cis and trans and drives internalization according to ...

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Prepublished online November 13, 2013; doi:10.1182/blood-2013-04-490821

Inhibitory FcγRIIb (CD32b) becomes activated by therapeutic mAb in both cis and trans and drives internalization according to antibody specificity Andrew T. Vaughan, Chisako Iriyama, Stephen A. Beers, Claude H.T. Chan, Sean H. Lim, Emily L. Williams, Vallari Shah, Ali Roghanian, Bjorn Frendéus, Martin J. Glennie and Mark S. Cragg

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Blood First Edition Paper, prepublished online November 13, 2013; DOI 10.1182/blood-2013-04-490821

TITLE: Inhibitory FcγRIIb (CD32b) becomes activated by therapeutic mAb in both cis and trans and drives internalization according to antibody specificity

Short title: Vaughan et al. mAb specificity determines internalization

Andrew T. Vaughan1, Chisako Iriyama1,2, Stephen A. Beers1, Claude HT. Chan1, Sean H. Lim1, Emily L. Williams1, Vallari Shah1, Ali Roghanian1, Bjorn Frendéus3, ‡Martin J. Glennie1 and ‡Mark S. Cragg 1

From: 1Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton, Faculty of Medicine, General Hospital, Southampton, SO16 6YD, UK. 2 Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan, 3 BioInvent International AB, Lund, Sweden

‡ These authors are the senior authors and contributed equally to the study.

Corresponding author : Mark S Cragg, Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton, Faculty of Medicine, General Hospital, Southampton, SO16 6YD, UK (FAX: +44 (0) 23 80704061; e-mail: [email protected])

The online version of the article contains a data supplement.

Section Designation: Article; Scientific Heading: Immunobiology

1 Copyright © 2013 American Society of Hematology


Key points 1) FcgRIIb dependent internalization of therapeutic mAb is dependent on antibody specificity 2) FcgRIIb can be activated in both cis and trans configurations

Abstract

A major feature that distinguishes type I from type II anti-CD20 mAb and reduces their therapeutic efficacy is the tendency to internalize from the cell surface. We have shown previously that the extent of internalization correlates with the capacity of type I mAb to simultaneously engage both CD20 and the inhibitory Fc γ receptor, FcγRIIb, in a bipolar configuration. Here we investigated whether mAb directed at other B-cell surface receptors also engaged Fc γRIIb and whether this interaction promoted internalization. Most mAb engaged and activated Fc γRIIb, with the strength of activation related to the level of mAb bound to the cell surface. However, engagement did not affect internalization of most mAb-ligated receptors, either in cell lines or primary chronic lymphocytic leukaemia (CLL) cells with the exception of CD19 and CD38. Furthermore, at high cell concentrations/density both cis and trans interactions between cell surface bound mAb and Fc γRIIb were evident, but trans interactions did not inhibit type I anti-CD20 mAb-mediated internalization. These data identify that Fc γRIIb is engaged by many mAb in both cis and trans configurations, triggering its activation, but that internalization via Fc γRIIb occurs for only a select subset. These findings have implications when designing new antibody-based therapeutics.

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Introduction

Anti-CD20 monoclonal antibodies (mAb) are classified as type I (rituximab(RTX)-like) or type II (tositumomab-like) based on functional differences that they mediate on B-cells in vitro. 1 We have recently observed that type I mAb induce appreciable internalization of CD20 from the surface of Bcells, in contrast to type II mAb that remain largely cell-surface localized. 2 The rate of internalization directly correlated with expression of the inhibitory Fc γ receptor (FcγR) IIb (CD32b), and was largely abrogated when target cells were treated with a F(ab’) 2 fragment of RTX or co-incubated with an anti-FcγRII blocking mAb.3 We demonstrated that type I anti-CD20 mAb were able to engage and activate FcγRIIb on the surface of B-cells via a process termed antibody bipolar bridging 4 where CD20 and FcγRIIb were engaged by the antibody in a cis configuration which was sufficient to induce internalization of the mAb:CD20:Fc γRIIb complex.3 Despite the widespread success of RTX in the treatment of follicular lymphoma and diffuse large Bcell lymphoma,5 it has proven less successful in the treatment of mantle cell lymphoma (MCL) 6 and chronic lymphocytic leukaemia (CLL) 7 in which larger doses are required 8. Reduced responses in MCL and CLL appear to correspond to a higher expression of Fc γRIIb and a faster rate of type I antiCD20 mAb-mediated internalization of CD20. Furthermore, in two separate retrospective studies, higher expression of Fc γRIIb on tumor cells was associated with reduced survival or response in patients treated with RTX therapy. 3,9 These observations together with the lower efficacy observed when using type I anti-CD20 mAb in murine models of B-cell depletion, 2,10 has led us to propose that type I anti-CD20 mAb-mediated internalization may contribute to treatment failure of mAb therapy in the clinic3,11 supporting the use of non-internalizing type II mAb for more refractory tumor-types.

