Fc-ReceptorInteractionsRegulateBothCytotoxic and ...€¦ · ative regulatory pathways. In this Masters of Immunology primer, we discuss FcR biology and the mechanisms of action of

Masters of Immunology

Fc-Receptor InteractionsRegulateBothCytotoxicand Immunomodulatory Therapeutic AntibodyEffector FunctionsDavid J. DiLillo and Jeffrey V. Ravetch

Abstract

Antibodies are now recognized as key therapeutic tools tocombat most forms of malignancy. Although the first wave oftherapeutic antibodies that emerged over two decades ago directlytarget tumor cells for killing, a new class of antibody therapiestargeting immunoregulatory pathways to boost antitumorimmune responses by activating the immune system is poisedfor clinical success. A notable common characteristic of bothclasses of therapeutic antibodies is the importance of the IgG Fcdomain, which connects the fine specificity of an antibody withimmune cells that mediate antibody-triggered effector functions

through their engagement of Fc receptor (FcR) familymembers. Itis now clear that multiple variables, including the nature of thetarget molecules, the local presence of effector cells, and theexpression patterns of FcRs, will dictate whether and how anantibody will necessitate interactions with FcRs to mediate opti-mal therapeutic effects. Thus, through careful in vivo mechanisticanalyses of individual therapeutic antibodies, Fc domains engi-neered for optimal engagement of the appropriate cellularFcRsmust be designed tomaximize clinical success. Cancer ImmunolRes; 3(7); 704–13. �2015 AACR.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

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

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

Learning ObjectivesAntibodies 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 thefine-specificity of the antibodywith 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 SupportThis activity does not receive commercial support.

IntroductionAntibodies have become a major therapeutic tool for the

treatment of all classes of malignancy. A well-defined but

often-overlooked characteristic of antibodies is their bifunctionalnature. The variable Fab regionof an antibodymediates specificityand dictates to what antigen and with what affinity the antibodywill bind its target. However, antibodies also contain a constantregion, termed the Fc domain,which engages a diversity of cellularreceptors, thereby triggering antibody-mediated effector func-tions. The Fc domain acts as a bridge between the specificitydictated by the Fab region and cells of the innate and adaptiveimmune system.

The first therapeutic antibodies designed almost three decadesago targeted tumor antigens, and were developed based on theirintrinsic activity to mediate tumoricidal effects by Fab-mediatedcross-linking of target molecules to trigger proapoptotic signaling

The Laboratory of Molecular Genetics and Immunology, The Rock-efeller University, New York, New York.

Corresponding Author: Jeffrey V. Ravetch, Rockefeller University, 1230 YorkAvenue, New York, NY 10065. Phone: 212-327-7321; Fax: 212-327-7319; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-15-0120

�2015 American Association for Cancer Research.

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cascades or through inhibition of autocrine loops. Little con-sideration was placed on the potential contributions from theFc domains and Fc receptors (FcR). However, it is now clear thatthe Fc domain plays an instrumental role in the effectormechanisms elicited by multiple classes of therapeutic anti-bodies, whether they directly target tumor cells or alternativelytarget the immune system, modulating either positive or neg-ative regulatory pathways. In this Masters of Immunologyprimer, we discuss FcR biology and the mechanisms of actionof cytotoxic antitumor antibodies as well as newer classes ofagonistic/antagonistic immunomodulatory antibodies, with anemphasis on how they differentially engage various membersof the FcR family to mediate their effector functions. New datademonstrating that cytotoxic antitumor antibodies can stimu-late long-term antitumor T-cell memory responses through anFcR-dependent "vaccinal effect" are also discussed. Through athoughtful analysis of their respective mechanisms of action,engineering Fc domains of therapeutic antibodies for optimalengagement of appropriate members of the FcR family will lead

to next-generation therapies with enhanced antitumor activitiescapable of sustained responses.

Fc Receptor Expression and FunctionUpon binding their cognate antigens, IgG antibodies mediate

downstream effector functions by interacting with either type I ortype II FcRs. Type I FcRs aremembers of the immunoglobulin (Ig)superfamily and include the canonical FcRs for IgG (FcgR; Fig. 1;ref. 1). Type II FcRs are members of the C-type lectin receptorfamily and currently include CD209 (also known as DC-SIGN,which is homologous to SIGN-R1 in mice) and CD23. Whethertype I or type II FcRs are engaged by an antibody is determined bythe conformational state of its Fc domain, which is regulated bythe complex, biantennary N-linked glycan attached to Asn297(1, 2). Thus, differences in Fc glycosylation at Asn297 can lead tomodulated affinities for FcRs: sialylated Fc domains adopt amoreflexible, "closed" conformation that allows engagement of type IIFcRs and reduces binding to type I FcRs (2, 3). By contrast,

© 2015 American Association for Cancer Research

A

FcgRI(Fcgr1)

FcgRI(FCGR1A)

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FcgRIV(Fcgr4)

FcgRIII(Fcgr3)

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Type I FcgRs in mice

Type I FcgRs in humans

Figure 1.Mouse (A) and human (B) type I FcgRfamily members and expressionpatterns. � , inducible upon cytokinestimulation; �� , inducible uponcytokine stimulation in immature DCs;�� , functional FcgRIIC is expressed by10% of individuals.

