Upregulation of FcγRIIb on monocytes is necessary

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Blood First Edition Paper, prepublished online November 13, 2014; DOI 10.1182/blood-2014-08-593061
Upregulation of FcγRIIb on monocytes is necessary to
promote the superagonist activity of TGN1412
Running title: FcγRIIb is required for TGN1412 activity
Khiyam Hussain1, Chantal E. Hargreaves1, Ali Roghanian1, Robert J. Oldham1, H. T.
Claude Chan1, C. Ian Mockridge1, Ferdousi Chowdhury1, Bjorn Frendéus2
Kirsty S. Harper3, Jonathan C. Strefford4, Mark S. Cragg1, Martin J. Glennie1,
Anthony P. Williams5* and Ruth R. French1*
1
Antibody and Vaccine Group, 4Cancer Genomics Group, and 5 Southampton Experimental
Cancer Medicine Centre, Cancer Sciences Unit, Faculty of Medicine, University of
Southampton, Southampton, United Kingdom; 2 Preclinical Research, BioInvent
International AB, Lund, Sweden; 3Huntingdon Life Sciences Ltd, Woolley Road, Alconbury,
Huntingdon, Cambridgeshire, U.K.
Corresponding author: Ruth R. French, MP88, Antibody and Vaccine Group, Cancer
Sciences Unit, Southampton General Hospital, Southampton, SO16 6YD, U.K.
[email protected]; Tel, +44(0)23-81208768; FAX, +44(0)2380704061
Abstract word count: 200 words
Text word count: 3968 words
Figure/table word count: 880
Reference word count: 1094
1
Copyright © 2014 American Society of Hematology
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Key Points
•
Cytokine release syndrome can be screened for with in vitro assays utilising high
density preculture.
•
The mechanism underlying this appears to be upregulation of FcγRIIb expression on
monocytes cultured at high density.
Abstract
The anti-CD28 superagonist TGN1412 caused life threatening cytokine release
syndrome (CRS) in healthy volunteers which had not been predicted by pre-clinical
testing. T cells in fresh PBMCs do not respond to soluble TGN1412, but do respond
following high density (HD) preculture. We show for the first time that this response is
dependent on FcγRIIb expression on monocytes. This was unexpected, since unlike B
cells, circulating monocytes express little or no FcγRIIb. However, FcγRIIb expression
is logarithmically increased on monocytes during HD preculture and this upregulation
is necessary and sufficient to explain TGN1412 potency after HD preculture. B-cell
FcγRIIb expression is unchanged by HD preculture, but they can support TGN1412mediated T-cell proliferation when added at a frequency higher than that in PBMCs.
Although low density (LD) precultured PBMCs do not respond to TGN1412, T cells
from LD preculture are fully responsive when co-cultured with FcγRIIb-expressing
monocytes from HD preculture showing that they are fully able to respond to
TGN1412-mediated activation. Our novel findings demonstrate that cross-linking by
FcγRIIb is critical for the superagonist activity of TGN1412 after HD preculture and
this may contribute to CRS in humans due to the close association of FcγRIIb bearing
cells with T cells in lymphoid tissues.
2
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Introduction
Immunostimulatory monoclonal antibodies (mAb) targeting T-cell co-stimulatory molecules
are an emerging class of therapeutics designed to promote either endogenous or vaccinemediated anti-cancer T-cell immunity. While cited as a major leap forward in the clinical use
of mAb, they are often associated with severe side effects including autoimmunity and
inflammatory reactions resulting from cytokine release syndrome (CRS)1.
CD28 is a key T-cell co-stimulatory molecule on antigen presenting cells (APC) and drives
T-cell activation alongside TCR engagement. There has been a keen focus on the
development of therapeutic anti-CD28 mAb for a range of diseases including autoimmunity
and cancer1-3. However, anti-CD28 mAb suffered a major setback when the first-in-man trial
of TGN1412 caused life-threatening CRS1. TGN1412, a so-called ‘superagonist’, is able to
stimulate T-cell activation without TCR engagement4. Preclinical in vitro testing using
human PBMCs and in vivo testing in cynomolgus macaques failed to predict this toxicity.
Later evaluations revealed that macaques lack CD28 on effector memory T-cells and so were
unable to respond 5. In addition, further species differences were observed in rodent models
where CD28 superagonist mAb had been found to preferentially activate regulatory T cells 3,6.
