Immunotherapy Targeting Inhibitory Fcγ Receptor IIB (CD32b) in the Mouse Is Limited by Monoclonal Consumption and Receptor Internalization This information is current as of September 29, 2021. Emily L. Williams, Alison L. Tutt, Stephen A. Beers, Ruth R. French, Claude H. T. Chan, Kerry L. Cox, Ali Roghanian, Christine A. Penfold, Cherié L. Butts, Peter Boross, J. Sjef Verbeek, Mark S. Cragg and Martin J. Glennie

J Immunol 2013; 191:4130-4140; Prepublished online 11 Downloaded from September 2013; doi: 10.4049/jimmunol.1301430 http://www.jimmunol.org/content/191/8/4130 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Immunotherapy Targeting Inhibitory Fcg Receptor IIB (CD32b) in the Mouse Is Limited by Consumption and Receptor Internalization

Emily L. Williams,* Alison L. Tutt,* Stephen A. Beers,* Ruth R. French,* Claude H. T. Chan,* Kerry L. Cox,* Ali Roghanian,* Christine A. Penfold,* Cherie´ L. Butts,† Peter Boross,‡ J. Sjef Verbeek,‡ Mark S. Cragg,*,1 and Martin J. Glennie*,1

Genetic deficiency of the inhibitory Fc receptor, FcgRIIB (CD32b), has been shown to augment the activity of activatory FcgR and promote mAb immunotherapy. To investigate whether mAbs capable of blocking FcgRIIB have similar capacity, we recently Downloaded from generated a panel of specific anti-mouse FcgRIIB mAbs that do not cross-react with other FcRs, allowing us to study the potential of FcgRIIB as a therapeutic target. Previous work revealed a number of these mAbs capable of eliciting programmed cell death of targets, and in the present study we demonstrated their ability to promote target cell . However, in a variety of murine tumor models, anti-FcgRIIB mAbs demonstrated limited therapeutic activity despite optimized treatment regimens. Unexpectedly, we observed that the anti-FcgRIIB mAbs are rapidly and extensively consumed in vivo, both by the tumor and host cells, including B cells, leading to a precipitous loss from the circulation. Closer analysis revealed that the anti-FcgRIIB mAbs http://www.jimmunol.org/ become extensively internalized from the cell surface within 24 h in vivo, likely explaining their suboptimal efficacy. Subsequent studies revealed that anti-FcgRIIB mAb immunotherapy was effective when used against FcgRIIB+ tumors in FcgRIIB2/2 recipients, indicating that consumption of the mAb by nontumor cells is the primary limitation of these reagents. Importantly, similar rates of internalization were not seen on human target cells, at least in vitro. These studies further highlight the need to determine the propensity of mAb therapeutics to internalize target receptors and also identify potential key differences between human and mouse cells in this respect. The Journal of Immunology, 2013, 191: 4130–4140.

herapeutic Abs such as rituximab, trastuzumab, and bev- the activatory FcgR (FcgR I, III, and IV in the mouse) occurs via acizumab are now established treatments for various an ITAM-containing cytoplasmic tail or association with a com- by guest on September 29, 2021 T cancers (1, 2). A major component of their therapeutic mon ITAM-containing g-chain. Conversely, inhibitory signaling is efficacy is thought to derive from their interaction with FcgRs (3). afforded by the ITIM-containing tail of FcgRIIB/CD32b. With the FcgRs are expressed on a wide range of immune cells such as exception of FcgRI (and IgG2a binding to FcgRIV), the FcgRs 6 –1 macrophages, neutrophils, and NK cells, as well as B cells and have relatively low affinity for monomeric IgG (Kd of ∼10 M ) dendritic cells (DCs) (4, 5). Both human and mouse FcgRs fall and so are unlikely to engage IgG in free solution. into two subgroups; activatory and inhibitory. Signaling through FcgR function varies with the cell type on which they are expressed. Triggering activatory FcgRs on effector cells, such as or macrophages, results in the release of a spectrum of *Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton, inflammatory immune modulators, such as cytokines. Conversely, Faculty of Medicine, General Hospital, Southampton SO16 6YD, United Kingdom; †Immunology Research, Biogen Idec, Cambridge, MA 02142; and ‡Department of triggering of FcgRIIB on B cells by IgG-containing immune Human Genetics, Leiden University Medical Center, 2333 ZA Leiden, The Nether- complexes (ICs) serves to inhibit signaling through the BCR, re- lands ducing proliferation, Ab production, and potentially delivering 1 M.S.C. and M.J.G. are cosenior authors and contributed equally to this study. apoptotic signals to the cells (reviewed in Ref. 4). More recently it Received for publication May 30, 2013. Accepted for publication August 7, 2013. has been shown that FcgRIIB is also expressed on plasma cells This work was supported by Leukaemia and Lymphoma Research Grants 07010, (and myeloma cells). In such cases, engagement can also induce 08014, and 09009. with potentially important implications for controlling E.L.W. performed experiments, analyzed results, produced figures, and wrote the long-lived plasma cells and shaping the Ab repertoire (6). The paper with M.S.C.; R.R.F. performed experiments, produced figures, and edited the manuscript; A.L.T., S.A.B., K.L.C., C.H.T.C., C.A.P., C.L.B., P.B., and J.S.V. pro- coexpression of both inhibitory and activatory FcgR by cells pro- duced and provided critical reagents; S.A.B. and A.R. performed experiments; M.J.G. vides a mechanism of fine control for cellular activation, where designed the research, analyzed results, and edited the manuscript; and M.S.C. de- signed the research, analyzed results, and wrote the manuscript with E.L.W. engagement of the FcgRIIB counterbalances activatory FcgR ef- fector function. Address correspondence and reprint requests to Prof. Mark Cragg, Antibody and Vaccine Group, Cancer Sciences Unit, Southampton University Faculty of Medicine, Within the field of Ab-based immunotherapy, growing evidence General Hospital, Southampton, SO16 6YD, U.K. E-mail address: [email protected] indicates that FcgRs on innate effector cells are critical for con- The online version of this article contains supplemental material. trolling therapeutic efficacy (7–10). In mouse models the thera- Abbreviations used in this article: ADCC, Ab-dependent cellular cytotoxicity; peutic activity of a range of anti-cancer mAbs correlates with their BMDM, bone marrow–derived macrophage; DC, dendritic cell; h, human; IC, im- ability to bind to activatory FcgR (8, 11–16), and deletion of the mune complex; PCD, programmed cell death; Tg, transgenic; WT, wild-type. FcgR g-chain abrogates therapeutic activity of most anti-cancer Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 mAbs (7). In contrast, in their seminal paper, Clynes et al. (7) www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301430 The Journal of Immunology 4131 demonstrated that deleting FcgRIIB enhances the therapeutic ac- human peripheral B cells were purified by negative selection using MACS tivity of a number of different therapeutic mAbs, including 4D5 isolation kits (Miltenyi Biotec). and trastuzumab (17), further alluding to the balance between Generation of bone marrow–derived macrophages FcgRIIB and activatory FcgR being critical for mAb-induced effector function. Murine bone marrow–derived macrophages (BMDMs) were generated Evidence for similar activity in humans has been more difficult from cells isolated from the bone marrow of the femur and tibia of mice. Cells were cultured in RPMI 1640 (Life Technologies Invitrogen, Paisley, to obtain and is largely derived from genetic studies, taking ad- U.K.) enriched with 10% FCS, 2 mM glutamine and 1 mM pyruvate, vantage of the fact that certain FcgRs have polymorphisms that penicillin and streptomycin (each at 100 mg/ml), and 20% L929 cell– affect their affinity for various IgG classes. Polymorphisms that conditioned medium (containing M-CSF). Cells were cultured at 37˚C, 5% increase the interaction between IgG and FcgRIIA or FcgRIIIA CO2 for 10–14 d prior to use. Macrophage differentiation was routinely confirmed by morphological examination and/or flow cytometry for CD11b have been shown to be associated with improved therapeutic efficacy expression. (18–21). Similarly, defucosylated IgG, which binds with a marked increased affinity to the activatory FcgRIIIA, shows ∼50-fold in- Abs and reagents crease in Ab-dependent cellular cytotoxicity (ADCC) activity, which mAbs were typically produced from the culture supernatant of hybridoma is expected to deliver significant improvement in clinical efficacy. In cells or stably transfected Chinese hamster ovary-K1 (AT128 m2a). IgG was both mice and humans, polymorphisms that result in reduced ex- purified on A with purity assessed by electrophoresis (Beckman EP pression or activity of the inhibitory FcgRIIB are associated with system; Beckman Coulter) and lack of aggregation was confirmed by increased susceptibility to autoimmune disease (22–28). HPLC. F(ab9)2 fragments were produced as described previously (40). The anti-mouse FcgRIIB mAbs were raised using conventional hybridoma For these reasons, FcgRIIB has long been seen as a potential technology and were recently reported (32). AT130-2 and AT128 m2a are Downloaded from target for immunotherapy, both as a means of augmenting effector of IgG2a isotype, whereas AT130-5 and AT128 are of IgG1 isotype. The cell activation and as a way of transducing death signals to target anti-human FcgRIIB mAbs AT10 (41), IV3 (42), and KB61 (43) were cells. Unfortunately, suitable mAb reagents capable of specifically previously described. Rituximab was gifted by Southampton General Hospital oncology pharmacy. Rit m2a was produced as described previ- and exclusively binding mouse FcgRIIB have largely been lack- ously (33). The anti-mouse CD20 mAb 18B12 m2a was made by com- ing, making specific in vivo manipulation of FcgRIIB impossible. bining VH and VL regions of the 18B12 IgG1 hybridoma with mouse g 2A