Fc-Receptors in Tumor Immunotherapy

www.aacrjournals.org Cancer Immunol Res; 3(7) July 2015 705



nonsialylated Fc domains assume an "open" conformation, there-by allowing binding to type I FcRs and preventing engagement oftype II FcRs. Detailed biochemical mechanisms regulating type Iand type II FcR engagement, as well as the anti-inflammatorydownstream consequences of type II FcR engagement, havebeen reviewed recently (1, 4, 5). Engagement of type II FcRs bysialylated IgG typically results in suppression of antibody- and Tcell–mediated inflammation (6), as exemplified by the use ofintravenous immunoglobulin (IVIG) in patients suffering fromvarious autoimmune diseases. By contrast, engagement of type IFcRs results in antibody-dependent cellular cytotoxicity andphagocytosis (ADCC and ADCP), demonstrating the ability ofan IgG to bridge target cells/pathogens and FcgR-expressingeffector cells to mediate cytotoxicity or phagocytosis (7). Type IFcRs also engage antigen–antibody immune complexes (IC) andmediate their downstream immunomodulatory effects on anti-gen-presenting cells (APC) and B cells. Immunomodulatoryeffects mediated by ICs are observed on dendritic cells (DC),where ICs can enhance antigen uptake and regulate DC matura-tion in an FcgR-dependent fashion, thereby shaping T-cellresponses (8). ICs can also regulate affinity maturation of Bcells in the germinal center, thus setting thresholds for B-cellactivation (1, 9).

Type I FcR family members comprise the canonical FcgRs,which can be classified into two functionally defined sub-classes: activating and inhibitory FcgRs (Fig. 1). The activatingFcgRs, which include murine FcgRI, FcgRIII, and FcgRIV, as wellas human FcgRI, FcgRIIA, and FcgRIIIA, initiate cellular activa-tion through their intracellular immunoreceptor tyrosine–based activation motif (ITAM), while inhibitory signaling path-ways are triggered by the intracellular immunoreceptor tyro-sine–based inhibitory motifs (ITIM) of the mouse and humaninhibitory FcgR, FcgRIIB (10). Both activating and inhibitoryFcgRs are expressed by cells of the innate immune system.Mouse natural killer (NK) cells exclusively express FcgRIII, andhuman NK cells primarily express FcgRIIIA; B cells of bothspecies exclusively express the inhibitory FcgRIIB. Murine DCsexpress the full array of activating and inhibitory FcgRs, whilehuman DCs only express a single activating FcgR, huFcgRIIA,and the inhibitory huFcgRIIB (Fig. 1; ref. 7). With the exceptionof FcgRI, type I FcRs are of intermediate or low affinity and thuswill not bind monomeric IgG at normal physiologic concen-trations. These receptors have evolved to engage immunecomplexes or IgG-coated targets, resulting in receptor cross-linking and triggering of cellular responses. The cellular out-come of IgG interactions with FcgRs is governed by the affinityof the Fc for the specific FcgR and the expression pattern ofthose receptors on the effector cells.

Because most effector cells coexpress activation and inhibitoryFcgRs, it is the relative ratio of the binding affinities of a specificIgG Fc to these receptors that will determine the outcome of theIgG–FcgR interaction. These binding affinities are determined bythe amino acid sequences of the different IgG Fc subclasses and theN-linked glycan patterns of the IgG Fc domains. Thus, the IgG Fccomposition can dramatically influence the in vivo outcome of anantibody–antigen complex engaging FcgRs on an innate cell, bydirecting the cell into either a proinflammatory or an anti-inflam-matory state. This is best exemplified by isotype-specific activity inthe murine FcgR system. Mouse (ms)IgG2a demonstrates a 2-loggreater affinity for the activating msFcgRIV receptor as comparedwith the inhibitory msFcgRIIB receptor, while msIgG1 preferen-

