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Antibody responses during infection and vaccination typically undergo affinity maturation to achieve high-affinity binding for efficient neutralization of pathogens 1,2 . Similarly, high affinity is routinely the goal for therapeutic antibody generation. However, in contrast to naturally occurring or direct-targeting therapeutic antibodies, immunomodulatory antibodies, which are designed to modulate receptor signalling, have not been widely examined for their affinity-function relationship. Here we examine three separate immunologically important receptors spanning two receptor superfamilies: CD40, 4-1BB and PD-1. We show that low rather than high affinity delivers greater activity through increased clustering. This approach delivered higher immune cell activation, in vivo T cell expansion and antitumour activity in the case of CD40. Moreover, an inert anti-4-1BB monoclonal antibody was transformed into an agonist. Low-affinity variants of the clinically important antagonistic anti-PD-1 monoclonal antibody nivolumab also mediated more potent signalling and affected T cell activation. These findings reveal a new paradigm for augmenting agonism across diverse receptor families and shed light on the mechanism of antibody-mediated receptor signalling. Such affinity engineering offers a rational, efficient and highly tuneable solution to deliver antibody-mediated receptor activity across a range of potencies suitable for translation to the treatment of human disease., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Antibodies protect from infection, underpin successful vaccines and elicit therapeutic responses in otherwise untreatable cancers and autoimmune conditions. The human IgG2 isotype displays a unique capacity to undergo disulfide shuffling in the hinge region, leading to modulation of its ability to drive target receptor signaling (agonism) in a variety of important immune receptors, through hitherto unexplained molecular mechanisms. To address the underlying process and reveal how hinge disulfide orientation affects agonistic activity, we generated a series of cysteine to serine exchange variants in the hinge region of the clinically relevant monoclonal antibody ChiLob7/4, directed against the key immune receptor CD40. We report how agonistic activity varies with disulfide pattern and is afforded by the presence of a disulfide crossover between F(ab) arms in the agonistic forms, independently of epitope, as observed in the determined crystallographic structures. This structural "switch" affects directly on antibody conformation and flexibility. Small-angle x-ray scattering and ensemble modeling demonstrated that the least flexible variants adopt the fewest conformations and evoke the highest levels of receptor agonism. This covalent change may be amenable for broad implementation to modulate receptor signaling in an epitope-independent manner in future therapeutics.

Background: Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb., Methods: We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription., Results: We report that TAMs are FcγRIIb bright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb +/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes., Conclusion: Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies., (© 2022. The Author(s).)

Monoclonal antibodies (mAb) and natural ligands targeting costimulatory tumor necrosis factor receptors (TNFR) exhibit a wide range of agonistic activities and antitumor responses. The mechanisms underlying these differential agonistic activities remain poorly understood. Here, we employ a panel of experimental and clinically-relevant molecules targeting human CD40, 4-1BB and OX40 to examine this issue. Confocal and STORM microscopy reveal that strongly agonistic reagents induce clusters characterized by small area and high receptor density. Using antibody pairs differing only in isotype we show that hIgG2 confers significantly more receptor clustering than hIgG1 across all three receptors, explaining its greater agonistic activity, with receptor clustering shielding the receptor-agonist complex from further molecular access. Nevertheless, discrete receptor clustering patterns are observed with different hIgG2 mAb, with a unique rod-shaped assembly observed with the most agonistic mAb. These findings dispel the notion that larger receptor clusters elicit greater agonism, and instead point to receptor density and subsequent super-structure as key determinants.

