T cells from mAb/tumor-primed mice transfers the vaccinal The next phase of antitumor immunotherapy will not only effect to na€ve animals (61). Using transgenic mice in which consist of antitumor and immunomodulatory antibodies with þ activating FcgRs are conditionally deleted on CD11c cells, it has engineered Fc domains for optimal effector functions, but it been demonstrated that FcgR expression by DCs is required for the should also incorporate logical combinations of Fc-optimized generation of the vaccinal effect (21). Collectively, these results antitumor antibodies, Fc-optimized immunomodulatory antibo- demonstrate that cytotoxic clearance (ADCC/ADCP) of tumor dies, and traditional radiotherapies and chemotherapies (Fig. 3). cells by anti-huCD20 mAbs induces the generation of huCD20 Because preclinical studies have suggested synergistic effects of immune complexes, which engage FcgRs expressed by DCs to immunomodulatory mAbs with distinct mechanisms of action initiate a huCD20-specific T-cell vaccinal effect (Fig. 3, left). (e.g., combining anti–CTLA-4 and anti–PD-1 checkpoint inhibi- Mechanistically understanding the antitumor vaccinal effect in tors), combinations of various immunomodulatory antibodies the context of the human FcgR system is vital for designing are being tested in clinical trials for synergy (62). Combining optimal antitumor antibody therapeutics. As discussed earlier, immunomodulatory mAbs with traditional chemotherapy and expression patterns of FcgRs differ between mouse and man. radiation treatments has also shown some benefit in early trials, While ADCC-mediating human monocytes/macrophages utilize most likely because cell death exposes tumor-specific antigens or only huFcgRIIIA for their cytotoxic effects, vaccinal effect-medi- neoantigens for recognition by an unencumbered immune sys- ating human DCs only express huFcgRIIA (21). To dissect the tem (Fig. 3, right; refs. 63, 64). Combining checkpoint inhibitors vaccinal effect mechanistically in the context of the huFcgR with antitumor vaccination may also lead to enhanced results. system, FcgR-humanized mice were used in combination with Thus, a logical extension would be to test cytotoxic antitumor huIgG1 anti-huCD20 mAbs, as well as mutant anti-huCD20 mAbs in combination with immunomodulatory mAbs. One mAbs that selectively engage either huFcgRIIA, huFcgRIIIA, or would predict that cytotoxic killing of tumors would generate both of these receptors. During the primary tumor challenge, immune complexes for vaccinal effect induction as well as poten- engagement of huFcgRIIIA was both necessary and sufficient for tially expose other tumor antigens; memory T-cell responses ADCC-mediated clearance of EL4-huCD20 tumor cells; would likely be boosted in combination with immunomodula- huFcgRIIA played no role. By contrast, engagement of huFcgRIIA tory mAb treatment (Fig. 3, right). was required for optimal induction of the vaccinal effect in FcgR- Although multiple classes of therapeutic antibodies are now humanized mice, consistent with this being the sole activating available to treat the full array of human malignancies as single huFcgR expressed by human antigen-presenting DCs (21). Thus, agents or in logical combinations, one common theme cannot be differential human FcgR requirements exist for antitumor mAb- ignored: The effector functions mediated by the Fc domain of each mediated cytotoxicity versus induction of a memory cellular therapeutic antibody must be carefully evaluated and mechanis- immune response during the vaccinal effect. tically understood. Through thoughtful analysis of each antibody's mechanism(s) of action in the context of the human FcgR Looking toward the Future in Therapeutic system, more potent therapies can be engineered through opti- Antibodies in Malignancy mized engagement of the appropriate FcgRs. These new mechanistic insights into the antitumor vaccinal in vivo effect shed important light on the effector mechanisms Acknowledgments triggered by cytotoxic antitumor antibodies in the context of D.J. DiLillo received a postdoctoral career development fellowship from the human IgG and human FcgRs. First, Fc engagement of huFcgRIIIA Leukemia and Lymphoma Society of America. expressed by monocytes and macrophages is absolutely vital for immediate cell-mediated cytotoxicity and phagocytosis triggered Received April 29, 2015; accepted May 20, 2015; published online July 2, by antitumor mAbs. Second, immune-complex engagement of 2015.

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David J. DiLillo and Jeffrey V. Ravetch

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