Although human FcgRIIB-specific mAbs have recently been pro- and k constant regions, respectively, and expressing the two polypeptide http://www.jimmunol.org/ duced (29–31), these have only been tested in xenograft systems chains in Chinese hamster ovary cells (44). Anti-idiotype mAbs MC106A5 to date, making their true therapeutic potential unclear. (BCL1) (45) and Mc39/16 (A31) (46) were generated in-house as previ- ously described. Following an extensive immunization protocol using FcgRIIB2/2 mice, we recently produced unique mAbs that specifically rec- Flow cytometry ognize FcgRIIB, block binding, and are ca- Fluorescently conjugated mAbs were purchased from BD Biosciences, AbD pable of regulating its activity. In particular, the agonistic mAbs Serotec, or made in-house. Flow cytometry was as described previously (46) (AT130-2 and AT130-5) were able to phosphorylate the receptor, with data analyzed with CellQuest Pro or FACSDiva (both BD Bio- inhibit BCR-induced Ca2+ flux, and trigger programmed cell sciences). To assay anti-FcgRIIB mAb (mouse IgG2a subclass) concen- + death (PCD) in B lymphoma cells (32). In contrast, the antag- trations, sera were incubated with pBCL1 lymphoma cells (FcgRIIB ) and by guest on September 29, 2021 cell-bound anti-FcgRIIB was detected using PE-labeled goat anti-mouse onistic mAbs (AT128 and AT128 m2a, along with the FcgRIIB/III Fc (Jackson ImmunoResearch Laboratories) with reference to a standard dual-specific mAb 2.4G2) evoked relatively little PCD and aug- curve obtained with known concentrations of anti-FcgRIIB mAbs. To mented Ca2+ responses. Having previously characterized these determine surface expression of FcgRIIB after mAb treatment in vivo or mAbs (32), in this study we addressed the potential efficacy of in vitro, available Ag was saturated with excess anti-FcgRIIB mAb (10 mg/ these reagents in vivo in fully syngeneic model systems. Using ml) and cells were washed and then surface anti-FcgRIIB detected using PE-conjugated goat anti-mouse Fc (Jackson ImmunoResearch Laborato- a variety of these models we have revealed a hitherto unexpected ries). Alternatively, to detect pre-existing surface accessible mAbs, cells limitation of these reagents that is associated with their propen- were washed and incubated with the PE-conjugated goat anti-mouse Fc sity to become modulated from the cell surface. alone before analysis. B cells or macrophages were then identified with allophycocyanin-labeled anti-mouse CD19 and CD11b. Human cells were monitored in the same manner using mouse mAb and anti-mouse sec- Materials and Methods ondary detection reagents. Animals and cells Measurement of anti-FcgRIIB mAb levels in serum Mice were bred and maintained in local facilities. Human (h) CD20 transgenic (Tg), g-chain2/2, and FcgRIIB2/2 mice have been described Anti-FcgRIIB mAb serum levels were monitored using a sandwich ELISA. previously (33, 34), as have the FcgRIIBfl/flCD19Cre+ mice (34–36). Briefly, 96-well Immuno MaxiSorb plates (Fisher Scientific, Leicester- Genotypes were confirmed by PCR and/or flow cytometry. Ten- to 12-wk- shire, U.K.) were coated with mouse anti-rat CD4 (OX68) F(ab9)2 frag- old female BALB/c and CBA/H mice were supplied by Harlan UK ments (5 mg/ml) in ELISA coating buffer (15 mM Na2CO3, 28.5 mM NaHCO at pH 9.6) and incubated for 1 h at 37˚C, then at 4˚C overnight, (Blackthorn, Oxon, U.K.) and maintained in local animal facilities. BCL1 3 (37) and A31 (38) mouse B lymphoma models and the 5T-33 myeloma followed by addition of FcgRIIB-CD4 fusion protein diluted 1:2 with 1% (gift from Dr. N. Savelyeva) were maintained by in vivo passage in BALB/C, BSA/PBS and incubated for 1 h. After 1 h the ELISA plate was washed CBA, and C57BL/6 KA mice, respectively. Animal experiments were with PBS/Tween 20 and the diluted sample was added to the wells for 90 cleared through the local ethical committee and performed under Home min. The ELISA plate was then washed and goat anti-mouse FcgR-HRP (Sigma-Aldrich, Dorset, U.K.) was added for a further 90 min before Office licenses PPL30/2450 and 30/2451. pBCL1 is a variant of BCL1 further washing and detection with o-phenylenediamine. The reaction was that arose from the wild-type BCL1 and is capable of growth both in culture and also in vivo (39). Cell culture was performed in supplemented halted with 2.5 M H2SO4 and absorbance detected at 495 nm on the RPMI 1640 (RPMI 1640 containing 2-ME [50 mM], glutamine [2 mM], Dynatech MR400 plate reader, with the serum concentration of mAb pyruvate [1 mM], penicillin and streptomycin [100 IU/ml], fungizone calculated against a standard curve of known concentrations of anti- [2 mg/ml], and 20% FCS [Myoclone]) (Life Technologies BRL, Paisley, FcgRIIB mAb. U.K.). Mouse splenic B cells were purified by negative selection using MACS B cell isolation kits (Miltenyi Biotec) and cultured in supple- In vivo internalization assay mented RPMI 1640. Human cell lines (Daudi, Raji) were obtained from 7 Fresh BCL1 tumor cells (2 3 10 ) were injected into female 8- to 12-wk- the European Cell Culture Collection (Porton Down, U.K.) and were old wild-type (WT) or g-chain2/2 BALB/c mice i.v. on day 0. Mice re- maintained in antibiotic-free RPMI 1640 containing glutamine (2 mM), ceived 0.5 mg mAb (anti-FcgRIIB or anti-idiotype) 4 d after tumor in- pyruvate (1 mM), and 10% FCS (Myoclone) (Life Technologies). Normal oculation. Forty-eight hours later mice were sacrificed and spleens were 4132 MODULATION AND CONSUMPTION LIMIT ANTI-FcgRIIB mAb THERAPY removed and assessed by flow cytometry for surface expression of the serum mAb levels. Statistical analysis was performed using GraphPad Prism receptor along with surface-accessible mAbs as described above. software (version 5 for Windows).