Background: Previous data suggests that anti-OX40 mAb can elicit anti-tumor effects in mice through deletion of Tregs. However, OX40 also has powerful costimulatory effects on T cells which could evoke therapeutic responses. Human trials with anti-OX40 antibodies have shown that these entities are well tolerated but to date have delivered disappointing clinical responses, indicating that the rules for the optimal use of anti-human OX40 (hOX40) antibodies is not yet fully understood. Changes to timing and dosages may lead to improved outcomes; however, here we focus on addressing the role of agonism versus depleting activity in determining therapeutic outcomes. We investigated a novel panel of anti-hOX40 mAb to understand how these reagents and mechanisms may be optimized for therapeutic benefit., Methods: This study examines the binding activity and in vitro activity of a panel of anti-hOX40 antibodies. They were further evaluated in several in vivo models to address how isotype and epitope determine mechanism of action and efficacy of anti-hOX40 mAb., Results: Binding analysis revealed the antibodies to be high affinity, with epitopes spanning all four cysteine-rich domains of the OX40 extracellular domain. In vivo analysis showed that their activities relate directly to two key properties: (1) isotype-with mIgG1 mAb evoking receptor agonism and CD8+ T-cell expansion and mIgG2a mAb evoking deletion of Treg and (2) epitope-with membrane-proximal mAb delivering more powerful agonism. Intriguingly, both isotypes acted therapeutically in tumor models by engaging these different mechanisms., Conclusion: These findings highlight the significant impact of isotype and epitope on the modulation of anti-hOX40 mAb therapy, and indicate that CD8+ T-cell expansion or Treg depletion might be preferred according to the composition of different tumors. As many of the current clinical trials using OX40 antibodies are now using combination therapies, this understanding of how to manipulate therapeutic activity will be vital in directing new combinations that are more likely to improve efficacy and clinical outcomes., Competing Interests: Competing interests: MSC is a retained consultant for BioInvent International and has performed educational and advisory roles for Baxalta and Boehringer Ingleheim. He has received research funding from Roche, Gilead, Bioinvent International and GSK. BF, IT, LM and MS are employees of Bioinvent International., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress toward identifying the function of this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAbs) was generated. LILRB3-specific mAbs bound to discrete epitopes in Ig-like domain 2 or 4. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.

Anti-CD40 monoclonal antibodies (mAbs) comprise agonists and antagonists, which display promising therapeutic activities in cancer and autoimmunity, respectively. We previously showed that epitope and isotype interact to deliver optimal agonistic anti-CD40 mAbs. The impact of Fc engineering on antagonists, however, remains largely unexplored. Here, we show that clinically relevant antagonists used for treating autoimmune conditions can be converted into potent FcγR-independent agonists with remarkable antitumor activity by isotype switching to hIgG2. One antagonist is converted to a super-agonist with greater potency than previously reported highly agonistic anti-CD40 mAbs. Such conversion is dependent on the unique disulfide bonding properties of the hIgG2 hinge. This investigation highlights the transformative capacity of the hIgG2 isotype for converting antagonists to agonists to treat cancer., Competing Interests: Declaration of Interests M.S.C. acts as a consultant for a number of biotech companies, being retained as a consultant for BioInvent and has received research funding from BioInvent, GSK, UCB, iTeos, and Roche. M.J.G. acts as a consultant to a number of biotech companies and receives institutional payments and royalties from antibody patents and licenses. This work is related to patent Family WO 2015/145360 protecting antibodies containing modified hIgG2 domains which elicit agonist or antagonistic properties., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Immune compromised mice are increasingly used for the preclinical development of monoclonal antibodies (mAb). Most common are non-obese diabetic (NOD) severe combined immunodeficient (SCID) and their derivatives such as NOD SCID interleukin-2 γ-/- (NSG), which are attractive hosts for patient-derived xenografts. Despite their widespread use, the relative biological performance of mAb in these strains has not been extensively studied., Methods: Clinically relevant mAb of various isotypes were administered to tumor and non-tumor-bearing SCID and NOD SCID mice and the mAb clearance monitored by ELISA. Expression analysis of surface proteins in both strains was carried out by flow cytometry and immunofluorescence microscopy. Further analysis was performed in vitro by surface plasmon resonance to assess mAb affinity for Fcγ receptors (FcγR) at pH 6 and pH 7.4. NOD SCID mice genetically deficient in different FcγR were used to delineate their involvement., Results: Here, we show that strains on the NOD SCID background have significantly faster antibody clearance than other strains leading to reduced antitumor efficacy of clinically relevant mAb. This rapid clearance is dependent on antibody isotype, the presence of Fc glycosylation (at N297) and expression of FcγRII. Comparable effects were not seen in the parental NOD or SCID strains, demonstrating the presence of a compound defect requiring both genotypes. The absence of endogenous IgG was the key parameter transferred from the SCID as reconstituting NOD SCID or NSG mice with exogenous IgG overcame the rapid clearance and recovered antitumor efficacy. In contrast, the NOD strain was associated with reduced expression of the neonatal Fc Receptor (FcRn). We propose a novel mechanism for the rapid clearance of certain mAb isotypes in NOD SCID mouse strains, based on their interaction with FcγRII in the context of reduced FcRn., Conclusions: This study highlights the importance of understanding the limitation of the mouse strain being used for preclinical evaluation, and demonstrates that NOD SCID strains of mice should be reconstituted with IgG prior to studies of mAb efficacy., Competing Interests: Competing interests: MSC is a retained consultant for Bioinvent and has performed educational and advisory roles for Boehringer Ingelheim, Merck KGaA, Baxalta and GLG. He has received research funding from Bioinvent, Roche, Gilead, Iteos, UCB and GSK., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated cancer. Many hematologic and solid tumors are resistant to therapeutic antibodies in the bone marrow (BM), but not in the periphery (e.g., spleen). We previously showed that cyclophosphamide (CTX) sensitizes the BM niche to antibody therapeutics. Here, we show that (i) BM resistance was induced not only by the tumor but also by the intrinsic BM microenvironment; (ii) CTX treatment overcame both intrinsic and extrinsic resistance mechanisms by augmenting macrophage activation and phagocytosis, including significant upregulation of activating Fcγ receptors (FcγRIII and FcγRIV) and downregulation of the inhibitory receptor, FcγRIIB; and (iii) CTX synergized with cetuximab (anti-EGFR) and trastuzumab (anti-Her2) in eliminating metastatic breast cancer in the BM of humanized mice. These findings provide insights into the mechanisms by which CTX synergizes with antibody therapeutics in resistant niche-specific organs and its applicability in treating BM-resident tumors., (©2019 American Association for Cancer Research.)