Following these failures, there has been a concerted effort to develop predictive in vitro
assays that allow a better understanding of the in vivo action of superagonists and their
preclinical prediction. The immobilisation of TGN1412 onto plastic or the addition of antiIgG antibody have been shown to induce cytokine release in PBMC assays 6,7. Both
approaches provide extensive cross-linking of the mAb on the T-cell surface. In assays with
soluble TGN1412, such cross-linking could be provided by co-engagement of the mAb Fc
3
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region with Fcγ receptors (FcγR) expressed on various PBMC cell subsets. The inability of
soluble TGN1412 to mediate cytokine release suggests that unmanipulated PBMCs lack
sufficient capacity to allow TGN1412 to induce T-cell activation. Co-culture of T cells with
human umbilical vein endothelial cells (HUVECs) has also been shown to induce T-cell
activation in response to TGN1412, although the level of cytokine release was low and
surprisingly the interaction of the mAb Fc region with FcγRs was not required 8.
In an interesting alternative approach to demonstrating TGN1412 activity in vitro, Romer et
al9 showed that soluble TGN1412 is able to stimulate cytokine release after preculturing
PBMCs at high density (HD) for 48 hours, and proposed that this was due to an increase in
the ‘tonic activation’ of the responder T cells which decreases their threshold for stimulation.
In our study we have used this HD preculture protocol to investigate the role of FcγRs in the
activation of T cells by a TGN1412. We show that HD preculture induces a remarkable
increase in the expression of FcγRIIb on monocytes, but not B cells, and that this provides
sufficient interactions with the Fc region of TGN1412 to induce T-cell activation. In contrast
to previous studies9,10 our observations indicate that no enhancement of T-cell sensitivity is
required, whereas co-engagement with FcγRIIb is crucial to the agonistic activity of soluble
TGN1412. These findings provide an insight into the cellular and molecular requirements for
superagonistic activity in the context of targeting CD28 with mAb and have important
implications for mAb designed to enhance T-cell responses.
4
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Materials and Methods
Donors and PBMC preparation
Anonymised leukocyte cones were from the National Blood Service (Southampton, UK) and
used within 4 hours for preparation of PBMCs by density gradient centrifugation
(Lymphoprep). Use of human samples was approved by local ethical committee, in
accordance with the Declaration of Helsinki.
Antibodies
OKT3 (ATCC); UCHT1 (eBioscience); 28.1 (Ancell). Anti-FcγR mAb: 10.1 (anti-FcγR1), a
gift from Nancy Hogg (London Research Institute, CRUK, London); E05 and 6G11 (antiFcγRIIa a and b respectively) with Fc regions mutated to eliminate FcγR binding were
produced by BioInvent International AB (Malmo, Sweden) using phage display technology
( 11; Roghanian et al, manuscript in revision; Tutt et al manuscript in preparation).
TGN1412 was produced using published sequences (US patent number US 7,585,960).
Variable regions were sub-cloned into expression vectors (pEE6.4 heavy chain and pEE12.4
light chain, Lonza) containing constant regions of human IgG4. Heavy and light chain
vectors were sub-cloned together before transfection into 293F cells for transient or CHO-K1
cells for stable production. mAb was purified on Protein A-Sepharose and aggregates
removed by gel filtration. Preparations were endotoxin low (<1 ng/mg protein) (EndosafePTS, Charles River Laboratories). Preparation of F(ab’)2 from IgG was as described
previously12.
5
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Cell culture and T-cell proliferation assays
Cell culture was in serum-free medium (CTL-Test™ Medium, CTL Europe GmbH, Bonn)
supplemented with glutamine (2 mM), pyruvate (1 mM), penicillin and streptomycin (100
IU/ml) at 37 °C, 5 % CO2 .
Fresh PBMCs were labelled with 2 μM carboxyfluorescein succinimidyl ester (CFSE). For
HD preculture, cells were cultured in a 24-well plate at 1 x 107/ml as described by Romer et
al9 for 48 hours prior to the stimulation assays. For low density (LD) preculture, cells were
cultured at 1 x 106/ml. For the PBMC stimulation, cells were transferred into roundbottomed 96-well plates at 1 x 105/well. On day 4 cells were labelled with anti-CD8-APC
(Biolegend) and anti-CD4-PE (in-house) and proliferation assessed by CFSE dilution on a
FACSCalibur or FACSCanto (BD Biosciences).
T cell, B cell and monocyte isolation
Cell fractions were isolated from CFSE-labelled PBMCs by negative selection using EasySep
for B cells and T cells (STEMCELL Technologies) and MACS for monocytes (Miltenyi
Biotec).