In vitro internalization assay and Alexa 488 labeling Results 2 2 mAbs were labeled with Alexa 488 (Invitrogen) according to the manu- Augmented immunotherapy in FcgRIIB / mice facturer’s instructions. To determine internalization, a quenching assay was 2/2 performed as detailed previously (47). In brief, cell samples (2–4 3 105 In xenograft models, Clynes et al. (7) showed that FcgRIIB mice cells/well) were incubated with Alexa 488–labeled mAbs (5 mg/ml) for 2, 6, demonstrate an enhanced capacity to reject tumor cells following or 24 h at 37˚C, washed, resuspended, and incubated at 4˚C for 30 min in mAb immunotherapy. To confirm these finding using a syngeneic the presence or absence of anti-Alexa 488 quenching Ab (Invitrogen). mouse tumor model, we compared anti-idiotype immunotherapy of Samples were then assessed by flow cytometry as described above. Only 2/2 the BCL1 tumor in WT and FcgRIIB mice. In both WT and mAb that is remaining on the cell surface is accessible for quenching by 2/2 anti-Alexa 488. Therefore, the results are represented as percentage surface- FcgRIIB mice, anti-idiotype mAb improved survival in com- accessible anti-FcgRIIB, which is inversely proportional to the amount of parison with the nontreated mice (p , 0.01 for both). Nevertheless, mAb internalized. in the FcgRIIB2/2 mice there was a significant increase in survival compared with the same treatment in WT mice (p , 0.05) (Fig. 1). RT-PCR for FcgRIIB isoforms FcgRIIB mAbs elicit phagocytosis Purified B cells or BMDMs were generated as indicated above and total RNA was isolated before conversion to cDNA SuperScript II first-strand We next examined the efficacy of our panel of recently developed anti- synthesis system for RT-PCR (Invitrogen). PCR was performed using FcgRIIB mAbs in a variety of mouse models of immunotherapy. We appropriate dilutions of cDNA and primers specific for murine FcgRIIB or Downloaded from murine b-actin as a control. Primers used were as follows: FcgRIIB, for- previously demonstrated that these mAbs were specific for FcgRIIB ward, 59-CCC AAG TCC AGC AGG TCT TTA CCA GTA TTG-39, re- and able to manipulate FcgRIIB signaling in ways potentially leading verse, 59-TGC TTG AGA AGT GAG TAG GTG ATC GTA TTC-39. to PCD of target cells (32). In addition to direct PCD, these mAbs also have the capacity to engender ADCC or phagocytosis of target Phagocytosis assay cells. To investigate the ability of our anti-FcgRIIB mAbs to elicit BMDMs were produced as detailed above and the phagocytosis assays were phagocytosis, we generated BMDMs, which express all four FcgRs, performed largely as detailed previously (47). Briefly, on the day of the and performed in vitro phagocytosis assays. Using CD20 Tg B cells http://www.jimmunol.org/ assay, macrophages were harvested using trypsin/EDTA (Invitrogen), resus- as targets we were able to demonstrate that AT130-2 and AT130-5 3 pended in RPMI 1640, and plated into a 96-well tissue culture plate (5 were able to elicit phagocytosis but to a lesser extent than anti-CD20 104 cells/well) and incubated for 2–4 h at 37˚C. Target B cells were isolated from hCD20 Tg mice, labeled with 5 mM CFSE, washed, and then incu- mAbs, with AT128 m2a showing relatively little efficacy (Fig. 2A), bated with anti-FcgRIIB or anti-CD20 mAb for 30 min at room tempera- probably due to its lower level of binding compared with AT130-2 ture. The B cells were then added to the BMDMs at a ratio of 5:1, the plates (32). As expected, phagocytosis was fully dependent on the expres- were incubated for 1 h at 37˚C, and then allophycocyanin-anti-F4/80 Ab sion of activatory FcgR (ablated with g-chain2/2 macrophages) and (AbD Serotec) was added to distinguish the macrophages. Cells were har- vested and analyzed by flow cytometry. The percentage of cells that stained was augmented by the loss of FcgRIIB on the macrophages (Sup- double positive for CFSE and allophycocyanin-anti-F480, which includes plemental Fig. 1). Interestingly, however, phagocytosis was not de- macrophages with both surface-bound and internalized B cells, was deter- pendent on the isotype of the anti-FcgRIIB mAbs, with both IgG2a by guest on September 29, 2021 mined as a measure of phagocytic potential. Confirmation of phagocytosis (AT130-2) and IgG1 (AT130-5) giving equivalent levels (Fig. 2A). was routinely provided by confocal microscopy (47). Furthermore, blockade of FcgRIIB on the macrophages with Immunotherapy AT130-2 or AT130-5 also resulted in augmentation of phagocytosis of anti-CD20 opsonized B cells, with AT128 m2a displaying a less 3 4 Fresh murine syngeneic B lymphoma cells (1 10 ), either BCL1 or A31, robust effect (Fig. 2B and data not shown). were injected i.v. into cohorts of female 8- to 12-wk-old BALB/c or CBA/H mice, respectively (day 0). Each mouse ($4 mice/group) received 0.5 mg of FcgRIIB mAbs do not elicit significant immunotherapy in either an irrelevant isotype control (WR17) or anti-FcgRIIB mAb (AT130-2 syngeneic mouse models or AT128 m2a) on day 3. On day 4, mice received 125 mg anti-idiotype mAb (Mc106A5 or Mc39/16 for the BCL1 or A31 tumors, respectively). We next examined the efficacy of our panel of recently developed The same mice then received further 125-mg doses of anti-idiotype or anti-FcgRIIB mAbs in a variety of mouse models of immuno- isotype control on days 5, 6, and 7. Alternatively, BCL1-inoculated mice received 125 mg anti-CD20 (18B12) or control mAb on days 1–4. 5T-33 cells (2 3 104) were injected i.v. into C57BL/6 KA mice on day 0. Mice then received 0.5 mg AT130-2, AT128 m2a, or irrelevant isotype control (WR17) on day 7. In the adoptive transfer assay, 2 3 107 splenocytes/ml from WT (target cells) and FcgRIIB2/2 (nontarget) BALB/c mice were stained with 5 and 0.5 mM CFSE, respectively, for 10 min at room temperature. FCS of the same volume was then added for 1 min to quench further labeling. Cells were washed twice with PBS (450 3 g for 5 min), combined in a 1:1 nontarget/target cell ratio, and injected on day 21 i.v. into recipient mice. WT and FcgRIIB2/2 BALB/c mice (three mice per group) were then injected i.v. with either 100 mg or 0.5 mg isotype control (WR17) or anti- FcgRIIB mAb on day 0. Mice were culled on day 1 and splenocytes were stained with allophycocyanin-conjugated anti-CD19 for 15 min at room temperature before analysis by flow cytometry. B cells were depleted from hCD20 Tg mice with 250 mg anti-hCD20 FIGURE 1. Anti-idiotype BCL1 immunotherapy shows improved effi- 2/2 4 mAb (Rit m2a), administered 7 d prior to tumor inoculation. cacy in FcgRIIB compared with WT mice. BCL1 cells (13 10 ) were inoculated i.v. into WT or FcgRIIB2/2 BALB/c mice (day 0). Mice then Statistical analysis received 125 mg/day control (Mc39/12) or anti-idiotype (Mc106A5; anti- To compare differences between experimental groups in vitro, two-tailed t test id) mAbs on days 4–7 and survival was monitored. Data represent a typical analysis was performed. To assess survival differences between experimental result from three independent experiments where n = 4–5 mice/group. groups in vivo, Kaplan–Meier curves were produced and analyzed by log- Median time of survival was increased following anti-idiotype treatment in testing. ANOVA analysis was used to determine differences between FcgRIIB2/2 compared with WT mice (p , 0.05). The Journal of Immunology 4133