While anti-angiogenic therapies for wet age-related macular degeneration (AMD) are effective for many patients, they require multiple injections and are expensive and prone to complications. Gene therapy could be an elegant solution for this problem by providing a long-term source of anti-angiogenic proteins after a single administration. Another potential issue with current therapeutic proteins containing a fragment crystallizable (Fc) domain (such as whole antibodies like bevacizumab) is the induction of an unwanted immune response. In wet AMD, a low level of inflammation is already present, so to avoid exacerbation of disease by the therapeutic protein, we propose single-chain fragment variable (scFv) antibodies, which lack the Fc domain, as a safer alternative. To investigate the feasibility of this, anti-vascular endothelial growth factor (VEGF)-blocking antibodies in two formats were produced and tested in vitro and in vivo . The scFv transgene was then cloned into an adeno-associated virus (AAV) vector. A therapeutic effect in a mouse model of choroidal neovascularization (CNV) was demonstrated with antibodies in both scFv and immunoglobulin G1 (IgG1) formats (p < 0.04). Importantly, the scFv anti-VEGF antibody expressed from an AAV vector also had a significant beneficial effect (p = 0.02), providing valuable preclinical data for future translation to the clinic.

The costimulatory receptor 4-1BB is expressed on activated immune cells, including activated T cells. Antibodies targeting 4-1BB enhance the proliferation and survival of antigen-stimulated T cells in vitro and promote CD8 T cell-dependent anti-tumor immunity in pre-clinical cancer models. We found that T regulatory (Treg) cells infiltrating human or murine tumors expressed high amounts of 4-1BB. Intra-tumoral Treg cells were preferentially depleted by anti-4-1BB mAbs in vivo. Anti-4-1BB mAbs also promoted effector T cell agonism to promote tumor rejection. These distinct mechanisms were competitive and dependent on antibody isotype and FcγR availability. Administration of anti-4-1BB IgG2a, which preferentially depletes Treg cells, followed by either agonistic anti-4-1BB IgG1 or anti-PD-1 mAb augmented anti-tumor responses in multiple solid tumor models. An antibody engineered to optimize both FcγR-dependent Treg cell depleting capacity and FcγR-independent agonism delivered enhanced anti-tumor therapy. These insights into the effector mechanisms of anti-4-1BB mAbs lay the groundwork for translation into the clinic., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Anti-CD40 monoclonal antibodies (mAbs) that promote or inhibit receptor function hold promise as therapeutics for cancer and autoimmunity. Rules governing their diverse range of functions, however, are lacking. Here we determined characteristics of nine hCD40 mAbs engaging epitopes throughout the CD40 extracellular region expressed as varying isotypes. All mAb formats were strong agonists when hyper-crosslinked; however, only those binding the membrane-distal cysteine-rich domain 1 (CRD1) retained agonistic activity with physiological Fc gamma receptor crosslinking or as human immunoglobulin G2 isotype; agonistic activity decreased as epitopes drew closer to the membrane. In addition, all CRD2-4 binding mAbs blocked CD40 ligand interaction and were potent antagonists. Thus, the membrane distal CRD1 provides a region of choice for selecting CD40 agonists while CRD2-4 provides antagonistic epitopes., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