tially engages the inhibitorymsFcgRIIB receptor (11). Anactivating:inhibitory (A:I) ratio can be assigned to each antibody isotype bycreating a ratio of the Fc's affinity for the relevant activating FcgRversus the Fc's affinity for the inhibitory FcgR. Thus, msIgG2aantibodies have an A:I ratio of approximately 70, whereas msIgG1antibodies show anA:I ratio of 0.1. The in vivo activities ofmsIgG2aand msIgG1 antibodies tightly correlate with their A:I ratios, asantitumor antibodies of the msIgG2a subclass potently activateeffector cells to kill msIgG2a antibody-opsonized target cells,whereas msIgG1 antibodies demonstrate little tumoricidal activityin vivo (11). Human IgG isotypes also demonstrate preferentialengagement of FcgRs, but to a lesser degree than the murine FcgRsystem, and without selective engagement of either activating orinhibitory receptors. Human (hu)IgG2 and IgG4 interact poorlywith huFcgRs, whereas huIgG1 and huIgG3 interact more strongly(12). Further, allelic variants of huFcgRs exist in the human pop-ulation that can considerably affect binding affinities to the huIgG1and huIgG3 subclasses. For example, the FcgRIIIA158V allele has ahigher affinity for huIgG1 than the FcgRIIIA158F allele, and theFcgRIIA131H allele demonstrates increased binding to huIgG1 thanFcgRIIA131R (13). The in vivo consequences of these allelic variantsare clearly evident, as patients with cancer carrying the FcgRIIIA158V

andFcgRIIA131Halleles showgreater responses toantitumormono-clonal antibody (mAb) therapies (14, 15). This is discussed inmoredetail below.

Monoclonal Antibody Therapy forMalignancies

Antibodies have become an important tool in the battle againstall forms of malignancy, and the number of therapeutic antibo-dies in clinical use or under investigation has dramaticallyincreased in recent years. There are several classes of therapeuticantibodies, each with the ultimate effect of priming either theinnate or the adaptive arm of the immune system to target tumorcells for destruction: (i) antitumor antibodies that directly engagetumor antigens and target the tumor for destruction by the innateimmune system; (ii) agonistic immunomodulatory antibodiesthat target immune system costimulatory molecules to potentiateantitumor immune responses; and (iii) antagonistic immuno-modulatory antibodies that target immune system regulatorymolecules to "release the brakes" on antitumor immuneresponses (Fig. 2).

Antitumor antibodiesThe fine specificity demonstrated by mAbs has enabled the

targeting of tumor-specific or tumor-associated antigens (TAA)restricted tomalignant cells, thereby allowing for precise targetingof tumor cells. Although radionucleotide-conjugated or toxin-conjugated antibodies have demonstrated therapeutic activities,"naked" Fc-engineered antibodies have the potential to recruitimmune effector pathways that may amplify and extend theactivity of antitumor therapies already in clinical use. Severalpotential mechanisms had been proposed by which antitumormAbs mediate their tumoricidal effects in vivo, including cross-linking–mediated activation of signaling cascades that lead to celldeath, blockade of ligands required for tumor cell survival,recruitment of the complement cascade, or recruitment of FcgRþ

cytotoxic or phagocytic effector cells (16). Rituximab, an anti-CD20antibody,was thefirstmAb tobe approved tofight cancer inlymphoma patients, and its mechanism of action has been

DiLillo and Ravetch

Cancer Immunol Res; 3(7) July 2015 Cancer Immunology Research706



studied extensively. Although in vitro studies indicated that ritux-imab is capable of activating complement and inducing apopto-sis, in vivo studies, both in animalmodels andpatients undergoingtreatment, have clearly demonstrated that engagement of FcgRson innate cell populations is crucial for this mAb to mediate itsantitumor cytotoxic effects (1, 17, 18).

Experiments in immunodeficient mice with xenografts ofhuCD20þ human tumor cells were the first in vivo studies todemonstrate that expression of murine activating FcgRs isrequired for the clearance of lymphoma cells by rituximab(17). Subsequent studies in immunocompetentmice treatedwithanti-msCD20 mAbs further demonstrated that macrophagesexpressing activating FcgRsmediated clearance (ADCC/ADCP) ofCD20þ B cells or syngeneic lymphoma cells (18, 19). No con-tributions by FcgRþ NK cells, complement pathways, or theinduction of apoptotic signaling pathways could be detectedin vivo. These results are consistent with results from studies inhumans demonstrating that functional polymorphisms inhuFcgRIIIA correlate with response rates in lymphoma patientstreated with rituximab (14, 15). Investigators have begun toperform therapeutic studies utilizing FcgR-humanized mice, inwhich the full array of huFcgRs is expressed in fully immuno-competent mice that lack all murine FcgRs (20). These mice fullyrecapitulate huFcgR expression patterns and function, and dem-onstrate that huIgG1mAb-mediatedADCCof tumor cells or othercellular populations is mediated by Fc–FcgR interactions in vivo

(20, 21). Specifically, huFcgRIIIA expressed bymonocytes/macro-phages is both necessary and sufficient for huIgG1mAb-mediatedclearance of lymphoma cells (21).