FcgRIIB mAb is rapidly lost from the serum through modulation We previously reported that the efficacy of the type I anti-CD20 mAb rituximab (and its mouse-optimized m2a derivative Rit m2a) becomes limited in vivo due to its modulation from the target cell surface and consumption from the sera (47). Additionally, we recently observed that FcgRIIB is a key regulator of this internalization process (48), leading us to hypothesize that FcgRIIB might also be rapidly in- ternalized upon binding. To address this we transferred BCL1 lym- phoma cells into recipient mice, allowed tumor to establish in the spleens, then treated with anti-FcgRIIB mAbs, and at 48 h measured the level of FcgRIIB and mAbs on the surface of the splenic tumor cells. Both the anti-FcgRIIB mAbs and the receptor were rapidly lost from the cell surface (Fig. 4A). Importantly, the same response was observed in g-chain2/2 mice, indicating that this loss was independent of activatory FcgRs and was therefore likely not a consequence of the shaving phenomenon reported by Beum et al. (49), although it has recently been proposed that shaving may also Downloaded from be driven by FcgRIIB (50) (Fig. 4A). Similar modulation was observed in the A31 model (data not shown). In the case of rit- uximab and CD20, the loss of mAbs occurred as a result of re- ceptor internalization upon mAb engagement (47), and so to determine whether the same phenomenon was occurring we in- vestigated lymphoma cells as well as normal B cells and BMDMs http://www.jimmunol.org/ for their ability to internalize FcgRIIB after treatment with AT130- 2. Using both a conventional cell surface binding assay (Fig. 4B) and an Alexa 488–quenching assay (47) (Fig. 4C) to measure in- FIGURE 2. Anti-FcgRIIB mAbs elicit phagocytosis and can augment ternalization, we were able to show cell surface modulation in each phagocytosis of opsonized target cells. (A) Purified hCD20 Tg B cells were case. Similar results were seen with AT130-5 (data not shown). CFSE labeled and then opsonized with 10 mg/ml anti-CD20 or anti- Previously, it has been reported that the FcgRIIB-1 isoform, FcgRIIB mAbs. These were then incubated with BMDMs at a 5:1 ratio for expressed predominantly on B cells, does not internalize, whereas 1 h at 37˚C. Cells were then harvested, stained with allophycocyanin-F480, the FcgRIIB-2 isoform expressed on macrophages does (51–54). washed once, and analyzed by flow cytometry with the proportion of However, in our in vitro and in vivo experiments we noted clear by guest on September 29, 2021 CFSE/allophycocyanin double-positive macrophages used as a measure of modulation of the surface-bound mAb and receptor in both normal phagocytosis. (B) BMDMs were pretreated for 1 h at 37˚C with PBS, isotype control (WR17), or AT130-5 (10 mg/ml). These cells were then and malignant B cells as well as macrophages. Therefore, we incubated for 1 h with CFSE-labeled CD20 Tg B cells preopsonized with assessed the nature of receptors expressed in these cell types by no mAbs, rituximab (Ritux), or Rit m2a. Cells were harvested and ana- RT-PCR and confirmed the expected pattern of expression with lyzed as in (A). *p , 0.05, **p , 0.001. B cells and tumors expressing predominantly the FcgRIIB-1 isoform and macrophages the FcgRIIB-2 isoform (Supplemental Fig. 3). therapy. Given our previous results demonstrating potent antitu- We also examined the potential internalization of anti-human mor activities in vitro (32), we investigated the potential of our FcgRIIB mAbs on human cells. None of the three anti-human agonistic (AT130-2) and antagonistic (AT128 m2a) mAbs (both FcgRIIB mAbs were modulated from either human cell lines or m2a isotype, which would favor immunotherapy) in vivo. Some- purified B cells (Fig. 4D), whereas IgM modulated rapidly as what surprisingly, the anti-FcgRIIB mAbs alone did not result in expected. any substantial improvement in survival in lymphoma (BCL1 or One consequence of Rit m2a internalization is a comparatively A31) or myeloma (5T-33) models (Fig. 3A–C), although FcgRIIB rapid loss of mAbs from the serum of treated mice. Therefore, we was well expressed in each case (Supplemental Fig. 2). This is in assessed the serum levels of anti-FcgRIIB mAbs (AT130-2) or contrast to the anti-mouse CD20 mAb (18B12), which was ther- anti-idiotype mAbs in tumor-bearing mice from Fig. 4A. These apeutic in the BCL1 model (p , 0.001) (Fig. 3A). Furthermore, experiments revealed that anti-FcgRIIB mAbs were lost rapidly the anti-FcgRIIB mAbs did not augment the anti-idiotype im- from the sera in comparison with the anti-idiotype mAb, which was munotherapy in the BCL1 model (Fig. 3D), previously shown by maintained at a much higher level (Fig. 5A). Notably, the BCR, us to be enhanced in the FcgRIIB2/2 mice (Fig. 1). We speculated which the anti-idiotype recognizes, is expressed at a higher level on that the tumor models used might have some additional regulatory BCL1 cells than FcgRIIB (Supplemental Fig. 2A, 2B) and also mechanism able to counteract the anti-FcgRIIB mAbs and so becomes internalized upon engagement (55, 56). These data indicate examined the ability of the anti-FcgRIIB mAbs to deplete normal that the engagement and modulation of the less highly expressed B cells in vivo using the hCD20 Tg mouse model. A 0.5-mg dose FcgRIIB on the tumor is unlikely to explain the precipitous drop in of the anti-FcgRIIB mAbs was unable to elicit any substantial sera mAb levels. Instead, they indicate that FcgRIIB expressed depletion of peripheral B cells (Fig. 3E), splenic B cells, macro- on other cells may play a dominant role, presumably through phages, or DCs, even when the treatment was repeated three times a similar internalization process. This hypothesis would also (1.5 mg total) (Fig. 3F). In contrast, a single 250-mg dose of the explain the inability of the anti-FcgRIIB mAbs to deplete host anti-CD20 mAb Rit m2a resulted in profound depletion of pe- cells efficiently (Fig. 3F and data not shown). To examine this ripheral B cells for .40 d (Fig. 3E) and substantial depletion of specifically we treated WT, FcgRIIB2/2,andg-chain2/2 mice B cells in the spleen and lymph nodes (47). (which retain FcgRIIB expression) with AT130-2 and deter- 4134 MODULATION AND CONSUMPTION LIMIT ANTI-FcgRIIB mAb THERAPY Downloaded from http://www.jimmunol.org/