CD134 (OX40) is a member of the tumour necrosis factor receptor superfamily (TNFRSF). It acts as a costimulatory receptor on T cells, but its role on NK cells is poorly understood. CD137, another TNFRSF member has been shown to enhance the anti-tumour activity of NK cells in various malignancies. Here, we examine the expression and function of CD134 on human and mouse NK cells in B-cell lymphoma. CD134 was transiently upregulated upon activation of NK cells in both species. In contrast to CD137, induction of CD134 on human NK cells was dependent on close proximity to, or cell-to-cell contact with, monocytes or T cells. Stimulation with an agonistic anti-CD134 mAb but not CD134 ligand, increased IFNγ production and cytotoxicity of human NK cells, but this was dependent on simultaneous antibody:Fcγ receptor binding. In complementary murine studies, intravenous inoculation with BCL 1 lymphoma into immunocompetent syngeneic mice resulted in transient upregulation of CD134 on NK cells. Combination treatment with anti-CD20 and anti-CD134 mAb produced a synergistic effect with durable remissions. This therapeutic benefit was abrogated by NK cell depletion and in Fcγ chain -/- mice. Hence, anti-CD134 agonists may enhance NK-mediated anti-tumour activity in an Fcγ receptor dependent fashion.

Monoclonal antibodies (mAbs) can destroy tumors by recruiting effectors such as myeloid cells, or targeting immunomodulatory receptors to promote cytotoxic T cell responses. Here, we examined the therapeutic potential of combining a direct tumor-targeting mAb, anti-CD20, with an extended panel of immunomodulatory mAbs. Only the anti-CD27/CD20 combination provided cures. This was apparent in multiple lymphoma models, including huCD27 transgenic mice using the anti-huCD27, varlilumab. Detailed mechanistic analysis using single-cell RNA sequencing demonstrated that anti-CD27 stimulated CD8 + T and natural killer cells to release myeloid chemo-attractants and interferon gamma, to elicit myeloid infiltration and macrophage activation. This study demonstrates the therapeutic advantage of using an immunomodulatory mAb to regulate lymphoid cells, which then recruit and activate myeloid cells for enhanced killing of mAb-opsonized tumors., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: A proportion of RA and SLE patients treated with standard doses of rituximab (RTX) display inefficient B cell deletion and poor clinical responses that can be augmented by delivering higher doses, indicating that standard-dose RTX is a sub-optimal therapy in these patients. This study aimed to investigate whether better responses could be achieved with mechanistically different anti-CD20 mAbs., Methods: We compared RTX with obinutuzumab (OBZ), a new-generation, glycoengineered type II anti-CD20 mAb, in a series of in vitro assays measuring B cell cytotoxicity in RA and SLE patient samples., Results: We found that OBZ was at least 2-fold more efficient than RTX at inducing B-cell cytotoxicity in in vitro whole blood assays. Dissecting this difference, we found that RTX elicited more potent complement-dependent cellular cytotoxicity than OBZ. In contrast, OBZ was more effective at evoking Fc gamma receptor-mediated effector mechanisms, including activation of NK cells and neutrophils, probably due to stronger interaction with Fc gamma receptors and the ability of OBZ to remain at the cell surface following CD20 engagement, whereas RTX became internalized. OBZ was also more efficient at inducing direct cell death. This was true for all CD19 + B cells as a whole and in naïve (IgD + CD27 - ) and switched (IgD - CD27 + ) memory B cells specifically, a higher frequency of which is associated with poor clinical response after RTX., Conclusion: Taken together, these data provide a mechanistic basis for resistance to rituximab-induced B-cell depletion, and for considering obinutuzumab as an alternative B-cell depleting agent in RA and SLE., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Tumors routinely attract and co-opt macrophages to promote their growth, angiogenesis, and metastasis. Macrophages are also the key effector cell for mAb therapies. Here we report that the tumor microenvironment creates an immunosuppressive signature on tumor-associated macrophages (TAM), which favors expression of inhibitory rather than activating Fcγ receptors (FcγR), thereby limiting the efficacy of