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The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer

The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer Antibodies targeting T-cell inhibitory pathways, such as CTLA-4 and PD-1/PD-L1, are emerging as an important class of cancer therapeutics, and a next generation of immunomodulatory therapies targeting alternative inhibitory (e.g. TIM-3, LAG-3, B7-H4, B7-H3, VISTA, A2aR), as well as co-stimulatory (e.g. CD27, OX40, GITR, CD137), pathways are poised to join them. Most of these immunomodulatory antibodies are of IgG isotypes that have low, or no, binding to the Fc gamma receptors (FcγRs) that trigger cell-mediated cytotoxic effector functions such as antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). These isotypes were selected to minimise the risk of depleting the T cells upon which such antibodies depend for their mechanism of action. However, recent preclinical data highlight a potential role for FcγR engagement in the activity of such antibodies. Here we review the biology of the FcγRs and IgG isotypes in both humans and mice, detail the potential roles that FcγR interactions can play in the activity of monoclonal antibodies in general, and of immunomodulatory antibodies in particular, and discuss how preclinical studies on these interactions might be best interpreted and translated to a human setting. Keywords: Fc gamma receptors, Anti-CTLA-4, Anti-GITR, Immunomodulatory antibodies Introduction TIM-3, LAG-3, B7-H4, B7-H3, VISTA, A2aR), as well as Antibodies of the IgG sub-class are bi-functional molecules, co-stimulatory (e.g. CD27, OX40, GITR, CD137), pathways possessing a F(ab) domain, variable in sequence and respon- are poised to join them. In contrast to agents such as rituxi- sible for the binding of antigen, and an Fc domain, constant mab, most of these immunomodulatory antibodies are of in sequence and responsible for mediating a range of anti- IgG isotypes that have low, or no, binding to the Fc gamma body effector functions [1]. These functions are primarily receptors (FcγRs), and effects such as ADCC have not been triggered through interaction with the complement compo- considered to be critical to their activity. Recent studies in nent C1q or with a family of FcγRs expressed, primarily, on mice have, however, highlighted a potential role for FcγR the surface of leukocytes. Each IgG isotype has a different binding, and FcγR mediated effector functions in the activity binding profile to the various FcγRs, and each FcγR has a of antibodies targeting a number of these pathways. different cellular expression pattern. These differences en- Below we review the findings of these recent studies and able the breadth, flexibility and control of function required discusshow they maychangeour understandingofthe role to mount an effective and regulated humoral response. FcγR interactions play in the activity of immunomodulatory Effector functions mediated by the FcγRs, such as ADCC antibodies in mice. We begin, however, by reviewing the and ADCP, are believed to play an important role in the ac- biology of the FcγRs and IgG isotypes in mice and humans, tivity of several therapeutic antibodies; including a number highlighting the key similarities and differences, which need used in Oncology, for example rituximab and trastuzumab. to be appreciated when thinking about how to translate Antibodies targeting T-cell inhibitory pathways, such as these findings to a human setting, in order to select the op- CTLA-4 and PD-1/PD-L1, are emerging as an important timal isotype for any potential therapeutic. class of cancer therapeutics, and a next generation of immu- nomodulatory therapies targeting alternative inhibitory (e.g. Review The Fc gamma receptor family * Correspondence: stewartr@medimmune.com Humans and mice possess two classes of FcγRs, the acti- MedImmune, AKB Building, Granta Park, CB21 6GH England, UK Full list of author information is available at the end of the article vating and inhibitory receptors. In both species FcγRI is © 2014 Stewart et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 2 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 an activating receptor with high affinity for IgG, and is immunogenicity risk respectively) and will not be con- expressed on monocytic DCs and on monocytes/macro- sidered further in this review. Of the remaining isotypes phages broadly in humans but in select locations in hIgG1 demonstrates the highest affinity for FcγRs, with a mouse (Table 1) [2,3]. FcγRI is the only FcγR with a nM or low μM binding affinity to all receptors (estimated functionally meaningful binding affinity for monomeric by surface plasmon resonance [SPR]), and is considered a IgG, the remaining FcγRs all exhibit such low affinity for potent inducer of all cell mediated effector functions, such the Fc region of IgG that functional engagement is only as ADCC and ADCP. hIgG4 demonstrates a nM affinity expected to occur upon binding of antibody complexed for FcγRI but μM affinities for all other receptors and is through antigen engagement (Table 2). Both species pos- considered a poor inducer of such effector functions. sess a single inhibitory FcγR, FcγRIIb, which is expressed hIgG2 has no measurable affinity for FcγRI, a high nM on B cells, DCs and basophils in both species, and found affinity for the H131 form FcγRIIa and μM affinities for additionally on monocytes, macrophages and all granulo- all other FcγRs (Table 2). cyte populations in mice (Table 1). The remaining FcγRs These absolute affinities are a useful guide as to the are all activating, but more divergent between species. binding of the hIgG isotypes to hFcγRs relative to each In mice there are only 2 additional activating receptors other. However, as noted above, the functional inter- FcγRIII, which is expressed broadly on NK cells, mono- action between IgG and most FcγRs is not monomeric cytes, macrophages, granulocytes and DCs, and FcγRIV, in nature, as is modelled by SPR. In a study [5] examin- which is found only on monocytes, macrophages and neu- ing the more functionally relevant binding of complexed trophils. In humans there are four additional activating IgG to FcγRs on cells, the relative ranking of the isotypes FcγRs, the best characterised being FcγRIIa and FcγRIIIa. with respect to FcγR binding was retained, however no FcγRIIa is expressed on granulocytes, monocytes and binding of IgG2 to cells expressing the inhibitory FcγRIIb monocyte-derived cells such as macrophages and DCs. or the F158 form of the activating FcγRIIIa could be de- FcγRIIIa is expressed primarily on NK cells, but is also tected using this methodology, despite the interaction be- found on monocytes and macrophages under some cir- tween IgG2 and these proteins demonstrated by SPR. cumstances (Table 1). Each of these receptors exists as Mice also have four IgG isotypes but they are not two allotypic variants with differing binding affinity for equivalent to those of human in either nomenclature or IgG (Table 2). For FcγRIIIa the more common F158 allo- properties. Mouse (m)IgG1, in contrast to hIgG1, is con- type has a lower affinity than the V158 allotype. For sidered a low effector function isotype, since while it FcγRIIa the more common H131 allotype has a higher binds inhibitory FcγRIIb and activating FcγRIII with nM affinity than the R131 allotype [4]. The other two acti- affinity, it has no measurable binding to activating FcγRIV. vating receptors are far less well characterised. FcγRIIc In mice it is the two IgG2 isotypes, mIgG2a and mIgG2b, is expressed in only 20% of people and is closely related to, that have the highest binding affinities to FcγRs, particu- but expressed more restrictedly than, FcγRIIa. FcγRIIIb is larly to FcγRIV (Table 2). In mice there is no IgG4 isotype, a GPI linked receptor expressed mainly on the sur- and mIgG3 has no measurable FcγR interactions [6]. face of neutrophils. These differences in FcγR and antibody isotype inter- Mice and humans also have a divergent family of IgG actions and expression, summarised in Tables 1 and 2, isotypes. In humans there are 4 isotypes, IgG1-4, each of mean that there are no direct homologs between human which has differing binding affinities to the various and mouse with respect to FcγR and antibody isotype FcγRs. Human (h)IgG3 demonstrates relatively high af- biology. However, it is possible to draw some parallels finity binding to most FcγRs, but is not used routinely between the species, for example mIgG2a and hIgG1 are as a therapeutic format (due to its long hinge region often considered equivalent functionally due to their and polymorphic nature which represent a stability and broad and high FcγR binding, while the generally lower Table 1 Expression pattern of the major FcγRs in human and mouse Cell type FcγRI FcγRIIb hFcγRIIa mFcγRIII hFcγRIIIa mFcγRIV hm h m B cells + + DCs + + + + + + NK cells ++ Monocytes/Macrophages + ~ ~ + + + + + Neutrophils (+) ~ + + + + Italics indicates an inhibitory receptor. Normal text indicates an activating receptor. Human receptors are denoted by h and mouse receptors by m. + indicates expression, (+) indicates inducible expression and ~ indicates expression on only a sub-set or on cells at specific physiological locations [2,3]. Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 3 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 Table 2 Relative affinity of binding between the major Nevertheless a significant effort has been made to engineer FcγRs and IgG Isotypes in human and mouse antibodies with improved affinity for the activating FcγRs Human Mouse [10-12], with the aim of enhancing these cytotoxic func- tions and improving therapeutic efficacy, and the outcome FcγR IgG1 IgG2 IgG4 FcγR IgG1 IgG2a IgG2b of clinical trials with such agents may provide further clar- I ++++ - ++++ I - ++++ ++++ ity as to the true contribution of FcγR mediated cytotox- IIa H +++ ++ ++ III ++ ++ ++ icity in activity. R +++ + ++ An alternative role for FcγRs is in the activity of anti- IIb ++ - ++ IIB +++ ++ +++ bodies that function by agonising cell surface receptors, IIIa V +++ + ++ IV - +++ ++ for example those of the tumor necrosis factor receptor F ++ - ++ (TNFR) super family. In this context binding of IgG Fc to FcγRs results in higher order clustering of antibody. Italics indicates an inhibitory receptor. Normal text indicates an activating 5 −1 receptor. Rankings are based on K x10 M for each interaction reported in This in turn results in higher order clustering of the target 5 −1 published studies [1,5,6]. ++++ indicates a K greater than 100 x 10 M , +++ 5 −1 receptor when bound by the F(ab) domain, which triggers indicates a K between 20 and 99 x 10 M , ++ indicates a K between 1 and A A 5 −1 5 −1 19 × 10 M , + indicates a K between 0.5 and 0.9 × 10 M , − indicates a K A A downstream signalling (Figure 1, right). This clustering 5 −1 that was undetectable or less than 0.5 × 10 M . effect has been shown to be critical to the activity of antibodies specific for the TNFR family members CD40 and more select binding profiles of mIgG1 and hIgG4 [13,14] death receptor 5 (DR5) [15,16] and CD95 (Fas) [17] make them close functional analogs. Additionally the ac- in preclinical mouse models, and has been suggested to tivating mFcγRIII and hFcγRIIa are often considered play a similar role in the activity of other TNFR agonists analogous based on affinities and expression pattern, as such as anti-OX40 and anti-CD27 [18]. are activating mFcγRIV and hFcγRIIIa. These parallels are frequently imperfect though, for example: hFcγRIIIa The canonical role of FcγRs in immunomodulatory is expressed at high levels on NK cells, while mFcγRIV antibodies is not; hFcγRIIa is not expressed on NK cells while Immunomodulatory antibodies mediate their beneficial mFcγRIII is; mIgG1 is a more functionally restricted iso- effects by promoting anti-tumor immunity. There are a type than hIgG4, based on their relative affinities for ac- number of potential ways to achieve this, but one area tivating FcγRI, mFcγRIV and hFcγRIIa. These differences showing particular promise is the use of antibodies that need to be kept in mind, and pose challenges, when try- antagonise inhibitory receptors on the surface of T cells. ing to translate findings in mouse preclinical models to One such antibody, the anti-CTLA-4 ipilimumab, was humans. approved based on its ability to improve survival in metastatic melanoma patients [19], while a number of The potential roles of FcγRs in the activity of therapeutic antibodies targeting PD-1 are showing highly encour- antibodies aging results in phase 1 clinical trials [20,21]. An alterna- The FcγR mediated functions most commonly associated tive approach, which is earlier in clinical development with therapeutic antibodies are those that mediate target but also showing promise, is the use of antibodies that cell elimination; ADCC, or the related ADCP. These agonise co-stimulatory receptors. The agonistic anti-CD40 functions are triggered when antibody binding to antigen antibody, CP870,893, has demonstrated early signs of activ- on the surface of a target cell generates sufficient avidity ity in pancreatic cancer [22], and tumor shrinkage was also to trigger signalling through FcγRs on effector cells such observed in some patients treated with agonistic antibodies as NK cells and macrophages, which then eliminate tar- directed against OX40 [23] and CD137 [24]. get cells through direct killing or phagocytosis (Figure 1, As mentioned above FcγR engagement has been left). Preclinical models show that these forms of FcγR- shown to be critical for the activity of antibodies that ag- mediated cytotoxicity are a significant component of the onise the TNFR CD40 in mice. The anti-tumor activity of mechanism of action for tumor targeted antibodies such anti-mouse CD40 in these studies is lost upon introduc- as the anti-CD20 antibody rituximab and the anti-HER2 tion of the D265A mutation into its Fc domain [13], antibody trastuzumab [7]. The clinical evidence for the which results in an antibody with no measurable FcγR role of such interactions, however, is more mixed. A binding [6]. It has also been shown that the in vitro activ- number of relatively small studies have shown improved ity of antibodies directed against other TNFRs in human outcomes in patients that are homozygous for the higher cells, such as OX40, GITR, CD137 and CD27, requires, affinity allotypic forms of human FcγRIIIa and FcγRIIa or is greatly enhanced by FcγR engagement or immobil- [8,9], supporting a role for FcγR-mediated cytotoxicity isation [25,26]. While the evidence suggests that a re- in this setting, but several larger, randomized studies quirement for cross linking is a common property of have generally failed to show the same associations [4]. antibodies targeting TNFRs receptors, there are some Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 4 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 Figure 1 The two primary forms of interaction between therapeutic IgGs and FcγRs. On the left: when IgG is bound to high levels of antigen on target cells, such as tumor cells, the resulting clustered Fc domains engage with FcγRs on immune effector cells such as NK cells and macrophages. This drives signals through those FcγRs triggering the initiation of cell-mediated effector functions such as ADCC and ADCP. On the right: when IgG is bound by its Fc domain to FcγRs it can act to cluster low levels of target receptors, such as members of the TNFR family. This clustering acts to trigger signals though the TNFR into target cells, such as T cells. exceptions. For example the hCD27.15 anti-human CD27 cells (APCs) critical to mounting an adaptive immune mAb has demonstrable agonistic properties in the soluble response [13]. In the case of the second hypothesis, the phase [27], the anti-CD40 antibody CP870,893 has been balance of FcγR engagement would become critical to shown to function in vitro in solution and as a F(ab)’ the activity of anti-CD40. Too little engagement, as fragment that lacks any Fc domain [28] and the anti- seen with alglycosyl antibodies, and no agonistic signal- OX40 antibody 9B12, which is a mouse IgG1 with very ling is triggered so no anti-tumor activity is observed. low affinity interactions with FcγRs, has shown early Too much FcγR engagement on effector cells and deple- signs of clinical activity and demonstrated the ability to tion of critical immune cells begins to occur and anti- activate T cells in patients [23]. tumor activity is once again lost. Such a trade off in FcγR Two studies that further explored the role of FcγR en- affinity could also apply to other immunomodulatory anti- gagement for the activity of anti-mouse CD40 demon- bodies that target TNFRs (CD27, OX40, GITR, CD137), strated that it is the inhibitory mouse FcγRIIb that plays which are also expressed on critical immune cells. In an essential role [13,14]. One of these studies [14] went addition it has been shown that for CD137 antibody medi- on to demonstrate that other mouse FcγRs are capable ated internalisation of receptor can play a role in driving of cross linking anti-CD40 to drive activity in vitro, and signalling [29], and it is unclear how changes in Fc isotype proposed that the dominant role of FcγRIIb in vivo was might affect this property. due primarily to its bioavailability at the site of action For those antibodies that target inhibitory receptors for anti-CD40, namely the secondary lymphoid organs. like PD-1 and CTLA-4 there has been little reason to Both studies also went on to show that the activity of believe, based on their antagonistic mechanism of action, anti-mouse CD40 was lost upon class switching from a that FcγR engagement plays an important role in their mouse (m)IgG1 to a mIgG2a. Two alternative, and non- activity, and no significant evidence to contradict this mutually exclusive, hypotheses exist to explain this obser- belief exists in the clinical setting. Since all of