The requirement for Fc–FcgR interactions during the cyto-toxic clearance of tumor cells by antitumor mAbs in vivo hasbeen confirmed for other antitumor mAbs. The anti-Her2 mAbtrastuzumab requires Fc–FcgR interactions to mediate cytotox-icity of xenografts of Her2þ tumor cells in mice (17). Accord-ingly, functional polymorphisms in huFcgRIIIA also correlatewith efficacy in breast cancer patients treated with trastuzumab(22). In addition to blocking downstream epidermal growthfactor receptor (EGFR) signaling, the efficacy of the anti-EGFRmAb cetuximab also correlates with huFcgRIIA and FcgRIIIApolymorphisms (23). Thus, multiple antitumor mAbs mediatetheir cytotoxic antitumor effects through engaging FcgRsexpressed by innate cell populations, including monocytes andmacrophages.

Fc-optimization of cytotoxic antitumor antibodiesBecause both activating and inhibitory FcgRs are coexpressed

by the majority of innate immune cells that mediate antitumorADCC/ADCP upon engagement of cell-bound mAbs or immunecomplexes, a threshold for immune cell activation exists. Thereby,modulation of an antibody's relative engagement of activatingversus inhibitory FcgRs (the A:I ratio) represents a key approachfor augmenting the in vivo activity of a therapeutic mAb. This


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

NKB

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&Fc-independent

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Figure 2.Schematic diagram of therapeuticantibodies with FcgR-dependent andFcgR-independent mechanisms ofaction for treating malignancies.





rationale is demonstrated by studies in which genetic deletion ofthe inhibitory FcgR, FcgRIIB, results in augmentedmAb-mediatedcytotoxicity (17, 19). Alternatively, two routes for engineering thehuIgGFc region for selectively enhanced engagement of activatingFcgRs are under investigation in both preclinical and clinicalstudies: (i) modification of Fc–FcgR interactions through manip-ulating the Fc glycan at Asn297; and (ii) modification of Fc–FcgRinteractions through the introduction of Fc domain pointmutants.

Although N-linked glycosylation of the IgG Fc is absolutelyrequired for FcR binding (1), modification of specific carbohy-drates, such as fucose and branching GNAc, can modulate the Fc'saffinity for FcgR. For example, afucosylated IgG demonstratesenhanced affinity specifically for huFcgRIIIA, thereby increasingthe A:I ratio and resulting in increased ADCC/ADCP effectorfunction both in vitro and in vivo (24). Thus, the next-generationantitumor mAb therapeutics lacking fucose residues for enhancedeffector functions are exploiting this technique (25, 26). A remark-able example of the in vivo consequences of selectively engagingactivating FcgRs and increasing the A:I ratio of a mAb's Fc is theclinical success of the FDA-approved anti-CD20 mAb obinutuzu-mab (GA101). Obinutuzumab's N-linked Fc glycan has beenengineered to contain a bisecting N-acetylglucosamine (GlcNAc),which precludes the addition of fucose residues. Thus, Fc-opti-mized, afucosylated obinutuzumab plus chemotherapy extendedprogression-free survival by almost a full year and showedmarked-ly higher rates of complete responses in patients with chroniclymphocytic leukemia compared with treatment with unmodifiedanti-CD20 rituximab plus chemotherapy (27). Preclinical studieswith an afucosylated anti-EGFR antibody also demonstratedincreased efficacy compared with unmodified cetuximab (28).Therefore, it is likely that many of the next-generation Fc-opti-mized, afucosylated antitumor antibodies nowunder examinationin clinical trials, includingmAbs targeting CD70, c-Met, EGFR, M1

of IgE, CD157, CCR4, or EphA3, will demonstrate significantefficacy against the tumors they target (Table 1).

Afucosylation of mAbs offers a limited approach to Fc-engi-neering, because this class of mAb shows augmented engagementof only huFcgRIIIA. By contrast, the introduction of Fc domainpoint mutations may be used to manipulate interactions withany and/or multiple FcgR family members. For example, knownmutations are capable of allowing the Fc domain to selectivelyengagehuFcgRIIA, huFcgRIIB, huFcgRIIIA, or bothhuFcgRIIA/IIIAsimultaneously (20, 21). Animal studies using FcgR-humanizedmice have demonstrated that appropriate Fc point mutations candramatically modulate the in vivo efficacy of anti-melanoma andanti-CD20 mAbs (20, 21), as well as antibodies that neutralizepathogens (29–31). Preclinical studies have also demonstratedthat an anti-CD19 mAb Fc-optimized using point mutationsshowed augmented antitumor activity in vivo (32). Thus, the nextgeneration of point-mutated, Fc-optimized antitumor mAbs nowin clinical testing, including mAbs reactive with B7-H3, HER2,CD30, CD19, and CD20, are likely to show superior efficacy inpatients (Table 1).