Cytokine determination
Supernatants were taken 48 hours post-stimulation and cytokines determined using the Vplex Proinflammatory Panel 1 (human) kit (Meso Scale Discovery, Rockville, MD).
Flow cytometry
FcγRIIb on monocytes and B cells was determined using anti-CD19-APC-Cy7, anti-CD14Pacific Blue (BD Biosciences), anti-FcγRIIb-APC and human IgG1 isotype control
6
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(BioInvent International AB). FcγRIIb expression was determined using a FACSCanto™ II
or FACSCalibur and analysed using FCS Express (De Novo Software) or Cellquest (BD
Biosciences).
Western Blot
Monocytes were isolated from HD and LD precultured PBMCs, resuspended in lysis buffer
and processed as previously13. Membranes were probed with rabbit anti-human FcγRIIb
(Abcam), goat anti-rabbit IgG HRP F(ab’)2 and the signal visualised using enhanced
chemiluminescence (GE Healthcare Lifesciences).
Transfection of CHO-K1 cells
CHO-K1 cells were transfected with FcγRIIb in plasmid pcDNA3, selected using 1mg/ml
geneticin (Life Technologies) and screened by flow cytometry using the pan-FcγRII mAb
AT10 F(ab’)2-FITC (in-house). Positive colonies were expanded and sorted using a
FACSAria II (BD Biosciences).
Statistics
Statistical analysis was performed using a 2-tailed Spearman-Rank correlation on Graphpad
Prism version 6 software.
7
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Results
T-cell proliferation and cytokine responses induced by anti-CD28 and anti-CD3 mAb
differ before and after high density preculture
CD28 superagonistic mAb induce polyclonal T-cell activation in vitro, independent of TCR
engagement14,15. TGN1412 was produced from the patented sequence. PBMCs from a large
panel of donors were used to assess T-cell proliferation induced by soluble TGN1412
(hIgG4), a second anti-CD28 superagonist, 28.1 (mIgG1), and the anti-CD3 mAb OKT3
(mIgG2a) and UCHT1 (mIgG1) before and after high density (HD) preculture. With fresh
PBMCs, anti-CD3 mAb induced predominantly CD8+ T-cell division whereas 28.1 induced
predominantly CD4+ T-cell division (Figure 1A). TGN1412 induced a very low or no
response in fresh PBMCs (Figure 1A), as has been reported previously6. However, after
PBMCs were precultured at HD for 48 hours as described by Romer et al9, CD4+ T-cell
division in response to TGN1412 was dramatically increased. The predominantly CD4
response to anti-CD28 mAb may be as a consequence of the differential expression of CD28
on CD4+ and CD8+ T-cells, with only around 50% of CD8+ cells expressing CD28, compared
with most CD4 cells (Supplementary Figure 1).
A comparison of the proliferative responses in fresh and precultured PBMCs from the panel
of donors is shown in Figure 1B. To reflect the predominant T-cell response, in this and
subsequent figures the percentage division refers to CD8 division for anti-CD3 mAb and
CD4 division for anti-CD28 mAb. There was considerable variation between donors in the
level of proliferation induced by TGN1412 and the anti-CD3 mAb, with a small proportion of
donors showing poor responses.
Supernatants were taken after 48 hours for measuring IFNγ, IL-2, IL-10, IL-6, IL-8 and
TNFα (Figure 2) and these confirmed the release of inflammatory cytokines in response to
8
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TGN1412 after HD preculture, with the level and range of TNFα and IFNγ release
comparable to those reported previously9. Thus our proliferation assays showed the same
response profile as shown by the measurement of cytokine release with the additional
discrimination of T-cell subset responsiveness. As a consequence we used T-cell
proliferation as a robust surrogate readout for cytokine release to assess the functional
requirements of TGN1412.
It has previously been shown that the function of anti-CD3 mAb is dependent on their
interaction with FcγRs16. To investigate the requirement of FcγR interactions for TGN1412
activity, F(ab’)2 was produced free from contaminating IgG. Biacore analysis confirmed that
its binding to CD28 matched that of the parent IgG (Supplementary Figure 2). The failure of
TGN1412 F(ab`)2 to induce T-cell proliferation in (Figure 1B middle panel) indicated that
Fc:FcγR interaction was probably necessary for activity. This was reinforced by the
inability of TGN1412 IgG to induce responses in T cells isolated following HD preculture
(Figure 1B right panel). In contrast, the anti-CD28 superagonist 28.1 induced proliferation in
isolated T cells, indicating that this mAb does not require interaction with FcγRs on accessory
cells, as previously reported17.