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Given the difficulty of finding effective mAb targets on cancer cells, including malignant B-cells, we embarked on a study of how Fc γRIIb might influence internalization of various target:mAb complexes from the cell surface. It is now established that certain B-cell targets such as CD22 and the BCR are prone to rapid and extensive internalization either as a result of constitutive turnover

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or crosslinking-induced reorganisation 13 and such reagents are best suited for toxin delivery. In contrast, the utility of other molecules, such as CD19 and CD40 which continue to be explored as targets for mAb that recruit natural effectors, might also be influenced by Fc γRIIB. Given the detrimental effect of bipolar Fc γRIIB interactions on mAb efficacy for Type I anti-CD20 mAb2,3, determining the potential impact of this interaction on other antigenic targets poses an important question for future drug development.

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Methods Patient samples and cell-lines

CLL or MCL samples were stored in the University of Southampton Cancer Sciences Unit Tumor Bank under Human Tissue Authority licensing. 3 Cells were thawed, resuspended in complete media ((RPMI 1640, 4mM L-glutamine, 1mM sodium pyruvate and 10% (v/v) FCS (all from Life Technologies)) and used on the same day. Cell lines were obtained from the European Collection of Cell Cultures and maintained in complete media. This study was conducted in accordance with the Declaration of Helsinki.

Generation of human FcγRIIb1 and truncated FcγRIIb plasmid vectors

Human FcγRIIb1 cDNA was amplified from Daudi cells using specific primers and cloned into the pCDNA3.1 expression vector (Life Technologies). To generate a truncated mutant version of Fc γRIIb lacking the intracellular domain, a stop mutation at residue 250 was introduced using the QuickChange Multi Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions.

Cell transfection and generation of stable transfectants

Cells were transfected by electroporation using Nucleofector kit V on the Nucleofector I device according to the manufacturer’s instructions (Lonza). Ramos and RPMI 8226 cells were transfected using programmes O-06 and G-15, respectively. Stable transfectants were selected with 1mg/ml G418 (Geneticin, Life Technologies) for Ramos cells or 0.5mg/ml G418 for RPMI 8226 cells. Colonies were screened for cell surface Fc γRIIb expression using AT10-PE (AbD Serotec) by flow cytometry. FcγRIIb2 transfected Ramos cells were generated previously. 3

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RT-PCR of FcγRIIb isoforms

Total RNA was isolated using the RNeasy minikit (Qiagen), cDNA prepared using the Superscript III first-strand synthesis system (Life Technologies) before PCR using primers specific to Fc γRIIb1/b2 and GAPDH as a loading control.

Monoclonal antibody production and labelling

GA101-gly (anti-CD20, human IgG1 and mouse IgG2a) are the non-glycoengineered parent versions of GA101 and were produced in-house from patent published sequences. Rituximab was gifted by Southampton General Hospital oncology pharmacy. AT13/5h (anti-CD38, chimeric human (h) IgG1 14), RFB9 (anti-CD19, mouse (m) IgG1 15), LOB 7-4 (anti-CD40, mIgG1), LOB 7-6 (anti-CD40, mIgG1), F3.3 (anti-MHC II, mIgG1 16), M15/8 (anti-µ, mIgG115), AT10 (anti-FcγRII, mIgG117), MB1/7 (anti-CD37, mIgG118) and 4KB128 (anti-CD22, mIgG119) were produced in-house. Antibodies were labelled with Alexa fluor 488 (A488)-TFP ester according to the manufacturer’s instructions (Life Technologies).