Immunomodulatory AntibodiesTumors have the unique ability to shield themselves from

attack from the immune system through a variety of immuno-suppressive mechanisms that occur in the tumor microenviron-ment (33). Thus, amajor focus in cancer research is to understandthe underlying mechanisms of this immunosuppression anddesign therapies to "release the brakes" on antitumor immuneresponses. Therapeutic success has been demonstrated in bothpreclinical and clinical studies of antibodies targeting not thetumors but the immunoregulatory pathways mediated by mole-cules expressed by cells of both the innate and adaptive immunesystem. Initially, it was assumed that using agonistic mAbs to

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 (solidtumors)

MacroGenics Fc domain point mutants

MGAH22 (margetuximab) HER2 Breast cancer Macrogenics Fc domain point mutantsXmAb-2513 CD30 Hodgkin lymphoma Xencor Fc domain point mutantsXmAb-5574 CD19 Leukemia/lymphoma (CLL) Xencor Fc domain point mutantsAME-133v (ocaratuzumab, LY2469298) CD20 Leukemia/lymphoma (NHL:

CLL, FL)Mentrik/Eli Lilly Fc domain point mutants

Obinutuzumab (GA101) CD20 Leukemia/lymphoma (NHL:CLL, FL)

Roche Afucosylated Fc domain

GA201 (RG7160) EGFR Various carcinomas Roche Afucosylated Fc domainMEDI-551 CD19 Leukemia/lymphoma (CLL) Medimmune Afucosylated Fc domainKW2871 GD3 ganglioside Melanoma Life Science

PharmaceuticalsAfucosylated Fc domain

ARGX-110 CD70 CD70þ tumors Argen-x Afucosylated Fc domainARGX-111 c-Met cMetþ tumors Argen-x Afucosylated Fc domainQuilizumab M1 of IgE IgEþ B cells Genentech Afucosylated Fc domainOBT357 (MEN1112) Bst1 (CD157) Acute myeloid leukemia Menarini Group/Oxford

BioTherapeuticsAfucosylated Fc domain

BIW-8692 Ganglioside GM2 Lung cancer Kyowa Hakko Kirin Afucosylated Fc domainMogamulizumab (KW-0761) CCR4 Peripheral and cutaneous

T-cell lymphomasKyowa Hakko Kirin Afucosylated Fc domain

CetuGEX EGFR Various carcinomas Glycotope Afucosylated Fc domainPankomab-GEX MUC1 Ovarian cancer Glycotope Afucosylated Fc domainTras-GEX HER2 Breast cancer Glycotope Afucosylated Fc domainKB004 EphA3 Hematologic and solid

tumorsKaloBios Pharaceuticals Afucosylated Fc domain

Abbreviations: CLL, chronic lymphocytic leukemia; FL, follicular lymphoma; NHL, non-Hodgkin lymphoma.

DiLillo and Ravetch




simply cross-link activator immune molecules (such as costimu-latory molecules) or using antagonistic mAbs to purely blockligand–receptor interactions for negative regulatory immunemolecules (such as CTLA-4)would potentiate antitumor immuneresponses. Unexpectedly, it has nowbeen demonstrated that bothagonistic and antagonistic immunomodulatory mAbs oftenrequire interactions with various FcgR family members for opti-mal in vivo antitumor activities (Fig. 2).

Agonistic immunomodulatory antibodiesThe costimulatory receptor CD40, a member of the tumor-

necrosis factor receptor (TNFR) superfamily, has been targetedusing agonistic mAbs due to its important role as a positiveregulator of innate and adaptive immunity (34). Animal studieshave demonstrated an unexpected and absolute requirement forinteractions with the inhibitory FcgR, FcgRIIB, for anti-CD40

mAbs to mediate their proinflammatory, T-cell priming, andantitumor effects (35, 36). This finding has been extended tomAbs targeting other members of the TNFR superfamily, includ-ing CD95 (Fas), and death receptors (DR)4, and DR5 (37–40).Furthermore, it is now known that anti-TNFR agonistic mAbsfunction in trans by engaging FcgRIIB on a cell other than theantibody-bound target cell; distinct cellular populations provideFcgRIIB depending on the TNFR target molecule and the localmicroenvironment (41). Because FcgRIIB signaling is not requiredfor the agonistic effect of these antibodies, engagement of FcgRIIBprovides a scaffold to mediate clustering of TNFR molecules toinitiate downstream signaling pathways (41).

Because clustering of these molecules is absolutely required forthe agonistic effect of CD40, Fas, DR4, and DR5 mAbs, reengi-neering the Fc for augmented engagement of FcgRIIB represents anapproach for enhancing the agonistic effect of these mAbs.


