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The majority of depleting monoclonal antibody (mAb) drugs elicit responses via Fc-FcγR and Fc-C1q interactions. Optimal C1q interaction is achieved through hexameric Fc:Fc interactions at the target cell surface. Herein is described an approach to exploit the tailpiece of the naturally multimeric IgM to augment hexamerisation of IgG. Fusion of the C-terminal tailpiece of IgM promoted spontaneous hIgG hexamer formation, resulting in enhanced C1q recruitment and complement-dependent cytotoxicity (CDC) but with off-target complement activation and reduced in-vivo efficacy. Mutation of the penultimate tailpiece cysteine to serine (C575S) ablated spontaneous hexamer formation, but facilitated reversible hexamer formation after concentration in solution. C575S mutant tailpiece antibodies displayed increased complement activity only after target binding, in-line with the concept of ‘on-target hexamerisation’, whilst retaining efficient in-vivo efficacy and augmented target cell killing in the lymph node. Hence, C575S-tailpiece technology represents an alternative format for promoting on-target hexamerisation and enhanced CDC., Sopp et al describe an approach, which exploits the tailpiece of the naturally multimeric IgM to augment hexamerisation of IgG. Their approach provides a newly engineered format of antibodies for promoting hexamerisation and enhanced complement-dependent cytotoxicity only when cell surface bound.

Many therapeutic antibodies deplete target cells and elicit immunotherapy by engaging activating Fc gamma receptors (FcγRs) on host effector cells. These antibodies are negatively regulated by the inhibitory FcγRIIB (CD32B). Dogma suggests inhibition is mediated through the FcγRIIB immunoreceptor tyrosine-based inhibition motif (ITIM), negatively regulating immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling from activating FcγR. To assess this, we generated experimental models expressing human (h)FcγRIIB on targets or effectors, lacking or retaining ITIM signaling capacity. We demonstrate that signaling through the hFcγRIIB ITIM is dispensable for impairing monoclonal antibody (mAb)-mediated depletion of normal and malignant murine target cells through three therapeutically relevant surface receptors (CD20, CD25, and OX40) affecting immunotherapy. We demonstrate that hFcγRIIB competition with activating FcγRs for antibody Fc, rather than ITIM signaling, is sufficient to impair activating FcγR engagement, inhibiting effector function and immunotherapy.

The majority of monoclonal antibody (mAb) therapeutics possess the ability to engage innate immune effectors through interactions mediated by their fragment crystallizable (Fc) domain. By delivering Fc-Fc gamma receptor (FcγR) and Fc-C1q interactions, mAb are able to link exquisite specificity to powerful cellular and complement-mediated effector functions. Fc interactions can also facilitate enhanced target clustering to evoke potent receptor signaling. These observations have driven decades-long research to delineate the properties within the Fc that elicit these various activities, identifying key amino acid residues and elucidating the important role of glycosylation. They have also fostered a growing interest in Fc-engineering whereby this knowledge is exploited to modulate Fc effector function to suit specific mechanisms of action and therapeutic purposes. In this review, we document the insight that has been generated through the study of the Fc domain; revealing the underpinning structure-function relationships and how the Fc has been engineered to produce an increasing number of antibodies that are appearing in the clinic with augmented abilities to treat cancer.

BackgroundImmune 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.MethodsClinically 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.ResultsHere, 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.ConclusionsThis 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.

Idelalisib is a highly selective oral inhibitor of PI3Kδ indicated for the treatment of patients with relapsed chronic lymphocytic leukemia in combination with rituximab. Despite additive clinical effects, previous studies have paradoxically demonstrated that targeted therapies potentially negatively affect anti-CD20 mAb effector mechanisms. To address these potential effects, we investigated the impact of PI3Kδ inhibition by idelalisib on the effector mechanisms of rituximab and obinutuzumab. At clinically relevant concentrations, idelalisib minimally influenced rituximab- and obinutuzumab-mediated Ab-dependent cellular cytotoxicity and phagocytosis on human lymphoma cell lines, while maintaining the superiority of obinutuzumab-mediated Ab-dependent cellular cytotoxicity. Consistent with this, idelalisib did not influence obinutuzumab-mediated B cell depletion in whole-blood B cell–depletion assays. Further, idelalisib significantly enhanced obinutuzumab-mediated direct cell death of chronic lymphocytic leukemia cells. In murine systems, in vivo inhibition of PI3Kδ minimally interfered with maximal rituximab- or obinutuzumab-mediated depletion of leukemic targets. In addition, the duration of rituximab- and obinutuzumab-mediated depletion of leukemia cells was extended by combination with PI3Kδ inhibition. Collectively, these data demonstrate that PI3Kδ inhibition does not significantly affect the effector mechanisms induced by rituximab or obinutuzumab and provides an effective in vivo therapeutic combination. Therefore, combinations of obinutuzumab and idelalisib are currently being assessed in clinical studies.