FIGURE 3. Anti-FcgRIIB mAbs do not elicit robust responses in mouse models of immunotherapy. WT BALB/c (A)orCBA(B) mice were inoc- 4 ulated i.v. with 1 3 10 BCL1 or A31 cells, respectively, on day 0. Mice were then treated with 0.5 mg AT130-2, AT128 m2a, or WR17 (irrelevant isotype control) on day 3. Concurrently, mice inoculated with BCL1 cells were treated with 125 mg 18B12 m2a on days 1–4, and mice inoculated with A31 were treated with 125 mg anti-idiotype (Mc39/16) on days 1–4. (C) WT C57BL/6 KA mice were inoculated with 2 3 104 5T-33 cells on day 0. Mice

were then treated with 0.5 mg AT130-2, AT128 m2a, or WR17 (irrelevant isotype control) on day 7. In each case AT130-2 and AT128 m2a did not by guest on September 29, 2021 increase survival of tumor-inoculated mice. 18B12 did improve survival of BCL1-inoculated mice (p , 0.05). (D) WT BALB/c mice were inoculated 4 with 1 3 10 BCL1 as in (A) and treated with 0.5 mg AT130-2, AT128 m2a, or WR17 (irrelevant control) on day 3, followed by 125 mg anti-idiotype (Mc106A5) on days 4–7. In this instance, AT130-2 and AT128 m2a did not increase survival either alone or in combination with anti-idiotype im- munotherapy. (E) WT C57BL/6 mice received 0.5 mg AT130-2 or irrelevant control (WR17) i.v. weekly for 4 wk. Circulating B cells were determined and expressed as a percentage of pretreatment B cell numbers. For comparison the level of B cell depletion is shown when a single 250-mg dose of an isotype control version of rituximab (Rit m2a) is used in congenic CD20 Tg mice. (F) WT BALB/c mice received 0.5 mg AT130-2 i.v. daily for 1, 2, or 3 d and the spleens were excised on days 2, 3, or 4, respectively. Splenocytes were stained for B cells (CD19 and B220), macrophages (F480 and CD11b), or DCs (CD11c and MHC class II) and analyzed by flow cytometry. Data are representative of at least three similar experiments, where n =4–5 animals/group. Error is expressed as 6SEM. mined the mAb half-life. In both WT and g-chain2/2 mice, anti- splenocytes into either WT or FcgRIIB2/2 mice and examined the FcgRIIB was rapidly lost from the serum, whereas mAb levels ability of the target cells to become depleted by the anti-FcgRIIB remained far higher in the absence of the receptor (FcgRIIB2/2 mAbs. These experiments revealed that anti-FcgRIIB mAbs were mice) (Fig. 5B). This is in contrast to the hCD20 Tg model, where only able to efficiently deplete FcgRIIB+ target B cells in the endogenous expression of the hCD20 throughout the B cell com- FcgRIIB2/2 recipients (Fig. 6A). Moreover, increasing the dose partment does not affect the ability of the mAb to deplete target of mAb from 100 to 500 mg did not yield appreciable depletion in cells or reduce its half-life to such an extent (Ref. 47 and data not the WT host. Furthermore, by assessing the remaining CFSE- shown). Increasing the dose of anti-FcgRIIB mAbs from a single labeled target cells 24 h after treatment, we were able to dem- 0.5-mg dose to multiple doses on sequential days was unable to onstrate that the modulation of FcgRIIB was much less in WT significantly reverse this effect (Fig. 5C). Genetic deletion of compared with FcgRIIB2/2 recipient mice. This indicates that in FcgRIIB on B cells (FcgRIIBfl/flCD19Cre+ mice) revealed that the context of an FcgRIIB competent mouse, the anti-FcgRIIB B cells only make a modest contribution to mAb consumption mAb is being consumed before it reaches the target B cells (Fig. in vivo with a small effect on day 1 (Fig. 5D). 6B). Interestingly, there was little discrimination between the

2/2 agonistic or antagonistic nature of the anti-FcgRIIB mAbs in these Anti-FcgRIIB mAbs are efficacious in FcgRIIB mice assays, with both AT130-2 and AT128 m2a giving equivalent Having established that the anti-FcgRIIB mAbs were being con- in vivo depletion of targets. sumed by endogenous cells, we next assessed whether FcgRIIB- Subsequently, having demonstrated that mAb consumption by expressing target cells could be depleted in the absence of FcgRIIB the cells of the host mouse could be the basis for impaired depletion in the host. First, using an in vivo depletion assay (47) we transferred of target cells, we repeated our immunotherapy experiments with + 2/2 2/2 CFSE-labeled FcgRIIB splenocytes and nontarget FcgRIIB BCL1 tumor cells in FcgRIIB mice. In an attempt to overcome The Journal of Immunology 4135 Downloaded from http://www.jimmunol.org/

FIGURE 4. Anti-FcgRIIB mAbs are modulated from the cell surface both in vitro and in vivo. (A)WTorg-chain2/2 BALB/c mice were inoculated 7 with 2 3 10 fresh BCL1 cells i.v. on day 0. Mice received 0.5 mg anti-FcgRIIB (AT130-2), anti-idiotype (Mc106A5), or isotype control (WR17) i.p. on by guest on September 29, 2021 day 4. Spleens were excised 48 h later and stained with CD19-allophycocyanin (top right panel) and gated on live cell events (top left panel) to identify B cells. Surface Ag expression and surface-accessible mAbs were then determined by flow cytometry as detailed in Materials and Methods.Maximal receptor expression was determined on control-treated samples. (B) Purified B cells, macrophages, and pBCL1 lymphoma cells were treated with 10 mg/ ml anti-FcgRIIB (AT130-2) or anti-idiotype (Mc106A5) and then cultured in vitro for 0–24 h. Cells were washed once and remaining surface Ag was detected as in (A). Surface expression was then calculated as a percentage of expression at 0 h. (C) Purified CD20 Tg B cells were incubated in vitro with Rit m2a-Alexa 488 (Rit-488) and Tosit-Alexa 488 (Tosit-488) mAbs, as well as AT130-2-Alexa 488 (5 mg/ml) for 2–24 h. Cells were washed once and then incubated in the presence or absence of anti-Alexa 488–quenching Ab. The fluorescence remaining after quenching indicates the proportion of internalized mAbs. (D) Malignant (Raji and Daudi) or normal B cells from PBLs were treated with 10 mg/ml AT10, IV.3, KB61 (anti-human FcgRII), or M15/8 (anti-IgM) and then cultured in vitro for 0–24 h. Surface Ag was determined as in (A) and (B). In (A) data are representative of at least three similar experiments, where n = 3 animals/group. In (B)–(D) data are representative of least three independent experiments, where n = 3 samples/group. Error is expressed as 6SEM. the effects of mAb consumption and provide effective immuno- B cell–depleted mice. Under these circumstances depletion of therapy, mice were treated with either one, two, or three 0.5-mg B cells lead to improved survival (Fig. 7D, right panel), although 2 2 doses of anti-FcgRIIB mAbs. As expected, and in contrast to the not comparable to that seen in FcgRIIB / mice. effects in WT mice (Fig. 3A), we were able to demonstrate a clear and statistically significant improvement in survival with the anti- Discussion FcgRIIB mAbs (Fig. 7A). Likewise, the anti-FcgRIIB mAbs were mAb immunotherapy is a rapidly evolving treatment modality, with also able to further augment immunotherapy of anti-idiotype mAb multiple new mAbs entering the clinical arena each year (57–59). 2 2 in the FcgRIIB / model (Fig. 7B). In some cases, multiple doses Surprisingly, given the almost exponential rate of new mAb reagents, of AT130-2 in combination with anti-idiotype mAbs resulted in relatively little is still known regarding what makes an effective target long-term survival (Fig. 7B). Ag when developing mAbs to recruit cytotoxic effectors. Currently, As B cells are a potential sink for the anti-FcgRIIB mAbs, we CD20 and CD52 stand out as the best available. subsequently investigated whether deleting B cells might augment In the present study, we have examined the therapeutic potential immunotherapy. First, to determine whether the absence of B cells of manipulating inhibitory FcgRIIB (CD32B). Initially, we in- would improve mAb half-life in vivo, we depleted host B cells vestigated the effect of deleting FcgRIIB in an mAb immuno- from CD20 Tg mice using Rit m2a and monitored serum mAb therapy model of lymphoma and confirmed that lack of FcgRIIB levels. This resulted in enhanced half-life of AT130-2 (Fig. 7C). augmented the ability of anti-idiotype to treat BCL1 lymphoma- Next, anti-FcgRIIB mAb was combined with anti-idiotype mAb bearing mice and for anti-CD20 mAb to deplete normal B cells, and administered to WT (Fig. 7D, left panel for comparison) or respectively. Our results are in keeping with the seminal findings 4136 MODULATION AND CONSUMPTION LIMIT ANTI-FcgRIIB mAb THERAPY Downloaded from http://www.jimmunol.org/