Mf

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complexes(IC)

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anti–PD-L1)

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blockade)

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memory T cells

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Figure 3.Left, mechanism of antitumor vaccinaleffect mediated by cytotoxicantitumor antibody. Antitumor mAbopsonizes tumor cells and targetsthem for killing by FcgR-mediatedantibody-dependent cellularcytotoxicity or phagocytosis (ADCCor ADCP), a process that generatesantibody–tumor antigen immunecomplexes (IC). These ICs engageactivating FcgRs expressed by DCs,which results in stimulation of DCmaturation and presentation of tumorantigens to T cells, thereby leading tolong-term antitumor cellular memoryformation. Right, tumor cell death byothermeansmay trigger the release ofTAAs, which could be captured,processed, and presented to T cells tostimulate antitumor cellular immunity.Blue boxes represent steps at whichantibody therapeutics may interveneand augment antibody-mediatedcytotoxicity or stimulation ofantitumor T-cell memory responses.





Alternatively, clustering can be achieved through Fc-independentmeans, as has been reported for the human IgG2 subclass that canadopt a "B" form structure that enhances CD40 signaling (42).Indeed, introduction of Fc domain point mutations that selec-tively enhance the affinity of the Fc for huFcgRIIB dramaticallyaugments the antitumor potency of agonistic anti-CD40mAbs inpreclinical FcgR-humanized mouse models (35). Thus, futureantibody-based agonistic therapeutics must incorporate ideal Fcdomains or other strategies for clustering these trimeric receptorsfor optimal efficacy.

Antagonistic immunomodulatory antibodiesAlthough most tumors contain some immunogenic antigens,

antitumor immune responses are hampered by multiple immu-nosuppressive mechanisms used by malignant cells in thetumor microenvironment. Immunomodulatory antagonisticmAbs (also known as checkpoint inhibitors) target regulatorymolecules expressed by immune cells in order to disrupt theirimmunoregulatory pathways and promote antitumor immuneresponses (43). Examples of the regulatory pathways currentlybeing targeted by mAbs include CTLA-4, GITR, OX40, and thePD-1/PD-L1 axis. As with the agonistic mAbs above, it wasunexpectedly found that engagement of activating FcgRs isrequired for optimal antitumor activity in preclinical modelsusing anti–CTLA-4 (44, 45), anti-GITR (46), and anti-OX40(47) mAbs. Mechanistically, each of these immunomodulatorymolecules is highly expressed by intratumoral regulatory T cells(Treg), which, upon opsonization by mAbs, are targeted forcytotoxic depletion by activating FcgR-expressing innate cells(intratumoralmacrophages). The removal of Tregs from the tumormicroenvironment thereby enhances the ability of the remainingeffector T cells to kill the tumor. By contrast, anti–PD-1mAbs nowin clinical use have been engineered to abrogate interactions withFcgRs, because PD-1 is broadly expressed by T cells, includingeffector CD8þ T cells that mediate antitumor activities; depletionof this T-cell subset would likely inhibit antitumor immunity andaugment tumor growth (Fig. 2). Whether Fc–FcgR interactions arerequired for the in vivo activity of anti–PD-L1, anti-CD137, anti-CD27, and anti-KIR mAbs now in clinical trials is the subject ofactive investigation.

The unexpected requirement for interactions with the inhibi-tory FcgRIIB for agonistic anti-TNFR family immunomodulatoryantibodies, or with activating FcgRs for some antagonistic anti-bodies, demonstrates how essential it is to understand the mech-anism of action with regard to FcgR engagement of each agonisticor antagonistic immunomodulatory antibody on a case-by-casebasis. It is likely that the expression levels of the therapeutic targeton effector immune cells, regulatory immune cells, or the tumoritself will dictate whether engagement of FcgRs is necessary toaugment or inhibit the effectiveness of a therapeutic antibody invivo. Expression patterns of activating and inhibitory FcgRs, aswellas the presence of the appropriate effector cells in the tumormicroenvironment, will further dictate whether and which FcgRsmay be involved. Because the complex conditions of the tumormicroenvironment cannot be recreated in vitro, careful in vivomechanistic analyses of each immunomodulatory antibodymustbe performed to inform the proper engineering of the Fc domainfor ideal interaction with the appropriate FcgRs for optimumin vivo efficacy. Thus, future generations of immunomodulatorymAbs will undoubtedly incorporate engineered Fc domains foroptimal activity in vivo.

Antibodies Prime Antitumor ImmuneResponses: The Vaccinal Effect

Although most mechanistic studies of antitumor antibodieshave focused on their short-term cytotoxic properties, an alternateconcept has emerged by which antitumor antibodies may func-tion to activate the immune system to stimulate a potent, long-term antitumor memory immune response. This phenomenon isknown as a vaccinal effect.