CD28 expression on CCR7- effector memory T cells did not alter during HD preculture
(Supplementary Figure 3) and therefore we next investigated the role of specific FcγRs in
facilitating TGN1412 activity.
Inhibition of T-cell proliferation by anti-FcγR mAb
To investigate the requirement for FcγR co-engagement by TGN1412, we used a panel of
anti-FcγR mAb to block specific FcγR:Fc interactions; anti-FcγRI and, anti-FcγRIII, and two
9
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novel anti-FcγRII mAb, E05 and 6G11, binding to FcγRIIa and FcγRIIb, respectively. E05
and 6G11 were engineered with the N297Q mutation to abrogate binding of their Fc region to
FcγRs (11; Tutt et al. manuscript in preparation). The observed inhibition of the responses to
OKT3 and UCHT1 was in agreement with their known FcγR requirements 16,18: OKT3
(mIgG2a) requires FcγRI, whereas UCHT1 (mIgG1) requires FcγRIIa (Figure 3A). In
contrast, we found that TGN1412 activity was inhibited by the anti-FcγRIIb mAb 6G11
(Figure 3A) and this was confirmed in 8 donors (Figure 3B).
Monocytes and B cells from high density cultures confer responsiveness to TGN1412
To investigate which immune cells were capable of restoring TGN1412-responsiveness to
isolated T cells, T cells from HD precultured PBMCs were co-cultured with monocytes or B
cells from the same HD precultures, and their response to anti-CD3 mAb and TGN1412
determined. Under these conditions monocytes restored the responsiveness of T cells to
OKT3, UCHT1 and TGN1412; at a monocyte:T cell ratio of 0.3:1, the proliferation was
comparable with that in total PBMCs (Figure 3C). In contrast, B cells isolated from HD
preculture were unable to restore the activity of OKT3 and UCHT1, but did restore activity to
TGN1412 (Figure 3C). The restoration of TGN1412 responsiveness with both monocytes and
B cells was almost totally blocked by anti-FcγRIIb mAb (Figure 3C). The ability of
monocytes to confer responsiveness to OKT3 and UCHT1 is consistent with their expression
of FcγRI and FcγRIIa and the requirement of cross-linking by these FcγR. In contrast, B cells
express only FcγRIIb so are unable to restore responsiveness to OKT3 and UCHT1. In donors
showing a medium-high response to TGN1412, a titration of monocytes or B cells from HD
PBMCs into isolated T cells showed that whereas with B cells the threshold for a TGN1412
10
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response was not reached until a B:T cell ratio of more than 0.1:1, monocytes induced a
response at a ratio of 0.02:1 (Figure 3D).
Expression of FcγRIIb is increased on high density precultured monocytes
The finding that the TGN1412-response could be restored by monocytes as well as B cells,
and that this response was blocked by anti-FcγRIIb was unexpected as FcγRIIb is expressed
on only a fraction of monocytes and at very low density19. We then compared the expression
of FcγRIIb on B cells and monocytes from fresh and HD precultured PBMCs. FcγRIIb
expression was similar on fresh and HD B cells. In contrast, we found that FcγRIIb
expression on monocytes was up to 50-fold greater post HD preculture compared with
monocytes in fresh PBMCs (Figure 4A); when PBMCs were precultured at LD, FcγRIIb on
monocytes was only modestly increased (Figure 4A). This increase in FcγRIIb expression
was detectable by 10 hours of HD preculture and reached a maximum by 36 hours (Figure
4B). Western blotting confirmed that the upregulation of FcγRIIb was due to the increased
expression of FcγRIIb2 isoform (Figure 4C).
Analysis of the monocyte expression of the other FcγRs showed that FcγRIIa and FcγRI were
unchanged by preculture, whereas FcγRIIIa was high on a small population of monocytes
from fresh PBMCs, but at an intermediate density on most monocytes after preculture (data
not shown).
The upregulation of FcγRIIb was seen consistently over the panel of donors (Figure 4D and
Supplementary Figure 4). It should be noted that even in donors showing the lowest FcγRIIb
expression after HD preculture there was still at least a 10-fold increase relative to fresh
monocytes. There was a statistically significant positive correlation between FcγRIIb
expression on monocytes and T-cell proliferation and TNFα release (Figure 4E). Together
11
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with the blocking results (Figure 3), these data explain how monocytes from HD precultures
are able to engage with and cross-link TGN1412 through FcγRIIb.