mAb immunotherapy. We assessed a panel of TLR and STING agonists (a) for their ability to reprogram macrophages to a state optimal for mAb immunotherapy. Both STINGa and TLRa induced cytokine release, modulated FcγR expression, and augmented mAb-mediated tumor cell phagocytosis in vitro However, only STINGa reversed the suppressive FcγR profile in vivo , providing strong adjuvant effects to anti-CD20 mAb in murine models of lymphoma. Potent adjuvants like STINGa, which can improve FcγR activatory:inhibitory (A:I) ratios on TAM, are appealing candidates to reprogram TAM and curb tumor-mediated immunosuppression, thereby empowering mAb efficacy. Cancer Res; 77(13); 3619-31. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Immunotherapy using mAbs, such as rituximab, is an established means of treating hematological malignancies. Abs can elicit a number of mechanisms to delete target cells, including complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity, and Ab-dependent cellular phagocytosis. The inherent properties of the target molecule help to define which of these mechanisms are more important for efficacy. However, it is often unclear why mAb binding to different epitopes within the same target elicits different levels of therapeutic activity. To specifically address whether distance from the target cell membrane influences the aforementioned effector mechanisms, a panel of fusion proteins consisting of a CD20 or CD52 epitope attached to various CD137 scaffold molecules was generated. The CD137 scaffold was modified through the removal or addition of cysteine-rich extracellular domains to produce a panel of chimeric molecules that held the target epitope at different distances along the protein. It was shown that complement-dependent cytotoxicity and Ab-dependent cellular cytotoxicity favored a membrane-proximal epitope, whereas Ab-dependent cellular phagocytosis favored an epitope positioned further away. These findings were confirmed using reagents targeting the membrane-proximal or -distal domains of CD137 itself before investigating these properties in vivo, where a clear difference in the splenic clearance of transfected tumor cells was observed. Together, this work demonstrates how altering the position of the Ab epitope is able to change the effector mechanisms engaged and facilitates the selection of mAbs designed to delete target cells through specific effector mechanisms and provide more effective therapeutic agents., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Purpose: Cutaneous squamous cell carcinoma (cSCC) is the most common human cancer with metastatic potential. Despite T cells accumulating around cSCCs, these tumors continue to grow and persist. To investigate reasons for failure of T cells to mount a protective response in cSCC, we focused on regulatory T cells (Tregs) as this suppressive population is well represented among the infiltrating lymphocytes., Experimental Design: Flow cytometry was conducted on cSCC lymphocytes and in vitro functional assays were performed using sorted tumoral T cells. Lymphocyte subsets in primary cSCCs were quantified immunohistochemically., Results: FOXP3(+) Tregs were more frequent in cSCCs than in peripheral blood (P < 0.0001, n = 86 tumors). Tumoral Tregs suppressed proliferation of tumoral effector CD4(+) (P = 0.005, n = 10 tumors) and CD8(+) T cells (P = 0.043, n = 9 tumors) and inhibited IFNγ secretion by tumoral effector T cells (P = 0.0186, n = 11 tumors). The costimulatory molecule OX40 was expressed predominantly on tumoral Tregs (P < 0.0001, n = 15 tumors) and triggering OX40 with an agonist anti-OX40 antibody overcame the suppression exerted by Tregs, leading to increased tumoral effector CD4(+) lymphocyte proliferation (P = 0.0098, n = 10 tumors). Tregs and OX40(+) lymphocytes were more abundant in primary cSCCs that metastasized than in primary cSCCs that had not metastasized (n = 48 and n = 49 tumors, respectively)., Conclusions: Tregs in cSCCs suppress effector T-cell responses and are associated with subsequent metastasis, suggesting a key role for Tregs in cSCC development and progression. OX40 agonism reversed the suppressive effects of Tregs in vitro, suggesting that targeting OX40 could benefit the subset of cSCC patients at high risk of metastasis. Clin Cancer Res; 22(16); 4236-48. ©2016 AACR., (©2016 American Association for Cancer Research.)