Fc gamma receptors (FcγR) are involved in multiple aspects of immune cell regulation, are central to the success of monoclonal antibody (mAb) therapeutics and underpin the pathology of several autoimmune diseases. However, reliable assays capable of accurately measuring FcγR interactions with their physiological ligands, immunoglobulin G (IgG) immune complexes (IC), are limited. A method to study and detect IC interactions with FcγRs was therefore developed. This method, designed to model the signalling pathway of the inhibitory FcγRIIB (CD32B), utilised NanoLuc® Binary Interaction Technology (NanoBiT™) to measure recruitment of the Src homology 2 (SH2) domain-containing inositol phosphatase 1 (SHIP-1) to the immunoreceptor tyrosine-based inhibitory motif (ITIM) of this receptor. Such recruitment required prior crosslinking of an immunoreceptor tyrosine-based activation motif (ITAM)-containing activatory receptor, and evoked luciferase activity in discrete clusters at the cell surface, recapitulating the known biology of CD32B signalling. The assay detected varying forms of experimental IC, including heat-aggregated IgG, Rituximab:anti-idiotype complexes and anti-trinitrophenol (TNP)-TNP complexes in a sensitive manner (≤1µg/ml), and discriminated between complexes of varying size and isotype. Proof-of-concept for the detection of circulating ICs in autoimmune disease was provided, as responses to sera from patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) were detected in small pilot studies. Finally, the method was translated to a stable cell line system. In conclusion, a rapid and robust method for the detection of IC was developed, which has numerous potential applications including the monitoring of IC in autoimmune diseases and the study of underlying FcγR biology.

Therapeutic antibodies have improved survival of both hematologic and solid cancer patients, inducing long-lasting responses and even cures. Clinically successful antibodies exert antitumor activity by targeting tumor cells directly e.g. anti-CD20 mAb in B cell cancer, or by targeting and activating immune cells that seek and destroy cancer cells in the tumor microenvironment (immune checkpoint blocking antibodies;ICB). Still, many patients fail to respond or acquire resistance to these therapies. Understanding mechanisms and overcoming resistance to distinct classes of antibody drugs, is therefore clearly warranted and has the potential to further improve cancer outcomes. The inhibitory Fc gamma receptor (FcγR) IIB may precipitate resistance to cancer immunotherapy by several mechanisms, acting both on tumor and immune effector cells. We previously developed an FcγRIIB blocking antibody (BI-1206). Based on its cytolytic activity against B cell cancer cells and ability to block rituximab internalization from tumor cells, as well as enhance rituximab therapeutic activity in mice humanized for CD20 and FcγRIIB or bearing relapsed/refractory CLL in vivo, BI-1206 is being explored in clinical trials (NCT03571568; NCT04219254). Emerging data demonstrate that FcγRs can modulate the therapeutic activity of ICB antibodies (anti-PD-1/PD-L1 and anti-CTLA-4). While both anti-CTLA-4 and anti-PD-1/L1 mAb are effective and approved for cancer therapy, and combined checkpoint blockade improves therapeutic responses, anti-CTLA-4 therapies are associated with tolerability issues. Means of enhancing the anti-CTLA-4 therapeutic window are therefore desirable in order to deliver this therapy to more patients. Recent preclinical and retrospective clinical data indicate that anti-CTLA-4 antibodies may promote antitumor immunity by dual mechanisms; “releasing the brakes” on T effector cells and depleting immune suppressive intratumoral T regulatory (Treg) cells. Anti-CTLA-4 induced Treg depletion was shown to be FcγR-dependent. Here, we report the generation of a novel, fully human FcγRIIB-blocking antibody (BI-1607) engineered to eliminate Fc-mediated FcγR binding and function (Fc-null anti-FcγRIIB). Using a mechanism-of-action-matched surrogate antibody, we demonstrate that Fc-null anti-FcγRIIB enhances therapeutic efficacy of submaximally efficacious doses of anti-CTLA-4 in responsive (MC38) or resistant (CT26) syngeneic immune competent mouse models. Of relevance to the clinical setting where low dose anti-CTLA-4/anti-PD-1 combination therapies have been approved for different indications, we further demonstrate that triplet therapy significantly enhanced survival in the checkpoint blockade-poorly responsive B16 tumor model. Collectively our results provide proof-of-concept for Fc-null anti-FcγRIIB to enhance anti-CTLA-4 ICB in the treatment of cancer. Citation Format: Linda Mårtensson, Robert Oldham, Marie Borggren, Mathilda Kovacek, Therese Blidberg, Ali Roghanian, Ulla-Carin Thornberg, Ingrid Karlsson, Stephen A. Beers, Mark S. Cragg, Ingrid Teige, Bjorn L. Frendéus. A novel FcγRIIB-blocking antibody to enhance FcγR-dependent antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1642.