T cells do not require high density preculture to respond to TGN1412 in the presence of
high density precultured monocytes
It has previously been suggested that changes in the activation state of T cells themselves
might be responsible for the responsiveness to TGN1412 after HD preculture9. We next
examined whether the changes we observed in the monocytes alone were sufficient. T cells
were isolated from PBMCs after HD preculture and their response to TGN1412 in the
presence of monocytes from autologous HD and LD precultured PBMCs was compared
(Figure 5A). With monocytes from HD precultures, TGN1412 induces T-cell proliferation
comparable with that in total PBMCs whereas it induces only a very low level of stimulation
with monocytes from LD preculture. In contrast, T cells responded to OKT3 with both LD
and HD precultured monocytes, and in many donors responses were higher with the LD
monocytes. We also determined the response of T cells isolated from PBMCs after LD
preculture in the presence of the HD and LD monocytes (Figure 5B). Importantly, with
monocytes from HD preculture, T cells from LD preculture gave responses to TGN1412 that
were comparable with those of the HD T cells, but they gave no response with monocytes
from LD preculture. In both cases, the response was blocked by anti-FcγRIIb mAb. These
results strongly point to the ability of T cells to respond to TGN1412 after HD preculture
being primarily due to an increase in t20he availability of FcγRIIb on monocytes, rather than
an increase in the responsiveness of the T cells themselves. This was supported by the
finding that T cells isolated from fresh PBMCs were responsive to TGN1412 in the presence
of autologous HD but not LD precultured monocytes (Supplementary Figure 5).
Fresh T cells respond to TGN1412 in the presence of sufficient FcγRIIb
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In Figure 3 we showed that when co-cultured with B cells at a high B:T cell ratio, T cells
isolated from HD preculture were responsive toTGN1412 . Since the level of FcγRIIb on B
cells is unchanged by HD preculture (Figure 4), we next determined whether fresh T cells
were also responsive to TGN1412 when co-cultured with B cells at a similarly high ratio.
The results in Figure 6A show clearly that in the presence of B cells, fresh T cells were
responsive to TGN1412. In contrast fresh T cells did not respond to TGN1412 when cocultured with fresh monocytes, consistent with the monocytes lack of FcγRIIb. However,
these monocytes were able to induce a T-cell response to OKT3, consistent with its
requirement for FcγRI.
Like monocytes in fresh PBMCs, those in LD precultured PBMCs express little FcγRIIb
(Figure 4A). Titrating monocytes or B cells from HD preculture into LD precultured PBMCs
restored the T-cell mitogenic capacity of TGN1412 (Figure 6B), showing that the
unresponsiveness to TGN1412 was due to a lack of FcγRIIb availability in the LD
precultures. Furthermore, in comparison to T cells co-cultured with untransfected CHO-K1
cells, marked T-cell proliferation, IL-2, IFNγ and TNFα release were observed in response to
TGN1412 when T cells were co-cultured with CHO-K1 cells transfected with FcγRIIb
(Figure 6C and 6D).
Discussion
In this study we show that HD preculture of PBMCs induces an exponential increase in the
expression of FcγRIIb by monocytes, and that this is a prerequisite for mediating the
superagonistic activity of TGN1412 in vitro. Our findings add to the growing body of
evidence showing the importance of Fc-FcγRIIb interactions for the activity of agonistic mAb
13
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targeting TNFR superfamily members such as CD40, TNF-related apoptosis-inducing ligand
(TRAIL) and death receptor 5 (DR5), where Fc-FcγRIIb interactions are crucial for activity2123
. In these experiments we did not observe any change in the sensitivity of the T cells
themselves to TGN1412 following HD preculture, in contrast to previous studies which
suggested the HD preculture resulted in enhanced tonic TCR signalling causing a reduction in
the activation threshold of the T cells in vitro24,25. On-going studies show that other
immunostimulatory mAb, including anti-OX40 and -4-1BB, show similar HD preculture
dependency for increased activatory activity (data not shown) suggesting that this assay will
have considerable utility for predicting the potency and perhaps toxicity for many FcγRIIbdependent agonistic mAb.
Lühder et al, proposed that anti-CD28 superagonists oligomerize CD28 on the T-cell surface
and that the position of the recognised epitope influences the proximity of intracellular
effector molecules leading to TCR-independent T-cell activation4. We postulate that this
increase in FcγRIIb following HD preculture enables efficient clustering of CD28 on the Tcell surface which presumably mimics the activity of CD80/CD86 at the APC: T cell synapse
leading to T-cell activation.