the anti- vation. The first is that the reduced affinity of mIgG2a for gens targeted by these antibodies are expressed either on FcγRIIb results in reduced cross linking at the relevant site T cells or on APCs, the preference has been to select iso- of action and so limits activity [14]. The second is that the types that will avoid the risk of depleting these cells loss of activity occurs due to increased depletion of CD40 through Fc mediated mechanisms; an outcome that would positive cells, which are predominantly antigen presenting likely negate any beneficial immune activation. As a result, Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 5 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 acommonfeatureofthe immunomodulatory antibodies engagement of the activating FcγRs in mouse tumor currently in clinical development is that they are low or models. Further examination of the mechanism for this null effector function isotypes (Table 3). Main exceptions dependence revealed that in the lymph nodes anti-mouse include ipilimumab (anti-CTLA-4), MSB0010718C (anti- CTLA-4 acted to increase the number of CD4 T cells, in- PD-L1) and antibodies like MGA271 (anti-B7-H3), CDX- cluding regulatory T cells (T-regs), but that within the 1127 (anti-CD27) and alemtuzumab (anti-CD52) which tumor it acted to selectively deplete only T-regs [33]. In employ ADCC of tumor cells as a part of their mechanism one of the three studies, a comparison of several different of action. anti-mouse CTLA-4 antibodies showed that the extent of However, several recent preclinical studies are calling T-reg depletion observed correlated with activity [33]. into question the view that all TNFR agonist antibodies The depletion of T-regs was mediated predominantly will behave as anti-CD40 does, or that all antibodies tar- by mouse FcγRIV, but was independent of NK cells, and geting inhibitory receptors will be maximally effective as considered likely to rely on the activity of intratumoral a low or null effector function isotype. CD11b + macrophages [33]. In all three studies the level of CTLA-4 expression was shown to be increased on intratu- The evolving role of FcγRs in immunomodulatory moral T cells relative to those in lymph nodes, and was antibodies higher on T-regs than on other T cell sub-sets. Three recently published studies [31-33] have examined In their study Bulliard et al. [31] also examined the the role of mouse FcγRs in the activity of anti-mouse role of mouse FcγRs in the activity of the anti-mouse CTLA-4 antibodies. In these studies anti-mouse CTLA-4 GITR antibody DTA-1. In contrast to the observations antibodies were studied in a range of syngeneic tumor made for anti-mouse CD40, DTA-1 activity was minim- models where they have previously shown activity, includ- ally affected by the absence of inhibitory FcγRIIb, but ing MC38 (colon), CT26 (colon) and B16 (melanoma). The was dependent on the presence of the activating mouse approach taken to examine the role of FcγRs varied be- FcγRs [31]. In further contrast to anti-CD40, a mouse tween studies and either involved the use of FcγRknock (m)IgG2a version of DTA-1 was equal in activity to the out mice [31,33] or reformatting of the anti-mouse CTLA- parental rat IgG2b version; although no mIgG1 was 4 to a variety of different antibody isotypes [32]. Irrelevant tested. Analogous to the mechanism observed for anti- of the approach taken the result was the same; the activity mouse CTLA-4, it was shown that while mIgG2a or rat of anti-mouse CTLA-4 was shown to be dependent upon IgG2b forms of DTA-1 treatment resulted in expansion of Table 3 List of immunomodulatory antibodies currently in clinical development and their respective isotypes Antibody Target Company Latest active clinical phase Isotype Ipilimumab CTLA-4 BMS Approved IgG1 Tremelimumab CTLA-4 MedImmune/AstraZeneca II IgG2 Nivolumab PD-1 BMS Approved IgG4 MK-3475 PD-1 Merck III IgG4 CT-011 PD-1 CureTech II IgG1 MPDL3280A PD-L1 Genentech/Roche III Aglycosyl IgG1 MEDI4736 PD-L1 MedImmune/AstraZeneca I IgG1 TM MDX-1105 PD-L1 BMS I IgG4 MSB0010718C PD-L1 Merck KGaA I IgG1 BMS-663513 CD137 BMS I IgG4 PF-05082566 CD137 Pfizer I IgG2 TRX518 GITR GITR Inc I Aglycosyl IgG1 MEDI6469 OX40 MedImmune/AstraZeneca/AgonOX I Mouse IgG1 CP-870,893 CD40 Roche I IgG2 CDX-1127 CD27 CellDex I IgG1 Lirilumab; IPH2102 KIR BMS/Innate Pharma II IgG4 MGA271 B7-H3 Macrogenics/Servier I IgG1 Alemtuzumab CD52 Genzyme/Bayer Approved IgG1 IgG1 TM = IgG1 triple mutant, which contains 3 point mutations in the Fc domain that reduce the binding affinity of the mAb to FcγRs [30]. Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 6 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 all T cell populations in the lymph node, they acted to de- effector function is entirely dependent upon the pres- plete T cells within the tumor. Also similar to anti-mouse ence of FcγR-bearing effector cells at an appropriate CTLA-4, this depletion preferentially occurred in the T-reg density. The studies by both Simpson et al. and Bulliard compartment and, although some reduction in both con- et al. highlight what many of us will have observed in ventional CD4+ and CD8+ T cells was also observed, re- our own work, that there are very few effector cells, such sulted in an increase in the CD8:T-reg ratio within the as NK cells, neutrophils and macrophages, present in tumor. In a more recent study published earlier this year lymph nodes, which are dominated by T, B and dendritic Builliard et al. [34] demonstrated a comparable mechan- cells. What both studies additionally show is that mouse ism, and dependence on activating mouse FcγRs for an tumors are extremely rich in FcγR-expressing effector anti-mouse OX40 antibody. It is however worth noting cells. Simpson et al. evidence greater than 20% NK cells that alternative studies have proposed, and evidenced, and greater than 40% CD11b + cells, which are predom- a mechanism of regulatory T cell re-programming for inantly macrophages, in B16 tumors and Bulliard et al. DTA-1 [35] and for anti-human OX40 mAbs [36], sug- highlight very similar levels of the same cells in CT26 gesting that a loss of FOXP3 expressing T cells following tumors. The coincident presence of high levels of target treatment with agents targeting either pathway may not antigen and a high density of effector cells creates a per- necessarily equate to depletion. fect storm for Fc mediated effector function within the These preclinical studies have brought into question tumor, while the lower target expression and relative two generally held beliefs regarding the role of FcγRs in paucity of effector cells in the periphery makes the same the activity of immunomodulatory antibodies. The first effector function harder to achieve in the lymph node. It is the idea that inhibitory FcγRIIb is the critical receptor is however worth noting that peripheral depletion of im- for the activity of all TNFR agonist antibodies. The data mune cells is possible, as demonstrated by the data on generated for the anti-mouse GITR antibody DTA-1 anti-CD40 [13] and the well documented effects of anti- clearly demonstrate FcγRIIb independent activity, and CD20 [39], suggesting that the nature or expression level our own unpublished data demonstrates similar inde- of the target are likely critical. pendence for other TNFR targeting antibodies. These data suggest, as previously proposed by White et al., that Translating preclinical data to man the specific mouse FcγRs involved in cross linking of As outlined at the start of this review, the significant dif- TNFR agonists may vary depending on the pathway be- ferences between human and mouse FcγR and IgG iso- ing targeted and on the bioavailability of each of the type biology make translation of findings between species FcγRs at the primary site of action. The second is the extremely challenging, and several factors need to be con- idea that effector function enabled antibodies represent sidered and better studied in the human setting before a risk to activity due to depletion of key immune cell these findings can be confidently applied to the activity of sub-sets. Importantly the data highlight the fact that per- therapeutic antibodies in man. ipheral and intratumoral antibody mediated effector Taking anti-CTLA-4 as an example, the first signifi- function are far from equivalent. This difference appears cant translational challenge is the key role for mouse to hinge on two critical differences; the level of target FcγRIV in the T-reg depleting activity demonstrated for antigen expression and the presence of appropriate ef- anti-mouse CTLA-4. FcγRIV does not exist in humans, fector cells (Figure 2). and its closest analog, human (h)FcγRIIIa, is expressed The extent of effector function triggered by antibody predominantly on NK cells, which played no role in the binding has been shown in a number of studies to be mainly macrophage mediated T-reg depletion observed strongly influenced by the level of target antigen expres- with anti-mouse