Stephen A Beers

University of Southampton, Southampton, England, United Kingdom

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Publications (34)245.88 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Therapeutic antibodies have transformed cancer therapy, unlocking mechanisms of action by engaging the immune system. Unfortunately, cures rarely occur and patients display intrinsic or acquired resistance. Here, we demonstrate the therapeutic potential of targeting human (h) FcγRIIB (CD32B), a receptor implicated in immune cell desensitization and tumor cell resistance. FcγRIIB-blocking antibodies prevented internalization of the CD20-specific antibody rituximab, thereby maximizing cell surface accessibility and immune effector cell mediated antitumor activity. In hFcγRIIB-transgenic (Tg) mice, FcγRIIB-blocking antibodies effectively deleted target cells in combination with rituximab, and other therapeutic antibodies, from resistance-prone stromal compartments. Similar efficacy was seen in primary human tumor xenografts, including with cells from patients with relapsed/refractory disease. These data support the further development of hFcγRIIB antibodies for clinical assessment. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cancer cell 04/2015; 27(4):473-88. DOI:10.1016/j.ccell.2015.03.005 · 23.89 Impact Factor
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    ABSTRACT: Following the success of rituximab, two other anti-CD20 monoclonal antibodies (mAb) ofatumumab and obinutuzumab have entered clinical use. Ofatumumab has enhanced capacity for complement dependent cytotoxicity (CDC) whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glyco-engineered for augmented antibody dependent cellular-cytotoxicity (ADCC). We previously showed that type I mAb such as rituximab have a propensity to undergo enhanced antigenic modulation compared to type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20 (hCD20) expressing B-cells in the mouse less effectively than glyco-engineered and WT forms of obinutuzumab, particularly when human IgG1 (hIgG1) mAb were compared. In contrast to mouse IgG2a (mIgG2a), hIgG1 mAb were ineffective in ADCC assays with murine NK cells as effectors, whereas ADCP was equivalent for mIgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo.
    Blood 01/2015; 125(12). DOI:10.1182/blood-2014-07-588376 · 10.43 Impact Factor
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    ABSTRACT: Type I anti-CD20 mAb such as rituximab and ofatumumab engage with the inhibitory FcγR, FcγRIIb on the surface of B cells, resulting in immunoreceptor tyrosine-based inhibitory motif (ITIM) phosphorylation. Internalisation of the CD20:mAb:FcγRIIb complex follows, the rate of which correlates with FcγRIIb expression. In contrast, although type II anti-CD20 mAb such as tositumomab and obinutuzumab also interact with and activate FcγRIIb, this interaction fails to augment the rate of CD20:mAb internalisation, raising the question of whether ITIM phosphorylation plays any role in this process. We have assessed the molecular requirements for the internalisation process and demonstrate that in contrast to internalisation of IgG immune complexes, FcγRIIb-augmented internalisation of rituximab-ligated CD20 occurs independently of the FcγRIIb ITIM, indicating that signalling downstream of FcγRIIb is not required. In transfected cells, activatory FcγRI, FcγRIIa and FcγRIIIa augmented internalisation of rituximab-ligated CD20 in a similar manner. However, FcγRIIa mediated a slower rate of internalisation than cells expressing equivalent levels of the highly homologous FcγRIIb. The difference was maintained in cells expressing FcγRIIa and FcγRIIb lacking cytoplasmic domains and in which the transmembrane domains had been exchanged. This difference may be due to increased degradation of FcγRIIa, which traffics to lysosomes independently of rituximab. We conclude that the cytoplasmic domain of FcγR is not required for promoting internalisation of rituximab-ligated CD20. Instead, we propose that FcγR provide a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 01/2015; 290(9). DOI:10.1074/jbc.M114.593806 · 4.60 Impact Factor
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    ABSTRACT: Monoclonal antibody (mAb) drugs that stimulate antitumor immunity are transforming cancer treatment but require optimization for maximum clinical impact. Here, we show that, unlike other immunoglobulin isotypes, human IgG2 (h2) imparts FcγR-independent agonistic activity to immune-stimulatory mAbs such as anti-CD40, -4-1BB, and -CD28. Activity is provided by a subfraction of h2, h2B, that is structurally constrained due its unique arrangement of hinge region disulfide bonds. Agonistic activity can be transferred from h2 to h1 by swapping their hinge and CH1 domains, and substitution of key hinge and CH1 cysteines generates homogenous h2 variants with distinct agonistic properties. This provides the exciting opportunity to engineer clinical reagents with defined therapeutic activity regardless of FcγR expression levels in the local microenvironment. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cancer Cell 12/2014; 27(1). DOI:10.1016/j.ccell.2014.11.001 · 23.89 Impact Factor