In our experiments TGN1412 F(ab’)2 failed to induce T-cell proliferation or cytokine release
in accordance with findings by Ball et al 26. However, a recent study reported that TGN1412
F(ab’)2 still induces residual intracellular TNFα expression in T cells at 0.5ug/mL10. The
reason for this discrepancy is unclear. Taken together, these findings suggest that TGN1412
should be defined as an FcγR-dependent superagonist. In contrast, the activity of a second
anti-CD28 mAb, 28.1, appears to be independent of FcγR interaction and so should be
considered a “true” superagonist.
14
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Our data supports a mechanism by which HD monocytes mediate TGN1412 activity as a
result of their dramatically increased FcγRIIb expression and the extent of proliferation and
cytokine release correlates with the level of FcγRIIb. This upregulation was observed on both
monocytes from HD PBMC cultures and on HD precultured isolated monocytes (data not
shown). FcγRIIb expression on HD monocytes was higher than on HD B cells (Figure 4B)
and predominantly of the b2 isoform (Figure 4C). In vitro, both IL-4 and IL-10 have been
shown to drive the upregulation of FcγRIIb on monocytes although they increased expression
of both the b1 and b2 isoforms27-29. However, we did not see any increase in the levels of
IL-4 or IL-10 when comparing supernatants from LD and HD precultures (data not shown)
and it remains to be seen what is responsible for the increase in monocyte FcγRIIb expression.
In the presence of monocytes from HD precultures, T cells from LD precultures gave
responses to TGN1412 that were comparable with those of HD T cells. This strongly
suggests that the HD preculture enhances responses through a direct effect upon the
monocyte population independent of any change in the responsiveness of the T cells
themselves9. This is further supported by experiments using fresh PBMCs in which T cells
were responsive to TGN1412 in the presence of autologous HD but not LD monocytes
(Supplementary Figure 5). This is in agreement with a recent study in which cross-linking
anti-IgG modified responsiveness to TGN1412 irrespective of whether the T cells were
isolated from HD or LD PBMCs10. Together these observations suggest that enhanced tonic
TCR signalling during HD preculture24,25 is not critical for responsiveness to TGN1412 in
vitro. However, our observations are in accordance with previous findings in which
monocyte maturation in HD PBMC cultures mediates full T-cell responsiveness to
TGN14129.
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FcγRIIb is the only ITIM-bearing FcγR. Although generally inhibitory, it may also act as a
positive regulator independent of any signalling activity by providing cross-linking and
confers activity for several agonistic mAbs (reviewed in20). Here, we show a similar FcγRIIb
requirement for T-cell proliferation and cytokine release in response to TGN1412. Although
human IgG4 mAb show little or no binding to FcγRIIb as monomers, Bruhns et al have
shown they are able to bind as immune complexes30; their binding to FcγRIIb when presented
as an array on the T-cell may be similarly favourable. Whether this requirement is due to
some inherent property of FcγRIIb or whether any FcγR would serve this function if
expressed at the right level, the right time, and with high enough IgG binding affinity, was
discussed recently20. Work with CD40 has shown that simply cross-linking the receptors is
sufficient for activity and that no downstream signalling is required 31,32,33. Whether the same
holds true for TGN1412 and CD28 remains to be confirmed.
Although in HD precultured PBMCs TGN1412 activity was dependent on monocytes,
activity could also be mediated by B cells when added at a high enough frequency. However,
monocytes were more active on a cell:cell basis. While this may be due to the higher
expression of FcγRIIb on monocytes, it may be associated with qualitative differences
between the two cell types. One intrinsic difference is their expression of alternate FcγRIIb
isoforms, with B cells expressing predominantly b1 and monocytes b2 (Figure 4C). The b1
isoform prevents association with clathrin-coated pits and internalization when co-ligated
with the BCR34,35. In contrast, the b2 isoform localises to clathrin-coated pits and adopts a
more clustered appearance (Manfredi et al., unpublished data). It is possible that the more
clustered b2 isoform on monocytes is a more efficient cross-linker giving more potent CD28
signalling.