Introduction Administration of therapeutic monoclonal antibodies (mAb) is often accompanied by infusion related reactions (IRR), of ill-determined mechanism. During the clinical development of BI-1206 (NCT02933320 and NCT03571568), a fully human mAb that binds specifically to hFcγRIIB (hCD32B), frequent IRR were observed, independent of sustained FcγRIIB blockade. At 100mg/patient, BI-1206 showed transient receptor saturation for up to 72h, whereas IRR most often resolved within 24h. Administration of BI-1206 was aslo associated with transient thrombocytopenia but not increased bleeding, and most episodes resolved within a week. Elevated transaminases (i.e. alanine transaminase (ALT) and aspartate transaminase (AST)) and a transient cytokine release was also observed alongside thrombocytopenia. Methods Six to eight-week-old C57BL/6 mice were injected either through intravenous (i.v.), intra-peritoneal (i.p.) or subcutaneous (s.c.) routes with a surrogate mouse anti-FcγRIIB (AT130-2 IgG2a) in doses ranging from 1-400μg/mouse. Mice were monitored post injection for changes in behavior and macroscopic symptoms such as isolation, mobility, and fur condition. Serum concentration of AT130-2 were quantified using an in-house ELISA. Platelet counts were analysed in fresh blood using a Vetscan (Triolab). Transaminases were analysed by IDEXX BioResearch. Cytokines were analysed with V-plex Proinflammatory Panel 1 Mouse kit (MesoScale Discovery), including interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL, 4, IL-5, IL-6, IL-10, IL-12p70, KC/GRO and tumor necrosis factor (TNF)-α. Results Following administration of 200μg AT130-2 i.v., rapid onset of IRR was seen within 5-7 minutes. Blood sampling of these mice indicated reduced blood pressure. The mice recovered 10-15 minutes post IRR onset and no macroscopic symptoms were observed by 1h. Tolerability was dose-dependent and showed a clear pattern of increasing IRR with regards route of administration in the order s.c. A decrease in platelet count was seen at the same time as IRRs after both i.v. and i.p. administration. The decrease was transient and normalised within 8h post injection. AST, ALT, IL-5, IL-6, IL-10 KC/GRO, TNF-α showed a transient increase, all peaking 1-3 post injection, except for IL-5. IL-5 showed a delayed peak 3-8h post injection. To investigate whether premedication with corticosteroids could inhibit the IRR and associated toxicities, mice were premedicated with betamethasone or dexamethasone 16-24h s.c. and 1h i.v. pre injection of AT130-2. Development of IRR and platelet reduction was completely inhibited with premedication. Also, the increase in liver transaminases and cytokine release was significantly reduced. A single dose of premedication at 1h pre injection did not inhibit the IRR nor prevent the decrease in platelet count, Whilst a single dose premedication 16-24h pre injection reduced the IRR and platelet decrease, it not completely block the changes, indicating that two doses of corticosteroids are optimal. Conclusions Here we demonstrate anin vivomodel that recapitulates the tolerability profile seen with BI-1206 in human cancer subjects, including IRR, decreased platelet count, elevated transaminases and transient cytokine release. In the mouse model, there was a correlation between IRR with high dose and rapid increase in serum concentration, rather than FcγRIIB saturation following administration of anti-FcγRIIB without IRR. This pre-medication regimen has been implemented in clinical trials and shown to improve tolerability to BI-1206. Disclosures Karlsson: BioInvent International AB:Current Employment.Mårtensson:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Borggren:BioInvent International AB:Current Employment.Kovacek:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Roos:BioInvent International AB:Current Employment.Blidberg:BioInvent International AB:Current Employment.Oldham:BioInvent International AB:Research Funding.Jerkeman:Roche:Research Funding;Abbvie:Research Funding;Janssen:Research Funding;Gilead:Research Funding;Celgene:Research Funding.Dyer:BioInvent International AB:Research Funding.Davies:Roche, Celgene, Kite Pharma, Acerta, Karyopharma, Regeneron, Incyte:Consultancy;Roche, Acerta Pharma, AstraZeneca, Celgene, Gilead, ADC Therapeutics, Gilead:Research Funding;Celegene, Roche, Kite Pharma, Celegene:Honoraria;Roche:Other: TRAVEL, ACCOMMODATIONS, EXPENSES.Córdoba:Roche:Honoraria, Other: travel and accommodation;Abbvie:Honoraria, Other: travel and accommodation;Janssen:Honoraria, Other: travel and accommodation;Takeda Farmacéutica España S.A.:Speakers Bureau;Gilead:Honoraria, Other: travel and accommodation.Abrisqueta:AbbVie:Consultancy, Honoraria, Speakers Bureau;Janssen:Consultancy, Honoraria, Speakers Bureau;Celgene:Consultancy, Honoraria;Roche:Consultancy, Honoraria, Speakers Bureau.Beers:BioInvent International AB:Research Funding.Teige:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.McAllister:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Lim:BioInvent International AB:Patents & Royalties, Research Funding.Cragg:BioInvent International AB:Patents & Royalties, Research Funding.Frendéus:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.