Antibody internalization assay

Internalization of A488-labelled mAb was quantified as reported previously 2,3 using the following formula: % cell-surface mAb remaining on B-cells = (MFI of unquenched-MFI of quenched)/MFI of unquenched x 100. The MFI of unstained cells was subtracted as background. To investigate the effect of cell-surface Fc γRIIb on mAb internalization, Fc γRIIb was blocked with AT10 (50 μg/ml) for 30 minutes at 37oC as previously described.3 In co-culture experiments, Ramos

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FcγRIIb1 high transfectants were pre-stained with PKH26 (Sigma-Aldrich) and mixed 1:1 with transfected RPMI 8226 cells.

Western blotting

Cells plated at 4 x 106/ml were pre-incubated at 37oC for 30 minutes before the addition of A488labelled mAb (5µg/ml) for 30 minutes, then washed in ice cold PBS, resuspended in lysis buffer and processed as described previously. 20 Membranes were probed with EP926Y (rabbit anti-human FcγRIIb (phospho-specific), Cambridge Bioscience) in TBS-Tween 0.05 %/5% BSA/ 0.05% sodium azide at 4oC overnight, then with donkey anti-rabbit IgG HRP-linked F(ab’) 2 for 1 hour at room temperature, washed and the signal visualised using ECL reagents and light sensitive film (all GE Healthcare Lifesciences). In co-culture experiments, cells were stained with A488-labelled mAb (5µg/ml) for 30 minutes at 4oC, washed 3 times with ice-cold PBS, then mixed 1:1 with unlabelled cells, incubated at 37 oC for 30 minutes and processed for western blotting.

Statistical analysis

Analyses were performed using non-parametric tests with a two-tailed hypothesis. Unpaired samples were analysed using the Mann-Whitney U test and paired samples were analysed using the Wilcoxon signed-ranks test using Graphpad Prism version 6.00 for Windows (Graphpad Software).

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Results FcγRIIb1 and -2 augment the internalization of mAb on the surface of B-cells

In a previous study with a small number of CLL samples, we showed that the predominant isoform of FcγRIIb expressed was b23 and that transfection of Ramos cells with the b2 isoform augmented the internalization of type I anti-CD20 mAb. 3 However, the predominant isoform expressed on nonmalignant B-cells is b1 which has an extra 19 amino acids in its intracellular tail that prevents its internalization when co-ligated with the BCR. 21 Subsequent analysis of a further 5 CLL samples and 3 MCL samples revealed that Fc γRIIb1 was also expressed at substantial levels on malignant cells, albeit variably between patients (Figure 1A). To investigate whether the b1 isoform could also promote type I anti-CD20 mAb-mediated internalization, Fc γRIIb-ve Ramos cells were transfected with human Fc γRIIb1. Colonies expressing low, medium or high levels of the receptor were selected (Figure 1B), reflecting the expression of FcγRIIb1 on normal human B -cells, primary lymphoma cells overexpressing the receptor, and an even higher (likely non-physiological) level of Fc γRIIb1, respectively. Empty vector transfected cells were used as controls. Cells were cultured with A488-labelled anti-CD20 mAb and the proportion of total mAb remaining on the cell surface quantified after 1 and 6 hours (Figure 1C and Supplementary Figure 1, respectively). Expression of Fc γRIIb1 at a physiological level (Fc γRIIb1-low) resulted in a significant increase in internalized RTX compared with controls at both time-points. Internalization was confirmed by both confocal microscopy and reduced detection of human IgG at the cell surface by flow cytometry (Supplementary Figure 2). Neither CD20 shedding nor shaving appeared to occur (data not shown). Using transfectants expressing almost identical physiological levels of the b1 and b2 isoforms (MFI 152; b2 as used previously 3) we were also able to confirm that both accelerate internalization of surface bound mAb to a similar extent (Figure 1C). There was no significant internalization of the

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type II anti-CD20 mAb tositumomab (Tosit) or non-glycomodified forms of GA101 (GA101-gly) of hIgG1 or mouse IgG2 (m2a) isotypes (Figure 1C and Supplementary Figure 1). As before with FcγRIIb2-transfectants3, there was a dose- and time-dependent increase in the internalization of RTX in cells expressing higher levels of Fc γRIIb1. There was also increased internalization of type II antiCD20 mAb with high levels of Fc γRIIb1, but to a far lesser extent than RTX, with tositumomab being the most resistant to internalization (Supplementary Figure 1A). As before 3, internalization was also related to isotype, with mouse 2a promoting less internalization than hIgG1 and mouse IgG1 (Supplementary Figure 1B). Equivalent activity was also observed with mouse Fc γRIIb1 and FcγRIIb2 isoforms transfected into Ramos cells (Supplementary Figure 3).