Tumor cells dying after cytotoxic therapy are a potential source of antigen for T-cell priming. Antigen-presenting cells (APC) can cross-present MHC I-restricted peptides after the uptake of dying cells. Depending on the nature of the surrounding environmental signals, APCs then orchestrate a spectrum of responses ranging from immune activation to inhibition. Previously, we had demonstrated that combining radiation with either agonistic monoclonal antibody (mAb) to CD40 or a systemically administered TLR7 agonist could enhance CD8 T-cell-dependent protection against syngeneic murine lymphoma models. However, it remains unknown how individual APC populations affect this antitumor immune response. Using APC depletion models, we now show that dendritic cells (DC), but not macrophages or B cells, were responsible for the generation of long-term immunologic protection following combination therapy with radiotherapy and either agonistic CD40 mAb or systemic TLR7 agonist therapy. Novel immunotherapeutic approaches that augment antigen uptake and presentation by DCs may further enhance the generation of therapeutic antitumor immune responses, leading to improved outcomes after radiotherapy. Cancer Immunol Res; 4(7); 621-30. ©2016 AACR., (©2016 American Association for Cancer Research.)

Monoclonal antibody (mAb) therapeutics are revolutionizing cancer treatment; however, not all tumors respond, and agent optimization is essential to improve outcome. It has become clear over recent years that isotype choice is vital to therapeutic success with agents that work through different mechanisms, direct tumor targeting, agonistic receptor engagement, or receptor-ligand blockade, having contrasting requirements. Here we summarize how isotype dictates mAb activity and discuss ways in which this information can be used for the development of enhanced therapeutics., (© 2016 by The American Society of Hematology.)

An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a -mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies.

CD70-mediated stimulation of CD27 is an important cofactor of CD4(+) T-cell licensed dendritic cells (DCs). However, it is unclear how CD70-mediated stimulation of T cells is integrated with signals that emanate from signal 3 pathways, such as type-1 interferon (IFN-1) and IL-12. We find that while stimulation of CD27 in isolation drives weak Eomesodermin(hi) T-bet(lo) CD8(+) T-cell responses to OVA immunization, profound synergistic expansion is achieved by cotargeting TLR. This cooperativity can substantially boost antiviral CD8(+) T-cell responses during acute infection. Concomitant stimulation of TLR significantly increases per cell IFN-γ production and the proportion of the population with characteristics of short-lived effector cells, yet also promotes the ability to form long-lived memory. Notably, while IFN-1 contributes to the expression of CD70 on DCs, the synergy between CD27 and TLR stimulation is dependent upon IFN-1's effect directly on CD8(+) T cells, and is associated with the increased expression of T-bet in T cells. Surprisingly, we find that IL-12 fails to synergize with CD27 stimulation to promote CD8(+) T-cell expansion, despite its capacity to drive effector CD8(+) T-cell differentiation. Together, these data identify complex interactions between signal 3 and costimulatory pathways, and identify opportunities to influence the differentiation of CD8(+) T-cell responses., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

FcγRs are key regulators of the immune response, capable of binding to the Fc portion of IgG Abs and manipulating the behavior of numerous cell types. Through a variety of receptors, isoforms, and cellular expression patterns, they are able to fine-tune and direct appropriate responses. Furthermore, they are key determinants of mAb immunotherapy, with mAb isotype and FcγR interaction governing therapeutic efficacy. Critical to understanding the biology of this complex family of receptors are reagents that are robust and highly specific for each receptor. In this study, we describe the development and characterization of mAb panels specific for both mouse and human FcγR for use in flow cytometry, immunofluorescence, and immunocytochemistry. We highlight key differences in expression between the two species and also patterns of expression that will likely impact on immunotherapeutic efficacy and translation of therapeutic agents from mouse to clinic., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Cancer immunotherapy has been revolutionised by the use monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics.