Introduction: Phosphatidylinositol-3-kinase δ (PI3Kδ)-signaling provides key maintenance, proliferation, and survival cues during both normal and malignant B-lymphocyte development. Consequently, isoform-selective PI3Kδ inhibitors (PI3Kδi) have generated huge interest as a potential treatment for lymphoid malignancies. In particular, PI3Kδi demonstrate impressive clinical efficacy in combination with anti-CD20 monoclonal antibodies (mAbs) for relapsed chronic lymphocytic leukemia (CLL). However, these combinations function primarily to delay disease progression, but are not curative. With the ever-rising number of new targeted therapeutics, the challenge is to identify combinations that will ultimately deliver curative regimes. In order to guide these selections, a detailed mechanistic understanding is required. To date, only limited data are available regarding the exact in vivo therapeutic mechanism of PI3Kδi. Currently, studies identify immunomodulation, inhibition of BCR-, chemokine/cytokine-signaling, and induction of apoptosis as putative therapeutic mechanisms. Here we characterize the molecular mechanisms responsible for PI3Kδi-induced cytotoxicity and determine the relative contribution toward in vivo therapeutic responses utilizing the Eµ-TCL1-Tg mouse model of CLL alongside human CLL samples. Methods: The molecular mechanisms of PI3Kδi alone or in combination with anti-CD20 mAbs were assessed using the Eµ-TCL1-Tg mouse model, an in vivo model system of CLL. To inhibit PI3Kδ, the δ isoform-selective inhibitor GS-9820 was chosen, as it is highly structurally related to idelalisib, and critically demonstrates improved pharmacokinetic properties in the mouse in comparison to idelalisib. In vitro GS-9820 IC50 are as follows: PI3Kδ 27 nM; PI3Kα 83,424 nM; PI3Kβ 14,899 nM; and PI3Kγ 15,606 nM. Assays to measure its effects on BCR-mediated kinase activation, chemokine signaling/chemotaxis, and cytokine- and cell-mediated support were performed. GS-9820 was administered in vivo at 10 mg/kg per os BID (formulated in 0.5% methylcellulose, 0.05% tween-80), once leukemias were detected, and maintained throughout the treatment period (GS-9820 Cmax 3114 nM, Ctrough 48.6 nM). Results: GS-9820 induced substantive in vitro cell death and disruptedBCR-mediated kinase activation, chemokine signaling/chemotaxis, and inhibited both cytokine- and cell-mediated support in murine (Eµ-TCL1) and human CLL cells. In vivo administration of GS-9820 imparted significant therapeutic responses in Eµ-TCL1-bearing animals, reducing leukemic burden by 75% and splenic tumor deposits by 66% 4 weeks posttreatment. GS-9820 appeared well tolerated in recipient animals with no obvious toxicity apparent (e.g. weight loss or behavioral symptoms). When in combination with anti-CD20 mAbs, GS-9820 extended leukemia depletion by several weeks. GS-9820 enhanced overall survival by 66% in comparison with vehicle control-recipient animals and enhanced the survival benefit of anti-CD20 mAb therapy. These therapeutic responses were associated with a 2-fold increase in expression of the pro-apoptotic BH3-only Bcl-2 family member Bim and a 3-fold increase in the extent of Bim/Bcl-2 interaction. Accordingly, Bim-/- Eµ-TCL1-Tg leukaemias exhibited profound resistance to PI3Kδi-induced cytotoxicity, were refractory to PI3Kδi in vivo, and failed to display combination efficacy with anti-CD20 mAbs. These findings informed the rational design of a GS-9820 + ABT199 (Venetoclax) complementary drug combination strategy. Combinations of GS-9820 and ABT199 were well tolerated with an absence of weight loss or altered behavioral symptoms. The GS-9820 + ABT199 combination effectively halted leukemia progression in vivo with increased efficacy compared to monotherapy regimes, resulting in a 90% reduction in leukemic burden at the end of the treatment period. Conclusions: Bim-dependent apoptosis represents the key in vivo effector mechanism for PI3Kδi in the Eµ-TCL1-Tg mouse model, both alone and in combination with anti-CD20 mAbs. As such, combinations of PI3Kδ and Bcl-2 inhibitors may represent an efficacious drug combination strategy. Disclosures Tannheimer: Gilead Sciences: Employment. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding. Cragg:Baxalta: Consultancy; Roche: Consultancy, Research Funding; Bioinvent International: Consultancy, Research Funding; Gilead Sciences: Research Funding; GSK: Research Funding.