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Peripheral blood comprises 1-7% B and 7-24% T cells, however, in lymphoid tissues the B:T
cell ratio may be as high as 2:1. Pre-clinical mAb testing typically relies on PBMCs in which
the FcγR availability and distribution is very different to that in lymphoid tissues (Tutt et al.,
manuscript in preparation). Increasing the B:T cell ratio of PBMCs to 0.5:1 restored
TGN1412 activity regardless of their preculture status indicating that in conventional PBMC
cultures the availability of FcγRIIb is insufficient purely due to low B cell frequency, rather
than to the FcγRIIb expression per se. This is supported by the observation that culturing T
cells isolated from fresh PBMCs with FcγRIIb-transfected CHO-K1 cells also restored
TGN1412 activity.
In the TGN1412 clinical trial the most pronounced feature was the early onset of respiratory
distress and pulmonary infiltrates1. It is plausible that the tissue architecture of the bronchialassociated lymphoid tissue, in which large numbers of FcγRIIb-expressing B cells reside in
close proximity to T cells, provided the ideal environment for the rapid cross-linking of
TGN1412 leading to T-cells activation and associated events. Other lymphoid organs such as
the spleen and lymph nodes show similar architecture in which B cell rich areas such as
germinal centres and primary follicles are surrounded by T cell dense periarteriolar lymphoid
sheaths and therefore these may be additional sites in which TGN1412 rapidly induces T-cell
activation.
Our study offers an insight on how TGN1412 may be cross-linked in vivo leading to adverse
events and has implications for in vitro testing of a wider range of therapeutic mAb targeting
T cell co-stimulatory molecules. Furthermore, we propose that TGN1412 can be specifically
re-defined as an FcγR-dependent superagonist, and together our observations indicate that
FcγRIIb-targeted Fc-engineering of mAb such as TGN1412 which target T-cell costimulatory molecules may be employed in order to improve their safe clinical use.
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Author contributions
K.H. designed and performed experiments, analysed and interpreted results, wrote paper;
C.E.H., A.R., C.I.M. and R.J.O. performed experiments; H.T.C.C. and B.F. produced and
provided vital reagents; F.C., K.S.H. and J.C.S. advised on assays and useful discussion;
M.S.C. and M.J.G. designed research, evaluated results and edited paper; A.P.W. designed
research and wrote paper; R.R.F. designed and performed experiments, analysed and
interpreted results, prepared figures and wrote paper.
The authors declare no competing financial interests
Acknowledgements
We thank Christine Penfold, Jinny Kim and Elizabeth Potter for their excellent technical
support.
This work was funded by Cancer Research UK, Leukaemia & Lymphoma Research grants
08014 and 12050 and the UK National Centre for the Replacement, Refinement and
Reduction of Animals in Research (NC3Rs) CRACK IT Programme. R.J.O. is the recipient
of a MRC-funded CASE studentship with HLS.
18
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Figure legends
Figure 1 T-cell proliferation in response to anti-CD3 and anti-CD28 mAb with fresh
and precultured PBMCs. Freshly prepared PBMCs were CFSE-labelled and used in
proliferation assays either immediately or after HD preculture for 48 hours. A) and B)
PBMCs were incubated with OKT3 and UCHT1 (anti-CD3; 0.1 μg/ml), TGN1412 (5 μg/ml),
TGN1412 F(ab’)2 ( 5 μg/ml, precultured PBMCs only) and CD28.1 superagonist (SA) (1
μg/ml) for 4 days. Cells were then labelled with anti-CD8-APC and anti-CD4-PE mAb and
analysed by flow cytometry to determine proliferation by CFSE-dilution. Results are
expressed as the percentage of cells having undergone one or more divisions. A) shows
representative dot plots and B) the responses from a panel of donors; n= 5 to 7 for fresh
PBMCs and n=30 for precultured PBMCs. Proliferation of T cells in response to OKT3 and
UCHT1 (anti-CD3; 0.1 μg/ml), TGN1412 and CD28.1 SA (anti-CD28; 5 and 1 mg/ml
respectively) was determined as in A)
Figure 2 Cytokine release in response to anti-CD3 and anti-CD28 mAb with fresh and
precultured PBMCs.
Supernatants from assays described in Figure 1 were taken at 48
hours for the determination of cytokine concentrations using the using the V-plex
Proinflammatory Panel 1 (human) kit (Meso Scale Discovery, Rockville, MD); n=5 for fresh
and n=7 for HD precultured PBMCs.