Immunotherapy is a promising strategy for the treatment of Alzheimer's disease (AD). Antibodies directed against Amyloid Beta (Aβ) are able to successfully clear plaques and reverse cognitive deficits in mouse models. Excitement towards this approach has been tempered by high profile failures in the clinic, one key issue has been the development of inflammatory side effects in the brain (ARIAs). New antibodies are entering the clinic for Alzheimer's disease; therefore, it is important to learn all we can from the current generation. In this study, we directly compared 3 clinical candidates in the same pre-clinical model, with the same effector function, for their ability to clear plaques and induce inflammation in the brain. We produced murine versions of the antibodies: Bapineuzumab (3D6), Crenezumab (mC2) and Gantenerumab (chGantenerumab) with an IgG2a constant region. 18-month transgenic APP mice (Tg2576) were injected bilaterally into the hippocampus with 2 µg of each antibody or control. After 7 days, the mice tissue was analysed for clearance of plaques and neuroinflammation by histology and biochemical analysis. 3D6 was the best binder to plaques and in vitro, whilst mC2 bound the least strongly. This translated into 3D6 effectively clearing plaques and reducing the levels of insoluble Aβ, whilst chGantenerumab and mC2 did not. 3D6 caused a significant increase in the levels of pro-inflammatory cytokines IL-1β and TNFα, and an associated increase in microglial expression of CD11B and CD68. chGantenerumab increased pro-inflammatory cytokines and microglial activation, but minimal changes in CD68, as an indicator of phagocytosis. Injection of mC2 did not cause any significant inflammatory changes. Our results demonstrate that the ability of an antibody to clear plaques and induce inflammation is dependent on the epitope and affinity of the antibody.

In this issue of Cancer Cell, Dahan and colleagues demonstrate that the Fc region has a significant impact on the therapeutic capacity of checkpoint inhibitor antibodies targeting the PD-1/PD-L1 axis in pre-clinical tumor models. This work provides important insights with respect to the further clinical development of checkpoint inhibitors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: Rituximab, a type I anti-CD20 monoclonal antibody (mAb), induces incomplete B cell depletion in some patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), thus contributing to a poor clinical response. The mechanisms of this resistance remain elusive. The purpose of this study was to determine whether type II mAb are more efficient than type I mAb at depleting B cells from RA and SLE patients, whether internalization influences the efficiency of depletion, and whether Fcγ receptor type IIb (FcγRIIb) and the B cell receptor regulate this internalization process., Methods: We used an in vitro whole blood B cell-depletion assay to assess the efficiency of depletion, flow cytometry to study cell surface protein expression, and surface fluorescence-quenching assays to assess rituximab internalization, in samples from patients with RA and patients with SLE. Paired t-test or Mann-Whitney U test was used to compare groups, and Spearman's rank correlation test was used to assess correlation., Results: We found that type II mAb internalized significantly less rituximab than type I mAb and depleted B cells from patients with RA and SLE at least 2-fold more efficiently than type I mAb. Internalization of rituximab was highly variable between patients, was regulated by FcγRIIb, and inversely correlated with cytotoxicity in whole blood B cell-depletion assays. The lowest levels of internalization were seen in IgD- B cells, including postswitched (IgD-CD27+) memory cells. Internalization of type I anti-CD20 mAb was also partially inhibited by anti-IgM stimulation., Conclusion: Variability in internalization of rituximab was observed and was correlated with impaired B cell depletion. Therefore, slower-internalizing type II mAb should be considered as alternative B cell-depleting agents for the treatment of RA and SLE., (© 2015 The Authors. Arthritis & Rheumatology is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

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.)

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. Internalization 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 internalization, raising the question of whether ITIM phosphorylation plays any role in this process. We have assessed the molecular requirements for the internalization process and demonstrate that in contrast to internalization of IgG immune complexes, FcγRIIb-augmented internalization of rituximab-ligated CD20 occurs independently of the FcγRIIb ITIM, indicating that signaling downstream of FcγRIIb is not required. In transfected cells, activatory FcγRI, FcγRIIa, and FcγRIIIa augmented internalization of rituximab-ligated CD20 in a similar manner. However, FcγRIIa mediated a slower rate of internalization 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 internalization of rituximab-ligated CD20. Instead, we propose that FcγR provides a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.)

The anti-CD28 superagonist antibody TGN1412 caused life-threatening cytokine release syndrome (CRS) in healthy volunteers, which had not been predicted by preclinical testing. T cells in fresh peripheral blood mononuclear cells (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 crystallizable fragment gamma receptor IIb (FcγRIIb) expression on monocytes. This was unexpected because, 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 B cells can support TGN1412-mediated 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 cocultured with FcγRIIb-expressing monocytes from HD preculture, which shows 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 because of the close association of FcγRIIb-bearing cells with T cells in lymphoid tissues., (© 2015 by The American Society of Hematology.)