  • Immunology 12/2014; 143:124-124. · 3.74 Impact Factor
  • T. Tipton, M. S. Cragg, S. A. Beers
    Immunology 12/2014; 143:131-131. · 3.74 Impact Factor
  • Immunology 12/2014; 143:128-129. · 3.74 Impact Factor
  • Immunology 12/2014; 143:125-126. · 3.74 Impact Factor
  • Immunology 12/2014; 143:138-139. · 3.74 Impact Factor
  • Immunology 12/2014; 143:139-139. · 3.74 Impact Factor
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    ABSTRACT: Immunomodulatory mAbs, led by the anti-CTLA4 mAb ipilimumab, are an exciting new class of drugs capable of promoting anticancer immunity and providing durable control of some tumors. Close analysis of a number of agents has revealed a critical yet variable role for Fcγ receptors in their efficacy. In this article, we reveal that agonistic anti-CD40 mAbs have an absolute requirement for cross-linking by inhibitory FcγRIIB when used systemically to treat established BCL1 syngeneic lymphoma, and therapy is lost when using a mouse IgG2a mAb not cross-linked by FcγRIIB. Furthermore, in FcγRIIB-deficient mice the lymphoma itself can provide FcγRIIB to cross-link anti-CD40 on neighboring cells, and only when this is blocked does therapy fail. The dependence on FcγRIIB for immunostimulatory activity was not absolute, however, because when anti-CD40 mAbs were administered systemically with the TLR3 agonist polyinosinic:polycytidylic acid or were given subcutaneously, activatory FcγR could also provide cross-linking. Using this mechanistic insight, we designed multimeric forms of anti-CD40 mAb with intrinsic FcγR-independent activity that were highly effective in the treatment of lymphoma-bearing mice. In conclusion, FcγR-independent anti-CD40 activation is a viable strategy in vivo. These findings have important translational implications, as humans, unlike mice, do not have IgG that binds strongly to FcγRIIB; therefore FcγR-independent derivatives represent an attractive therapeutic option.
    The Journal of Immunology 07/2014; 193(4). DOI:10.4049/jimmunol.1303204 · 5.36 Impact Factor
  • Ann L White, Stephen A Beers, Mark S Cragg
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    ABSTRACT: Fc gamma Receptor (FcγR) IIB (CD32B) is an immunoreceptor tyrosine inhibitory motif (ITIM)-bearing Fc receptor that is involved in abrogating the signalling and function delivered from other receptors; archetypally those arising from other, activatory, FcγR and from the B cell receptor (BCR) for antigen. In the context of immunotherapy, it has convincingly been shown to limit a variety of clinically important therapeutic monoclonal antibodies (mAb) such as rituximab and trastuzumab in preclinical models. However, recent exploration of so-called immunomodulatory mAb, for example agonist mAb directed against various members of the TNFR super-family, has cast new light on the ability of FcγRIIB to regulate immune responses and immunotherapy. These data, accumulated by several independent groups, have shown the seemingly paradoxical ability of FcγRIIB to augment or even be absolutely required for the activity of this class of mAb. In this review we highlight the key role of FcγRIIB in regulating agonistic mAb, detail the likely mechanism of action and propose new ways in which this information may be exploited therapeutically.
    Current topics in microbiology and immunology 01/2014; 382:355-72. DOI:10.1007/978-3-319-07911-0_16 · 3.47 Impact Factor
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    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 Fcγ receptor, FcγRIIb, in a bipolar configuration. Here we investigated whether mAb directed at other B-cell surface receptors also engaged FcγRIIb and whether this interaction promoted internalization. Most mAb engaged and activated FcγRIIb, 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 FcγRIIb were evident, but trans interactions did not inhibit type I anti-CD20 mAb-mediated internalization. These data identify that FcγRIIb is engaged by many mAb in both cis and trans configurations, triggering its activation, but that internalization via FcγRIIb occurs for only a select subset. These findings have implications when designing new antibody-based therapeutics.