CTLA-4 [33]. In humans macrophages sion [37,38], with antibodies directed against both hu- can express hFcγRIIIa following certain stimuli, such as man CCR4 and CD20 failing to elicit in vitro ADCC IL-10, but primarily express hFcγRIIa/b and hFcγRI, and against human tumor cell lines expressing anything less it is feasible that in human tumors these receptors would than 10 antigen molecules per cell and reaching optimal play the same role as mouse FcγRIV does in mouse tu- 5 6 activity only in the presence of 10 -10 antigen mole- mors. Further study of the intratumoral effector cell and cules [37]. Many of the targets for immunomodulatory FcγR constituent within human tumors would be ex- antibodies are expressed at low to undetectable levels on tremely helpful in understanding which human FcγR resting, peripheral immune cells, but are up-regulated might play the role of mouse FcγRIV in humans. significantly in the presence of pro-inflammatory stimuli, The second challenge for translation lies in the differ- such as those present in the tumor microenvironment. ences between IgG isotypes across species. In mice only As such, and in the light of recent studies, it is clear that mouse (m)IgG2a and mIgG2b demonstrate any measur- in mouse models it is significantly easier to elicit effector able binding to mouse FcγRIV; mIgG1 can therefore function against intratumoral immune cells. Additionally show none of the T-reg depleting activity observed for Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 7 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 Figure 2 Critical differences between effector function in the periphery relative to the tumor. On the left: in lymph nodes, relatively low levels of cell activation and pro-inflammatory cytokines result in low levels of expression for most targets of immunomodulatory antibodies, which are generally low on resting immune cells. This low level of target expression, together with a paucity of effector cells in lymph nodes means depletion of cells through cell mediated effector functions do not readily occur. On the right: high levels of pro-inflammatory cytokines within the tumor microenvironment trigger up-regulation of many targets. This increased target expression together with a high density of effector cells, such as macrophages, enables depletion of cells through cell-mediated effector functions. The level of target expression, and the sensitivity to depletion varies across cell types and favours depletion of T-regs for targets such as CTLA-4 and GITR. anti-mouse CTLA-4 in mice. In contrast, while human(h) 3 year survival rate seen in melanoma patients treated IgG1 has a significantly higher binding affinity to all human with the two agents. FcγRs, hIgG2 and hIgG4 are both capable of interact- The third translational challenge relates to the key ing with human FcγRIIa andIIIa, andwhile human driver for differential depletion of T-regs relative to FcγRI cannot bind hIgG2, its high affinity for monomeric other cell types in all three of the published studies, that IgG suggests that it would be heavily occupied by serum is target expression level. It is clear from the published IgG and less available to partake in effector function [40]. studies that the levels of GITR and CTLA-4 on mouse Critically, while hIgG2 and hIgG4 are both very poor T-regs is higher than that observed on mouse effector mediators of NK cell driven ADCC, they are both cap- T cells, in particular CD8s. However there are no studies able of mediating ADCC via PBMCs, monocytes and available comparing how the level of target expression neutrophils [41,42]. It is especially conceivable that in a varies between mice and humans, few that compare the tumor microenvironment, with its increased target anti- differential expression of target on human T-regs relative gen levels and high density of effector cells, that a hIgG2 to effectors, and none that examine how this expression or hIgG4 could begin to mediate some degree of ADCC changes quantitatively in the tumor versus the periphery. or ADCP. If one were to assume that a mouse IgG2a These are all significant variables that must be studied in were equivalent to human IgG1 and mouse IgG1 equiva- humans before a direct translational conclusion can be lent to human IgG2 then the data of Selby et al. would drawn. predict significant differences in the two anti-CTLA-4 antibodies that have been tested clinically one of which, Conclusions tremelimumb, is a human IgG2 and the other of which, Future directions ipilimumab, is a human IgG1 currently registered for the Immunomodulatory antibodies are showing positive re- treatment of metastatic melanoma. The complex differ- sults in early clinical trials in cancer. These antibodies ences in FcγR and IgG isotype biology that exist between generally fall into two classes. Those that antagonise in- species, outlined above, suggest however that such as- hibitory receptors, such as CTLA-4 and PD-1, and those sumptions about the comparability of mouse and human that agonise stimulatory receptors, such as CD27, OX40, IgG isotypes may be too reductionist. In relation to GITR and CD137. this, while no definitive comparison of the two agents Many groups, including our own, have been working has been conducted, the toxicity profiles of ipilimumab to study the requirement for FcγRs in the activity of TNFR and tremelimumab are comparable [19,43] and a recent agonist antibodies, but the recent study of Bulliard et al. commentary in the Journal of Clinical Oncology [44] [31] has further highlighted the potential complexity of this highlighted the very limited differences observed in the requirement by providing evidence that inhibitory mouse Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 8 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 FcγRIIb is not required for the activity of all TNFR agonist Competing interests Ross Stewart and Robert Wilkinson are salaried employees of MedImmune Ltd. antibodies in mice, as has been previously suggested. Fur- Scott Hammond and Mike Oberst are salaried employees of MedImmune LLC. ther work is required to pull apart the target specific nature All four authors hold shares in the AstraZeneca group. of FcγR requirements for mAb activity and the relative contributions of pathway agonism versus targeted deple- Authors’ contributions tion of cells. In the case of TNFR agonists pulling these RS, SH and MO conceived of, planned and drafted the initial content of the article. RS provided all figures. RW provided substantive review. All authors apart is made very difficult by the inability to divorce FcγR read and approved the final manuscript. cross linking, and therefore agonism, from effector func- tion for the majority of such antibodies. Authors’ information The study by Bulliard et al. [31], together with that of RS, SH, MO and RW are part of the Oncology research group within Selby et al. and Simpson et al. [32,33], also highlighted MedImmune where they are involved in the discovery and development of a range of immunomodulatory antibody approaches. the previously underappreciated difference between FcγR- mediated effector function in the periphery as compared to Acknowledgements the tumor microenvironment in mice and highlighted the The authors thank Norman Greenberg for careful review of the final unexpected potential role of FcγRs in the activity of immu- manuscript. nomodulatory antibodies that target the inhibitory receptor Author details CTLA-4 in mice. Based on these studies it will be interest- MedImmune, AKB Building, Granta Park, CB21 6GH England, UK. ing to see how FcγR interactions may impact antibodies MedImmune, One Medimmune Way, Gaithersburg, MD 20878, USA. targeting PD-1 or PD-L1, which have very different expres- Received: 14 February 2014 Accepted: 16 July 2014 sion patterns to CTLA-4, despite their shared inhibitory function. The most important future studies however will be References those that address the translatability of these findings in 1. Nimmerjahn F, Ravetch JV: Fcgamma receptors as regulators of immune mouse preclinical models to man. While their exact responses. Nat Rev Immunol 2008, 8:34–47. 2. Bruhns P: Properties of mouse and human IgG receptors and their value remains unconfirmed [4], studies exploring the im- contribution to disease models. Blood 2012, 119:5640–5649. pact of human FcγR polymorphisms on clinical activity 3. 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In Book Role of the immunoglobulin constant region in the antitumor activity of antibodies to cytotoxic T-lymphocyte antigen-4 (CTLA-4), Journal of Clinical Oncology, Volume 15_suppl. 31st edition. 2013:9055. doi:10.1186/s40425-014-0029-x Cite this article as: Stewart et al.: The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer. Journal for ImmunoTherapy of Cancer 2014 2:29. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal for ImmunoTherapy of Cancer Springer Journals

The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer

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Copyright © 2014 by Stewart et al.; licensee BioMed Central
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Abstract

Antibodies targeting T-cell inhibitory pathways, such as CTLA-4 and PD-1/PD-L1, are emerging as an important class of cancer therapeutics, and a next generation of immunomodulatory therapies targeting alternative inhibitory (e.g. TIM-3, LAG-3, B7-H4, B7-H3, VISTA, A2aR), as well as co-stimulatory (e.g. CD27, OX40, GITR, CD137), pathways are poised to join them. Most of these immunomodulatory antibodies are of IgG isotypes that have low, or no, binding to the Fc gamma receptors (FcγRs) that trigger cell-mediated cytotoxic effector functions such as antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). These isotypes were selected to minimise the risk of depleting the T cells upon which such antibodies depend for their mechanism of action. However, recent preclinical data highlight a potential role for FcγR engagement in the activity of such antibodies. Here we review the biology of the FcγRs and IgG isotypes in both humans and mice, detail the potential roles that FcγR interactions can play in the activity of monoclonal antibodies in general, and of immunomodulatory antibodies in particular, and discuss how preclinical studies on these interactions might be best interpreted and translated to a human setting. Keywords: Fc gamma receptors, Anti-CTLA-4, Anti-GITR, Immunomodulatory antibodies Introduction TIM-3, LAG-3, B7-H4, B7-H3, VISTA, A2aR), as well as Antibodies of the IgG sub-class are bi-functional molecules, co-stimulatory (e.g. CD27, OX40, GITR, CD137), pathways possessing a F(ab) domain, variable in sequence and respon- are poised to join them. In contrast to agents such as rituxi- sible for the binding of antigen, and an Fc domain, constant mab, most of these immunomodulatory antibodies are of in sequence and responsible for mediating a range of anti- IgG isotypes that have low, or no, binding to the Fc gamma body effector functions [1]. These functions are primarily receptors (FcγRs), and effects such as ADCC have not been triggered through interaction with the complement compo- considered to be critical to their activity. Recent studies in nent C1q or with a family of FcγRs expressed, primarily, on mice have, however, highlighted a potential role for FcγR the surface of leukocytes. Each IgG isotype has a different binding, and FcγR mediated effector functions in the activity binding profile to the various FcγRs, and each FcγR has a of antibodies targeting a number of these pathways. different cellular expression pattern. These differences en- Below we review the findings of these recent studies and able the breadth, flexibility and control of function required discusshow they maychangeour understandingofthe role to mount an effective and regulated humoral response. FcγR interactions play in the activity of immunomodulatory Effector functions mediated by the FcγRs, such as ADCC antibodies in mice. We begin, however, by reviewing the and ADCP, are believed to play an important role in the ac- biology of the FcγRs and IgG isotypes in mice and humans, tivity of several therapeutic antibodies; including a number highlighting the key similarities and differences, which need used in Oncology, for example rituximab and trastuzumab. to be appreciated when thinking about how to translate Antibodies targeting T-cell inhibitory pathways, such as these findings to a human setting, in order to select the op- CTLA-4 and PD-1/PD-L1, are emerging as an important timal isotype for any potential therapeutic. class of cancer therapeutics, and a next generation of immu- nomodulatory therapies targeting alternative inhibitory (e.g. Review The Fc gamma receptor family * Correspondence: stewartr@medimmune.com Humans and mice possess two classes of FcγRs, the acti- MedImmune, AKB Building, Granta Park, CB21 6GH England, UK Full list of author information is available at the end of the article vating and inhibitory receptors. In both species FcγRI is © 2014 Stewart et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 2 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 an activating receptor with high affinity for IgG, and is immunogenicity risk respectively) and will not be con- expressed on monocytic DCs and on monocytes/macro- sidered further in this review. Of the remaining isotypes phages broadly in humans but in select locations in hIgG1 demonstrates the highest affinity for FcγRs, with a mouse (Table 1) [2,3]. FcγRI is the only FcγR with a nM or low μM binding affinity to all receptors (estimated functionally meaningful binding affinity for monomeric by surface plasmon resonance [SPR]), and is considered a IgG, the remaining FcγRs all exhibit such low affinity for potent inducer of all cell mediated effector functions, such the Fc region of IgG that functional engagement is only as ADCC and ADCP. hIgG4 demonstrates a nM affinity expected to occur upon binding of antibody complexed for FcγRI but μM affinities for all other receptors and is through antigen engagement (Table 2). Both species pos- considered a poor inducer of such effector functions. sess a single inhibitory FcγR, FcγRIIb, which is expressed hIgG2 has no measurable affinity for FcγRI, a high nM on B cells, DCs and basophils in both species, and found affinity for the H131 form FcγRIIa and μM affinities for additionally on monocytes, macrophages and all granulo- all other FcγRs (Table 2). cyte populations in mice (Table 1). The remaining FcγRs These absolute affinities are a useful guide as to the are all activating, but more divergent between species. binding of the hIgG isotypes to hFcγRs relative to each In mice there are only 2 additional activating receptors other. However, as noted above, the functional inter- FcγRIII, which is expressed broadly on NK cells, mono- action between IgG and most FcγRs is not monomeric cytes, macrophages, granulocytes and DCs, and FcγRIV, in nature, as is modelled by SPR. In a study [5] examin- which is found only on monocytes, macrophages and neu- ing the more functionally relevant binding of complexed trophils. In humans there are four additional activating IgG to FcγRs on cells, the relative ranking of the isotypes FcγRs, the best characterised being FcγRIIa and FcγRIIIa. with respect to FcγR binding was retained, however no FcγRIIa is expressed on granulocytes, monocytes and binding of IgG2 to cells expressing the inhibitory FcγRIIb monocyte-derived cells such as macrophages and DCs. or the F158 form of the activating FcγRIIIa could be de- FcγRIIIa is expressed primarily on NK cells, but is also tected using this methodology, despite the interaction be- found on monocytes and macrophages under some cir- tween IgG2 and these proteins demonstrated by SPR. cumstances (Table 1). Each of these receptors exists as Mice also have four IgG isotypes but they are not two allotypic variants with differing binding affinity for equivalent to those of human in either nomenclature or IgG (Table 2). For FcγRIIIa the more common F158 allo- properties. Mouse (m)IgG1, in contrast to hIgG1, is con- type has a lower affinity than the V158 allotype. For sidered a low effector function isotype, since while it FcγRIIa the more common H131 allotype has a higher binds inhibitory FcγRIIb and activating FcγRIII with nM affinity than the R131 allotype [4]. The other two acti- affinity, it has no measurable binding to activating FcγRIV. vating receptors are far less well characterised. FcγRIIc In mice it is the two IgG2 isotypes, mIgG2a and mIgG2b, is expressed in only 20% of people and is closely related to, that have the highest binding affinities to FcγRs, particu- but expressed more restrictedly than, FcγRIIa. FcγRIIIb is larly to FcγRIV (Table 2). In mice there is no IgG4 isotype, a GPI linked receptor expressed mainly on the sur- and mIgG3 has no measurable FcγR interactions [6]. face of neutrophils. These differences in FcγR and antibody isotype inter- Mice and humans also have a divergent family of IgG actions and expression, summarised in Tables 1 and 2, isotypes. In humans there are 4 isotypes, IgG1-4, each of mean that there are no direct homologs between human which has differing binding affinities to the various and mouse with respect to FcγR and antibody isotype FcγRs. Human (h)IgG3 demonstrates relatively high af- biology. However, it is possible to draw some parallels finity binding to most FcγRs, but is not used routinely between the species, for example mIgG2a and hIgG1 are as a therapeutic format (due to its long hinge region often considered equivalent functionally due to their and polymorphic nature which represent a stability and broad and high FcγR binding, while the generally lower Table 1 Expression pattern of the major FcγRs in human and mouse Cell type FcγRI FcγRIIb hFcγRIIa mFcγRIII hFcγRIIIa mFcγRIV hm h m B cells + + DCs + + + + + + NK cells ++ Monocytes/Macrophages + ~ ~ + + + + + Neutrophils (+) ~ + + + + Italics indicates an inhibitory receptor. Normal text indicates an activating receptor. Human receptors are denoted by h and mouse receptors by m. + indicates expression, (+) indicates inducible expression and ~ indicates expression on only a sub-set or on cells at specific physiological locations [2,3]. Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 3 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 Table 2 Relative affinity of binding between the major Nevertheless a significant effort has been made to engineer FcγRs and IgG Isotypes in human and mouse antibodies with improved affinity for the activating FcγRs Human Mouse [10-12], with the aim of enhancing these cytotoxic func- tions and improving therapeutic efficacy, and the outcome FcγR IgG1 IgG2 IgG4 FcγR IgG1 IgG2a IgG2b of clinical trials with such agents may provide further clar- I ++++ - ++++ I - ++++ ++++ ity as to the true contribution of FcγR mediated cytotox- IIa H +++ ++ ++ III ++ ++ ++ icity in activity. R +++ + ++ An alternative role for FcγRs is in the activity of anti- IIb ++ - ++ IIB +++ ++ +++ bodies that function by agonising cell surface receptors, IIIa V +++ + ++ IV - +++ ++ for example those of the tumor necrosis factor receptor F ++ - ++ (TNFR) super family. In this context binding of IgG Fc to FcγRs results in higher order clustering of antibody. Italics indicates an inhibitory receptor. Normal text indicates an activating 5 −1 receptor. Rankings are based on K x10 M for each interaction reported in This in turn results in higher order clustering of the target 5 −1 published studies [1,5,6]. ++++ indicates a K greater than 100 x 10 M , +++ 5 −1 receptor when bound by the F(ab) domain, which triggers indicates a K between 20 and 99 x 10 M , ++ indicates a K between 1 and A A 5 −1 5 −1 19 × 10 M , + indicates a K between 0.5 and 0.9 × 10 M , − indicates a K A A downstream signalling (Figure 1, right). This clustering 5 −1 that was undetectable or less than 0.5 × 10 M . effect has been shown to be critical to the activity of antibodies specific for the TNFR family members CD40 and more select binding profiles of mIgG1 and hIgG4 [13,14] death receptor 5 (DR5) [15,16] and CD95 (Fas) [17] make them close functional analogs. Additionally the ac- in preclinical mouse models, and has been suggested to tivating mFcγRIII and hFcγRIIa are often considered play a similar role in the activity of other TNFR agonists analogous based on affinities and expression pattern, as such as anti-OX40 and anti-CD27 [18]. are activating mFcγRIV and hFcγRIIIa. These parallels are frequently imperfect though, for example: hFcγRIIIa The canonical role of FcγRs in immunomodulatory is expressed at high levels on NK cells, while mFcγRIV antibodies is not; hFcγRIIa is not expressed on NK cells while Immunomodulatory antibodies mediate their beneficial mFcγRIII is; mIgG1 is a more functionally restricted iso- effects by promoting anti-tumor immunity. There are a type than hIgG4, based on their relative affinities for ac- number of potential ways to achieve this, but one area tivating FcγRI, mFcγRIV and hFcγRIIa. These differences showing particular promise is the use of antibodies that need to be kept in mind, and pose challenges, when try- antagonise inhibitory receptors on the surface of T cells. ing to translate findings in mouse preclinical models to One such antibody, the anti-CTLA-4 ipilimumab, was humans. approved based on its ability to improve survival in metastatic melanoma patients [19], while a number of The potential roles of FcγRs in the activity of therapeutic antibodies targeting PD-1 are showing highly encour- antibodies aging results in phase 1 clinical trials [20,21]. An alterna- The FcγR mediated functions most commonly associated tive approach, which is earlier in clinical development with therapeutic antibodies are those that mediate target but also showing promise, is the use of antibodies that cell elimination; ADCC, or the related ADCP. These agonise co-stimulatory receptors. The agonistic anti-CD40 functions are triggered when antibody binding to antigen antibody, CP870,893, has demonstrated early signs of activ- on the surface of a target cell generates sufficient avidity ity in pancreatic cancer [22], and tumor shrinkage was also to trigger signalling through FcγRs on effector cells such observed in some patients treated with agonistic antibodies as NK cells and macrophages, which then eliminate tar- directed against OX40 [23] and CD137 [24]. get cells through direct killing or phagocytosis (Figure 1, As mentioned above FcγR engagement has been left). Preclinical models show that these forms of FcγR- shown to be critical for the activity of antibodies that ag- mediated cytotoxicity are a significant component of the onise the TNFR CD40 in mice. The anti-tumor activity of mechanism of action for tumor targeted antibodies such anti-mouse CD40 in these studies is lost upon introduc- as the anti-CD20 antibody rituximab and the anti-HER2 tion of the D265A mutation into its Fc domain [13], antibody trastuzumab [7]. The clinical evidence for the which results in an antibody with no measurable FcγR role of such interactions, however, is more mixed. A binding [6]. It has also been shown that the in vitro activ- number of relatively small studies have shown improved ity of antibodies directed against other TNFRs in human outcomes in patients that are homozygous for the higher cells, such as OX40, GITR, CD137 and CD27, requires, affinity allotypic forms of human FcγRIIIa and FcγRIIa or is greatly enhanced by FcγR engagement or immobil- [8,9], supporting a role for FcγR-mediated cytotoxicity isation [25,26]. While the evidence suggests that a re- in this setting, but several larger, randomized studies quirement for cross linking is a common property of have generally failed to show the same associations [4]. antibodies targeting TNFRs receptors, there are some Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 4 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 Figure 1 The two primary forms of interaction between therapeutic IgGs and FcγRs. On the left: when IgG is bound to high levels of antigen on target cells, such as tumor cells, the resulting clustered Fc domains engage with FcγRs on immune effector cells such as NK cells and macrophages. This drives signals through those FcγRs triggering the initiation of cell-mediated effector functions such as ADCC and ADCP. On the right: when IgG is bound by its Fc domain to FcγRs it can act to cluster low levels of target receptors, such as members of the TNFR family. This clustering acts to trigger signals though the TNFR into target cells, such as T cells. exceptions. For example the hCD27.15 anti-human CD27 cells (APCs) critical to mounting an adaptive immune mAb has demonstrable agonistic properties in the soluble response [13]. In the case of the second hypothesis, the phase [27], the anti-CD40 antibody CP870,893 has been balance of FcγR engagement would become critical to shown to function in vitro in solution and as a F(ab)’ the activity of anti-CD40. Too little engagement, as fragment that lacks any Fc domain [28] and the anti- seen with alglycosyl antibodies, and no agonistic signal- OX40 antibody 9B12, which is a mouse IgG1 with very ling is triggered so no anti-tumor activity is observed. low affinity interactions with FcγRs, has shown early Too much FcγR engagement on effector cells and deple- signs of clinical activity and demonstrated the ability to tion of critical immune cells begins to occur and anti- activate T cells in patients [23]. tumor activity is once again lost. Such a trade off in FcγR Two studies that further explored the role of FcγR en- affinity could also apply to other immunomodulatory anti- gagement for the activity of anti-mouse CD40 demon- bodies that target TNFRs (CD27, OX40, GITR, CD137), strated that it is the inhibitory mouse FcγRIIb that plays which are also expressed on critical immune cells. In an essential role [13,14]. One of these studies [14] went addition it has been shown that for CD137 antibody medi- on to demonstrate that other mouse FcγRs are capable ated internalisation of receptor can play a role in driving of cross linking anti-CD40 to drive activity in vitro, and signalling [29], and it is unclear how changes in Fc isotype proposed that the dominant role of FcγRIIb in vivo was might affect this property. due primarily to its bioavailability at the site of action For those antibodies that target inhibitory receptors for anti-CD40, namely the secondary lymphoid organs. like PD-1 and CTLA-4 there has been little reason to Both studies also went on to show that the activity of believe, based on their antagonistic mechanism of action, anti-mouse CD40 was lost upon class switching from a that FcγR engagement plays an important role in their mouse (m)IgG1 to a mIgG2a. Two alternative, and non- activity, and no significant evidence to contradict this mutually exclusive, hypotheses exist to explain this obser- belief exists in the clinical setting. Since all of the anti- vation. The