7 FIGURE 5. Anti-FcgRIIB mAbs are consumed from the serum in vivo. (A) WT BALB/c mice were inoculated i.v. with 2 3 10 fresh BCL1 cells on day 0. Mice received 0.5 mg AT130-2, AT128 m2a, or anti-idiotype (Mc106A5) i.p. on day 4. Forty-eight hours later the level of mAbs remaining in the serum was determined using a bioassay. (B)WT,FcgRIIB2/2,org-chain2/2 BALB/c mice were injected i.v. with 0.5 mg AT130-2. Serial serum samples were then collected and the levels of mAbs determined by ELISA. (C) WT BALB/c mice received 0.5 mg AT130-2 i.v. every other day for a total of one, two, or three doses with serum mAb levels determined as in (B) 48 h after the final injection. mAb remaining in the serum after a single 0.5-mg dose of AT130-2 in by guest on September 29, 2021 FcgRIIB2/2 mice is shown for comparison. (D) C57BL/6 WT, FcgRIIBfl/fl, CD19-Cre FcgRIIBfl/fl, and FcgRIIB2/2 mice were injected with 0.5 mg AT130-5 mAb on day 0. Serum samples were collected on days 1, 2, and 4 and mAb concentrations were determined as in (B). Data are representative of at least three independent experiments, where n = 2–3 mice/group. Error is expressed as 6SEM. *p , 0.05, ***p , 0.001. of Clynes et al. (7), albeit we observed less marked enhancement were injected s.c. into nude mice and treated with 4D5 or tras- of the therapeutic activity with the anti-idiotype mAb. Those tuzumab. Clearly, nude and SCID mice lack components of the authors showed that mAb therapy of B16 melanoma by TA99 and adaptive immune system, and so do not mimic the complex rela- BT474MI human breast cancer cells by 4D5 and trastuzumab in tionship between the tumor and its microenvironment. Furthermore, FcgRIIB2/2 mice abrogated tumor growth far more efficiently the human breast cancer cells may stimulate a nonphysiological than did WT littermate controls. Why the BCL1 model should be anti-human response within the mouse through the remaining innate less readily augmented is not known but likely reflects the dif- immune effectors, which may alter the extent of FcgRIIB involve- ferent models used. For example, growth in the B16 model was ment, potentially providing more impressive responses. examined by scoring metastases after day 14, whereas in this We subsequently explored the use of mAbs directed toward study we established the effect of the mAb on overall animal FcgRIIB to elicit and augment immunotherapy. These mAbs were survival in WT and FcgRIIB2/2 mice. Furthermore, in the B16 specific solely for FcgRIIB, elicited a number of potentially im- model, mAb treatment occurred on the same day as administration portant effector functions such as PCD (32) and phagocytosis, and of the tumor load, which might have prevented the cells from yet were unexpectedly poor in deleting target cells in a number of engrafting in the lungs, where in contrast in the present study we immunotherapy models, including mouse models of lymphoma, treated mice at least 4 d after tumor inoculation. Interestingly, myeloma, and normal B cell depletion. This is in stark contrast to others have also demonstrated little improvement in mAb therapy the levels of efficacy reported by Rankin et al. (29) with an anti- in FcgRIIB2/2 mice. Takeda et al. (60) treated TRAIL-sensitive human FcgRIIB mAbs. In those studies, the human Burkitt’s 4T1 murine mammary carcinoma with an anti-mouse anti-DR5 lymphoma cell line Daudi was injected into immunodeficient nude mAb (MD5-1). They showed that MD5-1 almost completely ab- mice with the mAb given either on day 0 or after 7 d. In both rogated tumor growth in WT mice, but that this therapy was not cases, impressive tumor retardation was demonstrated, with the improved in FcgRIIB2/2 mice, perhaps because it relies on pro- anti-FcgRIIB mAb (h2B6) inducing greater antitumor effects than apoptotic signaling rather than on cytotoxic effectors. Very recent the current gold standard rituximab. In addition to the caveats findings indicate that this apparent discrepancy may relate to the inherent in treating tissue-adapted human cell lines in immuno- requirement of certain classes of mAb to be cross-linked on the deficient mice, there are a number of additional issues relating to cell surface by FcgRIIB (61–63). The second model Clynes et al. this approach. First, unlike CD20, FcgRIIB is not B cell–specific used was a xenograft model of human breast cancer where cells and is expressed on a number of other lymphoid cell types such as The Journal of Immunology 4137

cytosis (69–71). By preventing coligation of the activatory FcgRs with FcgRIIB it is likely that anti-FcgRIIB mAbs prevent the appropriate recruitment of these phosphatases to the intracellular domains of the activatory FcgRs, thus allowing uninhibited phago- cytosis to occur. This is despite the fact that the blocking mAb used (AT130-5) has previously been shown to phosphorylate FcgRIIB, trigger activation of SHIP-1 and SHP-1, and inhibit BCR signaling responses (32). These data presumably indicate that preventing coaggregation of FcgRIIB with activatory FcgRin macrophages is the key factor in preventing inhibition of phago- cytosis through FcgRIIB. They may also indicate that the inhib- itory signaling induced by the anti-FcgRIIB mAb is only transient and not sufficiently potent to block phagocytosis. Our subsequent experiments revealed that FcgRIIB expression on the host cells is a major limitation toward immunotherapy with anti- FcgRIIB mAb, at least in the mouse. As such, the anti-FcgRIIB mAbs were able to elicit significant target cell depletion and im- munotherapy of lymphoma in FcgRIIB2/2 but not FcgRIIB-replete