    Blood 11/2013; 123(5). DOI:10.1182/blood-2013-04-490821 · 10.43 Impact Factor
  • Stephen A Beers, Martin J Glennie
    Blood 10/2013; 122(18):3093-4. DOI:10.1182/blood-2013-09-525451 · 10.43 Impact Factor
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    ABSTRACT: Genetic deficiency of the inhibitory Fc receptor, FcγRIIB (CD32b), has been shown to augment the activity of activatory FcγR and promote mAb immunotherapy. To investigate whether mAbs capable of blocking FcγRIIB have similar capacity, we recently generated a panel of specific anti-mouse FcγRIIB mAbs that do not cross-react with other FcRs, allowing us to study the potential of FcγRIIB 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 phagocytosis. However, in a variety of murine tumor models, anti-FcγRIIB mAbs demonstrated limited therapeutic activity despite optimized treatment regimens. Unexpectedly, we observed that the anti-FcγRIIB 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-FcγRIIB mAbs become extensively internalized from the cell surface within 24 h in vivo, likely explaining their suboptimal efficacy. Subsequent studies revealed that anti-FcγRIIB mAb immunotherapy was effective when used against FcγRIIB(+) tumors in FcγRIIB(-/-) 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 09/2013; 191(8). DOI:10.4049/jimmunol.1301430 · 5.36 Impact Factor
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    ABSTRACT: Atomic Force Microscopy (AFM) has been used to interrogate the bio-chemical and interfacial mechanical properties of cells surfaces[1]. However, the surface nature, micro and nanoscale cell movement often limits AFM imaging and sensing, particularly for suspension cells. To improve AFM probing of these cells, substrates that improve surface to cell adhesion and cell trapping microstructures are recommended. This work investigates three different fabrication processes of glass and SU-8 polymer-based materials to provide a low cost cell trapping template platform suitable for suspension cells such as malignant Ramos B cells. In this study, micro-cavity templates were fabricated on 150 mm diameter glass substrates using three different techniques, i.e. hydrofluoric (HF) wet etch, inductively coupled plasma (ICP) etch of using Octafluorocyclobutane/Trifluoromethane/Oxygen gas chemistry and photolithography pattern transfer to SU-8 3005 series polymer[2]. The target thickness for this template was 5 µm with an array of micro-cavity diameters ranging from 15 to 25 µm. These dimensions were compatible for the suspension cells of interest. Initial fabricated micro-cavities using the HF etch was unacceptable as the cavities' undercut was ~50 µm for a 5 µm depth. The templates fabricated using 5 µm thick SU-8 and ICP etch technique had good sidewall profiles and were then used to trap the test lymphocyte suspension cells.
    7th International Conference on Materials for Advanced Technologies (ICMAT), Singapore; 06/2013
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    ABSTRACT: Isotype plays a crucial role in therapeutic monoclonal antibody (mAb) function, mediated in large part through differences in Fcγ receptor (FcγR) interaction. Monoclonal Abs such as rituximab and alemtuzumab, which bind target cells directly, are designed for efficient recruitment of immune effector cells through their activatory FcγR engagement to mediate maximal target cell killing. In this setting, binding to inhibitory FcγRIIB is thought to inhibit function, making mAbs with high activatory/inhibitory (A/I) FcγR binding ratios, such as mouse IgG2a and human IgG1, the first choice for this role. In contrast, exciting new data show that agonistic mAbs directed against the tumour necrosis factor receptor superfamily member CD40 require interaction with FcγRIIB for in vivo function. Such ligation activates antigen-presenting cells, promotes myeloid and CTL responses and potentially stimulates effective anti-cancer immunity. It appears that the role of FcγRIIB is to mediate mAb hyper-crosslinking to allow CD40 downstream intracellular signalling. Previous work has shown that mAbs directed against other TNFR family members, Fas and death receptor 5 and probably death receptor 4, also require FcγRIIB hyper-crosslinking to promote target cell apoptosis, suggesting a common mechanism of action. In mouse models, IgG1 is optimal for these agents as it binds to FcγRIIB with tenfold higher affinity than IgG2a and hence has a relatively low A:I FcγR binding ratio. In contrast, human IgG isotypes have a universally low affinity for FcγRIIB, but in the case of human IgG1, engineering the Fc to increase its affinity for FcγRIIB can potentially overcome this problem. Thus, modifying the A/I binding ratio of human IgG Fc can be used to optimise different types of therapeutic activity by enhancing cytotoxic or hyper-crosslinking function.