We next asked if Fc γRIIb expression affected the internalization of mAb recognising other targets (CD19, CD22, CD37, CD38, CD40, MHC Class II (MHCII) and the BCR (IgM)). As we have observed differences in the ability of different IgG isotypes to activate FcγRIIb (Supplementary Figure 4), all the mAb we chose were either human or mouse IgG1 , which give equivalent activity in internalization assays with anti-CD20 mAb (Supplementary figure 1B), to minimise this effect. Using the low, medium and high Fc γRIIb1 transfectants, there was a significant increase in internalization of antiCD19 and anti-CD38 mAb in the presence of Fc γRIIb1, albeit to a lesser extent than with RTX (Figure 2A). The anti-CD19 mAb (RFB9) showed increased internalization with cells expressing Fc γRIIb1 but, unlike RTX, an obvious dose-response to the different levels of expression was not observed. The anti-MHC II, - CD37 and -CD40 mAb remained largely on the cell surface although a slow rate of internalization of anti-MHCII and anti-CD37 in particular was observed. However, all three specificities were unaffected by over-expression of Fc γRIIb1. As expected12,13 the anti-BCR and -CD22 mAb were rapidly internalized independently of Fc γRIIb1 expression. The results suggest that the effect of FcγRIIb expression on internalization is confined to a subset of mAb, most prominently, the type I anti-CD20 mAb. 9


Having established proof of concept using transfectants, we extended our study to primary tumor cells using a cohort of 10 CLL samples. For this analysis we used a blocking anti-Fc γRII mAb (AT10) which we have previously shown capable of impairing the internalization of RTX from Fc γRIIb2transfected cell-lines and CLL cells. 3 However, prior to our experiments on CLL cells, we also assessed the ability of AT10 to block the internalization of mAb targets from Ramos Fc γRIIb1 transfectants (Figure 2B). As expected, the data broadly paralleled that in Figure 1C and Figure 2A and confirmed the ability of AT10 to impair Fc γRIIb1-mediated internalization of RTX, CD19 and CD38 mAb. We then compared the internalization of mAb on CLL cells with and without AT10 (Figure 2C and D). In agreement with previous observations3 and despite the highly variable levels of RTX and AT10 binding observed between patients (Supplementary Table 1), AT10 treatment significantly reduced the RTX internalization (Figure 2C) but had a much smaller, though still significant effect on the internalization of both type II anti-CD20 mAb. There was no effect on internalization of RTX F(ab) 2. Consistent with the results from Fc γRIIb1 transfectants (Figure 2A), AT10 treatment significantly decreased internalization of CD19 and CD38 mAb, suggesting that Fc γRIIb promotes their internalization in primary CLL cells (Figure 2D). Of note, the internalization of the anti-CD37 mAb MB1/7 by CLL cells was also significantly inhibited by AT10 even though expression of FcγRIIb1 had no effect on its internalization by Ramos transfectants. Internalization of mAb specific for CD19, CD22, CD40, MHC Class II (MHCII) and the BCR (IgM)) were unaffected by AT10.

Monoclonal antibody ligation of B-cell surface receptors activates Fc γRIIb

The failure of FcγRIIb to affect the internalization of most mAb, and the subtle effect on the internalization of others led us to speculate whether type I anti-CD20 mAb were unique in their ability to engage and activate Fc γRIIb. We have previously demonstrated that in Daudi cells RTX but 10


not tositumomab induces phosphorylation of Fc γRIIb.3 To investigate whether ligation of other receptors results in Fc γRIIb activation, Raji, Daudi and Ramos Fc γRIIb transfected cells were treated with A488-labelled mAb and probed for phosphorylated Fc γRIIb (pFcγRIIb) (Figure 3A). Most of the mAb displayed some ability to phosphorylate Fc γRIIb but at lower levels than RTX. The anti-MHCII mAb induced pronounced phosphorylation in Raji and Daudi cells but not Ramos-Fc γRIIb transfectants. We have previously observed that Ramos cells express low levels of MHCII and so explored a possible link between expression and ability to phosphorylate Fc γRIIb. In general, the level of phosphorylation corresponded to the level of surface bound mAb (Table 1), with RTX and MHCII mAb binding at high levels (except for MHCII on Ramos cells) and inducing most phosphorylation, and the CD22 mAb the least. Similar results were obtained with primary CLL cells (Figure 3B).