hosts. At least in part, the reduction in immunotherapy was due to Downloaded from the loss of the mAb from the serum, presumably through mAb consumption in WT mice, similar to that reported by us previously fortypeIanti-CD20mAb(47). Our studies have revealed a role for B cells in anti-FcgRIIB mAb consumption. However, serum mAb was not entirely rescued in the fl/fl + + FcgRIIB CD19Cre , indicating that other FcgRIIB cell types http://www.jimmunol.org/ (such as macrophages, DCs, Kupffer cells, and certain endothelial cells) (72) may be additional consumers of mAb in vivo. Intrigu- ingly, deletion of B cells with anti-CD20 mAbs provided a more substantial improvement in serum half-life of anti-FcgRIIB mAb than seen in FcgRIIBfl/flCD19Cre+ mice. Potentially, this differ- ence is attributed to the engagement of FcgRIIB on other endog- enous cell types by the anti-CD20 mAb-opsonized B cells during the deletion process. In this setting, deletion of B cells was able to augment the efficacy of combining anti-FcgRIIB and anti-idiotype by guest on September 29, 2021 mAbs in the BCL1 model (Fig. 7D). However, the improvement in survival seen following combination therapy was not as pro- FIGURE 6. Anti-FcgRIIB mAbs deplete FcgRIIB-expressing targets in 2/2 2/2 nounced as that seen when anti-idiotype mAb was administered in FcgRIIB mice. (A) WT (target [T]) and FcgRIIB nontarget (NT) 2 2 the FcgRIIB / mice (Fig. 7B), providing further evidence that splenocytes were labeled with 5 and 0.5 mM CFSE, respectively, and injected i.v. into WT and FcgRIIB2/2 BALB/c mice on day 0. Mice re- other cell types are consuming mAbs in vivo. ceived 100 mg or 0.5 mg AT130-2, AT128 m2a, or irrelevant control (Irr; As with type I anti-CD20 mAb, FcgRIIB mAb consumption WR17) i.v. on day 1. Twenty-four hours later, spleens were excised and appears linked to receptor modulation. The current data confirm stained with anti–CD19-allophycocyanin before analysis by flow cytom- that AT130-2 and AT130-5 reduce cell surface levels of FcgRIIB etry. The ratio of CD19+ target/nontarget cells was determined and ex- and that this reduction occurs because the receptor is internalized. pressed as a normalized target/nontarget cell ratio. Data represent three On which cells and through which receptor (FcgRIIB-1 or -2) the experiments, where n = 3 mice/group. ***p , 0.001. (B) Splenocytes from modulation is occurring is the subject of our ongoing studies. A ( ) were incubated with or without 10 mg/ml unconjugated anti-FcgRIIB Previously, it has been reported that the FcgRIIB-1 isoform, prior to incubation with anti-mouse Fc-PE as previously described. Max- expressed predominantly on B cells, does not internalize, whereas imal receptor expression was determined on control-treated samples. the FcgRIIB-2 isoform expressed on macrophages and DCs does Target cells were identified as in (A). Data are representative of three similar independent experiments. (51, 52). However, in our in vitro and in vivo experiments we noted clear modulation of the surface-bound mAb and receptor in both normal and malignant B cells as well as macrophages and plasma cells, macrophages, DCs, Kupffer cells, Langerhans cells, confirmed the expected expression pattern of FcgRIIB isoforms in , mast cells (4), sinusoidal lining cells in the liver (64), these cells. Note that the previous conclusions were based on dermal microvascular endothelial cells (65), and placental villus experiments examining effects of IC binding (53, 73) as opposed endothelium (66). Furthermore, in the xenograft model, FcgRs are to direct receptor engagement by mAbs as in this study, where not blocked by endogenous IgG and the endogenous host cells do not both Fab and Fc engagement are possible. In this scenario, as we express the human target, meaning that all of the available mAb previously proposed (32), higher levels of receptor cross-linking injected is available for binding to the few tumor cell targets injected. may occur, resulting in endocytosis. Furthermore, those initial In relation to our phagocytosis experiments, it was interesting experiments were performed using human FcgRIIB constructs that blocking macrophage FcgRIIB augmented activity. It is expressed in an FcgRIIB-deficient derivative of the mouse lym- known that coaggregation of FcgRIIB with activatory FcgRs phoma cell line A20 (IAII.6), or in Chinese hamster ovary and results in the phosphorylation of the FcgRIIB ITIM, leading to COS-1 transfectants containing mouse FcgRIIB. Additionally, the activation of SHP-1 and SHIP-1 (67, 68), dephosphorylation of so-called nonendocytic FcgRIIB-1 isoform was previously studied Syk, PI3K, and Rac, and inhibition of cell activation and phago- during a very short time scale (within 30 min) (53), whereas in this 4138 MODULATION AND CONSUMPTION LIMIT ANTI-FcgRIIB mAb THERAPY Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 7. Augmentation of FcgRIIB mAb immunotherapy in FcgRIIB2/2 mice. (A)FcgRIIB2/2 BALB/c mice were inoculated i.v. with 1 3 104 2/2 BCL1 cells on day 0 and treated with 0.5 mg AT130-2 every other day for a total of one, two, or three doses starting from day 2. (B)FcgRIIB BALB/c 4 mice were inoculated with 1 3 10 BCL1 cells on day 0 and treated with 0.5 mg AT130-2 or an isotype control (WR17) on days 2, 4, and 6 followed by 125 mg/day of either an isotype control (Mc39-16) or anti-idiotype (Mc106A5) on days 4–7. Combined AT130-2 and anti-idiotype therapy gave improved survival compared with Irr (p , 0.0001), AT130-2 alone (p , 0.0001), or anti-idiotype alone (p = 0.0009). (C)WT,FcgRIIB2/2, or B cell–depleted (250 mg Rit m2a given on day 27) CD20 Tg BALB/c mice were injected with 0.5 mg AT130-2 on day 0. Serum mAb concentrations were determined from 4 serial samples as previously described. (D) WT or B cell–depleted CD20 Tg BALB/c mice were inoculated with 1 3 10 BCL1 cells on day 0. Mice then received immunotherapy as in (B). Data in (A)–(D) are representative of three similar experiments, where n = 5 mice/group with the exception of (B), where data represent a composite of three separate experiments. In both strains, anti-idiotype therapy significantly increased survival (p , 0.01). The addition of AT130-2 only further augmented survival in the B cell–depleted CD20 Tg mice (p , 0.05). study, we examined receptor modulation at 2, 6, and 24 h, perhaps It was previously reported that levels of FcgRIIB expression do in part explaining the difference in these studies. Although Clynes not alter in human lymphoma cell lines after treatment in vivo and colleagues (74) showed that FcgRIIB expressed on mouse with a humanized anti-FcgRIIB mAb. Therefore, the modulation DCs is competent to internalize IC, uniquely on plasmacytoid we detect on the normal and malignant lymphoma cells after DCs, it is still unclear from the existing literature whether murine treatment with murine anti-FcgRIIB mAbs both in vitro and FcgRIIB on murine cells possesses the same endocytic properties in vivo may indeed be a species-dependent observation. Poten- as described for the human receptor. There is a subtle difference tially, the lack of modulation reported by Rankin et al. (29) may between the structures of murine and human FcgRIIB, demon- reflect the cell line nature of the human tumor cell assessed, as we strated by a shortened cytoplasmic tail of the human receptor (51), also observed that cell lines routinely modulated type I anti-CD20 possibly accounting for the proposed differences in endocytosis. mAbs relatively slowly, compared with primary tumor cells (47). Alternatively, the observed differences in internalization may However, in our own studies, using anti-human FcgRII mAb on relate to the species that is, mouse versus human B cells. In support both cell lines and primary B cells, we also observed that all three of this notion, we have observed that hCD20 Tg mouse B cells of the mAbs tested did not internalize, in agreement with the data appear to internalize anti-CD20 mAb more rapidly than their reported by Rankin et al. (29) and in contrast to our results with normal human B cell counterparts (47). Furthermore, our recent anti-mouse FcgRIIB mAbs. As discussed above, whether these unpublished work using the A20 IAII.6 cells has indicated that data reflect inherent differences in the human and mouse recep- these mouse cells also internalize surface mAb unusually rapidly tors, differences in the mouse and human cells or differences in (V. Sha, A. Vaughan, and A. Roghanian, unpublished data). the individual mAbs remain to be determined and is the subject of The Journal of Immunology 4139 our ongoing studies. In an attempt to address this question, we are 14. Uchida, J., Y. Hamaguchi, J. A. Oliver, J. V. Ravetch, J. C. Poe, K. M. Haas, and T. F. Tedder. 2004. The innate mononuclear phagocyte network depletes currently generating transgenic mice that express either the human B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 FcgRIIB alone, or in the presence of the mouse receptor. Using antibody immunotherapy. J. Exp. Med. 199: 1659–1669. these mice we will be able to directly address whether the human 15. Bevaart, L., J. Goldstein, L. Vitale, C. Russoniello, J. Treml, J. Zhang, R. F. Graziano, J. H. Leusen, J. G. van de Winkel, and T. Keler. 2006. Direct receptor modulates when bound by mAb (in vitro and in vivo) in targeting of genetically modified tumour cells to FcgRI triggers potent tumour primary mouse cells and observe whether it results in rapid con- cytotoxicity. Br. J. Haematol. 132: 317–325. sumption of the anti-human FcgRIIb mAbs in vivo. Clearly, the 16. Nimmerjahn, F., and J. V. Ravetch. 2005. Divergent subclass activity through selective Fc receptor binding. Science 310: 1510–1512. findings from these experiments will have important consequences 17. Fossati-Jimack, L., A. Ioan-Facsinay, L. Reininger, Y. Chicheportiche, N. Watanabe, for the use of these reagents in the clinic. T. Saito, F. M. Hofhuis, J. E. Gessner, C. Schiller, R. E. Schmidt, et al. 2000. In summary, in this study we have assessed the ability of FcgRIIB- Markedly different pathogenicity of four immunoglobulin G isotype-switch variants of an antierythrocyte autoantibody is based on their capacity to interact in vivo with specific mAbs to treat fully syngeneic mouse tumor models and the low-affinity Fcg receptor III. J. Exp. Med. 191: 1293–1302. concluded they are ineffective due to their rapid consumption by 18. Shields, R. L., A. K. Namenuk, K. Hong, Y. G. Meng, J. Rae, J. Briggs, D. Xie, FcgRIIB-expressing cells of the host alongside target cell internal- J. Lai, A. Stadlen, B. Li, et al. 2001. High resolution mapping of the binding site on human IgG1 for FcgRI, FcgRII, FcgRIII, and FcRn and design of IgG1 ization. This novel and unexpected mode of resistance highlights the variants with improved binding to the FcgR. J. Biol. Chem. 276: 6591–6604. need to carefully examine the mechanisms of action and resistance 19. Wu, J., J. C. Edberg, P. B. Redecha, V. Bansal, P. M. Guyre, K. Coleman, J. E. Salmon, and R. P. Kimberly. 1997. A novel polymorphism of FcgRIIIa with therapeutic mAbs. Furthermore, these data are in marked (CD16) alters receptor function and predisposes to autoimmune disease. J. Clin. contrast to those reported with mAbs directed to human FcgRIIB Invest. 100: 1059–1070. in xenograft studies, and so it remains to be seen whether similar 20. Cartron, G., L. Dacheux, G. Salles, P. Solal-Celigny, P. Bardos, P. Colombat, and H. Watier. 2002. Therapeutic activity of humanized anti-CD20 monoclonal an- limitations will exist in human cells. The current in vitro data indi- tibody and polymorphism in IgG Fc receptor FcgRIIIa gene. Blood 99: 754–758. Downloaded from cate a species-specific difference in the activity of FcgRIIB in mice 21. Weng, W. K., and R. Levy. 2003. Two immunoglobulin G fragment C receptor and humans that provides optimism that anti-human FcgRIIB may polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J. Clin. Oncol. 21: 3940–3947. not be affected by the same issues, although this requires examina- 22. Tsuchiya, N., Z. Honda, and K. Tokunaga. 2006. Role of B cell inhibitory re- tion in appropriate model systems and the molecular details remain ceptor polymorphisms in systemic erythematosus: a negative times a negative makes a positive. J. Hum. Genet. 51: 741–750. to be elucidated. 23. Olferiev, M., E. Masuda, S. Tanaka, M. C. Blank, and L. Pricop. 2007. The role