    Cancer Immunology and Immunotherapy 03/2013; 62(5). DOI:10.1007/s00262-013-1398-6 · 3.94 Impact Factor
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    ABSTRACT: Cells of the monocytic lineage play fundamental roles in the regulation of health, ranging from the initiation and resolution of inflammation to bone homeostasis. In rheumatoid arthritis (RA), the inflamed synovium exhibits characteristic infiltration of macrophages along with local osteoclast maturation, which, together, drive chronic inflammation and downstream articular destruction. The aim of this study was to explore an entirely novel route of immunoglobulin-mediated regulation, involving simultaneous suppression of the inflammatory and erosive processes in the synovium. Using in vivo and in vitro studies of human cells and a murine model of RA, the ability of staphylococcal protein A (SPA) to interact with and modulate cells of the monocytic lineage was tested. In addition, the efficacy of SPA as a therapeutic agent was evaluated in murine collagen-induced arthritis (CIA). SPA showed a capacity to appropriate circulating IgG, by generating small immunoglobulin complexes that interacted with monocytes, macrophages, and preosteoclasts. Formation of these complexes resulted in Fcγ receptor type I-dependent polarization of macrophages to a regulatory phenotype, rendering them unresponsive to activators such as interferon-γ. The antiinflammatory complexes also had the capacity to directly inhibit differentiation of preosteoclasts into osteoclasts in humans. Moreover, administration of SPA in the early stages of disease substantially alleviated the clinical and histologic erosive features of CIA in mice. These findings demonstrate the overarching utility of immunoglobulin complexes for the prevention and treatment of inflammatory diseases. The results shed light on the interface between immunoglobulin complex-mediated pathways, osteoclastogenesis, and associated pathologic processes. Thus, therapeutic agents designed to harness all of these properties may be an effective treatment for arthritis, by targeting both the innate inflammatory response and prodestructive pathways.
    Arthritis & Rheumatology 12/2011; 63(12):3897-907. DOI:10.1002/art.30629 · 7.87 Impact Factor
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    ABSTRACT: A high activatory/inhibitory FcγR binding ratio is critical for the activity of mAb such as rituximab and alemtuzumab that attack cancer cells directly and eliminate them by recruiting immune effectors. Optimal FcγR binding profiles of other anti-cancer mAb, such as immunostimulatory mAb that stimulate or block immune receptors, are less clear. In this study, we analyzed the importance of isotype and FcγR interactions in controlling the agonistic activity of the anti-mouse CD40 mAb 3/23. Mouse IgG1 (m1) and IgG2a (m2a) variants of the parental 3/23 (rat IgG2a) were engineered and used to promote humoral and cellular responses against OVA. The mouse IgG1 3/23 was highly agonistic and outperformed the parental Ab when promoting Ab (10-100-fold) and T cell (OTI and OTII) responses (2- to >10-fold). In contrast, m2a was almost completely inactive. Studies in FcγR knockout mice demonstrated a critical role for the inhibitory FcγRIIB in 3/23 activity, whereas activatory FcγR (FcγRI, -III, and -IV) was dispensable. In vitro experiments established that the stimulatory effect of FcγRIIB was mediated through Ab cross-linking delivered in trans between neighboring cells and did not require intracellular signaling. Intriguingly, activatory FcγR provided effective cross-linking of 3/23 m2a in vitro, suggesting the critical role of FcγRIIB in vivo reflects its cellular distribution and bioavailability as much as its affinity for a particular Ab isotype. In conclusion, we demonstrate an essential cross-linking role for the inhibitory FcγRIIB in anti-CD40 immunostimulatory activity and suggest that isotype will be an important issue when optimizing reagents for clinical use.
    The Journal of Immunology 08/2011; 187(4):1754-63. DOI:10.4049/jimmunol.1101135 · 5.36 Impact Factor
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    ABSTRACT: The anti-CD20 mAb rituximab is central to the treatment of B-cell malignancies, but resistance remains a significant problem. We recently reported that resistance could be explained, in part, by internalization of rituximab (type I anti-CD20) from the surface of certain B-cell malignancies, thus limiting engagement of natural effectors and increasing mAb consumption. Internalization of rituximab was most evident in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), but the extent of internalization was heterogeneous within each disease. Here, we show that the inhibitory FcγRIIb on target B cells promotes this process and is largely responsible for the observed heterogeneity across a range of B-cell malignancies. Internalization correlated strongly with FcγRIIb expression on normal and malignant B cells, and resulted in reduced macrophage phagocytosis of mAb-coated targets. Furthermore, transfection of FcγRIIb into FcγRIIb negative Ramos cells increased internalization of rituximab in a dose-dependent manner. Target-cell FcγRIIb promoted rituximab internalization in a cis fashion and was independent of FcγRIIb on neighboring cells. It became phosphorylated and internalized along with CD20:anti-CD20 complexes before lysosomal degradation. In MCL patients, high FcγRIIb expression predicted less durable responses after rituximab-containing regimens. Therefore, target-cell FcγRIIb provides a potential biomarker of response to type I anti-CD20 mAb.
    Blood 07/2011; 118(9):2530-40. DOI:10.1182/blood-2011-01-330357 · 10.43 Impact Factor