first is that the reduced affinity of mIgG2a for gens targeted by these antibodies are expressed either on FcγRIIb results in reduced cross linking at the relevant site T cells or on APCs, the preference has been to select iso- of action and so limits activity [14]. The second is that the types that will avoid the risk of depleting these cells loss of activity occurs due to increased depletion of CD40 through Fc mediated mechanisms; an outcome that would positive cells, which are predominantly antigen presenting likely negate any beneficial immune activation. As a result, Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 5 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 acommonfeatureofthe immunomodulatory antibodies engagement of the activating FcγRs in mouse tumor currently in clinical development is that they are low or models. Further examination of the mechanism for this null effector function isotypes (Table 3). Main exceptions dependence revealed that in the lymph nodes anti-mouse include ipilimumab (anti-CTLA-4), MSB0010718C (anti- CTLA-4 acted to increase the number of CD4 T cells, in- PD-L1) and antibodies like MGA271 (anti-B7-H3), CDX- cluding regulatory T cells (T-regs), but that within the 1127 (anti-CD27) and alemtuzumab (anti-CD52) which tumor it acted to selectively deplete only T-regs [33]. In employ ADCC of tumor cells as a part of their mechanism one of the three studies, a comparison of several different of action. anti-mouse CTLA-4 antibodies showed that the extent of However, several recent preclinical studies are calling T-reg depletion observed correlated with activity [33]. into question the view that all TNFR agonist antibodies The depletion of T-regs was mediated predominantly will behave as anti-CD40 does, or that all antibodies tar- by mouse FcγRIV, but was independent of NK cells, and geting inhibitory receptors will be maximally effective as considered likely to rely on the activity of intratumoral a low or null effector function isotype. CD11b + macrophages [33]. In all three studies the level of CTLA-4 expression was shown to be increased on intratu- The evolving role of FcγRs in immunomodulatory moral T cells relative to those in lymph nodes, and was antibodies higher on T-regs than on other T cell sub-sets. Three recently published studies [31-33] have examined In their study Bulliard et al. [31] also examined the the role of mouse FcγRs in the activity of anti-mouse role of mouse FcγRs in the activity of the anti-mouse CTLA-4 antibodies. In these studies anti-mouse CTLA-4 GITR antibody DTA-1. In contrast to the observations antibodies were studied in a range of syngeneic tumor made for anti-mouse CD40, DTA-1 activity was minim- models where they have previously shown activity, includ- ally affected by the absence of inhibitory FcγRIIb, but ing MC38 (colon), CT26 (colon) and B16 (melanoma). The was dependent on the presence of the activating mouse approach taken to examine the role of FcγRs varied be- FcγRs [31]. In further contrast to anti-CD40, a mouse tween studies and either involved the use of FcγRknock (m)IgG2a version of DTA-1 was equal in activity to the out mice [31,33] or reformatting of the anti-mouse CTLA- parental rat IgG2b version; although no mIgG1 was 4 to a variety of different antibody isotypes [32]. Irrelevant tested. Analogous to the mechanism observed for anti- of the approach taken the result was the same; the activity mouse CTLA-4, it was shown that while mIgG2a or rat of anti-mouse CTLA-4 was shown to be dependent upon IgG2b forms of DTA-1 treatment resulted in expansion of Table 3 List of immunomodulatory antibodies currently in clinical development and their respective isotypes Antibody Target Company Latest active clinical phase Isotype Ipilimumab CTLA-4 BMS Approved IgG1 Tremelimumab CTLA-4 MedImmune/AstraZeneca II IgG2 Nivolumab PD-1 BMS Approved IgG4 MK-3475 PD-1 Merck III IgG4 CT-011 PD-1 CureTech II IgG1 MPDL3280A PD-L1 Genentech/Roche III Aglycosyl IgG1 MEDI4736 PD-L1 MedImmune/AstraZeneca I IgG1 TM MDX-1105 PD-L1 BMS I IgG4 MSB0010718C PD-L1 Merck KGaA I IgG1 BMS-663513 CD137 BMS I IgG4 PF-05082566 CD137 Pfizer I IgG2 TRX518 GITR GITR Inc I Aglycosyl IgG1 MEDI6469 OX40 MedImmune/AstraZeneca/AgonOX I Mouse IgG1 CP-870,893 CD40 Roche I IgG2 CDX-1127 CD27 CellDex I IgG1 Lirilumab; IPH2102 KIR BMS/Innate Pharma II IgG4 MGA271 B7-H3 Macrogenics/Servier I IgG1 Alemtuzumab CD52 Genzyme/Bayer Approved IgG1 IgG1 TM = IgG1 triple mutant, which contains 3 point mutations in the Fc domain that reduce the binding affinity of the mAb to FcγRs [30]. Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 6 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 all T cell populations in the lymph node, they acted to de- effector function is entirely dependent upon the pres- plete T cells within the tumor. Also similar to anti-mouse ence of FcγR-bearing effector cells at an appropriate CTLA-4, this depletion preferentially occurred in the T-reg density. The studies by both Simpson et al. and Bulliard compartment and, although some reduction in both con- et al. highlight what many of us will have observed in ventional CD4+ and CD8+ T cells was also observed, re- our own work, that there are very few effector cells, such sulted in an increase in the CD8:T-reg ratio within the as NK cells, neutrophils and macrophages, present in tumor. In a more recent study published earlier this year lymph nodes, which are dominated by T, B and dendritic Builliard et al. [34] demonstrated a comparable mechan- cells. What both studies additionally show is that mouse ism, and dependence on activating mouse FcγRs for an tumors are extremely rich in FcγR-expressing effector anti-mouse OX40 antibody. It is however worth noting cells. Simpson et al. evidence greater than 20% NK cells that alternative studies have proposed, and evidenced, and greater than 40% CD11b + cells, which are predom- a mechanism of regulatory T cell re-programming for inantly macrophages, in B16 tumors and Bulliard et al. DTA-1 [35] and for anti-human OX40 mAbs [36], sug- highlight very similar levels of the same cells in CT26 gesting that a loss of FOXP3 expressing T cells following tumors. The coincident presence of high levels of target treatment with agents targeting either pathway may not antigen and a high density of effector cells creates a per- necessarily equate to depletion. fect storm for Fc mediated effector function within the These preclinical studies have brought into question tumor, while the lower target expression and relative two generally held beliefs regarding the role of FcγRs in paucity of effector cells in the periphery makes the same the activity of immunomodulatory antibodies. The first effector function harder to achieve in the lymph node. It is the idea that inhibitory FcγRIIb is the critical receptor is however worth noting that peripheral depletion of im- for the activity of all TNFR agonist antibodies. The data mune cells is possible, as demonstrated by the data on generated for the anti-mouse GITR antibody DTA-1 anti-CD40 [13] and the well documented effects of anti- clearly demonstrate FcγRIIb independent activity, and CD20 [39], suggesting that the nature or expression level our own unpublished data demonstrates similar inde- of the target are likely critical. pendence for other TNFR targeting antibodies. These data suggest, as previously proposed by White et al., that Translating preclinical data to man the specific mouse FcγRs involved in cross linking of As outlined at the start of this review, the significant dif- TNFR agonists may vary depending on the pathway be- ferences between human and mouse FcγR and IgG iso- ing targeted and on the bioavailability of each of the type biology make translation of findings between species FcγRs at the primary site of action. The second is the extremely challenging, and several factors need to be con- idea that effector function enabled antibodies represent sidered and better studied in the human setting before a risk to activity due to depletion of key immune cell these findings can be confidently applied to the activity of sub-sets. Importantly the data highlight the fact that per- therapeutic antibodies in man. ipheral and intratumoral antibody mediated effector Taking anti-CTLA-4 as an example, the first signifi- function are far from equivalent. This difference appears cant translational challenge is the key role for mouse to hinge on two critical differences; the level of target FcγRIV in the T-reg depleting activity demonstrated for antigen expression and the presence of appropriate ef- anti-mouse CTLA-4. FcγRIV does not exist in humans, fector cells (Figure 2). and its closest analog, human (h)FcγRIIIa, is expressed The extent of effector function triggered by antibody predominantly on NK cells, which played no role in the binding has been shown in a number of studies to be mainly macrophage mediated T-reg depletion observed strongly influenced by the level of target antigen expres- with anti-mouse CTLA-4 [33]. In humans macrophages sion [37,38], with antibodies directed against both hu- can express hFcγRIIIa following certain stimuli, such as man CCR4 and CD20 failing to elicit in vitro ADCC IL-10, but primarily express hFcγRIIa/b and hFcγRI, and against human tumor cell lines expressing anything less it is feasible that in human tumors these receptors would than 10 antigen molecules per cell and reaching optimal play the same role as mouse FcγRIV does in mouse tu- 5 6 activity only in the presence of 10 -10 antigen mole- mors. Further study of the intratumoral effector cell and cules [37]. Many of the targets for immunomodulatory FcγR constituent within human tumors would be ex- antibodies are expressed at low to undetectable levels on tremely helpful in understanding which human FcγR resting, peripheral immune cells, but are up-regulated might play the role of mouse FcγRIV in humans. significantly in the presence of pro-inflammatory stimuli, The second challenge for translation lies in the differ- such as those present in the tumor microenvironment. ences between IgG isotypes across species. In mice only As such, and in the light of recent studies, it is clear that mouse (m)IgG2a and mIgG2b demonstrate any measur- in mouse models it is significantly easier to elicit effector able binding to mouse FcγRIV; mIgG1 can therefore function against intratumoral immune cells. Additionally show none of the T-reg depleting activity observed for Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 7 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 Figure 2 Critical differences between effector function in the periphery relative to the tumor. On the left: in lymph nodes, relatively low levels of cell activation and pro-inflammatory cytokines result in low levels of expression for most targets of immunomodulatory antibodies, which are generally low on resting immune cells. This low level of target expression, together with a paucity of effector cells in lymph nodes means depletion of cells through cell mediated effector functions do not readily occur. On the right: high levels of pro-inflammatory cytokines within the tumor microenvironment trigger up-regulation of many targets. This increased target expression together with a high density of effector cells, such as macrophages, enables depletion of cells through cell-mediated effector functions. The level of target expression, and the sensitivity to depletion varies across cell types and favours depletion of T-regs for targets such as CTLA-4 and GITR. anti-mouse CTLA-4 in mice. In contrast, while human(h) 3 year survival rate seen in melanoma patients treated IgG1 has a significantly higher binding affinity to all human with the two agents. FcγRs, hIgG2 and hIgG4 are both capable of interact- The third translational challenge relates to the key ing with human FcγRIIa andIIIa, andwhile human driver for differential depletion of T-regs relative to FcγRI cannot bind hIgG2, its high affinity for monomeric other cell types in all three of the published studies, that IgG suggests that it would be heavily occupied by serum is target expression level. It is clear from the published IgG and less available to partake in effector function [40]. studies that the levels of GITR and CTLA-4 on mouse Critically, while hIgG2 and hIgG4 are both very poor T-regs is higher than that observed on mouse effector mediators of NK cell driven ADCC, they are both cap- T cells, in particular CD8s. However there are no studies able of mediating ADCC via PBMCs, monocytes and available comparing how the level of target expression neutrophils [41,42]. It is especially conceivable that in a varies between mice and humans, few that compare the tumor microenvironment, with its increased target anti- differential expression of target on human T-regs relative gen levels and high density of effector cells, that a hIgG2 to effectors, and none that examine how this expression or hIgG4 could begin to mediate some degree of ADCC changes quantitatively in the tumor versus the periphery. or ADCP. If one were to assume that a mouse IgG2a These are all significant variables that must be studied in were equivalent to human IgG1 and mouse IgG1 equiva- humans before a direct translational conclusion can be lent to human IgG2 then the data of Selby et al. would drawn. predict significant differences in the two anti-CTLA-4 antibodies that have been tested clinically one of which, Conclusions tremelimumb, is a human IgG2 and the other of which, Future directions ipilimumab, is a human IgG1 currently registered for the Immunomodulatory antibodies are showing positive re- treatment of metastatic melanoma. The complex differ- sults in early clinical trials in cancer. These antibodies ences in FcγR and IgG isotype biology that exist between generally fall into two classes. Those that antagonise in- species, outlined above, suggest however that such as- hibitory receptors, such as CTLA-4 and PD-1, and those sumptions about the comparability of mouse and human that agonise stimulatory receptors, such as CD27, OX40, IgG isotypes may be too reductionist. In relation to GITR and CD137. this, while no definitive comparison of the two agents Many groups, including our own, have been working has been conducted, the toxicity profiles of ipilimumab to study the requirement for FcγRs in the activity of TNFR and tremelimumab are comparable [19,43] and a recent agonist antibodies, but the recent study of Bulliard et al. commentary in the Journal of Clinical Oncology [44] [31] has further highlighted the potential complexity of this highlighted the very limited differences observed in the requirement by providing evidence that inhibitory mouse Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29 Page 8 of 10 http://www.immunotherapyofcancer.org/content/2/1/29 FcγRIIb is not required for the activity of all TNFR agonist Competing interests Ross Stewart and Robert Wilkinson are salaried employees of MedImmune Ltd. antibodies in mice, as has been previously suggested. Fur- Scott Hammond and Mike Oberst are salaried employees of MedImmune LLC. ther work is required to pull apart the target specific nature All four authors hold shares in the AstraZeneca group. of FcγR requirements for mAb activity and the relative contributions of pathway agonism versus targeted deple- Authors’ contributions tion of cells. In the case of TNFR agonists pulling these RS, SH and MO conceived of, planned and drafted the initial content of the article. RS provided all figures. RW provided substantive review. All authors apart is made very difficult by the inability to divorce FcγR read and approved the final manuscript. cross linking, and therefore agonism, from effector func- tion for the majority of such antibodies. Authors’ information The study by Bulliard et al. [31], together with that of RS, SH, MO and RW are part of the Oncology research group within Selby et al. and Simpson et al. [32,33], also highlighted MedImmune where they are involved in the discovery and development of a range of immunomodulatory antibody approaches. the previously underappreciated difference between FcγR- mediated effector function in the periphery as compared to Acknowledgements the tumor microenvironment in mice and highlighted the The authors thank Norman Greenberg for careful review of the final unexpected potential role of FcγRs in the activity of immu- manuscript. nomodulatory antibodies that target the inhibitory receptor Author details CTLA-4 in mice. Based on these studies it will be interest- MedImmune, AKB Building, Granta Park, CB21 6GH England, UK. ing to see how FcγR interactions may impact antibodies MedImmune, One Medimmune Way, Gaithersburg, MD 20878, USA. targeting PD-1 or PD-L1, which have very different expres- Received: 14 February 2014 Accepted: 16 July 2014 sion patterns to CTLA-4, despite their shared inhibitory function. The most important future studies however will be References those that address the translatability of these findings in 1. Nimmerjahn F, Ravetch JV: Fcgamma receptors as regulators of immune mouse preclinical models to man. While their exact responses. Nat Rev Immunol 2008, 8:34–47. 2. Bruhns P: Properties of mouse and human IgG receptors and their value remains unconfirmed [4], studies exploring the im- contribution to disease models. Blood 2012, 119:5640–5649. pact of human FcγR polymorphisms on clinical activity 3. 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In Book Role of the immunoglobulin constant region in the antitumor activity of antibodies to cytotoxic T-lymphocyte antigen-4 (CTLA-4), Journal of Clinical Oncology, Volume 15_suppl. 31st edition. 2013:9055. doi:10.1186/s40425-014-0029-x Cite this article as: Stewart et al.: The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer. Journal for ImmunoTherapy of Cancer 2014 2:29. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Journal for ImmunoTherapy of Cancer – Springer Journals

Published: Aug 19, 2014

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