Figure 3 TGN1412 requires interaction with FcγRIIb for activity and this can be
provided by B cells and monocytes. A) T-cell proliferation in response to OKT3, UCHT1
and TGN1412 in the presence of anti-FcγRI, IIa, IIb, and IIIa mAb. PBMCs were incubated
with stimulating mAb as described in Figure 1 in the absence or presence of anti-FcγRI (10.1),
IIa (E05), IIb, (6G11), and IIIa (3G8). blocking mAb (50 μg/ml). Error bars show mean and
22
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range of duplicate wells. B) Effect of anti-FcγRIIa and IIb on response to TGN1412.
Results are expressed as % of division with TGN1412 alone and error bars represent mean
and SD from 7 donors. C) Responsiveness of isolated T cells to TGN1412 is restored by B
cells and monocytes. T cells, monocytes and B cells were isolated from PBMCs after 48 h
HD preculture. T cells were incubated with OKT3, UCHT1 and TGN1412 either alone, or in
co-culture with B cells or monocytes at a monocyte or B cell:T cell ratio of 0.3:1; TGN1412
responses were determined in the absence or presence of anti-FcγRIIb blocking mAb (50
μg/ml). Results show mean and range of 2 donors. D) B cells and monocytes were added to
isolated T cells at mono or B:T cell ratios from 0.02:1 to 0.5:1. Results show mean and
range from 2 donors.
Figure 4 FcγRIIb expression on monocytes is up-regulated during HD preculture. A)
Histograms showing expression of FcγRIIb on monocytes from fresh PBMCs, and taken after
18 and 36 hours of HD preculture, and 36 hours of LD preculture, and on B cells from fresh
PBMCs and after HD preculture. B) Time course of the increase in FcγRIIb expression on
monocytes during HD preculture. In contrast, B cells show no change in FcγRIIb expression
during HD preculture. Bars show mean and range of 2 donors. Analysis in A and B was
using a FACSCantoII. C) Western blot showing an increase in the expression of the b2
isoform of FcγRIIb during HD preculture. D) Comparison of FcγRIIb expression on fresh and
HD precultured monocytes. E) Plots showing relationship between FcγRIIb expression on
monocytes after HD preculture and proliferation and TNFα release in response to TGN1412;
n=14, analysed using 2-tailed Spearman-Rank correlation (Graphpad Prism 6). FcγRIIb
expression and proliferation plots from all donors are shown in Supplementary Figure 4.
Analysis in D and E was using a FACSCalibur.
23
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Figure 5 T cells respond to TGN1412 in the presence of monocytes from high density
preculture. CFSE-labelled PBMCs were cultured either at HD or LD for 48 hours. T cells
and monocytes were then isolated from each culture. A) T cells isolated from HD preculture
were co-cultured with monocytes isolated from LD (light bars) and HD (dark bars) cultures in
the presence of OKT3 or TGN1412 at a monocyte:T cell ratio of 0.3:1. T-cell division was
determined on day 4. Responses to TGN1412 were determined in the absence and presence
of anti-FcγRIIb mAb. B) As in A) but with T cells isolated from LD preculture. The results
show mean and SD from 3 donors.
Figure 6 T cells from fresh and LD cultured PBMCs respond to TGN1412 in the
presence of sufficient FcγRIIb A) T and B cells and monocytes were isolated from fresh
PBMCs. T cells were then cultured with OKT3 or TGN1412 either alone or in co-culture
with B cells or monocytes at a B cell or monocyte:T cell ratio of 0.3:1. T-cell division was
determined on day 4. B) LD precultured PBMCs were co-cultured with B cells or monocytes
isolated from HD preculture at B cell or monocyte:T cell ratios of 0.02:1 to 1:1 in the
presence of TGN1412. Division was determined on day 4. Results show mean and range
from 2 donors. C) FcγRIIb expression on transfected CHO-K1 cells. D) T cells isolated from
fresh PBMCs were co-cultured with untransfected or FcγRIIb transfected CHO-K1 cells and
TGN1412. Supernatant was taken for cytokine determination at 48 hours, and proliferation
determined on day 4. Results from 5 donors are shown.
24
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Prepublished online November 13, 2014;
doi:10.1182/blood-2014-08-593061
Upregulation of FcγRIIb on monocytes is necessary to promote the
superagonist activity of TGN1412
Khiyam Hussain, Chantal E. Hargreaves, Ali Roghanian, Robert J. Oldham, H.T. Claude Chan, C. Ian
Mockridge, Ferdousi Chowdhury, Bjorn Frendéus, Kirsty S. Harper, Jonathan C. Strefford, Mark S. Cragg,
Martin J. Glennie, Anthony P. Williams and Ruth R. French
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