FcγRIIb activation is associated with the level of cell surface mAb binding

Although RTX was not unique in its ability to activate Fc γRIIb, the response was greater and more consistent than with other mAb. This may be due to more mAb bound to the cell surface (Table 1), or alternatively, RTX may be more potent at activating Fc γRIIb. To investigate this, we titrated RTX to provide a comparable level of cell-bound mAb to a range of other mAb (Figure 4A) and repeated probing for pFc γRIIb (Figure 4B). RTX consistently induced more phosphorylation than tositumomab and CD19 mAb, even with the same level of surface staining. LOB 7-4 (CD40) and AT13/5h (CD38) did not always induce phosphorylation of Fc γRIIb (Figures 3 and 4), but when they did, the levels were similar to those seen with RTX (Figure 4). In contrast, IgM and to a lesser extent CD22 mAb induced equivalent or more phosphorylation than RTX suggesting that type I mAb are not inherently more potent at activating Fc γRIIb but that their effect largely reflects surface binding.

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Cell surface-specific mAb interact with Fc γRIIb in cis via bipolar antibody bridging

At the high cell concentrations used in Figures 3 and 4 (4 x 10 6/ml), we have previously shown that cell-cell contact between adjacent cells may occur 3 allowing both cis and trans interactions between surface mAb and Fc γRIIb. We considered whether the susceptibility of type I anti-CD20 mAb to FcγRIIb-mediated internalization may be explained by a unique ability to engage Fc γRIIb in a cis fashion. Daudi cells (natively expressing Fc γRIIb) were labelled with a panel of A488-labelled mAb at an 80 times lower density (5 x 10 4 cells/ml). After extensive washing, these were mixed 1:1 with unlabelled FcγRIIb-ve Ramos cells at a final concentration of 1 x 10 5/ml to minimise cell-cell contact. 3 When probed for pFc γRIIb we found a similar pattern to that observed at high density (compare figure 5A, left panel with Figure 3A), suggesting that all mAb could engage Fc γRIIb in a cis fashion. In the reverse experiment mixing mAb-labelled Fc γRIIb-ve Ramos cells with unlabelled Daudi cells, none of the mAb induced Fc γRIIb phosphorylation (Figure 5A (right panel), confirming that at low cell density they interact with (and phosphorylate) Fc γRIIb through cis-interactions on the cell surface.

Cell surface-specific mAb interact with Fc γRIIb in cis and trans at high cell concentrations

We then determined whether mAb can also interact with adjacent cells in trans. Fc γRIIb-ve Ramos cells were labelled with A488-mAb then washed and mixed with Daudi cells at a final concentration of 4 x 106/ml. In this system phosphorylation is induced by trans interactions between surface mAb on Ramos and Fc γRIIb on Daudi cells. Only RTX induced robust phosphorylation (Figure 5 B, left panel) reflecting the level of mAb binding on Ramos cells. The reverse experiment with A488-labelled Daudi mixed with Ramos was performed as a control (Figure 5 B, right panel).

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In Figure 5A and B, B-cells were at low density or Fc γRIIb was only present on a single cell type, preventing trans and cis interactions from occurring, respectively. Next, we wished to investigate whether cis or trans interactions predominate at high density when Fc γRIIb is present on both populations. To examine if cis interactions predominate we used Ramos cells transfected with a truncated (Tr) version of Fc γRIIb which can bind to IgG Fc but cannot be phosphorylated as it lacks the intracellular domain. Transfectants expressing Tr Fc γRIIb at approximately the same level as Daudi cells (Ramos Tr Fc γRIIb low) or expressing a much higher level (Ramos Tr Fc γRIIb high) (Figure 5C) were used to repeat the experiments in Figure 5B but here pFc γRIIb represents a trans interaction between mAb bound to the Ramos transfectants and Fc γRIIb on Daudi cells. With the exception of RTX, pFcγRIIb was lower with A488-mAb labelled Ramos Tr Fc γRIIb low cells. With the Ramos Tr FcγRIIb high cells pFcγRIIb was also lower in response to RTX, compared with Fc γRIIb-ve Ramos cells (Figure 5B, left panel). These results suggest that cis interactions between Tr Fc γRIIb and surface mAb on Ramos cells were able to compete with trans interactions of Fc γRIIb on Daudi cells. The strong signal in response to RTX in Figure 5D (left panel) may be due to some RTX Fc still being available for trans interaction with Daudi Fc γRIIb after saturation of the low level of surface Tr FcγRIIb; this is supported by the reduced signal with the Tr Fc γRIIb high cells (Figure 5E, left panel).