As early as 1907, Emil von Behring demonstrated that immu-nization with a mixture of diphtheria toxin and antitoxin (anti-diphtheria antibody) produced safe and lasting immunity todiphtheria in animals and humans (48). We now understandthesemixtures tobe antigen–antibody immune complexes,whichenhance the immunogenicity of soluble molecules. Whileimmune complexes are excellent inducers of humoral immunity(antibody responses), their ability to engage FcgRs expressed byAPCsof the innate immune system toprime cellular immunity (T-cell responses) is most relevant in the context of tumor immunity(Fig. 3; ref. 49). DCs, the most potent of the immune system'sAPCs, primed with immune complexes are capable of processingand presenting the relevant antigen to both CD4 (through tradi-tional antigen presentation) and CD8 T cells (through cross-presentation; refs. 50, 51). Studies using various antigen–anti-body immune complexes have demonstrated that DCs primedwith immune complexes cross-present antigens and generatemore potent CD8 T-cell responses compared with DCs primedwith antigen alone. Activating FcgRs are required for this process,and modulating the balance of activating versus inhibitory FcgRengagement (A:I ratio) toward activating FcgRs augments CD8immune responses and tumor immunity in vivo (8, 52). Mecha-nistically, immune-complex engagement of FcgRs leads to DCmaturation and upregulation of MHC molecules and costimula-tory molecules, including CD80, CD86, and CD40 (52, 53).Thus, immune complexes are potent inducers of cell-mediatedimmunity.

Passive administration of therapeutic antitumor cytotoxic anti-bodies likely results in the in vivo generation of immune com-plexes (Fig. 3), and recent studies have investigated whether suchantibodies initiate a vaccinal effect that is characterized by anantitumor cellular immune response. Examples of cytotoxic anti-body-induced vaccinal effects from the clinic include patientstreated with anti-MUC1 mAbs or anti-HER2/Neu mAbs, whogenerated MUC1-specific and HER2-specific T-cell responses,respectively (54, 55). Because a single course of treatment withanti-CD20 mAb (rituximab) can result in long-lasting, durableresponses, it has been hypothesized that treatment with this mAbmay also induce a vaccinal effect in patients with lymphoma (56).In support of this, lymphoma-specific anti-idiotype T-cellresponses were detected in some patients treated with anti-CD20rituximab (57, 58). Anti-CD20mAb treatment of lymphoma cellsin vitro stimulates DCmaturation and CD8 T-cell activation (59),and vaccination with huCD20þ tumor cells and anti-huCD20mAb treatment showeda synergistic antitumor effect inmice (60).Studies using mouse models have also demonstrated that passiveadministration of anti-CD20 mAbs can initiate antitumor T-cellresponses and vaccinal effects (21, 61).

Recent studies have elucidatedhowanantitumor vaccinal effectcould be generated upon treatment with a cytotoxic antitumormAb using a murine model in which syngeneic EL4 lymphomacells express huCD20 (EL4-huCD20 cells) as a tumor neoantigen

DiLillo and Ravetch




(21, 61). Mice given EL4-huCD20 cells and subsequently treatedwith anti-huCD20 mAbs cleared their tumors in an activatingFcgR-dependent manner, again demonstrating that the initialcytotoxic effect of antitumor mAbs requires Fc–FcgR interactions(21). Anti-huCD20 mAb-treated mice that survived this primarytumor challenge were rested for 90 days and then rechallengedwithout additionalmAb treatment. ThemAb/tumor-primedmicesurvived rechallenge with EL4-huCD20 cells as well as a distincttumor cell line expressing huCD20, but did not survive rechal-lenge with wild-type EL4 cells (21). Thus, mice primed with EL4-hCD20 and mAb generate a vaccinal effect memory immuneresponse against theCD20antigen and subsequently reject rechal-lenge with EL4-huCD20 cells, but not wild-type EL4 cells. Thisprocess relies on both CD4 and CD8 T cells, and adoptive transferof T cells from mAb/tumor-primed mice transfers the vaccinaleffect to na€�ve animals (61). Using transgenic mice in whichactivating FcgRs are conditionally deleted on CD11cþ cells, it hasbeendemonstrated that FcgRexpressionbyDCs is required for thegeneration of the vaccinal effect (21). Collectively, these resultsdemonstrate that cytotoxic clearance (ADCC/ADCP) of tumorcells by anti-huCD20 mAbs induces the generation of huCD20immune complexes, which engage FcgRs expressed by DCs toinitiate a huCD20-specific T-cell vaccinal effect (Fig. 3, left).