Nicotinamide adenine dinucleotide (NAD⁺) is an essential co-enzyme reported to operate both intra- and extracellularly. In the extracellular space, NAD⁺ can elicit signals by binding purinergic P2 receptors or it can serve as the substrate for a chain of ectoenzymes. As a substrate, it is converted to adenosine (ADO) and then taken up by the cells, where it is transformed and reincorporated into the intracellular nucleotide pool. Nucleotide-nucleoside conversion is regulated by membrane-bound ectoenzymes. CD38, the main mammalian enzyme that hydrolyzes NAD⁺, belongs to the ectoenzymatic network generating intracellular Ca(2+)-active metabolites. Within this general framework, the extracellular conversion of NAD⁺ can vary significantly according to the tissue environment or pathological conditions. Accumulating evidence suggests that tumor cells exploit such a network for migrating and homing to protected areas and, even more importantly, for evading the immune response. We report on the experience of this lab to exploit human multiple myeloma (MM), a neoplastic expansion of plasma cells, as a model to investigate these issues. MM cells express high levels of surface CD38 and grow in an environment prevalently represented by closed niches hosted in the bone marrow (BM). An original approach of this study derives from the recent use of the clinical availability of therapeutic anti-CD38 monoclonal antibodies (mAbs) in perturbing tumor viability and enzymatic functions in conditions mimicking what happens in vivo.

Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with 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-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a 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.

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.

NOD SCID (NS) mice are commonly used for the assessment of monoclonal antibody (mAb) therapeutics and form the genetic background of NSG mice widely used in human patient derived xenograft models. However, relatively little is known regarding how the NS background effects monoclonal antibody (mAb) pharmacokinetics and how this might influence therapeutic response. To address this we examined mAb half-life and clearance of hCD20 transgenic EμTCL-1 tumours in NS mice following administration of anti-hCD20 hIgG1 mAb and showed mAb half-life to be significantly shorter in NS versus SCID mice, correlating with reduced therapy. Reduced half-life was also seen with mIgG2a but not mIgG1 or hIgG2 isotypes. Additionally, NS mice lacking CD32 or the FcR γ-chain demonstrated this rapid loss was dependent on the expression of CD32 and not activatory FcγRs. Sequencing confirmed the NOD strains carry the rare ly17.1 variant of CD32. Recombinant ly17.1 displayed increased affinity for hIgG1 compared to the more common ly17.2 variant, providing a potential mechanism for its more rapid removal from serum. Importantly, the reduced half-life was dependent on the presence of both SCID and NOD mutations. SCID mice lack endogenous IgG and so we reasoned this might accentuate the effects of the NOD CD32 polymorphic variant. Accordingly, reconstitution of NS mice with physiological levels of mIgG restored normal half-life. These findings highlight the importance of examining mAb pharmacokinetics in complex mouse strains and demonstrate that therapeutic studies in mice on the NS background should be performed in the presence of exogenous IgG.

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.

SummaryTherapeutic 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.