The level of pFc γRIIb was also lower when mAb-labelled Daudi cells were mixed with Ramos Tr FcγRIIb cells (Figure 5D and E, right panel) suggesting that trans binding of the Fc with Tr Fc γRIIb on the transfectants was able to compete with cis binding on the Daudi cell.

Trans interactions do not inhibit type I anti-CD20 mAb-mediated internalization

Having established that competition from Fc γRIIb expressed in trans impairs cis-mediated Fc γRIIb phosphorylation, we hypothesized that it may affect the Fc γRIIb-mediated internalization of CD20 13


mAb. To investigate this we transfected CD20-ve Fc γRIIb-ve RPMI 8226 cells with Fc γRIIb1 and selected colonies expressing low or high levels (Figure 6A). Cells transfected with empty vector were used as controls. The transfectants were stained with PKH26 and mixed 1:1 with Ramos Fc γRIIb1high cells and cultured with A488-labelled anti-CD20 mAb for 1 hour (Figure 6C). The proportion of mAb remaining on the cell surface of Ramos Fc γRIIb1 high cells (PKH26-ve (Figure 6B)) was compared. RTX internalization was the same in Ramos cells cultured with RPMI 8226 Fc γRIIb1-high cells and Fc γRIIbve controls, inferring that Fc γRIIb1 expressed on adjacent cells (with the potential for trans interaction) does not impair internalization even when expressed at high levels. Furthermore, this lack of effect was seen over a range of RTX concentrations and levels of Fc γRIIb1.

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Discussion

We have previously demonstrated that type I anti-CD20 mAb engage Fc γRIIb by bipolar antibody bridging, resulting in internalization of the mAb:CD20:Fc γRIIb complex.2,3 Here we confirmed that both b1 and b2 isoforms of Fc γRIIb are expressed in malignant B-cells (CLL and MCL) and demonstrated that the native B-cell b1 isoform of Fc γRIIb augments the internalization of RTX in the same manner and to an equivalent extent to Fc γRIIb2. Given that only Fc γRIIb2 has previously been shown to elicit efficient internalization 21,22 this is perhaps surprising. Previous studies examined immune complex internalization whereas here we investigated internalization of mAb:target:Fc γRIIb complexes. These findings indicate that alternative trafficking mechanisms, perhaps not based upon coated pits are required for mAb:target:Fc γRIIb internalization. We are currently studying these mechanisms in further detail. Over-expression of either isoform is therefore able to potentiate the internalization of type I anti-CD20 mAb, rendering them less effective. As previously observed 2,3 type II anti-CD20 mAb were less efficiently internalized than type I. Interestingly, at very high levels of FcγRIIb1 expression (far greater than typically expressed on normal B-cells), internalization of certain type II mAb was induced. The degree of internalization appeared related to both the epitope specificity and isotype of the mAb as GA101 m2a was less readily internalized than GA101 hIgG1 (which interacts more strongly with human Fc γRIIb23) but more readily internalized than tositumomab (m2a), which was only very modestly affected by Fc γRIIb expression. These observations may have implications for further refinements of anti-CD20 mAb.

We also investigated the effect of Fc γRIIb on other mAb specificities. With most mAb, expression of FcγRIIb had no effect on the rate of internalization. The two exceptions were anti-CD19 and antiCD38 mAb which showed small but statistically significant increases in internalization with physiological levels of Fc γRIIb. A second anti-CD19 mAb had similar activity (data not shown) indicating that therapeutic mAb directed at this target may also be reduced in efficacy through this 15


mechanism, particularly in tumors expressing a high level of Fc γRIIb. Of interest, two anti-CD40 mAb (LOB 7-4 and LOB 7-6) remained almost entirely at the cell surface in the presence or absence of FcγRIIb on both CLL cells and Ramos transfectants, suggesting that anti-CD40 mAb may hold promise for directly targeting CD40 expressing tumors as suggested previously. 24 One of these, LOB 7-4 has recently been chimerized and is completing phase I trials. 25