of activating protein 1 in the transcriptional regulation of the human FCGR2B http://www.jimmunol.org/ promoter mediated by the -343 G → C polymorphism associated with systemic Acknowledgments lupus erythematosus. J. Biol. Chem. 282: 1738–1746. We thank Prof. Mark Shlomchik for the provision of hCD20 Tg mice and 24. McGaha, T. L., M. C. Karlsson, and J. V. Ravetch. 2008. FcgRIIB deficiency Dr. N Savelyeva for the gift of the 5T-33 myeloma cells. We thank Dr. Robert leads to autoimmunity and a defective response to apoptosis in Mrl-MpJ mice. J. Dunn (Biogen Idec) for helpful comments and initial provision of the anti- Immunol. 180: 5670–5679. 25. Kono, H., C. Kyogoku, T. Suzuki, N. Tsuchiya, H. Honda, K. Yamamoto, mouse CD20 mAbs. We thank all members of the Antibody and Vaccine K. Tokunaga, and Z. Honda. 2005. 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Figure S2: BCL1, A31 and 5T-33 tumor cells express FcȖRIIB. BCL1 (A) and A31 (B) were incubated with 10 ʅg/ml AT130-2 or anti-idiotype, before bound mAb was detected using an anti-mouse Fc-PE conjugated mAb before analysis by flow cytometry. C, 5T-33 cells were stained with anti-CD138-PE and either AT130-2 or 2.4G2-FITC before analysis by flow cytometry. All three tumor lines expressed good levels of FcɶRIIB.

Figure S3: Expression of FcɶRIIB isoforms in normal murine B cells, macrophages and lymphoma cells. RNA was extracted and purified from approximately 5 x 106 normal murine B cells (B), BMDMs (MɌ) and ʋBCL1 cells and converted to cDNA. RT-PCR was performed as described and demonstrated that B cells, including lymphoma cells in this instance, predominately express mFcɶRIIB-1, whilst BMDMs express mFcɶRIIB-2. As expected, no PCR product was observed from FcɶRIIB -/- splenocytes. A third PCR product was also visible after electrophoresis, it is believed this was the CD32-1’ isoform as described by Latour et al (1996).