Mechanistically understanding the antitumor vaccinal effect inthe context of the human FcgR system is vital for designingoptimal antitumor antibody therapeutics. As discussed earlier,expression patterns of FcgRs differ between mouse and man.While ADCC-mediating human monocytes/macrophages utilizeonly huFcgRIIIA for their cytotoxic effects, vaccinal effect-medi-ating human DCs only express huFcgRIIA (21). To dissect thevaccinal effect mechanistically in the context of the huFcgRsystem, FcgR-humanized mice were used in combination withhuIgG1 anti-huCD20 mAbs, as well as mutant anti-huCD20mAbs that selectively engage either huFcgRIIA, huFcgRIIIA, orboth of these receptors. During the primary tumor challenge,engagement of huFcgRIIIA was both necessary and sufficient forADCC-mediated clearance of EL4-huCD20 tumor cells;huFcgRIIA played no role. By contrast, engagement of huFcgRIIAwas required for optimal induction of the vaccinal effect in FcgR-humanized mice, consistent with this being the sole activatinghuFcgR expressed by human antigen-presenting DCs (21). Thus,differential human FcgR requirements exist for antitumor mAb-mediated cytotoxicity versus induction of a memory cellularimmune response during the vaccinal effect.

Looking toward the Future in TherapeuticAntibodies in Malignancy

These new mechanistic insights into the antitumor vaccinaleffect shed important light on the in vivo effector mechanismstriggered by cytotoxic antitumor antibodies in the context ofhuman IgG and human FcgRs. First, Fc engagement of huFcgRIIIAexpressed by monocytes and macrophages is absolutely vital forimmediate cell-mediated cytotoxicity and phagocytosis triggeredby antitumor mAbs. Second, immune-complex engagement of

huFcgRIIA expressed by human DCs induces DCmaturation andupregulation of costimulatory molecules for optimal antigenpresentation and cross-presentation to stimulate long-term anti-tumor T-cell memory (Fig. 3, left). As discussed above, the currentcollection of next-generation antitumor antibodies are beingoptimized through glycoengineering or with point mutations forsuperior huFcgRIIIA engagement to augment their cytotoxiceffects. However, these new antibodies have not been optimizedfor huFcgRIIA engagement, as defucosylation does not affectbinding to this receptor. Therefore, an ideal antitumor antibodywill need to be optimized for both effector functions: optimizedhuFcgRIIIA engagement for augmented immediate cytotoxicityand optimized huFcgRIIA binding for more potent induction oflong-term antitumor vaccinal effect T-cell responses.

The next phase of antitumor immunotherapy will not onlyconsist of antitumor and immunomodulatory antibodies withengineered Fc domains for optimal effector functions, but itshould also incorporate logical combinations of Fc-optimizedantitumor antibodies, Fc-optimized immunomodulatory antibo-dies, and traditional radiotherapies and chemotherapies (Fig. 3).Because preclinical studies have suggested synergistic effects ofimmunomodulatory mAbs with distinct mechanisms of action(e.g., combining anti–CTLA-4 and anti–PD-1 checkpoint inhibi-tors), combinations of various immunomodulatory antibodiesare being tested in clinical trials for synergy (62). Combiningimmunomodulatory mAbs with traditional chemotherapy andradiation treatments has also shown some benefit in early trials,most likely because cell death exposes tumor-specific antigens orneoantigens for recognition by an unencumbered immune sys-tem (Fig. 3, right; refs. 63, 64). Combining checkpoint inhibitorswith antitumor vaccination may also lead to enhanced results.Thus, a logical extension would be to test cytotoxic antitumormAbs in combination with immunomodulatory mAbs. Onewould predict that cytotoxic killing of tumors would generateimmune complexes for vaccinal effect induction as well as poten-tially expose other tumor antigens; memory T-cell responseswould likely be boosted in combination with immunomodula-tory mAb treatment (Fig. 3, right).

Although multiple classes of therapeutic antibodies are nowavailable to treat the full array of human malignancies as singleagents or in logical combinations, one common theme cannot beignored: The effector functionsmediated by the Fc domain of eachtherapeutic antibody must be carefully evaluated and mechanis-tically understood. Through thoughtful analysis of each anti-body's mechanism(s) of action in the context of the human FcgRsystem, more potent therapies can be engineered through opti-mized engagement of the appropriate FcgRs.

AcknowledgmentsD.J. DiLillo received a postdoctoral career development fellowship from the

Leukemia and Lymphoma Society of America.

Received April 29, 2015; accepted May 20, 2015; published online July 2,2015.

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2015;3:704-713. Cancer Immunol Res David J. DiLillo and Jeffrey V. Ravetch Immunomodulatory Therapeutic Antibody Effector FunctionsFc-Receptor Interactions Regulate Both Cytotoxic and

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