The lack of internalization was not due to an inability of bound mAb to engage Fc γRIIb, because most mAb were able to phosphorylate Fc γRIIb in cis, similar to RTX. In contrast to our previous observations3, we also saw activation of Fc γRIIb in response to tositumomab, albeit at a reduced level than by RTX. Type II mAb bind at approximately half the surface density of type I mAb 26, possibly explaining their weaker signalling. To address this we titrated RTX to achieve equivalent cell surface binding and then repeated our signalling experiments. The results revealed a clear doseresponse effect with less phosphorylation of Fc γRIIb at lower doses of RTX, perhaps explaining why some targets (e.g. CD22) expressed at low levels do not elicit extensive phosphorylation. Certain mAb such as LOB 7-4 and AT13/5h evoked equivalent levels of phosphorylation to RTX, but others including tositumomab, did not. This may be due to the m2a Fc being less able to activate Fc γRIIb than hIgG1 Fc (Supplementary Figure 3). Alternatively, type I and II anti-CD20 mAb have been shown to have different orientations when bound to CD20 27, possibly affecting their ability to interact with and activate Fc γRIIb.

The finding that the internalization of most anti-B cell mAb was not increased by Fc γRIIb whereas most were able to phosphorylate Fc γRIIb, suggests that the ability to activate Fc γRIIb will not predict whether a new therapeutic candidate will remain cell-surface localized. Furthermore, the lack of

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correlation between internalization and phosphorylation of Fc γRIIb may indicate that activation of the receptor is not required for internalization.

At high cell density there was competition for mAb Fc region binding between Fc γRIIb expressed in cis and trans, which may have implications for the success of mAb therapeutics in vivo. Antibody bipolar bridging between mAb bound to herpes simplex virus I antigens and the virally encoded Fc γR, protects infected cells from ADCC. 28,29 Thus, cis FcγRIIb may compete for mAb with activatory Fc γR expressed on NK cells and macrophages in trans, reducing effector activity. Also, cis interactions with FcγRIIb may reduce complement fixation and CDC by competing with C3b for Fc binding as has been demonstrated for Fc γRIII.30,31 As some mAb, including the type I anti-CD20 mAb efficiently fix complement32, inhibition of this activity may further reduce their therapeutic efficacy. Conversely, although our results indicate that trans competition for anti-CD20 mAb by inhibitory FcγR has no effect on the rate of CD20 internalization, it is possible that trans competition by cells expressing higher affinity activatory FcγR may inhibit internalization. However, reduced internalization as a result of trans engagement between type I anti-CD20 mAb on malignant B cells and high affinity activatory FcγR on cells such as monocytes could promote loss of the mAb:CD20 complex via trogocytosis as discussed by Beum et al 33; and may not improve therapeutic efficacy.

There are also other potential downstream effects of Fc γRIIb activation. By virtue of its role as an inhibitory ITIM-bearing receptor, co-ligation with Fc γRIIb often results in termination of downstream signalling34,35, so in situations where agonistic mAb have been chosen for their ability to activate cell surface receptors, co-ligation with Fc γRIIb may be detrimental. Conversely, we 36 and others37 have shown that FcγRIIb can transmit apoptotic signals. It is possible therefore that cis engagement of FcγRIIb by cell surface targeted mAb may contribute to therapy.

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Indirect interactions between the BCR and Fc γRIIb may also play a role in determining the efficacy of type I anti-CD20 mAb therapy. We have previously shown that type I mAb ligation causes CD20 to become physically associated with the BCR, resulting in activation. 20 BCR activation is a prerequisite for antigen processing and loading of peptides onto MHCII 38 and so may promote adaptive antitumor immune responses after type I mAb treatment. 39 However, FcγRIIb expression inhibits BCR activation20,40 reducing antigen-presentation 41, potentially impairing the development of adaptive immune responses upon treatment with RTX.

The data presented here provide evidence that preventing Fc γRIIb engagement has the potential to improve the efficacy of direct-targeting mAb. Thus, engineering the Fc region to reduce binding or blocking FcγRIIb with anti-Fc γRIIb mAb should be considered. mAb-mediated blocking of Fc γRIIb is a particularly promising approach, as it could reduce internalization, increase both ADCC and CDC and potentially augment vaccine effects. In addition, mAb-ligation of Fc γRIIb has been considered as a potential therapy for treating B-cell malignancies in its own right. 42

One area in which FcγRIIb-dependent internalization might have a positive effect on therapeutic efficacy is in the treatment of autoimmune disease. It has been proposed that type I anti-CD20 mAb promote a regulatory B-cell response that can suppress autoimmunity. 43 FcγRIIb is downregulated on B-cells in patients with SLE44, but is upregulated on a subset of regulatory B-cells 45. Therefore FcγRIIb-mediated internalization of CD20 in response to type I mAb-ligation may result in preferential clearance of pathogenic Fc γRIIb-low cells in SLE, whilst sparing Fc γRIIb-high regulatory Bcells, leading to amelioration of autoimmune symptoms. In addition, the negative signalling transduced by Fc γRIIb itself may also be beneficial in the context of autoimmunity. 18


In summary, we have shown that Fc γRIIb expressed in cis and trans can compete for binding to cell surface bound mAb. Although competition from Fc γRIIb expressed in trans does not affect the rate of CD20 internalization, cis-trans competition for Fc binding by Fc γRIIb and other FcγR may affect therapeutic efficacy via other mechanisms.

19


Acknowledgements

We are grateful to I. Henderson, A. Tilbury and K.N. Potter for provision and assistance with clinical material, and R.R French for critical review of the manuscript. We are also grateful to Esther Porée, Giusi Manfredi, Robert Oldham and Sonya James for technical assistance. We thank the Experimental Cancer Medicine Centre (ECMC) funded University of Southampton, Faculty of Medicine Human Tissue Bank (Human Tissue Authority licence 12009) for tissue collection and preparation. We thank David Johnston (Biomedical Imaging Unit, Southampton) for assistance with confocal microscopy. Funding was provided by Leukaemia and Lymphoma Research grants 09009, 12050; CRUK grants C328/A2738 and C328/A2737 and a research grant from Bioinvent international.

Authorship

A.V. performed research, analyzed and interpreted data and wrote the manuscript; C.I., S.H.L., E.L.W., V.S., and A.R. performed research. S.A.B. and C.H.T.C. contributed vital new reagents; B.F. designed research; M.J.G. designed research, analyzed and interpreted data and edited the manuscript; M.S.C. designed research, analyzed and interpreted data and wrote the manuscript with A.V.

Disclosure of Conflicts of Interest

B.F. is a paid employee of Bioinvent International. M.J.G. acts as a consultant to a number of biotech companies to write general antibody expert reports; he receives institutional payments and royalties from antibody patents and licenses. All other co-authors have no conflict of interest to declare. M.S.C. serves as a consultant for Bioinvent International, has received grant funding from them and has previously served as an ad hoc consultant for Roche.

20


References

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Tables

Table 1. Level of binding of fluorochrome–labelled mAb on the surface of B cells (MFI) mAb RTX A488 Tosit A488 MHCII (F3.3) A488 CD38 (AT13/5h) A488 BCR/IgM (M15/8) A488 CD22 (4KB128) A488 CD40 (LOB 7-4) A488 CD19 (RFB9) A488 CD32 (AT10) PE

Ramos

Raji

Daudi

CLL #571

CLL #564

500 226 290 129 358 52 208 92 104

786 313 2142 289 80 51 323 298 97

1017 527 1983 346 107 93 402 365 93

185 80 3504 145 127 55 130 354 128

2037 1624 4314 134 1189 99 140 524 172

24


Figure Legends

Figure 1. Expression and activity of Fc γRIIb1 and b2 isoforms in regulating the rate of internalization of anti-CD20 mAb. (A) mRNA from CLL and MCL samples alongside Ramos Fc γRIIb1 or b2 transfectants as controls, was converted to cDNA and analysed by PCR for the expression of

FcγRIIb1 and FcγRIIb2. (B) Ramos cells were transfected with empty vector, Fc γRIIb2, or FcγRIIb1 and stable transfectants selected expressing different levels of Fc γRIIb. Control cells (filled histogram), FcγRIIb2 low (solid black line), Fc γRIIb1 low, (solid grey line) Fc γRIIb1 medium (dotted line) and FcγRIIb1 high (dashed line) were labelled with AT10-PE and assessed by flow cytometry. (C) Ramos transfectants were cultured with 5µg/ml A488-labelled anti-CD20 mAb for 1 hour. The proportion of total mAb remaining on the cell surface was assessed by flow cytometry after treatment of cells with anti-A488 to quench cell-surface fluorescence. Transfectants were compared with control cells using the Mann-Whitney U test, ** = p

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